bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification in rat liver and serum of water-soluble class I MHC molecules possibly homologous to the murine Q10 gene product.
Authors: Spencer S C , Fabre J W
Published: June 2004

We have identified large quantities of a water-soluble, non-RT1.A class I MHC molecule in the serum of the DA rat strain, with a similar molecule being found in aqueous extracts of DA liver. The non-RT1.A class I molecules have heavy chains of 41 kD, which is smaller than RT1.A class I molecules isolated from liver membranes (45 kD) but larger than water-soluble RT1.A class I molecules previously identified in serum and aqueous extracts of liver and kidney (40 kD). NH3-terminal amino acid sequencing of bulk-purified RT1.A class I molecules and of this novel non-RT1.A class I molecule revealed two substitutions, in the first 25 amino acids, Tyr----His at position 9, and Ala----Ser at position 24. The non-RT1.A class I molecule did not react with any of the well-characterized polymorphic and monomorphic antibodies directed against RT1.Aa class I molecules, but did react with the MRC OX18 antibody. A similar class I molecule could not be identified on liver membranes. The non-RT1.A class I molecule was found in large quantities (approximately 20 micrograms/ml) in the serum of the DA rat strain, and similarly large quantities appeared to be present in the sera of BN, PVG, and LEW.RT1a rats. WAG and LEW.RT1u rats had readily detectable but lower amounts of this molecule in their serum, while LEW and SHR rats had little if any present. This molecule probably represents the rat homologue of the murine Q10 gene product, and is the major class I product in the serum of the DA rat strain.
Differentiation-linked secretion of urokinase and tissue plasminogen activator by normal human hemopoietic cells.
Authors: Wilson E L , Francis G E
Published: June 2004

Previous studies have shown that the response of patients with acute myeloid leukemia to induction chemotherapy can be predicted by the species of plasminogen activator that their cells secrete. Patients whose cells secreted tissue plasminogen activator (tPA) only failed to respond to combination chemotherapy. Individuals whose leukemic cells display features of the early progenitor phenotype also respond poorly to therapy. This suggested that the two species of plasminogen activator secreted by leukemic cells might be produced by normal cells at distinct stages of differentiation. These results indicate that the secretion of the two enzyme types is a differentiation-linked property of normal cells with tPA being produced by granulocyte/macrophage progenitors and urokinase by more differentiated cells and by mature neutrophils and macrophages.
Genetic control of diabetes and insulitis in the nonobese diabetic (NOD) mouse.
Authors: Wicker L S , Miller B J , Coker L Z , McNally S E , Scott S , Mullen Y , Appel M C
Published: June 2004

Genetic analysis of the development of diabetes and insulitis has been performed in the nonobese diabetic (NOD) mouse strain, a model of insulin-dependent (type I) diabetes mellitus. (NOD X C57BL/10)F1, F2, and (F1 X NOD) first-, second-, and third-backcross generations were studied. The data obtained were consistent with the hypothesis that diabetes is controlled by at least three functionally recessive diabetogenic genes, or gene complexes, one of which is linked to the MHC of the NOD. In contrast, pancreatic inflammation leading to insulitis was found to be controlled by a single incompletely dominant gene. One of the two diabetogenic loci that is not linked to the MHC appears to be essential for the development of severe insulitis. This diabetogenic gene may be identical to the gene that controls the initiation of the autoimmune response that progresses to insulitis. Although this gene appears to be functionally recessive in its control of diabetes, it is incompletely dominant in its control of insulitis. The MHC-linked diabetogenic gene, although not required for the development of insulitis, apparently influences the progression of the autoimmune response since NOD MHC homozygotes in the backcross generations displayed the highest incidence and most severe cases of insulitis. Interestingly, we have found two MHC heterozygous backcross females that have become diabetic, suggesting that either the MHC-linked diabetogenic gene is not strictly recessive or that a recombination event has occurred between the diabetogenic gene and the K or I-A regions of the MHC. The third diabetogenic locus appears to influence the progression of severe insulitis to overt diabetes. In animals homozygous at this locus, diabetes may result from a decreased ability to develop a protective suppressor response to the autoimmune process.
The autoantigen-binding B cell repertoires of normal and of chronically graft-versus-host-diseased mice.
Authors: Rolink A G , Radaszkiewicz T , Melchers F
Published: June 2004

A quantitative analysis of the frequencies of autoantibody-producing B cells in GVHD and in normal mice has been undertaken by generating collections of hybridomas of activated B cells. These hybridomas secreted sufficient quantities of Ig to allow binding analyses on a panel of autoantigens. B cells have been activated in a variety of ways. In vivo they were activated by injection of alloreactive T cells of one parent, leading to GVHD by a foreign antigen, sheep erythrocytes, in a secondary response, or by the polyclonal activator LPS. B cells from an experimentally unstimulated animal were used for an analysis of the normal background. In vitro B cells were activated by alloreactive T cells or by LPS. The frequencies of hybridomas and, therefore, of activated B cells producing autoantibodies to DNA or to kidney were not significantly different in mice activated by a graft-vs.-host T cell response as compared with B cell populations activated by any of the other procedures. They were found to compose 7.1-17.1% of the total repertoire of activated B cells. Moreover, the frequencies of autoantibody-producing activated B cells does not change with time after induction of the graft-vs.-host reaction. The pattern and frequencies of autoantigen-binding specificities to cytoskeleton, smooth muscle, nuclei, mitochondria, and DNA were not found to be different in any of the groups of hybridomas. The single notable exception, found in GVHD mice, were hybridomas producing autoantibodies to kidney proximal tubular brush border. These results allow the conclusion that autoantigen-binding B cells exist in an activated state in GVHD mice, as well as in mice activated by a foreign antigen or by a polyclonal activator, in B cell populations activated in vitro either by alloreactive T cells or by a polyclonal activator, and even in the background of experimentally unstimulated animals. T cell-mediated graft-vs.-host activation, in large part, does not lead to a selective expansion of autoantigen-binding B cells. The main difference between the graft-vs.-host-activated B cell repertoire and all others is that approximately 90% of teh autoantibodies were of the IgG class, whereas al autoantibodies found in the other groups were IgM.
Human autoantibodies against a desmosomal protein complex with a calcium-sensitive epitope are characteristic of pemphigus foliaceus patients.
Authors: Eyre R W , Stanley J R
Published: June 2004

