bluuebunny/crossref_metadata_2025 huggingface dataset:
Synergistic transcriptional activation of the IL-8 gene by NF-kappa B p65 (RelA) and NF-IL-6.
Authors: Kunsch C , Lang R K , Rosen C A , Shannon M F
Published: December 2022

Transcriptional activation of the IL-8 gene by several inflammatory mediators, including the cytokines IL-1 and TNF-alpha, is mediated through sequences located between nucleotide -94 and -71 of the IL-8 promoter. Because adjacent binding sites for the inducible transcription factors NF-kappa B and NF-IL-6 are located within this region, we examined the functional interaction of these two transcription factor families in IL-8 gene regulation. Maximal transcriptional activation by PMA in Jurkat T lymphocytes was shown to require intact binding sites for both NF-kappa B and NF-IL-6. Electrophoretic mobility shift analysis indicates that NF-IL-6, as well as other related members of this family, bind specifically to the NF-IL-6 site in the IL-8 promoter. In addition, NF-kappa B p65 (RelA), but not NF-kappa B p50 (NFKB1), binds specifically to the NF-kappa B site. When incubated together, RelA and NF-IL-6/C/EBP form a ternary complex with this region of the IL-8 promoter; this binding is dependent on intact binding sites for both NF-IL-6 and RelA. Transient cotransfection analyses indicate that the cooperative association of NF-IL-6 and RelA with the IL-8 promoter results in synergistic transcriptional activation. Mutational analyses of RelA demonstrate that the C-terminal transactivation domain and the DNA binding domain are required for synergistic activation with NF-IL-6. In addition, overexpression of the NF-kappa B inhibitor molecule, I kappa B, abolished the RelA- and RelA/NF-IL-6-dependent synergistic activation. These data demonstrate that RelA and members of the C/EBP/NF-IL-6 family can functionally cooperate in transcriptional activation of the IL-8 gene and suggest a common mechanism for inducible regulation of cytokine gene expression.
Transcriptional regulation of the TCA3 gene in mast cells after Fc epsilon RI cross-linking.
Authors: Oh C K , Metcalfe D D
Published: December 2022

TCA3 is a pro-inflammatory cytokine expressed in mast cells after activation. To examine how this gene is regulated, TCA3 mRNA levels were examined and nuclear run-on experiments and promoter analysis were performed. Northern blot analysis showed that TCA3 message appeared in mast cells within 2 h after induction, with a t1/2 of 30 min. Nuclear run-on experiments revealed that the appearance of TCA3 mRNA occurred in large part because of an increase in the level of de novo transcription. Analysis of the promoter region demonstrated that inducible gene expression was directed by a region extending between 1.324 kb and 0.082 kb upstream from the transcription start site. There was a 60- to 80-fold induction with TCA3 CAT constructs extending between 1.324 kb and 0.324 kb upstream from the transcription start site after treatment with PMA/A23187 and a 30- to 40-fold induction with Fc epsilon RI cross-linking. There was a seven- to eightfold induction with the region extending between 0.136 kb and 0.082 kb upstream from the transcription start site. TCA3 CAT constructs containing regions encompassing either the 0.042 kb or 2.0 kb sequence from the transcription start site were not able to direct CAT-protein synthesis. The TCA3 5' flanking sequence contained negative regulatory activity. Electrophoretic mobility shift assays revealed that protein in nuclear extracts of activated mast cells bound to an NF-kappa B element of the TCA3 gene. These findings demonstrate that the TCA3 gene is regulated transcriptionally in mast cells, with minimal promoter sequences contained within the 0.082 kb upstream region of the TCA3 gene; a putative enhancer NF-kappa B element is contained between 0.324 kb and 0.136 kb and a putative inhibitory element is contained between 1.324 kb and 2.0 kb upstream from the transcription start site.
Functional platelet-activating factor receptors are expressed by monocytes and granulocytes but not by resting or activated T and B lymphocytes from normal individuals or patients with asthma.
Authors: Simon H U , Tsao P W , Siminovitch K A , Mills G B , Blaser K
Published: December 2022

Platelet-activating factor (PAF) may play a role in the regulation of immune responsiveness and is a potent mediator in asthmatic inflammation. However, as yet, the mechanisms whereby PAF mediates its pleiomorphic effects on immune cells have not been elucidated. Because PAF is a potent chemotactic factor for eosinophils, the presence of receptors for PAF (PAFR) on lymphocytes may provide a mechanism for the concurrent recruitment of both eosinophils and T lymphocytes into the airways of asthmatic patients. To address this issue, we have examined freshly isolated PBMC and granulocytic cells as well as various T and B lymphocyte lines with regards to PAFR expression and PAF-induced changes in intracellular calcium concentration. Using two-color immunofluorescence techniques and highly purified cell populations, it was not possible to detect surface PAFR protein or functional PAFR on resting and in vivo or in vitro activated T and B cells derived from nonallergic individuals or patients with allergic asthma. In addition, we were unable to detect PAFR mRNA, protein, or functional response to PAF in human or murine T cell lines. In contrast, we found functional PAFR in most B lymphoblastoid cell lines. Within the PBMC population, CD14+ cells respond to PAF. These results suggest that PAF does not interact directly with lymphocytes and thus that previous observations suggestive of such an interaction likely reflect the effects of PAF on monocytes. PAF-induced increases in intracellular calcium concentration were also detected in neutrophils and eosinophils, but were lower in granulocytes relative to the levels detected in monocytes.
Production, selection, and maturation of thymocytes with high surface density of TCR.
Authors: Lucas B , Vasseur F , Penit C
Published: December 2022

