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Biomedical subjects

A L Smith

Publications and source records attributed to A L Smith.

At least 163 records · Page 9Linked to original sources
PubMed1993

Virulence of a Bordetella pertussis strain expressing a mutant adenylyl cyclase with decreased calmodulin affinity.

Bordetella pertussis, the pathogen responsible for whooping cough, produces a toxic calmodulin-sensitive adenylyl cyclase which enters animal cells and increases intracellular cAMP. A point mutant of B. pertussis with abolished adenylyl cyclase catalytic activity was over 1000-fold less pathogenic to newborn mice than wild-type bacteria, demonstrating the importance of the adenylyl cyclase for B. pertussis virulence (Gross et al.). The B. pertussis adenylyl cyclase is highly sensitive to calmodulin with an apparent Kd for calmodulin of approximately 1 nM. The importance of this high-affinity calmodulin binding for virulence in vivo was examined by the creation of a B. pertussis point mutant (Trp-242 to Glu-242) with 200-fold lower calmodulin affinity than the native enzyme. This mutant B. pertussis strain retained its virulence in a newborn mouse model of pertussis, but the time course for establishment of a lethal infection in vivo was significantly delayed for the mutant strain. These data illustrate that high-affinity calmodulin binding is not obligatory for the activity of this toxin but is important for the rate for establishment of a lethal infection.

Adenylyl Cyclases↗
PubMed1993

Trophectoderm microbiopsy in murine blastocysts: comparison of four methods.

PURPOSE: This study compares four trophectoderm microbiopsy techniques for removal of blastomeres from murine blastocysts: (1) aspiration, trophectoderm pipetted through the zona; (2) incision, trophectoderm excised with a microrazor; (3) slit/excision, the zona slit and herniating trophectoderm excised; and (4) hatch/excision, trophectoderm cells excised after spontaneous hatching. RESULTS: Murine blastocysts were comparatively biopsied using one of four methods and contrasted to zona slit and nonmicromanipulated controls. Operative cellular injury was assessed by uptake of fluorescein isothiocyanate (FITC). Postoperative embryonic viability was assessed by blastocoele reexpansion and hatching inner cell mass development and trophectoderm plating. All techniques yielded cells available for genetic analysis. CONCLUSIONS: The slit/excision technique and hatch/excision techniques exhibited lower operative injury and the higher postoperative viability than aspiration or incision. The slit/excision and the hatch/excision techniques, though requiring two operative steps, appear to be the least damaging of the four methods. Therefore, they should be most applicable to human blastocysts obtained either by extended culture in vitro or by uterine lavage.

Animals↗
PubMed1993

Development and optimization of plaque assays for rat coronaviruses.

Plaque assays under Sephadex or agarose overlays are described for rat coronaviruses (RCVs) grown in L2 mouse fibroblasts. A plaque assay using Sephadex was simple; however, viable plaques could not be collected for propagation, and fixation was necessary before evaluation. Plaque formation under agarose was optimized using diethylaminoethyl-dextran (DEAE-D) in the pre-treatment and absorption media and trypsin added to the absorption media and agarose overlay. The use of DEAE-D alone, trypsin alone or trypsin combined with DEAE-D significantly increased plaque numbers and visibility. Plaque numbers were highest when pre-treatment media contained DEAE-D, absorption media contained DEAE-D and trypsin, and the agarose overlay contained trypsin. The assay was useful for plaque isolation and quantification of sialodacryoadenitis virus (SDA), Parker's rat coronavirus (PRCV) and other coronavirus isolates from rats and its specificity was demonstrated by plaque-reduction neutralization testing. These methods will facilitate production of cloned virus stocks for study of RCV biology and virus quantification for in vitro and in vivo studies of RCVs.

Animals↗
PubMed1993

Characterization of acute rat parvovirus infection by in situ hybridization.

