Lack of associations of microRNAs with severe NAFLD in people living with HIV: discovery case-control study.

Background & objective: Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in people living with HIV (PLWH) and the expression of some microRNAs could be useful as biomarkers for the diagnosis of NAFLD. The aim of this study was to identify patterns of differential expression of microRNAs in PLWH and assess their diagnostic value for NALFD. Methods: A discovery case-control study with PLWH was carried out. The expression of miRNAs was determined using HTG EdgeSeq technology. Cases were defined as patients with severe NAFLD and controls as patients without NAFLD, characterized using the controlled attenuation parameter (CAP). Cases and controls were matched 1:1 for age, sex, BMI, CD4+ lymphocyte count, active HCV infection, and ART regimen. Results: Serum 2,083 simultaneous microRNA transcripts were analyzed using HTG technology and compared between cases and controls. Forty-five patients, 23 cases, and 22 controls were included in the study. In the analysis of the expression pattern of the 2,083 microRNAs, no differential expression patterns were found between both groups of patients included in the study. Conclusion: Analysis of the microRNA transcriptome profile of nonobese PLWH with severe NAFLD did not appear to differ from that of patients without NAFLD. Thus, microRNA might not serve as a proper biomarker for predicting severe NALFD in this population.

Specific IgY anti-group 1 dust mite allergens induced by unglycosylated synthetic oligopeptides

Introduction: The use of specific antibodies capable of detecting allergens of the group 1 of house dust mites represents a potential strategy to reduce exposure and clinical symptomatology associated with asthma and allergic rhinitis. Objective: To produce and purify chicken antibodies specific for the dust mites Dermatophagoides sp. and B. tropicalis using the IgY technology. Materials and methods: We designed and synthesized oligopeptides showing immunogenic epitopes of Der p1, Der f1, and Blo t1. These were used to produce IgY antibodies in Hy Line Brown chickens. IgY were extracted from egg yolk using thiophilic chromatography. The immunogenicity and specificity were assayed by indirect ELISA and Dot Blot. Results: We obtained high reactivity of IgY antibodies against epitopes of allergens present in whole body mites extracts of D. farinae, D. pteronyssinus, and B. tropicalis. The highest IgY levels were registered between days 32 and 40 after immunization. The antibodies showed high immunoreactivity and specificity towards D. farinae proteins with detection limits above 0.03 µg of mite proteins under the experimental conditions used. Purified IgY did not show significant reactivity when binding to Periplaneta americana extract. Conclusion: The IgY technology allowed the production of specific antibodies against house dust mites group 1 allergens using non-glycosylated synthetic peptides. To our knowledge, this is the first time that this immunochemicals are used in the detection of mites of medical relevance.

IgY antialérgenos específicos del grupo 1 de ácaros del polvo doméstico inducidos por oligopéptidos sintéticos no glicosilados / Specific IgY anti-group 1 dust mite allergens induced by unglycosylated synthetic oligopeptides

Resumen Introducción. La obtención de anticuerpos específicos capaces de detectar alérgenos del grupo 1 de ácaros del polvo doméstico representa una estrategia potencial de salud pública para reducir la exposición y la sintomatología clínica asociada con el asma y la rinitis alérgica. Objetivo. Producir y purificar anticuerpos aviares antialérgenos específicos del grupo 1 de los ácaros Dermatophagoides sp.y Blomia tropicalis utilizando la tecnología IgY. Materiales y métodos. Se diseñaron y sintetizaron oligopéptidos que evidenciaran epítopes inmunogénicos de los alérgenos Der p1, Der f1 y Blo t1 empleados posteriormente para producir anticuerpos IgY policlonales en gallinas Hy Line Brown. Las IgY presentes en las yemas de los huevos se purificaron mediante cromatografía tiofílica. Su inmunorreactividad y especificidad se determinaron mediante un inmunoensayo ELISA indirecto y Dot Blot. Resultados. Se obtuvo una reactividad elevada de las IgY contra epítopes de alérgenos presentes en extractos de cuerpo entero de D. farinae, D. pteronyssinus y B. tropicalis. Los niveles más altos de IgY se produjeron entre los días 32 y 40 de inmunización. Los anticuerpos mostraron mayor inmunorreactividad y especificidad en el reconocimiento de proteínas de D. farinae, con un límite de detección mayor de 0,03 µg de proteína total delcaroajo las condiciones experimentales analizadas. Las IgY purificadas no mostraron reactividad significativa frente al extracto de Periplaneta americana. Conclusión. La tecnología IgY permitió la producción de anticuerpos específicos contra alérgenos del grupo 1 de los ácaros del polvo al utilizar oligopéptidos sintéticos no glicosilados. Hasta donde se sabe, esta es la primera vez que se usan estos reactivos inmunológicos para la detección de ácaros de importancia médica.

