bluuebunny/crossref_metadata_2025 huggingface dataset:
Limited TCR repertoire of infiltrating T cells in the kidneys of Sjögren's syndrome patients with interstitial nephritis.
Authors: Murata H , Kita Y , Sakamoto A , Matsumoto I , Matsumura R , Sugiyama T , Sueishi M , Takabayashi K , Iwamoto I , Saitoh Y , Nishioka K , Sumida T
Published: December 2022

To analyze the mechanism of interstitial nephritis in patients with Sjörgren's syndrome (SS), we examined the TCR repertoire of infiltration T cells in kidney, labial salivary glands, and PBLs using a PCR. The repertoire of the TCR V beta gene on infiltrating T cells from the kidneys of SS patients was more restricted than those on infiltrating T cells in labial salivary glands and PBL. The TCR V beta 2 gene was expressed predominantly in six of seven (86%) SS patients. Junctional sequences of cDNAs encoding the V beta 2 gene on infiltrating T cells in the kidneys of five SS patients showed that some of the cells expanded clonally, indicating Ag-driven stimulation rather than superantigen-induced proliferation. The same V beta 2 clones in the kidney were not detected in labial salivary glands of the same SS patients; the conserved amino acid (arginine at position 96) in the CDR3 region of the V beta 2 gene was found at a frequency of 48.0% in the kidney, whereas it was detected in only 15.4% of the clones in lips. In conclusion, these findings suggest the possibility that T cells that infiltrate the kidneys of SS patients with interstitial nephritis might recognize different autoantigens than those that infiltrate labial salivary glands.
Lack of induction of antibodies specific for conserved, discontinuous epitopes of HIV-1 envelope glycoprotein by candidate AIDS vaccines.
Authors: VanCott T C , Bethke F R , Burke D S , Redfield R R , Birx D L
Published: December 2022

We examined the humoral immune response in both HIV-1 infected and uninfected volunteers immunized with candidate HIV-1 recombinant envelope subunit vaccines (Genentech gp120IIIB, MicroGeneSys gp160IIIB, or ImmunoAG gp160IIIB). Immunization of both HIV-1 infected and uninfected volunteers with these immunogens resulted in the induction of Abs preferentially reactive with epitopes accessible on a denatured form of gp120. While sera from HIV-1 uninfected gp120/gp160IIIB vaccinees bound gp120/gp41, which was expressed on the surface of H9 cells infected with HIV-1IIIB, minimal binding to HIV-1MN or HIV-1RF infected cells was obtained. Induction of qualitatively similar immune responses by these immunogens would not have been predicted based on their different tertiary structures. These data indicate a restriction of the immune response to linear, conserved epitopes poorly accessible on both monomeric gp120 and cell-surface expressed oligomeric gp120/gp41 and a lack of Abs specific for conformational epitopes conserved across divergent HIV-1 strains. Poor recognition of HIV-1 envelope tertiary and quaternary structure may explain the restricted neutralization profiles of vaccinee sera against laboratory-adapted strains of HIV-1 and their inability to neutralize primary HIV-1 isolates. Alternate immunogens or reformulations with the capacity to elicit Abs that preferentially bind to natively folded gp120 should be investigated and correlated with their ability to neutralize more diverse laboratory-adapted and primary HIV-1 isolates.
Structure and expression of a divergent canine class I gene.
Authors: Burnett R C , Geraghty D E
Published: December 2022

We have isolated and characterized a canine class I MHC (dog leukocyte Ag, DLA) gene, DLA-79. The deduced protein sequence shares only 65% identity with a previously published canine class I cDNA, designated DLA-A, and exhibits 64% amino acid identity with the HLA-A, -B, -C consensus. The peptide-binding region of DLA-79 is unusual. Three of four highly conserved tyrosine residues (Tyr7, 59, 159, and 171), proposed to interact with the N terminus of peptide-Ag, are substituted. Additionally, the long alpha-helix lining the peptide-binding region in the alpha 1 domain contains one more amino acid residue than that observed in typical class I. Together, these features suggest that DLA-79 binds a distinct subset of peptides or other ligands. This gene has been expressed in a class I null human lymphoblastoid cell line, and the encoded heavy chain associated with beta 2-microglobulin and was transported to the cell surface. Ribonuclease protection analysis detected low levels of gene-specific mRNA in a broad variety of dog tissues. The highest levels were found in skeletal muscle, a tissue expressing relatively low levels of classical class I Ag. These data suggest that DLA-79 is functional and plays a specialized role in the immune response. Nucleotide sequence analysis of second exon sequences (encoding the alpha 1 domain) identified only two alleles in five dogs of different breeds; a third variant was found in a coyote. The divergent structure, relatively low mRNA expression, and limited polymorphism of this gene suggest that DLA-79 is not a classical or class Ia gene, but rather, an analogue of the MHC class Ib genes of humans and rodents.
Molecular analysis of a heteroclitic T cell response to the immunodominant epitope of sperm whale myoglobin. Implications for peptide partial agonists.
Authors: England R D , Kullberg M C , Cornette J L , Berzofsky J A
Published: December 2022

