B5, a new B cell-restricted activation antigen

The characterization of a new human B cell-restricted activation antigen (B5) is described in this report. With the use of a monoclonal antibody to B5, we show that B5 can be detected on peripheral blood or splenic B cells after 1 day of stimulation with either anti-immunoglobulin, protein A, Epstein Barr virus, or pokeweed mitogen. In contrast, B5 was not expressed on resting B, T, or myeloid cells. More important, B5 could not be detected on activated T cells or monocytes. The B5 antigen was expressed on some lymphoblastoid B cell lines and B cell neoplasms but was not expressed on leukemias or lymphomas of T or myeloid origin. The B5 antigen is distinct from previously reported B cell activation antigens by its m.w. and pattern of cellular expression. These studies suggest that B5 is a novel B cell-restricted activation antigen, which may be useful to study the events of early human B cell activation.     

Mouse NKR-P1B, a novel NK1.1 antigen with inhibitory function

The mouse NK1.1 Ag originally defined as NK cell receptor (NKR)-P1C (CD161) mediates NK cell activation. Here, we show that another member of the mouse CD161 family, NKR-P1B, represents a novel NK1.1 Ag. In contrast to NKR-P1C, which functions as an activating receptor, NKR-P1B inhibits NK cell activation. Association of NKR-P1B with Src homology 2-containing protein tyrosine phosphatase-1 provides a molecular mechanism for this inhibition. The existence of these two NK1.1 Ags with opposite functions suggests a potential role for NKR-P1 molecules, such as those of the Ly-49 gene family, in regulating NK cell function.     

A unique antigen on mature B cells defined by a monoclonal antibody

A novel 42,000 dalton antigen (MB-1) expressed by mature human B cells in blood and tonsil was identified and characterized by utilizing a hybridoma monoclonal antibody. A comparison of MB-1 with other known B cell antigens suggests that the MB-1 antigen has not been previously identified. From one-and two-color immunofluorescence studies, it appears that the MB-1 antigen is found on all normal immunoglobulin (Ig)-expressing cells, but not on T cells, thymocytes, granulocytes, or platelets. Studies of malignant B cell tumors reveal that the antigen is expressed by virtually all Ig-expressing B cell tumors but only 10% of SIg- B-lineage leukemias. Data from these studies suggest that the MB-1 antigen is expressed late in B cell ontogeny before the expression of SIg.     

B7, a B-cell-restricted antigen that identifies preactivated B cells

After activation with antigen or mitogen, a number of cell surface proteins appear that are not expressed on resting B cells. To date, a number of B lineage restricted and associated activation antigens have been reported that appear at distinct intervals after in vitro activation. In this report, we describe a new B lineage restricted activation antigen (B7) that appears within 24 hr of in vitro stimulation. The expression of B7 antigen, which is detected on a minor subpopulation of B cells isolated from peripheral blood and lymphoid tissues, is strongly induced following stimulation with either anti-immunoglobulin or Epstein-Barr virus. In contrast, B7 was not detected on resting or activated T cells or monocytes. The B7 antigen was expressed on a subset of B cell lines and B cell neoplasms, but was not detected on leukemias and lymphomas of T cell or myeloid origin. B7 was distinguished from other B cell restricted and associated activation antigens by its unique pattern of expression on a variety of hemopoietic cell lines. The biochemical characterization of B7, that it is a single chain protein of 60 kDa, further distinguishes it from other B cell activation antigens. The functional importance of the B7 antigen was demonstrated when splenic B cells were fractionated into the B7+ and B7- populations. The peak of proliferation in response to anti-Ig, appeared earlier within the B7+ population. These studies suggest that B7 antigen identifies a subpopulation of B cells that are preactivated or primed in vivo, and have an accelerated response to subsequent activation via cross-linking of surface Ig.     

