bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the promoter region of the membrane cofactor protein (CD46) gene of the human complement system and comparison to a membrane cofactor protein-like genetic element.
Authors: Cui W , Hourcade D , Post T , Greenlund A C , Atkinson J P , Kumar V
Published: December 2022

Membrane cofactor protein (MCP; CD46) is a widely expressed C regulatory protein that inhibits C activation on self-tissue. MCP binds C3b and C4b deposited on autologous cells and then serves as a cofactor for their inactivation by limited proteolytic cleavage. To characterize the DNA sequence elements responsible for controlling MCP expression, the 5' flanking region of the human MCP gene was cloned. Sequencing of 1350 nucleotides upstream from the ATG codon revealed a GC-rich region in the initial 500 nucleotides that is especially rich in the CpG dinucleotide. A CAAT box in reverse orientation, surrounded by four putative SP1 binding sites but lacking a typical TATA element, was within the first 200 nucleotides of this GC-rich region. The major transcriptional initiation site for HeLa cells, determined by primer extension and S1 nuclease protection analyses, was located 105 nucleotides from the translational start site. This overall orientation of the promoter region is characteristic of "housekeeping" genes. The MCP promoter region was further examined in HEp-2 cells by the chloramphenicol acetyltransferase (CAT) reporter gene assay, using various constructs derived from the 5' region of the MCP gene. The MCP promoter activity was confined to the GC-rich region from -624 to +96 (start site of transcription being +1). Inclusion of an AT-rich sequence from -624 to -1204 resulted in a 42% reduction in CAT activity suggesting that an inhibitor is present among the AT-rich sequences. The 5' flanking region of a highly homologous partial duplication of the MCP gene was also cloned and sequenced, and various constructs were assessed in the CAT reporter system. Many of the functionally relevant sequences seen in MCP are also found in the MCP-like 5' UT region, which is 85% homologous to MCP. The most striking difference was a 224 nucleotide deletion that was upstream from the corresponding MCP region harboring most of the promoter activity. Although expression of an MCP-like protein has not been reported, the MCP-like promoter region produced promoter activity comparable with that of MCP. These results serve as a basis for subsequent analyses of the expression of MCP in various cells and tissues and for understanding the mechanism of its modulation in inflammatory conditions. Also, through a comparison of the 5' region of MCP with other genes in the regulators of C activation gene cluster (at 1 q32), we propose a model for the evolution of the promoters in this tight linkage group.(ABSTRACT TRUNCATED AT 400 WORDS)
Prevention of autoimmune type I diabetes by CD4+ suppressor T cells in superantigen-treated non-obese diabetic mice.
Authors: Kawamura T , Nagata M , Utsugi T , Yoon J W
Published: December 2022

The development of autoimmune type I diabetes in the NOD mouse appears to be controlled by both genetic and environmental factors. This investigation was initiated to determine whether exogenous superantigens, as environmental factors, can influence the development of diabetes. Several staphylococcal enterotoxins (SE) (SEA, SEC1, SEC2, or SEC3), which are known superantigens, were injected i.v. into female NOD mice at 4 and 10 wk of age. At 32 wk of age, the incidence of diabetes in the SE-treated mice ranged from 6 to 12.5%; this was significantly lower than that of mice treated with PBS--64%. There was no significant difference in effectiveness among the various SE used. SE induced a modest decrease in T lymphocytes bearing specific V beta TCR 2 wk after injection, but this effect did not persist past 4 wk. To elucidate the mechanism of the SE effect, suppressor activity in SE-treated mice was evaluated. Splenocytes from SE-treated mice inhibited the transfer of diabetes by splenocytes from acutely diabetic NOD mice when injected into irradiated young NOD mice; only 10% became diabetic. In contrast, 83% of the mice receiving splenocytes from PBS-treated control mice became diabetic. Suppressor activity of splenocytes from SE-treated mice was diminished by the depletion of CD4+ T cells, but not by the depletion of CD8+ T cells, indicating that the suppressor cells belonged to the CD4+ T class of lymphocytes. On the basis of these observations, we conclude that exogenous superantigens activate CD4+ suppressor T cells, leading to the prevention of autoimmune type I diabetes in NOD mice.
Increased expression of epidermal IL-8 receptor in psoriasis. Down-regulation by FK-506 in vitro.
Authors: Schulz B S , Michel G , Wagner S , Süss R , Beetz A , Peter R U , Kemény L , Ruzicka T
Published: December 2022

IL-8 is a chemotactic cytokine with proinflammatory and growth-promoting activities. Recently it has been shown to influence several functions of keratinocytes, including HLA-DR expression, chemotaxis, and proliferation by binding to a specific receptor. Because psoriasis vulgaris is characterized by epidermal hyperproliferation and infiltration of inflammatory cells, we investigated the expression of IL-8 and its receptor in normal and psoriatic epidermis using semiquantitative reverse-transcriptase-polymerase chain reaction. In addition the mRNA levels of the proto-oncogenes c-ras, c-raf, c-myc, and HER-2 were also investigated as potential growth-promoting stimuli in psoriatic epidermis. IL-8 mRNA was only detected in lesional psoriatic epidermis, and IL-8R-specific mRNA was found to be 10 times increased in lesional psoriatic epidermis. There was no significant difference in the protooncogene mRNA levels. In order to test the relevance of the massively increased IL-8R levels in psoriatic epidermis, we investigated the effect of the antipsoriatic drug FK-506 on specific IL-8 and IL-8R mRNA expression. FK-506 dose dependently inhibited IL-8R expression and function. Our data suggest that in psoriatic skin, elevated IL-8 levels and markedly increased IL-8R expression may act in concert to induce the cardinal signs of psoriasis--epidermal hyperproliferation and leukocyte infiltration. IL-8R may prove a molecular target for antipsoriatic drugs such as FK-506.
Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains.
Authors: Parker K C , Bednarek M A , Coligan J E
Published: December 2022

