bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel chromosome translocation caused by fusion of immunoglobulin heavy and light chain V genes in a human B lymphoblastoid cell line.
Authors: Kennedy M A
Published: June 2004

The chromosome breakpoints of a translocation, t(2;14), from an Epstein-Barr virus-transformed human B lymphoblastoid cell line were isolated and analyzed. This unusual translocation arose as a result of the fusion of two immunoglobulin (Ig) variable (V) genes, one from the heavy chain cluster on chromosome 14, the other from the light chain (k) cluster on chromosome 2. The chromosome breaks occurred within the coding sequence of each gene, and there was no obvious evidence for lymphoid V(D)J recombinase involvement in the translocation. This suggests that breakage and rejoining of the involved V genes occurred by some process other than that which normally rearranges Ig genes.
Surface antigen expression on Plasmodium falciparum-infected erythrocytes is modified in alpha- and beta-thalassemia.
Authors: Luzzi G A , Merry A H , Newbold C I , Marsh K , Pasvol G , Weatherall D J
Published: June 2004

In an attempt to determine the mechanism whereby thalassemia in its milder forms may protect against malaria, we have examined the expression of neoantigen at the surface of Plasmodium falciparum-parasitized thalassemic red cells. Neoantigen expression was estimated by measurement of antibody bound after incubation in serum from adults living in a malaria-endemic area, using a quantitative radiometric antiglobulin assay. We found that P. falciparum-parasitized alpha- and beta-thalassemic red cells bind greater levels of antibody from endemic serum than controls: mean binding ratios (+/- SE), respectively, for alpha- and beta-thalassemia compared with controls were 1.69 +/- 0.12 and 1.23 +/- 0.06 on a cell for cell basis, and 1.97 +/- 0.11 and 1.47 +/- 0.08 after a correction for surface area differences. Binding of antibody increased exponentially during parasite maturation. In addition, we found a small but significant degree of binding of naturally occurring antibody to parasitized red cells, the extent of which was also greater in thalassemia. The apparent protective effect of thalassemia against malaria may be related to enhanced immune recognition and hence clearance of parasitized erythrocytes.
Immunity to a pulmonary Cryptococcus neoformans infection requires both CD4+ and CD8+ T cells.
Authors: Huffnagle G B , Yates J L , Lipscomb M F
Published: June 2004

The role of CD4+ and CD8+ T cells in mediating pulmonary clearance of a cryptococcal infection was investigated. Intratracheal inoculation of BALB/c and C.B-17 mice with a moderately virulent strain of Cryptococcus neoformans (52D) resulted in a pulmonary infection, which was cleared by a T cell-dependent mechanism. During this clearance, there was a significant influx of both CD4+ and CD8+ T cells into the lungs. Depletion of CD4+ T cells by injections of CD4-specific monoclonal antibody (mAb) prevented pulmonary clearance and also resulted in significant colonization of the brain and spleen of infected mice. CD4 depletion did not prevent the influx of CD8+ T cells into the lungs. Surprisingly, depletion of CD8+ T cells by mAb also ablated pulmonary clearance. CD8-depleted mice also had a small but significant increase in brain and spleen colony-forming unit compared to control mice by the end of the study. CD4+ T cell pulmonary influx was independent of the presence of CD8+ T cells. The lungs of T cell-depleted mice were examined histologically. CD4+ and CD8+ T cells each mediated a degree of inflammatory influx seen in the lungs of infected mice and raised the possibility that CD4+ and CD8+ T cells may synergize to generate the inflammatory response in the lungs. Numerous phagocytized but intact cryptococci were seen in the inflammatory foci of CD8-depleted mice but not in control or CD4-depleted mice. We propose that CD4+ T cells may recruit and activate effector phagocytes while CD8+ T cells predominantly function to lyse cryptococcus-laden unactivated phagocytes similar to the function of CD8+ T cells during listeria and mycobacteria infections.
Differential tumor necrosis factor alpha expression by astrocytes from experimental allergic encephalomyelitis-susceptible and -resistant rat strains.
Authors: Chung I Y , Norris J G , Benveniste E N
Published: June 2004

There is evidence that the cytokine tumor necrosis factor alpha (TNF-alpha) contributes to the pathogenesis of neurological autoimmune diseases such as multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). TNF-alpha exerts damaging effects on oligodendrocytes, the myelin-producing cell of the central nervous system (CNS), and myelin itself. We have recently demonstrated TNF-alpha expression from astrocytes induced by lipopolysaccharide (LPS), interferon gamma (IFN-gamma), and interleukin 1 beta (IL-1 beta). Astrocytes secrete TNF-alpha in response to LPS alone, and can be primed by IFN-gamma to enhance LPS-induced TNF-alpha production. IFN-gamma and IL-1 beta, cytokines known to be present in the CNS during neurological disease states, do not induce TNF-alpha production alone, but act synergistically to stimulate astrocyte TNF-alpha expression. Inbred Lewis and Brown-Norway (BN) rats differ in genetic susceptibility to EAE, which is controlled in part by major histocompatibility complex (MHC) genes. We examined TNF-alpha gene expression by astrocytes derived from BN rats (resistant to EAE) and Lewis rats (highly susceptible). Astrocytes from BN rats express TNF-alpha mRNA and protein in response to LPS alone, yet IFN-gamma does not significantly enhance LPS-induced TNF-alpha expression, nor do they express appreciable TNF-alpha in response to the combined stimuli of IFN-gamma/IL-1 beta. In contrast, astrocytes from Lewis rats express low levels of TNF-alpha mRNA and protein in response to LPS, and are extremely responsive to the priming effect of IFN-gamma for subsequent TNF-alpha gene expression. Also, Lewis astrocytes produce TNF-alpha in response to IFN-gamma/IL-1 beta. The differential TNF-alpha production by astrocytes from BN and Lewis strains is not due to the suppressive effect of prostaglandins, because the addition of indomethacin does not alter the differential pattern of TNF-alpha expression. Furthermore, Lewis and BN astrocytes produce another cytokine, IL-6, in response to LPS, IFN-gamma, and IL-1 beta in a comparable fashion. Peritoneal macrophages and neonatal microglia from Lewis and BN rats are responsive to both LPS and IFN-gamma priming signals for subsequent TNF-alpha production, suggesting that differential TNF-alpha expression by the astrocyte is cell type specific. Taken together, these results suggest that differential TNF-alpha gene expression in response to LPS and IFN-gamma is strain and cell specific, and reflects both transcriptional and post-transcriptional control mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)
Structure of the mouse pore-forming protein (perforin) gene: analysis of transcription initiation site, 5' flanking sequence, and alternative splicing of 5' untranslated regions.
Authors: Youn B S , Liu C C , Kim K K , Young J D , Kwon M H , Kwon B S
Published: June 2004

