Biomedical subjects

N A Bos

Publications and source records attributed to N A Bos.

45 records · Page 3Linked to original sources

PubMed1991

Isolation and molecular characterization of the B cells producing the paraprotein in a case of benign monoclonal gammapathy in C57BL mice.

Benign monoclonal gammapathy (BMG) is defined as a benign monoclonal B cell proliferative disorder characterized by the presence of a persisting component of homogenous immunoglobulins (H-Ig) in the serum. A possible role of antigenic stimulation in the development of BMG has been suggested. From a C57BL mouse, a murine model for BMG, we have isolated clonally related B cells in order to investigate the occurrence of somatic mutations in the variable heavy chain (VH) region of the genes of H-Ig-producing B cell clones. Therefore, B cells were immortalized by hybridoma technology. The hybridomas were screened for resemblance of the serum H-Ig component by Wieme agar electrophoresis, followed by immunoblotting and isoelectrofocusing. Clonal relationship was investigated by Southern blot analysis using a JH probe. In this way we isolated five hybridomas producing an IgG2a, kappa that was identical to the original serum H-Ig component according to testing with anti-idiotypic antisera. mRNA sequencing of four hybridomas showed only one base pair difference in the VH genes. This particular gene belonged to the J558 VH gene family. When compared to the most closely related known VH sequence, three base pair differences were found. The almost complete absence of base pair differences in the VH genes of the four sequenced hybridomas, compared with an independently derived hybridoma, suggests that the same germ-line VH gene has been used and that somatic mutations were infrequent in our BMG clone.

Animals↗

PubMed1989

B cell repertoire in adult antigen-free and conventional neonatal BALB/c mice. I. Preferential utilization of the CH-proximal VH gene family PC7183.

Early in ontogeny B cells preferentially use VH gene families which are most adjacent to the genes coding for the constant part of the immunoglobulin molecule. In conventional adult mice, however, a random usage of VH gene families has been found. We investigated the role of exogenous antigenic stimulation on this normalization of VH gene usage by B cells. Therefore, we made use of adult germ-free BALB/c mice fed a chemically defined ultrafiltered antigen-free diet (GF-CD) and neonatal conventional BALB/c mice. Both the adult GF-CD and the newborn conventional mice represent situations with minimal exogenous antigenic stimulation. The results obtained with RNA dot blot hybridization with probes specific for the different VH gene families showed in hybridomas from adult GF-CD BALB/c mice a preferential usage of the CH-proximal VH gene family PC7183. In hybridomas from 5-day-old conventional BALB/c mice a less frequent usage of the J558 VH gene family was found and an increased usage of the PC7183 VH gene family than what would be expected from random usage. Evidence is presented that the RNA giving a positive signal with the PC7183 probe represents functional messages for IgM production.

Animals↗

PubMed1989

B cell repertoire in adult antigen-free and conventional neonatal BALB/c mice. II. Analysis of antigen-binding capacities in relation to VH gene usage.

Hybridomas were derived from lipopolysaccharide-reactive splenic B cells of adult germ-free BALB/c mice fed a chemically defined ultrafiltered "antigen-free" diet (GF-CD) and from splenic B cells of 5-day-old conventional (CV-NEO) BALB/c mice. The monoclonal antibodies (mAb) from both collections of hybridomas were tested for reactivity against a large panel of antigens of exogenous and endogenous origin. As a source of natural exogenous antigens 36 different bacteria and 9 different viruses were used, while as endogenous antigens frozen tissue sections of stomach, liver and kidney, the Hep-2 cell line and the anti-idiotopic mAb Ac38 and Ac146 were used. In both collections of mAb approximately 70% reacted with one or more bacterial antigens, while no reactivity could be detected against the viral antigens. Of the GF-CD and CV-NEO hybridomas, 16% and 19%, respectively, reacted with one or more frozen tissue sections. Overall 56% and 68% of the GF-CD and CV-NEO hybridomas, respectively, were producing multireactive antibodies reactive to several exogenous and/or endogenous antigens. Among the GF-CD hybridomas a correlation was found between multireactivity and the usage of the VH gene family PC7183. In CV-NEO hybridomas, however, the preferential utilization of the VH gene family PC7183 was found among both mono- and multireactive hybridomas. The results suggest (a) that the actual B cell repertoire of neonatal mice consists of a large proportion of multireactive B cells which are reactive with autoantigens and bacterial antigens, but not viral antigens and (b) that in antigen-deprived mice the neonatal repertoire is largely preserved during maturation of the mice.

