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BACKGROUND The goal of this study was to define the role of T-cell sub-sets in the pathogenesis of autoimmunity-induced obliterative airway disease by passive transfer of CD8+ or CD4+ T cells. METHODS Antibodies to major histocompatibility complex (MHC) class I were administered intrabronchially into C57BL/6 animals. Lungs were analyzed by histopathology and immunohistochemistry. The CD8+ and CD4+ T-cell sub-sets were purified from the lung-infiltrating cells and intrabronchially transferred. Frequency of cells secreting interleukin-17, interferon-γ, or interleukin-10 to self-antigens was enumerated by enzyme-linked immunospot assay. Myeloperoxidase and antibodies to self-antigens were determined by enzyme-linked immunosorbent assay. Cytokine and growth factor expression was determined by quantitative reverse-transcription polymerase chain reaction. RESULTS Passive transfer of lung-infiltrating CD8 T cells isolated after anti-MHC class I administration, along with sub-optimal dose, induced significantly higher cellular infiltration (89.3% ± 7.9% vs 62.8% ± 10.1%, p p p CONCLUSION Ligation of MHC molecules by its specific antibodies induced early injury with neutrophils, macrophages, and CD8 T cells, which leads to exposure of cryptic self-antigens and their presentation by the infiltrating CD4+ T cells and B cells, leading to the development of immune responses to self-antigens and culminating in obliterative airway disease.

Organ transplantation, an accepted treatment for end stage organ failure, is often complicated by allograft rejection and disease recurrence. In this review we will discuss the potential role of microRNAs in allograft immunity especially leading to rejection of the transplanted organ. microRNAs (miRNAs), originally identified in C. elegans, are short non-coding 21-24 nucleotide sequences that bind to its complementary sequences in functional messenger RNAs and inhibits post-translational processes through RNA duplex formation resulting in gene silencing (Lau et al., 2001). Gene specific translational silencing by miRNAs regulates pathways for immune responses such as development of innate immunity, inflammation, T-cell and B-cell differentiation and signaling that are implicated in various stages of allograft rejection. miRNAs also play a role in development of post-transplant complicacies like fibrosis, cirrhosis, carcinogenesis often leading to graft loss and poor patient outcome. Recent advancements in the methods for detecting and quantifying miRNA in tissue biopsies, as well as in serum and urine samples, has led to identification of specific miRNA signatures in patients with allograft rejection and have been utilized to predict allograft status and survival. Therefore, miRNAs play a significant role in post-transplant events including allograft rejection, disease recurrence and tumor development impacting patient outcome.

Transcriptional regulation is a complex process that requires the integrated action of many multi-protein complexes. The way in which a living cell coordinates the action of these complexes in time and space is still poorly understood. Recent work has shown that nuclear pores, well known for their role in 3′ processing and export of transcripts, also participate in the control of transcriptional initiation. We have recently begun to explore how nuclear pores interface with the well-described machinery that regulates initiation. This work led to the discovery that specific nucleoporins are required for binding of the repressor protein Mig1 to its site in target promoters. Nuclear pores are therefore involved in repressing, as well as activating, transcription. Here we discuss in detail the main models explaining our result and consider what each implies about the roles that nuclear pores play in the regulation of gene expression.

NUP98 is a nucleoporin that plays complex roles in the nucleocytoplasmic trafficking of macromolecules. Rearrangements of the NUP98 gene in human leukemia result in the expression of numerous fusion oncoproteins whose effect on nucleocytoplasmic trafficking is poorly understood. The present study was undertaken to determine the effects of leukemogenic NUP98 fusion proteins on CRM1-mediated nuclear export. NUP98-HOXA9, a prototypic NUP98 fusion, inhibited the nuclear export of two known CRM1 substrates: mutated cytoplasmic nucleophosmin and HIV-1 Rev. In vitro binding assays revealed that NUP98-HOXA9 binds CRM1 through the FG repeat motif in a Ran-GTP-dependent manner similar to but stronger than the interaction between CRM1 and its export substrates. Two NUP98 fusions, NUP98-HOXA9 and NUP98-DDX10, whose fusion partners are structurally and functionally unrelated, interacted with endogenous CRM1 in myeloid cells as shown by co-immunoprecipitation. These leukemogenic NUP98 fusion proteins interacted with CRM1, Ran, and the nucleoporin NUP214 in a manner fundamentally different from that of wild-type NUP98. NUP98-HOXA9 and NUP98-DDX10 formed characteristic aggregates within the nuclei of a myeloid cell line and primary human CD34+ cells and caused aberrant localization of CRM1 to these aggregates. These NUP98 fusions caused nuclear accumulation of two transcription factors, NFAT and NFkappaB, that are regulated by CRM1-mediated export. The nuclear entrapment of NFAT and NFkappaB correlated with enhanced transcription from promoters responsive to these transcription factors. Taken together, the results suggest a new mechanism by which NUP98 fusions dysregulate transcription and cause leukemia, namely, inhibition of CRM1-mediated nuclear export with aberrant nuclear retention of transcriptional regulators.

