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This first‐in‐human study evaluated the safety, tolerability, single‐ and multiple‐dose pharmacokinetic profiles with dietary influence, and pharmacodynamics (PD) of DFV890, an oral NLRP3 inhibitor, in healthy participants. In total, 122 participants were enrolled into a three‐part trial including single and 2‐week multiple ascending oral doses (SAD and MAD, respectively) of DFV890, and were randomized (3:1) to DFV890 or placebo (SAD [3–600 mg] and MAD [fasted: 10–200 mg, once‐daily or fed: 25 and 50 mg, twice‐daily]). DFV890 was generally well‐tolerated. Neither deaths nor serious adverse events were reported. A less than dose‐proportional increase in exposure was observed with the initially used crystalline suspension (3–300 mg); however, an adjusted suspension formulation using spray‐dried dispersion (SDD; 100–600 mg) confirmed dose‐proportional increase in exposure. Relative bioavailability between crystalline suspension and tablets, and food effect were evaluated at 100 mg. Under fasting conditions, Cmax of the tablet yielded 78% compared with the crystalline suspension, and both formulations showed comparable AUC. The fed condition led to a 2.05‐ and 1.49‐fold increase in Cmax and AUC0–last compared with the fasting condition. The median IC50 and IC90 for ex‐vivo lipopolysaccharide‐stimulated interleukin IL‐1β release inhibition (PD) were 61 (90% CI: 50, 70) and 1340 ng/mL (90% CI: 1190, 1490). Crystalline tablets of 100 mg once‐daily or 25 mg twice‐daily were sufficient to maintain ~90% of the IL‐1β release inhibition over 24 h at steady state. Data support dose and formulation selection for further development in diseases, in which an overactivated NLRP3 represents the underlying pathophysiology.

Background Chronic mucocutaneous candidiasis disease (CMCD) may result from various inborn errors of interleukin (IL)-17-mediated immunity. Twelve of the 13 causal mutations described to date affect the coiled-coil domain (CCD) of STAT1. Several mutations, including R274W in particular, are recurrent, but the underlying mechanism is unclear. Objective To investigate and describe nine patients with CMCD in Eastern and Central Europe, to assess the biochemical impact of STAT1 mutations, to determine cytokines in supernatants of Candida-exposed blood cells, to determine IL-17-producing T cell subsets and to determine STAT1 haplotypes in a family with the c.820C>T (R274W) mutation. Results The novel c.537C>A (N179K) STAT1 mutation was gain-of-function (GOF) for γ-activated factor (GAF)-dependent cellular responses. In a Russian patient, the cause of CMCD was the newly identified c.854 A>G (Q285R) STAT1 mutation, which was also GOF for GAF-dependent responses. The c.1154C>T (T385M) mutation affecting the DNA-binding domain (DBD) resulted in a gain of STAT1 phosphorylation in a Ukrainian patient. Impaired Candida-induced IL-17A and IL-22 secretion by leucocytes and lower levels of intracellular IL-17 and IL-22 production by T cells were found in several patients. Haplotype studies indicated that the c.820C>T (R274W) mutation was recurrent due to a hotspot rather than a founder effect. Severe clinical phenotypes, including intracranial aneurysm, are presented. Conclusions The c.537C>A and c.854A>G mutations affecting the CCD and the c.1154C>T mutation affecting the DBD of STAT1 are GOF. The c.820C>T mutation of STAT1 in patients with CMCD is recurrent due to a hotspot. Patients carrying GOF mutations of STAT1 may develop multiple intracranial aneurysms by hitherto unknown mechanisms.

Neurotropic herpesviruses can establish lifelong infection in humans and contribute to severe diseases including encephalitis and neurodegeneration. However, the mechanisms through which the brain’s immune system recognizes and controls viral infections propagating across synaptically linked neuronal circuits have remained unclear. Using a well-established model of alphaherpesvirus infection that reaches the brain exclusively via retrograde transsynaptic spread from the periphery, and in vivo two-photon imaging combined with high resolution microscopy, we show that microglia are recruited to and isolate infected neurons within hours. Selective elimination of microglia results in a marked increase in the spread of infection and egress of viral particles into the brain parenchyma, which are associated with diverse neurological symptoms. Microglia recruitment and clearance of infected cells require cell-autonomous P2Y12 signalling in microglia, triggered by nucleotides released from affected neurons. In turn, we identify microglia as key contributors to monocyte recruitment into the inflamed brain, which process is largely independent of P2Y12. P2Y12-positive microglia are also recruited to infected neurons in the human brain during viral encephalitis and both microglial responses and leukocyte numbers correlate with the severity of infection. Thus, our data identify a key role for microglial P2Y12 in defence against neurotropic viruses, whilst P2Y12-independent actions of microglia may contribute to neuroinflammation by facilitating monocyte recruitment to the sites of infection.

