bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fab Chains As an Efficient Heterodimerization Scaffold for the Production of Recombinant Bispecific and Trispecific Antibody Derivatives
Authors: Schoonjans Reinhilde , Willems An , Schoonooghe Steve , Fiers Walter , Grooten Johan , Mertens Nico 
Published: April 2014

Abstract
Due to their multispecificity and versatility, bispecific Abs (BsAbs) are promising therapeutic tools in tomorrow’s medicine. Especially intermediate-sized BsAbs that combine body retention with tissue penetration are valuable for therapy but necessitate expression systems that favor heterodimerization of the binding sites for large-scale application. To identify heterodimerization domains to which single-chain variable fragments (scFv) can be fused, we compared the efficiency of heterodimerization of CL and CH1 constant domains with complete L and Fd chains in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete L and Fd chains were used, secretion of L:Fd heterodimers was highly successful. Because these Fab chains contribute a binding moiety, C-terminal fusion of a scFv molecule to the L and/or Fd chains generated BsAbs or trispecific Abs (TsAbs) of intermediate size (75–100 kDa). These disulfide-stabilized bispecific Fab-scFv (“bibody”) and trispecific Fab-(scFv)2 (“tribody”) heterodimers represent up to 90% of all secreted Ab fragments in the mammalian expression system and possess fully functional binding moieties. Furthermore, both molecules recruit and activate T cells in a tumor cell-dependent way, whereby the trispecific derivative can exert this activity to two different tumor cells. Thus we propose the use of the disulfide-stabilized L:Fd heterodimer as an efficient platform for production of intermediate-sized BsAbs and TsAbs in mammalian expression systems.
Induced κ Receptor Editing Shows No Allelic Preference in a Mouse Pre-B Cell Line
Authors: Liu Xiangdong , Linden Michael , Van Ness Brian 
Published: April 2014

AbstractB cell Ag receptor editing is a process that can change κ antigen recognition specificity of a B cell receptor through secondary gene rearrangements on the same allele. In this study we used a model mouse pre-B cell line (38B9) to examine factors that might affect allelic targeting of secondary rearrangements of the κ locus. We isolated clones that showed both productive and nonproductive rearrangements of one κ allele, while retaining the other κ allele in the germline configuration (κ+/κ° or κ−/κ°). In the absence of any selective pressures, subsequent rearrangement of the germline alleles occurred at the same frequency as secondary rearrangement of the productive or nonproductive rearranged alleles. Because 38B9 cells lack Ig heavy chains, we stably expressed μ heavy chain protein in 38B9 cells to determine whether heavy-light pairing might affect allelic targeting of secondary κ rearrangements. Although the expression of heavy chain was found to both pair with and stabilize κ protein in these cells, it had no effect on preferential targeting Vκ-Jκ receptor editing compared with rearrangement of a germline allele. These studies suggest that in the absence of selection to eliminate autoreactive Vκ-Jκ genes, there is no allelic preference for secondary rearrangement events in 38B9 cells.
Immunization with a Recombinant Stage-Regulated Surface Protein fromLeishmania donovaniInduces Protection Against Visceral Leishmaniasis
Authors: Stäger Simona , Smith Deborah F. , Kaye Paul M. 
Published: April 2014

AbstractVaccination against visceral leishmaniasis has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine against visceral leishmaniasis is pressing. In this study, we demonstrate for the first time that a recombinant stage-specific hydrophilic surface protein of Leishmania donovani, recombinant hydrophilic acylated surface protein B1 (HASPB1), is able to confer protection against experimental challenge. Protection induced by rHASPB1 does not require adjuvant and, unlike soluble Leishmania Ag + IL-12, extends to the control of parasite burden in the spleen, an organ in which parasites usually persist and are refractory to a broad range of immunological and chemotherapeutic interventions. Both immunohistochemistry (for IL-12p40) and enzyme-linked immunospot assay (for IL-12p70) indicate that immunization with rHASPB1 results in IL-12 production by dendritic cells, although an analysis of Ab isotype responses to rHASPB1 suggests that this response is not sufficient in magnitude to induce a polarized Th1 response. Although both vaccinated and control-infected mice have equivalent frequencies of rHASPB1-specific CD4+ T cells producing IFN-γ, vaccine-induced protection correlates with the presence of rHASPB1-specific, IFN-γ-producing CD8+ T cells. Thus, we have identified a novel vaccine candidate Ag for visceral leishmaniasis, which appears to operate via a mechanism similar to that previously associated with DNA vaccination.
A Mechanism for the Impaired IFN-γ Production in C-C Chemokine Receptor 2 (CCR2) Knockout Mice: Role of CCR2 in Linking the Innate and Adaptive Immune Responses
Authors: Peters Wendy , Dupuis Marc , Charo Israel F. 
Published: April 2014

