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A Platform for Testing the Biocompatibility of Implants: Silicone Induces a Proinflammatory Response in a 3D Skin Equivalent
No full textBiocompatibility testing of materials is carried out in 2D cell cultures or animal models
despite serious limitations. 3D skin equivalents are advanced in vitro models for human skin. Sil-
icone has been shown to be noncytotoxic but capable of eliciting an immune response. Our aim
was to (1) establish a 3D skin equivalent to (2) assess the proinflammatory properties of silicone.
We developed a coculture of keratinocytes and fibroblasts resulting in a 3D skin equivalent with
an implant using samples from a breast implant. Samples with and without the silicone implant
were studied histologically and immunohistochemically in comparison to native human skin sam-
ples. Cytotoxicity was assessed via LDH-assay, and cytokine response was assessed via ELISA.
Histologically, our 3D skin equivalents had a four-layered epidermal and a dermal component. The
presence of tight junctions was demonstrated in immunofluorescence. The only difference in 3D
skin equivalents with implants was an epidermal thinning. Implanting the silicone samples did not
cause more cell death, however, an inflammatory cytokine response was triggered. We were able to
establish an organotypical 3D skin equivalent with an implant, which can be utilised for studies on
biocompatibility of materials. This first integration of silicone into a 3D skin equivalent confirmed
previous findings on silicone being non-cell-toxic but capable of exerting a proinflammatory effect
Accelerating development of engineered T cell therapies in the EU: current regulatory framework for studying multiple product versions and T2EVOLVE recommendations
No full textTo accelerate the development of Advanced Therapy Medicinal Products
(ATMPs) for patients suffering from life-threatening cancer with limited
therapeutic options, regulatory approaches need to be constantly reviewed,
evaluated and adjusted, as necessary. This includes utilizing science and risk-
based approaches to mitigate and balance potential risks associated with early
clinical research and a more flexible manufacturing paradigm. In this paper,
T2EVOLVE an Innovative Medicine Initiative (IMI) consortium explores
opportunities to expedite the development of CAR and TCR engineered T cell
therapies in the EU by leveraging tools within the existing EU regulatory
framework to facilitate an iterative and adaptive learning approach across
different product versions with similar design elements or based on the same
platform technology. As understanding of the linkage between product quality
attributes, manufacturing processes, clinical efficacy and safety evolves through
development and post licensure, opportunities are emerging to streamline
regulatory submissions, optimize clinical studies and extrapolate data across
product versions reducing the need to perform duplicative studies. It is worth
noting that this paper is focusing on CAR- and TCR-engineered T cell therapies
but the concepts may be applied more broadly to engineered cell therapy
products (e.g., CAR NK cell therapy products)
Binding Properties of Small Electrophilic Anions [B6X5]− and [B10X9]− (X=Cl, Br, I): Activation of Small Molecules Based on π-Backbonding
No full textSuperelectrophilic anions constitute a special class of molecular anions that show strong binding of weak nucleophiles despite their negative charge. In this study, the binding characteristics of smaller gaseous electrophilic anions of the types [B6X5]− and [B10X9]− (with X=Cl, Br, I) were computationally and experimentally investigated and compared to those of the larger analogues [B12X11]−. The positive charge of vacant boron increases from [B6X5]− via [B10X9]− to [B12X11]−, as evidenced by increasing attachment enthalpies towards typical σ-donor molecules (noble gases, H2O). However, this behavior is reversed for σ-donor–π-acceptor molecules. [B6Cl5]− binds most strongly to N2 and CO, even more strongly than to H2O. Energy decomposition analysis confirms that the orbital interaction is responsible for this opposite trend. The extended transition state natural orbitals for chemical valence method shows that the π-backdonation order is [B6X5]−>[B10X9]−>[B12X11]−. This predicted order explains the experimentally observed red shifts of the CO and N2 stretching fundamentals compared to those of the unbound molecules, as measured by infrared photodissociation spectroscopy. The strongest red shift is observed for [B6Cl5N2]−: 222 cm−1. Therefore, strong activation of unreactive σ-donor–π-acceptor molecules (commonly observed for cationic transition metal complexes) is achieved with metal-free molecular anions
Brain metastasis and survival outcomes after first-line therapy in metastatic melanoma: a multicenter DeCOG study on 1704 patients from the prospective skin cancer registry ADOREG
No full textBackground Despite the availability of effective systemic therapies, a significant number of advanced melanoma patients develops brain metastases. This study investigated differences in incidence and time to diagnosis of brain metastasis and survival outcomes dependent on the type of first-line therapy.
