Biomedical subjects

Andrea Ferreira-Gonzalez

Publications and source records attributed to Andrea Ferreira-Gonzalez.

At least 19 recordsLinked to original sources

PubMed2005

Study of mRNA growth factors in urinary cells of kidney transplant recipients as predictors of chronic allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy (CAN) is responsible for a significant proportion of graft loss. Current diagnostic methods for CAN are inadequate, and noninvasive assays for detecting allograft dysfunction/rejection and predicting long-term outcomes are a priority in transplantation. METHODS: Urine samples were collected from 48 kidney transplant recipients (KTR): 18 recipients with stable graft function (creatinine levels<2.0 mg/dL) and proteinuria of less than 500 mg/24 hr (Group 1); 18 recipients with stable graft function and proteinuria of greater than 500 mg/24 hr (Group 2); and 12 recipients with biopsy confirmed CAN. Urinary cell levels of transforming growth factor-beta1 (TGF-beta1) mRNA or epidermal growth factor (EGF) mRNA were measured using real-time quantitative PCR assay, and levels were correlated with renal allograft status. The integrity of RNA isolated from urine sediments was also assessed using the Agilent 2100 Bioanalyzer. RESULTS: Urinary cell TGF-beta1 mRNA levels were higher in the CAN group compared to Group 1 (P<0.0001) or Group 2 (P<0.0001). Urinary cell EGF mRNA levels were higher in Group 1 compared to Group 2 (P<0.0001) or the CAN group (P<0.0001). There were no significant differences in the urinary cell levels of EGF mRNA between patients with greater than 500 mg/24 hr proteinuria (Group 2) and the CAN group (P=0.75). CONCLUSION: These results demonstrate that urinary cell TGF-beta1 mRNA levels distinguish CAN patients from long-term transplant patients with stable renal function and varied levels of proteinuria. Urinary cells may be a good resource for the noninvasive diagnosis of CAN.

Adult↗

PubMed2005

Apolipoprotein E genotypes as predictors of high-risk groups for developing hyperlipidemia in kidney transplant recipients undergoing sirolimus treatment.

BACKGROUND: Hypercholesterolemia (HCHL) and hypertriglyceridemia (HTRG) have emerged as the most significant metabolic consequences of therapy with sirolimus. Lipid status can be exacerbated by a variety of factors in the posttransplant setting, including genetic factors. Apoliprotein E (Apo E) polymorphism is an established genetic risk factor for hyperlipidemia. We studied the association between Apo E gene polymorphisms and lipids after kidney transplantation in patients undergoing sirolimus treatment. METHODS: We studied 98 kidney transplant patients (KTP) with stable renal allograft undergoing sirolimus treatment: 39 with HCHL and HTRG within 90 days postsirolimus treatment (PST) and 59 without hyperlipidemia PST. Apo E genotyping was performed using INNO-LiPA-ApoE. RESULTS: The cholesterol and the triglyceride values between the groups were 323.3+/-71.6 vs. 180.9+/-31.2 mg/dL (P<0.001) and 318.9+/-97.2 vs. 159.7+/-38.7 mg/dL (P<0.001). There was a significant difference in the genotype distribution of the hyperlipidemia and normal groups (P=0.009) with the percentages in each group as follows: E2/2 and E3/2: 12.8 vs. 5.1%; E3/3: 69.2% vs. 86.4%; and E4/3 and E4/4: 18.0% vs. 8.5%. We observed a higher number of patients with the genotype E3/3 in the group without hyperlipidemia PST (P=0.039). E3/2 and E4/4 genotype frequencies were higher in patients with hyperlipidemia PST. LDL levels in the hyperlipidemia PST group was statistical significant higher (P<0.001) and we observed an association between Apo E allelic distribution and LDL (P=0.005). CONCLUSIONS: Genetic factors, as Apo E genotypes, could allow the early identification of patients who are at a high risk for developing hyperlipidemia PST.

