bluuebunny/crossref_metadata_2025 huggingface dataset:
Extracellular calcium results in a conformational change in Mac-1 (CD11b/CD18) on neutrophils. Differentiation of adhesion and phagocytosis functions of Mac-1.
Authors: Graham I L , Brown E J
Published: December 2022

Mac-1 is a leukocyte integrin involved in multiple adhesion phenomena and also in the phagocytosis of particles that are bound via CR1 and the IgG FcR. We examined the divalent cation requirements for the Mac-1-dependent processes of adhesion and receptor-mediated phagocytosis. With phorbol ester stimulation, both processes occurred in the absence of extracellular Ca+2. In Ca+2-containing buffer, IB4, an anti-beta 2 mAb, inhibited both adhesion and CR1-mediated ingestion. In the absence of Ca+2, IB4 no longer had any effect on ingestion, although it continued to inhibit adhesion to protein-coated plastic completely. This demonstrates that the role of Mac-1 in adhesion is distinct from its role in phagocytosis. In addition, 1) IB4 did not change intracellular Ca+2 homeostasis; 2) as little as 300 nM free extracellular Ca+2 could restore the ability of IB4 to inhibit ingestion; and 3) in the absence of extracellular Ca+2, Mac-1 was more susceptible to enzymatic cleavage. Together these data suggest a Ca+2-dependent conformational change in Mac-1, which allows distinction of the roles of Mac-1 in phagocytosis and adhesion.
Modulation of monocyte chemotactic function in inflammatory lesions. Role of inflammatory mediators.
Authors: Katona I M , Ohura K , Allen J B , Wahl L M , Chenoweth D E , Wahl S M
Published: December 2022

Monocyte recruitment and accumulation in the synovial tissue is pivotal in the evolution of rheumatoid arthritis (RA). In the present study we examined the chemotactic potential of monocytes obtained from synovial fluid (SF) of patients with RA. Functionally, SF monocytes exhibited greatly diminished chemotactic activity to C5a compared with monocytes from the peripheral blood. In contrast, their chemotactic responsiveness to the synthetic peptide, FMLP, was nearly normal. To define a mechanism for this differential chemotactic dysfunction, cell-surface receptors for C5a (C5aR) and FMLP (FMLP-R) were evaluated. Whereas FMLP-R expression was similar on both blood and inflammatory monocytes, C5aR expression was markedly reduced on SF cells. Because decreased C5a binding in certain RA SF samples could not be attributed to free C5a, known or suspected components of inflammatory SF were evaluated for their ability to modulate chemotactic ligand receptors. Bacterial products including LPS and streptococcal cell walls, which are potent monocyte activators, down-regulated C5aR without affecting FMLP-R. Moreover, the cytokines IFN-gamma and granulocyte-macrophage-CSF selectively decreased C5aR in parallel with decreased in vitro chemotactic activity to C5a. Thus, these data indicate that 1) synovial effusions may contain C5a and/or inflammatory mediators that modulate phenotypic and functional changes in monocytes, 2) chemotactic ligand receptors are independently regulated in inflammatory lesions, and 3) decreased C5aR expression and chemotactic potential likely provide a mechanism whereby monocyte-macrophages persist within the inflamed synovium.
The role of tyrosine phosphorylation in signal transduction through surface Ig in human B cells. Inhibition of tyrosine phosphorylation prevents intracellular calcium release.
Authors: Lane P J , Ledbetter J A , McConnell F M , Draves K , Deans J , Schieven G L , Clark E A
Published: December 2022

Cross-linking surface Ig on human B cells, or the TCR complex on T cells leads to the rapid appearance of newly tyrosine phosphorylated proteins. This is associated with inositol phospholipid turnover and a rise in intracellular calcium. Incubation of human B or T lymphocytes with the tyrosine kinase inhibitors, herbimycin and genistein, inhibits new tyrosine phosphorylation after receptor-linked activation. This is associated with complete abrogation of the increase in intracellular calcium in these lymphocytes and inhibition of inositol phospholipid turnover. Herbimycin- and genistein-treated lymphocytes are nevertheless still capable of responding to aluminum fluoride with a rise in intracellular calcium. These data support the contention that a B cell-associated protein tyrosine kinase regulates signal transduction via phospholipase C. CD45, the membrane associated protein tyrosine phosphatase, and PMA that activates protein kinase C, both inhibit the calcium response in B lymphocytes induced by receptor cross-linking. PMA and cross-linking CD45 both induced the appearance of tyrosine phosphorylated proteins in human B cells, although the pattern is quite distinct from that seen when surface lg is cross-linked. However, the induction of new tyrosine phosphorylation by anti-mu does not appear to be affected by these reagents. Although this may reflect an insensitivity of the tyrosine phosphorylation assay, it could indicate that regulation of the calcium response and regulation of the tyrosine kinase can be independent processes.
Neutrophil adhesion to xenogeneic endothelium via iC3b.
Authors: Vercellotti G M , Platt J L , Bach F H , Dalmasso A P
Published: December 2022

