Nanoparticles Stokes radius assessment through permeability coefficient determination within a new stratified epithelium on-chip model.

Tissue barrier permeability plays a crucial role in determining the selective transport of substances across epithelial tissues, including drugs, cosmetic substances, and chemicals. The ability of these substances to cross through tissue barriers affects their absorption into the bloodstream and ultimately their effectiveness. Therefore, the determination of their permeability on these type of tissue barriers represents a useful tool for pharmaceutical and cosmetic industries as well as for toxicological studies.In this regard, microfluidic devices and organ-on-chip technologies are becoming more important to generate reliable data. We have designed and performed an alternative new stratified epithelia-on-chip model that allows to correlate the Stokes radius and the diffusion of molecules and/or nanoformulations through the in vitro generated barrier and establish a system suitable for the analysis of diffusion through stratified epithelium. Thus, extrapolating from experimental data we can predict the Stokes radius for unknown fluorescent labelled particles within a molecular size range, such as gold nanoparticles.

Development, characterization and sterilisation of Nanocellulose-alginate-(hyaluronic acid)- bioinks and 3D bioprinted scaffolds for tissue engineering.

3D-bioprinting is an emerging technology of high potential in tissue engineering (TE), since it shows effective control over scaffold fabrication and cell distribution. Biopolymers such as alginate (Alg), nanofibrillated cellulose (NC) and hyaluronic acid (HA) offer excellent characteristics for use as bioinks due to their excellent biocompatibility and rheological properties. Cell incorporation into the bioink requires sterilisation assurance, and autoclave, ß-radiation and γ-radiation are widely used sterilisation techniques in biomedicine; however, their use in 3D-bioprinting for bioinks sterilisation is still in their early stages. In this study, different sterilisation procedures were applied on NC-Alg and NC-Alg-HA bioinks and their effect on several parameters was evaluated. Results demonstrated that NC-Alg and NC-Alg-HA bioinks suffered relevant rheological and physicochemical modifications after sterilisation; yet, it can be concluded that the short cycle autoclave is the best option to sterilise both NC-Alg based cell-free bioinks, and that the incorporation of HA to the NC-Alg bioink improves its characteristics. Additionally, 3D scaffolds were bioprinted and specifically characterized as well as the D1 mesenchymal stromal cells (D1-MSCs) embedded for cell viability analysis. Notably, the addition of HA demonstrates better scaffold properties, together with higher biocompatibility and cell viability in comparison with the NC-Alg scaffolds. Thus, the use of MSCs containing NC-Alg based scaffolds may become a feasible tissue engineering approach for regenerative medicine.

Type 1 Diabetes Mellitus reversal via implantation of magnetically purified microencapsulated pseudoislets.

Microencapsulation of pancreatic islets for the treatment of Type I Diabetes Mellitus (T1DM) generates a high quantity of empty microcapsules, resulting in high therapeutic graft volumes that can enhance the host's immune response. We report a 3D printed microfluidic magnetic sorting device for microcapsules purification with the objective to reduce the number of empty microcapsules prior transplantation. In this study, INS1E pseudoislets were microencapsulated within alginate (A) and alginate-poly-L-lysine-alginate (APA) microcapsules and purified through the microfluidic device. APA microcapsules demonstrated higher mechanical integrity and stability than A microcapsules, showing better pseudoislets viability and biological function. Importantly, we obtained a reduction of the graft volume of 77.5% for A microcapsules and 78.6% for APA microcapsules. After subcutaneous implantation of induced diabetic Wistar rats with magnetically purified APA microencapsulated pseudoislets, blood glucose levels were restored into normoglycemia (<200 mg/dL) for almost 17 weeks. In conclusion, our described microfluidic magnetic sorting device represents a great alternative approach for the graft volume reduction of microencapsulated pseudoislets and its application in T1DM disease.

Cell Microencapsulation and Cryopreservation with Low Molecular Weight Hyaluronan and Dimethyl Sulfoxide.

