Blood mRNA expression of REV3L and TYMS as potential predictive biomarkers from platinum-based chemotherapy plus pemetrexed in non-small cell lung cancer patients.

PURPOSE: Therapeutic options for cancer patients have increased in the last years, although drugs resistance problem remains unresolved. Genetic background in individual susceptibility to cancer treatment could influence the therapy responses. The aim of this study was to explore the feasibility of using blood 4 genes (AEG-1, BRCA-1, REV3L and TYMS) expression levels as a predictor of the efficacy of pemetrexed therapy in patients with advanced non-small cell lung cancer. METHODS: Sixteen patients from the Medical Oncology Department at "12 de Octubre" Hospital, were included in the study. Total mRNA was isolated from blood samples, and gene expression was analyzed by RT-qPCR. A panel of lung tumor cell lines were used in cell proliferation tests and siRNA-mediated silencing assays. RESULTS: Similarity between blood gene expression levels and protein expression in matched tumor tissue was observed in 54.54% (REV3L) and 81.81% (TYMS) of cases. Gene expression of REV3L and TYMS in blood correlated directly and inversely, respectively, with progression-free survival and overall survival in the patients from our cohort. In tumor cell lines, the knockdown of REV3L conferred resistance to pemetrexed treatment, and the TYMS silencing increased the pemetrexed sensitivity of tumor cells. CONCLUSIONS: The use of peripheral blood samples for expression quantification of interest genes is an affordable method with promising results in the evaluation of response to pemetrexed treatment. Therefore, expression levels of REV3L and TYMS genes might be used as predictive biomarkers in advanced NSCLC patients.

A composite peripheral blood gene expression measure as a potential diagnostic biomarker in bipolar disorder.

Gene expression in peripheral blood has the potential to inform on pathophysiological mechanisms and has emerged as a viable avenue for the identification of biomarkers. Here, we aimed to identify gene expression candidate genes and to explore the potential for a composite gene expression measure as a diagnostic and state biomarker in bipolar disorder. First, messenger RNA levels of 19 candidate genes were assessed in peripheral blood mononuclear cells of 37 rapid cycling bipolar disorder patients in different affective states (depression, mania and euthymia) during a 6-12-month period and in 40 age- and gender-matched healthy control subjects. Second, a composite gene expression measure was constructed in the first half study sample and independently validated in the second half of the sample. We found downregulation of POLG and OGG1 expression in bipolar disorder patients compared with healthy control subjects. In patients with bipolar disorder, upregulation of NDUFV2 was observed in a depressed state compared with a euthymic state. The composite gene expression measure for discrimination between patients and healthy control subjects on the basis of 19 genes generated an area under the receiver-operating characteristic curve of 0.81 (P < 0.0001) in sample 1, which was replicated with a value of 0.73 (P < 0.0001) in sample 2, corresponding with a moderately accurate test. The present findings of altered POLG, OGG1 and NDUFV2 expression point to disturbances within mitochondrial function and DNA repair mechanisms in bipolar disorder. Further, a composite gene expression measure could hold promise as a potential diagnostic biomarker.

Evaluation of the activity of thermostable DNA polymerases in the presence of heme, as a key inhibitor in the real time PCR method in diagnostics of sepsis.

The study aim was evaluation of the usefulness of several thermostable DNA polymerases in real time PCR conducted in the presence of the heme. Our study had the advantage of testing several different polymerases, one of which proved to be the least sensitive to heme activity. We also found that there is no need of supplementing the reaction mixture with protective substances like BSA. Selection of the appropriate polymerase can increase the efficiency of the PCR reaction which is very important for diagnosis of sepsis and for other analyses performed on DNA template isolated from the blood.

Comparison of sxx commercially-available dna polymerases for direct ccr / Comparacao de seis polimerases de DNA disponiveis comercialmente para o PCR direto

SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens. .

