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Biomedical subjects

D Chalmers

Publications and source records attributed to D Chalmers.

At least 19 recordsLinked to original sources
PubMed2001

Molecular mechanism for ganciclovir resistance in human T lymphocytes transduced with retroviral vectors carrying the herpes simplex virus thymidine kinase gene.

The herpes simplex virus thymidine kinase gene type 1 (HSV-Tk) ganciclovir (GCV) system is a novel therapeutic strategy for the modulation of graft-versus-host disease (GVHD), a major complication of allogeneic stem cell transplantation (allo-SCT). Retroviral-mediated gene transfer of the HSV-Tk gene into donor T lymphocytes before allo-SCT may allow their in vivo selective depletion after treatment with GCV. The expression of the HSV-Tk gene was analyzed in vitro in CEM cells, a human lymphoblastoid cell line, transduced with 2 different vectors, each containing the HSV-Tk gene and a selectable marker gene. GCV-resistant clones were identified within the clones expressing the marker gene. Characterization of the molecular events leading to this resistance revealed a 227-bp deletion in the HSV-Tk gene due to the presence of cryptic splice donor and acceptor sites within the HSV-Tk gene sequence. Furthermore, it was confirmed that this deletion was present in human primary T cells transduced with either vector and in 12 patients who received transduced donor T cells, together with a T-cell-depleted allo-SCT. In vivo circulating transduced T cells containing the truncated HSV-Tk gene were identified in all patients immediately after infusion and up to 800 days after transplantation. In patients who received GCV as treatment for GVHD, a progressive increase in the proportion of transduced donor T cells carrying the deleted HSV-Tk gene was observed. These results suggest that the limitations within the HSV-Tk/GCV system can be improved by developing optimized retroviral vectors to ensure maximal killing of HSV-Tk-transduced cells.

Antiviral Agents↗
PubMed2001

Elimination of the truncated message from the herpes simplex virus thymidine kinase suicide gene.

Introduction of the Herpes simplex virus thymidine kinase (HSV-tk) gene into target cells renders them susceptible to killing by ganciclovir (GCV). We are studying the use of HSV-tk-transduced T lymphocytes in the context of hematopoietic stem cell transplantation. We have previously shown, in vitro and in vivo, the occurrence of transduced cells resistant to GCV due to a deletion within HSV-tk. This deletion, a consequence of the presence of cryptic splice donor and acceptor sites, originates in the retroviral producer cell. Here we adopt two different methods that introduce third-base degenerate changes at the cryptic splice sites and so prevent splicing. Consequently, the HSV-tk protein is unaltered and the sensitivity of the target cells to GCV is preserved. The use of this mutated HSV-tk should reduce the likelihood of the development of resistant genetically modified cells during clinical trials.

Antiviral Agents↗
PubMed2001

How safe is adventure tourism in New Zealand? An exploratory analysis.

The paper reports findings from a multidisciplinary programme of research, the major aims of which were to determine the nature and extent of the New Zealand adventure tourism injury problem. Analysis of hospital discharge and mortality data for a 15-year period identified adventure tourism-related activities as contributing to approximately 20% of overseas visitor injuries, and 22% of fatalities. Activities that commonly involve independent-unguided adventure tourism, notably mountaineering, skiing and tramping, contributed most to injury and fatality incidence. Horse riding and cycling activities were identified from hospital discharge data and adventure tourism operators' reported client injury-incidence, as the commercial adventure tourism activities most frequently involved in client injuries. Falls were the most common injury events, and a range of client, equipment, environmental and organisational risk factors were identified. Possible interventions to reduce injury risk among overseas and domestic adventure tourists are discussed.

Accidents↗
PubMed2001

Distinct hematopoietic support by two human stromal cell lines.

OBJECTIVE: The hematopoietic microenvironment is complex, and the role of myofibroblast in its function is crucial. In order to obtain a stable model reflecting this particular cell type, we have previously established human bone marrow cell lines from primary myofibroblastic Stro1(+) population (pStro1(+)). We placed HPV16 E6 and E7 expression under the control of different promoters. Here, we have characterized and studied the hematopoietic support for two cell lines corresponding to the promoters alpha-SM (alphaSM-56 line) and SV40 (SV40-56 line). MATERIALS AND METHODS: The expression profile was analyzed at the RNA level by gene array and at the protein level by Western blot, flow cytometry, and ELISA. Hematopoietic support determined using colony-forming unit (CFU) and stroma-adherent colony-forming cell (SA-CFC) assays. RESULTS: The phenotype of cell lines was not significantly modified compared with primary myofibroblastic cells. They secreted a broad spectrum of hematopoietic cytokines and nonspecific mediators. The two lines allowed the growth of hematopoietic precursors and had different support capabilities. CONCLUSIONS: We have extensively characterized two novel human bone marrow stromal cell lines. They retained a myofibroblastic phenotype and have substantial but different hematopoietic support capabilities. These lines provided a basis for determining stromal factors involved in stem-cell regulation.

