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N Macdonald

Publications and source records attributed to N Macdonald.

At least 37 records · Page 2Linked to original sources
PubMed2000

National assessment of prevalent diagnosed HIV infections.

HIV infection is associated with high treatment and care costs and subject to large differences in prevalence between health districts. Equitable distribution of resources requires information provided by an annual national survey of prevalent diagnosed HIV infections (SOPHID). This measures HIV caseloads by health district of residence throughout England, Wales, and Northern Ireland and is used to inform local public health professionals and to improve allocation of government funding for HIV prevention and care. Survey totals are adjusted by underreporting and non-attendance factors to produce a more accurate assessment of the total caseloads. On average the combined adjustments increase the reported caseload by 14.7% annually. Adjusted prevalence estimates ranged from 14,164 in 1995 to 18,460 in 1998, an increase of 30%.

CD4 Lymphocyte Count↗
PubMed1999

Species differences in sequence and activity of the peroxisome proliferator response element (PPRE) within the acyl CoA oxidase gene promoter.

In rats and mice, peroxisome proliferators (PP) cause liver enlargement, hepatocarcinogenesis and peroxisome proliferation associated with induction of enzymes such as acyl CoA oxidase (ACO). However, humans appear to be non-responsive to the adverse effects of PPs such as ACO induction. PPs activate the peroxisome proliferator activated receptor alpha (PPARalpha) that binds to DNA at peroxisome proliferator response elements (PPREs) within the promoters of PP-responsive genes. When the human ACO promoter was cloned previously (Varanasi et al., 1996. Journal of Biological Chemistry, 271, 2147-2155), it was reported to contain a PPRE (5' AGGTCA C TGGTCA 3') that bound PPARalpha and could be activated in vitro by Wyeth-14,643 (at >1 mM) or DEHP (at > 1.5 mM). In contrast, when we cloned the ACO gene promoter from a human liver biopsy, it was non-responsive to PPs and differed at three positions (5' AGGTCA G CTGTCA 3') from that reported previously (Woodyatt et al., 1999. Carcinogenesis, 20, 369-375). Subsequent to this, Varanasi et al. re-sequenced their constructs and obtained the same sequence as we have described (Varanasi et al., 1998. Journal of Biological Chemistry, 273, 30832). However, the observation that the errant sequence (5' AGGTCA C TGGTCA 3') was able to bind PPARalpha still remained since it appears that this sequence was used by Varanasi et al. (1996) to design oligonucleotides for their DNA binding analyses. Thus, if the 5' AGGTCA C TGGTCA 3' sequence did exist in some individuals, it could be active. To address this, we used site-directed mutagenesis to create a promoter fragment that contained the errant sequence. This reporter gene was transfected into NIH3T3 cells together with a plasmid expressing mPPARalpha, and assessed for its ability to drive PP-mediated gene transcription using a non-toxic concentration of Wyeth-14,643 (100 microM). This human ACO promoter was also inactive, unlike the equivalent rat ACO promoter fragment used as a positive control. Next, we used site directed mutagenesis to convert the PPRE found in the active rat ACO promoter (3' AGGACA A AGGTCA 5') to our inactive human sequence (AGGTCA G CTGTCA). This human PPRE was unable to drive PP-induced gene transcription even in the context of the rat ACO promoter suggesting that the activity of the rat promoter is conferred principally by the PPRE sequence, even though it may be enhanced by flanking sequences. These data confirm that neither the native nor the errant human ACO gene PPRE can respond to PPs. The absence of a responsive PPRE contributes to our understanding of the lack of response of humans to some of the adverse effects of the PP class of non-genotoxic hepatocarcinogens.

3T3 Cells↗
PubMed1999

Addition of peroxisome proliferator-activated receptor alpha to guinea pig hepatocytes confers increased responsiveness to peroxisome proliferators.

