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26.  Cell of origin determines clinically relevant subtypes of MLL-rearranged AML 
Leukemia  2012;27(4):852-860.
MLL-fusion proteins can induce acute myeloid leukemias (AML) from either hematopoietic stem cells (HSC) or granulocyte macrophage progenitors (GMP), but it remains unclear if the cell of origin influences the biology of the resultant leukemia. MLL-AF9 transduced single HSC or GMP could be continuously replated, but HSC-derived clones were more likely than GMP-derived clones to initiate AML in mice. Leukemia stem cells derived from either HSC or GMP had a similar immunophenotype consistent with a maturing myeloid cell (LGMP). Gene expression analyses demonstrated that LGMP inherited gene expression programs from the cell of origin including high-level Evi-1 expression in HSC derived LGMP. The gene expression signature of LGMP derived from HSC was enriched in poor prognosis human MLL-rearranged AML in three independent data sets. Moreover, global 5’-mC levels were elevated in HSC-derived leukemias as compared to GMP-derived leukemias. This mirrored a difference seen in 5-mC between MLL-rearranged human leukemias that are either EVI1-positive or EVI1-negative. Finally, HSC derived leukemias were more resistant to chemotherapy than GMP-derived leukemias. These data demonstrate that the cell of origin influences the gene expression profile, the epigenetic state, and the drug response in AML, and that these differences can account for clinical heterogeneity within a molecularly defined group of leukemias.
Human AMLs are heterogeneous even within subtype defined by a specific genetic lesion such as MLL-translocations and this leads to variable clinical outcomes. The developmental stage (or epigenetic state) of the cell in which leukemogenic transformation is initiated may contribute to the ultimate disease phenotype. We used a well established model of MLL-AF9 mediated AML and transformation of single cells to test the relevance of the leukemia cell of origin on AML development, gene expression profiles, DNA methylation and chemotherapy response. We show that HSC derived AML models human high-risk/poor outcome AML pool, and that we can recapitulate important clinical subtypes of human MLL-rearranged AML through the transformation of different cell types. These data demonstrate that the cell of origin can influence the phenotype of a resultant leukemia even if the initiating genetic lesion is the same.
PMCID: PMC4693300  PMID: 23235717
MLL; leukemia; cell of origin; gene expression; DNA methylation; chemotherapy; drug resistance
27.  Pluripotent Stem Cells: Current Understanding and Future Directions 
Stem Cells International  2015;2016:9451492.
Pluripotent stem cells have the ability to undergo self-renewal and to give rise to all cells of the tissues of the body. However, this definition has been recently complicated by the existence of distinct cellular states that display these features. Here, we provide a detailed overview of the family of pluripotent cell lines derived from early mouse and human embryos and compare them with induced pluripotent stem cells. Shared and distinct features of these cells are reported as additional hallmark of pluripotency, offering a comprehensive scenario of pluripotent stem cells.
PMCID: PMC4699068  PMID: 26798367
29.  Distribution of Residual Cancer Cells in the Bowel Wall after Neoadjuvant Chemoradiation in Patients with Rectal Cancer 
Diseases of the colon and rectum  2013;56(2):142-149.
The standard treatment for locally advanced rectal cancer is pre-operative chemoradiation and total mesorectal excision. After surgery, tumors are classified according to the depth of tumor invasion, nodal involvement, and tumor regression grade. However, these staging systems do not provide information about the distribution of residual cancer cells within the bowel wall.
To determine the distribution of residual cancer cells in each layer of the bowel wall in rectal cancer specimens.
Prospective Phase II study.
153 patients with stage II or stage III rectal cancer.
Patients were treated with chemoradiation and surgery. Surgical specimen tumor tissue was analyzed and the distribution of residual cancer cells in each layer of the bowel wall was determined.
Main Outcome Measures
Statistical analysis was used to examine the correlation of residual cancer cells in each layer of the bowel wall with the clinical/pathological stage and tumor regression grade.
Forty-two of 153 (27%) patients had complete response in the bowel wall (ypT0). Of the remaining 111 patients who had residual cancer cells, 5 (3%) were ypTis, 12 (8%) were ypT1, 41 (27%) were ypT2, 50 (33%) were ypT3, and 3 (2%) were ypT4. Of the 94 patients with ypT2-4 tumors, 12 (13%) had cancer cells in the mucosa and 53 (56%) had cancer cells in the submucosa; 92 (98%) had cancer cells in the muscularis propria. Pretreatment cT stage correlated with distribution of residual cancer cells. Tumor regression grade was not associated with distribution of residual cancer cells after chemoradiation.
Patients received different chemotherapy regimens.
