bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoantibody specific for a thyroglobulin epitope inducing experimental autoimmune thyroiditis or its anti‐idiotype correlates with the disease
Authors: Tang Haïwen , Bedin Catherine , Texier Béatrice , Charreire Jeannine
Published: March 2007

In order to elucidate the role of anti‐thyroglobulin (Tg) autoantibodies (A‐Ab) in experimental autoimmune thyroiditis (EAT), a kinetic study was conducted in EAT‐susceptible (CBA/J) and non‐susceptible (C57BL/6) strains of mice. From day 0 to 70 post‐Tg immunization, titers of A‐Ab to Tg and to the linear 5‐10‐kDa Tg tryptic fragment inducing EAT as well as anti‐idiotypic A‐Ab representing the internal image of the thyroidogeneic antigen were measured.

EAT onset, development and recovery correlate with the presence of two subsets of A‐Ab only in susceptible strains of mice. First, with the presence of anti‐Tg A‐Ab to one determinant borne by the linear 5‐10‐kDa Tg tryptic fragment, and second with the presence of anti‐idiotypic A‐Ab specific for the monoclonal anti‐Tg A‐Ab (3B8G9) paratope which binds to Tg determinant inducing EAT.
Interleukin 1‐induced lymphocyte binding to endothelial cells. Role of cAMP as a second messenger
Authors: Renkonen Risto , Mattila Pirkko , Häyry Pekka , Ustinov Jarkko
Published: March 2007

Interleukin 1 (IL 1) is a potent protein mediator of inflammation. Among other things it increases the number of lymphocytes adhering to endothelial cell monolayers. We analyzed the signal transduction during IL 1‐induced lymphocyte binding. Dibutyryl cyclic AMP, which is a cAMP analog able to penetrate into the cytosol, increased lymphocyte binding to the same extent as IL 1. Direct activation of adenylate cyclase by forskolin enhanced also lymphocyte binding. IL 1 increased the level of cytosolic cAMP in a time‐ and dose‐dependent manner measured with radioimmunoassay. 2′, 5′‐Dideoxyadenosine, which is an inhibitor of adenylate cyclase, decreased both the IL 1‐induced lymphocyte binding to endothelial cells and elevation in cytosolic cAMP levels. Lymphocyte binding increased with cytosolic cAMP levels in accordance with elevation of IL 1 concentration. These results suggest that cAMP is essential in signal transduction during IL 1‐induced lymphocyte binding to cultured endothelial cell monol‐layers.
A bidirectional regulatory network involving IL 2 and IL 4 in the alternative CD2 pathway of T cell activation
Authors: Lorré Katrien , Van Damme Jo , Ceuppens Jan L.
Published: March 2007

In this study we examined the effect of interleukin 4 (IL 4) on T cell activation and proliferation via the alternative CD2 pathway. To this end highly purified human resting T cells were cultured with a stimulating pair of anti‐CD2 monoclonal antibodies in the absence of accessory signals from monocytes. Addition of either recombinant (r)IL 2 or rIL 4 resulted in proliferation of the anti‐CD2‐stimulated T cells. The growth‐promoting effect of rIL 4 on preactivated T cells was shown to be partly a direct effect. rIL 4 also induced IL 2 production and, as a consequence, the effect of rIL 4 on T cell growth was enhanced by endogenously produced IL 2. Moreover, rIL 4 acted in synergy with exogenously added rIL 2 in promoting growth of anti‐CD2‐stimulated T cells. The synergistic effect of IL 2 and IL 4 could be explained by IL 2‐induced up‐regulation of IL 4 responsiveness. In contrast, preincubation with rIL 4 did not enhance IL2 responsiveness and rIL2 but not rIL4 up‐regulated IL 2R expression on anti‐CD2‐stimulated T cells. Finally we could demonstrate that monocyte‐produced cytokines (IL 1 and IL 6) enhance the proliferative response to rIL 4 of anti‐CD2‐stimulated T cells. It can be concluded that IL 4 can act as a paracrine growth factor for T cells activated in the alternative CD2 pathway, and that it acts synergistically with IL 2, IL 1 and IL 6. Moreover, IL 4 is a helper signal for IL 2 production. Thus, IL 2 and IL 4 are involved in a bidirectional regulatory network, with IL 4 as an inducer of IL 2 production, and IL 2 as an enhancer of IL 4 responsiveness.
CD4+ T cells in murine acquired immunodeficiency syndrome: Evidence for an intrinsic defect in the proliferative response to soluble antigen
Authors: Cerny Andreas , Hügin Ambros W. , Holmes Kevin L. , Morse Herbert C.
Published: March 2007

C57BL/6 mice inoculated with LP‐BM5 murine leukemia viruses develop an immunodeficiency syndrome, termed murine acquired immunodeficiency syndrome (MAIDS), characterized by a variety of functional abnormalities of T and B cells. In the present study, we have analyzed the ability of lymph node cells from infected mice to generate secondary in vitro proliferative responses to soluble antigens. Our data demonstrate that the ability of lymph node cells to proliferate in response to soluble antigen or T cell mitogens declines progressively during the course of MAIDS. Impaired proliferative responses were shown to be characteristic of purified CD4+ but not CD8+ cells from infected mice when stimulated in the presence of normal accessory cells. In addition, the impaired responses of unseparated lymph node cells from infected mice could be reconstituted by the addition of purified CD4+ T cells from nodes of primed normal animals. These results strongly suggest that an intrinsic CD4+ T cell defect developing during the course of MAIDS contributes significantly to impaired responses to mitogens and to impaired secondary in vitro proliferative responses to soluble antigen.
Simultaneous production of interleukin 2, interleukin 4 and interferon‐γ by activated human blood lymphocytes
Authors: Andersson Ulf , Andersson Jan , Lindfors Anders , Wagner Kathrin , Möller Goran , Heusser Christoph H.
Published: March 2007

