bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of CTL by ecto-nictinamide adenine dinucleotide (NAD) involves ADP-ribosylation of a p56lck-associated protein.
Authors: Wang J , Nemoto E , Dennert G
Published: December 2022

Receptor-mediated activation of T lymphocytes involves protein phosphorylation by several protein tyrosine kinases, among those the src-related enzymes p56lck and p59fyn. Accumulating evidence supports the notion that these enzymes are regulated by tyrosine phosphorylation and dephosphorylation, but much is yet to be learned about regulation of their activity. Here we demonstrate that p56lck but not p59fyn exists as a complex with a 40-kDa protein, which in its ADP-ribosylated form inhibits p56lck kinase activity. ADP-ribosylation of this protein is mediated by an arginine-specific mono-ADP-ribosyltransferase, which makes use of extracellular nicotinamide adenine dinucleotide (NAD). This enzyme is a glycosyl-phosphatidylinositol-anchored protein releasable from the surface of cytotoxic T cells by glycosyl-phosphatidylinositol-specific phospholipase C. Release of arginine-specific mono-ADP-ribosyltransferase results in failure of extracellular NAD to downmodulate p56lck kinase activity. Concomitant to suppression of the kinase by NAD, CD8 mediated transmembrane signaling and p56lck kinase activation are inhibited.
Humanization and molecular modeling of the anti-CD4 monoclonal antibody, OKT4A.
Authors: Pulito V L , Roberts V A , Adair J R , Rothermel A L , Collins A M , Varga S S , Martocello C , Bodmer M , Jolliffe L K , Zivin R A
Published: December 2022

OKT4A, a murine mAb that recognizes an epitope on the CD4 receptor, is a potent immunosuppressive agent in vitro and in a variety of nonhuman primate models of graft rejection and autoimmune disease. Initial human cardiac transplant trials suggest that OKT4A does not cause either cytokine release syndrome or CD4+ cell depletion, but does induce a human anti-mouse Ab (HAMA) response despite strong concurrent immunosuppression. To further investigate the potential of OKT4A as an immunomodulator, it was necessary to decrease its immunogenicity. Therefore, we developed a humanized version of this Ab (gOKT4A-4), which has the same binding affinity and in vitro immunosuppressive properties of OKT4A, but retains only three murine sequence-derived amino acid residues outside of the complementarity-determining regions (CDRs). Detailed computer modeling of both OKT4A and gOKT4A-4 provided a computational rationale for the changes necessary to regain activity after humanization. This has also provided a plausible representation of the Ag binding site. Preliminary clinical results with gOKT4A-4 suggest that we have eliminated the immunogenicity observed in the parent murine Ab.
Effect of the IgM and IgA secretory tailpieces on polymerization and secretion of IgM and IgG.
Authors: Sørensen V , Rasmussen I B , Norderhaug L , Natvig I , Michaelsen T E , Sandlie I
Published: December 2022

Pentameric IgM and dimeric IgA both contain disulfide bonds between cysteines located in the secretory tailpieces of the heavy chains. To compare the influences of the mu and alpha tailpieces on the polymeric structure, we have replaced amino acids in the tailpiece of the human mu-chain with amino acids found in the corresponding positions in the human alpha tailpiece. We show that an IgM with an alpha tailpiece (IgM L561H, Y562V, L566V, S569A, D570E, T571V, and A572D) as well as IgM L561H, Y562V, and IgM A572D have a size distribution similar to that of wild-type IgM. However, one IgM mutant with a mu/alpha hybrid tailpiece (IgM L566V, S569A, D570E, T571V, and A572D) is secreted as a mixture of mainly hexamers, pentamers, tetramers, and dimers. The tetramers and dimers are specifically formed and secreted at the expense of pentamers and hexamers; no alterations in polymerization or secretion rates were observed. We have also incorporated the mu, alpha, and hybrid mu/alpha tailpieces to a human IgG3 or IgGL309C mutant. The IgG-tailpiece mutants are poorly secreted, but the secreted fractions contain multimeric molecules. Each of the mutants that contain both the L309C mutation and a secretory tailpiece forms mainly hexamers; however, small differences in polymer distribution exist for the different tailpieces. Comparison of the influence of different tailpieces on IgM and IgG polymeric structures suggests that the function of a specific tailpiece is dependent on other parts of the heavy chain, which can vary for different isotypes.
A novel Plasmodium falciparum sporozoite and liver stage antigen (SALSA) defines major B, T helper, and CTL epitopes.
Authors: Bottius E , BenMohamed L , Brahimi K , Gras H , Lepers J P , Raharimalala L , Aikawa M , Meis J , Slierendregt B , Tartar A , Thomas A , Druilhe P
Published: December 2022

In the search for subunit vaccines that are able to induce the type of sterile, protective immunity achieved by irradiated sporozoites, there is increasing evidence that defense mechanisms directed at the intrahepatic stage and Ags expressed at this stage are critical. We have initiated a systematic search for such molecules and report here the identification and partial characterization of a novel Plasmodium falciparum gene encoding a 70-kDa protein, expressed in both sporozoite and liver stages (SALSA), with a vaccine potential that stems from its antigenic features. Antigenicity and immunogenicity studies were conducted in individuals exposed to malaria, in immunized mice, and in chimpanzees, using a recombinant protein and two synthetic peptides. Results show that the SALSA nonrepetitive sequence defines 1) major B cell epitopes, as shown by a high prevalence of Abs to each peptide in three African areas differing in their level of endemicity; 2) Th epitopes, as demonstrated by lymphoproliferation and IFN-gamma secretion in cells from the individuals from one of the low transmission areas, as well as helper effect upon Ab secretion in mice; and 3) epitopes for cytolytic lymphocytes, demonstrated in immunized and sporozoite-challenged chimpanzees, and associated with MHC class I leukocyte Ags. The latter are of particular importance, because this is the only part of the malaria life cycle in which the parasite is located in a cell expressing class I Ags and because CD8+ lymphocytes were found to be responsible for protection in experimental models.
Brucella species release a specific, protease-sensitive, inhibitor of TNF-alpha expression, active on human macrophage-like cells.
Authors: Caron E , Gross A , Liautard J P , Dornand J
Published: December 2022

