bluuebunny/crossref_metadata_2025_split huggingface dataset:
DNA Binding by the VH Domain of Anti-Z-DNA Antibody and Its Modulation by Association of the VL Domain
Authors: Chen Yan , Stollar B. David 
Published: January 2023

AbstractmAb Z22 is a highly selective IgG anti-Z-DNA Ab from an immunized C57BL/6 mouse. Previous studies showed that heavy chain CDR3 amino acids are critical for Z-DNA binding by the single chain variable fragment (scFv) comprising both V region heavy chain (VH) and V region light chain (VL) of mAb Z22 and that the VH domain alone binds Z-DNA with an affinity similar to that of whole variable fragment (Fv). To determine whether Z-DNA binding by VH alone and by Fv involves identical complementarity determining region residues, we tested effects of single or multiple amino acid substitutions in recombinant VH, scFv, and associated VH-VL heterodimers. Each recombinant product was a fusion protein with a B domain of Staphylococcal protein A (SPA). Z22VH-SPA alone was not highly selective; it bound strongly to other polynucleotides, particularly polypyrimidines, and ssDNA as well as to Z-DNA. In contrast, scFv-SPA or associated VH-VL dimers bound only to Z-DNA. VL-SPA domains bound weakly to Z-DNA; SPA alone did not bind. Introduction of multiple substitutions revealed that the third complementarity determining region of the heavy chain (CDR3H) was critical for both VH and scFv binding to Z-DNA. However, single substitutions that eliminated or markedly reduced Z-DNA binding by scFv instead caused a modest increase or no reduction in binding by VH alone. Association of VH-SPA with Z22VL-SPA restored both the effects of single substitutions and Z-DNA selectivity seen with Fv and intact Ab. Polypyrimidine and ssDNA binding by the isolated VH domain of immunization-induced anti-Z-DNA Ab resembles the activity of natural autoantibodies and suggests that VH-dependent binding to a ligand mimicked by polypyrimidines may play a role in B cell selection before immunization with Z-DNA.
Specific Proteolytic Cleavages Limit the Diversity of the Pool of Peptides Available to MHC Class I Molecules in Living Cells
Authors: Serwold Thomas , Shastri Nilabh 
Published: January 2023

AbstractMHC class I molecules display peptides selected from a poorly characterized pool of peptides available in the endoplasmic reticulum. We analyzed the diversity of peptides available to MHC class I molecules by monitoring the generation of an OVA-derived octapeptide, OVA257–264 (SL8), and its C-terminally extended analog, SL8-I. The poorly antigenic SL8-I could be detected in cell extracts only after its conversion to the readily detectable SL8 with carboxypeptidase Y. Analysis of extracts from cells expressing the minimal precursor Met-SL8-I by this method revealed the presence of SL8/Kb and the extended SL8-I/Kb complexes, indicating that the peptide pool contained both peptides. In contrast, cells expressing full length OVA generated only the SL8/Kb complex, demonstrating that the peptide pool generated from the full length precursor contained only a subset of potential MHC-binding peptides. Deletion analysis revealed that SL8-I was generated only from precursors lacking additional C-terminal flanking residues, suggesting that the generation of the C terminus of the SL8 peptide involves a specific endopeptidase cleavage. To investigate the protease responsible for this cleavage, we tested the effect of different protease inhibitors on the generation of the SL8 and SL8-I peptides. Only the proteasome inhibitors blocked generation of SL8, but not SL8-I. These findings demonstrate that the specificities of the proteases in the Ag-processing pathway, which include but are not limited to the proteasome, limit the diversity of peptides available for binding by MHC class I molecules in the endoplasmic reticulum.
Enhancement of the Listeria monocytogenes p60-Specific CD4 and CD8 T Cell Memory by Nonpathogenic Listeria innocua
Authors: Geginat Gernot , Nichterlein Thomas , Kretschmar Marianne , Schenk Simone , Hof Herbert , Lalic-Mülthaler Mio , Goebel Werner , Bubert Andreas 
Published: January 2023

