Increased expression of urokinase-type plasminogen activator mRNA determines adverse prognosis in ErbB2-positive primary breast cancer.

PURPOSE: To evaluate and validate mRNA expression markers capable of identifying patients with ErbB2-positive breast cancer associated with distant metastasis and reduced survival. PATIENTS AND METHODS: Expression of 60 genes involved in breast cancer biology was assessed by quantitative real-time PCR (qrt-PCR) in 317 primary breast cancer patients and correlated with clinical outcome data. Results were validated subsequently using two previously published and publicly available microarray data sets with different patient populations comprising 295 and 286 breast cancer samples, respectively. RESULTS: Of the 60 genes measured by qrt-PCR, urokinase-type plasminogen activator (uPA or PLAU) mRNA expression was the most significant marker associated with distant metastasis-free survival (MFS) by univariate Cox analysis in patients with ErbB2-positive tumors and an independent factor in multivariate analysis. Subsequent validation in two microarray data sets confirmed the prognostic value of uPA in ErbB2-positive tumors by both univariate and multivariate analysis. uPA mRNA expression was not significantly associated with MFS in ErbB2-negative tumors. Kaplan-Meier analysis showed in all three study populations that patients with ErbB2-positive/uPA-positive tumors exhibited significantly reduced MFS (hazard ratios [HR], 4.3; 95% CI, 1.6 to 11.8; HR, 2.7; 95% CI, 1.2 to 6.2; and, HR, 2.8; 95% CI, 1.1 to 7.1; all P < .02) as compared with the group with ErbB2-positive/uPA-negative tumors who exhibited similar outcome to those with ErbB2-negative tumors, irrespective of uPA status. CONCLUSION: After evaluation of 898 breast cancer patients, uPA mRNA expression emerged as a powerful prognostic indicator in ErbB2-positive tumors. These results were consistent among three independent study populations assayed by different techniques, including qrt-PCR and two microarray platforms.

Medical image registration based on random line sampling

One of the key aspects in 3D-image registration is the computation of the joint intensity histogram. We propose a new approach to compute this histogram using uniformly distributed random lines to sample stochastically the overlapping volume between two 3D-images. The intensity values are captured from the lines at evenly spaced positions, taking an initial random offset different for each line. This method provides us with an accurate, robust and fast mutual information-based registration. The interpolation effects are drastically reduced, due to the stochastic nature of the line generation, and the alignment process is also accelerated. The results obtained show a better performance of the introduced method than the classic computation of the joint histogram

Kallikrein genes are associated with lupus and glomerular basement membrane-specific antibody-induced nephritis in mice and humans.

Immune-mediated nephritis contributes to disease in systemic lupus erythematosus, Goodpasture syndrome (caused by antibodies specific for glomerular basement membrane [anti-GBM antibodies]), and spontaneous lupus nephritis. Inbred mouse strains differ in susceptibility to anti-GBM antibody-induced and spontaneous lupus nephritis. This study sought to clarify the genetic and molecular factors that maybe responsible for enhanced immune-mediated renal disease in these models. When the kidneys of 3 mouse strains sensitive to anti-GBM antibody-induced nephritis were compared with those of 2 control strains using microarray analysis, one-fifth of the underexpressed genes belonged to the kallikrein gene family,which encodes serine esterases. Mouse strains that upregulated renal and urinary kallikreins exhibited less evidence of disease. Antagonizing the kallikrein pathway augmented disease, while agonists dampened the severity of anti-GBM antibody-induced nephritis. In addition, nephritis-sensitive mouse strains had kallikrein haplotypes that were distinct from those of control strains, including several regulatory polymorphisms,some of which were associated with functional consequences. Indeed, increased susceptibility to anti-GBM antibody-induced nephritis and spontaneous lupus nephritis was achieved by breeding mice with a genetic interval harboring the kallikrein genes onto a disease-resistant background. Finally, both human SLE and spontaneous lupus nephritis were found to be associated with kallikrein genes, particularly KLK1 and the KLK3 promoter, when DNA SNPs from independent cohorts of SLE patients and controls were compared. Collectively, these studies suggest that kallikreins are protective disease-associated genes in anti-GBM antibody-induced nephritis and lupus.

