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ABSTRACT: We have shown that overexpression of heme oxygenase-1 (HO-1) prevents the liver inflammation response leading to ischemia and reperfusion injury (IRI). This study was designed to explore the precise function and mechanism of HO-1 cytoprotection in liver IRI by employing a small interfering RNA (siRNA) that effectively suppresses HO-1 expression both in vitro and in vivo. Using a partial lobar liver warm ischemia model, mice were injected with HO-1 siRNA/nonspecific control siRNA or Ad-HO-1/Ad-beta-gal. Those treated with HO-1 siRNA showed increased serum glutamic-oxaloacetic transaminase levels, significant liver edema, sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis. In contrast, Ad-HO-1-pretreated animals revealed only minimal sinusoidal congestion without edema/vacuolization or necrosis. Administration of HO-1 siRNA significantly increased local neutrophil accumulation and the frequency of apoptotic cells. Mice treated with HO-1 siRNA were characterized by increased caspase-3 activity and reduced HO-1 expression, whereas those given Ad-HO-1 showed decreased caspase-3 activity and increased HO-1/Bcl-2/Bcl-x(L), data confirmed by use of an in vitro cell culture system. Thus, by using an siRNA approach this study confirms that HO-1 provides potent cytoprotection against hepatic IRI and regulates liver apoptosis. Indeed, siRNA provides a powerful tool with which to study gene function in a wide range of liver diseases.
Human gene therapy 07/2009; 20(10):1133-42. · 4.20 Impact Factor
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ABSTRACT: This study investigated the effects of dual endothelin (ET) receptor blockade in rat models of liver ischemia and reperfusion injury (IRI). Three models of IRI were used: (1) in vivo total hepatic warm ischemia with portal shunting for 60 minutes with control (saline) and treatment groups (15 mg/kg tezosentan intravenously prior to reperfusion), (2) ex vivo hepatic perfusion after 24 hours of cold storage in University of Wisconsin solution with control and treatment groups (10 mg/kg tezosentan in the perfusate), and (3) syngeneic liver transplantation (LT) after 24 hours of cold storage in University of Wisconsin solution with control and treatment groups (10 mg/kg tezosentan intravenously prior to reperfusion). Tezosentan treatment significantly improved serum transaminase and histology after IRI in all 3 models. This correlated with reduced vascular resistance, improved bile production, and an improved oxygen extraction ratio. Treatment led to a reduction in neutrophil infiltration and interleukin-1 beta and macrophage inflammatory protein 2 production. A reduction in endothelial cell injury as measured by purine nucleoside phosphorylase was seen. Survival after LT was significantly increased with tezosentan treatment (90% versus 50%). In conclusion, this is the first investigation to examine dual receptor ET blockade in 3 models of hepatic IRI and the first to use the parenterally administered agent tezosentan. The results demonstrate that in both warm and cold IRI tezosentan administration improves sinusoidal hemodynamics and is associated with improved tissue oxygenation and reduced endothelial cell damage. In addition, reduced tissue inflammation, injury, and leukocyte chemotactic signaling were seen. These results provide compelling data for the further investigation of the use of tezosentan in hepatic IRI.
Liver Transplantation 12/2008; 14(12):1737-44. · 3.39 Impact Factor
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ABSTRACT: Gene therapy represents a promising treatment for hepatic disease. Most approaches today use viral methods to target tissues. While nonviral gene therapy is less prominent, hydrodynamic gene delivery represents a promising approach to direct gene expression to the liver. The purpose of the present study was to evaluate promoters for efficient gene expression in hepatocytes in vivo by hydrodynamic delivery and to test the findings in a model of hemophilia A.
Human cytomegalovirus (hCMV), chicken beta-actin/CMV enhancer (CAG), elongation factor-1 alpha (EF1alpha), and phosphoglycerokinase (PGK) promoters were subcloned into plasmids with a luciferase reporter gene. In vitro calcium phosphate-mediated transfection of 2 x 10(5) HEK 293 cells was followed by in vivo whole animal bioluminescence and luminometry after hydrodynamic tail vein injection of plasmid DNA. Six-month-old FVB factor VIII (FVIII)-deficient mice were similarly injected with CBA- or EF1alpha-promoted constructs containing the FVIII heavy and light chains and expression was examined.
