Most children diagnosed with community-acquired pneumonia (CAP) are treated in the outpatient setting. The objective of this study was to determine the comparative clinical effectiveness of beta-lactam monotherapy and macrolide monotherapy in this population.
Children, 1–18 years old, with a clinical diagnosis of CAP at an outpatient practice affiliated (n=71) with Geisinger Health System during January 1, 2008 to January 31, 2010 were eligible. The primary exposure was receipt of beta-lactam or macrolide monotherapy. The primary outcome was treatment failure defined as change in antibiotic prescription within 14 days of the initial pneumonia diagnosis. Propensity scores were used to determine the likelihood of receiving macrolide monotherapy. Treatment groups were matched 1:1, based on propensity score, age group and asthma status. Multivariable conditional logistic regression models estimated the association between macrolide monotherapy and treatment failures.
Of 1,999 children with CAP, 1,164 were matched. In the matched cohorts, 24% of children had asthma. Patients who received macrolide monotherapy had no statistical difference in treatment failure regardless of age when compared with patients who received beta-lactam monotherapy.
Our findings suggest that children diagnosed with CAP in the outpatient setting and treated with beta-lactam or macrolide monotherapy have the same likelihood to fail treatment regardless of age.
pneumonia; child; pediatric
Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns.
•The 3D genome topology of four somatic cell types varies greatly and differs from ESCs•The 3D genomes of iPSCs from different founders and of ESCs are overall highly similar•Early-passage iPSCs show subtle but reproducible founder-dependent 3D differences•The distinctive topology features of iPSCs are acquired during reprogramming
Krijger et al. report that the reprogramming of four somatic cell types with highly distinct 3D genomes results in pluripotent cells with largely identical, ESC-like, genome conformations carrying founder-dependent topological hallmarks. The latter are not remnants of somatic chromosome topologies but are acquired during reprogramming in a cell-of-origin-dependent manner.
Characterizing the mechanisms underlying follicle development in the ovary is crucial to understanding female fertility and is an area of increasing research interest. The RNA binding protein Musashi is essential for post-transcriptional regulation of oocyte maturation in Xenopus and is expressed during ovarian development in Drosophila. In mammals Musashi is important for spermatogenesis and male fertility, but its role in the ovary has yet to be characterized. In this study we determined the expression of mammalian Musashi proteins MSI1 and MSI2 during mouse folliculogenesis, and through the use of a MSI2-specific knockout mouse model we identified that MSI2 is essential for normal follicle development. Time-course characterization of MSI1 and MSI2 revealed distinct differences in steady-state mRNA levels and protein expression/localization at important developmental time-points during folliculogenesis. Using a gene-trap mouse model that inactivates Msi2, we observed a significant decrease in ovarian mass, and change in follicle-stage composition due to developmental blocking of antral stage follicles and pre-antral follicle loss through atresia. We also confirmed that hormonally stimulated Msi2-deficient mice produce significantly fewer MII oocytes (60.9% less than controls, p < 0.05). Furthermore, the majority of these oocytes are of poor viability (62.2% non-viable/apoptotic, p < 0.05), which causes a reduction in female fertility evidenced by decreased litter size in Msi2-deficient animals (33.1% reduction to controls, p < 0.05). Our findings indicate that MSI1 and MSI2 display distinct expression profiles during mammalian folliculogenesis and that MSI2 is required for pre-antral follicle development.
fertility; Musashi; oocyte; granulosa cell; oogenesis
Transcription-factor-induced somatic cell conversions are highly relevant for both basic and clinical research yet their mechanism is not fully understood and it is unclear whether they reflect normal differentiation processes. Here we show that during pre-B-cell-to-macrophage transdifferentiation, C/EBPα binds to two types of myeloid enhancers in B cells: pre-existing enhancers that are bound by PU.1, providing a platform for incoming C/EBPα; and de novo enhancers that are targeted by C/EBPα, acting as a pioneer factor for subsequent binding by PU.1. The order of factor binding dictates the upregulation kinetics of nearby genes. Pre-existing enhancers are broadly active throughout the hematopoietic lineage tree, including B cells. In contrast, de novo enhancers are silent in most cell types except in myeloid cells where they become activated by C/EBP factors. Our data suggest that C/EBPα recapitulates physiological developmental processes by short-circuiting two macrophage enhancer pathways in pre-B cells.
