Zongdong Li, Ph.D.

The inhibition effect of anti-GPIIIa49-66 antibody on megakaryocyte differentiation
Pan, R; Wang, J; Nardi, M A; Li, Z
2011 Aug 31;106(3):484-490, Thrombosis & haemostasis
We previously reported that patients with early-onset HIV-1 ITP developed a unique anti-platelet integrin GPIIIa antibody against the GPIIIa49-66 epitope. Anti-GPIIIa49-66 antibody-induced platelet fragmentation requires sequential activation of the platelet 12-lipoxygenase (12-LO) and NADPH oxidase to release reactive oxygen species (ROS). 12-LO is upstream of the NADPH oxidase pathway and 12(S)-HETE, the product of 12-LO, induces the same oxidative platelet fragmentation as anti-GPIIIa49-66. Since the megakaryocyte (MK) is the progenitor cell for platelets, we have investigated the effect of anti-GPIIIa49-66 on MK differentiation and, in particular, the potential role of anti-GPIIIa49-66 induced ROS in this process. We first show that polyclonal anti-GPIIIa49-66 antibody isolated from HIV-1 ITP patients inhibits MK proliferation 2.5-fold in in vitro culture of human cord blood CD34+ cells driven by thrombopoietin (TPO). We also observe a three-fold decrease in the number of MK colony-forming units in the presence of a human monoclonal anti-GPIIIa49-66 antibody. However, we could not detect ROS release in DCFH-loaded mouse megakaryoblastic cells L8057 treated with anti-GPIIIa49-66 antibody. In addition, 12(S)-HETE does not inhibit the in vitro differentiation of L8057 cells induced by TPO. In fact, we found a dose dependent increase in the percentage of CD41 positive cells (from 17.1% to 48.7%) in in vitro culture of L8057 cells treated with various concentrations of H2O2 (from 5 to 20 muM). We therefore conclude that the anti-GPIIIa49-66 antibody inhibits MK differentiation through beta3 integrin signalling independent of ROS release
— id: 136997, year: 2011, vol: 106, page: 484, stat: Journal Article,

The effect of decitabine on megakaryocyte maturation and platelet release
Wang, J; Yi, Z; Wang, S; Li, Z
2011 Aug 1;106(2):337-343, Thrombosis & haemostasis
Thrombocytopenia is a common feature of myelodysplastic syndromes (MDS). 5-aza-2'-deoxycytidine (decitabine) has been used to treat MDS with an approximately 20% response rate in thrombocytopenia. However, the mechanism of how decitabine increases platelet count is not clear. In this study, we investigated the effect of decitabine on megakaryocyte maturation and platelet release in the mouse. The effect of decitabine on megakaryocyte maturation was studied in an in vitro megakaryocyte differentiation model utilising mouse bone marrow cells and mouse megakaryoblastic cell line L8057. Decitabine (2.5 muM) is able to induce L8057 cells to differentiate into a megakaryocyte-like polyploidy cells with positive markers of acetylcholinesterase and alphaIIb integrin (CD41). Higher expression of alphaIIb integrin was also found in primary mouse bone marrow cells and human cord blood CD34+ cells cultured with both thrombopoietin and decitabine as compared to thrombopoietin alone. In addition, we noted a 30% platelet count increase in Balb/c mice 12 hours after the injection of decitabine at a clinically relevant dose (15 mg/m2), suggesting a rapid platelet release from the spleen or bone marrow. Our data suggest that decitabine increases platelet counts by enhancing platelet release and megakaryocyte maturation
— id: 135560, year: 2011, vol: 106, page: 337, stat: Journal Article,

HIV-1 Tat-induced platelet activation and release of CD154 contribute to HIV-1-associated autoimmune thrombocytopenia
Wang, J; Zhang, W; Nardi, M A; Li, Z
2011 Mar;9(3):562-573, Journal of thrombosis & haemostasis : JTH
Summary. Background: Enhanced platelet activation in human immunodeficiency virus (HIV)-1-infected patients has been reported and shown to strongly correlate with plasma viral load. Activated platelets are known to express and to release a variety of proteins that can modulate the immune system. Specifically, platelet-derived CD154 has been shown to be directly involved in the development of autoimmune thrombocytopenia (ITP). The mechanism by which HIV-1 infection leads to platelet activation and the effect of this activation on the development of HIV-1 ITP, however, is not fully understood. Objective: We have investigated the effect of HIV-1 Trans activating factor (Tat) on platelet activation. Results: We report that HIV-1 Tat directly interacts with platelets and induces platelet activation resulting in platelet micro-particle release. This activation by Tat requires the chemokine receptor CCR3 and beta3-integrin expression on platelets, as well as calcium flux. Tat-induced activation of platelets releases platelet CD154, an immune modulator. Enhanced B-cell activity is found in mouse spleen B cells co-cultured with platelets treated with Tat in vitro. An early antibody response against adenovirus is found in Tat-injected mouse immunized with adenovirus, suggesting an enhanced immune response in vivo. Conclusions: We have described a role of Tat-induced platelet activation in the modulation of the immune system, with implications for the development of HIV-1-associated thrombocytopenia
— id: 127226, year: 2011, vol: 9, page: 562, stat: Journal Article,