Brucella species can establish themselves and cause disease in humans, but the mechanisms by which brucellae evade the antibacterial defenses of their host remain largely unknown. We have previously reported that, unlike Escherichia coli K12, intracellular pathogens from the genus Brucella survive and multiply within U937-derived phagocytes, and live Brucella organisms failed to induce TNF-alpha release upon infection. Moreover, exogenously added TNF-alpha restricted intracellular growth of Brucella species. Herein, we demonstrate that Brucella-infected U937 cells are activated to express IL-1 beta and IL-6 at both the mRNA and protein levels, while they cannot accumulate TNF-alpha mRNA. When physically separated from macrophages, live brucellae impaired TNF-alpha production in E. coli-infected cells. Moreover, in agonist-activated macrophages, supernatants from Brucella cultures promoted an inhibition of the induction of both TNF-alpha expression and release, without affecting IL-1 beta or IL-6 induction. These phenomena, observed whatever the Brucella strain assayed, show that brucellae release some high m.w. factor(s) that specifically inhibits TNF-alpha expression in activated human macrophages. The proteic nature of the factor(s) was demonstrated by its heat and protease sensitiveness, and this could explain why U937-derived macrophages did release TNF-alpha when infected with chloramphenicol-treated brucellae. We also found that the Brucella factor(s) specifically acts on human macrophagic cells, but not on murine macrophage-like cells. Our findings provide direct evidence that a secreted Brucella virulence factor(s) inhibiting TNF-alpha expression might contribute to the evasion of Brucella organisms from human antimicrobial defenses.
Genetically resistant (Ityr) and susceptible (Itys) congenic mouse strains show similar cytokine responses following infection with Salmonella dublin.
Authors: Eckmann L , Fierer J , Kagnoff M F
Published: December 2022

IFN-gamma, TNF-alpha, IL-1, and granulocyte-macrophage CSF (GM-CSF) play an important role in host resistance to infection with nontyphoid Salmonella. In mice, resistance to Salmonella is determined by alleles of the susceptibility gene, Nramp, which maps to the Ity/Lsh/Bcg locus and is expressed in macrophages. In vitro studies suggested that macrophages from Salmonella-susceptible mice (Itys phenotype) are impaired functionally in their ability to produce, or stimulate the production of, cytokines such as TNF-alpha and IFN-gamma. BALB/c and BALB/c.DBA2 Idh-lb-Ityr-Pep-3b mice are congenic strains that differ at the Ity/Lsh/Bcg locus and in their susceptibility to Salmonella infection. These strains were used to question whether differences in the host cytokine response determine the outcome of Salmonella infection in genetically susceptible and resistant mice. As reported in this work, the in vivo response to Salmonella dublin infection in both Itys and Ityr mice was characterized by increased expression of IFN-gamma, TNF-alpha, GM-CSF, IL-1 alpha, IL-2, IL-6, IL-10, and IL-12 p40. In contrast, expression of IL-4, IL-5, and TGF-beta 1 was not altered, or decreased, during the course of infection. Moreover, the kinetics and magnitude of the cytokine response following S. dublin infection were similar in susceptible Itys and resistant Ityr mice, even though the former group died while the latter survived the infection. Thus, in vivo cytokine responses that are associated with survival of Ityr mice following S. dublin infection do not confer protection in mice of the Itys phenotype.
Induction of antitumor immunity using bone marrow-generated dendritic cells.
Authors: Porgador A , Snyder D , Gilboa E
Published: December 2022

We have previously shown that bone marrow-generated dendritic cells (DC) pulsed with a class I-restricted peptide are potent inducers of CD8+ CTL. In the present study we have investigated whether bone marrow-generated DC are capable of inducing antitumor immunity. We show that a single immunization with DC pulsed with OVA peptide was highly effective in eliciting a protective immune response against a challenge with tumor cells expressing the OVA gene (E.G7-OVA), more so than immunization with irradiated E.G7-OVA cells, OVA peptide-pulsed RMA-S cells, or free OVA peptide mixed with adjuvant. The addition of free OVA protein to day 4 or day 7 bone marrow cultures, but not to day 9 mature DC, was also effective in eliciting CTL and engendering antitumor immunity, but was less effective than peptide-pulsed DC. Induction of CTL and antitumor immunity by bone marrow-generated DC pulsed with the class I-restricted OVA peptide correlated with the expression of syngeneic MHC class II molecules on the DC. This and the fact that induction of tumor immunity was dependent on CD4+ T cells suggest that in vivo priming of CTL and induction of antitumor immunity by bone marrow-generated DC also require the presentation of MHC class II-restricted epitopes and activation of CD4+ T cells. This observation has potentially important implications to the use of peptide-pulsed DC in clinical immunotherapy.
Differential effects of viable and killed bacteria on IL-12 expression of macrophages.
Authors: Song F , Matsuzaki G , Mitsuyama M , Nomoto K
Published: December 2022

