Evaluation of Socioeconomic and Racial/Ethnic Disparities in All-Cause Mortality in Adolescents and Young Adults With Cancer.

BACKGROUND: Adolescent and young adult (AYA) patients with cancer have historically been understudied. Few studies have examined survival disparities associated with racial/ethnic and socioeconomic status (SES) and do not account for the influence of insurance status and access to care. We evaluated the association of SES and race/ethnicity with overall mortality for AYA patients who were members of an integrated health system with relatively equal access to care. METHODS: AYA patients diagnosed with the 15 most common cancer types during 2010 through 2018 at Kaiser Permanente Southern California were included. Neighborhood Deprivation Index (NDI) quartile (Q1: least deprived; Q4: most deprived) was used as a measure of SES. Mortality rate per 1,000 person-years was calculated for each racial/ethnic and NDI subgroup. Multivariable Cox model was used to estimate hazard ratios (HRs) for all-cause mortality adjusting for sex, age and stage at diagnosis, cancer type, race/ethnicity, and NDI. RESULTS: Data for 6,379 patients were tracked for a maximum of 10 years. Crude mortality rates were higher among non-White racial/ethnic patients compared with non-Hispanic (NH)-White patients. In the Cox model, Hispanic (HR, 1.31; P=.004) and NH-Black (HR, 1.34; P=.05) patients experienced significantly higher all-cause mortality risk compared with NH-White patients. Patients from more deprived neighborhoods had higher mortality risk. In the Cox model, there was no significant difference in all-cause mortality between Q1 and Q2 through Q4 (Q2: HR, 0.88; P=.26, Q3: HR, 0.94; P=.56, and Q4: HR, 0.95; P=.70). CONCLUSIONS: For AYAs with cancer with similar access to care, Hispanic and NH-Black patients have higher risk of all-cause mortality than NH-White patients, whereas no significant SES-associated survival disparities were observed. These findings warrant further investigation, awareness, and intervention to address inequities in cancer care among vulnerable populations.

Spatial localization of CD16a at the human NK cell ADCC lytic synapse.

Natural Killer (NK) cells utilize effector functions, including antibody-dependent cellular cytotoxicity (ADCC), for the clearance of viral infection and cellular malignancies. NK cell ADCC is mediated by FcγRIIIa (CD16a) binding to the fragment crystallizable (Fc) region of immunoglobulin G (IgG) within immune complexes on a target cell surface. While antibody-induced clustering of CD16a is thought to drive ADCC, the molecular basis for this activity has not been fully described. Here we use MINFLUX nanoscopy to map the spatial distribution of stoichiometrically labeled CD16a across the NK cell membrane, revealing the presence of pairs of CD16a molecules with intra-doublet distance of approximately 17 nm. NK cells activated on supported lipid bilayers by Trastuzumab results in an increase of synaptic regions with greater CD16a density. Our results provide the highest spatial resolution yet described for CD16a imaging, offering new insight into how CD16a organization within the immune synapse could influence ADCC activity. MINFLUX holds great promise to further unravel the molecular details driving CD16a-based activation of NK cells.

Imidazo[1,2-b]pyridazines as inhibitors of DYRK kinases.

Selective inhibitors of DYRK1A are of interest for the treatment of cancer, Type 2 diabetes and neurological disorders. Optimization of imidazo [1,2-b]pyridazine fragment 1 through structure-activity relationship exploration and in silico drug design efforts led to the discovery of compound 17 as a potent cellular inhibitor of DYRK1A with selectivity over much of the kinome. The binding mode of compound 17 was elucidated with X-ray crystallography, facilitating the rational design of compound 29, an imidazo [1,2-b]pyridazine with improved kinase selectivity with respect to closely related CLK kinases.

Reduced executive and reward connectivity is associated with smoking cessation response to repetitive transcranial magnetic stimulation: A double-blind, randomized, sham-controlled trial.