In situ hybridization and virus titration were used to characterize early stages of rat virus (RV) infection of rat pups after oronasal inoculation. Results suggest that virus enters through the lung and that early viremia leads rapidly to pantropic infection. Cells derived from all three germ layers were infected with RV, but those of endodermal and mesodermal origin were the predominant targets. Infection of vascular endothelium was widespread and was associated with hemorrhage and infarction in the brain. Convalescence from acute infection was accompanied by mononuclear cell infiltrates at sites containing RV DNA. Viral DNA was also detected in endothelium, fibroblasts and smooth muscle myofibers four weeks after inoculation. Further examination of these cells as potential sites of persistent infection is warranted.

Animals↗
PubMed1993

Prevalence of viral antibodies and helminths in field populations of house mice (Mus domesticus) in southeastern Australia.

A 13-month study of wild mice (Mus domesticus) in wheatlands in southeastern Australia contrasted changes in the seroprevalence of antibody to 13 viruses and the occurrence of helminths with changes in their population dynamics. Mice were seropositive for mouse hepatitis virus (MHV), rotavirus, minute virus of mice (MVM), mouse adenovirus (MAdV), reovirus (reo 3), and murine cytomegalovirus (MCMV). The seroprevalences of all but rotavirus varied significantly with time and increased with host density. Near the end of the study, host density declined rapidly and the seroprevalence of MVM and reo 3 increased significantly. These two viruses had low seroprevalence when host survival was high and high seroprevalence when host survival was low, indicating they may play a role in regulating mouse populations. In the case of MVM, there was evidence of a viral epizootic during the decline in mouse abundance. The prevalence of four helminths (Taenia taeniaeformis, Syphacia obvelata, and Vampirolepis spp.) differed significantly with time but showed no apparent association with host density. These findings highlight the need for further study on the effect of viruses on the population dynamics of mice.

Animals↗
PubMed1993

Effect of age on distribution of zidovudine (azidothymidine) into the cerebrospinal fluid of Macaca nemestrina.

The brain tissue is an important target for anti-HIV drug therapy. Since the permeability of the blood-brain and blood-cerebrospinal fluid (CSF) barriers may differ between neonates and adults, we have determined the effect of age on the distribution of zidovudine (ZDV or azidothymidine) into the CSF in the macaque (M. nemestrina). Five newborn macaques were administered ZDV (iv bolus, 5 mg/kg) at various ages (2 days to 4 months). Both CSF (cisternal) and venous blood samples were obtained at approximately 60 and 90 min after drug administration. In another series of experiments, adult female macaques received ZDV as either an iv bolus (5 and 10 mg/kg) or an infusion for at least 12 hr. CSF (lumbar) and venous blood samples were obtained at approximately 60 and 90 min after iv bolus and at more than 12 hr after iv infusion. ZDV concentration in the CSF and the plasma samples was determined by high-performance liquid chromatography. The CSF/plasma concentration ratio of ZDV in the newborn and adult macaques, after iv bolus administration, was independent of time. In addition, no significant (P > 0.05) difference was observed in the pooled iv bolus ZDV CSF/plasma concentration ratio between the adult group (0.236 +/- 0.058) and the newborns (0.213 +/- 0.039). Moreover, the ZDV CSF/plasma concentration ratio in the adults and the newborns, after iv bolus administration, was found not to be significantly (P > 0.05) different from the ratio obtained at steady state in the adults (0.224 +/- 0.094). These data indicate that the distribution of ZDV into the CSF in macaque neonates and adults is similar.

Aging↗
PubMed1993

Disposition of drugs in cystic fibrosis. IV. Mechanisms for enhanced renal clearance of ticarcillin.

