bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of target cell DNA release by the cytotoxic T lymphocyte granule protease granzyme A.
Authors: Hayes M P , Berrebi G A , Henkart P A
Published: June 2004

The rapid breakdown of target cell DNA during CTL-mediated lysis has been difficult to explain by the granule exocytosis model of cytotoxicity. The involvement of CTL granule proteases in this process was strongly suggested by experiments in which CTL were pretreated with the serine protease inhibitor PMSF, in combination with agents that raise the pH of acidic intracellular compartments. While PMSF pretreatment alone had little effect on target lysis or DNA breakdown, the combination of PMSF and NH4Cl or monensin profoundly reduced target cell DNA release, while little effect was observed on target lysis, as measured by 51Cr release. CTL granule extracts cause release of 125I-DNA from detergent-permeabilized cells. This nuclear DNA-releasing (NDR) activity is inhibited by serine esterase inhibitors that also inhibit the granule BLT-esterase activity, and is specifically immunoabsorbed by antibodies to the CTL granule protease granzyme A. The NDR activity comigrates with BLT-esterase activity during subcellular fractionation, solubilization, gel filtration, and aprotinin-Sepharose affinity chromatography. SDS-PAGE analysis of the affinity-purified product indicates a molecular mass of 60,000 daltons under non-reducing conditions, which moves to 30,000 daltons upon reduction, consistent with previously reported behavior of granzyme A. When the purified material was reduced and alkylated, both esterase and NDR activities comigrated at 30,000 daltons upon gel filtration. Although fully lytic concentrations of purified LGL granule cytolysin alone failed to induce target cell DNA release, a combination of purified granzyme A and the cytolysin induces substantial DNA release.
Xenogeneic skin graft rejection is especially dependent on CD4+ T cells.
Authors: Pierson R N , Winn H J , Russell P S , Auchincloss H
Published: June 2004

B6 mice were treated in vivo with anti-CD4, anti-CD8, or both anti-T cell antibodies together in an effort to prolong xenogeneic compared with allogeneic skin graft survival. Mice treated with anti-CD4 antibody showed prolonged survival of xenogeneic monkey or rabbit skin even after they had rejected whole MHC-disparate allogeneic mouse skin. Furthermore, the addition of cyclosporine was synergistic with the anti-CD4 antibody in prolonging graft survival. These results suggest that the cell-mediated response to xenogeneic antigens is especially dependent on CD4+ lymphocytes, a feature shared by the response to allogeneic minor histocompatibility antigens. In addition, the results suggest a possible approach to clinical immunosuppression for some forms of xenogeneic transplantation.
Structure of idiotopes associated with antiphenylarsonate antibodies expressing an intrastrain crossreactive idiotype.
Authors: Haba S , Lascombe M B , Poljak R J , Nisonoff A
Published: June 2004

We have explored the structural basis of idiotopes associated with the major idiotype (CRIA) of A/J anti-p-azobenzenearsonate antibodies, with emphasis on the regions of contact with anti-idiotypic antibody. The analysis was facilitated by a recent description of the three-demensional structure of the Fab portion of a CRIA-related antibody molecule. Direct binding measurements failed to reveal idiotopes associated exclusively with the L chain. However, the L chain participated in the formation of approximately 80% of the idiotopes recognized by polyclonal anti-Id. This indicates that multiple complementarity-determining regions (CDRs) participate in the formation of idiotopes. The affinity of anti-Id for CDRs on L chains must be appreciable but insufficient to permit direct binding (i.e., less than approximately 10(4) M-1). Approximately 20-35% of polyclonal anti-Id reacted with high affinity with H chains recombined with non-CRIA-related L chains. This interaction was found to involve the D region as well as one or both CDRs in the VH segment, again indicating the contribution of multiple CDRs. It is suggested that a typical idiotope may be similar in size to that of protein epitopes whose three-dimensional structures are known; such epitopes comprise a substantial fraction of the surface area occupied by the CDRs of an antibody. The expression of an idiotope recognized by the mAb AD8, which interacts with the VH segment, was found to be unaffected by major changes in the neighboring D and VL regions. This observation is relevant to efforts to predict three-dimensional structure from the amino acid sequence of CRIA+ molecules.
Molecular analysis of original antigenic sin. I. Clonal selection, somatic mutation, and isotype switching during a memory B cell response.
Authors: Fish S , Zenowich E , Fleming M , Manser T
Published: June 2004

To determine how the memory B cell population elicited to one epitope might be used in immune responses to other, structurally related epitopes, we explored the phenomenon of original antigenic sin. Strain A/J mice reproducibly respond to immunization with p-azophenylarsonate (Ars) by production of anti-Ars antibodies encoded predominantly by a single VH gene segment (VHIdCR). The structural analogue of Ars p-azophenylsulfonate (Sulf) fails alone to elicit such V regions, but can do so in A/J mice previously immunized with Ars, providing a means to specifically examine B cells capable of responding secondarily to a crossreactive antigen (i.e., memory cells). VHIdCR-expressing hybridomas were derived from the Ars-primed, Sulf-boosted original antigenic sin response of A/J mice at various times after Ars priming, and the properties of the antibodies they express and the structure of the genes encoding these antibodies were characterized. The data obtained support the following conclusions: (a) The Ars-induced memory B cell population capable of being crossreactively stimulated by Sulf is largely formed from a small fraction of all B cells participating in the anti-Ars primary response that express somatically mutated V regions; (b) the antibody repertoire and clonal composition of this population are stable over long periods of time; (c) memory B cells are capable of clonal expansion in the absence of a high rate of V gene somatic mutation; (d) the activation requirements for clonal selection of memory, versus naive B cells appear to differ; and (e) a major fraction of Ars-induced memory B cells express either IgM or IgG3 prior to and during the initial stages of the sin response.
MHC antigen induction by interferon gamma on cultured mouse pancreatic beta cells and macrophages. Genetic analysis of strain differences and discovery of an "occult" class I-like antigen in NOD/Lt mice.
Authors: Leiter E H , Christianson G J , Serreze D V , Ting A T , Worthen S M
Published: June 2004

