bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vitro Comparison of the Biologic Activities of Monoclonal Monomeric IgA, Polymeric IgA, and Secretory IgA
Authors: Renegar Kathryn B. , Jackson Graham D. F. , Mestecky Jiri
Published: December 2022

Secretory IgA (S-IgA), a major humoral mediator of mucosal immunity, is a polymeric Ig containing an unusual extra polypeptide, secretory component (SC), added during transcytosis through epithelial cells. Polymeric S-IgA is more effective than monomeric IgA (mIgA) and IgG in neutralizing viruses. It is not known whether this increased efficacy is due solely to the polymeric structure of the molecule or whether SC itself makes S-IgA more efficient; a quantitative in vitro comparison of the biologic activities of S-IgA and pIgA has not been reported. We prepared purified pIgA and mIgA mAbs directed toward the H1 hemagglutinin of PR8 influenza virus and purified monoclonal S-IgA (made from monoclonal pIgA injected into a Lewis rat and collected as S-IgA from bile) and compared their abilities to carry out hemagglutination inhibition (HI) and neutralization of the infectivity of PR8 influenza virus in vitro. The polymeric Igs (pIgA and S-IgA) were 5 times more effective than mIgA in HI and 7 to 10 times more effective than mIgA in virus neutralization. Addition of SC to pIgA did not modify its ability to mediate HI and had only a minimal effect (S-IgA was 1.4 times more effective) on its ability to neutralize influenza virus in vitro. Trypsin preincubation partially abolished mIgA- or pIgA-mediated, but not S-IgA-mediated, viral neutralization. Thus, although S-IgA is more stable functionally than pIgA, the addition of SC does not influence, positively or negatively, the biologic activity associated with the Fab of S-IgA.
Structure, Diversity, and Repertoire of VH Families in the Mexican Axolotl
Authors: Golub Rachel , Charlemagne Jacques
Published: December 2022

The Mexican axolotl VH segments associated with the Igh Cμ and Cυ isotypes were isolated from anchored PCR libraries prepared from spleen cell cDNA. The eight new VH segments found bring the number of VH families in the axolotl to 11. Each VH had the canonical structural features of vertebrate VH segments, including residues important for the correct folding of the Ig domain. The distribution of ser AGC/T (AGY) and TCN codons in axolotl VH genes was biased toward AGY in complementarity-determining region-1 (CDR1) and TCN in framework region-1 (FR1); there were no ser residues in the FR2 region. Thus, the axolotl CDR1 region is enriched in DNA sequences forming potential hypermutation hot spots and is flanked by DNA sequences more resistant to point mutation. There was no significant bias toward AGY in CDR2. Southern blotting using family-specific VH probes showed restriction fragments from 1 (VH9) to 11–19 (VH2), and the total number of VH genes was 44 to 70, depending on the restriction endonuclease used. The VH segments were not randomly used by the Hμ and Hυ chains; VH1, VH6, and VH11 were underutilized; and the majority of the VH segments belonged to the VH7, VH8, and VH9 families. Most of the nine JH segments seemed to be randomly used, except JH6 and JH9, which were found only once in 79 clones.
Eosinophils Bind Rhinovirus and Activate Virus-Specific T Cells
Authors: Handzel Zeev T. , Busse William W. , Sedgwick Julie B. , Vrtis Rose , Lee Wai Ming , Kelly E. A. B. , Gern James E.
Published: December 2022

Episodes of virus-induced exacerbations of asthma are accompanied by increased eosinophils (EOS) in respiratory secretions and evidence of EOS degranulation. Although rhinoviruses (RV) are the viruses most often implicated in exacerbations of asthma in both children and adults, little is known about the immune response to this group of viruses and, in particular, EOS-RV interactions. To define such interactions, we incubated human rhinovirus type 16 (RV16), a serotype using ICAM-1 as a receptor, with EOS purified from PBMC, and measured EOS-RV binding, EOS-mediated Ag presentation and T cell activation, and EOS cell surface marker expression and superoxide production. Significant RV16 binding occurred to EOS that were pretreated with granulocyte-macrophage CSF, and this binding was inhibited by anti-ICAM-1 mAb. EOS also presented viral Ags to RV16-specific T cells, causing T cell proliferation and secretion of IFN-γ. RV16 induced a significant shift from CD18dim to CD18bright, but did not affect EOS expression of CD54, CD69, or HLA-DR. Finally, RV16 did not induce superoxide production from peripheral blood EOS. These findings suggest that RV16 also binds to airway EOS, which resemble granulocyte-macrophage CSF-treated blood EOS in terms of high expression of ICAM-1. Furthermore, our findings suggest that EOS could participate in RV-induced immune responses through Ag presentation and T cell activation. By activating RV-specific T cells, EOS may play an important role in the initiation of antiviral T cell responses, and these effects could also contribute to enhanced airway inflammation and increased asthma symptoms in susceptible individuals.
Protective Effect onLeishmania majorInfection of Migration Inhibitory Factor, TNF-α, and IFN-γ Administered Orally via AttenuatedSalmonella typhimurium
Authors: Xu Damo , McSorley Stephen J. , Tetley Lawrence , Chatfield Stephen , Dougan Gordon , Chan W. Ling , Satoskar Abhay , David John R. , Liew Foo Y.
Published: December 2022

