bluuebunny/crossref_metadata_2025 huggingface dataset:
A model of human cytokine regulation based on transfection of gamma interferon gene fragments directly into isolated peripheral blood T lymphocytes.
Authors: Chrivia J C , Wedrychowicz T , Young H A , Hardy K J
Published: June 2004

An approach has been optimized permitting measurement of human cytokine reporter gene expression after transient transfection directly into purified human peripheral blood T lymphocytes. Comparing the expression of interleukin 2 (IL-2) CAT with a series of specially engineered gamma interferon (IFN-gamma) constructs, a fundamental difference in the molecular mechanisms regulating these two cytokines has been suggested. A potent, tissue-specific, constitutive-acting positive regulatory element was located between sequences -215 and -53 in the human IFN-gamma gene. Deletion analyses suggested that sequences slightly upstream, between positions -251 to -215, exerted a powerful dominant suppressive influence over that positive element. Negative elements appear to play a major role in controlling the regulation of human IFN-gamma gene expression. We thus propose a model of cytokine gene regulation in which selective derepression may be an important fundamental mechanism of induction and/or positive modulation.
Induction of peripheral tolerance to class I major histocompatibility complex (MHC) alloantigens in adult mice: transfused class I MHC-incompatible splenocytes veto clonal responses of antigen-reactive Lyt-2+ T cells.
Authors: Heeg K , Wagner H
Published: June 2004

The efficacy and the mode of action of pretransplant transfusion with class I major histocompatibility complex (MHC)-disparate splenocytes in establishing a state of peripheral tolerance in adult mice is analyzed. Adult mice injected intravenously with a critical number of approximately 5 x 10(7) allogenic splenocytes accept skin grafts and develop chimerism in the peripheral lymphatic tissues, but not in thymus and bone marrow. In parallel, a split tolerance evolves: the frequency of class I MHC-reactive Lyt-2+ cytotoxic T lymphocyte precursor (CTL-p)- and interleukin 2 (IL-2)-producing T cells falls off in the peripheral lymphoid tissue, but remains unaltered intrathymically. In particular, high affinity CTL-p become clonally undetectable. In vivo generation of tolerant cells is cyclosporin A resistant, but dependent on recipient L3T4+ T cells. Loss of Lyt-2+ CTL-p- and IL-2-producing T cell precursors is not due to active suppression, but is caused by clonal anergy. Donor-derived chimeric cells positively selected 7 d after intravenous transfusion exhibit in vitro the hallmarks of veto cells, i.e., paralyze CTL-p reactive to donor-type class I MHC alloantigens. We conclude that the peripheral (split) tolerance induced in vivo by pretransplant transfusion operates because donor-type cells develop in vivo efficiently into "veto cells," which in turn induce a state of clonal anergy within antigen-reactive Lyt-2+ T lymphocytes.
Transforming growth factor beta is a potent inhibitor of interleukin 1 (IL-1) receptor expression: proposed mechanism of inhibition of IL-1 action.
Authors: Dubois C M , Ruscetti F W , Palaszynski E W , Falk L A , Oppenheim J J , Keller J R
Published: June 2004

Transforming growth factor beta (TGF-beta) acts as a potent inhibitor of the growth and functions of lymphoid and hemopoietic progenitor cells. Cell proliferation depends not only on the presence of growth factors, but also on the development of specific receptor-signal transducing complexes. We therefore investigated whether the inhibitory actions of TGF-beta could be mediated by inhibition of growth factor receptors. TGF-beta inhibited the constitutive level of interleukin 1 receptor (IL-1R) expression on several murine lymphoid and myeloid progenitor cell lines, as well as IL-1R expression induced by interleukin 3 (IL-3) on normal murine and human bone marrow cells. Furthermore, treatment of bone marrow progenitor cells with TGF-beta concomitantly inhibited the ability of IL-1 to promote high proliferative potential (HPP) colony formation as well as blocked IL-1-induced IL-2 production by EL-4 6.1 cells. These findings provide the first evidence that the inhibitory action of TGF-beta on the growth and functional activities of hematopoietic and T cells is associated with a reduction in the cell surface receptor expression for IL-1.
Variants selected by treatment of human immunodeficiency virus-infected cells with an immunotoxin.
Authors: Pincus S H , Wehrly K , Tschachler E , Hayes S F , Buller R S , Reitz M
Published: June 2004

An immunotoxin has been made by coupling anti-human immunodeficiency virus (HIV) envelope antibody 907 to ricin A chain (907-RAC). 907 recognizes an epitope within the immunodominant PB-1 loop of gp120. Variant cells were selected by cloning persistently infected H9/human T lymphocyte virus IIIB cells in the presence of the immunotoxin. Clones resistant to 907-RAC arose at a frequency of 0.1-1.0%. Seven clones were selected for intensive analysis. When studied, these clones fell into two distinct groups, members of which appeared to be identical, suggesting that the variation arose before the selection process. In contrast to the parent cells, none of the cloned variants produced infectious HIV. The first set of clones, designated the "E" variants, expressed decreased levels of the HIV envelope on the cell surface. However, levels of intracellular HIV antigens and reverse transcriptase were equal to or greater than that of the parental cell line. Radioimmunoprecipitation demonstrated that the gp160 was truncated to 145 kD (gp120 was normal length), capable of binding to CD4, and, unlike normal gp160, was released in its unprocessed form into the cellular supernatant. Sequence analysis demonstrated that a deletion at codon 687 of the envelope gene resulted in the production of this truncated protein. Ultrastructural analysis of E variants demonstrated some budding forms of virus, but also large numbers of HIV within intracellular vesicles. The second set of variants, the "F" series, produced no HIV antigens, reverse transcriptase, nor was there ultrastructural evidence of virus. However, proviral DNA was present. Virus could not be induced with agents known to activate latent HIV. These cells also lacked cell surface CD4 and could not be infected with HIV. These studies demonstrate that variation in HIV can affect the phenotype of the cells carrying the altered virus, allowing for escape from immunologic destruction. The E variants may serve as prototypes for attenuated HIV, which could be used as a vaccine. We have reconstructed the mutation found in the E variants within the infectious HIV clone HXB-2 and demonstrated that the resulting virus retains its noninfectious phenotype.
Involvement of a phospholipase D in the mechanism of action of granulocyte-macrophage colony-stimulating factor (GM-CSF): priming of human neutrophils in vitro with GM-CSF is associated with accumulation of phosphatidic acid and diradylglycerol.
Authors: Bourgoin S , Plante E , Gaudry M , Naccache P H , Borgeat P , Poubelle P E
Published: June 2004

