bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tolerance is overcome in beef insulin‐transgenic mice by activation of low‐affinity autoreactive T cells
Authors: Poplonski Lorraine , Vukusic Brian , Pawling Judy , Clapoff Susan , Roder John , Hozumi Nobu , Wither Joan 
Published: March 2007

AbstractTo gain insight into the factors controlling the maintenance or loss of T cell self tolerance we produced beef insulin (BI)‐transgenic BALB/c mice. Transgenic mice express BI under control of the human insulin promoter and secrete physiological amounts of beef insulin. Although these mice are tolerant to BI, as evidenced by the lack of insulin‐specific IgG antibody production following intraperitoneal immunization, tolerance is not complete. Footpad immunization results in a weak antigen‐specific T cell proliferative response, indicating the presence of self‐ reactive BI‐specific T cells in the periphery. These T cells are functional in vivo, providing support for IgG1, IgG2a, and IgG2b BI‐specific antibody production, but require higher concentrations of antigen than nontransgenic T cells (both in vivo and following recall responses in vitro) to become activated. In vitro, BI‐specific T cell proliferation in BI‐transgenic mice can be largely restored by addition of interleukin‐2, indicating that a significant component of T cell tolerance is mediated by anergy. To characterize the autoreactive T cells that become activated when tolerance is broken, BI‐specific T cell hybridomas were generated from transgenic mice and compared to a panel of hybridomas previously derived from nontransgenic BALB/c mice. The majority of BI‐transgenic hybridomas recognized the immunodominant A1–14 beef insulin peptide but with lower avidity than BALB/c hybridomas. Consistent with this, none of the dominant T cell receptor rearrangements found in the BALB/c BI‐specific T cell receptor repertoire were found in the transgenic hybridomas. These results indicate that, despite evidence for clonal inactivation of many BI‐specific T cells in BI‐transgenic mice, loss of tolerance results from activation of low‐affinity antigen‐specific T cells that appear to have escaped this process.
Negative feedback mechanism suppresses interleukin‐12 production by antigen‐presenting cells interacting with T helper 2 cells
Authors: Hino Ayako , Nariuchi Hideo 
Published: March 2007

AbstractInterleukin‐12 (IL‐12) has been shown to be produced by monocytes by the ligation of CD40. In the present experiments, IL‐12 is shown to be produced by murine spleen antigen‐presenting cells (APC) by interaction with T helper 1 (Th1) 1 clones through CD40‐CD40 ligand (CD40L) interaction, but not with Th2 clones. The IL‐12 production induced by the Th1 clone interaction was inhibited by the addition of exogenous IL‐10. Th2 clones were shown to produce a sufficient amount of IL‐10 to inhibit the IL‐12 production induced by Th1 clones. In the presence of anti‐IL‐10 monoclonal antibodies splenic APC interacting with Th2 clones produced IL‐12. These results indicate that IL‐10 produced by Th2 cells stimulated with antigen suppress IL‐12 production of APC interacting with Th2 cells. IL‐12 is composed of two subunits, p35 and p40. In our experiments, p40 mRNA accumulation was shown to be affected by IL‐10 more severely than the accumulation of p35 mRNA, indicating that IL‐10 regulates IL‐12 production by APC mainly by affecting p40 mRNA accumulation.
Expression of bcl‐x during mouse B cell differentiation and following activation by various stimuli
Authors: Choi Michael S. K. , Holman Mary , Atkins Chris J. , Klaus Gerry G. B. 
Published: March 2007

AbstractWe have studied the expression of the novel anti‐apoptotic protein bcl‐x during mouse B cell differentiation and activation. We find that bcl‐x is expressed throughout all stages of B cell differentiation in the bone marrow, and is only down‐regulated in mature (sIgD+) B cells. Immature peripheral B cells express low levels of bcl‐x even in adult animals, whereas mature resting B cells do not. Mature B cells re‐express the protein following activation, achieving maximal levels after 36–48 h. The highest levels of bcl‐x are observed with potent comitogenic stimuli (such as anti‐CD40 + anti‐Ig): B cells first express bcl‐x in the G1 phase of the cell cycle and contain maximal levels in S phase. In addition, B cells from CBA/N mice, which do not proliferate when stimulated with anti‐Ig, anti‐CD40, or both, exhibited only low levels of the protein following culture with these stimuli. To investigate the functional significance of bcl‐x in activated B cells, we tested their sensitivity to apoptosis induced by the Ca2+ ATPase inhibitor thapsigargin: B cell blasts activated with anti‐CD40 and anti‐Ig were resistant to this agent. The available data therefore suggest that bcl‐x fulfils two roles in B cells: it promotes survival of immature B cells (which lack bcl‐2) and secondly, it apparently plays an additional role in protecting activated mature B cells (perhaps those in germinal centers) from apoptotic stimuli.
Expression of high‐ and low‐affinity receptors for C3a on the human mast cell line, HMC‐1
Authors: Legler Daniel F. , Loetscher Marcel , Jones Simon A. , Dahinden Clemens A. , Arock Michel , Moser Bernhard 
Published: March 2007

