Circulating metabolic profile in idiopathic pulmonary fibrosis: data from the IPF-PRO Registry.

BACKGROUND: The circulating metabolome, reflecting underlying cellular processes and disease biology, has not been fully characterized in patients with idiopathic pulmonary fibrosis (IPF). We evaluated whether circulating levels of metabolites correlate with the presence of IPF, with the severity of IPF, or with the risk of clinically relevant outcomes among patients with IPF. METHODS: We analyzed enrollment plasma samples from 300 patients with IPF in the IPF-PRO Registry and 100 individuals without known lung disease using a set of targeted metabolomics and clinical analyte modules. Linear regression was used to compare metabolite and clinical analyte levels between patients with IPF and controls and to determine associations between metabolite levels and measures of disease severity in patients with IPF. Unadjusted and adjusted univariable Cox regression models were used to evaluate associations between circulating metabolites and the risk of mortality or disease progression among patients with IPF. RESULTS: Levels of 64 metabolites and 5 clinical analytes were significantly different between patients with IPF and controls. Among analytes with greatest differences were non-esterified fatty acids, multiple long-chain acylcarnitines, and select ceramides, levels of which were higher among patients with IPF versus controls. Levels of the branched-chain amino acids valine and leucine/isoleucine were inversely correlated with measures of disease severity. After adjusting for clinical factors known to influence outcomes, higher levels of the acylcarnitine C:16-OH/C:14-DC were associated with all-cause mortality, lower levels of the acylcarnitine C16:1-OH/C14:1DC were associated with all-cause mortality, respiratory death, and respiratory death or lung transplant, and higher levels of the sphingomyelin d43:2 were associated with the risk of respiratory death or lung transplantation. CONCLUSIONS: IPF has a distinct circulating metabolic profile characterized by increased levels of non-esterified fatty acids, long-chain acylcarnitines, and ceramides, which may suggest a more catabolic environment that enhances lipid mobilization and metabolism. We identified select metabolites that were highly correlated with measures of disease severity or the risk of disease progression and that may be developed further as biomarkers. TRIAL REGISTRATION: ClinicalTrials.gov; No: NCT01915511; URL: www. CLINICALTRIALS: gov .

Exposure of humans to the zoonotic nematode Dirofilaria immitis in Northern Portugal.

Dirofilariosis caused by Dirofilaria immitis (heartworm) is a zoonosis, considered an endemic disease of dogs and cats in several countries of Western Europe, including Portugal. This study assesses the levels of D. immitis exposure in humans from Northern Portugal, to which end, 668 inhabitants of several districts belonging to two different climate areas (Csa: Bragança, Vila Real and Csb: Aveiro, Braga, Porto, Viseu) were tested for anti-D. immitis and anti-Wolbachia surface proteins (WSP) antibodies. The overall prevalence of seropositivity to both anti-D. immitis and WSP antibodies was 6.1%, which demonstrated the risk of infection with D. immitis in humans living in Northern Portugal. This study, carried out in a Western European country, contributes to the characterisation of the risk of infection with D. immitis among human population in this region of the continent. From a One Health point of view, the results of the current work also support the close relationship between dogs and people as a risk factor for human infection.

Synthesis and secretion of volatile short-chain fatty acids in Triatoma infestans infected with Beauveria bassiana.

Physically disturbed Triatoma infestans (Hemiptera: Reduviidae) adults, as well as adults of other Chagas' disease vectors, secrete a mix of volatile organic compounds (VOCs) with alarm and possible sexual and defence functions. The aim of the present research was to test whether infection with the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales: Clavicipitaceae) has an effect on VOC secretion in disturbed T. infestans and on the expression of two genes (Ti-brnq and Ti-bckdc) potentially involved in VOC biosynthesis. The volatiles released by insects at different time periods after fungal treatment were identified and their relative amounts measured. Isobutyric acid was the most abundant volatile found in both healthy and fungus-infected insects and underwent no significant relative changes through the infection process. The secretion of propionic acid, however, was significantly higher at 1-4 days post-infection (d.p. i.) compared with that in controls. A slight induction of both Ti-brnq and Ti-bckdc genes was found by real-time polymerase chain reaction at 4 d.p. i., with expression values reaching up to three-fold those in controls. The early stages of fungal infection seem to affect the composition of the alarm pheromone by changing the expression pattern of both genes analysed. These results help to elucidate the impact of fungal infections on the chemical ecology of triatomine bugs.