This study provides a basis for understanding the wide variations reported in the literature in IFN-gamma inducibility of class II MHC antigens on murine beta cells. Inducibility is not an intrinsic property of all mouse beta cells, but instead depends upon strain- (and tissue-) specific response modifying factors. This was demonstrated by comparison of constitutive and IFN-gamma-induced class I and class II MHC gene products on cultured islet cell monolayers. Islet cultures were established from autoimmune diabetes-prone NOD/Lt mice, diabetes-resistant NON/Lt and CBA/J mice, as well as F1 hybrids between these latter two strains and NOD/Lt. Cultures of peritoneal macrophages (M phi) from each strain were established as controls. After 3 wk of culture (with incubation in the presence or absence of IFN-gamma during the last 6 d), constitutive expression as well as IFN-gamma induction of class I MHC antigen expression was demonstrated on NOD/Lt and NON/Lt islet cells by antibody plus complement-mediated cytotoxicity. Although CBA/J islets and M phi did not maintain constitutive class I or class II antigen expression in culture in the absence of IFN-gamma, class I H-2Kk antigen was IFN-gamma inducible. Whereas IFN-gamma-induced class II I-Ak antigen on CBA/J M phi, it failed to induce this antigen on CBA/J islets. In contrast, I-A antigens were IFN-gamma inducible on NOD/Lt and NON/Lt islets and M phi. In (CBA x NOD)F1 hybrids, loss of IFN-gamma inducibility of the I-ANOD product established that suppression was mediated by a trans-acting factor from the CBA/J genome. In the course of these studies, IFN-gamma inducibility of a crossreactive occult class I-like antigen on both NOD/Lt islet cell and M phi cultures was unexpectedly detected when mAb 28-13-3 (public specificity 39, reactive with H-2Kb,f) was used as a negative control. Although not detectable by cytofluorographic analysis of freshly isolated NOD/Lt splenic leukocytes, occult antigen could be induced on NOD/Lt peritoneal macrophages (M phi) cultured for 3 d in IFN-gamma. Time course of induction showed the occult antigen to be distinct from NOD/Lt class I and II gene products. In both islet cell and M phi cultures established from (CBA x NOD)F1 hybrids, trans-suppressive factor(s) from the CBA/J genome not only suppressed IFN-gamma-induced expression of I-ANOD, but additionally suppressed occult antigen induction. Backcross of F1 to both parental strains indicated that the occult locus was on Chr 17, tightly linked to MHC.(ABSTRACT TRUNCATED AT 400 WORDS)
Structural analysis of the human immunodeficiency virus-binding domain of CD4. Epitope mapping with site-directed mutants and anti-idiotypes.
Authors: Sattentau Q J , Arthos J , Deen K , Hanna N , Healey D , Beverley P C , Sweet R , Truneh A
Published: June 2004

The CD4 molecule, a differentiation marker expressed primarily by T lymphocytes, plays an important role in lymphocyte activation. CD4 is also the receptor for HIV. A number of recent studies have localized the high affinity binding site of the HIV envelope glycoprotein, gp120, to the NH2-terminal (V1) domain of CD4, a region with sequence and predicted structural homology with Ig kappa chain V domains (V kappa). In this report, we show that V1 bears structural similarities with V kappa regions through detailed epitope mapping of 26 CD4 mAbs. The binding sites of these mAbs were initially defined relative to one another by crossblocking analysis and were then localized to specific domains of CD4 in blocking studies with truncated, soluble CD4 proteins. The epitopes within the V1 domain were mapped in detail with a panel of 17 substitution mutants, and the specificities of several mAbs that appear to recognize very similar epitopes were examined in crossblocking studies with anti-idiotype antibodies. The location of the epitopes is consistent with a V kappa-like structure of V1. Most of the epitopes lie within regions of predicted exposed loops. A number of these epitopes span discontinuous residues in the linear sequence that lies in close proximity in an Ig fold. Alignment of CD4 V1 with the Ig V kappa chains places these epitopes within stretches corresponding to the complimentarity-determining regions. This epitope analysis is relevant for a vaccine strategy for HIV based on anti-idiotype antibodies to CD4 mAbs and for studies with CD4 antibodies on the role of CD4 in T lymphocyte activation.
Class I major histocompatibility complex-restricted T lymphocyte recognition of the influenza hemagglutinin. Overlap between class I cytotoxic T lymphocytes and antibody sites.
Authors: Sweetser M T , Braciale V L , Braciale T J
Published: June 2004

The influenza hemagglutinin is a critical regulator of disease expression during influenza virus infection and serves as a major target for the host immune response to this pathogen. In this report, we have analyzed an immunodominant site on the hemagglutinin (residues 202-221) recognized by murine class I MHC-restricted T lymphocytes. This analysis has revealed evidence for the duplication of a T cell recognition site within the region 202-221. We have also identified critical amino acids necessary for class I-restricted T cell recognition within these two epitopes. In addition, we provide evidence that a site on the influenza hemagglutinin recognized by neutralizing antibody directly overlaps with an epitope recognized by class I MHC-restricted CTL.
Soluble cytokine receptors are present in normal human urine.
Authors: Novick D , Engelmann H , Wallach D , Rubinstein M
Published: June 2004

