Molecular assay optimized by Taguchi experimental design method for venous thromboembolism investigation.

Two mutations - Factor V Leiden (1691G > A) and the 20210G > A on the Prothrombin gene - are key risk factors for a frequent and potentially fatal disorder called Venous Thromboembolism. These molecular alterations can be investigated using real-time Polymerase Chain Reaction (PCR) with Fluorescence Resonance Energy Transfer (FRET) probes and distinct DNA pools for both factors. The objective of this paper is to present an application of Taguchi Experimental Design Method to determine the best parameters adjustment of a Molecular Assays Process in order to obtain the best diagnostic result for Venous Thromboembolism investigation. The complete process contains six three-level factors which usually demands 729 experiments to obtain the final result, if using a Full Factorial Array. In this research, a Taguchi L27 Orthogonal Array is chosen to optimize the analysis and reduce the number of experiments to 27 without degrading the final result accuracy. The application of this method can lessen the time and cost necessary to achieve the best operation condition for a required performance. The results is proven in practice and confirmed that the Taguchi method can really offer a good approach for clinical assay efficiency and effectiveness improvement even though the clinical diagnostics can be based on the use of qualitative techniques.

Differential expression of apoptosis-related genes from death receptor pathway in chronic myeloproliferative diseases.

BACKGROUND: Chronic myeloproliferative disorders (MPDs) are clonal haematopoietic stem cell malignancies characterised by an accumulation of mature myeloid cells in bone marrow and peripheral blood. Deregulation of the apoptotic machinery may be associated with MPD physiopathology. AIMS: To evaluate expression of death receptors' family members, mononuclear cell apoptosis resistance, and JAK2 allele burden. SUBJECTS AND METHODS: Bone marrow haematopoietic progenitor CD34 cells were separated using the Ficoll-hypaque protocol followed by the Miltenyi CD34 isolation kit, and peripheral blood leukocytes were separated by the Haes-Steril method. Total RNA was extracted by the Trizol method, the High Capacity Kit was used to synthesise cDNA, and real-time PCR was performed using SybrGreen in ABIPrism 7500 equipment. The results of gene expression quantification are given as 2(-ΔΔCt). The JAK2 V617F mutation was detected by real-time allelic discrimination PCR assay. Peripheral blood mononuclear cells (PBMCs) were isolated by the Ficoll-hypaque protocol and cultured in the presence of apoptosis inducers. RESULTS: In CD34 cells, there was mRNA overexpression for fas, faim and c-flip in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), as well as fasl in PMF, and dr4 levels were increased in ET. In leukocytes, fas, c-flip and trail levels were increased in PV, and dr5 expression was decreased in ET. There was an association between dr5 and fasl expression and JAK2V617F mutation. PBMCs from patients with PV, ET or PMF showed resistance to apoptosis inducers. CONCLUSIONS: The results indicate deregulation of apoptosis gene expression, which may be associated with MPD pathogenesis leading to accumulation of myeloid cells in MPDs.

Lithium reduces Gsk3b mRNA levels: implications for Alzheimer Disease.

BACKGROUND: There is evidence of increased systemic expression of active GSK3B in Alzheimer's disease patients, which apparently is associated with the formation of senile plaques and neurofibrillary tangles. Due to its central role in the pathogenesis of AD, GSK3B is currently a promising target of the pharmaceutical industry. Whilst trials with specific GSK inhibitors in AD are under way, major attention has been focused on the neuroprotective effects of lithium. Whereas the direct and indirect inhibitory effects of lithium over GSK3 activity have been documented by several groups, its effects over Gsk3 transcription have not yet been addressed. METHODS: We used quantitative PCR to evaluate the transcriptional regulation of Gsk3a and Gsk3b in lithium-treated primary cultures of rat cortical and hippocampal neurons. RESULTS: We found a significant and dose-dependent reduction in the expression of Gsk3b, which was specific to hippocampal cells. This same effect was further confirmed in vivo by measuring Gsk3 expression in different brain regions and in peripheral leukocytes of adult rats treated with lithium. CONCLUSION: Our studies show that LiCl can modulate Gsk3b transcription in vitro and in vivo. This observation suggest new regulatory effects of lithium over Gsk3b, contributing to the better understanding of its mechanisms of action, offering a new and complementary explanation for Gsk3b modulation and reinforcing its potential for the inhibition of key pathological pathways in Alzheimer's disease.