Pemphigus foliaceus (PF) patients have antibodies against a tightly, but noncovalently bound complex of polypeptides, which consists of desmoglein I (DGI) and other, possibly desmosomal, proteins. Most PF antibodies bind a calcium-sensitive epitope on this complex and chelation of calcium destroys the reactivity of these sera with the complex, but not the complex itself. The PF sera that do bind the desmosomal complex in the absence of calcium are those sera capable of binding denatured DGI on immunoblotting, and these same sera also immunoprecipitate only DGI when the desmosomal complex is dissociated with SDS. These findings demonstrate that autoantibodies against a complex of desmosome-associated proteins are characteristic of PF and define a calcium-sensitive conformational epitope on this complex.
Production of a T cell hybridoma that expresses the T cell receptor gamma/delta heterodimer.
Authors: Yokoyama W M , Koning F , Stingl G , Bluestone J A , Coligan J E , Shevach E M
Published: June 2004

We have produced a T cell hybridoma line by fusion of an IL-2-dependent, long-term T cell receptor (TCR) gamma/delta+ Thy-1+, bone marrow-derived, dendritic epidermal cell line to the BW5147 tumor line. The resultant hybridoma was rapidly growing, lymphokine independent, and expressed T3 in association with the TCR gamma/delta heterodimer. Several subclones of the hybridoma line produced easily detectable levels of IL-2 after stimulation by anti-T3 or Con A. The availability of these cloned cell lines should greatly facilitate further functional, biochemical, and molecular studies of the TCR delta chain.
Functional T lymphocytes are required for a murine retrovirus-induced immunodeficiency disease (MAIDS).
Authors: Mosier D E , Yetter R A , Morse H C
Published: June 2004

Athymic nu/nu mice were found to be resistant to the immunodeficiency disease and lethality induced in normal mice by the injection of the LP-BM5 mixture of murine retroviruses (LP-BM5 MuLV). Susceptibility to disease induction was reconstituted by injection of nu/nu mice with purified, mature T lymphocytes. The extent of viral replication of both the ecotropic and mink cell focus forming (MCF) components of LP-BM5 MuLV was equivalent in both nu/nu and normal animals. Retrovirally-induced immunodeficiency disease in mice (MAIDS) is thus dependent upon the presence of functional T lymphocytes, and high virus titers in athymic mice have little or no effect on the immune system.
Identification and cloning of a prethymic precursor T lymphocyte from a population of common acute lymphoblastic leukemia antigen (CALLA)-positive fetal bone marrow cells.
Authors: Hokland P , Hokland M , Daley J , Ritz J
Published: June 2004

We have cloned common acute lymphoblastic leukemia (CALLA)-positive cells from human fetal bone marrow containing less than 1 in 10,000 E-RFC in round-bottomed microtiter wells (one cell per well) using the autocloning unit of an EPICS-V cell sorter. Expansion of such cells (with IL-2 and heavily irradiated autologous thymocytes as feeder cells) resulted in growth in 6-14% of the wells (mean, 11%) with cells with mature T lymphocyte phenotype. Two-color fluorescence analysis of outgrowing cultures furthermore ascertained that these cells had differentiated through a phase of simultaneous expression of T4 and T8 antigens and at the same time expression of the thymocyte-associated T6 antigens. Thus, given the fact that 10-20% of T cell acute lymphoblastic leukemia (T-ALLs) are CALLA+, we have been able to identify a human prethymic T lymphocyte population that might be the normal counterpart of precursor cell to the CALLA+ T-ALL cell.
Single germline VH and V kappa genes encode predominating antibody variable regions elicited in strain A mice by immunization with p-azophenylarsonate.
Authors: Wysocki L J , Gridley T , Huang S , Grandea A G , Gefter M L
Published: June 2004

We have cloned and sequenced the predominant germline V kappa gene segment expressed by B cells of strain A origin that synthesize antibodies with specificity for Ars. In hybridomas synthesizing anti-Ars antibodies, this V kappa gene segment (V kappa IdCR) has been found exclusively associated with the J kappa 1 gene segment without exhibiting junctional sequence variation. Sequence comparisons of the germline V kappa IdCR gene with expressed derivatives reveals that the latter frequently contain somatically introduced amino acid replacements. Taken together with results of previous structural analyses, these results show that the predominant population of IdCR+ V regions elicited in the secondary immune response is encoded by one or two combinations of V gene segments, has little junctional diversity, and is extensively diversified by somatic mutation in both heavy and light chains.
Molecular, cellular, and functional properties of bone marrow T lymphocyte progenitor clones.
Authors: Palacios R , Kiefer M , Brockhaus M , Karjalainen K , Dembić Z , Kisielow P , von Boehmer H
Published: June 2004

