bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum factor requirement for reactive oxygen intermediate release by rabbit alveolar macrophages.
Authors: Gerberick G F , Willoughby J B , Willoughby W F
Published: June 2004

Alveolar macrophages (AM) from pathogen-free rabbits were unable to release reactive oxygen intermediates (ROI) unless they were conditioned in serum for 24-48 h before triggering with membrane-active agents. The degree of serum conditioning of AM depended upon the concentration of serum used; optimal ROI release was obtained at or above 7.5% fetal bovine serum (FBS). FBS, autologous rabbit serum, pooled rabbit serum, and pooled human serum were each capable of conditioning AM for release of ROI. Serum conditioning of AM requires synthesis of new protein(s); and the enzyme required for ROI production, NADPH oxidase, was only detectable in serum-conditioned cells. Moreover, serum-conditioned cells lost their ability to release ROI after transfer to serum-free medium, while cells maintained in serum-free medium acquired the capacity to release ROI after their transfer to serum-containing medium, demonstrating the reversibility of the phenomenon. Initial purification data indicate that conditioning is mediated by a discrete serum constituent, which precipitates 40-80% saturated ammonium sulfate, does not bind to Cibacron Blue columns, and has a molecular weight of 30,000 to 50,000, as determined by molecular exclusion chromatography. Unlike gamma interferon, which also enhances ROI release by macrophages, our serum-conditioning factor is not acid labile, retaining 67% of its activity after 120 min incubation at pH 2.0. Moreover, it does not appear to be a contaminating endotoxin, since LPS neither conditioned AM for ROI production, nor triggered ROI production by serum-conditioned AM. We propose that such a conditioning requirement may normally protect the lung against ROI-mediated tissue injury. However, during a pulmonary inflammatory reaction initiated by other mediator systems, the resulting transudation of plasma proteins into the alveolar spaces may condition AM in situ for ROI production.
Location of variable and conserved epitopes among the multiple serotypes of streptococcal M protein.
Authors: Jones K F , Manjula B N , Johnston K H , Hollingshead S K , Scott J R , Fischetti V A
Published: June 2004

In studies primarily confined to the amino-terminal region of the fibrillar group A streptococcal M protein, only limited immunological crossreactions have been observed among M serotypes. In this investigation, two monoclonal antibodies generated against nearly the entire M6 molecule (LysM6) were used to determine the extent of crossreactions among M serotyping strains and to localize their epitopes on the M molecule. Colony blot and immunoblot analyses revealed that an epitope responsible for crossreactions among 5 of the 56 strains of different M serotypes tested is located in the amino-terminal half of the molecule, distal to the cell surface. In contrast, a more common crossreactive epitope, reacting with 20 of the 56 strains, is located near the middle of the M molecule. These studies also reveal that the more conserved determinant, located more proximally to the cell surface, is accessible to the immune system, even on the whole organism, and, thus, may be useful in devising means to protect against infections by multiple group A streptococcal M serotypes.
Major histocompatibility complex-restricted antigen receptor on T cells. VIII. Role of the LFA-1 molecule.
Authors: Golde W T , Kappler J W , Greenstein J , Malissen B , Hood L , Marrack P
Published: June 2004

We show that the LFA-1 molecule on T cells does not play a role in the stimulation of T cell hybridomas by certain targets, namely antigen presented by L cell derivatives or polyvalent anti-receptor antibody. These results suggest that LFA-1 may act by binding to ligands that are not present on all cells. We hope this result will help us and others to establish the true role of LFA-1 in T cell responses.
Isotype-specific immunoregulation. IgA-binding factors produced by Fc alpha receptor-positive T cell hybridomas regulate IgA responses.
Authors: Kiyono H , Mosteller-Barnum L M , Pitts A M , Williamson S I , Michalek S M , McGhee J R
Published: June 2004

T-T hybridomas, produced by fusions between R1.1 T lymphoma and cloned T helper cells that promote IgA responses (Th A cells) were characterized in this study. A total of 85 cloned cell lines were produced, and their supernatants were assessed for support of antigen-dependent IgA (and IgM and IgG) responses. 16 of 85 culture fractions supported IgA anti-sheep red blood cell, -horse red blood cell, or -trinitrophenyl responses in either lipopolysaccharide-triggered splenic B cell, or normal Peyer's patch B cell cultures, and the responses were specific for the antigen used for in vitro immunization. None of the supernatants from the cell lines induced significant polyclonal responses in these B cell cultures. Interestingly, the 16 hybridomas that produced supernatants with IgA-promoting properties had Fc receptors for IgA (Fc alpha R), but did not express Fc mu R or Fc gamma R. When supernatants from Fc alpha R+ T cell lines were subjected to IgA affinity chromatography, the IgA-promoting activity bound to IgA (IBF alpha) and was recovered in the eluate. No binding of active fractions occurred when supernates were passed through IgM or IgG immunoadsorbent columns. High concentrations of purified IBF alpha suppressed T-dependent IgA responses, while an optimal level was required for enhancement of this isotype response. These results suggest that Fc alpha R+ hybridomas derived from Th A cells release IBF alpha into the culture medium, and that these molecules regulate IgA responses to various T-dependent antigens.
Characterization of 1 alpha-hydroxylation of vitamin D3 sterols by cultured alveolar macrophages from patients with sarcoidosis.
Authors: Adams J S , Gacad M A
Published: June 2004

