The effect of laparoscopic pre- and postconditioning on pneumoperitoneum induced injury of the peritoneum.

INTRODUCTION: Pneumoperitoneum, widely used in laparoscopic surgery increases the intraabdominal pressure, leading to hypoperfusion of the abdominal organs, and promoting the buildup of reactive oxygen species(ROS) and inflammatory cytokines which in turn impairs the body postoperatively. Aim of our investigation was to evaluate the potential protective effects of short ischemic episodes on ischemic damages. METHODS: 70 Wistar rats were used, divided into 7 groups: 1st group sham, 2nd group pneumoperitoneum with 5 mmHg, 3rd group preconditioning with 5 mmHg, 4th group: postconditioning with 5 mmHg, 5th group pneumoperitoneum with 10 mmHg, 6th group: preconditioning with 10 mmHg, 7th group postconditioning with 10 mmHg. Pneumoperitoneum was created by Veres needle. Oxidative stress parameters: malondialdehyde (MDA) concentration, reduced glutathione (GSH), sulfhydril (SH-), myeloperoxidase (MPO) levels and superoxide-dismutase (SOD) activity were measured. We measured TNF-α and IL-6 concentrations. We monitored the activation of anti- and proapoptotic common signaling pathways (bax, bcl-2, p53) during the early phase of reperfusion. Histology was made from kidney samples. RESULTS: GSH concentrations were significantly reduced, MDA concentrations were significantly higher in each group compared to the Sham. SOD enzyme: a pressure of 10 mmHg elicited significantly greater damage than 5 mmHg. There was a significantly higher SOD activity in group 10 mmHg IPC compared to 10 mmHg. We found that the expression of bax was considerably higher in the none conditioned groups. Noticeably higher expression of anti-apoptotic bcl-2 level was measured in 10 mmHg IPC and 10 mmHg IPoC groups. In case of p53 expression: significant decrease could be seen in groups 10 mmHg IPC and 10 mmHg IPoC compared to the 10 mmHg group. CONCLUSION: Based on our results, the elevated intraabdominal pressure due to pneumoperitoneum induces oxidative stress, which is dependent on the applied pressure. Mostly precondiotioning - but also postconditioning - reduces surgical stress following laparoscopic procedures. In order to explore its mechanism it requires further investigations.

Note: 4-bounce neutron polarizer for reflectometry applications.

A neutron polarizer using four successive reflections on m = 2.5 supermirrors was built and installed at the GINA neutron reflectometer at the Budapest Neutron Centre. This simple setup exhibits 99.6% polarizing efficiency with 80% transmitted intensity of the selected polarization state. Due to the geometry, the higher harmonics in the incident beam are filtered out, while the optical axis of the beam remains intact for easy mounting and dismounting the device in an existing experimental setup.

Polymer-based microfluidic chip for rapid and efficient immunomagnetic capture and release of Listeria monocytogenes.

Infections caused by foodborne pathogens such as Listeria monocytogenes pose a threat to public health while timely detection is challenging due to pathogen low numbers. The development of robust and efficient sample preparation techniques is crucial to improve detection sensitivity and workflow. Immunomagnetic separation using magnetic nanoparticles (MNPs) is attractive, as it can efficiently capture target cells. For food safety applications, a platform is needed to rapidly process large sample volumes, allowing capture and release of target bacteria conjugated to immunomagnetic nanoparticles (IMNPs). Herein, we demonstrate a method for magnetic capture and release of bacteria-IMNPs complex based on a 3D magnetic trap integrated on a polymeric microfluidic device. The 3D magnetic capture region consist of a dense array of high-aspect ratio (3 : 1) cylindrical pillars embossed in thermoplastic polymer and coated with soft ferromagnetic nickel by an electroless deposition technique. This allows the generation of strong and switchable magnetic capture regions due to the very low remanence of the nickel shell. We propose and validate an optimized configuration of capture regions for efficient localized capture and rapid release of MNPs and IMNPs conjugated to L. monocytogenes. A maximum recovery rate for MNPs corresponded to 91% while a maximum capture efficiency of 30% was obtained for live bacteria, with a minimum detectable sample concentration of ~10 cfu ml(-1) in 1 ml volume using plate-culture method. We believe that the flexible design and low-cost fabrication process of the proposed system will allow rapid sample preparation for applications beyond food and water safety, including point-of-care diagnosis.