The Effect of Anti GPIIIa49 66 Antibody on Megakaryocyte Differentiation
Wang, Jianhui; Pan, Ruimin; Nardi, Michael A.; Li, Zongdong
2010 NOV 19 ;116(21):615-615, Blood
— id: 130853, year: 2010, vol: 116, page: 615, stat: Journal Article,

Specific cross-reaction of anti-dsDNA antibody with platelet integrin GPIIIa49-66
Zhang, Wei; Dang, Suying; Wang, Jianhui; Nardi, Michael A; Zan, Hong; Casali, Paolo; Li, Zongdong
2010 Dec;43(8):682-689, Autoimmunity
Anti-platelet autoantibodies are frequently found in systemic lupus erythematosus (SLE) patients and contribute to the development of SLE-associated immunologic thrombocytopenia (SLE-ITP). Although the correlation of anti-dsDNA autoantibody with platelet-associated antibody has been reported, the potential mechanism underlying such a correlation is incompletely understood. We have reported that anti-platelet integrin GPIIIa49-66 (CAPESIEFPVSEARVLED) autoantibodies play a major role in the development of HIV-1-related thrombocytopenia (HIV-1-ITP). The strong negative charge of GPIIIa49-66 prompts us to investigate whether GPIIIa49-66 can be an epitope mimicking dsDNA. We report here that anti-GPIIIa49-66 antibodies are found in three out of nine SLE-ITP patients. Double-stranded (ds) DNA competitively inhibited the binding of purified patient anti-dsDNA antibodies to GPIIIa49-66 peptide. Both polyclonal and monoclonal anti-GPIIIa49-66 antibodies are able to cross-react with dsDNA. Consistent with previous reports, the DNA binding activities of anti-GPIIIa49-66 antibodies are mainly dependent on the positively charged amino acid in the heavy-chain complementarity-determining region 3 (HCDR3). The HCDR3 of human SLE anti-dsDNA monoclonal antibody (mAb) 412.67 demonstrates a similar positively charged amino acid chain orientation compared with that of anti-GPIIIa49-66 mAb A11, and it cross-reacts with GPIIIa49-66 peptide. Purified anti-GPIIIa49-66 antibodies from SLE-ITP patients are able to induce platelet fragmentation in vitro and to induce thrombocytopenia in vivo. Thus, our data suggest that specific epitope cross-reaction between GPIIIa49-66 and dsDNA could be a mechanism involved in the development of SLE-associated thrombocytopenia
— id: 114820, year: 2010, vol: 43, page: 682, stat: Journal Article,

Dissolution of arterial platelet thrombi in vivo with a bifunctional platelet GPIIIa49-66 ligand which specifically targets the platelet thrombus
Zhang, Wei; Li, Yong-Sheng; Nardi, Michael A; Dang, Suying; Yang, Jing; Ji, Yong; Li, Zongdong; Karpatkin, Simon; Wisniewski, Thomas
2010 Sep 30;116(13):2336-2344, Blood
Patients with HIV-1 immune-related thrombocytopenia have a unique antibody (Ab) against integrin GPIIIa49-66 capable of inducing oxidative platelet fragmentation via Ab activation of platelet nicotinamide adenine dinucleotide phosphate oxidase and 12-lipoxygenase releasing reactive oxygen species. Using a phage display single-chain antibody (scFv) library, we developed a novel human monoclonal scFv Ab against GPIIIa49-66 (named A11) capable of inducing fragmentation of activated platelets. In this study, we investigated the in vivo use of A11. We show that A11 does not induce significant thrombocytopenia or inhibit platelet function. A11 can prevent the cessation of carotid artery flow produced by induced artery injury and dissolve the induced thrombus 2 hours after cessation of blood flow. In addition, A11 can prevent, as well as ameliorate, murine middle cerebral artery stroke, without thrombocytopenia or brain hemorrhage. To further optimize the antithrombotic activity of A11, we produced a bifunctional A11-plasminogen first kringle agent (SLK), which homes to newly deposited fibrin strands within and surrounding the platelet thrombus, reducing effects on nonactivated circulating platelets. Indeed, SLK is able to completely reopen occluded carotid vessels 4 hours after cessation of blood flow, whereas A11 had no effect at 4 hours. Thus, a new antithrombotic agent was developed for platelet thrombus clearance
— id: 114164, year: 2010, vol: 116, page: 2336, stat: Journal Article,

