bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental regulation of lck gene expression in T lymphocytes.
Authors: Wildin R S , Garvin A M , Pawar S , Lewis D B , Abraham K M , Forbush K A , Ziegler S F , Allen J M , Perlmutter R M
Published: June 2004

In the mouse and human, mRNA transcripts encoding the lymphocyte-specific protein tyrosine kinase p56lck are derived from two separate promoters resulting in heterogeneity in the 5' untranslated region sequence. The proximal promoter lies just 5' to the coding region for the gene and is active only in thymocytes. In contrast, the distal promoter lies 34 kilobases (kb) 5' in the human, and is active both in thymocytes and mature peripheral T cells. As previously reported, transgenic mice bearing functional proximal promoter sequence juxtaposed with the SV40 large T antigen gene invariably develop lymphoid tumors confined to the thymus. In the current work, transgenic mice bearing a 2.6-kb fragment of the human distal promoter fused to the SV40 large T antigen gene express large T antigen in thymocytes and in peripheral lymphoid cells, and develop tumors of both the thymus and the peripheral lymphoid organs. The ability of the human distal promoter to function appropriately in transgenic mice is consistent with the strong similarity observed between the mouse and human distal promoter sequences. With the exception of a single short interval that serves as a target for binding of nuclear factors, significant sequence similarity is not seen when the distal and proximal promoter sequences are compared. Hence, developmentally regulated, lineage-specific transcription of the lck gene is mediated by distinct promoter sequences that appear to be capable of functioning independently.
Molecules and structures involved in the adhesion of natural killer cells to vascular endothelium.
Authors: Allavena P , Paganin C , Martin-Padura I , Peri G , Gaboli M , Dejana E , Marchisio P C , Mantovani A
Published: June 2004

The present study was designed to define molecules and structures involved in the interaction of natural killer (NK) cells with the vascular endothelium in vitro. Resting and interleukin 2 (IL-2)-activated NK cells were studied for their capacity to adhere to resting and IL-1-treated human umbilical vein endothelial cells (EC). In the absence of stimuli, NK cells showed appreciable adhesion to EC, with levels of binding intermediate between polymorphs and monocytes. The binding ability was increased by pretreatment of NK cells with IL-2. Using the appropriate monoclonal antibody, the beta 2 leukocyte integrin CD18/CD11a was identified as the major adhesion pathway of NK cells to unstimulated EC. Activation of EC with IL-1 increased the binding of NK cells. In addition to the CD18-CD11a/intercellular adhesion molecule pathway, the interaction of resting or IL-2-activated NK cells to IL-1-activated EC involved the VLA-4 (alpha 4 beta 1)-vascular cell adhesion molecule 1 receptor/counter-receptor pair. No evidence for appreciable involvement of endothelial-leukocyte adhesion molecule was obtained. Often, NK cells interacted either with the culture substrate or with the EC surface via dot-shaped adhesion structures (podosomes) protruding from the ventral surface and consisting of a core of F-actin surrounded by a ring of vinculin and talin. The identification of molecules and microanatomical structures involved in the interaction of NK cells with EC may provide a better understanding of the regulation of NK cell recruitment from blood, their extravasation, and their migration to tissues.
The specific binding of peptide ligand to Ld class I major histocompatibility complex molecules determines their antigenic structure.
Authors: Lie W R , Myers N B , Connolly J M , Gorka J , Lee D R , Hansen T H
Published: June 2004

To better understand the biological implications of the association of ligand with major histocompatibility complex class I molecules, we have studied the Ld molecule of the mouse. The culturing of various nonselected cell lines with three different known Ld peptide ligands resulted in a two- to fourfold specific increase in surface Ld expression as detected by 10 of 11 different monoclonal antibodies (mAbs) recognizing Ld epitopes. These findings suggest that Ld molecules are not saturated with endogenous peptide ligands and thus have accessible binding sites. Exploiting this feature of Ld we demonstrate that the physical association of Ld with ligand is exquisitely specific, indicating that they function in determinant selection. In addition, a non-peptide-bound antigenic variant of Ld was specifically detected with an exceptional mAb designated 64-3-7. In comparison with other Ld molecules, 64-3-7+ Ld molecules are not peptide ligand inducible, are more susceptible to proteolysis, lack beta 2 microglobulin association, and display a slower rate of oligosaccharide maturation. In spite of their deficiencies, the non-ligand-associated 64-3-7 Ld molecules were detected on the surface of all cell types tested; however, they appear not to be recognized by alloreactive cytotoxic T lymphocytes.
Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation.
Authors: Guy-Grand D , Cerf-Bensussan N , Malissen B , Malassis-Seris M , Briottet C , Vassalli P
Published: June 2004

