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Biomedical subjects

K Stewart

Publications and source records attributed to K Stewart.

At least 19 recordsLinked to original sources
PubMed2003

MRI as a biomarker of disease progression in a therapeutic trial of milameline for AD.

OBJECTIVE: To assess the feasibility of using MRI measurements as a surrogate endpoint for disease progression in a therapeutic trial for AD. METHODS: A total of 362 patients with probable AD from 38 different centers participated in the MRI portion of a 52-week randomized placebo-controlled trial of milameline, a muscarinic receptor agonist. The therapeutic trial itself was not completed due to projected lack of efficacy on interim analysis; however, the MRI arm of the study was continued. Of the 362 subjects who underwent a baseline MRI study, 192 subjects underwent a second MRI 1 year later. Hippocampal volume and temporal horn volume were measured from the MRI scans. RESULTS: The annualized percent changes in hippocampal volume (-4.9%) and temporal horn volume (16.1%) in the study patients were consistent with data from prior single-site studies. Correlations between the rate of MRI volumetric change and change in behavioral/cognitive measures were greater for the temporal horn than for the hippocampus. Decline over time was more consistently seen with imaging measures, 99% of the time for the hippocampus, than behavioral/cognitive measures (p < 0.001). Greater consistency in MRI than behavioral/clinical measures resulted in markedly lower estimated sample size requirements for clinical trials. The estimated number of subjects per arm required to detect a 50% reduction in the rate of decline over 1 year are: AD Assessment Scale-cognitive subscale 320; Mini-Mental Status Examination 241; hippocampal volume 21; temporal horn volume 54. CONCLUSION: The consistency of MRI measurements obtained across sites, and the consistency between the multisite milameline data and that obtained in prior single-site studies, demonstrate the technical feasibility of using structural MRI measures as a surrogate endpoint of disease progression in therapeutic trials. However, validation of imaging as a biomarker of therapeutic efficacy in AD awaits a positive trial.

Adolescent↗
PubMed2003

IGF-I does not affect the proliferation or early osteogenic differentiation of human marrow stromal cells.

The ability of insulin-like growth factor-I (IGF-I) to regulate the proliferation and differentiation of primitive osteogenic precursors (CFU-F) has been investigated in cultures of bone marrow stromal cells (BMSC) derived from a large cohort of adult human donors. Treatment with IGF-I (0.1-20 ng/mL, days 0-28) had no consistent effect on the number or size of colonies that formed or the proportion of colonies that expressed the developmental marker alkaline phosphatase (AP). At the end of primary culture, similar numbers of cells were harvested from the control and IGF-I-treated groups and there was no detectable difference in the expression of AP (activity or percentage of positive cells) or the developmental marker STRO-1. This was found to be the case whether IGF-I was added alone or in combination with 10 nM dexamethasone (Dx), a known inducer of osteogenic differentiation in this cell culture system. In contrast, cells derived from the same cohort of donors responded to treatment with fibroblast growth factor-2 (FGF-2) with an increase in the number and size of the colonies that formed, in proliferation and in the number of cells recovered in STRO-1(+)/AP(+) (osteoprogenitor) fraction. Further analysis revealed that the majority of BMSC expressed the alpha and beta subunits of the type 1 receptor for IGF-I (IGF-IR), in the expected 1:1 ratio. Treatment with Dx did not affect the expression of these receptor subunits (percentage of positive cells or number of sites per cell) but did increase the proportion of cells present in the IGF-I(+)/AP(+) fraction. The results of this investigation suggest that the beneficial effects of IGF-I on the skeleton are not mediated primarily via an effect on osteoprogenitor fraction and are thus consistent with the hypothesis that the effects of IGF-I are differentiation dependent and restricted largely to the more mature cells of the osteoblast lineage.

Adolescent↗
PubMed2002

Active management of term prelabour rupture of membranes with oral misoprostol.

