Dysregulated copper transport in multiple sclerosis may cause demyelination via astrocytes.

Demyelination is a key pathogenic feature of multiple sclerosis (MS). Here, we evaluated the astrocyte contribution to myelin loss and focused on the neurotrophin receptor TrkB, whose up-regulation on the astrocyte finely demarcated chronic demyelinated areas in MS and was paralleled by neurotrophin loss. Mice lacking astrocyte TrkB were resistant to demyelination induced by autoimmune or toxic insults, demonstrating that TrkB signaling in astrocytes fostered oligodendrocyte damage. In vitro and ex vivo approaches highlighted that astrocyte TrkB supported scar formation and glia proliferation even in the absence of neurotrophin binding, indicating TrkB transactivation in response to inflammatory or toxic mediators. Notably, our neuropathological studies demonstrated copper dysregulation in MS and model lesions and TrkB-dependent expression of copper transporter (CTR1) on glia cells during neuroinflammation. In vitro experiments evidenced that TrkB was critical for the generation of glial intracellular calcium flux and CTR1 up-regulation induced by stimuli distinct from neurotrophins. These events led to copper uptake and release by the astrocyte, and in turn resulted in oligodendrocyte loss. Collectively, these data demonstrate a pathogenic demyelination mechanism via the astrocyte release of copper and open up the possibility of restoring copper homeostasis in the white matter as a therapeutic target in MS.

Erythroblast apoptosis and microenvironmental iron restriction trigger anemia in the VK*MYC model of multiple myeloma.

Multiple myeloma is a malignant disorder characterized by bone marrow proliferation of plasma cells and by overproduction of monoclonal immunoglobulin detectable in the sera (M-spike). Anemia is a common complication of multiple myeloma, but the underlying pathophysiological mechanisms have not been completely elucidated. We aimed to identify the different determinants of anemia using the Vk*MYC mouse, which spontaneously develops an indolent bone marrow localized disease with aging. Affected Vk*MYC mice develop a mild normochromic normocytic anemia. We excluded the possibility that anemia results from defective erythropoietin production, inflammation or increased hepcidin expression. Mature erythroid precursors are reduced in Vk*MYC bone marrow compared with wild-type. Malignant plasma cells express the apoptogenic receptor Fas ligand and, accordingly, active caspase 8 is detected in maturing erythroblasts. Systemic iron homeostasis is not compromised in Vk*MYC animals, but high expression of the iron importer CD71 by bone marrow plasma cells and iron accumulation in bone marrow macrophages suggest that iron competition takes place in the local multiple myeloma microenvironment, which might contribute to anemia. In conclusion, the mild anemia of the Vk*MYC model is mainly related to the local effect of the bone marrow malignant clone in the absence of an overt inflammatory status. We suggest that this reproduces the initial events triggering anemia in patients.

Protein profiling reveals energy metabolism and cytoskeletal protein alterations in LMNA mutation carriers.

Nuclear envelope-related muscular dystrophies, in particular those referred to as laminopathies, are relatively novel and unclear diseases, also considering the increasing number of mutations identified so far in genes of the nuclear envelope. As regard LMNA gene, only tentative relations between phenotype, type and localization of the mutations have been established in striated muscle diseases, while laminopathies affecting adipose tissue, peripheral nerves or progerioid syndromes could be linked to specific genetic variants. This study describes the biochemical phenotype of neuromuscular laminopathies in samples derived from LMNA mutant patients. Since it has been reported that nuclear alterations, due to LMNA defects, are present also in fibroblasts from Emery-Dreifuss muscular dystrophy and familial partial lipodystrophy patients, we analyzed 2D-maps of skin fibroblasts of patients carrying 12 different LMNA mutations spread along the entire gene. To recognize distinctive proteins underlying affected biochemical pathways, we compared them with fibroblasts from healthy controls and, more importantly, fibroblasts from patients with non-lamin related neuromuscular disorders. We found less abundance of cytoskeletal/structural proteins, confirming a dominant role for Lamin A/C in structural support of nuclear architecture. Interestingly, we also established significant changes in the expression of proteins involved in cellular energy production and oxidative stress response. To our knowledge, this is the first report where proteomics was applied to characterize ex-vivo cells from LMNA patients, suggesting that this may represent a new approach to better understand the molecular mechanisms of these rare diseases and facilitate the development of novel therapeutic treatments.