Mouse gut intraepithelial lymphocytes (IEL) consist mainly (90%) of two populations of CD8+ T cells. One bears heterodimeric alpha/beta CD8 chains (Lyt-2+, Lyt-3+), a T cell receptor (TCR) made of alpha/beta chains, and is Thy-1+; it represents the progeny of T blasts elicited in Peyer's patches by antigenic stimulation. The other bears homodimeric alpha/alpha CD8+ chains, contains no beta chain mRNA, and is mostly Thy-1- and TCR-gamma/delta + or -alpha/beta +; it is thymo-independent and does not require antigenic stimulation, as shown by its presence: (a) in nude and scid mice; (b) in irradiated and thymectomized mice repopulated by T-depleted bone marrow cells bearing an identifiable marker; (c) in thymectomized mice treated by injections of monoclonal anti-CD8 antibody, which lead to total depletion of peripheral CD8+ T lymphocytes; and (d) in germ-free mice and in suckling mice. In young nude mice, alpha/alpha CD8 chains, CD3-TCR complexes, and TCR mRNAs (first gamma/delta) are found on IEL, while they are not detectable on or in peripheral or circulating lymphocytes or bone marrow cells. IEL, in contrast to mature T cells, contain mRNA for the RAG protein, which is required for the rearrangement of TCR and Ig genes. We propose that the gut epithelium (an endoderm derivative, as the thymic epithelium) has an inductive property, attracting progenitors of bone marrow origin, and triggering their TCR rearrangement and alpha/alpha CD8 chains expression, thus giving rise to a T cell population that appears to belong to the same lineage as gamma/delta thymocytes and to recognize an antigenic repertoire different from that of alpha/beta CD8+ IEL.
Human immunodeficiency virus (HIV) infection in CD4+ T lymphocytes genetically deficient in LFA-1: LFA-1 is required for HIV-mediated cell fusion but not for viral transmission.
Authors: Pantaleo G , Butini L , Graziosi C , Poli G , Schnittman S M , Greenhouse J J , Gallin J I , Fauci A S
Published: June 2004

In the present study, we demonstrated that expression of the LFA-1 molecule is necessary for cell fusion and syncytia formation in human immunodeficiency virus (HIV)-infected CD4+ T lymphocytes. In contrast, the lack of expression of LFA-1 does not influence significantly cell-to-cell transmission of HIV. In fact, LFA-1- T lymphocytes obtained from a leukocyte adhesion deficiency patient were unable to fuse and form syncytia when infected with HIV-1 or HIV-2, despite the fact that efficiency of HIV infection (i.e., virus entry, HIV spreading, and levels of virus replication) was comparable with that observed in LFA-1+ T lymphocytes. In addition, we provide evidence that LFA-1 by mediating cell fusion contributes to the depletion of HIV-infected CD4+ T lymphocytes in vitro.
Distinct mechanisms of neonatal tolerance induced by dendritic cells and thymic B cells.
Authors: Inaba M , Inaba K , Hosono M , Kumamoto T , Ishida T , Muramatsu S , Masuda T , Ikehara S
Published: June 2004

To assess the role of different types of antigen-presenting cells (APC) in the induction of tolerance, we isolated B cells, macrophages, and dendritic cells from thymus and spleen, and injected these into neonatal BALB/c mice across an Mls-1 antigenic barrier. One week after injection of APC from Mls-1-incompatible mice or from control syngeneic mice, we measured the number of thymic, Mls-1a-reactive, V beta 6+ T cells and the capacity of thymocytes to induce a graft-vs.-host (GVH) reaction in popliteal lymph nodes of Mls-1a mice. Injection of thymic but not spleen B cells deleted thymic, Mls-1a-reactive V beta 6+ T cells and induced tolerance in the GVH assay. The thymic B cells were primarily of the CD5+ type, and fluorescence-activated cell sorter-purified CD5+ thymic B cells were active. Injection of dendritic cells from spleen or thymus also induced tolerance, but the V beta 6 cells were anergized rather than deleted. Macrophages from thymus did not induce tolerance. Dendritic cells and thymic B cells were also effective in inducing tolerance even when injected into Mls-, major histocompatibility complex-incompatible, I-E- mice, but only thymic B cells depleted V beta 6-expressing T cells. Therefore, different types of bone marrow-derived APC have different capacities for inducing tolerance, and the active cell types (dendritic cells and CD5+ thymic B cells) can act by distinct mechanisms.
T cell receptor-mediated selection of functional rat CD8 T cells from defined immature thymocyte precursors in short-term suspension culture.
Authors: Hünig T , Mitnacht R
Published: June 2004

Recent results have indicated that positive and negative repertoire selection act on the major population of CD4,8 double-positive (DP) thymocytes that express 5-10-fold less T cell receptor (TCR) than mature T cells (i.e., they are TCRlow). Since DP cells obtained ex vivo are heterogeneous with regard to their stage within thymic selection, a homogeneous population of virgin DP cells suitable for selection studies was generated in vitro from their immediate precursors, the CD8 single-positive (SP) immature blast cells. To mimic TCR-mediated selection signals, these virgin DP cells were then cultured for another 2 d in the presence of immobilized anti-TCR monoclonal antibodies with or without interleukin 2 (IL-2). Daily monitoring of recovery and phenotype showed that without TCR stimulation, the cells remained DP and became small, TCRlow cells that were lost with a half-life of 1 d, regardless of the presence of IL-2. TCR stimulation resulted in rapid downregulation of CD4 and CD8, maintenance of a larger cell size, and induction of the CD53 antigen that marks mature and CD4,8 double-negative rat thymocytes. In the absence of IL-2, viability decreased as rapidly as without TCR stimulation. Addition of IL-2 rescued TCR-stimulated virgin DP cells and prevented CD8 downregulation, so that 50-80% of input DP cells were recovered after 2 d as CD4-8+53+ cells. After release from modulation, these in vitro generated CD8 SP cells quantitatively upregulated the TCR to the TCRhigh phenotype and were readily induced to proliferate and exhibit cytotoxic T lymphocyte (CTL) activity in a polyclonal readout. Evidence is presented implicating an IL-2 receptor (IL-2R) not containing the p55 chain (i.e., most likely the p70 intermediate affinity IL-2R) in the TCR plus IL-2-driven in vitro differentiation of virgin DP cells towards the mature CD8 SP phenotype.
Transforming growth factor beta suppresses human immunodeficiency virus expression and replication in infected cells of the monocyte/macrophage lineage.
Authors: Poli G , Kinter A L , Justement J S , Bressler P , Kehrl J H , Fauci A S
Published: June 2004