C-terminal ADAMTS-18 fragment induces oxidative platelet fragmentation, dissolves platelet aggregates and protects against carotid artery occlusion and cerebral stroke
Li, Zongdong; Nardi, Michael A; Li, Yong-Sheng; Zhang, Wei; Pan, Ruimin; Dang, Suying; Yee, Herman; Quartermain, David; Jonas, Saran; Karpatkin, Simon
2009 Jun 11;113(24):6051-6060, Blood
Anti-platelet integrin GPIIIa49-66 Ab induces complement-independent platelet oxidative fragmentation and death by generation of platelet peroxide following NADPH oxidase activation. A C-terminal 385 amino acid fragment of ADAMTS-18 (A Disintegrin Metalloproteinase with Thrombospondin motifs produced in endothelial cells) induces oxidative platelet fragmentation in an identical kinetic fashion as anti-GPIIIa49-66 Ab. Endothelial cell ADAMTS-18 secretion is enhanced by thrombin and activated by thrombin cleavage to fragment platelets. Platelet aggregates produced ex vivo with ADP and fibrinogen are destroyed by the C-terminal ADAMTS-18 fragment. Anti-ADAMTS-18 Ab shortens the tail vein bleeding time. The C-terminal fragment protects against FeCl3 induced carotid artery thrombosis as well as cerebral infarction in a post-ischemic stroke model. Thus, a new mechanism is proposed for platelet thrombus clearance, via platelet oxidative fragmentation induced by thrombin cleavage of ADAMTS-18
— id: 93983, year: 2009, vol: 113, page: 6051, stat: Journal Article,

Decitabine Increase Platelet Count by Two Mechanisms
Wang, JH; Li, ZD
2009 NOV 20 ;114(22):1361-1361, Blood
— id: 109998, year: 2009, vol: 114, page: 1361, stat: Journal Article,

Platelet Derived CD154 Induced by Tat Contributes to HIV-1 Associated Autoimmune Thrombocytopenia
Wang, JH; Zhang, W; Li, ZD
2009 NOV 20 ;114(22):1046-1046, Blood
— id: 109993, year: 2009, vol: 114, page: 1046, stat: Journal Article,

Role of molecular mimickry of hepatitis C-virus (HCV) protein with platelet GPIIIa in hepatitis C-related immunologic thrombocytopenia
Zhang, Wei; Nardi, Michael A; Borkowsky, William; Li, Zongdong; Karpatkin, Simon
2009 Apr 23;113(17):4086-4093, Blood
HIV-1-ITP patients have a unique Ab against platelet GPIIIa49-66 capable of inducing oxidative platelet fragmentation in the absence of complement. HIV-1-seropositive drug abusers are more prone to develop immune thrombocytopenia (HIV-1-ITP) than non-drug abusers and have a higher coinfection with Hepatitis C virus than non-drug abusers (90% vs 30%). Molecular mimickry with a Hepatitis C protein was sought by screening a phage peptide library with anti-GPIIIa49-66 antibody as bait for peptides sharing homology sequences with HCV protein. Several phage peptide clones had 70% homology with HCV protein. Sera from dually infected thrombocytopenic patients with HCV and HIV-ITP reacted strongly with 4 non-conserved peptides from HCV core-envelope 1. Reactivity correlated inversely with platelet count, r(2)=0.7, p<0.01. Ab raised against peptide PHC09 in GPIIIa(-/-) mice induced severe thrombocytopenia in wild-type mice. Affinity-purified IgG against PHC09 induced oxidative platelet fragmentation in vitro. Drug abusers dually infected with HCV and HIV-1 had a greater incidence and severity of thrombocytopenia as well as greater incidence and titer of anti-GPIIIa49-66/PHC09 Ab. NZB/W F1 mice injected with recombinant core envelope 1 developed Ab vs PHC09 and significantly decreased their platelet count, p<0.001. Thus, HCV core envelope 1 can induce thrombocytopenia by molecular mimicry with GPIIIa49-66
— id: 95182, year: 2009, vol: 113, page: 4086, stat: Journal Article,

