Patients and mice with deficiency in the SNARE protein SYNTAXIN-11 have a secondary B cell defect.

SYNTAXIN-11 (STX11) is a SNARE protein that mediates the fusion of cytotoxic granules with the plasma membrane at the immunological synapses of CD8 T or NK cells. Autosomal recessive inheritance of deleterious STX11 variants impairs cytotoxic granule exocytosis, causing familial hemophagocytic lymphohistiocytosis type 4 (FHL-4). In several FHL-4 patients, we also observed hypogammaglobulinemia, elevated frequencies of naive B cells, and increased double-negative DN2:DN1 B cell ratios, indicating a hitherto unrecognized role of STX11 in humoral immunity. Detailed analysis of Stx11-deficient mice revealed impaired CD4 T cell help for B cells, associated with disrupted germinal center formation, reduced isotype class switching, and low antibody avidity. Mechanistically, Stx11-/- CD4 T cells exhibit impaired membrane fusion leading to reduced CD107a and CD40L surface mobilization and diminished IL-2 and IL-10 secretion. Our findings highlight a critical role of STX11 in SNARE-mediated membrane trafficking and vesicle exocytosis in CD4 T cells, important for successful CD4 T cell-B cell interactions. Deficiency in STX11 impairs CD4 T cell-dependent B cell differentiation and humoral responses.

Antibody-dye Conjugates Targeting EGFR and HER2 for the Photoimmunotherapy of Bladder Cancer.

BACKGROUND/AIM: Although there are curative treatment options for non-muscle-invasive bladder cancer, the recurrence of this tumor is high. Therefore, novel targeted therapies are needed for the complete removal of bladder cancer cells in stages of localized disease, in order to avoid local recurrence, to spare bladder cancer patients from stressful and expensive treatment procedures and to increase their quality of life and life expectancy. This study tested a new approach for the photoimmunotherapy (PIT) of bladder cancer. MATERIALS AND METHODS: We generated a cysteine modified recombinant version of the antibody cetuximab targeting the epidermal growth factor receptor (EGFR) on the surface of bladder cancer cells. Then, we coupled the novel photoactivatable phthalocyanine dye WB692-CB1 via a maleimide linker to the free cysteines of the antibody. PIT was performed by incubating bladder cancer cells with the antibody dye conjugate followed by irradiation with visible red light. RESULTS: The conjugate was able to induce specific cytotoxicity in EGFR-positive bladder cancer cells in a light dose-dependent manner. Enhanced cytotoxicity in RT112 bladder cancer cells was evoked by addition of a second antibody dye conjugate targeting HER2 or by repeated cycles of PIT. CONCLUSION: Our new antibody dye conjugate targeting EGFR-expressing bladder cancer cells is a promising candidate for the future PIT of bladder cancer patients.

Formation of dsRNA by-products during in vitro transcription can be reduced by using low steady-state levels of UTP.

Introduction: Exogeneous messenger ribonucleic acid (mRNA) can be used as therapeutic and preventive medication. However, during the enzymatic production process, commonly called in vitro transcription, by-products occur which can reduce the therapeutic efficacy of mRNA. One such by-product is double-stranded RNA (dsRNA). We therefore sought to limit the generation of dsRNA by-products during in vitro transcription. Materials and methods: In vitro transcription was performed with a DNA template including a poly(A)-tail-encoding region, dinucleotide or trinucleotide cap analogs for cotranscriptional capping, and relevant nucleoside triphosphates. Concentrations of UTP or modified UTP (m1ΨTP) and GTP were reduced and fed over the course of the reaction. mRNA was analyzed for dsRNA contamination, yield of the reaction, RNA integrity, and capping efficiency before translational activity was assessed. Results: Limiting the steady-state level of UTP or m1ΨTP during the enzymatic reaction reduced dsRNA formation, while not affecting mRNA yield or RNA integrity. Capping efficiency was optimized with the use of a combined GTP and UTP or m1ΨTP feed, while still reducing dsRNA formation. Lower dsRNA levels led to higher protein expression from the corresponding mRNAs. Discussion: Low steady-state concentrations of UTP and GTP, fed in combination over the course of the in vitro transcription reaction, produce mRNA with high capping and low levels of dsRNA formation, resulting in high levels of protein expression. This novel approach may render laborious purification steps to remove dsRNA unnecessary.

