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Biomedical subjects

S Harper

Publications and source records attributed to S Harper.

At least 19 recordsLinked to original sources
PubMed1999

A rapid method for determination of cell survival in primary neuronal DRG cultures.

A simple and rapid enzyme-linked immunosorbent assay (ELISA) has been developed to provide an alternative to cell counting to detect increases in cell survival in primary neuronal cultures. This sensitive assay has the advantage of being less time consuming and labour intensive than cell counting, can be used to quantify cell survival and is more accurate than estimation methods of counting. The ELISA uses an antibody raised to GAP-43, a growth-associated protein which is strongly expressed by developing and regenerating neurones. The effects of nerve growth factor (NGF), neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) on GAP-43 immunoreactivity in dissociated primary cultures of rat and chick dorsal root ganglia have been compared to results obtained by cell counting. Data show that human NGF produced the greatest increase in GAP-43-immunoreactive neurones in both species; this increase in immunoreactivity correlated well with the increased survival shown by cell count data. Results prove that the ELISA can also be used to accurately detect small changes in cell survival as seen with NT-3 and BDNF, or potentiation of the effects obtained with the trophic factor NT-3. In conclusion, this ELISA may be a useful tool to detect neurotrophic effects of unknown agents or novel neurotrophins.

Animals↗
PubMed1999

A rapid method for semi-quantitative analysis of neurite outgrowth from chick DRG explants using image analysis.

Neurite outgrowth from dorsal root ganglion (DRG) explants is a method of evaluating neurotrophic activity of growth factors and neurotrophin mimetics. The drawbacks to this approach are the difficulties in quantifying the response. Neurite counts are time consuming and labour intensive, and the accuracy is often questionable due to branching and fasciculation of the neurites. We report here a method of semi-quantitative analysis of neurite outgrowth from chick DRG explants, using image analysis to quantify the area occupied by neurites emanating from the ganglion. This method is rapid, takes into account both the length and number of neurites, and is unaffected by neurite fasciculation or branching. Primary explants of chick DRGs were treated with the neurotrophins nerve growth factor (NGF) or neurotrophin-3 (NT-3) and with the compound K252a. K252b was tested for potentiation of the response to NT-3. The results show a dose dependent outgrowth of neurites from explants treated with NGF, NT-3 and K252a, and potentiation of the NT-3 response by K252b. These responses were quantified by neurite area quantification using image analysis. We conclude that neurite area measurement using image analysis provides a robust means of evaluating neurotrophic activity of growth factors and neurotrophin mimetics in vitro.

Animals↗
PubMed1999

A randomized trial comparing the effect of rofecoxib, a cyclooxygenase 2-specific inhibitor, with that of ibuprofen on the gastroduodenal mucosa of patients with osteoarthritis. Rofecoxib Osteoarthritis Endoscopy Study Group.

BACKGROUND & AIMS: Prostaglandin production in the normal gastrointestinal tract, believed to be critical for mucosal integrity, is mediated by cyclooxygenase (COX)-1, whereas prostaglandin production at inflammatory sites seems to occur via induction of COX-2. We hypothesized that COX-2-specific inhibition with rofecoxib (25 mg once daily) in the treatment of patients with osteoarthritis would cause fewer gastroduodenal ulcers than an equally effective dose of ibuprofen (800 mg 3 times a day), a nonspecific COX inhibitor. METHODS: A total of 742 osteoarthritis patients without ulcers on baseline endoscopy were randomly assigned to receive rofecoxib (25 or 50 mg once daily), ibuprofen (800 mg 3 times daily), or placebo. Endoscopy was repeated at 6, 12, and 24 weeks. At 16 weeks, by study design, 95% of the placebo group and 5% of the other groups were discontinued. RESULTS: The cumulative incidence of gastroduodenal ulcers >/=3 mm with rofecoxib (25 or 50 mg once daily) was significantly (P < 0.001) lower than with ibuprofen and was statistically equivalent to placebo at week 12 (placebo, 9.9%; 25 mg rofecoxib, 4.1%; 50 mg rofecoxib, 7.3%; and ibuprofen, 27.7%). At 24 weeks, ulcer rates were 25 mg rofecoxib, 9. 6%; 50 mg rofecoxib, 14.7%; and ibuprofen, 45.8% (P < 0.001, ibuprofen vs. 25 and 50 mg rofecoxib). CONCLUSIONS: Rofecoxib, at doses 2-4 times the dose demonstrated to relieve symptoms of osteoarthritis, caused significantly less gastroduodenal ulceration than ibuprofen, with ulcer rates comparable to placebo.

