To reveal the clonal architecture of melanoma and associated driver mutations, whole genome sequencing (WGS) and targeted extension sequencing were used to characterize 124 melanoma cases. Significantly mutated gene analysis using 13 WGS cases and 15 additional paired extension cases identified known melanoma genes such as BRAF, NRAS, and CDKN2A, as well as a novel gene EPHA3, previously implicated in other cancer types. Extension studies using tumors from another 96 patients discovered a large number of truncation mutations in tumor suppressors (TP53 and RB1), protein phosphatases (e.g., PTEN, PTPRB, PTPRD, and PTPRT), as well as chromatin remodeling genes (e.g., ASXL3, MLL2, and ARID2). Deep sequencing of mutations revealed subclones in the majority of metastatic tumors from 13 WGS cases. Validated mutations from 12 out of 13 WGS patients exhibited a predominant UV signature characterized by a high frequency of C->T transitions occurring at the 3′ base of dipyrimidine sequences while one patient (MEL9) with a hypermutator phenotype lacked this signature. Strikingly, a subclonal mutation signature analysis revealed that the founding clone in MEL9 exhibited UV signature but the secondary clone did not, suggesting different mutational mechanisms for two clonal populations from the same tumor. Further analysis of four metastases from different geographic locations in 2 melanoma cases revealed phylogenetic relationships and highlighted the genetic alterations responsible for differential drug resistance among metastatic tumors. Our study suggests that clonal evaluation is crucial for understanding tumor etiology and drug resistance in melanoma.
The sensitivity of massively-parallel sequencing has confirmed that most cancers are oligoclonal, with subpopulations of neoplastic cells harboring distinct mutations. A fine resolution view of this clonal architecture provides insight into tumor heterogeneity, evolution, and treatment response, all of which may have clinical implications. Single tumor analysis already contributes to understanding these phenomena. However, cryptic subclones are frequently revealed by additional patient samples (e.g., collected at relapse or following treatment), indicating that accurately characterizing a tumor requires analyzing multiple samples from the same patient. To address this need, we present SciClone, a computational method that identifies the number and genetic composition of subclones by analyzing the variant allele frequencies of somatic mutations. We use it to detect subclones in acute myeloid leukemia and breast cancer samples that, though present at disease onset, are not evident from a single primary tumor sample. By doing so, we can track tumor evolution and identify the spatial origins of cells resisting therapy.
Sequencing the genomic DNA of cancers has revealed that tumors are not homogeneous. As a tumor grows, new mutations accumulate in individual cells, and as these cells replicate, the mutations are passed on to their offspring, which comprise only a portion of the tumor when it is sampled. We present a method for identifying the fraction of cells containing specific mutations, clustering them into subclonal populations, and tracking the changes in these subclones. This allows us to follow the clonal evolution of cancers as they respond to chemotherapy or develop therapy resistance, processes which may radically alter the subclonal composition of a tumor. It also gives us insight into the spatial organization of tumors, and we show that multiple biopsies from a single breast cancer may harbor different subclones that respond differently to treatment. Finally, we show that sequencing multiple samples from a patient's tumor is often critical, as it reveals cryptic subclones that cannot be discerned from only one sample. This is the first tool that can efficiently leverage multiple samples to identify these as distinct subpopulations of cells, thus contributing to understanding the biology of the tumor and influencing clinical decisions about therapy.
The Drug-Gene Interaction database (DGIdb) mines existing resources that generate hypotheses about how mutated genes might be targeted therapeutically or prioritized for drug development. It provides an interface for searching lists of genes against a compendium of drug-gene interactions and potentially druggable genes. DGIdb can be accessed at dgidb.org.
First-generation molecular profiles for human breast cancers have enabled the identification of features that can predict therapeutic response; however, little is known about how the various data types can best be combined to yield optimal predictors. Collections of breast cancer cell lines mirror many aspects of breast cancer molecular pathobiology, and measurements of their omic and biological therapeutic responses are well-suited for development of strategies to identify the most predictive molecular feature sets.
