Dobutamine stress echocardiogram: emergency department evaluation of chest pain

Secondary immune responses to T-independent antigens are characterized by little or no affinity maturation, a phenomenon attributed to limited somatic hypermutation. In the human immune response to Haemophilus influenzae type b capsular polysaccharide, however, there are numerous differences between rearranged heavy chain variable region gene segments and candidate germline genes, irrespective of antigen presentation in a T-independent or T-dependent form. To determine the characteristics of somatic hypermutation in this response, we analyzed rearranged heavy chain variable region segments and associated 3' untranslated JH4-JH5 introns from monoclonal anti-Hib PS antibodies. Mutation of untranslated introns and heavy chain variable segments in both T-independent and T-dependent responses resembles that described in murine and unselected human immune responses. Although mutation is frequent in both T-independent and T-dependent anti-Hib PS responses, there is little evidence of antigen-driven selection, suggesting that ongoing pressure to conserve the variable segment germline configuration limits affinity maturation in this immune response.     

Deletional mapping of fifteen mouse VH gene families reveals a common organization for three Igh haplotypes

In addition to the content of germ-line variable gene segments, the organization of V genes has been implicated in the development of the Ab repertoire. We have searched the expressed VH genes of BALB/c mice for additional VH gene families and utilized deletion mapping to explore the extent of VH gene family interspersion. We have identified and characterized one new VH gene family (VH15) and extended our previous studies of the Igha and Ighb haplotypes to include a third haplotype (Ighj) using a newly developed panel of pre-B cell lines (CXCB cell lines). We conclude that the Igha, Ighb, and Ighj haplotypes have a similar Igh-V locus structure. A refined deletional map for 15 VH gene families and an individual member of the VHSM7 family (H10) has been constructed based on the deletion profiles of 72 rearranged heavy chain loci. These results demonstrate previously unrecognized examples of interspersion among members of the VHS107, VH10, and VHSM7 families.     

V region diversity in human anti-insulin antibodies. Preferential use of a VHIII gene subset

Data on structures used by human antibody repertoires are derived principally from lymphoid malignancies and from autoantibodies that often express VH genes from the developmentally regulated fetal repertoire. To determine whether human immune responses generated by exogenous Ag use a pool of VH genes distinct from the fetal repertoire, nucleotide and predicted amino acid sequences were determined for five anti-insulin B cell clones from a type I diabetic patient treated with human insulin. The data show that a set of VHIII genes is preferentially used by human anti-insulin B cells. Structural features indicate that these expressed VH are derived from germ-line genes that are not frequent in fetal repertoires and these genes have undergone Ag-driven somatic mutation. The preferential use of related VH segments contrasts with the BALB/c anti-insulin response, which uses multiple V genes elements largely unmutated from germ-line sequences. In addition, long CDRH3 structures in human anti-insulin mAb are generated by complex gene interaction mechanisms that are not seen in murine anti-insulin mAb. Interestingly, similar potential insulin-binding structures are used by antibodies from both species. These findings suggest that human responses to exogenous insulin may express a limited number of VH genes and depend upon somatic mutation and complex D gene interactions in CDRH3 to expand the repertoire. Although these antibodies bind autologous insulin, VH gene usage and structural features that predominate in the response are not characteristic of the fetal repertoire.     

Germline structure and differential utilization of Igha and Ighb VH10 genes

Ab heavy chains encoded by mouse VH10 genes have been of particular interest due to their frequent association with DNA binding. We reported previously that VH10 sequences are over-represented in the preimmune repertoire considering the apparent number of germline-encoded VH10 gene segments. In this report, we show that the VH10 family consists of three and two germline genes in the Igha and Ighb haplotypes, respectively. The complete nucleotide sequences of these five genes, including promoters and recombination signal sequences, were determined and allow unambiguous assignment of allelic relationships. The usage of individual VH10 genes varied significantly and ranged from 0.2% to an extraordinary 7.2% of the VH genes expressed by splenic B cells. Since the promoter and recombination signal sequence elements of all five VH10 genes are identical, we suggest that the few amino acid differences encoded by these five germline VH10 genes determine their representation in the preimmune repertoire. Rearrangements of the most frequently used VH10 gene have an apparent bias for histidine at position 95 of complementarity-determining region-3 (CDR3). These CDR3s are also biased for asparagine, an amino acid associated with the CDRs of DNA binding Abs. Together, these results suggest that high VH10 gene use is the result of B cell receptor-mediated selection and may involve DNA and/or ligands that share antigenic features with DNA.     

