bluuebunny/crossref_metadata_2025 huggingface dataset:
Coordinate transcriptional control of murine endogenous retrovirus and Ig genes during B cell differentiation.
Authors: Hummell D S , Dooley J S , Khan A S , Lawton A R
Published: December 2022

Proliferation and differentiation of B lymphocytes are usually concurrent but independently regulated events. Anti-mu treatment of murine B lymphocytes stimulated with LPS provides a model system in which proliferation and differentiation may be independently studied. This treatment causes enhanced proliferation but with coordinate suppression of transcription of a family of unrelated genes including those for Ig heavy and light chains, J chain, and endogenous murine leukemia virus (MuLV) sequences. We show that in comparison to B lymphocytes stimulated with LPS alone cells stimulated with a combination of anti-mu and LPS exhibit relatively increased amounts of a nuclear binding factor(s), NF mu E1, which interacts with the B (mu E1) site of the IgH enhancer; binding is strongly inhibited by a synthetic probe of the B sequence. A negative regulatory sequence contained within the upstream conserved region (UCR) of the MuLV long terminal repeat (LTR) is identical to the complement of mu E1 in eight of nine bases and inhibits binding of NF mu E1 to the IgH enhancer probe. The mu E1 site is also present 3' to the kappa-light chain gene; binding of this sequence to a repressor protein may coordinately suppress the transcription of mu, kappa, and MuLV genes. Others have reported that the cDNA encoding NF mu E1, also known as mu EBP-B, CF-1, and YY-1, predicts a protein with structural features consistent with variable function as either a transcriptional activator or repressor.
CD3 zeta/eta/theta locus is colinear with and transcribed antisense to the gene encoding the transcription factor Oct-1.
Authors: Lerner A , D'Adamio L , Diener A C , Clayton L K , Reinherz E L
Published: December 2022

CD3 zeta and eta are signal-transducing components of the TCR and are derived from alternative splicing of transcripts from a single genetic locus that also encodes CD30 theta. We have isolated two murine cDNA clones that appear to result from antisense transcription through CD3 theta-specific exon 10 and CD3 eta-specific exon 9. The sequence of these clones shows no open reading frame. Northern analysis with single stranded probes confirms the existence of a ubiquitously expressed > 12-kb polyadenylated mRNA antisense to CD3 eta. A "genomic walk," which extended 32 kb distal to murine CD3 eta exon 9, provided genomic DNA containing a more 5' portion of the antisense transcript. This probe identified two murine thymic cDNA with 91% sequence homology to the human transcription factor Oct-1. Five exons of murine Oct-1 map in an antisense orientation to the CD3 zeta/eta/theta locus on the cloned genomic sequences. The murine Oct-1 cDNA and exon 9 of CD3 eta hybridize to the same > 12-kb mRNA. Similarly, human Oct-1 and previously characterized human genomic sequences homologous to murine CD3 eta exon 9 each hybridize to the same > 15-kb human mRNA. Thus, the CD3 zeta/eta/theta and Oct-1 gene loci are partially overlapping and transcribed in opposite directions. The potential functional implications of these findings are discussed.
HLA DR4w4-binding motifs illustrate the biochemical basis of degeneracy and specificity in peptide-DR interactions.
Authors: Sette A , Sidney J , Oseroff C , del Guercio M F , Southwood S , Arrhenius T , Powell M F , Colón S M , Gaeta F C , Grey H M
Published: December 2022

In the present study, we describe the definition of a DRB1*0401 (DR4w4)-specific motif. The strategy used entailed a three-step process: 1) screening a large set of unrelated peptide ligands to identify good MHC binders; 2) truncation analysis of several DR4w4 binding peptides of high affinity to identify the crucial core-binding regions; 3) the use of single amino acid substitutions of the DR4w4-binding peptide hemagglutinin (HA) 307-319 to elucidate the specific residues crucial for binding. The DR4w4 motif is characterized by the presence of a hydrophobic or aromatic (F, W, Y, L, I, V, M) anchor residue in position 1, and a second hydroxyl (S, T) or aliphatic (L, I, V, or M) anchor residue in position 6. Furthermore, positive charges (R, K) are not allowed in positions 4, 7, and 9, and negative charges (D, E) are not allowed in position 9. This motif was present in 92% of good (IC50 < or = 100 nM) DR4w4-binding peptides, but less than 25% of the negative (IC50 > 45 microM) binders, indicating that the presence of the motif is necessary, but not sufficient for good DR4w4 binding capacity. The results of the present study are discussed in relation to previous work defining binding motifs and rules for other DR alleles, illustrating how different DR alleles bind variations on a similar structural theme. Finally, using two different peptide ligands [tetanus toxoid 830-843 and HA 307-319] as model systems, it is demonstrated how the fine allelic specificity of the DR binders can be predictably modulated by introducing subtle changes in the primary amino acid sequence.
Neuropeptides induce rapid expression of endothelial cell adhesion molecules and elicit granulocytic infiltration in human skin.
Authors: Smith C H , Barker J N , Morris R W , MacDonald D M , Lee T H
Published: December 2022

