AGGREGATION BY LECTIN-METHODICAL APPROACH FOR EFFECTIVE ISOLATION OF EXOSOMES FROM CELL CULTURE SUPERNATANT FOR PROTEOME PROFILING.

Exosomes are small membrane vesicles secreted by most cell types in vivo and in vitro. Exosomes are found in cell culture media, blood, urine, amniotic fluid, malignant ascite fluids and contain distinct subsets of microRNAs and proteins depending upon the tissue from which they are secreted. Thus exosomes constitute potential biomarkers of human diseases, such as cancer. A major bottleneck in the development of exosome-based diagnostic assays is the challenging purification of these vesicles; this requires time-consuming and instrument-based procedures. Isolation of exosomes can be a tedious, non-specific, and difficult process. Here, we provide a preparative technique for isolation of exosomes based on their ability to aggregate in the presence of lectins. The new method for lectin-based isolation of exosomes from cell culture media was developed as a sample preparation step for exosome-based protein biomarker research.

[The pathochemical characteristics of oligomeric forms of Tamm-Horsfall protein under urolithiasis].

The role of Tamm-Horsfall protein in pathogenesis of urolithiasis was analyzed. The study of oligomeric forms of protein was carried out using technique of dynamic light scattering. The sampling of 57 patients with urolithiasis and 51 patients of control group of comparative age and gender were examined. The degree of purification of Tamm-Horsfall protein was controlled using denaturant electrophoresis in polyacridine amyl gel. The reversing change of oligomeric form of protein with molecule size 2 Mda in polymeric form 28 Mda under impact of guanidinhydrochloride. Under urolithiasis, the form of protein associated with non-organic components and with size of macromolecular complex larger than 1500 nm was detected. The diagnostic criterion of urolithiasis was proposed based on totality of biochemical and biophysical analyses of urine.

[Verification of a decrease in the rigidity of the phage lambda DNA polymeric chain in low ionic strength aqueous solutions by testing the polymer-polymer interlink interactions].

Changes in the rigidity of the polymetric chain of phage lambda double-strand DNA have been studied by laser correlation spectroscopy. It was shown that, as the ionic strength increases, the effect of the screening of the hydrodynamic interaction of the links of the polymeric chain specific for polymeric coils arises in a DNA solution. It is assumed that the screening occurs when the threshold of the overlapping of DNA coils is achieved. The overlapping of coils is the result of a previously observed significant rise of DNA coil size from abnormally small DNA coils in low ionic strength buffers (about 10(-2) M Na+ or less) to maximum possible large coils in the 5SSC and 5SSC-like buffers. Further analysis of the far interlink interactions in linear lambda phage DNA coils in similar buffers at pH 7 and 4 confirms the earlier proposal about the role of H+ ions in the appearance of abnormally small DNA coils. The abnormal decrease in the DNA coil size in low ionic strength buffers is not a specific feature of lambda phage DNA only.

[Investigation of exosomes secreted by different normal and malignant cells in vitro and in vivo].

Laser correlation spectroscopy, atomic force microscopy, and immunoaffinity chromatography were used to characterize exosomes produced by different human cells. The exosomes secreted into a culture medium by normal fibroblasts, dendritic cells, lymphocytes, as well as malignant cells obtained from tumors of various tissue origins. The similar investigations were made for exosomes detectable in plasma and cerebrospinal fluid. The dynamic light scattering technique has demonstrated that the exosomes from different sources are homogenous and similar in size of the order of 20 and 90 nm. The exceptional homogeneity of exosomes was confirmed by atomic force microscopy. The immunoaffinity method has shown that all the exosomes under study carry antigenic determinants recognizable by antibodies to the major histocompatibility complex of type 1 (HLA-ABC). A method is proposed for evidence-based detection of exosomes in various biological fluids. For this, dynamic light scattering detects 20- and 90-nm particles and whether they can be removed by immunoaffinity chromatography with HLA-ABC antibodies is checked.

[Validation of an instrument for measuring chronic pain in nursing homes]. / Validación de un instrumento para la medición del dolor crónico en centros asistenciales de la tercera edad.