Residual cancer cells in rectal cancer specimens after chemoradiation are preferentially located close to the invasive front. This should be considered when designing strategies to diagnose complete pathologic response and when investigating the mechanisms of tumor resistance to chemoradiation.
PMCID: PMC4674069  PMID: 23303141
Rectal cancer; Residual cancer cells; Bowel wall; Neoadjuvant chemoradiation
30.  Comparison of T1rho Measurements in Agarose Phantoms and Human Patellar Cartilage Using 2D Multislice Spiral and 3D Magnetization Prepared Partitioned k-Space Spoiled Gradient-Echo Snapshot Techniques at 3 T 
AJR. American journal of roentgenology  2011;196(2):W174-W179.
To compare in vitro T1ρ measurements in agarose phantoms and articular cartilage specimens using a 2D multi-slice spiral and 3D MAPSS MRI sequences.
Materials and Methods
Six phantoms (agarose concentration: 2%, 3%, 4%; n=2 each) and ten axially-sliced patellar specimens from five cadaveric donors were scanned at 3T. T1ρ weighted images were acquired using a 2D spiral and 3D MAPSS sequence. Regions of interest were analyzed to measure T1ρ values centrally within phantoms to evaluate effects of pulse sequence and agarose concentration. In patellar specimens, ROI’s were analyzed to measure T1ρ values with respect to anatomic location (the medial and lateral facets, and the median ridge in deep and superficial halves of the cartilage) as well as location that exhibited magic angle effect in proton density weighted images) to evaluate the effects of pulse sequence, anatomical location and magic angle.
In phantoms, T1ρ values were similar (p=0.9) between sequences but decreased significantly (p<0.001) from ~55 to ~29 ms, with increasing agarose concentration from 2% to 4%. In cartilage specimens, T1ρ values were also similar between sequences (p=0.3) but were significantly higher (p<0.001) in the superficial layer (95~120 ms) compared to the deep layer (45~75 ms).
T1ρ measurements of human patellar cartilage specimens and agarose phantoms using 2D spiral and 3D MAPSS sequences gave similar values. Lower T1ρ values of higher concentration phantoms, and deeper layer of cartilage where proteoglycan concentration is higher than superficial layer, suggests sensitivity of our methods to concentration of macromolecules in biological tissues.
PMCID: PMC4674086  PMID: 21257859
31.  Discovery, Synthesis, and Optimization of Diarylisoxazole-3-carboxamides as Potent Inhibitors of the Mitochondrial Permeability Transition Pore 
ChemMedChem  2015;10(10):1655-1671.
The mitochondrial permeability transition pore (mtPTP) is a Ca2+-requiring mega-channel which, under pathological conditions, leads to the deregulated release of Ca2+ and mitochondrial dysfunction, ultimately resulting in cell death. Although the mtPTP is a potential therapeutic target for many human pathologies, its potential as a drug target is currently unrealized. Herein we describe an optimization effort initiated around hit 1, 5-(3-hydroxyphenyl)-N-(3,4,5-trimethoxyphenyl)isoxazole-3-carboxamide, which was found to possess promising inhibitory activity against mitochondrial swelling (EC50 < 0.39 µm) and showed no interference on the inner mitochondrial membrane potential (rhodamine 123 uptake EC50 > 100 µm). This enabled the construction of a series of picomolar mtPTP inhibitors that also potently increase the calcium retention capacity of the mitochondria. Finally, the therapeutic potential and in vivo efficacy of one of the most potent analogues, N-(3-chloro-2-methylphenyl)-5-(4-fluoro-3-hydroxyphenyl)isoxazole-3-carboxamide (60), was validated in a biologically relevant zebrafish model of collagen VI congenital muscular dystrophies.
PMCID: PMC4674087  PMID: 26286375
calcium retention capacity; mitochondria; muscular dystrophy; permeability transition; zebrafish
32.  Transcription Factor Networks Directing the Development, Function, and Evolution of Innate Lymphoid Effectors 
Annual review of immunology  2015;33:505-538.
Mammalian lymphoid immunity is mediated by fast and slow responders to pathogens. Fast innate lymphocytes are active within hours after infections in mucosal tissues. Slow adaptive lymphocytes are conventional T and B cells with clonal antigen receptors that function days after pathogen exposure. A transcription factor (TF) regulatory network guiding early T cell development is at the core of effector function diversification in all innate lymphocytes, and the kinetics of immune responses is set by developmental programming. Operational units within the innate lymphoid system are not classified by the types of pathogen-sensing machineries but rather by discrete effector functions programmed by regulatory TF networks. Based on the evolutionary history of TFs of the regulatory networks, fast effectors likely arose earlier in the evolution of animals to fortify body barriers, and in mammals they often develop in fetal ontogeny prior to the establishment of fully competent adaptive immunity.