Inflammatory cell infiltrates and cell adhesion molecule expression have been examined in normal human skin after intradermal injection of sensory neuropeptides substance P (n = 6), vasoactive intestinal polypeptide (n = 6), and calcitonin gene-related peptide (n = 6) together with PBS as control (n = 4). Each neuropeptide induced rapid, time-dependent neutrophil influx into dermis, which was initially observed at 15 min and persisted for 8 h after injection. Increases in numbers of neutrophils with time after substance P, vasoactive intestinal polypeptide and calcitonin gene-related peptide were highly significant when compared with controls p < 0.005, p < 0.005, p < 0.005, respectively (analysis of variance). Substance P additionally induced marked eosinophilic accumulation at 4 and 8 h in four of six subjects. These changes paralleled rapid translocation of P-selectin from cytoplasmic Weibel-Palade granules to luminal membranes by 15 min, and significant up-regulation of E-selectin expression at 4 and 8 h. Increases in percentage of E-selectin positive vessels with respect to time after each neuropeptide were highly significant when compared with controls, p < 0.005, p < 0.005, p < 0.005 (ANOVA), respectively, and were significantly correlated with neutrophil infiltrates, r = 0.55, p < 0.001. VCAM-1 was not expressed, and constitutive ICAM-1 expression on dermal endothelium was unchanged at all time points examined (0-8 h). Induction of endothelial adhesion molecule expression by neuropeptides provides a mechanism for neutrophil accumulation in neurogenic inflammation. Substance P-induced eosinophil accumulation in the absence of VCAM-1 expression suggests that mechanisms distinct from VCAM-1/very late antigen-4 binding mediate selective tissue eosinophilia.
Induction of circulating and erythrocyte-bound IL-8 by IL-2 immunotherapy and suppression of its in vitro production by IL-1 receptor antagonist and soluble tumor necrosis factor receptor (p75) chimera.
Authors: Tilg H , Shapiro L , Atkins M B , Dinarello C A , Mier J W
Published: December 2022

The objective of this study was 1) to investigate the in vivo production of IL-8 in patients undergoing IL-2 immunotherapy and 2) to study the influence of IL-1Ra, soluble TNF receptor p75 (TNFsRp75), and a TNFsRp75-Fc fusion protein on IL-2-induced IL-8 production in vitro. Circulating IL-8 was assessed both in plasma and erythrocyte lysates prepared from patients undergoing IL-2 immunotherapy. IL-8 was detectable in the plasma within 2-4 h after the first IL-2 infusion, reached a peak level after 4 h, and declined rapidly to undetectable within 8 h. Erythrocyte-bound IL-8 was also detected within 4 h of the first IL-2 dose, but levels were higher than those measured in plasma and remained elevated long after the plasma levels had become undetectable. On day 4 of therapy, the increases in both plasma and the erythrocyte-lysate IL-8 levels induced by an IL-2 injection were less pronounced than on day 1. Although IL-1Ra and TNFsRp75-Fc individually had only a modest suppressive effect on IL-2-induced IL-8 production by PBMC in vitro, the combination of IL-1Ra and TNFsRp75-Fc markedly down-regulated IL-2-induced IL-8 synthesis and steady-state mRNA levels. TNFsRp75 had no effect on IL-2-induced IL-8 synthesis. Our studies suggest that the transient detection of IL-8 in plasma early in the course of IL-2 treatment is due to erythrocyte sequestration and that suppressed synthesis, due in part to high levels of circulating IL-1 and TNF antagonists, may play a role later in the course of treatment.
Ligand specificity of purified complement receptor type three (CD11b/CD18, alpha m beta 2, Mac-1). Indirect effects of an Arg-Gly-Asp (RGD) sequence.
Authors: Van Strijp J A , Russell D G , Tuomanen E , Brown E J , Wright S D
Published: December 2022

We have purified CR3 to homogeneity by affinity chromatography on C3bi-Sepharose and elution with EDTA. C3bi-coated erythrocytes bound to this purified CR3, and binding was dependent on the concentration of both C3bi and CR3, as well as on temperature and the presence of divalent cations. Moreover, binding could be blocked by mAb against CR3 or C3bi and could be enhanced by the addition of integrin modulating factor-1. We used the purified CR3 to test whether several putative ligands of CR3 directly bound the receptor. The interaction of purified CR3 with fibrinogen, filamentous hemagglutinin of Bordetella pertussis, lipophosphoglycan and glycoprotein 63 of Leishmania mexicana, and lipopolysaccharide from Escherichia coli was confirmed. However the interaction of CR3 with zymosan or its major component, beta-glucan, was not observed in these assays. Previous studies showed that binding of C3bi to PMN could be blocked with Arg-Gly-Asp (RGD) containing peptides and were interpreted to indicate that the RGD sequence in C3bi interacts directly with CR3. We found, however, that RGD containing peptides were unable to block the interaction of C3bi with purified CR3, yet retained the ability to block binding of C3bi to cells. We conclude that RGD-peptides do not directly bind CR3, but instead indirectly effect CR3 function. Inasmuch as the effect of RGD-peptides could be mimicked with antibodies against leukocyte response integrin, we suggest that RGD-peptides may bind to leukocyte response integrin on polymorphonuclear leukocytes and influence CR3 activity via this receptor.
Regulation of hapten-specific memory IgE responses induced in vitro. Requirement for both Thy-1+ AsGM1+ and Thy-1+ AsGM1- cells and for IFN-alpha and IL-4.
Authors: Herrick C A , Carucci J A , Kleiner G I , Durkin H G
Published: December 2022

