Increased Adhesive Potential of Antiphospholipid Syndrome Neutrophils Mediated by ß2 Integrin Mac-1.

OBJECTIVE: While the role of antiphospholipid antibodies in activating endothelial cells has been extensively studied, the impact of these antibodies on the adhesive potential of leukocytes has received less attention. This study was undertaken to investigate the extent to which antiphospholipid syndrome (APS) neutrophils adhere to resting endothelial cells under physiologic flow conditions and the surface molecules required for that adhesion. METHODS: Patients with primary APS (n = 43), patients with a history of venous thrombosis but negative test results for antiphospholipid antibodies (n = 11), and healthy controls (n = 38) were studied. Cells were introduced into a flow chamber and perfused across resting human umbilical vein endothelial cells (HUVECs). Surface adhesion molecules were quantified by flow cytometry. Neutrophil extracellular trap release (NETosis) was assessed in neutrophil-HUVEC cocultures. RESULTS: Upon perfusion of anticoagulated blood through the flow chamber, APS neutrophils demonstrated increased adhesion as compared to control neutrophils under conditions representative of either venous (n = 8; P < 0.05) or arterial (n = 15; P < 0.0001) flow. At the same time, APS neutrophils were characterized by up-regulation of CD64, CEACAM1, ß2 -glycoprotein I, and activated Mac-1 on their surface (n = 12-18; P < 0.05 for all markers). Exposing control neutrophils to APS plasma or APS IgG resulted in increased neutrophil adhesion (n = 10-11; P < 0.0001) and surface marker up-regulation as compared to controls. A monoclonal antibody specific for activated Mac-1 reduced the adhesion of APS neutrophils in the flow-chamber assay (P < 0.01). The same monoclonal antibody reduced NETosis in neutrophil-HUVEC cocultures (P < 0.01). CONCLUSION: APS neutrophils demonstrate increased adhesive potential, which is dependent upon the activated form of Mac-1. In patients, this could lower the threshold for neutrophil-endothelium interactions, NETosis, and possibly thrombotic events.

Adenosine receptor agonism protects against NETosis and thrombosis in antiphospholipid syndrome.

Potentiation of neutrophil extracellular trap (NET) release is one mechanism by which antiphospholipid antibodies (aPL Abs) effect thrombotic events in patients with antiphospholipid syndrome (APS). Surface adenosine receptors trigger cyclic AMP (cAMP) formation in neutrophils, and this mechanism has been proposed to regulate NETosis in some contexts. Here we report that selective agonism of the adenosine A2A receptor (CGS21680) suppresses aPL Ab-mediated NETosis in protein kinase A-dependent fashion. CGS21680 also reduces thrombosis in the inferior vena cavae of both control mice and mice administered aPL Abs. The antithrombotic medication dipyridamole is known to potentiate adenosine signaling by increasing extracellular concentrations of adenosine and interfering with the breakdown of cAMP. Like CGS21680, dipyridamole suppresses aPL Ab-mediated NETosis via the adenosine A2A receptor and mitigates venous thrombosis in mice. In summary, these data suggest an anti-inflammatory therapeutic paradigm in APS, which may extend to thrombotic disease in the general population.

Cryptic conspirators: a conversation about thrombocytopenia and antiphospholipid syndrome.