We investigated the 1 alpha-hydroxylation of vitamin D3 sterols by cultured pulmonary alveolar macrophages (PAM) from patients with sarcoidosis with or without clinically abnormal calcium homeostasis. Like the naturally occurring renal 1 alpha-hydroxylase, the PAM 1 alpha-hydroxylation reaction exhibited a high affinity for 25-hydroxyvitamin D3 (25-OH-D3) and a preference for substrates containing a 25-hydroxyl group in the side chain of the sterol. Unlike the renal enzyme, the PAM 1 alpha-hydroxylating mechanism was not accompanied by 24-hydroxylating activity, even after preincubation with 75 nM 1,25-dihydroxyvitamin D3 [1,25-(OH)2-D3] or exposure to high concentrations of substrate (500 nM 25-OH-D3). The PAM 25-OH-D3-1 alpha-hydroxylation reaction was stimulated by gamma interferon and inhibited by exposure to the glucocorticoid dexamethasone. The characteristics of the PAM hydroxylation process in vitro appear to reflect the efficiency of the extrarenal production of 1,25-(OH)2-D3 and the therapeutic efficacy of glucocorticoids in patients with sarcoidosis and disordered calcium metabolism.
A subset of Ly-1 inducer T cell clones activates B cell proliferation but directly inhibits subsequent IgG secretion.
Authors: Friedman S , Sillcocks D , Rao A , Faas S , Cantor H
Published: June 2004

We find that a fraction of Ly-1+2- inducer T cell clones inhibits differentiation of memory B cells into IgG-secreting plaque-forming cells. Inhibition of secondary antibody responses was not the result of induction of Ly-2+ T suppressors. Instead, inducer cells directly inactivated B cells, requiring an antigen bridge as well as identity at the major histocompatibility complex (I-A) locus. The interaction between the inducer T cell clone and hapten-specific B memory cells results in an early proliferative response and subsequent failure of B cells to secrete antibody in response to T helper cell signals. Possible mechanisms for this novel type of B cell inactivation are explored.
Impaired Kupffer cell function precedes development of secondary amyloidosis.
Authors: Fuks A , Zucker-Franklin D
Published: June 2004

It has been demonstrated previously that the acute phase reactant, serum amyloid A (SAA), is subject to degradation by surface membrane-associated proteinases of peripheral blood monocytes. However, monocytes obtained from the blood of patients with amyloidosis degraded SAA incompletely, leaving a cleavage product that, biochemically and immunologically, resembled the amyloid protein A (AA) deposited in their tissues. To investigate the role of fixed macrophages in amyloidogenesis and to establish more definitively that amyloid deposition is attributable to faulty processing of the precursor protein rather than aberrant synthesis, secondary amyloidosis was induced in C57BL/6J mice by serial injections of casein. Kupffer cells (KC) were isolated from livers of mice that had received 0, 8, 13, 18, and greater than 30 injections of the stimulant. The cells were cultured with SAA for 4, 8, and 18 h and then subjected to electron microscopy and enzyme analyses. The medium was analyzed by SDS-PAGE to determine the amount of residual SAA and/or the appearance of AA. KC of healthy animals degraded SAA completely whereas KC of stimulated mice showed increasing amounts of residual SAA and the appearance of the AA cleavage product. The AA peptide appeared in KC cultures early during the course of casein injections and before any amyloid could be demonstrated in the organs of the stimulated mice. The addition of KC isolated from healthy mice to cultures that had produced AA eliminated the abnormal peptide. The results, indicate that defective KC function precedes amyloidosis. The abnormal AA cleavage product formed by such cells is still susceptible to hydrolysis by normal cells. In addition, ultrastructural evidence is presented that suggests that KC may also play a role in fibrillogenesis of the AA protein.
Inhibition of bone marrow colony formation by human natural killer cells and by natural killer cell-derived colony-inhibiting activity.
Authors: Degliantoni G , Perussia B , Mangoni L , Trinchieri G
Published: June 2004

Incubation of human peripheral blood lymphocytes with bone marrow cells resulted in significant inhibition of colony formation by committed myeloid and erythroid cells. Using positively selected homogeneous natural killer (NK) cell preparations and lymphocyte subpopulations depleted of or enriched for NK cells, we definitively characterize as NK cells the cells in normal peripheral blood that are responsible for inhibition of bone marrow colony growth. The inhibitory effect of NK cells on hematopoiesis can be mediated by a soluble factor that is produced only by NK cells upon culture with HLA-DR+ hematopoietic cells and with NK-sensitive cell lines. Both NK cells and the NK-produced, colony-inhibiting activity (NK-CIA) are suppressive for allogeneic and autologous bone marrow CFU-GEMM (colony-forming units, granulocyte, erythroid, monocyte, megakaryocyte), CFU-E (CFU, erythroid), and early CFU-GM (CFU, granulocyte, monocyte), but not for either BFU-E (burst-forming units, erythroid) or late CFU-GM. [3H]Thymidine incorporation was inhibited by NK-CIA-containing supernatants in HLA-DR+ but not HLA-DR- bone marrow cell populations stimulated to proliferative by colony-stimulating factor (CSF). These data suggest that the NK cell-mediated inhibitory effect on proliferation and differentiation of hematopoietic precursor cells is mediated in part or completely by the secreted NK-CIA. The concentration of NK-CIA reached in the supernatant of the mixture of NK cell-containing lymphocyte populations with bone marrow cells is sufficient to account for the inhibitory effect mediated by NK cells. Our data support the hypothesis that human NK cells play a major role in the control of hematopoiesis, down-regulating it under conditions in which the NK cells are functionally activated.
Human genes for three complement components that regulate the activation of C3 are tightly linked.
Authors: Rodriguez de Cordoba S , Lublin D M , Rubinstein P , Atkinson J P
Published: June 2004