Adult↗

PubMed2005

BCRABL transcript detection by quantitative real-time PCR : are correlated results possible from homebrew assays?

BACKGROUND: Quantitative real-time PCR has become the predominant molecular technique to monitor BCRABL levels in response to treatment in Ph(+) leukemia patients. However, without some form of standardized methodology between laboratories, the correlation of results is difficult. METHODS: Using TaqMan-based assays, parallel quantitative real-time PCR analysis was performed on 70 clinical specimens at Vanderbilt University Medical Center and Virginia Commonwealth University. While the same positive control cell line (K562) and quality control gene (BCR) were used, the RNA isolation technique, cDNA synthesis, BCR control cell line, and PCR primer and probe sequences were different. RESULTS: The detection of BCRABL-positive results spanned a dynamic range from 10(0) to 10(5)/100,000 cells. Forty-three samples were negative at both facilities. A Spearman rank correlation analysis was performed for the 22 BCRABL-positive paired results. The correlation coefficient, r(s), was 0.9435 (p < 0.00001), suggesting a strong correlation of the results. One discordant result was obtained for consecutive samples from one patient with a low BCRABL copy number as a result of a minimal RNA yield at one laboratory. CONCLUSIONS: These results suggest that quantitative real-time PCR assays for BCRABL detection can be comparable between laboratories despite significant differences in methodologies if the same positive control cell line and quality control gene are used. It is imperative that some level of assay standardization be adopted between laboratories, not only for patients who are monitored at different facilities, but also for larger investigative studies in which hematologic, cytogenetic and molecular responses are to be compared.

Adolescent↗

PubMed2005

Developing a sustainable process to provide quality control materials for genetic testing.

PURPOSE: To provide a summary of the outcomes of two working conferences organized by the Centers for Disease Control and Prevention (CDC), to develop recommendations for practical, sustainable mechanisms to make quality control (QC) materials available to the genetic testing community. METHODS: Participants were selected to include experts in genetic testing and molecular diagnostics from professional organizations, government agencies, industry, laboratories, academic institutions, cell repositories, and proficiency testing (PT)/external Quality Assessment (EQA) programs. Current efforts to develop QC materials for genetic tests were reviewed; key issues and areas of need were identified; and workgroups were formed to address each area of need and to formulate recommendations and next steps. RESULTS: Recommendations were developed toward establishing a sustainable process to improve the availability of appropriate QC materials for genetic testing, with an emphasis on molecular genetic testing as an initial step. CONCLUSIONS: Improving the availability of appropriate QC materials is of critical importance for assuring the quality of genetic testing, enhancing performance evaluation and PT/EQA programs, and facilitating new test development. To meet the needs of the rapidly expanding capacity of genetic testing in clinical and public health settings, a comprehensive, coordinated program should be developed. A Genetic Testing Quality Control Materials Program has therefore been established by CDC in March 2005 to serve these needs.

Centers for Disease Control and Prevention, U.S.↗

PubMed2004

Evaluation of quality-control criteria for microarray gene expression analysis.

BACKGROUND: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. METHODS: In the present studies we developed quality-control criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A(260)/A(280) ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. RESULTS: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01). CONCLUSIONS: These results confirm the need to establish defined quality-control criteria for sample quality to distinguish between analytical and biological variability.

Analysis of Variance↗

PubMed2004

Molecular and clinical response to angiotensin II receptor antagonist in kidney transplant patients with chronic allograft nephropathy.