A single predominantly expressed polymorphic immunoglobulin VH gene family, related to mammalian group, I, clan, II, is identified in cattle

In order to understand the generation of antibody diversity in cattle, seven cDNAs, from heterohybridomas secreting bovine IgM and IgG1 antibodies, were cloned and structurally analyzed for rearranged bovine VDJ genes. All of the seven bovine VH genes, together with four available bovine VH gene sequences, shared a high nucleotide sequence homology (84.2-93.5%). Based upon the criteria of nucleic acid homology > or =80%, all of the bovine VH gene sequences isolated from the expressed antibody repertoire constitute a single VH gene family, which we have designated as bovine VH1 (Bov VH1). An analysis of 44 bovine IgM-secreting mouse x cattle heterohybridomas, originating from polyclonally-activated PBLs from bovine leukemia virus-infected cattle, revealed that all of these expressed Bov VH1 (100%) based upon DNA sequencing and Northern dot blot. The bovine VH genes showed highest DNA sequence similarity, ranging between 81.5 and 87.6%, with a single sheep VH gene family (related to human VH4) and are, thus, closest to the VH genes from another ruminant species. The Bov VH1 gene family is most homologous to the murine VH Q-52 (71.8-78%) and human VH4 (67.4-69.8%) gene families, which belong to mammalian group, I, clan, II, VH genes. The CDR3 length of rearranged bovine VDJ genes is characteristically long (15-23 amino acids). The bovine JH gene segments were most homologous to human JH4 (82.1-87.2%) and JH5 (84.6-89.7%) genes, suggesting the existence of at least two JH gene segments. An analysis of CDRs provides evidence that somatic hypermutations contribute significantly to the generation of antibody diversity in cattle. Southern blot analysis of BamH I, EcoR I and Hind III digested genomic DNA from four cattle breeds (Holstein, Jersey, Hereford and Charolais) revealed three RFLP patterns; the genomic complexity of Bov VH1 ranged between 13 and 15 genes. These observations provide evidence for polymorphism at the bovine Ig-VH locus, similar to that seen in mice and humans.     

Analysis of the genes encoding the variable regions of human IgG rheumatoid factor

OBJECTIVE: To better understand the immunoglobulin variable (V) region repertoire of rheumatoid factors (RF). METHODS: We characterized the heavy (H) and light (L) chain gene segments utilized in a monospecific IgG RF secreting hybridoma (AEE111F) which were derived from a patient with rheumatoid arthritis (RA). The hybridoma was established by fusion of a mouse myeloma cell line with bone marrow derived mononuclear cells from a patient with RA. First strand complementary DNA (cDNA) was generated and used for a polymerase chain reaction amplification of the H and L chain V domains. The amplified V domains were sequenced and compared with an extensive database of germline and cDNA V gene segments. RESULTS: The VH sequence was found to be 96% homologous to a previously described fetal VH3 cDNA (60P2). The VL sequence was also highly homologous to the previously described V lambda II gene (96%) derived from a patient with systemic lupus erythematosus which correlated with an 8.12 idiotype (Id), and to an antibacterial antibody against the Haemophilus influenzae type b capsular polysaccharide (94.7%). CONCLUSION: The overlap among this RF VL gene and the 2 reported V lambda sequences of antibodies that expressed anti-DNA related Id and an environmental pathogen specificity suggests that a part of the IgG RF isolated from patients with RA may thus be derived from the physiological natural antibody repertoire during an abnormal immune response and then develop high affinity, monospecific RF by the selection of an antigen driven mechanism.     