Neutrophils are thought to play an important role in the pathogenesis of hyperacute rejection, a dramatic form of tissue injury caused by the reaction of antigraft antibodies with endothelial cells of an organ allograft or xenograft. We asked whether the interactions of human polymorphonuclear leucocytes (PMN) with xenogeneic endothelium might be promoted by the binding of natural anti-endothelial antibodies and complement by using porcine aortic endothelial cells (PAEC), human serum, and human PMN in an in vitro model of hyperacute rejection. Pretreatment of PAEC with 10% human serum followed by washing markedly increased PMN adhesion from 15.7 +/- 1.8% to 62.5 +/- 3.6% (p less than 0.001). Complement and anti-endothelial antibodies were necessary for the increase, because heat-inactivated serum or serum depleted of IgM did not significantly increase PMN adhesion to treated endothelium. The induction of increased PMN adhesion to PAEC by human serum was observed within 1 min. The essential role of complement was defined using complement-depleted serum. Ten percent C2-deficient serum did not increase PMN adhesion whereas 10% C5-depleted or 10% C8-depleted serum caused the same increase in PMN adhesion as observed with normal human serum. These results suggested that C3 might play a critical role in enhanced neutrophil adhesion. In fact, PAEC treated with 10% human serum for 15 min and incubated with an F(ab')2 antihuman C3 for 10 min completely abolished the enhanced adhesion. PAEC treated with 10% human serum or C5-depleted serum displayed fluorescence of iC3b whereas monolayers treated with heat-inactivated serum or C2-deficient serum were non-reactive. The enhanced PMN adhesion to serum-treated PAEC was mediated through neutrophil receptors binding iC3b because mAb directed against CD11b/CD18 inhibited the serum-enhanced adhesion of PMN. We conclude that PMN adhesion to endothelium can be significantly enhanced by the endothelial deposition of iC3b.
Cytokine regulation of complement receptor-mediated ingestion by mouse peritoneal macrophages. M-CSF and IL-4 activate phagocytosis by a common mechanism requiring autostimulation by IFN-beta.
Authors: Sampson L L , Heuser J , Brown E J
Published: December 2022

Macrophage CSF (M-CSF, CSF-1) and IL-4 are two cytokines known to have effects on mature monocytic phagocytes in vitro. In this report we show that M-CSF and IL-4 activate resident mouse peritoneal macrophages to ingest particles via their C3b and C3bi receptors, which are not capable of mediating ingestion in resting cells. IgG-mediated ingestion was also increased by IL-4 and M-CSF. IL-1, IL-2, TNF-alpha, and IFN-gamma were not able to stimulate C receptor-mediated ingestion. Stimulation by IL-4 and M-CSF is dependent upon high cell density and greater than 24-h exposure to the cytokine. Interestingly, antibody to IFN-alpha/beta and mAb to IFN-beta inhibited the enhanced ingestion caused by both M-CSF and IL-4. However, neither IFN-alpha nor IFN-beta alone stimulated C receptor-mediated ingestion. M-CSF did not affect the ligand-independent distribution of CR3 on the macrophage surface. We conclude that two apparently unrelated cytokines, M-CSF and IL-4, both enhance macrophage phagocytosis of C and IgG-coated targets via a common pathway in which autocrine stimulation with IFN-alpha/beta is necessary but not sufficient.
Reduction in the number of UCHL-1+ cells and IL-2 production in the peripheral blood of patients with visceral leishmaniasis.
Authors: Cillari E , Milano S , Dieli M , Maltese E , Di Rosa S , Mansueto S , Salerno A , Liew F Y
Published: December 2022

PBMC from patients with visceral leishmaniasis (VL), before and after successful antimony therapy, were analyzed for their phenotypes and for their ability to produce IL-2 and IFN-gamma and to proliferate against PHA and leishmanial Ag. In agreement with results of earlier studies, PBMC from active VL patients showed a markedly reduced proliferative response and IL-2 and IFN-gamma production, compared with those of healthy controls. The levels of CD4+ and CD8+ T cells were within the normal range, but there was a significant decrease in UCHL-1+ cells (helper-inducer), compared with healthy individuals. The inhibited cellular responses, and lymphokine secretion and decreased level of UCHL-1+ cells in the PBMC of the VL patients returned to the normal range after successful chemotherapy. PBMC from active VL patients were fractionated into adherent cells and nonadherent cells, and the non-adherent were further fractionated into UCHL-1+ and UCHL-1- subpopulations. Results from cell depletion and reconstitution experiments suggest that the IL-2 production by nonadherent cells stimulated with PHA was inhibited by adherent cells, but the IL-2 production by nonadherent cells in response to specific Ag was not. In contrast, UCHL-1- cells seem to mediate the inhibition of Ag-driven IL-2 production by nonadherent cells but not mitogen-stimulated IL-2 secretion by nonadherent cells. Ag-specific IL-2 production principally involves UCHL-1+ cells.
Transient expression of the CD2 cell surface antigen as a sortable marker to monitor high frequency transfection of human primary B cells.
Authors: Pilon M , Gullberg M , Lundgren E
Published: December 2022

We have used the T cell surface molecule CD2 gene, expressed from the human cytomegalovirus promoter as a reporter to optimize a transfection system for human primary B cells. The CD2-encoding DNA was transfected into cells by electroporation and transient expression was monitored by flow cytometric analysis. By using our optimal electroporation conditions on activated primary B cells, more than 30% of the resulting viable cells expressed CD2 on the cell surface. Moreover, unactivated primary B cells could also be transfected using this system but subsequent expression of CD2 required cellular activation. Magnetic beads or plastic culture bottles coated with anti-CD2 antibodies have been used to selectively purify transfected cells. The high transfection efficiency combined with the ability to specifically purify transfected cells may allow future studies on specific genes transiently expressed in human primary B cells.
Induction of DNA fragmentation in chronic B-lymphocytic leukemia cells.
Authors: McConkey D J , Aguilar-Santelises M , Hartzell P , Eriksson I , Mellstedt H , Orrenius S , Jondal M
Published: December 2022