The recruitment model for gene activation presumes that DNA is a platform on which the requisite components of the transcriptional machinery are assembled. In contrast to this idea, we show here that Rap1/Gcr1/Gcr2 transcriptional activation in yeast cells occurs through a large anchored protein platform, the Nup84 nuclear pore subcomplex. Surprisingly, Nup84 and associated subcomplex components activate transcription themselves in vivo when fused to a heterologous DNA-binding domain. The Rap1 coactivators Gcr1 and Gcr2 form an important bridge between the yeast nuclear pore complex and the transcriptional machinery. Nucleoporin activation may be a widespread eukaryotic phenomenon, because it was first detected as a consequence of oncogenic rearrangements in acute myeloid leukemia and related syndromes in humans. These chromosomal translocations fuse a homeobox DNA-binding domain to the human homolog (hNup98) of a transcriptionally active component of the yeast Nup84 subcomplex. We conclude that Rap1 target genes are activated by moving to contact compartmentalized nuclear assemblages, rather than through recruitment of the requisite factors to chromatin by means of diffusion. We term this previously undescribed mechanism “reverse recruitment” and discuss the possibility that it is a central feature of eukaryotic gene regulation. Reverse recruitment stipulates that activators work by bringing the DNA to an nuclear pore complex-tethered platform of assembled transcriptional machine components.

Hepatitis C virus (HCV)-mediated liver diseases are one of the major health issues in the United States and worldwide. HCV infection has been reported to modulate microRNAs (miRNAs) that control various cell surface receptors and gene-regulatory complexes involved in hepatic inflammation and liver diseases. We report here that specific downregulation of miRNA-107 and miRNA-449a following HCV infection in patients with HCV-mediated liver diseases modulates expression of CCL2, an inflammatory chemokine upregulated in patients with chronic liver diseases, by targeting components of the interleukin-6 receptor (IL-6R) complex. Computational analysis for DNA-bound transcription factors in the CCL2 promoter identified adjacent binding sites for CCAAT/CEBPα, spleen focus-forming virus, proviral integration oncogene (SPI1/PU.1), and STAT3. We demonstrate that CEBPα, PU.1, and STAT3 interacted with each other physically to cooperatively bind to the promoter and activate CCL2 expression. Analysis of IL-6R and JAK1 expression in HCV patients by quantitative PCR showed significant upregulation when there was impaired miRNA-107 and miRNA-449a expression, along with upregulation of PU.1 and STAT3, but not CEBPα. miRNA-449a and miRNA-107 target expression of IL-6R and JAK1 , respectively, in vitro and also inhibit IL-6 signaling and impair STAT3 activation in human hepatocytes. Taken together, our results demonstrate a novel gene-regulatory mechanism in which HCV-induced changes in miRNAs (miRNA-449a and miRNA-107) regulate CCL2 expression by activation of the IL-6-mediated signaling cascade, which we propose will result in HCV-mediated induction of inflammatory responses and fibrosis. IMPORTANCE Hepatitis C virus (HCV)-induced hepatitis is a major health concern worldwide. HCV infection results in modulation of noncoding microRNAs affecting major cellular pathways, including inflammatory responses. In this study, we have identified a microRNA-regulated pathway for the chemokine CCL2 in HCV-induced hepatitis. Understanding microRNA-mediated transcriptional-regulatory pathways will result in development of noninvasive biomarkers for better disease prediction and development of effective therapeutics.