A new cluster of complement component genes, including C4BP, C3bR, and FH, is described. Family segregation data indicate that FH is linked to the genes for C4-bp and C4bR, previously reported to be linked and to maintain linkage disequilibrium. This cluster is not linked to the major histocompatibility complex, which contains the genes for the complement components, C4, C2, and factor B, or to the C3 locus. These data further suggest that the organization of genes for functionally related proteins in clusters may be a rule for the complement system.
Anti-liver-kidney microsome antibody recognizes a 50,000 molecular weight protein of the endoplasmic reticulum.
Authors: Alvarez F , Bernard O , Homberg J C , Kreibich G
Published: June 2004

Children with autoimmune chronic active hepatitis may have high titers of antibodies detected by immunofluorescence staining of hepatocytes and tubular cells in rat liver and kidney sections, respectively. These antibodies are directed against antigens contained in microsomal fractions prepared from these two organs. We have found that sera from these patients recognized a 50,000 mol wt protein present in higher concentration in smooth microsome subfractions compared with rough microsome subfractions. This protein is an integral membrane protein and is not glycosylated. It is exposed on the cytoplasmic face of the endoplasmic reticulum and is rather resistant to proteolysis with proteinase K. Since patients with liver disease of different etiology and similar severity of cell lysis do not give rise to liver-kidney microsome antibody (LKMA), lysis of hepatocytes is apparently not a sufficient condition for their development.
T cell receptor alpha chain genes are located on chromosome 14 at 14q11-14q12 in humans.
Authors: Caccia N , Bruns G A , Kirsch I R , Hollis G F , Bertness V , Mak T W
Published: June 2004

A cDNA clone encoding the alpha chain of the human T cell receptor was used in connection with somatic cell human-rodent hybrids to determine that the genes coding for the alpha chain are located on chromosome 14 in humans. In situ hybridization confirms this result and further localizes these genes to 14q11-14q12 on this chromosome. Since this region of chromosome has been shown to be nonrandomly involved in a number of T cell neoplasias, this assignment raises a number of interesting questions as to the possible involvement of the T cell receptor alpha chain genes in tumorigenesis.
Connective tissue proteins and phagocytic cell function. Laminin enhances complement and Fc-mediated phagocytosis by cultured human macrophages.
Authors: Bohnsack J F , Kleinman H K , Takahashi T , O'Shea J J , Brown E J
Published: June 2004

Brief exposure of culture-derived human macrophages to laminin, a glycoprotein component of all mammalian basement membranes that has a molecular weight of 1,000,000, led to enhancement of subsequent macrophage phagocytosis of EAC4b, EAC3bi, and EAIgG (sheep erythrocytes sensitized with IgG anti-Forssman antibody). This effect on macrophage phagocytosis occurred with both substrate-adherent and fluid phase laminin. Preincubation of macrophages, but not of EAC4b, with laminin led to augmentation of phagocytosis, suggesting that interaction with the phagocytic cell, but not with the opsonized particle, was required for laminin's effect. Laminin-stimulated phagocytosis of EAC4b was blocked entirely by a monoclonal antibody to CR1. Direct comparison of the phagocytic ability of macrophages adherent to laminin- and fibronectin-coated glass slides showed that fibronectin had a somewhat greater enhancing effect on phagocytosis. Nonetheless, the phagocytosis-enhancing effect of laminin was not due to contamination of the purified laminin preparation by fibronectin, since the laminin preparation was free of fibronectin, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay; in addition, laminin-enhanced phagocytosis was decreased in the presence of laminin-specific antibodies. Laminin inhibited macrophage adherence and spreading, but selection of a laminin-binding macrophage subpopulation could not account for the laminin-induced increases in phagocytosis. We hypothesize that interaction with extracellular matrix proteins may represent an important activation stimulus both to the macrophages normally present in the extravascular compartment and to the phagocytic cells that have emigrated from the blood-stream into areas of inflammation.
C-reactive protein mediates the solubilization of nuclear DNA by complement in vitro.
Authors: Robey F A , Jones K D , Steinberg A D
Published: June 2004

We have studied the interaction of C-reactive protein (CRP)-chromatin complexes with serum. The amount of chromatin solubilized by serum is directly proportional to the amount of CRP present. Serum minus C3 did not appreciably solubilize chromatin within the time allowed in these experiments regardless of the amount of CRP present. This indicates that, in addition to CRP, complement is critical to the solubilization process. Studies using genetically C2-deficient serum and purified C2 indicate that the classical complement pathway is primarily involved in the solubilization, however, there may be minor involvement by the alternative pathway. We used an enzyme-linked immunosorbent assay to determine the amounts of CRP in plasma from eight patients with systemic lupus erythematosus; two of the eight had levels of CRP far lower than previously reported for normal individuals, and an additional sample had antibodies reactive with CRP. Together, these results suggest that one of the functions of CRP is to mediate the removal of exposed nuclear DNA by complement-dependent solubilization of chromatin. A defect in this mechanism could (a) facilitate the production of antibodies against chromatin components exposed due to tissue damage or (b) contribute to immune complexes containing the chromatin components released from damaged tissue because they are not rapidly cleared.
Stimulation of a subset of normal resting T lymphocytes by a monoclonal antibody to a crossreactive determinant of the human T cell antigen receptor.
Authors: Bigler R D , Posnett D N , Chiorazzi N
Published: June 2004