A method to predict the relative binding strengths of all possible nonapeptides to the MHC class I molecule HLA-A2 has been developed based on experimental peptide binding data. These data indicate that, for most peptides, each side-chain of the peptide contributes a certain amount to the stability of the HLA-A2 complex that is independent of the sequence of the peptide. To quantify these contributions, the binding data from a set of 154 peptides were combined together to generate a table containing 180 coefficients (20 amino acids x 9 positions), each of which represents the contribution of one particular amino acid residue at a specified position within the peptide to binding to HLA-A2. Eighty peptides formed stable HLA-A2 complexes, as assessed by measuring the rate of dissociation of beta 2m. The remaining 74 peptides formed complexes that had a half-life of beta 2m dissociation of less than 5 min at 37 degrees C, or did not bind to HLA-A2, and were included because they could be used to constrain the values of some of the coefficients. The "theoretical" binding stability (calculated by multiplying together the corresponding coefficients) matched the experimental binding stability to within a factor of 5. The coefficients were then used to calculate the theoretical binding stability for all the previously identified self or antigenic nonamer peptides known to bind to HLA-A2. The binding stability for all other nonamer peptides that could be generated from the proteins from which these peptides were derived was also predicted. In every case, the previously described HLA-A2 binding peptides were ranked in the top 2% of all possible nonamers for each source protein. Therefore, most biologically relevant nonamer peptides should be identifiable using the table of coefficients. We conclude that the side-chains of most nonamer peptides to the first approximation bind independently of one another to the HLA-A2 molecule.
Tyrosine kinase-dependent assembly of actin plaques linking Fc epsilon R1 cross-linking to increased cell substrate adhesion in RBL-2H3 tumor mast cells.
Authors: Pfeiffer J R , Oliver J M
Published: December 2022

RBL-2H3 rat tumor mast cells form monolayers on various surfaces without assembling specialized adhesion structures at the cell substrate interface. Incubation of RBL-2H3 cells with Ag that cross-link the high affinity IgE receptor, Fc epsilon R1, activates at least two receptor-associated protein tyrosine kinases, Syk and Lyn, and elicits secretion and F-actin assembly, membrane ruffling, and increased spreading and adhesion. Herein, we report that Fc epsilon R1 cross-linking also causes the assembly in cell monolayers of a network of F-actin-rich plaques that form footlike processes at contact sites between the plasma membrane and the underlying substratum. Sheets of F-actin-rich ventral plasma membrane-bearing actin plaques are left on the substrate when monolayers of activated cells are displaced by incubation with ZnCl2 followed by shearing in a stream of buffer; in contrast, most unstimulated cells are completely displaced or leave only fragile membrane fragments. These observations link actin plaque assembly to increased cell substrate adhesion. Actin plaques disassemble rapidly in the presence of monovalent hapten, indicating their dependence on continued Fc epsilon R1 cross-linking. They accumulate antibody to phosphotyrosine and disassemble rapidly in the presence of the protein tyrosine kinase inhibitor, piceatannol, indicating their additional dependence on tyrosine kinase activation. Structures resembling actin plaques form when RBL-2H3 cell monolayers are incubated with the protein tyrosine phosphatase inhibitor, vanadyl hydroperoxide, in the presence of PMA, which increases actin polymerization. It is likely that the tyrosine kinase-dependent assembly of actin plaques plays an important role in linking the activation of signaling receptors to adhesive responses in RBL-2H3 and other immune system cells.
Specificity and class distribution of Fc gamma R-specific autoantibodies in patients with autoimmune disease.
Authors: Boros P , Odin J A , Chen J , Unkeless J C
Published: December 2022

To investigate the prevalence of autoantibodies directed against Fc gamma RII (CD32) and Fc gamma RIII (CD16), 151 serum samples from patients with different autoimmune diseases and 25 samples obtained from healthy individuals were assayed by ELISA on microtiter plates coated with recombinant truncated Fc gamma RII and Fc gamma RIII protein. Class specificity was defined with anti-IgG, anti-IgM, and anti-IgA reagents. High titers of circulating IgM autoantibodies reacting with both Fc gamma RII and Fc gamma RIII were characteristic for SLE and rheumatoid arthritis patients. Sera from patients with Raynaud's syndrome showed predominantly IgG reactivity with Fc gamma RIII. Sera from patients with progressive systemic sclerosis showed both IgG and IgM Fc gamma RII and Fc gamma RIII reactivity. Many patients diagnosed with degenerative osteoarthritis also had IgG autoantibodies, directed primarily against Fc gamma RII with lesser reactivity toward Fc gamma RIII. Further study is needed to correlate these findings to clinical characteristics of the different diseases.
Effects of syngeneic anti-IgE antibodies on the development of IgE memory and on the secondary IgE response.
Authors: Haba S , Nisonoff A
Published: December 2022

