[Measurement of exhaled nitric oxide fraction in lung diseases]. / Fracción de óxido nítrico exhalado: una herramienta clínica para las enfermedades pulmonares.

Exhaled Nitric Oxide fraction measurement is a new method for the evaluation of respiratory diseases. It has good correlation with airway inflammation and decreases with the administration of corticosteroids. It is useful as a complement for the diagnosis of asthma, Chronic Obstructive Pulmonary Disease, Cystic Fibrosis and Primary Ciliary Dyskinesia among other respiratory diseases that generate inflammation in the airway. Its assessment is easy, non-invasive, and safe, and the result is obtained immediately. It can be used routinely to evaluate the response and adherence to treatments. This article reviews the biology of Nitric Oxide, and the measurement, interpretation, and main clinical uses of Exhaled Nitric Oxide Fraction.

Neosaxitoxin Inhibits the Expression of Inflammation Markers of the M1 Phenotype in Macrophages.

(1) Background: Neosaxitoxin (NeoSTX) has been used as a local anesthetic, but its anti-inflammatory effects have not been well defined. In the present study, we investigate the effects of NeoSTX on lipopolysaccharide (LPS)-activated macrophages. (2) Methods: Raw 264.7 and equine PBMC cells were incubated with or without 100 ng/mL LPS in the presence or absence of NeoSTX (1µM). The expression of inflammatory mediators was assessed: nitric oxide (NO) content using the Griess assay, TNF-α content using the ELISA assay, and mRNA of inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) using a real-time polymerase chain reaction. (3) Results: NeoSTX (1 µM) significantly inhibited the release of NO, TNF-α, and expression of iNOS, IL-1ß, and TNF-α in LPS-activated macrophages of both species studied. Furthermore, our study shows that the LPS-induced release of inflammatory mediators was suppressed by NeoSTX. Additionally, NeoSTX deactivated polarized macrophages to M1 by LPS without compromising its polarization towards M2. (4) Conclusions: NeoSTX inhibits LPS-induced release of inflammatory mediators from macrophages, and these effects may be mediated by the blockade of voltage-gated sodium channels (VGSC).

[Sepsis hyperglycemia in the ICU: from the mechanism to the clinic]. / Hiperglicemia por sepsis: del mecanismo a la clínica.

Stress hyperglycemia is frequently diagnosed in septic patients in critical care units (ICU) and it is associated with greater illness severity and higher morbimortality rates. In response to an acute injury, high levels of counterregulatory hormones such as glucocorticoids and catecholamines are released causing increased hepatic gluconeogenesis and insulin resistance. Furthermore, during sepsis, proinflammatory cytokines also participate in the pathogenesis of this phenomenon. Septic patients represent a subtype of the critical ill patients in the ICU: this metabolic disarrangement management strategies and insulin therapy recommendations had been inconsistent. In this article, we describe the pathophysiological mechanisms of stress hyperglycemia in critical patients including the action of hormones, inflammatory cytokines and tissue resistance to insulin. In addition, we analyzed the main published studies for the treatment of acute hyperglycemia in critical patients.

High Prevalence of Pneumocystis jirovecii Dihydropteroate Synthase Gene Mutations in Patients with a First Episode of Pneumocystis Pneumonia in Santiago, Chile, and Clinical Response to Trimethoprim-Sulfamethoxazole Therapy.

Mutations in the dihydropteroate synthase (DHPS) gene of Pneumocystis jirovecii are associated with the failure of sulfa prophylaxis. They can develop by selection in patients receiving sulfa drugs or be acquired via person-to-person transmission. DHPS mutations raise concern about the decreasing efficacy of sulfa drugs, the main available therapeutic tool for Pneumocystis pneumonia (PCP). The prevalence of Pneumocystis DHPS mutations was examined in Pneumocystis isolates from 56 sulfa-prophylaxis-naive adults with a first episode of PCP from 2002 to 2010 in Santiago, Chile. Their clinical history was reviewed to analyze the effect of these mutations on response to trimethoprim-sulfamethoxazole (TMP-SMX) therapy and outcome. Mutant genotypes occurred in 22 (48%) of 46 HIV-infected patients and in 5 (50%) of 10 HIV-uninfected patients. Compared to patients with a wild-type genotype, those with mutant genotypes were more likely to experience sulfa treatment-limiting adverse reactions and to have a twice-longer duration of mechanical ventilation if mechanically ventilated. Specific genotypes did not associate with death, which occurred in none of the HIV-infected patients and in 50% of the non-HIV-infected patients. Chile has a high prevalence of DHPS mutations, which were presumably acquired through interhuman transmission because patients were not on sulfa prophylaxis. These results contrast with the low prevalence observed in other Latin American countries with similar usage of sulfa drugs, suggesting that additional sources of resistant genotypes may be possible. The twice-longer duration of mechanical ventilation in patients with mutant DHPS genotypes suggests a decreased efficacy of TMP-SMX and warrants collaborative studies to assess the relevance of DHPS mutations and further research to increase therapeutic options for PCP.

