bluuebunny/crossref_metadata_2025 huggingface dataset:
A selective defect of interferon alpha production in human immunodeficiency virus-infected monocytes.
Authors: Gendelman H E , Friedman R M , Joe S , Baca L M , Turpin J A , Dveksler G , Meltzer M S , Dieffenbach C
Published: June 2004

Interferon alpha (IFN-alpha) induces significant antiretroviral activities that affect the ability of human immunodeficiency virus (HIV) to infect and replicate in its principal target cells, CD4+ T cells and macrophages. A major endogenous source of IFN-alpha during any infection is the macrophage. Thus, macrophages have the potential to produce both IFN-alpha and HIV. In this study, we examined the production of IFN-alpha and other cytokines by macrophage colony-stimulating factor (M-CSF)-treated cultured monocytes during HIV infection. Tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), IL-6, IFN-omega, or IFN-beta were not detected nor was the mRNA expressed in either uninfected or HIV-infected monocytes. However, both uninfected and HIV-infected monocytes produced high levels of each of these cytokines after treatment with synthetic double-stranded RNA [poly(I).poly(C)]. Uninfected monocytes also produced high levels of IFN-alpha after treatment with poly(I).poly(C), Newcastle disease virus, or herpes simplex virus. In marked contrast to the preceding observations, HIV-infected monocytes produced little or no IFN-alpha before or after treatment with any of these agents. The absence of detectable IFN-alpha activity and mRNA in poly(I).poly(C)-treated HIV-infected monocytes was coincident with high levels of 2',5' oligoadenylate synthetase and complete ablation of HIV gene expression. The antiviral activity induced by poly(I).poly(C) may be a direct effect of this synthetic double-stranded RNA or secondary to the low levels of IFN-beta and IFN-omega produced by infected cells. The markedly diminished capacity of HIV-infected monocytes to produce IFN-alpha may reflect a specific adaptive mechanism of virus to alter basic microbicidal functions of this cell. The inevitable result of this HIV-induced cytokine dysregulation is virus replication and persistence in mononuclear phagocytes.
Formation of lipoxins and leukotrienes during receptor-mediated interactions of human platelets and recombinant human granulocyte/macrophage colony-stimulating factor-primed neutrophils.
Authors: Fiore S , Serhan C N
Published: June 2004

The generation of lipoxygenase products of arachidonic acid is considered an important event in inflammation. This study demonstrates the levels of both lipoxins and leukotrienes (LTC4, LTD4, LTB4, and omega-oxidized LTB4) generated from endogenous sources of arachidonate by PMN primed with recombinant human granulocyte/macrophage colony-stimulating factor and in coincubations with platelets (1:1 to 1:100 ratio). Upon exposure to receptor-mediated stimuli (FMLP and thrombin), the levels of lipoxins generated were within the range of both LTB4 and LTC4. Co-incubation of [1-14C]arachidonate-labeled platelets with primed polymorphonuclear leukocytes (PMN) followed by addition of thrombin and FMLP led to the formation of both 5- and 15-LO products that carried 14C label. Thus, in addition to the transcellular conversion of LTA4 to platelet-derived lipoxins and LTC4, PMN can use platelet-derived arachidonate to generate lipoxygenase products. These results are the first to document the relationship between the levels of lipoxins and leukotrienes generated by receptor-mediated activation of cytokineprimed PMN interacting with platelets. Moreover, they indicate that PMN-platelet interactions utilize bidirectional transcellular routes to contribute to lipoxin formation.
Interferon gamma is involved in the recovery of athymic nude mice from recombinant vaccinia virus/interleukin 2 infection.
Authors: Karupiah G , Blanden R V , Ramshaw I A
Published: June 2004

Athymic nude mice recover from an infection with recombinant vaccinia virus (VV) encoding murine interleukin 2 (IL-2), but treatment with a mAb to IL-2 accentuated infection. Administration of a mAb against interferon gamma (IFN-gamma) to mice infected with the IL-2-encoding virus completely prevented the IL-2-induced mechanisms of recovery. Both asialo-GM1+ (NK) and asialo-GM1- (non-NK) cells were participants in the IFN-gamma-mediated recovery of nude mice from infection with the IL-2-encoding VV recombinant. Depletion of asialo-GM1+ cells exacerbated infection, though not as much as anti-IFN-gamma mAb. In vitro, both asialo-GM1+ and asialo-GM1- nude mouse splenocytes produced IFN-gamma in response to IL-2.
Widespread intradermal accumulation of mononuclear leukocytes in lepromatous leprosy patients treated systemically with recombinant interferon gamma.
Authors: Nathan C , Squires K , Griffo W , Levis W , Varghese M , Job C K , Nusrat A R , Sherwin S , Rappoport S , Sanchez E
Published: June 2004

Intradermal administration of recombinant interferon gamma (rIFN-gamma) to lepromatous leprosy patients has converted the local histology toward a tuberculoid pattern. However, such changes have been confined to the site of injection. In contrast, in the present study, marked, intradermal accumulation of CD3+, CD4+, CD8+, and CD1a+ T cells and Leu-M5+ mononuclear phagocytes was induced at a distance from the sites of administration, in a dose-dependent manner, by 10 daily intramuscular injections of 10-30 micrograms rIFN-gamma/m2. Mononuclear cell infiltration began within 3 d of onset of rIFN-gamma therapy and persisted at least 8 wk. Intramuscular administration of rIFN-gamma to lepromatous patients receiving concurrent chemotherapy can safely induce widespread histologic features of an upgrading reaction.
Binding and regulation of cellular functions by monoclonal antibodies against human tumor necrosis factor receptors.
Authors: Shalaby M R , Sundan A , Loetscher H , Brockhaus M , Lesslauer W , Espevik T
Published: June 2004

