bluuebunny/crossref_metadata_2025 huggingface dataset:
Deficient protein kinase C-dependent Na+/H+ exchanger activity in T cells from bone marrow transplantation recipients.
Authors: Izquierdo M , Redondo J M , Balboa M A , López-Rivas A , Cragoe E J , Vázquez L , Fernández-Rañada J M , López-Botet M
Published: December 2022

The early Na+/H+ exchanger-mediated alkalinization of intracellular pH (pHi) was analyzed in peripheral blood T cells from 23 bone marrow transplantation (BMT) recipients (17 allogeneic and 6 autologous) and a group of 13 healthy controls, in response to stimulation of protein kinase C (PKC) with a phorbol ester. In parallel we evaluated the proliferative response of peripheral blood T cells to an anti-CD3 mAb in the presence of either IL-2 or PMA. The pHi increase (delta pHi) observed in control samples ranged from 0.14 to 0.23 pH units (X +/- SD = 0.17 +/- 0.03). In 10 allogeneic and four autologous BMT recipients the delta pHi was under the lower limit of the control range (range: 0.01 to 0.09, X +/- SD = 0.05 +/- 0.02), whereas the remaining nine cases responded similarly to control samples (range: 0.14 to 0.24, X +/- SD = 0.17 +/- 0.04). The response of the Na+/H+ antiporter to a PKC-independent osmotic stimulation appeared to be normal, thus indicating that the intrinsic Na+/H+ exchanger activity was unaltered. The anti-CD3 induced proliferative response of the group of samples displaying a suboptimal delta pHi, was significantly lower (p less than 0.01) than that detected in control samples. T cell proliferation in samples from BMT recipients displaying a normal delta pHi was undistinguishable from the control group (p greater than 0.05). Our results provide the first evidence for a defective early metabolic event, closely related to PKC activity, in T cells from BMT recipients displaying a low proliferative response to T cell mitogens.
Synergistic activation of granulocyte-macrophage colony-stimulating factor production by IL-1 and IL-2 in murine Th1 cells.
Authors: Quill H , Gaur A , Brown D , Infante A J , Phipps R P
Published: December 2022

Recent reports indicate that murine CD4+ Th1-type cloned T cells are insensitive to IL-1 because specific IL-1R are not detected on these cells and IL-1 does not modulate proliferative responses. However, we have determined that Th1 clones can respond to IL-1, because they function synergistically with IL-2 to induce granulocyte-macrophage-CSF secretion. This response to IL-1 plus IL-2 could be induced by IL-1 alpha or IL-1 beta and by membrane-bound IL-1 on macrophages. However, IL-1R could not be detected, and Th1 cells did not respond to IL-4 in the presence or absence of IL-1, as measured by either proliferation or granulocyte-macrophage-CSF production. Therefore, IL-1 functioned as a cofactor in Th1 cells stimulated with IL-2, but not with IL-4. A possible mechanism whereby IL-1 activates Th1 cells is discussed.
Epitope mapping of the cloned human autoantigen, histidyl-tRNA synthetase. Analysis of the myositis-associated anti-Jo-1 autoimmune response.
Authors: Ramsden D A , Chen J , Miller F W , Misener V , Bernstein R M , Siminovitch K A , Tsui F W
Published: December 2022

Autoantibodies that bind aminoacyl-tRNA synthetases are strongly associated with the human inflammatory myopathies polymyositis and dermatomyositis, but their molecular origins and relationship to pathogenesis are not known. To address these issues, we wished to identify the autoantigenic epitopes which react with these autoantibodies and to this end, we previously isolated a full length cDNA clone encoding the target Ag recognized most frequently by myositis sera, histidyl-tRNA synthetase (HRS). In the present study, we have analyzed the HRS autoepitopes by two amino acid insertion linker mutagenesis of HRS proteins expressed in Cos 1 cells. A series of mutant HRS cDNA were constructed and the expressed proteins were tested for enzyme activity and for immune reactivity with a panel of sera with anti-Jo-1 antibodies. Immunoblotting and immunoprecipitation analyses revealed that anti-Jo-1 antibodies recognize multiple conformation-dependent and independent epitopes on HRS and that the autoepitopes vary among different myositis patients.
Lipoxygenase inhibitors enhance the proliferation of human B cells.
Authors: Behrens T W , Lum L G , Lianos E A , Goodwin J S
Published: December 2022

The addition of drugs which inhibit the lipoxygenase pathways of arachidonic acid metabolism to 5 day cultures of mitogen-stimulated human B cells enhanced the proliferative response more than 10-fold. Several chemically dissimilar lipoxygenase inhibitors increased proliferation in this system, whereas the specific cyclooxygenase inhibitor indomethacin had no effect. A lipoxygenase inhibitor could be added as late as 48 to 72 h after the initiation of culture and still cause a significant increase in B cell proliferation. These drugs increased the proliferation of both peripheral blood B cells and tonsillar B cells activated by Staphylococcus aureus Cowan I or anti-Ig M antibodies, in combination with a crude T cell supernate, a commercial B cell growth factor preparation, or recombinant lymphotoxin. A similar effect was observed in tonsillar B cells purified by counterflow centrifugal elutriation to remove esterase positive accessory cells, suggesting this is a direct effect on the B cell. Lipoxygenase blockade also caused a greater than twofold increase in polyclonal Ig production. The enhanced proliferation caused by lipoxygenase blockade could not be reversed by adding back exogenous leukotrienes or hydroxyeicosatetraenoic acids to the cultures. Furthermore, B cells prelabeled with [3H]arachidonic acid did not produce radiolabeled lipoxygenase metabolites of arachidonic acid under the same culture conditions in which the addition of lipoxygenase inhibitors had a profound effect on proliferation. Thus, lipoxygenase inhibitors markedly stimulate B cell proliferation under a variety of experimental conditions, although the mechanism responsible for this action has not yet been elucidated.
Free fatty acid perturbation of transmembrane signaling in cytotoxic T lymphocytes.
Authors: Richieri G V , Kleinfeld A M
Published: December 2022