In this study, the authors use measures of carbon-14 in neuronal DNA from human stroke patient cortical tissue samples to show that, unlike previous studies done in rodents, they do not find any evidence of increased neurogenesis after an ischemic injury. In addition, DNA damage assays suggest that there is no increase in DNA rearrangement after this insult. It has been unclear whether ischemic stroke induces neurogenesis or neuronal DNA rearrangements in the human neocortex. Using immunohistochemistry; transcriptome, genome and ploidy analyses; and determination of nuclear bomb test–derived 14 C concentration in neuronal DNA, we found neither to be the case. A large proportion of cortical neurons displayed DNA fragmentation and DNA repair a short time after stroke, whereas neurons at chronic stages after stroke showed DNA integrity, demonstrating the relevance of an intact genome for survival.

Both patients carried the heterozygous germ line mutation detected by genomic DNA sequencing in the STAT1 gene, which encodes signal transducer and activator of transcription (STAT) 1 (see figure, appendix). The mechanism underlying reactivation disease in patients with STAT1 mutations and chronic mucocutaneous candidosis could involve the impairment of memory T cells, as reported in patients with hyper-IgE syndrome and STAT3 deficiency.5 The effect of recurrent viral infections on the prognosis of chronic mucocutaneous candidosis remains to be elucidated.

Non-muscle myosin II (NMII)-induced multicellular contractility is essential for development, maintenance and remodeling of tissue morphologies. Dysregulation of the cytoskeleton can lead to birth defects or enable cancer progression. We demonstrate that the Matrigel patterning assay, widely used to characterize endothelial cells, is a highly sensitive tool to evaluate cell contractility within a soft extracellular matrix (ECM) environment. We propose a computational model to explore how cell-exerted contractile forces can tear up the cell-Matrigel composite material and gradually remodel it into a network structure. We identify measures that are characteristic for cellular contractility and can be obtained from image analysis of the recorded patterning process. The assay was calibrated by inhibition of NMII activity in A431 epithelial carcinoma cells either directly with blebbistatin or indirectly with Y27632 Rho kinase inhibitor. Using Matrigel patterning as a bioassay, we provide the first functional demonstration that overexpression of S100A4, a calcium-binding protein that is frequently overexpressed in metastatic tumors and inhibits NMIIA activity by inducing filament disassembly, effectively reduces cell contractility.

Abdominal aortic aneurysm (AAA) is a lethal vascular disease characterized by intramural hemorrhage. This study delineates the signatures of heme and its metabolic imbalance related to progression and inflammation in AAA. Clinical analyses of patients undergoing open AAA surgery show that AAA patients exhibit vascular inflammation, with elevated serum CRP, IL-6, and heme levels correlating with the expression of heme-regulated gene Hmox1/HO-1 (heme oxygenase-1) in the affected aortic wall. Oxidation of hemoglobin to ferri state leading to accumulation of methemoglobin readily releasing heme occurs in human AAA and in angiotensin II (AngII)-induced AAA in apolipoprotein E-deficient mice. Transcriptomic analysis for AngII-induced AAA identifies upregulated genes predominantly enriched in inflammatory signaling, extracellular matrix degradation, oxidative stress pathways, and altered expression of genes related to heme metabolism including Hmox1. Immunohistochemistry for IL1β and TNFα confirms inflammatory activation within AAA tissues. The signatures of heme-responsive gene inductions, enhanced expression of HO-1 and H-ferritin, are detected. Mechanistic studies employing endothelial cells and smooth muscle cells reveal that heme exposure of resident cells markedly enhances the expression of IL1β and ICAM1, as well as the inflammasome component NLRP3, and such inflammatory response is controlled by HO-1. Intervention with Normosang (heme arginate), an HO-1 inducer, attenuates aneurysm progression, whereas HO-1 inhibition by Tin protoporphyrin IX abolishes this protection. Induction of HO-1 accompanied by elevated H-ferritin level also mitigated aortic wall inflammation as reflected by lowering IL1β and TNFα. These findings highlight the heme-HO-1-H-ferritin axis as an element of AAA pathogenesis and a potential therapeutic target.

