Karin Grunebaum Cancer Research Foundation Fellow Profile

Benjamin Gewurz Jamaica Plain, MA

M.D., Ph.D.; Instructor, Department of Internal Medicine, Harvard Medical School

Fellow Profile

Current Site Of Practice: Brigham and Women's Hospital
Hospital Affiliation: Brigham and Womens
Focus of Research: Lymphoma Oncogene/Tumor Suppressor Gene Discovery
Fellowship Year: 2010 – 2011
Attended: Harvard Medical School


Localization of sequence-determined neoepitopes and neutrophil digestion fragments of C-reactive protein utilizing monoclonal antibodies and synthetic peptides.

Co-Authors Ying, SC, Shephard, E, de Beer, FC, Siegel, JN, Harris, D, Fridkin, M, Gewurz H

We recently described 17 anti-CRP mAb, seven to native- (or conformational) and 10 to neo- (or sequence-determined) epitopes, including several anti-neo-CRP mAb specific for CRP peptide 199-206. In the present study, four new anti-native- and four new anti-neo-CRP mAb were generated and characterized by ELISA reactivity with native and modified human and rabbit CRP, as well as binding to pronase fragments of human CRP in Western blots. Assays with 17 synthetic CRP peptides identified anti-neo-CRP mAb specific for peptides 1-16, 14-24 and 137-152, respectively. The anti-neo-CRP mAb were reacted with fragments obtained by digesting CRP with multiple additional enzymes, including Staphylococcal V8 protease, trypsin, elastase, plasmin, thrombin and alpha-chymotrypsin. Native CRP was remarkably resistant to enzymic digestion, particularly in the presence of calcium, but was readily cleavable upon denaturation. Twenty-three informative fragments served to further distinguish mAb reactivity with at least four additional neo-CRP epitopes, which presumptively included residues in the regions of amino acids 22-45, 41-61, 114-121 and 130-138, respectively. The eight epitopes identified corresponded well with predicted regions of CRP antigenicity. In addition, at least six distinct native or conformation-determined epitopes were delineated. Reactivity of the anti-neo-CRP mAb with fragments of CRP generated by PMN enzymes indicated that regions sensitive to cleavage by neutrophil enzymes are located at approximately 3, 10 and 16 kD from the amino terminus of the CRP subunit. We expect that the anti-CRP mAb described and mapped herein will be useful tools for the elucidation of CRP structure and function

Mol Immunol. 1992 May;29(5):677-87

More: http://www.ncbi.nlm.nih.gov/pubmed/1374844

Reactivity of anti-human C-reactive protein (CRP) and serum amyloid P component (SAP) monoclonal antibodies with limulin and pentraxins of other species

Co-Authors Ying, SC, Marchalonis, JJ, Gewurz, AT, Siegel, JN, Jiang, H, Gewurz, H

Limulus polyphemus C-reactive protein (CRP) (limulin) has approximately 30% amino acid sequence homology and shares at least one idiotypic determinant associated with ligand-binding activity with human CRP (hCRP); limulin also shares amino acid sequence homology and lectin activity with human serum amyloid P component (hSAP). In the present study panels of 14 anti-hCRP monoclonal antibodies (mAb) directed to distinct hCRP epitopes and 11 anti-hSAP mAb directed to distinct epitopes of hSAP were tested for reactivity with limulin and pentraxins of other species including rabbit CRP (raCRP), rat CRP and hamster female protein (FP) by ELISA and Western blot analyses. None of the anti-human pentraxin mAb showed strong cross-reactivity with limulin; only five mAb reacted with limulin at all, and cross-reactivities of these mAb with the other pentraxins, when present, also were weak. Cross-reactivity of limulin with hCRP and hSAP was similar, and in light of comparable amino acid sequence homology, suggests this molecule can be considered the limulus SAP as well as the limulus CRP. Several anti-hCRP mAb cross-reacted strongly with rabbit CRP and rat CRP; a few anti-hSAP cross-reacted strongly with FP; and weak cross-reactions were observed between hCRP and hSAP, but cross-reactivities between the pentraxins generally were limited and weak. A rabbit polyclonal antibody raised to highly conserved limulin peptide 141-156 and strongly reactive with limulin reacted weakly with hCRP and raCRP but failed to react with rat CRP, hSAP or FP. These studies emphasize a limited but distinct antigenic similarity between limulin, hCRP and other pentraxins, and identify mAb reactive with potential regions of shared structure and/or function between pentraxins of different species.

