Attenuation of cytomegalovirus-induced endothelial intercellular adhesion molecule-1 mRNA/protein expression and T lymphocyte adhesion by a 2'-O-methoxyethyl antisense oligonucleotide.

Background: Intercellular adhesion molecule-1 (ICAM-1) is strongly induced under inflammatory conditions associated with allograft rejection, thereby promoting leukocyte recruitment and activation at the site of inflammation. Enhancement of ICAM-1 expression can also be the result of viral infection, in particular human cytomegalovirus (CMV), a frequent source of complications in the transplant recipient. In vitro studies have shown that CMV infection of endothelial cells (EC) results in the direct enhancement of ICAM-1 expression and consequent leukocyte adhesion/activation suggesting mechanisms by which CMV exacerbates graft vascular disease.

Suppression of src-induced transformed phenotype by expression of tropomyosin-1.

Suppression of high M(r) tropomyosins (TMs) is a common feature of transformed cells. Previous work from this laboratory has demonstrated that the isoform 1 of TM, TM1, acts as an anti-oncogene in ras-transformed murine fibroblasts. In this study, we have investigated whether TM1 is a ras-specific suppressor, or a general suppressor protein of the cellular transformation.

The human elk-1 gene family: the functional gene and two processed pseudogenes embedded in the IgH locus.

Elk-1 is a transcription factor whose activation by several mitogen-activated protein kinases (MAPKs) mediates the immediate early responses of the c-fos promoter to growth factors and other stimuli. Here, we report the structure of the human elk-1 gene, which we have localized about 6.5kb upstream of the properdin gene on the X chromosome.

CD40 ligand and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center and plasmacytoid phenotypic differentiation in a human monoclonal IgM+IgD+ B cell line.

B lymphocytes are induced to undergo Ig class switching and a complex phenotypic differentiation by the milieu of the germinal center. Partly as a result of the lack of a suitable in vitro B cell model, the relationship between these processes in the humans has never been formally established in vitro. We have identified a human monoclonal B cell line, CL-01, that expresses surface IgM and IgD and, upon induction with CD40 ligand, IL-4, and IL-10, switches to all seven downstream isotypes, showing typical DNA switch recombination preceded by germline transcription of targeted CH regions.

Enhancer complexes located downstream of both human immunoglobulin Calpha genes.

To investigate regulation of human immunoglobulin heavy chain expression, we have cloned DNA downstream from the two human Calpha genes, corresponding to the position in the mouse IgH cluster of a locus control region (LCR) that includes an enhancer which regulates isotype switching. Within 25 kb downstream of both the human immunoglobulin Calpha1 and Calpha2 genes we identified several segments of DNA which display B lymphoid-specific DNase I hypersensitivity as well as enhancer activity in transient transfections. The corresponding sequences downstream from each of the two human Calpha genes are nearly identical to each other.

Cleavage of oligodeoxyribonucleotides from controlled-pore glass supports and their rapid deprotection by gaseous amines.

A novel method for the deprotection of oligodeoxyribonucleotides has been developed. Gaseous amines such as ammonia or methylamine were employed under pressure to achieve mild and rapid deprotection conditions. For example, oligodeoxyribonucleotides having a (tert-butyl)phenoxyacetyl group for the protection of the exocyclic amino function of cytosine, adenine and guanine were released from controlled-pore glass supports and fully deprotected by ammonia or methylamine under gas phase conditions, at room temperature, within 35 or 2 min respectively.

Interleukin-10 induces immunoglobulin G isotype switch recombination in human CD40-activated naive B lymphocytes.

Upon activation, B lymphocytes can change the isotype of the antibody they express by immunoglobulin (Ig) isotype switch recombination. In previous studies on the regulation of human IgG expression, we demonstrated that interleukin 10 (IL-10) could stimulate IgG1 and IgG3 secretion by human CD40-activated naive (sIgD+) tonsillar B cells. To assess whether IL-10 actually promotes the DNA recombination underlying switching to these isotypes, we examined the effect of IL-10 on the generation of reciprocal products that form DNA circles as by-products of switch recombination.

Human Ig S gamma regions and their participation in sequential switching to IgE.

