bluuebunny/crossref_metadata_2025 huggingface dataset:
Cooperative effect of interferon‐γ and tumor necrosis factor‐α on the induction of the class II antigen‐associated invariant chain expression
Authors: Pessara Ulrich , Momburg Frank , Koch Norbert
Published: March 2007

The regulation of the invariant chain (Ii) expression was studied in the human colon carcinoma cell line HT‐29 that constitutively expressed neither Ii nor class II antigens. Upon stimulation of HT‐29 cells with a combination of human recombinant tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ), expression of mRNA and protein of the invariant chain were induced. In contrast, administration of TNF‐α or IFN‐γ alone had no effect. A delayed induction of Ii mRNA, which was first detected 10–12 h after stimulation, was observed; this suggests an indirect regulatory mechanism. Stimulation with both IFN‐γ and TNF‐α led to the co‐expression of class II antigens with the invariant chain. In order to study the genetic basis for this stimulation the murine invariant chain gene (800 bp 5′ flanking sequences and the structural gene) was transfected into HT‐29 cells and transfected cells were tested for the ability to respond to IFN‐γ and TNF‐α. Simultaneous application of both cytokines had a strong effect on the induction of the murine invariant chain. IFN‐γ alone had no effect and TNF‐α only marginally stimulates murine invariant chain expression. The transfection experiment indicates that the murine invariant chain gene construct contains the structural elements which are responsible for regulation with IFN‐γ and TNF‐α. We determined whether the cooperative effect of TNF‐α and IFNγ is also found in vivo. Stimulations of mice were performed with TNF‐α, IFN‐γ and a combination of both. The immunohistological analysis of kidney tissue sections revealed that TNF‐α had no effect on Ii and Ia expression. Upon IFN‐Y treatment a minor subset of renal tubules showed staining for Ii, and less prominently also for Ia. However, simultaneous application of both cytokines led a strong induction of both Ii and Ia antigens in renal epithelial cells, thus suggesting that this synergistic effect potentially occurs under physiological conditions.
Clonal analysis of human T cell activation by the Mycoplasma arthritidis mitogen (MAS)
Authors: Matthes Michaela , Schrezenmeier Hubert , Homfeld JÖRg , Fleischer Sabine , Malissen Bernard , Kirchner Holger , Fleischer Bernhard
Published: March 2007

Mycoplasma arthritidis produces an as yet undefined soluble molecule (MAS) that has a potent mitogenic effect on T cells of several species. We have used cloned human cytotoxic and proliferative T lymphocytes to dissect the molecular mechanism of T cell activation by this mitogen. Reactivity to MAS is clonally expressed among T cell receptor (TcR) α/β chain‐expressing T cell clones of CD4+ or CD8+ phenotype, as well as CD4−8− TcRα/β chain‐negative T lymphocyte clones expressing the CD3‐associated TcRγ chain. MAS is able to induce cytotoxicity and/or proliferation in these T cell clones. For triggering of these T cells, regardless of their phenotype of specificity, the presence of autologous, allogeneic or xenogeneic major histocompatibility complex (MHC) class II molecules on accessory cells or target cells is necessary. However, T cells do not immunologically recognize MAS on class II molecules, since a direct action of MAS on the T cells themselves can be demonstrated. Triggering of T cells by MAS can be blocked by monoclonal antibodies against CD2, CD3 and the TcRα/β chain dimer. We discuss as a possible explanation that MAS is a functionally bivalent molecule cross‐linking TcR and MHC class I1 molecules. Thus, the mechanism of T cell activation by MAS has striking similarities to the mechanisms by which Staphylococcal enterotoxins activate T cells. It is intriguing that a similar mitogenic principle has been developed by two evolutionary distinct pathogenic microorganisms.
Functional interaction between B cell subpopulations defined by CD23 expression
Authors: Armitage Richard J. , Goff Lindsey K.
Published: March 2007

Using the CD23 monoclonal antibody (mAb) MHM6 and sheep anti‐mouse Ig bound to magnetic beads we have obtained highly purified populations of MHM6+ and MHM6− tonsil B cells. We have found that the increased expression of MHM6 reactivity seen on B cells after activation results from up‐regulation of antigen on cells already weakly positive and not from expression of new antigen on the previously negative population. The strong proliferative responses of MHM6+ cells seen in the presence of anti‐IgM (αμ) and interleukin 4 (IL4) or the CDw40 mAb G28‐5, and with Staphylococcus aureus Cowan I (SAC), and to a lesser extent with the phorbol ester 12‐0‐tetradecanoylphorbol 13‐acetate (TPA), resemble that seen among unfractionated B cells. In contrast, the MHM6− population cultured alone responds only weakly to αμ+ G28‐5 or SAC and exhibits virtually no response to αμ + IL4 or TPA

With all these mitogenic stimuli, tritiated thymidine uptake by the MHM6− population is augmented three‐ to sixfold by the addition of mitomycin C (MC)‐treated MHM6+ cells. Pretreatment of cells with anti‐leukocyte functional antigen 1 mAb has little effect on the subsequent proliferation of the MHM6− population but shows cell contact to be critical for the proliferation of MHM6′ cells. Such pretreatment has revealed that the functional interaction observed between MHM6+ and MHM6− cells is dependent on both cell contact and the presence of an MHM6+ cell‐derived soluble component

We have found that addition of soluble CD23, purified from Epstein‐Barr virus‐transformed lymphoblastoid cell line supernatant, increases the proliferative response of MHM6− tonsil B cells to mitogenic stimuli in the presence of inactivated MHM6+ cells but has no effect on proliferation when MHM6+ cells are absent