The main steps in intrathymic T cell differentiation have been defined using bromodeoxyuridine as a postmitotic cell tracer. Thymocytes with a high surface expression of the TCR are generated in the first 24 h after DNA synthesis. The phenotype of these TCR(high) cells was studied during 10 days by using pairs of surface markers associated with BrdUrd. During the first 2 days, TCR(high) cells were of the CD4+CD8+HSA(high) phenotype, transiently expressed the early activation marker CD69, and contained a high percentage of cycling cells. This activation step preceded the transition from CD4+CD8+ to CD4+CD8- and then to CD4-CD8+ cells, followed by progressive HSA down regulation and increase in the expression of H-2K, Qa-2, and CD45RB. The phenotypic maturation was completed in 9 days. In Mls-1a mice, negative selection of V beta 6+ cells was observed at the earliest step of TCR(high) cell generation, and positive selection of V beta 8.2+ and V beta 14+ cells took place later and was correlated to the activation step. These data suggest that high TCR expression and cell activation are necessary for positive selection and subsequent T cell maturation.
Evidence for protein tyrosine kinase involvement in ligand-induced TCR/CD3 internalization and surface redistribution.
Authors: Luton F , Buferne M , Davoust J , Schmitt-Verhulst A M , Boyer C
Published: December 2022

Triggering of the TCR/CD3 complex can lead to its internalization and modulation from the cell surface. In the present study, we address the question of the dependence of internalization on protein tyrosine kinase (PTK) activation. With use of an activating anti-clonotypic (anti-Ti) mAb on a CTL clone, we have shown that the PTK inhibitors genistein and tyrphostin 25 delayed anti-Ti-induced internalization, but did not affect fluid phase protein uptake or transferrin receptor cycling. Confocal microscopy with use of fluorescent anti-Ti mAb revealed that the inhibition of TCR internalization corresponded to the induction of large patches that were localized in cell membrane areas depleted of polymerized actin, the formation of which was dependent on the combined action of the anti-Ti mAb and the PTK inhibitors. In contrast to the effect of these PTK inhibitors, depletion of Src-like PTKs by T cell pretreatment with herbimycin A led to an increased rate of anti-Ti-induced internalization. Internalization induced by the monovalent Fab fraction of anti-Ti mAb was similarly affected by the PTK inhibitors, although the extent of induced internalization was less by approximately one-half. An analysis of substrates phosphorylated in kinase assays on TCR/CD3 immunoprecipitates of the CTL, which were activated by anti-Ti mAb in both the absence and presence of genistein, identified protein bands in which phosphorylation or association with CD3 was inhibited in the presence of genistein. Thus, a genistein-sensitive PTK activity seems to control ligand-induced TCR/CD3 complex redistribution and internalization.
TNF-alpha inhibits the further development of committed progenitors while stimulating multipotential progenitors in mouse long-term bone marrow cultures.
Authors: Rogers J A , Berman J W
Published: December 2022

We studied simultaneously the effect of TNF on both relatively primitive and more mature hematopoietic progenitors by using a modification of the mouse long-term bone marrow culture system of Dexter. Mycophenolic acid-treated long-term bone marrow cultures were charged with stromally depleted bone marrow cells along with 10 to 500 U/ml recombinant human TNF-alpha, and assayed each week for 5 wk for the presence of primitive (CFU-S and high proliferative potential colony-forming cell) and mature (CFU-C) colony-forming units. The results demonstrate that TNF-alpha has differential effects on hematopoiesis; it virtually eliminates CFU-C, significantly lowers high proliferative potential colony-forming cell (CFC) numbers and increases CFU-S numbers. This effect appears to be independent of CSF production in the cultures as shown by protein assays. Northern blot analyses showed no differences in expression of CSF genes or any expression of those encoding for IL-1, IL-3, or IL-6. These results, obtained in a long-term culture system that closely mimics in vivo bone marrow, demonstrate that many of the apparently contradictory effects of TNF observed in vitro occur concomitantly and are attributable to the presence of cells at various stages of maturity in the cultures. The data suggest a mechanism of this activity in which TNF is able to block the differentiation of primitive progenitors even as they are stimulated to proliferate.
Regulation of monocyte integrin expression by beta-family chemokines.
Authors: Vaddi K , Newton R C
Published: December 2022

In the present study we investigated the ability of three monocyte chemokines (MCP-1, MIP-1 alpha, and RANTES) to modulate monocyte adhesion molecules in an attempt to evaluate their potential to induce tissue infiltration of macrophages in vivo. All three chemokines tested induced increased expression of the alpha-chains of two members of beta 2 family of integrins, CD11b and CD11c, and their common beta-chain (CD18). They had no effect on CD11a expression. Enhancement of CD11b and CD11c was dose dependent and followed a distinct time course with peak levels at 4 h. Levels declined to reach basal levels by 24 h. In contrast, IL-1 induced enhancement remained high after 24 h of stimulation. However, the increases caused by chemokines were not mediated by IL-1 as indicated by lack of inhibition by the IL-1R antagonist. Studies on the mechanism of integrin up-regulation showed that mobilization of cytosolic free calcium is an important signaling event in this response and that up-regulation is associated with mobilization from intracellular pools mediated by microtubules. Enhanced CD11b and CD11c expression by chemokines was also found to result in enhancement of monocyte binding to endothelial cells. Further studies indicated that monocyte binding to endothelial cells follows similar dose-response kinetics as the up-regulation of integrins and can be partially blocked by Abs to CD11b and CD11c. These results suggest that modulation of the integrin expression by chemokines may facilitate the tissue trafficking of monocytes during inflammation.
Lung monocyte chemoattractant protein-1 gene expression in bleomycin-induced pulmonary fibrosis.
Authors: Zhang K , Gharaee-Kermani M , Jones M L , Warren J S , Phan S H
Published: December 2022

