Effect of docosahexaenoic acid as an anti-inflammatory for Caco-2 cells and modulating agent for gut microbiota in children with obesity (the DAMOCLE study).

PURPOSE: Docosahexaenoic acid (DHA) is a long-chain omega-3 polyunsaturated fatty acid. We investigated the dual health ability of DHA to modulate gut microbiota in children with obesity and to exert anti-inflammatory activity on human intestinal Caco-2 cells. METHODS: In a pilot study involving 18 obese children (8-14 years), participants received a daily DHA supplement (500 mg/day) and dietary intervention from baseline (T0) to 4 months (T1), followed by dietary intervention alone from 4 months (T1) to 8 months (T2). Fecal samples, anthropometry, biochemicals and dietary assessment were collected at each timepoint. At preclinical level, we evaluated DHA's antioxidant and anti-inflammatory effects on Caco-2 cells stimulated with Hydrogen peroxide (H2O2) and Lipopolysaccharides (LPS), by measuring also Inducible nitric oxide synthase (iNOS) levels and cytokines, respectively. RESULTS: Ten children were included in final analysis. No major changes were observed for anthropometric and biochemical parameters, and participants showed a low dietary compliance at T1 and T2. DHA supplementation restored the Firmicutes/Bacteroidetes ratio that was conserved also after the DHA discontinuation at T2. DHA supplementation drove a depletion in Ruminococcaceae and Dialisteraceae, and enrichment in Bacteroidaceae, Oscillospiraceae, and Akkermansiaceae. At genus level, Allisonella was the most decreased by DHA supplementation. In Caco-2 cells, DHA decreased H2O2-induced reactive oxygen species (ROS) and nitric oxide (NO) production via iNOS pathway modulation. Additionally, DHA modulated proinflammatory (IL-1ß, IL-6, IFN-γ, TNF-α) and anti-inflammatory (IL-10) cytokine production in LPS-stimulated Caco-2 cells. CONCLUSION: An improvement in gut dysbiosis of children with obesity seems to be triggered by DHA and to continue after discontinuation. The ability to modulate gut microbiota, matches also with an anti-inflammatory effect of DHA on Caco-2 cells.

Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor roGFP2-Orp1.

Accurate yet efficient high-throughput screenings have emerged as essential technology for enzyme engineering via directed evolution. Modern high-throughput screening platforms for oxidoreductases are commonly assisted by technologies such as surface display and rely on emulsification techniques to facilitate single-cell analysis via fluorescence-activated cell sorting. Empowered by the dramatically increased throughput, the screening of significantly larger sequence spaces in acceptable time frames is achieved but usually comes at the cost of restricted applicability. In this work, we tackle this problem by utilizing roGFP2-Orp1 as a fluorescent one-component detection system for enzymatic H2O2 formation. We determined the kinetic parameters of the roGFP2-Orp1 reaction with H2O2 and established an efficient immobilization technique for the sensor on Saccharomyces cerevisiae cells employing the lectin Concanavalin A. This allowed to realize a peroxide-sensing shell on enzyme-displaying cells, a system that was successfully employed to screen for H2O2 formation of enzyme variants in a whole-cell setting.

Phenylketonuric diet negatively impacts on butyrate production.