Animals↗

PubMed1989

Serum immunoglobulin levels and naturally occurring antibodies against carbohydrate antigens in germ-free BALB/c mice fed chemically defined ultrafiltered diet.

This study investigates the influence of exogenous antigenic stimulation on the serum immunoglobulin levels and the levels of circulating natural antibodies against carbohydrate antigens. Thus, BALB/c mice, raised in a germ-free environment and fed a chemically defined, ultrafiltered diet (GF-CD), were employed. These mice had normal serum IgM levels, but IgG and IgA levels were approximately 5% of conventionally reared littermates. The concentrations of all four IgG isotypes were equally low. The variable part of the heavy chains of naturally occurring BALB/c antibodies against a number of carbohydrate antigens, including 3-fucosyllactosamine (3-FL), levan and dextran, are encoded by VH441, and these antibodies express cross-reactive idiotopes recognized by the monoclonal antibodies 6C4 and 6B1. Antibodies against levan and dextran were lower in GF-CD than in conventional mice, but levels of anti-3FL antibodies, and 6C4 and 6B1 idiotopes, were comparable to those in conventional animals. Peptidoglycan polysaccharide complexes (PPC) are carbohydrate antigens of bacterial origin, like levan and galactan. Naturally occurring antibodies against PPC were found in the serum of conventional mice, but were severely reduced in GF-CD mice. The results indicate that most naturally occurring antibodies against carbohydrate antigens of bacterial origin found in conventional mice are caused by exogenous stimulation.

Animals↗

PubMed1988

The influence of exogenous antigenic stimulation on the specificity repertoire of background immunoglobulin-secreting cells of different isotypes.

The total number of spontaneously occurring ("background") IgM-, IgG-, and IgA-secreting cells and the frequency of antigen-specific IgM-, IgG-, and IgA-secreting cells were determined in germ-free BALB/c mice fed a chemically defined ultrafiltered diet (GF-CD), in specific pathogen-free BALB/c mice fed an autoclaved natural ingredient diet (SPF-NI), and in conventional BALB/c mice fed nonautoclaved natural ingredients (CV-NI). This was done by means of the ELISA-plaque assay. The results did not show differences among the various groups of mice with regard to the total numbers of IgM-secreting cells in the various lymphoid organs. Also the frequencies of IgM-secreting cells specific for DNP27-BSA and the anti-idiotypic monoclonal antibodies Ac38 and Ac146 did not differ significantly among GF-CD, SPF-NI, and CV-NI mice. GF-CD mice, however, did show substantially decreased numbers of IgG- and IgA-secreting cells in their lymphoid organs. Furthermore, there were striking differences in the frequencies of antigen-specific IgG- and IgA-secreting cells between GF-CD mice and the two other groups of mice. These results indicate that exogenous antigenic stimulation has a great effect on both the total numbers and the specificity repertoires of background IgG- and IgA-secreting cells. Such an influence could not be detected with regard to the background IgM-secreting cells. This suggests two distinct compartments of background Ig-secreting cells: a very stable, endogenously regulated compartment consisting mainly of IgM-secreting cells, and another compartment, consisting mainly of IgG- and IgA-secreting cells, whose numbers and specificity repertoire appeared to be influenced by exogenous antigenic stimulation.

Animals↗

PubMed1988

Clonal analysis of the synergistic mitogenic effect of lipopolysaccharide and dextran sulphate on B cell activation, growth, and differentiation into Ig-secreting cells.

Splenic B cells of BALB/c mice were stimulated in vitro either with lipopolysaccharide (LPS), dextran sulphate (DxS), or with both LPS and DxS. The absolute frequency of B cells that differentiate into clones of immunoglobulin (Ig)-secreting cells upon activation with these mitogens was determined by means of limiting dilution analysis. Determination of the number of Ig-secreting cells in DxS-stimulated cultures with the protein A plaque assay proved to be difficult because of the anti-complementary activity of DxS. Therefore, we assayed the number of Ig-secreting cells with a reverse ELISA-plaque assay. This assay is complement-independent and is at least as sensitive as the protein A plaque assay. The results showed that LPS, DxS, and the combination of LPS and DxS stimulate 1 in 27, less than 1 in 500 and 1 in 15 nucleated spleen cells of BALB/c mice to proliferation and differentiation into a clone of Ig-secreting cells, respectively, indicating that these mitogens have a synergistic effect on B cells at the precursor cell level. Analysis of the clone size of the activated B cells showed that the combination of both mitogens also caused a larger clone size. Thus, the synergistic effect of LPS and DxS that was previously observed in mass cultures is due to two separate effects. Quantitatively most important, however, is that more precursors are activated by the combination of the two mitogens.

