T-cell specific in vivo gene delivery with DART-AAVs targeted to CD8.

One of the biggest challenges for in vivo gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs (DART-AAVs) displaying DARPins specific for human and murine CD8. Insertion of DARPins into the GH2/GH3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in high selectivity for CD8-positive T cells with unimpaired gene delivery activity. Remarkably, the capsid core structure was unaltered with protruding DARPins detectable. In complex primary cell mixtures, including donor blood or systemic injections into mice, the CD8-targeted AAVs were by far superior to unmodified AAV2 and AAV6 in terms of selectivity, target cell viability and gene transfer rates. In vivo, up to 80% of activated CD8+ T cells were hit upon a single vector injection into conditioned humanized or immunocompetent mice. While gene transfer rates decreased significantly under non-activated conditions, genomic modification selectively in CD8+ T cells was still detectable upon Cre delivery into indicator mice. In both mouse models, selectivity for CD8+ T cells was close to absolute with exceptional detargeting from liver. The CD8-AAVs described here expand strategies for immunological research and in vivo gene therapy options.

Magnetic order and disorder environments in superantiferromagnetic [Formula: see text] nanoparticles.

Magnetic nanoparticles exhibit two different local symmetry environments, one ascribed to the core and one corresponding to the nanoparticle surface. This implies the existence of a dual spin dynamics, leading to the presence of two different magnetic arrangements governed by different correlation lengths. In this work, two ensembles of [Formula: see text] nanoparticles with mean sizes of 18 nm and 13 nm have been produced to unravel the magnetic couplings established among the magnetic moments located within the core and at the nanoparticle surface. To this end, we have combined neutron diffraction measurements, appropriate to investigate magnetically-ordered spin arrangements, with time-dependent macroscopic AC susceptibility measurements to reveal memory and aging effects. The observation of the latter phenomena are indicative of magnetically-frustrated states. The obtained results indicate that, while the [Formula: see text] magnetic moments located within the nanoparticle core keep the bulk antiferromagnetic commensurate structure in the whole magnetic state, the correlations among the surface spins give rise to a collective frustrated spin-glass phase. The interpretation of the magnetic structure of the nanoparticles is complemented by specific-heat measurements, which further support the lack of incommensurability in the nanoparticle state.

Small-angle neutron scattering by spatially inhomogeneous ferromagnets with a nonzero average uniaxial anisotropy.

Micromagnetic small-angle neutron scattering theory is well established for analyzing spin-misalignment scattering data of bulk ferromagnets. Here, this theory is extended to allow for a global uniaxial magnetic anisotropy (texture) of the material, in addition to the already included random zero-average local anisotropy. Macroscopic cross sections and spin-misalignment response functions are computed analytically for several practically relevant mutual anisotropy and external magnetic field orientations in both parallel and perpendicular scattering geometries for field magnitudes both above and below the rotational saturation. Some of these expressions are tested on published experimental data of magnetic-field-annealed Vitroperm and plastically deformed Ni, allowing determination of the corresponding global uniaxial anisotropy quality factors.

Immune checkpoint inhibitor-induced Type 1 diabetes: a systematic review and meta-analysis.

AIM: To conduct a systematic review and meta-analysis to understand the timing and factors associated with anti-programmed cell death protein-1 (PD-1)/anti-programmed cell death protein-1 ligand (PD-L1) inhibitor-induced Type 1 diabetes. METHODS: We searched MEDLINE, EMBASE, SCOPUS and Cochrane databases (August 2000-2018) for studies of any design on immune checkpoint inhibitors. A total of 71 cases were reviewed from 56 publications. Comparisons were made using Fisher's exact and Student's t-tests. RESULTS: The mean ± sd age at Type 1 diabetes presentation was 61.7±12.2 years, 55% of cases were in men, and melanoma (53.5%) was the most frequent cancer. The median time to Type 1 diabetes onset was 49 (5-448) days with ketoacidosis in 76% of cases. The average ± sd HbA1c concentration was 62 ± 0.3 mmol/mol (7.84±1.0%) at presentation. All cases had insulin deficiency and required permanent exogenous insulin treatment. Half of the cases had Type 1 diabetes-associated antibodies at presentation, and those with antibodies had a more rapid onset (P=0.005) and higher incidence of diabetic ketoacidosis (P=0.02) compared to people without antibodies. CONCLUSIONS: Many people developed Type 1 diabetes within 3 months of initial PD-1/PD-L1 inhibitor exposure. People presenting with Type 1 diabetes-associated antibodies had a more rapid onset and higher incidence of ketoacidosis than those without antibodies. Healthcare providers caring for people receiving these state-of-the-art therapies need to be aware of this potential severe adverse event.