We studied the 5' untranslated regions (UTRs) of the mouse lymphocyte pore-forming protein (PFP, perforin, and cytolysin). 5' UTRs were determined by primer extension analysis, sequencing PFP cDNA clone PFP-7, ribonuclease protection assays, and amplification of poly(A)+ RNA of cytolytic T lymphocyte using polymerase chain reaction (PCR). Two alternatively spliced 5' UTRs, designated type I and type II, of 222 and 115 bp, respectively, were found associated with PFP. Type II is identical to type I, except for being 107 bp shorter in the second exon. This deletion was generated by the use of alternative acceptor splice sites. The mouse PFP gene (Pfp) encodes three exons, is separated by two small introns, and spans a chromosomal region of approximately 7 kb. The first exon contains 79 bp of 5' UTR, the second exon contains 143 or 36 bp of 5' UTR (type I or type II UTR, respectively) plus the NH2-terminal region of the mouse PFP, and the third exon contains the rest of the COOH-terminal mouse PFP. The organization of the mouse Pfp is similar to that of the human gene. Moreover, the 5' flanking sequence of the mouse Pfp is highly homologous to that of the human Pfp. In contrast to the human sequence, the more immediate 5' flanking sequence of mouse Pfp contains two tandem "TATA" box-related elements and a GC box, but lacks a typical CAAT box-related sequence. Several other enhancer elements were found further upstream, including cAMP-, phorbol ester-, interferon-gamma-, and UV-responsive elements, and PU box-like and NFkB binding site-like elements. In addition, we found a nuclear inhibitory protein-like element, a transcriptional silencer, and a pair of purine-rich sequence motifs that were found in other T cell-specific genes, and three repeats of GGCCTG that may be a variation of a highly repetitious GCCCTG consensus sequence found in human Pfp.
Generation of T cells with lytic specificity for atypical antigens. I. A mitochondrial antigen in the rat.
Authors: Davies J D , Wilson D H , Hermel E , Lindahl K F , Butcher G W , Wilson D B
Published: June 2004

F1 rats primed with normal parental strain lymphocyte populations and restimulated in culture with parental lymphoblasts generate potent cytotoxic T cell responses to unusual antigen systems. Here we describe in the Lewis (L)/DA anti-DA combination an antigen system most likely of mitochondrial origin with the following properties: it is transmitted maternally from DA strain females, inherited in an extra-chromosomal manner, restricted by class I RT1Aa major histocompatibility complex gene products, extinguished on target cells treated with chloramphenicol, and its pattern of expression in different rat strains correlates with restriction fragment-length polymorphisms of mitochondrial DNA. Sequence analysis of the rat ND1 gene indicates that the maternally transferred factor in the rat is not a homologue of the maternally transmitted factor responsible for the mitochondrial antigen in mice. In keeping with its inheritance from DA females, this antigen is present on target cells from (DA female x L male)F1 donors and all other F1 combinations derived from DA female parents, but absent from target cells from some F1 combinations (L/DA and Wistar-Furth [WF]/DA) derived from DA strain males. The presence of this antigen in other F1 combinations (Brown Norway [BN]/DA, August 2880 [AUG]/DA, and PVG/DA) indicates that this mitochondrial antigen system is shared by the DA, BN, and PVG strains, but not by the L and WF strains.
Generation of T cells with lytic specificity for atypical antigens. II. A novel antigen system in the rat dependent on homozygous expression of major histocompatibility complex genes of the class I-like RT1C region.
Authors: Davies J D , Wilson D H , Butcher G W , Wilson D B
Published: June 2004

Lymphocytes from parental strain DA rats can induce potent killer cell responses to atypical antigen systems in F1 Lewis (L)/DA and DA/L recipients. Here, we describe an antigen system, H, present on homozygous parental target cells, but not on F1 cells. This antigen system is unusual in several respects: it does not involve class I RT1A gene products usually used by killer cell responses in the rat, it maps to the major histocompatibility complex (MHC) class I-like RT1C region, and it requires homozygous expression of RT1Cav1 alleles. This may be another example, this time involving the RT1C region, of an MHC gene product antigenically altered by an MHC-linked trans-activating modifier gene.
Generation of T cells with lytic specificity for atypical antigens. III. Priming F1 animals with antigen-bearing cells also having reactivity for host alloantigens allows for potent lytic T cell responses.
Authors: Davies J D , Wilson D H , Wilson D B
Published: June 2004

