Biomedical subjects

K Meek

Publications and source records attributed to K Meek.

At least 37 records · Page 2Linked to original sources

PubMed1995

Equine severe combined immunodeficiency: a defect in V(D)J recombination and DNA-dependent protein kinase activity.

V(D)J rearrangement is the molecular mechanism by which an almost infinite array of specific immune receptors are generated. Defects in this process result in profound immunodeficiency as is the case in the C.B-17 SCID mouse or in RAG-1 (recombination-activating gene 1) or RAG-2 deficient mice. It has recently become clear that the V(D)J recombinase most likely consists of both lymphoid-specific factors and ubiquitously expressed components of the DNA double-strand break repair pathway. The deficit in SCID mice is in a factor that is required for both of these pathways. In this report, we show that the factor defective in the autosomal recessive severe combined immunodeficiency of Arabian foals is required for (i) V(D)J recombination, (ii) resistance to ionizing radiation, and (iii) DNA-dependent protein kinase activity.

Animals↗

PubMed1995

Regulation of human B cell function by recombinant CD40 ligand and other TNF-related ligands.

To assess the potential of CD40 ligand (CD40L) and the related molecules CD27 ligand (CD27), CD30 ligand (CD30L), and membrane TNF-alpha to stimulate B cell responses, expression of these proteins in the baculovirus system was performed. Sf9 cells expressing these membrane molecules were cultured with normal human B cells and a variety of B cell lines to assess the functional outcome. The signal provided by CD40L promotes aggregation of B cells, stimulates vigorous proliferation, and induces germ-line transcription of downstream heavy chain constant region genes in the absence of cytokine costimulation. In contrast, CD27L, CD30L, and TNF-alpha had no effects on B cell proliferation. CD27L and TNF-alpha had no effect on the induction of germ-line transcripts, whereas CD30L consistently inhibited constitutive and CD40L-induced germ-line transcription of the epsilon gene by B cell lines that express CD30. These results demonstrate the various members of the TNF family exert specific effects on human B cell function, with CD40L and CD30L providing powerful, but opposing, effects on l epsilon transcription.

Antigens, CD↗

PubMed1995

CD27-CD27 ligand/CD70 interactions enhance alloantigen-induced proliferation and cytolytic activity in CD8+ T lymphocytes.

To study the role of CD27-CD27 ligand (CD27L)/CD70 interactions in the generation of murine allospecific T cell responses, SF9 cells or cell membranes expressing recombinant human CD70 were added to in vitro MLC containing C57BL/6 (H-2b) responder cells and class I and II MHC disparate H-2b/d stimulator cells. Alloantigen-specific CTL generation, CD8+ T cell proliferation, and levels of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl esterase activity were enhanced in the presence of human CD27L/CD70 expressed on SF9 cell membranes. Enhancement of CD8+ T cell responses occurred in the absence of any discernible effects on CD4+ T cell proliferation or IL-2 responses. Additional studies demonstrated that CD27L/CD70-expressing membranes enhanced proliferative responses to class I MHC differences but had no effect on proliferative responses to class II MHC disparities. Enhancement of allospecific CTL generation was also observed when CD27L/CD70-expressing membranes were added only during the last 24 to 48 h of 5-day MLC. Thus, the present studies suggest that CD27-CD27L/CD70 interactions can selectively enhance differentiation of Ag-specific CD8+ T cell effector mechanisms under conditions in which Th cell responses are not altered.

Animals↗

PubMed1995

Recombination potential of the human DIR elements.

The human DIR genes are putative DH elements that are GC rich and are found between DN and DM DH gene segments. The DIR genes each have consensus recombination signal sequences (RSS) that could conceivably generate DIR coding regions of about 126 nucleotides. These RSS should allow for DH-DH rearrangements that do not violate the 12/23 recombination rule. Several Ig CDR3 sequences have been assigned to DIR usage; however, there are frequent gaps and mismatches associated with these assignments. In some instances these CDR3 sequences might be better explained by GC rich N segment addition. This report analyzes the recombination potential of the human DIR elements by PCR. Though DH-JH rearrangement of the DH genes flanking the DIR regions (DM and DN) are easily demonstrated, very little evidence of DIR-JH rearrangement could be documented. Furthermore, in amplifications that should concurrently detect both DH-DIR rearrangements (which maintain the 12/23 recombination rule) and DH-DH rearrangements (which violate the 12/23 recombination rule), DH-DH rearrangements predominate. We conclude that the DIR-associated RSS participate minimally in both DH-DH and DH-JH rearrangement. In addition, we describe several conventional DH-DH rearrangement intermediates, demonstrating unequivocally that this phenomena occurs during human Ig rearrangement.

