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Sample records for flere unge til

  1. Structural and biophysical analysis of interactions between cod and human uracil-DNA N-glycosylase (UNG) and UNG inhibitor (Ugi).

    PubMed

    Assefa, Netsanet Gizaw; Niiranen, Laila; Johnson, Kenneth A; Leiros, Hanna-Kirsti Schrøder; Smalås, Arne Oskar; Willassen, Nils Peder; Moe, Elin

    2014-08-01

    Uracil-DNA N-glycosylase from Atlantic cod (cUNG) shows cold-adapted features such as high catalytic efficiency, a low temperature optimum for activity and reduced thermal stability compared with its mesophilic homologue human UNG (hUNG). In order to understand the role of the enzyme-substrate interaction related to the cold-adapted properties, the structure of cUNG in complex with a bacteriophage encoded natural UNG inhibitor (Ugi) has been determined. The interaction has also been analyzed by isothermal titration calorimetry (ITC). The crystal structure of cUNG-Ugi was determined to a resolution of 1.9 Å with eight complexes in the asymmetric unit related through noncrystallographic symmetry. A comparison of the cUNG-Ugi complex with previously determined structures of UNG-Ugi shows that they are very similar, and confirmed the nucleotide-mimicking properties of Ugi. Biophysically, the interaction between cUNG and Ugi is very strong and shows a binding constant (Kb) which is one order of magnitude larger than that for hUNG-Ugi. The binding of both cUNG and hUNG to Ugi was shown to be favoured by both enthalpic and entropic forces; however, the binding of cUNG to Ugi is mainly dominated by enthalpy, while the entropic term is dominant for hUNG. The observed differences in the binding properties may be explained by an overall greater positive electrostatic surface potential in the protein-Ugi interface of cUNG and the slightly more hydrophobic surface of hUNG. PMID:25084329

  2. Structural and biophysical analysis of interactions between cod and human uracil-DNA N-glycosylase (UNG) and UNG inhibitor (Ugi)

    SciTech Connect

    Assefa, Netsanet Gizaw; Niiranen, Laila; Johnson, Kenneth A.; Leiros, Hanna-Kirsti Schrøder; Smalås, Arne Oskar; Willassen, Nils Peder; Moe, Elin

    2014-08-01

    A structural and biophysical study of the interactions between cod and human uracil-DNA N-glycosylase (UNG) and their inhibitor Ugi is presented. The stronger interaction between cod UNG and Ugi can be explained by a greater positive electrostatic surface potential. Uracil-DNA N-glycosylase from Atlantic cod (cUNG) shows cold-adapted features such as high catalytic efficiency, a low temperature optimum for activity and reduced thermal stability compared with its mesophilic homologue human UNG (hUNG). In order to understand the role of the enzyme–substrate interaction related to the cold-adapted properties, the structure of cUNG in complex with a bacteriophage encoded natural UNG inhibitor (Ugi) has been determined. The interaction has also been analyzed by isothermal titration calorimetry (ITC). The crystal structure of cUNG–Ugi was determined to a resolution of 1.9 Å with eight complexes in the asymmetric unit related through noncrystallographic symmetry. A comparison of the cUNG–Ugi complex with previously determined structures of UNG–Ugi shows that they are very similar, and confirmed the nucleotide-mimicking properties of Ugi. Biophysically, the interaction between cUNG and Ugi is very strong and shows a binding constant (K{sub b}) which is one order of magnitude larger than that for hUNG–Ugi. The binding of both cUNG and hUNG to Ugi was shown to be favoured by both enthalpic and entropic forces; however, the binding of cUNG to Ugi is mainly dominated by enthalpy, while the entropic term is dominant for hUNG. The observed differences in the binding properties may be explained by an overall greater positive electrostatic surface potential in the protein–Ugi interface of cUNG and the slightly more hydrophobic surface of hUNG.

  3. Opinion: uracil DNA glycosylase (UNG) plays distinct and non-canonical roles in somatic hypermutation and class switch recombination

    PubMed Central

    Yousif, Ashraf S.; Stanlie, Andre; Begum, Nasim A.

    2014-01-01

    Activation-induced cytidine deaminase (AID) is essential to class switch recombination (CSR) and somatic hypermutation (SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair complex, is required for CSR. The role of UNG in CSR and SHM is extremely controversial. AID deficiency in mice abolishes both CSR and SHM, while UNG-deficient mice have drastically reduced CSR but augmented SHM raising a possibility of differential functions of UNG in CSR and SHM. Interestingly, UNG has been associated with a CSR-specific repair adapter protein Brd4, which interacts with acetyl histone 4, γH2AX and 53BP1 to promote non-homologous end joining during CSR. A non-canonical scaffold function of UNG, but not the catalytic activity, can be attributed to the recruitment of essential repair proteins associated with the error-free repair during SHM, and the end joining during CSR. PMID:24994819

  4. Opinion: uracil DNA glycosylase (UNG) plays distinct and non-canonical roles in somatic hypermutation and class switch recombination.

    PubMed

    Yousif, Ashraf S; Stanlie, Andre; Begum, Nasim A; Honjo, Tasuku

    2014-10-01

    Activation-induced cytidine deaminase (AID) is essential to class switch recombination (CSR) and somatic hypermutation (SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair complex, is required for CSR. The role of UNG in CSR and SHM is extremely controversial. AID deficiency in mice abolishes both CSR and SHM, while UNG-deficient mice have drastically reduced CSR but augmented SHM raising a possibility of differential functions of UNG in CSR and SHM. Interestingly, UNG has been associated with a CSR-specific repair adapter protein Brd4, which interacts with acetyl histone 4, γH2AX and 53BP1 to promote non-homologous end joining during CSR. A non-canonical scaffold function of UNG, but not the catalytic activity, can be attributed to the recruitment of essential repair proteins associated with the error-free repair during SHM, and the end joining during CSR. PMID:24994819

  5. Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed

    PubMed Central

    Weeks, L D; Zentner, G E; Scacheri, P C; Gerson, S L

    2014-01-01

    Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG+/+ and UNG−/− cells. We report that despite identical growth rates and DSB repair capacities, UNG−/− cells accumulated significantly greater uracil and DSBs compared with UNG+/+ cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG+/+ cells. Furthermore, DSBs in UNG+/+ and UNG−/− cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG−/− cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG+/+ and UNG−/− cells treated with IC50-level pemetrexed. However, following pemetrexed exposure, UNG−/− but not UNG+/+ cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG−/− cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death. PMID:24503537

  6. Uracil-DNA Glycosylase UNG Promotes Tet-mediated DNA Demethylation.

    PubMed

    Xue, Jian-Huang; Xu, Gui-Fang; Gu, Tian-Peng; Chen, Guo-Dong; Han, Bin-Bin; Xu, Zhi-Mei; Bjørås, Magnar; Krokan, Hans E; Xu, Guo-Liang; Du, Ya-Rui

    2016-01-01

    In mammals, active DNA demethylation involves oxidation of 5-methylcytosine (5mC) into 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by Tet dioxygenases and excision of these two oxidized bases by thymine DNA glycosylase (TDG). Although TDG is essential for active demethylation in embryonic stem cells and induced pluripotent stem cells, it is hardly expressed in mouse zygotes and dispensable in pronuclear DNA demethylation. To search for other factors that might contribute to demethylation in mammalian cells, we performed a functional genomics screen based on a methylated luciferase reporter assay. UNG2, one of the glycosylases known to excise uracil residues from DNA, was found to reduce DNA methylation, thus activating transcription of a methylation-silenced reporter gene when co-transfected with Tet2 into HEK293T cells. Interestingly, UNG2 could decrease 5caC from the genomic DNA and a reporter plasmid in transfected cells, like TDG. Furthermore, deficiency in Ung partially impaired DNA demethylation in mouse zygotes. Our results suggest that UNG might be involved in Tet-mediated DNA demethylation. PMID:26620559

  7. HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins.

    PubMed

    Hrecka, Kasia; Hao, Caili; Shun, Ming-Chieh; Kaur, Sarabpreet; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Skowronski, Jacek

    2016-07-01

    HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4(DCAF1) E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4(DCAF1) E3. Thus, separate functions of HIV-1 Vpr usurp CRL4(DCAF1) E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host. PMID:27335459

  8. Comparative study on the larvicidal activity of drimane sesquiterpenes and nordrimane compounds against Drosophila melanogaster til-til.

    PubMed

    Montenegro, Ivan; Pino, Luis; Werner, Enrique; Madrid, Alejandro; Espinoza, Luis; Moreno, Luis; Villena, Joan; Cuellar, Mauricio

    2013-01-01

    Natural compounds from Drimys winteri Forst and derivatives exhibited larvicidal effects against Drosophila melanogaster til-til. The most active compound was isodrimenin (4). The highest lethal concentration to the larvae of D. melanogaster was 4.5 ± 0.8 mg/L. At very low concentrations drimenol (1), confertifolin (3), and drimanol (5) displayed antifeedant and larvae growth regulatory activity. The antifeedant results of nordrimanic and drimanic compounds were better in first instar larvae. The EC₅₀ value of polygodial (2) was 60.0 ± 4.2 mg/L; of diol 15 45.0 ± 2.8 mg/L, and of diol 17 36.9 ± 3.7 mg/L, while the new nordrimane compound 12 presented a value of 83.2 ± 3.5 mg/L. PMID:23612472

  9. Hearing in Drosophila Requires TilB, a Conserved Protein Associated With Ciliary Motility

    PubMed Central

    Kavlie, Ryan G.; Kernan, Maurice J.; Eberl, Daniel F.

    2010-01-01

    Cilia were present in the earliest eukaryotic ancestor and underlie many biological processes ranging from cell motility and propulsion of extracellular fluids to sensory physiology. We investigated the contribution of the touch insensitive larva B (tilB) gene to cilia function in Drosophila melanogaster. Mutants of tilB exhibit dysfunction in sperm flagella and ciliated dendrites of chordotonal organs that mediate hearing and larval touch sensitivity. Mutant sperm axonemes as well as sensory neuron dendrites of Johnston's organ, the fly's auditory organ, lack dynein arms. Through deficiency mapping and sequencing candidate genes, we identified tilB mutations in the annotated gene CG14620. A genomic CG14620 transgene rescued deafness and male sterility of tilB mutants. TilB is a 395-amino-acid protein with a conserved N-terminal leucine-rich repeat region at residues 16–164 and a coiled-coil domain at residues 171–191. A tilB-Gal4 transgene driving fluorescently tagged TilB proteins elicits cytoplasmic expression in embryonic chordotonal organs, in Johnston's organ, and in sperm flagella. TilB does not appear to affect tubulin polyglutamylation or polyglycylation. The phenotypes and expression of tilB indicate function in cilia construction or maintenance, but not in intraflagellar transport. This is also consistent with phylogenetic association of tilB homologs with presence of genes encoding axonemal dynein arm components. Further elucidation of tilB functional mechanisms will provide greater understanding of cilia function and will facilitate understanding ciliary diseases. PMID:20215474

  10. A phase I trial of repeated tumour-infiltrating lymphocyte (TIL) infusion in metastatic melanoma.

    PubMed Central

    Ravaud, A.; Legrand, E.; Delaunay, M. M.; Bussières, E.; Coulon, V.; Cany, L.; Huet, S.; Verdier, D.; Kind, M.; Chomy, F.

    1995-01-01

    The aim of the protocol was to evaluate the side-effects induced by repeated tumour-infiltrating lymphocyte (TIL) infusions in patients with metastatic melanoma (MM). Patients were to receive four TIL infusions at given intervals: every 3 weeks (two patients), every 2 weeks (3 patients) and weekly (4 patients). All patients were evaluated and received a total of 34 TIL infusions. The total number of TILs administered varied from 0.65 to 2.34 x 10(11) cells. TIL phenotypes were predominantly CD8+ (two patients), CD4+ (4 patients), CD4+ then CD8+ (two patients) or CD56+ (two patient). Autocytotoxicity was only observed for one culture. Six patients presented at least one WHO grade 3 side-effect: hypotension (5 patients), dyspnoea (two patients), fever (one patient), fatigue (one patient), chills (two patients), diarrhoea (one patient), agitation (one patient), locoregional pain (two patients). Hypotension was constantly seen in patients who were given TILs every week. Two cases of minor pericarditis were recorded. No objective response to treatment was observed; 1 stable disease occurred in one patient and progression in eight. However, five patients presented a partial response on a tumour site for 1-4 months. Three patients presented signs of inflammation or softening at one tumour site. Plasma tumour necrosis factor alpha (TNF-alpha) levels were increased 1.2- to 22-fold after TIL infusion. TILs could be produced in sufficient quantity to perform this study, so repetitive infusions of TIL became possible on a weekly basis. However, no objective response was observed even when TIL infusions were performed weekly. An increase in circulating TNF-alpha was noted after TIL infusion. PMID:7530984

  11. The Educational Thought of Cornelius Van Til: Philosophical Foundations of the Contemporary Christian School Movement.

    ERIC Educational Resources Information Center

    Maffet, Gregory J.; Dye, Charles M.

    This paper gives an account of the thoughts of Cornelius Van Til on the contemporary Christian school movement. An account of the historical development of Christian compromise is given, followed by a critique of the compromise among contemporary Christian educators. Van Til claims that any educational position which falls short of being founded…

  12. Alcohol and drug use among adolescents: and the co-occurrence of mental health problems. Ung@hordaland, a population-based study

    PubMed Central

    Skogen, Jens Christoffer; Sivertsen, Børge; Lundervold, Astri J; Stormark, Kjell Morten; Jakobsen, Reidar; Hysing, Mari

    2014-01-01

    Objectives The use of alcohol and drugs is prevalent among adolescents, but too little is known about the association between debut of alcohol and drug use, problematic use and concurrent mental health. The aim of the study was to investigate the cross-sectional association between debut of any alcohol or drug use and alcohol-related and drug-related problems and mental health. We also wanted to examine potential interactions between gender and age, and alcohol-related and drug-related variables. Design Cross-sectional study. Setting Population-based sample of Norwegian adolescents. Participants Data stem from the large population-based ung@hordaland study (N=9203), where all adolescents aged 17–19 years living in Hordaland county (Norway) were invited to participate. The main independent variables were debut of alcohol and drug use, alcohol consumption and the presence of alcohol and drug problems as measured by CRAFFT. Outcomes The dependent variables were self-reported symptoms of anxiety, depression, inattention and hyperactivity. Statistical analyses included logistic regression models. Results Debut of alcohol and drug use were associated with symptoms of depression, inattention and hyperactivity (crude ORs 1.69–2.38, p<0.001), while only debut of drug use was associated with increased symptoms of anxiety (OR=1.33, CI 95% 1.05 to 1.68, p=0.017). Alcohol-related and drug-related problems as measured by CRAFFT were associated with all mental health problems (crude ORs 1.68–3.24, p<0.001). There was little evidence of any substantial age or gender confounding on the estimated associations between alcohol-related and drug-related measures and mental health problems. Conclusions Early debut of alcohol and drug use and drug problems is consistently associated with more symptoms of mental health problems, indicating that these factors are an important general indicator of mental health in adolescence. PMID:25245403

  13. Aircraft borne combined measurements of the Fukushima radionuclide Xe-133 and fossil fuel combustion generated pollutants in the TIL - Implications for Cyclone induced lift and TIL physical-chemical processes

    NASA Astrophysics Data System (ADS)

    Arnold, Frank; Schlager, Hans; Simgen, Hardy; Aufmhoff, Heinfried; Baumann, Robert; Lindemann, Sigfried; Rauch, Ludwig; Kaether, Frank; Pirjolla, Liisa; Schumann, Ulrich

    2013-04-01

    The radionuclide Xe-133, released by the March 2011 nuclear disaster at Fukushima/Daiichi (hereafter FD), represents an ideal tracer for atmospheric transport. We report the, to our best knowledge, only aircraft borne measurements of FD Xe-133 in the Tropopause Inversion Layer (TIL), indicating rapid lift of Xe-133 rich planetary boundary layer air to the TIL. On the same research aircraft (FALCON), we have also conducted on-line measurements of fossil fuel combustion generated pollutant gases (SO2, NOx, HNO3,NOy), which were found to have increased concentrations in the TIL. In addition, we have conducted supporting model simulations of transport, chemical processes, and aerosol processes. Our investigations reveal a potentially important influence of East-Asian cyclone induced pollutants transport to the TIL, particularly influencing aerosol formation in the TIL.

  14. [On atrophy. A doctoral dissertation by Elias Til-landz in 1670].

    PubMed

    Kallinen, M

    1998-01-01

    Elias Til-landz is known as the most industrious professor of medicine at the Academy of Turku (Abo) during the 17th century. This article describes his student years in Uppsala and Leiden, and surveys the contents of his hitherto unanalysed doctoral thesis De Athrophia as evidence of his intellectual connections. Til-landz (originally called Tillander) studied philosophy at the Academy of Turku and medicine in Uppsala. Medicine attracted only a handful of students at Turku, most probably because of the low status of the occupation and the scarce opportunities for career. At the University of Uppsala - the biggest university of 17th-century Sweden - the professors of medicine were prominent men in their field and well versed in the new philosophical currents as well. It was neverless usual for Swedish medical students in the 17th century to complete their studies abroad, especially at the famous University of Leiden. It is probable that Til-landz had written his doctoral thesis himself. He defended it in Leiden on 11th March 1670 under the formal guidance of J.F. Gronovius, who was professor of the Greek language and history. In his thesis Til-landz shows intimate knowledge of the new medical authorities of the age, such as Walter Charleton, Francis Glisson, Santorio Santorio, etc. His approach to physiology follows Franciscus De le Boe Sylvius's iatrochemical theories. In Til-landz's view atrophy can affect either the whole body (general atrophy) or some parts only (special atrophy). It is caused by the lack or poor quality of blood and is mostly cured by diet, emesis or bathing. PMID:11625410

  15. [The life of Choe Ung-sok: with a focus on his design for and role in the health care system immediately after the liberation].

    PubMed

    Shin, Young-Joen; Kim, Jinhyouk

    2014-12-01

    Born in Pyongyang in 1914, Choe Ung-sok was a physician who lived through the Japanese colonial era (1910-1945), rule by the United States Army Military Government in Korea (USAMGIK; 1945-1948), and national division (1948). Influenced by socialism and social hygiene/social medicine during his studies in Japan, he played the role of representing the socialist camp in the discussions related to the construction of a heath care system immediately following the Liberation (1945). His key arguments were: first, the nationalization of the medical system and the implementation of nationwide programs to eradicate diseases; second, the provision of free medical services through the expansion of social insurance; third, the reeducation of the medical personnel; fourth, the provision of social sciences education to the medical personnel and the reorganization of medicine into preventive medicine; fifth, the nationalization of pharmaceutics; sixth, the laborers' establishment of autonomous medical organs (affordable clinics, medical consumers' unions through cooperatives); and seventh, the reduction of work hours to 6-8 hours, technical improvement, respite from research, and guarantee of economic life for the medical personnel. Influenced by the medical systems of the Soviet Union and Japan, such arguments stood in opposition to the right wing's plan for the construction of a relatively passive health care system at the time but, in the end, failed to be realized in southern part of Korea under the USAMGIK. Subsequently, he defected to northern part of Korea and came to participate in the task of constructing North Korea's health care system. Choe's life and design for a health care system provide examples through which one can confirm the nature of social hygiene/social medicine both during the Japanese colonial era and before and after the Liberation and the contents of the design related to a health care system as held by the socialist faction. In addition, they show that

  16. Antigen-specific TIL therapy for melanoma: A flexible platform for personalized cancer immunotherapy.

    PubMed

    Kelderman, Sander; Heemskerk, Bianca; Fanchi, Lorenzo; Philips, Daisy; Toebes, Mireille; Kvistborg, Pia; van Buuren, Marit M; van Rooij, Nienke; Michels, Samira; Germeroth, Lothar; Haanen, John B A G; Schumacher, N M

    2016-06-01

    Tumor infiltrating lymphocyte (TIL) therapy has shown objective clinical response rates of 50% in stage IV melanoma patients in a number of clinical trials. Nevertheless, the majority of patients progress either directly upon therapy or after an initial period of tumor control. Recent data have shown that most TIL products that are used for therapy contain only low frequencies of T cells reactive against known melanoma-associated epitopes. Because of this, the development of a technology to create T-cell products that are enriched for reactivity against defined melanoma-associated antigens would seem valuable, both to evaluate the tumoricidal potential of T cells directed against different antigen classes and to potentially increase response rates. Here, we developed and validated a conditional MHC streptamer-based platform for the creation of TIL products with defined antigen reactivities. We have used this platform to successfully enrich both high-frequency (≥1%) and low-frequency (<1%) tumor-specific CD8(+) T-cell populations, and thereby created T-cell products with enhanced tumor recognition potential. Collectively, these data demonstrate that selection of antigen-specific T-cell populations can be used to create defined T-cell products for clinical use. This strategy thus forms a highly flexible platform for the development of antigen-specific cell products for personalized cancer immunotherapy. PMID:27005018

  17. Simplified Method of the Growth of Human Tumor Infiltrating Lymphocytes (TIL) in Gas-Permeable Flasks to Numbers Needed for Patient Treatment

    PubMed Central

    Jin, Jianjian; Sabatino, Marianna; Somerville, Robert; Wilson, John R.; Dudley, Mark E.; Stroncek, David F.; Rosenberg, Steven A.

    2012-01-01

    Adoptive cell therapy (ACT) of metastatic melanoma with autologous tumor infiltrating lymphocytes (TIL) is clinically effective, but TIL production can be challenging. Here we describe a simplified method for initial TIL culture and rapid expansion in gas-permeable flasks. TIL were initially cultured from tumor digests and fragments in 40 mL capacity flasks with a 10 cm2 gas-permeable silicone bottom, G-Rex10. A TIL rapid expansion protocol (REP) was developed using 500 mL capacity flasks with a 100 cm2 gas-permeable silicone bottom, G-Rex100. TIL growth was successfully initiated in G-Rex10 flasks from tumor digests from 13 of 14 patients and from tumor fragments in all 11 tumor samples tested. TIL could then be expanded to 8–10×109 cells in a two-step REP which began by seeding 5 × 106 TIL into a G-Rex100 flask, followed by expansion at day 7 into 3 G-Rex100 flasks. To obtain the 30 to 60 × 109 cells used for patient treatment we seeded 6 G-Rex100 flasks with 5×106 cells and expanded into 18 G-Rex100 flasks. Large scale TIL REP in gas-permeable flasks requires approximately 9 to 10 liters of media, about 3 to 4 times less than other methods. In conclusion, TIL initiation and REP in gas-permeable G-Rex flasks require fewer total vessels, less media, less incubator space and less labor than initiation and REP in 24-well plates, tissue culture flasks and bags. TIL culture in G-Rex flasks will facilitate the production of TIL at the numbers required for patient treatment at most cell processing laboratories. PMID:22421946

  18. Evaluation of particulate filtering respirators using inward leakage (IL) or total inward leakage (TIL) testing--Korean experience.

    PubMed

    Han, Don-Hee; Lee, Jinheon

    2005-10-01

    Korean certification regulation for particulate filtering respirators requires inward leakage (IL) or total inward leakage (TIL) testing according to European Standard EN 13274-1, and the standard levels of compliance are similar to those of the European Standard. This study was conducted to evaluate particulate filtering respirators being commercially used in the Korean market using an IL or TIL test and the validity of standard level in Korea. Three half masks and 10 filtering facepieces (two top class, four 1st class and four 2nd class)-a total of 13 brand name respirators-were selected for the test with panels of 10 subjects. Each subject was classified with nine facial dimension grid squares in accordance with face length and lip length. IL or TIL testing was conducted at the laboratory of the 3M Innovation Center in which the experimental instruments and systems were established in compliance with European standards. The testing procedure followed EN 13274-1 (2001). As expected, leakages of half masks were less than those of filtering facepieces and the latter were significantly different among brands. TILs of the 1st class filtering facepieces were found to be much more than those of the 2nd class and the result may cause a wearer to get confused when selecting a mask. The main route leakage for filtering facepieces may not be the filter medium but the face seal. Therefore, it is necessary to develop well-fitting filtering facepieces for Koreans. Because leakages were significantly different for different facial dimensions, a defined test panel for IL or TIL testing according to country or race should be developed. A more precise method to demonstrate fit, for example, fit testing such as in the US regulations, will be needed before IL or TIL testing or when selecting a respirator. Another finding implies that geometric mean of five exercises for IL or TIL may be better than arithmetic mean to establish a standard individual subject mean. PMID:16126767

  19. Checkpoint Antibodies but not T Cell-Recruiting Diabodies Effectively Synergize with TIL-Inducing γ-Irradiation.

    PubMed

    Hettich, Michael; Lahoti, Jayashree; Prasad, Shruthi; Niedermann, Gabriele

    2016-08-15

    T cell-recruiting bispecific antibodies (bsAb) show promise in hematologic malignancies and are also being evaluated in solid tumors. In this study, we investigated whether T cell-recruiting bsAbs synergize with hypofractionated tumor radiotherapy (hRT) and/or blockade of the programmed death-1 (PD-1) immune checkpoint, both of which can increase tumor-infiltrating lymphocyte (TIL) numbers. Unexpectedly, large melanomas treated with hRT plus bsAb (AC133×CD3) relapsed faster than those treated with hRT alone, accompanied by massive TIL apoptosis. This fast relapse was delayed by the further addition of anti-PD-1. Mechanistic investigations revealed restimulation-induced cell death mediated by BIM and FAS as an additional cause of bsAb-mediated TIL depletion. In contrast, the double combination of hRT and anti-PD-1 strongly increased TIL numbers, and even very large tumors were completely eradicated. Our study reveals the risk that CD3-engaging bsAbs can induce apoptotic TIL depletion followed by rapid tumor regrowth, reminiscent of tolerance induction by CD3 mAb-mediated T-cell depletion, warranting caution in their use for the treatment of solid tumors. Our findings also argue that combining radiotherapy and anti-PD-1 can be quite potent, including against very large tumors. Cancer Res; 76(16); 4673-83. ©2016 AACR. PMID:27302161

  20. A temperature induced lipocalin gene from Medicago falcata (MfTIL1) confers tolerance to cold and oxidative stress.

    PubMed

    He, Xueying; Sambe, Mame Abdou Nahr; Zhuo, Chunliu; Tu, Qinghua; Guo, Zhenfei

    2015-04-01

    Temperature-induced lipocalins (TIL) are plasmalemma-localized proteins and responsive to environmental stresses. Physiological functions of MfTIL1 from Medicago sativa subsp. falcata (L.) Arcang. (hereafter falcata), a forage legume with cold and drought tolerance, were investigated in this study. MfTIL1 expression was greatly induced by 4-96 h of cold treatment, while transcript levels of the orthologs in Medicago truncatula, a model legume plant with lower cold tolerance than falcata, were reduced or not altered within 48-96 h. MfTIL1 expression was not responsive to dehydration and salinity. Compared to the wild type, transgenic tobacco plants overexpressing MfTIL1 had lower temperature (LT50) that resulted in 50 % lethal and elevated survival rate in response to freezing, elevated F v/F m and decreased ion leakage after treatments with chilling, high light and methyl viologen (MV). H2O2 and O2 (-) were less accumulated in transgenic plants than in the wild type after treatments with chilling, high light and MV, while antioxidant enzyme activities showed no difference between the two types of plants prior to or following treatments. Higher transcript levels of NtDREB3 and NtDREB4 genes were observed in transgenic plants than in the wild type under non-stressed conditions, but higher transcript levels of NtDREB1, NtDREB2, NtDREB4 and NtCOR15a genes under chilling conditions. It is suggested that MfTIL1 plays an important role in plant tolerance to cold and oxidative stress through promoted scavenging of reactive oxygen species and up-regulating expression of multiple cold responsive genes. PMID:25744207

  1. The Value of Tumor Infiltrating Lymphocytes (TILs) for Predicting Response to Neoadjuvant Chemotherapy in Breast Cancer: A Systematic Review and Meta-Analysis

    PubMed Central

    Mao, Yan; Qu, Qing; Zhang, Yuzi; Liu, Junjun; Chen, Xiaosong; Shen, Kunwei

    2014-01-01

    Background We carried out a systematic review and meta-analysis to evaluate the predictive roles of tumor infiltrating lymphocytes (TILs) in response to neoadjuvant chemotherapy (NAC) in breast cancer. Method A PubMed and Web of Science literature search was designed. Random or fixed effect models were adopted to estimate the summary odds ratio (OR). Heterogeneity and sensitivity analyses were performed to explore heterogeneity among studies and to assess the effects of study quality. Publication bias was evaluated using a funnel plot, Egger's test and Begg's test. We included studies where the predictive significance of TILs, and/or TILs subset on the pathologic complete response (pCR) were determined in NAC of breast cancer. Results A total of 13 published studies (including 3251 patients) were eligible. In pooled analysis, the detection of higher TILs numbers in pre-treatment biopsy was correlated with better pCR to NAC (OR = 3.93, 95% CI, 3.26–4.73). Moreover, TILs predicted higher pCR rates in triple negative (OR = 2.49, 95% CI: 1.61–3.83), HER2 positive (OR = 5.05, 95% CI: 2.86–8.92) breast cancer, but not in estrogen receptor (ER) positive (OR = 6.21, 95%CI: 0.86–45.15) patients. In multivariate analysis, TILs were still an independent marker for high pCR rate (OR = 1.41, 95% CI: 1.19–1.66). For TILs subset, higher levels of CD8+ and FOXP3+ T-lymphocytes in pre-treatment biopsy respectively predicted better pathological response to NAC (OR = 6.44, 95% CI: 2.52–16.46; OR = 2.94, 95% CI: 1.05–8.26). Only FOXP3+ lymphocytes in post-NAC breast tissue were a predictive marker for low pCR rate in univariate (OR = 0.41, 95% CI: 0.21–0.80) and multivariate (OR = 0.36, 95% CI: 0.13–0.95) analysis. Conclusion Higher TILs levels in pre-treatment biopsy indicated higher pCR rates for NAC. TILs subset played different roles in predicting response to NAC. PMID:25501357

  2. Distribution and prognostic relevance of tumor-infiltrating lymphocytes (TILs) and PD-1/PD-L1 immune checkpoints in human brain metastases

    PubMed Central

    Harter, Patrick N.; Bernatz, Simon; Scholz, Alexander; Zeiner, Pia S.; Zinke, Jenny; Kiyose, Makoto; Blasel, Stella; Beschorner, Rudi; Senft, Christian; Bender, Benjamin; Ronellenfitsch, Michael W.; Wikman, Harriet; Glatzel, Markus; Meinhardt, Matthias; Juratli, Tareq A.; Steinbach, Joachim P.; Plate, Karl H.; Wischhusen, Jörg; Weide, Benjamin; Mittelbronn, Michel

    2015-01-01

    The activation of immune cells by targeting checkpoint inhibitors showed promising results with increased patient survival in distinct primary cancers. Since only limited data exist for human brain metastases, we aimed at characterizing tumor infiltrating lymphocytes (TILs) and expression of immune checkpoints in the respective tumors. Two brain metastases cohorts, a mixed entity cohort (n = 252) and a breast carcinoma validation cohort (n = 96) were analyzed for CD3+, CD8+, FOXP3+, PD-1+ lymphocytes and PD-L1+ tumor cells by immunohistochemistry. Analyses for association with clinico-epidemiological and neuroradiological parameters such as patient survival or tumor size were performed. TILs infiltrated brain metastases in three different patterns (stromal, peritumoral, diffuse). While carcinomas often show a strong stromal infiltration, TILs in melanomas often diffusely infiltrate the tumors. Highest levels of CD3+ and CD8+ lymphocytes were seen in renal cell carcinomas (RCC) and strongest PD-1 levels on RCCs and melanomas. High amounts of TILs, high ratios of PD-1+/CD8+ cells and high levels of PD-L1 were negatively correlated with brain metastases size, indicating that in smaller brain metastases CD8+ immune response might get blocked. PD-L1 expression strongly correlated with TILs and FOXP3 expression. No significant association of patient survival with TILs was observed, while high levels of PD-L1 showed a strong trend towards better survival in melanoma brain metastases (Log-Rank p = 0.0537). In summary, melanomas and RCCs seem to be the most immunogenic entities. Differences in immunotherapeutic response between tumor entities regarding brain metastases might be attributable to this finding and need further investigation in larger patient cohorts. PMID:26517811

  3. TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi.

    PubMed

    Li, Youshan; Zhao, Ping; Liu, Huawei; Guo, Xiaomeng; He, Huawei; Zhu, Rui; Xiang, Zhonghuai; Xia, Qingyou

    2015-02-01

    Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields. PMID:25453359

  4. Tumor-infiltrating lymphocytes (TILs) are a powerful prognostic marker in patients with triple-negative breast cancer enrolled in the IBCSG phase III randomized clinical trial 22-00.

    PubMed

    Pruneri, Giancarlo; Gray, Kathryn P; Vingiani, Andrea; Viale, Giuseppe; Curigliano, Giuseppe; Criscitiello, Carmen; Láng, István; Ruhstaller, Thomas; Gianni, Lorenzo; Goldhirsch, Aron; Kammler, Roswitha; Price, Karen N; Cancello, Giuseppe; Munzone, Elisabetta; Gelber, Richard D; Regan, Meredith M; Colleoni, Marco

    2016-07-01

    The purpose of this study was to assess the prognostic and predictive value of tumor-infiltrating lymphocytes (TILs) in the triple-negative breast cancer (TNBC) cohort of the phase III IBCSG trial 22-00, comparing low-dose oral 'metronomic' cyclophosphamide-methotrexate maintenance chemotherapy (CM-maintenance) to no-CM-maintenance in early breast cancer. TILs were evaluated in full-face hematoxylin-and-eosin-stained sections of tumor samples confirmed centrally as TNBC (< 1 % of ER and PgR immunoreactivity and absence of HER2 overexpression or amplification). Mononuclear cells were evaluated in the stromal area within the borders of the invasive tumor. The primary endpoint was breast cancer-free interval (BCFI). Cox proportional hazards regression model assessed the association of BCFI and secondary endpoints with TILs score. In the 647 tumor samples, the median percentage of TILs was 18 % (IQR = 8-40 %), with 18 % having TILs ≥ 50 % (lymphocyte-predominant breast cancer, LPBC). At a median follow-up of 6.9 years, TILs were associated with better prognosis. For every 10 % increase of TILs, BCFI risk reduction was 13 % (HR 0.87, 95 % CI 0.79-0.95,P = 0.003). DFS, DRFI, and OS risk reductions were 11 % (P = 0.005), 16 % (P = 0.003), and 17 % (P < 0.001), respectively. Multivariable analysis confirmed the independent prognostic value of TILs. No significant TILs-by-treatment interaction was observed (P = 0.39) for associations of TILs with BCFI, although patients with LPBC receiving CM-maintenance had a greater breast cancer risk reduction (HR 0.64,95 % CI 0.23-1.78) than those with non-LPBC (TILs < 50 %) (HR 0.96, 95 % CI 0.67-1.40). TILs score is a potent prognostic factor in patients with TNBC. Low-dose chemotherapy confers a greater (not statistically significant) clinical benefit in patients with LPBC. PMID:27372069

  5. New anatase-type Til-2xNbxAlxO2 solid solution nanoparticles: direct formation, phase stability, and photocatalytic performance.

    PubMed

    Hirano, Masanori; Ito, Takaharu

    2006-12-01

    New anatase-type titania solid solutions co-doped with niobium and aluminum (Til-2xNbxAIlxO2 (X = 0 -0.20)) were synthesized as nanoparticles from precursor solutions of TiOSO4, NbCl5, and Al(NO3)3 under mild hydrothermal conditions at 180 degrees C for 5 h using the hydrolysis of urea. The lattice parameters a0 and c0 of anatase slightly and gradually increased, when the content of niobium and aluminum increased from X = 0 to 0.20. The crystallite size of anatase increased from 12 to 28 nm with increasing the value of X from 0 to 0.20. Their photocatalytic activity and adsorptivity were evaluated separately by the measurement of the concentration of methylene blue (MB) remained in the solution in the dark or under UV-light irradiation. The adsorptivity of TiO2 was improved by the formation of anatase-type Til-2xNbxAlxO2 solid solutions. The photocatalytic activity of anatase-type Til-2xNbxAlxO2 solid solutions was superior to that of commercially available anatase-type pure TiO2 (ST-01) and anatase-type pure TiO2 hydrothermally prepared. The new anatase phase of Til-2xNbxAlxO2 (X = 0-0.20) solid solutions existed stably up to 850 0C during heat treatment in air. In comparison with hydrothermal pure TiO2, the starting temperature of anatase-to-rutile phase transformation was delayed by the formation of Ti1-2xNbxAlxO, (X = 0-0.20) solid solutions, although its completing temperature was accelerated. PMID:17256336

  6. Seminal plasma induces inflammation in the uterus through the γδ T/IL-17 pathway

    PubMed Central

    Song, Zhi-Hui; Li, Zhong-Yin; Li, Dan-Dan; Fang, Wen-Ning; Liu, Hai-Yan; Yang, Dan-Dan; Meng, Chao-Yang; Yang, Ying; Peng, Jing-Pian

    2016-01-01

    After insemination, a large number of leukocytes migrate into the uterus, which is accompanied by intense inflammation. However, the details of how seminal plasma interacts with the uterus are still not very clear. Here, we present that neutrophils migrate and accumulate around the uterine epithelium following insemination, which is accompanied by an increase in interleukin (IL) 17A levels. Additionally, we find that γδ T cells are the major source of IL-17A, and the seminal plasma could induce the γδ T cells to secret IL-17A. Blocking IL-17A could reduce the number of neutrophils in the uterus and prevent them from migrating to the epithelium by decreasing the chemokines CXCL1, CXCL2 and CXCL5. Blocking IL-17A did not affect the Th1/Th2 balance but actually diminished the inflammation in the uterus by reducing the expression of IL-1β and TNF-α. In summary, we found a new mechanism by which seminal plasma could influence the inflammation in the uterus through the γδ T/IL-17 pathway to regulate the expression of various chemokines and cytokines. PMID:27109934

  7. Seminal plasma induces inflammation in the uterus through the γδ T/IL-17 pathway.

    PubMed

    Song, Zhi-Hui; Li, Zhong-Yin; Li, Dan-Dan; Fang, Wen-Ning; Liu, Hai-Yan; Yang, Dan-Dan; Meng, Chao-Yang; Yang, Ying; Peng, Jing-Pian

    2016-01-01

    After insemination, a large number of leukocytes migrate into the uterus, which is accompanied by intense inflammation. However, the details of how seminal plasma interacts with the uterus are still not very clear. Here, we present that neutrophils migrate and accumulate around the uterine epithelium following insemination, which is accompanied by an increase in interleukin (IL) 17A levels. Additionally, we find that γδ T cells are the major source of IL-17A, and the seminal plasma could induce the γδ T cells to secret IL-17A. Blocking IL-17A could reduce the number of neutrophils in the uterus and prevent them from migrating to the epithelium by decreasing the chemokines CXCL1, CXCL2 and CXCL5. Blocking IL-17A did not affect the Th1/Th2 balance but actually diminished the inflammation in the uterus by reducing the expression of IL-1β and TNF-α. In summary, we found a new mechanism by which seminal plasma could influence the inflammation in the uterus through the γδ T/IL-17 pathway to regulate the expression of various chemokines and cytokines. PMID:27109934

  8. 'Til the Needle Breaks

    ERIC Educational Resources Information Center

    Hawkins, B. Denise

    2011-01-01

    After more than half a century, the music of Motown not only thrives, it transcends generations. The iconic sound of Motown has led a handful of scholars to write, teach, lecture and share the music, history and business of Motown on their campuses. In its golden age, from 1959 to 1972, the sound Berry Gordy pioneered at Motown Records in Detroit…

  9. Erschließung eines Marmorkarstvorkommens als mitteltiefer Erdwärmesondenspeicher im Tuxertal, Tirol

    NASA Astrophysics Data System (ADS)

    Sass, Ingo; Heldmann, Claus-Dieter; Lehr, Clemens

    2016-05-01

    Borehole heat exchangers can be economically beneficial for meeting heating and cooling demands of houses or buildings. In karst aquifers development of thermal storage and exchange systems may be problematic in terms of groundwater protection and storage design, due to possibly high groundwater velocities. The new development of the Hochstegen marble unit in the Tux Valley (Zillertal, Austria) was designed in two stages for the requested cooling and heating demands. An enhanced geothermal response test was completed using optical frequency domain reflectometry in an exploration drillhole. Additional studies focussing on local geology and hydrology were also conducted. Geothermal parameters obtained at precise depths allowed differentiating between conductive and convective heat flow and were correlated with the lithostratigraphically-conditioned karst characteristics. The borehole heat exchanger field was developed with nine 400 m deep dual U-shaped tube probes in 2013 for 1 GWh/a extraction and 400 MWh/a induction. Along with borehole geophysics and geothermal response tests, the study has provided relevant geothermal data for improving storage design and exploration.

  10. Erschließung eines Marmorkarstvorkommens als mitteltiefer Erdwärmesondenspeicher im Tuxertal, Tirol

    NASA Astrophysics Data System (ADS)

    Sass, Ingo; Heldmann, Claus-Dieter; Lehr, Clemens

    2016-06-01

    Borehole heat exchangers can be economically beneficial for meeting heating and cooling demands of houses or buildings. In karst aquifers development of thermal storage and exchange systems may be problematic in terms of groundwater protection and storage design, due to possibly high groundwater velocities. The new development of the Hochstegen marble unit in the Tux Valley (Zillertal, Austria) was designed in two stages for the requested cooling and heating demands. An enhanced geothermal response test was completed using optical frequency domain reflectometry in an exploration drillhole. Additional studies focussing on local geology and hydrology were also conducted. Geothermal parameters obtained at precise depths allowed differentiating between conductive and convective heat flow and were correlated with the lithostratigraphically-conditioned karst characteristics. The borehole heat exchanger field was developed with nine 400 m deep dual U-shaped tube probes in 2013 for 1 GWh/a extraction and 400 MWh/a induction. Along with borehole geophysics and geothermal response tests, the study has provided relevant geothermal data for improving storage design and exploration.