OBJECTIVE: To compare the active management of term prelabour rupture of membranes with oral misoprostol with conservative management for 24 hours followed by induction with oxytocin or prostaglandin E(2) (PGE(2)) gel. DESIGN: A non-blinded randomised controlled trial. SETTING: Induction and labour wards, Aberdeen Maternity Hospital. POPULATION: Sixty-one women with confirmed prelabour rupture of the membranes at > or =36 weeks of gestation. METHODS: The women were randomised to 50 microg of oral misoprostol repeated every 4 hours, if required, to a maximum of five doses (active group), or to induction of labour with PGE(2) gel or oxytocin only if not in spontaneous labour 24 hours after prelabour rupture of membranes (conservative group). MAIN OUTCOME MEASURES: Number of women in active labour within 24 hours of the prelabour rupture of membranes, preference of women for any one particular method of management in any subsequent pregnancy with prelabour rupture of membranes. RESULTS: 93.3% of the active group and 54.8% of the conservative group were in spontaneous labour within 24 hours of the prelabour rupture of membranes (RR 1.7, 95% CI 1.2 to 2.4). Of those achieving a vaginal delivery, 72% of the active group did so within 24 hours of the prelabour rupture of membranes as compared with 26.9% of the conservative group (RR 2.7, 95% CI 1.4 to 5.3, P = 0.002). There were no significant differences in the neonatal or maternal outcomes. In the active group, 78% felt they would have the same method of induction as compared with 40% in the conservative group (RR 1.9, 95% CI 1.1 to 3.3, P = 0.03). CONCLUSIONS: Active management with oral misoprostol resulted in more women going into labour and delivering within 24 hours of the prelabour rupture of membranes with no increase in maternal or neonatal complications. Women tended to view active management of prelabour rupture of membranes more positively. Oral misoprostol might be an option to consider in those wishing active management.

Administration, Oral↗
PubMed2001

Species specificity of amidine-based urokinase inhibitors.

Inhibition of the proteolytic activity of urokinase has been shown to inhibit the progression of tumors in rodent models and is being investigated for use in human disease. Understanding the rodent/human species-specificity of urokinase inhibitors is therefore critical for interpretation of rodent cancer progression models that use these inhibitors. We report here studies with a panel of 11 diverse urokinase inhibitors in both human and mouse enzymatic assays. Inhibitors such as amiloride, B428, and naphthamidine, that occupy only the S1 subsite pocket were found to be nearly equipotent between the human and the murine enzymes. Inhibitors that access additional, more distal, pockets were significantly more potent against the human enzyme but there was no corresponding potency increase against the murine enzyme. X-ray crystallographic structures of these compounds bound to the serine protease domain of human urokinase were solved and examined in order to explain the human/mouse potency differences. The differences in inhibitor potency could be attributed to four amino acid residues that differ between murine and human urokinases: 60, 99, 146, and 192. These residues are Asp, His, Ser, and Gln in human and Gln, Tyr, Glu, and Lys in mouse, respectively. Compounds bearing a cationic group that interacts with residue 60 will preferentially bind to the human enzyme because of favorable electrostatic interactions. The hydrogen bonding to residue 192 and steric considerations with residues 99 and 146 also contribute to the species specificity. The nonparallel human/mouse enzyme inhibition observations were extended to a cell-culture assay of urokinase-activated plasminogen-mediated fibronectin degradation with analogous results. These studies will aid the interpretation of in vivo evaluation of urokinase inhibitors.

Amidines↗
PubMed2001

Design, synthesis, and structural analysis of influenza neuraminidase inhibitors containing pyrrolidine cores.