Listeria monocytogenes-specific, IFN-gamma-producing CD4+ T cells are induced in 5-day in vitro cultures of naive spleen cells with viable L. monocytogenes (VLM) but are not induced in cultures with heat killed L. monocytogenes (HKLM). Anti-IL-12 Abs abrogated the induction of the IFN-gamma-producing CD4+ T cells by VLM, suggesting an importance of IL-12 in the induction of IFN-gamma-producing CD4+ T cells. Accordingly, IL-12p40 was expressed by macrophages (M phi) from the cultures of whole spleen cells with VLM but not with HKLM, although HKLM induced IL-12p40 expression on M phi when nonadherent cells were removed before culture. In vivo analysis also showed that VLM induced IL-12p40 expression by M phi, whereas HKLM failed to induce it. On the other hand, the addition of anti-IL-10 mAb into cultures of whole spleen cells and HKLM resulted in an increase of IL-12p40 expression by M phi. Furthermore, we detected an expression of IFN-gamma by both adherent and nonadherent spleen cells at the early stage of stimulation with VLM before the appearance of IFN-gamma-producing CD4+ T cells. These data suggest that HKLM induce nonadherent spleen cells to produce IL-10 which may down-regulate IFN-gamma-producing CD4+ T cells by blocking IL-12 production by M phi. In contrast, VLM support IFN-gamma production by CD4+ T cells by stimulating M phi to produce IL-12 and IFN-gamma.
Platelet activating factor and endotoxin increase the enzyme activity and gene expression of type II phospholipase A2 in the rat intestine. Role of polymorphonuclear leukocytes.
Authors: Tan X D , Wang H , Gonzalez-Crussi F X , Chang H , Gonzalez-Crussi F , Hsueh W
Published: December 2022

Type II phospholipase A2 (PLA2-II) may regulate eicosanoid synthesis; is involved in various inflammatory processes, septic shock, and inflammatory bowel disease; and is up-regulated by LPS and inflammatory cytokines in vitro. We have previously shown that the platelet-activating factor (PAF)-induced intestinal injury is attenuated by peptido-leukotriene antagonists, suggesting a role of PLA2. The purpose of this study is to examine the regulation of intestinal PLA2-II by PAF and LPS. Using synthesized competitor RNA as competitor, we quantified PLA2-II transcripts by competitive reverse transcription-PCR. We found that PLA2-II gene is constitutively expressed in the rat ileum. PAF at a dose (1.5 micrograms/kg) below that causing intestinal injury rapidly up-regulated intestinal PLA2-II at both transcriptional and post-transcriptional levels, almost tripling its transcripts and significantly increasing enzyme activity in 30 min. LPS (5 mg/kg) also up-regulated PLA2-II. This effect could not be blocked by PAF antagonist. Depletion of circulating polymorphonuclear leukocytes (PMNs) abolished the effect of PAF on PLA2-II gene expression and enzyme activation. In contrast, PMN depletion prevented LPS-induced PLA2-II enzyme activity but enhanced the gene expression. We conclude that: 1) both PAF and LPS induce gene transcription and enzyme activation of PLA2-II in the small intestine; 2) PAF up-regulates PLA2-II via PMN activation; 3) LPS effect is independent of endogenous PAF formation; and 4) different pathways exist for PAF and LPS in the regulation of intestinal PLA2-II gene expression in vivo.
Localization of monocyte chemoattractant peptide-1 expression in the central nervous system in experimental autoimmune encephalomyelitis and trauma in the rat.
Authors: Berman J W , Guida M P , Warren J , Amat J , Brosnan C F
Published: December 2022

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine beta family of chemoattractants that has been shown to play a major role in the initiation of monocyte and T cell inflammation to sites of tissue injury. In this study, we have examined the distribution of MCP-1 expression in inflammation in the central nervous system (CNS) associated with the autoimmune disease experimental autoimmune encephalomyelitis (EAE) and compared the results with those detected in inflammation associated with trauma. In EAE, MCP-1 expression was detected at the onset of inflammation, prior to clinical expression of disease, in lymphocytes and endothelial cells in subarachnoid locations. Monocyte infiltration into these areas appeared 24 h later. After the onset of clinical signs, MCP-1 expression was widely distributed in the spinal cord with levels increasing and decreasing in association with disease activity. Lymphocytes, macrophages, astrocytes, and endothelial cells could be identified as sources of MCP-1 by immunoreactivity and in situ hybridization. A similar close correlation between macrophage infiltration and the levels of mRNA for MCP-1 was found in the CNS of rats subjected to trauma, and in these animals MCP-1 was detected by immunohistochemistry in macrophages and endothelial cells. The results support the conclusion that MCP-1 is an important mediator of inflammation in the CNS.
Functional differentiation of dendritic cells in rheumatoid arthritis: role of CD86 in the synovium.
Authors: Thomas R , Quinn C
Published: December 2022

Dendritic cells (DC) are the major APC in human peripheral blood (PB) and rheumatoid synovium. We previously identified in PB a population of CD33dim-CD14dim DC precursors, as well as a smaller population of CD33bright CD14dim mature DC. Neither PB DC population expressed the CD28/CTLA4 ligands, suggesting that additional signals are required for full functional DC differentiation. Because rheumatoid synovium is characterized by an ongoing immune response, the expression and function of CD80, CD86, and other markers of DC differentiation by rheumatoid arthritis synovial APC were examined. The phenotype of a large subset of freshly isolated rheumatoid arthritis synovial fluid (SF) DC resembled that of the mature PB DC. These DC expressed CD45R0, CD11c, CMRF-44, and high levels of CD33. Whereas CD80 expression by rheumatoid SF DC and monocytes was minimal, CD86 was expressed by a subset of SF monocytes and by CMRF-44+SF DC. Furthermore, sorted CD86- SF DC spontaneously up-regulated CD86 in vitro. CD80 was expressed diffusely and at low levels by rheumatoid synovial tissue cells, whereas CD86 was expressed by perivascular HLA-DR+HLA-DQ+CD80+CMRF-44+ DC, and by some CD14+ monocytes. Anti-CD86 mAb and CTLA4 Ig, but not anti-CD80 mAb, inhibited the MLR stimulated by SF DC. Both CMRF-44+ and CMRF-44- SF DC were efficient stimulators of the allogeneic MLR, which was in each case blocked by CTLA4 Ig. The data indicate that rheumatoid synovial DC can undergo full functional differentiation, associated with CD86 expression, in vitro and in situ. Synovial DC expressing high levels of MHC molecules and CD86 are strategically located to present arthritogenic Ag to T cells after transendothelial migration.
Continuous administration of Il-13 to mice induces extramedullary hemopoiesis and monocytosis.
Authors: Lai Y H , Heslan J M , Poppema S , Elliott J F , Mosmann T R
Published: December 2022

