bluuebunny/crossref_metadata_2025 huggingface dataset:
Histidine-rich glycoprotein inhibits the antiproliferative effect of heparin on smooth muscle cells.
Authors: Hajjar D P , Boyd D B , Harpel P C , Nachman R L
Published: June 2004

Histidine-rich glycoprotein (HRGP), an alpha-glycoprotein in human plasma that is also present in platelets and macrophages, binds heparin with high affinity and neutralizes its anticoagulant activity. We now report that HRGP specifically inhibits the antiproliferative effect of heparin on arterial smooth muscle cells while other heparinoid-binding proteins do not influence mitogenesis. The multicellular inflammatory response to endothelial injury characterized, in part, by the influx of platelets and macrophages, may be associated with HRGP release into the arterial microenvironment. This release of HRGP may allow smooth muscle cell proliferation and atherogenesis by inhibiting the action of endothelial cell-derived heparinoid substances.
Identification of the human 26-kD protein, interferon beta 2 (IFN-beta 2), as a B cell hybridoma/plasmacytoma growth factor induced by interleukin 1 and tumor necrosis factor.
Authors: Van Damme J , Opdenakker G , Simpson R J , Rubira M R , Cayphas S , Vink A , Billiau A , Van Snick J
Published: June 2004

A factor that promotes the growth of certain B cell hybridomas and of plasmacytomas is shown to be produced by normal human fibroblasts and by a line of human osteosarcoma cells (MG-63) after treatment with IL-1 or TNF. The hybridoma-plasmacytoma growth factor (HPGF) is identified with a 26 kD protein whose mRNA was previously shown to be induced in the same cells by the same inducers. First, poly(A)-rich RNA extracted from IL-1-treated cells could be enriched in HPGF-mRNA content by hybridization to 26 kD cDNA. Second, MG-63-derived HPGF purified to electrophoretic homogeneity was subjected to amino acid sequence analysis, whereby the NH2-terminal sequence was found to match the nucleotide sequence of a 26 kD cDNA clone.
Expression of the 6C3 antigen on murine hematopoietic neoplasms. Association with expression of abl, ras, fes, src, erbB, and Cas NS-1 oncogenes but not with myc.
Authors: Morse H C , Tidmarsh G F , Holmes K L , Frederickson T F , Hartley J N , Pierce J H , Langdon W Y , Dailey M O , Weissman I L
Published: June 2004

The monoclonal antibody 6C3 was used to test a wide variety of murine hematopoietic neoplasms for cell surface expression of a 160 kD glycoprotein (gp160(6C3)) previously shown to be expressed by neoplastic pre-B and some B lymphocytes transformed by Abelson murine leukemia virus (A-MuLV). This antigen was expressed on many pre-B and B cell lymphomas, but not on A-MuLV-transformed fibroblasts, T cell lymphomas, or myelomonocytic leukemias, gp160(6C3) was expressed by most early B-lineage spontaneous tumors, and early B tumors induced by replication-defective MuLV-containing oncogenes the products of which are associated with the cytoplasmic aspect of the plasma membrane, i.e., fes, abl, H-ras, bas, src, erbB, and Cas NS-1. By comparison, none of the early B lineage lymphomas induced by the "nuclear" oncogene avian v-myc MuLV, or arising in mice transgenic for a murine c-myc gene, or later B cell lineage stages bearing translocations of the c-myc locus expressed this antigen.
Human T cell gamma genes are frequently rearranged in B-lineage acute lymphoblastic leukemias but not in chronic B cell proliferations.
Authors: Chen Z , Le Paslier D , Dausset J , Degos L , Flandrin G , Cohen D , Sigaux F
Published: June 2004

T cell rearranging gene gamma (TRG gamma) and T cell antigen receptor beta (TCR beta) chain gene rearrangement and transcription were studied in a series of patients with B-lineage acute lymphoblastic leukemia (ALL), in which the Ig H chain genes are rearranged and the surface phenotype reproduces the stages of normal pre-B maturation. For comparison, polyclonal T cells from peripheral blood of healthy donors and blast cells from 19 cases of T lineage ALL were also studied. In this study we demonstrate the presence of a clonal rearrangement of the TRG gamma in 18 of the 22 B-lineage ALL cases and establish that this rearrangement, which generally involves the J gamma 1 region, is often monoallelic and appears different from the biallelic J gamma 2 rearrangement frequently seen in T-cell ALLs. In 9 of 22 cases, we found rearrangement of the genes of the TCR beta chain, which never involved the J beta 1 region. Conversely, the TRG gamma were seen in germline configuration in all 19 cases of B chronic lymphoid malignancies. In none of the 9 AML cases studied was TRG gamma and TCR beta chain gene rearrangement found. The TCR beta chain genes were rearranged in one B cell chronic lymphocytic leukemia (CLL). We also show that in B-lineage ALL, the cells probably use the same V gamma genes for TRG gamma rearrangements as the malignant cells in T-ALL and the polyclonal T cells. In none of the 13 B-lineage ALL cases investigated by Northern analysis was TCR beta mRNA expression detected, whereas a weak expression of TRG gamma transcripts was found in two of these cases. The correlations between surface phenotype, rearrangement of TRG gamma, TCR beta, and Ig H chain genes were analyzed. The significance of rearrangement of TRG gamma and TCR beta chain genes in B or pre-B cells is also discussed.
Gene conversion variations generate structurally distinct pilin polypeptides in Neisseria gonorrhoeae.
Authors: Swanson J , Robbins K , Barrera O , Koomey J M
Published: June 2004