The pleiotropic immunoregulatory cytokine transforming growth factor beta (TGF-beta) potently suppresses production of the human immunodeficiency virus (HIV), the causative agent of the acquired immunodeficiency syndrome, in the chronically infected promonocytic cell line U1. TGF-beta significantly (50-90%) inhibited HIV reverse transcriptase production and synthesis of viral proteins in U1 cells stimulated with phorbol myristate acetate (PMA) or interleukin 6 (IL-6). Furthermore, TGF-beta suppressed PMA induction of HIV transcription in U1 cells. In contrast, TGF-beta did not significantly affect the expression of HIV induced by tumor necrosis factor alpha (TNF-alpha). These suppressive effects were not mediated via the induction of interferon alpha (IFN-alpha). TGF-beta also suppressed HIV replication in primary monocyte-derived macrophages infected in vitro, both in the absence of exogenous cytokines and in IL-6-stimulated cultures. In contrast, no significant effects of TGF-beta were observed in either a chronically infected T cell line (ACH-2) or in primary T cell blasts infected in vitro. Therefore, TGF-beta may play a potentially important role as a negative regulator of HIV expression in infected monocytes or tissue macrophages in infected individuals.
Induced rearrangement of kappa genes in the BLIN-1 human pre-B cell line correlates with germline J-C kappa and V kappa transcription.
Authors: Martin D , Huang R Q , LeBien T , Van Ness B
Published: June 2004

The human pre-B acute lymphoblastic leukemia cell line, BLIN-1, has been previously shown to undergo kappa light chain rearrangement in vitro, making it a valuable resource for analyzing pre-B to B cell differentiation. We have examined the recombination potential of BLIN-1 by characterizing several independently derived kappa-expressing subclones for DNA rearrangement and V kappa gene usage. Analysis of five kappa-expressing subclones (all having the same heavy chain rearrangement) demonstrated independent kappa light chain rearrangement events by DNA hybridization analysis. Northern blot analysis using probes recognizing the four different V kappa families revealed that two subclones used the most proximal V kappa (V kappa IV), one subclone used a V kappa I, and one subclone used a V kappa II. By polymerase chain reaction analyses, we detected transcripts from rearranged V-J-C kappa genes as well as transcripts from germline J-C kappa and V kappa in BLIN-1 cells induced to rearrange the kappa locus. kappa germline transcripts were also detected in normal developing B cell populations in fetal liver and bone marrow. Our collective results indicate that: (a) BLIN-1 can be induced to rearrange the kappa locus, and this correlates with the expression of germline kappa locus transcripts that may play a role in activating or targeting gene rearrangement; and (b) active rearrangement and usage of V genes representing different kappa families suggest that, like in the mouse, repertoire diversification in humans occurs in the presence of a fixed heavy chain rearrangement.
The immunodeficient scid mouse as a model for human lymphatic filariasis.
Authors: Nelson F K , Greiner D L , Shultz L D , Rajan T V
Published: June 2004

The C.B.-17-scid/scid mouse (hereafter referred to as the scid mouse) is homozygous for a recessive mutation at a locus that influences the assembly of intact immunoglobulin and T cell receptor genes. Therefore, scid mice cannot generate functional B or T lymphocytes, are profoundly immunodeficient, and have been reported to be receptive to reconstitution with human immune cells. In the present study, we injected scid mice with infective larvae of the human filarial parasite Brugia malayi. Within 6-10 wk after subcutaneous injection of infective L3 larvae, both male and female worms were observed in various stages of development in 90% of the mice. In animals tested 8 weeks or more after infection, microfilariae were detected in the blood or peritoneal cavity of 52% of the mice examined. Adult worms were observed in the lymphatics of the infected scid mice, where their presence was associated with lymphangitis and lymphangiectasia. These results suggest that the scid mouse model of lymphatic filariasis may be important in investigation of the interaction of the murine, and possibly the human, immune system with the lymphatic filarial parasite.
Different epitope structures select distinct mutant forms of an antibody variable region for expression during the immune response.
Authors: Fish S , Fleming M , Sharon J , Manser T
Published: June 2004

Antibody variable (V) regions that initially differ from one another by only single amino acid residues at VH-D and D-JH segment junctions (termed canonical V regions) can be elicited in strain A/J mice by three different haptens. Among such V regions an amino acid substitution due to somatic mutation is recurrently observed at VH CDR2 position 58, regardless of which of these haptens is used for immunization. This substitution confers upon a canonical V region a generic increase in affinity for all the haptens. Conversely, the type of amino acid substitution at VH position 59 resulting from somatic mutation that is recurrently observed among such V regions changes with the eliciting hapten, in a manner that correlates directly with the cognate affinity increases (or decreases) for hapten conferred by the observed substitutions. This small subregion of VH CDR2 therefore plays a major role in determining both affinity and specificity for antigen. The data confirm that affinity for antigen is of pivotal importance in determining the degree of selection of different mutant forms of a V region. Moreover, during an immune response a sufficiently diverse mutant repertoire can be generated from a single canonical V region to allow adaptation to increase affinity for three different epitopes.
Thalidomide selectively inhibits tumor necrosis factor alpha production by stimulated human monocytes.
Authors: Sampaio E P , Sarno E N , Galilly R , Cohn Z A , Kaplan G
Published: June 2004