Recent studies indicate that monocyte chemoattractant protein-1 (MCP-1) may play an important role in pulmonary inflammation. In vitro studies show that a number of cell types are capable of producing MCP-1. In this study, MCP-1 expression in lungs of rats with bleomycin (BLM)-induced pulmonary fibrosis is examined to evaluate its cellular origin and potential role in pathogenesis. Lung fibrosis was induced in male Fisher 344 rats by endotracheal injection on day 0. On selected days after injection, lungs were harvested for in situ and Northern hybridization analyses for MCP-1 mRNA expression, immunochemical and histochemical analyses for MCP-1 protein expression, and identification of cell type. Northern analysis revealed significant elevation in lung MCP-1 mRNA expression beginning on day 3 post-BLM treatment, increasing to a peak on day 7, and then decreasing toward control levels after day 21. In situ hybridization combined with histochemical staining with chromotrope 2R indicate that most of the cells expressing MCP-1 mRNA at these time points are primarily eosinophils. A few scattered reactive fibroblasts, some mononuclear cells, epithelial cells, and cells of certain blood vessel walls also express this mRNA. Increased MCP-1 protein expression also was found to be predominantly within and adjacent to eosinophils. The eosinophils expressing this mRNA were found predominantly within areas of active fibrosis. The kinetics of increase in the number of cells expressing significant MCP-1 mRNA in lung sections paralleled that for MCP-1 mRNA expression, as assessed by Northern analysis. These results, for the first time, demonstrate that MCP-1 is up-regulated significantly in this rat animal model, and that infiltrating eosinophils represent the major cellular source for this increased MCP-1 expression.
Usage of a novel class of germ-line Ig variable region gene for cationic anti-DNA autoantibodies in human lupus nephritis and its role for the development of the disease.
Authors: Harada T , Suzuki N , Mizushima Y , Sakane T
Published: December 2022

It has been shown that cationic anti-DNA autoantibodies have nephritogenic potential in murine models of lupus nephritis. We have recently reported the close relationship between the presence of cationic anti-DNA Abs and the development of lupus nephritis in humans. To investigate underlining mechanisms responsible for the production of pathogenic autoantibodies, we have isolated a cDNA clone (SC17) encoding cationic anti-DNA Ab of human systemic lupus erythematosus with severe nephritis that was present at the onset of disease but disappeared after disease remission with corticosteroids. We have also cloned a counterpart Ig VL germ-line gene (SG3) from purified neutrophils of the patient and found the presence of replacement mutations only in the CDR of SC17. Surprisingly, predicted isoelectric point (pI) of deduced protein encoded by SG3 was the most cationic one among those encoded by previously reported human V kappa germ-line genes in the DNA database. These results raise the possibility that the use of specific germ-line genes may confer a cationic charge on the anti-DNA Ab, whereas somatic mutations induce affinity maturation of anti-DNA Ab in human lupus nephritis. Anti-DNA Ab-secreting B cells, but not DNA nonbinding B cells of the same patient, constitutively expressed SC17 mRNA. This mRNA is also expressed by B cells from a vast majority of patients at the onset of disease or exacerbation of lupus nephritis. However, the mRNA is absent in B cells from patients with lupus nephritis during disease remission, systemic lupus erythematosus patients without renal involvements, and normal individuals. It is suggested that the SC17 mRNA expression of B cells is rather restricted to systemic lupus erythematosus patients with active renal involvements.
Expression of adhesion molecules, platelet-activating factor, and chemokines by Kaposi's sarcoma cells.
Authors: Sciacca F L , Stürzl M , Bussolino F , Sironi M , Brandstetter H , Zietz C , Zhou D , Matteucci C , Peri G , Sozzani S
Published: December 2022

The present study was designed to investigate whether cells cultured from Kaposi's sarcoma (KS), a vascular tumor with a prominent leukocyte infiltration, express molecules important for the recruitment and activation of leukocytes. KS cells expressed intercellular adhesion molecule-1, which was augmented by exposure to IL-1 beta or TNF-alpha. Unlike endothelial cells, resting or cytokine-activated KS cells did not express appreciable levels of intercellular adhesion molecule-2, vascular cell adhesion molecule-1, and E-selectin on their surface. Weak expression of vascular cell adhesion molecule-1 mRNA was detectable by Northern blot analysis and, most clearly, by PCR analysis. Upon exposure to inflammatory cytokines, KS cells produced the attractant/activating lipid platelet-activating factor. KS cells expressed appreciable levels of the chemotactic cytokines, monocyte chemotactic protein-1 (MCP-1) and IL-8, as determined by Northern blot analysis, immunoassay, or bioassay. Chemokine production was augmented by IL-1 beta or TNF-alpha. MCP-1 expression was also detected in KS lesions by in situ hybridization. The set of molecules identified in the present study is probably important in determining the prominent leukocyte infiltration observed in KS. Tumor-associated leukocytes may amplify autocrine/paracrine circuits that sustain KS proliferation and contribute to recruitment of host vascular cells.
Cloned human CD8+ cytotoxic T lymphocytes protect human peripheral blood leukocyte-severe combined immunodeficient mice from HIV-1 infection by an HLA-unrestricted mechanism.
Authors: van Kuyk R , Torbett B E , Gulizia R J , Leath S , Mosier D E , Koenig S
Published: December 2022