BACKGROUND AND AIMS: Phenylalanine (Phe) restricted diet, combined with Phe-free l-amino acid supplementation, is the mainstay of treatment for phenylketonuria (PKU). Being the diet a key factor modulating gut microbiota composition, the aim of the present paper was to compare dietary intakes, gut microbiota biodiversity and short chain fatty acids (SCFAs) production in children with PKU, on low-Phe diet, and in children with mild hyperphenylalaninemia (MHP), on unrestricted diet. METHODS AND RESULTS: We enrolled 21 PKU and 21 MHP children matched for gender, age and body mass index z-score. Dietary intakes, including glycemic index (GI) and glycemic load (GL), and fecal microbiota analyses, by means of denaturing gradient gel electrophoresis (DGGE) and Real-time PCR were assessed. Fecal SCFAs were quantified by gas chromatographic analysis. RESULTS: We observed an increased carbohydrate (% of total energy), fiber and vegetables intakes (g/day) in PKU compared with MHP children (p = 0.047), as well a higher daily GI and GL (maximum p < 0.001). Compared with MHP, PKU showed a lower degree of microbial diversity and a decrease in fecal butyrate content (p = 0.02). Accordingly, two of the most abundant butyrate-producing genera, Faecalibacterium spp. and Roseburia spp., were found significantly depleted in PKU children (p = 0.02 and p = 0.03, respectively). CONCLUSION: The low-Phe diet, characterized by a higher carbohydrate intake, increases GI and GL, resulting in a different quality of substrates for microbial fermentation. Further analyses, thoroughly evaluating microbial species altered by PKU diet are needed to better investigate gut microbiota in PKU children and to eventually pave the way for pre/probiotic supplementations.

Microbiologic and clinical characteristics of biofilm-forming Candida parapsilosis isolates associated with fungaemia and their impact on mortality.

OBJECTIVES: Biofilm formation (BF) by fungal isolates may dramatically complicate infection. We determined the ability of Candida parapsilosis isolates from single fungaemia episodes to form biofilms and we analysed biofilm subgroups for antifungal susceptibility and pathogenic potential. We then correlated BF with clinical characteristics and outcomes of the episodes. METHODS: BF was measured using the crystal violet biomass assay. Antifungal susceptibility of preformed biofilms was assessed, and virulence was studied using the Galleria mellonella model. A retrospective analysis of patients' clinical records was performed. RESULTS: Of 190 patient-unique isolates, 84, 38 and 68 were identified as having high BF (HBF), moderate BF (MBF) or low BF (LBF), respectively. Among 30 randomly selected isolates, nine (eight HBF and one MBF), six (all HBF) and one (HBF) isolates had elevated sessile minimum inhibitory concentrations to fluconazole, anidulafungin or amphotericin B; all HBF and MBF isolates had elevated voriconazole sessile minimum inhibitory concentrations. G. mellonella killing rates of HBF isolates were significantly greater than MBF (or LBF) isolates (50% vs. 20%, 2 days from infection). By comparing HBF/MBF (106 patients) and LBF (84 patients) groups, we found that HBF/MBF patients had more central venous catheter-related fungaemias (62/106 (58.5%) vs. 29/84 (34.5%), p 0.001) and were more likely to die at 30 days from fungaemia onset (61/106 (57.5%) vs. 28/84 (33.3%), p 0.01). In the HBF/MBF group, azole antifungal therapy and central venous catheter removal were significantly associated with a higher and lower 30-day mortality rate, respectively. CONCLUSIONS: C. parapsilosis BF influences the clinical outcome in patients with fungaemia.

Does peripheral arterial occlusive disease influence muscle strength and exercise capacity in COPD patients? / A doença arterial periférica obstrutiva influencia a força muscular e a capacidade de exercício nos portadores de doença pulmonar obstrutiva crônica?

Abstract Background The pathophysiology of chronic obstructive pulmonary disease (COPD) is complex and understanding of it has been changing in recent years, with regard to its multisystemic manifestations, especially peripheral dysfunction and its influence on intolerance to exercise. Objectives To evaluate the relationship between peripheral arterial occlusive disease (PAOD) and peripheral muscle strength and exercise capacity in COPD patients. Methods We conducted a cross-sectional study of 35 patients with COPD who were evaluated with the Ankle-Brachial Index, handgrip strength test, 1 repetition maximum (1RM) of knee extensors and flexors, and distance covered in the incremental shuttle walking test (dISWT). Results COPD patients with coexisting PAOD had lower dominant handgrip strength test results (33.00 vs. 26.66 kgf, p = 0.02) and worse performance in the dISWT (297.32 vs. 219.41 m, p = 0.02) when compared to the COPD patients without PAOD. Strong correlations were found between the result of the handgrip strength test and both the dISWT (r = 0.78; p < 0.001) and the 1RM/knee extension (r = 0.71; p = 0.03); and also between the dISWT and both the 1RM/knee extension (r = 0.72; p = 0.02) and the 1RM/knee flexion (r = 0.92; p < 0.001). The linear regression model showed that the dISWT variable alone explains 15.3% of the Ankle-Brachial Index result (p = 0.01). Conclusion COPD patients with PAOD exhibit reduced muscle strength and lower exercise capacity than COPD patients without PAOD.

