Search PubMed⌕ Search

Biomedical subjects

M A Miles

Publications and source records attributed to M A Miles.

At least 55 records · Page 3Linked to original sources
PubMed1994

Diagnosis of amebic dysentery by detection of Entamoeba histolytica fecal antigen by an invasive strain-specific, monoclonal antibody-based enzyme-linked immunosorbent assay.

An invasive strain-specific monoclonal antibody against Entamoeba histolytica has been used in a capture enzyme-linked immunosorbent assay (ELISA) for the detection of invasive E. histolytica fecal antigen in clinical specimens and for the diagnosis of amebic dysentery in patients from Bangladesh. The fecal antigen capture ELISA (FAC-ELISA) did not cross-react with other parasite species in the clinical specimens or with noninvasive E. histolytica present in those specimens and in experimentally seeded stools. The limit of detection of the assay for invasive E. histolytica crude antigen diluted in phosphate-buffered saline or in stools was 0.58 and 3.9 micrograms/ml, respectively, which is the equivalent of approximately 72 and 487 E. histolytica trophozoites per well, respectively. The sensitivity, specificity, and efficiency of the FAC-ELISA were 87, 100, and 98%, respectively, for the detection of invasive E. histolytica antigens and 100, 100, and 100%, respectively, for the diagnosis of amebic dysentery. The FAC-ELISA is a potential alternative for the field diagnosis of amebic dysentery and for epidemiological studies to define the distribution of invasive E. histolytica.

Animals↗
PubMed1994

Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica.

AIMS: To assess the reliability of the detection of erythrophagocytic amoebic trophozoites in stool samples in the diagnosis of dysentery associated with invasive Entamoeba histolytica. METHODS: Amoebic culture was carried out on single stool samples collected from patients from Mexico, Colombia, and Bangladesh. The stools had been examined by light microscopy. Amoebic dysentery was diagnosed when erythrophagocytic E histolytica trophozoites were observed in a case of bloody diarrhoea. E histolytica isolates were characterised by isoenzyme electrophoresis and results correlated with microscopical findings in stools. Statistical analysis was performed using the chi 2 test. RESULTS: Where erythrophagocytic amoebae had been observed in dysenteric stool specimens the E histolytica phenotype was invariably invasive (p < 0.0001). Observation of erythrophagocytic amoebae in dysentery is 100% specific and predictive of infection with invasive E histolytica. When amoebic culture-positive cases only are considered it is 96% sensitive. In this study E histolytica of zymodeme XIV was more commonly associated with amoebic dysentery than zymodeme II. There was no significant difference between the carriage rate of invasive and non-invasive E histolytica in non-dysenteric diarrhoea. Asymptomatic subjects carried non-invasive E histolytica more frequently than invasive E histolytica. Patients with non-amoebic dysentery, when shown to be infected with E histolytica, carried non-invasive strains (12%). CONCLUSIONS: Sensitivity and specificity of microscopical examination of a single stool specimen for diagnosing amoebic dysentery is very high; intestinal carriage of invasive E histolytica detected by culture is not necessarily an indication of active disease as patients with diarrhoea and asymptomatic subjects shed invasive and non-invasive E histolytica. There are possibly two subpopulations of invasive E histolytica with different pathogenic potential which can be differentiated by zymodeme analysis.

Animals↗
PubMed1993

Feed intake affects metabolic clearance rate of progesterone in sheep.

To study the inverse relationship between feed level and concentrations of peripheral plasma progesterone, 36 ovariectomised ewes were fed rations at levels calculated to maintain liveweight (M). On the seventh day, the ewes were given an intravenous infusion of progesterone and the metabolic clearance rate (MCR) was calculated. The ewes were then randomly allotted to receive either 1/2M, M or 2M rations for seven days at which time the infusion and blood sampling schedules were repeated. The mean (SE) MCR of infused progesterone in ovariectomised ewes fed either 1/2M, M or 2M rations for seven days was 7.1 (0.59), 9.9 (1.64 and 13.0 (1.19) litre h-1 kg-1 of liveweight, respectively. Differences in MCR of progesterone between ewes fed 1/2M and 2M rations were significant (P < 0.05). The inverse relationship between level of feed intake and plasma progesterone concentration was attributed to differences in clearance rate of progesterone rather than to changes in the entry rate of the hormone into the blood.

Animals↗
PubMed1993

Liver blood flow and metabolic clearance rate of progesterone in sheep.

