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A J Simpson

Publications and source records attributed to A J Simpson.

At least 91 records · Page 5Linked to original sources
PubMed1998

Adenovirus-mediated expression of an elastase-specific inhibitor (elafin): a comparison of different promoters.

This report describes the design and construction of three recombinant adenoviruses of serotype 5 (Ad5) expressing elafin (EL), also called elastase-specific inhibitor. Three promoters were chosen to drive the synthesis of elafin: the small (380 bp) human cytomegalovirus promoter (HCMV), the Ad2 major late promoter (MLP) and the mouse cytomegalovirus (MCMV) promoter. Human alveolar epithelial cells (A549), as well as rat and human primary pulmonary fibroblasts were infected with Ad5-HCMV-EL, Ad5-MLP-EL, Ad5-MCMV-EL and with the control Ad5-dl70/3. The MCMV promoter was the most efficient promoter in all cells studied. MLP was the least efficient promoter Intermediate between MCMV and MLP was HCMV which was able to induce significant amounts of elafin, particularly in human A549 cells. When compared in vivo in rat lungs, results were similar; MCMV was the only promoter which induced significant amounts of elafin as assessed by Northern blot analysis and ELISA, even with a low dose of virus (3 x 10(8) p.f.u.). Our data indicate that the MCMV promoter is the promoter of choice for the strong induction of adenovirus-mediated transgenes in the lung and suggest its suitability both in rodent experimental models and in humans for investigative and therapeutic purposes.

Adenoviridae↗
PubMed1998

Not everything acid fast is Mycobacterium tuberculosis--a case report.

The Ziehl-Neelsen (ZN) stain is important in identifying organisms that are acid fast, principally Mycobacterium tuberculosis. However, decolorisation with a weaker acid concentration (for example 1% hydrochloric acid), often used in ZN staining in histology, can result in a wider variety of organisms appearing acid fast and can be a cause of misidentification. To illustrate this point, a patient is described with pulmonary nocardiosis who was misdiagnosed as having tuberculous empyema on pleural biopsy.

Aged↗
PubMed1998

Genetic variability and identification of the intermediate snail hosts of Schistosoma mansoni.

Studies based on shell or reproductive organ morphology and genetic considerations suggest extensive intraspecific variation in Biomphalaria snails. The high variability at the morphological and genetic levels, as well as the small size of some specimens and similarities between species complicate the correct identification of these snails. Here we review our work using methods based on polymerase chain reaction (PCR) amplification for analysis of genetic variation and identification of Biomphalaria snails from Brazil, Argentina, Uruguay and Paraguay. Arbitrarily primed-PCR revealed that the genome of B. glabrata exhibits a remarkable degree of intraspecific polymorphism. Low stringency-PCR using primers for 18S rRNA permitted the identification of B. glabrata, B. tenagophila and B. occidentalis. The study of individuals obtained from geographically distinct populations exhibits significant intraspecific DNA polymorphism, however, specimens from the same species, exhibit some species specific LSPs. We also showed that PCR-restriction fragment of length polymorphism of the internal transcribed spacer region of Biomphalaria rDNA, using Ddel permits the differentiation of the three intermediate hosts of Schistosoma mansoni. the molecular biological techniques used in our studies are very useful for the generation of new knowledge concerning the systematics and population genetics of Biomphalaria snails.

Animals↗
PubMed1998

Molecular identification of similar species of the genus Biomphalaria (Mollusca: Planorbidae) determined by a polymerase chain reaction-restriction fragment length polymorphism.

The freshwater snails Biomphalaria straminea, B. intermedia, B. kuhniana and B. peregrina, are morphologically similar; based on this similarity the first three species were therefore grouped in the complex B. straminea. The morphological identification of these species is based on characters such as vaginal wrinkling, relation between prepuce: penial sheath:deferens vas and number of muscle layers in the penis wall. In this study the polymerase chain reaction restriction fragment length polymorphism technique was used for molecular identification of these molluscs. This technique is based on the amplification of the internal transcribed spacer regions ITS1 e ITS2 of the ribosomal RNA gene and subsequent digestion of these fragments by restriction enzymes. Six enzymes were tested: Dde I, Mn1 I, Hae III, Rsa I, Hpa II e Alu I. The restriction patterns obtained with DdeI presented the best profile for separation of the four species of Biomphalaria. The profiles obtained with all the enzymes were used to estimate the genetic distances among the species through analysis of common banding patterns.

Animals↗
PubMed1997

Characterization of a Schistosoma mansoni gene encoding a homologue of the Y-box binding protein.