The discovery of (+/-)-(2S,3R,4R)-2-(trifluoroacetamido)methyl-3-amino-1-(N'-ethyl-N'-isopropylcarbamyl)pyrrolidine-4-carboxylic acid (A-192558, 20e) as a potent inhibitor of influenza neuraminidase (NA) is described. Efficient syntheses of two core structures, cis-3-(allyloxycarbonyl)amino-1-(9'-fluorenylmethoxycarbonyl)pyrrolidine-4-carboxylic acid (7) and tert-butyl (+/-)-(2S,3R,4R)-2-aminomethyl-3-bis(tert-butyloxycarbonyl)amino-1-(N'-ethyl-N'-isopropylcarbamyl)pyrrolidine-4-carboxylate (18b), were developed. Starting with these core structures and using available structural information of the NA active site as the guide, analogues were synthesized in both the tri- and tetrasubstituted pyrrolidine series by means of high-throughput parallel synthesis in solid or solution phase for expeditious SAR. These studies accelerated the identification of (+/-)-(2S,3R,4R)-2-(trifluoroacetamido)methyl-3-amino-1-(N-ethyl-N-isopropylcarbamyl)pyrrolidine-4-carboxylate (20e, A-192558) as the most potent NA inhibitor in this series (IC50 = 0.2 microM against NA A and 8 microM against NA B). The X-ray crystallographic structure of A-192558 bound to NA revealed the predicted interaction of the carboxylic group with the positively charged pocket (Arg118, Arg292, Arg371) and interaction of the trifluoroacetamino residue with the hydrophobic pocket (Ile222, Trp178) of the enzyme active site. Surprisingly, the ethyl and isopropyl groups of the urea functionality induced a conformational change of Glu276, turning the Glu276/Glu277 hydrophilic pocket, which normally accommodates the triglycerol side chain of substrate sialic acid, into an induced hydrophobic pocket.

Antiviral Agents↗
PubMed2001

Care management and the care programme approach: towards integration in old age mental health services.

OBJECTIVE: To examine the relationship between care management arrangements and the Care Programme Approach (CPA) in the context of old age mental health services and, particularly, dementia services. METHOD: The information reported is from a national study of care management arrangements, funded by the Department of Health. A response rate of 77% was obtained from local authority social services departments. RESULTS: In old age mental health services over half of the respondents reported joint screening arrangements for health and social care, almost four-fifths reported both joint criteria for the allocation of key workers and a clear definition of monitoring responsibilities. Of the latter over two-fifths were reported as being the same in care management and the CPA. Forty-six per cent of respondents provided a specialist service for people with dementia. Three-fifths of respondents reported that they did not apply CPA to people with dementia who were in receipt of care management or did so in less than 20% of cases. Where the CPA was applied it was more likely that a priority would be accorded to care management. A quarter of respondents reported the shared use of assessment documentation for people with dementia. DISCUSSION: The findings are set in the context of service developments to date and the implementation of the two systems of community based coordinated care for older people with mental health problems. Inter-authority variations are noted and the potential for greater service integration within the current legislative framework assessed.

Aged↗
PubMed2001

Intensive care-management at home: an alternative to institutional care?

BACKGROUND: Care management and assessment of need are the cornerstones of the community care reforms in the UK. Although much of the research base has been on highly vulnerable older people, in practice, care management has been implemented for a wider group. OBJECTIVE: To examine how intensive care-management at home has developed. DESIGN: Postal survey of all local authority social services departments in England. METHOD: We used an overview questionnaire (85% response) and an old-age services questionnaire (77% response). We classified local authorities according to the presence or absence of seven indicators of intensive care management at home. RESULTS: 97% of social services departments had a goal of providing a community-based alternative to residential and nursing-home care. However, only 5% had specialist intensive care-management services for older people. Other key indicators of intensive care-management, such as devolved budgets, health service care managers, small caseloads and clear eligibility criteria, were uncommon. CONCLUSIONS: There was little evidence of intensive care-management at home in older peoples' services. This is of concern, given the move towards community-based provision for frail older people. Closer links between secondary health-care services (such as geriatric medicine) and intensive care-management at home may promote more effective care at home for those who are most vulnerable.

Aged↗
PubMed2001

High concentrations of dexamethasone suppress the proliferation but not the differentiation or further maturation of human osteoblast precursors in vitro: relevance to glucocorticoid-induced osteoporosis.

