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ABSTRACT: B cells perform various immunological functions that include production of Abs, presentation of Ags, secretion of multiple cytokines, and regulation of immune responses mainly via their secretion of IL-10. While the liver is regarded both as an important immune organ and a tolerogenic environment, little is known about the functional biology of hepatic B cells. In this study, we demonstrate that, following LPS stimulation in vivo, normal mouse hepatic B cells rapidly increase their surface expression of CD39, CD40, CD80, and CD86, and produce significantly elevated levels of proinflammatory IFN-γ, IL-6, and TNFα compared with splenic B cells. Moreover, LPS-activated hepatic B cells produce very low levels of IL-10 compared with activated splenic B cells that produce comparatively high levels of this immunosuppressive cytokine. Splenic, but not hepatic B cells inhibited the activation of liver conventional myeloid dendritic cells (mDCs). Furthermore, compared with the spleen, the liver exhibited significantly smaller proportions of B1a and marginal zone-like B cells, which have been shown to produce IL-10 upon LPS stimulation. These data suggest that, unlike in the spleen, IL-10-producing regulatory B cells in the liver are not a prominent cell type. Consistent with this, when compared with liver conventional mDCs from B cell-deficient mice, those from B cell-competent wild-type mice displayed enhanced expression of the cell surface costimulatory molecule CD86, greater production of proinflammatory cytokines (IFN-γ, IL-6, IL-12p40), and reduced secretion of IL-10. These findings suggest that hepatic B cells have the potential to initiate rather than regulate inflammatory responses.
Clinical & Experimental Immunology 04/2013; · 3.36 Impact Factor
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Matthew Zhang,
Shinya Ueki,
Shoko Kimura,
Osamu Yoshida,
Antonino Castellaneta,
Kikumi S Ozaki,
Anthony J Demetris,
Mark Ross,
Yoram Vodovotz,
Angus W Thomson, Donna Beer Stolz,
David A Geller,
Noriko Murase
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ABSTRACT: Dendritic cells (DC) induce and regulate both innate and adaptive immune responses; however, theirin vivo functional importance in hepatic ischemia/reperfusion (IR) injury is perplexing. We hypothesized that liver-resident DC and locally recruited blood-borne DC might have distinctive roles in hepatic IR injury. We tested this hypothesis by utilizing DC-deficient, fms-like tyrosine kinase 3 ligand (Flt3L) KO mice in hepatic warm (70% partial clamping for 60 min) and cold IR injury (liver transplant [LTx] with 24 hr cold storage). Flt3L KO liver and lymphoid organs contained virtually no CD11c(+) F4/80(-) DC. Hepatic warm IR injury was significantly lower in Flt3L KO than in WT mice with lower ALT levels, reduced hepatic necrosis, and lower neutrophil infiltration. Hepatic mRNA and protein levels for inflammatory cytokines (TNFα, IL-6) and chemokines (CCL2, CXCL2) also were significantly lower in Flt3L KO than in WT mice, indicating that lack of both liver-resident and blood-borne DC ameliorated hepatic warm IR injury. Adoptive transfer of splenic or hepatic WT DC into Flt3L KO or WT mice increased hepatic warm IR injury, suggesting injurious roles of DC infusion. When Flt3LKO liver was transplanted into WT mice, ALT levels were significantly higher than in WT to WT LTx, with enhanced hepatic necrosis and neutrophil infiltration, indicating a protective role of liver-resident DC. Conclusions: Using both warm and cold hepatic IR models, this study suggests differential roles of liver-resident versus blood-borne DC, and points to the importance of the local microenvironment in determining DC function during hepatic IR injury. (HEPATOLOGY 2012.).
Hepatology 11/2012; · 11.66 Impact Factor
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ABSTRACT: Autocrine and paracrine cell communication can be conveyed by multiple mediators, including membrane-associate proteins, secreted proteins and exosomes. Exosomes are 30-100 nm endosome-derived vesicles consisting in cytosolic material surrounded by a lipid bilayer containing transmembrane proteins. We have previously shown that dendritic cells (DCs) express on their surface multiple TNF superfamily ligands (TNFSFLs), by which they can induce the apoptotic demise of tumor cells as well as the activation of natural killer (NK) cells. In the present study, we demonstrate that, similar to DCs, DC-derived exosomes (DCex) express on their surface TNF, FasL and TRAIL, by which they can trigger caspase activation and apoptosis in tumor cells. We also show that DCex activate NK cells and stimulate them to secrete interferonγ (IFNγ) upon the interaction of DCex TNF with NK-cell TNF receptors. These data demonstrate that DCex can mediate essential innate immune functions that were previously ascribed to DCs.