The roles of Thy-1+ and AsGM1+ spleen cells and cytokines (IL-4, IL-5, IL-6, IFN-alpha, and IFN-gamma) in regulation of hapten-specific memory IgE antibody-forming cell (AFC) responses induced in vitro were examined. BALB/c mice, injected i.p. with benzylpenicilloyl-keyhole limpet hemocyanin (BPO-KLH) (10 micrograms) on days 0 and 21, were killed on day 60 or day 120. Numbers of BPO-specific IgGI, IgE, and IgA AFC in spleen were determined by enzyme-linked immunosorbent spot assay after 0 to 6 days of culture +/- BPO-KLH. BPO-specific AFC of all isotypes were detected in spleen on day 60, but not on day 120. Day 60 AFC responses did not persist in culture in that no AFC were detected by day 2 of culture +/- BPO-KLH. When either day 60 or day 120 cells were cultured for 3 days with BPO-KLH, BPO-specific AFC responses were induced, and peaked on day 5, with similar numbers of AFC of each isotype induced with day 60 and day 120 cells. On day 60, spleen contained two subsets of Thy-1+ cells: AsGM1- (approximately 32% of total cells) and AsGM1+ (approximately 4%). Depletion and reconstitution experiments established that both subsets were required for induction of BPO-specific IgE AFC responses. Cytokines could not substitute for the Thy-1(+)-depleted cells. However, when unfractionated day 60 cells were cultured with cytokines or anti-cytokine antibodies, BPO-specific IgE AFC responses induced were both IFN-alpha and IL-4 dependent; either increased or decreased by IFN-gamma, depending on its concentration, and unaffected by IL-5 or IL-6.
Combination IL-2 and IL-4 reduces glucocorticoid receptor-binding affinity and T cell response to glucocorticoids.
Authors: Kam J C , Szefler S J , Surs W , Sher E R , Leung D Y
Published: December 2022

The mechanisms contributing to persistent T cell activation and poor response to glucocorticoids in chronic inflammatory illnesses such as steroid resistant (SR) asthma are poorly defined. We examined the possibility that certain cytokines, specifically IL-2 and IL-4, could affect T cell response to glucocorticoids. A [3H]dexamethasone radioligand-binding assay was used to measure the number of glucocorticoid receptors (GR) and dissociation constant (Kd) in PBMC from normal donors and patients with SR asthma, cultured in the absence and presence of these cytokines. PBMC from normal donors incubated for 48 h in the presence of combination IL-2 + IL-4 had nuclear GR with significantly reduced binding affinity (GR Kd = 36.1 +/- 1.63 nM, mean +/- SEM; p = 0.0001) as compared with PBMC incubated with medium alone (GR Kd = 6.74 +/- 0.46 nM). The cytosolic GR Kd remained unchanged. However, when PBMC were incubated with IL-2 alone or IL-4 alone, no change in GR-binding affinity was observed. Furthermore, when T cells and non-T cells were individually stimulated with combination IL-2 + IL-4, a significant reduction in GR-binding affinity was observed only in the T cell population (p = 0.0001). The IL-2 + IL-4-induced alteration in PBMC GR Kd was associated with an increase in GR number (8348 +/- 964 vs 1710 +/- 228 sites/cell; p = 0.0003). More importantly, the alteration in PBMC GR-binding affinity with IL-2 + IL-4 was associated with a functional change in T cell response to methylprednisolone MPN, i.e., a reduced inhibitory effect of MPN on PMA/ionomycin-induced T cell proliferation. These effects of IL-2 + IL-4 on PBMC GR affinity and response to MPN were blocked by co-incubation with IFN-gamma. Freshly isolated PBMC from four patients with SR asthma had a significantly reduced GR-binding affinity (Kd = 40.0 +/- 2.68 nM; p = 0.0001) when compared with seven normal subjects (7.15 +/- 0.41 nM). The altered PBMC GR binding from patients with SR asthma reversed to normal when incubated with medium alone, but was sustained with IL-2 + IL-4. These observations suggest that with persistent inflammation certain cytokines may contribute to an impaired response to glucocorticoids. Furthermore, the effects of IL-2 and IL-4 were blocked by IFN-gamma.
Murine T helper cell-2 lymphocytes express type I and type II IL-1 receptors, but only the type I receptor mediates costimulatory activity.
Authors: McKean D J , Podzorski R P , Bell M P , Nilson A E , Huntoon C J , Slack J , Dower S K , Sims J
Published: December 2022

The role of IL-1 in augmenting the Ag receptor-initiated activation program was evaluated in IL-4-producing (Th2) CD4+ murine T lymphocytes. Northern blot and 125I-labeled IL-1 alpha cross-linking analyses demonstrated that Th2 lymphocytes express both type I and type II IL-1R. The expression of both IL-1R isoforms on the surface of the Th2 cells is coordinately up-regulated in response to anti-CD3 cross-linking in the absence of detectable accessory cells. Analyses of the kinetics of IL-1R acquisition demonstrated that the peak level of type I and type II IL-1R mRNA expression occurs after the peak expression of mRNA encoding IL-2R alpha and IL-4, which are two IL-1-responsive events in the Th2 activation program. Type I IL-1R ligand-binding antagonists, IL-1R antagonist and anti-type I mAb, were used to evaluate the functional significance of Th2 cell expression of two IL-1R isoforms. The addition of either IL-1R antagonist or anti-type I mAb completely inhibited the IL-1 alpha-augmented component of the proliferative response stimulated by anti-CD3 plus exogenous IL-1 alpha. Together, these studies indicate that, although Th2 clones express inducible levels of both type I and type II IL-1R isoforms, the IL-1-induced intracellular signals involved in augmenting an anti-CD3-stimulated proliferative response are mediated solely through the type I IL-1R.
Studies of the adjuvant-independent antibody response to immunotargeting. Target structure dependence, isotype distribution, and induction of long term memory.
Authors: Skea D L , Barber B H
Published: December 2022