Genetic polymorphisms of the enzymes involved in DNA methylation and synthesis in elite athletes.

Physical exercise induces adaptive changes leading to a muscle phenotype with enhanced performance. We first investigated whether genetic polymorphisms altering enzymes involved in DNA methylation, probably responsible of DNA methylation deficiency, are present in athletes' DNA. We determined the polymorphic variants C667T/A1298C of 5,10-methylenetetrahydrofolate reductase (MTHFR), A2756G of methionine synthase (MTR), A66G of methionine synthase reductase (MTRR), G742A of betaine:homocysteine methyltransferase (BHMT), and 68-bp ins of cystathionine ß-synthase (CBS) genes in 77 athletes and 54 control subjects. The frequency of MTHFR (AC), MTR (AG), and MTRR (AG) heterozygous genotypes was found statistically different in the athletes compared with the control group (P=0.0001, P=0.018, and P=0.0001), suggesting a reduced DNA methylating capacity. We therefore assessed whether DNA hypomethylation might increase the expression of myogenic proteins expressed during early (Myf-5 and MyoD), intermediate (Myf-6), and late-phase (MHC) of myogenesis in a cellular model of hypomethylated or unhypomethylated C2C12 myoblasts. Myogenic proteins are largely induced in hypomethylated cells [fold change (FC)=Myf-5: 1.21, 1.35; MyoD: 0.9, 1.47; Myf-6: 1.39, 1.66; MHC: 1.35, 3.10 in GMA, DMA, respectively] compared with the control groups (FC=Myf-5: 1.0, 1.38; MyoD: 1.0, 1.14; Myf-6: 1.0, 1.44; MHC: 1.0, 2.20 in GM, DM, respectively). Diameters and length of hypomethylated myotubes were greater then their respective controls. Our findings suggest that DNA hypomethylation due to lesser efficiency of polymorphic MTHFR, MS, and MSR enzymes induces the activation of factors determining proliferation and differentiation of myoblasts promoting muscle growth and increase of muscle mass.

Comparison of different depletion strategies for improving resolution of the human urine proteome.

BACKGROUND: Urine, being an ultrafiltrate of plasma, is a rich source for biomarker discovery. Since potential new disease markers are often present in low concentrations, a prefractionation/enrichment step could be useful in the discovery process. To enhance the detection of low-abundance proteins, three immuno-affinity depletion approaches were evaluated. METHODS: To remove the most abundant proteins from a human urine sample, GenWay Spin IgY-12 kit, HPLC Agilent Hu-PL7 and a home-made column vs. human serum albumin [immuno-affinity column (IAC)] were compared. Quantification of total proteins, 2-D gel electrophoresis (2-DE), Progenesis gel images analysis and mass spectrometric proteins identification were applied to evaluate these strategies. RESULTS: Reproducibility of depletion columns, by estimating protein content of unbound fractions, were: 343+/-20.0 microg, 5.8%; 186.3+/-13.3 microg, 7.2%; 292+/-20.6 microg, 8.8% [mean+/- standard deviation (SD), CV%], for GenWay, Agilent and IAC methods, respectively. To isolate urinary protein after depletion, ethanol precipitation provided the highest recovery (80%). Applying 2-DE and Progenesis analysis, the number of spots visualized on the gels was 468+/-21, 331+/-7, 368+/-22 and 304+/-7 (mean+/-SD) for GenWay, Agilent, IAC, and the undepleted urine pool sample, respectively, with a significant difference p<0.001 compared to the GenWay procedure. CONCLUSIONS: The sequential procedure of urine samples using multi-protein immuno-affinity depletion represents a valid tool for simplifying 2-DE analysis of the urine proteome. Particularly, the GenWay kit followed by ethanol precipitation was found to be the most efficient method for exploring the urine proteome.