Resumo Contexto A doença pulmonar obstrutiva crônica (DPOC) apresenta uma complexa fisiopatologia e sua compreensão vem se modificando nos últimos anos, com atenção para as manifestações multissistêmicas, em especial a disfunção periférica e sua influência na intolerância ao exercício físico. Objetivo Avaliar o impacto da doença arterial periférica obstrutiva (DAOP) na força muscular periférica e na capacidade de exercício dos portadores de DPOC. Métodos Estudo transversal realizado com 35 portadores de DPOC, que foram avaliados pelo índice tornozelo-braquial, teste de força de preensão palmar (FPP), uma repetição máxima (1RM) de extensores e flexores de joelho, e a distância no incremental shuttle walking test (dISWT). Resultados Portadores de DPOC com DAP coexistente apresentaram menor FPP da mão dominante (33,00 versus 26,66 kgf, p = 0,02) e pior desempenho no dISWT (297,32 versus 219,41 m, p = 0,02) quando comparados aos portadores de DPOC sem DAP. Fortes correlações foram encontradas entre a medida da FPP e a dISWT (r = 0,78; p < 0,001) e a 1RM/extensão de joelho (r = 0,71; p = 0,03); entre a dISWT e a 1RM/extensão de joelho (r = 0,72; p = 0,02) e a 1RM/flexão de joelho (r = 0,92; p < 0,001). O modelo de regressão linear identificou que a variável dISWT explica isoladamente 15,3% do resultado do índice tornozelo braquial (p = 0,01). Conclusão Portadores de DPOC com DAOP coexistente apresentam maior perda da força muscular periférica e pior desempenho da capacidade de exercício quando comparados aos portadores de DPOC sem DAOP.

Does peripheral arterial occlusive disease influence muscle strength and exercise capacity in COPD patients?

BACKGROUND: The pathophysiology of chronic obstructive pulmonary disease (COPD) is complex and understanding of it has been changing in recent years, with regard to its multisystemic manifestations, especially peripheral dysfunction and its influence on intolerance to exercise. OBJECTIVES: To evaluate the relationship between peripheral arterial occlusive disease (PAOD) and peripheral muscle strength and exercise capacity in COPD patients. METHODS: We conducted a cross-sectional study of 35 patients with COPD who were evaluated with the Ankle-Brachial Index, handgrip strength test, 1 repetition maximum (1RM) of knee extensors and flexors, and distance covered in the incremental shuttle walking test (dISWT). RESULTS: COPD patients with coexisting PAOD had lower dominant handgrip strength test results (33.00 vs. 26.66 kgf, p = 0.02) and worse performance in the dISWT (297.32 vs. 219.41 m, p = 0.02) when compared to the COPD patients without PAOD. Strong correlations were found between the result of the handgrip strength test and both the dISWT (r = 0.78; p < 0.001) and the 1RM/knee extension (r = 0.71; p = 0.03); and also between the dISWT and both the 1RM/knee extension (r = 0.72; p = 0.02) and the 1RM/knee flexion (r = 0.92; p < 0.001). The linear regression model showed that the dISWT variable alone explains 15.3% of the Ankle-Brachial Index result (p = 0.01). CONCLUSION: COPD patients with PAOD exhibit reduced muscle strength and lower exercise capacity than COPD patients without PAOD.

