Pre-eclampsia is a hypertensive disorder of pregnancy characterised by hypertension and sodium retention by the kidneys. To identify changes in sodium uptake proteins in the tubules of the distal nephron, we studied their expression in urinary extracellular vesicles or exosomes (uEVs). Urine was collected from women with pre-eclampsia or during normal pregnancy, and from healthy non-pregnant controls. uEVs were isolated by centrifugation and analyzed by Western blot. Expression, proteolytic cleavage and phosphorylation was determined by densitometric analysis normalized to the exosome marker CD9. Results showed a significant increase in phosphorylation of the activating S130 site in NKCC2, the drug target for frusemide, in women with pre-eclampsia compared with normal pregnant women. Phosphorylation of the activating sites T101/105 in NKCC2 was similar but the activating T60 site in NCC, the drug target for thiazide diuretics, showed significantly less phosphorylation in pre-eclampsia compared with normal pregnancy. Expression of the larger forms of the α subunit of ENaC, the drug target for amiloride, was significantly greater in pre-eclampsia, with more fragmentation of theγ subunit. The differences observed are predicted to increase the activity of NKCC2 and ENaC while reducing that of NCC. This will increase sodium reabsorption, and so contribute to hypertension in pre-eclampsia.

Stress, and the chronic overactivation of major stress hormones, is associated with several neuropsychiatric disorders. However, clinical literature on the exact role of stress either as a causative, triggering, or modulatory factor to mental illness remains unclear. We suggest that the impact of stress on the brain and behavior is heavily dependent on the developmental timing at which the stress has occurred, and as such, this may contribute to the overall variability reported on the association of stress and mental illness. Here, animal models provide a way to comprehensively assess the temporal impact of stress on behavior in a controlled manner. This review particularly focuses on the long-term impact of stress on behavior in various rodent stress models at three major developmental time points: early life, adolescence, and adulthood. We characterize the various stressor paradigms into physical, social, and pharmacological, and discuss commonalities and differences observed across these various stress-inducing methods. In addition, we discuss here how sex can influence the impact of stress at various developmental time points. We conclude here that early postnatal life and adolescence represent particular periods of vulnerability, but that stress exposure during early life can sometimes lead to resilience, particularly to fear-potentiated memories. In the adult brain, while shorter periods of stress tended to enhance spatial memory, longer periods caused impairments. Overall, males tended to be more vulnerable to the long-term effects of early life and adolescent stress, albeit very few studies incorporate both sexes, and further well-powered sex comparisons are needed.

Primary non-Hodgkins lymphoma (NHL) of the breast, and its extranodal spread to the breast resulting from systemic lymphoma, are recognised albeit uncommon conditions. However, lymphoma involving the axilla, presenting with the clinical appearance of inflammatory breast carcinoma (IBC) without infiltration of breast dermal lymphatics has not been reported previously.As highlighted by the two cases presented here, this entity should be considered in the differential diagnosis of patients presenting with clinical IBC. The cases highlight the importance of careful histological analysis to distinguish IBC from NHL, since management strategies and prognosis are quite different.

INTRODUCTION: Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a rare immunodeficiency disorder with an autosomal-dominant pattern of inheritance and low fatality rate but significant lifelong morbidity. MATERIALS AND METHODS: A 27-year-old mother of two children has been suffering from severe neutropenia and recurrent infections with the diagnosis of sporadic WHIM syndrome established by sequencing the CXCR4 gene and the finding of a heterozygous 1000 C-->T nonsense mutation in the second CXCR4 exon. The first child was an apparently healthy boy delivered at full term. Umbilical cord blood cells were obtained for genetic analysis. Peripheral blood cells were also analyzed at 8 months of life. Both analyses revealed the same mutation as that of his mother. The child was in a good condition, manifesting neutropenia without infections until 11 months of life. He subsequently developed pneumonia requiring a more aggressive treatment. After that, the regular substitution of immunoglobulins (IVIGs) and G-CSF has been preventing serious infections. Six months ago the second boy was delivered who also demonstrated neutropenia without severe infections. Genetic studies using cord blood and also peripheral blood cells in the fourth month showed an identical mutation of the CXCR4 gene as in his mother. Moreover, the mother and her first son demonstrated monocytopenia. RESULTS: The results indicate that genetic defects connected with WHIM syndrome may influence not only the granulocyte, but also the monocytic lineage. Moreover, a perinatal diagnosis of WHIM syndrome made by sequencing the CXCR4 gene should be performed in cases where either parent is known to be affected with this disease. CONCLUSIONS: This would facilitate an earlier detection of the deficiency in children, thereby allowing a more comprehensive follow-up and administration of appropriate therapy.

