Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis.

Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.

RANK links thymic regulatory T cells to fetal loss and gestational diabetes in pregnancy.

Successful pregnancies rely on adaptations within the mother1, including marked changes within the immune system2. It has long been known that the thymus, the central lymphoid organ, changes markedly during pregnancy3. However, the molecular basis and importance of this process remain largely obscure. Here we show that the osteoclast differentiation receptor RANK4,5 couples female sex hormones to the rewiring of the thymus during pregnancy. Genetic deletion of Rank (also known as Tnfrsf11a) in thymic epithelial cells results in impaired thymic involution and blunted expansion of natural regulatory T (Treg) cells in pregnant female mice. Sex hormones, in particular progesterone, drive the development of thymic Treg cells through RANK in a manner that depends on AIRE+ medullary thymic epithelial cells. The depletion of Rank in the mouse thymic epithelium results in reduced accumulation of natural Treg cells in the placenta, and an increase in the number of miscarriages. Thymic deletion of Rank also results in impaired accumulation of Treg cells in visceral adipose tissue, and is associated with enlarged adipocyte size, tissue inflammation, enhanced maternal glucose intolerance, fetal macrosomia, and a long-lasting transgenerational alteration in glucose homeostasis, which are all key hallmarks of gestational diabetes. Transplantation of Treg cells rescued fetal loss, maternal glucose intolerance and fetal macrosomia. In human pregnancies, we found that gestational diabetes also correlates with a reduced number of Treg cells in the placenta. Our findings show that RANK promotes the hormone-mediated development of thymic Treg cells during pregnancy, and expand the functional role of maternal Treg cells to the development of gestational diabetes and the transgenerational metabolic rewiring of glucose homeostasis.

Systemic Inflammation and Normocytic Anemia in DOCK11 Deficiency.

BACKGROUND: Increasing evidence links genetic defects affecting actin-regulatory proteins to diseases with severe autoimmunity and autoinflammation, yet the underlying molecular mechanisms are poorly understood. Dedicator of cytokinesis 11 (DOCK11) activates the small Rho guanosine triphosphatase (GTPase) cell division cycle 42 (CDC42), a central regulator of actin cytoskeleton dynamics. The role of DOCK11 in human immune-cell function and disease remains unknown. METHODS: We conducted genetic, immunologic, and molecular assays in four patients from four unrelated families who presented with infections, early-onset severe immune dysregulation, normocytic anemia of variable severity associated with anisopoikilocytosis, and developmental delay. Functional assays were performed in patient-derived cells, as well as in mouse and zebrafish models. RESULTS: We identified rare, X-linked germline mutations in DOCK11 in the patients, leading to a loss of protein expression in two patients and impaired CDC42 activation in all four patients. Patient-derived T cells did not form filopodia and showed abnormal migration. In addition, the patient-derived T cells, as well as the T cells from Dock11-knockout mice, showed overt activation and production of proinflammatory cytokines that were associated with an increased degree of nuclear translocation of nuclear factor of activated T cell 1 (NFATc1). Anemia and aberrant erythrocyte morphologic features were recapitulated in a newly generated dock11-knockout zebrafish model, and anemia was amenable to rescue on ectopic expression of constitutively active CDC42. CONCLUSIONS: Germline hemizygous loss-of-function mutations affecting the actin regulator DOCK11 were shown to cause a previously unknown inborn error of hematopoiesis and immunity characterized by severe immune dysregulation and systemic inflammation, recurrent infections, and anemia. (Funded by the European Research Council and others.).

Biallelic NFATC1 mutations cause an inborn error of immunity with impaired CD8+ T-cell function and perturbed glycolysis.

