659 TLR4 and TRIF Play Positive and Negative Roles in Ad Induced Innate Immunity In Vivo 657 Construction of a High Performance Lentiviral cDNA Library Derived from Human Fetal Liver Ryo Kurita,'''' Tats[.]
657 Construction of a High Performance Lentiviral cDNA Library Derived from Human Fetal Liver Ryo Kurita,' Tatsuo Oikawa,' Michiyo Okada,I Tomoko Yokoo,' Norio Komatsu,' Yoshikuni Tanioka,' Erika Sasaki,' Kenzaburo Tani.' I Department ofMolecular Genetics, Inst ofBioregulation, KYlls11/I Univ., Fukuoka, Japan; 2Department ofHematology, Yamanashi University School ofMedicine, Kohfu, Japan; J Division ofAnimal Experimentation, Central Institute ofExperimental Animals, Kawasaki, Japan (Background) Recent developmentof the gene therapeutic strategies is remarkable and will certainly open new era to cure various intractable diseases Based on current gene therapeutic strategies, regenerative medicine has also been developed Namely, some specific gene transfer has been reported to facilitate the differentiation of stem cells including embryonic stem (ES) cells to various functional cells We have recently reported lentiviral gene transfer oftaillscl gene differentiate nonhuman primate common marmoset ES cells to hematopoietic cells efficiently without the support of any stromal cells (Kurita et ai, Stem Cells 2006) Translation of the result to human ES cells, however, requires further in vitro and in vivo experiments Also, we will possibly need to add the second or third gene to more efficiently differentiate primate ES cells to various hematopoietic cells and functionalgene screening targeting all-inclusive candidate genes strongly associated with early hematopoiesis of primates For this purpose, in this study we constructed high performance human fetal liver eDNA expression library using Icntiviralvector.(Materials and Methods) Human fetal liver mRNA was purchased from BD clonetech and lentiviral human fetal liver eDNA librarywas constructedutilizinggateway stystem(Invitrigen) following the manufacturer's protocol 293'1' cells, adenovirus E1A transformed human renal epithelial cell line, were tranduced by the lentiviral human fetal liver eDNA library Transduced 293 'I' cells were plated by limiting-dilution method and each clone was picked up and examined for the integrated eDNA using standard genomicPCR method (Results) The library consisted of more than IOA8 individual clones and the average insert size was more than kb DNAsequence analysis for each inserted cDNA revealed that more than 60% of them contained full-length coding region of proteins includingcytokine receptors, cytoplasmicproteins,proteininhibitors and nuclear factors Transduction efficiency targeting 293 'I' cells was 100% and average size ofthc integrated cDNAs was about 1.1 kb (for the present) (0 iseussion)Our previousstudies demonstrated the lentivirus vector can transducer human hematopoietic stem cells as well as nonhuman primate ES cells Also, lentivirus vector can transduce proliferating cells as well as donn ant cells, and our Ientivirus human fetal liver eDNA expression library is considered to be very helpful tool to accelerate the discovery of novel genes strongly related to early hematopoiesis and possibly hepatopoiesis Our nexttarget cells includehuman ES cells and cytokinc-depcndent cell lines to identify novel hematopoiesis-related genes 658 The Erythrocyte Immunity Research on Chronic Venous Insufficiency of Lower Limb Zhang Lan, Zhang Baigen I Vascular Department, Jiaotong University School ofMedicine, Shanghai, China Chronic venous insufficiency(CVI) of lower limb is among the most prevalent of vascular discases.A large body of subsequent work indicates that CVI is the result of the systemic inflammatory processes Otherwise,there have been rheological abnormalities reported in CVI,mainly an increase of erythrocyte aggregability,which probably take part in the pathophysiology of S252 the disease.It is clear that erythrocytes.as the largest quanitity blood cells in ciucluation,expressingvarious innate immune receptor,play an important role in innate immune reaction and specific immune reaction.additionally participate in controlling immunity Blood cell innate immune reaction and mechanism of CVI To determine the CD35,Fy6,CD44 expression on erythrocytes,CD I Ib expression on neutrophils,CD 14 expression on rnonocytes in patients with CVI by FCM,then compare them with the normal control IL-8 and IL-IO levels were measured by ELISA analysis from blood plasma.These changes will have great clinical value tojudge the genesis,development and turnover of CVI.The interest in the inflammatory reaction participation in the mechanism of CVI is helpful to offer theoretic basic for the research of venous active drug on therapeutic effect Our result shows that the fluorescence intensity expression of CD35 and Fy6 on erythrocytes are lower associated with the development of CVI Meanwhile,CD II b expression on neutrophils and CDI4 expression on monocytes are higher at the later stage of CVI,accompanied by an inbalance of proinflammatory mediator IL-8 and anti-inflammatory mediator IL-IO.These excessive inflammatory response may lead to the local tissue and microvascular damage of lower limb;The symptom oflipodennatosclerosis belong to C4 can play an important role to devide the severity degree of CVI.On the other hand,the capability of erythrocytes innate immunity in CVI is lower than in normal control,it become more obvious by added with inactive S.gn tumor cell which acted as antigen.Less expression of CD35 and Fy6 on erythrocytes weakened the modulation function leading to impropriety inflammatory intensity of neutrophils and cytokine.Vcnous endothelium and venous valve were damaged progressively.Once deep venous system occurred reflux,theimmuneadherence function and inflammatory regulating capacity of erythrocyte were reduced Higer CD14expression of'rnonocytes and the inbalance oflL-8 and IL-IO aggravately injured vein valve function IMMUNE RESPONSES TO AoENOVIRAL VECTORS 659 TLR4 and TRIF Play Positive and Negative Roles in Ad Induced Innate Immunity In Vivo Daniel M Appledorn,' Zachary Hartman,' Jeannine M Scott,' Andrea Amalfitano.' 