Affinity chromatography of crude human urinary proteins on either human rIL-6, human rIFN-gamma, or anti-IFN-gamma-R mAb yielded the two respective soluble receptors in significant quantities. A single sequence of 30 amino acid residues was obtained by NH2-terminal microsequencing of the protein peak purified in tandem by affinity chromatography on an IL-6 column and reversed-phase HPLC. This sequence was identical to the predicted NH2-terminal sequence of IL-6-R as previously reported. Analysis of the eluted proteins from both IFN-gamma and anti-IFN-gamma-R columns by inhibition of solid phase RIA, ELISA, SDS-PAGE, and Western blotting proved the existence of soluble IFN-gamma-R in normal urine. Our finding, together with the already known presence of urinary TNF binding proteins and a soluble IL-2-R both in plasma and in urine, indicates that release of soluble cytokine receptors into body fluids is a general phenomenon that occurs under normal physiological conditions.
Stimulation of mast cell chemotaxis by interleukin 3.
Authors: Matsuura N , Zetter B R
Published: June 2004

PWM-activated spleen cell-conditioned medium (SCCM) and a variety of purified hematopoietic growth factors were tested for their ability to stimulate chemotaxis of mouse connective tissue mast cells (CTMC). Of the agents tested, only IL-3 and SCCM promoted mast cell chemotaxis. Neither IL-2, IL-4, GM-CSF, nor endotoxin had any significant mast cell chemotactic activity. Neutralizing antibodies to mouse IL-3 blocked greater than 90% of the chemotactic activity of SCCM, suggesting that IL-3 is the predominant mast cell chemotactic factor produced by activated spleen cells. Our results demonstrate that mature connective tissue type mast cells are capable of moving toward a gradient of spleen cell-derived IL-3 and suggest that movement of mature mast cells toward lymphokines may influence the accumulation of mast cells at sites of inflammatory or immune reactions.
The severe combined immunodeficiency (scid) mouse. A laboratory model for the analysis of Lyme arthritis and carditis.
Authors: Schaible U E , Kramer M D , Museteanu C , Zimmer G , Mossmann H , Simon M M
Published: June 2004

We report that the spirochete B. burgdorferi induces progressive polyarthritis and carditis in mice with severe combined immunodeficiency syndrome (scid) but not in normal C.B-17 mice. The onset and severity of the disease were dependent on (a) the viability; (b) the infectivity; and (c) the dose of inoculated B. burgdorferi organisms. Infective spirochetes were isolated from both blood and joints of inoculated scid mice. These findings suggest that B. burgdorferi-induced chronic arthritis and carditis in mice develops independently of lymphocyte function and makes the scid mouse an attractive laboratory model to study the role of the immune system in experimental Lyme Borreliosis.
Analysis of peptide binding patterns in different major histocompatibility complex/T cell receptor complexes using pigeon cytochrome c-specific T cell hybridomas. Evidence that a single peptide binds major histocompatibility complex in different conformations.
Authors: Bhayani H , Paterson Y
Published: June 2004

The interaction of TCR, antigen, and MHC complex has been analyzed using synthetic peptide antigens and a series of single amino acid-substituted analogues. Two similar antigens, mouse cytochrome c (mcyt c) and pigeon cytochrome c (pcyt c), elicit T cell responses in strains of mice bearing MHC class II Ek beta Ek alpha (B10.A), Eb beta Ek alpha [B10.A(5R)], and Es beta Ek alpha [B10.S(9R)]. The immunogenic regions of these antigens are located in the peptide sequence p88-104 for pcyt c and m88-103 for mcyt c. The limited T cell repertoire for these antigens is comprised of four groups of T cell phenotypes that have very few differences in their TCR gene make up. In this paper, we examine the diversity in their fine specificity for each of the antigens, m88-103 and p88-104, complexed with each of the I-Ek haplotypes. Epitopes, i.e., residues that interact with the TCR, and agretopes, i.e., residues in the MHC-binding site, were assigned for the two peptide antigens in the presence of APC bearing E beta kEk alpha, Eb beta Ek alpha, or Eb beta Ek alpha using T cell hybridomas of the phenotypes I, IIIa, and IV. From our results, we conclude that first, the substitution of any residue between 95 and 104 of the cytochrome c peptide changed the antigenic potency of the peptide for at least one of the hybridomas. Second, each T cell type has a different recognition pattern of epitopes and agretopes for a particular antigen-MHC complex, thus, ruling out a static model of T cell recognition, which assigns certain, invariant agretopic residues to the peptide by which it interacts with the MHC molecule independently of the TCR. Third, the same T cell hybridoma responded to the antigens differently when presented on various MHC molecules, implying that overall changes in the MHC groove, as displayed by the three haplotypes, may affect the efficiency in binding the peptide. Fourth, since most of the residues are used as epitopes by at least one of the T cell specificities, the peptide appears to be recognized in a different conformation by each T cell hybridoma phenotype; and, finally, the epitopic and agretopic residues do not segregate, for any one of the T cell specificities, in such a way that suggests they are recognized in a helical conformation. In summary, our results suggest that a single peptide may generate diversity in the T cell response by virtue of its conformational flexibility within the TCR-MHC-antigen complex.
Macrophage colony-stimulating factor (rM-CSF) stimulates pinocytosis in bone marrow-derived macrophages.
Authors: Racoosin E L , Swanson J A
Published: June 2004

