COX-2: a link between airway inflammation and disordered chloride secretion in cystic fibrosis?
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Chlorides↗
Biomedical subjects
A Clayton
Publications and source records attributed to A Clayton.
The rationale for combined chemo/immunotherapy using a Toll-like receptor 3 (TLR3) agonist and tumour-derived exosomes in advanced ovarian cancer.
A clinical trial employing an immunotherapeutic approach based on the use of a Toll-like receptor 3 (TLR3) agonist and tumour-derived exosomes carrying tumour-associated antigens is planned in advanced ovarian cancer in conjunction with conventional first line chemotherapy. Most patients with ovarian cancer present with advanced disease and despite high initial response rate to chemotherapy the majority will relapse within 2 years with poor overall survival. Tumour antigen-specific T cells are naturally occurring in ovarian cancer patients and T cell infiltration of the tumour is highly prognostic. Novel immunotherapy to expand and activate tumour antigen-specific T cells combined with adjuvant treatment to overcome tumour-induced immunosuppression is considered to be therapeutically beneficial. The rationale for adopting such a combined approach is discussed here.
Animals↗
Interleukin-1beta, transforming growth factor-beta1, and bradykinin attenuate cyclic AMP production by human pulmonary artery smooth muscle cells in response to prostacyclin analogues and prostaglandin E2 by cyclooxygenase-2 induction and downregulation of adenylyl cyclase isoforms 1, 2, and 4.
Increased levels of inflammatory cytokines contribute to the pathophysiology of pulmonary hypertension. Prostacyclin (PGI2) analogues, which relax pulmonary vessels mainly through cAMP elevation, have a major therapeutic role. In this study, we show that prolonged incubation with bradykinin (BK), interleukin-1beta (IL-1beta), and transforming growth factor-beta1 (TGF-beta1) markedly impairs cAMP accumulation in human pulmonary artery smooth muscle cells in response to short-term incubation with prostaglandin E2 (PGE2) and the PGI2 analogues iloprost and carbaprostacyclin. A similar reduction in cAMP accumulation in response to a direct adenylyl cyclase activator, forskolin, suggested that the effect was attributable to downregulation of adenylyl cyclase. Reverse transcriptase-polymerase chain reaction studies showed downregulation of adenylyl cyclase isoforms 1, 2, and 4. The effect of IL-1beta, BK, and TGF-beta1 on cAMP levels was abrogated by the selective COX-2 inhibitor NS398. Furthermore, it was mimicked by prolonged incubation with the COX-2 product PGE2 and PGI2 analogues or the COX substrate arachidonic acid, suggesting that it was mediated by endogenous prostanoids produced by COX-2. Consistent with this, IL-1beta, BK, and TGF-beta1 all induced COX-2 and PGE2 release. These results show that BK, IL-1beta, and TGF-beta1 downregulate adenylyl cyclase in human pulmonary artery smooth muscle cells via COX-2 induction and prostanoid release. This suggests a novel mechanism whereby mediators and cytokines produced in pulmonary hypertension may impair the therapeutic effects of prostacyclin analogues such as iloprost and carbaprostacyclin.
Adenylyl Cyclases↗
Medical students' perceptions of sexual health issues prior to a curriculum enhancement.
The objectives were to compare first, second and third year medical students on self-perceived sexual health knowledge, comfort in addressing sexual health problems, and attitudes towards the importance of addressing sexual health issues with patients as part of a sexual health medical curriculum enhancement project. A paper-and-pencil questionnaire survey was designed and administered to first and second year medical students at the start of the fall semester, resulting in high participation rates for both years (98% and 86%, respectively). Third year students were surveyed through an on-line version of the questionnaire yielding a lower response rate (52%). Multivariate statistical analyses were used to compare knowledge, comfort and attitudes by year in medical school. Results were as follows: As might be expected, sexual health knowledge and comfort in addressing sexual health problems increased linearly from first to third year (P<0.01) for all questions. Unexpectedly, second year students had significantly higher scores on questions assessing attitudes towards the importance of addressing sexual health issues than either first or third year students (P<0.001). Female medical students reported that addressing sexual health issues with patients was significantly more important than did male medical students; however, male students reported higher levels of self-reported knowledge and comforting related to sexual health issues than did female students in a number of areas. In conclusion, knowledge gained from this survey was used to finalize the design of an enhanced, integrated curriculum on sexual health for medical students. Further investigation of gender differences related to training medical students in this area is suggested.