A previous study from this laboratory described a monoclonal antibody, S511, that reacted with the T cell antigen receptor on a human T cell leukemia and also on 1-2% of circulating T lymphocytes in all normal individuals tested. The data presented in the present study demonstrate that, when normal T lymphocytes are cultured with or without irradiated non-T cells in the presence of soluble S511 antibody, a concentration- and time-dependent proliferation of the S511-reactive population occurred. Proliferation indices as high as 184 times greater than control were observed, which represents a major stimulatory effect on the initially minor S511+ subset. When S511+ cells were studied for evidence of prior activation, they were shown to be unresponsive to interleukin 2 (IL-2) unless exposed to S511 antibody, and were shown to be in the G0/G1 phase of the cell cycle. Thus, the S511 antibody activated resting normal T cells in a manner analogous to specific antigen binding to the T cell antigen receptor. The leukemic S511 molecule has been shown previously to differ from most other antigen receptors in the mobility of the two chains at 43 and 38 kD and the neutral isoelectric point of each chain. Expansion of reactive normal cells by S511-Sepharose permitted the development of IL-2-dependent T cell lines enriched for S511-bearing cells. The antigen receptor molecules on one such polyclonal S511-enriched T cell line were immunoprecipitated with S511 antibody and shown to have comparable mobility to that present on the leukemic cells, but to possess a greater heterogeneity of mobility. Thus, the leukemic cells and normal cells express similar T cell receptor molecules. The differences in the S511 T cell antigen receptor molecule possibly relate to differences in glycosylation or polypeptide structure.
Phenotype of recovering lymphoid cell populations after marrow transplantation.
Authors: Ault K A , Antin J H , Ginsburg D , Orkin S H , Rappeport J M , Keohan M L , Martin P , Smith B R
Published: June 2004

Four patients who received bone marrow transplants were studied sequentially during the posttransplant period to define the pattern of recovering lymphoid cell types. Three patients received T cell-depleted, HLA-matched marrow, and one received untreated marrow from an identical twin. Blood lymphoid cells were labeled with 25 different pairs of monoclonal antibodies. In each sample, one antibody was conjugated to fluorescein and one to phycoerythrin, thus allowing simultaneous assessment of the expression of the two markers using the fluorescence activated cell sorter. A total of 14 antibodies were used, routinely including HLE, Leu-M3, Leu-4, Leu-1, Leu-5, Leu-9, Leu-6, Leu-2, Leu-3, HLA-DR, Leu-7, Leu-11, Leu-15, and Leu-12. Other antibodies were used to further define some populations. This study has allowed us to define six distinct cell types that have appeared in all four patients by day 90 posttransplantation, and which account for 90-100% of all circulating lymphoid cells. These cell types are (a) T helper cells expressing Leu-1, Leu-4, Leu-9, Leu-5, Leu-3, and variable amounts of HLA-DR; (b) T suppressor cells expressing Leu-1, Leu-4, Leu-9, Leu-5, Leu-2, and variable amounts of HLA-DR; (c) B cells expressing Leu-12, B1, HLA-DR, IgD, and IgM, but none of the T cell antigens; (d) an unusual B cell phenotype (Leu-1 B) expressing all of the B cell markers, and also having low amounts of Leu-1, but none of the other T cell antigens; (e) natural killer (NK) cells expressing Leu-11, Leu-15, Leu-5 but none of the other T cell or B cell markers; (f) NK cells expressing Leu-11, Leu-15, Leu-5, and low levels of Leu-2. Both NK types also express Leu-7 on some, but not all cells. The relative frequencies of these cell types varied among the patients and with time, but the striking findings were the presence of relatively few mature T cells, large numbers of NK cells, and the preponderance of the unusual Leu-1 B cell over conventional B cells in all three patients who developed B cells. Sorting experiments confirmed the NK activity of the major NK cell phenotypes, and DNA analysis confirmed that all of the cells studied were of donor origin. In addition, analysis of Ig genes in one patient showed that the Leu-1 B cells were not clonally rearranged.(ABSTRACT TRUNCATED AT 400 WORDS)
Sequential expression of genes involved in human T lymphocyte growth and differentiation.
Authors: Krönke M , Leonard W J , Depper J M , Greene W C
Published: June 2004

Nuclear transcription assays were performed with isolated nuclei from human peripheral blood T lymphocytes stimulated with phytohemagglutinin and phorbol myristate acetate to determine the kinetics of transcriptional activity of various genes occurring in T cell activation. Although silent in resting T cells, the genes encoding c-myc and the interleukin 2 (IL-2) receptor were induced early, preceding gamma interferon (IFN-gamma), IL-2, and transferrin receptor gene transcription. Transcriptional activity of these genes fell after their respective peaks, indicating that the expression of these genes is a transient event during T cell activation. With the exception of the transferrin receptor gene, the kinetics of induction of these genes were not altered by concentrations of cycloheximide that inhibited protein synthesis. These data indicate that the induction of genes encoding c-myc, IL-2, IL-2 receptor, and IFN-gamma occur independently of the sequential production of the proteins they encode.
Association of lpr gene with graft-vs.-host disease-like syndrome.
Authors: Theofilopoulos A N , Balderas R S , Gozes Y , Aguado M T , Hang L M , Morrow P R , Dixon F J
Published: June 2004