Pilus+ to pilus- phenotype change occurs in Neisseria gonorrhoeae through gene conversion of the gonococcus' complete, expressed pilin gene by nucleotides homologous to the pilS1 copy 5 partial pilin gene; assembly missense pilin is synthesized but pili are not. Reversion to pilus+ occurs by a subsequent recombinational event that replaces the complete pilin gene's pilS1 copy 5-like sequence with nucleotides from a different partial gene to effect expression of an orthodox (i.e., pilus producing) pilin. Sibling pilus+ revertants of common parentage can carry different sequences in their expressed pilin genes because they have undergone nonidentical gene conversion events such as recombinations with sequences from different partial genes, or recombinations with different length nucleotide stretches of the same partial gene; either can yield structurally and antigenically variant pilin polypeptides.
Purification and characterization of the major iron-regulated protein expressed by pathogenic Neisseriae.
Authors: Mietzner T A , Bolan G , Schoolnik G K , Morse S A
Published: June 2004

This report describes a method to purify the major iron-regulated protein (MIRP) expressed by N. gonorrhoeae and N. meningitidis. This purification procedure involves maximal expression of the MIRP by growing the organisms on iron-limited media; cellular disruption by sonication followed by centrifugal fractionation; selective solubilization of the MIRP with the cationic detergent hexadecyltrimethylammonium bromide; cation-exchange chromatography in the presence of this detergent; and gel filtration chromatography. The MIRP purified by this technique migrates as a single band when analyzed by SDS-PAGE. The purified MIRP displayed an unusually basic isoelectric point, this value being greater than 9.35. Further biochemical analysis revealed the highly conserved nature of this protein isolated from the two pathogenic species of the genus Neisseria. For example, the amino acid composition of the meningococcal and gonococcal MIRPs were nearly identical and amino terminal sequence analysis showed that both shared the identical primary sequence through residue 48. Surprisingly, the first five NH2-terminal residues of the MIRPs exhibited homology with the first five residues of the gonococcal porin, protein I. Purified preparations of the MIRP exhibited a characteristic pink color reminiscent of the basic iron-binding protein lactoferrin. This observation coupled with the property of iron-regulation prompted us to analyze purified MIRP for iron-content. Approximately 0.5 mol iron per 1 mol of MIRP was detected. This study is the first to show that iron is associated with the MIRP, a property that may implicate this protein as playing a direct role in neisserial iron assimilation. While the precise function of the MIRP is not known, the availability of this protein in pure and biologically relevant quantities will allow further studies to elucidate its pathobiologic function.
Experimental allergic encephalomyelitis in the absence of a classical delayed-type hypersensitivity reaction. Severe paralytic disease correlates with the presence of interleukin 2 receptor-positive cells infiltrating the central nervous system.
Authors: Sedgwick J , Brostoff S , Mason D
Published: June 2004

One characteristic of experimental allergic encephalomyelitis (EAE) in all species is the presence of a considerable leukocyte infiltrate in the central nervous system (CNS). By adoptive transfer of EAE into irradiated or nonirradiated Lewis strain rats we now show that the bulk (greater than 90%) of infiltrating cells in the CNS are superfluous to the induction of disease, as lethally irradiated recipients, despite having very few infiltrating cells in the CNS, acquire severe paralytic EAE. The reduction in the level of infiltration in irradiated recipients is selective, however, as both irradiated and nonirradiated diseased animals have very similar numbers of cells expressing IL-2-R. Disease in irradiated recipient animals is associated with substantial submeningeal hemorrhage in the spinal cord and brain stem and similar hemorrhages are found in recipients rendered leukopenic with cytotoxic drugs. Clinical signs of disease and hemorrhage are preventable, however, by administration to the recipient rats of mAbs specific for the CD4 antigen. Classic delayed-type hypersensitivity (DTH) reactions are transferable with the same cells that produce EAE in both irradiated and nonirradiated recipient rats, but such transfer of DTH is observed only in nonirradiated recipient animals and not in irradiated rats. Collectively, the findings reported herein support the conclusion that the paralysis characteristic of acute EAE is mediated by the direct action of very small numbers of activated CD4+ lymphocytes that infiltrate the CNS and produce their effects by inducing vascular damage. The findings are not consistent with reports that the lesions in EAE are produced by a classic DTH reaction.
Human T cell receptor V beta gene polymorphism.
Authors: Concannon P , Gatti R A , Hood L E
Published: June 2004

Southern blot hybridizations with human T cell receptor V beta gene probes were used to determine the sizes of the various V beta gene subfamilies. An analysis of DNA samples from 100 unrelated individuals identified a single individual who lacked one V beta gene segment. A second individual had an apparently different repertoire of V beta gene segments in one subfamily, as assayed by hybridization, possibly due to a gene conversion event. An analysis with four restriction enzymes of DNA from 30 consanguineous donors detected restriction fragment length polymorphisms associated with 12 of 14 V beta gene segment subfamilies examined. In an analysis of DNAs from a large panel of unrelated individuals, some alleles at these loci were found to be in linkage disequilibrium, indicating a potentially close physical linkage. The segregation of three polymorphisms, two associated with V beta gene segment loci and one associated with the C beta genes, was compatible with Mendelian inheritance, and demonstrated that highly informative haplotypes could be generated. The high degree of polymorphism observed in the human T cell receptor beta chain complex should allow exploration of possible associations between T cell receptor genes and inherited diseases involving the immune system.
Endocytosis and recycling of the T3-T cell receptor complex. The role of T3 phosphorylation.
Authors: Krangel M S
Published: June 2004

