Is Metabolic Flexibility Altered in Multiple Sclerosis Patients?

Objectives

Metabolic flexibility is defined as ability to adjust fuel oxidation to fuel availability. Multiple sclerosis (MS) results in reduced muscle strength and exercise intolerance. We tested the hypothesis that altered metabolic flexibility contributes to exercise intolerance in MS patients.

Methods

We studied 16 patients (all on glatiramer) and 16 matched healthy controls. Energy expenditure (EE), and carbohydrate (COX) and lipid oxidation (LOX) rates were determined by calorimetry, before and after an oral glucose load. We made measurements either at rest (canopy device) or during 40 min low-grade (0.5 W/kg) exercise (metabolic chamber). We also obtained plasma, and adipose tissue and skeletal muscle dialysate samples by microdialysis to study tissue-level metabolism under resting conditions.

Results

At rest, fasting and postprandial plasma glucose, insulin, and free fatty acid levels did not differ between patients and controls. Fasting and postprandial COX was higher and LOX lower in patients. In adipose, fasting and postprandial dialysate glucose, lactate, and glycerol levels were higher in patients vs. controls. In muscle, fasting and postprandial dialysate metabolite levels did not differ significantly between the groups. During exercise, EE did not differ between the groups. However, COX increased sharply over 20 min in patients, without reaching a steady state, followed by an immediate decrease within the next 20 min and fell even below basal levels after exercise in patients, compared to controls.

Conclusions

Glucose tolerance is not impaired in MS patients. At rest, there is no indication for metabolic inflexibility or mitochondrial dysfunction in skeletal muscle. The increased adipose tissue lipolytic activity might result from glatiramer treatment. Autonomic dysfunction might cause dysregulation of postprandial thermogenesis at rest and lipid mobilization during exercise.     

Effects of lycopene on the initial state of atherosclerosis in New Zealand White (NZW) rabbits

Background

Lycopene is the main carotenoid in tomatoes, where it is found in high concentrations. Strong epidemiological evidence suggests that lycopene may provide protection against cardiovascular diseases. We therefore studied the effects of lycopene on diet-induced increase in serum lipid levels and the initiation of atherosclerosis in New Zealand White (NZW) rabbits.

Methodology/Principal Findings

The animals, divided into four groups of 9 animals each, were fed either a standard diet, a high-cholesterol diet containing 0.5% cholesterol, a high-cholesterol diet containing placebo beadlets, or a high-cholesterol diet plus 5 mg/kg body weight/day of lycopene (in the form of lycopene beadlets), for a period of 4 weeks. We found significantly elevated lycopene plasma levels in the animal group treated with lycopene beadlets. Compared to the high-cholesterol and the placebo group, this was associated with a significant reduction of 50% in total cholesterol and LDL cholesterol serum levels in the lycopene group. The amount of cholesteryl ester in the aorta was significantly decreased by lycopene. However, we did not observe a significant decrease in the extent of aortic surface lipid accumulation in the lycopene group. In addition, no differences in the intima-media thickness among groups were observed. Endothelial-dependent and endothelial-independent vasodilation in isolated rabbit aortic and carotid rings did not differ among any of the animal groups.

Conclusions

Lycopene supplementation for 4 weeks increased lycopene plasma levels in the animals. Although we found strongly reduced total and LDL cholesterol serum levels as well as significantly lower amounts of cholesteryl ester in the aortae in the lycopene-treated group, no significant differences in initial lesions in the aortae were detected.     

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation

Background

Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis.

Methodology/Principal Findings

We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis.

Conclusions/Significance

We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.     