The present study was undertaken to further characterize the interaction of monoclonal antibodies (mAbs) against tumor necrosis factor (TNF) receptors with different targets, and to assess their ability to influence TNF effects on U937 and human endothelial cell (HEC) functions. Actions of recombinant TNF-alpha on U937 and HEC were effectively inhibited by Htr-5 and Utr-1, and to a greater extent by a combination of both mAbs. These observations indicate that TNF interaction with antigenically different components of membrane receptors (p55 and p75) represents a crucial step in transduction of signals for TNF toxicity against U937 and TNF activation of HEC functions.
Aspergillus fumigatus allergen I, a major IgE-binding protein, is a member of the mitogillin family of cytotoxins.
Authors: Arruda L K , Platts-Mills T A , Fox J W , Chapman M D
Published: June 2004

A major 18-kD IgE-binding protein from Aspergillus fumigatus (Asp fI) has been purified. Partial amino acid sequencing of Asp f I showed extensive sequence homology (95%) between Asp fI and a cytotoxin (mitogillin) produced by A. restrictus. Crossinhibition radioimmunoassay using murine monoclonal antibody and human IgG and IgE antibodies showed that Asp fI and mitogillin were antigenically indistinguishable. Furthermore, both proteins inhibited protein synthesis in vitro by greater than 90%. Asp fI was expressed in A. fumigatus but not in seven other Aspergillus species. The results suggest that Asp fI could play a dual role in the pathogenesis of A. fumigatus-related diseases by promoting colonization through cytotoxic activity and by causing inflammatory reactions involving IgE antibodies.
Identification of four subsets of human CD3-CD16+ natural killer (NK) cells by the expression of clonally distributed functional surface molecules: correlation between subset assignment of NK clones and ability to mediate specific alloantigen recognition.
Authors: Moretta A , Bottino C , Pende D , Tripodi G , Tambussi G , Viale O , Orengo A , Barbaresi M , Merli A , Ciccone E
Published: June 2004

In previous studies we identified a surface molecule (termed GL183) capable of mediating cell activation and selectively expressed by a subset of human CD3-CD16+ natural killer (NK) cells. In this study we analyzed whether other subset-specific functional molecules were expressed in GL183- NK cells. To this end, mice were immunized with the PE29 (CD3-CD16+GL183-) NK clone. Monoclonal antibodies (mAbs) were selected by screening the hybridoma supernatants for their ability to trigger the cytolytic activity of clone PE29 against the human myelomonocytic leukemia U937. The EB6 mAb (IgG1) triggered the PE29 clone, but not a GL183+ clone used as a control. EB6+ cells ranged between 1 and 13% of peripheral blood lymphocytes and were largely included in the CD3-CD16+CD56+ cell populations (only less than 2% of EB6+ cells were CD3+). Analysis of resting or activated CD3-CD16+ populations, or clones for the expression of EB6 or GL183 mAbs, allowed us to identify four distinct, phenotypically stable, NK subsets (EB6+GL183-; EB6+GL183+; EB6-GL183+; EB6-GL183-). Similar to GL183 mAb, the EB6 mAb selectively triggered the NK subset expressing the corresponding surface antigen to lyse human tumor cell lines including U937, IGROV-I, M14, and A549. In addition, EB6 mAb sharply inhibited the cytolytic activity of EB6+ clones against P815, M12, and P3U1 murine target cells. In EB6+GL183+ ("double-positive") clones both EB6 and GL183 mAb inhibited the redirected killing of P815 cells induced by anti-CD16, anti-CD2 mAbs and phytohemagglutinin (PHA). Similar to GL183 molecules, molecules precipitated by EB6 mAb were represented by either single 58-kD chain or double chains of 55 and 58 kD (with no detectable differences in EB6+GL183- or EB6+GL183+ clones). In sequential immunoprecipitation experiments using the double-positive clones CEG52 and CA25.50, preclearing of cell lysates with EB6 or GL183 mAb removed only EB6 or GL183 molecules, respectively, thus indicating that the two antigenic determinants are carried by two distinct molecules. Peptide map analysis indicated that EB6 (or GL183) molecules precipitated from double-positive clones were identical to the corresponding molecules isolated from single-positive ones. On the other hand, comparison of the EB6 and GL183 maps revealed peptides that were unique to each molecule, although most of the major peptides migrated to identical positions. We further investigated whether correlation existed between the phenotypic assignment of NK clones and their ability to mediate specific lysis of normal allogeneic cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Complement-mediated tumor cell damage induced by antibodies against membrane cofactor protein (MCP, CD46).
Authors: Seya T , Hara T , Matsumoto M , Sugita Y , Akedo H
Published: June 2004

We have developed polyclonal and monoclonal antibodies against human membrane cofactor protein (MCP) to use as tools to investigate the functions of MCP on intact nucleated cells. Two human T cell lines, CEM and TALL, are CR1- and DAF-. Pretreatment of these cell lines with M177 and polyclonal anti-MCP, which inhibit cofactor activity almost completely, resulted in effective C3 deposition immediately following addition of these cells to Mg2+/EGTA/human sera. The deposited C3 remained expressed partly on the cell surface and most of them were gradually converted to C3bi. Some of the deposited C3 were complexed with membrane proteins, since 140- and 250-kD bands became significantly accumulated on SDS-PAGE by treatment with the antibodies. We next tested whether these C3-coated cells were damaged by complement-mediated cytolysis. p18, an inhibitor of membrane attack complex (MAC) formation, was negative in TALL but positive in CEM. TALL was lysed efficiently only by treatment with the polyclonal anti-MCP, while CEM showed only slight lysis with the same treatment. Monoclonal antibodies to MCP, including M177, caused only minimal cell destruction. Based on these results, together with the fact that decay-accelerating factor (DAF) serves as a factor for preventing C3 attack on human cells, we conclude that MCP and DAF cooperatively protect host cells from C3 targeting and, in these T cell lines, MCP is sufficient for preventing C3 deposition even without DAF. After all, human cells undergo almost no autologous complement-mediated cytolysis if they express at least one of the functionally active inhibitors, MCP, DAF, or p18.
Boundaries of somatic mutation in rearranged immunoglobulin genes: 5' boundary is near the promoter, and 3' boundary is approximately 1 kb from V(D)J gene.
Authors: Lebecque S G , Gearhart P J
Published: June 2004