AbstractWe have recently shown that mice with a targeted disruption of CCR2, the receptor for monocyte chemoattractant protein-1, have markedly impaired recruitment of macrophages to sites of inflammation. An unexpected finding in the CCR2−/− mice was a dramatic decrease in the production of IFN-γ after challenge with purified protein derivative of Mycobacterium bovis. In this study, we have investigated the mechanism of this cytokine production defect. In vitro, direct activation of splenocytes with CD3/CD28 Abs failed to reveal any differences in IFN-γ production between CCR2+/+ and CCR2−/− mice. However, after immunization, the number of Ag-specific, IFN-γ-producing cells in the draining lymph nodes was decreased by 70% in the CCR2−/− mice, suggesting an in vivo trafficking defect. Direct measurement of cell trafficking with fluorescently labeled CFA revealed a marked decrease in the number of monocytes/macrophages migrating to the site of immunization and to the draining lymph nodes in the CCR2−/− mice. The data suggest that impaired trafficking of APCs in the CCR2−/− mice contributes to the defect in IFN-γ production. These data support the idea that CCR2-positive monocytes/macrophages are critical in linking the innate and adaptive immune responses.
The Importance of Exogenous Antigen in Priming the Human CD8+ T Cell Response: Lessons from the EBV Nuclear Antigen EBNA1
Authors: Blake Neil , Haigh Tracey , Shaka’a Ghadeer , Croom-Carter Debbie , Rickinson Alan 
Published: April 2014

Abstract
Mouse models suggest that the processing of exogenous Ag by dendritic cells can be important for priming the CD8+ CTL response. To study the situation in humans, we have exploited the CTL response to EBV infection. In this context EBV expresses eight latent proteins, of which EBV-encoded nuclear Ag (EBNA) 3A, 3B, and 3C appear to be immunodominant for CTL responses, whereas another nuclear Ag, EBNA1, which is completely protected from endogenous presentation via the MHC class I pathway, is thought to induce responses rarely, if ever. Here, using EBNA1 peptides and/or EBNA1 protein-loaded dendritic cells as in vitro stimuli, we have identified memory CTL responses to HLA-B*3501, -B7, and -B53-restricted EBNA1 epitopes that can be as strong as those seen in immunodominant epitopes from the “conventionally processed” EBNA3 Ags. Furthermore, we used HLA-peptide tetramers to show that the primary response to one such EBNA1 epitope constituted up to 5% of the CD8+ T cells in infectious mononucleosis blood, the strongest latent Ag-specific response yet detected in this setting. We conclude that exogenous protein represents a significant source of Ag for priming the human CTL response.
Human CD8+ CTL Specific for the Mycobacterial Major Secreted Antigen 85A
Authors: Smith Steven M. , Brookes Roger , Klein Michèl R. , Malin Adam S. , Lukey Pauline T. , King Abigail S. , Ogg Graham S. , Hill Adrian V. S. , Dockrell Hazel M. 
Published: April 2014

AbstractThe role of CD8+ CTL in protection against tuberculosis in human disease is unclear. In this study, we stimulated the peripheral blood mononuclear cells of bacillus Calmette-Guérin (BCG)-vaccinated individuals with live Mycobacterium bovis BCG bacilli to establish short-term cell lines and then purified the CD8+ T cells. A highly sensitive enzyme-linked immunospot (ELISPOT) assay for single cell IFN-γ release was used to screen CD8+ T cells with overlapping peptides spanning the mycobacterial major secreted protein, Ag85A. Three peptides consistently induced a high frequency of IFN-γ responsive CD8+ T cells, and two HLA-A*0201 binding motifs, P48–56 and P242–250, were revealed within the core sequences. CD8+ T cells responding to the 9-mer epitopes were visualized within fresh blood by ELISPOT using free peptide or by binding of HLA-A*0201 tetrameric complexes. The class I-restricted CD8+ T cells were potent CTL effector cells that efficiently lysed an HLA-A2-matched monocyte cell line pulsed with peptide as well as autologous macrophages infected with Mycobacterium tuberculosis or recombinant vaccinia virus expressing the whole Ag85A protein. Tetramer assays revealed a 6-fold higher frequency of peptide-specific T cells than IFN-γ ELISPOT assays, indicating functional heterogeneity within the CD8+ T cell population. These results demonstrate a previously unrecognized, MHC class I-restricted, CD8+ CTL response to a major secreted Ag of mycobacteria and supports the use of Ag85A as a candidate vaccine against tuberculosis.
Synergy and Cross-Tolerance Between Toll-Like Receptor (TLR) 2- and TLR4-Mediated Signaling Pathways
Authors: Sato Shintaro , Nomura Fumiko , Kawai Taro , Takeuchi Osamu , Mühlradt Peter F. , Takeda Kiyoshi , Akira Shizuo 
Published: April 2014

