Functional interaction between Sequestosome-1/p62 and Autophagy-Linked FYVE-containing protein WDFY3 in human osteoclasts

Peer reviewedPostprin

Signal peptide mutations in RANK prevent downstream activation of NF-κB

Familial expansile osteolysis and related disorders are caused by heterozygous tandem duplication mutations in the signal peptide region of the gene encoding receptor activator of NF-κB (RANK), a receptor critical for osteoclast formation and function. Previous studies have shown that overexpression of these mutant proteins causes constitutive activation of NF-κB signaling in vitro, and it has been assumed that this accounts for the focal osteolytic lesions that are seen in vivo. We show here that constitutive activation of NF-κB occurred in HEK293 cells overexpressing wild-type or mutant RANK but not in stably transfected cell lines expressing low levels of each RANK gene. Importantly, only cells expressing wild-type RANK demonstrated ligand-dependent activation of NF-κB. When overexpressed, mutant RANK did not localize to the plasma membrane but localized to extensive areas of organized smooth endoplasmic reticulum, whereas, as expected, wild-type RANK was detected at the plasma membrane and in the Golgi apparatus. This intracellular accumulation of the mutant proteins is probably the result of lack of signal peptide cleavage because, using two in vitro translation systems, we demonstrate that the mutations in RANK prevent cleavage of the signal peptide. In conclusion, signal peptide mutations lead to accumulation of RANK in the endoplasmic reticulum and prevent direct activation by RANK ligand. These results strongly suggest that the increased osteoclast formation/activity caused by these mutations cannot be explained by studying the homozygous phenotype alone but requires further detailed investigation of the heterozygous expression of the mutant RANK proteins. © 2011 American Society for Bone and Mineral Researc

RANK-Dependent Autosomal Recessive Osteopetrosis: Characterization of Five New Cases With Novel Mutations

Autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder attributed to reduced bone resorption by osteoclasts. Most human AROs are classified as osteoclast rich, but recently two subsets of osteoclast-poor ARO have been recognized as caused by defects in either TNFSF11 or TNFRSF11A genes, coding the RANKL and RANK proteins, respectively. The RANKL/RANK axis drives osteoclast differentiation and also plays a role in the immune system. In fact, we have recently reported that mutations in the TNFRSF11A gene lead to osteoclast-poor osteopetrosis associated with hypogammaglobulinemia. Here we present the characterization of five additional unpublished patients from four unrelated families in which we found five novel mutations in the TNFRSF11A gene, including two missense and two nonsense mutations and a single-nucleotide insertion. Immunological investigation in three of them showed that the previously described defect in the B cell compartment was present only in some patients and that its severity seemed to increase with age and the progression of the disease. HSCT performed in all five patients almost completely cured the disease even when carried out in late infancy. Hypercalcemia was the most important posttransplant complication. Overall, our results further underline the heterogeneity of human ARO also deriving from the interplay between bone and the immune system, and highlight the prognostic and therapeutic implications of the molecular diagnosis. © 2012 American Society for Bone and Mineral Researc

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

Meeting abstrac

Real-time point of care microcirculatory assessment of shock: design, rationale and application of the point of care microcirculation (POEM) tool

Background: Despite over a decade of research and technological advances, sublingual microcirculatory monitoring has not yet reached clinical utility. Offline analysis is time consuming and occurs away from the patient. A system to assess the microcirculation at the point of care is desirable. We present a novel 5-point grading system (the point of care microcirculation (POEM) scoring system) that can be used at the point of care during non-invasive sublingual microcirculatory monitoring. Methods: The POEM score is an ordinal scale from 1 (worst) to 5 (best), based on a composite assessment of flow and heterogeneity of four individual sublingual video-microscopy clips. Thirty-two healthcare professionals were trained in how to assign POEM scores. Following training they assigned scores to five test sequences (each consisting of four video clips). They were blinded to clinical status. Inter-user consistency and agreement were assessed using intra-class correlation coefficient (ICC) analysis. In addition, blinded expert scores for 68 video clips were compared to offline computer analysis using traditional microcirculatory parameters including total vessel density (TVD), perfused vessel density (PVD), proportion of perfused vessels (PPV), microcirculatory flow index (MFI) and microcirculatory heterogeneity index (MHI). The time taken to assign each was recorded. Results: Participants showed good inter-rater consistency (ICC 0.83, 95 % CI 0.626, 0.976) and agreement (ICC 0.815, 95 % CI 0.602, 0.974) for assigned POEM scores. Expert scoring of videos correlated with offline values for PVD (R = 0.39; p < 0.05), PPV (R = 0.71; p < 0.001), MFI (R = 0.75; p < 0.001), and MHI (R = 0.68; p < 0.001). POEM scores took less time to assign than conventional offline computer analysis (2 minutes versus 44 minutes). Conclusion: We present for the first time a novel 5-point ordinal scale of microcirculatory flow and heterogeneity that can be used at the point of care. It has minimal inter-user variability amongst healthcare professionals after just 1 hour of training. POEM scores take a short time to assign, and correspond well to traditional offline computer-analyzed parameters

Microcirculatory Impairment Is Associated With Multiple Organ Dysfunction Following Traumatic Hemorrhagic Shock: The MICROSHOCK Study.