In vitro transfection demonstrated a hierarchy of expression: hCMV-intron>CAG>EF1alpha>hCMV>PGK. In vivo luminometry demonstrated that the CAG construct produced 2.6x, 3.0x, 3.4x, and >1000x the expression of the hCMV-intron, EF1alpha, hCMV, and PGK constructs respectively. FVIII plasmid injected hemophilic mice demonstrated higher levels of FVIII expression with CAG versus EF1alpha, confirming the reporter gene studies. All FVIII-deficient mice injected with EF1alpha-FVIII or CAG-FVIII plasmids survived after tail clipping.
The CAG promoter/enhancer combination is an excellent alternative to the human CMV promoter for hydrodynamic gene delivery to the liver.
Journal of Surgical Research 08/2008; 148(1):60-6. · 2.25 Impact Factor
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Carlos Barcia,
Clare E Thomas,
James F Curtin,
Gwendalyn D King,
Kolja Wawrowsky,
Marianela Candolfi,
Wei-Dong Xiong,
Chunyan Liu,
Kurt Kroeger,
Olivier Boyer, Jerzy Kupiec-Weglinski,
David Klatzmann,
Maria G Castro,
Pedro R Lowenstein
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ABSTRACT: The microanatomy of immune clearance of infected brain cells remains poorly understood. Immunological synapses are essential anatomical structures that channel information exchanges between T cell-antigen-presenting cells (APC) during the priming and effector phases of T cells' function, and during natural killer-target cell interactions. The hallmark of immunological synapses established by T cells is the formation of the supramolecular activation clusters (SMACs), in which adhesion molecules such as leukocyte function-associated antigen 1 segregate to the peripheral domain of the immunological synapse (p-SMAC), which surrounds the T cell receptor-rich or central SMAC (c-SMAC). The inability so far to detect SMAC formation in vivo has cast doubts on its functional relevance. Herein, we demonstrate that the in vivo formation of SMAC at immunological synapses between effector CD8+ T cells and target cells precedes and mediates clearance of virally infected brain astrocytes.
Journal of Experimental Medicine 10/2006; 203(9):2095-107. · 13.85 Impact Factor
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Sumia Ali,
Gwendalyn D King,
James F Curtin,
Marianela Candolfi,
Weidong Xiong,
Chunyan Liu,
Mariana Puntel,
Queng Cheng,
Jesus Prieto,
Antoni Ribas, Jerzy Kupiec-Weglinski,
Nico van Rooijen,
Hans Lassmann,
Pedro R Lowenstein,
Maria G Castro
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ABSTRACT: In spite of preclinical efficacy and recent randomized, controlled studies with adenoviral vectors expressing herpes simplex virus-1 thymidine kinase (HSV1-TK) showing statistically significant increases in survival, most clinical trials using single therapies have failed to provide major therapeutic breakthroughs. Because glioma is a disease with dismal prognosis and rapid progression, it is an attractive target for gene therapy. Preclinical models using microscopic brain tumor models (e.g., < or =0.3 mm3) may not reflect the pathophysiology and progression of large human tumors. To overcome some of these limitations, we developed a syngeneic large brain tumor model. In this model, administration of single therapeutic modalities, either conditional cytotoxicity or immunostimulation, fail. However, when various immunostimulatory therapies were delivered in combination with conditional cytotoxicity (HSV1-TK), only the combined delivery of fms-like tyrosine kinase ligand (Flt3L) and HSV1-TK significantly prolonged the survival of large tumor-bearing animals (> or =80%; P < or = 0.005). When either macrophages or CD4+ cells were depleted before administration of viral therapy, TK + Flt3L therapy failed to prolong survival. Meanwhile, depletion of CD8+ cells or natural killer cells did not affect TK + Flt3L efficacy. Spinal cord of animals surviving 6 months after TK + Flt3L were evaluated for the presence of autoimmune lesions. Whereas macrophages were present within the corticospinal tract and low levels of T-cell infiltration were detected, these effects are not indicative of an overt autoimmune disorder. We propose that combined Flt3L and HSV1-TK adenoviral-mediated gene therapy may provide an effective antiglioma treatment with increased efficacy in clinical trials of glioma.