Repetitive transcranial magnetic stimulation (rTMS) can reduce cue-elicited craving, decrease cigarette consumption, and increase the abstinence rate in tobacco use disorders (TUDs). We used functional magnetic resonance imaging (fMRI) to investigate the effect of 10 sessions of rTMS on cortical activity and neural networks in treatment-seeking smokers. Smoking cue exposure fMRI scans were acquired before and after the 10 sessions of active or sham rTMS (10 Hz, 3000 pulses per session) to the left dorsal lateral prefrontal cortex (DLPFC) in 42 treatment-seeking smokers (≥ 10 cigarettes per day). Brain activity and functional connectivity were compared before and after 10 sessions of rTMS. Ten sessions of rTMS significantly reduced the number of cigarettes consumed per day (62.93%) compared to sham treatment (39.43%) at the end of treatment (p = 0.027). fMRI results showed that the rTMS treatment increased brain activity in the dorsal anterior cingulate cortex (dACC) and DLPFC, but decreased brain activity in the bilateral medial orbitofrontal cortex (mOFC). The lower strength of dACC and mOFC connectivity was associated with quitting smoking (Wald score = 5.00, p = 0.025). The reduction of cigarette consumption significantly correlated with the increased brain activation in the dACC (r = 0.76, p = 0.0001). By increasing the brain activity in the dACC and prefrontal cortex and decreasing brain activity in the mOFC, 10 sessions of rTMS significantly reduced cigarette consumption and increased quit rate. Reduced drive-reward and executive control functional connectivity was associated with the smoking cessation effect from rTMS. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02401672.

Direct observation of the conformational states of PIEZO1.

PIEZOs are mechanosensitive ion channels that convert force into chemoelectric signals1,2 and have essential roles in diverse physiological settings3. In vitro studies have proposed that PIEZO channels transduce mechanical force through the deformation of extensive blades of transmembrane domains emanating from a central ion-conducting pore4-8. However, little is known about how these channels interact with their native environment and which molecular movements underlie activation. Here we directly observe the conformational dynamics of the blades of individual PIEZO1 molecules in a cell using nanoscopic fluorescence imaging. Compared with previous structural models of PIEZO1, we show that the blades are significantly expanded at rest by the bending stress exerted by the plasma membrane. The degree of expansion varies dramatically along the length of the blade, where decreased binding strength between subdomains can explain increased flexibility of the distal blade. Using chemical and mechanical modulators of PIEZO1, we show that blade expansion and channel activation are correlated. Our findings begin to uncover how PIEZO1 is activated in a native environment. More generally, as we reliably detect conformational shifts of single nanometres from populations of channels, we expect that this approach will serve as a framework for the structural analysis of membrane proteins through nanoscopic imaging.

Cocaine-induced granulomatosis with polyangiitis-an under-recognized condition.

Objectives: Cocaine and cocaine mixed with levamisole are increasingly used in the UK and result in significant direct nasal damage in addition to promoting vasculitis. Our aims were as follows: (1) to identify the main symptoms and presentation of cocaine-induced vasculitis; (2) to provide evidence regarding the best practice for the investigation and diagnosis of cocaine-induced vasculitis; and (3) to analyse the clinical outcomes of patients in order to understand the optimal management for the condition. Methods: We performed a retrospective case series analysis of patients presenting with cocaine-induced midline destructive lesions or vasculitis compatible with granulomatosis with polyangiitis (GPA) from two large tertiary vasculitis clinics between 2016 and 2021. Results: Forty-two patients (29 Birmingham, 13 London) with cocaine-induced midline lesions or systemic disease were identified. The median age was 41 years (range 23-66 years). Current cocaine use was common, and 20 of 23 samples provided were positive when routine urine toxicology was performed; 9 patients who denied ever using cocaine were identified as using cocaine based on urine toxicology analysis, and 11 who stated they were ex-users still tested positive. There was a high incidence of septal perforation (75%) and oronasal fistula (15%). Systemic manifestations were less common (27%), and only one patient had acute kidney injury. Fifty-six per cent of our patients were PR3-ANCA positive, with none testing positive for MPO-ANCA. Symptom remission required cocaine discontinuation even when immunosuppression was administered. Conclusion: Patients with destructive nasal lesions, especially young patients, should have urine toxicology performed for cocaine before diagnosing GPA and considering immunosuppressive therapy. The ANCA pattern is not specific for cocaine-induced midline destructive lesions. Treatment should be focused on cocaine cessation and conservative management in the first instance in the absence of organ-threatening disease.