The ability to infect human peripheral blood leukocyte-reconstituted severe combined immunodeficient (hu-PBL-SCID) mice with HIV has allowed evaluation of several strategies for preventing or treating infection. In one study, hu-PBL-SCID mice derived from HIV gp160-vaccinated donors were shown to resist HIV infection, and resistance correlated best with in vitro assays of cellular immunity. We have assessed directly the importance of cellular immunity to HIV in the present experiments by the adoptive transfer of HLA-A3-restricted HIV-1 Nef-specific or HLA-B14-restricted Gag-specific CD8+ CTL clones to SCID mice bearing HLA-matched or mismatched PBL grafts. Multiple inoculations of CTL before and after HIV-1 exposure protected HLA-matched hu-PBL-SCID mice from infection, but initiation of CTL therapy on the same day as HIV infection was much less effective. However, at the high numbers of CTL required for complete protection from HIV infection, many HLA-mismatched hu-PBL-SCID mice were also protected by pre-exposure CTL transfer. Transfer of CTL with a different specificity (HTLV-1 Tax) to HLA-matched hu-PBL-SCID mice also afforded partial protection. These results suggest that HLA-restricted cytotoxicity may be less important than other nonspecific effector mechanisms for the inhibition of HIV-1 infection in vivo.
IL-12 is expressed and released by human keratinocytes and epidermoid carcinoma cell lines.
Authors: Aragane Y , Riemann H , Bhardwaj R S , Schwarz A , Sawada Y , Yamada H , Luger T A , Kubin M , Trinchieri G , Schwarz T
Published: December 2022

IL-12 is a 70-kDa heterodimeric cytokine composed of two covalently linked chains, p40 and p35. IL-12 has multiple effects on T and NK cells and recently was found to be required for optimal Th1 cell development. In a variety of inflammatory skin disorders including delayed type hypersensitivity and contact hypersensitivity, Th1 cells appear to be critically involved. Because keratinocytes are well known to exhibit the capacity to release a variety of proinflammatory and immunologic cytokines, and thereby to be able to modulate inflammatory and immune reactions within the skin, it was investigated whether human keratinocytes and keratinocyte cell lines can release IL-12. Supernatants of phorbol-12,13-dibutyrate (PDBu) stimulated human epidermoid carcinoma cell lines (KB, A431) induced IFN-gamma production by PBL. This activity could be completely blocked by the addition of an anti-IL-12 Ab directed against the p40 subunit of IL-12 (C8.6). Release of IL-12 by keratinocyte cell lines was further confirmed by an RIA specific for p40. Immunoprecipitation under reducing conditions using the C8.6 Ab yielded specific bands at 40 kDa in supernatants of both KB cells and normal human keratinocytes. Northern blot analysis revealed IL-12-specific mRNA transcripts in PDBu-treated KB cells using cDNA probes encoding for p35 and p40. Moreover, by reverse transcription PCR specific transcripts for p35 and p40 were found in keratinocytes. These data indicate that keratinocyte cell lines and, although to a much lesser degree, normal human keratinocytes, exhibit the capacity to make IL-12; thus demonstrating for the first time IL-12 production by nonhemopoietic cells. Thus, one may speculate that keratinocytes via this capacity may influence the fate of Th-mediated immune responses, favoring Th1 responses by enhanced production of IL-12 or Th2 responses by reduced IL-12 release.
CD44 is a cytotoxic triggering molecule in human peripheral blood NK cells.
Authors: Sconocchia G , Titus J A , Segal D M
Published: December 2022

Previous studies have shown that target cells that bind to CD44 adhesion molecules on cloned cytotoxic T cells are lysed by the CTL. To determine whether CD44 is also a cytotoxic trigger molecule in human PBL, we tested a bispecific Ab consisting of anti-CD44 Fab cross-linked to a Fab against a target cell Ag, in cytotoxicity assays using PBL as effectors. We found that PBL mediated lysis in the presence of the anti-CD44 bispecific Ab provided that the effector cells were stimulated with either IL-2 or IL-12. Cell fractionation experiments showed that CD44-directed cytolysis was mediated exclusively by CD56+ low buoyant density cells, mainly by NK (CD16+) cells, but also to a lesser extent by CD56+ T cells. CD44-directed cytolysis appeared in these subsets 24 to 48 h after addition of IL-2 and paralleled the acquisition of Ab-independent (LAK) activity; in contrast, these cells mediated Ab-dependent cellular cytotoxicity and CD3 redirected lysis before stimulation with IL-2. Unstimulated CD56+ cells uniformly expressed high levels of CD44 that increased modestly after incubation with IL-2. No changes in isoform expression in the extracellular domain of CD44 could be detected upon activation with the use of isoform-specific mAbs. Thus, lymphokine stimulation caused CD44 to become a cytotoxic trigger in subsets of PBL that mediated other forms of cytotoxicity and expressed CD44 before activation, suggesting that activation of these cells was accompanied by a coupling of CD44 to their lytic machinery.
TCR selection and allelic exclusion in RAG transgenic mice that exhibit abnormal T cell localization in lymph nodes and lymphatics.
Authors: Wayne J , Suh H , Sokol K A , Petrie H T , Witmer-Pack M , Edelhoff S , Disteche C M , Nussenzweig M C
Published: December 2022

