Biomedical subjects

D R Smith

Publications and source records attributed to D R Smith.

At least 91 records · Page 5Linked to original sources

PubMed1999

Experimental inoculation of adult dairy cows with bovine coronavirus and detection of coronavirus in feces by RT-PCR.

A reverse transcriptase PCR (RT-PCR) targeting a 407 bp fragment of the nucleocapsid gene of bovine coronavirus (BCV) was developed for detection of BCV RNA in feces of experimentally inoculated cattle. The sensitivity and specificity of the RT-PCR were confirmed using tissue culture-adapted BCV strains and feces of 2 calves inoculated with BCV. Ten nonpregnant, BCV seropositive, adult dairy cows were inoculated with winter dysentery (WD) (n = 8) or calf diarrhea (CD) (n = 2) strains of BCV intranasally and orally (n = 2) or through a surgically-placed duodenal catheter (n = 8) with and without dexamethasone treatment or feeding ice water. The 6 cows inoculated with BCV intranasally and through a duodenal catheter (2 of 2 cows given CD BCV and 4 of 6 cows given WD BCV) developed mild diarrhea, and BCV was detected in diarrheal feces by RT-PCR, ELISA or immune electron microscopy. These results suggest that CD and WD strains of BCV can cause diarrhea in adult cows in conjunction with host or environmental factors and that RT-PCR might be useful to diagnose BCV infections in calves and adult cows.

Animals↗

PubMed1999

c-Ki-ras mutations in colorectal adenocarcinomas from a country with a rapidly changing colorectal cancer incidence.

We have examined the incidence of mutation of the c-Ki-ras proto-oncogene in colorectal adenocarcinomas from two different time periods, namely 1962-1966 and 1994-1996. The first cohort of samples consisted of formalin-fixed, archival paraffin block and represent the oldest colorectal cancer samples for which ras mutation has been examined, while the second cohort of tumours were fresh, flash-frozen samples representative of genetic events occurring in contemporary times. Analysis of mutation status was undertaken by a mismatch-specific oligonucleotide hybridization analysis of exon 1 of the c-Ki-ras proto-oncogene after amplification by the polymerase chain reaction. Mutations in codon 12 or 13 of c-Ki-ras were detected in 28% (14/50) of contemporary samples, a figure consistent with locally established mutation rates. In contrast no mutation was detected in any of the 18 samples from the earlier period, a result that is statistically significant (P = 0.007). Age-standardized rates of colorectal cancer in Singapore have seen a marked increase over the last 30 years, and for the first time we have shown that such an increase in colorectal cancer is associated, at least in part with an increase in incidence of a specific mutagenic change.

Adenocarcinoma↗

PubMed1999

HIT family genes: FHIT but not PKCI-1/HINT produces altered transcripts in colorectal cancer.

Forty-five colorectal adenocarcinomas were examined for alterations in the HIT family genes FHIT and PKCI-1/HINT by a combination of reverse transcriptase polymerase chain reaction and DNA sequencing. In all cases a single transcript corresponding to the reported sequence was detected using primers specific for the PKCI-1/HINT gene. In contrast multiple transcripts were detected using primers specific for the FHIT gene transcript. 6% (3/45) of tumours evinced no detectable expression of any FHIT transcript and a further 12% (6/45) produced only the normal full length transcripts. Ninety-six aberrant transcripts were characterized from the remaining tumours. Deviations from the normal full length sequence characterized included deletions, insertions of novel sequences, a point mutation as well as the usage of a putative alternate splice site in exon 10. Message variants were detected with approximately equal frequency in all tumour stages with the exception that templates with insertions were found solely in Dukes' stage B tumours (P < 0.001). With the exception of the putative alternate splice site, aberrant transcripts were not detected in matched normal mucosa. These results suggest that members of the HIT family of genes are only selectively involved in tumorigenesis and that perturbation of FHIT gene expression is an early event in colorectal tumorigenesis.

Acid Anhydride Hydrolases↗

PubMed1999

Postnatal growth responses to insulin-like growth factor I in insulin receptor substrate-1-deficient mice.