The generation of diradylglycerol (DRG) and phosphatidic acid (PdtOH) was investigated in neutrophils primed with granulocyte-macrophage colony-stimulating factor (GM-CSF). Mass accumulation of DRG and PdtOH was measured using reversed-phase high performance liquid chromatography and thin layer chromatography, respectively. GM-CSF had no direct effect on the levels of PdtOH and DRG, but it increased PdtOH generation and the late phase of DRG accumulation in human neutrophils stimulated with FMLP. The elevation of the mass of PdtOH peaked approximately 100 s and clearly preceded that of DRG, which peaked at 150 s. The diacylglycerol kinase inhibitor R59022 enhanced the sustained increase in DRG but did not produce a parallel inhibition in PdtOH production. GM-CSF was without effect on the level of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] and did not affect the liberation of Ins(1,4,5)P3 induced by FMLP. These findings exclude the involvement of the PtdIns(4,5)P2-specific phospholipase C/diacylglycerol pathway in the sustained phase of DRG accumulation. The early (30-s) appearance of PdtOH clearly suggests that GM-CSF enhanced FMLP receptor-linked phospholipase D (PLD) generation of PdtOH. PLD was assessed more directly by formation of labeled phosphatidylethanol (PEt) through PLD capacity of catalyzing a trans-phosphatidylation in presence of ethanol. The formation of PEt associated with a concomitant decrease in PdtOH directly demonstrated that the mechanism by which GM-CSF enhances PdtOH production is activation of a PLD active on phosphatidylcholine. This study provides evidence that the mechanism of action of GM-CSF involves upregulation of PLD activity leading to enhanced generation of PdtOH and DRG in FMLP-stimulated neutrophils. These findings may provide the basis for several of the priming effects of GM-CSF.
Cytotoxic T cell recognition of an endogenous class I HLA peptide presented by a class II HLA molecule.
Authors: Chen B P , Madrigal A , Parham P
Published: June 2004

Human leukocytes were stimulated in vitro with peptides corresponding in sequence to the highly variable helix of the alpha 1 domain of various HLA-B and -C molecules. A CD4+ CD8- cytotoxic T cell line, CTL-AV, that is specific for the HLA-B7 peptide presented by HLA-DR11.1 was obtained. The HLA-DR11.2 molecule, which only differs at three residues from HLA-DR11.1, did not present the HLA-B7 peptide to CTL-AV. Peptides from the alpha 1 domain helix of other HLA-A and HLA-B molecules, but not HLA-C molecules, competed with the HLA-B7 peptide for binding to HLA-DR11.1. A cell line (WT50) that coexpresses HLA-B7 and HLA-DR11.1 was killed by CTL-AV in the absence of any added HLA-B7 peptide. The processing and presentation of HLA-B7 in these cells appears to be through the endogenous, and not the exogenous, pathway of antigen presentation. Thus, Brefeldin A inhibits presentation and chloroquine does not. Furthermore, introduction of purified HLA-B7 molecules into HLA-DR11.1+, HLA-B7- cells by cytoplasmic loading via osmotic lysis of pinosomes, but not by simple incubation, rendered them susceptible to CTL-AV killing. These results provide an example of class II major histocompatibility complex (MHC) presentation of a constitutively synthesized self protein that uses the endogenous pathway of antigen presentation. They also emphasize the capacity for presentation of MHC peptides by MHC molecules.
Distinct antibody specificities to a 64-kD islet cell antigen in type 1 diabetes as revealed by trypsin treatment.
Authors: Christie M R , Vohra G , Champagne P , Daneman D , Delovitch T L
Published: June 2004

Type 1 diabetes is associated with antibodies that immunoprecipitate a 64-kD islet cell membrane protein from detergent extracts of pancreatic islets. In this study we have determined whether mild trypsin treatment of islet membranes can release fragments of the antigen that bind antibodies in the serum of Type 1 diabetic patients. Partial tryptic proteolysis of [35S]methionine-labeled 64-kD antigen immunoprecipitated from detergent extracts of rat islets resulted in the formation of 50-, 40-, and 37-kD fragments. Similar sized fragments were recovered when sera from diabetic patients were employed to immunoprecipitate polypeptides solubilized by mild trypsin treatment of a particulate fraction of radiolabeled rat islets. Of 27 diabetic patients, 22 possessed antibodies to the 50-kD polypeptide and 21 to the 40- and 37-kD polypeptides. A positive association was found between 64k antibodies and antibodies to the 50-kD fragment but not between 64k antibodies and antibodies to the 40- or 37-kD fragments. Some 64k antibody negative patients possessed antibodies that efficiently immunoprecipitated the latter fragments. Serum from 25 of 27 (93%) diabetic patients immunoprecipitated at least one of the three tryptic polypeptides. One of 20 nondiabetic controls immunoprecipitated a 50-kD polypeptide and all controls were negative for antibodies to 40- and 37-kD fragments. Thus, Type 1 diabetes is associated with the presence of at least two antibody reactivities to distinct determinants of the 64-kD antigen, and some patients may possess antibodies to a cryptic epitope on the detergent-solubilized molecule. These data suggest that the detection of antibodies (present in 93% of patients) to epitopes on tryptic polypeptides of the 64-kD antigen may be of even greater diagnostic value for the onset of Type 1 diabetes than analyses of antibodies reactive with the intact 64-kD antigen.
Evidence of widespread binding of HLA class I molecules to peptides.
Authors: Frelinger J A , Gotch F M , Zweerink H , Wain E , McMichael A J
Published: June 2004