Cancer Research 09/2005; 65(16):7194-204. · 7.86 Impact Factor
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Douglas G Farmer,
Xiu-Da Shen,
Farin Amersi,
Dean Anselmo,
Jeffrey P Ma,
Bibo Ke,
Feng Gao,
Sarah Dry,
Susan Fernandez,
Gray D Shaw,
Sue V McDiarmid,
Ronald W Busuttil, Jerzy Kupiec-Weglinski
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ABSTRACT: Intestinal transplantation (ITx) is severely limited by ischemia-reperfusion (I/R) injury. This study investigates I/R injury and ameliorates its consequences by using a recombinant protein targeted against selectins (recombinant P-selectin glycoprotein ligand-immunoglobulin [rPSGL-Ig]).
An isogeneic model of ITx was undertaken with control animals (no therapy) and treatment animals (rPSGL-Ig). Survival was assessed. Separate groups underwent an analysis examining tissue at multiple time points after I/R injury including histopathology; myeloperoxidase staining; immunostaining for CD3 and ED2; polymerase chain reaction analysis of interleukin (IL)-8/cytokine-inducible neutrophil chemoattractant, IL1beta, IL-6, interferon-gamma, IL-2, IL-4, and IL10; and western blots for hemoxygenase-1, BCL-2, and BCL-xl. Standard statistical analysis was undertaken.
Treatment with rPSGL-Ig resulted in significantly improved survival after ITx. Analysis demonstrated diminished injury on histopathology and reduced tissue infiltration of neutrophils and lymphocytes. Significant differences in the cytokine profile after ITx were seen between the two groups including the production of inflammatory cytokines at 24 hr and the Th1 and Th2 cytokines at 2 and 4 hr. Last, treatment resulted in increased production of hemoxygenase, BCL-2, and BCL-xl.
The results of this investigation of I/R injury after ITx revealed that rPSGL-Ig treatment led to marked improvement in outcome. The mechanism of action seems to involve the blockade of neutrophil and lymphocyte infiltration leading to a decreased inflammatory response possibly driven by Th2 cytokines. The results not only lend insight into the mechanisms behind I/R injury after ITx but also demonstrate a potential therapeutic modality to ameliorate its consequences.
Transplantation 02/2005; 79(1):44-51. · 4.00 Impact Factor
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Steven K Takemoto,
Adriana Zeevi,
Sandy Feng,
Robert B Colvin,
Stanley Jordan,
Jon Kobashigawa, Jerzy Kupiec-Weglinski,
Arthur Matas,
Robert A Montgomery,
Peter Nickerson,
Jeffrey L Platt,
Hamid Rabb,
Richard Thistlethwaite,
Dolly Tyan,
Francis L Delmonico
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ABSTRACT: The process of humoral rejection is multifaceted and has different manifestations in the various types of organ transplants. Because this process is emerging as a leading cause of graft loss, a conference was held in April 2003 to comprehensively address issues regarding humoral rejection. Though humoral rejection may result from different factors, discussion focused on a paradigm caused by antibodies, typically against donor HLA antigens, leading to the term 'antibody-mediated rejection' (AMR). Conference deliberations were separated into four workgroups: The Profiling Workgroup evaluated strengths and limitations of different methods for detecting HLA reactive antibody, and created risk assessment guidelines for AMR; The Diagnosis Workgroup reviewed clinical, pathologic, and serologic criteria for assessing AMR in renal, heart and lung transplant recipients; The Treatment Workgroup discussed advantages, limitations and possible mechanisms of action for desensitization protocols that may reverse AMR; and The Basic Science Workgroup presented animal and human immunologic models for humoral rejection and proposed potential targets for future intervention. This work represents a comprehensive review with contributions from experts in the fields of Transplantation Surgery, Medicine, Pathology, Histocompatibility, Immunology, and clinical trial design. Immunologic barriers once considered insurmountable are now consistently overcome to enable more patients to undergo organ transplantation.
American Journal of Transplantation 08/2004; 4(7):1033-41. · 6.39 Impact Factor