Platelet fragmentation requires a specific structural conformation of human monoclonal antibody against beta3 integrin
Li, Zongdong; Nardi, Michael A; Wu, Jing; Pan, Ruimin; Zhang, Wei; Karpatkin, Simon
2008 Feb 8;283(6):3224-3230, Journal of biological chemistry
We have described an autoantibody against beta3 (GPIIIa49-66), a region of platelet integrin alphaIIbbeta3 that is unique. It induces platelet fragmentation in the absence of complement via antibody activation of platelet NADPH oxidase and 12-lipoxygenase to release reactive oxygen species, which destroy platelets. To study the mechanism of anti-GPIIIa antibody-induced platelet fragmentation, we screened a human single chain Fv antibody library with the GPIIIa49-66 peptide. Nine monoclonal antibodies were identified that were capable of binding to GPIIIa49-66. Surprisingly, binding avidity for GPIIIa49-66 did not correlate with activity of induction of platelet fragmentation. We therefore investigated the requirements for platelet fragmentation. Mutations were introduced into the heavy chain complementary-determining region-3 of clones 11, 43, and 54 by site-directed mutagenesis. The capability of these clones to induce platelet fragmentation or bind to GPIIIa49-66 subsequently changed. Molecular modeling of these clones with their mutants revealed that the ability to induce platelet fragmentation is affected by the side chain orientation of positively charged amino acids in the heavy chain of residues 99-102. Thus, a structural change in the conformation of anti-GPIIIa49-66 antibody contributes to its binding to the beta3 integrin and subsequent antibody-induced platelet fragmentation and aggregate dissolution
— id: 76762, year: 2008, vol: 283, page: 3224, stat: Journal Article,

A new mechanism of platelet activation and oxidative death induced by ADAMTS-18 and regulating bleeding time
Li, ZD; Nardi, M; Pan, RM; Yee, H; Karpatkin, S
2007 NOV 16 ;110(11):47A-48A, Blood
— id: 76172, year: 2007, vol: 110, page: 47A, stat: Journal Article,

Accumulation of miR-155 and BIC RNA in human B cell lymphomas
Eis, Peggy S; Tam, Wayne; Sun, Liping; Chadburn, Amy; Li, Zongdong; Gomez, Mario F; Lund, Elsebet; Dahlberg, James E
2005 Mar 8;102(10):3627-3632, Proceedings of the National Academy of Sciences of the United States of America
We show that the microRNA miR-155 can be processed from sequences present in BIC RNA, a spliced and polyadenylated but non-protein-coding RNA that accumulates in lymphoma cells. The precursor of miR-155 is likely a transient spliced or unspliced nuclear BIC transcript rather than accumulated BIC RNA, which is primarily cytoplasmic. By using a sensitive and quantitative assay, we find that clinical isolates of several types of B cell lymphomas, including diffuse large B cell lymphoma (DLBCL), have 10- to 30-fold higher copy numbers of miR-155 than do normal circulating B cells. Similarly, the quantities of BIC RNA are elevated in lymphoma cells, but ratios of the amounts of the two RNAs are not constant, suggesting that the level of miR-155 is controlled by transcription and processing. Significantly higher levels of miR-155 are present in DLBCLs with an activated B cell phenotype than with the germinal center phenotype. Because patients with activated B cell-type DLBCL have a poorer clinical prognosis, quantification of this microRNA may be diagnostically useful
— id: 64444, year: 2005, vol: 102, page: 3627, stat: Journal Article,

A new mechanism of platelet activation, oxidation and death induced by ADAMTS-18
Li, ZD; Nardi, MA; Feinmark, SJ; Karpatkin, S
2005 NOV 16 ;106(11):193A-193A, Blood
— id: 61462, year: 2005, vol: 106, page: 193A, stat: Journal Article,

Candidate PET radioligands for cannabinoid CB1 receptors: [18F]AM5144 and related pyrazole compounds
Li, Zizhong; Gifford, Andrew; Liu, Qian; Thotapally, Rajesh; Ding, Yu-Shin; Makriyannis, Alexandros; Gatley, S John
2005 May;32(4):361-366, Nuclear medicine & biology
INTRODUCTION: The mammalian brain contains abundant G protein-coupled cannabinoid CB(1) receptors that respond to Delta(9)-tetrahydrocannabinol, the active ingredient of cannabis. The availability of a positron emission tomography (PET) radioligand would facilitate studies of the addictive and medicinal properties of compounds that bind to this receptor. Among the known classes of ligands for CB(1) receptors, the pyrazoles are attractive targets for radiopharmaceutical development because they are antagonists and are generally less lipophilic than the other classes. METHODS: A convenient high-yield synthesis of N-(4-[(18)F]fluorophenyl)-5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-1H-pyra zole-3-carboxamide (AM5144) was devised by coupling the appropriate pyrazole-3-carboxyl chloride compound with 4-[(18)F]fluoroaniline. The labeled precursor was synthesized from 1-[(18)F]fluoro-4-nitrobenzene in 60% radiochemical yield for 10 min using an improved procedure involving sodium borohydride reduction with cobalt chloride catalysis. The product was purified by HPLC to give a specific activity >400 mCi/micromol and a radiochemical purity >95%, and a PET study was conducted in a baboon. RESULTS: Although the regional uptake of AM5144 in baboon brain was consistent with binding to cannabinoid CB(1) receptors, absolute uptake at <0.003% injected radioactivity per cubic centimeter was lower than the previously reported uptake of the radioiodinated pyrazole AM281. CONCLUSIONS: The relatively poor brain uptake of AM5144 and other pyrazole CB(1) receptor ligands is not surprising because of their high lipophilicity as compared with most brain PET radiotracers. However, for nine pyrazole compounds for which rodent data are available, brain uptake and calculated logP values are not correlated. Thus, high logP values should not preclude evaluation of radiotracers for targets such as the CB(1) receptor that may require very lipophilic ligands
— id: 149030, year: 2005, vol: 32, page: 361, stat: Journal Article,