Transmembrane signaling in CTL is found to be extremely sensitive to short term exposure to long chain free fatty acids (FFA). Both alloantigen specific target cells and the lectin Con A were used to stimulate cloned murine CTL. This stimulation was monitored by changes in intracellular calcium concentrations ([Ca2+]i) using the fluorescence indicator fura-2. Treatment of the CTL cells with oleic acid (18:1) at concentrations corresponding to less than 10% (mol/mol) bound to the cell, completely inhibits target cell or Con A-mediated rise in [Ca2+]i. The inhibitory effect of oleic acid is observed within seconds of addition and the inhibition is completely reversed by treating cells with fatty acid free BSA. In addition, using the fluorescence indicator 2',7'-bis(carboxyethyl)carboxyfluorescein to monitor intracellular pH, it was found that oleic acid itself acidifies the cytosol by about 0.3 to 0.4 pH units. Acidification is probably necessary, but is not sufficient to inhibit the calcium rise. Stearic acid (18:0), even at concentrations that correspond to a factor of two to three more bound to the cell than for oleic acid, had no effect on either the [Ca2+]i or intracellular pH responses. Oleic acid was found to bind to cells with single site kinetics and with a number of sites and affinity corresponding to membrane lipid binding sites. Esterification of added oleic acid was negligible in the time (seconds to minutes) required to induce inhibition of the [Ca2+]i response. Inasmuch as added FFA primarily binds to membrane lipid, is not appreciably esterified, and the inhibition is reversed by treatment with fatty acid free BSA, it is likely that the oleic acid effects are due to a physical perturbation of membrane lipid. Furthermore, oleic acid does not affect Con A binding or the production of inositol phosphate metabolites, suggesting that the inhibition of the response is distal to surface recognition events or receptor-phospholipase C coupling. Given the relatively low levels of FFA at which these effects occur it is possible, under conditions in which FFA levels are elevated, that FFA perturbation may modulate CTL activity.
Laminin promotes mast cell attachment.
Authors: Thompson H L , Burbelo P D , Segui-Real B , Yamada Y , Metcalfe D D
Published: December 2022

Tissue mast cells often localize in close proximity to the basement membrane of endothelial cells and increase at sites of inflammation. The reason for this unique tissue distribution is unknown. We report here that both the murine mast cell line PT18 and mouse bone marrow-derived mast cells possess functional receptors for laminin, and exhibit adhesion, spreading and redistribution of histamine-containing granules on a laminin substratum. This adherence is enhanced in the presence of purified IL-3 and can be inhibited by antibodies to laminin and by antibodies to laminin receptors. Northern analysis showed a high level of mRNA for a 32-kDa laminin receptor in PT18 mast cells. Mouse bone marrow-derived cultures initially exhibited a low level of the mRNA expression. However, the expression of the laminin receptor mRNA is induced rapidly within 1 wk of culture with IL-3. Thus, mast cells exhibit functional laminin receptors that may explain the tissue distribution of mast cells and their accumulation at sites of tissue injury.
Delineation of determinants on HLA-B7 and HLA-B27 that are necessary for cytolytic T cell recognition by using inter- and intra-domain recombinants.
Authors: Healy F , Toubert A , Gomard E , Jordan B R , Levy J P
Published: December 2022

We have used bulk culture HLA-B7 and HLA-B27 specific CTL lines derived from 11 donors, and a series of rHLA-B7/HLA-B27 genes transfected into and expressed on the surface of the murine cell P815, to determine the amino acid residues on these HLA class I molecules that are critical for allospecific CTL recognition. The results obtained indicate that for four of six HLA-B7-specific CTL lines the alpha-1 domain for CTL recognition. Furthermore, we found that residues 77 and/or 80 had a critical effect on recognition for all of the CTL lines tested. The region 97-156 in the alpha-2 domain was also important for some of these CTL lines. Furthermore, by using five bulk culture HLA-B27-specific CTL lines we were able to show that residues 77 and/or 80 and residue 152 are also essential for recognition of HLA-B27 by HLA-B27-specific CTL. The strong influence exerted by these residues is discussed in terms of the three-dimensional structure of class I molecules. Finally, a selection was regularly observed in the bulk cultures such that the CTL that were preferentially influenced by either the alpha-1 or the alpha-2 domain were lost after 4 to 7 wk of culture resulting in CTL cell lines which were extremely sensitive to sequence modifications of HLA-B7 or HLA-B27. The possible reasons for this selection, which we have previously observed with both anti-HLA-A2 and anti-HLA-A3 cell lines and is therefore not unique to HLA-B7 or HLA-B27, are discussed.
Locus-specific regulation of Kd, Dd, and Ld class I genes in the BALB/c S49 lymphoma sublines.
Authors: Beck Keeney J , Hedayat M , Myers N M , Connolly J M , Hansen T H
Published: December 2022