VEPdgets: Towards Richer Interaction Elements Based on Visually Evoked Potentials.

For brain-computer interfaces, a variety of technologies and applications already exist. However, current approaches use visual evoked potentials (VEP) only as action triggers or in combination with other input technologies. This paper shows that the losing visually evoked potentials after looking away from a stimulus is a reliable temporal parameter. The associated latency can be used to control time-varying variables using the VEP. In this context, we introduced VEP interaction elements (VEP widgets) for a value input of numbers, which can be applied in various ways and is purely based on VEP technology. We carried out a user study in a desktop as well as in a virtual reality setting. The results for both settings showed that the temporal control approach using latency correction could be applied to the input of values using the proposed VEP widgets. Even though value input is not very accurate under untrained conditions, users could input numerical values. Our concept of applying latency correction to VEP widgets is not limited to the input of numbers.

Synergistic Cytotoxicity of a Toxin Targeting the Epidermal Growth Factor Receptor and the Glycosylated Triterpenoid SO1861 in Prostate Cancer.

Treatment of advanced prostate cancer lacks specificity and curative intent. Therefore, the need for new targeted therapeutic approaches is high. In the present study, we generated the new targeted toxin EGF-PE24mutΔREDLK binding to the epidermal growth factor receptor (EGFR) on the surface of prostate cancer cells. It consists of the human epidermal growth factor (EGF) as binding domain and a de-immunized variant of Pseudomonas Exotoxin A (PE), called PE24mutΔREDLK, as toxin domain. The toxin domain contains a deletion of the C-terminal KDEL-like motif REDLK to prevent its transport from sorting endosomes via the KDEL receptor mediated pathway into the cytosol, where it can inhibit cellular protein biosynthesis and induce apoptosis. Indeed, REDLK deletion resulted in a strong decrease in cytotoxicity of the targeted toxin in prostate cancer cells compared to the parental targeted toxin EGF-PE24mut. However, addition of the plant glycosylated triterpenoid SO1861, which is known to mediate the release of biomolecules from endolysosomal compartments into the cytosol, resulted in an up to almost 7,000-fold enhanced synergistic cytotoxicity. Moreover, combination of PE24mutΔREDLK with SO1861 led to a cytotoxicity that was even 16- to 300-fold enhanced compared to that of EGF-PE24mut. Endolysosomal entrapment of the non-toxic targeted toxin EGF-PE24mutΔREDLK followed by activation through enhanced endosomal escape therefore represents a new promising approach for the future treatment of advanced prostate cancer with high efficacy and diminished side effects.

Enhanced cytotoxicity of a Pseudomonas Exotoxin A based immunotoxin against prostate cancer by addition of the endosomal escape enhancer SO1861.

