Detection in Malaysia of a Borrelia sp. From Haemaphysalis hystricis (Ixodida: Ixodidae).

Spirochetes from the Borrelia genus are known to cause diseases in humans, namely Lyme disease and relapsing fever. These organisms are commonly transmitted to humans by arthropod vectors including ticks, mite, and lice. Here, we report the molecular detection of a Borrelia sp. from a Haemaphysalis hystricis Supino tick collected from wildlife in an Orang Asli settlement in Selangor, Malaysia. Phylogenetic analyses of partial 16s rRNA and flaB gene sequences revealed that the Borrelia sp. is closely related to the relapsing fever group borreliae, Borrelia lonestari, Borrelia miyamotoi, and Borrelia theileri, as well as a number of uncharacterized Borrelia sp. from ticks in Portugal and Japan. To our knowledge, this is the first report of a Borrelia sp. detected in H. hystricis, and in Malaysia. The zoonotic potential of this Borrelia sp. merits further investigation.

Repetitive transgenes in C. elegans accumulate heterochromatic marks and are sequestered at the nuclear envelope in a copy-number- and lamin-dependent manner.

Chromatin is nonrandomly distributed in nuclear space, yet the functional significance of this remains unclear. Here, we make use of transgenes carrying developmentally regulated promoters to study subnuclear gene positioning during the development of Caenorhabditis elegans. We found that small transgenes (copy number ≤50) are randomly distributed in early embryonic nuclei, independent of promoter activity. However, in differentiated tissues, these same transgenes occupied specific subnuclear positions: When promoters are repressed, transgenes are found at the nuclear periphery, whereas active, developmentally regulated promoters are enriched in the nuclear core. The absence of specific transgene positioning in embryonic nuclei does not reflect an absence of proteins that mediate perinuclear sequestration: Embryonic nuclei are able to sequester much larger transgene arrays (copy number 300-500) at the periphery. This size-dependent peripheral positioning of gene arrays in early embryos correlates with the accumulation of heterochromatic marks (H3K9me3 and H3K27me3) on large arrays. Interestingly, depletion of nuclear lamina components caused release of arrays from the nuclear envelope and interfered with their efficient silencing. Our results suggest that developmentally silenced chromatin binds the nuclear lamina in a manner correlated with the deposition of heterochromatic marks. Peripheral sequestration of chromatin may, in turn, support the maintenance of silencing.

DNA methylation profiles in diffuse large B-cell lymphoma and their relationship to gene expression status.

In an initial epigenetic characterization of diffuse large B-cell lymphoma (DLBCL), we evaluated the DNA methylation levels of over 500 CpG islands. Twelve CpG islands (AR, CDKN1C, DLC1, DRD2, GATA4, GDNF, GRIN2B, MTHFR, MYOD1, NEUROD1, ONECUT2 and TFAP2A) showed significant methylation in over 85% of tumors. Interestingly, the methylation levels of a CpG island proximal to FLJ21062 differed between the activated B-cell-like (ABC-DLBCL) and germinal center B-cell-like (GCB-DLBCL) subtypes. In addition, we compared the methylation and expression status of 67 genes proximal (within 500 bp) to the methylation assays. We frequently observed that hypermethylated CpG islands are proximal to genes that are expressed at low or undetectable levels in tumors. However, many of these same genes were also poorly expressed in DLBCL tumors where their cognate CpG islands were hypomethylated. Nevertheless, the proportional reductions in BNIP3, MGMT, RBP1, GATA4, IGSF4, CRABP1 and FLJ21062 expression with increasing methylation suggest that epigenetic processes strongly influence these genes. Lastly, the moderate expression of several genes proximal to hypermethylated CpG tracts suggests that DNA methylation assays are not always accurate predictors of gene silencing. Overall, further investigation of the highlighted CpG islands as potential clinical biomarkers is warranted.

Role of the N-terminal forkhead-associated domain in the cell cycle checkpoint function of the Rad53 kinase.

