Aseptic loosening around total joint replacement in humans is regulated by miR-1246 and miR-6089 via the Wnt signalling pathway.

BACKGROUND: Total joint replacement for osteoarthritis is one of the most successful surgical procedures in modern medicine. However, aseptic loosening continues to be a leading cause of revision arthroplasty. The diagnosis of aseptic loosening remains a challenge as patients are often asymptomatic until the late stages. MicroRNA (miRNA) has been demonstrated to be a useful diagnostic tool and has been successfully used in the diagnosis of other diseases. We aimed to identify differentially expressed miRNA in the plasma of patients with aseptic loosening. METHODS: Adult patients undergoing revision arthroplasty for aseptic loosening and age- and gender-matched controls were recruited. Samples of bone, tissue and blood were collected, and RNA sequencing was performed in 24 patients with aseptic loosening and 26 controls. Differentially expressed miRNA in plasma was matched to differentially expressed mRNA in periprosthetic bone and tissue. Western blot was used to validate protein expression. RESULTS: Seven miRNA was differentially expressed in the plasma of patients with osteolysis (logFC >|2|, adj-P < 0.05). Three thousand six hundred and eighty mRNA genes in bone and 427 mRNA genes in tissue samples of osteolysis patients were differentially expressed (logFC >|2|, adj-P < 0.05). Gene enrichment analysis and pathway analysis revealed two miRNA (miR-1246 and miR-6089) had multiple gene targets in the Wnt signalling pathway in the local bone and tissues which regulate bone metabolism. CONCLUSION: These results suggest that aseptic loosening may be regulated by miR-1246 and miR-6089 via the Wnt signalling pathway.

Hydroxyurea therapy in UK children with sickle cell anaemia: A single-centre experience.

INTRODUCTION: Despite the demonstrated efficacy of hydroxyurea therapy, children with sickle cell anaemia in the UK are preferentially managed with supportive care or transfusion. Hydroxyurea is reserved for children with severe disease phenotype. This is in contrast to North America and other countries where hydroxyurea is widely used for children of all clinical phenotypes. The conservative UK practice may in part be due to concerns about toxicity, in particular marrow suppression with high doses, and growth in children. METHODS AND RESULTS: We monitored 37 paediatric patients with sickle cell anaemia who were treated with hydroxyurea at a single UK treatment centre. Therapy was well tolerated and mild transient cytopenias were the only toxicity observed. Comparative analysis of patients receiving ≥26 mg/kg/day versus <26 mg/kg/day demonstrates increasing dose has a significant positive effect on foetal haemoglobin (Hb; 29.2% vs. 20.4%, P = 0.0151), mean cell volume (94.4 vs. 86.5, P = 0.0183) and reticulocyte count (99.66 × 109 /l vs. 164.3 × 109 /l, P = 0.0059). Marrow suppression was not a clinical problem with high-dose treatment, Hb 92.25 g/l versus 91.81 g/l (ns), neutrophil count 3.3 × 109 /l versus 4.8 × 109 /l (ns) and platelet count 232.4 × 109 /l versus 302.2 × 109 /l (ns). Normal growth rates were maintained in all children. Good adherence to therapy was a significant factor in reducing hospitalisations. CONCLUSION: This study demonstrates the effectiveness and safety in practice of high-dose hydroxyurea as a disease-modifying therapy, which we advocate for all children with sickle cell anaemia.

Non-tumour bone marrow lymphocytes correlate with improved overall survival in childhood acute lymphoblastic leukaemia.

Composition of tumour immune cell infiltrates correlates with response to treatment and overall survival (OS) in several cancer settings. We retrospectively examined immune cells present in diagnostic bone marrow aspirates from paediatric patients with B-cell acute lymphoblastic leukaemia. Our analysis identified a sub-group (∼30% of patients) with >2.37% CD20 and >6.05% CD7 expression, which had 100% OS, and a sub-group (∼30% of patients) with ≤2.37% CD20 and ≤6.05% CD7 expression at increased risk of treatment failure (66.7% OS, P < 0.05). Immune cell infiltrate at diagnosis may predict treatment response and could provide a means to enhance immediate treatment risk stratification.

Inflammatory cytokines induce NOTCH signaling in nucleus pulposus cells: implications in intervertebral disc degeneration.

The objective of the study was to investigate how inflammatory cytokines, IL-1ß, and TNF-α control NOTCH signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective NOTCH receptors (NOTCH1 and -2), ligand (JAGGED2), and target genes (HES1, HEY1, and HEY2) was observed in NP cells following cytokine treatment. A concomitant increase in NOTCH signaling as evidenced by induction in activity of target gene HES1 and HEY1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2-kb NOTCH2 promoter. Treatment of cells with NF-κB and MAPK inhibitors abolished the inductive effect of cytokines on NOTCH2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on NOTCH2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting promoter studies, lentiviral delivery of sh-p65, and sh-IKKß significantly decreased cytokine dependent change in NOTCH2 expression. Interestingly, MAPK signaling showed an isoform-specific control of NOTCH2 promoter; p38α/ß2/δ, ERK1, and ERK2 contributed to cytokine dependent induction, whereas p38γ played no role. Analysis of human NP tissues showed that NOTCH1 and -2 and HEY2 expression correlated with each other. Moreover, expression of NOTCH2 and IL-1ß as well as the number of cells immunopositive for NOTCH2 significantly increased in histologically degenerate discs compared with non-degenerate discs. Taken together, these results explain the observed dysregulated expression of NOTCH genes in degenerative disc disease. Thus, controlling IL-1ß and TNF-α activities during disc disease may restore NOTCH signaling and nucleus pulposus cell function.

