Biomedical subjects

G L McLaughlin

Publications and source records attributed to G L McLaughlin.

At least 37 records · Page 2Linked to original sources

PubMed1993

Acanthamoeba binds to extracellular matrix proteins in vitro.

PURPOSE: To identify host-tissue amoeba interactions that may be important in the pathogenesis of Acanthamoeba keratitis, the ability of the opportunistic pathogen Acanthamoeba polyphaga to bind various components of the extracellular matrix (collagen type IV, laminin, or fibronectin) was examined in vitro. METHODS: A polyphaga, isolated from a case of human amoebic keratitis, was used in the studies. In the experiments, 96-well plates were coated with 0-, 5-, 10-, 20-, or 50-micrograms/ml solutions of the basal lamina proteins laminin or collagen type IV, the extracellular matrix protein fibronectin, or casein (control). Amoeba were metabolically labeled with 35[S]-methionine, and 1x 10(4) labeled amoeba in phosphate buffered saline (PBS) were seeded per well and allowed to bind for 20 min. After washing with PBS, bound amoeba were solubilized with 1% sodium dodecyl sulphate (SDS) and scintillation counting was used to determine the number of bound amoeba. RESULTS: Counts from casein and protein-free controls were not significantly different from each other (P > 0.05). There was a significant increase in the binding of 35[S]-labeled A. polyphaga to collagen IV, laminin, and fibronectin over controls (P < 0.0001) and the binding was concentration-dependent. The rank order of binding was collagen > or = laminin >> fibronectin. Alpha-methyl-mannopyranoside, but not fucose, inhibited binding of labeled A. polyphaga to collagen IV, laminin, and fibronectin in a concentration-dependent manner. CONCLUSION: In summary, the binding assays show that Acanthamoeba bind preferentially to collagen, laminin, and fibronectin, in that order, and that the adherence process is inhibited by mannose.

Acanthamoeba↗

PubMed1992

Use of the polymerase chain reaction for the sensitive detection of St. Louis encephalitis viral RNA.

Polymerase chain reaction (PCR) assays were developed for the detection of RNA from the St. Louis encephalitis (SLE) virus. Using computer-assisted analysis of the MSI-7 strain SLE virus genome, two primer pairs were selected from the capsid-coding and the membrane associated protein-coding genes, and one from the envelope-coding gene. Reverse transcription was primed with either specific oligomers or with random hexamers; these methods were compared for cDNA synthesis and subsequent PCR amplification with the oligomeric pairs. Random hexamers provided more sensitive detection of viral RNA. Each primer pair specifically amplified the expected sized fragment from the Parton SLE strain grown in Aedes albopictus cells, but did not amplify Aedes albopictus cell RNA controls. The technique also detected SLE virus RNA in 1 pg of total cellular RNA added to a background of 1 microgram boiled brain tissue, and in 0.5 pg of total RNA added to homogenized mosquito abdomen. PCR-based assays may be adaptable to detect SLE virus RNA in naturally infected mosquitoes, birds, and human cerebrospinal fluid and brain.

Animals↗

PubMed1992

Molecular approaches to malaria and babesiosis diagnosis.

The development of additional methods for detecting and identifying Babesia and Plasmodium infections may be useful in disease monitoring, management and control efforts. The preliminary evaluate synthetic peptide-based serodiagnosis, a hydrophilic sequence (DDESEFDKEK) was selected from the published BabR gene of B. bovis. Immunization of rabbits and cattle with the hemocyanin-conjugated peptide elicited antibody responses that specifically detected both P. falciparum and B. bovis antigens by immunofluorescence and Western blots. Using a dot-ELISA with this peptide, antisera from immunized and naturally-infected cattle, and immunized rodents, were specifically detected. Reactivity was weak and correlated with peptide immunization or infection. DNA-based detection using repetitive DNA was species-specific in dot-blot formats for B. bovis DNA, and in both dot-blot and in situ formats for P. falciparum; a streamlined enzyme-linked synthetic DNA assay for P. falciparum detected 30 parasites/mm3 from patient blood using either colorimetric (2-15 h color development) or chemiluminescent detection (0.5-6-min exposures). Serodiagnostic and DNA hybridization methods may be complementary in the respective detection of both chronic and acute infections. However, recent improvements in the polymerase chain reaction (PCR) make feasible a more sensitive and uniform approach to the diagnosis of these and other infectious disease complexes, with appropriate primers and processing methods. An analysis of ribosomal DNA genes of Plasmodium and Toxoplasma identified Apicomplexa-conserved sequence regions. Specific and distinctive PCR profiles were obtained for primers spanning the internal transcribed spacer locus for each of several Plasmodium and Babesia species.

