Human apolipoprotein B. Evidence for its immunochemical heterogeneity using monoclonal antibodies and an immunoenzymometric assay

Predefined monoclonal antibodies (Mabs) were used in an immunoenzymometric assay to study the immunochemical heterogeneity of lipoproteins and to search for potential epitopes with pathological importance. By measuring apolipoprotein B (apo B) epitopes in patients with and without angiographically documented coronary artery disease and in patients with type IIa hyperlipoproteinemia, we have found that both types of patients have a significant increase in Apo B-containing particles specifically recognized by one Mab (BL3). We have also observed that the effects of fenofibrate on type IIa patients vary greatly depending on the plasma concentrations of various Apo B-containing lipoproteins. The greatest effects occurred in patients with epitopes recognized by BL3. Lastly, by sequential precipitation of specific epitopes by BL3, we have obtained evidence that the residual epitope(s) may be related to one or more lipoprotein particles.     

Differential sensitivity of human B cell subsets to activation signals delivered by anti-mu antibody and proliferative signals delivered by a monoclonal B cell growth factor

The present study demonstrates the minimal, optimal, and synergistic signals involved in the activation of normal human peripheral blood and tonsillar B cells to proliferation. Initial activation signals were delivered to B cells by low concentrations of anti-mu antibody which did not induce proliferation by themselves. However, marked synergy was seen when anti-mu antibody was added to cultures in the presence of monoclonal B cell growth factor (BCGF) obtained from a human T-T cell hybrid such that the B cells underwent substantial proliferation. This latter proliferation was seen without maturation into Ig-secreting cells, which indicates that the BCGF is not a differentiation signal but a signal that drives the cell up to but not beyond the proliferative phase. Of note was the fact that B cells reflected differential sensitivity on the basis of size to either the activation signal delivered by anti-mu antibody or the proliferative signal delivered by BCGF. BCGF directly stimulated the larger B cells in the normal tonsillar B cell repertoire to proliferate without the requirement for an in vitro activation signal, which indicates that the cells had already received some form of activation signal in vivo. Indeed, these cells expressed the 4F2 antigen found on activated but not resting lymphocytes. In contrast, the smaller tonsillar B lymphocytes did not express the 4F2 activation antigen and required activation by anti-mu antibody, which did not of itself induce proliferation, but which acted in synergy with BCGF for substantial proliferation of the B cells. These studies thus provide a useful model of human B cell activation, proliferation, and differentiation and allow a more precise delineation of each phase in this cascade.     

Role of Ly-6 in lymphocyte activation. II. Induction of T cell activation by monoclonal anti-Ly-6 antibodies

The Ly-6 locus controls the expression and/or encodes for alloantigenic specificities found primarily on subpopulations of murine T and B lymphocytes. We have recently identified and characterized a new rat mAb, D7, that recognizes a nonpolymorphic Ly-6 specificity. After crosslinking by anti-Ig reagents or by Fc receptor-bearing accessory cells, mAb D7 could induce IL-2 production from T cell hybridomas, and in the presence of PMA could trigger a vigorous proliferative response in resting peripheral T cells. The addition of mAb D7 to cultures of antigen- and alloantigen-, but not mitogen-stimulated T cells resulted in a marked augmentation of the proliferative response. A number of other well-characterized mAbs to Ly-6 locus products could also stimulate a T cell proliferative response after crosslinking by anti-Ig and in the presence of PMA. These results strongly suggest that Ly-6 molecules may play a critical role in the T cell activation cascade, either as receptors for an unidentified soluble or cell-associated ligand or as transducing molecules that modulate signals initiated by antigen stimulation of the T3-Ti complex.     

