Mature DC from skin and skin-draining LN retain the ability to acquire and efficiently present targeted antigen

Skin-draining LN contain several phenotypically distinguishable DC populations, which may be immature or mature. Mature DC are generally considered to have lost the capacity to acquire and present newly encountered Ag. Using antibody-opsonized liposomes as Ag carriers, we show that mature DC purified from skin explants are able to efficiently capture liposomes, process Ag encapsulated within them and activate Ag-specific CD4(+) T cells. Explant DC from mice with Langerhans cells (LC) expressing the primate diphtheria toxin receptor that were exposed to diphtheria toxin in vivo presented Ag as well as explant DC from wild-type mice, indicating that LC are not required and dermal DC are probably responsible for this presentation. We further show that all DC subtypes from LN that capture opsonized Ag are capable of cross-presenting it to CD8(+) T cells. Induction of additional maturation in vivo by LPS or treatment with double-stranded RNA did not alter the Ag presentation capacity of the skin or LN DC subtypes. These results suggest that mature DC present in skin-draining LN may play an important role in the induction of primary and/or secondary immune responses against Ag delivered to the LN that they take up by receptor-mediated endocytosis.     

Long-term effectiveness and safety of interleukin-1 receptor antagonist (anakinra) in Schnitzler's syndrome: A french multicenter study

The aim of this study is to assess the long-term effectiveness and safety of IL1Ra in Schnitzler syndrome (SchS). Between 2010 and 2012, we performed a nationwide survey among French internal medicine departments to identify SchS patients. We retrospectively analyzed the long-term efficacy and safety of IL1Ra and the outcome of patients that did not receive this treatment. Forty-two patients were included in the study, 29 of whom received IL1Ra. The mean age at disease onset was 59.9 years. Disease manifestations included urticaria (100%), fever (76%), bone/joint pain (86%), bone lesions (76%), anemia (67%), and weight loss (60%). The monoclonal gammopathy was overwhelmingly IgM kappa (83%). The mean follow-up was 9.5 years (range: 1.6-35). Two patients developed Waldenström's macroglobulinemia and one developed AA amyloidosis. All of the 29 patients who received IL1Ra responded dramatically. After a median follow-up of 36 months (range: 2-79), the effectiveness remained unchanged. All patients remained on anti-IL-1 therapy. Twenty-four patients (83%) went into complete remission and five (17%) into partial remission. Three patients experienced grade 3-4 neutropenia. Six patients developed severe infections. No lymphoproliferative diseases occurred while on IL1Ra. When last seen, all patients without anakinra had an active disease with variable impact on their quality of life. Their median corticosteroids dosage was 6 mg/d (range: 5-25). IL1Ra is effective in SchS, with a sharp corticosteroid-sparing effect. Treatment failures should lead to reconsider the diagnosis. Long-term follow-up revealed no loss of effectiveness and a favorable tolerance profile. The long-term effects on the risk of hemopathy remain unknown.     

Biocompatible surface functionalization architecture for a diamond quantum sensor

Quantum metrology enables some of the most precise measurements. In the life sciences, diamond-based quantum sensing has led to a new class of biophysical sensors and diagnostic devices that are being investigated as a platform for cancer screening and ultrasensitive immunoassays. However, a broader application in the life sciences based on nanoscale NMR spectroscopy has been hampered by the need to interface highly sensitive quantum bit (qubit) sensors with their biological targets. Here, we demonstrate an approach that combines quantum engineering with single-molecule biophysics to immobilize individual proteins and DNA molecules on the surface of a bulk diamond crystal that hosts coherent nitrogen vacancy qubit sensors. Our thin (sub–5 nm) functionalization architecture provides precise control over the biomolecule adsorption density and results in near-surface qubit coherence approaching 100 μs. The developed architecture remains chemically stable under physiological conditions for over 5 d, making our technique compatible with most biophysical and biomedical applications.