FLT3 internal tandem duplication during myelodysplastic syndrome follow-up: a marker of transformation to acute myeloid leukemia.

Myelodysplastic syndrome (MDS) is a clonal hematopoietic stem cell disorder characterized by ineffective hematopoiesis and risk for evolving to acute leukemia. Some molecular abnormalities related to acute myeloid leukemia (AML) transformation have been reported, such as FLT3 (FMS-like tyrosine kinase 3) mutations. FLT3, a member of the class 3 receptor tyrosine kinase family, mediates stem cell proliferation and differentiation, and its mutations, internal tandem duplication (ITD) and Asp835, have been reported in rare MDS patients. We studied FLT3 ITD, prospectively, in 50 MDS patients at diagnosis, at 6 and 12 months follow-up, and at any other time-point if AML transformation was detected. FLT3 ITD was not observed at diagnosis, but during follow-up the mutation was present in 2 of 50 patients (4%). Of these, one case exhibited FLT3 ITD at the end of the 6 months of follow-up in approximately 8% of bone marrow cells; this case evolved into AML at 8 months, at which time FLT3 ITD was present in approximately 85% of bone marrow cells. The other case exhibited FLT3 ITD in 68% of bone marrow cells at 7 months, precisely at the time of AML transformation. Although rare in MDS, FLT3 ITD is associated with a high probability of evolution to AML.

LyM: a tool to reach the best factor in gene expression comparison.

We developed a Perl-based tool called LyM to determine the best factor for changes in the expression level for each transcript across two sets of expression libraries. LyM includes a Bayesian framework that analyzes the prior and posterior probability density function for each transcript considering the size of the libraries. To find out the best factor for change in each distribution, LyM was implemented with a binary search. In this work we aimed to validate the performance of LyM tool using SAGE libraries from different human tissues. The results were compared with those generated by DGED (Digital Gene Expression Displayer), which worked as the gold standard, on the same data set, to assess accuracy. SAGE libraries were selected from CGAP for the following tissues (normal versus tumor): breast, colon, lung and stomach, consisting of eight SAGE libraries and 381,569 tags. DGED analyses were performed with five arbitrary factors for gene expression in two expression libraries: 2, 4, 8, 16 and 32. The results were confronted using the ratio between LyM and DGED factors and were quantitatively well-matched. LyM was capable of retrieving the best value of F, a factor that represents the fold difference in the expression of a specific gene between two expression libraries, represented by its SAGE tags. However, the optimal value of F is only shown in DGED output after multiple manual interactions. As a result, there was a significant economy of time with the LyM binary search algorithm. In some anecdotal cases we observed that the differential expression levels reached values above 100-fold for a fixed value of P = 0.05, an information that initially remained hidden in DGED. Finally, LyM proved to be relatively fast, portable to the standard workstation present in the molecular biology laboratory, assisting accurate and convenient gene search in expression experiments with minimal user interactions.

FLT3 mutation and AML/ETO in a case of Myelodysplastic syndrome in transformation corroborates the two hit model of leukemogenesis.

The aim of this report is to present a case of Myelodysplastic syndrome (MDS) who presented, during AML transformation, a step-wise genetic progression that corroborates the two hit model of leukemogenesis. A RCDM-RS (WHO)/RARS (FAB) patient with normal karyotype at diagnosis, evolved into AML after six months of follow up. At transformation, AML/ETO fusion was detected, although marrow blast cells were not increased until 21 days later, when FLT3-ITD was also demonstrated pointing out that the overgrowth of the FLT3/ITD clone was concomitant with the outburst of marrow blasts. These findings corroborates the two hit model of leukemogenesis in which one class of mutations (Class I) (FLT3/ITD) confers a proliferative or survival advantage to cells, and a second class of mutations (Class II) (AML/ETO) interferes with hematopoietic differentiation.

An outbreak of keratitis caused by Mycobacterium immunogenum.