Thalidomide selectively inhibits the production of human monocyte tumor necrosis factor alpha (TNF-alpha) when these cells are triggered with lipopolysaccharide and other agonists in culture. 40% inhibition occurs at the clinically achievable dose of the drug of 1 micrograms/ml. In contrast, the amount of total protein and individual proteins labeled with [35S]methionine and expressed on SDS-PAGE are not influenced. The amounts of interleukin 1 beta (IL-1 beta), IL-6, and granulocyte/macrophage colony-stimulating factor produced by monocytes remain unaltered. The selectivity of this drug may be useful in determining the role of TNF-alpha in vivo and modulating its toxic effects in a clinical setting.
Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation.
Authors: Linsley P S , Brady W , Grosmaire L , Aruffo A , Damle N K , Ledbetter J A
Published: June 2004

A successful immune response requires intercellular contact between T and B lymphocytes. We recently showed that CD28, a T cell surface protein that regulates an activation pathway, could mediate intercellular adhesion with activated B cells by interaction with the B7 antigen. Here we show that CD28 is the primary receptor for B7 on activated peripheral blood T cells, that CD28 binds to B7 in the absence of other accessory molecules, and that interaction between CD28 and B7 is costimulatory for T cell activation. To characterize the binding of CD28 to B7, we have produced genetic fusions of the extracellular portions of B7 and CD28, and immunoglobulin (Ig) C gamma 1 chains. 125I-labeled B7 Ig bound to CD28-transfected Chinese hamster ovary (CHO) cells, and to immobilized CD28 Ig with a Kd approximately 200 nM. B7 Ig also inhibited CD28-mediated cellular adhesion. The function of CD28-B7 interactions during T cell activation was investigated with soluble fusion proteins and with B7-transfected CHO cells. Immobilized B7 Ig and B7+ CHO cells costimulated T cell proliferation. Stimulation of T cells with B7+ CHO cells also specifically increased levels of interleukin 2 transcripts. These results demonstrate that the CD28 signaling pathway could be activated by B7, resulting in increased T cell cytokine production and T cell proliferation. Cellular interactions mediated by B7 and CD28 may represent an important component of the functional interactions between T and B lymphoid cells.
Human B cell clones can be induced to proliferate and to switch to IgE and IgG4 synthesis by interleukin 4 and a signal provided by activated CD4+ T cell clones.
Authors: Gascan H , Gauchat J F , Roncarolo M G , Yssel H , Spits H , de Vries J E
Published: June 2004

In the present study, it is demonstrated that cloned surface IgM-positive human B cells can be induced to proliferate and to switch with high frequencies to IgG4 and IgE production after a contact-mediated signal provided by T cell clones and interleukin 4 (IL-4). This T cell signal is antigen nonspecific and is provided by activated CD4+ cells, whereas activated CD8+ or resting CD4+ T cell clones are ineffective. 15-35% of the B cell clones cultured with cloned CD4+ T cells and IL-4 produced antibodies; 35-45% of those wells in which antibodies were produced contained IgE and IgG4. In addition to B cell clones that produced IgG4 or IgE only, B cell clones producing multiple isotypes were observed. Simultaneous production of IgG4 and IgE, IgM, IgE, and IgM, or IgG4 and IgE was detected, suggesting that during clonal expansion switching might occur in successive steps from IgM to IgG4 and IgE. In addition, production of only IgM, IgG4, and IgE during clonal expansion indicates that this isotype switching is directed by the way a B cell is stimulated and that it is not a stochastic process.
Analysis of the HLA-restricted influenza-specific cytotoxic T lymphocyte response in transgenic mice carrying a chimeric human-mouse class I major histocompatibility complex.
Authors: Vitiello A , Marchesini D , Furze J , Sherman L A , Chesnut R W
Published: June 2004

Transgenic murine lines have been constructed that express a chimeric class I molecule composed of the alpha 1 and alpha 2 domains of HLA-A2.1 and the alpha 3, transmembrane, and cytoplasmic domains of H-2Kb. Upon immunization with influenza virus, transgenic mice developed a strong A2.1Kb-restricted cytotoxic T lymphocyte (CTL) response specific for the same matrix protein epitope that serves as the dominant A2.1-restricted determinant in the equivalent human response. Fine specificity analysis of CTL clones using truncated peptides revealed strong similarity between the response repertoire of transgenic mice and that previously reported using influenza-specific A2.1-restricted CTL clones from humans. This suggests that even when considering T cell responses by different species, the alpha 1 and alpha 2 domains of the restriction element play a dominant role in determining the CTL specific repertoire. Thus, substituting the alpha 3 domain of A2.1 with a murine counterpart has permitted development of a transgenic strain that should serve as an excellent model system in studies of HLA-restricted responses.
Detection of interleukin 8 and tumor necrosis factor in normal humans after intravenous endotoxin: the effect of antiinflammatory agents.
Authors: Martich G D , Danner R L , Ceska M , Suffredini A F
Published: June 2004