The continuous proliferating bone marrow clones C4-77, C4-86, and C4-95 express low levels of Thy-1 and Ly-1 surface antigens, but no detectable surface antigens normally present on thymocytes, peripheral mature T lymphocytes, cells of the B lymphocyte or myeloid lineages. They contain the T cell antigen receptor genes alpha, beta, and the T cell-specific gene gamma in the germline configuration, and they express functional receptors for IL-3 and nonfunctional receptors for IL-2. The C4 clones are able to home and undergo differentiation in the thymus of sublethally irradiated mice and give rise in vivo to phenotypically and functionally mature peripheral T lymphocytes displaying several antigen specificities. In vitro 5-Azacytidine induces the C4 clones to express Lyt-2 and L3T4 T cell differentiation antigens, and renders them amenable to be switched from IL-3 to IL-2 dependence. However, the C4 clones seem incapable of giving rise to B lymphocytes either in vivo or in vitro. They self-renew in vitro in the presence of IL-3 every 12-14 h. We conclude that the C4 clones represent cells at the earliest stage of T cell development, i.e., Pro-T lymphocytes.
Difference in the structural features of streptococcal M proteins from nephritogenic and rheumatogenic serotypes.
Authors: Khandke K M , Fairwell T , Manjula B N
Published: June 2004

The association of only certain M protein serotypes of group A streptococci with acute glomerulonephritis is very well recognized. Structural information on the M protein, a dimeric alpha-helical coiled-coil molecule, has come so far from three rheumatogenic serotypes, 5, 6, and 24. However, M proteins from the nephritogenic serotypes have not been well characterized. In the present study, we have isolated a biologically active 20,000 Mr pepsin fragment of type 49 M protein (PepM49), a nephritogenic serotype, and purified it to homogeneity using DEAE-Sephadex and gel filtration. The amino acid composition of PepM49 is similar to those of the rheumatogenic M protein serotypes PepM5, PepM6, and PepM24. However, the sequence of the NH2-terminal 60 residues of PepM49 shows little homology to any of these M protein serotypes, although the latter have significant homology among themselves. Nevertheless, PepM49 exhibits a strong heptad periodicity in its nonpolar residues, suggesting its overall conformational similarity with the other M molecules. During the course of the present studies, Moravek et al. (17) reported the NH2-terminal sequence of another M protein serotype, PepM1, which also does not exhibit much homology with the PepM5, PepM6, and PepM24 proteins. Our analysis of this sequence revealed that the PepM1 protein also exhibits a heptad periodicity of the nonpolar amino acids. A closer examination has revealed that the pattern of heptad periodicity in PepM49 and PepM1 proteins is more regular and more similar to each other than has been previously seen for the PepM5, PepM6, and PepM24 proteins. PepM1 is also a nephritogenic serotype. Taken together, these findings indicate an underlying conservation of the tertiary structure of the various M protein serotypes, despite the complexity in their antigenic variation and suggest that the nephritogenic M protein serotypes M1 and M49 may be further apart evolutionarily from the rheumatogenic serotypes 5, 6, and 24. The distinct differences in the structural features of the PepM1 and PepM49 proteins relative to the PepM5, PepM6, and PepM24 proteins are also suggestive of a correlation with the earlier broader classification of the group A streptococci into rheumatogenic and nephritogenic serotypes.
Autoantibodies to HLA B27 in the sera of HLA B27 patients with ankylosing spondylitis and Reiter's syndrome. Molecular mimicry with Klebsiella pneumoniae as potential mechanism of autoimmune disease.
Authors: Schwimmbeck P L , Yu D T , Oldstone M B
Published: June 2004

Ankylosing spondylitis (AS) and Reiter's syndrome (RS) both show a strong correlation with the HLA B27 haplotype. We studied whether sharing of homologous amino acid sequences in the HLA B27 antigen with an invading microbe might occur, and if so, what is the biological significance of such homology. In a computer search of the Dayhoff data bank, we found a homology of six consecutive amino acids between HLA B27.1 antigen residues 72-77 and Klebsiella pneumoniae nitrogenase residues 188-193. These shared sequences are hydrophilic, suggesting locations on molecules exposed to the cell surface. Immunochemical analysis showed that 18 of 34 sera from patients with RS (53%) and 7 of 24 sera from patients with AS (29%) contained antibodies that bound to a synthesized peptide sequence representing residues 69-84 of HLA B27.1. In contrast, only 1 of 22 sera from healthy, B27+ controls (5%) contained antibodies to this peptide (p less than 0.01). Sera from three HLA B27- patients with RS did not possess antibodies to the HLA B27 peptide. Additionally, greater than 40% of HLA B27 patients with AS or RS had antibodies to Klebsiella residues 184-193, while none of the normal nonarthritic HLA B27 haplotype subjects did. Our results suggest that an autoimmune response(s) directed against HLA B27.1 may be a pathogenic mechanism in a subset of patients with AS and RS. Further, this response may initially be induced against Klebsiella pneumoniae, a microorganism that shares sequence homology with HLA B27.
Direct activation of CD8+ cytotoxic T lymphocytes by dendritic cells.
Authors: Inaba K , Young J W , Steinman R M
Published: June 2004