RAG-1 and RAG-2 are developmentally regulated genes that are essential for V(D)J recombination and lymphocyte development. Expression of RAG-1 and RAG-2 by thymocytes is normally limited to cells that have not completed selection. We have previously documented that persistent expression of the recombinase activating genes (RAG) in transgenic mice results in aberrant thymic development, altered lymphatic microanatomy, and a profound immunodeficiency. Here we further document the pathologic changes found in TG.RAG-1,2 mice and examine the role of TCR recombination and positive and negative thymic selection, as well as allelic exclusion, in the etiology of the phenotype. We find that neither selection nor TCR allelic exclusion can be overcome by transgenic expression RAG-1 and RAG-2 under the control of an lck promoter.
CD43 (S7) expression identifies peripheral B cell subsets.
Authors: Wells S M , Kantor A B , Stall A M
Published: December 2022

CD43 (leukosialin) expression has previously been demonstrated on the surface of developing B cells in mouse bone marrow and on plasma cells induced in vitro, but not on peripheral B cells in spleen. Here we show that CD43, as recognized by mAb S7, is indeed expressed on a small population of splenic B cells. Flow cytometric phenotyping of normal mice and radiation chimeras reveals that CD43/S7 is expressed on virtually all (> 90 to 95%) splenic B-1 cells and the majority of peritoneal B-1 cells, but not on conventional B cells. The expression of CD43/S7, in conjunction with other cell surface markers, clearly distinguishes B-1 cells from follicular, marginal zone, and immature B cells in the unstimulated adult spleen and permits further phenotyping of these subsets. The phenotype of splenic and peritoneal B-1 cells in normal BALB/c and BAB/25 mice is essentially identical with the exception that all peritoneal B-1 cells express CD11b (Mac-1) and some lack CD43/S7 and heat stable Ag (as detected by the mAb 53-10) expression. Although splenic B-1, marginal zone, and immature B cells share many phenotypic characteristics, these studies show that, in addition to CD43, they differ with respect to the expression levels of a variety of Ags including heat stable Ag, B220, and the B cell activation Ag B7.
Novel allele-specific, post-translational reduction in HLA class I surface expression in a mutant human B cell line.
Authors: Greenwood R , Shimizu Y , Sekhon G S , DeMars R
Published: December 2022

Human B cell line .220 has a novel defect in HLA class I cell surface expression. Mutant .220 was derived from .184TGr, from which both copies of HLA-A and -B are deleted, and has less surface HLA-C than .184TGr. Transfer of class I genes into .220 revealed allele-specific reductions in surface expression: HLA-A1 and -B8 were 1 to 21% of normal; HLA-A11, -A24, and -B5 were moderately reduced; and HLA-A2, -A3, and -B7 were reduced little, if at all. Class I mRNA in .220(A1) and .220(B8) transferents is normal in size and at least normal in quantity. Surface expression of class I molecules was restored by fusing .220 transferents with mutant .174, which lacks the TAP-1 and -2 genes needed for transport of class I-binding peptides. Fusion of .220(A1) cells with beta 2-microglobulin-deficient Daudi cells also fully restored surface expression of class I molecules encoded by both parental cells, indicating beta 2-microglobulin is functional in .220. Pulse-chase experiments showed transgene-encoded HLA-A1 and -B8 alpha-chains are made in apparently normal amounts and associate with beta 2-microglobulin in .220. However, post-translational processing of the HLA-A1 and -B8 molecules is retarded in or before the Golgi apparatus, and immunoprecipitable HLA-A1 molecules disappear after their synthesis. The effects of these abnormalities on surface expression of class I molecules were reversed by incubating .220(A1) and .220(B8) cells at 21 degrees C, which greatly increased the amounts of cell surface HLA-A1 and -B8.
Expressed swine VH genes belong to a small VH gene family homologous to human VHIII.
Authors: Sun J , Kacskovics I , Brown W R , Butler J E
Published: December 2022

The sequences of 34 swine H chain V-regions expressed with either IgG, IgA, or IgM C regions in adult swine and newborn piglets are described and compared. Sixteen of these V-regions were cloned by using a specific leader primer whereas 18 were cloned by anchored PCR. According to the operational criterion of VH family classification, i.e., > 80% DNA sequence similarity, all sequences belong to a homogeneous VH gene family. A total of 31 additional VH-bearing C mu clones obtained by anchored PCR from colostrum-deprived newborn piglets hybridized equally with a probe for C mu and a pan-VH probe. When the consensus sequence of the expressed swine VH genes were compared with those of VH gene for humans (VHI, II, III), mouse (VHI, II, III), rabbit, and chicken, swine VH genes seem to have common ancestry with the human VHIII family, rabbit VH genes, and the single functional VH gene of the chicken. The leader peptides of all clones obtained by anchored PCR showed < 1% variability, and the deduced amino acid sequences from aa4 to aa25 in all 34 clones are identical. Framework (FR)1 and FR2 are conserved whereas FR3 shows greater variability. A total of 23 of 30 JH sequences were identical, suggesting preferential use of one JH. This frequent, putative JH sequence is not similar to any JH gene in humans. Identical genomic Southern hybridization patterns, each with 13 bands of differing intensity and regardless of stringency, were obtained when either a leader or a pan-specific VH probe was used. Analyses of individual genomic bands by single strand conformational polymorphism and sequence analysis suggest that the number of VHIII-related genes in the swine genome is < 20.
Construction, expression, and immunogenicity of multiple tandem copies of the Schistosoma mansoni peptide 115-131 of the P28 glutathione S-transferase expressed as C-terminal fusions to tetanus toxin fragment C in a live aro-attenuated vaccine strain of Salmonella.
Authors: Khan C M , Villarreal-Ramos B , Pierce R J , Demarco de Hormaeche R , McNeill H , Ali T , Chatfield S , Capron A , Dougan G , Hormaeche C E
Published: December 2022