BACKGROUND: Assessment of chronic pain is frequently done in care centers. The aim of this study was to analyze the psychometric properties of the Chronic Pain Grade Scale (CPGS) - that measures both the intensity of chronic pain and its incapacitating effects on the everyday activities of the elderly. METHOD: Cross-sectional study of 185 people with chronic pain from two nursing homes. The questionnaire was adapted by back-translation and administered as a structured interview. It was assessed for reliability, internal consistency, construct validity, convergent and divergent validity (regarding the SF-12 score) and discriminant validity. Studied variables were compared among the pain groups derived from applying the scale. RESULTS: The scale showed sufficient reliability (a=0.90), bidimensionality (intensity and disability), good convergent and divergent validity and sufficient discriminant validity. Elderly people in groups I and II had better physical health than those in groups III and IV, but group II people had similar mental health to those from group IV (highest/limiting disability). Males, people with no education and pensioners were more frequently classified as group IV. CONCLUSION: The Spanish version of the CPGS has proved to be valid and reliable for evaluating both intensity and disability related to chronic pain in older people living in nursing homes.

Impact of body mass index on minimally invasive ventral hernia repair: an ACS-NSQIP analysis.

PURPOSE: Body mass index (BMI) ≥ 35 kg/m2 is a known independent risk factor for complications following open ventral hernia repair (VHR). We sought to examine the relationship between BMI and minimally invasive VHR. METHODS: The ACS-NSQIP database was queried for all patients age ≥ 18 years undergoing minimally invasive VHR (2005-2015). Patients were stratified into seven BMI classes: underweight (BMI < 18.5 kg/m2), normal weight (BMI 18.5-24.9), overweight (25-29.9), obese (30-34.5), severely obese (35-39.9), morbidly obese (40-49.9), and super obese (BMI ≥ 50), as well as by hernia type (reducible vs. strangulated) and time of repair (initial vs. recurrent). Multivariate logistic regression was employed to assess the risk of complication by BMI class. RESULTS: A total of 55,180 patients met inclusion criteria, and 61.4% had a BMI > 30 kg/m2. When stratified by BMI class, we found significant differences in age, gender, race, comorbidities, and pre-operative characteristics across groups. The overall complication rate was 4.0%, ranging from 3.0% for normal BMI patients, to 6.9% for patients with a BMI ≥ 50 kg/m2. Recurrent repairs and strangulated hernias both demonstrated higher complication rates. All complications (surgical and medical) were significantly associated with BMI class after adjustment (p < 0.0001). Patients with a BMI ≥ 50 kg/m2 had a 1.4 times greater risk for complications than patients with normal BMIs (18-24.9 kg/m2, p = 0.01). CONCLUSION: BMI ≥ 50 kg/m2 was determined to be an independent risk factor for surgical and medical complications after minimally invasive VHR.

Specific features of immune complexes in patients with sarcoidosis and pulmonary tuberculosis.

Clinical and radiological features of tuberculosis and sarcoidosis are quite overlapping, and therefore, a diagnostic dilemma often persists. There are no commonly accepted criteria for the diagnosis of sarcoidosis due to the lack of data on the etiology of the disease. The exclusion of tuberculosis in every patient with suspected sarcoidosis is a mandatory stage of diagnosis, especially in countries with a high burden of tuberculosis. A prospective study was conducted with two groups of patients: group I (n = 50)-patients with pulmonary sarcoidosis established according to standard criteria; group II (n = 28)-patients with pulmonary tuberculosis with bacterial excretion. The control group (n = 24) was presented by healthy subjects. The examination complex included x-ray, bacteriological, immunological (Mantoux test with 2 TE, TB.SPOT test), and histological methods. All patients and healthy subjects were assessed for immune complexes with the use of the dynamic light scattering (DLS) method and adding of "healthy lung tissue extract" antigens and specific tuberculosis antigens ESAT-6 and SFP-10 in vitro. Significant differences were found in determining specific immune complexes in patients with pulmonary sarcoidosis and pulmonary tuberculosis. Registration of specific immune complex formation with "healthy lung tissue extract" in 100% cases may indicate the autoimmune nature of sarcoidosis. The absence of the immune complex formation in response to ESAT-6/SFP-10 antigens can be used for the differential diagnosis of two diseases. The diagnostic significance of the DLS method was 100% for sarcoidosis and 92.2% for tuberculosis. The data obtained in the study allows not only understanding the etiology of sarcoidosis, but also obtaining new criteria for the differential diagnosis of tuberculosis and pulmonary sarcoidosis.