To study the effects of feeding on concentrations of peripheral plasma progesterone, ovariectomised ewes, given exogenous progesterone, were fed 750 g of chopped lucerne hay at either 09.00 (group A, n = 5) or at 15.00 (group B, n = 5) or were fed ad libitum regularly through the experimental period (group C, n = 5). Peripheral blood samples were taken from each ewe at 09.00, 11.00, 15.00, 17.00 and 23.00. In ewes of groups A and B, mean plasma progesterone concentrations declined significantly (P < 0.05) after feeding. Mean progesterone concentrations of group C ewes remained low and were significantly different (P < 0.05) from the pre-feeding values of group A and B ewes. These results showed that the metabolic clearance rate of progesterone changed with the act of feeding. In a second experiment, portal, hepatic and mesenteric vein cannulae were placed in ovariectomised ewes (n = 17). They were then fed a ration calculated to maintain liveweight for seven days (M ration). The ewes were then infused with progesterone into a jugular vein and with p-amino hippuric acid into a mesenteric vein. Ewes were then allotted to receive either half M or twice M rations for seven days after which the infusions and blood sampling procedures were repeated. The mean rate of blood flow in the portal vein was directly related to the level of feed offered when ewes received either 1/2M, M or 2M rations. The liver and gut region removed a mean of 96 per cent of the progesterone entering these tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania.

A Trypanosoma cruzi expression vector has been constructed using sequences derived from the flanking regions of the glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) genes. The neomycin phosphotransferase (neor) gene was incorporated as a selectable marker. Using electroporation we have introduced this vector into both T. cruzi and Leishmania cells and conferred G418 resistance. Transformation is mediated by large extrachromosomal circular elements composed of head-to-tail tandem repeats of the vector. The transformed phenotype is stable for at least 6 months in the absence of G418 and can be maintained during passage through the T. cruzi life-cycle. Foreign genes inserted into an expression site within the vector (pTEX) can be expressed at high levels in transformed cells. To our knowledge this paper describes the first trypanosome shuttle vector and the first vector which functions in both trypanosomes and Leishmania.

Animals↗
PubMed1992

Adaptation of a radioactive L. donovani complex DNA probe to a chemiluminescent detection system gives enhanced sensitivity for diagnostic and epidemiological applications.

The cDNA probe, Lmet2, was labelled with digoxigenin and used in a chemiluminescent system to detect fewer than 100 membrane-immobilized Leishmania parasites. The probe was found to hybridize primarily with members of the L. donovani complex but a slight cross-reaction was also observed with greater than 5 x 10(4) L. major. This cross-reaction was reduced by hybridizations in 50% formamide at 37 degrees C. Formamide also significantly reduced non-specific binding of the digoxigenin-labelled probe to the membrane support which, in hybridizations without formamide, masked the specific hybridization signal. This background was not observed with the corresponding radio-isotope labelled probe. With hybridizations in formamide the sensitivity achieved by the chemiluminescent system after exposure to film for 3 h was greater than that achieved by the isotopic system even after autoradiography for 24 h.

Animals↗
PubMed1992

Mapping of a visceral leishmaniasis-specific immunodominant B-cell epitope of Leishmania donovani Hsp70.

We have shown that a member of the 70-kDa heat shock protein (Hsp70) family is a major target of the humoral immune response during Leishmania donovani infection. A recombinant fusion protein was recognized by sera from 92% (35 of 38) of patients with visceral leishmaniasis, including representatives from each of the major regions where it is endemic. Serological analysis of recombinant Hsp70, expressed by a series of deletion constructs, identified the carboxy-terminal region as the immunodominant site. This region, which is the most evolutionarily divergent part of the molecule, was recognized by all sera from patients with visceral leishmaniasis which exhibited an anti-Hsp70 response. Purified recombinant L. donovani Hsp70 was not recognized by sera from patients with cutaneous leishmaniasis, Chagas' disease, leprosy, malaria, or schistosomiasis. To determine the regions involved in antibody recognition, a series of overlapping peptides were synthesized on polyethylene pins by the Pepscan method, and a hexamer, EADDRA, was identified by the visceral leishmaniasis serum samples as an immunodominant B-cell epitope.

Amino Acid Sequence↗
PubMed1992

Further diagnostic use of an invasive-specific monoclonal antibody against Entamoeba histolytica.

Genotypic differences between invasive and non-invasive E. histolytica could explain the 1:10 ratio of symptomatic/asymptomatic infection worldwide. Currently, zymodeme analysis is used to differentiate invasive from non-invasive E. histolytica strains but the technique is cumbersome and expensive. In accordance with the WHO research priorities for amebiasis we report here the further use of an invasive-specific monoclonal antibody against E. histolytica in immunofluorescence, to identify isolates cultured from stool samples of patients from three geographically distant endemic regions: Bangladesh, Colombia and Mexico. We tested 107 E. histolytica isolates and the correlation between zymodeme characterization and the immunofluorescence assay was 100%.

Animals↗
PubMed1991

A sensitive repetitive DNA probe that is specific to the Leishmania donovani complex and its use as an epidemiological and diagnostic reagent.