Hemopoietic cells have been reciprocally transferred between two lines of mice (MRL lpr/lpr and MRL +/+) that are congenic, differing only at the lpr (lymphoproliferation) and possibly closely linked genes. The lpr strain develops a significantly more severe and fast-paced lupus-like syndrome than +/+ strain, along with a substantially larger lymphoid mass. The results showed that: (a) hemopoietic cells of such mice were sufficient to induce the respective disease phenotypes in lethally irradiated syngeneic recipients; (b) cells of MRL +/+ mice maturing in an MRL lpr/lpr environment essentially retained the disease-producing characteristics of the donor, i.e., they induced late-life lupus without lymphadenopathy; but (c) MRL lpr/lpr cells transferred into irradiated MRL +/+ recipients unexpectedly failed to induce the early-life severe lupus and lymphoid hyperplasia of the donor, instead they caused a severe wasting syndrome resembling, in many respects, graft-vs.-host disease (GVHD). This GVHD-like syndrome developed after transfer of MRL lpr/lpr fetal liver, bone marrow, or spleen cells, and was not abrogated by elimination of T cells from the inocula. Thymectomy of the MRL +/+ recipients retarded, but did not prevent, the wasting disease. The unidirectional nature of this disease suggests that the lpr mutation conferred either a structural or regulatory defect that interfered, blocked, or altered the expression or structure of certain lymphocyte antigen(s). As a result, the MRL +/+ cells that did express this antigen(s) were recognized as foreign, and stimulated a graft-vs.-host reaction. These findings may allow definition of a new kind of rejection phenomenon caused by non-H-2 products, and may extend our understanding of the means by which the lpr gene adversely affects lymphocyte regulation and homeostasis.
Pertussis toxin inhibition of chemotactic factor-induced calcium mobilization and function in human polymorphonuclear leukocytes.
Authors: Goldman D W , Chang F H , Gifford L A , Goetzl E J , Bourne H R
Published: June 2004

Chemotactic factors stimulate a rapid increase in the cytosolic concentration of intracellular calcium ions ([Ca2+]in) in human polymorphonuclear leukocytes (PMNL), which may be an event that is critical to the expression of chemotaxis and other PMNL functions. Treatment of PMNL with pertussis toxin catalyzes ADP-ribosylation of a protein similar or identical to the inhibiting regulatory protein of adenylate cyclase, Gi, and suppresses the increase in [Ca2+]in elicited by leukotriene B4(LTB4) and formyl-methionyl-leucyl-phenylalanine. Chemotactic migration and lysosomal enzyme release elicited by chemotactic factors were inhibited by pertussis toxin with a concentration-dependence similar to that for inhibition of the increase in [Ca2+]in, without an effect on lysosomal enzyme release induced by the ionophore A23187 and phorbol myristate acetate. Activated pertussis toxin catalyzed the [32P]ADP-ribosylation of a 41 kD protein in homogenates of PMNL. The extent of [32P]ADP-ribosylation of this protein was reduced 59% by pretreatment of intact PMNL with pertussis toxin. Pertussis toxin selectively decreased the number of high-affinity receptors for LTB4 on PMNL by 60% without altering the number or binding properties of the low-affinity subset of receptors. Pertussis toxin modification of a membrane protein of PMNL analogous to Gi thus simultaneously alters chemotactic receptors and attenuates the changes in cytosolic calcium concentration and PMNL function caused by chemotactic factors.
Syngeneic antiidiotypic immune responses to a B cell lymphoma. Comparison between heavy chain hypervariable region peptides and intact Ig as immunogens.
Authors: Thielemans K , Rothbard J B , Levy S , Levy R
Published: June 2004

The nucleic acid sequence of the heavy chain variable region (VH) expressed by 38C13, a B cell tumor of C3H origin, was determined by a combination of direct (messenger RNA) mRNA sequencing by primer extension and complementary DNA (cDNA) isolation and sequencing in M13. The VH amino acid sequence was deduced, and hypervariable regions were identified. From an analysis of predicted secondary structure, regions of predicted antigenicity were chosen, and a series of synthetic peptides corresponding to CDR2 and CDR3 (complementarity-determining region) were produced. These peptides were coupled to protein carriers and used to immunize syngeneic C3H mice. All peptides gave rise to a vigorous antibody response. However, only the CDR3 peptides induced antibodies that crossreacted with the isolated H chain protein. Only one CDR3 peptide induced antibody-producing clones, isolated as hybridomas, that reacted with the intact IgM protein. However, the appearance of these clones was a low-frequency event. All antibodies reacting with the H chain or the intact IgM protein were idiotypically specific for 38C13. These monoclonal antiidiotype (anti-Id) antibodies, raised against CDR3 peptides, gave strong reactions in enzyme-linked immunosorbent assays and immunoblots, but they were of low affinity compared to syngeneic anti-Id raised against the intact IgM protein. Moreover, while the intact IgM was capable of inducing tumor immunity, the CDR peptides were not able to do so.
Tubular antigen-derivatized cells induce a disease-protective, antigen-specific, and idiotype-specific suppressor T cell network restricted by I-J and Igh-V in mice with experimental interstitial nephritis.
Authors: Neilson E G , McCafferty E , Mann R , Michaud L , Clayman M
Published: June 2004

The nephritogenic effector T cell response producing interstitial nephritis in mice can be largely inhibited by the adoptive transfer of suppressor T cells before or after the induction of disease. These suppressor T cells are harvested from donor mice primed with tubular antigen-derivatized syngeneic lymphocytes, and two subsets of suppressor cells can be characterized within this donor cell population. The first suppressor cell in this network is an L3T4+, I-J+, RE-Id+ cell (Ts-1). Ts-1 cells are antigen-binding suppressor cells that inhibit afferent phase immune responses and, in the presence of tubular antigen, specifically induce Lyt-2+, I-J+ cells (Ts-2) that are antiidiotypic (RE-Id-binding) suppressors. The Ts-2 cell is functionally restricted in its suppressive effect by I-J and Igh-V gene products, and acts on the effector limb of the cell-mediated anti-tubular basement membrane immune response. These studies provide an experimental basis for further efforts to use immunoregulatory modulation in the control of autoimmune renal disease.
Allospecific cytolytic T lymphocytes recognize conformational determinants on hybrid mouse transplantation antigens.
Authors: Bluestone J A , Foo M , Allen H , Segal D , Flavell R A
Published: June 2004