Here, we explore the conditions required for generating two different highly potent F1 antiparental killer cell populations to unusual antigens in rats. The first, L/DA anti-DA, has lytic specificity for two antigen systems: MTA, a mitochondrial antigen expressed on DA and DA Lewis (L) target cells restricted by RT1A class I molecules; and H, an antigen that maps to the class I-like RT1C region and is present only on parental target cells from donors homozygous at the major histocompatibility complex. The second killer population is generated in the reciprocal DA/L anti-DA combination and has lytic specificity only for the H antigen system. We show that the killer cells are T cells, and that generation of these F1 cytotoxic T lymphocytes (CTL) requires an in vivo priming step in which it is essential that the inoculated parental cells bear the relevant target antigens and possess alloreactivity for F1 host antigens. The requirement for alloreactivity and antigen on the same priming cell population suggests that these potent lytic responses depend on a situation akin to a hapten-carrier effect that bypasses otherwise ineffective helper responses by the host to these unusual antigens. Restimulation of F1 lymphocytes in culture is also necessary, requiring the presence of antigen on irradiated lymphoblast stimulator cells, but alloreactivity to responder cell antigens is not necessary; normal, nonactivated lymph node cells are completely ineffective as stimulators. For effective lysis, the target cells need not possess the potential for alloreactivity to responder F1 CTL. We also demonstrate in a preliminary way additional antigen systems defined by killer populations raised with other F1 antiparental strain combinations.
Induction of interferon gamma production by natural killer cell stimulatory factor: characterization of the responder cells and synergy with other inducers.
Authors: Chan S H , Perussia B , Gupta J W , Kobayashi M , Pospísil M , Young H A , Wolf S F , Young D , Clark S C , Trinchieri G
Published: June 2004

We previously reported that natural killer cell stimulatory factor (NKSF), a heterodimeric lymphokine purified from the conditioned medium of human B lymphoblastoid cell lines, induces interferon gamma (IFN-gamma) production from resting peripheral blood lymphocytes (PBL) and synergizes with interleukin 2 in this activity. In this study, we show that human NKSF induces IFN-gamma production from both resting and activated human PBL and from freshly isolated murine splenocytes. Human T and NK cells produce IFN-gamma in response to NKSF, but resting PBL require the presence of nonadherent human histocompatibility leukocyte antigens DR+ (HLA-DR+) accessory cells to respond to NKSF. The mechanism(s) by which NKSF induces IFN-gamma production results in accumulation of IFN-gamma mRNA, is insensitive to cyclosporin A, and synergizes with those mediated by phytohemagglutinin, phorbol diesters, anti-CD3 antibodies, and allogeneic antigens, but not by Ca2+ ionophores. The ability of NKSF to directly induce IFN-gamma production and to synergize with other physiological IFN-gamma inducers, joined with the previously described ability to enhance lymphocyte cytotoxicity and proliferation, indicates that this lymphokine is a powerful immunopotentiating agent.
Sequential immunizations with rgp120s from independent isolates of human immunodeficiency virus type 1 induce the preferential expansion of broadly crossreactive B cells.
Authors: Klinman D M , Higgins K W , Conover J
Published: June 2004

The gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is a dominant target against which the host's humoral immune response is directed. Unfortunately, gp120 proteins from different isolates of HIV are antigenically distinct, complicating the use of the envelope glycoprotein in vaccines designed to prevent acquired immunodeficiency syndrome. Using an enzyme-linked immunosorbent spot assay (ELISA), BALB/c mice immunized and boosted with recombinant purified gp120 were studied at the single cell level for their humoral immune response to HIV-1 envelope proteins. Approximately 90% of responding B cells produced antibodies reactive with the immunizing form of gp120 but not with gp120s from other strains of HIV. A novel sandwich ELISA was then used to analyze the frequency with which individual in vivo activated B cells produced antibodies that crossreacted with heterologous gp120s. Repeated immunizations with a single gp120 or with a mixture of different gp120s resulted in the activation of primarily mono-specific (noncrossreactive) B cells. In contrast, the sequential immunization of mice with recombinant purified envelope proteins from different strains of HIV (IIIB, SF2, and Zr6) induced the selective expansion of B cells producing highly crossreactive antibodies.
Granulocyte colony-stimulating factor gene transfer suppresses tumorigenicity of a murine adenocarcinoma in vivo.
Authors: Colombo M P , Ferrari G , Stoppacciaro A , Parenza M , Rodolfo M , Mavilio F , Parmiani G
Published: June 2004

