Resistin regulates pituitary lipid metabolism and inflammation in vivo and in vitro.

The adipokine resistin is an insulin-antagonizing factor that also plays a regulatory role in inflammation, immunity, food intake, and gonadal function and also regulates growth hormone (GH) secretion in rat adenopituitary cells cultures with the adipokine. Although adipose tissue is the primary source of resistin, it is also expressed in other tissues, including the pituitary. The aim of this study is to investigate the possible action of resistin on the lipid metabolism in the pituitary gland in vivo (rats in two different nutritional status, fed and fast, treated with resistin on acute and a chronic way) and in vitro (adenopituitary cell cultures treated with the adipokine). Here, by a combination of in vivo and in vitro experimental models, we demonstrated that central acute and chronic administration of resistin enhance mRNA levels of the lipid metabolic enzymes which participated on lipolysis and moreover inhibiting mRNA levels of the lipid metabolic enzymes involved in lipogenesis. Taken together, our results demonstrate for the first time that resistin has a regulatory role on lipid metabolism in the pituitary gland providing a novel insight in relation to the mechanism by which this adipokine can participate in the integrated control of lipid metabolism.

Obesity and survival in operable breast cancer patients treated with adjuvant anthracyclines and taxanes according to pathological subtypes: a pooled analysis.

INTRODUCTION Obesity is an unfavorable prognostic factor in breast cancer (BC) patients regardless of menopausal status and treatment received. However, the association between obesity and survival outcome by pathological subtype requires further clarification. METHODS We performed a retrospective analysis including 5,683 operable BC patients enrolled in four randomized clinical trials (GEICAM/9906, GEICAM/9805, GEICAM/2003-02, and BCIRG 001) evaluating anthracyclines and taxanes as adjuvant treatments. Our primary aim was to assess the prognostic effect of body mass index (BMI) on disease recurrence, breast cancer mortality (BCM), and overall mortality (OM). A secondary aim was to detect differences of such prognostic effects by subtype. RESULTS Multivariate survival analyses adjusting for age, tumor size, nodal status, menopausal status, surgery type, histological grade, hormone receptor status, human epidermal growth factor receptor 2 (HER2) status, chemotherapy regimen, and under-treatment showed that obese patients (BMI 30.0 to 34.9) had similar prognoses to that of patients with a BMI < 25 (reference group) in terms of recurrence (Hazard Ratio [HR] = 1.08, 95% Confidence Interval [CI] = 0.90 to 1.30), BCM (HR = 1.02, 0.81 to 1.29), and OM (HR = 0.97, 0.78 to 1.19). Patients with severe obesity (BMI ≥ 35) had a significantly increased risk of recurrence (HR = 1.26, 1.00 to 1.59, P = 0.048), BCM (HR = 1.32, 1.00 to 1.74, P = 0.050), and OM (HR = 1.35, 1.06 to 1.71, P = 0.016) compared to our reference group. The prognostic effect of severe obesity did not vary by subtype. CONCLUSIONS Severely obese patients treated with anthracyclines and taxanes present a worse prognosis regarding recurrence, BCM, and OM than patients with BMI < 25. The magnitude of the harmful effect of BMI on survival-related outcomes was similar across subtypes.

Computational and Biological Evaluation of N-octadecyl-N'-propylsulfamide, a Selective PPARα Agonist Structurally Related to N-acylethanolamines.

To further understand the pharmacological properties of N-oleoylethanolamine (OEA), a naturally occurring lipid that activates peroxisome proliferator-activated receptor alpha (PPARα), we designed sulfamoyl analogs based on its structure. Among the compounds tested, N-octadecyl-N'-propylsulfamide (CC7) was selected for functional comparison with OEA. The performed studies include the following computational and biological approaches: 1) molecular docking analyses; 2) molecular biology studies with PPARα; 3) pharmacological studies on feeding behavior and visceral analgesia. For the docking studies, we compared OEA and CC7 data with crystallization data obtained with the reference PPARα agonist GW409544. OEA and CC7 interacted with the ligand-binding domain of PPARα in a similar manner to GW409544. Both compounds produced similar transcriptional activation by in vitro assays, including the GST pull-down assay and reporter gene analysis. In addition, CC7 and OEA induced the mRNA expression of CPT1a in HpeG2 cells through PPARα and the induction was avoided with PPARα-specific siRNA. In vivo studies in rats showed that OEA and CC7 had anorectic and antiobesity activity and induced both lipopenia and decreases in hepatic fat content. However, different effects were observed when measuring visceral pain; OEA produced visceral analgesia whereas CC7 showed no effects. These results suggest that OEA activity on the PPARα receptor (e.g., lipid metabolism and feeding behavior) may be dissociated from other actions at alternative targets (e.g., pain) because other non cannabimimetic ligands that interact with PPARα, such as CC7, do not reproduce the full spectrum of the pharmacological activity of OEA. These results provide new opportunities for the development of specific PPARα-activating drugs focused on sulfamide derivatives with a long alkyl chain for the treatment of metabolic dysfunction.