Abstract Introduction: The use of specific antibodies capable of detecting allergens of the group 1 of house dust mites represents a potential strategy to reduce exposure and clinical symptomatology associated with asthma and allergic rhinitis. Objective: To produce and purify chicken antibodies specific for the dust mites Dermatophagoides sp. and B. tropicalis using the IgY technology. Materials and methods: We designed and synthesized oligopeptides showing immunogenic epitopes of Der p1, Der f1, and Blo t1. These were used to produce IgY antibodies in Hy Line Brown chickens. IgY were extracted from egg yolk using thiophilic chromatography. The immunogenicity and specificity were assayed by indirect ELISA and Dot Blot. Results: We obtained high reactivity of IgY antibodies against epitopes of allergens present in whole body mites extracts of D. farinae, D. pteronyssinus, and B. tropicalis. The highest IgY levels were registered between days 32 and 40 after immunization. The antibodies showed high immunoreactivity and specificity towards D. farinae proteins with detection limits above 0.03 µg of mite proteins under the experimental conditions used. Purified IgY did not show significant reactivity when binding to Periplaneta americana extract. Conclusion: The IgY technology allowed the production of specific antibodies against house dust mites group 1 allergens using non-glycosylated synthetic peptides. To our knowledge, this is the first time that this immunochemicals are used in the detection of mites of medical relevance.

The impact of university provided nurse electronic medical record training on health care organizations: an exploratory simulation approach.

Training providers appropriately, particularly early in their caregiving careers, is an important aspect of electronic medical record (EMR) implementation. Considerable time and resources are needed to bring the newly hired providers 'up to speed' with the actual use practices of the organization. Similarly, universities lose valuable clinical training hours when students are required to spend those hours learning organization-specific EMR systems in order to participate in care during clinical rotations. Although there are multiple real-world barriers to university/health care organization training partnerships, the investment these entities share in training care providers, specifically nurses, to use and understand EMR technology encourages a question: What would be the cumulative effect of integrating a mutually agreed upon EMR system training program in to nursing classroom training on downstream hospital costs in terms of hours of direct caregiving lost, and benefits in terms of number of overall EMR trained nurses hired? In order to inform the development of a large scale study, we employed a dynamic systems modeling approach to simulate the theoretical relationships between key model variables and determine the possible effect of integrating EMR training into nursing classrooms on hospital outcomes. The analysis indicated that integrating EMR training into the nursing classroom curriculum results in more available time for nurse bedside care. Also, the simulation suggests that efficiency of clinical training can be potentially improved by centralizing EMR training within the nursing curriculum.

Effect of culture conditions and signal peptide on production of human recombinant N-acetylgalactosamine-6-sulfate sulfatase in Escherichia coli BL21.

The production and characterization of an active recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21(DE3) has been previously reported. In this study, the effect of the signal peptide (SP), inducer concentration, process scale, and operational mode (batch and semi-continuous) on GALNS production were evaluated. When native SP was presented, higher enzyme activity levels were observed in both soluble and inclusion bodies fractions, and its removal had a significant impact on enzyme activation. At shake scale, the optimal IPTG concentrations were 0.5 and 1.5 mM for the strains with and without SP, respectively, whereas at bench scale, the highest enzyme activities were observed with 1.5 mM IPTG for both strains. Noteworthy, enzyme activity in the culture media was only detected when SP was presented and the culture was carried out under semi-continuous mode. We showed for the first time that the mechanism that in prokaryotes recognizes the SP to mediate sulfatase activation can also recognize a eukaryotic SP, favoring the activation of the enzyme, and could also favor the secretion of the recombinant protein. These results offer significant information for scaling-up the production of human sulfatases in E. coli.

