bluuebunny/crossref_metadata_2025 huggingface dataset:
Polymorphic DQ alpha and DQ beta interactions dictate HLA class II determinants of allo-recognition.
Authors: Kwok W W , Mickelson E , Masewicz S , Milner E C , Hansen J , Nepom G T
Published: June 2004

18 transfected cell lines were generated that expressed distinct DQ molecules related to the serologically defined HLA-DQw3 specificity. These transfectants were constructed using site-directed mutagenesis to introduce nucleotide substitutions into DQ3.2 beta cDNA, followed by retrovirus-mediated gene expression of the mutagenized genes in human B cell lines with different endogenous DQ alpha chains. The capacity of particular class II dimers to stimulate alloreactive T cell clones was investigated. T cell activation was found to be dependent on both DQ alpha and DQ beta chains. In some cases, single amino acid substitutions at codons 13, 26, 45, or 57 of the DQ beta chain were sufficient to dramatically alter T cell reactivity; T cell recognition of these substitutions, however, was strongly influenced by the alpha chain polymorphisms present in the stimulatory class II dimer. Both gain and loss of major serologic and cellular specificities associated with specific DQw3+ alleles were observed with a limited array of site-directed substitutions.
5-Hydroxyeicosanoids selectively stimulate the human neutrophil 15-lipoxygenase to use endogenous substrate.
Authors: Nichols R C , Vanderhoek J Y
Published: June 2004

When human neutrophils, prelabeled with [3H]arachidonic acid, were incubated with 5S,15S-dihydroxyeicosatetraenoic acid (5,15-diHETE), a dose-dependent increase in the 15-lipoxygenase product [3H]-15-HETE was observed relative to untreated cells. Typically, a fivefold increase in [3H]-15-HETE formation was obtained upon exposure of these cells to 3 muM 5,15-diHETE. There was no appreciable enhancement of the 5-lipoxygenase metabolite [3H]-5-HETE. Product identities were confirmed by comparing retention times on straight- and reversed-phase HPLC with authentic standards, and RIA. Other 5-hydroxyeicosanoids, such as 5-HETE, 5-HETE methyl ester, and leukotriene B4(5S,12R-diHETE), were equally effective in stimulating the formation of [3H]-15-HETE, but exogenously added lipoxin A4, lipoxin B4, 15-HETE, and 12-HETE were much less potent, whereas stearic acid was ineffective. The diHETEs also showed a greater selectivity in activating the 15-lipoxygenase relative to the 5-lipoxygenase. A likely source of substrate for the 15- and 5-lipoxygenases is a pool of cell-associated but noncovalently bound arachidonic acid. In [3H]arachidonic acid-prelabeled neutrophils, the amount of free [3H]arachidonic acid ranged between 50 and 700 fmol/10(7) cells, whereas unlabeled neutrophils contained 100-2,200 pmol/10(7) cells of nonesterified arachidonic acid. The exogenously added hydroxyeicosanoids induce a 0.5-3% conversion of this substrate pool to product. These findings indicate that the 15-lipoxygenase in human neutrophils is a cryptic enzyme that needs to be stimulated in order to metabolize endogenous substrate. It is possible that 5-hydroxyeicosanoids may mimic an as yet unidentified physiological activator of the 15-lipoxygenase.
Class I-restricted processing and presentation of exogenous cell-associated antigen in vivo.
Authors: Carbone F R , Bevan M J
Published: June 2004

MHC class I-restricted T lymphocyte responses are usually directed to cellular antigenic components resulting from endogenous gene expression. Exogenous, non-replicating antigens, such as soluble proteins, usually fail to enter the class I pathway of antigen processing and presentation. Consistent with this notion, we have recently shown that soluble, exogenous proteins can be efficiently processed for class I presentation in vitro only if they are introduced directly into the target cell cytoplasm. In this report we extend this work to the in vivo situation by introducing soluble protein into the cytoplasm of mouse splenocytes via the osmotic lysis of pinosomes and then using these cells for in vivo immunization. Our results show that cytoplasmic loading of OVA and beta-GAL into H-2b and H-2d splenocytes respectively, resulted in effective in vivo immunogens for class I-restricted CTL. To our surprise, control spleen cell preparations simply incubated with the exogenous, native protein for 10 min at 37 degrees C in isotonic medium and then washed could also induce a comparable class I-restricted CTL response following intravenous injection. Experiments using (H-2b X H-2d)F1 mice showed that protein pulsed splenocytes from one parental strain could effectively "cross prime" T cells restricted to the MHC of the other parental strain. In all cases, target cell recognition by the effector CTL generated by immunization with spleen cell-associated antigen required the antigen to be present in the cell cytoplasm. Thus the CTL do not recognize target cells exposed to soluble, exogenous antigen. These results, reminiscent of analogous experiments with cross priming by minor histocompatibility antigens, argue that class I-restricted processing and presentation of exogenous antigen can occur in vivo following immunization with cell-associated antigen.
Growth autonomy and tumorigenicity of interleukin 6-dependent B cells transfected with interleukin 6 cDNA.
Authors: Tohyama N , Karasuyama H , Tada T
Published: June 2004

We introduced an IL-6 cDNA expression vector into a murine B cell line, the growth of which definitely required the presence of exogenous IL-6. The transfected cells secreted substantial amounts of IL-6, to which they themselves responded by proliferating without further requirement of exogenous IL-6. The proliferation was a direct function of cell density and was inhibitable by antibodies to IL-6, indicating the autocrine nature of the growth. The IL-6 cDNA-transfected cells displayed greatly enhanced tumorigenicity when inoculated into syngeneic and nude mice. Our data suggest that an IL-6 autocrine self stimulation confers on B cells a selective growth advantage and results in the induction of progression of the malignant state of B cells.
Characterization of binding and biological effects of monoclonal antibodies against a human tumor necrosis factor receptor.
Authors: Espevik T , Brockhaus M , Loetscher H , Nonstad U , Shalaby R
Published: June 2004