We have tested the binding of HLA class I proteins to peptides using a solid-phase binding assay. We tested 102 peptides, mostly derived from the HIV gag and HIV pol sequences. Most peptides did not bind to any class I protein tested. The pattern of binding among the three class I proteins tested, HLA-A2, -B27, and -B8, was approximately 85% concordant. Further, all five of the known HIV-1 gag T cell epitopes detected by human CTL bound at least one class I protein. Binding of class I to the peptides could be detected either by directly iodinated class I proteins, or indirectly using monoclonal antibodies specific for class I. The binding to the plates could be blocked with MA2.1, which binds in the alpha 1 region of A2, but not by W6/32, which binds elsewhere. The data presented here show that binding of class I to peptides is specific, but that many peptides bind to more than a single class I protein.
Metabolic, humoral, and cellular responses in adult volunteers immunized with the genetically inactivated pertussis toxin mutant PT-9K/129G.
Authors: Podda A , Nencioni L , De Magistris M T , Di Tommaso A , Bossù P , Nuti S , Pileri P , Peppoloni S , Bugnoli M , Ruggiero P
Published: June 2004

PT-9K/129G, a nontoxic mutant of pertussis toxin (PT) obtained by genetic manipulation, has been shown in animal models to be a promising candidate for new vaccines against whooping cough. To assess the safety and the immunogenicity of PT-9K/129G in humans, a pilot study has been performed in adult volunteers. The protein was found to be safe, capable of inducing high titers of toxin-neutralizing antibodies, and capable of generating immunological memory. In fact, vaccination caused an increase of cell-mediated response to PT, PT-9K/129G, S1 subunit, and B oligomer, indicating that memory T cells are induced by the vaccine. Since PT-9K/129G is mitogenic for T lymphocytes in vitro, it was investigated whether this activity is also present in vivo. No variation was observed in the proportion of T cells (CD3+), T helper cells (CD4+), and cytotoxic T cells (CD8+), as well as in that of other lymphoid populations, by FACS analysis. Interestingly, no thorough correlation was found between humoral and cellular responses. In one case, a very high cellular response was present in absence of detectable antibodies, suggesting that the antibody response, which is the only parameter measured in most clinical trials, may not give a complete picture of the response induced by a vaccine.
Analysis of physical interactions between peptides and HLA molecules and application to the detection of human immunodeficiency virus 1 antigenic peptides.
Authors: Choppin J , Martinon F , Gomard E , Bahraoui E , Connan F , Bouillot M , Lévy J P
Published: June 2004

The physical association of 40 antigenic peptides and purified HLA class I and class II molecules was monitored using a direct peptide binding assay (PBA) in solid phase and an inhibition peptide binding assay (IPBA) in which the competing peptide was present in a soluble phase. We also examined the ability of different peptides to inhibit the lytic activity of human antiviral cytolytic T cells towards cells incubated with the corresponding target peptide. Our results showed that: (a) Binding of a given human T cell-recognized peptide to several HLA class I and class II molecules occurred frequently. Nevertheless, preferential binding of peptides to their respective restriction molecules was also observed. (b) Binding of HLA molecules to peptides recognized by murine T cells occurred less frequently. (c) 11 of 24 (46%) randomly selected HIV-1 peptides contained agretopic residues allowing their binding to HLA molecules. (d) The kinetics of HLA/peptide association depended on the peptide tested and were faster than or similar to those reported for Ia molecules. Dissociation of these complexes was very low. (e) Peptide/HLA molecule binding was dependent on length, number of positive charges, and presence of hydrophobic residue in the peptide. (f) A correlation was demonstrated between a peptide inhibitory effect in the IPBA and its blocking effect in the cytolytic test. Our data indicated that the restriction phenomenon observed in T cell responses was not strictly related to either an elective HLA/peptide association, or a high binding capacity of a peptide to HLA molecules. These data also showed that the PBA and IPBA are appropriate for the detection of agretopic residues within HIV-1 proteins.
Cloning of self-major histocompatibility complex antigen-specific suppressor cells from adult bone marrow.
Authors: Takahashi T , Mafune K , Maki T
Published: June 2004

