Anna Espinal

Obesity, Adiponectin and Inflammation as Predictors of New‐Onset Diabetes Mellitus After Kidney Transplantation

The high incidence of new‐onset diabetes mellitus after transplantation (NODAT) suggests the need to find new factors to explain the pathogenesis. Our objectives were (1) to confirm that low levels of pre‐transplant adiponectin are an independent risk factor for the development of NODAT in a larger transplanted population; (2) to analyze whether adiponectin is a better predictor of NODAT than other inflammatory markers (C‐reactive protein (CRP), interleukin‐6 (IL‐6), tumor necrosis factor‐alpha (TNF‐α) and pregnancy‐associated plasma protein A (PAPP‐A)) and (3) to assess the relationship between obesity, inflammatory markers and NODAT. One hundred ninety‐nine non‐diabetic patients (128 men; age: 53 ± 11 years; body mass index (BMI) 24.98 ± 3.76 kg/m2) were included. Pre‐transplant plasma glucose, insulin, adiponectin, CRP, TNF‐α, IL‐6 and PAPP‐A were measured. Forty‐five patients developed NODAT. Patients with NODAT had a greater BMI (p = 0.005). Adiponectin was lower (p < 0.001) and CRP higher (p = 0.032) in patients with NODAT. Multivariate logistic regression and Cox analysis showed that the calcineurin inhibitor used, pre‐transplant BMI and adiponectin were predictors of NODAT. ROC analysis showed that an adiponectin concentration of 11.4 μg/mL had a significant negative prediction for NODAT risk (sensitivity: 81% and specificity: 70%). Of the inflammatory markers studied, adiponectin proved to be an independent predictor of NODAT.

Ultrastructural dynamics of proteins involved in endocytic budding

Fluorescence live-cell imaging has temporally resolved the conserved choreography of more than 30 proteins involved in clathrin and actin-mediated endocytic budding from the plasma membrane. However, the resolution of these studies is insufficient to unveil how the endocytic machinery actually drives membrane deformation in vivo. In this study, we use quantitative immuno-EM to introduce the temporal dimension to the ultrastructural analysis of membrane budding and define changes in the topography of the lipid bilayer coupled to the dynamics of endocytic proteins with unprecedented spatiotemporal resolution. Using this approach, we frame the emergence of membrane curvature with respect to the recruitment of endocytic factors and show that constriction of the invaginations correlates with translocation of membrane-sculpting proteins. Furthermore, we show that initial bending of the plasma membrane is independent of actin and clathrin polymerization and precedes building of an actin cap branched by the Arp2/3 complex. Finally, our data indicate that constriction and additional elongation of the endocytic profiles require the mechanochemical activity of the myosins-I. Altogether, this work provides major insights into the molecular mechanisms driving membrane deformation in a cellular context.

Messenger RNA expression of B7-1 and NPHS1 in urinary sediment could be useful to differentiate between minimal change disease and focal segmental glomerulosclerosis in adult patients

BACKGROUND Podocyte proteins are involved in the pathogenesis of glomerular kidney disease (GKD). However, there is little information on messenger RNA (mRNA) expression patterns of B7-1 and NPHS1 in urinary sediment of patients with GKD. The objective of this study was to analyse the gene expression of B7-1 in urinary sediment and correlate it with the expression of podocyte-specific genes in patients with GKD. METHODS Adult patients with proliferative and non-proliferative GKD, proteinuria and stable renal function, were included. A group of healthy subjects was used to determine normal levels of urinary markers and to obtain reference RNA. Biochemical, clinical and experimental procedures included measurement of creatinine level and total urinary protein, renal biopsy, identification of urinary podocytes, gene expression analysis of B7-1, NPHS1, NPHS2 and SyNPO genes and urinary B7-1 protein analysis by enzyme-linked immunosorbent assay. RESULTS Between June 2006 and November 2009, 69 patients with GKD (median age: 46 ± 15 years, 64% men) and 14 healthy subjects (median age: 34 ± 12 years, 43% men) were included. In both groups, urinary mRNA levels of B7-1 and NPHS1 were significantly higher in patients with GKD compared to healthy subjects (P = 0.050 and P = 0.008, respectively). Regarding GKD subtypes, patients with focal segmental glomerulosclerosis (FSGS), but not patients with minimal change disease (MCD), had a significantly higher mRNA expression of B7-1 and NPHS1 than healthy subjects (P = 0.012 and P = 0.030, respectively). Patients with MCD had a significantly lower NPHS1 mRNA expression than patients with FSGS (P = 0.012). The B7-1:NPHS1 urinary mRNA ratio was significantly higher in patients with MCD compared with patients with FSGS (P = 0.027). CONCLUSION mRNA expression analysis of B7-1 and NPHS1 in urinary sediment may be useful to differentiate between different histologic subtypes of GKD, particularly between MCD and FSGS.