Recent experiments (11-13) have shown that antigen-specific, CD8+, CD4- T lymphocytes can be induced to proliferate and become killer cells in the absence of a second population of "helper" CD8-, CD4+ cells. We have studied early events in the activation of CD4+ and CD8+ T cell subsets in the primary mixed leukocyte reaction. Dendritic cells are a major if not essential accessory cell for the activation of both subpopulations. Antigen-bearing macrophages fail to stimulate unprimed CD8+ cells, but act as targets for the sensitized cytolytic lymphocytes that are induced by dendritic cells. The initial proliferative response is comparable for CD4+ and CD8+ lymphocyte subsets. For both subpopulations, dendritic cells efficiently cluster the responding lymphocytes on the first day and induce the release of IL-2. The data indicate that CD4+ and CD8+ lymphocytes can be activated by a similar mechanism, and illustrate the special role of dendritic cells in the sensitization stage of cell-mediated immunity.
T cell antigen receptor expression in athymic (nu/nu) mice. Evidence for an oligoclonal beta chain repertoire.
Authors: MacDonald H R , Lees R K , Bron C , Sordat B , Miescher G
Published: June 2004

The expression of T cell antigen receptors (TCR) in congenitally athymic (nude) mice has been investigated. Lymph node T cells from 4-5-mo-old athymic mice expressed full-length transcripts for the TCR alpha and beta chains at a level two-to three-fold lower than normal littermate (nu/+) controls. Low levels of expression of TCR protein at the surface of a proportion of nude T cells was demonstrated by staining with monoclonal antibodies KJ16-133 and F23.1 (directed against protein products of a family of TCR beta chain variable region genes known as V beta 8). Immunoprecipitation studies confirmed that F23.1 reacted with a similar molecular species on nude and nu/+ T cells. Studies with individual nude mice revealed a striking heterogeneity in the proportion of T cells expressing KJ16/F23.1 that was not seen in normal animals. This heterogeneity correlated with the expression of mRNA specific for V beta 8 but not with total expression of full-length beta chain transcripts. Analysis of Lyt-2+ and L3T4+ T cell subsets in individual nude mice further demonstrated that F23.1 expression was frequently associated with only one subset, and several cases were seen in which all L3T4+ cells expressed F23.1. In contrast, a similar (and constant) proportion of Lyt-2+ or L3T4+ T cells expressed F23.1 in control mice. Southern blotting of Hind III-digested DNA from nude T cells with a C beta probe revealed a more restricted pattern of TCR beta chain rearrangements than was seen for normal T cells. Taken together, these data provide compelling evidence that TCR gene rearrangement and expression can occur extrathymically. Furthermore, they suggest a model according to which the restricted functional repertoire of T cells previously observed in individual nude mice results from an oligoclonal expansion of T cells that have randomly rearranged and expressed TCR beta chain genes.
Expression of human adenosine deaminase in nonhuman primates after retrovirus-mediated gene transfer.
Authors: Kantoff P W , Gillio A P , McLachlin J R , Bordignon C , Eglitis M A , Kernan N A , Moen R C , Kohn D B , Yu S F , Karson E
Published: June 2004

Primate bone marrow cells were infected with a retroviral vector carrying the genes for human adenosine deaminase (h-ADA) and bacterial neomycin resistance (neor). The infected cells were infused back into the lethally irradiated donor animals. Several monkeys fully reconstituted and were shown to express the h-ADA and neor genes at low levels in their recirculating hematopoietic cells for short periods of time.
Monokines regulate glycosylation of acute-phase proteins.
Authors: Mackiewicz A , Ganapathi M K , Schultz D , Kushner I
Published: June 2004

The acute-phase response to inflammatory stimuli, characterized by increased synthesis of acute-phase proteins (APP), is often accompanied by changes in the glycosylation patterns of some of these proteins. While expression of APP genes in hepatocytes is regulated by monokines, mechanisms governing changes in glycosylation are not known. Exposure of human hepatoma cell line Hep 3B to conditioned medium from LPS-activated human monocytes and to medium from the keratocarcinoma cell line COLO-16 led to increased synthesis of alpha 1 proteinase-inhibitor and ceruloplasmin and to alterations of their glycosylation patterns similar to those seen in human serum in various inflammatory states. IL-1, tumor necrosis factor, and hepatocyte stimulating factor I increased synthesis of ceruloplasmin without alterations in the pattern of its glycosylation. These findings demonstrate that altered glycosylation seen in plasma in some inflammatory states can be explained by the effects of monokines on glycosylation in hepatocytes and that gene expression and glycosylation of some APP during the acute-phase response may be regulated by different mechanisms.
A new variable region in mouse immunoglobulin lambda light chains.
Authors: Sanchez P , Cazenave P A
Published: June 2004

A series of lambda+ murine hybridomas were derived from a BALB/c mouse after a single injection of anti-lambda 2 antibodies coupled to LPS. Nine lambda B cell clones (five lambda 2 and four lambda 3) were expected and seven reacted with antibodies specific for the C lambda 2 constant region but showed a particular isoelectric spectrum. Their RNA products did not hybridize with the V lambda probe. The partial DNA sequence of gene segments coding the unexpected light chain of one hybridoma shows that the V gene segment has only 55% homology with the V lambda 2 gene segment sequence and that J lambda 2 and probably C lambda 2 gene segments are used. Taken together, these results demonstrate the existence of a new lambda light chain.
Characterization of CD3+, CD4-, CD8- clones expressing the putative T cell receptor gamma gene product. Analysis of the activation pathways leading to interleukin 2 production and triggering of the lytic machinery.
Authors: Ferrini S , Bottino C , Biassoni R , Poggi A , Sekaly R P , Moretta L , Moretta A
Published: June 2004