We have investigated the molecular basis for binding and Ag presentation of an immunodominant Th cell determinant of sperm whale myoglobin, a prototype amphipathic helical structure in the native protein. A series of peptides with three different substitutions at each position were evaluated for binding to the class II MHC molecule I-Ad and for activation of two T cell clones with distinct fine specificity, to determine the role of each residue. The assignment of MHC binding and TCR binding residues is consistent with a peptide bound as a twisted beta-strand, with 130 degrees twist similar to that of the influenza hemagglutinin peptide crystallized in the groove of HLA-DR1. This twist gives the peptide amphipathicity, with a periodicity similar to an alpha-helix without its being a helix. Two substituted peptides were discovered to be heteroclitic, but by different molecular mechanisms, one involving gain of a favorable residue and one involving loss of an unfavorable one. Complexes of both peptides with I-Ad had enormously higher affinity for the TCR, but peptide affinity for the MHC molecule was not increased, such that the wild-type peptide acted as a partial agonist and inhibited the response to the heteroclitic ones. Moreover, the magnitude of response was elevated in a way that could not be mimicked by the wild-type peptide even at higher concentration. These results suggest a TCR dwell time requirement for optimal signal transduction that may help explain the mechanism of partial agonism.
IL-1 beta is required for IL-12 to induce production of IFN-gamma by NK cells. A role for IL-1 beta in the T cell-independent mechanism of resistance against intracellular pathogens.
Authors: Hunter C A , Chizzonite R , Remington J S
Published: December 2022

Mice with the severe combined immunodeficiency (SCID) possess an IFN-gamma-dependent mechanism of resistance to the intracellular pathogens Toxoplasma gondii and Listeria monocytogenes that is dependent on IL-12-induced production of IFN-gamma by NK cells. In this report we demonstrate that IL-1 beta is required for IL-12 to stimulate production of IFN-gamma by NK cells, and that IL-1 is important in IL-12-mediated resistance to T. gondii in vivo. Stimulation of SCID mouse splenocytes with tachyzoites of T. gondii resulted in production of IFN-gamma. Addition of neutralizing Ab specific for IL-1 beta to these cultures inhibited completely the production of IFN-gamma. Similar results were obtained when LPS or L. monocytogenes were used to stimulate production of IFN-gamma by SCID mouse splenocytes. Addition of a neutralizing Ab to IL-1 alpha did not affect production of IFN-gamma by SCID mouse splenocytes stimulated with T. gondii, L. monocytogenes, or LPS. Stimulation of SCID mouse splenocytes with IL-1 beta or IL-1 alpha did not result in production of IFN-gamma but enhanced remarkably the ability of T. gondii or IL-12 to stimulate production of IFN-gamma. Furthermore, production of IFN-gamma by SCID mouse splenocytes stimulated with IL-12 plus TNF-alpha was completely ablated by anti-IL-1 beta, but not by anti-IL-1 alpha. Analysis of the culture supernatants of spleen cells from SCID mice stimulated with T. gondii or IL-12 plus TNF-alpha detected low levels of IL-1 beta; addition of a neutralizing Ab to IFN-gamma resulted in a 5- to 10-fold increase in levels of IL-1 beta. Furthermore, stimulation of SCID mouse splenocytes with IL-12, in the presence of anti-IFN-gamma, resulted in an increase in detectable levels of IL-1 beta. To determine the in vivo relevance of our in vitro data, SCID mice were infected with T. gondii and treated with IL-12 alone or IL-12 in combination with an Ab specific for the type I IL-1 receptor. This Ab reduced production of IFN-gamma by SCID mouse splenocytes stimulated with either T. gondii, LPS, L. monocytogenes, or IL-12 plus IL-1 beta. In vivo administration of this Ab antagonized significantly the ability of exogenous IL-12 to delay the time to death of SCID mice infected with T. gondii.(ABSTRACT TRUNCATED AT 400 WORDS)
Inhibition of macrophage inflammatory protein-1 alpha expression by IL-10. Differential sensitivities in human blood monocytes and alveolar macrophages.
Authors: Berkman N , John M , Roesems G , Jose P J , Barnes P J , Chung K F
Published: December 2022

IL-10 is a pleiotropic cytokine produced by monocytes and T cells that has potent inhibitory effects on monocyte/macrophage function. Because monocytes and macrophages are capable of releasing the C-C chemokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), which is a potent chemoattractant for activated T cells, we studied the effects of IL-10 on the expression of MIP-1 alpha in these cells. Low levels of MIP-1 alpha were detectable in resting monocytes and macrophages. Both LPS (1 micrograms/ml) and IL-1 beta (10 ng/ml) induced the expression of MIP-1 alpha mRNA and the release of MIP-1 alpha protein from these cells. The addition of exogenous human rIL-10 inhibited induced MIP-1 alpha mRNA expression as well as the release of MIP-1 alpha protein measured after 24 h. This inhibition was significantly higher in monocytes compared with alveolar macrophages. In monocytes, IL-10-induced inhibition of MIP-1 alpha was only partially accounted for by alterations in mRNA stability and was dependent on de novo protein synthesis. In the presence of an anti-human IL-10-neutralizing Ab, the release of MIP-1 alpha induced by LPS and IL-1 beta was further enhanced in monocytes but unchanged in alveolar macrophages. MIP-1 alpha mRNA was also increased in monocytes. There was no detectable release of IL-10 from alveolar macrophages after LPS or IL-1 beta in contrast to modest amounts released from monocytes. Thus, IL-10 is an inhibitor of the induced transcription of MIP-1 alpha mRNA and of the release of MIP-1 alpha protein, with a greater effect on monocytes as compared with alveolar macrophages. IL-10 may indirectly regulate effects on cells such as activated T lymphocytes partly through the inhibition of MIP-1 alpha expression from monocytes and macrophages.
Analysis of the role of MHC class II presentation in the stimulation of cytotoxic T lymphocytes by antigens targeted into the exogenous antigen-MHC class I presentation pathway.
Authors: Rock K L , Clark K
Published: December 2022

