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Biomedical subjects

D Shepherd

Publications and source records attributed to D Shepherd.

At least 19 recordsLinked to original sources
PubMed2001

Mature dendritic cells prime functionally superior melan-A-specific CD8+ lymphocytes as compared with nonprofessional APC.

Priming of melan-A(26/27-35)-specific CTL occurs only in a fraction of late stage melanoma patients, whereas during the early stages of the disease and in healthy volunteers, melan-A CTL have functional and phenotypic markers consistent with a naive phenotype. To study the requirements for expansion of naive melan-A CTL from healthy donors, we set up an in vitro priming protocol and, using tetramer assays, we demonstrate that the activity and phenotype of the expanded melan-A CTL are profoundly influenced by the type of APC used. Priming by nonprofessional APC leads to expansion of melan-A CTL with reduced cytolytic activity and low level of IFN-gamma secretion. In contrast, mature dendritic cells (DC) expand cytolytic and IFN-gamma-producing melan-A CTL. Priming by mature DC is also efficient at low peptide concentration and requires only one round of stimulation. Finally, we observed that a significant fraction of CD45RO(+) melan-A CTL primed by mature DC expresses high levels of the homing receptor CD62L, whereas CTL primed by nonprofessional APC express CD62L in lower percentages and at lower levels. These results suggest that suboptimal priming by nonprofessional APC could account for the presence in vivo of dysfunctional cells and strongly support the immunotherapeutic use of mature DC for expansion of effector and memory Ag-specific CTL.

Antigen-Presenting Cells↗
PubMed2001

Cytometric and functional analyses of NK and NKT cell deficiencies in NOD mice.

Defects in NK and NKT cell activities have been implicated in the etiology of type 1 (autoimmune) diabetes in NOD mice on the basis of experiments performed using surrogate phenotypes for the identification of these lymphocyte subsets. Here, we have generated a congenic line of NOD mice (NOD.b-Nkrp1(b)) which express the allelic NK1.1 marker, enabling the direct study of NK and NKT cells in NOD mice. Major deficiencies in both populations were identified when NOD.b-Nkrp1(b) mice were compared with C57BL/6 and BALB.B6-Cmv1(r) mice by flow cytometry. The decrease in numbers of peripheral NK cells was associated with an increase in their numbers in the bone marrow, suggesting that a defect in NK cell export may be involved. In contrast, the most severe deficiency of NKT cells found was in the thymus, indicating that defects in thymic production were probably responsible. The deficiencies in NK cell activity in NOD mice could only partly be accounted for by the reduced numbers of NK cells, and fewer NKT cells from NOD mice produced IL-4 following stimulation, suggesting that NK and NKT cells from NOD mice shared functional deficiencies in addition to their numerical deficiencies. Despite the relative lack of IL-4 production by NOD NKT cells, adoptive transfer of alpha beta TCR(+)NK1.1(+) syngeneic NKT cells into 3-week-old NOD recipients successfully prevented the onset of spontaneous diabetes. As both NK and NKT cells play roles in regulating immune responses, we postulate that the synergistic defects reported here contribute to the susceptibility of NOD mice to autoimmune disease.

Animals↗
PubMed2000

Tau and tau reporters disrupt central projections of sensory neurons in Drosophila.

In this paper, the authors report that the expression of tau-based reporter genes causes severe defects in the morphology of sensory neurons in adult Drosophila. Targeted expression of tau-green fluorescent protein (tau-GFP) in sensory neurons, using the galactosidase-4 (GAL4) system, produced a range of characteristic defects in expressing neurons. The defects observed included loss of axons, abnormal axon bundling, reduced sensory arborisations, and axonal swellings (beads). Blind comparisons of adult sensory neurons labelled with tau-GFP or CD8-GFP showed that tau-GFP neurons exhibited many more defects than CD8-GFP-expressing neurons. CD8-GFP was found to induce no significant defects on sensory neuron morphology. Expression of tau-lacZ and human tau in sensory neurons produced defects comparable to those seen with tau-GFP. A developmental study showed that tau-expressing axons grow normally and innervate the correct regions of the neuropil. The absence of these axons later in development suggests that tau-expressing axons are lost after initial ingrowth. Examination of silver-stained sections suggests that the absence of axons is due to axon loss rather than failure of the expression system to label the neurons. The results suggest that the expression of tau-based reporter constructs causes severe defects in sensory neurons, resulting in degeneration. The results also indicate that Drosophila may provide a useful model system for examining the role of tau in neurodegenerative disorders.

Afferent Pathways↗
PubMed2000

Confocal quality imaging of afferent neurons from semi-thin sections of Drosophila ganglia.