The prolonged inhibition of IgE synthesis in mice caused by perinatal inoculation of IgE is attributable, at least in part, to the formation of anti-IgE antibodies. The induction of unresponsiveness with respect to IgE synthesis requires that IgE be administered during a brief interval (2 to approximately 10) days after birth, that corresponds with the time period during which anti-IgE antibodies are induced. Passive administration of syngeneic anti-IgE also inhibits IgE synthesis. We have now investigated the effect of anti-IgE on the induction of memory for IgE production and on secondary IgE responses. Syngeneic anti-IgE antibodies were found to inhibit secondary IgE responses directly during immunization or after adoptive transfer of primed cells. Anti-IgE did not, however, prevent the induction of memory cells for IgE synthesis or cause the loss of memory for IgE synthesis. Inhibition of a secondary IgE response was found to require the presence of anti-IgE and was lost when anti-IgE antibodies were cleared from the mouse. After the transfer of primed cells and secondary challenge anti-IgE was inhibitory only when given during the first 3 to 5 days, after which the primed cells became resistant to inhibition. The failure of anti-IgE to prevent the induction of IgE memory cells is discussed in terms of class switches that occur during the transition from IgM to IgE production.
Immunobiologic and biochemical properties of mutants of toxic shock syndrome toxin-1.
Authors: Murray D L , Prasad G S , Earhart C A , Leonard B A , Kreiswirth B N , Novick R P , Ohlendorf D H , Schlievert P M
Published: December 2022

Toxic shock syndrome (TSS) is a multisystem illness caused mainly by Staphylococcus aureus producing TSS toxin-1 (TSST-1). A variant of TSST-1 has been isolated from ovine mastitis S. aureus. This toxin, TSST-ovine (TSST-O) is only weakly T cell mitogenic, is nonpyrogenic, does not enhance endotoxin shock, and does not cause TSS in the miniosmotic pump model. The sequence of the ovine gene (tstO) differs from the TSST-1 gene (tstH) by 14 nucleotides that change seven amino acids in the mature protein of which two are in the C-terminal half. A gene fusion containing half of both tstH and tstO was made and cloned into S. aureus. The fusion protein contained the two C-terminal amino acid differences that are in TSST-O at residues 132 and 140. The fusion protein was not T cell mitogenic and did not elicit TSS in two rabbit models. Additional experiments used mutagenesis to change the lysine residue at position 132 of TSST-O to glutamate (TSST-OK132E), as exists in TSST-1, and to change the lysine residue of the human-ovine fusion at position 132 to glutamate (TSST-11140T). Both mutants were pyrogenic, enhanced endotoxin shock, and caused TSS in the miniosmotic pump model. However, the proteins were only partially T cell mitogenic. The restoration of lethality of TSST-O and the human-ovine fusion by changing the lysine to glutamate, as exists in TSST-1, indicates that residue 132 is important in lethality. The failure to regenerate complete T cell mitogenicity of the same mutants indicates that residues 132 and 140 are important for that activity.
Serine phosphorylation of a 67-kDa protein in human T lymphocytes represents an accessory receptor-mediated signaling event.
Authors: Henning S W , Meuer S C , Samstag Y
Published: December 2022

Phosphorylation on serine residues of a 67-kDa cytoplasmic protein (p67) occurs as an early signaling event after stimulation of resting human T cells via CD2 but not via TCR/CD3. Phosphorylation of p67 is, however, not restricted to CD2 stimulation because it can also be induced by costimulation through CD4 and CD8 coreceptors when approximated to the TCR-CD3 complex, suggesting a common signaling mechanism for these accessory receptors of the Ig superfamily. Because late functional responses of T cell activation like IL-2 production and DNA synthesis correlate with phosphorylation of p67, this intracellular event may represent an accessory receptor-mediated costimulatory second signal for human T cell activation. The biochemical characteristics (m.w. and isoelectric point) of p67 are identical with those of the actin binding protein L-plastin (Fimbrin), a cytoplasmic protein that contains two Ca2+ binding sites and a calmodulin binding domain. Moreover, p67 reacts specifically with an L-Plastin (Fimbrin) antiserum.
Two processing pathways for the MHC class II-restricted presentation of exogenous influenza virus antigen.
Authors: Pinet V , Malnati M S , Long E O
Published: December 2022

The natural Ag influenza virus A was used to test the requirements for the HLA-DR1-restricted presentation of the epitopes 18-29 in the matrix protein and 307-318 in the hemagglutinin protein. CD4+ cytotoxic T cell clones of similar efficiency were used to detect presentation of these two epitopes. Presentation of the matrix epitope by APC pulsed with either inactivated virus particles or purified soluble protein followed the classical pathway in that 1) it required invariant chain expression, 2) it was blocked by inhibition of protein synthesis, and 3) it was dependent on a function(s) encoded in the MHC class II region. These characteristics suggest that peptides corresponding to the matrix epitope can only load onto newly synthesized class II molecules that were targeted to a processing compartment by the invariant chain. In contrast, presentation of the hemagglutinin epitope processed from virus particles followed a different pathway. First, presentation of hemagglutinin was independent of invariant chain expression. Second, a human B lymphoblastoid cell line in which protein synthesis was inhibited for 9 h was still able to present hemagglutinin even at very low doses of Ag. Third, a DR1-transfected mutant B cell line missing the MHC class II region was able to present hemagglutinin. Thus, mature class II alpha beta molecules can acquire immunogenic peptides derived from intact natural Ags for presentation to CD4+ T cells. This pathway may be useful for the binding of peptides derived from Ags that are rapidly degraded upon uptake into APC.
Second signal activity of IL-12 on the proliferation and IL-2R expression of T helper cell-1 clone.
Authors: Yanagida T , Kato T , Igarashi O , Inoue T , Nariuchi H
Published: December 2022