AbstractThe proteolytic cleavage product of complement component 3, (C3a), like C4a and C5a, is a potent anaphylatoxin and induces the production of inflammatory mediators in phagocytes. Notably, mast cells respond to C3a with the release of vasoactive substances, including histamine. We have examined the function and receptor binding of C3a in a human leukemic mast cell line, HMC‐1. Similar to chemoattractant agonists in leukocytes, C3a induced rapid cytosolic free calcium concentration increases in HMC‐1 cells. EGTA did not diminish this response, indicating that mobilizable Ca2+ was from intracellular stores. Receptors for C3a in HMC‐1 cells couple in part to Bordetella pertussis toxin‐sensitive G‐proteins and, therefore, appear to belong to the family of serpentine receptors that require G‐proteins for signal transduction. HMC‐1 cells express two types of C3a receptors, C3aR1 and C3aR2, that were shown to bind 125I‐C3a with high‐(Kd1 = 2.1–4.8 nM) or low‐affinity (Kd2 = 30–150 nM), and both receptors are expressed at high level: 3 × 105–6 × 105 C3aR1/cell and 5 × 105–2.3 × 106 C3aR2/cell. Results from cross‐linking experiments with 125I‐C3a fully agree with the presence of two different classes of C3a receptors in HMC‐1 cells. Two membrane proteins with apparent molecular masses of 54–61 kDa (p57) and 86–107 kDa (p97) could be covalently modified with 125I‐C3a, and this cross‐linking was inhibited with an excess of unlabeled C3a. Many of the known agonists for leukocytes including 13 chemokines (IL‐8, NAP‐2, GROα, ENA‐78, IP10, PF4, MCP‐1, 2 and 3, RANTES, MIP‐1α, MIP‐1β and 1309), three neuropeptides (neuropeptide Y, somatostatin and calcitonin), as well as C5a, did not activate HMC‐1 cells, indicating that C3a is one of a few protein ligands for which this cell line expresses specific receptors. The apparent selectivity for C3a and the abundant expression of C3a receptors make the HMC‐1 cell line an excellent choice for the cloning of the receptor genes.
Murine epidermal Langerhans cells do not express inducible nitric oxide synthase
Authors: Blank Christine , Bogdan Christian , Bauer Carmen , Erb Klaus , Moll Heidrun 
Published: March 2007

AbstractIn Leishmania‐infected macrophages (MΦ), the formation of reactive nitrogen intermediates by the inducible isoform of nitric oxide synthase (iNOS) is critical for the killing of the intracellular parasites. We have recently shown that, in addition to MΦ, epidermal Langerhans cells (LC) can phagocytose Leishmania major, but they do not allow parasite replication. Therefore, we analyzed whether LC and MΦ display the same leishmanicidal effector mechanism. Unlike MΦ, stimulation of unselected epidermal cells with interferon‐γ/lipopoly‐saccharide did not lead to the release of nitric oxide (NO), and inhibition of NO production had no effect on the rate of infection of LC. iNOS mRNA was clearly detectable in MΦ as well as unselected epidermal cells (the majority of which consists of keratinocytes) after stimulation with different cytokines. In contrast, pure LC obtained by single‐cell picking from cytokine‐activated or L. major‐infected epidermal cells did not express iNOS mRNA. Addition of the NO donor S‐nitroso‐N‐acetylpenicillamine to already‐infected LC did not alter their rate of infection, indicating that LC do not utilize exogenous NO for the control of intracellular Leishmania. These results suggest that in the L. major‐infected skin, activated MΦ and keratinocytes, but not LC have the ability to express iNOS activity. Therefore, an as yet unidentified, NO‐independent mechanism appears to be responsible for the control of parasite replication in LC.
Evidence that induction of tolerance in vivo involves active signaling via a B7 ligand‐dependent mechanism: CTLA4‐Ig protects Vβ8+ T cells from tolerance induction by the superantigen staphylococcal enterotoxin B
Authors: Lane Peter , Haller Corinne , McConnell Fiona 
Published: March 2007

