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Biomedical subjects

M A Miles

Publications and source records attributed to M A Miles.

At least 37 records · Page 2Linked to original sources
PubMed1998

On the molecular taxonomy of Trypanosoma cruzi using riboprinting.

In order to investigate the molecular taxonomy within Trypanosoma cruzi, the ribosomal small subunit (18S) gene was amplified by polymerase chain reaction (PCR) from a selection of 21 stocks and 3 outgroup taxa. Amplification products were digested with 10 restriction enzymes; restriction fragments were separated by polyacrylamide gel electrophoresis and profiles were visualized by silver staining. Upon analysis of such riboprint profiles, an estimate of pairwise phenetic distance between stocks of T. cruzi was calculated. Upon principal coordinate analysis of this data matrix, a tendency towards a bi-polar grouping of stocks was observed. These 2 groups were predominantly either zymodeme 1 stocks or zymodeme 2 stocks. The position of zymodeme 3 stocks remained intermediate between the 2 groups but did not form a coherent group by themselves. It would therefore appear premature to warrant division of T. cruzi into 2 discrete taxa or subspecies until the relationships of further zymodeme 3 stocks are elucidated.

Animals↗
PubMed1998

Input and central processing expressed in ERP and heart rate changes to rare target and rare nontarget stimuli.

Whether late positive components of event-related potentials (ERPs) parallel changes in heart rate (HR) indicative of attention/orienting to rare stimuli has been debated. In the present study, a three-stimulus design was used, with rare target, rare nontarget, and frequent standard stimuli delivered under identical conditions except that instructions to subjects described the targets to which subjects should respond but did not describe the nontargets. In Experiment 1, stimuli varied among modalities; in Experiment 2, auditory stimuli were employed. Both ERPs and HR were consistent with automatic processing preceding two stages of controlled processing. Rare stimuli evoked larger parietal P300 and initial HR deceleration than standards. Presumably because of load-reducing effects of long interstimulus intervals, targets and nontargets were not distinguished before a late slow wave and a late phase of HR acceleration. Neither rare stimulus elicited a recognizable frontal P3a.

Acoustic Stimulation↗
PubMed1998

Random amplification of polymorphic DNA as a tool for taxonomic studies of triatomine bugs (Hemiptera: Reduviidae).

Eleven of 27 decameric primers were found to be suitable for random amplification of polymorphic DNA (RAPD) from triatomine bugs on the basis that they produced discrete profiles and distinguished among Panstrongylus megistus (Burmeister), Rhodnius prolixus Stål, and Triatoma infestans (Klug). The legs, or single leg segments, of individual bugs were used as the source of DNA so that the taxonomic value of the bug was conserved. Within the scope of the specimens studied, RAPD profiles allowed assignment to species even when bugs were kept dry for up to 12 mo. Profiles for individuals within a species were not identical. RAPD profiles, with the specimens tested, distinguished among species of 3 pairs considered to be morphologically similar and closely related, namely, Rhodnius ecuadorensis Lent & León and Rhodnius pictipes Stål; Rhodnius nasutus Stål, and Rhodnius neglectus Lent; Rhodnius prolixus Stål and Rhodnius robustus Larrousse. RAPD data conformed with the perceived affinities among these species. RAPD polymorphisms were seen with T. infestans from 3 different localities, but none of the polymorphisms was confined to 1 source. RAPD provided a molecular basis to reassess taxonomic relationships within the Triatomine subfamily. The accurate distinction of triatomine species and of intraspecific bug populations may contribute to elimination of vector-borne Chagas disease from the Americas.

Animals↗
PubMed1998

Chagas disease in the Amazon Basin: association of Panstrongylus geniculatus (Hemiptera: Reduviidae) with domestic pigs.