Three different antibodies against a human TNF receptor (htr-1, htr-5, and htr-9) have been examined for their binding pattern to U937 cells and ability to mimic TNF-alpha activity in U937 cells, Fs4 fibroblasts, and human endothelial cells. Flow cytometric analysis revealed that htr-5 and htr-9 bound specifically to a TNF receptor on U937 cells that could be blocked by pretreatment with rTNF-alpha. Pretreatment of U937 cells with rTNF-beta blocked the binding of htr-9, but to a lesser extent htr-5 binding. Pretreatment with htr-5 inhibited the binding of htr-9 to U937 cells while pretreatment with htr-9 did not inhibit htr-5 binding. These results indicate that htr-5 and htr-9 recognize distinct but overlapping epitopes of a human TNF receptor on U937 cells and that htr-5 may be close to a TNF-alpha-specific domain of the binding site. Pretreatment with htr-5 or htr-9 only minimally reduced binding of BrTNF-alpha to U937 cells; however, these antibodies were much more effective in inhibiting BrTNF-alpha binding to HL-60 cells. Furthermore, it was found that htr-1 and htr-9, but not htr-5, had TNF-alpha activity on U937 cells, Fs4 fibroblasts, and endothelial cells and that the TNF-alpha activity induced by htr-9 was completely inhibited by htr-5. However, the cytotoxic activity of TNF-alpha was only partially inhibited by htr-5 on U937 cells while htr-5 had no effect on TNF-alpha activity on Fs4 cells. The data suggest that a common epitope is involved in inducing TNF-alpha activity in three different cell systems.
The role of cytokines in the generation of inflammation and tissue damage in experimental gram-positive meningitis.
Authors: Saukkonen K , Sande S , Cioffe C , Wolpe S , Sherry B , Cerami A , Tuomanen E
Published: June 2004

Cytokines mediate many host responses to bacterial infections. We determined the inflammatory activities of five cytokines in the central nervous system: TNF-alpha, IL-1 alpha, IL-1 beta, macrophage inflammatory protein 1 (MIP-1), and macrophage inflammatory protein 2 (MIP-2). Using a rabbit model of meningeal inflammation, each cytokine (except IL-1 beta) induced enhanced blood brain barrier permeability, leukocytosis in cerebrospinal fluid, and brain edema. Homologous antibodies to each mediator inhibited leukocytosis and brain edema, and moderately decreased blood brain barrier permeability. In rabbits treated with anti-CD-18 antibody to render neutrophils dysfunctional for adhesion, each cytokine studied lost the ability to cause leukocytosis and brain edema. After intracisternal challenge with pneumococci, antibodies to TNF or IL-1 prevented inflammation, while anti-MIP-1 or anti-MIP-2 caused only a 2-h delay in the onset of inflammation. We suggest these cytokines have multiple inflammatory activities in the central nervous system and contribute to tissue damage during pneumococcal meningitis.
The toxicity of staphylococcal enterotoxin B in mice is mediated by T cells.
Authors: Marrack P , Blackman M , Kushnir E , Kappler J
Published: June 2004

Staphylococcal enterotoxin B (SEB) has been shown in the past to be a potent T cell stimulant in mouse or man. The toxin acts as a superantigen that is, it binds to class II MHC proteins and, as such a complex, stimulates T cells bearing particular V beta s as part of their receptors. The toxin also has several pathological effects, causing, in mice, rapid weight loss, thymus atrophy, immunosuppression, and, at high doses, death. The data in this paper show that at least one of these effects, weight loss, is T cell mediated. Staphylococcal enterotoxin-mediated weight loss is MHC dependent, and is almost absent in animals expressing MHC class II molecules, which, complexed with SEB, are poor T cell stimulants. Also, mice that lack T cell function, genetically or because of cyclosporin A treatment, lose no or less weight than controls in response to SEB. Finally, animals bred such that they express few T cells bearing V beta s with which SEB can interact lose much less weight in response to the toxin than littermate controls that have higher numbers of reactive T cells. It is therefore suggested that the pathological effects of the staphylococcal, T cell-stimulating toxins in mouse and man may be partially or wholly the consequence of massive T cell stimulation.
The chromosome translocation (11;14)(p13;q11) associated with T cell acute leukemia. Asymmetric diversification of the translocational junctions.
Authors: Cheng J T , Yang C Y , Hernandez J , Embrey J , Baer R
Published: June 2004

The t(11;14)(p13;q13) translocation associated with T cell acute lymphocytic leukemia generates two abnormal chromosomes, designated 11p+ and 14q-. To investigate the mechanism of t(11;14)(p13;q11) formation, we analyzed the translocation junctions of 11p+ and 14q- from two patients. The 11p+ junctions consisted of precise fusions of a pseudo recombination signal from chromosome 11 and the downstream recombination signal of the TCR D delta 2 gene segment from chromosome 14. In contrast, the 14q- junctions from both patients were diversified by random loss and addition of nucleotides at the translocation site. This asymmetric pattern of junctional diversification is typical of normal Ig/TCR gene rearrangement, and therefore implies that the t(11;14)(p13;q11) translocation arose due to aberrant activity of the Ig/TCR recombinase.
Autoantibodies in chronic graft versus host result from cognate T-B interactions.
Authors: Morris S C , Cheek R L , Cohen P L , Eisenberg R A
Published: June 2004