To investigate why somatic mutations are spatially restricted to a region around the rearranged V(D)J immunoglobulin gene, we compared the distribution of mutations flanking murine V gene segments that had rearranged next to either proximal or distal J gene segments. 124 nucleotide substitutions, nine deletions, and two insertions were identified in 32,481 bp of DNA flanking the coding regions from 17 heavy and kappa light chain genes. Most of the mutations occurred within a 2-kb region centered around the V(D)J gene, regardless of which J gene segment was used, suggesting that the structural information for mutation is located in sequences around and within the V(D)J gene, and not in sequences downstream of the J gene segments. The majority of mutations were found within 300 bp of DNA flanking the 5' side of the V(D)J gene and 850 bp flanking the 3' side at a frequency of 0.8%, which was similar to the frequency in the coding region. The frequency of flanking mutations decreased as a function of distance from the gene. There was no evidence for hot spots in that every mutation was unique and occurred at a different position. No mutations were found upstream of the promoter region, suggesting that the promoter delimits a 5' boundary, which provides strong evidence that transcription is necessary to generate mutation. The 3' boundary was approximately 1 kb from the V(D)J gene and was not associated with a DNA sequence motif. Occasional mutations were located in the nuclear matrix association and enhancer regions. The pattern of substitutions suggests that there is discrimination between the two DNA strands during mutation, in that the four bases were mutated with different frequencies on each strand. The high frequency of mutations in the 3' flanking region and the uniqueness of each mutation argues against templated gene conversion as a mechanism for generating somatic diversity in murine V(D)J genes. Rather, the data support a model for random point mutations where the mechanism is linked to the transcriptional state of the gene.
Effects of interferon alpha on autocrine growth factor loops in B lymphoproliferative disorders.
Authors: Heslop H E , Bianchi A C , Cordingley F T , Turner M , Chandima W , De Mel C P , Hoffbrand A V , Brenner M K
Published: June 2004

The B lymphoproliferative disorders B chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL) produce a number of autocrine growth factors, including tumor necrosis factor (TNF), interleukin 6 (IL-6), and IL-1, all of which may induce positive feedback growth loops. If such malignancies depend on these autocrine growth loops for survival, their interruption may be therapeutically valuable. Interferon alpha (IFN-alpha) abrogates TNF- or IL-6-induced proliferation of HCL and B-CLL cells in vitro and has therapeutic activity in these diseases. We have investigated the possibility that IFN-alpha may act by interrupting autocrine growth factor loops. If purified B-CLL or HCL cells are cultured in the presence of TNF, there is induction of mRNA for TNF, IL-1 alpha, IL-1 beta, and IL-6. However, culture in the presence of IFN-alpha in addition to TNF reduced the level of mRNA for all these cytokines, compared with cells cultured in TNF alone. While cytokine mRNA levels were diminished, levels of mRNA for the ribonuclease activator 2-5A synthetase were increased. Analysis of the kinetics of cytokine mRNA production showed that levels fall shortly after the rise of 2-5A synthetase mRNA. IFN-alpha may produce these effects by shortening the half-life of cytokine mRNA, since TNF mRNA half-life in B-CLL and HCL cells is substantially reduced when the cells are cultured with IFN-alpha. These data suggest that IFN-alpha may mediate its therapeutic effects in these malignancies by blocking autocrine growth factor loops.
Stimulation of fibroblast chemotaxis by human recombinant tumor necrosis factor alpha (TNF-alpha) and a synthetic TNF-alpha 31-68 peptide.
Authors: Postlethwaite A E , Seyer J M
Published: June 2004

Macrophages are a major source of fibrogenic factors that promote healing of injured tissue. The recruitment of fibroblasts to sites of tissue injury is a prerequisite for optimal repair of tissue damage. In the present study, human recombinant tumor necrosis factor alpha (hrTNF-alpha), a major macrophage-derived cytokine, was demonstrated to be a potent fibroblast chemoattractant, inducing migration at picomolar concentrations. Anti-hrTNF-alpha monoclonal antibody neutralized most of the fibroblast chemotactic activity generated during short-term culture of human peripheral blood monocytes stimulated with bacterial lipopolysaccharide, suggesting that TNF-alpha is a major monocyte-derived fibroblast chemoattractant. The portion of the human TNF-alpha molecule responsible for its chemotactic stimulation of fibroblasts appears to reside in residues 31-68. This region is highly conserved between TNF-alpha and lymphotoxin. This peptide is not only itself chemotactic but is also able to block the chemotactic response of fibroblasts to hrTNF-alpha and vice versa, suggesting that they each mediate fibroblast migration through similar mechanisms. These data further underscore the potential importance of TNF-alpha in modulating a variety of fibroblast functions, including chemotaxis and synthesis of collagen, glycosaminoglycans, interleukin 1 alpha (IL-1 alpha) and -beta, human histocompatibility leukocyte antigen A and B antigens, collagenase, prostaglandin E2, and IL-6.
Transgenic rearranged T cell receptor gene inhibits lymphadenopathy and accumulation of CD4-CD8-B220+ T cells in lpr/lpr mice.
Authors: Mountz J D , Zhou T , Eldridge J , Berry K , Blüthmann H
Published: June 2004