CONTEXTO: A doença pulmonar obstrutiva crônica (DPOC) apresenta uma complexa fisiopatologia e sua compreensão vem se modificando nos últimos anos, com atenção para as manifestações multissistêmicas, em especial a disfunção periférica e sua influência na intolerância ao exercício físico. OBJETIVO: Avaliar o impacto da doença arterial periférica obstrutiva (DAOP) na força muscular periférica e na capacidade de exercício dos portadores de DPOC. MÉTODOS: Estudo transversal realizado com 35 portadores de DPOC, que foram avaliados pelo índice tornozelo-braquial, teste de força de preensão palmar (FPP), uma repetição máxima (1RM) de extensores e flexores de joelho, e a distância no incremental shuttle walking test (dISWT). RESULTADOS: Portadores de DPOC com DAP coexistente apresentaram menor FPP da mão dominante (33,00 versus 26,66 kgf, p = 0,02) e pior desempenho no dISWT (297,32 versus 219,41 m, p = 0,02) quando comparados aos portadores de DPOC sem DAP. Fortes correlações foram encontradas entre a medida da FPP e a dISWT (r = 0,78; p < 0,001) e a 1RM/extensão de joelho (r = 0,71; p = 0,03); entre a dISWT e a 1RM/extensão de joelho (r = 0,72; p = 0,02) e a 1RM/flexão de joelho (r = 0,92; p < 0,001). O modelo de regressão linear identificou que a variável dISWT explica isoladamente 15,3% do resultado do índice tornozelo braquial (p = 0,01). CONCLUSÃO: Portadores de DPOC com DAOP coexistente apresentam maior perda da força muscular periférica e pior desempenho da capacidade de exercício quando comparados aos portadores de DPOC sem DAOP.

Efficacy of non-invasive mechanical ventilation in the general ward in patients with chronic obstructive pulmonary disease admitted for hypercapnic acute respiratory failure and pH < 7.35: a feasibility pilot study.

AIM: To date non-invasive (NIV) mechanical ventilation use is not recommended in chronic obstructive pulmonary disease (COPD) patients with acute respiratory failure (ARF) and pH < 7.30 outside a 'protected environment'. We assessed NIV efficacy and feasibility in improving arterial blood gases (ABG) and in-hospital outcome in patients with ARF and severe respiratory acidosis (RA) admitted to an experienced rural medical ward. METHODS: This paper is a prospective pilot cohort study conducted in the General Medicine Ward of Budrio's District Hospital. Two hundred and seventy-two patients with ARF were admitted to our Department, 112, meeting predefined inclusion criteria (pH < 7.35, PaCO2 > 45 mmHg). Patients were divided according to the severity of acidosis into: group A (pH < 7.26), group B (7.26 ≤ pH < 7.30) and group C (7.30 ≤ pH < 7.35). ABG were assessed at admission, at 2-6 h, 24 h, 48 h and at discharge. RESULTS: Group A included 55 patients (24 men, mean age: 80.8 ± 8.3 years), group B 31 (12 men, mean age: 80.3 ± 9.4 years) and group C 26 (15 men, mean age: 78.6 ± 9.9 years). ABG improved within the first hours in 92/112 (82%) patients, who were all successfully discharged. Eighteen percent (20/112) of the patients died during the hospital stay, no significant difference emerged in mortality rate (MR) within the groups (23%, 16% and 8%, for groups A, B and C, respectively) and between patients with or without pneumonia: 8/29 (27%) versus 12/83 (14%). On multivariable analysis, only age and Glasgow Coma Scale had an impact on the clinical outcome. CONCLUSION: In a non-'highly protected' environment such as an experienced medical ward of a rural hospital, NIV is effective not only in patients with mild, but also with severe forms of RA. MR did not vary according to the level of initial pH.

A histological procedure to study fungal infection in the wax moth Galleria mellonella.