Organ weight was compared in adult mice with deletion of one (IRS-1-/+) or both (IRS-1-/-) copies of the insulin receptor substrate-1 (IRS-1) gene and IRS-1+/+ littermates. IRS-1-/+ mice showed modest reductions in weight of most organs in proportion to a decrease in body weight. IRS-1-/- mice showed major reductions in weight of heart, liver, and spleen that were directly proportional to a decrease in body weight. In IRS-1-/- mice, kidney and particularly small intestine and brain exhibited proportionately smaller weight reductions, and gastrocnemius muscle showed a proportionately greater weight reduction than the decrease in body weight. Growth deficits in IRS-1-/- mice could reflect impaired actions of multiple hormones or cytokines that activate IRS-1. To assess the requirement for IRS-1 in insulin-like growth factor I (IGF-I)-dependent postnatal growth, IRS-1-/+ mice were cross-bred with mice that widely overexpress a human IGF-I transgene (IGF+) to generate IGF+ and wild-type mice on an IRS-1+/+, IRS-1-/+, and IRS-1-/- background. IGF-I overexpression increased body weight and weight of brain, small intestine, kidney, spleen, heart, and gastrocnemius muscle in IRS-1+/+ mice. IGF-I overexpression could not completely reverse the body growth retardation in IRS-1-/- mice. Absolute or partial IRS-1 deficiency impaired IGF-I-induced body overgrowth more in females than in males. In males and females, IGF-I stimulated similar overgrowth of brain regardless of IRS-1 status, and intestine and spleen showed dose dependence on IRS-1 for IGF-I-induced growth. IGF-I-induced growth of gastrocnemius muscle had an absolute requirement for IRS-1. IGF-I-induced growth of kidney and heart was impaired by IRS-1 deficiency only in females. In vivo, therefore, most organs do not require IRS-1 for IGF-I-induced growth and can use alternate signaling molecules to mediate IGF-I action. Other organs, such as gastrocnemius muscle, require IRS-1 for IGF-I-induced growth in vivo.

Animals↗

PubMed1999

p53 point mutation and survival in colorectal cancer patients: effect of disease dissemination and tumour location.

The relationship between p53 point mutation and patient survival was examined in 328 colorectal cancer patients. Point mutation was detected in 51% (166/328) of cases and was associated with a poorer prognosis in univariate and multivariate analysis. However, subcohort analysis showed that this relationship was restricted to patients with lymphatic dissemination, patients without evident distant metastatic lesions at the time of presentation and in tumours confined to the distal colorectum. These results suggest that the utility of p53 point mutation as a specific, patient based prognostic marker may be restricted to certain classes of patients.

Adenocarcinoma↗

PubMed1999

Surgical sterilization of free-ranging wolves.

The objective of the study was to determine whether surgical sterilization of both males and females in wolf pairs alters basic wolf social and territorial behaviors. Wolves were located from the air by snow-tracking methods and were tranquilizer-darted from a helicopter. Surgeries were performed either in a tent at the capture site or in a heated building in a nearby village. Six vasectomies and seven uterine horn ligations were performed in January and February of 1996 and 1997. Two females died: one likely related to the capture procedure, the other of a peritonitis unrelated to the surgery. One wolf had a litter. None of the wolves have shown changes in behavioral patterns. Surgical sterilization can be effective, but other, less invasive, fertility control techniques should be investigated.

Animals↗

PubMed1999

Prognostic factors in patients with inferior vena cava injuries.

