Inflammatory breast cancer defined: proposed common diagnostic criteria to guide treatment and research.

PURPOSE: Inflammatory breast cancer is a deadly and aggressive type of breast cancer. A key challenge relates to the need for a more detailed, formal, objective definition of IBC, the lack of which compromises clinical care, hampers the conduct of clinical trials, and hinders the search for IBC-specific biomarkers and treatments because of the heterogeneity of patients considered to have IBC. METHODS: Susan G. Komen, the Inflammatory Breast Cancer Research Foundation, and the Milburn Foundation convened patient advocates, clinicians, and researchers to review the state of IBC and to propose initiatives to advance the field. After literature review of the defining clinical, pathologic, and imaging characteristics of IBC, the experts developed a novel quantitative scoring system for diagnosis. RESULTS: The experts identified through consensus several "defining characteristics" of IBC, including factors related to timing of onset and specific symptoms. These reflect common pathophysiologic changes, sometimes detectable on biopsy in the form of dermal lymphovascular tumor emboli and often reflected in imaging findings. Based on the importance and extent of these characteristics, the experts developed a scoring scale that yields a continuous score from 0 to 48 and proposed cut-points for categorization that can be tested in subsequent validation studies. CONCLUSION: To move beyond subjective 'clinical diagnosis' of IBC, we propose a quantitative scoring system to define IBC, based on clinical, pathologic, and imaging features. This system is intended to predict outcome and biology, guide treatment decisions and inclusion in clinical trials, and increase diagnostic accuracy to aid basic research; future validation studies are necessary to evaluate its performance.

Decoration of trastuzumab with short oligonucleotides: synthesis and detailed characterization.

Trastuzumab (Herceptin®) is an FDA-approved therapeutic antibody currently employed in the treatment of metastatic stages of breast cancer. Herein, we propose a simple, fast and cost-effective methodology to conjugate trastuzumab with 22-mer 5' thiol-modified oligonucleotides using a bifunctional crosslinker. The conjugates were successfully characterized by MALDI-ToF MS and SDS-PAGE, obviating the need for enzymatic digestion and difficult chromatographic separations. Furthermore, ELISA was performed to ensure that trastuzumab activity is not affected by oligonucleotide conjugation.

Quantification of cells with specific phenotypes II: determination of CD4 expression level on reconstituted lyophilized human PBMC labelled with anti-CD4 FITC antibody.

This report focuses on the characterization of CD4 expression level in terms of equivalent number of reference fluorophores (ERF). Twelve different flow cytometer platforms across sixteen laboratories were utilized in this study. As a first step the participants were asked to calibrate the fluorescein isothiocyanate (FITC) channel of each flow cytometer using commercially available calibration standard consisting of five populations of microspheres. Each population had an assigned value of equivalent fluorescein fluorophores (EFF denotes a special case of the generic term ERF with FITC as the reference fluorophore). The EFF values were assigned at the National Institute of Standards and Technology (NIST). A surface-labelled lyophilized cell preparation was provided by the National Institute of Biological Standards and Control (NIBSC), using human peripheral blood mononuclear cells (PBMC) pre-labeled with a FITC conjugated anti-CD4 monoclonal antibody. Three PBMC sample vials, provided to each participant, were used for the CD4 expression analysis. The PBMC are purported to have a fixed number of surface CD4 receptors. On the basis of the microsphere calibration, the EFF value of the PBMC samples was measured to characterize the population average CD4 expression level of the PBMC preparations. Both the results of data analysis performed by each participant and the results of centralized analysis of all participants' raw data are reported. Centralized analysis gave a mean EFF value of 22,300 and an uncertainty of 750, corresponding to 3.3% (level of confidence 68%) of the mean EFF value. The next step will entail the measurement of the ERF values of the lyophilized PBMC stained with labels for other fluorescence channels. The ultimate goal is to show that lyophilized PBMC is a suitable biological reference cell material for multicolor flow cytometry and that it can be used to present multicolor flow cytometry measurements in terms of ABC (antibodies bound per cell) units.

Interleukin-17 enhances immunosuppression by mesenchymal stem cells.

