TDP2 is a regulator of estrogen-responsive oncogene expression.

With its ligand estrogen, the estrogen receptor (ER) initiates a global transcriptional program, promoting cell growth. This process involves topoisomerase 2 (TOP2), a key protein in resolving topological issues during transcription by cleaving a DNA duplex, passing another duplex through the break, and repairing the break. Recent studies revealed the involvement of various DNA repair proteins in the repair of TOP2-induced breaks, suggesting potential alternative repair pathways in cases where TOP2 is halted after cleavage. However, the contribution of these proteins in ER-induced transcriptional regulation remains unclear. We investigated the role of tyrosyl-DNA phosphodiesterase 2 (TDP2), an enzyme for the removal of halted TOP2 from the DNA ends, in the estrogen-induced transcriptome using both targeted and global transcription analyses. MYC activation by estrogen, a TOP2-dependent and transient event, became prolonged in the absence of TDP2 in both TDP2-deficient cells and mice. Bulk and single-cell RNA-seq analyses defined MYC and CCND1 as oncogenes whose estrogen response is tightly regulated by TDP2. These results suggest that TDP2 may inherently participate in the repair of estrogen-induced breaks at specific genomic loci, exerting precise control over oncogenic gene expression.

IL-1 receptor 1 signaling shapes the development of viral antigen-specific CD4+ T cell responses following COVID-19 mRNA vaccination.

BACKGROUND: The innate immune cytokine interleukin (IL)-1 can affect T cell immunity, a critical factor in host defense. In a previous study, we identified a subset of human CD4+ T cells which express IL-1 receptor 1 (IL-1R1). However, the expression of such receptor by viral antigen-specific CD4+ T cells and its biological implication remain largely unexplored. This led us to investigate the implication of IL-1R1 in the development of viral antigen-specific CD4+ T cell responses in humans, including healthy individuals and patients with primary antibody deficiency (PAD), and animals. METHODS: We characterized CD4+ T cells specific for SARS-CoV-2 spike (S) protein, influenza virus, and cytomegalovirus utilizing multiplexed single cell RNA-seq, mass cytometry and flow cytometry followed by an animal study. FINDINGS: In healthy individuals, CD4+ T cells specific for viral antigens, including S protein, highly expressed IL-1R1. IL-1ß promoted interferon (IFN)-γ expression by S protein-stimulated CD4+ T cells, supporting the functional implication of IL-1R1. Following the 2nd dose of COVID-19 mRNA vaccines, S protein-specific CD4+ T cells with high levels of IL-1R1 increased, likely reflecting repetitive antigenic stimulation. The expression levels of IL-1R1 by such cells correlated with the development of serum anti-S protein IgG antibody. A similar finding of increased expression of IL-1R1 by S protein-specific CD4+ T cells was also observed in patients with PAD following COVID-19 mRNA vaccination although the expression levels of IL-1R1 by such cells did not correlate with the levels of serum anti-S protein IgG antibody. In mice immunized with COVID-19 mRNA vaccine, neutralizing IL-1R1 decreased IFN-γ expression by S protein-specific CD4+ T cells and the development of anti-S protein IgG antibody. INTERPRETATION: Our results demonstrate the significance of IL-1R1 expression in CD4+ T cells for the development of viral antigen-specific CD4+ T cell responses, contributing to humoral immunity. This provides an insight into the regulation of adaptive immune responses to viruses via the IL-1 and IL-1R1 interface. FUNDING: Moderna to HJP, National Institutes of Health (NIH) 1R01AG056728 and R01AG055362 to IK and KL2 TR001862 to JJS, Quest Diagnostics to IK and RB, and the Mathers Foundation to RB.

Downregulation of IRF8 in alveolar macrophages by G-CSF promotes metastatic tumor progression.