Oncoimmunology. 10/2012; 1(7):1074-1083.
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Yugo Tanaka,
Norihisa Shigemura,
Tomohiro Kawamura,
Kentaro Noda,
Kumiko Isse, Donna Beer Stolz,
Timothy R Billiar,
Yoshiya Toyoda,
Christian A Bermudez,
James Lyons-Weiler,
Atsunori Nakao
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ABSTRACT: We previously demonstrated that donor treatment with inhaled hydrogen protects lung grafts from cold ischemia/reperfusion (I/R) injury during lung transplantation. To elucidate the mechanisms underlying hydrogen's protective effects, we conducted a gene array analysis to identify changes in gene expression associated with hydrogen treatment.
Donor rats were exposed to mechanical ventilation with 98% oxygen and 2% nitrogen or 2% hydrogen for 3h before harvest; lung grafts were stored for 4h in cold Perfadex. Affymetrix gene array analysis of mRNA transcripts was performed on the lung tissue prior to implantation.
Pretreatment of donor lungs with hydrogen altered the expression of 229 genes represented on the array (182 upregulated; 47 downregulated). Hydrogen treatment induced several lung surfactant-related genes, ATP synthase genes and stress-response genes. The intracellular surfactant pool, tissue adenosine triphosphate (ATP) levels and heat shock protein 70 (HSP70) expression increased in the hydrogen-treated grafts. Hydrogen treatment also induced the transcription factors C/EBPα and C/EBPβ, which are known regulators of surfactant-related genes.
Donor ventilation with hydrogen significantly increases expression of surfactant-related molecules, ATP synthases and stress-response molecules in lung grafts. The induction of these molecules may underlie hydrogen's protective effects against I/R injury during transplantation.
Biochemical and Biophysical Research Communications 08/2012; 425(4):873-9. · 2.48 Impact Factor
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ABSTRACT: Sterile inflammatory insults, such as ischemia-reperfusion (I/R) injury, result from pathogenic factors, including damage-associated molecular pattern signaling, activation of innate immunity, and upregulation of proinflammatory cytokines. At the same time, a number of protective, or prosurvival, pathways are also activated, and the extent of end-organ damage is ultimately determined by the balance between these two systems. In liver I/R, members of the calcium/calmodulin-dependent protein kinase (CaMK) family are known to be activated, but their individual roles are largely unknown. In this study, we show that one CaMK member, CaMKIV, is protective in hepatic I/R by activating the prosurvival pathway of autophagy in hepatocytes. CaMKIV knockout mice experience significantly worse organ damage after I/R and are deficient in hepatocyte autophagic signaling. Restoration of autophagic signaling with rapamycin reduces organ damage in CaMKIV knockout mice to wild-type levels. In vitro, we show that CaMKIV activation induces autophagy in mouse hepatocytes, and that CaMKIV activation protects hepatocytes from oxidative stress-induced cell death. In conclusion, the protective autophagic signaling pathway serves to reduce organ damage following I/R and is regulated by activation of CaMKIV signaling in hepatocytes.