Methods Patients with metastatic, non-resectable melanoma (AJCCv8 stage IIIC–V) without brain metastasis at start of first-line therapy (1L-therapy) were identified from the prospective multicenter real-world skin cancer registry ADOREG. Study endpoints were incidence of brain metastasis, brain metastasis-free survival (BMFS), progression-free survival (PFS), and overall survival (OS).
Results Of 1704 patients, 916 were BRAF wild-type (BRAFwt) and 788 were BRAF V600 mutant (BRAFmut). Median follow-up time after start of 1L-therapy was 40.4 months. BRAFwt patients received 1L-therapy with immune checkpoint inhibitors (ICI) against CTLA-4+PD-1 (n=281) or PD-1 (n=544). In BRAFmut patients, 1L-therapy was ICI in 415 patients (CTLA-4+PD-1, n=108; PD-1, n=264), and BRAF+MEK targeted therapy (TT) in 373 patients. After 24 months, 1L-therapy with BRAF+MEK resulted in a higher incidence of brain metastasis compared with PD-1±CTLA-4 (BRAF+MEK, 30.3%; CTLA-4+PD-1, 22.2%; PD-1, 14.0%). In multivariate analysis, BRAFmut patients developed brain metastases earlier on 1L-therapy with BRAF+MEK than with PD-1±CTLA-4 (CTLA-4+PD-1: HR 0.560, 95% CI 0.332 to 0.945, p=0.030; PD-1: HR 0.575, 95% CI 0.372 to 0.888, p=0.013). Type of 1L-therapy, tumor stage, and age were independent prognostic factors for BMFS in BRAFmut patients. In BRAFwt patients, tumor stage was independently associated with longer BMFS; ECOG Performance status (ECOG-PS), lactate dehydrogenase (LDH), and tumor stage with OS. CTLA-4+PD-1 did not result in better BMFS, PFS, or OS than PD-1 in BRAFwt patients. For BRAFmut patients, multivariate Cox regression revealed ECOG-PS, type of 1L-therapy, tumor stage, and LDH as independent prognostic factors for PFS and OS. 1L-therapy with CTLA-4+PD-1 led to longer OS than PD-1 (HR 1.97, 95% CI 1.122 to 3.455, p=0.018) or BRAF+MEK (HR 2.41, 95% CI 1.432 to 4.054, p=0.001), without PD-1 being superior to BRAF+MEK.
