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The human neonatal B cell response to respiratory syncytial virus uses a biased antibody variable gene repertoire that lacks somatic mutations.
Williams JV, Weitkamp JH, Blum DL, LaFleur BJ, Crowe JE
(2009) Mol Immunol 47: 407-14
MeSH Terms: Adult, Antibodies, Antibody Specificity, B-Lymphocytes, Cell Separation, Child, Complementarity Determining Regions, Flow Cytometry, Humans, Immunoglobulin Heavy Chains, Immunoglobulin Light Chains, Immunoglobulin Variable Region, Infant, Infant, Newborn, Lymphocyte Count, Mutagenesis, Insertional, Mutation, Respiratory Syncytial Viruses, Species Specificity
Show Abstract · Added February 27, 2014
The human Ab repertoire exhibits restrictions during fetal life characterized by biases of variable gene segment usage and lack of junctional diversity. We tested the hypotheses that Ab repertoire restriction persists in the early postnatal period and contributes to the observed poor quality of specific Ab responses made by neonates to viruses and vaccines. We analyzed the molecular determinants of B cell responses in humans to respiratory syncytial virus (RSV). Analysis of the variable gene segment usage of adult RSV-specific B cells revealed a repertoire profile in these cells similar to that seen in randomly selected B cells, which was V(H)3-dominant. Four gene segments (V(H)3-23, V(H)3-30, V(H)3-33 and V(H)4-04) accounted for almost half of the V(H) genes used. In contrast, very young infant RSV-specific antibodies exhibited a biased repertoire characterized by comparable use of the V(H)1, V(H)3, and V(H)4 families, and less common use of the four immunodominant gene segments. Infants and children older than three months used an antibody repertoire similar to that of adults. Mutational analysis revealed that the antibody variable genes of infants under three months of age also possessed significantly fewer somatic mutations in both framework and complementarity-determining region (CDR) regions than those of adults, even in a child with recurrent RSV infection. These data suggest that neonates use a biased antibody gene repertoire that is less V(H)3-focused and that possesses a dramatically lower frequency of somatic mutations. These biased features of the RSV-specific repertoire likely contribute to the poor functional Ab response in very young infants.
0 Communities
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19 MeSH Terms
Functional maturation of the human antibody response to rotavirus.
Kallewaard NL, McKinney BA, Gu Y, Chen A, Prasad BV, Crowe JE
(2008) J Immunol 180: 3980-9
MeSH Terms: Antibodies, Viral, Antigens, Viral, Binding Sites, Antibody, Capsid Proteins, Humans, Immunodominant Epitopes, Immunoglobulin D, Immunoglobulin Fab Fragments, Immunoglobulin Heavy Chains, Immunoglobulin Variable Region, Kinetics, Respiratory Syncytial Viruses, Rotavirus, Somatic Hypermutation, Immunoglobulin
Show Abstract · Added August 6, 2012
Infant Abs induced by viruses exhibit poor functional activity compared with those of adults. The human B cell response to rotavirus is dominated by use of the V(H)1-46 gene segment in both adults and infants, but only adult sequences are highly mutated. We investigated in detail the kinetic, structural, and functional advantage conferred by individual naturally occurring somatic mutations in rotavirus-specific human Abs encoded by the immunodominant V(H)1-46 gene segment. Adult Abs achieved enhanced binding through naturally occurring somatic mutations in the H chain CDR2 region that conferred a markedly prolonged off-rate and a desirable increase in antiviral potency. Three-dimensional cryoelectron microscopy studies of Ag-Ab complexes revealed the mechanism of viral inhibition to be the binding of high-affinity Abs at the viral RNA release pore in the double-layer particle. These structure-function studies suggest a molecular basis for the poor quality of Abs made in infancy following virus infection or immunization.
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14 MeSH Terms
Immunodominance of the VH1-46 antibody gene segment in the primary repertoire of human rotavirus-specific B cells is reduced in the memory compartment through somatic mutation of nondominant clones.
