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Biomedical subjects

P E Harris

Publications and source records attributed to P E Harris.

At least 19 recordsLinked to original sources
PubMed2001

Hemochromatosis mutations C282Y and H63D in 'cis' phase.

Homozygosity for the C282Y mutation of the HFE gene is a highly significant risk factor for the development of hereditary hemochromatosis (HH) and the majority of patients with HH have this genotype. An Irish/Belgian female with an elevated serum ferritin level and a family history of hemochromatosis was tested for the presence of the C282Y and H63D mutations. Results of digested PCR products have shown the patient to be homozygous for C282Y mutation and heterozygous for H63D mutation. Sequencing confirmed these findings. Genotyping of the patient's offspring and husband has also indicated the inheritance of both C282Y and H63D in 'cis'. Implications of this finding are: 1) the compound heterozygous state is by far the most common, but not the universal, phase for individuals found to be heterozygous for the two mutations, C282Y and H63D; 2) the C282Y and H63D mutations in the 'cis' phase may account for some cases of questionable parentage.

Aged↗
PubMed2001

Transcript profiling of human dendritic cells maturation-induced under defined culture conditions: comparison of the effects of tumour necrosis factor alpha, soluble CD40 ligand trimer and interferon gamma.

Using cDNA arrays, we characterized patterns of gene expression in populations of human dendritic cells (DCs) produced for clinical use. Culture and maturation induction of myeloid adherent cells under serum-free conditions yielded DCs with phenotypes similar to those described in serum-based systems. Analysis of gene expression in DCs treated with tumour necrosis factor alpha, soluble CD40L trimer or interferon gamma, however, showed specific patterns for each factor examined. Our studies document the expression of several transcripts that have not hitherto been described in DCs and/or differentially regulated according to the differentiation state of the DCs, and suggest important functional differences among the DC populations examined. In addition, DC maturation directs changes in the levels of mRNA specific for transcriptional regulators that effect the production of cytokines (e.g. BCL-6, c-rel). Other changes observed, including alteration in the gene expression profile of adhesion molecules and chemokine receptors such as CD44H, CD 49B, Rantes R, CXCR5 and CD37, suggest differences in trafficking potential between the populations studied. This broad-based description of DC populations, produced under serum-free conditions, has enabled us to better define intermediate stages of DC maturation as well as the differentiation-inducing effects of cytokines on these cells.

Base Sequence↗
PubMed2000

Specific T cell deletion by transfected human monocytes expressing fas ligand and antigen.

The aim of our experiments was to determine whether deletion of antigen specific T helper cells could be accomplished by delivering the antigenic peptide to antigen presenting cells. Tetanus toxin peptide residues 830-843 was chosen for these experiments. Two mammalian expression vectors carrying the genes for human Fas ligand and a chimeric invariant chain-tetanus toxin peptide construct were designed. The T cell proliferative response to tetanus toxoid was inhibited when the antigen was presented by autologus monocytes transfected with Fas ligand. T cell mixture experiments using two syngeneic T cell lines specific either for tetanus toxoid or for pertussis toxin demonstrated that the killing effect elicited by the antigen pulsed/Fas ligand-transfected antigen presenting cells was antigen specific. Finally, we demonstrated that transient expression of antigen delivered by plasmid DNA can substitute for soluble antigen in the induction of antigen-specific T cell responses. Antigen presenting cells transfected with the vector carrying Fas ligand and the vector carrying the chimeric invariant chain-peptide antigen gene were shown to inhibit antigen specific T cell reactivity. This strategy may be useful for the induction of apoptosis in allopeptide reactive T cells driving chronic rejection.

Apoptosis↗
PubMed2000

Cushing's syndrome associated with a chemodectoma and a carcinoid tumour.

We present a case of Cushing's syndrome where 111In-octreotide scanning provided evidence for the presence of two neuroendocrine tumours. Uptake in the right neck corresponded to a chemodectoma, but there was no change in the clinical condition or fall in ACTH levels following surgical resection. Uptake in the left chest was assumed to relate to a bronchial carcinoid, but a tumour could not initially be localized on magnetic resonance imaging (MRI), spiral CT scanning or on selective venous sampling. A 1 cm bronchial carcinoid tumour was identified post-mortem which immunostained for ACTH. This case demonstrates that 111ln-octreotide scanning is a useful technique for identifying the source of ectopic ACTH production in difficult cases of Cushing's syndrome. Reliance should not be placed solely on standard imaging techniques to localize the tumour prior to surgery. Although rare, the possibility of a non-ACTH secreting neuroendocrine tumour should also be considered in patients with ectopic ACTH syndrome, who have positive 111In-octreotide scans.

