IU Indianapolis ScholarWorks :: Browsing by Author "Burlak, Christopher"

Browsing by Author "Burlak, Christopher"

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Blocking porcine sialoadhesin improves extracorporeal porcine liver xenoperfusion with human blood
(Wiley, 2013-07) Waldman, Joshua P.; Vogel, Thomas; Burlak, Christopher; Coussios, Constantin; Dominguez, Javier; Friend, Peter; Rees, Michael A.; Surgery, School of Medicine
BACKGROUND: Patients in fulminant hepatic failure currently do not have a temporary means of support while awaiting liver transplantation. A potential therapeutic approach for such patients is the use of extracorporeal perfusion with porcine livers as a form of "liver dialysis". During a 72-h extracorporeal perfusion of porcine livers with human blood, porcine Kupffer cells bind to and phagocytose human red blood cells (hRBC) causing the hematocrit to decrease to 2.5% of the original value. Our laboratory has identified porcine sialoadhesin expressed on Kupffer cells as the lectin responsible for binding N-acetylneuraminic acid on the surface of the hRBC. We evaluated whether blocking porcine sialoadhesin prevents the recognition and subsequent destruction of hRBCs seen during extracorporeal porcine liver xenoperfusion. METHODS: Ex vivo studies were performed using wild type pig livers perfused with isolated hRBCs for 72-h in the presence of an anti-porcine sialoadhesin antibody or isotype control. RESULTS: The addition of an anti-porcine sialoadhesin antibody to an extracorporeal porcine liver xenoperfusion model reduces the loss of hRBC over a 72-h period. Sustained liver function was demonstrated throughout the perfusion. CONCLUSIONS: This study illustrates the role of sialoadhesin in mediating the destruction of hRBCs in an extracorporeal porcine liver xenoperfusion model.
Caspase-1 causes truncation and aggregation of the Parkinson's disease-associated protein α-synuclein
(National Academy of Sciences, 2016-08-23) Wang, Wei; Nguyen, Linh T. T.; Burlak, Christopher; Chegini, Fariba; Guo, Feng; Chataway, Tim; Ju, Shulin; Fisher, Oriana S.; Miller, David W.; Datta, Debajyoti; Wu, Fang; Wu, Chun-Xiang; Landeru, Anuradha; Wells, James A.; Cookson, Mark R.; Boxer, Matthew B.; Thomas, Craig J.; Gai, Wei Ping; Ringe, Dagmar; Petsko, Gregory A.; Hoang, Quyen Q.; Department of Biochemistry & Molecular Biology, IU School of Medicine
The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well.
Erythrocytes from GGTA1/CMAH knockout pigs: implications for xenotransfusion and testing in non-human primates
(Wiley, 2014-07) Wang, Zheng-Yu; Burlak, Christopher; Estrada, Jose L.; Li, Ping; Tector, Matthew F.; Tector, A. Joseph; Department of Surgery, IU School of Medicine
Background Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement mediated lysis. Methods Distribution of anti Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. Results The pooled human AB serum contained 0.38 μg/ml anti Neu5Gc IgG and 0.085 μg/ml anti Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutinaion of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared to GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared to GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared to GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared to GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. Conclusions Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantion researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplation to the clinic.