We used least squares-support vector machines and random forest algorithms to identify molecular features associated with responses of a collection of 70 breast cancer cell lines to 90 experimental or approved therapeutic agents. The datasets analyzed included measurements of copy number aberrations, mutations, gene and isoform expression, promoter methylation and protein expression. Transcriptional subtype contributed strongly to response predictors for 25% of compounds, and adding other molecular data types improved prediction for 65%. No single molecular dataset consistently out-performed the others, suggesting that therapeutic response is mediated at multiple levels in the genome. Response predictors were developed and applied to TCGA data, and were found to be present in subsets of those patient samples.
These results suggest that matching patients to treatments based on transcriptional subtype will improve response rates, and inclusion of additional features from other profiling data types may provide additional benefit. Further, we suggest a systems biology strategy for guiding clinical trials so that patient cohorts most likely to respond to new therapies may be more efficiently identified.
Brain tumors (gliomas) contain large populations of infiltrating macrophages and recruited microglia, which in experimental murine glioma models promote tumor formation and progression. Among the barriers to understanding the contributions of these stromal elements to high-grade glioma (glioblastoma; GBM) biology is the relative paucity of tools to characterize infiltrating macrophages and resident microglia. In this study, we leveraged multiple RNA analysis platforms to identify new monocyte markers relevant to GBM patient outcome.
High-confidence lists of mouse resident microglia- and bone marrow-derived macrophage-specific transcripts were generated using converging RNA-seq and microarray technologies and validated using qRT-PCR and flow cytometry. Expression of select cell surface markers was analyzed in brain-infiltrating macrophages and resident microglia in an induced GBM mouse model, while allogeneic bone marrow transplantation was performed to trace the origins of infiltrating and resident macrophages. Glioma tissue microarrays were examined by immunohistochemistry, and the Gene Expression Omnibus (GEO) database was queried to determine the prognostic value of identified microglia biomarkers in human GBM.
We generated a unique catalog of differentially-expressed bone marrow-derived monocyte and resident microglia transcripts, and demonstrated that brain-infiltrating macrophages acquire F11R expression in GBM and following bone-marrow transplantation. Moreover, mononuclear cell F11R expression positively correlates with human high-grade glioma and additionally serves as a biomarker for GBM patient survival, regardless of GBM molecular subtype.
These studies establish F11R as a novel monocyte prognostic marker for GBM critical for defining a subpopulation of stromal cells for future potential therapeutic intervention.
Age at onset of diagnostic motor manifestations in Huntington disease (HD) is strongly correlated with an expanded CAG trinucleotide repeat. The length of the normal CAG repeat allele has been reported also to influence age at onset, in interaction with the expanded allele. Due to profound implications for disease mechanism and modification, we tested whether the normal allele, interaction between the expanded and normal alleles, or presence of a second expanded allele affects age at onset of HD motor signs.
We modeled natural log-transformed age at onset as a function of CAG repeat lengths of expanded and normal alleles and their interaction by linear regression.
An apparently significant effect of interaction on age at motor onset among 4,068 subjects was dependent on a single outlier data point. A rigorous statistical analysis with a well-behaved dataset that conformed to the fundamental assumptions of linear regression (e.g., constant variance and normally distributed error) revealed significance only for the expanded CAG repeat, with no effect of the normal CAG repeat. Ten subjects with 2 expanded alleles showed an age at motor onset consistent with the length of the larger expanded allele.
Normal allele CAG length, interaction between expanded and normal alleles, and presence of a second expanded allele do not influence age at onset of motor manifestations, indicating that the rate of HD pathogenesis leading to motor diagnosis is determined by a completely dominant action of the longest expanded allele and as yet unidentified genetic or environmental factors. Neurology® 2012;78:690–695
Adenocarcinomas of the tongue are rare and represent the minority (20 to 25%) of salivary gland tumors affecting the tongue. We investigated the utility of massively parallel sequencing to characterize an adenocarcinoma of the tongue, before and after treatment.
