Design, fabrication, and characterization of kinetic-inductive force sensors for scanning probe applications.

We describe a transducer for low-temperature atomic force microscopy based on electromechanical coupling due to a strain-dependent kinetic inductance of a superconducting nanowire. The force sensor is a bending triangular plate (cantilever) whose deflection is measured via a shift in the resonant frequency of a high-Q superconducting microwave resonator at 4.5 GHz. We present design simulations including mechanical finite-element modeling of surface strain and electromagnetic simulations of meandering nanowires with large kinetic inductance. We discuss a lumped-element model of the force sensor and describe the role of an additional shunt inductance for tuning the coupling to the transmission line used to measure the microwave resonance. A detailed description of our fabrication is presented, including information about the process parameters used for each layer. We also discuss the fabrication of sharp tips on the cantilever using focused electron beam-induced deposition of platinum. Finally, we present measurements that characterize the spread of mechanical resonant frequency, the temperature dependence of the microwave resonance, and the sensor's operation as an electromechanical transducer of force.

Cross-reactive EBNA1 immunity targets alpha-crystallin B and is associated with multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system, for which and Epstein-Barr virus (EBV) infection is a likely prerequisite. Due to the homology between Epstein-Barr nuclear antigen 1 (EBNA1) and alpha-crystallin B (CRYAB), we examined antibody reactivity to EBNA1 and CRYAB peptide libraries in 713 persons with MS (pwMS) and 722 matched controls (Con). Antibody response to CRYAB amino acids 7 to 16 was associated with MS (OR = 2.0), and combination of high EBNA1 responses with CRYAB positivity markedly increased disease risk (OR = 9.0). Blocking experiments revealed antibody cross-reactivity between the homologous EBNA1 and CRYAB epitopes. Evidence for T cell cross-reactivity was obtained in mice between EBNA1 and CRYAB, and increased CRYAB and EBNA1 CD4+ T cell responses were detected in natalizumab-treated pwMS. This study provides evidence for antibody cross-reactivity between EBNA1 and CRYAB and points to a similar cross-reactivity in T cells, further demonstrating the role of EBV adaptive immune responses in MS development.

Identification of four novel T cell autoantigens and personal autoreactive profiles in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), in which pathological T cells, likely autoimmune, play a key role. Despite its central importance, the autoantigen repertoire remains largely uncharacterized. Using a novel in vitro antigen delivery method combined with the Human Protein Atlas library, we screened for T cell autoreactivity against 63 CNS-expressed proteins. We identified four previously unreported autoantigens in MS: fatty acid-binding protein 7, prokineticin-2, reticulon-3, and synaptosomal-associated protein 91, which were verified to induce interferon-γ responses in MS in two cohorts. Autoreactive profiles were heterogeneous, and reactivity to several autoantigens was MS-selective. Autoreactive T cells were predominantly CD4+ and human leukocyte antigen-DR restricted. Mouse immunization induced antigen-specific responses and CNS leukocyte infiltration. This represents one of the largest systematic efforts to date in the search for MS autoantigens, demonstrates the heterogeneity of autoreactive profiles, and highlights promising targets for future diagnostic tools and immunomodulatory therapies in MS.

Positive Expiratory Pressure Therapy on Oxygen Saturation and Ventilation After Abdominal Surgery: A Randomized Controlled Trial.

Objective: To evaluate the immediate effects of positive expiratory pressure therapy on oxygen saturation and ventilation after abdominal surgery. Background: Positive expiratory pressure therapy to treat postoperative hypoxia is widespread, despite a lack of evidence of effect. Methods: This randomized, sham-controlled, crossover trial investigated adults 1-2 days after abdominal surgery at Umeå University Hospital, Sweden. The intervention was positive expiratory pressure of 10-15 cm H2O. The control was a sham device. The investigations were ended with deep-breathing maneuvers. Outcomes were the gradient of changes in peripheral oxygen saturation and transcutaneous carbon-dioxide partial pressure (PtcCO2). Results: Eighty patients were included and randomized and 76 patients were analyzed. Oxygen saturation increased from a baseline mean of 92% to 95%, P < 0.001, during positive expiratory pressure breathing, while PtcCO2 decreased from a mean of 36 to 33 mm Hg, P < 0.001. This was followed by apnea, oxygen desaturations to a mean of 89%, P < 0.001, and increased PtcCO2 before returning to baseline values. The changes in oxygen saturation and PtcCO2 did not differ from sham breathing or deep-breathing maneuvers. Conclusions: Positive expiratory pressure breathing after abdominal surgery improves oxygen saturation during the maneuver because of hyperventilation, but it is followed by apnea, hypoventilation, and oxygen desaturation. The effect is not different from the expiration to a sham device or hyperventilation. It is time to stop positive expiratory pressure therapy after abdominal surgery, as there is no evidence of effect in previous trials, apart from the adverse effects reported here.