Just over 100 autochthonous cases of Chagas disease are reported from the Brazilian Amazon Basin. Panstrongylus geniculatus (Latreille) occurs throughout the region and is the known vector of Trypanosoma cruzi, principal zymodeme 3 (Z3) to the armadillo Dasypus novemcinctus. In the small riverine community of Furo do Rio Pau Grande, pigsties adjoining houses were heavily infested with P. geniculatus, which repeatedly attacked local inhabitants. Palm trees in the immediate vicinity were also infested. T. cruzi principal zymodeme 1 (Z1) was isolated from P. geniculatus, domestic pigs, and opossums, but no human infections were detected. The threat of endemic Chagas disease to the Amazon Basin from either domiciliation of local silvatic triatomine species, or from migration of domestic vectors, demands a program of vigilance and plans of action to eliminate household triatomine colonies.

Animals↗
PubMed1998

Clinical recovery and limited cure in canine visceral leishmaniasis treated with aminosidine (paromomycin).

Three groups of three, six, and 12 dogs with parasitologically proven clinical visceral leishmaniasis (Leishmania chagasi infection) were treated with intramuscular aminosidine sulfate at doses of 20 mg/kg/day for 15 days; 80 mg/kg/day for 20 days, and 40 mg/kg/day for 30 days, respectively. Follow-up was by parasitologic examination of bone marrow and skin, serology using the indirect immunofluorescent antibody test, and clinical examination for signs of visceral leishmaniasis or adverse effects of treatment. In animals treated with 20 mg/kg/day, for 15 days, there was dramatic clinical improvement with disappearance of conjunctivitis, increase in appetite, weight gain, and recovery of normal skin condition and a healthy coat, but parasitologic relapse occurred between 50 and 100 days after initiation of treatment. Adverse effects were seen with treatment with 80 mg/kg/day for 20 days; three dogs died during or just after treatment, two showed temporary recovery, and one showed total clinical and parasitologic cure that was maintained for four years. Although adverse effects and relapses were seen in some dogs treated with 40 mg/kg/day for 30 days, three of 12 dogs showed complete parasitologic and clinical cure that was sustained for at least four years. Aminosidine treatment cannot be recommended as an alternative to the humane destruction of dogs for the control of canine visceral leishmaniasis because ineffective treatment may prolong carrier status or encourage development of drug resistance. This drug may be a therapeutic option if there is no danger of a dog acting as a reservoir of infection. Achievement of clinical recovery and limited cure with aminosidine suggests that further trials would be of value, possibly in combination with other anti-leishmanial drugs and with supportive measures to reduce adverse effects.

Animals↗
PubMed1997

Overexpression of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, is associated with enhanced metacyclogenesis.

Cruzipain, the major cysteine proteinase of Trypanosoma cruzi has been proposed as a target for chemotherapy against Chagas' disease. To investigate the role of cruzipain we transfected T. cruzi epimastigotes with a recombinant cosmid containing approximately 20 tandemly repeated cruzipain genes. Transformed cells had multiple episomal copies of the vector and exhibited considerable overexpression of cruzipain activity. The upregulation was maintained throughout the parasite life-cycle, and electrophoretic detection techniques indicated that overexpression was correlated with correctly processed enzyme. Immunoelectron microscopy demonstrated that cruzipain had the same developmentally regulated subcellular localisation in transformed and non-transformed cells. In the insect epimastigote form, the enzyme was restricted to vesicles of the endosomal/lysosomal system, whereas in the intracellular forms it was also readily detectable on the cell surface. Phenotypic analysis of the transformed parasites showed that they had an enhanced ability to undergo metacyclogenesis and suggested an association between overexpression of cruzipain and increased resistance to the cysteine proteinase inhibitor Cbz-Phe-Phe-CHN2 (where Cbz is benzoyloxycarbonyl). The increased resistance, however, was less than might be expected if cruzipain was the primary target of the inhibitor. Transgenic parasites did not exhibit increased infectivity.

Animals↗
PubMed1997

Use of polymerase chain reaction-based single strand conformational polymorphism and denaturing gradient gel electrophoresis methods for detection of sequence variation of ribosomal DNA of Trypanosoma cruzi.