Forkhead-associated (FHA) domains are multifunctional phosphopeptide-binding modules and are the hallmark of the conserved family of Rad53-like checkpoint protein kinases. Rad53-like kinases, including the human tumor suppressor protein Chk2, play crucial roles in cell cycle arrest and activation of repair processes following DNA damage and replication blocks. Here we show that ectopic expression of the N-terminal FHA domain (FHA1) of the yeast Rad53 kinase causes a growth defect by arresting the cell cycle in G(1). This phenotype was highly specific for the Rad53-FHA1 domain and not observed with the similar Rad53-FHA2, Dun1-FHA, and Chk2-FHA domains, and it was abrogated by mutations that abolished binding to a phosphothreonine-containing peptide in vitro. Furthermore, replacement of the RAD53 gene with alleles containing amino acid substitutions in the FHA1 domain resulted in an increased DNA damage sensitivity in vivo. Taken together, these data demonstrate that the FHA1 domain contributes to the checkpoint function of Rad53, possibly by associating with a phosphorylated target protein in response to DNA damage in G(1).

FHA domain boundaries of the dun1p and rad53p cell cycle checkpoint kinases.

Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead-associated (FHA) domains. Here, we demonstrate that these FHA domains contain 130-140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55-75 residues). In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks. This indicates that the non-catalytic FHA domain plays an important role in the transcriptional function of the Dun1p protein kinase.

Genomic mapping of chromosomal region 2p15-p21 (D2S378-D2S391): integration of Genemap'98 within a framework of yeast and bacterial artificial chromosomes.

The region of chromosome 2 encompassed by the polymorphic markers D2S378 (centromeric) and D2S391 (telomeric) spans an approximately 10-cM distance in cytogenetic bands 2p15-p21. This area is frequently involved in cytogenetic alterations in human cancers. It also harbors the genes for several genetic disorders, including Type I hereditary nonpolyposis colorectal cancer (HNPCC), familial male precocious puberty (FMPP), Carney complex (CNC), Doyne's honeycomb retinal dystrophy (DHRD), and one form of familial dyslexia (DYX-3). Only a handful of known genes have been mapped to 2p16. These include MSH2, which is responsible for HNPCC, FSHR, the gene responsible for FMPP, EFEMP-1, the gene mutated in DHRD, GTBP, a DNA repair gene, and SPTBN1, nonerythryocytic beta-spectrin. The genes for CNC and DYX-3 remain unknown, due to lack of a contig of this region and its underrepresentation in the existing maps. This report presents a yeast- and bacterial-artificial chromosome (YAC and BAC, respectively) resource for the construction of a sequence-ready map of 2p15-p21 between the markers D2S378 and D2S391 at the centromeric and telomeric ends, respectively. The recently published Genemap'98 lists 146 expressed sequence tags (ESTs) in this region; we have used our YAC-BAC map to place each of these ESTs within a framework of 40 known and 3 newly cloned polymorphic markers and 37 new sequence-tagged sites. This map provides an integration of genetic, radiation hybrid, and physical mapping information for the region corresponding to cytogenetic bands 2p15-p21 and is expected to facilitate the identification of disease genes from the area.

Mutations in the human Jagged1 gene are responsible for Alagille syndrome.

Alagille syndrome (AGS) is an autosomal-dominant disorder characterized by intrahepatic cholestasis and abnormalities of heart, eye and vertebrae, as well as a characteristic facial appearance. Identification of rare AGS patients with cytogenetic deletions has allowed mapping of the gene of 20p12. We have generated a cloned contig of the critical region and used fluorescent in situ hybridization on cells from patients with submicroscopic deletions to narrow the candidate region to only 250 kb. Within this region we identified JAG1, the human homologue of rat Jagged1, which encodes a ligand for the Notch receptor. Cell-cell Jagged/Notch interactions are known to be critical for determination of cell fates in early development, making this an attractive candidate gene for a developmental disorder in humans. Determining the complete exon-intron structure of JAG1 allowed detailed mutational analysis of DNA samples from non-deletion AGS patients, revealing three frame-shift mutations, two splice donor mutations and one mutation abolishing RNA expression from the altered allele. We conclude that AGS is caused by haploinsufficiency of JAG1.

