Apolipoprotein E ε4 allele and malondialdehyde level are independent risk factors for Alzheimer's disease.

BACKGROUND: The ε4 allele of Apolipoprotein E is involved in lipid metabolism. Oxidative stress produces an increase in lipid peroxidation that has been implicated in the pathogenic cascade in Alzheimer's disease. This study estimated the effect of the ε4 allele, malondialdehyde and lipid levels on the risk for Alzheimer's disease. METHODS: A total of 41 control subjects and 73 patients with Alzheimer's disease were recruited. The Apolipoprotein E genotype was determined by amplification of exon 4 of the Apolipoprotein E by polymerase chain reaction (PCR); malondialdehyde concentration was determined by high-pressure liquid chromatography, and serum lipids were measured by routine photometric techniques. RESULTS: Malondialdehyde levels were significantly higher in Alzheimer's disease patients independent of the Apolipoprotein E genotype and ε4 allele. The ε4 allele increases the risk of Alzheimer's disease by 5.114 times and elevated malondialdehyde levels increase the risk by 9.342. CONCLUSION: The presence of ε4 allele and elevated malondialdehyde levels are independent risk factors for Alzheimer's disease. These findings support the hypothesis that lipid peroxidation and ε4 allele contribute to the pathogenic cascade in Alzheimer's disease by different pathways.

HGUE-C-1 is an atypical and novel colon carcinoma cell line.

BACKGROUND: Colorectal carcinoma is a common cause of cancer. Adjuvant treatments include: 5-fluorouracil administered together with folinic acid, or more recently, oral fluoropyrimidines such as capecitabine, in combination with oxaliplatin or irinotecan. Metastatic colorectal cancer patients can benefit from other additional treatments such as cetuximab or bevacizumab. METHODS: Using cell culture techniques, we isolated clonal populations from primary cultures of ascitic effusion derived from a colon cancer patient and after several passages an established cell line, HGUE-C-1, was obtained. Genetic analysis of HGUE-C-1 cells was performed by PCR of selected exons and sequencing. Cell proliferation studies were performed by MTT assays and cell cycle analyses were performed by flow cytometry. Retinoblastoma activity was measured by luciferase assays and proteins levels and activity were analysed by Western blot or immunohistochemistry. RESULTS: We have established a new cell line from ascitic efussion of a colon cancer patient who did not respond to 5-fluorouracil or irinotecan. HGUE-C-1 cells did not show microsatellite instability and did not harbour mutations in KRAS, BRAF, PI3KCA or TP53. However, these cells showed loss of heterozygosity affecting Adenomatous Polyposis Coli and nuclear staining of ß-catenin protein. The HGUE-C-1 cell line was sensitive to erlotinib, gefitinib, NVP-BEZ235, rapamycin and trichostatin, among other drugs, but partially resistant to heat shock protein inhibitors and highly resistant to AZD-6244 and oxaliplatin, even though the patient from which this cell line was derived had not been exposed to these drugs. Molecular characterization of this cell line revealed low expression levels and activity of Retinoblastoma protein and elevated basal levels of Erk1/2 activity and p70S6K expression and activity, which may be related to chemoresistance mechanisms. CONCLUSIONS: HGUE-C-1 represents a novel and peculiar colon carcinoma model to study chemoresistance to chemotherapeutic agents and to novel anti-neoplasic drugs that interrupt signalling pathways such as the APC/ßcatenin, Ras/Raf/Mek/Erk, PI3K/mTOR/p70S6K pathways as well as histone regulation mechanisms.

Imaging cytometry for counting circulating tumor cells: comparative analysis of the CellSearch vs ImageStream systems.

Circulating tumor cell (CTC) enumeration is important clinically for identifying prognostic and predictive factors in patients with solid cancers. The CellSearch device (Veridex) is an immunomagnetic CTC selection and enumeration system used in clinical practice. The ImageStream (Amnis) combines the strengths of flow cytometry and fluorescent microscopy in a single platform and has potential application for CTC counting. The performance in CTC enumeration was compared between the ImageStream and CellSearch systems. Various numbers of PANC-1 tumor cells were spiked into 7.5 mL of peripheral blood from a healthy donor. Before cell analysis by the ImageStream, tumor cell enrichment was performed by immunomagnetic selection with anti-EpPCAM. Anti-CD45 and anti-CK markers were used to discriminate between tumor cells and leukocytes. The ratios of tumor cells recovered from each dilution were calculated for both methods. The Wilcoxon rank test was applied to compare the results of the two methods and the reference value. The results of the two tested methods differed significantly from the reference value, but did not differ between them. Nevertheless, lower level of trueness and precision was observed in ImageStream when fewer numbers of CTCs were analyzed. Our results suggest that ImageStream platform for CTC enumeration has a potential value for the early diagnosis of disseminated disease, but needs an improvement of precision for the enumeration of low number of CTC.

Prevalence of methicillin-resistant Staphylococcus aureus : effect of different criteria for elimination of duplicates.