Human cytotrophoblastic antigens defined by monoclonal antibodies

Monoclonal antibodies have been raised against cytotrophoblast. Two different antigens, defined on cytotrophoblast but not on syncytiotrophoblast were designated ACT-1 and ACT-2, respectively. Chorionic villi were taken from normal early pregnancy and processed for immunization by two different procedures. ACT-1 was demonstrated to be present in lung alveolar cells, endothelial mucosa of the jejunum, colon, ureter, urinary bladder and the fallopian tube, and endometrial gland of the pregnant uterus. On the other hand, ACT-2 was present in the endothelial mucosa of the stomach, endothelium of the renal vessel, and the decidua of the pregnant uterus. Although the monoclonal antibodies did not react with such established cell lines as Bewo, SCH, OVK-18, HHUA, MK-01, FL, BHK and P3 X 63Ag 8 . 653, they did react with some of the cell lines when the cell membrane was destroyed with Triton X-100. Each antibody, therefore, may recognize the antigen not on the cell membrane but in the cytoplasm. The antigens might be shed or may disappear in the process of differentiation from cytotrophoblast to syncytiotrophoblast.     

Human coronary endothelial cell activation by endotoxin is characterized by NF-kappa B activation and TNF-alpha synthesis.

Tumor necrosis factor-alpha (TNF-alpha), an early inflammatory mediator typically regulated by nuclear factor kappa B (NF-kappa B), plays a critical role in the development of cardiovascular dysfunction in sepsis. While several myocardial cell types synthesize TNF-alpha, the importance of the myocardial endothelium in sepsis-related cardiac cytokine production is unclear. To determine the role of the human coronary artery endothelial cell (HCA-EC) in the cytokine response to endotoxin we measured in vitro TNF-alpha synthesis, TNF-alpha mRNA, and the associated NF-kappa B response to LPS. To determine the magnitude of the HCA-EC response we assessed the TNF-alpha and NF-kappa B response to LPS in a human monocytic cell line (THP-1) as well. We observed an increase in supernatant TNF-alpha from LPS-stimulated HCA-EC (12 h) that was ablated by the proteosome inhibitor, ALLN (N-acetyl-Leu-Leu-norleucinal). Similarly, ALLN-sensitive TNF-alpha was produced by monocytes following LPS, although at concentrations 100-fold higher than HCA-EC. TNF-alpha mRNA from HCA-EC was detected at 60 min in LPS-stimulated cells, but not in unstimulated cells or cells pretreated with ALLN. NF-kappa B p50/p65 subunits were detectable in endothelial nuclear protein 60 min following LPS. In contrast, NF-kappa B subunits from monocytes were detected at 15 min. Also, while ALLN only attenuated endothelial NF-kappa B translocation, monocyte NF-kappa B translocation was completely inhibited. These data suggest endotoxin-stimulated human coronary endothelial cells express TNF-alpha, which is regulated in part by NF-kappa B activation, in a manner and degree distinct from human monocytes.     

Human T cell activation. I. Monocyte-independent activation and proliferation induced by anti-T3 monoclonal antibodies in the presence of tumor promoter 12-o-tetradecanoyl phorbol-13 acetate

Three monoclonal antibodies (mAb), of IgG1, IgG2a, and IgM isotypes, raised against the T3 complex, were used to probe the activation of human T cells. The IgM antibody 235 was not mitogenic for peripheral blood mononuclear cells (PMC). It efficiently blocked the proliferation of PMC induced by T cell mitogens, alloantigens, and soluble antigens. The other two antibodies were mitogenic, and behaved similarly to Leu 4 and OKT3, respectively. In T cell preparations with less than 0.1% monocytes (as assayed by nonspecific esterase staining), all three mAb were not mitogenic. They failed to induce either interleukin 2 (IL-2) receptor expression or IL-2 secretion. Addition of IL-1 failed to collaborate with anti-T3 mAb to induce these T cells to proliferate, but IL-2 enhanced T cell proliferation slightly. Monocyte-depleted T cells, however, proliferated in response to all three anti-T3 mAb, when TPA was added, in a dose-dependent manner. TPA induced a low level of IL-2 receptor expression in monocyte-depleted T cells, without inducing IL-2 secretion. Anti-T3 plus TPA induced a marked enhancement in both quantity and intensity of IL-2 receptor expression. IL-2 secretion was also detected. These results indicate that anti-T3 IgM can deliver an inductive signal despite its blockage of T cell proliferation, and that two signals are necessary and perhaps sufficient to induce human T cell activation and proliferation.     