Hepatic safety of antibiotics used in primary care

Antibiotics used by general practitioners frequently appear in adverse-event reports of drug-induced hepatotoxicity. Most cases are idiosyncratic (the adverse reaction cannot be predicted from the drug's pharmacological profile or from pre-clinical toxicology tests) and occur via an immunological reaction or in response to the presence of hepatotoxic metabolites. With the exception of trovafloxacin and telithromycin (now severely restricted), hepatotoxicity crude incidence remains globally low but variable. Thus, amoxicillin/clavulanate and co-trimoxazole, as well as flucloxacillin, cause hepatotoxic reactions at rates that make them visible in general practice (cases are often isolated, may have a delayed onset, sometimes appear only after cessation of therapy and can produce an array of hepatic lesions that mirror hepatobiliary disease, making causality often difficult to establish). Conversely, hepatotoxic reactions related to macrolides, tetracyclines and fluoroquinolones (in that order, from high to low) are much rarer, and are identifiable only through large-scale studies or worldwide pharmacovigilance reporting. For antibiotics specifically used for tuberculosis, adverse effects range from asymptomatic increases in liver enzymes to acute hepatitis and fulminant hepatic failure. Yet, it is difficult to single out individual drugs, as treatment always entails associations. Patients at risk are mainly those with previous experience of hepatotoxic reaction to antibiotics, the aged or those with impaired hepatic function in the absence of close monitoring, making it important to carefully balance potential risks with expected benefits in primary care. Pharmacogenetic testing using the new genome-wide association studies approach holds promise for better understanding the mechanism(s) underlying hepatotoxicity.

Neuropathologie et pathologie moléculaire des gliomes [Neuropathology and molecular pathology of gliomas]

Gliomas are the most frequent primary brain tumours. The WHO classification is essentially based on histological and immunohistochemical criteria. More recently multiple cytogenetic and molecular alterations associated with initiation and progression have been shown and the genetic profiles of tumour entities have been incorporated in the WHO classifiacation. Molecular testing of the MGMT promotor methylation in glioblastoma, predictive for the response to combined radio-/chimiothérapie, and the LOH 1p/19q in oligodendroglial tumours, as prognostic factor supplements the histopathological diagnosis. In the near futur array-based profiling techniques will contribute to a refinement of glioma classification and identify targets for more individualized glioma therapies.

An unexpected role of semaphorin3a-neuropilin-1 signaling in lymphatic vessel maturation and valve formation.

RATIONALE: Lymphatic vasculature plays important roles in tissue fluid homeostasis maintenance and in the pathology of human diseases. Yet, the molecular mechanisms that control lymphatic vessel maturation remain largely unknown. OBJECTIVE: We analyzed the gene expression profiles of ex vivo isolated lymphatic endothelial cells to identify novel lymphatic vessel expressed genes and we investigated the role of semaphorin 3A (Sema3A) and neuropilin-1 (Nrp-1) in lymphatic vessel maturation and function. METHODS AND RESULTS: Lymphatic and blood vascular endothelial cells from mouse intestine were isolated using fluorescence-activated cell sorting, and transcriptional profiling was performed. We found that the axonal guidance molecules Sema3A and Sema3D were highly expressed by lymphatic vessels. Importantly, we found that the semaphorin receptor Nrp-1 is expressed on the perivascular cells of the collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1 but not with an antibody that blocks VEGF-A binding to Nrp-1 resulted in a complex phenotype of impaired lymphatic vessel function, enhanced perivascular cell coverage, and abnormal lymphatic vessel and valve morphology. CONCLUSIONS: Together, these results reveal an unanticipated role of Sema3A-Nrp-1 signaling in the maturation of the lymphatic vascular network likely via regulating the perivascular cell coverage of the vessels thus affecting lymphatic vessel function and lymphatic valve development.

MicroRNA-200 family modulation in distinct breast cancer phenotypes.