Ki-67 and PCNA Expression in Canine Mammary Tumors and Adjacent Nonneoplastic Mammary Glands: Prognostic Impact by a Multivariate Survival Analysis.

The assessment of tumor proliferation has been considered a determining prognostic factor in canine mammary tumors (CMTs). However, no studies have assessed the prognostic importance of proliferation in adjacent nonneoplastic mammary glands. We included 64 CMTs (21 benign and 43 malignant) and studied the proliferation index (PI) of Ki-67 and proliferating cell nuclear antigen (PCNA) together with several clinicopathological characteristics. A positive and statistically significant correlation between the PI of Ki-67 and PCNA in tumors and adjacent nonneoplastic mammary glands was observed in benign and malignant tumors. Tumor size, skin ulceration, histological type, mitotic index, nuclear grade, differentiation grade, histological grade of malignancy, lymph node metastasis, Ki-67, and PCNA expression in tumors and adjacent nonneoplastic mammary glands were statistically associated with overall survival by univariate analysis in malignant cases (n = 43). Histological grade of malignancy and high intratumoral PCNA retained their significance by multivariate analysis arising as independent predictors of overall survival. Interestingly, the PI of Ki-67 and PCNA of adjacent nontumoral mammary glands were associated with clinicopathological features of tumor aggressiveness and shorter overall survival, demonstrating the need to better explore this adjacent non-neoplastic tissue.

Comparison between Clinical Aspects and Salivary Microbial Profile of Children with and without Early Childhood Caries: A Preliminary Study.

PURPOSE: To compare the clinical aspects and microbial profile of children with and without early childhood caries (ECC). STUDY DESIGN: 14 patients (7 without caries and 7 with ECC) were submitted to anamnesis, clinical exam and saliva collection for microbiological analyses. Counts of Streptococcus mutans, Lactobacillus spp. Candida spp., and total microorganisms were performed by culture methods. Microbial diversity was characterized by PCR-DGGE. Demographic/clinical data and salivary microbial counts were compared between groups. RESULTS: Habits of hygiene and breastfeeding presented no association with ECC. Use of pacifiers was associated with absence of caries (p=0.035). Counts of total microorganisms and Candida spp. did not differ between the groups. The ECC group presented larger quantity of S. mutans (p=0.026) and Lactobacillus spp. (p=0.002). There was no correlation between microorganisms and breastfeeding and pacifier use. The dmf-t of ECC Group was 10.5 ± 1.9 and the modified dmf-t was 11.3 ± 3.6. The DGGE demonstrated difference in the pattern of bands between the groups. CONCLUSION: Pacifiers usage was associated with the absence of ECC and microorganism number was higher in the caries group. The PCR-DGGE revealed a characteristic microbial diversity in the ECC Group, being an excellent tool for observing the dynamics of the salivary microbial community in these patients.

New insights on the effectiveness of Metarhizium anisopliae formulation and application against Aedes aegypti eggs.

