Biomedical subjects

Christina Chan

Publications and source records attributed to Christina Chan.

10 recordsLinked to original sources

PubMed2003

Direct extraction of Sabin poliovirus genomes from human fecal samples using a guanidine thiocyanate extraction method.

To permit rapid and efficient detection of Sabin poliovirus type 3 from human fecal samples, we developed a guanidine thiocyanate (GuSCN) extraction and reverse transcriptase polymerase chain reaction (RT-PCR) method. Using 10-fold serial dilutions from stock Sabin-Leon 12 a1b poliovirus type 3 at 10(7) TCID(50) per 0.1 ml, genome was detected to a dilution of 10(3) TCID(50) per 0.1 ml. A total of 40 archived fecal samples were examined using this GuSCN extraction method followed by RT-PCR. Fourteen of 20 poliovirus type 3 tissue culture-positive specimens (70%) and two of 20 tissue culture-negative specimens (10%) were detected by GuSCN extraction and RT-PCR. All positive and negative extraction and RT-PCR controls were identified accurately. This GuSCN extraction and RT-PCR technique is rapid, inexpensive, and can be readily adapted to identify genome sequences of other enterovirus types in large numbers of fecal samples. Moreover, the GuSCN technique extracts viral RNA directly from fecal samples, allowing observation of in vivo alterations of genome sequences. Further studies are underway to examine the development of revertant point mutations in the Sabin poliovirus type 3 genome following oral administration of trivalent Sabin Oral Poliovirus Vaccine to humans.

Animals↗

PubMed2003

Splinter removal.

Splinter injuries are common, but larger and deeper splinters are often difficult and painful to remove at home. These splinters often present as a foreign body embedded in the superficial or subcutaneous soft tissues. Whenever possible, reactive objects like wood, thorns, spines, and vegetative material should be removed immediately, before inflammation or infection occurs. Superficial horizontal splinters are generally visible on inspection or easily palpated. A horizontal splinter is exposed completely by incising the skin over the length of the long axis of the splinter, and removed by lifting it out with forceps. A subungual splinter may be removed by cutting out a V-shaped piece of the nail. The point of the V is at the proximal tip of the splinter, which is grasped and removed, taking particular care not to push the splinter further into the nail bed. Removal of an elusive splinter can be challenging and may require the use of imaging modalities for better localization. Deeper splinters, especially those close to important structures such as nerves, tendons, blood vessels, or vital organs, should be referred for removal.

Foreign Bodies↗

PubMed2003

Metabolic flux analysis of cultured hepatocytes exposed to plasma.

Hepatic metabolism can be investigated using metabolic flux analysis (MFA), which provides a comprehensive overview of the intracellular metabolic flux distribution. The characterization of intermediary metabolism in hepatocytes is important for all biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. During BAL operation, hepatocytes are exposed to plasma or blood from the patient, at which time they are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. In a prior study, we found that preconditioning the primary rat hepatocytes in culture medium containing physiological levels of insulin, as opposed to the typical supraphysiological levels found in standard hepatocyte culture media, reduced lipid accumulation during subsequent plasma exposure. Furthermore, supplementing the plasma with amino acids restored hepatospecific functions. In the current study, we used MFA to quantify the changes in intracellular pathway fluxes of primary rat hepatocytes in response to low-insulin preconditioning and amino acid supplementation. We found that culturing hepatocytes in medium containing lower physiological levels of insulin decreased the clearance of glucose and glycerol with a concomitant decrease in glycolysis. These findings are consistent with the general notion that low insulin, especially in the presence of high glucagon levels, downregulates glycolysis in favor of gluconeogenesis in hepatocytes. The MFA model shows that, during subsequent plasma exposure, low-insulin preconditioning upregulated gluconeogenesis, with lactate as the primary precursor in unsupplemented plasma, with a greater contribution from deaminated amino acids in amino acid-supplemented plasma. Concomitantly, low-insulin preconditioning increased fatty acid oxidation, an effect that was further enhanced by amino acid supplementation to the plasma. The increase in fatty acid oxidation reduced intracellular triglyceride accumulation. Overall, these findings are consistent with the notion that the insulin level in medium culture presets the metabolic machinery of hepatocytes such that it directly impacts on their metabolic behavior during subsequent plasma culture.

Amino Acids↗

PubMed2003

Measurement of cell-mediated immunity with a Varicella-Zoster Virus-specific interferon-gamma ELISPOT assay: responses in an elderly population receiving a booster immunization.