The production of interleukin 2 (IL 2), IL 4 and interferon‐γ (IFN‐γ) by in vitro activated unselected human blood mononuclear cells was studied at a single‐cell level. Individual lymphokine‐synthesizing cells were identified by intracellular immunofluorescent staining using cytokine‐specific monoclonal or polyclonal antibodies. Cultures from adult blood donors revealed a biphasic kinetic production pattern for IL 2 and IFN‐γ with peaks occurring 4–6 and 24–30 h after initiation of the cultures. Approximately 20%–40% of the lymphocytes produced IL 2 and IFN‐γ. In contrast, only 1%–3% of the lymphocytes synthesized IL 4 with maximal frequency after 6 h of culture. CD4+ as well as CD8+ T cells contributed to the synthesis of all three lymphokines studied. CD4+CD45R− T cells were the major producers of IL 2 and IL 4, while CD8+CD45R− T cells were the most common phenotype of IFN‐γ‐synthesizing cells. By performing two‐color immunofluorescence studies we observed that among IL 4‐producing cells every second one made simultaneously IL 2 and every fourth one made IFN‐γ. Mononuclear cells from umbilical cord blood could be stimulated to make IL 2 to the same extent as cells from adult blood donors. No IL 4 production and a strikingly reduced frequency of IFN‐γ producers were noted in cell cultures from neonates. IL 2, IL 4 and IFN‐γ accumulated in the Golgi system, which resulted in a characteristic morphology of the staining, eliminating problems with evaluation of background signals.
C4 from C4‐high and C4‐low mouse strains have identical sequences in the region corresponding to the isotype‐specific segment of human C4
Authors: Ogata Ronald T. , Zepf Nancy E.
Published: March 2007

The human complement component C4 isotypes, C4A and C4B, show a substantial and biologically important difference in chemical reactivity. Murine C4 from different mouse strains has recently been reported to have a comparable difference in reactivity. In human C4, the difference in reactivity has been attributed to the effect(s) of one or more of only four amino acid residues, within a six‐residue‐long segment of the α subunit, which distinguish the two isotypes. In the present study, we sought to assess the role of the corresponding four amino acids in mouse C4 in the strain‐specific modulation of C4 reactivity. In order to compare the sequences of the corresponding region in murine C4 among different mouse strains, we used the polymerase chain reaction method to amplify an approximately 229‐bp segment of the murine gene that includes the codons for these four amino acid residues. Because the difference in chemical reactivity of murine C4 has been reported to be between C4 from strains which express different levels of C4, we examined sequences from mouse strains C57BL/6, DBA/2, C3H/He, B10.BR and CBA/J; these represent two C4‐high strains and three C4‐low strains. The amplified segments were cloned into the pUC19 vector and 20 independent clones from each mouse strain were sequenced. Across the entire amplified segment, our results revealed expected isotypic differences between C4 and its nonfunctional isotype in the mouse, Slp, as well as allelic differences among the C4 and Slp genes. However, all of these differences were quite distant from the amino acid residues corresponding to the human isotype‐specific residues and those corresponding residues were identical in all five mouse strains. This result indicates that any strain‐specific difference in chemical reactivity of murine C4 must be due to a mechanism distinct from that operating in human C4.
Anti‐CD4 modifies the kinetics but not the magnitude of anti‐CD3‐induced interleukin 2 production in peripheral T cells
Authors: Jönsson Jan‐Ingvar , Eichmann Klaus
Published: March 2007

We have studied the effect of T cell receptor (TcR) cross‐linking and TcR‐CD4 cross‐linking on the induction of interleukin (IL) 2 and IL 2 receptor mRNA expression in peripheral T cells. We confirm our previous results using bioassays, suggesting that CD4 is involved in T cell activation by up‐regulating the expression of IL 2 receptors. Furthermore, we show that this also concerns other functional T cell genes such as IL 4 and interferon‐γ. In contrast, similar levels of IL 2 mRNA are found in T cells activated with anti‐CD3 with and without anti‐CD4 antibody, suggesting that the induction of the IL 2 gene is regulated by TcR cross‐linking alone and not influences by CD4. However, anti‐CD4 appears to accelerate the induction of IL 2 mRNA in the T cell populations, presumably by a more rapid adhesion of the cells to the antibody‐coated surface.
Macrophages and the activity of high endothelial venules. The effect of interferon‐γ
Authors: Mebius Reina E. , Hendriks Hans R. , Brevé John , Kraal Georg
Published: March 2007

Interruption of the afferent lymphatic vessels of rat popliteal lymph nodes led to the disappearance of the monoclonal antibody ED3‐reactive subcapsular sinus macrophages within 3 weeks, but had no effect on the ED1+ macrophages in the paracortical area. This disconnection of the afferent lymph flow to the popliteal lymph node also reduced the capacity of high endothelial venules (HEV) to bind lymphocytes and led to a flattening of the HEV. Activating factors or cells in the incoming lymph might be responsible for the maintenance and function of several different cell populations and we therefore wished to determine if the effects of interruption could be restored by injection of recombinant rat interferon‐γ (rIFN‐γ). Injection of rIFN‐γ directly into operated lymph nodes could mediate an apparent increase of ED1+ cells within 24 h but rIFN‐γ could not restore the macrophage subpopulation in the subcapsular sinus, as recognized by monoclonal antibody ED3. Restoration of the decreased binding capacity of HEV could not be observed with the doses and time points tested, suggesting that HEV are a distinct type of endothelium.
Disulfide bridge formation between C1q and IgG in vitro
Authors: Martin Harry , Kaul Marcus , Loos Michael
Published: March 2007

