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Biomedical subjects

D P Hughes

Publications and source records attributed to D P Hughes.

18 recordsLinked to original sources
PubMed2006

Hairworm anti-predator strategy: a study of causes and consequences.

One of the most fascinating anti-predator responses displayed by parasites is that of hairworms (Nematomorpha). Following the ingestion of the insect host by fish or frogs, the parasitic worm is able to actively exit both its host and the gut of the predator. Using as a model the hairworm, Paragordius tricuspidatus, (parasitizing the cricket Nemobius sylvestris) and the fish predator Micropterus salmoïdes, we explored, with proteomics tools, the physiological basis of this anti-predator response. By examining the proteome of the parasitic worm, we detected a differential expression of 27 protein spots in those worms able to escape the predator. Peptide Mass Fingerprints of candidate protein spots suggest the existence of an intense muscular activity in escaping worms, which functions in parallel with their distinctive biology. In a second step, we attempted to determine whether the energy expended by worms to escape the predator is traded off against its reproductive potential. Remarkably, the number of offspring produced by worms having escaped a predator was not reduced compared with controls.

Animals↗
PubMed2004

Tiny genomes and endoreduplication in Strepsiptera.

Using flow cytometry, the genome sizes of two species of Strepsiptera were studied: that of male Caenocholax fenyesi texensis Kathirithamby & Johnston (Myrmecolacidae) at 108 Mb, which is the smallest insect genome documented to date; and those of male and female Xenos vesparum Rossi (Stylopidae), which are 1C = 130 and 133 Mb, respectively. The genome sizes of the following were analysed for comparative purposes: (a) the Hessian fly, Mayetiola destructor (Say), which was previously reported to be the smallest among insects: the male measured at 1C = 121 Mb and the female at 1C = 158 Mb; and (b) the female parasitic, haplodiploid, microhymenopteran wasp, Trichogramma brassicae Bezdenko, which measured at 1C = 246 Mb. The hosts of the strepsipterans were also measured: male Solenopsis invicta Buren, the red imported fire ant (host of male C. f. texensis), which is 1C = 753.3 Mb, and female Polistes dominulus Christ, the paper wasp (host of X. vesparum), is 1C = 301.4 Mb. Endoreduplication (4C) of the genome of the thorax of the male strepsipteran, and higher levels of endoduplication (4, 8, 16C) in the body of the larger female was observed. In contrast, little or no endoreduplication was observed, either in the Hessian fly, or in the parasitic wasp.

Animals↗
PubMed2001

Correction of DiGeorge anomaly with EBV-induced lymphoma by transplantation of organ-cultured thymus and Epstein-Barr-specific cytotoxic T lymphocytes.

A young woman with DiGeorge anomaly showed normal immune tests as a child and did not experience the symptoms of profound T cell immunodeficiency. However, she had chronic pulmonary infections which led to bronchiectasis. At age 14, she developed an Epstein-Barr virus-induced lymphoma and her T cell function tests were markedly abnormal. After intensive chemotherapy, she received an organ-cultured thymus transplant but because of an abnormally high EBV DNA titer was also given autologous EBV-specific cytotoxic T cells, prepared prior to transplant. Titers fell from 80,000 genome copies/mg DNA to 2000 within 6 weeks. She was clinically well and her T cell tests improved. Sixteen months after the transplant, however, her tumor returned; EBV DNA levels had risen to 40,000 copies/mg DNA. She again received autologous EBV-specific cytotoxic T lymphocytes and valcyclovir and Cytogam as well. Her tumor resolved on this therapy and she has remained well to this date, 29 months after the recurrence. T cell tests, which had deteriorated with the recurrence of the tumor, now show normal responses. This experience records a number of unique features of thymus transplantation. This is the first recorded successful thymus transplant in a patient with partial T cell immunity and thus expands the potential of this treatment modality to patients other than infants with complete DiGeorge anomaly. The patient demonstrates cytotoxic activity against mouse cells, demonstrating the ability to respond to a new antigen which requires host antigen presenting cells. Measurement of alpha 1 TRECs (T cell receptor excision circles) shows evidence of increasing and sustained thymopoiesis since the transplant at a level consistent with the age of the transplant donor rather than that of the recipient.

Biopsy↗
PubMed2000

Evidence for heterotypic interaction between the receptor tyrosine kinases TIE-1 and TIE-2.