The invertebrate model Galleria mellonella is a widely used factitious host to study the microbial pathogenesis in vivo. However, a specific procedure for the recovery and the processing of the infected tissues, important for a better understanding of the host-pathogen interactions, has not been reported to our knowledge. In the present study we describe a new procedure of fixation and processing of larval tissue that allows studying the larval topographic anatomy and assessing the morphological changes due to the fungal infection. Lepidopteran larvae were infected with Candida albicans strains displaying various biofilm-forming abilities. The whole larvae were then examined for tissue changes by histological techniques. We show that comparing cutting planes, serial transversal sections of paraffin-embedded larva result in better accuracy and information recovering. Using this technique, it was possible to preserve the integrity of G. mellonella internal structures allowing the detailed analysis of morphological differences in different experimental groups (i.e., healthy vs infected larvae). We were also able to study strain-related differences in the pathogenesis of C. albicans by observing the immune response elicited and the invasiveness of two isolates within the larval tissues. In general, by processing the whole larva and optimizing routinely histochemical stainings, it is possible to visualize and analyse infected tissues. Various degrees of pathogenicity (strain- or inoculum-related), and the infection time course can be described in details. Moreover, the host immune response events can be followed throughout the infectious process leading to a comprehensive picture of the studied phenomenon.

Candidemia in intensive care unit: a nationwide prospective observational survey (GISIA-3 study) and review of the European literature from 2000 through 2013.

BACKGROUND: Candida bloodstream infections (BSI) represent an important problem in Intensive Care Units (ICUs). The epidemiology of candidemia is changing with an increase in the proportion of Candida (C.) non-albicans. OBJECTIVES: An Italian 2-year observational survey on ICU was conducted to evaluate the species distribution and possible differences between BSI caused by C. albicans and C. non-albicans. For comparative purposes, we performed a European literature-based review to evaluate distribution and frequency of Candida spp. causing ICU candidemia, during the period 2000-2013. MATERIALS AND METHODS: This laboratory-based survey involved 15 microbiology centers (GISIA-3 study). All candidemia episodes in adult patients were considered. Data were prospectively collected from 2007 to 2008. PubMed was searched for peer-reviewed articles. RESULTS: In total, 462 candidemia episodes were collected. C. albicans accounted for 49.4% of the isolates, followed by C. parapsilosis (26.2%) and C. glabrata (10.4%). Mortality was higher in patients with C. non-albicans than C. albicans (47.3% vs. 32.4 %, p > 0.05). Among risk factors, parenteral nutrition was more common (p = 0.02) in non-albicans candidemia, while surgery was more frequent (p = 0.02) in C. albicans candidemia. Twenty-four relevant articles were identified. C. albicans was the predominant species in almost all studies (range 37.9% -76.3%). C. glabrata was commonly isolated in the German-speaking countries, France, UK and North Europe; C. parapsilosis in Turkey, Greece and Spain. CONCLUSIONS: Although C. non-albicans BSI is increasing, our study shows that C. albicans is still the predominant species in ICU candidemia. There are differences in the epidemiology of Candida BSI among European countries, with a prevalence of C. glabrata and C. parapsilosis in Northern and Southern countries, respectively.

Reliability and validity of the Italian version of the Consensus Auditory-Perceptual Evaluation of Voice (CAPE-V).

OBJECTIVES: To evaluate the reliability and validity of the Italian version of the Consensus Auditory-Perceptual Evaluation of Voice (CAPE-V). PATIENTS AND METHODS: Eighty dysphonic patients and 120 asymptomatic subjects were enrolled. The voice signal of each participant was recorded, listened to and rated by 3 licensed speech-language pathologists using the GRBAS scale and the Italian version of the CAPE-V. The intra- and interrater reliability of the CAPE-V was assessed as well as the degree of association between the CAPE-V and GRBAS judgments. The CAPE-V values were also compared between the patients with dysphonia and the asymptomatic subjects. RESULTS: The intra- and interrater reliability appeared to be good for all the parameters except for the strain parameter. The attributes 'consistent' and 'intermittent' demonstrated optimal intra- and interrater reliability. The difference between pathological and control groups was significant for six perceptual parameters. The highest average correlation between GRBAS and CAPE-V judgments was found between overall severity and grade while the lowest was found between the two strain scales. CAPE-V profiles differed significantly between different pathological groups. CONCLUSION: The Italian version of CAPE-V appears to be a reliable and valid tool for the perceptual analysis of the voice signal.