Inferior vena cava (IVC) injuries are potentially devastating insults that continue to be associated with high mortality despite advances in prehospital and in-hospital critical care. Between 1987 and 1996, 37 patients (32 males and 5 females; average age, 30 years) were identified from the trauma registry as having sustained IVC trauma. Overall mortality was 51 per cent (n = 19), with 13 intraoperative deaths and five patients dying within the first 48 hours. Blunt IVC injuries (n = 8) had a higher associated mortality than penetrating wounds (63% versus 48%). Of the 29 patients with penetrating IVC trauma, the wounding agent influenced mortality (shotgun-100% versus gunshot-43% versus stab-0%). Anatomical location of injury was also predictive of death [suprahepatic (n = 3)-100% versus retrohepatic (n = 9)-78% versus suprarenal (n = 6)-33% versus juxtarenal (n = 2)-50% versus infrarenal (n = 15)-33%]. A direct relationship existed between outcome and the number of associated injuries: nonsurvivors averaged four and survivors averaged three. Eighty per cent of patients sustaining four or more associated injuries died, by contrast to a 33 per cent mortality in those suffering less than four injuries. Physiological factors were also predictive of outcome. Patients in shock (systolic blood pressure < 80) on arrival had a higher mortality than those who were hemodynamically stable (76% versus 30%). Preoperative lactate levels were of prognostic value for death (> or = 4.0-59% versus < 4.0-0%), as was base deficit (< 4-22%, > or = 4, and < 10-36%, > or = 10-73%). Interestingly, neither time from injury to hospital arrival (47.4 minutes versus 33.0 minutes) nor time in the emergency department before surgery (45.6 minutes versus 42.6 minutes) differed between survivors and fatalities. Mortality remained high in the 34 patients who had operative control of their IVC injuries [lateral repair (n = 27)-44% versus ligation (n = 6)-66% versus Gortex graft (n = 1)-0%]. As wounding agent, anatomical location, associated injuries, and physiological status seem to most directly impact mortality, future efforts must focus both on establishing prevention programs directed at reducing the incidence of this injury, as well as on advancing the management of those who do survive to hospitalization, if we are to improve on the outcome of these devastating injuries.

Adolescent↗

PubMed1999

Lessons learned from a review of the development of selected vaccines. National Vaccine Advisory Committee.

BACKGROUND: Although the vaccine research and development network in the United States remains vibrant, its continued success requires maintaining harmonious interaction among its many components. Changing one component is likely to affect the system overall. An examination of case studies of the development of selected vaccines would allow an examination of the network as a whole. This article presents conclusions drawn from the case study review undertaken. OBJECTIVE: Successful development of vaccines is a time-intensive process requiring years of commitment from a network of scientists and a continuum of regulatory and manufacturing entities. We undertook this work to shed light on how well the vaccine development system in the United States performs. METHOD: The National Vaccine Advisory Committee examined the research and development pathways of several vaccines that reached licensure expeditiously (hepatitis B vaccine, Haemophilus influenzae type b conjugate vaccines); some that became licensed only after considerable delay (oral typhoid Ty21a vaccine, varicella vaccine); some that are at the point of imminent or recent licensure (reassortant Rhesus rotavirus vaccine, which was licensed by the Food and Drug Administration on August 30, 1998) or near submission for licensure (intranasal cold adapted influenza vaccine); and one for which clinical development is slow because of hurdles that must be overcome (respiratory syncytial virus vaccines). RESULTS: Some common themes emerged from the reviews of these vaccine "case histories": the expediting influence of a strong scientific base and rationale; the need for firm quantitation of disease burden and clear identification of target populations; the critical role played by individuals or teams who act as "champions" to overcome the inevitable obstacles; availability of relevant animal models, high-quality reagents and standardized assays to measure immune response; the absolute requirement for well designed, meticulously executed clinical trials of vaccine safety, immunogenicity, and efficacy; postlicensure measurements of the public health impact of the vaccine and a track record of the vaccine's safety and acceptance with large-scale use; and the critical need for international collaborations to evaluate vaccines against diseases of global importance that are rare in the United States (eg, typhoid fever). It was clear that the critical step-up from bench scale to pilot lots and then to large-scale production, which depends on a small group of highly trained individuals, is often a particularly vulnerable point in the development process. CONCLUSIONS: One fundamental lesson learned is that within the varied and comprehensive US vaccine development infrastructure, multiple and rather distinct paths can be followed to reach vaccine licensure. The National Vaccine Advisory Committee review process should be conducted periodically in the future to ascertain that the US vaccine development network, which has been enormously productive heretofore and has played a leadership role globally, is adapting appropriately to ensure that new, safe, and efficacious vaccines become available in a timely manner.

Drug Approval↗

PubMed1998

High-affinity renal lead-binding proteins in environmentally-exposed humans.