Immunotoxins consist of an antibody or antibody fragment that binds to a specific cell surface structure and a cytotoxic domain that kills the cell after cytosolic uptake. Pseudomonas Exotoxin A (PE) based immunotoxins directed against a variety of tumor entities have successfully entered the clinic. PE possesses a KDEL-like motif (REDLK) that enables the toxin to travel from sorting endosomes via the KDEL-receptor pathway to the endoplasmic reticulum (ER), from where it is transported into the cytosol. There, it ADP-ribosylates the eukaryotic elongation factor 2, resulting in ribosome inhibition and finally apoptosis. One major problem of immunotoxins is their lysosomal degradation causing the need for much more immunotoxin molecules than finally required for induction of cell death. The resulting dose limitations and substantially increased side effects require new strategies to achieve improved cytosolic uptake. Here we generated an immunotoxin consisting of a humanized single chain variable fragment (scFv) targeting the prostate specific membrane antigen (PSMA) and the de-immunized PE variant PE24mut. This immunotoxin, hD7-1(VL-VH)-PE24mut, showed high and specific cytotoxicity in PSMA-expressing prostate cancer cells. We deleted the REDLK sequence to prevent transport to the ER and achieve endosomal entrapment. The cytotoxicity of this immunotoxin, hD7-1(VL-VH)-PE24mutΔREDLK, was greatly reduced. To restore activity, we added the endosomal escape enhancer SO1861 and observed an up to 190,000-fold enhanced cytotoxicity corresponding to a 57-fold enhancement compared to the initial immunotoxin with the REDLK sequence. A biodistribution study with different routes of administration clearly showed that the subcutaneous injection of hD7-1(VL-VH)-PE24mutΔREDLK in mice resulted in the highest tumor uptake. Treatment of mice bearing prostate tumors with a combination of hD7-1(VL-VH)-PE24mutΔREDLK plus SO1861 resulted in inhibition of tumor growth and enhanced overall survival compared to the monotherapies. The endosomal entrapment of non-toxic anti-PSMA immunotoxins followed by enhanced endosomal escape by SO1861 provides new therapeutic options in the future management of prostate cancer.

S2k guideline for diving accidents.

For the purposes of this guideline, a diving accident is defined as an event that is either potentially life-threatening or hazardous to health as a result of a reduction in ambient pressure while diving or in other hyperbaric atmospheres with and without diving equipment. This national consensus-based guideline (development grade S2k) presents the current state of knowledge and recommendations on the diagnosis and treatment of diving accident victims. The treatment of a breath-hold diver as well as children and adolescents does not differ in principle. In this regard only unusual tiredness and itching without visible skin changes are mild symptoms. The key action statements: on-site 100% oxygen first aid treatment, immobilization/no unnecessary movement, fluid administration and telephone consultation with a diving medicine specialist are recommended. Hyperbaric oxygen therapy (HBOT) remains unchanged as the established treatment in severe cases, as there are no therapeutic alternatives. The basic treatment scheme recommended for diving accidents is hyperbaric oxygenation at 280 kPa.

Inhibitor of apoptosis proteins as therapeutic targets in bladder cancer.

Evasion from apoptosis is a hallmark of cancer. Inhibitor of apoptosis proteins (IAPs) contribute to this hallmark by suppressing the induction of cell death. IAPs were found to be overexpressed in cancerous tissues and to contribute to therapeutic resistance. The present review focuses on the IAP members cIAP1, cIAP2, XIAP, Survivin and Livin and their importance as potential therapeutic targets in bladder cancer.

Prostate Cancer Stem Cells: Clinical Aspects and Targeted Therapies.

Despite decades of research and successful improvements in diagnosis and therapy, prostate cancer (PC) remains a major challenge. In recent years, it has become clear that PC stem cells (PCSCs) are the driving force in tumorigenesis, relapse, metastasis, and therapeutic resistance of PC. In this minireview, we discuss the impact of PCSCs in the clinical practice. Moreover, new therapeutic approaches to combat PCSCs are presented with the aim to achieve an improved outcome for patients with PC.

Pro-apoptotic complexes of BAX and BAK on the outer mitochondrial membrane.

In multicellular organisms the regulated cell death apoptosis is critically important for both ontogeny and homeostasis. Mitochondria are indispensable for stress-induced apoptosis. The BCL-2 protein family controls mitochondrial apoptosis and initiates cell death through the pro-apoptotic activities of BAX and BAK at the outer mitochondrial membrane (OMM). Cellular survival is ensured by the retrotranslocation of mitochondrial BAX and BAK into the cytosol by anti-apoptotic BCL-2 proteins. BAX/BAK-dependent OMM permeabilization releases the mitochondrial cytochrome c (cyt c), which initiates activation of caspase-9. The caspase cascade leads to cell shrinkage, plasma membrane blebbing, chromatin condensation, and apoptotic body formation. Although it is clear that ultimately complexes of active BAX and BAK commit the cell to apoptosis, the nature of these complexes is still enigmatic. Excessive research has described a range of complexes, varying from a few molecules to several 10,000, in different systems. BAX/BAK complexes potentially form ring-like structures that could expose the inner mitochondrial membrane. It has been suggested that these pores allow the efflux of small proteins and even mitochondrial DNA. Here we summarize the current state of knowledge for mitochondrial BAX/BAK complexes and the interactions between these proteins and the membrane.