Fluoride Ion Release of Self-Adhesive Resin Cements and Their Potential to Inhibit In Situ Enamel and Dentin Demineralization.

OBJECTIVE: This study evaluated in situ the potential of a glass ionomer and self-adhesive resin cements to inhibit enamel and dentin demineralization around indirect restorations exposed to cariogenic challenge. The cumulative fluoride release (CFR) of materials was measured in water and acid. METHODS: Seventy blocks cut from human molars received two indirect composite restorations (one in enamel and another in dentin) luted with Ketac Cem EasyMix (GIC, positive control), SeT (SeT), Maxcem Elite (Max), Smart Cem2 (Smart), and RelyX Unicem 2 (Unicem2). Fourteen volunteers wore palatal appliances containing five blocks exposed to a cariogenic challenge (20% sucrose solution, eight times per day, seven days). Knoop microhardness (KH) at two distances from the margins and three depths from the outer surface determined enamel and dentin demineralization. Disc-shape specimens of materials were immersed in daily-replaced deionized water or lactic acid solutions. KH and CFR data were analyzed by analysis of variance, Games-Howell test, and Tukey test (α=0.05). RESULTS: The overall KH ranking was GIC > SeT > Max > Smart = Unicem2 in both enamel and dentin (">" means p<0.05). SeT was the only resin cement that resulted in enamel and dentin KH comparable to that of GIC at most distances and depths. In water, CFR rank of materials was GIC > SeT = Max > Smart = Unicem2. In acid, the rank was similar, except that Set was significantly superior to Max. CONCLUSION: SeT inhibited demineralization in enamel and dentin quite comparably to GIC. All resin cements released lower cumulative amounts of fluoride than the glass ionomer cement.

Histones link inflammation and thrombosis through the induction of Weibel-Palade body exocytosis.

Essentials Dysregulated DNA and histone release can promote pathological immunothrombosis. Weibel-Palade bodies (WPBs) are sentinel-like organelles that respond to proinflammatory stimuli. Histones induce WPB exocytosis in a caspase, calcium and charge-dependent mechanism. A targetable axis may exist between DNA/histones and WPBs in inflammation and immunothrombosis. SUMMARY: Background Damage-associated molecular patterns (DAMPs), including molecules such as DNA and histones, are released into the blood following cell death. DAMPs promote a procoagulant phenotype through enhancement of thrombin generation and platelet activation, thereby contributing to immunothrombosis. Weibel-Palade bodies (WPBs) are dynamic endothelial cell organelles that contain procoagulant and proinflammatory mediators, such as von Willebrand factor (VWF), and are released in response to cell stresses. VWF mediates platelet adhesion and aggregation, and has been implicated as a procoagulant component of the innate immune response. Objective To determine the influence of histones and DNA on WPB release, and characterize their association in models of inflammation. Methods We treated C57BL/6J mice and cultured endothelial cells with histones (unfractionated, lysine-rich or arginine-rich) and DNA, and measured WPB exocytosis. We used inhibitors to determine a mechanism of histone-induced WPB release in vitro. We characterized the release of DAMPs and WPBs in response to acute and chronic inflammation in human and murine models. Results and conclusions Histones, but not DNA, induced the release of VWF (1.46-fold) from WBPs and caused thrombocytopenia (0.74-fold), which impaired arterial thrombus formation in mice. Histones induced WPB release from endothelial cells in a caspase-dependent, calcium-dependent and charge-dependent manner, and promoted platelet capture in a flow chamber model of VWF-platelet string formation. The levels of DAMPs and WPB-released proteins were elevated during inflammation, and were positively correlated in chronic inflammation. These studies showed that DAMPs can regulate the function and level of VWF by inducing its release from endothelial WPBs. This DAMP-WPB axis may propagate immunothrombosis associated with inflammation.