Steatotic liver grafts tolerate ischemia–reperfusion (I/R) injury poorly, contributing to poor survival following transplantation. However the molecular mechanisms leading to I/R injury still remain to be defined. We have previously reported that the protective effect of bortezomib towards inhibiting cold induced I/R injury in obese rat liver transplant model is through NF-κB down modulation. In this report using an orthotopic liver transplant (OLT) model in Zucker rats (from obese, leptin deficient donor, to lean recipient) we defined the mechanisms of steatotic liver injury, and characterized the role of bortezomib in inhibiting MMP activation and YKL-40, both of which are involved in extracellular matrix deposition and fibrosis, the key pathological features of liver allograft failure. Obese donor rats were treated with bortezomib (i.v., 0.1 mg/kg immediately prior to liver procurement) to assess the role of MMP and YKL-40 in steatotic liver I/R injury. I/R injury in steatotic livers resulted in significant increases in expression of YKL-40 (9 fold), and activation of MMP-2 (15 fold)/MMP-9 (12 fold). Bortezomib treatment reduced the expression of YKL-40 and MMP to basal levels. Bortezomib also inhibited the pro-fibrotic (VEGF, HGF, bFGF, TGF-β) and pro-inflammatory (IL-1β, TNF-α and IFN-γ) cytokines significantly in comparison to untreated animals with I/R injury. These results demonstrate that I/R injury in steatotic livers following transplantation are associated with MMP activation and YKL-40 upregulation resulting in pro-fibrotic and pro-inflammatory cytokine release. Administration of the proteosomal inhibitor, bortezomib, effectively attenuated the I/R injury by inhibiting MMP and YKL-40 expression and therefore support the clinical utility of this drug in donor management for preventing I/R injury and its sequelae.

Immune responses to lung-associated self-antigens (SAgs) have been implicated in chronic lung allograft rejection. The goals of this study were to determine the prevalence of pre-existing antibodies (Abs) to the SAgs in pulmonary diseases and the association between pre-existing Abs to SAgs and the development of primary graft dysfunction (PGD), donor-specific antibodies (DSA), and chronic rejection.Pre- and post-transplant sera were analyzed from 317 lung transplant (LTx) recipients between 2000 and 2011 with diagnosis of chronic obstructive disease (n = 161), idiopathic pulmonary fibrosis (IPF; n = 50), cystic fibrosis (CF; n = 55), and others (n = 51). Samples were analyzed for Abs to SAgs by enzyme-linked immunosorbent assay, and DSA and cytokines by Luminex. The clinical diagnosis of PGD and bronchiolitis obliterans syndrome (BOS) was based on International Society for Heart and Lung Transplantation guidelines.The overall prevalence of Abs to SAgs was 22.71%, including 18% in chronic obstructive pulmonary disease (p = 0.033), 34% in IPF (p = 0.0006), 29% in CF (p = 0.0023), and 19.6% in other diagnoses (p = 0.044). The incidence of PGD (88% vs 54%, p0.05), DSA (70% vs 45%, p0.01), and BOS (90% vs 38% (p0.001) after LTx was significantly higher in patients with pre-LTx Abs to SAgs than without. Pro-inflammatory cytokines (interleukin-1β, interleukin-17, and interferon-γ) were elevated in patients who had pre-LTx Abs to SAgs, along with a reduction in anti-inflammatory interleukin-10.Patients with IPF and CF have the highest prevalence of Abs to SAgs. Patients with pre-existing Abs to SAgs are at increased risk for development of PGD, DSA, and BOS. Strategies to remove pre-existing Abs to SAgs should be considered to improve lung allograft outcome.

The enzyme-linked immunosorbent assay (ELISA) is a widely used technique for detecting antibodies (Abs) and is employed in clinical laboratories to identify Abs against various self-antigens-autoAb development and quantitation. This method relies on specific antigen-Ab interactions where one of the components is immobilized on a solid surface. Using this method, the concentrations of antigens or Ab present in the serum can be quantified with high specificity and accuracy. Here, we describe the detection of autoAbs to various self-antigens with different tissue restriction patterns which includes collagens, k-α1 tubulin, vimentin, and myosin. We also discuss their relevance in monitoring for rejection following solid organ transplantation.