The genes encoding murine macrophage migration inhibitory factor (MIF), IL-2, IFN-γ or TNF-α were cloned individually into an expression plasmid under the control of the inducible promoter nirB and transfected into the aroA−aroD− deletion mutant strain of Salmonella typhimurium (BRD509). These S. typhimurium derivatives (henceforward called constructs and termed GIDMIF, GIDIL2, GIDIFN and GIDTNF) expressed their respective cytokines in vitro under anaerobic conditions and stably colonized BALB/c mice up to 14 days after oral administration. The highly susceptible BALB/c mice that had received the constructs orally and that had been subsequently infected via the footpad with Leishmania major, developed significantly reduced disease compared with control mice administered the untransfected Salmonella strain (BRD509). Importantly, a combination of GIDMIF, GIDIFN, and GIDTNF administered orally after L. major infection was able to significantly limit lesion development and reduced parasite loads by up to three orders of magnitude. Spleen and lymph node cells of mice administered this combination expressed markedly higher levels of inducible nitric oxide synthase (iNOS) compared with those from mice receiving an equivalent dose of the control strain of Salmonella (BRD509). These data therefore demonstrate the feasibility of therapeutic treatment in an infectious disease model using cytokines delivered by attenuated Salmonella. The protective effect observed correlates with the induction of inducible nitric oxide synthase in vivo.
Activation of Mitogen-Activated Protein Kinase Pathways byMycoplasma fermentansMembrane Lipoproteins in Murine Macrophages: Involvement in Cytokine Synthesis
Authors: Rawadi Georges , Ramez Valerie , Lemercier Brigitte , Roman-Roman Sergio
Published: December 2022

Stimulation of monocytes and resident macrophages by mycoplasmas induces production of numerous cytokines. We have previously reported that membrane lipoproteins derived from Mycoplasma fermentans are responsible for the induction of proinflammatory cytokines by monocytic cells and that triggering protein tyrosine kinase activation is an essential requirement for this biologic effect. In the present study, we have investigated the effect of M. fermentans-derived membrane lipoproteins (LAMPf) on mitogen-activated protein kinase (MAPK) cascades in the murine macrophage cell line RAW 264.7 and have analyzed the contribution of these pathways to the cytokine induction mediated by this agent. Treatment of murine macrophages with LAMPf resulted in significant activation of MAPK family members extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), and p38. Unlike LPS, these effects were demonstrated to be independent of the presence of serum. The activation of MAPKs paralleled the tyrosine kinase activation and peaked at 30 min after stimulation. The specific p38 inhibitor SB203580 abrogated the mycoplasma-induced IL-6, IL-1β, and TNF-α synthesis. The selective MAPK/extracellular signal-regulated kinase 1 (MEK-1) inhibitor PD-98059 blocked both IL-1β and TNF-α but not IL-6 production by RAW 264.7 cells in response to LAMPf. Additionally, transfection of murine macrophages with a JNK dominant negative mutant significantly reduced only IL-6 production. These data underscore the role of MAPKs as signal transduction molecules controlling the expression of cytokines upon mycoplasma stimulation.
Activation of Protein Kinase C-ζ and Phosphatidylinositol 3′-Kinase and Promotion of Macrophage Differentiation by Insulin-Like Growth Factor-I
Authors: Liu Qiang , Ning Wei , Dantzer Robert , Freund Gregory G. , Kelley Keith W.
Published: December 2022

Phosphoinositides that are phosphorylated at the D3 position have been reported to activate an atypical, Ca2-independent protein kinase C (PKC) isoform designated PKC-ζ, and overexpression of this enzyme leads to monocytic differentiation. In this study, we cultured human HL-60 promyeloid cells with vitamin D3 and insulin-like growth factor-I (IGF-I), a 70-amino-acid peptide that activates phosphatidylinositol 3′-kinase (PI 3-kinase) in murine promyeloid cells. Two days later, the proportion of cells differentiating into macrophages in serum-free medium, as assessed by expression of the α-subunit of the β2 integrin CD11b, increased from 5 ± 1% to 25 ± 3%. Addition of IGF-I increased the proportion of cells differentiating into CD11b-positive macrophages to 78 ± 5%. In the absence of vitamin D3, IGF-I did not induce expression of CD11b (6 ± 1%). The IGF-I-promoted macrophage differentiation was blocked specifically by preincubation of HL-60 cells with a mAb (αIR3) directed against the IGF type I receptor. Similarly, pretreatment of cells with either αIR3 or an IGF-binding protein, IGFBP-3, led to a 75% inhibition of CD11b expression when cells were cultured with vitamin D3 in serum-containing medium. IGF-I, but not vitamin D3, caused a sevenfold increase in the enzymatic activity of both PI 3-kinase and atypical PKC-ζ. Inhibition of IGF-I-inducible PI 3-kinase with either wortmannin or LY294002 abrogated the IGF-I-induced activation of PKC-ζ and totally blocked the enhancement in macrophage differentiation caused by IGF-I. These data establish that PKC-ζ is a putative downstream target of PI 3-kinase that is activated during IGF-I-promoted macrophage differentiation.
Role of the Mitogen-Activated Protein Kinases in the Expression of the Kinin B1 Receptors Induced by Tissue Injury
Authors: Larrivée Jean-François , Bachvarov Dimcho R. , Houle François , Landry Jacques , Huot Jacques , Marceau François
Published: December 2022