  11. Zeroing in on Tumor-Reactive TILs.

    PubMed

    Ohashi, Pamela S

    2016-09-01

    Adoptive cell transfer of tumor-specific T cells provides an effective strategy for cancer immunotherapy. An article in this issue provides a novel approach to refine this technology by identifying tumor-reactive T cells based on frequency and PD-1 expression. Cancer Immunol Res; 4(9); 719. ©2016 AACRSee article by Ascierto et al., p. 726. PMID:27590279

  12. William Van Til: The Last Progressive?

    ERIC Educational Resources Information Center

    Beineke, John A.

    2010-01-01

    In 1999, the changing goals of American schools were explored in "Education Week" through the events, achievements, and personalities that had formed United States education in the 20th century. First a series of articles, the collection was later published in book form as "Lessons of a Century: A Nation's Schools Come of Age." The chapter titled…

  13. Erkundung und Beweissicherung für eine geothermale Erschließung eines Alpinen Karstaquifers im Tuxertal, Österreich

    NASA Astrophysics Data System (ADS)

    Sass, Ingo; Heldmann, Claus-Dieter; Schäffer, Rafael

    2016-06-01

    Karst aquifers may on one hand improve the efficiency of geothermal systems due to increased permeabilities, but on the other hand, high groundwater velocities can reduce the efficiency of the underground heat storage capacity. The marble karst aquifer of the Hochstegen formation was explored and developed for the first time as an intermediate-depth geothermal energy storage system at Finkenberg, Tux valley (Tyrol, Austria). Geological field studies and a spring monitoring program for the project revealed characteristic hydro-chemical signatures related to the catchments in specific tectonic units depending on their lithology. Observations showed that the catchment area of the Hochstegen formation karst aquifer extends up to 2650 m a.s.l. southwest of Finkenberg. In the boreholes, karstification was detected to 400 m below surface (Sass et al., 2016). A monitoring program involving seven springs downgradient of the boreholes has shown that the geothermal project has had no long-term impact on groundwater quality.

  14. Erkundung und Beweissicherung für eine geothermale Erschließung eines Alpinen Karstaquifers im Tuxertal, Österreich

    NASA Astrophysics Data System (ADS)

    Sass, Ingo; Heldmann, Claus-Dieter; Schäffer, Rafael

    2016-02-01

    Karst aquifers may on one hand improve the efficiency of geothermal systems due to increased permeabilities, but on the other hand, high groundwater velocities can reduce the efficiency of the underground heat storage capacity. The marble karst aquifer of the Hochstegen formation was explored and developed for the first time as an intermediate-depth geothermal energy storage system at Finkenberg, Tux valley (Tyrol, Austria). Geological field studies and a spring monitoring program for the project revealed characteristic hydro-chemical signatures related to the catchments in specific tectonic units depending on their lithology. Observations showed that the catchment area of the Hochstegen formation karst aquifer extends up to 2650 m a.s.l. southwest of Finkenberg. In the boreholes, karstification was detected to 400 m below surface (Sass et al., 2016). A monitoring program involving seven springs downgradient of the boreholes has shown that the geothermal project has had no long-term impact on groundwater quality.

  15. Registration of TIL:383.13, TIL:625 and TIL:634, three long grain tropical Japonica Rice (Oryza sativa L.) germplasm lines containing novel Indica Alleles that increase tiller production and grain yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    These three breeding lines were from a set of 123 progeny lines that were released by the USDA-ARS in 2012 as a mapping population. Chromosomal regions containing genes for increased tiller number under greenhouse conditions were subsequently identified in this population. We used the molecular an...

  16. Absence of the Uracil DNA Glycosylase of Murine Gammaherpesvirus 68 Impairs Replication and Delays the Establishment of Latency In Vivo

    PubMed Central

    Minkah, Nana; Macaluso, Marc; Oldenburg, Darby G.; Paden, Clinton R.; White, Douglas W.; McBride, Kevin M.

    2015-01-01

    ABSTRACT Uracil DNA glycosylases (UNG) are highly conserved proteins that preserve DNA fidelity by catalyzing the removal of mutagenic uracils. All herpesviruses encode a viral UNG (vUNG), and yet the role of the vUNG in a pathogenic course of gammaherpesvirus infection is not known. First, we demonstrated that the vUNG of murine gammaherpesvirus 68 (MHV68) retains the enzymatic function of host UNG in an in vitro class switch recombination assay. Next, we generated a recombinant MHV68 with a stop codon in ORF46/UNGUNG) that led to loss of UNG activity in infected cells and a replication defect in primary fibroblasts. Acute replication of MHV68ΔUNG in the lungs of infected mice was reduced 100-fold and was accompanied by a substantial delay in the establishment of splenic latency. Latency was largely, yet not fully, restored by an increase in virus inoculum or by altering the route of infection. MHV68 reactivation from latent splenocytes was not altered in the absence of the vUNG. A survey of host UNG activity in cells and tissues targeted by MHV68 indicated that the lung tissue has a lower level of enzymatic UNG activity than the spleen. Taken together, these results indicate that the vUNG plays a critical role in the replication of MHV68 in tissues with limited host UNG activity and this vUNG-dependent expansion, in turn, influences the kinetics of latency establishment in distal reservoirs. IMPORTANCE Herpesviruses establish chronic lifelong infections using a strategy of replicative expansion, dissemination to latent reservoirs, and subsequent reactivation for transmission and spread. We examined the role of the viral uracil DNA glycosylase, a protein conserved among all herpesviruses, in replication and latency of murine gammaherpesvirus 68. We report that the viral UNG of this murine pathogen retains catalytic activity and influences replication in culture. The viral UNG was impaired for productive replication in the lung. This defect in expansion at the

  17. FcγRIII in ITP: it ain't over 'til it's over.

    PubMed

    McCrae, Keith R

    2016-01-01

    In this issue of Blood, Yu et al describe a novel anti–Fcγ receptor III (FcγRIII)-albumin fusion protein that inhibits the development of thrombocytopenia in a murine model of immune thrombocytopenia (ITP).1 The unique aspect of this protein is that it blocks FcγRIII-mediated uptake of antibody-coated platelets without activating FcγRIII and the associated inflammatory response. PMID:26744437

  18. ''Fake It 'Til You Make It'': Why Community College Students' Aspirations ''Hold Steady''

    ERIC Educational Resources Information Center

    Nielsen, Kelly

    2015-01-01

    Sociologists of education have explored the relationship between students' postsecondary aspirations and their propensity to get "cooled out" in community colleges. However, researchers have directed little attention to students whose aspirations remain stable over long periods of time or to the different roles that college degree goals…

  19. The Repeal of Section 28: It Ain't over 'til It's over

    ERIC Educational Resources Information Center

    Greenland, Katy; Nunney, Rosalind

    2008-01-01

    Section 28 (part of the Local Government Act of 1988) was a notorious piece of legislation that sought to prevent local education authorities in the UK from "promoting homosexuality". The effect of Section 28 was to create uncertainty and fear among teachers as to what was (and what was not) permitted in schools. Over time practitioners and…

  20. 'Til death parts us: women’s domestic partnerships in eighteenth-century Brittany.

    PubMed

    Locklin, Nancy

    2011-01-01

    This article investigates the legal provision for two adult, unmarried women to create a “perpetual society” with one another found in the customary code of 1725 for the French province of Brittany. This arrangement allowed women who shared a household to designate one another as primary heir and to protect their community property from the claims of others. Evidence of this arrangement demonstrates that single women in some places had options outside of marriage and the convent. The contracts filed by the women also reveal the extent to which this arrangement went beyond considerations of property to express both affection and loyalty. Available to siblings as well as to pairs of unrelated women, this union is likely not the equivalent of same-sex marriage. It does however broaden our knowledge of the meaning of marriage, partnership, and kin in early modern Europe. PMID:22250309

  1. The Nozoe Autograph Books: "It ain't over 'til it's over".

    PubMed

    Seeman, Jeffrey I; Johnson, Brian P

    2015-02-01

    All are welcome! This issue contains the final of the 15 segments of the original Nozoe Autograph Books. But this project couldn't just stop here. A modern extension of the autograph books-a 16(th) segment-will be published and will include signatures from around today's chemistry community. All are welcome to participate and add their mark on the legacy of the Nozoe Autograph Books. See the Essay for details. This Essay and the interactive website that accompanies the Nozoe Autograph Book project are available free-access for at least a three-year period at http://www.tcr.wiley-vch.de/nozoe. PMID:25690992

  2. X4 and R5 HIV-1 have distinct post-entry requirements for uracil DNA glycosylase during infection of primary cells.

    PubMed

    Jones, Kate L; Roche, Michael; Gantier, Michael P; Begum, Nasim A; Honjo, Tasuku; Caradonna, Salvatore; Williams, Bryan R G; Mak, Johnson

    2010-06-11

    It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is approximately 10-20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations. PMID:20371602

  3. X4 and R5 HIV-1 Have Distinct Post-entry Requirements for Uracil DNA Glycosylase during Infection of Primary Cells

    PubMed Central

    Jones, Kate L.; Roche, Michael; Gantier, Michael P.; Begum, Nasim A.; Honjo, Tasuku; Caradonna, Salvatore; Williams, Bryan R. G.; Mak, Johnson

    2010-01-01

    It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is ∼10–20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations. PMID:20371602

  4. Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase

    PubMed Central

    Yousif, Ashraf S.; Stanlie, Andre; Mondal, Samiran; Honjo, Tasuku; Begum, Nasim A.

    2014-01-01

    Activation-induced cytidine deaminase (AID) is essential to class-switch recombination (CSR) and somatic hypermutation (SHM) in both V region SHM and S region SHM (s-SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair (BER) complex, is required for CSR. Strikingly, however, UNG deficiency causes augmentation of SHM, suggesting involvement of distinct functions of UNG in SHM and CSR. Here, we show that noncanonical scaffold functions of UNG regulate s-SHM negatively and CSR positively. The s-SHM suppressive function of UNG is attributed to the recruitment of faithful BER components at the cleaved DNA locus, with competition against error-prone polymerases. By contrast, the CSR-promoting function of UNG enhances AID-dependent S-S synapse formation by recruiting p53-binding protein 1 and DNA-dependent protein kinase, catalytic subunit. Several loss-of-catalysis mutants of UNG discriminated CSR-promoting activity from s-SHM suppressive activity. Taken together, the noncanonical function of UNG regulates the steps after AID-induced DNA cleavage: error-prone repair suppression in s-SHM and end-joining promotion in CSR. PMID:24591630

  5. Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase.

    PubMed

    Yousif, Ashraf S; Stanlie, Andre; Mondal, Samiran; Honjo, Tasuku; Begum, Nasim A

    2014-03-18

    Activation-induced cytidine deaminase (AID) is essential to class-switch recombination (CSR) and somatic hypermutation (SHM) in both V region SHM and S region SHM (s-SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair (BER) complex, is required for CSR. Strikingly, however, UNG deficiency causes augmentation of SHM, suggesting involvement of distinct functions of UNG in SHM and CSR. Here, we show that noncanonical scaffold functions of UNG regulate s-SHM negatively and CSR positively. The s-SHM suppressive function of UNG is attributed to the recruitment of faithful BER components at the cleaved DNA locus, with competition against error-prone polymerases. By contrast, the CSR-promoting function of UNG enhances AID-dependent S-S synapse formation by recruiting p53-binding protein 1 and DNA-dependent protein kinase, catalytic subunit. Several loss-of-catalysis mutants of UNG discriminated CSR-promoting activity from s-SHM suppressive activity. Taken together, the noncanonical function of UNG regulates the steps after AID-induced DNA cleavage: error-prone repair suppression in s-SHM and end-joining promotion in CSR. PMID:24591630

  6. Defective repair of uracil causes telomere defects in mouse hematopoietic cells.

    PubMed

    Vallabhaneni, Haritha; Zhou, Fang; Maul, Robert W; Sarkar, Jaya; Yin, Jinhu; Lei, Ming; Harrington, Lea; Gearhart, Patricia J; Liu, Yie

    2015-02-27

    Uracil in the genome can result from misincorporation of dUTP instead of dTTP during DNA synthesis, and is primarily removed by uracil DNA glycosylase (UNG) during base excision repair. Telomeres contain long arrays of TTAGGG repeats and may be susceptible to uracil misincorporation. Using model telomeric DNA substrates, we showed that the position and number of uracil substitutions of thymine in telomeric DNA decreased recognition by the telomere single-strand binding protein, POT1. In primary mouse hematopoietic cells, uracil was detectable at telomeres, and UNG deficiency further increased uracil loads and led to abnormal telomere lengthening. In UNG-deficient cells, the frequencies of sister chromatid exchange and fragility in telomeres also significantly increased in the absence of telomerase. Thus, accumulation of uracil and/or UNG deficiency interferes with telomere maintenance, thereby underscoring the necessity of UNG-initiated base excision repair for the preservation of telomere integrity. PMID:25572391

  7. It ain`t over `til it`s over - building the auto/oil fuel quality bridge

    SciTech Connect

    Colucci, J.M.

    1995-05-01

    The nation`s situation regarding clean air, and the involvement of vehicles and fuels in providing cleaner air are discussed. There is no question that much has been done by both the automotive and fuels industries. But much remains to be done. In the early 1960s GM developed the first emission control device, the PCV valve. This device recycled crank-case vapors into the intake manifold and prevented them from escaping into the air. After that came engine controls in the late 1960s, and charcoal evaporation canisters and oxidizing catalytic converters in the 1970s. The catalytic converter made the greatest contribution to reducing emissions. Yet, is would not have been possible without unleaded gasoline. The oil industry made that happen and should take credit for it. In hindsight, it would have been smart to reformulate gasoline at the same time that it was made unleaded. Total cost would have been lower than doing the jobs separately, the air would have gotten cleaner quicker, and some of the more costly options for cleaning the air might have been avoided.

  8. Give 'til it hurts: trade-offs between immunity and male reproductive effort in the decorated cricket, Gryllodes sigillatus.

    PubMed

    Gershman, S N; Barnett, C A; Pettinger, A M; Weddle, C B; Hunt, J; Sakaluk, S K

    2010-04-01

    Trade-offs between life-history variables can be manifested at either the phenotypic or genetic level, with vastly different evolutionary consequences. Here, we examined whether male decorated crickets (Gryllodes sigillatus) from eight inbred lines and the outbred founder population from which they were derived, trade-off immune effort [lytic activity, phenoloxidase (PO) activity or encapsulation] to produce spermatophylaxes: costly nuptial food gifts essential for successful sperm transfer. Canonical correlation analysis of the outbred population revealed a trade-off between spermatophylax mass and lytic activity. Analysis of our inbred lines, however, revealed that although PO activity, encapsulation, body mass, spermatophylax mass and ampulla (sperm capsule) mass were all highly heritable, lytic activity was not, and there was, therefore, no negative genetic correlation between lytic activity and spermatophylax mass. Thus, males showed a phenotypic but not a genetic trade-off between spermatophylax mass and lytic activity, suggesting that this trade-off is mediated largely by environmental factors. PMID:20210833

  9. Fish gotta swim, Birds gotta fly, I gotta do Feynmann Graphs 'til I die: A continuum Theory of Flocking

    NASA Astrophysics Data System (ADS)

    Toner, John; Tu, Yu-Hai

    2002-05-01

    We have developed a new continuum dynamical model for the collective motion of large "flocks" of biological organisms (e.g., flocks of birds, schools of fish, herds of wildebeest, hordes of bacteria, slime molds, etc.) . This model does for flocks what the Navier-Stokes equation does for fluids. The model predicts that, unlike simple fluids, flocks show huge fluctuation effects in spatial dimensions d < 4 that radically change their behavior. In d=2, it is only these effects that make it possible for the flock to move coherently at all. This explains why a million wildebeest can march together across the Serengeti plain, despite the fact that a million physicists gathered on the same plane could NOT all POINT in the same direction. Detailed quantitative predictions of this theory agree beautifully with computer simulations of flock motion.

  10. Stream primary producers relate positively to watershed natural gas measures in north-central Arkansas streams.

    PubMed

    Austin, Bradley J; Hardgrave, Natalia; Inlander, Ethan; Gallipeau, Cory; Entrekin, Sally; Evans-White, Michelle A

    2015-10-01

    Construction of unconventional natural gas (UNG) infrastructure (e.g., well pads, pipelines) is an increasingly common anthropogenic stressor that increases potential sediment erosion. Increased sediment inputs into nearby streams may decrease autotrophic processes through burial and scour, or sediment bound nutrients could have a positive effect through alleviating potential nutrient limitations. Ten streams with varying catchment UNG well densities (0-3.6 wells/km(2)) were sampled during winter and spring of 2010 and 2011 to examine relationships between landscape scale disturbances associated with UNG activity and stream periphyton [chlorophyll a (Chl a)] and gross primary production (GPP). Local scale variables including light availability and water column physicochemical variables were measured for each study site. Correlation analyses examined the relationships of autotrophic processes and local scale variables with the landscape scale variables percent pasture land use and UNG metrics (well density and well pad inverse flow path length). Both GPP and Chl a were primarily positively associated with the UNG activity metrics during most sample periods; however, neither landscape variables nor response variables correlated well with local scale factors. These positive correlations do not confirm causation, but they do suggest that it is possible that UNG development can alleviate one or more limiting factors on autotrophic production within these streams. A secondary manipulative study was used to examine the link between nutrient limitation and algal growth across a gradient of streams impacted by natural gas activity. Nitrogen limitation was common among minimally impacted stream reaches and was alleviated in streams with high UNG activity. These data provide evidence that UNG may stimulate the primary production of Fayetteville shale streams via alleviation of N-limitation. Restricting UNG activities from the riparian zone along with better enforcement of

  11. Effects of vaccinia virus uracil DNA glycosylase catalytic site and deoxyuridine triphosphatase deletion mutations individually and together on replication in active and quiescent cells and pathogenesis in mice

    PubMed Central

    De Silva, Frank S; Moss, Bernard

    2008-01-01

    Background Low levels of uracil in DNA result from misincorporation of dUMP or cytosine deamination. Vaccinia virus (VACV), the prototype poxvirus, encodes two enzymes that can potentially reduce the amount of uracil in DNA. Deoxyuridine triphosphatase (dUTPase) hydrolyzes dUTP, generating dUMP for biosynthesis of thymidine nucleotides while decreasing the availability of dUTP for misincorporation; uracil DNA glycosylase (UNG) cleaves uracil N-glycosylic bonds in DNA initiating base excision repair. Studies with actively dividing cells showed that the VACV UNG protein is required for DNA replication but the UNG catalytic site is not, whereas the dUTPase gene can be deleted without impairing virus replication. Recombinant VACV with an UNG catalytic site mutation was attenuated in vivo, while a dUTPase deletion mutant was not. However, the importance of the two enzymes for replication in quiescent cells, their possible synergy and roles in virulence have not been fully assessed. Results VACV mutants lacking the gene encoding dUTPase or with catalytic site mutations in UNG and double UNG/dUTPase mutants were constructed. Replication of UNG and UNG/dUTPase mutants were slightly reduced compared to wild type or the dUTPase mutant in actively dividing cells. Viral DNA replication was reduced about one-third under these conditions. After high multiplicity infection of quiescent fibroblasts, yields of wild type and mutant viruses were decreased by 2-logs with relative differences similar to those observed in active fibroblasts. However, under low multiplicity multi-step growth conditions in quiescent fibroblasts, replication of the dUTPase/UNG mutant was delayed and 5-fold lower than that of either single mutant or parental virus. This difference was exacerbated by 1-day serial passages on quiescent fibroblasts, resulting in 2- to 3-logs lower titer of the double mutant compared to the parental and single mutant viruses. Each mutant was more attenuated than a revertant

  12. Breeding Value of the qSB9b and qSB12a QTLs in RiceBreeding Value of the qSB9b and qSB12a QTLs in Rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sheath blight (SB) caused by Rhizoctonia solani Kuhn is a serious rice disease worldwide. The results of 123 TeQing-into-Lemont (TILs) showed those with introgressions containing qSB9b and/or qSB12a were among the most SB resistant TILs. TIL:615, TIL:642 and TIL:567 have consistently appeared modera...

  13. Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase

    PubMed Central

    Endres, Matthias; Biniszkiewicz, Detlev; Sobol, Robert W.; Harms, Christoph; Ahmadi, Michael; Lipski, Andreas; Katchanov, Juri; Mergenthaler, Philipp; Dirnagl, Ulrich; Wilson, Samuel H.; Meisel, Andreas; Jaenisch, Rudolf

    2004-01-01

    Uracil-DNA glycosylase (UNG) is involved in base excision repair of aberrant uracil residues in nuclear and mitochondrial DNA. Ung knockout mice generated by gene targeting are viable, fertile, and phenotypically normal and have regular mutation rates. However, when exposed to a nitric oxide donor, Ung–/– fibroblasts show an increase in the uracil/cytosine ratio in the genome and augmented cell death. After combined oxygen-glucose deprivation, Ung–/– primary cortical neurons have increased vulnerability to cell death, which is associated with early mitochondrial dysfunction. In vivo, UNG expression and activity are low in brains of naive WT mice but increase significantly after reversible middle cerebral artery occlusion and reperfusion. Moreover, major increases in infarct size are observed in Ung–/– mice compared with littermate control mice. In conclusion, our results provide compelling evidence that UNG is of major importance for tissue repair after brain ischemia. PMID:15199406

  14. Uracil DNA Glycosylase Is Dispensable for Human Immunodeficiency Virus Type 1 Replication and Does Not Contribute to the Antiviral Effects of the Cytidine Deaminase Apobec3G

    PubMed Central

    Kaiser, Shari M.; Emerman, Michael

    2006-01-01

    It is well established that many host factors are involved in the replication of human immunodeficiency virus (HIV) type 1. One host protein, uracil DNA glycosylase 2 (UNG2), binds to multiple viral proteins and is packaged into HIV type 1 virions. UNG initiates the removal of uracils from DNA, and this has been proposed to be important both for reverse transcription and as a mediator to the antiviral effect of virion-incorporated Apobec3G, a cytidine deaminase that generates numerous uracils in the viral DNA during virus replication. We used a natural human UNG−/− cell line as well as cells that express a potent catalytic active-site inhibitor of UNG to assess the effects of removing UNG activity on HIV infectivity. In both cases, we find UNG2 activity and protein to be completely dispensable for virus replication. Moreover, we find that virion-associated UNG2 does not affect the loss of infectivity caused by Apobec3G. PMID:16378989

  15. Electrostatic interactions play an essential role in DNA repair and cold-adaptation of uracil DNA glycosylase.

    PubMed

    Olufsen, Magne; Smalås, Arne O; Brandsdal, Bjørn O

    2008-03-01

    Life has adapted to most environments on earth, including low and high temperature niches. The increased catalytic efficiency and thermoliability observed for enzymes from organisms living in constantly cold regions when compared to their mesophilic and thermophilic cousins are poorly understood at the molecular level. Uracil DNA glycosylase (UNG) from cod (cUNG) catalyzes removal of uracil from DNA with an increased k(cat) and reduced K(m) relative to its warm-active human (hUNG) counterpart. Specific issues related to DNA repair and substrate binding/recognition (K(m)) are here investigated by continuum electrostatics calculations, MD simulations and free energy calculations. Continuum electrostatic calculations reveal that cUNG has surface potentials that are more complementary to the DNA potential at and around the catalytic site when compared to hUNG, indicating improved substrate binding. Comparative MD simulations combined with free energy calculations using the molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) method show that large opposing energies are involved when forming the enzyme-substrate complexes. Furthermore, the binding free energies obtained reveal that the Michaelis-Menten complex is more stable for cUNG, primarily due to enhanced electrostatic properties, suggesting that energetic fine-tuning of electrostatics can be utilized for enzymatic temperature adaptation. Energy decomposition pinpoints the residual determinants responsible for this adaptation. PMID:18196298

  16. Don't Know What You've Got 'Til It's Gone? Skills-Led Qualifications, Secondary School Attainment and Policy Choices

    ERIC Educational Resources Information Center

    Harrison, Neil; James, David; Last, Kathryn

    2015-01-01

    In the name of curriculum breadth and raising standards, recent government policy in England has removed a large number of non-academic qualifications from the list of those that secondary schools can count in league tables, discouraging their use. Most of these were vocational qualifications, but they also include skills-led qualifications. This…

  17. Using TeQing-into-Lemont introgression lines (TILs) to dissect sheath blight resistance QTLs and fine-map a spreading culm gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have been using a set of TeQing-into-Lemont introgression lines to fine-map sheath blight resistance QTL. In the course of dissection a QTL region previously mapped on chromosome 9, we discovered a gene for spreading culms (or open plant-type) residing in one half of the QTL region, while the she...

  18. 'Faking til you make it': social capital accumulation of individuals on low incomes living in contrasting socio-economic neighbourhoods and its implications for health and wellbeing.

    PubMed

    Browne-Yung, Kathryn; Ziersch, Anna; Baum, Fran

    2013-05-01

    People on low-income living in low socio-economic neighbourhoods have poorer health in comparison with those living in advantaged neighbourhoods. To explore neighbourhood effects on health and social capital creation, the experiences of low-income people living in contrasting socio-economic neighbourhoods were compared, in order to examine how low-income status and differing levels of neighbourhood resources contributed to perceived health and wellbeing. Quantitative and qualitative data were analysed: survey data from 601 individuals living in contrasting socio-economic areas and in-depth interviews with a new sample of 24 individuals on low-incomes. The study was guided by Bourdieu's theory of practice, which examines how social inequalities are created and reproduced through the relationship between individuals' varying resources of economic, social and cultural capital. This included an examination of individual life histories, cultural distinction and how social positions are reproduced. Participants' accounts of their early life experience showed how parental socio-economic position and socially patterned events taking place across the life course, created different opportunities for social network creation, choice of neighbourhood and levels of resources available throughout life, all of which can influence health and wellbeing. A definition of poverty by whether an individual or household has sufficient income at a particular point in time was an inadequate measure of disadvantage. This static measure of 'low income' as a category disguised a number of different ways in which disadvantage was experienced or, conversely, how life course events could mitigate the impact of low-income. This study found that the resources necessary to create social capital such as cultural capital and the ability to socially network, differed according to the socio-economic status of the neighbourhood, and that living in an advantaged area does not automatically guarantee access to potentially beneficial social networks. PMID:23540360

  19. Mutations at Arginine 276 transform human uracil-DNA glycosylase into a single-stranded DNA-specific uracil-DNA glycosylase

    PubMed Central

    Chen, Cheng-Yao; Mosbaugh, Dale W.; Bennett, Samuel E.

    2011-01-01

    To investigate the role of Arginine 276 in the conserved leucine-loop of human uracil-DNA glycosylase (UNG), the effects of six R276 amino acid substitutions (C, E, H, L, W, and Y) on nucleotide flipping and enzyme conformational change were determined using transient and steady state, fluorescence-based, kinetic analysis. Relative to UNG, the mutant proteins exhibited a 2.6- to 7.7-fold reduction in affinity for a doubled-stranded oligonucleotide containing a pseudouracil residue opposite 2-aminopurine, as judged by steady-state DNA binding-base flipping assays. An anisotropy binding assay was utilized to determine the Kd of UNG and the R276 mutants for carboxyfluorescein-labeled uracil-containing single- and double-stranded oligonucleotides; the binding affinities varied 11-fold for single-stranded uracil-DNA, and 43-fold for double-stranded uracil-DNA. Productive uracil-DNA binding was monitored by rapid quenching of UNG intrinsic protein fluorescence. Relative to UNG, the rate of intrinsic fluorescence quenching of five mutant proteins for binding double-stranded uracil-DNA was reduced approximately 50%; the R276E mutant exhibited 1% of the rate of fluorescence quenching of UNG. When reacted with single-stranded uracil-DNA, the rate of UNG fluorescence quenching increased. Moreover, the rate of fluorescence quenching for all the mutant proteins, except R276E, was slightly faster than UNG. The kcat of the R276 mutants was comparable to UNG on single-stranded DNA and differentially affected by NaCl; however, kcat on double-stranded DNA substrate was reduced 4–12-fold and decreased sharply at NaCl concentrations as low as 20 mM. Taken together, these results indicate that the effects of mutations at Arg276 were largely limited to enzyme interactions with double-stranded uracil-containing DNA, and suggested that mutations at Arg276 effectively transformed UNG into a single-stranded DNA-specific uracil-DNA glycosylase. PMID:15970468

  20. Impact of Marcellus Shale natural gas development in southwest Pennsylvania on volatile organic compound emissions and regional air quality.

    PubMed

    Swarthout, Robert F; Russo, Rachel S; Zhou, Yong; Miller, Brandon M; Mitchell, Brittney; Horsman, Emily; Lipsky, Eric; McCabe, David C; Baum, Ellen; Sive, Barkley C

    2015-03-01

    The Marcellus Shale is the largest natural gas deposit in the U.S. and rapid development of this resource has raised concerns about regional air pollution. A field campaign was conducted in the southwestern Pennsylvania region of the Marcellus Shale to investigate the impact of unconventional natural gas (UNG) production operations on regional air quality. Whole air samples were collected throughout an 8050 km(2) grid surrounding Pittsburgh and analyzed for methane, carbon dioxide, and C1-C10 volatile organic compounds (VOCs). Elevated mixing ratios of methane and C2-C8 alkanes were observed in areas with the highest density of UNG wells. Source apportionment was used to identify characteristic emission ratios for UNG sources, and results indicated that UNG emissions were responsible for the majority of mixing ratios of C2-C8 alkanes, but accounted for a small proportion of alkene and aromatic compounds. The VOC emissions from UNG operations accounted for 17 ± 19% of the regional kinetic hydroxyl radical reactivity of nonbiogenic VOCs suggesting that natural gas emissions may affect compliance with federal ozone standards. A first approximation of methane emissions from the study area of 10.0 ± 5.2 kg s(-1) provides a baseline for determining the efficacy of regulatory emission control efforts. PMID:25594231

  1. Methane Emissions from Conventional and Unconventional Natural Gas Production Sites in the Marcellus Shale Basin.

    PubMed

    Omara, Mark; Sullivan, Melissa R; Li, Xiang; Subramanian, R; Robinson, Allen L; Presto, Albert A

    2016-02-16

    There is a need for continued assessment of methane (CH4) emissions associated with natural gas (NG) production, especially as recent advancements in horizontal drilling combined with staged hydraulic fracturing technologies have dramatically increased NG production (we refer to these wells as "unconventional" NG wells). In this study, we measured facility-level CH4 emissions rates from the NG production sector in the Marcellus region, and compared CH4 emissions between unconventional NG (UNG) well pad sites and the relatively smaller and older "conventional" NG (CvNG) sites that consist of wells drilled vertically into permeable geologic formations. A top-down tracer-flux CH4 measurement approach utilizing mobile downwind intercepts of CH4, ethane, and tracer (nitrous oxide and acetylene) plumes was performed at 18 CvNG sites (19 individual wells) and 17 UNG sites (88 individual wells). The 17 UNG sites included four sites undergoing completion flowback (FB). The mean facility-level CH4 emission rate among UNG well pad sites in routine production (18.8 kg/h (95% confidence interval (CI) on the mean of 12.0-26.8 kg/h)) was 23 times greater than the mean CH4 emissions from CvNG sites. These differences were attributed, in part, to the large size (based on number of wells and ancillary NG production equipment) and the significantly higher production rate of UNG sites. However, CvNG sites generally had much higher production-normalized CH4 emission rates (median: 11%; range: 0.35-91%) compared to UNG sites (median: 0.13%, range: 0.01-1.2%), likely resulting from a greater prevalence of avoidable process operating conditions (e.g., unresolved equipment maintenance issues). At the regional scale, we estimate that total annual CH4 emissions from 88 500 combined CvNG well pads in Pennsylvania and West Virginia (660 Gg (95% CI: 500 to 800 Gg)) exceeded that from 3390 UNG well pads by 170 Gg, reflecting the large number of CvNG wells and the comparably large fraction of

  2. [Research progress of tumor infiltrating lymphocytes in breast cancer].

    PubMed

    Huang, Jiahui; Chen, Xiaosong; Shen, Kunwei

    2015-09-01

    Breast cancer is a heterogeneous disease. The formation and progression of tumor and the sensitivity to treatment differs from patient to patient. In addition to the widely used molecular subtype, novel markers are needed to better personalize the treatment of breast cancer. Tumor infiltrating lymphocyte (TIL) have been consistently documented in breast cancer lesions especially in triple negative and human epidermal growth factor receptor-2 positive breast cancer. Several clinical trials have revealed that TIL are associated with prognosis and can predict therapeutic efficacy of special therapy. TIL could be divided to different subtypes including CD8 + TIL, CD4 + TIL, cytotoxic T lymphocyte-associated antigen-4 + TIL, programmed death-1 + TIL. They play different roles in the process of anti-tumor immunity and can predict different prognosis. Screening out special TIL subtype which is well associated with prognosis and therapeutic efficacy and developing targeting immunotherapy can help to improve outcomes of breast cancer patients. PMID:26654152

  3. Postsecondary Education Employment and Independent Living Outcomes of Persons with Autism and Intellectual Disability

    ERIC Educational Resources Information Center

    Ross, Jeffrey; Marcell, Jamia; Williams, Paula; Carlson, Dawn

    2013-01-01

    The aim of this study is to report employment and independent living outcomes of 125 graduates from the Taft College Transition to Independent Living (TIL) program. The TIL program has served students with intellectual and developmental disabilities, including autism spectrum disorder, since 1995. The TIL program follows graduates from the time of…

  4. Human gene transfer: Characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man

    SciTech Connect

    Kasid, A.; Morecki, S.; Aebersold, P.; Cornetta, K.; Culver, K.; Freeman, S.; Director, E.; Lotze, M.T.; Blaese, R.M.; Anderson, W.F.; Rosenberg, S.A. )

    1990-01-01

    Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible. The authors transduced human TILs with the bacterial gene for neomycin-resistance (Neo{sup R}) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact Neo{sup R} gene integration and expressed high levels of neomycin phosphotransferase activity. The Neo{sup R} gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for {beta}- and {gamma}-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the {beta} and {gamma} chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors ({alpha} and {beta}) and interleukin 2 receptor P55. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs.

  5. Efficient and reproducible generation of tumour-infiltrating lymphocytes for renal cell carcinoma

    PubMed Central

    Baldan, V; Griffiths, R; Hawkins, R E; Gilham, D E

    2015-01-01

    Background: Tumour-infiltrating lymphocyte (TIL) therapy is showing great promise in the treatment of patients with advanced malignant melanoma. However, the translation of TIL therapy to non-melanoma tumours such as renal cell carcinoma has been less successful with a major constraint being the inability to reproducibly generate TILs from primary and metastatic tumour tissue. Methods: Primary and metastatic renal cell carcinoma biopsies were subjected to differential tumour disaggregation methods and procedures that stimulate the specific expansion of TILs tested to determine which reliably generated TIL maintained antitumour specificity. Results: Enzymatic or combined enzymatic/mechanical disaggregation resulted in equivalent numbers of TILs being liberated from renal cell carcinoma biopsies. Following mitogenic activation of the isolated TILs with anti-CD3/anti-CD28-coated paramagnetic beads, successful TIL expansion was achieved in 90% of initiated cultures. The frequency of T-cell recognition of autologous tumours was enhanced when tumours were disaggregated using the GentleMACS enzymatic/mechanical system. Conclusion: TILs can be consistently produced from renal cell carcinoma biopsies maintaining autologous tumour recognition after expansion in vitro. While the method of disaggregation has little impact on the success of TIL growth, methods that preserve the cell surface architecture facilitate TIL recognition of an autologous tumour, which is important in terms of characterising the functionality of the expanded TIL population. PMID:25867267

  6. PRENATAL DEXAMETHASONE ADMINISTRATION DISRUPTS THE PATTERN OF CELLULAR DEVELOPMENT IN RAT LUNG

    EPA Science Inventory

    To examine whether prenatal exposure to glucocorticoids could adversely affect subsequent cellular development of the lung, we administered 0.2 mg/kg of dexamethasone to pregnant rats on gestational days 17, 18, and 19. ungs of the offspring were then examined for patterns of cel...

  7. ANALYSIS OF UNCERTAINTIES IN DOSE RECONSTRUCTION FROM BIOMARKERS: IMPACT ON STUDY DESIGN

    EPA Science Inventory

    The absorbed dose is defined as the quantity which has passed through the barriers (skin, GI tract, The absorbed dose of a pesticide can be estimated from its established urinary biomarker. ungs). For an exposure study, there are several options for biomarker collection, each w...

  8. Hepatitis B - Multiple Languages: MedlinePlus

    MedlinePlus

    ... ung thư gan - Tiếng Việt (Vietnamese) PDF Stanford University, Asian Liver Center Characters not displaying correctly on this page? See language display issues . Return to the MedlinePlus Health Information ...

  9. Liver Cancer - Multiple Languages: MedlinePlus

    MedlinePlus

    ... ung thư gan - Tiếng Việt (Vietnamese) PDF Stanford University, Asian Liver Center Characters not displaying correctly on this page? See language display issues . Return to the MedlinePlus Health Information ...

  10. Polymorphisms in uracil-processing genes, but not one-carbon nutrients, are associated with altered DNA uracil concentrations in an urban Puerto Rican population

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background. Five genes - UNG, SMUG1, MBD4, TDG and DUT - are involved in repair or prevention of uracil misincorporation into DNA, an anomaly that can cause mutagenic events leading to cancer. Little is known about the determinants of uracil misincorporation, including the effects of single-nucleoti...

  11. Oral Cancer - Multiple Languages: MedlinePlus

    MedlinePlus

    ... Oral Cancer English Ung Thư Miệng - Tiếng Việt (Vietnamese) PDF California Dental Association Characters not displaying correctly on this page? See language display issues . Return to the MedlinePlus Health Information ...

  12. Prostate Cancer - Multiple Languages: MedlinePlus

    MedlinePlus

    ... Cancer Ung Thư Tuyến Tiền Liệt - Tiếng Việt (Vietnamese) Bilingual PDF Health Information Translations Characters not displaying correctly on this page? See language display issues . Return to the MedlinePlus Health Information ...

  13. Single nucleotide polymorphisms in uracil-processing genes, intake of one-carbon nutrients and breast cancer risk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background/Objectives: The misincorporation of uracil into DNA leads to genomic instability. In a previous study, some of us identified four common single nucleotide polymorphisms (SNPs) in uracil-processing genes (rs2029166 and rs7296239 in SMUG1, rs34259 in UNG and rs4775748 in DUT) that were asso...

  14. Relevance of underground natural gas storage to geologic sequestration of carbon dioxide

    SciTech Connect

    Lippmann, Marcelo J.; Benson, Sally M.

    2002-07-01

    The practice of underground natural gas storage (UNGS), which started in the USA in 1916, provides useful insight into the geologic sequestration of carbon dioxide--the dominant anthropogenic greenhouse gas released into the atmosphere. In many ways, UNGS is directly relevant to geologic CO{sub 2} storage because, like CO{sub 2}, natural gas (essentially methane) is less dense than water. Consequently, it will tend to rise to the top of any subsurface storage structure located below the groundwater table. By the end of 2001 in the USA, about 142 million metric tons of natural gas were stored underground in depleted oil and gas reservoirs and brine aquifers. Based on their performance, UNGS projects have shown that there is a safe and effective way of storing large volumes of gases in the subsurface. In the small number of cases where failures did occur (i.e., leakage of the stored gas into neighboring permeable layers), they were mainly related to improper well design, construction, maintenance, and/or incorrect project operation. In spite of differences in the chemical and physical properties of the gases, the risk-assessment, risk-management, and risk-mitigation issues relevant to UNGS projects are also pertinent to geologic CO{sub 2} sequestration.

  15. Social Influence, Health Variables and Criminal Behaviours Associated with Substance Use among Rural Norwegian Adolescents

    ERIC Educational Resources Information Center

    Nordfjaern, Trond; Dahl, Hilde; Flemmen, Grete

    2013-01-01

    Aims: To investigate social influence, health, criminality and substance use in a sample of 1288 Norwegian rural adolescents. Relations between these factors and substance use were examined. Methods: Data were obtained from the "UngData" study. A cross-sectional questionnaire survey was conducted among adolescents (n = 740) in nine secondary…

  16. Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

    PubMed Central

    Shalhout, Sophia; Haddad, Dania; Sosin, Angela; Holland, Thomas C.; Al-Katib, Ayad; Martin, Alberto

    2014-01-01

    Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG−/− cells, uracil levels increase by 11- to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG−/−splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels. PMID:25154417

  17. Integrating the UAS in Undergraduate Teaching and Research - Opportunities and Challenges at University of North Georgia

    NASA Astrophysics Data System (ADS)

    Sharma, J. B.; Hulsey, D.

    2014-11-01

    The University of North Georgia (UNG) has begun to evaluate both fixed and rotary UAS platforms across the departments to evaluate their potential for furthering both student learning experiences and undergraduate research. A research project of the Institute for Spatial Analysis (IESA) at UNG has acquired the fixed wing eBee UAS and is currently piloting its integration into the undergraduate geospatial science curriculum. Limited very low altitude, line of sight calibration runs within areas of our campus have help us understand the capabilities that this technology brings to learning and research opportunities at UNG. In our pilot area of study on the UNG Gainesville Campus, we will collect overlapping imagery and generate 3-D models. These models will be compared with models based on LiDAR data. Geographic Object Based Image Analysis (GEOBIA) methods are essential to the analysis of voluminous high resolution UAS data and the associated computational and regulatory issues are discussed. Several future interdisciplinary projects are envisaged with the eBee UAS and this project helps establish their viability.

  18. Telling Their Stories: Women Construct/Instruct through Survival Rhetoric.

    ERIC Educational Resources Information Center

    Meagher, Eileen M.

    Malika Oufkir of Morocco recounts her story in "Stolen Lives." Loung Ung of Cambodia relates her story in "First, They Killed My Father." Susan McDougal of Arkansas, USA, tells her story in the aptly named, "The Woman Who Wouldn't Talk." This paper looks at the struggles of these three very different women from very different cultures, struggles…

  19. Increased CD4 and CD8-positive T cell infiltrate signifies good prognosis in a subset of triple-negative breast cancer.