Interleukin 8 (IL-8), a potent activator of neutrophils, may be important in the early host response to serious Gram-negative infections. IL-8 was measured with other acute phase cytokines (tumor necrosis factor alpha [TNF-alpha], IL-6 and IL-1 beta) in 25 normal humans randomized to receive either intravenous endotoxin alone or endotoxin after oral administration of ibuprofen or pentoxifylline, agents that alter some of the inflammatory responses induced by endotoxin in vitro. TNF immunoreactivity was maximum at 1.5 h, and total TNF (area under the curve) was 4.2- and 4.5-fold greater in subjects given endotoxin/ibuprofen compared to subjects given endotoxin alone (p = 0.026) or endotoxin/pentoxifylline (p = 0.004), respectively. IL-6 levels were maximum at 2-3 h and did not differ among the three groups. No IL-1 beta was detected in any subject. IL-8 levels peaked at 2 h in subjects given either endotoxin alone or endotoxin/pentoxifylline, falling towards baseline by 5 h. Subjects given endotoxin/ibuprofen had a more sustained rise in IL-8 with peak levels 2.8- and 2.5-fold higher at 3 h compared to endotoxin alone (p = 0.048) or endotoxin/pentoxifylline (p = 0.023), respectively. Differences in total IL-8 release among groups approached statistical significance (ANOVA, p = 0.07). This trend reflected the increased release of IL-8 by the subjects receiving ibuprofen compared to pentoxifylline (1.9-fold higher; p = 0.024). This suggests that cyclooxygenase products may provide important negative feedback loops for cytokine production in vivo. Increases in circulating IL-8 are part of the acute inflammatory response of humans to endotoxin. Altered cytokine responses caused by antiinflammatory therapy may have important implications for both host defense and injury during septicemia.
The differential ability of human IgG1 and IgG4 to activate complement is determined by the COOH-terminal sequence of the CH2 domain.
Authors: Tao M H , Canfield S M , Morrison S L
Published: June 2004

Using domain switch chimeric antibodies, we confirm the important role of CH2 in complement activation. In addition, we demonstrate that the structures responsible for the differential ability of human IgG1 and IgG4 to activate complement are located at the COOH-terminal part (from residue 292 to 340) of the CH2 domain. The amino acids in CH2 that might be involved in complement interaction are discussed. While CH3 contributes to efficient complement activation, CH3 from IgG2 and CH3 IgG3 are equally effective.
Correlation between CD8 dependency and determinant density using peptide-induced, Ld-restricted cytotoxic T lymphocytes.
Authors: Alexander M A , Damico C A , Wieties K M , Hansen T H , Connolly J M
Published: June 2004

We have taken advantage of some unique properties of H-2Ld to investigate the determinant density requirements for cytotoxic T lymphocyte (CTL) priming versus effector function and to correlate the determinant density requirements with CD8 dependency. In a previous study (Lie, W.-R., N. B. Myers, J. Gorka, R. J. Rubocki, J. M. Connolly, and T. H. Hansen. 1990. Nature [Lond.]. 344:439), we demonstrated that culturing normal cells with peptides known to be restricted by H-2Ld led to a two- to fourfold increase in surface Ld expression. In the present study, we demonstrate the generation of Ld-restricted, peptide-specific in vitro primary CTL by culturing spleen cells with murine cytomegalovirus or tum- peptide at concentrations previously shown to result in maximum induction of Ld expression. Target cells can be sensitized for recognition by these CTL with lower dose of peptide than are required for the primary sensitization. This demonstrates differences in the determinant density requirements for priming versus effector function. The in vitro primary CTL generated with peptide can weakly lyse target cells that express the determinant endogenously, and CTL lines and clones capable of strong lysis of endogenous expressors are easily obtained. In both cases, target cells treated with exogenous peptide are lysed better than target cells expressing antigen endogenously. This suggested that there are differences in the determinant density of peptide-fed versus endogenous targets. This interpretation was substantiated when it was observed that the level of lysis of target cells expressing endogenous determinants correlated inversely with the amount of peptide required to sensitize targets for recognition by various tum- -specific CTL clones. Furthermore, simultaneous titration of both the peptide used to treat target cells and the antibody to CD8 revealed that the various CTL clones analyzed displayed widely disparate CD8 dependencies. In each case, the CD8 dependency correlated inversely with the determinant density requirement. Therefore, CD8 dependency of CTL is relative, but shows an absolute and quantitative correlation with their dependency on determinant density. These findings suggest that under physiologic conditions, where only low determinant densities are likely to be encountered, all CTL clones will show at least partial CD8 dependency.
Induction of severe granulomatous experimental autoimmune thyroiditis in mice by effector cells activated in the presence of anti-interleukin 2 receptor antibody.
Authors: Braley-Mullen H , Sharp G C , Bickel J T , Kyriakos M
Published: June 2004

Spleen cells from CBA/J mice immunized with mouse thyroglobulin (MTg) and the adjuvant lipopolysaccharide induce experimental autoimmune thyroiditis (EAT) after transfer to recipient mice if they are first activated in vitro with MTg. EAT induced by cells cultured with MTg is generally moderate in severity and is characterized by a thyroid infiltration consisting primarily of mononuclear cells. Addition of the anti-interleukin 2 receptor (IL-2R) monoclonal antibodies (mAbs) M7/20, 3C7, or 7D4 to spleen cell cultures with MTg resulted in a cell population capable of inducing a more severe type of EAT characterized by extensive follicular destruction, granuloma formation, and the presence of multinucleated giant cells. Recipients of cells cultured with MTg and anti-IL-2R mAb also had higher anti-MTg autoantibody responses than recipients of cells cultured with MTg alone. Activation of cells capable of transferring severe granulomatous EAT and increased anti-MTg autoantibody responses required both MTg and M7/20 in culture and required addition of M7/20 within the first 8 h of the 72-h culture period. CD4+ T cells were required for the expression of both the severe granulomatous EAT lesions and the mononuclear cell infiltrates typically observed in murine EAT. The increased anti-MTg autoantibody responses in recipients of cells cultured with MTg and anti-IL-2R mAbs were not restricted to a particular immunoglobulin G (IgG) subclass and included antibody of the IgG1, IgG2A, and IgG2B subclasses. These results suggest that a subset of CD4+ T cells capable of inducing severe granulomatous EAT and increased anti-MTg autoantibody responses is preferentially activated when cells are cultured in the presence of anti-IL-2R mAb. Anti-IL-2R mAb may either prevent activation of cells that induce classical lymphocytic EAT or prevent activation of cells that normally function to downregulate EAT effector T cell activity.
The major histocompatibility complex class II-linked cim locus controls the kinetics of intracellular transport of a classical class I molecule.
Authors: Powis S J , Howard J C , Butcher G W
Published: June 2004