Abstract
The contact of T cells to cross-reactive antigenic determinants expressed by nonpathogenic environmental micro-organisms may contribute to the induction or maintenance of T cell memory. This hypothesis was evaluated in the model of murine Listeria monocytogenes infection. The influence of nonpathogenic L. innocua on the L. monocytogenes p60-specific T cell response was analyzed. We show that some CD4 T cell clones raised against purified p60 from L. monocytogenes cross-react with p60 purified from L. innocua. The L. monocytogenes p60-specific CD4 T cell clone 1A recognized the corresponding L. innocua p60 peptide QAAKPAPAPSTN, which differs only in the first amino acid residue. In vitro experiments revealed that after L. monocytogenes infection of APCs, MHC class I-restricted presentation of p60 occurs, while MHC class II-restricted p60 presentation is inhibited. L. innocua-infected cells presented p60 more weakly but equally well in the context of both MHC class I and MHC class II. In contrast to these in vitro experiments the infection of mice with L. monocytogenes induced a strong p60-specific CD4 and CD8 T cell response, while L. innocua infection failed to induce p60-specific T cells. L. innocua booster infection, however, expanded p60-specific memory T cells induced by previous L. monocytogenes infection. In conclusion, these findings suggest that infection with a frequently occurring environmental bacterium such as L. innocua, which is nonpathogenic and not adapted to intracellular replication, can contribute to the maintenance of memory T cells specific for a related intracellular pathogen.
DNA Vaccination Against the Idiotype of a Murine B Cell Lymphoma: Mechanism of Tumor Protection
Authors: Syrengelas Athanasia D. , Levy Ronald 
Published: January 2023

AbstractSeveral studies have shown that immunization with DNA, which encodes the idiotypic determinants of a B cell lymphoma, generates tumor-specific immunity. Although induction of antiidiotypic Abs has correlated with tumor protection, the effector mechanisms that contribute to tumor protection have not been clearly identified. This study evaluated the tumor protective effects of humoral and cellular immune mechanisms recruited by idiotype-directed DNA vaccines in the 38C13 murine B cell lymphoma model. Antiidiotypic Abs induced by DNA vaccination supported in vitro complement-mediated cytotoxicity of tumor cells, and simultaneous transfer of tumor cells and hyperimmune sera protected naive animals against tumor growth. However, in vitro stimulation of immune splenocytes with tumor cells failed to induce idiotype-specific cytotoxicity, and following vaccination, depletion of CD4 or CD8 T cell subsets did not compromise protection. Furthermore, protection of naive recipients against tumor challenge could not be demonstrated either by a Winn assay approach or by adoptive transfer of spleen and lymph node cells. Thus, in this experimental model, current evidence suggests that the tumor-protective effects of DNA vaccination can be largely attributed to idiotype-specific humoral immunity.
Endotoxin-Stimulated Monocytes Release Multiple Forms of IL-1β, Including a ProIL-1β Form Whose Detection Is Affected by Export
Authors: Wewers2 Mark D. , Winnard Alissa V. , Dare Heidi A. 
Published: January 2023

Abstract
The processing and release of 31-kDa proIL-1β to the mature 17-kDa form of IL-1β are still poorly understood. To help elucidate the mechanisms involved in IL-1β processing and release, we measured IL-1β forms released from endotoxin-stimulated monocytes by immunoprecipitation of [35S]methionine-labeled protein, by Western blots, and by our recently developed ELISA specific for proIL-1β. Our studies demonstrate that in addition to the 17-kDa mature IL-1β, IL-1β is also released as 31-, 28-, and 3-kDa molecules. The 31-kDa-released form of proIL-1β represented 20–40% of the total released IL-1β, as measured by SDS-PAGE with densitometry. This released proIL-1β was susceptible to ICE processing; however, this proIL-1β was not detectable by either a mature or proIL-1β-specific ELISA, suggesting that release induces a conformational change. The ELISA inability to detect proIL-1β was not due to inadequate sensitivity or subsequent degradation in the ELISA. Furthermore, while immunoaffinity-purified cytosolic proIL-1β could complex the type II IL-1R, released proIL-1β did not. Finally, the absence of a band shift in nondenaturing gel electrophoresis excluded proIL-1β binding to another protein. These findings imply that IL-1β is exported from monocytes as 3-, 17-, 28-, and 31-kDa forms and that the released 31-kDa form differs from cytosolic proIL-1β.
IL-3 and IL-4 Activate Cyclic Nucleotide Phosphodiesterases 3 (PDE3) and 4 (PDE4) by Different Mechanisms in FDCP2 Myeloid Cells
Authors: Ahmad Faiyaz , Gao Guang , Wang Ling Mei , Landstrom Tova Rahn , Degerman Eva , Pierce Jacalyn H. , Manganiello Vincent C. 
Published: January 2023