    PubMed

    Matsumoto, Hirofumi; Thike, Aye Aye; Li, Huihua; Yeong, Joe; Koo, Si-Lin; Dent, Rebecca Alexandra; Tan, Puay Hoon; Iqbal, Jabed

    2016-04-01

    Tumour-infiltrating lymphocytes (TILs) signify immune response to tumour in a variety of cancers including breast cancer. However, earlier studies examining the clinical significance of TILs in breast cancers have generated mixed results. There are only a few that address the relationship between TILs and clinical outcomes in triple-negative breast cancers (TNBC). The aim of this study is to evaluate the clinical significance of TILs that express CD4 + and CD8 + , in TNBC. Immunohistochemical staining of CD4 and CD8 was performed on tissue microarrays of 164 cases of TNBC. TILs were counted separately as intratumoral when within the cancer cell nests (iTILs) and as stromal when within cancer stroma (sTILs). High CD8 + iTILs and sTILs, and CD4 + iTILs correlated with histologic grade. On Kaplan-Meier analysis, a significantly better survival rate was observed in high CD8 + iTIL (disease-free survival, DFS: P = 0.004, overall survival, OS: P = 0.02) and both high CD4 + iTILs (DFS: P = 0.025, OS: P = 0.023) and sTILs (DFS: P = 0.01, OS: P = 0.002). In multivariate analysis, CD8 + iTILs (DFS: P = 0.0095), CD4 + sTILs (DFS: P = 0.0084; OS: P = 0.0118), and CD4 (high) CD8 (high) CD8 iTILs (DFS: P = 0.0121; OS: P = 0.0329) and sTILs (DFS: P = 0.0295) showed significantly better survival outcomes. These results suggest that high levels of both CD8 + iTILs and CD4 + sTILs as well as CD4 (high) CD8 (high) iTILs and sTILs are independent prognostic factors in TNBC. PMID:26960711

  20. Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA.

    PubMed

    Burmeister, Wim P; Tarbouriech, Nicolas; Fender, Pascal; Contesto-Richefeu, Céline; Peyrefitte, Christophe N; Iseni, Frédéric

    2015-07-17

    Vaccinia virus polymerase holoenzyme is composed of the DNA polymerase catalytic subunit E9 associated with its heterodimeric co-factor A20·D4 required for processive genome synthesis. Although A20 has no known enzymatic activity, D4 is an active uracil-DNA glycosylase (UNG). The presence of a repair enzyme as a component of the viral replication machinery suggests that, for poxviruses, DNA synthesis and base excision repair is coupled. We present the 2.7 Å crystal structure of the complex formed by D4 and the first 50 amino acids of A20 (D4·A201-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison of the viral complex with its human counterpart revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar, suggesting a dominant role of the DNA conformation for UNG function. In contrast to human UNG, D4 appears rigid, and we do not observe a conformational change upon DNA binding. We also studied the interaction of D4·A201-50 with different DNA oligomers by surface plasmon resonance. D4 binds weakly to nonspecific DNA and to uracil-containing substrates but binds abasic sites with a Kd of <1.4 μm. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action. PMID:26045555

  1. Growth of tumor-infiltrating lymphocytes from human solid cancers: summary of a 5-year experience.

    PubMed

    Yannelli, J R; Hyatt, C; McConnell, S; Hines, K; Jacknin, L; Parker, L; Sanders, M; Rosenberg, S A

    1996-02-01

    Between 1989 and 1993, 255 tumor biopsies representing 4 tumor histologies (melanoma, breast cancer, colon cancer and renal cell cancer) were received by the Surgery Branch of the National Cancer Institute. Tumor-infiltrating lymphocytes (TIL) were grown from single-cell suspensions of tumor biopsies over the course of 30-45 days. The TIL were grown in medium containing IL-2. To obtain numbers suitable for therapy (>10(11)), TIL were expanded using a large-scale system of cell culture and harvesting. While the largest number of biopsies was obtained from melanoma patients, TIL were successfully grown from 160 of 255 tumor biopsies representing all 4 histologies. Under the culture conditions employed, several characteristics of TIL expansion were observed. The cell surface phenotype of TIL which grew out from the tumor biopsies was generally a mix of CD3+/CD4+ or CD3+/CD8+ lymphocytes. Only TIL from melanoma biopsies were found to be consistently cytolytic and, in many cases, lysed autologous tumor cells preferentially. Interestingly, TIL derived from extra-nodal sites of metastatic melanoma biopsies (subcutaneous, lung, bowel; 36 of 67, 54%) were more likely to have these cytolytic characteristics than TIL derived from tumor-involved lymph node biopsies (7 of 39, 18%). The present study summarizes 5 years of laboratory effort and validates the technologies developed for the large-scale growth and harvesting of TIL. In addition, it summarizes the laboratory effort supporting previously published clinical reports on TIL from our group. PMID:8621219

  2. Synoptic-scale behavior of the extratropical tropopause inversion layer

    NASA Astrophysics Data System (ADS)

    Pilch Kedzierski, Robin; Matthes, Katja; Bumke, Karl

    2015-11-01

    High-resolution GPS radio occultation temperature profiles from the COSMIC satellite mission (2007-2013) are used to obtain daily snapshots of the strength of the extratropical tropopause inversion layer (TIL). Its horizontal structure and day-to-day variability are linked to the synoptic situation at near-tropopause level. The strength of the TIL in cyclonic as well as anticyclonic conditions is investigated by separating relative vorticity into curl and shear terms. The analysis shows that the TIL has high zonal variability, and its strength is instantaneously adjusted to the synoptic situation at near-tropopause level. Our key finding is that the TIL within midlatitude ridges in winter is as strong as or stronger than the TIL in polar summer. The strongest TIL in anticyclonic conditions is related to the shear term, while the weaker TIL in cyclonic conditions is enhanced by the curl term.

  3. Adhesion of tumour-infiltrating lymphocytes to endothelium: a phenotypic and functional analysis.

    PubMed Central

    Adams, D. H.; Yannelli, J. R.; Newman, W.; Lawley, T.; Ades, E.; Rosenberg, S. A.; Shaw, S.

    1997-01-01

    Efficacy of cancer immunotherapy with cultured tumour-infiltrating lymphocytes (TILs) depends upon infused TILs migrating into tumour-bearing tissue, in which they mediate an anti-tumour response. For TILs to enter a tumour, they must first bind to tumour endothelium, and this process depends on TILs expressing and regulating the function of relevant cell-surface receptors. We analysed the cell-surface phenotype and endothelial binding of TILs cultured from human melanoma and compared them with peripheral blood T cells and with allostimulated T cells cultured under similar conditions. Compared with peripheral blood T cells, TILs expressed high levels of five integrins, two other adhesion molecules, including the skin homing molecule CLA, and several activation markers and showed markedly enhanced integrin-mediated adhesion to a dermal microvascular endothelial cell line in vitro. Compared with the allostimulated T cells, TILs expressed higher levels of the cutaneous lymphocyte antigen (CLA), the adhesion molecule CD31 and the activation markers CD30 and CD69, but lower levels of several other adhesion and activation molecules. These phenotypic and functional properties of TILs should have complex effects on their migration in vivo. Expression of CLA, the skin homing receptor, may increase migration to melanoma (a skin cancer), whereas integrin activation may cause non-specific binding of TILs to other endothelium. Manipulation of the culture conditions in which TILs are expanded might result in a phenotype that is more conducive to selective tumour homing in vivo. Images Figure 1 PMID:9166933

  4. Tumor infiltrating lymphocytes in ovarian cancer

    PubMed Central

    Santoiemma, Phillip P; Powell, Daniel J

    2015-01-01

    The accumulation of tumor infiltrating lymphocytes (TILs) in ovarian cancer is prognostic for increased survival while increases in immunosuppressive regulatory T-cells (Tregs) are associated with poor outcomes. Approaches that bolster tumor-reactive TILs may limit tumor progression. However, identifying tumor-reactive TILs in ovarian cancer has been challenging, though adoptive TIL therapy in patients has been encouraging. Other forms of TIL immunomodulation remain under investigation including Treg depletion, antibody-based checkpoint modification, activation and amplification using dendritic cells, antigen presenting cells or IL-2 cytokine culture, adjuvant cytokine injections, and gene-engineered T-cells. Many approaches to TIL manipulation inhibit ovarian cancer progression in preclinical or clinical studies as monotherapy. Here, we review the impact of TILs in ovarian cancer and attempts to mobilize TILs to halt tumor progression. We conclude that effective TIL therapy for ovarian cancer is at the brink of translation and optimal TIL activity may require combined methodologies to deliver clinically-relevant treatment. PMID:25894333

  5. Structural basis for translational fidelity ensured by transfer RNA lysidine synthetase.

    PubMed

    Nakanishi, Kotaro; Bonnefond, Luc; Kimura, Satoshi; Suzuki, Tsutomu; Ishitani, Ryuichiro; Nureki, Osamu

    2009-10-22

    Maturation of precursor transfer RNA (pre-tRNA) includes excision of the 5' leader and 3' trailer sequences, removal of introns and addition of the CCA terminus. Nucleotide modifications are incorporated at different stages of tRNA processing, after the RNA molecule adopts the proper conformation. In bacteria, tRNA(Ile2) lysidine synthetase (TilS) modifies cytidine into lysidine (L; 2-lysyl-cytidine) at the first anticodon of tRNA(Ile2) (refs 4-9). This modification switches tRNA(Ile2) from a methionine-specific to an isoleucine-specific tRNA. However, the aminoacylation of tRNA(Ile2) by methionyl-tRNA synthetase (MetRS), before the modification by TilS, might lead to the misincorporation of methionine in response to isoleucine codons. The mechanism used by bacteria to avoid this pitfall is unknown. Here we show that the TilS enzyme specifically recognizes and modifies tRNA(Ile2) in its precursor form, thereby avoiding translation errors. We identified the lysidine modification in pre-tRNA(Ile2) isolated from RNase-E-deficient Escherichia coli and did not detect mature tRNA(Ile2) lacking this modification. Our kinetic analyses revealed that TilS can modify both types of RNA molecule with comparable efficiencies. X-ray crystallography and mutational analyses revealed that TilS specifically recognizes the entire L-shape structure in pre-tRNA(Ile2) through extensive interactions coupled with sequential domain movements. Our results demonstrate how TilS prevents the recognition of tRNA(Ile2) by MetRS and achieves high specificity for its substrate. These two key points form the basis for maintaining the fidelity of isoleucine codon translation in bacteria. Our findings also provide a rationale for the necessity of incorporating specific modifications at the precursor level during tRNA biogenesis. PMID:19847269

  6. Prognostic Value of Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancers From Two Phase III Randomized Adjuvant Breast Cancer Trials: ECOG 2197 and ECOG 1199

    PubMed Central

    Adams, Sylvia; Gray, Robert J.; Demaria, Sandra; Goldstein, Lori; Perez, Edith A.; Shulman, Lawrence N.; Martino, Silvana; Wang, Molin; Jones, Vicky E.; Saphner, Thomas J.; Wolff, Antonio C.; Wood, William C.; Davidson, Nancy E.; Sledge, George W.; Sparano, Joseph A.; Badve, Sunil S.

    2014-01-01

    Purpose Recent studies suggest that tumor-infiltrating lymphocytes (TILs) are associated with disease-free (DFS) and overall survival (OS) in operable triple-negative breast cancer (TNBC). We seek to validate the prognostic impact of TILs in primary TNBCs in two adjuvant phase III trials conducted by the Eastern Cooperative Oncology Group (ECOG). Patients and Methods Full-face hematoxylin and eosin–stained sections of 506 tumors from ECOG trials E2197 and E1199 were evaluated for density of TILs in intraepithelial (iTILs) and stromal compartments (sTILs). Patient cases of TNBC from E2197 and E1199 were randomly selected based on availability of sections. For the primary end point of DFS, association with TIL scores was determined by fitting proportional hazards models stratified on study. Secondary end points were OS and distant recurrence–free interval (DRFI). Reporting recommendations for tumor marker prognostic studies criteria were followed, and all analyses were prespecified. Results The majority of 481 evaluable cancers had TILs (sTILs, 80%; iTILs, 15%). With a median follow-up of 10.6 years, higher sTIL scores were associated with better prognosis; for every 10% increase in sTILs, a 14% reduction of risk of recurrence or death (P = .02), 18% reduction of risk of distant recurrence (P = .04), and 19% reduction of risk of death (P = .01) were observed. Multivariable analysis confirmed sTILs to be an independent prognostic marker of DFS, DRFI, and OS. Conclusion In two national randomized clinical trials using contemporary adjuvant chemotherapy, we confirm that stromal lymphocytic infiltration constitutes a robust prognostic factor in TNBCs. Studies assessing outcomes and therapeutic efficacies should consider stratification for this parameter. PMID:25071121

  7. Enzymatic Excision of Uracil Residues in Nucleosomes Depends on Local DNA Structure and Dynamics†

    PubMed Central

    Ye, Yu; Stahley, Mary R.; Xu, Jianqing; Friedman, Joshua I.; Sun, Yan; McKnight, Jeffrey N.; Gray, Jeffrey J.; Bowman, Gregory D.; Stivers, James T.

    2012-01-01

    The excision of uracil bases from DNA is accomplished by the enzyme uracil DNA glycosylase (UNG). Recognition of uracil bases in free DNA is facilitated by uracil base pair dynamics, but it is not known whether this same mechanistic feature is relevant for detection and excision of uracil residues embedded in nucleosomes. Here we investigate this question using nucleosome core particles (NCPs) generated from X. laevis histones and the high-affinity “Widom 601” positioning sequence. The reactivity of uracil residues in NCPs under steady-state multiple turnover conditions was generally decreased as compared to free 601 DNA, mostly due to anticipated steric effects of histones. However, some sites in NCPs had equal or even greater reactivity than free DNA, and the observed reactivities were not readily explained by simple steric considerations, or by global DNA unwrapping models for nucleosome invasion. In particular, some reactive uracils were found in occluded positions, while some unreactive uracils were found in exposed positions. One feature of many exposed reactive sites is a wide DNA minor groove, which allows penetration of a key active site loop of the enzyme. In single-turnover kinetic measurements, multi-phasic reaction kinetics were observed for several uracil sites, where each kinetic transient was independent of the UNG concentration. These kinetic measurements, and supporting structural analyses, support a mechanism where some uracils are transiently exposed to UNG by local, rate-limiting nucleosome conformational dynamics, followed by rapid trapping of the exposed state by the enzyme. We present structural models and plausible reaction mechanisms for the reaction of UNG at three distinct uracil sites in the NCP. PMID:22784353

  8. Metabolic programming of puberty: sexually dimorphic responses to early nutritional challenges.

    PubMed

    Sánchez-Garrido, M A; Castellano, J M; Ruiz-Pino, F; Garcia-Galiano, D; Manfredi-Lozano, M; Leon, S; Romero-Ruiz, A; Diéguez, C; Pinilla, L; Tena-Sempere, M

    2013-09-01

    Body energy stores and metabolic cues influence the onset of puberty. However, the pubertal impact of early nutritional challenges has been only fragmentarily addressed. We evaluated here the consequences, in terms of pubertal timing and hormonal markers, of various nutritional manipulations during pre- or postnatal maturation in rats of both sexes. Males and females were submitted to gestational undernutrition (UNG) or peripubertal (SUB) subnutrition or were raised in large (LL; underfeeding) or small (SL; overfeeding) litters. In addition, groups of UNG, LL, and SL rats were fed on a high-fat diet (HFD) after weaning. Postnatal overfeeding resulted in higher body weights (BWs) during pubertal transition in both sexes, but only SL males displayed overtly advanced external signs of puberty. Postnatal underfeeding persistently decreased BW gain during puberty, yet the magnitude of pubertal delay was greater in LL males. In contrast, regardless of postnatal nutrition, HFD tended to advance the onset of puberty in females but did not alter pubertal timing in males. Likewise, SUB females displayed a marked delay in BW gain and puberty onset, whereas despite similar reduction in BW, SUB males showed normal timing of puberty. These sex divergences were also detected in various hormonal and metabolic indices so that postnatal overnutrition consistently increased LH, FSH, leptin, and insulin levels only in pubertal females, whereas HFD decreased gonadotropin levels in SL females but increased them in SL males. Notably, UNG rats did not show signs of delayed puberty but displayed a striking sex dimorphism in serum insulin/glucose levels, regardless of the diet, so that only UNG males had signs of presumable insulin resistance. Our data disclose important sex differences in the impact of various early nutritional challenges on the timing of puberty, which may help to explain the different trends of altered puberty and related comorbidities between sexes. PMID:23751873

  9. Role of uracil-DNA glycosylase in mutation avoidance by Streptococcus pneumoniae

    SciTech Connect

    Chen, Jau-Der; Lacks, S.A. )

    1991-01-01

    Uracil-DNA glycosylase activity was found in Streptococcus pneumoniae, and the enzyme was partially purified. An ung mutant lacking the activity was obtained by positive selection of cells transformed with a plasmid containing uracil in its DNA. The effects of the ung mutation on mutagenic processes in S. pneumoniae were examined. The sequence of several malM mutations revertible by nitrous acid showed them to correspond to A {center dot}T{r arrow}G {center dot} C transitions. This confirmed a prior deduction that nitrous acid action on transforming DNA gave only G {center dot} C{r arrow}A {center dot} T mutations. Examination of malM mutant reversion frequencies in ung strains indicated that G {center dot} C{r arrow}A {center dot} T mutation rates generally were 10-fold higher than in wild-type strains, presumably owing to lack of repair of deaminated cytosine residues in DNA. No effect of ung on mutation avoidance by the Hex mismatch repair system was observed, which means that uracil incorporation and removal from nascent DNA cannot be solely responsible for producing strand breaks that target nascent DNA for correction after replication. One malM mutation corresponding to an A {center dot} T{r arrow}G {center dot} C transition showed a 10-fold-higher spontaneous reversion frequency than other such transitions in a wild-type background. This hot spot was located in a directly repeated DNA sequence; it is proposed that transient slippage to the wild-type repeat during replication accounts for the higher reversion frequency.

  10. Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

    PubMed Central

    Weil, Amy F.; Ghosh, Devlina; Zhou, Yan; Seiple, Lauren; McMahon, Moira A.; Spivak, Adam M.; Siliciano, Robert F.; Stivers, James T.

    2013-01-01

    HIV-1 reverse transcriptase discriminates poorly between dUTP and dTTP, and accordingly, viral DNA products become heavily uracilated when viruses infect host cells that contain high ratios of dUTP:dTTP. Uracilation of invading retroviral DNA is thought to be an innate immunity barrier to retroviral infection, but the mechanistic features of this immune pathway and the cellular fate of uracilated retroviral DNA products is not known. Here we developed a model system in which the cellular dUTP:dTTP ratio can be pharmacologically increased to favor dUTP incorporation, allowing dissection of this innate immunity pathway. When the virus-infected cells contained elevated dUTP levels, reverse transcription was found to proceed unperturbed, but integration and viral protein expression were largely blocked. Furthermore, successfully integrated proviruses lacked detectable uracil, suggesting that only nonuracilated viral DNA products were integration competent. Integration of the uracilated proviruses was restored using an isogenic cell line that had no detectable human uracil DNA glycosylase (hUNG2) activity, establishing that hUNG2 is a host restriction factor in cells that contain high dUTP. Biochemical studies in primary cells established that this immune pathway is not operative in CD4+ T cells, because these cells have high dUTPase activity (low dUTP), and only modest levels of hUNG activity. Although monocyte-derived macrophages have high dUTP levels, these cells have low hUNG activity, which may diminish the effectiveness of this restriction pathway. These findings establish the essential elements of this pathway and reconcile diverse observations in the literature. PMID:23341616

  11. Tumour-infiltrating lymphocytes bear the 75 kDa tumour necrosis factor receptor.

    PubMed Central

    Trentin, L.; Zambello, R.; Bulian, P.; Cerutti, A.; Enthammer, C.; Cassatella, M.; Nitti, D.; Lise, M.; Agostini, C.; Semenzato, G.

    1995-01-01

    Tumour necrosis factor alpha (TNF-alpha) is a cytokine with a variety of immunological properties. The identification of two receptors for this molecule, i.e. the 75 kDa and the 55 kDa TNF receptors (TNF-R), recently clarified the mechanisms through which this cytokine provides its wide range of immunomodulatory activities. In this study we have investigated the expression and the functional properties of these receptors on tumour-infiltrating lymphocytes (TILs) recovered from 17 patients with solid cancers (melanoma, colorectal carcinoma and lung cancer). To this end, TIL lines and freshly isolated TILs were evaluated for (a) the expression and the functional role of TNF receptors following culture in the presence of interleukin 2 (IL-2) and (b) the production of TNF-alpha following culture with IL-2 and the role of this cytokine in IL-2-driven TIL proliferation. Flow cytometry analysis demonstrated that TILs bear the 75 kDa TNF-R. Moreover, TIL lines express detectable messages for TNF-alpha and release this cytokine. Functional in vitro studies have shown that anti-TNF-alpha, as well as anti-75 kDa TNF-R antibodies, are able to inhibit the IL-2-induced TIL proliferation. These data demonstrate that TILs are equipped with a fully functional TNF-R system and suggest a putative role for this receptor and its ligand in the activation and expression of TILs following immunotherapy with IL-2. Images Figure 2 PMID:7841036

  12. High PD-1 expression and suppressed cytokine signaling distinguish T cells infiltrating follicular lymphoma tumors from peripheral T cells.

    PubMed

    Myklebust, June H; Irish, Jonathan M; Brody, Joshua; Czerwinski, Debra K; Houot, Roch; Kohrt, Holbrook E; Timmerman, John; Said, Jonathan; Green, Michael R; Delabie, Jan; Kolstad, Arne; Alizadeh, Ash A; Levy, Ronald

    2013-02-21

    Defects in T-cell function in patients with cancer might influence their capacity to mount efficient antitumor immune responses. Here, we identified highly reduced IL-4-, IL-10-, and IL-21-induced phosphorylation of STAT6 and STAT3 in tumor-infiltrating T cells (TILs) in follicular lymphoma (FL) tumors, contrasting other non-Hodgkin lymphoma TILs. By combining phospho-protein-specific flow cytometry with several T-cell markers, we identified that CD4(+)CD45RO(+)CD62L(-) FL TILs were largely nonresponsive to cytokines, in contrast to the corresponding autologous peripheral blood subset. We observed differential expression of the inhibitory receptor PD-1 in FL TILs and peripheral blood T cells. Furthermore, CD4(+)PD-1(hi) FL TILs, containing T(FH) and non-T(FH) cells, had lost their cytokine responsiveness, whereas PD-1 TILs had normal cytokine signaling. However, this phenomenon was not tumor specific, because tonsil T cells were similar to FL TILs. FL tumor cells were negative for PD-1 ligands, but PD-L1(+) histiocytes were found within the T cell-rich zone of the neoplastic follicles. Disruption of the microenvironment and in vitro culture of FL TILs could restore cytokine signaling in the PD-1(hi) subset. Because FL TILs in vivo probably receive suppressive signals through PD-1, this provides a rationale for testing PD-1 Ab in combination with immunotherapy in patients with FL. PMID:23297127

  13. Carbon starvation increases endoglycosidase activities and production of "unconjugated N-glycans" in Silene alba cell-suspension cultures.

    PubMed Central

    Lhernould, S; Karamanos, Y; Priem, B; Morvan, H

    1994-01-01

    We previously reported the occurrence of oligomannosides and xylomannosides corresponding to unconjugated N-glycans (UNGs) in the medium of a white campion (Silene alba) cell suspension. Attention has been focused on these oligosaccharides since it was shown that they confer biological activities in plants. In an attempt to elucidate the origin of these oligosaccharides, we studied two endoglycosidase activities, putative enzymes involved in their formation. The previously described peptide-N4-(N-acetyl-glucosaminyl) asparagine amidase activity and the endo-N-acetyl-beta-D-glucosaminidase activity described in this paper were both quantified in white campion cells during the culture cycle with variable initial concentrations of sucrose. The lower the sucrose supply, the higher the two activities. Furthermore, endoglycosidase activities were greatly enhanced after the disappearance of sugar from the medium. The production of UNGs in the culture medium rose correlatively. These data strongly suggest that the production of UNGs in our white campion cell-suspension system is due to the increase of these endoglycosidase activities, which reach their highest levels of activity during conditions of carbon starvation. PMID:7991689

  14. Interplay between Target Sequences and Repair Pathways Determines Distinct Outcomes of AID-Initiated Lesions.

    PubMed

    Chen, Zhangguo; Eder, Maxwell D; Elos, Mihret T; Viboolsittiseri, Sawanee S; Chen, Xiaomi; Wang, Jing H

    2016-03-01

    Activation-induced deaminase (AID) functions by deaminating cytosines and causing U:G mismatches, a rate-limiting step of Ab gene diversification. However, precise mechanisms regulating AID deamination frequency remain incompletely understood. Moreover, it is not known whether different sequence contexts influence the preferential access of mismatch repair or uracil glycosylase (UNG) to AID-initiated U:G mismatches. In this study, we employed two knock-in models to directly compare the mutability of core Sμ and VDJ exon sequences and their ability to regulate AID deamination and subsequent repair process. We find that the switch (S) region is a much more efficient AID deamination target than the V region. Igh locus AID-initiated lesions are processed by error-free and error-prone repair. S region U:G mismatches are preferentially accessed by UNG, leading to more UNG-dependent deletions, enhanced by mismatch repair deficiency. V region mutation hotspots are largely determined by AID deamination. Recurrent and conserved S region motifs potentially function as spacers between AID deamination hotspots. We conclude that the pattern of mutation hotspots and DNA break generation is influenced by sequence-intrinsic properties, which regulate AID deamination and affect the preferential access of downstream repair. Our studies reveal an evolutionarily conserved role for substrate sequences in regulating Ab gene diversity and AID targeting specificity. PMID:26810227

  15. Interplay between Target Sequences and Repair Pathways Determines Distinct Outcomes of AID-Initiated Lesions

    PubMed Central

    Chen, Zhangguo; Eder, Maxwell D.; Elos, Mihret T.; Viboolsittiseri, Sawanee S.; Chen, Xiaomi

    2016-01-01

    Activation-induced deaminase (AID) functions by deaminating cytosines and causing U:G mismatches, a rate-limiting step of Ab gene diversification. However, precise mechanisms regulating AID deamination frequency remain incompletely understood. Moreover, it is not known whether different sequence contexts influence the preferential access of mismatch repair or uracil glycosylase (UNG) to AID-initiated U:G mismatches. In this study, we employed two knock-in models to directly compare the mutability of core Sμ and VDJ exon sequences and their ability to regulate AID deamination and subsequent repair process. We find that the switch (S) region is a much more efficient AID deamination target than the V region. Igh locus AID-initiated lesions are processed by error-free and error-prone repair. S region U:G mismatches are preferentially accessed by UNG, leading to more UNG-dependent deletions, enhanced by mismatch repair deficiency. V region mutation hotspots are largely determined by AID deamination. Recurrent and conserved S region motifs potentially function as spacers between AID deamination hotspots. We conclude that the pattern of mutation hotspots and DNA break generation is influenced by sequence-intrinsic properties, which regulate AID deamination and affect the preferential access of downstream repair. Our studies reveal an evolutionarily conserved role for substrate sequences in regulating Ab gene diversity and AID targeting specificity. PMID:26810227

  16. dUTP pyrophosphatase is an essential enzyme in Saccharomyces cerevisiae.

    PubMed Central

    Gadsden, M H; McIntosh, E M; Game, J C; Wilson, P J; Haynes, R H

    1993-01-01

    dUTP pyrophosphatase (dUTPase; EC 3.6.1.23) catalyses the hydrolysis of dUTP to dUMP and PPi and thereby prevents the incorporation of uracil into DNA during replication. Although it is widely believed that dUTPase is essential for cell viability because of this role, direct evidence supporting this assumption has not been presented for any eukaryotic system. We have analysed the role of dUTPase (DUT1) in the life cycle of yeast. Using gene disruption and tetrad analysis, we find that DUT1 is necessary for the viability of S. cerevisiae; however, under certain conditions dut1 null mutants survive if supplied with exogenous thymidylate (dTMP). Analyses with isogenic uracil-DNA-glycosylase (UNG1) deficient or proficient strains indicate that in the absence of dUTPase, cell death results from the incorporation of uracil into DNA and the attempted repair of this damage by UNG1-mediated excision repair. However, in dut1 ung1 double mutants, starvation for dTMP causes dividing cells to arrest and die in all phases of the cell cycle. This latter effect suggests that the extensive stable substitution of uracil for thymine in DNA leads to a general failure in macromolecular synthesis. These results are in general agreement with previous models in thymine-less death that implicate dUTP metabolism. They also suggest an alternative approach for chemotherapeutic drug design. Images PMID:8223452

  17. Cloning of a new gene encoding an antigen recognized by melanoma-specific HLA-A24-restricted tumor-infiltrating lymphocytes

    SciTech Connect

    Robbins, P.F.; El-Gamil, M.; Li, Y.F.

    1995-06-01

    The role of tumor-specific T cells in mediating the regression of metastatic melanoma has been suggested by the clinical response of patients to treatment with tumor-infiltrating lymphocytes (TIL). A number of Ags recognized by class I-restricted melanoma-specific T cells have recently been isolated, raising the hope that this will lead to the development of improved therapies. In this study, we report the cloning of a tumor Ag recognized by T cells from melanoma patient 888. Previously, we reported that TIL 888, grown from the tumor of this patient, recognized tyrosinase in an HLA-A24 -restricted fashion. This line, when infused into the autologous patient, resulted in complete regression of multiple metastases. Three years later, a second TIL line, TIL 1290, was isolated from a recurrent pelvic tumor. Infusion of a mixture of TIL 888 and TIL 1290 cell lines into the patient resulted in complete regression of a residual abdominal mass and the patient remains disease-free 2 yr later. The TIL 1290 cell line, which recognized melanoma in an HLAA-A24-restricted manner, failed to recognize tyrosinase. TIL 1290 was then used to screen an 888 melanoma cDNA library, and an Ag was isolated that did not correspond to any found in sequence databases. This gene, termed p15, was found to be expressed in a variety of normal tissues, and a peptide epitope recognized by TIL 1290 was found to represent the product of an nonmutated gene. Screening of additional cDNA pools resulted in the isolation of a second clone which stimulated TIL 1290. This clone also appeared to represent a transcript of the p15 gene, indicating that this gene may encode the predominant Ag recognized by TIL 1290. 27 refs., 4 figs., 5 tabs.

  18. Molecular Profile of Tumor-Specific CD8+ T Cell Hypofunction in a Transplantable Murine Cancer Model.

    PubMed

    Waugh, Katherine A; Leach, Sonia M; Moore, Brandon L; Bruno, Tullia C; Buhrman, Jonathan D; Slansky, Jill E

    2016-08-15

    Mechanisms of self-tolerance often result in CD8(+) tumor-infiltrating lymphocytes (TIL) with a hypofunctional phenotype incapable of tumor clearance. Using a transplantable colon carcinoma model, we found that CD8(+) T cells became tolerized in <24 h in an established tumor environment. To define the collective impact of pathways suppressing TIL function, we compared genome-wide mRNA expression of tumor-specific CD8(+) T cells from the tumor and periphery. Notably, gene expression induced during TIL hypofunction more closely resembled self-tolerance than viral exhaustion. Differential gene expression was refined to identify a core set of genes that defined hypofunctional TIL; these data comprise the first molecular profile of tumor-specific TIL that are naturally responding and represent a polyclonal repertoire. The molecular profile of TIL was further dissected to determine the extent of overlap and distinction between pathways that collectively restrict T cell functions. As suggested by the molecular profile of TIL, protein expression of inhibitory receptor LAG-3 was differentially regulated throughout prolonged late-G1/early-S phase of the cell cycle. Our data may accelerate efficient identification of combination therapies to boost anti-tumor function of TIL specifically against tumor cells. PMID:27371726

  19. A New Approach to the Adoptive Immunotherapy of Cancer with Tumor-Infiltrating Lymphocytes

    NASA Astrophysics Data System (ADS)

    Rosenberg, Steven A.; Spiess, Paul; Lafreniere, Rene

    1986-09-01

    The adoptive transfer of tumor-infiltrating lymphocytes (TIL) expanded in interleukin-2 (IL-2) to mice bearing micrometastases from various types of tumors showed that TIL are 50 to 100 times more effective in their therapeutic potency than are lymphokine-activated killer (LAK) cells. Therefore the use of TIL was explored for the treatment of mice with large pulmonary and hepatic metastatic tumors that do not respond to LAK cell therapy. Although treatment of animals with TIL alone or cyclophosphamide alone had little impact, these two modalities together mediated the elimination of large metastatic cancer deposits in the liver and lung. The combination of TIL and cyclophosphamide was further potentiated by the simultaneous administration of IL-2. With the combination of cyclophosphamide, TIL, and IL-2, 100% of mice (n = 12) bearing the MC-38 colon adenocarcinoma were cured of advanced hepatic metastases, and up to 50% of mice were cured of advanced pulmonary metastases. Techniques have been developed to isolate TIL from human tumors. These experiments provide a rationale for the use of TIL in the treatment of humans with advanced cancer.

  20. Spreading culm locus discovered while dissecting a sheath blight resistance QTL within a set of TeQing-into-Lemont introgression lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new gene-mapping population comprised of 123 TeQing-into-Lemont backcross introgression lines (TILs) was developed for the purpose of providing a uniquely efficient avenue for verifying, molecularly tagging, and incorporating desired TeQing QTL into improved U.S. rice varieties. The TILs were bei...

  1. Mechanisms of the tRNA wobble cytidine modification essential for AUA codon decoding in prokaryotes.

    PubMed

    Numata, Tomoyuki

    2015-01-01

    Bacteria and archaea have 2-lysylcytidine (L or lysidine) and 2-agmatinylcytidine (agm(2)C or agmatidine), respectively, at the first (wobble) position of the anticodon of the AUA codon-specific tRNA(Ile). These lysine- or agmatine-conjugated cytidine derivatives are crucial for the precise decoding of the genetic code. L is synthesized by tRNA(Ile)-lysidine synthetase (TilS), which uses l-lysine and ATP as substrates. Agm(2)C formation is catalyzed by tRNA(Ile)-agm(2)C synthetase (TiaS), which uses agmatine and ATP for the reaction. Despite the fact that TilS and TiaS synthesize structurally similar cytidine derivatives, these enzymes belong to non-related protein families. Therefore, these enzymes modify the wobble cytidine by distinct catalytic mechanisms, in which TilS activates the C2 carbon of the wobble cytidine by adenylation, while TiaS activates it by phosphorylation. In contrast, TilS and TiaS share similar tRNA recognition mechanisms, in which the enzymes recognize the tRNA acceptor stem to discriminate tRNA(Ile) and tRNA(Met). PMID:25348586

  2. Tumors with high-density tumor infiltrating lymphocytes constitute a favorable entity in breast cancer: a pooled analysis of four prospective adjuvant trials

    PubMed Central

    Kotoula, Vassiliki; Chatzopoulos, Kyriakos; Lakis, Sotiris; Alexopoulou, Zoi; Timotheadou, Eleni; Zagouri, Flora; Pentheroudakis, George; Gogas, Helen; Galani, Eleni; Efstratiou, Ioannis; Zaramboukas, Thomas; Koutras, Angelos; Aravantinos, Gerasimos; Samantas, Epaminontas; Psyrri, Amanda; Kourea, Helen; Bobos, Mattheos; Papakostas, Pavlos; Kosmidis, Paris; Pectasides, Dimitrios; Fountzilas, George

    2016-01-01

    Background Tumor infiltrating lymphocytes (TILs) are considered in the prognosis of breast cancer (BC) patients. Here, we investigated the prognostic/predictive effect of TILs in patients treated in the frame of four prospective trials with adjuvant anthracycline-based chemotherapy in the pre- and post-trastuzumab era. Methods TILs density was histologically assessed as percentage of stromal area on whole routine sections of 2613 BC (1563 Luminal A/B; 477 Luminal HER2; 246 HER2-enriched; 327 triple negative [TNBC]) and were evaluated as high/low at three cut-offs (c/o; 50% [lymphocytic predominance, LP], 35% and 25%), in separate training and validation sets. Results High TILs were present in 3.5%, 6.5% and 11.5% of all tumors, using the 50%, 35% and 25% c/o, respectively. TILs status did not interact with BC subtypes or trastuzumab treatment. LPBC patient outcome was not affected by nodal status, while high TILs were favorable in TNBC with unfavorable nodal status. When adjusted for standard clinicopathological parameters and treatment, high TILs independently predicted for favorable outcome, e.g., disease-free survival with the 35% c/o in the entire cohort (HR = 0.44, 95% CI 0.28-0.69, p < 0.001) and in specific subtypes. Conclusions High TILs tumors, especially LPBC seem worthy validating as a separate entity of favorable prognosis in breast cancer. PMID:26506242

  3. Tumour-infiltrating lymphocytes in melanoma prognosis and cancer immunotherapy.

    PubMed

    Lee, Nayoung; Zakka, Labib R; Mihm, Martin C; Schatton, Tobias

    2016-02-01

    The field of systemic cancer therapy for metastatic disease has entered an exciting era with the advent of novel immunomodulatory strategies targeting immune checkpoints. At the heart of these promising efforts are the tumour-infiltrating lymphocytes (TILs). As the reports demonstrating efficacy of modulating TIL effector function in patients with advanced stage cancer continue to accrue, it has become essential to better understand TIL immunobiology in order to further improve clinical outcome. In addition to providing an overview of the current immunotherapies available for metastatic melanoma, this review will briefly introduce the history and classification of TILs. Moreover, we will dissect the multifaceted roles of TILs in tumour-specific immunity and melanoma immune escape. The significance of TILs in melanoma prognosis and cancer immunotherapy will also be discussed, with a particular focus on their potential utility as biomarkers of patient response. The goal of personalised medicine appears to be in realistic sight, as new immunomodulatory techniques and technological innovations continue to advance the field of cancer immunotherapy. In light of recent studies highlighting the possible utility of TILs in determining therapeutic outcome, further characterisation of TIL phenotype and function has the potential to help translate individualised care into current medical practice. PMID:27020390

  4. Current Issues and Clinical Evidence in Tumor-Infiltrating Lymphocytes in Breast Cancer

    PubMed Central

    Ahn, Sung Gwe; Jeong, Joon; Hong, SoonWon; Jung, Woo Hee

    2015-01-01

    With the advance in personalized therapeutic strategies in patients with breast cancer, there is an increasing need for biomarker-guided therapy. Although the immunogenicity of breast cancer has not been strongly considered in research or practice, tumor-infiltrating lymphocytes (TILs) are emerging as biomarkers mediating tumor response to treatments. Earlier studies have provided evidence that the level of TILs has prognostic value and the potential for predictive value, particularly in triple-negative and human epidermal growth factor receptor 2–positive breast cancer. Moreover, the level of TILs has been associated with treatment outcome in patients undergoing neoadjuvant chemotherapy. To date, no standardized methodology for measuring TILs has been established. In this article, we review current issues and clinical evidence for the use of TILs in breast cancer. PMID:26278518

  5. Characterization of Ex Vivo Expanded Tumor Infiltrating Lymphocytes from Patients with Malignant Melanoma for Clinical Application

    PubMed Central

    Junker, Niels; thor Straten, Per; Andersen, Mads Hald; Svane, Inge Marie

    2011-01-01

    Clinical trials of adoptive transfer of autologous tumor infiltrating lymphocytes (TILs) to patients with advanced malignant melanoma have shown remarkable results with objective clinical responses in 50% of the treated patients. In order to initiate a clinical trial in melanoma, we have established a method for expanding TILs to clinical relevant quantities in two steps with in 8 weeks. Further characterization of expanded TILs revealed an oligoclonal composition of T-cells with an effector memory like phenotype. When autologous tumor was available, TILs showed specific activity in all patients tested. TIL cultures contained specificity towards tumor cells as well as peptides derived from tumor-associated antigens (TAAs) during expansion procedures. PMID:21773037

  6. Syngeneic syrian hamster tumors feature tumor-infiltrating lymphocytes allowing adoptive cell therapy enhanced by oncolytic adenovirus in a replication permissive setting.