PMCID: PMC4674156  PMID: 25650177
T cells; transcription factor regulatory network; evolution of immunity; fetal lymphopoiesis; γδ T cells; cytokines
33.  Extragenital Gonorrhea and Chlamydia Testing and Infection Among Men Who Have Sex With Men—STD Surveillance Network, United States, 2010–2012 
Gonorrhea (GC) and chlamydia (CT) are the most commonly reported notifiable diseases in the United States. The Centers for Disease Control and Prevention recommends that men who have sex with men (MSM) be screened for urogenital GC/CT, rectal GC/CT, and pharyngeal GC. We describe extragenital GC/CT testing and infections among MSM attending sexually transmitted disease (STD) clinics.
The STD Surveillance Network collects patient data from 42 STD clinics. We assessed the proportion of MSM attending these clinics during July 2011–June 2012 who were tested and positive for extragenital GC/CT at their most recent visit or in the preceding 12 months and the number of extragenital infections that would have remained undetected with urethral screening alone.
Of 21 994 MSM, 83.9% were tested for urogenital GC, 65.9% for pharyngeal GC, 50.4% for rectal GC, 81.4% for urogenital CT, 31.7% for pharyngeal CT, and 45.9% for rectal CT. Of MSM tested, 11.1% tested positive for urogenital GC, 7.9% for pharyngeal GC, 10.2% for rectal GC, 8.4% for urogenital CT, 2.9% for pharyngeal CT, and 14.1% for rectal CT. More than 70% of extragenital GC infections and 85% of extragenital CT infections were associated with negative urethral tests at the same visit and would not have been detected with urethral screening alone.
Extragenital GC/CT was common among MSM attending STD clinics, but many MSM were not tested. Most extragenital infections would not have been identified, and likely would have remained untreated, with urethral screening alone. Efforts are needed to facilitate implementation of extragenital GC/CT screening recommendations for MSM.
PMCID: PMC4666527  PMID: 24647015
extragenital gonorrhea; extragenital chlamydia; men who have sex with men (MSM); STD
34.  Humans can integrate feedback of discrete events in their sensorimotor control of a robotic hand 
Experimental brain research  2014;232(11):3421-3429.
Providing functionally effective sensory feedback to users of prosthetics is a largely unsolved challenge. Traditional solutions require high band-widths for providing feedback for the control of manipulation and yet have been largely unsuccessful. In this study, we have explored a strategy that relies on temporally discrete sensory feedback that is technically simple to provide. According to the Discrete Event-driven Sensory feedback Control (DESC) policy, motor tasks in humans are organized in phases delimited by means of sensory encoded discrete mechanical events. To explore the applicability of DESC for control, we designed a paradigm in which healthy humans operated an artificial robot hand to lift and replace an instrumented object, a task that can readily be learned and mastered under visual control. Assuming that the central nervous system of humans naturally organizes motor tasks based on a strategy akin to DESC, we delivered short-lasting vibrotactile feedback related to events that are known to forcefully affect progression of the grasp-lift-and-hold task. After training, we determined whether the artificial feedback had been integrated with the sensorimotor control by introducing short delays and we indeed observed that the participants significantly delayed subsequent phases of the task. This study thus gives support to the DESC policy hypothesis. Moreover, it demonstrates that humans can integrate temporally discrete sensory feedback while controlling an artificial hand and invites further studies in which inexpensive, noninvasive technology could be used in clever ways to provide physiologically appropriate sensory feedback in upper limb prosthetics with much lower band-width requirements than with traditional solutions.
PMCID: PMC4666528  PMID: 24992899
Sensorimotor control; Human; Hand; Tactile afferents; Sensory substitution; Sensory feedback
35.  Biosynthesis of the Common Polysaccharide Antigen of Pseudomonas aeruginosa PAO1: Characterization and Role of GDP-d-Rhamnose:GlcNAc/GalNAc-Diphosphate-Lipid α1,3-d-Rhamnosyltransferase WbpZ 
Journal of Bacteriology  2015;197(12):2012-2019.