The nuclear factor of activated T cells (NFAT) family of transcription factors plays central roles in adaptive immunity in murine models; however, their contribution to human immune homeostasis remains poorly defined. In a multigenerational pedigree, we identified 3 patients who carry germ line biallelic missense variants in NFATC1, presenting with recurrent infections, hypogammaglobulinemia, and decreased antibody responses. The compound heterozygous NFATC1 variants identified in these patients caused decreased stability and reduced the binding of DNA and interacting proteins. We observed defects in early activation and proliferation of T and B cells from these patients, amenable to rescue upon genetic reconstitution. Stimulation induced early T-cell activation and proliferation responses were delayed but not lost, reaching that of healthy controls at day 7, indicative of an adaptive capacity of the cells. Assessment of the metabolic capacity of patient T cells revealed that NFATc1 dysfunction rendered T cells unable to engage in glycolysis after stimulation, although oxidative metabolic processes were intact. We hypothesized that NFATc1-mutant T cells could compensate for the energy deficit due to defective glycolysis by using enhanced lipid metabolism as an adaptation, leading to a delayed, but not lost, activation responses. Indeed, we observed increased 13C-labeled palmitate incorporation into citrate, indicating higher fatty acid oxidation, and we demonstrated that metformin and rosiglitazone improved patient T-cell effector functions. Collectively, enabled by our molecular dissection of the consequences of loss-of-function NFATC1 mutations and extending the role of NFATc1 in human immunity beyond receptor signaling, we provide evidence of metabolic plasticity in the context of impaired glycolysis observed in patient T cells, alleviating delayed effector responses.

K+ Efflux-Independent NLRP3 Inflammasome Activation by Small Molecules Targeting Mitochondria.

Imiquimod is a small-molecule ligand of Toll-like receptor-7 (TLR7) that is licensed for the treatment of viral infections and cancers of the skin. Imiquimod has TLR7-independent activities that are mechanistically unexplained, including NLRP3 inflammasome activation in myeloid cells and apoptosis induction in cancer cells. We investigated the mechanism of inflammasome activation by imiquimod and the related molecule CL097 and determined that K+ efflux was dispensable for NLRP3 activation by these compounds. Imiquimod and CL097 inhibited the quinone oxidoreductases NQO2 and mitochondrial Complex I. This induced a burst of reactive oxygen species (ROS) and thiol oxidation, and led to NLRP3 activation via NEK7, a recently identified component of this inflammasome. Metabolic consequences of Complex I inhibition and endolysosomal effects of imiquimod might also contribute to NLRP3 activation. Our results reveal a K+ efflux-independent mechanism for NLRP3 activation and identify targets of imiquimod that might be clinically relevant.

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation.

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.

Failure to launch commercially-approved mesenchymal stromal cell therapies: what's the path forward? Proceedings of the International Society for Cell & Gene Therapy (ISCT) Annual Meeting Roundtable held in May 2023, Palais des Congrès de Paris, Organized by the ISCT MSC Scientific Committee.

Mesenchymal stromal cells (MSCs) are promising cell therapy candidates, but their debated efficacy in clinical trials still limits successful adoption. Here, we discuss proceedings from a roundtable session titled "Failure to Launch Mesenchymal Stromal Cells 10 Years Later: What's on the Horizon?" held at the International Society for Cell & Gene Therapy 2023 Annual Meeting. Panelists discussed recent progress toward developing patient-stratification approaches for MSC treatments, highlighting the role of baseline levels of inflammation in mediating MSC treatment efficacy. In addition, MSC critical quality attributes (CQAs) are beginning to be elucidated and applied to investigational MSC products, including immunomodulatory functional assays and other potency markers that will help to ensure product consistency and quality. Lastly, next-generation MSC products, such as culture-priming strategies, were discussed as a promising strategy to augment MSC basal fitness and therapeutic potency. Key variables that will need to be considered alongside investigations of patient stratification approaches, CQAs and next-generation MSC products include the specific disease target being evaluated, route of administration of the cells and cell manufacturing parameters; these factors will have to be matched with postulated mechanisms of action towards treatment efficacy. Taken together, patient stratification metrics paired with the selection of therapeutically potent MSCs (using rigorous CQAs and/or engineered MSC products) represent a path forward to improve clinical successes and regulatory endorsements.

A phase 1b study of glasdegib + azacitidine in patients with untreated acute myeloid leukemia and higher-risk myelodysplastic syndromes.