The S49 tumor sublines are variants isolated from a single parent BALB/c tumor which demonstrate locus-specific shut-off of their Kd, Dd, and/or Ld surface molecules. Four phenotypically different sublines were characterized: wild-type (Kd Dd Ld)+, a null phenotype (Kd Dd Ld)-, and two intermediates (Kd Dd)+ (Ld)- and (Kd)+ (Dd, Ld)-. Nonexpressed class I Ag were not induced with IFN-gamma even though the expressed antigens were found to be appropriately responsive. Southern blot analysis using probes specific for the 5', exonic or 3' portions of the Kd, Dd, and Ld genes indicated that these S49 sublines have no major chromosomal aberrancies. Northern blot analysis of RNA from each S49 subline using locus-specific oligonucleotide probes revealed message only for the expressed Ag. Thus, the locus-specific shut-off of class I expression in these tumors appears to be acting at the transcriptional level. RNase protection analysis confirmed this result and furthermore demonstrated that the repression is exquisitely specific for the Kd, Dd, and Ld genes as other "class I-like" messages were detected in each cell line. These findings are discussed in the context of the various cis- and trans-acting mechanisms that have been proposed to regulate class I expression.
Protein kinase C regulation of terminal deoxynucleotidyl transferase.
Authors: Tillinghast J P , Russell J H , Fields L E , Loh D Y
Published: December 2022

Terminal deoxynucleotidyl transferase (TdT) is a nuclear enzyme found in early lymphocytes which is thought to increase junctional diversity of TCR and Ig genes by the addition of N regions. TdT is normally found only in immature lymphoid cells and is turned off in both mature B and T cells. To investigate the regulation of TdT gene expression, pre-B and pre-T cells were treated with PMA or three of its analogs and its effects on steady-state TdT mRNA levels determined. Rapid and reversible decline in steady-state TdT mRNA levels was observed within 6 h with PMA. This rapid decline can be blocked by pretreatment of the cells with a relatively selective protein kinase C inhibitor implicating the role of protein kinase C activation in the decline of TdT mRNA. Nuclear run-off studies demonstrate that TdT transcription is rapidly down-regulated within 45 min after PMA treatment, indicating that this regulation occurs mainly at the level of transcription. Furthermore, the TdT mRNA decline is blocked in the presence of cycloheximide, showing that new protein synthesis is required for inactivation of the gene.
Expression of human IFN-gamma genomic DNA in transgenic mice.
Authors: Young H A , Komschlies K L , Ciccarone V , Beckwith M , Rosenberg M , Jenkins N A , Copeland N G , Durum S K
Published: December 2022

We have introduced an 8.6-kb fragment of human genomic DNA containing the full length IFN-gamma gene into the mouse germline. The transgenic animals had no biologic or developmental defects as human IFN-gamma does not bind to the mouse IFN R. Regulation of the transgene paralleled that of the endogenous murine IFN-gamma gene in that: 1) it is not expressed constitutively in any tissue examined thus far, 2) it can be induced in thymus and spleen cells by T cell mitogens, 3) it is not expressed in B cells stimulated by LPS, and 4) it produces normal mRNA and biologically active IFN protein. Whereas expression of the transgene is likely restricted to T cells, we had observed that both fibroblasts and B cell lines could express the same DNA when transfected in vitro; this indicates that in vivo, developmental factors restrict expression of the IFN-gamma gene to T cells. These findings also indicate that the 8.6-kb fragment contains the regulatory elements necessary for normal tissue specific expression in vivo. Moreover, they indicate that the regulatory elements for this gene are completely preserved over the phylogenetic distance separating mouse and man, even though substantial drift has occurred in the structural gene, and probably in the IFN-gamma R as well.
Maturational steps of bone marrow-derived dendritic murine epidermal cells. Phenotypic and functional studies on Langerhans cells and Thy-1+ dendritic epidermal cells in the perinatal period.
Authors: Elbe A , Tschachler E , Steiner G , Binder A , Wolff K , Stingl G
Published: December 2022

The adult murine epidermis harbors two separate CD45+ bone marrow (BM)-derived dendritic cell systems, i.e., Ia+, ADPase+, Thy-1-, CD3- Langerhans cells (LC) and Ia-, ADPase-, Thy-1+, CD3+ dendritic epidermal T cells (DETC). To clarify whether the maturation of these cells from their ill-defined precursors is already accomplished before their entry into the epidermis or, alternatively, whether a specific epidermal milieu is required for the expression of their antigenic determinants, we studied the ontogeny of CD45+ epidermal cells (EC). In the fetal life, there exists a considerable number of CD45+, Ia-, ADPase+ dendritic epidermal cells. When cultured, these cells become Ia+ and, in parallel, acquire the potential of stimulating allogeneic T cell proliferation. These results imply that CD45+, Ia-, ADPase+ fetal dendritic epidermal cells are immature LC precursors and suggest that the epidermis plays a decisive role in LC maturation. The day 17 fetal epidermis also contains a small population of CD45+, Thy-1+, ADPase-, CD3- round cells. Over the course of 2 to 3 wk, they are slowly replaced by an ever increasing number of round and, finally, dendritic CD45+, Thy-1+, CD3+ EC. Thus, CD45+, Thy-1+, ADPase-, CD3- fetal EC may either be DETC precursors or, alternatively, may represent a distinctive cell system of unknown maturation potential. According to this latter theory, these cells would be eventually outnumbered by newly immigrating CD45+, Thy-1+, CD3+ T cells--the actual DETC.
A role for endogenous retroviral sequences in the regulation of lymphocyte activation.
Authors: Krieg A M , Gause W C , Gourley M F , Steinberg A D
Published: December 2022