Attitude of Health Personnel↗
Induction of viral and tumour specific CTL responses using antibody targeted HLA class I peptide complexes.
The production of cytotoxic T cells with specificity for cancer cells is a rapidly evolving branch of cancer therapeutics. A variety of approaches aim to amplify anti-tumour cytotoxic T cell responses using purified peptides, tumour cell lysates or recombinant HLA/peptide complexes in differing antigen presenting systems. Using a two-step biotin-streptavidin antibody targeting system, recombinant HLA-class I/peptide complexes were attached to the surface of B cells via the anti-CD20 B9E9-scFvSA antibody-streptavidin fusion protein. Flow cytometry with a conformation dependant monoclonal antibody to HLA class I indicated that targeted HLA-class I/peptide complexes remain on the surface of B cells in culture for periods in excess of 72 h. PBMCs were stimulated in vitro for 8-14 days using the autologous B cells as antigen presenting cells. Following a single cycle of stimulation specific cytotoxic T cell responses to targeted HLA-A2 complexes containing the M1, BMLF1 and Melan A peptides could be demonstrated by tetramer staining and Cr release assays. With the HLA-A2/BMLF1 complex up to 2.99% of CD8+ve cells were tetramer positive producing 20% lysis (E : T 10 : 1) of CIR-A2 target cells in an in vitro cytotoxicity assay compared to baseline levels of 0.09% tetramer +ve and 2% lysis in the unstimulated population. PBMCs from a healthy donor treated with two cycles of stimulations with targeted HLA-A2/Melan A complexes, demonstrated expansion of the melanA tetramer +ve population from 0.03% to 1.4% producing 15% lysis of Melan A pulsed target cells. With further consideration to the key variables of HLA/peptide complex density, the ratio of stimulator to effector cells and optimum cytokine support, this system should offer an easy and effective method for the in vitro amplification of specific cytotoxic T cell responses and warrants development for the in vivo induction of cytotoxic T cell responses in cancer therapy.
Antibodies↗
Compound effects of point mutations causing campomelic dysplasia/autosomal sex reversal upon SOX9 structure, nuclear transport, DNA binding, and transcriptional activation.
Human mutations in the transcription factor SOX9 cause campomelic dysplasia/autosomal sex reversal. Here we identify and characterize two novel heterozygous mutations, F154L and A158T, that substitute conserved "hydrophobic core" amino acids of the high mobility group domain at positions thought to stabilize SOX9 conformation. Circular dichroism studies indicated that both mutations disrupt alpha-helicity within their high mobility group domain, whereas tertiary structure is essentially maintained as judged by fluorescence spectroscopy. In cultured cells, strictly nuclear localization was observed for wild type SOX9 and the F154L mutant; however, the A158T mutant showed a 2-fold reduction in nuclear import efficiency. Importin-beta was demonstrated to be the nuclear transport receptor recognized by SOX9, with both mutant proteins binding importin-beta with wild type affinity. Whereas DNA bending was unaffected, DNA binding was drastically reduced in both mutants (to 5% of wild type activity in F154L, 17% in A158T). Despite this large effect, transcriptional activation in cultured cells was only reduced to 26% in F154L and 62% in A158T of wild type activity, suggesting that a small loss of SOX9 transactivation activity could be sufficient to disrupt proper regulation of target genes during bone and testis formation. Thus, clinically relevant mutations of SOX9 affect protein structure leading to compound effects of reduced nuclear import and reduced DNA binding, the net effect being loss of transcriptional activation.
Abnormalities, Multiple↗
Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry.