Tissue-resident macrophages (TRMs) are abundant immune cells within pre-metastatic sites, yet their functional contributions to metastasis remain incompletely understood. Here, we show that alveolar macrophages (AMs), the main TRMs of the lung, are susceptible to downregulation of the immune stimulatory transcription factor IRF8, impairing anti-metastatic activity in models of metastatic breast cancer. G-CSF is a key tumor-associated factor (TAF) that acts upon AMs to reduce IRF8 levels and facilitate metastasis. Translational relevance of IRF8 downregulation was observed among macrophage precursors in breast cancer and a CD68hiIRF8loG-CSFhi gene signature suggests poorer prognosis in triple-negative breast cancer (TNBC), a G-CSF-expressing subtype. Our data highlight the underappreciated, pro-metastatic roles of AMs in response to G-CSF and identify the contribution of IRF8-deficient AMs to metastatic burden. AMs are an attractive target of local neoadjuvant G-CSF blockade to recover anti-metastatic activity.

Aging gene signature of memory CD8+ T cells is associated with neurocognitive functioning in Alzheimer's disease.

BACKGROUND: Memory CD8+ T cells expand with age. We previously demonstrated an age-associated expansion of effector memory (EM) CD8+ T cells expressing low levels of IL-7 receptor alpha (IL-7Rαlow) and the presence of its gene signature (i.e., IL-7Rαlow aging genes) in peripheral blood of older adults without Alzheimer's disease (AD). Considering age as the strongest risk factor for AD and the recent finding of EM CD8+ T cell expansion, mostly IL-7Rαlow cells, in AD, we investigated whether subjects with AD have alterations in IL-7Rαlow aging gene signature, especially in relation to genes possibly associated with AD and disease severity. RESULTS: We identified a set of 29 candidate genes (i.e., putative AD genes) which could be differentially expressed in peripheral blood of patients with AD through the systematic search of publicly available datasets. Of the 29 putative AD genes, 9 genes (31%) were IL-7Rαlow aging genes (P < 0.001), suggesting the possible implication of IL-7Rαlow aging genes in AD. These findings were validated by RT-qPCR analysis of 40 genes, including 29 putative AD genes, additional 9 top IL-7R⍺low aging but not the putative AD genes, and 2 inflammatory control genes in peripheral blood of cognitively normal persons (CN, 38 subjects) and patients with AD (40 mild cognitive impairment and 43 dementia subjects). The RT-qPCR results showed 8 differentially expressed genes between AD and CN groups; five (62.5%) of which were top IL-7Rαlow aging genes (FGFBP2, GZMH, NUAK1, PRSS23, TGFBR3) not previously reported to be altered in AD. Unbiased clustering analysis revealed 3 clusters of dementia patients with distinct expression levels of the 40 analyzed genes, including IL-7Rαlow aging genes, which were associated with neurocognitive function as determined by MoCA, CDRsob and neuropsychological testing. CONCLUSIONS: We report differential expression of "normal" aging genes associated with IL-7Rαlow EM CD8+ T cells in peripheral blood of patients with AD, and the significance of such gene expression in clustering subjects with dementia due to AD into groups with different levels of cognitive functioning. These results provide a platform for studies investigating the possible implications of age-related immune changes, including those associated with CD8+ T cells, in AD.

Complement C1q essential for aeroallergen sensitization via CSF1R+ conventional dendritic cells type 2.

BACKGROUND: Dendritic cells (DCs) are heterogeneous, comprising multiple subsets with unique functional specifications. Our previous work has demonstrated that the specific conventional type 2 DC subset, CSF1R+cDC2s, plays a critical role in sensing aeroallergens. OBJECTIVE: It remains to be understood how CSF1R+cDC2s recognize inhaled allergens. We sought to elucidate the transcriptomic programs and receptor-ligand interactions essential for function of this subset in allergen sensitization. METHODS: We applied single-cell RNA sequencing to mouse lung DCs. Conventional DC-selective knockout mouse models were employed, and mice were subjected to inhaled allergen sensitization with multiple readouts of asthma pathology. Under the clinical arm of this work, human lung transcriptomic data were integrated with mouse data, and bronchoalveolar lavage (BAL) specimens were collected from subjects undergoing allergen provocation, with samples assayed for C1q. RESULTS: We found that C1q is selectively enriched in lung CSF1R+cDC2s, but not in other lung cDC2 or cDC1 subsets. Depletion of C1q in conventional DCs significantly attenuates allergen sensing and features of asthma. Additionally, we found that C1q binds directly to human dust mite allergen, and the C1q receptor CD91 (LRP1) is required for lung CSF1R+cDC2s to recognize the C1q-allergen complex and induce allergic lung inflammation. Lastly, C1q is enriched in human BAL samples following subsegmental allergen challenge, and human RNA sequencing data demonstrate close homology between lung IGSF21+DCs and mouse CSF1R+cDC2s. CONCLUSIONS: C1q is secreted from the CSF1R+cDC2 subset among conventional DCs. Our data indicate that the C1q-LRP1 axis represents a candidate for translational therapeutics in the prevention and suppression of allergic lung inflammation.