RESUMO O propósito deste estudo foi avaliar 6 polimerases de DNA disponíveis comercialmente que são resistentes aos inibidores do PCR para uma amplificação potencial de DNA de amostras de sangue total. O DNA genômico do parasita humano da malária, Plasmodium falciparum, foi analisado sob condições que incluíram os componentes inibidores do sangue extraído de sangue ressacado em papel de filtro. Nossos resultados sugerem que a polimerase KOD FX DNA é superior a outras polimerases. .

Comparison of six commercially-available DNA polymerases for direct PCR.

The use of a "direct PCR" DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

Comparison of human memory CD8 T cell responses to adenoviral early and late proteins in peripheral blood and lymphoid tissue.

Treatment of invasive adenovirus (Ad) disease in hematopoietic stem cell transplant (SCT) recipients with capsid protein hexon-specific donor T cells is under investigation. We propose that cytotoxic T cells (CTLs) targeted to the late protein hexon may be inefficient in vivo because the early Ad protein E3-19K downregulates HLA class I antigens in infected cells. In this study, CD8+ T cells targeted to highly conserved HLA A2-restricted epitopes from the early regulatory protein DNA polymerase (P-977) and late protein hexon (H-892) were compared in peripheral blood (PB) and tonsils of naturally infected adults. In tonsils, epitope-specific pentamers detected a significantly higher frequency of P-977+CD8+ T cells compared to H-892+CD8+ T cells; this trend was reversed in PB. Tonsil epitope-specific CD8+ T cells expressed IFN-γ and IL-2 but not perforin or TNF-α, whereas PB T cells were positive for IFN-γ, TNF-α, and perforin. Tonsil epitope-specific T cells expressed lymphoid homing marker CCR7 and exhibited lower levels of the activation marker CD25 but higher proliferative potential than PB T cells. Finally, in parallel with the kinetics of mRNA expression, P-977-specific CTLs lysed targets as early as 8 hrs post infection. In contrast, H-892-specific CTLs did not kill unless infected fibroblasts were pretreated with IFN-γ to up regulate HLA class I antigens, and cytotoxicity was delayed until 16-24 hours. These data show that, in contrast to hexon CTLs, central memory type DNA polymerase CTLs dominate the lymphoid compartment and kill fibroblasts earlier after infection without requiring exogenous IFN-γ. Thus, use of CTLs targeted to both early and late Ad proteins may improve the efficacy of immunotherapy for life-threatening Ad disease in SCT recipients.

Measurement of kinetic parameters of human platelet DNA polymerase gamma.

Synthesis of mitochondrial DNA is performed by DNA polymerase gamma. Mutations in POLG, the gene encoding the catalytic subunit of DNA polymerase gamma, are a major cause of neurological disease. A large proportion of patients carry rare nucleotide substitutions leading to single amino acid changes. Confirming that these replacements are pathogenic can be problematic without biochemical evidence. Here, we provide a hands-on protocol for an in vitro kinetic assay of DNA polymerase gamma which allows assessment of the K(m) and V(max) for the incoming nucleotide of the polymerization reaction. To avoid measurement of contaminating nuclear DNA polymerases, platelet extracts are used since platelets do not contain a nucleus. Moreover, platelets have the advantage of being obtainable relatively non-invasively. Polymerization activity is determined by measurement of the incorporation of radioactive thymidine 5'-triphosphate (dTTP) on the homopolymeric RNA substrate poly(rA).oligo(dT)(12-18). To further minimize nuclear DNA polymerase activity, aphidicolin, an inhibitor of most nuclear DNA polymerases, is included in the reaction. In addition, reactions are carried out in the absence and presence of the competitive inhibitor of DNA polymerase gamma, 2',3'-dideoxythymidine 5'-triphosphate (ddTTP), to allow calculation of the ddTTP-sensitive incorporation. With this method, platelets from healthy control subjects extracted with 3% Triton X-100 showed a K(m) for dTTP of 1.42 microM and a V(max) of 0.83 pmol min(-1)mg(-1).

[Serum proteomics in patients with RAEB myelodysplastic syndromes].