A 2.8-Mb clone contig of the multiple endocrine neoplasia type 1 (MEN1) region at 11q13.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder that results in parathyroid, anterior pituitary, and pancreatic and duodenal endocrine tumors in affected individuals. The MEN1 locus is tightly linked to the marker PYGM on chromosome 11q13, and linkage analysis has placed the MEN1 gene within a 2-Mb interval flanked by D11S1883 and D11S449. As a step toward cloning the MEN1 gene, we have constructed a 2.8-Mb clone contig consisting of YAC and bacterial clones (PAC, BAC, and P1) for the D11S480 to D11S913 region. The bacterial clones alone represent nearly all of the 2.8-Mb contig. The contig was assembled based on a high-density STS-content analysis of 79 genomic clones (YAC, PAC, BAC, and P1) with 118 STSs. The STSs included 22 polymorphic markers and 20 transcripts, with the remainder primarily derived from the end sequences of the genomic clones. An independent cosmid contig for the 1-Mb PYGM-SEA region was also generated. Support for correctness of the 2.8-Mb contig map comes from an independent ordering of the clones by fiber-FISH. This sequence-ready contig will be a useful resource for positional cloning of MEN1 and other disease genes whose loci fall within this region.

Interferon-gamma downregulates the proliferative response of hapten-specific B cells stimulated by antigen and cytokines.

IFN-gamma plays a role in many aspects of cellular interactions, both positive and negative. Among its functions during the immune response, the antagonistic effects of IFN-gamma and IL-4 are well documented. Observations in our laboratory suggested that IFN-gamma could also interfere with the activation of single, antigen-specific B cells by antigen and other cytokines. Closer examination revealed that IFN-gamma reduced the number of proliferating cell clones in response to antigen and a variety of cytokines, alone or in combination. Cell viability remained at the initial level and the cells were still able to produce Ig, albeit to a lesser extent than in the absence of IFN-gamma. On the other hand, the frequency of IgM secreting clones was not affected, whereas the total amount of secreted IgM was lower in the presence of IFN-gamma, probably due to the reduced cell number and a decrease in Ig production. In addition, proliferation was prevented when B cells were pre-incubated with IFN-gamma and then stimulated by other cytokines. Kinetic studies revealed that INF-gamma had to be present from the onset of culture because delayed addition did not inhibit the proliferation of the B cells. After its initial action, IFN-gamma could be removed without abolishing the negative signal for proliferation. From these results it can be concluded that IFN-gamma transmits a signal that causes B cells to stop proliferating and prevents them from forming large clones.

Effects of combinations of cytokines on murine antigen-specific B lymphocytes.

Cytokines are among the most important mediators in the immune response. They act on B lymphocytes at various stages along the activation pathway. To study the effects of combinations of cytokines, we have used an antigen-specific single B cell-cloning system devised in this laboratory. We report here that IL-1 can enhance the proliferative response and IgM secretion by B cells induced by IL-6. The results of a similar study demonstrated that IL-1 can also enhance the effects of IL-5 on B cell proliferation and IgM secretion in an additive manner. Kinetic analyses showed that IL-1 had to be present from the beginning of the culture for an optimum cooperative effect, whereas the addition of IL-6 could be delayed up to 2 days without a significant reduction of the response. In contrast, IL-5 had to be added together with IL-1 at the onset of culture to promote an optimum response. The responses elicited by IL-1 plus IL-6, IL-1 plus IL-5, and IL-4 plus IL-5 were almost identical. The addition of further cytokines to these cultures gave no enhancement above the effects observed with the two-cytokine combinations.

Spontaneous and cytokine-inducible 'natural' immunoglobulin secreting cells in organized lymphoid tissues of mice.