Chronic lymphocytic leukemia of B cell type (B-CLL) is a neoplastic disorder characterized by the accumulation of small resting lymphocytes in the periphery. The phenotype of these cells suggests that they are "frozen" at an early stage of maturation. Glucocorticoid hormones are commonly used to treat patients with B-CLL, resulting in a reduction in the peripheral lymphocyte count by an undefined mechanism. Here we report that glucocorticoids stimulate DNA fragmentation characteristic of a suicide process known as apoptosis or programmed cell death (PCD) in suspensions of cells from patients with B-CLL. The effects can be mimicked by Ca2+ ionophore and involve a sustained increase in the cytosolic Ca2+ concentration. Specific antibodies binding to membrane-associated IgM on the leukemic cells can also induce PCD by a similar mechanism. Phorbol esters block DNA fragmentation and cell killing in response to all of the agents, suggesting that activation of protein kinase C desensitizes the cells to PCD. Targeting the B-CLL cells with antibodies that induce an unbalanced, sustained Ca2+ increase may therefore represent a rational strategy for the destruction of leukemic cells.
Transforming growth factor-beta-induced inhibition of T cell function. Susceptibility difference in T cells of various phenotypes and functions and its relevance to immunosuppression in the tumor-bearing state.
Authors: Tada T , Ohzeki S , Utsumi K , Takiuchi H , Muramatsu M , Li X F , Shimizu J , Fujiwara H , Hamaoka T
Published: December 2022

The present study investigates the nature of humoral component(s) generated in tumor-bearing hosts to induce immune dysfunction of T cells. Cell-free ascitic fluid and culture supernatant (SN) were obtained from the ascites and cultures allowing MH134 hepatoma cells to grow. These ascites and SN samples were tested for their abilities to influence the generation of CTL responses to TNP and alloantigens. The generation of the anti-TNP CTL responses that require self H-2-restricted CD4+ Th cells was markedly suppressed by addition of the ascites or SN under conditions in which these samples did not inhibit anti-allo CTL responses capable of using alternate pathways of allo-restricted CD4+ and CD8+ Th. The activation of CD8+ CTL precursors and CTL activity were also resistant to the ascites or SN. The ascites- or SN-induced suppressive effect to which CD4+ Th were most susceptible was found to be mediated by transforming growth factor-beta (TGF-beta) activity, because: 1) the TGF-beta activity was detected in the MH134 ascites and culture SN; 2) the suppression of CD4+ Th function required for anti-TNP CTL responses was almost completely prevented by addition of anti-TGF-beta antibody to cultures and; 3) rTGF-beta also induced similar patterns of immunosuppression to those observed by ascites or SN. These results indicate that TGF-beta produced by tumor cells induces deleterious effects on T cell, especially on the CD4+ Th subset, and provide an explanation for the molecular mechanism underlying the previously observed CD4+ Th-selective suppression in the tumor-bearing state.
TraT: a powerful carrier molecule for the stimulation of immune responses to protein and peptide antigens.
Authors: Croft S , Walsh J , Lloyd W , Russell-Jones G J
Published: December 2022

A number of integral membrane proteins (Imps) isolated from Escherichia coli have been examined for their ability to generate serum antibody responses in the absence of adjuvant. These proteins were found to stimulate high titers of serum antibody when injected into rabbits or mice in saline. The antibody titers elicited were not significantly increased by the addition of a powerful adjuvant such as IFA. Covalent conjugation of BSA, of the DNP group, and of a peptide Ag from Plasmodium falciparum to these protein carriers resulted in a significant enhancement of the immune response to the conjugated material in comparison with the response elicited when the immunogen was injected without adjuvant or was not conjugated to Imps. The antibody response to these conjugates could not be significantly increased by the addition of IFA. Thus, the Imps of E. coli represent powerful carrier molecules which, when injected into mice and rabbits, are not only capable of generating high titers of antibody to themselves, but also to molecules conjugated to them. Immunization with immunogens coupled to these proteins results in the production of high titers of antibody without the need for oil-based adjuvants, thereby avoiding the unwanted side effects of such adjuvants.
Growth regulation of a human mature B cell line, B104, by anti-IgM and anti-IgD antibodies.
Authors: Kim K M , Yoshimura T , Watanabe H , Ishigami T , Nambu M , Hata D , Higaki Y , Sasaki M , Tsutsui T , Mayumi M
Published: December 2022

An EBNA- human B lymphoma cell line, B104, was established. B104 cells express IgD as well as IgM on their surface, which is thought to be a basic characteristic of mature B cells. The growth of B104 cells was inhibited by treatment with a panel of anti-IgM antibodies. Cell cycle analyses revealed that the transition of B104 cells from the G2/M to the G0/G1 phase of the cell cycle was markedly inhibited by treatment with anti-IgM antibodies. Progression of B104 cells to the M phase of the cell cycle was found to be suppressed in the presence of anti-IgM antibodies. In contrast, both the entrance of G0/G1 phase cells into the S phase and the progression of S phase cells to the G2/M phase of the cell cycle did not seem to be inhibited significantly by treatment with anti-IgM antibodies. These results indicate that the mechanism of the inhibition of growth of B104 cells by anti-IgM antibodies is blockage of the transition from the G2 to the M phase of the cell cycle. In contrast to anti-IgM antibodies, anti-IgD antibodies could not cause growth inhibition of B104 cells at all. B cell growth factors such as IL-4 and IL-6 had no effect on the inhibition of growth of B104 cells by anti-IgM antibody. IFN-alpha and -beta, which have no B cell growth factor activity, did increase the number of cells that survived the treatment with anti-IgM antibodies. B104 is an excellent experimental model for the study of the mechanism of signal transduction through sIg as well as the functional difference between sIgM and sIgD.
Mechanism of hybrid resistance. The role of a natural antibody in parental bone marrow cell rejection.
Authors: Cabaniols J P , Bolonakis-Tsilivakos I , Cibotti R , Churaqui E , Duc H T , Halle-Pannenko O , Kosmatopoulos K
Published: December 2022