By conjugation of proteins to beads, Ags can be selectively targeted into the MHC class I pathway of phagocytes in vivo and can stimulate CTL responses. Because phagocytes also present particulate Ag on MHC class II molecules, we examined whether these Ags stimulated concomitant CD4 T cell immunity. Although the priming of CD4 T cells with soluble OVA required adjuvants, particulate Ag was stimulatory when injected in saline. We next examined whether CD4 T cell responses played a role in the generation of CTL to particulate Ag. At low concentrations of Ag, OVA primed CTLs in wild-type mice but not in MHC class II-deficient animals, indicating that MHC class II presentation of Ag was essential for CTL generation. These data both support a model where CD4 T cells collaborate with CTLs as part of a three-cell interaction and identify a phagocyte as the third cell in this reaction. Interestingly, injection of higher concentrations of the same Ag primed equivalent CTL responses in both wild-type and MHC class II-deficient mice. These results indicate that a key variable in determining whether CTL generation is helper cell dependent or independent is the dose of immunogen. This may explain in part why CTL responses to abundant Ags, such as viruses, tend to be helper independent, while responses to less abundant Ags, such as minor histocompatibility Ags, require T helper cells. In addition, these results also point to the potential of using particulate Ags to prime or boost responses in settings with CD4 immunodeficiency.
Rejection of MHC class II-transfected tumor cells requires induction of tumor-encoded B7-1 and/or B7-2 costimulatory molecules.
Authors: Baskar S , Clements V K , Glimcher L H , Nabavi N , Ostrand-Rosenberg S
Published: December 2022

Many tumor cells that have been transfected with genes encoding B7 costimulatory molecules become effective cellular vaccines against wild-type tumor. The improved immunity is dependent on newly induced tumor-specific CD8+ and/or CD4+ T cells and presumably occurs because the B7 transfectants provide the requisite second signal for activation of T cells in conjunction with tumor cell-presented MHC class I/tumor peptide and/or MHC class II/tumor peptide complexes, respectively. Since B7 expression is such a potent enhancer of tumor immunity, and yet some tumors are immunogenic in the absence of B7 transfection, we have used class I+ class-II-transfected tumors to investigate whether costimulatory molecules are also involved in rejection of immunogenic, non-B7-transfected tumor. Blocking studies with B7 mAbs demonstrate that induction of tumor immunity in naive mice requires B7-1 and/or B7-2 expression, while experiments with tumor-primed mice indicate that once antitumor immunity is established, expression of B7 is not necessary. Flow cytometry analyses demonstrate that costimulatory molecules are expressed by the tumor cells via an in vivo induction process. Experiments with class II genes with truncated cytoplasmic tails indicate that the cytoplasmic region of the tumor-expressed class II heterodimer is involved in induction of B7. We therefore conclude that for this class I+ class II-transfected tumor, generation of tumor immunity requires induction of tumor cell-encoded B7 molecules that are mediated by the cytoplasmic region of the transfected class II heterodimer.
Stem cell factor (c-kit ligand) influences eosinophil recruitment and histamine levels in allergic airway inflammation.
Authors: Lukacs N W , Strieter R M , Lincoln P M , Brownell E , Pullen D M , Schock H J , Chensue S W , Taub D D , Kunkel S L
Published: December 2022

The increased reactivity of mast cells during allergic airway inflammation has been linked to several aspects of pulmonary disease. A primary inducer of mast cell differentiation, proliferation, and activation has been identified as c-kit ligand or stem cell factor (SCF). In the present study, we used an established murine model of allergic eosinophilic airway inflammation to examine the role of SCF during an Ag-specific airway response. Initial data demonstrates increased SCF protein production at 8 h postchallenge in both lungs and serum of allergen-challenged, but not vehicle-challenged, mice. The immunolocalization of SCF in Ag-challenged lungs suggested that macrophage populations were the primary source of SCF, while epithelial cell regions also stained positive. Intense immunohistochemical staining of macrophages in bronchoalveolar lavage samples recovered from Ag-sensitized mice indicate that these cells may be a significant source of SCF in the lungs. Alveolar macrophages from the airways of normal mice stimulated with either TNF (0.1-10 ng/ml) or IL-4 (10 ng/ml) produced significant levels of SCF. Furthermore, neutralization studies demonstrated that the inhibition of airway SCF during allergen challenge significantly decreased eosinophil, but not neutrophil, infiltration throughout the response. Furthermore, when mice were treated with anti-SCF Ab, histamine levels were significantly reduced at 8 h postchallenge, the time of significant SCF production. Together, these data indicate that the production of SCF during Ag-induced lung inflammation by alveolar macrophages can play a significant role in the subsequent recruitment of eosinophils, possibly via mast cell activation and degranulation.
Binding of HIV-1 virions or gp120-anti-gp120 immune complexes to HIV-1-infected quiescent peripheral blood mononuclear cells reveals latent infection.
Authors: Briant L , Coudronnière N , Robert-Hebmann V , Benkirane M , Devaux C
Published: December 2022

