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Dive into the research topics where Christopher J. Lees is active.

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Featured researches published by Christopher J. Lees.


Journal of Investigative Dermatology | 2011

Induced Pluripotent Stem Cells from Individuals with Recessive Dystrophic Epidermolysis Bullosa

Jakub Tolar; Lily Xia; Megan Riddle; Christopher J. Lees; Cindy R. Eide; Ron T. McElmurry; Matthias Titeux; Mark J. Osborn; Troy C. Lund; Alain Hovnanian; John E. Wagner; Bruce R. Blazar

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7), the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions, and currently, there are no effective forms of therapy. Nevertheless, some advances in patient therapy are being made, and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized, gene-corrected, patient-specific cell transfer, we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly, human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis.


Journal of Immunology | 2004

B7 Expression on T Cells Down-Regulates Immune Responses through CTLA-4 Ligation via R-T Interactions

Patricia A. Taylor; Christopher J. Lees; Sylvie Fournier; James P. Allison; Arlene H. Sharpe; Bruce R. Blazar

Although B7 on APCs has a well-recognized role in T cell costimulation, little is known about the functional significance of constitutive and activation-induced B7 expression that also occurs on T cells. To analyze the role of B7 on T cells, B7-1/B7-2-deficient mice (B7 double knockout) and mice overexpressing B7-2 exclusively on T cells (B7-2 transgenic) were used as T cell donors for allogeneic transplant recipients, and graft-vs-host disease (GVHD) was assessed. B7 double-knockout T cells resulted in significant GVHD acceleration compared with wild-type T cells. Conversely, B7-2 transgenic donor T cells mediated reduced GVHD mortality compared with wild-type T cells. Data indicated that B7 expression on T cells down-regulated alloresponses through CTLA-4 ligation. This study is the first to provide definitive in vivo data illustrating the importance of T cell-associated B7 as a negative regulator of immune responses in a clinically relevant murine model of GVHD. The up-regulation of B7 on T cells may be an important component of normal immune homeostasis.


Molecular Therapy | 2008

Sleeping Beauty transposon-mediated engineering of human primary T cells for therapy of CD19+ lymphoid malignancies.

Xin Huang; Hongfeng Guo; Johnthomas Kang; Suet Choi; Tom C. Zhou; Syam Tammana; Christopher J. Lees; Zhong Ze Li; Michael C. Milone; Bruce L. Levine; Jakub Tolar; Carl H. June; R. Scott McIvor; John E. Wagner; Bruce R. Blazar; Xianzheng Zhou

We have reported earlier that the non-viral Sleeping Beauty (SB) transposon system can mediate genomic integration and long-term reporter gene expression in human primary peripheral blood (PB) T cells. In order to test whether this system can be used for genetically modifying both PB T cells and umbilical cord blood (UCB) T cells as graft-versus-leukemia effector cells, an SB transposon was constructed to coexpress a single-chain chimeric antigen receptor (CAR) for human CD19 and CD20. PB and UCB were nucleofected with the transposon and a transposase plasmid, activated and then expanded in culture using anti-CD3/CD28 beads. Stable dual-gene expression was confirmed in both T-cell types, permitting enrichment by positive selection with Rituxan. The engineered CD4(+) T cells and CD8(+) T cells both exhibited specific cytotoxicity against CD19(+) leukemia and lymphoma cell lines, as well as against CD19 transfectants, and produced high-levels of antigen-dependent Th1 (but not Th2) cytokines. The in vivo adoptive transfer of genetically engineered T cells significantly reduced tumor growth and prolonged the survival of the animal. Taken together, these data indicate that T cells from PB and UCB can be stably modified using a non-viral DNA transfer system, and that such modified T cells may be useful in the treatment of refractory leukemia and lymphoma.


Molecular Therapy | 2011

Minicircle DNA-based gene therapy coupled with immune modulation permits long-term expression of α-L-iduronidase in mice with mucopolysaccharidosis type I.