Cryopreservation is commonly used for the storage of cells, tissues, organs or 3D cell-based products using ultra-low temperatures, which involves the immersion in liquid nitrogen for their long-term preservation. The cryopreservation of several microencapsulated cells is usually performed by the slow freezing with the dimethyl sulfoxide (DMSO) as a cryoprotectant agent (CPA). In this study, we cryopreserved several microencapsulated cells with the natural, non-toxic low molecular-weight hyaluronan (LMW-HA) at 5% and DMSO 10% solution assessing cell viability and metabolic activity after thawing. The cryopreservation of microencapsulated D1 mesenchymal stem cells (D1MSC) and murine myoblast cells (C2C12) with the LMW-HA 5% presented similar outcomes after thawing compared to the DMSO solution, showing the low molecular weight hyaluronan as a natural, non-toxic CPA that can be used preventing the DMSO related adverse effects after the implantation of the cryopreserved cell-based products.

Low molecular-weight hyaluronan as a cryoprotectant for the storage of microencapsulated cells.

The low-temperature storage of therapeutic cell-based products plays a crucial role in their clinical translation for the treatment of diverse diseases. Although dimethylsulfoxide (DMSO) is the most successful cryoprotectant in slow freezing of microencapsulated cells, it has shown adverse effects after cryopreserved cell-based products implantation. Therefore, the search of alternative non-toxic cryoprotectants for encapsulated cells is continuously investigated to move from bench to the clinic. In this work, we investigated the low molecular-weight hyaluronan (low MW-HA), a natural non-toxic and non-sulfated glycosaminoglycan, as an alternative non-permeant cryoprotectant for the slow freezing cryopreservation of encapsulated cells. Cryopreservation with low MW-HA provided similar metabolic activity, cell dead and early apoptotic cell percentage and membrane integrity after thawing, than encapsulated cells stored with either DMSO 10% or Cryostor 10. However, the beneficial outcomes with low MW-HA were not comparable to DMSO with some encapsulated cell types, such as the human insulin secreting cell line, 1.1B4, maybe explained by the different expression of the CD44 surface receptor. Altogether, we can conclude that low MW-HA represents a non-toxic natural alternative cryoprotectant to DMSO for the cryopreservation of encapsulated cells.

Advances in the slow freezing cryopreservation of microencapsulated cells.

Over the past few decades, the use of cell microencapsulation technology has been promoted for a wide range of applications as sustained drug delivery systems or as cells containing biosystems for regenerative medicine. However, difficulty in their preservation and storage has limited their availability to healthcare centers. Because the preservation in cryogenic temperatures poses many biological and biophysical challenges and that the technology has not been well understood, the slow cooling cryopreservation, which is the most used technique worldwide, has not given full measure of its full potential application yet. This review will discuss the different steps that should be understood and taken into account to preserve microencapsulated cells by slow freezing in a successful and simple manner. Moreover, it will review the slow freezing preservation of alginate-based microencapsulated cells and discuss some recommendations that the research community may pursue to optimize the preservation of microencapsulated cells, enabling the therapy translate from bench to the clinic.

Use of Flow Focusing Technique for Microencapsulation of Myoblasts.

Alginate cell microencapsulation implies the immobilization of cells within a polymeric membrane that allows the bidirectional diffusion of nutrients and oxygen inside the microcapsules and the release of waste and therapeutic molecules outside them. This technology has been applied to several cell types and it has been extensively described with pancreatic islets. However, other cells such as myoblasts are being currently studied and showing high interest. Moreover, different systems and approaches have been developed for cell encapsulation such as electrostatic extrusion and Flow focusing technology. When Flow focusing technology is applied for myoblast encapsulation, several factors should be considered, such as the pressure, the flow of the system, or the diameter size of the nebulizer, which will determine the final diameter size and shape of the microcapsules containing the myoblasts. Finally, viability of encapsulated myoblasts needs to be assessed before further studies are performed.

Microencapsulated Cells for Cancer Therapy.

The microencapsulation of different types of cells that are able to produce therapeutic factors is being investigated for the treatment of several human diseases. Most efforts are focused on chronic and degenerative diseases as this strategy could become an alternative to some commonly used parenteral treatments that need to be repeatedly administered. But, this approach has also been investigated in the field of oncology with the aim of providing immunomodulatory antibodies that are able to enhance the patient's inherent immune response against the tumor. These kind of treatments would provide the patient with the therapeutic drug produced in situ, de novo, and in a sustained way, making the therapy more comfortable.Although different devices are nowadays available to produce cell-enclosing alginate-microcapsules, here, we describe the most important steps and advices in order to fabricate alginate-poly-L-lysine-alginate microcapsules containing hybridoma cells for cancer management using an electrostatic bead generator, and how to evaluate the viability of those cells over the time.