Incubation of murine bone marrow-derived macrophages (BMM) in medium containing recombinant macrophage colony-stimulating factor (rM-CSF) stimulated influx, efflux, and the net accumulation of the fluid-phase pinocytic marker, lucifer yellow (LY). Stimulation was dose dependent, occurred within 5 min of addition of the growth factor, and was sustained. Previous experiments had shown that BMM treated with PMA were stimulated to accumulate LY, but compared with rM-CSF-treated cells, the onset of stimulation in PMA-treated macrophages was slower. In further comparisons of rM-CSF- and PMA-stimulated LY accumulation, it was found that rM-CSF-stimulated pinocytosis could be abolished by pretreatment with 0.5 mg/ml trypsin, whereas neither unstimulated nor PMA-stimulated LY accumulation was affected by trypsin pretreatment. These findings indicate that the rM-CSF response was initiated at the cell surface, while the PMA response occurred via intracellular (or trypsin-resistant) receptors. However, once initiated, the pinocytic responses elicited by either agent were very similar. First, rM-CSF-treated cells, like PMA-treated cells, showed extensive ruffling and formation of large phase-bright pinosomes. Second, both rM-CSF- and PMA-stimulated LY accumulation could be inhibited by treatment of cells with the cytoskeleton destabilizing drugs nocodazole, colchicine, or cytochalasin D. Finally, rM-CSF, like PMA, was found to stimulate efflux of LY from cells preloaded with the dye. Thus, both rM-CSF and PMA stimulate the net rate of solute flow through the macrophage endocytic compartment.
Colony morphology of piliated Neisseria meningitidis.
Authors: Blake M S , MacDonald C M , Klugman K P
Published: June 2004

An association between piliation and colony morphology has not been observed for the meningococcus. We have found that growth of meningococci overnight at 30 degrees C in a candle extinction jar allows observation of distinct colonial phenotypes correlated to the presence or absence of piliation and the expression of opacity-associated proteins. These phenotypes are similar to those observed in gonococci grown overnight at 37 degrees C.
Analysis of a novel VHS107 haplotype in CLA-2 and WSA mice. Evidence for gene conversion among IgVH genes in outbred populations.
Authors: Ferguson S E , Cancro M P , Osborne B A
Published: June 2004

Gene conversion has been suggested as the basis for many VH allelic differences, particularly in the murine VHS107 family. Whether conversion among IgVH genes is likely to have occurred in outbred populations has not been directly addressed. The CLA-2/Cn and WSA strains, which were recently and independently derived from a feral population exhibiting low responsiveness to PC, provide the opportunity to approach this question. In previous studies, the heavy chain cDNA sequence of a PC-specific hybridoma derived from CLA-2/Cn suggested gene conversion events within the VHS107 family. Accordingly, we have examined the germline VHS107 genes of CLA-2/Cn and WSA. The results indicate that: (a) The CLA-2 and WSA strains bear an identical but novel VHS107 family haplotype, which lacks a V3 element and contains a V1, a V13, and two V11 genes; (b) low PC responsiveness in these populations is unlikely due to an inability to express the V1 member of the VHS107 gene family; and (c) when compared with the other known VHS107 haplotypes, the proportion of differences consistent with gene conversion greatly exceeds that expected by random base substitution. Thus, gene conversion events appear to have occurred with considerable frequency in the evolution of the murine VHS107 family, especially among the V3, V13, and V11 members.
Prevention of endogenous leukotriene production during anaphylaxis in the guinea pig by an inhibitor of leukotriene biosynthesis (MK-886) but not by dexamethasone.
Authors: Guhlmann A , Keppler A , Kästner S , Krieter H , Brückner U B , Messmer K , Keppler D
Published: June 2004

Leukotriene C4 (LTC4) underwent rapid elimination from the circulating blood and was extensively converted to LTD4 within the vascular space of the guinea pig. To mimic the elimination and metabolism of endogenous LTC4 generated during anaphylaxis, 14,15-3H-labeled LTC4 was infused intravenously over a period of 15 min, leading to a recovery in bile of 85% of the infused LT radioactivity within 2 h. Corresponding to the tracer studies, LTD4 and, to a lesser extent, LTC4 were the predominant endogenous cysteinyl LTs in guinea pig bile. The biliary production rate of endogenous LTD4 increased from 0.3 +/- 0.1 to 6.2 +/- 1.8 pmol x min-1 x kg-1 (p less than 0.001) during anaphylactic shock induced by intravenous injection of OVA (0.2 mg/kg) into sensitized guinea pigs. A novel LT biosynthesis inhibitor (MK-886; 10 mg/kg, i.v., 15 min before antigen challenge) suppressed the antigen-induced cysteinyl LT production by greater than 92% (p less than 0.001). This inhibition of systemic LTC4 formation was associated with a complete protection against lethal anaphylactic shock in animals pretreated in addition with the H1 receptor antagonist pyrilamine. Pretreatment with either the inhibitor of LT synthesis or the histamine receptor antagonist reduced the lethality during anaphylactic shock from 100 to 60 and 78%, respectively. In artificially ventilated, pyrilamine-pretreated animals, the antigen-induced decrease in dynamic lung compliance and the rise in hematocrit were significantly reduced (p less than 0.05) by pretreatment with the inhibitor of LT synthesis. Dexamethasone at high doses (10 mg/kg, i.p., once daily for 7 d, or in a single dose of 10 mg/kg, i.v., 3.5 h before challenge) had no inhibitory effect on LT generation during anaphylaxis in vivo. However, in resident peritoneal macrophages, harvested from these dexamethasone-treated sensitized guinea pigs and stimulated with zymosan, both cysteinyl LT and 6-keto-PGF1 alpha formation were strongly suppressed. These studies indicate an important role of cysteinyl LTs in systemic anaphylaxis in vivo and demonstrate the blockade of anaphylactic LT generation by a novel inhibitor of LT biosynthesis (MK-886) but not by dexamethasone.
Identification of an endogenous membrane anchor-cleaving enzyme for group A streptococcal M protein. Its implication for the attachment of surface proteins in gram-positive bacteria.
Authors: Pancholi V , Fischetti V A
Published: June 2004