Adult↗
PubMed1999

Prevalence of Ras mutations in thyroid neoplasia.

OBJECTIVE: Mutations at codons 12, 13 or 61 of ras which result in constitutive activation occur frequently in human malignancies. There have been varied reports on their prevalence and hence their likely significance in the pathogenesis of primary thyroid neoplasia. To address this, we have examined a large series of benign and malignant thyroid tumours for ras mutations. DESIGN: Genomic DNA was analysed for the presence of mutations at codons 12, 13 and 61 of H-ras, K-ras and N-ras by allele-specific oligonucleotide hybridization. Direct DNA sequencing was used to confirm the mutations. PATIENTS: A total of 90 samples with benign (66) and malignant (24) thyroid disease were investigated. RESULTS: A total of 14/90 (15.5%) samples had a ras mutation. All mutations were at codon 61 of either N-ras or K-ras. The positive cases were 1/25 (4%) nodular goitre, 7/38 (18%) follicular adenoma, 4/9 (44%) follicular carcinoma, 1/1 anaplastic carcinoma, 1/1 follicular variant of papillary carcinoma, and 1 metastatic follicular carcinoma in which the primary tumour had the same mutation. CONCLUSIONS: Our data demonstrate a relatively low overall prevalence of ras mutations in thyroid neoplasia, with a predominance in follicular neoplasms. Their presence in follicular adenomas suggests that they may have an early aetiological role in the development of thyroid neoplasia.

Adenocarcinoma, Follicular↗
PubMed1999

A novel thyrotropin receptor mutation in an infant with severe thyrotoxicosis.

An infant girl was born at 37 weeks gestation and found to be clinically thyrotoxic at 9 months of age. Thyroid autoantibodies were negative, and thyroid function failed to normalize with medical treatment. The patient underwent a total thyroidectomy. DNA obtained from her thyroid gland and leukocytes was analyzed for thyrotropin receptor (TSHR) mutations using single strand conformation polymorphism and direct sequencing. A mobility shift of polymerase chain reaction (PCR)-amplified DNA was detected on single strand conformation polymorphism gel. Direct sequencing identified a novel point mutation in the fifth transmembrane domain of the TSH receptor at codon 597 (GTC to CTC), resulting in the amino acid substitution of leucine for valine. The mutation was heterozygous and germline, and was not identified in DNA from either of her parents. Expression of the V597L mutant is transiently transfected COS 7 cells displayed increased constitutive cyclic adenosine monophosphate (cAMP) production compared with the wild-type receptor. The mutant is expressed at very low levels on the surface of COS cells, and its response to TSH is marginal.

Animals↗
PubMed1999

Mutation analysis of protein kinase A catalytic subunit in thyroid adenomas and pituitary tumours.

OBJECTIVE: The adenylyl cyclase system plays an important role in the control of both thyroid follicular and anterior pituitary cell function. Activating mutations affecting important pathway components such as the TSH receptor and Gsalpha occur in the majority of autonomously functioning thyroid nodules. Only a small proportion of other types of thyroid tumours, however, have been reported to harbour these mutations. Activating mutations of Gsalpha have been reported to occur in up to 40% of pituitary somatotroph adenomas. As the majority of cold thyroid nodules and pituitary tumours are unaffected by these mutations, we have investigated the possibility of activating mutations occurring in protein kinase A (PKA), which is another key component of the adenylyl cyclase pathway. DESIGN: Genomic DNA and cDNA were analysed for the presence of PKA Calpha mutations by allele-specific oligonucleotide hybridisation and single strand conformation polymorphism analysis. PATIENTS: A total of 171 tissue samples were investigated. These comprised 66 benign and 24 malignant thyroid neoplasms, 21 somatotroph adenomas, 35 non-functioning pituitary adenomas, 2 corticotroph adenomas, 1 malignant prolactinoma, and 22 normal pituitary tissue samples. RESULTS: No mutations of PKA Calpha were identified using either allele-specific oligonucleotide hybridisation or single strand conformation polymorphism analysis. CONCLUSIONS: It appears that PKA Calpha mutations at the codons investigated do not represent an oncogenetic mechanism in the development of thyroid and pituitary neoplasms.

Adenoma↗
PubMed1999

Wild-type p53 epitope naturally processed and presented by an HLA-B haplotype on human breast carcinoma cells.