We present a simple yet powerful method for the isolation and analysis of exosomes released by antigen-presenting cells (APC). Exosomes are small vesicles (40-90 nm) released by APC, and may have an immuno-regulatory function in vivo. Such exosomes originate from MHC class II peptide loading compartments and, as such, express high levels of MHC Class II. We have utilised magnetic beads, coated with monoclonal antibodies specific for HLA DP, DQ, DR for the specific isolation of exosomes from cell-free supernatants. Beads coated with exosomes are subsequently stained with conjugated antibodies, and analysed by flow cytometry. Characterisation of exosomes by this method demonstrated that exosomes derived from B-lymphocytes express abundant MHC Class I and II molecules. Other immunologically important molecules detected included the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86). The adhesion molecule ICAM-1 (CD54) was also detected. These exosomes also expressed the B cell marker CD20, and the complement inhibitory protein CD59. The expression of CD63, a lysosomal marker, was variable, and there was no detectable expression of transferrin receptor (CD71). Monocyte derived dendritic cells (cultured for 7 days in GM-CSF/IL-4), demonstrated an immature phenotype, and secreted exosomes with a similar phenotype, with abundant MHC molecules. The expression of CD63 was consistently strong, and the MHC Class I-like molecule CD1a was also present, suggesting a possible function in the presentation of lipid antigens. Again CD59 was expressed suggesting a possible role for APC exosomes in complement regulation. There was no detectable CD71, CD40, CD14, CD20 or CD83. Modification of the extraction protocol allowed a comparative analysis of exosome secretion under various conditions. Treatment of cells with calcium ionophore, or phorbol ester resulted in apparent increases in exosome release, while the phosphatidyl inositol 3-kinase inhibitor, wortmannin, reduced exosome secretion. The immuno-magnetic isolation and analysis of exosomes is a versatile and rapid tool for the analysis of APC exosomes, and may prove a valuable tool for the study of exosome biology.
Androstadienes↗
Cell surface heparan sulfate proteoglycans control the response of renal interstitial fibroblasts to fibroblast growth factor-2.
BACKGROUND: While the progression of renal disease to end stage is strongly correlated with tubulointerstitial changes, the control of the fibrotic process within the interstitium is poorly understood. Basic fibroblast growth factor (FGF-2) has been implicated as a major growth factor involved in fibroblast activation and extracellular matrix synthesis. Furthermore, in many cells, the activity of FGF-2 is controlled by a low-affinity but high-capacity interaction with heparan sulfate (HS) proteoglycans (PGs), such as members of the syndecan family. These molecules are likely to be central to the control of interstitial fibrosis, but as yet, there has been no characterization of their synthesis by interstitial cells. METHODS: The expression of HSPG on the surface of NRK 49F fibroblasts was demonstrated by immunohistochemistry and by metabolic labeling with [(35)S]-sulfate. HSs were characterized by specific enzymatic digestion, size exclusion chromatography, and anion exchange chromatography. The mRNA for syndecan 1 through syndecan 4 in the fibroblasts was detected by semiquantitative reverse transcription-polymerase chain reaction. Fibroblast proliferation was measured by the MTT assay. RESULTS: Immunohistochemistry and [(35)S]-sulfate-labeling demonstrated that renal fibroblasts expressed HSPGs on their surface. Furthermore, enzymatic removal of these HS (but not chondroitin sulfate) glycosaminoglycan (GAG) chains, or inhibition of GAG sulfation, abolished the proliferative response of both NRK cells and primary human cortical fibroblasts to FGF-2 but not to platelet-derived growth factor. The addition of conditioned medium, containing HS-GAG fragments, restored the proliferative response to FGF-2, confirming the specificity of the interaction. Finally, the mRNA for all four syndecans was detected in the fibroblasts, and that for syndecan 1 in particular was up-regulated by FGF-2. CONCLUSIONS: The present study demonstrates that the expression of cell surface HSPG was essential for the proliferation of renal fibroblasts in response to FGF-2, and therefore may play a major role in the development and persistence of a proliferating phenotype during interstitial nephritis.