Amino Acid Sequence↗

PubMed1992

Detection of St. Louis encephalitis virus in mosquitoes by use of the polymerase chain reaction.

We recently developed an assay using the polymerase chain reaction (PCR) for the specific detection of St. Louis encephalitis (SLE) virus RNA. This assay was tested in a blind study on 7 samples of pooled mosquitoes (50 mosquitoes/pool) which were also characterized for SLE virus by plaque assay in Vero cell culture. One sample was positive for the SLE virus as determined by both the PCR assay and a combination of the plaque assay and the indirect fluorescent antibody assay. The remaining 6 samples were negative for the presence of SLE virus as determined by both methods. These data indicate that this PCR assay can be used to monitor for the presence of SLE virus in pools of homogenized mosquitoes. This approach could provide early data on which to base disease control decisions.

Animals↗

PubMed1991

Effects of temperature, amebic strain, and carbohydrates on Acanthamoeba adherence to corneal epithelium in vitro.

An in vitro coincubation assay was used to measure adhesion of radiolabeled Acanthamoeba trophozoites to corneal epithelium. Adhesion of amebae to corneal epithelium was higher at 25 degrees C than at 37 or 4 degrees C, did not consistently correlate with the reported pathogenicity of the strain of Acanthamoeba, and was inhibited by mannose and by methyl-alpha-D-mannopyranoside.

Acanthamoeba↗

PubMed1991

Amplification of repetitive DNA for the specific detection of Naegleria fowleri.

By using hybridization at low C0t values, a genomic library on Naegleria fowleri was screened for clones containing repetitive DNA. Partial sequence information from a repetitive clone, Nf9, showed sequence homologies with the mitochondrial ATPase 6 subunit from yeasts and other organisms. Synthetic DNA primers were selected and tested in amplification reactions. Nonstringent hybridization conditions were defined which allowed amplification of N. fowleri DNA and reduced amplification of DNA from nonpathogenic Naegleria species. Stringent conditions were selected which allowed detection only of N. fowleri. Identity of the amplified DNA was confirmed by using internal restriction sites and an internal primer. In a blind study, tissue from mice experimentally infected with N. fowleri was specifically detected by using stringent hybridization conditions.

Amebiasis↗

PubMed1991

Optimization of a rapid nonisotopic DNA probe assay for Plasmodium falciparum in the Gambia.

An enzyme-linked synthetic DNA probe which hybridizes to repetitive DNA of Plasmodium falciparum was used in conjunction with a microtiter-based lysis and filtration blood processing procedure. An assay protocol was developed that is more sensitive and robust than previous protocols, which use stored blood and phenol extraction. In comparison with thick smear examination, 33% positive, 60% negative, and 7% conflicting scores were recorded from 390 analyzed clinical samples, and the sensitivity threshold was about 30 parasites per mm3 of blood.

Animals↗

PubMed1991

Cocarcinogenesis between cadmium and Aroclor 1254 in planarians is enhanced by inhibition of glutathione synthesis.