Genetic fusions have been constructed between the highly immunogenic but atoxic fragment C of tetanus toxin and a guest peptide, aa115-131, from the protective 28-kDa glutathione S-transferase Ag of Schistosoma mansoni. Fusions have been assembled to express one, two, four, and eight tandem copies of the peptide. The recombinant vectors have been electroporated into the nonvirulent aroA strain of Salmonella typhimurium SL3261. The fusion proteins are soluble and stably expressed in Salmonella as evaluated by Western blotting with fragment C and glutathione S-transferase antisera. Mice have been immunized i.v. with a single dose of the live recombinant salmonellae. The strains are stable in mice and elicit Ab responses directed against fragment C, as determined by enzyme-linked immunosorbent assays. Ab responses were also detected against the guest peptide. The Ab responses improved dramatically toward the aa115-131 peptide with increasing copy number, with the octameric "repitope" fusion displaying the greatest potency. This approach may represent a general strategy for eliciting immune responses against peptides in live bacterial vaccines.
IL-2-dependent NK cell responses discovered in virus-infected beta 2-microglobulin-deficient mice.
Authors: Su H C , Orange J S , Fast L D , Chan A T , Simpson S J , Terhorst C , Biron C A
Published: December 2022

In vivo NK cell responses to lymphocytic choriomeningitis virus were studied in CD8+ T cell-deficient mice. On day 7 after infection, dramatically elevated splenic NK cell activities were observed in both beta 2-microglobulin-negative (beta 2-m-/-) mice deficient in CD8+ T cells and anti-CD8-treated C57BL/6 animals. The enhanced responses could be attributed to increased numbers of activated NK1.1+CD3- cells. The day 7 NK cell responses in beta 2-m-/- mice, but not in normal C57BL/6 animals, were cyclosporin A sensitive and coincided with IL-2 production and high affinity IL-2R expression on NK cells. Proof that IL-2 played an essential role in day 7 responses was provided by the observation that IL-2-/- x beta 2-m-/- mice lacked the late NK cell activation. Taken together, these results showed that NK cells can be activated and expanded by an IL-2-dependent pathway. Because these responses can only be measured in the absence of CD8+ T lymphocytes, an exciting model of networking between T and NK cells in response to viruses is postulated.
LPS induces selective translocation of protein kinase C-beta in LPS-responsive mouse macrophages, but not in LPS-nonresponsive mouse macrophages.
Authors: Shinji H , Akagawa K S , Yoshida T
Published: December 2022

Translocation of protein kinase C (PKC) after PMA or LPS stimulation has been studied in thioglycolate (TGC)-elicited murine peritoneal macrophages. Among the PKC subtypes we examined (alpha, beta, gamma, delta, and epsilon) by indirect immunostaining and immunoblot analysis, conventional PKC-beta, as well as novel PKC-delta and PKC-epsilon were found to exist in TGC-elicited C3H/HeN mouse macrophages. Translocation of PKC-beta to the Triton-stable cytoskeleton could be seen in macrophages after stimulation by both PMA and LPS. On the other hand, novel PKCs redistributed only after PMA stimulation. Macrophages obtained from LPS-nonresponsive C3H/HeJ mice also exhibited PKC-beta, and the m.w., cellular distribution, and cellular content of this enzyme could not be distinguished from those of C3H/HeN macrophages. These macrophages exhibited PKC-delta and PKC-epsilon, as did the C3H/HeN macrophages. In these macrophages, however, LPS did not induce any remarkable change in the intracellular distribution of PKC-delta and PKC-epsilon or PKC-beta, whereas PMA was able to induce the translocation of PKC-beta to the cytoskeleton. These results suggest that LPS stimulation induces selective redistribution of PKC-beta in LPS-responsive macrophages, whereas a defect related to LPS unresponsiveness exists in C3H/HeJ mouse macrophages before the PKC activation. Translocation of PKC-beta can be understood to be an important event in LPS signaling in macrophages.
IL-10 is induced during HIV-1 infection and is capable of decreasing viral replication in human macrophages.
Authors: Akridge R E , Oyafuso L K , Reed S G
Published: December 2022

IL-10 has been shown to be capable of down-regulating several aspects of macrophage function. This study was undertaken to define the association between IL-10 and HIV-1 infection in human macrophages. Infection of macrophages with a monocytotropic strain of the human immunodeficiency virus, HIV-BaL, resulted in expression of IL-10 mRNA within 3 to 12 h after infection, as determined by the reverse transcriptase PCR. Biologically active IL-10 was detected in supernatants from HIV-1-infected macrophages as early as 12 h post-infection. The addition of human rIL-10 to HIV-1-infected macrophage cultures resulted in a significant decrease in the viral replication. In addition, exogenous IL-10 blocked the ability of TNF-alpha to elevate viral replication. To determine whether IL-10 was associated with in vivo infection, lymph nodes from AIDS patients were examined for the presence of IL-10 mRNA by using PCR. IL-10 mRNA was evident in all lymph node tissue examined, but was absent in normal lymph node biopsies. These in vitro and in vivo findings demonstrate a strong and heterogeneous association between HIV-1 infection and IL-10.
The role of CD40-CD40 ligand interaction in human T cell-B cell collaboration.
Authors: Nishioka Y , Lipsky P E
Published: December 2022