HIV-1-infected quiescent CD4+ cells harbor the virus in an inactive state until subsequent activation. The possibility that HIV-1 itself and the virus envelope glycoprotein 120 (gp120) might be important agents of this activation was investigated. The present data indicate that binding of heat-inactivated HIV-1 (iHIV-1) to infected resting PBMCs was sufficient to activate NF-kappa B and AP-1, to induce transition from the G0/G1 stage of the cell cycle to the S/G2/M stage, to induce cell surface expression of CD25, to stimulate provirus integration, and to commit cells to produce virus. The cumulative amount of HIV-1 produced by iHIV-1-stimulated cells strictly depended on the concentration of p24gag in the virion preparations used for stimulation. Moreover, virus production was not evidenced in infected resting cells exposed to iHIV-1 previously incubated with soluble CD4 (sCD4), indicating that activation requires a contact between HIV-1 envelope glycoproteins and cell surface CD4. Although soluble gp120 did not stimulate virus production, we found that transition to the S/G2/M stage of the cell cycle, cell surface expression of activation Ags, and virus production were stimulated by cross-linking of CD4 by gp120-anti-gp120 immune complexes. Finally, incubation of gp120-anti-gp120 immune complexes with sCD4 inhibited these effects. These findings suggest that virions and gp120 anti-gp120 immune complexes found in infected patients at all times of infection can stimulate virus production in CD4+ cells harboring HIV-1 in an inducible state.
Immunization with short peptides from the sequence of the systemic lupus erythematosus-associated 60-kDa Ro autoantigen results in anti-Ro ribonucleoprotein autoimmunity.
Authors: Scofield R H , Henry W E , Kurien B T , James J A , Harley J B
Published: December 2022

Systemic lupus erythematosus is characterized by the production of Abs to ribonucleoproteins, including Ro (or SS-A) and La (or SS-B). The mechanism by which these autoantibodies arise is unknown. We have immunized rabbits with peptides from the sequence of the 60-kDa Ro. Animals so immunized not only produce Abs to the immunogen peptide, but also develop an autoimmune response to the entire 60-kDa Ro Ag. In about 20% of these animals, autoimmunity also spreads to the La Ag. Anti-native DNA was present in one of nine rabbits. All animals had anti-nuclear Ab, but even among those immunized with the same peptides, different patterns of immunofluorescence were found. Thus, among these outbred animals immunized with peptides from 60-kDa Ro, distinct serologic manifestations were present, showing heterogeneity in the development of autoimmunity.
Inability to produce IL-6 is a functional feature of human germinal center B lymphocytes.
Authors: Burdin N , Galibert L , Garrone P , Durand I , Banchereau J , Rousset F
Published: December 2022

In response to Ag encounter, B lymphocytes undergo a complex maturation process yielding phenotypically distinct subpopulations that are located in highly organized compartments of secondary lymphoid organs. This study describes the patterns of cytokine secretion of naive, memory, and germinal center (GC) human tonsillar B lymphocytes, activated either through CD40 or B cell receptor or with Staphylococcus aureus Cowan I particles. The three B cell subpopulations produced comparable levels of IL-10 and TNF-alpha, regardless of the stimulation pathway. Interestingly, activated GC B lymphocytes fail to express IL-6, as determined both at mRNA and at protein levels, whereas both naive and memory B cells can be induced to secrete IL-6. Likewise, naive B lymphocytes undergoing dual ligation of CD40 and B cell receptor fail to express IL-6, since they acquire a GC-like phenotype. IL-6 receptors are up-regulated on both ex vivo-purified GC B lymphocytes and in vitro generated GC-like B cells, following CD40 activation. Consistent with this, addition of exogenous IL-6 sustains growth of CD40-stimulated GC B lymphocytes. Taken together, these results demonstrate that loss of IL-6 secretion is a functional characteristic of human GC B lymphocytes. The swap from an autocrine to a paracrine IL-6 response may permit a better control of B cell growth and differentiation during the germinal center reaction.
Distinct effects of recombinant cholera toxin B subunit and holotoxin on different stages of class II MHC antigen processing and presentation by macrophages.
Authors: Matousek M P , Nedrud J G , Harding C V
Published: December 2022

Cholera toxin (CT) is a potent mucosal adjuvant with enhancing effects on Ag presentation, although the mechanisms of its adjuvanticity remain poorly understood. Using an in vitro Ag presentation assay, we found CT and recombinant B subunit (rCTB) to have distinct effects on different stages of processing and class II MHC (MHC-II)-restricted presentation of hen egg lysozyme (HEL). CT treatment of macrophages resulted in enhanced presentation of soluble HEL(48-61) peptide to3A9 hybridoma cells. However, CT had inhibitory effects on intracellular processing of soluble native Ag. Thus, CT inhibited presentation when added prior to HEL, whereas presentation was enhanced when CT was added after HEL exposure and the generation of peptide-MHC-II complexes. Pretreatment of macrophages with CT also markedly inhibited phagocytic processing of a Crl-HEL fusion protein (containing the HEL(48-61) epitope) expressed in intact bacteria (Escherichia coli HB101.Crl-HEL or Salmonella typhimurium 14028s.Crl-HEL), whereas addition of CT to macrophages after a 2-h incubation with the bacteria again enhanced presentation. CT produced little effect on overall uptake and catabolism of radiolabeled HEL or HB101.Crl-HEL. In contrast to the holotoxin, purified rCTB subunit did not inhibit intracellular processing of soluble or bacterial Ag, although it similarly enhanced the presentation of surface HEL-(48-61)-I-Ak complexes to 3A9 cells. These data suggest that the inhibitory effects of CT on Ag processing are mediated by the A subunit.
Regulation of CTLA-4 expression during T cell activation.
Authors: Perkins D , Wang Z , Donovan C , He H , Mark D , Guan G , Wang Y , Walunas T , Bluestone J , Listman J , Finn P W
Published: December 2022