The dominant trans-acting major histocompatibility complex (MHC)-linked class I modifier (cim) locus, previously recognized through its ability to determine altered alloantigenicity of a rat class I molecule, RT1.A3, is shown here to influence class I intracellular transport. The MHC recombinant laboratory rat strains PVG.R1 and PVG.R8 display unusually long retention of RT1.Aa within the endoplasmic reticulum or cis-Golgi. In appropriate F1 hybrid cells heterozygous for RT1.Aa and another class I MHC allele, RT1.Ac, only the RT1.Aa protein is subject to slow transport. The cim gene product therefore shows class I allele specificity in its action, cim appears to be a polymorphic locus whose product is directly involved in the processes of class I MHC assembly and/or intracellular transport.
Inhibition of interleukin 1 (IL-1) binding and bioactivity in vitro and modulation of acute inflammation in vivo by IL-1 receptor antagonist and anti-IL-1 receptor monoclonal antibody.
Authors: McIntyre K W , Stepan G J , Kolinsky K D , Benjamin W R , Plocinski J M , Kaffka K L , Campen C A , Chizzonite R A , Kilian P L
Published: June 2004

Recombinant human interleukin 1 receptor antagonist (IL-1ra) and 35F5, a neutralizing monoclonal antibody (mAb) to the type I mouse IL-1 receptor, were examined for their ability to bind to IL-1 receptors (IL-1Rs) on various types of mouse cells and to block immune and inflammatory responses to IL-1 in vitro and in mice. IL-1ra competed for binding of 125I-IL-1 alpha to type I IL-1R present on EL-4 thymoma cells, 3T3 fibroblasts, hepatocytes, and Chinese hamster ovary cells expressing recombinant mouse type I IL-1R. The IC50 values for IL-1ra binding (ranging from 2 to 4 ng/ml) were similar to those of IL-1 alpha. In contrast, IL-1ra bound with very low affinity (IC50 values ranging from 10 to 200 micrograms/ml) to cells expressing type II IL-1R, i.e., 70Z/3 pre-B cell line and polymorphonuclear leukocytes (PMN) derived from bone marrow and acute inflammatory exudates. The mAb 35F5 bound specifically to type I IL-1R; no inhibition of 125I-IL-1 alpha binding to cells having type II IL-1R was observed with very high concentrations of antibody. While neither IL-1ra nor 35F5 had intrinsic activity in bioassays using T helper D10.G4.1 cells and mouse thymocytes, both agents blocked the ability of IL-1 to stimulate proliferation of these cells. The effects of IL-1ra and 35F5 on acute inflammatory responses in mice were also evaluated. IL-1ra and 35F5 blocked the local accumulation of PMN after intraperitoneal injection of rIL-1 alpha. The response to IL-1 was inhibited when IL-1ra or 35F5 was administered simultaneously with or before administration of IL-1. IL-1ra and 35F5 also blocked PMN accumulation after intraperitoneal injection of lipopolysaccharide or proteose peptone, suggesting IL-1 is important in mediating responses to these agents. In addition, IL-1ra and 35F5 significantly blocked the ability of IL-1 to stimulate egress of PMN from bone marrow, to induce a transient neutrophilia, and to elevate serum levels of hepatic acute phase proteins, IL-6, and corticosterone. Thus, IL-1ra and 35F5 competitively inhibit the binding of IL-1 to the IL-1R on certain cell types. These two IL-1 receptor antagonists act to inhibit biological responses induced by IL-1 and other inflammatory agents.
Molecular characterization of an autoantigen of PM-Scl in the polymyositis/scleroderma overlap syndrome: a unique and complete human cDNA encoding an apparent 75-kD acidic protein of the nucleolar complex.
Authors: Alderuccio F , Chan E K , Tan E M
Published: June 2004

About 50% of patients with the polymyositis/scleroderma (PM-Scl) overlap syndrome are reported to have autoantibodies to a nuclear/nucleolar particle termed PM-Scl. The particle is composed of several polypeptides of which two have been identified as autoantigens. In this report, human cDNA clone coding for the entire 75-kD autoantigen of the PM-Scl particle (PM-Scl 75) was isolated from a MOLT-4 lambda gt-11 library. The deduced amino acid sequence of the cDNA clone represented a protein of 355 amino acids and 39.2 kD; the in vitro translation product of this cDNA migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at approximately 70 kD. The aberrant migration of the polypeptide in SDS-PAGE was shown to be related to the COOH half that was rich in acidic residues. Authenticity of the cDNA coding for PM-Scl 75 was shown by immunoreactivity of PM-Scl sera with in vitro translation products and recombinant fusion proteins encoded by the cDNA. In addition, rabbit antibodies raised to recombinant fusion protein reacted in immunofluorescence, immunoblotting, and immunoprecipitation with the characteristic features displayed by human anti-PM-Scl sera.
The systemic influence of recombinant interleukin 2 on the manifestations of lepromatous leprosy.
Authors: Kaplan G , Britton W J , Hancock G E , Theuvenet W J , Smith K A , Job C K , Roche P W , Molloy A , Burkhardt R , Barker J
Published: June 2004