Increasing needs for innovative control tools against the dengue vector Aedes aegypti have prompted investigations into the development of specific mycoinsecticides. The entomopathogenic fungus Metarhizium anisopliae attacks both larval and adult stages, but its ovicidal activity against A. aegypti is still little explored. This study reports important findings about the effectiveness of conidia formulated in water and oil-in-water emulsions and of direct and indirect application techniques against A. aegypti eggs. The ovicidal activity of M. anisopliae increased with higher conidial concentrations regardless of the application technique, and larvae elimination concentrations were lowest with oil-in-water-formulated conidia (LEC50 ≤ 4·8 × 10(3) conidia cm(-2) and LEC90 ≤ 1·9 × 10(5) conidia cm(-2), respectively). Conidia eventually stimulated larval eclosion. Consequently, the indirect application of oil-based fungal formulations onto substrates where oviposition will later occur appears to be a more efficient means to infect those eggs than the direct fungal application to previously deposited eggs.

Poor tolerability of cystic fibrosis transmembrane conductance regulator modulator therapy in lung transplant recipients.

Elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) is a highly effective therapy for patients with cystic fibrosis (CF) with potential benefits in lung transplant recipients (LTRs) for extrapulmonary CF manifestations; however, tolerability and efficacy in this population are largely unknown. We report our experience with ELX/TEZ/IVA in LTRs for extrapulmonary complications of CF including tolerability, drug-drug interactions, and therapeutic benefit. All LTRs at a single center initiated on ELX/TEZ/IVA were reviewed. Adverse events and patient-reported outcomes attributed to ELX/TEZ/IVA were documented. Pulmonary function, tacrolimus requirements in mg/kg/dl, body mass index (BMI), and reason for initiation were assessed at the initiation of ELX/TEZ/IVA, and at 12 months post-initiation or at the time of discontinuation for those in whom therapy was discontinued. Thirteen LTRs were initiated on ELX/TEZ/IVA at a mean of 115 ± 92 months post-transplant. All were initiated on ELX/TEZ/IVA for sinus or sinus and gastrointestinal CF manifestations. Five (38.4%) patients discontinued therapy due to declining pulmonary function (2/5, 40%), mood disturbances (2/5, 40%), or lack of benefit (1/5, 20%). Of the eight patients who remain on ELX/TEZ/IVA, four reported adverse effects and three LTRs temporarily held therapy. Six (46.2%) LTRs reported improvement in sinus symptoms, while four (30.7%) reported improved gastrointestinal symptoms. Weight declined in the cohort overall. Tacrolimus dose requirements decreased following initiation of ELX/TEZ/IVA therapy, with a 50% decline in dose requirements observed. In our experience, ELX/TEZ/IVA in LTRs is poorly tolerated with modest perceived extrapulmonary benefit and a significant effect on tacrolimus dose requirements. More data are needed to determine the benefits of ELX/TEZ/IVA therapy in LTRs.

Polymer screening by radiofrequency glow discharge time-of-flight mass spectrometry.

The aim of this work is to optimise and evaluate radiofrequency glow discharge (RF GD) time-of-flight mass spectrometry (TOFMS) for identification of organic polymers. For this purpose, different polymers including poly[methylmethacrylate], poly[styrene], polyethylene terephthalate-co-isophthalate and poly[alpha-methylstyrene] have been deposited on silicon wafers and the RF GD-TOFMS capabilities for qualitative identification of these polymeric layers by molecular depth profiling have been investigated. Although some molecular information using the RF continuous mode is available, the pulsed mode offers a greater analytical potential to characterise such organic coatings. Some formed polyatomic ions have proved to be useful to identify the different polymer layers, confirming that layers having similar elemental composition but different polymer structure could be also differentiated and identified.

Histologic characterization of dilated cardiomyopathy in Estrela mountain dogs.