Uromodulin and α1-Antitrypsin Urinary Peptide Analysis to Differentiate Glomerular Kidney Diseases

Background/Aims: Glomerular kidney disease (GKD) is suspected in patients based on proteinuria, but its diagnosis relies primarily on renal biopsy. We used urine peptide profiling as a noninvasive means to link GKD-associated changes to each glomerular entity. Methods: Urinary peptide profiles of 60 biopsy-proven glomerular patients and 14 controls were analyzed by combining magnetic bead peptide enrichment, MALDI-TOF MS analysis, and ClinProTools v2.0 to select differential peptides. Tentative identification of the differential peptides was carried out by HPLC-MS/MS. Results: The HPLC-MS/MS results suggest that uromodulin (UMOD; m/z: 1682, 1898 and 1913) and α1-antitrypsin (A1AT; m/z: 1945, 2392 and 2505) are differentially expressed urinary peptides that distinguish between GKD patients and healthy subjects. Low UMOD and high A1AT peptide abundance was observed in 80–92% of patients with GKD. Proliferative forms of GKD were distinguished from nonproliferative forms, based on a combination of UMOD and A1AT peptides. Nonproliferative forms correlated with higher A1AT peptide levels – focal segmental glomerulosclerosis was linked more closely to high levels of the m/z 1945 peptide than minimal change disease. Conclusion: We describe a workflow – urinary peptide profiling coupled with histological findings – that can be used to distinguish GKD accurately and noninvasively, particularly its nonproliferative forms.

Selected Swine viral pathogens in indoor pigs in Spain. Seroprevalence and farm-level characteristics.

A serosurvey on porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), Aujeszkys disease virus gE protein (ADV gE), porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2) was carried out in Spanish pig herds. The serosurvey consisted of two studies. First, a retrospective study assessed the proportion of seropositive boar, sow and fattening pig herds and their seroprevalences to PRRSV, SIV, ADV gE and PPV from 2003 to 2005 and to PCV2 from 2000 to 2005. Such information was obtained from routine serologic analyses from two veterinary diagnostic laboratory services. Second, a cross-sectional study in sow and fattening pig herds from 44 farms (without vaccination interferences on serologic analyses) was performed to provide information on seroprevalences and co-seropositivity to PRRSV, SIV, ADV gE and PCV2 (PPV was excluded because of widespread vaccination) and to elucidate their relationships with farm characteristics, management and productive parameters. Similar seroprevalences were observed in both studies, although some variations were obtained, probably because of vaccination schedules, number of tested sera, sampling age and regional variations. Percentage of PRRSV and SIV seropositive herds was over 85% for sows, around 80% for fatteners and around 50% for boar studs. The proportion of ADV gE seropositive sow herds decreased from 41% to 30% between 2003 and 2005, whereas such decrease was from 41% to 33% in fattening pig herds and from 13% to 4% in boar studs PCV2 antibodies were widespread as well as those against PPV; in the latter case, if antibodies were elicited by infection and/or vaccination was not assessed. Concurrent presence of PCV2, PRRSV and SIV antibodies was found in 89% and 66% sow and fattening herds, respectively. No statistical associations were obtained between seroprevalences or co-seropositivity and farm characteristics, management or productive parameters.

Post-weaning multisystemic wasting syndrome (PMWS) clinical expression under field conditions is modulated by the pig genetic background

Post-weaning multisystemic wasting syndrome (PMWS) is a worldwide distributed disease of multifactorial origin and porcine circovirus type 2 (PCV2) has been identified as its essential infectious aetiology. Pig genetic background has been pointed to influence disease expression. In the present study, three different boar lines, namely A (100% Pietrain), B (50% Large White × 50% Pietrain) and C (25% Large White × 75% Duroc), were used to inseminate sows from the same genetic line (37.5% Large White × 37.5% Duroc × 25% Landrace) located on two PMWS-affected farms (farm-1 and farm-2). The PMWS clinical expression of their offspring was studied from weaning to slaughter, evaluating three parameters: total post-weaning mortality (PWM), PWM associated to PMWS (PMWS-PWM) and body weight (BW) evolution. The effect of other variables potentially related with PMWS, including sow and piglet PCV2 exposure, sow parity, piglet gender and piglet BW at weaning, were also considered in the study design. Overall, a total of 6.5% PWM and 4.3% PMWS-PWM occurred in the monitored farms. Pigs from boar line C showed the highest PWM (16.3%) and PMWS-PWM (12.4%), and the lowest BW; pigs from boar line A showed the lowest PWM (1.8%) and the highest BW. Furthermore, PWM was also higher in piglets from farm-2 and from multiparous sows. In farm-2, PMWS-PWM was higher in piglets from multiparous sows. Finally, BW was influenced by interactions between genetics and both farm and pig age, and was lower in piglets from farm-2. This study represents a consistent observation of the genetic background effect on PMWS clinical expression under field conditions.

