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.     

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.     

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.     

B cell triggering properties of a nontoxic derivative of amphotericin B

The immunomodulating properties of amphotericin B (AMB), an antifungal polyene antibiotic, have been reported in multiple studies. However, many findings on the subject are conflicting, and the precise mechanism of AMB action on the immune system is yet unknown. Because toxicity and limited solubility of AMB are likely to be responsible for these discrepancies, we synthesized a nontoxic derivative of AMB (AMBSH), and we investigated its immune modulating effects on murine B cells. Our results show that AMBSH induces a strong proliferative response under conditions where AMB is weakly efficient or toxic, and that AMBSH supports maturation to Ig secretion. When suboptimal doses of LPS (or BCGF) are present together with AMBSH, a synergistic effect on B cell proliferation occurs. Frequency analyses reveal that, although only a limited number of B cells respond to AMBSH alone, a large population of B cells will respond to subthreshold doses of LPS in the presence of this polyene. Finally, we show that incubation of spleen cells with AMBSH results in an increase in Ia expression. These results are discussed in terms of the membrane disorganizing properties of polyene antibiotics.     

Expression of a V region-less B cell receptor confers a tolerance-like phenotype on transgenic B cells

Neoplastic B cells from H chain disease patients express a truncated B cell receptor (BCR), comprising a membrane Ig that lacks part of its extracellular domain. It has been speculated that deletion of the Ag binding domain would confer a constitutive activity on the BCR, as it has been shown for oncogenic growth factor receptors. A V region-less BCR has constitutive activity, because in transgenic mice it causes inhibition of endogenous H chain gene rearrangements and relieves the requirement for surrogate L chain in pre-B cell development. However, it has been speculated that normal Ag receptors also display constitutive activity. Here we show that transgenic B cells expressing a membrane H chain disease protein on their surface are phenotypically and functionally similar to B cells developing in the presence of their cognate Ag and that cells with normal levels of mutant BCR are eliminated in spleen via a bcl-2 sensitive pathway while progressing toward the mature stage. In contrast, cells with lower levels of mutant receptors develop as mature B cells. These findings support the view that the truncated BCR has a constitutive activity that mimics ligand binding, in analogy to what has been shown for oncogenic growth factor receptors.     

MurA as a Primary Target of Tulipalin B and 6-Tuliposide B

(?)-Tulipalin B and (+)-6-tuliposide B were confirmed to inhibit MurA in vitro. However, contrary to fosfomycin, these compounds showed potent inhibitory activities against MurA overexpressing Escherichia coli, especially in the presence of UDP-GlcNAc. These observations suggest that these compounds induced bacterial cell death not through a MurA malfunction, but in such a MurA-mediated indirect manner as the inhibition of other Mur enzymes.     

Development and maintenance of a B220- memory B cell compartment

We have recently demonstrated that a novel somatically mutated B220(-) memory B cell subset rapidly dominates the secondary immune response to (4-hydroxy-3-nitrophenyl) acetyl (NP). Upon adoptive transfer with Ag, B220(+)NP(+) memory B cells produce large numbers of B220(-)NP(+) B cells that can rapidly differentiate into plasma cells. Therefore, it is not clear whether the novel B220(-) memory compartment is a consequence of secondary Ag challenge or whether it develops as a stable memory subset after initial Ag challenge. In this study, we demonstrate the gradual emergence of B220(-)NP(+) B cells in the spleen to maximal numbers 3 wk after initial Ag exposure. Like their B220(+) counterparts, the B220(-) B cells initially appear unmutated at days 5-7; however, the majority rapidly accumulate affinity increasing mutations by days 9-14 of the primary immune response. More extensive cell surface phenotype (GL7(-)BLA-1(-)CD24(-)CD43(+)) argues strongly against germinal center localization and direct analysis in situ places a cohort of B220(-)CD11b(+)NP(+) B cells in the red pulp of the spleen and not in the MZs. These data provide direct evidence for the development of B220(-) memory B cells as a unique cellular consequence of primary Ag exposure. The cellular dynamics and molecular attributes of these unique memory B cells suggest they are distinct cellular products of the germinal center reaction in the primary response and are maintained long-term in the spleen and bone marrow.     

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.     

Emericella astellata, a new producer of aflatoxin B, B and sterigmatocystin

AIMS: To report on aflatoxin B(1) and B(2) production from a species of Emericella. METHODS AND RESULTS: Aflatoxins and sterigmatocystin were determined by high-pressure liquid chromatography (HPLC) with diode array detection and confirmed by HPLC with mass spectrometry detection. Among 30 known species of Emericella only one species produced aflatoxin. Strains originating from the same geographical source material had different patterns of aflatoxin and sterigmatocystin production on different media, indicating that epigenetic factors may be involved in the regulation of aflatoxin production. However, two cultures from the same original genet were very similar. CONCLUSIONS: Emericella astellata can produce small amounts of sterigmatocystin and aflatoxin B(1) and B(2). SIGNIFICANCE AND IMPACT OF THE STUDY: Emericella has been used extensively in genetic studies and therefore the isolates producing aflatoxin can be used to elucidate the genetic, evolutionary and maybe ecological role of aflatoxins using molecular genetic methods.     

Splenic B cells and antigen-specific B cells process anti-Ig in a similar manner

B lymphocytes can process and present antigen to T cells. However, the fate of native antigen after its binding to specific B cells, i.e., the intracellular events involved in the processing and recycling of the antigenic fragments to the cell surface for antigen presentation, are not well understood. In the present study, we demonstrate that murine B cells degrade anti-Ig molecules bound to their surface and release acid soluble fragments into the supernatant. We also demonstrate that the kinetics of this process are identical for anti-mu, anti-delta, and anti-light chain antibodies, indicating that both surface IgM and surface IgD are equally effective in binding antigen and directing its processing. We also describe the effects of azide, chloroquine, and irradiation on this process. To extend these studies to the processing of specifically bound antigen, we demonstrate that highly purified trinitrophenyl antigen-binding cells degrade anti-Ig molecules with the same kinetics as unpurified splenic B cells. Thus, this purified population provides a suitable model system for the analysis of antigen degradation by antigen-specific cells.     

