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Biomedical subjects

M J Evans

Publications and source records attributed to M J Evans.

At least 37 records · Page 2Linked to original sources
PubMed2001

Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disordered macrophage inflammatory responses and reduced clearance of the pathogen, Staphylococcus aureus.

Tartrate-resistant acid phosphatase (TRAP) is a lysosomal di-iron protein of mononuclear phagocytes and osteoclasts. Hitherto, no role for the enzyme in immunity has been identified; however, knockout mice lacking TRAP have a skeletal phenotype caused by an intrinsic osteoclast defect. To investigate a putative function for TRAP in macrophages (Mphi), we investigated proinflammatory responses and systemic microbial clearance in knockout mice compared with age- and gender-matched congenic wild-type mice. Phorbol 12-myristate 13-acetate (PMA)-stimulated and interferon-gamma (IFN-gamma)-induced superoxide formation was enhanced in peritoneal Mphi lacking TRAP; nitrite production in response to stimulation with lipopolysaccharide (LPS) and IFN-gamma was also increased. In addition, secretion of the proinflammatory cytokines, tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta and IL-12, was significantly greater in TRAP-deficient Mphi when stimulated with LPS, with or without addition of either TNF-alpha or IFN-gamma. The activity of tartrate-sensitive (lysosomal) acid phosphatase was increased in Mphi from the knockout mice but activities of the lysosomal hydrolases N-acetyl beta-glucosaminidase and acid beta-glucuronidase were unchanged, indicating selective activation of compensatory acid phosphatase activity. Evidence of impaired Mphi function in vivo was obtained in TRAP knockout mice, which showed delayed clearance of the microbial pathogen, Staphylococcus aureus, after sublethal intraperitoneal inoculation. After microbial challenge, peritoneal exudates obtained from TRAP knockout mice had a reduced population of Mphi. As peritoneal Mphi and neutrophils lacking TRAP were able to phagocytose and kill S. aureus normally in vitro, TRAP may directly or indirectly influence recruitment of Mphi to sites of microbial invasion. Our study shows that TRAP participates in the inflammatory response of the Mphi and influences effector signalling pathways in innate immunity.

Acid Phosphatase↗
PubMed2001

Prior exposure to aged and diluted sidestream cigarette smoke impairs bronchiolar injury and repair.

The bronchiolar injury/repair response to naphthalene (NA) in mice includes acute distal airway epithelial injury that is followed by epithelial proliferation and redifferentiation, which result in repair of the epithelium within 14 days. To test whether prior exposure to aged and diluted sidestream cigarette smoke (TS) would alter the injury/repair response of the airway epithelium, adult mice were exposed to either filtered air (FA) or smoke for 5 days before injection with either corn oil carrier (CO) or naphthalene. Mice were killed 1 and 14 days after naphthalene injury. Lung and lobar bronchus were examined and measured using high-resolution epoxyresin sections. The control group (FACOFA) that was exposed to filtered air/corn oil/filtered air contained airway epithelium similar to untreated controls at all airway levels. The group exposed to tobacco smoke/corn oil/filtered air (TSCOFA) contained some rounded cells in the small airways and some expansion of the lateral intercellular space in the larger airways. Necrotic or vacuolated cells were not observed. As expected, the epithelium in the group exposed to filtered air/naphthalene/filtered air (FANAFA) contained many light-staining vacuolated Clara cells and squamated ciliated cells within distal bronchioles during the acute injury phase. Repair (including redifferentiation of epithelial cells and restoration of epithelial thickness) was nearly complete 14 days after injury. The extent of Clara cell injury, as assessed in lobar bronchi, was not different between the four groups. Although the FANAFA group contained greater initial injury in the distal airways at 1 day, the group exposed to tobacco smoke/naphthalene/filtered air (TSNAFA) had the least amount of epithelial repair at 14 days after naphthalene treatment; many terminal bronchioles contained abundant squamated undifferentiated epithelium. We conclude that tobacco smoke exposure prior to injury (1) does not change the target site or target cell type of naphthalene injury, since Clara cells in terminal bronchioles are still selectively injured; (2) results in slightly diminished acute injury from naphthalene in distal bronchioles; and (3) delays bronchiolar epithelial repair.