The Ig isotype switch from IgM to IgE is accompanied by a DNA recombination that joins S mu, the highly repetitive "switch" region upstream of the C mu gene, to the S epsilon region upstream of C epsilon, thereby creating a composite S mu-S epsilon region. In human B cells cultured in vitro with IL-4 to promote the switch to IgE, we previously described evidence for S mu-S gamma-S epsilon structures, suggesting that some B cells can switch sequentially from mu to gamma and then to epsilon; similar sequential switching to epsilon occurs routinely in the mouse. To identify which of the four human gamma genes might be involved in this mu-gamma-epsilon switching pathway, we cloned and analyzed nine S mu-S gamma-S epsilon composite switch regions and studied S epsilon-S gamma junctions from reciprocal deletion circles.

Human polyreactive and monoreactive antibodies: effect of glycosylation on antigen binding.

The present experiments were initiated to determine whether the carbohydrate portions of antibody molecules contribute to polyreactivity. Cell lines making human monoclonal polyreactive or monoreactive antibodies of the immunoglobulin (Ig) M, IgG and IgA isotypes were treated with tunicamycin to block N-linked glycosylation of the proteins. Analysis of the secreted native and non-glycosylated proteins revealed a > 95% inhibition of [3H]mannose incorporation.

Half-life of polyreactive antibodies.

Monoclonal polyreactive antibodies bind to a variety of self and foreign antigens. In contrast, monoclonal monoreactive antibodies bind to a single or restricted number of known antigens. The rate at which polyreactive antibodies are removed from the circulation compared to monoreactive antibodies has not been determined.

Structure of the VH and VL segments of polyreactive and monoreactive human natural antibodies to HIV-1 and Escherichia coli beta-galactosidase.

B lymphocytes committed to the production of antibodies binding to antigens on pathogenic bacteria and viruses (natural antibodies) are common components of the normal human B cell repertoire. A major proportion of natural antibodies is capable of binding multiple antigens (polyreactive antibodies). Using B cells from three HIV-1 seronegative healthy subjects, and purified HIV-1 and beta-galactosidase from Escherichia coli as selecting antigen, we generated three natural IgM mAb to HIV-1 and a natural IgM mAb to beta-galactosidase.

Recombination activating genes-1 and -2 of the rabbit: cloning and characterization of germline and expressed genes.

The recombination activating genes RAG-1 and RAG-2 appear to be necessary components of the machinery needed for the Ig or TCR gene rearrangements that occur in developing B and T lymphocytes. In addition RAG-2 has been implicated in the process of V-gene diversification by somatic gene conversion in the chicken. Because gene conversion may be an important mechanism for V-gene diversification in the rabbit, we cloned the rabbit RAG locus and characterized the coding regions of the genomic RAG-1 and RAG-2.

Clonal analysis of a human antibody response. II. Sequences of the VH genes of human IgM, IgG, and IgA to rabies virus reveal preferential utilization of VHIII segments and somatic hypermutation.

The construction of mAb-producing cell lines has been instrumental in dissecting the fine specificities and genetic makeup of murine antibodies to exogenous and self Ag. The analysis of the genetic composition of human antibody responses has been hampered by the difficulty in generating human mAb of predetermined class and specificity. Using B lymphocytes from three healthy subjects vaccinated with inactivated rabies virus vaccine, we generated nine human mAb binding to rabies virus and analyzed the genes encoding their VH regions.

Complete sequence of the genes encoding the VH and VL regions of low- and high-affinity monoclonal IgM and IgA1 rheumatoid factors produced by CD5+ B cells from a rheumatoid arthritis patient.

We have characterized the VH and VL genes of three low-affinity polyreactive and two high-affinity monoreactive IgM and IgA1 rheumatoid factor (RF) mAb generated using circulating CD5+ B cells from a single rheumatoid arthritis patient. We found that four and one RF mAb utilized genes of the VHIV and VHIII families, respectively. The VHIV gene usage by these RF mAb differs from the preferential VHIII, VHI, and, to a lesser extent, VHII gene usage by the IgM with RF activity found in patients with mixed cryoglobulinemia, Waldenstrom's macroglobulinemia, and other monoclonal gammopathies.

Evolutionarily conserved organization and sequences of germline diversity and joining regions of the rabbit T-cell receptor beta 2 chain.