Safety and immunomodulatory effects of three probiotic strains isolated from the feces of breast-fed infants in healthy adults: SETOPROB study.

We previously described the isolation and characterization of three probiotic strains from the feces of exclusively breast-fed newborn infants: Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036. These strains were shown to adhere to intestinal mucus in vitro, to be sensitive to antibiotics and to resist biliary salts and low pH. In the present study, a multicenter, randomized, double-blind, placebo-controlled trial with 100 healthy volunteers in three Spanish cities was carried out to evaluate the tolerance, safety, gut colonization and immunomodulatory effects of these three probiotics. Volunteers underwent a 15-day washout period, after which they were randomly divided into 5 groups that received daily a placebo, a capsule containing one of the 3 strains or a capsule containing a mixture of two strains for 30 days. The intervention was followed by another 15-day washout period. Patients did not consume fermented milk for the entire duration of the study. Gastrointestinal symptoms, defecation frequency and stool consistency were not altered by probiotic intake. No relevant changes in blood and serum, as well as no adverse events occurred during or after treatment. Probiotic administration slightly modified bacterial populations in the volunteers' feces. Intestinal persistence occurred in volunteers who received L. rhamnosus CNCM I-4036. Administration of B. breve CNCM I-4035 resulted in a significant increase in fecal secretory IgA content. IL-4 and IL-10 increased, whereas IL-12 decreased in the serum of volunteers treated with any of the three strains. These results demonstrate that the consumption of these three bacterial strains was safe and exerted varying degrees of immunomodulatory effects.

Rationale and design of a randomized, double-blind, placebo controlled multicenter trial to study efficacy, security, and long term effects of intermittent repeated levosimendan administration in patients with advanced heart failure: LAICA study.

BACKGROUND Advanced heart failure (HF) is associated with high morbidity and mortality; it represents a major burden for the health system. Episodes of acute decompensation requiring frequent and prolonged hospitalizations account for most HF-related expenditure. Inotropic drugs are frequently used during hospitalization, but rarely in out-patients. The LAICA clinical trial aims to evaluate the effectiveness and safety of monthly levosimendan infusion in patients with advanced HF to reduce the incidence of hospital admissions for acute HF decompensation. METHODS The LAICA study is a multicenter, prospective, randomized, double-blind, placebo-controlled, parallel group trial. It aims to recruit 213 out-patients, randomized to receive either a 24-h infusion of levosimendan at 0.1 μg/kg/min dose, without a loading dose, every 30 days, or placebo. RESULTS The main objective is to assess the incidence of admission for acute HF worsening during 12 months. Secondarily, the trial will assess the effect of intermittent levosimendan on other variables, including the time in days from randomization to first admission for acute HF worsening, mortality and serious adverse events. CONCLUSIONS The LAICA trial results could allow confirmation of the usefulness of intermittent levosimendan infusion in reducing the rate of hospitalization for HF worsening in advanced HF outpatients.

Phenotypes and endotypes of uncontrolled severe asthma: new treatments.

Severe asthma is a heterogeneous disease that affects only 5%-10% of asthmatic patients, although it accounts for a significant percentage of the consumption of health care resources. Severe asthma is characterized by the need for treatment with high doses of inhaled corticosteroids and includes several clinical and pathophysiological phenotypes. To a large extent, this heterogeneity restricts characterization of the disease and, in most cases, hinders the selection of appropriate treatment. In recent years, therefore, emphasis has been placed on improving our understanding of the various phenotypes of severe asthma and the identification of biomarkers for each of these phenotypes. Likewise, the concept of the endotype has been gaining acceptance with regard to the various subtypes of the disease, which are classified according to their unique functional or pathophysiological mechanism. This review discusses the most relevant aspects of the clinical and inflammatory phenotypes of severe asthma, including severe childhood asthma and the various endotypes of severe asthma. The main therapeutic options available for patients with uncontrolled severe asthma will also be reviewed.