Systemic inflammation in 222.841 healthy employed smokers and nonsmokers: white blood cell count and relationship to spirometry

BackgroundSmoking has been linked to low-grade systemic inflammation, a known risk factor for disease. This state is reflected in elevated white blood cell (WBC) count.ObjectiveWe analyzed the relationship between WBC count and smoking in healthy men and women across several age ranges who underwent preventive medical check-ups in the workplace. We also analysed the relationship between smoking and lung function.MethodsCross-sectional descriptive study in 163 459 men and 59 382 women aged between 16 and 70 years. Data analysed were smoking status, WBC count, and spirometry readings.ResultsTotal WBC showed higher counts in both male and female smokers, around 1000 to 1300 cell/ml (t test, P < 0.001). Forced expiratory volume in 1 second (FEV1%) was higher in nonsmokers for both sexes between 25 to 54 years (t test, P < 0.001). Analysis of covariance showed a multiple variable effect of age, sex, smoking status, body mass index on WBC count. The relationship between WBC blood count and smoking status was confirmed after the sample was stratified for these variables. Smokers with airway obstruction measured by FEV1% were found to have higher WBC counts, in comparison to smokers with a normal FEV1% among similar age and BMI groups.ConclusionsSmoking increases WBC count and affects lung function. The effects are evident across a wide age range, underlining the importance of initiating preventive measures as soon as an individual begins to smoke.

Exploratory study on the influence of climatological parameters on Mycoplasma hyopneumoniae infection dynamics

The objective of the present work was to elucidate the potential relationship between Mycoplasma hyopneumoniae infection and seroconversion dynamics and climatological conditions in four groups of pigs from the same farm born in different seasons of the year. Nasal swabs and blood samples were taken from 184 pigs at 1, 3, 6, 9, 12, 15, 18, 22 and 25 (slaughter age) weeks of age. Outside climatologic parameters, including temperature (°C), relative humidity (%), precipitation (l/m2) and wind speed (m/s) were recorded weekly from January 2003 to June 2004. Percentage of nPCR detection of M. hyopneumoniae in nasal swabs was associated significantly with the weekly precipitation rate [P = 0.0018, OR = 1.31 (IC = 1.11–1.55)]; the higher the precipitation rate, the higher the probability of being M. hyopneumoniae nPCR-positive. On the other hand, the percentage of seropositive pigs had a significant association with mean weekly temperature rate [P = 0.0012, OR = 0.89 [IC = 0.84–0.95]); the lower the temperature, the higher the probability of being M. hyopneumoniae seropositive. Animals born in autumn (when higher precipitations rates were recorded), entering finishing units in winter (when lower temperatures were recorded), and reaching slaughter in spring, had the highest probability of being infected by M. hyopneumoniae and the highest probability of being M. hyopneumoniae seropositive.

Changes in Bacterial Population of Gastrointestinal Tract of Weaned Pigs Fed with Different Additives

This study aimed to provide novel insights into the gastrointestinal microbial diversity from different gastrointestinal locations in weaning piglets using PCR-restriction fragment length polymorphism (PCR-RFLP). Additionally, the effect of different feed additives was analyzed. Thirty-two piglets were fed with four different diets: a control group and three enriched diets, with avilamycin, sodium butyrate, and a plant extract mixture. Digesta samples were collected from eight different gastrointestinal segments of each animal and the bacterial population was analysed by a PCR-RFLP technique that uses 16S rDNA gene sequences. Bacterial diversity was assessed by calculating the number of bands and the Shannon-Weaver index. Dendrograms were constructed to estimate the similarity of bacterial populations. A higher bacterial diversity was detected in large intestine compared to small intestine. Among diets, the most relevant microbial diversity differences were found between sodium butyrate and plant extract mixture. Proximal jejunum, ileum, and proximal colon were identified as those segments that could be representative of microbial diversity in pig gut. Results indicate that PCR-RFLP technique allowed detecting modifications on the gastrointestinal microbial ecology in pigs fed with different additives, such as increased biodiversity by sodium butyrate in feed.

Comparative differential proteomic analysis of minimal change disease and focal segmental glomerulosclerosis

BackgroundMinimal change disease (MCD) and primary focal segmental glomerulosclerosis (FSGS) are glomerular diseases characterized by nephrotic syndrome. Their diagnosis requires a renal biopsy, but it is an invasive procedure with potential complications. In a small biopsy sample, where only normal glomeruli are observed, FSGS cannot be differentiated from MCD. The correct diagnosis is crucial to an effective treatment, as MCD is normally responsive to steroid therapy, whereas FSGS is usually resistant.The purpose of our study was to discover and validate novel early urinary biomarkers capable to differentiate between MCD and FSGS.MethodsForty-nine patients biopsy-diagnosed of MCD and primary FSGS were randomly subdivided into a training set (10 MCD, 11 FSGS) and a validation set (14 MCD, 14 FSGS). The urinary proteome of the training set was analyzed by two-dimensional differential gel electrophoresis coupled with mass spectrometry. The proteins identified were quantified by enzyme-linked immunosorbent assay in urine samples from the validation set.ResultsUrinary concentration of alpha-1 antitrypsin, transferrin, histatin-3 and 39S ribosomal protein L17 was decreased and calretinin was increased in FSGS compared to MCD. These proteins were used to build a decision tree capable to predict patient’s pathology.ConclusionsThis preliminary study suggests a group of urinary proteins as possible non-invasive biomarkers with potential value in the differential diagnosis of MCD and FSGS. These biomarkers would reduce the number of misdiagnoses, avoiding unnecessary or inadequate treatments.