The aim of this study was to develop protocols for computer imaging of the thoraco-abdominal ganglion of Drosophila from serial semi-thin sections, in which specific neurons were stained and related to neuropilar structures. The central projections of a subset of transgenically labelled sensory neurons were revealed by immunohistochemistry, while Nomarski optics were used to show motor neuron targets in the neuropil. Digital photomicrographs of each section were aligned and the resultant image stacks rendered into three-dimensional (3D) images that can be rotated in real time. The result is a detailed, in-depth visualization of labelled neurons at a resolution comparable with that in confocal reconstructions, which also allows investigation of their relationships with other components of the neuropil.

Animals↗
PubMed2000

A shift in the phenotype of melan-A-specific CTL identifies melanoma patients with an active tumor-specific immune response.

In a significant proportion of melanoma patients, CTL specific for the melan-A(26/7-35) epitope can be detected in peripheral blood using HLA-A2/peptide tetramers. However, the functional capacity of these CTL has been controversial, since although they prove to be effective killers after in vitro expansion, in some patients they have blunted activation responses ex vivo. We used phenotypic markers to characterize melan-A tetramer(+) cells in both normal individuals and melanoma patients, and correlated these markers with ex vivo assays of CTL function. Melanoma patients with detectable melan-A tetramer(+) cells in peripheral blood fell into two groups. Seven of thirteen patients had a CCR7(+) CD45R0(-) CD45RA(+) phenotype, the same as that found in some healthy controls, and this phenotype was associated with a lack of response to melan-A peptide ex vivo. In the remaining six patients, melan-A tetramer(+) cells were shifted toward a CCR7(-) CD45R0(+) CD45RA(-) phenotype, and responses to melan-A peptide could be readily demonstrated ex vivo. When lymph nodes infiltrated by melan-A-expressing melanoma cells were examined, a similar dichotomy emerged. These findings demonstrate that activation of melan-A-specific CTL occurs in only some patients with malignant melanoma, and that only patients with such active immune responses are capable of responding to Ag in ex vivo assays.

Adult↗
PubMed2000

Glial dependent survival of neurons in Drosophila.

According to the classical model of insect neurogenesis, neuron fate and survival is determined largely by cell autonomous mechanisms with no requirement for cell-cell interactions to control the total number of neurons. In a recent paper by Booth et al.,(1) however, the central tenet of this model has been called into question. Using a combination of mutations and targeted glial ablation, this paper shows that, contrary to common thinking, neuron survival in the embryonic nervous system of Drosophila is dependent upon normal glial function. This surprising result suggests that insect neurogenesis may have more in common with vertebrate neurogenesis than previously thought.

Animals↗
PubMed2000

Capgras syndrome: possibly more common among the Maori of New Zealand.

OBJECTIVE: The report describes an apparently greater incidence of Capgras syndrome among the Maori population compared with the European population, in the most easterly catchment area served by Tauranga Hospital in New Zealand's Bay of Plenty. METHOD: Over the last year we have become aware of five cases of Capgras syndrome in our catchment area. This area (population nearly 21,000) consists of a rapidly expanding new suburb of the city of Tauranga and a rural area extending 55 km east of the city. These figures were compared with those of the westerly catchment area served by Tauranga Hospital, where the psychiatric team is not aware of any examples of Capgras syndrome among their population. The 1996 census figures were obtained in order to calculate a population ethnicity breakdown. RESULTS: Five cases of Capgras syndrome were identified in the most easterly catchment area where 19% of the population identified as Maori, 75% as European and 6% as other or non-specified. All of the cases occurred in Maori patients. This compares with no identified cases of Capgras syndrome in the most westerly catchment area where 12% of the population identified as Maori, 87% as European and 1% as other or non-specified. Four out of five cases were female. Two cases had a history of cannabis use. Three cases had exhibited dangerous behaviour towards family members. CONCLUSIONS: There is an apparently greater incidence of Capgras syndrome among the New Zealand Maori population compared with the European population in the most easterly catchment area served by Tauranga Hospital. In our population Capgras syndrome is a common, not rare, feature of psychotic illness, and the cases support a previously reported association of this syndrome with dangerousness.

Adult↗
PubMed2000

Methods for imaging labeled neurons together with neuropil features in Drosophila.

We describe staining protocols for serial semithin sections of Drosophila central ganglia that allow visualization of gene expression in particular neurons with counterstaining to display the ganglion architecture. Green fluorescent protein (GFP), expressed in a subset of sensory neurons from a selected enhancer trap line, is visualized by conventional immunohistochemistry with a peroxidase-linked antibody, and neural architecture is revealed by reduced silver staining. This makes visible in histological sections the same GFP-labeled cells seen with confocal microscopy, but with the especial advantage that neuropil structures are also revealed at the level of individual cells and neuron processes. Not only does this allow the physical relationships among intracellularly labeled neurons to be determined by reference to specific features in the neuropil but it also enables a function to be ascribed provisionally to particular regions of neuropil. These methods have particular utility for mapping morphological information on specific neurons in the context of central nervous system architecture, both in adult Drosophila and during development.