The lpr gene in homozygous form induces development of CD4-CD8-B220+ T cells and lymphadenopathy in MRL and C57BL/6 mice. Although the propensity for excessive production of T cells is related to an intrinsic T cell defect, a thymus is also required because neonatal thymectomy eliminates lymphadenopathy. Recent evidence suggests that excessive production and release of autoreactive T cells from the thymus of lpr/lpr mice might lead to downregulation of CD4 and CD8 as a "fail safe" tolerance mechanism that occurs during late thymic or post-thymic development. To test this hypothesis, T cell receptor (TCR) transgenic mice that produce large numbers of immature thymocytes recognizing the H-2Db and male H-Y antigens were backcrossed with C57BL/6-lpr/lpr mice and MRL-lpr/lpr mice. It was predicted that Db male lpr/lpr mice would produce large numbers of autoreactive T cells during early thymic development that would lead to an accelerated lymphoproliferative disease. In contrast, Db female lpr/lpr mice would produce large numbers of Db H-Y-reactive T cells, but might not develop lymphadenopathy because the male H-Y antigen would not be present. Unexpectedly, there was complete elimination of lymphadenopathy in both male and female TCR transgenic lpr/lpr mice. The elimination of lymphadenopathy was not due to a failure of thymic maturation since the thymus of H-2Db female lpr/lpr mice contained nearly normal numbers of mature thymocytes. Elimination of lymphadenopathy was also not due to a lack of autoreactive T cells in the peripheral lymph nodes (LN) since there was an increased syngeneic mixed lymphocyte proliferative response of LNT cells from transgenic lpr/lpr compared with +/+ mice in vitro. Hypergammaglobulinemia and autoantibody production in the transgenic lpr/lpr was present at levels comparable with or higher than control nontransgenic lpr/lpr mice, suggesting a dissociation of autoantibody production from the lymphoproliferative disease in the TCR transgenic mice. Conversely, the development of lymphadenopathy and production of CD4-CD8-B220+ T cells appear to be intimately linked, as both were completely eliminated in T cells expressing the transgenic TCR. We propose that lymphoproliferation and production of CD4-CD8-6B2+ T cells in lpr/lpr mice is related to decreased expression of the TCR, and providing the T cells with a rearranged TCR transgene overcomes this defect.
CD40 and IgE: synergism between anti-CD40 monoclonal antibody and interleukin 4 in the induction of IgE synthesis by highly purified human B cells.
Authors: Jabara H H , Fu S M , Geha R S , Vercelli D
Published: June 2004

A novel pathway of IgE-B cell differentiation has been identified. Engagement of the B cell antigen CD40 by F(ab')2 fragments of monoclonal antibody (mAb) 626.1 in the presence of recombinant interleukin 4 (rIL-4) induced intense IgE synthesis, but modest IgG synthesis, by highly purified human B cells. Surface IgE- B cells isolated by cell sorting were induced to produce IgE by mAb 626.1 and IL-4. Thus, IgE synthesis is unlikely to result from expansion of a B cell population precommitted to IgE in vivo. A neutralizing anti-IL-6 antibody strongly, but not completely, inhibited the IgE response. This indicates that autocrine production of IL-6 plays an important amplification role in IgE synthesis triggered by anti-CD40 mAb and IL-4. Although the exact role played by CD40 in IgE responses in vivo remains to be established, this T cell-independent system represents a useful model to characterize the biochemical and molecular events leading to IgE synthesis in human B cells.
Fibroblasts mediate T cell survival: a proposed mechanism for retention of primed T cells.
Authors: Scott S , Pandolfi F , Kurnick J T
Published: June 2004

This report describes a salvage pathway whereby activated T lymphocytes revert to nonproliferating cells in the absence of antigen or mitogenic signals. After the removal of mitogenic cytokines, cultured T lymphocytes cease dividing and rapidly begin to undergo cell death. However, the addition of fibroblasts to interleukin 2 (IL-2)-propagated T cells results in prolonged survival of the previously activated T lymphocytes in the absence of proliferation. The prevention of cell death is also achieved by conditioned medium from the fibroblasts. T lymphocytes cultured with fibroblasts or the conditioned medium retain the ability to be restimulated if mitogenic stimuli are added to the culture. The activity is not accounted for by IL-1-7. The studies suggest a stromal cell-mediated, nonspecific mechanism for survival of primed T lymphocytes in a nonproliferating state.
Minor histocompatibility antigen-specific cytotoxic T cell lines, capable of lysing human hematopoietic progenitor cells, can be generated in vitro by stimulation with HLA-identical bone marrow cells.
Authors: Marijt W A , Veenhof W F , Brand A , Goulmy E , Fibbe W E , Willemze R , van Rood J J , Falkenburg J H
Published: June 2004