This phase 1b study evaluated glasdegib (100 mg once daily) + azacitidine in adults with newly diagnosed acute myeloid leukemia (AML), higher-risk myelodysplastic syndromes (MDS), or chronic myelomonocytic leukemia (CMML) who were ineligible for intensive chemotherapy. Of 72 patients enrolled, 12 were in a lead-in safety cohort (LIC) and 60 were in the AML and MDS (including CMML) expansion cohorts. In the LIC, the safety profile of glasdegib + azacitidine was determined to be consistent with those of glasdegib or azacitidine alone, with no evidence of drug-drug interaction. In the expansion cohort, the most frequently (≥ 10%) reported non-hematologic Grade ≥ 3 treatment-emergent adverse events were decreased appetite, electrocardiogram QT prolongation, and hypertension in the AML cohort and sepsis, diarrhea, hypotension, pneumonia, and hyperglycemia in the MDS cohort. Overall response rates in the AML and MDS cohorts were 30.0% and 33.3%, respectively; 47.4% and 46.7% of patients who were transfusion dependent at baseline achieved independence. Median overall survival (95% confidence interval) was 9.2 (6.2-14.0) months and 15.8 (9.3-21.9) months, respectively, and response was associated with molecular mutation clearance. Glasdegib + azacitidine in patients with newly diagnosed AML or MDS demonstrated an acceptable safety profile and preliminary evidence of clinical benefits.Trial registration: ClinicalTrials.gov NCT02367456.

PD-L1 overexpression correlates with JAK2-V617F mutational burden and is associated with 9p uniparental disomy in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are chronic stem cell disorders characterized by enhanced proliferation of myeloid cells, immune deregulation, and drug resistance. JAK2 somatic mutations drive the disease in 50-60% and CALR mutations in 25-30% of cases. Published data suggest that JAK2-V617F-mutated MPN cells express the resistance-related checkpoint PD-L1. By applying RNA-sequencing on granulocytes of 113 MPN patients, we demonstrate that PD-L1 expression is highest among polycythemia vera patients and that PD-L1 expression correlates with JAK2-V617F mutational burden (R = 0.52; p < .0001). Single nucleotide polymorphism (SNP) arrays showed that chromosome 9p uniparental disomy (UPD) covers both PD-L1 and JAK2 in all MPN patients examined. MPN cells in JAK2-V617F-positive patients expressed higher levels of PD-L1 if 9p UPD was present compared to when it was absent (p < .0001). Moreover, haplotype-based association analyses provided evidence for germline genetic factors at PD-L1 locus contributing to MPN susceptibility independently of the previously described GGCC risk haplotype. We also found that PD-L1 is highly expressed on putative CD34+ CD38- disease-initiating neoplastic stem cells (NSC) in both JAK2 and CALR-mutated MPN. PD-L1 overexpression decreased upon exposure to JAK2 blockers and BRD4-targeting agents, suggesting a role for JAK2-STAT5-signaling and BRD4 in PD-L1 expression. Whether targeting of PD-L1 can overcome NSC resistance in MPN remains to be elucidated in forthcoming studies.

Vectorial Catalysis in Surface-Anchored Nanometer-Sized Metal-Organic Frameworks-Based Microfluidic Devices.

Vectorial catalysis-controlling multi-step reactions in a programmed sequence and by defined spatial localization in a microscale device-is an enticing goal in bio-inspired catalysis research. However, translating concepts from natural cascade biocatalysis into artificial hierarchical chemical systems remains a challenge. Herein, we demonstrate integration of two different surface-anchored nanometer-sized metal-organic frameworks (MOFs) in a microfluidic device for modelling vectorial catalysis. Catalyst immobilization at defined sections along the microchannel and a two-step cascade reaction was conducted with full conversion after 30 seconds and high turnover frequencies (TOF≈105  h-1 ).

Mutational landscape of the transcriptome offers putative targets for immunotherapy of myeloproliferative neoplasms.