Core-shell nanoparticles as prodrugs: possible cytotoxicological and biomedical impacts of batch-to-batch inconsistencies.

Numerous samples of magnetite@silica and magnetite@silica@silane core-shell nanoparticles, previously used as prodrugs, were prepared by an experienced chemist, using the same identical equipment and the same lots of reagents. Their surface analyses showed batch-to-batch chemical variations: no two batches were found to have the same surface chemistries, showing unexpected Si-O bond scission and amine oxidation. Because the preparations used reactions recognized to be mild, and bond scission and oxidation were never previously reported for similar reactions on larger surfaces, the Fe(3)O(4) nanoparticles that form the nanoparticle core appear to have acted as catalysts for these side reactions. The intended use of these nanoparticles, as drug carriers, is discussed in terms of cytotoxicological and biomedical consequences.

Nitric oxide delivery by core/shell superparamagnetic nanoparticle vehicles with enhanced biocompatibility.

We report the synthesis of Fe(3)O(4)/silica core/shell nanoparticles and their functionalization with S-nitrosothiols. These nanoparticles are of immense interest because of their nitric oxide (NO) release capabilities in human alveolar epithelial cells. Moreover, they act as large storage reservoirs of NO that can be targeted magnetically to the specific site with a sustainable release of NO for up to 50 h. Such nanoparticles provide an enhancement of the biocompatibility with released NO while allowing intracellular accumulation ascribed to their small size.

Manejo de la hipotermia accidental severa / Management of severe accidental hypothermia

La hipotermia accidental es una patología ambiental con unos principios básicos de clasificación y reanimación que sirven tanto para el medio montañoso, marítimo o urbano. Esta patología ha formado parte, junto a la acidosis y la coagulopatía, de la famosa «tríada letal» de las víctimas traumáticas en situación crítica. En su manejo y asistencia está implicada toda una cadena asistencial que se extiende desde la medicina de urgencia prehospitalaria hasta la medicina intensiva, llegando incluso hasta la cirugía cardiaca y/o a los programas de circulación extracorpórea. Una buena clasificación prehospitalaria del grado de hipotermia facilitará su manejo inicial y evitará traslados interhospitalarios o secundarios innecesarios. Lo fundamental es trasladar, con la mayor urgencia posible, a las víctimas hipotérmicas en asistolia o fibrilación ventricular hasta aquellos hospitales que tengan la capacidad tecnológica adecuada para el tratamiento de estas especiales situaciones clínicas. Este artículo, trata de sentar las bases que faciliten un manejo adecuado de la hipotermia accidental desde la primera asistencia prehospitalaria hasta tratamiento final hospitalario, incluyendo la reanimación y el recalentamiento con circulación extracorpórea (AU)

Accidental hypothermia is an environmental condition with basic principles of classification and resuscitation that apply to mountain, sea or urban scenarios. Along with coagulopathy and acidosis, hypothermia belongs to the lethal triad of trauma victims requiring critical care. A customized healthcare chain is involved in its management, extending from on site assistance to intensive care, cardiac surgery and/or the extracorporeal circulation protocols. A good classification of the degree of hypothermia preceding admission contributes to improve management and avoids inappropriate referrals between hospitals. The most important issue is to admit hypothermia victims in asystolia or ventricular fibrillation to those hospitals equipped with the medical technology which these special clinical scenarios require. This study attempts to establish the foundations for optimum management of accidental hypothermia from first emergency care on site to treatment in hospital including, resuscitation and rewarming with extracorporeal circulation (AU)

In vitro biocompatibility assessment of functionalized magnetite nanoparticles: biological and cytotoxicological effects.