By way of contrast to normal B lymphocytes, we have examined functional responses of prolymphocytic leukemia (PLL) B cells. Although these cells, when freshly isolated, show comparable levels of CD23 expression to normal B cells, this expression is not increased upon activation. In addition, in contrast to normal B cells, the PLL MHM6− population cultured alone shows a strong proliferative response to various mitogenic stimuli, comparable to that of MHM6+ or unfractionated cells, and this response is not augmented by the addition of MC‐treated MHM6+ cells. Thus, a novel functional interaction is described between normal, but not leukemic, B cell populations defined by their expression of CD23. This interaction involves two components, one mediated through cell contact and the other by the presence of soluble factor(s) derived from the MHM6+ cells with neither component alone being effective.
B lymphopoiesis on stromal cell clone: stromal cell clones acting on different stages of B cell differentiation*
Authors: Nishikawa Shin‐Ichi , Ogawa Minetaro , Nishikawa Satomi , Kunisada Takahiro , Kodama Hiroaki
Published: March 2007

B lymphopoiesis supporting activities of two stromal cell clones, MC3T3‐G2/PA6 (PA6) and ST2, were compared. When normal bone marrow cells were cultured in these clones under Whitlock‐Witte‐type condition, mature B cells were generated only in the culture with the ST2 layer. The cells maintained on the PA6 layer, however, contained the precursor cells giving rise to mature B cells when transferred to the ST2 layer. Thus, PA6 is a stromal cell clone capable of supporting the early B progenitors but cannot support a further maturation step into pre‐B cells. The immunoglobulin heavy chain gene configuration of B progenitors maintained on the PA6 layer diversified after their transfer onto ST2 layer. This suggests that they are actually the earliest progenitors. This marked difference in the stromal cell activities between PA6 and ST2 could also be distinguished by stromal cell‐dependent pre‐B cell lines. Among four ST2‐dependent pre‐B cell lines tested, two grew only on the ST2 layer, which is capable of supporting B lymphopoiesis, while the others grew both on the ST2 and PA6 layers. These results strongly suggest that the process of intra‐marrow B cell development is controlled by more than one signal acting on different stages of B cell differentiation.
Immune responses to an adjuvant‐free native syngeneic myeloma protein (M315)
Authors: Kristoffersen Gunn , Hannestad Kristian
Published: March 2007

Myeloma protein 315 (M315; isotype IgA, λ2) is used in this report as a model to explore the immunogenicity of a syngeneic Ig under nearly physiological conditions. We have previously shown that a synthetic peptide spanning the mutated HV3 loop of the L‐315 chain, when emulsified in complete Freund's adjuvant, elicits T helper cells (Th) that respond to a boost with L‐315 or M315, indicating that M315 is recognized as a processed protein antigen. We now show that the adjuvant‐free 7 S monomer of native or of mildly reduced and alkylated M315, given in divided doses totalling 300 or 800 μg to BALB/c mice, induced persistent anti‐M315 antibodies (Ab), a large part of which was IgG1 directed mainly to idiotypes (Id) associated with M315′s haptenbinding site. Polymers of M315 IgA (800 μg) failed to induce Ab, due probably to their rapid clearance into bile. Short‐term treatment with anti‐CD4 monoclonal Ab GK1.5 at the time of priming with 7s M315 inhibited the responses almost completely. The spleens of M315‐immune mice contained Th that recognized the L‐chain subunit of M315 as a carrier indicating that these Th did not require an assembled (VH‐VL) pair of 315 V regions to be activated. We also observed low amounts of Ab specific for epitopes of the Cα region. This evidence opens the possibility that a distinct autoimmune pathway exists for elicitation of rheumatoid factor (RF; auto Ab to Fcγ) that involves help to RF‐producing B cells by Id‐specific Th. We suggest that these Th recognize V‐region peptides from IgG that have been captured, processed and presented by these B cells.
Excessive production of interleukin 6/B cell stimulatory factor‐2 in rheumatoid arthritis
Authors: Hirano Toshio , Matsuda Tadashi , Turner Martin , Miyasaka Nobuyuki , Buchan Glenn , Tang Bo , Sato Kazuto , Shimi Masatoshi , Maid Ravinder , Feldmann Marc , Kishimoto Tadamitsu
Published: March 2007

High levels of interleukin 6 (IL 6/B cell stimulatory factor‐2) were detected in synovial fluids from the joints of patients with active rheumatoid arthritis (RA). The cells found in freshly isolated synovial fluid constitutively expressed IL6 mRNA. The synovial tissues obtained by joint biopsy were also found to produce IL6 in vitro. Immunohistochemical analysis demonstrated that CD2+ T cells as well as CD20+ blastoid B cells in the synovial tissues produce IL6. The data indicate that IL6 is generated constitutively in RA and its overproduction may explain the local as well as the generalized symptoms of RA, since IL6 can function as B cell growth and differentiation factor as well as hepatocyte‐stimulating factor.
Decreased TcR‐CD3+ T cell numbers in healthy aged humans. Evidence that T cell defects are masked by a reciprocal increase of TcR‐CD3−CD2+ natural killer cells
Authors: Alés‐Martínez JoséE. , Alvarez‐Mon Melchor , Merino Francisco , Bonilla FéLix , Martínez‐A Carlos , Durántez Alberto , De La Hera Antonio
Published: March 2007