We have investigated the effect of granulocyte colony-stimulating factor (G-CSF) delivery at the site of tumor growth by transducing, via retroviral vector, the human (hu) G-CSF gene into the colon adenocarcinoma C-26 and assaying the ability of transduced cells to form tumors when injected into syngeneic mice. As a control, the same tumor cells were infected with retroviruses engineered to transduce an unrelated gene, the human nerve growth factor receptor, or carry the neomycin resistance gene only. Only cells transduced with the huG-CSF were unable to develop tumors, although huG-CSF was expressed and produced at low level as estimated by both RNA analysis and enzyme-linked immunosorbent assay, indicating that G-CSF can exert an antitumor effect at a physiological dose. Implication of G-CSF as mediator of tumor inhibition was proven by reversing the nontumorigenic phenotype of G-CSF-expressing cells with anti-huG-CSF monoclonal antibody injected at the tumor site. No tumors were formed by injecting C-26 infected cells into nu/nu mice, while neoplastic nodules appeared after injection into sublethally irradiated mice; such tumors, however, regressed when mice normalized their leukocyte counts after irradiation. Tumors were also formed after injection of a mixture of infected and uninfected C-26 cells, although critical delay in tumor formation occurred when infected cells were 10 times more represented in the mixture. Histological examination of tissues surrounding the site of injection showed infiltration of neutrophilic granulocytes, whose number correlated with that of G-CSF-expressing C-26 cells in the injected mixture. These results indicate that G-CSF may have a potent antitumoral activity when released, even at low doses, at the tumor site. The antitumoral effect is mediated by recruitment and targeting of neutrophilic granulocytes to G-CSF-releasing cells.
Structural assignment of novel and immunodominant antigenic sites in the neutralizing antibody response of CBA/Ca mice to influenza hemagglutinin.
Authors: Smith C A , Barnett B C , Thomas D B , Temoltzin-Palacios F
Published: June 2004

Information on the antigenic structure of influenza hemagglutinin (HA) has been deduced previously from sequence analyses of laboratory mutant viruses selected, in vitro, with neutralizing monoclonal antibody (mAb) established exclusively from BALB/c (H-2d) mice; and there has been no attempt to investigate the influence of host genetic background, or natural route of infection, on the protective antibody repertoire. CBA/Ca mice are extremely sensitive to X31 virus infection, and in the present study a structural analysis was made of the antibody repertoire, by direct sequencing of the HA genes of laboratory mutant viruses selected, in ovo with mAb from CBA/Ca mice primed by natural infection with X31 virus at two different infectious doses. Single nucleotide substitutions in the HA genes of mutant viruses identified both novel and immunodominant antigenic sites on the HA1 subunit: a majority of mAbs, from different donors, were of the IgG2a isotype and were specific for HA1 158 Gly. In addition, novel laboratory mutants were obtained containing substitutions in the HA1 subunit that had not been reported previously for H3 subtype viruses, either natural variants or laboratory mutants, at residues: HA1 62 Ile----Arg; HA1 165 Asn----Ser (resulting in the loss of a N-glycosylation site); and HA1 273 Pro----Leu. Our findings suggest that host genetic background and/or a natural route of infection may be significant factors in the selection of different and distinct neutralizing antibody responses to influenza HA and therefore be of some relevance in our further understanding of the immune pressure for antigenic drift, and the immunogenic features of a protective antigen.
Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum: a catabolic pathway initiated by a specific aspartic protease.
Authors: Goldberg D E , Slater A F , Beavis R , Chait B , Cerami A , Henderson G B
Published: June 2004

Hemoglobin is an important nutrient source for intraerythrocytic malaria organisms. Its catabolism occurs in an acidic digestive vacuole. Our previous studies suggested that an aspartic protease plays a key role in the degradative process. We have now isolated this enzyme and defined its role in the hemoglobinolytic pathway. Laser desorption mass spectrometry was used to analyze the proteolytic action of the purified protease. The enzyme has a remarkably stringent specificity towards native hemoglobin, making a single cleavage between alpha 33Phe and 34Leu. This scission is in the hemoglobin hinge region, unraveling the molecule and exposing other sites for proteolysis. The protease is inhibited by pepstatin and has NH2-terminal homology to mammalian aspartic proteases. Isolated digestive vacuoles make a pepstatin-inhibitable cleavage identical to that of the purified enzyme. The pivotal role of this aspartic hemoglobinase in initiating hemoglobin degradation in the malaria parasite digestive vacuoles is demonstrated.
Macrophage- and astrocyte-derived transforming growth factor beta as a mediator of central nervous system dysfunction in acquired immune deficiency syndrome.
Authors: Wahl S M , Allen J B , McCartney-Francis N , Morganti-Kossmann M C , Kossmann T , Ellingsworth L , Mai U E , Mergenhagen S E , Orenstein J M
Published: June 2004

The multifunctional cytokine, transforming growth factor beta (TGF-beta), was identified by immunocytochemistry in the brain tissues of four patients with acquired immune deficiency syndrome (AIDS), but not in control brain tissue. The TGF-beta staining was localized to cells of monocytic lineage as well as astrocytes, especially in areas of brain pathology. In addition, the brain tissues from the AIDS patients contained transcripts for human immunodeficiency virus 1 (HIV-1) by in situ hybridization, suggesting a correlation between the presence of HIV-1 in the brain and the expression of TGF-beta. However, the expression of TGF-beta was not limited to HIV-1-positive cells, raising the possibility of alternative mechanisms for the induction of TGF-beta in these AIDS patients' brains. To investigate these mechanisms, purified human monocytes were infected in vitro with HIV-1 and were shown to secrete increased levels of TGF-beta. In addition, HIV-1-infected monocytes released a factor(s) capable of triggering cultured astrocytes that are not infected with HIV-1 to secrete TGF-beta. The release of TGF-beta, which is an extremely potent chemotactic factor, may contribute to the recruitment of HIV-1-infected monocytic cells, enabling viral spread to and within the central nervous system (CNS). Moreover, TGF-beta augments cytokine production, including cytokines known to be neurotoxic. The identification of TGF-beta within the CNS implicates this cytokine in the immunopathologic processes responsible for AIDS-related CNS dysfunction.
Reentry of T cells to the adult thymus is restricted to activated T cells.
Authors: Agus D B , Surh C D , Sprent J
Published: June 2004