In these experiments, effects of IL-12 on the proliferation and IL-2R expression of Th1 and Th2 clones were studied. Although neither Th1 nor Th2 clones proliferated on an Ag stimulation with B cell APC, Th1 clones but not Th2 clones, exhibited IL-2-dependent proliferation in the presence of IL-12 in response to the Ag stimulation. The IL-2R alpha-chain was also shown to be induced on Th1 clones when they were stimulated with B cell APC in the presence of IL-12. Effects of IL-12 on these T cell functions were indicated to be exerted in concert with IL-2, although IL-12 did not enhance IL-2 production of Th1 clones. Cytokines produced by Th1 clones such as IFN-gamma and TNF-alpha were indicated not to be involved in induction of the IL-2R alpha-chain expression or proliferation. IL-12 also induced proliferation and IL-2R alpha-chain expression of Th1 clone stimulated with anti-CD3 in the absence of APC, indicating that IL-12 exerted the effect on Th1 cells directly and other costimulator signal from APC is not required for the function of IL-12. In contrast to IL-12, IL-1 induced proliferation and IL-2R alpha-chain expression of Th2 clones stimulated with Ag on B cell APC. The failure of IL-12 in the induction of IL-2R alpha-chain expression on Th2 clone seemed not to be caused by the IL-4 produced by the clone. These results suggest that IL-12 plays an important role in IL-2R alpha-chain expression and proliferation of Th1 clones, but not Th2 clones, as a second signal.
MHC class I and non-MHC-linked capacity for generating an anti-viral CTL response determines susceptibility to CTL exhaustion and establishment of virus persistence in mice.
Authors: Moskophidis D , Lechner F , Hengartner H , Zinkernagel R M
Published: December 2022

The influence of the murine MHC gene complex H-2 class I alleles or of the genetic background and the role of virus variants on establishment of a persistent infection with lymphocytic choriomeningitis virus (LCMV) was analyzed in immunocompetent mice of H-2d haplotype by evaluation of cytotoxic T cell responses. Susceptibility to establishment of a virus carrier state increased in various H-2d mice with lower CTL response and slower CTL kinetics. Low responder BALB/c-H-2dm2, lacking H-2Ld molecules to present the major T cell epitope amino acids 118-126 of the LCMV nucleoprotein or DBA/2 mice possessing relatively few CD8+ T cells, were more susceptible than high responder BALB/c mice expressing H-2Ld. Additional critical factors were LCMV isolate and dose of infection. The rapidly replicating LCMV-DOCILE and Cl 13 Armstrong induced viral persistence readily, whereas the slowly replicating parental virus strains WE or Armstrong did not. The presented findings illustrate a model of MHC-linked or MHC-unlinked susceptibility for virus persistence and may help to explain pathogeneses of chronic virus infections in humans that are often associated with slowly progressing immunopathology.
Regulation of leukemia inhibitory factor synthesis in cultured human astrocytes.
Authors: Aloisi F , Rosa S , Testa U , Bonsi P , Russo G , Peschle C , Levi G
Published: December 2022

We have examined the ability of human astrocytes to synthesize and secrete leukemia inhibitory factor (LIF), which is a multifunctional cytokine that controls cell proliferation and differentiation in many tissues, including the nervous system. Astrocyte-enriched cultures, prepared from 8- to 9-wk-old embryonic brains, expressed LIF mRNA and secreted LIF protein. LIF synthesis was significantly increased by the cytokines IL-1 beta, TNF-alpha, and TGF-beta 1, but not by IFN-gamma, IL-6, or LPS. No major differences in basal and cytokine-inducible LIF production were detected among astrocyte populations obtained from different brain areas. LIF synthesis was lower in serum-free than in serum-containing astrocyte cultures. A role for protein kinase C in the regulation of astrocyte LIF production was suggested by the findings that phorbol esters induced both LIF mRNA and protein and that the cytokine-induced LIF increase was partially antagonized by relatively selective inhibitors of protein kinase C such as H7 and staurosporine. Human leptomeningeal fibroblasts also expressed LIF gene and protein. Astrocytes produced LIF and responded to cytokines with increased LIF synthesis only after being subcultured, and not when grown in primary cultures in close contact with neurons. Our findings suggest that in vivo induction of astrocyte LIF secretion might occur in pathologic conditions as a consequence of both alterations of neuronal-glial interactions and a local increase in the level of inflammatory cytokines.
IL-1 beta amplifies bradykinin-induced prostaglandin E2 production via a phospholipase D-linked mechanism.
Authors: Angel J , Audubert F , Bismuth G , Fournier C
Published: December 2022

The proinflammatory cytokine IL-1 was shown to increase the responsiveness of synovial cells to the potent inflammatory peptide bradykinin (BK). We have investigated the biochemical events linked to this amplifying action of IL-1. Stimulation of synoviocytes with only BK elicited a rapid increase in inositol phosphates and a concomitant accumulation of diacylglycerol (DAG), monoacylglycerol, and free arachidonic acid (AA). In contrast, IL-1 did not stimulate any of these events. Thus, BK can induce AA release via the hydrolysis of phosphatidylinositols by a phospholipase C (PLC). BK also activated a phospholipase D (PLD) to cleave phosphatidylcholine (PC), because it caused an increase in phosphatidic acid (PA) content and a sustained DAG formation, which both were inhibited by ethanol in [3H]myristic acid-labeled cells. Moreover, the addition of ethanol diverted PLD into the formation of phosphatidylethanol (PEt) thus inhibiting the amounts of PA and DAG formed. Priming of synovial cells with rIL-1 beta 24 h before exposure to BK in the presence of ethanol further enhanced the BK-induced formation of PEt. Conversely, preincubation with IL-1 did not influence the BK-induced PLC activation nor did it alter the liberation of AA. Finally, we demonstrated that the IL-1-mediated amplification of PGE2 release in response to BK was reduced by the presence of ethanol in the culture medium, suggesting that part of the synergistic action of IL-1 and BK on prostanoid production was dependent on the activation of the PC-specific PLD pathway.
Recombinant IL-1 receptor antagonist protects against TNF-induced lethality in mice.
Authors: Everaerdt B , Brouckaert P , Fiers W
Published: December 2022