[Understanding cortisol action in acute inflammation: A view from the adrenal gland to the target cell]. / Para entender la acción de cortisol en inflamación aguda: una mirada desde la glándula suprarrenal hasta la célula blanco.

Glucocorticoids (cortisol in humans) are essential for numerous biological functions. Among critically ill patients, therapy with cortisol has gained strength in recent years, but clinical results have been mixed. A series of events, that may explain the diversity of clinical responses, occur from the synthesis of cortisol in the adrenal gland to the activation of the cortisol receptor by the hormone when it enters the nucleus of the target cell. Some of these events are revised; a proposition for identifying critically ill patients who may benefit with this therapy is suggested.

Cerebral and Cutaneous Involvements of Xanthoma Disseminatum Successfully Treated with an Interleukin-1 Receptor Antagonist: A Case Report and Minireview.

A young male presented with panhypopituitarism (including diabetes insipidus) and temporal lobe epilepsy. A histology specimen of cutaneous papules was diagnostic of non-Langerhans histiocytosis. The diagnosis of xanthoma granulomata was considered based on the clinical and brain MRI findings. Brain lesions significantly worsened over time despite radiotherapy until anakinra induced a complete clinical and radiological remission of all active lesions. Although a single case, the outcome of this patient with xanthoma disseminatum treated with an interleukin-1 receptor antagonist opens and strengthens new and recent physiopathogenic and treatment perspectives for the otherwise difficult-to-treat non-Langerhans cell histiocytosis. Similar results with anakinra have been observed in patients with Erdheim-Chester disease and in multicentric reticulohistiocytosis.

Septic serum induces glucocorticoid resistance and modifies the expression of glucocorticoid isoforms receptors: a prospective cohort study and in vitro experimental assay.

BACKGROUND: A protective role for glucocorticoid therapy in animal models of sepsis was shown many decades ago. In human sepsis, there is new interest in glucocorticoid therapy at a physiological dose after reports of improved response to vasopressor drugs and decreased mortality in a selected group of patients. However, other reports have not confirmed these results. Cellular glucocorticoid resistance could explain a possible cause of that. To evaluate this hypothesis, we evaluated the expression of glucocorticoid receptor beta, the dominant negative isoform of glucocorticoid receptor, in peripheral mononuclear cells of septic patients and the effect of serum septic patients over glucocorticoid receptor expression and glucocorticoid sensitivity in immune cells culture. METHODS: A prospective cohort study and an in vitro experimental study with matched controls were developed. Nine patients with septic shock and nine healthy controls were prospectively enrolled. Mononuclear cells and serum samples were obtained from the patients with sepsis on admission to the Intensive Care Unit and on the day of discharge from hospital, and from healthy volunteers matched by age and sex with the patients. Glucocorticoid receptor alpha and beta expression from patients and from immune cell lines cultured in the presence of serum from septic patients were studied by western blot. Glucocorticoid sensitivity was studied in control mononuclear cells cultured in the presence of serum from normal or septic patients. A statistical analysis was performed using a Mann-Whitney test for non-parametric data and analysis of variance for multiple comparison; P<0.05 was considered significant. RESULTS: The patients' glucocorticoid receptor beta expression was significantly higher on admission than on discharge, whereas the alpha receptor was not significantly different. In vitro, septic serum induced increased expression of both receptors in T and B cells in culture, with a greater effect on receptor beta than the control serum. Septic serum induced glucocorticoid resistance in control mononuclear cells. CONCLUSION: There is a transient increased expression of glucocorticoid receptor beta in mononuclear cells from septic patients. Serum from septic patients induces cell glucocorticoid resistance in vitro. Our findings support a possible cell glucocorticoid resistance in sepsis.

