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This work proposes a displacement-based finite element model for large strain analysis of isotropic compressible and nearly-incompressible hyperelastic materials. Constitutive law is written in terms of invariants of the right Cauchy-Green tensor; coupled and decoupled formulations of strain energy functions are presented, whereas a penalty function is used to impose an incompressibility constraint. Based on a total Lagrangian formulation, the nonlinear governing equations are thus obtained by employing the principle of virtual displacements. Analytic expression of both internal forces vector and tangent matrix of linear and high-order hexahedral finite elements are derived by adopting a three-dimensional formalism based on the Carrera Unified Formulation. Popular benchmark problems in hyperelasticity are analyzed to establish the capabilities of the present implementation of fully-nonlinear solid elements in the case of compressible and nearly-incompressible beams, cylindrical shells, and curved structures.
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This paper presents the numerical damage analysis of concrete structures using higher-order beam theories based on Carrera Unified Formulation (CUF). The concrete constitutive relation is modeled using continuum damage mechanics based on a modified Mazars concrete damage model, in which both the tensile and compressive softening behaviors are regularized with classical fracture energy methodology. An expression is proposed to estimate the characteristic length in higher-order beam theories, to prevent mesh dependency. Both softening constitutive laws and fracture energy calculations are obtained according to Model Code 2010. To assess the efficiency of the proposed model, three classical benchmark quasi-static experiments are taken for validation. From the comparison between numerical and experimental results, the proposed CUF model using continuum damage mechanics can present 3D accuracy with low computational costs and reduce the mesh dependency.
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Based on the Carrera unified formulation (CUF) and first-invariant hyperelasticity, this work proposes a displacement-based high order one-dimensional (1 D) finite element model for the geometrical and physical nonlinear analysis of isotropic, slightly compressible soft material structures. Different strain energy functions are considered and they are decomposed in a volumetric and an isochoric part, the former acting as penalization of incompressibility. Given the material Jacobian tensor, the nonlinear governing equations are derived in a unified, total Lagrangian form by expanding the three-dimensional displacement field with arbitrary cross-section polynomials and using the virtual work principle. The exact analytical expressions of the elemental internal force vector and tangent matrix of the unified beam model are also provided. Several problems are addressed, including uniaxial tension, bending of a slender structure, compression of a three-dimensional block, and a thick pinched cylinder. The proposed model is compared with analytical solutions and literature results whenever possible. It is demonstrated that, although 1 D, the present CUF-based finite element can address simple to complex nonlinear hyperelastic phenomena, depending on the theory approximation order.
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Static and modal responses of representative biomechanical structures are investigated in this paper by employing higher-order theories of structures and finite element approximations. Refined models are implemented in the domain of the Carrera unified formulation (CUF), according to which low- to high-order kinematics can be postulated as arbitrary and, eventually, hierarchical expansions of the generalized displacement unknowns. By using CUF along with the principle of virtual work, the governing equations are expressed in terms of fundamental nuclei of finite element arrays. The fundamental nuclei are invariant of the theory approximation order and can be opportunely employed to implement variable kinematics theories of bio-structures. In this work, static and free-vibration analyses of an atherosclerotic plaque of a human artery and a dental prosthesis are discussed. The results from the proposed methodologies highlight a number of advantages of CUF models with respect to already established theories and commercial software tools. Namely, (i) CUF models can represent correctly the higher-order phenomena related to complex stress/strain field distributions and coupled mode shapes; (ii) bio-structures can be modeled in a component-wise sense by only employing the physical boundaries of the problem domain and without making any geometrical simplification. This latter aspect, in particular, can be currently accomplished only by using three-dimensional analysis, which may be computationally unbearable as complex bio-systems are considered.