AbstractA family of Toll-like receptor (TLR) mediates the cellular response to bacterial cell wall components; murine TLR2 and TLR4 recognize mycoplasmal lipopeptides (macrophage-activating lipopeptides, 2 kDa (MALP-2)) and LPS, respectively. Costimulation of mouse peritoneal macrophages with MALP-2 and LPS results in a marked increase in TNF-α production, showing the synergy between TLR2- and TLR4-mediated signaling pathways. Macrophages pretreated with LPS show hyporesponsiveness to the second LPS stimulation, termed LPS tolerance. The LPS tolerance has recently been shown to be primarily due to the down-regulation of surface expression of the TLR4-MD2 complex. When macrophages were treated with MALP-2, the cells showed hyporesponsiveness to the second MALP-2 stimulation, like LPS tolerance. Furthermore, macrophages pretreated with MALP-2 showed reduced production of TNF-α in response to LPS. LPS-induced activation of both NF-κB and c-Jun NH2-terminal kinase was severely impaired in MALP-2-pretreated cells. However, MALP-2-pretreated macrophages did not show any reduction in surface expression of the TLR4-MD2 complex. These findings indicate that LPS-induced LPS tolerance mainly occurs through the down-regulation of surface expression of the TLR4-MD2 complex; in contrast, MALP-2-induced LPS tolerance is due to modulation of the downstream cytoplasmic signaling pathways.
Granulysin, a T Cell Product, Kills Bacteria by Altering Membrane Permeability
Authors: Ernst William A. , Thoma-Uszynski Sybille , Teitelbaum Rachel , Ko Christine , Hanson Dennis A. , Clayberger Carol , Krensky Alan M. , Leippe Matthias , Bloom Barry R. , Ganz Tomas , Modlin Robert L. 
Published: April 2014

Abstract
Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four α helical bundle motif, with the α helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the α helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic β-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.
IL-12-Activated NK Cells Reduce Lung Eosinophilia to the Attachment Protein of Respiratory Syncytial Virus But Do Not Enhance the Severity of Illness in CD8 T Cell-Immunodeficient Conditions
Authors: Hussell Tracy , Openshaw Peter J. M. 
Published: April 2014

AbstractBronchiolitis caused by respiratory syncytial virus (RSV) infection is a major cause of hospitalization in children under 1 year of age. RSV causes common colds in older children and adults, but can cause serious disease in immunodeficient patients and the elderly. Development of effective vaccines and treatments for RSV infection is therefore a priority. Because bronchiolitis and vaccine-augmented disease are thought to be caused by exuberant T cell activation, attention has focused on the use of immunomodulators that affect T cell responses. In mice, IL-12 treatment down-regulates type 2 cytokine responses to the attachment protein G of RSV, reducing lung eosinophilia but further enhancing illness. We now show that CD8+ T cells are responsible for enhanced weight loss, whereas IL-12-activated NK cells express high levels of IFN-γ and inhibit lung eosinophilia without causing illness. Moreover, unlike immunocompetent mice, virus is detected in the mediastinal lymph nodes after elimination of both CD8+ T cells and NK cells. These studies show that innate immune responses to viral infections direct the pattern of subsequent specific immunity and are critical to the development of nonpathogenic antiviral effects. We speculate that IL-12 treatment might be beneficial and safe in T cell-deficient patients with RSV pneumonitis.
T Cell-Mediated Tumor Rejection Displays Diverse Dependence Upon Perforin and IFN-γ Mechanisms That Cannot Be Predicted From In Vitro T Cell Characteristics
Authors: Peng Liaomin , Krauss John C. , Plautz Gregory E. , Mukai Shigehiko , Shu Suyu , Cohen Peter A. 
Published: April 2014