Animals↗
Development and clinical assessment of an artificial cornea.
Keratoprosthesis research has been a gradual, rather fragmentary process with advances being made by isolated groups of researchers. This has arisen partly because of poor funding in the area; research groups which have achieved commercial support have often had constraints upon the full disclosure of their findings. Despite these difficulties there has been real progress over the last decade by several independent groups. This article concentrates upon our own development of a hydrogel core-and-skirt keratoprosthesis, the Chirila KPro, in order to illustrate the scientific and clinical problems common to keratoprosthesis research. Pilot data from a clinical trial is presented and the priorities for future research are discussed.
Animals↗
Deliberate self-harm and antidepressant drugs. Investigation of a possible link.
BACKGROUND: It is not clear if the frequency of deliberate self-harm (DSH) is the same in patients taking different pharmacological classes of antidepressant drugs. AIMS: To compare the frequency of DSH in patients who had been prescribed a tricyclic antidepressant (TCA) or a selective serotonin reuptake inhibitor (SSRI) prior to the DSH event. METHOD: This was a prospective study in 2776 consecutive DSH cases attending an accident and emergency department. The incidence of DSH in TCA-treated cases and SSRI-treated cases is expressed as number of DSH events per 10 000 prescriptions of each antidepressant. RESULTS: Significantly more DSH events occurred following the prescription of an SSRI than that of a TCA (P<0.001). The occurrence of DSH was highest with fluoxetine and lowest with amitriptyline. CONCLUSIONS: Merely prescribing safer-in-overdose antidepressants is unlikely to reduce the overall morbidity from DSH.
Adolescent↗
A pilot study with simvastatin and folic acid/vitamin B12 in preparation for the Study of the Effectiveness of Additional Reductions in Cholesterol and Homocysteine (SEARCH).
BACKGROUND AND AIM: This study was conducted in preparation for the Study Evaluating Additional Reduction in Cholesterol and Homocysteine (SEARCH). SEARCH is a 12,000 patient 2X2 factorial study in post-myocardial infarction patients that will compare simvastatin 20 mg with simvastatin 80 mg to evaluate whether greater LDL-C reductions with simvastatin provide greater coronary event reductions. SEARCH will also test the hypothesis that lowering plasma homocysteine with folic acid and vitamin B12 will reduce coronary events. This pilot study was performed to determine whether any clinically meaningful interaction between simvastatin and folic acid/vitamin B12 exists. METHODS AND RESULTS: Following a 2-week diet/placebo run-in period, 141 patients with primary hypercholesterolaemia were randomised to one of three treatments for 6 weeks: 80 mg/day simvastatin and 2 mg folic acid/0.8 mg vitamin B12 daily (combination group); or 80 mg/day simvastatin and placebo vitamins (simvastatin alone group); or 2 mg folic acid/0.8 mg vitamin B12 daily and placebo simvastatin (vitamins alone group). The combination group and simvastatin alone group experienced similar serum lipid changes with reductions in LDL-cholesterol of 55.2% and 51.5% respectively. The combination group and vitamins alone group experienced similar homocysteine lowering with reductions in homocysteine of 25.3% and 23.1% respectively. All therapies were well tolerated. CONCLUSIONS: There was no detectable antagonistic effect when simvastatin and folic acid/vitamin B12 were administered concomitantly.
Cholesterol↗
Compulsive voyeurism and exhibitionism: a clinical response to paroxetine.
The compulsive behaviors seen in sexual paraphilias may be related to those of obsessive-compulsive disorder (OCD). Based primarily upon case reports as well as studies indicating the effectiveness of serotonin reuptake inhibitors in the treatment of sexual paraphilias, it has been speculated that sexual paraphilias lie within the obsessive-compulsive spectrum. There have been no reports of the use of paroxetine in the treatment of sexual paraphilias. This is a report of two patients, the first a voyeur and the second an exhibitionist, both of whom responded to treatment with paroxetine. The discussion addresses the need for further comparative studies investigating the role of the serotonin system in the pathogenesis of sexual paraphilias and OCD as well as the effect of serotonin reuptake inhibitors in comparison to other pharmacologic modalities used in the treatment of paraphilias.
