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Flow cytometric analysis of T-independent antigen binding to dinitrophenyl-specific cells.

Binding of Ag to membrane Ig (mIg) can lead to either activation or desensitization of the B cell. For thymus-independent (TI) Ags the nature and concentration of the Ag determines what type of signal is delivered to the cell. These Ags are capable of directly activating B lymphocytes and are an important model system for the study of mechanisms involved in B cell responses. In this study, we quantified TI Ag binding and B cell receptor involvement as functions of TI Ag structure, concentration, and epitope density. Various epitope densities of two structurally different TI Ags, DNP-polymerized flagellin (pol) and DNP-dextran (dex), were labeled with tetramethylrhodamine isothiocyanate (TRITC) and reacted with DNP-specific murine splenic B lymphocytes and with cells of a cloned DNP-specific cell line. The amount of Ag bound to the cell surface at various doses was measured directly by flow cytometry. For each Ag and dose, FITC-labeled DNP-L-papain was used to quantitate receptor sites not occupied by Ag. Approximately 5% receptor occupancy was observed for immunogenic doses of Ag. Higher Ag concentrations that can induce tolerance caused a substantial increase in the fraction of occupied receptors. This suggests that tolerogenic responses result from an overly restrictive cross-linking of surface receptors. By comparing these data to previously published data on biologic activity of the Ags, we are able to more clearly define those conditions of Ag binding that lead to B cell activation.

Animals↗

Acquired myasthenia gravis. Immunopathology.

Much of the remarkable advance in our understanding of the immunopathology of MG relates to the availability of two gifts of nature that permit the ready purification of the antigen, AChR. Immunization of experimental animals with AChR has led to the development of the extremely faithful animal model, EAMG. Analysis of both EAMG and MG has revealed that the effector agents in this autoimmune disease are anti-AChR antibodies, whose production is regulated by anti-AChR CD4+ T cells. The pathogenic effects on neuromuscular transmission are mediated by antibody-induced antigenic modulation of end-plate AChR, end-plate membrane destruction through complement fixation and recruitment of inflammatory cells, and antibody-induced blockade of the function of the remaining AChR molecules. The origin of MG remains unknown. One theory proposes that dysregulation of the thymic control of tolerance plays an important role. An alternative hypothesis is that tolerance is broken as the result of a vigorous immune response directed against an invading microorganism that expresses a molecule that is similar to AChR, so-called molecular mimicry. This "normal" response eventually cross-reacts with self-AChR, resulting in the autoimmune damage. Our current knowledge of MG has suggested a number of possible sites of therapeutic intervention that are under active study. Future information concerning the origin of the disease will likely be useful in the design of more effective treatment for this and other related autoimmune diseases.

Animals↗

Cell-mediated cytotoxicity against measles virus in SSPE. I. Enhancement by antibody.

In patients with subacute sclerosing panencephalitis (SSPE) virus persistency has been explained by a blocking factor of specific cell-mediated immunity in SSPE serum and cerebrospinal fluid (CSF). In the present study the effect of SSPE serum and CSF on cell-mediated cytotoxicity against 51Cr-labeled allogenic measles virus-infected target cells was tested. Pretreatment of SSPE peripheral lymphoid cells by serum or CSF neither blocked nor increased measles-specific cytotoxicity when cells were subsequently tested in FCS containing tissue culture medium. However, killing activity was always enhanced after pretreatment of target cells or direct addition of serum or CSF to the cytotoxic assay. Enhancement was also observed with non-SSPE-derived measles antibodies. The effect was dependent on measles antibody concentrations. These results suggest that antibody-dependent killing by K cells is intact in patients with SSPE. It is possible that virus-infected brain cells are protected from an immune attack in vivo by antibody-induced antigenic modulation.

Antibodies, Viral↗

Dietary fatty acid modulates actions of nucleotides on humoral immune responses.