How streptococcal M protein or other surface proteins of gram-positive bacteria are anchored to the cell is poorly understood. Previously, we reported that M protein released after cell wall removal with a muralytic enzyme lacked the COOH terminal hydrophobic amino acids and charged tail predicted from DNA sequence. An endogenous membrane anchor-cleaving enzyme has now been identified with the ability to release M protein from isolated streptococcal protoplasts. At pH 5.5 in the presence of 30% raffinose, the streptococcal cell wall may be removed with a muralytic enzyme without releasing M protein from the resulting protoplasts indicating that the M molecule is attached through the bacterial cytoplasmic membrane. Release of M molecules occurs when the M protein-charged protoplasts are placed in raffinose buffer at pH 7.4. Although Zn2+, Cd2+, Ca2+, PHMB, and pHMPS inhibit the activity of the releasing enzyme, the blocking activity of Zn2+, Cd2+, and Ca2+ are reversible while PHMB and pHMPS are irreversible. PHMB-treated protoplasts are unable to release M protein at pH 7.4. However, M protein is liberated from these protoplasts when mixed with those prepared from M- streptococci serving as an enzyme source. The supernatant from M- protoplasts is unable to release M protein from PHMB-inactivated M+ protoplasts, confirming that the anchor-cleaving enzyme is membrane bound. Thus, the M protein releasing activity appears to be the result of a thiol-dependent anchor-cleaving enzyme. Streptococcal membranes treated with sodium carbonate and Triton X-114 still retain the M protein verifying that it is an integral membrane molecule. Evidence also is presented indicating significant sequence similarity between M protein and certain GPI-anchored proteins in the region responsible for protein anchoring.
Gamma interferon limits access of Listeria monocytogenes to the macrophage cytoplasm.
Authors: Portnoy D A , Schreiber R D , Connelly P , Tilney L G
Published: June 2004

The effect of rIFN-gamma and rTNF on the fate of hemolytic and nonhemolytic (hly-) Listeria monocytogenes in cultured mouse peritoneal macrophages was investigated. In untreated macrophages, approximately 80% of the hemolytic bacteria were killed during the first 2 h of incubation, but the survivors doubled between two and three times. In rIFN-gamma-treated macrophages, although the bacterial killing was identical to the controls during the first 2 h, there was no subsequent bacterial growth, and bactericidal activity continued for the duration of the experiment. rTNF has no affect by itself, but acted synergistically with rIFN-gamma to promote bacterial killing. Infected macrophages with or without rIFN-gamma were examined by EM. The results clearly showed that the role of rIFN-gamma was to prevent access of L. monocytogenes to the macrophage cytoplasm, which would prevent cell-to-cell spread of the bacteria. In addition, rIFN-gamma-treated macrophages exhibited enhanced digestive capacity of the intracellular bacteria.
CD19 is functionally and physically associated with surface immunoglobulin.
Authors: Pesando J M , Bouchard L S , McMaster B E
Published: June 2004

The pan-B and B cell-specific sIg and CD19 antigens are functionally and physically associated in the presence of anti-Ig mAb. Incubation of B cells with anti-Ig antibodies causes rapid, specific, reversible, concentration-dependent, and unidirectional comodulation of CD19 on every mature B cell studied. Comodulation is produced by mAbs specific for the gamma, mu, kappa, and lambda chains of Ig, and by at least one idiotype-specific mAb. Comodulation is observed using 15 CD19-specific mAbs that detect at least three different CD19 epitopes. Of 18 surface antigens studied, only CD19 is comodulated. Loss of sIg and CD19 occurs concurrently during anti-Ig modulation and demonstrates a comparable dependence on anti-Ig concentration, suggesting that these are parallel rather than serial events. Incubation with anti-Ig specifically cocaps and suggests internalization of anti-CD19 mAb. Comodulation of sIg and CD19 by anti-Ig but not anti-CD19 mAbs suggests that ligand binding enables sIg to then interact with CD19. We propose that CD19 is a component of the B cell antigen receptor and suggest that it could facilitate signal transduction by sIg-antigen complexes.
In individual T cells one productive alpha rearrangement does not appear to block rearrangement at the second allele.
Authors: Kumar V , Urban J L , Hood L
Published: June 2004

The T cell lymphoma line BW5147 has rearranged TCR alpha chain genes segments on both the homologous chromosomes: one is functional (V alpha 1) and the second (V alpha 16.1) is a pseudogene. The extreme 3' position of the V alpha 16.1 gene segment in the V alpha locus allows us to recognize rearrangements of most V alpha gene segments using the V alpha 16 probe as a marker. The absence of the genomic V alpha 16.1 gene fragment in mature thymocytes, antigen-specific T cells, and in more than two-thirds of the peripheral T cells suggests that most T lymphocytes rearrange both alpha loci. It appears that productive alpha chain rearrangement on one allele probably does not block a subsequent rearrangement on the other alpha locus.
In vivo treatment with monoclonal antibody 3.2.3 selectively eliminates natural killer cells in rats.
Authors: van den Brink M R , Hunt L E , Hiserodt J C
Published: June 2004