Hybrid resistance (HR) to parental bone marrow growth is specifically directed against hemopoietic histocompatibility (Hh-1) Ag that are present in parental bone marrow cells (bmc). The mechanism of HR seems to be a multistep process. According to a model we proposed earlier, a T cell recognizes the Hh-1 Ag and stimulates a macrophage to secrete IFN-alpha/beta (recognition phase). IFN-alpha/beta activates a NK-like cell that specifically kills the parental bmc (effector phase). We have also described in a previous paper that serum from resistant F1 hybrids contains a humoral factor that seems to be involved in the effector phase of HR. In the present work, we study the role and the nature of this humoral factor. Our results show that this humoral factor: 1) is present in all resistant H-2Db heterozygous F1 hybrids we have tested but not in nonresistant H-2Db homozygous mice; 2) seems to recognize the Hh-1b Ag because it is absorbed on bmc from Hh-1b mice but not on bmc from Hh-1d and Hh-1- mice; and 3) is an IgG1 Ig (natural antibody). These results could help us to explain the specificity of HR at the effector phase by supposing that this natural antibody recognize the Hh-1 Ag and enable NK-like cells to kill parental bmc cells in Hh-1 specific manner.
Hyperphosphorylation of CD20 in hairy cells. Alteration by low molecular weight B cell growth factor and IFN-alpha.
Authors: Genot E , Valentine M A , Degos L , Sigaux F , Kolb J P
Published: December 2022

Hairy cell leukemia (HCL) is a B cell tumor affecting the pre-plasma stage of B cell differentiation. Hairy cells produce B cell growth factor (BCGF)-related growth factor(s) and we have previously shown that low mol wt (LMW)-BCGF-induced proliferation of hairy cells is inhibited in vitro and in vivo by IFN-alpha. We therefore suggested that this effect might contribute to the exquisite sensitivity of HCL to IFN-alpha therapy. To elucidate the mechanism involved in the therapeutic effect of IFN-alpha, we have analyzed the pattern of phosphorylated proteins in hairy cells. We detected the presence of a hyperphosphorylated protein with a molecular mass of about 35 kDa. This protein was identified as the CD20 molecule (B1), which is a structurally unique phosphoprotein exclusively detected on B cells and expressed during most stages of B cell development. Incubation of hairy cells with mitogenic concentrations of LMW-BCGF induces an additional increase in CD20 protein phosphorylation. In contrast, preincubation of cells with IFN-alpha, but not IFN-gamma, decreases both basal and LMW-BCGF-induced CD20 phosphorylation. CD20 phosphorylation in hairy cells is also reduced after in vivo IFN-alpha administration. In contrast, in one case of a patient unresponsive to IFN-alpha therapy, CD20 phosphorylation is not altered by in vitro IFN-alpha treatment, whereas LMW-BCGF still elicits CD20 phosphorylation stimulation. Our results suggest that IFN-alpha may act in HCL, at least in part, by inhibiting leukemic cell proliferation via regulation of phosphorylation, since CD20 phosphorylation is thought to be associated with cellular proliferation. A model involving dysregulation of CD20 is discussed.
Characterization of fragments of the murine Ia-associated invariant chain.
Authors: Mehringer J H , Harris M R , Kindle C S , McCourt D W , Cullen S E
Published: December 2022

It has been proposed that invariant chain (Ii), a nonpolymorphic, transmembrane glycoprotein found in noncovalent association with Ia molecules, may function to protect the Ia Ag-binding site from association with self-peptides during Ia synthesis. Selective binding of foreign antigenic peptides could then be allowed by the dissociation of Ii molecules from Ia in the appropriate intracellular compartment. In this study, we have examined the structure and intracellular trafficking patterns of a putative proteolytic product of Ii, p25. We found that p25 is a non-membrane-bound fragment of Ii with an N terminus beginning at Met98 of the Ii sequence. p25 is formed at a very early stage of Ii synthesis in the rough endoplasmic reticulum rather than in a post-Golgi Ag-processing compartment. We have also characterized a second Ii-related species, p28, which has not been reported previously. The p28 form of Ii, unlike p25, is generated under acidic conditions similar to those found during Ag processing.
Paratope- and framework-related cross-reactive idiotopes on anti-acetylcholine receptor antibodies.
Authors: Verschuuren J J , Graus Y M , Tzartos S J , van Breda Vriesman P J , De Baets M H
Published: December 2022