An interferon-gamma ELISPOT assay has been developed for assessment of cellular immune responses to Varicella-Zoster Virus (VZV) in large, multi-center clinical vaccine trials. We show that the assay performed best when testing peripheral blood mononuclear cells (PBMCs) that had been isolated and then frozen on the same day as blood was drawn, and that freezing PBMCs from blood that was stored overnight before processing resulted in dramatically reduced responses. This assay was used to monitor cell-mediated immunity (CMI) in response to a booster immunization with an investigational live, attenuated VZV vaccine in an elderly population that had been vaccinated 8-10 years previously. The booster vaccine elicited a 1.6- to 1.7-fold rise in the VZV-specific cellular immune response as measured by the ELISPOT assay. The increase from pre to post booster vaccination response was more pronounced (approximately 2.2-fold rise) in a subset of subjects who had received two prior immunizations with a live, attenuated vaccine.

Aged↗

PubMed2003

Metabolic flux analysis of hepatocyte function in hormone- and amino acid-supplemented plasma.

Understanding the metabolic and regulatory pathways of hepatocytes is important for biotechnological applications involving liver cells. Previous attempts to culture hepatocytes in plasma yielded poor functional results. Recently we reported that hormone (insulin and hydrocortisone) and amino acid supplementation reduces intracellular lipid accumulation and restores liver-specific function in hepatocytes exposed to heparinized human plasma. In the current study, we performed metabolic flux analysis (MFA) using a simplified metabolic network model of cultured hepatocytes to quantitively estimate the changes in lipid metabolism and relevant intracellular pathways in response to hormone and amino acid supplementation. The model accounts for the majority of central carbon and nitrogen metabolism, and assumes pseudo-steady-state with no metabolic futile cycles. We found that beta-oxidation and tricarboxylic acid (TCA) cycle fluxes were upregulated by both hormone and amino acid supplementation, thus enhancing the rate of lipid oxidation. Concomitantly, hormone and amino acid supplementation increased gluconeogenic fluxes. This, together with an increased rate of glucose clearance, caused an increase in predicted glycogen synthesis. Urea synthesis was primarily derived from ammonia and aspartate generated through transamination reactions, while exogenous ammonia removal accounted for only 3-6% of the urea nitrogen. Amino acid supplementation increased the endogenous synthesis of oxaloacetate, and in turn that of aspartate, a necessary substrate for the urea cycle. These findings from MFA provide cues as to which genes/pathways relevant to fatty acid oxidation, urea production, and gluconeogenesis may be upregulated by plasma supplementation, and are consistent with current knowledge of hepatic amino acid metabolism, which provides further credence to this approach for evaluating the metabolic state of hepatocytes under various environmental conditions.

Albumins↗

PubMed2003

Varicella vaccination in children with nephrotic syndrome: a report of the Southwest Pediatric Nephrology Study Group.

OBJECTIVE: To evaluate the safety and immunogenicity of varicella vaccine in children with nephrotic syndrome, including those taking low-dose, alternate-day prednisone. STUDY DESIGN: Prospective, open-label, multicenter clinical trial of varicella vaccine in a 2-dose regimen in US and Canadian children (12 months to <18 years) with nephrotic syndrome. Varicella Zoster Virus (VZV) antibody levels were measured after the first and second vaccine dose and yearly for 2 years. Patients were monitored for adverse reactions to vaccine, exposure to varicella, dermatomal zoster, and chickenpox. RESULTS: Twenty-nine children, mean age 4.9 (SD 1.9) years, 45% receiving every-other-day steroids, received 2 vaccine doses. All patients seroconverted and had VZV antibody levels considered protective against breakthrough varicella (>or=5 gpELISA units) after 2 doses. At 2-year follow-up, all patients retained detectable antibody, and 91% (21 of 23) had levels >or=5 gpELISA units. There were no adverse events associated with vaccination. CONCLUSIONS: Varicella vaccine was generally well tolerated and highly immunogenic in children with nephrotic syndrome, including those on low-dose, alternate-day prednisone.

Anti-Inflammatory Agents↗

PubMed2002

Use of statistical models for evaluating antibody response as a correlate of protection against varicella.