Several cytokines and LPS regulate the population of the B1 receptors (B1Rs) for kinins; these are responsive to des-Arg9-bradykinin (BK) and Lys-des-Arg9-BK. B1R activation contributes to inflammatory vascular changes and pain. Aortic rings isolated from normal rabbits and incubated in vitro in Krebs physiological medium were used as a model of tissue injury. From a null level of response, these rings exhibit a time- and protein synthesis-dependent increase in the maximal contractile response to des-Arg9-BK. Exposure to exogenous IL-1β or epidermal growth factor (EGF) considerably increases the process of sensitization to the kinins. Freshly isolated control aortic rings showed high mitogen-activated protein (MAP) kinase activities (persistent activation of p38, but less prolonged for extracellular signal-regulated kinase and c-Jun-N-terminal kinase/stress-activated protein kinase pathways) relatively to the basal activities found in various types of cultured cells. IL-1β or EGF further increased the activities of the extracellular signal-regulated kinase and c-Jun-N-terminal kinase/stress-activated protein kinase MAP kinases. The inhibitor of the p38 MAP kinase, SB 203580 (10 μM), massively (∼75%) and selectively inhibited the spontaneous sensitization to des-Arg9-BK over 6 h. SB 203580 also significantly reduced the development of the response to des-Arg9-BK as stimulated by IL-1 or EGF. Both spontaneous and IL-1β-stimulated up-regulation of responsiveness to des-Arg9-BK were significantly inhibited by the MAP kinase extracellular signal-regulated kinase kinase 1 inhibitor PD 98059 (∼40%). The protein kinase inhibitors failed to inhibit protein synthesis and to acutely inhibit the contractile effect of des-Arg9-BK, suggesting that they do not influence B1 receptor transduction mechanisms. In cultured aortic smooth muscle cells stimulated with EGF, MAP kinase activation preceded B1R mRNA induction. Protein kinase inhibitors reveal the role of cell injury-controlled MAP kinase pathways, and singularly of the p38 pathway, in the induction of B1R.
Liver-Derived CTL in Hepatitis C Virus Infection: Breadth and Specificity of Responses in a Cohort of Persons with Chronic Infection
Authors: Wong David K. H. , Dudley Darryll D. , Afdhal Nezam H. , Dienstag Jules , Rice Charles M. , Wang Liping , Houghton Michael , Walker Bruce D. , Koziel Margaret J.
Published: December 2022

Hepatitis C virus (HCV)-specific CTL have been found within the inflammatory infiltrate of the liver of chronically infected individuals, but the breadth and specificity of the CTL response in relation to viral load are less well characterized. In this study, we analyzed the intrahepatic CTL response in liver biopsy specimens from 44 chronically infected subjects. Liver-infiltrating lymphocytes were expanded polyclonally in bulk cultures, and multiple clones were derived by limiting dilution. HCV-specific CTL responses directed at genotype 1a structural proteins were assessed in all subjects, and 22 subjects were tested more comprehensively using vectors expressing all structural and nonstructural HCV Ags. CTL responses were further characterized to determine the HLA restriction and optimal epitopes recognized. In those persons screened for recognition of all HCV Ags, HLA class I-restricted CTL were detected in 45%. Nineteen different CTL epitopes were identified, which were distributed throughout the genome; only one epitope was targeted by more than one person. In those persons with CTL responses, the breadth of response ranged from one to five epitopes. There was no correlation between the presence of a detectable CTL response and viral load. These results indicate considerable heterogeneity in detectable HCV-specific CTL responses in chronically infected persons. The mechanisms by which HCV persists during chronic infection remain to be clarified.
Intracellular and Surface Expression of the HIV-1 Coreceptor CXCR4/Fusin on Various Leukocyte Subsets: Rapid Internalization and Recycling Upon Activation
Authors: Förster Reinhold , Kremmer Elisabeth , Schubel Andreas , Breitfeld Dagmar , Kleinschmidt Andrea , Nerl Christoph , Bernhardt Günter , Lipp Martin
Published: December 2022

We describe the expression and regulation of the HIV-1 coreceptor CXCR4/fusin. Using anti-CXCR4 mAb, we demonstrate that this chemokine receptor is highly expressed on neutrophils, monocytes, B cells, and naive T cells among peripheral blood cells. In secondary lymphoid organs CXCR4 was found to be expressed on B cells. However, individual variations with regard to surface expression could be observed on T cells. Expression of the receptor is not confined to the cell surface, as large amounts of intracellular stores can be found on various leukocytes. Upon activation with phorbol esters the amount of cell surface-expressed CXCR4 on lymphocytes increases twofold within 30 s before it is completely down-regulated within the next 2 min. Incubation of leukocytes with stroma derived factor-1α, the natural ligand for CXCR4, induces down-regulation of up to 60% of surface-expressed receptors in a pertussis toxin-insensitive manner. Interestingly, receptor cross-linking caused by incubation of cells with anti-CXCR4 mAb triggers receptor trafficking, in that the receptor is rapidly internalized and recycled to the cell surface. Therefore, receptor internalization and recycling may regulate the functional interaction of the receptor with envelope proteins during an initial step of HIV-1 infection.
Molecular Analysis of the Third Component of Canine Complement (C3) and Identification of the Mutation Responsible for Hereditary Canine C3 Deficiency
Authors: Ameratunga Rohan , Winkelstein Jerry A. , Brody Lawrence , Binns Matthew , Cork Linda C. , Colombani Paul , Valle David
Published: December 2022

Genetically determined deficiency of the third component of complement (C3) in the dog is characterized by a predisposition to recurrent bacterial infections and to type 1 membranoproliferative glomerulonephritis. The current studies were undertaken to characterize the cDNA for wild-type canine C3 and identify the molecular basis for hereditary canine C3 deficiency. Amplification, cloning, and sequence analysis indicated that canine C3 is highly conserved in comparison with human, mouse, and guinea pig C3. Southern blot analysis failed to show any gross deletions or rearrangements of DNA from C3-deficient animals. Northern blot analysis indicated that the livers of these animals contain markedly reduced quantities of a normal length C3 mRNA. The full-length 5.1-kb canine C3 cDNA was amplified in overlapping PCR fragments. Sequence analysis of these fragments has shown a deletion of a cytosine at position 2136 (codon 712), leading to a frameshift that generates a stop codon 11 amino acids downstream. The deletion has been confirmed in genomic DNA, and its inheritance has been demonstrated by allele-specific oligonucleotide hybridization.
Delineation of Selective Influences Shaping the Mutated Expressed Human Ig Heavy Chain Repertoire
Authors: Dörner Thomas , Brezinschek Hans-Peter , Foster Sandra J. , Brezinschek Ruth I. , Farner Nancy L. , Lipsky Peter E.
Published: December 2022