A chronic graft-versus-host reaction (GVH) induced in nonautoimmune mice causes a syndrome that closely resembles SLE. In this model, donor T cells react against incompatible host Ia structures and generate excessive help, which activates a subpopulation of self-reactive B cells. We have studied whether these self-reactive B cells are activated by direct interaction with alloreactive T cells or by nonspecific bystander effects. Two types of chimeras were made: double-parental chimeras, differing at both Ia and Igh allotype [B6.C20 + bm12----(B6.C20 x bm12)F1]; and control chimeras [(B6.C20 x bm12)F1----(B6.C20 x bm12)F1]. A chronic GVH syndrome was induced in the chimeras by infusion of B6 or bm12 spleen cells. Coombs and antichromatin autoantibodies were measured using Igh allotype-specific immunoassays. The double-parental chimeras that received bm12 cells made autoantibodies principally of the Igha allotype, indicating that the bm12 T cells interacted only with the Iab-bearing host B cells. Conversely, double-parental chimeras that received B6 cells made mostly Ighb autoantibodies, indicating direct cognate interaction with the Iabm12-bearing host B cells. The control chimeras made autoantibodies of both allotypes. These results indicate that autoantibodies in chronic GVH result from direct T-B interactions and not from nonspecific T cell-derived factors.
A new allele of the lpr locus, lprcg, that complements the gld gene in induction of lymphadenopathy in the mouse.
Authors: Matsuzawa A , Moriyama T , Kaneko T , Tanaka M , Kimura M , Ikeda H , Katagiri T
Published: June 2004

Several mice with generalized lymphadenopathy were found in the CBA/KlJms (CBA) colony maintained at our institute. A new mutant strain of mice that develop massive lymphoid hyperplasia at 100% incidence within 5 mo after birth was established by crossing these diseased mice. Genetic studies on lymphadenopathy were conducted in F1, F2, and backcross populations from crosses between mutant CBA (CBA-m) and various inbred strains of mice. The results supported the control of lymphadenopathy by a single autosomal recessive gene. Since C3H/He-gld/gld (C3H-gld), MRL/MpJ-lpr/lpr (MRL-lpr), and C3H/HeJ-lpr/lpr (C3H-lpr) mice develop the same type of lymphoid hyperplasia, allelism of the mutant gene with gld or lpr was tested by investigating lymphadenopathy in F1 and backcross populations from crosses between CBA-m and C3H-gld, MRL-lpr, or C3H-lpr mice. The gene was confirmed to be allelic with lpr but not with gld. Interestingly, however, the mutant gene interacted with gld to induce less severe lymphadenopathy. Thus, the mutant gene was named lprcg, an lpr gene complementing gld in induction of lymphoproliferation. The genetic conclusion was supported by the same profile of surface markers of lymphoid cells with gld/gld, lpr/lpr, lprcg/lprcg, lprcg/lpr, and +/gld +/lprcg genotypes, as well as by massive lymph node hyperplasia and high titers of autoantibodies in the first four genotypes, but slight hyperplasia and insignificant autoantibody production in the last. The discovery of lprcg provided strong genetic evidence for the parallels between anomalous phenotypes of gld and lpr, and CBA/KlJms-lprcg/lprcg mice will contribute to elucidation of the mechanism of induction of the same abnormal differentiation and functions of lymphocytes by gld and lpr.
Genomic organization of the mouse pore-forming protein (perforin) gene and localization to chromosome 10. Similarities to and differences from C9.
Authors: Trapani J A , Kwon B S , Kozak C A , Chintamaneni C , Young J D , Dupont B
Published: June 2004

Genomic clones encompassing the entire coding region of the mouse lymphocyte pore-forming protein gene (Pfp) have been isolated and used to determine its intron-exon organization. In contrast to C9, Pfp has a simple structure, consisting of only three exons (two of which encode polypeptide), a large 5' intron, and a single, smaller intron that is situated approximately one-third of the way through the protein-coding portions of the gene. The regions encoding the homologous domains of PFP and C9 are encoded on exons 7, 8, 9, and 10 of C9, but form only approximately half of the open reading frame of exon III in Pfp. Although encoding polypeptides with related functions, the two genes possess such sharply contrasting structures as to suggest that their analogous regions may have risen independently, by a process of convergent evolution. Using a panel of somatic cell hybrid cell lines, Pfp has been mapped to chromosome 10.
Autoantibodies against triosephosphate isomerase. A possible clue to pathogenesis of hemolytic anemia in infectious mononucleosis.
Authors: Ritter K , Brestrich H , Nellen B , Kratzin H , Eiffert H , Thomssen R
Published: June 2004

In sera from patients with acute EBV, infection and the clinical symptoms of infectious mononucleosis antibodies of the Ig class M were found that are directed against two cellular proteins. The molecular mass of these proteins was determined to be 29 (p29) and 26 kD (p26), respectively, in SDS-PAGE. P29 was identified as part of the glycolytic enzyme triosephosphate isomerase (TPI) by comparison of the NH2-terminal amino acid sequences. A purified antibody against TPI induces a 51Cr release from human erythrocytes. Possibly, anti-TPI causes hemolysis, which is an infrequent but serious symptom of infectious mononucleosis.
Cellular distribution, regulation, and biochemical nature of an Fc alpha receptor in humans.
Authors: Monteiro R C , Kubagawa H , Cooper M D
Published: June 2004

