Synthesis of C-5" and C-6"-modified α-GalCer analogues as iNKT-cell agonists

Alpha-Galactosyl Ceramide (α-GalCer) is a prototypical synthetic ligand of invariant natural killer T (iNKT) cells. Upon presentation by the MHC class I-like molecule CD1d, this glycolipid stimulates iNKT cells to secrete a vast amount of both pro-inflammatory Th1 and anti-inflammatory Th2 cytokines. Recently, we discovered that selected 6″-modified α-GalCer analogues may produce markedly Th1-biased responses due to the formation of either an additional anchor with CD1d or by establishing extra interactions with the T-cell receptor of iNKT cells. Here, we report a practical synthesis towards 6″-O-carbamate and galacturonamide analogues of α-GalCer and their evaluation as iNKT cell agonists in mice

Penetration enhancing effect of phytoceramides

Ceramides are essential components in the stratum corneum barrier function. Different classes of ceramides are present in human skin, differing in the nature of sphingosine and acyl moieties with respect to chain length, degree of saturation and the presence of an OH group [1]. Ceramides with a saturated sphingosine base containing a hydroxyl function at C4 are known as phytoceramides. A few studies demonstrated the penetration enhancing properties of ceramides [2-5], however, systematic studies using phytoceramides are lacking. This led us to assess the penetration enhancing effect of phytosphingosine and a series of nine phytoceramides via transdermal experiments using in vitro Franz diffusion cells. As transdermal model compounds, testosterone, caffeine and ibuprofen were tested in a 50:50 (V/V) EtOH:H2O dose formulation [6]. Results showed that the penetration enhancing effect of the phytoceramides depends on the used model compound. Selected phytoceramides exhibited a penetration enhancing ratio of more than two. References [1] Janůšová, B.; Zbytovská, J.; Lorenc, P.; Vavrysová, H.; Palát K.; Hrabálek, A.; Vávrová, K. (2011). Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes. Biochimica et Biophysica, 1811, 129–137. [2] Vávrová, K.; Hrabálek, A.; Dolezal, P.; Holas, T.; Zbytovská, J. (2003). L-serine and glycine based ceramide analogues as transdermal permeation enhancers: polar head size and hydrogen bonding. Bioorganic & medicinal chemistry letters, 13, 2351-2353. [3] Vávrová, K.; Zbytovská, J.; Hrabálek, A. (2005). Amphiphilic transdermal permeation enhancers: structure-activity relationships. Current Medicinal Chemistry, 12, 2273-2291. [4] Novotý, J.; Janůšová, B.; Novotý, M.; Hrabálek, A.; Vávrová, K. (2009). Short-chain ceramides decrease skin barrier properties. Skin pharmacology and Physiology, 22, 22-30. [5] Sinko, B.; Kökösi, J.; Avdeef, A.; Takács-Novák, K. (2009). A PAMPA study of the permeability-enhancing effect of new ceramide analogues. Chemistry & Biodiversity, 6, 1867-1874. [6] Baert, B.; Deconinck, E.; Van Gele, M.; Slodicka, M.; Stoppie, P.; Bode, S.; Slegers, G.; Vander Heyden, Y.; Lambert, J.; Beetens, J.; De Spiegeleer, B. (2007). Transdermal penetration behaviour of drugs: CART-clustering, QSPR and selection of model compounds. Bioorganic & medicinal chemistry, 15(22), 6943-6955

Galactosylsphingamides : new α-GalCer analogues to probe the F’-pocket of CD1d

Invariant Natural Killer T-cells (iNKT-cells) are an attractive target for immune response modulation, as upon CD1d-mediated stimulation with KRN7000, a synthetic alpha-galactosylceramide, they produce a vast amount of cytokines. Here we present a synthesis that allows swift modification of the phytosphingosine side chain by amidation of an advanced methyl ester precursor. The resulting KRN7000 derivatives, termed alpha-galactosylsphingamides, were evaluated for their capacity to stimulate iNKT-cells. While introduction of the amide-motif in the phytosphingosine chain is tolerated for CD1d binding and TCR recognition, the studied alpha-galactosylsphingamides showed compromised antigenic properties

Synthesis of C6′′-modified α-C-GalCer analogues as mouse and human iNKT cell agonists

alpha-GalCer analogues that combine known Th1 polarizing C6''-modifications with a C-glycosidic linkage were synthesized. We employed a protecting group strategy that allowed the preparation of both saturated and unsaturated derivatives with variable C6''-substituents. Selected analogues demonstrate promising activity in mice. Interestingly, the introduction of a 6''-O-pyridinylcarbamoyl substituent to alpha-C-GalCer restores its antigenicity in human iNKT cells

Synthesis and structure-activity relationships of cerebroside analogues as substrates of cerebroside sulphotransferase and discovery of a competitive inhibitor

Metachromatic leukodystrophy (MLD) is a rare genetic disease characterised by a dysfunction of the enzyme arylsulphatase A leading to the lysosomal accumulation of cerebroside sulphate (sulphatide) causing subsequent demyelination in patients. The enzyme galactosylceramide (cerebroside) sulphotransferase (CST) catalyses the transfer of a sulphate group from 3 '-phosphoadenosine-5'-phosphosulphate (PAPS) to cerebrosides producing sulphatides. Substrate reduction therapy for arylsulphatase A by inhibition of CST was proposed as a promising therapeutic approach. To identify competitive CST inhibitors, we synthesised and investigated analogues of the substrate galactosylceramide with variations at the anomeric position, the acyl substituent and the carbohydrate moiety, and investigated their structure-activity relationships. While most of the compounds behaved as substrates, alpha-galactosylceramide16was identified as the first competitive CST inhibitor. Compound16can serve as a new lead structure for the development of drugs for the treatment of this devastating disease, MLD, for which small molecule therapeutics are currently not available