Cross-reactive idiotopes are a possible target for therapeutical interventions in autoimmune diseases. To investigate their role in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG) we analyzed the Id of rat anti-AChR mAb 6, 35, 61, 65 and a control myeloma protein IR27. Anti-Id 6, 35, 61, 65 bound in a direct binding assay with various affinity to all rat anti-AChR mAb that were tested. Anti-Id IR27 recognized none of the anti-AChR mAb. The specificity of these crossreactions was confirmed by inhibition studies with anti-AChR mAb and two control rat myeloma proteins (IR27 and IR241). In addition, the Id expression on mAb D6, a mouse anti-human AChR mAb was recognized by anti-Id 6, 35, and 65. Anti-Id, except anti-Id IR27, bound to affinity purified IgG from the sera of rats with EAMG, but not to preimmune Lewis IgG. These results suggest extensive sharing of idiotopes among anti-AChR mAb, which are also present in EAMG serum. Anti-AChR mAb against the main immunogenic region (6, 35, 65) from different rat strains, shared at least one paratope-related cross-reactive idiotopes. In the view of the fact that anti-main immunogenic region antibodies might form a predominant fraction of the polyclonal response against AChR, it is conceivable that an anti-Id recognizing these antibodies could have therapeutical applications as for example a selective immune absorbent or in immunotoxin therapy.
Immobilization and internalization of mutated IgE receptors in transfected cells.
Authors: Mao S Y , Varin-Blank N , Edidin M , Metzger H
Published: December 2022

Earlier studies have shown that the mast cell receptor IgE (Fc epsilon RI) for is expressed on COS-7 cells transfected with the cDNA for each of the three types of subunits that form the tetrameric, alpha beta gamma 2, receptor. Although such transfected COS cells fail to exhibit some of the early biochemical perturbations initiated by aggregation of the receptor on normal mast cells and related tumor lines, we show here that other characteristics of the endogenous Fc epsilon RI are retained. Thus, the unaggregated transfected wild-type receptors were found to have a restricted translational diffusion similar to that observed for endogenous receptors on mast cells as assessed by fluorescence photobleaching and recovery. Similarly, as with endogenous receptors the mobility of transfected receptors was sharply reduced when the receptors were aggregated by reaction with small oligomers of IgE. In addition, aggregation of the transfected Fc epsilon RI caused them to be internalized by the COS cells by a cytochalasin-sensitive mechanism, albeit at a considerably slower rate than was seen with endogenous receptors on mast cells or with transfected receptors in a line of receptor-deficient mast cells. We also examined the mobility and internalization before and after aggregation, of some 13 different combinations of receptor subunit mutants in which one or more of the five cytoplasmic domains of the receptor had been truncated. Our results show that whatever interactions between the receptor and cellular components may account for the phenomena we studied, such interactions do not critically depend upon the bulk of the cytoplasmic domains of the receptor.
Endothelial cell adhesiveness for human T lymphocytes is inhibited by transforming growth factor-beta 1.
Authors: Gamble J R , Vadas M A
Published: December 2022

Recombinant human transforming growth factor-beta (TGF-beta) was found to inhibit the adhesive phenotype of human umbilical vein endothelial cells for human PBL, purified T lymphocytes, and PHA-activated lymphoblasts. TGF-beta inhibited lymphocyte attachment to resting human umbilical vein endothelial cells and also to endothelial monolayers stimulated with the pro-inflammatory cytokines TNF-alpha and IL-1 beta. Our investigations also show that the ability of endothelial cells to respond to TGF-beta by altering their adhesiveness is lost with prolonged culture of the cells. However, this loss is selective as TGF-beta inhibits cell proliferation in both early and late passage endothelial cells. These results suggest that in vivo TGF-beta may inhibit the adhesive phenotype of endothelial cells and also may limit the immunologic response occurring at the endothelial cell barrier.
Accumulation of Th-2-like helper T cells in the conjunctiva of patients with vernal conjunctivitis.
Authors: Maggi E , Biswas P , Del Prete G , Parronchi P , Macchia D , Simonelli C , Emmi L , De Carli M , Tiri A , Ricci M
Published: December 2022

A total number of 132 T cell clones (TCC) were obtained by PHA-stimulation of single T cells from mononuclear cell suspensions of conjunctival flogistic infiltrates of three patients with vernal conjunctivitis (VC). The phenotype and functional properties of these TCC were compared with those of 122 TCC contemporarily established from PB mononuclear cell suspensions of the same patients, 120 TCC established from lymph nodes of three patients with nonspecific hyperplastic lymphoadenitis and 159 TCC established from thyroid lymphocyte infiltrates of three patients with Graves' disease. The great majority of conjunctival TCC displayed the CD4+ CD8- phenotype (CD4/CD8 ratios ranging from 6.1 to 7.0), whereas the mean CD4/CD8 ratios for control TCC ranged from 0.9 to 2.4. After stimulation with either PHA or PMA plus anti-CD3 mAb, conjunctival TCC differed from control TCC for their ability to produce cytokines. In particular, a large number of conjunctival TCC produced IL-4, but no, or limited amounts of, IFN-gamma, whereas no difference was observed between conjunctival and control TCC with regard to the production of IL-2. The failure of IFN-gamma production by conjunctival TCC was apparently not caused by delay or block in cytokine production, but actually reflected the lack of IFN-gamma transcription. Virtually all conjunctival TCC able to produce IL-4, but not IFN-gamma, as well as most of those producing both cytokines, provided helper function for IgE synthesis in allogeneic normal B cells. The accumulation in the conjunctiva of patients with vernal conjunctivitis of CD4+ T cells that, apart from the production of IL-2, resembles murine Th2 cells for their profile of cytokine production and helper function suggests a possible role for these cells in the pathogenesis of the disease.
Characterization of a conserved T cell epitope in HIV-1 gp41 recognized by vaccine-induced human cytolytic T cells.
Authors: Hammond S A , Obah E , Stanhope P , Monell C R , Strand M , Robbins F M , Bias W B , Karr R W , Koenig S , Siliciano R F
Published: December 2022