Chronic allograft nephropathy (CAN) represents an important cause of graft loss after kidney transplantation. TGF-beta1 is a key factor in fibrogenesis, and the angiotensin II receptor antagonist losartan may decrease the intra-graft synthesis of TGF-beta1. The aim of this study was to determine the clinical and molecular effect of losartan in kidney transplant patients (KTPs) with CAN. We studied nine KTPs, after the first year of transplantation, with proteinuria (more than 500 mg/24 h), stable renal function, and histological signs of CAN. Immunosuppression was cyclosporine, azathioprine, and corticoids. Kidney biopsy was performed in all patients at the beginning of the study and 12 weeks after treatment with 50 mg/day of losartan. Quantitation of intra-graft expression of TGF-beta1 was performed in all biopsies, by real-time PCR. After losartan treatment there were no differences in patients' BP and blood creatinine level. The proteinuria significantly dropped to 414.2+/-377 mg/24 h, P=0.001. Intra-graft expression of TGF-beta1 was decreased after treatment. In conclusion, losartan significantly decreases the intra-graft expression of TGF-beta1 and proteinuria in KTPs with CAN.

Adult↗

PubMed2004

Evaluation of a linear amplification method for small samples used on high-density oligonucleotide microarray analysis.

High-density oligonucleotide microarray analysis has proven to be an excellent approach for gene expression profiling in human cancers. This technique assesses the expression of thousands of genes simultaneously, from at least 5 microg of total RNA per sample per experiment. This total RNA requirement poses a challenge when studying small, unique clinical samples, like biopsies. Recently, a new standardized protocol for small samples was released by Affymetrix, which includes a linear amplification step. To evaluate the impact of such amplification in the gene expression profiling of human ovarian cancer, we compared results obtained from 5 microg and 100 ng of total RNA from the same tumor sample, using the standard Affymetrix protocol and the new linear RNA amplification protocol, respectively. We identified a small bias in gene expression data caused by linear amplification, potentially due to shorter elongation products leading to misclassification of probe sets directed to the middle-5' region of the transcripts. Interestingly, the magnitude of the bias varied when different normalization and expression summary algorithms were used. However, this bias does not affect tumor gene expression profiling. Consequently, linear amplification may be of utility in cases of extremely low RNA recovery from critical and unique samples, such as small biopsies.

Cluster Analysis↗

PubMed2004

Hepatocellular carcinoma in HCV-infected patients awaiting liver transplantation: genes involved in tumor progression.

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death in the world. The present study aimed to investigate the genes involved in viral carcinogenesis and tumor progression in liver transplant recipients with hepatitis C virus (HCV) and HCC. To accomplish this, we performed the analysis of hepatic gene expression in HCV-infected liver recipient patients with stages of disease ranging from early cirrhosis with preserved volume and function to late cirrhosis with diminished volume and function with and without HCC. We found consistent differences between the gene expression patterns in HCV-HCC and those of early HCV-cirrhosis, late HCV cirrhosis, and normal control livers. The expression patterns in HCC were also readily distinguished between early and advanced HCC tumor stages. Moreover, we found different gene expression patterns between early cirrhosis and late cirrhosis. In conclusion, these findings confirm the presence of multiple molecular alterations during HCV-HCC hepatocarcinogenesis and, clinically, indicate the possibility for identifying prognostic factors associated with HCC progression in liver transplant patients waiting for a donor and/or posttransplantation recurrence.

Adult↗

PubMed2004

Use of diagnostic testing for managing hepatitis C virus infection.

Serological and virological testing for the hepatitis C virus (HCV) is essential in the management of patients with chronic HCV infection. Recent advances in molecular virology tests and the development of a new international unit standard have greatly simplified the use and interpretation of these tests. In the absence of treatment, baseline serum HCV RNA does not change over time and does not correlate with the severity of hepatic inflammation or fibrosis on liver biopsy. In contrast, changes in serum HCV RNA level are important predictors of virological response to treatment with pegylated interferon and ribavirin.

DNA, Viral↗

PubMed2004

Four-year follow-up of a prospective randomized trial of mycophenolate mofetil with cyclosporine microemulsion or tacrolimus following liver transplantation.