T cell activation requires at least two distinct signals, including signaling via the Ag-specific TCR and a costimulatory pathway. The best characterized costimulatory pathway involves the CD28 molecule, which is expressed constitutively on T cells and binds the family of B7 counter-receptors on APCs. Inhibition of this costimulatory pathway prevents T cell activation and can lead to long-term T cell unresponsiveness or anergy. In contrast, CTLA4, which is homologous to CD28, has been shown to be a negative regulator of T cell activation. The CTLA4 molecule is not expressed on resting T cells, but is induced after the initial steps of T cell activation. To address the regulation of CTLA4 expression, we have analyzed CTLA4 at the level of cell surface expression, mRNA, rate of transcription, and rate of decay of message. Nuclear runoff results show an increase in the rate of transcription following T cell activation. Our analyses of non-T cells, including B cells, mastocytoma, and fibroblasts, by Northern blot analysis detect only T cell expression of CTLA4. Reporter gene analysis indicates that 335 bp of upstream CTLA4 sequence are sufficient to control inducibility. We have identified important regulatory regions that control inducible and cell-specific CTLA4 expression. These results also suggest that both positive and negative response elements modulate the transcriptional regulation of CTLA4 gene expression. Understanding the regulation of CTLA4 should provide insight into the regulation of T cell activation at the molecular level.
TAP-independent selection of CD8+ intestinal intraepithelial lymphocytes.
Authors: Sydora B C , Brossay L , Hagenbaugh A , Kronenberg M , Cheroutre H
Published: December 2022

Intestinal intraepithelial lymphocytes (IEL) are mostly CD8 single positive T cells. IEL with a TCR-alpha(beta) that are CD8 single positive are absent from beta(2)-microglobulin (beta(2)m)-deficient mice, consistent with the idea that these IEL, like other TCR-alpha(beta)+, CD8+ T cells, require class I molecules for positive selection. In contrast, here we show that substantial numbers of TCR-alpha(beta)+, CD8 single positive IEL are present in mice deficient for the transporter associated with Ag processing 1 (TAP 1) gene, although T cells with this phenotype are absent from thymus, spleen, and lymph nodes of these same mice. The majority of TCR-alpha(beta)+, CD8 single positive IEL in TAP-deficient mice expresses CD8 molecules composed of alpha(alpha) homodimers and they express a diverse set of V(beta) gene segments. In addition, the number of TCR-alpha(beta)+, CD4/CD8 double positive IEL is decreased in beta(2)m-deficient mice but not in TAP-deficient mice. The dependence of the two TCR-alpha(beta)+ IEL populations that express CD8alpha(alpha) homodimers on beta(2)m as opposed to TAP molecules is striking. It suggests that TAP-independent but beta(2)m-requiring nonclassical class I molecules expressed by cells in the intestine, such as the thymus leukemia Ag and CD1, could play a pivotal role in the development and/or the accumulation of major subpopulations of TCR-alpha(beta)+ IEL.
Stimulation of Fc(alpha) receptors induces tyrosine phosphorylation of phospholipase C-gamma(1), phosphatidylinositol phosphate hydrolysis, and Ca2+ mobilization in rat and human mesangial cells.
Authors: Gómez-Guerrero C , Duque N , Egido J
Published: December 2022

Although knowledge of IgA Fc receptor (Fc(alpha)R) structure and gene organization has progressed in the past few years, signal transduction pathways elicited by its activation have hardly been studied. Previously, we have demonstrated that mesangial cells (MC) possess Fc(alpha)R stimulation triggers several biologic responses. In this work, we studied the early biochemical signals triggered by Fc(alpha)R stimulation in MC. MC incubation with aggregated IgA (AIgA) elicited a dose-dependent increase in cytosolic Ca2+ ([Ca2+]i). The response was rapid and transient, and slowly fell to the original baseline. Ca2+ mobilization was dependent on the Fc region of the IgA, because Fc, but neither Fab fragment nor carbohydrates, inhibited the [Ca2+] rise. The initial induction of [Ca2+]i rise was due to Ca2+ mobilization from inositol trisphosphate (IP3)-sensitive intracellular stores, while sustained levels were maintained through extracellular Ca2+ influx. Stimulation of Fc(alpha)R also resulted in production of IP3, temporally correlated with Ca2+ mobilization. Protein tyrosine kinase inhibitors abolished [Ca2+]i rise, indicating that tyrosine phosphorylation of some substrates is required for Ca2+ mobilization. Stimulation through Fc(alpha)R gave rise to a marked increase in tyrosine phosphorylation of several proteins, including the 147-kDa band, similar in size to phospholipase C-gamma(1) (PLC-gamma(1)). Tyrosine phosphorylation of PLC-gamma(1) reached a maximum 30 s after stimulation, as determined by immunoprecipitation and Western blot. Collectively, these results indicate that the Fc(alpha)R signaling pathway in MC involves PLC-(gamma(1) activation, IP3 formation, and Ca2+ mobilization, and is linked to activation of tyrosine kinases.
Role of IL-12 in peripheral blood mononuclear cell responses to fungi in persons with and without HIV infection.
Authors: Harrison T S , Levitz S M
Published: December 2022