Chronic low level lead (Pb) exposure is associated with decrements in renal function in humans, but the molecular mechanisms underlying toxicity are not understood. We investigated cytosolic Pb-binding proteins (PbBP) in kidney of environmentally-exposed humans to identify molecular targets of Pb and elucidate mechanisms of toxicity. This study is unique in that it localized PbBPs based on physiologic Pb that was bound in vivo. Two Pb-binding polypeptides were identified, thymosin beta 4 (T beta 4, 5 kDa) and acyl-CoA binding protein (ACBP, 9 kDa, also known as diazepam binding inhibitor, DBI). These polypeptides, which have not been previously recognized for their metal-binding capabilities, were shown to bind Pb with high affinity (Kd approximately 14 nM) and to account for an estimated > 35% of the total Pb in kidney cortex tissue. Both T beta 4 and ACBP (DBI) occur across animal species from invertebrates to mammals and in all major tissues, serving multiple possible functions (e.g. regulation of actin polymerization, calmodulin-dependent enzyme activity, acyl-CoA metabolism, GABA-A/benzodiazepine receptor modulation, steroidogenesis, etc.). Thus, these data provide the first evidence of specific molecular targets of Pb in kidney of environmentally-exposed humans, and they suggest that low-level Pb toxicity may occur via alteration of T beta 4 and ACBP (DBI) function in renal and other tissues, including the central nervous system.

Amino Acid Sequence↗

PubMed1998

Results of an oral rabies vaccination program for coyotes.

OBJECTIVE: To determine effectiveness of large-scale distribution of an oral rabies vaccine contained in a palatable bait for halting expansion of a canine rabies epizootic in coyotes (Canis latrans). DESIGN: Prospective study. ANIMALS: 98 coyotes during prevaccination surveillance and 449 coyotes and 60 other wild animals during postvaccination surveillance. PROCEDURE: A vaccinia recombinant oral rabies vaccine was inserted into an edible bait for coyotes that also contained tetracycline as a biomarker. Vaccine units were then distributed via aircraft, using automated distribution equipment and flight plans developed by incorporating global positioning system equipment. The target area was along the northern edge of an area that had an epizootic of canine rabies. This area was identified through previously conducted epidemiologic surveillance of rabies cases. During postvaccination surveillance, dental specimens were examined for biomarker evidence of bait acceptance, and serum samples were analyzed for rabies neutralizing antibodies. RESULTS: Samples from 449 coyotes were obtained during postvaccination surveillance. Seroconversion was detected in 39 of 96 (40.6%) coyotes that had evidence of tetracycline biomarker. Additionally, the number of rabies cases in the target area decreased, and expansion of the epizootic area ceased. CLINICAL IMPLICATIONS: Mass distribution of an oral rabies vaccine in a palatable bait is an effective means to halt expansion of a rabies epizootic involving coyotes.

Administration, Oral↗

PubMed1998

Methodological considerations for the accurate determination of lead in human plasma and serum.

Studies which accurately measure plasma or serum lead (Pb) are needed to evaluate the 'biologically active' fraction of Pb in the circulation, and to clarify the role of plasma in the transportation of Pb between different compartments of the body. We evaluated several methodological aspects which influence the determination of Pb in plasma and serum. Generally, venous blood was obtained by different sampling methods (routine and ultraclean) from 3 subjects without history of Pb exposure. After centrifugation (800 g) for 10 min, the plasma or serum was analyzed by inductively coupled plasma-high-resolution mass spectrometry (ICP-MS). Several evaluations were conducted, including 1) comparison of an ultraclean serum collection method with a plasma collection method that used a commercial Vacutainer-type tube for trace metals (EDTA anticoagulant); 2) the effect of whole blood standing time prior to centrifugation on plasma or serum Pb concentration; and 3) comparison of a method using commercial heparinized Vacutainer tubes to an ultraclean plasma sampling method that utilized a low-Pb heparin anticoagulant. Plasma or serum iron (Fe) levels were also measured to evaluate hemolysis. The 3 subjects had whole blood Pb (blood-Pb) levels of 1.8, 2.0, and 2.7 micrograms/dl. Their corresponding ultraclean serum-Pb levels were 0.40%, 0.30%, and 0.48% of their whole blood-Pb levels, respectively. By comparison, the EDTA Vacutainer method plasma-Pb values were 1.7%, 1.5%, and 2.4% of whole blood-Pb, respectively. Whole blood standing (clotting) times of 15, 40, and 70 min before centrifugation resulted in increasing ultraclean serum-Pb levels of 0.21%, 0.81%, and 1.2% of whole blood-Pb (1.8 micrograms/dl), respectively. Whole blood standing time had no effect on plasma-Pb levels when heparin Vacutainers were used, or when a low-Pb heparin was used to obtain ultraclean plasma. However, plasma collected using the commercial heparin Vacutainer method contained consistently higher and more variable Pb levels than samples collected using the ultraclean plasma-Pb method. Hemolysis, when present, contributed significantly to both plasma-Pb and serum-Pb levels. In conclusion, plasma-Pb and serum-Pb levels are dependent upon methodologic processing techniques, including Pb contamination control, redistribution due to EDTA anticoagulant, hemolysis, and time dependency in sample processing. While true plasma-Pb and serum-Pb levels by any method have yet to be defined, these data provide a methodological basis from which to investigate variation in Pb partitioning between whole blood and plasma within individuals.