IL-13, a recently identified Th2 cytokine, shares some, but not all, IL-4 functions, including inhibition of monocyte and macrophage activation, stimulation of human B cells, and induction of growth and differentiation of mouse bone marrow cells in vitro. We have now tested the in vivo effects of recombinant mouse IL-13 (rIL-13) from stably transfected, high expressing BW5147 thymoma cells. After purification by anion exchange chromatography, rIL-13 was administered in the peritoneal cavity of BALB/c mice via osmotic pump for 7 days. Spleens from the rIL-13-treated mice were significantly enlarged compared with control spleens due to increased cellularity. In particular, increased numbers of immature erythroblasts and megakaryocytes were observed in splenic sections after rIL-13 treatment. Spleen cells from rIL-13-treated mice showed greatly increased responsiveness in vitro to recombinant forms of mouse IL-3, mouse granulocyte-macrophage CSF, or human CSF-1 and, to a lesser extent, to mouse IL-4 or IL-13. Moreover, the rIL-13-treated mice also showed significant increases in CFU-E, CFU-C, and erythroid burst colonies in the spleen, further indicating the presence of increased numbers of hemopoietic precursors. Hematologic analyses indicated that rIL-13 treatment induced slight anemia and striking monocytosis. Finally, spleen cells from rIL-13-treated mice produced significantly more IL-6 upon LPS stimulation. Interestingly, the strong Th2 response induced by Nippostrongylus brasiliensis infection was also accompanied by an increase in hemopoietic precursor frequencies in the spleen. Collectively, these data indicate that exogenous rIL-13 induces extramedullary hemopoiesis in mice and suggest that endogenous IL-13 may contribute to replenishment of effector cells during strong Th2 responses.
Kinetically coordinated induction of TAP1 and HLA class I by IFN-gamma: the rapid induction of TAP1 by IFN-gamma is mediated by Stat1 alpha.
Authors: Min W , Pober J S , Johnson D R
Published: December 2022

Transporter associated with Ag processing-1 (TAP1) is induced by IFN-gamma more rapidly than is HLA class I. Kinetic analysis of transfectants reveals that IFN-gamma activates the TAP1 promoter more rapidly than the HLA-B7 class I promoter. A gamma-activating sequence (GAS) in the TAP1 promoter is necessary for the rapid induction by IFN-gamma. Two overlapping IFN consensus sequences contribute to the constitutive and TNF-induced expression of TAP1 but are not necessary for the IFN-gamma response. Moreover, IFN-gamma activates the GAS-binding protein Stat1 alpha much more rapidly than it induces the IFN consensus sequence-binding protein IRF-1, which mediates the response of the HLA-B7 class I promoter. We conclude that IFN-gamma uses different transcription factors to regulate the sequence of appearance of these interacting gene products.
HLA-DQ8 transgenic mice lacking endogenous class II molecules respond to house dust allergens: identification of antigenic epitopes.
Authors: Neeno T , Krco C J , Harders J , Baisch J , Cheng S , David C S
Published: December 2022

We have introduced HLA-DQ8 (HLADQB*0302 and HLA-DQA*0301) genes into A beta 0 knockout mice. Transgenic animals were immunized with a whole body extract of Dermatophagoides pteronyssinus (Der p), one of the causative agents of house dust mite allergy. Transgenic mice expressing HLA-DQ8 genes elicited HLA-DQ8-restricted responses driven by CD4+ T cells. Synthetic-overlapping peptides representing a major allergen of house dust mite (Der p 2) were synthesized and used as immunogens. HLA-DQ8+ mice responded to three peptides: 8 (residues 61-80), 11 (residues 91-110), and 13 (residues 111-129). The mice produced IL-2, IL-4, and IL-6 response to Der p challenge, suggesting a mixed Th1/Th2 response. These mice represent a new model for studies of the immune basis of allergy.
Tolerance of single, but not multiple, amino acid replacements in antibody VH CDR 2: a means of minimizing B cell wastage from somatic hypermutation?
Authors: Brown M , Rittenburg M B , Chen C , Roberts V A
Published: December 2022

Mutations in the heavy chain complementarity determining region 2 (CDR2) of the phosphocholine-specific T15 Ab can have a dramatic effect on the ability of the Ab to bind Ag. A panel of multisite mutants that had lost detectable binding to phosphocholine-containing Ags was previously created by saturation mutagenesis of the CDR2 region of T15. Based on the predicted importance of amino acid changes represented in the multisite mutants, we have created single-site mutations, yielding a panel of Abs with which to test 17 of the 19 CDR2 residues. Of the 17 positions examined, only one, Arg52, is intolerant to change, yielding a nonbinder phenotype even with conservative amino acid replacement. Mutation at two other sites, Ala50 and Tyr55, can yield a nonbinder phenotype depending on the amino acid replacement. Single-site mutations of the remaining 14 positions allowed retention of binding ability. Thus, except for positions 50, 52, and 55, multiple mutations must be introduced into the CDR2 region to create a nonbinder phenotype. We provide a newly refined model of T15, illustrating the structure and the interactions of the CDR2 region. Our results imply that introduction of point mutations would not normally delete Ag-binding ability until two or more mutations had accumulated. This would minimize potentially harmful effects of somatic mutation on Ig V region genes and improve the chance of survival for an Ab such as T15, which in its unmutated form is already well suited to bind Ag.
MHC class I-dependent presentation of exoerythrocytic antigens to CD8+ T lymphocytes is required for protective immunity against Plasmodium berghei.
Authors: White K L , Snyder H L , Krzych U
Published: December 2022