The fibrate drugs, such as nafenopin and fenofibrate, show efficacy in hyperlipidemias but cause peroxisome proliferation and liver tumors in rats and mice via nongenotoxic mechanisms. However, humans and guinea pigs appear refractory to these adverse effects. The peroxisome proliferator (PP)-activated receptor alpha (PPAR alpha) mediates the effects of PPs by heterodimerizing with the retinoid X receptor (RXR) to bind to DNA at PP response elements (PPREs) upstream of PP-regulated genes, such as acyl-CoA oxidase. Hepatic expression of PPAR alpha in guinea pigs and humans is low, suggesting that species differences in response to PPs may be due at least in part to quantity of PPAR alpha. To test this hypothesis, we introduced mouse PPAR alpha and its heterodimerization partner, RXR alpha, into guinea pig hepatocytes by transient transfection and determined responsiveness to the PP nafenopin by cyanide-insensitive palmitoyl-CoA oxidation (CIPCO). Expression of the mRNA for mouse PPAR alpha in transfected guinea pig hepatocytes was verified using species-specific PCR. In guinea pig hepatocytes transfected with control plasmids and treated with 50 microM nafenopin in the absence or presence of the RXR ligand, 9-cis-retinoic acid (5 microM) gave only a 1.7 +/- 1.5- or 3.3 +/- 1.5-fold induction in CIPCO, respectively. However, addition of ligands to hepatocytes co-transfected with both mPPAR alpha and RXR gave a strong induction of CIPCO (14.8 +/- 8.6-fold). Mouse, human, and guinea pig PPAR alpha showed equivalent function in the CIPCO assays. Thus, quantity of PPAR alpha plays a significant role in the lack of response to PPs in guinea pigs. In humans, however, lack of PPAR alpha may be only one factor dictating lack of response because recent data show that the human acyl-CoA oxidase gene lacks a functional PP response element.

Alitretinoin↗
PubMed1999

Suppression of apoptosis and induction of DNA synthesis in vitro by the phthalate plasticizers monoethylhexylphthalate (MEHP) and diisononylphthalate (DINP): a comparison of rat and human hepatocytes in vitro.

Diethylhexylphthalate (DEHP) and diisononylphthalate (DINP) are plasticizers with many important commercial, industrial and medical applications. However, both DEHP and DINP are rodent peroxisome proliferators (PPs), a class of compounds that cause rodent liver tumours associated with peroxisome proliferation, induction of hepatic DNA synthesis and the suppression of apoptosis. Despite these effects in the rodent, humans appear to be nonresponsive to the adverse effects of PPs. Previously, we have shown that the fibrate hypolipidaemic peroxisome proliferator, nafenopin, induced DNA synthesis and suppressed apoptosis in rat but not in human hepatocytes. In this work, we have examined species differences in the response of rat and human hepatocytes to DEHP and DINP in vitro. In rat hepatocytes in vitro, both DINP and MEHP (a principle metabolite of DEHP and the proximal peroxisome proliferator) caused a concentration-dependent induction of DNA synthesis and suppression of both spontaneous and transforming growth factor beta1 (TGFbeta1)-induced apoptosis. Similarly, both MEHP and DINP caused a concentration-dependent induction of peroxisomal beta-oxidation although the response to DINP was less robust. In contrast to the pleiotropic response noted in rat hepatocytes, neither DINP nor MEHP caused an induction of beta-oxidation, stimulation of DNA synthesis and suppression of apoptosis in human hepatocytes cultured from three separate donors. These data provide evidence for species differences in the hepatic response to the phthalates DEHP and DINP, confirming that human hepatocytes appear to be refractory to the hepatocarcinogenic effects of PPs first noted in rodents.

Animals↗
PubMed1999

Induction of DNA replication by peroxisome proliferators is independent of both tumour necrosis factor (alpha) priming and EGF-receptor tyrosine kinase activity.