Dilated cardiomyopathy (DCM) is the second-most-important acquired cardiovascular disease in dogs (excluding heartworm disease in some geographic regions) and a major cause of morbidity and mortality in Estrela Mountain Dogs. The objective of this study is to describe the histologic features of DCM in Estrela Mountain Dogs, with special attention to the localization and quantification of attenuated wavy fibers (AWFs), fibrosis, and fatty infiltration. Myocardial samples from 10 areas were collected from the hearts of 10 dogs with DCM and 7 dogs without signs of cardiac disease-namely, the basal, middle, and apical portions of the free wall of both cardiac ventricles and the interventricular septum, as well as the left ventricular papillary muscle. In each sample, the presence or absence of AWFs was noted, and fatty infiltration and fibrosis were quantified. Fatty infiltration, fibrosis, and AWFs were observed in the myocardium of all dogs with DCM, in contrast to what has been described in other breeds. The left ventricular myocardium was the best tissue for diagnosis of DCM, based on these histologic features. The authors concluded that quantification of fibrosis and observation of AWFs in the left ventricular myocardium are useful in the histologic diagnosis of DCM in Estrela Mountain dogs.

Plasma proprotein convertase subtilisin/kexin type 9 inhibitors and cataract risk: A systematic review and meta-analysis. / Inhibidores de la proproteína convertasa plasmática subtilisina kexina tipo 9 y riesgo de cataratas: revisión sistemática y metaanálisis.

BACKGROUND: The marked decrease in LDL-C levels produced by the inhibitors of the plasma proprotein convertase subtilisin/kexin type 9 (iPCSK9) could be associated with an increased risk of cataracts. METHODS: A meta-analysis was performed that included randomised clinical trials controlled with iPCSK9, alone, or in combination with other lipid-lowering drugs, which reported new cases of cataracts, by searching PubMed/Medline, databases of EMBASE and Cochrane Clinical Trials. A fixed-effect model was used, and a meta-regression was carried out evaluating the relationship between intra-treatment LDL-C and the risk of developing cataracts. RESULTS: Five eligible studies of iPCSK9 including 83,492 patients were taken into account for the analysis, and 531 new cases of cataracts in iPCSK9 group vs. 532 in placebo group were diagnosed. The iPCSK9 therapy was not associated with an increased risk of cataracts [OR: 0.96, 95% CI: 0.85-1.08; P=.86, I2: 0%]. Likewise, no significant association was found between on-treatment LDL-C levels, differences between study arms, and new cases of cataracts. CONCLUSION: In this analysis, the use of iPCSK9 was not associated with an increased risk of cataracts.

High COX-2 expression is associated with increased angiogenesis, proliferation and tumoural inflammatory infiltrate in canine malignant mammary tumours: a multivariate survival study.

COX-2 expression affects mammary tumourigenesis by promoting angiogenesis and cell proliferation, encouraging metastatic spread and tumour-associated inflammation. Samples of canine mammary tumours (n = 109) were submitted to immunohistochemistry to detect COX-2, CD31, VEGF, Ki-67, CD3 and MAC387 expression. Concurrent high expression of COX-2/CD31, COX-2/VEGF, COX-2/Ki-67, COX-2/CD3 and COX-2/MAC was associated with elevated grade of malignancy, presence of intravascular emboli and presence of lymph node metastasis. Tumours with high COX-2 (P < 0.001) and tumours with concurrent expression of high COX-2 and high CD31 (P = 0.008); high VEGF (P < 0.001); high Ki-67 (P < 0.001); high CD3+ T-lymphocytes (P = 0.002) and elevated MAC387 macrophages (P = 0.024) were associated with shorter overall survival (OS) time. Interestingly the groups with high COX-2/CD31 and high COX-2/VEGF retained their significance after multivariate analysis arising as independent predictors of OS. Present data highlight the importance of COX-2 in canine mammary tumourigenesis.

Combined effects of sphingomyelin and cholesterol on the hydrolysis of emulsion particle triolein by lipoprotein lipase.