Evidence for immunoglobulin heavy chain variable region gene replacement in a patient with B cell chronic lymphocytic leukemia

Immunoglobulin heavy chain variable (V) gene replacement is an unusual recombinatorial event characterized by rearrangement of a germline V gene to a preformed VDJ gene complex. This phenomenon has occasionally been implicated in the emergence of clonal subpopulations during the course of acute lymphoblastic leukemia; it has also been found in murine precursor B cell lines. V gene replacement has never been described in lymphoproliferative disorders corresponding to more differentiated stages of B cell ontogeny. The present communication provides evidence for the operation of the same mechanism in B cell chronic lymphocytic leukemia (B-CLL). Genomic DNA and total cellular RNA extracted from peripheral blood mononuclear cells of a 48-year-old female patient diagnosed as having typical B-CLL were subjected to polymerase chain reaction (PCR) amplification aiming to detect rearranged clonal heavy and light chain variable genes (VH and VL, respectively). PCR consistently gave two VH amplification products, both at the DNA and the RNA level; similar analysis for the VL region revealed the presence of a single rearranged VK gene. Direct sequence analysis of the PCR products revealed that, except for a number of silent mutations, the single rearranged VK gene was identical to the germline A1-A17 VK gene. The two rearranged VH gene segments belong to the VHl and VHIII gene families and are closely homologous, respectively, to the germline gene segments V1-18 and V3-30, which have been shown to be used by autoantibodies. Both rearranged VH genes showed identical in-frame D-N-JH junctions and JH gene usage (JH5b), whereas the VH-N-D junctions were different. The above findings indicate that, during the course of the disease of our patient, VH gene replacement took place giving rise to two different clonally related subpopulations. This raises the intriguing possibility that the recombinase machinery, which governs Ig recombinatorial processes, might be operative even at more advanced stages in B cell ontogeny.     

Molecular analysis of V gene sequences encoding cytotoxic anti-streptococcal/anti-myosin monoclonal antibody 36.2.2 that recognizes the heart cell surface protein laminin

Anti-streptococcal/anti-myosin mAb 36.2.2 is unique among the cross-reactive anti-streptococcal mAbs due to its cytotoxicity for rat heart cells and its ability to strongly label the surface of heart cells in indirect immunofluorescence assays. In this study, cytotoxic mAb 36.2.2 was found to react strongly with the extracellular matrix protein laminin in immunoblots and inhibition assays, while 11 other cross-reactive anti-streptococcal mAbs did not react with laminin and were not cytotoxic. Cytotoxicity appeared to correlate with the presence of laminin on the surface of cells. Heavy and light chain variable region genes encoding mAb 36.2.2 were highly homologous to other V genes encoding anti-carbohydrate and/or autoantibodies. VH, JH, and Jkappa segments of mAb 36.2.2 may be encoded by germline gene segments. The VH segment may be identical with an as yet unidentified VH7183 family germline sequence, and the 36.2.2 Vkappa region gene is encoded by a Vkappa8 family member.     

Passively acquired anti-tetanus and anti-Haemophilus antibodies and the response to Haemophilus influenzae type b-tetanus toxoid conjugate vaccine in infancy

OBJECTIVE: To study the influence of maternally inherited tetanus antitoxin (anti-TT) antibodies on the response to the Haemophilus influenzae type b (Hib) capsular polysaccharide (PS)-tetanus toxoid conjugate (PRP-T) vaccine. DESIGN: One hundred thirty healthy infants received their first dose of PRP-T in the same syringe with diphtheria-tetanus-pertussis vaccine (DTP) at 1 to 2 months, and 66 of them received a second dose at 3 to 4 months of age. RESULTS: Maternal anti-TT antibodies did not interfere with the anti-Hib PS response to the first PRP-T vaccination; the geometric mean concentration (GMC) of anti-Hib PS was 0.14 microgram/ml in those with the lowest preimmunization anti-TT (< 0.3 IU/ml, n = 15) and 0.13 microgram/ml in those with the highest anti-TT (> or = 3 IU/ml, n = 25). After the second dose of PRP-T there was a positive correlation (r = 0.37, P = 0.004) between the anti-Hib PS response and the preimmunization anti-TT; those with the lowest preimmunization anti-TT (< 0.3 IU/ml, n = 9) achieved GMC of anti-Hib PS of 1.22 micrograms/ml and those with anti-TT > or = IU/ml (n = 22) anti-Hib PS GMC of 2.67 micrograms/ml. High preimmunization anti-Hib PS antibodies did not interfere with the final antibody concentrations; the GMC of anti-Hib PS after the second dose of PRP-T was 1.60 micrograms/ml in those with a preimmunization titer > or = 1.0 microgram/ml (n = 12) and 1.57 micrograms/ml in those with a titer of < 1.0 microgram/ml (n = 53). CONCLUSION: The data suggest that infants can be safely vaccinated with PRP-T even though they have received high concentrations of anti-TT from their mother.     