The epithelial to mesenchymal transition (EMT) contributes to tumor invasion and metastasis in a variety of cancer types. In human breast cancer, gene expression studies have determined that basal-B/claudin-low and metaplastic cancers exhibit EMT-related characteristics, but the molecular mechanisms underlying this observation are unknown. As the family of miR-200 microRNAs has been shown to regulate EMT in normal tissues and cancer, here we evaluated whether the expression of the miR-200 family (miR-200f) and their epigenetic state correlate with EMT features in human breast carcinomas. We analyzed by qRT-PCR the expression of miR-200f members and various EMT-transcriptional inducers in a series of 70 breast cancers comprising an array of phenotypic subtypes: estrogen receptor positive (ER+), HER2 positive (HER2+), and triple negative (TN), including a subset of metaplastic breast carcinomas (MBCs) with sarcomatous (homologous or heterologous) differentiation. No MBCs with squamous differentiation were included. The DNA methylation status of miR-200f loci in tumor samples were inspected using Sequenom MassArray® MALDI-TOF platform. We also used two non-tumorigenic breast basal cell lines that spontaneously undergo EMT to study the modulation of miR-200f expression during EMT in vitro. We demonstrate that miR-200f is strongly decreased in MBCs compared with other cancer types. TN and HER2+ breast cancers also exhibited lower miR-200f expression than ER+ tumors. Significantly, the decreased miR-200f expression found in MBCs is accompanied by an increase in the expression levels of EMT-transcriptional inducers, and hypermethylation of the miR-200c-141 locus. Similar to tumor samples, we demonstrated that downregulation of miR-200f and hypermethylation of the miR-200c-141 locus, together with upregulation of EMT-transcriptional inducers also occur in an in vitro cellular model of spontaneous EMT. Thus, the expression and methylation status of miR-200f could be used as hypothetical biomarkers to assess the occurrence of EMT in breast cancer.

The involvement of thaumatin-like proteins in plant food cross-reactivity: a multicenter study using a specific protein microarray

Cross-reactivity of plant foods is an important phenomenon in allergy, with geographical variations with respect to the number and prevalence of the allergens involved in this process, whose complexity requires detailed studies. We have addressed the role of thaumatin-like proteins (TLPs) in cross-reactivity between fruit and pollen allergies. A representative panel of 16 purified TLPs was printed onto an allergen microarray. The proteins selected belonged to the sources most frequently associated with peach allergy in representative regions of Spain. Sera from two groups of well characterized patients, one with allergy to Rosaceae fruit (FAG) and another against pollens but tolerant to food-plant allergens (PAG), were obtained from seven geographical areas with different environmental pollen profiles. Cross-reactivity between members of this family was demonstrated by inhibition assays. Only 6 out of 16 purified TLPs showed noticeable allergenic activity in the studied populations. Pru p 2.0201, the peach TLP (41%), chestnut TLP (24%) and plane pollen TLP (22%) proved to be allergens of probable relevance to fruit allergy, being mainly associated with pollen sensitization, and strongly linked to specific geographical areas such as Barcelona, Bilbao, the Canary Islands and Madrid. The patients exhibited >50% positive response to Pru p 2.0201 and to chestnut TLP in these specific areas. Therefore, their recognition patterns were associated with the geographical area, suggesting a role for pollen in the sensitization of these allergens. Finally, the co-sensitizations of patients considering pairs of TLP allergens were analyzed by using the co-sensitization graph associated with an allergen microarray immunoassay. Our data indicate that TLPs are significant allergens in plant food allergy and should be considered when diagnosing and treating pollen-food allergy.

Avaluació de vitamines i antioxidants en aliments vegetals mitjançant tècniques HPLC-DAD i UPLC-MS/MS

El programa experimental s’ha portat a terme dins el marc de les activitats del projecte TRUEFOOD, finançat per la UE per als anys 2007-2010. L’objectiu principal d’aquesta activitat ha estat l’avaluació dels continguts en àcid ascòrbic (vitamina C), polifenols totals, àcids fenòlics i flavonoides en mostres de tomàquet i enciam, produïts sota diferents condicions de camp (producció ecològica i convencional). Per aconseguir els resultats s’han utilitzat mètodes analítics basats en tècniques de cromatografia líquida d’alta eficàcia (HPLC) i d’ultra-alta eficàcia (UHPLC) acoblades a sistemes de detecció de diode array (DAD) i espectrometria de masses (MSn). Per a l’àcid ascòrbic, s’ha desenvolupat un mètode ràpid que ha permès la determinació d’aquest compost en diferents matrius vegetals amb el mínim pretractament de les mostres, utilitzant una fase estacionària HILIC (Fluorinated Stationary Phase). Els mètodes desenvolupats d’anàlisi de compostos fenòlics han permès realitzar les anàlisi de forma ràpida, a fi de processar el màxim nombre de mostres per a obtenir resultats representatius. S’ha realitzat una completa caracterització dels extractes de tomàquet i enciam, ampliant el coneixement descrit en la bibliografia sobre la seva composició fenòlica. En el cas de l’enciam, s’ha identificat quatre compostos fenòlics que mai abans han estat descrits i quantificats en aquesta hortalissa. La definició, amb precisió, dels continguts en vitamina C i compostos fenòlics en les mostres analitzades ha permès comparar els efectes de diferents tècniques de cultiu sobre les característiques nutricionals dels vegetals objecte de l’estudi. Els mètodes d’anàlisi desenvolupats i els resultats derivats del projecte seran publicats properament en revistes científiques de reconegut prestigi.