The genomes of most vertebrates contain numerous retroviral sequences, the great majority of which are non-infectious. These endogenous retroviral sequences are transcribed and translated in many host tissues, and are induced by mitogens. The function, if any, of endogenous retroviruses has been unclear. The transmembrane envelope proteins of some infectious type C retroviruses suppress lymphocyte activation, but it is unknown whether any endogenous type C retroviruses share this suppressive activity. To study the possible effects of murine endogenous retroviral expression, specific antisense oligonucleotides were synthesized complementary to type C retroviral sequences, and were cultured with murine spleen cells. If any of these endogenous retroviruses are suppressing lymphocyte activation, then inhibiting such endogenous retroviral-mediated suppression with antisense might result in lymphocyte stimulation. Three classes of endogenous type C retroviral sequences may be distinguished by antisense oligonucleotides (based on their homology to infectious retroviruses): ecotropic, xenotropic, and mink cell focus-forming (MCF). Antisense oligonucleotides to endogenous MCF envelope gene (env) initiation regions caused: i) doubling or tripling of spleen cell RNA synthesis, and ii) marked increases in lymphocyte surface Ia and Ig expression relative to control oligonucleotides. Antisense oligos to xenotropic or ecotropic env sequences or to endogenous MCF non-envelope sequences had no effect. These data suggest that endogenous MCF sequences exert an inhibitory influence on the murine immune system. Because endogenous MCF expression is inducible by immune stimuli, such expression could constitute an inhibitory feedback circuit that participates in the regulation of immune homeostasis.
Modulation of colony-stimulating factor-1 receptors on macrophages by tumor necrosis factor.
Authors: Shieh J H , Peterson R H , Warren D J , Moore M A
Published: December 2022

The effect of murine rTNF-alpha on the binding of human 125I-rCSF-1 to murine thioglycolate-elicited peritoneal exudate macrophages (PEM) was investigated. At 4 degrees C, 125I-CSF-1 binding to PEM was inhibited by preincubation with human rCSF-1, but not by other cytokines. When PEM were incubated with various cytokines at 37 degrees C, murine rTNF-alpha caused greater than 90% decrease in 125I-CSF-1 binding. This decrease was time, temperature and TNF dose dependent, and was not affected by preincubation with cycloheximide. The reduction in CSF-1-binding activity was reversed by prolonged incubation at 37 degrees C even in the presence of TNF. However, PEM preincubated with TNF subsequently washing free of residual TNF resulted in a rapid recovery of CSF-1 binding. This recovery of CSF-1-binding activity required protein synthesis. Binding studies suggested that the decrease in 125I-CSF-1 binding was most likely caused by a reduction in the number of CSF-1 receptors. In addition, preincubation with TNF at 37 degrees C inhibited 125I-CSF-1 binding on mononuclear phagocytes, including the macrophage cell line J774, bone marrow-derived macrophages, and nonelicited macrophages from three different strains of mice. In contrast, 125I-murine rTNF-alpha binding to PEM was not inhibited by preincubation with CSF-1 at 4 degrees C or 37 degrees C. These data suggest that TNF may play a role in the modulation of receptor expression on blood cells, and may point to a role for this pleiotropic cytokine in the regulation of hemopoiesis.
A specific Fc gamma receptor on cultured rat mesangial cells.
Authors: Santiago A , Satriano J , DeCandido S , Holthofer H , Schreiber R , Unkeless J , Schlondorff D
Published: December 2022

Mesangial cells represent specialized pericytes in the renal glomerulus that contribute to the regulation of a variety of glomerular functions. Recently we and others have shown that cultured mesangial cells bind and take up immune complexes in an Fc-dependent manner leading in turn to generation of PGE2, reactive oxygen, and platelet-activating factor. The present studies were designed to further characterize potential Fc-gamma R on mesangial cells. Binding assays with either monomeric or heat aggregated (HA) [125I] labeled rat subclass-specific IgG were performed at 4 degrees C for 2 h on subcultured rat mesangial cells. Monomeric rat IgG2a, IgG2b, IgG1 and HA IgG2a bound only nonspecifically. Saturable Fc-dependent binding occurred for HA IgG2b and HA IgG1 though maximal binding and affinity were much higher for IgG2b. The presence of an Fc-gamma R was confirmed by surface protein iodination of mesangial cells (MC) and immunoprecipitation with either a polyclonal or mAb 2.4G2 prepared against murine Fc-gamma R. Both antibodies precipitated a 45-kDa iodinated protein band from cultured rat MC that comigrated with that from murine macrophage J774 cells on SDS-PAGE. This protein band also reacted with the polyclonal anti Fc-gamma R antibody on immunoblots. In contrast rat renal papillary epithelial cells were negative. The 45-kDa protein recognized by the rat anti-Fc-gamma R antibody 2.4G2 probably represents the binding site for HA IgG2b, as the 2.4G2 antibody also blocked binding of HA IgG2b. By immunofluorescence microscopy all MC stained positively with the polyclonal anti-Fc-gamma R antibody. A cDNA probe for the Fc-gamma RII-alpha on murine macrophages hybridized to mRNA from cultured rat MC which was of the same size (though less abundant) as that from J774 macrophages. These results further characterize the Fc-gamma R on cultured rat MC, and raise the possibility that the mesangial Fc-gamma R may play a role in the handling of immune-complexes by the renal glomerulus.
Recognition of hepatitis B virus envelope proteins by liver-infiltrating T lymphocytes in chronic HBV infection.
Authors: Barnaba V , Franco A , Alberti A , Balsano C , Benvenuto R , Balsano F
Published: December 2022