We examined if suppressor cell clones may be established from adult bone marrow that contains a population of cells capable of specifically downregulating the immune response directed toward self-major histocompatibility complex (MHC) antigens. Freshly prepared adult C3H (H-2k) marrow cells were cultured in medium containing interleukin 2 (IL-2), IL-3, or a mixture of IL-2 and IL-3. After 7-10 d, cells grown in IL-3-containing medium were screened for their capacity to suppress cytotoxic T lymphocyte (CTL) generation against self-MHC antigens in allogeneic mixed lymphocyte cultures. Cells capable of suppressing anti-C3H CTL generation were cloned by limiting dilution. Several suppressor clones were established that exhibited strong suppression of anti-H-2k, anti-H-2Kk/Ik, and anti-H-2Dk CTL generation, but failed to suppress anti-H-2d and anti-H-2b responses. When tested in a skin allograft model, intravenous injections of these bone marrow-derived anti-self suppressor cells (2.5 x 10(7) cells) together with IL-3 induced prolongation of C3H skin allografts in anti-mouse lymphocyte serum-treated B6AF1 mice. Injection of IL-3 alone had no effect on allograft survival. Moreover, these cells failed to prolong B10.AKM skin allografts on B6AF1 recipients. Northern blot analysis showed that these cells express full-length transcripts of the T cell receptor (TCR) gamma gene, but not those of TCR alpha, beta, or delta genes. However, no rearrangement of gamma gene was observed by Southern blot analysis. Flow cytometric analysis revealed that bone marrow-derived suppressor cells are strongly positive for Thy-1 antigen but negative for CD3, CD4 (L3T4), and CD8 (Lyt-2) surface markers, and express only class I MHC antigens. Suppressor cells derived from adult bone marrow may play an important role in extrathymic induction of self-tolerance.
Cloning and characterization of cDNAs for murine macrophage inflammatory protein 2 and its human homologues.
Authors: Tekamp-Olson P , Gallegos C , Bauer D , McClain J , Sherry B , Fabre M , van Deventer S , Cerami A
Published: June 2004

A cDNA clone of murine macrophage inflammatory protein 2 (MIP-2) has been isolated from a library prepared from lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and the nucleotide sequence determined. This cDNA was used to clone cDNAs for human homologues of MIP-2 from a library prepared from phorbol myristate acetate-treated and LPS-stimulated U937 cells. Two homologues were isolated and sequenced. Human MIP-2 alpha and MIP-2 beta are highly homologous to each other and to a previously isolated gene, human gro/melanoma growth-stimulating activity (MGSA). These three human genes, MIP-2 alpha, MIP-2 beta, and gro/MGSA, constitute a sub-family within the cytokine family represented by platelet factor 4 and interleukin 8.
An endogenous processing pathway in vaccinia virus-infected cells for presentation of cytoplasmic antigens to class II-restricted T cells.
Authors: Jaraquemada D , Marti M , Long E O
Published: June 2004

The recognition of virus-infected cells by class I MHC-restricted cytotoxic T cells requires endogenous processing of antigen for presentation. It is still unclear whether endogenous processing of antigen can be utilized by class II MHC molecules for presentation. To test this possibility, a human B cell line expressing HLA-A2 and HLA-DR1 was infected with a recombinant vaccinia virus expressing the Influenza A virus M1 matrix protein (VAC-M1) and was assayed for lysis by different M1-specific cytolytic T cell lines, restricted by either HLA-A2 or by HLA-DR1. Class II-restricted lysis of VAC-M1-infected cells did occur. This lysis required de novo M1 synthesis and was not due to exogenous antigen. Several properties of the endogenous processing pathway for class II-restricted presentation were different from those of the pathway utilized by class I molecules. First, class II-mediated recognition of VAC-M1 infected cells was less efficient, requiring higher doses of virus and longer infection times, than the class I-mediated recognition. Second, chloroquine completely blocked presentation of endogenous M1 to class II-restricted T cells but had no effect on the class I-restricted presentation. Third, the class II-restricted presentation of M1 was only mildly affected by Brefeldin A, a drug that prevents transport from the endoplasmic reticulum to the Golgi, whereas the class I-restricted presentation of M1 was completely abrogated by this drug. These data demonstrate the existence of an endogenous processing pathway for the presentation of cytosolic antigen by class II molecules and show that this pathway is distinct from the one used for presentation by class I molecules.
Identification and characterization of autoantibodies against the nuclear envelope lamin B receptor from patients with primary biliary cirrhosis.
Authors: Courvalin J C , Lassoued K , Worman H J , Blobel G
Published: June 2004

We have identified autoantibodies from two patients with primary biliary cirrhosis (PBC) that recognize the nuclear envelope of mammalian cells on indirect immunofluorescence microscopy. These antibodies bind to a 58-kD integral membrane protein (p58) of the turkey erythrocyte nuclear envelope, which has been previously identified as a membrane receptor for lamin B (Worman, H. J., J. Yuan, G. Blobel, and S. D. Georgatos. 1988. Proc. Natl. Acad. Sci. USA. 85:8531). The antibodies also bind to a 61-kD integral membrane protein (p61) of the rat liver nuclear envelope. Affinity-purified antibodies eluted from turkey p58 bind to rat p61, showing that the two proteins share an epitope(s) and that p61 is likely the rat liver lamin B receptor. In human nuclear envelopes, the antigen recognized has an apparent molecular mass close to that of avian protein. These findings, along with the previous discovery of autoantibodies against an integral membrane glycoprotein (gp210) of the nuclear pore membrane in patients with PBC, suggest that antibodies against integral membrane proteins of the nuclear envelope are characteristic of a subset of patients with PBC.
The proteins encoded by the VpreB and lambda 5 pre-B cell-specific genes can associate with each other and with mu heavy chain.
Authors: Karasuyama H , Kudo A , Melchers F
Published: June 2004