An assay has been developed to assess the dynamics of cell surface glycoproteins, in which neuraminidase digestion of intact cells is used to determine the fate of cell surface molecules initially labelled via lactoperoxidase-catalyzed iodination. This approach has been used to demonstrate the constitutive endocytosis and recycling of the T3-T cell receptor (T CR) complex on the human T leukemic cell line HPB-MLT. Stable populations of both phosphorylated and nonphosphorylated forms of the T3 gamma peptide have been identified in these cells. Whereas the former are constitutively endocytosed, the latter appear to be excluded from this pathway. The results presented indicate that T3 gamma phosphorylation may control the endocytosis and recycling of the T3-TCR complex on this cell line.
Development of B lymphocytes in mice heterozygous for the X-linked immunodeficiency (xid) mutation. xid inhibits development of all splenic and lymph node B cells at a stage subsequent to their initial formation in bone marrow.
Authors: Forrester L M , Ansell J D , Micklem H S
Published: June 2004

CBA/N mice were crossed with CBA/Ca-Pgk-1a to produce female F1 hybrids that were heterozygous for both xid and the phosphoglycerate kinase 1 (PGK-1) allozymes. PGK acted as a quantifiable marker for the frequency of cells in which the xid-bearing X chromosome was active in lymphocytic and other cell populations. In adults, such cells (termed xid cells) were virtually absent in FACS-sorted splenic and lymph node B cells, and in all three splenic subpopulations distinguished on the basis of their relative expression of membrane mu and delta chains. Thus, the xid mutation appeared to compromise the development of all B cells. Erythrocytes, thymocytes, T cells, and granulocytes were unaffected. Selection against xid cells was less pronounced in the spleens of 2-6-wk-old mice. In the bone marrow, there was evidence for selection against xid in the production of B cells (except at 2 wk of age), but not at the pre-B cell level. These data suggest that, in competition with normal non-xid cells, newly-formed xid B cells were less likely to be incorporated into the peripheral B cell pool.
Tyrosine phosphorylation of an interleukin 2 receptor-like protein in cells transformed by human T cell leukemia virus type I.
Authors: Ogawa R , Sugamura K , Watanabe Y
Published: June 2004

Three interleukin 2 (IL-2)-independent human T cell lines transformed with human T cell leukemia virus type I were analyzed for o-phosphotyrosine-containing proteins (Ptyr proteins). Membrane and intracellular immunofluorescence was positive with antibody to Ptyr (Ptyr antibody). 10 size classes of Ptyr proteins with molecular masses of 81-28 kD were isolated with Ptyr antibodies. Among these, proteins of 64 kD (pI 4.5 and 4.8-5.3) and 45 kD (pI 4.3) were found on the outer cell surface. The Ptyr protein of 64 kD (pI 4.5) had the characteristics of the IL-2 receptor (IL-2-R) in that it was recognized by two monoclonal antibodies directed against different epitopes on the IL-2-R.
The contribution of NZW genes to lupus-like disease in (NZB x NZW)F1 mice.
Authors: Kotzin B L , Palmer E
Published: June 2004

Unlike parental NZB or NZW mice, (NZB X NZW)F1 mice exhibit a lupus-like disease characterized by high serum levels of IgG antinuclear antibodies and a fatal immune-complex glomerulonephritis. At least three unlinked gene loci can be distinguished in NZW mice that conceivably contribute to a T cell-dependent autoimmune disease, including the MHC (H-2z) and the T cell receptor alpha and beta chain gene complexes. We undertook an (NZB X NZW)F1 X NZB backcross to determine the relative contribution of these NZW genes to lupus-like renal disease and autoantibody production in F1 mice. The incidence of severe renal disease and elevated levels of IgG antibodies to dsDNA and histone in the backcross mice was approximately half of that observed in (NZB X NZW)F1 mice. Furthermore, there was a strong correlation between the presence of the NZW H-2z haplotype and lupus-like disease in backcross mice. Approximately 90% of backcross mice with disease carried the NZW H-2z locus compared with 16% of mice without disease; nearly 90% of H-2d/z mice expressed severe autoimmune disease. In contrast, no association was apparent between the presence of the NZW T cell receptor alpha chain gene complex or beta chain gene complex and severe renal disease or autoantibody production. Thus, the NZW MHC or gene(s) linked to this locus appear to be the only dominant NZW genetic contribution to F1 disease.
Induction of a cationic shift in IgG anti-DNA autoantibodies. Role of T helper cells with classical and novel phenotypes in three murine models of lupus nephritis.
Authors: Datta S K , Patel H , Berry D
Published: June 2004

We investigated the underlying mechanisms of systemic autoimmune disease in MRL-+/+, (NZB X NZW)F1, and (NZB X SWR)F1 mice, since these strains develop glomerulonephritis without the superimposition of any secondary lupus-accelerating genes. All three strains manifested a common immunoregulatory defect specific for the production of pathogenic anti-DNA autoantibodies that are of IgG class and cationic in charge. At or just before the age they began to develop lupus nephritis, spleen cells of the mice contained a subpopulation of Th cells that selectively induced their B cells in vitro to produce highly cationic IgG autoantibodies to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). By contrast, T cells from younger preautoimmune mice were incapable of providing this help. Moreover, only B cells of the older lupus mice could be induced to secrete cationic anti-DNA antibodies of IgG class. B cells of young lupus mice could not produce the cationic autoantibodies even with the help of T cells from the older mice, nor upon stimulation with mitogens. In the older lupus mice we found two sets of Th cells that spontaneously induced the cationic shift in autoantibodies; one set belonged to the classical Th category with L3T4+,Lyt-2- phenotype, whereas the other surprisingly belonged to a double-negative (L3T4-,Lyt-2-), Lyt-1+ subpopulation. The latter set of unusual Th cells were unexpected in these lupus mice since they lacked the lpr (lympho-proliferation) gene. Thus three apparently different murine models of systemic lupus erythematosus possess a common underlying mechanism specific for the spontaneous production of pathogenic anti-DNA autoantibodies.
MHC-restricted minimal regulatory circuit initiated by a class II-autoreactive T cell clone.
Authors: Sano K , Fujisawa I , Abe R , Asano Y , Tada T
Published: June 2004

