Proteinuria Increases Plasma Phosphate by Altering Its Tubular Handling.

Proteinuria and hyperphosphatemia are cardiovascular risk factors independent of GFR. We hypothesized that proteinuria induces relative phosphate retention via increased proximal tubule phosphate reabsorption. To test the clinical relevance of this hypothesis, we studied phosphate handling in nephrotic children and patients with CKD. Plasma fibroblast growth factor 23 (FGF-23) concentration, plasma phosphate concentration, and tubular reabsorption of phosphate increased during the proteinuric phase compared with the remission phase in nephrotic children. Cross-sectional analysis of a cohort of 1738 patients with CKD showed that albuminuria≥300 mg/24 hours is predictive of higher phosphate levels, independent of GFR and other confounding factors. Albuminuric patients also displayed higher plasma FGF-23 and parathyroid hormone levels. To understand the molecular mechanisms underlying these observations, we induced glomerular proteinuria in two animal models. Rats with puromycin-aminonucleoside-induced nephrotic proteinuria displayed higher renal protein expression of the sodium-phosphate co-transporter NaPi-IIa, lower renal Klotho protein expression, and decreased phosphorylation of FGF receptor substrate 2α, a major FGF-23 receptor substrate. These findings were confirmed in transgenic mice that develop nephrotic-range proteinuria resulting from podocyte depletion. In vitro, albumin did not directly alter phosphate uptake in cultured proximal tubule OK cells. In conclusion, we show that proteinuria increases plasma phosphate concentration independent of GFR. This effect relies on increased proximal tubule NaPi-IIa expression secondary to decreased FGF-23 biologic activity. Proteinuria induces elevation of both plasma phosphate and FGF-23 concentrations, potentially contributing to cardiovascular disease.

Impact of platelet rich plasma and adipose stem cells on lymphangiogenesis in a murine tail lymphedema model.

BACKGROUND: Lymphedema is an underdiagnosed pathology which in industrialized countries mainly affects cancer patients that underwent lymph node dissection and/or radiation. Currently no effective therapy is available so that patients' life quality is compromised by swellings of the concerned body region. This unfortunate condition is associated with body imbalance and subsequent osteochondral deformations and impaired function as well as with an increased risk of potentially life threatening soft tissue infections. METHODS: The effects of PRP and ASC on angiogenesis (anti-CD31 staining), microcirculation (Laser Doppler Imaging), lymphangiogenesis (anti-LYVE1 staining), microvascular architecture (corrosion casting) and wound healing (digital planimetry) are studied in a murine tail lymphedema model. RESULTS: Wounds treated by PRP and ASC healed faster and showed a significantly increased epithelialization mainly from the proximal wound margin. The application of PRP induced a significantly increased lymphangiogenesis while the application of ASC did not induce any significant change in this regard. CONCLUSIONS: PRP and ASC affect lymphangiogenesis and lymphedema development and might represent a promising approach to improve regeneration of lymphatic vessels, restore disrupted lymphatic circulation and treat or prevent lymphedema alone or in combination with currently available lymphedema therapies.

Neuroenergetic Response to Prolonged Cerebral Glucose Depletion after Severe Brain Injury and the Role of Lactate.

Lactate may represent a supplemental fuel for the brain. We examined cerebral lactate metabolism during prolonged brain glucose depletion (GD) in acute brain injury (ABI) patients monitored with cerebral microdialysis (CMD). Sixty episodes of GD (defined as spontaneous decreases of CMD glucose from normal to low [<1.0 mmol/L] for at least 2 h) were identified among 26 patients. During GD, we found a significant increase of CMD lactate (from 4±2.3 to 5.4±2.9 mmol/L), pyruvate (126.9±65.1 to 172.3±74.1 μmol/L), and lactate/pyruvate ratio (LPR; 27±6 to 35±9; all, p<0.005), while brain oxygen and blood lactate remained normal. Dynamics of lactate and glucose supply during GD were further studied by analyzing the relationships between blood and CMD samples. There was a strong correlation between blood and brain lactate when LPR was normal (r=0.56; p<0.0001), while an inverse correlation (r=-0.11; p=0.04) was observed at elevated LPR >25. The correlation between blood and brain glucose also decreased from r=0.62 to r=0.45. These findings in ABI patients suggest increased cerebral lactate delivery in the absence of brain hypoxia when glucose availability is limited and support the concept that lactate acts as alternative fuel.

Distribution of monocarboxylate transporters in the peripheral nervous system suggests putative roles in lactate shuttling and myelination.