Recent developments in quantum engineering and diamond processing have brought us considerably closer to performing nanoscale NMR and electron paramagnetic resonance (EPR) spectroscopy of small ensembles and even individual biomolecules. Notably, these advances have enabled the detection of the nuclear spin noise from a single ubiquitin protein (1) and the probing of the EPR spectrum of an individual paramagnetic spin label conjugated to a protein (2) or DNA molecule (3). More recently, lock-in detection and signal reconstruction techniques (4, 5) have enabled one- and multidimensional NMR spectroscopy with 0.5-Hz spectral resolution (6–8). More advanced control sequences at cryogenic temperatures have further enabled mapping the precise location of up to 27 ¹³C nuclear spins inside of diamond (9). Yet biologically meaningful spectroscopy on intact biomolecules remains elusive. One of the main outstanding challenges, which is required to perform nanoscale magnetic resonance spectroscopy of biomolecules, is the need to immobilize the target molecules within the 10- to 30-nm sensing range (2, 3, 7) of a highly coherent nitrogen vacancy (NV) qubit sensor. Immobilization is necessary because an untethered molecule would otherwise diffuse out of the detection volume within a few tens of microseconds.Various avenues to the functionalization of high-quality, single-crystalline diamond chips have been pursued over the last decade (10–12). However, none of the currently known approaches has led to the desired results of interfacing a coherent quantum sensor with target biomolecules. For example, hydrogen-terminated diamond surfaces can be chemically modified and form biologically stable surfaces (10, 13); but near-surface NV centers are generally charge-unstable under hydrogen termination (14), posing open challenges for NV sensing. On the other hand, oxygen-terminated diamond surfaces have been used to create charge stable NV^– centers with exceptional coherence times within 10 nm from the diamond surface (15). However, perfectly arranged, ether-terminated diamond surfaces generally lack chemically functionalizable surface groups (such as carboxyl or hydroxyl groups), making it difficult to control immobilization density and surface passivation. Other platforms such as diamond nanocrystals can generally be functionalized (16, 17) because of their heterogeneous surface chemistry, but they do not possess the coherence times needed for nanoscale magnetic resonance spectroscopy. Our approach (Fig. 1A) overcomes these limitations by utilizing a 2-nm-thick Al₂O₃ layer deposited onto an oxygen-terminated diamond surface by atomic layer deposition (ALD). This Al₂O₃ “adhesion” layer is silanized by N-[3-(trimethoxysilyl)propyl]ethylenediamine to create an amine (–NH₂) -terminated surface, which in turn is then grafted with a monolayer of heterobifunctional polyethylene glycol (PEG) via an N-hydroxysuccinimide (NHS) reaction, a process also referred as PEGylation. The PEG layer serves two purposes. First, it passivates the diamond surface to prevent nonspecific adsorption of biomolecules. Second, by adjusting the density of PEG molecules with functional groups (e.g., biotin or azide), we can control the immobilization density of proteins or DNA target molecules on the diamond surface. Furthermore, the small persistence length of the PEG linker (∼0.35 nm) allows the immobilized biomolecules to undergo rotational diffusion (18). This tumbling motion is the basis for motional averaging of the NMR spectra and helps to prevent immobilization of molecules in biologically inactive orientations.Open in a separate windowFig. 1.Architecture and characterization of the diamond functionalization approach. (A) Schematic illustration of the functionalization process. A thin layer of Al₂O₃ (gray) was deposited to the pristine, oxygen-terminated diamond surfaces (blue), followed by silanization (purple) and PEGylation (green). Functional groups (biotin, yellow circle; azide, red triangle) allow for cross-linking with target biomolecules. AFM characterization of the surfaces (B) and XPS Al2p signal after each step of the functionalization (C). (D) Illustration of the overall chemical functionalization architecture (not to scale), with corresponding thicknesses. (E) Illustration of SPAAC reaction. (F) A lithographically fabricated Al₂O₃ pattern on the diamond surface by lift-off, with a thickness of ∼2.1 nm. The Al₂O₃ layer is uniform without the presence of pin holes. The elevated edges originate from lift-off combined with ALD deposition.     

Early prediction of acute pancreatitis: Prospective study comparing computed tomography scans,ranson, glasgow,acute physiology and chronic health evaluation II scores,and various serum markers

The aim of this study was to assess the predictability of the outcome of acute pancreatitis using the Ranson, Glascow, and Acute Physiology and Chronic Health Evaluation (APACHE) II scores, the computed tomography (CT) scan, and several serum markers. Altogether, 137 consecutive patients with acute pancreatitis confirmed by CT scan were prospectively included. Blood samples were obtained daily for 6 days. The predictive value of each parameter was studied by univariate and multivariate analyses comparing mild and severe pancreatitis. A total of 111 attacks were graded as mild (81%) and 26 as severe (19%). Ranson (p = 0.3) and APACHE II (p = 0.049) scores appeared insufficiently predictive in the univariate analysis. Pancreatic imaging by CT scan was insufficiently predictive (p > 0.05), whereas the presence of extrapancreatic fluid collections was more indicative of outcome (p <0.05). With the univariate analysis, the four most reliable serum markers were pancreatic amylase (p <0.001), neutrophil elastase (p <0.05), albumin (p <0.002), and C-reactive protein (p <0.001). Results became homogeneous when the CT results were added; serum albumin plus extrapancreatic fluid collections (negative predictive value 92%-96% and positive predictive value 67%-100%) comprised the best indicator of severity. None of the parameters tested achieved sufficient predictability when used alone. Serum albumin plus extrapancreatic fluid collections comprise the best indicator of severity at the time of admission.