•C/EBPα activates two classes of prospective myeloid enhancers in B cells•Pre-existing enhancers are bound by PU.1 and become hyper-activated by C/EBPα•C/EBPα acts as a pioneer factor with delayed kinetics on de novo enhancers•The two types of enhancers direct myeloid cell fate in B cells and hematopoiesis
In this study, Graf and colleagues show that, during B-cell-to-macrophage transdifferentiation, C/EBPα activates two types of myeloid enhancers in B cells. The two enhancer types are differentially activated, dictating gene expression levels of macrophage genes during the conversion of B cells into macrophages and normal hematopoiesis.
Electronic medical records and insurance claims data from the Geisinger Health System were examined to assess the real-world healthcare costs of being overweight or obese at different glycemic stages, including normal glycemia, pre-diabetes (PreD), and type 2 diabetes (T2D).
The medical history of the sample subjects was segmented into different glycemic stages via diagnosis codes, glycosylated hemoglobin A1c or fasting plasma glucose laboratory results, and use of antidiabetic drugs. Healthcare resource utilization captured by the claims and associated costs (in 2013 values) were examined for each glycemic stage. The association between costs and body mass index (BMI) was estimated by regressions, and adjusted for sociodemographics. We predicted the adjusted incremental annual costs associated with high BMI, relative to normal BMI (18.5–24.9 kg/m2).
We identified 48,344 adults in normal glycemic stage, 3,085 in the PreD stage, and 9,526 in the T2D stage (mean age 46, 58, and 60 years, respectively; mean BMI 29, 32, and 33 kg/m2, respectively). The adjusted incremental annual costs associated with high BMI relative to normal BMI ranged from $336 for overweight (25–29.9 kg/m2) to $1,850 for class III obesity (≥40 kg/m2) during normal glycemic stage; were only significant for class III ($2,434) during the PreD stage; and ranged from $1,139 for overweight to $4,649 for class III during the T2D stage (all p < 0.05).
Positive associations between healthcare costs and BMI levels were observed within each glycemic stage. Management of body weight is important in reducing the overall healthcare costs, especially for subjects with PreD or T2D.
MicroRNAs (miRNAs) exert negative effects on gene expression and influence cell lineage choice during hematopoiesis. C/EBPa-induced pre-B cell-to-macrophage transdifferentiation provides an excellent model to investigate the contribution of miRNAs to hematopoietic cell identity, especially because the two cell types involved fall into separate lymphoid and myeloid branches. In this process, efficient repression of the B cell-specific program is essential to ensure transdifferentation and macrophage function. miRNA profiling revealed that upregulation of miRNAs is highly predominant compared with downregulation and that C/EBPa directly regulates several upregulated miRNAs. We also determined that miRNA 34a (miR-34a) and miR-223 sharply accelerate C/EBPa-mediated transdifferentiation, whereas their depletion delays this process. These two miRNAs affect the transdifferentiation efficiency and activity of macrophages, including their lipopolysaccharide (LPS)-dependent inflammatory response. miR-34a and miR-223 directly target and downregulate the lymphoid transcription factor Lef1, whose ectopic expression delays transdifferentiation to an extent similar to that seen with miR-34a and miR-223 depletion. In addition, ectopic introduction of Lef1 in macrophages causes upregulation of B cell markers, including CD19, Pax5, and Ikzf3. Our report demonstrates the importance of these miRNAs in ensuring the erasure of key B cell transcription factors, such as Lef1, and reinforces the notion of their essential role in fine-tuning the control required for establishing cell identity.