Incidence and Characteristics of Bacteremia among Children in Rural Ghana

The objective of the study was to describe systemic bacterial infections occurring in acutely ill and hospitalized children in a rural region in Ghana, regarding frequency, incidence, antimicrobial susceptibility patterns and associations with anthropometrical data.Blood cultures were performed in all children below the age of five years, who were admitted to Agogo Presbyterian Hospital (APH), Asante Region, Ghana, between September 2007 and July 2009. Medical history and anthropometrical data were assessed using a standardized questionnaire at admission. Incidences were calculated after considering the coverage population adjusted for village-dependent health-seeking behavior.Among 1,196 hospitalized children, 19.9% (n = 238) were blood culture positive. The four most frequent isolated pathogens were nontyphoidal salmonellae (NTS) (53.3%; n = 129), Staphylococcus aureus (13.2%; n = 32), Streptococcus pneumoniae (9.1%; n = 22) and Salmonella ser. Typhi (7.0%; n = 17). Yearly cumulative incidence of bacteremia was 46.6 cases/1,000 (CI 40.9–52.2). Yearly cumulative incidences per 1,000 of the four most frequent isolates were 25.2 (CI 21.1–29.4) for NTS, 6.3 (CI 4.1–8.4) for S. aureus, 4.3 (CI 2.5–6.1) for S. pneumoniae and 3.3 (CI 1.8–4.9) for Salmonella ser. Typhi. Wasting was positively associated with bacteremia and systemic NTS bloodstream infection. Children older than three months had more often NTS bacteremia than younger children. Ninety-eight percent of NTS and 100% of Salmonella ser. Typhi isolates were susceptible to ciprofloxacin, whereas both tested 100% susceptible to ceftriaxone. Seventy-seven percent of NTS and 65% of Salmonella ser. Typhi isolates were multi-drug resistant (MDR). Systemic bacterial infections in nearly 20% of hospitalized children underline the need for microbiological diagnostics, to guide targeted antimicrobial treatment and prevention of bacteremia. If microbiological diagnostics are lacking, calculated antimicrobial treatment of severely ill children in malaria-endemic areas should be considered.     

Comparison of Two Commercial PCR Methods for Methicillin-Resistant Staphylococcus aureus (MRSA) Screening in a Tertiary Care Hospital

Nose/throat-swabs from 1049 patients were screened for MRSA using CHROMagar MRSA, LightCycler Advanced MRSA, and Detect-Ready MRSA. Results were compared to the CHROMagar MRSA results, which was set as reference system. MRSA was detected in 3.05% of the patients with CHROMagar MRSA. LightCycler MRSA Advanced showed a higher clinical sensitivity (84.38%) than Detect-Ready MRSA (57.69%).The negative predictive values were high for both tests (>98%). The specificity and the positive predictive value were higher for the Detect-Ready MRSA test than for the LightCycler MRSA test (99.59% and 78.95% versus 98.52% and 64.29%). For routine screening LightCycler MRSA Advanced proved to be more efficient in our clinical setting as the clinical sensitivity was much higher than the sensitivity of Detect-Ready MRSA. CHROMagar MRSA detected more MRSA positive samples than both PCR methods, leading to the conclusion that the combination of PCR with cultural screening is still the most reliable way for the detection of MRSA. LightCycler MRSA Advanced was faster and needed less hands-on time. The advantage of Detect-Ready MRSA was the additional identification of methicillin-sensitive S.aureus (here in 34.63% of the samples), an information which can be possibly used for reducing the risk of postoperative infections in surgical patients in future.     

Biochemical characterization and N-terminomics analysis of leukolysin, the membrane-type 6 matrix metalloprotease (MMP25): chemokine and vimentin cleavages enhance cell migration and macrophage phagocytic activities

The neutrophil-specific protease membrane-type 6 matrix metalloproteinase (MT6-MMP)/MMP-25/leukolysin is implicated in multiple sclerosis and cancer yet remains poorly characterized. To characterize the biological roles of MT6-MMP, it is critical to identify its substrates for which only seven are currently known. Here, we biochemically characterized MT6-MMP, profiled its tissue inhibitor of metalloproteinase inhibitory spectrum, performed degradomics analyses, and screened 26 chemokines for cleavage using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. MT6-MMP processes seven each of the CXC and CC chemokine subfamilies. Notably, cleavage of the neutrophil chemoattractant CXCL5 activates the chemokine, thereby increasing its agonist activity, indicating a feed-forward mechanism for neutrophil recruitment. Likewise, cleavage also activated CCL15 and CCL23 to increase monocyte recruitment. Utilizing the proteomics approach proteomic identification of cleavage site specificity (PICS), we identified 286 peptidic cleavage sites spanning from P6 to P6' from which an unusual glutamate preference in P1 was identified. The degradomics screen terminal amine isotopic labeling of substrates (TAILS), which enriches for neo-N-terminal peptides of cleaved substrates, was used to identify 58 new native substrates in fibroblast secretomes after incubation with MT6-MMP. Vimentin, cystatin C, galectin-1, IGFBP-7, and secreted protein, acidic and rich in cysteine (SPARC) were among those substrates we biochemically confirmed. An extracellular "moonlighting" form of vimentin is a chemoattractant for THP-1 cells, but MT6-MMP cleavage abolished monocyte recruitment. Unexpectedly, the MT6-MMP-cleaved vimentin potently stimulated phagocytosis, which was not a property of the full-length protein. Hence, MT6-MMP regulates neutrophil and monocyte chemotaxis and by generating "eat-me" signals upon vimentin cleavage potentially increases phagocytic removal of neutrophils to resolve inflammation.     

