Integration of multiple flexible electrodes for real-time detection of barrier formation with spatial resolution in a gut-on-chip system.

In healthy individuals, the intestinal epithelium forms a tight barrier to prevent gut bacteria from reaching blood circulation. To study the effect of probiotics, dietary compounds and drugs on gut barrier formation and disruption, human gut epithelial and bacterial cells can be cocultured in an in vitro model called the human microbial crosstalk (HuMiX) gut-on-a-chip system. Here, we present the design, fabrication and integration of thin-film electrodes into the HuMiX platform to measure transepithelial electrical resistance (TEER) as a direct readout on barrier tightness in real-time. As various aspects of the HuMiX platform have already been set in their design, such as multiple compressible layers, uneven surfaces and nontransparent materials, a novel fabrication method was developed whereby thin-film metal electrodes were first deposited on flexible substrates and sequentially integrated with the HuMiX system via a transfer-tape approach. Moreover, to measure localized TEER along the cell culture chamber, we integrated multiple electrodes that were connected to an impedance analyzer via a multiplexer. We further developed a dynamic normalization method because the active measurement area depends on the measured TEER levels. The fabrication process and system setup can be applicable to other barrier-on-chip systems. As a proof-of-concept, we measured the barrier formation of a cancerous Caco-2 cell line in real-time, which was mapped at four spatially separated positions along the HuMiX culture area.

Two-Photon Polymerization Printing with High Metal Nanoparticle Loading.

Two-photon polymerization (2PP) is an efficient technique to achieve high-resolution, three-dimensional (3D)-printed complex structures. However, it is restricted to photocurable monomer combinations, thus presenting constraints when aiming at attaining functionally active resist formulations and structures. In this context, metal nanoparticle (NP) integration as an additive can enable functionality and pave the way to more dedicated applications. Challenges lay on the maximum NP concentrations that can be incorporated into photocurable resist formulations due to the laser-triggered interactions, which primarily originate from laser scattering and absorption, as well as the limited dispersibility threshold. In this study, we propose an approach to address these two constraints by integrating metallic Rh NPs formed ex situ, purposely designed for this scope. The absence of surface plasmon resonance (SPR) within the visible and near-infrared spectra, coupled with the limited absorption value measured at the laser operating wavelength (780 nm), significantly limits the laser-induced interactions. Moreover, the dispersibility threshold is increased by engineering the NP surface to be compatible with the photocurable resin, permitting us to achieve concentrations of up to 2 wt %, which, to our knowledge, is significantly higher than the previously reported limit (or threshold) for embedded metal NPs. Another distinctive advantage of employing Rh NPs is their role as promising contrast agents for X-ray fluorescence (XRF) bioimaging. We demonstrated the presence of Rh NPs within the whole 2PP-printed structure and emphasized the potential use of NP-loaded 3D-printed nanostructures for medical devices.

Pre-oxygenation using high-flow nasal oxygen in parturients undergoing caesarean section in general anaesthesia: A prospective, multi-centre, pilot study.