To broaden the clinical applicability of peptide-based immunotherapy in breast cancer, there is a need to identify further tumor-associated peptide epitopes that are specific for HLA alleles, in addition to HLA-A2. The HLA-B44 haplotype is one of the most common HLA-B haplotypes, occurring in 10-20% of the population. We performed the structural characterization of HLA class I-bound self-peptides presented by a human breast cancer cell line with a HLA-A68, A32, B40, B44 haplotype, to identify potential tumor-specific antigens. Of 13 sequenced peptides, 1 peptide had the HLA-A68 peptide binding motif and 12 peptides had the HLA-B40, B44 peptide binding motif. One of the latter peptides, FEVRVCACPG, shared 100% homology to residues 270-279 of wild-type P53 protein. Our study, thus, provides direct evidence for the natural processing and presentation of p53 epitope 270-279 by HLA-B40, B44-bearing human breast tumor cells. Epitopes spanning this region of P53 may have potential use for immunotherapy in patients expressing HLA-A2 and -B44 supertypes.

Adenocarcinoma↗
PubMed1999

Indirect allorecognition in solid organ transplantation.

Recipient T cell recognition of donor major histocompatibility complex (MHC) alloantigens plays a central role in both acute and chronic rejection of human organ allografts. Two different pathways of T cell recognition of donor MHC alloantigens have been described. The direct pathway involves T cell recognition of intact MHC molecules expressed by donor antigen-presenting cells (APCs). The second, or indirect pathway, operates via T helper cell recognition of peptides derived from the processing and presentation of allogeneic MHC molecules on self-APCs. At the onset of primary acute rejection, recipient CD4+ T cell responses to donor HLA-DR alloantigens are limited to a single dominant determinant present on one of the disparate alloantigens and restricted by one of the responder's HLA-DR molecules. In allograft recipients with recurring episodes of rejection, and/or at the onset of chronic rejection, recipient T cell reactivity may spread to other epitopes within the allogeneic MHC molecule, as well as to other alloantigens expressed by graft tissue. Both quantitative and qualitative alterations in T cell allopeptide reactivity are associated with increased risk of cellular and/or humoral rejection. These studies provide a basis for the design of new therapeutic strategies and for immunologic monitoring of transplant recipients.

Animals↗
PubMed1998

Peptides bound to major histocompatibility complex molecules.

Peptides are the means by which immune effector T cells recognize and defend against the foreign proteins of pathogens. T cell recognition of these molecules, however, is strictly dependent on peptide binding to the receptor-like molecules of the major histocompatibility complex (MHC) locus. The basic unit of recognition is a trimolecular complex consisting of the T cell antigen receptor, the MHC molecule, and the MHC-bound peptide ligand. The multistep process that culminates in MHC presentation of peptides to T cells begins in the last phases of protein catabolism. While the individual roles of many key molecules involved in peptide presentation have recently been defined, there still remain many questions regarding processing of proteins into MHC-bound peptides. This review summarizes the recent developments in peptide antigen processing for MHC molecules, with focus on how proteins are believed to be sampled and selected for degradation into peptides.

Animals↗
PubMed1998

Ligation of HLA class I molecules on smooth muscle cells with anti-HLA antibodies induces tyrosine phosphorylation, fibroblast growth factor receptor expression and cell proliferation.

The development of transplant atherosclerosis, a manifestation of chronic rejection, is the major obstacle to long-term survival of cardiac and renal allografts. The incidence of transplant atherosclerosis is increased in transplant recipients producing antidonor HLA antibodies following transplantation, suggesting that anti-HLA antibodies play a role in the pathogenesis of the disease. We have postulated that anti-HLA antibodies mediate the development of transplant atherosclerosis by binding to class I molecules on the endothelium and smooth muscle of the graft and transducing signals which stimulate cell proliferation. In this report we demonstrate that anti-HLA class I antibodies transduce signals in smooth muscle cells stimulating increased tyrosine phosphorylation of intracellular proteins and up-regulation of fibroblast growth factor (FGF) receptors. Antibody binding to class I molecules on smooth muscle cells is also accompanied by increased responsiveness to basic FGF and augmented cell proliferation. These findings may explain the increased occurrence of transplant atherosclerosis in recipients producing anti-donor HLA antibodies.

Antibodies↗
PubMed1998

Intramolecular and intermolecular spreading during the course of organ allograft rejection.