Autonomous action and cooperativity between the ONECUT2 transcription factor and its 3' untranslated region.

The transcription factor ONECUT2 (OC2) is a master transcriptional regulator operating in metastatic castration-resistant prostate cancer that suppresses androgen receptor activity and promotes neural differentiation and tumor cell survival. OC2 mRNA possesses an unusually long (14,575 nt), evolutionarily conserved 3' untranslated region (3' UTR) with many microRNA binding sites, including up to 26 miR-9 sites. This is notable because miR-9 targets many of the same genes regulated by the OC2 protein. Paradoxically, OC2 expression is high in tissues with high miR-9 expression. The length and complex secondary structure of OC2 mRNA suggests that it is a potent master competing endogenous RNA (ceRNA) capable of sequestering miRNAs. Here, we describe a novel role for OC2 3' UTR in lethal prostate cancer consistent with a function as a ceRNA. A plausible ceRNA network in OC2-driven tumors was constructed computationally and then confirmed in prostate cancer cell lines. Genes regulated by OC2 3' UTR exhibited high overlap (up to 45%) with genes driven by the overexpression of the OC2 protein in the absence of 3' UTR, indicating a cooperative functional relationship between the OC2 protein and its 3' UTR. These overlapping networks suggest an evolutionarily conserved mechanism to reinforce OC2 transcription by protection of OC2-regulated mRNAs from miRNA suppression. Both the protein and 3' UTR showed increased polycomb-repressive complex activity. The expression of OC2 3' UTR mRNA alone (without protein) dramatically increased the metastatic potential by in vitro assays. Additionally, OC2 3' UTR increased the expression of Aldo-Keto reductase and UDP-glucuronyl transferase family genes responsible for altering the androgen synthesis pathway. ONECUT2 represents the first-described dual-modality transcript that operates as both a key transcription factor driving castration-resistant prostate cancer and a master ceRNA that promotes and protects the same transcriptional network.

Phospholipid Encapsulation of an Anti-Fibrotic Endopeptide to Enhance Cellular Uptake and Myocardial Retention.

BACKGROUND: Cardioprotective effects of N-acetyl-ser-asp-lys-pro (Ac-SDKP) have been reported in preclinical models of myocardial remodeling. However, the rapid degradation of this endogenous peptide in vivo limits its clinical use. METHOD: To prolong its bioavailability, Ac-SDKP was encapsulated by phosphocholine lipid bilayers (liposomes) similar to mammalian cell membranes. The physical properties of the liposome structures were assessed by dynamic light scattering and scanning electron microscopy. The uptake of Ac-SDKP by RAW 264.7 macrophages and human and murine primary cardiac fibroblasts was confirmed by fluorescence microscopy and flow cytometry. Spectrum computerized tomography and competitive enzyme-linked immunoassays were performed to measure the ex vivo cardiac biodistribution of Ac-SDKP. The biological effects of this novel synthetic compound were examined in cultured macrophages and cardiac fibroblasts and in a murine model of acute myocardial infarction induced by permanent coronary artery ligation. RESULTS: A liposome formulation resulted in the greater uptake of Ac-SDKP than the naked peptide by cultured RAW 264.7 macrophages and cardiac fibroblasts. Liposome-delivered Ac-SDKP decreased fibroinflammatory genes in cultured cardiac fibroblasts co-treated with TGF-ß1 and macrophages stimulated with LPS. Serial tissue and serum immunoassays showed the high bioavailability of Ac-SDKP in mouse myocardium and in circulation. Liposome-delivered Ac-SDKP improved cardiac function and reduced myocardial fibroinflammatory responses in mice with acute myocardial infarction. CONCLUSION: Encapsulation of Ac-SDKP in a cell membrane-like phospholipid bilayer enhances its plasma and tissue bioavailability and offers cardioprotection against ischemic myocardial injury. Future clinical trials can use this novel approach to test small protective endogenous peptides in myocardial remodeling.