Conclusions In BRAFmut patients 1L-therapy with PD-1±CTLA-4 ICI resulted in a delayed and less frequent development of brain metastasis compared with BRAF+MEK TT. 1L-therapy with CTLA-4+PD-1 showed superior OS compared with PD-1 and BRAF+MEK. In BRAFwt patients, no differences in brain metastasis and survival outcomes were detected for CTLA-4+PD-1 compared with PD-1
Brain structure and function: a multidisciplinary pipeline to study hominoid brain evolution
No full textTo decipher the evolution of the hominoid brain and its functions, it is essential to conduct comparative studies in primates, including our closest living relatives. However, strong ethical concerns preclude in vivo neuroimaging of great apes. We propose a responsible and multidisciplinary alternative approach that links behavior to brain anatomy in non-human primates from diverse ecological backgrounds. The brains of primates observed in the wild or in captivity are extracted and fixed shortly after natural death, and then studied using advanced MRI neuroimaging and histology to reveal macro- and microstructures. By linking detailed neuroanatomy with observed behavior within and across primate species, our approach provides new perspectives on brain evolution. Combined with endocranial brain imprints extracted from computed tomographic scans of the skulls these data provide a framework for decoding evolutionary changes in hominin fossils. This approach is poised to become a key resource for investigating the evolution and functional differentiation of hominoid brains
Fundamental Change Beneath the Surface: The Supranationalisation of Rule of Law Protection in the European Union
No full textWhereas most studies on the European Union's (EU) responses to the rule of law crisis stress the underenforcement of EU law, this article offers a different perspective. Focusing on the long-term dynamics concerning rule of law protection, we detect a gradual trend towards supranationalism. The Rule of Law Conditionality mechanism adopted in 2020 is the first instance of ‘effective supranationalism’, that is, a rule of law tool that combines supranational decision-making procedures with binding and enforceable consequences. To explain this development in an area that has been marked by considerable resistance to efforts to strengthen supranational oversight, we draw on the agent-centric historical institutionalist approach. Our qualitative study shows that supranationalisation has become possible through two interrelated factors: the joint strategies of community bodies, which have promoted competence transfer to the EU level, and the increasing marginalisation of sovereigntist positions within the Council, given the escalating conflicts with Hungary and Poland
Immunoproteomics reveal increased serum IgG3/5 binding to Dermatophagoides and yeast protein antigens in severe equine asthma in a preliminary study
No full textIntroduction: Severe equine asthma (SEA) is a common, chronic respiratory disease of horses characterized by hyperreactivity to hay dust which has many similarities to severe neutrophilic asthma in humans. SEA-provoking antigens have not been comprehensively characterized, but molds and mites have been suggested as relevant sources. Here, we identified relevant antigen candidates using immunoproteomics with IgG isotype-binding analyses.
Methods: Proteins from Dermatophagoides pteronyssinus (Der p) were separated by two-dimensional gel electrophoresis followed by immunoblotting (2D immunoblots) resulting in a characteristic pattern of 440 spots. After serum incubation, antibody (Ig)-binding of all Ig (Pan-Ig) and IgG isotypes (type-2-associated IgG3/5, type-1-associated IgG4/7) was quantified per each spot and compared between asthmatic and healthy horses’ sera (n=5 per group).
Results: Ig binding differences were detected in 30 spots. Pan-Ig binding was higher with asthmatics compared to healthy horses’ sera on four spots, and IgG3/5 binding was higher on 18 spots. Small IgG4/7 binding differences were detected on 10 spots with higher binding with asthmatics’ sera on four but higher binding with healthy horses’ sera on six spots. Proteins from the spots with group differences including mite and yeast proteins were identified by liquid chromatography mass spectrometry. The latter likely originated from the feeding substrate of the Der p culture. Prioritized antigen candidates amongst the proteins identified were Der p 1, Der p 11, group 15 allergens, myosin heavy chain, and uncharacterized Der p proteins. Additionally, yeast enolases, alcohol dehydrogenase (ADH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase, and heat shock proteins were prioritized. Eleven antigen candidates were tested for confirmation by ELISAs using the respective proteins separately. Differences in asthmatics vs. healthy horses’ serum Ig binding to Der p 1, Der p 18, and three yeast enzymes (enolase, ADH, and PGK) confirmed these as promising antigens of immune responses in SEA.
Discussion: Antigens with relevance in SEA were newly identified by immunoproteomics, and yeast antigens were considered for SEA for the first time. Serum IgG3/5 binding to relevant antigens was increased in SEA and is a novel feature that points to increased type-2 responses in SEA but requires confirmation of the corresponding cellular responses
Control of Intermediates and Products by Combining Droplet Reactions and Ion Soft-Landing
No full textDespite being recognized primarily as an
analytical technique, mass spectrometry also has a large
potential as a synthetic tool, enabling access to advanced
synthetic routes by reactions in charged microdroplets
or ionic thin layers. Such reactions are special and
proceed primarily at surfaces of droplets and thin layers.