Evidence for Chemokine-mediated Coalescence of Preformed Flotillin Hetero-oligomers in Human T-cells

We have shown previously that endogenous flotillin-1 and -2, closely related proteins implicated in scaffolding of membrane microdomains, are rapidly recruited to the uropods of chemoattractant-stimulated human neutrophils and T-cells and are involved in cell polarization. Coexpressed flotillin-1 and -2, but not singly expressed proteins, are also targeted to the uropod of T-cells and neutrophils. Biochemical studies suggest formation of flotillin homo- and hetero-oligomers in other cell types, but so far knowledge is lacking on in situ flotillin organization in leukocytes. We have now analyzed flotillin organization in human T-cells using fluorescence resonance energy transfer (FRET). Coexpressed C-terminally tagged flotillin-1-mCherry and flotillin-2-enhanced green fluorescent protein (EGFP) show significant FRET when analyzed in intact human T-cells in the absence and presence of chemokine. In contrast, little FRET was observed between coexpressed flotillin-1-mCherry and flotillin-1-EGFP before or after chemokine addition, indicating predominant formation of heterodimers and/or -oligomers. Interestingly coexpression of untagged flotillin-2 strongly enhanced FRET between differently tagged flotillin-1 molecules in resting and chemokine-stimulated cells, indicating that close contacts of flotillin-1 molecules only occur in flotillin-2-containing hetero-oligomers. Comparable results were obtained for tagged flotillin-2. We further show that disruption of the actin network, depletion of intracellular calcium, and inhibition of phospholipase C all result in suppression of chemokine-induced polarization and flotillin cap formation, but do not abolish FRET between tagged flotillin-1 and -2. Our results support predominant formation of flotillin-1 and -2 hetero-oligomers in resting and chemokine-stimulated human T-cells which may importantly contribute to structuring of the uropod.     

Regulation of retinal proteome by topical antiglaucomatous eye drops in an inherited glaucoma rat model

Examination of the response of the retinal proteome to elevated intraocular pressure (IOP) and to the pharmacological normalization of IOP is crucial, in order to develop drugs with neuroptorective potential. We used a hereditary rat model of ocular hypertension to lower IOP with travaprost and dorzolamide applied topically on the eye surface, and examine changes of the retinal proteome. Our data demonstrate that elevated IOP causes alterations in the retinal protein profile, in particular in high-mobility-group-protein B1 (HMGB1), calmodulin, heat-shock-protein (HSP) 70 and carbonic anhydrase II expression. The changes of the retinal proteome by dorzolamide or travoprost are different and independent of the IOP lowering effect. This fact suggests that the eye drops exert a direct IOP-independent effect on retinal metabolism. Further investigations are required to elucidate the potential neuroprotective mechanisms signaled through changes of HMGB1, calmodulin, HSP70 and carbonic anhydrase II expression in glaucoma. The data may facilitate development of eye drops that exert neuroprotection through direct pharmacological effect.     

Quantification of Ligand Binding to G-Protein Coupled Receptors on Cell Membranes by Ellipsometry

G-protein-coupled receptors (GPCRs) are prime drug targets and targeted by approximately 60% of current therapeutic drugs such as β-blockers, antipsychotics and analgesics. However, no biophysical methods are available to quantify their interactions with ligand binding in a native environment. Here, we use ellipsometry to quantify specific interactions of receptors within native cell membranes. As a model system, the GPCR-ligand CXCL12α and its receptor CXCR4 are used. Human-derived Ishikawa cells were deposited onto gold coated slides via Langmuir-Schaefer film deposition and interactions between the receptor CXCR4 on these cells and its ligand CXCL12α were detected via total internal reflection ellipsometry (TIRE). This interaction could be inhibited by application of the CXCR4-binding drug AMD3100. Advantages of this approach are that it allows measurement of interactions in a lipid environment without the need for labelling, protein purification or reconstitution of membrane proteins. This technique is potentially applicable to a wide variety of cell types and their membrane receptors, providing a novel method to determine ligand or drug interactions targeting GPCRs and other membrane proteins.