Lactate, a product of glycolysis, has been shown to play a key role in the metabolic support of neurons/axons in the CNS by both astrocytes and oligodendrocytes through monocarboxylate transporters (MCTs). Despite such importance in the CNS, little is known about MCT expression and lactate function in the PNS. Here we show that mouse MCT1, MCT2, and MCT4 are expressed in the PNS. While DRG neurons express MCT1, myelinating Schwann cells (SCs) coexpress MCT1 and MCT4 in a domain-specific fashion, mainly in regions of noncompact myelin. Interestingly, SC-specific downregulation of MCT1 expression in rat neuron/SC cocultures led to increased myelination, while its downregulation in neurons resulted in a decreased amount of neurofilament. Finally, pure rat SCs grown in the presence of lactate exhibited an increase in the level of expression of the main myelin regulator gene Krox20/Egr2 and the myelin gene P0. These data indicate that lactate homeostasis participates in the regulation of the SC myelination program and reveal that similar to CNS, PNS axon-glial metabolic interactions are most likely mediated by MCTs.

Plasma cell and terminal B-cell differentiation in mantle cell lymphoma mainly occur in the SOX11-negative subtype.

Mantle cell lymphoma is a mature lymphoid neoplasm characterized by the t(11;14)(q13;q32) and cyclin D1 overexpression. SOX11 is a transcription factor commonly overexpressed in these tumors but absent in most other mature B-cell lymphomas whose function is not well understood. Experimental studies have shown that silencing of SOX11 in mantle cell lymphoma cells promotes the shift from a mature B cell into an early plasmacytic differentiation phenotype, suggesting that SOX11 may contribute to tumor development by blocking the B-cell differentiation program. The relationship between SOX11 expression and terminal B-cell differentiation in primary mantle cell lymphoma and its relationship to the plasmacytic differentiation observed in occasional cases is not known. In this study we have investigated the terminal B-cell differentiation phenotype in 60 mantle cell lymphomas, 41 SOX11-positive and 19 SOX11-negative. Monotypic plasma cells and lymphoid cells with plasmacytic differentiation expressing cyclin D1 were observed in 7 (37%) SOX11-negative but in none of 41 SOX11-positive mantle cell lymphomas (P<0.001). Intense cytoplasmic expression of a restricted immunoglobulin light chain was significantly more frequent in SOX11-negative than -positive tumors (58 vs 13%) (P=0.001). Similarly, BLIMP1 and XBP1 expression was also significantly more frequent in SOX11-negative than in -positive cases (83 vs 34% and 75 vs 11%, respectively) (P=0.001). However, no differences in the expression of IRF4/MUM1 were observed among these subtypes of mantle cell lymphoma. In conclusion, these results indicate that SOX11-negative mantle cell lymphoma may be a particular subtype of this tumor characterized by more frequent morphological and immunophenotypic terminal B-cell differentiation features that may be facilitated by the absence of SOX11 transcription factor.

The mediterranean diet pattern and its main components are associated with lower plasma concentrations of tumor necrosis factor receptor 60 in patients at high risk for cardiovascular disease

Adherence to aMediterranean diet (MD) is associated with a reduced risk of coronary heart disease. However, themolecular mechanisms involved are not fully understood. The aim of this studywas to compare the effects of 2MD with those of a lowfat- diet (LFD) on circulating inflammatory biomarkers related to atherogenesis. A total of 516 participants included in the PreventionwithMediterraneanDiet Studywere randomized into 3 intervention groups [MD supplementedwith virgin olive oil (MD-VOO); MD supplemented with mixed nuts (MD-Nuts); and LFD]. At baseline and after 1 y, participants completed FFQ and adherence to MD questionnaires, and plasma concentrations of inflammatory markers including intercellular adhesion molecule-1(ICAM-1), IL-6, and 2 TNF receptors (TNFR60 and TNFR80) were measured by ELISA. At 1 y, the MD groups had lower plasma concentrations of IL-6, TNFR60, and TNFR80 (P , 0.05), whereas ICAM-1, TNFR60, and TNFR80 concentrations increased in the LFD group (P , 0.002). Due to between-group differences, participants in the 2 MD groups had lower plasma concentrations of ICAM-1, IL-6, TNFR60, and TNFR80 compared to those in the LFD group (P # 0.028). When participants were categorized in tertiles of 1-y changes in the consumption of selected foods, those in the highest tertile of virgin olive oil (VOO) and vegetable consumption had a lower plasma TNFR60 concentration compared with those in tertile 1 (P,0.02).Moreover, the only changes in consumption thatwere associated with 1-y changes in the geometricmean TNFR60 concentrations were those of VOO and vegetables (P = 0.01). This study suggests that a MD reduces TNFR concentrations in patients at high cardiovascular risk.