Association of HbA1c and cardiovascular and renal disease in an adult Mediterranean population

BACKGROUND: Increasing evidence suggests a mechanistic link between the glycemic environment and renal and cardiovascular events, even below the threshold for diabetes. We aimed to assess the association between HbA1c and chronic kidney disease (CKD) and cardiovascular disease (CVD). METHODS: A cross-sectional study involving a random representative sample of 2270 adults from southern Spain (Malaga) was undertaken. We measured HbA1c, serum creatinine and albuminuria in fasting blood and urine samples. RESULTS: Individuals without diabetes in the upper HbA1c tertile had an unfavorable cardiovascular and renal profile and shared certain clinical characteristics with the patients with diabetes. Overall, a higher HbA1c concentration was strongly associated with CKD or CVD after adjustment for traditional risk factors. The patients with known diabetes had a 2-fold higher odds of CKD or CVD. However, when both parameters were introduced in the same model, the HbA1c concentration was only significantly associated with clinical endpoints (OR: 1.4, 95% CI, 1.1-1.6, P = 0.002). An increase in HbA1c of one percentage point was associated with a 30% to 40% increase in the rate of CKD or CVD. This relationship was apparent in persons with and without known diabetes. ROC curves illustrated that a HbA1c of 37 mmol/mol (5.5%) was the optimal value in terms of sensitivity and specificity for predicting endpoints in this population. CONCLUSION: HbA1c levels were associated with a higher prevalence of CKD and CVD cross-sectionally, regardless of diabetes status. These data support the value of HbA1c as a marker of cardiovascular and renal disease in the general population.

Pattern of recurrence of early breast cancer is different according to intrinsic subtype and proliferation index.

INTRODUCTION Recurrence risk in breast cancer varies throughout the follow-up time. We examined if these changes are related to the level of expression of the proliferation pathway and intrinsic subtypes. METHODS Expression of estrogen and progesterone receptor, Ki-67, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR) and cytokeratin 5/6 (CK 5/6) was performed on tissue-microarrays constructed from a large and uniformly managed series of early breast cancer patients (N = 1,249). Subtype definitions by four biomarkers were as follows: luminal A (ER + and/or PR+, HER2-, Ki-67 <14), luminal B (ER + and/or PR+, HER2-, Ki-67 ≥14), HER2-enriched (any ER, any PR, HER2+, any Ki-67), triple-negative (ER-, PR-, HER2-, any Ki-67). Subtype definitions by six biomarkers were as follows: luminal A (ER + and/or PR+, HER2-, Ki-67 <14, any CK 5/6, any EGFR), luminal B (ER + and/or PR+, HER2-, Ki-67 ≥14, any CK 5/6, any EGFR), HER2-enriched (ER-, PR-, HER2+, any Ki-67, any CK 5/6, any EGFR), Luminal-HER2 (ER + and/or PR+, HER2+, any Ki-67, any CK 5/6, any EGFR), Basal-like (ER-, PR-, HER2-, any Ki-67, CK5/6+ and/or EGFR+), triple-negative nonbasal (ER-, PR-, HER2-, any Ki-67, CK 5/6-, EGFR-). Each four- or six-marker defined intrinsic subtype was divided in two groups, with Ki-67 <14% or with Ki-67 ≥14%. Recurrence hazard rate function was determined for each intrinsic subtype as a whole and according to Ki-67 value. RESULTS Luminal A displayed a slow risk increase, reaching its maximum after three years and then remained steady. Luminal B presented most of its relapses during the first five years. HER2-enriched tumors show a peak of recurrence nearly twenty months post-surgery, with a greater risk in Ki-67 ≥14%. However a second peak occurred at 72 months but the risk magnitude was greater in Ki-67 <14%. Triple negative tumors with low proliferation rate display a smooth risk curve, but with Ki-67 ≥14% show sharp peak at nearly 18 months. CONCLUSIONS Each intrinsic subtype has a particular pattern of relapses over time which change depending on the level of activation of the proliferation pathway assessed by Ki-67. These findings could have clinical implications both on adjuvant treatment trial design and on the recommendations concerning the surveillance of patients.

Recommendations of the GARIN group for managing non-critically ill patients with diabetes or stress hyperglycaemia and artificial nutrition.

BACKGROUND & AIMS By means of this update, the GARIN working group aims to define its position regarding the treatment of patients with diabetes or stress hyperglycaemia and artificial nutrition. In this area there are many aspects of uncertainty, especially in non-critically ill patients. METHODS Bibliographical review, and specific questions in advance were discussed and answered at a meeting in the form of conclusions. RESULTS We propose a definition of stress hyperglycaemia. The indications and access routes for artificial nutrition are no different in patients with diabetes/stress hyperglycaemia than in non-diabetics. The objective must be to keep pre-prandial blood glucose levels between 100 and 140 mg/dl and post-prandial levels between 140 and 180 mg/dl. Hyperglycemia can be prevented through systematic monitoring of capillary glycaemias and adequately calculate energy-protein needs. We recommend using enteral formulas designed for patients with diabetes (high monounsaturated fat) to facilitate metabolic control. The best drug treatment for treating hyperglycaemia/diabetes in hospitalised patients is insulin and we make recommendations for adapt the theoretical insulin action to the nutrition infusion regimen. We also addressed recommendations for future investigation. CONCLUSIONS This recommendations about artificial nutrition in patients with diabetes or stress hyperglycaemia can add value to clinical work.