In the biomedical field, nanomaterials have the potential for use in the targeted delivery of drugs in the human body and in the diagnosis and therapy of certain diseases. In the category of targeted delivery, magnetite (Fe(3)O(4)) nanoparticles have received much attention. As with any similar new therapy, when such nanoparticles are functionalized with chemical groups designed to permit the specific attachment of drugs, cytotoxicological testing is necessary before moving to animal models. Here, we consider several variously functionalized magnetite nanoparticles, including those prepared with (1) a monolayer of oleic acid (Fe(3)O(4)@OA), which is subsequently converted to (2) a shell of amine-containing silane (Fe(3)O(4)@NH(2)), (3) a shell of silica (Fe(3)O(4)@SiO(2)), and (4) a shell of amine-containing silane over a shell of silica (Fe(3)O(4)@SiO(2)@NH(2)). These latter three functionalities were evaluated for biocompatibility, cellular morphology, mitochondrial function (MTT assay), lactate dehydrogenase membrane leakage (LDH assay), and proinflammatory potential through enzyme linked immunosorbent assay (ELISA) for interleukin 6 (IL-6). Controlled tests were performed over a period of 72 h, with results showing LDH leakage and abnormal Il-6 secretion at high concentrations (>50 µg/mL). The tests showed that, in addition to the surface characteristics of the nanoparticles, both the nutrient medium and the time of suspension before exposure to cells also contribute to nanoparticle cytotoxicity.

[Management of severe accidental hypothermia]. / Manejo de la hipotermia accidental severa.

Accidental hypothermia is an environmental condition with basic principles of classification and resuscitation that apply to mountain, sea or urban scenarios. Along with coagulopathy and acidosis, hypothermia belongs to the lethal triad of trauma victims requiring critical care. A customized healthcare chain is involved in its management, extending from on site assistance to intensive care, cardiac surgery and/or the extracorporeal circulation protocols. A good classification of the degree of hypothermia preceding admission contributes to improve management and avoids inappropriate referrals between hospitals. The most important issue is to admit hypothermia victims in asystolia or ventricular fibrillation to those hospitals equipped with the medical technology which these special clinical scenarios require. This study attempts to establish the foundations for optimum management of accidental hypothermia from first emergency care on site to treatment in hospital including, resuscitation and rewarming with extracorporeal circulation.

Use of a dual-labelled oligonucleotide as a DNA dosemeter for radiological exposure detection.

A reporter molecule consisting of a synthetic oligonucleotide is being characterised for a novel damage detection scenario for its potential use as a field-deployable, personal deoxyribonucleic acid (DNA) dosemeter for radiation detection. This dosemeter is devoid of any biological properties other than being naked DNA and therefore has no DNA repair capabilities. It supports biodosimetry techniques, which require lengthy analysis of cells from irradiated individuals, and improves upon inorganic dosimetry, thereby providing for a more relevant means of measuring the accumulated dose from a potentially mixed-radiation field. Radiation-induced single strand breaks (SSBs) within the DNA result in a quantifiable fluorescent signal. Proof of concept has been achieved over 250 mGy-10 Gy dose range in radiation fields from 6°Co, with similar results seen using a linear accelerator X-ray source. Further refinements to both the molecule and the exposure/detection platform are expected to lead to enhanced levels of detection for mixed-field radiological events.

The neuropeptide calcitonin gene-related peptide affects allergic airway inflammation by modulating dendritic cell function.

BACKGROUND: The neuropeptide calcitonin gene-related peptide (CGRP) is released in the lung by sensory nerves during allergic airway responses. Pulmonary dendritic cells (DC) orchestrating the allergic inflammation could be affected by CGRP. OBJECTIVE: To determine the immunomodulatory effects of CGRP on DC function and its impact on the induction of allergic airway inflammation. METHODS: CGRP receptor expression on lung DC was determined by RT-PCR and immunofluorescence staining. The functional consequences of CGRP receptor triggering were evaluated in vitro using bone marrow-derived DC. DC maturation and the induction of ovalbumin (OVA)-specific T cell responses were analysed by flow cytometry. The in vivo relevance of the observed DC modulation was assessed in a DC-transfer model of experimental asthma. Mice were sensitized by an intrapharyngeal transfer of OVA-pulsed DC and challenged with OVA aerosol. The impact of CGRP pretreatment of DC on airway inflammation was characterized by analysing differential cell counts and cytokines in bronchoalveolar lavage fluid (BALF), lung histology and cytokine responses in mediastinal lymph nodes. RESULTS: RT-PCR, immunofluorescence and cAMP assay demonstrated the expression of functionally active CGRP receptors in lung DC. RT-PCR revealed a transcriptional CGRP receptor down-regulation during airway inflammation. CGRP specifically inhibited the maturation of in vitro generated DC. Maturation was restored by blocking with the specific antagonist CGRP(8-37) . Consequently, CGRP-pretreated DC reduced the activation and proliferation of antigen-specific T cells and induced increased the numbers of T regulatory cells. The transfer of CGRP-pretreated DC diminished allergic airway inflammation in vivo, shown by reduced eosinophil numbers and increased levels of IL-10 in BALF. CONCLUSIONS AND CLINICAL RELEVANCE: CGRP inhibits DC maturation and allergen-specific T cell responses, which affects the outcome of the allergic airway inflammation in vivo. This suggests an additional mechanism by which nerve-derived mediators interfere with local immune responses. Thus, CGRP as an anti-inflammatory mediator could represent a new therapeutic tool in asthma therapy.