In the pre-treatment tumor we identified 7,629 genes within regions of copy number gain. There were 1,078 genes that exhibited increased expression relative to the blood and unrelated tumors and four genes contained somatic protein-coding mutations. Our analysis suggested the tumor cells were driven by the RET oncogene. Genes whose protein products are targeted by the RET inhibitors sunitinib and sorafenib correlated with being amplified and or highly expressed. Consistent with our observations, administration of sunitinib was associated with stable disease lasting 4 months, after which the lung lesions began to grow. Administration of sorafenib and sulindac provided disease stabilization for an additional 3 months after which the cancer progressed and new lesions appeared. A recurring metastasis possessed 7,288 genes within copy number amplicons, 385 genes exhibiting increased expression relative to other tumors and 9 new somatic protein coding mutations. The observed mutations and amplifications were consistent with therapeutic resistance arising through activation of the MAPK and AKT pathways.
We conclude that complete genomic characterization of a rare tumor has the potential to aid in clinical decision making and identifying therapeutic approaches where no established treatment protocols exist. These results also provide direct in vivo genomic evidence for mutational evolution within a tumor under drug selection and potential mechanisms of drug resistance accrual.
ORegAnno is an open-source, open-access database and literature curation system for community-based annotation of experimentally identified DNA regulatory regions, transcription factor binding sites and regulatory variants. The current release comprises 30 145 records curated from 922 publications and describing regulatory sequences for over 3853 genes and 465 transcription factors from 19 species. A new feature called the ‘publication queue’ allows users to input relevant papers from scientific literature as targets for annotation. The queue contains 4438 gene regulation papers entered by experts and another 54 351 identified by text-mining methods. Users can enter or ‘check out’ papers from the queue for manual curation using a series of user-friendly annotation pages. A typical record entry consists of species, sequence type, sequence, target gene, binding factor, experimental outcome and one or more lines of experimental evidence. An evidence ontology was developed to describe and categorize these experiments. Records are cross-referenced to Ensembl or Entrez gene identifiers, PubMed and dbSNP and can be visualized in the Ensembl or UCSC genome browsers. All data are freely available through search pages, XML data dumps or web services at: http://www.oreganno.org.
We describe here the derivation, characterization, and use of clonal cadmium-resistant (Cdr) strains of the Chinese hamster cell line CHO which differ in their metallothionein (MT) induction capacity. By nondenaturing polyacrylamide gel electrophoresis, we showed that the stable Cdr phenotype is correlated with the augmented expression of both isometallothioneins (MTI and MTII). In cells resistant to concentrations of CdCl2 exceeding 20 microM, coordinate amplification of genes encoding both isometallothioneins was demonstrated by using cDNA MT-coding sequence probes and probes specific for 3'-noncoding regions of Chinese hamster MTI and MTII genes. Molecular and in situ hybridization analyses supported close linkage of Chinese hamster MTI and MTII genes, which we have mapped previously to Chinese hamster chromosome 3. This suggests the existence of a functionally related MT gene cluster in this species. Amplified Cdr variants expressing abundant MT and their corresponding Cds parental CHO cells should be useful for future studies directed toward elucidating the mechanisms that regulate expression of the isometallothioneins.
Polyadenylated RNA was extracted from a cadmium resistant Chinese hamster (CHO) cell line, enriched for metal-induced, abundant RNA sequences and cloned as double-stranded cDNA in the plasmid pBR322. Two cDNA clones, pCHMT1 and pCHMT2, encoding two Chinese hamster isometallothioneins were identified, and the nucleotide sequence of each insert was determined. The two Chinese hamster metallothioneins show nucleotide sequence homologies of 80% in the protein coding region and approximately 35% in both the 5' and 3' untranslated regions. Interestingly, an 8 nucleotide sequence (TGTAAATA) has been conserved in sequence and position in the 3' untranslated regions of each metallothionein mRNA sequenced thus far. Estimated nucleotide substitution rates derived from interspecies comparisons were used to calculate a metallothionein gene duplication time of 45 to 120 million years ago.