Mark J. Osborn; Ron T. McElmurry; Christopher J. Lees; Anthony P. Defeo; Zhi-Ying Chen; Mark A. Kay; Luigi Naldini; Gordon J. Freeman; Jakub Tolar; Bruce R. Blazar

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease characterized by mutations to the α-L-iduronidase (IDUA) gene resulting in inactivation of the IDUA enzyme. The loss of IDUA protein results in the progressive accumulation of glycosaminoglycans within the lysosomes resulting in severe, multi-organ system pathology. Gene replacement strategies have relied on the use of viral or nonviral gene delivery systems. Drawbacks to these include laborious production procedures, poor efficacy due to plasmid-borne gene silencing, and the risk of insertional mutagenesis. This report demonstrates the efficacy of a nonintegrating, minicircle (MC) DNA vector that is resistant to epigenetic gene silencing in vivo. To achieve sustained expression of the immunogenic IDUA protein we investigated the use of a tissue-specific promoter in conjunction with microRNA target sequences. The inclusion of microRNA target sequences resulted in a slight improvement in long-term expression compared to their absence. However, immune modulation by costimulatory blockade was required and permitted for IDUA expression in MPS I mice that resulted in the biochemical correction of pathology in all of the organs analyzed. MC gene delivery combined with costimulatory pathway blockade maximizes safety, efficacy, and sustained gene expression and is a new approach in the treatment of lysosomal storage disease.


Blood | 2011

Hematopoietic differentiation of induced pluripotent stem cells from patients with mucopolysaccharidosis type I (Hurler syndrome)

Jakub Tolar; In-Hyun Park; Lily Xia; Christopher J. Lees; Brandon Peacock; Beau R. Webber; Ron T. McElmurry; Cindy R. Eide; Paul J. Orchard; Michael Kyba; Mark J. Osborn; Troy C. Lund; John E. Wagner; George Q. Daley; Bruce R. Blazar

Mucopolysaccharidosis type I (MPS IH; Hurler syndrome) is a congenital deficiency of α-L-iduronidase, leading to lysosomal storage of glycosaminoglycans that is ultimately fatal following an insidious onset after birth. Hematopoietic cell transplantation (HCT) is a life-saving measure in MPS IH. However, because a suitable hematopoietic donor is not found for everyone, because HCT is associated with significant morbidity and mortality, and because there is no known benefit of immune reaction between the host and the donor cells in MPS IH, gene-corrected autologous stem cells may be the ideal graft for HCT. Thus, we generated induced pluripotent stem cells from 2 patients with MPS IH (MPS-iPS cells). We found that α-L-iduronidase was not required for stem cell renewal, and that MPS-iPS cells showed lysosomal storage characteristic of MPS IH and could be differentiated to both hematopoietic and nonhematopoietic cells. The specific epigenetic profile associated with de-differentiation of MPS IH fibroblasts into MPS-iPS cells was maintained when MPS-iPS cells are gene-corrected with virally delivered α-L-iduronidase. These data underscore the potential of MPS-iPS cells to generate autologous hematopoietic grafts devoid of immunologic complications of allogeneic transplantation, as well as generating nonhematopoietic cells with the potential to treat anatomical sites not fully corrected with HCT.


Blood | 2008

TLR agonists regulate alloresponses and uncover a critical role for donor APCs in allogeneic bone marrow rejection

Patricia A. Taylor; Michael J. Ehrhardt; Christopher J. Lees; Angela Panoskaltsis-Mortari; Arthur M. Krieg; Arlene H. Sharpe; William J. Murphy; Jonathan S. Serody; Hiroaki Hemmi; Shizuo Akira; Robert B. Levy; Bruce R. Blazar

Cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs) are synthetic ODNs with unmethylated DNA sequences that mimic viral and bacterial DNA and protect against infectious agents and tumor challenge. We show that CpG ODNs markedly accelerated graft-versus-host disease (GVHD) lethality by Toll-like receptor 9 (TLR9) ligation of host antigen-presenting cells (APCs), dependent upon host IFNgamma but independent of host IL-12, IL-6, or natural killer (NK) cells. Imaging studies showed significantly more green fluorescent protein-positive (GFP(+)) effector T cells in lymphoid and nonlymphoid organs. In engraftment studies, CpG ODNs promoted allogeneic donor bone marrow (BM) rejection independent of host IFNgamma, IL-12, or IL-6. During the course of these studies, we uncovered a previously unknown and critical role of donor BM APCs in modulating the rejection response. CpG ODNs promoted BM rejection by ligation of donor BM, but not host, TLR9. CpG ODNs did not impair engraftment of TLR9(-/-) BM unless wild-type myeloid (CD11b(+)) but not B-lineage (CD19(+)) BM cells were added to the donor inoculum. The importance of donor BM APCs in modulating the strength of the host antidonor rejection response was underscored by the finding that B7-1/B7-2(-/-) BM was less likely than wild-type BM to be rejected. Collectively, these data offer new insight into the mechanism of alloresponses regulating GVHD and BM rejection.