PURPOSE OF REVIEW: Although antiphospholipid syndrome (APS) is best known for conveying increased risk of thrombotic events and pregnancy morbidity, thrombocytopenia is also recognized as a common association. In this review, we will explore the relationship between thrombocytopenia and APS, highlighting our evolving understanding - and persistent knowledge gaps - through clinically oriented questions and answers. RECENT FINDINGS: A history of thrombocytopenia likely portends a more severe APS phenotype (including increased risk of thrombosis). Although the pathophysiology underlying thrombocytopenia in APS has yet to be definitively revealed, mechanisms that play a role (at least in subsets of patients) include: immune thrombocytopenic purpura/ITP-like autoantibodies against platelet glycoproteins; antiphospholipid antibody (aPL)-mediated platelet activation and consumption; and potentially life threatening thrombotic microangiopathy. Although thrombocytopenia is often 'mild' in APS (and therefore, may not require specific therapy), there are causes of acute-onset thrombocytopenia that mandate emergent work-up and treatment. When APS-related thrombocytopenia does require therapy, the approach must be individualized (requiring an understanding of pathophysiology in the particular APS patient). For patients with ITP-like disease, rituximab is emerging as a popular approach to treatment; in contrast, there are hints that thrombopoietin mimetics may be associated with elevated thrombotic risk. SUMMARY: Thrombocytopenia is common in APS, and is likely associated with more severe disease. Improved understanding of thrombocytopenia in APS has the potential to improve risk stratification, reveal novel aspects of APS pathophysiology, and lead to treatments that are more individualized and holistic.

Rheumatologists Modestly More Likely to Counsel Smokers in Visits Without Rheumatoid Arthritis Control: An Observational Study.

BACKGROUND: Among patients with rheumatoid arthritis (RA), smoking increases risk of severe RA and pulmonary and cardiovascular disease. Despite this, little is known about smoking cessation counseling by rheumatologists. OBJECTIVES: We examined predictors of tobacco counseling in RA patients who smoke including the effect of perceived RA control. We hypothesized that patients with controlled RA would receive more counseling according to the competing demands model, which explains that preventive care gaps occur as a result of competing provider, patient, and clinic factors. METHODS: This secondary data analysis involved RA patients with an additional cardiovascular disease risk factor identified in an academic medical center 2004-2011. Trained abstractors assessed documented smoking counseling and rheumatologists' impression of RA control in clinic notes. We used multivariable logistic regression to predict having received smoking cessation counseling, including sociodemographics and comorbidity in models. RESULTS: We abstracted 3396 RA visits, including 360 visits (10%) with active smokers. Perceived controlled RA was present in 31% of visits involving smokers (39% in nonsmokers). Beyond nurse documentation, providers documented smoking status in 39% of visit notes with smokers and smoking cessation counseling in 10%. Visits with controlled versus active RA were less likely to include counseling (odds ratio, 0.3; confidence interval, 0.1-0.97). Counseling was more likely in visits with prevalent cardiovascular, pulmonary, and psychiatric disease, but decreased with obesity. CONCLUSIONS: Smoking cessation counseling was documented in 10% of visits and was less likely when RA was controlled. Given smoking's impact on RA and long-term outcomes, systematic cessation counseling efforts are needed.

Antiphospholipid syndrome: an update for clinicians and scientists.

PURPOSE OF REVIEW: Antiphospholipid syndrome (APS) is a leading acquired cause of thrombosis and pregnancy loss. Upon diagnosis (which is unlikely to be made until at least one morbid event has occurred), anticoagulant medications are typically prescribed in an attempt to prevent future events. This approach is not uniformly effective and does not prevent associated autoimmune and inflammatory complications. The goal of this review is to update clinicians and scientists on mechanistic and clinically relevant studies from the past 18 months, which have especially focused on inflammatory aspects of APS pathophysiology. RECENT FINDINGS: How antiphospholipid antibodies leverage receptors and signaling pathways to activate cells is being increasingly defined. Although established mediators of disease pathogenesis (like endothelial cells and the complement system) continue to receive intensive study, emerging concepts (such as the role of neutrophils) are also receiving increasing attention. In-vivo animal studies and small clinical trials are demonstrating how repurposed medications (hydroxychloroquine, statins, and rivaroxaban) may have clinical benefit in APS, with these concepts importantly supported by mechanistic data. SUMMARY: As anticoagulant medications are not uniformly effective and do not comprehensively target the underlying pathophysiology of APS, there is a continued need to reveal the inflammatory aspects of APS, which may be modulated by novel and repurposed therapies.

Can serum isotope levels accurately measure intestinal calcium absorption compared to gold-standard methods?