Clinical trials of IL-12 in persons infected with HIV have been proposed based on recent evidence suggesting IL-12 plays a critical role in the development of protective immune responses, and the HIV infection is associated with a deficiency of IL-12. As fungal infections are among the most common opportunistic infections associated with AIDS, we examined whether IL-12 p40 gene expression and p70 release in response to Cryptococcus neoformans and Candida albicans were deficient in monocyte-enriched PBMC from HIV-seropositive donors and whether rIL-12 could augment the proliferation of PBMC from HIV-seropositive donors in response to these fungi and to Pneumocystis carinii. PBMC from HIV-seronegative donors expressed IL-12 p40 mRNA in response to C. neoformans, C. albicans, and the positive control Staphylococcus aureus Cowan strain 1 (SAC), although the induction of IL-12 p40 mRNA was later and more prolonged with C. neoformans as the stimulus. Expression of IL-12 p40 mRNA in response to the three stimuli was similar in cells from HIV-seropositive and HIV-seronegative donors. However, when stimulated with SAC, cells from HIV-seropositive donors released significantly less IL-12, suggesting HIV infection induces a post-transcriptional defect in IL-12 release in response to SAC. While PBMC from HIV-seropositive donors had impaired proliferative responses to the three fungi tested, addition of rIL-12 did not enhance proliferation. These studies do not lend further support for the therapeutic use of IL-12 to prevent or treat fungal infections in persons infected with HIV.
Structural requirements for CD28-mediated costimulation of IL-2 production in Jurkat T cells.
Authors: Truitt K E , Nagel T , Suen L F , Imboden J B
Published: December 2022

Although under certain conditions an association with phosphatidylinositol 3'-kinase (PI3-K) appears to be critical for CD28 signaling, mutation of the PI3-K binding site (Tyr 170) does not alter the costimulatory ability of murine CD28 (mCD28) in Jurkat T cells. To define the structural requirements for this PI3-K-independent signaling, we expressed a series of mCD28 mutants in Jurkat. Mutation to Phe of all four cytoplasmic Tyr residues together (ALL F mutant) greatly reduced the ability of mCD28 to augment IL-2 production. Isolated re-constitution of Tyr 188, but not 170, 185, or 197, restored the ability of ALL F mCD28 to deliver a costimulus. Thus, a signal based upon Tyr 188 can deliver a costimulus for the enhancement of IL-2 production by Jurkat cells.
Phosphatidylinositol 3-kinase activation in normal human B lymphocytes.
Authors: Aagaard-Tillery K M , Jelinek D F
Published: December 2022

A variety of signals, mediated via either the B cell Ag receptor (BCR), or non-BCR molecules such as CD40 or cytokine receptors, have been shown to be crucial for the regulation of B cell survival, growth, and differentiation. Although it is clear that a variety of signaling pathways can be activated in B cells in a stimulus-dependent manner, it remains unknown whether differential activation of these signaling pathways is the underlying mechanism controlling B cell fate, i.e., growth vs differentiation. Initial studies reported here indicated that stimulation of highly purified peripheral blood B cells with the polyclonal B cell activators Staphylococcus aureus and CD40 ligand (CD40L) resulted in the rapid induction of phosphatidylinositol 3-kinase (PI 3-kinase) activity. Moreover, pretreatment of B cells with wortmannin, a specific inhibitor of PI 3-kinase, resulted in a complete block in induction of PI 3-kinase activity. The effects of wortmannin, as well as a second PI 3-kinase inhibitor, LY294002, on the induction of both B cell growth and differentiation were therefore investigated. Although these PI 3-kinase inhibitors variably inhibited B cell DNA synthesis in a stimulus-dependent manner, both drugs effected a near-complete block of the ability of each of these stimuli to induce Ig production. Furthermore, separation of B cells into naive IgD+ and postswitch IgD- B cells failed to reveal differential sensitivity of these populations to wortmannin. These results suggest that activation of PI 3-kinase, or other wortmannin- and LY294002-sensitive targets, is a crucial event that occurs during the differentiation of normal human B lymphocytes. The differential sensitivity of B cell responses to inhibitors of PI 3-kinase supports the notion that distinct signal transduction pathways are involved in differentiation vs proliferation of normal human B lymphocytes.
Immature stage B cells enter but do not progress beyond the early G1 phase of the cell cycle in response to antigen receptor signaling.
Authors: Carman J A , Wechsler-Reya R J , Monroe J G
Published: December 2022

In contrast to mature B cells, immature stage B cells do not proliferate following Ag receptor cross-linking with anti-Ig Abs. To determine where in the cell cycle immature B cells arrest, we have examined the expression of specific G, cell cycle regulators. Following surface IgM (sIgM) cross-linking on mature B cells, we observed increased expression of the early G1 kinase, cyclin-dependent kinase 4 (cdk4), and one of its regulatory subunits, cyclin D2. Mature B cells also showed increased expression of components required for G1/S transition, including cyclin E and cdk2. Whereas immature stage B cells increased expression of cyclin D2 and cdk4 after anti-IgM stimulation, unlike mature stage B cells they failed to express cyclin E and cdk2. Expression of cyclin D2 and cdk4 indicates that these cells can exit G0 and enter the initial G1 phase following sIgM ligation. Interestingly, IL-4, which by itself does not stimulate proliferation of immature B cells, induced expression of cyclin E and cdk2. These latter results suggest that IL-4 complements sIgM, signaling for proliferation by increasing the basal levels of late G1 cell cycle regulators. Consistent with this idea, IL-4 synergizes with anti-Ig Abs to promote cell cycle progression and proliferation of immature B cells. Finally, c-myc, a transcriptional regulator of some members of the cell cycle machinery, is not induced following sIgM cross-linking of immature cells. This lack of inducible expression contrasts with that seen in mature stage B cells, and in immature stage cells stimulated to proliferate with LPS. These results suggest that c-myc may be a component of the signaling pathway that induces cyclin E and cdk2 expression.
Is there a role for nitric oxide in regulation of T cell secretion of IL-2?
Authors: Marcinkiewicz J , Grabowska A , Chain B M
Published: December 2022