Recent studies strongly suggest an increasing role for immune responses against self-antigens (Ags) which are not encoded by the major histocompatibility complex in the immunopathogenesis of allograft rejection. Although, improved surgical techniques coupled with improved methods to detect and avoid sensitization against donor human leukocyte antigen (HLA) have improved the immediate and short term function of transplanted organs. However, acute and chronic rejection still remains a vexing problem for the long term function of the transplanted organ. Immediately following organ transplantation, several factors both immune and non immune mechanisms lead to the development of local inflammatory milieu which sets the stage for allograft rejection. Traditionally, development of antibodies (Abs) against mismatched donor HLA have been implicated in the development of Ab mediated rejection. However, recent studies from our laboratory and others have demonstrated that development of humoral and cellular immune responses against non-HLA self-Ags may contribute in the pathogenesis of allograft rejection. There are reports demonstrating that immune responses to self-Ags especially Abs to the self-Ags as well as cellular immune responses especially through IL17 has significant pro-fibrotic properties leading to chronic allograft failure. This review summarizes recent studies demonstrating the role for immune responses to self-Ags in allograft immunity leading to rejection as well as present recent evidence suggesting there is interplay between allo- and autoimmunity leading to allograft dysfunction.

Significant progress has been made in preventing acute allograft rejection following solid organ transplantation resulting in improved allograft survival. However, long term function still remains disappointing primarily due to chronic allograft rejection. Alloimmune responses primarily defined by the development of antibodies (Abs) to donor mismatched major histocompatibility antigens during the post-transplantation period have been strongly correlated to the development of chronic rejection. In addition, recent studies have demonstrated an important role for autoimmunity including the development of Abs to organ specific self-antigens in the pathogenesis of chronic allograft rejection. Based on this, a new paradigm has evolved indicating a possible cross-talk between the alloimmune responses and autoimmunity leading to chronic rejection. In this review, we will discuss the emerging concept for the role of cellular and humoral immune responses to self-antigens in the immunopathogenesis of chronic allograft rejection which has the potential to develop new strategies for the prevention and/or treatment of chronic rejection.

NUP98-HOXA9 is the prototype of NUP98 fusion oncoproteins that cause acute myeloid leukemia. It consists of an N-terminal FG-rich portion of the nucleoporin NUP98 fused to the homeodomain region of the homeobox protein HOXA9, and acts as an aberrant transcription factor. To identify interacting partners of NUP98-HOXA9, we used a cytoplasmic yeast two-hybrid assay to avoid the nonspecific trans-activation that would occur with the traditional yeast two-hybrid assay due to the transactivating properties of NUP98-HOXA9. We identified amino-terminal enhancer of split (AES), a transcriptional regulator of the transducin-like enhancer/Groucho family as a novel interaction partner of NUP98-HOXA9. The interaction was confirmed by in vitro pulldown and co-immunoprecipitation assays and was shown to require the FG repeat region of NUP98-HOXA9. Immunofluorescence analysis showed that AES localizes primarily to the interior of the nucleus. AES also showed a strong interaction with wild-type NUP98. AES augmented the transcriptional activity of NUP98-HOXA9. In the presence of NUP98-HOXA9, AES caused an increase in long-term proliferation of primary human CD34+ cells with a marked increase in the numbers of primitive cells. These effects of AES were not observed in the absence of NUP98-HOXA9. AES knockdown diminished the transcriptional and proliferative effects of NUP98-HOXA9. AES caused a shift away from the erythroid lineage in cells expressing NUP98-HOXA9. These data establish AES as an interacting partner of NUP98-HOXA9 and show that it cooperates with NUP98-HOXA9 in transcriptional regulation and cell transformation.