Base Sequence↗

PubMed1995

Evidence for a CD40 response element, distinct from the IL-4 response element, in the germline epsilon promoter.

Engagement of CD40 by its ligand induces transcription of unrearranged Ig heavy chain genes, an initial step in switch recombination. The following studies were undertaken to understand the molecular basis of this response. Co-culture of S19 cells expressing membrane-bound CD40 ligand (CD40L) encoded by recombinant baculovirus with EBV-transformed B cell lines induced germline transcription of the epsilon gene in the absence of cytokines. To identify a putative CD40 response element, a reporter construct consisting of the 777 bp of the 5' flank of the human l epsilon region linked to the chloramphenicol acetyl transferase (CAT) gene was stably transfected into B cell lines. Stimulation with either CD40L-expressing Sf9 cells or IL-4 induced CAT activity. Deletional analysis of this promoter region confirmed that an IL-4 response element was identified within a 63 bp segment 3' to the IL-4-responsive element that was responsive to CD40 ligation. These results indicate that the germline epsilon promoter contains a CD40 response element that is distinct from that accounting fro IL-4 responsiveness. Activity of this response element may explain the capacity of ligation of CD40 to induce germline epsilon transcripts in the absence of cytokines.

Animals↗

PubMed1994

Ligation of CD40 induces sterile transcripts of multiple Ig H chain isotypes in human B cells.

Stimulation of human B cells with mAb to CD40 in the presence of IL-4 induces proliferation and differentiation into Ig-secreting cells. To delineate the molecular events leading to Ig secretion after stimulation via the CD40 molecule, the induction of germ-line transcripts of Ig H chain isotypes was analyzed by polymerase chain reaction. The results document that costimulation with mAb to CD40 and IL-4 induces sterile transcription of all Ig H chain isotypes. Of importance, stimulation with mAb to CD40 without the addition of IL-4 induced germ-line transcription of most downstream isotypes, suggesting that this signal is sufficient to initiate the first step in switch recombination.

Antigens, CD↗

PubMed1993

Molecular characterization of monoclonal CRIA-positive anti-arsonate antibodies derived from idiotype-negative mice bearing a light chain polymorphism.

We have elicited anti-arsonate antibodies bearing the major cross-reactive idiotype (CRIA) in a double congenic idiotype-negative strain (C.C58.AL-20) bearing a light chain polymorphism that has previously been shown serologically not to complement idiotype-positive heavy chains. Using the idiotype cascade (Ab1-->Ab2-->Ab3-->-->Ab1'), CRIA-positive antibodies were raised and monoclonal antibodies were isolated and characterized serologically and by nucleotide sequence analysis. Two types of idiotype-positive anti-arsonate antibodies were generated in the C.C58.AL-20 strain. One group of hybridomas used the canonical VH1.8 heavy chain gene segment with V kappa 10 variant light chains. A second group used a VHGAM3.8 heavy chain with V kappa 10 variant light chains. This latter heavy-light pairing has been observed in CRIA-like responses previously in BALB/c mice after idiotypic manipulation (or rarely after antigen alone). These studies demonstrate the plasticity of the immune response when manipulated with idiotype reagents as well as its structural variability. Additionally, they provide important insights into the potentials of idiotype vaccines.

Amino Acid Sequence↗

PubMed1993

T cell receptor V beta gene bias in rheumatoid arthritis.