Ph-negative myeloproliferative neoplasms (MPNs) are hematological cancers that can be subdivided into entities with distinct clinical features. Somatic mutations in JAK2, CALR, and MPL have been described as drivers of the disease, together with a variable landscape of nondriver mutations. Despite detailed knowledge of disease mechanisms, targeted therapies effective enough to eliminate MPN cells are still missing. In this study of 113 MPN patients, we aimed to comprehensively characterize the mutational landscape of the granulocyte transcriptome using RNA sequencing data and subsequently examine the applicability of immunotherapeutic strategies for MPN patients. Following implementation of customized workflows and data filtering, we identified a total of 13 (12/13 novel) gene fusions, 231 nonsynonymous single nucleotide variants, and 21 insertions and deletions in 106 of 113 patients. We found a high frequency of SF3B1-mutated primary myelofibrosis patients (14%) with distinct 3' splicing patterns, many of these with a protein-altering potential. Finally, from all mutations detected, we generated a virtual peptide library and used NetMHC to predict 149 unique neoantigens in 62% of MPN patients. Peptides from CALR and MPL mutations provide a rich source of neoantigens as a result of their unique ability to bind many common MHC class I molecules. Finally, we propose that mutations derived from splicing defects present in SF3B1-mutated patients may offer an unexplored neoantigen repertoire in MPNs. We validated 35 predicted peptides to be strong MHC class I binders through direct binding of predicted peptides to MHC proteins in vitro. Our results may serve as a resource for personalized vaccine or adoptive cell-based therapy development.

Mitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial Cells.

RATIONALE: Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. OBJECTIVE: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. METHODS AND RESULTS: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. CONCLUSIONS: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.

Hematopoietic expression of a chimeric murine-human CALR oncoprotein allows the assessment of anti-CALR antibody immunotherapies in vivo.

Myeloproliferative neoplasms (MPNs) are characterized by a pathologic expansion of myeloid lineages. Mutations in JAK2, CALR and MPL genes are known to be three prominent MPN disease drivers. Mutant CALR (mutCALR) is an oncoprotein that interacts with and activates the thrombopoietin receptor (MPL) and represents an attractive target for targeted therapy of CALR mutated MPN. We generated a transgenic murine model with conditional expression of the human mutant exon 9 (del52) from the murine endogenous Calr locus. These mice develop essential thrombocythemia like phenotype with marked thrombocytosis and megakaryocytosis. The disease exacerbates with age showing prominent signs of splenomegaly and anemia. The disease is transplantable and mutCALR stem cells show proliferative advantage when compared to wild type stem cells. Transcriptome profiling of hematopoietic stem cells revealed oncogenic and inflammatory gene expression signatures. To demonstrate the applicability of the transgenic animals for immunotherapy, we treated mice with monoclonal antibody raised against the human mutCALR. The antibody treatment lowered platelet and stem cell counts in mutant mice. Secretion of mutCALR did not constitute a significant antibody sink. This animal model not only recapitulates human MPN but also serves as a relevant model for testing immunotherapeutic strategies targeting epitopes of the human mutCALR.

Health-related quality of life and utility outcomes with selinexor in relapsed/refractory diffuse large B-cell lymphoma.

Aim: Evaluate health-related quality of life (HRQoL) and health utility impact of single-agent selinexor in heavily pretreated patients with relapsed/refractory diffuse large B-cell lymphoma. Patients & methods: Functional Assessment of Cancer Therapy (FACT) - Lymphoma and EuroQoL five-dimensions five-levels data collected in the single-arm Phase IIb trial SADAL (NCT02227251) were analyzed with mixed-effects models. Results: Treatment responders maintained higher FACT - Lymphoma (p ≤ 0.05), FACT - General (p < 0.05) and EuroQoL five-dimensions five-levels index scores (p < 0.001) beginning in cycle 3. The estimated difference in health state utilities for treatment response and progressive disease was both statistically significant and clinically meaningful (mean difference: 0.07; p = 0.001). Conclusion: In patients with relapsed/refractory diffuse large B-cell lymphoma, objective response to selinexor was associated with HRQoL maintenance, reduction in disease-related HRQoL decrements and higher health utilities.

Lay abstract This work examined quality of life (QoL) among patients with relapsed/refractory diffuse large B-cell lymphoma with two to five prior therapies who received single-agent selinexor in the SADAL clinical trial. Analysis of patient-reported Functional Assessment of Cancer Therapy ­ Lymphoma and EuroQoL five-dimensions five-levels data showed that patients who had objective clinical response to selinexor maintained their QoL over the course of treatment. Grade ≥3 adverse events and serious adverse events were not associated with clinically meaningful negative QoL impacts. Clinical trial registration: NCT02227251 (ClinicalTrials.gov).