Comparison of the Anti-inflammatory Properties of Two Nicotinic Acetylcholine Receptor Ligands, Phosphocholine and pCF3-diEPP.

Activation of nicotinic acetylcholine receptors (nAChRs) expressed by innate immune cells can attenuate pro-inflammatory responses. Silent nAChR agonists, which down-modulate inflammation but have little or no ionotropic activity, are of outstanding clinical interest for the prevention and therapy of numerous inflammatory diseases. Here, we compare two silent nAChR agonists, phosphocholine, which is known to interact with nAChR subunits α7, α9, and α10, and pCF3-N,N-diethyl-N'-phenyl-piperazine (pCF3-diEPP), a previously identified α7 nAChR silent agonist, regarding their anti-inflammatory properties and their effects on ionotropic nAChR functions. The lipopolysaccharide (LPS)-induced release of interleukin (IL)-6 by primary murine macrophages was inhibited by pCF3-diEPP, while phosphocholine was ineffective presumably because of instability. In human whole blood cultures pCF3-diEPP inhibited the LPS-induced secretion of IL-6, TNF-α and IL-1ß. The ATP-mediated release of IL-1ß by LPS-primed human peripheral blood mononuclear leukocytes, monocytic THP-1 cells and THP-1-derived M1-like macrophages was reduced by both phosphocholine and femtomolar concentrations of pCF3-diEPP. These effects were sensitive to mecamylamine and to conopeptides RgIA4 and [V11L; V16D]ArIB, suggesting the involvement of nAChR subunits α7, α9 and/or α10. In two-electrode voltage-clamp measurements pCF3-diEPP functioned as a partial agonist and a strong desensitizer of classical human α9 and α9α10 nAChRs. Interestingly, pCF3-diEPP was more effective as an ionotropic agonist at these nAChRs than at α7 nAChR. In conclusion, phosphocholine and pCF3-diEPP are potent agonists at unconventional nAChRs expressed by monocytic and macrophage-like cells. pCF3-diEPP inhibits the LPS-induced release of pro-inflammatory cytokines, while phosphocholine is ineffective. However, both agonists signal via nAChR subunits α7, α9 and/or α10 to efficiently down-modulate the ATP-induced release of IL-1ß. Compared to phosphocholine, pCF3-diEPP is expected to have better pharmacological properties. Thus, low concentrations of pCF3-diEPP may be a therapeutic option for the treatment of inflammatory diseases including trauma-induced sterile inflammation.

How Do Hexokinases Inhibit Receptor-Mediated Apoptosis?

The regulated cell death apoptosis enables redundant or compromised cells in ontogeny and homeostasis to remove themselves receptor-dependent after extrinsic signaling or after internal stress by BCL-2 proteins on the outer mitochondrial membrane (OMM). Mitochondrial BCL-2 proteins are also often needed for receptor-mediated signaling in apoptosis. Then, the truncated BH3-only protein BID (tBID) blocks retrotranslocation of the pro-apoptotic BCL-2 proteins BAX and BAK from the mitochondria into the cytosol. BAX and BAK in turn permeabilize the OMM. Although the BCL-2 proteins are controlled by a complex regulatory network, a specific mechanism for the inhibition of tBID remained unknown. Curiously, it was suggested that hexokinases, which channel glucose into the metabolism, have an intriguing function in the regulation of apoptosis. Recent analysis of transient hexokinase interactions with BAX revealed its participation in the inhibition of BAX and also BAK by retrotranslocation from mitochondria to the cytosol. In contrast to general apoptosis inhibition by anti-apoptotic BCL-2 proteins, hexokinase I and hexokinase 2 specifically inhibit tBID and thus the mitochondrial apoptosis pathway in response to death receptor signaling. Mitochondrial hexokinase localization and BH3 binding of cytosolic hexokinase domains are prerequisites for protection against receptor-mediated cell death, whereas glucose metabolism is not. This mechanism protects cells from apoptosis induced by cytotoxic T cells.

