Biomedical subjects

I C Nicholson

Publications and source records attributed to I C Nicholson.

13 recordsLinked to original sources

PubMed2001

Novel control motif cluster in the IgH delta-gamma 3 interval exhibits B cell-specific enhancer function in early development.

The majority of the human Ig heavy chain (IgH) constant (C) region locus has been cloned and mapped. An exception is the region between C delta and C gamma 3, which is unstable and may be a recombination hot spot. We isolated a pBAC clone (pHuIgH3'delta-gamma 3) that established a 52-kb distance between C delta and C gamma 3. Sequence analysis identified a high number of repeat elements, explaining the instability of the region, and an unusually large accumulation of transcription factor-binding motifs, for both lymphocyte-specific and ubiquitous transcription activators (IKAROS, E47, Oct-1, USF, Myc/Max), and for factors that may repress transcription (Delta EF1, Gfi-1, E4BP4, C/EBP beta). Functional analysis in reporter gene assays revealed the importance of the C delta-C gamma 3 interval in lymphocyte differentiation and identified independent regions capable of either enhancement or silencing of reporter gene expression and interaction with the IgH intron enhancer E mu. In transgenic mice, carrying a construct that links the beta-globin reporter to the novel delta-gamma 3 intron enhancer (E delta-gamma 3), transgene transcription is exclusively found in bone marrow B cells from the early stage when IgH rearrangement is initiated up to the successful completion of H and L locus recombination, resulting in Ab expression. These findings suggest that the C delta-C gamma 3 interval exerts regulatory control on Ig gene activation and expression during early lymphoid development.

Amino Acid Motifs↗

PubMed2001

Increased yield and activity of soluble single-chain antibody fragments by combining high-level expression and the Skp periplasmic chaperonin.

The success of recombinant antibody fragments as diagnostic reagents and therapeutic agents depends on the availability of sufficient functional material. We have produced a bacterial expression vector that combines high-level expression driven by a modified Shine-Dalgarno sequence with the periplasmic chaperonin Skp. Using this vector, we are able to obtain higher yields of soluble antibody fragments from cultures without the need for supplementation of the culture medium during expression. The fragments produced in the presence of the Skp show improved antigen binding activity compared to when the chaperonin is absent.

Animals↗

PubMed2001

Antibody against CD20 in patients with B cell malignancy.

Cancer patients may make antibodies against antigens on the surface of their malignant cells due either to the expression of unique antigens or to dysregulated responses to self antigens. Patients with B cell malignancy frequently produce autoantibodies and may therefore be a source of immunoglobulin genes for the production of phage display antibody libraries directed against tumour-associated antigens. Patients with autoimmune disease have circulating antibodies against lymphocyte surface antigens, and may also provide a good starting point for the production of a library of lymphocyte-reactive antibody structures. In this study, plasma and serum samples from patients with B cell malignancy or Sjogren's syndrome and from healthy controls were screened for antibodies against the B cell membrane antigens CD20. While the majority of samples showed very low reactivity, some individuals did show significant and reproducible binding to CD20. To identify a good donor for library construction, it would be advisable to screen donors for antibody against the antigens of interest.

Antigens, CD20↗

PubMed1999

Antibody repertoires of four- and five-feature translocus mice carrying human immunoglobulin heavy chain and kappa and lambda light chain yeast artificial chromosomes.

We have produced mice that carry the human Ig heavy (IgH) and both kappa and lambda light chain transloci in a background in which the endogenous IgH and kappa loci have been inactivated. The B lymphocyte population in these translocus mice is restored to about one-third of normal levels, with preferential (3:1) expression of human lambda over human kappa. Human IgM is found in the serum at levels between 50 and 400 microg/ml and is elevated following immunization. This primary human Ab repertoire is sufficient to yield diverse Ag-specific responses as judged by analysis of mAbs. The use of DH and J segments is similar to that seen in human B cells, with an analogous pattern of N nucleotide insertion. Maturation of the response is accompanied by somatic hypermutation, which is particularly effective in the light chain transloci. These mice therefore allow the production of Ag-specific repertoires of both IgM,kappa and IgM,lambda Abs and should prove useful for the production of human mAbs for clinical use.

Animals↗

PubMed1999

B-cell tumorigenesis in mice carrying a yeast artificial chromosome-based immunoglobulin heavy/c-myc translocus is independent of the heavy chain intron enhancer (Emu).

We have used YAC (yeast artificial chromosome) technology to create large translocation regions where the c-myc proto-oncogene is coupled to the core region of the human immunoglobulin heavy chain (IgH) locus (from VH2-5 through to Cdelta). Chimeric mice were obtained from embryonic stem cells carrying a single copy of the 240-kb IgH/c-myc translocation region. B-cell tumorigenesis occurs in the translocus mice, even when the entire Emu intron enhancer region between the joining segments and switch mu is deleted. This demonstrates that as yet unidentified regulatory elements in the IgH locus, independent from the known enhancers, are sufficient to cause B-cell specific activation of c-myc after translocation. The phenotype of tumors from IgH/c-myc YAC transgenic mice with or without Emu (B220+, IgM+/IgD+) is reminiscent of Burkitt's lymphoma. A rapidly expanding abnormal B-cell population is present at birth and accumulates in bone marrow, periphery, and spleen, well before discrete tumor establishment. Molecular analysis identified a clonal origin, with rearrangement of one mouse heavy chain allele retained in tumor cells from different sites, whereas subsequent rearrangements of heavy or light chain loci can be diverse. These mice routinely develop mature B-cell tumors early in life and may provide an invaluable resource of a B-cell lymphoma model.