Telomere length in peripheral blood mononuclear cells is associated with folate status in men.

Human chromosomes are capped by telomeres, which consist of tandem repeats of DNA and associated proteins. The length of the telomeres is reduced with increasing cell divisions except when the enzyme telomerase is active, as in stem cells and germ cells. Telomere dysfunction has been associated with development of age-related pathologies, including cancer, cardiovascular disease, Alzheimer's disease, and Parkinson's disease. DNA damage in the telomeric region causes attrition of telomeres. Because folate provides precursors for nucleotide synthesis and thus affects the integrity of DNA, including that of the telomeric region, folate status has the potential to influence telomere length. Telomere length is epigenetically regulated by DNA methylation, which in turn could be modulated by folate status. In this study, we determined whether folate status and the 677C > T polymorphism of the methylene tetrahydrofolate reductase (MTHFR) gene are associated with the telomere length of peripheral blood mononuclear cells in healthy men. The results of our study showed that plasma concentration of folate was associated with telomere length of peripheral blood mononuclear cells in a nonlinear manner. When plasma folate concentration was above the median, there was a positive relationship between folate and telomere length. In contrast, there was an inverse relationship between folate and telomere length when plasma folate concentration was below the median. The MTHFR 677C > T polymorphism was weakly associated (P = 0.065) with increased telomere length at below-median folate status. We propose that folate status influences telomere length by affecting DNA integrity and the epigenetic regulation of telomere length through DNA methylation.

No adjustment vs. adjustment formula as input weight for propofol target-controlled infusion in morbidly obese patients.

BACKGROUND AND OBJECTIVE: The purpose of this prospective, randomized, double-blind study was to determine the predictive performance of target-controlled infusions of propofol in morbidly obese patients using the 'Marsh' pharmacokinetic parameter set. METHODS: Twenty-four patients (ASA II or III, age 25-62 years, BMI 35.5-61.7) were randomly allocated to receive propofol target-controlled infusion based on a weight adjustment formula (group adjusted) or without adjustment [group total body weight (TBW)]. Anaesthesia was induced by a propofol-targeted concentration of 6 microg ml that was subsequently adapted to maintain stable bispectral index values ranging between 40 and 50. Arterial blood samples were collected before the start of the infusion and every 15 min thereafter to determine the predictive performances. RESULTS: There were no statistically significant differences between the groups with regard to performance errors, divergence and wobble. Results are presented as median (interquartiles). Median performance error and median absolute performance error were -31.7 (-35.9, -19.4) and 31.7% (20.2, 35.9) for group adjusted and -16.3 (-26.3, 2.2) and 20.6% (14.8, 26.9) for group TBW, respectively. Wobble median value was 7.4% (3.8, 8.4) for group adjusted and 8.2% (7.0, 9.6) for group TBW. As for wobble and divergence, no statistically significant differences were found between groups. CONCLUSION: Weight adjustment causes a clinically unacceptable performance bias, which is not corrected when TBW is used as an input to the 'Marsh' model. It is, therefore, advisable to administer propofol to morbidly obese patients by titration to targeted processed-EEG values.

Identification of nephropathy predictors in urine from children with a recent diagnosis of type 1 diabetes.