Notch2 haploinsufficiency results in diminished B1 B cells and a severe reduction in marginal zone B cells

Recent studies have implicated a role for Notch in the generation of marginal zone (MZ) B cells. To further investigate the role of Notch in the B cell lineage, we have analyzed the effects of reduced Notch2 signaling in mice expressing one functional allele of Notch2 (Notch2(+/-)). Notch2(+/-) mice have reduced B1 B cells of the peritoneal cavity and show a severe reduction in MZ B cells of the spleen. The reduction in MZ B cells was not due to the disruption of splenic architecture, disregulated terminal differentiation, nor to increased apoptosis within the MZ B cell compartment. Rather, our data suggest that Notch2 haploinsufficiency leads to impaired development of MZ B cells, possibly by impacting the formation of immediate MZ B precursors. These results provide evidence that Notch2 plays a determining role in the development and/or the maintenance of B1 B and MZ B cells.     

Characterization of a B cell-derived growth-enhancing factor produced by a human B cell line established from a patient with rheumatoid arthritis

A human B cell line, TKS-1, which was established from the peripheral blood of a patient with rheumatoid arthritis, was found to spontaneously produce a factor which enhances the activity of interleukin 1 (IL-1). This factor, designated B cell-derived growth-enhancing factor (BGEF), enhanced IL-1-induced proliferation of peanut agglutinin nonagglutinated thymocytes. BGEF also enhanced IL-1-induced production of interleukin 2 (IL-2) by both thymocytes and a human T cell clone, HSB.2 C5B2. BGEF alone did not induce the production of IL-2. BGEF failed to induce proliferation of the IL-2-dependent T cell clone, and did not enhance its response to IL-2. The activity of BGEF was not blocked by antisera against human IL-1-alpha or human IL-1-beta. Gel filtration analysis revealed that BGEF has a m.w. of 60,000 to 65,000 in its native state. We concluded that BGEF differed from IL-1 and IL-2, but is a novel factor produced by TKS-1 cells. In addition, we found that partially purified B cells from patients with rheumatoid arthritis produced factors which enhanced the activity of IL-1.     

Virus-like display of a neo-self antigen reverses B cell anergy in a B cell receptor transgenic mouse model

The ability to distinguish between self and foreign Ags is a central feature of immune recognition. For B cells, however, immune tolerance is not absolute, and factors that include Ag valency, the availability of T help, and polyclonal B cell stimuli can influence the induction of autoantibody responses. Here, we evaluated whether multivalent virus-like particle (VLP)-based immunogens could induce autoantibody responses in well-characterized transgenic (Tg) mice that express a soluble form of hen egg lysozyme (HEL) and in which B cell tolerance to HEL is maintained by anergy. Immunization with multivalent VLP-arrayed HEL, but not a trivalent form of HEL, induced high-titer Ab responses against HEL in both soluble HEL Tg mice and double Tg mice that also express a monoclonal HEL-specific BCR. Induction of autoantibodies against HEL was not dependent on coadministration of strong adjuvants, such as CFA. In contrast to previous data showing the T-independent induction of Abs to foreign epitopes on VLPs, the ability of HEL-conjugated VLPs to induce anti-HEL Abs in tolerant mice was dependent on the presence of CD4(+) Th cells, and could be enhanced by the presence of pre-existing cognate T cells. In in vitro studies, VLP-conjugated HEL was more potent than trivalent HEL in up-regulating surface activation markers on purified anergic B cells. Moreover, immunization with VLP-HEL reversed B cell anergy in vivo in an adoptive transfer model. Thus, Ag multivalency and T help cooperate to reverse B cell anergy, a major mechanism of B cell tolerance.     

Biological Activity of Aflatoxin B2a

The toxicity of alfatoxin B(2a) (hydroxydihydro-aflatoxin B(1)) was studied in several biological systems. Aflatoxin B(2a) is the monohydroxylated derivative obtained from addition of water to the double bond of the terminal furan of B(1). Examination of the sensitivity of a group of microorganisms to B(2a) demonstrated that the inhibitory spectrum was similar to aflatoxin B(1). However, the toxicity of B(2a) was markedly lower than B(1), as measured by the initiation of bile duct hyperplasia in ducklings. Binding of aflatoxin to deoxyribonucleic acid (DNA) was determined by measuring the hypochromicity produced by the nucleic acid at 363 nm and the capacity of increasing amounts of DNA to quench the fluorescence of the toxin was also used as a measure of the binding of toxin to nucleic acid. These tests showed that the DNA-binding capacity of B(2a) was lower than B(1).     

Characterization of a human B lymphocyte-specific antigen

A human B lymphocyte-specific antigen (B1) was identified and characterized by the use of a monoclonal antibody. By indirect immunofluorescence, cytotoxicity, and quantitative absorption, B1 was present on approximately 9% of the peripheral blood mononuclear cell fraction and >95% of B cells from blood and lymphoid organs in all individuals tested. Monocytes, resting and activated T cells, null cells, and tumors of T cell and myeloid origin were B1 negative. B1 was distinct from standard B cell phenotypic markers, including Ig and Ia antigen. Removal of the B1 positive population in peripheral blood eliminated all B cells capable or responding to pokeweed mitogen by maturation to Ig-producing cells.