Four clones were derived from human peripheral blood T lymphocytes from which CD4+ and CD8+ cells had been removed by treatment with specific mAbs and complement. All expressed the CD2+, 3+, 4-, 8-, T44- phenotype, and did not react with the WT31 mAb, which is specific for a framework determinant of the CD3-associated alpha/beta heterodimer which serves as receptor for antigen on most human T lymphocytes. Surface iodination followed by crosslinking with dithiobis-succinimidyl propionate (DSP) and immunoprecipitation with anti-CD3 mAbs indicated that, in all four clones, the CD3-associated molecules consisted of a major 45 kD band and a minor band of 43 kD. Northern blot analysis showed that mRNA for the gamma chain was expressed at high levels, whereas mRNA for the alpha chain was missing; beta chain mRNA was present in a defective form (1 kb instead of 1.3 kb). These data support the concept that these clones may express, in association with CD3, the molecular product of the T cell receptor gamma genes instead of the typical alpha/beta heterodimer. CD3+, WT31- clones lysed the NK-sensitive K562 target cells and produced IL-2 upon stimulation with PHA. In addition, they released IL-2 after triggering with soluble anti-CD3 mAbs or with an appropriate combination of anti-CD2 mAbs (in the presence of adherent cells). When CD3+, WT31- clones were incubated with an anti-CD3 producing hybridoma as triggering target, the latter was efficiently lysed. Target cell lysis also occurred when a suitable combination of anti-CD2 mAbs-producing hybridomas was used. Therefore, CD3+, WT31- cells appear to use two pathways of cell activation that function also in conventional CD3+, WT31+ T cells, but they lack a third putative pathway initiated by T44 surface molecules.
In vitro mutagenesis at a single residue introduces B and T cell epitopes into a class I HLA molecule.
Authors: Salter R D , Clayberger C , Lomen C E , Krensky A M , Parham P
Published: June 2004

We have studied the interaction of HLA class I antigens with alloreactive cytotoxic T lymphocytes and monoclonal antibodies using site-directed mutagenesis and expression of an HLA-Aw68.1 gene. Two mutants containing distinct substitutions at polymorphic residues near the NH2-terminal end of the alpha 2 domain were made. One mutant with substitutions at positions 95 and 97 corresponding to residues found in HLA-A2.1 showed no alterations in binding of HLA-Aw68- or HLA-A2-specific monoclonal antibodies, but was reactive with some HLA-A2-specific CTL clones. A second mutant, in which glycine at position 107 was replaced with tryptophan found at that position in HLA-A2.1, was recognized by HLA-A2-specific CTL clones and HLA-A2, Aw69-specific monoclonal antibodies. Thus, substitution of a single amino acid residue at position 107 of the HLA-Aw68.1 molecule generates an allospecific determinant shared with HLA-A2.1 and recognized by both B and T lymphocytes.
A nonstructural polypeptide encoded by immediate-early transcription unit 1 of murine cytomegalovirus is recognized by cytolytic T lymphocytes.
Authors: Koszinowski U H , Keil G M , Schwarz H , Schickedanz J , Reddehase M J
Published: June 2004

We have constructed target cells by cotransfection of the MHC gene Ld and fragments of murine cytomegalovirus (MCMV) DNA coding for nonstructural immediate-early (IE) proteins. Transfectants were tested by using CTL clone IE1 with specificity for an IE epitope presented in association with Ld. Data show that clone IE1 recognizes a product of the ie1 transcription unit of MCMV, and that its specificity is shared by approximately 25% of polyclonal IE-specific CTL. The results provide the first definite evidence that expression of a herpes virus IE gene encoding a regulatory protein gives rise to antigen expression detectable by specific CTL.
Antigenic and immunogenic properties of cyanogen bromide peptides from gonococcal outer membrane protein IB. Evidence for the existence of a surface-exposed conserved epitope.
Authors: Teerlink T , Versantvoort H , Beuvery E C
Published: June 2004

Two distinct species of gonococcal porin proteins exist that differ with regard to surface exposure. Protein IB, expressed by strains of the WII/III serogroup, has both termini buried in the outer membrane, leaving a central region of the molecule exposed at the cell surface. We have attempted to define this region of protein IB in detail by studying the antigenic and immunogenic properties of peptides derived from protein IB. Treatment of gonococcal protein IB (serotype 5) with cyanogen bromide resulted in cleavage of protein IB into three major fragments of Mr of 15,000, 13,000, and 8,000. The location of these peptides in the intact protein was determined by analysis of partial cleavage products. The 8,000 Mr peptide (CB2) was found to be located in the central region of the protein. Chymotrypsin cleavage of protein IB revealed a cleavage site near one of the cyanogen bromide cleavage sites. Trypsin was found to cleave the protein, either in outer membranes complexes (OMC) or in detergent micelles, in the central CB2 fragment. These results suggest that CB2 is a part of the surface-exposed region of protein IB. Immunization of mice with purified protein IB (serotype 5) induced antibodies against all three CB-peptides. Absorption of the sera with homologous OMC resulted in a complete removal of antibodies against CB2, supplying further evidence for its surface-exposed nature. Antibodies against the 13,000 Mr peptide (CB1) could not be absorbed with intact OMC, suggesting that this peptide is buried within the outer membrane. Antisera raised against CB2 of serotype 5 demonstrated a considerable cross-reactivity with heterologous outer membranes. On the contrary, intact OMC induced mainly type-specific antibodies. These data demonstrate the presence of conserved epitopes on the surface-exposed CB2 peptide. These conserved epitopes are generally not very immunogenic when present in intact OMC.
Widespread transcription of a Qa region gene in adult mice.
Authors: Palmer M J , Frelinger J A
Published: June 2004