Design Strategies of Li-Si Alloy Anode for Mitigating Chemo-Mechanical Degradation in Sulfide-Based All-Solid-State Batteries.

Composite anodes of Li3 PS4  glass+Li-Si alloy (Type 1) and Li3 N+LiF+Li-Si alloy (Type 2) are prepared for all-solid-state batteries with Li3 PS4 (LPS) glass electrolyte and sulfur/LPS glass/carbon composite cathode. Using a three-electrode system, the anode and cathode potentials are separated, and their polarization resistances are individually traced. Even under high-cutoff-voltage conditions (3.7 V), Type 1 and 2 cells are stably cycled without voltage noise for >200 cycles. Although cathode polarization resistance drastically increases after 3.7 V charge owing to LPS oxidation, LPS redox behavior is fairly reversible upon discharge-charge unlike the non-composite alloy anode cell. Time-of-flight secondary ion mass spectrometry analysis reveals that the enhanced cyclability is attributed to uniform Li-Si alloying throughout the composite anode, providing more pathways for lithium ions even when these ions are over-supplied via LPS oxidation. These results imply that LPS-based cells can be reversibly cycled with LPS redox even under high-cutoff voltages, as long as non-uniform alloying (lithium dendrite growth) is prevented. Type 1 and 2 cells exhibit similar performance and stability although reduction product is formed in Type 1. This work highlights the importance of alloy anode design to prevent chemo-mechanical failure when cycling the cell outside the electrochemical stability window.

Risk stratification models for predicting preventable hospitalization in commercially insured late middle-aged adults with depression.

BACKGROUND: A significant number of late middle-aged adults with depression have a high illness burden resulting from chronic conditions which put them at high risk of hospitalization. Many late middle-aged adults are covered by commercial health insurance, but such insurance claims have not been used to identify the risk of hospitalization in individuals with depression. In the present study, we developed and validated a non-proprietary model to identify late middle-aged adults with depression at risk for hospitalization, using machine learning methods. METHODS: This retrospective cohort study involved 71,682 commercially insured older adults aged 55-64 years diagnosed with depression. National health insurance claims were used to capture demographics, health care utilization, and health status during the base year. Health status was captured using 70 chronic health conditions, and 46 mental health conditions. The outcomes were 1- and 2-year preventable hospitalization. For each of our two outcomes, we evaluated seven modelling approaches: four prediction models utilized logistic regression with different combinations of predictors to evaluate the relative contribution of each group of variables, and three prediction models utilized machine learning approaches - logistic regression with LASSO penalty, random forests (RF), and gradient boosting machine (GBM). RESULTS: Our predictive model for 1-year hospitalization achieved an AUC of 0.803, with a sensitivity of 72% and a specificity of 76% under the optimum threshold of 0.463, and our predictive model for 2-year hospitalization achieved an AUC of 0.793, with a sensitivity of 76% and a specificity of 71% under the optimum threshold of 0.452. For predicting both 1-year and 2-year risk of preventable hospitalization, our best performing models utilized the machine learning approach of logistic regression with LASSO penalty which outperformed more black-box machine learning models like RF and GBM. CONCLUSIONS: Our study demonstrates the feasibility of identifying depressed middle-aged adults at higher risk of future hospitalization due to burden of chronic illnesses using basic demographic information and diagnosis codes recorded in health insurance claims. Identifying this population may assist health care planners in developing effective screening strategies and management approaches and in efficient allocation of public healthcare resources as this population transitions to publicly funded healthcare programs, e.g., Medicare in the US.

Aging gene signature of IL-7 receptor alpha low effector memory CD8+ T cells is associated with neurocognitive functioning in Alzheimer's disease.