After Ag exposure, somatic hypermutation and subsequent selection play significant roles in shaping the peripheral B cell repertoire. However, the disparate impact of each process has not been completely delineated. To address this, the mutational patterns of a large panel of productive VHDJH rearrangements of individual human B cells were analyzed and compared with those of a previously reported panel of nonproductive VHDJH rearrangements. The productive VH rearrangements exhibited a significantly lower mutational frequency and a significantly smaller number of replacement mutations than the nonproductively rearranged genes, suggesting that structural constraints of the Ig molecule and selective influences both impacted the repertoire, militating against replacement mutations. Positive selection favored a mean of four to six replacements in complementarity-determining region 1 (CDR1) and CDR2, and less than two replacements in the framework regions (FRs). In contrast, the negative impact of replacement mutations generated an increased number of silent mutations within both the CDRs and FRs of the productive repertoire accompanied by a net increase in the ratio of replacement to silent mutations in the CDRs compared with that in the FRs. Moreover, there was a negative influence on the distribution of amino acid changes resulting from mutations of highly mutable codons, such as AGY, TAY, GTA, and GCT, preferentially leading to conservative changes in the expressed Ig repertoire. The results are consistent with the conclusion that the expressed repertoire is limited, compared with the potential generated by the mutational machinery, by the dual requirements of avoiding autoreactivity and satisfying structural constraints of an intact Ig molecule.
A Class I MHC-Restricted Recall Response to a Viral Peptide Is Highly Polyclonal Despite Stringent CDR3 Selection: Implications for Establishing Memory T Cell Repertoires in “Real-World” Conditions
Authors: Naumov Yuri N. , Hogan Kevin T. , Naumova Elena N. , Pagel Jeminah T. , Gorski Jack
Published: December 2022

In this study, we analyze the recall response to influenza A matrix peptide M1(58-66) restricted by HLA-A2 in one individual and find a strict CDR3 selection as well as a high degree of polyclonality. The TCR β-chain repertoire of memory T cells specific for this Ag system has been shown previously to be constrained by the use of the BV17 family and the I/sRSA/S amino acid motif in the CDR3 region. Our sequence analysis of BV17 TCR from a CTL line showed the repertoire to be highly polyclonal, as 95 distinct CDR3 sequences (clonotypes) were identified expressing this CDR3 motif. The clonotype frequencies showed a power law distribution with an extensive low-frequency tail. The clonotypes present in the high-frequency component of the distribution could be measured directly in the PBMC. This measurement showed that the relative frequencies of these clonotypes before stimulation were similar to their frequencies after culturing. Analysis of short-term cultures showed that the responding clonotypes have a similar ability to proliferate, which is independent of TCR β-chain CDR3 sequence or precursor frequency. These data indicate that the memory T cell repertoire is composed of a surprisingly diverse set of T cell clonotypes with a limited potential for expansion. We propose that the high-frequency component represents T cells that have existed the longest. In keeping with this hypothesis, these clonotypes were measured over a 2-year period, during which their precursor frequency did not change.
Superinduction of IL-8 in T Cells by HIV-1 Tat Protein Is Mediated Through NF-κB Factors
Authors: Ott Melanie , Lovett Jennie Lee , Mueller Laurel , Verdin Eric
Published: December 2022

Elevated levels of circulating IL-8, a potent chemotactic factor for granulocytes and T lymphocytes, are found in HIV-infected individuals. The HIV-1 transactivator protein Tat increased IL-8 secretion in T cell lines following CD3- and CD28-mediated costimulation. Full-length Tat (Tat101) enhanced IL-8 transcription through up-regulated transcription factor binding to the CD28-responsive element (CD28RE) in the IL-8 promoter. Expression of the Tat splice variant Tat72 (72 amino acids) also enhanced IL-8 production following T cell stimulation via a different, most likely post-transcriptional, mechanism. The CD28RE in the IL-8 promoter was characterized as a low-affinity NF-κB binding site recognized by the transcription factors p50 (NF-κB1), p65 (RelA) and c-rel. Transcription factor binding to “classical” NF-κB sites in the HIV-1, the human IL-2, and lymphotoxin promoters, recognized by p50 and p65 following CD3+28-mediated costimulation, was unaffected by Tat101 as was binding to the AP-1 motif in the IL-8 promoter. These experiments identify the CD28RE in the IL-8 promoter as a c-rel recognition site and a Tat101-responsive element. The effect of Tat101 on CD28REs in the IL-8 promoter and the subsequent up-regulation of IL-8 secretion is likely to contribute to the immune dysregulation observed during HIV-1 infection.
Site-Directed C3a Receptor Antibodies from Phage Display Libraries
Authors: Hawlisch Heiko , Frank Ronald , Hennecke Meike , Baensch Melanie , Sohns Bettina , Arseniev Lubomir , Bautsch Wilfried , Kola Axel , Klos Andreas , Köhl Jörg
Published: December 2022