Tumor necrosis factor-α (TNF) is an inflammatory cytokine that induces context-dependent proliferation, survival, and apoptosis responses in hepatocytes. TNF stimulates and enhances growth factor-mediated hepatocyte proliferation and survival following partial hepatectomy, but also acts in concert with other inflammatory cytokines of the innate immune response during viral infection to induce apoptosis in hepatocytes. In other epithelial cell types, TNF has recently been shown to stimulate autocrine release of transforming growth factor-α (TGF-α) and interleukin-1 (IL-1) family ligands. Here, we examine the role of these autocrine ligands in modulating TNF-induced proliferation and apoptosis in primary hepatocytes. We show that TNF-induced hepatocyte proliferation is regulated by an inducible, coupled, and self-antagonizing autocrine cascade involving the pro-proliferative TGF-α and IL-1 receptor antagonist (IL-1ra) ligands and antiproliferative IL-1α/β ligands. Moreover, cooperative stimulation of hepatocyte proliferation by combined TNF and TGF-α; treatment is self-limited through antiproliferative autocrine IL-1α/β feedback We show that TNF potently induces apoptosis of adenovirus-infected hepatocytes in a manner similarly determined through the integrated activity of a coupled TGF-α–IL-1α/β–IL-1ra autocrine cascade. Exogenous TGF-α can either enhance or diminish apoptosis in adenoviral vector-treated and TNF-treated hepatocytes, in a biphasic relationship also mediated by autocrine IL-1α/β feedback.
We demonstrate that TNF-induced hepatocyte proliferation and apoptosis are both governed by a self-antagonizing TGF-α–IL-1α/β–IL-1ra autocrine cascade in vitro, and thus identify multiple molecular targets for control of TNF-regulated hepatocyte phenotypic responses related to liver regeneration and adenoviral gene therapy.
The neutrophil-derived granular protein, CAP37, an innate immune system molecule, has antibiotic and immunomodulatory effects on host cells, including corneal epithelial cells. We previously showed that CAP37 modulates corneal epithelial cell migration, adhesion, and proliferation, and that protein kinase C delta (PKCδ) mediates CAP37-induced chemotaxis of these cells. The objective of this study was to investigate the hypothesis that CAP37 facilitates corneal wound healing through the PKC signaling pathway.
The standard “scratch” assay performed on monolayers of corneal epithelial cells was used to measure the in vitro effect of CAP37 on wound closure. In vivo wound healing in response to CAP37 was measured using a mouse corneal epithelium abrasion model. In vitro and in vivo wound closure were monitored over 48 hours. The PKCδ was visualized during wound closure in cell monolayers and corneal epithelium by immunohistochemistry. The importance of PKCδ in CAP37-induced corneal wound healing was assessed by siRNA.
We found that CAP37 accelerated wound closure in vitro and in vivo. Maximal closure occurred with concentrations of CAP37 between 250 and 500 ng/mL. Topical applications on mouse cornea led to re-epithelialization of the cornea by 24 hours. Immunohistochemistry of in vitro and in vivo wounds revealed a local increase of PKCδ along the wound edge in CAP37-treated cell monolayers and corneas, compared to untreated controls. CAP37-induced corneal wound healing was significantly reduced in vivo upon treatment with PKCδ siRNA.
These findings support the hypothesis that CAP37 facilitates corneal wound healing through the activation of PKCδ.
The significance of this work is the demonstration that a neutrophil-derived granule protein, CAP37, originally thought to act primarily as an antimicrobial peptide on Gram-negative bacteria, is able to promote corneal wound healing and that the mechanism is through PKC-delta signaling pathway.