The orphan receptor tyrosine kinase Tie-1 is expressed in endothelial cells and is essential for vascular development. Nothing is known about the signaling pathways utilized by this receptor. In this study we have used chimeric receptors composed of the TrkA ectodomain fused to the transmembrane and intracellular domains of Tie-1, or the related receptor Tie-2, to examine Tie-1 signaling capacity. In contrast to TrkA/Tie-2, the Tie-1 chimera was unable to phosphorylate cellular proteins or undergo autophosphorylation. Consistent with this Tie-1 exhibited negligible kinase activity. Co-immunoprecipitation analysis revealed Tie-1 was present in endothelial cells bound to Tie-2. Full-length Tie-1 and truncated receptor, formed by regulated endoproteolytic cleavage, were found to complex with Tie-2. Association was mediated by the intracellular domains of the receptors and did not require Tie-1 to be membrane-localized. Tie-1 bound to Tie-2 was not tyrosine-phosphorylated under basal conditions or following Tie-2 stimulation. This study provides the first evidence for the existence of a pre-formed complex of Tie-1 and Tie-2 in endothelial cells. The data suggest Tie-1 does not signal via ligand-induced kinase activation involving homo-oligomerization. The physical association between Tie-1 and Tie-2 is consistent with Tie-1 having a role in modulating Tie-2 signaling.

Cation Transport Proteins↗
PubMed2000

Tie-1 receptor tyrosine kinase endodomain interaction with SHP2: potential signalling mechanisms and roles in angiogenesis.

The endothelial receptor tyrosine kinase plays an essential role in vascular development where it is thought to be required for vessel maturation and stabilization. The ligands responsible for activating Tie-1, its signalling pathways and specific cellular functions are however not known. As with some other receptor tyrosine kinases, Tie-1 is subject to extracellular proteolytic cleavage generating a membrane bound receptor fragment comprising the intracellular and transmembrane domains. Here we examine the signalling potential of this Tie-1 endodomain. We show that the Tie-1 endodomain has poor ability to induce tyrosine phosphorylation. However, on formation the endodomain physically associates with a number of tyrosine phosphorylated signalling intermediates including the tyrosine phosphatase and adaptor protein SHP2. The assembly of this multimolecular complex is consistent with the endodomain having a ligand-independent signalling role in the endothelial cell. The potential roles of ectodomain cleavage and cleavage activated signalling in regulating microvessel stability in angiogenesis, vessel remodelling and regression are considered.

Animals↗
PubMed1999

An investigation into the distribution of different collagen types within adult and juvenile porcine pancreata.

One of the difficulties of porcine islet isolation is their fragility during collagenase digestion. The object of this study was to determine the distribution of 4 different collagen types within adult and juvenile porcine pancreata. Five different areas from each pancreas were analysed by light microscopy. The distribution of collagen types I, IV, V and VI were measured within the interlobar, intralobular (acinar), peri-insular and intra-islet regions. Their was an abundance of collagen type VI compared to I, IV and V in both the interlobar and intralobular septa in both juveniles (P<0.001) and adults (P<0.001). The periinsular collagen content also showed diversity. This was mainly attributable to the distribution of collagen type I (weak) and type VI (intense) in both adults and juveniles. In general, the peri-insular capsule was fragmentary and contained less than 50% of the total islet circumference. The latter finding taken together with the distributions of different collagen types can partly explain some of the variability of porcine islet isolation.

Animals↗
PubMed1999

A comparison of cross sectional surface area densities between adult and juvenile porcine islets of Langerhans.

The object of this study was to evaluate differences in islet diameters, their distribution and both cross sectional surface areas and densities of insulin containing islets between adult and juvenile porcine pancreata using a computerised image analysis system (Improvision). Five adult (A) (2-3 yrs) and 5 juvenile (J) (< 12 mths) Large White pancreata were assessed. Biopsies were taken from 5 different regions (posterior lobe, duodenal lobe, along with the head, body and tail regions of the splenic lobe) of the pancreas and tissue sections stained for insulin. In both A and J pancreata islet numbers increased with decreasing islet diameter, showing a skewed distribution. There was no statistical significance between the cross sectional surface area within A (mean 5.04 x 10(3) microm2) or J (mean 5.99 x 10(3) microm2) pancreata. Assuming islets are spherical, extrapolation from pir2 showed that the mean diameter for A was 80 microm and 87 microm in J. These compared with A 77 microm and J 86 microm diameters using conventional microscopic techniques. The percentage islet volume density relative to exocrine tissue, derived from the principle of Delesse (Area density = volume density), did not significantly differ between each of the 5 areas studied, either in A or J. The percentage islet volume densities did show a significant difference between A (mean 1.83%) (P = 0.001) but not between J pancreata (mean 2.13 %). In conclusion poor islet yields can be attributed to differences in islet volume density of islets within porcine pancreata. These results also suggest that the posterior and duodenal lobes should be used along with the splenic lobe in order to improve porcine islet yields. Furthermore, the current practise of reporting porcine islet yields and the isolation index relative to 150 microm (IEQs) needs to be redefined, based on the assumption that the average size of an adult porcine islet is 80 microm.