Sphingomyelin (SM) is one of the major lipids in lipoproteins. However, its function in lipoprotein metabolism is unknown. In an attempt to understand the role that this lipid plays in modulation of lipoprotein lipase (LPL)-mediated hydrolysis, triolein-based emulsion particles containing 15% (physiological concentration) and 30% of the phospholipid content as SM together with phosphatidyl choline were used as substrate for the enzyme. Using a continuous fluorescence displacement assay to measure triglyceride (triolein) hydrolysis, it is shown that LPL activity was not modified by physiological concentrations of SM. However, under these assay conditions the presence of 30% SM inhibited LPL hydrolysis. SM and cholesterol (a normal component of the lipoprotein surface monolayer) become closely associated in phospholipid monolayers and bilayers. Incorporation of cholesterol into emulsion particles containing only PC increased LPL activity, but this increase was reduced by the additional presence of a physiological concentration (15%) of SM. These model studies suggest that the ratio, cholesterol:SM, in the monolayer may regulate the hydrolytic activity of the LPL. The production of ceramide by sphingomyelinase pre-treatment of emulsion particles containing SM leads to a two- to three-fold increase in LPL activity. This effect was dependent on sphingomyelinase concentration and time of pre-incubation and was not seen with cholesterol containing substrates. The ability of apolipoprotein CII to enhance LPL-catalysed triolein hydrolysis was not affected by the presence of SM; however, the stimulatory effect of this apolipoprotein was attenuated by pre-treatment of emulsion particles with sphingomyelinase. In summary, physiological concentrations of SM can inhibit the hydrolysis of cholesterol-containing emulsion particles; while pre-treatment of SM containing emulsion particles with sphingomyelinase in the absence of cholesterol can increase LPL-mediated triglyceride hydrolysis.

Effects of alcohol on sleep parameters of sleep-deprived healthy volunteers.

Both partial and total sleep deprivation frequently result in a rebound of paradoxical sleep (PS), as well as of slow-wave or delta sleep. Acute administration of ethanol inhibits PS in normal volunteers. This effect is dose-dependent and consists of increased latency to and reduced duration of paradoxical sleep. It has also been shown that PS rebound may occur on the same night, as blood alcohol concentration (BAC) declines. The present study examined the effects of sleep deprivation prior to ethanol administration on nocturnal-sleep parameters in healthy male volunteers. Polysomnograms were performed with a randomized, crossover design on baseline, placebo, post-placebo, ethanol (0.9 g/kg), and post-ethanol recovery nights. Subjects were submitted to partial (PSD) (n = 6) or total (TSD) (n = 6) sleep deprivation for 40 hours before placebo or ethanol conditions. Results evidenced a PS inhibition after ethanol treatment in both deprivation groups, despite their sleep debt (PSD, placebo = 103.7 minutes and ethanol = 72.7 minutes; TSD, placebo = 111.8 minutes and ethanol = 76.6 minutes). This inhibition was BAC-dependent and specific to PS, since delta sleep remained unaltered. These effects could be due to the reduction of cholinergic release and/or glutamatergic inhibition, both of which modulate acetylcholine release.

Sleep deprivation induces brain region-specific decreases in glutathione levels.

Rats were deprived of sleep for 96 h by the platform technique and total glutathione (GSHtau) levels were measured in seven different brain areas. Glutathione levels were found to be significantly reduced in the hypothalamus of sleep-deprived animals when compared with large platform (-18%) or home cage (-31%) controls. Deprived rats also had reduced GSHtau levels in thalamus compared with home cage controls only. Glutathione levels did not differ among the three groups in any of the other brain areas examined. These results indicate that specific brain areas may be differentially susceptible to oxidative stress after sleep deprivation. The apparent vulnerability of the hypothalamus to these effects may contribute to some of the functional effects of sleep deprivation.

Hypoprolactinemic rats under conditions of constant darkness or constant light. Effects on the sleep-wake cycle, cerebral temperature and sulfatoxymelatonin levels.