Adult↗

PubMed1998

Seasonal variation of interferon-gamma production in progressive multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system. There is increased interferon (IFN)-gamma secretion in MS patients in vitro, and IFN-gamma administration induces exacerbations of disease suggesting a link between IFN-gamma and disease activity. We observed significantly increased IFN-gamma production in the autumn and winter months compared with the spring and summer months in chronic progressive MS, and this increase was linked to endogenous interleukin (IL)-12 production. Increased seasonal IFN-gamma was not observed in normal control subjects, and there were no seasonal changes in IL-10 in progressive MS. These results suggest a potential environmental link between dysregulated IFN-gamma production and MS disease progression and pathogenesis.

Cells, Cultured↗

PubMed1998

Pulse cyclophosphamide plus methylprednisolone induces myelin-antigen-specific IL-4-secreting T cells in multiple sclerosis patients.

Multiple sclerosis (MS) is a presumed cell-mediated Th1-type autoimmune disease. Thus therapies which decrease T cells secreting IFN-gamma production or increase IL-4 production would be expected to have an ameliorating effect on MS. We have previously reported increased anti-CD3-induced IL-4 secretion by T cells in progressive MS patients treated with cyclophosphamide plus methylprednisolone (CY/MP) which was associated with eosinophilia. To investigate whether the increased IL-4 secretion was myelin antigen specific, we generated 3990 short-term T cell lines to myelin basic protein (MBP), proteolipid protein (PLP), or tetanus toxoid (TT) from 31 progressive MS patients: 11 MS patients treated with CY/MP, 10 MS patients treated with MP alone, and 10 untreated MS patients. We found increased frequencies of both MBP- and PLP-specific IL-4-secreting T cell lines in CY/MP-treated patients compared to untreated MS patients. However, no change in the frequency of TT-specific IL-4-secreting T cells was observed. MP treatment alone did not increase the frequency of antigen-specific IL-4-secreting T cell lines. These results demonstrate immune deviation favoring Th2-type responses specific to autoantigens following pulse cyclophosphamide therapy in MS patients.

Adult↗

PubMed1998

Behavioural assessment of mice lacking D1A dopamine receptors.

Dopamine D1A receptor-deficient mice were assessed in a wide variety of tasks chosen to reflect the diverse roles of this receptor subtype in behavioural regulation. The protocol included examination of exploration and locomotor activity in an open field, a test of sensorimotor orienting, both place and cue learning in the Morris water maze, and assessment of simple associative learning in an olfactory discrimination task. Homozygous mice showed broad-based impairments that were characterized by deficiencies in initiating movement and/or reactivity to external stimuli. Data obtained from flash evoked potentials indicated that these deficits did not reflect gross visual impairments. The partial reduction in D1A receptors in the heterozygous mice did not affect performance in most tasks, although circumscribed deficits in some tasks were observed (e.g., failure to develop a reliable spatial bias in the water maze). These findings extend previous behavioural studies of null mutant mice lacking D1A receptors and provide additional support for the idea that the D1A receptor participates in a wide variety of behavioural functions. The selective impairments of heterozygous mice in a spatial learning task suggest that the hippocampal/cortical dopaminergic system may be uniquely vulnerable to the partial loss of the D1A receptor.

Animals↗

PubMed1998

An efficient, automatable template preparation for high throughput sequencing.