The globular heads of C1q are known to possess free‐SH groups. Here we show that these groups, which are concealed in the native molecule, are exposed by interaction of C1q with dialysis membrane. During iodination, I+ and I2 oxidize these sulfhydryls to produce disulfide‐linked C1q aggregates. Approximately 15% of C1q bound to immunoglobulin aggregates is resistant to high conductivity elution and reducing agent is required to release it. These data show that dialysis, adsorption to Ig and iodination of C1q result in structural and functional changes in the molecule, and suggest a mechanism by which these changes occur. Disulfide bridging between C1q and IgG in vitro suggests that this may be a normal physiological function of C1q for which the free cysteines of human, mouse and guinea pig C1q have been conserved.
Effect of activation of protein kinase C on CD45 isoform expression and CD45 protein tyrosine phosphatase activity in T cells
Authors: Yamada Akira , Streuli Michel , Saito Haruo , Rothstein David M. , Schlossman Stuart F. , Morimoto Chikao
Published: March 2007

The T200/leukocyte common antigen (CD45) is a family of at least five large‐molecular weight glycoproteins, which are differentially expressed on T cell subsets. The CD45 antigen consists of a variable heavily glycosylated exterior domain, a single membrane‐spanning region, and a large cytoplasmic domain that has protein tyrosine phosphatase (PTPase) activity. In this study, we examined the effects of activation of protein kinase C (PKC) on the phosphorylation and expression of CD45 isoforms and PTPase activity in human T cells. After activation of PKC by phorbol 12‐myristate 13‐acetate (PMA), CD45RA expression rapidly increased within the first 24 h, whereas CD45RO expression did not change within this time. However by 48 h, expression of CD45RO also began to increase. Metabolic labeling showed that the rapid increment in CD45RA expression observed after PMA stimulation is primarily due to increased de novo synthesis of the 205‐kDa and not the 220‐kDa molecule. PMA treatment resulted in the phosphorylation of each CD45 isoform to a degree corresponding to its relative surface expression. Significantly, we found that the phosphorylation of CD45 by PKC activation down‐regulated CD45 PTPase activity.
Divergency in the specificity of the induction and maintenance of neonatal suppression
Authors: Mota‐Santos Tomas , Masmoudi Hatem , Voegtlé Danielle , Freitas Antònio , Coutinho Antònio , Cazenave Pierre‐André
Published: March 2007

Neonatal treatment of BALB/c mice with anti‐VHT15 antibodies suppresses serum expression of VH T15 immunoglobulins in adult animals (2 months) which remains for over 8 months in half of the cases. Suppressed mice, however, contain control numbers of B cells expressing genes of the S107 VH family and producing VH T15 after mitogenic stimulation. Furthermore, immunization with phosphorylcholine (PC) breaks suppression and stimulates the production of VH T15 anti‐PC antibodies. These animals, however, contain no detectable B lymphocytes expressing the T15 idiotype and produce no T15 idiotype‐positive antibodies in response to PC. These results are discussed in the context of lymphocyte repertoire selection.
Natural killer cells activated with interleukin 2 in vitro can be adoptively transferred and mediate hematopoietic histocompatibility‐1 antigen‐specific bone marrow rejection in vivo
Authors: Murphy William J. , Kumar Vinay , Bennett Michael
Published: March 2007

An adoptive transfer model was developed for examining natural killer (NK) cell‐mediated bone marrow cell (BMC) graft rejection. Homogeneous populations of NK cells were obtained by culturing spleen cells from mice with severe combined immune deficiency in the presence of recombinant interleukin 2 for 7 days. These cells maintained a phenotypic (CD3−CD4−CD8−NK‐2.1+ and asialo GM1+) and lytic pattern consistent with that of activated NK cells. The cells were infused into lethally irradiated recipients whose own NK cells were depleted by anti‐NK‐1.1 antibody treatment and could no longer reject marrow allografts. The transferred NK cells restored the ability of the hosts to reject BMC in a hematopoietic histocompatibility‐1 (Hh‐1) antigen‐specific manner. Immunogenetic studies demonstrated that the rejection was a function of the donor, not host, NK cells. These studies demonstrate that NK cells can be cultured in vitro with recombinant interleukin 2 and are capable of mediating Hh‐1‐specific BMC rejection in vivo in the absence of other immune cell types.
Human natural killer cells and mature T lymphocytes express identical CD3ζ subunits as defined by cDNA cloning and sequence analysis
Authors: Moingeon Philipe , Stebbins Christopher C. , D'Adamio Lucciano , Lucich Jeanne , Reinherz Ellis L.
Published: March 2007

In order to characterize the CD3 ζ‐related protein found in human natural killer (NK) cells and compare it with CD3 ζ expressed in T lymphocytes, the present study was performed. A polyclonal CD3−CD16+NK population displaying a strong non‐major histocompatibility complex‐restricted cytotoxic activity against the NK target K‐562 was isolated and a product corresponding to CD3 ζ amplified using the polymerase chain reaction method. This 0.6‐kb product was present in similar amounts in NK cells and T cells. In contrast, a product corresponding to CD3 ζ was amplified from T lymphocytes exclusively. Thus, the CD3 ζ product detected in NK cells did not originate from contaminating T cells. DNA sequence analysis of two independent polymerase chain reaction products from the NK cells demonstrates that human NK cells and mature T cells share a CD3 ζ subunit with an identical primary amino acid sequence. The nucleotide sequence of a third NK‐derived cDNA revealed an insertion of a CAG triplet encoding an additional glutamine residue in the cytoplasmic domain. Since this residue is encoded by nucleotides at a putative RNA splice junction, it possibly results from a difference in pre‐mRNA splicing. Taken together, these data show that CD3 ζ is not structurally distinct in NK cells and in T lymphocytes.
Structural diversity of Plasmodium falciparum gp200 is detected by T cells
Authors: Simitsek Phaedra D. , Ramirez Elbe , Perrin Luc H.
Published: March 2007