Polynucleotides enhance T cell-dependent antibody production in culture. Impaired antibody production in mice fed a nucleotide-free diet can be easily restored by in vivo supplementation of both a mononucleotide-nucleoside mixture (OG-VI) and polynucleotides. Polynucleotides appear to act partly by modulating antigen presentation processes mediated by cell surface molecules. We examined whether dietary fatty acid manipulation alters nucleotides' actions on humoral immunity. Antibody production was studied in C57B1/6 mice fed I) a nucleotide-free diet high in saturated fatty acid (SFA diet), 2) a nucleotide-free diet high in polyunsaturated fatty acid (PUFA diet), and 3) a regular nucleotide-free diet (control). In vivo and in vitro T cell-dependent antibody production decreased in all groups, but mice fed the SFA diet produced more antibody in vivo than did mice in the other diet groups. Spleen cells from mice fed the SFA diet also produced more interferon-alpha when stimulated with mitogens than did those from mice fed the control diet. In contrast, polynucleotides enhanced in vitro antibody production much less efficiently in mice fed the SFA diet than in the other mice and in vivo supplementation of OG-VI was also less effective in restoring impaired antibody production in these mice. A diet with a high content of SFA may alter nucleotides' action on humoral immune responses, in addition to its direct effects on immune functions.

Animals↗

Immunohistochemical study in breast carcinoma. S100-protein positive cells and neuroendocrine differentiation.

In this study S100-protein positive cells were found in all 12 cases of benign dysplastic changes and in all of the investigated 53 cases of breast carcinomas. These cells belong to interdigitating cells (IDC) and were located in a regular pattern in the basal cell layer of the ducts and alveolar nodules in benign lesions. However, in breast carcinomas S100-protein positive IDC were in various pattern of distribution; only in the stroma, in basal cell layer, between cancer cells and S100-protein positivity of IDC and epithelial cancer cells. This phenomenon suggests the antigenic modulation of cancer cells transferring them the property of S100-protein positive cells or it may be the consequence of fusion. Immunoreactivity for chromogranin A (ChgA) and synaptophysin (Syn) was found in epithelial cells in some cases of benign dysplastic changes. However, in some carcinoma cases ChgA and Syn were revealed in carcinoma cells, in myoepithelial cells, in some stromal mesenchymal cells and endothelial cells. Immunoreactivity to smooth muscle actin was shown in the endothelial and smooth muscle cells of the vessel's wall, in myoepithelial cells and was also sporadic in carcinoma cells.

Actins↗

Immunologic advances in monoclonal antibody therapy: implications for oncology nursing.

PURPOSE/OBJECTIVES: To provide an overview of monoclonal antibody (MoAb) formation, therapeutic and diagnostic uses of MoAbs, and the implications for oncology nurses. DATA SOURCES: Books and Journal articles (including research studies). DATA SYNTHESIS: Clinical trials have demonstrated the diagnostic and therapeutic potential of MoAb therapy. Advances in hybridoma technology and gene-splicing techniques have led to the formation of chimeric MoAbs, which exhibit decreased immunogenicity in the recipient. Clinical limitations with MoAb therapy include cross-reactivity with normal tissues, heterogeneity of antigen expression, presence of circulating antigen, antigenic modulation, tumor size and vascularity, and the anti-antibody response. CONCLUSIONS: MoAbs currently are used for diagnostic purposes and in phase I, II, and III clinical trials for cancer treatment. As research progresses, MoAbs are likely to be incorporated into the mainstream of cancer therapy as have other biologic response modifiers. IMPLICATIONS FOR NURSING PRACTICE: Current uses of MoAb therapy in clinical trials involve nurses in many roles, including clinical nurse specialist, staff nurse, and research nurse. As more oncology nurses encounter MoAb therapy in practice, they will have to have an increased understanding of basic immunologic principles and the expertise to manage the unique toxicities associated with MoAb therapy.

Antibodies, Monoclonal↗

Expression of a 33-kDa antigen Tm 1 on lymphocyte surface after modulation of cell surface antigens by IgM monoclonal antibody.