Sensitive detection of antigen-specific T-cells using bead-bound antigen for in vitro re-stimulation.

Reliable and sensitive detection of antigen specific cells is essential in several fields of research, whether it concerns monitoring responses to infectious agents or exploring the auto-antigen repertoire in autoimmune diseases. Identification of these cells is however difficult, especially when the cells often are rare and methods not sensitive, specific or practical enough. We propose a novel method of processing antigens before stimulation of cells which consists of covalently binding protein antigen to superparamagnetic micro-beads and using denaturing washes to remove contaminants. Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated using both cytomegalovirus and tetanus-diphtheria antigen-beads as well as non-antigenic protein-beads as negative control in an IFNγ FluoroSpot assay in order to detect Th1 and CD8+ responses. The responses toward the antigen beads were both antigen specific and sensitive, with a detection threshold of 1 IFNγ producing T-cell per 18,000 PBMCs. •Covalently binding antigen to paramagnetic beads allows for harsh denaturing washes without loss of antigen.•Microbeads are phagocytosed by antigen presenting cells, resulting in efficient uptake, processing and presentation of the antigens.•The method allows the usage of relatively impure starting antigen material and whole PBMC samples without high background levels in follow up cellular assays.

Myelin oligodendrocyte glycoprotein revisited-sensitive detection of MOG-specific T-cells in multiple sclerosis.

Autoreactive CD4+ T-cells are believed to be a main driver of multiple sclerosis (MS). Myelin oligodendrocyte glycoprotein (MOG) is considered an autoantigen, yet doubted in recent years. The reason is in part due to low frequency and titers of MOG autoantibodies and the challenge to detect MOG-specific T-cells. In this study we aimed to analyze T-cell reactivity and frequency utilizing a novel method for detection of antigen-specific T-cells with bead-bound MOG as stimulant. Peripheral blood mononuclear cells (PBMCs) from natalizumab treated persons with MS (n = 52) and healthy controls (HCs) (n = 24) were analyzed by IFNγ/IL-22/IL-17A FluoroSpot. A higher number of IFNγ (P = 0.001), IL-22 (P = 0.003), IL-17A (P < 0.0001) as well as double and triple cytokine producing MOG-specific T-cells were detected in persons with MS compared to HCs. Of the patients, 46.2-59.6% displayed MOG-reactivity. Depletion of CD4+ T-cells or monocytes or blocking HLA-DR completely eliminated the MOG specific response. Anti-MOG antibodies did not correlate with T-cell MOG-responses. In conclusion, we present a sensitive method to detect circulating autoreactive CD4+ T-cells producing IFNγ, IL-22 or IL-17A using MOG as a model antigen. Further, we demonstrate that MOG-specific T-cells are present in approximately half of persons with MS.

Allergens in dog extracts: Implication for diagnosis and treatment.

BACKGROUND: Five to ten percent of the population in affluent countries are allergic to dog. Diagnosis and treatment is based on allergen extracts from natural sources where composition and concentration are poorly defined. OBJECTIVE: We aimed to quantify six dog allergens (Can f 1-6) in commercial skin prick test (SPT) solutions and to determine individual allergen profiles in dogs. METHOD: The allergen content of SPT solutions from five vendors and allergen source material from three anatomical sites were analyzed. Fur and saliva samples were collected from a mixed population of 120 dogs. Can f 1-6 were quantified by inhibition ELISA using purified recombinant or natural allergens and polyclonal or monoclonal antibodies. Allergenicity was analyzed by basophil activation test. RESULTS: Extensive variation in allergen composition was observed in commercial SPT vials resulting in a patient-dependent ability to activate basophils. Extract heterogeneity depended on collection site and allergen composition in individual dogs and source materials. Can f 2 and Can f 6 exhibited low levels in fur and SPT solutions, whereas Can f 4, which was the dominating allergen in fur samples, did not display similar high proportions in SPT solutions. Can f 3 varied most among SPT solutions. CONCLUSION: There is a great variation of dog allergens in natural extracts raising questions of source, sampling, processing and ultimately of standardization and minimum allergen levels for accurate diagnosis and treatment.