Recent cloning of the human C3a receptor (C3aR) revealed that this receptor belongs to the large family of rhodopsin-type receptors. A unique feature of the C3aR is the large second extracellular loop comprising about 175 amino acid residues. We constructed combinatorial phage Ab libraries expressing single chain Fv Abs from BALB/c mice immunized with the affinity-purified second extracellular loop of the C3aR, fused to glutathione-S-transferase. A panel of anti-C3aR single chain Fv fragments (scFvs) was selected after four rounds of panning using the second extracellular loop of the C3aR, fused to the maltose binding protein as Ag. Sequencing of the clones obtained revealed three different groups of scFvs, the epitopes of which were mapped to two distinct regions within the loop, i.e., positions 185 to 193 and 218 to 226, representing the immunodominant domains of the loop. By flow cyotmetric analyses, the scFvs bound to RBL-2H3 cells transfected with the C3aR, but not to cells transfected with the C5aR or to nontransfected RBL-2H3 cells. In addition, the scFvs bound to the human mast cell line HMC-1. Immunofluorescence studies showed C3aR expression on polymorphonuclear granulocytes and monocytes, but not on lymphocytes. In addition, no C3aR expression was observed on human erythrocytes or platelets. Surprisingly, none of the scFvs alone or in combination inhibited C3a-induced Ca2+ mobilization from RBL-2H3 cells transfected with the C3aR. In addition, C3a did not displace binding of the scFvs to the receptor, strongly suggesting that the N-terminal part of the second extracellular loop is not involved in ligand binding.
Involvement of IL-16 in the Induction of Airway Hyper-Responsiveness and Up-Regulation of IgE in a Murine Model of Allergic Asthma
Authors: Hessel Edith M. , Cruikshank William W. , Van Ark Ingrid , De Bie Joris J. , Van Esch Betty , Hofman Gerard , Nijkamp Frans P. , Center David M. , Van Oosterhout Antoon J. M.
Published: December 2022

Experiments were designed to investigate the role of IL-16 in a mouse model of allergic asthma. OVA-sensitized mice were repeatedly exposed to OVA or saline aerosols. Bronchoalveolar lavage fluid (BALF) was collected after the last aerosol, and the presence of IL-16 was evaluated using a migration assay with human lymphocytes. Migration of lymphocytes was significantly increased in the presence of cell-free BALF from OVA-challenged mice compared with BALF from saline-challenged controls. This response was significantly inhibited after addition of antibodies to IL-16, demonstrating the presence of IL-16 in BALF of OVA-challenged animals. Immunohistochemistry was performed and revealed IL-16 immunoreactivity particularly in airway epithelial cells but also in cellular infiltrates in OVA-challenged mice. IL-16 immunoreactivity was absent in nonsensitized animals; however, some reactivity was detected in epithelial cells of sensitized but saline-challenged mice, suggesting that sensitization induced IL-16 expression in airway epithelium. Treatment of mice with antibodies to IL-16 during the challenge period significantly suppressed up-regulation of OVA-specific IgE in OVA-challenged animals. Furthermore, antibodies to IL-16 significantly inhibited the development of airway hyper-responsiveness after repeated OVA inhalations, whereas the number of eosinophils in bronchoalveolar lavage or airway tissue was not affected. In conclusion, IL-16 immunoreactivity is present in the airways after sensitization. After repeated OVA inhalation, IL-16 immunoreactivity is markedly increased and IL-16 is detectable in BALF. Furthermore, IL-16 plays an important role in airway hyper-responsiveness and up-regulation of IgE but is not important for eosinophil accumulation in a mouse model of allergic asthma.
Superdominance Among Immunodominant H-2Kb-Restricted Epitopes and Reversal by Dendritic Cell-Mediated Antigen Delivery
Authors: Sandberg Johan K. , Grufman Per , Wolpert Elisabeth Z. , Franksson Lars , Chambers Benedict J. , Kärre Klas
Published: December 2022

To examine possible interference patterns between immunodominant CTL Ags, we analyzed the response to mixtures of five well-characterized H-2Kb-restricted epitopes, each of which had earlier been described as immunodominant within its antigenic system. Clear patterns of dominance were observed between peptides in the mixture, with the CTL response focusing on the Sendai virus nucleoprotein 324–332 and vesicular stomatitis virus nucleoprotein 52–59 epitopes. The dominance of these epitopes correlated with high CTL availability. Subdominance of the OVA257–264 and the MCF1233 murine leukemia virus envelope 574–581 peptides could not be explained by inferior ability to bind and stabilize MHC class I molecules. Interestingly, immunodominance was broken if the peptide mixture was pulsed on bone marrow-derived dendritic cells, a mode of immunization allowing efficient recognition of a broader set of specificities. Our results show that immunodominance is neither an absolute feature of a given epitope nor does it apply only in relation to other epitopes within the same protein, micro-organism, or cell. Novel “superdominant” hierarchies emerge in the response against multiple “dominant” epitopes. A T cell competition model to explain the data in terms of a balance influenced by CTL frequencies and available APC capacity is discussed.
Differential Susceptibility to Activation-Induced Apoptosis Among Peripheral Th1 Subsets: Correlation with Bcl-2 Expression and Consequences for AIDS Pathogenesis
Authors: Ledru Eric , Lecoeur Hervé , Garcia Sylvie , Debord Thierry , Gougeon Marie-Lise
Published: December 2022

It has been proposed that HIV infection is associated with an imbalance in Th1 and Th2 subsets. Recent reports indicate that Th1 and Th2 effectors differ in their susceptibility to activation-induced apoptosis. To determine whether increased T cell apoptosis in HIV-infected patients contributes to alterations in cytokine synthesis, we performed single-cell analysis of type 1 and type 2 cytokine production by CD4 and CD8 T cells, simultaneously with detection of apoptosis. We demonstrate that a differential alteration in representation of Th1 subsets, rather than commitment of T cells to secrete Th2 cytokines, occurs throughout HIV infection. A significant decrease in the number of IL-2- or TNF-α-producing T cells was observed, whereas those producing IFN-γ remained preserved. Furthermore, there is a gradient of susceptibility to activation-induced apoptosis (IL-2 < IFN-γ < TNF-α) among the different Th1 subsets. This gradient was detected in both CD4 and CD8 subsets, as well as in control donors and HIV-infected patients, in whom the susceptibility to apoptosis of IL-2 and IFN-γ producers was increased compared with controls. This differential intrinsic apoptosis susceptibility of Th1 effectors was found to be tightly regulated by Bcl-2 expression. In HIV-infected persons, disappearance of IL-2-producing T cells was a good indicator of disease progression and was correlated with the progressive shrinkage of the CD4+CD45RA+ T cell compartment and a gradual increased susceptibility to activation-induced apoptosis of the IL-2-producing subset. This close relationship between the CD45RA/CD45R0 ratio, the level of type 1 cytokine production, and susceptibility to apoptosis should be considered in HIV-infected patients under antiviral or immune-based therapies.
Maturation of Qa-1b Class I Molecules Requires β2-Microglobulin But Is TAP Independent
Authors: Robinson Peter J. , Travers Paul J. , Stackpoole Arthur , Flaherty Lorraine , Djaballah Hakim
Published: December 2022