Association between the IL28B genotype and hepatitis C viral kinetics in the early days of treatment with pegylated interferon plus ribavirin in HIV/HCV co-infected patients with genotype 1 or 4.

OBJECTIVES: To evaluate the effect of the interleukin 28B (IL-28B) genotype on hepatitis C virus (HCV) viral kinetics in the first 4 weeks from start of treatment with pegylated interferon plus ribavirin (PEG-IFN/RBV) in HIV/HCV co-infected patients. METHODS: HIV/HCV co-infected patients naive to PEG-IFN/RBV treatment were enrolled in a prospective study. HCV RNA plasma viral loads were measured at baseline and at weeks 1, 2 and 4 after commencement of treatment. Patients were grouped by HCV genotype (genotype 1/4 versus 3) and by IL-28B genotype (CC versus non-CC). Differences in viral load reduction were evaluated by IL-28B genotype between baseline, week 1, week 2 and week 4. RESULTS: One hundred and nineteen HIV/HCV patients were included in the study. HCV patients with genotype 1/4 and bearing the IL-28 CC genotype showed the greatest reductions in HCV RNA plasma levels between baseline and weeks 1 (B-1), 2 (B-2) and 4 (B-4) than did those with non-CC genotypes (B-1: 1.06 ± 0.89 versus 0.48 ± 0.48 log IU/mL, P = 0.009; B-2: 1.36 ± 0.72 versus 0.77 ± 0.66 log IU/mL, P = 0.01; and B-4: 1.91 ± 0.64 versus 1.38 ± 0.96 log IU/mL, P = 0.03). However, differences between weeks 1 and 2 (W1-2) and between weeks 2 and 4 (W2-4) were not associated with the IL-28B genotype (W1-2: CC 0.48 ± 0.42 versus non-CC 0.38 ± 0.38 log IU/mL, P = 0.62; W2-4: CC 0.32 ± 0.23 versus non-CC 0.39 ± 0.31 log IU/mL, P = 0.67). No differences in decline of HCV RNA viral load were found in HCV genotype 3 patients. CONCLUSIONS: The IL-28B genotype impacts on viral kinetics during the first week of treatment with PEG-IFN/RBV in patients with HCV genotype 1/4.

Development of a sandwich enzyme linked immunosorbent assay (ELISA) for the quantification of iduronate-2-sulfate sulfatase.

Iduronate-2-sulfate sulfatase (IDS; EC 3.1.6.13) is an enzyme that belongs to human sulfatases. IDS deficiency causes the Hunter syndrome or mucopolysaccharidosis type II (MPS II; OMIM 309900). We have been developing an expression system for human recombinant IDS (hrIDS) in Pichia pastoris, therefore a method was required for its detection during production and purification processes, which could be used also to measure the enzyme in human fluids. In this study, an immunoquantification assay for human and recombinant IDS was developed with the combination of two antibodies. Rabbit IgG and chicken IgY were used as IDS capture and detection antibodies, respectively. Chicken IgY antibodies were developed against specific amino acid sequences present in IDS but absent in other human sulfatases. hrIDS produced in P. pastoris, commercial hrIDS, and normal human plasma samples were used as antigens and immunoquantification results were compared to enzyme activity. The technique was linear over the range 8 to 500 ng mL(-1) using commercial hrIDS. The concentration range detected for IDS in normal human plasma was 14.43 to 287.88 ng mL(-1). The hrIDS was detected in P. pastoris cultures even when the enzyme was inactive, which is convenient for monitoring the production of recombinant proteins. These results show that chicken site-specific antibodies provide a good alternative, as a substitute of monoclonal antibodies, for the detection of human proteins. This is the first report on the development of an ELISA system to detect and quantify IDS with IgY antibodies.

Evaluation of toxicity and degradation of a chlorophenol mixture by the laccase produced by Trametes pubescens.