Animals↗
PubMed2000

A comparison of individual and population smoking data from a postal survey and general practice records.

BACKGROUND: Data on smoking held by general practitioners (GPs) may contribute to clinical care and to an assessment of population health. However, these data are prone to several biases and their validity has not been tested. AIM: To examine the accuracy of general practice data as an estimate for population prevalence of smoking and to estimate the accuracy of GP data on individuals' smoking habit compared with self-report. METHODS: A postal questionnaire on smoking habit over the past six years was sent to a random sample of individuals aged 15 to 74 years and registered with five out of seven general practices in one part of Leicester. GP records of those sampled were examined for an entry of smoking status over this period. RESULTS: Response rate to the postal questionnaire was 1906 out of 2490 (76.5%). Reported smoking prevalence was 35.2%. Of those notes sampled, 1784 out of 2432 (73.4%) had an entry about smoking recorded between 1991 and 1996. Patients recorded as smokers were less likely to respond to the postal questionnaire than non-smokers. Using practice data to ascribe smoking status to non-responders produced an estimated prevalence of 38.6%. Using questionnaire data alone as the 'gold standard', the last practice record collected since 1991 overestimated current smoking prevalence by a factor of 1.22; using questionnaire data supplemented by practice data for non-responders as the 'gold standard' meant that the overestimate was by a factor of 1.11. Data from notes and the questionnaire were available for 1398 individuals and 2188 observations. Levels of agreement were high (kappa = 0.83). CONCLUSION: GP-held data are valid for individuals but over-estimate smoking prevalence at a population level.

Adolescent↗
PubMed1999

Persistent larval sensory neurons in adult Drosophila melanogaster.

Using a combination of lineage tracing and laser ablation, we have identified a segmentally repeated array of embryonically produced sensory neurons that persist through metamorphosis into adult stages of Drosophila development. The persistent sensory neurons are found in all unfused abdominal segments, but there is segment-specific variation in the number of neurons observed. There are 12 persistent neurons in the first abdominal segment (A1), 18 in the second (A2), and 16 in segments A3-A7. Most are internal sensory neurons (dendritic arborization neurons and bipolar dendrite neurons), but two are associated with external sensilla on the sternite. All of these neurons and their axons define specific adult sensory pathways in the periphery and their locations and persistence through metamorphosis suggest a role in guiding the growth of adult sensory and motor axons.

Age Factors↗
PubMed1998

Disconnected mutants show disruption to the central projections of proprioceptive neurons in Drosophila melanogaster.

We used a P[GAL4] enhancer-trap line, C161, in conjunction with the UAS-lacZ reporter construct to visualize the central projections of a defined set of thoracic and abdominal sensory neurons in a disconnected (disco) mutant background. The results show defects in the organization of sensory axons in the larval and adult central nervous system. The defects are indicative of problems with axon growth and development and include (a) poor axon fasciculation, (b) aberrant axon growth, (c) excessive terminal branching, and (d) ectopic innervation. Sensory neuron identity appears to be normal. The defects are comparable to those previously described for larval photoreceptor and adult retinular cells in disco mutants and extend the known effects of this mutation. Reduced larval and adult viability are likely to result from locomotory defects related to the disruption of the sensory system.

Animals↗
PubMed1998

Isolation and characterisation of smallminded, a Drosophila gene encoding a new member of the Cdc48p/VCP subfamily of AAA proteins.

Smallminded (smid) encodes a new member of the cdc48p/VCP subfamily of AAA proteins in Drosophila. The gene was isolated by plasmid rescue from a GAL4 enhancer trap line which shows reporter gene expression in neuroblasts, imaginal disks and a subset of sensory neurons. Larvae homozygous for the insert arrest development as second instar larvae and die without pupating. The most obvious defect in these larvae is a significantly reduced CNS, hence the naming of the gene as smallminded. The deduced amino acid sequence of smid contains a tandem duplication of the AAA nucleotide binding domain characteristic of the cdc48p/VCP subfamily. Overall, smid shares 33% identical residues with its closest relative, yeast L0919-chrXII and 26-29% with other members of the cdc48p/VCP subfamily. The most highly conserved regions of the predicted protein structure are found in and around the nucleotide binding domains. The gene is expressed at all developmental stages.

Amino Acid Sequence↗
PubMed1998

Dynamic developmental expression of smallminded, a Drosophila gene required for cell division.

Here we describe the expression pattern of the smallminded (smid) gene during Drosophila development and investigate the phenotype of a null mutant. In situ hybridisation reveals the ubiquitous expression of smid transcript throughout early embryonic stages until the extended germ band stage, after which expression becomes localised to the neurogenic ectoderm and gonad. Post-embryonic expression is restricted to tissues engaged in the developmental programme of the adult fly: the re-enlarged neuroblasts; imaginal disks; histoblast nests; and precursors of adult muscles. The correlation of smid expression with mitotic activity suggests a cell cycle function which is confirmed by the observed phenotype of a smid null mutant characterised by an abnormally small CNS, due to defective mitosis of post-embryonic neuroblasts and their subsequent death by apoptosis.