OBJECTIVE: To screen the molecular markers for refractory anemia with excess blasts in transformation (RAEB) in myelodysplastic syndromes (MDS) by serum proteome profiling. METHODS: The serum protein were isolated from patients with RAEB, acute myeloid leukemia or normal subjects by 2-dimensional electrophoresis (2-DE), and the electrophoresis gels were obtained to identify the differentially reacting protein spots. The replica gels of the differentially reacting proteins were analyzed to locate the matching protein spots, which were identified by peptide mass fingerprint based on matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF-MS) and database searching. RESULTS: Seven differentially expressed proteins in RAEB were found by 2-DE. Of the 7 proteins, 4 were identified by MALDI-TOF-MS to have significantly differential expression in RAEB, including dipeptidyl peptidase (DPP/CD26), polymerase (DNA directed) kappa, PRO2044 and an albumin-like protein. CONCLUSION: 2-DE-based serum proteome profiling helps identify serum proteomic biomarkers related to MDS. DDP/CD26 has increased expression in the serum in RAEB subtype MDS, suggesting its possible role in advanced MDS.

Short-term intravenous interferon therapy for chronic hepatitis B.

AIM: To investigate the therapeutic efficacy of short-term, multiple daily dosing of intravenous interferon (IFN) in patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B. METHODS: IFN-beta was intravenously administered at a total dose of 102 million international units (MIU) over a period of 28 d in 26 patients positive for HBeAg and HBV-DNA. IFN-beta was administered at doses of 2 MIU and 1 MIU on d 1, 3 MIU twice daily from d 2 to d 7, and 1 MIU thrice daily from d 8 to d 28. Patients were followed up for 24 wk after the end of treatment. RESULTS: Six months after the end of the treatment, loss of HBV-DNA occurred in 13 (50.0%) of the 26 patients, loss of HBeAg in 9 (34.6%), development of anti-HBe in 10 (38.5%), HBeAg seroconversion in 8 (30.8%), and normalization of alanine aminotransferase (ALT) levels in 11 (42.0%). CONCLUSION: This 4-wk long IFN-beta therapy, which was much shorter than conventional therapy lasting 12 wk or even more than 1 year, produced therapeutic effects similar to those achieved by IFN-alpha or pegylated-IFN-alpha (peg-IFN). Fewer adverse effects, greater efficacy, and a shorter treatment period led to an improvement in patients' quality of life. IFN-beta is administered intravenously, whereas IFN-alpha is administered intramuscularly or subcutaneously. Because both interferons are known to bind to an identical receptor and exert antiviral effects through intracellular signal transduction, the excellent results of IFN-beta found in this study may be attributed to the multiple doses allowed by the intravenous route.

Comparison of mass spectrometric analysis and TRUGENE HBV genotyping for monitoring lamivudine resistance in chronic hepatitis B patients.

BACKGROUND: There is an increasing need for the early detection of emerging mutations in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of hepatitis B virus (HBV) DNA polymerase with using sensitive molecular methods. METHODS: We evaluated the usefulness of monitoring lamivudine resistance using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based assay (the restriction fragment mass polymorphism; RFMP) in comparison with the direct sequencing assay, the TRUGENE ' HBV genotyping kit. We also investigated the treatment responses in relation to the presence of YMDD mutants. The sera from 50 chronic HBVs patients were analysed for the presence of YMDD mutants by performing RFMP and TRUGENE. The results at codons 180 and 204 were compared for 46 patients. RESULTS: The concordance rate between the two assays was 65.2% (30/46). All the discordance corresponded to the detection of additional virus populations by RFMP. Early detection of mutants before viral breakthrough was accomplished by RFMP in two patients. Persistence of very low viraemia was observed in five patients who harboured mutant virus populations. Additional information was provided by TRUGENE in eight patients. CONCLUSIONS: RFMP showed a superior ability for detecting minor mutant virus populations compared with TRUGENE. However, the results of highly sensitive RFMP should be interpreted carefully because lamivudine could be effective despite the presence of mutants. RFMP could be a practical tool in conjunction with regular measurements of the HBV viral load for the early detection of lamivudine resistance and the timely introduction of new antiviral drugs.

Quantitation of hepatitis B surface antigen by an automated chemiluminescent microparticle immunoassay.