BACKGROUND: Parturients undergoing caesarean section in general anaesthesia have an increased risk of desaturating during anaesthesia induction. Pre- and peri-oxygenation with high-flow nasal oxygen prolong the safe apnoea time but data on parturients undergoing caesarean section under general anaesthesia are limited. This pilot study aimed to investigate the clinical effects and frequency of desaturation in parturients undergoing caesarean section in general anaesthesia pre- and peri-oxygenated with high-flow nasal oxygen and compare this to traditional pre-oxygenation using a facemask. METHODS: In this prospective, non-randomised, multi-centre study we included pregnant women with a gestational age ≥30 weeks undergoing caesarean section under general anaesthesia. All parturients were asked to participate in the intervention group consisting of pre-oxygenation using high-flow nasal oxygen. Parturients declining participation were pre-oxygenated with a traditional facemask. Primary outcome was the proportion of parturients desaturating below 93% from start of pre-oxygenation until 1 min after tracheal intubation. Secondary outcomes investigated end-tidal oxygen concentrations after tracheal intubation and the proportion of parturients with signs of regurgitation. RESULTS: A total of 34 parturients were included, 25 pre- and peri-oxygenated with high-flow nasal oxygen and 9 pre-oxygenated with facemask. No difference in patient or airway characteristics could be seen except for a higher BMI in the high-flow nasal oxygen group (31.4 kg m-2 [4.7] vs. 27.7 kg m-2 [3.1]; p = .034). No woman in any of the two groups desaturated below 93%. The lowest peripheral oxygen saturation observed, in any parturient, was 97%. There was no difference detected in end-tidal oxygen concentration after tracheal intubation, 87% (6) in the high-flow nasal oxygen group vs 80% (15) in the facemask group (p = .308). No signs of regurgitation, in any parturient, were seen. CONCLUSION: Pre- and peri-oxygenation with high-flow nasal oxygen maintain adequate oxygen saturation levels during induction of anaesthesia also in parturients. Regurgitation of gastric content did not occur in any parturient and no other safety concerns were observed in this pilot study.

Photophysiological response of Symbiodiniaceae single cells to temperature stress.

Photosynthetic dinoflagellates in the family Symbiodiniaceae engage in symbiosis with scleractinian corals. As coral 'bleaching' is partly governed by the thermal sensitivity of different Symbiodiniaceae lineages, numerous studies have investigated their temperature sensitivity. However, the systematic identification of single-cells with increased temperature resistance among these dinoflagellates has remained inaccessible, mostly due to a lack of technologies operating at the microscale. Here, we employed a unique combination of microfluidics, miniaturized temperature control, and chlorophyll fluorometry to characterize the single-cell heterogeneity among five representative species within the Symbiodiniaceae family under temperature stress. We monitored single-cell maximum quantum yields (Fv/Fm) of photosystem (PS) II under increasing temperature stress (22‒39 °C, + 1 °C every 15 min), and detected a significant Fv/Fm reduction at lineage-specific temperatures ranging from 28 °C to 34 °C alongside a 40- to 180- fold increase in intraspecific heterogeneity under elevated temperatures (>31 °C). We discovered that the initial Fv/Fm of a cell could predict the same cell's ability to perform PSII photochemistry under moderate temperature stress (<32 °C), suggesting its use as a proxy for measuring the thermal sensitivity among Symbiodiniaceae. In combination, our study highlights the heterogeneous thermal sensitivity among photosynthetic Symbiodiniaceae and adds critical resolution to our understanding of temperature-induced coral bleaching.

Dynamics and Sorption Kinetics of Methanol Monomers and Clusters on Nopinone Surfaces.

Organic-organic interactions play important roles in secondary organic aerosol formation, but the interactions are complex and poorly understood. Here, we use environmental molecular beam experiments combined with molecular dynamics simulations to investigate the interactions between methanol and nopinone, as atmospheric organic proxies. In the experiments, methanol monomers and clusters are sent to collide with three types of surfaces, i.e., graphite, thin nopinone coating on graphite, and nopinone multilayer surfaces, at temperatures between 140 and 230 K. Methanol monomers are efficiently scattered from the graphite surface, whereas the scattering is substantially suppressed from nopinone surfaces. The thermal desorption from the three surfaces is similar, suggesting that all the surfaces have weak or similar influences on methanol desorption. All trapped methanol molecules completely desorb within a short experimental time scale at temperatures of 180 K and above. At lower temperatures, the desorption rate decreases, and a long experimental time scale is used to resolve the desorption, where three desorption components are identified. The fast component is beyond the experimental detection limit. The intermediate component exhibits multistep desorption character and has an activation energy of Ea = 0.18 ± 0.03 eV, in good agreement with simulation results. The slow desorption component is related to diffusion processes due to the weak temperature dependence. The molecular dynamics results show that upon collisions the methanol clusters shatter, and the shattered fragments quickly diffuse and recombine to clusters. Desorption involves a series of processes, including detaching from clusters and desorbing as monomers. At lower temperatures, methanol forms compact cluster structures while at higher temperatures, the methanol molecules form layered structures on the nopinone surface, which are visible in the simulation. Also, the simulation is used to study the liquid-liquid interaction, where the methanol clusters completely dissolve in liquid nopinone, showing ideal organic-organic mixing.