Animals↗
PubMed1997

Two types of ACh receptors contribute to fast channel gating on mouse skeletal muscle.

Single-channel recordings from mouse C2 myotubes indicate that maturation of skeletal muscle is accompanied by the appearance of two types of fast acetylcholine (ACh) receptor channels that are each functionally distinct from the embryonic receptor type present at early stages of differentiation. The embryonic receptor type has a low conductance (45 pS) and long channel open time, rendering slowly decaying synaptic currents. One fast channel type that appears during muscle maturation is distinguished from the embryonic receptor type on the basis of both higher conductance (65 pS) and shorter open time. However, single-channel recordings from differentiated mouse skeletal muscle cell line (C2) point to the existence of a second fast receptor type, which has a conductance similar to the embryonic receptor type (45 pS), yet significantly reduced mean channel open time. Analyses of individual channel function at high ACh concentrations directly demonstrate the coexistence of two kinetically distinct types of 45 pS ACh receptors. Openings by fast type and slow embryonic type of 45 pS receptors occurred in bursts, allowing distinction on the basis of both mean open time and open probability for individual receptors. The embryonic type of 45 pS receptor has an open time approximately twofold longer than the fast-receptor counterpart. Additional differences were reflected in the open probability distributions for fast and slow 45 pS receptor types. Both types of 45 pS receptor were kinetically distinguishable from the 65 pS receptor. We found no support for the idea that the slow and fast 45 pS receptor types result from the interconversion of dual gating modes involving the same receptor protein. Our results are consistent with the idea that the acquisition of fast synaptic current decay, required at mature neuromuscular synapses, is the result of the up-regulation of two distinct fast types of nicotinic ACh receptors during skeletal muscle development.

Animals↗
PubMed1997

Prostaglandins induce vascular endothelial growth factor in a human monocytic cell line and rat lungs via cAMP.

Prostaglandins have emerged as a therapeutic option for patients with peripheral vascular disease as well as pulmonary hypertension as a means to increase blood flow. We tested the hypothesis that prostaglandins regulate vascular endothelial growth factor (VEGF) expression in the human monocytic THP-1 cell line and in isolated perfused rat lungs. Our data show that the stable PGI2-analogue iloprost induces VEGF gene expression (predominantly VEGF121, but also VEGF165 isoforms) and VEGF protein synthesis in THP-1 cells. This effect is abolished by dexamethasone and by Rp-cAMP, a specific inhibitor of cAMP-dependent protein kinase (PKA) activation. The calcium channel blocker diltiazem has no effect on the iloprost-induced VEGF gene expression, and depletion of intracellular Ca2+ stores by long-term exposure (16 h) of THP-1 cells to thapsigargin does not inhibit iloprost-induced VEGF gene expression, suggesting that an increase in intracellular Ca2+ is not essential for VEGF gene induction by iloprost. However, an increase of intracellular Ca2+ by a short-term (2 h) exposure of THP-1 cells to thapsigargin or to the calcium-ionophore A23187 increases VEGF mRNA levels, indicating that a change in intracellular Ca2+ by itself can alter VEGF gene expression. The effects of thapsigargin or A23187 on VEGF gene expression are also mediated via cAMP-PKA since they are inhibited by Rp-cAMP. In isolated perfused rat lungs, PGI2 and PGE2 increases VEGF mRNA abundance whereas Rp-cAMP inhibits the prostaglandin-induced VEGF gene activation. Thus, our data suggest that prostaglandins stimulate VEGF gene expression in monocytic cells and in rat lungs via a cAMP-dependent mechanism.

Animals↗
PubMed1996

Central afferent projections of proprioceptive sensory neurons in Drosophila revealed with the enhancer-trap technique.

We have used a GAL4 enhancer-trap line coupled with an upstream activation sequence (UAS)-linked lacZ reporter construct to visualise and describe the central projections of proprioceptive sensory neurons of the thorax and abdomen in Drosophila. In the legs, lacZ expression is restricted to sensory neurons associated with hair plates, a subset of campaniform sensilla, and with the femoral chordotonal organ; whereas, in the wing, expression is seen only in subsets of campaniform sensilla. In the abdomen, expression is seen in Wheeler's organ and in a segmentally repeated array of internal sensory neurons that have not been previously described. The central projections from all of these neurons are described. The results confirm and expand upon our knowledge of the organisation of sensory neuropils in insects. The enhancer-trap technique provides a potentially powerful tool for describing the organisation of the central nervous system of Drosophila.

Afferent Pathways↗