The opportunistic pathogen Pseudomonas aeruginosa produces two major cell surface lipopolysaccharides, characterized by distinct O antigens, called common polysaccharide antigen (CPA) and O-specific antigen (OSA). CPA contains a polymer of d-rhamnose (d-Rha) in α1-2 and α1-3 linkages. Three putative glycosyltransferase genes, wbpX, wbpY, and wbpZ, are part of the CPA biosynthesis cluster. To characterize the enzymatic function of the wbpZ gene product, we chemically synthesized the donor substrate GDP-d-Rha and enzymatically synthesized GDP-d-[3H]Rha. Using nuclear magnetic resonance (NMR) spectroscopy, we showed that WbpZ transferred one d-Rha residue from GDP-d-Rha in α1-3 linkage to both GlcNAc- and GalNAc-diphosphate-lipid acceptor substrates. WbpZ is also capable of transferring d-mannose (d-Man) to these acceptors. Therefore, WbpZ has a relaxed specificity with respect to both acceptor and donor substrates. The diphosphate group of the acceptor, however, is required for activity. WbpZ does not require divalent metal ion for activity and exhibits an unusually high pH optimum of 9. WbpZ from PAO1 is therefore a GDP-d-Rha:GlcNAc/GalNAc-diphosphate-lipid α1,3-d-rhamnosyltransferase that has significant activity of GDP-d-Man:GlcNAc/GalNAc-diphosphate-lipid α1,3-d-mannosyltransferase. We used site-directed mutagenesis to replace the Asp residues of the two DXD motifs with Ala. Neither of the mutant constructs of wbpZ (D172A or D254A) could be used to rescue CPA biosynthesis in the ΔwbpZ knockout mutant in a complementation assay. This suggested that D172 and D254 are essential for WbpZ function. This work is the first detailed characterization study of a d-Rha-transferase and a critical step in the development of CPA synthesis inhibitors.
IMPORTANCE This is the first characterization of a d-rhamnosyltransferase and shows that it is essential in Pseudomonas aeruginosa for the synthesis of the common polysaccharide antigen.
PMCID: PMC4438205  PMID: 25845842
36.  Distinct Paths for Basic Amino Acid Export in Escherichia coli: YbjE (LysO) Mediates Export of l-Lysine 
Journal of Bacteriology  2015;197(12):2036-2047.
In Escherichia coli, argO encodes an exporter for l-arginine (Arg) and its toxic analogue canavanine (CAN), and its transcriptional activation and repression, by Arg and l-lysine (Lys), respectively, are mediated by the regulator ArgP. Accordingly argO and argP mutants are CAN supersensitive (CANss). We report the identification of ybjE as a gene encoding a predicted inner membrane protein that mediates export of Lys, and our results confirm the previous identification with a different approach of YbjE as a Lys exporter, reported by Ueda and coworkers (T. Ueda, Y. Nakai, Y. Gunji, R. Takikawa, and Y. Joe, U.S. patents 7,629,142 B2 [December 2009] and 8,383,363 B1 [February 2013] and European patent 1,664,318 B1 [September 2009]). ybjE was isolated as a multicopy suppressor of the CANss phenotype of a strain lacking ArgO. The absence of YbjE did not confer a CANss phenotype but instead conferred hypersensitivity to the lysine antimetabolite thialysine and led to growth inhibition by the dipeptide lysylalanine, which is associated with elevated cellular Lys content. YbjE overproduction resulted in Lys excretion and syntrophic cross-feeding of a Lys auxotroph. Constitutive overexpression of argO promoted Lys cross-feeding that is indicative of a latent Lys export potential of ArgO. Arg modestly repressed ybjE transcription in an ArgR-dependent manner, and ArgR displayed Arg-sensitive binding to the ybjE promoter region in vitro. Our studies suggest that the reciprocal repression of argO and ybjE, respectively, by Lys and Arg confers the specificity for basic amino acid export by distinct paths and that such cross-repression contributes to maintenance of cytoplasmic Arg/Lys balance. We propose that YbjE be redesignated LysO.
IMPORTANCE This work ascribes a lysine export function to the product of the ybjE gene of Escherichia coli, leading to a physiological scenario wherein two proteins, ArgO and YbjE, perform the task of separately exporting arginine and lysine, respectively, which is distinct from that seen for Corynebacterium glutamicum, where the ortholog of ArgO, LysE, mediates export of both arginine and lysine. Repression of argO transcription by lysine is thought to effect this separation. Accordingly, ArgO mediates lysine export when repression of its transcription by lysine is bypassed. Repression of ybjE transcription by arginine via the ArgR repressor, together with the lysine repression of argO effected by ArgP, is indicative of a mechanism of maintenance of arginine/lysine balance in E. coli.
PMCID: PMC4438211  PMID: 25845847
37.  Expression of Neurogenin 1 in mouse embryonic stem cells directs the differentiation of neuronal precursors and identifies unique patterns of down-stream gene expression 
Delineating the cascades of growth and transcription factor expression that shape the developing nervous system will improve our understanding of its molecular histogenesis and suggest strategies for cell replacement therapies. In the current investigation, we examined the ability of the proneural gene, Neurogenin1 (Neurog1; also Ngn1, Neurod3), to drive differentiation of pluripotent embryonic stem cells (ESC).