CSF lactate for accurate diagnosis of community-acquired bacterial meningitis.

CSF lactate measurement is recommended when nosocomial meningitis is suspected, but its value in community-acquired bacterial meningitis is controversial. We evaluated the diagnostic performance of lactate and other CSF parameters in a prospective cohort of adult patients with acute meningitis. Diagnostic accuracy of lactate and other CSF parameters in patients with microbiologically documented episodes was assessed by receiver operating characteristic (ROC) curves. The cut-offs with the best diagnostic performance were determined. Forty-five of 61 patients (74%) had a documented bacterial (n = 18; S. pneumoniae, 11; N. meningitidis, 5; other, 2) or viral (n = 27 enterovirus, 21; VZV, 3; other, 3) etiology. CSF parameters were significantly different in bacterial vs. viral meningitis, respectively (p < 0.001 for all comparisons): white cell count (median 1333 vs. 143/mm(3)), proteins (median 4115 vs. 829 mg/l), CSF/blood glucose ratio (median 0.1 vs. 0.52), lactate (median 13 vs. 2.3 mmol/l). ROC curve analysis showed that CSF lactate had the highest accuracy for discriminating bacterial from viral meningitis, with a cutoff set at 3.5 mmol/l providing 100% sensitivity, specificity, PPV, NPV, and efficiency. CSF lactate had the best accuracy for discriminating bacterial from viral meningitis and should be included in the initial diagnostic workup of this condition.

In Vivo Profiling and Distribution of Known and Novel Phase I and Phase II Metabolites of Efavirenz in Plasma, Urine, and Cerebrospinal Fluid.

Efavirenz (EFV) is principally metabolized by CYP2B6 to 8-hydroxy-efavirenz (8OH-EFV) and to a lesser extent by CYP2A6 to 7-hydroxy-efavirenz (7OH-EFV). So far, most metabolite profile analyses have been restricted to 8OH-EFV, 7OH-EFV, and EFV-N-glucuronide, even though these metabolites represent a minor percentage of EFV metabolites present in vivo. We have performed a quantitative phase I and II metabolite profile analysis by tandem mass spectrometry of plasma, cerebrospinal fluid (CSF), and urine samples in 71 human immunodeficiency virus patients taking efavirenz, prior to and after enzymatic (glucuronidase and sulfatase) hydrolysis. We have shown that phase II metabolites constitute the major part of the known circulating efavirenz species in humans. The 8OH-EFV-glucuronide (gln) and 8OH-EFV-sulfate (identified for the first time) in humans were found to be 64- and 7-fold higher than the parent 8OH-EFV, respectively. In individuals (n = 67) genotyped for CYP2B6, 2A6, and CYP3A metabolic pathways, 8OH-EFV/EFV ratios in plasma were an index of CYP2B6 phenotypic activity (P < 0.0001), which was also reflected by phase II metabolites 8OH-EFV-glucuronide/EFV and 8OH-EFV-sulfate/EFV ratios. Neither EFV nor 8OH-EFV, nor any other considered metabolites in plasma were associated with an increased risk of central nervous system (CNS) toxicity. In CSF, 8OH-EFV levels were not influenced by CYP2B6 genotypes and did not predict CNS toxicity. The phase II metabolites 8OH-EFV-gln, 8OH-EFV-sulfate, and 7OH-EFV-gln were present in CSF at 2- to 9-fold higher concentrations than 8OH-EFV. The potential contribution of known and previously unreported EFV metabolites in CSF to the neuropsychological effects of efavirenz needs to be further examined in larger cohort studies.

Blood pressure reductions following catheter-based renal denervation are not related to improvements in adherence to antihypertensive drugs measured by urine/plasma toxicological analysis.