Asexual Dugesia dorotocephala planaria continuously exposed to 50 micrograms/beaker of Aroclor 1254 (A1254) and cadmium (Cd) developed tumors after 11 days of continuous exposure to 0.65 or 1.3 mg/liter Cd and after 23 days to 0.13 mg/liter Cd. The tumor rate at 14 days averaged 53% for the survivors in both 0.65 and 1.3 mg/liter Cd + A1254 and 40% at 0.13 mg/liter Cd + A1254. Other groups were either preexposed or coexposed to L-buthionine-S,R-sulfoximine (BSO), a specific inhibitor of glutathione synthesis. In the presence of A1254 (50 micrograms/beaker), animals continuously exposed to BSO and 0.13 mg/liter Cd first developed tumors at 18 days, with a tumor yield equal to 41% of the survivors. Tumors developed by 6 days at 0.65 mg/liter Cd, with a yield equal to 75% of survivors. At 1.3 mg/liter Cd, BSO did not change the rate or frequency of planarian tumor production. Continuous BSO always produced high mortality over 20 days. In contrast, a 24-h preexposure to BSO caused little mortality. In these groups, tumor yields increased with cadmium concentration (0.13, 0.25, 0.65 mg/liter), and the large cocarcinogenic effect of A1254 was clearly evident. No tumors developed in animals exposed only to 50 micrograms/beaker of Aroclor 1254 or continuously only to 1 mM BSO.

Animals↗

PubMed1990

Thermal ecology of Naegleria fowleri from a power plant cooling reservoir.

The pathogenic, free-living amoeba Naegleria fowleri is the causative agent of human primary amebic meningoencephalitis. N. fowleri has been isolated from thermally elevated aquatic environments worldwide, but temperature factors associated with occurrence of the amoeba remain undefined. In this study, a newly created cooling reservoir (Clinton Lake, Illinois) was surveyed for Naegleria spp. before and after thermal additions from a nuclear power plant. Water and sediment samples were collected from heated and unheated arms of the reservoir and analyzed for the presence of thermophilic Naegleria spp. and pathogenic N. fowleri. Amoebae were identified by morphology, in vitro cultivation, temperature tolerance, mouse pathogenicity assay, and DNA restriction fragment length analysis. N. fowleri was isolated from the thermally elevated arm but not from the ambient-temperature arm of the reservoir. The probability of isolating thermophilic Naegleria and pathogenic N. fowleri increased significantly with temperature. Repetitive DNA restriction fragment profiles of the N. fowleri Clinton Lake isolates and a known N. fowleri strain of human origin were homogeneous.

Amebiasis↗

PubMed1989

Intravenous administration of d-tubocurarine and pancuronium in fetal limbs.

The purpose of this study was to assess the effects of intravenous administration of d-tubocurarine and pancuronium in fetal lambs. Eighteen experiments were performed in seven chronically instrumented pregnant ewes between 0.8 and 0.9 of timed gestation. Each fetus received 6 ml of study drug intravenously at 1.2 ml/min over 5 minutes (d-tubocurarine 3.0 mg/kg, pancuronium 0.5 mg/kg, or saline solution control). Pancuronium (n = 7) significantly increased both fetal heart rate and mean arterial pressure. d-Tubocurarine (n = 5) significantly decreased both fetal heart rate and mean arterial pressure. Saline solution control (n = 6) did not significantly alter fetal heart rate or mean arterial pressure. The present study suggests that pancuronium is preferable to d-tubocurarine for those intrauterine procedures when an increase in fetal heart rate is desired.

Animals↗

PubMed1989

Comparison of thick films, in vitro culture and DNA hybridization probes for detecting Plasmodium falciparum malaria.

Using blood from volunteers with sporozoite induced malaria, a comparison was made of the sensitivity and specificity of Giemsa stained thick film examination, in vitro culture, and 4 different DNA probes for detecting parasitemia. Between 9 and 13 days after sporozoite inoculation, patent parasitemia (4-550 parasites/microliters) was detected by thick film examination of 0.5 microliters blood in 7 volunteers. Cultures of 1 ml blood obtained 7 days after sporozoite inoculation were positive in all volunteers who eventually developed patent parasitemia. The DNA hybridization probes detected parasites in only 5-28% of smear- or culture-positive samples.

Animals↗

PubMed1988

Does the intravenous infusion of ritodrine or magnesium sulfate alter the hemodynamic response to hemorrhage in gravid ewes?