While there is accumulating evidence to indicate the presence of functional abnormalities in T cells from aged healthy humans, their cellular basis remains unclear. By using two‐color immunofluorescence and multiparameter flow cytometry we show that (a) the number of peripheral blood antigen receptor‐positive (TcR‐CD3+) T cells is significantly lower in aged than in young adults; (b) the numbers of E‐rosette‐forming (CD2+) cells are maintained in the elderly due to a reciprocal increase in the frequency of TcR‐CD3− cells, which constitute only a minor lymphocyte subpopulation in young adults, and (c) TcR‐CD3−CD2+5− lymphocytes exhibit the phenotypic features of natural killer (NK) cells. By using functional assays we show the TcR‐CD3−CD2+16+ lymphocytes are indeed NK cells because they are activated by and lyse NK targets. In contrast, they are unresponsive to either phytohemagglutinin or mitogenic CD2 monoclonal antibody stimulation, which in turn activates TcR‐CD3+CD2+16− T cells. We conclude that the increase in TcR‐CD3−CD2+ NK cells masks the T cell reduction in aged humans by normalizing CD2+ cell frequencies. However, NK cells cannot functionally substitute the thymus‐derived lymphocytes they replace.
B cell precursors in chick embryos surgically bursectomized at 72 h of incubation
Authors: Lassila Olli , Alanen Anna , Vainio Olli , Houssaine Elisabeth , Pink J. Richard L. , Weber Wilfried T.
Published: March 2007

To determine the presence of precursor B cells in chick embryos surgically bursectomized at 72 h of incubation (E‐Bx) we studied chick chimeras that were produced by establishing parabiotic connections between blood vessels of chorioallantoic membranes of normal and surgically bursectomized chick embryos. Using sex chromosomes and a B cell alloantigen (Bu‐1a) as markers we showed that chick embryos bursectomized at 72 h of incubation contain B cell precursors capable of colonizing the bursa of Fabricius and developing into B lymphocytes. The repopulation capacity of 14‐day‐old embryonic spleen cells from E‐Bx recipients was tested by transferring them into age‐matched X‐irradiated Bu‐1‐disparate embryos. The results show that B cell precursors are present in 14‐day spleen of chick embryos bursectomized at 72 h of incubation. These precursors carry the Bu‐1 B cell alloantigen, suggesting that commitment to the B cell lineage can take place in the absence of bursa.
Identification of anti‐acetylcholinesterase and anti‐idiotype antibodies in human and experimental Chagas+ disease: pathological implications
Authors: Ouaissi Ali , Cornette Jocelyne , And Phillipe Velge , Capron Andre
Published: March 2007

This report presents evidence that human acetylcholinesterase (AChE; acetylcholine hydrolase, EC 3.1.1.7) exhibits immunological cross‐reactivity with the protozoan parasite Trypanosoma cruzi. The immunological probes used indicate that the cross‐reactive determinant is an oligosaccharidic epitope. Antibodies to AChE were detected in a high proportion of T. cruzi‐infected patients sera and during the experimental infection of BALB/c mice. Moreover, anti‐idiotypic antibodies against an anti‐AChE rabbit antibody or a monoclonal antibody to a parasite surface antigen of 80–85 kDa were detected in sera of patients presenting the chronic cardiac form of the disease. The antibodies were less frequently found in sera from individuals with asymptomatic chronic infection. Our data may provide a biochemical basis for denervation hypersensitivity in Chagas' disease. In addition, it may support the notion of an idiotype‐anti‐idiotype regulation of conducting tissue damage during the course of T. cruzi infection.
Human Ig superfamily CTLA‐4 gene: chromosomal localization and identity of protein sequence between murine and human CTLA‐4 cytoplasmic domains
Authors: Dariavach Piona , Mattéi Marie‐Geneviève , Golstein Pierre , Lefranc Marie‐Paule
Published: March 2007

The mouse CTLA‐4 gene has been shown to code for an activated lymphocyte‐associated sequence belonging to the Ig gene superfamily. We now report on the molecular cloning and study of the human corresponding gene isolated from a genomic library and designated Hu‐CTLA‐4. The Hu‐CTLA‐4 gene exists as a single copy per human haploid genome and maps to band q33 of chromosome 2. It comprises 3 exons notwithstanding the leader sequence. The first exon encodes a V‐like domain of 116 amino acids, the second one a hydrophobic putative transmembrane region of 37 amino acids and the third one a 34 amino acid putative cytoplasmic domain. Whereas the overall homology between the human and murine CTLA‐4 proteins is 76%, there is, remarkably, a complete identity of their cytoplasmic domains. This complete interspecies conservation comes in support of an important role for this domain in CTLA‐4 function.
Molecular analysis of T cell receptor gamma gene expression in allo‐activated splenic T cells of adult mice
Authors: Jones Barry , Carding Simon , Kyes Susan , Mjolsness Shelley , Janeway Charles , Hayday Adrian
Published: March 2007

Northern analysis, hybridization in situ and cDNA sequence analysis have been used to demonstrate that the induction of T cell γ‐gene expression is a general occurrence when primary splenic T cells of adult mice are cultured in short‐term mixed lymphocyte reactions (MLR). Splenic T cells from nine strains of mice examined in eleven different MLR all showed significant induction of γ‐RNA, even when the primary T cell response was to only a three amino acid mismatch in a major histocompatibility complex class I antigen. In MLR examined in detail, the expression is highly enriched for in CD3+ “double‐negative” T cells (lacking both CD4 and CD8 expression). A cDNA sequence analysis, constituting the first such analysis of any size of γ‐gene transcripts from circulating, peripheral cells of adult mice, revealed transcription to be frequently of productively rearranged genes. These genes display extensive junctional diversity.
Tolerization during pregnancy: impact on the development of antigen‐specific help and suppression
Authors: Zöller Margot
Published: March 2007