The Ag specificity and cytotoxic function of human T cell clones, generated from lymphocytes infiltrating the liver of a chronic hepatitis B patient, were studied. Both class I- and class II-restricted T clones specifically proliferated to hepatitis B virus envelope proteins, but not to hepatitis B core Ag. The fine specificity of T cells was studied by using rAg having different composition in relation to HBV-envelope proteins or synthetic peptides of preS regions. The antigenic determinant recognized by T cell clones mapped to the preS2 region based on the response to r(preS1+preS2+S) and to r(preS2+S) and the failure to respond to S or preS1 alone. More precise epitope mapping was based on synthetic preS2 peptides 120-150 or 120-134, which stimulated both class I- and class II-restricted T clones, whereas preS2 153-171 or preS1 1-110 peptides did not; thus, the preS2 120-134 appears to contain both the residues binding to class I molecules and the residues binding to class II molecules. Moreover, strong and specific cytotoxic responses of these clones were observed only when HLA-matched EBV-lines, used as target cells, were previously sensitized with r(preS1+preS2+S) or preS2 peptides, which were shown to stimulate the clones. Thus, a preS2 epitope can represent a target Ag for liver-infiltrating T cells, which could kill the hepatocytes expressing the Ag plus the appropriate MHC molecule.
I-Ad restricted T cell recognition of influenza hemagglutinin. Synthetic peptides identify multiple epitopes corresponding to antibody-binding regions of the HA1 subunit.
Authors: Barnett B C , Burt D S , Graham C M , Warren A P , Skehel J J , Thomas D B
Published: December 2022

In a recent study, we reported extensive diversity in the Iak-restricted T cell repertoire for the hemagglutinin molecule (HA) of influenza A viruses (H3 subtype). Synthetic peptides identified six nonoverlapping epitopes on the HA1 subunit, and CD4+ T cell clones, specific for these regions, discriminated between natural variant viruses that had accumulated amino acid substitutions during antigenic drift. Here, we demonstrate similar specificity and diversity for the Iad haplotype and have identified multiple T cell epitopes within the sequences HA1 56-76, 71-91, 81-97, 177-199, 186-205, and 206-227. These also include recognition sites for neutralizing antibodies and correlations can be made between antigenic drift substitutions in H3 subtype viruses and the specificity of individual CD4+ clones for mutant HA. Moreover, these peptides appear not to exhibit structural homology and fail to compete for Ag presentation, indicating heterogeneity in peptide-Ia interaction. To explain the observation that CD4+ T cells, from two major haplotypes, recognize antibody binding regions of the HA molecule, we propose that surface Ig receptors of the Ag-specific B memory cell exert a direct effect on the processing of HA peptides and subsequent selection of the class II-restricted T cell memory repertoire after natural infection.
Nucleotide sequence of channel catfish heavy chain cDNA and genomic blot analyses. Implications for the phylogeny of Ig heavy chains.
Authors: Ghaffari S H , Lobb C J
Published: December 2022

Our prior analyses defined the cDNA sequence on part of the CH2 domain, the complete CH3 and CH4 domains, and the 3'-untranslated region of a catfish H chain. To complete the catfish H chain mRNA sequence, a primer-extended H chain cDNA library was constructed. Analysis of this library has resulted in the definition of full-length clones encoding a 61-bp 5' untranslated region, a 51-bp leader sequence, the V region and the complete CH1 and CH2 domains. The high similarity defined with other vertebrate V regions readily allowed the catfish sequence to be divided into FR and CDR regions. Sequence comparisons with mammalian VH and JH genes strongly suggest that the catfish V region is the product of multiple genes. Using a catfish VH cDNA probe, at least 25 different genomic VH members were defined. Because this probe does not hybridize with other full-length H chain cDNA clones, additional VH families will likely be defined in catfish. Phylogenetic sequence comparisons of the catfish C region domains indicated that the CH1 and CH4 were the most highly conserved. In addition several important features were defined in genomic Southern blot analyses of catfish DNA. Gene titration experiments established that the catfish CH gene is represented by a single genomic copy. This finding provides clear evidence that the genomic organization of H chain genes in catfish must be different from that defined in sharks and suggests that the phylogeny of single copy CH genes may have been established at the level of the bony fishes. It is also likely that there is an additional CH gene in catfish. This gene is also represented by a single genomic copy, and based upon its relative signal intensity when compared with the known CH gene it appears to share higher similarity with the known CH1 domain than it does with the CH2 domain.
VH gene family repertoire of resting B cells. Preferential use of D-proximal families early in development may be due to distinct B cell subsets.
Authors: Jeong H D , Teale J M
Published: December 2022

The fetal VH gene repertoire was shown previously to be characterized by overrepresentation of D-proximal families, VH 7183 and VH Q52, compared with adult bone marrow B cells in which VH genes were expressed in a more stochastic fashion. To determine the underlying mechanisms of these findings, adult vs fetal progenitors were placed in the same supportive microenvironment and the resulting B lineage cells analyzed for VH gene family expression. The supportive microenvironment was provided by established adult bone marrow stromal cell layers. In this way the relative importance of environmental vs genetic influences could be determined. The fetal B cells and pre-B cells that developed on adult stromal cells maintained a fetal-like VH gene family repertoire with preference for D-proximal families VH 7183 and Q52. In contrast, adult cultured B cells maintained the adult-like repertoire with predominance of the largest family VH J558. Only after long-term incubation was there a change in the expression of particular VH gene families. These findings suggest that the D-proximal VH gene family preference observed early in ontogeny is associated more with the inherent genetic potential of B cell progenitors that predominate during fetal life and less with environmental influences.
Functional heterogeneity among CD4+ encephalitogenic T cells in recruitment of CD8+ T cells in experimental autoimmune encephalomyelitis.
Authors: Sun D , Klinkert W E
Published: December 2022