Proteinase 3 promotes formation of multinucleated giant cells and granuloma-like structures in patients with granulomatosis with polyangiitis.

OBJECTIVES: Granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are autoimmune vasculitides associated with antineutrophil cytoplasm antibodies that target proteinase 3 (PR3) or myeloperoxidase (MPO) found within neutrophils and monocytes. Granulomas are exclusively found in GPA and form around multinucleated giant cells (MGCs), at sites of microabscesses, containing apoptotic and necrotic neutrophils. Since patients with GPA have augmented neutrophil PR3 expression, and PR3-expressing apoptotic cells frustrate macrophage phagocytosis and cellular clearance, we investigated the role of PR3 in stimulating giant cell and granuloma formation. METHODS: We stimulated purified monocytes and whole peripheral blood mononuclear cells (PBMCs) from patients with GPA, patients with MPA or healthy controls with PR3 or MPO and visualised MGC and granuloma-like structure formation using light, confocal and electron microscopy, as well as measuring the cell cytokine production. We investigated the expression of PR3 binding partners on monocytes and tested the impact of their inhibition. Finally, we injected zebrafish with PR3 and characterised granuloma formation in a novel animal model. RESULTS: In vitro, PR3 promoted monocyte-derived MGC formation using cells from patients with GPA but not from patients with MPA, and this was dependent on soluble interleukin 6 (IL-6), as well as monocyte MAC-1 and protease-activated receptor-2, found to be overexpressed in the cells of patients with GPA. PBMCs stimulated by PR3 formed granuloma-like structures with central MGC surrounded by T cells. This effect of PR3 was confirmed in vivo using zebrafish and was inhibited by niclosamide, a IL-6-STAT3 pathway inhibitor. CONCLUSIONS: These data provide a mechanistic basis for granuloma formation in GPA and a rationale for novel therapeutic approaches.

GPR97 triggers inflammatory processes in human neutrophils via a macromolecular complex upstream of PAR2 activation.

Neutrophils play essential anti-microbial and inflammatory roles in host defense, however, their activities require tight regulation as dysfunction often leads to detrimental inflammatory and autoimmune diseases. Here we show that the adhesion molecule GPR97 allosterically activates CD177-associated membrane proteinase 3 (mPR3), and in conjugation with several protein interaction partners leads to neutrophil activation in humans. Crystallographic and deletion analysis of the GPR97 extracellular region identified two independent mPR3-binding domains. Mechanistically, the efficient binding and activation of mPR3 by GPR97 requires the macromolecular CD177/GPR97/PAR2/CD16b complex and induces the activation of PAR2, a G protein-coupled receptor known for its function in inflammation. Triggering PAR2 by the upstream complex leads to strong inflammatory activation, prompting anti-microbial activities and endothelial dysfunction. The role of the complex in pathologic inflammation is underscored by the finding that both GPR97 and mPR3 are upregulated on the surface of disease-associated neutrophils. In summary, we identify a PAR2 activation mechanism that directs neutrophil activation, and thus inflammation. The PR3/CD177/GPR97/PAR2/CD16b protein complex, therefore, represents a potential therapeutic target for neutrophil-mediated inflammatory diseases.

Differential dysregulation of granule subsets in WASH-deficient neutrophil leukocytes resulting in inflammation.

Dysregulated secretion in neutrophil leukocytes associates with human inflammatory disease. The exocytosis response to triggering stimuli is sequential; gelatinase granules modulate the initiation of the innate immune response, followed by the release of pro-inflammatory azurophilic granules, requiring stronger stimulation. Exocytosis requires actin depolymerization which is actively counteracted under non-stimulatory conditions. Here we show that the actin nucleator, WASH, is necessary to maintain azurophilic granules in their refractory state by granule actin entrapment and interference with the Rab27a-JFC1 exocytic machinery. On the contrary, gelatinase granules of WASH-deficient neutrophil leukocytes are characterized by decreased Rac1, shortened granule-associated actin comets and impaired exocytosis. Rac1 activation restores exocytosis of these granules. In vivo, WASH deficiency induces exacerbated azurophilic granule exocytosis, inflammation, and decreased survival. WASH deficiency thus differentially impacts neutrophil granule subtypes, impairing exocytosis of granules that mediate the initiation of the neutrophil innate response while exacerbating pro-inflammatory granule secretion.

