Doris Wiener

Correlation between UDP-Glucuronosyltransferase Genotypes and 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone Glucuronidation Phenotype in Human Liver Microsomes

The nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is one of the most potent and abundant procarcinogens found in tobacco and tobacco smoke, and glucuronidation of its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), is an important mechanism for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone detoxification. Substantial interindividual variability in urinary NNAL glucuronide formation has been observed in smokers and tobacco chewers. To determine whether genetic variations may play a role in this interindividual variability, NNAL-glucuronidating activities were analyzed in 78 human liver microsomal specimens and compared with the prevalence of missense polymorphisms in the two major NNAL-glucuronidating enzymes UGT1A4 and UGT2B7. In vitro assays using liver microsomal specimens from individual subjects demonstrated a 70- and 50-fold variability in NNAL-N-Gluc and NNAL-O-Gluc formation, respectively, and a 20-fold variability in the ratio of NNAL-N-Gluc:NNAL-O-Gluc formation. Microsomes from subjects with a homozygous polymorphic UGT1A424Thr/UGT1A424Thr genotype exhibited a significantly higher (P < 0.05) level of NNAL-N-Gluc activity compared with microsomes from subjects with the wild-type UGT1A424Pro/UGT1A424Pro genotype, and a significantly higher (P < 0.05) number of subjects with liver microsomes having high NNAL-N-Gluc formation activity contained the UGT1A424Thr/UGT1A424Thr genotype. Microsomes from subjects with the homozygous polymorphic UGT2B7268Tyr/UGT2B7268Tyr genotype exhibited a significantly lower level (P < 0.025) of NNAL-O-Gluc activity when compared with microsomes from subjects with the wild-type UGT2B7268His/UGT2B7268His genotype, and a significantly (P < 0.05) higher number of subjects with liver microsomes having low NNAL-O-Gluc formation activity contained the UGT2B7268Tyr/UGT2B7268Tyr genotype. These data suggest that the UGT1A4 codon 24 and UGT2B7 codon 268 polymorphisms may be associated with altered rates glucuronidation and detoxification of NNAL in vivo.

The Effects of Maternal Dietary Docosahexaenoic Acid Intake on Rat Pup Myelin and the Auditory Startle Response

We investigated the effects of maternal docosahexanoic acid (DHA) supplementation on pups’ auditory startle responses and the composition of brain myelin. Methods: Timed-pregnant rats were fed throughout pregnancy and lactation diets that contained 0, 0.3, 0.7 or 3% of total fatty acids as DHA. Milk was collected from culled pups’ stomachs on postnatal day (PND) 3, latency of the auditory startle reflex was measured on PND 15, and pups were killed and brains collected on PND 24. Results: Higher levels of DHA in maternal diet were reflected in milk and in pups’ myelin. The latency of the auditory startle response was significantly longer in offspring of dams fed higher levels of DHA. There was a positive correlation between the myelin content of DHA and the latency of the startle response (p = 0.044), and a negative correlation between the myelin content of DHA and the myelin content of cholesterol (p = 0.005). Conclusion: High levels of maternal DHA intake alter the lipid composition of rat pup myelin, and are associated with longer latencies of the auditory startle response – a myelin-dependent electrophysiologic response.

The fatty acid composition of maternal diet affects the response to excitotoxic neural injury in neonatal rat pups.

Fatty acids and their derivatives play a role in the response to neural injury. The effects of prenatal and postnatal dietary fatty acid composition on excitotoxic neural injury were investigated in neonatal rat pups. Dams were fed during gestation and lactation a diet whose fat source was either corn oil or menhaden fish oil. On postnatal day 3, litters were culled to 10 per dam. On postnatal day 4, excitotoxic neural injury was induced by infusion of the glutamate analog N-methyl-DL-aspartate (NMA) into the left cerebral hemisphere. Three days later, pups were killed and brains were removed for histological and volume assessments. Levels of arachidonic acid were 2.3-fold higher in cerebrums of pups in the corn oil group than in the fish oil group. Left cerebral hemispheres among all pups were atrophic. Right cerebral hemispheres of pups in the corn oil group showed more histological evidence of edema, and had significantly higher volumes than pups in the fish oil group (66 vs. 42 mm2, p=0.007). These data suggest that the fatty acid composition of prenatal and/or postnatal diet can affect the neonatal response to excitotoxic neural injury.