T lymphocytes are believed to play a major role in protection against malaria. Previous experiments using in vivo depletion of CD8+ T cells, reconstitution with CD8+ T splenic cells, and adoptive transfer of CD8+ CTL clones demonstrated that protection against the exoerythrocytic stage of the murine strain, Plasmodium berghei malaria, was CD8+ T cell-dependent. Despite evidence for the critical role of CD8+ CTL, neither the cellular nor the molecular requirements for CD8+ T cell induction or for recognition of malaria Ags are known. In this study, we wished to define the role of CD8+ T cells and MHC class I molecules by using the P. berghei malaria attenuated sporozoites (SPZ) protection model in beta 2-microglobulin (beta 2m) knockout (-/-) mice. In contrast to observations that beta 2m-/- mice are resistant to many infectious diseases by compensatory mechanisms involving non-class I-restricted T cells, we found that beta 2m-/- mice failed to be protected against P. berghei SPZ, although immunization with attenuated SPZ induced production of IL-2, INF-gamma, anti-circumsporozoite protein IgG, and proliferative T cells. The lack of compensatory mechanisms involving non-CD8+ T cells was particularly evident in the failure to adoptively transfer protective immunity with wild-type SPZ-immune splenic T cells. From our data it can be concluded that CD8+ T cells induced during immunization with attenuated SPZ must recognize liver-expressed Ags presented by class I molecules to engage effectively in a response leading to destruction of the malaria parasites.
Chemotactic factors stimulate CD18-dependent canine neutrophil adherence and motility on lung fibroblasts.
Authors: Burns A R , Simon S I , Kukielka G L , Rowen J L , Lu H , Mendoza L H , Brown E S , Entman M L , Smith C W
Published: December 2022

The mechanisms by which neutrophils migrate through the alveolar interstitium during acute lung inflammation are unknown. We wished to determine whether platelet-activating factor (PAF) and IL-8, two important mediators in neutrophil transendothelial migration, stimulated neutrophil adherence and motility on lung fibroblasts. Canine fibroblasts grown from lung explants were characterized by light and electron microscopy, and flow cytometry. Unstimulated neutrophils adhered poorly (less than 2%) to cultured fibroblasts. However, neutrophils stimulated with PAF (20-200 nM) showed a dose-dependent increase in adherence that was largely (70%) mediated by the beta 2 (CD11/CD18) integrins; adherence was less dependent (50%) on fibroblast intercellular adhesion molecule-1. Conversely, neutrophils stimulated with canine rIL-8 did not increase their adherence to fibroblasts. PAF-stimulated neutrophils were nonmotile on the surface of the fibroblast, but subsequent addition of rIL-8 (10(-8) M) induced motility that was entirely CD1 8 dependent. Fibroblasts stimulated with human rTNF-alpha or Escherichia coli endotoxin (LPS) were a significant source of IL-8 mRNA. In response to rTNF-alpha (50 U/ml), IL-8 mRNA was detected at 2 h by northern blot analysis; it peaked at 6 h and returned to baseline by 24 h. Fibroblasts stimulated with rTNF-alpha secreted IL-8 protein into the culture medium; secreted IL-8 was chemotactic for neutrophils. These data suggest that fibroblasts can function not only as an adhesive substrate, but also as a source of stimulation for neutrophil migration through the inflamed alveolar interstitium.
Heat shock mimics glucocorticoid effects on IFN-gamma-induced Fc gamma RI and Ia messenger RNA expression in mouse peritoneal macrophages.
Authors: Sivo J , Harmon J M , Vogel S N
Published: December 2022

Glucocorticoids activate or repress the expression of different genes. In murine macrophages, glucocorticoids exert opposing effects on the IFN-gamma-induced expression of Fc gamma RI and Ia mRNA and cell surface expression; they enhance IFN-gamma-induced Fc gamma RI mRNA and protein expression, yet inhibit IFN-gamma-induced Ia mRNA and protein expression. Recently, in transfected cell lines, heat shock (HS) has been shown to promote nuclear accumulation of the glucocorticoid receptor (GR), resulting in potentiation of certain GR-mediated responses. In this study, we compared the effects of HS and dexamethasone (DEX) treatment on the IFN-gamma induction of Fc gamma RI and Ia mRNA in murine primary peritoneal macrophages. Our results show that HS exerted the same opposing effects on these IFN-gamma-responsive genes as DEX at 37 degrees C. The glucocorticoid antagonist RU 486 blocked both DEX and HS-induced enhancement of IFN-gamma induction of Fc gamma RI, suggesting a common GR-mediated mechanism. While RU 486 also reversed DEX-induced repression of Ia mRNA expression, supporting a GR-mediated action, it did not affect HS-mediated repression, raising the possibility of a ligand-independent HS response pathway.
Regulation of cytokine production during development of autoimmune diabetes induced with multiple low doses of streptozotocin.
Authors: Herold K C , Vezys V , Sun Q , Viktora D , Seung E , Reiner S , Brown D R
Published: December 2022