Transient expression of Neurog1 in ESC was sufficient to initiate neuronal differentiation, and produced neuronal subtypes reflecting its expression pattern in vivo. To begin to address the molecular mechanisms involved, we employed microarray analysis to identify potential down-stream targets of Neurog1 expressed at sequential stages of neuronal differentiation.
ESC expressing Neurogenin1 begin to withdraw from cycle and form precursors that differentiate exclusively into neurons. This work identifies unique patterns of gene expression following expression of Neurog1, including genes and signaling pathways involved in process outgrowth and cell migration, regional differentiation of the nervous system, and cell cycle.
PMCID: PMC4646168  PMID: 23288605
bHLH; BMP4; ES cell; FGF; microarray; neural crest cell; Neurod3; neural induction; neuronal differentiation; Ngn1; noggin; sonic hedgehog; Tet-on
38.  Palmitoylation and trafficking of GAD65 is impaired in a cellular model of Huntington disease 
The Biochemical journal  2012;442(1):39-48.
Huntington disease (HD) is caused by an expanded polyglutamine repeat in the huntingtin protein. GABAergic medium spiny neurons in the striatum are mostly affected in HD. However, mutant huntingtin (mhtt)-induced molecular changes in these neurons remain largely unknown. This study focuses on the effect of mhtt on the subcellular localization of glutamic acid decarboxylase (GAD), the enzyme responsible for synthesizing GABA. In this study, we report that the subcellular distribution of GAD is significantly altered in two neuronal cell lines that express either the N-terminus or full length mhtt. GAD65 is predominantly associated with Golgi membrane in cells expressing normal htt. However, it diffuses in the cytosol of cells expressing mhtt. As a result, vesicle-associated GAD65 trafficking is impaired. Since palmitoylation of GAD65 is required for GAD65 trafficking, we then demonstrate that palmitoylation of GAD65 is reduced in the HD model. Furthermore, overexpression of huntingtin-interacting protein 14, the enzyme responsible for palmitoylating GAD65 in vivo, could rescue GAD65 palmitoylation and vesicle-associated GAD65 trafficking. Taken together, our data support the idea that GAD65 palmitoylation is important for the delivery of GAD65 to inhibitory synapses and suggest that impairment of GAD65 palmitoylation by mhtt may lead to altered inhibitory neurotransmission in HD.
PMCID: PMC4646170  PMID: 22103299
huntingtin; protein palmitoylation; huntingtin-interacting protein 14; GAD65; axonal transport; protein aggregates
39.  Microenvironmental effects limit efficacy of thymoquinone treatment in a mouse model of ovarian cancer 
Molecular Cancer  2015;14:192.
Ovarian cancer is the most lethal gynecologic malignancy, with limited treatment options for chemoresistant disease. An important link between inflammation and peritoneal spread of ovarian cancer is NF-κB signaling. Thymoquinone (TQ) exerts multiple anti-tumorigenic cellular effects, including NF-κB inhibition. We aimed to investigate the therapeutic potential of TQ in an established murine syngeneic model of ovarian cancer.
ID8-NGL mouse ovarian cancer cells stably expressing an NF-κB reporter transgene were injected intra-peritoneally into C57BL/6 mice, and mice were treated with TQ or vehicle for 10 or 30 days. TQ was combined with the macrophage depleting drug, liposomal clodronate, in selected experiments. Effects on peritoneal tumor burden were measured by volume of ascites, number of peritoneal implants and mesenteric tumor mass. NF-κB reporter activity and markers of proliferation and apoptosis were measured in tumors and in confirmatory in vitro experiments. Protein or mRNA expression of M1 (anti-tumor) and M2 (pro-tumor) macrophage markers, and soluble cytokine profiles, were examined from harvested ascites fluid, peritoneal lavages and/or tumor sections. 2-tailed Mann–Whitney tests were used for measuring differences between groups in in vivo experiments.
Consistent with its effects in vitro, TQ reduced proliferation and increased apoptosis in ID8-NGL tumors after 10 and 30 day treatment. Prolonged TQ treatment did not significantly alter tumor number or mass compared to vehicle, but rather exerted an overall deleterious effect by stimulating ascites formation. Increased ascites was accompanied by elevated NF-κB activity in tumors and macrophages, increased pro-tumor M2 macrophages and expression of pro-tumorigenic soluble factors such as VEGF in ascites fluid, and increased tumor infiltration of M2 macrophages. In contrast, a 10 day exposure to TQ produced no ascites, and reduced tumor NF-κB activity, M2 macrophages and soluble VEGF levels. Peritoneal macrophage depletion by clodronate significantly reduced tumor burden. However, TQ-stimulated ascites was further enhanced by co-treatment with clodronate, with macrophages present overwhelmingly of the M2 phenotype.