Aged↗
PubMed1999

Analysis of the neurotrophic effects of GPI-1046 on neuron survival and regeneration in culture and in vivo.

The putative neurotrophic effects of the immunophilin ligand GPI-1046 were evaluated in established experimental systems of neuron survival and axon growth in vitro and in vivo. GPI-1046 marginally increased neurite outgrowth of chick dorsal root ganglia in culture under conditions where a very robust effect of nerve growth factor was seen. GPI-1046 failed to protect dopaminergic neurons from 1-methyl-4-phenylpyridinium in culture or to protect cultured cortical neurons from experimentally induced apoptosis in vitro. In adult rats in vivo, daily administration of GPI-1046 (10 mg/kg, s.c.) for three days enhanced the maximal regeneration distance of both motor and large myelinated sensory axons measured using an electrophysiological assay. However, detailed morphometric analysis of these animals failed to provide evidence for an increase in axon numbers in GPI-1046-treated animals. The ability of GPI-1046 to promote the recovery of dopaminergic function following unilateral 6-hydroxydopamine lesions of the substantia nigra was also tested in rats. In the first study, the duration of amphetamine (3 mg/kg, s.c.)-induced circling, but not the maximal number of rotations, was significantly reduced in animals treated with GPI-1046 for five days (10 mg/kg/day). In a second study, testing the effects of delayed GPI-1046 administration, chronic treatment with GPI-1046 (10 mg/kg/day) for two weeks, beginning one month after surgery, did not alter circling responses. Morphometric analysis failed to reveal any changes in either the density of tyrosine hyroxylase-positive fibres in dopaminergic target areas or in cell numbers in the substantia nigra in both experiments. Thus, while GPI-1046 produced marginal effects on neurite outgrowth in dorsal root ganglia cultures and on functional paramaters of nerve regeneration in vivo, we failed to obtain evidence in support of the notion of a general neuroprotective effect of the compound or for an effect on morphologic nerve regeneration in vivo.

1-Methyl-4-phenylpyridinium↗
PubMed1999

The staurosporine-like compound L-753,000 (NB-506) potentiates the neurotrophic effects of neurotrophin-3 by acting selectively at the TrkA receptor.

K-252b, a member of the staurosporine family of protein kinase inhibitors, selectively potentiates the activation of the nerve growth factor receptor, TrkA, by a nonpreferred ligand, neurotrophin-3 (NT-3), in a variety of cell types. At higher (micromolar) concentrations of K-252b, an inhibitory effect occurs because of the inhibitory action of K-252b on the Trk kinase. By examining analogs of K-252b, we identified the compound L-753,000 (NB-506), which potentiates the action of NT-3 on TrkA but is devoid of the inhibitory action of K-252b. L-753,000 was effective at nanomolar concentrations in a Chinese hamster ovary cell line that expressed TrkA but was devoid of p75, the low-affinity neurotrophin receptor. L-753,000 also potentiated the activation of mitogen-activating protein kinase signaling (downstream from Trk activation) by NT-3 in this cell line. Although L-753,000, like K-252b, had a negligible effect in the absence of NT-3, the compound was found to potentiate NT-3-induced survival in both rat and chick primary cultures of dissociated dorsal root ganglia (DRG) and on neurite outgrowth of chick DRG explants. Unlike K-252b, which at micromolar concentrations inhibits the survival response of NT-3 in dissociated rat DRG, L-753,000 continued to potentiate the actions of NT-3 up to a concentration of 10 microM. Furthermore, the compound, unlike K-252b, did not inhibit an unrelated protein kinase, protein kinase C, at concentrations up to 10 microM. Because L-753, 000 selectively potentiates the NT-3-induced stimulation of TrkA without inhibiting Trks and other protein kinases, it represents a novel class of selective modifiers of neurotrophin actions.