The possible role of induced IL-1 in a number of in vivo actions of TNF was investigated. We were particularly interested to know whether a species-specific induction of IL-1 might explain the important differences observed between murine TNF and human TNF in systems such as lethal shock and the induction of long lasting, high levels of circulating IL-6 in mice. We also studied the possible involvement of IL-1 in the sensitization to TNF observed in tumor-bearing mice or in combination with D(+)-galactosamine or RU38486, particularly because such sensitization results in the loss of species-specific differences between both TNF. Using a specific rIL-1R antagonist (IL-1ra), which inhibits the binding of IL-1 to the IL-1R type I, we were able to protect mice against a lethal murine TNF injection. However, the induction of high levels of circulating IL-6 by murine TNF was not affected, although IL-1ra almost completely blocked the induction of IL-6 by exogenously administered IL-1. Furthermore, the increased susceptibility of tumor-bearing mice to human TNF, relative to control mice, or the sensitization by co-administration of RU38486 or D(+)-galactosamine do not seem to be mediated by IL-1.
HIV-1 Nef activity in murine T cells. CD4 modulation and positive enhancement.
Authors: Rhee S S , Marsh J W
Published: December 2022

Immediately after infection of the targeted cell by HIV-1, proviral gene expression is limited to the three regulatory proteins, Tat, Rev, and Nef, with the nef transcript representing nearly 80% of total expression. Additionally, simian immunodeficiency virus Nef has been shown to be essential for high in vivo titer and the development of immunodeficiency. Recent findings demonstrate that the negative effects of Nef expression, as first defined in transformed T cell lines, are not present when Nef is expressed in primary human T cells or in T cells from transgenic mice, in which one sees moderate positive enhancements of HIV replication and the T cell activation process, respectively. We find that Nef expression in an Ag-specific murine T cell hybridoma results in both the down-modulation of CD4, as seen in primary cells and human T cell lines, and a positive enhancement of the TCR response to stimuli. Examination of a CD4- cell demonstrated that the positive enhancement is independent of CD4 expression or modulation. CD4 down-modulation is shown to be caused by a post-Golgi, acid-dependent process, which dramatically decreases the lifespan of the CD4 molecule. The TCR, Thy Ag, and CD45 remained unchanged in their surface expression. These findings suggest that Nef alters the normal routing and residencies of the CD4 molecule and that the positive effect of Nef on T cell activation is independent of this modulation.
An H2-T MHC class Ib molecule presents Listeria monocytogenes-derived antigen to immune CD8+ cytotoxic T cells.
Authors: Bouwer H G , Lindahl K F , Baldridge J R , Wagner C R , Barry R A , Hinrichs D J
Published: December 2022

Mouse spleen T cells can adoptively transfer immunity to Listeria monocytogenes; this activity was markedly enhanced by stimulation with Con A in vitro before transfer. The enhanced and prolonged protection against L. monocytogenes in vivo was correlated with enhanced lysis in vitro of target cells infected with strains of L. monocytogenes that produce listeriolysin O (LLO). One of the targets of such cytotoxic cells from BALB/c (H2d) mice was a peptide that corresponded to amino acids 91 to 99 (p91-99) of the LLO molecule, which satisfies the binding motif of H2-Kd. Listeria-immune CD3+CD8+, but not CD3+CD8-, cells could also lyse H-2-incompatible, infected target cells. Immune cells from C57BL/6 (H2b) mice lysed allogeneic H-2d target cells infected with L. monocytogenes or a Bacillus subtilis transformant that secretes LLO, but did not lyse targets pulsed with p91-99. This H2-unrestricted cytolysis was therefore directed at a fragment of the LLO molecule other than p91-99. Listeria-infected bone marrow macrophages from congenic and recombinant strains of mice were lysed only when they shared the H2-T region or were Qa1-compatible with the immune cytotoxic cells; sharing of the H2-D, Q, or M region was insufficient. Thus, the immune response to L. monocytogenes included cytolytic CD8+ cells that recognized endogenously processed Listeria-derived Ags in the context of the class Ia H2-K molecule, as well as a class Ib H2-T molecule.
Efficient tumor cell lysis mediated by a bispecific single chain antibody expressed in Escherichia coli.
Authors: Gruber M , Schodin B A , Wilson E R , Kranz D M
Published: December 2022