BACKGROUND: This is a 4-yr follow-up of a trial using mycophenolate mofetil (MMF) induction in orthotopic liver transplantation (OLT). The goal of this study was to evaluate a multidrug approach that would reduce both early and long-term morbidity related to immunosuppression while maintaining an acceptable freedom from rejection. METHODS: This was a prospective, randomized, intent to treat study designed to compare the primary endpoints of rejection and infection, and secondary endpoints of liver function, renal function, bone marrow function, cardiovascular risk factors, and the recurrence of hepatitis C. Ninety-nine consecutive patients with end stage liver disease who underwent OLT were randomized to receive either cyclosporine microemulsion (N) (50 patients) or tacrolimus (FK) (49 patients) starting on postoperative day 2, with MMF and an identical steroid taper begun preoperatively. RESULTS: Ninety of 99 patients (N 46, FK 44) completed the 4-yr follow-up. The overall 4-yr patient and graft survivals were 93 and 89%, respectively. There was no significant difference in 4-yr patient (N 96% vs. FK 90%, p = ns) or graft (N, 90% vs. FK, 88%, p = ns) survival between groups. The 4-yr rejection rate was not significantly different in either arm (N = 34%, FK = 24%; p = 0.28). There were no differences in infection rates in either arm. The patients with hepatitis C had no differences in the viral titers or Knodell biopsy scores between groups. However, in the hepatitis C subgroup (37 patients), the FK patients had a significantly lower rejection rate (p = 0.0097) and a significantly lower clinically recurrent hepatitis C rate (p = 0.05) than the N patients. No difference was seen in the percent of patients weaned off of steroids after 4 yr (N 51%, FK 49%). There were no differences in the incidences of diabetes mellitus and hypertension. When renal dysfunction was analyzed, a significant difference in the number of patients whose creatinine had increased twofold since transplant was seen (N 63%, FK 38%, p = 0.04). CONCLUSIONS: Use of MMF induction and maintenance following OLT in conjunction with either N or FK and an identical steroid taper, resulted in an acceptable long-term incidence of rejection and infection, without an increase in long-term graft or patient morbidity.

Cyclosporine↗

PubMed2004

Engraftment measurement in human liver tissue after liver cell transplantation by short tandem repeats analysis.

Hepatocyte transplantation has been proposed as a technique for bridging patients to whole-organ transplantation, for providing metabolic support during liver failure, and for replacing whole-organ transplantation in certain metabolic liver diseases. Assessment of hepatocyte engraftment has been difficult to measure, and the degree of engraftment needed to correct various liver disorders is still unknown. A sensitive, simple, and specific method of monitoring engraftment of transplanted hepatocytes for the purpose of bridging human liver failure to native regeneration using short tandem repeats (STRs) was evaluated. The analytical sensitivity of the test was evaluated using DNA mixing curves and established as 0.5% (percentage of donor DNA/ recipient DNA). Sex-matched and mismatched cases were included during the validation. The clinical evaluation of the assay was performed using liver samples from two patients who underwent hepatocyte transplantation. We concluded from this study that the AmpFLSTR Profiler Plus PCR Amplification Kit, a well-established technique in forensic medicine, is specific, sensitive, and a reproducible assay for measurement of engraftment after hepatocyte transplantation in both sex-matched and sex-mismatched cases.

Adult↗

PubMed2003

Hepatic artery thrombosis after liver transplantation and genetic factors: prothrombin G20210A polymorphism.

Hepatic artery thrombosis (HAT) after liver transplantation is associated with a high incidence of graft failure. The incidence ranges between 2% and 25%, with an overall incidence of approximately 7%. Different risk factors have been associated, but the participation of genetic factors in the cause of HAT is less well studied. A single-base change (G to A) at position 20210 in the 3' untranslated region of the prothrombin gene is associated with increased plasma levels of prothrombin and might therefore increase the risk for thrombosis. We reviewed our HAT experience in 11 years at Medical College of Virginia hospitals of 491 patients undergoing 533 liver transplantations. There were 14 liver grafts with documented HAT (2.62%) in 13 patients. Prothrombin G20210A polymorphism was found in the DNA obtained from 2 of 14 liver allograft tissues (14.2%) but not in the DNA from leukocytes obtained from the peripheral blood of recipients with HAT.