Analysis of splenic B cells in the offspring of BALB/c mice which were treated with 2,4,6‐trinitrobenzenesulfonic acid (TNBS) throughout the gestational period revealed that reduced responsiveness was predominantly reflecting changes in regulatory cells. Since development of T cells takes place in the thymus, postnatal maturation of helper (Th) and suppressor (Ts,) T cells was evaluated in the thymus as well as in the spleen. To elaborate the impact of prenatal TNBS‐treatment on the trinitrophenyl (TNP) repertoire, TNP‐sheep red blood cell‐ and fluorescein isothiocyanate‐specific regulatory elements were determined in parallel. It was found that the thymus contained activated helper and suppressor cells during the first 2 weeks of postnatal life, the majority of which appeared to be self‐specific. Self reactivity in thymus and periphery disappeared within 3 weeks. Prenatally TNBS‐treated mice differed in as much as they apparently recognized TNP‐self as a self component, TNP‐self reactivity being down‐regulated similar to self reactivity in prenatally untreated mice. This explains why prenatally TNBS‐treated mice responded poorly to stimulation with conventional T‐dependent TNP‐carrier conjugates. Yet, they responded transiently to TNP‐anti‐TNP conjugates. It could be shown that this was due to down‐regulation of putatively idiotype‐specific Tswhich were highly increased in prenatally TNBS‐treated mice. Data are interpreted in the sense that the establishment of self tolerance is preceded by activation of self‐reactive Th, which become partially eliminated. Remaining autoreactivity is controlled by the establishment on an equilibrium between idiotypic and anti‐idiotypic elements.
Modulation on immunogenicity by HLA‐B27 subtype polymorphism
Authors: Jaraquemada Dolores , Galocha Begoña , Aparicio Pedro , Rojo Susana , And Victor Calvo , Castro José A.LÓPez De
Published: March 2007

Cells from the same HLA‐B27− individual, PA, were stimulated in vitro in primary mixed lymphocyte culture, with either B*2705+ or B*2704+ lymphoblastoid cell lines, in independent experiments. Cytolytic T lymphocytes (CTL) were cloned at limiting dilution and the clones obtained were screened for anti‐B27 alloreactivity. Most of the CTL clones generated against the B*2705+ stimulator cells were directed against the B*2705 +antigen. In contrast, no anti‐B27 CTL clones were found among those derived against the B*2704+ stimulator cells. This was not due to a poor cytotoxic response against these cells because a large proportion of the T cell clones derived from this stimulation were cytotoxic. B2704 differs from B705 by only two amino acid changes at positions 77 and 152. Previous studies (Aparacio, P. et al., Eur. J. Immunol. 1988. 18:203) have shown that none of the anti‐B*2705 CTL clones derived from donor PA and amenable to detailed characterization cross‐reacted with B*2704, suggesting that most of this cytotoxic response was directed against an immunodominant determinant contributed for by residues 77 and/or 152 from B*2705. The present results further suggest that the changes at these positions in B*2704 alter this determinant in such a way that B*2704 becomes less immunogenic for the particular individual PA. Furthermore, a similar poor anti‐B*2704 CTL response was obtained from a second B27− responder individual, AE, stimulated with another B2704+ cell line. The single anti‐B*2704 CTL clone, 64.8P, isolated from this second individual, displayed an unusual reaction pattern in that it cross‐reacted with all B27 subtypes with changes only at or close to positions 77 and 152, including B*2705. Significantly, the only HLA‐B27 subtype that was not recognized by CTL 64.8P was B*2703, which differs from B*2705 only at residue 59. This residue is located in the three‐dimensional structure at the opposite end from residues 77 and 152 at the surface of the antigen‐binding groove of the class I molecule. Thus, the area around residues 77 and 152 is not an essential part of the epitope recognized by CTL 64.8P.
Glucocorticosteroid‐dependent synergy between interleukin 1 and interleukin 6 for human B lymphocyte differentiation
Authors: Emilie Dominique , Crevon Marie‐Claude , Auffredou Marie Thérèse , Galanaud Pierre
Published: March 2007

In order to analyze the effects of interleukin (IL) 6 on human in vitro Ig production B lymphocytes were activated by Staphylococcus aureus Cowan strain I (SAC) in the presence of low concentrations of IL2 (1 U/ml) and dexamethasone (10−7 M). Previously we showed that this model of B cell response is completely monocyte dependent. We here demonstrate that, under these experimental conditions, IL6 is able to replace monocytes and stimulate Ig production provided IL1 is also present. Dose‐effect curves show that these two monokines act synergistically. This synergy is demonstrable only in the presence of dexamethasone, when B lymphocytes are activated (by SAC) and when T cell help (provided by IL2) is present. It results in the production of both IgM and IgG. Both IL 1 and IL6 have to be present during the first 48 h of culture to exert an optimal effect. These results show that IL6 may act on early (as well as on late) stages of normal B lymphocyte differentiation. Moreover, glucocorticosteroids potentiate the synergistic effect of IL1 and IL6 on their B lymphocyte target, an effect comparable to that exerted on hepatocytes.
At least three distinct B cell epitopes reside in the C‐terminal half of La protein, as determined by a recombinant DNA approach
Authors: Rauh Andreas Johann Gallus , Hornig Horst , Lührmann Reinhard
Published: March 2007

