Nod2 Suppresses Borrelia burgdorferi Mediated Murine Lyme Arthritis and Carditis through the Induction of Tolerance

The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen. The intracellular, cytosolic receptor Nod2 has been shown to play varying roles in either enhancing or attenuating inflammation in response to different infectious agents. We examined the role of Nod2 in responses to B. burgdorferi. In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells. However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice. We explored multiple potential mechanisms for the paradoxical response in in vivo versus in vitro systems and found that prolonged stimulation with a Nod2 ligand, muramyl dipeptide (MDP), resulted in tolerance to stimulation by B. burgdorferi. This tolerance was lost with stimulation of Nod2 deficient cells that cannot respond to MDP. Cytokine patterns in the tolerance model closely paralleled cytokine profiles in infected Nod2 deficient mice. We propose a model where Nod2 has an enhancing role in activating inflammation in early infection, but moderates inflammation after prolonged exposure to the organism through induction of tolerance

The Relationship between Independent Transfer Skills and Upper Limb Kinetics in Wheelchair Users

Transfers are one of the most physically demanding wheelchair activities. The purpose of this study was to determine if using proper transfer skills as measured by the Transfer Assessment Instrument (TAI) is associated with reduced loading on the upper extremities. Twenty-three wheelchair users performed transfers to a level-height bench while a series of forces plates, load cells, and a motion capture system recorded the biomechanics of their natural transferring techniques. Their transfer skills were simultaneously evaluated by two study clinicians using the TAI. Logistic regression and multiple linear regression models were used to determine the relationships between TAI scores and the kinetic variables on both arms across all joints. The results showed that the TAI measured transfer skills were closely associated with the magnitude and timing of joint moments ( < .02, model R 2 values ranged from 0.27 to 0.79). Proper completion of the skills which targeted the trailing arm was associated with lower average resultant moments and rates of rise of resultant moments at the trailing shoulder and/or elbow. Some skills involving the leading side had the effect of increasing the magnitude or rate loading on the leading side. Knowledge of the kinetic outcomes associated with each skill may help users to achieve the best load-relieving effects for their upper extremities

Increased serum kallistatin levels in type 1 diabetes patients with vascular complications

BACKGROUND: Kallistatin, a serpin widely produced throughout the body, has vasodilatory, anti-angiogenic, anti-oxidant, and anti-inflammatory effects. Effects of diabetes and its vascular complications on serum kallistatin levels are unknown. METHODS: Serum kallistatin was quantified by ELISA in a cross-sectional study of 116 Type 1 diabetic patients (including 50 with and 66 without complications) and 29 non-diabetic controls, and related to clinical status and measures of oxidative stress and inflammation. RESULTS: Kallistatin levels (mean(SD)) were increased in diabetic vs. control subjects (12.6(4.2) vs. 10.3(2.8) μg/ml, p = 0.007), and differed between diabetic patients with complications (13.4(4.9) μg/ml), complication-free patients (12.1(3.7) μg/ml), and controls; ANOVA, p = 0.007. Levels were higher in diabetic patients with complications vs. controls, p = 0.01, but did not differ between complication-free diabetic patients and controls, p > 0.05. On univariate analyses, in diabetes, kallistatin correlated with renal dysfunction (cystatin C, r = 0.28, p = 0.004; urinary albumin/creatinine, r = 0.34, p = 0.001; serum creatinine, r = 0.23, p = 0.01; serum urea, r = 0.33, p = 0.001; GFR, r = -0.25, p = 0.009), total cholesterol (r = 0.28, p = 0.004); LDL-cholesterol (r = 0.21, p = 0.03); gamma-glutamyltransferase (GGT) (r = 0.27, p = 0.04), and small artery elasticity, r = -0.23, p = 0.02, but not with HbA1c, other lipids, oxidative stress or inflammation. In diabetes, geometric mean (95%CI) kallistatin levels adjusted for covariates, including renal dysfunction, were higher in those with vs. without hypertension (13.6 (12.3-14.9) vs. 11.8 (10.5-13.0) μg/ml, p = 0.03). Statistically independent determinants of kallistatin levels in diabetes were age, serum urea, total cholesterol, SAE and GGT, adjusted r2 = 0.24, p < 0.00001. CONCLUSIONS: Serum kallistatin levels are increased in Type 1 diabetic patients with microvascular complications and with hypertension, and correlate with renal and vascular dysfunction

The multifunctional Ca²⁺/calmodulin-dependent protein kinase II delta (CaMKIIδ) phosphorylates cardiac titin’s spring elements