In these studies, we characterize an Fc receptor (FcR) for IgA that is present on human granulocytes, monocyte/macrophages, and their corresponding cell lines. Receptor expression appears to be constitutive but can be selectively upregulated on monocyte cell lines by stimulation with a phorbol ester and polymeric IgA. Both the induction requirements and ligand specificity of the IgA receptor differ from the IgG receptors, Fc gamma R I, II, and III, that are also expressed on monocytes and granulocytes. IgA binding to the cell surface receptor is mediated via the Fc alpha region. The Fc alpha R is a heterogenously charged, approximately 60-kD molecule with an isoelectric point of 4.5-5.6 that binds monomeric or polymeric IgA1 and IgA2 molecules. This transmembrane glycoprotein appears to be composed of 32- and 36-kD protein cores with multiple N-linked carbohydrate moieties. We conclude that this Fc alpha R represents a novel member of the FcR family that may have a distinctive role in host defense.
Antitumor activity of recombinant interleukin 6 in mice.
Authors: Mulé J J , McIntosh J K , Jablons D M , Rosenberg S A
Published: June 2004

IL-6 possesses multiple biologic activities that affect a broad range of cells including those directly involved in immune responses as well as cells important in the systemic response to infection or trauma. We now show that purified human rIL-6, when administered alone at relatively high doses that are comparable to therapeutic levels of IL-2, mediated substantial reductions in the number of pulmonary and hepatic micrometastases from four distinct syngeneic tumors. Unlike IL-2, IL-6 injections resulted in neither observable toxicity nor death of the treated mice at the dose regimens used. Host immunosuppression by sublethal total-body irradiation before the initiation of therapy prevented the IL-6 antitumor effect, thus suggesting that IL-6 acted through a radiosensitive host component rather than directly on the tumor itself. Moreover, the systemic administration of relatively low doses of IL-6 in combination with subtherapeutic doses of TNF to mice bearing an established weakly immunogenic, syngeneic tumor at a subcutaneous site resulted in marked tumor regression and cure rates. These studies represent the first demonstration of tumor regression mediated by recombinant IL-6 in vivo.
In vivo administration of interleukin 2 plus T cell-depleted syngeneic marrow prevents graft-versus-host disease mortality and permits alloengraftment.
Authors: Sykes M , Romick M L , Hoyles K A , Sachs D H
Published: June 2004

Previous work from this laboratory has demonstrated that T cell-depleted (TCD) syngeneic marrow can delay, but not prevent, the mortality from acute graft-vs.-host disease (GVHD) caused by MHC-mismatched lymphoid cells administered to lethally irradiated mice. We demonstrate here that a protective effect against GVHD is also observed after in vivo treatment with IL-2. Administration of 10,000-50,000 U of IL-2 twice daily for the first 5 d after bone marrow transplantation markedly reduced the mortality from both acute and chronic GVHD induced across complete MHC barriers in lethally irradiated mice, and frequently led to long-term survival. Complete allogeneic reconstitution was demonstrated in all long-term survivors of this treatment regimen. While either IL-2 or TCD syngeneic marrow administered alone was protective in some experiments, the maximal protective effect was observed after administration of both IL-2 and TCD syngeneic marrow, especially when the effects of IL-2 were suboptimal. The timing of IL-2 administration was critical to this beneficial effect, since a delay of 7 d in commencing IL-2 treatment was associated with accelerated GVHD mortality. This new approach to the prevention of GVHD permits the administration of allogeneic T cells, and may therefore avoid the increased incidence of graft failure and loss of antileukemic effects associated with the T cell depletion of allogeneic marrow, which is otherwise required for the prevention of GVHD.
A novel surface antigen expressed by a subset of human CD3- CD16+ natural killer cells. Role in cell activation and regulation of cytolytic function.
Authors: Moretta A , Tambussi G , Bottino C , Tripodi G , Merli A , Ciccone E , Pantaleo G , Moretta L
Published: June 2004

The GL183 mAb was obtained by immunizing BALB/c mice with the E57 clone (CD7+CD2+CD3-CD16+CD56+) derived from human peripheral blood NK cells. In human peripheral blood, GL183-reactive cells ranged between 2 and 12% (mean 6.5%) in 10 different donors. Double fluorescence and FACS analysis showed that GL183+ cells were consistently included in the CD56+ or CD16+ cell populations. Moreover, since only a fraction of CD56+ or CD16+ cells (approximately 40%) coexpressed GL183 surface antigen, reactivity with GL183 mAb appears to define two subsets within the CD3- lymphocyte population expressing NK cell markers. Although, the majority of GL183+ cells were CD3-, approximately 1% expressed CD3 surface antigens. As shown by clonal analysis, these infrequent CD3+GL183+ cells coexpressed CD56 and CD16 antigens. Cloning of CD3-GL183+ or CD3-GL183- cell populations under limiting dilution conditions yielded clonal progenies that maintained their original surface phenotype. Therefore, expression or lack of expression of GL183 surface antigens represents a stable phenotypic property of a subset of human CD3- NK cells. Immunoprecipitation experiments and two-dimensional PAGE analysis indicated that GL183-reactive molecules were represented in different clones either by a single 58-kD chain or, more frequently, by two chains of approximately 55 and approximately 58 kD, respectively. Analysis of GL183+ or GL183- NK clones for their ability to lyse human (IGROV I) or murine (P815) tumor target cells indicated that GL183- clones were, on average, fivefold more efficient in inducing target cell lysis. GL183+ and GL183- clones produced comparable levels of TNF-alpha in response to PHA plus PMA or anti-CD16 mAb plus PMA. Importantly, production of TNF-alpha was also induced by stimulation of GL183+ clones with GL183 mAb plus PMA. These data indicated that GL183 antigen could mediate cell triggering. This concept was confirmed by the analysis of Ca2+ mobilization, as GL183 mAb induced (in GL183+ clones) increments of [Ca2+]i comparable with those induced by PHA. Moreover, GL183 mAb, or its F(ab')2 fragments, strongly enhanced the cytolytic activity of GL183+ clones against a panel of human tumor target cells, including U937, Raji, IGROV I, M14, and A549. In contrast, GL183 mAb, but not the F(ab')2 fragments, sharply inhibited the cytolytic activity of the same clones against P815, M12, and P3U1 murine target cells. In this case, the effect of GL183 mAb (inhibition) was opposite that of PHA or of stimulatory anti-CD2 or anti-CD16 mAbs, which consistently enhanced the target cell lysis.(ABSTRACT TRUNCATED AT 400 WORDS)
Downregulation of tumor necrosis factor receptors on macrophages and endothelial cells by microtubule depolymerizing agents.
Authors: Ding A H , Porteu F , Sanchez E , Nathan C F
Published: June 2004