In this study, the biodegradation of a mixture of 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP) and pentachlorophenol (PCP) using the laccase produced by the white-rot fungus Trametes pubescens CBS 696.94 was evaluated. Two laccase isoenzymes with molecular weights of about 60 and 120 kDa were identified in the enzymatic crude extract. The highest laccase activity with syringaldazine was observed with pH 6.0 and 60°C, while with 2,2-azino-bis(3-ethylbenzothiazoline-6) sulphonic acid the highest activity was observed between 50 and 60°C and 3.0-4.0 pH. A biodegradation of 100%, 99%, 82.1% and 41.1% for 2-CP, 2,4-DCP, 2,4,6-TCP and PCP, respectively, was observed after 4h of reaction. The reduction in chlorophenols concentration allowed 90% reduction in mixture toxicity. In summary, these results show the feasibility of a laccase enzymatic crude extract from T. pubescens for the reduction of concentration and toxicity of chlorophenols.

Enzyme replacement therapy for Morquio A: an active recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in Escherichia coli BL21.

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Currently no effective therapies exist for MPS IVA. In this work, production of a recombinant GALNS enzyme (rGALNS) in Escherichia coli BL21 strain was studied. At shake scale, the effect of glucose concentration on microorganism growth, and microorganism culture and induction times on rGALNS production were evaluated. At bench scale, the effect of aeration and agitation on microorganism growth, and culture and induction times were evaluated. The highest enzyme activity levels at shake scale were observed in 12 h culture after 2-4 h induction. At bench scale the highest enzyme activity levels were observed after 2 h induction. rGALNS amounts in inclusion bodies fraction were up to 17-fold higher than those observed in the soluble fraction. However, the highest levels of active enzyme were found in the soluble fraction. Western blot analysis showed the presence of a 50-kDa band, in both soluble and inclusion bodies fractions. These results show for the first time the feasibility and potential of production of active rGALNS in a prokaryotic system for development of enzyme replacement therapy for MPS IVA disease.

Estudio y tratamiento de las neumonías de adquisición comunitaria en adultos / Clinical management of community- acquired pneumonia in adults

No disponible

No disponible

Effect of elongation factor 1alpha promoter and SUMF1 over in vitro expression of N-acetylgalactosamine-6-sulfate sulfatase.

Morquio A is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to the lysosomal accumulation of keratan-sulfate and chondroitin-6-sulfate. We evaluated in HEK293 cells the effect of the cytomegalovirus immediate early enhancer/promoter (CMV) or the elongation factor 1alpha (EF1alpha) promoters, and the coexpression with the sulfatase modifying factor 1 (SUMF1) on GALNS activity. Four days postransfection GALNS activity in transfected cells with CMV-pIRES-GALNS reached a plateau, whereas in cells transfected with EF1alpha-pIRES-GALNS continued to increase until day 8. Co-transfection with pCXN-SUMF1 showed an increment up to 2.6-fold in GALNS activity. Finally, computational analysis of transcription factor binding-sites and CpG islands showed that EF1alpha promoter has long CpG islands and high-density binding-sites for Sp1 compared to CMV. These results show the advantage of the SUMF1 coexpression on GALNS activity and indicate a considerable effect on the expression stability using EF1alpha promoter compared to CMV.

[Variability in coronary risk assessment in HIV-infected patients]. / Variabilidad en la valoración del riesgo coronario en pacientes infectados por el virus de la inmunodeficiencia humana.

BACKGROUND AND OBJECTIVE: Antiretroviral treatment of human immunodeficiency virus (HIV)-infected patients seems to increase the coronary risk (CR) in these patients. Adequate assessment of CR has significant implications for the management of these patients. Our objective was to compare 2 systems for assessing 10-year CR in HIV-infected patients. PATIENTS AND METHOD: CR was calculated in a prospective cohort of 205 HIV-infected patients using Framingham tables and REGICOR adapted tables. Prevalence of cardiovascular risk factors in these patients was evaluated. RESULTS: Mean age (standard deviation) was 41.4 (8.2) years. Most patients were taking antiretrovirals and had a good immunological status. Current smoking was reported by 77.1% of patients, while a history of dyslipidemia, hypertension, or diabetes was found in 29.3%, 7.3%, and 4.9% of patients, respectively. Lipodystrophy was seen in 41% of patients, abdominal obesity in 21.5%, and a sedentary lifestyle in 50.7% Mean values obtained were 6.55 (6.36) in the Framingham scale and 2.85 (2.31) in the REGICOR scale. A 10-year CR greater than 10% was found in 26 patients (12.9%) with the Framingham tables and in 4 patients (2.0%) with the REGICOR tables. The difference between both methods was significant (p < 0.001). CONCLUSIONS: Application of the Framingham tables to our cohort may overestimate the CR. Studies aimed at identifying the most adequate method for measuring CR in HIV-infected patients are required. Until such data are available, estimation of CR in these patients should be taken with caution.