The number and frequency of spontaneous and cytokine-inducible 'natural' immunoglobulin-secreting cells (ISC) were determined in bone marrow (BM), spleen and Peyer's patch (PP), in vitro. Cells were cultured at limiting dilution in the presence or absence of exogenous recombinant cytokines and supernatants then assayed for total immunoglobulin (Ig) and Ig isotype using an ELISA. Most spontaneous ISC were found in the spleen and BM, with fewer in PP. The addition of recombinant interleukin 5 (rIL-5) promoted a marked increase in both the ISC frequency and the amount of Ig secreted/ISC whereas recombinant IL-6 (rIL-6) promoted only a marginal increase. Recombinant IL-4 (rIL-4) promoted a marginal increase in ISC frequency only. The isotype profile of ISC was in the order IgM greater than IgG2 greater than IgA greater than IgG3 greater than IgG1. The exposure of cells to 1200 rad of gamma-radiation resulted in decreased numbers of spontaneous ISC in all tissues, but the addition of rIL-5 or rIL-6 to the irradiated cells increased both the ISC frequency and Ig secreted. The Ig isotype profile was similar to that of non-irradiated ISC with a few minor exceptions. This large population of potential cytokine-inducible ISC could contribute to 'natural' Ig secretion in vivo.

The response of human B cells to interleukin 4 is determined by their stage of activation and differentiation.

The effect of purified recombinant human interleukin 4 (IL-4) on proliferation and IgM secretion of normal and malignant human B cells was studied. IL-4 was found to co-stimulate the proliferation of splenic B cells in the presence of anti-Ig coupled to polyacrylamide beads (anti-Ig beads) for a period of 4 days. In contrast, IL-4 had little co-stimulatory effect on the proliferative response of splenic B cells to the more potent mitogen Staphylococcus aureus Cowan strain 1 (SAC). Moreover, IL-4 inhibited interleukin 2 (IL-2)-induced proliferation of cells co-stimulated with SAC. Mitogen-induced pre-activation of B cells in the presence of IL-4 resulted in a reduction in subsequent IL-2-induced IgM secretion without significantly affecting proliferation. Human B-cell tumours were also cultured over a 2-3 day period in the presence of anti-Ig beads plus IL-2, or IL-4 or both IL-2 and IL-4. IL-4 inhibited IL-2-induced proliferation in all cases of B-cell chronic lymphocytic leukaemia (B-CLL) and the majority of cases of low-grade lymphoma (LGL) and hairy cell leukaemia (HCL). These findings suggest that IL-4 has stimulatory actions on resting B cells, most evident in the presence of submaximal co-mitogenic signals, and inhibitory actions on activated B cells, especially antagonism of the effects of IL-2.

Clonal repertoire analysis of murine B cells specific for repeat sequence antigens of Plasmodium falciparum.

Clonal analysis of the murine B-cell repertoire has been used to investigate the possible role of tandem repeat sequence epitopes of Plasmodium falciparum in immune evasion. A limiting dilution culture system was used whereby murine spleen cells were stimulated with the B-cell mitogen lipopolysaccharide (LPS) in the presence of 3T3 fibroblast filler cells. One in three B cells were shown to produce clones secreting immunoglobulin measurable by an ELISA. The frequency of antibody forming cell precursors (AFCp) specific for the 3' repeat epitopes of the ring injected erythrocyte surface antigen (RESA) was estimated in non-primed mice and found to be low. However, an accurate frequency determination was not possible using this method since the detection of the few positive cultures was found to depend on the presence of more than one AFCp or its products. Limiting dilution analysis was used to assess the frequency and repertoire of splenic AFCp at various times after immunization with a synthetic peptide of the RESA 3' repeat epitope (8 x 4-mer), presented in various ways. There was no marked increase in LPS-responsive AFCp specific for this antigen at the level of either IgM or IgG secretion. This was in marked contrast to the antibody response in vivo, where moderate IgG antibody titres, normally indicative of a secondary response, were seen in the serum of the same mice used for AFCp assay. This discrepancy between serum titre and AFCp frequency following immunization was not apparent with a non-malarial antigen, keyhole limpet haemocyanin (KLH). It was concluded that the LPS-stimulated limiting dilution culture system was not registering RESA-specific memory AFCp. These results raise the possibility that the malarial antigens are deficient in memory B-cell generation, or that secondary responses to these determinants may arise from a distinct B-cell progenitor which is non-responsive to LPS in vitro.