Immunology. 1992 Jun;76(2):324-30

More: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1421529/?tool=pubmed

Phenotypic switching of Haemophilus influenzae

Co-Authors Moxon, ER, Richards, JC, Inzana, T, Jennings, MP, Hood, DW

Mol Microbiol. 1996 Mar;19(5):1149-50.

More: http://www.ncbi.nlm.nih.gov/pubmed/8830271

B7 costimulation is critical for antibody class switching and CD8(+) cytotoxic T-lymphocyte generation in the host response to vesicular stomatitis virus

Co-Authors McAdam, AJ, Farkash, EA, Sharpe, AH

Antibody and cytotoxic T-lymphocyte (CTL) responses have critical roles in eliminating many viral infections. In addition to stimulation of the T-cell receptor, T cells require costimulatory signals to respond optimally. We evaluated the role of B7 costimulatory molecules (B7-1 and B7-2) in the immune response to viral infection using vesicular stomatitis virus (VSV) and mice lacking either B7-1 or B7-2 or both molecules. Mice lacking both B7-1 and B7-2 had essentially no anti-VSV immunoglobulin G1 (IgG1) response, decreased IgG2a responses, and normal IgM responses, while mice lacking either B7-1 or B7-2 had unaltered anti-VSV antibody responses compared to wild-type mice. Depletion of CD4(+) cells further reduced the IgG2a response in mice lacking both B7 molecules, suggesting that CD4(-) cells may supply help for IgG2a in the absence of B7 costimulation. The absence of both B7 molecules profoundly reduced generation of both primary and secondary VSV-specific class I major histocompatibility complex (MHC)-restricted CTL, whereas VSV-specific CTL responses in mice lacking either B7-1 or B7-2 were similar to those of wild-type animals. Class I MHC-restricted CTL in wild-type mice were not dependent on CD4(+) cells, suggesting that the failure of CTL in the absence of B7s is due to a lack of B7 costimulation directly to the CD8(+) CTL. These data demonstrate that B7-1 and B7-2 have critical, overlapping functions in the antibody and CTL responses to this viral infection.

J Virol. 2000 Jan;74(1):203-8

More: http://jvi.asm.org/cgi/content/full/74/1/203?view=long&pmid=10590107

Viral subversion of the immune system

Co-Authors Tortorella D, Furman, MH, Schust, DJ, Ploegh, HL

This review describes the diverse array of pathways and molecular targets that are used by viruses to elude immune detection and destruction. These include targeting of pathways for major histocompatibility complex-restricted antigen presentation, apoptosis, cytokine-mediated signaling, and humoral immune responses. The continuous interactions between host and pathogens during their coevolution have shaped the immune system, but also the counter measures used by pathogens. Further study of their interactions should improve our ability to manipulate and exploit the various pathogens.

Annu Rev Immunol. 2000;18:861-926

More: http://www.ncbi.nlm.nih.gov/pubmed/10837078

Either B7 costimulation or IL-2 can elicit generation of primary alloreactive CTL

Co-Authors McAdam, AJ, Farkash, EA, Sharpe, AH

B7-1 and B7-2 are important costimulatory molecules in the activation of T cell immunity. We have used mice made genetically deficient in either or both B7 molecules to determine the role of B7 molecules in activation of primary alloreactive CTL. The absence of either B7-1 or B7-2 did not alter generation of CTL from unfractionated lymphocytes, but the absence of B7-2 greatly decreased CTL generation from purified CD8+ responder cells. However, if B7-1 was induced on the stimulating cells then CTL generation was restored to wild-type levels. Absence of both B7-1 and B7-2 from MLR using whole splenocytes resulted in a profound reduction in generation of CTL. This could completely be reversed by the addition of IL-2. B7 molecules could directly costimulate CD8+ cells, as purified CD8+ cells developed into mature CTL when stimulated with wild-type APC, but not with B7-deficient APC. Again, IL-2 could drive CTL generation from purified CD8+ cells, even in the absence of B7 molecules. Taken together, these results demonstrate an important role for B7 costimulation in CTL generation.