The purpose of this study was to determine whether the intravenous infusion of ritodrine or magnesium sulfate alters the hemodynamic response to maternal hemorrhage in gravid ewes. Twenty-seven experiments were performed in 12 chronically instrumented animals at 0.8 of timed gestation. Each animal was subjected to hemorrhage (20 ml/kg over 60 minutes) during infusion of ritodrine (0.004 mg/kg/min), magnesium sulfate (4 gm/hour), or saline solution control. Infusion of magnesium sulfate increased the mean (+/- SEM) maternal serum magnesium concentration to 4.8 +/- 0.2 mg/dl before hemorrhage and 5.3 +/- 0.3 mg/dl after hemorrhage. At the end of hemorrhage maternal mean arterial pressures were 63% +/- 4%, 82% +/- 2%, and 79% +/- 6% of baseline in the magnesium sulfate, ritodrine, and control groups, respectively. The maternal mean arterial pressure response in the magnesium sulfate group differed significantly from the maternal mean arterial pressure responses in the ritodrine and control groups (p less than 0.01). Fetal pH was decreased significantly only in the magnesium sulfate group (p = 0.0001). Fetal PO2 was decreased significantly in the magnesium sulfate and ritodrine groups (p less than 0.001) but not in the control group. We conclude that magnesium sulfate but not ritodrine, worsened the maternal hypotensive response to hemorrhage in gravid ewes.

Animals↗

PubMed1988

Restriction fragment length polymorphisms of the DNA of selected Naegleria and Acanthamoeba amebae.

Fourteen strains of Naegleria fowleri, two strains of N. gruberi, and one strain each of N. australiensis, N. jadini, N. lovaniensis, Acanthamoeba sp., A. castellanii, A. polyphaga, and A. comandoni isolated from patients, soil, or water were characterized by restriction fragment length polymorphisms. Total cellular DNA (1 microgram) was digested with either HindIII, BglII, or EcoRI; separated on agarose gels; and stained with ethidium bromide. From 2 to 15 unusually prominent repetitive restriction fragment bands, totaling 15 to 50 kilobases in length and constituting probably more than 30% of the total DNA, were detected for all ameba strains. Each species displayed a characteristic pattern of repetitive restriction fragments. Digests of the four Acanthamoeba spp. displayed fewer, less intensely staining repetitive fragments than those of the Naegleria spp. All N. fowleri strains, whether isolated from the cerebrospinal fluid of patients from different parts of the world or from hot springs, had repetitive restriction fragment bands of similar total lengths (ca. 45 kilobases), and most repetitive bands displayed identical mobilities. However, polymorphic bands were useful in identifying particular isolates. Restriction fragment length polymorphism analysis generally was consistent with taxonomy based on studies of infectivity, morphology, isoenzyme patterns, and antibody reactivity and suggests that this technique may help classify amebae isolated from clinical specimens or from the environment.

Acanthamoeba↗

PubMed1988

DNA hybridization for assessment of response of Plasmodium falciparum to chloroquine therapy.

We studied the accuracy of PFR1-AP, a synthetic DNA hybridization probe conjugated to alkaline phosphatase, in monitoring Plasmodium falciparum parasitemia during in vivo drug susceptibility surveys. Duplicate blood samples were collected from six children enrolled in a 14-day in vivo chloroquine study in Rwanda. Results obtained by microscopic examination of Giemsa-stained thick blood smears and by DNA hybridization were compared. Both techniques successfully monitored an infection with chloroquine-susceptible parasites and infections exhibiting various levels of resistance to treatment. For each patient, temporal evolution of the microscopic parasite counts and the DNA hybridization signals were closely parallel, although a wide range of rapidly changing levels of parasitemia occurred through the course of the study. This suggests that DNA hybridization assay using PFR1-AP detects P. falciparum parasites sensitively and specifically and is a valuable tool for drug resistance surveys.

Animals↗

PubMed1987

Use of enzyme-linked synthetic DNA in diagnosis of falciparum malaria.

Plasmodium falciparum contains a family of 21-base-long repetitive DNA sequences in its genome. A 21-base synthetic DNA oligomer, formerly labelled with phosphorus-32 for autoradiographic detection of P falciparum DNA, was covalently coupled to alkaline phosphatase for histochemical detection. The conjugate (PFR1-AP) detected purified P falciparum DNA with a sensitivity and specificity equal to that of 32P-labelled probes after 2-day exposures. PFR1-AP did not detect host DNA or DNA of other Plasmodium species. In African blood specimens PFR1-AP specifically detected P falciparum infections of 100 parasites/microliter. This sensitive, rapid, nonisotopic probe will allow more widespread use of DNA hybridisation in the diagnosis of malaria.