OBJECTIVE: The use of glucocorticoids (GCs) in the treatment of RA is a frequent cause of bone loss. In vitro, however, this same class of steroids has been shown to promote the recruitment and/or maturation of primitive osteogenic precursors present in the colony forming unit-fibroblastic (CFU-F) fraction of human bone and marrow. In an effort to reconcile these conflicting observations, we investigated the effects of the synthetic GC dexamethasone (Dx) on parameters of growth and osteogenic differentiation in cultures of bone marrow stromal cells derived from a large cohort of adult human donors (n=30). METHODS: Marrow suspensions were cultured in the absence and presence of Dx at concentrations between 10 pm and 1 microm. After 28 days we determined the number and diameter of colonies formed, the total number of cells, the surface expression of receptors for selected growth factors and extracellular matrix proteins and, based on the expression of the developmental markers alkaline phosphatase (AP) and the antigen recognized by the STRO-1 monoclonal antibody, the proportion of cells undergoing osteogenic differentiation and their extent of maturation. RESULTS: At a physiologically equivalent concentration, Dx had no effect on the adhesion of CFU-F or on their subsequent proliferation, but did promote their osteogenic differentiation and further maturation. These effects were independent of changes in the expression of the receptors for fibroblast growth factors, insulin-like growth factor 1, nerve growth factor, platelet-derived growth factors and parathyroid hormone/parathyroid hormone-related protein, but were associated with changes in the number of cells expressing the alpha(2) and alpha(4), but not beta(1), integrin subunits. At supraphysiological concentrations, the effects of Dx on the osteogenic recruitment and maturation of CFU-F and their progeny were maintained but at the expense of a decrease in cell number. CONCLUSIONS: A decrease in the proliferation of osteogenic precursors, but not in their differentiation or maturation, is likely to be a key factor in the genesis of GC-induced bone loss.

Adult↗
PubMed2001

Thy1 glomerulonephritis induced in young lewis rats accelerates age-related glomerulosclerosis.

Age-related and disease-induced glomerulosclerosis (GS) in rats have both been well defined in a number of strains and experimental models, but the inter-relationship between the two is not clear. The present study was undertaken to compare the pattern of glomerular injury in these two types of GS. One- and two-shot Thy1 glomerulonephritis (GN) was induced at 2 months of age and followed for 12 months. At 12 months histological injury in proteinuric rats was characterized by segmental hyaline lesions. Two-shot Thy1 GN resulted in accelerated, but morphologically identical injury at 8 months. Histological lesions predictive of subsequent accelerated GS were evaluated at 1, 2, 4 and 6 months. In this regard, glomerular hypercellularity, rather than hypertrophy or matrix increase, was the most consistent histological index of later accelerated disease. The profibrotic cytokines transforming growth factor (TGF)-beta(1) and -beta(3) were localized distinctly to segmental hyaline lesions, but not to areas of matrix increase within the glomerular tuft. This study reveals that GS after Thy1 GN represents acceleration of an age-related disease, presents evidence for use of prolonged glomerular hypercellularity as the best histological index of future disease progression, and correlates the key lesion of GS in these animals, the segmental hyaline lesion, with the presence of TGF-beta peptides.

Aging↗
PubMed2001

Candida proteases and their inhibition: prospects for antifungal therapy.

The rationale for inhibition of the secreted acid proteases (SAP) of Candida fungal species as a novel antifungal strategy is outlined. Enzyme structure-activity relationship data regarding the inhibitor A-70450 are described along with results from in vivo antifungal assays. Developments from protein X-ray crystallographic studies, SAP knock-out genetic studies, and the importance of these new results for drug discovery are reviewed. Finally, inhibition of the Candida SAP enzymes with HIV protease inhibitors and a proposed role in candidiasis of AIDS patients is discussed.

Antifungal Agents↗
PubMed2000

Structure-directed discovery of potent non-peptidic inhibitors of human urokinase that access a novel binding subsite.