Human breast cancer-associated antigen, mammaglobin-A (Mam-A), potentially offers a novel therapeutic target as a breast cancer vaccine. In this study, we define the CD8(+) cytotoxic T lymphocyte (CTL) response to Mam-A-derived candidate epitopes presented in the context of HLA-A24 (A*2402). HLA-A24 has a frequency of 72% in Japanese, 27% in Asian Indian, and 18% in Caucasian populations. Using a human leukocyte antigen (HLA)-binding prediction algorithm we identified 7 HLA-A24-restricted Mam-A-derived candidate epitopes (MAA24.1-7). Membrane stabilization studies with TAP-deficient T2 cells transfected with HLA-A2402 (T2.A24) indicated that MAA24.2 (CYAGSGCPL) and MAA24.4 (ETLSNVEVF) have the highest HLA-A24 binding affinity. Further, 2 CD8(+) CTL cell lines generated in vitro against T2.A24 cells individually loaded with Mam-A-derived candidate epitopes demonstrated significant cytotoxic activity against MAA24.2 and MAA24.4. In addition, the same CD8(+) CTL lines lysed the HLA-A24(+)/Mam-A(+) stable transfected human breast cancer cell lines AU565 and MDA-MB-361. However, these CTLs had no cytotoxicity against HLA-A24(-)/Mam-A(+) and HLA-A24(+)/Mam-A(-) breast cancer cell lines. In summary, our results define HLA-A24-restricted, Mam-A-derived, CD8(+) CTL epitopes that can potentially be employed for Mam-A-based breast cancer vaccine therapy to breast cancer patients with HLA-A24 phenotype.

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages. We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Regulation of gene transcription is a key feature of developmental, homeostatic, and oncogenic processes. The reverse recruitment model of transcriptional control postulates that eukaryotic genes become active by moving to contact transcription factories at nuclear substructures; our previous work showed that at least some of these factories are tethered to nuclear pores. We demonstrate here that the nuclear periphery is the site of key events in the regulation of glucose-repressed genes, which together compose one-sixth of the Saccharomyces cerevisiae genome. We also show that the canonical glucose-repressed gene SUC2 associates tightly with the nuclear periphery when transcriptionally active but is highly mobile when repressed. Strikingly, SUC2 is both derepressed and confined to the nuclear rim in mutant cells where the Mig1 repressor is nuclear but not perinuclear. Upon derepression all three subunits (α, β, and γ) of the positively acting Snf1 kinase complex localize to the nuclear periphery, resulting in phosphorylation of Mig1 and its export to the cytoplasm. Reverse recruitment therefore appears to explain a fundamental pathway of eukaryotic gene regulation.

Aim Bronchiolitis Obliterans Syndrome (BOS), clinically diagnosed as chronic rejection, has remained a major setback following lung transplantation (LT). However, its pathogenesis is poorly understood and no predictive biomarkers have been identified. Expression of microRNA (miRNA) in blood is deregulated in physiopathological conditions including immunological disorders. Methods In this study, we hypothesized that miRNA profiles from peripheral blood mononuclear cells (PBMCs) in LT recipients (LTxR) could identify patients at risk of BOS. MiRNA expression profiling was performed using TaqMan Human MicroRNA Array in PBMCs collected from three groups of LTxR: 10 stable, 10 who developed DSA and 10 who developed BOS. Results There were 98 miRNAs upregulated in LTxR with BOS compared to that of stable transplants (relative fold > 2.5); and 32 miRNAs downregulated with BOS (relative fold > 2.5). In addition, we found that there were 16 miRNAs upregulated (including miR-99a, miR-147, and miR-124 ∗ ) with BOS compared to that of DSA; and 5 miRNAs downregulated (including miR-34b, miR-363 ∗ , and miR-155 ∗ ) in BOS compared to DSA, indicating that these miRNAs may play a role in BOS with DSA. We validated those differential miRNAs in independent PBMCs and broncheoalveolar lavage cells using Taqman real-time PCR. In addition we focused on one miRNA, miR-99a, which caused upregulation of 2.8-fold in LTxR with BOS compared to that of DSA. Bioinformatics prediction indicated that miR-99a can mediate gene-expression regulation of TNIK (TRAF2 and NCK interacting kinase) and TRAF4 (TNF receptor-associated factor 4), which were involved in regulating NF- κ B signaling pathway. Conclusions In conclusion our results demonstrate that differential miRNA expression profile has the potential to predict BOS and development of DSA following LT.