Association between bone mineral density at different anatomical sites and both mortality and fracture risk in patients receiving renal replacement therapy: a longitudinal study.

Background: The clinical utility of bone mineral density (BMD) measurement by dual-energy X-ray absorptiometry (DXA) is debated in end-stage kidney disease (ESKD). We assessed the ability of BMD measured at different anatomical sites to predict mortality and fracture risk in patients requiring renal replacement therapy (RRT). Methods: We reviewed all-cause mortality as well as incident hip and overall fracture risk in RRT patients who had BMD measured at the femoral neck, lumbar spine, arm, head, pelvis and total body as part of their routine follow-up between January 2004 and June 2012 at a single university centre. Results: A total of 588 patients were included. The median follow-up was 6.5 years, the mean age was 59.6 years and 57.9% were males. Femoral neck BMD (FNBMD) (normal/high versus low) was negatively associated with mortality in univariate and multivariate analyses (P < .001 and P = .048, respectively). Other sites of BMD measurements were not associated with mortality. In multivariate analysis, FNBMD was negatively associated with hip and any fracture risk (P = .004 and P = .013, respectively). No significant interaction was found between FNBMD and gender or parathyroid hormone (PTH) (P = .112 and P = .794, respectively). Conclusions: BMD measured at the femoral neck is predictive of mortality in patients requiring RRT, regardless of modality. Low BMD might be a marker of global patient frailty rather than a direct causal factor in this setting. FNBMD is also a strong predictor of hip and any fracture risk in this population, regardless of bone turnover as assessed by PTH levels. FNBMD is thus an overall prognostic marker in patients requiring RRT.

Disparity in peripheral and renal B-cell depletion with rituximab in systemic lupus erythematosus: an opportunity for obinutuzumab?

OBJECTIVES: To investigate key factors that may contribute to the variability of rituximab-mediated peripheral and renal B cell depletion (BCD) in SLE. METHODS: We analysed: (i) CD19+ B cell counts in patients with SLE before and 1, 2, 3 and 6 months after treatment with rituximab, comparing them with RA patients; (ii) the presence of B cells in renal biopsies after rituximab therapy; (iii) whether the duration of BCD correlated with patient demographics and B cell expression of CD20 and FcγRIIb; and (iv) the effect of B cell activation factor (BAFF) on the efficiency of rituximab and obinutuzumab at inducing BCD in whole blood assays, in vitro. RESULTS: In SLE (n = 71), the duration of BCD was shorter compared with RA (n = 27). B cells were detectable in renal biopsy samples (n = 6) after treatment with rituximab in all patients with poor response while peripheral blood B cells remained low or undetectable in the same patients. There were no significant relationships between peripheral BCD and patient age, disease duration, serum C3 levels or the level of expression of B cell surface proteins CD20 and FcγRIIb. Obinutuzumab was more efficient than rituximab at inducing BCD in whole blood assays, regardless of excess BAFF. CONCLUSIONS: BCD in SLE is less efficient than in RA. Renal B cell presence following rituximab treatment was associated with poor outcomes. No significant relationships between any measured B cell related, clinical or laboratory parameters and the efficiency of BCD by rituximab was found. Obinutuzumab was superior to rituximab at inducing BCD.

Neutrophils are key mediators in crescentic glomerulonephritis and targets for new therapeutic approaches.

Crescentic glomerulonephritis (CGN) results from a diverse set of diseases associated with immune dysregulation and the breakdown of self-tolerance to a wide range of autoantigens, some known and some that remain unknown. Experimental data demonstrate that neutrophils have an important role in the pathogenesis of CGN. Upon activation, neutrophils generate reactive oxygen species, release serine proteases and form neutrophil extracellular traps (NETs), all of which can induce direct tissue damage. In addition, serine proteases such as myeloperoxidase and proteinase 3, presented on NETs, can be processed and recognized as autoantigens, leading to the generation and maintenance of autoimmune responses in susceptible individuals. The basis of the specificity of autoimmune responses in different patients to NET proteins is unclear, but relates at least in part to differences in human leucocyte antigen expression. Conditions associated with CGN are often characterized by aberrant neutrophil activation and NETosis and, in some, impaired NET degradation. Targeting neutrophil degranulation and NETosis is now possible using a variety of novel compounds and may provide a promising therapeutic alternative to glucocorticoid use, which has been a mainstay of management in CGN for decades and is associated with significant adverse effects. In this review, we discuss the evidence supporting the role of neutrophils in the development of CGN and the pathways identified in neutrophil degranulation and NETosis that may translate to novel therapeutic applications.