Systematic review of Spanish outcome measures on motor imagery ability: use in physical rehabilitation. / Revision sistematica sobre instrumentos de valoracion de la imaginacion motora para poblacion hispanohablante: su uso en rehabilitacion.

INTRODUCTION: Motor imagery is a mental representation of movement without any body movement and its training accelerates motor learning and improves motor skills. A thorough understanding of how to manipulate mental images is necessary before using motor imagery in physical rehabilitation. This systematic review analyzes the psychometric properties of the outcome measures on motor imagery ability for the Spanish-speaking people and discusses its usefulness in people with motor disabilities. MATERIALS AND METHODS: A review was conducted, using the COSMIN checklist to appraise 19 articles on measurement properties of motor imagery ability assessments found in reviewed databases. The criteria for grading the usefulness of instruments to measure motor imagery was established depending on the sensory modality assessed. We found 17 questionnaires. RESULTS: Methodological quality was mostly fair in reliability and validity. Four tests have been considered highly useful in assessing motor imagery. MIQ (alpha = 0.90; EFA=2) and MIQ-R (alpha = 0.84; EFA=2) are the best suited to evaluate motor imagery in Spanish-speaking population. To handle spatial images, MASMI (alpha = 0.93) or MARMI (alpha = 0.90) tests may be more beneficial. CONCLUSIONS: MIQ and MIQ-R evaluate visual and kinesthetic imagery, but these are difficult to use in the physical rehabilitation of people with motor disabilities. Currently, there are no valid Spanish translations of studies regarding motor imagery outcome measures for people with disabilities.

TITLE: Revision sistematica sobre instrumentos de valoracion de la imaginacion motora para poblacion hispanohablante: su uso en rehabilitacion.Introduccion. La imaginacion motora es el acto de imaginar una accion sin realizar el movimiento fisico. Su practica acelera el aprendizaje y mejora las destrezas motrices. Previamente a la rehabilitacion fisica utilizando la imaginacion motora, es necesario evaluar la capacidad de los individuos para formar y manipular imagenes mentales. Esta revision sistematica analiza las propiedades psicometricas de las herramientas existentes que miden la imaginacion motora en la comunidad hispanohablante y discute su utilidad clinica en personas con discapacidad motora. Materiales y metodos. Se hallaron 19 articulos en diferentes bases de datos, y se aplico la escala COSMIN para evaluar los 17 instrumentos de medida hallados sobre imaginacion mental. El criterio utilizado para graduar la utilidad clinica de estas herramientas fue establecido en funcion de la modalidad sensorial evaluada. Resultados. La calidad metodologica de los estudios fue aceptable en terminos de fiabilidad y validez. Cuatro cuestionarios se consideraron de utilidad alta en imaginacion motora. El Movement Imagery Questionnaire (MIQ) (alfa = 0,90; AFE = 2) y su version revisada (MIQ-R) (alfa = 0,84; AFE=2) son los cuestionarios autoadministrables que mejor se ajustan para evaluar la imaginacion motora en la poblacion hispanohablante. Entre los tests espaciales es posible utilizar la medida de la aptitud para formar imagenes mentales espaciales (alfa = 0,93) o la medida de la aptitud para rotar imagenes mentales (alfa = 0,90). Conclusiones. Aunque el MIQ y el MIQ-R evaluan la imaginacion visual y cinestesica, su aplicacion en la rehabilitacion con personas con discapacidad motora es complicada. Actualmente no hay instrumentos validados en castellano para poblacion con discapacidad fisica.