A fully automated chemiluminescent microparticle immunoassay (Architect HBsAg QT) was used for the detection and quantitation of hepatitis B surface antigen (HBsAg). The assay is capable of processing up to 800 HBsAg tests per hour. The concentration of HBsAg is determined by utilizing a previously generated Architect HBsAg calibration curve. Architect HBsAg QT sensitivity was found to be around 0.2ng/ml which is equivalent or superior to other known and commercially available enzyme immunoassays and/or chemiluminescent immunoassays. We performed a quantitative study of HBsAg, HBeAg, HBV-DNA and HBV-DNA polymerase in over 733 sera obtained from 43 chronic hepatitis B carriers. Serum HBsAg levels detected by Architect HBsAg QT were found to be higher in HBeAg-positive than in anti-HBe-positive HBV chronic carriers and correlated with the level of serum HBV-DNA and HBV-DNA polymerase.

Characterization of hepatitis B virus surface antigen and polymerase mutations in liver transplant recipients pre- and post-transplant.

We evaluated serum samples from 18 chronic hepatitis B virus (HBV) patients who underwent liver transplantation for the presence of HBV polymerase and S gene mutations and HBV genotype using a new commercially available sequencing assay. All three patients with hepatitis B immune globulin (HBIG) treatment failure followed by nucleoside analogue treatment failure were infected with HBV genotype C; a pre-existing HBV S antigen (HBsAg) mutation (sD144A) was identified in one patient pretransplant, while sG145R mutations emerged in the other two patients post-transplant. These HBsAg mutations persisted for the duration of the study (5-6 years), despite the absence of HBIG administration for a 4-5-year period. Significant viral polymerase mutations (rtL180M and rtM204I/V) also emerged in all of these patients following treatment with lamivudine and/or famciclovir. Four of six patients with HBIG breakthrough without nucleoside analogue treatment failure yielded potentially significant HBsAg mutations post transplant. These data do not support previous reports highlighting the disappearance of HBsAg mutants in liver transplant recipients after discontinuation of HBIG. Determination of HBV genotype, as well as identification of HBV polymerase and S gene mutations in liver transplant candidates may be warranted to optimize HBV management strategies post transplant.

[Hepatitis B virus DNA polymerase]. / Polimeraza DNA wirusa zapalenia watroby typu B.

Our aim was to discuss the practical value of HBV DNA polymerase (DNAp) determination. DNAp activity is a good marker of HBV high replication in patients with chronic hepatitis B (HBeAg+), very useful during monitoring of immunostimulation and/or antiviral treatment.

Heterogeneous response for a mammalian hepadnavirus infection to acyclovir: drug-arrested intermediates of minus-strand viral DNA synthesis are enveloped and secreted from infected cells as virion-like particles.

Three woodchucks infected persistently with the woodchuck hepatitis virus (WHV) were treated with acyclovir (ACV) to investigate the effect of inhibiting viral DNA synthesis upon the replication of an orthohepadnavirus in vivo. Normal viraemia was reduced during the treatment period in all three animals, but each responded with a distinct serum phenotype. In the most provocative case, the profile of the WHV DNAs in both the liver and serum provided a simple and novel description of the orthohepadnaviral infection for this ACV protocol. The pre-drug viraemia was rapidly cleared from the serum and replaced by virion-like particles containing predominantly minus-strand WHV DNAs. These serum DNA species had the character of replicative intermediates arrested in their elongation by ACV-mediated chain termination and were contained in particles with a buoyant density in CsCl essentially identical with virions. However, in infected hepatocytes, initiation of reverse transcription within newly formed core particles was not inhibited by the ACV treatment. Instead, an heterogeneous array of minus-strand DNAs were synthesised, each presumed to be truncated by the incorporation of one molecule of ACV monophosphate. An approximately normal level of core particles was present in the liver of this woodchuck after 26 days of the ACV protocol; excess drug-arrested nucleocapsids were steadily removed throughout the dosing period upon their envelopment and secretion as virion-like particles into the circulation. These data suggest that plus-strand DNA synthesis may not be absolutely required prior to secretion of virus from the infected cell.