Exposure of murine and human macrophages and human umbilical vein endothelial cells to micromolar concentrations of five microtubule (MT)-depolymerizing agents (colchicine, nocodazole, podophyllotoxin, vincristine, and vinblastine) resulted in a loss of binding sites for iodinated TNF-alpha. The reduction amounted to 40-60% by 1 h and approximately 75% by 2-4 h. In 1 h, specific binding was reduced 50% by 0.1-5 microM of these drugs at 37 degrees C, but not at 4 degrees C. Inactive isomers of colchicine were ineffective, as were microfilament-destabilizing cytochalasins. The active agents did not compete with TNF-alpha R for binding. Antiserum against TNF-alpha did not neutralize the effect of colchicine and nocodazole. PGE1 and dibutyryl-cAMP could not mimic, and cyclooxygenase inhibitors could not prevent the drug effects. All the binding sites were regenerated within 3 h after removal of nocodazole, which binds tubulin reversibly, whereas little recovery was found even 18 h after the removal of colchicine, which binds tubulin irreversibly. These findings suggested that MT disassembly was responsible for the observed downregulation of TNF-alpha R. The protein synthesis inhibitor cycloheximide inhibited binding of TNF-alpha to a similar extent and with a similar time course as colchicine in the absence of added ligand. Neither drug affected binding of IFN-gamma to macrophages, nor binding of TNF-alpha to human polymorphonuclear leukocytes. Thus, an intact MT network appears to be important in maintenance of the steady state of TNF-alpha R on those cells in which TNF-alpha R turns over rapidly in the absence of ligand. The antiinflammatory actions of MT-depolymerizing agents may result in part from their interference with the ability of such cells to respond to TNF-alpha.
Cyclosporin A inhibition of interleukin 2 gene expression, but not natural killer cell proliferation, after interferon induction in vivo.
Authors: Kasaian M T , Biron C A
Published: June 2004

The IFN inducer, poly(I:C), elicits acute NK cell blastogenesis and proliferation in vivo. The role of IL-2 in mediating this proliferation was investigated in the studies presented here. Blast NK cells were isolated from poly(I:C)-treated, T cell-deficient athymic mice. Dividing cells, incorporating [3H]thymidine, were enriched in the J11d- low density populations isolated from poly(I:C)-treated mice, and were characterized as NK by the following criteria: (a) they were eliminated by treatment with anti-AGM1 in vivo; and (b) they directly mediated lysis of NK-sensitive target cells in a single cell cytotoxicity assay with autoradiography. These poly(I:C)-induced blast NK cells were responsive to IL-2, but, when compared with in vivo activated T cells, responsiveness required 1,000-fold higher concentrations of the factor. The technique of in situ hybridization was used to evaluate induction of IL-2 gene expression after poly(I:C) treatment in vivo. Treatment of euthymic, athymic, and severe combined immunodeficient mice with poly(I:C) activated IL-2 gene expression in a small percentage of spleen leukocytes. The transcription-positive cells were enriched in low density cell populations. These findings demonstrate that IL-2 transcription occurs after IFN induction in vivo, and suggest that an endogenous source of IL-2 exists other than the mature T cell. To assess the IL-2 dependence of in vivo NK cell expansion, poly(I:C)-treated athymic mice were given cyclosporin A (CsA), an agent that regulates IL-2 production at the level of gene transcription. The drug resulted in an 85-100% reduction in the percentages of cells transcribing IL-2. In contrast, CsA administration did not block IFN-enhanced NK cell cytolytic activity, expansion of large granular lymphocyte numbers, or NK cell proliferation. These findings demonstrate that although the proliferation of blast NK cells can be supported by IL-2, IL-2 is not an important mediator of IFN-induced NK cell expansion. Moreover, they establish that the acute proliferation of NK cells in response to IFNs is CsA insensitive.
Cytotoxic T cells recognize a peptide from the circumsporozoite protein on malaria-infected hepatocytes.
Authors: Weiss W R , Mellouk S , Houghten R A , Sedegah M , Kumar S , Good M F , Berzofsky J A , Miller L H , Hoffman S L
Published: June 2004

Irradiated malaria sporozoites can induce CD8+ T cells that are required for protection against infection. However, the parasite antigens targeted by this immune response are unknown. We have discovered a 16-amino acid epitope from the Plasmodium yoelii circumsporozoite (CS) protein that is recognized by cytotoxic T cells from immune mice. Lymphocytes stimulated with this peptide can kill P. yoelii liver stage parasites in vitro in an MHC-restricted, antigen-specific manner. Thus, epitopes from the CS protein are presented on the surface of infected hepatocytes and can be targets for T cells, even though intact CS protein has not been detected on the surface of the infected hepatocyte. A vaccine that induced CTL to parasite antigens might protect humans against malaria by eliminating liver stage parasites.
Intracellular application of guanosine-5'-O-(3-thiotriphosphate) induces exocytotic granule fusion in guinea pig eosinophils.
Authors: Nüsse O , Lindau M , Cromwell O , Kay A B , Gomperts B D
Published: June 2004