T lymphocyte clones (TLC) specific for P. falciparum gp200 (a glycoprotein precursor of the main merozoite surface component) were obtained from two individuals with past exposure to malaria. The 25 established TLC carried the CD4 antigen and proliferated in the presence of immunopurified gp200, crude lysate of the parasite and intact infected red blood cells. They were further tested in proliferation assays for their capacity to recognize the structural diversity displayed by gp200. The stimulating antigen used in these assays was either sonicated or viable preparations of schizonts from five P. falciparum isolates differing in their gp200. The majority of the TLC proliferated similarly in the presence of each of the isolates. One third of the TLC proliferated to a different extent depending on the isolate used for stimulation, while two clones gave isolate‐specific responses. These results indicate that the majority of human TLC raised in vitro against gp200, is directed against common determinants. This also suggests that immunization with full length gp200 will not lead predominantly to T cell help restricted to isolate‐specific determinant.
Expression of CD1 in the mouse thymus
Authors: Bradbury Andrew , Calabi Franco , Milstein Cesar
Published: March 2007

The CD1 antigens are a family of differentiation antigens found predominantly, but not exclusively, in the human thymus. Although three antigens (CD1a–c) are described by monoclonal antibodies, five genes (CD1A–E) are found in the human genome. The cloning of the mouse CD1 genes (Bradbury, A., Belt, K. T., Nery, T. M., Milstein, C. and Calabi, F., EMBO J. 1988. 7: 3081) demonstrated the presence of homologues to human CD1D, but not to any of the other human CD1 genes. In this work we have examined the expression of mouse CD1D mRNA in the thymus and shown that it is predominantly cortical, as is the expression of the CD1 antigens in man. Somewhat surprisingly, we also find that most CD1D mRNA in the mouse thymus is unspliced. Despite this, we have also been able to show, using a polyclonal antiserum directed against a bacterial fusion protein, the existence of the expected protein product.
Autoantibody production by severe combined immunodeficient mice reconstituted with synovial cells from rheumatoid arthritis patients
Authors: Tighe Helen , Silverman Gregg J. , Kozin Franklin , Tucker Rebecca , Gulizia Richard , Peebles Carol , Lotz Martin , Rhodes Gary , Machold Klaus , Mosier Donald E. , Carson Dennis A.
Published: March 2007

In an attempt to characterize the heterogeneity of the human autoantibody response, mice with severe combined immunodeficiency were reconstituted with synovial or blood lymphocytes from patients with rheumatoid arthritis (RA). Mononuclear cells extracted from synovial fluid or tissue (SMC) were a greatly enriched source of IgM rheumatoid factor (RF)‐producing cells compared to the peripheral blood mononuclear cells (PBMC) of rheumatoid arthritis patients or normal donors. Six to nine weeks after reconstitution of mice with synovial mononuclear cells, 0%‐39.3% (mean = 11.4%) of total IgM consisted of IgM RF compared to 0%‐0.15% (mean = 0.02%) in mice given RA PBMC and 0%‐1.2% (mean = 0.34%) in mice given normal PBMC. Detectable levels of IgM RF were maintained in some mice for as long as 20 weeks after transfer. Mice reconstituted with synovial membrane or synovial fluid lymphocytes produced a heterogeneous mixture of immunoglobulins. These included other autoantibodies, such as anti‐nuclear and anti‐cytoplasmic antibodies, and antibodies to exogenous antigens such as the Epstein‐Barr virus nuclear antigen‐1 (EBNA‐1). This heterogeneity is further illustrated by the demonstration that the sera from mice given synovial cells also contained IgG antibodies possessing all three major VH families (VH, VH3 and VH4) and the four major Vx families (Vx1 to Vx4). Autoantibody production gradually decreased with time even under circumstances where total immunoglobulin levels increased, and elevated production could not be induced by antigenic stimulation. These findings describe a new model for the analysis of human autoantibody production.
The Vϰ gene repertoire in the human germ line
Authors: Meindl Alfons , Klobeck H.‐Gustav , Ohnheiser Rita , Zachau Hans G.
Published: March 2007

The question of how many Vϰ gene segments exist in the human germ line was addressed. Seventy‐five Vϰ genes of the x locus and twenty‐five Vϰ genes localized outside of the locus (“orphons”) had been cloned previously; 67 of the genes and 19 of the orphons had already been sequenced yielding 36 and 1 potentially functional Vϰ genes, respectively, the remaining ones being pseudogenes. We now (a) determined the relative hybridization intensities of the cloned Vϰ genes and orphons, (b) identified the bands in blot hybridizations of genomic DNA digests with the cloned genes and orphons, (c) determined the band intensities in the genomic DNA digests from two individuals and one cell line, (d) normalized the results with the help of the Cϰ gene segment which is present in the haploid genome in one copy, (e) compared the genomic blot hybridization patterns with patterns of equimolar mixtures of the cloned Vϰ genes and orphons, and (f) defined the bands and fractional intensities in bands that could not be assigned to cloned genes or orphons. From the resulting data we conclude that there are 5‐7 still uncloned Vϰ genes in germ‐line DNA in addition to the 75 known Vϰ genes and in addition to the 25 orphons 12‐15 orphon candidates. It appears that the rheumatoid factor light chains of the Wa and 6B6.6 idiotypes are coded for by one VϰIII gene each. It is concluded that the x locus comprises no more than 50 potentially functional genes and no more than 85 Vϰ genes altogether.
Restoration of H‐2b expression and processing of endogenous antigens in the MHC class I pathway by fusion of a lymphoma mutant to L cells of the H‐2k haplotype
Authors: Öhlén Claes , Bastin Judy , Ljunggren Hans‐Gustaf , Imreh Stephan , Klein George , Townsend Main R. M. , Kärre Has
Published: March 2007