    PubMed

    Siurala, Mikko; Vähä-Koskela, Markus; Havunen, Riikka; Tähtinen, Siri; Bramante, Simona; Parviainen, Suvi; Mathis, J Michael; Kanerva, Anna; Hemminki, Akseli

    2016-05-01

    Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown promising yet sometimes suboptimal results in clinical trials for advanced cancer, underscoring the need for approaches improving efficacy and safety. Six implantable syngeneic tumor cell lines of the Syrian hamster were used to initiate TIL cultures. TIL generated from tumor fragments cultured in human interleukin-2 (IL-2) for 10 d were adoptively transferred into tumor-bearing hamsters with concomitant intratumoral injections of oncolytic adenovirus (Ad5-D24) for the assessment of antitumor efficacy. Pancreatic cancer (HapT1) and melanoma (RPMI 1846) TIL exhibited potent and tumor-specific cytotoxicity in effector-to-target (E/T) assays. MHC Class I blocking abrogated the cell killing of RPMI 1846 TIL, indicating cytotoxic CD8(+) T-cell activity. When TIL were combined with Ad5-D24 in vitro, HapT1 tumor cell killing was significantly enhanced over single agents. In vivo, the intratumoral administration of HapT1 TIL and Ad5-D24 resulted in improved tumor growth control compared with either treatment alone. Additionally, splenocytes derived from animals treated with the combination of Ad5-D24 and TIL killed autologous tumor cells more efficiently than monotherapy-derived splenocytes, suggesting that systemic antitumor immunity was induced. For the first time, TIL of the Syrian hamster have been cultured, characterized and used therapeutically together with oncolytic adenovirus for enhancing the efficacy of TIL therapy. Our results support human translation of oncolytic adenovirus as an enabling technology for adoptive T-cell therapy of solid tumors. PMID:27467954

  7. Analysis of the hippo transducers TAZ and YAP in cervical cancer and its microenvironment.

    PubMed

    Buglioni, Simonetta; Vici, Patrizia; Sergi, Domenico; Pizzuti, Laura; Di Lauro, Luigi; Antoniani, Barbara; Sperati, Francesca; Terrenato, Irene; Carosi, Mariantonia; Gamucci, Teresa; Vincenzoni, Cristina; Mariani, Luciano; Vizza, Enrico; Venuti, Aldo; Sanguineti, Giuseppe; Gadducci, Angiolo; Barba, Maddalena; Natoli, Clara; Vitale, Ilio; Mottolese, Marcella; De Maria, Ruggero; Maugeri-Saccà, Marcello

    2016-06-01

    Hippo is a tumor-suppressor pathway that negatively regulates the oncoproteins TAZ and YAP. Moreover, Hippo affects the biology of a variety of non-neoplastic cells in the tumor microenvironment, even including immune cells. We herein assessed the predictive role of TAZ and YAP, assessed by immunohistochemistry, in 50 cervical cancer patients prevalently treated with neoadjuvant chemotherapy. Tumors were classified as positive or negative according to the percentage of tumor-expressing cells and cellular localization. TAZ/YAP were also evaluated in non-neoplastic cells, namely endothelial cells, non-lymphocytic stromal cells and tumor-infiltrating lymphocytes (TILs). TAZ expression in cancer cells (TAZ(pos)) was associated with a reduced pathological complete response (pCR) rate (p = 0.041). Conversely, the expression of TAZ and YAP in TILs (TAZ(TIL+) and YAP(TIL+)) seemed to be associated with increased pCRs (p = 0.083 and p = 0.018, respectively). When testing the predictive significance of the concomitant expression of TAZ in cancer cells and its absence in TILs (TAZ(pos)/TAZ(TIL-)), patients with TAZ(pos)/TAZ(TIL-) showed lower pCR rate (p = 0.001), as confirmed in multivariate analysis (TAZ(pos)/TAZ(TIL-): OR 8.67, 95% CI: 2.31-32.52, p = 0.001). Sensitivity analysis carried out in the 41 patients treated with neoadjuvant chemotherapy yielded comparable results (TAZ(pos)/TAZ(TIL-): OR 11.0, 95% CI: 2.42-49.91, p = 0.002). Internal validation carried out with two different procedures confirmed the robustness of this model. Overall, we found evidence on the association between TAZ expression in cervical cancer cells and reduced pCR rate. Conversely, the expression of the Hippo transducers in TILs may predict increased treatment efficacy, possibly mirroring the activation of a non-canonical Hippo/MST pathway necessary for T-cells activation and survival. PMID:27471633

  8. Modulating Effects of Spirulina platensis against Tilmicosin-Induced Cardiotoxicity in Mice

    PubMed Central

    Ibrahim, Abdelaziz E.; Abdel-Daim, Mohamed Mohamed

    2015-01-01

    Objective Tilmicosin (TIL) is a long-acting macrolide antibiotic used to treat cattle for pathogens that cause bovine respiratory disease. However, overdoses of this medication have been reported to induce cardiac damage. Our experimental objective was to evaluate the protective effects of Spirulina platensis (SP) administration against TIL-induced cardiotoxicity in mice. Materials and Methods Our experimental in vivo animal study used 40 male albino mice that were divided into five groups of eight mice per group. The first group served as a control group and was injected with saline. The second group received SP at dose of 1000 mg/kg body weight for five days. The third group received a single dose of TIL (75 mg/kg, subcutaneously). Groups 4 and 5 were given SP at doses of 500 and 1000 mg/kg body weight for five consecutive days just before administration of TIL at the same dose and regimen used for group 3. Results TIL treated animals showed a significant increase in serum cardiac injury biomarkers as well as cardiac lipid peroxidation, however they had evidence of an inhibition in antioxidant biomarkers. SP normalized elevated serum levels of lactate dehydrogenase (LDH), creatine kinase (CK), and CK-MB. Furthermore, SP reduced TIL-induced lipid peroxidation and oxidative stress in a dose-dependent manner. Conclusion Administration of SP minimized the toxic effects of TIL by its free radicalscavenging and potent antioxidant activity. PMID:25870843

  9. Expansion of human tumor infiltrating lymphocytes for use in immunotherapy trials.

    PubMed

    Topalian, S L; Muul, L M; Solomon, D; Rosenberg, S A

    1987-08-24

    The potential utility of tumor-infiltrating lymphocytes (TIL) in the adoptive immunotherapy of human tumors has been suggested by murine experiments showing these cells to be 50-100 times more powerful than LAK cells in treating advanced metastatic disease. A method for the large-scale expansion of human TIL for the use of these cells in clinical trials is described in this report. TIL were successfully expanded on an experimental scale from 24 of 25 consecutive human tumors, including six melanomas, ten sarcomas, and eight adenocarcinomas. Tumors were digested enzymatically to yield single cell suspensions which were cultured in RPMI 1640 medium with 10% human serum and 1000 U/ml recombinant interleukin-2. Lymphocytes constituted from 3% to 74% of single cell tumor suspensions, and expanded from 2.9-fold to 9.1 X 10(8)-fold over a culture period ranging from 14 to 100 days. Nine of 24 TIL cultures lysed fresh autologous tumor targets in 4 h chromium release assays. Cell surface phenotyping identified cultured TIL as activated cytotoxic/suppressor T cells. Subsequently, large-scale expansion of TIL was successful in generating more than 10(10) lymphocytes in five of eight consecutive cases. Clinical trials employing the adoptive transfer of expanded TIL to patients with metastatic disease have begun. PMID:3305708

  10. Changes in Immunogenicity during the Development of Urinary Bladder Cancer: A Preliminary Study

    PubMed Central

    Jóźwicki, Wojciech; Brożyna, Anna A.; Siekiera, Jerzy; Slominski, Andrzej T.

    2016-01-01

    In the present study, we evaluated tumor-infiltrating lymphocytes (TILs) and blood regulatory T lymphocyte (Tregs, CD4+/CD25+/FoxP3+) expression in bladder cancer patients. The number of CD4+, CD8+, CD25+, FoxP3+ and CD20+ TILs was analyzed in association with clinico-pathomorphological features. In more advanced metastasizing tumors, showing non-classic differentiation (ND) and a more aggressive tissue invasion type (TIT), the number of TILs decreased. A low number of CD4+ TILs was associated with poor prognosis. Similarly, Treg frequency before surgery and after surgical treatment was significantly lower in more advanced tumors. The changes in TILs, as well as of local and systemic Tregs, were accompanied by changes in the histological phenotype of urothelial carcinoma regarding pT stage, NDs, TIT, and clinical outcomes. The number of TILs and the frequency of blood Tregs (indicators of antitumor response) may be essential for choosing an immunotherapy that is adjusted to the immune status according to the phase of tumor growth. Moreover, a significant reduction in the number of CD4+ and CD8+ TILs with the development of NDs in more advanced tumors may be associated with lower tumor immunogenicity, resulting in immune tolerance towards tumor tissue. These observations and the tendency of urothelial bladder carcinoma to undergo NDs in a heterogeneous manner during tumor progression suggest complex interactions between bladder cancer immunogenicity and stages of tumor progression. PMID:26927070

  11. Synergistic protective role of mirazid (Commiphora molmol) and ascorbic acid against tilmicosin-induced cardiotoxicity in mice.

    PubMed

    Abdel-Daim, Mohamed M; Ghazy, Emad W; Fayez, Mostafa

    2015-01-01

    Tilmicosin (TIL) is a long-acting macrolide antibiotic approved for the treatment of cattle with Bovine Respiratory Disease. However, overdose of TIL has been reported to induce cardiotoxicity. The purpose of our experiment was to evaluate the protective effects of Commiphora molmol (mirazid (MRZ); myrrh) and (or) ascorbic acid (AA) against TIL-induced cardiotoxicity in mice. MRZ and AA were orally administered using stomach gavage, either alone or in combination for 5 consecutive days, followed with a single TIL overdose. TIL overdose induced a significant increase in serum levels of cardiac damage biomarkers (AST, LDH, CK, CK-MB, and cTnT), as well as cardiac lipid peroxidation, but cardiac levels of antioxidant biomarkers (GSH, SOD, CAT, and TAC) were decreased. Both MRZ and AA tended to normalize the elevated serum levels of cardiac injury biomarkers. Furthermore, MRZ and AA reduced TIL-induced lipid peroxidation and oxidative stress parameters. MRZ and AA combined produced a synergistic cardioprotective effect. We conclude that myrrh and (or) vitamin C administration minimizes the toxic effects of TIL through their free-radical-scavenging and potent antioxidant activities. PMID:25429612

  12. Progressive upregulation of PD-1 in primary and metastatic melanomas associated with blunted TCR signaling in infiltrating T lymphocytes.

    PubMed

    Chapon, Maxime; Randriamampita, Clotilde; Maubec, Eve; Badoual, Cécile; Fouquet, Stéphane; Wang, Shu-Fang; Marinho, Eduardo; Farhi, David; Garcette, Marylène; Jacobelli, Simon; Rouquette, Alexandre; Carlotti, Agnès; Girod, Angélique; Prévost-Blondel, Armelle; Trautmann, Alain; Avril, Marie-Françoise; Bercovici, Nadège

    2011-06-01

    Programmed death-1 (PD-1) is involved in T-cell tolerance to self-antigens. For some cancers, it has been suggested that the expression of a ligand of PD-1, namely PD-L1, could contribute to tumor escape from immune destruction. Nevertheless, the relationship between PD-1 expression on tumor-infiltrating T lymphocytes (TILs), disease stage, and TIL responsiveness is still poorly documented. In this study, we show that freshly isolated CD4(+) and CD8(+) TILs express substantial levels of PD-1 in primary melanomas. The expression of PD-1 was further increased at later stages in distant cutaneous metastases, especially on CD8(+) TILs. The expression of PD-1 ligands was frequent only in metastases, on both tumor cells and tumor-derived myeloid cells. TILs isolated from these cutaneous tumors are poorly reactive ex vivo, with blunted calcium response and IFN-γ production after TCR stimulation. Surprisingly, in distinct parts of a primary melanoma, either invasive or regressing, we show that TILs similarly express PD-1 and remain dysfunctional. The expressions of PD-1 and PD-L1 in metastatic melanoma lesions could be considered as witnesses of an unsuccessful anti-tumoral immune response, but the direct involvement of PD-1 in the severity of the disease, and the importance of TILs in tumor regression, remain to be established. PMID:21346771

  13. Total inward leakage measurement of particulates for N95 filtering facepiece respirators--a comparison study.

    PubMed

    Rengasamy, Samy; Walbert, Gary F; Newcomb, William E; Faulkner, Kimberly; Rengasamy, Mathi M; Brannen, Jeremy J; Szalajda, Jonathan V

    2014-03-01

    National Institute for Occupational Safety and Health (NIOSH) certified particulate respirators need to be properly fit tested before use to ensure workers' respiratory protection. However, the effectiveness of American National Standards Institute-/Occupational Safety and Health Administration (ANSI-/OSHA)-accepted fit tests for particulate respirators in predicting actual workplace protection provided to workers is lacking. NIOSH addressed this issue by evaluating the fit of half-mask particulate filtering respirators as a component of a program designed to add total inward leakage (TIL) requirements for all respirators to Title 42 Code of Federal Regulations Part 84. Specifically, NIOSH undertook a validation study to evaluate the reproducibility of the TIL test procedure between two laboratories. A PortaCount® was used to measure the TIL of five N95 model filtering facepiece respirators (FFRs) on test subjects in two different laboratories. Concurrently, filter efficiency for four of the five N95 FFR models was measured using laboratory aerosol as well as polydisperse NaCl aerosol employed for NIOSH particulate respirator certification. Results showed that two N95 models passed the TIL tests at a rate of ~80-85% and ~86-94% in the two laboratories, respectively. However, the TIL passing rate for the other three N95 models was 0-5.7% in both laboratories combined. Good agreement (≥83%) of the TIL data between the two laboratories was obtained. The three models that had relatively lower filter efficiency for laboratory aerosol as well as for NaCl aerosol showed relatively low TIL passing rates in both laboratories. Of the four models tested for penetration, one model with relatively higher efficiency showed a higher passing rate for TIL tests in both laboratories indicating that filter efficiency might influence TIL. Further studies are needed to better understand the implications of the data in the workplace. PMID:24107745

  14. Density of tumor-infiltrating lymphocytes correlates with extent of brain edema and overall survival time in patients with brain metastases

    PubMed Central

    Berghoff, Anna S; Fuchs, Elisabeth; Ricken, Gerda; Mlecnik, Bernhard; Bindea, Gabriela; Spanberger, Thomas; Hackl, Monika; Widhalm, Georg; Dieckmann, Karin; Prayer, Daniela; Bilocq, Amelie; Heinzl, Harald; Zielinski, Christoph; Bartsch, Rupert; Birner, Peter; Galon, Jerome; Preusser, Matthias

    2016-01-01

    The immune microenvironment of the brain differs from that of other organs and the role of tumor-infiltrating lymphocytes (TILs) in brain metastases (BM), one of the most common and devastating complication of cancer, is unclear. We investigated TIL subsets and their prognostic impact in 116 BM specimens using immunohistochemistry for CD3, CD8, CD45RO, FOXP3, PD1 and PD-L1. The Immunoscore was calculated as published previously. Overall, we found TIL infiltration in 115/116 (99.1%) BM specimens. PD-L1 expression was evident in 19/67 (28.4%) BM specimens and showed no correlation with TIL density (p > 0.05). TIL density was not associated with corticosteroid administration (p > 0.05). A significant difference in infiltration density according to TIL subtype was present (p < 0.001; Chi Square); high infiltration was most frequently observed for CD3+ TILs (95/116; 81.9%) and least frequently for PD1+ TILs (18/116; 15.5%; p < 0.001). Highest TIL density was observed in melanoma, followed by renal cell cancer and lung cancer BM (p < 0.001). The density of CD8+ TILs correlated positively with the extent of peritumoral edema seen on pre-operative magnetic resonance imaging (p = 0.031). The density of CD3+ (15 vs. 6 mo; p = 0.015), CD8+ (15 vs. 11 mo; p = 0.030) and CD45RO+ TILs (18 vs. 8 mo; p = 0.006) showed a positive correlation with favorable median OS times. Immunoscore showed significant correlation with survival prognosis (27 vs. 10 mo; p < 0.001). The prognostic impact of Immunoscore was independent from established prognostic parameters at multivariable analysis (HR 0.612, p < 0.001). In conclusion, our data indicate that dense TILs infiltrates are common in BM and correlate with the amount of peritumoral brain edema and survival prognosis, thus identifying the immune system as potential biomarker for cancer patients with CNS affection. Further studies are needed to substantiate our findings. PMID:26942067

  15. Electrostatic properties of complexes along a DNA glycosylase damage search pathway.

    PubMed

    Cravens, Shannen L; Hobson, Matthew; Stivers, James T

    2014-12-01

    Human uracil DNA glycosylase (hUNG) follows an extended reaction coordinate for locating rare uracil bases in genomic DNA. This process begins with diffusion-controlled engagement of undamaged DNA, followed by a damage search step in which the enzyme remains loosely associated with the DNA chain (translocation), and finally, a recognition step that allows the enzyme to efficiently bind and excise uracil when it is encountered. At each step along this coordinate, the enzyme must form DNA interactions that are highly specialized for either rapid damage searching or catalysis. Here we make extensive measurements of hUNG activity as a function of salt concentration to dissect the thermodynamic, kinetic, and electrostatic properties of key enzyme states along this reaction coordinate. We find that the interaction of hUNG with undamaged DNA is electrostatically driven at a physiological concentration of potassium ions (ΔGelect = -3.5 ± 0.5 kcal mol(-1)), with only a small nonelectrostatic contribution (ΔGnon = -2.0 ± 0.2 kcal mol(-1)). In contrast, the interaction with damaged DNA is dominated by the nonelectrostatic free energy term (ΔGnon = -7.2 ± 0.1 kcal mol(-1)), yet retains the nonspecific electrostatic contribution (ΔGelect = -2.3 ± 0.2 kcal mol(-1)). Stopped-flow kinetic experiments established that the salt sensitivity of damaged DNA binding originates from a reduction of kon, while koff is weakly dependent on salt. Similar findings were obtained from the salt dependences of the steady-state kinetic parameters, where the diffusion-controlled kcat/Km showed a salt dependence similar to kon, while kcat (limited by product release) was weakly dependent on salt. Finally, the salt dependence of translocation between two uracil sites separated by 20 bp in the same DNA chain was indistinguishable from that of kon. This result suggests that the transition-state for translocation over this spacing resembles that for DNA association from bulk solution and that hUNG

  16. Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex

    PubMed Central

    Su, Mei-Tzu; Liu, I-Hua; Wu, Chia-Wei; Chang, Shu-Ming; Tsai, Ching-Hwa; Yang, Pei-Wen; Chuang, Yu-Chia; Lee, Chung-Pei

    2014-01-01

    ABSTRACT Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmunoprecipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. IMPORTANCE Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme

  17. Electrostatic Properties of Complexes along a DNA Glycosylase Damage Search Pathway

    PubMed Central

    2015-01-01

    Human uracil DNA glycosylase (hUNG) follows an extended reaction coordinate for locating rare uracil bases in genomic DNA. This process begins with diffusion-controlled engagement of undamaged DNA, followed by a damage search step in which the enzyme remains loosely associated with the DNA chain (translocation), and finally, a recognition step that allows the enzyme to efficiently bind and excise uracil when it is encountered. At each step along this coordinate, the enzyme must form DNA interactions that are highly specialized for either rapid damage searching or catalysis. Here we make extensive measurements of hUNG activity as a function of salt concentration to dissect the thermodynamic, kinetic, and electrostatic properties of key enzyme states along this reaction coordinate. We find that the interaction of hUNG with undamaged DNA is electrostatically driven at a physiological concentration of potassium ions (ΔGelect = −3.5 ± 0.5 kcal mol–1), with only a small nonelectrostatic contribution (ΔGnon = −2.0 ± 0.2 kcal mol–1). In contrast, the interaction with damaged DNA is dominated by the nonelectrostatic free energy term (ΔGnon = −7.2 ± 0.1 kcal mol–1), yet retains the nonspecific electrostatic contribution (ΔGelect = −2.3 ± 0.2 kcal mol–1). Stopped-flow kinetic experiments established that the salt sensitivity of damaged DNA binding originates from a reduction of kon, while koff is weakly dependent on salt. Similar findings were obtained from the salt dependences of the steady-state kinetic parameters, where the diffusion-controlled kcat/Km showed a salt dependence similar to kon, while kcat (limited by product release) was weakly dependent on salt. Finally, the salt dependence of translocation between two uracil sites separated by 20 bp in the same DNA chain was indistinguishable from that of kon. This result suggests that the transition-state for translocation over this spacing resembles that for DNA association from bulk solution and

  18. Targeting Transcriptional Regulators of CD8+ T Cell Dysfunction to Boost Anti-Tumor Immunity

    PubMed Central

    Waugh, Katherine A.; Leach, Sonia M.; Slansky, Jill E.

    2015-01-01

    Transcription is a dynamic process influenced by the cellular environment: healthy, transformed, and otherwise. Genome-wide mRNA expression profiles reflect the collective impact of pathways modulating cell function under different conditions. In this review we focus on the transcriptional pathways that control tumor infiltrating CD8+ T cell (TIL) function. Simultaneous restraint of overlapping inhibitory pathways may confer TIL resistance to multiple mechanisms of suppression traditionally referred to as exhaustion, tolerance, or anergy. Although decades of work have laid a solid foundation of altered transcriptional networks underlying various subsets of hypofunctional or “dysfunctional” CD8+ T cells, an understanding of the relevance in TIL has just begun. With recent technological advances, it is now feasible to further elucidate and utilize these pathways in immunotherapy platforms that seek to increase TIL function. PMID:26393659

  19. Improved Personalized Cancer Immunotherapy: Rapid Selection of Tumor-Reactive T Cells based on Expression of Specific Cell Surface Markers | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute’s Surgery Branch seeks partners interested in collaborative research to co-develop adoptive transfer of tumor infiltrating leukocytes (TIL) for cancers other than melanoma.

  20. Development of tunable high pressure CO2 laser for lidar measurements of pollutants and wind velocities

    NASA Technical Reports Server (NTRS)

    Levine, J. S.; Guerra, M.; Javan, A.

    1980-01-01

    The problem of laser energy extraction at a tunable monochromatic frequency from an energetic high pressure CO2 pulsed laser plasma, for application to remote sensing of atmospheric pollutants by Differential Absorption Lidar (DIAL) and of wind velocities by Doppler Lidar, was investigated. The energy extraction principle analyzed is based on transient injection locking (TIL) at a tunable frequency. Several critical experiments for high gain power amplification by TIL are presented.

  1. Synoptic-Scale Behavior of the Extratropical Tropopause Inversion Layer

    NASA Astrophysics Data System (ADS)

    Pilch Kedzierski, Robin; Matthes, Katja; Bumke, Karl

    2015-04-01

    The Tropopause Inversion Layer (TIL) is a climatological feature of the tropopause region, characterized by enhanced static stability and strong temperature inversion in a thin layer (about 1km deep) right above the tropopause. It was discovered recently via tropopause-based averaging [Birner 2002]. The sharp static stability, temperature and wind shear gradients of the TIL theoretically shall inhibit stratosphere-troposphere exchange and influence the vertical propagation of planetary scale Rossby and small-scale gravity waves. High vertically resolved radiosonde and GPS radio occultation measurements show that the strength of the TIL is positively correlated with the tropopause height and anticyclonic conditions, and that it reaches its maximum strength in polar regions during summer [Birner 2006] [Randel and Wu, 2007 and 2010]. Our study takes advantage of the high density of vertical profiles (~2000 measurements per day, globally) measured by the COSMIC satellites (2007-present), in order to describe the synoptic-scale structures of the TIL and the differences between the seasonal climatologies from earlier studies and the real-time TIL. Also, using ERA-Interim reanalysis wind fields, we split relative vorticity into shear and curl terms and study separately their relation to TIL strength in cyclonic-anticyclonic conditions. We find that the TIL has a rich zonal structure, especially in midlatitude winter, and that its strength is instantly adjusted to the synoptic situation at near-tropopause level. The peaks of strongest TIL at midlatitude ridges in winter are stronger and much more frequent than any peaks found in polar summer. The roles of shear and curl vorticity differ substantially towards higher values of relative vorticity (both cyclonic and anticyclonic).

  2. The tropopause inversion layer in baroclinic life-cycle experiments: the role of diabatic processes

    NASA Astrophysics Data System (ADS)

    Kunkel, D.; Hoor, P.; Wirth, V.

    2016-01-01

    Recent studies on the formation of a quasi-permanent layer of enhanced static stability above the thermal tropopause revealed the contributions of dynamical and radiative processes. Dry dynamics leads to the evolution of a tropopause inversion layer (TIL), which is, however, too weak compared to observations and thus diabatic contributions are required. In this study we aim to assess the importance of diabatic processes in the understanding of TIL formation at midlatitudes. The non-hydrostatic model COSMO (COnsortium for Small-scale MOdelling) is applied in an idealized midlatitude channel configuration to simulate baroclinic life cycles. The effect of individual diabatic processes related to humidity, radiation, and turbulence is studied first to estimate the contribution of each of these processes to the TIL formation in addition to dry dynamics. In a second step these processes are stepwise included in the model to increase the complexity and finally estimate the relative importance of each process. The results suggest that including turbulence leads to a weaker TIL than in a dry reference simulation. In contrast, the TIL evolves stronger when radiation is included but the temporal evolution is still comparable to the reference. Using various cloud schemes in the model shows that latent heat release and consecutive increased vertical motions foster an earlier and stronger appearance of the TIL than in all other life cycles. Furthermore, updrafts moisten the upper troposphere and as such increase the radiative effect from water vapor. Particularly, this process becomes more relevant for maintaining the TIL during later stages of the life cycles. Increased convergence of the vertical wind induced by updrafts and by propagating inertia-gravity waves, which potentially dissipate, further contributes to the enhanced stability of the lower stratosphere. Finally, radiative feedback of ice clouds reaching up to the tropopause is identified to potentially further affect

  3. Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma

    PubMed Central

    Beane, Joal D; Lee, Gary; Zheng, Zhili; Mendel, Matthew; Abate-Daga, Daniel; Bharathan, Mini; Black, Mary; Gandhi, Nimisha; Yu, Zhiya; Chandran, Smita; Giedlin, Martin; Ando, Dale; Miller, Jeff; Paschon, David; Guschin, Dmitry; Rebar, Edward J; Reik, Andreas; Holmes, Michael C; Gregory, Philip D; Restifo, Nicholas P; Rosenberg, Steven A; Morgan, Richard A; Feldman, Steven A

    2015-01-01

    Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9–84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500–2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted. PMID:25939491

  4. Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma.

    PubMed

    Beane, Joal D; Lee, Gary; Zheng, Zhili; Mendel, Matthew; Abate-Daga, Daniel; Bharathan, Mini; Black, Mary; Gandhi, Nimisha; Yu, Zhiya; Chandran, Smita; Giedlin, Martin; Ando, Dale; Miller, Jeff; Paschon, David; Guschin, Dmitry; Rebar, Edward J; Reik, Andreas; Holmes, Michael C; Gregory, Philip D; Restifo, Nicholas P; Rosenberg, Steven A; Morgan, Richard A; Feldman, Steven A

    2015-08-01

    Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted. PMID:25939491

  5. Immune Checkpoint Blockade to Improve Tumor Infiltrating Lymphocytes for Adoptive Cell Therapy

    PubMed Central

    Kodumudi, Krithika N.; Siegel, Jessica; Weber, Amy M.; Scott, Ellen; Sarnaik, Amod A.; Pilon-Thomas, Shari

    2016-01-01

    Tumor-infiltrating lymphocytes (TIL) has been associated with improved survival in cancer patients. Within the tumor microenvironment, regulatory cells and expression of co-inhibitory immune checkpoint molecules can lead to the inactivation of TIL. Hence, there is a need to develop strategies that disrupt these negative regulators to achieve robust anti-tumor immune responses. We evaluated the blockade of immune checkpoints and their effect on T cell infiltration and function. We examined the ability of TIL to induce tumor-specific immune responses in vitro and in vivo. TIL isolated from tumor bearing mice were tumor-specific and expressed co-inhibitory immune checkpoint molecules. Administration of monoclonal antibodies against immune checkpoints led to a significant delay in tumor growth. However, anti-PD-L1 antibody treated mice had a significant increase in T cell infiltration and IFN-γ production compared to other groups. Adoptive transfer of in vitro expanded TIL from tumors of anti-PD-L1 antibody treated mice led to a significant delay in tumor growth. Blockade of co-inhibitory immune checkpoints could be an effective strategy to improve TIL infiltration and function. PMID:27050669

  6. Tumor-infiltrating lymphocyte activity is enhanced in tumors with low IL-10 production in HBV-induced hepatocellular carcinoma

    SciTech Connect

    Shi, Yang Song, Qingwei; Hu, Dianhe; Zhuang, Xiaohu; Yu, Shengcai

    2015-05-22

    Hepatocellular carcinoma (HCC) is one of the most common cancers and can be induced by chronic HBV infection. The role of HBV-specific immune responses in mediating tumorigenesis and HCC prognosis is debated. The effect of intratumoral microenvironment on tumor-infiltrating lymphocytes (TILs) is also unclear. Here, we examined resected tumor tissue from 36 patients with HBV-induced HCC. We categorized study cohort based on ex vivo IL-10 secretion by tumor cells into high IL-10-secreting (Hi10) and low IL-10-secreting (Lo10) groups, and found that the Lo10 group was less sensitive to TLR ligand stimulation. TILs from the Lo10 group contained higher frequencies of HBV-specific IFN-g-producing cells and total IFN-g-producing cells, and possessed higher proliferative capacity. Moreover, the proliferative capacity of TILs from the Hi10 group was negatively correlated with IL-10 secretion from tumor cells. Together, our data demonstrated that low IL-10-producing capacity in HBV-induced HCC tumors is associated with enhanced TIL activity. - Highlights: • We examined intratumoral IL-10 production in HBV-induced HCC. • We grouped HCC tumors into Hi10 and Lo10 groups based on their IL-10 production. • Lo10 groups had better IFN-g response by TILs. • Lo10 groups had better TIL proliferative capacity. • Lo10 group tumor cells were refractory to TLR ligand stimulation.

  7. Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells.

    PubMed

    Saito, Hidehito; Okita, Keisuke; Fusaki, Noemi; Sabel, Michael S; Chang, Alfred E; Ito, Fumito

    2016-01-01

    Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) that express programmed cell death protein-1 (PD-1) are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate in vitro and in vivo. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy. PMID:27057178

  8. Sunitinib pretreatment improves tumor-infiltrating lymphocyte expansion by reduction in intratumoral content of myeloid-derived suppressor cells in human renal cell carcinoma.

    PubMed

    Guislain, Aurelie; Gadiot, Jules; Kaiser, Andrew; Jordanova, Ekaterina S; Broeks, Annegien; Sanders, Joyce; van Boven, Hester; de Gruijl, Tanja D; Haanen, John B A G; Bex, Axel; Blank, Christian U

    2015-10-01

    Targeted therapy with sunitinib, pazopanib or everolimus has improved treatment outcome for patients with metastatic renal cell carcinoma patients (RCC). However, despite considerable efforts in sequential or combined modalities, durable remissions are rare. Immunotherapy like cytokine therapy with interleukin-2, T cell checkpoint blockade or adoptive T cell therapies can achieve long-term benefit and even cure. This raises the question of whether combining targeted therapy with immunotherapy could also be an effective treatment option for RCC patients. Sunitinib, one of the most frequently administered therapeutics in RCC patients has been implicated in impairing T cell activation and proliferation in vitro. In this work, we addressed whether this notion holds true for expansion of tumor-infiltrating lymphocytes (TILs) in sunitinib-treated patients. We compared resected primary RCC tumor material of patients pretreated with sunitinib with resection specimen from sunitinib-naïve patients. We found improved TIL expansion from sunitinib-pretreated tumor digests. These TIL products contained more PD-1 expressing TIL, while the regulatory T cell infiltration was not altered. The improved TIL expansion was associated with reduced intratumoral myeloid-derived suppressor cell (MDSC) content. Depletion of MDSCs from sunitinib-naïve RCC tissue-digest improved TIL expansion, proving the functional relevance of the MDSC alteration by sunitinib. Our in vivo results do not support previous in vitro observations of sunitinib inhibiting T cell function, but do provide a possible rationale for the combination of sunitinib with immunotherapy. PMID:26105626

  9. Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells

    PubMed Central

    Saito, Hidehito; Okita, Keisuke; Fusaki, Noemi; Sabel, Michael S.; Chang, Alfred E.; Ito, Fumito

    2016-01-01

    Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) that express programmed cell death protein-1 (PD-1) are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate in vitro and in vivo. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy. PMID:27057178

  10. Early T cell signalling is reversibly altered in PD-1+ T lymphocytes infiltrating human tumors.

    PubMed

    Wang, Shu-Fang; Fouquet, Stéphane; Chapon, Maxime; Salmon, Hélène; Regnier, Fabienne; Labroquère, Karine; Badoual, Cécile; Damotte, Diane; Validire, Pierre; Maubec, Eve; Delongchamps, Nicolas B; Cazes, Aurélie; Gibault, Laure; Garcette, Marylène; Dieu-Nosjean, Marie-Caroline; Zerbib, Marc; Avril, Marie-Françoise; Prévost-Blondel, Armelle; Randriamampita, Clotilde; Trautmann, Alain; Bercovici, Nadège

    2011-01-01

    To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL. PMID:21408177

  11. Hyper-IgM, neutropenia, mild infections and low response to polyclonal stimulation: hyper-IgM syndrome or common variable immunodeficiency?

    PubMed

    Rosado, M M; Picchianti Diamanti, A; Cascioli, S; Ceccarelli, S; Caporuscio, S; D'Amelio, R; Carsetti, R; Lagana, B

    2011-01-01

    A young woman presenting respiratory infections, polyarthritis, severe neutropenia, and increased serum IgM was treated with intravenous immunoglobulin (IVIG) with good clinical and laboratory outcome followed by a loss of efficacy. The increased serum IgM associated to recurrent infections and autoimmune manifestations suggested the diagnosis of a hyper-IgM syndrome (HIGMs). The frequency of peripheral T cells, the expression of CD40 on the patients' B cells and CD40L on T cells and the activation-induced cytidine deaminase (AID) and uracil-DNA glycosylase (UNG) at mRNA level was comparable to controls. In contrast, the frequency of B cells was one half of the healthy control and all cells showed an atypical phenotype. Although AID and UNG were normal, class-switch recombination was not very efficient because circulating switched memory were reduced and, once stimulated with CpG, generated less antibody-secreting cells than controls. An increase in serum B Lymphocytes stimulator (BLyS) was also found. The patient presented a peculiar clinical and immunological phenotype fitting for many aspects of both HIGM4 and Common Variable Immunodeficiency (CVID). These findings underline the need to better explore the complex link between these two diseases. PMID:22230404

  12. Non-canonical uracil processing in DNA gives rise to double-strand breaks and deletions: relevance to class switch recombination.

    PubMed

    Bregenhorn, Stephanie; Kallenberger, Lia; Artola-Borán, Mariela; Peña-Diaz, Javier; Jiricny, Josef

    2016-04-01

    During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions, repetitive sequences flanking the CH loci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks (DSBs), which trigger CSR. Surprisingly, genetic experiments revealed that CSR is dependent not only on AID and UNG, but also on mismatch repair (MMR). To elucidate the role of MMR in CSR, we studied the processing of uracil-containing DNA substrates in extracts of MMR-proficient and -deficient human cells, as well as in a system reconstituted from recombinant BER and MMR proteins. Here, we show that the interplay of these repair systems gives rise to DSBs in vitro and to genomic deletions and mutations in vivo, particularly in an S-region sequence. Our findings further suggest that MMR affects pathway choice in DSB repair. Given its amenability to manipulation, our system represents a powerful tool for the molecular dissection of CSR. PMID:26743004

  13. Folate Deficiency Induces Neurodegeneration and Brain Dysfunction in Mice Lacking Uracil DNA Glycosylase

    PubMed Central

    Kronenberg, Golo; Harms, Christoph; Sobol, Robert W.; Cardozo-Pelaez, Fernando; Linhart, Heinz; Winter, Benjamin; Balkaya, Mustafa; Gertz, Karen; Gay, Shanna B.; Cox, David; Eckart, Sarah; Ahmadi, Michael; Juckel, Georg; Kempermann, Gerd; Hellweg, Rainer; Sohr, Reinhard; Hörtnagl, Heide; Wilson, Samuel H.; Jaenisch, Rudolf

    2008-01-01

    Folate deficiency and resultant increased homocysteine levels have been linked experimentally and epidemiologically with neurodegenerative conditions like stroke and dementia. Moreover, folate deficiency has been implicated in the pathogenesis of psychiatric disorders, most notably depression. We hypothesized that the pathogenic mechanisms include uracil misincorporation and, therefore, analyzed the effects of folate deficiency in mice lacking uracil DNA glycosylase (Ung−/−) versus wild-type controls. Folate depletion increased nuclear mutation rates in Ung−/− embryonic fibroblasts, and conferred death of cultured Ung−/− hippocampal neurons. Feeding animals a folate-deficient diet (FD) for 3 months induced degeneration of CA3 pyramidal neurons in Ung−/− but not Ung+/+ mice along with decreased hippocampal expression of brain-derived neurotrophic factor protein and decreased brain levels of antioxidant glutathione. Furthermore, FD induced cognitive deficits and mood alterations such as anxious and despair-like behaviors that were aggravated in Ung−/− mice. Independent of Ung genotype, FD increased plasma homocysteine levels, altered brain monoamine metabolism, and inhibited adult hippocampal neurogenesis. These results indicate that impaired uracil repair is involved in neurodegeneration and neuropsychiatric dysfunction induced by experimental folate deficiency. PMID:18614692

  14. Mitochondrial DNA Toxicity in Forebrain Neurons Causes Apoptosis, Neurodegeneration, and Impaired Behavior ▿

    PubMed Central

    Lauritzen, Knut H.; Moldestad, Olve; Eide, Lars; Carlsen, Harald; Nesse, Gaute; Storm, Johan F.; Mansuy, Isabelle M.; Bergersen, Linda H.; Klungland, Arne

    2010-01-01

    Mitochondrial dysfunction underlying changes in neurodegenerative diseases is often associated with apoptosis and a progressive loss of neurons, and damage to the mitochondrial genome is proposed to be involved in such pathologies. In the present study we designed a mouse model that allows us to specifically induce mitochondrial DNA toxicity in the forebrain neurons of adult mice. This is achieved by CaMKIIα-regulated inducible expression of a mutated version of the mitochondrial UNG DNA repair enzyme (mutUNG1). This enzyme is capable of removing thymine from the mitochondrial genome. We demonstrate that a continual generation of apyrimidinic sites causes apoptosis and neuronal death. These defects are associated with behavioral alterations characterized by increased locomotor activity, impaired cognitive abilities, and lack of anxietylike responses. In summary, whereas mitochondrial base substitution and deletions previously have been shown to correlate with premature and natural aging, respectively, we show that a high level of apyrimidinic sites lead to mitochondrial DNA cytotoxicity, which causes apoptosis, followed by neurodegeneration. PMID:20065039

  15. Silencing-associated and meiosis-specific small RNA pathways in Paramecium tetraurelia.

    PubMed

    Lepère, Gersende; Nowacki, Mariusz; Serrano, Vincent; Gout, Jean-François; Guglielmi, Gérard; Duharcourt, Sandra; Meyer, Eric

    2009-02-01

    Distinct small RNA pathways are involved in the two types of homology-dependent effects described in Paramecium tetraurelia, as shown by a functional analysis of Dicer and Dicer-like genes and by the sequencing of small RNAs. The siRNAs that mediate post-transcriptional gene silencing when cells are fed with double-stranded RNA (dsRNA) were found to comprise two subclasses. DCR1-dependent cleavage of the inducing dsRNA generates approximately 23-nt primary siRNAs from both strands, while a different subclass of approximately 24-nt RNAs, characterized by a short untemplated poly-A tail, is strictly antisense to the targeted mRNA, suggestive of secondary siRNAs that depend on an RNA-dependent RNA polymerase. An entirely distinct pathway is responsible for homology-dependent regulation of developmental genome rearrangements after sexual reproduction. During early meiosis, the DCL2 and DCL3 genes are required for the production of a highly complex population of approximately 25-nt scnRNAs from all types of germline sequences, including both strands of exons, introns, intergenic regions, transposons and Internal Eliminated Sequences. A prominent 5'-UNG signature, and a minor fraction showing the complementary signature at positions 21-23, indicate that scnRNAs are cleaved from dsRNA precursors as duplexes with 2-nt 3' overhangs at both ends, followed by preferential stabilization of the 5'-UNG strand. PMID:19103667

  16. Molecular mechanism for generation of antibody memory

    PubMed Central

    Shivarov, Velizar; Shinkura, Reiko; Doi, Tomomitsu; Begum, Nasim A.; Nagaoka, Hitoshi; Okazaki, Il-Mi; Ito, Satomi; Nonaka, Taichiro; Kinoshita, Kazuo; Honjo, Tasuku

    2008-01-01

    Activation-induced cytidine deaminase (AID) is the essential enzyme inducing the DNA cleavage required for both somatic hypermutation and class switch recombination (CSR) of the immunoglobulin gene. We originally proposed the RNA-editing model for the mechanism of DNA cleavage by AID. We obtained evidence that fulfils three requirements for CSR by this model, namely (i) AID shuttling between nucleus and cytoplasm, (ii) de novo protein synthesis for CSR, and (iii) AID–RNA complex formation. The alternative hypothesis, designated as the DNA-deamination model, assumes that the in vitro DNA deamination activity of AID is representative of its physiological function in vivo. Furthermore, the resulting dU was removed by uracil DNA glycosylase (UNG) to generate a basic site, followed by phosphodiester bond cleavage by AP endonuclease. We critically examined each of these provisional steps. We identified a cluster of mutants (H48A, L49A, R50A and N51A) that had particularly higher CSR activities than expected from their DNA deamination activities. The most striking was the N51A mutant that had no ability to deaminate DNA in vitro but retained approximately 50 per cent of the wild-type level of CSR activity. We also provide further evidence that UNG plays a non-canonical role in CSR, namely in the repair step of the DNA breaks. Taking these results together, we favour the RNA-editing model for the function of AID in CSR. PMID:19022739

  17. Non-canonical uracil processing in DNA gives rise to double-strand breaks and deletions: relevance to class switch recombination

    PubMed Central

    Bregenhorn, Stephanie; Kallenberger, Lia; Artola-Borán, Mariela; Peña-Diaz, Javier; Jiricny, Josef

    2016-01-01

    During class switch recombination (CSR), antigen-stimulated B-cells rearrange their immunoglobulin constant heavy chain (CH) loci to generate antibodies with different effector functions. CSR is initiated by activation-induced deaminase (AID), which converts cytosines in switch (S) regions, repetitive sequences flanking the CH loci, to uracils. Although U/G mispairs arising in this way are generally efficiently repaired to C/Gs by uracil DNA glycosylase (UNG)-initiated base excision repair (BER), uracil processing in S-regions of activated B-cells occasionally gives rise to double strand breaks (DSBs), which trigger CSR. Surprisingly, genetic experiments revealed that CSR is dependent not only on AID and UNG, but also on mismatch repair (MMR). To elucidate the role of MMR in CSR, we studied the processing of uracil-containing DNA substrates in extracts of MMR-proficient and –deficient human cells, as well as in a system reconstituted from recombinant BER and MMR proteins. Here, we show that the interplay of these repair systems gives rise to DSBs in vitro and to genomic deletions and mutations in vivo, particularly in an S-region sequence. Our findings further suggest that MMR affects pathway choice in DSB repair. Given its amenability to manipulation, our system represents a powerful tool for the molecular dissection of CSR. PMID:26743004

  18. Comparative study of various subpopulations of cytotoxic cells in blood and ascites from patients with ovarian carcinoma

    PubMed Central

    Lukesova, Sarka; Vroblova, Vladimira; Tosner, Jindrich; Kopecky, Jindrich; Sedlakova, Iva; Čermáková, Eva; Vokurkova, Doris

    2015-01-01

    Aim of the study A number of observations have indicated that the immune system plays a significant role in patients with epithelial ovarian cancer (EOC). In cases of EOC, the prognostic significance of tumour infiltrating lymphocytes has not been clearly explained yet. The aim is to determine the phenotype and activation molecules of cytotoxic T cell and NK cell subpopulations and to compare their representation in malignant ascites and peripheral blood in patients with ovarian cancer. Material and methods Cytotoxic cells taken from blood samples of the cubital vein and malignant ascites were obtained from 53 patients with EOC. Their surface and activation characteristics were determined by means of a flow cytometer. Immunophenotype multiparametric analysis of peripheral blood lymphocytes (PBLs) and tumour infiltrating lymphocytes (TILs) was carried out. Results CD3+ T lymphocytes were the main population of TILs (75.9%) and PBLs (70.9%). The number of activating T cells was significantly higher in TILs: CD3+/69+ 6.7% vs. 0.8% (p < 0.001). The representation of (CD3–/16+56+) NK cells in TILs was significantly higher: 11.0% vs. 5.6% (p = 0.041); likewise CD56bright and CD–56bright from CD56+ cells were higher in TILs (both p < 0.001). The activation receptor NKG2D was present in 45.1% of TILs vs. 32.3% of PBLs (p = 0.034), but we did not find a significant difference in the numbers of CD56+/NKG2D+ in TILs and PBLs. Conclusions These results prove that the characteristics and intensity of anti-tumour responses are different in compared compartments (ascites/PBLs). The knowledge of phenotype and functions of effector cells is the basic precondition for understanding the anti-tumour immune response. PMID:26557777

  19. Tumor-infiltrating lymphocytes in glioblastoma are associated with specific genomic alterations and related to transcriptional class

    PubMed Central

    Rutledge, W. Caleb; Kong, Jun; Gao, Jingjing; Gutman, David A.; Cooper, Lee A.D.; Appin, Christina; Park, Yuna; Scarpace, Lisa; Mikkelsen, Tom; Cohen, Mark L.; Aldape, Kenneth D.; McLendon, Roger E.; Lehman, Norman L.; Miller, C. Ryan; Schniederjan, Matthew J.; Brennan, Cameron W.; Saltz, Joel H.; Moreno, Carlos S.; Brat, Daniel J.