Recipient-antidonor alloreactivity before HLA genotypically identical bone marrow transplantation (BMT) between donor-recipient pairs that are negative in the mixed lymphocyte reaction (MLR), the cell-mediated lympholysis (CML) assay, and the lymphocyte crossmatch was not detectable in the majority of cases, using recipient peripheral blood lymphocytes (PBL) collected before BMT as responder cells and donor PBL as stimulator cells. However, when donor bone marrow mononuclear cells (BMMNC) instead of PBL were used as stimulator cells, we could detect donor-specific alloreactivity in 7 of 10 HLA genotypically identical donor-recipient pairs. To demonstrate that this alloreactivity was minor histocompatibility (mH) antigen specific and not directed against HLA class I splits or variants, two cytotoxic T lymphocyte (CTL) lines were tested in further detail against phytohemagglutinin (PHA) blasts from pairs of HLA genotypically identical siblings positive for the HLA class I restriction molecule. Both CTL lines recognized mH antigens, as illustrated by the differential recognition of PHA blasts of one of the two siblings from several pairs. The potential role of these mH antigen-specific CTLs in bone marrow graft rejection was demonstrated by the mH antigen-specific growth inhibition of hematopoietic progenitor cells from the original bone marrow donor and from HLA class I restriction molecule-positive individuals who expressed the mH antigens on their PBL and BMMNC. Our assay can be used in HLA genotypically identical BMT to detect a recipient-antidonor response, directed against cellularly defined mH antigens expressed on donor HPC, BMMNC, and PBL, before transplantation.
Defect in negative selection in lpr donor-derived T cells differentiating in non-lpr host thymus.
Authors: Matsumoto K , Yoshikai Y , Asano T , Himeno K , Iwasaki A , Nomoto K
Published: June 2004

Transplantation of bone marrow cells of lpr/lpr mice into irradiated normal mice fails to develop massive lymphadenopathy or autoimmunity but causes severe graft-vs.-host-like syndrome. To elucidate an abnormality of lpr/lpr bone marrow-derived T cells, we transplanted bone marrow cells of Mlsb lpr/lpr mice into H-2-compatible Mlsa non-lpr mice. Although lpr/lpr T cell precursors repopulated the host thymus as well as +/+ cells, a proportion of CD4+CD8+ cells decreased, and that of both CD4- and CD8- single-positive cells increased compared with those of +/+ recipients. Notably, in MRL/lpr----AKR and C3H/lpr----AKR chimeras, CD4 single-positive thymocytes contained an increased number of V beta 6+ cells in spite of potentially deleting alleles of Mlsa, whereas V beta 6+ mature T cells were deleted in the MRL/+ ----AKR and C3H/+ ----AKR chimeras. There was no difference between MRL/+ ----AKR and MRL/lpr----AKR chimeras in their proportion of V beta 3+ cells because both host and donor strain lack the deleting alleles. Interleukin 2 receptor expression of mature T cells, in the thymus and lymph node, was obviously higher in the MRL/lpr----AKR chimeras, in particular in the "forbidden" V beta 6+ subset. Moreover, lpr donor-derived peripheral T cells showed vigorous anti-CD3 response. These results indicate that lpr-derived T cells escape not only tolerance-related clonal deletion but also some induction of unresponsiveness in the non-lpr thymus.
cDNA cloning of the B cell membrane protein CD22: a mediator of B-B cell interactions.
Authors: Wilson G L , Fox C H , Fauci A S , Kehrl J H
Published: June 2004

We have cloned a full-length cDNA for the B cell membrane protein CD22, which is referred to as B lymphocyte cell adhesion molecule (BL-CAM). Using subtractive hybridization techniques, several B lymphocyte-specific cDNAs were isolated. Northern blot analysis with one of the clones, clone 66, revealed expression in normal activated B cells and a variety of B cell lines, but not in normal activated T cells, T cell lines, Hela cells, or several tissues, including brain and placenta. One major transcript of approximately 3.3 kb was found in B cells although several smaller transcripts were also present in low amounts (approximately 2.6, 2.3, and 1.6 kb). Sequence analysis of a full-length cDNA clone revealed an open reading frame of 2,541 bases coding for a predicted protein of 847 amino acids with a molecular mass of 95 kD. The BL-CAM cDNA is nearly identical to a recently isolated cDNA clone for CD22, with the exception of an additional 531 bases in the coding region of BL-CAM. BL-CAM has a predicted transmembrane spanning region and a 140-amino acid intracytoplasmic domain. Search of the National Biological Research Foundation protein database revealed that this protein is a member of the immunoglobulin super family and that it had significant homology with three homotypic cell adhesion proteins: carcinoembryonic antigen (29% identity over 460 amino acids), myelin-associated glycoprotein (27% identity over 425 amino acids), and neural cell adhesion molecule (21.5% over 274 amino acids). Northern blot analysis revealed low-level BL-CAM mRNA expression in unactivated tonsillar B cells, which was rapidly increased after B cell activation with Staphylococcus aureus Cowan strain 1 and phorbol myristate acetate, but not by various cytokines, including interleukin 4 (IL-4), IL-6, and gamma interferon. In situ hybridization with an antisense BL-CAM RNA probe revealed expression in B cell-rich areas in tonsil and lymph node, although the most striking hybridization was in the germinal centers. COS cells transfected with a BL-CAM expression vector were immunofluorescently stained positively with two different CD22 antibodies, each of which recognizes a different epitope. Additionally, both normal tonsil B cells and a B cell line were found to adhere to COS transfected with BL-CAM in the sense but not the antisense direction.(ABSTRACT TRUNCATED AT 400 WORDS)
Inducibility of class II major histocompatibility complex antigens by interferon gamma is associated with reduced tumorigenicity in C3H mouse fibroblasts transformed by v-Ki-ras.
Authors: Bateman W J , Fiera R , Matthews N , Morris A G
Published: June 2004