In-Line Analysis of Organ-on-Chip Systems with Sensors: Integration, Fabrication, Challenges, and Potential.

Organ-on-chip systems are promising new in vitro research tools in medical, pharmaceutical, and biological research. Their main benefit, compared to standard cell culture platforms, lies in the improved in vivo resemblance of the cell culture environment. A critical aspect of these systems is the ability to monitor both the cell culture conditions and biological responses of the cultured cells, such as proliferation and differentiation rates, release of signaling molecules, and metabolic activity. Today, this is mostly done using microscopy techniques and off-chip analytical techniques and assays. Integrating in situ analysis methods on-chip enables improved time resolution, continuous measurements, and a faster read-out; hence, more information can be obtained from the developed organ and disease models. Integrated electrical, electrochemical, and optical sensors have been developed and used for chemical analysis in lab-on-a-chip systems for many years, and recently some of these sensing principles have started to find use in organ-on-chip systems as well. This perspective review describes the basic sensing principles, sensor fabrication, and sensor integration in organ-on-chip systems. The review also presents the current state of the art of integrated sensors and discusses future potential. We bring a technological perspective, with the aim of introducing in-line sensing and its promise to advance organ-on-chip systems and the challenges that lie in the integration to researchers without expertise in sensor technology.

A microscopy-compatible temperature regulation system for single-cell phenotype analysis - demonstrated by thermoresponse mapping of microalgae.

This work describes a programmable heat-stage compatible with in situ microscopy for the accurate provision of spatiotemporally defined temperatures to different microfluidic devices. The heat-stage comprises an array of integrated thin-film Joule heaters and resistance temperature detectors (RTDs). External programming of the heat-stage is provided by a custom software program connected to temperature controllers and heater-sensor pairs. Biologically relevant (20-40 °C) temperature profiles can be supplied to cells within microfluidic devices as spatial gradients (0.5-1.5 °C mm-1) or in a time-varying approach via e.g. step-wise or sinusoidally varying profiles with negligible temperature over-shoot. Demonstration of the device is achieved by exposing two strains of the coral symbiont Symbiodinium to different temperature profiles while monitoring their single-cell photophysiology via chlorophyll fluorometry. This revealed that photophysiological responses to temperature depended on the exposure duration, exposure magnitude and strain background. Moreover, thermal dose analysis suggested that cell acclimatisation occurs under longer temperature (6 h) exposures but not under shorter temperature exposures (15 min). As the thermal sensitivity of Symbiodinium mediates the thermal tolerance in corals, our versatile technology now provides unique possibilities to research this interdependency at single cell resolution. Our results also show the potential of this heat-stage for further applications in fields such as biotechnology and ecotoxicology.

Organ-on-a-chip technology: a novel approach to investigate cardiovascular diseases.

The development of organs-on-chip (OoC) has revolutionized in vitro cell-culture experiments by allowing a better mimicry of human physiology and pathophysiology that has consequently led researchers to gain more meaningful insights into disease mechanisms. Several models of hearts-on-chips and vessels-on-chips have been demonstrated to recapitulate fundamental aspects of the human cardiovascular system in the recent past. These 2D and 3D systems include synchronized beating cardiomyocytes in hearts-on-chips and vessels-on-chips with layer-based structures and the inclusion of physiological and pathological shear stress conditions. The opportunities to discover novel targets and to perform drug testing with chip-based platforms have substantially enhanced, thanks to the utilization of patient-derived cells and precise control of their microenvironment. These organ models will provide an important asset for future approaches to personalized cardiovascular medicine and improved patient care. However, certain technical and biological challenges remain, making the global utilization of OoCs to tackle unanswered questions in cardiovascular science still rather challenging. This review article aims to introduce and summarize published work on hearts- and vessels-on chips but also to provide an outlook and perspective on how these advanced in vitro systems can be used to tailor disease models with patient-specific characteristics.