Renal denervation can reduce blood pressure in patients with uncontrolled hypertension. The adherence to prescribed antihypertensive medication following renal denervation is unknown. This study investigated adherence to prescribed antihypertensive treatment by liquid chromatography-high resolution tandem mass spectrometry in plasma and urine at baseline and 6 months after renal denervation in 100 patients with resistant hypertension, defined as baseline office systolic blood pressure ≥140 mmHg despite treatment with ≥3 antihypertensive agents. At baseline, complete adherence to all prescribed antihypertensive agents was observed in 52 patients, 46 patients were partially adherent, and two patients were completely non-adherent. Baseline office blood pressure was 167/88 ± 19/16 mmHg with a corresponding 24-h blood pressure of 154/86 ± 15/13 mmHg. Renal denervation significantly reduced office and ambulatory blood pressure at 6-month follow-up by 15/5 mmHg (p < 0.001/p < 0.001) and 8/4 mmHg (p < 0.001/p = 0.001), respectively. Mean adherence to prescribed treatment was significantly reduced from 85.0 % at baseline to 80.7 %, 6 months after renal denervation (p = 0.005). The blood pressure decrease was not explained by improvements in adherence following the procedure. Patients not responding to treatment significantly reduced their drug intake following the procedure. Adherence was highest for angiotensin-converting enzyme inhibitors/angiotensin receptor blockers and beta blockers (>90 %) and lowest for vasodilators (21 %). In conclusion, renal denervation can reduce office and ambulatory blood pressure in patients with resistant hypertension despite a significant reduction in adherence to antihypertensive treatment after 6 months.

Optimized exosome isolation protocol for cell culture supernatant and human plasma.

Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.

A probable dual mode of action for both L- and D-lactate neuroprotection in cerebral ischemia.

Lactate has been shown to offer neuroprotection in several pathologic conditions. This beneficial effect has been attributed to its use as an alternative energy substrate. However, recent description of the expression of the HCA1 receptor for lactate in the central nervous system calls for reassessment of the mechanism by which lactate exerts its neuroprotective effects. Here, we show that HCA1 receptor expression is enhanced 24 hours after reperfusion in an middle cerebral artery occlusion stroke model, in the ischemic cortex. Interestingly, intravenous injection of L-lactate at reperfusion led to further enhancement of HCA1 receptor expression in the cortex and striatum. Using an in vitro oxygen-glucose deprivation model, we show that the HCA1 receptor agonist 3,5-dihydroxybenzoic acid reduces cell death. We also observed that D-lactate, a reputedly non-metabolizable substrate but partial HCA1 receptor agonist, also provided neuroprotection in both in vitro and in vivo ischemia models. Quite unexpectedly, we show D-lactate to be partly extracted and oxidized by the rodent brain. Finally, pyruvate offered neuroprotection in vitro whereas acetate was ineffective. Our data suggest that L- and D-lactate offer neuroprotection in ischemia most likely by acting as both an HCA1 receptor agonist for non-astrocytic (most likely neuronal) cells as well as an energy substrate.

Comission 49: Interplanetary plasma and heliosphere. TRIENNIAL REPORT 2006 - 2009

Commission 49 covers research on the solar wind, shocks and particle acceleration, both transient and steady-state, e.g., corotating, structures within the heliosphere, and the termination shock and boundary of the heliosphere. The present triennal report is particularly rich in important results and events. The crossing of the solar wind termination shock by Voyager 2 in 2007 is a highlight and a milestone that will certainly have important consequences for astrophysical processes in general (Section 7). The fiftieth anniversary of the International Geophysical Year (19571958), which is also the fiftieth anniversary of the birth of the Space Age, was marked not only by celebrations and a strong Education and Public Outreach Program, but also by efforts in coordinating present observations and in starting new scientific programs, particularly implying developing countries (Section 8). Studies of solar energetic particles (Section 3) and the related radio bursts (Section 4) benefited from new data from a number of spacecraft. The STEREO mission was launched in October 2006 and has obtained new results on 3-D aspects of the inner heliosphere. Meanwhile, solar cycle 24 is expected to become active soon, following what is already the deepest solar minimum of the space age...

L-Lactate protects neurons against excitotoxicity: implication of an ATP-mediated signaling cascade.

Converging experimental data indicate a neuroprotective action of L-Lactate. Using Digital Holographic Microscopy, we observe that transient application of glutamate (100 μM; 2 min) elicits a NMDA-dependent death in 65% of mouse cortical neurons in culture. In the presence of L-Lactate (or Pyruvate), the percentage of neuronal death decreases to 32%. UK5099, a blocker of the Mitochondrial Pyruvate Carrier, fully prevents L-Lactate-mediated neuroprotection. In addition, L-Lactate-induced neuroprotection is not only inhibited by probenicid and carbenoxolone, two blockers of ATP channel pannexins, but also abolished by apyrase, an enzyme degrading ATP, suggesting that ATP produced by the Lactate/Pyruvate pathway is released to act on purinergic receptors in an autocrine/paracrine manner. Finally, pharmacological approaches support the involvement of the P2Y receptors associated to the PI3-kinase pathway, leading to activation of KATP channels. This set of results indicates that L-Lactate acts as a signalling molecule for neuroprotection against excitotoxicity through coordinated cellular pathways involving ATP production, release and activation of a P2Y/KATP cascade.