    2013-01-01

    Purpose Tumor-infiltrating lymphocytes (TILs) have prognostic significance in many cancers, yet their roles in glioblastoma (GBM) have not been fully defined. We hypothesized TILs in GBM are associated with molecular alterations, histologies and survival. Experimental Design We used data from The Cancer Genome Atlas (TCGA) to investigate molecular, histologic and clinical correlates of TILs in GBMs. Lymphocytes were categorized as absent, present or abundant in histopathologic images from 171 TCGA GBMs. Associations were examined between lymphocytes and histologic features, mutations, copy number alterations, CpG island methylator phenotype, transcriptional class and survival. We validated histologic findings using CD3G gene expression. Results We found a positive correlation between TILs and GBMs with gemistocytes, sarcomatous cells, epithelioid cells and giant cells. Lymphocytes were enriched in the mesenchymal transcriptional class and strongly associated with mutations in NF1 and RB1. These mutations are frequent in the mesenchymal class and characteristic of gemistocytic, sarcomatous, epithelioid and giant cell histologies. Conversely, TILs were rare in GBMs with small cells and oligodendroglioma components. Lymphocytes were depleted in the classical transcriptional class and in EGFR-amplified and homozygous PTEN-deleted GBMs. These alterations are characteristic of GBMs with small cells and GBMs of the classical transcriptional class. No association with survival was demonstrated. Conclusions TILs were enriched in GBMs of the mesenchymal class, strongly associated with mutations in NF1 and RB1 and typical of histologies characterized by these mutations. Conversely, TILs were depleted in the classical class, EGFR-amplified and homozygous PTEN-deleted tumors and rare in histologies characterized by these alterations. PMID:23864165

  20. Prognostic and predictive value of tumor-infiltrating lymphocytes for clinical therapeutic research in patients with non-small cell lung cancer

    PubMed Central

    Zeng, Dong-Qiang; Yu, Yun-Fang; Ou, Qi-Yun; Li, Xiao-Yin; Zhong, Ru-Zhi; Xie, Chuan-Miao; Hu, Qiu-Gen

    2016-01-01

    Background Previous preclinical and clinical studies have shown that levels of tumor-infiltrating lymphocytes (TILs) significantly correlated with prognosis in non-small cell lung cancer (NSCLC), and survival after therapy; however, this finding remains controversial. We performed a meta-analysis, to evaluate, systematically, the clinical utilization of TIL subtypes in patients with NSCLC. Methods The PubMed, ISI Web of Science, EMBASE, and Cochrane Library databases were searched to identify relevant studies. We pooled estimates of treatment effects, and hazards were summarized using random or fixed effects models to evaluate survival outcomes. Results A total of 24 relevant studies involving 7,006 patients were eligible. The median percentage of lymph node positivity was 45.7% (95% confidence interval [CI], 37.1–56.4%). Pooled analysis shows that high levels of CD8+ TILs had a good prognostic effect on survival with a hazard ratio (HR) of 0.91 (P = 0.013) for death and 0.74 (P = 0.001) for recurrence, as did high levels of CD3+ and CD4+ TILs, with HRs of 0.77 (P = 0.009) and 0.78 (P = 0.005) for death, respectively. By contrast, high levels of FoxP3+ regulatory TILs had a worse prognostic effect for overall and recurrence-free survival, with HRs of 1.69 (P = 0.042) and 1.79 (P = 0.001), respectively. No individual study affected the results, and no publication bias was found. Conclusions Our findings support the hypothesis that TILs could be a prognostic marker in NSCLC. High-quality randomized studies are needed to verify statistically the effect of TILs on prognosis in future research. PMID:26871598

  1. Surface Proteome Analysis of a Natural Isolate of Lactococcus lactis Reveals the Presence of Pili Able to Bind Human Intestinal Epithelial Cells*

    PubMed Central

    Meyrand, Mickael; Guillot, Alain; Goin, Mélodie; Furlan, Sylviane; Armalyte, Julija; Kulakauskas, Saulius; Cortes-Perez, Naima G.; Thomas, Ginette; Chat, Sophie; Péchoux, Christine; Dupres, Vincent; Hols, Pascal; Dufrêne, Yves F.; Trugnan, Germain; Chapot-Chartier, Marie-Pierre

    2013-01-01

    Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells

  2. Surface proteome analysis of a natural isolate of Lactococcus lactis reveals the presence of pili able to bind human intestinal epithelial cells.

    PubMed

    Meyrand, Mickael; Guillot, Alain; Goin, Mélodie; Furlan, Sylviane; Armalyte, Julija; Kulakauskas, Saulius; Cortes-Perez, Naima G; Thomas, Ginette; Chat, Sophie; Péchoux, Christine; Dupres, Vincent; Hols, Pascal; Dufrêne, Yves F; Trugnan, Germain; Chapot-Chartier, Marie-Pierre

    2013-12-01

    Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells

  3. Prognostic value of tumor-infiltrating lymphocytes for patients with completely resected stage IIIA(N2) non-small cell lung cancer

    PubMed Central

    Shen, Lei; Cai, Xu-Wei; Zhu, Zheng-Fei; Chang, Jian-Hua; Xiang, Jia-Qing; Zhang, Ya-Wei; Chen, Hai-Quan; Fu, Xiao-Long

    2016-01-01

    Background The patient prognosis after complete resection for pathologic stage IIIA(N2) non-small cell lung cancer (NSCLC) remains a significant concern. The clinical relevance of the host immune response to NSCLC has yet to be established. We aimed to investigate the prognostic value of tumor-infiltrating lymphocytes (TILs) in a uniform cohort of patients with completely resected stage IIIA(N2) NSCLC. Methods From 2005 to 2012, consecutive patients with pathologic stage IIIA(N2) NSCLC who underwent complete resection at our institution were reviewed. For each case, full-face hematoxylin and eosin-stained sections from surgical specimens were evaluated for the TIL density. A published, recommended TIL scoring scale was followed. The patients were stratified into the TIL− or TIL+ group based on pathologic evaluation. Results Data from 320 patients were included in the analysis. Based on a median follow-up duration of 30.8 months, a higher density of TILs was associated with an improved postoperative survival time (P = 0.06). Subgroup analyses indicated that this positive effect was the greatest for patients with squamous cell carcinoma (SCC; P = 0.03). Among those with SCC, the TIL+ patients experienced a significantly increased 3-year distant metastasis-free survival (DMFS) compared to the TIL− patients (60.6% versus 42.7%, P = 0.02). Multivariate analyses of the 93 patients with SCC tumors confirmed that TIL+ was an independent prognostic factor for an increased DMFS (HR = 0.39, 95%CI 0.17–0.87, P = 0.02) and a prolonged overall survival (OS; HR = 0.47, 95%CI 0.22–1.00, P = 0.05). Conclusions Our data suggest a potential role of TILs in predicting the survival of patients with completely resected stage IIIA(N2) NSCLC. The beneficial effects of TILs were more pronounced in the prediction of the DMFS and the OS in patients with SCC. This parameter should be considered for prospective inclusion in clinical trials. PMID:26811495

  4. The Effects of Isoproterenol and Propranolol on Cytokine Profile Secretion by Cultured Tumor-infiltrating Lymphocytes Derived from Colorectal Cancer Patients

    PubMed Central

    Seyedi, Shahram; Andalib, Alireza; Rezaei, Abbas; Hosseini, Seyed Mohsen; Mohebbi, Seyed Reza; Zali, Mohammad Reza; Vafai, Mohamad; Behboo, Roubik; Tabatabaei, Seyed Abbas; Shahabi, Shahram

    2012-01-01

    Objective: Anti-tumor immunity and cytokine profiles have important roles in the development of cancer. Norepinephrine (NE) release due to sympathetic activation leads to a Th2 deviation via the beta-2 adrenergic receptor Beta-2 adrenergic receptor (β-2AR) and could increase cancer progression. This study intends to determine the effects of isoproterenol (ISO; beta-agonist) and propranolol (PRO; beta-antagonist) on the production of IFN-γ, IL-4, and IL-17. Cytokine levels have been examined in tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) of patients with colorectal cancer (CRC). The β-2AR expression on lymphocyte subsets was also assessed. Materials and Methods: In this experimental study, TILs were isolated from fresh CRC tissue and patient PBMCs were obtained just prior to surgery. The cells were cultured in medium for 72 hours. Concomitantly, cells were stimulated with 10 µg/ml phytohemagglutinin (PHA) alone or in the presence of either 1 µmol/L of PRO or 1 µmol/L ISO. The concentration of cytokines in the supernatants was measured by ELISA. Three-color flow cytometry was used to determine the expression of β-2AR on the lymphocyte subsets. Statistical analyses were performed via paired or independent t-test. Results: Levels of IFN-γ, IL-4 and IL-17 were elevated after PHA-stimulation of PBMCs and TILs. However, the elevation of IFN-γ and IL-17 production by TILs in response to PHA was significantly lower than PBMCs. In the presence of ISO, the IFN-γ/IL-4 ratio reduced in all groups, but this reduction was very low in TILs. Interestingly, the effects of PRO on cytokine production were, at least partially, comparable to those of ISO. Depressed levels of β-2AR expression were demonstrated on CD4+IFN-γ+ and CD4+IL-17+ lymphocytes in patients' PBMCs and TILs. Conclusion: This study has demonstrated the effects of ISO and PRO on cytokine production by TILs and determined β-2AR expression on these cells. ISO failed

  5. Dynamics of uracil and 5-fluorouracil in DNA.

    PubMed

    Parker, Jared B; Stivers, James T

    2011-02-01

    The prodrug 5-fluorouracil (5-FU), after activation into 5-F-dUMP, is an extensively used anticancer agent that inhibits thymidylate synthase and leads to increases in dUTP and 5-F-dUTP levels in cells. One mechanism for 5-FU action involves DNA polymerase mediated incorporation of dUTP and 5-F-dUTP into genomic DNA leading to U/A, 5-FU/A, or 5-FU/G base pairs. These uracil-containing lesions are recognized and excised by several human uracil excision repair glycosylases (hUNG2, hSMUG2, and hTDG) leading to toxic abasic sites in DNA that may precipitate cell death. Each of these enzymes uses an extrahelical base recognition mechanism, and previous studies with UNG have shown that extrahelical recognition is facilitated by destabilized base pairs possessing kinetically enhanced base pair opening rates. Thus, the dynamic properties of base pairs containing 5-FU and U are an important unknown in understanding the role of these enzymes in damage recognition and prodrug activation. The pH dependence of the (19)F NMR chemical shift of 5-FU imbedded in a model trinucleotide was used to obtain a pK(a) = 8.1 for its imino proton (10 °C). This is about 1.5 units lower than the imino protons of uracil or thymine and indicates that at neutral pH 5-FU exists significantly as an ionized tautomer that can mispair with guanine during DNA replication. NMR imino proton exchange measurements show that U/A and 5-FU/A base pairs open with rate constants (k(op)) that are 6- and 13-fold faster than a T/A base pair in the same sequence context. In contrast, these same base pairs have apparent opening equilibrium constants (αK(op)) that differ by less than a factor of 2, indicating that the closing rates (k(cl)) are enhanced by nearly equal amounts as k(op). These dynamic measurements are consistent with the previously proposed kinetic trapping model for extrahelical recognition by UNG. In this model, the enhanced intrinsic opening rates of destabilized base pairs allow the bound

  6. A major secretory defect of tumour-infiltrating T lymphocytes due to galectin impairing LFA-1-mediated synapse completion

    PubMed Central

    Petit, Anne-Elisabeth; Demotte, Nathalie; Scheid, Benoît; Wildmann, Claude; Bigirimana, René; Gordon-Alonso, Monica; Carrasco, Javier; Valitutti, Salvatore; Godelaine, Danièle; van der Bruggen, Pierre

    2016-01-01

    Surface galectin has been shown to contribute to dysfunctions of human tumour-infiltrating lymphocytes (TILs). We show here that galectin-covered CD8 TILs produce normal amounts of intracellular cytokines, but fail to secrete them because of defective actin rearrangements at the synapse. The non-secreting TILs also display reduced adhesion to their targets, together with defective LFA-1 recruitment and activation at the synapse. These defects are relieved by releasing surface galectin. As mild LFA-1 blockade on normal blood T cells emulate the defects of galectin-covered TILs, we conclude that galectin prevents the formation of a functional secretory synapse by preventing optimal LFA-1 triggering. Our results highlight a major secretory defect of TILs that is not revealed by widely used intracellular cytokine immunomonitoring assays. They also provide additional insights into the T-cell response, by showing that different thresholds of LFA-1 triggering are required to promote the intracellular production of cytokines and their secretion. PMID:27447355

  7. Akt inhibition enhances expansion of potent tumor-specific lymphocytes with memory cell characteristics.

    PubMed

    Crompton, Joseph G; Sukumar, Madhusudhanan; Roychoudhuri, Rahul; Clever, David; Gros, Alena; Eil, Robert L; Tran, Eric; Hanada, Ken-Ichi; Yu, Zhiya; Palmer, Douglas C; Kerkar, Sid P; Michalek, Ryan D; Upham, Trevor; Leonardi, Anthony; Acquavella, Nicolas; Wang, Ena; Marincola, Francesco M; Gattinoni, Luca; Muranski, Pawel; Sundrud, Mark S; Klebanoff, Christopher A; Rosenberg, Steven A; Fearon, Douglas T; Restifo, Nicholas P

    2015-01-15

    Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) results in complete regression of advanced cancer in some patients, but the efficacy of this potentially curative therapy may be limited by poor persistence of TIL after adoptive transfer. Pharmacologic inhibition of the serine/threonine kinase Akt has recently been shown to promote immunologic memory in virus-specific murine models, but whether this approach enhances features of memory (e.g., long-term persistence) in TIL that are characteristically exhausted and senescent is not established. Here, we show that pharmacologic inhibition of Akt enables expansion of TIL with the transcriptional, metabolic, and functional properties characteristic of memory T cells. Consequently, Akt inhibition results in enhanced persistence of TIL after adoptive transfer into an immunodeficient animal model and augments antitumor immunity of CD8 T cells in a mouse model of cell-based immunotherapy. Pharmacologic inhibition of Akt represents a novel immunometabolomic approach to enhance the persistence of antitumor T cells and improve the efficacy of cell-based immunotherapy for metastatic cancer. PMID:25432172

  8. Identification and Functional Analysis of Interleukin-1β in the Chinese Soft-Shelled Turtle Pelodiscus sinensis

    PubMed Central

    Liang, Quan; Li, Weifen; Guo, Ningning; Tong, Chao; Zhou, Yingshan; Fang, Weihuan; Li, Xiaoliang

    2016-01-01

    Chinese soft-shelled turtle (Pelodiscus sinensis) is commercially cultured in East and Southeast Asia for its nutritional and medicinal values. In this study, we identified interleukin-1β (IL-1β) from Chinese soft-shelled turtle. The full-length cDNA of Pelodiscus sinensis IL-1β (tIL-1β) consists of 1529 base pairs with an 831-base-pair open reading frame, encoding 277 amino acids. The guanine-cytosine (GC) content in the coding sequence and 3’ untranslated region of tIL-1β is considerably higher than that of other vertebrates. Its mRNA expression level increased significantly during Aeromonas hydrophila infection. The tIL-1β lacks the typical IL-1β-converting enzyme (ICE) cut site found in mammalian IL-1β, but still could be cleaved by turtle caspase-1. By mutating the potential cleavage sites, we identified aspartic acid (Asp/D) 130 as the ICE cut site in tIL-1β. The peptide truncated at D130 was expressed using the baculovirus expression system; its bioactivity is confirmed by the ability to induce cyclooxygenase-2 (COX-2) and tIL-1β itself in peripheral blood monocytes. In conclusion, we characterized IL-1β from Chinese soft-shelled turtle and identified its D130 as a non-typical ICE cut size. PMID:27153094

  9. Tumor-Infiltrating Lymphocytes in Triple Negative Breast Cancer: The Future of Immune Targeting

    PubMed Central

    García-Teijido, Paula; Cabal, María Luque; Fernández, Ignacio Peláez; Pérez, Yolanda Fernández

    2016-01-01

    Triple negative breast cancer (TNBC) is a highly heterogeneous tumor. There is increasing evidence of the role of tumor lymphocytic immune infiltrates in this subtype of breast cancer. Robust levels of tumor infiltrating lymphocytes (TILs) have been associated with improved disease-free and overall survival rates in TNBC patients with and without any treatment. Recent efforts have been made to develop a standardized methodology for evaluating TILs. The presence of TILs in the breast tumor microenvironment can also predict responses not only to neoadjuvant but also to adjuvant chemotherapy treatments. High numbers of TILs correlate with increased pathological complete responses (pCR) in TNBC. TILs are prognostic and predictive of response to standard therapies; thus, the immune system appears to play an active role in a subgroup of breast cancer. There is an increasing interest in directly targeting the immune system as part of breast cancer therapy, mainly in patients with TNBC. New immune modulatory agents, including immune checkpoints inhibitors, have shown promising activity in a subgroup of metastatic TNBC. Increased programmed cell death protein 1 ligand (PD-L1) expression on the surface of TNBC provides the rationale for implementing therapeutic strategies targeting the PD-1/PD-L1 axis in TNBC. The programmed cell death protein 1 (PD-1) inhibitor pembrolizumab, and the PD-L1 inhibitor atezolizumab have shown promising results in clinical trials. PMID:27081325

  10. Identification and Functional Analysis of Interleukin-1β in the Chinese Soft-Shelled Turtle Pelodiscus sinensis.

    PubMed

    Liang, Quan; Li, Weifen; Guo, Ningning; Tong, Chao; Zhou, Yingshan; Fang, Weihuan; Li, Xiaoliang

    2016-01-01

    Chinese soft-shelled turtle (Pelodiscus sinensis) is commercially cultured in East and Southeast Asia for its nutritional and medicinal values. In this study, we identified interleukin-1β (IL-1β) from Chinese soft-shelled turtle. The full-length cDNA of Pelodiscus sinensis IL-1β (tIL-1β) consists of 1529 base pairs with an 831-base-pair open reading frame, encoding 277 amino acids. The guanine-cytosine (GC) content in the coding sequence and 3' untranslated region of tIL-1β is considerably higher than that of other vertebrates. Its mRNA expression level increased significantly during Aeromonas hydrophila infection. The tIL-1β lacks the typical IL-1β-converting enzyme (ICE) cut site found in mammalian IL-1β, but still could be cleaved by turtle caspase-1. By mutating the potential cleavage sites, we identified aspartic acid (Asp/D) 130 as the ICE cut site in tIL-1β. The peptide truncated at D130 was expressed using the baculovirus expression system; its bioactivity is confirmed by the ability to induce cyclooxygenase-2 (COX-2) and tIL-1β itself in peripheral blood monocytes. In conclusion, we characterized IL-1β from Chinese soft-shelled turtle and identified its D130 as a non-typical ICE cut size. PMID:27153094

  11. A phase I clinical trial utilizing autologous tumor-infiltrating lymphocytes in patients with primary hepatocellular carcinoma

    PubMed Central

    Weng, De-Sheng; Zhou, Zhong-Guo; Pan, Ke; Pan, Qiu-Zhong; Wang, Qi-Jing; Liu, Qing; He, Jia; Zhao, Jing-Jing; Li, Jiang; Chen, Min-Shan; Chang, Alfred E.; Li, Qiao; Xia, Jian-Chuan

    2015-01-01

    This report describes an ongoing Phase I clinical trial testing the safety of adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) in patients with primary hepatocellular carcinoma (HCC). Fifteen HCC patients were treated with their activated and expanded TILs following tumor resection. From a total of 17 patients with HCC, TIL were successfully expanded from 15 patients (88%), whereas two patients showed minimal or no expansion of TIL. Transient increase in the frequency of T cells was observed after adoptive transfer who was found only associated with grade I flu-like symptoms and malaise. After a median follow-up of 14 months, 15 patients (100%) were alive; and 12 patients (80%) showed no evidence of disease, 3 patients (patient 1,11,12) had tumor recurrence. The time to the diagnosis of tumor recurrence following therapy ranged from 105 to 261 days. These results indicate that immunotherapy with activated and expanded autologous TIL could be successfully performed with low toxicity, thus would serve as a novel treatment modality for patients with HCC. PMID:26515587

  12. TGF-β modulates the functionality of tumor-infiltrating CD8+ T cells through effects on TCR signaling and Spred1 expression

    PubMed Central

    di Bari, Maria Giovanna; Lutsiak, M.E. Christine; Takai, Shinji; Mostböck, Sven; Farsaci, Benedetto; Semnani, Roshanak Tolouei; Wakefield, Lalage M.; Schlom, Jeffrey; Sabzevari, Helen

    2012-01-01

    This study demonstrates that CD8+ T cells in the tumor microenvironment display reduced functionality and hyporesponsiveness. TGF-β contributed markedly to the tumor-infiltrating CD8+ T cells’ (TILs) reduced functionality, which could be reversed using a small molecule TGF-β inhibitor. Upon T-cell receptor (TCR) activation, the activation of ITK and ERK kinases were reduced in CD8+ TILs, as compared to splenic CD8+ T cells: TGF-β inhibitor could reverse this phenomenon. This study demonstrates for the first time the association of the Spred-1 gene, an inhibitor of the Ras/MAPK pathway, with CD8+ TILs and TGF-β activity. Spred-1 was upregulated in CD8+ TILs and TGF-β enhanced the expression of Spred-1 in effector/memory CD8+ T cells and not in rested/memory CD8+ T cells. Based on these findings, this study supports the hypothesis that TGF-β mediates an inhibitory mechanism on CD8+ TILs involving TCR-signaling blockade and the upregulation of Spred-1, thus implicating Spred-1 as a potential new target for future anti-tumor immune studies. PMID:19319531

  13. TGF-beta modulates the functionality of tumor-infiltrating CD8+ T cells through effects on TCR signaling and Spred1 expression.

    PubMed

    di Bari, Maria Giovanna; Lutsiak, M E Christine; Takai, Shinji; Mostböck, Sven; Farsaci, Benedetto; Semnani, Roshanak Tolouei; Wakefield, Lalage M; Schlom, Jeffrey; Sabzevari, Helen

    2009-11-01

    This study demonstrates that CD8+ T cells in the tumor microenvironment display reduced functionality and hyporesponsiveness. TGF-beta contributed markedly to the tumor-infiltrating CD8+ T cells' (TILs) reduced functionality, which could be reversed using a small molecule TGF-beta inhibitor. Upon T-cell receptor (TCR) activation, the activation of ITK and ERK kinases were reduced in CD8+ TILs, as compared to splenic CD8+ T cells: TGF-beta inhibitor could reverse this phenomenon. This study demonstrates for the first time the association of the Spred-1 gene, an inhibitor of the Ras/MAPK pathway, with CD8+ TILs and TGF-beta activity. Spred-1 was upregulated in CD8+ TILs and TGF-beta enhanced the expression of Spred-1 in effector/memory CD8+ T cells and not in rested/memory CD8+ T cells. Based on these findings, this study supports the hypothesis that TGF-beta mediates an inhibitory mechanism on CD8+ TILs involving TCR-signaling blockade and the upregulation of Spred-1, thus implicating Spred-1 as a potential new target for future anti-tumor immune studies. PMID:19319531

  14. High-throughput sequencing of T cell receptors reveals a homogeneous repertoire of tumor-infiltrating lymphocytes in ovarian cancer

    PubMed Central

    Rieder, Mark J.; Guenthoer, Jamie; Williamson, David W.; Carlson, Christopher S.; Drescher, Charles W.; Tewari, Muneesh; Bielas, Jason H.; Robins, Harlan S.

    2014-01-01

    The cellular adaptive immune system mounts a response to many solid tumors mediated by tumor infiltrating T lymphocytes (TILs). Basic measurements of these TILs, including total count, show promise as prognostic markers for a variety of cancers, including ovarian and colorectal. In addition, recent therapeutic advances are thought to exploit this immune response to effectively fight melanoma with promising studies showing efficacy in additional cancers. However, many of the basic properties of TILs are poorly understood including specificity, clonality, and spatial heterogeneity of the T cell response. We utilize deep sequencing of rearranged T-cell receptor beta (TCRB) genes to characterize the basic properties of TILs in ovarian carcinoma. Due to somatic rearrangement during T cell development, the TCR beta chain sequence serves as a molecular tag for each T cell clone. Using these sequence tags, we assess similarities and differences between infiltrating T cells in discretely sampled sections of large tumors and compare to T cells from peripheral blood. Within the limits of sensitivity of our assay, the TIL repertoires show strong similarity throughout each tumor and are distinct from the circulating T cell repertoire. We conclude that the cellular adaptive immune response within ovarian carcinomas is spatially homogeneous and distinct from the T cell compartment of peripheral blood. PMID:24027095

  15. Treating Cancer with Genetically Engineered T Cells

    PubMed Central

    Park, Tristen S.; Rosenberg, Steven A.; Morgan, Richard A.

    2011-01-01

    Administration of ex-vivo cultured, naturally occurring tumor-infiltrating lymphocytes (TILs) have been shown to mediate durable regression of melanoma tumors. However, the generation of TIL is not possible in all patients and there has been limited success in generating TIL in other cancers. Advances in genetic engineering have overcome these limitations by introducing tumor-antigen-targeting receptors into human T lymphocytes. Physicians can now genetically engineer lymphocytes to express highly active T-cell receptors (TCRs) or chimeric antigen receptors (CARs) targeting a variety of tumor antigens expressed in cancer patients. In this review we discuss the development of TCR and CAR gene transfer technology and the expansion of these therapies into different cancers with the recent demonstration of the clinical efficacy of these treatments. PMID:21663987

  16. In vitro augmentation of the cytotoxic activity of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes by famotidine in cancer patients.

    PubMed

    Tsunoda, T; Tanimura, H; Yamaue, H; Iwahashi, M; Tani, M; Tamai, M; Arii, K; Noguchi, K

    1992-01-01

    We investigated the in vitro effects of famotidine on the cytotoxic activity of peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TILs). The cytotoxic activity of PBMC was augmented by famotidine at a concentration of 10 ng/ml, which is equivalent to the serum level achieved by the intravenous administration of a dose of 20 mg. This response to famotidine was seen only in cancer patients. Both the cytotoxic activity and DNA synthesis of activated TILs were increased by the combination of interleukin-2 and 1 microgram/ml of famotidine. Augmentation of cytotoxic activity by famotidine occurred independently of any decrease in the population of suppressor T-cells. Thus, famotidine may have the potential to be used in adoptive immunotherapy with TILs for cancer patients. PMID:1582736

  17. Transfection of Tumor-Infiltrating T Cells with mRNA Encoding CXCR2.

    PubMed

    Idorn, Manja; Thor Straten, Per; Svane, Inge Marie; Met, Özcan

    2016-01-01

    Adoptive T-cell therapy based on the infusion of patient's own immune cells after ex vivo culturing is among the most potent forms of personalized treatment among recent clinical developments for the treatment of cancer. However, despite high rates of successful initial clinical responses, only about 20 % of patients with metastatic melanoma treated with tumor-infiltrating lymphocytes (TILs) enter complete and long-term regression, with the majority either relapsing after initial partial regression or not benefiting at all. Previous studies have shown a positive correlation between the number infused T cells migrating to the tumor and the clinical response, but also that only a small fraction of adoptively transferred T cells reach the tumor site. In this chapter, we describe a protocol for transfection of TILs with mRNA encoding the chemokine receptor CXCR2 transiently redirecting and improving TILs migration toward tumor-secreted chemokines in vitro. PMID:27236805

  18. Analysis of nuclear uracil-DNA glycosylase (nUDG) turnover during the cell cycle.

    PubMed

    Fischer, Jennifer A; Caradonna, Salvatore

    2011-01-01

    Uracil-DNA glycosylases (UDG/UNG) are enzymes that remove uracil from DNA and initiate base-excision repair. These enzymes play a key role in maintaining genomic integrity by reducing the mutagenic events caused by G:C to A:T transition mutations. The recent finding that a family of RNA editing enzymes (AID/APOBECs) can deaminate cytosine in DNA has raised the interest in these base-excision repair enzymes. The methodology presented here focuses on determining the regulation of the nuclear isoform of uracil-DNA glycosylase (nUDG), a 36,000 Da protein. In synchronized HeLa cells, nUDG protein levels decrease to barely detectable levels during the S phase of the cell cycle. Immunoblot analysis of immunoprecipitated or affinity-isolated nUDG reveals ubiquitin-conjugated nUDG when proteolysis is inhibited by agents that block proteasomal-dependent protein degradation. PMID:21755446

  19. Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions

    PubMed Central

    Maul, Robert W; Saribasak, Huseyin; Martomo, Stella A; McClure, Rhonda L; Yang, William; Vaisman, Alexandra; Gramlich, Hillary S; Schatz, David G; Woodgate, Roger; Wilson, David M; Gearhart, Patricia J

    2013-01-01

    Activation-induced deaminase (AID) initiates diversity of immunoglobulin genes through deamination of cytosine to uracil. Two opposing models have been proposed for the deamination of DNA or RNA by AID. Although most data support DNA deamination, there is no physical evidence of uracil residues in immunoglobulin genes. Here we demonstrate their presence by determining the sensitivity of DNA to digestion with uracil DNA glycosylase (UNG) and abasic endonuclease. Using several methods of detection, we identified uracil residues in the variable and switch regions. Uracil residues were generated within 24 h of B cell stimulation, were present on both DNA strands and were found to replace mainly cytosine bases. Our data provide direct evidence for the model that AID functions by deaminating cytosine residues in DNA. PMID:21151102

  20. Campus Single Sign-On und hochschulübergreifendes Identity Management

    NASA Astrophysics Data System (ADS)

    Hommel, Wolfgang

    Das im Rahmen von IntegraTUM für die TUM geschaffene Identity & Access Management System setzt das Paradigma unified login um, d. h. ein Benutzer kann alle für ihn relevanten Dienste innerhalb der Hochschule mit derselben Loginname-/Passwortkombination nutzen. Dieser Artikel zeigt, wie auf Basis der Software Shibboleth und der deutschlandweiten Hochschulföderation DFN-AAI als weitere Mehrwerte das campusweite web single sign-on und die nahtlose Nutzung zahlreicher externer Web-Anwendungen erreicht werden. Als Beispiel für die Abläufe bei der Erschließung neuer Dienste für die hochschulübergreifende Nutzung wird die Anbindung von Learning Management Systemen auf Basis des DFN-AAI E-Learning-Profils diskutiert. Den umfassenden Vorteilen werden schließlich die aktuellen technischen Grenzen bei der Umsetzung des hochschulübergreifenden Identity Management gegenübergestellt.

  1. Identification and characterization of a tumor infiltrating CD56(+)/CD16 (-) NK cell subset with specificity for pancreatic and prostate cancer cell lines.

    PubMed

    Frankel, Timothy L; Burns, William; Riley, John; Morgan, Richard A; Davis, Jeremy L; Hanada, Kenichi; Quezado, Martha; Rosenberg, Steven A; Royal, Richard E

    2010-12-01

    In a recent clinical trial, a patient exhibited regression of several pancreatic cancer metastases following the administration of the immune modulator Ipilimumab (anti-CTLA-4 antibody). We sought to characterize the immune cells responsible for this regression. Tumor infiltrating lymphocytes (TIL-2742) and an autologous tumor line (TC-2742) were expanded from a regressing metastatic lesion excised from this patient. Natural killer (NK) cells predominated in the TIL (92% CD56(+)) with few T cells (12% CD3(+)). A majority (88%) of the NK cells were CD56(bright)CD16(-). TIL-2742 secreted IFN-γ and GM-CSF following co-culture with TC-2742 and major histocompatibility complex mismatched pancreatic tumor lines. After sorting TIL-2742, the purified CD56(+)CD16(-)CD3(-) subset showed reactivity similar to TIL-2742 while the CD56(-)CD16(-)CD3(+) cells exhibited no tumor recognition. In co-culture assays, TIL-2742 and the NK subset expressed high reactivity to several pancreatic and prostate cancer cell lines and could lyse the autologous tumor as well as pancreas and prostate cancer lines. Reactivity was partially abrogated by blockade of TRAIL. We thus identified a unique subset of NK cells (CD56(bright)CD16(dim)) isolated from a regressing metastatic pancreatic cancer in a patient responding to Ipilimumab. This represents the first report of CD56(+)CD16(-) NK cells with apparent specificity for pancreatic and prostate cancer cell lines and associated with tumor regression following the treatment with an immune modulating agent. PMID:20734041

  2. Immunomodulation by MYB is associated with tumor relapse in patients with early stage colorectal cancer.

    PubMed

    Millen, Rosemary; Malaterre, Jordane; Cross, Ryan S; Carpinteri, Sandra; Desai, Jayesh; Tran, Ben; Darcy, Phillip; Gibbs, Peter; Sieber, Oliver; Zeps, Nikolajs; Waring, Paul; Fox, Stephen; Pereira, Lloyd; Ramsay, Robert G

    2016-07-01

    The presence of tumor immune infiltrating cells (TILs), particularly CD8(+) T-cells, is a robust predictor of outcome in patients with colorectal cancer (CRC). We revisited TIL abundance specifically in patients with microsatellite stable (MSS) CRC without evidence of lymph node or metastatic spread. Examination of the density of CD8(+) T-cells in primary tumors in the context of other pro-oncogenic markers was performed to investigate potential regulators of TILs. Two independent cohorts of patients with MSS T2-4N0M0 CRC, enriched for cases with atypical relapse, were investigated. We quantified CD8(+) and CD45RO(+) -TILs, inflammatory markers, NFkBp65, pStat3, Cyclo-oxygenase-2 (COX2) and GRP78 as well as transcription factors (TF), β-catenin and MYB. High CD8(+) TILs correlated with a better relapse-free survival in both cohorts (p = 0.002) with MYB and its target gene, GRP78 being higher in the relapse group (p = 0.001); no difference in pSTAT3 and p65 was observed. A mouse CRC (CT26) model was employed to evaluate the effect of MYB on GRP78 expression as well as T-cell infiltration. MYB over-expressing in CT26 cells increased GRP78 expression and the analysis of tumor-draining lymph nodes adjacent to tumors showed reduced T-cell activation. Furthermore, MYB over-expression reduced the efficacy of anti-PD-1 to modulate CT26 tumor growth. This high MYB and GRP78 show a reciprocal relationship with CD8(+) TILs which may be useful refining the prediction of patient outcome. These data reveal a new immunomodulatory function for MYB suggesting a basis for further development of anti-GRP78 and/or anti-MYB therapies. PMID:27622014

  3. Tumor-infiltrating lymphocytes predict cutaneous melanoma survival.

    PubMed

    Fortes, Cristina; Mastroeni, Simona; Mannooranparampil, Thomas J; Passarelli, Francesca; Zappalà, Alba; Annessi, Giorgio; Marino, Claudia; Caggiati, Alessio; Russo, Nicoletta; Michelozzi, Paola

    2015-08-01

    Understanding differences in survival across distinct subgroups of melanoma patients may help with the choice of types of therapy. Tumor-infiltrating lymphocytes (TILs) are considered a manifestation of the host immune response to tumor, but the role of TILs in melanoma mortality is controversial. The aim of this study was to investigate independent prognostic factors for melanoma mortality. We carried out a 10-year cohort study on 4133 melanoma patients from the same geographic area (Lazio) with primary cutaneous melanoma diagnosed between January 1998 and December 2008. The probability of survival was estimated using Kaplan-Meier methods and prognostic factors were evaluated by multivariate analysis (Cox proportional hazards model). The 10-year survival rate for melanoma decreased with increasing Breslow thickness (Pfor trend<0.0001) and with age (Pfor trend<0.0001) whereas survival increased with increasing levels of TILs (Pfor trend=0.0001). The 10-year survival rate for melanoma divided into TILs intensity as scanty, moderate, and marked was 88.0, 92.2, and 97.0%, respectively. In the multivariate Cox model, the presence of high levels of TILs in primary invasive melanomas was associated with a lower risk of melanoma death (hazard ratio 0.32; 95% confidence interval 0.13-0.82) after controlling for sex, age, Breslow thickness, histological type, mitotic rate, and ulceration. After including lymph node status in the multivariate analysis, the protective effect of marked TILs on melanoma mortality remained (hazard ratio 0.37; 95% confidence interval 0.15-0.94). The results of this study suggest that the immune microenvironment affects melanoma survival. PMID:25933208

  4. The Prognostic Value of Tumor-Infiltrating Lymphocytes in Breast Cancer: A Systematic Review and Meta-Analysis

    PubMed Central

    Mao, Yan; Qu, Qing; Chen, Xiaosong; Huang, Ou; Wu, Jiayi; Shen, Kunwei

    2016-01-01

    Background The prognostic values of tumor-infiltrating lymphocytes (TILs) and TILs subsets in breast cancer (BC) are uncertain. Methods A systematic literature search (MEDLINE, Web of Science, EMBASE, and the Cochrane Library to August 2014) was conducted for studies which met the eligibility criteria. The primary clinical outcome was defined as disease-free survival (DFS), overall survival (OS), and BC-specific survival (BCSS). Random or fixed-effects model was adopted to estimate the summary hazard ratio (HR). Results Twenty-five published studies comprising 22,964 patients were reviewed. Pooled analysis indicated that TILs were not prognostic markers for DFS and OS in overall population, but related to improved DFS (HR, 0.82; 95% CI, 0.76–0.88) and OS (HR, 0.79; 95% CI, 0.71–0.87) in triple negative breast cancer (TNBC) patients. For TILs subsets, CD8+ lymphocytes were associated with improved DFS (HR, 0.69; 95% CI, 0.56–0.84) and BCSS (HR, 0.78; 95% CI, 0.71–0.86) in overall population, while FOXP3+ lymphocytes were associated with reduced DFS (HR, 1.47; 95% CI, 1.01–2.05) and OS (HR, 1.50; 95% CI, 1.15–1.97). In estrogen receptor (ER) negative patients, CD8+ lymphocytes was also related to better BCSS. In addition, the high density of CD20+, CD3+ or low level of PD-1+ or γδ T lymphocytes indicated increased OS in limited studies. Conclusion TILs and TILs subsets are promising prognostic biomarkers in breast cancer, especially in TNBC. PMID:27073890

  5. The ratio of CD8 to Treg tumor-infiltrating lymphocytes is associated with response to cisplatin-based neoadjuvant chemotherapy in patients with muscle invasive urothelial carcinoma of the bladder

    PubMed Central

    Baras, Alexander S.; Drake, Charles; Liu, Jen-Jane; Gandhi, Nilay; Kates, Max; Hoque, Mohamed O.; Meeker, Alan; Hahn, Noah; Taube, Janis M.; Schoenberg, Mark P.; Netto, George; Bivalacqua, Trinity J.

    2016-01-01

    ABSTRACT Introduction: Randomized controlled trials of platinum-based neoadjuvant chemotherapy (NAC) for bladder cancer have shown that patients who achieve a pathologic response to NAC exhibit 5 y survival rates of approximately 80–90% while NAC resistant (NR) cases exhibit 5 y survival rates of approximately 30–40%. These findings highlight the need to predict who will benefit from conventional NAC and the need for plausible alternatives. Methods: The pre-treatment biopsy tissues from a cohort of 41 patients with muscle invasive bladder who were treated with NAC were incorporated in tissue microarray and immunohistochemistry for PD-L1, CD8, and FOXP3 was performed. Percentage of PD-L1 positive tumor cells was measured. Tumor-infiltrating lymphocytes (TIL) densities, along with CD8 and Treg-specific TILs, were measured. Results: TIL density was strongly correlated with tumor PD-L1 expression, consistent with the mechanism of adaptive immune resistance in bladder cancer. Tumor cell PD-L1 expression was not a significant predictor of response. Neither was the CD8 nor Treg TIL density associated with response. Intriguingly though, the ratio of CD8 to Treg TIL densities was strongly associated with response (p = 0.0003), supporting the hypothesis that the immune system plays a role in the response of bladder cancer to chemotherapy. Discussion: To our knowledge, this is the first report in bladder cancer showing that the CD8 to Treg TIL density in the pre-treatment tissues is predictive for conventional NAC response. These findings warrant further investigations to both better characterize this association in larger cohorts and begin to elucidate the underlying mechanism(s) of this phenomenon.

