Revisiting amebiasis.
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Antigens, Protozoan↗
Biomedical subjects
D Lyerly
Publications and source records attributed to D Lyerly.
Estimating the impact of amebiasis on health.
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Animals↗
Toxin gene analysis of a variant strain of Clostridium difficile that causes human clinical disease.
A toxin variant strain of Clostridium difficile was isolated from two patients with C. difficile-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis. This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for C. difficile toxin A. Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay. Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (tcdA) and toxin B (tcdB) genes of type CF2 isolate 5340. Type CF2 5340 tcdA exhibited a 1,821-bp truncation, due to three deletions in the 3' end of the gene, and a point mutation in the 5' end of the gene, resulting in a premature stop codon at tcdA position 139. Type CF2 5340 tcdB exhibited multiple nucleotide base substitutions in the 5' end of the gene compared to tcdB of the standard toxigenic strain VPI 10463. Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant C. difficile strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans. REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2. A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD. PCR amplification of the 3' end of tcdA demonstrated identical 1. 8-kb deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of C. difficile that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A.
Aged↗
Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile responsible for a nosocomial outbreak of Clostridium difficile-associated diarrhea.
Clostridium difficile-associated diarrhea (CAD) is a very common nosocomial infection that contributes significantly to patient morbidity and mortality as well as to the cost of hospitalization. Previously, strains of toxin A-negative, toxin B-positive C. difficile were not thought to be associated with clinically significant disease. This study reports the characterization of a toxin A-negative, toxin B-positive strain of C. difficile that was responsible for a recently described nosocomial outbreak of CAD. Analysis of the seven patient isolates from the outbreak by pulsed-field gel electrophoresis indicated that this outbreak was due to transmission of a single strain of C. difficile. Our characterization of this strain (HSC98) has demonstrated that the toxin A gene lacks 1.8 kb from the carboxy repetitive oligopeptide (CROP) region but apparently has no other major deletions from other regions of the toxin A or toxin B gene. The remaining 1.3-kb fragment of the toxin A CROP region from strain HSC98 showed 98% sequence homology with strain 1470, previously reported by M. Weidmann in 1997 (GenBank accession number Y12616), suggesting that HSC98 is toxinotype VIII. The HSC98 strain infecting patients involved in this outbreak produced the full spectrum of clinical illness usually associated with C. difficile-associated disease. This pathogenic spectrum was manifest despite the inability of this strain to alter tight junctions as determined by using in vitro tissue culture testing, which suggested that no functional toxin A was produced by this strain.
Adolescent↗
Diagnosis of amebic liver abscess and intestinal infection with the TechLab Entamoeba histolytica II antigen detection and antibody tests.
A noninvasive diagnostic test for amebic liver abscess is needed, because amebic and bacterial abscesses appear identical on ultrasound or computer tomography and because it is rarely possible to identify Entamoeba histolytica in stool specimens from patients with amebic liver abscess. Here we report a method of detection in serum of circulating E. histolytica Gal/GalNAc lectin to diagnose amebic liver abscess, which was used in patients from Dhaka, Bangladesh. The TechLab E. histolytica II test (which differentiates the true pathogen E. histolytica from Entamoeba dispar) detected Gal/GalNAc lectin in the sera of 22 of 23 (96%) amebic liver abscess patients tested prior to treatment with the antiamebic drug metronidazole and 0 of 70 (0%) controls. After 1 week of treatment with metronidazole, 9 of 11 (82%) patients became serum lectin antigen negative. The sensitivity of the E. histolytica II antigen detection test for intestinal infection was also evaluated. Antigen detection identified E. histolytica infection in 50 samples from 1, 164 asymptomatic preschool children aged 2 to 5 years, including 16 of 16 (100%) culture-positive specimens. PCR analysis of stool specimens was used to confirm that most antigen-positive but culture-negative specimens were true-positive: PCR identified parasite DNA in 27 of 34 (79%) of the antigen-positive, culture-negative stool specimens. Antigen detection was a more sensitive test for infection than antilectin antibodies, which were detected in only 76 of 98 (78%) amebic liver abscess patients and in 26 of 50 (52%) patients with intestinal infection. We conclude that the TechLab E. histolytica II kit is a sensitive means to diagnose hepatic and intestinal amebiasis prior to the institution of metronidazole treatment.