J Immunol. 2000 Sep 15;165(6):3088-93.

More: http://www.jimmunol.org/cgi/content/full/165/6/3088

Human cytomegalovirus US2 endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class I in a locus-specific manner

Co-Authors Wang, EW, Tortorella, D, Schust,, DJ, Ploegh, HL

The human cytomegalovirus-encoded US2 glycoprotein targets endoplasmic reticulum-resident major histocompatibility complex (MHC) class I heavy chains for rapid degradation by the proteasome. We demonstrate that the endoplasmic reticulum-lumenal domain of US2 allows tight interaction with class I molecules encoded by the HLA-A locus. Recombinant soluble US2 binds properly folded, peptide-containing recombinant HLA-A2 molecules in a peptide sequence-independent manner, consistent with US2's ability to broadly downregulate class I molecules. The physicochemical properties of the US2/MHC class I complex suggest a 1:1 stoichiometry. These results demonstrate that US2 does not require additional cellular proteins to specifically interact with soluble class I molecules. Binding of US2 does not significantly alter the conformation of class I molecules, as a soluble T-cell receptor can simultaneously recognize class I molecules associated with US2. The lumenal domain of US2 can differentiate between the products of distinct class I loci, as US2 binds several HLA-A locus products while being unable to bind recombinant HLA-B7, HLA-B27, HLA-Cw4, or HLA-E. We did not observe interaction between soluble US2 and either recombinant HLA-DR1 or recombinant HLA-DM. The substrate specificity of US2 may help explain the presence in human cytomegalovirus of multiple strategies for downregulation of MHC class I molecules.

J Virol. 2001 Jun;75(11):5197-204

More: http://jvi.asm.org/cgi/content/full/75/11/5197?view=long&pmid=11333901

Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6794-9.

Co-Authors Gaudet, R, Tortorella, D, Wang, EW, Ploegh, HL, Wiley, DC

Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cell-surface expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2/Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the alpha3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation.

Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6794-9.

More: http://www.pnas.org/cgi/pmidlookup?view=long&pmid=11391001

Virus subversion of immunity: a structural perspective

Co-Authors Gaudet R, Tortorella D, Wang EW, Ploegh HL

Over the past year, we have witnessed the discovery of further virus immuno-evasins--proteins that alter the host immune response. Although many of these factors have been described over the past decade, the structural basis underlying their biology has lagged behind. Structural data have now been obtained for several such proteins. Major advances of the past year include the structures of a viral chemokine-binding protein, of an intact viral regulator of complement activation and of an immuno-evasin with its cellular target.

Curr Opin Immunol. 2001 Aug;13(4):442-50

More: http://www.ncbi.nlm.nih.gov/pubmed/11498300

US2, a human cytomegalovirus-encoded type I membrane protein, contains a non-cleavable amino-terminal signal peptide.

Co-Authors Tortorella, D, Ploegh, H

The human cytomegalovirus US2 gene product targets major histocompatibility class I molecules for degradation in a proteasome-dependent fashion. Degradation requires interaction between the endoplasmic reticulum (ER) lumenal domains of US2 and class I. While ER insertion of US2 is essential for US2 function, US2 lacks a cleavable signal peptide. Radiosequence analysis of glycosylated US2 confirms the presence of the NH(2) terminus predicted on the basis of the amino acid sequence, with no evidence for processing by signal peptidase. Despite the absence of cleavage, the US2 NH(2)-terminal segment constitutes its signal peptide and is sufficient to drive ER translocation of chimeric reporter proteins, again without further cleavage. The putative US2 signal peptide c-region is responsible for the absence of cleavage, despite the presence of a suitable -3,-1 amino acid motif for signal peptidase recognition. In addition, the US2 signal peptide affects the early processing events of the nascent polypeptide, altering the efficiency of ER insertion and subsequent N-linked glycosylation. To our knowledge, US2 is the first example of a membrane protein that does not contain a cleavable signal peptide, yet otherwise behaves like a type I membrane glycoprotein.