Effect of acute and chronic arecoline treatment on cerebral metabolism and blood flow in the conscious rat

To investigate the structural contribution of the light chain of anti-DNA antibodies to fine specificity, the VKappa genes of two monoclonal anti-DNA antibodies, termed H241 and H102, were cloned and sequenced. H102 and H241 are independently derived from MRL-lpr/lpr mice and differ in their fine specificity: H241 binds dsDNA and normal glomeruli in vitro and deposits in the kidney in vivo, whereas H102 binds only ssDNA and does not deposit in the kidney. Both are encoded by nearly identical VH genes but different N and D regions. Our previous results have demonstrated that the VH gene for H102 and H241 encodes eight other anti-DNA antibodies that also differed in fine specificity. This suggested that the gene product encoded by the VH 102/241 gene, may have intrinsic affinity for DNA, but is unlikely to determine fine specificity or nephritogenicity. In the present study we examined whether the VKappa gene might account for the difference in nephritogenicity. The complete nucleotide and deduced amino acid sequence of VK 102 and VK241 revealed that they are very dissimilar to each other (< 60% homology). VK 241 defined a new member of the VKappa gene family and was moderately homologous to two other VK genes encoding anti-DNA antibodies and to one VK gene encoding an anti-histone antibody all from lupus strains of mice. In addition, sequence diversity in the VK CDR1 region and position 96 of the CDR3 region was observed that may be of significance in determining fine specificity. VK 102 was highly homologous to two other VKappa genes, VKs17.2 and VK C8.5, both encoding anti-DNA antibodies and members of the VK20 gene family. It was striking that all three members of the VK 20 gene family code for DNA reactivity. This suggests that certain VKappa genes may also be used to repeatedly code for anti-DNA reactivity.     

Determination of gene organization in individual haplotypes by analyzing single DNA fragments from single spermatozoa

To determine human Ig heavy chain variable region (VH) gene segment organization on individual homologous chromosomes, an efficient approach has been developed. Single spermatozoa were used as subjects for the study. Upon sperm lysis, VH regions in each sperm were randomly sheared into fragments by the random Brownian force. The fragments were separated from each other by aliquoting the lysate into a certain number of tubes. The gene segments in the VH1 and VH4 families in each tube were identified by denaturing gradient gel electrophoresis after PCR amplification. The polymorphic VH sequences were used to determine the parental origins of the analyzed sperm. VH segment organization in the parental haplotypes was determined by aligning the overlapping fragments from the spermatozoa with the corresponding haplotypes. Based on this comparison between the resulting haplotype maps and the composite map reported previously, the VH region on chromosome 14 could be subdivided into four portions. The numbers and compositions of the VH gene segments differ considerably among the maps in two portions, but are highly conserved in the other two. The data also indicate that the VH region on chromosome 15 may contain a large duplicated block with copy number varying among haplotypes. The approach used in the present study may be used to construct high-resolution haplotype maps without molecular cloning.     