Peroxisome proliferators (PPs) cause hepatocyte proliferation and tumorigenesis in rodent liver. PPs induce hepatocyte DNA synthesis although the mechanism is unclear. Tumour necrosis factor (alpha) (TNF(alpha)) and epidermal growth factor (EGF) have been implicated in mediating this growth response since these factors induce a threefold and 17.2-fold increase, respectively, in DNA synthesis in rat primary hepatocyte cultures. Previously, others have suggested that TNF(alpha) acts as a primer to sensitise hepatocytes to the proliferative effects of growth factors. Indeed, here we show that costimulation with TNF(alpha) and a suboptimal (4-20% of optimal) concentration of EGF permits an 11.7-fold increase in DNA synthesis in rat primary hepatocyte cultures. The PP nafenopin induced a 2. 3-fold increase in DNA synthesis but there was no further increase upon co-administration of either TNF(alpha) or a suboptimal concentration of EGF. Furthermore, there was no gross dysregulation of the CDK and cyclin protein expression profile upon stimulation with nafenopin. Using a specific epidermal growth factor receptor tyrosine kinase inhibitor (4-(3-chloro-4-fluorophenylamino)-7-methoxy-6- (3-¿1-pyrolidino)-propoxyquinazoline, EGFR-TKI), we show that signalling through EGF-R is not required for nafenopin-induced DNA synthesis. The EGFR-TKI also prevented progression into S phase upon stimulation with TNF(alpha), but DNA synthesis was not reduced to control levels, indicating that TNF(alpha) has a mitogenic activity in the absence of EGF signalling. Therefore, although TNF(alpha) can act as a priming factor for growth factors such as EGF, nafenopin does not appear to act via this mechanism.

Animals↗
PubMed1998

Peroxisome proliferator-activated receptor (PPAR) alpha-regulated growth responses and their importance to hepatocarcinogenesis.

Peroxisome proliferators (PPs) are a class of non-genotoxic rodent hepatocarcinogens that act by perturbing liver growth regulation. We have demonstrated previously that PPs suppress both spontaneous rat hepatocyte apoptosis and that induced by exogenous stimuli such as transforming growth factor-beta1 (TGF beta1). More recently, we have demonstrated that PPs can suppress apoptosis induced by more diverse stimuli such as DNA damage or ligation of Fas, a receptor related to the tumour necrosis factor alpha (TNF alpha) family of cell surface receptors. PPs transcriptionally activate the peroxisome proliferator activated receptor-alpha, PPAR alpha, a member of the nuclear hormone receptor superfamily. We investigated whether activation of PPAR alpha mediates the suppression of rat hepatocyte apoptosis induced by PPs. We isolated a naturally occurring variant form of PPAR alpha (hPPAR alpha-6/29) from human liver by PCR cloning. hPPAR alpha-6/29 shared the ability of mPPAR alpha to bind to DNA but, unlike mPPAR alpha, could not be activated by PPs. Furthermore, hPPAR alpha-6/29 could act as a dominant negative regulator of PPAR-mediated gene transcription. When introduced into primary rat liver cell cultures by transient transfection, hPPAR alpha-6/29 prevented the suppression of hepatocyte apoptosis by the PP nafenopin, but not that seen in response to phenobarbitone (PB), a non-genotoxic carcinogen whose action does not involve PPAR alpha. The suppression of hepatocyte apoptosis was abrogated completely even though only 30% of hepatocytes were transfected, suggesting the involvement of a soluble factor. Recent data have suggested that TNF alpha, perhaps released by liver Kupffer cells in response to PPs, may play a key role in mediating the effects of PPs on hepatocyte growth regulation.

Animals↗
PubMed1998

Evidence for the suppression of apoptosis by the peroxisome proliferator activated receptor alpha (PPAR alpha).