A molecular switch in mouse CD1d modulates natural killer T cell activation by α-galactosylsphingamides

Type I natural killer T (NKT) cells are a population of innate like T lymphocytes that rapidly respond to α-GalCer presented by CD1d via the production of both pro- and anti-inflammatory cytokines. While developing novel α-GalCer analogs that were meant to be utilized as potential adjuvants because of their production of pro-inflammatory cytokines (Th1 skewers), we generated α-galactosylsphingamides (αGSA). Surprisingly, αGSAs are not potent antigens in vivo despite their strong T-cell receptor (TCR)–binding affinities. Here, using surface plasmon resonance (SPR), antigen presentation assays, and X-ray crystallography (yielding crystal structures of 19 different binary (CD1d-glycolipid) or ternary (CD1d-glycolipid-TCR) complexes at resolutions between 1.67 and 2.85 Å), we characterized the biochemical and structural details of αGSA recognition by murine NKT cells. We identified a molecular switch within murine (m)CD1d that modulates NKT cell activation by αGSAs. We found that the molecular switch involves a hydrogen bond interaction between Tyr-73 of mCD1d and the amide group oxygen of αGSAs. We further established that the length of the acyl chain controls the positioning of the amide group with respect to the molecular switch and works synergistically with Tyr-73 to control NKT cell activity. In conclusion, our findings reveal important mechanistic insights into the presentation and recognition of glycolipids with polar moieties in an otherwise apolar milieu. These observations may inform the development αGSAs as specific NKT cell antagonists to modulate immune responses

One-time deformable thermoplastic devices based on flexible circuit board technology

This contribution describes an efficient process flow for production of one-time deformable electronic devices based on standard circuit board technology and demonstrates multiple devices fabricated using this technique. The described technology has the potential to streamline and simplify the production of complex user interfaces which typically require extensive mechanical design and many components. The employed technique allows for the production of complex 3D shapes without the need to modify existing circuit board manufacturing equipment or processes significantly. To achieve this the device is manufactured in a flat state, encapsulated in a thermoplastic polymer laminate and deformed afterwards. This allows the usage of standard electronic components in surface mount packages, which are assembled using lead-free high-temperature solder. The circuit is deformed using a high-volume cost-effective thermoforming approach, where the encapsulating polymer is heated above its glass transition temperature and forced against a mold where it is allowed to cool down again. To enable significant out-of-plane deformations stretchable meandering interconnects are used, which were traditionally developed for dynamically stretchable devices. Fabrication of the circuit starts using a standard flexible copper clad laminate which is processed using the default techniques, the resulting circuit is then attached to a carrier board coated with a reusable high-temperature pressure sensitive adhesive. The interconnect and circuit outline is then defined using laser routing or punching, cutting the flexible circuit without damaging the carrier. The residuals not part of the circuit are removed, in a process akin to protective film removal, and solder paste is stencil printed on the circuit. Afterwards components are placed using a pick-and-place machine and the boards are reflow soldered. After functional testing and repair (if necessary) the circuits are placed in a vacuum press with a thermoplastic laminate, consisting of a thermoplastic elastomer and a rigid thermoplastic sheet. During this lamination the components are protected by a highly conforming press pad. Because the adhesion between the elastomer and the circuit far exceeds that between the circuit and the carrier the circuit is released readily as the thermoplastic laminate is peeled away. The resulting laminate is built up further using thermoplastic films and sheets, and finally deformed using a vacuum forming machine. The resulting device, which is trimmed to remove the clamping edges, can then be mounted in the final assembly. The advantages of this approach are demonstrated using a series of test vehicles, demonstrating the integration of complex circuits, connectors, and power circuitry. Finally, a series of design considerations that became apparent after initial reliability testing are discussed, together with the resulting design rules

Daily allergy burden and heart rate characteristics in adults with allergic rhinitis based on a wearable telemonitoring system

Background: Allergic rhinitis includes a certain degree of autonomic imbalance. However, no information is available on how daily changes in allergy burden affect autonomic imbalance. We aimed to estimate associations between daily allergy burden (allergy symptoms and mood) and daily heart rate characteristics (resting heart rate and sample entropy, both biomarkers of autonomic balance) of adults with allergic rhinitis, based on real-world measurements with a wearable telemonitoring system. Methods: Adults with a tree pollen allergy used a smartphone application to self-report daily allergy symptoms (score 0–44) and mood (score 0–4), and a Mio Alpha 2 wristwatch to collect heart rate characteristics during two pollen seasons of hazel, alder and birch in Belgium. Associations between daily allergy burden and heart rate characteristics were estimated using linear mixed effects distributed lag models with a random intercept for individuals and adjusted for potential confounders. Results: Analyses included 2497 participant-days of 72 participants. A one-point increase in allergy symptom score was associated with an increase in next-day resting heart rate of 0.08 (95% CI: 0.02–0.15) beats per minute. A one-point increase in mood score was associated with an increase in same-day sample entropy of 0.80 (95% CI: 0.34–1.26) × 10−2. No associations were found between allergy symptoms and heart rate sample entropy, nor between mood and resting heart rate. Conclusion: Daily repeated measurements with a wearable telemonitoring system revealed that the daily allergy burden of adults with allergic rhinitis has systemic effects beyond merely the respiratory system.</p