BACKGROUND: Several recent studies have shown that screen detection remains an independent prognostic factor after adjusting for disease stage at presentation. This study compares the molecular characteristics of screen-detected with symptomatic breast cancers to identify if differences in tumour biology may explain some of the survival benefit conferred by screen detection. METHODS: A total of 1379 women (aged 50-70 years) with invasive breast cancer from a large population-based case-control study were included in the analysis. Individual patient data included tumour size, grade, lymph node status, adjuvant therapy, mammographic screening status and mortality. Immunohistochemistry was performed on tumour samples using 11 primary antibodies to define five molecular subtypes. The effect of screen detection compared with symptomatic diagnosis on survival was estimated after adjustment for grade, nodal status, Nottingham Prognostic Index (NPI) and the molecular markers. RESULTS: Fifty-six per cent of the survival benefit associated with screen-detected breast cancer was accounted for by a shift in the NPI, a further 3-10% was explained by the biological variables and more than 30% of the effect remained unexplained. CONCLUSION: Currently known biomarkers remain limited in their ability to explain the heterogeneity of breast cancer fully. A more complete understanding of the biological profile of breast tumours will be necessary to assess the true impact of tumour biology on the improvement in survival seen with screen detection.

BACKGROUND: Tissue micro-arrays (TMAs) are increasingly used to generate data of the molecular phenotype of tumours in clinical epidemiology studies, such as studies of disease prognosis. However, TMA data are particularly prone to missingness. A variety of methods to deal with missing data are available. However, the validity of the various approaches is dependent on the structure of the missing data and there are few empirical studies dealing with missing data from molecular pathology. The purpose of this study was to investigate the results of four commonly used approaches to handling missing data from a large, multi-centre study of the molecular pathological determinants of prognosis in breast cancer. PATIENTS AND METHODS: We pooled data from over 11,000 cases of invasive breast cancer from five studies that collected information on seven prognostic indicators together with survival time data. We compared the results of a multi-variate Cox regression using four approaches to handling missing data - complete case analysis (CCA), mean substitution (MS) and multiple imputation without inclusion of the outcome (MI-) and multiple imputation with inclusion of the outcome (MI+). We also performed an analysis in which missing data were simulated under different assumptions and the results of the four methods were compared. RESULTS: Over half the cases had missing data on at least one of the seven variables and 11 percent had missing data on 4 or more. The multi-variate hazard ratio estimates based on multiple imputation models were very similar to those derived after using MS, with similar standard errors. Hazard ratio estimates based on the CCA were only slightly different, but the estimates were less precise as the standard errors were large. However, in data simulated to be missing completely at random (MCAR) or missing at random (MAR), estimates for MI+ were least biased and most accurate, whereas estimates for CCA were most biased and least accurate. CONCLUSION: In this study, empirical results from analyses using CCA, MS, MI- and MI+ were similar, although results from CCA were less precise. The results from simulations suggest that in general MI+ is likely to be the best. Given the ease of implementing MI in standard statistical software, the results of MI+ and CCA should be compared in any multi-variate analysis where missing data are a problem.

The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (copy number variants and single nucleotide polymorphisms) and acquired somatic copy number aberrations (CNAs) were associated with expression in ~40% of genes, with the landscape dominated by cis- and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP and MAP2K4. Unsupervised analysis of paired DNA–RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, oestrogen-receptor-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the ‘CNA-devoid’ subgroup and a basal-specific chromosome 5 deletion-associated mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic CNAs on the transcriptome.

Cancer immunoediting, the process by which the immune system controls tumour outgrowth and shapes tumour immunogenicity, is comprised of three phases: elimination, equilibrium and escape. Although many immune components that participate in this process are known, its underlying mechanisms remain poorly defined. A central tenet of cancer immunoediting is that T-cell recognition of tumour antigens drives the immunological destruction or sculpting of a developing cancer. However, our current understanding of tumour antigens comes largely from analyses of cancers that develop in immunocompetent hosts and thus may have already been edited. Little is known about the antigens expressed in nascent tumour cells, whether they are sufficient to induce protective antitumour immune responses or whether their expression is modulated by the immune system. Here, using massively parallel sequencing, we characterize expressed mutations in highly immunogenic methylcholanthrene-induced sarcomas derived from immunodeficient Rag2(-/-) mice that phenotypically resemble nascent primary tumour cells. Using class I prediction algorithms, we identify mutant spectrin-β2 as a potential rejection antigen of the d42m1 sarcoma and validate this prediction by conventional antigen expression cloning and detection. We also demonstrate that cancer immunoediting of d42m1 occurs via a T-cell-dependent immunoselection process that promotes outgrowth of pre-existing tumour cell clones lacking highly antigenic mutant spectrin-β2 and other potential strong antigens. These results demonstrate that the strong immunogenicity of an unedited tumour can be ascribed to expression of highly antigenic mutant proteins and show that outgrowth of tumour cells that lack these strong antigens via a T-cell-dependent immunoselection process represents one mechanism of cancer immunoediting.