Peroxisome proliferators (PPs) are a class of nongenotoxic rodent hepatocarcinogens. We have demonstrated previously that PPs suppress both spontaneous rat hepatocyte apoptosis and that induced by exogenous stimuli such as transforming growth factor-beta1 (TGFbeta1). PPs transcriptionally activate the peroxisome proliferator activated receptor-alpha (PPAR alpha), a member of the nuclear hormone receptor superfamily. Here, we investigate whether activation of PPAR alpha mediates the suppression of rat hepatocyte apoptosis induced by PPs. We isolated a naturally occurring variant form of PPAR alpha (hPPAR alpha-6/29) from human liver by PCR cloning. Electrophoretic mobility shift assays (EMSA) demonstrated that hPPAR alpha-6/29 shared the ability of mPPAR alpha to heterodimerise with the retinoid X receptor (RXR) and bind to DNA. When hPPAR alpha-6/29 was transfected into Hepa1c1c7 cells together with a reporter plasmid containing a PPAR response element (PPRE), hPPAR alpha-6/29, unlike mPPAR alpha, could not be activated by PPs. Furthermore, hPPAR alpha-6/29 could act as a dominant negative regulator of PPAR-mediated gene transcription since increasing concentrations of hPPAR alpha-6/29 abrogated the activation of co-transfected mPPAR alpha. When introduced into primary rat liver cell cultures by transient transfection, hPPAR alpha-6/29 prevented the suppression of hepatocyte apoptosis by the PP nafenopin, but not that seen in response to phenobarbitone (PB), a nongenotoxic carcinogen whose action does not involve PPAR alpha. The suppression of hepatocyte apoptosis was abrogated completely even though only 30% of hepatocytes were transfected, suggesting the involvement of a soluble factor. These data indicate that activation of rat liver PPAR alpha provides a survival signal for hepatocytes, preventing their death in response to apoptotic stimuli.

Amino Acid Sequence↗
PubMed1997

Sexually transmitted diseases among teenagers in England and Wales.

A profile of sexually transmitted diseases (STDs) and HIV infections among teenagers in England and Wales was obtained from reports of newly diagnosed STDs among teenagers attending genitourinary medicine (GUM) clinics in 1995, laboratory reports of newly diagnosed HIV infections between 1985 when reporting began and the end of 1995, and the prevalence of HIV (unlinked anonymous programme) among teenagers attending genitourinary medicine clinics and antenatal clinics in 1994 and 1995. STD reports were analysed by sex, age group, and place of residence of patients--whether in the NHS Thames regions or elsewhere in England and Wales. High rates of STDs were reported in teenagers, particularly in girls. The incidences of gonorrhoea, chlamydia infection, and first attack genital wart infections were higher in teenage girls than in any other age group. Boys under 16 years of age had substantially higher rates of infection with all STDs in the Thames regions than elsewhere. Rates of gonorrhoea in teenagers of both sexes in the Thames regions were more than twice those in the rest of the country. Infection rates for genital herpes, and chlamydia in girls, were also higher in the Thames regions, although the geographical differences were less marked. The seroprevalence of HIV among heterosexual teenagers was very low. In contrast, 226 HIV infections among teenage boys had probably been acquired through sexual intercourse with other males. Unlinked anonymous testing revealed HIV antibody in 7.5% of routinely collected serology specimens taken from teenage homosexual or bisexual males attending GUM clinics in London. The high rates of STDs among teenage girls and all teenagers in the Thames regions make these groups a high priority for sexual health promotion, with special consideration given to homo/bisexual male teenagers. Detailed surveillance of risk factors for STDs, and further studies of teenage sexual behaviour will help to effectively target resources to improve the sexual health of teenagers in England and Wales.

Adolescent↗
PubMed1997

Amino acid residues in both the DNA-binding and ligand-binding domains influence transcriptional activity of the human peroxisome proliferator-activated receptor alpha.