BACKGROUND: The purpose of this study is to compare the effect of different systems for eliminating duplicates in order to optimize the calculation of the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infection. METHODS: We compare the Clinical and Laboratory Standards Institute (CLSI) criterion, time criteria and the criterion recommended by the European Antimicrobial Surveillance System (EARSS). RESULTS: Multiple isolates of MRSA are frequently recovered from successive cultures from the same patient (the average isolation rate of MRSA is 2.72), which demonstrates the importance of eliminating duplicates. When CLSI criterion data are compared to those obtained using other criteria, a significant increase in the number of S. aureus isolates was found applying time criteria (up to 36%) or the EARSS criterion (13%). There is also an increase in the methicillin resistance rate (between 3.31 and 3.96%; p < 0.01). CONCLUSIONS: We believe that the EARSS method, with the proper quality controls and latest software tools available, is the best for determining the true situation of MRSA.

Análisis comparativo de tres métodos de ELISA frente aInmunodot para la determinación de antígenos extraíbles del núcleo (ENAs) / Comparative analysis of three ELISA methods versus simple dot-blot method for the determination of extractable nuclear antigens (ENA)

La detección y la especificidad de los autoanticuerpos contra antígenos extraíbles del núcleo (ENA) tienen un importante papel en la ayuda aldiagnóstico de ciertas enfermedades autoinmunes. Existen diversos métodos para detectar estos autoanticuerpos entre los que se encuentran el enzimoinmunoanálisis (ELISA) y el “Inmunodot” como los más utilizados.En este estudio evaluamos cuatro métodos para la detección de ENAs,comparando un ensayo Inmunodot frente a tres ensayos ELISA. Se analizaron muestras de 26 pacientes en las que fueron determinados los autoanticuerpos antinucleares (ANAs) por Inmunofluorescencia (IFI) y los anticuerpos frente a ENAs (Ro, La, Sm-RNP, Sm, Scl-70 y Jo-1) por Inmunodot y porlos tres ensayos de ELISA. Se obtuvo que en un 27% de los casos todas lastécnicas eran coincidentes en el resultado y se correlacionaron con los síntomas clínicos del paciente. La concordancia entre las técnicas de ELISA evaluadas fue de un 88% para ELISA 1 vs 2, 73% para ELISA 1 vs 3 y 73% paraELISA 2 vs 3, y la de los métodos de ELISA frente el método de Inmunodotfueron de un 38%, 42% y 27% (vs ELISA1, 2 y 3 respectivamente).Las técnicas de ELISA tienen un porcentaje de concordancia entre sí másalto que con Inmunodot, y además se correlacionan mejor con los síntomasclínicos y el patrón ANA del paciente. Los resultados muestran que la presencia de autoanticuerpos ENA no siempre tiene relación con la presentación clínica del paciente, por ello el laboratorio y el clínico deben ser conscientes de la sensibilidad y la especificidad de cada método empleado en ellaboratorio clínico (AU)

The detection and the specificity of the autoantibodies to extractablenuclear antigens (ENAs) play a critical role in the development of the diagnosis of certain autoimmune diseases. There are several methods to detectthese autoantibodies, and enzyme-linked immunosorbent assay (ELISA)or the simple dot-blot are the most frequently used.Here, we evaluate four methods to detect ENAs, namely an Immunodot test along with three different ELISA tests. Twenty six samples ofpatients were analysed. Antibodies to nuclear antigens (ANAS) weredetermined by indirect immunofluorescence (IFI), whereas antibodies todifferent ENAs (Ro, La, Sm-RNP, Sm, Scl-70 and Jo-1) were determinedby simple dot-blot and by the three ELISA tests. In 27% of the cases all thetechniques yielded similar results, and were related to the clinical symptoms of the patient. The concordance among the different ELISA techniques tested was 88% for ELISA 1 vs 2, 73% for ELISA 2 vs 3, and 73% forELISA 2 vs 3. The concordance of the ELISA methods with the Immunodot was 38%, 42% and 27% for ELISA 1, 2, and 3, respectively. The ELISA techniques obtained a higher percentage of concordanceamong them than with Immunodot and, moreover, they related better tothe clinical symptoms and ANA pattern of the patients. The results alsoshow that the presence or the specific type of ENA autoantibody does notalways have a relation to the clinical status of the patient, so that the laboratory and the clinic must be conscious of the sensitivity and specificitylimits of each method used in the clinical laboratory (AU)

Optimization of the trace element determination by ICP-MS in human blood serum.

The ICP-MS for simultaneous trace element determination in human blood has prevailed as the most suitable methodology for clinical aims because of its rapidity, detection limits and minimal sample quantity needed for analysis. As the proteic matrix is high, it is necessary to fine-tune the ICP-MS Agilent 7500i with autosampler CETAC ASX-500 and ISIS System connected, and further we have to the sample pre-treatment in order to obtain good results. The study of the results shows that the best pre-treatment for blood serum samples consists of a basic treatment by 1/10 dilution with a solution of EDTA and NH(4)OH, with a detection limit of the order of mug/L and a reduction of the necessary patient sample volume to 2 mL.