Tian C, Luskin GK, Dischert KM, Higginbotham JN, Shepherd BE, Crowe JE
(2008) J Immunol 180: 3279-88
MeSH Terms: Adult, Antibodies, Viral, Antigens, Viral, B-Lymphocyte Subsets, Capsid Proteins, Clone Cells, Epitopes, B-Lymphocyte, Humans, Immunodominant Epitopes, Immunoglobulin Class Switching, Immunoglobulin Heavy Chains, Immunoglobulin Variable Region, Immunologic Memory, Middle Aged, Resting Phase, Cell Cycle, Rotavirus, Somatic Hypermutation, Immunoglobulin
Show Abstract · Added August 6, 2012
Detailed characterization of Ag-specific naive and memory B cell Ab repertoires elucidates the molecular basis for the generation of Ab diversity and the optimization of Ab structures that bind microbial Ags. In this study, we analyzed the immunophenotype and VH gene repertoire of rotavirus (RV) VP6-specific B cells in three circulating naive or memory B cell subsets (CD19+IgD+CD27-, CD19+IgD+CD27+, or CD19+IgD-CD27+) at the single-cell level. We aimed to investigate the influence of antigenic exposure on the molecular features of the two RV-specific memory B cell subsets. We found an increased frequency of CD19+IgD+CD27+ unclass-switched memory B cells and a low frequency of somatic mutations in CD19+IgD-CD27+ class-switched memory B cells in RV-specific memory B cells, suggesting a reduced frequency of isotype switching and somatic mutation in RV VP6-specific memory B cells compared with other memory B cells. Furthermore, we found that dominance of the VH1-46 gene segment was a prominent feature in the VH gene repertoire of RV VP6-specific naive B cells, but this dominance was reduced in memory B cells. Increased diversity in the VH gene repertoire of the two memory B cell groups derived from broader usage of VH gene segments, increased junctional diversity that was introduced by differential TdT activities, and somatic hypermutation.
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17 MeSH Terms
Tertiary lymphoid structures in the pancreas promote selection of B lymphocytes in autoimmune diabetes.
Kendall PL, Yu G, Woodward EJ, Thomas JW
(2007) J Immunol 178: 5643-51
MeSH Terms: Amino Acid Sequence, Animals, Autoimmune Diseases, B-Lymphocytes, Diabetes Mellitus, Type 1, Germinal Center, Immunoglobulin Heavy Chains, Immunoglobulin Variable Region, Islets of Langerhans, Mice, Mice, Inbred NOD, Molecular Sequence Data, Mutation, Pancreas, Transgenes
Show Abstract · Added December 10, 2013
Autoimmune diabetes occurs when invading lymphocytes destroy insulin-producing beta cells in pancreatic islets. The role of lymphocytic aggregates at this inflammatory site is not understood. We find that B and T lymphocytes attacking islets in NOD mice organize into lymphoid structures with germinal centers. Analysis of BCR L chain genes was used to investigate selection of B lymphocytes in these tertiary lymphoid structures and in draining pancreatic lymph nodes. The pancreatic repertoire as a whole was found to be highly diverse, with the profile of L chain genes isolated from whole pancreas differing from that observed in regional lymph nodes. A Vkappa14 L chain predominated within the complex pancreatic repertoire of NOD mice. Skewing toward Vkappa4 genes was observed in the pancreas when the repertoire of NOD mice was restricted using a fixed Ig H chain transgene. Nucleotide sequencing of expressed Vkappas identified shared mutations in some sequences consistent with Ag-driven selection and clonal expansion at the site of inflammation. Isolated islets contained oligoclonal B lymphocytes enriched for the germinal center marker GL7 and for sequences containing multiple mutations within CDRs, suggesting local T-B interactions. Together, these findings identify a process that selects B lymphocyte specificities within the pancreas, with further evolution of the selected repertoire at the inflamed site. This interpretation is reinforced by Ag-binding studies showing a large population of insulin-binding B lymphocytes in the pancreas compared with draining lymph nodes.
3 Communities
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15 MeSH Terms
Evidence for preferential Ig gene usage and differential TdT and exonuclease activities in human naïve and memory B cells.
Tian C, Luskin GK, Dischert KM, Higginbotham JN, Shepherd BE, Crowe JE
(2007) Mol Immunol 44: 2173-83
MeSH Terms: Adult, Amino Acid Motifs, Animals, Antigens, CD19, B-Lymphocytes, Blood Donors, Cell Separation, Clone Cells, Complementarity Determining Regions, DNA Nucleotidylexotransferase, Exonucleases, Flow Cytometry, Gene Expression Regulation, Genes, Immunoglobulin, Humans, Immunoglobulin Heavy Chains, Immunoglobulin Isotypes, Immunoglobulin Variable Region, Immunologic Memory, Mice, Mutation, Nucleotides, Tumor Necrosis Factor Receptor Superfamily, Member 7
Show Abstract · Added August 6, 2012
Memory B cells and the antibodies they encode are important for protective immunity against infectious pathogens. Characterization of naïve and memory B cell antibody repertoires will elucidate the molecular basis for the generation of antibody diversity in human B cells and the optimization of antibody structures that bind microbial antigens. In this study we aimed to investigate the influence of antigenic selection on the antibody genes of the two CD27+ memory B cell subsets, comparing them with the naïve repertoire in CD27- cells. We analyzed and compared the Ig heavy chain gene transcripts in three recently defined circulating naïve and memory B cell subsets (CD19+IgD+CD27- [naïve], CD19+IgD+CD27+ [un-class-switched memory] or CD19+IgD- CD27+ [class-switched memory]) at the single cell level. We found similar biased patterns of variable, diversity and joining heavy chain gene usages in all three groups of cells. CD19+IgD+CD27+ memory B cells harbored as diverse an antibody gene repertoire as CD19+IgD-CD27+ memory B cells. Interestingly, CD19+IgD+CD27+ memory B cells possessed a lower frequency of somatic mutations, a higher incidence of exonuclease activity at the 3' end of D regions, and a lower frequency of N and P nucleotide additions at both VH-D and D-JH junctions of CDR3 regions compared to CD19+IgD-CD27+ memory B cells. These data suggest distinct functional mechanisms underlying selection of this unique subset of un-class-switched memory B cells.