Role of molecular mimickry to HIV-1 peptides in HIV-1-related immunologic thrombocytopenia
Li, Zongdong; Nardi, Michael A; Karpatkin, Simon
2005 Jul 15;106(2):572-576, Blood
Patients with early HIV-1 infection develop an autoimmune thrombocytopenia in which Ab is directed against an immunodominant epitope of the beta3 (GPIIIa) integrin, GPIIIa49-66. This Ab induces thrombocytopenia by a novel complement independent mechanism in which platelets are fragmented by Ab-induced generation of H2O2 derived from the interaction of platelet NADPH oxidase and 12-lipoxygenase. To examine whether sharing of epitope between host and parasite may be responsible for this immunodominant epitope, we screened for Ab-reactive peptides capable of inhibiting platelet lysis and oxidation in vitro, utilizing a filamentous-phage display 7 mer peptide library. Fourteen of these phage-peptide clones were identified. Five shared close sequence similarity with GPIIIa49-66, as expected. Nine were molecular mimicks with close sequence similarity to HIV-1 proteins nef, gag, env and pol. Seven were synthesized as 10 mers from their known HIV-1 sequence and found to inhibit anti-GPIIIa49-66 induced platelet oxidation/fragmentation in vitro. Three rabbit antibodies raised against these peptides induced platelet oxidation/fragmentation in vitro and thrombocytopenia in vivo when passively transferred into mice. One of the peptides shared a known epitope region with HIV-1 protein nef and was derived from a mutant region of the protein. These data provide strong support for molecular-mimickry in HIV-1-ITP within polymorphic regions of HIV-1 proteins. A known epitope of nef is particularly incriminated
— id: 56068, year: 2005, vol: 106, page: 572, stat: Journal Article,

Complement-independent Ab-induced peroxide lysis of platelets requires 12-lipoxygenase and a platelet NADPH oxidase pathway
Nardi, Michael; Feinmark, Steven J; Hu, Liang; Li, Zongdong; Karpatkin, Simon
2004 Apr;113(7):973-980, Journal of clinical investigation
Antiplatelet GPIIIa49-66 Ab of HIV-related thrombocytopenic patients induces thrombocytopenia and platelet fragmentation by the generation of peroxide and other reactive oxygen species (ROS). Here we report the presence of a functional platelet NADPH oxidase pathway that requires activation by the platelet 12-lipoxygenase (12-LO) pathway to fragment platelets. A new Ab-mediated mechanism is described in which the platelet 12-LO product, 12(S)-HETE activates the NADPH oxidase pathway to generate ROS
— id: 44809, year: 2004, vol: 113, page: 973, stat: Journal Article,

Role of molecular Mimickry to HIV-1 peptides in HIV-1-related thrombocytopenia
Li, ZD; Nardi, MS; Karpatkin, S
2003 NOV 16 ;102(11):125A-126A, Blood
— id: 42492, year: 2003, vol: 102, page: 125A, stat: Journal Article,

Synthesis of structurally identical fluorine-18 and iodine isotope labeling compounds for comparative imaging
Li, Zizhong; Ding, Yu-Shin; Gifford, Andrew; Fowler, Joanna S; Gatley, John S
2003 Mar-Apr;14(2):287-294, Bioconjugate chemistry
The synthesis of a benzophenone-based labeling compound designed for comparative imaging studies with both in vivo positron emission tomograph (PET) and single-photon computed tomography (SPECT) and ex vivo autoradiography is described. The new compound can be labeled with either F-18 or iodine radioisotopes to give two different radioisotopmers: N-[2-fluoro-5-(3-[I-131]iodobenzoyl)benzyl]-2-bromoacetamide (1) and N-[2-[F-18]fluoro-5-(3-iodobenzoyl)benzyl]-2-bromoacetamide (2). Compound 1 and 2 have a 2-bromoacetyl group, which can be used to conjugate with biomolecules through a nucleophilic substitution reaction. Compound 1 was synthesized from the corresponding tributyltin derivatives via an oxidative destannylation reaction, and compound 2 was prepared via a four-step radiosynthesis (nucleophilic aromatic substitution, reduction, oxidation, and alkylation) starting from 4-(N,N,N-trimethylammonio)-3-cyano-3'-iodobenzophenone triflate. A remarkably high radiochemical yield (>90%) was achieved for the F-18 nucleophilic aromatic substitution under mild conditions (room temperature in less than 10 min), indicating the structural advantage of the designed molecule to facilitate the F-18 for trimethylammonium substitution in the presence of two electron-withdrawing groups (nitrile and carbonyl). The overall radiosynthesis time for compound 2 is less than 3 h after end of bombardment (EOB) with an unoptimized radiochemical yield of about 2% (not decay corrected) and specific activity of 0.8 Ci/micromol at EOB. The radiolabeling precursors for compound 1 and 2 were synthesized via a carbon-carbon bond-forming reaction between 2-substituted-5-lithiobenzonitrile and 3-substituted benzaldehyde derivatives. Compounds 1 and 2 should allow us to label biomolecules with F-18 or iodine isotopes and gives structurally identical products, which are expected to have identical biological properties and should be useful for comparative imaging studies
— id: 149034, year: 2003, vol: 14, page: 287, stat: Journal Article,