The in vivo administration of a self-class II-reactive Th clone MS202 derived from C3H into syngeneic mice resulted in the suppression of both primary and early secondary antibody responses against T cell-dependent antigens. The suppression was due to the generation of antigen-nonspecific Ts cells in the recipient, as the splenic T cells from the mice treated with MS202 were able to strongly suppress the in vitro secondary antibody response of primed syngeneic spleen cells. The dose-response curve of suppression indicated the generation of an effector type Ts that directly suppressed Th. The surface phenotype of Ts was Ly-1+,2-, L3T4+, I-J-. The presence of Ly-1+,2+ T cells was not required to induce the suppression. The suppression was strictly restricted to H-2k, as F1 Ts cells were able to suppress the response of C3H but not of B6 B cells helped by the same F1 Th cells. The experiments with chimeric mice indicated that the direct target of Ts is an MHC-restricted Th but not a B cell or APC. The results indicate the existence of a minimal regulatory circuit where an MHC-restricted Th induces a preprogrammed Ts that in turn directly suppresses Th with the same MHC-restriction specificity. The induction of and suppression by Ts appeared to be due to the direct recognition of MHC restriction sites of Th cells.
Human interleukin 1 induces interleukin 1 gene expression in human vascular smooth muscle cells.
Authors: Warner S J , Auger K R , Libby P
Published: June 2004

The recognition that cells of the vascular wall can secrete cytokines such as IL-1 suggests new mechanisms for initiating or sustaining inflammatory responses in blood vessels. We report that purified human monocyte-derived IL-1 or recombinant human IL-1 (rIL-1 beta and rIL-1 alpha) induce cultured human smooth muscle cells derived from veins or arteries to synthesize IL-1 beta mRNA and produce and release biologically active IL-1. rIL-1 beta also stimulated the production of PGE2 by smooth muscle cells. Exposure to rIL-1 beta (1-100 ng/ml), or rIL-1 alpha (0.01-10 ng/ml) increased IL-1 beta mRNA levels within 30 min. Actinomycin D (1 microgram/ml) prevented the induction of IL-1 beta mRNA by rIL-1. IL-1 alpha mRNA was detected in SMC treated with cycloheximide (1 microgram/ml) and rIL-1 beta, or cycloheximide alone. rIL-1 alpha and rIL-1 beta produced maximal levels of IL-1 beta mRNA after 4 h, and intracellular IL-1 biological activity after 6 h of exposure. Release of IL-1 activity in the extracellular medium began after 1 h of incubation with rIL-1 beta or rIL-1 alpha, and continued for up to 24 h. Anti-TNF antiserum that neutralized the biological activity of rTNF did not affect rIL-1-induced production of IL-1 beta mRNA or IL-1 release, suggesting that the release of TNF does not mediate these processes. Several experimental approaches indicated that the release of IL-1 by smooth muscle cells was not due to endotoxin contamination of the IL-1 preparations. Anti-IL-1 antiserum blocked the induction of smooth muscle cell IL-1 gene expression by rIL-1 beta. Polymyxin B did not prevent IL-1-induced IL-1 expression by these cells, but blocked the effect of endotoxin. Heat treatment destroyed the stimulatory capacity of rIL-1 beta, but did not affect the ability of bacterial endotoxin to induce IL-1 expression. The production of IL-1 by human vascular smooth muscle cells was not due to contamination of the cell cultures with blood monocytes, inasmuch as treatment with an antimonocyte antibody (anti-Mo2) and complement did not alter IL-1 beta mRNA content or the amount of IL-1 released from the cells in response to endotoxin, rIL-1 alpha, or rIL-1 beta.(ABSTRACT TRUNCATED AT 400 WORDS)
Tissue-specific expression of cell-surface Qa-2 antigen from a transfected Q7b gene of C57BL/10 mice.
Authors: Waneck G L , Sherman D H , Calvin S , Allen H , Flavell R A
Published: June 2004

We screened a cDNA library prepared from a BALB.B10 CTL clone that expresses Qa-2 antigen, and isolated four clones derived from Q7b, a Qa region gene of C57BL/10. One of these Q7b cDNAs and the Q7b chromosomal gene were subcloned into expression vectors and transfected into L cells and R1.1 thymoma cells. We found that the chromosomal Q7b gene expresses Qa-2 on the surface of R1.1 cells, but not on L cells while the Q7b cDNA expresses protein on the surface of both cell types. The levels of Qa-2 expression do not correlate with the total levels of Q7b mRNA in these transfectants. Our results suggest that the tissue-specific expression of Qa-2 may be controlled, in part, by mechanisms of alternate RNA splicing. By using hybrid gene constructs, we have mapped the tissue-specific element to the 3' part of the gene, downstream of a site near the middle of exon 4. The hybrid polypeptides differ significantly in their transmembrane and cytoplasmic regions. These portions of the protein also may play a role in the tissue-specific expression of Qa-2.
Monoclonal antibodies to LFA-1 and to CD4 inhibit the mixed leukocyte reaction after the antigen-dependent clustering of dendritic cells and T lymphocytes.
Authors: Inaba K , Steinman R M
Published: June 2004