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23 MeSH Terms
Rotavirus-specific CD5+ B cells in young children exhibit a distinct antibody repertoire compared with CD5- B cells.
Weitkamp JH, Lafleur BJ, Crowe JE
(2006) Hum Immunol 67: 33-42
MeSH Terms: Antibodies, Viral, Antibody Formation, B-Lymphocyte Subsets, Base Sequence, CD5 Antigens, Complementarity Determining Regions, Female, Humans, Immunoglobulin Heavy Chains, Immunoglobulin Variable Region, Infant, Male, Molecular Sequence Data, Mutation, Rotavirus, Rotavirus Infections
Show Abstract · Added February 27, 2014
Antiviral antibody responses in infants are limited in quality. One reason for this finding could be that the majority of B cells in infants are CD5+ cells, a subset of B cells that is thought to contain cells expressing polyreactive, low-affinity B cell receptors. We analyzed the rotavirus (RV)-specific antibody heavy chain variable region (VH) repertoire in CD5+ and CD5- B cells of four RV-infected children between 10 and 19 months of age. We found that the RV-specific B cell repertoire in CD5+ cells was VH3 family biased, in contrast to the VH1/VH4 dominance seen in CD5- B cells. The immunodominant RV-specific gene segment in CD5- B cells was VH1-46, which is the dominant segment used in RV-specific peripheral blood B cells from infants and adults. In contrast, the immunodominant gene segment was VH3-23 in RV-specific CD5+ B cells, which is the dominant gene segment in randomly selected B cells. Both RV-specific CD5+ and RV-specific CD5- B cells from all children studied demonstrated very low frequencies of somatic mutations. In conclusion, CD5+ B cells in infants responding to RV use an antibody gene repertoire that differs from the virus-specific repertoire of CD5- B cells, and both CD5+ and CD5- RV-specific B cells exhibit a low frequency of somatic mutations.
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2 Members
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16 MeSH Terms
Natural evolution of a human virus-specific antibody gene repertoire by somatic hypermutation requires both hotspot-directed and randomly-directed processes.
Weitkamp JH, Lafleur BJ, Greenberg HB, Crowe JE
(2005) Hum Immunol 66: 666-76
MeSH Terms: Adult, Amino Acid Motifs, Antibodies, Viral, B-Lymphocytes, Base Pairing, Clone Cells, Evolution, Molecular, Humans, Immunoglobulin Heavy Chains, Immunoglobulin Variable Region, Infant, RNA, Messenger, Rotavirus, Rotavirus Infections, Somatic Hypermutation, Immunoglobulin
Show Abstract · Added February 27, 2014
Somatic hypermutation of antibody genes is mediated by activation-induced cytidine deaminase and targets primarily hotspot motifs. We tested the hypothesis that the antibody variable genes of virus-specific B cells from infants exhibit a decreased frequency of somatic mutations compared with adults. We also sought to determine whether virus-specific B cells exhibit predominantly hotspot or randomly directed processes. We analyzed somatic mutations in rotavirus (RV)-specific B cells from otherwise healthy but recently RV-infected infants or adults in comparison with B cells from healthy volunteers not recently infected. We compared these antibody variable gene sequences with those derived from RV-specific B cells from an adult patient with X-linked hyper-IgM syndrome (XHIM). We found that the overall mutational frequency within the antibody variable region was lowest in RV-specific B cells from RV-infected infants, followed by randomly selected B cells, followed by RV-specific B cells from the patient with XHIM. RV-specific memory B cells from healthy adults exhibited the highest frequency of mutations. Approximately half of mutations in random or RV-specific B cells from adults or infants occurred at the DGYW/WRCH or WA/TW hotspot motifs. These findings suggest that virus-specific antibodies require both hotspot and randomly-directed processes.
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2 Members
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15 MeSH Terms
Monoclonal immunoglobin deposition disease, heavy chain deposition type.