Titin-based passive stiffness is post-translationally regulated by several kinases that phosphorylate specific spring elements located within titin's elastic I-band region. Whether titin is phosphorylated by calcium/calmodulin dependent protein kinase II (CaMKII), an important regulator of cardiac function and disease, has not been addressed. The aim of this work was to determine whether CaMKIIδ, the predominant CaMKII isoform in the heart, phosphorylates titin, and to use phosphorylation assays and mass spectrometry to study which of titin's spring elements might be targeted by CaMKIIδ. It was found that CaMKIIδ phosphorylates titin in mouse LV skinned fibers, that the CaMKIIδ sites can be dephosphorylated by protein phosphatase 1 (PP1), and that under baseline conditions, in both intact isolated hearts and skinned myocardium, about half of the CaMKIIδ sites are phosphorylated. Mass spectrometry revealed that both the N2B and PEVK segments are targeted by CaMKIIδ at several conserved serine residues. Whether phosphorylation of titin by CaMKIIδ occurs in vivo, was tested in several conditions using back phosphorylation assays and phospho-specific antibodies to CaMKIIδ sites. Reperfusion following global ischemia increased the phosphorylation level of CaMKIIδ sites on titin and this effect was abolished by the CaMKII inhibitor KN-93. No changes in the phosphorylation level of the PEVK element were found suggesting that the increased phosphorylation level of titin in IR (ischemia reperfusion) might be due to phosphorylation of the N2B element. The findings of these studies show for the first time that titin can be phosphoryalated by CaMKIIδ, both in vitro and in vivo, and that titin's molecular spring region that determines diastolic stiffness is a target of CaMKIIδ.Centro de Investigaciones Cardiovasculare

Longitudinal analysis of low-molecular weight fluorophores in type 1 diabetes mellitus

Objectives : Circulating low molecular weight (<10 kDa) fluorophores (LMW-F) measured by non-specific fluorescence spectroscopy may detect small advanced glycation end-products (AGEs) not recognized by other assays. This longitudinal study assessed correlates of LMW-F and predictive power of LMW-F levels for vascular health in Type 1 diabetes (T1DM) patients. Methods : Fasting patients with T1DM (n=37) were studied twice at intervals of 12-60 months (mean±SD, 33±15 months). LMW-F levels were also measured once in 112 healthy control subjects. Results : Relative to controls, LMW-F levels were higher in diabetic subjects at initial and final time points (mean±SD), 5.4±1.9 AU/ml and 4.5±1.8 AU/ml respectively vs. 3.8±2.1 AU/ml p=0.0001 and p=0.06). Baseline LMW-F levels predicted subsequent hs-CRP and oxLDL/LDL values. LMW-F levels decreased significantly over time in diabetes (5.4±1.9 vs. 4.5±1.8 AU/ml p=0.02). Rises in LMW-F levels in individual diabetic subjects correlated significantly with worsening renal function (BUN), glycemia (HbA1c) and with vascular dysfunction (systemic vascular resistance). Conclusions : LMW-F levels predict levels of inflammation and oxidation in T1DM. Changes in LMW-F levels in T1DM reflect variations in glycemia and renal function. Biochemical characterization of LMW-F would facilitate understanding of the potential utility of LMW-F as a therapeutic target

Relationship between tobacco, cagA and vacA i1 virulence factors and bacterial load in patients infected by Helicobacter pylori

Background and Aim Several biological and epidemiological studies support a relationship between smoking and Helicobacter pylori (H. pylori) to increase the risk of pathology. However, there have been few studies on the potential synergistic association between specific cagA and vacA virulence factors and smoking in patients infected by Helicobacter pylori. We studied the relationship between smoking and cagA, vacA i1 virulence factors and bacterial load in H. pylori infected patients. Methods Biopsies of the gastric corpus and antrum from 155 consecutive patients in whom there was clinical suspicion of infection by H. pylori were processed. In 106 patients H. pylori infection was detected. Molecular methods were used to quantify the number of microorganisms and presence of cagA and vacA i1 genes. A standardized questionnaire was used to obtain patients’ clinical data and lifestyle variables, including tobacco and alcohol consumption. Adjusted Odds Ratios (ORadjusted) were estimated by unconditional logistic regression. Results cagA was significantly associated with active-smoking at endoscope: ORadjusted 4.52. Evidence of association was found for vacA i1 (ORadjusted 3.15). Bacterial load was higher in active-smokers, although these differences did not yield statistical significance (median of 262.2 versus 79.4 copies of H. pylori per cell). Conclusions The association between smoking and a higher risk of being infected by a virulent bacterial population and with higher bacterial load, support a complex interaction between H. pylori infection and environmental factors

Cudraflavone C induces tumor-specific apoptosis in colorectal cancer cells through inhibition of the phosphoinositide 3-kinase (PI3K)-AKT pathway