The mAb Tm 1 was obtained from a fusion of SP2/O tumor cells with spleen cells from CF1 mouse immunized with T cells modulated by an IgM anti-CD3 mAb.mAb Tm 1 reacted with IgM anti-CD3 modulated T cells (66.6%) but not with unmodulated T cells (4.4%). Tm 1 was not expressed on T cells modulated with either IgG2a or IgG1 anti-CD3 mAb. Immunoprecipitation from 125I-labeled CD3-modulated T cells showed that Tm 1 Ag is a single polypeptide of 33 kDa under reducing and nonreducing conditions. Kinetic studies revealed that Tm 1 was detectable on T cells 10 min after incubation and maximally expressed after 4 h of incubation with IgM anti-CD3 mAb. CD3 expression was markedly modulated by this anti-CD3 mAb after the same period of incubation. Studies with cycloheximide revealed that Tm 1 expression on T cells does not require new protein synthesis. Tm 1 expression persisted long after CD3-reexpression 24 h later. Tm 1 was present on a small fraction of circulating T cells, B cells, and monocytes and absent from granulocytes, platelets, E, and thymocytes. Tm 1 was not expressed on T cells after various activation stimuli but was expressed on B cells upon activation. Additional studies indicate that IgM mAb against other T cell differentiation Ag and IgM mAb against B cell Ag also lead to the expression of Tm 1 on these cells. Thus, modulation of surface Ag by IgM mAb externalizes this cytoplasmic Ag. However, one exception has been noted. Purified mAb Tm 1 was not mitogenic and was unable to block either the T cell proliferation induced by 12-O-tetradecanoyl phorbol-13-acetate plus anti-CD3 mAb and other T cell stimuli, or the B cell proliferation induced by B cell mitogens. The role of Tm 1 on lymphocyte function remains to be determined.

Animals↗

Antigen-based immune modulation: DNA vectors and beyond.

The ultimate goal for autoimmune immunotherapy is to achieve a specific downregulation or modification of autoaggressive immune responses while leaving in place the normal repertoire, capable of mediating antimicrobial responses. A multitude of preclinical studies, particularly during the last 15 years, raised hopes that self-antigens could be used to achieve the goal of specific immune modulation. Difficulties associated with the translation of this concept to the clinic revealed inherent limitations of antigen-based immune modulation. To increase the efficiency of antigen-dependent immune modulation, researchers started to investigate novel vectors for antigen delivery. Plasmid vectors, as opposed to protein antigens or peptides, have the ability to trigger prolonged production of limited amounts of antigen in the periphery. However, one complicating factor may be the inherent "danger" signal stimulated by the nature of the unmethylated CpG motifs on bacterial plasmid. Currently, various approaches are being explored to improve the efficacy of response while ameliorating the safety concerns of plasmids as immunotherapeutic tools. This manuscript offers a perspective on such efforts and outlines how the knowledge accumulated in the process will help scientists advance to the next generation of immunotherapeutics.

Adjuvants, Immunologic↗

Passive modulation of blood-group antigens.

Rh-positive erythrocytes were enriched and depleted of membrane cholesterol, and the mediated change in the degree of exposure of the D antigens was determined by fluorescence-activated cell sorting, using indirect fluorescent antibody labeling. The results are compatible with a model in which the expression of the D antigens can be modulated significantly by the lipid microviscosity (eta). At a high cholesterol-to-phospholipid ratio (C/PL) of 1.55, which corresponds to eta (25 degrees C) = 7.5 poise (1 poise = 0.1 Pa.sec), the relative detectable number of D antigens was about double than that at C/PL = 0.65, eta (25 degrees C) = 4.1 poise. In analogous experiments similar fluidity changes resulted in only about 20% modulation of expression of the A1 antigen, suggesting that in the native state this antigen is already well exposed on the erythrocyte surface. This type of antigenic modulation may also operate in vivo, and may thus bear some fundamental implications on tumor immunology and autoimmune diseases.

ABO Blood-Group System↗

Immunity to sexual stages of human malaria parasites: immune modulation during natural infections, antigenic determinants, and the induction of transmission-blocking immunity.