Despite research progresses, the chance to accurately predict the risk for diabetic nephropathy (DN) is still poor. So far, the first evidence of DN is micro-albuminuria, which is detected only 10-20 years after the onset of diabetes. Our goal is to develop new predictive tools of nephropathy starting from urine, which can be easily obtained using noninvasive procedures and it is directly related to kidney. Since it is reasonable to suppose that, in predisposed patients, the mechanisms leading to nephropathy start acting since the diabetes onset, urine from children with recent diagnosis of type 1 diabetes was subjected to proteomic analysis in comparison to age-matched controls. Targeted confirmation was performed on children with a longer history of diabetes using Western Blotting and applying a urinary lipidomic approach. To definitively understand whether the observed alterations could be related to diabetic nephropathy, urine from diabetic adults with or without albuminuria was also examined. For the first time, lipid metabolisms of prostaglandin and ceramide, which are significantly and specifically modified in association with DN, are shown to be already altered in children with a recent diabetes diagnosis. Future studies on larger cohorts are needed to improve the validity and generalizability of these findings. Data are available via ProteomeXchange with identifier PXD011183 Submission details: Project Name: Urinary proteomics by 2DE and LC-MS/MS. Project accession: PXD011183 Project DOI: https://doi.org/10.6019/PXD011183 SIGNIFICANCE: Nephropathy is a very common diabetic complication. Once established, its progression can only be slowed down but full control or remission is achieved in very few cases, thus posing a large burden on worldwide health. The first evidence of diabetic nephropathy (DN) is micro-albuminuria, but only 30% of patients with micro-albuminuria progress to proteinuria, while in some patients it spontaneously reverts to normo-albuminuria. Thus, there is clear need for biomarkers that can accurately predict the risk to develop DN. Herein, by applying proteomic and lipidomic approaches on urine samples, we show that alteration of prostaglandin and ceramide metabolisms specifically occurs in association with DN. Interestingly, we demonstrate that the modification of these metabolic pathways is an early event in diabetic patients, suggesting the identified changed proteins as possible predictive biomarkers of diabetes-induced renal function decline.

Tight glycemic control does not affect asymmetric-dimethylarginine in septic patients.

OBJECTIVE: We investigated whether preventing hyperglycemia in septic patients affected the plasma concentration of asymmetric-dimethylarginine and if this was associated with clinical benefit. DESIGN: A prospective, multicenter, randomized, controlled, clinical study. SETTING: Intensive care units (ICU) in three university hospitals. PATIENTS: A total of 72 patients admitted for severe sepsis or septic shock, who stayed at least 3 days in the ICU. At admission the patients were assigned to receive either tight or conventional glycemic control. INTERVENTIONS: Determination of circulating levels of asymmetric-dimethylarginine, arginine, interleukin-6, C-reactive-protein and tumor-necrosis-factor-alpha. MEASUREMENTS AND RESULTS: Blood was sampled at admission (no differences between groups), and on the 3rd, 6th, 9th, and 12th (T12) days. Sequential organ failure assessment was scored at each sampling time. All the data were analyzed on an intention-to-treat basis. The control and treatment groups received the same energy intake, glycemia (110.4 +/- 17.3 vs. 163.0 +/- 28.9 mg/dL, P < 0.001) and insulin (P = 0.02) supply differed. No differences were found in high plasma levels of asymmetric-dimethylarginine (P = 0.812) at any time during the ICU stay. The clinical course, as indicated by markers of inflammation, average and maximum organ failure score, ICU stay and ICU and 90-day mortality, was the same. CONCLUSIONS: Intensive insulin treatment, while achieving glucose control, did not reduce asymmetric-dimethylarginine in high-risk septic patients fed with no more than 25 kcal/kg per day to limit ventilatory demand and to simplify glucose control. DESCRIPTOR: 45 (SIRS/sepsis: clinical studies).

Characterization of collagenase blend enzymes for human islet transplantation.