Characterization of a human B cell-specific antigen (B2) distinct from B1

A human B lymphocyte-specific antigen (B2) was identified and characterized by the use of a monoclonal antibody. By indirect immunofluorescence and quantitative absorption, B2 was shown to be expressed exclusively on Ig+ B cells isolated from peripheral blood and lymphoid tissues. In contrast, B2 was not found on monocytes, resting and activated T cells, Null cells, or granulocytes, nor was it found on cell lines or tumor cells of T cell or myeloid origin. Functional studies demonstrated that only B2 antigen-positive splenocytes could be induced to differentiate into plasma cells under the stimulus of pokeweed mitogen, further confirming the B cell specificity of B2. It was then demonstrated that the B2 antigen was distinct from the previously described B cell-surface determinants including surface immunoglobulin, Ia-like antigens, and Fc and C3 receptors. More importantly, the B2 antigen has been clearly shown to be distinct from the previously described B cell-specific antigen, B1, by its m.w. and expression on normal and malignant B lymphocytes. The distinct distribution of B2 on normal and malignant lymphocytes supports the notion of B cell heterogeneity and provides further evidence for existence of subpopulations of human B lymphocytes.     

Studies of a tumor-associated antigen,COX-1, recognized by a monoclonal antibody

Summary Monoclonal antibodies against an ovarian tumor cell line, OC-3-VGH, were generated using modified hybridoma technology. Among the seven that were selected for their high specificity and affinity to ovarian cancer cells and low cross-reactivity to most normal human tissues, RP 215 was shown to react specifically with a tumor-associated antigen, COX-1, from certain ovarian/cervical cancer cell lines. By Western blot assay, COX-1 was shown to have a subunit molecular mass of about 60 kDa and exist as an aggregate in the native state. COX-1 could also be detected in the shed medium of certain cultured tumor cells. A solid-phase sandwich enzyme-immunoassay procedure was designed for quantitative determinations of COX-1 in the shed medium or in patients' sera using RP 215 for both well-coating and the signal detection. Highly purified COX-1 was obtained from the shed medium of cultured OC-3-VGH tumor cells mainly by hydroxyapatite and immunoaffinity chromatography with RP 215 as the affinity ligand. At neutral pH, purified COX-1 also exists as an aggregate and is relatively stable at temperatures below 50°C. Its immunoactivity was found to decrease with time in the presence of trypsin. However, the immunoactivity of COX-1 was not affected upon incubation with carbohydrate-digestive enzymes or concanavalin A and only partially inactivated in the presence of NaIO₄ or iodoacetamide. Treatments of COX-1 with dithiothreitol and guanidine thiocyanate resulted in a complete loss of activity. Furthermore, rabbit antisera raised against purified COX-1 exhibited similar immunospecificity to that of RP 215. The results of this study suggest that COX-1 is a glycoprotein consisting of a 60 kDa subunit, which is recognized by RP 215 through its peptide determinant. Preliminary retrospective clinical studies were performed to assess the utility of a COX-1 enzyme immunoassay kit for detection and monitoring of patients with ovarian and cervical cancers.     

Activation of human B lymphocytes by a particulate antigen

A specific IgM antibody response toward the trinitrophenol (TNP) hapten can be induced in mononuclear blood cell suspensions upon culture with a particulate antigen: polyacrylamide beads conjugated with the TNP hapten (TNP-PAA). The response, and its specificity, are demonstrated by an increase in the number of TNP binding B lymphocytes (specific rosette forming cells), by the appearance of cells producing anti-TNP antibody at a high rate (haemolytic plaques), (ELISA test). The anti-TNP response requires monocytes, the role of which is to produce interleukin-1 (IL-1) and T lymphocytes (belonging to the T4 helper subset) the role of which is to produce interleukins (the characterization of which is under study). We propose a model or B cell activation based on the following signals: an early specific signal, provided by the particulate antigen; several non specific signals, provided by T derived interleukins. The anti-TNP response is negatively regulated by monocytes, the functional states of which can be modified in certain situations (autoimmunity, aging) or influenced by glucocorticoids. Suppressor T lymphocytes of this response (not exclusively of the T8 phenotype) can be induced and this can allow the evaluation of T suppressor cell function. This was used in adult idiopathic thrombocytopenic purpura treated with high doses of intra-venous gammaglobulins.     