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Arterias/patología , Aterosclerosis/patología , Implantes Dentales , Análisis de Elementos Finitos , Modelos Anatómicos , Vibración , Huesos/patología , Encía/patología , Humanos , Masticación , Análisis Numérico Asistido por Computador , Estrés MecánicoRESUMEN
INTRODUCTION: Pulmonary artery sarcoma (PAS) is a rare tumor, whose therapeutic approach is mainly based on surgery, either pneumonectomy or pulmonary endarterectomy (PEA). The prognosis reported in published series is very poor, with survival of 1.5 months without any kind of treatment. PATIENTS AND METHODS: From January 2010 to January 2016, 1027 patients were referred to our hospital for symptoms of acute or chronic pulmonary thromboembolic disease. Twelve patients having a confirmed diagnosis of PAS underwent PEA. Median age was 64.5 years. Most patients had a long history of symptoms, having a median time of 7.5 months from onset of symptoms to surgery. RESULTS: Following PEA and cardiopulmonary rehabilitation, 10 patients received conventional chemotherapy with doxorubicin and ifosfamide, starting at a median of 42 days from surgery. Four patients also received radiotherapy. Four patients have died due to disease progression, while 7 are still alive, with 5 being disease-free at 4-55+ months from diagnosis. CONCLUSIONS: In patients with PAS, a multimodal approach including PEA, CT, and RT is feasible but it should be evaluated individually, according to the tumor extension and the patient's clinical condition. Apart from improving quality of life mainly by reducing or delaying symptoms due to PH, it may improve life expectancy.
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BACKGROUND: Iron is essential for hemoglobin synthesis during terminal erythropoiesis. To supply adequate iron the carrier transferrin is required together with transferrin receptor endosomal cycle and normal mitochondrial iron utilization. Iron and iron protein deficiencies result in different types of anemia. Iron-deficiency anemia is the commonest anemia worldwide due to increased requirements, malnutrition, chronic blood losses and malabsorption. Mutations of transferrin, transferrin receptor cycle proteins, enzymes of the first step of heme synthesis and iron sulfur cluster biogenesis lead to rare anemias, usually accompanied by iron overload. Hepcidin plays an indirect role in erythropoiesis by controlling plasma iron. Inappropriately high hepcidin levels characterize the rare genetic iron-refractory iron-deficiency anemia (IRIDA) and the common anemia of chronic disease. Iron modulates both effective and ineffective erythropoiesis: iron restriction reduces heme and alpha-globin synthesis that may be of benefit in thalassemia. MATERIAL AND METHODS: This review relies on the analysis of the most recent literature and personal data. RESULTS: Erythropoiesis controls iron homeostasis, by releasing erythroferrone that inhibits hepcidin transcription to increase iron acquisition in iron deficiency, hypoxia and EPO treatment. Erythroferrone, produced by EPO-stimulated erythropoiesis, inhibits hepcidin only when the activity of BMP/SMAD pathway is low, suggesting that EPO somehow modulates the latter signaling. Erythroblasts sense circulating iron through the second transferrin receptor (TFR2) that, in animal models, modulates the sensitivity of the erythroid cells to EPO. DISCUSSION: The advanced knowledge of the regulation of systemic iron homeostasis and erythropoiesis-mediated hepcidin regulation is leading to the development of targeted therapies for anemias and iron disorders.
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Eritropoyesis/fisiología , Hierro/metabolismo , Anemia Ferropénica/etiología , Animales , Hepcidinas/antagonistas & inhibidores , Hepcidinas/metabolismo , Homeostasis/fisiología , Humanos , Hierro/fisiología , Deficiencias de HierroRESUMEN
BACKGROUND: Tyrosine kinase inhibitors (TKIs) are associated with prolongation of the QTc interval on the electrocardiogram (ECG). The QTc-interval prolongation increases the risk of life-threatening arrhythmias. However, studies evaluating the effects of TKIs on QTc intervals are limited and only consist of small patient numbers. METHODS: In this multicentre trial in four centres in the Netherlands and Italy we screened all patients who were treated with any TKI. To evaluate the effects of TKIs on the QTc interval, we investigated ECGs before and during treatment with erlotinib, gefitinib, imatinib, lapatinib, pazopanib, sorafenib, sunitinib, or vemurafenib. RESULTS: A total of 363 patients were eligible for the analyses. At baseline measurement, QTc intervals were significantly longer in females than in males (QTcfemales=404 ms vs QTcmales=399 ms, P=0.027). A statistically significant increase was observed for the individual TKIs sunitinib, vemurafenib, sorafenib, imatinib, and erlotinib, after the start of treatment (median ΔQTc ranging from +7 to +24 ms, P<0.004). The CTCAE grade for QTc intervals significantly increased after start of treatment (P=0.0003). Especially patients who are treated with vemurafenib are at increased risk of developing a QTc of ⩾470 ms, a threshold associated with an increased risk for arrhythmias. CONCLUSIONS: These observations show that most TKIs significantly increase the QTc interval. Particularly in vemurafenib-treated patients, the incidence of patients at risk for arrhythmias is increased. Therefore, especially in case of combined risk factors, ECG monitoring in patients treated with TKIs is strongly recommended.