BACKGROUND: Efficient islet isolation represents a necessary requirement for successful islet transplantation as a treatment for type 1 diabetes. The choice of collagenase for pancreas digestion is critical for the isolation outcome, and Liberase is the most widely used enzyme, although large intra-batched variability in activity and efficiency has been observed. METHODS: The aim of this study was to characterize Liberase components and their relative role in pancreas digestion. Liberase batches were characterized by microelectrophoresis. RESULTS: By means of microelectrophoresis, we identified three main proteins each with different prevalences between batches. Two proteins were found to correspond to class I (CI) and one to class II (CII) collagenase. In a series of 163 islet isolations, we observed that the CII correlated with islet yield (P<0.001) and digestion time (P<0.001); additionally, CI directly correlated with purity (P=0.028). Finally, when CII and one of the CI isoforms were >50 percentile, 15 of 36 preparations were transplanted, with 27 of 127 transplanted in the other cases (P=0.013). CONCLUSION: These results represent an important step toward the characterization of enzymes, with the final aim of identifying key components for a standardized product.

Lactulose and mannitol intestinal permeability detected by capillary electrophoresis.

Aim of this study was to set up a method by capillary electrophoresis to detect lactulose and mannitol in urine after an oral load, and to estimate the intestinal permeability in controls and in type I diabetes patients. The underivatized carbohydrates were monitored by indirect UV detection using sorbate, cetyltrimethylammonium bromide and LiOH as background electrolyte. Urines were purified by solid phase extraction, shaken with cation exchange resin, filtered and analysed. Carbohydrates migrated in <10 min in relation to their pK(a) and M(r). Controls (n = 33) and patients (n = 23) had an excretion ratio lactulose/mannitol 0.025 (0.018-0.051) and 0.067 (0.050-0.127), respectively (p < 0.01, median, interquartile range).

Acute intravenous L-arginine infusion decreases endothelin-1 levels and improves endothelial function in patients with angina pectoris and normal coronary arteriograms: correlation with asymmetric dimethylarginine levels.

BACKGROUND: We tested the hypothesis that asymmetric dimethylarginine (ADMA) levels could be elevated and influence endothelin-1 and nitric oxide release and action in patients with cardiac syndrome X (CSX). In addition, we evaluated whether an intravenous infusion of L-arginine would improve endothelial function in these subjects. METHODS AND RESULTS: Nine patients with CSX and 14 control subjects underwent a continuous infusion of L-arginine (0.125 g/min) or saline for 120 minutes. Sixty minutes after L-arginine or saline infusions, an intravenous insulin bolus (0.1 U/kg) combined with a euglycemic clamp was performed. Basal ADMA and endothelin-1 levels were higher in patients with CSX than in controls. At the end of the first hour of infusion, compared with saline, L-arginine infusion increased basal forearm blood flow, nitrite and nitrate (NOx), and forearm cGMP release and decreased endothelin-1. After insulin bolus, during saline, insulin-induced NOx, endothelin-1, and forearm cGMP release was almost abolished. Conversely, L-arginine restored a physiological profile of all endothelial variables compared with control subjects. In control subjects, compared with saline infusion, L-arginine infusion did not modify any parameter. ADMA levels were positively correlated with basal endothelin-1 levels and negatively correlated with insulin-induced incremental levels of NOx and forearm cGMP release. CONCLUSIONS: Plasma ADMA levels are increased in patients with CSX, and they are correlated with increases in endothelin-1 and reductions in insulin-induced increments in plasma NOx and cGMP, effects that are reversed by intravenous L-arginine. These data suggest that increased ADMA levels play a role in the abnormal vascular reactivity that is observed in patients with CSX.

Denaturing HPLC analysis of DNA deletions and insertions.