Abstract
In FDCP2 myeloid cells, IL-4 activated cyclic nucleotide phosphodiesterases PDE3 and PDE4, whereas IL-3, granulocyte-macrophage CSF (GM-CSF), and phorbol ester (PMA) selectively activated PDE4. IL-4 (not IL-3 or GM-CSF) induced tyrosine phosphorylation of insulin-receptor substrate-2 (IRS-2) and its association with phosphatidylinositol 3-kinase (PI3-K). TNF-α, AG-490 (Janus kinase inhibitor), and wortmannin (PI3-K inhibitor) inhibited activation of PDE3 and PDE4 by IL-4. TNF-α also blocked IL-4-induced tyrosine phosphorylation of IRS-2, but not of STAT6. AG-490 and wortmannin, not TNF-α, inhibited activation of PDE4 by IL-3. These results suggested that IL-4-induced activation of PDE3 and PDE4 was downstream of IRS-2/PI3-K, not STAT6, and that inhibition of tyrosine phosphorylation of IRS molecules might be one mechnism whereby TNF-α could selectively regulate activities of cytokines that utilized IRS proteins as signal transducers. RO31-7549 (protein kinase C (PKC) inhibitor) inhibited activation of PDE4 by PMA. IL-4, IL-3, and GM-CSF activated mitogen-activated protein (MAP) kinase and protein kinase B via PI3-K signals; PMA activated only MAP kinase via PKC signals. The MAP kinase kinase (MEK-1) inhibitor PD98059 inhibited IL-4-, IL-3-, and PMA-induced activation of MAP kinase and PDE4, but not IL-4-induced activation of PDE3. In FDCP2 cells transfected with constitutively activated MEK, MAP kinase and PDE4, not PDE3, were activated. Thus, in FDCP2 cells, PDE4 can be activated by overlapping MAP kinase-dependent pathways involving PI3-K (IL-4, IL-3, GM-CSF) or PKC (PMA), but selective activation of PDE3 by IL-4 is MAP kinase independent (but perhaps IRS-2/PI3-K dependent).
A Fatal Cytokine-Induced Systemic Inflammatory Response Reveals a Critical Role for NK Cells
Authors: Carson William E. , Yu Haixin , Dierksheide Julie , Pfeffer Klaus , Bouchard Page , Clark Reed , Durbin Joan , Baldwin Albert S. , Peschon Jacques , Johnson Philip R. , Ku George , Baumann Heinz , Caligiuri Michael A. 
Published: January 2023

AbstractThe mechanism of cytokine-induced shock remains poorly understood. The combination of IL-2 and IL-12 has synergistic antitumor activity in vivo, yet has been associated with significant toxicity. We examined the effects of IL-2 plus IL-12 in a murine model and found that the daily, simultaneous administration of IL-2 and IL-12 resulted in shock and 100% mortality within 4 to 12 days depending on the strain employed. Mice treated with IL-2 plus IL-12 exhibited NK cell apoptosis, pulmonary edema, degenerative lesions of the gastrointestinal tract, and elevated serum levels of proinflammatory cytokines and acute phase reactants. The actions of TNF-α, IFN-γ, macrophage-inflammatory protein-1α, IL-1, IL-1-converting enzyme, Fas, perforin, inducible nitric oxide synthase, and STAT1 did not contribute to the observed toxicity, nor did B or T cells. However, toxicity and death from treatment with IL-2 plus IL-12 could be completely abrogated by elimination of NK cells. These results suggest that the fatal systemic inflammatory response induced by this cytokine treatment is critically dependent upon NK cells, but does not appear to be mediated by the known effector molecules of this cellular compartment. These data may provide insight into the pathogenesis of cytokine-induced shock in humans.
4-1BBL Cooperates with B7-1 and B7-2 in Converting a B Cell Lymphoma Cell Line into a Long-Lasting Antitumor Vaccine
Authors: Guinn Barbara-ann , DeBenedette Mark A. , Watts Tania H. , Berinstein Neil L. 
Published: January 2023