The RMA‐S lymphoma mutant cannot process and present antigens to H‐2‐restricted cytotoxic T lymphocytes. It synthesizes major histocompatibility complex class I heavy (H‐2KbDb) and light β2‐microglobulin (β2mb) chains of normal size and charge, but only a fraction of these assemble and reach the cell surface. As a first step investigating the genetic defect of this line, we have fused it to a L cell fibroblast line (H‐2KkDk/β2ma). The fusion restored H‐2Kb, Db and β2mb expression as well as the ability to process and present internally derived (minor histocompatibility and influenza virus nucleoprotein) antigens in RMA‐S. This shows that the mutation(s) responsible for the phenotype of RMA‐S is (are) not located within the MHC class I heavy and light chain genes. Other cellular factors, derived from the L cell fusion partner, can control antigen processing and transport of MHC class I molecules. These findings are discussed in relation to the observation that assembly and transport of MHC class I molecules can be induced in the mutant by H‐2b‐restricted peptides. The recessive nature of the defect and its independence of MHC class I genes in the mutant has important implications for future transfection studies, of this and similar mutants, aiming at establishing cells containing non‐assembled MHC class I molecules of different alleles and identifying the gene(s) controlling processing of endogenous antigens.
The G protein coupling T cell antigen receptor/CD3‐complex and phospholipase C in the human T cell lymphoma Jurkat is not a target for cholera toxin
Authors: Sommermeyer Henning , Schwinzer Reinhard , Kaever Volkhard , Behl Berthold , Resch Klaus
Published: March 2007

Intact Jurkat cells could be stimulated by monoclonal antibodies against the T cell antigen receptor complex (OKT3 directed against the CD3 complex, BMA031 directed against constant framework epitopes in the α/β heterodimer). The accumulation of inositol phosphates was inhibited by prior incubation of the cells with cholera holotoxin. The inhibitory effect of cholera toxin (CT) was not cAMP mediated because forskolin (a direct activator of adenylate cyclase) did not mimic the inhibitory effect. When measuring phospholipase C (PLC) in a cell‐free assay system by using [3H]inositol‐labeled membranes, the enzyme could be stimulated by the poorly hydrolyzable GTP analogue guanosine 5′‐O‐thiotriphosphate (GTPγS). Both anti‐receptor antibodies augmented the GTPγS stimulatory effect, while the antibodies alone had no stimulatory capacity. In membranes from CT‐pretreated Jurkat cells GTPγS had the same stimulatory effect on PLC as in untreated cells, whereas the antibodies lost their stimulatory capacity in the presence of GTPγS. These data imply that CT exerts its inhibitory effect on signaling by acting at the receptor level while the PLC regulating G protein is not a target for CT‐mediated alterations. This assumption is supported by the finding that in intact Jurkat cells CT, which ADP ribosylated only the α‐subunit of the stimulatory G protein of the adenylate cyclase, led to a loss of the T cell antigen receptor complex from the cell surface as demonstrated by a decrease of receptor density using flow cytometry analysis. Receptor loss could not be achieved by forskolin treatment or incubation of the cells with the binding subunit of the toxin alone.
Dual effects of interleukin 4 on antigen‐activated human B cells: induction of proliferation and inhibition of interleukin 2‐dependent differentiation
Authors: Llorente Luis , Mitjavila Francesca , Crevon Marie‐Claude , Galanaud Pierre
Published: March 2007

We analyzed the effects of interleukin 2 (IL 2) and IL 4 isolated and in association on the specific response of human B cells triggered by trinitrophenylated polyacrylamide beads (TNP‐PAA). IL 2 induced an increase (more than 10 times) in the number of hapten‐binding cells [detected by a rosette‐forming cell (RFC) assay] as well as the generation of antibody‐producing cells [detected by a plaque‐forming cell (PFC) assay]. IL 4 induced an isolated RFC response without PFC response. We verified that the IL 4 (as well as I2)‐induced RFC were hapten specific and mediated through membrane IgM. Density fractionation experiments showed that IL 4‐induced RFC were equally distributed between highdensity and intermediate‐density B cells. IL 2 appeared to drive more B cells into the intermediate density fraction. IL 2‐induced PFC belonged to the RFC population and were intermediate‐size B cells. IL 2 drove more RFC into an activated stage and it induced the differentiation of a number of them into antibody‐producing cells. The evaluation of the proportion of RFC able to incorporate thymidine showed that both IL induced a substantial proliferation of antigen‐activated B cells. However, IL 4 inhibited the IL 2‐dependent PFC without affecting the number of RFC nor the proportion of proliferating RFC induced by this IL. These results directly demonstrate that human IL 4 triggers the expansion of antigen‐activated B cells and selectively inhibits the IL 2‐induced differentiation.
Mast cell heterogeneity: two‐dimensional gel electrophoretic analyses of rat peritoneal and intestinal mucosal mast cell
Authors: Abe Tatsuya , Swieter Mark , Imai Toru , Hollander Neal Den , Befus A. Dean
Published: March 2007