Animals↗
PubMed1997

Different treatment regimens to reduce microbial contamination of purified porcine islets of Langerhans.

BACKGROUND: Different treatment regimens for minimizing microbial contamination of purified porcine islets were analyzed. METHODS: Purified islets, prepared from abattoir-excised pancreata (n=26), were cultured in Hams F-12 with or without antibiotics after islet isolation. Aliquots of pancreas transport media were all contaminated with microbes (100%). Of those islets cultured with no antibiotics, 70% were contaminated, compared with 42% of the islets cultured in penicillin and streptomycin (P/S) (P>0.05). A further 20 consecutive pancreata were then randomized to saline washing (N=10) before islet isolation. Islets were then cultured in either Hams F-12 with P/S (groups C and E) or Hams F-12 with gentamicin, penicillin, and amphotericin B (G/P/A) (groups D and F). RESULTS: None of those purified islets prepared from washed pancreata and then undergoing culture in Hams F-12 with G/P/A (group F) were contaminated. This compared with 30% contamination when islets were cultured in P/S (group E). Neither antibiotic regimen compromised stimulated insulin release (P=0.13). CONCLUSIONS: In conclusion pancreas washing in saline and culture in G/P/A was shown to eradicate detectable microbial contamination of purified porcine islets isolated from abattoir-excised pancreata.

Animals↗
PubMed1996

Murine lupus in the absence of alpha beta T cells.

To investigate the possibility that non-alpha beta T cell-dependent mechanisms can induce systemic autoimmune disease, and to address the roles of alpha beta T cells in murine lupus, we analyzed lupus-prone MRL mice congenitally deficient in alpha beta T cells. Surprisingly, TCR-alpha-/- MRL mice developed several characteristics of human systemic lupus erythematosus, including hypergammaglobulinemia, autoantibodies against DNA and small nuclear ribonucleoproteins, and immune deposits in kidneys. These results, which contrast with past studies concluding that MRL autoimmunity requires CD4+ alpha beta T cells, demonstrate that non-alpha beta T cell-dependent mechanisms are capable of inducing lupus phenomena, and further suggest that MRL disease may consist of both alpha beta T cell-independent and alpha beta T cell-dependent mechanisms.

Animals↗
PubMed1996

Influence of different collagenase solvents and timing of their delivery on porcine islet isolation.

Methods to minimize the effect of cold ischaemia on porcine islet isolation were investigated. Forty pancreata were randomized to intraductal collagenase delivery in University of Wisconsin solution (UW) or Hanks balanced salt solution (HBSS) (control) both before and after 65 min of cold pancreas storage. Collagenase was also administered in a specially designed cold storage solution (University of Leicester solution, ULEIC), before cold storage. Median islet yield was significantly greater if the pancreas was distended with collagenase in either UW (21524 islet equivalents (IEQ) per g) or ULEIC (19814 IEQ/g) before cold storage, compared with that after distension with HBSS (6924 IEQ/g) following cold storage (P < 0.05). Islet fragmentation, islet purification and glucose-stimulated insulin release were not significantly different after collagenase delivery in either UW or ULEIC compared with those after administration in HBSS. It is concluded that porcine islet yields can be improved significantly by intraductal collagenase administration in either UW or ULEIC immediately after excision of the pancreas.

Adenosine↗
PubMed1995

A naturally occurring soluble isoform of murine Fas generated by alternative splicing.

We report a soluble isoform of mouse Fas, which is generated by alternative splicing of Fas mRNA to a newly identified exon located between exons 2 and 3 of the previously published Fas sequence. This splicing event creates a novel Fas transcript, Fas beta, with the potential to encode a truncated form of the extracellular domain, termed Fas B. In vitro, P815 mastocytoma cells transfected with Fas B become resistant to Fas ligand-induced apoptosis, and the resistance is mediated by a secreted product of the transfected cells. In vivo, Fas beta mRNA expression is correlated inversely with apoptosis among subsets of intrahepatic T lymphocytes, a cell population in which activation-induced T cell apoptosis occurs. We propose that Fas B is a new cytokine that acts physiologically to limit apoptosis induced by Fas ligand.

Amino Acid Sequence↗
PubMed1995

T cells with gamma/delta T cell receptors (TCR) of intestinal type are preferentially expanded in TCR-alpha-deficient lpr mice.