Abstract
A20 is a B cell lymphoma that constitutively expresses the costimulatory molecule B7-2 yet grows readily as a tumor in syngeneic BALB/c mice. We have compared the tumorigenicity of A20 variants expressing either B7-1 (A20/B7-1) or B7-2 (A20/B7-2) with an A20 variant expressing B7-1 and B7-2 with 4-1BBL (A20/4-1BBL), a costimulatory member of the TNF family. Mice injected with tumors expressing the vector backbone (A20/CMV) or B7-1 developed tumors within 25 days of s.c. injection. In contrast, mice injected with A20/4-1BBL were tumor free for the 150-day follow-up period, while 25% of mice injected with A20/B7-2 developed tumors. Tumorigenicity experiments using nude mice indicated the requirement for T cells for variant rejection. Almost all mice that resisted the initial tumor challenge were resistant to further challenge with the parental tumor. Splenocytes from these mice showed high CTL lytic activity against the parental tumor, A20, as well as the syngeneic BALB/c lymphoma K46J, but showed background levels of lytic activity against the congenic SCID thymoma line ST-D2 or the allogeneic EL4 thymoma. In vitro blocking experiments with anti-B7-1 plus anti-B7-2 and/or soluble 4-1BB receptor showed B7-1, B7-2, and 4-1BBL all contributed to the CTL activity. Thus, the data show that neither B7-1 or B7-2 alone can confer full immunogenicity to the A20 lymphoma but that the addition of 4-1BBL results in a tumor that is highly immunogenic and can confer long-lasting protection against challenge with parental tumor in vivo.
Induction of Cyclooxygenase-2 on Activated T Lymphocytes: Regulation of T Cell Activation by Cyclooxygenase-2 Inhibitors
Authors: Iñiguez Miguel A. , Punzón Carmen , Fresno Manuel 
Published: January 2023

AbstractCyclooxygenase (COX), known to exist in two isoforms, COX-1 and COX-2, is a key enzyme in prostaglandin synthesis and the target for most nonsteroidal anti-inflammatory drugs. In this study, we show that human T lymphocytes express the COX-2 isoenzyme. COX-2 mRNA and protein were induced in both Jurkat and purified T cells stimulated by TCR/CD3 or PMA activation. COX-2 mRNA was induced very early after activation and superinduced by protein synthesis inhibitors, whereas it was inhibited by the immunosuppressive drug cyclosporin A, identifying it as an early T cell activation gene. Interestingly, treatment with COX-2-specific inhibitors such as NS398 or Celecoxib severely diminished early and late events of T cell activation, including CD25 and CD71 cell surface expression, IL-2, TNF-α, and IFN-γ production and cell proliferation, but not the expression of CD69, an immediate early gene. COX-2 inhibitors also abolished induced transcription of reporter genes driven by IL-2 and TNF-α promoters. Moreover, induced transcription from NF-κB- and NF-AT-dependent enhancers was also inhibited. These results may have important implications in anti-inflammatory therapy and open a new field on COX-2-selective nonsteroidal anti-inflammatory drugs as modulators of the immune activation.
Immunogenicity of Apoptotic Cells In Vivo: Role of Antigen Load, Antigen-Presenting Cells, and Cytokines
Authors: Ronchetti Anna , Rovere Patrizia , Iezzi Giandomenica , Galati Giacomo , Heltai Silvia , Protti Maria Pia , Garancini Maria Paola , Manfredi Angelo A. , Rugarli Claudio , Bellone Matteo 
Published: January 2023