Two conformationally distinct and stable forms of Qa-1b, one strongly associated with β2-microglobulin (β2m) and the other associated with a novel molecule, gp44, were observed during immunochemical studies on the expression of Qa-1b molecules in mouse spleen cells. Both forms are efficiently processed and expressed at the cell surface. However, a large proportion of Qa-1b was found to be disulfide linked to gp44 without any detectable β2m. In TAP1-deficient mice, both forms undergo carbohydrate processing and are expressed on the cell surface, suggesting that they may traffic using a pathway not requiring a TAP association step. Consistent with this, size exclusion chromatography of newly synthesized class I molecules shows that high molecular mass complexes containing H-2Kk do not contain Qa-1b. Although Qa-1b can be stably expressed without β2m, there was no maturation of either form in cells from β2m-deficient mice where heavy chains were rapidly degraded. These results suggest that Qa-1b, like most other class I molecules, requires β2m for an initial folding step. However, β2m is not essential for subsequent processing of Qa-1b molecules.
Phenotype-Dependent Differences in Proteasome Subunit Composition and Cleavage Specificity in B Cell Lines
Authors: Frisan Teresa , Levitsky Victor , Polack Axel , Masucci Maria G.
Published: December 2022

We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from Burkitt’s lymphoma (BL) cells and in vitro EBV-transformed lymphoblastoid cell lines (LCLs) of normal B cell origin. Low expression of the IFN-γ-regulated β low molecular mass polypeptide (Lmp)2, Lmp7, and MECL-1 was demonstrated in a panel of seven BL lines that express the germinal center cell phenotype of the original tumor. Coexpression of Lmp2 and Lmp7 with the constitutively expressed subunits δ and MB1 was demonstrated in the BL lines by immunoprecipitation and two-dimensional gel fractionation of the 20S proteasomes. Coexpression of these subunits correlated with reduced levels of chymotrypsin- and trypsin-like activities detected by the cleavage of fluorogenic substrates. Down-regulation of Lmp2 and Lmp7 and decreased chymotrypsin- and trypsin-like activities were also observed in purified proteasomes from a c-myc-transfected subline of the ER/EB2–5 LCL that has adopted a BL-like phenotype. A synthetic peptide analogue of the immunodominant epitope from the EBV nuclear Ag 4 (E4416–424Y) was cleaved by proteasomes from BLs and A1, while proteasomes from LCLs were inactive. Cleavage of the E4416–424Y peptide was not affected by treatment of the BL cells with IFN-γ despite both significant up-regulation of Lmp2 and Lmp7 and reconstitution of chymotrypsin and trypsin-like activities against fluorogenic substrates to LCL-like levels. The results demonstrate that B cell lines representing different stages of B cell activation and differentiation express proteasomes with different subunit compositions and enzymatic activity. This may result in the generation of a distinct set of endogenous peptides and influence the immunogenicity of these cells.
Epitope-Specific Antibody Response to IgE by Mimotope Immunization
Authors: Rudolf Michael P. , Vogel Monique , Kricek Franz , Ruf Christine , Zürcher Adrian W. , Reuschel Roland , Auer Manfred , Miescher Sylvia , Stadler Beda M.
Published: December 2022

We have previously described a mouse monoclonal anti-human IgE antibody (BSW17) capable of recognizing receptor-bound IgE without inducing mediator release from human basophils or mast cells. Moreover, immune complexes of IgE and BSW17 are not able to bind to the IgE receptor. An initial attempt to map the precise epitope recognized by this mAb by using Fcε-derived peptides of variable length was unsuccessful. However, by screening random peptide phage display libraries we isolated circular nona- and octapeptides specifically recognized by BSW17. These constrained peptides mimic at least a part of a conformational epitope and are thus called mimotopes. These mimotopes, either phage displayed or synthetically synthesized, did not react with any other anti-human IgE antibody tested, but efficiently inhibited the binding of human IgE to BSW17 only. The use of Rhodol-Green-labeled free cyclic peptide proved that these interactions were not carrier dependent. Immunization of rabbits with phage clones displaying the specific peptides on the surface induced an anti-human IgE response specific for the epitope of BSW17. Therefore, we conclude that such mimotopes or mimotope-derived peptides might be used for vaccination to induce in vivo a beneficial anti-IgE response as a novel immunotherapy.
Several Common HLA-DR Types Share Largely Overlapping Peptide Binding Repertoires
Authors: Southwood Scott , Sidney John , Kondo Akihiro , del Guercio Marie-France , Appella Ettore , Hoffman Stephen , Kubo Ralph T. , Chesnut Robert W. , Grey Howard M. , Sette Alessandro
Published: December 2022