Similar biological activity in skin prick test for Oralair® (8200 BAU) and Grazax® (6200 BAU) reinforces effective SLIT dosing level.

In Europe, allergen extracts are standardized based on skin prick wheal size in 20-30 allergic subjects. To understand the biological activity of clinically effective Sublingual immunotherapy, we used this method to determine the biological activity of solution and tablet Timothy grass pollen (TIM) extracts, compared to an FDA-approved extract (Reference) of 10 000 BAU/ml. Blinded, quadruplicate skin prick tests with concentrate and three serial half-log dilutions allowed the construction of a semilogarithmic regression line per extract. Bioequivalent allergy units (BAU) values were obtained from the comparison with reference. Extracts and dilutions showed a neat linear dose response (all: R2 > 0.98) in 33 rhinitis patients. Relative potencies: Staloral® 12 000 BAU/ml, Soluprick® 10 300 BAU/ml, Oralair® 8200 BAU, and Grazax® 6200 BAU. Even though all extract concentrates differed in wheal size (P = 0.01-0.001), Grazax® producing a 25% smaller wheal size than Oralair® , and the biological activity of these clinically effective TIM tablets led in the same range (6200-8200 BAU; 0.92-1.23 cm2 ). SLIT dose-finding studies for other pollens might start with allergen extracts producing 1.1 cm2 wheal surface.

An experimental study on the influence of trace impurities on ionization of atmospheric noble gas dielectric barrier discharges.

While the influence of trace impurities in noble gas discharges is well established in theoretical work, experimental approaches are difficult. Particularly the effects of trace concentrations of N2 on He discharges are complicated to investigate due to the fact that for He 5.0 the purity of He is only 99.999%. This corresponds to a residual concentration of 10 ppm, thereof 3 ppm of N2, in He. Matters are made difficult by the fact that He DBD plasmajets are normally operated under an ambient atmosphere, which has a high abundance of N2. This work tackles these problems from two sides. The first approach is to operate a DBD plasmajet under a quasi-controlled He atmosphere, therefore diminishing the effect of atmospheric N2 and making a defined contamination with N2 possible. The second approach is using Ar as the operating gas and introducing propane (C3H8) as a suitable substitute impurity like N2 in He. As will be shown both discharges in either He or Ar, with their respective impurity show the same qualitative behaviour.

Validation of a rapid type 1 diabetes autoantibody screening assay for community-based screening of organ donors to identify subjects at increased risk for the disease.

The Network for Pancreatic Organ donors with Diabetes (nPOD) programme was developed in response to an unmet research need for human pancreatic tissue obtained from individuals with type 1 diabetes mellitus and people at increased risk [i.e. autoantibody (AAb)-positive] for the disease. This necessitated the establishment of a type 1 diabetes-specific AAb screening platform for organ procurement organizations (OPOs). Assay protocols for commercially available enzyme-linked immunosorbent assays (elisas) determining AAb against glutamic acid decarboxylase (GADA), insulinoma-associated protein-2 (IA-2A) and zinc transporter-8 (ZnT8A) were modified to identify AAb-positive donors within strict time requirements associated with organ donation programmes. These rapid elisas were evaluated by the international islet AAb standardization programme (IASP) and used by OPO laboratories as an adjunct to routine serological tests evaluating donors for organ transplantation. The rapid elisas performed well in three IASPs (2011, 2013, 2015) with 98-100% specificity for all three assays, including sensitivities of 64-82% (GADA), 60-64% (IA-2A) and 62-68% (ZnT8A). Since 2009, nPOD has screened 4442 organ donors by rapid elisa; 250 (5·6%) were identified as positive for one AAb and 14 (0.3%) for multiple AAb with 20 of these cases received by nPOD for follow-up studies (14 GADA+, two IA-2A(+) , four multiple AAb-positive). Rapid screening for type 1 diabetes-associated AAb in organ donors is feasible, allowing for identification of non-diabetic, high-risk individuals and procurement of valuable tissues for natural history studies of this disease.

Aging and ABO blood type influence von Willebrand factor and factor VIII levels through interrelated mechanisms.