Cytokines have been shown to play an important role in regulating tolerance to islet Ags and provoking destructive islet lesions. However, data from a number of experimental systems have been conflicting, and the role of cytokines produced by T lymphocytes at various stages of diabetes has not been clearly defined. We have studied the production of cytokines in the pancreas during the development of autoimmune diabetes induced in mice by administration of (5) low doses of streptozotocin (STZ) (MDSDM). Diabetes in this model is T lymphocyte dependent. We used techniques of semiquantitative PCR to identify and quantitate cytokines that are produced. We have found that IL-2, IL-4, TNF-alpha, and IFN-gamma are expressed by the time the fourth dose of STZ is given. In the same pancreas, all of these cytokines (including IL-4) may be found. However, expression of IFN-gamma, but not IL-4, was limited to intrapancreatic lymphocytes and was not detectable at extrapancreatic lymphoid sites. Moreover, mAbs against IFN-gamma, but not against IL-4 or IL-2, prevent hyperglycemia and insulitis in MDSDM, suggesting that IFN-gamma regulates development of disease. Cells in the pancreases of nondiabetic mice treated with anti-IFN-gamma mAb and STZ show enhanced expression of IL-4, but the prevention of disease is due to blockade of the IFN-gamma itself, and not due to secretion of IL-4, because systemic administration of IL-4 does not prevent MDSDM. Thus, our findings indicate that cytokines produced by Th1 (or T cytolytic 1) and Th2 (or T cytolytic 2) cells are found in the pancreases of mice developing autoimmune diabetes. IFN-gamma is responsible for progression to diabetes, and its production is limited to lymphocytes only at that site.
Blockade of platelet endothelial cell adhesion molecule-1 protects against myocardial ischemia and reperfusion injury in cats.
Authors: Murohara T , Delyani J A , Albelda S M , Lefer A M
Published: December 2022

Polymorphonuclear leukocytes have been shown to play an important role in myocardial ischemia (MI) and reperfusion (R) injury. Since blockade of platelet-endothelial cell adhesion molecules (PECAM-1) inhibits neutrophil transmigration in vitro and in vivo, the effects of a polygonal Ab directed against PECAM-1 were examined in a feline model of Ml/R. We established cross-reactivity of our anti-human PECAM-1 Ab to cat coronary vasculature and neutrophils by immunohistochemistry and flow cytometry. The anti-PECAM-1 Ab markedly blocked leukocyte transmigration into the peritoneal cavity of cats after glycogen-induced peritonitis. Then, anti-PECAM-1 Ab (1 mg/kg) was tested to determine whether it attenuates MI/R injury in a well characterized feline model of Ml and R. Anti-PECAM-1 Ab administered 10 min before R significantly inhibited the myocardial necrosis seen 4.5 h post-R compared with that in MI/R cats treated with control isotype rabbit IgG (12 +/- 2 vs 29 +/- 4% of area at risk; p less than 0.01) and significantly attenuated the rise in plasma creatine kinase activity (p less than 0.05). The Ab did not prevent increases in cardiac myeloperoxidase activity within the affected regions and did not significantly inhibit autologous neutrophil adhesion to coronary endothelium after stimulation of either neutrophils (by leukotriene B4) or coronary endothelium (by thrombin) in vitro. These results indicate that in vivo blockade of PECAM-1 significantly attenuates MI/R injury, presumably by inhibiting transendothelial migration of neutrophils.
Human CD4-internal antigen anti-idiotypic monoclonal antibody: induction of a CD4-specific response in humans.
Authors: Perosa F , Scudeletti M , Imro M A , Dammacco F , Indiveri F
Published: December 2022

We previously showed that anti-idiotypic mAb (mAb2) F16-16D7 (16D7) to the paratope (or paratope-related idiotope) of the anti-CD4 mAb HP2/6 induces anti-CD4 Abs in BALB/c mice. In view of the potential ability of 16D7 to induce anti-CD4 Ab in humans and the potential benefits of anti-CD4 Abs in the treatment of autoimmune diseases, we evaluated the immunologic response to and assessed the safety of four 2-mg 16D7 s.c. injections in one patient with systemic lupus erythematosus (SLE) and one with rheumatoid arthritis (RA). 16D7 induced anti-isotypic and anti-anti-idiotypic Abs (Ab3), which were almost exclusively of the IgG isotype. Ab3 specifically reacted with 16D7 as they inhibited its binding to mAb HP2/6. Although Ab3 did not react with cellular or recombinant CD4 (rCD4), single-cell enzyme-linked immunospot assays of anti-CD4 Ab production revealed many more spot-forming cells in rCD4- and 16D7-coated wells than in wells coated with BSA or 16D7 isotype-matched MK2-23. Spot-forming anti-CD4 Abs were specifically induced by 16D7, since rCD4-dependent spot formation 1) was not observed with PBL from one patient with SLE, one with mixed connective tissue disease, and one with melanoma immunized with MK2-23; and 2) was inhibited by 16D7 and not by MK2-23. Spot-forming anti-CD4 Abs recognize a CD4 epitope identical (or closely related) to that seen by HP2/6, since this specifically inhibited spot formation. A substantial, although transient, CD4+ T cell depletion was only observed in the RA patient. Local and/or general toxicity and laboratory and/or clinical signs indicative of immunodepression or diseases relapse were not observed during an 18-month follow-up.
Rheumatoid factor idiotypic and antigenic specificity is strongly influenced by the light chain VJ junction.
Authors: Zhang M , Spey D , Ackerman S , Majid A , Davidson A
Published: December 2022