Experimental and Computational Study of Molecular Water Interactions with Condensed Nopinone Surfaces Under Atmospherically Relevant Conditions.

Water and organics are omnipresent in the atmosphere, and their interactions influence the properties and lifetime of both aerosols and clouds. Nopinone is one of the major reaction products formed from ß-pinene oxidation, a compound emitted by coniferous trees, and it has been found in both gas and particle phases in the atmosphere. Here, we investigate the interactions between water molecules and nopinone surfaces by combining environmental molecular beam (EMB) experiments and molecular dynamics (MD) simulations. The EMB method enables detailed studies of the dynamics and kinetics of water interacting with solid nopinone at 170-240 K and graphite coated with a molecularly thin nopinone layer at 200-270 K. MD simulations that mimic the experimental conditions have been performed to add insights into the molecular-level processes. Water molecules impinging on nopinone surfaces are efficiently trapped (≥97%), and only a minor fraction scatters inelastically while maintaining 35-65% of their incident kinetic energy (23.2 ± 1.0 kJ mol-1). A large fraction (60-80%) of the trapped molecules desorbs rapidly, whereas a small fraction (20-40%) remains on the surface for more than 10 ms. The MD calculations confirm both rapid water desorption and the occurrence of strongly bound surface states. A comparison of the experimental and computational results suggests that the formation of surface-bound water clusters enhances water uptake on the investigated surfaces.

Pair Correlation Analysis Maps the Dynamic Two-Dimensional Organization of Natural Killer Cell Receptors at the Synapse.

In living systems, the contact between cells is the basis of recognition, differentiation, and orchestration of an immune response. Obstacles and barriers to biomolecular motion, especially for receptors at cellular synapses, critically control these functions by creating an anisotropic environment. Whereas conventional fluorescence fluctuation methods, such as fluorescence correlation spectroscopy or fluorescence recovery after photobleaching, can only measure the isotropic diffusion of molecules, the two-dimensional pair correlation function (2D-pCF) approach probes the anisotropic paths at different spatial locations within an image, allowing the creation of high-resolution maps that can visualize and quantify how molecules move in a living cell. In this work, we show how the 2D-pCF method maps the environment in cellular synapses as perceived by natural killer (NK) cell receptors. In cultured human HLA null 721.221 cells, 2D-pCF reveals the motion of inhibitory receptor HLA-Cw4-YFP coexpressed with KIR3DL1 to be highly directional around specific loci, while these restrictions were absent in the case of HLA-B51-YFP coexpressed with KIR2DL1. Further, in freshly isolated educated (H-2Dd) and uneducated (MHC-/-) primary murine NK cells, the 2D-pCF method shows significant differences in the paths taken by activating receptor NKp46 and inhibitory receptor Ly49A in educated compared to uneducated cells. Altogether, we demonstrate that the 2D-pCF method is very powerful in informing about the spatial organization of motion in cells. Our data support the hypothesis that flexibility in the spatial arrangement of membrane receptors, that is, the absence of barriers, is crucial for NK cell function.

Understanding water interactions with organic surfaces: environmental molecular beam and molecular dynamics studies of the water-butanol system.