Our findings show that pro-tumorigenic microenvironmental effects limited the efficacy of TQ in a syngeneic mouse model of ovarian cancer, and provide caution regarding its potential use in clinical trials in ovarian cancer patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s12943-015-0463-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4640396  PMID: 26552746
NF-κB activity; Ovarian cancer; Syngeneic mouse model; Macrophages; Thymoquinone; VEGF
40.  Detection and Biological Activities of Carboxyethylpyrrole Ethanolamine Phospholipids (CEP-EPs) 
Chemical Research in Toxicology  2014;27(12):2015-2022.
Oxidation of docosahexaenoate phospholipids produces 4-hydroxy-7-oxo-hept-5-eonyl phospholipids (HOHA-PLs) that react with protein lysyl ε-amino residues to generate 2-ω-carboxyethylpyrrole (CEP) derivatives, endogenous factors that induce angiogenesis in the retina and tumors. It seemed likely, but remained unproven, that HOHA-PLs react with ethanolamine phospholipids (EPs) in vivo to generate CEP-EPs. We now show that CEP-EPs are present in human blood at 4.6-fold higher levels in age-related macular degeneration plasma than in normal plasma. We also show that CEP-EPs are pro-angiogenic, inducing tube formation by human umbilical vein endothelial cells by activating Toll-like receptor 2. CEP-EP levels may be a useful biomarker for clinical assessment of AMD risk and CEP-associated tumor progression and a tool for monitoring the efficacy of therapeutic interventions.
PMCID: PMC4269404  PMID: 25380349
41.  Use mouse biobanks or lose them 
Nature  2015;522(7555):151-153.
Now that genetic engineering of mice is so easy, centralized repositories are essential, argue Kent Lloyd and colleagues.
PMCID: PMC4636083  PMID: 26062496
43.  Peritoneal Resting with Heparinized Lavage Reverses Peritoneal Type I Membrane Failure. A Comparative Study of the Resting Effects on Normal Membranes 
♦ Background: Ultrafiltration failure (UFF) is a serious complication of long-term peritoneal dialysis (PD). Peritoneal rest (PR) has been demonstrated as a valid treatment to reverse the functional changes that occur in UFF. The effects of PR on a normally functioning human peritoneum are unknown but are expected to be neutral. Our hypothesis was that PR positively modifies peritoneal function in patients with UFF, in contrast to the absence of effects when PR is applied under normal conditions.
♦ Patients and Methods: We studied 84 PR periods, comparing 35 patients with UFF and 49 controls (resting for abdominal surgery with temporary discontinuation of PD). We analyzed peritoneal transport pre-PR and post-PR by calculating the mass transfer coefficients of creatinine (Cr-MTAC), the dialysate/plasma creatinine ratio (D/P Cr) and the ultrafiltration (UF).
♦ Results: Baseline data was similar for the 2 groups, although the UFF group had a longer median time in PD (39 [18 - 60] vs 10 [5 - 23] months; p = 0.00001). Peritoneal rest induced a decrease in D/P Cr, Cr-MTAC and an increase in UF capacity in the UFF group (p = 0.0001, p = 0.004 and p = 0.001, respectively), without causing changes in the control group. Peritoneal rest in patients with more than 6 months of UFF was not able to reduce peritoneal solute transport or improve UF capacity. Response to PR did not differ among UFF patients with or without a previous history of peritonitis. Peritoneal rest enabled patients with UFF to continue on PD for a median time of 23 months (range, 13 - 46 months).
♦ Conclusions: Peritoneal rest induces functional changes in patients with UFF but not in those with no functional abnormalities. This demonstrates that PR works only when abnormal but reversible functional conditions are present. However, the effect is highly dependent on how early PR is applied.
PMCID: PMC4269495  PMID: 25292401
Peritoneal transport; peritoneal dialysis; peritoneal rest; heparin; ultrafiltration failure
44.  Utilization of Viral Molecular Diagnostics Among Children Hospitalized With Community Acquired Pneumonia 
Hospital pediatrics  2014;4(6):372-376.
To examine whether results of a polymerase chain reaction–based respiratory viral panel (RVP) are associated with changes in antibiotic use or differential clinical outcomes among children hospitalized with pneumonia.
We retrospectively identified otherwise healthy children hospitalized over a 3-year period at a single institution with community-acquired pneumonia who had an RVP performed within 24 hours of admission. We examined associations between RVP results and clinical outcomes as well as management decisions including initiation and duration of intravenous antibiotics.
Among 202 children, a positive RVP (n = 127, 63%) was associated with a more complicated clinical course, although this was due largely to more severe disease seen in younger children and those with respiratory syncytial virus (n = 38, 30% of positive detections). Detection of a virus did not influence antibiotic therapy. Included children were younger and had more severe illness than children hospitalized with pneumonia at the same institution without an RVP obtained.