To seek information on the capacity of mature T cells to migrate to the thymus, mice were injected with Thy-1-marked populations enriched for resting T cells or T blast cells; localization of the donor cells in the host thymus was assessed by staining cryostat sections of thymus and by FACS analysis of cell suspensions. With injection of purified resting T cells, thymic homing was extremely limited, even with injection of large doses of cells. By contrast, in vivo generated T blast cells migrated to the thymus in substantial numbers. Thymic homing by T blasts was greater than 50-fold more efficient than with resting T cells. Blast cells localized largely in the medulla and remained in the thymus for at least 1 mo post-transfer. Interestingly, localization of T blasts in the thymus was 10-fold higher in irradiated hosts than normal hosts. Thymic homing was especially prominent in mice injected with T blasts incubated in vitro with the DNA precursor, 125I-5-iodo-2'deoxyuridine (125IDUR); with transfer of 125IDUR-labeled blasts to irradiated hosts, up to 5% of the injected counts localized in the host thymus. These data suggest that thymic homing by T blasts might be largely restricted to cells in S phase. The physiological significance of blast cell entry to the thymus is unclear. The possibility that these cells participate in intrathymic tolerance induction is discussed.
Natural killer (NK) cell response to virus infections in mice with severe combined immunodeficiency. The stimulation of NK cells and the NK cell-dependent control of virus infections occur independently of T and B cell function.
Authors: Welsh R M , Brubaker J O , Vargas-Cortes M , O'Donnell C L
Published: June 2004

The activation, proliferation, and antiviral properties of natural killer (NK) cells were examined in severe combined immunodeficiency (SCID) mice to determine the influence of mature T or B cells on virus-induced NK cell functions and to more conclusively determine the antiviral properties of prototypical CD3- NK cells. NK cells were activated to high levels of cytotoxicity 3 d after infection of mice with lymphocytic choriomeningitis virus (LCMV) or murine cytomegalovirus (MCMV). Analyses of spleen leukocytes from LCMV-infected mice by a variety of techniques indicated that the NK cells proliferated and increased in number during infection. Propidium iodide staining of the DNA of cycling cells revealed that the great majority of proliferating spleen leukocytes 3 d after LCMV infection was of the NK cell phenotype (CD3-, Ig-, Mac-1+, CZ1+, 50% Thy-1+), in contrast to uninfected mice, whose proliferating cells were predominantly of other lineages. Analyses of the NK cell responses over a 2 wk period in control CB17 mice infected with MCMV indicated a sharp rise in serum interferon (IFN) and spleen NK cell activity early (days 3-5) in infection, followed by sharp declines at later stages. In SCID mice the IFN levels continued to rise over a 10-d period, whereas the NK cell response peaked on day 3-5 and gradually tapered. In contrast to the immunocompetent CB17 mice, SCID mice did not clear the MCMV infection and eventually succumbed. SCID mice, again in contrast to immunocompetent CB17 mice, also failed to clear infections with LCMV and Pichinde virus (PV); these mice, infected as adults, did not die but instead developed long-term persistent infections. Depletion of the NK cells in vivo with antiserum to asialo GM1 rendered both SCID and CB17 control mice much more sensitive to MCMV infection, as shown by titers of virus in organs and by survival curves. In contrast, similar depletions of NK cells did not enhance the titers of the NK cell-resistant virus, LCMV. Two variants of PV, one sensitive to NK cells and the other selected for resistance to NK cells by in vivo passage, were also tested in NK cell-depleted SCID mice. The NK-sensitive PV replicated to higher titers in NK cell-depleted SCID mice, whereas the titers of the NK cell-resistant PV were the same, whether or not the mice had NK cells. These experiments support the concept that CD3- prototypical NK cells mediate resistance to NK cell-sensitive viruses via a mechanism independent of antiviral or "natural" antibody.(ABSTRACT TRUNCATED AT 400 WORDS)
Deletions of immunoglobulin C kappa region characterized by the circular excision products in mouse splenocytes.
Authors: Shimizu T , Iwasato T , Yamagishi H
Published: June 2004

We have identified circular DNAs containing the kappa light chain constant region (C kappa), as well as the excision products of V kappa-J kappa and V lambda-J lambda joining in adult mouse splenocytes. Analysis of C kappa-positive circular DNA clones revealed two recombination sites (intron recombining sequence [IRS]1 and -2) within the germline J kappa-C kappa intron region and the recombining sequence (RS) located downstream of the C kappa exon. While RS contains a conserved heptamer and nonamer separated by a 23-bp spacer on the 5' side, IRS1 sequence is an isolated heptamer without an obvious nonamer, and IRS2 contains a variant heptamer, CACAAAA. Since IRS1 and IRS2 recombined with both RS (23-bp spacer signal) and V kappa (12-bp spacer signal) with significant frequency, intron recombination sites seem to have dual recombination signals. These findings provide direct evidence that C kappa deletion preceding lambda gene rearrangement can occur by looping out and excision. Increased accessibility of inefficient recombinational loci within the intron may enable recombinase to accept wide signal sequence variation.
T cell receptor V gene usage of islet beta cell-reactive T cells is not restricted in non-obese diabetic mice.
Authors: Nakano N , Kikutani H , Nishimoto H , Kishimoto T
Published: June 2004