[Perioperative Atrial Fibrillation]. / Perioperatives Vorhofflimmern.

New atrial fibrillation is a risk factor for increased morbidity and mortality in the perioperative phase. The prevalence and incidence of atrial fibrillation depend on age, previous illnesses and the intercurrent diagnoses in the perioperative phase. Pathomechanisms for both permanent and acute forms of atrial fibrillation relate to electrophysiological, histopathological and other pathophysiological abnormalities. In the perioperative phase, decompensated heart failure, systemic inflammation, sepsis and cardiac surgery are risk factors for conversion to atrial fibrillation and/or rapid transition. The consequences of newly occurring and/or rapidly transferred atrial fibrillation are cardiac instability due to a drop in blood pressure or cardiac output volume or coronary underperfusion due to tachycardia and the risk of thromboembolism. Associated with this is a significantly increased long-term mortality risk in the elderly if atrial fibrillation occurs again in the perioperative phase. The diagnosis of atrial fibrillation is made in the monitor or 12-channel electrocardiogram. Echocardiography and the adenosine test can be included in the differential diagnosis in cases of doubt. Important current guidelines for the treatment of atrial fibrillation are the ESC (atrial fibrillation) and the ERC (Advanced Cardiac Life Support) guidelines. The conversion into the sinus rhythm can be done electrically or medicinally. It is indicated in cases of acute instability or symptomatic atrial fibrillation despite frequency control after cardiac thrombi have been excluded by echocardiography. A step-by-step concept for thromboembolism prophylaxis from initial parenteral and secondary oral therapy must take place up to four weeks after conversion to sinus rhythm or permanently in the case of permanent atrial fibrillation. The individual indication for thromboembolism prophylaxis results from the CHA2-DS2-VASc score.

Host-Guest Interactions in a Metal-Organic Framework Isoreticular Series for Molecular Photocatalytic CO2 Reduction.

A strategy to improve homogeneous molecular catalyst stability, efficiency, and selectivity is the immobilization on supporting surfaces or within host matrices. Herein, we examine the co-immobilization of a CO2 reduction catalyst [ReBr(CO)3 (4,4'-dcbpy)] and a photosensitizer [Ru(bpy)2 (5,5'-dcbpy)]Cl2 using the isoreticular series of metal-organic frameworks (MOFs) UiO-66, -67, and -68. Specific host pore size choice enables distinct catalyst and photosensitizer spatial location-either at the outer MOF particle surface or inside the MOF cavities-affecting catalyst stability, electronic communication between reaction center and photosensitizer, and consequently the apparent catalytic rates. These results allow for a rational understanding of an optimized supramolecular layout of catalyst, photosensitizer, and host matrix.

Looking at Silver-Based Nanoparticles in Environmental Water Samples: Repetitive Cloud Point Extraction Bridges Gaps in Electron Microscopy for Naturally Occurring Nanoparticles.

The growing use of silver-based nanoparticles (Ag-b-NPs) in everyday products goes hand in hand with their release into the environment, resulting in ng L-1 traces in natural water bodies. In order to assess their fate, possible transformations and ecotoxicology-essential information to proper risk assessment-particle size, shape, and chemical composition have to be determined. Transmission electron microscopy coupled with energy dispersive X-ray spectroscopy (TEM-EDX) is a powerful tool for determining these particle characteristics, but it requires high particle concentrations in order to produce statistically reliable results. In this study, we will present the extraction of Ag-b-NPs at environmentally relevant concentrations down to 5 ng L-1 from artificial as well as environmental water samples via cloud point extraction on a repetitive basis. The combination with an on-grid centrifugation technique ensures an efficient concentration and deposition of the extracted particles onto the TEM grid for subsequent TEM-EDX measurements. Furthermore, electron microscopy investigations were supplemented by single particle inductively coupled plasma mass spectrometry (sp-ICP-MS) measurements. Ag-b-NPs were successfully visualized and characterized at environmentally relevant concentrations of 5 ng L-1 with TEM-EDX and sp-ICP-MS measurements. Their size, shape, and chemical composition were not affected by the sample preparation.