antimicrobial proteins; corneal epithelium; corneal wound healing; protein kinase C
Kaposi's sarcoma-associated herpesvirus (KSHV) ORF6 is homologous to the herpes simplex virus 1 (HSV-1) ICP8 and Epstein-Barr virus (EBV) BALF2 proteins. Here, we describe its single-stranded DNA (ssDNA) binding properties. Based on previous findings with ICP8 and BALF2, a 60-amino-acid C-terminal deletion mutant of Orf6 was generated, and the protein was purified to explore the function of the C terminus in ssDNA binding. We showed that full-length ORF6 binds cooperatively to M13 ssDNA, disrupting its secondary structure and extending it to a length equivalent to that of duplex M13 DNA. The width of the ORF6-ssDNA filament is 9 nm, and a 7.3-nm repeat can be distinguished along the filament axis. Fluorescence polarization analysis revealed that the wild-type and C-terminal mutant ORF6 proteins bind equally well to short ssDNA substrates, with dissociation constant (Kd) values of 2.2 × 10−7M and 1.5 × 10−7M, respectively. These values were confirmed by electrophoretic mobility shift assay (EMSA) analysis, which also suggested that binding by the full-length protein may involve both monomers and small multimers. While no significant difference in affinities of binding between full-length ORF6 and the C-terminal deletion mutant were observed with the short DNAs, binding of the C-terminal mutant protein to M13 ssDNA showed a clear lack of cooperativity as seen by electron microscopy (EM). Incubation of a duplex DNA containing a long single-stranded tail with double-helical ORF6 protein filaments revealed that the ssDNA segment can be enveloped within the protein filament without disrupting the filament structure.
IMPORTANCE This work describes the biochemical characterization of the single-stranded DNA binding protein of KSHV, ORF6, central to viral DNA replication in infected cells. A C-terminal deletion mutant protein was generated to aid in understanding the role of the C terminus in DNA binding. Here we analyze the binding of the wild-type and mutant proteins to short oligomeric and longer genomic ssDNA substrates. Although it is capable of interacting with the short substrates, the inability of mutant ORF6 to form oligomers in solution hindered it from fully covering the long genomic substrates. We previously showed that ORF6 forms long filaments in solution, and we showed here that these can absorb ssDNA without disruption of the filament structure. This work will provide an important basis for future studies by us and/or others.
Neonatal immune responses to infection and vaccination are biased toward TH2 at the cost of proinflammatory TH1 responses needed to combat intracellular pathogens. However, upon appropriate stimulation, the neonatal immune system can induce adult-like TH1 responses. Here we report that a new class of vaccine adjuvant is especially well suited to enhance early life immunity. The GVI3000 adjuvant is a safe, nonpropagating, truncated derivative of Venezuelan equine encephalitis virus that targets dendritic cells (DCs) in the draining lymph node (DLN) and produces intracellular viral RNA without propagating to other cells. RNA synthesis strongly activates the innate immune response so that in adult animals, codelivery of soluble protein antigens induces robust humoral, cellular, and mucosal responses. The adjuvant properties of GVI3000 were tested in a neonatal BALB/c mouse model using inactivated influenza virus (iFlu). After a single immunization, mice immunized with iFlu with the GVI3000 adjuvant (GVI3000-adjuvanted iFlu) had significantly higher and sustained influenza virus-specific IgG antibodies, mainly IgG2a (TH1), compared to the mice immunized with antigen only. GVI3000 significantly increased antigen-specific CD4+ and CD8+ T cells, primed mucosal immune responses, and enhanced protection from lethal challenge. As seen in adult mice, the GVI3000 adjuvant increased the DC population in the DLNs, caused activation and maturation of DCs, and induced proinflammatory cytokines and chemokines in the DLNs soon after immunization, including gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6). In summary, the GVI3000 adjuvant induced an adult-like adjuvant effect with an influenza vaccine and has the potential to improve the immunogenicity and protective efficacy of new and existing neonatal vaccines.
IMPORTANCE The suboptimal immune responses in early life constitute a significant challenge for vaccine design. Here we report that a new class of adjuvant is safe and effective for early life immunization and demonstrate its ability to significantly improve the protective efficacy of an inactivated influenza virus vaccine in a neonatal mouse model. The GVI3000 adjuvant delivers a truncated, self-replicating viral RNA into dendritic cells in the draining lymph node. Intracellular RNA replication activates a strong innate immune response that significantly enhances adaptive antibody and cellular immune responses to codelivered antigens. A significant increase in protection results from a single immunization. Importantly, this adjuvant also primed a mucosal IgA response, which is likely to be critical for protection during many early life infections.