IL-17 is one of the most potent and most actively investigated proinflammatory cytokines. In this study, we examined the effect of IL-17 on mesenchymal stem cells (MSCs) under the influence of inflammatory cytokines. Ironically, IL-17 dramatically enhanced the immunosuppressive effect of MSCs induced by IFNγ and TNFα, revealing a novel role of IL-17 in immunosuppression. Interestingly, we found that this action of IL-17 was dependent on the promoted expression of a key immune suppressive molecule, inducible nitric oxide synthase (iNOS), in MSCs. In a concanavalin A (ConA)-induced hepatitis mouse model, we found that IL-17 also enhanced the in vivo immunosuppressive effect of MSCs in an iNOS-dependent manner. Moreover, this promoting effect of IL-17 was found to be exerted through enhancing mRNA stability by modulating the protein level of ARE/poly(U)-binding/degradation factor 1 (AUF1), a well-known factor that promotes mRNA decay. In auf1(-/-) MSCs, IFNγ and TNFα could induce maximal immunosuppressive effect, both in vitro and in vivo, without the need for IL-17. Thus, our studies demonstrated that in the presence of MSCs, IL-17 promotes immunosuppression.

Salvia divinorum Epling & Játiva (Maria Pastora) e Salvinorina A: crescente uso recreacional e potencial de abuso / Salvia divinorum Epling & Játiva (ska María Pastora) and Salvinorin A: increasing recreational use and abuse potential

A planta Salvia divinorum Epling & Játiva (SDI), da família Lamiaceae, tem sido usada por séculos pela cultura mazateca e vem ganhando popularidade como droga recreacional nos últimos anos. Seu princípio ativo - Salvinorina A (SA) - é agonista dos receptores opióides kappa, com potencial psicotrópico. A utilização da planta vem crescendo na Europa e na América do Norte, apesar de ainda não existirem provas concretas sobre abuso. A presente revisão da literatura contemporânea aborda as evidências sobre o potencial de abuso de SDI, bem como o crescente uso recreacional, ainda que seja alucinógeno permitido legalmente e de fácil compra em muitos países.

The plant Salvia divinorum Epling & Játiva (SDI), of the Lamiaceae family, has been used for centuries by the Mazateca culture and has gained popularity as a recreational drug in the last years. Its active principle, Salvinorin A (SA), is a potentially psychotropic agonist of the kappa opioid receptors. The use of SDI has increased in Europe and North America, although there are no concrete proofs about abuse. The present review discusses current evidence on potential SDI abuse, as well as its increasing recreational use, although it is considered a legalized hallucinogen easily acquired in many countries.

Effect of long term organic amendments on adsorption-desorption of thiram onto a luvisol soil derived from loess.

The objective of this work was to assess the influence of soil organic amendments on the sorption properties of the fungicide thiram. The organic amendments studied were organic household compost (COM), sewage sludge from municipal water treatment facilities (SLU) and farmyard manure (FYM), which were compared to mineral fertilizer application (MIN). Sorption-desorption experiments were performed using the batch method and the results indicated that the adsorption isotherms were non-linear and were found to conform to the Brunauer-Emmett-Teller (BET) model, suggesting multilayer adsorption and adsorbate-adsorbate interactions after the saturation of the surface layer. In general, distribution coefficient values, K(D), are dependent on, but not proportional to, the initial concentration of thiram. For a fixed thiram initial concentration, a significant correlation (r(2)>0.851; p<0.001) between K(D) values and the soil organic carbon content (OC) was observed. The highest value of K(D) was observed for the soil amended with compost, which is the one with the highest organic carbon content. K(D) values were divided by the soil organic carbon contents in order to obtain organic carbon partition coefficients K(OC). Comparing K(OC) means from 3 (initial concentrations) x 4 (soil organic matter compositions) x 3 (replicates) factorial ANOVA allow us to conclude that there is a significant but not proportional influence of the initial concentration of thiram on those values, but changes in the soil organic matter composition, associated to different soil amendments, have no significant influence on adsorption of thiram. To evaluate the reversibility of thiram adsorption, two consecutive desorption cycles were performed with CaCl(2) 0.01 mol L(-1). The desorption K(D) values were consistently higher (approximately twice) than those for adsorption at the same equilibrium concentrations for all soil samples supporting the existence of hysteresis in the adsorption-desorption behavior of thiram. Despite the fact that the adsorption K(D) values were proportionally increased with increasing total organic carbon content, this was not the case for the desorption K(D) values.

Inhibition of Cap-initiation complexes linked to a novel mechanism of eIF4G depletion in acute myocardial ischemia.