Cooperative Effect of Chemical and Physical Processes for Flame Retardant Additives in Recycled ABS.

In the present work, the effectiveness of four non-halogenated flame retardants (FR) (aluminium trihydroxide (ATH), magnesium hydroxide (MDH), Sepiolite (SEP) and a mix of metallic oxides and hydroxides (PAVAL)) in blends with recycled acrylonitrile-butadiene-styrene (rABS) was studied in order to develop a more environmentally friendly flame-retardant composite alternative. The mechanical and thermo-mechanical properties of the obtained composites as well as their flame-retardant mechanism were evaluated by UL-94 and cone calorimetric tests. As expected, these particles modified the mechanical performance of the rABS, increasing its stiffness at the expense of reducing its toughness and impact behavior. Regarding the fire behavior, the experimentation showed that there is an important synergy between the chemical mechanism provided by MDH (decomposition into oxides and water) and the physical mechanism provided by SEP (oxygen barrier), which means that mixed composites (rABS/MDH/SEP) can be obtained with a flame behavior superior to that of the composites studied with only one type of FR. In order to find a balance between mechanical properties, composites with different amounts of SEP and MDH were evaluated. The results showed that composites with the composition rABS/MDH/SEP: 70/15/15 wt.% increase the time to ignition (TTI) by 75% and the resulting mass after ignition by more than 600%. Furthermore, they decrease the heat release rate (HRR) by 62.9%, the total smoke production (TSP) by 19.04% and the total heat release rate (THHR) by 13.77% compared to unadditivated rABS; without compromising the mechanical behavior of the original material. These results are promising and potentially represent a greener alternative for the manufacture of flame-retardant composites.

Star-Branched Polyamides as the Matrix in Thermoplastic Composites.

The aim of this study is the preparation of star-shaped branched polyamides (sPA6) with low melt viscosity, but also with improved mechanical properties by reactive extrusion. This configuration has been obtained by grafting a tri-functional, three-armed molecule: 5-aminoisophthalic-acid, used as a linking agent (LA). The balance between the fluidity, polarity and mechanical properties of sPA6s is the reason why these materials have been investigated for the impregnation of fabrics in the manufacture of thermoplastic composites. For these impregnation processes, the low viscosity of the melt has allowed the processing parameters (temperature, pressure and time) to be reduced, and its new microstructure has allowed the mechanical properties of virgin thermoplastic resins to be maintained. A significant improvement in the ultrasonic welding processes of the composites was also found when an energy director based on these materials was applied at the interface. In this work, an exhaustive microstructural characterization of the obtained sPAs is presented and related to the final properties of the composites obtained by film stacking.

Rheological Method for Determining the Molecular Weight of Collagen Gels by Using a Machine Learning Technique.

This article presents, for the first time, the results of applying the rheological technique to measure the molecular weights (Mw) and their distributions (MwD) of highly hierarchical biomolecules, such as non-hydrolyzed collagen gels. Due to the high viscosity of the studied gels, the effect of the concentrations on the rheological tests was investigated. In addition, because these materials are highly sensitive to denaturation and degradation under mechanical stress and temperatures close to 40 °C, when frequency sweeps were applied, a mathematical adjustment of the data by machine learning techniques (artificial intelligence tools) was designed and implemented. Using the proposed method, collagen fibers of Mw close to 600 kDa were identified. To validate the proposed method, lower Mw species were obtained and characterized by both the proposed rheological method and traditional measurement techniques, such as chromatography and electrophoresis. The results of the tests confirmed the validity of the proposed method. It is a simple technique for obtaining more microstructural information on these biomolecules and, in turn, facilitating the design of new structural biomaterials with greater added value.

Effects of methotrexate on the expression of the translational isoforms of glucocorticoid receptors α and ß: correlation with methotrexate efficacy in rheumatoid arthritis patients.