Alterations of mRNA splicing in primary effusion lymphomas
Li, Zongdong; Pan, Langxing; Cesarman, Ethel; Knowles, Daniel M
2003 May;44(5):833-840, Leukemia & lymphoma
Primary effusion lymphoma (PEL) is a unique form of malignant lymphoma associated with infection by the Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8). The majority of PELs also contain the EBV genome. Although viral infection is believed to play a critical role in the pathogenesis of PEL, it has been suggested that additional molecular lesions are required for the development of PEL. Alternative splicing of pre-mRNA is an important mechanism in the regulation of cellular and viral gene expression. Deregulation of pre-mRNA splicing may shift the gene expression balance and lead to the development of cancer. In order to investigate mRNA splicing in PELs, we examined mRNA splicing of three genes, DNA polymerase beta (pol beta), Bcl-x and CD45, in eight PEL cell lines. We found that the average variant percentage of pol beta in PEL cell lines is two times higher than in peripheral blood mononuclear cells (PBMC) and that the variant pattern of genes bcl-x and CD45 is quite different in PEL cell lines than in PBMC. In addition, we also found that the percentage of variant pol beta increased two-fold in PBMC following Epstein-Barr virus (EBV) infection. Therefore, viral infection may contribute to mRNA alternative splicing in PEL. In order to explore the mechanism by which viral infection affects mRNA splicing, we also examined the roles of genes KS-SM, SM and EBERs and viral copies in mRNA splicing. Our findings indicate that various factors acting as positive or negative regulators may be involved in mRNA alternative splicing caused by viral infection. In conclusion, mRNA splicing in PEL can be altered by viral infection and this alteration may contribute to the pathogenesis of PEL
— id: 64445, year: 2003, vol: 44, page: 833, stat: Journal Article,

DNA polymerase mu gene expression in B-cell non-Hodgkin's lymphomas: an analysis utilizing in situ hybridization
Chiu, April; Pan, Langxing; Li, Zongdong; Ely, Scott; Chadburn, Amy; Knowles, Daniel M
2002 Oct;161(4):1349-1355, American journal of pathology
DNA polymerase mu (pol mu) is a novel error-prone DNA repair enzyme bearing significant structural homology with terminal deoxynucleotidyltransferase. Whereas other human error-prone DNA polymerases identified thus far show no preferential lymphoid tissue distribution, the highest levels of pol mu mRNA have been detected in peripheral lymphoid tissues, particularly germinal center B cells. Conceivably, up-regulation of the pol mu gene may be biologically significant in lymphomagenesis, especially in the development of B-cell non-Hodgkin's lymphomas (B-NHLs), because of enhanced error-prone DNA repair activities. To explore this possibility, we generated a digoxigenin-labeled riboprobe to pol mu mRNA and used the probe and in situ hybridization to examine the expression pattern of the pol mu gene in formalin-fixed, paraffin-embedded tissue sections of 37 B-NHLs. This included eight chronic lymphocytic leukemia/small lymphocytic lymphomas, six mantle cell lymphomas, seven follicular lymphomas, nine diffuse large B-cell lymphomas, three splenic marginal zone lymphomas, two Burkitt's lymphomas, and two precursor B-lymphoblastic lymphomas. We also correlated the pol mu mRNA expression levels with the tumor proliferation index, which was assessed in each case by image analysis of Ki-67 immunostained slides. Nineteen of 21 (90%) B-NHLs arising from postgerminal center B cells (follicular lymphomas, diffuse large B-cell lymphomas, splenic marginal zone lymphomas, and Burkitt's lymphomas) exhibited high expression of pol mu mRNA. In contrast, only 2 of 16 (13%) B-NHLs arising from pregerminal center B cells (chronic lymphocytic leukemia/small lymphocytic lymphomas, mantle cell lymphomas, and precursor B-lymphoblastic lymphomas) expressed significant levels of pol mu mRNA. Pol mu gene expression did not seem to correlate with the proliferation index, especially because a significant level of pol mu mRNA was not detected in either case of precursor B-lymphoblastic lymphomas. In conclusion, pol mu gene expression is highly associated with B-NHLs of postgerminal center B-cell derivation. Furthermore, the expression level is independent of the proliferation rate and thus is unrelated to the biological aggressiveness of the tumors. These findings, along with the error-prone nature of the enzyme, suggest that up-regulation of pol mu gene expression may be a contributing factor to the pathogenesis of a subset of B-NHLs through DNA repair-associated genomic instability
— id: 64446, year: 2002, vol: 161, page: 1349, stat: Journal Article,