The aim of this study was to define the structural basis for rheumatoid factor (RF) specificity and for the expression of the RF light chain-associated Ids, 4C9 and 6B6.6, by determining the reactivity of recombined heavy and light chains of Ig derived from monoclonal B cell lines of patients with rheumatoid arthritis and of light chains with site-directed mutations. We found that expression of the 4C9 and 6B6.6 Ids resulted from use of the VkIIIa genes Humkv 328 and Vg, but only in the presence of a permissive VJ junction. Expression of the Ids was independent of heavy chain use for the Humkv328-encoded light chains, but was highly dependent on the associated heavy chain for the Vg-encoded light chains. The RF specificity of the Abs was primarily heavy chain dependent, but the light chain VJ junction was critical in determining the relative avidity of the Abs for Fc. Our study points to the critical contribution of the somatically generated VJ junction to RF autoantibody specificity and to the expression of the two RF-associated Ids studied.
CD45 monoclonal antibodies inhibit TCR-mediated calcium signals, calmodulin-kinase IV/Gr activation, and oncoprotein 18 phosphorylation.
Authors: Shivnan E , Clayton L , Allridge L , Keating K E , Gullberg M , Alexander D R
Published: December 2022

The effects of a pan-CD45 mAb (CD45.2) on TCR-mediated signaling pathways were investigated in Jurkat T cells. The simultaneous addition of CD45 mAb with an activating OKT3 mAb had little effect on TCR-stimulated signals. However, when Jurkat cells were exposed to the CD45 mAb for 10 to 20 min before the addition of OKT3, a partial uncoupling of the TCR from intracellular signals was observed. The maximal increase in intracellular calcium was inhibited 47 +/- 10% (n = 11, range 33-67%), whereas no inhibition of inositol trisphosphate production was detected. The transient TCR-mediated activation of the Ca2+/calmodulin-activated kinase IV/Gr was also inhibited by the CD45 mAb, and this was reflected in a 50 to 60% inhibition in the TCR-stimulated generation of the p21 and p23 phosphoisomers of oncoprotein 18, a Ca2+/calmodulin-activated kinase IV/Gr substrate recently implicated in cell cycle regulatory events. Oncoprotein 18 is also a substrate for mitogen- activated protein kinase, but no inhibition by the CD45 mAb of TCR-triggered mitogen-activated protein kinase activation was observed. The CD45 mAb was therefore selective in causing the uncoupling of the TCR from calcium signals and calcium-regulated events without promoting a general inhibition of all TCR-mediated signals. Confocal microscopy revealed that binding of the CD45 mAb caused patching of CD45 molecules at the cell surface and, unexpectedly, a marked redistribution of intracellular CD45. However, no change was observed in the total level of CD45 expressed at the cell surface. Aggregation of CD45 at the cell surface may result in its sequestration from its tyrosine kinase substrates, with a consequent selective uncoupling of the TCR from intracellular signaling pathways.
Suppression of murine allergic contact dermatitis by CTLA4Ig. Tolerance induction of Th2 responses requires additional blockade of CD40-ligand.
Authors: Tang A , Judge T A , Nickoloff B J , Turka L A
Published: December 2022

Blockade of costimulation through the B7-CD28 pathway by CTLA4Ig can lead to Ag-specific T cell tolerance. Most models studied to date involve a Th1-dependent response. To investigate whether the tolerizing effects of CTLA4Ig might vary depending upon the cytokine nature of the immune response, we studied its effects on contact hypersensitivity (CHS) in response to two allergens. In BALB/c mice, both 2,4-dinitrofluorobenzene (DNFB) and FITC induce CHS. However, the DNFB response is Th1-predominant, while the FITC response is Th2 predominant. CTLA4Ig treatment during primary sensitization induced long-lasting unresponsiveness to DNFB, with 88% and 76% inhibition of primary (first challenge) and secondary (re-sensitization and re-challenge) CHS, respectively. In contrast, CTLA4Ig inhibited primary CHS to FITC by over 82% but had little effect on secondary CHS. Consistent with its effects on CHS, the suppressive effect of CTLA4Ig on Th2 cells was short-lived in FITC-sensitized mice, while Th1-like cytokine-secreting cells remained reduced in DNFB-sensitized mice, even when the animals were rechallenged with DNFB. The addition of anti-CD40L Ab to CTLA4Ig was able to induce long-lasting unresponsiveness to FITC, indicating the ability of cells mounting this Th2 response to receive costimulatory signals through either pathway. In conclusion, CHS can be mediated by both Th1 and Th2 cells, and the ability of CTLA4Ig to lead to long-standing nonresponsiveness in this model depends on the nature (i.e., cytokine profile) of the immune response.
Epitope mapping by mass spectrometry: determination of an epitope on HIV-1 IIIB p26 recognized by a monoclonal antibody.
Authors: Parker C E , Papac D I , Trojak S K , Tomer K B
Published: December 2022

Matrix-assisted laser desorption mass spectrometry in combination with proteolytic protection assays has been used to identify the functional epitope on HIV-1 IIIB p26 recognized by a mAb. In this procedure, the intact protein is affinity bound to an immobilized mAb under physiologic conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Protected residues were identified by matrix-assisted laser desorption mass spectrometry based on their m.w. With this approach, an 11-residue sequence was identified as the most tightly affinity-bound fragment. in addition, two less tightly bound segments were observed. These latter two residues may contain elements of a discontinuous epitope or may be residues involved in a wider contact area. The combination of matrix-assisted laser desorption and proteolytic epitope footprinting has been applied to the determination of the epitope on a recombinant protein recognized by a mAb but should be equally applicable to the definition of an epitope on a native protein in its natural folded conformation.
Stimulation of phosphatidylinositol turnover is a key event for Fas-dependent, activation-induced apoptosis in human T lymphocytes.
Authors: Izquierdo M , Ruiz-Ruiz M C , López-Rivas A
Published: December 2022

