Sepsis, a life‐threatening clinical condition affecting more than 1.5 million Americans per year, is defined as an over‐exuberant immune response to infection. Currently, sepsis is the leading cause of death in U.S. hospitals, and the incidence of sepsis caused by Gram‐negative bacteria, such as Escherichia coli (E. coli), has been steadily increasing since the late 1990's. While the detailed mechanism of sepsis is not fully understood, several bacterial components are thought to contribute to the hyper‐inflammatory response in humans. Past studies suggest that one E. coli lipoprotein, Peptidoglycan‐Associated Lipoprotein (Pal), may contribute to inflammation and the pathogenesis of sepsis. This work describes our efforts to elucidate the role of Pal in E. coli sepsis and the effect of antibiotics in Pal release from E. coli using a mouse model of sepsis. Our preliminary results corroborate the hypothesis that Pal releases from E. coli and contributes to inflammation and sepsis.Support or Funding InformationRochester Institute of TechnologyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Purpose: Epidemiological data links lack of breastfeeding with increased risk of breast cancer, particularly triple negative breast cancer (TNBC). The involution process is characterized by mammary tissue remodeling, including adipocyte repopulation and re-differentiation fueled by metabolic rewiring. Our mouse model mimicking short-term breast feeding that leads to abrupt involution (AI) revealed a chronic inflammatory state in the mammary gland (MG) (Basree, et al. Breast Cancer Research; 2019). As metabolic dysfunction is linked to increased BC risk, we sought to elucidate the effects of AI and gradual involution (GI) on MG energy metabolism and associated oxidative stress. In addition, we evaluated the impact of AI and GI on whole body glucose metabolism and insulin response/resistance. Methods: FVB/n mice (8week old) were paired for breeding. At partum, dams were randomized to AI or GI cohort and standardized to 6 pups per dam. AI mice had pups removed on postpartum day 7 (d7) to mimic short-term breastfeeding. GI mice had 3 pups removed on day 28 and 31 each to mimic gradual weaning. MGs were harvested on d28, 56, and 120 postpartum. Prior to harvest, mice underwent an echoMRI and insulin tolerance test (ITT). At harvest, mice were fasted for 4 hours, and blood was collected for serum insulin measurement using Ultra-Sensitive Insulin ELISA. Lipid peroxidation (MDA) and DNA adduct formation (8-OHdG), which represent chronic oxidative stress, were measured in MG by ELISA. Whole MG RNA were subjected to affymetrix followed by gene set enrichment analyses (GSEA) and qPCR for target validation. MGs were also subjected to untargeted metabolomics analysis and Mitofuel flex assay to assess substrate dependence by Seahorse Bioanalyzer. Results: GSEA and qPCR revealed enrichment of fatty acid oxidation (FAO) and mitochondrial oxidative phosphorylation (OXPHOS) pathways in AI MGs at d28, further validated by expression of genes (PGC-1α, Cpt-2, Srebp1c, and Chrebp). Metabolites associated with FAO were enriched in AI MGs at d28 and 56. Mitofuel flex assay indicated a significantly higher dependence on FAO in AI MGs. On d56, fasted blood glucose was significantly higher in AI mice with serum insulin and HOMA-IR trending to be higher than in GI mice. Level of 8-OHdG was elevated in AI MGs at d120. AI mice trended to be heavier, have greater body fat, and lower lean mass than GI Mice at d120. Body weight and percent body fat on d120 were positively correlated to MDA concentration in the mammary gland. Conclusion: Our mouse models of AI and GI revealed early MG specific metabolic shift towards increased FAO and OXPHOS that persists over time in AI glands. Similarly, increased oxidative stress and its association with adiposity at d120 indicates continued effect of AI. This change in adiposity, altered systemic glucose metabolism and metabolic shift seen in AI mice may contribute to the higher risk of breast cancer. [K.O. and K.K. are co-first authors.] Citation Format: Kate Ormiston, Kirti Kaul, Neelam Shinde, Allen Zhang, Morgan Bauer, Hee Kyung Kim, Ramesh K. Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Lack of breast feeding may contribute to increased breast cancer risk by altering metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5814.