To investigate the hypothesis that renal secretion of penicillins is enhanced in cystic fibrosis the maximal tubular secretion rate (Tmax) of ticarcillin and the serum concentration of ticarcillin at half-maximal secretion rate (TC50) were determined in patients with cystic fibrosis (n = 6) and control subjects (n = 6). Each subject received three consecutive constant-rate intravenous infusions of ticarcillin (4, 13, and 70 mg/kg/hr; 2 1/2 hours each) simultaneously with a constant-rate (30 mg/kg/hr) infusion of insulin. Urine samples were collected at 1/2-hour intervals and serum samples at the midpoint of the urine collections. Ticarcillin and inulin concentrations in serum and urine were determined by high-performance liquid chromatographic and a spectrophotometric method, respectively. Ticarcillin serum protein binding was determined by ultrafiltration. Steady-state ticarcillin serum concentrations were achieved at all three infusion rates. The TC50 was significantly lower (p < 0.05) in patients with cystic fibrosis (33.7 +/- 12.2 micrograms/ml) compared with that in control subjects (77.6 +/- 38.4 micrograms/ml). In contrast, the Tmax was similar (cystic fibrosis, 0.25 +/- 0.12 mg/min/kg; control, 0.22 +/- 0.14 mg/min/kg; p > 0.05). These data indicate that renal clearance of penicillins is enhanced in cystic fibrosis because of greater affinity of the renal secretory system for these drugs.

Adolescent↗
PubMed1993

Disposition of drugs in cystic fibrosis. V. In vivo CYP2C9 activity as probed by (S)-warfarin is not enhanced in cystic fibrosis.

Enhanced metabolism of theophylline in subjects with cystic fibrosis suggests that the activity of certain cytochrome P450 isoforms is affected in subjects with this genetic disease. To determine whether this effect on the P450 enzymes is selective, the in vivo activity of the cytochrome P450 isoform CYP2C9 was determined in adult subjects with cystic fibrosis (n = 6) and in control subjects (n = 8). Subjects were administered (S)-warfarin as a single intravenous bolus dose (0.375 mg/kg), and urine and plasma samples were collected for 96 hours. Plasma (S)-warfarin concentrations were determined by HPLC; urinary concentrations of (S)-warfarin and its metabolites were determined by gas chromatography-mass spectrometry. The total plasma clearance of (S)-warfarin (subjects with cystic fibrosis, 3.6 +/- 0.48 ml/hr/kg; control subjects, 3.82 +/- 0.73 ml/hr/kg), elimination half-life (subjects with cystic fibrosis, 29.5 +/- 4.2 hours; control subjects, 25.9 +/- 5.4 hours); and steady-state volume of distribution (subjects with cystic fibrosis, 153 +/- 18 ml/kg; control subjects, 138 +/- 22 ml/kg) were similar in the two groups (p > 0.05). The metabolic clearance of (S)-warfarin to its major metabolites mediated by CYP2C9, 6-hydroxywarfarin and 7-hydroxywarfarin, was not significantly (p > 0.05) different between the two groups (6-hydroxywarfarin: subjects with cystic fibrosis, 0.33 +/- 0.1 ml/hr/kg; control subjects, 0.41 +/- 0.1 ml/hr/kg; 7-hydroxywarfarin: subjects with cystic fibrosis, 1.34 +/- 0.49 ml/hr/kg; control subjects, 1.8 +/- 0.45 ml/hr/kg). On the basis of these data, we conclude that the in vivo cytochrome P450 activity is selectively affected in persons with cystic fibrosis.

Adult↗
PubMed1993

Localization of the A1.12/9 antigen family to the neurones, putative sensory receptors and tegument of Schistosoma mansoni.

The A1.12/9 antigen family were localized to the periphery of vesicle-like structures in neurones and in the putative sensory receptors of cercariae by immuno-electron microscopy. After transformation into schistosomula, the antigens were rapidly lost until, after 18 h, no immunoreactivity could be detected. Expression resumed at approximately 36 h post-transformation and had returned to high levels by 4 days post-infection. This level was maintained through to the adult worm stage. In 4 day lung schistosomula and in 14 day and 21 day liver schistosomula immunolabelling was observed within the matrix of the tegument itself and also in association with the tegumental membrane and vesicles within the tegument. These properties suggest that the A1.12/9 antigens may be involved in neuropeptide processing pathways. In particular, this family may represent the schistosome homologue of the granin family with which they share common properties and some sequence homology.

Animals↗
PubMed1993

Variant responses of mice to Borrelia burgdorferi depending on the site of intradermal inoculation.