From 8 October to 12 November 2003, 36 patients underwent surgical correction of myopia in a São Paulo, Brazil, clinic. Five patients had clinical signs of infectious keratitis, and a Mycobacterium species with previously unreported patterns determined by PCR restriction enzyme analysis of the hsp65 gene and PCR restriction enzyme analysis of the 16S-23S rRNA internal transcribed spacer (ITS) was isolated from corneal scrapings from four of these patients. Subsequent evaluation by phenotypic tests and partial sequencing of the hsp65, sodA, rpoB, and 16S rRNA genes and the ITS supported the species identification as a variant of Mycobacterium immunogenum. The source of infection was not determined. The outbreak was caused by a single clone, as evidenced by identical pulsed-field gel electrophoresis and enterobacterial repetitive intergenic consensus-PCR profiles. This is the first report of an outbreak where this species was isolated from infected tissues.

Low HBV-DNA levels in end-stage renal disease patients with HBeAg-negative chronic hepatitis B.

In end-stage renal disease patients treated by hemodialysis with HBeAg-negative chronic hepatitis B virus (HBV) infection, the evaluation of the presence of viral replication is essential in the assessment for renal transplantation. Data on HBV viral load, prevalence of precore mutations, as well as the influence of HCV coinfection on HBV-DNA levels in this group of patients is scarce. The aim of this study was to determine the HBV viral load in HBsAg-positive/HBeAg-negative hemodialysis patients; to compare HBV-DNA levels between isolated HBV infection carriers and HBV-HCV coinfected patients, and to evaluate the prevalence of precore mutations in these patients. Fifty hemodialysis patients with chronic HBeAg-negative HBV infection were studied. Viral load was determined by PCR (Amplicor HBV Monitor-Roche). The detection of precore mutations was made by sequencing. Of a total of 50 patients, 76% were male, with a mean age of 44 +/- 11 years. Anti-HCV was positive in 56% of patients. HBV-DNA was undetectable in 58% of patients; 24% had HBV-DNA <10,000 copies/ml, 12% between 10,000-100,000 copies/ml, and only 6% had HBV-DNA >100,000 copies/ml. There was no difference in the viral load of patients infected only by HBV and HBV-HCV co-infected patients (P = 0.96). Precore mutations were detected in only 8% of cases. In conclusion, hemodialysis patients with HBeAg-negative HBV infection had a low viral load. Precore mutations were infrequent and the presence of anti-HCV has not influenced the levels of HBV-DNA.

Long-term hydroxyurea therapy in beta-thalassaemia patients.

OBJECTIVE: The study aimed to investigate the use of hydroxyurea (HU) for the treatment of beta-thalassaemia (beta-thal) patients. METHODS: We examined the haematological effects of orally administered HU (10-20 mg/kg/d) in 11 patients, including four beta-thal major and seven beta-thal intermedia patients. Complete blood count and levels of foetal haemoglobin (HbF), liver enzymes and serum creatinine were evaluated before and during HU. Response to therapy was evaluated at 6 months of treatment. RESULTS: A substantial increase in haemoglobin (Hb) level (4.1 g/dL), leading to complete withdrawal from a regular transfusion programme, was observed in one unique beta-thal major patient. In the beta-thal intermedia patients, increases in Hb level of 1.3, 1.9 and 2.0 g/dL were observed in three of seven (42.9%) patients during HU therapy. The mean values of Hb, mean corpuscular haemoglobin (MCH), and HbF were higher during HU treatment than baseline values (8.7 vs. 7.7 g/dL, P = 0.05; 26.7 vs. 22.9 pg, P = 0.05; 57.2 vs. 44.9%, P = 0.04; respectively). In contrast, the mean reticulocyte count measured during therapy decreased (97.0 x 10(9) vs. 632.0 x 10(9)/L, P = 0.03). No correlations were observed between levels of Hb and HbF (r = 0.77, P = 0.10), and levels of Hb and reticulocyte counts (r = 0.26, P = 0.31). No significant toxicity was observed in our patients. CONCLUSION: These results suggest that HU may improve Hb levels in beta-thal. Thus, we may conclude that a large trial concerning the response to HU in these patients should be carried out to clarify this issue.