Five islet-reactive T cell clones were established from islet-infiltrating T cells of non-obese diabetic (NOD) mice. All clones expressed CD4, but not CD8, and responded to islet cells from various strains of mice in the context of I-ANOD. They could induce insulitis when transferred into disease-resistant I-E+ transgenic NOD mice. The T cell receptor (TCR) sequences utilized by the clones were determined. Their usage of TCR V and J segments was not restricted but was rather diverse. One of the clones utilized V beta 16. The expression of V beta 16 was significantly reduced in I-E+ transgenic NOD, suggesting the possibility that the islet-reactive T cell clone expressing V beta 16 may be deleted or inactivated by I-E molecules. This clone might be one of the candidates that triggers insulitis.
Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes.
Authors: Peters P J , Borst J , Oorschot V , Fukuda M , Krähenbühl O , Tschopp J , Slot J W , Geuze H J
Published: June 2004

Cytotoxic T lymphocytes (CTL) contain granules that are exocytosed during specific interaction with target cells (TC). In this process, the granule contents, including the lethal protein perforin, as well as granzymes, a family of serine esterases, are delivered to the TC. Information regarding the routing of these proteins towards the granule and their exact localization within the granule is of primary importance to resolve the mechanism of granule-mediated TC killing. In this study, the subcellular localization of perforin, granzymes, and known endosomal and lysosomal marker proteins was determined in human and murine CTL, by immunogold labeling of ultrathin cryosections followed by electron microscopy. Perforin and granzymes can be detected in rough endoplasmic reticulum, Golgi complex, trans-Golgi reticulum, and in all cytotoxic granules. Within the granules, they have a similar distribution and are localized not only in the so-called dense core but also over the region containing small internal vesicles. This finding implies that perforin and granzymes can be released in membrane-enveloped and/or -associated form into the intercellular cleft formed upon CTL-TC interaction. On the basis of the present evidence, additional release of these molecules in soluble form cannot be excluded. The lysosomal membrane glycoproteins lamp-1, lamp-2, and CD63, are abundantly present on the granule-delimiting outer membrane, which becomes incorporated into the CTL plasma membrane during lethal hit delivery. In contrast, the cation-dependent mannose 6-phosphate receptor, known to be present in endosomes and absent from lysosomes, is found only in a minority of the granules. Together with our previous findings that the granules are acidic and connected to the endocytic pathway, these observations define CTL granules as secretory lysosomes.
Human lung tumor-associated antigen identified as an extracellular matrix adhesion molecule.
Authors: Chen F A , Repasky E A , Bankert R B
Published: June 2004

A single chain glycoprotein with an estimated molecular mass of 160 kD (gp160) was previously identified as a human lung tumor-associated antigen. This tumor marker is shown here to be associated noncovalently with a second 130-kD protein. Sequential immunoprecipitation studies of surface iodinated lung tumor cell lysates reveal that this heterodimeric complex is indistinguishable serologically and structurally from the integrin VLA-2, found originally on activated T lymphocytes and platelets. The VLA-2-like complex expressed on the lung tumors possesses similar characteristic Mg2+ dependent binding of collagen and laminin as observed with VLA-2 on normal cells. RNA analysis indicates that human lung tumors express at least 20 times more VLA-2 alpha chain message than normal adult human lung tissue. The results presented here raise the possibility that the overproduction of VLA-2 may be involved in the pathogenesis of human lung tumors by modulating the invasive and metastatic potential of the tumor.
Transforming growth factor beta 1 (TGF-beta 1) induced neutrophil recruitment to synovial tissues: implications for TGF-beta-driven synovial inflammation and hyperplasia.
Authors: Fava R A , Olsen N J , Postlethwaite A E , Broadley K N , Davidson J M , Nanney L B , Lucas C , Townes A S
Published: June 2004

We have studied the consequences of introducing human recombinant transforming growth factor beta 1 (hrTGF-beta 1) into synovial tissue of the rat, to begin to better understand the significance of the fact that biologically active TGF-beta is found in human arthritic synovial effusions. Within 4-6 h after the intra-articular injection of 1 microgram of hrTGF-beta 1 into rat knee joints, extensive recruitment of polymorphonuclear leukocytes (PMNs) was observed. Cytochemistry and high resolution histological techniques were used to quantitate the influx of PMNs, which peaked 6 h post-injection. In a Boyden chamber assay, hrTGF-beta 1 at 1-10 fg/ml elicited a chemotactic response from PMNs greater in magnitude than that evoked by FMLP, establishing that TGF-beta 1 is an effective chemotactic agent for PMNs in vitro as well as in vivo. That PMNs may represent an important source of TGF-beta in inflammatory infiltrates was strongly suggested by a demonstration that stored TGF-beta 1 was secreted during phorbol myristate acetate-stimulated degranulation in vitro. Acid/ethanol extracts of human PMNs assayed by ELISA contained an average of 355 ng of TGF/beta 1 per 10(9) cells potentially available for secretion during degranulation of PMNs. [3H]Thymidine incorporation in vivo and autoradiography of tissue sections revealed that widespread cell proliferation was triggered by TGF-beta 1 injection. Synovial lining cells and cells located deep within the subsynovial connective tissue were identified as sources of at least some of the new cells that contribute to TGF-beta 1-induced hyperplasia. Our results demonstrate that TGF-beta is capable of exerting pathogenic effects on synovial tissue and that PMNs may represent a significant source of the TGF-beta present in synovial effusions.
Cloning of murine TCF-1, a T cell-specific transcription factor interacting with functional motifs in the CD3-epsilon and T cell receptor alpha enhancers.
Authors: Oosterwegel M , van de Wetering M , Dooijes D , Klomp L , Winoto A , Georgopoulos K , Meijlink F , Clevers H
Published: June 2004