Antigens, CD34↗
PubMed2001

Comparative analysis of the nonA region in Drosophila identifies a highly diverged 5' gene that may constrain nonA promoter evolution.

A genomic fragment from Drosophila virilis that contained all the no-on-transientA (nonA) coding information, plus several kilobases of upstream material, was identified. Comparisons of nonA sequences and the gene nonA-like in D. melanogaster, a processed duplication of nonA, suggest that it arose before the split between D. melanogaster and D. virilis. In both species, another gene that lies <350 bp upstream from the nonA transcription starts, and that probably corresponds to the lethal gene l(1)i19, was identified. This gene encodes a protein that shows similarities to GPI1, which is required for the biosynthesis of glycosylphosphatidylinositol (GPI), a component for anchoring eukaryotic proteins to membranes, and so we have named it dGpi1. The molecular evolution of nonA and dGpi1 sequences show remarkable differences, with the latter revealing a level of amino acid divergence that is as high as that of transformer and with extremely low levels of codon bias. Nevertheless, in D. melanogaster hosts, the D. virilis fragment rescues the lethality associated with a mutation of l(1)i19e, as well as the viability and visual defects produced by deletion of nonA(-). The presence of dGpi1 sequences so close to nonA appears to have constrained the evolution of the nonA promoter.

Amino Acid Sequence↗
PubMed2000

Loss of Fas (CD95/APO-1) expression by antigen-specific cytotoxic T cells is reversed by inhibiting DNA methylation.

Elimination of clonally expanded peripheral CD8 T cells was thought to involve apoptosis induction mediated principally by TNF, but recently Fas (CD95/APO-1) has been shown to play a role in certain responses. Here we study Fas expression and sensitivity to its ligation on murine CD8 cells specific for the CW3 antigen expressed by transfected P815 cells. Fas was progressively downregulated after successive in vitro restimulations of antigen-specific CD8 cells, until clones became Fas negative and totally resistant to the effects of recombinant Fas ligand. In contrast, Fas expression by in vivo restimulated antigen-specific cells did not diminish. Loss of Fas expression in vitro was not totally irreversible, since it could be reinduced by inhibition of DNA methylation. Understanding how Fas may be differentially regulated in vivo and in vitro is an important issue for the optimal manipulation of T cells for adoptive immunotherapy protocols.

Animals↗
PubMed2000

Health-related quality of life in survivors of Wilms' tumor and advanced neuroblastoma: aA cross-sectional study.

PURPOSE: In pediatric oncology, Wilms' tumor and advanced neuroblastoma represent opposite ends of the spectra of survival probability and therapeutic intensity. Consequently, it was envisaged that survivors of Wilms' tumor would enjoy better health status and health-related quality of life (HRQL) than survivors of advanced neuroblastoma. PATIENTS AND METHODS: Health status questionnaires were sent to the parents of all eligible children and to the children themselves if they were > or = 8 years of age. Responses were received from 84% of 93 eligible families. Responses were converted by established algorithms into levels of two multiattribute health status classification systems known as Health Utilities Index Mark 2 and Mark 3. These systems are linked to measures of preference, in the form of multiattribute utility functions, which provide scores of morbidity for single-attribute levels and of global HRQL for comprehensive health states. RESULTS: A greater burden of morbidity was identified in the survivors of advanced neuroblastoma than in survivors of Wilms' tumor based on the assessments of the parents of these children. In particular, survivors of advanced neuroblastoma exhibited deficits in hearing and speech. It is possible that this morbidity burden reflects the prevalent use of platinum compounds (causing ototoxicity) in this group. Within parent-child dyads there was a high level of percentage agreement on responses in all attributes except cognition. CONCLUSION: Extension of this study to a larger sample size of patients will provide clarification of these observations.

Adolescent↗
PubMed2000

Development of a competitive PCR method for in vitro and in vivo quantification of herpes simplex virus thymidine kinase and neomycin resistance-expressing cells used in a clinical trial.