Adult↗
Pharmacological characterization and selectivity of the NPY antagonist GR231118 (1229U91) for different NPY receptors.
Neuropeptide Y (NPY) is widely distributed throughout the central and peripheral nervous system and exerts a wide range of physiological responses by activating specific receptors. In this study we have characterized the potency of the high affinity peptide dimer antagonist, GR231118, to displace radiolabeled NPY/PYY from different tissues and cell lines expressing Y1 or Y2 receptors and from CHO cells stably transfected with human cDNA encoding for Y1, Y2 and Y4 receptors. GR231118 displays high affinity for Y1 and Y4 receptors, equal or better than that of NPY itself, while its activity is several fold weaker for Y2 receptors. Displacement of radiolabeled PYY from rat hypothalamic membranes by GR231118, reveals the existence of high and low affinity binding sites which may be equated to Y1 and Y2 receptors respectively suggesting that the compound maybe used as a tool to dissect central NPY receptors.
Animals↗
Preclinical neurochemical and electrophysiological profile of 1192U90, a potential antipsychotic.
11192U90 was submitted to receptor binding and monoamine uptake assays. It bound potently at serotonin 5-HT2, dopaminergic D2, serotonin 5-HT1A, and adrenergic alpha 1 and alpha 2 receptors. It also bound to dopaminergic D1, serotonin 5-HT3, serotonin 5-HT4, and sigma sites, albeit with lower affinity. It was essentially inactive at 22 other sites, including those for cholinergic M1 and M2. It weakly inhibited uptake of 3H-norepinephrine, 3H-serotonin and 3H-dopamine. Acute doses of 1192U90 (5 and 20 mg/kg P.O.) increased whole-brain levels of dopamine metabolites but did not affect levels of norepinephrine, dopamine, and serotonin. Subcutaneous injection of 1192U90 (0.8 mg/kg/day) and clozapine (20 mg/kg/day) for 28 days preferentially decreased the number of spontaneously active dopamine cells in the ventral tegmental area (VTA) but not the substantia nigra (SN) of rats, as measured by population sampling. This outcome is characteristics of atypical antipsychotics like clozapine. Acute injections of 1192U90 reversed the rate-inhibiting effects of microiontophoretically applied dopamine and intravenously injected apomorphine and d-amphetamine on dopamine cell firing. Intravenous injection or iontophoretic application of 1192U90 or the 5-HT1A agonist (+/-)8-OH-DPAT inhibited the firing rates of dorsal raphe nucleus (DRN) neurons in rats, and the effects of both compounds were blocked by iontophoretically applied S(-) propranolol, a 5-HT1A antagonist. The results suggest that 1192U90 is a preferential dopamine D2 antagonist as well as a 5-HT1A agonist that may prove to be an atypical antipsychotic.
Amphetamine↗
Evaluation of a New Latex Agglutination Test for Fecal Lactoferrin in Travelers' Diarrhea.