Inoculation of Lewis rats with live or attenuated (irradiated or paraformaldehyde-fixed) CD4+ encephalitogenic T cells (S1 line) protects the recipients from transferred experimental autoimmune encephalomyelitis (tEAE) induced by S1 cells. A CD8+ T lymphocyte population specifically activated against the EAE-inducing S1 cells can be readily isolated from the lymphoid organs of pretreated animals. We show, in the present study, that encephalitogenic T cell lines derived from Lewis rats differ in their ability to induce resistance against tEAE in vivo and to stimulate CD8+ cell proliferation in vitro. We also demonstrate that the S19 line of encephalitogenic T cells, in combination with myelin basic protein (MBP), can stimulate CD8+ cell proliferation in vitro. The CD8+ cells generated in this way strongly suppress MBP-specific T cell proliferation in vitro. This combined effect of T cells and MBP was also evident in vivo. Neither S19 cells nor MBP alone induced resistance against S19-mediated tEAE, rather coinjection of these cells and MBP was required. Our results suggest that resistance to EAE is mediated by distinct populations of encephalitogenic T cells that activate Ts cells through different mechanisms. In some instances, both autoreactive T cells and their relevant autoantigen(s) may be needed to activate Ts cells in vivo.
The role of IFN-gamma in delayed-type hypersensitivity mediated by Th1 clones.
Authors: Fong T A , Mosmann T R
Published: December 2022

One type of Th cell clone, Th1, causes delayed-type hypersensitivity (DTH) when injected with the appropriate Ag into the footpads of naive mice. Because IFN-gamma, one of the Th1-specific cytokines, has been reported to produce some of the manifestations of DTH, we have investigated the role of IFN-gamma in the DTH reaction induced by Th1 cells by using a mAb which neutralizes the biologic activity of IFN-gamma. Anti IFN-gamma inhibited up to 55% of the swelling response induced by seven Th1 clones in BALB/c and CBA/J mice suggesting that IFN-gamma is an important mediator of Th1-induced DTH. This inhibition was consistently observed during the peak phase of the DTH response and could be partly but not entirely explained by decreases in vascular leakage. The DTH responses induced by two Th1 clones in C57BL/6 mice were not inhibited by anti IFN-gamma. Taken together, these data suggest that other inflammatory mediators also play a role in Th1-induced DTH responses. Based on the gross description and kinetics of the response and the numbers of polymorphonuclear neutrophilic granulocytes in the cellular infiltrate, Th1-induced DTH appears to be similar to the Jones-Mote type of hypersensitivity.
Evidence that tumor necrosis factor has an important role in antibacterial resistance.
Authors: Havell E A
Published: December 2022

TNF is produced in the spleens of Listeria-infected mice during the first 3 days of a sublethal immunizing infection. The production of Listeria-induced TNF coincides with the time when peak numbers of bacteria are present in the liver and spleen. Evidence suggesting that TNF produced in Listeria-infected organs functions in a T cell-independent resistance mechanism comes from results showing that listeriosis is exacerbated in both T cell-intact mice and T cell-deficient (athymic nude) mice treated with a monospecific anti-murine TNF IgG. Listeriosis is exacerbated, however, only if the anti-TNF IgG is given during the first 3 days of infection, i.e., at the time when TNF is being produced in the spleen. Anti-TNF IgG administered on the first day of infection neutralized all the cytotoxic activity of endogenously produced TNF in the spleen. Additional evidence that TNF plays an important role in antibacterial defenses was obtained by showing that administration of pure murine rTNF protects mice against a normally lethal Listeria challenge given 1 to 24 h later.
Human monoclonal anti-La antibodies. The La protein resides on a subset of Ro particles.
Authors: Mamula M J , Silverman E D , Laxer R M , Bentur L , Isacovics B , Hardin J A
Published: December 2022

We have addressed the problem of anti-La autoimmune responses by defining the specific binding sites of human mAb to the La protein. Two human anti-La mAb were developed; one an IgM (kappa) (designated 8G3) and the second an IgG1 (kappa) (9A5) isotype. The mAb 8G3 immunoprecipitated the La RNA and La protein from crude human cell lysates; bound the 50-kDa La protein and a 28-kDa digestion fragment in immunoblots, and recognized a small defined internal segment from the cloned La protein. In contrast, the IgG isotype (9A5) failed to precipitate native La from cell lysates but bound the same segment of digested La protein and the same polypeptide of 131 amino acids in length from the cloned La protein. Immunoprecipitation experiments performed with these mAb demonstrated that the La protein is a component of a subset of Ro particles. The data suggest that the La protein is not present on the hY RNA in the absence of the Ro polypeptide. These observations may define functional subsets or maturation states of hY RNA based on their association with Ro or Ro and La polypeptides.
Human IgM monoclonal proteins that bind 3-fucosyllactosamine, asialo-GM1, and GM1.
Authors: Marcus D M , Perry L , Gilbert S , Preud'homme J L , Kyle R
Published: December 2022