Human monoclonal antibodies to a genus-specific chlamydial antigen, produced by EBV-transformed B cells

Stable B cell lines producing human monoclonal antibodies to Chlamydia were established from salpingitis patients in the early convalescence phase. The antibody-producing cells were immortalized by Epstein Barr virus (EBV) transformation. Specific antibody-secreting clones were enriched by a stepwise microtiter plate cloning procedure. The selected B cell clones showed stable antibody production for more than 1 yr in continuous culture. Serologic specificity was demonstrated by micro-immunofluorescence (micro-IF) tests against a panel of Chlamydia reference strains. The antibodies were of the IgG1 subclass, and complement fixation could be demonstrated for one clone. There was no cross-reactivity against a large number of other bacteria. The monoclonal antibodies are directed against a common genus-specific surface antigen of the Chlamydia organism. Infected McCoy cells showed a brilliant, punctuated fluorescence surrounded by an inclusion membrane. Compared with conventional antisera, the monoclonal antibodies showed a clearer fluorescence pattern with very low background.     

B4, a human B lymphocyte-associated antigen expressed on normal, mitogen-activated, and malignant B lymphocytes

The characterization of a new B cell-specific antigen (B4) is described in this report. With the use of a monoclonal antibody to B4, it was shown that B4 is present on B cells isolated from peripheral blood and lymphoid organs, on cell lines derived from normal and malignant B cells, and on tumor cells isolated from patients with B cell-derived neoplasms. B4, in contrast, was not detected on normal, activated, or malignant cells of T or myeloid origin. The B4 antigen is distinct from known B cell antigens, including sIg, Ia, B1, B2, Fc, and C3. Examination of mitogen-stimulated B lymphocytes suggests that the B4 antigen initially increases with B cell activation and then is lost at the terminal stage of B cell differentiation. Moreover, the observation that B4 is expressed on almost all early B cell tumors suggests that it may precede B1, CALLA, cytoplasmic mu, and B2 in early B cell ontogeny.     

A mathematical model of B lymphocyte differentiation: Control by antigen

A mathematical model of B lymphocyte differentiation, based on experimental results, has been developed. The model focuses on the role of antigen in initiating and regulating B cell differentiation while other mechanisms, acting in concert with antigen but the functioning of which can be circumvented under appropriate conditions, are not considered. The importance of presence of antigen at individual stages of B cell differentiation was studied in experiments with an easily metabolizable antigen. Immunocompetent cells (ICC), arising by antigen-independent differentiation of stem cells, are activated by antigen (they become immunologically activated cells — IAC). Excess of antigen drives IAC into the terminal stage (antibody-forming cells — AFC) thereby restricting proliferation. Exhaustive terminal differentiation results in tolerance. A low primary dose permits IAC to escape antigen; IAC proliferate and later give rise to resting memory cells (MC) which are amenable to reactivation. MC have higher avidity for antigen (due to higher affinity, number and density of receptors) and the effect of different doses of antigen on MC is diverse. A very low secondary dose induces tolerance, a medium dose secondary response, and the administration of a high dose of antigen also brings about tolerance. The model suggests that the fate of memory cells is controlled by the ratio RAg, of the number of immunoglobulin receptors on B cells (R) to the number of available antigenic molecules (Ag), low values of RAg favouring stimulation to differentiation while high values of RAg favouring inactivation. A nonlinear system of ordinary differential equations, describing the development of the populations involved in antigen driven B cell differentiation, was used to simulate experiments and good qualitative agreement was achieved.     