In our study, only respiratory syncytial virus was associated with a more severe clinical course compared with RVP-negative children. Regardless of the virus detected, RVP positivity did not influence antibiotic usage. However, RVP use focused primarily on children with severe pneumonia. Whether similar testing influences management decisions among children with less severe illness deserves further study.
PMCID: PMC4269521  PMID: 25362079
molecular diagnostics; pneumonia; length of stay; pediatric; outcomes; RSV
46.  An approach to the immunophenotypic features of circulating CD4+NKG2D+ T cells in invasive cervical carcinoma 
NKG2D, an activating immunoreceptor, is primarily restricted to NK cells and CD8+ T cells. The existence of an atypical cytotoxic CD4+NKG2D+ T cell population has also been found in patients with autoimmune dysfunctions. Nonetheless, contradictory evidence has categorized this population with a regulatory rather than cytotoxic role in other situations. These confounding data have led to the proposal that two distinct CD4+NKG2D+ T cell subsets might exist. The immune response elicited in cervical cancer has been characterized by apparent contradictions concerning the role that T cells, in particular T-helper cells, might be playing in the control of the tumor growth. Interestingly, we recently reported a substantial increase in the frequency of CD4+NKG2D+ T cells in patients with cervical intraepithelial neoplasia grade-1. However, whether this particular population is also found in patients with more advanced cervical lesions or whether they express a distinctive phenotype remains still to be clarified. In this urgent study, we focused our attention on the immunophenotypic characterization of CD4+NKG2D+ T cells in patients with well-established cervical carcinoma and revealed the existence of at least two separate CD4+NKG2D+ T cell subsets defined by the co-expression or absence of CD28.
Patients with diagnosis of invasive cervical carcinoma were enrolled in the study. A group of healthy individuals was also included. Multicolor flow cytometry was used for exploration of TCR alpha/beta, CD28, CD158b, CD45RO, HLA-DR, CD161, and CD107a. A Luminex-based cytokine kit was used to quantify the levels of pro- and anti-inflammatory cytokines. We found an increased percentage of CD4+NKG2D+ T cells in patients with cervical cancer when compared with controls. Accordingly with an increase of CD4+NKG2D+ T cells, we found decreased CD28 expression. The activating or degranulation markers HLA-DR, CD161, and CD107a were heterogeneously expressed. The levels of IL-1beta, IL-2, TNF-alpha, and IL-10 were negatively correlated with the percentages of CD4+NKG2D+ T cells in patients with cervical carcinoma.
Taken together, our results reveal the existence of two separate CD4+NKG2D+ T cell subsets defined by the co-expression or absence of CD28, the latter more likely to be present in patients with cervical cancer.
PMCID: PMC4615321  PMID: 26486970
NKG2D; CD4+NKG2D+ T cells; Cervical carcinoma
47.  Projection of participant recruitment to primary care research: a qualitative study 
Trials  2015;16:473.
Recruitment to clinical trials remains a challenge, particularly in primary care settings. Initial projections of participant recruitment need to be as accurate as possible in order to avoid the financial, clinical and ethical costs of trial extensions or failures. However, estimation of recruitment rates is challenging and often poorly executed, if attempted at all. We used qualitative methods to explore the experiences and views of researchers on the planning of recruitment in this setting.
Participants had registered accrual to a UK-based primary care research study between April 2009 and March 2012. We conducted nine interviews with chief investigators or study managers, using a semi-structured topic guide. Analysis was conducted using the framework approach.
Three themes are presented: 1) the factors affecting recruitment rates, 2) the use of planning techniques, and 3) influences on poor estimation. 1) A large number of factors affecting recruitment rates were discussed, including those relating to the study protocol, the clinical setting and the research setting. Use of targeted mail-outs to invite apparently eligible individuals to participate was preferred in order to eliminate some of the uncertainty in the recruitment rate associated with opportunistic clinician referrals. 2) The importance of pilot work was stressed. We identified significant uncertainty as to how best to schedule trial timelines to maximise efficiency. 3) Several potential sources of bias involved in the estimation of recruitment rates were explored and framed as technological, psychological or political factors.
We found a large number of factors that interviewees felt impact recruitment rates to primary care research and highlighted the complexity of realistic estimation. Suitable early planning of the recruitment process is essential, and there may be potential to improve the projection of trial timelines by reducing biases involved in the process. Further research is needed to develop formal approaches that would be suitable for use in this setting.