Amino Acid Substitution↗

PubMed2003

Effects of stress management on PNI-based outcomes in persons with HIV disease.

A pretest-posttest, repeated-measures design was used to evaluate the effects of two stress management interventions on a battery of outcomes derived from a psychoneuroimmunological (PNI) framework. The effects of cognitive-behavioral relaxation training groups (CBSM) and social support groups (SSG) were compared with a WAIT-listed control group on the outcomes of psychosocial functioning, quality of life, neuroendocrine mediation, and somatic health. Participants were 148 individuals (119 men, 29 women), diagnosed with HIV disease; 112 (76%) completing the study groups. Using analysis of covariance, the CBSM group was found to have significantly higher postintervention emotional well-being and total quality-of-life scores than did either the SSG or WAIT groups. SSG participants had significantly lower social/family well-being scores immediately postintervention and lower social support scores after 6 months. The findings point to a pressing need for further, well-controlled research with these common intervention modalities.

Adaptation, Psychological↗

PubMed2003

Genome-wide detection of LOH in prostate cancer using human SNP microarray technology.

Loss of heterozygosity (LOH) of chromosomal regions is crucial in tumor progression. In this study we assessed the potential of the Affymetrix GeneChip HuSNP mapping assay for detecting genome-wide LOH in prostate tumors. We analyzed two human prostate cell lines, P69SV40Tag (P69) and its tumorigenic subline, M12, and 11 prostate cancer cases. The M12 cells showed LOH in chromosomes 3p12.1-p22.1, 11q22.1-q24.2, 19p13.12, and 19q13.42. All of the prostate cases with informative single-nucleotide polymorphism (SNP) markers showed LOH in 1p31.2, 10q11.21, 12p13.1, 16q23.1-q23.2, 17p13.3, 17q21.31, and 21q21.2. Additionally, a high percentage of cases showed LOH at 6p25.1-p25.3 (75%), 8p22-p23.2, and 10q22.1 (70%). Several tumor suppressor genes (TSGs) have been mapped in these loci. These results demonstrate that the HuSNP mapping assay can serve as an alternative to comparative genomic hybridization for assessing genome-wide LOH and can identify chromosomal regions harboring candidate TSGs implicated in prostate cancer.

Adult↗

PubMed2003

Comparison of three commercially available assays for HCV RNA using the international unit standard: implications for management of patients with chronic hepatitis C virus infection in clinical practice.

OBJECTIVES: The present study was performed to evaluate the impact of the international unit standard for measuring HCV RNA in the management of patients with chronic hepatitis C virus (HCV) infection. METHODS: The three assays used were Amplicor Monitor PCR, the National Genetics Institute PCR assay, and branched chain DNA. HCV RNA was measured at four time points (baseline, 3 months after the start of therapy, at the end of treatment, and 6 months after discontinuation of therapy) in 106 consecutive patients who received interferon and ribavirin for chronic HCV. RESULTS: The mean age of the patients was 44 yr. Of the patients, 62% were male, 24% were African American, 38% had bridging fibrosis or cirrhosis, and 75% were HCV genotype 1. Of the 424 samples analyzed, 82-89% of values were within 1 log unit and 85-92% were within 2 log units by the various assays. This variability was not dependent upon HCV genotype. HCV RNA was undetectable in 1.4-6.8% of samples when virus was detected by another assay. The mean HCV RNA in these discordant samples was 1.47-6.33 log IU/ml (30-2,100,000 IU/ml). CONCLUSIONS: These data demonstrate that approximately 90% of serum values for HCV RNA were within 1 log unit by the international unit standard regardless of which virological assay was used. However, false positive and false negative results as well as variations in the HCV RNA level of more than 1 to 2 log units can occur with any of the assays, and these results may have an impact upon the management of patients receiving interferon therapy. It is therefore unwise in clinical practice to base important treatment decisions upon a single HCV RNA determination.