BACKGROUND: Low fractional calcium absorption (FCA) contributes to osteoporosis but is not measured clinically, as the gold-standard method requires administration of two calcium tracers and a subsequent 24-h urine collection. We evaluated alternate methods to measure FCA, compared to the gold standard method. METHODS: We administered two stable calcium isotope tracers (~8 mg oral (44)Ca and ~3 mg intravenous (42)Ca) with breakfast to 20 fasting post-menopausal women (Cohort 1) 59 ± 7 years old with vitamin D insufficiency. We measured subsequent calcium isotope concentrations in 24-h urine samples and serum collected 1, 3 and 5 h post tracer administration during an inpatient research stay. We assessed the candidate serum estimates in a second cohort of 9 women with similar characteristics. Methods of measuring FCA were compared using correlation coefficients and Bland-Altman tests. RESULTS: FCA estimated from a 3-h serum sample correlated highest with the levels from the 24-h urine collection (ρ 0.78, p < 0.001), but explained only 58 % of the variance in FCA. The total variance explained by 3-h estimates improved to 61 % with incorporation of glomerular filtration rate (GFR). FCA estimates from the 3-h serum measurement were assessed in a second group of nine women (Cohort 2) 60 ± 7 years old. In this cohort, however, FCA estimated by 3-h serum isotope levels did not correlate with gold-standard FCA measurements, whether determined with (ρ 0.02, p = 0.97) or without GFR values (ρ 0.03, p = 0.93). By contrast, FCA in Cohort 2 correlated best with 5-h serum isotope levels (ρ 0.75, p = 0.02). CONCLUSIONS: We conclude that serum isotope levels correlate with true fractional calcium absorption, but do not reliably estimate FCA when analyzed using Bland-Altman tests, compared to gold-standard methods. TRIAL REGISTRATION: ClinicalTrials.gov.Identifier: NCT00933244.

Characterization of the decision network for wing expansion in Drosophila using targeted expression of the TRPM8 channel.

After emergence, adult flies and other insects select a suitable perch and expand their wings. Wing expansion is governed by the hormone bursicon and can be delayed under adverse environmental conditions. How environmental factors delay bursicon release and alter perch selection and expansion behaviors has not been investigated in detail. Here we provide evidence that in Drosophila the motor programs underlying perch selection and wing expansion have different environmental dependencies. Using physical manipulations, we demonstrate that the decision to perch is based primarily on environmental valuations and is incrementally delayed under conditions of increasing perturbation and confinement. In contrast, the all-or-none motor patterns underlying wing expansion are relatively invariant in length regardless of environmental conditions. Using a novel technique for targeted activation of neurons, we show that the highly stereotyped wing expansion motor patterns can be initiated by stimulation of N(CCAP), a small network of central neurons that regulates the release of bursicon. Activation of this network using the cold-sensitive rat TRPM8 channel is sufficient to trigger all essential behavioral and somatic processes required for wing expansion. The delay of wing expansion under adverse circumstances thus couples an environmentally sensitive decision network to a command-like network that initiates a fixed action pattern. Because N(CCAP) mediates environmentally insensitive ecdysis-related behaviors in Drosophila development before adult emergence, the study of wing expansion promises insights not only into how networks mediate behavioral choices, but also into how decision networks develop.

Loss of myotubularin function results in T-tubule disorganization in zebrafish and human myotubular myopathy.