Abstract
Apoptosis allows the clearance of unwanted cells from living tissues without causing inflammation. Processing of phagocytosed apoptotic cells yields Ags that access the cytosol and the MHC class I pathway of engulfing cells and are recognized by Ag-specific CTL. We show here that injection of apoptotic RMA cells, a syngeneic T cell lymphoma, into C57BL/6 mice results in priming of a functional and long-lasting tumor-specific immune response. Cross-priming of CTLs by apoptotic cells requires CD4+ T cell help. Apoptotic cells, however, are at least 20-fold less immunogenic than nonreplicating live cells. Immunogenicity of apoptotic cells is proportional to the number of cells injected, correlates with the serum concentration of IL-10 and IL-1β cytokines, and is enhanced in IL-10 knockout mice. Moreover, immunization with dendritic cells (DCs), but not macrophages (Mφ), pulsed with apoptotic cells primes tumor-specific CTLs and confers protection against a tumor challenge. Our findings demonstrate that tumor cells undergoing apoptosis are, though scarcely, immunogenic in vivo, outline the different roles of Mφ and DCs in the physiologic clearance of unwanted cells, and have implications in designing immunomodulating vaccines.
Anergic CD8+ T Cells Can Persist and Function In Vivo
Authors: Blish Catherine A. , Dillon Stacey R. , Farr Andrew G. , Fink Pamela J. 
Published: January 2023

Abstract
Using a mouse model system, we demonstrate that anergic CD8+ T cells can persist and retain some functional capabilities in vivo, even after the induction of tolerance. In TCR Vβ5 transgenic mice, mature CD8+Vβ5+ T cells transit through a CD8lowVβ5low deletional intermediate during tolerance induction. CD8low cells are characterized by an activated phenotype, are functionally compromised in vitro, and are slated for deletion in vivo. We now demonstrate that CD8low cells derive from a proliferative compartment, but do not divide in vivo. CD8low cells persist in vivo with a t1/2 of 3–5 days, in contrast to their in vitro t1/2 of 0.5–1 day. During this unexpectedly long in vivo life span, CD8low cells are capable of producing IFN-γ in vivo despite their inability to proliferate or to kill target cells in vitro. CD8low cells also accumulate at sites of inflammation, where they produce IFN-γ. Therefore, rather than withdrawing from the pool of functional CD8+ T cells, anergic CD8low cells retain a potential regulatory role despite losing their capacity to proliferate. The ability of anergic cells to persist and function in vivo adds another level of complexity to the process of tolerance induction in the lymphoid periphery.
Predicting Regional Mutability in Antibody V Genes Based Solely on Di- and Trinucleotide Sequence Composition
Authors: Shapiro Gary S. , Aviszus Katja , Ikle David , Wysocki Lawrence J. 
Published: January 2023

AbstractSomatic mutations are not distributed randomly throughout Ab V region genes. A sequence-specific target bias is revealed by a defined hierarchy of mutability among di- and trinucleotide sequences located within Ig intronic DNA. Here we report that the di- and trinucleotide mutability preference pattern is shared by mouse intronic JH and Jκ clusters and by human VH genes, suggesting that a common mutation mechanism exists for all Ig V genes of both species. Using di- and trinucleotide target preferences, we performed a comprehensive analysis of human and murine germline V genes to predict regional mutabilities. Heavy chain genes of both species exhibit indistinguishable patterns in which complementarity-determining region 1 (CDR1), CDR2, and framework region 3 (FR3) are predicted to be more mutable than FR1 and FR2. This prediction is borne out by empirical mutation data from nonproductively rearranged human VH genes. Analysis of light chain genes in both species also revealed a common, but unexpected, pattern in which FR2 is predicted to be highly mutable. While our analyses of nonfunctional Ig genes accurately predicts regional mutation preferences in VH genes, observed relative mutability differences between regions are more extreme than expected. This cannot be readily accounted for by nascent mRNA secondary structure or by a supplemental gene conversion mechanism that might favor nucleotide replacements in CDR. Collectively, our data support the concept of a common mutation mechanism for heavy and light chain genes of mice and humans with regional bias that is qualitatively, but not quantitatively, accounted for by short nucleotide sequence composition.
Surfactant Protein D Binds to Mycobacterium tuberculosis Bacilli and Lipoarabinomannan via Carbohydrate-Lectin Interactions Resulting in Reduced Phagocytosis of the Bacteria by Macrophages1
Authors: Ferguson J. Scott , Voelker Dennis R. , McCormack Francis X. , Schlesinger Larry S. 
Published: January 2023