IL-2 production by B cells stimulated with a specific antigen

The ability of a specific antigen (Ag) to stimulate B cells to produce IL-2 was examined with a murine B lymphoma line, A20-HL, which expressed surface IgM specific for trinitrophenyl (TNP). The culture supernatant of A20-HL cells stimulated with TNP3.9-ovalbumin (-OVA) or anti-IgM goat IgG contained an activity which supported the proliferation of an IL-2-dependent T cell line, CTLL-2. Neither TNP3.9-OVA nor anti-IgM antibody stimulated the parent line, A20.2J, which did not bear TNP-specific sIg, whereas anti-mouse Ig rabbit IgG F(ab)2 did stimulate both A20-HL cells and A20.2J cells. The active material in the culture supernatant was identified as IL-2 based on the experiments in which the activity was inhibited by anti-IL-2 mAb, and IL-2 mRNA was expressed in A20-HL cells stimulated with TNP3.9-OVA or anti-IgM antibody. These results support the conclusion that a specific Ag can stimulate A20-HL cells to produce IL-2. For IL-2 production, TNP receptors on A20-HL cells have to be appropriately cross-linked, inasmuch as either TNP3.9-OVA or TNP6.7-OVA was much more effective than TNP1.2-OVA and TNP22.9-OVA in the induction of IL-2 production by A20-HL cells.     

Regulation of B cell receptor-mediated MHC class II antigen processing by FcgammaRIIB1

The processing and presentation of Ag by Ag-specific B cells is highly efficient due to the dual function of the B cell Ag receptor (BCR) in both signaling for enhanced processing and endocytosing bound Ag. The BCR for IgG (FcgammaRIIB1) is a potent negative coreceptor of the BCR that blocks Ag-induced B cell proliferation. Here we investigate the influence of the FcgammaRIIB1 on BCR-mediated Ag processing and show that coligating the FcgammaRIIB1 and the BCR negatively regulates both BCR signaling for enhanced Ag processing and BCR-mediated Ag internalization. Treatment of splenic B cells with F(ab')2 anti-Ig significantly enhances APC function compared with the effect of whole anti-Ig; however, whole anti-Ig treatment is effective when binding to the FcgammaRIIB1 was blocked by a FcgammaRII-specific mAb. Processing and presentation of Ag covalently coupled to anti-Ig were significantly decreased compared with Ag coupled to F(ab')2anti-Ig; however, the processing of the two Ag-Ab conjugates was similar in cells that did not express FcgammaRIIB1 and in splenic B cells treated with a FcgammaRII-specific mAb to block Fc binding. Internalization of monovalent Ag by B cells was reduced in the presence of whole anti-Ig as compared with F(ab')2 anti-Ig, but the internalized Ag was correctly targeted to the class II peptide loading compartment. Taken together, these results indicate that the FcgammaRIIB1 is a negative regulator of the BCR-mediated Ag-processing function.     

Galactose-regulated expression of hepatitis B surface antigen by a recombinant yeast

Summary A yeast expressing the hepatitis B surface antigen (HBsAg) gene under the control of a regulated promoter was grown in batch and fed-batch modes. In batch fermentations, the addition of galactose caused the rapid production of HBsAg. Cells grown in fed-batch mode did not produce HBsAg unless the conditioned medium was replaced prior to induction.     

Cloning of a complementary DNA encoding a new mouse B lymphocyte differentiation antigen, homologous to the human B1 (CD20) antigen, and localization of the gene to chromosome 19