Paired lines of C3H mouse fibroblasts transformed with murine sarcoma virus (Kirsten strain) were prepared that express high or low levels of class II major histocompatibility complex antigen after treatment with interferon gamma (IFN-gamma). Here, we described a comparison of the tumorigenicity of these lines in euthymic syngeneic and thymus-deficient nu/nu mice and in mice depleted of IFN-gamma. The class II-inducible cells are clearly less tumorigenic than the noninducible cells in syngeneic mice, but of similar tumorigenicity in nu/nu mice and in mice treated with antibodies to deplete IFN-gamma. We propose that in this system, IFN-gamma induction of class II antigens on the tumor cell surface operates to limit tumor growth; ras expression, which inhibits induction of class II antigens, prevents this and so allows tumor growth.
The vitronectin receptor serves as an accessory molecule for the activation of a subset of gamma/delta T cells.
Authors: Roberts K , Yokoyama W M , Kehn P J , Shevach E M
Published: June 2004

Constitutive production of cytokines was observed in 3 of 12 gamma/delta T cell lines derived from murine epidermis and correlated with the expression of the C gamma 4, V delta 6 T cell receptor (TCR). After adaptation of one of the lines (T195/BW) to serum-free culture conditions, cessation of the "spontaneous" production of interleukin 4 (IL-4) was observed and IL-4 production could then by induced by the addition of RGD-containing extracellular matrix (ECM) proteins to the culture. The response to the ECM proteins could be completely inhibited by a mAb to the murine vitronectin receptor (VNR). However, the induction of IL-4 production could also be inhibited by anti-CD3 and by an anti-clonotypic mAb to the TCR-gamma/delta of T195/BW. As TCR-gamma/delta loss mutants of T195/BW also failed to respond to ECM proteins, these data demonstrate that engagement of the VNR by its ligand is necessary, but not sufficient, for the induction of IL-4 production. Furthermore, the VNR is expressed by many other T cell clones (both gamma/delta and alpha/beta), none of which produce lymphokines constitutively. Taken together, these observations strongly favor the view that not only is coexpression of the VNR and TCR required for the induction of IL-4 production, but that the TCR must also be engaged by its ligand, most likely a cell surface antigen expressed by the hybridoma itself.
Adoptive transfer of neonatal T lymphocytes rescues immunoglobulin production in mice with severe combined immune deficiency.
Authors: Riggs J E , Stowers R S , Mosier D E
Published: June 2004

Mice with the autosomal recessive severe combined immune deficiency (scid) mutation lack mature lymphocytes because of defective joining of T cell receptor and immunoglobulin (Ig) gene segments. Penetrance of this mutation is incomplete since 10-25% of SCID mice produce some T or B lymphocytes. This "leaky" phenotype could be due to a reversion of the mutation in some mice or to a constant, low frequency of functional lymphocytes generated in all SCID mice with variable survival of such cells. We report here that all SCID mice can be stimulated to produce functional B cells by the transfer of normal neonatal, but not adult, T cells. T cell-induced rescue of C.B-17scid B cells results in high levels of Ig expressing the Ighb allotype of the SCID recipient. These results show that all SCID mice generate some functional B cells, the majority of which do not survive in the absence of a subset of T cells present in high frequency in the neonate.
Congenital osteoclast deficiency in osteopetrotic (op/op) mice is cured by injections of macrophage colony-stimulating factor.
Authors: Kodama H , Yamasaki A , Nose M , Niida S , Ohgame Y , Abe M , Kumegawa M , Suda T
Published: June 2004

Osteopetrotic (op/op) mice have a severe deficiency of osteoclasts, monocytes, and peritoneal macrophages because of a defect in the production of functional macrophage colony-stimulating factor (M-CSF) resulting from a mutation within the M-CSF gene. In this study, we examined whether daily 5-microgram injections of purified recombinant human M-CSF (rhM-CSF) for 14 d would cure these deficiencies in the mutant mice. Monocytes in the peripheral blood of the op/op mice were significantly increased in number after subcutaneous injections of the factor two or three times a day. In contrast, osteopetrosis in the long bones of op/op mice was completely cured by only one injection of rhM-CSF per day. Bone trabeculae in the diaphyses were removed. Many osteoclasts were detected on the surface of bone trabeculae in the metaphyses. Although development of tooth germs of uninjected op/op mice was impaired, rhM-CSF injection restored the development of molar tooth germs and led to tooth eruption as a consequence of the recovery of bone-resorbing activity. These results demonstrate that M-CSF is one of the factors responsible for the differentiation of osteoclasts and monocyte/macrophages under physiological conditions.
Human T lymphocytes recognize a peptide of single point-mutated, oncogenic ras proteins.
Authors: Jung S , Schluesener H J
Published: June 2004

P21ras proteins are thought to play an important role in cell proliferation and differentiation. Single nucleotide mutations in the encoding cellular proto-oncogenes often result in p21ras proteins with transforming activity. Such activated ras oncogenes have been demonstrated in a variety of human malignancies and also in preneoplastic changes. Using a synthetic peptide corresponding to amino acids 5-16 of mutated p21ras proteins with an exchange of the normal glycine at position 12 by valine, it is shown here that human CD4+ T cells specifically recognize the mutated protein sequence and can be generated as antigen-specific T lymphocyte lines. The fact that these T lines did not crossreact to the sequence of normal p21ras proteins offers new perspectives for specific immunotherapy of human malignancies and even precancerous lesions.
Effect of antigen/antibody ratio on macrophage uptake, processing, and presentation to T cells of antigen complexed with polyclonal antibodies.
Authors: Manca F , Fenoglio D , Li Pira G , Kunkl A , Celada F
Published: June 2004