The impact of a statewide training to increase child care providers' knowledge of nutrition and physical activity rules in Delaware.

BACKGROUND: Childhood obesity has been recognized as a national problem of epidemic proportions. Child care represents an ideal venue in which to address this problem, as many young children spend a significant amount of time and consume the majority of their meals in this setting. Recognizing this opportunity, Delaware recently enacted reforms to statewide licensing regulations designed to improve the quality of the nutrition-, physical activity-, and screen viewing-related environments in child care settings. METHODS: To facilitate the translation of these regulations into practices, a series of broad-scale trainings was held throughout the state. Attendance was required for all Child & Adult Care Food Program (CACFP)-participating facilities, although child care providers from non-CACFP facilities also attended. Pre- and posttraining surveys were used to assess changes in providers' knowledge of the regulations and satisfaction with the training. RESULTS: In total 1094 presurveys and 1076 postsurveys were received. Participants were highly satisfied with the training format and content, including the instructors, materials, and schedule. Data analysis demonstrates improved knowledge of all 26 regulation components from presurvey to postsurvey. Family child care providers, providers with more years of experience, CACFP-participating facilities, and facilities with food service personnel scored significantly higher than their center staff, less experienced and non-CACFP counterparts, as well as those without food service personnel. CONCLUSIONS: Broad-scale, in-person training can effectively increase child care providers' knowledge of the regulations and is well received by this audience. Other states and jurisdictions seeking to improve nutrition, physical activity, and screen-viewing practices in child care settings should consider this model of quality improvement.

Tumor necrosis factor α- and interleukin-1ß-dependent induction of CCL3 expression by nucleus pulposus cells promotes macrophage migration through CCR1.

OBJECTIVE: To investigate tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) regulation of CCL3 expression in nucleus pulposus (NP) cells and in macrophage migration. METHODS: Quantitative reverse transcription-polymerase chain reaction and immunohistochemistry were used to measure CCL3 expression in NP cells. Transfections were used to determine the role of NF-κB, CCAAT/enhancer binding protein (C/EBPß), and MAPK on cytokine-mediated CCL3 promoter activity. The effect of NP-conditioned medium on macrophage migration was measured using a Transwell system. RESULTS: An increase in CCL3 expression and promoter activity was observed in NP cells after TNFα or IL-1ß treatment. Treatment of cells with NF-κB and MAPK inhibitors abolished the effect of the cytokines on CCL3 expression. The inductive effect of p65 and C/EBPß on the CCL3 promoter was confirmed through gain-of-function and loss-of-function studies. Notably, cotransfection with p50 completely blocked cytokine- and p65-dependent induction. In contrast, c-Rel and RelB had little effect on promoter activity. Lentiviral transduction with short hairpin RNA for p65 (shp65) and shIKKß significantly decreased the TNFα-dependent increase in CCL3 expression. Analysis of degenerated human NP tissue samples showed that CCL3, but not CCL4, expression correlated positively with the grade of tissue degeneration. Importantly, treatment of macrophages with conditioned medium of NP cells treated with TNFα or IL-1ß promoted their migration. Pretreatment of macrophages with an antagonist of CCR1, the primary receptor for CCL3 and CCL4, blocked cytokine-mediated migration. CONCLUSION: Our findings indicate that TNFα and IL-1ß modulate the expression of CCL3 in NP cells by controlling the activation of MAPK, NF-κB, and C/EBPß signaling. The CCL3-CCR1 axis may play an important role in promoting macrophage infiltration in degenerated, herniated discs.

Doxorubicin in vivo rapidly alters expression and translation of myocardial electron transport chain genes, leads to ATP loss and caspase 3 activation.

BACKGROUND: Doxorubicin is one of the most effective anti-cancer drugs but its use is limited by cumulative cardiotoxicity that restricts lifetime dose. Redox damage is one of the most accepted mechanisms of toxicity, but not fully substantiated. Moreover doxorubicin is not an efficient redox cycling compound due to its low redox potential. Here we used genomic and chemical systems approaches in vivo to investigate the mechanisms of doxorubicin cardiotoxicity, and specifically test the hypothesis of redox cycling mediated cardiotoxicity. METHODOLOGY/PRINCIPAL FINDINGS: Mice were treated with an acute dose of either doxorubicin (DOX) (15 mg/kg) or 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) (25 mg/kg). DMNQ is a more efficient redox cycling agent than DOX but unlike DOX has limited ability to inhibit gene transcription and DNA replication. This allowed specific testing of the redox hypothesis for cardiotoxicity. An acute dose was used to avoid pathophysiological effects in the genomic analysis. However similar data were obtained with a chronic model, but are not specifically presented. All data are deposited in the Gene Expression Omnibus (GEO). Pathway and biochemical analysis of cardiac global gene transcription and mRNA translation data derived at time points from 5 min after an acute exposure in vivo showed a pronounced effect on electron transport chain activity. This led to loss of ATP, increased AMPK expression, mitochondrial genome amplification and activation of caspase 3. No data gathered with either compound indicated general redox damage, though site specific redox damage in mitochondria cannot be entirely discounted. CONCLUSIONS/SIGNIFICANCE: These data indicate the major mechanism of doxorubicin cardiotoxicity is via damage or inhibition of the electron transport chain and not general redox stress. There is a rapid response at transcriptional and translational level of many of the genes coding for proteins of the electron transport chain complexes. Still though ATP loss occurs with activation caspase 3 and these events probably account for the heart damage.