The mechanism of eosinophil secretion was studied in guinea pig eosinophils by measuring release of hexosaminidase from cell suspensions (greater than 98% pure) permeabilized with streptolysin-O and by whole-cell patch-clamp capacitance measurements. It is shown that release of eosinophil granule components occurs by an exocytotic mechanism in which individual granules fuse with the plasma membrane. Exocytosis can be induced by intracellular application of the nonhydrolyzable GTP analog guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting the involvement of a GTP-binding protein. The activation is modulated by the intracellular calcium concentration, with activation by GTP-gamma-S inducing transient elevations in the concentration of Ca2+. Thus, the nature and regulation of the release mechanism appear to be very similar to that of the mast cell and neutrophil.
Human anti-p68 autoantibodies recognize a common epitope of U1 RNA containing small nuclear ribonucleoprotein and influenza B virus.
Authors: Guldner H H , Netter H J , Szostecki C , Jaeger E , Will H
Published: June 2004

Autoantibodies from patients with systemic rheumatic diseases were used to map antigenic sites on the 68-kD autoantigen (p68) associated with (U1)RNA-containing small nuclear ribonucleoprotein (snRNP) particles. With truncated recombinant fusion proteins and synthetic peptides, a subset of anti-p68 autoantibodies was found to recognize the amino acid sequence motif Glu-Arg-Lys-Arg-Arg (ERKRR). To investigate the possible involvement of epitopes shared by microbial antigens and host self-components in initiation of autoimmunity (molecular mimicry), a sequence data bank was screened for proteins containing an amino acid motif identical or related to ERKRR. The identical motif was found on the M1 matrix protein of influenza B viruses, and affinity-purified human anti-ERKRR autoantibodies recognized this epitope also in the viral amino acid sequence context. The common epitope recognized by human autoantibodies suggests that influenza B virus infection may play a role in initiation of the anti-p68 and anti-(U1)RNP autoimmune response.
Recognition of a mycobacteria-specific epitope in the 65-kD heat-shock protein by synovial fluid-derived T cell clones.
Authors: Gaston J S , Life P F , Jenner P J , Colston M J , Bacon P A
Published: June 2004

Adjuvant arthritis in rats is induced by a T cell clone specific for amino acids 180-188 of the mycobacterial 65-kD heat-shock protein, and synovial T cell responses to this same Ag have been noted in human arthritis. We have isolated 65-kD Ag-specific T cell clones from synovial fluid mononuclear cells of a patient with acute arthritis, which, unlike the corresponding PBMC, showed a marked proliferative response to the 65-kD Ag. Using synthetic peptides corresponding to the whole sequence of the 65-kD Ag, all the clones were shown to recognize an epitope present in the first NH2-terminal peptide (amino acids 1-15), with no response to the adjacent peptide (amino acids 6-22) or to any other peptide. The complete dominance of this epitope in the response to the 65-kD Ag was shown by documenting responses to the peptide in PBMC obtained after recovery from the arthritis. This epitope, like that recognized by the rat arthritogenic T cell clone, is in a portion of the 65-kD sequence that is not conserved between bacteria and eukaryotes, so that in this case, joint inflammation could not be attributed to bacteria-induced T cell clones cross-reacting with the self 65-kD Ag.
Association between protective efficacy of anti-lipopolysaccharide (LPS) antibodies and suppression of LPS-induced tumor necrosis factor alpha and interleukin 6. Comparison of O side chain-specific antibodies with core LPS antibodies.
Authors: Baumgartner J D , Heumann D , Gerain J , Weinbreck P , Grau G E , Glauser M P
Published: June 2004

Two-core LPS antibodies, the rabbit J5 polyclonal antiserum and the human anti-lipid A IgM mAb HA-1A, did not improve the survival of mice challenged with E. coli O111 or P. aeruginosa 3, or with the LPS extracted from them, and did not decrease the incidence of Shwartzman reactions in rabbits challenged with O111 LPS. In contrast, O side chain-specific rabbit antisera were protective in these models. The protection afforded by O side chain-specific antisera against endotoxin lethality was associated with decreased LPS-induced serum TNF and IL-6 levels, whereas core LPS antibodies had no effect on TNF or IL-6 levels. The absence of reduction of LPS-induced cytokines levels by core LPS antibodies suggests that these antibodies are not able to prevent the interactions between LPS and target cells.
Specialized functions of MHC class I molecules. I. An N-formyl peptide receptor is required for construction of the class I antigen Mta.
Authors: Shawar S M , Cook R G , Rodgers J R , Rich R R
Published: June 2004

Maternally transmitted factor (Mtf) is a mitochondrial gene that controls the antigenic polymorphism of the MHC class I maternally transmitted antigen (Mta). Synthetic peptides from the NH2 terminus of the mitochondrially encoded NADH dehydrogenase subunit 1 (ND1) mimic Mtf peptide activity in an allele-specific manner. We show that the minimal ND1-alpha peptide length recognized by Mtaa-specific polyclonal CTLs was between 8 and 12 amino acids, while some Mtaa-specific CTL clones recognized a six amino acid peptide. The N-formyl group at the NH2 terminus of ND1 was essential for Mta activity. Competition experiments using N-substituted ND1-alpha peptides showed that an N-formyl peptide receptor on the target cell, which differs from the chemotactic peptide receptor, was required for Mta expression. The specificity of this receptor can account for the distinct immune restriction of Mta in which Mtf peptides are uniquely restricted by Hmt. It is possible that the Hmt gene product is the N-formyl peptide receptor itself and that it represents a class I antigen presentation molecule specialized for binding, transport, and immune presentation of N-formyl-peptide antigens of mitochondrial and prokaryotic origin.
Antiinflammatory peptides (antiflammins) inhibit synthesis of platelet-activating factor, neutrophil aggregation and chemotaxis, and intradermal inflammatory reactions.
Authors: Camussi G , Tetta C , Bussolino F , Baglioni C
Published: June 2004