Analysis of monoclonal human Ig that occur in association with lymphoproliferative diseases has provided valuable information about antibody structure and idiotypes. We analyzed 940 human sera that contained monoclonal IgM proteins for their ability to bind to four carbohydrate epitopes. Ten sera bound asialo-GM1, five of these sera also bound GM1, 10 bound to 3-fucosyllactosamine (3-FL), and one each bound to levan and galactan. Although the antibody activity in each serum was associated with a single L chain isotype, both kappa and lambda isotypes were represented among the proteins that bound to asialo-GM1 and to 3-FL. Some antibodies against asialo-GM1 were highly specific for this compound, whereas others cross-reacted with the structurally related gangliosides GM1 and GD1b. The antibodies to asialo-GM1 also varied considerably in their ability to lyse liposomes that contain asialo GM1. An association of IgM mAb against gangliosides with peripheral neuropathies has been reported recently, but only one of five patients whose antibodies reacted with GM1 ganglioside had a neuropathy. The antibodies that bound 3-FL exhibited narrower specificity, and less than 10% cross reactivity was noted with structurally related carbohydrates. The frequency of monoclonal proteins that bound 3-FL and asialo-GM1, approximately 1:100 sera for each specificity, was surprisingly high in view of the fact that both of these epitopes are expressed in human tissues. We suggest that these antibodies may be poly-specific and/or that the subset of B lymphocytes that synthesizes these anti-carbohydrate antibodies undergoes malignant transformation more frequently than other B lymphocytes.
A human milk galactosyltransferase is specific for secreted, but not plasma, IgA.
Authors: McGuire E J , Kerlin R , Cebra J J , Roth S
Published: December 2022

IgA from human milk and colostrum is a substrate for a galactosyltransferase also present in milk and colostrum. The secreted IgA that serves as the best acceptor for the transferase activity is the IgA that fails to bind readily to jacalin lectin. Upon becoming galactosylated by the transferase, however, the IgA shows an increased affinity for jacalin. Glycosidase and electrophoretic results indicate that the transferred galactose is beta-linked to the alpha-chain of the IgA. The IgA:transferase activity can be purified by gel filtration and cation exchange chromatographies, as well as by affinity chromatography on Sepharose derivatized with UDP or IgM. The enzyme has an apparent Mr of about 64 kDa, is prevalent in both milk and colostrum, but has a sixfold higher sp act in colostrum.
Generation of biologically active C-reactive protein peptides by a neutral protease on the membrane of phorbol myristate acetate-stimulated neutrophils.
Authors: Shephard E G , Beer S M , Anderson R , Strachan A F , Nel A E , de Beer F C
Published: December 2022

The association of human C-reactive protein (CRP) with nonstimulated and PMA-stimulated human neutrophils and the concomitant degradation of CRP (monitored by TCA-soluble peptides and SDS-PAGE analysis) has been studied. Maximum association of 125I-labeled CRP with neutrophils and 125I-labeled CRP degradation during association with these cells was achieved by stimulating the neutrophils with PMA at 10 ng/ml; a concentration in which azurophil granule release was not significant. For PMA-stimulated neutrophils, the association of 125I-labeled CRP was 1.8 times higher and PMA-stimulated neutrophil-mediated degradation of the ligand was three times faster than that for nonstimulated cells. The neutrophil-associated 125I-labeled CRP in the absence and presence of PMA proved on SDS-PAGE analysis to be approximately 50% degraded. There was a positive correlation between the extent of CRP degradation and the association of 125I-labeled CRP with neutrophils. In addition to generation of neutrophil associated CRP intermediates, small soluble CRP peptides were generated during association of CRP with neutrophils. These peptides inhibited superoxide production from opsonized zymosan-activated neutrophils by approximately 40% at 10 micrograms/ml. 125I-labeled CRP degradation mediated by nonstimulated neutrophils, and neutrophil-conditioned medium (from both non-stimulated and PMA-stimulated cells) was inhibitable by alpha 1-antitrypsin and approximately seven times less at 1 h than that occurring during 125I-labeled CRP-association with PMA-stimulated neutrophils. The degradation of 125I-labeled CRP mediated by PMA-stimulated neutrophils was not fully inhibitable by alpha 1-antitrypsin. The data point to the involvement of a membrane-associated serine protease, which is maximally activated by PMA, in the degradation of 125I-labeled CRP during association with neutrophils. Our results indicate that at an inflammatory site CRP-derived peptides can be produced that inhibit the action of activated neutrophils.
Analysis of foot-and-mouth disease virus-neutralizing idiotypes from immune bovine and swine with anti-murine idiotype antibody probes.
Authors: Garmendia A E , Borca M V , Morgan D O , Baxt B
Published: December 2022

Rabbit anti-idiotypic antibodies (a-IdAb) induced by foot-and-mouth disease virus (FMDV) neutralizing mAb were used as probes to identify anti-FMDV Id in immune serum from bovine and swine. In a competitive RIA, at least two of the a-IdAb exhibited a dose-dependent capacity to compete with labeled virus for anti-FMDV antibodies from a convalescent bovine serum. These a-IdAb were immobilized on activated Sepharose and used to isolate anti-viral Id from bovine, swine, and murine FMDV immune sera. Both the bovine and swine antibodies recovered from the a-IdAb/Sepharose columns reacted with virus, and to a lesser extent with corresponding mAb-resistant virus variants. The binding of affinity isolated bovine and swine antibodies to virus was specifically inhibited by the homologous a-IdAb, and in addition, both were capable of neutralizing FMDV in suckling mouse protection and plaque reduction neutralization assays. Therefore, by means of a-IdAb probes generated against FMDV murine Id, two neutralizing Id were identified in bovine and swine. These results suggest that FMDV-neutralizing epitopes recognized by murine systems play a role in the overall immunity of foot-and-mouth disease-susceptible animals.
Generation and characterization of IL-2-activated bone marrow cells as a potent graft vs tumor effector in transplantation.
Authors: Agah R , Malloy B , Kerner M , Mazumder A
Published: December 2022