N-linked glycan profiling of GGTA1/CMAH knockout pigs identifies new potential carbohydrate xenoantigens
(Wiley Online Library, 2015-10) Burlak, Christopher; Bern, Marshall; Brito, Alejandro E.; Isailovic, Dragan; Wang, Zheng-Yu; Estrada, Jose L.; Li, Ping; Tector, A. Joseph; Department of Surgery, IU School of Medicine
BACKGROUND: The temporary or long-term xenotransplantation of pig organs into people would save thousands of lives each year if not for the robust human antibody response to pig carbohydrates. Genetically engineered pigs deficient in galactose α1,3 galactose (gene modified: GGTA1) and N-glycolylneuraminic acid (gene modified: CMAH) have significantly improved cell survival when challenged by human antibody and complement in vitro. There remains, however, a significant portion of human antibody binding. METHODS: To uncover additional xenoantigens, we compared the asparagine-linked (N-linked) glycome from serum proteins of humans, domestic pigs, GGTA1 knockout pigs, and GGTA1/CMAH knockout pigs using mass spectrometry. Carbohydrate structures were determined with assistance from GlycoWorkbench, Cartoonist, and SimGlycan software by comparison to existing database entries and collision-induced dissociation fragmentation data. RESULTS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of reduced and solid-phase permethylated glycans resulted in the detection of high-mannose, hybrid, and complex type N-linked glycans in the 1000-4500 m/z ion range. GGTA1/CMAH knockout pig samples had increased relative amounts of high-mannose, incomplete, and xylosylated N-linked glycans. All pig samples had significantly higher amounts of core and possibly antennae fucosylation. CONCLUSIONS: We provide for the first time a comparison of the serum protein glycomes of the human, domestic pig, and genetically modified pigs important to xenotransplantation.
Potential pharmacological chaperones targeting cancer-associated MCL-1 and Parkinson disease-associated α-synuclein
(PNAS, 2014-07-29) Oh, Misook; Le, Ji Hoon; Wang, Wei; Sun Lee, Hui; Sirl Lee, Woo; Burlak, Christopher; Im, Wonpil; Hoang, Quyen; Lim, Hyun-Suk; Department of Biochemistry & Molecular Biology, IU School of Medicine
Pharmacological chaperones are small molecules that bind to proteins and stabilize them against thermal denaturation or proteolytic degradation, as well as assist or prevent certain protein-protein assemblies. These activities are being exploited for the development of treatments for diseases caused by protein instability and/or aberrant protein-protein interactions, such as those found in certain forms of cancers and neurodegenerative diseases. However, designing or discovering pharmacological chaperones for specific targets is challenging because of the relatively featureless protein target surfaces, the lack of suitable chemical libraries, and the shortage of efficient high-throughput screening methods. In this study, we attempted to address all these challenges by synthesizing a diverse library of small molecules that mimic protein α-helical secondary structures commonly found in protein-protein interaction surfaces. This was accompanied by establishing a facile "on-bead" high-throughput screening method that allows for rapid and efficient discovery of potential pharmacological chaperones and for identifying novel chaperones/inhibitors against a cancer-associated protein, myeloid cell leukemia 1 (MCL-1), and a Parkinson disease-associated protein, α-synuclein. Our data suggest that the compounds and methods described here will be useful tools for the development of pharmaceuticals for complex-disease targets that are traditionally deemed "undruggable."
Previously differentiated medial vascular smooth muscle cells contribute to neointima formation following vascular injury
(BioMed Central, 2014-10-01) Herring, Brian Paul; Hoggatt, April M.; Burlak, Christopher; Offermanns, Stefan; Department of Cellular & Integrative Physiology, School of Medicine
Background The origins of neointimal smooth muscle cells that arise following vascular injury remains controversial. Studies have suggested that these cells may arise from previously differentiated medial vascular smooth muscle cells, resident stem cells or blood born progenitors. In the current study we examined the contribution of the previously differentiated vascular smooth muscle cells to the neointima that forms following carotid artery ligation. Methods We utilized transgenic mice harboring a cre recombinase-dependent reporter gene (mTmG). These mice express membrane targeted tandem dimer Tomato (mTomato) prior to cre-mediated excision and membrane targeted EGFP (mEGFP) following excision. The mTmG mice were crossed with transgenic mice expressing either smooth muscle myosin heavy chain (Myh11) or smooth muscle α-actin (Acta2) driven tamoxifen regulated cre recombinase. Following treatment of adult mice with tamoxifen these mice express mEGFP exclusively in differentiated smooth muscle cells. Subsequently vascular injury was induced in the mice by carotid artery ligation and the contribution of mEGFP positive cells to the neointima determined. Results Analysis of the cellular composition of the neointima that forms following injury revealed that mEGFP positive cells derived from either Mhy11 or Acta2 tagged medial vascular smooth muscle cells contribute to the majority of neointima formation (79 ± 17% and 81 ± 12%, respectively). Conclusion These data demonstrate that the majority of the neointima that forms following carotid ligation is derived from previously differentiated medial vascular smooth muscle cells.
