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Interplay between IFNα and COX-2/PGE2 system in the regulation of and suppressive inflammatory mediators.

Presence of cytotoxic CD8+ T cells (CTL) in tumor microenvironments (TME) is critical for the effectiveness of immune therapies and patients' outcome, whereas regulatory T(reg) cells promote cancer progression. Immune adjuvants, including double-stranded (ds)RNAs, which signal via Toll-like receptor-3 (TLR3) and helicase (RIG-I/MDA5) pathways, all induce intratumoral production of CTL-attractants, but also Treg attractants and suppressive factors, raising the question of whether induction of these opposing groups of immune mediators can be separated. Here, we use human tumor explant cultures and cell culture models to show that the (ds) RNA Sendai Virus (SeV), poly-I:C, and rintatolimod (poly-I:C12U) all activate the TLR3 pathway involving TRAF3 and IRF3, and induce IFNα, ISG-60, and CXCL10 to promote CTL chemotaxis to ex vivo–treated tumors. However, in contrast with SeV and poly I:C, rintatolimod did not activate the MAVS/helicase pathway, thus avoiding NFκB– and TNFα-dependent induction of COX2, COX2/PGE2-dependent induction of IDO, IL10, CCL22, and CXCL12, and eliminating Treg attraction. Induction of CTL-attractants by either poly I:C or rintatolimod was further enhanced by exogenous IFNα (enhancer of TLR3 expression), whereas COX2 inhibition enhanced the response to poly-I:C only. Our data identify the helicase/NFκB/TNFα/COX2 axis as the key suppressive pathway of dsRNA signaling in human TME and suggest that selective targeting of TLR3 or elimination of NFκB/TNFα/COX2-driven suppression may allow for selective enhancement of type-1 immunity.Significance: This study characterizes two different poly-I:C-induced signaling pathways in their induction of immunostimulatory and suppressive factors and suggests improved ways to reprogram the TME to enhance the antitumor efficacy of immunotherapies. Cancer Res; 78(15); 4292–302. ©2018 AACR.

Dose-dependent response to rintatolimod (RINT) and poly-I:C (pIC) in macrophage cultures.

IFNα enhances the response to TLR3 triggering by either poly-I:C or rintatolimod, but prostaglandin inhibition is selectively needed to optimize the pattern of inflammation induced by poly-I:C.

The model: Molecular separation of pro-inflammatory and suppressive pathways of dsRNA signaling in cancer TME.

Uniform activation of immunostimulatory and suppressive inflammatory mediators, including COX-2, by different TLR ligands.

Ex vivo tumor tissue explant culture system

PDF file - 212KB, Representative gene expression studies demonstrating overexpression of PPP2R2B in human cancers. Data obtained from the ONCOMINE database (2).

PDF file - 84KB, Cyclin E1 protein levels do not correlate with B56beta (A), B56delta (B), or Fbxw7alpha (C) expression in breast cancers. Fresh-frozen breast cancer specimens were analyzed for cyclin E1 protein by Western blotting and B56beta, B56delta, Fbxw7alpha mRNA by qPCR. Linear regression analysis was used to identify possible correlations.

Cyclin E1 regulates the initiation of S-phase in cellular division. However, in many cancers, cyclin E1 is aberrantly overexpressed and this molecular phenotype correlates with increased tumor aggressiveness and poor patient survival. The molecular cause(s) of cyclin E1 abnormalities in cancers is poorly understood. Here, we show that cyclin E1 overexpression in cancer is promoted by dysregulation of the protein phosphatase PP2A-B55β. PP2A-B55β targets the N- and C-terminal phosphodegrons of cyclin E1 for dephosphorylation, thus protecting it from degradation mediated by the SCFFbxw7 ubiquitin ligase. Augmented B55β expression stabilizes cyclin E1 and promotes its overexpression in cancer-derived cell lines and breast tumors. Conversely, B55β ablation enforces the degradation of cyclin E1 and inhibits cancer cell proliferation in vitro and tumor formation in vivo. Therefore, PP2A-B55β promotes cyclin E1 overexpression by antagonizing its degradation and its inhibition could represent a therapeutic mechanism for abrogating cyclin E1 function in cancers. Cancer Res; 74(7); 2006–14. ©2014 AACR.