OBJECTIVES: To test the effect of MTX on the expression of glucocorticoid receptor (GR) α and ß isoforms AB, C and D in peripheral blood mononuclear cells (PBMCs) in culture, from newly diagnosed RA patients and to evaluate whether the test results correlate with patients' subsequent response to MTX treatment. METHODS: Twenty patients with early active RA were enrolled. Patients who had previously received any DMARD or cytotoxic agent, or who had received CSs in the 6 months before enrolment were excluded. PBMCs from all patients were obtained and cultured in the presence and absence of MTX (10(-4), 10(-6) and 10(-8) M). The expression of GR isoforms was evaluated by western blot. After blood samples were taken, patients entered a 24-week study receiving MTX, diclofenac and prednisone (10 mg/day). At Week 24, the ACR core set of disease activity measures was calculated and a correlation between the MTX effect on patients' PBMC GR expression in vitro and the ACR response was evaluated. RESULTS: MTX 10(-6) M in the culture medium induced the expression of the PBMC isoform AB of GRα (P = 0.009). Other GR isoforms were unaffected. The magnitude of the induced expression correlated with the ACR response to treatment at Week 24 of therapy (r = 0.92, P = 0.00003). CONCLUSION: MTX in vitro induces greater expression of GRαAB isoform in PBMC from RA patients who later respond to MTX treatment than in non-responding patients. This may have clinical applications for predicting MTX efficacy in RA patients.

In vitro Phenotype Induction of Circulating Monocytes: CD16 and CD163 Analysis.

INTRODUCTION: CD14 (monocyte differentiation antigen, LPS binding protein - endotoxin receptor) and CD16 (FcγRIII, Low-affinity receptor for IgG) define three subpopulations of circulating monocytes with different inflammatory and phagocytic capabilities. Contradictory reports exist regarding both in vivo monocyte phenotype-disease association and response of these circulating monocytes to in vitro stimulation. We analyzed phenotypic changes in circulating monocytes when stimulated with LPS (pro-inflammatory stimulus) and IL-4 (alternative inflammatory stimulus). METHODS: Mononuclear cells from nine healthy donors were extracted and studied for surface and intracellular markers using flow cytometry. PBMC were extracted using Ficoll technic and immediately analyzed using flow cytometry. Pro-inflammatory interleukin IL-1ß and IL-6 were measured by intracellular cytometry. Mononuclear cells were stimulated using LPS and IL-4 as previously described. Changes against non-stimulated populations were statistically analyzed. RESULTS: Compared to non-stimulated and IL-4 stimulated monocytes, LPS-stimulated cells display a singular pattern of markers, with higher levels of intracellular IL-1ß and IL-6 directly correlating with CD14+CD163- cell frequency and diminishing membrane CD163 fluorescence. CD14+CD16- classical monocytes show greater percentage of CD163- cells upon LPS stimulation. CD86 levels on monocytes' surface did not change with LPS or IL-4 stimulation. CONCLUSIONS AND DISCUSSION: We showed that CD14+CD16- classical monocytes display higher sensitivity to LPS stimulation, with more IL-1ß and IL-6 levels than intermediate and non-classical monocytes. This subset also diminishes its CD163 levels on the membrane after LPS stimulation with a contemporary raise in CD163- cells, suggesting that classical monocytes preferentially acquire CD163- defined M1 characteristics upon in vitro LPS stimulation. Intermediate and non-classical monocytes respond with lower levels of interleukins and display surface proteins in an M2-type profile (CD163+).

Physalis peruviana L. Pulp Prevents Liver Inflammation and Insulin Resistance in Skeletal Muscles of Diet-Induced Obese Mice.

A chronic high-fat diet (HFD) produces obesity, leading to pathological consequences in the liver and skeletal muscle. The fat in the liver leads to accumulation of a large number of intrahepatic lipid droplets (LD), which are susceptible to oxidation. Obesity also affects skeletal muscle, increasing LD and producing insulin signaling impairment. Physalis peruviana L. (PP) (Solanaceae) is rich in peruvioses and has high antioxidant activity. We assessed the ability of PP to enhance insulin-dependent glucose uptake in skeletal muscle and the capacity to prevent both inflammation and lipoperoxidation in the liver of diet-induced obese mice. Male C57BL/6J mice were divided into groups and fed for eight weeks: control diet (C; 10% fat, 20% protein, 70% carbohydrates); C + PP (300 mg/kg/day); HFD (60% fat, 20% protein, 20% carbohydrates); and HFD + PP. Results suggest that PP reduces the intracellular lipoperoxidation level and the size of LD in both isolated hepatocytes and skeletal muscle fibers. PP also promotes insulin-dependent skeletal muscle glucose uptake. In conclusion, daily consumption of 300 mg/kg of fresh pulp of PP could be a novel strategy to prevent the hepatic lipoperoxidation and insulin resistance induced by obesity.