The detection and diagnosis of travelers' diarrhea by fecal culture method, often insensitive, expensive, and time consuming, could well be replaced with the latex agglutination test to detect fecal lactoferrin. As an initial screening, this test could be useful to differentiate between those patients who are more likely to have an invasive enteropathogen and those patients requiring antidiarrheal chemotherapy only. Fecal samples from 92 patients with travelers' diarrhea were tested for occult blood, fecal leukocytes, and fecal lactoferrin at stool dilutions of 1:50 and 1:200. The results were compared with findings from fecal cultures for enteropathogens. Statistical analyses were performed measuring the performance of the latex agglutination test for fecal lactoferrin to calculate its sensitivity, specificity, and positive and negative predictive values. Invasive pathogens were identified in 36 (39%), and a noninvasive pathogen was found in 18, or 20%, of the cases. At the stool dilution of 1:50, fecal lactoferrin showed more sensitivity than did leukocytes or occult blood in detecting the presence of invasive enteropathogens and, with a negative predictive value of 94%, was superior in predicting their absence. At the 1:200 dilution, a lower sensitivity of 55%, but a higher specificity of 82%, compared to 55% with the 1:50 dilution, was demonstrated. The determination of fecal lactoferrin might prove to be more useful and less expensive and time consuming as an initial screening for patients presenting with travelers' diarrhea. Further evaluation and antibacterial treatment could, therefore, be reserved for those with a positive lactoferrin test.
Journal Article↗
Diagnosis of pathogenic Entamoeba histolytica infection using a stool ELISA based on monoclonal antibodies to the galactose-specific adhesin.
Monoclonal antibodies (MAbs) directed against pathogen-specific epitopes of the galactose adhesin of Entamoeba histolytica were used in an ELISA to detect antigen from pathogenic E. histolytica. Single stool specimens from 74 patients in Bangladesh were used. The ELISA for pathogenic E. histolytica was positive in all 12 stool specimens with pathogenic amebae subsequently cultured, in no stool specimens with nonpathogenic E. histolytica and in 2 of 40 stools with other or no intestinal parasites detected. Specificity and sensitivity of the assay for pathogenic E. histolytica were 97% and 100%, respectively. These preliminary data offer promise for an ELISA using MAbs to the galactose adhesin as a rapid and sensitive means to detect the presence of pathogenic E. histolytica infection in stool specimens.
Animals↗
Protection against experimental pseudomembranous colitis in gnotobiotic mice by use of monoclonal antibodies against Clostridium difficile toxin A.
The pathogenicity of Clostridium difficile is due to the production of two toxins (toxins A and B). We prepared monoclonal antibodies against toxin A and determined whether axenic mice passively immunized with the monoclonal antibodies were protected against C. difficile disease. The mice were kept in an isolator and were given ascites fluid intravenously prior to challenge with a toxinogenic strain of C. difficile. Control mice and mice receiving ascites fluid devoid of toxin antibody died within 2 days and had high levels of toxins A and B in their feces. Mice that received ascites fluid containing high amounts of toxin A monoclonal antibodies directed against the repeating units of the toxin survived. In protected mice, toxin B levels were similar to those in dying mice, but toxin A levels were greatly reduced. These data show that passive immunity induced by monoclonal antibodies against toxin A was effective against pseudomembranous cecitis.
Animals↗
Triphenyl phosphite: in vivo and in vitro inhibition of rat neurotoxic esterase.
Organophosphorus compounds which, after acute administration, inhibit neurotoxic esterase (NTE) by greater than or equal to 65% and undergo a subsequent "aging" reaction, produce a delayed neuropathy characterized by degeneration of large and long nerve fibers (OPIDN). The present studies examine in detail the NTE-inhibiting properties of triphenyl phosphite (TPP), a plasticizer which produces ataxia and degeneration of the spinal cord in animals. A neurotoxic dosing regimen (1184 mg/kg/week, sc, for 2 weeks) inhibited both brain and spinal cord NTE (less than or equal to 40%) only marginally 4 and 48 hr postdosing. By contrast, TPP was shown in vitro to be a potent (150 = 0.98 microM) inhibitor of rat brain NTE relative to Mipafox or diisopropyl phosphorofluoridate. Compounds structurally related to TPP (i.e., triphenyl phosphate, triphenyl phosphine, trimethyl phosphite, and phenol) failed to inhibit NTE in vitro at less than 10 microM concentrations. Close examination of the TPP inhibition of NTE showed a nonlinear relationship between the duration of incubation time and loss of log(NTE activity). Preincubation of 10 microM TPP in buffer (37 degrees C) resulted in a time-dependent loss of TPP's ability to inhibit NTE. In summary, TPP is a powerful NTE inhibitor in vitro, but only a marginal NTE inhibitor after in vivo administration. These results raise questions as to the causal events mediating TPP-induced neuropathy in the rat.