Mechanisms of Damage to the Gastrointestinal Tract From Nonsteroidal Anti-Inflammatory Drugs.

Nonsteroidal anti-inflammatory drugs (NSAIDs) can damage the gastrointestinal tract, causing widespread morbidity and mortality. Although mechanisms of damage involve the activities of prostaglandin-endoperoxide synthase 1 (PTGS1 or cyclooxygenase [COX] 1) and PTGS1 (COX2), other factors are involved. We review the mechanisms of gastrointestinal damage induction by NSAIDs via COX-mediated and COX-independent processes. NSAIDs interact with phospholipids and uncouple mitochondrial oxidative phosphorylation, which initiates biochemical changes that impair function of the gastrointestinal barrier. The resulting increase in intestinal permeability leads to low-grade inflammation. NSAID inhibition of COX enzymes, along with luminal aggressors, results in erosions and ulcers, with potential complications of bleeding, protein loss, stricture formation, and perforation. We propose a model for NSAID-induced damage to the gastrointestinal tract that includes these complex, interacting, and inter-dependent factors. This model highlights the obstacles for the development of safer NSAIDs.

Anoctamin 2 identified as an autoimmune target in multiple sclerosis.

Multiple sclerosis (MS) is the most common chronic inflammatory disease of the central nervous system and also is regarded as an autoimmune condition. However, the antigenic targets of the autoimmune response in MS have not yet been deciphered. In an effort to mine the autoantibody repertoire within MS, we profiled 2,169 plasma samples from MS cases and population-based controls using bead arrays built with 384 human protein fragments selected from an initial screening with 11,520 antigens. Our data revealed prominently increased autoantibody reactivity against the chloride-channel protein anoctamin 2 (ANO2) in MS cases compared with controls. This finding was corroborated in independent assays with alternative protein constructs and by epitope mapping with peptides covering the identified region of ANO2. Additionally, we found a strong interaction between the presence of ANO2 autoantibodies and the HLA complex MS-associated DRB1*15 allele, reinforcing a potential role for ANO2 autoreactivity in MS etiopathogenesis. Furthermore, immunofluorescence analysis in human MS brain tissue showed ANO2 expression as small cellular aggregates near and inside MS lesions. Thus this study represents one of the largest efforts to characterize the autoantibody repertoire within MS. The findings presented here demonstrate that an ANO2 autoimmune subphenotype may exist in MS and lay the groundwork for further studies focusing on the pathogenic role of ANO2 autoantibodies in MS.

Designing a multimer allergen for diagnosis and immunotherapy of dog allergic patients.

BACKGROUND: Dog dander extract used for diagnosis and allergen-specific immunotherapy is often of variable and of poor quality. OBJECTIVE: To assemble four well-established dog allergen components into one recombinant folded protein for improved diagnosis and vaccination of allergy to dog. METHODS: A linked molecule, comprising the four dog lipocalin allergens Can f 1, Can f 2, Can f 4 and Can f 6 was constructed. The tetrameric protein was structurally characterized by small angle X-ray scattering, and compared with each single recombinant lipocalin allergen or an equimolar mix of the four allergens by analytical size exclusion chromatography, circular dichroism, allergen-specific IgE in serum by ELISA and allergen-dependent capacity to activate basophils. The immunogenicity of the fusion protein was evaluated in immunized mice by assessing splenocyte proliferation and antibody production. RESULTS: The linked tetrameric construct was produced as a soluble fusion protein, with the specific folds of the four individual allergens conserved. This multi-allergen molecule was significantly more efficient (p<0.001) than each single recombinant allergen in binding to dog-specific IgE, and the epitope spectrum was unaffected compared to an equimolar mix of the four allergens. Basophil degranulation revealed that the biologic activity of the linked molecule was retained. Immunization of mice with the linked construct induced comparable allergen-specific IgG responses with blocking capacity towards all included allergens and generated comparably low T-cell responses. CONCLUSION: We provide the first evidence for a linked recombinant molecule covering the major dog allergens for potential use in diagnostics and allergy vaccination of dog allergic patients.