The Primary Immune Deficiency Treatment Consortium was formed to analyze the results of hematopoietic-cell transplantation in children with severe combined immunodeficiency (SCID) and other primary immunodeficiencies. Factors associated with a good transplantation outcome need to be identified in order to design safer and more effective curative therapy, particularly for children with SCID diagnosed at birth.
We collected data retrospectively from 240 infants with SCID who had received transplants at 25 centers during a 10-year period (2000 through 2009).
Survival at 5 years, freedom from immunoglobulin substitution, and CD3+ T-cell and IgA recovery were more likely among recipients of grafts from matched sibling donors than among recipients of grafts from alternative donors. However, the survival rate was high regardless of donor type among infants who received transplants at 3.5 months of age or younger (94%) and among older infants without prior infection (90%) or with infection that had resolved (82%). Among actively infected infants without a matched sibling donor, survival was best among recipients of haploidentical T-cell–depleted transplants in the absence of any pretransplantation conditioning. Among survivors, reduced-intensity or myeloablative pre-transplantation conditioning was associated with an increased likelihood of a CD3+ T-cell count of more than 1000 per cubic millimeter, freedom from immunoglobulin substitution, and IgA recovery but did not significantly affect CD4+ T-cell recovery or recovery of phytohemagglutinin-induced T-cell proliferation. The genetic subtype of SCID affected the quality of CD3+ T-cell recovery but not survival.
Transplants from donors other than matched siblings were associated with excellent survival among infants with SCID identified before the onset of infection. All available graft sources are expected to lead to excellent survival among asymptomatic infants. (Funded by the National Institute of Allergy and Infectious Diseases and others.)
Sleep promotes memory consolidation in humans and many other species, but the physiological and anatomical relationships between sleep and memory remain unclear. Here, we show the dorsal paired medial (DPM) neurons, which are required for memory consolidation in Drosophila, are sleep-promoting inhibitory neurons. DPMs increase sleep via release of GABA onto wake-promoting mushroom body (MB) α'/β' neurons. Functional imaging demonstrates that DPM activation evokes robust increases in chloride in MB neurons, but is unable to cause detectable increases in calcium or cAMP. Downregulation of α'/β' GABAA and GABABR3 receptors results in sleep loss, suggesting these receptors are the sleep-relevant targets of DPM-mediated inhibition. Regulation of sleep by neurons necessary for consolidation suggests that these brain processes may be functionally interrelated via their shared anatomy. These findings have important implications for the mechanistic relationship between sleep and memory consolidation, arguing for a significant role of inhibitory neurotransmission in regulating these processes.
Sleep affects memory: if you do not sleep well after a learning task, chances are you will not be able to recall whatever you tried to learn earlier. This is seen in almost all animals ranging from the fruit fly Drosophila, to mice and humans. However, the precise details of how memory and sleep are connected remain unclear.
Drosophila is an excellent model for teasing out the connections between memory and sleep. This is because its brain has a simple and well-studied memory region that contains a pair of nerve cells called the dorsal paired medial neurons. These neurons enable memories to be stored for the long term. Here, Haynes et al. asked whether these neurons can also affect sleep, and if so, how.
The experiments show that the dorsal paired medial neurons promote sleep in fruit flies. The neurons release a signaling molecule called GABA, which is detected by a type of neighboring ‘mushroom body’ neuron that usually promotes wakefulness. This leads to increases in the levels of chloride ions in the mushroom body neurons, but no change in the levels of calcium ions and a molecule called cAMP, which indicates that GABA inhibits these cells. Flies that have lower levels of two receptor proteins that detect GABA sleep less than normal flies.
Haynes et al.'s findings suggest that dorsal paired medial neurons deactivate their neighbors to promote sleep in fruit flies. This result was unexpected because current models of memory formation propose that dorsal paired medial neurons can activate the mushroom body neurons. Understanding how inhibiting mushroom body neurons influences memory will require researchers to reassess these models.
learning and memory; circuits; sleep; D. melanogaster
In The HIV Prevention Trials Network 061 study, 155 human immunodeficiency virus (HIV)–infected men reported no prior HIV diagnosis; 83 of those men had HIV RNA levels of <1000 copies/mL at enrollment. Antiretroviral drug testing revealed that 65 of the 83 (78.3%) men were on antiretroviral treatment. Antiretroviral drug testing can help distinguish between newly diagnosed and previously diagnosed HIV infection.