The translesion DNA polymerase zeta plays a major role in Ig and bcl-6 somatic hypermutation
Zan H; Komori A; Li Z; Cerutti A; Schaffer A; Flajnik MF; Diaz M; Casali P
2001 May;14(5):643-653, Immunity
Ig somatic mutations would be introduced by a polymerase (pol) while repairing DNA outside main DNA replication. We show that human B cells constitutively express the translesion pol zeta, which effectively extends DNA past mismatched bases (mispair extender), and pol eta, which bypasses DNA lesions in an error-free fashion. Upon B cell receptor (BCR) engagement and coculture with activated CD4+ T cells, these lymphocytes upregulated pol zeta, downregulated pol eta, and mutated the Ig and bcl-6 genes. Inhibition of the pol zeta REV3 catalytic subunit by specific phosphorothioate-modified oligonucleotides impaired Ig and bcl-6 hypermutation and UV damage-induced DNA mutagenesis, without affecting cell cycle or viability. Thus, pol zeta plays a critical role in Ig and bcl-6 hypermutation, perhaps facilitated by the downregulation of pol eta
— id: 64521, year: 2001, vol: 14, page: 643, stat: Journal Article,

Structure-function analysis of a lupus anti-DNA autoantibody: central role of the heavy chain complementarity-determining region 3 Arg in binding of double- and single-stranded DNA
Li Z; Schettino EW; Padlan EA; Ikematsu H; Casali P
2000 Jul;30(7):2015-2026, European journal of immunology
To determine the contribution of the somatic point mutations and that of the complementarity-determining region (CDR)3 Arg to DNA binding, we engineered the germline V(H) and V(kappa) gene revertant and site-mutagenized the CDR3 Arg residues of the mutated and 'antigen-selected' mAb 412.67. This anti-DNA autoantibody was derived from B-1 cells of a lupus patient and bore two H-CDR3 Arg, Arg105 and Arg107, encoded by N segment additions, and one kappa-CDR3 Arg, Arg97, resulting from a point mutation (Kasaian et al. 1994. J. Immunol. 152: 3137-3151; Kasaian et al. 1995. Ann. N.Y Acad. Sci. 764: 410-423). The germ-line revertant bound double-stranded (ds) DNA and single-stranded (ss) DNA as effectively as its wild-type counterpart (relative avidity: 6.4x10(-7) and 9.9x10(-9) vs. 6.7x10(-7) and 9.1 x10(-9) g/microl), raising the possibility that an antigen other than DNA was responsible for the selection of the mAb 412.67 V(H) and V(kappa) point mutations. H-CDR3 Arg105 and Arg107 were both required for dsDNA binding, but either Arg105 or Arg107 was sufficient for ssDNA binding. The central role of Arg105 and Arg107 in DNA binding reflected their solvent-exposed orientation at the apex of the H-CDR3 main loop. Consistent with its inward orientation afar from the antigen-binding surface, the kappa-CDR3 Arg97 played no role in either dsDNA or ssDNA binding
— id: 64522, year: 2000, vol: 30, page: 2015, stat: Journal Article,

B cell receptor engagement and T cell contact induce Bcl-6 somatic hypermutation in human B cells: identity with Ig hypermutation
Zan H; Li Z; Yamaji K; Dramitinos P; Cerutti A; Casali P
2000 Jul 15;165(2):830-839, Journal of immunology
The human bcl-6 proto-oncogene has been found to be mutated in both neoplastic and normal B cells. We used CL-01 cells, our monoclonal model of germinal center differentiation, and normal human B cells to explore the induction requirements and the modalities of bcl-6 hypermutation. As we have previously shown, CL-01 cells are IgM+ IgD+ and effectively mutate the expressed Ig VHDJH and V lambda J lambda genes and switch to IgG, IgA, and IgE upon B cell receptor engagement and contact with CD4+ T cells through CD40:CD154 and CD80:CD28 coengagement. In this paper we showed that the same stimuli induce somatic hypermutation of bcl-6 in CL-01 and normal IgM+ IgD+ B cells. bcl-6 hypermutation was not accompanied by translocation of this proto-oncogene or hypermutation of the beta-actin gene, and it did mimic Ig hypermutation. It was associated with transcription initiation, in that it targeted the first exon and a 696-bp sequence immediately downstream (approximately 0.6 kb) of the transcription initiation site while sparing further downstream (approximately 2.5 kb) and upstream (approximately 0.1 kb) areas. bcl-6 hypermutation displayed an overall rate of 2.2 x 10-4 changes/base/cell division with characteristic nucleotide preferences and showed strand polarity. These findings show that B cell receptor engagement promotes hypermutation in genes other than Ig, and suggest that cis-regulating elements similar to those of the Ig locus exist in bcl-6
— id: 64523, year: 2000, vol: 165, page: 830, stat: Journal Article,