The aim of this study was to set up a sensitive and specific method to quantify the number of gene-modified cells in a gene therapy clinical trial currently underway at our institution. This trial involves the use of retrovirally transduced allogeneic T cells expressing the herpes simplex-1 thymidine kinase (HSV-TK) and neomycin-phosphotransferase (NeoR) resistance gene. Quantification by competitive PCR was performed, with two homologous internal standards (deltaTK, deltaNeoR), 30 bp shorter than the target sequences (TK, NeoR), coupled to fluorescent laser-based detection. Assessment of the amplification systems procedures was carried out for each sequence. The 30-bp deletion did not affect the amplification efficiency significantly. Determination of the plateau phase of both amplified sequences demonstrated that each sample must be quantified during the predetermined exponential phase. Finally, a blinded study of a transduced cell dilutions panel validated the overall methodology. The competitive PCR was applied to quantification of the retroviral transduction process by quantifying the NeoR gene in transduced PBMC samples (prior to G418 selection) from 18 donors in our clinical trial. A mean transduction efficiency of 9.78% +/- 1.37% was observed. We also quantified TK-expressing donor transgenic T cells in a murine GvHD model. Results demonstrated on initial expansion of donor HSV-TK- expression T cells as well as a significant ganciclovir (GCV)-induced decrease correlated with the number of circulating gene-modified T cells. Therefore, we have developed an efficient gene quantification tool that should be useful for in vivo monitoring of gene-modified cells.

Animals↗
PubMed2000

The clock gene period of the housefly, Musca domestica, rescues behavioral rhythmicity in Drosophila melanogaster. Evidence for intermolecular coevolution?

In Drosophila, the clock gene period (per), is an integral component of the circadian clock and acts via a negative autoregulatory feedback loop. Comparative analyses of per genes in insects and mammals have revealed that they may function in similar ways. However in the giant silkmoth, Antheraea pernyi, per expression and that of the partner gene, tim, is not consistent with the negative feedback role. As an initial step in developing an alternative dipteran model to Drosophila, we have identified the per orthologue in the housefly, Musca domestica. The Musca per sequence highlights a pattern of conservation and divergence similar to other insect per genes. The PAS dimerization domain shows an unexpected phylogenetic relationship in comparison with the corresponding region of other Drosophila species, and this appears to correlate with a functional assay of the Musca per transgene in Drosophila melanogaster per-mutant hosts. A simple hypothesis based on the coevolution of the PERIOD and TIMELESS proteins with respect to the PER PAS domain can explain the behavioral data gathered from transformants.

Amino Acid Sequence↗
PubMed1999

Endoscopic and histological healing with infliximab anti-tumor necrosis factor antibodies in Crohn's disease: A European multicenter trial.

BACKGROUND & AIMS: Tumor necrosis factor (TNF) is a pivotal cytokine in intestinal inflammation. Controlled trials using a chimeric anti-TNF antibody (infliximab) have shown its efficacy in refractory Crohn's disease. METHODS: Endoscopic and histological response to infliximab was investigated in a multicenter, randomized, double-blind, and placebo-controlled trial including 30 patients with active Crohn's disease undergoing ileocolonoscopy before and 4 weeks after intravenous administration of 5, 10, or 20 mg/kg of infliximab or placebo as a single infusion. Lesions were scored by means of the validated Crohn's Disease Endoscopic Index of Severity (CDEIS). Endoscopic biopsy specimens were taken during both procedures from 9 of 30 patients and scored by a single gastrointestinal pathologist. RESULTS: CDEIS scores decreased significantly in most infliximab-treated patients without an apparent dose response. No endoscopic improvement was observed in the placebo group. The changes in CDEIS correlated highly with those of the Crohn's Disease Activity Index. At a histological level, disappearance of the inflammatory infiltrate was observed in infliximab-treated patients but not in placebo-treated ones; however, architectural changes persisted in most patients. Strictures developed in several patients. CONCLUSIONS: Clinical improvement after infliximab therapy in active Crohn's disease is accompanied by significant healing of endoscopic lesions and disappearance of the mucosal inflammatory infiltrate.

Adult↗
PubMed1997

Amelioration of relapsing experimental autoimmune encephalomyelitis with altered myelin basic protein peptides involves different cellular mechanisms.

T-cells specific for a region of human myelin basic protein, amino acids 87-99 (hMBP87-99), have been implicated in the development of multiple sclerosis (MS) a demyelinating disease of the central nervous system. Administration of soluble altered peptide ligand (APL), made by substituting native residues with alanine at either positions 91(91K > A or A91) or 97 (97R > A or A97) in the hMBP87-99 peptide, blocked the development of chronic relapsing experimental autoimmune encephalomyelitis (R-EAE), in the SJL mouse. The non-encephalitogenic APL A91, appears to induce cytokine shifts from Th1 to Th2 in the target T-cells, whereas the encephalitogenic superagonist APL A97 causes deletion of the MBP87-99 responsive cells. Thus, single amino acid changes at different positions in the same peptide epitope can lead to APL capable of controlling auto-immune disease by different mechanisms.