AbstractExperimental pulmonary metastases have been successfully treated by adoptive transfer of tumor-sensitized T cells from perforin knockout (KO) or Fas/APO-1 ligandKO mice, suggesting a prominent role for secretion of cytokines such as IFN-γ. In the present study we confirmed that rejection of established methylcholanthrene-205 (MCA-205) pulmonary metastases displayed a requirement for T cell IFN-γ expression. However, this requirement could be obviated by transferring larger numbers of tumor-sensitized IFN-γ KO T cells or by immunosensitizing sublethal irradiation (500 rad) of the host before adoptive therapy. Extrapulmonary tumors (MCA-205 s.c. and intracranial) that required adjunct sublethal irradiation for treatment efficacy also displayed no requirement for host or T cell expression of IFN-γ. Nonetheless, rejection of MCA-205 s.c. tumors and i.p. EL-4 tumors, but not MCA-205 pulmonary or intracranial tumors, displayed a significant requirement for T cell perforin expression (i.e., CTL participation). The capacity of T cells to lyse tumor targets and secrete IFN-γ in vitro before adoptive transfer was nonpredictive of the roles of these activities in subsequent tumor rejection. Adoptive therapy studies employing KO mice are therefore indispensable for revealing a diversity of tumor rejection mechanisms that may lack in vitro correlation due to delays in their induction. Seemingly contradictory KO data from different studies are reconciled by the capacity of anti-tumor T cells to rely on alternative mechanisms when treated in larger numbers, the variable participation of CTL at different anatomic locations of tumor, and the apparent capacity of sublethal irradiation to provide a therapeutic alternative to host or T cell IFN-γ production.
Evidence for an Accessory Protein Function for Toll-Like Receptor 1 in Anti-Bacterial Responses
Authors: Wyllie D. H. , Kiss-Toth E. , Visintin A. , Smith S. C. , Boussouf S. , Segal D. M. , Duff G. W. , Dower S. K. 
Published: April 2014

AbstractMembers of the Toll-like receptor (TLR) family are components of the mammalian anti-microbial response, signaling with a domain closely related to that of IL-1 receptors. In this report the expression and function of TLR1, a TLR of unknown function, are examined. TLR1 is expressed by monocytes, as demonstrated using a novel mAb. Monocytes also express TLR2. TLR1 transfection of HeLa cells, which express neither TLR1 nor TLR2, was not sufficient to confer responsiveness to several microbial extracts. However, cotransfection of TLR1 and TLR2 resulted in enhanced signaling by HeLa cells to soluble factors released from Neisseria meningitidis relative to the response with either TLR alone. This phenomenon was also seen with high concentrations of some preparations of LPS. The N. meningitidis factors recognized by TLR1/TLR2 were not released by N. meningitidis mutant in the LpxA gene. Although LpxA is required for LPS biosynthesis, because cooperation between TLR1 and TLR2 was not seen with all LPS preparations, the microbial component(s) TLR1/2 recognizes is likely to be a complex of LPS and other molecules or a compound metabolically and chemically related to LPS. The functional IL-1R consists of a heterodimer; this report suggests a similar mechanism for TLR1 and TLR2, for certain agonists. These data further suggest that mammalian responsiveness to some bacterial products may be mediated by combinations of TLRs, suggesting a mechanism for diversifying the repertoire of Toll-mediated responses.
Vγ9Vδ2 T Cells Impair Intracellular Multiplication ofBrucella suisin Autologous Monocytes Through Soluble Factor Release and Contact-Dependent Cytotoxic Effect
Authors: Ottones Florence , Dornand Jacques , Naroeni Aroem , Liautard Jean-Pierre , Favero Jean 
Published: April 2014