Alkaline Phosphatase↗

PubMed1987

Comparison of genomic, plasmid, synthetic, and combined DNA probes for detecting Plasmodium falciparum DNA.

Total genomic Plasmodium falciparum DNA, the plasmid clone pRepHind, and a 21-base-long synthetic DNA probe (PFR1), the sequence of which was derived from pRepHind, were hybridized with DNA from various species of the phylum Apicomplexa. The genomic probe hybridized with P. reichenowi and P. falciparum DNA and significantly cross-hybridized with DNA of all the other Plasmodium species tested. The synthetic and plasmid probes hybridized to P. falciparum DNA and at reduced levels to P. reichenowi but did not hybridize to P. vivax, P. malariae, P. ovale, P. fragile, P. inui, P. knowlesi, Babesia bovis, B. microti, B. bigemina, Anopheles sp., Pan sp., Aotus sp., or human DNA. Southern blot analysis indicated that approximately 60 distinct restriction enzyme fragments from P. falciparum DNA were similarly detected by PFR1 and pRepHind. A method was developed by using a second brief hybridization with synthetic DNA to amplify signals from samples that were previously hybridized with plasmid-borne repetitive DNA. This amplification procedure was shown to allow the detection of 0.005% P. falciparum parasitemias from 10-microliter samples of blood from patients in Kenya.

Animals↗

PubMed1987

Repeated immunogenic amino acid sequences of Plasmodium species share sequence homologies with proteins from humans and human viruses.

The use of recombinant peptides based upon the repeated amino acid sequences of Plasmodium has been proposed for malaria vaccines. By reducing homologies of such peptide vaccines to host proteins, the possibility of autoimmune complications may be reduced, and the effective immune response may be enhanced. The Wilbur and Lipman Wordsearch algorithm was used to identify homologous amino acid sequences between tandemly repeated Plasmodium amino acid sequences and the human and human viral sequences compiled in the National Biomedical Research Foundation database. Six published repetitive immunogenic amino acid sequences from the circumsporozoite (CS) antigen, ring-infected erythrocyte surface antigen (RESA), soluble (S) antigen, and falciparum interspersed repetitive antigen (FIRA) of P. falciparum, and the CS protein of P. vivax, were analyzed by computer. Matches of at least 4 amino acids were found for all sequences. In the database, 29 matches were found for human proteins and 26 matches were found for human viruses with the 6 antigen sequences. Most of the matched proteins, and many of the matched human viruses, are found in blood. The biological significance of these matches remains to be clarified.

Amino Acid Sequence↗

PubMed1987

Assessment of a synthetic DNA probe for Plasmodium falciparum in African blood specimens.

Synthetic DNA oligomers homologous to 21-base long repetitive sequences of Plasmodium falciparum DNA were labeled with 32P using T4 kinase, and were hybridized with purified DNA and with processed blood samples from Africa. The sequence PFR1, its antiparallel oligomer PFR1R, and PFR1 covalently attached to biotin hybridized similarly to P. falciparum DNA. One-microliter aliquots of blood from Zaire spotted on prewet nylon filters and hybridized with PFR1 gave detectable autoradiogram signals from samples with parasitemias as low as 1,000 parasites/mm3. Blood lysis and protein digestion followed by alkylation allowed dot-blot processing of larger aliquots of blood. After hybridization with PFR 1 and autoradiography, 26 samples were scored positive visually, compared with 34 scored positive by microscopy. The effective sensitivity for processed 10-microliter samples was about 500 parasites/mm3. Signals from hybridized probes were quantitated by liquid scintillation counting and densitometry, and were proportional to the amounts of purified P. falciparum DNA applied to the filter. Autoradiogram signals also were roughly proportional (correlation coefficient, r = 0.77) to the number of parasites/mm3 of blood from field samples as determined by microscopic examination.

Animals↗