Tissue morphogenesis and patterning during development involve the segregation of cell types. Segregation is driven by differential tissue surface tensions generated by cell types through controlling cell-cell contact formation by regulating adhesion and actomyosin contractility-based cellular cortical tensions. We use vertebrate tissue cell types and zebrafish germ layer progenitors as in vitro models of 3-dimensional heterotypic segregation and developed a quantitative analysis of their dynamics based on 3D time-lapse microscopy. We show that general inhibition of actomyosin contractility by the Rho kinase inhibitor Y27632 delays segregation. Cell type-specific inhibition of non-muscle myosin2 activity by overexpression of myosin assembly inhibitor S100A4 reduces tissue surface tension, manifested in decreased compaction during aggregation and inverted geometry observed during segregation. The same is observed when we express a constitutively active Rho kinase isoform to ubiquitously keep actomyosin contractility high at cell-cell and cell-medium interfaces and thus overriding the interface-specific regulation of cortical tensions. Tissue surface tension regulation can become an effective tool in tissue engineering. In vertebrate tissue cell types and zebrafish germ layer progenitors, interface-specific regulation of the actomyosin contractile system governs tissue surface tension generation and the geometry and dynamics of cell aggregation and segregation.

(1) Background: The mechanism of platinum resistance in ovarian cancer is not fully clarified, but the properly functioning DNA repair mechanism can counteract the effect of conventional anticancer treatment. The objective of our study was to evaluate the expression of an important DNA repair enzyme, the Poly (ADP-Ribose) Polymerase (PARP) expression in epithelial ovarian cancer (EOC) tissues depending on BRCA status and to assess its relationship with platinum resistance. (2) Methods: Immunostaining to highlight PARP protein expression was performed using a rabbit polyclonal anti-PARP antibody. The intensity and distribution of immunostaining were assessed by light. Somatic BRCA1 or BRCA2 mutation carriers were identified with bidirectional sequencing of DNA from archived tumor tissue, if the test could not be performed due to technical reasons from tumor cells, the sequencing was done from peripheral blood cells to identify germline mutation carriers. The median progression-free survival (PFS) was generated for each semiquantitative group of PARP expression among chemotherapy-naive cases at the time of PARP immunohistochemistry. (3) Results: In the overall population, negative PARP immunohistochemistry predicted significant PFS (20.1 vs. 11.9 months, p = 0.001) and OS (49 vs. 114 months, p = 0.014) benefit. Genotype-stratified subgroup analysis in BRCA-negative cases confirmed the role of PARP positivity indicating an unfavorable prognosis in the entire population (relapsed 73.91% vs. 92%; OR: 4.06; p = 0.04). In the cases of the subgroup carrying the BRCA mutation, the presence of PARP expression was not associated with less favorable relapse rates, but with marginal significance for overall survival predicted a lower chance of survival (OS more than 32 months 72.73% vs. 35%; OR: 0.2; p = 0.05). (4) Conclusion: The BRCA wild type patients with strong expression of PARP enzymes before the first set of chemotherapy have a poor prognosis.

Both patients carried the heterozygous germline mutation detected by genomic DNA sequencing in the STAT1 gene, which encodes signal transducer and activator of transcription (STAT) 1 (see figure, appendix). [...]to previously reported patients with STAT1 mutations and chronic mucocutaneous disease only, our patients presented with recurrent shingles and cutaneous lesions due to the reactivation of varicella and HSV infection.1,2 These unusual reactivations followed the normal courses of chickenpox and uneventful primary HSV infections in childhood. The mechanism underlying reactivation disease in patients with STAT1 mutations and chronic mucocutaneous candidosis could involve the impairment of memory T cells, as reported in patients with hyper-IgE syndrome and STAT3 deficiency.5 The effect of recurrent viral infections on the prognosis of chronic mucocutaneous candidosis remains to be elucidated.