DNA mutation data currently reside in many online databases, which differ markedly in the terminology used to describe or define the mutation and also in completeness of content, potentially making it difficult both to locate a mutation of interest and to find sought-after data (eg phenotypic effect). To highlight the current deficiencies in the accessibility of web-based genetic variation information, we examined the ease with which various resources could be interrogated for five model mutations, using a set of simple search terms relating to the change in amino acid or nucleotide. Fifteen databases were investigated for the time and/or number of mouse clicks; clicks required to find the mutations; availability of phenotype data; the procedure for finding information; and site layout. Google and PubMed were also examined. The three locus-specific databases (LSDBs) generally yielded positive outcomes, but the 12 genome-wide databases gave poorer results, with most proving not to be searchable and only three yielding successful outcomes. Google and PubMed searches found some mutations and provided patchy information on phenotype. The results show that many web-based resources are not currently configured for fast and easy access to comprehensive mutation data, with only the isolated LSDBs providing optimal outcomes. Centralising this information within a common repository, coupled with a simple, all-inclusive interrogation process, would improve searching for all gene variation data.

Gamma-secretase is involved in the production of Aβ amyloid peptides. It cleaves the transmembrane domain of the amyloid precursor protein (APP) at alternative sites to produce Aβ and the APP intracellular domain (AICD). Metal ions play an important role in Aβ aggregation and metabolism, thus metal chelators and ligands represent potential therapeutic agents for AD treatment. A direct effect of metal chelators on γ-secretase has not yet been investigated. The authors used an in vitro  γ-secretase assay consisting of cleavage of APP C100-3XFLAG by endogenous γ-secretase from rodent brains and human neuroblastoma SH-SY5Y, and detected AICD production by western blotting. Adding metalloprotease inhibitors to the reaction showed that clioquinol, phosphoramidon, and zinc metalloprotease inhibitors had no significant effect on γ-secretase activity. In contrast, phenanthroline, EDTA, and EGTA markedly decreased γ-secretase activity that could be restored by adding back calcium and magnesium ions. Mg(2+) stabilized a 1,000 kDa presenilin 1 complex through blue native gel electrophoresis and size-exclusion chromatography. Data suggest that Ca(2+) and Mg(2+) stabilize γ-secretase and enhance its activity.

Specific regions of the Alzheimer's disease (AD) brain are burdened with extracellular protein deposits, the accumulation of which is concomitant with a complex cascade of overlapping events. Many of these pathological processes produce oxidative stress. Under normal conditions, oxidative stress leads to the activation of defensive gene expression that promotes cell survival. At the forefront of defence is the nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor that regulates a broad spectrum of protective genes. Glycogen synthase kinase-3β (GSK-3β) regulates Nrf2, thus making this kinase a potential target for therapeutic intervention aiming to boost the protective activation of Nrf2. This paper aims to review the neuroprotective role of Nrf2 in AD, with special emphasis on the role of GSK-3β in the regulation of the Nrf2 pathway. We also examine the potential of inducing GSK-3β by small-molecule activators, dithiocarbamates, which potentially exert their beneficial therapeutic effects via the activation of the Nrf2 pathway.

BACKGROUND: Knowing a young woman with newly diagnosed breast cancer has a germline BRCA1 mutation informs her clinical management and that of her relatives. We sought an optimal strategy for identifying carriers using family history, breast cancer morphology and hormone receptor status data. METHODS: We studied a population-based sample of 452 Australian women with invasive breast cancer diagnosed before age 40 years for whom we conducted extensive germline mutation testing (29 carried a BRCA1 mutation) and a systematic pathology review, and collected three-generational family history and tumour ER and PR status. Predictors of mutation status were identified using multiple logistic regression. Areas under receiver operator characteristic (ROC) curves were estimated using five-fold stratified cross-validation. RESULTS: The probability of being a BRCA1 mutation carrier increased with number of selected histology features even after adjusting for family history and ER and PR status (P<0.0001). From the most parsimonious multivariate model, the odds ratio for being a carrier were: 9.7 (95% confidence interval: 2.6-47.0) for trabecular growth pattern (P=0.001); 7.8 (2.7-25.7) for mitotic index over 50 mitoses per 10 high-powered field (P=0.0003); and 2.7 (1.3-5.9) for each first-degree relative with breast cancer diagnosed before age 60 years (P=0.01).The area under the ROC curve was 0.87 (0.83-0.90). CONCLUSION: Pathology review, with attention to a few specific morphological features of invasive breast cancers, can identify almost all BRCA1 germline mutation carriers among women with early-onset breast cancer without taking into account family history.