Acute Disease↗
PubMed2001

Inflammation in cystic fibrosis airways: relationship to increased bacterial adherence.

It is unclear whether inflammation in the cystic fibrosis (CF) lung relates predominantly to bacterial infection, or occurs as a direct consequence of mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. Interleukin (IL)-8 secretion from CF and non-CF cell lines, and from CF and non-CF human primary nasal epithelial cells incubated with or without Pseudomonas aeruginosa, was measured. Activation of nuclear factor-kappaB (NF-kappaB) in unstimulated CF and non-CF nasal epithelial cells, cell lines and murine tissues was measured by gel-shift assays. No significant difference in basal IL-8 production or NF-kappaB activation was observed between CF and non-CF primary nasal cells. However, CF cells exhibited a significantly (p<0.01) increased IL-8 secretion following P. aeruginosa stimulation. Equalization of the increased P. aeruginosa adherence observed in CF cells, to non-CF levels, resulted in comparable IL-8 secretion. Further, IL-8 production did not differ with mutations which result in either correctly localized CFTR, or in partial/total mislocalization of this protein. Similar levels of NF-kappaB activation were observed in a number of organs of wildtype and CF mice. Finally, IL-8 secretion and NF-kappaB activity were not consistently increased in CF cell lines. Cos-7 cell transfection with plasmids expressing deltaF508 or G551D mutant CFTR protein resulted in increased activation of a p50-containing NF-kappaB complex, but IL-8 secretion was similar to wild-type cells. The authors conclude that the stimulus produced by Pseudomonas aeruginosa is the predominant inflammatory trigger in their models.

Adolescent↗
PubMed2001

In vitro-activated human lupus T cells express normal estrogen receptor proteins which bind to the estrogen response element.

We have shown that estrogen receptor (ERalpha, ERbeta) transcripts are expressed in SLE and normal T cells. In this study, T cell nuclear extracts from female lupus patients and normal donors were tested for biologically active ER proteins capable of binding to the human estrogen response element (hERE) by electrophoretic mobility shift assays. When peripheral blood T cells were stimulated with 17beta-estradiol (E2), PMA and ionomycin, two major retarded bands in T cell nuclear extracts exhibited a migration pattern similar to slow migrating protein-ERE complexes in human breast cancer cell extracts. T cells cultured only with E2 did not have these complexes. The formation of the complexes was inhibited by competition with the hERE cold oligonucleotide and partially with anti-ERalpha antibodies. There was no notable difference in the migration pattern of ERE-binding proteins between the SLE and normal T cell extracts. Together, these results suggest that activated human T cells, whether lupus-derived or normal-derived, contain biologically active ERalpha proteins. Other factors may be responsible for differential sensitivity of lupus T cells to estrogen.

Adult↗
PubMed2001

Acetylcholine induces cytosolic Ca2+ mobilization in isolated distal colonic crypts from normal and cystic fibrosis mice.

In intestinal biopsies from cystic fibrosis (CF) patients acetylcholine fails to elicit a chloride secretion response, and this observation can be explained by a defect in the Ca2+ signalling pathway in CF secretory cells. We tested the hypothesis that in CF intestine, the generation of an intracellular Ca2+ signal upon cholinergic stimulation is absent. A transgenic CF mouse model was used. Electrical measurements on intact jejunum and unstripped colon were performed in Ussing chambers. Intact distal colonic crypts were isolated, and the intracellular Ca2+ concentration was monitored using the Ca2+-sensitive dye fura-2. Acetylcholine increased the short-circuit current generated by wild-type jejunum and colon, but failed to induce a response in CF tissues. Acetylcholine caused a transient elevation in the intracellular Ca2+ concentration in colonic crypts from both wild-type and CF mice; the amplitude and timing of the response in CF crypts was indistinguishable from that in wild-type crypts. The response to acetylcholine was also observed in the absence of extracellular calcium, indicating intracellular stores as the source from which the cytosolic Ca2+ concentration increased. We conclude that the absence of a cholinergically-induced secretory response in CF intestine is not due to a defect in the generation of a Ca2+ signal in intestinal cells upon cholinergic stimulation.