AJP Gastrointestinal and Liver Physiology 05/2012; 303(2):G189-98. · 3.43 Impact Factor
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Xiaoyan Liang,
Michael E De Vera,
William J Buchser,
Antonio Romo de Vivar Chavez,
Patricia Loughran, Donna Beer Stolz,
Per Basse,
Tao Wang,
Bennett Van Houten,
Herbert J Zeh,
Michael T Lotze
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ABSTRACT: Administration of high-dose interleukin-2 (HDIL-2) has durable antitumor effects in 5% to 10% of patients with melanoma and renal cell carcinoma. However, treatment is often limited by side effects, including reversible, multiorgan dysfunction characterized by a cytokine-induced systemic autophagic syndrome. Here, we hypothesized that the autophagy inhibitor chloroquine would enhance IL-2 immunotherapeutic efficacy and limit toxicity. In an advanced murine metastatic liver tumor model, IL-2 inhibited tumor growth in a dose-dependent fashion. These antitumor effects were significantly enhanced upon addition of chloroquine. The combination of IL-2 with chloroquine increased long-term survival, decreased toxicity associated with vascular leakage, and enhanced immune cell proliferation and infiltration in the liver and spleen. HDIL-2 alone increased serum levels of HMGB1, IFN-γ, IL-6, and IL-18 and also induced autophagy within the liver and translocation of HMGB1 from the nucleus to the cytosol in hepatocytes, effects that were inhibited by combined administration with chloroquine. In tumor cells, chloroquine increased autophagic vacuoles and LC3-II levels inhibited oxidative phosphorylation and ATP production and promoted apoptosis, which was associated with increased Annexin-V(+)/propidium iodide (PI)(-) cells, cleaved PARP, cleaved caspase-3, and cytochrome c release from mitochondria. Taken together, our findings provide a novel clinical strategy to enhance the efficacy of HDIL-2 immunotherapy for patients with cancer.
Cancer Research 04/2012; 72(11):2791-801. · 7.86 Impact Factor
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Angela Montecalvo,
Adriana T Larregina,
William J Shufesky, Donna Beer Stolz,
Mara L G Sullivan,
Jenny M Karlsson,
Catherine J Baty,
Gregory A Gibson,
Geza Erdos,
Zhiliang Wang,
Jadranka Milosevic,
Olga A Tkacheva,
Sherrie J Divito,
Rick Jordan,
James Lyons-Weiler,
Simon C Watkins,
Adrian E Morelli
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ABSTRACT: Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.
Blood 01/2012; 119(3):756-66. · 9.90 Impact Factor
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Peiyuan Li,
Qiang Du,
Zongxian Cao,
Zhong Guo,
John Evankovich,
Wei Yan,
Ying Chang,
Lifang Shao, Donna Beer Stolz,
Allan Tsung,
David A Geller
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ABSTRACT: Interferon-gamma (IFN-γ) is a pleiotropic cytokine with immunomodulatory, anti-viral, and anti-proliferative effects. In this study, we examined the effects of IFN-γ on autophagy and cell growth in human hepatocellular carcinoma (HCC) cells. IFN-γ inhibited cell growth of Huh7 cells with non-apoptotic cell death. IFN-γ induced autophagosome formation and conversion/turnover of microtubule associated protein 1 light chain 3 (LC3) protein. Furthermore, overexpression of IRF-1 also induced autophagy in Huh7 cells. Silencing IRF-1 expression with target small hairpin RNA blocked autophagy induced by IFN-γ. Silencing of the autophagy signals Beclin-1 or Atg5 attenuated the inhibitory effect of IFN-γ on Huh7 cells with decreased cell death. Additionally, IFN-γ activated autophagy in freshly cultured human HCC cells. Together, these findings show that IFN-γ induces autophagy through IRF-1 signaling pathway and the induction of autophagy contributes to the growth-inhibitory effect of IFN-γ with cell death in human liver cancer cells.