Animals↗
PubMed1996

Rat homolog of mouse interleukin-1 receptor accessory protein: cloning, localization and modulation studies.

A protein which facilitates the binding between interleukin-1 (IL-1) and the type I IL-1 receptor (designated as interleukin-1 receptor accessory protein, IL-1RAcP) has recently been cloned in mouse cells. In the present study, a rat homolog of the mouse IL-1RAcP was isolated from a rat superior cervical ganglion library. The deduced 570 amino acid sequences between rat and mouse IL-1RAcP have > 95% sequence identity to each other with similar predicted signal peptide sequence (20 amino acids), extracellular domain (339 amino acids), a single transmembrane domain (24 amino acids) and a long intracellular domain (187 amino acids). The rat IL-1RAcP has approximately 25% sequence identity to the rat type I IL-1 receptor and a predicted extracellular domain with three immunoglobulin-like loops. RNase protection assays demonstrated that rat IL-1RAcP is expressed in both brain and peripheral tissues with the highest densities present in liver and brain areas such as hypothalamus, cerebral cortex, hippocampus and cerebellum; significantly lower densities were present in lung and in immune tissues such as thymus and spleen. The presence of IL-1RAcP in brain was confirmed by in situ hybridization histochemical studies with a discrete localization present in the dentate gyrus of the hippocampus. The IL-1RAcp was down-regulated in parallel with the type I IL-1 receptor in the liver following endotoxin treatment in rats. These data demonstrating the presence and modulation of a rat homolog of a mouse IL-1RAcP, which is highly expressed in brain and peripheral tissues containing type I rat IL-1 receptor, further suggest the importance of the interaction between the two proteins in rat in modulating the actions of IL-1. On the other hand, the presence of the IL-1RAcP in brain areas which show an absence of type I IL-1 receptors suggests additional functions for this protein in the rat.

Amino Acid Sequence↗
PubMed1996

Incidence of death and hospitalization from assault occurring in and around licensed premises: a comparative analysis.

The aim of the research was to: determine the incidence of serious assault in and around licensed premises in New Zealand, and to compare the circumstances of assault with those that occurred in other locations. For the period 1978-87, inclusive, 49 assault fatalities occurred in or around licensed premises representing 9.4% of all homicides and 12.9% where a place was specified. The comparable figures for assaults resulting in hospitalization in 1988 were: 251, 10.2% and 18.4%, respectively. Further analyses suggests that our estimate of the incidence rate is likely to be an underestimate due to changes over time in the large number of assault cases which have no specific place of occurrence identified. In comparison with homes homicides in licensed premises were more likely to involve: males; Maori, unarmed fights and brawls; unknown assailants; alcohol; occur during the evening and toward the end of the week; and result in head injury. For non-fatal events similar differences were found. In comparison with homes non-fatal assaults were more likely to involve: males; young adults, Maori, the unemployed, unarmed fights and brawls, and head injury. There have been a number of significant policy changes in New Zealand since 1988 which may have resulted in a change to the situation reported here.

Adolescent↗
PubMed1996

Neurobiology of corticotropin releasing factor (CRF) receptors and CRF-binding protein: implications for the treatment of CNS disorders.

The actions of CRF in the brain and in the periphery are mediated through multiple binding sites. There are three receptors, CRF1, CRF2 alpha and CRF2 beta, which encode 411, 415 and 431 amino acid proteins and transduce signals via the stimulation of intracellular cAMP production. The recent identification of high-affinity non-peptide CRF receptor antagonists should allow for rapid progress in drug development of CRF receptor antagonists. In addition to the receptors, the actions of CRF in brain and in the periphery can also be modulated by a binding protein of 322 amino acids. Ligands of CRF-BP, such as CRF (6-33) can elevate brain levels of 'free' CRF and improve learning and memory without stress-like side effects of CRF receptor agonists. Urocortin, a mammalian CRF-related peptide with close sequence homology to fish urotensin, interacts with CRF1, CRF2 receptors and with CRF-BP. These data indicate that CRF receptor antagonists may be useful for the treatment of the disease states where CRF is elevated such as anxiety and depression, anorexia nervosa and stroke and that ligand inhibitors of CRF-BP may be used to elevate brain levels of 'free' urocortin and other CRF-related peptides.

Animals↗