The La antigen is a nuclear protein that is one of the major target antigens of autoantibodies found in the sera of patients with primary Sjögren's syndrome. The formation of such autoantibodies is therefore likely to reflect the basic immunopathogenesis of this disorder. A recombinant DNA strategy has been used to examine the La protein for sequences that encode autoimmunizing B cell epitopes. We have isolated and characterized a 1.2‐kb‐long cDNA from a human liver cDNA library encoding a region of the La protein; this region contains 296 amino acids, including the C terminus. A sub‐library of recombinant DNA in the expression vector pEX was made from portions of the La cDNA. Individual fusion proteins were tested by immunoblotting and enzyme‐linked immunosorbent assay for their ability to react with anti‐La autoantibodies contained in sera from patients with Sjögren's syndrome. In this way, we have identified at least three distinct epitopes in the C‐terminal half of the La protein. Every anti‐La serum tested contained antibodies against all three of the antigenic regions identified. Furthermore, most of the sera display similar ratios between the titers of antibodies with the three kinds of specificity. Our data suggest that the production of anti‐La autoantibodies may be antigen driven.
Functional and biochemical characterizations of avian T lymphocyte antigens identified by monoclonal antibodies
Authors: Lillehoj Hyun Soon , Lillehoj Erik Peter , Weinstock Daniel , Schat Kaiel Antoni
Published: March 2007

Seven monoclonal antibodies (mAb) were used to characterize antigens present on chicken T lymphocytes and on natural killer cells by flow cytometry, radioimmunoprecipitation and by effects on cell‐mediated cytotoxicity and mitogen‐induced proliferation. mAb CTLA8 and 5 stained 73% of thymus, 44% of spleen and 51% of peripheral blood lymphocytes (PBL), respectively, and immunoprecipitated 65‐ and 45‐kDa proteins from detergent extracts of 125I surface‐labeled thymocytes. Pretreatment of splenic lymphocytes with mAb CTLA5 and 8 in the presence of rabbit complement (C) eliminated the concanavalin A (Con A)‐induced T cell proliferative responses. mAb CTLA3, 4 and 9 stained 43% of thymus, 36% of spleen and 18% of PBL, and immunoprecipitated 33–35‐kDa proteins. Pretreatment of spleen cells with mAb 4 or 9 plus C reduced, but did not eliminate, the Con A‐induced proliferative response and significantly reduced both major histocompatibility complex (MHC)‐restricted and non‐MHC‐restricted cellular cytotoxicity. mAb CTLA1 and 6 stained 58% of thymus, 13% of spleen and 19% of PBL. mAb CTLA 1 and 6 immunoprecipitated a 65‐kDa protein. mAb CTLA l and 6 had no effect on the Con A‐induced blastogenesis and CTLA 6 caused no decrease in virus‐specific cytotoxic T lymphocyte and natural killer activity. These results indicate that (a) mAb CTLA 5 and 8 identify antigens on mature T lymphocytes that are similar in tissue distribution, molecular mass and function to the mammalian CD5 antigen; (b) mAb CTLA 3, 4 and 9 detect the avian homologue of CD8 antigen; and (c) mAb CTLA l and 6 identify the avian homologue of CD4 antigen.
Human IgG and IgA subclass response following immunization with a polyvalent Klebsiella capsular polysaccharide vaccine
Authors: Cryz Stanley J. , Cross Alan S. , Sadof Gerald C. , Que John U.
Published: March 2007

The human IgG and IgA response was determined after vaccination with an experimental 24‐valent Klebsiella capsular polysaccharide vaccine. The majority of natural antibody acquired prior to immunization was found in the IgG1, IgG2 and IgA1 subclasses. The immune response to vaccination was concentrated within the IgG1, IgG2, IgA1, and IgA2 subclasses with > 70% of subjects responding with a significant (2 fourfold) rise in titer. The IgG3 and IgG4 response was meager with few volunteers (0%‐40%) showing a significant titer rise. Therefore, vaccination with Klebsiella capsular polysaccharide induces a vigorous IgG and IgA response in humans which is not restricted to single antibody class or subclass.
Tumor necrosis factor α stimulates leukotriene production in vivo
Authors: Huber Michael , Beutler Bruce , Keppler Dietrich
Published: March 2007

Tumor necrosis factor α, or cachectin (TNF), is a polypeptide mediator with proinflammatory and antitumor actions. It is produced in large amounts by lipopolysaccharide (LPS)‐activated macrophages. TNF as well as LPS stimulated the arachidonate cascade leading to the synthesis of leukotrienes (LT) in vivo. Production of endogenous cysteinyl LT was measured in anesthetized rat using the biliary excretion of N‐acetyl‐LTE4 as an indicator. Infusion of TNF over a 1‐h period greatly increased the rate of cysteinyl LT prodcution during the subsequent 3 h. Pretreatment with anti‐TNF antibody F(ab′)2 fragments prevented enhanced LT generation as well as tachypnea (a sign of the in vivo action of TNF). LT production elicited by TNF was similar to that evoked by infusion of LPS. Our results indicate that lipoxygenase products are involved in the network of pathophysiological events induced by TNF. The proinflammatory and shock‐inducing LT may mediate many of the adverse effects of TNF in vivo as well as its antitumor action.
Cross‐linking of the T cell antigen receptor interferes with the generation of CD4+8+ thymocytes from their immediate CD4−8+ precursors
Authors: Hünig Thomas
Published: March 2007

The majority of thymocytes are immature cells co‐expressing the surface markers CD4 and CD8. About two thirds of these cells also express the T cell antigen receptor (TcR), though at a level distinctly lower than found on mature T cells. The direct precursors of these “double‐positive” thymocytes are cycling cortical blast cells of the CD4−8+ phenotype. Using a new monoclonal antibody to a constant determinant of the rat TcRα/β, it is shown here that (a) about 50% of these CD8 “single‐positive” committed precursor cells already express the TcRα/β, though at very low levels, (b) during short‐term suspension culture in medium supplemented only with fetal calf serum they not only acquire CD4 but also TcRα/β levels characteristic of CD4,8 “double‐positive” thymocytes, and (c) cross‐linking of the TcR during culture inhibits the acquisition of the CD4 antigen in the majority of these cells.
The tumor progression‐associated human melanoma antigen P3.58 mediates monocyte‐lymphocyte interactions in vitro
Authors: Johnson Judith P. , Lehmann Jurgen M. , Riethmiiller Gert
Published: March 2007