Alloreactive cytolytic T cell (CTL) lines and clones have been used to identify the sites of polymorphism of antigens of the major histocompatibility complex (MHC). Specific CTL were generated against wild-type H-2b products by cells from H-2b mutant mice that had one or a few amino acid changes in either the alpha 1 or alpha 2 domains of the Kb or Db class I molecules. These CTL populations, which might be expected to react with determinants expressed on single MHC domains, were examined for lytic activity on L cells expressing newly constructed hybrid class I molecules. Transformed cell lines expressing native class I molecules or hybrid class I molecules in which the alpha 1 and alpha 2 domains of H-2Kb had been substituted by those domains of H-2Db were lysed by H-2Db-specific CTL. Similarly, all H-2Kb-specific CTL recognized hybrid molecules in which the alpha 1 and alpha 2 domains of H-2Kb were inserted into the H-2Db molecule. In contrast, exchange of the alpha 1 domains of H-2Kb and H-2Db resulted in a total loss of recognition by Kb and Db-specific CTL. These results suggest that the allodeterminants recognized by H-2 mutant CTL are influenced by interactions between the alpha 1 and alpha 2 domains, findings similar to those seen using conventional alloreactive T cells (11). These results were compared to the binding of alloreactive mAbs, including 5 new mAbs specific for the Kb molecules. Finally, it was shown that primary and secondary CTL responses could be generated by direct sensitization against hybrid class I molecules, demonstrating that these molecules express neoantigenic determinants recognized by alloreactive CTL.
Ig heavy chain variable region gene complex of lupus mice exhibits normal restriction fragment length polymorphism.
Authors: Kofler R , Perlmutter R M , Noonan D J , Dixon F J , Theofilopoulos A N
Published: June 2004

B cell hyperactivity, hypergammaglobulinemia, and autoantibody expression, the hallmarks of systemic lupus erythematosus, might be associated with structural abnormalities within the Ig heavy chain variable region (Igh-V) gene complex. The Igh-V loci from several lupus-prone mouse strains, their ancestors, and other nonautoimmune mice were therefore analyzed by restriction fragment length polymorphisms with DNA probes corresponding to seven VH gene families. These seven families comprise the majority of the known polymorphic murine VH gene repertoire, including some involved in autoantibody generation. Our study showed that the Igh-V loci from lupus and haplotype-matched nonlupus mice resulted in essentially identical restriction fragment patterns, a finding which suggests that the Igh-V gene complex does not carry a primary defect responsible for autoimmune disease.
Stable expression of cDNA encoding the human interleukin 2 receptor in eukaryotic cells.
Authors: Greene W C , Robb R J , Svetlik P B , Rusk C M , Depper J M , Leonard W J
Published: June 2004

Human interleukin 2 (IL-2) receptor cDNA derived from HUT 102B2 cells was stably expressed in murine L cells. These L cell transfectants (a) displayed surface receptors of the aberrant size of the IL-2 receptors on HUT 102B2 cells, (b) did not respond to exogenous IL-2 with augmented proliferation, and (c) expressed low affinity but not high affinity receptors for IL-2.
Early events in Epstein-Barr virus infection provide a model for B cell activation.
Authors: Thorley-Lawson D A , Mann K P
Published: June 2004

We have used Epstein-Barr virus (EBV) infection in vitro to delineate two distinct stages in B cell activation. Previous studies have shown that the BLAST-2 (EBVCS) (EBV cell surface) activation antigen is expressed on a small fraction of B cells within 24 h of stimulation with a variety of agents, including mitogens and EBV. In this study, we have been able to isolate the BLAST-2 (EBVCS)+ cells early after activation/infection with EBV. These cells are small B cells that are actively synthesizing RNA but not DNA, and are, therefore, clearly distinct from large proliferating lymphoblasts. In addition, they contain multiple copies of the EBV genome, express the viral nuclear antigen (EBNA) and, most importantly, proceed to undergo transformation when placed back in culture. By comparison, the BLAST-2 (EBVCS)- population does not undergo transformation, even though a fraction of these cells are activated for RNA synthesis and express EBNA. Thus, using the EBV system, we have been able to show directly that an activated B cell first expresses the BLAST-2 (EBVCS) antigen concomitant with an increase in RNA synthesis, and then subsequently proceeds to differentiate into a proliferating lymphoblast.
T cell regulation of B cell activation. Lyt-1+,2-T cells modify the MHC-restricted function of heterogeneous and cloned T suppressor cells.
Authors: Asano Y , Hodes R J
Published: June 2004