Cancer letters 10/2011; 314(2):213-22. · 4.86 Impact Factor
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Siobhán Q Gregg,
Verónica Gutiérrez,
Andria Rasile Robinson,
Tyler Woodell,
Atsunori Nakao,
Mark A Ross,
George K Michalopoulos,
Lora Rigatti,
Carrie E Rothermel,
Irene Kamileri,
George A Garinis, Donna Beer Stolz,
Laura J Niedernhofer
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ABSTRACT: The liver changes with age, leading to an impaired ability to respond to hepatic insults and increased incidence of liver disease in the elderly. Therefore, there is critical need for rapid model systems to study aging-related liver changes. One potential opportunity is murine models of human progerias or diseases of accelerated aging. Ercc1(-/Δ) mice model a rare human progeroid syndrome caused by inherited defects in DNA repair. To determine whether hepatic changes that occur with normal aging occur prematurely in Ercc1(-/Δ) mice, we systematically compared liver from 5-month-old progeroid Ercc1(-/Δ) mice to old (24-36-month-old) wild-type (WT) mice. Both displayed areas of necrosis, foci of hepatocellular degeneration, and acute inflammation. Loss of hepatic architecture, fibrosis, steatosis, pseudocapillarization, and anisokaryosis were more dramatic in Ercc1(-/Δ) mice than in old WT mice. Liver enzymes were significantly elevated in serum of Ercc1(-/Δ) mice and old WT mice, whereas albumin was reduced, demonstrating liver damage and dysfunction. The regenerative capacity of Ercc1(-/Δ) liver after partial hepatectomy was significantly reduced. There was evidence of increased oxidative damage in Ercc1(-/Δ) and old WT liver, including lipofuscin, lipid hydroperoxides and acrolein, as well as increased hepatocellular senescence. There was a highly significant correlation in genome-wide transcriptional changes between old WT and 16-week-old, but not 5-week-old, Ercc1(-/Δ) mice, emphasizing that the Ercc1(-/Δ) mice acquire an aging profile in early adulthood. CONCLUSION: There are strong functional, regulatory, and histopathological parallels between accelerated aging driven by a DNA repair defect and normal aging. This supports a role for DNA damage in driving aging and validates a murine model for rapidly testing hypotheses about causes and treatment for aging-related hepatic changes.
Hepatology 09/2011; 55(2):609-21. · 11.66 Impact Factor
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Heth R Turnquist,
Zhenlin Zhao,
Brian R Rosborough,
Quan Liu,
Antonino Castellaneta,
Kumiko Isse,
Zhiliang Wang,
Megan Lang, Donna Beer Stolz,
Xin Xiao Zheng,
A Jake Demetris,
Foo Y Liew,
Kathryn J Wood,
Angus W Thomson
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ABSTRACT: IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.
The Journal of Immunology 09/2011; 187(9):4598-610. · 5.79 Impact Factor
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ABSTRACT: Tumor necrosis factor receptor 1 (TNFR1) activation in hepatocytes can trigger apoptotic or inflammatory signaling. The factors that determine which signaling pathway dominates are not clear and are thought to relate to the efficiency of death-inducing signaling complex (DISC) formation. However, the steps involved in DISC formation in hepatocytes are poorly understood. In characterizing DISC formation within cultured hepatocytes, we demonstrated that TNF-α exposure leads to the rapid formation of a DISC involving TNF-α, the TNFR-associated death domain adaptor molecule (TRADD), the Fas-associated death domain adaptor molecule (FADD), caspase-8, TNFR-associated factor 2 (TRAF2), and receptor-interacting protein (RIP). The inclusion of the sensitizing agent actinomycin D both accelerated and amplified the appearance of the DISC. Notably, TNFR1 along with some DISC components also appeared within mitochondria within 30 minutes. Whereas TNFR1 consistently co-localized with the TRADD, FADD, the caspase-8, and TRAF2 in the cytosolic fraction, TNFR1 in the mitochondria was associated only with caspase-8 after TNF-α exposure. Similar observations were made in vivo using TNF-α with D-galactosamine. Actinomycin D alone also enhanced the appearance of DISC components in both cytosol and the mitochondria. Thus the DISC that includes TNFR1 forms in the cytosol of hepatocytes under both survival and pro-apoptotic conditions. The observations also suggest that TNF-α-mediated signaling includes the translocation of TNFR1 to mitochondria.
American Journal Of Pathology 09/2011; 179(3):1221-9. · 4.89 Impact Factor
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ABSTRACT: Ischemia/reperfusion (I/R) injury in liver grafts, which is initiated by cold preservation and is augmented by reperfusion, is a major problem that complicates graft quality, posttransplant patient care, and outcomes of liver transplantation (LT). Kupffer cells (KCs) play important roles in I/R injury; however, little is known about their changes during cold preservation. We examined whether a pretreatment with carbon monoxide (CO), a cytoprotective product of heme degradation, could influence KC activity during cold storage and protect liver grafts against LT-induced I/R injury. In vitro, primary rat KCs were stimulated for 24 hours under hypothermic conditions (4°C, 20% O(2)), with lipopolysaccharide, or under hypoxic conditions (37°C, 5% O(2)) with or without a CO pretreatment. When rat KCs were exposed to hypothermic conditions, they produced reactive oxygen species (ROS), but they did not produce tumor necrosis factor α (TNF-α) or nitric oxide. The preincubation of KCs with CO up-regulated heat shock protein 70 (HSP70) and inhibited ROS generation. When liver grafts from donor rats exposed to CO (250 ppm) for 24 hours were transplanted after 18 hours of cold preservation in University of Wisconsin solution, HSP70 expression increased in these grafts versus control grafts, and serum aspartate aminotransferase and alanine aminotransferase levels as well as necrotic areas and inflammatory infiltrates were significantly reduced after LT. CO-pretreated liver grafts showed less up-regulation of TNF-α and inducible nitric oxide synthase messenger RNA (mRNA) and reduced expression of proapoptotic B cell lymphoma 2-associated X protein mRNA, cleaved caspase-3, and poly(adenosine diphosphate ribose) polymerase. In conclusion, the pretreatment of donors with CO ameliorates LT-associated I/R injury with increased hepatic HSP70 expression, particularly in the KC population.