CD45RA+ effector memory (EM) CD8+ T cell expansion was reported in Alzheimer's disease (AD). Such cells are IL-7 receptor alpha (IL-7Rα)low EM CD8+ T cells, which expand with age and have a unique aging gene signature (i.e., IL-7Rαlow aging genes). Here we investigated whether IL-7Rαlow aging genes and previously reported AD and memory (ADM) genes overlapped with clinical significance in AD patients. RT-qPCR analysis of 40 genes, including 29 ADM, 9 top IL-7Ralow aging and 2 control genes, showed 8 differentially expressed genes between AD and cognitively normal groups; five (62.5%) of which were top IL-7Rαlow aging genes. Over-representation analysis revealed that these genes were highly present in molecular and biological pathways associated with AD. Distinct expression levels of these genes were associated with neuropsychological testing performance in 3 subgroups of dementia participants. Our findings support the possible implication of the IL-7Rαlow aging gene signature with AD.

CX3CR1+ Macrophage Facilitates the Resolution of Allergic Lung Inflammation via Interacting CCL26.

Rationale: The resolution of inflammation is an active process coordinated by mediators and immune cells to restore tissue homeostasis. However, the mechanisms for resolving eosinophilic allergic lung inflammation triggered by inhaled allergens have not been fully elucidated. Objectives: Our objectives were to investigate the cellular mechanism of tissue-resident macrophages involved in the resolution process of eosinophilic lung inflammation. Methods: For the study, we used the institutional review board-approved protocol for human subsegmental bronchoprovocation with allergen, mouse models for allergic lung inflammation, and novel transgenic mice, including a conditional CCL26 knockout. The samples were analyzed using mass cytometry, single-cell RNA sequencing, and biophysical and immunological analyses. Measurements and Main Results: We compared alveolar macrophage (AM) subsets in the BAL before and after allergen provocation. In response to provocation with inhaled allergens, the subsets of AMs are dynamically changed in humans and mice. In the steady state, the AM subset expressing CX3CR1 is a relatively small fraction in bronchoalveolar space and lung tissue but drastically increases after allergen challenges. This subset presents unique patterns of gene expression compared with classical AMs, expressing high C1q family genes. CX3CR1+ macrophages are activated by airway epithelial cell-derived CCL26 via a receptor-ligand interaction. The binding of CCL26 to the CX3CR1+ receptor induces CX3CR1+ macrophages to secrete C1q, subsequently facilitating the clearance of eosinophils. Furthermore, the depletion of CX3CR1 macrophages or CCL26 in airway epithelial cells delays the resolution of allergic lung inflammation displaying prolonged tissue eosinophilia. Conclusions: These findings indicate that the CCL26-CX3CR1 pathway is pivotal in resolving eosinophilic allergic lung inflammation.

Stratifying non-small cell lung cancer patients using an inverse of the treatment decision rules: validation using electronic health records with application to an administrative database.

BACKGROUND: To validate a stratification method using an inverse of treatment decision rules that can classify non-small cell lung cancer (NSCLC) patients in real-world treatment records. METHODS: (1) To validate the index classifier against the TNM 7th edition, we analyzed electronic health records of NSCLC patients diagnosed from 2011 to 2015 in a tertiary referral hospital in Seoul, Korea. Predictive accuracy, stage-specific sensitivity, specificity, positive predictive value, negative predictive value, F1 score, and c-statistic were measured. (2) To apply the index classifier in an administrative database, we analyzed NSCLC patients in Korean National Health Insurance Database, 2002-2013. Differential survival rates among the classes were examined with the log-rank test, and class-specific survival rates were compared with the reference survival rates. RESULTS: (1) In the validation study (N = 1375), the overall accuracy was 93.8% (95% CI: 92.5-95.0%). Stage-specific c-statistic was the highest for stage I (0.97, 95% CI: 0.96-0.98) and the lowest for stage III (0.82, 95% CI: 0.77-0.87). (2) In the application study (N = 71,593), the index classifier showed a tendency for differentiating survival probabilities among classes. Compared to the reference TNM survival rates, the index classification under-estimated the survival probability for stages IA, IIIB, and IV, and over-estimated it for stages IIA and IIB. CONCLUSION: The inverse of the treatment decision rules has a potential to supplement a routinely collected database with information encoded in the treatment decision rules to classify NSCLC patients. It requires further validation and replication in multiple clinical settings.