Recombinant human interleukin 6 (B cell stimulatory factor 2) enhances immunoglobulin secretion by single murine hapten-specific B cells in the absence of cell division.

We have assessed the role of recombinant human IL-6 (r-hu-IL-6) in promotion of early activation, proliferation, and immunoglobulin (Ig) secretion amongst single hapten-specific murine splenic B cells in vitro. It was found that r-hu-IL-6 acting alone was able to induce early B cell activation in a proportion of B cells, as measured by a significant increase in cell diameter within 24 h. An enhanced effect was seen in the concomitant presence of a 'T-independent' antigen. None of the B cells activated by r-hu-IL-6 appeared to divide, as the frequencies of proliferating clones induced by either medium alone or antigen alone were virtually identical whether r-hu-IL-6 was present or absent. However, assay of the culture supernatants for the presence of Ig by ELISA revealed that r-hu-IL-6 effected a significant 2-fold increase in the frequency of B cells secreting Ig. Thus, the prime effect of r-hu-IL-6 appears to be to recruit more precursor B cells into Ig secretion, rather than to promote proliferation or to enhance the amount of Ig secreted by pre-committed but non-cycling B cells. Delayed addition experiments showed that r-hu-IL-6 enhanced Ig secretion late in the activation pathway. Kinetics studies demonstrated detectable Ig secretion as early as day 2, and when taken with its apparent ability to induce early activation, these findings suggest that IL-6 is not exclusively a late-acting interleukin. Studies with size fractionated hapten-specific B cells showed the larger B cells to be preferentially responsive to r-hu-IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of the B lymphocyte repertoire by polyclonal activation. Hindrance by clones yielding antibodies which bind promiscuously to plastic.

We here describe a form of 'noise' in the ELISA as commonly performed on antigen-coated microtiter trays that represents a major hindrance to the accurate enumeration of infrequent antibody-forming cell (AFC) precursors (AFCp) specific for epitopes on monomeric proteins. Supernatants from cultures of lipopolysaccharide-stimulated murine splenocytes, when split into aliquots and separately assayed, scored as positive in parallel on ELISA trays coated with unrelated proteins and on uncoated trays. Some properties of such coincident false positives (CFP) noted were: (1) optical density (OD) ranges for CFP and non-CFP overlapped; (2) different members of CFP triplets on differently coated assay trays usually had similar OD values; (3) CFP-generating culture supernatants did not contain unusually high immunoglobulin concentrations; and (4) numbers of CFP-forming supernatants increased with increasing input cells/culture consistent with causation by single AFCp present at an approximate mean frequency of 1 in 6600 CBA splenocytes. It is proposed that CFP are due to AFC clones that secrete antibody reactive with some epitope(s) present in the assay tray itself. Repertoire elements with such 'anti-plastic' characteristics are rarer than anti-keyhole limpet hemocyanin (KLH) AFCp, but at least as frequent as anti-bovine serum albumin (BSA) or anti-transferrin (TFN) AFCp.

Immunologic competence of B cells subjected to constitutive c-myc oncogene expression in immunoglobulin heavy chain enhancer myc transgenic mice.