Synthetic peptides corresponding to the region of highest similarity between human lipocortin I and rabbit uteroglobin inhibit phospholipase A2 and show potent antiinflammatory activity on the carrageenan-induced rat footpad edema. The peptide HDMNKVLDL (antiflammin-2) inhibits the synthesis of platelet-activating factor (PAF) induced by TNF or phagocytosis in rat macrophages and human neutrophils, and by thrombin in vascular endothelial cells. The peptide MQMKKVLDS (antiflammin-1) is less inhibitory than antiflammin-2 for macrophages and not inhibitory for neutrophils after a 5-min preincubation. This finding suggests that antiflammin-1 is inactivated by neutrophils secretory products, possibly oxidizing agents. Synthesis of PAF is inhibited by antiflammin-2 without an appreciable lag, but this inhibition is reversed when neutrophils or macrophages are washed and incubated in fresh medium. Therefore, antiflammins must be continuously present to inhibit PAF synthesis. Antiflammins block activation of the acetyltransferase required for PAF synthesis, suggesting that this enzyme is another target for the inhibitory activity of antiflammins. These peptides inhibit neutrophil aggregation and chemotaxis induced by complement component C5a. Antiflammin-2 suppresses the increase in vascular permeability and the leukocyte infiltration induced in rats by an Arthus reaction or by intradermal injection of rTNF and C5a.
Reovirus-induced liver disease in severe combined immunodeficient (SCID) mice. A model for the study of viral infection, pathogenesis, and clearance.
Authors: George A , Kost S I , Witzleben C L , Cebra J J , Rubin D H
Published: June 2004

Adult severe combined immunodeficient (SCID) mice can be infected by the oral route with reovirus, and a systemic infection can be established. Infectious virus is recovered from all internal organs, and the mice die in 4-6 wk. Chronic, discrete inflammatory lesions appear in the liver of infected mice, and are associated with hepatocytes containing demonstrable levels of viral antigen. The adoptive transfer of Peyer's patch (PP) cells from congenic mice before infection protects the SCID mice against disease and death. Immune donor PP cells can be distinguished from nonimmune cells by their ability to contain and resolve infection by 1 wk after challenge.
Response of mature unprimed CD8+ T cells to Mlsa determinants.
Authors: Webb S R , Sprent J
Published: June 2004

Contrary to existing dogma, evidence is presented that proliferative responses of mature unprimed T cells to Mlsa antigens involve CD8+ cells as well as CD4+ cells. The response of CD8+ cells to Mlsa antigens proved to be heavily dependent on help from CD4+ cells, and responses were stronger in three I-E+ strain combinations than in an I-E- combination. In I-E+ combinations, CD8+ blast cells accounted for 20-25% of the blasts generated from unseparated T cells responding to Mlsa-bearing stimulator cells in vitro; similar findings applied to blast cells generated in vivo. The observation that the majority (greater than or equal to 50%) of Mlsa-stimulated CD8+ cells (and CD4+ cells) were V beta 6+ indicated that CD8+ cells respond to Mlsa antigens, per se, rather than to nonspecific stimuli. Whether CD4+ and CD8+ cells use the same or different H-2-restricting elements to respond to Mlsa antigens has yet to be resolved.
Soluble CD23 (Fc epsilon RII) and interleukin 1 synergistically induce early human thymocyte maturation.
Authors: Mossalayi M D , Lecron J C , Dalloul A H , Sarfati M , Bertho J M , Hofstetter H , Delespesse G , Debre P
Published: June 2004

The ability of human thymus-derived CD7+CD2-CD3- cells to acquire mature T cell antigens was assessed. Purified CD7+ thymocytes were incubated with rIL-1, rIL-2, and/or recombinant soluble CD23 (rsCD23). Short-term incubation of these cells with only rsCD23 + rIL-1 induced mature T cell antigen expression on at least half of the cells. The induction of CD2 was functionally significant, as these cells became able to respond to CD2 triggering and could proliferate in response to IL-2. Possible sources of CD23 in the thymus are under investigation.
Cytotoxic T lymphocytes reactive against a syngeneic murine tumor and their specific suppressor T cells are both elicited by in vitro allosensitization.
Authors: Leshem B , Kedar E
Published: June 2004