The peptide binding specificities of HLA-DRB1*0401, DRB1*0101, and DRB1*0701 have been analyzed by the use of large collections of synthetic peptides corresponding to naturally occurring sequences. The results demonstrated that nearly all peptides binding to these DR molecules bear a motif characterized by a large aromatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, M) and a small, noncharged residue in position 6 (S, T, C, A, P, V, I, L, M). In addition, allele-specific secondary effects and secondary anchors were defined, and these parameters were utilized to derive allele-specific motifs and algorithms. By the combined use of such algorithms, peptides capable of degenerate DRB1*0101, DRB1*0401, and DRB1*0701 binding were identified. Additional experiments utilizing a panel of quantitative assays specific for nine additional common DR molecules identified a large set of DR molecules, which includes at least the DRB1*0101, DRB1*0401, DRB1*0701, DRB5*0101, DRB1*1501, DRB1*0901, and DRB1*1302 allelic products, characterized by overlapping peptide-binding repertoires. These results have implications for understanding the molecular interactions involved in peptide-DR binding, as well as the genetic and structural basis of MHC polymorphism. These results also have potential practical implications for the development of epitope-based prophylactic and therapeutic vaccines.
Revealing the In Vivo Behavior of CD4+ T Cells Specific for an Antigen Expressed inEscherichia coli
Authors: Chen Zong-ming , Jenkins Marc K.
Published: December 2022

The clonal expansion and anatomic location of microbe-specific CD4+ Th cells was studied by tracking the fate of adoptively transferred DO11.10 TCR transgenic T cells specific for OVA peptide 323–339/I-Ad in BALB/c mice infected s.c. with Escherichia coli expressing a MalE-OVA fusion protein. After infection, the DO11.10 T cells accumulated in the T cell-rich paracortical regions of the draining lymph nodes, proliferated there for several days, and then moved into the B cell-rich follicles before they slowly disappeared from the lymph nodes. These changes occurred despite the fact that viable organisms were never found in the lymph nodes. The DO11.10 T cells also accumulated in the s.c. infection site, but about 1 day later than in the draining lymph nodes. Injection of purified MalE-OVA fusion protein alone induced a transient accumulation of DO11.10 T cells in the paracortical regions, but these T cells never entered follicles and the mice did not produce anti-OVA antibodies. The DO11.10 T cells that survived in animals injected with MalE-OVA alone were hyporesponsive to in vitro Ag restimulation and did not produce IL-2 and IFN-γ, whereas DO11.10 T cells from mice infected with MalE-OVA-expressing bacteria produced both lymphokines. These results suggest that Ag-specific T cells are first activated in secondary lymphoid organs following primary bacterial infection and then migrate to the infection site. Furthermore, productive activation of the T cells during the primary response is dependent on bacterial components other than the Ag itself.
Ribavirin Inhibits Viral-Induced Macrophage Production of TNF, IL-1, the Procoagulant fgl2 Prothrombinase and Preserves Th1 Cytokine Production But Inhibits Th2 Cytokine Response
Authors: Ning Qin , Brown Deron , Parodo Jean , Cattral Mark , Gorczynski Reginald , Cole Edward , Fung Laisum , Ding Jin Wen , Liu Ming Feng , Rotstein Ori , Phillips M. James , Levy Gary
Published: December 2022

Ribavirin, a synthetic guanosine analogue, possesses a broad spectrum of activity against DNA and RNA viruses. It has been previously shown to attenuate the course of fulminant hepatitis in mice produced by murine hepatitis virus strain 3. We therefore studied the effects of ribavirin on murine hepatitis virus strain 3 replication, macrophage production of proinflammatory mediators including TNF, IL-1, and the procoagulant activity (PCA), fgl2 prothrombinase; and Th1/Th2 cytokine production. Although ribavirin had inhibitory effects on viral replication (<1 log), even at high concentrations complete eradication of the virus was not seen. In contrast, at physiologic concentrations (up to 500 μg/ml), ribavirin markedly reduced viral-induced parameters of macrophage activation. With ribavirin treatment, the concentrations of PCA, TNF-α and IL-1β all decreased to basal concentrations: PCA from 941 ± 80 to 34 ± 11 mU/106 cells; TNF-α from 10.73 ± 2.15 to 2.74 ± 0.93 ng/ml; and IL-1β from 155.91 ± 22.62 to 5.74 ± 0.70 pg/ml. The inhibitory effects of ribavirin were at the level of gene transcription as evidenced by Northern analysis. Both in vitro and in vivo, ribavirin inhibited the production of IL-4 by Th2 cells, whereas it did not diminish the production of IFN-γ in Th1 cells. In contrast, ribavirin had no inhibitory effect on TNF-α and IL-1β production in LPS-stimulated macrophages. These results suggest that the beneficial effects of ribavirin are mediated by inhibition of induction of macrophage proinflammatory cytokines and Th2 cytokines while preserving Th1 cytokines.
Cutting Edge: Ca2+-Dependent Exocytosis in Mast Cells Is Stimulated by the Ca2+ Sensor, Synaptotagmin I
Authors: Baram Dana , Linial Michal , Mekori Yoseph A. , Sagi-Eisenberg Ronit
Published: December 2022

Mast cells secrete a variety of biologically active substances that mediate inflammatory responses. Synaptotagmin(s) (Syts) are a gene family of proteins that are implicated in the control of Ca2+-dependent exocytosis. In the present study, we investigated the possible occurrence and functional involvement of Syt in the control of mast cell exocytosis. Here, we demonstrate that both connective tissue type and mucosal-like mast cells express Syt-immunoreactive proteins, and that these proteins are localized almost exclusively to their secretory granules. Furthermore, expression of Syt I, the neuronal Ca2+ sensor, in rat basophilic leukemia cells (RBL-2H3), a tumor analogue of mucosal mast cells, resulted in prominent potentiation and acceleration of Ca2+-dependent exocytosis. Therefore, these findings implicate Syt as a Ca2+ sensor that mediates regulated secretion in mast cells to calcium ionophore.
Monoclonal Antibodies to Mitochondrial E2 Components Define Autoepitopes in Primary Biliary Cirrhosis
Authors: Migliaccio Christopher , Nishio Akiyoshi , Van de Water Judy , Ansari Aftab A. , Leung Patrick S. C. , Nakanuma Yasuni , Coppel Ross L. , Gershwin M. Eric
Published: December 2022