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Síndrome de Jervell-Lange Nielsen/inducido químicamente , Inhibidores de Proteínas Quinasas/efectos adversos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Anciano , Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/epidemiología , Electrocardiografía/métodos , Femenino , Humanos , Incidencia , Italia/epidemiología , Síndrome de Jervell-Lange Nielsen/enzimología , Síndrome de Jervell-Lange Nielsen/epidemiología , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Estudios Retrospectivos , RiesgoRESUMEN
The wild boar is an important source of trichinellosis for people in European countries as a large number of hunted animals escape veterinary control. In November 2012, uncooked sausages made with meat from wild boar were consumed by 38 persons in a village of the Lucca province (Tuscany region, Italy). Of them, 34 were serologically positive, 32 developed clinical signs and symptoms of trichinellosis, and two were asymptomatic. Trichinella britovi larvae were detected in vacuum-packed sausages made with the same batch of sausages consumed raw which had been prepared with meat from wild boar hunted in the Lucca province. As no case of trichinellosis had been reported in this region during the last 20 years, the regional public health authority considered the risk for this zoonosis to be negligible and put in place a surveillance programme on Trichinella spp. in indicator animals (mainly foxes and including wild boar for private consumption), by testing only a percentage of heads. The experience from this outbreak shows that the definition of a region with a negligible risk for Trichinella infection is not applicable to wild boar and stresses the need to test all Trichinella-susceptible wild animals intended for human consumption and to implement risk communication to consumers and hunters.
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Brotes de Enfermedades , Enfermedades Transmitidas por los Alimentos/microbiología , Carne/microbiología , Sus scrofa/microbiología , Enfermedades de los Porcinos/transmisión , Triquinelosis/transmisión , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Niño , Femenino , Microbiología de Alimentos , Enfermedades Transmitidas por los Alimentos/epidemiología , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Porcinos , Enfermedades de los Porcinos/epidemiología , Trichinella/aislamiento & purificación , Triquinelosis/epidemiología , Adulto JovenRESUMEN
Spontaneous plants of Ipomoea acuminata ("morning glory") exhibiting white rust pustules were found in a field crop area of Planaltina, DF, in the fall season of 2010 and the disease causal agent was identified as Albugo ipomoea-panduratae (Oomycota). No reports of the association between A. ipomoea-panduratae and I. acuminata were known in Brazil previously to 2010. A reference specimen was deposited at the University of Brasilia Mycological Reference Collection.
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Enfermedades de las Plantas/etiología , Ferrobacterias , Ipomoea/crecimiento & desarrollo , Ipomoea/microbiología , Microbiología Ambiental , Métodos , MétodosRESUMEN
Spontaneous plants of Ipomoea acuminata ("morning glory") exhibiting white rust pustules were found in a field crop area of Planaltina, DF, in the fall season of 2010 and the disease causal agent was identified as Albugo ipomoea-panduratae (Oomycota). No reports of the association between A. ipomoea-panduratae and I. acuminata were known in Brazil previously to 2010. A reference specimen was deposited at the University of Brasilia Mycological Reference Collection.