Immunoconjugates composed of avidin linked to biotinylated antibodies specific for different surface determinants on cells of the immune system were evaluated for their ability to induce adjuvant-independent anti-avidin IgG responses in mice. Previously, we demonstrated that allele-specific murine anti-class II MHC-avidin immunoconjugates were immunogenic in mice bearing the appropriate haplotype. Herein we report the immunotargeting potential of heterologous anti-class II MHC antibodies specific for framework determinants, and extend the range of effective targets to include certain non-MHC structures present on APC (e.g., 33D1 on dendritic cells, 14.8 on B cells, and CD45--the leukocyte common antigen). However, antibodies with other specificities (e.g., leukocyte integrins and some macrophage markers) were not effective targeting vehicles. Surprisingly, immunoconjugates specific for CD3 and CD4 were immunogenic. The isotype distribution of the anti-avidin antibody response induced in mice by immunotargeting to class II MHC or 33D1 was similar to that induced by immunization with Ag emulsified in complete Freund's adjuvant. Most of the antibody induced was IgG1 (65-75%), but a significant proportion was IgG2a (20-30%). We also demonstrate that immunotargeting is able to prime for long-term immunologic memory in mice.
V-region-mediated binding of human Ig by protein A.
Authors: Ibrahim S , Seppälä I , Mäkelä O
Published: December 2022

The Fab-mediated "alternative" binding of Ig by staphylococcal protein A is a marker of a set of VH genes (a subset of family VH3 in man). We typed 115 monoclonal human Ig as alternative binders or nonbinders. The proportion of binders varied depending on the isotype, 35% in IgM but only 11-13% in IgA1 and IgG3. It was 28% among lambda-chain-bearing but 16% among kappa-bearing monoclonal Ig. Independent estimates of the proportions bound were obtained by studying polyclonal Ig of 10 healthy adults. The proportions bound were close to those observed in the study of monoclonal Ig (the means were IgM 32%, IgA1 13%, IgA2 24%, IgG3 14%). A higher proportion of infant than adult Ig was bound by protein A. Also, the proportion was less isotype-dependent in infants than in adults. At the age of 4 mo, 47% of IgM was bound (mean of 10 children), the values of other isotypes were: IgA1 35%, IgA2 39%, and IgG3 38%. At the age of 14 mo the proportion of alternative binders had decreased but was still far from adult values. We propose that ontogenically early ("virgin") B cells, besides being rich in IgM and lambda-chain producers, are rich in producers of alternative binders. A subsequent selection reduces the proportion of these B cells so that in ontogenically most developed B cell populations, e.g., those producing IgA1 kappa, such cells make up only about 10% of the total.
Characterization of the insulin A-chain major immunogenic determinant presented by MHC class II I-Ad molecules.
Authors: Williams D B , Ferguson J , Gariepy J , McKay D , Teng Y T , Iwasaki S , Hozumi N
Published: December 2022

Data are presented which demonstrate the minimal insulin peptide required to activate a large group of insulin-specific T hybrids following presentation by either live or fixed APC, is the N-terminal insulin-A(1-13) peptide. Functional activation and competition assays using both live and fixed APC with 19 synthesized variants of the N-terminal bovine insulin A-chain molecule permitted classification of peptide residues into MHC agretope and T cell epitope regions. Our findings indicate insulin A-chain peptide occupies the Ag binding groove of class II MHC in an extended conformation as a result of intracellular reduction of A-loop disulfide bonds. Insulin A-chain Cys7 and Cys11 residues represent two independent T cell epitopes N- and C-terminal to the A-loop region. Data are presented that demonstrate the unique residues associated with several insulin isoform molecules contribute to the peptide agretope region. Our findings may suggest peptide agretopes may subtly modify the peptide/MHC conformation presented to TCR.
Cytotoxic T cells from ovarian malignant tumors can recognize polymorphic epithelial mucin core peptides.
Authors: Ioannides C G , Fisk B , Jerome K R , Irimura T , Wharton J T , Finn O J
Published: December 2022

CTL isolated from tumor infiltrating lymphocytes/tumor associated lymphocytes (TAL) infiltrating ovarian tumors have been demonstrated to mediate lysis of tumor targets after in vitro culture. These effector cells are of particular interest as cellular probes to detect and define human tumor Ag and epitopes that stimulate cellular immunity to human tumors. Polymorphic epithelial mucin (PEM) core peptides are potential candidates as tumor specific Ag because of their preferential expression on epithelial tumors. We report here that ovarian CTL-TAL can recognize mucin (Muc-1) core peptide of PEM. Several ovarian CTL-TAL lines were developed that recognized in a non-MHC restricted fashion an Muc-1+ ovarian tumor, but not Muc-1-tumor. To define the specificity of these CTL-TAL and exclude cross-reactivity with other potential Ag, cytotoxicity experiments were performed using as targets EBV-transformed cell lines with an expression construct containing the Muc.1 cDNA. These ovarian CTL-TAL lysed mucin core-peptide transfected cells but not targets transfected with an expression construct containing a mucin frame-shift mutant cDNA as control. In addition, targets pulsed with short synthetic peptides composed of amino acids 1-14 of the Muc 1 core peptide repeat were also lysed by the same CTL-TAL. This lysis was inhibited by the mAb SM3 that recognize an epitope on the mucin core peptide. These results, which are a demonstration of a specific Ag recognized by ovarian CTL-TAL, may be of interest for specific immunotherapy of ovarian cancer.
Antigen-reactive gamma delta T cells in human leishmaniasis.
Authors: Russo D M , Armitage R J , Barral-Netto M , Barral A , Grabstein K H , Reed S G
Published: December 2022