Previous studies have shown the existence of both heterogeneous Lyt-1-,2+ suppressor (Ts) cells and cloned Lyt-1+,2- Ts cells which, despite the difference in their Lyt phenotypes, functioned in a similar antigen-specific and major histocompatibility complex (MHC)-restricted fashion to suppress the antibody responses generated by cloned helper T (Th) cells and hapten-primed B cells. Our studies were carried out to assess in further detail the genetically restricted cell interactions that mediate this immune response suppression. We show that the activation of both heterogeneous and cloned Ts cells is antigen-specific and MHC-restricted under our experimental conditions. After appropriate activation, the effector function of both cloned Lyt-1+,2-Ts cells and heterogeneous Lyt-1-,2+ Ts cells was also antigen-specific. In contrast, once activated, Ts cells suppressed the responses generated by cloned Th cells and hapten-primed B cells in an MHC-unrestricted fashion. We also showed, however, that a population of unprimed Lyt-1+,2-T cells was able to significantly alter the genetic restriction requirements for Ts cell function. The activity of this population was itself MHC-restricted, and was observed only when the unprimed Lyt-1+,2-T cells shared the MHC restriction specificity of the cloned Th cells functioning in a given response. When these requirements were satisfied, Lyt-1+,2- T cells significantly modified the suppression mediated by both heterogeneous and cloned Ts cells, resulting in suppression that was then MHC restricted in its effector function as well as in its activation requirements. Thus, our findings suggest that the observed MHC restriction in Ts function is the result of a complex interaction involving Ts cells, Th cells, and an additional population of MHC-restricted Lyt-1+,2- T cells. This newly characterized activity of Lyt-1+,2- T cells functionally resembles that of an MHC-restricted contrasuppressor population that selectively blocks a pathway of MHC-unrestricted Ts activity, while leaving intact susceptibility to MHC-restricted Ts effects.
Characterization of human rheumatoid factors with seven antiidiotypes induced by synthetic hypervariable region peptides.
Authors: Chen P P , Gõni F , Houghten R A , Fong S , Goldfien R , Vaughan J H , Frangione B , Carson D A
Published: June 2004

Recently, we have used synthetic peptides corresponding to the complementarity-determining regions (CDR) of Ig molecules to induce antiidiotypic antisera. Peptide PSH3, representing the third CDR of the IgM rheumatoid factor (RF) Sie heavy (H) chain, induced a private antiidiotype that reacted with only one out of five IgM-RF. Peptide PSL2, corresponding to the second CDR of Sie light (L) chain, induced an antibody against a crossreactive idiotype (CRI), expressed by 10 out of 12 human IgM-RF analyzed. Herein, we report that five additional antiidiotypic antibodies were generated by immunization with synthetic peptides identical to the third L chain CDR of IgM-RF Sie (PSL3), the second and third H chain CDR of IgM-RF Wol, and the second and third CDR of IgM-RF Pom. As analyzed by immunoblot assay, both anti-PSL3 and anti-PSL2 reacted with the majority of 16 IgM-RF. In contrast, all five antiidiotypes induced by the H chain peptides reacted only with the parent proteins, except anti-PSH3, which reacted weakly with one additional RF. These results suggest that one (or very few) VL gene(s), but a larger number of VH genes, are used to encode IgM-RF autoantibodies.
Structure of a gene encoding a murine thymus leukemia antigen, and organization of Tla genes in the BALB/c mouse.
Authors: Fisher D A , Hunt S W , Hood L
Published: June 2004

We have determined the DNA sequence of a gene encoding a thymus leukemia (TL) antigen in the BALB/c mouse, and have more definitively mapped the cloned BALB/c Tla-region class I gene clusters. Analysis of the sequence shows that the Tla gene is less closely related to the H-2 genes than H-2 genes are to one another or to a Qa-2,3-region genes. The Tla gene, 17.3A, contains an apparent gene conversion. Comparison of the BALB/c Tla genes with those from C57BL shows that BALB/c has more Tla-region class I genes, and that one of the genes absent in C57BL is gene 17.3A.
Cellular processing of bovine factors X and Xa by cultured bovine aortic endothelial cells.
Authors: Nawroth P P , McCarthy D , Kisiel W , Handley D , Stern D M
Published: June 2004

Previous studies have shown that Factor X and Factor Xa bind specifically to distinct sites on the endothelial cell surface. Since the coagulant activity of a cell-bound clotting protein is dependent on its remaining on the cell surface, endocytosis and degradation studies have been carried out. Cell-bound Factor X was internalized at 0.07 fmol/min/10(6) cells, a rate slower than its dissociation from the cell surface. Endocytosed Factor X was not degraded, but was returned to the cell surface. In contrast, Factor Xa was internalized at an initial rate of 0.38 fmol/min/10(6) cells and subsequently degraded at about the same rate. The degradation of Factor Xa was prevented by chloroquine. These results suggest that Factor Xa is internalized and degraded by a lysosomal-dependent pathway. Studies with Factor X- and Xa-colloidal gold conjugates showed endocytosis proceeding at coated pit regions, and accumulation of Factor Xa-gold particles in lysosome-like structures. Endocytosis was studied as a clearance pathway for cell-bound Factor Xa by activating Factor X with Factors IXa and VIII on the endothelial cell surface. Endocytosis of the Factor Xa formed was significant, as only 44% of the Factor Xa formed was released into the supernatant, whereas the remainder was internalized and degraded. Thus, endocytosis of Factor Xa bound to its specific endothelial cell sites may be an important factor in the balance of vessel wall hemostatic mechanisms.
Molecular genetic analysis of the V kappa Ser group associated with two mouse light chain genetic markers. Complementary DNA cloning and southern hybridization analysis.
Authors: Goldrick M M , Boyd R T , Ponath P D , Lou S Y , Gottlieb P D
Published: June 2004