Journal of Investigative Dermatology | 2014

Patient-Specific Naturally Gene-Reverted Induced Pluripotent Stem Cells in Recessive Dystrophic Epidermolysis Bullosa

Jakub Tolar; John A. McGrath; Lily Xia; Megan Riddle; Christopher J. Lees; Cindy R. Eide; Douglas R. Keene; Lu Liu; Mark J. Osborn; Troy C. Lund; Bruce R. Blazar; John E. Wagner

Spontaneous reversion of disease-causing mutations has been observed in some genetic disorders. In our clinical observations of severe generalized recessive dystrophic epidermolysis bullosa (RDEB), a currently incurable blistering genodermatosis caused by loss-of-function mutations in COL7A1 that results in a deficit of type VII collagen (C7), we have observed patches of healthy-appearing skin on some individuals. When biopsied, this skin revealed somatic mosaicism resulting from the self-correction of C7 deficiency. We believe this source of cells could represent an opportunity for translational “natural” gene therapy. We show that revertant RDEB keratinocytes expressing functional C7 can be reprogrammed into induced pluripotent stem cells (iPSCs) and that self-corrected RDEB iPSCs can be induced to differentiate into either epidermal or hematopoietic cell populations. Our results give proof in principle that an inexhaustible supply of functional patient-specific revertant cells can be obtained—potentially relevant to local wound therapy and systemic hematopoietic cell transplantation. This technology may also avoid some of the major limitations of other cell therapy strategies, e.g., immune rejection and insertional mutagenesis, which are associated with viral- and nonviral- mediated gene therapy. We believe this approach should be the starting point for autologous cellular therapies using “natural” gene therapy in RDEB and other diseases.


Journal of Investigative Dermatology | 2013

Keratinocytes from Induced Pluripotent Stem Cells in Junctional Epidermolysis Bullosa

Jakub Tolar; Lily Xia; Christopher J. Lees; Megan Riddle; Amber N. McElroy; Douglas R. Keene; Troy C. Lund; Mark J. Osborn; M. Peter Marinkovich; Bruce R. Blazar; John E. Wagner