The human B1 (CD20) molecule is a differentiation Ag found only on the surface of B lymphocytes. This structurally unique phosphoprotein plays a role in the regulation of human B cell proliferation and differentiation. In order to determine whether this structure is also expressed by murine B cells, cDNA clones that encode the mouse equivalent of the B1 molecule were isolated. The longest murine cDNA clone isolated, pmB1-1, contained a 1.4-kb insert with an 873 base pair open reading frame that encodes a protein of 32 kDa. The predicted mouse B1 protein contains three hydrophobic domains that may span the membrane four times and shares a 73% amino acid sequence homology with the human B1 protein. The pmB1-1 cDNA probe was used to examine mB1 mRNA expression. Northern blot analysis indicated that pmB1-1 hybridized with two mRNA species of 2.3 and 3.0 kb that were expressed only in murine spleen lymphocytes, in B lineage cell lines representing mature B cells, and were weakly expressed in one of two plasmacytoma cell lines. pmB1-1 failed to hybridize with RNA isolated from murine T cell lines, thymus, and nonlymphoid tissues. Southern blot analysis indicated that mB1 was encoded by a single copy gene. In situ hybridization localized the mB1 gene to chromosome 19 band B, a region that also contains the genes that encode the Ly-1, Ly-10, and Ly-12 Ag. These results suggest that only B cells express this heretofore undescribed murine cell-surface protein that is structurally homologous with the membrane-embedded human B1 Ag.     

Characterization and purification of acidocin 1B, a bacteriocin produced by Lactobacillus acidophilus GP1B

In the present study, acidocin 1B, a bacteriocin produced by Lactobacillus acidophilus GP1B, exhibited profound inhibitory activity against a variety of LAB and pathogens, including Gram-negative bacteria, and its mode of action was to destabilize the cell wall, thereby resulting in bactericidal lysis. Acidocin 1B was found to be heat stable, because it lost no activity when it was heated up to 95 degrees C for 60 min. It retained approximately 67% of the initial activity after storage for 30 days at 4 degrees C, and 50% of its initial activity after 30 days at 25 degrees C and 37 degrees C. The molecular mass of acidocin 1B was estimated to be 4214.65 Da by mass spectrometry. Plasmid curing results indicated that a plasmid, designated as pLA1B, seemed to be responsible for both acidocin 1B production and host immunity, and that the pLA1B could be transformed into competent cells of L. acidophilus ATCC 43121 by electroporation. Our findings indicate that the acidocin 1B and its producer strain may have potential value as a biopreservative in food systems.     

Isolation of Novel Antifungal Antibiotics,Ezomycins A1, A2, B1 and B2

From ezomycin complex produced by a strain of Streptomyces were isolated four components named ezomycins A₁ (C₂₆H₄₀N₈O₁₆S), A₂ (C₁₉H₂₈N₆O₁₃), B₁ (C₂₆H₃₉N₇O₁₇S) and B₂ (C₁₉H₂₇N₅O₁₄) which are new pyrimidine nucleosides. Ezomycins A₁ and B₁ containing l-cystathionine were found to be responsible for specific antimicrobial activity of the complex against Sclerotinia and Botrytis species.     

Cell vacuolization induced by Helicobacter pylori: Inhibition by bafilomycins A1, B1, C1 and D

Abstract All available bafilomycins (A1, B1, C1 and D) inhibit and revert macroscopic vacuolization induced by Helicobacter pylori cell-free extracts. Bafilomycin A1 displays the highest activity, followed by bafilomycin B1, C1 and D. The different potency of bafilomycins correlates with their ability to inhibit the vacuolar-type ATPase (V-ATPase) and to dissipate the membrane pH gradient of intracellular acidic organelles. These results suggest that bafilomycins should be considered as possible therapeutic agents in the treatment of gastritis.     

A unique DR-related B cell differentiation antigen

The Ia or class II molecules in both mouse and man are the basis for the genetic control of the immune response. In addition to DR, other class II antigens have been described in man. We describe a new human Ia antigen K19, recognized by three monoclonal antibodies (HK-9, HK-19, and HK-20). This antigen has the general biochemical characteristics of an Ia antigen but is different from a DR antigen. Further, this antigen is found only on mature B lymphocytes and not on monocytes and activated T cells. Thus, this antigen may represent a new Ia-like molecule that is preferentially expressed on mature B cells.