Prostate cancer extracellular vesicle digital scoring assay - a rapid noninvasive approach for quantification of disease-relevant mRNAs.

Optimizing outcomes in prostate cancer (PCa) requires precision in characterization of disease status. This effort was directed at developing a PCa extracellular vesicle (EV) Digital Scoring Assay (DSA) for detecting metastasis and monitoring progression of PCa. PCa EV DSA is comprised of an EV purification device (i.e., EV Click Chip) and reverse-transcription droplet digital PCR that quantifies 11 PCa-relevant mRNA in purified PCa-derived EVs. A Met score was computed for each plasma sample based on the expression of the 11-gene panel using the weighted Z score method. Under optimized conditions, the EV Click Chips outperformed the ultracentrifugation or precipitation method of purifying PCa-derived EVs from artificial plasma samples. Using PCa EV DSA, the Met score distinguished metastatic (n = 20) from localized PCa (n = 20) with an area under the receiver operating characteristic curve of 0.88 (95% CI:0.78-0.98). Furthermore, longitudinal analysis of three PCa patients showed the dynamics of the Met scores reflected clinical behavior even when disease was undetectable by imaging. Overall, a sensitive PCa EV DSA was developed to identify metastatic PCa and reveal dynamic disease states noninvasively. This assay may complement current imaging tools and blood-based tests for timely detection of metastatic progression that can improve care for PCa patients.

Carboplatin enhances lymphocyte-endothelial interactions to promote CD8+ T cell trafficking into the ovarian tumor microenvironment.

OBJECTIVES: Standard chemotherapy agents, including carboplatin, have known immunogenic properties. We sought to determine how carboplatin may influence lymphocyte trafficking to tumor sites. METHODS: Murine models of ovarian cancer were utilized to examine lymphocyte trafficking with common clinically used agents including carboplatin, anti-PD-1 antibody, or anti-VEGFR-2 antibody. Adhesion interactions of lymphocytes with tumor vasculature were measured using intravital microscopy, lymphocyte homing with immunohistochemistry, and treatment groups followed for overall survival. RESULTS: Carboplatin chemotherapy profoundly alters the tumor microenvironment to promote lymphocyte adhesive interactions with tumor vasculature and resultant improvement in lymphocyte trafficking. The measured results seen with carboplatin in the tumor microenvironment were superior to anti-PD-1 treatment or anti-VEGFR-2 which may have contributed to increased overall survival in carboplatin treated groups. CONCLUSIONS: These novel findings suggest a role for chemotherapeutic agents to broadly influence anti-tumor immune responses beyond the induction of immunogenic tumor cell death.

Understanding viscoelastic behavior of hybrid nanocellulose film based on rheological and electrostatic observation in blended suspension.

The effects of TEMPO-mediated oxidized cellulose nanofibril (TOCN) on the viscoelastic behavior and phase of cellulose nanocrystal (CNC) in suspension and film were investigated using polarized optical microscopy, rotational rheometry, and dynamic mechanical analysis. The sodium cation from TOCN changed the electrostatic state of CNC by screening the CNC surface charge. The volume inflation of TOCN locally increased the CNC concentration in the suspension. In turn, the CNC-CNC interactions increased the viscosity and the yield stress. Based on the experimental observation, the changing mechanisms of electrostatic state and particular interaction in the TOCN/CNC suspensions were suggested. In the hybrid film, the time dependency of complex moduli was changed owing to the different networking between CNCs and TOCNs. The CNC-CNC contacts easily collapsed by strain, while the TOCN-TOCN entanglements were slowly altered. This study provides a fundamental understanding of CNC behavior for optimizing processes and composite properties.

Antagonizing CD105 and androgen receptor to target stromal-epithelial interactions for clinical benefit.