Idiotypic markers of polyclonal B cell activation. Public idiotypes shared by monoclonal antibodies derived from patients with systemic lupus erythematosus or leprosy.

We investigated idiotypic markers of monoclonal antibodies derived from patients with polyclonal B-cell activation. Four monoclonal antibodies with different ligand binding specificities derived from a patient with lepromatous leprosy and three monoclonal anti-DNA antibodies from two patients with SLE were studied. Three new public idiotopes, which were common to monoclonal antibodies from all three patients, were defined by five polyclonal rabbit antiidiotypes, two monoclonal mouse antiidiotopes, and a monoclonal mouse antibody against a synthetic peptide that contains residues of the heavy chain CDR-1 of a monoclonal lupus anti-DNA antibody. The antibody against the synthetic idiotype was found to react with native immunoglobulins in solution. One idiotope was found to be consistently immunogenic in all animals tested. Since the three patients are of different ethnic origins, these shared idiotypes are probably encoded by germline V genes. These genes may be recurrently expressed in states of polyclonal B-cell activation, regardless of etiology. The results suggest that some autoantibodies arise by expansion of a pool of precursors in the normal antibody repertoire.     

Control of human B cell tumor growth in severe combined immunodeficiency mice by monoclonal anti-B cell antibodies.

Severe combined immunodeficiency (scid) mice develop EBV (+)B cell tumors after infusion of EBV(+)B cells or of B cells and EBV. In this study, scid mice were infused with B cell lines derived from three patients who developed a B lymphocyte proliferative disorder after bone marrow or organ transplantation. Intraperitoneal injection of 5 x 10(6) B cells induced tumor growth in all mice, leading to death within 60 d. Human B cells were identified in spleen and bone marrow by means of immunofluorescence or EBV genome amplification, and human IgM was detected in serum. Infusion of murine monoclonal antibodies specific for human B cell membrane antigens CD21, CD24, and CD23 was effective in 80% of animals, against two of the three cell lines preventing tumor development or inducing remission according to the time of treatment. The effect was antibody dose dependent and was optimal with four intravenous infusions of at least 0.1 mg 4 d apart. Human IgM in serum and human B cells in spleen and bone marrow became undetectable when peritoneal tumors regressed completely. Infusions of IgG1 isotype-matched anti-CD4 antibody or anti-CD3 antibody had no effect. Tumors developed or recurred in 50% of these animals injected with one of the B cell line 3 mo after treatment was stopped. The same anti-CD21 and anti-CD24 antibodies had been used to treat the three patients, and shown similar degrees of effectiveness as in the scid mouse model. These results indicate that scid mice may be suitable for assessing therapeutic approaches to human B cell proliferation.     

Structural analysis of hepatitis B surface antigen by monoclonal antibodies.

A method has been developed for the analysis of hepatitis B surface antigen (HBsAg) antigenic structure at the molecular level that creates "fingerprints" or "signatures" of various hepatitis B viral (HBV) strains. This technique employs high affinity IgM and IgG monoclonal antibodies (anti-HBs) directed against distinct and separate determinants on HBsAg. In performing this antigenic structural analysis, separate binding curves for different monoclonal anti-HBs are generated by measuring immunoreactivity in serial dilutions of HBsAg-positive serum by radioimmunoassay. Since the HBsAg concentration in serum is unknown, the binding profiles of groups of samples are aligned by an iterative least-squares procedure to generate the numerical signature characteristic of the viral strain. The numerical signatures are then displayed on a computer-graphic plot. The signature profiles of HBsAg subtypes are a true reflection of their antigenic structure, and in vertical and horizontal transmission studies the molecular characteristics of the viral epitopes are conserved. By signature analysis we found substantial antigenic heterogeneity among the ayw3 strain both in the U.S. and France, as well as in populations of the Far East and Africa. Populations in Ethiopia, Gambia, and the Philippines were infected with two antigenically distinct HBV strains. In some newly identified HBV strains, it was found that epitopes identified by some monoclonal antibodies were absent or substantially reduced, which suggested that a genetic mutation may have occurred. Thus this study suggests that there is far more antigenic heterogeneity in HBV than previously recognized. These variants are antigenically distinct from each other at the epitope level, and were heretofore unrecognized by polyvalent anti-HBsAg antibodies.     