Polymerase chain reaction (PCR) technology was employed to examine peripheral blood and synovial T cells in patients with rheumatoid arthritis (RA) for biased utilization of T cell receptor (TCR) variable region (V) genes. Oligonucleotide primers specific for individual TCR V beta gene families were used to amplify TCR gene products in a semiquantitative assay of their relative utilization in unselected T cell populations. Mean V beta expression in 24 RA peripheral blood samples was very similar to that in a panel of 15 normal subjects, except for a slight decrease in V beta 13.2 expression. V beta utilization in 8 RA synovial tissue samples and 13 synovial fluid samples was compared to simultaneously obtained blood samples. Although heterogeneous patterns of skewed V beta utilization were observed, several significant trends emerged. By a number of approaches to data analysis, a statistically significant increase in expression of V beta 6 and V beta 15 in synovial T cells was documented. In addition, increased synovial expression of V beta 14 was found, but only in the synovial fluid samples. Reduced expression of V beta 1, V beta 4, V beta 5.1, V beta 10, V beta 16, and V beta 19 was also observed in synovial T cells. These results indicate that biased V beta gene utilization in different peripheral compartments of RA patients can be observed in unselected T cell populations, and are consistent with the conclusion that populations of T cells expressing these V beta gene products may be involved in the pathogenesis of the disease.

Adult↗

PubMed1992

Assembly of IgH CDR3: mechanism, regulation, and influence on antibody diversity.

The most variable portion of immunoglobulin molecules is the third complementarity determining region (CDR3) of the heavy chain. This is simply because CDR3 encompasses the region of the rearranged gene where the three gene segments (VH-DH-JH) are joined. Since imprecisions exist in the recombinase reaction, significant differences can be generated at the sites of recombination. This results in the generation of antigen receptor molecules which can differ in their antigen specificity even though they derive from the same germline information. In sum, the significance of the inaccuracy in recombination is that antibodies which are reactive to different antigens can be derived from identical genetic information. This explains how the immune system (using only a limited amount of genetic information) can generate antibodies to virtually any antigen. Though the basic phenomenon of VH-DH-JH assembly has been appreciated for years, two recent findings demonstrate that the generation of CDR3 is more complex than originally believed. First, junctional modification is not a stochastic process as was initially presumed, but is in part developmentally regulated. Second, it has now been well documented that more complex recombinations (for example VH-DH-DH-JH, VH-DH-invDH-JH, etc.) are involved in generating the third hypervariable region of the heavy chain. Not only do these unusual rearrangements--which break the so-called "12/23" recombination rule--occur, but interestingly, certain predicted rearrangements (even some which do follow the "12/23" recombination rule) cannot be demonstrated and apparently do not occur. To date, there is no adequate explanation for the lack of these predicted recombinations. These results have important implications for both the generation of antibody diversity and the recombinase reaction itself.

Animals↗

PubMed1991

Conservation of the most JH proximal Ig VH gene segment (VHVI) throughout primate evolution.

The human VHVI gene segment, the sole member of the VHVI gene family, is remarkable in that it is the most D-proximal VH gene segment and is apparently nonpolymorphic. Here we report that the VHVI gene segment has been remarkably preserved in primate evolution. We were unable to detect RFLP among several primates, and nucleotide sequences of several VHVI gene segments showed remarkable conservation. No differences were detected in the nucleotide sequences of the VHVI gene segment from three unrelated chimpanzees. These findings suggest that the VHVI gene segment has been strongly selected for during primate evolution, suggesting an important immunologic role.

Amino Acid Sequence↗

PubMed1990

Analysis of junctional diversity during B lymphocyte development.

Immunoglobulin rearrangement is central to generating antibody diversity because of heterogeneity generated during recombination by deletion or addition of nucleotides at coding joints by the recombinase machinery. Examination of these junctional modifications revealed that the addition of nongermline-encoded nucleotides was more prevalent in adult versus fetal B cells, thus partially limiting the fetal antibody repertoire. In contrast, deletion of nucleotides occurs equivalently in B cells at different stages of development and at different points in B cell ontogeny. Finally, the bias in murine immunoglobulins for one DH segment reading frame occurs at the DHJH intermediate.

Aging↗

PubMed1990

Anti-peptide antibodies detect a lupus-related interspecies idiotype that maps to H chain CDR2.