An Activity to Promote Recognition of Unintentional Plagiarism in Scientific Writing in Undergraduate Biology Courses.

Unintentional plagiarism frequently occurs in undergraduate writing assignments because students are unaware of the complexity of correct paraphrasing and citation rules. There is often a lack of formal instruction in science courses on proper paraphrasing and citation to reduce plagiarism. To address this deficit, we developed a brief activity to teach students to recognize the range of paraphrasing and citation errors that can result in plagiarism. The activity was used in a biology-focused scientific literacy course, but it can be incorporated into different instructional settings, with undergraduate students of all levels. During this classroom activity, part 1 addresses the nuances associated with proper paraphrasing and citation in scientific writing and part 2 asks students to practice paraphrasing and properly citing a passage from a scientific source. Pretest results revealed that students were proficient at identifying plagiarism when a citation error occurred but were less proficient at recognizing improper paraphrasing (patchwriting or direct plagiarism). Posttest results indicated that the activity was effective at increasing the students' ability to recognize a paraphrasing error even when a correct citation was present. Students also reported higher confidence in their understanding of what constitutes plagiarism and that they are more confident in their ability to properly paraphrase and cite scientific source content.

Glucocorticoid Receptor-α and MKP-1 as Candidate Biomarkers for Treatment Response and Disease Activity in Vogt-Koyanagi-Harada Disease.

PURPOSE: To investigate the potential of utilizing the expression of genes for glucocorticoid receptor (GR) and mitogen-activated protein kinase phosphatase-1 (MKP-1) as biomarkers of corticosteroid (CS) refractoriness and disease activity in patients with Vogt-Koyanagi-Harada (VKH) disease. DESIGN: Prospective cohort study. METHODS: Twenty VKH patients receiving their first cycle of CS treatment in the absence of additional systemic immunosuppressive therapy and a control group of fifteen healthy volunteers were recruited from the University of Chile (Santiago, Chile) and US National Institutes of Health (Bethesda, United States). Intraocular inflammation was clinically quantified at enrolment and all follow-up visits. CS refractoriness was defined as an ocular reactivation of VKH upon CS withdrawal at a daily oral prednisone dose of 10 mg or more. Quantitative Reverse transcription polymerase chain reaction (qRT-PCR) was performed to measure the mRNA levels of the alpha (α) and beta (ß) isoforms of GR and MKP-1 in peripheral blood mononuclear cells (PBMC) after in vitro stimulation with either anti-CD3/anti-CD28 antibodies, lipopolysaccharide (LPS), or phytohemagglutinin (PHA), in the presence or absence of dexamethasone (Dex). RESULTS: After 6 hours of stimulation in the presence of Dex, PBMC from CS-refractory VKH patients had an impaired elevation in GRα expression (P = .03). Furthermore, inactive patients showed a significant Dex-induced upregulation of MKP-1 (P = .005). CONCLUSIONS: In this pilot study, the expression of GR isoforms and MKP-1 corresponded with patients' clinical response to systemic CS treatment and disease activity, respectively. Hence, these candidate biomarkers have potential clinical utility in the early identification of CS refractoriness and subclinical inflammation in patients with VKH disease.

LPS regulates the expression of glucocorticoid receptor α and ß isoforms and induces a selective glucocorticoid resistance in vitro.

