Inositol phosphatase SHIP1 is a primary target of miR-155

MicroRNA-155 (miR-155) has emerged as a critical regulator of immune cell development, function, and disease. However, the mechanistic basis for its impact on the hematopoietic system remains largely unresolved. Because miRNAs function by repressing specific mRNAs through direct 3′UTR interactions, we have searched for targets of miR-155 implicated in the regulation of hematopoiesis. In the present study, we identify Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1) as a direct target of miR-155, and, using gain and loss of function approaches, show that miR-155 represses SHIP1 through direct 3′UTR interactions that have been highly conserved throughout evolution. Repression of endogenous SHIP1 by miR-155 occurred following sustained over-expression of miR-155 in hematopoietic cells both in vitro and in vivo, and resulted in increased activation of the kinase Akt during the cellular response to LPS. Furthermore, SHIP1 was also repressed by physiologically regulated miR-155, which was observed in LPS-treated WT versus miR-155−/− primary macrophages. In mice, specific knockdown of SHIP1 in the hematopoietic system following retroviral delivery of a miR-155-formatted siRNA against SHIP1 resulted in a myeloproliferative disorder, with striking similarities to that observed in miR-155-expressing mice. Our study unveils a molecular link between miR-155 and SHIP1 and provides evidence that repression of SHIP1 is an important component of miR-155 biology

Coordination of tolerogenic immune responses by the commensal microbiota

All mammals are born ignorant to the existence of micro-organisms. Soon after birth, however, every mammal begins a lifelong association with a multitude of microbes that lay residence on the skin, mouth, vaginal mucosa and gastrointestinal (GI) tract. Approximately 500–1000 different species of microbes have highly evolved to occupy these bodily niches, with the highest density and diversity occurring within the intestine [1]. These organisms play a vital role in mammalian nutrient breakdown and provide resistance to colonization by pathogenic micro-organisms. More recently, however, studies have demonstrated that the microbiota can have a profound and long-lasting effect on the development of our immune system both inside and outside the intestine [2]. While our immune system has evolved to recognize and eradicate foreign entities, it tolerates the symbiotic micro-organisms of the intestine. How and why this tolerance occurs has remained unclear. Here we present evidence that the commensal microbes of the intestine actively induce tolerant responses from the host that coordinate healthy immune responses. Potentially, disruption of this dialogue between the host and microbe can lead to the development of autoimmune diseases such as inflammatory bowel disease (IBD), rheumatoid arthritis (RA), or Type I diabetes (TID). As a wealth of publications have focused on the impact of the microbiota on intestinal immune responses and IBD, this chapter will focus on the extra-intestinal impacts of the microbiota from development to disease and integrate the known mechanisms by which the microbiota is able to actively communicate with its host to promote health

Fas ligand expression in human and mouse cancer cell lines; a caveat on over-reliance on mRNA data

BACKGROUND: During carcinogenesis, tumors develop multiple mechanisms for evading the immune response, including upregulation of Fas ligand (FasL/CD95L) expression. Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of anti-tumor immune effector cells. Recently this idea has been challenged by studies reporting that tumor cells of varying origin do not express FasL. In the present study, we aimed to comprehensively characterize FasL expression in tumors of both murine and human origin over a 72 hour time period. METHODS: RNA and protein was extracted from six human (SW620, HT29, SW480, KM12SM, HCT116, Jurkat) and three mouse (CMT93, CT26, B16F10) cancer cell lines at regular time intervals over a 72 hour time period. FasL expression was detected at the mRNA level by RT-PCR, using intron spanning primers, and at the protein level by Western Blotting and immunofluorescence, using a polyclonal FasL- specific antibody. RESULTS: Expression of FasL mRNA and protein was observed in all cell lines analysed. However, expression of FasL mRNA varied dramatically over time, with cells negative for FasL mRNA at many time points. In contrast, 8 of the 9 cell lines constitutively expressed FasL protein. Thus, cells can abundantly express FasL protein at times when FasL mRNA is absent. CONCLUSION: These findings demonstrate the importance of complete analysis of FasL expression by tumor cells in order to fully characterize its biological function and may help to resolve the discrepancies present in the literature regarding FasL expression and tumor immune privilege

Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder

Mammalian microRNAs are emerging as key regulators of the development and function of the immune system. Here, we report a strong but transient induction of miR-155 in mouse bone marrow after injection of bacterial lipopolysaccharide (LPS) correlated with granulocyte/monocyte (GM) expansion. Demonstrating the sufficiency of miR-155 to drive GM expansion, enforced expression in mouse bone marrow cells caused GM proliferation in a manner reminiscent of LPS treatment. However, the miR-155–induced GM populations displayed pathological features characteristic of myeloid neoplasia. Of possible relevance to human disease, miR-155 was found to be overexpressed in the bone marrow of patients with certain subtypes of acute myeloid leukemia (AML). Furthermore, miR-155 repressed a subset of genes implicated in hematopoietic development and disease. These data implicate miR-155 as a contributor to physiological GM expansion during inflammation and to certain pathological features associated with AML, emphasizing the importance of proper miR-155 regulation in developing myeloid cells during times of inflammatory stress

MicroRNA-146a acts as a guardian of the quality and longevity of hematopoietic stem cells in mice

During inflammation and infection, hematopoietic stem and progenitor cells are stimulated to proliferate and differentiate into mature immune cells, especially of the myeloid lineage. MicroRNA-146a (miR-146a) is a critical negative regulator of inflammation. Deletion of miR-146a produces effects that appear as dysregulated inflammatory hematopoiesis, leading to a decline in the number and quality of hematopoietic stem cells (HSCs), excessive myeloproliferation, and, ultimately, to HSC exhaustion and hematopoietic neoplasms. At the cellular level, the defects are attributable to both an intrinsic problem in the miR-146a–deficient HSCs and extrinsic effects of lymphocytes and nonhematopoietic cells. At the molecular level, this involves a molecular axis consisting of miR-146a, signaling protein TRAF6, transcriptional factor NF-κB, and cytokine IL-6. This study has identified miR-146a to be a critical regulator of HSC homeostasis during chronic inflammation in mice and provided a molecular connection between chronic inflammation and the development of bone marrow failure and myeloproliferative neoplasms

Consent for routine neonatal procedures: A study of practices in Irish neonatal units. How do we compare with the gold standard BAPM guidelines?

The Irish National Consent Policy (NCP)¹ proposes that the legal requirement for consent extends to all forms of interventions, investigations and treatment, carried out on or behalf of the Health Service Executive (HSE). This study employs a quantitative descriptive approach to investigate the practices for obtaining consent for an identified group of routine neonatal procedures in neonatal facilities throughout Ireland. The BAPM (British Association of Perinatal Medicine)² guidelines were identified as ‘gold standard’ for the purposes of this study. The results indicated a lack of consistency between participating units pertaining to the modes of consent utilised and notable variances from ‘gold standard’ guidelines. Unanimity was evident for 3 procedures only (administering BCG, 6-in-1, and donor breast milk to infant). Significant findings related to EEG with video recordings, MRI/CT and gastro intestinal imaging, screening of an infant with suspected substance abuse or retinopathy of prematurity screening (ROP), administration of Vitamin K, and the carrying out of a lumbar puncture

Spatial and temporal variability in costs and effectiveness in phosphorus loss mitigation at farm scale: A scenario analysis

Current policy instruments under the EU Water Framework Directive (WFD) to mitigate phosphorus (P) loss require that P use on farms is managed through regulation of farm gate P balances. Regulation at farm scale does not account for spatial variability in nutrient use and soil fertility at field scale, affecting the costs and effectiveness of farm gate measures. This study simulated the implementation of a P loss mitigation measure coupled with improving soil fertility so that farm productivity would not be compromised. The measure was simulated at field scale and the costs and effectiveness assessed at farm scale. Effectiveness was expressed as the time taken for excessive soil P levels to decline to levels that matched off-takes and this varied temporally and spatially within and between farms ranging from 1 to 8 years. Sub-optimum soil fertility was corrected on all fields across both farms, with applications of other soil nutrients and lime to protect productivity. An increase in costs ranging from 1.5 to 116% was predicted in the first two years of the measure on both farms after-which savings of 15-31% were predicted for each subsequent year until the measure was effective in year 9. Despite initial cost increase, there was no statistically significant difference in costs over the time taken for the measure to be effective, when compared to baseline costs. Successful implementation of measures should consider the impact on farm costs and time taken for measures to environmentally effective. Adoption of measures could improve if demonstrating to farmers that costs will not vary significantly from current practice and in time may results in savings if measures are paired with correcting soil fertility and increasing yields. This 'win-win' approach could be used into the future to ensure successful implementation and uptake of measures within the farming community