We recently described a mAb 3.2.3 (IgG1), that recognizes a 60-kD dimeric molecule expressed exclusively on fresh and rIL-2-activated NK cells and polymorphonuclear cells. mAb 3.2.3 enhances cytolytic activity of NK cells against selected FcR+ tumor target cells by reverse antibody-dependent cellular cytotoxicity (ADCC), indicating that it recognizes an important triggering site on NK cells. The in vivo treatment of F344 rats with mAb 3.2.3 intraperitoneally completely and selectively eliminated NK/ADCC function in the spleen and peripheral blood for up to 10 d after treatment. Total numbers and percentages of T cells, monocytes, or PMN were not decreased and T cell function, as determined by Con A stimulation, was not affected. The reduction in NK function was associated with a decrease in the numbers of LGL and the expression of other NK-related cell surface markers including CD2, CD8, and asialo GM1. Depletion of NK cells with 3.2.3 markedly decreased the survival of F344 rats injected intravenously with MADB106 mammary adenocarcinoma cells, but did not affect the subcutaneous growth of MADB106 tumors. These results indicate that mAb 3.2.3 (in contrast to anti-asialo GM1 and OX8, which are less selective markers) will be useful for studies on the functional role of NK cells in vivo as well as their in vivo differentiation and origin from 3.2.3- precursors.
Anti-DNA antibodies from autoimmune mice arise by clonal expansion and somatic mutation.
Authors: Shlomchik M , Mascelli M , Shan H , Radic M Z , Pisetsky D , Marshak-Rothstein A , Weigert M
Published: June 2004

The proximate cause of autoantibodies characteristic of systemic autoimmune diseases has been controversial. One hypothesis is that autoantibodies are the result of polyclonal nonspecific B cell activation. Alternatively, autoantibodies could be the result of antigen-driven B cell activation, as observed in secondary immune responses. We have approached this question by studying monoclonal anti-DNA autoantibodies derived from unmanipulated spleen cells of the autoimmune MRL/lpr mouse strain. This analysis shows that anti-DNAs, like rheumatoid factors (19), are the result of specific antigen-driven stimulation. In addition, correlation of sequences with fine specificity shows that: (a) somatic mutations can cause specificity for dsDNA and that such mutations are selected for; (b) arginine residues play an important role in determining specificity; and (c) anti-idiotypes that recognize the majority of anti-DNA are probably not specific for any one family of V regions.
Anti-neutrophil cytoplasm antibodies in Wegener's granulomatosis recognize an elastinolytic enzyme.
Authors: Lüdemann J , Utecht B , Gross W L
Published: June 2004

The target antigen of anti-neutrophil cytoplasm antibodies (ACPA; also known as ANCA) was isolated by affinity chromatography from supernatants of human neutrophils, stimulated with phorbol ester to induce degranulation. Sequence analysis of the antigen revealed 17 NH2-terminal amino acids (IVGGHEAQPHIRPIYMA), which have considerable sequence homology with known serine proteinases. Investigation of the enzymatic activity showed that the antigen is a neutral serine proteinase that is able to cleave elastin. Since the molecular weight of the antigen, its substrate specificity, and its inhibitor profile reported in this study are identical with those reported recently for proteinase 3, we conclude that ACPA are most probably directed against proteinase 3.
Intrathymic clonal deletion of V beta 6+ T cells in cyclophosphamide-induced tolerance to H-2-compatible, Mls-disparate antigens.
Authors: Eto M , Mayumi H , Tomita Y , Yoshikai Y , Nomoto K
Published: June 2004

When C3H (H-2k, Mls-1b) mice were primed intravenously with 10(8) viable spleen cells from AKR (H-2k, Mls-1a) and treated intraperitoneally with 200 mg/kg of cyclophosphamide (CP) 2 d later, not only a long-lasting skin allograft tolerance but also a tolerance in mixed lymphocyte reaction to Mls-1a-encoded antigens was established. The cellular mechanisms of CP-induced tolerance were examined by assessing the V beta 6-bearing T cells that are strongly correlated with reactivity to Mls-1a-encoded antigens bound to MHC class II molecules. At the relatively early stage (2 or 5 wk) after the CP treatment, CD4+-V beta 6+ T cells of C3H origin were preferentially eliminated in the lymph nodes of the tolerant mice, whereas CD8+-V beta 6+ T cells remained. On the other hand, neither CD4+CD8- nor CD4-CD8+ thymocytes bearing a high density of V beta 6 was detected in the chimeric thymus. Namely, in the thymus of the tolerant C3H mice, neither mixed chimerism nor the clonal deletion of the V beta 6-bearing T cells was observed on day 14, whereas both of them were observed on day 35. The clonal deletion and mixed chimerism in the thymus were lasting for greater than 10 wk after the CP treatment. Expression of V beta 6 on the peripheral T cells in the tolerant C3H mice gradually reduced in the process of time. These results strongly suggested that the clonal deletion in the thymus was one of the essential mechanisms in the CP-induced tolerance system.
The exit of Trypanosoma cruzi from the phagosome is inhibited by raising the pH of acidic compartments.
Authors: Ley V , Robbins E S , Nussenzweig V , Andrews N W
Published: June 2004

The protozoan parasite Trypanosoma cruzi can infect many distinct mammalian cell types. The parasites enter cells through the formation of phagocytic vacuoles, but later are found free in the cytosol, where they multiply as amastigotes. Using transmission electron microscopy we found that within 2 h after infection 70% of the parasites, including examples of both mammalian forms (trypomastigotes and amastigotes), were inside partially disrupted vacuoles or free in the cytosol. We demonstrated that the pH of vacuoles containing recently interiorized parasites is acidic, through immunocytochemical localization of the acidotropic compound DAMP (18) in their interior. Increasing the vacuolar pH with chloroquine, ammonium chloride, methylamine, or monensin significantly inhibited the escape of the parasites into the cytosol. These results are compatible with the hypothesis that an acid-active hemolysin of T. cruzi (15) might be involved in the escape mechanism.
CD8 is required during positive selection of CD4-/CD8+ T cells.
Authors: Zúñiga-Pflücker J C , Jones L A , Longo D L , Kruisbeek A M
Published: June 2004