We have cloned and characterized a Schistosoma mansoni cDNA encoding a basic protein homologous to the human Y-box binding protein 1 (YB-1). The 1.3-kb S. mansoni YB-1 transcript, which was shown to be expressed in various stages of the parasite life cycle, codes for a protein of 217 amino acids containing, towards its N-terminus, a nucleic acid binding motif, known as the cold-shock domain (CSD). This domain is 64% identical to the cold-shock domain of other members of the Y-box binding protein family and 43% identical to the cold-shock protein CspA of Escherichia coli. In S. mansoni YB-1, the cold-shock domain possess some structural characteristics that permit dimer formation as occurs in the Bacillus subtilis cold-shock protein CspB. The C-terminal region of S. mansoni YB-1 differs from the other Y-box binding proteins because of the presence of tandem repeats of Arg and Gly, suggesting the formation of a fibroin-like beta-sandwich structure. This novel folding pattern for the C-terminus of S. mansoni YB-1 might suggest a distinct specific function for this protein in the parasite.

Amino Acid Sequence↗
PubMed1997

Minilibraries constructed from cDNA generated by arbitrarily primed RT-PCR: an alternative to normalized libraries for the generation of ESTs from nanogram quantities of mRNA.

The generation of expressed sequenced tags (ESTs) depends on the arbitrary selection of individual cDNA clones from libraries. The efficiency of this process reflects the clonal structure of the library used and can be significantly increased using size selected, directional, normalized cDNA libraries. This strategy, however, is not readily applicable when mRNA is limiting, as is the case in the study of complex microorganisms such as parasites, fetal tissues or tumor biopsies. We show here that the construction and systematic sequencing of minilibraries of cDNAs produced by arbitrarily primed PCR provides an alternative means of efficiently generating ESTs in situations where only nanogram quantities of RNA are available. This methodology greatly compensates for unequal message abundance, avoids the need for complex library construction, is equally applicable to the analysis of abundant or rare biological material and is ideally suited to multicenter programmes.

Animals↗
PubMed1997

Some molecular insights into schistosome evolution.

Robust phylogenies based on molecular data for species within the genus Schistosoma have been generated in recent years. The considerable progress made in understanding the relationships between many of the 19 recognised species of Schistosoma is reviewed with particular attention being given to the detection and analysis of parasite variation as shown by studies on ribosomal RNA genes, mitochondrial DNA and RAPDs. For the most part, molecular phylogenies agree with observations based on morphological or life-history characteristics. It is clear that the parasites of man do not form a monophyletic group and that close relationships exist between parasites within species groups, especially in the S. haematobium group of species. The S. japonicum group appears to be the most divergent of the species groups and yet little DNA sequence variation has been observed between various isolates of S: japonicum. Some of the less studied schistosomes have yet to be examined at the molecular level and may prove to be interesting links between the species groups as has recently been shown with S. hippopotami. The power of molecular approaches for the analysis of schistosomes at the population and individual level is now apparent, especially for S. mansoni. Important questions remain concerning the maintenance of parasite diversity and how schistosomes respond to selection pressures imposed either during natural progression through the life-cycle or through drug treatment or vaccination. Gene discovery and gene mapping projects are leading to a better understanding of the schistosome genome and can be expected to contribute significantly to future comparative evolutionary studies.

Animals↗
PubMed1997

The natural somatic mutation frequency and human carcinogenesis.

Much recent attention has been paid to the important role of the DNA mismatch repair system in controlling the accumulation of somatic mutations in human tissues and the association of mismatch repair deficiency with carcinogenesis. In the absence of an intact mismatch repair system, cells accumulate mutations at a rate some 1000 times faster than normal cells, and this mutator phenotype is easily measured by the detection of the formation of new variant alleles at microsatellite loci. However, the mismatch repair system is not 100% efficient, even when intact, and the pattern of microsatellite alterations in a wide variety of tumors is consistent with these being due to clonal amplification from tissues that are genetically heterogeneous at microsatellite loci rather than mismatch repair deficiency in the tumor itself. On this basis, it can be estimated that the mutation frequency of microsatellites in normal human tissues is approximately 10(-2) per locus per cell. Similarly, a frequency of mutation at minisatellite loci in normal tissues of around 10(-1) per locus per cell can be estimated. Such elevated levels of mutation are consistent with a recent study of the frequency of HPRT mutation in human kidneys that demonstrated these to be frequent (average 2.5 x 10(-4) in individuals of 70 years or more) and exponentially related to age. Taken as a whole, the data suggest that somatic mutation in human epithelial cells may be some 10-fold higher than in peripheral blood lymphocytes and that the underlying rate of spontaneous mutation is sufficient to account for a large proportion of human carcinogenesis without the need to evoke either stepwise alteration to a mutator phenotype of clonal expansion at all the mutation steps in carcinogenesis. The exponential increase in mutation frequency with age is predictable on the basis that the mutation rate is controlled at the level of repair and that mutation in genes that affect the efficiency of these processes will gradually increase the underlying rate. In addition, the age relatedness of mutation frequency strongly supports the concept that mutation is cell division dependent and that cellular proliferation per se is an important risk factor for cancer. Comparison of somatic mutations with those in the human germline mutation suggests common mechanistic origins and that the high levels of somatic mutation that occur are a direct reflection of the germline mutation rate selected over evolutionary time. Thus, the somatic accumulation of mutations can be seen as a natural process within the human body and cancer a normal part of the human life cycle. This point of view may explain why it has been so difficult to significantly reduce cancer incidence and suggests that, for this to be achieved, the means of altering the natural somatic mutation rate needs to be identified.

