Biomedical subjects

Devinder Sehgal

Publications and source records attributed to Devinder Sehgal.

4 recordsLinked to original sources

PubMed2004

Analysis of mutational lineage trees from sites of primary and secondary Ig gene diversification in rabbits and chickens.

Lineage trees of mutated rearranged Ig V region sequences in B lymphocyte clones often serve to qualitatively illustrate claims concerning the dynamics of affinity maturation. In this study, we use a novel method for analyzing lineage tree shapes, using terms from graph theory to quantify the differences between primary and secondary diversification in rabbits and chickens. In these species, Ig gene diversification starts with rearrangement of a single (in chicken) or a few (in rabbit) V(H) genes. Somatic hypermutation and gene conversion contribute to primary diversification in appendix of young rabbits or in bursa of Fabricius of embryonic and young chickens and to secondary diversification during immune responses in germinal centers (GCs). We find that, at least in rabbits, primary diversification appears to occur at a constant rate in the appendix, and the type of Ag-specific selection seen in splenic GCs is absent. This supports the view that a primary repertoire is being generated within the expanding clonally related B cells in appendix of young rabbits and emphasizes the important role that gut-associated lymphoid tissues may play in early development of mammalian immune repertoires. Additionally, the data indicate a higher rate of hypermutation in rabbit and chicken GCs, such that the balance between hypermutation and selection tends more toward mutation and less toward selection in rabbit and chicken compared with murine GCs.

Algorithms↗

PubMed2004

Demonstration that mast cells, T cells, and B cells bearing the activating kit mutation D816V occur in clusters within the marrow of patients with mastocytosis.

Mastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We hypothesized that these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells, and T cells, from both lesional and non-lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V mutation in their DNA, using HinfI restriction digestion of nested PCR products amplified from extracts of dissected cells. The D816V mutation was detected in mast cells, B cells, and T cells from lesional but not non-lesional areas of bone marrow tissues. B cells obtained from lesional areas of tissue were also assessed for clonality and were found to at least represent an oligoclonal population. Thus, mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the codon 816 activating mutation. Further, the B cell population is oligoclonal, suggesting that clonal proliferation is unlikely to be the basis of clustering.

Adult↗

PubMed2002

A comparison of hydraulic and laser capture microdissection methods for collection of single B cells, PCR, and sequencing of antibody VDJ.

During the development of B lymphocytes, a series of gene rearrangements assemble the sequences that encode immunoglobulin heavy and light chains (VDJ). Earlier studies of VDJ sequence diversification during expansion of cells in splenic or appendix germinal centers used hydraulic micromanipulation (HM) to collect single B cells for PCR amplification of rearranged antibody heavy and light chain genes. PCR products were directly sequenced without a cloning step. Hydraulic micromanipulation is a very tedious method. Once capability to collect single cells by laser capture microdissection (LCM) was developed, we modified previous tissue staining and fixation methods so that we could collect cells from a given stained tissue section by HM and LCM and directly compare our success rates using these two methods. Cells were alkaline lysed and after two rounds of nested PCR products were recovered and directly sequenced. Because each rearrangement of genomic DNA that occurs to form the immunoglobulin heavy-chain-encoding sequence in developing B cells is unique, this system allowed us to verify our success rate in recovering single lymphocytes from tissue sections and amplifying a single allele. The methods developed have now made LCM an efficient alternative to HM for the collection of single B cells.

Animals↗

PubMed2002

Distinct clonal Ig diversification patterns in young appendix compared to antigen-specific splenic clones.

The young rabbit appendix is a dynamic site for primary B cell repertoire development. To study diversification patterns during clonal expansion, we collected single appendix B cells from 3- to 9-wk-old rabbits and sequenced rearranged H and L chain genes. Single cells obtained by hydraulic micromanipulation or laser capture microdissection were lysed, PCR amplified, and products directly sequenced. Gene conversion-like changes occurred in rearranged H and L chain sequences by 3-4 wk of age. Somatic mutations were found in the D regions that lack known conversion donors and probably also occurred in the V genes. A few small sets of clonally related appendix B cells were found at 3-5 wk; by 5.5 wk, some larger clones were recovered. The diversification patterns in the clones from appendix were strikingly different from those found previously in splenic germinal centers where an immunizing Ag was driving the expansion and selection process toward high affinity. Clonally related appendix B cells developed different amino acid sequences in each complementarity-determining region (CDR) including CDR3, whereas dominant clones from spleen underwent few changes in CDR3. The variety of combining sites generated by diversification within individual clones suggests that at least some clonal expansion and selection, known to require normal gut flora, may be driven through indirect effects of microbial components rather than solely by their recognition as specific foreign Ags. This diversity of combining sites within B cell clones supports the proposed role of appendix in generating the preimmune repertoire.

Amino Acid Sequence↗