Four antigens of Plasmodium falciparum have so far been identified as targets of transmission-blocking antibodies; three of them (Pfs 230, 48/45) are detectable in gametocytes and expressed on gametes, the fourth (Pfs 25) appears only after fertilization. Epitope analyses of each antigen were made with competitive immunoassays, and the extent of antigenic diversity determined amongst numerous isolates of P. falciparum. There was minimal variation within one of the two epitopes on Pfs 230 both of which induce transmission-blocking antibodies. The epitopes on Pfs 25 to which blocking monoclonal antibodies respond showed a variability amongst different isolates by immunofluorescence which was unexpected in view of sequence data on the molecule. Five epitope regions have been identified on Pfs 48/45 and antibodies to them interact in a complex manner. Antigenic diversity affecting these epitopes was minimal. In P. vivax malaria much greater polymorphism was seen amongst gamete surface antigens. Natural P. falciparum infections induce antibody responses to gametocyte/gamete surface antigens that will suppress infectivity to mosquitoes but these responses may involve reactivity with any of a series of different epitopes, interactions between antibodies, and may be sequential. In P. vivax infections antibody to the sexual stage antigens may suppress or enhance transmission depending on the antibody level. Cytokine production induced by sexual stage antigens may also modulate transmission, by rendering gametocytes non-infective. Experimental studies showed marked MHC-restriction of immune responses to gamete antigens (but not to the Pfs 25 zygote antigen); the evidence from studies in humans is less convincing. Antibody responses to the sexual stage antigens seem to be more frequent in persons who have experienced only one or a few attacks of malaria as opposed to those who have been exposed frequently. Some form of down-regulation may therefore be occurring.

Animals↗

Granulomatous hypersensitivity to Schistosoma mansoni egg antigens in human schistosomiasis. I. Granuloma formation and modulation around polyacrylamide antigen-conjugated beads.

We have developed an in vitro model of granuloma formation for the purpose of studying the immunological components of delayed type hypersensitivity granuloma formation in patients infected with Schistosoma mansoni. Our data show that 1) granulomatous hypersensitivity can be studied by examining the cellular reactivity manifested as multiple cell layers surrounding the antigen conjugated beads; 2) this reactivity is a CD4 cell dependent, macrophage dependent, B cell independent response and 3) the in vitro granuloma response is antigenically specific for parasite egg antigens. Studies designed to investigate the immune regulation of granulomatous hypersensitivity using purified populations of either CD4 or CD8 T cells have demonstrated the complexity of cellular interactions in the suppression of granulomatous hypersensitivity. The anti-S. mansoni egg immune responses of individual patients with chronic intestinal schistosomiasis can be classified either as soluble egg antigen (SEA) hypersensitive with maximal granulomatous hypersensitivity or SEA suppressive with activation of the T cell suppressor pathway with effective SEA granuloma modulation. Our data suggest that T cell network interactions are active in the generation of effective granuloma modulation in chronic intestinal schistosomiasis patients.

Animals↗

Modulation of the antigenic reactivity of the citrus tristeza virus coat protein.

Trapping properties of a panel of monoclonal antibodies (Mabs) raised against citrus tristeza virus (CTV) were analyzed in an indirect double-antibody sandwich ELISA (I-DAS-ELISA). These antibodies had been previously assigned by serological specificity into five groups (I to V). Mabs from group V, which are directed to conformational epitopes, trapped significant amounts of virus antigen from CTV-infected plant tissue at IgG concentration above 10 ng/ml. Mabs from groups I to IV, which are directed to linear, continuous epitopes, performed poorly as coating antibodies, even at a 1 microgram/ml concentration of the IgG's, indicating that the respective linear epitopes were inaccessible. However, when Mabs from groups I to IV were combined with a small amount of Mabs from group V, a substantial increase in trapping of the CTV antigen was recorded. In this 'two antibody-binding assay' previously cryptic, linear epitopes of the CTV CP apparently became accessible to the Mabs from groups I to IV. Modulation of the antigenic reactivity of the CTV CP was also recorded upon binding of the Mabs directed to the conformational epitopes in solution. Induced exposure of the linear epitopes of the CTV CP was revealed in 'two antibody-binding assays' with pairwise combinations of different mouse Mabs and several rabbit and chicken polyclonal antisera with different serological specificities, including antisera to bacterially expressed CP fragments. This mixed coating in I-DAS-ELISA resulted in substantially increased efficiency of the virus antigen trapping by antisera produced against bacterially expressed protein fragments and an increased sensitivity of the CTV detection after optimization of the ratio between conformational and linear antibodies.