We have developed a 96-well format for DNA template isolation that can be readily automatable. The template isolation protocol involves simple alkaline lysis chemistry and reversible capture on a silica solid phase. After the cells are lysed, no centrifugation is necessary, as lysate purification, DNA binding, washing, and release occur in 96-well filter plates. Large numbers of templates prepared using the silica purification method have been sequenced and analyzed. The quality of sequence resulting from our method has been compared with that generated from several commercial plasmid preparation protocols. We found sequence quality of the silica bead preparations to be equivalent to or, in some cases, better than those prepared by other methods. This method offers many advantages over other protocols we have used. First, the silica purifications have allowed us to more than double overall laboratory throughput while decreasing our template isolation materials cost at least five-fold. Second, because we have eliminated all centrifugation steps in the protocol, automation has been much simpler. The protocol has also been adapted to purify PCR products for use as templates in subsequent sequencing reactions.

Automation↗

PubMed1998

Animal models: nutrition and lipoprotein metabolism.

Despite limitations, animal models continue to provide insight into the etiology of atherosclerosis. Hamsters, guinea-pigs, and domesticated pigs are being used more frequently to address the biochemical bases for the effects of dietary lipids on cholesterol metabolism.

Animals↗

PubMed1998

Further validation of antibody-capture and antigen-capture enzyme-linked immunosorbent assays for determining exposure of cattle to bovine coronavirus.

OBJECTIVE: To further validate an antibody-capture ELISA for measuring bovine coronavirus (BCV) exposure (antibody seroresponse) in cattle and to explain the apparent loss of sensitivity of a BCV antigen-capture ELISA when testing feces from adult versus neonatal cattle. ANIMALS: 98 adult cows from herds with and without winter dysentery; 10 gnotobiotic or colostrum-deprived calves. PROCEDURES: Results of an antibody-capture ELISA for BCV and a plaque reduction virus neutralization assay performed on paired serum samples from 24 cattle were compared with each other and with results of immunoelectron microscopy (IEM) of feces for BCV. For samples from 98 cattle, results of antibody-capture ELISA were compared with results of IEM. Calves were inoculated with feces ELISA-positive or IEM-positive for BCV and monitored for BCV infection. An ELISA was developed to detect BCV antigen-antibody complexes in feces and results were compared with results of an antigen-capture ELISA and IEM. RESULTS: Antibody-capture ELISA results correlated with neutralization assay results, but agreed more closely with results of IEM. Calves became infected with BCV following inoculation with either ELISA-positive or ELISA-negative but IEM-positive feces. Results of the antigen-antibody ELISA correlated with results of IEM and the antigen-capture ELISA. CLINICAL IMPLICATIONS: In adult cattle, testing of paired serum samples by use of an antibody-capture ELISA may be a better indicator of recent BCV exposure than results of virus neutralization tests. Antigen-antibody binding in feces may interfere with results of antigen-capture ELISA for BCV.

Animals↗

PubMed1998

Evaluation of cow-level risk factors for the development of winter dysentery in dairy cattle.

OBJECTIVE: To identify exposures to etiologic agents and to identify characteristics that could explain risk of disease for adult cattle in herds affected by winter dysentery (WD). ANIMALS: 229 lactating and nonlactating adult cattle (125 case and 104 control cattle) selected from 12 dairy herds. PROCEDURE: A case-control study, using multivariate conditional logistic regression and controlling for herd effects, was used to develop a model for risk factors associated with disease for each cow. RESULTS: Likelihood of developing disease increased as the ELISA value for bovine coronavirus (BCV) antigen detectable in feces increased (odds ratio [OR] = 2.94 for each 0.100 increase in BCV antigen ELISA value). Pregnant cattle were less likely to develop WD, compared with nonpregnant herdmates. Cows with high acute BCV antibody titers that seroresponded had greater odds of developing disease, compared with seroresponding cows with low acute titers. However, among those cows that did not serorespond, high acute antibody titers were associated with lower odds of developing the disease. CONCLUSION: In herds affected by WD, ill cows were more likely to shed detectable amounts of BCV antigen in their feces, and pregnancy appeared to protect cattle from the disease. The measured interaction between BCV seroresponse and acute BCV antibody titer may be evidence of an immunopathologic condition, but could also have been attributable to dynamics of the ELISA or study design. CLINICAL RELEVANCE: Factors that explained a cow's risk for illness within WD-affected herds may have been surrogate measures for that cow's nonspecific and BCV-specific immune profile.

Animals↗