CD3-epsilon gene expression is confined to the T cell lineage. We have recently identified and cloned a human transcription factor, TCF-1, that binds to a functional element in the T lymphocyte-specific enhancer of CD3-epsilon. In a panel of human cell lines, TCF-1 expression was restricted to T lineage cells. TCF-1 belonged to a novel family of genes that contain the so-called high mobility group 1 (HMG) box. Here we report the cloning of murine TCF-1. Two splice alternatives were identified that were not previously observed in human TCF-1. Murine and human TCF-1 displayed a 95.5% overall amino acid homology. Recombinant murine and human TCF-1 recognized the same sequence motif in the CD3-epsilon enhancer as judged by gel retardation and methylation interference assays. With the murine cDNA clones several aspects of TCF-1 were analyzed. First, deletion analysis revealed that a region of TCF-1 containing the HMG box was sufficient for sequence-specific binding. Second, by high stringency Northern blotting and in situ hybridization, TCF-1 expression was shown to be confined to the thymus and to the T cell areas of the spleen. Third, TCF-1 bound specifically to a functional T cell-specific element in the T cell receptor alpha (TCR-alpha) enhancer. The T lineage-specific expression and the affinity for functional motifs in the TCR-alpha and CD3-epsilon enhancers imply an important role for TCF-1 in the establishment of the mature T cell phenotype.
Complement mediates human immunodeficiency virus type 1 infection of a human T cell line in a CD4- and antibody-independent fashion.
Authors: Boyer V , Desgranges C , Trabaud M A , Fischer E , Kazatchkine M D
Published: June 2004

Incubation of the human T cell lymphotropic virus (HTLV)-IIIB and HTLV-RF strains of human immunodeficiency virus type 1 (HIV-1) with normal seronegative human serum under conditions that allow complement activation resulted in enhancement of infection of the MT2 human T cell line cultured in the presence of low amounts of virus. Infection of MT2 cells was assessed by measuring reverse transcriptase activity in supernatants at day 9 of culture. Complement activation by viral suspensions occurred through the alternative pathway. Opsonization of HTLV-RF viral particles with complement was sufficient to allow a productive infection to occur in cells exposed to suboptimal amounts of virus. Infection of MT2 cells with suboptimal amounts of serum-opsonized HIV-1 was suppressed by blocking the C3dg receptor (CR2, CD21) on MT2 cells with monoclonal anti-CR2 antibody and rabbit F(ab')2 anti-mouse immunoglobulin antibodies. Blocking of the gp120-binding site on CD4 under similar experimental conditions had no inhibitory effect on infection of MT2 cells with opsonized virus. Opsonization of HIV-1 with seronegative serum also resulted in a CR2-mediated enhancement of the infection of normal peripheral blood mononuclear cells and T lymphocytes. These results indicate that complement in the absence of antibody may enhance infection of C3 receptor-bearing T cells with HIV-1, and that the interaction of opsonized virus with the CR2 receptor may result by itself in the infection of target T cells in a CD4- and antibody-independent fashion.
In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations.
Authors: Jacob J , Kassir R , Kelsoe G
Published: June 2004

After primary immunization with an immunogenic conjugate of (4-hydroxy-3-nitrophenyl)acetyl, two anatomically and phenotypically distinct populations of antibody-forming cells arise in the spleen. As early as 2 d after immunization, foci of antigen-binding B cells are observed along the periphery of the periarteriolar lymphoid sheaths. These foci expand, occupying as much as 1% of the splenic volume by day 8 of the response. Later, foci grow smaller and are virtually absent from the spleen by day 14. A second responding population, germinal center B cells, appear on day 8-10 and persist at least until day 16 post-immunization. Individual foci and germinal centers represent discrete pauciclonal populations that apparently undergo somatic evolution in the course of the primary response. We suggest that foci may represent regions of predominantly interclonal competition for antigen among unmutated B cells, while germinal centers are sites of intraclonal clonal competition between mutated sister lymphocytes.
Role of cytokines (interleukin 1, tumor necrosis factor, and transforming growth factor beta) in natural and lipopolysaccharide-enhanced radioresistance.
Authors: Neta R , Oppenheim J J , Schreiber R D , Chizzonite R , Ledney G D , MacVittie T J
Published: June 2004

Studies of radioresistance and radioprotection provide an excellent in vivo model for dissection of the pathophysiological role of cytokines. The availability of neutralizing antibodies to cytokines has made it possible to assess the contribution of cytokines to host defense and repair processes involved in radioresistance and radioprotection. Administration of anti-interleukin 1 receptor (IL-1R) antibody (35F5) or anti-tumor necrosis factor (TNF) antibody (TN3 19.12) reduced survival of irradiated CD2F1 mice. These results demonstrate conclusively that natural levels of IL-1 and TNF contribute to radioresistance of normal mice. Furthermore, the radioprotective effect of administered IL-1 was blocked not only with anti-IL-1R antibody but also with anti-TNF antibody. Similarly, the radioprotective effect of TNF was reduced with anti-IL-1R antibody. These data suggest that cooperative interaction of both cytokines is necessary to achieve successful radioprotection. Finally, when LPS was used as a radioprotector, the combined administration of anti-IL-1R and anti-TNF not only blocked the radioprotection with LPS, but actually revealed LPS to have a radiosensitizing effect. This effect may be due to induction of TGF-beta, since administration of this cytokine results in reduced survival of irradiated mice.
Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow.
Authors: Hardy R R , Carmack C E , Shinton S A , Kemp J D , Hayakawa K
Published: June 2004