14 patients with lepromatous leprosy received twice daily injections of 10 micrograms recombinant interleukin 2 (rIL-2), by the intradermal route, in the skin of the back for 8 d (total dose, 160 micrograms). Lymphokine administration was accomplished without drug toxicity, or the development of acute nerve damage. The majority of patients developed nontender axillary lymphadenopathy during the course of treatment. Local injection sites showed progressively larger zones of induration, peaking at 24 h and persisting for many days. Early 12-h reactions were of a macular, erythematous nature and exhibited an increasingly striking diurnal variation. The morning injection sites were three- to fourfold larger in diameter than those placed in the evening (9 am to 9 pm). Systemic manifestations of intradermal rIL-2 administration were noted. Peripheral blood T cells, including CD4+ and CD8+ phenotypes, increased 2-2.5-fold and NK cells increased sixfold. Elevations in [3H]TdR incorporation into peripheral blood mononuclear cells occurred to a variety of mycobacterial antigens, but not to those of Mycobacterium leprae. Within 2 wk, biopsies at sites far removed from the back showed increased infiltration of mononuclear cells in 12 of 14 patients. Immunocytochemistry revealed the presence of newly emigrated CD4+ T cells, monocytes, and dermal CD1+ Langerhans cells. Endothelial cells of small dermal vessels expressed major histocompatibility complex class II determinants on their surface. Transmission electron microscopy of these specimens revealed markedly enlarged endothelial cells with many surface projections extending into the lumen as well as extravasating lymphoid cells. The numbers of acid-fast M. leprae in the peripheral sites were examined by slit smear and in biopsies of matched leprosy lesions taken before and after IL-2 administration. Within 2 mo, slit smears showed a 0.5 log or greater reduction in 12 of 14 patients, with a mean for all patients tested of 0.5 log units. Biopsy specimens showed a 1 log unit or greater reduction in the bacterial index (B.I.) in 6 of 14 patients. Historical controls in this Nepalese population showed a 0.5 log unit reduction after multidrug therapy over a period of 12 mo. Thus, after 8 d of IL-2 injections, a fivefold reduction in B.I. was observed during the first 2 mo of the study. Antibody levels against M. leprae phenolic glycolipid 1 (PGL-1) and lipoarabinomanan B were markedly elevated after IL-2 injections, while PGL-1 antigen levels were reduced. We conclude that the administration of rIL-2 has had a significant effect in decreasing the total body burden of M. leprae.(ABSTRACT TRUNCATED AT 400 WORDS)
Tumor suppression after tumor cell-targeted tumor necrosis factor alpha gene transfer.
Authors: Blankenstein T , Qin Z H , Uberla K , Müller W , Rosen H , Volk H D , Diamantstein T
Published: June 2004

The tumor necrosis factor alpha (TNF-alpha) gene was introduced by retroviral gene transfer into the TNF-alpha-insensitive tumor cell line J558L. Production of 40 pg/ml TNF-alpha by clone J2T12 consistently did not change the growth rate in vitro, but drastically suppressed tumor growth when injected into syngeneic BALB/c mice. Within 2 wk, 90% of the mice inoculated with J558L cells developed a tumor, but none of the mice injected with J2T12 did so. Within the observation period (greater than 3 mo), 60% of the mice inoculated with J2T12 did not develop a tumor. In the other 40% of the mice, tumor manifestation was significantly delayed. Mice injected simultaneously with J2T12 cells and an anti-TNF-alpha monoclonal antibody developed tumors similar to parental J558L cells. Similarly, the tumor-suppressive effects of TNF-alpha were abolished, e.g., by injection of an anti-type 3 complement receptor (CR3) monoclonal antibody that is known to prevent migration of inflammatory cells. These results and the observation of tumor-infiltrating macrophages suggest that lack of tumorigenicity of J2T12 cells is due to the TNF-alpha secretion by the tumor cells and that TNF-alpha acts indirectly by a mechanism that involves chemotactic recruitment and activation of cells, predominantly of macrophages. In contrast, the tumor growth was not affected when, instead of TNF-alpha, interleukin 6 was expressed by J558L cells. Together, our results support the concept of tumor cell-targeted cytokine gene transfer as a tool for cancer treatment, and particularly demonstrate that extremely low doses of TNF-alpha produced by tumor cells are sufficient to inhibit tumor growth without detectable side effects.
Structure of the gene coding for the alpha polypeptide chain of the human complement component C4b-binding protein.
Authors: Rodriguez de Cordoba S , Sanchez-Corral P , Rey-Campos J
Published: June 2004