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BACKGROUND AND AIMS: Cross-sectional studies have shown that chronic sub-clinical inflammation is associated with left ventricular hypertrophy (LVH), but results are conflicting. We investigated the association between baseline LVH and high-sensitivity C-reactive protein (CRP) values, both cross-sectionally and after a six-year-follow-up, in a population-based cohort (n = 1564) and a subgroup from this cohort (n = 515), without obesity, diabetes, metabolic syndrome or any drugs. METHODS AND RESULTS: ECG tracings at baseline were interpreted according to the Cornell voltage-duration product criteria: 166/1564 subjects (10.6%) showed LVH. Patients with baseline LVH showed increased BMI, waist circumference, blood pressure, and a worse metabolic pattern. Their CRP values both at baseline and at follow-up were almost two-fold higher than in patients without LVH. Similar results were found in the healthier sub-sample. In a multiple regression model, CRP at follow-up was directly associated with baseline LVH (expressed as Cornell voltage-duration product) in the whole cohort (ß = 0.0003; 95%CI 0.0002-0.0006; p < 0.001) and in the sub-sample (ß = 0.0003; 0.0002-0.0004; p < 0.001), after adjusting for age, sex, BMI, waist circumference, smoking, exercise levels, blood pressure and baseline CRP values. CONCLUSION: Baseline LVH, which is associated with systemic inflammation, predicts increased CRP values at follow-up, independently of cardiovascular and metabolic risk factors, both in a population-based cohort and a healthier sub-sample. The inflammatory consequences of LVH might be an intriguing subject for further researches.
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Proteína C-Reactiva/metabolismo , Hipertrofia Ventricular Izquierda/sangre , Mediadores de Inflamación/sangre , Inflamación/sangre , Biomarcadores/sangre , Distribución de Chi-Cuadrado , Estudios Transversales , Electrocardiografía , Femenino , Estudios de Seguimiento , Humanos , Hipertrofia Ventricular Izquierda/diagnóstico , Hipertrofia Ventricular Izquierda/epidemiología , Hipertrofia Ventricular Izquierda/inmunología , Inflamación/diagnóstico , Inflamación/epidemiología , Inflamación/inmunología , Italia/epidemiología , Modelos Logísticos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Oportunidad Relativa , Medición de Riesgo , Factores de Riesgo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
CONTEXT: Ghrelin is a peptide with multiple functions that circulates in acylated (AG) and unacylated (UAG) forms. However, the role of ghrelin in neonates (NN) remains to be clarified. OBJECTIVE: The aim of this study was to determine ghrelin concentrations of the two forms in NN to clarify their biological roles. As such, ghrelin levels at birth were compared with those in later life. SETTING AND DESIGN: Tertiary Care Center. In this cross-sectional study, we evaluated AG, UAG, AG/UAG ratio, and insulin levels in venous cord blood from NN and in fasted normal weight (NW) and obese (OB) children, both prepubertal and pubertal. SUBJECTS: We studied 82 NN, 82 NW, and 58 OB children. RESULTS: AG levels were lower in NN than in NW and OB children (P<0.0001), more specifically the prepubertal NW and OB children (P<0.0001). UAG levels were higher in NN than in NW and OB children (P<0.0001). Therefore, the AG/UAG ratio was lower in NN than in NW and OB children (P<0.0001). NN showed insulin levels similar to NW and lower than OB children (P<0.0001). At birth UAG was positively correlated with AG (Pearson: 0.425; P<0.0001) and negatively with insulin (-0.253; P<0.02). In NW and OB, UAG and AG were positively correlated to each other and negatively correlated with insulin and body mass index (-0.566; P<0.0001). CONCLUSIONS: NN compared with children, showed higher UAG and lower AG levels. The AG/UAG ratio showed a very different profile in NN, being lower than in NW and OB children, thus suggesting a different metabolic function for the two forms in NN. Further studies are needed to clarify the exact role of the different ghrelin forms in NN.