  6. Immunomodulation by MYB is associated with tumor relapse in patients with early stage colorectal cancer

    PubMed Central

    Millen, Rosemary; Malaterre, Jordane; Cross, Ryan S.; Carpinteri, Sandra; Desai, Jayesh; Tran, Ben; Darcy, Phillip; Gibbs, Peter; Sieber, Oliver; Zeps, Nikolajs; Waring, Paul; Fox, Stephen; Pereira, Lloyd; Ramsay, Robert G.

    2016-01-01

    ABSTRACT The presence of tumor immune infiltrating cells (TILs), particularly CD8+ T-cells, is a robust predictor of outcome in patients with colorectal cancer (CRC). We revisited TIL abundance specifically in patients with microsatellite stable (MSS) CRC without evidence of lymph node or metastatic spread. Examination of the density of CD8+ T-cells in primary tumors in the context of other pro-oncogenic markers was performed to investigate potential regulators of TILs. Two independent cohorts of patients with MSS T2-4N0M0 CRC, enriched for cases with atypical relapse, were investigated. We quantified CD8+ and CD45RO+ -TILs, inflammatory markers, NFkBp65, pStat3, Cyclo-oxygenase-2 (COX2) and GRP78 as well as transcription factors (TF), β-catenin and MYB. High CD8+ TILs correlated with a better relapse-free survival in both cohorts (p = 0.002) with MYB and its target gene, GRP78 being higher in the relapse group (p = 0.001); no difference in pSTAT3 and p65 was observed. A mouse CRC (CT26) model was employed to evaluate the effect of MYB on GRP78 expression as well as T-cell infiltration. MYB over-expressing in CT26 cells increased GRP78 expression and the analysis of tumor-draining lymph nodes adjacent to tumors showed reduced T-cell activation. Furthermore, MYB over-expression reduced the efficacy of anti-PD-1 to modulate CT26 tumor growth. This high MYB and GRP78 show a reciprocal relationship with CD8+ TILs which may be useful refining the prediction of patient outcome. These data reveal a new immunomodulatory function for MYB suggesting a basis for further development of anti-GRP78 and/or anti-MYB therapies. PMID:27622014

  7. How does breathing frequency affect the performance of an N95 filtering facepiece respirator and a surgical mask against surrogates of viral particles?

    PubMed

    He, Xinjian; Reponen, Tiina; McKay, Roy; Grinshpun, Sergey A

    2014-01-01

    Breathing frequency (breaths/min) differs among individuals and levels of physical activity. Particles enter respirators through two principle penetration pathways: faceseal leakage and filter penetration. However, it is unknown how breathing frequency affects the overall performance of N95 filtering facepiece respirators (FFRs) and surgical masks (SMs) against viral particles, as well as other health-relevant submicrometer particles. A FFR and SM were tested on a breathing manikin at four mean inspiratory flows (MIFs) (15, 30, 55, and 85 L/min) and five breathing frequencies (10, 15, 20, 25, and 30 breaths/min). Filter penetration (Pfilter) and total inward leakage (TIL) were determined for the tested respiratory protection devices against sodium chloride (NaCl) aerosol particles in the size range of 20 to 500 nm. "Faceseal leakage-to-filter" (FLTF) penetration ratios were calculated. Both MIF and breathing frequency showed significant effects (p < 0.05) on Pfilter and TIL. Increasing breathing frequency increased TIL for the N95 FFR whereas no clear trends were observed for the SM. Increasing MIF increased Pfilter and decreased TIL resulting in decreasing FLTF ratio. Most of FLTF ratios were >1, suggesting that the faceseal leakage was the primary particle penetration pathway at various breathing frequencies. Breathing frequency is another factor (besides MIF) that can significantly affect the performance of N95 FFRs, with higher breathing frequencies increasing TIL. No consistent trend of increase or decrease of TIL with either MIF or breathing frequency was observed for the tested SM. To potentially extend these findings beyond the manikin/breathing system used, future studies are needed to fully understand the mechanism causing the breathing frequency effect on the performance of respiratory protection devices on human subjects. PMID:24521067

  8. A quantitative assessment of the total inward leakage of NaCl aerosol representing submicron-size bioaerosol through N95 filtering facepiece respirators and surgical masks.

    PubMed

    Rengasamy, Samy; Eimer, Benjamin C; Szalajda, Jonathan

    2014-01-01

    Respiratory protection provided by a particulate respirator is a function of particle penetration through filter media and through faceseal leakage. Faceseal leakage largely contributes to the penetration of particles through a respirator and compromises protection. When faceseal leaks arise, filter penetration is assumed to be negligible. The contribution of filter penetration and faceseal leakage to total inward leakage (TIL) of submicron-size bioaerosols is not well studied. To address this issue, TIL values for two N95 filtering facepiece respirator (FFR) models and two surgical mask (SM) models sealed to a manikin were measured at 8 L and 40 L breathing minute volumes with different artificial leak sizes. TIL values for different size (20-800 nm, electrical mobility diameter) NaCl particles representing submicron-size bioaerosols were measured using a scanning mobility particle sizer. Efficiency of filtering devices was assessed by measuring the penetration against NaCl aerosol similar to the method used for NIOSH particulate filter certification. Results showed that the most penetrating particle size (MPPS) was ∼45 nm for both N95 FFR models and one of the two SM models, and ∼350 nm for the other SM model at sealed condition with no leaks as well as with different leak sizes. TIL values increased with increasing leak sizes and breathing minute volumes. Relatively, higher efficiency N95 and SM models showed lower TIL values. Filter efficiency of FFRs and SMs influenced the TIL at different flow rates and leak sizes. Overall, the data indicate that good fitting higher-efficiency FFRs may offer higher protection against submicron-size bioaerosols. PMID:24275016

  9. PD-L1 (B7-H1) expression and the immune tumor microenvironment in primary and metastatic breast carcinomas.

    PubMed

    Cimino-Mathews, Ashley; Thompson, Elizabeth; Taube, Janis M; Ye, Xiaobu; Lu, Yao; Meeker, Alan; Xu, Haiying; Sharma, Rajni; Lecksell, Kristen; Cornish, Toby C; Cuka, Nathan; Argani, Pedram; Emens, Leisha A

    2016-01-01

    Programmed death ligand 1 (PD-L1) expression by tumor-infiltrating lymphocytes (TILs) and tumor cells in breast cancer has been reported, but the relationships between PD-L1 expression by TIL, carcinoma cells, and other immunologic features of the breast tumor microenvironment remain unclear. We therefore evaluated the interrelationships between tumor cell surface and TIL PD-L1 expression, lymphocyte subpopulations, and patterns of immune cell infiltration in cohorts of treatment-naive, primary breast cancers (PBCs) (n = 45) and matched PBC and metastatic breast cancers (MBC) (n = 26). Seventy-eight percent of untreated PBCs contained PD-L1(+) TILs, but only 21% had PD-L1(+) carcinoma cells. Carcinoma PD-L1 expression localized to the tumor invasive front and was associated with high tumor grade (P = .04). Eighty-nine percent of PD-L1(+) carcinomas contained brisk TIL infiltrates, compared to only 24% of PD-L1(-) carcinomas; this included CD3(+) (P = .02), CD4(+) (P = .04), CD8(+) (P = .002), and FoxP3(+) T cells (P = .02). PD-L1(+) PBCs were more likely to contain PD-L1(+) TIL than PD-L1(-) PBCs (P = .04). Peripheral lymphoid aggregates were present in 100% of PD-L1(+) compared to 41% of PD-L1(-) PBC (P < .001). No patient with PD-L1(+) PBC developed distant recurrence, compared to 15% of patients with PD-L1(-) PBC. For the matched PBC and MBC cohort, 2 patients (8%) had PD-L1(+) tumors, with 1 case concordant and 1 case discordant for carcinoma PD-L1 expression in the PBC and MBC. Our data support PD-L1 expression by tumor cells as a biomarker of active breast tumor immunity and programmed death 1 blockade as a therapeutic strategy for breast cancer. PMID:26527522

  10. HLA association with response and toxicity in melanoma patients treated with interleukin 2-based immunotherapy.

    PubMed

    Marincola, F M; Venzon, D; White, D; Rubin, J T; Lotze, M T; Simonis, T B; Balkissoon, J; Rosenberg, S A; Parkinson, D R

    1992-12-01

    Peripheral blood lymphocytes from 146 patients with metastatic melanoma undergoing interleukin 2 (IL-2)-based immunotherapy were characterized for HLA A, B, Cw, DR, DQw, and DRw specificities. Patients had been enrolled into sequential treatment protocols with either IL-2 alone (28) or in combination with tumor-infiltrating lymphocytes (TILs) (86), alpha-interferon (26), lymphokine-activated killer cells (16), radiation therapy (7), cyclophosphamide (3), tumor necrosis factor (1), and interleukin 4 (1) for a total of 168 courses of therapy. HLA phenotype was then correlated with response rate and toxicity to IL-2. We noted: (a) a significant difference in the frequency of A11 (20.5% versus 10.2%; P < 0.05) allele between melanoma patients and the North American Caucasian population; (b) a significantly higher frequency of A11 phenotype among responders (40.5%) than in the melanoma patient population (20.5%; P < 0.01), which was even more obvious among patients responding to TIL therapy (47.4% versus 22.1%; P < 0.05); within TIL patients, responders also had an increased frequency of A19 (42.1% versus 25.6%; P < 0.05); (c) a correlation between the number of TILs received and response rate (P < 0.005); and (d) an association between DR4 haplotype and decreased tolerance to IL-2 among the patients receiving TILs (P = 0.01). These results suggest that, in melanoma patients, some HLA Class I specificities may predict for a greater likelihood of response to IL-2-based therapy, while HLA Class II phenotype correlates with tolerance to the combination of TIL and IL-2 therapy. PMID:1423301

  11. Differential expression of APE1 and APE2 in germinal centers promotes error-prone repair and A:T mutations during somatic hypermutation

    PubMed Central

    Stavnezer, Janet; Linehan, Erin K.; Thompson, Mikayla R.; Habboub, Ghaith; Ucher, Anna J.; Kadungure, Tatenda; Tsuchimoto, Daisuke; Nakabeppu, Yusaku; Schrader, Carol E.

    2014-01-01

    Somatic hypermutation (SHM) of antibody variable region genes is initiated in germinal center B cells during an immune response by activation-induced cytidine deaminase (AID), which converts cytosines to uracils. During accurate repair in nonmutating cells, uracil is excised by uracil DNA glycosylase (UNG), leaving abasic sites that are incised by AP endonuclease (APE) to create single-strand breaks, and the correct nucleotide is reinserted by DNA polymerase β. During SHM, for unknown reasons, repair is error prone. There are two APE homologs in mammals and, surprisingly, APE1, in contrast to its high expression in both resting and in vitro-activated splenic B cells, is expressed at very low levels in mouse germinal center B cells where SHM occurs, and APE1 haploinsufficiency has very little effect on SHM. In contrast, the less efficient homolog, APE2, is highly expressed and contributes not only to the frequency of mutations, but also to the generation of mutations at A:T base pair (bp), insertions, and deletions. In the absence of both UNG and APE2, mutations at A:T bp are dramatically reduced. Single-strand breaks generated by APE2 could provide entry points for exonuclease recruited by the mismatch repair proteins Msh2–Msh6, and the known association of APE2 with proliferating cell nuclear antigen could recruit translesion polymerases to create mutations at AID-induced lesions and also at A:T bp. Our data provide new insight into error-prone repair of AID-induced lesions, which we propose is facilitated by down-regulation of APE1 and up-regulation of APE2 expression in germinal center B cells. PMID:24927551

  12. DNA polymerases β and λ do not directly affect Ig variable region somatic hypermutation although their absence reduces the frequency of mutations

    PubMed Central

    Schrader, Carol E.; Linehan, Erin K.; Ucher, Anna J.; Bertocci, Barbara; Stavnezer, Janet

    2014-01-01

    During somatic hypermutation (SHM) of antibody variable (V) region genes, activation-induced cytidine deaminase (AID) converts dC to dU, and dUs can either be excised by uracil DNA glycosylase (UNG), by mismatch repair, or replicated over. If UNG excises the dU, the abasic site could be cleaved by AP-endonuclease (APE), introducing the single-strand DNA breaks (SSBs) required for generating mutations at A:T bp, which are known to depend upon mismatch repair and DNA Pol η. DNA Pol β or λ could instead repair the lesion correctly. To assess the involvement of Pols β and λ in SHM of antibody genes, we analyzed mutations in the VDJh4 3′ flanking region in Peyer’s patch germinal center (GC) B cells from polβ−/−polλ−/−, polλ−/−, and polβ−/− mice. We find that deficiency of either or both polymerases results in a modest but significant decrease in V region SHM, with Pol β having a greater effect, but there is no effect on mutation specificity, suggesting they have no direct role in SHM. Instead, the effect on SHM appears to be due to a role for these enzymes in GC B cell proliferation or viability. The results suggest that the BER pathway is not important during V region SHM for generating mutations at A:T bp. Furthermore, this implies that most of the SSBs required for Pol η to enter and create A:T mutations are likely generated during replication instead. These results contrast with the inhibitory effect of Pol β on mutations at the Ig Sμ locus, Sμ DSBs and class switch recombination (CSR) reported previously. We show here that B cells deficient in Pol λ or both Pol β and λ proliferate normally in culture and undergo slightly elevated CSR, as shown previously for Pol β-deficient B cells. PMID:24084171

  13. Glyphosate Resistant Palmer Amaranth - A Threat To Conservation Agriculture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glyphosate resistant Palmer amaranth is now present in throughout the Southeast. Hundreds of thousands of conservation tillage cotton acres, some currently under USDA Natural Resources Conservation Service (NRCS) conservation program contracts, are at risk of being converted to higher-intensity til...

  14. The Desire to Learn as a Kind of Love: Gardening, Cooking, and Passion in Outdoor Education

    ERIC Educational Resources Information Center

    Wistoft, Karen

    2013-01-01

    "Gardens for Bellies" ["Haver til Maver"] is an organic school gardens project at Krogerup farm in Northern Sealand, Denmark, which provides children with first-hand experiences in a natural, outdoor environment. The general intention of the project is to expand children's competences and their knowledge of nature, farming and food preparation.…

  15. Tumour infiltrating lymphocytes in squamous cell carcinoma of the oro- and hypopharynx: prognostic impact may depend on type of treatment and stage of disease.

    PubMed

    Distel, Luitpold V; Fickenscher, Rainer; Dietel, Katrin; Hung, Alexander; Iro, Heiner; Zenk, Johannes; Nkenke, Emeka; Büttner, Maike; Niedobitek, Gerald; Grabenbauer, Gerhard G

    2009-10-01

    he purpose of this study was to evaluate the prognostic influence of various subtypes of tumour infiltrating lymphocytes (TIL) in head and neck cancer, in particular the potential influence of regulatory T cells (Treg) in relation to different treatment modalities was addressed. A total of 115 patients with squamous cell carcinoma of the oro- and hypopharynx were selected. A low-risk group of 62 patients with early disease was treated by primary surgery followed by external radiotherapy. A high-risk group of 53 inoperable patients with advanced disease was treated by primary radiochemotherapy. Two-hundred and forty biopsy samples were evaluated by use of the tissue-micro-array technique employing the following markers: CD3, CD4, CD8, CD20, CD68, FOXP3, Granzyme B. In the low-risk group high CD20+ infiltration was associated with a significantly better NED-survival rate (p=0.02). Contrary, among high-risk patients low CD20+ counts indicated significantly better survival (p=0.03). Additionally, in the low-risk group higher numbers of intraepithelial CD8+ TIL (>66.6 per thousand) led to improved NED-survival of 95% vs. 52% (p=0.005). The impact of TIL on prognosis in patients with head and neck cancer may be affected by type of treatment and stage of disease. This finding will influence future studies on the role of TIL in human cancers. PMID:19576838

  16. Phenotypic gain from introgression of two QTLs, qSB9-2 and qSB12-1, for rice sheath blight resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    F2:3 families from crosses between three indica introgression lines and their common japonica recurrent parent were used to evaluate two quantitative trait loci (QTLs) for sheath blight (SB) resistance. Three selected ‘TeQing’-into-‘Lemont’ backcross introgression lines (TILs) were more resistant th...

  17. Differential annotation of tRNA genes with anticodon CAT in bacterial genomes

    PubMed Central

    Silva, Francisco J.; Belda, Eugeni; Talens, Santiago E.

    2006-01-01

    We have developed three strategies to discriminate among the three types of tRNA genes with anticodon CAT (tRNAIle, elongator tRNAMet and initiator tRNAfMet) in bacterial genomes. With these strategies, we have classified the tRNA genes from 234 bacterial and several organellar genomes. These sequences, in an aligned or unaligned format, may be used for the identification and annotation of tRNA (CAT) genes in other genomes. The first strategy is based on the position of the problem sequences in a phenogram (a tree-like network), the second on the minimum average number of differences against the tRNA sequences of the three types and the third on the search for the highest score value against the profiles of the three types of tRNA genes. The species with the maximum number of tRNAfMet and tRNAMet was Photobacterium profundum, whereas the genome of one Escherichia coli strain presented the maximum number of tRNAIle (CAT) genes. This last tRNA gene and tilS, encoding an RNA-modifying enzyme, are not essential in bacteria. The acquisition of a tRNAIle (TAT) gene by Mycoplasma mobile has led to the loss of both the tRNAIle (CAT) and the tilS genes. The new tRNA has appropriated the function of decoding AUA codons. PMID:17071718

  18. Short-term stover, tillage, and nitrogen management affect near-surface soil organic matter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Corn (Zea mays L.) stover removal for biofuel or forage has implications on soil organic C (SOC). The objective of this study was to evaluate short-term (3-yr) stover management (retained or removed [79 removed, across treatments and years]), tillage system (chisel tillage, strip-tillage, and no-til...

  19. MELOE-1 is a new antigen overexpressed in melanomas and involved in adoptive T cell transfer efficiency

    PubMed Central

    Godet, Yann; Moreau-Aubry, Agnès; Guilloux, Yannik; Vignard, Virginie; Khammari, Amir; Dreno, Brigitte; Jotereau, Francine; Labarriere, Nathalie

    2008-01-01

    A cytotoxic T lymphocyte (CTL) clone was derived from a tumor-infiltrating lymphocyte (TIL) population infused to a melanoma patient who remained relapse free for 10 yr after this adoptive transfer. This clone recognized all melanoma cell lines tested and, to a lower extent, melanocytes, in the context of human histocompatibility leukocyte antigen A2 (HLA-A2), but it did not recognize other tumor cell types. The gene coding for the antigen recognized by this clone was identified by the screening of a melanoma complementary DNA expression library. This antigen is overexpressed in melanomas, compared with other cancer cell lines and healthy tissues, and was thus called melanoma-overexpressed antigen (meloe). Remarkably, the structure of meloe was unusual, with multiple short open reading frames (ORFs). The peptide recognized by the CTL clone was encoded by one of these ORFs, called MELOE-1. Using a specific HLA-A2/peptide tetramer, we showed a correlation between the infusion of TILs containing MELOE-1–specific T cells and relapse prevention in HLA-A2 patients. Indeed, 5 out of 9 patients who did not relapse were infused with TILs that contained MELOE-1–specific T cells, whereas 0 out of the 21 patients who relapsed was infused with such TIL-containing lymphocytes. Overall, our results suggest that this new antigen is involved in immunosurveillance and, thus, represents an attractive target for immunotherapy protocols of melanoma. PMID:18936238

  20. 78 FR 35290 - Government-Owned Inventions; Availability for Licensing

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-12

    ... attacking TIL from a patient's tumor based on these markers prior to re-infusion, in vitro culture time is... specific cell surface markers will reduce the culture time for these T cells before re-infusion into the... for autologous tumor recognition before re- infusion into the patient Development Stage:...

  1. Programming for Adolescents with Behavioral Disorders.

    ERIC Educational Resources Information Center

    Braaten, Sheldon, Ed.; And Others

    This book presents 17 papers from a 1982 national multidisciplinary conference on services for behaviorally disordered adolescents. The following papers are included: "Programming for Youth in Secondary Schools and the Community," (W. Van Til); "Who's Crazy? II" (C. Michael Nelson); "Correlates of Successful Adaptive Behavior: Comparative Studies…

  2. Continuous and residual effects of broiler litter application to cotton on soil properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Application of broiler litter to soils provides nutrients for plant growth and improves soil quality characteristics. However, little is known about these effects after broiler litter application is terminated. Field studies were conducted at Cruger and Coffeeville in Mississippi representing no-til...

  3. Remote sensing of soil tilage intensity in central Iowa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Crop residues on the soil surface decrease soil erosion, increase soil organic matter, improve soil quality, and reduce the amount of nutrients and pesticides that reaches stream and rivers. Crop residue cover is often used to classify soil tillage intensity and assess the extent of conservation til...

  4. Genomic expression analysis by single-cell mRNA differential display of quiescent CD8 T cells from tumor-infiltrating lymphocytes obtained from in vivo liver tumours

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We performed a genomic approach combining single-cell mRNA differential display and RNA subtractive hybridization to elucidate the CD8 T-cell ignorance. By comparing the quiescent CD8 T-cell obtained from liver tumor TIL with a control at the single cell level, we identified candidate genes differen...

  5. Use of the tritium trick for analysis of the influence that hydrogen and helium exert on the mechanical properties of radiation-resistant precipitation-hardening FCC steels

    SciTech Connect

    Goshchitskii, B.N.; Sagaradze, V.V.; Arbuzov, V.L.; Zuev, Y.N.; Markelov, N.N.; Zimin, A.V.

    1995-10-01

    The aging austenitic stainless steel Crl6Nil5Mo3Til is shown to possess a high resistance to the radiation void formation under irradiation with fast neutrons (60 dpa, 753K). The influence of tritium introduced from the gaseous phase and radiogenic helium on the mechanical properties is analysed. 3 refs., 3 tabs.

  6. Hydrogeological analysis of the upper Dupi Tila Aquifer, towards the implementation of a managed aquifer-recharge project in Dhaka City, Bangladesh

    NASA Astrophysics Data System (ADS)

    Rahman, Mohammad Azizur; Wiegand, Bettina A.; Badruzzaman, A. B. M.; Ptak, Thomas

    2013-08-01

    A preliminary feasibility assessment of managed aquifer-recharge (MAR) techniques was undertaken for Dhaka City, Bangladesh. Considering the top impermeable-layer (TIL) thickness and the land-use classification, four primary MAR techniques have been suggested: (1) soil-aquifer treatment (SAT) for TIL thickness 0-8 m, (2) cascade-type recharge trenches/pits for TIL thickness 9-30 m, (3) aquifer storage, transfer and recovery (ASR/ASTR) for TIL thickness 31-52 m, and (4) use of natural wetlands to recharge water collected from open spaces. The study suggests that recharge trenches and pits will be the most appropriate MAR techniques, which can be implemented in most parts of the recharge area (ca. 277 km2). In case of a recharge trench, the lower parts (15-20 m) that are in direct contact with the aquifer can be backfilled with biosand filters with a reactive layer containing metallic iron (Fe0) to offer pre-treatment of the infiltrated water. In addition to the suggested four techniques, the regional groundwater flow direction, from the northwest and northeast towards Dhaka City, may allow use of the aquifer as a natural treatment and transport medium for groundwater, if spreading basins are installed in the greater Dhaka area.

  7. Total copper, manganese, and zinc levels in a Cecil soil during ten years of poultry litter application

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Heavy metals in poultry litter (PL) can cause environmental problems despite the cost-effectiveness of PL as source of plant nutrients. We compared total Cu, Mn, and Zn levels in a Cecil soil near Watkinsville, GA, in a 5-yr of cotton and 5-yr of corn study under conventional tillage (CT) and no-til...

  8. Starved and Asphyxiated: How Can CD8+ T Cells within a Tumor Microenvironment Prevent Tumor Progression

    PubMed Central

    Zhang, Ying; Ertl, Hildegund C. J.

    2016-01-01

    Although cancer immunotherapy has achieved significant breakthroughs in recent years, its overall efficacy remains limited in the majority of patients. One major barrier is exhaustion of tumor antigen-specific CD8+ tumor-infiltrating lymphocytes (TILs), which conventionally has been attributed to persistent stimulation with antigen within the tumor microenvironment (TME). A series of recent studies have highlighted that the TME poses significant metabolic challenges to TILs, which may contribute to their functional exhaustion. Hypoxia increases the expression of coinhibitors on activated CD8+ T cells, which in general reduces the T cells’ effector functions. It also impairs the cells’ ability to gain energy through oxidative phosphorylation. Glucose limitation increases the expression of programed cell death protein-1 and reduces functions of activated CD8+ T cells. A combination of hypoxia and hypoglycemia, as is common in solid tumors, places CD8+ TILs at dual metabolic jeopardy by affecting both major pathways of energy production. Recently, a number of studies addressed the effects of metabolic stress on modulating CD8+ T cell metabolism, differentiation, and functions. Here, we discuss recent findings on how different types of metabolic stress within the TME shape the tumor-killing capacity of CD8+ T cells. We propose that manipulating the metabolism of TILs to more efficiently utilize nutrients, especially during intermittent periods of hypoxia could maximize their performance, prolong their survival and improve the efficacy of active cancer immunotherapy. PMID:26904023

  9. Decoding system for the AUA codon by tRNAIle with the UAU anticodon in Mycoplasma mobile

    PubMed Central

    Taniguchi, Takaaki; Miyauchi, Kenjyo; Nakane, Daisuke; Miyata, Makoto; Muto, Akira; Nishimura, Susumu; Suzuki, Tsutomu

    2013-01-01

    Deciphering the genetic code is a fundamental process in all living organisms. In many bacteria, AUA codons are deciphered by tRNAIle2 bearing lysidine (L) at the wobble position. L is a modified cytidine introduced post-transcriptionally by tRNAIle-lysidine synthetase (TilS). Some bacteria, including Mycoplasma mobile, do not carry the tilS gene, indicating that they have established a different system to decode AUA codons. In this study, tRNAIle2 has been isolated from M. mobile and was found to contain a UAU anticodon without any modification. Mycoplasma mobile isoleucyl-tRNA synthetase (IleRS) recognized the UAU anticodon, whereas Escherichia coli IleRS did not efficiently aminoacylate tRNAIle2UAU. In M. mobile IleRS, a single Arg residue at position 865 was critical for specificity for the UAU anticodon and, when the corresponding site (W905) in E. coli IleRS was substituted with Arg, the W905R mutant efficiently aminoacylated tRNA with UAU anticodon. Mycoplasma mobile tRNAIle2 cannot distinguish between AUA and AUG codon on E. coli ribosome. However, on M. mobile ribosome, M. mobile tRNAIle2UAU specifically recognized AUA codon, and not AUG codon, suggesting M. mobile ribosome has a property that prevents misreading of AUG codon. These findings provide an insight into the evolutionary reorganization of the AUA decoding system. PMID:23295668

  10. Registration of a rice gene-mapping population consisting of 'TeQing'-into-'Lemont' backcross introgression lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A new rice (Oryza sativa L.) mapping population consisting of 123 'TeQing'-into-'Lemont' backcross introgression lines (TILs) (Reg. No. MP-5, NSL 477436 MAP) was developed by the USDA-ARS Rice Research Unit at the Texas A&M University System AgriLife Research and Extension Center at Beaumont, TX, in...

  11. Estrogenic Activity in Runoff – Effect of Animal Waste-Based Fertilizer Application to Frozen Fields

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The potential for contamination of surface waters with estrogenic activity from agricultural animal waste has been of recent concern. Application of manure to frozen fields is currently permitted in some states, but requires a 100 ft vegetative buffer between application area and any waterway or til...

  12. Closing the Student Achievement Gap in California's Elementary Schools: A Lead Teachers' Perspective on Transformational Instructional Leadership

    ERIC Educational Resources Information Center

    Hays, Kelli

    2010-01-01

    This study has tackled the thorny problem of closing the Student Achievement Gap (SAG) in California's elementary schools. To address that problem, an "Integrated" form of educational leadership called Transformational Instructional Leadership (TIL), a form grounded in "best practices" of Transformational and Instructional leadership, was…

  13. Fluid Fertilizer's Role in Sustainng Soils Used for Bio-Fuels Production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The short- and long-term effects on soil nutrient cycling, physical properties, and biological activity of striving for higher grain yields and removing crop residues for bio-fuels production must be understood to provide more quantitative crop and soil management guidelines. Studies focusing on til...

  14. Effector, Memory, and Dysfunctional CD8+ T Cell Fates in the Antitumor Immune Response

    PubMed Central

    2016-01-01

    The adaptive immune system plays a pivotal role in the host's ability to mount an effective, antigen-specific immune response against tumors. CD8+ tumor-infiltrating lymphocytes (TILs) mediate tumor rejection through recognition of tumor antigens and direct killing of transformed cells. In growing tumors, TILs are often functionally impaired as a result of interaction with, or signals from, transformed cells and the tumor microenvironment. These interactions and signals can lead to transcriptional, functional, and phenotypic changes in TILs that diminish the host's ability to eradicate the tumor. In addition to effector and memory CD8+ T cells, populations described as exhausted, anergic, senescent, and regulatory CD8+ T cells have been observed in clinical and basic studies of antitumor immune responses. In the context of antitumor immunity, these CD8+ T cell subsets remain poorly characterized in terms of fate-specific biomarkers and transcription factor profiles. Here we discuss the current characterization of CD8+ T cell fates in antitumor immune responses and discuss recent insights into how signals in the tumor microenvironment influence TIL transcriptional networks to promote CD8+ T cell dysfunction. PMID:27314056

  15. Manipulations of soil microbiota for C sequestration and mitigation of greenhouse gas emissions in managed systems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil microbes dominate processes that regulate soil trace gas emissions and soil C and N dynamics. Intensive management in agroecosystems provides unique opportunities to assess the effectiveness of microbial manipulations to enhance soil C retention and reduce trace gas emissions. While reduced til...

  16. Effects of TP53 and PIK3CA mutations in early breast cancer: a matter of co-mutation and tumor-infiltrating lymphocytes.

    PubMed

    Kotoula, Vassiliki; Karavasilis, Vasilios; Zagouri, Flora; Kouvatseas, George; Giannoulatou, Eleni; Gogas, Helen; Lakis, Sotiris; Pentheroudakis, George; Bobos, Mattheos; Papadopoulou, Kyriaki; Tsolaki, Eleftheria; Pectasides, Dimitrios; Lazaridis, Georgios; Koutras, Angelos; Aravantinos, Gerasimos; Christodoulou, Christos; Papakostas, Pavlos; Markopoulos, Christos; Zografos, George; Papandreou, Christos; Fountzilas, George

    2016-07-01

    The purpose of this study is to investigate whether the outcome of breast cancer (BC) patients treated with adjuvant chemotherapy is affected by co-mutated TP53 and PIK3CA according to stromal tumor-infiltrating lymphocytes (TILs). Paraffin tumors of all clinical subtypes from 1661 patients with operable breast cancer who were treated within 4 adjuvant trials with anthracycline-taxanes chemotherapy were informative for TP53 and PIK3CA mutation status (semiconductor sequencing genotyping) and for stromal TILs density. Disease-free survival (DFS) was examined. TP53 mutations were associated with higher (p < 0.001) and PIK3CA with lower (p = 0.004) TILs in an ER /PgR-specific manner (p < 0.001). Mutations did not affect the favorable DFS of patients with lymphocyte-predominant (LP) BC. Within non-LPBC, PIK3CA-only mutations conferred best, while TP53-PIK3CA co-mutations (6 % of all tumors) conferred worst DFS (HR 0.59; 95 % CI 0.44-0.79; p = 0.001 for PIK3CA-only). TP53-only mutations were unfavorable in patients with lower TILs, while patients with lower TILs performed worse if their tumors carried TP53-only mutations (interaction p = 0.046). Multivariate analysis revealed favorable PIK3CA-only mutations in non-LPBC (HR 0.64; 95 % CI 0.47-0.88; p = 0.007), and unfavorable TP53 mutations in ER/PgRpos/HER2neg (HR 1.55; 95 % CI 1.07-2.24; p = 0.021). Mutations did not interact with TILs in non-LP triple-negative and HER2-positive patients. TP53 and PIK3CA mutations appear to have diverse effects on the outcome of early BC patients, according to whether these genes are co-mutated or not, and for TP53 according to TILs density and ER/PgR-status. These findings need to be considered when evaluating the effect of these two most frequently mutated genes in the context of large clinical trials. PMID:27369359

  17. Total inward leakage of nanoparticles through filtering facepiece respirators.

    PubMed

    Rengasamy, Samy; Eimer, Benjamin C

    2011-04-01

    Nanoparticle (<100 nm size) exposure in workplaces is a major concern because of the potential impact on human health. National Institute for Occupational Safety and Health (NIOSH)-approved particulate respirators are recommended for protection against nanoparticles based on their filtration efficiency at sealed conditions. Concerns have been raised on the lack of information for face seal leakage of nanoparticles, compromising respiratory protection in workplaces. To address this issue, filter penetration and total inward leakage (TIL) through artificial leaks were measured for NIOSH-approved N95 and P100 and European certified Conformit'e Europe'en-marked FFP2 and FFP3 filtering facepiece respirator models sealed to a breathing manikin kept inside a closed chamber. Monodisperse sucrose aerosols (8-80 nm size) generated by electrospray or polydisperse NaCl aerosols (20-1000 nm size) produced by atomization were passed into the chamber. Filter penetration and TIL were measured at 20, 30, and 40 l min(-1) breathing flow rates. The most penetrating particle size (MPPS) was ∼50 nm and filter penetrations for 50 and 100 nm size particles were markedly higher than the penetrations for 8 and 400 nm size particles. Filter penetrations increased with increasing flow rates. With artificially introduced leaks, the TIL values for all size particles increased with increasing leak sizes. With relatively smaller size leaks, the TIL measured for 50 nm size particles was ∼2-fold higher than the values for 8 and 400 nm size particles indicating that the TIL for the most penetrating particles was higher than for smaller and larger size particles. The data indicate that higher concentration of nanoparticles could occur inside the breathing zone of respirators in workplaces where nanoparticles in the MPPS range are present, when leakage is minimal compared to filter penetration. The TIL/penetration ratios obtained for 400 nm size particles were larger than the ratios obtained for

  18. Tumor Infiltrating Lymphocytes Genetically Engineered with an Inducible Gene Encoding Interleukin-12 for the Immunotherapy of Metastatic Melanoma

    PubMed Central

    Zhang, Ling; Morgan, Richard A.; D.Beane, Joal; Zheng, Zhili; Dudley, Mark E.; Kassim, Sadik H.; Nahvi, Azam V.; Ngo, Lien T.; Sherry, Richard M.; Phan, Giao Q.; Hughes, Marybeth S.; Kammula, Udai S.; Feldman, Steven A.; Toomey, Mary Ann; Kerkar, Sid. P.; Restifo, Nicholas P.; Yang, James C.; Rosenberg, Steven A.

    2015-01-01

    Purpose Infusion of interleukin-12 (IL-12) can mediate anti-tumor immunity in animal models, yet its systemic administration to patients with cancer results in minimal efficacy and severe toxicity. Here, we evaluated the anti-tumor activity of adoptively transferred human tumor infiltrating lymphocytes (TIL) genetically engineered to secrete single-chain IL-12 selectively at the tumor site. Experimental design Thirty-three patients with metastatic melanoma were treated in a cell-dose escalation trial of autologous TIL transduced with a gene encoding a single chain IL-12 driven by a nuclear factor of activated T cells promoter (NFAT.IL12). No IL-2 was administered. Results The administration of 0.001-0.1 X 109 NFAT.IL12 transduced TIL to 17 patients resulted in a single objective response (5.9%). However, at doses between 0.3-3 X 109 cells, 10 of 16 patients (63%) exhibited objective clinical responses. The responses tended to be short and the administered IL-12 producing cells rarely persisted at one month. Increasing cell doses were associated with high serum levels of IL-12 and gamma-interferon as well as clinical toxicities including liver dysfunction, high fevers and sporadic life threatening hemodynamic instability. Conclusions In this first-in-man trial, administration of TIL transduced with an inducible IL-12 gene mediated tumor responses in the absence of IL-2 administration using cell doses 10-100 fold lower than conventional TIL. However, due to toxicities, likely attributable to the secreted IL-12, further refinement will be necessary before this approach can be safely utilized in the treatment of cancer patients. PMID:25695689

  19. Intratumoral regulatory T cells alone or in combination with cytotoxic T cells predict prognosis of hepatocellular carcinoma after resection.

    PubMed

    Chen, Kang-jie; Zhou, Lin; Xie, Hai-yang; Ahmed, Taki-eldin; Feng, Xiao-wen; Zheng, Shu-sen

    2012-09-01

    Tumor-infiltrating lymphocytes (TILs) represent the host immune response to cancer. CD8(+) cytotoxic T cells (CTLs) have a central role in the elimination of tumors, while regulatory T cells (Tregs) can suppress the immune reaction. The aim of this study was to investigate the prognostic value of TILs, especially Tregs and CTLs, in hepatocellular carcinoma (HCC) patients after resection. CD3(+), CD4(+), CD8(+), and FoxP3(+) TILs were assessed by immunohistochemistry in tumor tissue from 141 randomly selected HCC patients. Prognostic effects of low- or high-density TIL subsets were evaluated by Kaplan-Meier and Cox regression analysis using the median values as cutoff. The density of intratumoral Tregs (P = 0.040) and peritumoral CTLs (P = 0.004) were an independent factor for overall survival (OS), but not for disease-free survival (DFS). The density of CD3(+) and CD4(+) TILs, and the prevalence of Tregs and CTLs were associated with neither OS nor DFS. The presence of low intratumoral Tregs with high intratumoral CTLs was a negative independent prognostic factor for OS (P = 0.001), while that of low intratumoral Tregs and low peritumoral CTLs independently correlated with improved DFS (P = 0.008). Moreover, the combined analysis of Tregs and CTLs displayed better prognostic performances than any of them alone. Additionally, higher density of intratumoral Tregs correlated with both the presence of liver cirrhosis (P = 0.025) and increased tumor size (P = 0.050). Tregs within tumor environment are promising prognostic parameters for HCC patients, and their combination with CTLs can predict prognosis more effectively. PMID:21678026

  20. The double tropopause and its dynamical relationship to the tropopause inversion layer in storm track regions

    NASA Astrophysics Data System (ADS)

    Peevey, T. R.; Gille, J. C.; Homeyer, C. R.; Manney, G. L.

    2014-09-01

    Using High Resolution Dynamic Limb Sounder observations and ERA-Interim reanalysis this study demonstrates that the warm conveyor belt (WCB) is a mechanism responsible for the relationship between the double tropopause (DT) and the tropopause inversion layer (TIL), a relationship recently suggested in the literature based on idealized model simulations of baroclinic disturbances. Using these data sets, spatial and temporal characteristics of the DT-TIL relationship are examined over a 3 year period, 2005-2008. In the extratropics, results from satellite data show that as the TIL increases in strength, so does the frequency of the DT, regardless of season or hemisphere. The inverse relationship is found in the tropics. Using only DT profiles, zonal composites of wind, relative vorticity, and temperature from reanalysis data show that as the TIL increases in strength, the upper tropospheric circulation switches from cyclonic to anticyclonic, and the upward vertical motion increases. This result suggests the WCB as a mechanism since it is on the anticyclonic side of the jet and is characterized by the movement of tropical air poleward and upward from the surface. To verify this relationship, the vertical and horizontal development of a synoptic-scale baroclinic system is analyzed over a 4 day period. Results show the equatorward extension of the polar tropopause, and thus the formation of the DT, due to the strengthening of the TIL in the region of vertical motion associated with the WCB. Moreover, this result suggests that air movement within the DT could originate from high latitudes when associated with a baroclinic disturbance.

  1. Prognostic impact of programmed cell death-1 (PD-1) and PD-ligand 1 (PD-L1) expression in cancer cells and tumor-infiltrating lymphocytes in ovarian high grade serous carcinoma

    PubMed Central

    Kulbe, Hagen; Sehouli, Jalid; Wienert, Stephan; Lindner, Judith; Budczies, Jan; Bockmayr, Michael; Dietel, Manfred; Denkert, Carsten; Braicu, Ioana; Jöhrens, Korinna

    2016-01-01

    Aims Antibodies targeting the checkpoint molecules programmed cell death 1 (PD-1) and its ligand PD-L1 are emerging cancer therapeutics. We systematically investigated PD-1 and PD-L1 expression patterns in the poor-prognosis tumor entity high-grade serous ovarian carcinoma. Methods PD-1 and PD-L1 protein expression was determined by immunohistochemistry on tissue microarrays from 215 primary cancers both in cancer cells and in tumor-infiltrating lymphocytes (TILs). mRNA expression was measured by quantitative reverse transcription PCR. An in silico validation of mRNA data was performed in The Cancer Genome Atlas (TCGA) dataset. Results PD-1 and PD-L1 expression in cancer cells, CD3+, PD-1+, and PD-L1+ TILs densities as well as PD-1 and PD-L1 mRNA levels were positive prognostic factors for progression-free (PFS) and overall survival (OS), with all factors being significant for PFS (p < 0.035 each), and most being significant for OS. Most factors also had prognostic value that was independent from age, stage, and residual tumor. Moreover, high PD-1+ TILs as well as PD-L1+ TILs densities added prognostic value to CD3+TILs (PD-1+: p = 0.002,; PD-L1+: p = 0.002). The significant positive prognostic impact of PD-1 and PD-L1 mRNA expression could be reproduced in the TCGA gene expression datasets (p = 0.02 and p < 0.0001, respectively). Conclusions Despite their reported immune-modulatory function, high PD-1 and PD-L1 levels are indicators of a favorable prognosis in ovarian cancer. Our data indicate that PD-1 and PD-L1 molecules are biologically relevant regulators of the immune response in high-grade serous ovarian carcinoma, which is an argument for the evaluation of immune checkpoint inhibiting drugs in this tumor entity. PMID:26625204

  2. Programmed death-1 ligand 1 and 2 are highly expressed in pleomorphic carcinomas of the lung: Comparison of sarcomatous and carcinomatous areas.