Interactions between CD40 on B cells and its ligand on activated T cells have been reported to play an important role in T cell-B cell collaboration. In this current study, a mAb against the human CD40 ligand (5c8) was used to investigate the impact of CD40-CD40 ligand interactions in the initial activation of normal human peripheral blood B cells and in subsequent proliferation and differentiation. B cells were activated by co-culture with anti-CD3-stimulated normal T cells. mAb against CD40 ligand blocked initial T cell-dependent B cell activation, as assessed by [3H]uridine incorporation and IL-2R expression. Subsequent B cell proliferation and differentiation were also inhibited by this mAb. In addition to its effect on B cell activation, 5c8 also inhibited the capacity of B cells to augment IL-2 production by anti-CD3 activated T cells, implying a role for CD40-CD40 ligand interactions in the accessory function of B cells. Despite the importance of CD40-CD40 ligand interactions in T cell-B cell collaboration, CD40 ligand-deficient T cell clones were found to induce initial activation of B cells and support Ig production. This effect was only marginally effected by mAb to LFA-1 and ICAM-1, suggesting that additional interaction molecules play a role in T cell-B cell collaboration. Taken together, the data indicate that CD40-CD40 ligand interactions plays an important role in T cell-dependent B cell activation and subsequent differentiation but additional interaction structures are also involved in T cell-B cell collaboration.
Glycosyl phosphatidylinositol membrane anchor is not required for T cell activation through CD73.
Authors: Resta R , Hooker S W , Laurent A B , Shuck J K , Misumi Y , Ikehara Y , Koretzky G A , Thompson L F
Published: December 2022

Ecto-5'-nucleotidase (5'-NT,) CD73 is a glycosyl phosphatidylinositol (GPI)-anchored differentiation Ag and purine salvage enzyme expressed on the surface of subsets of human lymphocytes. CD73 mAbs, in combination with submitogenic PMA or OKT3, activate human T cells to proliferate, secrete IL-2, and express IL-2 receptors. For several GPI-anchored proteins implicated in T cell activation, activation is thought to occur through a mechanism that requires the GPI anchor. To investigate the role of the GPI anchor in CD73-mediated signal transduction, CD73 cDNA was transfected into the human T cell leukemia line Jurkat. Transfectants were stimulated to secrete IL-2 by immobilized OKT3 + PMA and by soluble CD73 mAb + PMA. The amount of IL-2 synthesized by individual clones in response to CD73 mAb + PMA was proportional to the IL-2 response to immobilized OKT3 + PMA. CD73 transfectants of Jurkat mutants defective in the TCR, in p56lck, or in the tyrosine phosphatase CD45 did not secrete IL-2 in response to CD73 mAb + PMA, suggesting a role for the CD3/TCR-signaling complex in CD73-mediated signal transduction. A transmembrane form of CD73 was expressed in Jurkat cells by fusing the extracellular portion of CD73 to the transmembrane domain of human tissue factor. Although as a group, clones expressing transmembrane CD73 synthesized less IL-2 in response to CD73 mAb + PMA than clones expressing the GPI-anchored form of the molecule, the responses of the two groups overlapped considerably. In contrast to previous studies with Ly-6, Qa-2, and CD55, the GPI anchor is not critical for activation through CD73.
Ly-49 multigene family expressed by IL-2-activated NK cells.
Authors: Smith H R , Karlhofer F M , Yokoyama W M
Published: December 2022

The susceptibility of a target cell to killing by NK cells is inversely correlated with target cell expression of MHC class I molecules. We have recently demonstrated that a murine NK cell surface molecule, Ly-49, is a primary determinant of IL-2-activated NK cell specificity by apparently acting as an inhibitory receptor. Ly-49 seems to interact with polymorphic regions of certain MHC class I molecules on target cells, yet is expressed only by a subset (15 to 20%) of NK cells. If the interaction between Ly-49 and MHC class I represents a paradigm for understanding IL-2-activated NK cell specificity and putative inhibitory IL-2-activated NK cell receptors, these effector cells should express other molecules, perhaps related to Ly-49, that provide the inhibitory receptor function. We now report the isolation of cDNAs, encoding polypeptides related to Ly-49, from a CD3-, IL-2-activated NK cell library. Full length PCR products were also cloned and sequenced, confirming expression in IL-2-activated NK cells. These cDNAs were homologous to the Ly-49 cDNA and seem to be derived from distinct genes. The new cDNAs encode type II integral membrane proteins having lectin superfamily homology and 66 to 79% amino acid identity with Ly-49A. In contrast to previous reports, these cDNAs cannot be allelic variants because they were all isolated from C57BL/6-derived cells. On the basis of nucleotide homology, the cDNAs could be segregated into five groups; one with Ly-49 itself (now termed Ly-49A) and four others containing distinct sequences. One of the new groups comprised three cDNAs that shared identical sequences, but differed from one another by deletions or insertions of short DNA segments in the predicted extracellular region of the polypeptides. Thus, IL-2-activated NK cells express mRNAs for multiple, Ly-49-related molecules.
Light chain isotype regulation in the horse. Characterization of Ig kappa genes.
Authors: Ford J E , Home W A , Gibson D M
Published: December 2022

Horse Ig kappa genes have been characterized to determine whether there may be a structural basis for the low level of kappa expression in this species. The overall organization of the J kappa-C kappa locus is remarkably similar to that of the mouse and human loci. A single C kappa exon is separated by 2.9 kb from five J kappa segments, four of which seem functional and three of which are associated with canonical recombination signal sequences. A highly conserved intron enhancer was identified upstream of the C kappa exon and a single restriction fragment in horse genomic DNA hybridized strongly with the mouse downstream kappa enhancer. Germ-line and splenic cDNA V kappa sequences were characterized and found to encode N-terminal amino acid sequences previously found by protein sequencing. Hybridization of horse genomic DNA with the horse V kappa-1 germ-line gene and a variety of mouse V kappa-region probes provided evidence for at least 20 V kappa gene segments. The results indicate that the horse possesses a functional kappa locus with a complement of variable region gene segments potentially as large as that previously found at the lambda locus although the exact numbers of functional variable region genes remains to be established for both loci. This finding suggests that the predominance of lambda-chains in horse Ig may not be simply a result of a lack of functional kappa genes or of a disproportionate number of lambda vs kappa variable region genes. The results are discussed in terms of various models of isotype regulation.
Bovine parainfluenza-3 virus selectively depletes a calcium-independent, phospholipid-dependent protein kinase C and inhibits superoxide anion generation in bovine alveolar macrophages.
Authors: Dyer R M , Majumdar S , Douglas S D , Korchak H M
Published: December 2022