Androgen receptor signaling inhibitors (ARSIs) are standard of care for advanced prostate cancer (PCa) patients. Eventual resistance to ARSIs can include the expression of androgen receptor (AR) splice variant, AR-V7, expression as a recognized means of ligand-independent androgen signaling. We demonstrated that interleukin (IL)-6-mediated AR-V7 expression requires bone morphogenic protein (BMP) and CD105 receptor activity in both PCa and associated fibroblasts. Chromatin immunoprecipitation supported CD105-dependent ID1- and E2F-mediated expression of RBM38. Further, RNA immune precipitation demonstrated RBM38 binds the AR-cryptic exon 3 to enable AR-V7 generation. The forced expression of AR-V7 by primary prostatic fibroblasts diminished PCa sensitivity to ARSI. Conversely, downregulation of AR-V7 expression in cancer epithelia and associated fibroblasts was achieved by a CD105-neutralizing antibody, carotuximab. These compelling pre-clinical findings initiated an interventional study in PCa patients developing ARSI resistance. The combination of carotuximab and ARSI (i.e., enzalutamide or abiraterone) provided disease stabilization in four of nine assessable ARSI-refractory patients. Circulating tumor cell evaluation showed AR-V7 downregulation in the responsive subjects on combination treatment and revealed a three-gene panel that was predictive of response. The systemic antagonism of BMP/CD105 signaling can support ARSI re-sensitization in pre-clinical models and subjects that have otherwise developed resistance due to AR-V7 expression.

Manipulating adrenergic stress receptor signalling to enhance immunosuppression and prolong survival of vascularized composite tissue transplants.

BACKGROUND: Vascularized composite tissue allotransplantation (VCA) to replace limbs or faces damaged beyond repair is now possible. The resulting clear benefit to quality of life is a compelling reason to attempt this complex procedure. Unfortunately, the high doses of immunosuppressive drugs required to protect this type of allograft result in significant morbidity and mortality giving rise to ethical concerns about performing this surgery in patients with non-life-threatening conditions. Here we tested whether we could suppress anti-graft immune activity by using a safe ß2 -adrenergic receptor (AR) agonist, terbutaline, to mimic the natural immune suppression generated by nervous system-induced signalling through AR. METHODS: A heterotopic hind limb transplantation model was used with C57BL/6 (H-2b) as recipients and BALB/c (H-2d) mice as donors. To test the modulation of the immune response, graft survival was investigated after daily intraperitoneal injection of ß2 -AR agonist with and without tacrolimus. Analyses of immune compositions and quantification of pro-inflammatory cytokines were performed to gauge functional immunomodulation. The contributions to allograft survival of ß2 -AR signalling in donor and recipient tissue were investigated with ß2 -AR-/- strains. RESULTS: Treatment with the ß2 -AR agonist delayed VCA rejection, even with a subtherapeutic dose of tacrolimus. ß2 -AR agonist decreased T-cell infiltration into the transplanted grafts and decreased memory T-cell populations in recipient's circulation. In addition, decreased levels of inflammatory cytokines (IFN-γ, IL-6, TNF-α, CXCL-1/10 and CCL3/4/5/7) were detected following ß2 -AR agonist treatment, and there was a decreased expression of ICAM-1 and vascular cell adhesion molecule-1 in donor stromal cells. CONCLUSIONS: ß2 -AR agonist can be used safely to mimic the natural suppression of immune responses, which occurs during adrenergic stress-signalling and thereby can be used in combination regimens to reduce the dose needed of toxic immunosuppressive drugs such as tacrolimus. This strategy can be further evaluated for feasibility in the clinic.

Discoidin Domain Receptor-Driven Gene Signatures as Markers of Patient Response to Anti-PD-L1 Immune Checkpoint Therapy.