The human gene coding for the 70-kD polypeptide of the complement regulatory component C4b-binding protein (C4BP alpha) spans over 40 kb of DNA and is composed of twelve exons. Upon transcription in liver, or in Hep-G2 cells, this gene produces a single transcript of 2,262 nucleotides, excepting the poly A tail, that presents an unusually long 5' untranslated region (5' UTR) of 223 nucleotides. The C4BP alpha gene is organized as follows: the first exon codes for the first 198 nucleotides of the 5' UTR. It is separated by a large intron from the second exon including the remaining of the 5' UTR and the coding region for the signal peptide. Each of the eight 60-amino acid repeats (short consensus repeats [SCRs]) that compose the C4BP alpha polypeptide chain is encoded by a single exon, except for the second SCR, which is split in two exons. At the 3' end of the C4BP alpha gene, the twelfth exon codes for the COOH-terminal 57 amino acids of the mature protein, which have no similarities to the SCRs, and the 245 nucleotides of the 3' UTR. Examination of the nucleotide sequence of the first exon revealed an interesting characteristic, strongly suggesting that this exon may specify a functional domain of the C4BP alpha transcript. It includes two in-phase ATG codons, in a different frame respect to that coding the C4BP alpha polypeptide, followed by an in-frame termination codon, also within the first exon. Comparison between mouse and human C4BP alpha transcripts indicates conservation of this structure within the 5' UTR. C4BP is expressed in the liver and is an acute phase protein. A computer search of the genomic sequences upstream the transcription start site demonstrates the presence of potential cis-acting regulatory elements similar to those found in the promoters of other liver-expressed and/or acute phase genes.
Integrin-mediated localization of Bordetella pertussis within macrophages: role in pulmonary colonization.
Authors: Saukkonen K , Cabellos C , Burroughs M , Prasad S , Tuomanen E
Published: June 2004

The adherence of Bordetella pertussis to human respiratory cilia is critical to the pathogenesis of whooping cough but the significance of bacterial attachment to macrophages has not been determined. Adherence to cilia and macrophages is mediated by two large, nonfimbrial bacterial proteins, filamentous hemagglutinin (FHA), and pertussis toxin (PT). PT and FHA both recognize carbohydrates on cilia and macrophages; FHA also contains an Arg-Gly-Asp (RGD) sequence which promotes bacterial association with the macrophage integrin complement receptor 3 (CR3). We determined that virulent B. pertussis enter and survive in mammalian macrophages in vitro and that CR3 is important for this uptake process. We then determined the relative contribution of CR3 versus carbohydrate-dependent interactions to in vivo pulmonary colonization using a rabbit model. B. pertussis colonized the lung as two approximately equal populations, one extracellular population attached to ciliary and macrophage surface glycoconjugates and another population within pulmonary macrophages. Loss of the CR3 interaction, either by mutation of FHA or treatment with antibody to CR3, disrupted accumulation of viable intracellular bacteria but did not prevent lung pathology. In contrast, elimination of carbohydrate-bound bacteria, either by a competitive receptor analogue or an anti-receptor antibody, was sufficient to prevent pulmonary edema. We propose that CR3-dependent localization of B. pertussis within macrophages promotes persistence of bacteria in the lung without pulmonary injury. On the other hand, the presence of extracellular bacteria adherent to cilia and macrophages in carbohydrate-dependent interactions is associated with pulmonary pathology.
The C3b/C4b receptor is recognized by the Knops, McCoy, Swain-langley, and York blood group antisera.
Authors: Moulds J M , Nickells M W , Moulds J J , Brown M C , Atkinson J P
Published: June 2004

Erythrocytes (E) lacking high incidence blood group antigens were screened by an antiglobulin test with a monoclonal antibody to human complement receptor type 1 (CR1; C3b/C4b receptor; CD35). Some examples of E lacking Knops, McCoy, Swain-Langley, and York antigens, a serologically related group, were not agglutinated. Moreover, E of the null phenotype for these same antigens were nonreactive. To further explore this relationship, E expressing these antigens were surface labeled, solubilized, and incubated with the corresponding blood group-specific antisera. CR1 was immunoprecipitated, indicating that the epitopes recognized by each of these antisera are expressed on CR1. E of two individuals, putative null phenotypes for the Knops, McCoy, and Swain-Langley blood group antigens, expressed a very low number of CR1 (less than 30/E; approximately 10% of the normal mean). This observation accounts for their lack of reactivity in the antiglobulin test and their prior designation as null phenotypes. Also, the previously reported low as well as variable expression of CR1 on E explains prior difficulties in the serologic analyses of these blood group antigens.
Analysis of the interaction site for the self superantigen Mls-1a on T cell receptor V beta.
Authors: Pullen A M , Bill J , Kubo R T , Marrack P , Kappler J W
Published: June 2004

Superantigen bound to major histocompatibility complex (MHC) products have been shown to stimulate T cells in a V beta-specific manner. Mouse T cells bearing V beta 8.1 usually respond to the self superantigen, Mls-1a, whereas T cells bearing V beta 8.2a do not. Previously, using site-directed mutational analysis, we identified the residues of natural variants of T cell receptor (TCR) V beta 8.2 that conferred Mls-1a reactivity. These residues are predicted to lie on a beta-pleated sheet of the TCR V beta element, well away from the expected binding site for antigen and MHC proteins. This study was undertaken to determine the effect of glycosylation on this beta-pleated sheet on Mls-1a reactivity and to map the extent of the interaction site on V beta 8.2 for Mls-1a. to Mls-1a, as well as to peptides derived from the conventional protein antigen, chicken ovalbumin. Here we demonstrate that first, N-linked carbohydrate on the lateral surface of V beta blocks the interaction of the TCR V beta with the self superantigen, Mls-1a, but has no effect on the TCR interaction with peptide antigen and MHC, second, that the interaction site for Mls-1a extends over the surface of the solvent-exposed beta-pleated sheet on the side of the TCR, and third, that mutations which affect both superantigen and peptide antigen reactivity lie at the beginning of the first complementarity determining region of V beta, consistent with models of the trimolecular complex of TCR-peptide-MHC.