T cell proliferation in response to many stimuli is known to occur in discrete clusters of dendritic cells (DC) and CD4+ helper lymphocytes. The role of lymphocyte function-associated antigen (LFA-1) and CD4 in the formation and function of these clusters has been evaluated in the mixed leukocyte reaction (MLR). By day 1 of the control MLR, most of the DC and responsive T cells are associated in discrete aggregates. Addition of anti-LFA-1 and CD4 reagents does not block DC-T aggregation but reduces the subsequent proliferative response by 80-90%. Anti-LFA-1 disassembles newly formed DC-T cell aggregates, whereas anti-CD4 inhibits blastogenesis without disrupting the cluster. Binding of DC to sensitized, antigen-specific CD4+ cells has been studied using lymphoblasts isolated at day 4 of the MLR. It has been shown previously that greater than 80% blasts rebind to DC in an antigen-specific fashion in rapid (10 min) binding assays. Antigen-dependent DC-T binding is blocked by anti-Ia but not by mAb to LFA-1 or CD4. However, the bound anti-CD4-coated lymphocytes are unable to release IL-2. Anti-LFA-1-coated T cells release IL-2 but are easily disaggregated after binding to DC. These findings lead to two conclusions. LFA-1 and CD4 are not involved in the initial steps whereby DC bind to T cells but exert an independent and subsequent role. LFA-1 acts to stabilize the DC-T cluster, while CD4 contributes to lymphocyte blastogenesis and IL-2 release. Because DC but not other presenting cells cluster unprimed lymphocytes, it seems likely that an antigen-independent mechanism distinct from LFA-1 and CD4 mediates aggregate formation at the onset of cell-mediated immunity.
A major immunogen in Schistosoma mansoni infections is homologous to the heat-shock protein Hsp70.
Authors: Hedstrom R , Culpepper J , Harrison R A , Agabian N , Newport G
Published: June 2004

A 70,000 mol wt protein of Schistosoma mansoni was shown to be a major immunogen that invariably elicited an antibody response in infected humans. The universality of the response to this abundant antigen was confirmed in experimental animals and included the antibody response associated with the protective irradiated cercarial vaccine. We identified the 70,000 mol wt antigen as an S. mansoni homologue of the major eukaryotic heat-shock protein hsp70 by DNA sequence analysis of a cDNA insert from a lambda gt11 clone expressing the antigen and located the immunodominant epitope near the COOH-terminus of the molecule. The antigenic relationship of hsp70 to schistosome infections suggested an important role for this protein in parasite development and pathogenesis.
The fifth component of complement (C5) in the mouse. Analysis of the molecular basis for deficiency.
Authors: Wheat W H , Wetsel R , Falus A , Tack B F , Strunk R C
Published: June 2004

C5-deficient mice differed from C5-sufficient mice both quantitatively and qualitatively in C5 protein, C5 mRNA, and the C5 gene. C5-deficient protein was present as decreased amounts of an unprocessed, single-chain precursor. C5-deficient mRNA was decreased in amount and present in two forms, the smaller of which was the same as the single form in normal cells. Nuclei from both normal and deficient cells contained the larger form of C5 mRNA, and C5-deficient DNA demonstrated differences from the normal pattern on Southern analysis for two restriction enzymes. These data suggest that the primary transcript of the C5-deficient gene is abnormal, retarding the processing of the C5 mRNA, and that the C5-deficient mRNA codes for an abnormal protein.
Functional gamma chain-associated T cell receptors on cerebrospinal fluid-derived natural killer-like T cell clones.
Authors: Ang S L , Seidman J G , Peterman G M , Duby A D , Benjamin D , Lee S J , Hafler D A
Published: June 2004

We have derived 33 independent T cell clones from the cerebrospinal fluid (CSF) of a patient with subacute sclerosing panencephalitis using a single T cell cloning method. 6% (2 of 33) of these clones express the T cell receptor gamma (TCR-gamma) protein and are called CSF TCR-gamma clones. Phenotypic analyses of the CSF TCR-gamma clones indicate that they are WT-31-, CD3+, CD4-, and CD8-. The TCR-gamma protein exists on the cell surface as part of an 85-kD disulphide-linked dimer noncovalently associated with the CD3 polypeptides. The CSF TCR-gamma clones have NK-like activity that can be inhibited by anti-CD3 mAbs. Both CSF TCR-gamma clones proliferated in response to anti-CD3 mAbs coupled to Sepharose beads and/or IL-2. Furthermore, stimulation of one of these clones with anti-CD3 mAbs results in a rapid rise in intracellular calcium. These data suggest that T cells bearing the CD3-TCR-gamma protein complex are functional and play a role in the human immune response.
Chemoattractant-induced activation of c-fos gene expression in human monocytes.
Authors: Ho Y S , Lee W M , Snyderman R
Published: June 2004

Human monocytes use the products of phosphoinositide hydrolysis (1,2-diacylglycerol and inositol 1,4,5-triphosphate) as second messengers to trigger rapid cellular activation during the occupancy of chemoattractant receptors. The effect of chemoattractants on modulation of gene expression in monocytes was examined in this study. The chemoattractants FMLP and platelet-activating factor induced the progressive increase of c-fos RNA to 6-15-fold over those of control within 30 min after treatment. Similar kinetics of c-fos gene activation was also observed when cells were treated with PMA or sn-1,2-dioctanoylglycerol, but not with the calcium mobilizer ionomycin, suggesting a role for protein kinase C in gene regulation by chemoattractant receptors. Activation of c-fos gene expression by FMLP is mediated through a pertussis toxin-sensitive G protein, since pertussis toxin treatment of the cells blocked the induction of the c-fos gene by FMLP but not PMA. The level of c-myc RNA was slightly decreased after 1 h of treatment with chemoattractants, but not with PMA or diacylglycerol. This implies that chemoattractant receptor occupancy generates signals beyond protein kinase C activation that are capable of selectively downregulating monocyte gene expression. The effect of FMLP and PMA on the accumulation of c-fos RNA appears to result from altering both the rate of transcription and message stability. These observations indicate that signals generated through chemoattractant receptor occupancy may regulate monocyte function at the genetic level.
Active lambda and kappa antibody gene rearrangement in Abelson murine leukemia virus-transformed pre-B cell lines.
Authors: Persiani D M , Durdik J , Selsing E
Published: June 2004