Fas-mediated apoptosis is essential for activation-induced cell death of alpha/beta T cells, but it is not clear what role, if any, it plays in regulating other components of the immune system. To study the role of Fas in gamma/delta T cell development, Fas-deficient lpr mice were bred with T cell receptor alpha gene-ablated (TCR-alpha-/-) mice to generate mice deficient in one or both genes. The TCR-alpha-/-, lpr/lpr mice had a nearly 10-fold increase in total lymph node cell (LNC) number compared with Fas-intact TCR-alpha-/- mice, because of expansion of TCR-gamma/delta+ and TCR-beta+ cells. In Fas-intact TCR-alpha-/- mice, approximately one third of the LNCs expressed TCR-gamma/delta. These were evenly divided between the CD4-, CD8-alpha+ and the CD4-, CD8- subsets, and rarely expressed the B220 epitope of CD45. In contrast, in TCR-alpha-/-, lpr/lpr mice, TCR-gamma/delta+ cells comprised half of the LNCs and were primarily CD4-, CD8-, and B220+. Moreover, Fas deficiency in TCR-alpha-/- mice caused a preferential expansion of gamma/delta T cells expressing variable region genes characteristic of intestinal intraepithelial lymphocytes. These results demonstrate a role for Fas in regulating the gamma/delta T cell contribution to peripheral lymph nodes. This mechanism may be most important in limiting the access of activated intestinal intraepithelial lymphocytes to the peripheral lymphoid system.

Animals↗
PubMedDate not supplied

The risks of total pancreatectomy and splenic islet autotransplantation.

The intraportal site is the most common site for islet transplantation. Many other sites have been tried experimentally, including the spleen, which has successfully lead to insulin independence in a number of animal models. Nevertheless, there are no detailed reports of total pancreatectomy and splenic islet autotransplantation in humans. Five patients underwent total pancreatectomy and splenic islet autotransplantation for chronic pancreatitis. Four patients had a pylorus-preserving total pancreatectomy and one patient a duodenal-preserving pancreatectomy. In three cases islets were embolized into both the portal vein and spleen. Two patients received splenic islet transplants alone. Islets were transplanted by retrograde venous infusion via the short gastric veins (n = 3), splenic vein stump (n = 1), and the left gastroepiploic vein (n = 1). The total volumes of transplanted pancreatic digest in those receiving combined intraportal and splenic autografts (n = 3) were 15.8, 13.0, and 13.5 ml. The volumes in those receiving a splenic-alone autograft (n = 2) were 12.0 and 5 ml. The mean rise in portal pressure was 18 cm of water. Complications related to the splenic autograft included a wedge splenic infarct, an emergency splenectomy, and a portal vein thrombosis in one patient having a combined intraportal and splenic autograft. Two patients developed insulin independence. two patients were still insulin independent at 1-year follow-up, and all had normal HbA1c levels (mean 5.6, range 5.2-6.3). Splenic islet autotransplantation, after total pancreatectomy, does lead to insulin independence. However, in our experience the combined procedure has a high morbidity because of splenic infarction and venous thrombosis.

Adult↗
PubMedDate not supplied

A preliminary study of the activation of endogenous pancreatic exocrine enzymes during automated porcine islet isolation.

The activation of endogenous pancreatic enzymes during automated pancreas digestion may be detrimental to islet isolation. In this report we assessed the activation of trypsin, chymotrypsin, elastase, carboxypeptidases A and B, phospholipase A2, and lipase using a porcine model. Four islet isolations were examined. Duplicate aliquots were taken from the automated circuit at 5-min time intervals up to the completion of pancreas digestion (approx 60 min). One aliquot was activated in vitro with exogenous trypsin in order to convert the enzymes into their active non-"proform," with the exception of trypsinogen, which was activated with exogenous enterokinase. This was done to assess the percentage activation of each individual enzyme (total potentially activatable enzyme release). The extent of activation between isolations was extremely variable. During the closed (recirculating) circuit phase of pancreas digestion there were both gradual and rapid increases in the levels of enzymes released. Peak activity of enzyme activation varied from 13 to 30 min; similarly, total potentially activatable peaks occurred between 13 and 38 min. Lipase and carboxypeptidase B showed greater than 70% activation, chymotrypsin, carboxypeptidase A, and phospholipase A2 between 50% and 70% activation, and trypsin and elastase less than 20%. There were up to 30-fold differences between the four islet preparations. In summary, it is unlikely that poor islet yields are soley explained by variations between collagenases; the variable activation of endogenous pancreatic exocrine enzymes is also likely to be influential to porcine islet yields.

Animals↗