Liver Transplantation 08/2011; 17(12):1457-66. · 3.39 Impact Factor
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ABSTRACT: The identification of resident stem cells in the mouse gallbladder is, to date, unexplored. In addition, the relationship between adult gallbladder stem cells and intrahepatic bile duct (IHBD) cells is not well understood. The aim of this study was to isolate stem cells from an adult mouse gallbladder and determine whether they were unique, compared to IHBD cells. By limiting dilution analyses and index sorts, we found that an EpCAM(+) CD49f(hi) epithelial cell subpopulation from primary gallbladder is enriched in colony-forming cells, compared to EpCAM(+) CD49f(lo) cells. EpCAM(+) CD49f(hi) cells expressed cluster of differentiation (CD)29, CD133, and stem cell antigen-1, but were negative for lineage markers CD31, CD45, and F4/80. Using a novel feeder cell-culture system, we observed long-term (>passage 20) and clonal expansion of the EpCAM(+) CD49f(hi) cells in vitro. In a matrigel differentiation assay, EpCAM(+) CD49f(+) cells expanding in vitro underwent organotypic morphogenesis forming ductular structures and cysts. These structures are similar to, and recapitulate a transport function of, primary gallbladder. EpCAM(+) CD49f(+) cells also engraft into the subcutaneous space of recipient mice. We compared primary gallbladder and IHBD cells by flow cytometry and found phenotypic differences in the expression of CD49f, CD49e, CD81, CD26, CD54, and CD166. In addition, oligonucleotide microarrays showed that the expanded EpCAM(+) CD49f(+) gallbladder cells and IHBD cells exhibit differences related to lipid and drug metabolism. Notable genes that were different are cytochrome P450, glutathione S-transferase, Indian hedgehog, and solute carrier family genes. CONCLUSION: We have isolated an epithelial cell population from primary mouse gallbladder with stem cell characteristics and found it to be unique, compared to IHBD cells.
Hepatology 07/2011; 54(5):1830-41. · 11.66 Impact Factor
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ABSTRACT: In the steady state, hepatic antigen (Ag)-presenting cells (APC) generally dampen systemic inflammatory responses to gut-derived Ags. Our studies focus on the role of specific liver APC populations, both non-parenchymal cells (dendritic cells [DC], Kupffer cells, and hepatic stellate cells [HSC]) and parenchymal cells, in the molecular regulation of tissue damage (ischemia and reperfusion [I/R] injury) and immunity following liver transplantation. We focus on factors that either promote or overwhelm the natural tendency of the liver to suppress inflammatory/immune responses. We are also examining molecular mechanisms that regulate liver DC maturation and function and that determine their role in the control of allogeneic T-cell function and the fate of the transplanted liver. Our studies are also aimed at elucidating mechanisms by which HSC regulate DC and T-cell function. These investigations may provide new targets for therapeutic intervention in liver inflammation.