Fogo AB, Rossini M
(2003) Am J Kidney Dis 42: A48, E1-2
MeSH Terms: Biopsy, Diagnosis, Differential, Edema, Epiglottis, Glomerular Mesangium, Humans, Hypergammaglobulinemia, Hypertension, Immunoglobulin G, Immunoglobulin Heavy Chains, Immunoglobulin lambda-Chains, Kidney Diseases, Kidney Glomerulus, Kidney Tubules, Male, Microscopy, Electron, Microscopy, Fluorescence, Middle Aged, Paraproteinemias
Added January 20, 2012
1 Communities
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19 MeSH Terms
Infant and adult human B cell responses to rotavirus share common immunodominant variable gene repertoires.
Weitkamp JH, Kallewaard N, Kusuhara K, Bures E, Williams JV, LaFleur B, Greenberg HB, Crowe JE
(2003) J Immunol 171: 4680-8
MeSH Terms: Adult, Antibody Diversity, Antibody Specificity, B-Lymphocyte Subsets, Clone Cells, Complementarity Determining Regions, Female, Gene Rearrangement, B-Lymphocyte, Heavy Chain, Humans, Immunodominant Epitopes, Immunoglobulin Heavy Chains, Immunoglobulin J-Chains, Immunoglobulin Light Chains, Immunoglobulin Variable Region, Infant, Lymphocyte Count, Male, Protein Structure, Tertiary, Random Allocation, Rotavirus
Show Abstract · Added February 27, 2014
Ab repertoires exhibit marked restrictions during fetal life characterized by biases of variable gene usage and lack of junctional diversity. We tested the hypothesis that Ab repertoire restriction contributes to the observed poor quality of specific Ab responses made by infants to viral infections. We analyzed the molecular determinants of B cell responses in humans to two Ags of rotavirus (RV), a common and clinically important infection of human infants. We sequenced Ab H and L chain V region genes (V(H) and V(L)) of clones expanded from single B cells responding to RV virus protein 6 or virus protein 7. We found that adults exhibited a distinct bias in use of gene segments in the V(H)1 and V(H)4 families, for example, V(H)1-46, V(H)4-31, and V(H)4-61. This gene segment bias differed markedly from the V(H)3 dominant bias seen in randomly selected adult B cells. Recombinant Abs incorporating any of those three immunodominant V(H) segments bound to RV-infected cells and also to purified RV particles. The RV-specific B cell repertoires of infants aged 2-11 mo and those of adults were highly related when compared by V(H), D, J(H), V(L), and J(L) segment selection, extent of junctional diversity, and mean H chain complementarity determining region 3 length. These data suggest that residual fetal bias of the B cell repertoire is not a limiting determinant of the quality of Ab responses to viruses of infants beyond the neonatal period.
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20 MeSH Terms
Generation of recombinant human monoclonal antibodies to rotavirus from single antigen-specific B cells selected with fluorescent virus-like particles.
Weitkamp JH, Kallewaard N, Kusuhara K, Feigelstock D, Feng N, Greenberg HB, Crowe JE
(2003) J Immunol Methods 275: 223-37
MeSH Terms: Adult, Antibodies, Monoclonal, Antibodies, Viral, Antigens, Viral, B-Lymphocytes, Base Sequence, Capsid Proteins, Cell Separation, DNA Primers, DNA, Recombinant, Gastroenteritis, Genes, Immunoglobulin, Genetic Vectors, Humans, Immunoglobulin Fab Fragments, Immunoglobulin Heavy Chains, Immunoglobulin Light Chains, Infant, Polymerase Chain Reaction, Recombinant Proteins, Rotavirus, Rotavirus Infections
Show Abstract · Added February 27, 2014
Technical difficulties have severely limited the yield of methods for the generation of human antiviral monoclonal antibodies (Mabs) in the past. We describe here a novel method for the efficient development of human Mabs against viruses. Rotavirus (RV) is a major cause of gastroenteritis in infants and adults worldwide. We generated fluorescent virus-like particles (VLPs) to identify and physically sort single RV-specific B cells from healthy adult blood donors, or RV-infected infants or adults. We expanded the sorted single B cells in culture, tested for RV-specific antibody secretion, and cloned and sequenced the antibody heavy and light chain variable region (VH and VL) genes. The percentage of wells that produced antibodies after sorting and expanding RV-specific adult B cell clones was high at 23%. The overall efficiency of RV-specific antibody gene recovery after the isolation, confirmation, and cloning of RV-specific VH segments was 1.3% of sorted cells in adults. RV-specific variable gene segments also were obtained from acutely infected infants, although infant B cells did not proliferate and differentiate in culture as well as adult B cells. We expressed recombinant Fabs incorporating the VH and VL genes from RV-specific B cell clones using a new modified bacterial Fab expression vector that we describe. Finally, we demonstrated binding of purified Fabs to RV proteins by immunofluorescence and ELISA. This method for the generation of recombinant human Mabs to RV from single antigen-specific B cell clones selected with fluorescent VLPs could be used to generate human Mabs to many other viruses whose proteins can self-assemble into VLPs.
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22 MeSH Terms