Aim Hepatitis C virus (HCV) mediated liver diseases are one of the major health issues in United States and worldwide. This study is to identify the role of HCV induced changes in microRNAs (miRNA) that controls various cell surface receptors and gene regulatory complexes involved in patients with HCV mediated inflammation and fibrosis. Methods Computational sequence analyses and reporter assays using the CCL2 promoter region were carried out to analyze transcription factors. Gene expression analyses were done using q-PCR. Gene overexpression and knockdowns in human hepatocytes were carried out using cDNA plasmids and siRNAs. Chromatin (ChIP) and protein (CoIP) immunoprecipitations were done to establish interactions between transcription factors and DNA. Results We report here that down-regulation of miRNA-107 and miRNA-449a following HCV infection in patients modulate expression of the CCL2, an inflammatory chemokine up-regulated in patients with chronic liver diseases, by targeting components of the IL6R complex. Computational analysis for DNA bound transcription factors in the CCL2 promoter identified adjacent binding sites for CCAAT/CEBP α , spleen focus forming virus, proviral integration oncogene (SPI1/PU.1), and STAT3. We demonstrate that CEBP α , PU.1 and STAT3 interacted with each other physically to co-operatively bind to the promoter and activate CCL2 expression. Analysis of IL6R and JAK1 expression in HCV patients showed significant up regulation when there is impaired miRNA-107 and miRNA-449a expression along with up regulation of PU.1 and STAT3 but not CEBP α . miRNA-449a and miRNA-107 target IL6R and JAK1 respectively, inhibit IL6 signaling and impair STAT3 activation. Conclusions Our results demonstrate a novel gene regulatory mechanism wherein HCV induced changes in miRNAs (miRNA-449a and miRNA-107) regulate CCL2 expression by activation of the IL6 mediated signaling cascade in HCV patients which we propose will result in HCV mediated induction of inflammatory responses and fibrosis.

The chimeric oncoprotein NUP98-HOXA9 results from the t(7;11)(p15;p15) chromosomal translocation and is associated with acute myeloid leukemia. It causes aberrant gene regulation and leukemic transformation through mechanisms that are not fully understood. NUP98-HOXA9 consists of an N-terminal portion of the nucleoporin NUP98 that contains many FG repeats fused to the DNA-binding homeodomain of HOXA9. We used a Cytotrap yeast two-hybrid assay to identify proteins that interact with NUP98-HOXA9. We identified Dynein Light Chain 1 (DYNLT1), an integral 14 KDa protein subunit of the large microtubule-based cytoplasmic dynein complex, as an interaction partner of NUP98-HOXA9. Binding was confirmed by in vitro pull down and co-immunoprecipitation assays and the FG repeat region of NUP98-HOXA9 was shown to be essential for the interaction. RNAi-mediated knockdown of DYNLT1 resulted in reduction of the ability of NUP98-HOXA9 to activate transcription and also inhibited the ability of NUP98-HOXA9 to induce proliferation of primary human hematopoietic CD34+ cells. DYNLT1 also showed a strong interaction with wild-type NUP98 and other nucleoporins containing FG repeats. Immunofluorescence analysis showed that DYNLT1 localizes primarily to the nuclear periphery, where it co-localizes with the nuclear pore complex, and to the cytoplasm. Deletion studies showed that the interactions of the nucleoporins with DYNLT1 are dependent predominantly on the C-terminal half of the DYNLT1. These data show for the first time that DYNLT1 interacts with nucleoporins and plays a role in the dysregulation of gene expression and induction of hematopoietic cell proliferation by the leukemogenic nucleoporin fusion, NUP98-HOXA9.