Electronic supplementary material
The online version of this article (doi:10.1186/s13063-015-1002-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4615323  PMID: 26482231
Qualitative research; Primary care; General practice; Participant recruitment; Recruitment projection; Planning; randomised controlled trials; Clinical trials
48.  Factors that determine local control with gamma knife radiosurgery: The role of primary histology† 
Journal of radiosurgery and SBRT  2015;3(4):281-286.
Stereotactic radiosurgery for the treatment of brain metastases is commonly delivered without regard to primary cancer histology. This study sought to determine if the primary site of origin for brain metastases affected the propensity for local failure.
A total of 83 patients with 200 brain metastases were examined retrospectively for predictors of infield failure. Tumor, patient, and treatment characteristics were analyzed including primary tumor histology, radiosurgical dose and age. Cox proportional hazards models, univariate and multivariate analyses were used to identify predictors of local failure.
Freedom from local failure for the entire population was 83% and 65% at 6 and 12 months, respectively. Multivariate analysis revealed that breast cancer brain metastases have a significantly lower risk of local failure than melanoma (HR = 0.31, p< 0.001). Additionally, multivariate analysis revealed that increasing dose lowered risk for local failure (HR = 0.87, p<0.001).
Melanoma histology leads to a higher rate of local failure. Higher prescription dose results in higher incidence of local control.
PMCID: PMC4605606  PMID: 26478823
brain metastasis; gamma knife; stereotactic radiosurgery; radiosensitivity; histology; predictive outcomes; breast neoplasms; proportional hazards models; retrospective studies; multivariate analysis; brain neoplasms; melanoma
49.  Heightened Delta Power during Slow-Wave-Sleep in Patients with Rett Syndrome Associated with Poor Sleep Efficiency 
PLoS ONE  2015;10(10):e0138113.
Sleep problems are commonly reported in Rett syndrome (RTT); however the electroencephalographic (EEG) biomarkers underlying sleep dysfunction are poorly understood. The aim of this study was to analyze the temporal evolution of quantitative EEG (qEEG) biomarkers in overnight EEGs recorded from girls (2–9 yrs. old) diagnosed with RTT using a non-traditional automated protocol. In this study, EEG spectral analysis identified high delta power cycles representing slow wave sleep (SWS) in 8–9h overnight sleep EEGs from the frontal, central and occipital leads (AP axis), comparing age-matched girls with and without RTT. Automated algorithms quantitated the area under the curve (AUC) within identified SWS cycles for each spectral frequency wave form. Both age-matched RTT and control EEGs showed similar increasing trends for recorded delta wave power in the EEG leads along the antero-posterior (AP). RTT EEGs had significantly fewer numbers of SWS sleep cycles; therefore, the overall time spent in SWS was also significantly lower in RTT. In contrast, the AUC for delta power within each SWS cycle was significantly heightened in RTT and remained heightened over consecutive cycles unlike control EEGs that showed an overnight decrement of delta power in consecutive cycles. Gamma wave power associated with these SWS cycles was similar to controls. However, the negative correlation of gamma power with age (r = -.59; p<0.01) detected in controls (2–5 yrs. vs. 6–9 yrs.) was lost in RTT. Poor % SWS (i.e., time spent in SWS overnight) in RTT was also driven by the younger age-group. Incidence of seizures in RTT was associated with significantly lower number of SWS cycles. Therefore, qEEG biomarkers of SWS in RTT evolved temporally and correlated significantly with clinical severity.
PMCID: PMC4596813  PMID: 26444000
50.  Home treatment teams and facilitated discharge from psychiatric hospital 
There has been little research into the facilitated discharge (FD) function of Home Treatment Teams (HTTs). We aimed to explore and describe the prevalence and associations of FD and to estimate its effects on bed days during the index admission (length of stay corrected for ward leave) and on readmission.
Descriptive and regression analyses of data collected by South London and Maudsley NHS Foundation Trust on discharges from its general psychiatric wards, with multiple imputation of missing covariate values.
Overall, 29% of our sample of 7891 hospital admissions involved a FD. FD was associated with female gender, diagnosis of a severe mental illness, previous home treatment, having a longer previous admission, neither being discharged to a new address nor to a care home, having no other community team and having HoNOS item scores consistent with an active depressive or psychotic mental illness. In the regression analysis, FD was associated with 4.0 fewer bed days (95% confidence interval −6.7 to −1.3; p = 0.0004). There was no effect on readmission.
Our analysis provides some support for the effectiveness of FD in slightly reducing the time spent in hospital and suggests that this may be achieved without increasing the rate of readmission. Further studies in this area are important, especially given existing research that suggests that the introduction of HTTs in England and Wales was associated with little or no change in service utilisation.
PMCID: PMC4594043  PMID: 24978136
Community mental health services; home care services; length of stay; mental disorders

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