A combinatorial screen of the CLOUD uncovers a synergy targeting the androgen receptor.

Approved drugs are invaluable tools to study biochemical pathways, and further characterization of these compounds may lead to repurposing of single drugs or combinations. Here we describe a collection of 308 small molecules representing the diversity of structures and molecular targets of all FDA-approved chemical entities. The CeMM Library of Unique Drugs (CLOUD) covers prodrugs and active forms at pharmacologically relevant concentrations and is ideally suited for combinatorial studies. We screened pairwise combinations of CLOUD drugs for impairment of cancer cell viability and discovered a synergistic interaction between flutamide and phenprocoumon (PPC). The combination of these drugs modulates the stability of the androgen receptor (AR) and resensitizes AR-mutant prostate cancer cells to flutamide. Mechanistically, we show that the AR is a substrate for γ-carboxylation, a post-translational modification inhibited by PPC. Collectively, our data suggest that PPC could be repurposed to tackle resistance to antiandrogens in prostate cancer patients.

Copper Drinking Water Pipes as a Previously Undocumented Source of Silver-Based Nanoparticles.

Wastewater streams are widely known to release silver-based nanoparticles (Ag-b-NPs) into the environment with a plethora of unknown consequences. Until recently, studies have commonly associated Ag-b-NP sources with products that contain these NPs for antimicrobial reasons, such as fabrics, cosmetics, and medical products. However, our study reveals that there is a thus far completely undocumented source of Ag-b-NPs: copper drinking water pipes. We applied cloud point extraction hyphenated to electrothermal atomic absorption spectrometry or single-particle inductively coupled plasma mass spectrometry to analyze the concentration and perform size-selective quantification of Ag-b-NPs in tap water passing through copper pipes. Up to 83 ng of total silver and 25 ng of Ag-b-NPs were present in tap water samples per liter, which resulted in an NP proportion of approximately 30% of total silver. In total, 96% of the measurable particle sizes ranged from 10 to 36 nm. Additionally, 53 µg of copper was released per liter tap water on average. The measurements included tap water from different sampling days and from four different buildings with varying ages, whereas Ag-b-NPs could be detected in the tap water of two buildings. Silver traces in the copper pipe material of 27.5 ± 4.4 µg g-1 were found to be responsible for the release of nanoparticulate silver into the tap water.

Inherited DOCK2 Deficiency in Patients with Early-Onset Invasive Infections.

Background Combined immunodeficiencies are marked by inborn errors of T-cell immunity in which the T cells that are present are quantitatively or functionally deficient. Impaired humoral immunity is also common. Patients have severe infections, autoimmunity, or both. The specific molecular, cellular, and clinical features of many types of combined immunodeficiencies remain unknown. Methods We performed genetic and cellular immunologic studies involving five unrelated children with early-onset invasive bacterial and viral infections, lymphopenia, and defective T-cell, B-cell, and natural killer (NK)-cell responses. Two patients died early in childhood; after allogeneic hematopoietic stem-cell transplantation, the other three had normalization of T-cell function and clinical improvement. Results We identified biallelic mutations in the dedicator of cytokinesis 2 gene (DOCK2) in these five patients. RAC1 activation was impaired in the T cells. Chemokine-induced migration and actin polymerization were defective in the T cells, B cells, and NK cells. NK-cell degranulation was also affected. Interferon-α and interferon-λ production by peripheral-blood mononuclear cells was diminished after viral infection. Moreover, in DOCK2-deficient fibroblasts, viral replication was increased and virus-induced cell death was enhanced; these conditions were normalized by treatment with interferon alfa-2b or after expression of wild-type DOCK2. Conclusions Autosomal recessive DOCK2 deficiency is a new mendelian disorder with pleiotropic defects of hematopoietic and nonhematopoietic immunity. Children with clinical features of combined immunodeficiencies, especially with early-onset, invasive infections, may have this condition. (Supported by the National Institutes of Health and others.).