Ramasamy Jagadeeswaran

University of Illinois at Chicago, Chicago, Illinois, United States

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Publications (6)23.38 Total impact

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    ABSTRACT: Background: Fetal swallowing of amniotic fluid, which contains several cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid might provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. Methods: We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (rHGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC-6 cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Results: Amniotic fluid supplementation of oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by rHGF and blocked by anti-HGF antibody or a PI3K inhibitor. rHGF transactivated several RTKs in IEC-6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, rHGF also reduced the frequency and severity of NEC-like injury in rat pups. Conclusions: Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF.
    AJP Gastrointestinal and Liver Physiology 01/2014; 306(5). DOI:10.1152/ajpgi.00272.2013 · 3.74 Impact Factor
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    ABSTRACT: Neonates and young infants exposed to extracorporeal circulation during extracorporeal membrane oxygenation (ECMO) and cardiopulmonary bypass are at risk of developing a systemic inflammatory response syndrome with multi-organ dysfunction. We used a piglet model of ECMO to investigate the hypothesis that epithelial apoptosis is an early event that precedes villous damage during ECMO-related bowel injury. Healthy 3-week-old piglets were subjected to ECMO for up to 8 h. Epithelial apoptosis was measured in histopathological analysis, nuclear imaging, and terminal deoxynucleotidyl transferase dUTP nick end labeling. Plasma intestinal fatty acid-binding protein (I-FABP) levels were measured by enzyme immunoassay. Intestinal mast cells were isolated by fluorescence-assisted cell sorting. Cleaved caspase-8, caspase-9, phospho-p38 MAPK, and fas ligand expression were investigated by immunohistochemistry, western blots, and reverse transcriptase-quantitative PCR. Piglet ECMO was associated with increased gut epithelial apoptosis. Extensive apoptotic changes were noted on villus tips and in scattered crypt cells after 2 h of ECMO. After 8 h, the villi were denuded and apoptotic changes were evident in a majority of crypt cells. Increased circulating I-FABP levels, a marker of gut epithelial injury, showed that epithelial injury occurred during ECMO. We detected increased cleaved caspase-8, but not cleaved caspase-9, in epithelial cells indicating that the extrinsic apoptotic pathway was active. ECMO was associated with increased fas ligand expression in intestinal mast cells, which was induced through activation of the p38 mitogen-activated protein kinase. We conclude that epithelial apoptosis is an early event that initiates gut mucosal injury in a piglet model of ECMO.Laboratory Investigation advance online publication, 23 December 2013; doi:10.1038/labinvest.2013.149.
    Laboratory Investigation 12/2013; DOI:10.1038/labinvest.2013.149 · 3.83 Impact Factor
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    ABSTRACT: Background: Human milk contains substantial amounts of TGF-β, particularly the isoform TGF-β2. We have previously shown in preclinical models that enterally-administered TGF-β2 can protect against necrotizing enterocolitis (NEC), an inflammatory bowel necrosis of premature infants. In this study, we hypothesized that premature infants remain at higher risk of NEC than term infants even when they receive their own mother's milk because preterm human milk contains less bioactive TGF-β than term milk. Objective: Compare TGF-β bioactivity in preterm vs. term milk and identify factors that activate milk-borne TGF-β. Design/Methods: Mothers who delivered between 23 0/7 and 31 6/7 weeks or at ≥37 weeks of gestation provided milk samples at serial time points. TGF-β bioactivity and NF-κB signaling was measured using specific reporter cells and in murine intestinal tissue explants. TGF-β1, TGF-β2, and TGF-β3 and various TGF-β activators were measured by real-time PCR, enzyme immunoassays, or established enzymatic activity assays. Results: Preterm human milk showed minimal TGF-β bioactivity in the native state but contained a large pool of latent TGF-β. TGF-β2 was the predominant isoform of TGF-β in preterm milk. Using a combination of several in vitro and ex vivo models, we show that neuraminidase is a key regulator of TGF-β bioactivity in human milk. Finally, we show that addition of bacterial neuraminidase to preterm human milk increased TGF-β bioactivity. Conclusions: Preterm milk contains large quantities of TGF-β but most of it is in an inactive state. Addition of neuraminidase can increase TGF-β bioactivity in preterm milk and enhance its anti-inflammatory effects.
    AJP Gastrointestinal and Liver Physiology 04/2013; 304(12). DOI:10.1152/ajpgi.00039.2013 · 3.74 Impact Factor