Peritoneal mast cells (PMC) and intestinal mucosal mast cells (IMMC) were purified from rats infected with the nematode Nippostrongylus brasiliensis. Overall protein constituents of both mast cell subtypes were analyzed by two‐dimensional gel electrophoresis using either nonequilibrium pH gradient electrophoresis (NEPHGE) or isoelectric focusing (IEF) in the first dimension and SDS‐PAGE (10%) in the second dimension followed by silver staining. PMC had seven dominant basic proteins (PB2–8; PI 9–9.5) with estimated molecular masses of 26 to 37 kDa, as well as 80 to 90 neutral or acidic proteins, most of which had pI 6 to 7.5 and estimated molecular masses of 20 to 100 kDa. All the basic proteins were granule‐associated. Three basic proteins, PB6 (29 kDa), PB7 (28 kDa) and PB8 (RMCP I, 26 kDa), bound [3H]diisopropyl fluorophosphate (DFP), suggesting that they are serine proteases. However, only PB8 was reactive with antibodies to RMCP I. Another basic component (< 14 kDa), perhaps a degradation product of PB6, PB7 or PB8, also bound [3H]DFP. By comparison, IMMC possessed nine basic proteins (IB1‐9) and, in general, they were more acidic (pI about 8.5–9) than those of PMC. Four major basic proteins (IB6–9) were all 24 kDa but were slightly different in isoelectric points. These and another 46‐kDa basic component (IB2) were reactive with antibodies to RMCP II and bound [3H]DFP. There were no other DFP‐binding proteins in IMMC. In spite of remarkable differences between basic granule‐associated proteins in PMC and basic proteins in IMMC, spots in the neutral‐acidic range were for the most part similar in the two mast cell subsets, although quantitative differences were evident for some spots. Thus, rat mast cell populations from the peritoneal cavity and intestinal mucosa exhibit marked heterogeneity in their protein constituents with basic pI, including in their granule‐associated proteins with serine protease activity.
The biological effects of IgM hexamer formation
Authors: Randall Troy D. , King Leslie B. , Corley Ronald B.
Published: March 2007

The inducible B cell lymphoma, CH12, and its in vitro adapted subclone, CH12‐LBK, produce immunoglobulins of identical sequence, specificity and isotype, with equivalent affinities for the hapten trimethyl ammonium. However, the hemolytic efficiencies of the antibody secreted by the two cell lines are quite different. Antibody preparations from lipopolysaccharide‐stimulated CH12 cells lyse erythrocytes six‐ to ten times more effectively than antibody preparations of the same concentration from CH12‐LBK cells. Both cell lines secrete polymeric IgM, but while CH12‐LBK cells secrete predominantly the canonical pentameric IgM, CH12 cells secrete a mixture of pentamers and hexamers. High‐efficiency complement‐dependent cytolysis is associated with hexameric IgM, which has a specific activity that is approximately twenty times higher than that of the pentameric form. J chain protein is found in the secreted IgM of both cell lines, but is associated only with the pentameric IgM and not with the hexameric form, nor with any intermediate polymers smaller than a pentamer. A deficit in, or the inaccessibility of, J chain protein appears to facilitate hexamer formation. These experiments confirm previously published data showing that J chain is not necessary either for assembly or secretion of polymeric IgM, and suggest instead that J chain may be important in regulating the lytic efficiency of polymeric IgM by controlling the IgM pentamer/hexamer ratio. The experiments further suggest a mechanism, in addition to isotype switching and somatic mutation, by which the biological efficiency of antibodies from a single clone of B cells can be regulated.
Requirements for the growth of TH1 lymphocyte clones
Authors: Germann Tieno , Partenheimer Andrea , Rüde Erwin
Published: March 2007

Besides the signal generated in a T lymphocyte after triggering the T cell receptor (TcR), most lymphocytes need a “second signal” to become fully activated. The necessity and nature of the “second signal” differs between different types of T cells. At the level of CD4‐positive T helper lymphocytes interleukin 1 (IL 1) serves as “second signal” for those of the TH2 subtype (IL 4, 5, 6 producer) but not for those of the TH1 subtype (IL 2, IFN‐γ producer). This correlates with the absence of the IL 1 receptor at the surface of TH1 clones. We report herein the further purification of T cell stimulating factor (TSF), a soluble mediator involved in the proliferation of TH1 lymphocytes. A preparation free of detectable IL 1, 2, 4 and IL 6 activity could act as “second signal” required for the growth of TH1 lymphocytes in a TcR‐mediated, as well as a TcR‐independent activation system. In addition, we suggest that IL 1 can influence the proliferation of TH1 clones in an indirect way, probably via the induction of TSF in accessory cells.
Rat bone marrow cells undergo thymopoiesis in mouse fetal thymic organ culture
Authors: Deugnier Marie‐Ange , Dargemont Catherine , Denoyelle Monique , Blanche Martine , Imhof Beat A. , Thiery Jean Paul
Published: March 2007

We have developed an in vitro differentiation assay allowing the study of thymopoiesis from rat bone marow cells. In this assay, Wistar rat bone marrow cells repopulated fetal Swiss mouse thymic lobes depleted in endogeneous lymphoid cells by deoxyguanosine treatment. Due to the xenogeneic situation, repopulating rat cells from any hemopoietic lineage could be easily recognized by anti‐rat monoclonal antibodies such as anti‐Thy‐1.1 that did not react with Swiss mouse thymocytes. After 15 days in vitro, 80% of the developing rat cells were Thy‐1.1+ lymphoid cells and about 70% of the Thy‐1.1+ cells expressed CD5, CD2 and leukosialin. The percentages of cells expressing pre‐B cell, B cell and myeloid determinants were < 20%. The developing thymocytes comprised CD4−CD8− T cell receptor (TcR) α/β−, CD4−CD8+TcR α/βlow and CD4+CD8+TcR α/βlow cells, indicating that the early stages of rat thymopoiesis occurred within mouse thymic lobes. Limiting dilution assays showed that 50% of positive assays were obtained with 3000 nucleated bone marrow cells, which is in good agreement with recent estimates derived from in vivo reconstitution after intrathymical transfer. Moreover the limiting dilution assays proved to be sensitive enough to evidence a tenfold enrichment of pre‐T cell activity in the low‐density fraction of rat bone marrow. This xenogeneic system might greatly facilitate studies on prethymic and intrathymic stages of rat T cell development and permit new in vitro approaches of the colonizing bone marrow T cell precursor properties.
Expression of T cell receptor genes in the thymus: localization of transcripts in situ and comparison of mature and immature subsets
Authors: Held Werner , Mueller Christoph , MacDonald H. Robson
Published: March 2007