The melanoma‐associated antigen P3.58 is rarely found on benign proliferating melanocytes but is consistently expressed on advanced malignant melanomas which have a high probability of metastasis. Previous studies have shown that its expression on normal tissues is limited to vascular endothelia and lymphoid follicle germinal centers and that it is also expressed by activated monocytes in vitro. In the studies reported here, anti‐P3.58 monoclonal antibodies (mAb) were shown to partially inhibit antigen‐specific and anti‐CD3‐induced T cell proliferation and to completely block a lymphocytelmonocyte clustering which occurs in the absence of added antigen. This inhibition is highly specific for P3.58 mAb and was not affected by mAb directed to major histocompatibility complex or T cell antigens. P3.58 therefore seems to be involved in an antigen‐independent attraction or adhesion of lymphocytes. P3.58 is the second example (HLA‐DR being the first) of an association between the expression of an immune function‐associated molecule and the development of metastatic disease in melanoma.
Identification of a new epitope of the 4F2/44D7 molecular complex present on sarcolemma and isolated cardiac fibers
Authors: Bellone Graziella , Alloatti Giuseppe , Levi Renzo , Geuna Massimo , Tetta Ciro , Peruzzi Licia , Letarte Michelle , Malavasi Fabio
Published: March 2007

The murine monoclonal antibody (mAb) CB43, raised against the K‐562 erythroleukemia line, reacts with monocytes, tissue macrophages, thymocytes and with all the human lines tested but not with resting lymphocytes, large granular lymphocytes, granulocytes and erythrocytes. However, activated lymphocytes and natural killer cells express the CB43 antigen. Embryonic and fetal fibroblasts are positive, while adult fibroblasts are negative. Proximal convoluted tubules in kidney, epithelial cells in esophagus and breast, and sarcolemma in skeletal muscle are reactive with mAb CB43. This antibody can also bind to isolated guinea pig cardiac myocytes, and, furthermore, can induce a transient inotropic effect on isolated atria.

The reactivity with different cell and tissue types and the functional effects of the CB43 mAb were reminescent of the 4F2/44D7 antibodies, shown previously to block Na/Ca2+ exchange in heart and skeletal muscle. Co‐immunoprecipitation studies with CB43 and 44D7 mAb, using radiolabeled Daudi cells, revealed co‐migration of polypeptides of 87 and 38 kDa.

However, the epitope recognized by CB43 is not present on the human heavy chain which bears the 44D7/4F2 epitope as demonstrated by the lack of reactivity of CB43 mAb with mouse L cells transfected with the 4F2 heavy chain gene. Thus, CB43 represents a newly described epitope present on the human light chain or dependent on the conformation of the human dimer.
Activation of the interleukin 2 pathway precedes CD3‐T cell receptor expression in thymic development. Differential growth requirements of early and mature intrathymic subpopulations
Authors: Toribio María Luisa , Hera Antonio De La , Marcos Miguel A. R. , Márquez Carlos , Martínez‐A Carlos
Published: March 2007

T cell activation induced via the CD3‐T cell receptor (TcR) complex, or by triggering of polyclonal antigen‐independent pathways, involves both interleukin 2 (IL 2) production and IL 2 receptor (IL 2R) expression and results in T cell proliferation. To assess the potential role of the IL 2 pathway in T cell development, growth and activation requirements of intrathymic T cell precursors were analyzed and correlated with the expression of IL 2R. In contrast to CD3‐TcR (either CD4 or CD8) mature thymic cells, CD3‐TcR‐ CD1‐4‐8‐ early prothymocytes constitutively expressed IL 2R and displayed IL 2‐mediated proliferation, which was inhibited by anti‐IL 2R monoclonal antibodies (mAb). Moreover, prothymocytes developed spontaneous proliferation in the absence of exogenous IL 2, which was also abrogated by blocking of IL 2R with specific mAb. The possibility that both IL 2 production and IL 2R expression are autonomously activated early in T cell development, before acquisition of the CD3‐TcR complex, led us to study the implication of alternative pathways of activation at this ontogenic stage. Triggering of the CD2 pathway with mAb against two different epitopes of the molecule (D66 and 9.6/T111) induced proliferation of CD3‐TcR‐ prothymocytes in the absence of exogenous IL 2, while proliferation of CD3‐TcR mature thymocytes required IL 2 supplementation. These data suggest that polyclonal activation of the IL 2 pathway may be selectively operative at early stages of T cell development, being involved in the growth and differentiation of intrathymic T cell precursors.
Evidences for protein kinase C. Activation in T lymphocytes by stimulation of either the CD2 or CD3 antigens
Authors: Friedrich Bengt , Cantrell Doreen A. , Gullberg Martin
Published: March 2007

Phosphorylation of protein kinase C (PKC) substrates in T lymphocytes was analyzed after stimulation with specific pairs of anti‐CD2 monoclonal antibodies (mAb) or an anti‐CD3 mAb. The results show that the appropriate stimulation of both CD2 or CD3 antigens results in phosphorylation of a 80‐kDa putative PKC substrate and that this phosphorylation event is sensitive to a PKC inhibitor, sphinganine. CD2‐ and CD3‐dependent phosphorylation was found to be strongly dependent on an extensive cross‐linking of surface antigens. The biological importance of cross‐linking of CD2 and CD3 was also evident for other biological responses such as interleukin 2 production and induction of an autocrine growth response. Finally, we also present evidence for interaction between the CD2 and CD3 signal transducing pathways.
Restriction fragment analysis of VpreB and λ5 within the genus Mus
Authors: D'Hoostelaere Lawrence A. , Bauer Steven R. , Melchers Fritz
Published: March 2007