Graphene oxide increases the viability of C2C12 myoblasts microencapsulated in alginate.

Cell microencapsulation represents a great promise for long-term drug delivery, but still several challenges need to be overcome before its translation into the clinic, such as the long term cell survival inside the capsules. On this regard, graphene oxide has shown to promote proliferation of different cell types either in two or three dimensions. Therefore, we planned to combine graphene oxide with the cell microencapsulation technology. We first studied the effect of this material on the stability of the capsules and next we analyzed the biocompatibility of this chemical compound with erythropoietin secreting C2C12 myoblasts within the microcapsule matrix. We produced 160 µm-diameter alginate microcapsules with increasing concentrations of graphene oxide and did not find modifications on the physicochemical parameters of traditional alginate microcapsules. Moreover, we observed that the viability of encapsulated cells within alginate microcapsules containing specific graphene oxide concentrations was enhanced. These results provide a relevant step for the future clinical application of graphene oxide on cell microencapsulation.

Use of B7 costimulatory molecules as adjuvants in a prime-boost vaccination against Visna/Maedi ovine lentivirus.

RNA transcripts of the B7 family molecule (CD80) are diminished in blood leukocytes from animals clinically affected with Visna/Maedi virus (VMV) infection. This work investigates whether the use of B7 genes enhances immune responses and protection in immunization-challenge approaches. Sheep were primed by particle-mediated epidermal bombardment with VMV gag and env gene recombinant plasmids together with plasmids encoding both CD80 and CD86 or CD80 alone, boosted with gag and env gene recombinant modified vaccinia Ankara virus and challenged intratracheally with VMV. Immunization in the presence of one or both of the B7 genes resulted in CD4+ T cell activation and antibody production (before and after challenge, respectively), but only immunization with CD80 and CD86 genes together, and not CD80 alone, resulted in a reduced number of infected animals and increased early transient cytotoxic T lymphocytes (CTL) responses. Post-mortem analysis showed an immune activation of lymphoid tissue in challenge-target organs in those animals that had received B7 genes compared to unvaccinated animals. Thus, the inclusion of B7 genes helped to enhance early cellular responses and protection (diminished proportion of infected animals) against VMV infection.

[LC-MS/MS analysis of herbicide residues in waters from Navarre]. / Análisis de residuos de herbicidas en aguas procedentes de Navarra mediante LC-MS/MS.

BACKGROUND: The aim of the study was to obtain information on the residues of herbicides (imazametabenz, simazine, isoproturon, clortoluron, metribuzine, atrazine, cianazine, terbutrin, propanil, terbutilazine, alachlor and pendimetalin) present in water proceeding from Navarre. MATERIAL AND METHODS: A total of 465 samples of water were taken: 378 were taken from 141 water supplies; the remaining 87 proceeded from fountains, springs and borings in agricultural areas in the south of Navarre. The technique employed was LC-MS/MS. RESULTS: Herbicide residues were detected in 33% of the water supplies, and their concentration exceeded the normative limit in 8% of them. Herbicide residues were found in 86% of the samples from wells and fountains of the agricultural areas of the south of Navarre. The number of water supplies with herbicide residues increased from north to south. However, the percentage of water supplies in which the concentration of herbicides exceeded the limits was greater in the centre of Navarre than in the north and south. Imazametabenz was the herbicide most detected in the samples from the northern, central and southern areas. Atrazine, terbutilazine and imazametabenz were most detected in the samples of the southern area. CONCLUSIONS: The differences amongst the three areas of Navarre can be explained as being due to the different agricultural practices of each area. Concentrations of herbicides exceeding the normative limits were not detected in any of the water samples proceeding from the larger towns of Navarre.

A genetic fusion GDNF-C fragment of tetanus toxin prolongs survival in a symptomatic mouse ALS model.