Previous studies (21) have shown that two mouse kappa light (L) chain variable (V) region polymorphisms, the IB-peptide and Efla markers, reflect expression of a characteristic group of V kappa regions, called V kappa Ser, by some inbred strains and not others. Expression of V kappa Ser is controlled by a locus on chromosome 6, the chromosome that contains the kappa locus. To further characterize this V kappa group and begin to analyze the basis for its strain-specific expression, full-length complementary DNA (cDNA) copies were produced of L chain mRNA from the M75 myeloma that had been induced in the C.C58 strain of mice, and which produces a V kappa Ser L chain. The C.C58 strain is congenic with BALB/cAn, differing in the region of chromosome 6 that controls expression of the V kappa polymorphisms and the Lyt-2 and Lyt-3 T cell alloantigens. The complete nucleotide sequence of this cloned cDNA was determined and compared with the nucleotide sequences the most closely related BALB/c myeloma L chains known. Results indicated significant differences throughout the variable region, but particularly toward the 5' portion of the sequence. A probe corresponding to 200 bp of the 5' end of the cloned V kappa Ser cDNA was used in Southern hybridizations of restriction digests of liver DNA from a number of inbred, recombinant, and recombinant inbred strains. Under stringent hybridization conditions, one strongly-hybridizing fragment was observed in Bam HI, Hind III, and Eco RI digests, and based on the size of the fragments, strains could be organized into two groups. The presence of strongly hybridizing Bam HI, Hind III, and Eco RI fragments of 3.2, 2.8, and 2.1 kb, respectively, was found to correlate completely with expression by the strain of the IB-peptide and Efla markers. All nonexpressor strains yielded hybridizing fragments of 7.8, 8.4, and 2.8 kb, respectively. Possible explanations for strain-specific expression of V kappa Ser-associated phenotypic markers are discussed.
Synthetic peptides corresponding to third hypervariable region of human monoclonal IgM rheumatoid factor heavy chains define an immunodominant idiotype.
Authors: Goldfien R D , Chen P P , Fong S , Carson D A
Published: June 2004

Synthetic peptides corresponding to eight individual heavy chain complementarity-determining regions (CDR) of three human monoclonal IgM anti-IgG (rheumatoid factor [RF]) paraproteins elicited rabbit antibodies with markedly different properties. All antisera recognized the immunizing peptide, and several reacted with the isolated IgM heavy chain on immunoblots. However, only the antisera against peptides representing the third CDR bound consistently and specifically to the intact IgM-RF molecule. These data indicate that the third CDR of human mu chains comprises an immunodominant idiotype, and suggest that the D gene segment may be especially important in creating idiotypic diversity. Synthetic peptides corresponding to the third heavy chain CDR of human paraproteins may be clinically useful for the specific induction of antiidiotypic antibodies.
Hereditary tyrosinemia. Formation of succinylacetone-amino acid adducts.
Authors: Manabe S , Sassa S , Kappas A
Published: June 2004

Succinylacetone (SA) (4,6-dioxoheptanoic acid) is an abnormal metabolite produced in patients with hereditary tyrosinemia as a consequence of an inherited deficiency of fumaryl acetoacetate hydrolase activity. Patients with this disease are associated with a number of abnormalities, including aminoaciduria, proteinuria, liver failure, commonly hepatoma, and decreased GSH concentration in the liver. In the course of our studies of tyrosinemia, we found that the urine of patients with this disorder contains material(s) that absorbs light at 315 nm. We investigated the nature of the 315 nm material in detail. SA was found to react with amino acids and protein nonenzymatically, to form stable adducts at physiological temperature and pH. All SA adducts with amino acids and/or proteins exhibited an absorption peak at 315 nm. Although all amino acids reacted with SA, the most reactive amino acid was lysine (Lys), followed, in order, by glycine, methionine, phenylalanine, serine, alanine, and glutamine. SA-adducts were unstable at pH below 6, while they were made considerably more stable after reduction with NaBH4, suggesting that SA forms an adduct via Schiff base formation. High-performance liquid chromatography (HPLC) analysis of urines from patients with tyrosinemia revealed the existence of SA-glycine, SA-methionine, SA-tyrosine, and SA-phenylalanine. After digestion of urines with proteinase K, three more HPLC peaks appeared, which all corresponded to SA-Lys adducts. TLC analysis of SA-Lys showed that SA-Lys could form as many as seven different adducts. No SA-adduct peaks were observed in HPLC in urines from normal subjects, patients with other forms of aminoaciduria, or patients with the nephrotic syndrome. In addition to amino acids and proteins, SA reacted with reduced glutathione (GSH) and formed a stable adduct. These findings suggest that SA adduct formation with amino acids, GSH, and proteins is a significant process occurring in tyrosinemia, and may account for certain of the pathologic findings in this hereditary disorder.
Human T cell clones express functional homing receptors required for normal lymphocyte trafficking.
Authors: Navarro R F , Jalkanen S T , Hsu M , Søenderstrup-Hansen G , Goronzy J , Weyand C , Fathman C G , Clayberger C , Krensky A M , Butcher E C
Published: June 2004

To function efficiently in vivo, lymphocytes must circulate from the blood into lymphoid tissues and other sites of immune reaction. Herein, we show that human cytotoxic and helper T cell clones and lines, maintained in vitro with IL-2, express the functional capacity to recognize and bind to high endothelial venules (HEV), a capacity essential for lymphocyte exit from the blood, and hence for normal lymphocyte trafficking. The expression of functional homing receptors distinguishes human T cell clones from their murine counterparts, which uniformly lack receptors for HEV and are unable to migrate normally from the blood in vivo. The results raise the possibility that human T cell clones may be more effective in mediating in vivo immune responses than is suggested by murine models.