    PubMed

    Kim, Sehui; Kim, Moon-Young; Koh, Jaemoon; Go, Heounjeong; Lee, Dong Soo; Jeon, Yoon Kyung; Chung, Doo Hyun

    2015-11-01

    Pleomorphic carcinoma (PC) of the lung is a rare type of poorly differentiated non-small cell lung carcinoma (NSCLC) that belongs to sarcomatoid carcinoma (SC). It exhibits aggressive behaviour and resistance to chemotherapy and radiotherapy. Recently, immunotherapy targeting the programmed death-1 (PD-1)/PD ligand 1 (PD-L1) pathway has demonstrated favourable clinical outcomes in NSCLC. However, the expression patterns of PD-1-related molecules in pulmonary PC remain elusive. PD-L1 and PD-L2 expression was estimated in 41 cases of PC using immunohistochemistry. CD8(+) and PD-1(+) tumour-infiltrating lymphocytes (TILs) were also evaluated. PD-L1 and PD-L2 were highly expressed in pulmonary PCs (90.2% [37/41)]; 87.8% [36/41]). The amount of CD8(+) or PD-1(+) TILs and the ratio of PD-1(+)/CD8(+) TILs in PC were higher in males, smokers and older patients. PD-L1-positive PCs were infiltrated by higher numbers of CD8(+) TILs compared to PD-L1-negative cases (P=0.006). Of note, PD-L1 expression in pulmonary PCs was significantly higher in sarcomatous areas than in the carcinomatous portion (P=0.006). PC patients with a high ratio of PD-1(+)/CD8(+) TILs showed a shorter progression-free survival (P=0.036), whereas PD-L1 and PD-L2 expression had no prognostic implications. Our study demonstrates that pulmonary PCs very frequently express PD-L1 and PD-L2. Moreover, their expression is higher in sarcomatous cells than in carcinomatous areas. Thus, targeting the PD-1/PD-L1 pathway may represent a potential therapeutic candidate for this aggressive tumour. PMID:26329973

  3. Correlation between Density of CD8+ T-cell Infiltrate in Microsatellite Unstable Colorectal Cancers and Frameshift Mutations: A Rationale for Personalized Immunotherapy.

    PubMed

    Maby, Pauline; Tougeron, David; Hamieh, Mohamad; Mlecnik, Bernhard; Kora, Hafid; Bindea, Gabriela; Angell, Helen K; Fredriksen, Tessa; Elie, Nicolas; Fauquembergue, Emilie; Drouet, Aurélie; Leprince, Jérôme; Benichou, Jacques; Mauillon, Jacques; Le Pessot, Florence; Sesboüé, Richard; Tuech, Jean-Jacques; Sabourin, Jean-Christophe; Michel, Pierre; Frébourg, Thierry; Galon, Jérôme; Latouche, Jean-Baptiste

    2015-09-01

    Colorectal cancers with microsatellite instability (MSI) represent 15% of all colorectal cancers, including Lynch syndrome as the most frequent hereditary form of this disease. Notably, MSI colorectal cancers have a higher density of tumor-infiltrating lymphocytes (TIL) than other colorectal cancers. This feature is thought to reflect the accumulation of frameshift mutations in sequences that are repeated within gene coding regions, thereby leading to the synthesis of neoantigens recognized by CD8(+) T cells. However, there has yet to be a clear link established between CD8(+) TIL density and frameshift mutations in colorectal cancer. In this study, we examined this link in 103 MSI colorectal cancers from two independent cohorts where frameshift mutations in 19 genes were analyzed and CD3(+), CD8(+), and FOXP3(+) TIL densities were quantitated. We found that CD8(+) TIL density correlated positively with the total number of frameshift mutations. TIL densities increased when frameshift mutations were present within the ASTE1, HNF1A, or TCF7L2 genes, increasing even further when at least one of these frameshift mutations was present in all tumor cells. Through in vitro assays using engineered antigen-presenting cells, we were able to stimulate peripheral cytotoxic T cells obtained from colorectal cancer patients with peptides derived from frameshift mutations found in their tumors. Taken together, our results highlight the importance of a CD8(+) T cell immune response against MSI colorectal cancer-specific neoantigens, establishing a preclinical rationale to target them as a personalized cellular immunotherapy strategy, an especially appealing goal for patients with Lynch syndrome. PMID:26060019

  4. Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection.

    PubMed Central

    Kawakami, Y; Eliyahu, S; Delgado, C H; Robbins, P F; Sakaguchi, K; Appella, E; Yannelli, J R; Adema, G J; Miki, T; Rosenberg, S A

    1994-01-01

    The cultured T-cell line TIL1200, established from the tumor-infiltrating lymphocytes (TILs) of a patient with advanced metastatic melanoma, recognized an antigen on most HLA-A2+ melanomas and on all HLA-A2+ cultured neonatal melanocytes in an HLA-A2 restricted manner but not on other types of tissues or cell lines tested. A cDNA encoding an antigen recognized by TIL1200 was isolated by screening an HLA-A2+ breast cancer cell line transfected with an expression cDNA library prepared from an HLA-A2+ melanoma cell line. The nucleotide and amino acid sequences of this cDNA were almost identical to the genes encoding glycoprotein gp100 or Pmel17 previously registered in the GenBank. Expression of this gene was restricted to melanoma and melanocyte cell lines and retina but was not expressed on other fresh or cultured normal tissues or other types of tumor tested. The cell line transfected with this cDNA also expressed antigen recognized by the melanoma-specific antibody HMB45 that bound to gp100. A synthetic 10-amino acid peptide derived from gp100 was recognized by TIL1200 in the context of HLA-A2.1. Since the administration of TIL1200 plus interleukin 2 resulted in regression of metastatic cancer in the autologous patient, gp100 is a possible tumor rejection antigen and may be useful for the development of immunotherapies for patients with melanoma. Images PMID:8022805

  5. TIGIT and PD-1 impair tumor antigen–specific CD8+ T cells in melanoma patients

    PubMed Central

    Chauvin, Joe-Marc; Pagliano, Ornella; Fourcade, Julien; Sun, Zhaojun; Wang, Hong; Sander, Cindy; Kirkwood, John M.; Chen, Tseng-hui Timothy; Maurer, Mark; Korman, Alan J.; Zarour, Hassane M.

    2015-01-01

    T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen–specific (TA-specific) CD8+ T cells and CD8+ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8+ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8+ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1+TIM-3+ TA-specific CD8+ T cells. PD-1+TIGIT+, PD-1–TIGIT+, and PD-1+TIGIT– CD8+ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand–expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8+ T cells and CD8+ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8+ T cells and CD8+ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8+ T cell responses in patients with advanced melanoma. PMID:25866972

  6. CXCR3/CCR5 pathways in metastatic melanoma patients treated with adoptive therapy and interleukin-2

    PubMed Central

    Bedognetti, D; Spivey, T L; Zhao, Y; Uccellini, L; Tomei, S; Dudley, M E; Ascierto, M L; De Giorgi, V; Liu, Q; Delogu, L G; Sommariva, M; Sertoli, M R; Simon, R; Wang, E; Rosenberg, S A; Marincola, F M

    2013-01-01

    Background: Adoptive therapy with tumour-infiltrating lymphocytes (TILs) induces durable complete responses (CR) in ∼20% of patients with metastatic melanoma. The recruitment of T cells through CXCR3/CCR5 chemokine ligands is critical for immune-mediated rejection. We postulated that polymorphisms and/or expression of CXCR3/CCR5 in TILs and the expression of their ligands in tumour influence the migration of TILs to tumours and tumour regression. Methods: Tumour-infiltrating lymphocytes from 142 metastatic melanoma patients enrolled in adoptive therapy trials were genotyped for CXCR3 rs2280964 and CCR5-Δ32 deletion, which encodes a protein not expressed on the cell surface. Expression of CXCR3/CCR5 in TILs and CXCR3/CCR5 and ligand genes in 113 available parental tumours was also assessed. Tumour-infiltrating lymphocyte data were validated by flow cytometry (N=50). Results: The full gene expression/polymorphism model, which includes CXCR3 and CCR5 expression data, CCR5-Δ32 polymorphism data and their interaction, was significantly associated with both CR and overall response (OR; P=0.0009, and P=0.007, respectively). More in detail, the predicted underexpression of both CXCR3 and CCR5 according to gene expression and polymorphism data (protein prediction model, PPM) was associated with response to therapy (odds ratio=6.16 and 2.32, for CR and OR, respectively). Flow cytometric analysis confirmed the PPM. Coordinate upregulation of CXCL9, CXCL10, CXCL11, and CCL5 in pretreatment tumour biopsies was associated with OR. Conclusion: Coordinate overexpression of CXCL9, CXCL10, CXCL11, and CCL5 in pretreatment tumours was associated with responsiveness to treatment. Conversely, CCR5-Δ32 polymorphism and CXCR3/CCR5 underexpression influence downregulation of the corresponding receptors in TILs and were associated with likelihood and degree of response. PMID:24129241

  7. OX40, PD-1 and CTLA-4 are selectively expressed on tumor-infiltrating T cells in head and neck cancer

    PubMed Central

    Montler, Ryan; Bell, R Bryan; Thalhofer, Colin; Leidner, Rom; Feng, Zipei; Fox, Bernard A; Cheng, Allen C; Bui, Tuan G; Tucker, Christopher; Hoen, Helena; Weinberg, Andrew

    2016-01-01

    The tumor microenvironment of squamous cell carcinoma of the head and neck (SCCHN) has been shown to be immune suppressive. Therefore, strategies aimed at overcoming this issue could have a positive therapeutic impact. Hence, we investigated the expression of the known immune-modulatory proteins OX40, programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in SCCHN on different T-cell subsets of tumor-infiltrating lymphocytes (TIL) to ascertain whether these proteins could potentially be targeted alone or in combination for future clinical trials. T cells from peripheral blood (PBL) and tumor were analyzed for the expression of OX40, PD-1 and CTLA-4 in 29 patients undergoing surgery. These proteins were all expressed significantly higher in T-cell subsets isolated from tumors compared with PBL of the same patient. OX40 expression was significantly greater in the TIL regulatory T-cell (Treg) population relative to conventional CD4 and CD8 TIL or the Treg isolated from PBL. PD-1 expression was increased in all T-cell subsets relative to PBL. CTLA-4 was also increased in all TIL subsets relative to blood, and similar to OX40, its highest level of expression was observed in the Treg TIL. The highest frequency of PD-1, CTLA-4 and OX40 triple-positive cells were found in the Treg population isolated from the tumor. We analyzed both human papilloma virus-positive and -negative patients and found similar levels and expression patterns of these two patient populations for all three proteins. These data suggest that there may be therapeutic advantages of targeting these pathways independently or in combination for patients with this disease. PMID:27195113

  8. Cancer immunotherapy utilizing gene-modified T cells: From the bench to the clinic.

    PubMed

    Duong, Connie P M; Yong, Carmen S M; Kershaw, Michael H; Slaney, Clare Y; Darcy, Phillip K

    2015-10-01

    The immune system plays a critical role in the elimination and suppression of pathogens. Although the endogenous immune system is capable of immune surveillance resulting in the elimination of cancer cells, tumor cells have developed a variety of mechanisms to escape immune recognition often resulting in tumor outgrowth. The presence of immune infiltrate in tumors has been correlated with a good prognosis following treatment (Sato et al., 2005; Loi et al., 2013; Clemente et al., 1996; Galon et al., 2006). As such, immune cells such as T cells, have been harnessed in order to target cancer. Tumor reactive lymphocytes, called tumor-infiltrating lymphocytes (TILs) have been isolated and expanded from the tumor and reinfused back into patients for the treatment of melanoma. The promise of adoptive immunotherapy utilizing TILs as a robust treatment for cancer has been highlighted in patients with advanced melanoma with greater than 50% of patients responding to treatment (Dudley et al., 2005). Although TIL therapy has shown promising results in melanoma patients, it has proved difficult to translate this approach to other cancers, given that the numbers of TILs that can be isolated are generally low. To broaden this therapy for other cancers, T cells have been genetically modified to endow them with tumor reactivity using either a T cell receptor (TCR) (Parkhurst et al., 2009, 2011; Chinnasamy et al., 2011) or a chimeric antigen receptor (CAR) (Grupp et al., 2013; Park et al., 2007). This review will outline the origins and development of adoptive immunotherapy utilizing TILs leading to genetic modification strategies to redirect T cells to cancer. Potential hurdles and novel strategies will be discussed for realizing the full potential of adoptive immunotherapy becoming a standard of care treatment for cancer. PMID:25595028

  9. Nanoparticle penetration through filter media and leakage through face seal interface of N95 filtering facepiece respirators.

    PubMed

    Rengasamy, Samy; Eimer, Benjamin C

    2012-07-01

    National Institute for Occupational Safety and Health recommends the use of particulate respirators for protection against nanoparticles (<100 nm size). Protection afforded by a filtering facepiece particulate respirator is a function of the filter efficiency and the leakage through the face-to-facepiece seal. The combination of particle penetration through filter media and particle leakage through face seal and any component interfaces is considered as total inward leakage (TIL). Although the mechanisms and extent of nanoparticle penetration through filter media have been well documented, information concerning nanoparticle leakage through face seal is lacking. A previous study in our laboratory measured filter penetration and TIL for specific size particles. The results showed higher filter penetration and TIL for 50 nm size particles, i.e. the most penetrating particle size (MPPS) than for 8 and 400 nm size particles. To better understand the significance of particle penetration through filter media and through face seal leakage, this study was expanded to measure filter penetration at sealed condition and TIL with artificially introduced leaks for 20-800 nm particles at 8-40 l minute volumes for four N95 models of filtering facepiece respirators (FFRs) using a breathing manikin. Results showed that the MPPS was ~45 nm for all four respirator models. Filter penetration for 45 nm size particles was significantly (P < 0.05) higher than the values for 400 nm size particles. A consistent increase in filter penetrations for 45 and 400 nm size particles was obtained with increasing breathing minute volumes. Artificial leakage of test aerosols (mode size ~75 nm) through increasing size holes near the sealing area of FFRs showed higher TIL values for 45 nm size particles at different minute volumes, indicating that the induced leakage allows the test aerosols, regardless of particle size, inside the FFR, while filter penetration determines the TIL for different size

  10. The prognostic advantage of preoperative intratumoral injection of OK-432 for gastric cancer patients

    PubMed Central

    Gochi, A; Orita, K; Fuchimoto, S; Tanaka, N; Ogawa, N

    2001-01-01

    To investigate, by a multi-institutional randomized trial, the prognostic significance of the augmentation of tumour-infiltrating lymphocytes (TILs) by preoperative intratumoral injection of OK-432 (OK-432 it), a bacterial biological response modifier, in patients with gastric cancer. The 10-year survival and disease-free survival were examined and analysis of the factors showing survival benefit was performed. 370 patients who had undergone curative resection of gastric cancer were enrolled in this study and followed up for 10 years postoperatively. Patients were randomized into either an OK-432 it group or a control group. Ten Klinishe Einheit (KE) of OK-432 was endoscopically injected at 1 to 2 weeks before the operation in the OK-432 it group. Both groups received the same adjuvant chemoimmunotherapy consisting of a bolus injection of mitomycin C (0.4 mg kg−1i.v.) and administration of tegafur and OK-432 from postoperative day 14 up to 1 year later. Tegafur (600 mg day−1) was given orally and OK-432 (5 KE/2 weeks) was injected intradermally for a maintenance therapy. The TILs grades in resected tumour specimens and presence of metastasis and metastatic pattern in dissected lymph nodes were examined. Multivariate analysis was performed to determine the efficacy of OK-432 it on prognostic factors. All patients were followed up for 10 years. The overall 5- and 10-year survival rates and disease-free survival rates of the OK-432 it group were not significantly higher than those of the control group. However, OK-432 it significantly increased the 5- and 10-year survival rates of patients with stage IIIA + IIIB, moderate lymph node metastasis (pN2), and positive TILs. OK-432 it was most effective at prolonging the survival of patients who had both positive TILs and lymph node metastasis. The OK-432 it group with positive TILs showed a significant decrease in metastatic lymph node frequency and in the number of lymph node micro- metastatic foci when compared to

  11. Association and prognostic significance of BRCA1/2-mutation status with neoantigen load, number of tumor-infiltrating lymphocytes and expression of PD-1/PD-L1 in high grade serous ovarian cancer

    PubMed Central

    Strickland, Kyle C.; Howitt, Brooke E.; Shukla, Sachet A.; Rodig, Scott; Ritterhouse, Lauren L.; Liu, Joyce F.; Garber, Judy E.; Chowdhury, Dipanjan; Wu, Catherine J.; D'Andrea, Alan D.; Matulonis, Ursula A.; Konstantinopoulos, Panagiotis A.

    2016-01-01

    Immune checkpoint inhibitors (e.g., anti-PD-1 and anti-PD-L1 antibodies) have demonstrated remarkable efficacy against hypermutated cancers such as melanomas and lung carcinomas. One explanation for this effect is that hypermutated lesions harbor more tumor-specific neoantigens that stimulate recruitment of an increased number of tumor-infiltrating lymphocytes (TILs), which is counterbalanced by overexpression of immune checkpoints such as PD-1 or PD-L1. Given that BRCA1/2-mutated high grade serous ovarian cancers (HGSOCs) exhibit a higher mutational load and a unique mutational signature with an elevated number of larger indels up to 50 bp, we hypothesized that they may also harbor more tumor-specific neoantigens, and, therefore, exhibit increased TILs and PD-1/PD-L1 expression. Here, we report significantly higher predicted neoantigens in BRCA1/2-mutated tumors compared to tumors without alterations in homologous recombination (HR) genes (HR-proficient tumors). Tumors with higher neoantigen load were associated with improved overall survival and higher expression of immune genes associated with tumor cytotoxicity such as genes of the TCR, the IFN-gamma and the TNFR pathways. Furthermore, immunohistochemistry studies demonstrated that BRCA1/2-mutated tumors exhibited significantly increased CD3+ and CD8+ TILs, as well as elevated expression of PD-1 and PD-L1 in tumor-associated immune cells compared to HR-proficient tumors. Survival analysis showed that both BRCA1/2-mutation status and number of TILs were independently associated with outcome. Of note, two distinct groups of HGSOCs, one with very poor prognosis (HR proficient with low number of TILs) and one with very good prognosis (BRCA1/2-mutated tumors with high number of TILs) were defined. These findings support a link between BRCA1/2-mutation status, immunogenicity and survival, and suggesting that BRCA1/2-mutated HGSOCs may be more sensitive to PD-1/PD-L1 inhibitors compared to HR-proficient HGSOCs. PMID

  12. [Development of rapid detection of infectious hypodermal and hematopoietic necrosis virus by loop-mediated isothermal amplification].

    PubMed

    He, Lin; Xu, Hai-Sheng; Wang, Mei-Zhen; Rong, Hua-Nan

    2010-11-01

    Loop-mediated isothermal amplification (LAMP) assay is a novel method of gene amplification with high specificity, sensitivity and rapidity, which can be applied for disease diagnosis in shrimp aquaculture. The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target. In the present study, according to the conservative regions of non-structural protein gene NS1, a set of four specific primers were designed, and a rapid detection of IHHNV was established by LAMP assay. The parameters of reaction time and temperature were optimized, and its specificity and sensitivity were assessed. The reactions were carried out at 60 degrees C, 62 degrees C, 63 degrees C, 64 degrees C, 65 degrees C, 66 degrees C, 67 degrees C, 68 degrees C for different time (0 min; 15 min; 30 min; 45 min; 60 min; 75 min). A plasmid pMDIHHNV carrying target sequence of LAMP detection was constructed. Ten-fold serially diluted pMDIHHNV (10(7)-10(0)copies/microL) was used as template for LAMP assay to investigate the detection limit. To determine the specificity, LAMP assays were carried out with DNA templates from other pathogens (White spot syndrome virus; WSSV, Taura Syndrome Virus; TSV, Aeromonas. hydrophila, V. alginolyticus, Vibrio. parahaemolytious, Escherichia. coli). The results showed the optimized LAMP assay for the rapid detection of IHHNV was performed at 65 degrees C for 60 min. The LAMP assay had an unequivocal detection limit of 100 copies/microL, and it was 1,000 times lower than that of PCR. The nucleic acids of other pathogens were not amplified by this LAMP system with the specific primers, which showed a good specificity. The resulting amplicons were detected using visual observation after the addition of SYBR Green I and gel electrophoresis. We investigated the efficacy of UNG (uracil-N-glycosylase) and dUTP in avoiding carry-over contamination in the LAMP assay procedure and explored its

  13. Charge sensing and real-time electron counting in quantum dots

    NASA Astrophysics Data System (ADS)

    Ihn, Thomas

    2012-02-01

    The charge state of a Coulomb blockaded quantum dot can be sensed at the single-electron level using charge sensors integrated on-chip [1]. Quantum point contacts or quantum dots have proven to work as reliable charge sensors. Coupling between the quantum dot's charge state and the sensor is provided by Coulomb interactions between electrons in the two systems. The technique is therefore independent of the material of quantum dot and sensor, and works for diverse systems such as GaAs [1] and graphene [2]. Different materials, such as InAs and GaAs have even been combined. Time-resolved charge sensing is of fundamental interest for studying the statistical properties of charge flow, like the full counting statistics. Single-shot charge and spin read-out schemes are also needed for the measurement of qubits. Some of our recent work has focused on the exploration and optimization of the read-out bandwidth limits. Towards this goal, we learned how to influence the dot-detector coupling [3], and how to employ high-frequency techniques in the range of a few hundred megahertz [4], or even up to a few gigahertz for improved performance. New experiments using the slower conventional sensing schemes have allowed us to explore non-equilibrium statistics and the fluctuation theorem. In our measurements, rare events can be detected, where electrons flow against the applied source-drain bias direction thereby consuming entropy in agreement with theoretical predictions. [4pt] [1] T. Ihn, S. Gustavsson, U. Gasser, R. Leturcq, I. Shorubalko, and K. Ensslin, Physica E: Low-dimensional Systems and Nanostructures 42, 803-808 (2010).[0pt] [2] J. G"uttinger, J. Seif, C. Stampfer, A. Capelli, K. Ensslin, and T. Ihn, Phys. Rev. B 83, 165445 (2011).[0pt] [3] C. R"ossler, B. K"ung, S. Dr"oscher, T. Choi, T. Ihn, K. Ensslin, and M. Beck, Appl. Phys. Lett. 97, 152109 (2010).[0pt] [4] T. M"uller, B. K"ung, S. Hellm"uller, P. Studerus, K. Ensslin, T. Ihn, M. Reinwald, and W. Wegscheider, Appl

  14. Cell Killing Mechanisms and Impact on Gene Expression by Gemcitabine and 212Pb-Trastuzumab Treatment in a Disseminated i.p. Tumor Model

    PubMed Central

    Yong, Kwon Joong; Milenic, Diane E.; Baidoo, Kwamena E.; Brechbiel, Martin W.

    2016-01-01

    In pre-clinical studies, combination therapy with gemcitabine and targeted radioimmunotherapy (RIT) using 212Pb-trastuzumab showed tremendous therapeutic potential in the LS-174T tumor xenograft model of disseminated intraperitoneal disease. To better understand the underlying molecular basis for the observed cell killing efficacy, gene expression profiling was performed after a 24 h exposure to 212Pb-trastuzumab upon gemcitabine (Gem) pre-treatment in this model. DNA damage response genes in tumors were quantified using a real time quantitative PCR array (qRT-PCR array) covering 84 genes. The combination of Gem with α-radiation resulted in the differential expression of apoptotic genes (BRCA1, CIDEA, GADD45α, GADD45γ, IP6K3, PCBP4, RAD21, and p73), cell cycle regulatory genes (BRCA1, CHK1, CHK2, FANCG, GADD45α, GTSE1, PCBP4, MAP2K6, NBN, PCBP4, and SESN1), and damaged DNA binding and repair genes (BRCA1, BTG2, DMC1, ERCC1, EXO1, FANCG, FEN1, MSH2, MSH3, NBN, NTHL1, OGG1, PRKDC, RAD18, RAD21, RAD51B, SEMA4G, p73, UNG, XPC, and XRCC2). Of these genes, the expression of CHK1, GTSE1, EXO1, FANCG, RAD18, UNG and XRCC2 were specific to Gem/212Pb-trastuzumab administration. In addition, the present study demonstrates that increased stressful growth arrest conditions induced by Gem/212Pb-trastuzumab could suppress cell proliferation possibly by up-regulating genes involved in apoptosis such as p73, by down-regulating genes involved in cell cycle check point such as CHK1, and in damaged DNA repair such as RAD51 paralogs. These events may be mediated by genes such as BRCA1/MSH2, a member of BARC (BRCA-associated genome surveillance complex). The data suggest that up-regulation of genes involved in apoptosis, perturbation of checkpoint genes, and a failure to correctly perform HR-mediated DSB repair and mismatch-mediated SSB repair may correlate with the previously observed inability to maintain the G2/M arrest, leading to cell death. PMID:27467592

  15. Immunogenicity of somatic mutations in human gastrointestinal cancers.

    PubMed

    Tran, Eric; Ahmadzadeh, Mojgan; Lu, Yong-Chen; Gros, Alena; Turcotte, Simon; Robbins, Paul F; Gartner, Jared J; Zheng, Zhili; Li, Yong F; Ray, Satyajit; Wunderlich, John R; Somerville, Robert P; Rosenberg, Steven A

    2015-12-11

    It is unknown whether the human immune system frequently mounts a T cell response against mutations expressed by common epithelial cancers. Using a next-generation sequencing approach combined with high-throughput immunologic screening, we demonstrated that tumor-infiltrating lymphocytes (TILs) from 9 out of 10 patients with metastatic gastrointestinal cancers contained CD4(+) and/or CD8(+) T cells that recognized one to three neo-epitopes derived from somatic mutations expressed by the patient's own tumor. There were no immunogenic epitopes shared between these patients. However, we identified in one patient a human leukocyte antigen-C*08:02-restricted T cell receptor from CD8(+) TILs that targeted the KRAS(G12D) hotspot driver mutation found in many human cancers. Thus, a high frequency of patients with common gastrointestinal cancers harbor immunogenic mutations that can potentially be exploited for the development of highly personalized immunotherapies. PMID:26516200

  16. Target-in-the-loop remote sensing of laser beam and atmospheric turbulence characteristics.

    PubMed

    Vorontsov, Mikhail A; Lachinova, Svetlana L; Majumdar, Arun K

    2016-07-01

    A new target-in-the-loop (TIL) atmospheric sensing concept for in situ remote measurements of major laser beam characteristics and atmospheric turbulence parameters is proposed and analyzed numerically. The technique is based on utilization of an integral relationship between complex amplitudes of the counterpropagating optical waves known as overlapping integral or interference metric, whose value is preserved along the propagation path. It is shown that the interference metric can be directly measured using the proposed TIL sensing system composed of a single-mode fiber-based optical transceiver and a remotely located retro-target. The measured signal allows retrieval of key beam and atmospheric turbulence characteristics including scintillation index and the path-integrated refractive index structure parameter. PMID:27409206

  17. Exploiting natural anti-tumor immunity for metastatic renal cell carcinoma

    PubMed Central

    Murphy, Katherine A; James, Britnie R; Guan, Yue; Torry, Donald S; Wilber, Andrew; Griffith, Thomas S

    2015-01-01

    Clinical observations of spontaneous disease regression in some renal cell carcinoma (RCC) patients implicate a role for tumor immunity in controlling this disease. Puzzling, however, are findings that high levels of tumor infiltrating lymphocytes (TIL) are common to RCC. Despite expression of activation markers by TILs, functional impairment of innate and adaptive immune cells has been consistently demonstrated contributing to the failure of the immune system to control RCC. Immunotherapy can overcome the immunosuppressive effects of the tumor and provide an opportunity for long-term disease free survival. Unfortunately, complete response rates remain sub-optimal indicating the effectiveness of immunotherapy remains limited by tumor-specific factors and/or cell types that inhibit antitumor immune responses. Here we discuss immunotherapies and the function of multiple immune system components to achieve an effective response. Understanding these complex interactions is essential to rationally develop novel therapies capable of renewing the immune system's ability to respond to these tumors. PMID:25996049

  18. HLA-E/β2 microglobulin overexpression in colorectal cancer is associated with recruitment of inhibitory immune cells and tumor progression.

    PubMed

    Bossard, Céline; Bézieau, Stéphane; Matysiak-Budnik, Tamara; Volteau, Christelle; Laboisse, Christian L; Jotereau, Francine; Mosnier, Jean-François

    2012-08-15

    The host immune response plays a major role in colorectal carcinoma (CRC) progression. A mechanism of tumor immune escape might involve expression of the human leucocyte antigen (HLA)-E/β2m on tumor cells. The inhibitory effect of HLA-E/β2m on CD8+ cytotoxic T lymphocytes and natural killer (NK) cells is mediated by the main HLA-E receptor CD94/NKG2A. As the pathophysiological relevance of this mechanism in CRC remains unknown, this prompted us to examine, in situ, in a series of 80 CRC (i) the HLA-E and β2m coexpression by tumor cells, (ii) the density of CD8+, cytotoxic, CD244+ and NKP46+ intraepithelial tumor-infiltrating lymphocyte (IEL-TIL) and (iii) the expression of CD94/NKG2 receptor on IEL-TIL. These data were then correlated to patient survival. We provided (i) the in situ demonstration of HLA-E/β2m overexpression by tumor cells in 21% of CRC characterized by an overrepresentation of signet ring cell carcinomas, mucinous carcinomas and medullary carcinomas, (ii) the significant association between HLA-E/β2m overexpression by tumor cells and increased density of CD8+ cytotoxic, CD244+ and CD94+ IEL-TIL and (iii) finally, the unfavorable prognosis associated with HLA-E/β2m overexpression by tumor cells. Our findings show that HLA-E/β2m overexpression is a surrogate marker of poor prognosis and point to a novel mechanism of tumor immune escape in CRC in restraining inhibitory IEL-TIL. PMID:21953582

  19. Immunophenotyping of patients with oral squamous cell carcinoma in peripheral blood and associated tumor tissue.

    PubMed

    Grimm, Martin; Feyen, Oliver; Hofmann, Heiko; Teriete, Peter; Biegner, Thorsten; Munz, Adelheid; Reinert, Siegmar

    2016-03-01

    The immune system is important for elimination of cancer cells. Tumors including oral squamous cell carcinoma (OSCC) are capable of escaping detection by host immune cells through apoptotic depletion of tumor-infiltrating lymphocytes (TILs). Circulating peripheral blood lymphocytes (PBLs) and corresponding TILs of tumor specimen were evaluated before and after curative tumor resection (n = 30) compared with PBLs of controls (n = 87). PBLs were characterized for the total number of T cells (CD3(+)), T helper cells (Th, CD3(+)/CD4(+)), regulatory T cells (Treg, CD4(+)/CD25(+)/CD127(low)), cytotoxic T cells (Tc, CD3(+)/CD8(+)), activated T cells (CD3(+)/HLA-DR(+)), and natural killer (NK) cells (CD3(-)/CD16(+)/CD56(+)). In tumor tissue, the prevalence of CD3(+), CD4(+), and CD8(+) TILs was assessed using immunohistochemistry, whereas the incidence of apoptosis was assessed using terminal deoxynucleotidyl transferase deoxyuridinetriphosphate nick-end labeling (TUNEL) assay. In PBLs of pretreated OSCC patients, a highly significant decrease in total number of T cells (p = 0.0001), Th cells (p < 0.0001), Treg cells (p < 0.0001), Tc cells (p < 0.0001), and NK cells (p = 0.0037) were found compared with controls. Decreased PBLs of OSCC patients were correlated with decreased numbers of corresponding TILs, which were associated with increased detection of apoptosis in the tumor tissue. Compared with the controls, the total number of T cells remained unchanged after surgery but the total number of NK cells significantly increased. Standardized immunophenotyping of OSCC may help to identify patients likely to benefit from cancer immunotherapy strategies and/or chemoradiation. Finally, future attempts to enhance an effective tumor-reactive immune response by immunotherapy or vaccination should be made by promoting tumor-specific Th and/or Tc cell/NK cell responses. PMID:26474587

  20. Tumor-Infiltrating γδ T Lymphocytes: Pathogenic Role, Clinical Significance, and Differential Programing in the Tumor Microenvironment

    PubMed Central

    Lo Presti, Elena; Dieli, Franceso; Meraviglia, Serena

    2014-01-01

    There is increasing clinical evidence indicating that the immune system may either promote or inhibit tumor progression. Several studies have demonstrated that tumors undergoing remission are largely infiltrated by T lymphocytes [tumor-infiltrating lymphocytes (TILs)], but on the other hand, several studies have shown that tumors may be infiltrated by TILs endowed with suppressive features, suggesting that TILs are rather associated with tumor progression and unfavorable prognosis. γδ T lymphocytes are an important component of TILs that may contribute to tumor immunosurveillance, as also suggested by promising reports from several small phase-I clinical trials. Typically, γδ T lymphocytes perform effector functions involved in anti-tumor immune responses (cytotoxicity, production of IFN-γ and TNF-α, and dendritic cell maturation), but under appropriate conditions they may divert from the typical Th1-like phenotype and polarize to Th2, Th17, and Treg cells thus acquiring the capability to inhibit anti-tumor immune responses and promote tumor growth. Recent studies have shown a high frequency of γδ T lymphocytes infiltrating different types of cancer, but the nature of this association and the exact mechanisms underlying it remain uncertain and whether or not the presence of tumor-infiltrating γδ T lymphocytes is a definite prognostic factor remains controversial. In this paper, we will review studies of tumor-infiltrating γδ T lymphocytes from patients with different types of cancer, and we will discuss their clinical relevance. Moreover, we will also discuss on the complex interplay between cancer, tumor stroma, and γδ T lymphocytes as a major determinant of the final outcome of the γδ T lymphocyte response. Finally, we propose that targeting γδ T lymphocyte polarization and skewing their phenotype to adapt to the microenvironment might hold great promise for the treatment of cancer. PMID:25505472

  1. Serum ferritin level changes in children with sickle cell disease on chronic blood transfusion are nonlinear and are associated with iron load and liver injury

    PubMed Central

    Abboud, Miguel R.; Paley, Carole; Olivieri, Nancy; Kirby-Allen, Melanie; Vichinsky, Elliott; Casella, James F.; Alvarez, Ofelia A.; Barredo, Julio C.; Lee, Margaret T.; Iyer, Rathi V.; Kutlar, Abdullah; McKie, Kathleen M.; McKie, Virgil; Odo, Nadine; Gee, Beatrice; Kwiatkowski, Janet L.; Woods, Gerald M.; Coates, Thomas; Wang, Winfred; Adams, Robert J.

    2009-01-01

    Chronic blood transfusion is increasingly indicated in patients with sickle cell disease. Measuring resulting iron overload remains a challenge. Children without viral hepatitis enrolled in 2 trials for stroke prevention were examined for iron overload (STOP and STOP2; n = 271). Most received desferrioxamine chelation. Serum ferritin (SF) changes appeared nonlinear compared with prechelation estimated transfusion iron load (TIL) or with liver iron concentrations (LICs). Averaged correlation coefficient between SF and TIL (patients/observations, 26 of 164) was r = 0.70; between SF and LIC (patients/observations, 33 of 47) was r = 0.55. In mixed models, SF was associated with LIC (P = .006), alanine transaminase (P = .025), and weight (P = .026). Most patients with SF between 750 and 1500 ng/mL had a TIL between 25 and 100 mg/kg (72.8% ± 5.9%; patients/observations, 24 of 50) or an LIC between 2.5 and 10 mg/g dry liver weight (75% ± 0%; patients/observations, 8 of 9). Most patients with SF of 3000 ng/mL or greater had a TIL of 100 mg/kg or greater (95.3% ± 6.7%; patients/observations, 7 of 16) or an LIC of 10 mg/g dry liver weight or greater (87.7% ± 4.3%; patients/observations, 11 of 18). Although SF changes are nonlinear, levels less than 1500 ng/mL indicated mostly acceptable iron overload; levels of 3000 ng/mL or greater were specific for significant iron overload and were associated with liver injury. However, to determine accurately iron overload in patients with intermediately elevated SF levels, other methods are required. These trials are registered at www.clinicaltrials.gov as #NCT00000592 and #NCT00006182. PMID:19721013

  2. Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor

    PubMed Central

    Schreiner, Jens; Thommen, Daniela S.; Herzig, Petra; Bacac, Marina; Klein, Christian; Roller, Andreas; Belousov, Anton; Levitsky, Victor; Savic, Spasenija; Moersig, Wolfgang; Uhlenbrock, Franziska; Heinzelmann-Schwarz, Viola A.; Umana, Pablo; Pisa, Pavel; Lardinois, Didier; Müller, Philipp; Karanikas, Vaios; Zippelius, Alfred

    2016-01-01

    ABSTRACT T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1hi TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1hi TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1int and PD-1neg T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1hi expressing cells; in contrast, patients with abundance of PD-1hi expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1hi T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction. PMID:27057429

  3. CD8+ tumour-infiltrating lymphocytes in relation to HPV status and clinical outcome in patients with head and neck cancer after postoperative chemoradiotherapy: A multicentre study of the German cancer consortium radiation oncology group (DKTK-ROG).

    PubMed

    Balermpas, Panagiotis; Rödel, Franz; Rödel, Claus; Krause, Mechthild; Linge, Annett; Lohaus, Fabian; Baumann, Michael; Tinhofer, Inge; Budach, Volker; Gkika, Eleni; Stuschke, Martin; Avlar, Melanie; Grosu, Anca-Lidia; Abdollahi, Amir; Debus, Jürgen; Bayer, Christine; Stangl, Stefan; Belka, Claus; Pigorsch, Steffi; Multhoff, Gabriele; Combs, Stephanie E; Mönnich, David; Zips, Daniel; Fokas, Emmanouil

    2016-01-01

    We examined the prognostic value of tumour-infiltrating lymphocytes (TILs) in patients with squamous cell carcinoma of the head and neck (SCCHN) after surgery and postoperative cisplatin-based chemoradiotherapy. FFPE-tissue originating from the surgery of 161 patients treated in 8 DKTK partner sites was immunohistochemically stained for CD3 and CD8. Their expression was correlated with clinicopathological characteristics as well as overall survival (OS), local progression-free survival (LPFS) and distant metastases free-survival (DMFS), also in the context of the HPV16-DNA/p16 status. After a median follow-up of 48 months (range: 4100 months), OS at 4 years was 46.5% for the entire cohort. In multivariate analysis, high CD8 expression was confirmed as an independent prognostic parameter for OS (p = 0.002), LPFS (p = 0.004) and DMFS (p = 0.006), while CD3 expression lacked significance. In multivariate analysis HPV16 DNA positivity was associated with improved OS (p = 0.025) and LPFS (p = 0.013) and p16-positive patients showed improved DMFS (p = 0.008). Interestingly, high CD8 expression was a prognostic parameter for the clinical outcome in both HPV16 DNA-positive and HPV16 DNA-negative patients. Similar findings were observed in the multivariate analysis for the combined HPV16 DNA/p16 status. Altogether, CD8+ TILs constitute an independent prognostic marker in SCCHN patients treated with adjuvant chemoradiotherapy. These data indicate that CD8-positive TILs have antitumour activity and could be used for treatment stratification. Further validation of the prognostic value of CD8+ TILs as a biomarker and its role in the immune response in SCCHN patients after adjuvant chemoradiotherapy is warranted and will be performed in the prospective DKTK-ROG study. PMID:26178914

  4. The plant Apolipoprotein D ortholog protects Arabidopsis against oxidative stress

    PubMed Central

    Charron, Jean-Benoit F; Ouellet, Francois; Houde, Mario; Sarhan, Fathey

    2008-01-01

    Background Lipocalins are a large and diverse family of small, mostly extracellular proteins implicated in many important functions. This family has been studied in bacteria, invertebrate and vertebrate animals but little is known about these proteins in plants. We recently reported the identification and molecular characterization of the first true lipocalins from plants, including the Apolipoprotein D ortholog AtTIL identified in the plant model Arabidopsis thaliana. This study aimed to determine its physiological role in planta. Results Our results demonstrate that the AtTIL lipocalin is involved in modulating tolerance to oxidative stress. AtTIL knock-out plants are very sensitive to sudden drops in temperature and paraquat treatment, and dark-grown plants die shortly after transfer to light. These plants accumulate a high level of hydrogen peroxide and other ROS, which causes an oxidative stress that is associated with a reduction in hypocotyl growth and sensitivity to light. Complementation of the knock-out plants with the AtTIL cDNA restores the normal phenotype. On the other hand, overexpression enhances tolerance to stress caused by freezing, paraquat and light. Moreover, this overexpression delays flowering and maintains leaf greenness. Microarray analyses identified several differentially-regulated genes encoding components of oxidative stress and energy balance. Conclusion This study provides the first functional evidence that a plant lipocalin is involved in modulating tolerance to oxidative stress. These findings are in agreement with recently published data showing that overexpression of ApoD enhances tolerance to oxidative stress and increases life span in mice and Drosophila. Together, the three papers strongly support a similar function of lipocalins in these evolutionary-distant species. PMID:18671872

  5. HLA ligandomics identifies histone deacetylase 1 as target for ovarian cancer immunotherapy

    PubMed Central

    Peper, Janet Kerstin; Bösmüller, Hans-Christian; Schuster, Heiko; Gückel, Brigitte; Hörzer, Helen; Roehle, Kevin; Schäfer, Richard; Wagner, Philipp; Rammensee, Hans-Georg; Stevanović, Stefan; Fend, Falko; Staebler, Annette

    2016-01-01

    abstract The recent approval of clincially effective immune checkpoint inhibitors illustrates the potential of cancer immunotherapy. A challenging task remains the identification of specific targets guiding immunotherapy. Facilitated by technical advances, the direct identification of physiologically relevant targets is enabled by analyzing the HLA ligandome of cancer cells. Since recent publications demonstrate the immunogenicity of ovarian cancer (OvCa), immunotherapies, including peptide-based cancer vaccines, represent a promising treatment approach. To identify vaccine peptides, we employed a combined strategy of HLA ligandomics in high-grade serous OvCa samples and immunogenicity analysis. Only few proteins were naturally presented as HLA ligands on all samples analyzed, including histone deacetylase (HDAC) 1 and 2. In vitro priming of CD8+ T cells demonstrated that two HDAC1/2-derived HLA ligands can induce T-cell responses, capable of killing HLA-matched tumor cells. High HDAC1 expression shown by immunohistochemistry in 136 high-grade serous OvCa patients associated with significantly reduced overall survival (OS), whereas patients with high numbers of CD3+ tumor-infiltrating lymphocytes (TILs) in the tumor epithelium and CD8+ TILs in the tumor stroma showed improved OS. However, correlating HDAC1 expression with TILs, high levels of TILs abrogated the impact of HDAC1 on OS. This study strengthens the role of HDAC1/2 as an important tumor antigen in OvCa, demonstrating its impact on OS in a large cohort of OvCa patients. We further identified two immunogenic HDAC1-derived peptides, which frequently induce multi-functional T-cell responses in many donors, suitable for future multi-peptide vaccine trials in OvCa patients.