A human CTL epitope located in a region of the HIV-1 envelope protein gp41 that is highly conserved among various HIV-1 strains was identified. This epitope was recognized by CD4+ CTL clones that were induced in seronegative humans by immunization with recombinant gp160. Fusion proteins carrying portions of the HIV-1 env gene and synthetic peptides were used to localize this epitope to amino acids 584-595 of the HIV-1 BRU env sequence. Only two positions within this epitope showed variation among North American HIV-1 isolates, and the substitutions were conservative in nature. The Lys to Arg substitution at position 593 abolished recognition, probably by interfering with the peptide-MHC interactions. This epitope was recognized in association with at least one subtype of the widely distributed human class II MHC specificity DPw4, namely DPw4.2. The relatively high frequency of this allele (27.2% among Caucasians) makes it likely that a larger fraction of the population would generate a response directed at this epitope than would be the case for epitopes recognized in the context of gene products of most other class II and class I loci. Interestingly, the closely related DP beta-chain allele types 4.1 and 2.1, which differ from 4.2 by 3 and 1 amino acids, respectively, were unable to present this gp41 peptide to DPw4.2-restricted clones. Comparison of the structure of this epitope with that of other peptides recognized in the context of DPw4.2 led to the identification of a consensus sequence for DPw4.2 binding peptides. Because the gp41 CTL epitope 584-595 identified here is highly conserved and is recognized in the context of a common DP allele, it may represent an important target region for vaccine development. Our results indicate that vaccines containing this epitope may induce in a significant fraction of those immunized CTL active against at least half of all HIV-1 strains.
Differential regulation of fibronectin receptor subunit gene and cell surface expression in human peripheral blood T lymphocytes.
Authors: Kohn F R , Grigg M E , Klingemann H G
Published: December 2022

Members of the beta 1 subfamily of heterodimeric integrins, such as the fibronectin receptors alpha 5 beta 1 and alpha 4 beta 1, are expressed on human T lymphocytes. The presence of these two adhesion receptors on T lymphocytes suggests an involvement in cell-cell and cell-extracellular matrix interactions that may be important for the development of immune and inflammatory reactions. We have examined the cell surface expression of alpha 5, alpha 4, and beta 1 subunits on purified peripheral blood T lymphocytes before and after activation with Con A and PMA. Freshly isolated T lymphocytes contained distinct fractions expressing high or low levels of alpha 5 and beta 1. Only a high expressing T lymphocyte population was present after 72-h culture with Con A and PMA. Time course analysis indicated that the shift in alpha 5 and beta 1 expression occurred during the first 24 h after addition of activating agents and occurred in the absence of proliferation. In contrast to alpha 5 and beta 1, essentially all freshly isolated T lymphocytes expressed high levels of alpha 4. After 72-h culture with Con A and PMA, a wide distribution of alpha 4 expression was observed. Further experiments showed that after activation, a proportion of CD4-positive cells decreased their surface expression of alpha 4, but increased their surface expression of alpha 5 and beta 1. In contrast, most CD8-positive cells increased their surface expression of alpha 5, beta 1, and alpha 4 upon activation. An examination of mRNA levels in pan-T lymphocyte cultures after activation indicated that alpha 5 and alpha 4 mRNA expression decreased, whereas beta 1 mRNA expression was unchanged, in Con A/PMA-activated cells as compared to those cultured in medium alone. Our results indicate that T lymphocyte activating agents may differentially affect the expression of alpha 5 beta 1 and alpha 4 beta 1, thus providing a mechanism for the selective regulation of binding interactions that occur at sites of immune reactions.
Generation of monoclonal antibodies to murine IL-1 beta and demonstration of IL-1 in vivo.
Authors: Hogquist K A , Nett M A , Sheehan K C , Pendleton K D , Schreiber R D , Chaplin D D
Published: December 2022

The role of murine IL-1 beta in vitro and in vivo has not been defined. We describe here the production of neutralizing and immunoprecipitating mAb and polyclonal antibodies specific for murine IL-1 beta and their application to a characterization of the murine IL-1 beta protein. Immunization of either hamsters or rabbits with the recombinant mature form of murine IL-1 beta emulsified in CFA elicited antisera and hamster mAb that only recognized denatured IL-1 beta. In contrast, immunization with rIL-1 beta adsorbed to alum resulted in the generation of neutralizing and immunoprecipitating rabbit and hamster antisera and hamster mAb. All of the mAb recognize both the pro-form of IL-1 beta and the mature bioactive form produced by cultures of murine peritoneal macrophages. Using these antibodies, we demonstrate that approximately half of the IL-1 activity present in supernatants of LPS-treated cultured mouse macrophages is composed of IL-1 beta. Additionally, IL-1 beta as well as IL-1 alpha can be detected in the plasma of LPS-treated mice. These studies, therefore, demonstrate the production of IL-1 beta both in vitro and in vivo.
Six epitopes reacting with human cytotoxic CD8+ T cells in the central region of the HIV-1 NEF protein.
Authors: Culmann B , Gomard E , Kiény M P , Guy B , Dreyfus F , Saimot A G , Sereni D , Sicard D , Lévy J P
Published: December 2022