The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is lengthy and inefficient. The development of a reprogramming system that allows rapid and synchronous reprogramming to pluripotency is imperative for understanding the mechanism of iPSC formation and for future therapeutic applications. We have recently reported that a short expression in mouse primary B cells of the transcription factor C/EBPα before the induction of pluripotency factors increases the iPSC reprogramming efficiency >100-fold, involving 95% of the cells within a week. Here we present a dataset containing the time course of gene expression during this process as determined by microarray and RNA-seq techniques.
At the 2011 American College of Medical Informatics (ACMI) Winter Symposium we studied the overlap between health IT and economics and what leading healthcare delivery organizations are achieving today using IT that might offer paths for the nation to follow for using health IT in healthcare reform. We recognized that health IT by itself can improve health value, but its main contribution to health value may be that it can make possible new care delivery models to achieve much larger value. Health IT is a critically important enabler to fundamental healthcare system changes that may be a way out of our current, severe problem of rising costs and national deficit. We review the current state of healthcare costs, federal health IT stimulus programs, and experiences of several leading organizations, and offer a model for how health IT fits into our health economic future.
Electronic Health Records/economics Health care Reform/trends*; cost-benefit analysis; information Systems/economics*; medical records systems; Computerized/economics; Developing/using computerized provider order entry; Natural-language processing; systems to support and improve diagnostic accuracy; other specific EHR applications (results review); patient safety; decision support; data exchange; system implementation and management issues; improving the education and skills training of health professionals; Developing/using clinical decision support (other than diagnostic) and guideline systems; Measuring/improving patient safety and reducing medical errors; clinical research informatics; information Retrieval; Collaborative technologies; methods for integration of information from disparate sources; Demonstrating return on IT investment; distributed systems; distributed systems
The methylcytosine hydroxylase Tet2 has been implicated in hematopoietic differentiation and the formation of myeloid malignancies when mutated. An ideal system to study the role of Tet2 in myelopoeisis is C/EBPa induced transdifferentiation of pre-B cells into macrophages. Here we found that C/EBPa binds to upstream regions of Tet2 and that the gene becomes activated. Tet2 knockdowns impaired the upregulation of macrophage markers as well as phagocytic capacity, suggesting that the enzyme is required for both early and late stage myeloid differentiation. A slightly weaker effect was seen in primary cells with a Tet2 ablation. Expression arrays of transdifferentiating cells with Tet2 knockdowns permitted the identification of a small subset of myeloid genes whose upregulation was blunted. Activation of these target genes was accompanied by rapid increases of promoter hydroxy-methylation. Our observations indicate that Tet2 helps C/EBPa rapidly de-repress myeloid genes during the conversion of pre-B cells into macrophages.
Epigenetics; Transdifferentiation; DNA methylation; DNA hydroxymethylation
National quality indicators show little change in the overuse of antibiotics for uncomplicated acute bronchitis. We compared the impact of two decision support strategies on antibiotic treatment of uncomplicated acute bronchitis.
We conducted a three-arm, cluster-randomized trial among 33 primary care practices belonging to an integrated health care system in central Pennsylvania. The printed intervention arm (n=11 practices) received decision support for acute cough illness through a print-based strategy, the computerized intervention group (n=11) received decision support through an electronic medical record-based strategy, and third group of practices (n=11) served as the control arm. Both intervention groups also received provider education and feedback on prescribing practices, and patient education brochures at check-in. Antibiotic prescription rates for uncomplicated acute bronchitis in the winter period (October 2009 – March 2010) following introduction of the intervention were compared with the previous three winter periods in an intent-to-treat analysis.
Compared with the baseline period, the percentage of adolescents and adults prescribed antibiotics during the intervention period decreased at the printed (from 80.0% to 68.3%) and computerized intervention sites (from 74.0% to 60.7%), but increased slightly at the control sites (from 72.5% to 74.3%). After controlling for patient and provider characteristics, and clustering of observations by provider and practice site, the differences for the intervention groups were statistically significant from control (control vs. printed P=0.003; control vs. computerized P=0.014) but no among themselves (printed vs. computerized P=0.67). Changes in total visits, proportion diagnosed as uncomplicated acute bronchitis and thirty-day return visit rates were similar between study groups.