Immunologic Research 06/2011; 50(2-3):221-7. · 3.03 Impact Factor
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ABSTRACT: Peripheral neuropathy is a common aging-related degenerative disorder that interferes with daily activities and leads to increased risk of falls and injury in the elderly. The etiology of most aging-related peripheral neuropathy is unknown. Inherited defects in several genome maintenance mechanisms cause tissue-specific accelerated aging, including neurodegeneration. We tested the hypothesis that a murine model of XFE progeroid syndrome, caused by reduced expression of ERCC1-XPF DNA repair endonuclease, develops peripheral neuropathy. Nerve conduction studies revealed normal nerve function in young adult (8 week) Ercc1(-/Δ) mice, but significant abnormalities in 20 week-old animals. Morphologic and ultrastructural analysis of the sciatic nerve from mutant mice revealed significant alterations at 20 but not 8 weeks of age. We conclude that Ercc1(-/Δ) mice have accelerated spontaneous peripheral neurodegeneration that mimics aging-related disease. This provides strong evidence that DNA damage can drive peripheral neuropathy and offers a rapid and novel model to test therapies.
Mechanisms of ageing and development 05/2011; 132(8-9):437-42. · 4.18 Impact Factor
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ABSTRACT: Dendritic cells (DC) are "professional" bone marrow-derived antigen (Ag)-presenting cells of interest both as therapeutic targets and potential cellular vaccines due to their ability to regulate innate and adaptive immunity. Harnessing the inherent tolerogenicity of DC is a promising and incompletely explored approach to the prevention of allograft rejection. Previously, we and others have reported the ability of pharmacologically-modified DC, that resist maturation, to inhibit CD4(+) T cell responses and prolong allograft survival. Here we evaluated the ability of murine myeloid DC conditioned with the immunosuppressive pro-drug rapamycin (RAPA) to acquire and directly present alloAg to syngeneic CD8(+) T cells. RAPA-conditioned DC (RAPA-DC) pulsed with allogeneic splenocyte lysate acquired and expressed donor MHC class I and enhanced the apoptotic death of directly-reactive donor Ag-specific CD8(+) T cells in vitro. Moreover, following their adoptive transfer, they reduced the survival of these T cells in vivo. The ability of RAPA-DC to inhibit the survival of alloAg-specific CD8(+) T cells provides a potential mechanism by which host-derived DC may act as negative regulators of T cell alloreactivity and support donor-specific unresponsiveness. Adoptive cell therapy with alloAg-pulsed RAPA-DC may offer an effective approach to suppression of alloimmunity, with reduced dependence on systemic immunosuppression.
Transplant Immunology 05/2011; 25(1):20-6. · 1.46 Impact Factor
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Alejandro Soto-Gutierrez,
Li Zhang,
Chris Medberry,
Ken Fukumitsu,
Denver Faulk,
Hongbin Jiang,
Janet Reing,
Roberto Gramignoli,
Junji Komori,
Mark Ross,
Masaki Nagaya,
Eric Lagasse, Donna Stolz,
Stephen C Strom,
Ira J Fox,
Stephen F Badylak
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ABSTRACT: The therapy of choice for end-stage liver disease is whole-organ liver transplantation, but this option is limited by a shortage of donor organs. Cell-based therapies and hepatic tissue engineering have been considered as alternatives to liver transplantation, but neither has proven effective to date. A regenerative medicine approach for liver replacement has recently been described that includes the use of a three-dimensional organ scaffold prepared by decellularization of xenogeneic liver. The present study investigates a new, minimally disruptive method for whole-organ liver decellularization and three different cell reseeding strategies to engineer functional liver tissue.
A combination of enzymatic, detergent, and mechanical methods are used to remove all cells from isolated rat livers. Whole-organ perfusion is used in a customized organ chamber and the decellularized livers are examined by morphologic, biochemical, and immunolabeling techniques for preservation of the native matrix architecture and composition. Three different methods for hepatocyte seeding of the resultant three-dimensional liver scaffolds are evaluated to maximize cell survival and function: (1) direct parenchymal injection, (2) multistep infusion, or (3) continuous perfusion.
The decellularization process preserves the three-dimensional macrostructure, the ultrastructure, the composition of the extracellular matrix components, the native microvascular network of the liver, and the bile drainage system, and up to 50% of growth factor content. The three-dimensional liver matrix reseeded with the multistep infusion of hepatocytes generated ∼90% of cell engraftment and supported liver-specific functional capacities of the engrafted cells, including albumin production, urea metabolism, and cytochrome P450 induction.
Whole-organ liver decellularization is possible with maintenance of structure and composition suitable to support functional hepatocytes.