We previously reported that there are three different copies of T-cell receptor beta chain constant region (C beta) genes in some rabbits, two of which are present on an approximately 16-kb and one on an approximately 6-kb Eco RI fragment. We also reported that one of the C beta genes on the approximately 16-kb fragment was chimeric, with a 5' cluster of J beta 2 segments and a 3' untranslated region of beta 1 type. Here we report the complete genomic sequences of the D beta 2 and J beta 2 segments associated with the chimeric C beta gene.

Identification of enhancer sequences 3' of the rabbit Ig kappa L chain loci.

The rabbit is useful for studies of Ig L chain gene expression because of a great disparity in expression of two isotypic forms of the kappa L chain. Normally, K1 is expressed at high levels and K2 is almost silent; expression of K2 increases in mutant or experimentally allotype-suppressed animals. The reasons for the preferential utilization of the K1 isotype have not been fully elucidated.

Clonal analysis of a human antibody response. Quantitation of precursors of antibody-producing cells and generation and characterization of monoclonal IgM, IgG, and IgA to rabies virus.

We quantitated and characterized the changes in the human B cell repertoire, at the clonal level, before and after immunization with rabies virus. Moreover, we generated 10 monoclonal cell lines producing IgM, IgG, and IgA antibodies to the virus. We found that in healthy subjects, not previously exposed to the virus, nearly 2% of the circulating B lymphocytes were committed to the production of antibodies that bound the virus.

Evolutionary conservation of splice sites in sterile C mu transcripts and of immunoglobulin heavy chain (IgH) enhancer region sequences.

The immunoglobulin JHC mu intron was cloned from genomic DNA of a VHa3 rabbit and a 1257 bp sequence which contains conserved enhancer and splice sites was determined. From positions 315 to 1257, there is approximately 72 and 67% similarity to available sequences of man and mouse, respectively (counting gaps as single changes at single positions). In earlier studies of rabbit cDNAs encoding immunoglobulin heavy chains, we found a C mu-encoding cDNA clone (pB3) derived from splenic mRNA of a Trypanosome-hyperimmunized rabbit (VHa1) which lacked VH, DH or JH sequences and had an unknown sequence 5' of that encoding C mu.

Allotypes of the constant region of the rabbit T cell receptor beta-2 chain.

Our laboratory previously reported that there was restriction fragment length polymorphism of TCR C beta genes in rabbits. EcoRI digests of DNA from different rabbits gave fragments of 9 and 6 kb (C beta a) or 14 and 6 kb (C beta b) that hybridized to a C beta cDNA probe. We also reported that the 9- and 14-kb types segregated as Mendelian traits and that there were allotypic differences in the first exon of the C beta 1 genes of C beta a and C beta b animals.

Genomic sequence and organization of rabbit Tcrb constant region genes.

We previously reported that some rabbits have three different copies of T-cell receptor b (Tcrb) constant region genes unlike man, mice, and rats who generally have two copies. Two of these C beta genes were found on an approximately 14 kilobase (kb) and one on an approximately 6 kb Eco RI fragment. The gene on the 6 kb fragment is of beta 2 type.

Genetic analyses of restriction fragment length polymorphisms using high molecular weight DNA from sperm or lymphocytes embedded in agarose.

A simple and efficient method for determining restriction fragment length polymorphism types on large numbers of individuals using small samples of peripheral blood or sperm cells is described. Whole cells embedded in low gelling/melting temperature agarose were treated with a series of enzyme, detergent, and washing steps to release high molecular weight DNA that was then digested with standard restriction enzymes such as EcoRI and PstI, electrophoresed, blotted, and probed as in normal Southern analyses. The technique should be readily adaptable to any application requiring DNA from small numbers of cells for Southern analyses or pulsed field gel electrophoresis.

Linked genetic markers of the rabbit kappa light chain are not linked to the Tcr beta chain genes.

In order to investigate linkage, we used serum allotypes of the two rabbit C kappa isotypes and restriction fragment length polymorphisms (RFLPs) of the genes for V kappa, C kappa, and T-cell receptor C beta. The inheritance of these genetic markers was studied through backcross and F2 matings. Southern analysis and hybridization of genomic DNA with a C kappa probe detected a 5 kb Pst I fragment linked to expression of the K2bas1 allotype and the presence of the kappa 1bbas gene and a 6.