The mouse MHC class I family includes genes encoded in four regions: H-2K, H-2D, Qa and Tla. While K/D genes are well characterized, relatively little is known about Qa or Tla genes. We have studied the transcription of a B10.P Qa region gene. DNA sequence comparisons of the transmembrane region, supported by Southern blot analysis of cosmid and genomic DNAs from BALB/c and C57BL/10, demonstrate the lambda 3a gene corresponds to Q4p. In both Northern blots and RNA protection experiments using probes derived from the 3' noncoding region, we found that Q4, like the H-2K and H-2D genes, is widely transcribed in B10.P tissues. These data demonstrate for the first time widespread transcription of a Qa gene.
Fine mapping of epitopes by intradomain Kd/Dd recombinants.
Authors: Abastado J P , Jaulin C , Schutze M P , Langlade-Demoyen P , Plata F , Ozato K , Kourilsky P
Published: June 2004

11 intradomain recombinants between H-2Kd and H-2Dd were produced using an original technique based on in vivo recombination in Escherichia coli. After transfection into mouse L cells, all these recombinants were expressed at high levels on the cell surface. The specificities of 77 mAbs were examined on these cell lines. mAbs could be organized in 12 groups. In each group, a small number of amino acids participating in the recognized epitope(s) were identified. In a few instances, noncontinuous epitopes comprising amino acids belonging to different domains of the antigen were found. The data thus obtained are compatible with those produced in previous exon-shuffling experiments, but permit a much more precise definition of recognized epitope(s).
A low polymorphic mouse H-2 class I gene from the Tla complex is expressed in a broad variety of cell types.
Authors: Transy C , Nash S R , David-Watine B , Cochet M , Hunt S W , Hood L E , Kourilsky P
Published: June 2004

We have previously described the isolation of pH-2d-37, a cDNA clone that encodes a so far unknown, poorly polymorphic, class I surface molecule. We report here the isolation of the corresponding gene, its nucleotide sequence, and its localization in the Tla region of the murine MHC. Using a RNase mapping assay, we have confirmed that the second domain coding region of the 37 gene displays very limited polymorphism, and that the gene is transcribed in a broad variety of cell types, in contrast to the genes encoding the known Qa and TL antigens. Possible functions are discussed.
Transmembrane signaling of interleukin 2 receptor. Conformation and function of human interleukin 2 receptor (p55)/insulin receptor chimeric molecules.
Authors: Hatakeyama M , Doi T , Kono T , Maruyama M , Minamoto S , Mori H , Kobayashi M , Uchiyama T , Taniguchi T
Published: June 2004

Chimeric genes were constructed which gave rise to the expression of novel receptor molecules consisting of the extracellular domain of the human interleukin 2 receptor (IL-2-R; p55 or Tac antigen) joined to the transmembrane domain and either full-length or truncated cytoplasmic domain of the human insulin receptor (Ins-R). Expression studies using mouse T cell line EL-4 revealed that the chimeric receptors are able to manifest properties indistinguishable from the authentic IL-2-R. On the other hand, stimulation of the tyrosine kinase activity by IL-2 was not observed in the chimeric receptor with the entire cytoplasmic domain of the Ins-R. These findings thus shed light on the structural conformation and functioning of the IL-2-R complex.
Regulated progression of B lymphocyte differentiation from cultured fetal liver.
Authors: Denis K A , Dorshkind K , Witte O N
Published: June 2004

Lymphoid fetal liver cultures (LFLC) are long-term, nontransformed cultures of early B lymphoid lineage cells which appear developmentally blocked at the pre-B stage in vitro. When injected into severe combined immunodeficient (SCID) mice, cells from LFLC could reconstitute splenic B lymphocytes and serum IgM. T lymphocyte reconstitution was not observed and serum IgG levels were very low. IgG3 was the predominant gamma subisotype in the serum of the LFLC-reconstituted mice, indicating impaired class switching in these B lymphocytes. When thymocytes were coinjected with LFLC, the B lymphocytes were able to class switch fully and respond to T-dependent antigens. These serological responses were heterogeneous. This experimental system allows separation of three B lymphocyte developmental stages: early differentiation in vitro, progression to IgM secretion in vivo, and late differentiation dependent upon mature T lymphocytes in vivo. The unique advantage of this system is the ability to regulate the B lymphocyte developmental pathway in a defined, stepwise manner.
Circulating antibodies to mouse laminin in Chagas disease, American cutaneous leishmaniasis, and normal individuals recognize terminal galactosyl(alpha 1-3)-galactose epitopes.
Authors: Towbin H , Rosenfelder G , Wieslander J , Avila J L , Rojas M , Szarfman A , Esser K , Nowack H , Timpl R
Published: June 2004