Animals↗

PubMed1987

Early development of Ig-secreting cells in young of germ-free BALB/c mice fed a chemically defined ultrafiltered diet.

The influence of antigenic stimulation on the early development of the "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells has been studied in mice. To evaluate the effect of such exogenous stimulation by an evolving microbial microflora, the young of BALB/c mice that were kept under germ-free conditions and fed a low molecular weight chemically defined synthetic diet (GF-CD) were compared with the young of conventional BALB/c mice fed natural ingredients (CV-NI). The young were first suckling maternal milk and between Days 15 and 18 changed to the same diet as their parents. Background Ig-secreting cells in the spleen were enumerated in the protein A plaque assay. The specificity repertoire of the IgM-secreting cells was determined with plaque assays specific for sheep red blood cells (SRBC) that were haptenized with different concentrations of nitroiodophenyl (NIP), 4-hydroxy-3.5-dinitrophenyl (NNP), and 2,4,6-trinitrophenyl (TNP). The results show that during the first few weeks of life the numbers of background IgM-, IgG-, and IgA-secreting cells in the spleen develop faster in CV-NI mice than in GF-CD mice. At 4 weeks of age equal numbers of IgM- and IgG-secreting cells were found in both groups of mice, but the number of IgA-secreting cells remained reduced in GF-CD mice during the whole period of observation. The frequencies of IgM-secreting cells specific for the differently haptenized SRBC were the same in both groups of mice during the observation period of 10 weeks. This suggests that the ontogenetic appearance of IgM-, IgG-, and IgA-secreting cells in the spleen, and the specificity repertoire of the IgM-secreting cells, as far as was tested in our panel, is independent of exogenous antigenic and/or mitogenic stimulation. However, during neonatal development the rate of development of the background Ig synthesis is enhanced by environmental antigenic stimulation.

Animal Feed↗

PubMed1986

Expression of the human immunoglobulin heavy chain gene of the 14q+ chromosome in a t(8;14)-positive Burkitt lymphoma cell line demonstrated in somatic cell hybrids.

A newly isolated Burkitt lymphoma cell line, ROS-1, carrying the specific translocation (8;14) has been studied using somatic cell hybridization techniques. As in other reported Burkitt cell lines, the oncogene c-myc was found to be translocated from the 8q- to the 14q+ chromosome. In contrast to other reports, expression of the mu immunoglobulin (Ig) heavy chain correlated with the presence of the 14q+ derivative and not of its normal homologue. These results indicate that the translocation (8;14) is not necessarily an abortive event for the production of the mu Ig heavy chain by the 14q+ derivative.

Burkitt Lymphoma↗

PubMed1986

'Background' Ig-secreting cells in pregnant germfree mice fed a chemically defined ultrafiltered diet.

During syngeneic pregnancy the numbers of 'spontaneously' occurring ('background') Ig-secreting cells were determined in the spleen, bone marrow (BM) and mesenteric lymph nodes (MLN) of BALB/c mice that were kept under germfree conditions and fed a low molecular weight synthetic diet (GF-CD), SPF BALB/c mice fed autoclaved natural ingredient (SPF-NI) and conventional BALB/c mice fed natural ingredient (CV-NI). 'Background' Ig-secreting cells were enumerated in the protein A plaque assay and the specificity repertoire of the IgM-secreting cells was determined with plaque assays specific for differently haptenized sheep red blood cells (SRBC). The numbers of 'background' Ig-secreting cells, especially the IgG- and IgA-secreting cells, are very much reduced in the BM and MLN of GF-CD mice as compared to SPF-NI and CV-NI mice. During pregnancy the total number of Ig-secreting cells increased in all lymphoid organs tested, but the proportional increase was most prominent for the IgG- and IgA-secreting cells in the BM and MLN of the GF-CD mice. This increase could only be due to their pregnant state since all environmental antigenic influences are excluded in GF-CD mice. No changes were found in the background specificity repertoire of the IgM-secreting cells during pregnancy. This suggests a polyclonal activation of the Ig-secreting cells during pregnancy. The reason for this activation remains obscure, but it has to be endogenous. Pregnancy apparently induces a new steady state of the immune system, which can be most properly investigated in GF-CD mice.

Animals↗