Translational control in the rat heart was characterized during acute myocardial ischemia introduced by left coronary artery ligature. Within 10 min of ischemia, eukaryotic (eIF)4E binds to its negative regulator, eIF4E-binding protein-1 (4E-BP1), but the levels of 4E-BP1 are insufficient to disrupt cap-dependent mRNA initiation complexes. However, by 1 h of ischemia, the abundance of the cap-initiation complex protein eIF4G is reduced by relocalization into TIAR protein complexes, triggering 4E-BP1 sequestration of eIF4E and disruption of cap-dependent mRNA initiation complexes. As the heart begins to fail at 6 h, proteolysis of eIF4G is observed, resulting in its depletion and accompanied by limited destruction of 4E-BP1 and eIF4E. eIF4G proteolysis and modest loss of 4E-BP1 are associated with caspase-3 activation and induction of cardiomyocyte apoptotic and necrotic death. Acute heart ischemia therefore downregulates cap-dependent translation through eIF4E sequestration triggered by eIF4G depletion.

Calcium signaling by HBx protein in hepatitis B virus DNA replication.

Hepatitis B virus (HBV) infects more than 300 million people and is a leading cause of liver cancer and disease. The HBV HBx protein is essential for infection; HBx activation of Src is important for HBV DNA replication. In our study, HBx activated cytosolic calcium-dependent proline-rich tyrosine kinase-2 (Pyk2), a Src kinase activator. HBx activation of HBV DNA replication was blocked by inhibiting Pyk2 or calcium signaling mediated by mitochondrial calcium channels, which suggests that HBx targets mitochondrial calcium regulation. Reagents that increased cytosolic calcium substituted for HBx protein in HBV DNA replication. Thus, alteration of cytosolic calcium was a fundamental requirement for HBV replication and was mediated by HBx protein.

Hepatitis B virus HBx protein activation of cyclin A-cyclin-dependent kinase 2 complexes and G1 transit via a Src kinase pathway.

Numerous studies have demonstrated that the hepatitis B virus HBx protein stimulates signal transduction pathways and may bind to certain transcription factors, particularly the cyclic AMP response element binding protein, CREB. HBx has also been shown to promote early cell cycle progression, possibly by functionally replacing the TATA-binding protein-associated factor 250 (TAF(II)250), a transcriptional coactivator, and/or by stimulating cytoplasmic signal transduction pathways. To understand the basis for early cell cycle progression mediated by HBx, we characterized the molecular mechanism by which HBx promotes deregulation of the G0 and G1 cell cycle checkpoints in growth-arrested cells. We demonstrate that TAF(II)250 is absolutely required for HBx activation of the cyclin A promoter and for promotion of early cell cycle transit from G0 through G1. Thus, HBx does not functionally replace TAF(II)250 for transcriptional activity or for cell cycle progression, in contrast to a previous report. Instead, HBx is shown to activate the cyclin A promoter, induce cyclin A-cyclin-dependent kinase 2 complexes, and promote cycling of growth-arrested cells into G1 through a pathway involving activation of Src tyrosine kinases. HBx stimulation of Src kinases and cyclin gene expression was found to force growth-arrested cells to transit through G1 but to stall at the junction with S phase, which may be important for viral replication.

Process-centered revenue-cycle management optimizes payment process.

By implementing a process-centered revenue cycle, healthcare organizations and group practices can achieve a seamless payment process with clear lines of accountability to achieve target outcomes. The integrated, end-to-end, revenue-cycle process involves four key components: jobs, skills, staffing, and structure; information and information systems; organizational alignment and accountability; and performance measures and evaluation measures. The Henry Ford Health System (HFHS), based in Detroit, Michigan, exemplifies the type of results that are achievable with this model. HFHS includes a group practice with more than 1,000 physicians in 40 specialties. After implementing a process-centered revenue cycle, HFHS dramatically improved registration and verification transactions and optimized revenues.

Role of NF-kappaB and myc proteins in apoptosis induced by hepatitis B virus HBx protein.