Abstract
Surfactant protein-D (SP-D) is a collectin produced in the distal lung airspaces that is believed to play an important role in innate pulmonary immunity. Naive immunologic responses to Mycobacterium tuberculosis (M.tb) are especially important in the lung, since entry of this inhaled pathogen into the alveolar macrophage is a pivotal event in disease pathogenesis. Here we investigated SP-D binding to M.tb and the effect of this binding on the adherence of M.tb to human macrophages. These studies demonstrate specific binding of SP-D to M.tb that is saturable, calcium dependent, and carbohydrate inhibitable. In addition to purified SP-D, SP-D in bronchoalveolar lavage fluids from healthy donors and patients with alveolar proteinosis also binds to M.tb. Incubation of M.tb with SP-D results in agglutination of the bacteria. In contrast to its binding to M.tb, SP-D binds minimally to the avirulent Mycobacterium smegmatis. SP-D binds predominantly to lipoarabinomannan from the virulent Erdman strain of M.tb, but not the lipoarabinomannan from M. smegmatis. The binding of SP-D to Erdman lipoarabinomannan is mediated by the terminal mannosyl oligosaccharides of this lipoglycan. Incubation of M.tb with subagglutinating concentrations of SP-D leads to reduced adherence of the bacteria to macrophages (62.7% of control adherence ± 3.3% SEM, n = 8), whereas incubation of bacteria with surfactant protein A leads to significantly increased adherence to monocyte-derived macrophages. These data provide evidence for specific binding of SP-D to M. tuberculosis and indicate that SP-D and surfactant protein A serve different roles in the innate host response to this pathogen in the lung.
P-Selectin Binding Promotes the Adhesion of Monocytes to VCAM-1 Under Flow Conditions
Authors: Yago Tadayuki , Tsukuda Mamoru , Minami Mutsuhiko 
Published: January 2023

AbstractThis study examined the adhesive interaction of peripheral blood monocytes with VCAM-1 and analyzed the effect of P-selectin binding to monocytes on the adhesive interaction with VCAM-1 under flow conditions. P-selectin glycoprotein ligand-1 is expressed on most monocytes. Furthermore, most monocytes bind soluble P-selectin derived from platelets. P-selectin binding to monocytes did not alter the amount of expression of α4 integrin on monocytes. However, the mean channel fluorescence value for binding Cy2-conjugated soluble VCAM-1 to P-selectin-bound monocytes was slightly more than that for binding Cy2-conjugated soluble VCAM-1 to untreated monocytes. Under flow conditions, the number of P-selectin-bound monocytes bound to VCAM-1 was much higher than that of untreated monocytes bound to VCAM-1. These bindings were abolished by pretreatment of untreated monocytes and P-selectin-bound monocytes with anti-VCAM-1 mAb or anti-α4 integrin mAb. Furthermore, P-selectin binding to monocytes increased shear resistance and thus increased the adhesive strength of monocytes to VCAM-1. These findings indicate that P-selectin binding to monocytes enhances the adhesive interaction of monocytes with VCAM-1. It is suggested that P-selectin glycoprotein ligand-1/P-selectin interaction and α4 integrin/VCAM-1 interaction can act sequentially in the adhesion cascade that regulates monocyte trafficking to inflammatory and atherosclerotic lesion.