HIV; antiretroviral; self-report; MSM; new diagnosis
Rationale: The 2007 American Thoracic Society (ATS) and Infectious Diseases Society of America (IDSA) recommend that patients with pulmonary nontuberculous mycobacterial (PNTM) disease caused by Mycobacterium avium complex (MAC) or M. abscessus be treated with a macrolide-based multidrug antibiotic regimen until sputum culture negative for 1 year. After 6 years, the degree of adherence to recommended guidelines among physicians remains unknown.
Objective: To describe antibiotic treatment practices among physicians treating patients with PNTM in the United States.
Methods: A nationally representative sample of 1,286 U.S. physicians was contacted in December 2011 through January 2012; 582 of the responding physicians were treating patients with PNTM and were eligible to participate. Physicians were asked to extract medical record data on the last four patients they treated in the past year with PNTM disease from either MAC or M. abscessus. Treatment patterns were assessed for all patients by NTM species and physician specialty, and compared with the 2007 recommended ATS/IDSA guidelines.
Main Results: Questionnaires were completed by 349 physicians on 915 patients with PNTM, including 744 (81%) with MAC and 174 (19%) with M. abscessus; 3 patients were positive for both. Physicians treated 76 (44%) patients with M. abscessus and 411 (55%) patients with MAC. Only 13% of antibiotic regimens prescribed to patients with MAC met ATS/IDSA guidelines, 56% did not include a macrolide, and 16% were for macrolide monotherapy. Among patients with M. abscessus, 64% of regimens prescribed did not include a macrolide.
Conclusions: Adherence to the 2007 ATS/IDSA guidelines for treating PNTM disease is poor. Across all physician specialties evaluated, suboptimal or potentially harmful antibiotic regimens were commonly prescribed.
Mycobacterium infection; nontuberculous; treatment; guideline adherence
Non-surgical bleeding (NSB) is a major complication among heart failure (HF) patients supported by CF-LVADs. Understanding the hemostatic defects contributing to NSB after CF-LVAD implantation is crucial for prevention of this adverse event. The aim of this study was to examine the link between platelet GPIbα ectodomain shedding and NSB in CF-LVAD recipients and to identify a potential biomarker of NSB.
Serial blood samples were collected from thirty five HF patients supported with CF-LVADs. Platelet function was evaluated by Platelet Function Analyzer 100® and thromboelastography (TEG). Platelet GPIbα shedding, von Villebrand factor (vWF) antigen and vWF collagen binding capacity were determined using enzyme-linked immunosorbent assays (ELISAs). The structural analysis of vWF was performed by gel electrophoresis. These platelet functional measures with vWF parameters of the patients who experienced NSB between 4 to 32 days after CF-LVAD implantation (bleeder) were analyzed against those without NSB (non-bleeder). Blood samples from seven healthy individuals were collected to obtain the healthy reference values for the laboratory assays.
Elevated GPIbα shedding was found to be a preexisting condition in all HF patients prior to CF-LVAD implantation. Post-operative level of GPIbα shedding increased and remained elevated in the bleeder group while a consistent decrease was found in the non-bleeder group. A receiver operating characteristic (ROC) analysis indicated that the level of GPIbα shedding has a predictive power of NSB in patients supported with CF-LVADs.