The importance of Ag-specific gamma delta T lymphocytes in human immune responses to pathogenic organisms is unknown. In the present study the expression of gamma delta TCR on T lymphocytes from patients with cutaneous, mucosal, or visceral leishmaniasis was examined. All of these patient groups had elevated levels of gamma delta T cells in peripheral blood. Patients' gamma delta T cells included CD8+ as well as null cells. The percentage of T cells expressing gamma delta TCR was increased significantly by stimulation in vitro with certain parasite Ag. T-cell lines generated by stimulation with promastigote lysates of Leishmania amazonensis or L. braziliensis typically contained 25 to 60% gamma delta T cells. In contrast, two immunodominant surface Ag of L. amazonensis, gp63 and gp42, did not expand gamma delta T cells from infected patients, although both Ag elicited strong alpha beta T-cell responses. gamma delta T cells isolated from a Leishmania-specific T-cell line responded to stimulation with promastigote lysate. Of particular interest, gamma delta T cells from PBMC of a patient with mucosal leishmaniasis responded to stimulation with a recombinant 70 kDa heat shock protein of L. chagasi. This study demonstrated that several clinical forms of leishmaniasis induced elevated numbers of gamma delta T cells that responded specifically to Leishmania Ag in vitro. Therefore, this component of the T-cell response to Leishmania may impact the outcome of clinical disease.
Inhibition of DNA repair with aphidicolin enhances sensitivity of targets to tumor necrosis factor.
Authors: Gera J F , Fady C , Gardner A , Jacoby F J , Briskin K B , Lichtenstein A
Published: December 2022

To test whether DNA injury contributes to TNF-induced cytotoxicity, we attempted to enhance DNA injury by inhibiting its repair and then assessing effects on cytotoxicity. DNA repair, assayed as unscheduled DNA synthesis, was first detected in TNF-sensitive targets by 2-3 h of incubation with TNF. Targets resistant to TNF cytotoxicity did not demonstrate significant repair replication. Repair preceded the detection of TNF-induced DNA injury, which was subsequently demonstrated by a double-stranded DNA fragmentation assay, sedimentation of DNA in neutral and alkaline sucrose gradients, and gel electrophoresis of extracted DNA. This suggested that early during exposure to TNF, DNA repair proceeds more rapidly than strand breakage. To inhibit repair, nontoxic concentrations of aphidicolin (inhibitor of DNA polymerase-alpha) and dideoxythymidine (inhibitor of DNA polymerase-beta and gamma) were used. Aphidicolin inhibited repair and consistently sensitized to TNF cytotoxicity, decreasing the ID50 for TNF at least 10- to 50-fold. In contrast, dideoxythymidine had no effect on repair or cytotoxicity. Deoxycytidine, which competitively inhibits binding of aphidicolin to DNA polymerase, blocked the sensitization in a concentration-dependent fashion. In targets sensitized with aphidicolin, TNF-induced strand breakage was accelerated, being detected by 4 h of culture in the sucrose gradient assay. Sensitization to TNF was not due to a heightened activation of poly (ADP-ribose) polymerase. These results indicate that TNF-induced strand breakage participates in TNF-induced cytotoxicity and that the level of DNA repair plays a role in determining relative sensitivity of targets.
Modulation of rat mast cell reactivity by IL-1 beta. Divergent effects on nitric oxide and platelet-activating factor release.
Authors: Hogaboam C M , Befus A D , Wallace J L
Published: December 2022

Release of inflammatory mediators by mast cells can be modulated by certain cytokines and by nitric oxide. An in vitro platelet aggregation bioassay was used to assess the effects of interleukin-1 beta (IL-1 beta) on the release of platelet-activating factor and nitric oxide from resting or ionophore-activated peritoneal mast cells (PMC) from rat. PMC spontaneously released a substance that inhibits thrombin-stimulated platelet aggregation. The activity of this substance is abolished by addition of hemoglobin to the platelet suspension and augmented by preincubation of the PMC with L-arginine, suggesting that it is nitric oxide. Within minutes, IL-1 beta concentration-dependently (1 pg/ml-100 ng/ml) enhanced the release from activated PMC of nitric oxide, as measured by its ability to inhibit thrombin-induced platelet aggregation, and as confirmed with a biochemical assay for nitrite. This action of IL-1 beta was inhibited by pretreatment of PMC with a calmodulin antagonist (calmidazolium), an IL-1 receptor antagonist, or either of two nitric oxide synthase inhibitors (L-NAME and LY-83583). IL-1 beta also inhibited the release of platelet-activating factor from PMC through a nitric-oxide-dependent mechanism. These results demonstrate that IL-1 beta is a potent and rapid-acting modulator of mast cell reactivity, stimulating nitric oxide release while inhibiting the production of platelet-activating factor.
Co-clustering of beta 1 integrins, cytoskeletal proteins, and tyrosine-phosphorylated substrates during integrin-mediated leukocyte aggregation.
Authors: Sánchez-Mateos P , Campanero M R , Balboa M A , Sánchez-Madrid F
Published: December 2022