Transcutaneous Absorption of Vitamin A in Newborn Rats

Very low birth weight neonates have low tissue concentrations of vitamin A, which may contribute to the development of lung disease. These infants, however, may not receive vitamin A supplementation for several days after birth. We determined if the relatively permeable skin of a newborn could be used to administer vitamin A. 25 control rat pups were killed and lungs and livers were collected. 20 μl (1,000 IU) of retinyl palmitate were applied to the skin surface of an additional 50 two-day-old pups. At 2.5 and 5 h after application, 25 pups were killed, and lungs and livers were collected. Concentrations of retinyl palmitate and retinol were significantly higher in the lungs of pups 5 h after administration of vitamin A compared with controls. There were no differences in concentrations of retinyl palmitate or retinol in livers. We conclude that transcutaneous administration may be an effective means of delivering vitamin A to the lungs of newborn rats.

DIETARY FATTY ACID (FA) EFFECTS UPON THYMOCYTE SUBSET DISTRIBUTION. † 1817

Alterations in dietary FAs are reported to affect immunity via effects upon cytokine gene expression, eicosanoid synthesis and membrane physicochemical properties. The effects of maternal diet FA composition upon thymocyte subset distribution were studied in neonatal rat pups. Methods: Timed pregnant dams were fed, beginning on day 2 of gestation and throughout lactation, either chow (control) or a purified diet whose fat source (22% of cals) was corn oil (high n6) or menhaden fish oil (high n3). On day 3 of life, pups were culled to 10 per dam and were randomly cross-fostered among dams fed the same diets to minimize litter effects. Culled pup thymuses were removed for FA analysis. On day 7 of life, pups were sacrificed and thymuses removed for flow cytometric analysis of thymocyte subsets; milk samples were collected for FA analysis. Results: Length of gestation and pup weights did not differ among diet groups. The FA composition of maternal milk and pup thymuses reflected maternal diet, with a significant enrichment of n6 and n3 FAs in the corn and fish groups, respectively (FA data shown for pup thymus only). The percent of double positive immature thymocytes (CD4+CD8+) was significantly lower in the fish versus corn group, while the percent of CD4+CD8-, CD3brtCD4brt and CD3brtCD8brt thymocytes was significantly higher in the fish versus corn and chow groups.Conclusion: The FA composition of pre- and/or postnatal diet affects the FA composition of neonatal thymus, and may influence thymocyte differentiation. (Funded in part by Ross Products Division)Table

Oncodriver inhibition and CD4 + Th1 cytokines cooperate through Stat1 activation to induce tumor senescence and apoptosis in HER2+ and triple negative breast cancer: implications for combining immune and targeted therapies

In patients with HER2-expressing breast cancer many develop resistance to HER2 targeted therapies. We show that high and intermediate HER2-expressing cancer cell lines are driven toward apoptosis and tumor senescence when treated with either CD4+ Th1 cells, or Th1 cytokines TNF-α and IFN-γ, in a dose dependent manner. Depletion of HER2 activity by either siRNA or trastuzumab and pertuzumab, and subsequent treatment with either anti-HER2 Th1 cells or TNF-α and IFN-γ resulted in synergistic increased tumor senescence and apoptosis in cells both sensitive and cells resistant to trastuzumab which was inhibited by neutralizing anti-TNF-α and IFN-γ. Th1 cytokines induced minimal senescence or apoptosis in triple negative breast cancer cells (TNBC); however, inhibition of EGFR in combination with Th1 cytokines sensitized those cells causing both senescence and apoptosis. TNF-α and IFN-γ led to increased Stat1 phosphorylation through serine and tyrosine sites and a compensatory reduction in Stat3 activation. Single agent IFN-γ enhanced Stat1 phosphorylation on tyrosine 701 and similar effects were observed in combination with TNF-α and EGFR inhibition. These results demonstrate Th1 cytokines and anti-oncodriver blockade cooperate in causing tumor senescence and apoptosis in TNBC and HER2-expressing breast cancer, suggesting these combinations could be explored as non-cross-reactive therapy preventing recurrence in breast cancer.