The murine pre-B cell-specific genes VpreB and lambda 5, as well as the murine gene for mu heavy chain, were introduced into Ltk- fibroblast cells which normally do not express these genes. Stable transfectants carrying these genes produced the corresponding proteins of 15.5, 21.5, and 75 kD. They secreted the three proteins as a triple complex that could be immunoprecipitated by mu heavy chain-specific antibodies, consisting of one VpreB, one lambda 5, and one mu heavy chain. The mu heavy chain and lambda 5 were disulfide-bonded with each other, while the VpreB protein was noncovalently associated. These experiments proved that the VpreB, lambda 5 and mu H chain proteins can form a heavy/light chain-like heterocomplex.
The products of pre-B cell-specific genes (lambda 5 and VpreB) and the immunoglobulin mu chain form a complex that is transported onto the cell surface.
Authors: Tsubata T , Reth M
Published: June 2004

We constructed expression vectors coding for the two pre-B-specific genes, VpreB and lambda 5, and transfected them together with a mu vector (mu tm) into Ig- myeloma cells. In a transfectant expressing all three introduced genes, the mu tm chain is transported on the cell surface. A biochemical analysis demonstrated that, in these cells, the mu tm chain is associated noncovalently with an 18-kD protein and covalently with a 22-kD protein, which are most likely the products of VpreB and lambda 5, respectively. Our results, thus, strongly suggest that the products of lambda 5 and VpreB bind to mu chains and have the same capacity as conventional Ig L chains to allow surface expression of mu chains.
Co-infection of macrophages modulates interferon gamma and tumor necrosis factor-induced activation against intracellular pathogens.
Authors: Black C M , Bermudez L E , Young L S , Remington J S
Published: June 2004

Co-infection of macrophages (M phi) with Toxoplasma gondii and Mycobacterium avium-intracellulare complex (MAC) has been observed in patients with acquired immunodeficiency syndrome (AIDS). In this study we have demonstrated that co-infected murine M phi respond differently to cytokine stimulation than M phi infected with either of the microorganisms alone. Whereas treatment with interferon gamma (IFN-gamma) activated both single and co-infected groups of M phi to kill T. gondii, treatment with TNF did not influence the rate of MAC growth in co-infected M phi, in contrast with the inhibition of growth observed in MAC-infected M phi. These results suggest that in AIDS patients suffering infection with multiple intracellular pathogens, the ability of cytokines to stimulate microbicidal or static activity in mononuclear phagocytes can be impaired by the presence of more than one of the intracellular organisms.
Transfer of human systemic lupus erythematosus in severe combined immunodeficient (SCID) mice.
Authors: Duchosal M A , McConahey P J , Robinson C A , Dixon F J
Published: June 2004

To study the role of peripheral blood leukocytes (PBL) in the pathogenesis of human systemic lupus erythematosus (SLE), we transferred PBL from 5 SLE patients into 15 severe combined immunodeficiency (SCID) mice. Such reconstituted mice showed long-term presence of auto-antibodies characteristic of the donor in their sera, as well as human immunoglobulin deposition, and in some cases mouse C3, in the renal glomeruli. SCID mice repopulated with PBLs from normal donors do not develop serologic abnormalities or immunodeposits. It is concluded that human SLE serology and some associated renal changes can be reproduced solely by PBL transferred from afflicted patients, and that SCID-human-SLE mice may serve as an in vivo laboratory model for the study of human SLE.
Protein kinase regulates tumor necrosis factor mRNA stability in virus-stimulated astrocytes.
Authors: Lieberman A P , Pitha P M , Shin M L
Published: June 2004

Infection of astrocytes with Newcastle disease virus stimulated the production of 1,2-diacylglycerol, and resulted in the kinase-dependent expression of mRNAs encoding tumor necrosis factor (TNF), interferon alpha and beta, and interleukin 6. The half-life of TNF mRNA was significantly decreased in the presence of protein kinase inhibitors H-7 and staurosporine, but not in the presence of HA1004. In contrast to the decay of TNF mRNA, the half-lives of other cytokine mRNAs were only minimally affected by the kinase inhibitors. These data indicated that the stability of TNF mRNA was regulated through a novel, kinase-dependent pathway.
Mouse plasmacytoma growth in vivo: enhancement by interleukin 6 (IL-6) and inhibition by antibodies directed against IL-6 or its receptor.
Authors: Vink A , Coulie P , Warnier G , Renauld J C , Stevens M , Donckers D , Van Snick J
Published: June 2004

Murine plasmacytomas show a striking dependence on interleukin 6 (IL-6) for their growth in vitro. Here, we present evidence suggesting that IL-6 also plays an essential role in the in vivo development of these tumors. This conclusion is based on the finding that the tumorigenicity of an IL-6-dependent plasmacytoma cell line was increased approximately 100-fold on transfection with an IL-6 expression vector, whereas it was inhibited in animals treated with monoclonal antibodies capable of blocking the binding of IL-6 to its receptor. Injection of these antibodies 1 d before tumor challenge protected greater than 50% of the mice and retarded tumor growth in all animals. Tumors arising in antibody-treated mice retained their IL-6 dependence in vitro, suggesting that the level of protection could be improved if stronger IL-6 antagonists were available.
Molecular basis of polymorphisms of human complement component C3.
Authors: Botto M , Fong K Y , So A K , Koch C , Walport M J
Published: June 2004