AbstractCo‐stimulation through CD28 is thought to be necessary for the activation of unprimed CD4+ T cells, which are otherwise rendered tolerant. However, we previously found that CD4+ T cell priming was normal or augmented in mice which overexpressed a soluble form of CTLA4 where co‐stimulation through CD28 was abrogated. To investigate this CD4+ T cell response, we exploited the capacity of the superantigen staphylococcal enterotoxin B to stimulate T lymphocytes bearing Vβ8+, which represent ϰ30% of all CD4+ T cells. In littermate controls of CTLA4‐Ig transgenic mice, immunization with staphylococcal enterotoxin B leads to expansion, followed by deletion of Vβ8+ T cells, and the remaining cells are tolerant when stimulated in vitro. Comparable expansion and deletion of Vβ8+ T cells occurs in CTLA4‐Ig transgenic mice. However, in contrast to normal mice, the remaining Vβ8+ T cells from CTLA4‐Ig transgenic mice are not anergic and remain responsive to superantigen in vitro.
Tracing interactions of thymocytes with individual stromal cell partners
Authors: Merkenschlager Matthias 
Published: March 2007

AbstractCellular interactions in T cell development can be analyzed using thymus chimeras prepared in vitro, in which stromal cells and T cell precursors are manipulated separately. In an earlier study, we showed that for optimal T cell maturation, most ‐ if not all ‐ stromal cells must display appropriate (selecting) major histocompatibility complex (MHC) molecules: the substituion of selecting by nonselecting stromal cells leads to a proportional decrease in mature T cell production. These data imply that the availability of selecting stromal micro‐environments is rate limiting for positive selection, and that in positive selection, each thymocyte engages only one (rather than multiple) stromal cell partners. To test this hypothesis, we developed a tracing sytem for thymocyte/stromal cell interactions, based on the acquisition by thymocytes of stroma‐derived MHC class II determinants. When MHC class II‐deficient precursors are placed in H‐2b × k F1 environments (where all stromal cells co‐express H‐2b and H‐2k), individual thymocytes acquire class II determinants of both haplotypes. In striking contrast, when placed in mosaic stromal environments (where stromal cells express either H‐2b or H‐2k evenly interspersed), individual thymocytes preferentially acquire MHC class II determinants of one or the other haplotypes, but rarely both. This provides strong evidence that thymocytes have intimate interactions with individual stromal cells: having engaged one stromal cell niche, thymocytes do not (or only rarely) have promiscuous liaisons with others.
IL‐12 inhibits endotoxin‐induced inflammation in the eye
Authors: Whitcup Scott M. , Rizzo Luiz V. , Lai James C. , Hayashi Seigi , Gazzinelli Ricardo , Chan Chi‐Chao 
Published: March 2007

AbstractInterleukin‐12 (IL‐12) is a heterodimeric cytokine that induces interferon (IFN)‐γ production and an increased generation of Th1 cells. Both IL‐12 and IL‐12 antagonists are being studied for the treatment of allergic reactions, autoimmune disease and malignancy. The goal of the present experiments was to examine the importance of IL‐12 in endotoxin‐induced ocular inflammation. The number of inflammatory cells infiltrating eyes with endotoxin‐induced uveitis (EIU) was significantly increased in animals treated with intraperitoneal anti‐IL‐12 antibody when compared to control animals, but there was no difference in infiltrating inflammatory cells in the eyes of animals treated with IL‐12 when compared to controls. In contrast, intraocular injection of IL‐12 significantly inhibited the development of endotoxin‐induced intraocular inflammation. The infiltrating inflammatory cells were reduced in the eyes of animals receiving intraocular IL‐12 when compared to controls. Cytokine analysis of the aqueous humor obtained from eyes with EIU showed increased levels of IFN‐γ and decreased levels of IL‐6 in eyes receiving intraocular IL‐12. These data show that IL‐12 has an inhibitory effect on endotoxin‐induced inflammation in the eye and suggest that IL‐12 can have an immunoregulatory function in some forms of inflammatory disease.
Use of monoclonal antibodies to study interleukin ‐1β‐converting enzyme expression: only precursor forms are detected in interleukin‐1β‐secreting cells
Authors: Miossec Christine , Decoen Marie‐Claude , Durand Laurence , Fassy Florence , Diu‐Hercend Anita 
Published: March 2007