The two Abelson murine leukemia virus (A-MuLV)-transformed cell lines, BM18-4 and ABC-1, undergo immunoglobulin L-chain gene recombination during passage in tissue culture. BM18-4 cells are capable of kappa gene recombination, whereas ABC-1 cells are capable of both kappa and lambda gene recombination. The expression of H chains is apparently not necessary for continuing L chain gene recombination in either of these cells, although H-chain expression may have been involved in the initiation of L-chain gene recombination. All ABC-1 cells that have lambda gene rearrangements also display recombined kappa alleles, supporting the hypothesis that kappa and lambda gene recombination are initiated in an ordered, developmentally regulated manner in maturing B cells. However, analyses of the ABC-1 line indicate that pre-B cells that have initiated lambda gene recombination do not terminate kappa gene rearrangement. The lambda gene recombinations that occur in the ABC-1 cell line indicate that the germline order of lambda gene segments is: 5' ... V lambda 2 ... J lambda 2C lambda 2-J lambda 4C lambda 4 ... V lambda 1 ... J lambda 3C lambda 3-J lambda 1C lambda 1 ... 3'. In addition, the frequencies of lambda 1, lambda 2, and lambda 3 gene recombinations among ABC-1 cells are quite different than the frequencies of B cells producing lambda 1, lambda 2, and lambda 3 L-chains in the mouse. RS DNA recombinations also occur in the BM18-4 and ABC-1 cell lines, supporting the notion that Ig gene recombinases are involved in RS rearrangement. Recombined RS segments are infrequent among BM 18-4 cells but common among ABC-1 cells, suggesting that RS recombinational events often occur in maturing pre-B cells just before initiation of lambda gene rearrangements. This developmental timing is consistent with the hypothesis that RS recombination may be involved in the initiation of lambda gene assembly.
Lipopolysaccharide induces recurrence of arthritis in rat joints previously injured by peptidoglycan-polysaccharide.
Authors: Stimpson S A , Esser R E , Carter P B , Sartor R B , Cromartie W J , Schwab J H
Published: June 2004

Rat ankle joints injected intraarticularly with 5 micrograms of group A streptococcal peptidoglycan-polysaccharide (PG-APS) developed an acute course of arthritis. Recurrence of arthritis was induced in 100% of these joints by intravenous injection of as little as 10 micrograms of Salmonella typhimurium lipopolysaccharide (LPS) 3 wk after intraarticular injection. This reaction was similar in athymic and euthymic rats. Buffalo rats were less susceptible than Lewis or Sprague-Dawley rats. Neisseria gonorrhoeae, Yersinia enterocolitica, and Escherichia coli LPS, and S. typhimurium Re mutant LPS, were also active. Re mutant LPS activity was greatly reduced by mixing with polymyxin B. E. coli lipid A was weakly active. An acute synovitis of much less incidence, severity, and duration was seen in contralateral joints injected initially with saline, and in ankle joints of naive, previously uninjected rats after intravenous LPS injection. The intravenous injection of the muramidase mutanolysin on day 0 or 7 after intraarticular PG-APS injection prevented LPS-induced recurrence of arthritis. These studies suggest that the phlogistic activities of lipid A and peptidoglycan might interact in an inflammatory disease process, and that LPS may play a role in recurrent episodes of rheumatoid arthritis or reactive arthritis.
Invasion of mouse erythrocytes by the human malaria parasite, Plasmodium falciparum.
Authors: Klotz F W , Chulay J D , Daniel W , Miller L H
Published: June 2004

Plasmodium falciparum malaria merozoites require erythrocyte sialic acid for optimal invasion of human erythrocytes. Since mouse erythrocytes have the form of sialic acid found on human erythrocytes (N-acetyl neuraminic acid), mouse erythrocytes were tested for invasion in vitro. The Camp and 7G8 strains of P. falciparum invaded mouse erythrocytes at 17-45% of the invasion rate of human erythrocytes. Newly invaded mouse erythrocytes morphologically resembled parasitized human erythrocytes as shown on Giemsa-stained blood films and by electron microscopy. The rim of parasitized mouse erythrocytes contained the P. falciparum 155-kD protein, which is on the rim of ring-infected human erythrocytes. Camp but not 7G8 invaded rat erythrocytes, indicating receptor heterogeneity. These data suggest that it may be possible to adapt the asexual erythrocytic stage of P. falciparum to rodents. The development of a rodent model of P. falciparum malaria could facilitate vaccine development.
The gene encoding decay-accelerating factor (DAF) is located in the complement-regulatory locus on the long arm of chromosome 1.
Authors: Lublin D M , Lemons R S , Le Beau M M , Holers V M , Tykocinski M L , Medof M E , Atkinson J P
Published: June 2004