Chronic infection with hepatitis B virus (HBV) promotes a high level of liver disease and cancer in humans. The HBV HBx gene encodes a small regulatory protein that is essential for viral replication and is suspected to play a role in viral pathogenesis. HBx stimulates cytoplasmic signal transduction pathways, moderately stimulates a number of transcription factors, including several nuclear factors, and in certain settings sensitizes cells to apoptosis by proapoptotic stimuli, including tumor necrosis factor alpha (TNF-alpha) and etopocide. Paradoxically, HBx activates members of the NF-kappaB transcription factor family, some of which are antiapoptotic in function. HBx induces expression of Myc protein family members in certain settings, and Myc can sensitize cells to killing by TNF-alpha. We therefore examined the roles of NF-kappaB, c-Myc, and TNF-alpha in apoptotic killing of cells by HBx. RelA/NF-kappaB is shown to be induced by HBx and to suppress HBx-mediated apoptosis. HBx also induces c-Rel/NF-kappaB, which can promote apoptotic cell death in some contexts or block it in others. Induction of c-Rel by HBx was found to inhibit its ability to directly mediate apoptotic killing of cells. Thus, HBx induction of NF-kappaB family members masks its ability to directly mediate apoptosis, whereas ablation of NF-kappaB reveals it. Investigation of the role of Myc protein demonstrates that overexpression of Myc is essential for acute sensitization of cells to killing by HBx plus TNF-alpha. This study therefore defines a specific set of parameters which must be met for HBx to possibly contribute to HBV pathogenesis.

Adenovirus-specific translation by displacement of kinase Mnk1 from cap-initiation complex eIF4F.

Translation of cellular mRNAs involves formation of a cap-binding translation initiation complex known as eIF4F, containing phosphorylated cap-binding protein eIF4E, eIF4E kinase Mnk1, eIF4A, poly(A)-binding protein and eIF4G. Adenovirus is shown to prevent cellular translation by displacing Mnk1 from eIF4F, thereby blocking phosphorylation of eIF4E. Over expression of an eIF4E mutant that cannot be phosphorylated by Mnk1 impairs translation of cellular but not viral late mRNAs. Adenovirus 100k protein is shown to bind the C-terminus of eIF4G in vivo and in vitro, the same region bound by Mnk1. In vivo, 100k protein displaces Mnk1 from eIF4G during adenovirus infection, or in transfected cells. Purified 100k protein also evicts Mnk1 from isolated eIF4F complexes in vitro. A mutant adenovirus with a temperature-sensitive 100k protein that cannot inhibit cellular protein synthesis at restrictive temperature no longer blocks Mnk1 binding to eIF4G, or phosphorylation of eIF4E. We describe a mechanism whereby adenovirus selectively inhibits the translation of cellular but not viral mRNAs by displacement of Mnk1 from eIF4G and inhibition of eIF4E phosphorylation.

Chaperone hsp27 inhibits translation during heat shock by binding eIF4G and facilitating dissociation of cap-initiation complexes.

Inhibition of protein synthesis during heat shock limits accumulation of unfolded proteins that might damage eukaryotic cells. We demonstrate that chaperone Hsp27 is a heat shock-induced inhibitor of cellular protein synthesis. Translation of most mRNAs requires formation of a cap-binding initiation complex known as eIF4F, consisting of factors eIF4E, eIF4A, eIF4E kinase Mnk1, poly(A)-binding protein, and adaptor protein eIF4G. Hsp27 specifically bound eIF4G during heat shock, preventing assembly of the cap-initiation/eIF4F complex and trapping eIF4G in insoluble heat shock granules. eIF4G is a specific target of Hsp27, as eIF4E, eIF4A, Mnk1, poly(A)-binding protein, eIF4B, and eIF3 were not bound by Hsp27 and were not recruited into insoluble complexes. Dissociation of eIF4F was enhanced during heat shock by ectopic overexpression of Hsp25, the murine homolog of human Hsp27. Overexpression of Hsc70, a constitutive homolog of Hsp70, prevented loss of cap-initiation complexes and maintained eIF4G solubility. Purified Hsp27 specifically bound purified eIF4G in vitro, prevented in vitro translation, eliminated eIF4G interaction with protein binding factors, and promoted eIF4G insolubilization. These results therefore demonstrate that Hsp27 is a heat-induced inhibitor of eIF4F-dependent mRNA translation.

Motivational versus confrontational interviewing: a comparison of substance abuse assessment practices at employee assistance programs.

The aim of this study was to conduct a quasi-experimental comparison of two employee assistance program (EAP) assessment approaches with substance abusers: confrontational interviewing (CI) and motivational interviewing (MI). A total of 176 EAP clients from 14 study sites met the study criteria, and 89 (51%) agreed to participate in the study. At three and nine months postassessment, both the MI and CI groups showed similar changes in readiness for change, completion of initial treatment plans, and subsequent treatment. Most important, both the MI and CI participants showed significant and comparable improvement on all of the substance abuse baseline measures as well as measures of family-social well-being and effects of drinking/drugging on work performance. The results open the door for EAP counselors to use an empirically supported assessment style that is at least as effective as the traditional confrontational approach.