In order to identify the target epitopes recognized by specific CTL in the NEF protein of HIV-1, 33 peptides derived from the HIV-BRU sequence were tested with NEF-specific CTL generated from HIV-seropositive donors. Six different epitopes were identified and several points were remarkable: 1) They were all located in two regions of the central part of the NEF protein corresponding to residues 73 to 94 and 113 to 147, respectively. 2) The CTL issued from a single donor could recognize several peptides of the NEF protein. 3) Some of these peptides could be recognized in association with at least two or three different HLA class I molecules. 4) Two different overlapping epitopes were present in a relatively short sequence of 15 amino acids. These results suggest that multiple epitopes corresponding to different HLA restrictions could coexist in a relatively small region of the NEF protein. The implications of these results in vaccine strategies using synthetic peptides bearing CTL epitopes are discussed.
An intracellular 50-kDa Fc gamma-binding protein is induced in human cells by alpha-IFN.
Authors: Thomas N S , Linch D C
Published: December 2022

This report describes a novel intracellular Ig Fc-binding protein that is induced by alpha-IFN in a wide range of human cell types. The protein has a m.w. of 45,000 to 50,000 and it will bind human IgG but not IgM, IgA, or IgE. The IgG-binding specificity of this protein (designated Fc gamma BP) is similar to that of the Fc gamma RII receptor in that it binds only to mouse IgG1 and IgG2b but not IgG2a. It is an intracellular protein and is detectable by immunoprecipitation from [35S]methionine-labeled cells but it is undetectable on the cell surface. Fc gamma BP is a phosphoprotein, and the amount of phosphorylated Fc gamma BP increases after treatment with alpha-IFN. Although the function of this protein has yet to be determined, the induction of an Fc-binding protein by alpha-IFN suggests a role in the immune response, particularly during infection by viruses.
Differential susceptibility of activated macrophage cytotoxic effector reactions to the suppressive effects of transforming growth factor-beta 1.
Authors: Nelson B J , Ralph P , Green S J , Nacy C A
Published: December 2022

We examined the effects of TGF-beta 1 on induction of several activated macrophage antimicrobial activities against the protozoan parasite Leishmania, and on induction of tumoricidal activity against the fibrosarcoma tumor target 1023. TGF-beta by itself did not affect the viability of either the intracellular or extracellular target in concentrations up to 200 ng/ml. As little as 1 ng/ml TGF-beta, however, suppressed more than 70% of the intracellular killing activity of macrophages treated with lymphokines. In contrast, more than 100 ng/ml TGF-beta was required to suppress intracellular killing by cells activated with an equivalent amount of recombinant IFN-gamma. Addition of TGF-beta for up to 30 min after exposure to activation factors significantly reduced macrophage killing of intracellular parasites. Pretreatment of macrophages with TGF-beta was even more effective: treatment of cells with TGF-beta for 4 h before addition of activation factors abolished all macrophage intracellular killing activity. Regardless of treatment sequence, however, TGF-beta had absolutely no effect, at any concentration tested, on activated macrophage resistance to infection induced by lymphokines or by the cooperative interaction of IFN-gamma and IL-4. Effects of TGF-beta on tumoricidal activity of activated macrophages was intermediate to that of its effects on intracellular killing or resistance to infection. Lymphokine-induced tumor cytotoxicity was marginally (25%) affected by TGF-beta; 200 ng/ml was able to suppress IFN-gamma-induced tumoricidal activity by 40%. Thus, TGF-beta dramatically suppressed certain activated macrophage cytotoxic effector reactions, but was only partially or not at all effective against others, even when the same activation agent (IFN-gamma) was used. The biochemical target for TGF-beta suppressive activity in these reactions may be the pathway for nitric oxide production from L-arginine, because TGF-beta also inhibited the generation of nitric oxide by cytokine-activated macrophages.
IFN-gamma retards the turnover of H-2Dd antigens by splenic lymphocytes.
Authors: Guha A , Cone R E
Published: December 2022

The effect of IFN-gamma on the rate of shedding and biosynthesis of H-2Dd was determined by culture of cell surface-radioiodinated BALB/c spleen cells with rIFN-gamma or spleen cells metabolically labeled with 35S-methionine in the presence of IFN-gamma. Radioiodinated or 35S-labeled H-2Dd was quantitated by immunoprecipitation of H-2Dd from detergent lysates of radiolabeled cells taken at different culture intervals. The loss of 125I-labeled H-2Dd was retarded 75 to 90% by IFN-gamma whereas the biosynthetic rate was unaffected during the first 10-h culture. The net result was a ninefold increase in newly synthesized cell-associated H-2Dd. The results were consistent with determination of the kinetics of increased expression of H-2Dd determined by immunofluorescence and suggest that an early effect of IFN-gamma on the expression of class I Ag is a retardation of catabolism leading to an increase of newly synthesized class I Ag.
In vivo response of secretory component in the rat uterus to antigen, IFN-gamma, and estradiol.
Authors: Wira C R , Bodwell J E , Prabhala R H
Published: December 2022