Antibodies, Viral↗

Membrane changes in malignant cells--modulation of receptors and antigens by lipids.

The dynamic characteristics of lipid regions in biological membranes can be expressed in terms of microviscosity parameters. These parameters not only determine the lateral and rotational movements of specific membrane sites but also their degree of exposure to the outer surrounding. Changes in microviscosity can thus reversibly displace the vertical position of antigens or receptor sites and modulate their active expression. The lipid mediated modulation of antigens and receptors may play an active role in the various physiological manifestations of malignant cells.

Antibody Formation↗

Induction of T cell adhesion by antigen stimulation and modulation by the coreceptor CD4.

T cell adhesion induced after physiological stimulation by antigen was investigated using murine T cell hybridomas specific for a tetanus toxin peptide. By employing a novel assay, the T cell hybridomas were shown to strongly adhere to peptide-pulsed APC in a dose-dependent fashion. Adhesion peaked at 30-60 min and declined thereafter. This assay allowed us to study the relationship between T cell adhesion and later activation responses using tetanus toxin peptide and alanine monosubstituted analogs. We show that the degree of peptide-induced T cell adhesion correlated with the magnitude of late functional responses. CD4, LFA-1 (CD11a/CD18), and CD28 were critical in the adhesion response. The enhancing role of CD4 was further demonstrated by reduced levels of T cell adhesion and late responses of CD4- T cell hybridomas. Reexpression of CD4 reversed these defects. Our data suggest a link between antigen-induced T cell adhesion and late responses and also suggest that signals mediated by TCR and CD4 coengagement may induce a greater activation and/or recruitment of molecules involved in T cell adhesion.

Animals↗

Modulation of antigen-specific immune responses by the oral administration of a traditional medicine, bo-yang-hwan-o-tang.

Bo-yang-hwan-o-tang (BHT) has long been used to treat cancer in traditional Korean medicine and is believed to have immune-modulating activity. This study investigated the effect of BHT on the induction of antigen-specific immune responses using hen egg-white lysozyme (HEL) as a model antigen system. Oral administration of BHT enhanced both HEL-specific humoral and lymphocyte proliferative responses in HEL low-responder mice. Feeding BHT to the mice increased INF-gamma levels, but did not change IL-4 levels. Interestingly, however, the oral BHT feeding significantly increased HEL-specific antibodies of the IgG1, IgG2b, and IgG3 subtypes, which are associated with the direct stimulation of B cells. This indicates that BHT treatment enhances anti-HEL-specific humoral immune responses via the direct stimulation of B lymphocytes rather than by selective priming of specific subtypes of the helper T-cell population. This conclusion was supported by in vitro experiments, in which the presence of BHT significantly augmented B-cell mitogen-mediated proliferation of mouse splenocytes. This augmentation was closely associated with a glycoprotein with a molecular weight of around 100 kDa. The results suggest that BHT modulates antigen-specific immune responses, and might be used as a therapeutic agent for patients who need enhanced immune function.

Adjuvants, Immunologic↗

Modulation of surface antigen expression by Klebsiella pneumoniae in response to growth environment.

Growth in pooled human body fluids [urine, serum and peritoneal dialysate (HPD)] modulated the expression of cell envelope antigens in virulent (serotype O1:K1) and avirulent (serotype O1:K66) Klebsiella pneumoniae strains. Marked variations in the outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles were noted when broth-grown cells were compared with those of bacteria cultured in body fluids. In particular, for the O1:K1 serotype strain, growth in the latter resulted in: (a) the expression of at least five iron-regulated OMPs in the 74-87 kDa range, the pattern of which was medium dependent; (b) alterations in the migration of the LPS core polysaccharide; and (c) the reversion of isogenic O-:K+ and O-:K- mutants to the O+ phenotype after growth in fresh serum but not in heat-inactivated serum, urine or HPD. Similar results were obtained for the O1:K66 serotype, although no variation in the migration of the LPS core was noted. For both O1:K1 and O1:K66 serotypes, neither the surface exposure of O1 serotype LPS nor the production of K-antigen (capsular polysaccharide) was affected by growth in body fluids. No reversion of K- mutants to the K+ phenotype was observed. These data illustrate the phenotype flexibility of this opportunistic pathogen and emphasise the crucial role of the O- rather than the K-antigen in protecting K. pneumoniae from complement-mediated serum killing.