[Use of laser correlation spectroscopy for evaluation of metabolic changes in workers engaged in radiation-dangerous industry].

The pattern of metabolic changes was studied in nuclear fuel plant workers by laser correlation spectroscopy (LCS) of biological fluids (blood serum and plasma, urine, oropharyngeal lavages (OPL). Plasma samples were divided into 3 groups: 1) control (unirradiated) samples; 2) those irradiated by below 100 mZv; 2) those irradiated by more than 100 mZv. With larger dose irradiation, the contribution of small particles (6-8 nm) to the dispersion of increased and the proportion of large components (300-400 nm) decreased. There was a correlation between the total accumulated dose, the dose in the past 9 months and the changes in the contribution of the above groups of particles to light diffusion. The found regulations in the changes of the subfraction composition of blood agree with earlier data on changes in the serum of nuclear fuel workers. Analysis of urine samples revealed an increase in the contribution of catabolic processes. That of OPL showed the preponderance of anabolic changes over catabolic ones in the presence of a considerable contribution of normologically similar LC spectra. Differences were found in the pattern of metabolic changes in relation to technological stages. Although the nature of the observed spectral transformations remains unknown, the simplicity and rapidity of the LCS technique may be considered as a suitable tool for detecting the effects caused by small dose irradiation and other factors.

[The light diffusion technique for estimation of the size of urine particles in patients with type 2 diabetes mellitus in the Tamm-Horsfall protein precipitation].

Tamm-Horsfall uroprotein accounts for more than 50% of the urinary proteins in healthy individuals. In abnormalities, it creates a favorable background for detecting smaller-sized uroproteins and for diagnosing pathological processes from the results of native urine tests. In this connection, there is a need for precipitating Tamm-Horsfall glycoprotein while applying laser correlation spectroscopy to analyze the size of urine particles in patients with type 2 diabetes mellitus. Eighty patients with this condition concurrent with different stages of diabetic nephropathy and 23 apparently healthy individuals were examined. The findings suggest that the subfraction urine composition before and after Tamm-Horsfall protein precipitation is different in apparently healthy individuals and patients with type 2 diabetes mellitus concurrent with diabetic nephropathy. This is most likely to be due to the change in the qualitative composition of protein as renal lesion progresses, to the specific features of protein excretion at different stages of a pathological process, and to different concentrations of other low and high molecular-weight proteins.

Association between obsessive-compulsive disorder and polymorphisms of genes encoding components of the serotonergic and dopaminergic pathways.

Obsessive-compulsive disorder (OCD) is a severe and disabling anxiety disorder with a marked genetic contribution. Pharmacological data indicated involvement of the serotonergic and dopaminergic systems. We studied the association between OCD and six candidate genes encoding important components of the serotonergic and dopaminergic pathways in 75 biologically unrelated patients and 172 ethnically matched controls (Ashkenazi and non-Ashkenazi Jews). Polymorphisms in the following genes were studied: tryptophan hydroxylase (TPH), serotonin 2A receptor (HTR2A), serotonin 2C receptor (HTR2C), serotonin transporter (5-HTT), dopamine receptor D4 (DRD4), and dopamine transporter (DAT1). The genotypic and allelic distribution of all polymorphisms tested did not show statistically significant differences between patients and controls. Our results suggest that these polymorphisms do not play a major role in the genetic predisposition to OCD, although a minor contribution cannot be ruled out.

"Peripheral" benzodiazepine receptor density is not altered by methylphenidate treatment in children with attention-deficit hyperactivity disorder.

ABSTRACT "Peripheral" benzodiazepine receptors (PBRs) are located in the central nervous system and the periphery, are involved in the release of dopamine, and are sensitive to agents active at the dopaminergic system. Because attention-deficit hyperactivity disorder (ADHD) is related to dysregulation of dopaminergic neurotransmission, and because benzodiazepines can potentially aggravate the symptoms of ADHD, we have assessed the possible involvement of PBRs in the pathophysiology and pharmacotherapy of ADHD. [(3)H]PK 11195 was used to label platelet PBRs. Platelet PBRs were measured in eight ADHD boys (aged 9-15 years) before and after 4 weeks of treatment with methylphenidate 10-20 mg/day. ADHD patients before treatment did not differ from age-matched healthy controls in [(3)H]PK 11195 binding values. Four weeks of methylphenidate treatment did not significantly affect platelet PBR density in children with ADHD, despite the beneficial clinical effects. These results do not support the involvement of the PBRs in the pathophysiology of ADHD or in the therapeutic effects of methylphenidate.