C3 exhibits two common allotypic variants that may be separated by gel electrophoresis and are called C3 fast (C3 F) and C3 slow (C3 S). C3 F, the less common variant, occurs at appreciable frequencies only in Caucasoid populations (gene frequency = 0.20). An increased prevalence of the C3 F allele has been reported in patients with partial lipodystrophy, IgA nephropathy, and Indian childhood hepatic cirrhosis. Studies of the genomic organization of the human C3 gene led to the identification of a single change (C to G) between C3 S and C3 F at nucleotide 364 in exon 3. This leads, at the translation level, to the substitution of an arginine residue (positively charged) in C3 S for a glycine residue (neutral) in C3 F. This substitution results in a polymorphic restriction site for the enzyme HhaI. The resulting restriction fragment length polymorphism (RFLP) was investigated using genomic DNA, amplified using the polymerase chain reaction; there was absolute concordance between the genomic polymorphism and the distribution of C3 S and C3 F in 50 normal subjects. The molecular basis of a second structural polymorphism, defined by the monoclonal antibody HAV 4-1, was also characterized. The polymorphic determinant was identified at codon 314 in the exon 9 of the beta chain where a leucine residue (HAV 4-1+) is substituted for a proline residue (HAV 4-1-). Identification of the amino acid sequences of these polymorphic variants will facilitate characterization of possible functional differences between different allotypes of C3. Three RFLPs (BamHI, EcoRI, and SstI) were located to introns in the C3 gene. There was no allelic association between these three RFLPs, or between the RFLPs and the C3 F/S polymorphic site. Genetic equilibration of these polymorphisms has occurred within a gene of 41 kb.
Suppression of experimental allergic encephalomyelitis in Lewis rats after elimination of macrophages.
Authors: Huitinga I , van Rooijen N , de Groot C J , Uitdehaag B M , Dijkstra C D
Published: June 2004

Almost 50% of the cells infiltrating the central nervous system (CNS) of animals with experimental allergic encephalomyelitis (EAE) are macrophages (M psi). To investigate the role of the M psi in the pathogenesis of EAE, we eliminated M psi by means of mannosylated liposomes containing dichloromethylene diphosphonate (Cl2MDP). Cl2MDP-containing liposomes injected intravenously eliminate M psi in spleen and liver. Incorporation of mannose into the lipid layers enables the liposomes to pass the blood-brain barrier (BBB). Injections of Cl2MDP-containing mannose liposomes intravenously shortly before the appearance of clinical signs, markedly suppressed the expression of clinical signs of EAE. This suppression was accompanied by a marked reduction of infiltrated M psi in the CNS. Cl2MDP-containing liposomes without mannose incorporated had no effect. Cl2MDP-containing mannosylated liposomes had no effect on plasma corticosterone levels compared with injections of saline; thus, the suppression of expression of EAE was not corticosterone mediated. These results show that the M psi within the CNS play an important role in the pathogenesis of EAE.
Reinfection results in accumulation of unintegrated viral DNA in cytopathic and persistent human immunodeficiency virus type 1 infection of CEM cells.
Authors: Pauza C D , Galindo J E , Richman D D
Published: June 2004

High levels of unintegrated viral DNA accumulate during human immunodeficiency virus type 1 (HIV-1) infection of CEM T cells. Reinfection of already infected cells is required to attain these levels and reinfection also promotes the development of HIV-induced cytopathology. Rates of virus production, however, are independent of the accumulation of unintegrated viral DNA. Neutralizing antibody added soon after infection reduced viral DNA levels without appreciably affecting the production of cell-free viral p24 antigen or reverse transcriptase activity. Only 50 pM AZT were required to reduce the accumulation of unintegrated viral DNA by 50% in contrast to the 25 nM required to inhibit virus production by 50%. Cytopathology, as measured by number of syncytia in infected cell cultures, was correlated with highly elevated levels of unintegrated viral DNA. The minimal levels of unintegrated viral DNA present constitutively in the persistently infected HCEM cell line were consonant with the absence of cytopathic effects in these cells. These data demonstrate that inhibiting the reinfection of already infected cells modulates cytopathic HIV-1 infection to a form that is persistent and noncytopathic.
Genetic basis of viral persistence: single amino acid change in the viral glycoprotein affects ability of lymphocytic choriomeningitis virus to persist in adult mice.
Authors: Matloubian M , Somasundaram T , Kolhekar S R , Selvakumar R , Ahmed R
Published: June 2004

This study has identified a single amino acid change in the viral glycoprotein that profoundly affects the ability of lymphocytic choriomeningitis virus (LCMV) to persist in its natural host. Adult immunocompetent mice infected with a variant of the Armstrong strain, spleen isolate clone 13 (svA/svA), harbor virus for several months and exhibit suppressed T cell responses. In contrast, adult mice infected with a reassortant virus (svA/wtA) that contains the L segment of the spleen variant and the S segment of the parental wt Armstrong, make potent LCMV-specific CTL responses and clear the infection within 2-4 wk. These two viruses, spleen variant clone 13 and the reassortant svA/wtA, are identical in their noncoding regions and show no amino acid changes in any of their viral genes except for one substitution in the glycoprotein. The reassortant virus svA/wtA has a phenylalanine at amino acid residue 260 of the glycoprotein, whereas the spleen variant clone 13 has a leucine at this position. This study constitutes one of the first reports defining the genetic basis of viral persistence at the whole animal level, and identifying a single mutation that markedly increases the ability of a virus to persist in its natural host.
Long-term human hematopoiesis in the SCID-hu mouse.
Authors: Namikawa R , Weilbaecher K N , Kaneshima H , Yee E J , McCune J M
Published: June 2004