UNLABELLED: Essentials von Willebrand factor (VWF) and factor VIII (FVIII) levels are modulated by age and ABO status. The effect of aging and ABO blood type on VWF and FVIII was assessed in 207 normal individuals. Aging and ABO blood type showed combined and bidirectional influences on VWF and FVIII levels. Aging and ABO blood type influence VWF levels through both secretion and clearance mechanisms. SUMMARY: Background The effect of aging and ABO blood type on plasma levels of von Willebrand factor (VWF) and factor VIII (FVIII) have been widely reported; however, a comprehensive analysis of their combined effect has not been performed and the mechanisms responsible for the age-related changes have not been determined. Objectives To assess the influence of aging and ABO blood type on VWF and FVIII levels, and to evaluate the contribution of VWF secretion and clearance to the age-related changes. Methods A cross-sectional observational study was performed in a cohort of 207 normal individuals, whose levels of VWF, FVIII, VWF propeptide (VWFpp), VWFpp/VWF:Ag ratio and blood type A antigen content on VWF (A-VWF) were quantified. Results Aging and ABO blood type exerted interrelated effects on VWF and FVIII plasma levels, because the age-related increase in both proteins was significantly higher in type non-O individuals (ß = 0.011 vs. 0.005). This increase with age in non-O subjects drove the differences between blood types in VWF levels, as the mean difference increased from 0.13 U/mL in the young to 0.57 U/mL in the old. Moreover, A-VWF was associated with both VWF antigen (ß = 0.29; 95% confidence interval [CI], 0.09, 0.50) and VWF clearance (ß = -0.15; 95% CI, -0.25, -0.06). We also documented an effect of ABO blood type on VWF secretion with aging, as old individuals with blood type non-O showed higher levels of VWFpp (mean difference 0.29 U/mL). Conclusions Aging and ABO blood type have an interrelated effect on VWF and FVIII levels, where the effect of one is significantly influenced by the presence of the other.

Reajustement des charges des appareils de traction cervicale au cours de l'arthrose cervicale a Kinshasa

Objectif : Reajuster les charges des appareils de traction cervicale par la mesure des ecarts entre les charges affichees et celles accrochees; en vue d'une correction eventuelle par rapport au poids du patient.Methodes : C'est une etude de cas portant sur 6 materiels de traction cervicale appartenant aux 4 centres de reeducation et aux Cliniques Universitaires de Kinshasa; de janvier 2005 a decembre 2010 dans la ville de Kinshasa. Le reajustement des charges de ces appareils de traction a permis de verifier a l'aide d'un dynamometre intercale entre la sangle de traction et l'autre bout de la corde; la concordance ou non des charges accrochees au bout libre de l'appareil et celles affichees au dynamometre lors de la traction. Nous avons calcule la variation de la charge de la tete sur la colonne cervicale en fonction du degre de la lordose et du niveau de la vertebre. Etant donne que la tete pese en moyenne 8;1% du poids corporel; la charge a accrocher devait etre superieure au poids de la tete pour exercer une reelle traction sur la colonne cervicale.Resultats : Le reajustement des charges des appareils de traction cervicale dans ces cinq services a montre des ecarts importants entre les charges accrochees et celles affichees au dynamometre; compris entre 1;5 et 10 kg. Il a ete aussi mis en evidence l'importance de la lordose cervicale et celle du niveau de la vertebre dans l'augmentation de la charge sur la colonne; les variations de charges obeissant a une progression arithmetique.Conclusion : Nos resultats peuvent donc permettre; dans un contexte des pays a faibles revenus; d'aider les praticiens a proceder au reajustement des appareils de traction avant la prise en charge

Magnetic field dependent small-angle neutron scattering on a Co nanorod array: evidence for intraparticle spin misalignment.

The structural and magnetic properties of a cobalt nanorod array have been studied by means of magnetic field dependent small-angle neutron scattering (SANS). Measurement of the unpolarized SANS cross section dΣ/dΩ of the saturated sample in the two scattering geometries where the applied magnetic field H is either perpendicular or parallel to the wavevector ki of the incoming neutron beam allows one to separate nuclear from magnetic SANS, without employing the usual sector-averaging procedure. The analysis of the SANS data in the saturated state provides structural parameters (rod radius and centre-to-centre distance) that are in good agreement with results from electron microscopy. Between saturation and the coercive field, a strong field dependence of dΣ/dΩ is observed (in both geometries), which cannot be explained using the conventional expression of the magnetic SANS cross section of magnetic nanoparticles in a homogeneous nonmagnetic matrix. The origin of the strong field dependence of dΣ/dΩ is believed to be related to intradomain spin misalignment, due to magnetocrystalline and magnetoelastic anisotropies and magnetostatic stray fields.