Sensitization of C57BL/6 (B6, H-2b) splenocytes against normal BALB/c (H-2d) leukocytes (B6 a/BALB) in bulk MLC induced CTL reactive against the syngeneic (H-2b) nonimmunogenic lymphoma PIR-2, in addition to the CTL directed against the corresponding (H-2d) allotargets. However, MLC-derived lymphocytes did not directly exhibit anti-PIR-2 cytotoxicity in spite of the high anti-PIR-2 CTL frequency (up to 1/20) among them, as demonstrated by the limiting dilution culture (LDC) technique. The present study was undertaken to resolve this contradiction. We found that anti-PIR-2 cytotoxicity could be detected only when B6 a/BALB MLC-derived responding cells were plated in LDC at low numbers (less than 200) of cells/well. In contrast, increasing the number of the plated cells to 500-5,000 resulted in a gradual decrease in the percentage of wells cytotoxically reactive against PIR-2, whereas the percentage of wells exhibiting cytotoxicity against the allotargets remained unchanged (100%). This decrease of anti-PIR-2 cytotoxicity in LDC and the lack of anti-PIR-2 reactivity among MLC-derived lymphocytes were shown by mixing experiments to result from the activity of radioresistant Thy-1+, Lyt-2+, L3T4- suppressor cells, blocking the anti-PIR-2 cytotoxicity at the effector phase. The suppression was specific as indicated by the following observations: (a) freshly obtained B6 splenocytes, cultured unsensitized B6 splenocytes, mitogen-induced B6 lymphoblasts, B6 LAK cells, or B6 a/B6 MLC-derived lymphocytes were not suppressive; (b) anti-PIR-2 cytotoxicity elicited in B6 a/BALB LDC was suppressed only by lymphocytes derived from B6 a/BALB MLC and not from B6 a/C3H (H-2k) MLC; and (c) B6 a/BALB MLC-induced suppressor cells could be adsorbed on monolayers of BALB/c but not of C3H lymphoblasts. Since both syngeneic tumor and allogeneic target cells were lysed by the same clonal cell population but only the antisyngeneic activity was suppressed, we suggest that a single CTL can exhibit two cytotoxic activities that are differentially affected by the described suppressor cells. This mode of suppression may play a role in controlling autoimmune reactivity.
Murine interleukin 7 (IL-7) receptor. Characterization on an IL-7-dependent cell line.
Authors: Park L S , Friend D J , Schmierer A E , Dower S K , Namen A E
Published: June 2004

A murine cell line (IxN/2b) absolutely dependent upon exogenous IL-7 for continued growth has been obtained that expresses lymphoid precursor and class I MHC antigens and also contains a rearranged mu heavy chain. This cell line has been used to define the binding and structural characteristics of the murine IL-7 receptor using 125I-labeled recombinant murine IL-7. 125I-IL-7 binding to IxN/2b cell was rapid and saturable at both 4 degrees and 37 degrees C. Equilibrium binding studies produced curvilinear Scatchard plots at both temperatures with high and low affinity Ka values of approximately 1 x 10(10) M-1 and 4 x 10(8) M-1, respectively, and a total of 2,000-2,500 IL-7 binding sites expressed per cell. Experiments measuring inhibition of binding of 125I-IL-7 by unlabeled IL-7 also produced data consistent with the existence of two classes of IL-7 receptors. Evidence concerning the possible molecular nature of two classes of IL-7 receptors was provided by dissociation kinetics and affinity crosslinking experiments. The dissociation rate of 125I-IL-7 was markedly increased when measured in the presence of unlabeled IL-7 at both 37 degrees and 4 degrees C, which is diagnostic of a receptor population displaying negative cooperativity. Crosslinking studies showed that under both reducing and nonreducing conditions, the major crosslinked species observed corresponded to a receptor size of 75-79 kD while a less intense higher molecular mass crosslinked species was also seen which corresponded to a receptor size approximately twice as large (159-162 kD). Both types of experiments suggest that the IL-7 receptor may form noncovalently associated dimers in the membrane. The IL-7 receptor was expressed on pre-B cells, but not detected on several murine B cell lines or primary mature B cells. It was also expressed on murine thymocytes, some T lineage cell lines, and on bone marrow-derived macrophage. All cells binding 125I-IL-7 exhibited curvilinear Scatchard plots. No cytokines or growth factors tested were able to inhibit binding of 125I-IL-7 to its receptor. These results define the initial binding and structural characteristics, and the cellular distribution, of the murine IL-7 receptor.
Strong T cell tolerance in parent----F1 bone marrow chimeras prepared with supralethal irradiation. Evidence for clonal deletion and anergy.
Authors: Gao E K , Lo D , Sprent J
Published: June 2004

T cell tolerance induction was examined in long-term H-2-heterozygous parent----F1 chimeras prepared with supralethal irradiation (1,300 rad). Although these chimeras appeared to be devoid of host-type APC, the donor T cells developing in the chimeras showed marked tolerance to host-type H-2 determinants. Tolerance to the host appeared to be virtually complete in four assay systems: (a) primary mixed lymphocyte reactions (MLR) of purified lymph node (LN) CD8+ cells (+/- IL-2); (b) primary MLR of CD4+ (CD8-) thymocytes; (c) skin graft rejection; and (d) induction of lethal graft-vs.-host disease by CD4+ cells. Similar tolerance was observed in chimeras given double irradiation. The only assay in which the chimera T cells failed to show near-total tolerance to the host was the primary MLR of post-thymic CD4+ cells. In this assay, LN CD4+ cells regularly gave a significant antihost MLR. The magnitude of this response was two- to fourfold less than the response of normal parental strain CD4+ cells and, in I-E(-)----I-E+ chimeras, was paralleled by approximately 70% deletion of V beta 11+ cells. Since marked tolerance was evident at the level of mature thymocytes, tolerance induction in the chimeras presumably occurred in the thymus itself. The failure to detect host APC in the thymus implies that tolerance reflected contact with thymic epithelial cells (and/or other non-BM-derived cells in the thymus). To account for the residual host reactivity of LN CD4+ cells and the incomplete deletion of V beta 11+ cells, it is suggested that T cell contact with thymic epithelial cells induced clonal deletion of most of the host-reactive T cells but spared a proportion of these cells (possibly low affinity cells). Since these latter cells appeared to be functionally inert in the thymus (in contrast to LN), we suggest that the thymic epithelial cells induced a temporary form of anergy in the remaining host-reactive thymocytes. This anergic state disappeared when the T cells left the thymus and reached LN.