Recent advances in the expression of Abs in Escherichia coli have raised the possibility that virtually any specificity can be obtained by either cloning Ab genes from characterized hybridomas or by de novo selection using Ab gene libraries. Bispecific Abs have been more difficult to engineer because of problems inherent in the proper folding and association of VH and VL domains. In this report, a model system for expressing and testing the activity of a single chain bispecific Ab was used. The Ab contained the VH and VL genes from the anti-TCR Ab 1B2 joined by a 25 amino acid residue linker to the VH and VL genes from the anti-fluorescein Ab 4420. The 57-kDa single chain bispecific Ab (scFv2) was purified in a single step by affinity chromatography through a fluorescein column at a yield of 1 mg/L of bacterial culture. Despite the presence of 1B2 V regions at the NH2-terminus and a 10-residue c-myc peptide at the COOH-terminus, the refolded protein had an affinity for fluorescein that was nearly identical with the monospecific single chain Ab. The scFv2 also bound the TCR of the mouse CTL clone 2C and redirected the lysis of human tumor cells that had fluorescein covalently linked to their surface. Lysis was mediated at scFv2 concentrations that were 100-fold lower than the concentrations of Ab that inhibited normal recognition by CTL 2C. These results show that single chain bispecific Abs can mediate CTL lysis of target cells without the immunosuppressive side effects associated with the use of anti-TCR Abs.
Regulation of neutrophil responses by phosphotyrosine phosphatase.
Authors: Cui Y , Harvey K , Akard L , Jansen J , Hughes C , Siddiqui R A , English D
Published: December 2022

By using immunofluorescent flow cytometry, we observed a profound up-regulation of CD45 on the plasma membrane of neutrophils exposed to low levels of a culture supernatant of the Gram-negative pathogen, Fusobacterium nucleatum (FN). Plasma membranes of neutrophils freshly prepared form human blood possessed little enzymatically active phosphotyrosine phosphatase. The activity of this enzyme was markedly potentiated in plasma membranes prepared from cells preexposed to the FN culture supernatant. This activity was vanadate sensitive and could be immunoprecipitated with anti-CD45 Ab. Cells preexposed to the FN culture supernatant were inhibited in their ability to release superoxide when challenged with the bacterial chemotactic factor, FMLP, but not PMA. The tyrosine kinase inhibitor, genistein, likewise inhibited FMLP but not PMA-induced superoxide release. Pretreatment of neutrophils with vanadate reversed FN-mediated inhibition of FMLP-triggered superoxide release but had no effect on genistein-mediated inhibition of FMLP-induced superoxide release. Of several proteins tyrosine phosphorylated in response to treatment of neutrophils with FMLP, Western analysis revealed one (m.w. approximately 93,000) that was lost when FMLP-stimulated cells were exposed to FN. This effect was inhibited when the cells were preexposed to vanadate. These results are consistent with the hypothesis that plasma membrane tyrosine phosphatase modulates FMLP-induced superoxide release by reversing the effects of tyrosine kinases activated in the initial phases of cell stimulation.
Transfection of thyroid autoantigens into EBV-transformed B cell lines. Recognition by Graves' disease thyroid T cells.
Authors: Mullins R J , Chernajovsky Y , Dayan C , Londei M , Feldmann M
Published: December 2022

Autologous EBV-transformed B cell lines (EBVL) from a patient with Graves' disease (GD) were transfected with the expression vector pREP4 encoding thyroid peroxidase (TPO), the thyroid-stimulating hormone receptor (TSHR), or chloramphenicol acetyl transferase. The relevant proteins were expressed. The capacity of EBVL to present transfected Ag was assessed by using EBVL transfected with TPO (EBVL-TPO) to stimulate TPO-specific T cells cloned from autologous thyroid tissue; stimulation indices greater than 100 were consistently obtained, even at low APC:T cell ratios. EBVL-TPO and EBVL transfected with TSHR (EBVL-TSHR) were used to characterize the Ag specificity of five thyroid epithelial cell-specific T cell clones. Despite previous unresponsiveness to recombinant TPO or purified thyroglobulin, four of these clones responded vigorously to EBVL-TPO but none responded to EBVL-TSHR. Experiments were then performed to determine whether transfected EBVL were presenting endogenously synthesized TPO, or were taking up and reprocessing exogenous Ag shed from the surface of adjacent cells. The stimulatory capacity of EBVL-TPO was not transferable to untransfected EBVL by cell-free culture supernatant and to only a minor degree by EBVL-TPO-derived cell membrane fragments, indicating that the majority of the TPO presented by the transfected EBVL is endogenously synthesized. The clear and reproducible proliferative responses obtained illustrate the usefulness and potency of this method of Ag presentation to T cells. Because this system obviates the need for large numbers of autologous APC or purified recombinant Ag, it will be useful for obtaining autoantigen-reactive T cells and to help define the autoantigenic epitopes recognized.
Antigen presentation by human dermal microvascular endothelial cells. Immunoregulatory effect of IFN-gamma and IL-10.
Authors: Vora M , Yssel H , de Vries J E , Karasek M A
Published: December 2022

Human dermal microvascular endothelial cells (HDMEC) play a central role in many aspects of the inflammatory and immune reactions in skin. HDMEC display a phenotypic diversity ranging from cells with an epithelioid morphology to those that show both morphologic and biochemical characteristics of macrophages. Here it is shown that HDMEC possess the capability to both process and present Ags. T lymphocyte clones specific for peptide p94-104, which are derived from the protein of group I allergen of Dermatophagoides pteronyssinus, a major house dust mite allergen, and restricted by HLADR11, proliferated specifically to stimulation with the group I allergen of D. pteronyssinus and with peptide p94-104 presented by HDMEC. Preincubation for 48 h with IFN-gamma enhanced the expression of class II MHC Ags on HDMEC, which in turn increased the capacity of HDMEC to present Ag. When HDMEC were primed with Ag in the presence of IL-10, a 75% inhibition of Ag-specific T cell proliferation was observed. IL-10 also inhibited T cell proliferation induced by IFN-gamma-stimulated HDMEC. These findings demonstrate that HDMEC possess the ability to process and present Ag to CD4+ T cells and that these reactions are stimulated by IFN-gamma and inhibited by IL-10. The reduced Ag-presenting capacity of HDMEC mediated by IL-10 is not associated with a down-regulation of class II MHC expression. No significant reduction of HLA-DR expression was detected either at the protein or gene level when HDMEC were incubated with IFN-gamma and IL-10 as compared with incubation with IFN-gamma alone. The profound down-regulatory effect of IL-10 on Ag presentation may provide a new pharmacologic approach to control inflammatory responses in skin.
Molecular cloning and characterization of a murine LPS-inducible cDNA.
Authors: Lee C G , Demarquoy J , Jackson M J , O'Brien W E
Published: December 2022