Induction of Ig somatic hypermutation and class switching in a human monoclonal IgM+ IgD+ B cell line in vitro: definition of the requirements and modalities of hypermutation
Zan H; Cerutti A; Dramitinos P; Schaffer A; Li Z; Casali P
1999 Mar 15;162(6):3437-3447, Journal of immunology
Partly because of the lack of a suitable in vitro model, the trigger(s) and the mechanism(s) of somatic hypermutation in Ig genes are largely unknown. We have analyzed the hypermutation potential of human CL-01 lymphocytes, our monoclonal model of germinal center B cell differentiation. These cells are surface IgM+ IgD+ and, in the absence of T cells, switch to IgG, IgA, and IgE in response to CD40:CD40 ligand engagement and exposure to appropriate cytokines. We show here that CL-01 cells can be induced to effectively mutate the expressed VHDJH-C mu, VHDJH-C delta, VHDJH-C gamma, VHDJH-C alpha, VHDJH-C epsilon, and V lambda J lambda-C lambda transcripts before and after Ig class switching in a stepwise fashion. In these cells, induction of somatic mutations required cross-linking of the surface receptor for Ag and T cell contact through CD40:CD40 ligand and CD80: CD28 coengagement. The induced mutations showed intrinsic features of Ig V(D)J hypermutation in that they comprised 110 base substitutions (97 in the heavy chain and 13 in the lambda-chain) and only 2 deletions and targeted V(D)J, virtually sparing CH and C lambda. These mutations were more abundant in secondary VHDJH-C gamma than primary VHDJH-C mu transcripts and in V(D)J-C than V lambda J lambda-C lambda transcripts. These mutations were also associated with coding DNA strand polarity and showed an overall rate of 2.42 x 10(-4) base changes/cell division in VHDJH-CH transcripts. Transitions were favored over transversions, and G nucleotides were preferentially targeted, mainly in the context of AG dinucleotides. Thus, in CL-01 cells, Ig somatic hypermutation is readily inducible by stimuli different from those required for class switching and displays discrete base substitution modalities
— id: 64524, year: 1999, vol: 162, page: 3437, stat: Journal Article,

Application of the study in immunogloblulin gene variable region
Li Z; Yeh M
1998 ;10(5):236-239, Sheng ming ke xue = Life science
— id: 64529, year: 1998, vol: 10, page: 236, stat: Journal Article,

Construction and diversity analysis of a murine IgE phage surface display library
Li ZD; Yeh M
1997 Dec;7(2):161-170, Cell research
To make further investigation of the IgE antibody repertoire in Trichosanthin (TCS) allergic responses, a murine IgE phage surface display library was constructed (3.0 x 10(5) independent clones). We first constructed the V epsilon cDNA library (4.6 x 10(5) independent clones) and V kappa cDNA library (3.0 x 10(5) independent clones). Then, the V epsilon and V kappa gene segments were amplified from both libraries by PCR respectively, and assembled into Fab fragment by SOE PCR. The phage library containing Fabs was thus constructed. The diversity of V epsilon from this library was analyzed and proved. Fab clones with high specificity to TCS have been screened out
— id: 64526, year: 1997, vol: 7, page: 161, stat: Journal Article,

Structural analysis and molecular modeling of two anti-trichosanthin IgE clones from phage antibody library
Li ZD; Yuan YR; Yeh M
1997 Dec;7(2):171-178, Cell research
Recently we constructed a murine IgE phage surface display library and screened out two IgE (Fab) clones with specific binding activity to Trichosanthin (TCS). In this work, the V epsilon and V kappa genes of the two clones were sequenced and their putative germline gene usages were studied. On the basis of the known 3D structure of Trichosanthin and antibody, molecular modeling was carried out to study the antigen-antibody interaction. The possible antigenic determinant sites on the surface of TCS recognized by both the clones were analyzed, and the reaction forces between TCS and two Fab fragments were also analyzed respectively
— id: 64525, year: 1997, vol: 7, page: 171, stat: Journal Article,