Tissue Engineering Part C Methods 03/2011; 17(6):677-86. · 4.64 Impact Factor
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ABSTRACT: Hepatocyte transplantation is a potential therapeutic approach for liver disease. However, most patients with chronic hepatic damage have cirrhosis and fibrosis, which limit the potential for cell-based therapy of the liver. The development of an ectopic liver as an additional site of hepatic function represents a new approach for patients with end-stage liver disease. We investigated the development and function of liver tissue in lymph nodes in mice with liver failure.
Hepatocytes were isolated from 8- to 12-week-old mice and transplanted by intraperitoneal injection into 8- to 12-week-old fumarylacetoacetate hydrolase mice (Fah(-/-)), a model of the human liver disease tyrosinemia type I. Survival was monitored and the locations and functions of the engrafted liver cells were determined.
Lymph nodes of Fah(-/-) mice were colonized by transplanted hepatocytes; Fah(+) hepatocytes were detected adjacent to the CD45(+) lymphoid cells of the lymphatic system. Ten weeks after transplantation, these mice had substantial improvements in serum levels of transaminases, bilirubin, and amino acids. Homeostatic expansion of donor hepatocytes in lymph nodes rescued the mice from lethal hepatic failure.
Functional ectopic liver tissue in lymph nodes rescues mice from lethal hepatic disease; lymph nodes therefore might be used as sites for hepatocyte transplantation.
Gastroenterology 11/2010; 140(2):656-666.e2. · 11.68 Impact Factor
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ABSTRACT: Extracellular matrix (ECM) scaffolds prepared from different tissue sources or using different methods have been demonstrated to have distinctive effects upon cell adhesion patterns and the ability to support and maintain differentiated phenotypes. It is unknown whether the molecular composition or the ultrastructure of the ECM plays a greater role in determining the phenotype of the cells with which it comes into contact. However, when implanted, the topology and ligand landscape of the material will determine the host molecules that bind and the type and behavior of cells that mediate the host response. Therefore, a comprehensive understanding of surface characteristics is essential in the design of scaffolds for specific clinical applications. The surface characteristics of ECM scaffolds derived from porcine urinary bladder, small intestine, and liver as well as the effects of two commonly used methods of chemical cross-linking upon UBM were investigated. Electron microscopy and time of flight secondary ion mass spectroscopy were used to examine the surface characteristics of the scaffolds. The results show that ECM scaffolds have unique morphologic and structural properties which are dependant on the organ or tissue from which the scaffold is harvested. Furthermore, the results show that the surface characteristics of an ECM scaffold are changed through chemical cross-linking.
Biomaterials 10/2009; 31(3):428-37. · 7.40 Impact Factor
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ABSTRACT: EP is a potent inhibitor of HMGB1 release that has significant anti-inflammatory activities and exerts a protective effect in animal models of inflammation. As inflammation is linked to cancer growth, we hypothesized that EP would have anti-tumor activity and explored its effects in a liver tumor model. Mice injected intraportally with MC38 colorectal cancer cells led to the growth of visible hepatic tumors within 2 weeks. Pretreatment with EP 30 min prior to infusion of tumor cells and continuing daily for 9 days inhibited tumor growth significantly in a dose-dependent manner, with 80 mg/kg EP achieving >70% reduction in the number of tumor nodules when compared with untreated animals. Delayed treatment with EP also suppressed tumor growth significantly, although to a lesser extent. Tumors had early, marked leukocytic infiltrates, and EP administration decreased innate (NK cells, monocytes) and adaptive (T and B cell lymphocytic) immune cell infiltrates acutely and significantly in the liver. Serum IL-6 and HMGB1 levels, which were elevated following tumor injection, were decreased significantly in EP-treated animals. Tumors showed an increase in apoptosis in EP treated mice, and tumor cells treated in vitro with EP had marked increases in LC3-II and cleaved PARP, consistent with enhanced autophagic flux and apoptosis. Thus, EP inhibition of tumor growth in the liver was mediated by tumor (induction of apoptosis) and host (decreased inflammation) effects. EP administration may have a therapeutic role in the treatment of cancer in conjunction with other therapeutic agents.
Journal of leukocyte biology 08/2009; 86(3):599-607. · 4.99 Impact Factor