Adult↗
ICAM-1 interactions in the renal interstitium: a novel activator of fibroblasts during nephritis.
Chronic renal diseases often degenerate towards end-stage failure, requiring replacement renal therapy. The progressive decline of such diseases is a highly complex, multi-factorial process, which is poorly understood. Indeed, not all chronic conditions take on a progressive course, some may recover to regain normal function, while others may remain functionally impaired yet stable. The structural features of progressive decline, however, show common histological features, despite the diverse nature of the primary injury. These aberrant structural alterations are characterised essentially by a dramatic expansion of the tubulointerstitium, with accompanying tubular atrophy, resulting from interstitial fibrosis. These changes are thought to be a uniform response to prolonged inflammation which may originate in the glomerulus, the vasculature or the interstitial space (Strutz et al., 1995). A histomorphometric analysis of renal diseases, initially performed by Risdon et al. (1968), and supported by Bohle et al. (1987) and others (Eknoyan et al., 1990), revealed that the severity of abnormal glomerular pathology did not always correlate directly with impaired renal function. The extent of interstitial inflammation and the degree of interstitial fibrosis, however, were both shown to be more accurate predictors of renal function (Bohle et al., 1992). Furthermore there was a high probability of irreversible functional decline, in the presence of interstitial fibrotic lesions and tubular atrophy. Interstitial fibrosis is therefore considered an important histological marker for end stage renal failure, and is believed to be functionally more significant than primary changes within the glomerulus. In most tissues, resident fibroblasts are believed to be the cells principally responsible for the synthesis and breakdown of extracellular matrix (ECM) within connective tissues. Indeed in fibrotic diseases of lung and skin, the resident fibroblast has been identified as the most important cell responsible for the abnormal deposition of ECM components during the disease process (Phan et al., 1985). In the kidney, there are probably several sources of matrix components during fibrosis including tubular epithelial cells, inflammatory macrophages (Vaage and Linbland, 1990) as well as interstitial fibroblasts. Although the precise cellular source of the bulk of this matrix requires clarification, there is mounting evidence supporting a significant contribution from resident or infiltrating fibroblasts (Rodemann and Muller, 1990, 1991a,b; Strutz and Muller, 1995).
Animals↗
CD34+AC133+ cells isolated from cord blood are highly enriched in long-term culture-initiating cells, NOD/SCID-repopulating cells and dendritic cell progenitors.
The AC133 antigen is a novel antigen selectively expressed on a subset of CD34+ cells in human fetal liver, bone marrow, and blood as demonstrated by flow cytometric analyses. In this study, we have further assessed the expression of AC133 on CD34+ cells in hemopoietic samples and found that there was a highly significant difference between normal bone marrow and cord blood versus aphereses (p <0.0001) but not between bone marrow and cord blood. Most of the clonogenic cells (67%) were contained in the CD34+AC133+ fraction. Compared with cultures of the CD34+AC133- cells, generation of progenitor cells in long-term culture on bone marrow stroma was consistently 10- to 100-fold higher in cultures initiated with CD34+AC133+ cells and was maintained for the 8-10 weeks of culture. Only the CD34+AC133+ cells were capable of repopulating NOD/SCID mice. Human cells were detectable as early as day 20, with increased levels (67%) apparent 40 days post-transplantation. Five thousand CD34+AC133+ cells engrafted about 20% of the mice, while no engraftment was observed in animals transplanted with up to 1.2 x 10(5) CD34+AC133- cells. The CD34+AC133+ population was also enriched (seven-fold) in dendritic cell precursors, and the dendritic cells generated were functionally active in a mixed lymphocyte reaction assay. AC133+ cells should be useful in the study of cellular and molecular mechanisms regulating primitive hemopoietic cells.
AC133 Antigen↗
Cellular activation through the ligation of intercellular adhesion molecule-1.