We have investigated the basis of the lack of activity of a natural variant human peroxisome proliferator-activated receptor alpha, hPPARalpha6/29. A subcloning approach was used to change the four variant amino acids in the hPPARalpha6/29 sequence, individually and in combination, to those found in an active human PPARalpha. Individual amino acid "back mutations" were unable to confer on hPPARalpha6/29 the ability to be activated by peroxisome proliferators in a transient transfection assay. Although hPPARalpha6/29 was able to bind specifically to DNA in the presence of the retinoid X receptor alpha (RXRalpha), the complete restoration of receptor transcriptional activity required two separate back mutations of the hPPARalpha6/29 sequence, namely amino acid 123 in the DNA binding domain, and amino acid 444 close to the C-terminus. This suggests that sequences in the PPARalpha DNA binding domain influence other receptor functions besides DNA binding.

Alanine↗
PubMed1996

Peroxisome proliferator-activated receptors: structures and function.

We have been attempting to elucidate the molecular mechanisms through which peroxisome proliferators exert their pleiotropic effects, with particular emphasis on understanding why humans appear unresponsive to these compounds. There is a wealth of data to implicate the peroxisome proliferator-activated receptor alpha (PPAR alpha) in mediating these effects in rodent species; PPAR alpha is expressed in tissues that show physiological changes in response to PPs, is transcriptionally activated in vitro by a variety of PPs, and it has been recently demonstrated that mice lacking this receptor are refractory to the effects of clofibrate and Wy-14,643, at least in the short term. It is conceivable that differences in PPAR alpha between responsive rodent and unresponsive human subjects may provide the key to understanding the basis of this species variation in response, and with this in mind we have been studying the biology of PPAR alpha in humans and looking at interindividual variation. There is already published evidence, albeit on only two sequences, for structural and functional polymorphism in human PPAR alphas. We have extended these findings, and shown that: There is considerable variation in hPPAR alpha cDNAs obtained from different individuals, both at the gross structural level (lack of a coding exon) and of a more subtle nature (single base changes leading to amino acid substitutions). One such cDNA, the sequence of which differs at only three amino acids from that published, encodes a receptor that is incapable of transcriptional activation by potent PPs. The degree to which hPPAR alpha transcripts are expressed in human livers can vary by up to an order of magnitude between individuals. The tissue-specific expression profile of PPAR alpha in humans is very different from that in rat and mouse. In particular, the human liver contains generally low levels of PPAR alpha in contrast to the responsive rodents, in which potent PPs cause liver tumors. Taken together, these data suggest first that human and rodent PPAR alphas differ according to a number of molecular and biochemical criteria, and secondly that there is a degree of interindividual variation in PPAR alpha structure and function. Studies are ongoing to clarify this further, but human polymorphism may go some way towards explaining the apparent paradox that active PPAR alpha receptors can be isolated from an "unresponsive" species.

Acyl-CoA Oxidase↗
PubMed1987

Comparative efficacies of erythromycin-sulfisoxazole and cefaclor in acute otitis media: a double blind randomized trial.

A prospective double blind trial compared the fixed combination of erythromycin-sulfisoxazole (E/S) with cefaclor in the treatment of acute otitis media. One hundred nineteen children in six centers across Canada were studied. Diagnostic tympanocentesis of 134 ears yielded 135 bacterial isolates: Streptococcus pneumoniae (42%); Haemophilus influenzae (21%); Branhamella catarrhalis (10%); Streptococcus pyogenes (5%); and other bacteria (22%). Seventy-seven percent of strains of B. catarrhalis and 14% of strains of H. influenzae were beta-lactamase producers. E/S exhibited greater in vitro activity against H. influenzae and B. catarrhalis. Twenty-three patients had bacteriologically sterile middle ear fluid. The overall clinical outcome at Days 10 and 31 was identical in both treatment groups. Otoscopic findings improved more rapidly in the E/S group than in the cefaclor group at 10 and 31 days (P less than or equal to 0.04). In cases where pre-treatment middle ear fluid was negative on routine bacterial culture, complete cure at 10 days was observed in 75% of patients treated with E/S but only in 14% of those treated with cefaclor (P = 0.02). Side effects were infrequent and comparable between the test drugs. E/S is at least as effective as cefaclor in the management of acute otitis media and may be superior, particularly for cases not yielding bacteria on routine culture.

Acute Disease↗