Polymerase chain reaction (PCR) was used to amplify the V1 region of the small sub-unit (18S) ribosomal DNA gene from representative strains of Trypanosoma cruzi. In order to screen for sequence variation, amplification products were subsequently analysed by single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) techniques. SSCP could not detect sequence variation within T. cruzi, although electrophoretic profiles were clearly distinct from both Leishmania donovani and Leishmania braziliensis. DGGE could differentiate strains of T. cruzi and it appears that there are at least 2 18S V1 sequences of this multi-gene family within each strain examined, contrasting with Leishmania spp. where only 1 was identified. This is the first application of PCR-linked SSCP and DGGE analysis for differentiating parasitic protozoa.

Animals↗
PubMed1996

Dissemination of Leishmania (Viannia) braziliensis.

Destructive human mucocutaneous leishmaniasis may appear many years after the primary cutaneous infection with Leishmania (Viannia) braziliensis. Hamsters (Mesocricetus auratus) were infected with metacyclic L. braziliensis promastigotes. It was found that secondary metastatic visceral lesions could arise from a primary cutaneous lesion, or secondary cutaneous lesions from a primary visceral lesion. Parasites in the viscera were shown to be viable, multiplying and capable of metastasis to either secondary visceral or cutaneous sites. The finding of an early metastasis in the wall of a small cutaneous vessel indicates that dissemination can occur by the haematogenous route. Slow growing organisms in viscera may thus be a source for late metastasis to mucocutaneous sites or for systemic relapse after immunosuppression.

Animals↗
PubMed1996

Identification of the C-terminal region of 70 kDa heat shock protein from Leishmania (Viannia) braziliensis as a target for the humoral immune response.

A Leishmania (Viannia) braziliensis (Lb) promastigote cDNA library in lambda gt11 was screened with patients' sera with the aim of identifying antigens specifically related to mucocutaneous leishmaniasis (MCL). One of the clones isolated, 133P, consistently reacted with MCL sera; it was sequenced and found to encode the C-terminal three-quarters of a protein belonging to the highly conserved Hsp70 family. Since Hsp70 proteins from different species tend to be less conserved through the C-terminal end, it was predicted that this region would be more antigenic and was likely to bear the discriminatory epitopes. In order to test this hypothesis, the N- and C-terminal halves of the polypeptide encoded by 133P, 133P-N and 133P-C, respectively, were expressed in Escherichia coli. Immunoblotting analysis demonstrated that 133P-C reacted more strongly with a pool of MCL sera than 133P-N, and both recombinant proteins reacted faintly with pools of cutaneous (CL) and visceral (VL) leishmaniasis sera. These results confirmed the predicted epitope location in the C-terminal region. The 133P-C fragment was also expressed as a fusion protein with glutathione-S-transferase (GST-133P-C), affinity-purified with glutathione-agarose and tested by ELISA with individual sera. From 46 Lb-infected patients, 41 sera (89%) were positive, no cross-reactivity was observed with healthy, Trypanosoma cruzior L. amazonensis-infected individuals. Despite a relatively high cross-reactivity with VL sera, the enhanced humoral response of MCL as compared with CL patients might be interesting for studies on disease aggravation.

Amino Acid Sequence↗
PubMed1996

Functional status in depressed patients: the relationship to disease severity and disease resolution.

BACKGROUND: We set out to measure the impact of depression and its clinical resolution on patients' functional status. METHOD: The Work and Social Disability Scale (WSDS), a five-category investigator-rated scale measuring patient functional status, was completed at baseline and study discontinuation in a 56-day, open, uncontrolled study evaluating the safety of a sustained release (SR) formulation of bupropion in 3167 patients at 105 sites. To be included in the study, patients had to be 18 years or older, have a diagnosis of depression, and be considered appropriate for treatment with bupropion SR. The proportion of patients in each WSDS category, for those patients taking more than 7 days of bupropion SR (N = 2915), was assessed at screen and study discontinuation. The percentage of patients with improved WSDS scores at 56 days was also measured for all patients and correlated with patient and treatment characteristics. RESULTS: Of the patients entering the trial, 61.8% were markedly or severely impaired in their work or social activities, and only 5.4% were mildly or not impaired. At study discontinuation, more than 54% of patients were judged by the investigator to have very much or much improvement in their clinical symptoms. Results on the WSDS correlated with the clinical improvements; only 22.3% were markedly or severely impaired; and 50.0% were mildly or not impaired at study discontinuation. In addition, 63.9% of patients had less work or social disability at the end of the trial than at study entry. Functional status improved more in patients who had not previously been treated for the episode, had more severe depression at study entry, and had a higher dose and duration of treatment with bupropion SR. CONCLUSION: The results show that depression results in significant impairment in patients' functional status. Functional status improved in patients treated with bupropion SR for up to 56 days. This improvement was highly correlated with improvement in clinical symptoms and was related to patient characteristics at study entry as well as to treatment patterns during the study.