Variabilidad en la valoración del riesgo coronario en pacientes infectados por el virus de la inmunodeficiencia humana / Variability in coronary risk assessment in HIV-infected patients

Fundamento y objetivo: El tratamiento antirretroviral de los pacientes infectados por el virus de la inmunodeficiencia humana (VIH) parece aumentar su riesgo coronario (RC). La correcta valoración de éste tiene importantes implicaciones en el tratamiento de estos pacientes. Nuestro objetivo ha sido comparar 2 sistemas de evaluación del RC a 10 años en pacientes infectados por el VIH. Pacientes y método: Se calculó el RC en una cohorte prospectiva de 205 pacientes infectados por el VIH utilizando las tablas de Framingham y las tablas adaptadas REGICOR. Se evaluó la prevalencia de factores de riesgo cardiovascular de estos pacientes. Resultados: La edad media (desviación estándar) fue de 41,4 (8,2) años. La mayoría de los pacientes tomaba antirretrovirales y tenía buena situación inmunológica. Presentaba tabaquismo activo el 77,1%, antecedentes de dislipemia el 29,3%, de hipertensión el 7,3%, de diabetes el 4,9%, lipodistrofia el 41%, obesidad abdominal el 21,5% y sedentarismo el 50,7%. La valoración media en la escala Framingham fue de 6,55 (6,36) y en la escala REGICOR de 2,85 (2,31). El RC a 10 años fue mayor del 10% en 26 pacientes (12,9%) con las tablas de Framingham y en 4 (2,0%) con las tablas REGICOR. La diferencia entre ambos métodos resultó significativa (p < 0,001). Conclusiones: Aplicar las tablas Framingham en nuestra cohorte podría suponer una sobrestimación del RC. Son necesarios estudios que tengan como objetivo identificar el método más adecuado para medir el RC en pacientes infectados por el VIH. Mientras no dispongamos de estos datos, debemos tomar con precaución la estimación del RC en estos pacientes

Background and objective: Antiretroviral treatment of human immunodeficiency virus (HIV)-infected patients seems to increase the coronary risk (CR) in these patients. Adequate assessment of CR has significant implications for the management of these patients. Our objective was to compare 2 systems for assessing 10-year CR in HIV-infected patients. Patients and method: CR was calculated in a prospective cohort of 205 HIV-infected patients using Framingham tables and REGICOR adapted tables. Prevalence of cardiovascular risk factors in these patients was evaluated. Results: Mean age (standard deviation) was 41.4 (8.2) years. Most patients were taking antiretrovirals and had a good immunological status. Current smoking was reported by 77.1% of patients, while a history of dyslipidemia, hypertension, or diabetes was found in 29.3%, 7.3%, and 4.9% of patients, respectively. Lipodystrophy was seen in 41% of patients, abdominal obesity in 21.5%, and a sedentary lifestyle in 50.7% Mean values obtained were 6.55 (6.36) in the Framingham scale and 2.85 (2.31) in the REGICOR scale. A 10-year CR greater than 10% was found in 26 patients (12.9%) with the Framingham tables and in 4 patients (2.0%) with the REGICOR tables. The difference between both methods was significant (p < 0.001). Conclusions: Application of the Framingham tables to our cohort may overestimate the CR. Studies aimed at identifying the most adequate method for measuring CR in HIV-infected patients are required. Until such data are available, estimation of CR in these patients should be taken with caution