Central mucoepidermoid carcinoma. Report of a case and review of the literature.

A case of central mucoepidermoid carcinoma (CMEC) occurring in a 68-year-old woman is reported. The tumor clinically appeared as a long-standing cortical swelling in the left angle of her mandible. As an intrabony lesion was radiologically evidenced, an odontogenic cyst was preoperatively diagnosed. Surgical curettage demonstrated a solid tumor the histological study of which allowed the final diagnosis. Immunohistochemical profile accorded with the patterns previously observed in salivary gland counterparts. Adjuvant radiotherapy was performed. Neither local recurrences nor metastases have been observed to date.

Hidradenitis suppurativa: patient satisfaction with wound healing by secondary intention.

Ariyan and Krizek, in 1976, reported on three patients with perineal hidradenitis suppurativa who, following excision, achieved satisfactory closure allowing spontaneous wound healing by secondary intention. We have used this approach and have considered it very satisfactory from the surgeon's point of view. We now report from the point of view of patients and review the records of 20 consecutive patients with perineal hidradenitis treated by excision and secondary healing. Surgical results and patient satisfaction were assessed 1 year following complete wound closure. All patients reported minimal inconvenience and interruption of daily activities from this method of management. Analgesic requirements were minimal, and little reinforcement was necessary to maintain vigorous wound care. Uncomplicated wound closure was uniformly achieved with unrestrictive, stable scars providing long-term relief of this disabling disease. Two patients expressed dissatisfaction as a result of a new onset of disease in previously uninvolved and therefore unresected apocrine tissue. With this exception, patient satisfaction has been great and hospitalization time and costs reduced.

Bone regeneration after radicular cyst removal with and without guided bone regeneration.

In order to determine the degree of bone regeneration after removal of radicular cysts using guided bone regeneration (GBR), a prospective, controlled and randomized clinical study was performed. Thirty patients with radicular cysts were divided into three groups. One group, the control group (n=10 patients), was treated by enucleation and primary closure. The other two groups were treated by enucleation and primary closure but GBR was used in addition, using a resorbable membrane (n=10) and a nonresorbable membrane (n=10). The membranes were fixed with nonresorbable Memfix System screws. The residual volume and the density of the newly formed tissue was measured by computer-assisted tomography and computer-assisted digital image analysis before enucleation and three and six months postoperatively. No statistical significance was found in density and residual volume between the three treatment groups after six months. These results suggest that GBR using membranes does not contribute to increased bone regeneration.

New approach to objective diagnostics of polyfunctional disorders of the neuromuscular regulation in children with various forms of spine deformities.

A combination of two methodical approaches (parallel evaluation of the psychomotor function and metabolic shifts) was used in examination of children with scoliosis. We evaluated adaptation of children at the certain stage of the disease for prediction of its further course and correction of the treatment.

[Polysystemic assessment of the state of sanogenesis in workers employed in nuclear fuel plants. The analysis of metabolism regulation processes]. / Polisistemnaia otsenka sostoianiia sanogeneza rabotnikov predpriiatiia iaderno-toplivnogo tsikla.Analiz reguliatsii obmennykh protsessov.

Using the method of laser correlation spectroscopy of biological fluids (blood serum, urine, oropharyngeal washout fluid) we studied the types of metabolic shifts in workers employed in nuclear fuel complex plant. In was found that the incidence of catabolic shifts considerably increased in workers with higher level of occupational exposure. In individuals contacting with open radiation sources we found the contribution of anabolic immunomodifying shifts with predominance of autoimmune sensibilization. A risk group for blood diseases was identified.