PURPOSE: Amyotrophic Lateral Sclerosis (ALS) is a paralyzing disorder that kills individuals within three to five years of onset without any possibility for effective treatment. One proposed therapy has been the use of neurotrophic factors to inhibit the apoptosis of motorneurones. At the present, one way to deliver neurotrophic factors after intramuscular injection to the motor neurones is through the use of adenoviral vectors. An alternative strategy is the use of the atoxic C fragment of tetanus toxin (TTC) as a neurotrophic factor carrier for motorneurones. METHODS: We have produced the recombinant protein fusion Glial Derived Neurotrophic Factor and C fragment of tetanus toxin (GDNF-TTC) and we have tested its antiapoptotic activity in degeneration culture cells and in the symptomatic SOD;{G93A} transgenic animal model for ALS. RESULTS: We demonstrated that GDNF-TTC induces the neuronal survival Akt kinase pathway in mouse cortical culture neurons and~maintains its antiapoptotic neuronal activity in Neuro2A cells. Moreover, we have found that genetic fusion is able to increase survival by 9 days and improves life quality in symptomatic ALS animal models. CONCLUSION: These results suggest that recombinant GDNF-TTC fusion protein intramuscular injections provide a potential therapy for ALS treatment.

[Organic pollutant residues of different origins in Navarra]. / Residuos de contaminantes orgánicos de diferentes orígenes en Navarra.

An analysis was made of residues of polychlorobyphenyls and trihalomethanes through GC-ECD and of herbicides through HPLC-PAD in samples proceeding from Navarra. Polychlorobyphenyls were detected (0.30 +/- 0.05 and 0.11 +/- 0.05 microg/l) in two of the 106 water samples analysed. Sixty-six food samples were analysed, and polychlorobyphenyls were only found in 8 samples of trout (dissimilar to dioxins: 21-194 microg/kg of fat; similar to dioxins: 41-139 microg/kg of fat). Of 107 fat samples analysed, polychlorobyphenyls dissimilar to dioxins were detected in two (27 +/- 5 and 30 +/- 5 microg/kg). Out of a total of 94 feed samples analysed, polychlorobyphenyls were detected in all the samples (12) of feed for aquaculture and their raw materials; the concentration of polychlorobyphenyls dissimilar to dioxins varied by an interval of 8-247 microg/kg of fat; polychlorobyphenyls similar to dioxins, between 18 and 107 microg/kg of fat. Contamination by polychlorobyphenyls of the fish from aquaculture could be due to the feed used in these exploitations. The average of trihalomethanes in the waters of the southern zone of Navarra (44 +/- 4 microg/l) was higher than those of the middle zone (16 +/- 1 microg/l) and the mountain zone (12 +/- 1 microg/l). The concentration of 99% of the samples fulfilled the norms on halomethanes. A relation was observed between muddiness and the concentration of trihalomethanes. Herbicide (cianazine) was only detected in one of the 135 samples of water analysed, with a concentration of (0.4 +/- 0.2 microg/l) which exceeded the established limit. The use of confirmation techniques (GC-MS, HPLC-MS/MS) would make it possible to validate these results and to expand the number of compounds analysed.

Gas chromatographic determination of organochlorine pesticides in cow milk.

A gas chromatographic method of use in determining the following organochlorine pesticides in cow milk has been adapted and validated: alpha-hexachlorocyclohexane (alpha-HCH), beta-HCH, lindane, delta-HCH, hexachlorobenzene, aldrin, heptachlor, heptachlor epoxide, mirex, 2,4-dichlorodiphenyltrichloroethane (2,4-DDT), 4,4-DDT, 2,4-dichlorodiphenyldichloroethane (2,4-DDD), 4,4-DDD, 2,4-dichlorodiphenylethane (2,4-DDE) and 4,4-DDE. The method studied consists of three steps: the extraction of fat by centrifugation, mixing with anhydrous sodium sulfate, and transfer to a glass column and elution with hexane; purification of the extract via concentrated sulfuric acid treatment; and determination of the organochlorine pesticides by gas chromatography equipped with an electron capture detector. The analytical parameters determined to estimate the quality of the method yielded the following results: correlation coefficients (r) of the calibration curves were greater than 0.996, the variation coefficients of the response factors were less than 4.90%--with the exception of aldrin (5.53%) and lindane (5.75%)--the relative standard deviation of the slope (Sm rel) being < or =1.52%. Zero was included in the confidence limits of the independent term range. The method repeatability and reproducibility values were < or =4.73% and < or =5.79%, respectively. The recovery values of the pesticides analyzed were in the range of 81.92% (alpha-HCH) to 105.49% (4,4-DDT), except in the case of aldrin and heptachlor epoxide, which yielded recovery values of under 40%. The quantification limits of the method were < or =1 ng/g, with the exception of mirex (5 ng/g).