Direct Transition from Ultrathin Orthorhombic Dinickel Silicides to Epitaxial Nickel Disilicide Revealed by In Situ Synthesis and Analysis.

Understanding phase transitions of ultrathin metal silicides is crucial for the development of nanoscale silicon devices. Here, the phase transition of ultrathin (3.6 nm) Ni silicides on Si(100) substrates is investigated using an in situ synthesis and characterization approach, supplemented with ex situ transmission electron microscopy and nano-beam electron diffraction. First, an ultrathin epitaxial layer and ordered structures at the interface are observed upon room-temperature deposition. At 290 °C, this structure is followed by formation of an orthorhombic δ-Ni2 Si phase exhibiting long-range order and extending to the whole film thickness. An unprecedented direct transition from this δ-Ni2 Si phase to the final NiSi2- x phase is observed at 290 °C, skipping the intermediate monosilicide phase. Additionally, the NiSi2- x phase is found epitaxial on the substrate. This transition process substantially differs from observations for thicker films. Furthermore, considering previous studies, the long-range ordered orthorhombic δ-Ni2 Si phase is suggested to occur regardless of the initial Ni thickness. The thickness of this ordered δ-Ni2 Si layer is, however, limited due to the competition of different orientations of the δ-Ni2 Si crystal. Whether the formed δ-Ni2 Si layer consumes all deposited nickel is expected to determine whether the monosilicide phase appears before the transition to the final NiSi2- x phase.

[Management of COVID-19 mass casualty incidents in nursing and retirement homes]. / Managementstrategie für den Massenanfall von Erkrankten/Infizierten in Alten- und Pflegeheimen im Kontext der COVID-19-Pandemie.

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, outbreaks in inpatient care facilities, which grow into a large-scale emergency scenario, are frequently observed. A standardized procedure analogous to algorithms for mass casualty incidents (MCI) is lacking. METHODS: Based on a case report and the literature, the authors present a management strategy for infectious MCI during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic and distinguish it from traumatic MCI deployment tactics. RESULTS: This management strategy can be divided into three phases, beginning with the acute emergency response including triage, stabilization of critical patients, and transport of patients requiring hospitalization. Phase 2 involves securing the facility's operational readiness, or housing residents elsewhere in case staff are infected or quarantined to a relevant degree. Phase 3 marks the return to regular operations. DISCUSSION: Phase 1 is based on usual MCI principles, phase 2 on hospital crisis management. Avoiding evacuation of residents to relieve hospitals is an important operational objective. The lack of mission and training experience with such situations, the limited applicability of established triage algorithms, and the need to coordinate a large number of participants pose challenges. CONCLUSION: This strategic model offers a practical, holistic approach to the management of infectious mass casualty scenarios in nursing facilities.

The effects of stature, age, gender, and posture preferences on preferred joint angles after real driving.

Few studies have investigated preferred driving posture involving real-world driving. For this purpose, seven preferred joint angles of 127 participants were recorded three times from sagittal plane photographs after a short, standardized drive on public roads. Participants' individual driving posture preferences were determined using a word-based questionnaire. The inter- and intraindividual joint angle differences were calculated and some significant effects of stature, age, and gender on these joint angle differences were found. Additionally, significant correlations were found between posture preferences and joint angles, except for the lower extremities. The results show that the factors stature, age, gender, and posture preferences have influence on the preferred joint angles. However, other human-, vehicle-, and context-related factors also seem relevant and should be considered in future studies. The results provide important field-based knowledge for an application-oriented understanding of driving posture and for the human-centered design process of future vehicle interior research and development.