Sera from patients with American cutaneous leishmaniasis and Chagas disease and from monkeys infected with either Trypanosoma cruzi or Trypanosoma rhodesiense show, in RIAs, strong binding to mouse laminin. A distinct although weaker binding activity is also detected in normal human sera. The antibodies recognize a common carbohydrate epitope present on mouse laminin, which was assigned to a terminal galactosyl(alpha 1-3)-galactose group. Distinct crossreactions were observed with some other basement membrane proteins, rabbit glycosphingolipids, defucosylated human B blood group substance and components produced by some human tumor cells. Only little activity was, however, found on laminin obtained from human placenta. The data indicate that the antibodies arising in infectious diseases are stimulated by similar carbohydrate epitopes present on the surface of parasites. Tissue-specific occurrence of such epitopes may exist and explain the involvement of distinct tissues in autoimmune disorders.
DNA sequence analysis of I-Ak beta mutants reveals serologically immunodominant region.
Authors: Beck B N , Pease L R , Bell M P , Buerstedde J M , Nilson A E , Schlauder G G , McKean D J
Published: June 2004

We have produced a series of in vitro serologically selected cell lines that express mutant I-Ak molecules. In this report we describe the DNA sequence analysis of the Ak beta gene of four cell lines that express serologically altered Ak beta polypeptides in association with wild-type Ak alpha polypeptides. Each of the major serologic epitopes on the Ak beta polypeptide has been altered in one or more of the four mutants. In addition, the four mutants exhibit a broad spectrum of functional defects when used to stimulate a panel of T hybridomas of various specificities. The DNA sequence analysis revealed that each mutant had sustained a single nucleotide substitution resulting in a single amino acid substitution. All four independent substitutions occurred within or near the third of the four variable regions defined in the beta 1 domain of the A beta polypeptide by allelic comparisons. These data strongly suggest that the third variable region is the major determinant of alloantigenicity on the Ak beta polypeptide.
Four Ia invariant chain forms derive from a single gene by alternate splicing and alternate initiation of transcription/translation.
Authors: O'Sullivan D M , Noonan D , Quaranta V
Published: June 2004

We determined the structural basis for the presence of electrophoretically-distinct, antigenically-related forms of invariant chains in Ia oligomers, and established the mechanisms by which they can be expressed from a single gene. S1 nuclease protection assays indicated that, in B cells, transcription of this gene initiates at a minimum of three sites. Thus, unlike previously thought, invariant chain mRNAs have heterogeneous 5' untranslated segments that may differentially affect initiation of translation. Further, restriction mapping and nucleotide sequencing of cDNAs revealed two kinds of invariant chain mRNAs differing by an internal coding segment of 192 bp. This segment represents an alternatively spliced exon, as demonstrated by nucleotide sequencing of corresponding genomic regions. The exon (exon X) encodes a cysteine-rich stretch of 64 amino acids near the COOH terminus that displays a striking and surprising homology to an internal amino acid repeat of thyroglobulin, suggesting an evolutionary mechanism of exon shuffling. Transient expression of cDNAs indicated that both types of alternatively spliced mRNAs contain two in-frame AUGs functioning as alternate start sites for translation. Thus, transfections with exon X-lacking cDNAs resulted in the expression of Mr 33,000 and 31,000 proteins, detected by immunoprecipitation with anti-invariant chain antisera, and identical by two-dimensional gel (2-D) analyses to the B cell invariant-chain forms gamma 1 (Mr 31,000), gamma 2, and gamma 3 (Mr 33,000). Similarly, exon X-containing cDNAs expressed Mr 43,000 and 41,000 proteins, also identical by 2-D migration to Ia-associated proteins. Thus, human Ia molecules contain four forms of invariant chain of closely related but nonidentical primary structure that are generated from a single gene by a complex pattern of alternate transcriptional start, exon splicing, and translational start.
Depletion of RT6.1+ T lymphocytes induces diabetes in resistant biobreeding/Worcester (BB/W) rats.
Authors: Greiner D L , Mordes J P , Handler E S , Angelillo M , Nakamura N , Rossini A A
Published: June 2004

To investigate the role of RT6+ T cells in the pathogenesis of diabetes in BB/W rats, we treated animals from the diabetes-resistant (DR) subline with anti-RT6.1 lymphocytotoxic mAb. This depleted greater than 95% of peripheral RT6+ T cells but did not substantially reduce levels of circulating T cells or the in vitro response of spleen cells to mitogen. Treatment of 30-d-old DR BB/W rats in this way: induced insulitis and diabetes, rendered nondiabetic RT6-depleted DR rats susceptible to the adoptive transfer of diabetes by spleen cells from acutely diabetic BB/W rats, and yielded DR spleen cell populations capable of the adoptive transfer of diabetes to diabetes-prone (DP) or DR recipients. Treatment of DR rats beginning at 60 d of age failed to produce these effects. These results suggest that both susceptibility and resistance to diabetes in the BB/W rat are in part regulated by the RT6+ T cell subset and provide evidence for the importance of regulatory T lymphocytes in the pathogenesis of autoimmunity and diabetes in BB/W rats.
Characterization of the human B cell stimulatory factor 1 receptor.
Authors: Park L S , Friend D , Sassenfeld H M , Urdal D L
Published: June 2004