J Biol Chem. 2002 Mar 29;277(13):11306-13

More: http://www.jbc.org/content/277/13/11306.long

Capravirine, a nonnucleoside reverse-transcriptase inhibitor in patients infected with HIV-1: a phase 1 study

Co-Authors Jacobs, M, Proper, JA, Dahl, TA, Fujiwara, T, Dezube, BJ

Capravirine is a nonnucleoside reverse-transcriptase inhibitor (NNRTI) with a unique resistance profile. Although single mutations allow resistance to established NNRTIs, human immunodeficiency virus (HIV)-1 must undergo multiple mutations to achieve resistance to capravirine. In the present phase 1 study, capravirine was administered orally for up to 28 days to 55 HIV-1-infected individuals with CD4+ T lymphocyte counts of 50-500 cells/microL. The most frequent adverse events were diarrhea (5%) and nausea (4%), with no drug-related rashes observed. The day 15 median (mean) HIV-1 load decreased by 1.34 (1.45) log(10) copies/mL in the patients receiving 25 mg/kg/day. Capravirine demonstrated potent antiviral activity, even in antiretroviral-experienced patients.

J Infect Dis. 2004 Dec 1;190(11):1957-61

More: http://www.ncbi.nlm.nih.gov/pubmed/15529260

DNA-damage control: Claspin destruction turns off the checkpoint

Co-Authors Harper, JW

The ATR-Claspin-Chk1 pathway is critical for turning on the cellular response to DNA damage and replication stress. Five recent reports uncover new mechanisms controlling the recovery phase of the checkpoint response, and introduce crucial roles for Claspin, Rad17 phosphorylation and the ubiquitin proteasome pathway in Chk1 signaling.

Curr Biol. 2006 Nov 7;16(21):R932-4

More: http://www.ncbi.nlm.nih.gov/pubmed/17084694

Persistent and relapsing babesiosis in immunocompromised patients

Co-Authors Krause PJ, Hill, D, Marty, FM, Vannier, E, Foppa, IM, Furman, RR, Neuhaus, E, Skowron, G, Gupta, S, McCalla, C, Pesanti, EL, Young, M, Heiman D, Hsue G, Gelfand JA, Wormser GP, Dickason J, Bia FJ, Hartman B, Telford SR 3rd, Christianson D, Dardick K, Cole

BACKGROUND: Human babesiosis is a tickborne malaria-like illness that generally resolves without complication after administration of atovaquone and azithromycin or clindamycin and quinine. Although patients experiencing babesiosis that is unresponsive to standard antimicrobial therapy have been described, the pathogenesis, clinical course, and optimal treatment regimen of such cases remain uncertain. METHODS: We compared the immunologic status, clinical course, and treatment of 14 case patients who experienced morbidity or death after persistence of Babesia microti infection, despite repeated courses of antibabesial treatment, with those of 46 control subjects whose infection resolved after a single course of standard therapy. This retrospective case-control study was performed in southern New England, New York, and Wisconsin. RESULTS: All case patients were immunosuppressed at the time of acute babesiosis, compared with <10% of the control subjects. Most case patients experienced B cell lymphoma and were asplenic or had received rituximab before babesial illness. The case patients were more likely than control subjects to experience complications, and 3 died. Resolution of persistent infection occurred in 11 patients after 2-10 courses of therapy, including administration of a final antimicrobial regimen for at least 2 weeks after babesia were no longer seen on blood smear. CONCLUSIONS: Immunocompromised people who are infected by B. microti are at risk of persistent relapsing illness. Such patients generally require antibabesial treatment for >or=6 weeks to achieve cure, including 2 weeks after parasites are no longer detected on blood smear.