Keratinocytes and dermal fibroblasts express adhesive proteins that ensure the epidermis remains attached to the skin basement membrane and to the papillary dermis. Congenital deficiency of any of at least 15 such proteins results in a blistering condition, termed epidermolysis bullosa (EB)(Fine et al., 2008). The most severe form of EB is the Herlitz variant of junctional EB (JEB-H) caused by loss-of-function mutations in one of the three genes (LAMA3, LAMB3, and LAMC2) encoding one of three chains of the heterotrimeric protein laminin 332 (LM-332)(Kiritsi et al., 2011). LM-332 is secreted by keratinocytes and interacts with integrin receptors α3β1 and α6β4 to form focal adhesions and stable anchoring contacts in the dermalepidermal junction (DEJ). Children with this autosomal recessive genodermatosis develop generalized skin blistering; extensive mucosal erosions in the upper respiratory, gastrointestinal, and genitourinary tracts; infections; and, despite supportive measures, typically die within the first year of life. Even though it has been largely accepted that JEB-H is untreatable(Yuen et al., 2012), evidence from a gene therapy trial for the less severe (non-Herlitz) form of JEB(Mavilio et al., 2006), and from LAMB3 gene correction of human JEB-H cells –(Robbins et al., 2001; Sakai et al., 2010), suggests novel treatment options. Prominent among these, the novel technology of reprogramming skin cells into pluripotent stem cells (iPSCs), already applied to EB (Bilousova et al., 2011; Itoh et al., 2011; Tolar et al., 2010) by a combination of specific transcription factors has the dual potential of generating patient-specific, highly proliferative cells for gene-correction strategies and of providing a tool for better understanding the biology of JEB-H. We hypothesized that such iPSCs can be derived from JEB-H individuals. Thus, in principle, an inexhaustible supply of patient-specific stem cells can be generated for local wound therapy and for systemic administration aimed at reaching both skin and internal mucosal membranes. To investigate this, we obtained skin biopsies from two individuals with JEB-H, who carried mutations in the LAMB3 gene: patient 1 P1; c.1365_1366del (p.Asn456ArgfsX7); c.2207C>A (p.Ser736X(Varki et al., 2006)) and patient 2 (P2; c.1903 C>T (p.Arg635X); c.1117C>T (p.Gln373X). Samples were obtained with written informed consent and following a protocol approved by the University of Minnesota Institutional Review Board and with adherence to the Helsinki Guidelines. All mutations create premature stop codons in the open reading frame, with expected nonsense-mediated decay of the mRNA or truncation of the protein product. The children experienced extensive areas of mucocutaneous lesions from birth, hoarseness and stridor, numerous infections, and progressive severe malnutrition. Examination of skin sections revealed the absence of laminin β3 chain at the DEJ. Electron microscopy examination showed infrequent and underdeveloped hemidesmosomes. Collectively these molecular, clinical, biochemical, and ultrastructural features were consistent with the diagnosis of JEB-H. To derive JEB-iPSCs, we transduced skin fibroblasts with the four transcription factors OCT4, SOX2, KLF4, and c-MYC, which are known to induce pluripotency in somatic cells(Tolar et al., 2010). Within three weeks of culture, the patient-specific JEB-iPSCs emerged as raised clusters of cells (Figure 1 a–c). To document the embryonic stem cell-like cellular state, we examined their mRNA and protein expression patterns. When compared with the parental fibroblasts, the JEB-iPSCs expressed the genes coding for nuclear, cytoplasmic, and cell surface proteins (e.g., TRA-1-60, TRA-1-81, stage-specific embryonic antigens 3 and 4, Lin28, Rex1, ABCG2 and DNMT3b, OCT4, and NANOG) in a pattern consistent with embryonic stem cell and iPSC phenotype (Figure 1 d–m). In support of the known activation of endogenous expression of stem cell genes by exogenous reprogramming factors, the maintenance of pluripotency became independent of the original exogenous reprogramming factors (data not shown). As expected in fully reprogrammed iPSCs, the epigenetic profiles showed that endogenous OCT4 and NANOG promoters were largely demethylated (Supplementary Figure 1). The JEB-iPSC lines were maintained for more than 20 passages, and they showed no evidence of genomic instability as evidenced by cytogenetic analysis (Supplementary Figure 2). To exclude the possibilities of cell contamination or the mosaicism observed in JEB(Pasmooij et al., 2007), we verified the authenticity of the JEB-iPSCs by genomic finger-typing with competitive polymerase chain reaction of a variable number of tandem repeat polymorphisms and by sequencing of LAMB3 gene mutations in the JEB-iPSCs (data not shown). To show that JEB-iPSCs are capable of differentiating into cells of endodermal, mesodermal, and ectodermal origin, we injected them into immune-deficient mice lacking T cells, B cells, and natural killer cells, and having a macrophage defect that makes them reliable recipients of human cells. In 6–8 weeks, cystic teratomas formed and cells derived from all three embryonic layers were seen (Figure 1 n). In aggregate, these data show that fully reprogrammed iPSCs can be derived from skin cells of JEB-H individuals. Figure 1 Expression profile of JEB-iPSCs JEB-iPSCs can provide means for drug screening and to model cellular interactions among various mucocutaneous cell types derived from the same individual. To our knowledge, this is a previously unreported use of iPSCs as a cellular tool to study the skin pathology in JEB. We showed first that skin-like structures formed in the process of in vivo JEB-iPSC differentiation (Supplementary Figure 3). In contrast to wild-type iPSCs, the skin-like structures arising from the JEB-iPSCs expressed no detectable laminin β3, but expressed collagen type VII, the DEJ protein deficient in distinct, dystrophic forms of EB (Figure 2 a–d). Next, to substantiate the proof-of-concept that skin cell cultures can be derived from JEB-iPSCs, we differentiated JEB-iPSCs into keratinocytes (Supplementary Figures 4 and 5). Lastly, to demonstrate that this operating procedure can serve as a platform for gene correction of these highly proliferative cells, we transduced the JEB-iPSCs with LAMB3 gene. After transduction, the JEB-iPSC-derived cells expressed and secreted laminin β3 protein (Figure 2 e). Figure 2 Skin cells derived from JEB-iPSCs In summary, we have shown that the LAMB3 defect does not preclude reprogramming into pluripotency, as has been observed in other genetic diseases(Raya et al., 2009). We have also shown that the JEB-iPSCs—in addition to establishing a reliable stem cell source for gene therapy interventions in JEB-H—can be used in the study of early human skin formation and compared to LM-332 in early development. With the ultimate clinical application of iPSC technology in mind, it is worth noting that strategies exist for genome-nonintegrating reprogramming, for depletion of tumor-inducing cells from differentiated iPSC cultures, and—as an JEB-H individual can develop anti-LM-332 antibody—for induction of immunological tolerance to disease-correcting transgenes(Vailly et al., 1998; Wu and Hochedlinger, 2011). Thus, gene-corrected JEB-iPSCs can inform medical advances in this severe and lethal blistering disease, as well as additional extracellular matrix disorders of the skin and other tissues(McGowan and Marinkovich, 2000).