Age Factors↗

PubMed1999

A human immunoglobulin lambda locus is similarly well expressed in mice and humans.

Transgenic mice carrying a 380-kb region of the human immunoglobulin (Ig) lambda light (L) chain locus in germline configuration were created. The introduced translocus on a yeast artificial chromosome (YAC) accommodates the most proximal Iglambda variable region (V) gene cluster, including 15 Vlambda genes that contribute to >60% of lambda L chains in humans, all Jlambda-Clambda segments, and the 3' enhancer. HuIglambdaYAC mice were bred with animals in which mouse Igkappa production was silenced by gene targeting. In the kappa-/- background, human Iglambda was expressed by approximately 84% of splenic B cells. A striking result was that human Iglambda was also produced at high levels in mice with normal kappa locus. Analysis of bone marrow cells showed that human Iglambda and mouse Igkappa were expressed at similar levels throughout B cell development, suggesting that the Iglambda translocus and the endogenous kappa locus rearrange independently and with equal efficiency at the same developmental stage. This is further supported by the finding that in hybridomas expressing human Iglambda the endogenous L chain loci were in germline configuration. The presence of somatic hypermutation in the human Vlambda genes indicated that the Iglambda-expressing cells function normally. The finding that human lambda genes can be utilized with similar efficiency in mice and humans implies that L chain expression is critically dependent on the configuration of the locus.

Animals↗

PubMed1997

Somatic hypermutation of immunoglobulin genes in human neonates.

The antibody response in the young infant is limited in several ways; in particular, responses generally are of low affinity and restricted to IgM. This raises the question whether the affinity maturation process, consisting of somatic mutation of immunoglobulin genes coupled with selection of high-affinity variants, is operative in the neonate. Re-arranged V(H)6 genes were amplified by polymerase chain reaction (PCR) from cord blood and from peripheral blood of infants. Heteroduplex analysis detected mutation in only 2/18 cord blood samples, while mutations were seen from about 10 days of age onwards. Cloning and sequencing of mutated neonatal V(H)6 genes showed that mutated sequences contained relatively few mutations (one to three mutations per sequence) compared with published values of about 10 in adult IgM sequences. Selection was not evident in the majority of neonatal samples. Thus mutation can occur in the human neonate, but is minimal and generally not accompanied by selection. The age at which affinity maturation develops effectively is yet to be defined.

Amino Acid Sequence↗

PubMed1995

Memory B lymphocytes in human tonsil do not express surface IgD.

To clarify the phenotype of memory B lymphocytes, we have determined the frequency of somatic hypermutations in purified tonsil B cell populations. Our particular interest was the controversial question of whether any memory B-cells express IgD. Ig heavy chain gene rearrangements that used the nonpolymorphic VH6 gene were amplified by PCR, cloned, and sequenced. All eight sequences obtained from the surface IgD (sIgD)-negative fraction contained point mutations, with frequency of one mutation in every 24 bases of sequence. In contrast, only 4 of 12 sequences obtained from the sIgD-positive fraction contained point mutations, with a mutation frequency of one in 600. This frequency was similar to that found for cord blood B cells (one in 550), a population that does not contain memory B cells. These results indicate that although memory B cells are present in the sIgD-negative fraction, no memory cells can be detected in the sIgD-positive fraction of tonsil B lymphocytes.

Amino Acid Sequence↗

PubMed1994

Rapid detection of immunoglobulin gene somatic hypermutation using heteroduplex formation.

In order to identify somatic hypermutation of rearranged human immunoglobulin genes, we have examined heteroduplex formation between cloned VH6 genes. In test systems, the presence of five or more point mutations could be detected by examining the formation of heteroduplexes between a known germline VH6 gene and other sequenced genes using polyacrylamide gel electrophoresis. If a mutated sequence was used, then the presence of two or more mutations could be detected. The method was used for rapid screening of VH6-D-JH rearrangements for the presence of point mutations before sequencing, and to distinguish between different highly mutated sequences, allowing clones containing the same rearrangement to be identified indirectly.

Base Sequence↗

PubMedDate not supplied

Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B lineage leukaemia and lymphoma.

The B cell specific antigen CD19 is a target for the immunotherapy of B lineage leukaemias and lymphomas. We have engineered a single chain Fv (scFv) fragment from the mouse hybridoma cell line FMC63 which produces monoclonal antibody specific for CD19. The genes encoding the FMC63 heavy and light chain variable regions were amplified from cDNA and a scFv was constructed by splice overlap extension PCR. Analysis of staining of lymphoblastoid cell lines, peripheral blood lymphocytes and tonsil sections demonstrated that the monovalent scFv fragment has the same cellular specificity as the parent hybridoma antibody. Kinetic studies with radiolabelled material showed that the scFv binds target cells with a Ka of 2.3 x 10(-9), compared with 4.2 x 10(-9) for the parent antibody. This CD19 scFv will be used in experimental models to test its therapeutic efficacy and immunogenicity, with a view to application in the diagnosis and treatment of human B cell cancers.

Animals↗

PubMedDate not supplied

CD32 (Fc gammaRII).

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Amino Acid Sequence↗

PubMedDate not supplied

CD20.

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Amino Acid Sequence↗

PubMedDate not supplied

CD62L (L-selectin).

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Amino Acid Sequence↗