Intrauterine immunization of ovariectomized rats with SRBC is known to elicit pronounced IgA and IgG antibody responses in uterine secretions of immunized uteri. To determine whether secretory component (SC), the receptor for transporting polymeric IgA from tissues to mucosal surfaces, was also influenced by Ag, ovariectomized rats were immunized and boosted by placing SRBC into the lumena of individual uterine horns. In response to Ag, the levels of polymeric IgA, as well as free SC and SC bound to polymeric IgA, increased in uterine secretions. When ovariectomized animals were treated with estradiol, a fivefold increase in SC levels was observed in the immunized horns, indicating that a hormone response is superimposed on the Ag-induced stimulation of uterine SC. To determine whether IFN-gamma influences the presence of SC in uterine secretions, IFN-gamma was placed in the uterine lumena of ovariectomized nonimmunized rats. When uterine secretions were analyzed, significantly higher levels of SC were found in IFN-gamma-exposed uteri than were present in saline treated control animals. In contrast, intrauterine instillation of IFN-gamma had no effect on the levels of IgA in uterine secretions. This response was specific for IFN-gamma in that IFN-alpha/beta had no effect on uterine SC or IgA levels. These results indicate that intrauterine instillation of Ag, in addition to evoking pronounced antibody responses, stimulates the production of SC, which may be responsible for the transport of polymeric IgA from tissue to uterine secretions. Furthermore, they indicate that IFN-gamma placed in the uterine lumen stimulates SC production and suggest that the uterine SC response to Ag may be mediated by the action of IFN-gamma on uterine epithelial cells.
Immune responses to the 18-kDa protein of Mycobacterium leprae. Similar B cell epitopes but different T cell epitopes seen by inbred strains of mice.
Authors: Doherty T M , Bäckström B T , Prestidge R L , Love S G , Harding D R , Watson J D
Published: December 2022

Antibody responses to the 18-kDa protein of Mycobacterium leprae have been analyzed in different strains of mice. High, intermediate, and low responder strains have been identified and these response patterns show clear linkage to genes encoded in the H-2 complex. Three peptides, residues 1-50, 51-100, and 101-148 have been synthesized, as well as a series of 20-mer peptides, which span the entire 18-kDa protein. Repeated immunization of different strains of mice with the 18-kDa protein resulted in IgG responses to epitopes found on all three synthetic peptides. Immunization of BALB/cJ and B10.BR mice, two high responder strains, with 18-kDa protein resulted in high levels of IgG antibody to epitopes found on peptides 1-20, 16-35, 31-50, 46-65, and 76-95. B10.BR mice also contained IgG that bound peptide 61-80 and BALB/cJ mice produced IgG that bound peptide 91-110. Although B10.BR mice produced IgG that bound the 50-mer peptide 101-148, this IgG was not detected by binding to peptides 91-110, 106-125, 121-140, and 131-148. Immunization of B10.BR mice with individual overlapping 20-mer peptides as Ag revealed that peptides 1-20, 16-35, 31-50, and 76-95 elicited high titers of IgG that bound both the immunizing peptide as well as 18-kDa protein. As these peptides induce antibody synthesis they must contain both B cell and T cell epitopes. By contrast, immunization of BALB/cJ mice with the same 20-mer peptides, all of which contain B cell epitopes for this strain, failed to elicit IgG responses with one exception. Peptide 91-110 induced IgG that bound peptide 91-110, but not the intact 18-kDa protein. We conclude that peptides 1-20, 16-35, 31-50, and 76-95 either lack T cell epitopes for BALB/cJ mice, or activate different T cell subpopulations in the two strains. We suggest that the induction of IgG responses to small peptide Ag is an in vivo assay of the activity of Th2 cell subpopulations.
Dominant low responsiveness in the IgG response of mice to the complex protein antigen type 1 fimbriae from Actinomyces viscosus T14V.
Authors: Haber J , Paradis G , Grinnell C M
Published: December 2022

Type 1 fimbriae from Actinomyces viscosus T14V, composed of a complex protein of Mr 65,000, mediate the adherence of A. viscosus T14V to the host, whereas type 1 fimbriae-specific antibodies inhibit adherence. Genetic control of the serum IgG response to type 1 fimbriae was evaluated in a series of inbred, hybrid, recombinant inbred, and back-cross mice. Mice were given i.p. injections of 10(8) A. viscosus T14V cells in saline on days 0 and 14, and IgG anti-type 1 fimbriae in sera obtained on day 26 were measured by ELISA. Segregation analysis of the responses of (BALB/cJ x A/J)F1 x A/J backcross mice suggested polygenic control. Linkage analysis in (BALB/cJ x A/J)F1 x A/J backcross and SWXL recombinant inbred mice suggested control by genes linked with H-2 and with Ly-17 and Akp-1. In several F1 hybrid strains derived from H-2-disparate high and low responder parental strains, low responsiveness was dominant. The F1 derived from the H-2-identical high and low responder strains CBA/J and C3H/HeJ was a low responder, suggesting that dominant low responsiveness was mediated by non-H-2-linked genes. A three-gene model is proposed for regulation of the type 1 fimbriae response, including an MHC-linked gene, a gene linked with Ly-17 and Akp-1 on the telomeric portion of chromosome 1, and a background gene whose linkage is unknown.
Comparison of D, JH, and junctional diversity in the fetal, adult, and aged B cell repertoires.
Authors: Bangs L A , Sanz I E , Teale J M
Published: December 2022

Polymerase chain reaction-amplified cDNA libraries of the IgH genes of fetal, young adult, and aged BALB/c mice were sequenced so that the complimentarity determining region 3 (CDR3) in each could be analyzed. The results show extensive diversity in the CDR3 region in all three libraries examined. A prominent feature of the fetal repertoire is the lack of nucleotide region additions and shorter germline-derived D segments compared with the adult repertoires. Also of interest were distinct differences in D family and JH usage in the three libraries representing different stages of ontogeny. The absence of DFL16.2 in the fetal sequences analyzed was of particular note. Also of note was a substantial underutilization of the largest D family, DSP2, in the aged repertoire. The study provides further evidence that the Ig repertoire is developmentally regulated. In addition, the results indicate that several aspects of the recombination process are different in adult and fetal B lineage cells, suggesting that B cells present early in ontogeny are distinct from those present in the adult.