B cell activation is associated with a marked transient rise in expression of the c-myc proto-onco-gene. A unique opportunity to examine the effects of constitutive c-myc expression upon B cell function is provided by transgenic mice in which the c-myc oncogene is regulated by the enhancer (E mu) from the immunoglobulin locus (E mu-myc mice). We have examined the immunologic competence of B cells from E mu-myc mice both in vitro and in vivo. Upon stimulation, many E mu-myc B cells can proliferate to form clones most of which contain antibody-forming cells. However, the frequency of responsive B cells from E mu-myc donors is only about 30% that of B cells from normal littermates. Thus, enforced myc expression is not sufficient to block the differentiation of all B cells, but a much larger fraction of the immunoglobulin-bearing cells from E mu-myc mice are incompetent. Upon immunization, E mu-myc mice mounted specific antibody responses, although some responses were delayed. Isotype switching can occur, since we observed hemolytic plaques of both IgM and IgG type and detected specific antibody of both classes in the serum. Moreover, the serum from nonimmunized E mu-myc mice contained normal levels of both IgM and IgG. Thus constitutive expression of the c-myc gene appears to retard B cell differentiation, but does not grossly impair immunologic function in the intact animal.

Recombinant T cell replacing factor (interleukin 5) acts with antigen to promote the growth and differentiation of single hapten-specific B lymphocytes.

The role of murine recombinant T cell replacing factor (rTRF) (interleukin 5) in the early activation, proliferation, and antibody-forming cell (AFC) clone formation of single fluorescein (FLU)-specific B cells was examined in vitro. FLU-specific B cells were selected by their adherence to FLU-gelatin and then cultured in 10-microliters wells with or without rTRF in the presence or absence of the T-independent antigen FLU-polymerized flagellin (FLU-POL). rTRF acting alone was unable to induce early B cell activation as assessed by significant cell enlargement after 24 hr in culture. When acting in the presence of FLU-POL, however, a greater number of B cells were induced to enlarge than with FLU-POL alone. When FLU-specific B cells were cultured in the presence of FLU-POL, the addition of rTRF markedly increased the frequencies of both proliferating clones and AFC clones above that induced by FLU-POL alone. Furthermore, in the presence of FLU-POL, the activity of rTRF was comparable to that seen with the mixture of B cell growth and differentiation factors contained within the supernatant from concanavalin A-stimulated EL4 cells. However, rTRF exerted little activity when acting alone in contrast to the medium conditioned by concanavalin A-stimulated EL4 cells which showed some activity in the absence of FLU-POL. rTRF acting with FLU-POL also promoted AFC clone development among single B cells stimulated in the presence of 3T3 fibroblast filler cells. Thus rTRF can be added to the list of B cell active factors (including recombinant murine interleukin 1 and recombinant human interleukin 2) that act in the concomitant presence of antigen to induce both growth and differentiation among single hapten-specific murine splenic B cells. This stands in contrast to the activity seen with interleukin 4 (formerly termed B cell stimulatory factor 1) which acts to promote early activation and proliferation but not IgM secretion.

T-independent activation of single B cells: an orderly analysis of overlapping stages in the activation pathway.

This review has three chief purposes. It describes a microculture system in which single, hapten-specific B lymphocytes can be microscopically observed, cultured and assayed for antibody production in isolation and thus are the unequivocal target of ligands present in the culture fluid. It defines the respective roles of antigens and cytokines acting singly or in combination in the four discernible phases of the immunoproliferative cascade, namely activation, clonal expansion, IgM antibody secretion and isotype switching. It then argues that this precise stepwise analysis can yield useful information concerning important immunological situations, such as experimentally induced immunological tolerance or the effects of constitutive expression of the c-myc oncogene. Evidence is presented that initial activation of the resting B cell in "T-independent" triggering can be achieved either by attachment of a molecule that has B-cell stimulatory properties, such as FLU-LPS or FLU-polymerized flagellin (FLU-POL) or by the lymphokine interleukin 4 (IL-4). IL-4 + FLU-POL is somewhat more effective than either agent alone. IL-4 alone or, better, FLU-POL + IL-4 can stimulate clonal proliferation of the B cell, but FLU-POL alone does not achieve this. Moreover, IL-4 or FLU-POL + IL-4 lead to very little antibody formation. None of IL-1, IL-2 or IL-5 acting alone causes either activation or proliferation. IgM antibody formation is stimulated most strongly by FLU-POL + IL-5, somewhat less strongly by FLU-POL + IL-1 + IL-2 and rather weakly with antigen plus only one of the latter cytokines. The cloning efficiency in the single cell system, and the median clone size can be markedly enhanced by the addition of small numbers of fibroblast or other filler cells to the cultures. While filler cell-free clones do not progress to the stage of isotype switching, filler cell-supported ones can do so in up to 30% of cases. The only cloned lymphokine which has so far been found to promote such switching is IL-4, and the fact that it is at least as powerful as a T-cell supernatant may mean that it is the only agent active in this particular system. However, the detailed pattern of secreted isotypes is different from that seen when MHC-restricted, carrier-specific T cells act on hapten-specific B cells. Hapten-specific B cells from animals rendered neonatally tolerant to FLU-HGG exhibit anergy in the single cell system.(ABSTRACT TRUNCATED AT 400 WORDS)