Acetylcholine↗
PubMed2001

Overlapping functions of lysosomal acid phosphatase (LAP) and tartrate-resistant acid phosphatase (Acp5) revealed by doubly deficient mice.

To date, two lysosomal acid phosphatases are known to be expressed in cells of the monocyte/phagocyte lineage: the ubiquitously expressed lysosomal acid phosphatase (LAP) and the tartrate-resistant acid phosphatase-type 5 (Acp5). Deficiency of either acid phosphatase results in relatively mild phenotypes, suggesting that these enzymes may be capable of mutual complementation. This prompted us to generate LAP/Acp5 doubly deficient mice. LAP/Acp5 doubly deficient mice are viable and fertile but display marked alterations in soft and mineralised tissues. They are characterised by a progressive hepatosplenomegaly, gait disturbances and exaggerated foreshortening of long bones. Histologically, these animals are distinguished by an excessive lysosomal storage in macrophages of the liver, spleen, bone marrow, kidney and by altered growth plates. Microscopic analyses showed an accumulation of osteopontin adjacent to actively resorbing osteoclasts of Acp5- and LAP/Acp5-deficient mice. In osteoclasts of phosphatase-deficient mice, vacuoles were frequently found which contained fine filamentous material. The vacuoles in Acp5- and LAP/Acp5 doubly-deficient osteoclasts also contained crystallite-like features, as well as osteopontin, suggesting that Acp5 is important for processing of this protein. This is further supported by biochemical analyses that demonstrate strongly reduced dephosphorylation of osteopontin incubated with LAP/Acp5-deficient bone extracts. Fibroblasts derived from LAP/Acp5 deficient embryos were still able to dephosphorylate mannose 6-phosphate residues of endocytosed arylsulfatase A. We conclude that for several substrates LAP and Acp5 can substitute for each other and that these acid phosphatases are essential for processing of non-collagenous proteins, including osteopontin, by osteoclasts.

Acid Phosphatase↗
PubMed2000

Eomesodermin is required for mouse trophoblast development and mesoderm formation.

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior-posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.

Amino Acid Sequence↗
PubMed2000

Design of a self-calibrating simulated acoustic emission source.

The use of conical piezoelectric transducers as point acoustic sources has been investigated. It has been shown that transducers based on a design originally developed at the National Institute for Standards and Technology in the USA can be used as point transmitters over the frequency range of interest in acoustic emission measurements (100 kHz to around 1 MHz). They should, therefore, be suitable for use in experiments to calibrate structures so that acoustic emission source strengths can be determined. It has also been shown that measurements of the response of the transmitting transducer backing can be used to assess the coupling efficiency, and hence to remove concerns about inconsistent coupling affecting the calibration measurements. The results indicate that the variation of the backing response with coupling is due to a shift in the resonance frequencies of the transducer with the mechanical load impedance. If other transducers can be shown to behave in a similar fashion this effect could be used to measure coupling in standard acoustic emission and ultrasonic transducers.

Calibration↗
PubMed2000

A murine tracheal culture system to investigate parameters affecting gene therapy for cystic fibrosis.