Coimplantation of small fragments of human fetal thymus and fetal liver into immunodeficient SCID mice resulted in the formation of a unique structure (Thy/Liv). Thereafter, the SCID-hu mice showed reproducible and long-term reconstitution of human hematopoietic activity. For periods lasting 5-11 mo after transplantation, active T lymphopoiesis was observed inside the grafts and cells that were negative for T cell markers were found to have colony-forming units for granulocyte/macrophage (CFU-GM) and erythroid burst-forming unit (BFU-E) activity in the methylcellulose colony assay. In addition, structures similar to normal human bone marrow were observed inside the Thy/Liv grafts, consisting of blast cells, mature and immature forms of myelomonocytic cells, and megakaryocytes. These data indicate long-term maintenance, in vivo, of human progenitor cells for the T lymphoid, myelomonocytic, erythroid, and megakaryocytic lineages. The role of the implanted fetal liver fragments was analyzed using HLA-mismatched Thy/Liv implants. The HLA type of the liver donor was found on T cells and macrophages in the graft. In addition, cells grown in the methylcellulose colony assay and cells in a bone marrow-like structure, the "thymic isle," expressed the HLA type of the liver donor. Thus, the Thy/Liv implants provided a microenvironment in which to follow human hematopoietic progenitor cells for multiple lineages. The formation of the Thy/Liv structures also results in a continuous source of human T cells in the peripheral circulation of the SCID-hu mouse. Though present for 5-11 mo, these cells did not engage in a xenograft (graft-versus-host) reaction. This animal model, the first in which multilineage human hematopoietic activity is maintained for long periods of time, should be useful for the analysis of human hematopoiesis in vivo.
Selective anergy of V beta 8+,CD4+ T cells in Staphylococcus enterotoxin B-primed mice.
Authors: Kawabe Y , Ochi A
Published: June 2004

The cellular basis of the in vitro and in vivo T cell responses to Staphylococcus enterotoxin B (SEB) has been investigated. The proliferation and cytotoxicity of V beta 8.1,2+,CD4+ and CD8+ T cells were observed in in vitro response to SEB. In primary cytotoxicity assays, CD4+ T cells from control spleens were more active than their CD8+ counterparts, however, in cells derived from SEB-primed mice, CD8+ T cells were dominant in SEB-specific cytotoxicity. In vivo priming with SEB abrogated the response of V beta 8.1,2+,CD4+ T cells despite the fact that these cells exist in significant number. This SEB-specific anergy occurred only in V beta 8.1,2+,CD4+ T cells but not in CD8+ T cells. These findings indicate that the requirement for the induction of antigen-specific anergy is different between CD4+ and CD8+ T cells in post-thymic tolerance, and the existence of coanergic signals for the induction of T cell anergy is suggested.
T cell target 1 (TCT.1): a novel target molecule for human non-major histocompatibility complex-restricted T lymphocytes.
Authors: Mami-Chouaib F , Miossec C , Del Porto P , Flament C , Triebel F , Hercend T
Published: June 2004

We have studied two gamma/delta T cell clones, E102 and E117, generated in a mixed lymphocyte culture using an allogeneic Epstein-Barr virus-transformed B cell line, E418. These clones were both found to express a molecular form of T cell receptor (TCR) infrequent in human peripheral blood, associating a V1-J1-C delta chain and a V3-JP2-C2 gamma chain. Functionally, they appeared as cytotoxic T lymphocytes (CTL) with non-major histocompatibility complex (MHC) (class I and II) requiring cytotoxicity, able to kill both the immunizing (i.e., E418) and unrelated (e.g., K562, REX, F601, and KAS) target cells. A monoclonal antibody, anti-10H3, able to selectively inhibit the cytotoxic activity of the clones has been produced. This reagent defines a 43-kD molecule, designated TCT.1, with broad distribution in the hematopoietic system, that appears to be distinct from class I MHC gene products. A series of functional experiments using various effector/target cell combinations strongly suggested that TCT.1 may represent a unique TCR ligand involved in the interaction between these particular CTL clones and certain of the target cells tested, while others were likely to be recognized and killed through a TCR-independent natural killer-like pathway. Although further experimentation will be needed to strengthen our interpretation of the present data, this study provides additional evidence that some T lymphocytes, in particular of the gamma/delta type, may interact specifically with target cells in a non-MHC class I/II-requiring fashion.
Oral Salmonella: malaria circumsporozoite recombinants induce specific CD8+ cytotoxic T cells.
Authors: Aggarwal A , Kumar S , Jaffe R , Hone D , Gross M , Sadoff J
Published: June 2004

Oral immunization with an attenuated Salmonella typhimurium recombinant containing the full-length Plasmodium berghei circumsporozoite (CS) gene induces protective immunity against P. berghei sporozoite challenge in the absence of antibody. We found that this immunity was mediated through the induction of specific CD8+ T cells since in vivo elimination of CD8+ cells abrogated protection. In vitro studies revealed that this Salmonella-P. berghei CS recombinant induced class I-restricted CD8+ cytotoxic T cells that are directed against the P. berghei CS peptide epitope spanning amino acids 242-253. This is the same peptide that previously was identified as the target of cytotoxic T lymphocytes (CTL) induced by sporozoite immunization. Salmonella-P. falciparum CS recombinants were constructed that contained either the full-length CS gene or a repeatless gene consisting of CS flanking sequences. Both of these vaccines were able to induce CD8+ CTL directed against P. falciparum CS peptide 371-390, which is identical to the target of CTL induced by sporozoites and vaccinia CS recombinants. These results directly demonstrate the ability of an intracellular bacteria such as Salmonella to induce class I-restricted CD8+ CTL and illustrate the importance of CD8+ CTL in immunity to malaria.
Inflammatory lipid mediator generation elicited by viable hemolysin-forming Escherichia coli in lung vasculature.
Authors: Grimminger F , Thomas M , Obernitz R , Walmrath D , Bhakdi S , Seeger W
Published: June 2004