Delay-accelerating factor (DAF) protects host cells from complement-mediated damage by regulating the activation of C3 convertases on host cell surfaces. Using a panel of hamster-human somatic cell hybrids, the DAF gene was mapped to human chromosome 1. In situ hybridization studies using human metaphase cells further localized the gene to bands 1q31-41, with the largest cluster of grains at 1q32. This establishes the close linkage of the DAF gene to genes for four other proteins (C3b/C4b receptor or complement receptor 1, C3d receptor or complement receptor 2, factor H, and C4-binding protein) that share 60-amino-acid homologous repeats as well as complement-regulatory or -receptor activity, thereby enlarging the complement-regulatory gene family on the long arm of human chromosome 1.
Clearance of Giardia muris infection requires helper/inducer T lymphocytes.
Authors: Heyworth M F , Carlson J R , Ermak T H
Published: June 2004

The aim of this work was to identify the T cell subset (helper/inducer or cytotoxic/suppressor) that plays a major part in the clearance of Giardia muris infection. BALB/c mice were selectively depleted of helper/inducer (Th/i) or cytotoxic/suppressor (Tc/s) T cells, by treatment with rat IgG monoclonal antibodies directed against the Th/i cell surface antigen L3T4, or against the Tc/s cell surface antigen Ly-2, and were infected with G. muris cysts. While mice depleted of Tc/s cells cleared G. muris infection at a normal rate, mice depleted of Th/i cells did not clear the infection. Depletion of the relevant T cell subset was confirmed by flow cytometry and by immunohistochemistry. The data indicate that helper/inducer T cells, but not cytotoxic T cells, are of major importance for elimination of G. muris from the mouse intestine.
Initial events in the formation of immune deposits in passive Heymann nephritis. gp330-anti-gp330 immune complexes form in epithelial coated pits and rapidly become attached to the glomerular basement membrane.
Authors: Kerjaschki D , Miettinen A , Farquhar M G
Published: June 2004

The nephritogenic antigen of Heymann's nephritis (HN), gp330, was previously demonstrated (4-9) to be a resident glycoprotein of coated pits in the glomerular and proximal tubule epithelium of rats, and anti-gp330 IgG given intravenously was found to form IDs in glomeruli (passive HN). The purpose of this study was to investigate the detailed events that occur in the formation of IDs in passive HN. HN was induced by the injection of either 125I-labeled or unlabeled anti-gp330 IgG. At various times after injection (15 min to 8 d) the kidneys of some of the injected rats were fixed by perfusion, and the distribution of the rabbit IgG was determined by immunofluorescence and by immunoelectron microscopy. Glomeruli were isolated from the kidneys of injected rats and used for isolation of GBM fractions or for elution of the bound IgG. At 15 min to 1 h after injection, the rabbit IgG was localized by immunocytochemistry exclusively in coated pits along the podocyte plasmalemma facing the GBM. By 1-8 d, anti-gp330 IgG was detected in larger electron-dense IDs often located under the slit diaphragms. Serial sectioning revealed that each of the IDs maintained contact with a coated pit at some level. When GBMs isolated from rats given radiolabeled anti-gp330 IgG were examined by electron microscopy, the IDs were found to remain attached to the GBMs as early as 15 min after injection and coisolated with them at all time points. By double-immunolabeling of the isolated GBMs with two sizes of gold particles, both the antigen (gp330) and the anti-gp330 IgG could be demonstrated in IDs at all time points. When the amount of radiolabeled anti-gp330 bound to GBM fractions was compared with that of isolated glomeruli, it was found that 20% of the radiolabel remained bound to the purified GBMs at 15 min after injection, and 90% at 3 d. The bound IgG was released only by treatments that disrupt antibody-antigen complexes (high and low pH), but not by the other treatments we tried (detergent, high salt, heparinase, or collagenase digestion). When the IgG bound to glomeruli was eluted with acid citrate buffer 3 d after injection, it was found to specifically immunoprecipitate only gp330 from detergent-solubilized 125I-labeled kidney microvillar vesicles. By isoelectric focusing the eluate was found to be enriched in IgGs with acidic isoelectric points.(ABSTRACT TRUNCATED AT 400 WORDS)
Regulation of human eosinophil viability, density, and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fibroblasts.
Authors: Owen W F , Rothenberg M E , Silberstein D S , Gasson J C , Stevens R L , Austen K F , Soberman R J
Published: June 2004

Normodense human peripheral blood eosinophils were isolated under sterile conditions from the 22/23 and 23/24% interfaces and the cell pellet of metrizamide gradients. After culture for 7 d in RPMI media in the presence of 50 pM biosynthetic (recombinant) human granulocyte/macrophage colony-stimulating factor (rH GM-CSF), 43 +/- 7% (mean +/- SEM, n = 8) of the cells were viable; in the absence of rH GM-CSF, no eosinophils survived. The rH GM-CSF-mediated viability was concentration dependent; increased survival began at a concentration of 1 pM, a 50% maximal response was attained at approximately 3 pM, and a maximal effect was reached at concentrations of greater than or equal to 10 pM rH GM-CSF. In the presence of rH GM-CSF and mouse 3T3 fibroblasts, 67 +/- 6% (mean +/- SEM, n = 8) of the eosinophils survived for 7 d. In a comparative analysis, there was no difference in eosinophil viability after 7 and 14 d (n = 3) in the presence of 50 pM GM-CSF and fibroblasts. Culture with fibroblasts alone did not support eosinophil survival. The addition of fibroblast-conditioned media to rH GM-CSF did not further improve eosinophil viability, indicating a primary role for GM-CSF in supporting these eosinophil cell suspensions ex vivo and a supplementary role for 3T3 fibroblasts. Eosinophils cultured for 7 d localized on density gradient sedimentation at the medium/18, 18/20, and 20/21 interfaces of metrizamide gradients, indicating a change to the hypodense phenotype from their original normodense condition. In addition, the cultured eosinophils generated approximately 2.5-fold more LTC4 than freshly isolated cells when stimulated with the calcium ionophore A23187 and manifested sevenfold greater antibody-dependent killing of S. mansoni larvae than the freshly isolated, normodense cells from the same donor. Thus we demonstrate the rH GM-CSF dependent conversion in vitro of normodense human eosinophils to hypodense cells possessing the augmented biochemical and biological properties characteristic of the hypodense eosinophils associated with a variety of hypereosinophilic syndromes. In addition, these studies provide a culture model of at least 14 d suitable for the further characterization of hypodense eosinophils.
Genotypic analysis of B cell colonies by in situ hybridization. Stoichiometric expression of three VH families in adult C57BL/6 and BALB/c mice.
Authors: Schulze D H , Kelsoe G
Published: June 2004