Long-term outcome of hepatitis B virus-related glomerulonephritis after therapy with interferon alfa.

BACKGROUND & AIMS: Glomerulonephritis is an uncommon complication of chronic hepatitis B virus (HBV) infection in adults. A high percentage of patients seem to have short-term response to interferon therapy with improvement of proteinuria. The aim of this study was to assess the long-term response of patients with HBV-related glomerulonephritis to interferon alfa therapy. METHODS: All patients with chronic hepatitis B and glomerulonephritis who were treated with interferon alfa at the National Institutes of Health between 1985 and 1993 were assessed. RESULTS: Of the 15 patients treated, 8 (53%) had a long-term serological response with sustained loss of serum hepatitis B e antigen and HBV DNA. After 1-7 years of follow-up, all 8 responders have normal serum aminotransferase levels and 5 are hepatitis B surface antigen negative. Seven of the responders also showed a gradual but marked improvement in proteinuria. In contrast, the 7 nonresponders continued to have evidence of active renal disease and 1 required long-term dialysis therapy. All 8 responders had membranous glomerulonephritis, whereas 4 of 7 nonresponders had membranoproliferative glomerulonephritis. CONCLUSIONS: Interferon alfa therapy resulted in long-term remission in liver disease in 8 of 15 patients with chronic hepatitis B and glomerulonephritis. This response was accompanied by significant improvement in markers of renal disease in the majority of patients.

Clinical evaluation of intramuscular administration of natural interferon-gamma in the treatment of chronic hepatitis B.

Natural interferon-gamma at a dose of 0.5 x 10(6) or 1 x 10(6) IU daily was intramuscularly administered daily for 4 weeks to 15 patients with chronic hepatitis B. The efficacy and safety of the treatment were evaluated for 24 weeks following the completion of the 4-week treatment period. Persistent disappearance of HBeAg was observed in 5 of 15 patients. Serum hepatitis B virus (HBV)-related DNA polymerase disappeared in 5 of 13 patients at the end of interferon therapy. On the other hand, serum ALT and beta 2-microglobulin levels showed a significant increase during the interferon therapy period. The side effects were completely reversible. These findings suggest that interferon-gamma has an antiviral effect in patients with chronic hepatitis B and that the main mechanism of the therapeutic effect may be associated with the elimination of HBV-infected hepatocytes due to the immunopotentiating effect of the substance.

Decrease in serum levels of markers of hepatic connective tissue turnover during and after treatment of chronic hepatitis B with interferon-alpha.

Interferon-alpha induces remission in 30% to 40% of patients with chronic hepatitis B, but its effect on hepatic connective tissue turnover has not been well documented. We studied the changes in serum procollagen III propeptide and laminin-P1 peptide (Lam-P1) in 33 patients with chronic hepatitis B (11 nontreated controls and 22 treated patients) during a 4-mo randomized trial of interferon-alpha. Liver biopsy specimens were obtained at the start of treatment and 12 mo later. Liver biochemical tests, procollagen III propeptide, laminin-P1 peptide and hepatitis B virus DNA polymerase were determined before treatment with interferon was begun (mo -3), at the initiation (0 time) and completion of treatment (mo 4) and also at 8, 12 and 18 mo. Treated patients were classified as "responders" and "nonresponders" on the basis of clearance of HBV e antigen from serum. There were no significant changes in the control group, whereas the responders had persistent decreases in ALT, AST, hepatitis B virus dna polymerase, procollagen III propeptide and laminin-P1 peptide. The nonresponders had transient ALT, AST and hepatitis B virus dna polymerase reductions that returned toward baseline levels during follow-up, but procollagen III propeptide and laminin-P1 peptide persisted below the baseline at mo 18. Significant correlations between procollagen III propeptide and laminin-P1 peptide with ALT, AST and liver histologic specimens were noted at baseline but not after 12 mo. Changes in procollagen III propeptide levels also correlated with changes in AST, ALT and liver histologic specimens.(ABSTRACT TRUNCATED AT 250 WORDS)