Analysis of Variance↗
PubMed1998

Evidence that P2Y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP.

1. Previous studies have shown that ATP and UTP are able to stimulate phospholipase C (PLC) and proliferation in cultured aortic smooth muscle cells. Here we set out to characterize the receptor responsible, and investigate a possible role for p42 and p44 mitogen activated protein kinase (MAPK) in the proliferative response. 2. The phospholipase C response of spontaneously hypertensive rat (SHR) derived aortic smooth muscle cells in culture showed that the response to ATP was partial compared to the response to UTP. 3. Further studies characterized the responses of the SHR derived cells. UTP was the only full agonist with the SHR cells; UDP gave a partial response while ADP, 2-methythio-ATP and alpha,beta-methylene ATP were essentially ineffective. The response to UDP was almost lost in the presence of hexokinase, consistent with this being due to extracellular conversion to UTP. These observations are inconsistent with the response being mediated by either P2Y1 or P2Y6 receptors. 4. When increasing concentrations of ATP were present with a maximally effective concentration of UTP, the size of the response diminished, consistent with UTP and ATP acting at a single population of receptors for which ATP was a partial agonist. This is inconsistent with a response mainly at P2Y2 receptors. 5. 1321N1 cells transfected with human P2Y4 receptors gave a similar agonist response profile, with ATP being partial compared to UTP, loss of response to UDP with hexokinase treatment, and with the response to UTP diminishing in the presence of increasing concentrations of ATP. 6. Use of the reverse transcriptase-polymerase chain reaction confirmed the presence of mRNA encoding P2Y4 receptors in SHR derived vascular smooth muscle cells. Transcripts for P2Y2, P2Y4 and P2Y6 receptors, but not P2Y1 receptors, were detected. 7. Stimulation of SHR derived cells with UTP enhanced the tyrosine phosphorylation of both p42 and p44 MAPK, and the incorporation of [3H]-thymidine into DNA. Both these responses were diminished in the presence of an inhibitor of activation of MAPK. 8 These results lead to the conclusion that in SHR derived cultured aortic smooth muscle cells, PLC responses to extracellular UTP and ATP are predominantly at P2Y4 receptors, and suggest that these receptors are coupled to mitogenesis via p42/p44 MAPK.

Adenosine Triphosphate↗
PubMed1997

Isolated microscopic haematuria in the genitourinary clinic: the value of renal biopsy.

Isolated microscopic haematuria is a common finding in the genitourinary clinic. The conventional approach to investigation includes urological referral for cystourethroscopy if renal imaging is normal. However the diagnostic yield is very low; in particular urothelial malignancy at age < 40 years is rare. Glomerular disease is increasingly recognized as a common cause of microscopic haematuria. In this study 50 patients with persistent microscopic haematuria detected at a genitourinary clinic underwent renal biopsy. Twelve (24%) had an abnormal biopsy--IgA nephropathy 6 (12%), thin membrane nephropathy 3 (6%), other glomerulonephritis 3 (6%). In 7 others no abnormality was found but information was incomplete as electron microscopy was unavailable. It is important to establish these diagnoses since some patients will develop progressive renal disease. In this clinical setting renal biopsy will give diagnostic and prognostic information, protects from repeated urological investigation, and allows reassurance if renal histology is normal. Renal biopsy is recommended for patients age < 40 years with persistent microscopic haematuria. An algorithm for the investigation of microscopic haematuria is presented.

Adult↗
PubMed1996

RS cyclophilins: identification of an NK-TR1-related cyclophilin.

We report the isolation of a large cyclophilin protein containing RS (arginine-serine) repeats from a yeast two-hybrid screen using ClK (CDC28/cdc2-like kinase) as a probe. This Clk associating RS-cyclophilin (CARS-Cyp) possesses 39% homology to the NK-TR1 (natural killer tumor recognition protein-1) we have previously characterized (Anderson et al. (1993) Proc. Natl. Acad. Sci. USA 90 (1993) 542-546). CARS-Cyp is expressed in a variety of tissues and cell types, and codes for a protein with a predicted mass of 89 kDa containing a cyclophilin-related domain, two Nopp140 (nucleolar phosphoprotein of 140 kDa)-related domains, and a large RS domain. The RS-cyclophilins, a novel class of proteins, may play an important role in the regulation of pre-mRNA splicing.