Prevalence of and risk factors for biliary carriage of bacteria showing worrisome and unexpected resistance traits.

Data on biliary carriage of bacteria and, specifically, of bacteria with worrisome and unexpected resistance traits (URB) are lacking. A prospective study (April 2010 to December 2011) was performed that included all patients admitted for <48 h for elective laparoscopic cholecystectomy in a Spanish hospital. Bile samples were cultured and epidemiological/clinical data recorded. Logistic regression models (stepwise) were performed using bactobilia or bactobilia by URB as dependent variables. Models (P < 0.001) showing the highest R(2) values were considered. A total of 198 patients (40.4% males; age, 55.3 ± 17.3 years) were included. Bactobilia was found in 44 of them (22.2%). The presence of bactobilia was associated (R(2) Cox, 0.30) with previous biliary endoscopic retrograde cholangiopancreatography (ERCP) (odds ratio [OR], 8.95; 95% confidence interval [CI], 2.96 to 27.06; P < 0.001), previous admission (OR, 2.82; 95% CI, 1.10 to 7.24; P = 0.031), and age (OR, 1.09 per year; 95% CI, 1.05 to 1.12; P < 0.001). Ten out of the 44 (22.7%) patients with bactobilia carried URB: 1 Escherichia coli isolate (CTX-M), 1 Klebsiella pneumoniae isolate (OXA-48), 3 high-level gentamicin-resistant enterococci, 1 vancomycin-resistant Enterococcus isolate, 3 Enterobacter cloacae strains, and 1 imipenem-resistant Pseudomonas aeruginosa strain. Bactobilia by URB (versus those by non-URB) was only associated (R(2) Cox, 0.19) with previous ERCP (OR, 11.11; 95% CI, 1.98 to 62.47; P = 0.006). For analyses of patients with bactobilia by URB versus the remaining patients, previous ERCP (OR, 35.284; 95% CI, 5.320 to 234.016; P < 0.001), previous intake of antibiotics (OR, 7.200; 95% CI, 0.962 to 53.906; P = 0.050), and age (OR, 1.113 per year of age; 95% CI, 1.028 to 1.206; P = 0.009) were associated with bactobilia by URB (R(2) Cox, 0.19; P < 0.001). Previous antibiotic exposure (in addition to age and previous ERCP) was a risk driver for bactobilia by URB. This may have implications in prophylactic/therapeutic measures.

Evaluation of three automated systems for susceptibility testing of enterobacteria containing qnrB, qnrS, and/or aac(6')-Ib-cr.

The accuracy of the MicroScan WalkAway, BD Phoenix, and Vitek-2 systems for susceptibility testing of quinolones and aminoglycosides against 68 enterobacteria containing qnrB, qnrS, and/or aac(6 ')-Ib-cr was evaluated using reference microdilution. Overall, one very major error (0.09%), 6 major errors (0.52%), and 45 minor errors (3.89%) were noted.

Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis.

Real-time PCR is a widely used tool for the diagnosis of many infectious diseases. However, little information exists about the influences of the different factors involved in PCR on the amplification efficiency. The aim of this study was to analyze the effect of boiling as the DNA preparation method on the efficiency of the amplification process of real-time PCR for the diagnosis of human brucellosis with serum samples. Serum samples from 10 brucellosis patients were analyzed by a SYBR green I LightCycler-based real-time PCR and by using boiling to obtain the DNA. DNA prepared by boiling lysis of the bacteria isolated from serum did not prevent the presence of inhibitors, such as immunoglobulin G (IgG), which were extracted with the template DNA. To identify and confirm the presence of IgG, serum was precipitated to separate and concentrate the IgG and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The use of serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of IgG in serum samples was not concentration dependent, and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite the lack of the complete elimination of the IgG from the template DNA, boiling does not require any special equipment and it provides a rapid, reproducible, and cost-effective method for the preparation of DNA from serum samples for the diagnosis of brucellosis.

Epidemiology and antifungal susceptibility of bloodstream fungal isolates in pediatric patients: a Spanish multicenter prospective survey.