Cell Division↗
PubMed1997

The effects of chronic smoking on the fibrinolytic potential of plasma and platelets.

We studied tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) in healthy individuals divided by smoking habit into current smokers, former smokers and non-smokers (who had never smoked). Plasma PAI-1 antigen was significantly higher in smokers than in non-smokers with intermediate levels in former smokers. A similar trend was observed for plasma PAI activity but this did not reach statistical significance. Platelet PAI-1 and plasma t-PA were not significantly different when comparing the three groups. After venous occlusion t-PA rose significantly in all groups; no significant change in plasma PAI-1 was observed. The ratio of t-PA to PAI-1 in plasma was similar in non-smokers and former smokers but lower in smokers, suggesting that there is at least partial restoration of plasma fibrinolytic potential after smoking cessation. Plasma PAI-1 antigen and PAI activity correlated with estimated pack-years of cigarettes smoked among smokers and former smokers. When all subjects were studied collectively, plasma PAI-1 correlated strongly with plasma t-PA and triglycerides: plasma t-PA also correlated strongly with triglycerdes. We conclude that chronic smoking is associated with impaired fibrinolysis in plasma and that this largely reflects elevated plasma PAI-1 in smokers. Smoking does not appear to affect the response to venous occlusion. The postulated effect of chronic smoking on plasma PAI-1 may be mediated by the influence of smoking on triglycerides and insulin resistance. Stopping smoking appears to return impaired fibrinolysis towards normal. Smoking does not quantitatively affect the platelet pool of PAI-1. Smoking habit should be controlled for in clinical analyses of PAI-1 and t-PA.

Adult↗
PubMed1997

Highly sensitive single-step PCR protocol for diagnosis and monitoring of human cytomegalovirus infection in renal transplant recipients.

A multiplex, single-step PCR protocol for the detection of human cytomegalovirus (HCMV) DNA is described. The protocol amplifies regions of the viral LA and IE genes and employs elevated temperatures for both reagent mixing and primer annealing together with product detection by silver staining on polyacrylamide gels. This assay detects one to five HCMV genomes in clinical samples containing up to 100 ng of human DNA, a level of sensitivity equivalent to that of more complex assays involving either nested PCR or postamplification hybridization. As well as being of importance in clinical situations where high-sensitivity qualitative diagnosis is required, this assay is also applicable to the monitoring of HCMV infection in renal transplant recipients. Due to its multiplex format the assay provides quantitative information, in that samples from which a single target is amplified contain on average sevenfold fewer viral genomes per 10(6) leukocytes than those from which both targets are amplified. When weekly blood leukocyte DNA preparations from renal transplant patients were assayed, findings of three consecutive tests in which both HCMV targets were amplified were highly indicative of patients who had developed very high loads of HCMV (100% sensitivity, 88% specificity). We thus show that the same simple PCR assay which permits highly sensitive HCMV diagnosis can also be used for the efficient identification of transplant recipients at risk of clinically significant infection.

Base Sequence↗
PubMed1997

PCR amplification of the mitochondrial DNA minisatellite region to detect Schistosoma mansoni infection in Biomphalaria glabrata snails.