C3H/He mice inoculated intradermally at one of two sites with Borrelia burgdorferi responded differently to infection. Shoulder-inoculated mice developed spirochetemia, B. burgdorferi-specific antibody, and arthritis earlier than foot-inoculated mice. Lymphocyte populations derived from spleen tissue were elevated in the shoulder- but not the foot-inoculated mice, and those from lymph nodes were increased in both groups. Lymphocytes derived from blood and spleen tissue showed impaired proliferative responses to all mitogens for shoulder-inoculated mice only, whereas proliferation of lymph node cells was not affected, regardless of route. These results demonstrate that the site of initial B. burgdorferi inoculation is an important determinant in the pathogenesis of B. burgdorferi infection.

Animals↗
PubMed1993

Exacerbation of Pneumocystis carinii pneumonia in immunodeficient (scid) mice by concurrent infection with a pneumovirus.

scid mice naturally infected with Pneumocystis carinii and inoculated with a normally apathogenic pneumovirus had significantly higher P. carinii cyst counts and developed significantly more severe P. carinii-related disease than did sham-inoculated, P. carinii-infected scid mice. P. carinii-free, virus-infected scid mice survived for 2 months despite high pulmonary virus titers. These results show that a respiratory virus infection can exacerbate P. carinii disease in an immunocompromised-rodent model.

Animals↗
PubMed1993

Long-term study of cell-mediated responses to Borrelia burgdorferi in the laboratory mouse.

Borrelia burgdorferi infection of disease-susceptible (C3H) and -resistant (BALB) mice resulted in impaired proliferation to both T- and B-cell mitogens up to 30 days after inoculation. Interleukin-2 and -4 production was also impaired, paralleling the T-cell response to concanavalin A. Impaired lymphocyte proliferation could not be attributed to diminished numbers of T or B cells and was found to depend on the lymphoid organ (spleen or lymph node) examined. Prostaglandin production accounted for part of this immune dysfunction. Attempts to assess antigen-specific proliferation to B. burgdorferi were inconsistent, and delayed-type hypersensitivity responses were not detected. Adoptive transfer of T-enriched cells from chronically infected donors failed to prevent infection and disease development in recipient C3H mice. The current study emphasizes caution in the study of B. burgdorferi antigen-specific assays and argues against the role of a vigorous T-cell response in Lyme borreliosis in infected laboratory mice.

Animals↗
PubMed1993

A serologic survey for viruses and Mycoplasma pulmonis among wild house mice (Mus domesticus) in southeastern Australia.

Plasma samples from 267 wild house mice (Mus domesticus) trapped at 14 sites in southeastern Australia were screened for antibody to 14 viruses normally associated with laboratory-reared rodents and to Mycoplasma pulmonis. Serologic prevalence was high for murine cytomegalovirus (99%, n = 94), murine coronavirus (95%), and murine rotavirus (74%). Samples from mice collected at all sites contained antibody to these viruses. The serologic prevalence was lower for mouse adenovirus, strain K87 (37%), parvovirus (33%), and reovirus type 3 (28%), with substantial site-to-site variation. Plasma from mice collected at 12 sites contained mouse adenovirus or reovirus antibody, and samples from mice at eight sites contained parvovirus antibody. Parvovirus-antibody positive mice were typically from high density populations or from low density populations that had recently declined from high density. Antibody to lymphocytic choriomeningitis virus (LCMV) and Sendai virus occurred at only three sites, and the serologic prevalence was very low (9.6% and 1.8%, respectively). All of the LCMV-positive mice were from northeastern New South Wales. The presence of this zoonotic virus in a mouse plague-prone region raises questions about human health risks resulting from cohabitation with large numbers of mice. It appeared that mouse populations at high density or declining from high density had higher prevalence of viral antibody than populations that had been at low or moderate density for some time. Thus, viral epizootics may occur among high-density populations and may be responsible for or precipitate declines in mouse density. These data raise the possibility of rodent viruses having potential as biological control agents.

Animals↗