Activation of a galactosidase-specific murine T hybridoma clone and of a human tetanus toxoid-specific T clone by antigen-presenting cells (APC) was used to evaluate the regulatory function of antibodies complexed with the relevant antigen. Complexed antigen, in fact, is taken up with high efficiency thanks to Fc receptors borne by APC. Antibody/antigen ratio in the complexes proved to be a critical parameter in enhancing antigen presentation. Complexes in moderate antibody excess provided optimal T cell activation independently of the physical state of the complexes (precipitated by a second antibody or solubilized by complement). Complexes in extreme antibody excess, on the contrary, did not yield T cell activation although taken up by APC efficiently. The effect of antibodies at extreme excess was observed with substimulatory dose of antigen (loss of potentiation) and with optimal dose of antigen (loss of stimulation). An excess of specific polyclonal antibodies hampers proteolytic degradation of antigen in vitro, supporting the view that a similar mechanism may operate within the APC that have internalized immune complexes in extreme antibody excess. The possibility that immune complex forming in extreme antibody excess may turn off the T cell response is proposed as a regulatory mechanism.
Structure of the T cell antigen receptor (TCR): two CD3 epsilon subunits in a functional TCR/CD3 complex.
Authors: de la Hera A , Müller U , Olsson C , Isaaz S , Tunnacliffe A
Published: June 2004

Transgenic mice carrying and expressing the human CD3 epsilon gene incorporate the corresponding protein product into T cell receptor (TCR)/CD3 complexes on thymocyte and T cell surfaces. The chimeric antigen receptors allow normal T cell development and selection of repertoires in vivo and are able to transduce activation signals in vitro. We have exploited the ability to distinguish mouse (m) and human (h)CD3 epsilon chains to analyze the stoichiometry of CD3 epsilon in transgenic mouse TCRs. Immunoprecipitation and fluorescence resonance energy transfer experiments demonstrate that such TCRs can contain both h- and mCD3 epsilon chains, implying that more than one CD3 epsilon subunit occurs per TCR. Antigen comodulation studies are consistent with a stochastic use of h- or mCD3 epsilon during receptor assembly, and further suggest a structure for the TCR/CD3 complex with two CD3 epsilon chains. The determination of CD3 epsilon subunit stoichiometry, together with existing biochemical data, allows the generation of a minimal model for the structure of the TCR and illustrates the potential value of the transgenic approach to the analysis of complex receptors.
Peripheral tolerance to alloantigen results from altered regulation of the interleukin 2 pathway.
Authors: Dallman M J , Shiho O , Page T H , Wood K J , Morris P J
Published: June 2004

Tolerance to alloantigen may be induced in rats by administration of blood followed by transplantation of a renal allograft. The mechanism of this tolerance was investigated by directly analyzing the functional activity of graft-infiltrating cells. We have previously shown cytotoxic T lymphocyte infiltration of, and major histocompatibility complex induction on, grafts of tolerant animals. We now report that cells isolated from the grafts of tolerant rats show a reduced expression of the p55 interleukin 2 receptor (IL-2R) chain on the cell surface compared with that seen on the cells of untreated animals. Scatchard analysis further reveals low expression of high affinity IL-2R. This is due to reduced transcription of both IL-2R alpha and beta chain mRNAs and results in a reduced ability of cells to proliferate in response to IL-2. Cells isolated from tolerant animals are unable to make biologically active IL-2 in culture, whereas cells from untreated animals make high levels. This is not reflected at the mRNA level as the IL-2 gene is induced in both tolerant and untreated animals to similar levels. The induction of tolerance is abrogated by administration of recombinant IL-2 to animals at the time of transplantation. Thus, we conclude that an altered regulation of the IL-2 pathway results in tolerance in these alloantigen-treated and transplanted animals.
A novel class of anti-DNA antibodies identified in BALB/c mice.
Authors: Shefner R , Kleiner G , Turken A , Papazian L , Diamond B
Published: June 2004

We have characterized four IgG monoclonal antibodies (mAbs) derived from BALB/c mice that bind double-stranded DNA (dsDNA) with high affinity. The hydridomas were selected for expression of a member of the VHS107 family. Three of the four cell lines use the VH11 gene and one uses the VH1 gene. These antibodies exhibit many characteristics of pathogenic anti-DNA antibodies. They are high affinity and not broadly crossreactive. Unlike the anti-DNA antibodies in autoimmune mice, they exhibit no somatic mutation in their VH genes. These results demonstrate that somatic mutation of VHS107 genes is not necessary for generating high affinity dsDNA binding. The fact that such antibodies have not previously been reported suggests that they are rare and that their expression may be downregulated in both nonautoimmune and autoimmune individuals.
Combined interleukin 1/interleukin 2 therapy of mice injected with highly metastatic Friend leukemia cells: host antitumor mechanisms and marked effects on established metastases.
Authors: Ciolli V , Gabriele L , Sestili P , Varano F , Proietti E , Gresser I , Testa U , Montesoro E , Bulgarini D , Mariani G
Published: June 2004

Peritumoral injection of recombinant human interleukin 1 beta (IL-1 beta) in mice transplanted subcutaneously with Friend erythroleukemia cells (FLC) resulted in a marked increase in survival time and inhibition of metastatic tumor growth in liver and spleen. In contract, IL-2 treatment alone did not significantly inhibit the development of FLC metastases. A synergistic antitumor effect was observed after combined IL-1/IL-2 therapy of these mice. The antitumor action of IL-1/IL-2 treatment was abolished or markedly reduced in mice treated with antibodies to CD4 or CD8 antigens, whereas antibodies to asialo-GM1 were ineffective. A clear-cut increase in the percentage of CD4+ cells was observed in the spleens of cytokine-treated mice on days 17 and 23. On day 23 of cytokine therapy, CD8+ cells were increased in both spleens and lymph nodes. On day 17, infiltrates of host-reactive cells (i.e., lymphocytes, granulocytes, and monocytes) were observed in both spleen and liver from FLC-injected mice treated with IL-1/IL-2, in association with tumor cells. On days 17 and 23, spleen cells and cells recovered from mesenteric lymph nodes of IL-1/IL-2-treated mice exerted a potent antitumor effect as determined by Winn assay experiments. This antitumor activity was abolished by preincubation of spleen cells with anti-CD8 antibody, but not by treatment with antibodies to asialo-GM1; antibodies to CD4 exerted only a slight effect. Combined IL-1/IL-2 therapy was more effective on established (i.e., 6-7-d) FLC tumors than on early (i.e., 1-d) tumor-transplanted mice. IL-1/IL-2 treatments were also highly effective in increasing survival time of mice from which the subcutaneous primary tumors were excised 7 d after FLC injection. These data indicate that in mice injected with FLC, the antitumor effects of IL-1/IL-2 are mediated by CD4+ and CD8+ cells (but not NK cells), and suggest that this combined cytokine treatment may be effective against established metastatic tumors.
The AU-rich sequences in the 3' untranslated region mediate the increased turnover of interferon mRNA induced by glucocorticoids.
Authors: Peppel K , Vinci J M , Baglioni C
Published: June 2004