Nitric oxide (NO) can have both effector (cytotoxic) and regulatory roles in immune function. In this study, we have re-examined the potential role of nitric oxide in mediating the macrophage-dependent suppression of IL-2 synthesis. In our model, TNP-specific CD4+ T cells are cocultured with Ag and either peritoneal or alveolar macrophages. Both populations of macrophages after in vitro stimulation with IFN-gamma can inhibit IL-2 release. In vitro stimulation also induces substantial levels of NO release by these macrophages, as well as high levels of prostaglandin E2 (PGE2). However, there was no correlation between NO levels and inhibitory activity. Furthermore, NG-monomethyl-L-arginine monoacetate, a specific inhibitor of NO release had no effect on IL-2 release, while indomethacin, which blocked prostaglandin synthesis, largely abrogated the suppressor activity of both macrophage populations. Although the addition of exogenous NO donors at high concentrations could inhibit IL-2 release by T cells, our data does not support the hypothesis that NO is a major macrophage mediator of suppression in this model.
An N-terminal domain shared by Fas/Apo-1 (CD95) soluble variants prevents cell death in vitro.
Authors: Papoff G , Cascino I , Eramo A , Starace G , Lynch D H , Ruberti G
Published: December 2022

Fas/Apo-1 molecule, also designated as CD95, is a member of the TNF receptor family. Fas cross-linking by its natural ligand or by agonistic mAbs results in rapid induction of apoptosis in susceptible cells. in addition to the Fas full-length mRNA, human activated PBMC and tumor cell lines express several mRNA Fas variants that derive from alternative splicing of the primary transcript. All five variants identified, two of which are newly described here, code for soluble proteins that, with the exception of FasTMDel, are truncated in the extracytoplasmic region and possess short C-terminal amino acid sequences corresponding to a different reading frame. We have identified Abs that recognize all splicing variants and established a sandwich ELISA by which the soluble Fas molecules could be detected in culture supernatants of transfected cell lines and in PBMC following T cell activation. Next, we have studied in detail the functional role of these variants by apoptosis inhibition studies. We found that all soluble proteins block the apoptosis induced by either an agonistic Ab or, more importantly, by the natural Fas ligand in Fas-positive sensitive cell lines. interestingly, this functional property can be assigned to the first 49 amino acids of the mature protein that is the only region shared by the five soluble Fas molecules.
Possible involvement of C5/C5a in the efferent and elicitation phases of contact sensitivity.
Authors: Tsuji R F , Kikuchi M , Askenase P W
Published: December 2022

The elicitation of 24-h contact sensitivity (CS) in mice requires a serotonin-dependent, 2-h response called CS initiation. We studied the role of complement (C) by comparing CS in DBA/1 (C5-normal) vs DBA/2 (C5-deficient) mice and found impaired 2-h, but not 24-h, CS. We showed previously that 2-h responses represent CS initiation and are required for elicitation of 24-h responses. Treatment of C5-deficient mice with absent macroscopic responses (ear swelling) with a selective serotonin antagonist inhibited 24-h CS, suggesting that C5-deficient mice had submacroscopic (i.e., microscopic), serotonin-dependent CS initiation. When normal mouse serum was used as a source of C5 to reconstitute C5-deficient mice, significant 2-h responses were restored. Furthermore, heat treatment of normal mouse serum to inactivate C abrogated restoration of 2-h responses. Thus, C5 was suggested to be involved in CS initiation. Using a suboptimal immunizing dose of Ag revealed an impaired 24-h component of CS in DBA/2 mice, but not in DBA/1 mice, and also in C5-deficient B10.D2/o mice compared with C5-normal B10.D2/n mice with a suboptimal eliciting dose of Ag. Again, reconstitution of B10.D2/o mice with normal mouse serum restored deficient 24-h CS responses. Thus, 2-h and classical 24-h CS probably depend in part on C5. These results imply that C5 may play a role in the elicitation of 24-h CS, probably via required preceding CS initiation.
C-reactive protein binds to a novel ligand on Leishmania donovani and increases uptake into human macrophages.
Authors: Culley F J , Harris R A , Kaye P M , McAdam K P , Raynes J G
Published: December 2022

C-reactive protein (CRP) is a major acute phase protein of man, with serum concentrations increasing dramatically following stimulation of hepatocytes by inflammatory cytokines. However, the role of CRP in inflammation and resistance to infection is still poorly understood. Here, the specificity of CRP binding to the surface of Leishmania donovani, an obligate intracellular parasite of mononuclear phagocytes, is described. CRP is shown to bind to promastigotes at the infectious metacyclic stage of development, at concentrations found in normal human serum. The presence of CRP on the surface of promastigotes substantially increases uptake into human monocyte-derived macrophages. Unusually, CRP does not bind via its characteristic ligand, phosphorylcholine. We show that CRP binds to the lipophosphoglycan (LPG) component of the promastigote cell surface, a molecule implicated in both uptake and survival of these parasites within the macrophage, and also to the major secreted protein of promastigotes, secreted acid phosphatase. Using mAb to LPG with known ligand specificities, we define a novel ligand for CRP as the repeating phosphorylated disaccharide units that form the backbone of LPG.
Cytokine control of parasite-specific anergy in human urinary schistosomiasis. IL-10 modulates lymphocyte reactivity.
Authors: King C L , Medhat A , Malhotra I , Nafeh M , Helmy A , Khaudary J , Ibrahim S , El-Sherbiny M , Zaky S , Stupi R J , Brustoski K , Shehata M , Shata M T
Published: December 2022