  6. Heterogeneous expansion of CD4+ tumor-infiltrating T-lymphocytes in clear cell renal cell carcinomas.

    PubMed

    Zhang, Qian; Jia, Qingzhu; Deng, Tianxing; Song, Bo; Li, Longkun

    2015-02-27

    Aberrant expression of tumor-associated antigens (TAAs) mediates the effective mounting of adaptive immunity in human solid tumors. The foundations of this tumor-host interaction strongly depend on specific recognition via TAA-cognate-receptors in T-cell repertoires. Previous studies focused on the phenotypic and functional properties of CD4+/CD8+ tumor-infiltrating T-lymphocytes (TILs), but the detailed composition of T-cell repertoires of these fundamental subsets remains largely unknown. This study recruited 10 clear cell renal cell carcinoma (ccRCC) patients and obtained samples from various tissues, including tumors, adjacent healthy renal tissue and peripheral blood. We utilized deep sequencing of T-cell receptor beta chains (TCRB), which serve as a unique identifier for each T clonotype, to characterize the CD4+/CD8+ TIL repertoire in ccRCC patients, assess the diversity and clonality of infiltrated T-cells in distinct tissues from patients and depict the clonal expansion events that occur in anti-tumor immune responses. We found that the CD4+ TIL repertoire exhibited signatures of heterogeneous T-cell expansion, which were characterized by divergent TRBV/J usage and an enrichment of expanded dominant clones. Taken together, our findings provide additional evidence of CD4+ T-cell-mediated anti-tumor immunity. The identification of the underlying molecular mechanisms of this process may provide novel avenues for targeted immunotherapeutic interventions. PMID:25637538

  7. Immune infiltrates are prognostic factors in localized gastrointestinal stromal tumors.

    PubMed

    Rusakiewicz, Sylvie; Semeraro, Michaela; Sarabi, Matthieu; Desbois, Mélanie; Locher, Clara; Mendez, Rosa; Vimond, Nadège; Concha, Angel; Garrido, Federico; Isambert, Nicolas; Chaigneau, Loic; Le Brun-Ly, Valérie; Dubreuil, Patrice; Cremer, Isabelle; Caignard, Anne; Poirier-Colame, Vichnou; Chaba, Kariman; Flament, Caroline; Halama, Niels; Jäger, Dirk; Eggermont, Alexander; Bonvalot, Sylvie; Commo, Frédéric; Terrier, Philippe; Opolon, Paule; Emile, Jean-François; Coindre, Jean-Michel; Kroemer, Guido; Chaput, Nathalie; Le Cesne, Axel; Blay, Jean-Yves; Zitvogel, Laurence

    2013-06-15

    Cancer immunosurveillance relies on effector/memory tumor-infiltrating CD8(+) T cells with a T-helper cell 1 (TH1) profile. Evidence for a natural killer (NK) cell-based control of human malignancies is still largely missing. The KIT tyrosine kinase inhibitor imatinib mesylate markedly prolongs the survival of patients with gastrointestinal stromal tumors (GIST) by direct effects on tumor cells as well as by indirect immunostimulatory effects on T and NK cells. Here, we investigated the prognostic value of tumor-infiltrating lymphocytes (TIL) expressing CD3, Foxp3, or NKp46 (NCR1) in a cohort of patients with localized GIST. We found that CD3(+) TIL were highly activated in GIST and were especially enriched in areas of the tumor that conserve class I MHC expression despite imatinib mesylate treatment. High densities of CD3(+) TIL predicted progression-free survival (PFS) in multivariate analyses. Moreover, GIST were infiltrated by a homogeneous subset of cytokine-secreting CD56(bright) (NCAM1) NK cells that accumulated in tumor foci after imatinib mesylate treatment. The density of the NK infiltrate independently predicted PFS and added prognostic information to the Miettinen score, as well as to the KIT mutational status. NK and T lymphocytes preferentially distributed to distinct areas of tumor sections and probably contributed independently to GIST immunosurveillance. These findings encourage the prospective validation of immune biomarkers for optimal risk stratification of patients with GIST. PMID:23592754

  8. Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma

    PubMed Central

    Giannakis, Marios; Mu, Xinmeng Jasmine; Shukla, Sachet A.; Qian, Zhi Rong; Cohen, Ofir; Nishihara, Reiko; Bahl, Samira; Cao, Yin; Amin-Mansour, Ali; Yamauchi, Mai; Sukawa, Yasutaka; Stewart, Chip; Rosenberg, Mara; Mima, Kosuke; Inamura, Kentaro; Nosho, Katsuhiko; Nowak, Jonathan A.; Lawrence, Michael S.; Giovannucci, Edward L.; Chan, Andrew T.; Ng, Kimmie; Meyerhardt, Jeffrey A.; Van Allen, Eliezer M.; Getz, Gad; Gabriel, Stacey B.; Lander, Eric S.; Wu, Catherine J.; Fuchs, Charles S.; Ogino, Shuji; Garraway, Levi A.

    2016-01-01

    Summary Large-scale genomic characterization of tumors from prospective cohort studies may yield new insights into cancer pathogenesis. We performed whole-exome sequencing of 619 incident colorectal cancers (CRCs) and integrated the results with tumor immunity, pathology, and survival data. We identified recurrently mutated genes in CRC, such as BCL9L, RBM10, CTCF, and KLF5, that were not previously appreciated in this disease. Furthermore, we investigated the genomic correlates of immune-cell infiltration and found that higher neoantigen load was positively associated with overall lymphocytic infiltration, tumor-infiltrating lymphocytes (TILs), memory T cells, and CRC-specific survival. The association with TILs was evident even within microsatellite-stable tumors. We also found positive selection of mutations in HLA genes and other components of the antigen-processing machinery in TIL-rich tumors. These results may inform immunotherapeutic approaches in CRC. More generally, this study demonstrates a framework for future integrative molecular epidemiology research in colorectal and other malignancies. PMID:27149842

  9. Functional Inactivation of EBV-Specific T-Lymphocytes in Nasopharyngeal Carcinoma: Implications for Tumor Immunotherapy

    PubMed Central

    Li, Jiang; Zeng, Xue-hui; Mo, Hao-yuan; Rolén, Ulrika; Gao, Yan-fang; Zhang, Xiao-shi; Chen, Qiu-yan; Zhang, Li; Zeng, Mu-sheng; Li, Man-zhi; Huang, Wen-lin; Wang, Xiao-ning; Zeng, Yi-Xin; Masucci, Maria G.

    2007-01-01

    Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated malignancy with high prevalence in Southern Chinese. In order to assess whether defects of EBV-specific immunity may contribute to the tumor, the phenotype and function of circulating T-cells and tumor infiltrating lymphocytes (TILs) were investigated in untreated NPC patients. Circulating naïve CD3+CD45RA+ and CD4+CD25− cells were decreased, while activated CD4+CD25+ T-cells and CD3−CD16+ NK-cells were increased in patients compared to healthy donors. The frequency of T-cells recognizing seven HLA-A2 restricted epitopes in LMP1 and LMP2 was lower in the patients and remained low after stimulation with autologous EBV-carrying cells. TILs expanded in low doses of IL-2 exhibited an increase of CD3+CD4+, CD3+CD45RO+ and CD4+CD25+ cells and 2 to 5 fold higher frequency of LMP1 and LMP2 tetramer positive cells compared to peripheral blood. EBV-specific cytotoxicity could be reactivated from the blood of most patients, whereas the TILs lacked cytotoxic activity and failed to produce IFNγ upon specific stimulation. Thus, EBV-specific rejection responses appear to be functionally inactivated at the tumor site in NPC. PMID:17987110

  10. Immune-mediated antitumor effect by type 2 diabetes drug, metformin

    PubMed Central

    Eikawa, Shingo; Nishida, Mikako; Mizukami, Shusaku; Yamazaki, Chihiro; Nakayama, Eiichi; Udono, Heiichiro

    2015-01-01

    Metformin, a prescribed drug for type 2 diabetes, has been reported to have anti-cancer effects; however, the underlying mechanism is poorly understood. Here we show that this mechanism may be immune-mediated. Metformin enabled normal but not T-cell–deficient SCID mice to reject solid tumors. In addition, it increased the number of CD8+ tumor-infiltrating lymphocytes (TILs) and protected them from apoptosis and exhaustion characterized by decreased production of IL-2, TNFα, and IFNγ. CD8+ TILs capable of producing multiple cytokines were mainly PD-1−Tim-3+, an effector memory subset responsible for tumor rejection. Combined use of metformin and cancer vaccine improved CD8+ TIL multifunctionality. The adoptive transfer of antigen-specific CD8+ T cells treated with metformin concentrations as low as 10 μM showed efficient migration into tumors while maintaining multifunctionality in a manner sensitive to the AMP-activated protein kinase (AMPK) inhibitor compound C. Therefore, a direct effect of metformin on CD8+ T cells is critical for protection against the inevitable functional exhaustion in the tumor microenvironment. PMID:25624476

  11. Diversity and divergence of the glioma-infiltrating T-cell receptor repertoire.

    PubMed

    Sims, Jennifer S; Grinshpun, Boris; Feng, Yaping; Ung, Timothy H; Neira, Justin A; Samanamud, Jorge L; Canoll, Peter; Shen, Yufeng; Sims, Peter A; Bruce, Jeffrey N

    2016-06-21

    Although immune signaling has emerged as a defining feature of the glioma microenvironment, how the underlying structure of the glioma-infiltrating T-cell population differs from that of the blood from which it originates has been difficult to measure directly in patients. High-throughput sequencing of T-cell receptor (TCR) repertoires (TCRseq) provides a population-wide statistical description of how T cells respond to disease. We have defined immunophenotypes of whole repertoires based on TCRseq of the α- and β-chains from glioma tissue, nonneoplastic brain tissue, and peripheral blood from patients. Using information theory, we partitioned the diversity of these TCR repertoires into that from the distribution of VJ cassette combinations and diversity due to VJ-independent factors, such as selection due to antigen binding. Tumor-infiltrating lymphocytes (TILs) possessed higher VJ-independent diversity than nonneoplastic tissue, stratifying patients according to tumor grade. We found that the VJ-independent components of tumor-associated repertoires diverge more from their corresponding peripheral repertoires than T-cell populations in nonneoplastic brain tissue, particularly for low-grade gliomas. Finally, we identified a "signature" set of TCRs whose use in peripheral blood is associated with patients exhibiting low TIL divergence and is depleted in patients with highly divergent TIL repertoires. This signature is detectable in peripheral blood, and therefore accessible noninvasively. We anticipate that these immunophenotypes will be foundational to monitoring and predicting response to antiglioma vaccines and immunotherapy. PMID:27261081

  12. Feature-based registration of historical aerial images by Area Minimization

    NASA Astrophysics Data System (ADS)

    Nagarajan, Sudhagar; Schenk, Toni

    2016-06-01

    The registration of historical images plays a significant role in assessing changes in land topography over time. By comparing historical aerial images with recent data, geometric changes that have taken place over the years can be quantified. However, the lack of ground control information and precise camera parameters has limited scientists' ability to reliably incorporate historical images into change detection studies. Other limitations include the methods of determining identical points between recent and historical images, which has proven to be a cumbersome task due to continuous land cover changes. Our research demonstrates a method of registering historical images using Time Invariant Line (TIL) features. TIL features are different representations of the same line features in multi-temporal data without explicit point-to-point or straight line-to-straight line correspondence. We successfully determined the exterior orientation of historical images by minimizing the area formed between corresponding TIL features in recent and historical images. We then tested the feasibility of the approach with synthetic and real data and analyzed the results. Based on our analysis, this method shows promise for long-term 3D change detection studies.

  13. Transient exposure to proteins SOX2, Oct-4, and NANOG immortalizes exhausted tumor-infiltrating CTLs.

    PubMed

    Bhadurihauck, Anjuli; Li, Lei; Li, Qianqian; Wang, Jianjun; Xiao, Zhengguo

    2016-05-13

    Adoptive cell transfer therapy (ACT) is one of the most promising immunotherapies against cancer, using tumor-infiltrating lymphocytes (TILs) expanded in vitro. Tumor-infiltrating cytotoxic T lymphocytes (TICTLs) play a prominent role in cancer control. TILs terminally differentiate in response to immunosuppressive environments within tumors, and thus are slow to expand and challenging to maintain both in vitro and in patients. To reverse this exhaustion, we utilize a nuclear protein delivery system that exposes TICTLs to the SOX2, Oct-4, and NANOG (SON) proteins. Unlike activated naïve CTLs (effector CTLs), TICTLs respond favorably to SON treatment, exhibiting steady proliferation and extended survivability independent of cytokine and antigen stimulation. Though TICTLs treated with SON (STICTLs) still express T cell receptors as well as other critical downstream components, they are unresponsive to antigen challenge, suggesting that SON treatment regresses TICTLs into a state similar to that of an early double negative T cell. Our findings indicate the TICTL response to SON proteins is unique when compared to effector CTLs, suggesting TICTLs may be sensitive to regulation by other lineage-specific transcription factors and opening a promising new avenue into cancer immunotherapy. To our knowledge, this is the first report on lineage reprogramming of TILs using protein stem cell transcription factors delivered directly to the nucleus. PMID:27084449

  14. Absence of Tumor-Infiltrating Lymphocyte Is a Reproducible Predictive Factor for Sentinel Lymph Node Metastasis: A Multicenter Database Study by the Brazilian Melanoma Group

    PubMed Central

    Duprat, João Pedreira; Brechtbülh, Eduard René; Costa de Sá, Bianca; Enokihara, Mauro; Fregnani, Jose Humberto; Landman, Gilles; Maia, Marcus; Riccardi, Felice; Belfort, Francisco Alberto; Wainstein, Alberto; Moredo, Luciana F.; Steck, Higino; Brandão, Miguel; Moreno, Marcelo; Miranda, Eduardo; Santos, Ivan Dunshee de Oliveira

    2016-01-01

    Aims The aim of this study is to confirm the function of tumor-infiltrating lymphocytes (TILs) in sentinel lymph node (SLN) metastasis. Materials and Methods This retrospective study included 633 patients with invasive melanoma who underwent sentinel lymph node biopsy in 7 referral centers certified by the Brazilian Melanoma Group. Independent risk factors of sentinel node metastasis (SNL) were identified by multiple logistic regression. Results SLN metastasis was detected in 101 of 633 cases (16.1%) and in 93 of 428 patients (21.7%) when melanomas ≤ 1mm were excluded. By multiple logistic regression, the absence of TILs was as an independent risk factor of SLN metastasis (OR = 1.8; 95%CI: 1.1–3.0), in addition to Breslow index (greater than 2.00 mm), lymph vascular invasion, and presence of mitosis. Conclusion SLNB can identify patients who might benefit from immunotherapy, and the determination of predictors of SLNB positivity can help select the proper population for this type of therapy. The absence of TILs is a reproducible parameter that can predict SLNB positivity in melanoma patients, since this study was made with several centers with different dermatopathologists. PMID:26859408

  15. EMAP-II-dependent lymphocyte killing is associated with hypoxia in colorectal cancer

    PubMed Central

    Youssef, M M S; Symonds, P; Ellis, I O; Murray, J C

    2006-01-01

    Endothelial-monocyte-activating polypeptide-II (EMAP-II) is a novel multifunctional polypeptide with proinflammatory activity. We have previously shown that the recombinant and native forms of EMAP-II can induce apoptosis in mitogen-stimulated lymphocytes, and that the release of this protein into the extracellular milieu is enhanced by hypoxia. We hypothesised that hypoxia may lead to death of tumour-infiltrating lymphocytes (TILs) via an EMAP-II-dependent mechanism, thereby assisting tumours to evade the immune system. In this study, we used immunohistochemistry to detect EMAP-II, active caspase-3 and cleaved Poly (ADP-ribose) Polymerase (PARP) as indicators of apoptosis in TILs, and carbonic anhydrase IX (CA IX) as a surrogate marker of hypoxia. EMAP-II expression is associated with regions of hypoxia, and furthermore there is a significant association between TILs apoptosis and the presence of hypoxia. Using a coculture model of colorectal cancer cell/lymphocyte interactions, we were also able to demonstrate lymphocyte apoptosis induced by tumour cells, with concomitant caspase-3 activity. Lymphocyte killing was enhanced by direct cell–cell contact, particularly by tumour cells exposed to hypoxic conditions. Our data support the hypothesis that hypoxia plays a role in immune evasion by tumour cells, through EMAP-II-dependent lymphocyte killing. PMID:16929248

  16. Posttraumatic responses to the July 22, 2011 Oslo Terror among Norwegian high school students.

    PubMed

    Nordanger, Dag Ø; Hysing, Mari; Posserud, Maj-Britt; Lundervold, Astri Johansen; Jakobsen, Reidar; Olff, Miranda; Stormark, Kjell Morten

    2013-12-01

    The July 22, 2011, Oslo Terror was defined as a national disaster. Former studies on terror attacks and mass shootings have shown elevated levels of posttraumatic complaints both in direct victims and in general populations. Little is known about how such extreme events in a generally safe society such as Norway would affect an adolescent population. This study examines posttraumatic stress reactions and changes in worldview in relationship to risk factors among 10,220 high school students using data from the ung@hordaland survey. One out of 5 respondents knew someone directly exposed, 55.7% experienced the events to some extent as threatening to their own or their close ones' lives, and 79.9% reported their worldview to be changed. For posttraumatic stress disorder (PTSD) DSM IV criteria, 0.8% reported substantial symptoms of reexperiencing (Criterion B), 4.9% of avoidance (Criterion C), and 1.1% of hyperarousal (Criterion D). Greater personal proximity to the events, higher levels of perceived life threat, and being a female or an immigrant predicted higher levels of PTSD symptom distress. Results indicate that the terror events made a deep impression on Norwegian adolescents, but without causing markedly elevated levels of PTSD symptomatology in the general young population. PMID:24243587

  17. Oxidative stress alters base excision repair pathway and increases apoptotic response in apurinic/apyrimidinic endonuclease 1/redox factor-1 haploinsufficient mice.

    PubMed

    Unnikrishnan, Archana; Raffoul, Julian J; Patel, Hiral V; Prychitko, Thomas M; Anyangwe, Njwen; Meira, Lisiane B; Friedberg, Errol C; Cabelof, Diane C; Heydari, Ahmad R

    2009-06-01

    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is the redox regulator of multiple stress-inducible transcription factors, such as NF-kappaB, and the major 5'-endonuclease in base excision repair (BER). We utilized mice containing a heterozygous gene-targeted deletion of APE1/Ref-1 (Apex(+/-)) to determine the impact of APE1/Ref-1 haploinsufficiency on the processing of oxidative DNA damage induced by 2-nitropropane (2-NP) in the liver tissue of mice. APE1/Ref-1 haploinsufficiency results in a significant decline in NF-kappaB DNA-binding activity in response to oxidative stress in liver. In addition, loss of APE1/Ref-1 increases the apoptotic response to oxidative stress, in which significant increases in GADD45g expression, p53 protein stability, and caspase activity are observed. Oxidative stress displays a differential impact on monofunctional (UNG) and bifunctional (OGG1) DNA glycosylase-initiated BER in the liver of Apex(+/-) mice. APE1/Ref-1 haploinsufficiency results in a significant decline in the repair of oxidized bases (e.g., 8-OHdG), whereas removal of uracil is increased in liver nuclear extracts of mice using an in vitro BER assay. Apex(+/-) mice exposed to 2-NP displayed a significant decline in 3'-OH-containing single-strand breaks and an increase in aldehydic lesions in their liver DNA, suggesting an accumulation of repair intermediates of failed bifunctional DNA glycosylase-initiated BER. PMID:19268524

  18. Effects of Short-Term Thermal Alteration on Organic Matter in Experimentally-Heated Tagish Lake Observed by Raman Spectroscopy

    NASA Technical Reports Server (NTRS)

    Chan, Q. H. S.; Nakato, A.; Zolensky, M. E.; Nakamura, T.; Kebukawa, Y.

    2007-01-01

    Carbonaceous chondrites exhibit a wide range of aqueous and thermal alteration characteristics. Examples of the thermally metamorphosed carbonaceous chondrites (TMCCs) include the C2-ung/CM2TIVs Belgica (B)-7904 and Yamato (Y) 86720. The alteration extent is the most complete in these meteorites and thus they are considered typical end-members of TMCCs exhibiting complete dehydration of matrix phyllosilicates [1, 2]. The estimated heating conditions are 10 to 10(sup 3) days at 700 C to 1 to 100 hours at 890 C, i.e. short-term heating induced by impact and/or solar radiation [3]. The chemical and bulk oxygen isotopic compositions of the matrix of the carbonate (CO3)-poor lithology of the Tagish Lake (hereafter Tag) meteorite bears similarities to these TMCCs [4]. We investigated the experimentally-heated Tag with the use of Raman spectroscopy to understand how short-term heating affects the maturity of insoluble organic matter (IOM) in aqueously altered meteorites.

  19. Strand-biased cytosine deamination at the replication fork causes cytosine to thymine mutations in Escherichia coli

    PubMed Central

    Bhagwat, Ashok S.; Hao, Weilong; Townes, Jesse P.; Lee, Heewook; Tang, Haixu; Foster, Patricia L.

    2016-01-01

    The rate of cytosine deamination is much higher in single-stranded DNA (ssDNA) than in double-stranded DNA, and copying the resulting uracils causes C to T mutations. To study this phenomenon, the catalytic domain of APOBEC3G (A3G-CTD), an ssDNA-specific cytosine deaminase, was expressed in an Escherichia coli strain defective in uracil repair (ung mutant), and the mutations that accumulated over thousands of generations were determined by whole-genome sequencing. C:G to T:A transitions dominated, with significantly more cytosines mutated to thymine in the lagging-strand template (LGST) than in the leading-strand template (LDST). This strand bias was present in both repair-defective and repair-proficient cells and was strongest and highly significant in cells expressing A3G-CTD. These results show that the LGST is accessible to cellular cytosine deaminating agents, explains the well-known GC skew in microbial genomes, and suggests the APOBEC3 family of mutators may target the LGST in the human genome. PMID:26839411

  20. Application of the Firefly and Luus-Jaakola algorithms in the calculation of a double reactive azeotrope

    NASA Astrophysics Data System (ADS)

    Mendes Platt, Gustavo; Pinheiro Domingos, Roberto; Oliveira de Andrade, Matheus

    2014-01-01

    The calculation of reactive azeotropes is an important task in the preliminary design and simulation of reactive distillation columns. Classically, homogeneous nonreactive azeotropes are vapor-liquid coexistence conditions where phase compositions are equal. For homogeneous reactive azeotropes, simultaneous phase and chemical equilibria occur concomitantly with equality of compositions (in the Ung-Doherty transformed space). The modeling of reactive azeotrope calculation is represented by a nonlinear algebraic system with phase equilibrium, chemical equilibrium and azeotropy equations. This nonlinear system can exhibit more than one solution, corresponding to a double reactive azeotrope. In a previous paper (Platt et al 2013 J. Phys.: Conf. Ser. 410 012020), we investigated some numerical aspects of the calculation of reactive azeotropes in the isobutene + methanol + methyl-tert-butyl-ether (with two reactive azeotropes) system using two metaheuristics: the Luus-Jaakola adaptive random search and the Firefly algorithm. Here, we use a hybrid structure (stochastic + deterministic) in order to produce accurate results for both azeotropes. After identifying the neighborhood of the reactive azeotrope, the nonlinear algebraic system is solved using Newton's method. The results indicate that using metaheuristics and some techniques devoted to the calculation of multiple minima allows both azeotropic coordinates in this reactive system to be obtains. In this sense, we provide a comprehensive analysis of a useful framework devoted to solving nonlinear systems, particularly in phase equilibrium problems.

  1. Calculation of a double reactive azeotrope using stochastic optimization approaches

    NASA Astrophysics Data System (ADS)

    Mendes Platt, Gustavo; Pinheiro Domingos, Roberto; Oliveira de Andrade, Matheus

    2013-02-01

    An homogeneous reactive azeotrope is a thermodynamic coexistence condition of two phases under chemical and phase equilibrium, where compositions of both phases (in the Ung-Doherty sense) are equal. This kind of nonlinear phenomenon arises from real world situations and has applications in chemical and petrochemical industries. The modeling of reactive azeotrope calculation is represented by a nonlinear algebraic system with phase equilibrium, chemical equilibrium and azeotropy equations. This nonlinear system can exhibit more than one solution, corresponding to a double reactive azeotrope. The robust calculation of reactive azeotropes can be conducted by several approaches, such as interval-Newton/generalized bisection algorithms and hybrid stochastic-deterministic frameworks. In this paper, we investigate the numerical aspects of the calculation of reactive azeotropes using two metaheuristics: the Luus-Jaakola adaptive random search and the Firefly algorithm. Moreover, we present results for a system (with industrial interest) with more than one azeotrope, the system isobutene/methanol/methyl-tert-butyl-ether (MTBE). We present convergence patterns for both algorithms, illustrating - in a bidimensional subdomain - the identification of reactive azeotropes. A strategy for calculation of multiple roots in nonlinear systems is also applied. The results indicate that both algorithms are suitable and robust when applied to reactive azeotrope calculations for this "challenging" nonlinear system.

  2. Crystal structures of two (±)-exo-N-isobornyl­acetamides

    PubMed Central

    Stepanovs, Dmitrijs; Posevins, Daniels; Turks, Maris

    2015-01-01

    The title compounds consist of a bornane skeleton with attached acetamide, C12H21NO (±)-(1) {systematic name: (±)-N-[(1RS,2RS,4RS)-1,7,7-tri­methylbi­cyclo­[2.2.1]heptan-2-yl]acetamide}, and chloro­acetamide, C12H20ClNO (±)-(2) {systematic name: (±)-2-chloro-N-[(1RS,2RS,4RS)-1,7,7-tri­methylbi­cyclo­[2.2.1]heptan-2-yl]­acetamide}, functionalities to the 2-exo-position. The crystal structure of the first monoclinic polymorph of (±)-(1) has been reported previously [Ung et al. (2014 ▸). Monatsh. Chem. 145, 983–992]. Compound (±)-(1) crystallizes in the space group P21/n with two independent mol­ecules in the asymmetric unit, in contrast to the above-mentioned polymorph which crystallized in the space group C2/c with one mol­ecule in the asymmetric unit. In the title compounds, the bicyclic bornane moieties have normal geometries. In the crystals of both compounds, mol­ecules are linked by N—H⋯O hydrogen bonds, reinforced by C—H⋯O contacts, forming trans-amide chains propagating along the a-axis direction. In the case of compound (±)-(1), neighbouring chains are linked by further C—H⋯O contacts, forming double-chain ribbons along [100]. PMID:26594386

  3. Genetic Variability in DNA Repair Proteins in Age-Related Macular Degeneration

    PubMed Central

    Blasiak, Janusz; Synowiec, Ewelina; Salminen, Antero; Kaarniranta, Kai

    2012-01-01

    The pathogenesis of age-related macular degeneration (AMD) is complex and involves interactions between environmental and genetic factors, with oxidative stress playing an important role inducing damage in biomolecules, including DNA. Therefore, genetic variability in the components of DNA repair systems may influence the ability of the cell to cope with oxidative stress and in this way contribute to the pathogenesis of AMD. However, few reports have been published on this subject so far. We demonstrated that the c.977C>G polymorphism (rs1052133) in the hOGG1 gene and the c.972G>C polymorphism (rs3219489) in the MUTYH gene, the products of which play important roles in the repair of oxidatively damaged DNA, might be associated with the risk of AMD. Oxidative stress may promote misincorporation of uracil into DNA, where it is targeted by several DNA glycosylases. We observed that the g.4235T>C (rs2337395) and c.–32A>G (rs3087404) polymorphisms in two genes encoding such glycosylases, UNG and SMUG1, respectively, could be associated with the occurrence of AMD. Polymorphisms in some other DNA repair genes, including XPD (ERCC2), XRCC1 and ERCC6 (CSB) have also been reported to be associated with AMD. These data confirm the importance of the cellular reaction to DNA damage, and this may be influenced by variability in DNA repair genes, in AMD pathogenesis. PMID:23202958

  4. Uracil excision repair in Mycobacterium tuberculosis cell-free extracts.

    PubMed

    Kumar, Pradeep; Bharti, Sanjay Kumar; Varshney, Umesh

    2011-05-01

    Uracil excision repair is ubiquitous in all domains of life and initiated by uracil DNA glycosylases (UDGs) which excise the promutagenic base, uracil, from DNA to leave behind an abasic site (AP-site). Repair of the resulting AP-sites requires an AP-endonuclease, a DNA polymerase, and a DNA ligase whose combined activities result in either short-patch or long-patch repair. Mycobacterium tuberculosis, the causative agent of tuberculosis, has an increased risk of accumulating uracils because of its G + C-rich genome, and its niche inside host macrophages where it is exposed to reactive nitrogen and oxygen species, two major causes of cytosine deamination (to uracil) in DNA. In vitro assays to study DNA repair in this important human pathogen are limited. To study uracil excision repair in mycobacteria, we have established assay conditions using cell-free extracts of M. tuberculosis and M. smegmatis (a fast-growing mycobacterium) and oligomer or plasmid DNA substrates. We show that in mycobacteria, uracil excision repair is completed primarily via long-patch repair. In addition, we show that M. tuberculosis UdgB, a newly characterized family 5 UDG, substitutes for the highly conserved family 1 UDG, Ung, thereby suggesting that UdgB might function as backup enzyme for uracil excision repair in mycobacteria. PMID:21371942

  5. Protective effects of Rabdosia japonica var. glaucocalyx extract on lipopolysaccharide-induced acute lung injury in mice.

    PubMed

    Xu, Nai-Yu; Chu, Chun-Jun; Xia, Long; Zhang, Jian; Chen, Dao-Feng

    2015-10-01

    The present study was designed to evaluate the protective effects of ethanol extracts of Rabdosia japonica var. glaucocalyx (Maxim.) Hara (RJ) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and the possible underlying mechanisms of action. The mice were orally administrated with RJ extract (16, 32 or 64 mg(kg(-1)) daily for consecutive7 days before LPS challenge. The ung specimens and the bronchoalveolar lavage fluid (BALF) were collected for histopathological examinations and biochemical analyses. Pretreatment with RJ significantly enhanced superoxide dismutase (SOD) activity and reduced the wet-to-dry weight (W/D) ratio, the levels of nitric oxide (NO) and protein leakage, and myeloperoxidase (MPO) activity in mice with ALI, in a dose-dependent manner. RJ reduced complement deposition and significantly attenuated LPS-induced ALI by reducing productions of inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). The results demonstrated that RJ may attenuate LPS-induced ALI via reducing the production of pro-inflammatory mediators, and reducing complement deposition and radicals. PMID:26481377

  6. DNA Damage Responses Are Induced by tRNA Anticodon Nucleases and Hygromycin B.

    PubMed

    Wemhoff, Sabrina; Klassen, Roland; Beetz, Anja; Meinhardt, Friedhelm

    2016-01-01

    Previous studies revealed DNA damage to occur during the toxic action of PaT, a fungal anticodon ribonuclease (ACNase) targeting the translation machinery via tRNA cleavage. Here, we demonstrate that other translational stressors induce DNA damage-like responses in yeast as well: not only zymocin, another ACNase from the dairy yeast Kluyveromyces lactis, but also translational antibiotics, most pronouncedly hygromycin B (HygB). Specifically, DNA repair mechanisms BER (base excision repair), HR (homologous recombination) and PRR (post replication repair) provided protection, whereas NHEJ (non-homologous end-joining) aggravated toxicity of all translational inhibitors. Analysis of specific BER mutants disclosed a strong HygB, zymocin and PaT protective effect of the endonucleases acting on apurinic sites. In cells defective in AP endonucleases, inactivation of the DNA glycosylase Ung1 increased tolerance to ACNases and HygB. In addition, Mag1 specifically contributes to the repair of DNA lesions caused by HygB. Consistent with DNA damage provoked by translation inhibitors, mutation frequencies were elevated upon exposure to both fungal ACNases and HygB. Since polymerase ζ contributed to toxicity in all instances, error-prone lesion-bypass probably accounts for the mutagenic effects. The finding that differently acting inhibitors of protein biosynthesis induce alike cellular responses in DNA repair mutants is novel and suggests the dependency of genome stability on translational fidelity. PMID:27472060

  7. DNA Damage Responses Are Induced by tRNA Anticodon Nucleases and Hygromycin B

    PubMed Central

    Beetz, Anja; Meinhardt, Friedhelm

    2016-01-01

    Previous studies revealed DNA damage to occur during the toxic action of PaT, a fungal anticodon ribonuclease (ACNase) targeting the translation machinery via tRNA cleavage. Here, we demonstrate that other translational stressors induce DNA damage-like responses in yeast as well: not only zymocin, another ACNase from the dairy yeast Kluyveromyces lactis, but also translational antibiotics, most pronouncedly hygromycin B (HygB). Specifically, DNA repair mechanisms BER (base excision repair), HR (homologous recombination) and PRR (post replication repair) provided protection, whereas NHEJ (non-homologous end-joining) aggravated toxicity of all translational inhibitors. Analysis of specific BER mutants disclosed a strong HygB, zymocin and PaT protective effect of the endonucleases acting on apurinic sites. In cells defective in AP endonucleases, inactivation of the DNA glycosylase Ung1 increased tolerance to ACNases and HygB. In addition, Mag1 specifically contributes to the repair of DNA lesions caused by HygB. Consistent with DNA damage provoked by translation inhibitors, mutation frequencies were elevated upon exposure to both fungal ACNases and HygB. Since polymerase ζ contributed to toxicity in all instances, error-prone lesion-bypass probably accounts for the mutagenic effects. The finding that differently acting inhibitors of protein biosynthesis induce alike cellular responses in DNA repair mutants is novel and suggests the dependency of genome stability on translational fidelity. PMID:27472060

  8. Expression of programmed death-1 ligand (PD-L1) in tumor-infiltrating lymphocytes is associated with favorable spinal chordoma prognosis

    PubMed Central

    Zou, Ming-Xiang; Peng, An-Bo; Lv, Guo-Hua; Wang, Xiao-Bin; Li, Jing; She, Xiao-Ling; Jiang, Yi

    2016-01-01

    Aberrant expression of programmed death-1 (PD-1) receptor/PD-1 ligand (PD-L1) proteins alters human immunoresponse and promotes tumor development and progression. We assessed the expression status of PD-1 and PD-L1 in spinal chordoma tissue specimens and their association with clinicopathological characteristics of patients. Formalin-fixed paraffin-embedded tumor samples from 54 patients with spinal chordoma were collected for immunohistochemical analysis of PD-1 and PD-L1 expression. The association of the expression levels of PD-1 and PD-L1 with clinicopathological variables and survival data were statistically analyzed. Lymphocyte infiltrates were present in all 54 patient samples. Of 54 samples, 37 (68.5%) had both positive PD-1 and PD-L1 expression in tumor cell membrane. Moreover, 38 (70.4%) and 12 (22.2%) had positive PD-1 and PD-L1 expression in tumor-infiltrating lymphocytes (TILs), respectively. Tumors with positive PD-L1 expression were significantly associated with advanced stages of chordoma (p = 0.041) and TIL infiltration (p = 0.005), and had a borderline association with tumor grade (p = 0.051). However, positive tumor PD-L1 expression was not significantly associated with local recurrence-free survival (LRFS) or overall survival (OS). PD-1 expression in TILs was associated with poor LRFS (χ2 = 10.051, p = 0.002, log-rank test). Multivariate analysis showed that PD-L1 expression only in TILs was an independent predictor for LRFS (HR = 0.298, 95% CI: 0.098-0.907, p = 0.033), and OS (HR = 0.188, 95% CI: 0.051-0.687, p = 0.011) in spinal chordoma patients. In conclusion, PD-L1 expression in TILs was an independent predictor for both LRFS and OS in spinal chordoma patients. Our findings suggest that the PD-1/PD-L1 pathway may be a novel therapeutic target for the immunotherapy of chordoma. PMID:27508049

  9. Th17-type cytokines, IL-6 and TNF-α synergistically activate STAT3 and NF-kB to promote colorectal cancer cell growth

    PubMed Central

    De Simone, V; Franzè, E; Ronchetti, G; Colantoni, A; Fantini, M C; Di Fusco, D; Sica, G S; Sileri, P; MacDonald, T T; Pallone, F; Monteleone, G; Stolfi, C

    2015-01-01

    Colorectal cancers (CRCs) often show a dense infiltrate of cytokine-producing immune/inflammatory cells. The exact contribution of each immune cell subset and cytokine in the activation of the intracellular pathways sustaining CRC cell growth is not understood. Herein, we isolate tumor-infiltrating leukocytes (TILs) and lamina propria mononuclear cells (LPMCs) from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of patients who underwent resection for sporadic CRC and show that the culture supernatants of TILs, but not of LPMCs, potently enhance the growth of human CRC cell lines through the activation of the oncogenic transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-kB). Characterization of immune cell complexity of TILs and LPMCs reveals no differences in the percentages of T cells, natural killer T cells, natural killer (NK) cells, macrophages and B cells. However, T cells from TILs show a functional switch compared with those from LPMCs to produce large amounts of T helper type 17 (Th17)-related cytokines (that is, interleukin-17A (IL-17A), IL-17F, IL-21 and IL-22), tumor necrosis factor-α (TNF-α) and IL-6. Individual neutralization of IL-17A, IL-17F, IL-21, IL-22, TNF-α or IL-6 does not change TIL-derived supernatant-driven STAT3 and NF-kB activation, as well as their proproliferative effect in CRC cells. In contrast, simultaneous neutralization of both IL-17A and TNF-α, which abrogates NF-kB signaling, and IL-22 and IL-6, which abrogates STAT3 signaling, reduces the mitogenic effect of supernatants in CRC cells. IL-17A, IL-21, IL-22, TNF-α and IL-6 are also produced in excess in the early colonic lesions in a mouse model of sporadic CRC, associated with enhanced STAT3/NF-kB activation. Mice therapeutically given BP-1-102, an orally bioavailable compound targeting STAT3/NF-kB activation and cross-talk, exhibit reduced colon tumorigenesis and diminished expression of

  10. Th17-type cytokines, IL-6 and TNF-α synergistically activate STAT3 and NF-kB to promote colorectal cancer cell growth.

    PubMed

    De Simone, V; Franzè, E; Ronchetti, G; Colantoni, A; Fantini, M C; Di Fusco, D; Sica, G S; Sileri, P; MacDonald, T T; Pallone, F; Monteleone, G; Stolfi, C

    2015-07-01

    Colorectal cancers (CRCs) often show a dense infiltrate of cytokine-producing immune/inflammatory cells. The exact contribution of each immune cell subset and cytokine in the activation of the intracellular pathways sustaining CRC cell growth is not understood. Herein, we isolate tumor-infiltrating leukocytes (TILs) and lamina propria mononuclear cells (LPMCs) from the tumor area and the macroscopically unaffected, adjacent, colonic mucosa of patients who underwent resection for sporadic CRC and show that the culture supernatants of TILs, but not of LPMCs, potently enhance the growth of human CRC cell lines through the activation of the oncogenic transcription factors signal transducer and activator of transcription 3 (STAT3) and nuclear factor-kappa B (NF-kB). Characterization of immune cell complexity of TILs and LPMCs reveals no differences in the percentages of T cells, natural killer T cells, natural killer (NK) cells, macrophages and B cells. However, T cells from TILs show a functional switch compared with those from LPMCs to produce large amounts of T helper type 17 (Th17)-related cytokines (that is, interleukin-17A (IL-17A), IL-17F, IL-21 and IL-22), tumor necrosis factor-α (TNF-α) and IL-6. Individual neutralization of IL-17A, IL-17F, IL-21, IL-22, TNF-α or IL-6 does not change TIL-derived supernatant-driven STAT3 and NF-kB activation, as well as their proproliferative effect in CRC cells. In contrast, simultaneous neutralization of both IL-17A and TNF-α, which abrogates NF-kB signaling, and IL-22 and IL-6, which abrogates STAT3 signaling, reduces the mitogenic effect of supernatants in CRC cells. IL-17A, IL-21, IL-22, TNF-α and IL-6 are also produced in excess in the early colonic lesions in a mouse model of sporadic CRC, associated with enhanced STAT3/NF-kB activation. Mice therapeutically given BP-1-102, an orally bioavailable compound targeting STAT3/NF-kB activation and cross-talk, exhibit reduced colon tumorigenesis and diminished expression of