BACKGROUND: This study was aimed to evaluate the effect of LPS in glucocorticoid receptor (GR) isoforms expression on different cell lines and PBMC from healthy donors in vitro and glucocorticoid sensitivity of PBMC in vitro. METHODS: U-2 OS cell lines expressing GR isoforms, different cell lines (CEM, RAJI, K562 and HeLa) or PBMC from healthy donors, were cultured or not with LPS. The expression of GRα and GRß was evaluated by Western blot. Glucocorticoid sensitivity was evaluated in PBMC treated with LPS, testing genes which are transactivated or transrepressed by glucocorticoid. For transactivated genes (MKP1, FKBP5) PBMC were treated with Dexamethasone 100 nM for 6 h. The mRNA expression was measured by RT-PCR. For transrepressed genes (IL-8, GM-CSF), PBMC were cultured in Dexamethasone 100 nM and LPS 10 µg/ml for 6 h and protein expression was measure by ELISA. RESULTS: GR isoforms were induced in U-2 OS cells with a greater effect on GRα expression. Both isoforms were also induced in CEM cells with a tendency to a greater effect on GRß. LPS induced only the expression of GRα in Raji and HeLa cells, and in PBMC, with no effect in K562 cells. LPS induced a loss of glucocorticoid inhibitory effect only on the secretion of GM-CSF. CONCLUSION: LPS in vitro differentially modulates the expression of GR isoforms in a cell specific manner. In PBMC from healthy donors LPS induces an approximately two times increase in the expression of GRα and a loss of the glucocorticoid inhibitory effect on the secretion of GM-CSF, without affecting other glucocorticoid responses evaluated.

Evaluation of the Glucocorticoid Receptor as a Biomarker of Treatment Response in Vogt-Koyanagi-Harada Disease.

Purpose: This study is aimed to investigate the role of glucocorticoid receptor (GR) isoforms in peripheral blood mononuclear cells (PBMC) as biomarkers of glucocorticoid (GC) resistance and to validate a set of clinical predictive factors in patients with Vogt-Koyanagi-Harada (VKH) disease. Methods: This was a prospective cohort study that included a total of 21 patients with VKH. A complete ophthalmologic evaluation was carried out at baseline that recorded the presence of any clinical predictive factors (visual acuity ≤ 20/200, tinnitus, chronic disease, and fundus depigmentation). Real-time quantitative PCR was performed to measure the mRNA levels of GR alpha (GRα) and beta (GRß) isoforms at baseline and at 2 weeks after prednisone therapy initiation. Results: There were no differences between GRα and GRß levels in GC-sensitive and GC-resistant patients at baseline before treatment initiation. After 2 weeks of prednisone treatment, GC-sensitive patients had a median 5.5-fold increase in levels of GRα, whereas GC-resistant patients had a median 0.7-fold decrease in levels of this isoform (P = 0.003). Similarly, GRß increased in GC-sensitive patients, in comparison with GR-resistant patients (6.49-fold versus 1.01 fold, respectively, I = 0.04). The mRNA levels of GR isoforms were independent of disease activity. Fundus depigmentation and chronic disease at diagnosis were associated with GC resistance (P = 0.03, odds ratio = 21.0; and P = 0.008, odds ratio = 37.8, respectively). However, associations with visual acuity or tinnitus were not confirmed in this study. Conclusions: The evaluation of clinical predictive factors and determination of the change in expression of GR isoforms as potential biomarkers can contribute to the early identification of GC-resistant patients with VKH.

Glucocorticoid receptor beta in acute and chronic inflammatory conditions: clinical implications.

Glucocorticoids (GC) are hormones with a wide variety of actions, including profound anti-inflammatory/immunosuppressive effects. Their actions are mediated by an intracellular receptor called the glucocorticoid receptor (GCR). The classical GCR that mediates the hormone response is called GCR alpha. Recently however, many GCR isotypes have been described. A defective GC action has been proposed as an etio-pathogenic mechanism for the development of inflammatory/autoimmune diseases. Inadequate GC actions may have multiple causes such as: defective hypothalamic-pituitary-adrenal axis function, GC export from cells, hormone metabolization into inactive compounds and modifications of the GC receptor, among others. In 1995, a dominant negative effect of a GC receptor isotype termed beta was described; starting a still unsolved controversy about the role of GCR beta as an inducer of GC resistance in certain pathological conditions. The present article will review the data about a possible role for GCR beta in the development of GC resistance in inflammatory diseases. This review will especially focus on the role of the GCR beta in rheumatoid arthritis and in septic shock as examples of a chronic inflammatory disease and an acute systemic inflammatory condition. Original data supporting possible hyperexpression of GCR beta in both conditions will be shown.