The involvement of the VLA-4 integrin in an alternative leukocyte homotypic adhesion mechanism that is LFA-1/ICAM-1 independent, has been previously reported. We describe here the localization of beta 1 and alpha 4 integrin subunits at sites of cell-cell contact, on both beta 1- and alpha 4-induced aggregates of B lymphoblastoid Ramos cells. Moreover, the distribution of different VLA-alpha subunits was also examined on beta 1-induced cell aggregates of alpha 2- and alpha 4-transfected K-562 cells. Both alpha 2 and alpha 4 integrin subunits were mainly localized at sites of intercellular boundaries, suggesting a possible role for these integrins in leukocyte intercellular adhesion. The fibronectin receptor alpha 5 subunit was either diffuse throughout the plasma membrane, or displayed some accumulation at sites of cell-cell contact. Even though homotypic aggregation of U-937 cells was induced with the anti-alpha 5 P1D6 mAb, the alpha 5 subunit showed only partial redistribution to regions of cell-cell contact, compared with the complete redistribution of the alpha 4 subunit in the alpha 4-induced aggregates. The reorganization of the actin-cytoskeleton was observed at sites of intercellular boundaries in both the anti-beta 1- and anti-alpha 4-induced cell aggregates. Hence, F-actin and the cytoskeletal protein talin co-localized with beta 1 and alpha 4 integrin clusters at sites of cell-cell contact. Signal transduction during VLA-mediated homotypic cell adhesion has also been investigated. We found co-localization of beta 1 and alpha 4 subunits with tyrosine-phosphorylated proteins at cell-cell contact regions during cell aggregation. These data indicate that VLA integrin-mediated leukocyte aggregation results in clustering of beta 1-integrins at sites of cell-cell contact, together with co-localization of cytoskeletal proteins. These results also suggest that protein tyrosine phosphorylation is an important signal transduction mechanism when VLA integrins participate in intercellular contacts.
Splenic uptake of immune complexes in man is complement-dependent.
Authors: Davies K A , Erlendsson K , Beynon H L , Peters A M , Steinsson K , Valdimarsson H , Walport M J
Published: December 2022

We have examined the effects of hereditary homozygous C2 deficiency on the processing of radiolabeled soluble immune complexes (IC). A patient with C2 deficiency was studied before and after treatment with fresh frozen plasma (FFP). Hepatitis B surface Ag (HBsAg):anti-HBsAg immune complexes were prepared in vitro using Ag radiolabeled with 123I, and injected intravenously. Dynamic and static gamma-scintigraphy was performed to delineate the sites and kinetics of complex clearance. The patient was initially studied when her C2 level and CH50 were zero, and again 1 wk later after treatment with 12 units of FFP, which normalized these parameters. Before treatment there was rapid uptake of complexes by the liver (t90% [time for 90% uptake] = 13.6 min) and rapid clearance from the circulation (t1/2 = 6.8 min). No splenic uptake was detected, and there was no binding of complexes to erythrocyte CR1. Between 30 and 60 min there was release of 11% of the tracer from the liver. In the second study, performed after normalization of classical pathway complement activity, the t1/2 of IC clearance increased to 9.8 min, and t90% was 27 min. Twenty percent of injected complexes now localized to the spleen, and there was no longer any release of complexes between 30 and 60 min. The kinetics of IC processing and the sites of uptake in this posttherapy study were closely similar to two normal subjects studied in parallel, with a maximum of 72% of injected complexes binding to erythrocytes. These observations indicate that the uptake of immune complexes in the spleen in humans is complement-dependent, and suggest that the observed predisposition to SLE in patients with complement deficiency may be related to abnormal processing of immune complexes.
Characterization of MHC class I restricted cytotoxic T cell responses to tax in HTLV-1 infected patients with neurologic disease.
Authors: Koenig S , Woods R M , Brewah Y A , Newell A J , Jones G M , Boone E , Adelsberger J W , Baseler M W , Robinson S M , Jacobson S
Published: December 2022

To understand the nature of the cytotoxic T cell response generated in human T lymphotropic virus type 1 (HTLV-1)-infected patients with HTLV-1-associated myelopathy/tropical spastic paraparesis, we cloned CTL from the peripheral blood and cerebrospinal fluid from patients with neurologic diseases and demonstrated the presence of HLA-A2, A3, and B14 restricted responses to the HTLV-1 p40x (tax) protein. We identified the minimal amino acid residues within the epitopes required for binding and recognition by HLA-A2- and B14-restricted CTL, identified the critical residues within the peptide sequence defining the HLA-A2-restricted response, and demonstrated that CTL can lyse T cells infected with HTLV-1. This study shows that the CTL response to HTLV-1 tax in patients with neurologic diseases is heterogenous in nature and is not confined to patients of a single HLA haplotype or to a specific region of the tax protein.
Specific antibody production to a recall or a neoantigen by SCID mice reconstituted with human peripheral blood lymphocytes.
Authors: Nonoyama S , Smith F O , Ochs H D
Published: December 2022

To explore the extent of immune reconstitution of SCID mice by human peripheral blood lymphocytes (hu-PBL-SCID mice), we studied the production of immunoglobulin isotypes and specific antibody (Ab) by the engrafted human cells. Human IgG was detectable in 94% of hu-PBL-SCID mice. IgE synthesis by hu-PBL-SCID mice correlated with the IgE levels observed in human donors. All SCID mice receiving PBL obtained from human donors previously immunized with the T-cell-dependent Ag, bacteriophage phi x 174 (phage), produced phage neutralizing antibody. Quantity and quality (Ig isotypes) of phage-specific Ab produced by hu-PBL-SCID mice correlated with that observed in the donor serum. Human B cells alone failed to engraft, and T cells were required for the production of Ig and anti-phage Ab. Phage-specific Ab production occurred without direct Ag exposure of the hu-PBL-SCID mice, suggesting that the specific Ab production was induced directly by polyclonal activation of the engrafted human cells. Intravenous phage injections given 4 wk after cell transfer failed to further increase the anti-phage Ab titer. Phage neutralizing Ab production could not be boosted if spleen cells obtained from hu-PBL-SCID mice were cultured in the presence of Ag. However, hu-PBL-SCID mice produced increased amounts of anti-phage Ab, providing they were injected with phage at the time of cell transfer. Injection of phage at the time of cell transfer, but not 4 wk later, to mice receiving PBL from nonimmunized donors induced production of minute amounts of anti-phage Ab. We conclude that human peripheral blood lymphocytes transferred into SCID mice become maximally stimulated presumably by xenogeneic murine Ag, resulting in polyclonal expansion of the graft and spontaneous production of Ab to Ag the human donor was previously exposed to, and in loss of responses to subsequent Ag exposure. Ab production to neoantigen, however, can be induced and that to recall Ag can be modified if PBL are exposed to Ag at the time of cell transfer.
A subset of mouse monoclonal antibodies cross-reactive with cytoskeletal proteins and group A streptococcal M proteins recognizes N-acetyl-beta-D-glucosamine.
Authors: Shikhman A R , Greenspan N S , Cunningham M W
Published: December 2022