Mechanism of antibody-mediated reduction of nasopharyngeal colonization by Haemophilus influenzae type b studied in an infant rat model

The mechanism of antibody-mediated reduction of Haemophilus influenzae type b (Hib) carriage was studied in the infant rat colonization model. Monoclonal Hib polysaccharide (PS) antibody (MAb) given intranasally or intraperitoneally and human secretory anti-Hib PS IgA given intranasally inhibited colonization by Hib during the entire follow-up period (2-48 h after challenge) but did not affect colonization by Hi, a noncapsulated variant of Hib. F(ab')2 fragments, prepared from the MAb or from human serum anti-Hib IgG reduced Hib colonization as efficiently as the uncleaved molecules. Complement depletion by cobra venom treatment had no effect on the antibody-mediated reduction of Hib colonization. These results indicate that Fc-mediated activities of immunoglobulins are not essential in the reduction of Hib colonization. Instead, antibodies to Hib most likely reduce colonization by a direct effect on growth of the bacteria or their adherence to the nasopharyngeal mucosa.     

Deletion and editing of B cells that express antibodies to DNA

We previously demonstrated that in mice transgenic for genes coding for an anti-ssDNA autoantibody B cells were functionally inactivated but not physically deleted. We have now extended this model by introducing an arginine into the CDR2 of the heavy chain transgene. This change alters the specificity of the Ab from anti-ssDNA to anti-dsDNA and increases the affinity for ssDNA. Mice carrying this transgene displayed a significant reduction of peripheral B cells and anti-dsDNA B cells were not recovered from the spleens. The remaining B cells escape deletion by revising their Ag receptors in several ways: 1) elimination of the transgenic heavy chain gene via intrachromosomal recombination, followed by rearrangement and expression of endogenous VH genes; 2) ongoing rearrangement of endogenous kappa light chain genes to generate a non-dsDNA-binding Ab; and 3) expression of a rare V lambda gene, V lambda x, to generate a non-DNA-binding Ab.     

Heavy chain variable region, light chain variable region, and heavy chain CDR3 influences on the mono- and polyreactivity and on the affinity of human monoclonal rheumatoid factors

Monoreactive high affinity pathologic autoantibodies were supposed previously to derive through somatic mutation from polyreactive low affinity autoantibodies that are encoded by a small set of unmutated V region genes in fetal and neonatal B cells. However, recent data exploring the physiologically expressed Ab repertoire and the importance of the stochastically generated heavy chain CDR3 (H-CDR3) in autoreactivity suggest that this scheme is incomplete. Here we analyzed via gene-swapping experiments and site-directed mutagenesis the relative contributions of the mutations in the light chain variable region (VL) and the heavy chain variable region (VH) domains and of the H-CDR3 in the autoreactivity of two IgM rheumatoid factors (RF), one a polyreactive low affinity Ab, the other a monoreactive high affinity Ab. These two RFs derived from the same V kappa III (humkv325) and VH1 (51p1) genes, but differed from each other by a few mutations and by the structure of the H-CDR3. The analysis of the reactivity patterns of different combinations of wild-type and in vitro engineered hybrid gene products clearly demonstrates the main influence of the H-CDR3 in the autoAb activity profiles. The results directly demonstrate the previously proposed hypothesis, namely, that the H-CDR3 plays a critical role in distinguishing poly- from monospecific RF. However, the data also indicate that self polyreactivity is a very fragile property and is dependent upon the primary structure of the VH segment.     

The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus

The complete nucleotide sequence of the 957-kb DNA of the human immunoglobulin heavy chain variable (VH) region locus was determined and 43 novel VH segments were identified. The region contains 123 VH segments classifiable into seven different families, of which 79 are pseudogenes. Of the 44 VH segments with an open reading frame, 39 are expressed as heavy chain proteins and 1 as mRNA, while the remaining 4 are not found in immunoglobulin cDNAs. Combinatorial diversity of VH region was calculated to be approximately 6,000. Conservation of the promoter and recombination signal sequences was observed to be higher in functional VH segments than in pseudogenes. Phylogenetic analysis of 114 VH segments clearly showed clustering of the VH segments of each family. However, an independent branch in the tree contained a single VH, V4-44.1P, sharing similar levels of homology to human VH families and to those of other vertebrates. Comparison between different copies of homologous units that appear repeatedly across the locus clearly demonstrates that dynamic DNA reorganization of the locus took place at least eight times between 133 and 10 million years ago. One nonimmunoglobulin gene of unknown function was identified in the intergenic region.     