The interactions between water molecules and condensed n-butanol surfaces are investigated at temperatures from 160 to 240 K using the environmental molecular beam experimental method and complementary molecular dynamics (MD) simulations. In the experiments hyperthermal water molecules are directed onto a condensed n-butanol layer and the flux from the surface is detected in different directions. A small fraction of the water molecules scatters inelastically from the surface while losing 60-90% of their initial kinetic energy in collisions, and the angular distributions of these molecules are broad for both solid and liquid surfaces. The majority of the impinging water molecules are thermalized and trapped on the surface, while subsequent desorption is governed by two different processes: one where molecules bind briefly to the surface (residence time τ < 10 µs), and another where the molecules trap for a longer time τ = 0.8-2.0 ms before desorbing. Water molecules trapped on a liquid n-butanol surface are substantially less likely to escape from the surface compared to a solid layer. The MD calculations provide detialed insight into surface melting, adsorption, absorption and desorption processes. Calculated angular distributions and kinetic energy of emitted water molecules agree well with the experimental data. In spite of its hydrophobic tail and enhanced surface organization below the melting temperature, butanol's hydrophilic functional groups are concluded to be surprisingly accessible to adsorbed water molecules; a finding that may be explained by rapid diffusion of water away from hydrophobic surface structures towards more strongly bound conformational structures.

Educated natural killer cells show dynamic movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A.

Educated natural killer (NK) cells have inhibitory receptors specific for self major histocompatibility complex (MHC) class I molecules and kill cancer cells more efficiently than do NK cells that do not have such receptors (hyporesponsive NK cells). The mechanism behind this functional empowerment through education has so far not been fully described. In addition, distinctive phenotypic markers of educated NK cells at the single-cell level are lacking. We developed a refined version of the image mean square displacement (iMSD) method (called iMSD carpet analysis) and used it in combination with single-particle tracking to characterize the dynamics of the activating receptor NKp46 and the inhibitory receptor Ly49A on resting educated versus hyporesponsive murine NK cells. Most of the NKp46 and Ly49A molecules were restricted to microdomains; however, individual NKp46 molecules resided in these domains for shorter periods and diffused faster on the surface of educated, compared to hyporesponsive, NK cells. In contrast, the movement of Ly49A was more constrained in educated NK cells compared to hyporesponsive NK cells. Either disrupting the actin cytoskeleton or adding cholesterol to the cells prohibited activating signaling, suggesting that the dynamics of receptor movements within the cell membrane are critical for the proper activation of NK cells. The faster and more dynamic movement of NKp46 in educated NK cells may facilitate a swifter response to interactions with target cells.

Central regional anaesthesia in patients with aortic stenosis - a systematic review.

INTRODUCTION: Aortic stenosis is a valvular lesion that poses several haemodynamic challenges for the anaesthesiologist. The use of central regional anaesthesia is traditionally regarded as contraindicated in patients with severe aortic stenosis due to its sympatholytic effect, potentially causing loss of vascular tone and ultimately diminished cardiac output. The aim of this paper was to review current literature to find evidence for or against the use of neuroaxial blockade in patients with aortic stenosis. METHODS: We searched PubMed for relevant articles, using the following MeSH terms: "aortic valve stenosis", "epidural anesthesia", "spinal anesthesia" and "epidural analgesia". Only English language literature was included. Papers concerning aortic stenosis and obstetrical anaesthesia were excluded. RESULTS: There are no randomised clinical trials on the subject, and existing literature is extremely sparse. Four retrospective studies and eight case reports counting a total of ten patients were found. All report successful use of neuroaxial blockade in patients with aortic stenosis, without severe haemodynamic alterations. In addition, data indicate that postepidural analgesia improves outcome compared with conventional analgesia. CONCLUSIONS: To the best of our knowledge, there is no clinical evidence supporting the notion that central regional anaesthesia has any adverse effects on patients with aortic stenosis. Carefully managed neuroaxial blockade could become a useful alternative to general anaesthesia in this patient group. However, evidence is sparse and of questionable quality. Large prospective randomised clinical trials are required to establish best practise.

The Dynamics and Kinetics of Water Interactions with a Condensed Nopinone Surface.