MsrR contributes to cell surface characteristics and virulence in Staphylococcus aureus.

MsrR, a factor contributing to methicillin resistance in Staphylococcus aureus, belongs to the LytR-CpsA-Psr family of cell envelope-associated proteins. Deletion of msrR increased cell size and aggregation, and altered envelope properties, leading to a temporary reduction in cell surface hydrophobicity, diminished colony-spreading ability, and an increased susceptibility to Congo red. The reduced phosphorus content of purified cell walls of the msrR mutant suggested a reduction in wall teichoic acids, which may explain some of the observed phenotypes. Microarray analysis of the msrR deletion mutant revealed only minor changes in the global transcriptome, suggesting that MsrR has structural rather than regulatory functions. Importantly, virulence of the msrR mutant was decreased in a nematode-killing assay as well as in rat experimental endocarditis. MsrR is therefore likely to play a role in cell envelope maintenance, cell separation, and pathogenicity of S. aureus.

13q deletion syndrome and retinoblastoma in identical dichorionic diamniotic monozygotic twins.

PURPOSE: To report the case of identical dichorionic diamniotic female twins with unilateral retinoblastoma in 13q deletion syndrome. METHODS: Clinical and ophthalmoscopic evaluation, combination of multiple ligation-dependent probe amplification, array-comparative genomic hybridization analyses, and magnetic resonance imaging were performed. RESULTS: Peculiar facial features, marked hypotonia, gastroesophageal reflux, interatrial septal defect with left to right shunt and light dilatation of right chambers, 5th finger hypoplasia, 3rd-5th toes clinodactyly, 2nd toe overlapped to 3rd toe, and cutis marmorata were found. Ophthalmoscopic evaluation revealed unilateral retinoblastoma in both girls. Magnetic resonance imaging detected corpus callosum hypoplasia in both twins. A 34.4-Mb deletion involving bands 13q13.2-q21.33 and including the RB1 gene was identified in both twins. The deletion was not present in the DNA of their parents and older brother. CONCLUSIONS: Dysmorphic features in children must be always suspicious of 13q deletion syndrome and a short ophthalmoscopic follow-up is necessary to detect the presence of a retinoblastoma.

Molecular characterization of 39 de novo sSMC: contribution to prognosis and genetic counselling, a prospective study.

Small supernumerary marker chromosomes (sSMCs) are structurally abnormal chromosomes that cannot be characterized by karyotype. In many prenatal cases of de novo sSMC, the outcome of pregnancy is difficult to predict because the euchromatin content is unclear. This study aimed to determine the presence or absence of euchromatin material of 39 de novo prenatally ascertained sSMC by array-comparative genomic hybridization (array-CGH) or single nucleotide polymorphism (SNP) array. Cases were prospectively ascertained from the study of 65,000 prenatal samples [0.060%; 95% confidence interval (CI), 0.042-0.082]. Array-CGH showed that 22 markers were derived from non-acrocentric markers (56.4%) and 7 from acrocentic markers (18%). The 10 additional cases remained unidentified (25.6%), but 7 of 10 could be further identified using fluorescence in situ hybridization; 69% of de novo sSMC contained euchromatin material, 95.4% of which for non-acrocentric markers. Some sSMC containing euchromatin had a normal phenotype (31% for non-acrocentric and 75% for acrocentric markers). Statistical differences between normal and abnormal phenotypes were shown for the size of the euchromatin material (more or less than 1 Mb, p = 0.0006) and number of genes (more or less than 10, p = 0.0009). This study is the largest to date and shows the utility of array-CGH or SNP array in the detection and characterization of de novo sSMC in a prenatal context.

A male with unilateral microphthalmia reveals a role for TMX3 in eye development.