BACKGROUND: Anti-programmed cell death 1 (anti-PD-1) and PD ligand 1 (PD-L1) immune checkpoint therapies (ICTs) provided durable responses only in a subset of cancer patients. Thus, biomarkers are needed to predict nonresponders and offer them alternative treatments. We recently implicated discoidin domain receptor tyrosine kinase 2 (DDR2) as a contributor to anti-PD-1 resistance in animal models; therefore, we sought to investigate whether this gene family may provide ICT response prediction. METHODS: We assessed mRNA expression of DDR2 and its family member DDR1. Transcriptome analysis of bladder cancer (BCa) models in which DDR1 and 2 were perturbed was used to derive DDR1- and DDR2-driven signature scores. DDR mRNA expression and gene signature scores were evaluated using BCa-The Cancer Genome Atlas (n = 259) and IMvigor210 (n = 298) datasets, and their relationship to BCa subtypes, pathway enrichment, and immune deconvolution analyses was performed. The potential of DDR-driven signatures to predict ICT response was evaluated and independently validated through a statistical framework in bladder and lung cancer cohorts. All statistical tests were 2-sided. RESULTS: DDR1 and DDR2 showed mutually exclusive gene expression patterns in human tumors. DDR2high BCa exhibited activation of immune pathways and a high immune score, indicative of a T-cell-inflamed phenotype, whereas DDR1high BCa exhibited a non-T-cell-inflamed phenotype. In IMvigor210 cohort, tumors with high DDR1 (hazard ratio [HR] = 1.53, 95% confidence interval [CI] = 1.16 to 2.06; P = .003) or DDR2 (HR = 1.42, 95% CI = 1.01 to 1.92; P = .04) scores had poor overall survival. Of note, DDR2high tumors from IMvigor210 and CheckMate 275 (n = 73) cohorts exhibited poorer overall survival (HR = 1.56, 95% CI = 1.20 to 2.06; P < .001) and progression-free survival (HR = 1.77 95%, CI = 1.05 to 3.00; P = .047), respectively. This result was validated in independent cancer datasets. CONCLUSIONS: These findings implicate DDR1 and DDR2 driven signature scores in predicting ICT response.

Characterizing molecular subtypes of high-risk non-muscle-invasive bladder cancer in African American patients.

BACKGROUND: We sought to determine whether differences in subtype distribution and differentially expressed genes exist between African Americans (AAs) and European Americans (EAs) in patients with high-risk nonmuscle-invasive bladder cancer (NMIBC). METHODS: We performed a retrospective cohort study including 26 patients (14 AAs and 12 EAs) from the University of Texas Medical Branch and the Durham Veterans Affair Health Care System from 2010 to 2020 among treatment naïve, high-risk NMIBC. Profiled gene expressions were performed using the UROMOL classification system. RESULTS: UROMOL racial subtype distributions were similar with class 2a being most common with 10 genes commonly upregulated in AAs compared to EAs including EFEMP1, S100A16, and MCL1 which are associated with progression to muscle-invasive bladder cancer, mitomycin C resistance, and bacillus Calmette-Guérin durability, respectively. We used single nuclei analysis to map the malignant cell heterogeneity in urothelial cancer which 5 distinct malignant epithelial subtypes whose presence has been associated with different therapeutic response prediction abilities. We mapped the expression of the 10 genes commonly upregulated by race as a function of the 5 malignant subtypes. This showed borderline (P = 0.056) difference among the subtypes suggesting AAs and EAs may be expected to have different therapeutic responses to treatments for bladder cancer. AAs were enriched with immune-related, inflammatory, and cellular regulation pathways compared to EAs, yet appeared to have reduced levels of the aggressive C3/CDH12 bladder tumor cell population. CONCLUSIONS: While premature, gene expression differed between AAs and EAs, supporting potential race-based etiologies for muscle-invasion, response to treatments, and transcriptome pathway regulations.

Rheological analysis of physical states of cellulose nanocrystal suspension and synergetic effect of aligned gel state.

The viscoelastic behavior of cellulose nanocrystals (CNCs) in highly concentrated suspension was studied using various rheological tools. As the concentration increased, the physical state of CNC suspension changed from an isotropic phase to an aligned gel state via a cholesteric phase. The physical network originating from hydrogen bond and van der Waals forces gave the suspension elastic and solid-like features. With large shear deformation, the CNC became aligned after network destruction. The aligned gel was not easily relaxed or destroyed by shear deformation or time. The aligned gel state of CNCs provides a better chance for a robust structure of films or composites.