Amino Acid Isomerases↗
PubMed1996

Does treatment with L-thyroxine influence health status in middle-aged and older adults with subclinical hypothyroidism?

OBJECTIVE: To determine if health-related quality of life (HRQL) in patients of middle age and older with elevated thyroid-stimulating hormone (TSH) and normal total thyroid hormone levels-subclinical hypothyroidism-improves with L-thyroxine replacement therapy. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: Outpatient clinic. PATIENTS: Thirty-seven patients with subclinical hypothyroidism, most with symptoms consistent with hypothyroidism, over 55 years of age. INTERVENTIONS: Placebo or L-thyroxine replacement therapy to achieve normal TSH level. MEASUREMENTS AND MAIN RESULTS: Disease-specific and general HRQL, cognitive function, bone mineral density, lipid levels. The mean daily dose of L-thyroxine replacement in the active group was 68 +/- 21 micrograms. TSH decreased by 8.6 mIU/L (95% confidence interval [CI] 4.1 to 13.1) and T4 increased by 27.9 nmol/L (95% CI 14.8 to 41.2). There was a statistically significant improvement in a composite psychometric memory score in treated versus control patients; all other outcomes showed similar findings in the two groups. Although confidence intervals for most measures did not exclude an important improvement in HRQL with thyroid replacement, no measure of symptoms or HRQL either showed clinically important trends in favor of treatment, or approached conventional levels of statistical significance. CONCLUSIONS: In middle-aged and older patients with elevated TSH and normal T4, it may not be harmful to follow biochemical and clinical status even in the presence of nonspecific symptoms potentially associated with hypothyroidism.

Aged↗
PubMed1995

Stem cell patterning and fate in human epidermis.

Within human epidermis there are two types of proliferating keratinocyte: stem cells, which have high proliferative potential, and transit-amplifying cells, which are destined to undergo terminal differentiation after a few rounds of division. We show that, in vivo, stem cells express higher levels of the alpha 2 beta 1 and alpha 3 beta 1 integrins than transit-amplifying cells and that this can be used both to determine the location of stem cells within the epidermis and to isolate them directly from the tissue. The distribution of stem cells and transit-amplifying cells is not random: patches of integrin-bright and integrin-dull cells have a specific location with respect to the epidermal-dermal junction that varies between body sites and that correlates with the distribution of S phase cells. Stem cell patterning can be recreated in culture, in the absence of dermis, and appears to be subject to autoregulation.

Adult↗
PubMed1994

Localization of nerve growth factor (NGF) and low-affinity NGF receptors in touch domes and quantification of NGF mRNA in keratinocytes of adult rats.

Touch domes are clearly delineated mechanoreceptors that are visible on the depilated skin of mammals. These structures consist of a sharply circumscribed disk of thickened epithelium surmounting a group of Merkel cells that are innervated by type I sensory neurons. These characteristic cutaneous structures provide an ideal opportunity for investigating whether the localization of nerve growth factor (NGF) in the skin is related to sites of sensory axon termination. For these reasons, we have used immunocytochemistry to study the distribution of NGF and the low-affinity NGF receptor (p75NGFR) in the touch domes of adult rat skin. Intense NGF-like immunoreactivity was sharply restricted to keratinocytes (excluding the stratum corneum) of the thickened epidermis of touch domes. The epidermis immediately surrounding touch domes and the epidermis of the tylotrich hair follicle associated with touch domes were not stained by anti-NGF antiserum. Merkel cells of the basal epidermis of touch domes were immunonegative for NGF but were immunopositive for p75NGFR as were the type I nerve endings innervating these cells. Quantitative Northern blotting revealed that the level of NGF mRNA was substantially higher in keratinocytes isolated from the stratum granulosum and stratum spinosum than in keratinocytes isolated from the stratum germinativum. These findings indicate that NGF synthesis in mature skin has a highly restricted regional distribution that is primarily associated with the innervation of a specialized touch receptor.