Extraction of explosives from soil followed by gas chromatography-mass spectrometry analysis with negative chemical ionization.

A new, simple and accurate method for extraction of explosives from soil was developed and validated. The method includes one hour gentle extraction of compounds from soil in acetonitrile:dichloromethane 50:50 at 30°C. Further analysis was made with GC-MS using cool on-column injection and negative chemical ionization. The method increased the recovery of the more volatile products, generated higher accuracy and was extensively time-saving compared to the conventional EPA (US Environmental Protection Agency) 8330 method. Applications are demonstrated on commercial reference materials.

Production of a truncated soluble human semicarbazide-sensitive amine oxidase mediated by a GST-fusion protein secreted from HEK293 cells.

Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in several human conditions such as congestive heart failure, diabetes mellitus, and inflammation. The reactive aldehydes and hydrogen peroxide produced by SSAO have been suggested to contribute to the progression of vascular complications associated with these conditions. In addition, SSAO activity has been shown to be involved in the leukocyte extravasation process at sites of inflammation. To facilitate characterization and development of specific and selective inhibitors of SSAO, we have developed a method for production of recombinant human SSAO. The extracellular region (residues 29-763) of human SSAO was expressed in HEK293 cells in fusion with a mutated Schistosoma japonicum glutathione S-transferase (GST) and secreted to the culture medium. The mutGST-SSAO fusion protein was purified in a single step by glutathione-affinity chromatography followed by site-specific cleavage using a GST-3C protease fusion protein to remove the mutGST fusion partner. A second glutathione-affinity chromatography step was then used to capture both the mutGST fusion partner and the GST-3C protease, resulting in milligram quantities of pure, enzymatically active, and soluble recombinant human SSAO.

Determination and characterization of organic explosives using porous graphitic carbon and liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

A new LC-MS method for the determination and characterization of three groups of commonly used organic explosives (nitroaromatic compounds, cyclic nitroamines and nitrate esters) was developed using a porous graphitic carbon (PGC) (Hypercarb) column. Twenty-one different explosive-related compounds--including 2,4,6-trinitrotoluene, its by-products and its degradation products--were chromatographically separated in a single analysis. This efficient separation facilitates the identification of the manufacturer of the explosive using the identified analytes as a fingerprint. A final, conclusive identification of the analytes can be obtained using LC-MS equipped with an atmospheric pressure chemical ionization (APCI) interface. Solvent effects on chromatographic behaviour were investigated, as were the effects of solvent mixtures and mobile phase additives. The number and the relative positions of the nitro groups within analyte molecules influence their order of elution; these effects were investigated. The data thus generated can be interpreted to support a hypothesis concerning the retention mechanism of nitro-containing compounds when using PGC. Limits of detection ranged from 0.5 to 41.2 ng. The new methodology described herein improves the sensitivity and selectivity of explosive detection. The effectiveness of the method is demonstrated by the analysis of soil samples containing explosives residue from test fields in Sweden and Afghanistan.

On-line coupling of supercritical fluid extraction with high-performance liquid chromatography for the determination of explosives in vapour phases.

An analytical method for determining nitroaromatic explosives in vapour phases is presented. Samples were collected by pumping air through glass fibre filters and polyurethane foam adsorbents, and an on-line extraction system combining supercritical fluid extraction (SFE) and high-performance liquid chromatography (HPLC) was developed. This allows analytes to be transferred from the adsorbent to the HPLC system via a porous graphitic carbon trap. When using gradient elution with a suitable mobile phase, most of the nitroaromatic isomers tested were separated. The proposed method is fully automated, allows a complete analysis to be processed in less than 30 min, and it is compatible with most of the organic solvents commonly used as SFE modifiers or additives. The method has been applied to the analysis of real samples obtained from headspace sampling of military-grade 2,4,6-trinitrotoluene and has been shown to constitute a promising alternative for assessing whether areas are mined in landmine-clearing operations.