We have analyzed the expression of T cell receptor (TcR) genes in the thymus using in situ RNA hybridizations with probes to the constant regions of the TcR α, β, γ and δ chains. Localization of transcripts revealed low TcR α mRNA levels in the thymus cortex and very low levels in the subcapsular region. In contrast, TcR β message was very abundant in the cortex. TcR γ or δ mRNA+ thymocytes showed a scattered, predominantly cortical localization. In contrast to γ, TcR δ transcripts were abundant in the subcapsular region. Control experiments with sorted TcR α/β or γ/δ cells revealed a detection efficiency of 75%–85% for the respective TcR mRNA and data on TcR gene expression in mature, CD3+ thymocytes were consistent with previous reports. The analysis of immature, CD3− thymocyte subsets, however, revealed a virtual absence of TcR α transcripts and an unexpectedly high proportion of cells (14%–29%) expressing the gene for the TcR δ chain. The data are discussed in view of current models of lineage relationships in the thymus.
Detection of interleukin 8 biological activity in synovial fluids from patients with rheumatoid arthritis and production of interleukin 8 mRNA by isolated synovial cells
Authors: Brennan Fionula M. , Zachariae Claus O. C. , Chantry David , Larsen Christian G. , Turner Martin , Maini Ravinder N. , Matsushima Kouji , Feldmann Marc
Published: March 2007

The presence of neutrophils in the synovial joint of patients with rheumatoid arthritis (RA) is thought to be due to the activity of chemotactic factors released by activated cells in the joint. We have shown in this report, for the first time, the abundance of one such factor, interleukin 8 (IL 8), in the synovial fluid of patients both with RA and other non‐RA joint diseases, and the spontaneous production of IL8 mRNA by RA synovial cells in culture. There was no correlation between the levels of chemotactic activity and IL8 protein, suggesting that other factors with similar neutrophil chemotactic activity are also present in the synovial fluid exudate. In support of this concept neither the level of chemotactic activity nor IL8 protein levels correlated with neutrophil or leukocyte infiltration, indicating that the mechanism of migration into the inflammatory environment of the joint is complex. Such migration is likely to be due to a number of chemotactic signals in addition to IL8, which may either synergize with, or inhibit, the action of IL8.
Analysis of the immune‐encodrine feedback loop in the avian system and its alteration in chickens with spontaneous autoimmune thyroiditis
Authors: Brezinschek Hans P. , Faessler Reinhard , Klocker Helmut , Kroemer Guido , Sgonc Roswitha , Dietrich Hermann , Jakober Rosanna , Wick Georg
Published: March 2007

In both mammals and chickens, immunization with exogenous antigens results in a surge of serum glucocorticoid hormone levels concomitant with the antibody response. This effect is mediated by glucocorticoid‐increasing factors (GIF) produced by cells of the immune system. In the avian system, GIF appear to act via the hypothalamo‐pituitary axis and not directly on the adrenal gland. Interleukin 1 is the main active substance responsible for GIF activity, as shown by molecular sieve and immunoaffinity chromatography studies. In contrast to data from mammals, we found no evidence that interleukin 2 elevates chicken corticosterone.

Obese strain chickens with spontaneous Hashimoto‐like autoimmune thyroiditis are deficient in their in vivo GIF response. Because no differences were found between autoimmune and healthy chickens in the corticosterone response of the adrenal gland after ACTH administration, and since autoimmune animals are able to react normally to immobilization stress, it is assumed that this deficiency is due to a specific defect rather than a general disturbance in the endocrine system.
The activation of resting lymphocytes is accompanied by the biogenesis of lysosomal organelles
Authors: Olsen Irwin , Bou‐Gharios George , Abraham David
Published: March 2007

The degradative activity of lymphocytes plays by important role in a number of essential immune functions. In the present study we have examined how the activation of resting lymphocytes, by the mitogen concanavalin A (Con A), affects three major components of the lysosomal compartment: the lysosomal enzyme β‐glucuronidase (Gus); an integral lysosomal membrane protein (LAMP‐1); and the mannose 6‐phosphate receptor (MPR) which directs lymphocyte enzyme transport. Resting T cells were found to contain only very low levels of these proteins, but they were actively synthesized by, and far more abundant in, stimulated lymphoblasts. Although the lysosomal antigens did not have a distinct cytoplasmic localization in the resting lymphocytes, in the activated T lymphoblasts they were present in several highly developed intracellular structures, including the rough endoplasmic reticulum and the Golgi complex. Furthermore, in these latter cells Gus was also found to be accumulated within the lumen of large vesicles which we characterized as lysosomes by the presence of LAMP‐1 at the periphery and by the absence of MPR. Subcellular fractionation confirmed that these organelles were present in the activated lymphocytes only, and not in the resting T cells. Our results demonstrate that lymphocyte activation is accompanied by the synthesis of the enzymic and structural components of the lysosomal compartment which are sorted and assembled into distinct organelles in the activated cell.
Human IgE mRNA expression by peripheral blood lymphocytes stimulated with interleukin 4 and pokeweed mitogen
Authors: Qiu Gang , Gauchat Jean‐Francois , Vogel Monique , Mandallaz Michele , de Weck Alain L. , Stadler Beda M.
Published: March 2007