The filter paper disc method for cloning inducible lymphocytes was used to census the splenic B cell population of C57BL/6 and BALB/c mice for the expression of three VH gene-families, VH X-24, -Q52, and -J558. B cell colonies, arising from single founder lymphocytes, were identified by in situ hybridization with VH family- and C mu-specific cDNA probes. Some 6.7 X 10(4) C mu+ colonies were screened. Among C57BL/6- or BALB/c-derived colonies, approximately 3% were VH X-24+, approximately 19% were VH Q52+, and approximately 54% were VH J558+. These frequencies are consistent with a process of equiprobable expression for individual VH segments, and provide direct evidence that normal splenic B lymphocytes use a process of random genetic combinatorics to generate the antibody repertoire.
Protection of bone marrow transplant recipients from lethal doses of methotrexate by the generation of methotrexate-resistant bone marrow.
Authors: Williams D A , Hsieh K , DeSilva A , Mulligan R C
Published: June 2004

To develop a highly efficient means for generating methotrexate resistant (MTXr) hematopoietic cells in vivo, a recombinant retroviral genome was constructed that encodes a MTXr dihydrofolate reductase (DHFRr). Cell lines producing high titers of virus capable of transmitting the DHFR gene were generated and used to infect mammalian cells in vitro. Analysis of infected fibroblasts indicated that the DHFRr gene was transmitted intact and conferred a high level of MTXr upon cells. Based on these findings, DHFRr-containing virus was used to infect murine bone marrow cells in vitro. Following infection, the transduced cells were introduced into lethally irradiated recipients via bone marrow transplantation techniques. The presence of the proviral sequences in cells of the spleen and bone marrow of engrafted recipients was associated with significantly increased survival of mice treated with otherwise lethal doses of MTX.
Tumor necrosis factor-mediated release of platelet-derived growth factor from cultured endothelial cells.
Authors: Hajjar K A , Hajjar D P , Silverstein R L , Nachman R L
Published: June 2004

Platelet-derived growth factor (PDGF) is a 30,000-Mr glycoprotein that is chemotactic and mitogenic for vascular smooth muscle cells (SMC). It is also a potent vasoconstrictor. In the present study, we found that the macrophage-derived polypeptide, tumor necrosis factor (TNF), releases a factor from human umbilical vein endothelial cells (EC) that is mitogenic for SMC. Postculture medium from TNF-stimulated EC induced a 90% increase in mitogenesis is compared with controls. This effect was half-maximal at a TNF dose of 114 pM, reflected a 2.5-fold increase in PDGF-specific mRNA synthesis, and peaked at 15 h of TNF stimulation. Mitogenic activity was completely abrogated by preincubation of postculture medium with antibody to platelet PDGF. Stimulation of EC with IL-1 (60-240 pM) led to the release of similar mitogenic activity. Thus, in addition to its effects on the hemostatic and adhesive properties of EC, TNF also promotes release of PDGF, which may serve to modulate proliferation of vascular SMC during wound healing, inflammation, and atherogenesis.
Decay-accelerating factor. Genetic polymorphism and linkage to the RCA (regulator of complement activation) gene cluster in humans.
Authors: Rey-Campos J , Rubinstein P , Rodriguez de Cordoba S
Published: June 2004

We have investigated the genetic relationships between the human decay-accelerating factor (DAF) and a group of complement components including the C3b/C4b receptor (CR1), C4-binding protein (C4bp), and factor H (H), to which DAF is structurally and functionally related. CR1, C4bp, and H were previously demonstrated to be encoded by a cluster of closely linked genes, which we have designated regulator of complement activation (RCA). Southern blot analysis of genomic DNA using a DAF cDNA probe unraveled the existence of restriction fragment length polymorphism (RFLP) for both Bam HI and Hind III restriction endonucleases. Segregation analysis of these polymorphic fragments in families informative for the segregation of alleles at the CR1, C4BP, and H loci (RCA-haplotypes), demonstrated that, in humans, the gene encoding DAF is located within the RCA gene cluster. No recombinants between DAF and C4BP/CR1 were encountered in 32 informative meioses. In addition, in two individuals showing recombination between the CR1/C4BP and H loci, DAF segregated with the CR1/C4BP segment. Thus, the DAF gene maps closer to the CR1/C4BP loci than to the H gene, from which it can be separated by genetic recombination.
High hepatic natural killer cell activity in murine lupus.
Authors: Magilavy D B , Steinberg A D , Latta S L
Published: June 2004

This study demonstrates a profound elevation of NK activity, as measured by cytotoxicity to YAC-1 targets in a 4-h incubation 51Cr-release assay, of freshly isolated hepatic NPC from both MRL/lpr and (NZB X NZW)F1 mice. This marked increase was not observed in splenic or peripheral blood NK. The hepatic NK were nonadherent, radioresistant, Ly-1-,2-, and AGM1+. Furthermore, biologic response modifiers can further augment hepatic NK activity in these autoimmune strains.