Partial solvation of the reactants is usually considered to
play an important role for reducing the activation
barrier, but many mechanistic details still need to be
clarified. In our study, we showcase the synergy between
two sequentially applied “preparative mass spectrometry”
methods: initiating accelerated reactions within
microdroplets during electrospray ionization to generate
gaseous ionic intermediates in high abundance, which
are subsequently mass-selected and soft-landed to react
with a provided reagent on a substrate. This allows the
generation of products at a nanomolar scale, amenable
to further characterization. In this proof-of-concept
study, the contrasting reaction pathways between intrinsically
neutral and pre-charged reagents, respectively,
both in microdroplets and in layers generated by
ion soft-landing are investigated. This provides new
insights into the role of partially solvated reagents at
microdroplet surfaces for increased reaction rates. Additionally,
further insights into reactions of ions of the
same polarity under various conditions is obtained
Interrater variability of ML-based CT-FFR during TAVR-planning: influence of image quality and coronary artery calcifications
No full textObjective: To compare machine learning (ML)-based CT-derived fractional flow reserve (CT-FFR) in patients before transcatheter aortic valve replacement (TAVR) by observers with differing training and to assess influencing factors.
Background: Coronary computed tomography angiography (cCTA) can effectively exclude CAD, e.g. prior to TAVR, but remains limited by its specificity. CT-FFR may mitigate this limitation also in patients prior to TAVR. While a high reliability of CT-FFR is presumed, little is known about the reproducibility of ML-based CT-FFR.
Methods: Consecutive patients with obstructive CAD on cCTA were evaluated with ML-based CT-FFR by two observers. Categorization into hemodynamically significant CAD was compared against invasive coronary angiography. The influence of image quality and coronary artery calcium score (CAC) was examined.
Results: CT-FFR was successfully performed on 214/272 examinations by both observers. The median difference of CT-FFR between both observers was −0.05(−0.12-0.02) (p 0.13).
Conclusion: Differences between CT-FFR values increased in values below the cut-off, having little clinical impact. Categorization into CAD differed in several patients, but ultimately only had a moderate influence on diagnostic accuracy. This was independent of image quality or CAC
Carboranes as Potent Phenyl Mimetics: A Comparative Study on the Reversal of ABCG2-Mediated Drug Resistance by Carboranylquinazolines and Their Organic Isosteres
No full textMultidrug resistance is a major challenge in clinical cancer
therapy. In particular, overexpression of certain ATP-binding
cassette (ABC) transporter proteins, like the efflux transporter
ABCG2, also known as breast cancer resistance protein (BCRP),
has been associated with the development of resistance to
applied chemotherapeutic agents in cancer therapies, and
therefore targeted inhibition of BCRP-mediated transport might
lead to reversal of this (multidrug) resistance (MDR). In a
previous study, we have described the introduction of a boroncarbon
cluster, namely closo-dicarbadodecaborane or carborane,
as an inorganic pharmacophore into a polymethoxylated
2-phenylquinazolin-4-amine backbone. In this work, the scope
was extended to the corresponding amide derivatives. As most
of the amide derivatives suffered from poor solubility, only the
amide derivative QCe and the two amine derivatives DMQCc
and DMQCd were further investigated. Carboranes are often
considered as sterically demanding phenyl mimetics or isosteres.
Therefore, the organic phenyl and sterically demanding
adamantyl analogues of the most promising carborane derivatives
were also investigated. The studies showed that the
previously described DMQCd, a penta-methoxylated N-carboranyl-
2-phenylquinazolin-4-amine, was by far superior to its
organic analogues in terms of cytotoxicity, inhibition of the
human ABCG2 transporter, as well as the ability to reverse
BCRP-mediated mitoxantrone resistance in MDCKII-hABCG2 and
HT29 colon cancer cells. Our results indicate that DMQCd is a
promising candidate for further in vitro as well as in vivo studies
in combination therapy for ABCG2-overexpressing cancers