Adult↗

PubMed2002

Analytical validation of a real-time reverse transcription-polymerase chain reaction quantitation of different transcripts of the Wilms' tumor suppressor gene (WT1).

Transcript variants of the same gene may play distinct functions in the tissue where they are expressed. Absolute quantitation of different transcript variants in malignant and normal tissues can address the specific role of each particular isoform in cancer development and progression. We have recently demonstrated differential expression of the wild-type Wilms' tumor transcript (wtWT1) and a novel truncated WT1 transcript (trWT1) which lacks the first five exons of wtWT1, among human prostate cancer, leukemia, and breast cancer cell lines. Here we report the analytical validation of a real-time RT-PCR assay for the absolute quantitation of these two different WT1 transcripts with specific primers and probes that ensure specificity for each WT1 variant. By cloning each WT1 transcript in a T3 promoter-containing plasmid, we obtained two WT1 transcript-specific in vitro-generated RNA calibrators for absolute quantitation. Serial dilution of each RNA calibrator demonstrated a 5 log linear dynamic range (5 x 10(1) to 5 x 10(6) copies/reaction, R(2)=0.9963 for wtWT1 and R(2)=0.9993 for trWT1). Dilution of the calibrators in total RNA from 1 x 10(3) non-WT1-expressing cells showed a decreased sensitivity without affecting the linear dynamic range. Precision studies for values within the linear dynamic range showed a coefficient of variation of less than 4% for both transcripts. The described method provides a sensitive and reliable technique for quantitating different WT1 mRNA transcripts.

Calibration↗

PubMed2002

Prospective validation of quantitative polymerase chain reaction for management of cytomegalovirus disease in solid-organ transplant patients.

This study evaluates the utility of quantitative polymerase chain reaction (QPCR) to determine duration of treatment of transplant patients with human cytomegalovirus (HCMV) disease. Eighteen patients with HCMV disease were prospectively evaluated and followed for recurrence using a QPCR assay. We used plasma samples from which nucleic acid was extracted. Quantification was determined by using an internal standard that contained the same primer sequences as for HCMV. During treatment, weekly QPCR assays were performed. Patients were treated with HCMV immunoglobulin-G for a finite period, but intravenous ganciclovir was continued until less than 100 viral copies (vc) per mL was detectable. After cessation of therapy, patients were followed for 6 months with monthly clinical assessment and QPCR. No patient developed recurrence of HCMV at a mean follow-up of 16 months. This preliminary study suggests that the use of QPCR to assess viral load is useful in deciding the length of HCMV treatment with ganciclovir but requires further randomized validation.

Adolescent↗

PubMed2002

Human immunodeficiency virus genotype and hypertriglyceridemia.

Many HIV patients develop a progressive syndrome of abnormal body fat distribution accompanied by hypertriglyceridemia. Antiretroviral agents are thought to be etiologic in the syndrome, often termed "highly active antiretroviral therapy (HAART)-associated lipodystrophy." In the course of clinical HIV genotype testing, we observed that our HIV patients with hypertriglyceridemia had viral genotypes that were more highly mutated than those of our therapy-matched control patients. Hypertriglyceridemia was statistically associated with predicted resistance for three nucleoside reverse transcriptase inhibitors: zidovudine, abacavir, and stavudine. Statistical analysis of 51 patients in retrospect revealed a strong association of mutations at reverse transcriptase codons M41 and T215 with hypertriglyceridemia (chi-square (chi(2)) = 8.375, P=.0038; and chi(2)=7.445, P=.0064, respectively). This was in contrast to silent mutations, which occurred at equivalent rates in retroviral genotypes of patients with and without hypertriglyceridemia. The findings imply that the HIV genotype itself may be a significant etiologic factor in antiretroviral-associated lipodystrophy.

Adult↗