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Sangre Fetal/metabolismo , Ghrelina/sangre , Antropometría , Índice de Masa Corporal , Femenino , Humanos , Recién Nacido , Insulina/metabolismo , Masculino , Obesidad/sangreRESUMEN
We report two cases, with overlapping cyto-histological characteristics, of invasive neuroendocrine carcinoma of the breast with associated stromal features distinctive of carcinoma with osteoclastic giant cells. Fine-needle aspiration cytology showed monomorphic, medium-sized, mildly atypical neoplastic cells, with interspersed multinucleated giant cells and lympho-histiocytic components; hemosiderin deposits were also appreciable. Macroscopic features were typical of invasive carcinoma, but with unusual brown staining. Light microscopy revealed moderately differentiated invasive carcinoma mainly composed of solid sheets of round to polygonal medium-sized cells with a tendency to produce peripheral palisading (carcinoid-like morphology); osteoclast-like multinucleated giant cells, lymphocytes and histiocytes were dispersed only among tumour cells in the distinctive stroma of the carcinoma with osteoclastic giant cells; this stroma was characterised by reactive/fibroblastic features, hypervascularization, extravasated blood cells and hemosiderin deposits that gave rise to the typical staining seen macroscopically. Immunohistochemically, cancer cells were diffusely positive for the neuroendocrine marker synaptophisin with partial chromogranin and NSE staining; the ostoclastic giant cell and histiocyte component were strongly positive for CD68. Both cases also had lymph node metastases; the epithelial neoplastic cells, with regards to neuroendocrine markers, were overlapping to the primitive tumour, the histiocytic/giant cell component appeared present, although with a lower degree, while the distinctive stroma was absent. Breast neuroendocrine carcinomas, associated with multinucleated giant cells and stroma typical of the carcinoma with osteoclastic-like cells, to our knowledge, have not been reported.
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Neoplasias de la Mama/patología , Adulto , Biopsia con Aguja Fina , Carcinoma Neuroendocrino/patología , Femenino , Humanos , Persona de Mediana Edad , Invasividad Neoplásica , OsteoclastosRESUMEN
The ligand deorphanization of TRP channels has a tremendous potential for biomedical and nutritional research, and this review highlights the role that natural products have played in the identification of ligands for these targets and their establishment as viable candidates for drug discovery. Specific ligands have so far been discovered only for some thermoTRPs, like TRPV1, TRPV3, TRPV4, TRPM8 and TRPA1, and the lack of selective pharmacology has been a major drawback for unraveling the biological role of TRPs. While genetic approaches (transgenic animal models) have partially compensate for the lack of ligands, the universal expression of TRPs in living systems and the success achieved with TRPV1 suggest that a systematic investigation of the natural products pool might alleviate this shortage, fostering adoption by small molecules within this class of still largely orphan biological targets.
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Productos Biológicos/farmacología , Sistemas de Liberación de Medicamentos , Canales Catiónicos TRPC/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Diseño de Fármacos , Expresión Génica , Humanos , Ligandos , Canales Catiónicos TRPC/metabolismoRESUMEN
OBJECTIVE: Data on the association of resistin levels with markers of insulin resistance are highly contrasting in humans and very few studies about its role in inflammation are available. This study investigates associations between serum resistin levels and markers of insulin resistance, inflammation (C-reactive protein (CRP)) and of oxidative stress (nytrotirosine (NT)). SUBJECTS: A randomly collected sample of 300 men from a population-based cohort was analysed, separated into two groups according to body mass index (BMI) and waist values. RESULTS: Correlations between resistin and BMI, waist, triglyceride, uric acid, fasting glucose, insulin and Homeostasis Model Assessment (HOMA) values were significant in subjects with normal BMI, but not in overweight/obese subjects. In a multiple regression model, after multiple adjustments and exclusion of diabetic patients, only fasting glucose remained significantly associated with resistin levels. Otherwise, resistin is associated to CRP levels in all individuals, after multiple adjustments and exclusion of diabetic patients (in normal BMI beta=0.82; 95% CI 0.21, 1.42; in overweight/obese beta=0.43; 95% CI 0.10, 0.76). In the same model, resistin values are negatively related to NT levels in normal weight individuals (beta=-1.61; 95% CI -0.77-2.45). CONCLUSIONS: Serum resistin is weakly associated with metabolic abnormalities in subjects with normal BMI, while in overweight/obese patients this correlation is not significant, perhaps due to the higher fat content in these subjects. Serum resistin is directly correlated with CRP and inversely to NT. An intriguing hypothesis, which needs to be tested, is that resistin is secreted in response to a chronic low-grade inflammation, and has antioxidant properties.