Murine macrophages respond to endotoxins by inducing a vast array of genes that play a major role in the host's response to infection and tumor growth. We have isolated and characterized a 1.8-kb cDNA, designated IRG2, from a cDNA library prepared from RNA isolated from the murine cell line, RAW 264.7, after bacterial LPS stimulation. The cDNA encodes a protein of 47 kDa that is the murine homologue of a small family of proteins described from IFN-induced human cells. The IRG2 message does not appear until 3 h after LPS exposure and its induction is dependent on new protein synthesis. IRG2 induction by LPS is slightly inhibited by the anti-inflammatory steroid, dexamethasone. Increasing cytosolic cAMP with either forskolin, dibutyryl cAMP, or 8-(4-chlorophenylthio)-cAMP caused marked inhibition of the LPS induction of IRG2. In contrast, activation of PKC with phorbol ester potentiated the LPS response. Removing extracellular Ca2+ with EGTA inhibited IRG2 induction; increasing intracellular calcium with the calcium ionophore A23187 led to enhanced levels of the IRG2 transcript. These data suggest that the induction of IRG2 occurs via a PKC pathway.
Modulation of Fc gamma and complement receptor function by the glycosyl-phosphatidylinositol-anchored form of Fc gamma RIII.
Authors: Edberg J C , Kimberly R P
Published: December 2022

Human neutrophils express two structurally distinct receptors for IgG, the transmembrane Fc gamma RII (CD32) and the glycosyl-phosphatidylinositol-linked Fc gamma RIII (CD16). To explore the respective functional roles of Fc gamma RII and Fc gamma RIII, we have used anti-receptor mAb fragments coupled with erythrocytes to quantify both individual and cooperative receptor functions. With individual receptor engagement, Fc gamma RII (E-IV.3) was much more efficient than Fc gamma RIIIB (E-3G8) in initiating phagocytosis (p < 0.001). However, when identical total numbers of receptors were engaged, co-ligation of Fc gamma RII and Fc gamma RIIIB resulted in a phagocytic response, which was: 1) greater than that for either receptor alone (> twofold Fc gamma RII alone (p < 0.001) and > 20-fold Fc gamma RIIIB alone (p < 0.001)); 2) greater than the sum of Fc gamma RII and Fc gamma RIIIB (p < 0.001); and 3) comparable to the phagocytic potential of Fc gamma RII in FMLP pre-activated neutrophils. This synergistic capacity of Fc gamma RIIIB also enabled CR phagocytosis. Furthermore, the capacity for Fc gamma RIIIB to interact synergistically with Fc gamma RII was preserved in FMLP-preactivated neutrophils. The activation of Fc gamma RII by Fc gamma RIIIB was associated with tyrosine phosphorylation of the Fc gamma RII cytoplasmic domain, which is essential for Fc gamma RII function and which, by analogy to the Ig alpha-chain of the B cell Ag receptor complex, the zeta-chain of CD3, and the gamma-chain of some Fc epsilon Rs and Fc gamma Rs, may enhance the binding and activation of Src or Syk family tyrosine kinases. Thus, Fc gamma RIIIB can affect multiple receptor families and play a role in achieving maximal Fc gamma R capacity, even in stimulated neutrophils in inflammatory sites.
Protective effect of an adenosine kinase inhibitor in septic shock.
Authors: Firestein G S , Boyle D , Bullough D A , Gruber H E , Sajjadi F G , Montag A , Sambol B , Mullane K M
Published: December 2022

Adenosine exhibits potent anti-inflammatory activities but its therapeutic use is limited by cardiovascular side effects. Inhibitors of an enzyme involved in adenosine metabolism, adenosine kinase (EC 2.7.1.20), were evaluated for their ability to enhance endogenous adenosine production. One novel adenosine kinase inhibitor, GP-1-515, was studied in two models of septic shock to assess its protective effects. GP-1-515 significantly decreased mortality in mice that received a lethal i.v. injection of endotoxin. The beneficial effect was accompanied by decreased neutrophil accumulation in the lungs and was reversed by an adenosine receptor antagonist, implying that the effects were mediated by endogenous adenosine. Plasma levels of TNF-alpha, but not IL-1 alpha or IL-6, were lower in the GP-1-515-treated animals. In a second model of sepsis, GP-1-515 increased survival in bacterial peritonitis in rats. The mechanism of action in both models was likely multifactorial, including adenosine-mediated inhibition of neutrophil adhesion, cytokine production, and oxygen radical generation. Adenosine kinase inhibitors have potent anti-inflammatory effects in vitro and in vivo and represent a novel therapeutic approach to the treatment of inflammatory diseases.
Transforming growth factor-beta 1 mediates mast cell chemotaxis.
Authors: Gruber B L , Marchese M J , Kew R R
Published: December 2022