125I-labeled recombinant human B cell stimulatory factor 1 (BSF-1) was used to characterize receptors specific for this lymphokine on in vitro cell lines representing human B, T, and hematopoietic lineages, as well as on adherent cell lines of epithelial and endothelial origin, and on primary human gingival fibroblasts. BSF-1 binding was extremely rapid and saturable at both 4 and 37 degrees C, with a slow dissociation rate. On all human cell types examined, BSF-1 bound to a single class of high-affinity receptor (less than 3,000 receptors per cell) with a Ka of 0.5-1.0 X 10(10)/M. Human BSF-1 also bound to cell lines of simian but not murine origin. Comparison of kinetic characteristics obtained with a yeast-derived hyperglycosylated form of BSF-1 (Mr 60,000) and N-glycanase-treated, sugar-free BSF-1 (Mr 15,000) showed no significant differences. Among a panel of lymphokines and growth hormones, only unlabeled human BSF-1 was able to compete for the binding of 125I-labeled human BSF-1. Affinity crosslinking experiments resulted in the identification on both Raji cells and on primary human gingival fibroblasts of a receptor subunit with an average Mr of 139,000. These studies show that the BSF-1 receptor on human cells has an extremely broad cellular distribution, while further supporting the notion that the ability of BSF-1 to mediate a spectrum of biological activities cannot be accounted for by overt differences in the receptor for this lymphokine on different cell lineages.
Platelet-mediated cytotoxicity. Role of antibody and C3, and localization of the cytotoxic system in membranes.
Authors: Slezak S , Symer D E , Shin H S
Published: June 2004

Platelets are one of several cell types capable of mediating antibody-dependent cellular cytotoxicity. We have developed a plasma-free system in which washed mouse platelets lyse washed antibody and complement-sensitized SRBC targets in the presence of EDTA. The dose-response curve is concave to the abscissa, indicating that lysis is a one-hit reaction. Determination of the actual number of platelets required to lyse a target shows that each platelet could lyse a single target. A limited degree of lysis is observed when platelets are incubated with SRBC sensitized with monoclonal IgG2a alone, but no lysis occurs with SRBC bearing comparable amounts of other isotypes. In the presence of C1 through C3, but not C1 through C2, efficient lysis is triggered by complement-fixing monoclonal IgG2a, IgG2b, and IgG3. In contrast, IgM and non-complement-fixing IgG1 and IgE are inactive. To achieve efficient lysis, it appears that platelets require both target cell-bound antibody and C3 fragments in close proximity. It is unlikely that proteases, pore-forming proteins, or toxic oxygen metabolites are involved in platelet-mediated lysis. Freezing and thawing of platelets, sonication, or sonication followed by hypotonic shock causes severe depletion of cytoplasmic and granular contents, as shown by electron microscopy and marker assays. However, the membrane fraction of these preparations retains cytolytic activity. When platelets are treated with trypsin or heated, lytic activity is eliminated, indicating that at least one component of this system is protein. These findings, as well as the fact that platelets do not lyse unsensitized innocent bystander SRBC, suggest that the complete cytotoxic system of platelets capable of specific recognition and lysis resides in their membranes.
T-independent and T-dependent steps in the murine B cell response to antiimmunoglobulin.
Authors: Birkeland M L , Simpson L , Isakson P C , Pure E
Published: June 2004

Sepharose-anti-Ig and purified populations of small, high-density B cells have been used to study the formation and function of B lymphoblasts. Sepharose-anti-Ig converts small, Ia-poor B cells with a high-buoyant density to large, Ia-rich, B blasts with a low-buoyant density. We find that this response proceeds efficiently in the absence of IL-4 (BSF-1) as well as most T cells, macrophages, and dendritic cells. Further development of the blasts requires an additional stimulus, such as LPS or the conditioned medium of stimulated EL-4 thymoma cells. Within 6 h, blasts begin to enter S phase and within 24 h most divide. At later times (48-72 h) most of the blasts are actively secreting IgM. Recombinant IL-1, -2, -3, and -4 have little or no effect on the B blasts, and a neutralizing mAb to IL-4 does not block the response to EL-4 Sn. We conclude that Sepharose-anti-Ig induces B cell blastogenesis in a T-independent fashion and that these blasts represent a highly enriched population of cells that respond to distinct, T cell-derived lymphokines.
Seeding of thymic microenvironments defined by distinct thymocyte-stromal cell interactions is developmentally controlled.
Authors: Kyewski B A
Published: June 2004

Seeding of distinct intrathymic microenvironments defined by direct thymocyte-stromal cell interactions was correlated with T cell development in situ using radiation and nonradiation chimeras of Thy-1.1/1.2 congenic mice. The results identify associations of thymocytes with I-A- macrophages in the cortex as the earliest discernible cell-cell interactions during thymopoiesis. After a significant delay, this recognition stage is followed by concomitant interactions of T cells with I-A+ epithelial cells in the cortex and bone marrow-derived I-A+ dendritic cells in the medulla. All three types of T cell-stromal cell interactions occur after seeding of the intrathymic precursor cell subset and before development of mature medullary-type T cells. The seeding kinetics imply that recognition of cortical epithelial cells by thymocytes in situ represents a relatively late stage of cortical T cell development, whereas thymocyte-dendritic cell interactions denote a very early stage of T cell development in the medulla. The relative positioning of these cell-cell recognition stages during the course of T cell maturation pertains to a putative role of these microenvironments in selection and tolerization of the T cell repertoire.