Platelet GPIbα ectodomain shedding which attenuates platelet reactivity is associated with NSB. Plasma GPIbα level may potentially be used to refine bleeding risk stratification in CF-LVAD patients.
heart failure; left ventricular assist device; non-surgical bleeding; platelet GPIbα shedding
Sepsis describes the life-threatening systemic inflammatory response (SIRS) of an organism to an infection and is the leading cause of mortality on intensive care units (ICU) worldwide. An acute episode of sepsis is characterized by the extensive release of cytokines and other mediators resulting in a dysregulated immune response leading to organ damage and/or death. This initial pro-inflammatory burst often transits into a state of immune suppression characterised by loss of immune cells and T-cell dysfunction at later disease stages in sepsis survivors. However, despite these appreciations, the precise nature of the evoked defect in T-cell immunity in post-acute phases of SIRS remains unknown. Here we present an in-depth functional analysis of T-cell function in post-acute SIRS/sepsis. We document that T-cell function is not compromised on a per cell basis in experimental rodent models of infection-free SIRS (LPS or CpG) or septic peritonitis. Transgenic antigen-specific T-cells feature an unaltered cytokine response if challenged in vivo and ex vivo with cognate antigens. Isolated CD4+/CD8+ T-cells from post-acute septic animals do not exhibit defects in T-cell receptor-mediated activation at the the level of receptor-proximal signalling, activation marker upregulation or expansion. However, SIRS/sepsis induced transient lymphopenia and gave rise to an environment of immune attenuation at post acute disease stages. Thus, systemic inflammation has an acute impact on T-cell numbers and adaptive immunity, but does not cause major cell-autonomous enduring functional defects in T-cells.
A detailed map of the neurons that carry information away from the mushroom bodies in the brains of fruit flies has improved our understanding of the ways in which experiences can modify behaviour.
mushroom body; olfactory learning; associative memory; behavioral valence; sleep; D. melanogaster
Few studies have examined social factors related to breast cancer screening in Asian Indian women in the Midwestern US. This cross-sectional, community-based survey utilized constructs of the Health Belief Model to examine factors associated with breast cancer screening among Asian Indian women in metropolitan Detroit, Michigan. Of the 160 participants, 63.8% reported receiving both a clinical breast exam and mammogram within the past 2 years. Women were more likely to screen for breast cancer if they had a college education, lived in the US for more years, perceived that breast cancer screening is useful in detecting breast cancer early, agreed that mammography was important, and received a recommendation by a healthcare provider to get a mammogram. These findings highlight the need for further research on regional differences in breast cancer screening knowledge, behaviors and predictors among Asian Pacific Islanders subgroups such as Asian Indian women who recently immigrated to the US.
Asian Indian; Asian Pacific Islander; Breast cancer; Cancer screening; Health belief model
The vast majority of cancer mortalities result from distant metastases. The metastatic microenvironment provides unique protection to ectopic tumors as the primary tumors often respond to specific agents. Although significant interventional progress has been made on primary tumors, the lack of relevant accessible model in vitro systems in which to study metastases has plagued metastatic therapeutic development - particularly among micrometastases. A real-time, all-human model of metastatic seeding and cancer cells that recapitulate metastatic growth and can be probed in real time by a variety of measures and challenges would provide a critical window into the pathophysiology of metastasis and pharmacology of metastatic tumor resistance. To achieve this we are advancing our microscale bioreactor that incorporates human hepatocytes, human nonparenchymal liver cells, and human breast cancer cells to mimic the hepatic niche in three dimensions with functional tissue. This bioreactor is instrumented with oxygen sensors, micropumps capable of generating diurnally varying profiles of nutrients and hormones, while enabling real-time sampling. Since the liver is a major metastatic site for a wide variety of carcinomas and other tumors, this bioreactor uniquely allows us to more accurately recreate the human metastatic microenvironment and probe the paracrine effects between the liver parenchyma and metastatic cells. Further, as the liver is the principal site of xenobiotic metabolism, this reactor will help us investigate the chemotherapeutic response within a metabolically challenged liver microenvironment. This model is anticipated to yield markers of metastatic behavior and pharmacologic metabolism that will enable better clinical monitoring, and will guide the design of clinical studies to understand drug efficacy and safety in cancer therapeutics. This highly instrumented bioreactor format, hosting a growing tumor within a microenvironment and monitoring its responses, is readily transferable to other organs, giving this work impact beyond the liver.
liver; mammary carcinoma; microenvironment; tumor dissemination