Cystic fibrosis (CF) is a life-threatening condition caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Delivery of the CFTR gene to the airways offers a potential treatment for CF but requires improvement in efficiency to obtain clinical benefit. We have developed a murine tracheal culture system that maintains tissue integrity as judged by normal histological appearance, high transepithelial resistance and electrophysiological responses similar to fresh tissue. This ex vivo system allows precise control of gene delivery parameters to a structure that retains the in vivo cellular architecture. We have demonstrated correction of CFTR-dependent Cl- secretion following ex vivo delivery of the CFTR gene to tracheas from CF null mice. We have used this system to examine parameters affecting liposome-mediated gene delivery to the upper airway such as plasmid dose. We have also found that a contact time of 1 min for the transfection mixture is sufficient to achieve significant DNA binding and maximal reporter gene expression.

Animals↗
PubMed2000

Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis.

The major cause of mortality in patients with cystic fibrosis (CF) is lung disease. Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene product in the airways is a potential treatment. Clinical studies in which the CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in modest, transient gene expression. It seems likely that repeated administration of the gene transfer vector will be required for long-term gene expression. We have undertaken a double-blinded study in which multiple doses of a DNA/liposome formulation were delivered to the nasal epithelium of CF patients. Ten subjects received plasmid DNA expressing the CFTR cDNA complexed with DC-Chol/DOPE cationic liposomes, whilst two subjects received placebo. Each subject received three doses, administered 4 weeks apart. There was no evidence of inflammation, toxicity or an immune response towards the DNA/liposomes or the expressed CFTR. Nasal epithelial cells were collected 4 days after each dose for a series of efficacy assays including quantitation of vector-specific DNA and mRNA, immunohistochemistry of CFTR protein, bacterial adherence, and detection of halide efflux ex vivo. Airway ion transport was also assessed in vivo by repeated nasal potential difference (PD) measurements. On average, six of the treated subjects were positive for CFTR gene transfer after each dose. All subjects positive for CFTR function were also positive for plasmid DNA, plasmid-derived mRNA and CFTR protein. The efficacy measures suggest that unlike high doses of recombinant adenoviral vectors, DNA/liposomes can be successfully re-administered without apparent loss of efficacy.

Adolescent↗
PubMed2000

Interleukin-1 potentiates basal and AVP-stimulated ACTH secretion in vitro--the role of CRH pre-incubation.

The acute-phase cytokine interleukin-1 (IL-1) is known to activate the hypothalamic pituitary adrenal axis, primarily via corticotropin releasing hormone (CRH). The aim of this study was to determine whether IL-1beta could directly stimulate ACTH secretion from perifused equine anterior pituitary cells, and whether CRH pre-incubation affected corticotroph responsiveness. Isolated equine anterior pituitary cells were pre-incubated with media containing 10 nM CRH or vehicle for 20 hours before being loaded onto columns and perifused with 0.02 nM CRH and 100 nM cortisol. Columns were given a 5-minute pulse of arginine vasopressin (AVP, 10 nM), perifused for 4 hours with 0 (control) or 1 nM IL-1beta, then given a further 5-minute pulse of AVP (10nM). ACTH was measured in 5 minute fractions. In the setting of CRH pre-incubation, cells perifused with IL-1beta for 4 hours showed increased basal ACTH secretion compared to control (114 +/- 6 pM vs. 86 +/- 4 pM [means +/- S.E.M.], p < 0.001) and a significantly greater ACTH response to the final AVP pulse (240 +/- 32% vs. 96 +/- 30%, p = 0.009, expressed as % of ACTH response to the initial AVP pulse). The potentiation of AVP-stimulated ACTH release by IL-1 was not observed in cells pre-incubated with vehicle alone. In conclusion, IL-1 increases ACTH release in equine corticotroph cells pre-incubated with CRH and potentiates responsivity to AVP.

Adrenocorticotropic Hormone↗
PubMed2000

Genistein activates CFTR-mediated Cl(-) secretion in the murine trachea and colon.