OBJECTIVES To assess the relationship between microcirculatory perfusion and multiple organ dysfunction syndrome in patients following traumatic hemorrhagic shock. DESIGN Multicenter prospective longitudinal observational study. SETTING Three U.K. major trauma centers. PATIENTS Fifty-eight intubated and ventilated patients with traumatic hemorrhagic shock. INTERVENTIONS Sublingual incident dark field microscopy was performed within 12 hours of ICU admission (D0) and repeated 24 and 48 hours later. Cardiac output was assessed using oesophageal Doppler. Multiple organ dysfunction syndrome was defined as Serial Organ Failure Assessment score greater than or equal to 6 at day 7 post injury. MEASUREMENTS AND MAIN RESULTS Data from 58 patients were analyzed. Patients had a mean age of 43 ± 19 years, Injury Severity Score of 29 ± 14, and initial lactate of 7.3 ± 6.1 mmol/L and received 6 U (interquartile range, 4-11 U) of packed RBCs during initial resuscitation. Compared with patients without multiple organ dysfunction syndrome at day 7, patients with multiple organ dysfunction syndrome had lower D0 perfused vessel density (11.2 ± 1.8 and 8.6 ± 1.8 mm/mm; p < 0.01) and microcirculatory flow index (2.8 [2.6-2.9] and 2.6 [2.2-2.8]; p < 0.01) but similar cardiac index (2.5 [± 0.6] and 2.1 [± 0.7] L/min//m; p = 0.11). Perfused vessel density demonstrated the best discrimination for predicting subsequent multiple organ dysfunction syndrome (area under curve 0.87 [0.76-0.99]) compared with highest recorded lactate (area under curve 0.69 [0.53-0.84]), cardiac index (area under curve 0.66 [0.49-0.83]) and lowest recorded systolic blood pressure (area under curve 0.54 [0.39-0.70]). CONCLUSIONS Microcirculatory hypoperfusion immediately following traumatic hemorrhagic shock and resuscitation is associated with increased multiple organ dysfunction syndrome. Microcirculatory variables are better prognostic indicators for the development of multiple organ dysfunction syndrome than more traditional indices. Microcirculatory perfusion is a potential endpoint of resuscitation following traumatic hemorrhagic shock

Transforming growth factor-β signaling pathway in patients with Kawasaki disease

Background-Transforming growth factor (TGF)-β is a multifunctional peptide that is important in T-cell activation and cardiovascular remodeling, both of which are important features of Kawasaki disease (KD). We postulated that variation in TGF-β signaling might be important in KD susceptibility and disease outcome. Methods and Results - We investigated genetic variation in 15 genes belonging to the TGF-β pathway in a total of 771 KD subjects of mainly European descent from the United States, the United Kingdom, Australia, and the Netherlands. We analyzed transcript abundance patterns using microarray and reverse transcriptase-polymerase chain reaction for these same genes, and measured TGF-β2 protein levels in plasma. Genetic variants in TGFB2, TGFBR2, and SMAD3 and their haplotypes were consistently and reproducibly associated with KD susceptability, coronary artery aneurysm formation, aortic root dilatation, and intravenous immunoglobulin treatment response in different cohorts. A SMAD3 haplotype associated with KD susceptibility replicated in 2 independent cohorts and an intronic single nucleotide polymorphism in a separate haplotype block was also strongly associated (A/G, rs4776338) (P=0.000022; odds ratio, 1.50; 95% confidence interval, 1.25 to 1.81). Pathway analysis using all 15 genes further confirmed the importance of the TGF-β pathway in KD pathogenesis. Whole-blood transcript abundance for these genes and TGF-β2 plasma protein levels changed dynamically over the course of the illness. Conclusions-These studies suggest that genetic variation in the TGF-β pathway influences KD susceptibility, disease outcome, and response to therapy, and that aortic root and coronary artery Z scores can be used for phenotype/genotype analyses. Analysis of transcript abundance and protein levels further support the importance of this pathway in KD pathogenesis