Water and organic molecules are omnipresent in the environment, and their interactions are of central importance in many Earth system processes. Here we investigate molecular-level interactions between water and a nopinone surface using an environmental molecular beam (EMB) technique. Nopinone is a major reaction product formed during oxidation of ß-pinene, a prominent compound emitted by coniferous trees, which has been found in both the gas and particle phases of atmospheric aerosol. The EMB method enables detailed studies of the dynamics and kinetics of D2O molecules interacting with a solid nopinone surface at 202 K. Hyperthermal collisions between water and nopinone result in efficient trapping of water molecules, with a small fraction that scatter inelastically after losing 60-80% of their incident kinetic energy. While the majority of the trapped molecules rapidly desorb with a time constant τ less than 10 µs, a substantial fraction (0.32 ± 0.09) form strong bonds with the nopinone surface and remain in the condensed phase for milliseconds or longer. The interactions between water and nopinone are compared to results for recently studied water-alcohol and water-acetic acid systems, which display similar collision dynamics but differ with respect to the kinetics of accommodated water. The results contribute to an emerging surface science-based view and molecular-level description of organic aerosols in the atmosphere.

A novel gas-vacuum interface for environmental molecular beam studies.

Molecular beam techniques are commonly used to obtain detailed information about reaction dynamics and kinetics of gas-surface interactions. These experiments are traditionally performed in vacuum and the dynamic state of surfaces under ambient conditions is thereby excluded from detailed studies. Herein we describe the development and demonstration of a new vacuum-gas interface that increases the accessible pressure range in environmental molecular beam (EMB) experiments. The interface consists of a grating close to a macroscopically flat surface, which allows for experiments at pressures above 1 Pa including angularly resolved measurements of the emitted flux. The technique is successfully demonstrated using key molecular beam experiments including elastic helium and inelastic water scattering from graphite, helium and light scattering from condensed adlayers, and water interactions with a liquid 1-butanol surface. The method is concluded to extend the pressure range and flexibility in EMB studies with implications for investigations of high pressure interface phenomena in diverse fields including catalysis, nanotechnology, environmental science, and life science. Potential further improvements of the technique are discussed.

IL-15, TIM-3 and NK cells subsets predict responsiveness to anti-CTLA-4 treatment in melanoma patients.

Despite the success of immune checkpoint blockade in melanoma, the majority of patients do not respond. We hypothesized that the T and NK cell subset frequencies and expression levels of their receptors may predict responses and clinical outcome of anti-CTLA-4 treatment. We thus characterized the NK and T cell phenotype, as well as serum levels of several cytokines in 67 melanoma patients recruited in Italy and Sweden, using samples drawn prior to and during treatment. Survival correlated with low expression of the inhibitory receptor TIM-3 on circulating T and NK cells prior to and during treatment and with the increased frequency of mature circulating NK cells (defined as CD3-CD56dim CD16+) during treatment. Survival also correlated with low levels of IL-15 in the serum. Functional experiments in vitro demonstrated that sustained exposure to IL-15 enhanced the expression of PD-1 and TIM-3 on both T and NK cells, indicating a causative link between high IL-15 levels and enhanced expression of TIM-3 on these cells. Receptor blockade of TIM-3 improved NK cell-mediated elimination of melanoma metastasis cell lines in vitro. These observations may lead to the development of novel biomarkers to predict patient response to checkpoint blockade treatment. They also suggest that induction of additional checkpoints is a possibility that needs to be considered when treating melanoma patients with IL-15.

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy.

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

Forty years of shunt surgery at Rigshospitalet, Denmark: a retrospective study comparing past and present rates and causes of revision and infection.