Purpose Immune responses to lung self-antigens (SAgs), Kα1Tubulin (Kα1T) and Collagen V (ColV), have been implicated as risk factors for chronic rejection following lung transplantation (LTx). The goal is to determine the prevalence of pre-existing antibodies (Abs) to these SAgs in diseases leading to organ failure and investigate their role in primary graft dysfunction (PGD) and chronic rejection. Methods and Materials Pre- and post-Tx sera from 317 LTx patients for diagnoses (COPD: 161, IPF: 50, CF: 55 and Other: 51) between 2000 and 2011 at Washington University (n: 296) and Cleveland Clinic (n: 21) were analyzed for donor specific Abs (DSA) by Flow PRA, Abs to ColV and Kα1T by ELISA and cytokine levels by LUMINEX assay. Subsequent diagnoses of PGD and bronchiolitis obliterans syndrome (BOS) were made in accordance with ISHLT guidelines. Results Pre-Tx, 18% with COPD, 44% with IPF (P 0.7) had Abs to Sags. The incidence of PGD was higher in patients with pre-Tx Abs to these Sags compared to those without (80-88% to 42-54%, p Conclusions Patients with CF and IPF demonstrated a significantly increased incidence of Abs to lung Sags, Kα1T and ColV. Patients with pre-Tx Abs to Sags were at increased risk for developing PGD, DSA and BOS, therefore strategies to deplete pre-existing Abs to Sags should be considered in order to improve outcomes after LTx.

Aim HCV mediated liver diseases is a major health problem both in the United States and worldwide. This study is to identify the role of HCV induced changes in microRNAs in mediating inflammation leading to autoimmunity and allograft fibrosis. Methods Using Genomewide microarray analyses and qPCR dysregulation of miR-449a and NOTCH1 in the livers of HCV patients were identified. Computational sequence analyses and reporter assays using the YKL40 promoter region were done to analyze transcription factors. Chromatin and protein immunoprecipitations were done to establish interactions between transcription factors and DNA. Results Ykl40 is up regulated in patients with chronic liver diseases with fibrosis and cirrhosis. YKL40 is regulated by miR-449a in human hepatocytes in a TNFα dependent manner. Putative binding sites for transcription factors were defined for YKL40 promoter which included NF Kappa B/P65 and CEBPα which play a key role in hepatic fibrosis. siRNA mediated knockdown of NOTCH1 , a direct target of miR-449a, significantly reduced YKL40 expression. NOTCH1 expression was found to be upregulated of HCV patients. Further, NOTCH1 facilitated nuclear localization of P65 in response to TNFα. Co-expression of P65 and CEBPα results in higher level of activation of the YKL40 promoter compared to either alone indicating co-operation between these two in regulating YKL40 . Immunoprecipitation experiments established interaction between P65 and CEBPα in hepatocytes and sequence specific binding of P65 and CEBPα to the YKL40 promoter. Conclusions Taken together our results demonstrate that miR-449a plays an important role in modulating expression of the inflammatory biomarker Ykl40 through targeting the components of the NOTCH signaling pathway in response to HCV infection. Therefore, defining transcriptional regulatory mechanisms which control inflammation following HCV will be important towards developing strategies to prevent hepatic fibrosis following liver transplantation of HCV recipients.

Fibrin sealants are used in surgical procedures as an adjunct to sutures and staples to prevent fluid leakage and bleeding. These applications require extended longevity of fibrin sealant. It takes about 2 weeks for a typical surgical wound to heal, whereas fibrin sealant typically survives only 2 to 3 days in vivo due to its degradation by proteases such as plasmin present in the tissue micro-environment. Tranexamic acid (TA) is a well-known inhibitor of proteases including plasmin present in the blood. However, water soluble TA rapidly diffuses out of the sealant under in vivo conditions. Therefore, an investigation aimed to study the mechanism of sustained release of TA has been undertaken using microspheres of an absorbable PLGA (poly-lactic-glycolic-acid) polymer. TA particles of average dimension 3 micron are impregnated at a loading of 5% and 30% in average 40 micron PLGA microspheres. These are verified by Scanning Electron Microscopy / Energy Dispersive X-Ray spectroscopy. Confocal Raman spectroscopy shows that TA is distributed desirably more towards the core of the microsphere. Release study of TA by LC-MS using direct auto-sampler injection into Mass Spectral detector with ESI MS positive ion mode ionization shows that about 70% of TA from 5% TA Loaded / PLGA microspheres is released continuously over a period of 144 hours (or 6 days). Rate of TA release significantly slows down after the initial 72 hours. A gravimetric in vitro method has been developed to monitor the degradation of the sealant in a plasmin containing medium at pH 7.4 and 37 0C. The plasmin medium has been changed each day continuously up to 4 days. The proof-of-principle of improving longevity of fibrin sealant has been demonstrated with both 5% and 30% TA loaded / PLGA microspheres compared to the control.