DNA from a panel of inbred strains of mice and colony bred mice, isolated from different geographical locations, was hybridized to mouse VpreB and λ5 probes under stringent conditions, indicating sequence similarities greater than 80%. The probe for λ5 detects one gene and the probe for VpreB detects two genes (VpreB1 and VpreB2) in the inbred strains of mice examined under the stringency used. No restriction endonuclease fragment length polymorphisms (RFLP) were detected with the VpreB and λ5 DNA probes among the inbred strains of mice using Bam HI and Hind III. Very few RFLP were detected among Mus musculus subspecies, and the intensity of the hybridization did not differ significantly with either DNA probe. The number of RFLP increased slightly when different species and subgenera were examined, and the intensity of the hybridization signal began to decrease in samples from the different subgenera, suggesting a slight decrease in sequence similarity for both VpreB genes with increased time of divergence. Fewer RFLP were detected with the λ5 DNA probe. DNA from 11 different Mus species representing 4 subgenera, genetically isolated from laboratory mice for approximately 1–12 million years, continued to hybridize under high stringency conditions using both DNA probes. A comigrating λ5 and VpreB restriction endonuclease fragment was detected in most of the samples examined, suggesting the close physical linkage of VpreB1 and λ5 is maintained within the genus Mus. These results suggest that VpreB1, VpreB2 and λ5 have been present for over 12 million years.
Efficient mapping and characterization of a T cell epitope by the simultaneous synthesis of multiple peptides
Authors: Zee Ruurd Van Der , Eden Willem Van , Meloen Rob H. , Noordzij Alie , Van Embden Jan D. A.
Published: March 2007

Prediction, identification and analysis of T cell epitopes in protein antigens has become a central theme in fundamental and applied immunology. However, while for the characterization of linear B cell epitopes the so‐called Pepscan procedure was found to be extremely effective, no such technique has so far been available for T cell studies. Recently, we described the identification and localization of a T cell epitope in a mycobacterial 65‐kDa shock protein in the model of adjuvant arthritis. This was done by molecular cloning and conventional solid‐phase synthesis techniques. We now show that the delineation of such a T cell epitope and its further characterization can be accomplished in a much more rapid and efficient manner by a modification of the existing Pepscan technique. We show for the first time that several hundreds of peptides, simultaneously synthesized in an automated way on activated polyethylene rods, can be easily recovered from these rods in adequate quantities, enabling a systematic analysis of T cell epitopes. Synthesis of sequentially overlapping peptides along the 65‐kDa protein revealed that the adjuvant arthritis T cell clones are fully stimulated by peptides that comprise a minimal sequence of seven residues, corresponding to positions 180–186 in the sequence of the 65‐kDa protein of M. bovis Bacillus Calmette Guerain (BCG). Detailed examination of the epitope by peptides containing a single amino acid substitution showed that, apart from one conservative replacement (Glu → Asp), the requirement for the native residue at all positions in peptide 180–186 was absolute for full T cell stimulation. Their indispensability was confirmed with deletion and insertion peptides. It is concluded that the occurrence of indifferent or spacer residues in a minimal stimulatory sequence, as observed by others, is not a general feature of T cell epitopes.
Effect of cyclosporin A on T cell immunity I. Dose‐dependent suppression of different murine T helper cell pathways
Authors: Fukuzawa Masahiro , Shearer Gene M.
Published: March 2007

Cyclosporin A (CsA) is used as a clinical immunosuppressive agent. Despite its immunosuppressive potential, some studies involving in vivo administration of cyclosporin have failed to verify the immunosuppressive activity of this agent. The present study investigates the effect of different concentrations of CsA added in vitro, or of different doses of CsA administered in vivo, on the ability of murine spleen cells to produce interleukin 2 and to generate cytotoxic T lymphocytes in vitro when stimulated with TNP‐self or H‐2 alloantigens. The results indicate that self Ia‐restricted T helper (Th) cells are more sensitive to lower doses of CsA than Th cells that are allorestricted. Thus, doses of CsA were found (15–30 mg/kg) that inactivated self Ia‐restricted Th function, but not other Th or effector function. This Th cell defect could be partially corrected in vitro by addition of Th cell factors to the sensitization cultures. A higher dose (75 mg/kg) of CsA inactivated all detectable T cell responses, and this defect was not corrected by addition of Th cell factors. T cell function returned to normal levels within two weeks of cessation of CsA at all three doses of CsA tested. The selective loss of L3T4 Th function at the lower doses of CsA was associated with a radiosensitive, Ly‐2 suppressor T cell that was selective in its action on self Ia‐restricted Th cell function. Loss of all T cell function at the higher dose of CsA was associated with a radioresistant non‐T suppressor cell that inactivated all T cell function tested. These results are discussed with respect to the selective dose‐dependent effects of CsA on Th subsets, on the activation of suppressor cells with similar selectivity, and the implications of these findings on the use of CsA to prevent rejection of tissue allografts.
SC5b‐7, SC5b‐8 and SC5b‐9 complexes of complement: ultrastructure and localization of the S‐protein (vitronectin) within the macromolecules
Authors: Preissner Klaus P. , Podack Eckhard R. , Müller‐Eberhard Hans J.
Published: March 2007