Interactions between self-MHC molecules and T cells are necessary for the proper development of mature T cells, in part due to an absolute requirement for self-MHC-TCR interactions. Recently, we showed that CD4-mediated interactions also participate in shaping the T cell repertoire during thymic maturation. We now examine the possible role of the CD8 molecule during in vivo T cell development. Our results demonstrate that perinatal thymi treated with intact anti-CD8 mAb fail to generate CD8 single-positive T cells, while the generation of the other main phenotypes remains unchanged. Most importantly, the use of F(ab')2 anti-CD8 mAb fragments gave identical results, i.e., lack of generation of CD4-/CD8+ cells, with no effect on the generation of CD4+/CD8+. Furthermore, selective blocking of one CD8 allele with F(ab')2 mAbs in F1 mice expressing both CD8 alleles did not interfere with the development of CD4-/CD8+ cells, demonstrating that the absence of CD8+ T cells in homozygous mice is not due to depletion, but rather is caused by a lack of positive selection. This is most likely attributable to a deficient CD8-MHC class I interaction. Our findings strongly advocate that CD8 molecules are vital to the selection process that leads to the development of mature single-positive CD8 T cells.
Generation of the neutrophil-activating peptide NAP-2 from platelet basic protein or connective tissue-activating peptide III through monocyte proteases.
Authors: Walz A , Baggiolini M
Published: June 2004

We studied the origin of the neutrophil-activating peptide NAP-2, a presumed 70 amino acid cleavage product of platelet basic protein (PBP) and connective tissue-activating peptide III (CTAP-III). Purified human blood monocytes or lymphocytes were cultured with or without stimuli (LPS or PHA) in the presence or absence of platelet-release supernatant, and the formation of NAP-2 and other neutrophil-activating peptides was monitored. NAP-2 was generated whenever monocytes and platelet release supernatant were present. When a monocyte stimulus was added, NAF/NAP-1 was also formed, and in the presence of LPS a third, less potent neutrophil-stimulating fraction, consisting of NAP-2 variants with 73, 74, and 75 residues, also appeared. Monocytes alone did not yield NAP-2 and no neutrophil-activating peptide was generated by lymphocytes. The monocyte-conditioned medium was found to cleave purified CTAP-III into NAP-2 through proteinases that were highly sensitive to PMSF, moderately sensitive to leupeptin and insensitive to EDTA.
Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long-term bone marrow cultures.
Authors: Miyake K , Medina K L , Hayashi S , Ono S , Hamaoka T , Kincade P W
Published: June 2004

A new panel of mAbs was prepared to a stromal cell line known to support lymphocytes in Whitlock-Witte type long-term bone marrow cultures. These antibodies were then screened with a cell adhesion assay and four were selected that inhibited the binding of B lineage cells to stromal cell monolayers. Immunofluorescent and biochemical analyses revealed that these new antibodies detected epitopes of the previously described Pgp-1/CD44 antigen complex. Addition of Pgp-1/CD44 antibodies to Dexter-type long-term bone marrow cultures completely prevented emergence of myeloid cells and they also blocked lymphocyte growth in Whitlock-Witte type cultures. mAbs MEL-14, LFA-1, and CD45R did not inhibit under the same conditions and there was no apparent relationship to Ig isotype. Adherent layers in treated cultures were not unusual in terms of morphology and the antibodies did not affect factor-dependent replication of lymphoid or myeloid progenitor cells. Therefore, the mechanism of inhibition may not involve direct toxicity to precursors or microenvironmental elements. Previous studies in humans and mice have implicated Pgp-1/CD44-related glycoproteins in the migration of peripheral lymphoid cells, as well as interactions of cells with the extracellular matrix. These findings suggest that they may also be critical for formation of lymphoid and myeloid cells within bone marrow.
Lymphokine gene expression in vivo is inhibited by cyclosporin A.
Authors: Granelli-Piperno A
Published: June 2004

Murine T cells were stimulated in vivo by administering allogeneic cells or mitogens into the foot pads and then examining the draining popliteal lymph nodes. Allogeneic spleen cells induced the expression of IL2 and IFN-gamma mRNAs in a time- and dose-dependent manner. Induction of these transcripts also was detected after administration of Con A and anti-CD3 mAb. An increase in DNA-synthesizing cells was observed by 48 h, and these were shown to be T cells because of their sensitivity to anti-Thy-1 but not anti-B220 mAb and complement, and because of their localization to the T-dependent areas of the lymph node. The in vivo administration of cyclosporin A (CSA) reduced several of these T cell responses. The level of DNA synthesis and the frequency of cells synthesizing DNA were decreased by approximately 75%, while the induction of IL-2 responsiveness was not substantially diminished. IL-2 and IFN-gamma transcripts were inhibited at least 70-90%, as determined by Northern blot and in situ hybridization. Although the inhibition by CSA was not as complete in animals as observed previously in tissue culture, our findings indicate that in both systems, a major site of action of CSA is to inhibit T cell growth by inhibiting lymphokine production.
Preferential linkage of bcl-2 to immunoglobulin light chain gene in chronic lymphocytic leukemia.
Authors: Adachi M , Tefferi A , Greipp P R , Kipps T J , Tsujimoto Y
Published: June 2004