Clonal analysis of the anti-DNA repertoire of murine B lymphocytes.

The present studies characterize at the clonal level the repertoire of lipopolysaccharide-responsive murine B lymphocytes committed to the production of antibodies reactive with denatured DNA. This repertoire is vast in normal mice as 1-5% of total mitogen-induced antibody-forming cell clones secreted denatured DNA-reactive antibodies when the splenocyte donors were CBA (Ighj), BALB/c (Igha), C57BL/6 (Ighb), CBA nu/nu, and C57BL/6 nu/nu athymic mice. The autoimmune NZB (Ighe) strain did not display elevated proportions of anti-denatured DNA antibody-forming cell precursors. Cross-reactions shown by CBA anti-denatured DNA antibodies suggest that many antibodies might derive significant binding energy from interaction with the bases or similar hydrophobic moieties. Cross-reactions with other tested polynucleotides were frequent, but cross-reactions with phospholipids and phosphocholine were undetectable. Most anti-DNA antibodies bound preferentially or exclusively to single-stranded denatured DNA as compared to double-stranded native DNA. The frequency of anti-denatured DNA antibody-forming cell precursors among CBA peritoneal cells was not elevated. Fluorescence-activated cell sorter-selected Ly-1-positive NZB splenic B cells were not enriched, and Ly-1 negative B cells were not depleted of anti-DNA antibody-forming cell precursors. These results show that antibody-forming cell precursors specific for denatured DNA are not restricted to the Ly-1 positive B-cell subset.

Single cell studies on the role of B-cell stimulatory factor 1 in B-cell activation.

The role of the T-cell-derived lymphokine B-cell stimulatory factor 1 (BSF-1) in the early activation, proliferation, and antibody-forming cell (AFC) clone formation of single fluorescein-specific B lymphocytes isolated from normal mouse spleens by hapten-gelatin adherence has been studied in vitro. BSF-1 acting alone induced early B-cell activation, as assessed by a significant increase in cell diameter of single B cells cultured for 24 hr. A small but significant number of these B cells formed proliferating clones, some of which secreted antibody. When acting with the specific antigen fluorescein-polymerized flagellin, BSF-1 augmented early cell enlargement and markedly enhanced proliferation, but it did not increase the frequency of AFC clones stimulated by fluorescein-polymerized flagellin alone. The further addition of recombinant murine interleukin 1 (IL-1) marginally enhanced proliferation caused by antigen plus BSF-1. No synergy was observed between BSF-1 and IL-1 for antibody formation. In the presence of fibroblast filler cells, BSF-1 substantially inhibited AFC clone development achieved by antigen plus IL-1. BSF-1 was also found to be inhibitory to AFC clone development stimulated by specific antigen acting with either recombinant human interleukin 2 (IL-2) or with IL-2 plus IL-1, both in the presence or absence of filler cells. The results suggest that BSF-1 plays a complex role in the regulation of the B-cell activation pathway by enhancing early activation and antigen-specific proliferation as well as inhibiting the effects of other B-cell factors on antibody formation. BSF-1 is the only cytokine so far tested in the single B-cell system that acts with antigen to promote proliferation without concomitant antibody production.