The Annals of otology, rhinology & laryngology. Supplement | 1994

Determining otitis media severity from middle ear fluid analysis

Steven K. Juhn; W. J. Garvis; Christopher J. Lees; Chap T. Le; Chong Sun Kim

Otitis media has a complex multifactorial pathogenesis, and the middle ear inflammatory response is typified by the accumulation of cellular and chemical mediators in middle ear effusion. However, specific biochemical and immunochemical factors that may be responsible for the severity or chronicity of otitis media have not been identified. Identification of factors involved in chronicity appears to be an essential step in the treatment and ultimate prevention of chronic otitis media. We analyzed 70 effusion samples from patients 1 to 10 years of age who had chronic otitis media with effusion for two cytokines (interleukrn-1β and tumor necrosis factor α) and total collagenase. The highest concentrations of all three inflammatory mediators were found in purulent otitis media, and concentrations were higher in younger than in older patients. Mediator concentrations were similar in samples obtained from patients having their first myringotomy for otitis media with effusion and in those who had had multiple previous myringotomies. The multiresponse star, which incorporates several biochemical parameters in one graphic illustration, may best characterize the complex nature of middle ear inflammation.


European Archives of Oto-rhino-laryngology | 1995

Effect of platelet-activating factor on secretion of mucous glycoprotein from chinchilla middle ear epithelial cells in vitro

Jizhen Lin; Youngki Kim; Christopher J. Lees; Steven K. Juhn

Platelet-activating factor (PAF) is a naturally occurring phospholipid that acts as a pleiotropic mediator and mediates cell-cell reactions under physiological and pathological conditions. Recently, it has been shown that PAF is a strong secretagogue of mucous glycoprotein in the airways, suggesting its role in mucous glycoprotein secretion and the pathogenesis of otitis media with effusion. In the current study, we examined the effect of PAF on mucous glycoprotein secretion in cultured chinchilla middle ear epithelial cells. PAF at 1 μM significantly stimulated mucous glycoprotein secretion from cultured chinchilla middle ear epithelial cells. This action was concentration-dependent, with secretions reaching near maximum when the cells were incubated with PAF at 100 μM. In a time-dependent study, PAF demonstrated an initial rapid stimulation of mucous glycoprotein secretion, followed by a gradual increase thereafter. A six-fold increase was seen in the first 2 h compared with controls. Cycloheximide, a protein synthesis inhibitor, demonstrated an inhibitory effect on PAF-stimulated mucous glycoprotein secretion in this study. These findings suggest that PAF plays an important role in the pathogenesis of otitis media with effusion by stimulating mucous glycoprotein secretion in vitro.

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Patricia A. Taylor

Medical College of Wisconsin

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Jakub Tolar

University of Minnesota

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Angela Panoskaltsis-Mortari

Science Applications International Corporation

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Megan Riddle

University of Minnesota

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Lily Xia

University of Minnesota

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