'Microbiology and Molecular Genetics, Michigan State University, East Lansing; 2Division ofMedical Genetics, Department of Pediatrics, Duke University Medical Center; Durham The use of Adenovirus (Ad) based vectors for both gene transfer applications and vaccine development continues to be promising Our recent studies have shown that aggressive innate immune responses following intravenous administration of adenovirus are dependent on both alternative and classical complement pathways as well as the TLR adaptor protein MyD88 This response is in part characterized by thrombocytopenia, and increased plasma levels of numerous cytokines and chemokines Recent studies in TLR9 and MyD88 deficient mice have revealed their partial role in regulating many of these Ad induced innate immune responses In this study, we show that TRIF, a TLR adaptor protein mediating both TLR3 and TLR4 signaling responses to other pathogens, also plays a significant role in Ad induced thrombocytopenia, induction of cytokine and chemokines, as well as profound changes in the mouse transcriptome response to Ad mediated gene transfer Specifically, in response to high titer adenovirus administration, we show that TRIF deficient mice have reduced serum levels of IL-Ia, IL-12, G-CSF, MCP-I and RANTES at both high (1.5 x 10" vp/mouse) and low (7.5 x 1010 vp/mousc) doses of adcnoviral administration, relative to identicalAd treatmentsofTRIF+ mice Wealso observed that TRIF is required for high serum IL-6 levels induced after high dose Ad treatment, but is not required for this response at low Molecular Therapy Yohunc 15 Supplcrncnr t, ~b )' 2007 Copyright © Oll ie 'uuericm Society o f Gen e Therapy doses Furthermore, we found that TRIF plays a significant role in attenuating secretion of KC shortly after higher dose Ad injection Ad induced thrombocytopenia was also avoided in TRIF deficient mice compared to TRIF+ mice inj ected at low doses Array based analysis ofthe mouse liver transcriptome after Ad injection revealed over 1900 gene expression differences in Ad treated TRIF deficient mice, relative to Ad treated normal mice These genes include many expected changes i.e TRAF and TAK-binding proteins as well as numerous transcripts not otherwise implicated in immune response pathways Since our previous publication confirmed MyD88 as another TLR adaptor protein important in several Ad induced immune responses', and the TLR4 receptor utilizes both TRIF and MyD88 as adaptor proteins for TLR4 mediated signaling after LPS exposure, we next asked whether TLR4 mediates the TRIF and MyD88 dependent Ad induced immune responses we were studying While thrombocytopenia and most cytokinc and chernokinc responses were not significantly altered in Ad treated mice deficient in TLR4 signaling, we actually observed an inhibitory role forTLR4 in secretion of both IL-6 and IL-12(p70) This study clearly reveals the complexity of innate immune responses to high titer Ad injection , and suggests negative roles of TLR4 , and both positive and negative roles for TRIF in these responses These roles must be taken into account when considering usc of inhibitors or activators ofTLR signaling in gene transfer applications or vaccine development generally, and specifically in the context of Ad based gene transfer approaches 'Hartman et.al: J Virology(81)pp:1796-1812:2007 660 Adenovirus Induced Innate Immune Responses Are Mediated in Part by the Presence of Natural Antibodies in Ad NaIve Mice Jeannine M Scott, I Tyler T Voss,I Daniel M Appledom, I Andrea Amalfitano 1.2 I Department ofMicrobiology and Molecular Genetics, Michigan State University, East Lansing, M/; 2Department ofPediatrics, Michigan State University, East Lansing, Ml Intravenous, high-dose Adenovirus (Ad) injection leads to a rapid and robust innate immune response, resulting in plasma elevations of several proinflammatory chemokines and cytokines, and thrombocytopenia Our previous studies have confirmed that the molecular basis of these responses is dependent upon Ad interactions with the complement system The complement dependent anti-Ad immune response is complex and dependent upon interactions with both the classical and alternative pathways of complement activation (see Scott et.al this meeting) It is known that antibodies (specific and natural) are involved in the activation of complement systems, eg antigen-antibody complexes interact with Clq and activate the elassical pathway of complement, while natural antibodies are involved in stab ilizing an alternative pathway C3 convertase We wished to determine if natural antibodies present in Ad narve mice may be directly involved in the innate immune response to Ad vectors We therefore injected an Ad vector expressing LacZ into immunoglobulin deficient (SCI D) mice, and assessed several known Ad induced innate immune responses shortly after infection, relative to identical injections into wild-type (WT) mice Interestingly, Ad injections into scm mice did not result in thrombocytopenia This correlated with significantly higher levels ofLacZ expression in the livers of scm mice, relative to Ad-injected WT mice Investigation of this latter phenomenon, found no significant difference in the Ad genome copy number per hepatocyte, (nor hepatotoxicity) suggesting the CMV promoter utilized to drive LacZ expression in this construct had enhanced activity in the livers of scm mice We also noted higher plasma levels of IL-6 at hpi in the Ad injected scm mice, but no increase in IL-12 or G-CSF at 1,6 or 24 hpi, again relative to Ad injected WT mice In an attempt to directly implicate immunoglobulins as causative in some ofthese responses, Molecular Therapy Volume15 Supplement I ~ br 2007 Copyright © The American Soci ety o r Gene Therapy we pretreated scm mice with immunoglobulins derived from Ad nalve mice Immunoglobulin reconstituted scm mice now developed thrombocytopenia after Ad injection , similar to Ad-injected WT mice, demonstrating a requirement for circulating antibody in this important Ad induced response Our previous studies have implicated the C3 and Factor B proteins ofthe alternative complement pathway as also necessary fortheAd induction ofthrombocytopenia These new results indirectly suggest that Ads are interacting with C3, Factor B, and natural antibodies to cause thrombocytopenia in Ad naive mice These studies suggest that natural antibodies present in Ad narve subjects may both positively and negatively regulate several pro-inflammatory innate immune responses rapidly elicited by Ad vectors 661 Dose-Limiting Toxicities Following Systemic Administration of Non-Human Primate Derived Adenoviral Vectors to Cynomolgus Macaques Suryanarayan Sornanathan,' Guang-Ping Gao,' Martin Lock; Peter Bell, I Rebecca Grant, I Soumitra Roy,' Arbansjust Sandhu,' James M Wilson: 'Gene Therapy Program Department ofPathology and Laboratory Medicine, University ofPennsylvania, Philadelphia PA Non-human primate derived adenoviral vectors are currently being pursued in pre-clinical trials as immunizing agents against diseases such as HIV, Ebola, and SARS However, adenoviral vectors based on human adenovirus type 5, a sub-group B virus , induce a dose-dependent systemic inflammatory response when administered intravenously in species ranging from mice to humans In the present study we compared the inflammatory response following intravenous administration of E lIE3-deleted non-human primate derived adenoviruses, AdC6 and AdC7, expressing b-galactosidase in cynomolgus macaques (Macaca fascicularis) The hematological, biochemical, histochemical and pathological findings were compared to those obtained from animals administered HuAd5 vectors Animals were dosed with increasing amounts ofAdC6 vectors and with higher doses of (2: 5* 1012 p/kg) ofAdC7 and HuAd5 vectors The actual administration of vee tor was uneventful and the only symptoms observed upon recovery from anesthesia were nausea, hypothermia or an elevated heart rate A striking difference between non-human primate and HuAd5 vector administered animals was the development of petechiae by 24 hr in the latter that resolved by day All vectors induced a robust inflammatory response characterized by transient leukopenia, thrombocytopenia, and elevations in serum levels of' pro-inflammatory cytokines, When compared to HuAd5, the non-human primate derived adenoviruses induced much higher levels of pro-inflammatory cytokines IL-6 and lFN-a Two animals that received high dose AdC6 vectors died or had to be euthanized within hrs of vector for reasons that were not clear; all other animals survived to the scheduled necropsy time Bio-distribution of vectors indicated that infectious AdC6 and AdC7 viral partieles were rapidly cleared from peripheral blood (24 hr) when compared to HuAd5 (>day 3) Furthermore, liver transaminases (AST, ALT) were elevated in all animals In addition , vector genomes could be detected in several tissues including bone marrow, spleen, skeletal muscle, heart and brain Histochemical analysis indicated that only AdC7 and HuAd5 vectors transduced cells in the liver and spleen , although , all three vectors induced infiltrates in liver (HuAd5>AdC7o;AdC6) Markers for disseminated intravascular coagulopathy (DIC) were increased following all three vector administrations These data indicate a disconnect between the extent of transduction and the onset ofa systemic inflammatory response and strongly suggest that the initial inflammatory response is primarily vector induced These studies represent an important step towards understanding the safety or non-human primate derived adenoviral vector administrations S253 ... titer Ad injection , and suggests negative roles of TLR4 , and both positive and negative roles for TRIF in these responses These roles must be taken into account when considering usc of inhibitors... as another TLR adaptor protein important in several Ad induced immune responses'', and the TLR4 receptor utilizes both TRIF and MyD88 as adaptor proteins for TLR4 mediated signaling after LPS exposure,... whether TLR4 mediates the TRIF and MyD88 dependent Ad induced immune responses we were studying While thrombocytopenia and most cytokinc and chernokinc responses were not significantly altered in Ad