Cudraflavone C (Cud C) is a naturally-occurring flavonol with reported anti-proliferative activities. However, the mechanisms by which Cud C induced cytotoxicity have yet to be fully elucidated. Here, we investigated the effects of Cud C on cell proliferation, caspase activation andapoptosis induction in colorectal cancer cells (CRC). We show that Cud C inhibits cell proliferation in KM12, Caco-2, HT29, HCC2998, HCT116 and SW48 CRC but not in the non-transformed colorectal epithelial cells, CCD CoN 841. Cud C induces tumorselective apoptosis via mitochondrial depolarization and activation of the intrinsic caspase pathway. Gene expression profiling by microarray analyses revealed that tumor suppressor genes EGR1, HUWE1 and SMG1 were significantly up-regulated while oncogenes such as MYB1, CCNB1 and GPX2 were down-regulated following treatment with Cud C. Further analyses using Connectivity Map revealed that Cud C induced a gene signature highly similar to that of protein synthesis inhibitors and phosphoinositide 3-kinase (PI3K)-AKT inhibitors, suggesting that Cud C might inhibit PI3K-AKT signaling. A luminescent cell free PI3K lipid kinase assay revealed that Cud C significantly inhibited p110?/p85? PI3K activity, followed by p120?, p110?/p85?, and p110?/p85? PI3K activities. The inhibition by Cud C on p110?/p85? PI3K activity was comparable to LY-294002, a known PI3K inhibitor. Cud C also inhibited phosphorylation of AKT independent of NF?B activity in CRC cells, while ectopic expression of myristoylated AKT completely abrogated the anti-proliferative effects, and apoptosis induced by Cud C in CRC. These findings demonstrate that Cud C induces tumor-selective cytotoxicity by targeting the PI3K-AKT pathway. These findings provide novel insights into the mechanism of action of Cud C, and indicate that Cud C further development of Cud C derivatives as potential therapeutic agents is warranted

Profiling the HER3/PI3K Pathway in Breast Tumors Using Proximity-Directed Assays Identifies Correlations between Protein Complexes and Phosphoproteins

The identification of patients for targeted antineoplastic therapies requires accurate measurement of therapeutic targets and associated signaling complexes. HER3 signaling through heterodimerization is an important growth-promoting mechanism in several tumor types and may be a principal resistance mechanism by which EGFR and HER2 expressing tumors elude targeted therapies. Current methods that can study these interactions are inadequate for formalin-fixed, paraffin-embedded (FFPE) tumor samples.Herein, we describe a panel of proximity-directed assays capable of measuring protein-interactions and phosphorylation in FFPE samples in the HER3/PI3K/Akt pathway and examine the capability of these assays to inform on the functional state of the pathway. We used FFPE breast cancer cell line and tumor models for this study. In breast cancer cell lines we observe both ligand-dependent and independent activation of the pathway and strong correlations between measured activation of key analytes. When selected cell lines are treated with HER2 inhibitors, we not only observe the expected molecular effects based on mechanism of action knowledge, but also novel effects of HER2 inhibition on key targets in the HER receptor pathway. Significantly, in a xenograft model of delayed tumor fixation, HER3 phosphorylation is unstable, while alternate measures of pathway activation, such as formation of the HER3PI3K complex is preserved. Measurements in breast tumor samples showed correlations between HER3 phosphorylation and receptor interactions, obviating the need to use phosphorylation as a surrogate for HER3 activation.This assay system is capable of quantitatively measuring therapeutically relevant responses and enables molecular profiling of receptor networks in both preclinical and tumor models

Nod2 Suppresses Borrelia burgdorferi Mediated Murine Lyme Arthritis and Carditis through the Induction of Tolerance

The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen. The intracellular, cytosolic receptor Nod2 has been shown to play varying roles in either enhancing or attenuating inflammation in response to different infectious agents. We examined the role of Nod2 in responses to B. burgdorferi. In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells. However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice. We explored multiple potential mechanisms for the paradoxical response in in vivo versus in vitro systems and found that prolonged stimulation with a Nod2 ligand, muramyl dipeptide (MDP), resulted in tolerance to stimulation by B. burgdorferi. This tolerance was lost with stimulation of Nod2 deficient cells that cannot respond to MDP. Cytokine patterns in the tolerance model closely paralleled cytokine profiles in infected Nod2 deficient mice. We propose a model where Nod2 has an enhancing role in activating inflammation in early infection, but moderates inflammation after prolonged exposure to the organism through induction of tolerance

Ih Current Is Necessary to Maintain Normal Dopamine Fluctuations and Sleep Consolidation in Drosophila

HCN channels are becoming pharmacological targets mainly in cardiac diseases. But apart from their well-known role in heart pacemaking, these channels are widely expressed in the nervous system where they contribute to the neuron firing pattern. Consequently, abolishing Ih current might have detrimental consequences in a big repertoire of behavioral traits. Several studies in mammals have identified the Ih current as an important determinant of the firing activity of dopaminergic neurons, and recent evidences link alterations in this current to various dopamine-related disorders. We used the model organism Drosophila melanogaster to investigate how lack of Ih current affects dopamine levels and the behavioral consequences in the sleep∶activity pattern. Unlike mammals, in Drosophila there is only one gene encoding HCN channels. We generated a deficiency of the DmIh core gene region and measured, by HPLC, levels of dopamine. Our data demonstrate daily variations of dopamine in wild-type fly heads. Lack of Ih current dramatically alters dopamine pattern, but different mechanisms seem to operate during light and dark conditions. Behaviorally, DmIh mutant flies display alterations in the rest∶activity pattern, and altered circadian rhythms. Our data strongly suggest that Ih current is necessary to prevent dopamine overproduction at dark, while light input allows cycling of dopamine in an Ih current dependent manner. Moreover, lack of Ih current results in behavioral defects that are consistent with altered dopamine levels