Data on fungemia epidemiology and antifungal susceptibility of isolates from children are scarce, leading frequently to pediatric empirical treatment based on available adult data. The present study was designed to update the epidemiological, mycological, and in vitro susceptibility data on fungal isolates from children with fungemia in Spain. All fungemia episodes were identified prospectively by blood culture over 13 months at 30 hospitals. Tests of susceptibility to amphotericin B, flucytosine, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, and micafungin were performed at participant institutions by a microdilution colorimetric method. New species-specific clinical breakpoints for fluconazole, voriconazole, and echinocandins were also applied. A total of 203 episodes of fungemia in 200 children were identified. A higher proportion of fungal isolates was from general wards than intensive care units (ICU). Candida parapsilosis (46.8%), Candida albicans (36.5%), Candida tropicalis (5.9%), Candida glabrata (3.9%), and Candida guilliermondii (2.5%) were the leading species. C. parapsilosis was the predominant species except in neonates. C. albicans was the most frequent in neonatal ICU settings (51.9%). Intravascular catheter (79.3%), surgery (35%), prematurity (30%), and neutropenia (11%) were the most frequent predisposing factors. Most Candida isolates (95.1%) were susceptible to all antifungals. When the new species-specific clinical breakpoints were applied, all C. parapsilosis isolates were susceptible to echinocandins except one, which was micafungin resistant. This is the largest published series of fungemia episodes in the pediatric setting. C. parapsilosis is the most prevalent species in Spain, followed by C. albicans and C. tropicalis. Resistance to azole and echinocandin agents is extremely rare among Candida species. The fluconazole resistance rate in Spain has decreased in the last 10 years.

Lack of association between the protein tyrosine phosphatase non-receptor type 22 R263Q and R620W functional genetic variants and endogenous non-anterior uveitis.

OBJECTIVE Endogenous uveitis is a major cause of visual loss mediated by the immune system. The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene encodes a lymphoid-specific phosphatase that plays a key role in T-cell receptor (TCR) signaling. Two independent functional missense single nucleotide polymorphisms (SNPs) located within the PTPN22 gene (R263Q and R620W) have been associated with different autoimmune disorders. We aimed to analyze for the first time the influence of these PTPN22 genetic variants on endogenous non-anterior uveitis susceptibility. METHODS We performed a case-control study of 217 patients with endogenous non-anterior uveitis and 718 healthy controls from a Spanish population. The PTPN22 polymorphisms (rs33996649 and rs2476601) were genotyped using TaqMan allelic discrimination assays. The allele, genotype, carriers, and allelic combination frequencies were compared between cases and controls with χ(2) analysis or Fisher's exact test. RESULTS Our results showed no influence of the studied SNPs in the global susceptibility analysis (rs33996649: allelic P- value=0.92, odds ratio=0.97, 95% confidence interval=0.54-1.75; rs2476601: allelic P- value=0.86, odds ratio=1.04, 95% confidence interval=0.68-1.59). Similarly, the allelic combination analysis did not provide additional information. CONCLUSIONS Our results suggest that the studied polymorphisms of the PTPN22 gene do not play an important role in the pathophysiology of endogenous non-anterior uveitis.

Claudin-19 mutations and clinical phenotype in Spanish patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is an autosomal recessive tubular disorder characterized by excessive renal magnesium and calcium excretion and chronic kidney failure. This rare disease is caused by mutations in the CLDN16 and CLDN19 genes. These genes encode the tight junction proteins claudin-16 and claudin-19, respectively, which regulate the paracellular ion reabsorption in the kidney. Patients with mutations in the CLDN19 gene also present severe visual impairment. Our goals in this study were to examine the clinical characteristics of a large cohort of Spanish patients with this disorder and to identify the disease causing mutations. We included a total of 31 patients belonging to 27 unrelated families and studied renal and ocular manifestations. We then analyzed by direct DNA sequencing the coding regions of CLDN16 and CLDN19 genes in these patients. Bioinformatic tools were used to predict the consequences of mutations. Clinical evaluation showed ocular defects in 87% of patients, including mainly myopia, nystagmus and macular colobomata. Twenty two percent of patients underwent renal transplantation and impaired renal function was observed in another 61% of patients. Results of the genetic analysis revealed CLDN19 mutations in all patients confirming the clinical diagnosis. The majority of patients exhibited the previously described p.G20D mutation. Haplotype analysis using three microsatellite markers showed a founder effect for this recurrent mutation in our cohort. We also identified four new pathogenic mutations in CLDN19, p.G122R, p.I41T, p.G75C and p.G75S. A strategy based on microsequencing was designed to facilitate the genetic diagnosis of this disease. Our data indicate that patients with CLDN19 mutations have a high risk of progression to chronic renal disease.