It is well known that antibodies to N-acetyl-beta-D-glucosamine (GlcNAc) cross-react with cardiac valves, skin, and other host tissues. However, molecular targets of these antibodies have not been identified. For this reason, anti-streptococcal mAb cross-reactive with group A streptococci and heart proteins were studied for their reactivity with GlcNAc, the immunodominant epitope of group A streptococcal carbohydrate. Characterization of the mAb that recognized GlcNAc revealed that each mAb had its own unique antigen-binding profile and pattern of immunofluorescence on rat heart cells. In the ELISA and Western blot these mAb reacted with cytoskeletal and heart proteins such as actin, keratin, myosin, and vimentin, as well as with streptococcal recombinant M5 and M6 proteins. Binding of the mAb to cytoskeletal proteins was inhibited by GlcNAc conjugated with BSA in a dose-dependent manner, and the mAb preferentially reacted with high-density GlcNAc-BSA conjugates. Antigenic determinants on the proteins recognized by the mAb were resistant to sodium periodate and N-acetylglucosaminidase treatment, suggesting reactivity with peptide and not carbohydrate structures. On reaction of the mAb with a panel of synthetic streptococcal, viral, and myosin peptides, one of the mAb, 49.8.9, was found to react most strongly with a synthetic peptide sequence synthesized from the coxsackievirus B3 capsid protein VP1, which shows homology with and cross-reacts with sequences in the streptococcal M6 protein and human cardiac myosin. This most interesting mAb, previously shown to neutralize coxsackie viruses, recognized the amino acid sequence RRKLEFF, which may mimic the GlcNAc epitope. The data collected show that we have identified a new group of multireactive autoantibodies that recognize GlcNAc and cytoskeletal proteins, as well as defined peptide epitopes.
Xenogeneic thyroid-stimulating hormone-like activity of the human natural anti-Gal antibody. Interaction of anti-Gal with porcine thyrocytes and with recombinant human thyroid-stimulating hormone receptors expressed on mouse cells.
Authors: Winand R J , Anaraki F , Etienne-Decerf J , Galili U
Published: December 2022

Anti-Gal is a natural polyclonal antibody that constitutes 1% of circulating IgG in all humans and that interacts specifically with the mammalian carbohydrate epitope Gal alpha 1-3Gal beta 1-4GlcNAc-R (termed the alpha-galactosyl epitope). This epitope is abundant on thyrocytes, as well as, on other cells of nonprimate mammals, prosimians and New World monkeys, but its expression is diminished in Old World monkey, ape, and human tissues. We hypothesized that anti-Gal may bind in vitro to alpha-galactosyl epitopes on xenogeneic TSH receptors (TSHR) and mimic the effect of TSH on xenogeneic thyrocytes. Assays performed with porcine thyrocytes have indicated that anti-Gal can mimic in vitro TSH effects in stimulation for cAMP synthesis, 125I uptake, and cell proliferation. Furthermore, depletion of anti-Gal from serum of patients with Graves' disease resulted in elimination of a large proportion of the thyroid stimulating immunoglobulin activity and half of the thyroglobulin binding inhibiting Ig activity, when the sera were assayed with porcine thyrocytes. The effect of anti-Gal binding to alpha-galactosyl epitopes on TSHR was further demonstrated by the antibody-mediated stimulation for cAMP synthesis in mouse 3T3 cells (cells expressing alpha-galactosyl epitopes), which were transfected with recombinant human TSHR. CHO cells (cells lacking alpha-galactosyl epitopes), transfected with recombinant human TSHR were not stimulated by anti-Gal. It is, therefore, suggested that in studies on antibodies in Graves' disease sera, the effect of anti-Gal may be excluded by using target cells that are devoid of alpha-galactosyl epitopes. Alternatively, anti-Gal could be removed from the tested sera, before the assay with xenogeneic thyrocytes.
Immune functions of tumor necrosis factor. I. Tumor necrosis factor induces apoptosis of mouse thymocytes and can also stimulate or inhibit IL-6-induced proliferation depending on the concentration of mitogenic costimulation.
Authors: Hernández-Caselles T , Stutman O
Published: December 2022