Engineering human hematopoietic stem/progenitor cells to produce a broadly neutralizing anti-HIV antibody after in vitro maturation to human B lymphocytes

Broadly neutralizing anti-HIV antibodies are rare and have proved hard to elicit with any immunogen. We have tested in vitro the notion that such antibodies or other antiviral proteins could be made by lentivirus-mediated gene transfer into human hematopoietic stem/progenitor cells (HSPCs), followed by differentiation of the transduced cells into B cells, the most potent antibody-producing cells. To do this, we have developed a highly efficient system for in vitro maturation of secreting B lymphocytes and plasma cells from CD34^+ HSPCs. It is a 3-stage, in vitro culture system that supports normal human B-lineage development from HSPCs to antibody-secreting plasmablasts (~36%) and plasma cells (~20%). By transducing human cord blood CD34^+ cells with lentiviral vectors encoding a secretory monoclonal anti-HIV antibody, b12 (IgG1), we were able to program human B cells to produce in vitro up to 1.5 µg/mL of this broadly neutralizing antibody. Our results suggest that an HIV vaccine might be delivered by autologous transplantation of in vitro–programmed HSPCs, which would develop into antibody-secreting B cells in vivo and provide a continuous supply of anti-HIV neutralizing antibodies

Epistasis between MicroRNAs 155 and 146a during T Cell-Mediated Antitumor Immunity

An increased understanding of antitumor immunity is necessary for improving cell-based immunotherapies against human cancers. Here, we investigated the roles of two immune system-expressed microRNAs (miRNAs), miR-155 and miR-146a, in the regulation of antitumor immune responses. Our results indicate that miR-155 promotes and miR-146a inhibits interferon γ (IFNγ) responses by T cells and reduces solid tumor growth in vivo. Using a double-knockout (DKO) mouse strain deficient in both miR-155 and miR-146a, we have also identified an epistatic relationship between these two miRNAs. DKO mice had defective T cell responses and tumor growth phenotypes similar to miR-155^(−/−) mice. Further analysis of the T cell compartment revealed that miR-155 modulates IFNγ expression through a mechanism involving repression of Ship1. Our work reveals critical roles for miRNAs in the reciprocal regulation of CD4^+ and CD8^+ T cell-mediated antitumor immunity and demonstrates the dominant nature of miR-155 during its promotion of immune responses

Mortality due to cardiovascular disease, respiratory disease and cancer in adults with cerebral palsy

Aim: To compare mortality rates for cardiovascular disease, cancer, and respiratory disease between adults with cerebral palsy (CP) and the general population. Method: A cohort study was conducted using data from adults with CP in England, identified through a primary care dataset (the Clinical Practice Research Datalink), with linked data on death registrations from the Office for National Statistics. Cause of death was categorised according to ICD codes. Standardised mortality ratios were calculated to compare mortality rates between adults with CP and the general population, adjusted for age, sex, and calendar-year. Results: 958 adults with CP were identified (median age at start of follow-up 31 yr; 52.5% males) and followed for a total of 7693 person-years. 142 patients (15%) died during follow-up. Adults with CP had an increased risk of death due to cardiovascular disease (SMR: 3.19, 95% CI 2.20 to 4.62) and respiratory disease (SMR: 13.59, 95% CI to 18.67), but not from malignant neoplasms (SMR: 1.42, 95% CI 0.83 to 2.45). Interpretation: We found that adults with CP in England have increased risk of death due to diseases of the circulatory and respiratory systems, supporting findings from two studies that compared cause-specific mortality rates between adults with CP in the US and the general population. Further research is required into primary and secondary prevention of cardiovascular and respiratory disease in people with CP worldwide. </p