Molecular imaging of glioblastoma multiforme using anti-insulin-like growth factor-binding protein-7 single-domain antibodies.

BACKGROUND: Insulin-like growth factor-binding protein 7 (IGFBP7) is an abundant, selective and accessible biomarker of glioblastoma multiforme (GBM) tumour vessels. In this study, an anti-IGFBP7 single-domain antibody (sdAb) was developed to target GBM vessels for molecular imaging applications. METHODS: Human GBM was modelled in mice by intracranial implantation of U87MG.EGFRvIII cells. An anti-IGFBP7 sdAb, isolated from an immune llama library by panning, was assessed in vitro for its binding affinity using surface plasmon resonance and by ex vivo immunobinding on mouse and human GBM tissue. Tumour targeting by Cy5.5-labelled anti-IGFBP7 sdAb as well as by anti-IGFBP7 sdAb conjugated to PEGylated Fe3O4 nanoparticles (NPs)-Cy5.5 were assessed in U87MG.EGFRvIII tumour-bearing mice in vivo using optical imaging and in brain sections using fluorescent microscopy. RESULTS: Surface plasmon resonance analyses revealed a medium affinity (K(D)=40-50 nM) binding of the anti-IGFBP7 sdAb to the purified antigen. The anti-IGFBP7 sdAb also selectively bound to both mouse and human GBM vessels, but not normal brain vessels in tissue sections. In vivo, intravenously injected anti-IGFBP7 sdAb-Cy5.5 bound to GBM vessels creating high imaging signal in the intracranial tumour. Similarly, the anti-IGFBP7 sdAb-functionalised PEGylated Fe3O4 NP-Cy5.5 demonstrated enhanced tumour signal compared with non-targeted NPs. Fluorescent microscopy confirmed the presence of anti-IGFBP7 sdAb and anti-IGFBP7 sdAb-PEGylated Fe3O4 NPs selectively in GBM vessels. CONCLUSIONS: Anti-IGFBP7 sdAbs are novel GBM vessel-targeting moieties suitable for molecular imaging.

Airway-specific recruitment of T cells is reduced in a CD26-deficient F344 rat substrain.

Asthma is a chronic inflammatory disease affecting the airways. Increased levels of T cells are found in the lungs after the induction of an allergic-like inflammation in rats, and flow cytometry studies have shown that these levels are reduced in CD26-deficient rats. However, the precise anatomical sites where these newly recruited T cells appear primarily are unknown. Therefore, we quantified the distribution of T cells in lung parenchyma as well as in large, medium and small airways using immunohistochemical stainings combined with morphometric analyses. The number of T cells increased after the induction of an allergic-like inflammation. However, the differences between CD26-deficient and wild-type rats were not attributable to different cell numbers in the lung parenchyma, but the medium- and large-sized bronchi revealed significantly fewer T cells in CD26-deficient rats. These sites of T cell recruitment were screened further using immunohistochemistry and quantitative real-time polymerase chain reaction with regard to two hypotheses: (i) involvement of the nervous system or (ii) expression of chemokines with properties of a T cell attractor. No topographical association was found between nerves and T cells, but a differential transcription of chemokines was revealed in bronchi and parenchyma. Thus, the site-specific recruitment of T cells appears to be a process mediated by chemokines rather than nerve-T cell interactions. In conclusion, this is the first report showing a differential site-specific recruitment of T cells to the bronchi in a CD26-deficient rat substrain during an asthma-like inflammation.

Magnetically controlled dielectrophoresis of metallic colloids.