Denaturing HPLC (DHPLC) is a useful technique for the fast screening of known and unknown heterozygous gene mutations. Most DNA mutations causing genetic disorders consist of nucleotide substitutions, but insertions and deletions occur, albeit less frequently. The heteroduplexes with insertions/deletions have gaps that may affect molecular stability differently from the mismatches caused by substitutions. Therefore, gaps and mismatches may be distinguished by DHPLC analysis, which is based on the differential thermal stability of amplicons with different characteristics. To verify this hypothesis, we examined 12 DNA samples containing insertions and deletions of different sizes (one to 29 residues) from four different genes (ABCA4, CFTR, FTL, and SLC11A3). We found that all of them were detected by DHPLC runs at 50 degrees C, which is considered a non-denaturing temperature, as well as by runs at the temperature optimized for mismatch recognition. The finding confirms that gaps reduce heteroduplex stability more than mismatches, and indicates that DHPLC analysis at low temperature may be applied to distinguish DNA deletions/insertions from substitutions.

Insulin resistance and endothelial function are improved after folate and vitamin B12 therapy in patients with metabolic syndrome: relationship between homocysteine levels and hyperinsulinemia.

OBJECTIVE: The purpose of this study was (a) to study whether a folate and vitamin B12 treatment, aimed at decreasing homocysteine levels, might ameliorate insulin resistance and endothelial dysfunction in patients with metabolic syndrome according to the National Cholesterol Education Program-Adult Treatment Panel-III criteria and (b) to evaluate whether, under these metabolic conditions, there is a relationship between hyperhomocysteinemia and insulin resistance. DESIGN AND METHODS: A double-blind, parallel, identical placebo-drug, randomized study was performed for 2 months in 50 patients. Patients were randomly allocated to two groups. In group 1, patients were treated with diet plus placebo for 2 months. In group 2, patients were treated with diet plus placebo for 1 month, followed by diet plus folic acid (5 mg/day) plus vitamin B12 (500 microg/day) for another month. RESULTS: In group 2, folate treatment significantly decreased homocysteine levels by 27.8% (12.2+/-1.2 vs 8.8+/-0.7 micromol/l; P<0.01). A significant decrement was observed for insulin levels (19.9+/-1.7 vs 14.8+/-1.6 microU/ml; P<0.01) accompanied by a 27% reduction in the homeostasis model assessment levels. A positive relationship was found between the decrement of homocysteine and insulin levels (r=0.60; P<0.002). In parallel, endothelial dysfunction significantly improved in the treated group, since post-ischemic maximal hyperemic vasodilation increased by 29.8% and cGMP by 13.6% while asymmetrical dimethylarginine levels decreased by 21.7%. On the contrary, in group 1 patients, treated with placebo, no changes were shown in any of the variables. CONCLUSIONS: Folate and vitamin B12 treatment improved insulin resistance and endothelial dysfunction, along with decreasing homocysteine levels, in patients with metabolic syndrome, suggesting that folic acid has several beneficial effects on cardiovascular disease risk factors.

Identification of two novel mutations in the 5'-untranslated region of H-ferritin using denaturing high performance liquid chromatography scanning.

BACKGROUND AND OBJECTIVES: Hereditary hyperferritinemia cataract syndrome is caused by mutations of the iron responsive elements (IREs) of L-ferritin mRNA. These alter the IRE structure and determine L-ferritin upregulation. IREs are located in 5'untranslated regions (5'UTR) of ferritin mRNAs. L-ferritin 5'UTR has been extensively studied and up to 21 different mutations have been identified. Only one mutation has been reported for H-ferritin 5'UTR; this mutation modified IRE structure and was apparently associated with high serum ferritin levels and iron overload. DESIGN AND METHODS: To identify other mutations in H ferritin 5'UTR we developed a fast DNA scanning method based on denaturing high performance liquid chromatography (HPLC). Five artificial DNA mutants were produced in order to validate the analytical conditions of the system for the identification of all mutations by single runs at 68 degrees C. The system was used to screen 660 DNA samples from subjects with high serum ferritin levels. RESULTS: Two abnormal patterns were identified carrying the mutations C20G and G34T. Structural data and the analysis of ferritin levels in red blood cells suggest that these mutations do not affect the functionality of the IRE. INTERPRETATION AND CONCLUSIONS: This large and first population analysis indicates that mutations in the H-ferritin 5'UTR are rare and do not seem to contribute to hyperferritinemia or iron overload.