Adolescent↗
PubMed1996

Genetic exchange as a possible source of genomic diversity in sylvatic populations of Trypanosoma cruzi.

Thirty six stocks of Trypanosoma cruzi isolated from sylvatic mammals (32 Didelphis marsupialis and one Philander opossum) and triatomine bugs (Rhodnius robustus and one unidentified bug) in the Amazonian forest by Carajas, Brazil were characterized by isoenzyme and random amplified polymorphic DNA (RAPD) analysis as belonging to principal zymodeme '1 (Z1). Two different homozygous phenotypes and the corresponding heterozygous phenotype were found for phosphoglucomutase with an observed frequency almost identical with that predicted by the theoretical Hardy-Weinberg distribution. Parental and hybrid profiles were also suggested by RAPD analysis, which allowed exclusion of mixed parental strains from the hybrids: isoenzyme and RAPD profiles of biological clones were also indistinguishable from those of uncloned stocks. Trypanosoma cruzi stocks from widely separated geographic origins in Central and South America gave similar RAPD profiles that allowed them to be recognized as being Z1. These results indicate that genetic exchange could contribute to the generation of genetic diversity during the sylvatic cycle of T. cruzi, and this may have epidemiologic and taxonomic implications.

Animals↗
PubMed1995

Estimating increases in outpatient dialysis costs resulting from scientific and technological advancement.

The Medicare program's base payment rate for outpatient dialysis services has never been adjusted for the effects of inflation, productivity changes, or scientific and technological advancement on the costs of treating patients with end-stage renal disease. In recognition of this, Congress asked the Prospective Payment Assessment Commission to annually recommend an adjustment to Medicare's base payment rate to dialysis facilities. One component of this adjustment addresses the cost-increasing effects of technological change--the scientific and technological advances (S&TA) component. The S&TA component is intended to encourage dialysis facilities to adopt technologies that, when applied appropriately, enhance the quality of patient care, even though they may also increase costs. We found the appropriate increase to the composite payment rate for Medicare outpatient dialysis services in fiscal year 1995 to vary from 0.18% to 2.18%. These estimates depend on whether one accounts for the lack of previous adjustments to the composite rate. Mathematically, the S&TA adjustment also depends on whether one considers the likelihood of missing some dialysis sessions because of illness or hospitalization. The S&TA estimates also allow for differences in the incremental costs of technological change that are based on the varying advice of experts in the dialysis industry. The major contributors to the cost of technological change in dialysis services are the use of twin-bag disconnect peritoneal dialysis systems, automated peritoneal dialysis cyclers, and the new generation of hemodialysis machines currently on the market. Factors beyond the control of dialysis facility personnel that influence the cost of patient care should be considered when payment rates are set, and those rates should be updated as market conditions change. The S&TA adjustment is one example of how the composite rate payment system for outpatient dialysis services can be modified to provide appropriate incentives for producing high-quality care efficiently.

Health Care Costs↗
PubMed1995

Detection of parasites of the Leishmania donovani-complex by a polymerase chain reaction-solution hybridization enzyme-linked immunoassay (PCR-SHELA).