We have analyzed the requirements for activation-induced apoptosis in Jurkat T cells expressing the heterologous human muscarinic type 1 receptor (HM1R; J-HM1-2.2 cells) that is coupled to phosphatidylinositol turnover through a protein tyrosine kinase-independent, G protein-regulated mechanism. Triggering of HM1R with the agonist carbachol is sufficient to induce apoptosis in J-HM1-2.2 cells. Apoptosis is also induced in J-HM1-2.2 cells by triggering of the TCR. Calcium influx, intracellular Ca2+ increase, calcineurin function, and de novo protein synthesis are necessary for receptor-controlled apoptosis. However, blocking protein kinase C with a specific inhibitor does not abrogate receptor-induced apoptosis. Furthermore, HM1R-induced apoptosis is inhibited by blocking Fas ligand/Fas interaction with an antagonist anti-Fas Ab, and Fas ligand mRNA and protein are expressed in J-HM1-2.2 cells stimulated through the HM1R. Therefore, protein tyrosine kinase activation is not an absolute requirement for receptor-controlled Fas ligand expression. Taken together, the results demonstrate that stimulation of phosphatidylinositol turnover can induce apoptosis through a Fas-dependent mechanism that requires calcineurin stimulation, but not protein kinase C activation.
Selection and binding of peptides to human transporters associated with antigen processing and rat cim-a and -b.
Authors: Wang P , Gyllner G , Kvist S
Published: December 2022

Cytotoxic T lymphocytes recognize antigenic peptides presented by MHC class I molecules. The peptides are generated in the cytosol by proteasomes, and probably also other proteases, and are then translocated into the endoplasmic reticulum (ER) lumen. The transporters associated with Ag processing (TAP) are key molecules for transporting peptides from the cytosol to the lumen of the ER. Using semipermeabilized cells, TAP-dependent peptide translocation was demonstrated, and the selectivity of peptide translocation was based on the carboxyl-terminal amino acid of peptides. We have examined peptide binding proteins in the ER membrane and the selection of peptides for binding to TAP by using a panel of peptides of different sequences and carboxyl-termini as well as peptides containing D amino acids. Peptides bound to TAP molecules in the absence of ATP. The presence of ATP induced binding of peptides to two additional membrane proteins (58 and 43 kDa). The selection of peptides by TAP molecules was based on peptide sequence and the carboxyl-terminal amino acid. Peptides containing D amino acid did not bind to TAP molecules. Rat cim-a and -b selected peptides differently, and selection was not only dependent on the carboxyl-terminal residue of the peptide, but included an influence of the peptide sequence. The different off-rates after peptide binding to TAP, indicated a dual binding step of peptide to TAP. ATP regulated the off-rate of peptides at a high affinity binding step. Our results demonstrate that the binding of peptides to TAP molecules is specific and most likely involves a multiple step pathway.
A synthetic peptide derived from the tumor-associated protein mdm2 can stimulate autoreactive, high avidity cytotoxic T lymphocytes that recognize naturally processed protein.
Authors: Dahl A M , Beverley P C , Stauss H J
Published: December 2022

Studies in melanoma patients have shown that unaltered self proteins can function as targets for tumor-reactive CTL. Here, we have investigated in a murine model whether autoreactive CTL can be found against the widely expressed proteins cyclin D1, mdm2, and p53, which are frequently overexpressed in transformed cells. Sixteen MHC class I binding peptides were identified in these proteins, and seven of them consistently stimulated primary CTL in vitro. Avidity measurements revealed that the avidity of peptide-induced CTL differed by >1000-fold. The highest avidity CTL were induced by a peptide derived from mdm2. These CTL recognized target cells expressing mdm2 endogenously, while CTL generated against the remaining peptides were of lower avidity and did not recognize cells expressing relevant proteins endogenously. Generation of high avidity anti-mdm2 CTL required several cycles of peptide stimulation, suggesting that the CTL precursor frequency was low. The data show the normal T cell repertoire contains small numbers of potentially autoreactive CTL. Expansion of these CTL may lead to beneficial autoimmunity against tumors, but, equally, it may be the basis of detrimental autoimmune diseases.
T cell vasoactive intestinal peptide receptor subtype expression differs between granulomas and spleen of schistosome-infected mice.
Authors: Metwali A , Elliott D , Blum A M , Li J , Sandor M , Weinstock J V
Published: December 2022

Granulomas form in the liver and intestines of mice infected with the parasite Schistosoma mansoni. Vasoactive intestinal peptide (VIP) is a neurokine that can modulate aspects of the immune response by acting through receptors within the granuloma. Cloned are two novel VIP receptor (VIPR) mRNAs (VIPR1 and VIPR2) that also bind a second neurokine called pituitary adenylated cyclase-activating polypeptide (PACAP). The objective of this study was to determine if granulomas express either VIPR1 or VIPR2. Using a radioligand-binding assay, it was established that PACAP is as effective as VIP at displacing radiolabeled VIP from splenocytes and granuloma cells, and that most if not all VIPRs in the spleen and granulomas bind PACAP. PCR amplification of reverse transcribed RNA determined that granulomas express both VIPR1 and VIPR2 mRNAs. Gel electrophoresis and nucleotide sequencing confirmed the authenticity of the PCR products. Also, both receptor subtypes were amplified from several granuloma CD4+ T cell lines; yet reverse transcribed RNA from T cell-depleted, dispersed granuloma cells had only VIPR1 RNA. It is notable that reverse transcriptase-PCR detected only VIPR1 in the thymus and spleen, which are organs rich in T lymphocytes. Thus, the granulomas and spleens from mice with schistosomiasis contain cells that display authentic VIP/PACAP receptors. Moreover, these data suggest that T cells in different compartments vary in VIPR subtype expression. VIPR1 and VIPR2 may have different physiologic roles in inflammation.