It remains unknown which factor(s) control mast cell recruitment in chronic immune reactions. Although TGF-beta has been shown to function as a potent chemotactic factor for monocytes, fibroblasts, and neutrophils, its effect on mast cells has not been previously determined. In this study, TGF-beta 1 was shown to cause directed migration of cultured mouse mast cells at femtomolar concentrations, with a maximal chemotactic response observed at 25 fM. Moreover, chemotaxis to TGF-beta was also seen using freshly isolated rat peritoneal mast cells. Addition of neutralizing Ab to TGF-beta abrogated its chemotactic activity for both freshly isolated rat peritoneal mast cells and cultured mouse mast cells, whereas an irrelevant species-matched control Ab had no effect. Checkerboard analysis confirmed the mast cell chemotactic activity after exposure to concentration gradients of TGF-beta. Mast cells were observed to undergo rapid and extensive shape changes on exposure to TGF-beta, assuming a polarized morphology in preparation for migration. Other known mast cell chemoattractants including laminin, c-kit ligand, and IL-3 were found to be considerably less potent on a molar basis in inducing directed migration. Affinity cross-linking studies identified TGF-beta binding proteins with M(r) at 70 and 288 kDa, consistent with types I and III TGF-beta receptors on the mast cells. In summary, TGF-beta is the most potent chemoattractant described for mast cells and conceivably relevant, because pathologic processes mediated by TGF-beta are often associated with mast cell accumulation.
Kirsten sarcoma virus-immortalized mast cell lines. Reversible inhibition of growth by dexamethasone and evidence for the presence of an autocrine growth factor.
Authors: Tchekneva E , Serafin W E
Published: December 2022

Expansion of mast cell numbers occurs in vivo during certain inflammatory reactions, including active fibrosis, parasite infestations, and immediate hypersensitivity reactions. T cell-produced cytokines, including IL-3 and IL-4, are thought to control this mast cell proliferation in part, and glucocorticoid regulation of T cell-produced cytokines is thought to account for diminished mast cell proliferation during administration of glucocorticoids in vivo. Here we show that glucocorticoids have a direct inhibitory effect on proliferation of Kirsten sarcoma virus-immortalized mast cells (KiSV-MC) in vitro, with an ID50 of 1.0 +/- 0.2 nM dexamethasone (mean +/- SD, n = 4). At 10 nM dexamethasone, KiSV-MC proliferation was inhibited by 83 +/- 5% (mean +/- SD, n = 4). As determined by trypan blue staining and [3H]TdR incorporation, the glucocorticoid-mediated growth inhibition was due to diminished mast cell proliferation rather than cell death and was completely reversible after 6 days of glucocorticoid treatment. By cell cycle analysis, glucocorticoids diminished the percentage of mast cells in S phase and increased the percentage in G0-G1 phase. Although we show that the KiSV-MC proliferate via an autocrine mechanism, glucocorticoid treatment of the KiSV-MC did not inhibit their production of the autocrine growth factor. During 6 days of treatment with 1 to 1000 nM dexamethasone, mast cell carboxypeptidase activity increased by a maximum of 3.5-fold. In contrast, total chymotryptic and tryptic esterase activities diminished by as much as 40% with dexamethasone treatment. We conclude that glucocorticoids directly affect mast cell growth and differentiation at levels equal to the reported Kd for glucocorticoid receptors on other immune cells.
TNF-beta produced by human T lymphotropic virus type I-infected cells influences the proliferation of human endothelial cells and fibroblasts.
Authors: Yu F , Itoyama Y , Kira J , Fujihara K , Kobayashi T , Kitamoto T , Suzumura A , Yamamoto N , Nakajima Y , Goto I
Published: December 2022

Human T lymphotropic virus type I (HTLV-I) is linked to adult T cell leukemia as well as to HTLV-I-associated myelopathy/tropical spastic paraparesis. In this report, we studied the effects of HTLV-I-infected cell supernatants on HUVEC, fibroblasts, and glioma cells. The HTLV-I-infected cell supernatants (HUT102 and MT-2) strongly inhibited the proliferation of HUVEC, although they enhanced the proliferation of the fibroblasts. Regarding the glioma cells, only the MT-2 supernatant showed weak inhibitory effects on the proliferation. However, the HTLV-I-uninfected cell supernatants showed no effects on these target cells. The biologic activities of both HUT102 and MT-2 supernatants were found to be dose dependent and were reduced by heat treatment at 100 degrees C for 5 min, but not at 56 degrees C for 30 min. These activities were not dependent on the concentrations of HTLV-I viral particles and were only minimally affected by the presence of anti-HTLV-I Abs. A bioassay of various cytokines revealed that the activity of TNF was much higher in the HUT102 and MT-2 supernatants than in the HTLV-I-uninfected cell supernatants (MOLT-4, Jurkat, and K-562). rTNF-alpha and rTNF-beta also showed strong inhibitory effects on HUVEC as well as on the enhancement of the fibroblast growth. With the use of Sephadex G-100 column chromatography, we obtained the highest activities from the 60- through 70-kDa fractions of the HUT102 supernatant and some activities from the 20- through 30-kDa fractions. The biologic activities of both the whole HUT102 supernatant and its active fractions were completely blocked by anti-TNF-beta mAb, although they were not blocked by anti-TNF-alpha mAb. In a Western blot assay, the 25- and 27-kDa bands of TNF-beta were shown clearly in the HUT102 supernatant, although no TNF-alpha bands appeared. These findings suggest that TNF-beta is present in either its oligomeric or monomeric form in the HTLV-I-infected cell supernatants and is also mainly responsible for the supernatants' effects on HUVECs and fibroblasts.