Humans chronically infected with schistosomiasis usually have impaired parasite Ag-specific lymphocyte proliferation and IFN-gamma production that may facilitate persistence of the parasite while producing little clinical disease. The mechanisms that contribute to the immunologic hyporesponsiveness in these patients remain undefined. IL-10 has been shown to exert an inhibitory effect on cell-mediated immunity. To determine whether endogenous IL-10 has a role in regulating parasite-specific anergy in schistosomiasis, neutralizing anti-IL-10 added to PBMC from Schistosoma haematobium patients' enhanced adult worm (SWAP)- or egg Ag (SEA)-driven lymphocyte proliferation and/or IFN-gamma production by 2- to >100-fold in 32 of 38 subjects. In contrast, anti-IL-10 failed to significantly augment the mycobacterial Ag, purified protein derivative (PPD)-driven lymphocyte proliferation, or IFN-gamma production in 9 or 10 of 14 individuals, respectively. SWAP or SEA triggered IL-10 release from PBMC of both patients and healthy individuals; however, CD4+ cells were a significant source of IL-10 only in infected subjects. PPD relative to SWAP induced fivefold less IL-10 release by CD4+ cells (p < 0.01). A possible mechanism whereby IL-10 suppressed Ag-specific T cell responses was demonstrated by the ability of SWAP and not PPD to suppress B7 expression on PBMC. Anti-IL-10 completely inhibited the parasite Ag-induced down-regulation of B7 expression. These studies indicate that IL-10 contributes to parasite Ag-induced T cell hyporesponsiveness observed in patients with chronic schistosomiasis hematobia.
Relation of oxidative stress and glutathione synthesis to CD95(Fas/APO-1)-mediated apoptosis of adult T cell leukemia cells.
Authors: Kohno T , Yamada Y , Hata T , Mori H , Yamamura M , Tomonaga M , Urata Y , Goto S , Kondo T
Published: December 2022

An IL-2 dependent adult T cell leukemia cell line (SO4) has been established that is sensitive to CD95-mediated apoptosis as well as a subline (R-SO4) that is resistant. Incubating SO4 cells with anti-CD95 IgM mAb caused concentration-dependent cell death. On the contrary, R-SO4 cells did not die even at 1000 ng/ml of anti-CD95 IgM mAb. The levels of CD95 expression on R-SO4 cells were one-third of those on SO4 cells. However a blocking Ab, anti-CD95 IgG mAb, did not induce complete resistance of SO4 cells to anti-CD95 IgM mAb as R-SO4 cells. As CD95 and TNF receptor are similar, and TNF/TNF receptor binding induces oxygen radicals, the involvement of oxidant and antioxidant systems in CD95-mediated apoptosis has been examined. The addition of anti-CD95 IgM mAb resulted in formation of intracellular oxygen radical species in the SO4 cells as measured using 2',5',-dichlorofluorescein as substrate. The oxygen radical production induced DNA damage as determined by formation of 8-hydroxydeoxyguanosine. No increase in the formation of oxygen radicals was observed in R-SO4 cells. Concentrations of the intracellular antioxidant, glutathione, and the key enzyme for its synthesis, gamma-glutamylcysteine synthetase, were 150% increased in R-SO4 cells in comparison with that of SO4 cells. Moreover, glutathione ester decreased the formation of 8-hydroxydeoxyguanosine. These results suggested that apoptosis mediated by CD95 in ATL cells is related to the production of oxygen radical species and cellular antioxidant systems, especially, glutathione synthesis.
Helicobacter-specific cell-mediated immune responses display a predominant Th1 phenotype and promote a delayed-type hypersensitivity response in the stomachs of mice.
Authors: Mohammadi M , Czinn S , Redline R , Nedrud J
Published: December 2022

Studies regarding the nature of cell-mediated immunity in Helicobacter pylori infection and its role in pathogenesis have yielded controversial results. To address this issue in a controlled manner, we have employed the well-characterized Helicobacter felis-mouse model. Immunized/challenged and nonimmunized/infected mice were evaluated for cellular proliferation, gastric inflammation, and cytokine and Ab production at various times after infection. We observed two types of cell-mediated immune responses depending on the nature of the Ag preparation. The first response is a Helicobacter-independent response, present in all experimental groups, which is directed toward Ags such as urease and heat shock proteins. The second is a Helicobacter-dependent cellular response restricted to mice previously exposed to Helicobacter Ags either by immunization or infection. This response was not seen in noninfected controls. The Helicobacter-dependent cellular response had a Th1 phenotype, as either infected or immunized/challenged mice demonstrated local and systemic production of IFN-gamma and undetectable levels of IL-4 or IL-5. Cellular proliferation correlated with the severity of gastric inflammation in both immunized/challenged (protected) and nonimmunized/infected mice. Finally, in vivo neutralization of IFN-gamma resulted in a significant reduction of gastric inflammation in H. felis-infected, as well as immunized/challenged, mice. This treatment also revealed the presence of Th2 cells, restricted to immunized/challenged mice, as demonstrated by local and systemic production of IL-4 in these mice. These data demonstrate that Helicobacter infection and/or immunization stimulate a predominantly Th1-type, Ag-specific response and promote a local delayed-type hypersensitivity response in the stomach that may be inhibited by depletion of IFN-gamma.