Cell retargeting by bispecific monoclonal antibodies. Evidence of bypass of intratumor susceptibility to cell lysis in human melanoma.

Intratumor heterogeneity for susceptibility to cytotoxic T lymphocytes (CTL)-mediated lysis represents a major obstacle to cancer adoptive immunotherapy. To overcome the heterogeneity observed in terms of susceptibility of target cells to cell-mediated lysis, in this study we used two purified bispecific monoclonal antibodies (bsmAbs) that recognize molecules expressed by cytotoxic effector cells (CD3 and IgG Fc receptorial molecules), as well as one high molecular weight melanoma-associated antigen (HMW-MAA). The ability of these reagents to enhance or induce a relevant in vitro cytotoxic activity by a CTL clone (CTL 49) isolated from PBL of a melanoma patient was tested on a large panel of autologous and allogeneic melanoma cell lines and clones. Functional studies revealed that the CTL 49 clone lysed all the HMW-MAA+ tumor lines in the presence of bsmAbs and that these reagents affected the target lysis in a cooperative fashion. The effectiveness of bsmAbs in overcoming the heterogeneous susceptibility of human melanoma cells to cell-mediated lysis may find practical implications in cancer adoptive immunotherapy.     

Anti-ampicillin monoclonal antibodies and their cross-reactivities to various beta-lactams.

Two cell lines producing monoclonal antibodies, Abp4 (IgM) and Abp7 (IgG1) against ampicillin were established. The epitopes and the cross-reactions of the antibodies with various beta-lactams were examined by enzyme-linked immunosorbent assay (ELISA) and ELISA inhibition test. Abp4 showed broad cross-reaction to human serum albumin (HSA) conjugates of several beta-lactams, 6-aminopenicillanic acid and 7-aminocephalosporanic acid. Abp7 reacted only with ampicillin and cephalexin, which have the same acyl side chain. In ELISA inhibition tests, Abp4 inhibited binding to ampicillin-HSA by benzylpenicilloyl-epsilon-amino-n-caproic acid, and strongly inhibited the binding by penicillamine. Abp7 strongly inhibited the reaction by aminobenzylpenicilloyl-epsilon-amino-n-caproic acid, but benzylpenicilloyl-epsilon-amino-n-caproic acid was less effected. These data suggest that Abp4 recognizes the thiazolidine ring and Abp7 recognizes the acyl side chain. Therefore, the thiazolidine ring-epitope acts in broad cross-reaction among beta-lactams, and the acyl side chain acts only in the cross-reactivity between penam and cephem, which have a similar acyl side chain.     

Immunohistological identification of B cell differentiation in human tonsillar follicles by using monoclonal antibodies

Using immunoperoxidase technique and various monoclonal antibodies, B1, B2, OKT9, OKT10 and Leu-7, we investigated B cell differentiation in the tonsillar follicle. Mantle zone was stained with B1 intensely and B2 faintly. In contrast, germinal center was stained with B1, OKT10 and OKT9, and more intensely with B2. In the intermediate part of the germinal center some large cells were stained with OKT10. With OKT9 antibody, most of cells in the lymphoepithelial symbiosis and some large dendritic cells considered to be tangible body macrophages or dendritic reticulum cells in the germinal center were intensely stained. Leu-7 positive cells were localized mainly in the intermediate part of the germinal center. Stages of B cell differentiation in the tonsillar follicle were discussed, considering these results.     