Abstract 2292: Reconstitution of myeloid derived suppressor cells after the induction of lymphopenia and TIL therapy

Factors such as T regulatory (Treg) cells and myeloid derived suppressor cells (MDSC) limit the effectiveness of current immunotherapy protocols. Several reports have shown that administration of chemotherapeutic agents or total body irradiation (TBI) before adoptive transfer of tumor-specific T cells reduces or eliminates the immunosuppressive populations such as Tregs and MDSC. A previous study in our lab has shown that MDSC that reconstitute after lymphodepletion are highly suppressive compared to the endogenous MDSC in a B16 melanoma model. Blockade of MDSC expansion in combination with adoptive T cell transfer delayed tumor growth and improved survival rate. However, there is very little known about the MDSC reconstitution after the adoptive transfer of tumor infiltrating lymphocytes (TIL) in melanoma patients. Melanoma patients were treated with non- myeloablative therapy that included cyclophosphamide and fludarabine. At the time of TIL transfer, no circulating lymphoid or myeloid cells were detected in the PBMC. One day after completion of chemotherapy, up to 100 billion TIL were transferred followed by high dose bolus IL-2. At weeks 1-4 after TIL transfer, PBMC were collected and we investigated the reconstitution and the suppressive function of MDSC. MDSC were phenotyped by flow cytometry as HLA-DR − Lin − CD14 + CD33 + or CD11b + CD14 − CD33 + . While 3-15% of MDSC was observed in the PBMC of melanoma patients as their baseline, after lymphodepletion and one week post TIL infusion, the percent of MDSC increased to 10-30% and gradually dropped to baseline by week 4. To examine suppressive function, MDSC were isolated from peripheral blood at 1-2 weeks after TIL infusion and co-cultured with healthy donor T cells or patient TIL. MDSC were highly suppressive and abrogated T cell proliferation and IFN-gamma secretion. Furthermore, in patients that responded to TIL therapy, the ratio of peripheral MDSC to total CD8+ T cells was lower. These studies demonstrate that MDSC that reconstitute post- lymphodepletion are suppressive and may potentially interfere with robust T cell function. Further studies are warranted to clarify the role of MDSC in the efficacy of TIL therapy. Blocking MDSC in combination with TIL therapies represent an improved strategy for the treatment of melanoma patients. Citation Format: Krithika N. Kodumudi, Doris Wiener, Amy Weber, Erica Royster, Linda Kelley, Amod Sarnaik, Shari Pilon-Thomas. Reconstitution of myeloid derived suppressor cells after the induction of lymphopenia and TIL therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2292.

THE EFFECT OF MATERNAL VITAMIN A SUPPLEMENTATION UPON PRETERM INFANT CORD BLOOD VITAMIN A LEVELS † 1361

THE EFFECT OF MATERNAL VITAMIN A SUPPLEMENTATION UPON PRETERM INFANT CORD BLOOD VITAMIN A LEVELS † 1361

DIETARY FATTY ACID (FA) EFFECTS UPON GROUP B STREPTOCOCCAL (GBS) INFECTION. † 1893

Dietary FA effects upon the immune response may be mediated in part by effects upon proinflammatory cytokines and eicosanoids. The impact of maternal diet FA composition upon mortality from GAS infection was studied in neonatal rat pups. Methods: Timed pregnant dams were fed, beginning on day 2 of gest. and throughout lactation, either chow (control) or a purified diet whose fat source (22% of cals) was either corn oil (high n6) or menhaden fish oil (high n3). On day 3 of life, pups were culled to 10 pups per dam and were randomly cross-fostered among dams fed the same diets to minimize litter effects. Milk was removed from culled pup stomachs for FA analysis. Exp 1: On day 7 of life, pups were injected i.p. with 0.1 ml of a suspension of GAS Type I as follows: one litter per diet with 106.5 organisms, and one litter per diet with 107.5 organisms. Data from both doses were combined for statistical analyses. Exp 2: On day 7 of life, pups (n=1 litter per diet group) were injected with 107 GAS organisms and sacrificed 48 hours later. Blood was collected for GAS culture and analysis of serum TNFα levels; spleen weights were determined. Results: FA composition of milk reflected maternal diet (Table). Exp 1: 100% of pups in the chow and corn oil groups died within 96 hours, while 33% survived in the fish oil group (p=0.002 fishers exact (permutation) test). Exp 2: Spleen weights (as% body weight) were significantly higher in the fish vs corn and chow groups. Preliminary cytokine data suggest lower serum levels of TNFα and a trend towards fewer positive blood culture in pups of the fish group. Conclusion: The FA composition pre- and/or postnatal diet may affect immune response to bacterial sepsis. Funded in part by Ross Laboratories.