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of antimitochondrial Abs (AMA). The autoantigens recognized by AMA are the E2 components of the pyruvate dehydrogenase complex (PDC-E2), the branched chain 2-oxoacid dehydrogenase complex E (BCOADC-E2), and the 2-oxoglutarate dehydrogenase complex E (OGDC-E2). Previous studies using murine monoclonal and human combinatorial Abs to PDC-E2 have demonstrated an intense linear staining pattern in the apical region of biliary epithelial cells (BEC) in PBC but not control liver. We therefore examined whether mAbs to the other mitochondrial autoantigens BCOADC-E2 and OGDC-E2 demonstrated disease-specific patterns of reactivity. Using an expressed recombinant “trihybrid” protein containing the lipoyl domains of PDC-E2, OGDC-E2, and BCOADC-E2, we immunized BALB/c mice to produce 35 mAbs specific for one or more of the above mitochondrial autoantigens. Seven of these mAbs uniquely stained the apical region of BEC in PBC. Of these seven, one was reactive to PDC-E2, two recognized BCOADC-E2, three were reactive to OGDC-E2, and one recognized all three Ags. Our current data demonstrate that, similar to our previous studies regarding PDC-E2, mAbs to BCOADC-E2 and OGDC-E2, or a molecule that cross-reacts with the inner lipoyl domain of all three enzymes, also show a uniquely intense staining pattern in the apical region of BEC in patients with PBC when compared with diseased controls. The abundance of such disease-specific determinants in the target cells of PBC raises interesting possibilities regarding the role of these autoantigens in the pathogenesis of this disease.
Acceleration oflprLymphoproliferative and Autoimmune Disease by Transgenic Protein Kinase CK2α
Authors: Rifkin Ian R. , Channavajhala Padma L. , Kiefer Heather L.B. , Carmack Adrienne J. , Landesman-Bollag Esther , Beaudette Britte C. , Jersky Brian , Salant David J. , Ju Shyr-Te , Marshak-Rothstein Ann , Seldin David C.
Published: December 2022

MRL-lpr/lpr mice have a Fas receptor mutation that leads to abnormalities of apoptosis, lymphoproliferation, and a lupus-like autoimmune disease associated with the production of autoantibodies. Other than Fas pathway defects, little is known about molecular abnormalities that predispose to autoimmunity. Protein kinase CK2 (also termed casein kinase II), a serine-threonine protein kinase whose targets include many critical regulators of cellular growth, is highly expressed in a lymphoproliferative disease of cattle and in many human cancers. Overexpression of the CK2α catalytic subunit in lymphocytes of transgenic mice leads to T cell lymphoma. We hypothesized that CK2 dysregulation and Fas mutation might cooperatively augment lymphocyte proliferation and transformation. We find that in MRL-lpr/lpr mice bearing the CK2α transgene, the lymphoproliferative process is dramatically exacerbated, as these mice develop massive splenomegaly and lymphadenopathy by 12 wk of age in association with increased autoantibody production and accelerated renal disease. The lymphoid organs are filled with the unusual B220+CD4−CD8− T cells typically seen in MRL-lpr/lpr mice, not the B220−CD4+CD8+ or B220−CD4−CD8+ T cells typically seen in CK2α transgenic lymphomas. The T cells do not fulfill the criteria for transformation, as they are polyclonal and not transplantable or immortal in cell culture. Thus, although the lpr lymphoproliferative and autoimmune syndrome is potentiated by the presence of the CK2α transgene, this combination of apoptotic and proliferative abnormalities appears to be insufficient to transform lymphoid cells.
CD40 Engagement Triggers Switching to IgA1 and IgA2 in Human B Cells Through Induction of Endogenous TGF-β: Evidence for TGF-β But Not IL-10-Dependent Direct Sμ→Sα and Sequential Sμ→Sγ, Sγ→Sα DNA Recombination
Authors: Zan Hong , Cerutti Andrea , Dramitinos Patricia , Schaffer András , Casali Paolo
Published: December 2022

IgA are major effectors of antimicrobial defense in the respiratory and digestive tracts. We have analyzed the requirements for and the modalities of switching to IgA using our recently identified monoclonal model of human germinal center differentiation, CL-01 B cells. CL-01 cells bear surface IgM (sIgM) and sIgD and switch to all seven downstream isotypes in response to physiologic stimuli. In these cells, CD40 engagement by CD40 ligand induces production of endogenous TGF-β and IL-10, expression of germline Iα1-Cα1 and Iα2-Cα2 transcripts, mature VHDJH-Cα1 and VHDJH-Cα2 transcripts, and IgA secretion. These events are associated with not only direct Sμ→Sα, but also sequential Sμ→Sγ, Sγ→Sα DNA recombination, and are ablated by neutralizing anti-TGF-β but not IL-10 Ab, and indicating that TGF-β, not IL-10, is a crucial mediator of the transcriptional activation and recombination of human Cα1 and Cα2 genes. Our findings in CL-01 cells were reproduced in freshly isolated naive sIgM+ sIgD+ B lymphocytes. Thus, engagement of CD40, in the absence of other (known) stimuli, is sufficient to effectively induce switching to IgA in human B cells. This is effected by direct and sequential DNA recombination events, which are both dependent upon endogenous TGF-β secreted by the CD40L-induced B cells.