PDF file - 167KB, Transfection of MDA-MB-231 cells with three different B55beta siRNAs decreases cyclin E1 protein level. Cells were harvested 48 hrs post-transfection. All subsequent experiments were performed using B55beta-siRNA-2. qPCR data showing efficiency of B55beta knockdown and potential effects on cyclin E1 expression are shown below. Expression was normalized to control (scrambled) siRNA.

PDF file - 73KB, B55beta expression knockdown inhibits the growth and decreases cyclin E1 protein levels in colorectal cancer cell lines containing hypermethylated PP2R2B alleles (3). A, Growth of HT29 and HCT116 colorectal carcinoma cells transfected with control or B55beta siRNA measured 96 hrs post-transfection. Experiments were performed in triplicate. B, Western blot of cyclin E1 in HT29 cells 48 hrs post-transfection. Ku86 is shown as loading control.

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PDF file - 235KB, Cell cycle analysis of MDA-MB-231 cells knocked down for B55beta expression. Cells were transfected with control or B55beta siRNA and 48 hrs later stained with propidium iodide and cell cycle kinetics determined by flow cytometry. Percentage of cells in G1, S and G2/M phases are shown on the right.

PDF file - 162KB, B56beta and B56delta do not associate with endogenous cyclin E1. HeLa cells were transfected with plasmids that express Flag-B56beta or Flag-B56delta and extracts (20 mg) immunoprecipitated with either IgG (control) or anti-Flag antibodies. Western blots were then probed with anti-cyclin E1 or anti-Flag antibodies. WCE indicates position of cyclin E1 in the gel.

PDF file - 48KB, Clinicopathological features of breast cancers analyzed for cyclin E1 protein, and CCNE1 and B55beta expression.

Objectives The aim of this study is to identify the peri-procedural risk factors and outcomes of hemodynamic instability (HI) after carotid artery stenting (CAS). Methods A single-center, retrospective study was performed in 168 patients who underwent CAS procedure between September 2017 and September 2020. The presence of HI, as defined by hypertension (systolic blood pressure >160 mmHg), hypotension (systolic blood pressure Results Among 168 patients (mean age, 68.2 ± 8.3 years; 81.5% male), the frequency of post-procedural long-period HI was noted in 42 patients (25.0%). Male was prone to experience HI (odds ratio, 9.156, p = 0.021). Aggressive inflation pressure (>7 atm) and 5 mm balloon for pre-dilatation were risk factors of long-period HI (OR, 7.372, p = 0.035; OR, 3.527, p = 0.023). Intraoperative peak blood pressure and larger-sized stents remained independent predictors for the development of HI (OR, 1.043, p = 0.027, and OR, 1.973, p = 0.015). Patients with prolonged HI were more likely to suffer TIA and stroke compared to other patients and significant difference was found in the occurrence of TIA ( p < 0.05). Non-significance was found in mortality rate and other outcomes. Conclusions CAS-induced HI occurs in a considerable percentage while several peri-procedural variables are determined as independent predictors to develop long-period HI. Patients with prolonged HI are associated with increased risk of neurologic events and thus standardized intervention as well as management of long-period HI are of critical importance during clinical process.

Objective. We aimed to evaluate the clinical characteristics and long-term prognosis of brain arteriovenous malformations (bAVMs) treated with multimodality management of one-staged hybrid operation. Methods. We identified bAVM patients treated with one-staged hybrid operation from a multicenter prospective cohort study (NCT03774017) between January 2016 and June 2020. Patients were divided into unruptured and ruptured groups by the hemorrhagic presentation. Long-term (>12 months) neurological disability, postoperative complications of stroke, and nidus obliteration were evaluated and compared between groups. Prognostic predictors associated with outcomes were analyzed. Results. A total of 130 patients were identified in the study receiving one-staged hybrid operations, including 61 unruptured cases and 69 ruptured cases. Mean age was 29.1 years old, with 78 (60.0%) being male. Patients included in the study were followed up for a mean period of 37.4 (11.07) months. The annual hemorrhagic risk was 4.2% per year. Thirteen postoperative stroke events were detected in 11 patients (8.5%). Long-term disability occurred in 6.9% of cases, and 86.2% of patients experienced an unchanged or improved neurological status at the last follow-up. All patients achieved complete obliteration on follow-up angiographies. Increased AVM volume was associated with a higher risk of postoperative stroke (odds ratio (OR) 1.021, 95% confidence interval (CI) 1.006-1.037, and P = 0.006 ). Poor neurological status (OR 6.461, 95% CI 1.309-31.889, and P = 0.022 ) and infratentorial location (OR 5.618, 95% CI 1.158-27.246, and P = 0.032 ) were independent predictors for long-term disability. Conclusions. One-staged hybrid operation of embolization combined microsurgical resection can be performed as a safe and effective strategy for bAVM treatments. Long-term prognosis of complete obliteration with low rates of morbidity and mortality can be achieved. Unruptured and ruptured bAVMs acquired similar favorable outcomes after the multimodality treatment.