Spin-coating process evolution and reproducibility for power-law fluids.

A distinct development of an exact analytical solution for power-law fluids during the spin-coating process is presented for temporal and spatial thickness evolution, after steady state conditions are attained. This solution leads to the definition of a characteristic time, related to the memory of the initial thickness profile. Previously obtained experimental data, for several rotation speeds and carboxymetilcellulose concentrations in water, are quantitatively analyzed through the evaluation of their characteristic times and compared with theoretical predictions, thus allowing better understanding of thickness profile evolution and of process reproducibility.

Critical behaviour of nanocrystalline gadolinium: evidence for random uniaxial dipolar universality class.

We report on how nanocrystal size affects the critical behaviour of the rare-earth metal Gd near the ferromagnetic-to-paramagnetic phase transition. The asymptotic critical behaviour of the coarse-grained polycrystalline sample (with an average crystallite size of L≅100 µm) is that of a (pure) uniaxial dipolar ferromagnet, as is the case with single crystal Gd, albeit the width of the asymptotic critical region (ACR) is reduced. As the grain size approaches ∼30 nm, the ACR is so narrow that it could not be accessed in the present experiments. Inaccessibly narrow ACR for L ∼ 30 nm and continuous increase in the width of the ACR as L decreases from 16 to 9.5 nm basically reflect a crossover to the random uniaxial dipolar fixed point caused by the quenched random exchange disorder prevalent at the internal interfaces (grain boundaries).

Objectifying the conjunctival provocation test: photography-based rating and digital analysis.

BACKGROUND: Patients with allergic rhinoconjunctivitis are susceptible to both nasal and ocular symptoms. The conjunctival provocation test (CPT) is an established diagnostic procedure used in allergic rhinoconjunctivitis, particularly to document a patient's current reactivity to allergens. To date, there are no international guidelines defining the CPT. No approved evaluation method exists for interpreting CPT results. This paper aims to establish the digital analysis of macroimages as an objective, validated and standardized method for interpreting CPT results. METHODS: In a clinical immunotherapy trial with 155 patients, treatment progress was documented based on the CPT. Local investigators used a symptom score to grade tearing, reddening and the patients' subjective perception of symptoms (mucosal irritation). A central observer rated conjunctival hyperemia via digital photography. Digital image analysis software was utilized to determine conjunctival hyperemia. RESULTS: Spearman's correlation between the local investigators' and the central observer's ratings was r = 0.729 (p < 0.001); the percentage of total agreement was 48% (based on 739 photos). Digital image analysis (based on 48 photos) had a high percentage of total agreement with the central observer's ratings (69%) but a low percentage of total agreement with the investigators' ratings (38%). The corresponding correlations were r = 0.264 and 0.064, respectively. CONCLUSION: Photography-based rating by a central observer may represent a valuable supplement to the local investigator's assessment for making an objective evaluation of CPT results. Digital image analysis possesses the potential of being an objective evaluation method compared to the wide-spread subjective evaluation by the investigators.

Current density and conductivity dependent electroporation of Escherichia coli C600.

Transformation and surviving of E. coli C600 have been evaluated in dependence on the electric field strength and current density by changing the conductivity of the bacteria suspension. In this context the impact of making bacteria electrocompetent and the addition of NaCl solution was examined. Transformation efficiency declines with increasing conductivity of the suspension. When washing bacteria differently, the transformation efficiency correlates with the number of survivors. In contrary, adding different concentrations of NaCl has no effect on the surviving of E. coli C600. In dependence on the electric field strength, the transformation efficiency shows no effect on changing the conductivity. Regarding the transformation efficiency in dependence of the current density, a clear shift of the transformation maximum was observed. For higher conductivities, higher current densities are needed to reach the transformation maximum.

Teplizumab treatment may improve C-peptide responses in participants with type 1 diabetes after the new-onset period: a randomised controlled trial.