OBJECTIVE: The objective of this study is to review our experience of shunt surgery by investigating 40 years of development in terms of rates of revision and infection, shunt survival and risk factors. DESIGN AND PARTICIPANTS: Medical records and operative reports were reviewed retrospectively for all patients who underwent primary shunt surgery at our department in the years 2010 to 2012. All results were compared with a previous study from our department. A mixed population consisting of 434 patients was included. Adults (≥15 years) accounted for 89.9% of all patients and the mean follow-up time was 1.71 years. RESULTS: Overall, 42.6% had a revision of which 65.4% fell within 6 months postoperatively. Low age, high-risk diagnoses and less severe brain injury were associated with a higher risk of revision. One and 5-year shunt survival probabilities were 66.2% (61.5-70.9) and 48.0% (41.1-54.9). Within 4 weeks postoperatively, 3.2% had an infection and overall infection rate was 5.5%. Short duration of surgery and the use of antibiotic prophylaxis were associated with a lower risk of infection. The most frequent causes of revision were valve defects (18.4%) and proximal defects or obstructions (15.7%). Compared to the previous study, no convincing improvement was found with regard to the revision rate (42.6% vs 48.3%, p 0.060) or overall infection rate (5.5% vs 7.4%, p 0.261). CONCLUSIONS: Regardless of changes in patient demographics, techniques and equipment, risk of revision and infection still constitutes a major challenge in shunt surgery. The absence of convincing improvements calls for more studies concerning strategies to reduce complications.

Cytokines Induce Faster Membrane Diffusion of MHC Class I and the Ly49A Receptor in a Subpopulation of Natural Killer Cells.

Cytokines have the potential to drastically augment immune cell activity. Apart from altering the expression of a multitude of proteins, cytokines also affect immune cell dynamics. However, how cytokines affect the molecular dynamics within the cell membrane of immune cells has not been addressed previously. Molecular movement is a vital component of all biological processes, and the rate of motion is, thus, an inherent determining factor for the pace of such processes. Natural killer (NK) cells are cytotoxic lymphocytes, which belong to the innate immune system. By fluorescence correlation spectroscopy, we investigated the influence of cytokine stimulation on the membrane density and molecular dynamics of the inhibitory receptor Ly49A and its ligand, the major histocompatibility complex class I allele H-2D(d), in freshly isolated murine NK cells. H-2D(d) was densely expressed and diffused slowly in resting NK cells. Ly49A was expressed at a lower density and diffused faster. The diffusion rate in resting cells was not altered by disrupting the actin cytoskeleton. A short-term stimulation with interleukin-2 or interferon-α + ß did not change the surface density of moving H-2D(d) or Ly49A, despite a slight upregulation at the cellular level of H-2D(d) by interferon-α + ß, and of Ly49A by IL-2. However, the molecular diffusion rates of both H-2D(d) and Ly49A increased significantly. A multivariate analysis revealed that the increased diffusion was especially marked in a subpopulation of NK cells, where the diffusion rate was increased around fourfold compared to resting NK cells. After IL-2 stimulation, this subpopulation of NK cells also displayed lower density of Ly49A and higher brightness per entity, indicating that Ly49A may homo-cluster to a larger extent in these cells. A faster diffusion of inhibitory receptors could enable a faster accumulation of these molecules at the immune synapse with a target cell, eventually leading to a more efficient NK cell response. It has previously been assumed that cytokines regulate immune cells primarily via alterations of protein expression levels or posttranslational modifications. These findings suggest that cytokines may also modulate immune cell efficiency by increasing the molecular dynamics early on in the response.

Depletion of IL-2 receptor ß-positive cells protects from diabetes in non-obese diabetic mice.

The destruction of ß-cells in type 1 diabetes (T1D) progresses silently until only a minor fraction of the ß-cells remain. A late acting therapy leading to the prevention of further ß-cell killing would therefore be desirable. CD122, the ß chain of the interleukin-2 receptor, is highly expressed on natural killer (NK) cells and on a subpopulation of CD8 T cells. In this study, we have treated non-obese diabetic (NOD) mice with a depleting antibody against CD122. The treatment protected from diabetes, even when initiated just before disease onset. The degree of leukocyte infiltration into islets was unaffected by the treatment, further supporting effectiveness late in the disease process. It effectively removed all NK cells from the spleen, pancreas and pancreatic lymph nodes and abolished NK cell activity. Interestingly, despite the lack of CD122 expression on CD8 T cells in the pancreas, the overall frequency of CD8 cells decreased in this organ, whereas it was unaffected in the spleen. T cells were also still capable to respond against a foreign antigen. Conclusively, targeting of CD122(+) cells could represent a novel treatment strategy against T1D.