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Proteína C-Reactiva/análisis , Resistencia a la Insulina , Obesidad/sangre , Resistina/sangre , Tirosina/análogos & derivados , Biomarcadores/sangre , Glucemia/análisis , Composición Corporal , Índice de Masa Corporal , Estudios de Casos y Controles , Homeostasis , Humanos , Insulina/sangre , Masculino , Persona de Mediana Edad , Análisis de Regresión , Triglicéridos/sangre , Tirosina/sangre , Ácido Úrico/sangreRESUMEN
Standardized procedures regarding the sampling of cervical conization are extensively described in the Literature. The cervical cone dimension depends on the surgical technical approach and on the extension of the dysplasic/neoplastic lesion. Current sampling procedures frequently turn out to be of problematic correct realization, with special regard to small size cervical cones and to fixed material with subsequent artefactual tissue coarctation. Here we describe a simple device, which makes the cone sampling procedure easier and more accurate, allowing better oriented and better recomposed samples. Perpendicular sections to the cervical lumen allow in fact an accurate evaluation of the lesion's depth and of the status of the resection surgical borders. After conization, preventive agreements with the surgeon provide that, before fixation, a 2-3 mm in diameter smooth plastic probe is inserted into the cervical channel, from the external cervical os to the conization apex (a fragment of the handle of an endocervical cytological brush can be used). The insertion of such a "specillum" must be performed with warning gently since superficial cervical epithelium, especially if dysplasic (overall in case of severe dysplasia), is particularly subjected to mechanical abrasions. The preventive insertion of the endocervical probe makes the cone sampling more manageable, in fact the cervical channel is slightly dilated and the subsequent fixation hardens the cone tissues. After the "specillum" extraction, the cervical lumen appears clearly visible; in such a situation, the endocervical cone apex will be exactly sampled, and the subsequent seriated clockwise sampling of the residual cone will be more smoothly performed, with following preparation of adequate histological sections 4. In our experience such a simple and easy-to-apply device, proves to be useful in making the uterine cone sampling more accurate and comfortable.
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Conización/instrumentación , Cuello del Útero/patología , Conización/métodos , Diseño de Equipo , Femenino , Humanos , Displasia del Cuello del Útero/diagnóstico , Displasia del Cuello del Útero/patologíaRESUMEN
House dust mite and other indoor allergens play a prominent role in the pathogenesis of asthma and other allergic diseases. Several studies have shown a close relationship between sensitisation and/or onset of asthmatic symptoms and levels of indoor allergen exposure. Aim of the study was to investigate the concentration of specific markers of the indoor allergenic pollution, such as Der p 1, Der f 1, Mite Group 2, Fel d 1 and Bla g 2. Dust samples were taken using a standard method by means of a 1200 W vacuum cleaner connected with a dust-sampling device (MITEST). A standard A4 size area has been vacuumed four times during 2 min. The concentrations of Der p 1, Der f 1, Mite Group 2, Fel d 1 and Bla g 2 were determined in dust samples from 53 different sources (office chair and carpet) using a commercial kit (DUSTSCREEN). House dust mite allergens were not always detectable in the offices. Indoor allergen concentrations (Der p 1, Der f 1, Mite Group 2, Fel d 1) were significant higher in the work station (chair) than in the carpet (p < 0.0001). Der 1 exceeded the current threshold for sensitization in about 1/4 of the samples. Der f 1 was predominant over Der p 1 according to other studies. A good correlation between the results of Der p 1 and Der f 1 was observed both in carpet and work station. Cat allergen was ubiquitous and predominantly detected in the chairs because of the employees' clothes. No appreciable levels for Mite Gr 2 and Bla g 2 were detected. Such an exposure for 8 hours in every working day may be an important occupational risk for the development of sensitization/elicitation symptoms to house dust mite. To reduce mite allergen levels are necessary preventive measure by means of specific techniques and products as barriers for preventing the direct contact with allergens.
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Contaminación del Aire Interior/análisis , Alérgenos/análisis , Antígenos Dermatofagoides/análisis , Lugar de Trabajo , HumanosRESUMEN
The mucolitic bromhexine [N-(2-amino-3,5-dibromobenzyl)-N-methylcyclohexylamine] has been determined in cotrimoxazole-containing tablets by partial least-squares (PLS-1) multivariate of spectrophotometric calibration data in the spectral range 310-350 nm. In the studied commercial tablets, cotrimoxazole is present in large excess (ca. 100:1 in weight) with respect to bromhexine, and a high degree of spectral overlapping exists among bromhexine and cotrimoxazole components. However, the obtained recoveries are reasonably good with the presently discussed technique.