Leishmania donovani (HU3 strain) metacyclic promastigotes generated in vitro were used to construct a cDNA library in the bacteriophage vector lambda gt10. A cDNA clone (Lmet 2), was isolated by differential screening with metacyclic-derived or log-phase promastigote-derived cDNA. The clone insert was comprised predominantly of four copies of an imperfect 60-bp repeat motif, which was represented in the genome by multiple tandem repeats distributed among at least six chromosomes. The corresponding mRNA transcript was a developmentally regulated 12-kb doublet. The Lmet 2 sequence was entirely specific to L. donovani donovani, L. donovani (East Africa), L. donovani infantum and L. donovani chagasi, even when genomic Southern blots and slot blots of other Leishmania species were washed at low stringencies. Twenty-two strains of L. donovani were clearly detected by radiolabelled Lmet 2 cDNA probe, with signals of approximately equal intensity, irrespective of geographical origin, which encompassed widely dispersed endemic regions. The probe could detect DNA from fewer than 100 organisms and identified small numbers of promastigotes in infected sand flies. Amastigotes were also detected in impression smears of organs from infected hamsters. The Lmet 2 probe is likely to be a valuable reagent for clinical diagnosis and epidemiological investigations.

Animals↗
PubMed1991

Cognitive remediation in pediatric head injury: a case study.

Little work has been done in delineating cognitive remediation approaches for pediatric survivors of head injury. The use of a cognitive remediation approach is described in a case study of a 16-year-old male, who was 1 1/2 years post severe traumatic brain injury. Specific techniques and treatment course are outlined to demonstrate the model offered as one meeting the unique needs of the pediatric survivor. This model emphasizes a behavioral, individually tailored approach with clear delineation of a functional academic skill goal of competence in mathematical computation. Results indicate accomplishment of goals and some generalization of skills obtained to a math domain that was not the major focus of the remediation attempts.

Achievement↗
PubMed1991

Cloning of microbial epitopes relevant for T- and B-cells.

This review summarises, and illustrates, the technology that is available for the molecular cloning and precise identification of T-cell and B-cell epitopes, particularly those of bacteria and parasites. Methods include: selective cloning following subtractive hybridization of nucleic acids; selective screening of expression libraries; analysis of subcloned "epitope libraries" or "deletion constructs"; scanning of multiple synthetic peptides, and computer enhanced prediction. The direct sequencing of polymerase chain reaction products allows the rapid analysis of epitope heterogeneity occurring among natural populations. Multiple epitopes can be assembled either by synthesis or by the expression of polymeric epitope-bearing peptides. Prospects for probing expression libraries with T-cells are bleak due to the complexities of antigen processing, presentation and T-cell recognition in vitro. Elucidation of the enzymic steps involved in processing, resolution of peptide/MHC II co-crystals, and pairing of a large number of known epitopes with their functional restriction elements will significantly improve the ability to predict T-cell epitopes.

Animals↗
PubMed1990

Identification and characterisation of a Leishmania donovani antigen belonging to the 70-kDa heat-shock protein family.

A Leishmania donovani promastigote cDNA library was screened with serum obtained from a patient infected with visceral leishmaniasis. Sequence analysis of a clone obtained from this library revealed that the 600-bp insert corresponded to the carboxy-terminal region of an antigen related to the 70-kDa heat-shock protein family. The full-length sequence of the corresponding gene (1959 nucleotides) was determined after isolation of genomic clones. Genes encoding the antigen are present on a single chromosome as a series of approximately twelve 3.7-kb direct tandem repeats. The antigen can be identified as a 70-kDa heat-shock cognate protein by virtue of its molecular mass, sequence and constitutive expression during heat shock. It is expressed at all stages of the parasite life-cycle. Antibodies against the lambda gt11 fusion protein were detected in more than 50% of serum samples obtained from patients with visceral leishmaniasis, but were not detected in sera from patients with cutaneous leishmaniasis or Chagas' disease.

Amino Acid Sequence↗
PubMed1990

Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase does not conform to the 'hotspot' topogenic signal model.

The genes which encode glycosomal glyceraldehyde-phosphate dehydrogenase (gGAPDH) of Trypanosoma cruzi are arranged as a tandemly repeated pair on a single chromosome and are identical at the level of nucleotide sequence. They are separated by an intergenic region which contains a 317 base pair sequence with the properties of a retroposon. The genes express a 1.5 kb mRNA and a 38 kd protein. The amino acid sequence contains features characteristic of glycosomal enzymes such as peptide insertions and a C-terminal extension. However, T. cruzi gGAPDH lacks one of the positively charged 'hotspot' motifs which have been proposed as topogenic signals for import into the glycosome, a unique microbody-like organelle. Molecular modelling of the T. cruzi and T. brucei enzymes suggests that neither structure would fulfil the requirements of the 'hotspot' glycosomal import model.

Amino Acid Sequence↗
PubMed1990

A comparison of the lectin-binding properties of glycoconjugates from a range of Leishmania species.

Constituent glycoconjugates of promastigotes of 14 different Leishmania strains from 6 different species were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently stained with 14 125I-labelled lectins of different specificities. Autoradiography of the gels revealed lectin-specific glycoproteins and other glycoconjugates of known molecular weight. Similarities and differences in antigens and glycoproteins present in the strains are described. The possibility of identification or characterisation of Leishmania species from their electrophoretic behaviour and lectin-binding patterns is unlikely, but these results should be an aid to purification of glycoconjugates from particular strains by lectin-affinity chromatography.

Animals↗