Myotubularin is a lipid phosphatase implicated in endosomal trafficking in vitro, but with an unknown function in vivo. Mutations in myotubularin cause myotubular myopathy, a devastating congenital myopathy with unclear pathogenesis and no current therapies. Myotubular myopathy was the first described of a growing list of conditions caused by mutations in proteins implicated in membrane trafficking. To advance the understanding of myotubularin function and disease pathogenesis, we have created a zebrafish model of myotubular myopathy using morpholino antisense technology. Zebrafish with reduced levels of myotubularin have significantly impaired motor function and obvious histopathologic changes in their muscle. These changes include abnormally shaped and positioned nuclei and myofiber hypotrophy. These findings are consistent with those observed in the human disease. We demonstrate for the first time that myotubularin functions to regulate PI3P levels in a vertebrate in vivo, and that homologous myotubularin-related proteins can functionally compensate for the loss of myotubularin. Finally, we identify abnormalities in the tubulo-reticular network in muscle from myotubularin zebrafish morphants and correlate these changes with abnormalities in T-tubule organization in biopsies from patients with myotubular myopathy. In all, we have generated a new model of myotubular myopathy and employed this model to uncover a novel function for myotubularin and a new pathomechanism for the human disease that may explain the weakness associated with the condition (defective excitation-contraction coupling). In addition, our findings of tubuloreticular abnormalities and defective excitation-contraction coupling mechanistically link myotubular myopathy with several other inherited muscle diseases, most notably those due to ryanodine receptor mutations. Based on our findings, we speculate that congenital myopathies, usually considered entities with similar clinical features but very disparate pathomechanisms, may at their root be disorders of calcium homeostasis.

Kindlin-2 is required for myocyte elongation and is essential for myogenesis.

BACKGROUND: Integrins are required for normal muscle differentiation and disruptions in integrin signaling result in human muscle disease. The intracellular components that regulate integrin function during myogenesis are poorly understood. Unc-112 is an integrin-associated protein required for muscle development in C. elegans. To better understand the intracellular effectors of integrin signaling in muscle, we examined the mammalian homolog of Unc-112, kindlin-2. RESULTS: Kindlin-2 expression is upregulated during differentiation and highly enriched at sites of integrin localization. RNAi knockdown of kindlin-2 in C2C12 cells results in significant abnormalities during the early stages of myogenesis. Specifically, differentiating myocytes lacking kindlin-2 are unable to elongate and fail to fuse into multinucleated myotubes. These changes are correlated with decreased cell substratum adhesion and increased cell motility. They are also associated with redistribution of a known kindlin-2 binding partner, integrin linked kinase (ILK), to the membrane insoluble subcellular fraction. CONCLUSION: In all, our study reveals kindlin-2 as a novel integrin adaptor protein important for muscle differentiation, and identifies it particularly as a critical regulator of myocyte elongation.

Lipopolysaccharide recognition protein, MD-2, facilitates cellular uptake of E. coli-derived plasmid DNA in synovium.

BACKGROUND: Several cell types are susceptible to transfection in vivo using naked plasmid DNA. The mechanisms involved in mediating in vivo transfection are incompletely known, but evidence suggests that receptor-mediated endocytosis is important for specific types of cells. In this study we tested the hypothesis that residual Escherichia coli lipopolysaccharide (LPS) forms a non-covalent complex with expression plasmid DNA, and host-cell-derived soluble LPS-binding proteins bind to the DNA-LPS complexes in order to facilitate receptor-mediated endocytosis. METHODS: Cells from the murine synovial lining were used as an in vivo model system and in vivo luciferase imaging was used to quantify levels of transgene expression. Using a series of gene-deleted mice, the roles of LPS recognition complex proteins, lipopolysaccharide-binding protein (LBP), CD14 and MD-2, in the process of in vivo transfection were determined. RESULTS: Luciferase expression assays revealed that mice lacking LBP or CD14 had increased luciferase expression (p < 0.023 and < 0.165, respectively), while mice deleted of MD-2 had significant reductions in luciferase expression (p < 0.001). Gene deletion of hyaluronic acid binding protein CD44 was used as a control and had no statistically significant effect on transgene expression in vivo. In muscle tissue, where neither cell surface nor soluble MD-2 is expressed, no MD-2 dependence of plasmid transfection was identified, suggesting the role of MD-2 is tissue or cell type specific. Additionally, depleting mice of macrophages showed that luciferase expression is occurring within fibroblast-like synoviocytes. CONCLUSIONS: Our data support a physical association between LPS and E. coli-derived plasmid DNA, and that in vivo transfection of fibroblast-like synoviocytes is dependent on the soluble form of the LPS-binding protein MD-2.