BACKGROUND: Human urokinase-type plasminogen activator has been implicated in the regulation and control of basement membrane and interstitial protein degradation. Because of its role in tissue remodeling, urokinase is a central player in the disease progression of cancer, making it an attractive target for design of an anticancer clinical agent: Few urokinase inhibitors have been described, which suggests that discovery of such a compound is in the early stages. Towards integrating structural data into this process, a new human urokinase crystal form amenable to structure-based drug design has been used to discover potent urokinase inhibitors. RESULTS: On the basis of crystallographic data, 2-naphthamidine was chosen as the lead scaffold for structure-directed optimization. This co-crystal structure shows the compound binding at the primary specificity pocket of the trypsin-like protease and at a novel binding subsite that is accessible from the 8-position of 2-napthamidine. This novel subsite was characterized and used to design two compounds with very different 8-substituents that inhibit urokinase with K(i) values of 30-40 nM. CONCLUSIONS: Utilization of a novel subsite yielded two potent urokinase inhibitors even though this site has not been widely used in inhibitor optimization with other trypsin-like proteases, such as those reported for thrombin or factor Xa. The extensive binding pockets present at the substrate-binding groove of these other proteins are blocked by unique insertion loops in urokinase, thus necessitating the utilization of additional binding subsites. Successful implementation of this strategy and characterization of the novel site provides a significant step towards the discovery of an anticancer clinical agent.

Binding Sites↗
PubMed2000

The expression and secretion of atrial natriuretic factor and brain natriuretic peptide by rat proximal tubular cells.

We examined the expression of both the natriuretic peptides and natriuretic peptide receptors (NPR) in primary cultures of rat proximal tubular (RPT) cells using Northern blot assay for peptides and receptors and radioimmunoassay and immunohistochemical analysis for atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide. Freshly isolated cells expressed mRNA coding for ANF, BNP, and the NPR-C. The presence of ANF and BNP in freshly isolated cells was confirmed by immunocytochemical staining. As cells approached confluence, there was a marked increase in mRNA expression for ANF and BNP. Immunocytochemical analysis and radioimmunoassay confirmed that both these peptides were co-localised in RPT cells and present in the cell supernatant. These changes in peptide expression were associated with a concurrent decrease in the expression of the NPR-C and the appearance of the NPR-A and -B. These results confirm that freshly isolated RPT cells possess the components of an autocrine natriuretic peptide system and that growth in primary culture is associated with changes in both peptide system and that growth in primary culture is associated with changes in both peptide and receptor subtype expression, raising the possibility that the endogenous production of ANF and BNP may be involved in the control of control cell growth.

Animals↗
PubMed2000

Follicular dendritic cells in scrapie pathogenesis.

Scrapie pathogenesis was studied in chimaeric mice that carried the prion protein (PrP) gene only in particular cells of the immune system. These mice were produced by grafting bone marrow from PrP expressing donors into PrP deficient recipients and vice versa. As follicular dendritic cells are not replaced significantly from the bone marrow in adult mice, this procedure resulted in a mismatch in PrP genotype between these cells and bone marrow derived cells such as lymphocytes. Using these models we obtained strong evidence that follicular dendritic cells produced high levels of the normal form of PrP in uninfected mice. Furthermore, the replication of a mouse-passaged scrapie strain in the spleen depended only on the presence of PrP expressing follicular dendritic cells. PrP expression by lymphocytes or other bone marrow derived cells had no influence on replication in spleen or on neuroinvasion in these models. These results indicate that the follicular dendritic cell is a potential target for prophylactic or therapeutic intervention in transmissible spongiform encephalopathies.

Animals↗
PubMed2000

Macrophages from inflamed but not normal glomeruli are unresponsive to anti-inflammatory cytokines.