Schistosoma infection in Biomphalaria glabrata can be detected by either exposing snails to light to induce cercarial shedding or by squeezing them between glass slides to detect parasites in the digestive gland and other regions. The methods available are inefficient for identification of prepatent infections and do not allow the diagnosis of infection in snails that die before arriving in the laboratory. Furthermore, infection is undetectable after migration of sporocysts from the head-foot region of the snail. We examined the use of polymerase chain reaction (PCR) amplification of minisatellite repeats from Schistosoma mansoni mitochondrial DNA (mtDNA) to identify snail infection. We found that amplification of mtDNA under low stringency conditions (LS-PCR) allowed for the identification of specific S. mansoni infection in Biomphalaria snails. To confirm these results, specific amplification reactions were performed using 2 sets of primers that allowed for the diagnosis of infection and an internal control of the reaction (multiplex PCR). Results obtained using multiplex PCR demonstrated the ability of the assay to detect S. mansoni-specific infection. Thus, LS-PCR as well as specific multiplex PCR allow for the detection of prepatent infections and show high specificity for S. mansoni in comparison with other trematode infections in either living or dead snails.

Animals↗
PubMed1997

Partially degraded DNA of parasitological interest serves as an adequate template for the production of random amplified polymorphic DNAs (RAPDs).

Genomic DNA was extracted from Schistosoma mansoni adult worms and deliberately degraded by sonication. Samples with varying average molecular weight were subjected to RAPD (randomly amplified polymorphic DNA) analysis using the primer 3307 (5'-AGTGCTACGT-3') and other primers. Reproducible and complex DNA banding patterns were obtained, irrespective of the extent of DNA degradation. The same amplification protocol was employed with naturally degraded Biomphalaria glabrata genomic DNA and the primer 3302 (5'-CTGATGCTAC-3'). Again, reproducible RAPD patterns resulted. The experiment shows that the partially degraded DNA samples can be safely compared in RAPD analysis without artifactual bands compromising the accuracy of genetic analysis. Thus RAPD analysis permits complex and reproducible DNA fingerprinting from degraded samples of parasitological interest.

Animals↗
PubMed1996

Evidence that cellular immune responses to soluble and membrane associated antigens are independently regulated during human schistosomiasis mansoni.

We have made a comparative analysis of human cellular and antibody responses to membrane associated adult worm antigens (Mb-A), soluble adult worm antigens (SWAP) and soluble egg antigens (SEA) derived from Schistosoma mansoni. Chronically infected patients with the intestinal (I) and hepatosplenic (HS) forms of the disease as well as non-infected putative immune 'endemic normals' (EN), were studied. We observed that the cellular responses, of individuals, to the two adult worm preparations, SWAP and Mb-A, may be distinct and can be related to the occurrence of resistance or pathology. The resistant group (EN) presented higher levels of both cellular proliferation, and IFN-gamma production, in response to Mb-A as compared with SWAP whereas HS individuals presented higher levels of cellular proliferation to SWAP as compared with Mb-A. Individuals with intestinal disease had similar levels of proliferation to both antigens. The response to SEA by all groups was generally similar, and not predictive of any clinical form. The specific antibody response to the three antigens were in general higher among infected patients than in resistant EN individuals. These results support the hypothesis that the response to adult worm antigens may be pivotal in determining both the development of resistance and severity of disease.

Adolescent↗
PubMed1996

A controlled clinical trial of dorzolamide: a single-centre subset of a multicentre study.

PURPOSE: To assess the safety and intraocular pressure (IOP)-lowering activity of 2% dorzolamide (topical carbonic anhydrase inhibitor), compared to 0.5% timolol and 0.5% betaxolol eyedrops. METHODS: A parallel, masked, randomised one-year clinical trial was conducted in 16 patients with open-angle glaucoma or ocular hypertension, being a subset of a multicentre study which enrolled 523 subjects. Patients had IOP > 22 mmHg in one eye at baseline following washout of ocular hypotensive medications and were then randomised in a 3:1:1 ratio to receive 2% dorzolamide thrice daily, 0.5% timolol twice daily or 0.5% betaxolol twice daily respectively. IOP was measured at Hour 2 (morning peak), Hour 5 and Hour 8 (afternoon trough for dorzolamide) at baseline, Weeks 2 and 4 and Months 2, 3, 6, 9 and 12. RESULTS: Topical dorzolamide 2% solution was well tolerated and safe. Mean IOP for dorzolamide at Hour 2 was 29.1 mmHg at baseline and 20.8 mmHg on treatment at one year, a 28.5% change. Mean IOP for dorzolamide at Hour 8 was 24.5 mmHg at baseline and 20.2 mmHg on treatment at one year, a 17.6% change. Comparable percent changes for timolol and betaxolol were 43.2/25.7 mmHg at Hour 2 and 21.9/13.5 mmHg at Hour 8 respectively. CONCLUSIONS: Dozolamide 2% given thrice daily was well tolerated and safe, with a clinically significant effect on IOP comparable to betaxolol 0.5% twice daily, but not as great as timolol 0.5% twice daily.

Adrenergic beta-Antagonists↗