Clin Infect Dis. 2008 Feb 1;46(3):370-6

More: http://www.ncbi.nlm.nih.gov/pubmed/18181735

Human babesiosis

Co-Authors Vannier, J, Krause, PJ

Human babesiosis is an emerging intraerythrocytic infection caused by protozoal parasites transmitted by ixodid ticks. Babesiosis is endemic in the northeastern and upper midwestern regions of the United States and is found sporadically in other parts of the United States, Europe, Asia, Africa, and South America. Babesial infections range from asymptomatic to severe and occasionally are fatal. Specific laboratory diagnosis of babesial infection is made by morphologic examination of Giemsa-stained blood smears, serology, and amplification of babesial DNA using polymerase chain reaction. The combination of atovaquone and azithromycin is the treatment of choice for mild-to-moderate illness, whereas clindamycin and quinine and exchange transfusion are indicated for severe disease.

Infect Dis Clin North Am. 2008 Sep;22(3):469-88

More: http://www.ncbi.nlm.nih.gov/pubmed/18755385

Human herpesvirus 6 encephalitis

Co-Authors Marty, FM, Baden, LR, Katz, JT

Human herpesvirus (HHV) 6, the etiologic agent of roseola, is nearly universally acquired during childhood. The virus establishes lifelong infection, including within the central nervous system (CNS), and replicates within several CNS cell types. HHV-6 has been linked to CNS disease during primary infection, including febrile seizures and possibly hippocampal injury. HHV-6 may also be associated with neurologic disease later in life, particularly in transplant patients. Recent reports offer evidence that HHV-6 reactivation may underlie a characteristic limbic encephalitis syndrome following hematopoietic cell transplant; the cardinal features of this syndrome include memory loss, insomnia, electroencephalographic evidence of temporal lobe seizure activity, MRI signal intensity abnormalities of the mesial temporal lobe, and the syndrome of inappropriate release of antidiuretic hormone. HHV-6 DNA is frequently detectable by nucleic acid amplification tests in the cerebrospinal fluid and peripheral blood upon symptom onset, which may provide a screening strategy in high-risk patients. Possible associations of HHV-6 with meningoencephalitis, mesial temporal lobe epilepsy, and multiple sclerosis in apparently immunocompetent hosts are under investigation.

Curr Infect Dis Rep. 2008 Jul;10(4):292-9.

More: http://www.ncbi.nlm.nih.gov/pubmed/18765102

IRF7 activation by Epstein-Barr virus latent membrane protein 1 requires localization at activation sites and TRAF6, but not TRAF2 or TRAF3

Co-Authors Song. YJ, Izumi, K, Shinners, N, Kieff, E

Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1), a constitutively aggregated and activated pseudoreceptor, activates IFN regulatory factor 7 (IRF7) through RIP1. We now report that the LMP1 cytoplasmic carboxyl terminal amino acids 379-386 bound IRF7 and activated IRF7. IRF7 activation required TRAF6 and RIP1, but not TRAF2 or TRAF3. LMP1 Y(384)YD(386), which are required for TRADD and RIP1 binding and for NF-kappaB activation, were not required for IRF7 binding, but were required for IRF7 activation, implicating signaling through TRADD and RIP1 in IRF7 activation. Association with active LMP1 signaling complexes was also critical for IRF7 activation because (i) a dominant-negative IRF7 bound to LMP1, blocked IRF7 association and activation, but did not inhibit LMP1 induced NF-kappaB or TBK1 or Sendai virus-mediated IFN stimulated response element activation; and (ii) two different LMP1 transmembrane domain mutants, which fail to aggregate, each bound IRF7 and prevented LMP1 from binding and activating IRF7 in the same cell, but did not prevent NF-kappaB activation. Thus, efficient IRF7 activation required association with LMP1 CTAR2 in proximity to LMP1 CTAR2 mediated kinase activation sites.

Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18448-53.

More: http://www.pnas.org/content/105/47/18448.long