There are two distinct pathways by which T cells may recognize MHC alloantigens. The direct pathway involves T-cell recognition of intact MHC molecules expressed by donor antigen-presenting cells (APCs). The second, or indirect, pathway describes T-cell recognition of peptides derived from the processing and presentation of allogeneic MHC molecules on self APCs. Recent data demonstrates that indirect recognition plays a central role in both acute and chronic rejection of human organ allografts. Our studies have shown that, at the onset of primary acute rejection, recipient T-cell responses to donor HLA-DR alloantigens are limited to a single dominant determinant present on one of the disparate alloantigens and restricted by one of the responder's HLA-DR molecules. In allograft recipients with recurring episodes of rejection, and/or at the onset of chronic rejection, recipient T-cell reactivity may spread to other epitopes within the allogeneic MHC molecule as well as to other alloantigens expressed by graft tissue. Both quantitative and qualitative alterations in T-cell allopeptide reactivity are associated with increased risk of cellular and/or humoral rejection. These studies provide a basis for the design of new therapeutic strategies and for immunologic monitoring of transplant recipients.

Graft Rejection↗
PubMed1997

Naturally processed tissue- and differentiation stage-specific autologous peptides bound by HLA class I and II molecules of chronic myeloid leukemia blasts.

Structural analysis of naturally processed peptides bound to the HLA class I and class II molecules of chronic myeloid leukemia (CML) blast cells was performed to characterize the antigen processing and autoantigen repertoire in this hematopoietic malignancy. Self-peptides derived from the carboxy-terminal end of the breakpoint cluster region (bcr) protein, as well as several differentiation stage- and tissue-specific self-antigens characteristic of early stages of myeloid differentiation, such as c-fes, c-pim, granulocyte-macrophage colony-stimulating factor receptor alpha chain, proteinase 3, and cathepsin G, were identified. A common characteristic of several of the high copy-number self-peptides identified in this study is the participation of their parent proteins in signal transduction or myeloid effector function. Because bcr-abl junctional peptides bind to a limited number of major histocompatibility complex (MHC) class I alleles, an effective peptide-based immunotherapy strategy for CML requires identification of further tumor-associated or tissue-specific peptide antigens binding to common MHC alleles such as HLA-A2. The differentiation stage- and tissue-specific MHC-bound peptides found in this study, as well as the naturally processed proteins from which they are derived, may represent autoantigens towards which T-cell responses may potentially be developed for immunotherapy of hematopoietic malignancies such as CML.

Amino Acid Sequence↗
PubMed1997

Induction of high affinity fibroblast growth factor receptor expression and proliferation in human endothelial cells by anti-HLA antibodies: a possible mechanism for transplant atherosclerosis.

The major limitation to long term survival of organ allografts is chronic rejection, which is manifested as atherosclerosis of the vessels of the transplanted organ. There is a significant association between transplant atherosclerosis and the development of Abs to the disparate HLA Ags present on the graft vasculature. We have investigated the effect of anti-HLA Abs on endothelial cells (EC) and smooth muscle cells cultured in vitro. Ab ligation of class I molecules on ECs results in increased high affinity fibroblast growth factor receptor mRNA expression, and enhanced basic fibroblast growth factor ligand binding. Ab binding to class I molecules on EC and smooth muscle cells is also accompanied by augmented cell proliferation. These results suggest that the intimal thickening observed in transplant atherosclerosis is the result of the proliferative effects of anti-HLA Abs.

Antibodies↗
PubMed1997

Polymorphism in the 5' terminal region of the mRNA of HLA-DQA1 gene: identification of four groups of transcripts and their association with polymorphism in the alpha 1 domain.

Relative to other loci in the MHC, the HLA-DQ locus exhibits an exceptional degree of polymorphism of both A1 and B1 genes, particularly in the region coding for alpha and beta chains. Diversification of the association between different alpha and beta molecules either in cis or in trans contributes to the structural diversity of the repertoire of cell-surface class II protein's in the population. In addition, structural allelic polymorphisms in the 5' regulatory region of both DQB1 and DQA1 shows several linkage groups with respect to the allelic coding sequence of the respective genes. We describe here the allelic polymorphism in the DQA1 mRNA structure located at the 5' untranslated terminal region. This portion of the mRNA molecule represents, in many genes, a cis-acting regulatory sequence playing a role in the posttranscriptional mechanisms by which gene expression can be modulated. Based on detailed transcriptional analysis, we have been able to define at least four groups of transcripts in DQA1. The mRNA variability was associated with the polymorphism of the second exon of the DQA1 gene, coding for the alpha 1 domain and not with the DNA polymorphism in the 5' regulatory region.

B-Lymphocytes↗