Implementation of a decision support strategy for acute bronchitis can help reduce overuse of antibiotics in primary care settings. The impact of printed and computerized strategies for providing decision support was equivalent. The study was registered with Clinical Trials.Gov prior to enrolling patients (NCT00981994).
Sphingosine 1-phosphate guides the elongation of megakaryocytic proplatelet extensions and triggers their shedding.
Millions of platelets are produced each hour by bone marrow (BM) megakaryocytes (MKs). MKs extend transendothelial proplatelet (PP) extensions into BM sinusoids and shed new platelets into the blood. The mechanisms that control platelet generation remain incompletely understood. Using conditional mutants and intravital multiphoton microscopy, we show here that the lipid mediator sphingosine 1-phosphate (S1P) serves as a critical directional cue guiding the elongation of megakaryocytic PP extensions from the interstitium into BM sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. Collectively, our findings uncover a novel function of the S1P–S1pr1 axis as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia.
B lymphopoiesis is the result of several cell-commitment, lineage-choice, and differentiation processes. Every differentiation step is characterized by the activation of a new, lineage-specific, genetic program and the extinction of the previous one. To date, the central role of specific transcription factors in positively regulating these distinct differentiation processes to acquire a B cell–specific genetic program is well established. However, the existence of specific transcriptional repressors responsible for the silencing of lineage inappropriate genes remains elusive. Here we addressed the molecular mechanism behind repression of non-lymphoid genes in B cells. We report that the histone deacetylase HDAC7 was highly expressed in pre-B cells but dramatically down-regulated during cellular lineage conversion to macrophages. Microarray analysis demonstrated that HDAC7 re-expression interfered with the acquisition of the gene transcriptional program characteristic of macrophages during cell transdifferentiation; the presence of HDAC7 blocked the induction of key genes for macrophage function, such as immune, inflammatory, and defense response, cellular response to infections, positive regulation of cytokines production, and phagocytosis. Moreover, re-introduction of HDAC7 suppressed crucial functions of macrophages, such as the ability to phagocytose bacteria and to respond to endotoxin by expressing major pro-inflammatory cytokines. To gain insight into the molecular mechanisms mediating HDAC7 repression in pre-B cells, we undertook co-immunoprecipitation and chromatin immunoprecipitation experimental approaches. We found that HDAC7 specifically interacted with the transcription factor MEF2C in pre-B cells and was recruited to MEF2 binding sites located at the promoters of genes critical for macrophage function. Thus, in B cells HDAC7 is a transcriptional repressor of undesirable genes. Our findings uncover a novel role for HDAC7 in maintaining the identity of a particular cell type by silencing lineage-inappropriate genes.
Through the hematopoietic system, all the distinct mature blood cell types are generated, thereby constituting one of the best-studied paradigms for cell lineage commitment and differentiation in biology. B lymphocytes are generated through several cell-commitment, lineage-choice, and differentiation processes. To date, the central role of lineage-specific transcription factors in positively regulating these distinct developmental steps is well established. However, in the absence of proper transcriptional repression, an “adolescent cell” will never be able to reach its “adulthood identity,” having a potential impact in the development of hematological malignancies. In this article, we examined the molecular mechanism responsible for the gene silencing of lineage undesirable genes in B cell precursors and uncovered the role played in this process by the histone deacetylase HDAC7. We show that HDAC7 is expressed in B cell precursors where it interacts with the transcription factor MEF2C and is recruited to the promoters of non-B cell genes. While HDAC7 is down-regulated during the lineage conversion of pre-B cells into macrophages, re-expression of HDAC7 interferes with both the acquisition of the myeloid gene transcriptional program and macrophage-specific cell functions. We therefore have identified a novel lineage-specific transcriptional repressor in the hematopoietic system.
Background and purpose
Metaphyseal anchored short-stem hip implants were designed to improve load transmission and preserve femoral bone stock. Until now, only few outcome data have been available and migration studies are one of the few ways of obtaining data that are predictive of implant survival. We therefore evaluated a metaphyseal anchored short-stem hip implant by Ein Bild Roentgen Analyse femoral component analysis (EBRA-FCA).