Islet transplantation stabilizes hemostatic abnormalities and cerebral metabolism in individuals with type 1 diabetes.

OBJECTIVE Islets after kidney transplantation have been shown to positively affect the quality of life of individuals with type 1 diabetes (T1D) by reducing the burden of diabetes complications, but fewer data are available for islet transplantation alone (ITA). The aim of this study was to assess whether ITA has a positive impact on hemostatic and cerebral abnormalities in individuals with T1D. RESEARCH DESIGN AND METHODS Prothrombotic factors, platelet function/ultrastructure, and cerebral morphology, metabolism, and function have been investigated over a 15-month follow-up period using ELISA/electron microscopy and magnetic resonance imaging, nuclear magnetic resonance spectroscopy, and neuropsychological evaluation (Profile of Mood States test and paced auditory serial addition test) in 22 individuals with T1D who underwent ITA (n = 12) or remained on the waiting list (n = 10). Patients were homogeneous with regard to metabolic criteria, hemostatic parameters, and cerebral morphology/metabolism/function at the time of enrollment on the waiting list. RESULTS At the 15-month follow-up, the group undergoing ITA, but not individuals with T1D who remained on the waiting list, showed 1) improved glucose metabolism; 2) near-normal platelet activation and prothrombotic factor levels; 3) near-normal cerebral metabolism and function; and 4) a near-normal neuropsychological test. CONCLUSIONS ITA, despite immunosuppressive therapy, is associated with a near-normalization of hemostatic and cerebral abnormalities.

Calcium signaling-related proteins are associated with broncho-pulmonary dysplasia progression.

Broncho-pulmonary dysplasia (BPD) is a chronic pulmonary disorder that follows premature birth. It is preceded by respiratory distress syndrome (RDS), characterized by acute respiratory failure due to deficiency of surfactant at birth. Clinical characteristics of infants affected by BPD have widely changed in the last decades: they are extraordinarly immature, with impaired alveolar and vascular lung development. To build up new therapeutic strategies for BPD babies, it is necessary to understand the pathogenic mechanisms, which are complicated by environmental risk factors and genetic predisposition. Therefore, the aim of this study was to highlight protein changes in the broncho-alveolar lavage fluid (BALF), thus providing an appropriate picture on what is happening in the locus of injury. We analyzed BALF samples from preterm babies, born at different stages of lung development. We confirmed that gestational age is relevant for BPD progression, but we also detected few de-regulated proteins in the younger babies; we discovered less abundant calcium signaling-related proteins, consistent with BPD severity, comparing severe to mild BPD babies with matched gestational age. In conclusion, this study suggests a subset of proteins to be investigated to better treat BPD babies and facilitate the definition of potential drug targets for novel therapies. BIOLOGICAL SIGNIFICANCE: Pulmonary biomarkers are needed to predict the clinical course of lung disease, status, progression and response to treatment. A key aspect in biomarker discovery is uncovering molecules that appear early during disease initiation, when the natural history of the disease can be modified. Using a proteomic-based approach we compared broncho-alveolar lavage fluid (BALF) protein profile from preterm neonates at different postmenstrual ages, to have a molecular description of broncho-pulmonary dysplasia (BPD) progression. BALF provided a snapshot of local molecular changes, which are relevant for early diagnosis, assessment and characterization of lung disorders. We showed that even if the studied patients had similar clinical phenotype (they all developed severe BPD and they were all cured in the same way in terms of mechanical ventilation, surfactant administration, antenatal steroid treatment and ibuprofen treatment for patent ductus arteriosus), however their BALF protein profiling displayed significant differences in a subset of proteins, which could be exploited to facilitate the development of novel effective therapies, distinct for age and severity of the disease.