In genetic hypoprolactinemic rats under light-dark (LD) conditions, the circadian rhythms of slow-wave (SWS) and paradoxical (PS) sleep display an alteration of their phase relationship. The aim of our study was to investigate the effects of constant darkness (DD) or constant light (LL) on the daily distribution and amounts of sleep-wake stages, cerebral temperature and concentrations of the urinary melatonin metabolite, 6-sulfatoxymelatonin, in prolactin-deficient rats. After 3 weeks of DD, the SWS period was 24 h 8+/-6 min and the acrophase occurred at 15:44+/-1:35, while for PS, the period was more stable than during LD (24 h 10+/-8 min vs. 24 h 55+/-43 min) and the acrophase occurred at 16:44+/-1:54. Under LL conditions, circadian sleep rhythms persisted during the first 3 days, then completely disappeared during the third week, to be replaced by ultradian rhythms (period of 4-6 h). Time-series analysis showed that the two sleep states became synchronous as early as the second day under constant conditions. The total amount of PS was increased under both conditions (LL and DD) at the expense of duration of waking. Under LD and constant conditions, the pattern of changes in cerebral temperature was similar to that for wakefulness (W). Sulfatoxymelatonin was rhythmically secreted under both LD and DD conditions, whereas, under LL conditions, its rhythm was abolished. The results show that, in IPL rats in the absence of a zeitgeber, the PS and SWS rhythms recover a synchronous phase relationship and PS amounts are increased.

Melatonin treatment does not prevent decreases in brain glutathione levels induced by sleep deprivation.

Recent findings from this laboratory revealed that sleep deprivation reduces total glutathione (GSH) levels in hypothalamus, suggesting an increased vulnerability to oxidative damage. Since melatonin has been shown to prevent oxidative damage in other experimental situations, the present study tested the effects of exogenous melatonin on sleep deprivation-induced GSH decreases. Rats were deprived of sleep for 96 h on small platforms, and melatonin (10 mg/kg body weight; i.p.) or vehicle was given twice a day. Hypothalamic GSH levels were significantly reduced in sleep-deprived groups, irrespective of melatonin treatment. Indeed, unexpectedly, melatonin treatment resulted in lower hypothalamic GSH levels in all groups, including cage controls. These results confirm that sleep deprivation reduces hypothalamic GSH and further indicate that melatonin treatment not only is ineffective in reversing this effect but may actually potentiate it.

Absence of oxidative stress following paradoxical sleep deprivation in rats.

Paradoxical sleep deprivation was performed on rats using platform technique to investigate the oxidative process associated with it. Levels of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total glutathione (GSH) and malondialdehyde production were measured in brain of rats under control conditions (C) and those on single large platforms (SLP), multiple large platforms (MLP), single small platforms (SSP) and multiple small platforms (MSP) groups. SOD, CAT and GPx brain activity and malondialdehyde production were not modified by any of the procedures. Brain GSH, however, was significantly reduced in both SSP and SLP groups. These results suggest that paradoxical sleep deprivation per se is not associated with oxidative damage. The observed alterations could be attributed to factors such as immobilization and social isolation present in the single platform techniques.

Cholinergic modulation of inhibitory avoidance impairment induced by paradoxical sleep deprivation.

1. Male Wistar rats were submitted to paradoxical sleep deprivation for 96 hr by a modified multiple platform technique. 2. Training of step-through inhibitory avoidance was performed immediately after the last day of paradoxical sleep deprivation. Twenty-four hr after training the animals were submitted to the retention test. 3. In Experiment 1, pilocarpine (4 mg/kg, i.p.) or atropine (4 mg/kg, i.p.) were administered daily during the paradoxical sleep deprivation period. Pilocarpine, but not atropine, reversed the impairment induced by PS deprivation. 4. In Experiment 2, pilocarpine (4, 8 and 12 mg/kg, i.p.) was injected 1 hr before training in order to verify if the reversal of memory impairment was an effect secondary to residual enhanced blood levels of pilocarpine during training. Acute treatment with pilocarpine, in any dose, did not reverse the impairment produced by paradoxical sleep deprivation 5. Activation of the cholinergic system during the period of deprivation is able to prevent memory deficits induced by paradoxical sleep deprivation.