Patients and methods
First, the EBRA-FCA method was validated for the short-stem hip implant. Then 80 of the first 100 consecutive implants were evaluated after at least 2 years. Clinical assessment was performed using the WOMAC and the UCLA score.
After 2.7 (2.0–4.2), years none of the implants had been revised and by that time the stems had subsided by a mean of 0.7 mm (SD 1.8) (95% CI: 0.3–1.1). Of the 80 implants, 78 were stable after 2 years, with 74 being primary stable and 4 showing secondary stabilization after initial subsidence. Continuous migration was seen in only 2 patients. The clinical outcome showed good results with a mean WOMAC of 11 (SD 13) and a mean UCLA score of 7.3 (SD 2.0). [OK?]
The metaphyseal anchored short-stem hip implant showed good functional results and a high degree of stability after 2 years. The outcome is comparable to that of clinically proven conventional hip implants and if the results are confirmed by long-term studies, short-stem hip arthroplasty might be an alternative for young patients requiring hip replacement.
Transcription factor-induced lineage reprogramming or transdifferentiation experiments are essential for understanding the plasticity of differentiated cells. These experiments helped to define the specific role of transcription factors in conferring cell identity and played a key role in the development of the regenerative medicine field. We here investigated the acquisition of DNA methylation changes during C/EBPα-induced pre-B cell to macrophage transdifferentiation. Unexpectedly, cell lineage conversion occurred without significant changes in DNA methylation not only in key B cell- and macrophage-specific genes but also throughout the entire set of genes differentially methylated between the two parental cell types. In contrast, active and repressive histone modification marks changed according to the expression levels of these genes. We also demonstrated that C/EBPα and RNA Pol II are associated with the methylated promoters of macrophage-specific genes in reprogrammed macrophages without inducing methylation changes. Our findings not only provide insights about the extent and hierarchy of epigenetic events in pre-B cell to macrophage transdifferentiation but also show an important difference to reprogramming towards pluripotency where promoter DNA demethylation plays a pivotal role.
One step closer to on-demand platelet production.
The era of induced pluripotent stem (iPS) cells carries with it the promise of virtually unlimited sources of autologous cells for regenerative medicine. However, efficiently differentiating iPS cells into fully functional mature cell types remains challenging. A new study reporting the formation of fully functional platelets from human iPS (hiPS) cells improves upon recent efforts to generate this enucleated cell type, which remains in high demand for therapeutic transfusions. Notably, their lack of nucleus renders platelets unable to retain the pluripotent or tumorigenic properties of iPS cells.
In this paper, we describe the unexpected outgrowth of B lineage cells from PU.1−/− fetal liver cultures. The cells express all early B cell genes tested, including the putative PU.1 target genes IL-7R and EBF but not B220, and can produce immunoglobulin M. However, we observed a delay in the PU.1−/− B cell outgrowth and reduced precursor frequencies, indicating that although PU.1 is not strictly required for B cell commitment, it facilitates B cell development. We also ablated PU.1 in CD19-expressing B lineage cells in vivo, using a Cre-lox approach that allows them to be tracked. PU.1 excision resulted in a shift from B-2 cells to B-1–like cells, which dramatically increased with the age of the mice. Our data indicate that this shift is predominantly caused by a B-2 to B-1 cell reprogramming. Furthermore, we found that B-2 cells express substantially more PU.1 than B-1 cells, which is consistent with the idea that maintenance of the B-2 cell phenotype requires relatively high levels of PU.1, but B-1 cells require little.