AbstractWe have generated a series of monoclonal antibodies (mAb) using recombinant interleukin (IL)‐1β‐converting enzyme (ICE) p20 and p10 subunits as immunogens. The mAb have been selected for further study based on their reactivity with ICE in transfected COS cells and their lack of cross‐reactivity with TX, the closest ICE homolog known to date. Two anti‐p20 and one anti‐p10 mAb have been used to study ICE expression by Western blotting and immunodetection. In ICE‐transfected COS cells, the mAb recognize the p45 ICE precursor and the maturation products (p20 or p10 subunits) for which they are specific. In monocytes and cell lines expressing ICE, only precursor forms are detected and intracellular immunostaining followed by confocal microscopy shows that they are located in the cytoplasm. Quantification experiments show that THP1 cells express approximately 67000 molecules of ICE precursor per cell, with an estimated precursor to mature ratio of at least 100. In these cells as well as in monocytes, lipopolysaccharide stimulation did not change the pattern of ICE expression, although efficient secretion of mature IL‐1β was measured. However, upon cell disruption, precursor maturation was observed. Our results, therefore, show that ICE is present in cells as a large pool of intracytoplasmic precursor, and that very limited amounts of mature ICE protein are present, but nevertheless sufficient to allow efficient IL‐1β cleavage. Altogether, these observations suggest that post‐translational maturation of the precursor protein could represent a specific step in the regulation of ICE enzymatic activity.
Analysis of hypermutation in immunoglobulin heavy chain passenger transgenes
Authors: Johnston J. Martin , Ihyer Sudhakar R. , Smith Robin S. , Tai Kuei‐Fang , Farmer Tara , Korsmeyer Stanley J. , Nadon Nancy L. , Carroll William L. 
Published: March 2007

AbstractSomatic hypermutation of immunoglobulin (Ig) genes plays a critical role in the maturation of the human antibody response. The molecular basis of this important process is, however, unknown. To identify cis‐acting sequences that initiate and target hypermutation, we have made three minitransgenes containing different portions of an Ig heavy chain (IgH) locus. Each transgene is a passenger, bearing a nonsense mutation preventing its translation; thus, transgene mutations reflect the endogenous mutational process and are not subject to affinity selection. To study transgenes after their circulation through the compartment associated with hypermutation in vivo, we rescued B cells as hybridomas after hyperimmunizing mice with the hapten 4‐hydroxy‐3‐nitrophenyl acetyl (NP). Hybridoma transgene and endogenous variable regions were amplified by polymerase chain reaction, subcloned, and sequenced. Endogenous anti‐NP VDJ regions show the expected, at times extensive degree of base substitution. In mice bearing the smallest construct, which includes 2.4 kb of 5′ IgH sequences, a rearranged VDJ region, the 5′ matrix attachment region, and the intron enhancer, one of four evaluable hybridomas demonstrates two base substitutions in the V segment of one transgene copy. The two larger constructs include additional 3′ IgH sequences (an α constant region and the 3′ enhancer) and either the original VDJ segment or a substituted T cell receptor β segment. Ten hybridomas derived from mice bearing these larger constructs demonstrate no evidence of targeted mutation, despite demonstrable transgene transcription in all hybridomas. In our system, mutation of a rearranged VDJ segment and surrounding promoter/enhancer regions is not increased by the juxtaposition of a constant region segment and the IgH 3′ enhancer.
The protein interactions of the immunoglobulin receptor family tyrosine‐based activation motifs present in the T cell receptor ζ subunits and the CD3 γ,δ and ϵ chains
Authors: Osman Narin , Turner Helen , Lucas Susan , Reif Karin , Cantrell Doreen A. 
Published: March 2007