This study examined the properties and responsiveness to cytokines of macrophages purified from normal and nephritic glomeruli to ascertain whether macrophages activated in vivo develop programmed unresponsiveness to cytokines as do bone marrow-derived macrophages in vitro when activated by interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), interleukin-4 (IL-4), or transforming growth factor-beta (TGF-beta). Macrophages from normal glomeruli did not generate nitric oxide (NO) spontaneously but only after treatment with IFN-gamma and TNF-alpha. NO generation by these macrophages was abrogated by administering IL-4, TGF-beta, or TNF-alpha before but not after IFN-gamma treatment. Glomerular macrophages also expressed beta-glucuronidase, which was increased by TGF-beta and decreased by IFN-gamma and TNF. By contrast, glomerular macrophages from rats with nephrotoxic nephritis did not express beta-glucuronidase even after exposure to TGF-beta. Furthermore, they generated NO spontaneously, and this spontaneous generation of NO was not suppressed by IL-4, TGF-beta, or TNF-alpha. Systemic treatment of nephritic rats with IL-4 reduced NO generation by 40% but did not prevent activation, which is similar to the effect of IL-4 on bone marrow-derived macrophages in vitro when given simultaneously with IFN-gamma. We conclude that macrophages infiltrating inflamed glomeruli have developed programmed unresponsiveness to activating cytokines. This may enable them to function appropriately in the complex conditions within an inflammatory focus.

Animals↗
PubMed2000

Expression of the developmental markers STRO-1 and alkaline phosphatase in cultures of human marrow stromal cells: regulation by fibroblast growth factor (FGF)-2 and relationship to the expression of FGF receptors 1-4.

Autologous marrow stromal cells have been proposed as an adjuvant in the treatment of bone defects and diseases. This will require the development of culture conditions that permit their rapid expansion ex vivo while retaining their potential for further differentiation. Fibroblast growth factor (FGF)-2 has been proposed as a candidate for the ex vivo expansion of cells with enhanced osteogenic potential, and we have explored this possibility further using cells obtained from a large cohort of adult human donors. Treatment with FGF-2 (0.001-2.5 ng/mL) had no detectable effect on colony formation, but markedly increased their proliferative potential and that of their immediate progeny, as shown by the increases in colony size and cell number. Based on the observed increase in the expression of the developmental markers STRO-1 and alkaline phosphatase (AP), a major target for the actions of FGF-2 appears to be the more primitive cells of the osteoblast lineage, and that, when added in combination with the synthetic glucocorticoid dexamethasone (Dx), it interacts positively to promote further cell maturation. The maintenance of adequate levels of ascorbate was shown to be a critical component in determining the nature of the effect of FGF-2 on AP expression. Variation in the response (predominantly in the magnitude and/or sensitivity) of the cultured cell populations to treatment with FGF-2 was apparent, but a preliminary analysis indicated that this was not due to differences in the age or gender of the donors used. The cultured cell populations were found to express multiple FGF receptors (FGFRs; 1-4) and the observed changes in the spectrum and abundance of FGFRs expressed in relation to that of STRO-1 and AP are consistent with their expression being developmentally regulated during the process of osteogenic differentiation. These results provide novel insights into the mechanism of action of FGF-2 on human cells of the osteoblast lineage and support the use of this factor, alone or in combination with Dx, for the rapid, ex vivo expansion of cell populations with enhanced osteogenic potential.

Adult↗
PubMed2000

Biphasic release of immunoreactive endothelins following acute pulmonary thromboembolism in pigs.

The purpose of this investigation was to study the role of endothelins (ETs) in the pathogenesis of acute pulmonary thromboembolism (PTE). Eighteen piglets (20 +/- 3 kg) were anesthetized and ventilated with 100% oxygen, five of them then served as controls. Acute thromboembolic injury in the lung was induced by injecting 15-25 ml of preformed clots into the left lower lobar pulmonary artery during thoracotomy. Pulmonary arterial pressure (Ppa) increased by at least 2.5-fold from baseline. During the subsequent 8 h, seven blood samples were collected from the left atrium and assayed for immunoreactive ETs. The results showed that following PTE: (1) Ppa remained elevated but cardiac output remained constant throughout the experiments; (2) plasma level of immunoreactive ETs increased in the embolized group compared to controls and the profile of immunoreactive ET release suggested a biphasic response. We conclude that the release of these vasocontractile and bronchoconstrictive mediators after PTE may contribute to ventilation perfusion mismatching and account for the pulmonary hypertension and deterioration of gas exchange that are often seen clinically.

Acute Disease↗