Translation by ribosome shunting on adenovirus and hsp70 mRNAs facilitated by complementarity to 18S rRNA.

Translation initiation on eukaryotic mRNAs involves 40S ribosome association with mRNA caps (m(7)GpppN), mediated by initiation factor eIF4F. 40S eukaryotic ribosomes and initiation factors undergo 5' scanning to the initiation codon, with no known role for complementarity between eukaryotic 18S rRNA and the 5' noncoding region of mRNAs. We demonstrate that the 5' noncoding region of human adenovirus late mRNAs, known as the tripartite leader, utilizes a striking complementarity to 18S rRNA to facilitate a novel form of translation initiation referred to as ribosome shunting, in which 40S ribosomes bind the cap and bypass large segments of the mRNA to reach the initiation codon. Related elements are also shown to promote ribosome shunting in adenovirus IVa2 intermediate phase mRNA during virus infection and in human heat shock protein 70 (hsp70) mRNA for selective translation during heat shock. The importance of mRNA complementarity to 18S rRNA suggests that ribosome shunting may involve either specific RNA structural features or a prokaryotic-like interaction between mRNA and rRNA.

Src kinases involved in hepatitis B virus replication.

Chronic infection by hepatitis B virus is a leading cause of human liver cancer and liver disease. The hepatitis B virus HBx protein is a regulatory factor that is essential for virus infection in mammals and is implicated in development of liver cancer and liver disease. Among the reported activities of HBx is the ability to stimulate Src tyrosine kinases, Ras-GTPases and transcriptional activation. We now demonstrate that HBx activation of Src tyrosine kinases, but not Ras, promotes a high level of viral replication in cell culture. HBx is shown to stimulate reverse transcription of the viral pregenomic mRNA into genomic DNA through a Src-mediated pathway in tissue culture cells. Targeted inhibition of Src tyrosine kinase activity, mutational inactivation of the HBx gene or retargeting of HBx to the nucleus to abolish cytoplasmic signal transduction activity, are shown to impair viral reverse transcription strongly. These studies implicate HBx stimulation of the Src family of tyrosine kinases in stimulation of viral polymerase activity.

Calcium-induced stabilization of AU-rich short-lived mRNAs is a common default response.

The AU-rich element (AUUUA)n, found in the 3' noncoding region of many short-lived cytokine and proto-oncogene mRNAs, is sufficient to specifically target these mRNAs for rapid degradation in mammalian cells. The mechanism by which the AU-rich element promotes rapid mRNA decay is not known. Previous studies have shown that release of intracellular stored calcium by ionophore treatment of thymocytes and mast cells inhibits the rapid turnover of AU-rich interleukin mRNAs. Increased cytoplasmic half-life of interleukin mRNAs was linked to calcium-induced activation of the N-terminal c-Jun kinase. In this report we have characterized the calcium-induced stabilization of AU-rich mRNAs. We show that calcium induces stabilization of mRNAs with canonical AU-rich elements in all cell types tested. These results indicate that short-lived mRNA stabilization by calcium is not unique to immune cells nor interleukin mRNAs, but is a widespread default response that includes generic AU-rich mRNAs. Stabilization is shown to be rapid but transient, and to act without altering nuclear transcription or cytoplasmic translation rates. These data support the view that calcium release likely stabilizes short-lived mRNAs by altering trans-acting decay factors that promote AU-rich mRNA turnover.

Control of mRNA decay by heat shock-ubiquitin-proteasome pathway.

Cytokine and proto-oncogene messenger RNAs (mRNAs) are rapidly degraded through AU-rich elements in the 3' untranslated region. Rapid decay involves AU-rich binding protein AUF1, which complexes with heat shock proteins hsc70-hsp70, translation initiation factor eIF4G, and poly(A) binding protein. AU-rich mRNA decay is associated with displacement of eIF4G from AUF1, ubiquitination of AUF1, and degradation of AUF1 by proteasomes. Induction of hsp70 by heat shock, down-regulation of the ubiquitin-proteasome network, or inactivation of ubiquitinating enzyme E1 all result in hsp70 sequestration of AUF1 in the perinucleus-nucleus, and all three processes block decay of AU-rich mRNAs and AUF1 protein. These results link the rapid degradation of cytokine mRNAs to the ubiquitin-proteasome pathway.