AbstractImmunoglobulin family tyrosine‐based activation motifs (ITAM), which define the conserved signaling sequence EX2YX2L/IX7YX2L/I, couple the T cell antigen receptor (TCR) to cellular proteins including protein tyrosine kinases (PTK) and adapter molecules. The TCR is a multichain complex with four invariant chains CD3γ, δ and ϵ that each contain a single ITAM and the TCR ζ chain that contains three ITAM. The present study explores the protein interactions of the doubly phosphorylated CD3 γ, δ, ϵ ITAM to determine whether they have common or unique biochemical properties. The data show that the doubly phosphorylated ITAM all bind the PTK ZAP‐70, but the ITAM also variably bind the PTK p59fyn and the adapters Shc, Grb‐2 and the p85 regulatory subunit of phosphoinositol 3′ kinase. The CD3 and ζ ITAM display a hierarchy of ZAP‐70 binding: ζ1 = γ = δ > ζ3 > ζ2 = ϵ. Shc, Grb‐2 and p85 could bind the ζ ITAM and the CD3 γ and δ ITAM, but not the CD3 ϵ ITAM. There were also subtle differences in the hierarchy of reactivity of these adapters for the CD3 γ,δ and ζ ITAM that show that the ζ, CD3 γ, δ and ϵ ITAM have different binding properties. The present study thus shows that the different ITAM of the TCR/CD3 complex can interact with different cytosolic effectors, indicating that differential ITAM phosphorylation during T cell activation could be a mechanism to generate signaling diversity by the TCR complex.
Induction of T cell unresponsiveness by anti‐CD3 antibodies occurs independently of co‐stimulatory functions
Authors: Andris Fabienne , Van Mechelen Marcelle , De Mattia Fabrizio , Baus Erika , Urbain Jacques , Leo Oberdan 
Published: March 2007

AbstractAntibodies to the T cell receptor (TcR)‐associated CD3 molecules represent potent immunosuppressive agents in vivo in both human and animals models, in spite of their well‐characterized mitogenic properties. We demonstrate in this report that antibodies to the B7.2 molecule inhibit IL‐2 production in vivo caused by anti‐CD3 administration, suggesting that anti‐CD3 monoclonal antibodies (mAb) stimulate naive T cells in vivo in a co‐stimulation‐dependent fashion. To characterize better the mechanisms by which antibodies to CD3 induce antigen unresponsiveness in naive T cells, we developed a model of activation‐induced T cell unresponsiveness in vitro. Our data indicate that following interaction with mitogenic anti‐CD3 mAb in vitro, naive purified CD4+ T cells become refractory to a further stimulus. This unresponsive state develops independently of co‐stimulatory functions, as neither B7‐expressing antigen‐presenting cells nor anti‐CD28 mAb are able to prevent anergy induction in this model. We therefore conclude that induction of unresponsiveness in naive T cells by anti‐CD3 mAb is not a consequence of co‐stimulus‐deficient stimulation, but may develop following a productive response both in vivo and in vitro. Unresponsive T cells display a defective calcium mobilization upon TcR triggering, suggesting that anergy is maintained in these cells through receptor desensitization. The potential role of co‐stimulation‐independent TcR desensitization in the down‐regulation of immune responses in vivo is briefly discussed.
Demonstration of local clonality of mucosal T cells in human colon using DNA obtained by microdissection of immunohistochemically stained tissue sections
Authors: Dogan Ahmet , Dunn‐Walters Deborah K. , MacDonald Thomas T. , Spencer Jo 
Published: March 2007

AbstractIntraepithelial lymphocytes have been shown to be oligoclonal and to be disseminated widely along the human intestine. However, studies using monoclonal antibodies have suggested that superimposed on the widespread clones, there is local variability in the mucosal T cell population. We have investigated the possibility that local dominant clones of T cells are present in the colonic mucosa by polymerase chain reaction (PCR) amplification of T cell receptor β chain junctional regions using DNA extracted from microdissected fragments of tissue sections. Colon from two right hemicolectomy specimens was sampled at 7‐cm intervals. Adjacent areas of mucosa were microdissected from sections from each colon sample. When the PCR products were separated according to size on polyacrylamide gels, bands of identical size were often observed when DNA extracted from adjacent fragments of mucosa had been used. Different bands were present when the different samples of colon had been studied. Sequencing of the PCR products confirmed that clonally related T cells were present in adjacent areas of mucosa, whereas different clones dominated at distant sites. DNA extracted from cells microdissected from the T cell zone of Peyer's patch was treated identically. The sequences obtained from the Peyer's patch, as expected, were diverse. However, one of the sequences identified was identical to that of one of the clones in the colon, implying that this clone was either trafficking through the Peyer's patch or possibly originated from the Peyer's patch. In this study, we also identified the Peyer's patches as a site of proliferation of CD4+ T cells. No T cell division was observed in the lamina propria. The molecular and immunohistochemical observations together support the hypothesis that the Peyer's patches are a source of mucosal T cells.