Escherichia coli hemolysin, a transmembrane pore-forming exotoxin, is considered an important virulence factor for E. coli-related extraintestinal infections and sepsis. The possible significance of hemolysin liberation for induction of inflammatory lipid mediators was investigated in isolated rabbit lungs infused with viable bacteria (concentration range, 10(4)-10(7)/ml). Hemolysin-secreting E. coli (E. coli-Hly+), but not an E. coli strain that releases an inactive form of the exotoxin, induced marked lung leukotriene (LT) generation with predominance of cysteinyl LTs. Eicosanoid synthesis was not inhibited in the presence of plasma with toxin-neutralizing capacity. Pre-application of 2 x 10(8) human granulocytes, which sequestered in the lung microvasculature, caused a severalfold increase in leukotriene generation in response to E. coli-Hly+ challenge both in the absence and presence of plasma. Data are presented indicating neutrophil-endothelial cell cooperation in arachidonic acid lipoxygenase metabolism as an underlying mechanism. We conclude that liberation of hemolysin from viable E. coli induces marked lipid mediator generation in lung vasculature, which is potentiated in the presence of neutrophil sequestration and may contribute to microcirculatory disturbances during the course of severe infections.
Effect of olfactory bulb ablation on spread of a neurotropic coronavirus into the mouse brain.
Authors: Perlman S , Evans G , Afifi A
Published: June 2004

Previous results suggested that, after intranasal inoculation, mouse hepatitis virus (MHV), a neurotropic coronavirus, entered the central nervous system (CNS) via the olfactory and trigeminal nerves. To prove this hypothesis, the effect of interruption of the olfactory pathway on spread of the virus was studied using in situ hybridization. Unilateral surgical ablation of this pathway prevented spread of the virus via the olfactory tract on the side of the lesion. MHV RNA could be detected, however, at distal sites on the operated side, indicating that the virus spread via well-described circuits involving the anterior commissure from the control (intact) side of the brain. Viral transport via the trigeminal nerve was not affected by removal of the olfactory bulb, showing that the surgical procedure was specific for the olfactory pathway. These results prove conclusively that MHV gains entry to the CNS via a transneuronal route, and spreads to additional sites in the brain via known neuroanatomic pathways.
Human immunodeficiency virus type 1-infected individuals make autoantibodies that bind to CD43 on normal thymic lymphocytes.
Authors: Ardman B , Sikorski M A , Settles M , Staunton D E
Published: June 2004

Sera from human immunodeficiency virus type 1 (HIV-1)-infected and -noninfected individuals were screened for antibodies that could bind to native T cell differentiation antigens. Antibodies that could immunoprecipitate CD43 (sialophorin, leukosialin) from a T cell lymphoma line were detected in sera from 27% of patients, and antibodies that could bind specifically to transfected cells expressing CD43 were detected in 47% of patients. The anti-CD43 antibodies were related to HIV-1 infection in that no patients with other chronic viral infections or systemic lupus erythematosus contained such antibodies in their sera. The anti-CD43 autoantibodies bound to a partially sialylated form of CD43 expressed by normal human thymocytes, but not by normal, circulating T lymphocytes. However, the determinant(s) recognized by the anti-CD43 autoantibodies was present on a large proportion of circulating T lymphocytes, but masked from antibody recognition by sialic acid residues. These results demonstrate that HIV-1 infection is specifically associated with the production of autoantibodies that bind to a native T cell surface antigen.
Biochemical nature of an Fc mu receptor on human B-lineage cells.
Authors: Ohno T , Kubagawa H , Sanders S K , Cooper M D
Published: June 2004

An IgM-binding protein of approximately 60 kD has been identified on activated B cells, but not on resting and activated T cells, monocytes, or granulocytes. Here, we characterize this IgM-binding protein as a receptor for the Fc portion (CH3 and/or CH4 domains) of IgM molecules (Fc microR). The Fc microR can be expressed as a cell surface activation antigen throughout the pre-B and B cell stages in differentiation. Receptor expression is not directly linked with IgM production, as both mu- pre-B cells and isotype-switched B cells may express the Fc microR. The receptor molecules produced by both pre-B and B cells are identical in size and are characterized as an acidic sialoglycoprotein with O-linked, but no N-linked, oligosaccharide. The Fc microR is anchored to the surface of B-lineage cells via a glycosyl phosphatidylinositol linkage. The Fc microR is thus the third member of a family of Fc receptors expressed on B-lineage cells, and its preferential expression on activated B cells suggests a potential role in the response to antigens.
Late dominance of the inflammatory process in murine influenza by gamma/delta + T cells.
Authors: Carding S R , Allan W , Kyes S , Hayday A , Bottomly K , Doherty P C
Published: June 2004

The inflammatory response in the lungs of mice infected with an influenza A virus consists largely of macrophages and CD3+ T cells. Most T lymphocytes recovered before day 7 after infection express mRNA for the T cell receptor alpha/beta (TCR-alpha/beta), while TCR-gamma/delta mRNA+ cells are found at much higher frequency over the next 7 d. The predominant surface phenotype for the TCR-gamma/delta mRNA+ population is CD3+4-8-TCR-alpha/beta-. Some lymphocytes expressing all the known V gamma genes are found in the inflammatory exudate, but V gamma 2+/V gamma 1+ and V gamma 4+ T cells are present at highest frequency. The response is staged, with maximal numbers of V gamma 4+ cells occurring on day 10 after infection, while the predominant phenotype on day 13 is V gamma 2/V gamma 1+. The emerging peak in numbers of V gamma 4+ lymphocytes is paralleled by increasing numbers of macrophages expressing hsp mRNA. The later maxima found for the V gamma 2+/V gamma 1+ T cells is consistent with the possibility that at least some of these lymphocytes are responding to the hsp+ cells and are functioning to resolve the inflammatory process.