The action of the isoflavone genistein on the cystic fibrosis transmembrane conductance regulator (CFTR) has been studied in many cell systems but not in intact murine tissues. We have investigated the action of genistein on murine tissues from normal and cystic fibrosis (CF) mice. Genistein increased the short-circuit current (I(sc)) in tracheal (16.4 +/- 2.8 microA/cm(2)) and colonic (40.0 +/- 4.4 microA/cm(2)) epithelia of wild-type mice. This increase was inhibited by furosemide, diphenylamine-2-carboxylate, and glibenclamide, but not by DIDS. In contrast, genistein produced no significant change in the I(sc) of the tracheal epithelium (0.9 +/- 1.1 microA/cm(2)) and decreased the I(sc) of colons from CF null (-13.1 +/- 2.3 microA/cm(2)) and DeltaF508 mice (-10.3 +/- 1.3 microA/cm(2)). Delivery of a human CFTR cDNA-liposome complex to the airways of CF null mice restored the genistein response in the tracheas to wild-type levels. Tracheas from DeltaF508 mice were also studied: 46% of trachea showed no response to genistein, whereas 54% gave an increase in I(sc) similar to that in wild type. We conclude that genistein activates CFTR-mediated Cl(-) secretion in the murine trachea and distal colon.

Animals↗
PubMed2000

Three-dimensional organization of the lamina reticularis in the rat tracheal basement membrane zone.

The airway basement membrane zone is a region specialized for the attachment of the epithelium with the matrix. The epithelium is attached to the lamina densa, which, in turn, is connected to types I and III collagen of the lamina reticularis with anchoring fibrils. The purpose of this study was to define the three-dimensional organization of the structural proteins of the lamina reticularis in the rat trachea. We approached this problem by using whole mounts to look down on the flat surface of the basement-membrane zone rather than a cross section of its thin profile. Fluorescent microscopy with long working distance water immersion objectives and scanning electron microscopy revealed that the structural proteins are arranged as a mat of large fibers oriented along the longitudinal axis of the airway. Smaller fibers are crosslinked with the larger fibers to complete this structure. Other small fibers are oriented around the large fibers and an amorphous material covers individual fibers. The large fibers oriented along the longitudinal axis of the airway are consistent with prior descriptions of fibers composed of collagen III with some collagen I and V; small fibers encircling the large fibers may be collagen VI. The crosslinking fibers are made up of elastin and probably elastin-associated microfibrils. The amorphous proteins covering the fibrous framework may contain proteoglycans and other nonstructural proteins reported to be in the lamina reticularis. The present studies demonstrate that the structural proteins of the lamina reticularis in the rat trachea are arranged as fibers in a highly organized manner.

Animals↗
PubMed2000

Sodium channel blockers and uridine triphosphate: effects on nasal potential difference in cystic fibrosis mice.

Sodium channel inhibitors block the enhanced Na+ reabsorption in cystic fibrosis (CF). Extracellular nucleotides facilitate Cl- secretion via Ca2+ gated Cl- channels. A combination of these effects may produce less viscid secretions in CF which are easier to expectorate. This study examined the effects of combining sodium channel blockers with uridine triphosphate (UTP) on nasal membrane potential difference (PD) in CF insertional null mutant mice (cftr(tm1HGU)), deltaF508 homozygous mice (cftr(tm1Cam)) and matched control animals. Median basal PD in the insertional CF mice and deltaF508 CF mice were -28 and -34 mV respectively. These values were significantly different to the control animals (-20 mV). Amiloride and loperamide reduced the PD in cftr(tm1HGU) CF mice (deltaPD 13 mV & 15 mV respectively) suggesting Na+ blockade. The subsequent addition of UTP in a chloride-free vehicle increased the PD (deltaPD -8- -12.5 mV). DeltaF508 mice showed significantly greater responses compared with CF insertional null mutant mice (p<0.05). The action of UTP was brief and not prolonged by the addition alpha-beta-methylene-adenosine 5' diphosphate. Suramin, a competitive antagonist of P2 purinoceptors blocked the action of UTP. In conclusion, this study demonstrated dose dependant nasal membrane potential changes in differences mice with uridine triphosphate in the presence of sodium channel blockers suggestive of chloride secretion. More stable analogues of uridine triphosphate in combination with long acting sodium channel blockers such as loperamide may have therapeutic potential in cystic fibrosis.