The potential of bone marrow transplantation as an immunotherapeutic modality, using biomodulation of the marrow cells has been ignored in autologous transplantation. Furthermore, many common cancers such as lung, colon, prostate, and pancreas are resistant to even transplant doses of conventional agents and hence require novel approaches such as biomodulation. This study shows that we can generate cytotoxic killer cells similar to lymphokine-activated killer cells capable of lysing NK-resistant tumor cells in vitro if we incubate human or murine bone marrow in IL-2. This was accomplished without affecting the ability of the bone marrow to fully reconstitute mice similar to that of fresh nonactivated bone marrow. Studies evaluating the IL-2 activated human bone marrow in vitro also indicated that these activated bone marrow have similar CFU to that of fresh human marrow. Furthermore, in murine in vivo studies, the activated bone marrow (ABM) caused significant tumor regression in tumor-bearing mice. Also, these ABM cells had similar or higher tumoricidal activity and longer kinetics than spleen lymphokine-activated killer cells in vitro. Also, the ABM had purging ability in vitro. Therefore this IL-2 ABM could be used as an active therapeutic tool and not just as a passive rescue element in the autologous bone marrow transplantation setting.
Mechanism of action of a streptococcal preparation (OK-432) in prevention of autoimmune diabetes in NOD mice. Suppression of generation of effector cells for pancreatic B cell destruction.
Authors: Shintani S , Satoh J , Seino H , Goto Y , Toyota T
Published: December 2022

We have previously reported that treatment with a streptococcal preparation (OK-432), one of the biologic response modifiers, inhibits insulitis and development of autoimmune diabetes in nonobese diabetic (NOD) mice and Bio-Breeding rats used as animal models of insulin-dependent diabetes mellitus (IDDM). We studied the mechanism by which OK-432 inhibited development of IDDM in NOD mice. In female NOD mice diabetes spontaneously developed from 10 to 15 wk of age and the cumulative incidence of diabetes amounted to 74.7% at 30 wk of age. NOD mice, however, never developed diabetes over the observation period of up to 45 wk of age when they were i.p. injected with 0.1 mg of OK-432 weekly from 4 to 15 wk of age. OK-432 treatment in younger age had a stronger inhibitory effect on the development of diabetes. Diabetes was transferred to young, irradiated mice by spleen cells of nontreated, adult mice, but barely transferred by those of OK-432-treated mice. Furthermore, these spleen cells of OK-432-treated mice did not suppress transfer of diabetes by diabetic mice spleen cells. Treatment with cyclophosphamide promoted development of diabetes in nontreated NOD mice due to removal of suppressor cells. However, cyclophosphamide did not show the promotive effect in OK-432-treated mice. Taken together, these results indicate that OK-432 treatment prevented development of diabetes mainly by suppression in generation of the effector cells for pancreatic B cell destruction. The same OK-432 treatment did not suppress the immune response to exogenous AG such as xenogeneic SRBC and allogeneic tumor cells. The study suggests that BRM in the natural environment such as streptococci may suppress induction and progression of autoimmunity and be useful for the immunotherapy of human IDDM.
Direct evidence for an intracellular role for tumor necrosis factor-alpha 1. Microinjection of tumor necrosis factor kills target cells.
Authors: Smith M R , Munger W E , Kung H F , Takacs L , Durum S K
Published: December 2022

TNF-alpha is a small peptide cytokine produced primarily by activated macrophages. One of the many biologic activities of TNF is the killing of diverse types of tumor cells. We considered the possibility that killing was mediated by TNF itself at an intracellular site, subsequent to receptor-mediated endocytosis. To test this hypothesis, we microinjected TNF into various murine normal cells and cell lines, some of which were killed by TNF given by the usual extracellular route, and others that were not. Cytotoxic effects of microinjected TNF were observed in several cell types 2 to 4 h after injection. L929 fibroblasts were killed by either extracellular or intracellular TNF. A TNF-resistant subline of L929 was insensitive to either extracellular or intracellular TNF. L6 fibroblasts were found to be resistant to high doses of TNF given either extracellularly or microinjected. Normal macrophages and the J774 macrophage-like cell line were not killed by extracellular TNF, but were rapidly killed by microinjected TNF. Thus, TNF, an extracellular peptide ligand, has an intracellular activity, suggesting that internalization of this ligand may have important intracellular biochemical roles.
Effect of isotypes and allelic polymorphism on the binding of staphylococcal exotoxins to MHC class II molecules.
Authors: Scholl P R , Diez A , Karr R , Sekaly R P , Trowsdale J , Geha R S
Published: December 2022

Interaction of staphylococcal exotoxins (SE) with MHC class II molecules plays a central role in the activation of immune cells by SE. We have recently demonstrated directly that toxic shock syndrome toxin-1 (TSST-1) binds to MHC class II molecules with high affinity, and similar results have been reported for SEA and SEB. The ability of T cells to respond to individual SE is associated with the expression of particular TCR-V beta gene elements. In the present study we have examined the effect of polymorphism on the ability of MHC class II molecules to bind SEB and TSST-1. We have used a panel of L cell transfectants that express different allelic forms of each of the three human class II isotypes. Radioligand binding assays detected binding of SEB and TSST-1 to most, but not all of the MHC class II molecules examined. Toxin-driven MHC class II-dependent T cell proliferation occurred with all transfectants examined even in the absence of detectable toxin binding. These results indicate that SE can bind to human MHC class II molecules of diverse phenotypes.