Orthogonal Peptide-Templated Labeling Elucidates Lateral ETA R/ETB R Proximity and Reveals Altered Downstream Signaling.

Fine-tuning of G protein-coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one-step labeling method of multiple membrane-embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this approach to four receptors of the cardiovascular system: the endothelin receptor A and B (ETA R and ETB R), angiotensin II receptor type 1, and apelin. Wild type-like G protein activation after N-terminal modification was demonstrated for all receptor species. Using FRET-competent dyes, a constitutive proximity between hetero-receptors was limited to ETA R/ETB R. Further, we demonstrate, that ETA R expression regulates the signaling of co-expressed ETB R. Our orthogonal peptide-templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling.

Targeted Toxins for the Treatment of Prostate Cancer.

Prostate cancer is the second most common cancer and the fifth leading cause of cancer deaths worldwide. Despite improvements in diagnosis and treatment, new treatment options are urgently needed for advanced stages of the disease. Targeted toxins are chemical conjugates or fully recombinant proteins consisting of a binding domain directed against a target antigen on the surface of cancer cells and a toxin domain, which is transported into the cell for the induction of apoptosis. In the last decades, targeted toxins against prostate cancer have been developed. Several challenges, however, became apparent that prevented their direct clinical use. They comprise immunogenicity, low target antigen binding, endosomal entrapment, and lysosomal/proteasomal degradation of the targeted toxins. Moreover, their efficacy is impaired by prostate tumors, which are marked by a dense microenvironment, low target antigen expression, and apoptosis resistance. In this review, current findings in the development of targeted toxins against prostate cancer in view of effective targeting, reduction of immunogenicity, improvement of intracellular trafficking, and overcoming apoptosis resistance are discussed. There are promising approaches that should lead to the clinical use of targeted toxins as therapeutic alternatives for advanced prostate cancer in the future.

The potential of CAR T cell therapy for prostate cancer.

Chimeric antigen receptor (CAR) T cell immunotherapy involves the genetic modification of the patient's own T cells so that they specifically recognize and destroy tumour cells. Considerable clinical success has been achieved using this technique in patients with lymphoid malignancies, but clinical studies that investigated treating solid tumours using this emerging technology have been disappointing. A number of developments might be able to increase the efficacy of CAR T cell therapy for treatment of prostate cancer, including improved trafficking to the tumour, techniques to overcome the immunosuppressive tumour microenvironment, as well as methods to enhance CAR T cell persistence, specificity and safety. Furthermore, CAR T cell therapy has the potential to be combined with other treatment modalities, such as androgen deprivation therapy, radiotherapy or chemotherapy, and could be applied as focal CAR T cell therapy for prostate cancer.

Ligand-binding and -scavenging of the chemerin receptor GPR1.

Tight regulation of cytokines is essential for the initiation and resolution of inflammation. Chemerin, a mediator of innate immunity, mainly acts on chemokine-like receptor 1 (CMKLR1) to induce the migration of macrophages and dendritic cells. The role of the second chemerin receptor, G protein-coupled receptor 1 (GPR1), is still unclear. Here we demonstrate that GPR1 shows ligand-induced arrestin3 recruitment and internalization. The chemerin C-terminus triggers this activation by folding into a loop structure, binding to aromatic residues in the extracellular loops of GPR1. While this overall binding mode is shared between GPR1 and CMKLR1, differences in their respective extracellular loop 2 allowed for the design of the first GPR1-selective peptide. However, our results suggest that ligand-induced arrestin recruitment is not the only mode of action of GPR1. This receptor also displays constitutive internalization, which allows GPR1 to internalize inactive peptides efficiently by an activation-independent pathway. Our results demonstrate that GPR1 takes a dual role in regulating chemerin activity: as a signaling receptor for arrestin-based signaling on one hand, and as a scavenging receptor with broader ligand specificity on the other.