The clinical condition of sepsis often results from a systemic bacterial infection, followed by an over‐exuberant immune response, which leads to widespread inflammation. Severe sepsis can result in organ failure or death. Past studies have proposed a role for bacterial Peptidoglycan‐Associated Lipoprotein (Pal) in the pathogenesis of Gram‐negative sepsis. In this study, we confirmed the ability of Escherichia coli (E. coli) to release Pal under certain conditions, and we employed both in vitro and in vivo (mouse) studies and protein detection methods to determine the effect of antibiotics on Pal's release from E. coli. Results from our studies suggest that antibiotics that target peptidoglycan may enhance Pal's release more so than other antibiotics.Support or Funding InformationRochester Institute of TechnologyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Objective: Epidemiological studies indicate a direct relation between length of breast feeding and protection against risk of triple negative breast cancer (TNBC), an aggressive subtype. While prolonged breastfeeding allows gradual involution (GI) of the breast, short-term or no breast feeding leads to abrupt involution (AI). We modelled AI and GI of breast in mice, and showed that mice subjected to GI have better protection from tissue remodeling associated injuries in the mammary gland. Our data revealed late development of ductal hyperplasia aided by pro-tumorigenic microenvironment in the AI glands1. Detailed mechanism of mammary gland involution immediately following cessation of lactation has been studied in the past, but only in the AI setting, that is after abrupt removal of pups at the peak of lactation2,3. The goal of this study is to conduct stepwise comparison of the mechanism of GI vs. AI early on to understand the protective effect of GI against breast cancer risk.Methods: Wild-type FVB mice were used in all our studies. Females were mated at 8 weeks of age and uniparous mice were allowed to nurse (6 pups/dam) for 7 days. Females were then assigned randomly to AI or GI cohort. All pups were removed from the AI dams on postnatal day7 (PND7). Three pups each were removed from GI dams on day28 and 31. Mammary glands were harvested intermittently between PND7 and PND35. H&E stained sections were used for histological studies. Unstained FFPE sections were used for immunohistochemistry and TUNEL assay. Total RNA and protein from whole mammary gland was used for qPCR and western blot respectively. Results: GI glands transitioned from fully active lactating to near involuted glands over a period of 8 days (PND17- PND25), while for AI glands it took < 4 days (PND8.5-PND12). The shrinkage and flattening of tall epithelia and loss of acini was gradual in GI glands as opposed to rapid breakdown of acini and adipocyte repopulation in AI gland. Apoptotic cell count peaked on PND11 (5%) in AI vs. PND25 (3%) in GI glands. The pStat3Y705+ cells were highest on PND8.5 (25%) in AI vs. on PND25 (11%) in GI glands. Macrophage infiltration (F4/80+) peaked on PND11 (35%) and remained elevated at ~24% till PND25, while in GI glands increase was gradual from PND17 through PND25 (27%). Expression of key genes identified in AI mice2,3 have markedly different expression pattern in GI mice (Table). While some peaked at a later time point in GI vs. AI coinciding with maximum cell death, expression of some are significantly low or undetectable in GI glands at both RNA and protein level. Conclusions: We show for the first time that kinetics of cell death, adipocyte repopulation, immune cell infiltration and inflammatory state of glands undergoing abrupt vs. gradual involution are markedly different. Several genes known to play a key role during AI are either not expressed or barely detectable in the GI glands at any time point during involution. These data suggests that not only the kinetics, but mechanism of GI and AI are not identical. We conclude that orchestrated cell death in GI protects from drastic lysosomal, and immune cell activities that predisposes mammary glands to higher risk of neoplastic changes.Significance: Epidemiological data highlights the benefits of prolonged breastfeeding in protecting against breast cancer, particularly, TNBC, an aggressive subtype prevalent in the African American women. Our study highlights the mechanism underlying the benefits of gradual involution of breast. GeneFold Change compared to PND7 (GI vs. AI) Peaked on (GI vs. AI)Stat34.0 vs. 5.9PND25 vs. PND8.5Ctsb2.8 vs. 3.4PND25 vs. PND8.5CD143.4 vs. 20PND25 vs. PND8.5Orm11.7 vs. 10PND25 vs. PND12Lrg130 vs. 216PND25 vs. PND12MMP215.8 vs. 27PND25 vs. PND12Chi3L11350 vs. 572, 859PND28 vs. PND11, 28Cebpδnone vs. 4.4None vs. PND8.5CtsLnone vs. 5.7None vs. PND8.5Orm2none vs. 370None vs. PND12Slpinone vs. 92None vs. PND8.5 Citation Format: Bhuvaneswari Ramaswamy, Neelam Shinde, Morgan Bauer, Maria Cuitino, Saba Mehra, Resham Mawalkar, Mustafa Basree, Allen Zhang, Kirti Kaul, Xiaoli Zhang, Ramesh Ganju, Sarmila Majumder. Mechanistic differences between abrupt and gradual involution of mouse mammary gland [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-08.
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