We have resolved B220+ IgM- B-lineage cells in mouse bone marrow into four fractions based on differential cell surface expression of determinants recognized by S7 (leukosialin, CD43), BP-1, and 30F1 (heat stable antigen). Functional differences among these fractions can be correlated with Ig gene rearrangement status. The largest fraction, lacking S7, consists of pre-B cells whereas the others, expressing S7, include B lineage cells before pre-B. These S7+ fractions, provisionally termed Fr. A, Fr. B, and Fr. C, can differentiate in a stromal layer culture system. Phenotypic alteration during such culture suggests an ordering of these stages from Fr. A to Fr. B to Fr. C and thence to S7- pre-B cells. Using polymerase chain reaction amplification with pairs of oligonucleotide primers for regions 5' of JH1, DFL16.1, and Jk1, we find that the Ig genes of Fr. A are in germline configuration, whereas Fr. B and C are pro-B cell stages with increasing D-J rearrangement, but no V-D-J. Finally, functional analysis demonstrates that the proliferative response to IL-7, an early B lineage growth factor, is restricted to S7+ stages and, furthermore, that an additional, cell contact-mediated signal is essential for survival of Fr. A.
Macrophage stimulating protein: purification, partial amino acid sequence, and cellular activity.
Authors: Skeel A , Yoshimura T , Showalter S D , Tanaka S , Appella E , Leonard E J
Published: June 2004

Macrophage stimulating protein (MSP) was purified to homogeneity from human blood plasma by selection of biologically active fractions obtained by sequential immunoaffinity and high pressure liquid ion exchange chromatography. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular mass of MSP was 70 kilodaltons (kD); under reducing conditions two gel bands were seen, at 47 and 22 kD. The disulfide-linked two-chain structure of MSP was confirmed by separation of reduced and alkylated MSP chains. A computer search comparison of six partial sequences of MSP digests showed that MSP has not been recorded in data banks of protein sequences. Two MSP fragments had greater than 80% identity in overlaps of 12-16 residues to sequences in the protein family that includes human prothrombin, plasminogen, and hepatocyte growth factor. The concentration of purified MSP required for half-maximal biological activity was the order of 10(-10) M. In addition to making mouse resident peritoneal macrophages responses to chemoattractants, MSP caused the appearance of long cytoplasmic processes and pinocytic vesicles in freshly plated macrophages. MSP also caused phagocytosis via the C3b receptor, CR1. Whereas resident peritoneal macrophages bind but do not ingest sheep erythrocytes opsonized with IgM anti-Forssman antibody and mouse C3b, addition of MSP caused ingestion. Thus, MSP causes direct or indirect activation of two receptors of the mouse resident peritoneal macrophage, CR1 and the C5a receptor.
Premature expression of the macrophage colony-stimulating factor receptor on a multipotential stem cell line does not alter differentiation lineages controlled by stromal cells used for coculture.
Authors: Kinashi T , Lee K H , Ogawa M , Tohyama K , Tashiro K , Fukunaga R , Nagata S , Honjo T
Published: June 2004

We are interested to know whether expression of a lineage-specific growth factor receptor is deterministic to lineage commitment during hematopoiesis. For this purpose, we introduced the human c-fms gene into the multipotential stem cell clone LyD9 and two myeloid progenitor clones, L-GM3 and L-G3, cells that differentiate in response to granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte (G)-CSF, respectively. Although LyD9 cells have differentiation potential to become macrophages, c-fms transfectants of LyD9 and L-GM3 cells did not differentiate in response to human macrophage (M)-CSF. However, c-fms transfectants of L-G3 cells differentiated to neutrophils in response to human M-CSF. These results indicate that the M-CSF receptor requires a specific signal transduction pathway to exert its differentiational and proliferative effects. Furthermore, the M-CSF receptor can convey a granulocyte-type differentiation signal possibly by cooperating with the G-CSF receptor signal transduction pathway. The c-fms-transfected LyD9 cells as well as the original LyD9 cells differentiated predominantly into GM-CSF- and G-CSF-responsive cells by coculturing with PA6 and ST2 stromal cells, respectively. The results indicate that differentiation lineage is not affected by premature expression of the M-CSF receptor. Instead, the stromal cell used for coculture apparently controls lineage-selective differentiation of the multi-potential stem cell line.
Activation of the adhesive capacity of CR3 on neutrophils by endotoxin: dependence on lipopolysaccharide binding protein and CD14.
Authors: Wright S D , Ramos R A , Hermanowski-Vosatka A , Rockwell P , Detmers P A
Published: June 2004

Tumor necrosis factor alpha, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, and formyl peptide were each found to cause a twofold increase in expression of CD14 on the surface of polymorphonuclear leukocytes (PMN). Upregulation of CD14 was complete by 20 min and thus appeared to result from expression of preformed stores of protein. The CD14 on the surface of PMN was shown to serve two biological functions. It bound particles coated with complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP). This binding activity was enhanced by agonists that upregulated CD14 expression and may serve in the clearance of Gram-negative bacteria opsonized with LBP. Interaction of CD14 with LPS in the presence of LBP or serum also caused a dramatic, transient increase in the adhesive activity of CR3 (CD11b/CD18) on PMN. Enhanced activity of CR3 and other members of the CD11/CD18 family underlies many of the known physiological responses of PMN to LPS and may be a central feature of the in vivo responses of PMN to endotoxin.