Animals↗
Gnotobiotic models for study of the microbial ecology of Clostridium difficile and Escherichia coli.
Hamster flora introduced into germfree mice reduced the cecum to conventional size, suppressed populations of Escherichia coli and Clostridium difficile to the same degree that mouse flora did, and corrected the hypocellularity that is characteristic of the small bowel of germfree mice. A highly toxigenic strain of C. difficile readily induced cecitis in germfree and antibiotic-treated conventional mice, and histological examination frequently revealed pseudomembranes. Toxins A and B were both detected in ceca of animals with colitis. Gnotobiotic mice provide a model in which to study the role of the indigenous microflora in protecting against antibiotic-associated colitis.
Animals↗
Effects of hypothermia on the in vivo measurement of rapid axonal transport in the rat: a cautionary note.
Rapid axonal transport of glycoproteins was examined in the retinofugal projections of hypothermic and normothermic adult male Long-Evans hooded rats previously receiving intraocular injections of [3H]fucose. The amount of retinal fucosylation appeared normal in the hypothermic animals 3.5 h after isotope injection, but glycoprotein transport was reduced relative to normothermic controls. This reduction was especially pronounced in the most distal structure of the retinofugal tract (superior colliculus). We conclude that rapid axonal transport decreases with reductions in mammalian body temperature. This finding emphasizes the importance of controlling body temperature in in vivo studies of mammalian axonal transport.
Animals↗
The correlation between neurotoxic esterase inhibition and mipafox-induced neuropathic damage in rats.
The correlation between neuropathic damage and inhibition of neurotoxic esterase or neuropathy target enzyme (NTE) was examined in rats acutely exposed to Mipafox (N, N'-diisopropylphosphorodiamidofluoridate), a neurotoxic organophosphate. Brain and spinal cord NTE activities were measured in Long-Evans male rats 1 hr post-exposure to various dosages of Mipafox (ip, 1-15 mg/kg). These data were correlated with histologically scored cervical cord damage in a separate group of similarly dosed rats sampled 14-21 days post-exposure. Those dosages (greater than or equal to 10 mg/kg) that inhibited mean NTE activity in the spinal cord greater than or equal to 73% and brain greater than or equal to 67% of control values produced severe (greater than or equal to 3) cervical cord pathology in 85% of the rats. In contrast, dosages of Mipafox (less than or equal to 5 mg/kg) which inhibited mean NTE activity in spinal cord less than or equal to 61% and brain less than or equal to 60% produced this degree of cord damage in only 9% of the animals. These data indicate that a critical percentage of NTE inhibition in brain and spinal cord sampled shortly after Mipafox exposure can predict neuropathic damage in rats several weeks later.
Animals↗
Clostridial toxins active locally in the gastrointestinal tract.
Clostridium difficile and Clostridium spiroforme have only in recent years been recognized as intestinal pathogens. They both produce toxins that are also produced by other clostridia. C. difficile toxins A and B are produced by C. sordellii and a few strains of C. perfringens whereas C. spiroforme produces the same toxins as C. perfringens Type E (iota toxin). Iota toxin activity may be the product of two proteins. Toxigenic strains of C. spiroforme and Type E produce two antigens which possess much more biological activity when administered together than when given alone. C. difficile was thought for some time to produce only a single toxin, but then the enterotoxic activity was shown to be due to a separate toxin (toxin A). This toxin increases the oral toxicity of toxin B (the main cytotoxin) and may increase the permeability of the colon. Toxin A binds to a specific receptor in hamster brush border membranes and in the membranes of rabbit erythrocytes. This receptor appears to be a glycoprotein. The receptor can be extracted from the membrane with Triton and binds to immobilized toxin A. The receptor can be extracted and used to coat plastic plates as a first phase in an ELISA assay. Another assay has been developed in which the toxin A binds to the red cells and then the erythrocytes are agglutinated with antitoxin. An even more sensitive assay consists of using rabbit erythrocyte ghosts to bind the toxin and then precipitating the ghosts with antibody to toxin A attached to latex beads. Monoclonal antibodies to toxin A also have been developed and are used in these and other assays.