In vaccine clinical trials, humoral antibody responses are often used to measure the effect of a vaccine because they correlate with a vaccine's protective efficacy against the target disease. While the concept of a correlate of protection usually refers to establishing a protective level of antibody titre, identifying a clear-cut value is often impossible because vaccine efficacy is not related solely to the antibody titre. We propose examining the relationship between disease protection and the whole distribution of antibody responses rather than a single cut-off level. In particular, we use failure-time models to estimate the relationship between long-term disease breakthroughs and primary antibody responses after vaccination. We apply these models to show that the varicella antibody response measured by glycoprotein enzyme-linked immunosorbent assay 6 weeks after vaccination strongly correlate with protection against varicella (chickenpox); we used 7-year follow-up data from children who received one dose of a live attenuated varicella (Oka/Merck) vaccine. In addition, we explore the potential use of these models to predict long-term disease breakthrough rates and to estimate the predicted vaccine efficacy of a similar varicella vaccine made with a modified manufacturing process.

Antibodies, Viral↗

PubMed2002

Varicella vaccination in children with chronic renal failure. A report of the Southwest Pediatric Nephrology Study Group.

Children with kidney disease are at risk for serious varicella-related complications. To evaluate the safety and immunogenicity of a two-dose regimen of varicella vaccine in children (aged 1-19 years) with chronic renal insufficiency and on dialysis, the Southwest Pediatric Nephrology Study Group (SPNSG) undertook an open-label, multi-center, prospective 3-year clinical trial. Ninety-six patients without history of varicella were enrolled. Fifty (mean age 4.2 years) had no detectable varicella zoster virus (VZV) antibody; 98% seroconverted after the two-dose regimen. At 1, 2, and 3 years' follow-up, all patients studied maintained VZV antibody, including 16 who received a transplant. No significant vaccine-associated adverse events were seen. One subject developed mild varicella (10-50 maculopapular lesions) 16 months post transplant. In multivariate regression analysis, patients vaccinated after age 6 years had VZV antibody levels 73% (95% confidence interval 33%-89%) lower than patients vaccinated before age 6 years after controlling for gender, estimated glomerular filtration rate, and dialysis treatment. Adjusted analysis also showed that VZV antibody levels were lower after kidney transplantation, but this appeared to be a transient phenomenon. In this study, varicella vaccination with a two-dose regimen of varicella vaccine was generally well tolerated and highly immunogenic in children with chronic kidney disease.

Adolescent↗

PubMed2002

Metabolic pre-conditioning of cultured cells in physiological levels of insulin: generating resistance to the lipid-accumulating effects of plasma in hepatocytes.

Understanding the regulation of hepatocyte lipid metabolism is important for several biotechnological applications involving liver cells. During exposure of hepatocytes to plasma, as is the case in extracorporeal bioartificial liver assist devices, it has been reported that hepatic-specific functions, e.g., albumin and urea synthesis and diazepam removal, are dramatically compromised and hepatocytes progressively accumulate cytoplasmic lipid droplets. We hypothesized that the composition of hepatocyte culture medium significantly affects lipid metabolism during subsequent plasma exposure. Rat hepatocytes were cultured in medium containing either physiological (50 microU/mL) or supra-physiological (500 mU/mL) insulin levels for 1 week and then exposed to human plasma supplemented with or without amino acids. We found that insulin's anabolic effects, such as stimulation of triglyceride storage, were carried over from the pre-conditioning to the plasma exposure period. While hepatocytes cultured in high insulin medium accumulated large quantities of triglycerides during subsequent plasma exposure, culture in low insulin medium largely prevented lipid accumulation. Urea and albumin secretion, as well as the ammonia removal rate, were largely unaffected by insulin but increased with amino acid supplementation. Thus, hepatocyte metabolism during plasma exposure can be modulated by medium pre-conditioning and supplements added to plasma.

Albumins↗

PubMedDate not supplied

Application of multivariate analysis to optimize function of cultured hepatocytes.

Understanding the metabolic and regulatory pathways of hepatocytes is important for biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. To characterize intermediary metabolism in the hepatocytes, metabolic flux analysis (MFA) was applied to elucidate the changes in intracellular pathway fluxes of primary rat hepatocytes exposed to human plasma and to provide a comprehensive snapshot of the hepatic metabolic profile. In the current study, the combination of preconditioning and plasma supplementation produced distinct metabolic states. Combining the metabolic flux distribution obtained by MFA with methodologies such as Fisher discriminant analysis (FDA) and partial least squares or projection to latent structures (PLS) provided insights into the underlying structure and causal relationship within the data. With the aid of these analyses, patterns in the cellular response of the hepatocytes that contributed to the separation of the different hepatic states were identified. Of particular interest was the recognition of distal pathways that strongly correlated with a particular hepatic function. The hepatic functions investigated were intracellular triglyceride accumulation and urea production. This study illustrates a framework for optimizing hepatic function and a possibility of identifying potential targets for improving hepatic functions.

Algorithms↗