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    ABSTRACT: BACKGROUND & AIMS: Preterm infants may be at risk of necrotizing enterocolitis (NEC) due to deficiency of transforming growth factor-beta 2 (TGF-β(2)) in the developing intestine. We hypothesized that low epithelial TGF-β(2) expression in preterm intestine and during NEC results from diminished autocrine induction of TGF-β(2) in these cells. METHODS: Premature baboons delivered at 67% gestation were treated per current norms for human preterm infants. NEC was diagnosed by clinical and radiological findings. Inflammatory cytokines, TGF-β(2), Smad7, Ski, and SnoN/SKI-like (SKIL) was measured using quantitative reverse transcriptase-polymerase chain reaction, immunoblots, and immunohistochemistry. Smad7 effects were examined in transfected IEC6 intestinal epithelial cells in vitro. Findings were validated in archived human tissue samples of NEC. RESULTS: NEC was recorded in 7 premature baboons. Consistent with existing human data, premature baboon intestine expressed less TGF-β(2) than term intestine. TGF-β(2) expression was regulated in epithelial cells in an autocrine fashion, which was interrupted in the premature intestine and during NEC due to increased expression of Smad7. LPS increased Smad7 binding to the TGF-β(2) promoter and was associated with dimethylation of the lysine H3K9, a marker of transcriptional silencing, on the nucleosome of TGF-β(2). Increased Smad7 expression in preterm intestine was correlated with the deficiency of SnoN/SKIL, a repressor of the Smad7 promoter. CONCLUSIONS: Smad7 inhibits autocrine expression of TGF-β(2) in intestinal epithelial cells in the normal premature intestine and during NEC. Increased Smad7 expression in the developing intestine may be due to a developmental deficiency of the SnoN/SKIL oncoprotein.
    AJP Gastrointestinal and Liver Physiology 11/2012; 304(2). DOI:10.1152/ajpgi.00141.2012 · 3.74 Impact Factor
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    ABSTRACT: Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. In tissue samples of NEC, we identified numerous macrophages and a few neutrophils but not many lymphocytes. We hypothesized that these pathoanatomic characteristics of NEC represent a common tissue injury response of the gastrointestinal tract to a variety of insults at a specific stage of gut development. To evaluate developmental changes in mucosal inflammatory response, we used trinitrobenzene sulfonic acid (TNBS)-induced inflammation as a nonspecific insult and compared mucosal injury in newborn vs. adult mice. Enterocolitis was induced in 10-day-old pups and adult mice (n = 25 animals per group) by administering TNBS by gavage and enema. Leukocyte populations were enumerated in human NEC and in murine TNBS-enterocolitis using quantitative immunofluorescence. Chemokine expression was measured using quantitative polymerase chain reaction, immunoblots, and immunohistochemistry. Macrophage recruitment was investigated ex vivo using intestinal tissue-conditioned media and bone marrow-derived macrophages in a microchemotaxis assay. Similar to human NEC, TNBS enterocolitis in pups was marked by a macrophage-rich leukocyte infiltrate in affected tissue. In contrast, TNBS-enterocolitis in adult mice was associated with pleomorphic leukocyte infiltrates. Macrophage precursors were recruited to murine neonatal gastrointestinal tract by the chemokine CXCL5, a known chemoattractant for myeloid cells. We also demonstrated increased expression of CXCL5 in surgically resected tissue samples of human NEC, indicating that a similar pathway was active in NEC. We concluded that gut mucosal injury in the murine neonate is marked by a macrophage-rich leukocyte infiltrate, which contrasts with the pleomorphic leukocyte infiltrates in adult mice. In murine neonatal enterocolitis, macrophages were recruited to the inflamed gut mucosa by the chemokine CXCL5, indicating that CXCL5 and its cognate receptor CXCR2 merit further investigation as potential therapeutic targets in NEC.
    AJP Gastrointestinal and Liver Physiology 04/2012; 303(1):G93-102. DOI:10.1152/ajpgi.00016.2012 · 3.74 Impact Factor
  • Uppoor G Bhat, Ramasamy Jagadeeswaran, Marianna Halasi, Andrei L Gartel
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    ABSTRACT: Using mass spectrometric analysis we found that oncogenic transcription factor FOXM1 that is overexpressed in a majority of human cancers interacts with multifunctional protein NPM, which is also overexpressed in a variety of human tumors. Coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrated that NPM forms a complex with FOXM1 and also identified the regions responsible for their interaction. Immunofluorescence microscopy confirmed the interaction between FOXM1 and NPM in cancer and immortal cells. Furthermore, knockdown of NPM in immortal and cancer cells led to significant down-regulation of FOXM1 similar to its levels in normal cells, suggesting that NPM might modulate FOXM1 level. In addition, in OCI/AML3 leukemia cells where mutant NPM is localized in the cytoplasm we found that typically nuclear FOXM1 was predominantly co-localized with NPM in the cytoplasm, while NPM knockdown led to the disappearance of FOXM1 from the cytoplasm, suggesting that NPM may also determine intracellular localization of FOXM1. Knockdown of FOXM1 or NPM in MIA PaCa-2 pancreatic cancer cells inhibited anchorage-dependent and independent growth in cell culture, and tumor growth in nude mice. In addition, over-expression of FOXM1 reversed the effect of NPM knockdown in vitro. Our data suggest that in cancer cells NPM interacts with FOXM1 and their interaction is required for sustaining the level and localization of FOXM1. Targeting the interaction between FOXM1 and NPM by peptides or small molecules may represent a novel therapeutic strategy against cancer.
    Journal of Biological Chemistry 12/2011; 286(48):41425-33. DOI:10.1074/jbc.M111.270843 · 4.60 Impact Factor