Antigens, Bacterial↗

Antigen-induced aggregation and modulation of receptors on hapten-specific B lymphocytes.

Mouse spleen cells were subjected to a fractionation procedure designed to enrich for 4-hydroxy-3-iodo-5-nitro-phenylacetyl (NIP)- or DNP-specific B lymphocytes, which depended on adherence of specific cells to a layer of hapten-gelatin at 4 degrees C, recovery of bound cells by melting, and digestion of adherent antigen by collagenase. A population of cells resulted which contained 90% typical B cells and 37% of cells capable of binding a fluorescent, haptenated polymeric protein. Fractionated cells were reacted in vitro with fluorescent conjugates of the specific haptens with polymerized flagellin [NIP-polymerized flagellin (POL)-tetramethylrhodamine isothiocyanate conjugate or DNP-POL-fluorescein isothiocyanate conjugate] under a variety of conditions, with the aim of investigating the behavior of Ig receptors on B lymphocytes after exposure to antigen; Experiments were performed with immunogenic and tolerogenic concentrations of antigen. Furthermore, four experimental designs were used, namely: (a) brief labeling with fluorescent antigen followed by culture without antigen (pulse design); (b) culture in the continuous presence of fluorescent antigen (continuous-labeling design); (c) culture in the continuous presence of nonlabeled antigen followed by labeling of unoccupied receptors by fluorescent antigen (receptor status design); and (d) culture with nonlabeled antigen for 2 h followed by incubation without further antigen for 20 h and labeling with fluorescent antigen (modulation design). Further insight into receptor occupancy and distribution was gained by the use of fluorescent antihapten and antiglobulin reagents. It was found that both immunogenic and tolerogenic antigen concentrations caused rapid patching and capping of the receptors to which they attached, followed by endocytosis and probably some shedding of Ig receptors. However, a proportion of cells continued to bear some cell surface antigen for 24 h. The immunogenic antigen concentration failed to completely remove the receptor coat from the cell surface. At all stages of immunogenesis, plentiful unoccupied receptors could be demonstrated. The tolerogenic concentration nearly saturated available receptors, and in its continuous presence, only few unoccupied or antigen-occupied surface receptors could be detected after 24 h of culture. Experiments of the modulation design showed that brief incubation with the tolerogenic concentration appeared to suppress receptor resynthesis, as few new receptors could be demonstrated after 20 h of further culture without antigen. Experiments were performed to determine whether fractionated cells prepared from spleens of 8-day-old mice showed an unusual tendency for modulation, even with immunogenic antigen concentrations. They were found to behave essentially like adult fractionated cells. The results are discussed in the framework of current theories of B-lymphocyte activation and tolerization.

Animals↗

Modulation of CALLA (CD10) antigen on cultured ALL (REH) cells: effect of various modulators.

Phorbol ester (TPA)-induced down-regulation of the common ALL (CALLA) antigen was studied by continuous flow immunocytometry with the aid of several CD10 monoclonal antibodies, including a new CD10 monoclonal antibody (DGH-10-1-A9), shown to be of IgG1 isotype, recognizing a 100 kDa cell surface protein and effectively inhibited by a series of reference CD10 monoclonal antibodies. The TPA-induced down-regulation of CALLA on REH cells was demonstrated with the aid of the following CD10 monoclonal antibodies: J-5, VIL-A1 and DGH-10-1-A9. No major modulations in cell surface expression of CALLA on REH cells were observed after induction with 1,25-(OH)2 vitamin D3, retinoic acid, recombinant interferon (IFN) alpha 2a and recombinant interleukin 2.

Antibodies, Monoclonal↗