Discovery and Characterization of Selective and Ligand-Efficient DYRK Inhibitors.

Dual-specificity tyrosine-regulated kinase 1A (DYRK1A) regulates the proliferation and differentiation of neuronal progenitor cells during brain development. Consequently, DYRK1A has attracted interest as a target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Down's syndrome. Recently, the inhibition of DYRK1A has been investigated as a potential treatment for diabetes, while DYRK1A's role as a mediator in the cell cycle has garnered interest in oncologic indications. Structure-activity relationship (SAR) analysis in combination with high-resolution X-ray crystallography leads to a series of pyrazolo[1,5-b]pyridazine inhibitors with excellent ligand efficiencies, good physicochemical properties, and a high degree of selectivity over the kinome. Compound 11 exhibited good permeability and cellular activity without P-glycoprotein liability, extending the utility of 11 in an in vivo setting. These pyrazolo[1,5-b]pyridazines are a viable lead series in the discovery of new therapies for the treatment of diseases linked to DYRK1A function.

Variability in the Occupancy of Escherichia coli O157 Integration Sites by Shiga Toxin-Encoding Prophages.

Escherichia coli O157:H7 strains often produce Shiga toxins encoded by genes on lambdoid bacteriophages that insert into multiple loci as prophages. O157 strains were classified into distinct clades that vary in virulence. Herein, we used PCR assays to examine Shiga toxin (Stx) prophage occupancy in yehV, argW, wrbA, and sbcB among 346 O157 strains representing nine clades. Overall, yehV was occupied in most strains (n = 334, 96.5%), followed by wrbA (n = 213, 61.6%), argW (n = 103, 29.8%), and sbcB (n = 93, 26.9%). Twelve occupancy profiles were identified that varied in frequency and differed across clades. Strains belonging to clade 8 were more likely to have occupied sbcB and argW sites compared to other clades (p < 0.0001), while clade 2 strains were more likely to have occupied wrbA sites (p < 0.0001). Clade 8 strains also had more than the expected number of occupied sites based on the presence of stx variants (p < 0.0001). Deletion of a 20 kb non-Stx prophage occupying yehV in a clade 8 strain resulted in an ~18-fold decrease in stx2 expression. These data highlight the complexity of Stx prophage integration and demonstrate that clade 8 strains, which were previously linked to hemolytic uremic syndrome, have unique Stx prophage occupancy profiles that can impact stx2 expression.

Discrimination between Functional and Non-functional Cellular Gag Complexes involved in HIV-1 Assembly.

HIV-1 Gag and Gag-Pol are responsible for viral assembly and maturation and represent a major paradigm for enveloped virus assembly. Numerous intracellular Gag-containing complexes (GCCs) have been identified in cellular lysates using sucrose gradient ultracentrifugation. While these complexes are universally present in Gag-expressing cells, their roles in virus assembly are not well understood. Here we demonstrate that most GCC species are predominantly comprised of monomeric or dimeric Gag molecules bound to ribosomal complexes, and as such, are not on-pathway intermediates in HIV assembly. Rather, these GCCs represent a population of Gag that is not yet functionally committed for incorporation into a viable virion precursor. We hypothesize that these complexes act as a reservoir of monomeric Gag that can incorporate into assembling viruses, and serve to mitigate non-specific intracellular Gag oligomerization. We have identified a subset of large GCC complexes, comprising more than 20 Gag molecules, that may be equivalent to membrane-associated puncta previously shown to be bona fide assembling-virus intermediates. This work provides a clear rationale for the existence of diverse GCCs, and serves as the foundation for characterizing on-pathway intermediates early in virus assembly.