Antibodies to the small nucleoprotein Sm occur spontaneously in human and murine systemic lupus erythematosus. Human and mouse monoclonal anti-Sm autoantibodies designated 4B4 and Y2 share an idiotype (Id) determinant located on the Ig H chain. To understand the molecular basis of this cross-reactivity, the VH regions of both antibodies were sequenced and analyzed for homology. The antibodies showed only 49.6% homology. The second complementary determining region (CDR2) was the most likely candidate for the Id site. To investigate this possibility, rabbit antiserum was made against a peptide corresponding to the CDR2 of 4B4. This antiserum was specific for the immunizing peptide and reacted weakly to a peptide corresponding to the CDR2 of Y2. Anti-CDR2 antibody bound to 4B4 and Y2 but not to other human and mouse mAb. Binding was directed at the H chain when analyzed by Western blots. Anti-CDR2 antibody blocked anti-Id antibody binding to 4B4 and Y2 by 58% and 24%, respectively. These studies suggest that this interspecies Id maps to the H chain CDR2 and that a conserved Id can occur within molecules that are otherwise radically different.

Amino Acid Sequence↗

PubMed1990

Organization of the murine immunoglobulin VH complex: placement of two new VH families (VH10 and VH11) and analysis of VH family clustering and interdigitation.

During B cell development, there is an ordered expression of heavy chain variable region (VH) genes during ontogeny such that JH proximal VH genes are rearranged and expressed before the more JH distal VH genes. Thus, the relative chromosomal position of VH genes is biologically significant. We have previously employed deletion mapping to order the nine described murine VH gene families as follows: 3609-J558-(J606/VGAM3-8/S107)-3660-(X24/Q52/7183 ). (Families within parentheses were not mapped relative to each other.) In this report we continue this analysis by mapping two recently described heavy chain variable region gene families (VH10 and VH11). VH10 is located at the JH proximal end of the major cluster of J558 VH gene segments. VH11 (a very small family) is intermingled with the 3660 family. Although in general VH genes are thought to be clustered, we and others have reported some interspersion between families. To further address this issue, we have analyzed 80 recombinant phage clones containing J558 VH gene segments for the presence of other VH family genes. Our data indicate that the J558 and 3609 VH families are extensively intermingled as has recently been described for the most JH proximal Q52 and 7183 families.

Animals↗

PubMed1990

Structural implications of AB2s. Are novel D segments involved with anti-idiotypic specificity?

For these studies it is clear that within an antigenic system, syngeneic anti-idiotypic antibodies are restricted in their use of germline gene segments. They differ considerably from either allogeneic Ab2s recognizing public idiotopes or syngeneic Ab2s recognizing private idiotopes which are structurally heterogeneous. The D segments of Ab2s in a variety of systems are novel in structure and cannot easily be explained by previously described germline D segments. D-D fusion contributes to the generation of the third hypervariable region in these antibodies. It is not completely clear whether or not this mechanism plays a more important role in generating Ab2s than it does in generating other antibodies. Finally, somatic mutation does occur in anti-idiotypic antibodies. It is unlikely that the idiotypic network, per se, is strictly germline encoded. On the contrary, somatic events (i.e. mutation, junctional diversity, etc.) are probably important in the generation of anti-idiotypic antibodies.

Animals↗

PubMed1989

Structural characterization of antiidiotypic antibodies. Evidence that Ab2s are derived from the germline differently than Ab1s.

We have found that syngeneic Ab2s in the antiarsonate system are serologically and structurally similar to one another. In contrast, the allogeneic Ab2 response is heterogeneous and derives from a large number of unrelated germline gene segments. The Ab2 response of the BALB/c strain to polyclonal A/J Ars A molecules can probably best be compared with a response to a foreign protein and might have been predicted in a strain that completely lacks the H chain V region gene from which the Ab1 derives. Partial variable region sequences of Ab2s from three other systems in addition to previously reported Ab2 structures indicates that this difference in allogeneic vs. syngeneic Ab2s may be a general phenomena. These data support Jerne's hypothesis of complementary V region genes existing in the germline. However, there is good evidence that these antiidiotypic antibodies are not derived directly from the germline, as somatic processes most likely play an important role in their generation. The D segments of Ab2s in the arsonate system as well as in other systems, are novel in structure and cannot easily be explained by previously described germline D segments. D-D fusion may play a role in the generation of the third hypervariable region in these antibodies.