Bovine parainfluenza-3 (PI-3) virus inhibits oxygen-dependent bacterial killing by phagocytes, a key pulmonary defense, thus predisposing the host to intrapulmonary bacterial superinfection. PI-3 virus inhibited opsonized zymosan or PMA-activated superoxide anion (O2-) generation in bovine alveolar macrophages. The respiratory virus influenza also inhibits O2- generation by phagocytes, however, the mechanisms(s) of viral inhibition differs from PI-3. PI-3 did not trigger O2- generation before inhibition, whereas influenza triggered O2- generation before desensitization of ligand-initiated respiratory burst. PI-3 modified the twin signals of calcium and protein kinase C in alveolar macrophages. PI-3 infection increased macrophage membrane permeability to extracellular calcium, but did not inhibit calcium mobilization triggered by opsonized zymosan. These effects further distinguish bovine PI-3 from human influenza, which triggers mobilization of cell-associated calcium and inhibits calcium mobilization activated by physiologic ligands. Macrophages possessed two classes of PKC activity, a calcium/phosphatidylserine/diglyceride (Ca/PS/DG))-dependent activity and a Ca-independent, PS/DG-dependent histone IIIS phosphorylating activity. PI-3 infection selectively depleted the Ca-independent, PS/DG-dependent kinase activity but not the classical Ca/PS/DG-dependent activity. Inhibition of Ca-independent, PS/DG-dependent kinase activity and inhibition of O2- generation by PI-3 occurred at a similar viral dose and time frame, suggesting a role for this kinase in activating the respiratory burst. Inhibition of the oxygen-dependent bactericidal function of alveolar macrophages and disturbances in signal transduction may contribute to the immunosuppression and bacterial superinfection accompanying viral respiratory disease.
T lymphocytes isolated from the hepatic granulomas of schistosome-infected mice express somatostatin receptor subtype II (SSTR2) messenger RNA.
Authors: Elliott D E , Metwali A , Blum A M , Sandor M , Lynch R , Weinstock J V
Published: December 2022

Schistosomiasis mansoni is a disease characterized by liver and intestinal granulomas. Previous investigations in our laboratory showed that nylon wool-adherent CD4+ T lymphocytes isolated from murine hepatic schistosome granulomas express receptors for somatostatin 1-14. Moreover, somatostatin 1-14 substantially decreased IFN-gamma and IgG2a, but not IL-5 secretion by dispersed granuloma cells. This paper extends these observations by defining the somatostatin receptor (SSTR) isoform most likely expressed by granuloma inflammatory lymphocytes. Amplification of mRNA by reverse transcription PCR shows SSTR1, SSTR2, and SSTR3 mRNA in normal mouse brain and other tissues. Nevertheless, only the SSTR2 PCR product was amplified from granuloma cell RNA. The nucleotide sequence of the granuloma SSTR2 PCR product from inflammatory cells is identical to the CBA murine brain SSTR2 cDNA sequence. Granuloma-derived T cell lines, FACS-isolated granuloma CD4+ T cells, thymocytes, splenocytes, and cloned T cell lines all contain mRNA for SSTR2 by reverse transcription PCR. Moreover, both SSTR2A and the splice variant SSTR2B can be amplified from dispersed granuloma cells, granuloma T cell lines, thymocytes, and splenocytes. This is the first demonstration that inflammatory cells produce mRNA for an authentic somatostatin receptor. It is probable that the lymphocyte SSTR2 receptor mediates somatostatin-induced modulation of IFN-gamma secretion.
Loss of HLA haplotype and B locus down-regulation in melanoma cell lines.
Authors: Marincola F M , Shamamian P , Alexander R B , Gnarra J R , Turetskaya R L , Nedospasov S A , Simonis T B , Taubenberger J K , Yannelli J , Mixon A
Published: December 2022

The expression of HLA class I molecules on tumor cells is vital for CD8+T cell recognition of tumor Ags. Loss of HLA class I Ag expression as a result of defective beta 2-microglobulin genes has been described in melanoma cells. To further evaluate mechanisms of tumor escape, HLA class I Ag expression was compared in 24 metastatic melanoma cell lines and 20 melanocyte strains by FACS analysis with use of allele-specific mAbs. Total loss of HLA class I Ag expression was not noted; instead, two relatively common phenomena were identified: 1) A variable degree of expression of HLA-B Ags by melanoma cell lines and melanocytes; however, HLA-A Ags were consistently expressed in all cell types. Furthermore, HLA-B locus Ag expression was detected in vivo in only one of six frozen section specimens obtained from six patients having metastatic melanoma. 2) Loss of allelic expression was noted in two of 14 HLA-A2 (14%) and one of three HLA-A29 (33%) melanoma cell lines and included a full haplotype, which suggests loss of a genomic fragment. Allele-specific PCR amplification demonstrated deletion of genes in linkage disequilibrium within the MHC class II, III, and I regions. Aberrations of HLA class I expression in tumor lines should be considered when assessing MHC-restricted phenomena in in vitro models.