Human nesidioblastosis tissue as an immunogen for generation of islet cell specific monoclonal antibodies

Nesidioblastosis pancreas was used as an immunogen in BALB/c-mice to generate monoclonal antibodies against structures of human islet cells. Seven clones were selected by screening 311 growing hybridomas for reactivity with the rat insulinoma cell line, RIN m5F, and with cryostat sections from human pancreas. None of the selected clones reacted with pancreatic hormones or endocrine-specific peptides. Monoclonal antibodies cross-reacting with the RIN-cell line also bound to different endocrine organs or cell lines, while some RIN-cell-negative clones bound only to Langerhans islets from human pancreas.     

Mechanism of suppression of lipopolysaccharide-driven B cell differentiation by anti-mu antibodies. Evidence for a trans-acting repressor of transcription

Bivalent anti-mu antibodies suppress LPS-driven B cell differentiation by inhibiting the coordinate activation of a family of differentiation-related genes, including those encoding the heavy, light, and J chains of IgM. We have shown that the presence of inhibitors of RNA or protein synthesis during a pulse with anti-mu can interfere with induction of suppression. We suggest that suppression is mediated by a trans-acting repressor protein with specificity for common motifs in regulatory regions of each of these genes.     

Quantitation of apolipoprotein B by polyclonal and monoclonal antibodies

A non-competitive sandwich enzyme linked immunosorbent assay for the quantitation of apolipoprotein B with polyclonal and monoclonal antibodies was developed. Polyclonal antibodies were used as 'coater'. In the assay with polyclonal antibodies, the same antibody was used as conjugate with alkaline phosphatase. For studies with monoclonal antibodies, enzyme conjugated anti-mouse immunoglobulin had to be used, since monoclonal antibodies lost their reactivity upon enzyme conjugation. Two murine monoclonal antibodies were employed: MAB B-1 with specificity for apolipoproteins (Apo) B-48 and B-100 and MAB B-5 with specificity for B-100 (Radioimmunoassay Inc.). In a reference group Apo B values of 0.82 +/- 0.20 g/l were measured with polyclonal antibodies, 0.68 +/- 0.19 g/l and 0.95 +/- 0.33 g/l with MAB B-1 and MAB B-5. In pure hypercholesterolemia, a similar increase was found with all three antibodies, while in combined hyperlipoproteinemia MAB B-5 gave greater than 40% lower values. Differences were also found with respect to the correlation between Apo B and cholesterol or triglycerides.     

Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies.

We used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. None of the monoclonal autoantibodies appeared to bind to a significant percentage of cells of relatively small cell size, either before or after culture. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Further experiments, including those using aggregated Ig to block antibody binding, strongly indicated that anti-histone antibody binding was not Fc receptor mediated. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations (0.25 micrograms/ml) of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases.     

B cell activation by cytomegalovirus

Human cytomegalovirus is shown to be a nonspecific polyclonal B cell activator. The B cell response is independent of virus replication and requires little, if any, T cell help.     

Human monoclonal antibodies to group B streptococcus. Reactivity and in vivo protection against multiple serotypes

Group B streptococcal (GBS) infections cause significant mortality and morbidity among infants. Passive antibody immunotherapy has been proposed as treatment for infected infants. To this end, two human mAb-secreting cell lines were produced by EBV immortalization of human B cells. The mAbs were specific for the group B polysaccharide and bound to strains of all five serotypes as demonstrated by ELISA and crossed immunoelectrophoresis. The mAbs reacted and opsonized 100% (132/132) of the clinical isolates tested which represented all four capsule types. Both prophylactic and therapeutic protection with these mAbs were demonstrated in neonatal rats given lethal infections of types Ia and III human clinical isolates. These data indicate that a single human mAb directed against the group B carbohydrate can protect against GBS infections caused by the different serotypes. This antibody may be useful in the passive immunotherapy of infants infected with GBS.