Anophthalmia and microphthalmia are important birth defects, but their pathogenesis remains incompletely understood. We studied a patient with severe unilateral microphthalmia who had a 2.7 Mb deletion at chromosome 18q22.1 that was inherited from his mother. In-situ hybridization showed that one of the deleted genes, TMX3, was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TMX3 in 162 patients with anophthalmia or microphthalmia, and found two missense substitutions in unrelated patients: c.116G>A, predicting p.Arg39Gln, in a male with unilateral microphthalmia and retinal coloboma, and c.322G>A, predicting p.Asp108Asn, in a female with unilateral microphthalmia and severe micrognathia. We used two antisense morpholinos targeted against the zebrafish TMX3 orthologue, zgc:110025, to examine the effects of reduced gene expression in eye development. We noted that the morphant larvae resulting from both morpholinos had significantly smaller eye sizes and reduced labeling with islet-1 antibody directed against retinal ganglion cells at 2 days post fertilization. Co-injection of human wild type TMX3 mRNA rescued the small eye phenotype obtained with both morpholinos, whereas co-injection of human TMX3(p.Arg39Gln) mutant mRNA, analogous to the mutation in the patient with microphthalmia and coloboma, did not rescue the small eye phenotype. Our results show that haploinsufficiency for TMX3 results in a small eye phenotype and represents a novel genetic cause of microphthalmia and coloboma. Future experiments to determine if other thioredoxins are important in eye morphogenesis and to clarify the mechanism of function of TMX3 in eye development are warranted.

Microarray profiling of mononuclear peripheral blood cells identifies novel candidate genes related to chemoradiation response in rectal cancer.

Preoperative chemoradiation significantly improves oncological outcome in locally advanced rectal cancer. However there is no effective method of predicting tumor response to chemoradiation in these patients. Peripheral blood mononuclear cells have emerged recently as pathology markers of cancer and other diseases, making possible their use as therapy predictors. Furthermore, the importance of the immune response in radiosensivity of solid organs led us to hypothesized that microarray gene expression profiling of peripheral blood mononuclear cells could identify patients with response to chemoradiation in rectal cancer. Thirty five 35 patients with locally advanced rectal cancer were recruited initially to perform the study. Peripheral blood samples were obtained before neaodjuvant treatment. RNA was extracted and purified to obtain cDNA and cRNA for hybridization of microarrays included in Human WG CodeLink bioarrays. Quantitative real time PCR was used to validate microarray experiment data. Results were correlated with pathological response, according to Mandard´s criteria and final UICC Stage (patients with tumor regression grade 1-2 and downstaging being defined as responders and patients with grade 3-5 and no downstaging as non-responders). Twenty seven out of 35 patients were finally included in the study. We performed a multiple t-test using Significance Analysis of Microarrays, to find those genes differing significantly in expression, between responders (n = 11) and non-responders (n = 16) to CRT. The differently expressed genes were: BC 035656.1, CIR, PRDM2, CAPG, FALZ, HLA-DPB2, NUPL2, and ZFP36. The measurement of FALZ (p = 0.029) gene expression level determined by qRT-PCR, showed statistically significant differences between the two groups. Gene expression profiling reveals novel genes in peripheral blood samples of mononuclear cells that could predict responders and non-responders to chemoradiation in patients with locally advanced rectal cancer. Moreover, our investigation added further evidence to the importance of mononuclear cells' mediated response in the neoadjuvant treatment of rectal cancer.

Differences in osteogenic and apoptotic genes between osteoporotic and osteoarthritic patients.

BACKGROUND Osteoporosis is a metabolic disorder characterized by a reduction in bone mass and deterioration in the microarchitectural structure of the bone, leading to a higher risk for spontaneous and fragility fractures.The main aim was to study the differences between human bone from osteoporotic and osteoarthritic patients about gene expression (osteogenesis and apoptosis), bone mineral density, microstructural and biomechanic parameters. METHODS We analyzed data from 12 subjects: 6 with osteoporotic hip fracture (OP) and 6 with hip osteoarthritis (OA), as the control group. All subjects underwent medical history, analytical determinations, densitometry, histomorphometric and biochemical study. The expression of 86 genes of osteogenesis and 86 genes of apoptosis was studied in pool of bone samples from patients with OP and OA by PCR array. RESULTS We observed that most of the genes of apoptosis and osteogenesis show a decrease in gene expression in the osteoporotic group in comparison with the osteoarthritic group. The histomorphometric study shows a lower bone quality in the group of patients with hip fractures compared to the osteoarthritic group. CONCLUSIONS The bone tissue of osteoporotic fracture patients is more fragile than the bone of OA patients. Our results showed an osteoporotic bone with a lower capacities for differentiation and osteoblastic activity as well as a lower rate of apoptosis than osteoarthritic bone. These results are related with structural and biochemical parameters.