AIMS/HYPOTHESIS: Type 1 diabetes results from a chronic autoimmune process continuing for years after presentation. We tested whether treatment with teplizumab (a Fc receptor non-binding anti-CD3 monoclonal antibody), after the new-onset period, affects the decline in C-peptide production in individuals with type 1 diabetes. METHODS: In a randomised placebo-controlled trial we treated 58 participants with type 1 diabetes for 4-12 months with teplizumab or placebo at four academic centres in the USA. A central randomisation centre used computer generated tables to allocate treatments. Investigators, patients, and caregivers were blinded to group assignment. The primary outcome was a comparison of C-peptide responses to a mixed meal after 1 year. We explored modification of treatment effects in subgroups of patients. RESULTS: Thirty-four and 29 subjects were randomized to the drug and placebo treated groups, respectively. Thirty-one and 27, respectively, were analysed. Although the primary outcome analysis showed a 21.7% higher C-peptide response in the teplizumab-treated group (0.45 vs 0.371; difference, 0.059 [95% CI 0.006, 0.115] nmol/l) (p = 0.03), when corrected for baseline imbalances in HbA(1c) levels, the C-peptide levels in the teplizumab-treated group were 17.7% higher (0.44 vs 0.378; difference, 0.049 [95% CI 0, 0.108] nmol/l, p = 0.09). A greater proportion of placebo-treated participants lost detectable C-peptide responses at 12 months (p = 0.03). The teplizumab group required less exogenous insulin (p < 0.001) but treatment differences in HbA(1c) levels were not observed. Teplizumab was well tolerated. A subgroup analysis showed that treatment benefits were larger in younger individuals and those with HbA(1c) <6.5% at entry. Clinical responders to teplizumab had an increase in circulating CD8 central memory cells 2 months after enrolment compared with non-responders. CONCLUSIONS/INTERPRETATIONS: This study suggests that deterioration in insulin secretion may be affected by immune therapy with teplizumab after the new-onset period but the magnitude of the effect is less than during the new-onset period. Our studies identify characteristics of patients most likely to respond to this immune therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00378508 FUNDING: This work was supported by grants 2007-502, 2007-1059 and 2006-351 from the JDRF and grants R01 DK057846, P30 DK20495, UL1 RR024139, UL1RR025780, UL1 RR024131 and UL1 RR024134 from the NIH.

Traumatic wound microbiome workshop.

On May 9-10, 2011, the Walter Reed Army Institute of Research, as the Army Center of Excellence for Infectious Disease, assembled over a dozen leaders in areas related to research into the communities of microorganisms which colonize and infect traumatic wounds. The objectives of the workshop were to obtain guidance for government researchers, to spur research community involvement in the field of traumatic wound research informed by a microbiome perspective, and to spark collaborative efforts serving the Wounded Warriors and similarly wounded civilians. During the discussions, it was made clear that the complexity of these infections will only be met by developing a new art of clinical practice that engages the numerous microbes and their ecology. It requires the support of dedicated laboratories and technologists who advance research methods such as community sequencing, as well as the kinds of data analysis expertise and facilities. These strategies already appear to be bearing fruit in the clinical management of chronic wounds. There are now funding announcements and programs supporting this area of research open to extramural collaborators.

Optical monitoring for power law fluids during spin coating.

Optical monitoring is applied, in situ and in real time, to non-newtonian, power law fluids in the spin coating process. An analytical exact solution is presented for thickness evolution that well fits to most measurement data. As result, typical rheological parameters are obtained for several CMC (carboximetilcelullose) concentrations and rotation speeds. Optical monitoring thus precisely indicates applicability of the model to power law fluids under spin coating.

Neutron spin-flip scattering of nanocrystalline cobalt.

We report results of longitudinal (one-dimensional) neutron polarization analysis on polycrystalline bulk Co with an average crystallite size of D = 10 nm. The spin-flip small-angle neutron scattering (SANS) data are analyzed in the approach-to-saturation regime within the framework of micromagnetic theory. In particular, we provide a closed-form expression for the spin-flip SANS cross section [Formula: see text]. From the data analysis, we find a room-temperature value of A = (2.6 ± 0.1) × 10( - 11) J m( - 1) for the exchange-stiffness constant, which agrees well with earlier data.