A polymerase chain reaction (PCR) based on the detection of the Lmet2 repeat sequence specific to members of the Leishmania donovani-complex is described. To improve PCR specificity, a post-PCR hybridization step is often performed but this usually involves an entirely new procedure with additional manipulations, expense and time. We have simplified this post-PCR hybridization by developing a strategy which includes the probe in the PCR and enables the hybridization to be performed automatically as part of the PCR programme. The hybrids are afterwards detected by capture in microtitre wells and colorimetric visualization. This method, which we have termed PCR-solution hybridization enzyme-linked immunoassay (PCR-SHELA), is rapid, able to detect less than 5 cultured parasites and is specific for parasites of the Leishmania donovani-complex. We also describe the application of PCR-SHELA to the detection of amastigotes in various tissues of infected laboratory animals.

Animals↗
PubMed1994

Precise location of sequential dengue virus subcomplex and complex B cell epitopes on the nonstructural-1 glycoprotein.

The reactions of a panel of 34 mouse monoclonal antibodies (MAbs) specific for the dengue-2 virus nonstructural-1 glycoprotein (NS1), were analysed using 174 overlapping synthetic nonameric peptides covering the entire sequence. Using this methodology, four epitopes were identified. One pair of MAbs, which defined a dengue-2/4 virus subcomplex epitope (24C: amino acids 299-309) using native NS1 proteins, showed the same reaction pattern with synthetic peptides containing the corresponding NS1 sequences of each virus serotype. One amino acid substitution, present in the sequences from the dengue-1/3 virus subcomplex abrogated almost all reaction by these MAbs. A dengue complex epitope (LX1: amino acids 111-121) was also located and peptides containing the sequences of each serotype were shown to contain only antigenically silent amino-acid substitutions. In contrast, MAbs which defined a dengue type-specific epitope (LD2: amino acids 25-33) and another dengue subcomplex epitope (24A: amino acids 61-69) failed to show the same reaction profiles using peptides of each serotype, suggesting that these determinants were partially dependent upon conformation. The LX1 epitope is a good candidate for further trials aimed at generating cross-protective immune responses to these viruses without the risk of antibody-dependent enhancement.

Amino Acid Sequence↗
PubMed1994

The structure, organization, and expression of the Leishmania donovani gene encoding trypanothione reductase.

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in the trypanosomatids. We report here the cloning by expression of the Leishmania donovani gene. It is single copy, expresses a 2.6-kb transcript and a 52-kDa protein and is located on a 1.1-Mbp chromosome. The 491 amino acid sequence has 76% similarity to Crithidia fasciculata and 67% similarity to Trypanosoma cruzi, Trypanosoma congolense and Trypanosoma brucei TR. Residues recognising the adenosine pyrophosphate moiety of NADPH and FAD, and residues in the catalytic site segment (A47-A67) involving electron transfer from TR to trypanothione disulphide (T(S)2) were completely conserved. Thus inhibitors of TR are likely to be active against the enzyme from all the parasitic trypanosomatids. Two peptide inserts (39-47, 131-140) seen in other TR genes and a C-terminal extension of 19 residues were also present. When the gene was introduced back into L. donovani at high copy number using the pTEX expression vector, we detected elevated expression of TR RNA and a 14-fold increase in TR activity. Transfection and overexpression of the TR gene will facilitate studies of gene function and of the dependence of trypanosomatids on TR for protection against oxidative stress.

Amino Acid Sequence↗
PubMed1994

A putative Leishmania panamensis/Leishmania braziliensis hybrid is a causative agent of human cutaneous leishmaniasis in Nicaragua.

As part of a survey of human leishmaniasis in Nicaragua we examined phenotypic and genotypic characteristics of 40 Leishmania isolates. We identified 3 distinct parasites associated with cutaneous disease in this area; Leishmania panamensis (40% of cases), Leishmania braziliensis (33%), and a strain which exhibits the heterozygous isoenzyme and DNA fingerprinting patterns expected of a L. panamensis/L. braziliensis hybrid (27%). There was complete correlation between the isoenzyme and DNA data for each of the putative hybrids examined. All of the 'hybrids' were obtained from foci in the northern region of the country where L. panamensis and L. braziliensis occur sympatrically. These observations provide strong evidence for sexual reproduction in New World Leishmania populations and suggest that it is of taxonomic and epidemiological significance.

Animals↗