Different vectors were constructed that expressed the human interferon-beta (IFN-beta) mRNA constitutively and contained various deletions in the 3' untranslated region (UTR). AU-rich sequences in the 3' UTR were specifically deleted in two vectors. Cell lines secreting human IFN-beta were established by transfecting murine L929 cells with the vectors. These cells showed similar levels of IFN-beta mRNA and secreted comparable amounts of IFN-beta, indicating that the deletion of AU-rich sequences had no effect on the stability and little effect on the efficiency of translation of this mRNA. The synthetic glucocorticoid dexamethasone was previously shown to increase the turnover of IFN-beta mRNA. This activity of dexamethasone was clearly observed only in cells expressing IFN-beta mRNA with AU-rich sequences in the 3' UTR. The increased turnover of this mRNA occurred in the presence of cycloheximide; therefore, it did not require synthesis of new proteins. These findings suggest that glucocorticoids may activate a ribonuclease that degrades mRNAs containing AU-rich sequences in the 3' UTR.
Hydrazine sulfate protects D-galactosamine-sensitized mice against endotoxin and tumor necrosis factor/cachectin lethality: evidence of a role for the pituitary.
Authors: Silverstein R , Turley B R , Christoffersen C A , Johnson D C , Morrison D C
Published: June 2004

In previously published studies, we had demonstrated that hydrazine sulfate pretreatment protected mice against the lethal effects of endotoxin and that this protection was accompanied by a sustained increase in hepatic phosphoenolpyruvate carboxykinase activity (Silverstein, R., C.A. Christoffersen, and D.C. Morrison. 1989. Infect. Immun. 57:2072). The same hydrazine sulfate pretreatment has now been found to protect mice against endotoxin in the D-galactosamine model with an increase in the endotoxin LD50 of approximately four orders of magnitude. Elimination of the pretreatment period, or administration of an additional dose of D-galactosamine at the time of hydrazine sulfate pretreatment, renders the mice refractory to the protection. Given the sensitivity of phosphoenolpyruvate carboxykinase regulation to several hormones, we investigated the possibility that protection may have been hormone mediated. In addition to determining the effect of hydrazine sulfate on the plasma levels of phosphoenolpyruvate carboxykinase regulating hormones, we have investigated the effects of hydrazine sulfate on endotoxin lethality in mice whose capacity to respond hormonally to external stimuli has been compromised by hypophysectomy. Our results show a significant enhancement in circulating levels of plasma corticosterone 30 min after hydrazine sulfate injection. Moreover, hypophysectomy results in a marked increase in sensitivity of mice to endotoxin challenge as well as an abrogation of the protection against endotoxin lethality mediated by hydrazine sulfate. Although hydrazine sulfate protection distinguishes between sensitivity brought on, individually, by D-galactosamine and by hypophysectomy, mice sensitized by both hypophysectomy and D-galactosamine are not protected against endotoxin lethality by hydrazine sulfate. We conclude that hydrazine sulfate protection against endotoxin lethality is endocrine dependent, with the available evidence implicating a pituitary/adrenal axis, with glucocorticoid involvement. In as much as D-galactosamine is known to act directly in the liver in disrupting protein synthesis, it is proposed that events in the liver are critical to the hydrazine sulfate-mediated protection against endotoxin and are possibly the target of the endocrine involvement. Hydrazine sulfate pretreatment also protects D-galactosamine-sensitized mice against the lethal effects of injected tumor necrosis factor/cachectin.
The CD4 molecule is not always required for the T cell response to bacterial enterotoxins.
Authors: Sékaly R P , Croteau G , Bowman M , Scholl P , Burakoff S , Geha R S
Published: June 2004

T cells respond in a V beta-restricted fashion to bacterial enterotoxins bound to major histocompatibility complex (MHC) class II molecules. The requirement for CD4 in MHC class II-restricted T cell responses is very well established. We have assessed the role of CD4 in the T cell response to the bacterial enterotoxins Staphylococcal enterotoxin A (SEA), SEB, and toxic shock syndrome toxin 1. Three CD4- murine T cell hybridomas were transfected with the human CD4 molecule and assayed for interleukin 2 production in the presence of accessory cells bearing human MHC class II molecules and of the appropriate enterotoxin. The results clearly indicate that CD4- cells responded even to suboptimal concentrations of enterotoxin(s) equally well as CD4+ cells. Furthermore, expression of CD4 did not result in the acquisition of previously undetectable reactivity to enterotoxins. These results suggest that unlike the case with antigen-specific responses, formation of a T cell receptor-CD3/CD4 supramolecular complex is not always essential for T cell activation by bacterial enterotoxins.