Dimethylarginines in complicated type 1 diabetes: roles of insulin, glucose, and oxidative stress.

To investigate the roles of insulin, glucose, and oxidative stress on plasma asymmetric and symmetric dimethylarginine (ADMA, SDMA) levels in complicated diabetes, we studied patients with type 1 diabetes (T1D; n = 20), T1D + end-stage renal disease under hemodialysis (T1D + ESRD; n = 12), T1D + ESRD who received kidney transplant (KD; n = 16), and T1D + ESRD who received kidney-pancreas transplant (KP; n = 20) and healthy controls (n = 50). Levels of ADMA, SDMA, and free and total malondialdehyde (MDA) were increased in all patients, with the highest rises for SDMA and free MDA in T1D+ESRD. In KP, the normalized glycemia contributes to the recovery of ADMA, SDMA, and MDA levels toward normal values. From the covariance analyses, both glucose and insulin relate significantly to ADMA in T1D + ESRD (beta = +0.004, beta = -0.038, respectively) and in KP (beta = +0.032, beta = +0.032, respectively). Creatinine clearance and insulin relate to SDMA in all patient groups (beta = -0.006). Our results provide evidence for the effect of kidney-pancreas transplant on the recovery of ADMA, SDMA, and indexes of oxidative stress toward normal values. Only free MDA allows one to discriminate the magnitude of the oxidative status, as increased total MDA could also be attributable to a reduced renal function.

Effects of growth hormone treatment on arginine to asymmetric dimethylarginine ratio and endothelial function in patients with growth hormone deficiency.

Patients with growth hormone deficiency (GHD) are known to have reduced life expectancy due to increased cardiovascular and cerebrovascular events. An increase in asymmetric dimethylarginine (ADMA) levels previously found in GHD patients could promote premature atherosclerosis. The aim of this study was to determine whether 6-month growth hormone (GH) replacement therapy was able to decrease ADMA levels and ameliorate endothelial dysfunction. Thirty-one GHD patients were studied before and after 6 months of GH (4 microg/[kg d], daily) replacement therapy. Reduced pretreatment levels of serum insulin-like growth factor (IGF) 1 were normalized during GH treatment (88.2 +/- 62.5 to 191.7 +/- 80.3 ng/mL, P < .0001). After 6 months of GH replacement, plasma cyclic guanosine monophosphate levels significantly increased (2.14 +/- 0.52 to 3.54 +/- 1.2 ng/mL, P < .0001), serum ADMA levels were significantly decreased (0.65 +/- 0.1 vs 0.59 +/- 0.11 mumol/L, P < .05), and arganine (Arg) to ADMA ratio was significantly higher (155 +/- 53 vs 193 +/- 61, P < .01). No changes were observed for plasma nitric oxide end products (nitrite and nitrate) levels after GH treatment (21.9 +/- 14.9 vs 24.1 +/- 19.0 mumol/L, not significant). Basal forearm blood flow remained unchanged, whereas reactive hyperemia increased from 7.30 +/- 5.31 mL/100 mL forearm per minute before GH therapy to 13.18 +/- 7.30 mL/100 mL forearm per minute after 6 months of therapy (P < .001). There was a positive correlation between IGF-1 and cyclic guanosine monophosphate (r = 0.73, P < .0001), IGF-1 and reactive hyperemia (r = 0.63, P < .0001), and IGF-1 and Arg/ADMA ratio (r = 0.44, P < .01). Conversely, a negative correlation was found between IGF-1 and ADMA levels (r = -0.41, P < .02). At the end of the study period, fat-free mass, plasma glucose, and hemoglobin A(1c) levels significantly increased, even if they were still in the reference range, suggesting moderate alteration of glucose metabolism. In conclusion, in GHD patients, GH replacement contributes to decreased, to some extent, cardiovascular risk, reducing ADMA levels and improving Arg/ADMA ratio and endothelial dysfunction.