In addition to its role in mediating leukocyte adherence to and migration across the endothelium, intercellular adhesion molecule (ICAM)-1 on the surface of interstitial cells has been implicated as a principal adhesion molecule controlling leukocyte infiltration at inflammatory sites. The present study demonstrates that leukocyte binding to fibroblasts isolated from both the human renal cortex and lung and to endothelial cells induced the de novo synthesis of ICAM-1 mRNA and protein through the ICAM-1-dependent activation of the cultured cells. This was mimicked by specifically cross-linking the ICAM-1 receptor with anti-ICAM-1 antibodies. Following cross-linking there was a two- to threefold increase in the steady state levels of ICAM-1 mRNA isolated from the cells. The expression of this mRNA peaked at 1-3 hours and was dose-dependent on the concentration of secondary cross-linking antibody. Peak protein expression was between 18 and 48 hours after cross-linking. Additional data demonstrated a similar increase in the expression of VCAM-1 following ICAM-1 cross-linking. In contrast, there was no reponse as a result of incubation with an isotype-matched control antibody. Both the binding of leukocytes and the cross-linking of ICAM-1 triggered a rise in cytosolic free calcium as the result, in part, of a calcium influx from the extracellular medium. Using BAPTA-AM to chelate intracellular calcium ions indicated that this increase in cytosolic free calcium was directly involved in the initiation of adhesion molecule upregulation. The present study demonstrates that both endothelial cells and fibroblasts can be stimulated by the direct cell surface interaction of ICAM-1 with its ligands on inflammatory cells. One phenotypic change resulting from this interaction is the upregulated synthesis and expression of more cellular adhesion molecules. This may have profound implications for the control and persistence of a leukocyte infiltrate and the progression of inflammatory disease.
Actins↗
Cells isolated from the human cortical interstitium resemble myofibroblasts and bind neutrophils in an ICAM-1--dependent manner.
Progressive renal disease is frequently accompanied by renal interstitial inflammation and fibrosis in which the activity of resident fibroblasts may be of central importance. Because there are relatively few fibroblasts in the normal cortical interstitium and there is no specific marker to permit their identification, these cells have proved difficult to characterize in vitro. In this study, these cells were isolated and established in culture, using CD90 as a positive selection marker. Antibodies to CD90 bound to tubular epithelial cells and fibroblasts, but not to glomerular cells in kidney sections. In culture, only fibroblasts were CD90-positive. These normal renal cortical fibroblasts (RCF) were alpha-smooth muscle actin- and vimentin-positive, but desmin-, cytokeratin-, and factor VIII-negative, identifying them as myofibroblasts. They expressed platelet-derived growth factor alpha and beta receptors; CD44; and alpha 2, beta 1, and beta 3 integrin chains: this combination of markers was also characteristic of fibroblasts in sections of normal cortex. These cells were positive for ICAM-1 but negative for VCAM-1. Similarly, proliferating or growth-arrested renal cortical fibroblasts (RCF) in culture expressed ICAM-1 but not VCAM-1. The expression of VCAM-1 was detected, however, and that of ICAM-1 was increased on fibroblasts associated with inflammatory infiltrates in sections from fibrotic kidneys, and ICAM-1 and VCAM-1 were up-regulated on RCF in culture after incubation with increasing doses of interleukin-1 beta or tumor necrosis factor alpha (maximum between 24 and 48 h). These adhesion molecules were functional, and neutrophils adhered to resting and cytokine-activated RCF. Binding was maximal between 24 and 48 h after cytokine treatment and was inhibited by anti-CD18 antibodies. ICAM-1 is the principal adhesion molecule controlling inflammatory cell infiltration of the interstitium. The study presented here suggests that cortical fibroblasts may be central to the control of this infiltration.
Cells, Cultured↗
IDNDR conference: protecting vulnerable communities, the Royal Society and the Institution of Civil Engineers, London, 13-15 October 1993.
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Disaster Planning↗
Fluoxetine and nortriptyline combination therapy.
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Adult↗