Most of human follicular lymphomas possess the t(14;18) chromosome translocation that juxtaposes the IgH gene to the 3' region of bcl-2 in a head-to-tail configuration. Here we show that the rearrangement of the bcl-2 gene occurs in a significant fraction (approximately of 10%) of B cell CLL. In all cases analyzed, breakpoints on chromosome 18 clustered at the 5' flanking region of the bcl-2 gene, and no rearrangements were found at the major or minor breakpoint clustering region (3' region of bcl-2 gene) typical of the t(14;18) chromosome translocation. All of the rearranged bcl-2 genes were juxtaposed with the Ig lambda or K genes in a head-to-head configuration. These results imply that the bcl-2 gene is preferentially linked to the IgL genes in CLL and could function in leukemogenesis.
An immunodominant class I-restricted cytotoxic T lymphocyte determinant of human immunodeficiency virus type 1 induces CD4 class II-restricted help for itself.
Authors: Takahashi H , Germain R N , Moss B , Berzofsky J A
Published: June 2004

We have observed that a peptide corresponding to an immunodominant epitope of the HIV-1 envelope protein recognized by class I MHC-restricted CD8+ CTL can also induce T cell help for itself. The help is necessary for restimulation of CTL precursors in vitro with peptide alone in the absence of exogenous lymphokines, can be removed by depletion of CD4+ T cells, and can be replaced by exogenous IL-2. Whereas the CTL in BALB/c or B10. D2 mice are restricted by the class I molecule Dd, the Th cells are restricted by the class II molecule Ad, and the help can be blocked by anti-Ad mAb. To examine the genetic regulation of the induction of help, we studied B10.A mice that share the class I Dd molecule, but have different class II molecules, Ak and Ek. Spleen cells of immune B10.A mice behave like CD4-depleted BALB/c spleen cells in that they cannot be restimulated in vitro by the peptide alone, but can with peptide plus IL-2. Therefore, in the absence of exogenous lymphokines, peptide-specific help is necessary for restimulation with this immunodominant CTL epitope peptide, and in H-2d mice, this peptide stimulates help for itself as well as CTL. We speculate on the implications of these findings for the immunodominance of this peptide in H-2d mice, and for the selective advantage of pairing certain class I and class II molecules in an MHC haplotype.
Antigen-reactive T cell clones restricted by mixed isotype A beta d/E alpha d class II molecules.
Authors: Matsunaga M , Seki K , Mineta T , Kimoto M
Published: June 2004

Mixed isotype A beta dE alpha d class II molecule-restricted antigen-reactive T cell clones were obtained from (BALB/c x B6E alpha d)F1 mice. These T cell clones responded to keyhole limpet hemocyanin in the presence of (BALB/c x B6E alpha d)F1 but not CBF1 APCs. Both anti-A beta d and anti-E alpha mAbs blocked the proliferative responses of these clones. The frequency of such mixed isotype A beta E alpha-restricted T cell clones in (BALB/c x B6E alpha d)F1 mice was estimated to be approximately 10% from our limiting dilution cloning. The existence of such mixed isotype class II molecule-restricted T cells would have important implications for the expansion of the T cell repertoire as well as the induction of autoimmunity.
N-linked glycan modification on antigen-presenting cells restores an allospecific cytotoxic T cell response.
Authors: Neefjes J J , De Bruijn M L , Boog C J , Nieland J D , Boes J , Melief C J , Ploegh H L
Published: June 2004

The B6 anti-bm6 allospecific CTL response is strictly dependent on CD4+ cells when using LPS blasts as stimulator cells. Altering the N-linked carbohydrates on stimulator cells by use of the N-linked trimming glycosidase inhibitors 1-deoxymannojirimycin and swainsonine, or by treatment with bacterial neuraminidase, results in a restoration of the B6 anti-bm6 response in the absence of CD4+ cells. The extent of restoration is inversely correlated with the number of sialic acids present on N-linked glycans of stimulator cells.
Structural requirements for pairing of alpha and beta chains in HLA-DR and HLA-DP molecules.
Authors: Karp D R , Teletski C L , Jaraquemada D , Maloy W L , Coligan J E , Long E O
Published: June 2004

To test for the assembly of human MHC class II molecules having an alpha chain from one isotype (HLA-DR, -DQ, or -DP) and the beta chain of another (mixed isotypic pairs), murine fibroblasts were transfected with expressible cDNAs encoding the different class II alpha and beta chains. A rapid and efficient transient transfection system was developed using a polyoma virus-based vector. Typically, 30-50% of cells transfected using this system expressed high levels of class II molecules on their surface, but only with matched isotypic pairs. Biochemical analysis of cells transfected with matched or mixed isotypic pairs of the DR and DP molecules revealed that only matched chains could pair efficiently inside the cell. Thus, the lack of expression of the two mixed isotypic pairs is due to inefficient primary assembly of the class II molecule and not to a processing or transport defect. To define what region of the beta chains controlled their assembly with alpha chains, a series of chimeric cDNA molecules containing both DR and DP beta chain sequences were constructed. Expression of these chimeric beta chains with DR and DP alpha chains was determined by cytofluorimetry and biochemical analysis. Both alpha chains paired with beta chains in which only the beta 1 domain was isotypically matched. In contrast, the pattern of expression of chimeras made at other points within the beta 1 domain was different for DR and DP. These data show that different areas of primary sequence are important for the assembly of different human class II isotypes, and suggest that HLA-DR and -DP molecules have different secondary or tertiary structures in their NH2-terminal domains.