Podocalyxin is a CD34-related sialomucin that is expressed at high levels by podocytes, and also by mesothelial cells, vascular endothelia, platelets, and hematopoietic stem cells. To elucidate the function of podocalyxin, we generated podocalyxin-deficient (podxl−/−) mice by homologous recombination. Null mice exhibit profound defects in kidney development and die within 24 hours of birth with anuric renal failure. Although podocytes are present in the glomeruli of the podxl−/− mice, they fail to form foot processes and slit diaphragms and instead exhibit cell–cell junctional complexes (tight and adherens junctions). The corresponding reduction in permeable, glomerular filtration surface area presumably leads to the observed block in urine production. In addition, podxl−/− mice frequently display herniation of the gut (omphalocele), suggesting that podocalyxin may be required for retraction of the gut from the umbilical cord during development. Hematopoietic and vascular endothelial cells develop normally in the podocalyxin-deficient mice, possibly through functional compensation by other sialomucins (such as CD34). Our results provide the first example of an essential role for a sialomucin in development and suggest that defects in podocalyxin could play a role in podocyte dysfunction in renal failure and omphalocele in humans.
sialomucin; umbilical hernia; podocyte; hematopoiesis; vascular endothelium
The sequenced gammaherpesviruses each contain a single viral bcl-2 homolog (v-bcl-2) which may encode a protein that functions in preventing the apoptotic death of virus-infected cells. Epstein-Barr virus (EBV), a gammaherpesvirus associated with several lymphoid and epithelial malignancies, encodes the v-Bcl-2 homolog BHRF1. In this report the previously uncharacterized BALF1 open reading frame in EBV is identified as having significant sequence similarity to other v-bcl-2 homologs and cellular bcl-2. Transfection of cells with a BALF1 cDNA conferred apoptosis resistance. Furthermore, a recombinant green fluorescent protein-BALF1 fusion protein suppressed apoptosis and associated with Bax and Bak. These results indicate that EBV encodes a second functional v-bcl-2.
MEP21 is an avian antigen specifically expressed on the surface of Myb-Ets–transformed multipotent hematopoietic precursors (MEPs) and of normal thrombocytes. Using nanoelectrospray tandem mass spectrometry, we have sequenced and subsequently cloned the MEP21 cDNA and named the gene thrombomucin as it encodes a 571–amino acid protein with an extracellular domain typical of the mucin family of proteoglycans. Thrombomucin is distantly related to CD34, the best characterized and most used human hematopoietic stem cell marker. It is also highly homologous in its transmembrane/intracellular domain to podocalyxinlike protein–1, a rabbit cell surface glycoprotein of kidney podocytes.
Single cell analysis of yolk sac cells from 3-d-old chick embryos revealed that thrombomucin is expressed on the surface of both lineage-restricted and multipotent progenitors. In the bone marrow, thrombomucin is also expressed on mono- and multipotent progenitors, showing an overlapping but distinct expression pattern from that of the receptor-type stem cell marker c-kit. These observations strengthen the notion that the Myb-Ets oncoprotein can induce the proliferation of thrombomucin-positive hematopoietic progenitors that have retained the capacity to differentiate along multiple lineages. They also suggest that thrombomucin and CD34 form a family of stem cell–specific proteins with possibly overlapping functions in early hematopoietic progenitors.
Several laboratories have reported the reprogramming of mouse and human fibroblasts into pluripotent cells, using retroviruses carrying the Oct4, Sox2, Klf4, and c-Myc transcription factor genes. In these experiments the frequency of reprogramming was lower than 0.1% of the infected cells, raising the possibility that additional events are required to induce reprogramming, such as activation of genes triggered by retroviral insertions. We have therefore determined by ligation-mediated polymerase chain reaction (LM-PCR) the retroviral insertion sites in six induced pluripotent stem (iPS) cell clones derived from mouse fibroblasts. Seventy-nine insertion sites were assigned to a single mouse genome location. Thirty-five of these mapped to gene transcription units, whereas 29 insertions landed within 10 kilobases of transcription start sites. No common insertion site was detected among the iPS clones studied. Moreover, bioinformatics analyses revealed no enrichment of a specific gene function, network, or pathway among genes targeted by retroviral insertions. We conclude that Oct4, Sox2, Klf4, and c-Myc are sufficient to promote fibroblast-to-iPS cell reprogramming and propose that the observed low reprogramming frequencies may have alternative explanations.
Induced pluripotent stem cells; Retroviral insertions; Cellular reprogramming; Retroviral tagged mouse genes; Insertional mutagenesis