Murine rTNF produces at least three effects on mouse thymocytes in vitro: 1) Is a modest co-stimulator of proliferation with low PHA-P doses. 2) Has a bi-directional interaction with rIL-6-depending on PHA concentration: at low PHA (5 to 10 micrograms/ml) TNF augments and at high PHA (20 to 30 micrograms/ml) inhibits IL-6-induced proliferation. A comparable bidirectional PHA dose-dependent TNF interaction was seen with IL-1 beta, whereas only inhibition at high PHA with IL-2 and only augmentation at low PHA with IL-4 were seen. 3) TNF induces direct thymocyte apoptosis (a property not shared by IL-1 beta, IL-2, IL-4, IL-6 and IL-7). Of the cytokines studied, only IL-7 reduced TNF apoptosis. Thymocyte apoptosis by TNF showed the same species specificity as costimulation (i.e., human TNF had no effect) and was not inhibited by CY. The thymocyte CD4-CD8 phenotype after 72-h cultures showed that TNF decreased mainly double negative (DN) and single positive (SP) subsets, whereas IL-6 with low or high PHA increased DN and SP, especially the SP CD8+ subset. The regulatory and apoptotic effects of TNF were seen only with thymocytes and not with peripheral splenic or lymph node T cells. Four mAb to mouse TNF (2E2, XT22, 1C6, and 1OD9) could abrogate TNF costimulation and the TNF effects on IL-6-induced thymocyte proliferation, at both augmenting and inhibitory PHA conditions. However, only the two antibodies that also neutralize TNF lytic activity (2E2, XT22) could inhibit TNF-mediated apoptosis, implying two different but neighboring functional domains in the TNF molecule mediating apoptosis/lysis and costimulation. Our studies show that TNF might have unique and complex regulatory effects on growth and death of thymocyte populations in adult mice quite different from its effects on T cells in periphery.
Vimentin expression is differentially regulated by IL-2 and IL-4 in murine T cells.
Authors: Hornbeck P V , Garrels J I , Capetanaki Y , Heimer S
Published: December 2022

IL-2 and IL-4 are T cell growth factors that are produced by different T cell subsets and have distinct roles in lymphocyte biology. Despite their importance in the immune system, little is known about the genes that these lymphokines may specifically control and the interaction of these lymphokines in regulating the expression of their target genes. In this paper, we use the factor-dependent murine T cell line (CT.4R) to investigate the interaction of IL-2 and IL-4 in regulating gene expression. We report that the intermediate filament protein vimentin is differentially regulated by these lymphokines. Cells grown in IL-2 typically express 10- to 20-fold more vimentin and vimentin RNA than those grown in IL-4, but express similar levels of other cytoskeletal proteins including actin and tubulin. Vimentin was specifically induced by IL-2 and apparently suppressed by IL-4 in normal lymph node T cells, suggesting that its differential regulation by these lymphokines is physiologically relevant. We investigated the synergy between IL-2 and IL-4 in regulating the expression of vimentin RNA and compared it to that of two other lymphokine-responsive genes, pancreatic lipase and the IL-2R alpha subunit. Complex regulatory interactions were revealed: IL-4 suppressed the ability of IL-2 to induce vimentin RNA but not IL-2R alpha RNA, whereas IL-2 inhibited the ability of IL-4 to induce lipase RNA. These results indicate that IL-2 and IL-4 can cross-regulate lymphokine-responsive genes and can simultaneously exert both positive and negative regulation of different genes within the same cell.
Characterization of murine T cell responses to peptides of the variable region of self T cell receptor beta-chains.
Authors: MacNeil D , Fraga E , Singh B
Published: December 2022

Immunization with peptides of the TCR may regulate cellular immune responses that are dominated by a particular TCR. However, no extensive study of the immunogenicity of peptides of different regions of the V beta chain of the TCR has been done. We have tested the immunogenicity of several V beta peptides in several strains of mice and characterized the cellular response to these peptides. We examined the ability of six strains of mice of H-2b, H-2d, or H-2k and of mouse lymphocyte stimulatory (Mls)-1a or Mls-1b haplotypes to respond to several peptides of the V beta 6 region and an NH2-terminal peptide of other V beta of the TCR. These include V beta 6 peptides 1-20, 32-48, 39-60, 48-75, 58-75, and V beta 3, V beta 8.1, and V beta 8.3 peptides 1-20. The various mouse strains respond to these peptides independently of deletion of V beta 6+ T cells from peripheral lymphocytes. All of the Mls deleting and nondeleting strains tested respond weakly to one peptide of V beta 6, V beta 6(39-60). Antibody titers were also demonstrated in BALB/c and DBA/2J to V beta 6(1-20), V beta 6(39-60) and V beta 6(48-75), but not to V beta 6(32-48). We demonstrated that T cells responding to V beta 6(32-48) produce IL-2 and IFN-gamma, consistent with the Th1 subset of T cells. None of the antipeptide antibodies recognized the intact V beta 6 TCR on the cell surface. In vitro antibody blocking studies with TCR peptides show that these peptides are presented by class II MHC to CD4+ T cells. We conclude that the T cell and B cell repertoires contain cells able to respond to peptides of self TCR and immunization with peptides induces CD4+ T cells and that these cells may have an immunoregulatory role.
Cytolytic activity of murine IL-2-producing CD4+ and CD8+ T cell clones cycles in response to IL-2.
Authors: McKisic M D , Lancki D W , Cronin D C , Fitch F W
Published: December 2022

Alloreactive or OVA-reactive cloned murine CD4+ or CD8+ T cells that produce IL-2 exhibit greatly reduced cytolytic activity after being cultured with high concentrations of rIL-2. Furthermore, such cells fail to produce lymphokines or proliferate when stimulated with Ag. The duration of this unresponsiveness to Ag correlates with the concentration of rIL-2 to which the cells were exposed; higher concentrations of rIL-2 prolong the period of unresponsiveness. The presence of ionomycin during the cytolytic assay restores lytic activity to cells rendered unresponsive by exposure to rIL-2. These results suggest that rIL-2-induced unresponsiveness to Ag is a consequence of impairment of a calcium-dependent signal important for cytolysis, proliferation, and lymphokine production. Thus, IL-2 appears to be an important lymphokine that regulates T cell responses downward as well as upward.