Expression of human IgE mRNA by peripheral blood lymphocytes (PBL) and an IgE‐producing myeloma cell line, U‐266, was examined by Northern blot hybridization and compared with IgE levels in culture supernatants. A 2.35‐kb IgE mRNA was detected in unstimulated atopic PBL and U‐266 cells but not in normal PBL, and its levels correlated with IgE protein levels in the supernatant. Upon stimulation with interleukin 4, a new 1.75‐kb transcript was revealed in both atopic and normal PBL but not in U‐266 cells. Its expression did not correlate with IgE levels in the supernatant. Pokeweed mitogen also induced the expression of the 1.75‐kb transcript without concomitant induction of IgE synthesis by normal PBL and even suppressed the spontaneous expression of the 2.35‐kb transcript and IgE protein synthesis by atopic PBL. Interferon‐γ, which suppressed both the 2.35‐kb transcript and IgE protein production, had no inhibitory effect on the 1.75‐kb transcript. Expression of the 1.75‐kb transcript was already high after 2 days of stimulation and peaked around day 4. The length of the transcript is smaller than that of mRNA coding for secreted human IgG and IgA and contains all four Cε exon sequences, suggesting it might be a truncated transcript without v region and might be a human counterpart of the murine germ‐line Cε transcript.
T‐T cell interactions are mediated by adhesion molecules
Authors: Brod Staley A. , Purvee Meghan , Benjamin Deborah , Hafler David A.
Published: March 2007

The mechanism by which T cells signal other T cells is not well defined. This was investigated by studying the ability of circulating T cells to induce the proliferation of autologous T cell clones. Peripheral blood T cells activated by cross‐linking of the CD3/T cell receptor complex, which increased the expression of cell adhesion molecules LFA‐1, LFA‐3 and ICAM‐1, induced the proliferation of autologous T cell clones. Irradiated antigen‐activated peripheral blood T cells could also induce the proliferation of T cell clones which could not recognize that antigen. T‐T cell activation required cell contact, was not major histocompatibility complex (MHC) restricted and was blocked by monoclonal antibodies directed against adhesion molecules CD2 and LFA‐3 but was not blocked by antibody to class II MHC determinants. As CD2 is the natural ligand for LFA‐3, increased expression of T cell surface adhesion molecules LFA‐1, ICAM‐1 and particularly LFA‐3 during an inflammatory response may rapidly recruit T cells that are activated through the CD2 pathway. These results allow a simplified model to explain how relatively few antigen/MHC‐specific T cells can recruit large numbers of non‐antigen‐specific T cells in the generation of an inflammatory response and postulates a novel role of the CD2 molecule in T cell immune function.
Mutations in the major histocompatibility complex class I antigen‐presenting groove affect both negative and positive selection of T cells
Authors: Jacobs Heinz , Von Boehmer Harald , Melief Cornelis J. M. , Berns Anton
Published: March 2007

In several transgenic mouse models T cell development was shown to be controlled by the binding of the α/β T cell receptor (TcR) to ligands in the thymus. In transgenic mice expressing a male‐specific TcR α/β, the presence of the restricting Db major histocompatibility complex (MHC) molecule plus the male specific peptide deleted thymocytes at an early stage of development. On the other hand, maturation of T cells required an interaction of the TcR with the thymic Db MHC molecules in the absence of specific peptides. This could imply that negative and positive selection of this receptor are affected differently by mutations in the HY peptide‐binding groove of the Db MHC molecule. Such mutants have been isolated and were shown to affect the response to HY antigen in that both the bm14 (residue Glu70 → Asp) and the bm13 (residue Leu114 → Glu, Phe116 → Tyr and Glu119 → Asp) strains do not normally mount cytotoxic responses to male cells. Here we show that these mutations affect antigenicity of male cells, as well as negative and positive selection of T cells in TcR α/β transgenic mice.
Differential responsiveness to CD3‐Ti vs. CD2‐dependent activation of human intestinal T lymphocytes
Authors: Pirzer Ursula C. , Schürmann Guido , Post Stefan , Betzler Michael , Meuer Stefan C.
Published: March 2007

Human lamina propria lymphocytes were recovered from surgical specimens of unaffected large bowel and compared with peripheral blood lymphocytes (PBL) from the same individual. We investigated their capacities to respond to triggering stimuli delivered by mitogenic monoclonal antibodies directed at the T cell receptor/CD3 complex or, alternatively, the CD2 glycoprotein. In marked contrast to PBL, expression of the interleukin 2/interleukin 2 receptor system was markedly reduced in LPL following activation through the CD3/T cell receptor. Perhaps, more important, responses to CD2 triggering were similar or even stronger in LPL as opposed to PBL. This indicates that the CD2‐dependent alternative pathway of T cell activation might serve a functional role in local immunity in the gut.
Polarity of immunogens: implications for vaccine design
Authors: Golvano José , Lasarte Juan J. , Sarobe Pablo , Gullón Arturo , Prieto Jesús , Borrás‐Cuesta Francisco
Published: March 2007

Peptide constructs have been engineered consisting of amino acid sequence determinant recognized by T cells (TD) co‐linearly linked to haptenic peptides. It was found that high anti‐hapten antibody titers were induced after immunization with those constructs which had the TD sequence in the N‐terminal position with respect to the hapten. Low or zero titers were elicited when the TD was in C‐terminal position. Also, a high anti‐hapten antibody titer corresponded to a low or zero anti‐TD antibody titer and vice versa. These results suggest that immunogens are polar and stress the relevance of searching the more adequate position of the TD within a peptide construct when designing immunogens or synthetic peptide vaccines.