Cefalea en un paciente joven. Neurocisticercosis / Migraine in a young patient. Neurocysticercosis

La cisticercosis cerebral es una forma de presentación de parasitosis cerebral causada por Tenia Soleum. A pesar de la escasa prevalencia lejos de las zonas endémicas, el incremento de los movimientos migratorios obliga a un mayor estado de alerta por parte del médico ante síntomas frecuentes como es una cefalea, sobre todo en pacientes con factores de riesgo epidemiológicos. El diagnóstico debe ser precoz y el tratamiento instaurarse lo antes posible, de lo contrario las consecuencias pueden ser fatales para el enfermo (AU)

[Study of the accumulation of lead in the primary teeth]. / Estudio de la acumulación de plomo en dientes primarios.

The purpose of the study was to determine the accumulation of lead in deciduous teeth in children living in the area of Pamplona (Spain). In this manner, we tried to make a relationship between the quantity of lead accumulated in the tooth against certain factors of exposure that were documented on a questionnaire carried out at the time that the tooth was presented. We analysed 457 deciduous teeth using a technique of microwave digestion follow by Atomic Absorption Spectrometry (AAS), to determine the concentration of lead in the sample. The mean lead concentration was 2.60+/-1.36 microg/g (range 0.25-10.71 microg/g). The lead concentration in our study is inferior to those observed in other European studies.

[Weight-loss drugs: composition of diet pills prescribed in Navarra]. / Fármacos adelgazantes: Composición de preparados magistrales prescritos en Navarra.

BACKGROUND: The prescription, dispensation and sale of "Magisterial formulas" for weight loss is a very extended practice in Spain, in spite of being strongly unadvised by different Administration Department as well as by medical and sanitary groups. The composition of these preparations is not labelled if so only in a generic way. METHODS: The content of 54 capsules from 6 different origins (3 medical consultations, 1 pharmacist consultation, 1 free sale in pharmacies and 1 free sale in stores) was analysed in 2 laboratories. The presence and quantity of 29 active principles was searched by spectrophotometry, chromatography and radioimmunoassay. RESULTS: The presence of some of the 12 following substances was confirmed in 42 samples (77.8%): hormones (levothyroxine, cortisol and cortisone), appetite suppression phenetilamines (amphetamine, amphepranone, fenfluramine and fenproporex), benzodiazepines (diazepam, clorazepato and chlordiazepoxide) and diuretics (triamterene and ciclotiazide). The quantities of active principles was very variable in and among the six groups. In 13 of the capsules 1 active principle and in 29 combinations of two or more was found. None component of the labels, when available, corresponded with their analysed components. In 12 samples (22.2%) none of the which may correspond to some substances different from the 29 sought ones. CONCLUSIONS: The usage of formulas as the studied here on the overweight and obesity treatment should be obviously unadvised.

[Orthopedic treatment of nocturnal acroparesthesias in primary care]. / Tratamiento ortopédico de las acroparestesias nocturnas en atención primaria.

OBJECTIVE: To report the experience gained from the systematic use of a splint immobilising at night the flexion or extension of the carpus of patients affected by nocturnal acroparaesthesiae. DESIGN: Descriptive, observational study. SETTING: Two urban Health Centres. PATIENTS OR OTHERS PARTICIPANTS: Twenty-two patients were studied. These were 6 men and 16 women who attended during the period from June 1991 and June 1992 because of nocturnal acroparaesthesiae in the area innervated by the median nerve. INTERVENTIONS: The treatment used was to immobilize with a splint the flexion and extension of the carpus during the night. After three weeks the results were evaluated. MEASUREMENTS AND MAIN RESULTS: Disappearance of the symptomatology occurred in 8 cases (36.4%). In 10 cases (45.5%), the intensity or frequency of the acroparaesthesiae lessened and in the remaining 4 (18.2%) the symptoms persisted. No significant differences in the therapeutic results could be shown when the patients were grouped in line with clinical or exploratory findings. CONCLUSIONS: On account of the absence of side-effects, its low cost and its being accessible and bloodless, we recommend this treatment as the initial model of therapeutic intervention in Primary Care, while other diagnostic and/or therapeutic possibilities are considered.