We present a finite-element/discrete-element numerical model for calculating full trajectories of cylindrical metallic colloids in liquid flows and subjected to non-uniform electric fields. The effect of the particle orientation relative to the liquid flow is investigated by considering barcode magnetic nanowires pinned in different directions by applying uniform magnetic fields. We compare the motion of free as well as vertically and horizontally pinned nanowires and demonstrate that their nanoassembly may accurately be tuned by magnetically controlling the orientation during the dielectrophoretic capture.

Ex vivo testing of immune responses in precision-cut lung slices.

The aim of this study was the establishment of precision-cut lung slices (PCLS) as a suitable ex vivo alternative approach to animal experiments for investigation of immunomodulatory effects. For this purpose we characterized the changes of cytokine production and the expression of cell surface markers after incubation of PCLS with immunoactive substances lipopolysaccharide (LPS), macrophage-activating lipopeptide-2 (MALP-2), interferon gamma (IFNgamma), and dexamethasone. Viability of PCLS from wild-type and CD11c-enhanced yellow fluorescent protein (CD11-EYFP)-transgenic mice was controlled by measurement of lactate dehydrogenase (LDH) enzyme activity and live/dead fluorescence staining using confocal microscopy. Cytokines and chemokines were detected with Luminex technology and ELISA. Antigen presenting cell (APC) markers were investigated in living mouse PCLS in situ using confocal microscopy. LPS triggered profound pro-inflammatory effects in PCLS. Dexamethasone prevented LPS-induced production of cytokines/chemokines such as interleukin (IL)-5, IL-1alpha, TNFalpha, IL-12(p40), and RANTES in PCLS. Surface expression of MHC class II, CD40, and CD11c, but not CD86 was present in APCs of naive PCLS. Incubation with LPS enhanced specifically the expression of MHC class II on diverse cells. MALP-2 only failed to alter cytokine or chemokine levels, but was highly effective in combination with IFNgamma resulting in increased levels of TNFalpha, IL-12(p40), RANTES, and IL-1alpha. PCLS showed characteristic responses to typical pro-inflammatory stimuli and may thus provide a suitable ex vivo technique to predict the immunomodulatory potency of inhaled substances.

Fabrication of large area nanoprism arrays and their application for surface enhanced Raman spectroscopy.

This work demonstrates the fabrication of metallic nanoprism (triangular nanostructure) arrays using a low-cost and high-throughput process. In the method, the triangular structure is defined by the shadow of a pyramid during angle evaporation of a metal etching mask. The pyramids were created by nanoimprint lithography in polymethylmethacrylate (PMMA) using a mould having an inverse-pyramid-shaped hole array formed by KOH wet etching of silicon. Silver and gold nanoprism arrays with a period of 200 nm and an edge length of 100 nm have been fabricated and used as effective substrates for surface enhanced Raman spectroscopy (SERS) detection of rhodamine 6G (R6G) molecules. Numerical calculations confirmed the great enhancement of electric field near the sharp nanoprism corners, as well as the detrimental effect of the chromium adhesion layer on localized surface plasmon resonance. The current method can also be used to fabricate non-equilateral nanoprism and three-dimensional (3D) nanopyramid arrays, and it can be readily extended to other metals.

Enhanced surface plasmon resonance imaging detection of DNA hybridization on periodic gold nanoposts.

We explore periodic gold nanoposts as substrates for the enhanced surface plasmon resonance imaging (SPRi) detection of DNA hybridization. Rigorous coupled-wave analysis was used to model and design the nanopost-based SPRi biosensor. Arrayed gold nanoposts on gold-coated glass substrate, with various widths and periodicity, were fabricated using electron-beam lithography and characterized with scanning electron and atomic force microscopy. A scanning-angle SPRi apparatus was used to conduct the kinetic analysis of DNA hybridization on nanopost-based sensor surface and assess the corresponding SPR signal amplification. Experimental results showed that both the nanostructure size and period influenced the SPR signal enhancement; the optimized 30 nm height, 50 nm size, and 110 nm period nanoposts provided a fivefold SPR signal amplification compared with the plain 50 nm thick gold film used as control.

Statistical study of effective anisotropy field in ordered ferromagnetic nanowire arrays.