Reduced human platelet uptake by pig livers deficient in the asialoglycoprotein receptor 1 protein
(Wiley, 2015-05) Paris, Leela L.; Estrada, Jose L.; Li, Ping; Blankenship, Ross L.; Sidner, Richard A.; Reyes, Luz M.; Montgomery, Jessica B.; Burlak, Christopher; Butler, James R.; Downey, Susan M.; Wang, Zheng-Yu; Tector, Matthew; Tector, A. Joseph; Surgery, School of Medicine
BACKGROUND: The lethal thrombocytopenia that accompanies liver xenotransplantation is a barrier to clinical application. Human platelets are bound by the asialoglycoprotein receptor (ASGR) on pig sinusoidal endothelial cells and phagocytosed. Inactivation of the ASGR1 gene in donor pigs may prevent xenotransplantation-induced thrombocytopenia. METHODS: Transcription activator-like effector nucleases (TALENs) were targeted to the ASGR1 gene in pig liver-derived cells. ASGR1 deficient pig cells were used for somatic cell nuclear transfer (SCNT). ASGR1 knock out (ASGR1-/-) fetal fibroblasts were used to produce healthy ASGR1 knock out piglets. Human platelet uptake was measured in ASGR1+/+ and ASGR1-/- livers. RESULTS: Targeted disruption of the ASGR1 gene with TALENs eliminated expression of the receptor. ASGR1-/- livers phagocytosed fewer human platelets than domestic porcine livers during perfusion. CONCLUSIONS: The use of TALENs in liver-derived cells followed by SCNT enabled the production of healthy homozygous ASGR1 knock out pigs. Livers from ASGR1-/- pigs exhibit decreased human platelet uptake. Deletion of the ASGR1 gene is a viable strategy to diminish platelet destruction in pig-to-human xenotransplantation.
Study of Physiologic and Immunologic Incompatibilities of Pig to Human Transplantation
(2014) Chihara, Ray K.; Burlak, Christopher; Tector, A. Joseph; Basile, David P.; Tune, Johnathan D.
Solid organ transplantation is limited by available donor allografts. Pig to human transplantation, xenotransplantation, could potentially solve this problem if physiologic and immunologic incompatibilities are overcome. Genetic modifications of pigs have proven valuable in the study of xenotransplantation by improving pig to human compatibility. More genetic targets must be identified for clinical success. First, this study examines platelet homeostasis incompatibilities leading to acute thrombocytopenia in liver xenotransplantation. Mechanisms for xenogeneic thrombocytopenia were evaluated using liver macrophages, Kupffer cells, leading to identification of CD18, beta-2 integrin, as a potential target for modification. When disruption of CD18 was accomplished, human platelet binding and clearance by pig Kupffer cells was inhibited. Further, human and pig platelet surface carbohydrates were examined demonstrating significant differences in carbohydrates known to be involved with platelet homeostasis. Carbohydrate recognition domains of receptors responsible for platelet clearance Macrophage antigen complex-1 (CD11b/CD18) and Asialoglycoprotein receptor 1 in pigs were found to be different from those in humans, further supporting the involvement of platelet surface carbohydrate differences in xenogeneic thrombocytopenia. Second, immunologic incompatibilities due to antibody recognition of antigens resulting in antibody-mediated rejection were studied. Identification of relevant targets was systematically approached through evaluation of a known xenoantigenic protein fibronectin from genetically modified pigs. N-Glycolylneuraminic acid, a sialic acid not found in humans, was expressed on pig fibronectin and was identified as an antigenic epitope recognized by human IgG. These studies have provided further insight into xenogeneic thrombocytopenia and antibody-mediated rejection, and have identified potential targets to improve pig to human transplant compatibility.

Browsing by Author "Burlak, Christopher"

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