Animals↗
PubMed1994

Spectrum of quality of life impairment in hypothyroidism.

The aim of this study is to evaluate the frequency and relative importance of different aspects of health related quality of life (HRQOL) impairment in patients with hypothyroidism. A survey of 200 patients with treated hypothyroidism was undertaken. Patients were identified and questionnaires distributed through family physicians, endocrinologists, and regional branches of the Thyroid Foundation of Canada. Frequency of importance of patients' symptoms attributed to hypothyroidism and resolved or improved with treatment were measured. Establishing the wide variety of patient-specific complaints associated with hypothyroidism provides guidance for clinicians dealing with hypothyroid patients, and investigators interested in HRQOL impairment in these patients.

Humans↗
PubMed1994

Effect of increasing doses of beta agonists on spirometric parameters, exercise capacity, and quality of life in patients with chronic airflow limitation.

BACKGROUND: A study was undertaken to determine the impact of different doses of inhaled terbutaline on peak flow rates, spirometric parameters, functional exercise capacity, and quality of life in patients with chronic airflow limitation. METHODS: A double blind, randomised, placebo controlled, multiple crossover trial was conducted with treatment periods of one week. Patients with a clinical diagnosis of chronic airflow limitation and FEV1 below 70% predicted after administration of bronchodilator were recruited from secondary care respiratory practices, and the effect of 500, 1000, and 1500 micrograms inhaled terbutaline four times daily on spirometric parameters (FEV1, FVC), maximum inspiratory pressures, six minute walking distance, and health-related quality of life (Chronic Respiratory Disease Questionnaire, Quality of Well Being, Standard Gamble) was measured. RESULTS: Twenty five patients completed the trial. Peak flow rates and FEV1 showed statistically significant but clinically trivial improvement on the higher drug doses. Results of maximum inspiratory pressure measurements, walk test distance, and quality of life measures showed minimal differences on the different dosages, and none of the differences approached conventional statistical significance. CONCLUSIONS: Regular use of beta agonists in doses higher than two puffs four times a day is very unlikely to provide additional functional or symptomatic benefit to patients with chronic airflow limitation.

Aged↗
PubMed1993

The effect of increasing doses of beta-agonists on airflow in patients with chronic airflow limitation.

OBJECTIVE: To determine the increase in FEV1 associated with increasing doses of inhaled terbutaline and salbutamol, the reproducibility of the increase in FEV1, and the reproducibility of the associated optimal bronchodilator dose, in patients with chronic airflow limitation (CAL). DESIGN: Double-blind, randomized, controlled trial examining spirometric response to cumulative doses of bronchodilators. PATIENTS AND SETTING: Patients with clinical diagnosis of CAL, FEV1 below 70% predicted, and FEV1 to FVC ratio less than 0.7 after administration of bronchodilator recruited from secondary care respirology practices. MEASURES OF OUTCOME: The estimates of maximum and optimal bronchodilation, as well as the associated drug dosages, were established in each patient on three occasions (twice on terbutaline and once on salbutamol). The 'optimal' drug dose was defined as the lowest dose associated with an FEV1 not exceeded by 50 ml on any other dose. MAIN RESULTS: Thirty-five patients completed the trial. FEV1 improved from 0.93 to a maximum of 1.191 with terbutaline (average of the two administrations) and from 0.951 to 1.141 with inhaled salbutamol (difference in increase in FEV1 between terbutaline and salbutamol P = 0.006). In less than 50% of cases administration of more than four puffs of bronchodilator resulted in FEV1 increase by more than 50 ml. The average dose of salbutamol and terbutaline associated with optimal bronchodilation were 430 micrograms and 1160 micrograms respectively. Patients varied widely in the optimal dose. Estimates of optimal dose were not reproducible (intraclass correlation coefficient < 0.5). CONCLUSION: Substantial incremental increase in FEV1 in response to increasing doses of beta-agonists beyond those commonly used in clinical practice is restricted to a minority of patients. Lack of reproducibility limits the clinical usefulness of establishing the optimal dose of beta-agonist for a given patient.

Administration, Inhalation↗