Soft ferromagnetic nanowire arrays were obtained by electrodeposition of Co-Fe-P alloy into the pores of high quality home-made anodized aluminum oxide (AAO) templates. Bath acidity and current density were the two parameters used in order to tailor the orientation of local anisotropy axes in individual nanowires. In order to quantify the influence of the induced anisotropies on the magnetization processes in individual nanowires, the in-plane magnetization loops of the arrays are modeled as log-normal distributions of Stoner-Wohlfarth transverse magnetization processes. Using the lognormal mean parameter as an approximation for the saturation applied field of the array, we compute the effective anisotropy of the nanowires, which is found to increase with the pH of the electrodeposition bath.

Enzymatically-generated fluorescent detection in micro-channels with internal magnetic mixing for the development of parallel microfluidic ELISA.

The Enzyme-Linked Immuno-Sorbent Assay, or ELISA, is commonly utilized to quantify small concentrations of specific proteins for a large variety of purposes, ranging from medical diagnosis to environmental analysis and food safety. However, this technique requires large volumes of costly reagents and long incubation periods. The use of microfluidics permits one to specifically address these drawbacks by decreasing both the volume and the distance of diffusion inside the micro-channels. Existing microfluidic systems are limited by the necessary control of extremely low flow rates to provide sufficient time for the molecules to interact with each other by diffusion only. In this paper, we describe a new microfluidic design for the realization of parallel ELISA in stop-flow conditions. Magnetic beads were used both as a solid phase to support the formation of the reactive immune complex and to achieve a magnetic mixing inside the channels. In order to test the detection procedure, the formation of the immune complex was performed off-chip before the reactive beads were injected into the reaction chamber. Anti-streptavidin antibodies were quantified with low picomolar sensitivity (0.1-6.7 pM), a linear range of 2 orders of magnitude and good reproducibility. This work represents the first step toward a new platform for simple, highly effective and parallel microfluidic ELISA.

Selective expression of histamine receptors H1R, H2R, and H4R, but not H3R, in the human intestinal tract.

BACKGROUND AND AIMS: Histamine is known as a regulator of gastrointestinal functions, such as gastric acid production, intestinal motility, and mucosal ion secretion. Most of this knowledge has been obtained from animal studies. In contrast, in humans, expression and distribution of histamine receptors (HR) within the human gastrointestinal tract are unclear. METHODS: We analysed HR expression in human gastrointestinal tissue specimens by quantitative reverse transcription-polymerase chain reaction and immunostaining. RESULTS: We found that H1R, H2R, and H4R mRNA were expressed throughout the gastrointestinal tract, while H3R mRNA was absent. No significant differences in the distribution of HR were found between different anatomical sites (duodenum, ileum, colon, sigma, and rectum). Immunostaining of neurones and nerve fibres revealed that H3R was absent in the human enteric nervous system; however, H1R and H2R were found on ganglion cells of the myenteric plexus. Epithelial cells also expressed H1R, H2R and, to some extent, H4R. Intestinal fibroblasts exclusively expressed H1R while the muscular layers of human intestine stained positive for both H1R and H2R. Immune cells expressed mRNA and protein for H1R, H2R, and low levels of H4R. Analysis of endoscopic biopsies from patients with food allergy and irritable bowel syndrome revealed significantly elevated H1R and H2R mRNA levels compared with controls. CONCLUSIONS: We have demonstrated that H1R, H2R and, to some extent, H4R, are expressed in the human gastrointestinal tract, while H3R is absent, and we found that HR expression was altered in patients with gastrointestinal diseases.

Microwave synthesis and characterization of Co-ferrite nanoparticles.

Stable CoFe(2)O(4) nanoparticles have been obtained by co-precipitation using a microwave heating system. Transmission electron microscopy images analysis shows an agglomeration of particles with an average size of about 5 nm, and X-ray diffraction reveals the presence of a pure ferrite nanocrystalline phase. X-ray photoelectron spectroscopy and thermal gravimetric analysis show the presence of organic matter in the range of about 16 wt%. The magnetic response in DC fields is typical for an assembly of single-domain particles. The measured saturation magnetization is slightly larger than the bulk value, probably due to the presence of small amounts of Co and Fe. AC magnetization data indicate the presence of magnetic interactions between the nanoparticles.