AbstractHuman Vγ9Vδ2 T cells are considered to play an important role in brucellosis, as this population is dramatically increased in peripheral blood of patients during the acute phase of the infection. This T lymphocyte population has been largely demonstrated to be activated by small m.w. nonpeptidic molecules from natural or synthetic origin. We recently identified a nonpeptidic fraction of Brucella suis that specifically activates human Vγ9Vδ2 T cells. Using a two-separate-chambers system, we showed that Brucella fraction, as well as isopentenyl pyrophosphate-activated Vγ9Vδ2 T cells, impaired the multiplication of B. suis in differentiated THP-1 cells through TNF-α and IFN-γ release. In the present study, using circulating Vγ9Vδ2 T cells and autologous monocytes infected with B. suis, we provide evidence that 1) intramonocytic multiplication of B. suis is impaired by supernatants of activated Vγ9Vδ2 T cells in part via TNF-α and IFN-γ, this impairment occurring without host cell lysis; 2) unstimulated Vγ9Vδ2 T cells can impair intracellular bacterial multiplication after their activation by soluble factors released by infected monocytes; and 3) activated Vγ9Vδ2 T cells lyse Brucella-infected monocytes in a contact-dependent manner. Taken together, these results provide evidence that Vγ9Vδ2 T cells, in addition to being directly activated by soluble nonpeptidic molecules, can be stimulated to become highly cytotoxic in the specific presence of infected monocytes; moreover, they suggest how Vγ9Vδ2 T cells could be triggered and respond as antibacterial effector cells in the early stages of Brucella infection.
T Cell Expression Cloning of aMycobacterium tuberculosisGene Encoding a Protective Antigen Associated with the Early Control of Infection
Authors: Skeiky Yasir A. W. , Ovendale Pamela J. , Jen Shyian , Alderson Mark R. , Dillon Davin C. , Smith Sherilyn , Wilson Christopher B. , Orme Ian M. , Reed Steven G. , Campos-Neto Antonio 
Published: April 2014

AbstractInfection of C57BL/6 mice with Mycobacterium tuberculosis results in the development of a progressive disease during the first 2 wk after challenge. Thereafter, the disease is controlled by the emergence of protective T cells. We have used this infection model in conjunction with direct T cell expression cloning to identify Ags involved with the early control of the disease. A protective M. tuberculosis-specific CD4 T cell line derived from mice at 3 wk postchallenge was used to directly screen an M. tuberculosis genomic expression library. This screen resulted in the identification of a genomic clone comprising two putative adjacent genes with predicted open reading frames of 10 and 41 kDa, MTB10 and MTB41, respectively (the products of Rv0916c and Rv0915c, respectively, in the TubercuList H37Rv database). MTB10 and MTB41 belong to the PE and PPE family of proteins recently identified to comprise 10% of the M. tuberculosis genome. Evaluation of the recombinant proteins revealed that MTB41, but not MTB10, is the Ag recognized by the cell line and by M. tuberculosis-sensitized human PBMC. Moreover, C57BL/6 mice immunized with MTB41 DNA developed both CD4- (predominantly Th1) and CD8-specific T cell responses to rMTB41 protein. More importantly, immunization of C57BL/6 mice with MTB41 DNA induced protection against infection with M. tuberculosis comparable to that induced by bacillus Calmette-Guérin. Thus, the use of a proven protective T cell line in conjunction with the T cell expression cloning approach resulted in the identification of a candidate Ag for a subunit vaccine against tuberculosis.
Inhibition of TNF-α Produced by Kupffer Cells Protects Against the Nonspecific Liver Toxicity of Immunotoxin Anti-Tac(Fv)-PE38, LMB-2
Authors: Onda Masanori , Willingham Mark , Wang Qing-cheng , Kreitman Robert J. , Tsutsumi Yasuo , Nagata Satoshi , Pastan Ira 
Published: April 2014

AbstractLMB-2 (anti-Tac(Fv)-PE38) is a recombinant immunotoxin composed of the Fv fragment of the anti-Tac Ab fused to a 38-kDa form of Pseudomonas exotoxin A. Recent clinical trials showed that LMB-2 is a promising agent for the treatment of patients with Tac-positive leukemia or lymphoma. One major side effect that needs to be overcome is nonspecific liver toxicity. In the current study, we have analyzed the mechanism of this toxicity using a mouse model. Mice that were injected with a lethal dose of LMB-2 showed severe hepatic necrosis. Immunohistochemistry revealed that LMB-2 accumulated in Kupffer cells in the liver, suggesting that the damage to the hepatocytes was indirect. When we examined the effects of LMB-2 on peritoneal macrophages, cells in the same lineage as Kupffer cells, we found that LMB-2 induced the production of TNF-α by these cells. Following LMB-2 administration to mice, the levels of TNF-α in the liver increased to very high levels, whereas the rise in serum levels was modest. In addition, the LMB-2-induced liver toxicity was blocked by a specific TNF binding protein (TNFsRp55). Liver toxicity was also blocked by indomethacin, which also blocked the rise of TNF-α in the liver. Both TNFsRp55 and indomethacin treatment protected mice against a lethal dose of LMB-2. These data indicate that TNF-α produced in the liver by Kupffer cells has an important causal role in the nonspecific liver toxicity of LMB-2. These findings have important clinical implications for the use of immunotoxins in the therapy of patients with cancer.