Amiloride↗
PubMed2000

Interactions of CRH, AVP and cortisol in the secretion of ACTH from perifused equine anterior pituitary cells: "permissive" roles for cortisol and CRH.

To further elucidate the interaction of CRH, AVP and cortisol in the control of ACTH secretion, we used an in vitro perifusion model with dispersed equine anterior pituitary cells. To approximate the in vivo milieu in the horse, CRH was perifused continuously (at 0, 2 and 20 pmol/L) and 5-min pulses of AVP (0, 1, 3 and 10 nmol/L) were given every 30 min in the presence of 0 or 100 nmol/L cortisol. Total (baseline + incremental) ACTH secretion increased as both the CRH (p<0.001) and the AVP (p<0.001) concentration increased and interaction between CRH and AVP was significant (p=0.042). Cortisol reduced total ACTH secretion in the presence of 2 pmol CRH/L (p=0.001) but not 0 or 20 pmol CRH/L. For incremental ACTH there was interaction between CRH and AVP (p<0.0001), with increased secretion at higher concentrations, and no significant main effect of cortisol. There was significant (p=0.001) interaction between cortisol and CRH, with cortisol attenuating ACTH release at 0 pmol CRH/L (p=0.008), having no effect at 2 pmol CRH/L and potentiating it at 20 pmol CRH/L (p=0.026). We conclude that (1) CRH at high physiological levels has a "permissive" role in preventing the cortisol inhibition of the ACTH response to AVP, and (2) basal cortisol levels have a "permissive" action in priming the HPA axis for maximal responsiveness to stimulated levels of CRH and AVP.

Adrenocorticotropic Hormone↗
PubMed1999

Human herpesviruses types 6 and 7 and febrile seizures.

The frequency was studied with which human herpesviruses types 6 and 7 (HHV-6 and HHV-7) occur in the cerebrospinal fluid (CSF) of patients with febrile seizures and matched control patients. CSF samples were prospectively collected from a case series of patients with febrile seizures and from age-, sex-, and race-matched control patients without febrile seizures, all of whom were evaluated in the emergency department of an urban, tertiary care, pediatric medical center. Using polymerase chain reaction, the samples were examined for the presence of viral DNA from HHV-6, HHV-7, herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), and cytomegalovirus (CMV). CSF from a subset of both groups was also examined for RNA from enteroviruses. During the 7-month, 2-week collection period, a total of 174 patients were evaluated for fever and seizures. Of these, 23 (13.2%) met the study criteria. Their mean age was 1.4 +/- 0.7 years. Sixteen (70%) of the 23 were male. The 23 patients were matched to 21 control subjects. None of the samples from the patients or control subjects had polymerase chain reaction evidence of HHV-6, HHV-7, HSV-1, or HSV-2. All samples from the patients were negative for CMV. One control subject was positive for CMV. The 10 patients and seven control subjects tested for enteroviral RNA were negative. Neither HHV-6 nor HHV-7 appears to be present in the CSF of patients with febrile seizures. What role, if any, they have in the pathogenesis of febrile seizures merits further study.

Case-Control Studies↗
PubMed1999

Mml, a mouse Mix-like gene expressed in the primitive streak.

A mouse Mix-like gene, Mml, related to the Xenopus Mix/Bix homeobox gene family and the chick CMIX gene has been identified. At E5.5, Mml is expressed symmetrically in the visceral endoderm but by E6.0 this expression is noticeably asymmetric. At E6.5, expression is restricted to the nascent primitive streak. Mml expression persists in the primitive streak through E7.5-E9.5, marking those cells fated to form extra-embryonic and lateral mesoderm.

Amino Acid Sequence↗