Animals↗
Inactivation of human plasma alpha 1-proteinase inhibitor by a metalloproteinase from Serratia marcescens.
The interaction of a Serratia marcescens metalloproteinase with human plasma alpha 1-proteinase inhibitor has been investigated. The enzyme was not inactivated by this inhibitor but, instead, converted the native plasma protein into an inactive form of decreased molecular weight. Amino terminal sequence analysis indicated that the interaction of the inhibitor and enzyme was at the reactive site of the inhibitor, with peptide-bond cleavage resulting in the inactivation. This process may be important in necrotic processes occurring during bacterial infiltration of host tissues.
Amino Acid Sequence↗
Proteolysis of human fixed, washed platelets by gram-negative bacterial metalloproteases: effect on von Willebrand factor-human platelet interactions.
Fixed, washed platelets (FWP) are usually stable to aggregation with von Willebrand factor (vWF) from human and certain animal plasmas over several months of storage. When one lot of FWP lost its stability in less than 1 week, studies demonstrated contamination with Serratia marcescens. Extracellular proteases produced by S. marcescens, as well as by Pseudomonas aeruginosa and Escherichia coli, were found to cause loss of FWP aggregability. Purified proteases were prepared from cell-free culture filtrates of S. marcescens and two different strains of P. aeruginosa. They were used to study the effect on the interaction of FWP and vWF. All three purified proteases destroyed FWP aggregability in a time- and concentration-dependent fashion. The protease produced by S. marcescens (SP) was found to be at least eight times more potent against FWP as a substrate than either of the two P. aeruginosa enzymes. The ability of SP to destroy FWP aggregability was prevented by EDTA and could be restored by the addition of Zn2+ in slight molar excess. When compared with trypsin and chymotrypsin, SP was found to be highly selective in digesting the FWP membrane, even at concentrations greater than that established to give a similar loss of FWP aggregability. SP does not induce aggregation of fresh, washed platelets or PRP, but renders them unaggregable with vWF. These proteases may be useful research tools for studying membranes and vWF-platelet interactions.
Blood Coagulation Factors↗
Characterization of rabbit corneal damage produced by Serratia keratitis and by a serratia protease.
The structural alterations elicited in the rabbit corneal stroma by experimental Serratia marcescens keratitis and by a highly purified serratia protease preparation were compared by gross observation, biochemical analyses, and electron microscopic examination of the affected tissue. Acute inflammation, liquefactive necrosis of the cornea, and descemetocele formation occurred during the development of the infection and after the intracorneal injection of submicrogram amounts of the protease. In vitro incubation of insoluble corneal stromal tissue with the bacterium or with the protease resulted in solubilization of the stromal proteoglycan ground substance; however, specific collagenase activity was not detected. Electron microscopic examination of corneas damaged by the bacterial infection and by the protease revealed loss of ruthenium red staining of the proteoglycan ground substance and dispersal of ultrastructurally normal collagen fibrils. Thus, our findings indicate that the major corneal damage which occurs during serratia keratitis and after the injection of the serratia protease is caused by solubilization and loss of the ground substance of the tissue. In addition, the observation that the major structural alterations observed during serratia keratitis can be reproduced by the bacterial protease supports the idea that the enzyme is involved, at least in part, with the production of severe corneal damage by the bacterium.
Animals↗