Amino Acid Sequence↗

PubMed1989

Nucleotide changes in sequential variants of influenza virus hemagglutinin genes and molecular structures of corresponding monoclonal antibodies specific for each variant.

We have generated four monoclonal antibodies specific for one or more members of a series of two sequentially derived PR8 influenza virus variants. Three of these antibodies share cross-reactive idiotypes. The amino acid sequences of these antibodies were determined, and it was found that two of these antibodies use genes from the VH7183 family, whereas the third uses a gene from the VHJ558 family. All four monoclonal antibodies derive from different families of genes encoding the variable region of the kappa chain. The RNA sequence of the parent PR8 virus as well as the RNA sequences of the sequential variants were also determined, and it was demonstrated that the variant hemagglutinin molecules differed from the parent molecule by only a single amino acid interchange. Despite these subtle differences in antigenic structures of hemagglutinin, and the cross-reactive idiotype of the antibodies, their primary structures were very different. These data reinforce the idea that a wide variety of antibody structures exist which are directed against subtly different structures in biologically important antigens.

Amino Acid Sequence↗

PubMed1989

Molecular basis for expression of the A48 regulatory idiotope on antibodies encoded by immunoglobulin variable-region genes from various families.

The idiotype defined by the levan-specific BALB/c myeloma protein ABPC48 (A48) has previously been encountered only in antibodies the variable regions of which derive from the VHX24 and V kappa 10 gene families. We have demonstrated expression of the idiotope recognized by the monoclonal anti-A48 idiotype antibody IDA10 on five monoclonal antibodies from different mouse strains, with different specificities including foreign and self antigens and deriving their variable regions from families other than VHX24 and V kappa 10. We analyzed variable region protein structure (deduced from nucleotide sequences) and hydrophilicity profiles of idiotype+ and idiotype- antibodies. We identified four surface-exposed areas (one in the heavy chain and three in the light chain) that may contribute to expression of the idiotope defined by antibody IDA10.

Animals↗

PubMed1988

Sequences of the VH and VL regions of murine monoclonal antibodies against 3-fucosyllactosamine.

Many mAb that bind the carbohydrate antigenic determinant 3-fucosyl-lactosamine (3-FL), Gal beta 1-4[Fuc alpha-3]GlcNAc-R have been raised in BALB/c mice, and we are studying the structure and regulation of these antibodies. In this report, we present the first information about their amino acid sequences and the Ig gene segments used to encode them. V regions of the H and L chains of three anti-3-FL antibodies, PMN6, PMN29, and PM81, were sequenced by a combination of mRNA and amino acid sequencing. The L chain sequences of PMN6 and PM81 antibodies indicate that their VK and JK regions are encoded by VK24B and JK1 germ-line genes, respectively. The nucleotide and amino acid sequences of the H chains suggest that the three anti-3-FL antibodies are encoded by the VH441 gene segment of the X24 VH family, and this conclusion was supported by Southern filter hybridization with VH441 and JH3-JH4 probes. PMN29 has at least 11 amino acid substitutions, which is an unusually large amount of somatic mutation for an IgM antibody. Previous analyses of BALB/c genomic libraries with VHX24 and VH441 probes make it unlikely that this VH family contains additional germ-line genes, but this possibility cannot be excluded. All three antibodies use the DQ52 and JH4 gene segments. The single VH and VL gene segments used to encode the anti-3-FL antibodies is in contrast to the multiple VH and VL segments used by antibodies against other carbohydrate Ag such as alpha 1-6 dextran and group A streptococcal carbohydrate. VH441 also encodes the VH regions of antibodies against galactan and levan (beta 2-6 fructosan). The similarities among VH segments of antibodies against 3-FL, levan, and galactan, and the striking differences in their CDR3 sequences, suggest that CDR3 plays an important role in the formation of the Ag binding site. The use of a single VH segment from the smallest VH gene family by antibodies against at least three different carbohydrate determinants is noteworthy. It raises the possibility that the amino acid sequence encoded by VH441 has some general structural features that make it particularly well adapted for binding to carbohydrate sequences.

Amino Acid Sequence↗