Structure and genomic organization of a second cluster of immunoglobulin heavy chain gene segments in the channel catfish

The structure, organization, and partial sequence of a 25-kb genomic region containing a second cluster of H chain gene segments in the channel catfish (Ictalurus punctatus) has been determined. Multiple VH gene segments, representing different VH families, are located upstream of a germline-joined VDJ. The VDJ segment has a split leader sequence and a single open reading consistent with that expressed in members of the VH1 family. Downstream of the germline-joined VDJ is a single JH segment and two pseudogene exons structurally similar to the Cmu1 and Cmu2 exons of the functional gene. Both pseudogene exons are multiply crippled with RNA splice sites destroyed, and open reading frames are interrupted by termination codons, insertions, and/or deletions. Sequence alignment of a 10.8-kb region within the second H chain cluster with the genomic sequence of the nine JH segments and the functional Cmu within the first H chain gene cluster indicates that the second H chain gene cluster probably arose by a massive duplication event. The JH region of the VDJ, the coding and flanking regions of the single JH segment, and the pseudogene Cmu exons were readily aligned with homologous segments in the first gene cluster. This duplication event may have extended to include the upstream VH segments. A member of the Tc1 mariner family of transposable elements is located downstream of the pseudogene Cmu2, which suggests that the transposition may have contributed to the evolution of the duplicated Cmu.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. V. In vivo expression of individual antibody clones is dependent on Ig CH haplotypes and the categories of antigen

Antibodies (Ab) to the polysaccharide capsule of Haemophilus influenzae type b (Hib-PS) provide protection against Haemophilus influenzae type b disease in children, and Hib-PS vaccines with different immunologic properties are widely used clinically. The repertoire of human anti-Hib-PS Ab induced by these vaccines is relatively restricted and can be divided into two types by the structure of the light chain V region. Ab using A2-V kappa II gene product, which account for the majority of anti-Hib-PS Ab response in most patients, show little somatic mutations. In contrast, non-Ab using A2-V kappa II gene product use VL genes from the V kappa I, V kappa II, V kappa III, V kappa IV, and V lambda subgroups, are variably expressed among patients, and contain somatic mutations. To further study the expression of these two types of anti-Hib-PS Ab, we have produced KB13, a mAb specific for V kappa II subgroup, and used mAb specific for various other VL subgroups to develop immunoassays specific for anti-Hib-PS Ab of each VL subgroup. When Ig allotypes were studied for the effect on the Ab repertoire, A2-V kappa II (A2) Ab were found to be expressed less in patients expressing fb or zag CH haplotypes (p < 0.05). When the T cell-independent Hib-PS carbohydrate vaccine was compared to two T cell-dependent Hib-PS protein conjugate vaccines for their effect on Ab repertoire, Ab using V kappa III VL were found to be more often elicited with the conjugate vaccines than with the Hib-PS carbohydrate vaccine (p < 0.01). Thus, individual members of the anti-Hib-PS Ab repertoire differ not only in their V region structure but also in the control of their expression.     

Two human neonatal IgM antibodies encoded by different variable-region genes bind the same linear peptide: evidence for a stereotyped repertoire of epitope recognition

Two monoclonal IgM Abs have been produced from lymphocytes isolated from two human umbilical cord bloods. These mAbs recognize a conformational epitope present in a CNBr digestion fraction of lactoferrin. Linear epitopes recognized by each mAb were selected from several phage display peptide libraries. In each case, phages displaying a peptide with a motif defined by [WF],G,[EQS],N were recovered. Phages displaying that motif bound equally well to either mAb but did not bind to control IgM. A peptide bearing this motif competed with the phage-displayed peptides for binding to either mAb. The same peptide also competes with a component of the CNBr digestion fraction of lactoferrin for Ab binding in ELISA. The Abs use different families of VH, JH, and VK gene cassettes but use the same JK cassette. All segments are virtually identical to their germline gene counterparts. This work provides further evidence that certain innate specificities are stereotyped among individuals.