Purified terminal components of the complement system were used together with purified S‐protein, the inhibitor of the membrane attack complex, to generate the soluble complexes SC5b‐7, SC5b‐8 and SC5b‐9. These complexes were purified by ultracentrifugation in sucrose density gradients with 50–70% yield, exhibiting sedimentation coefficients of 20 S, 21 S and 23 S, respectively. In Ouchterlony double‐diffusion analysis, the purified complexes gave a line of identity against all antisera of the precursor components indicating that complex formation had occurred. The identity of the complexes was also revealed by the appearance of all subunit components after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Since the inhibitor function of S‐protein in the terminal complement cascade should also be manifested in the morphology of the macromolecules generated, the ultrastructures of the three complexes were analyzed by electron microscopy. In contrast to aggregated (C5b‐7)n and (C5b‐8)n, negatively stained SC5b‐7 and SC5b‐8 imaged mostly as monomeric irregularly shaped cylindrical structures, whereas SC5b‐9 < 27 S) appeared as wedge‐shaped structure lacking the tubular polymerized C9. (All three complexes were also generated in the presence of biotinyl‐S‐protein and labeled with avidin‐gold conjugates as electron‐dense marker). Analysis of the modified complexes in electron micrographs demonstrated that the complexes were marked exclusively at one site of their ultrastructures, suggesting this region to be the location of S‐protein and the critical site for membrane binding of C5b‐7 or C5b‐8 and for initiation of C9 polymerization. These results support recent findings in which the function of S‐protein as complement inhibitor was dependent on conformational changes of the protein molecule with concomitant exposure of the heparin‐binding domain.
Peripheral tolerance mechanisms prevent the development of autoreactive T cells in chimeras grafted with two minor incompatible thymuses
Authors: Zamoyska Rose , Waldmann Herman , Matzinger Polly
Published: March 2007

The thymus has been shown to play an important role in the generation of T cell tolerance to self antigens. Developing T cells are readily tolerized to antigens which are expressed in the thymus, and it is generally thought that such thymic tolerance occurs by a mechanism of clonal deletion. We sought to examine whether T cells which initially encountered a “self antigen” post‐thymically would be rendered tolerant of that antigen, and if so whether the mechanism of tolerance induction would differ from that found for thymic antigens. We constructed bone marrow radiation chimeras which were grafted with two thymus lobes differing in minor histocompatibility antigens. T cells which matured in one thymus would be tolerized to the minor histocompatibility antigens expressed in that thymus but would not encounter, and would therefore have no early opportunity of being tolerized to the minor histocompatibility antigens expressed by the other thymus. The initial encounter with the minor antigens on the second thymus would occur post‐thymically. Would these T cells be tolerant or responsive to those minor histocompatibility antigens? We found that tolerance was dominant in these chimeras. The data further suggest that the mechanism responsible for tolerance induction in the periphery may differ from that which operates in the thymus.
Different mechanisms account for the suppression of interleukin 2 production and the suppression of interleukin 2 receptor expression in Trypanosoma brucei‐infected mice
Authors: Sileghem Maarten , Darji Ayub , Remels Linda , Hamers Raymond , Baetselier Patrick De
Published: March 2007

Lymph node cell populations derived from Trypanosoma brucei‐infected mice fail to produce interleukin 2 (IL 2) in response to a potent mitogenic trigger and suppress the potential of normal lymph node cells to secrete IL 2 in co‐culture assays. This suppression is promptly restored by the addition of indomethacin, which blocks prostaglandin synthesis, but is not markedly affected by the addition of catalase, which degrades H2O2. The suppression of the IL 2 receptor expression, on the other hand, is not restored by the addition of indomethacin, nor by the simultaneous supply of both indomethacin and catalase. This discrepancy is not caused by an extreme susceptibility of the receptor expression to low prostaglandin (PG) concentrations, but rather by the presence of suppressive cells that operate through a PG‐independent mechanism. This suppressive mechanism accounts for the loss of the IL 2 receptors on both the Ly‐2 and the L3T4 T cell compartment. The indomethacin‐treated co‐cultures, which manifest a normal IL 2 production but lack the IL 2 receptors, manifest an impaired DNA synthesis and contain a decreased number of T cell blasts.
Ki‐1 (CD30) antigen is released by Ki‐1‐positive tumor cells in vitro and in vivo. I. Partial characterization of soluble Ki‐1 antigen and detection of the antigen in cell culture supernatants and in serum by an enzyme‐linked immunosorbent assay
Authors: Josimovic‐Alasevic Olivera , Dürkop Horst , Schwarting Roland , Backé Eva , Stein Harald , Diamantstein Tibor
Published: March 2007

An enzyme‐linked immunosorbent assay (ELISA) has been developed that allows the quantitative determination of the Ki‐1 (CD30) antigen in soluble form. Similar levels of sensitivity of this new Ki‐1 ELISA and the ELISA previously described for measuring the soluble 55‐kDa chain of the interleukin 2 receptor were seen. As assessed with this ELISA, the investigated Ki‐1 permanent cell lines released the Ki‐1 antigen into the culture supernatant. In culture supernatants of concanavalin A‐activated human peripheral blood lymphocytes, however, this antigen could not be detected. The released Ki‐1 antigen has an apparent molecular weight (Mr) of 85000, whereas the cell‐associated Ki‐1 antigen has an Mr of 105000. We investigated sera from 30 normal donors, 15 patients with systemic infections, and 63 patients suffering from lymphomas for soluble Ki‐1 antigen. In all sera from normal donors and patients with systemic infectious diseases, soluble Ki‐1 antigen was below the detection limit (i.e., < 70 pg). In contrast, high amounts of the soluble Ki‐1 antigen were found in sera from 18 malignant lymphomas containing Ki‐1 tumor cells. This finding demonstrates that the release of the Ki‐1 antigen takes place not only in vitro, but in vivo as well. Moreover, these results imply that the Ki‐1 antigen may be used as a serum tumor marker.