Background. Intracranial vertebrobasilar trunk large (≥10 mm) aneurysms (IVBTLAs) are rare and challenging to manage. In this study, we describe the natural prognosis and evaluate the safety and efficacy of endovascular treatment of IVBTLAs compared with conservative therapy. Methods. This prospective multicenter cohort study included patients with IVBTLAs, who chose either endovascular treatment (endovascular group) or conservative therapy (conservative group) after discussion with their doctors. The primary endpoint was the incidence of serious adverse events (SAEs) related to the target vessel, while secondary endpoints included target vessel-related mortality, major stroke, other serious adverse events, and aneurysm occlusion rate. Results. In total, 258 patients were referred to our two centers for the management of vertebrobasilar aneurysms, and 69 patients had IVBTLAs. Among them, 51 patients underwent endovascular treatment, and 18 patients received conservative therapy. The incidence of target vessel-related SAEs was 15.7% (8/51) in the endovascular group and 44.4% (8/18) in the conservative group ( P = 0.031 ). The target vessel-related mortality was 2.0% (1/51) in the endovascular group and 38.9% (7/18) in the conservative group ( P < 0.001 ). The cumulative survival rates in the endovascular group and conservative group within 1-year, 3-year, and 5-year were 98.0% vs. 83.3%, P = 0.020 ; 98.0% vs. 66.7%, P = 0.001 ; and 98.0% vs. 35.6%, P < 0.001 , respectively. Multivariate analysis revealed conservative therapy, giant aneurysm, and ischemic onset as risks factor for SAEs. Conclusions. Compared with conservative treatment, endovascular treatment of the IVBTLAs may be associated with a lower incidence of SAEs, with higher 1-year, 3-year, and 5-year survival rates. Conservative therapy, giant aneurysm, and ischemic onset were associated with a high risk of SAEs.

The COVID-19 outbreak has been a public health crisis of international concern, causing huge impact on people’s lives. As an important part of social public crisis management, how to quickly and effectively raise resources to participate in emergency relief in the era of self-media is a common challenge faced by global charitable organizations. This article attempts to use empirical evidence from Tencent charitable crowdfunding platform, the largest charitable crowdfunding platform in China, to answer this question. We consider 205 COVID-19 charitable projects and 11,177,249 donors to assess the process by which non-profit organizations raise funds through the information about project descriptions. Based on the effects of information and emotional framing, we explore the effects of the readability (i.e., complexity and understandability) and negative tone of the project description on fundraising amount. We then investigate the mediating role of forwarding times, as affective response to the text might explain forwarding times, which in turn affects money raised by increasing the visibility of the campaign. On this basis, the moderating role of recipient’s crisis involvement is tested during this process. The empirical results indicate that the complexity of the description will reduce the fundraising amount, while understandability and negative tone help to improve it. Furthermore, we found that forwarding times played an important mediating role in this process. Then the buffer effect of crisis involvement on the negative effect of complexity was validated, and its amplification on the positive effects of understandability was also verified.

Objectives Patients with complex cerebral aneurysms can now be treated intravascularly with the help of flow-diverting stents. The primary purpose of this article is to document the clinical and angiographic outcomes in 80 patients who were treated with the Pipeline flow-diverting stent (PFS; Medtronic, Dublin, Ireland) and the obliteration mechanism was discussed. Patients and methods Between October 2015 and October 2019, 80 patients with 90 complex (undefined neck, large/giant, blood blister–like, and recurrent side-wall) cerebral aneurysms treated with the PFS were retrospectively reviewed. Forty-five patients were women and 35 were men, with a mean age of 52 years. Large or giant aneurysms were defined as 10 mm or larger and small aneurysms were defined as less than 10 mm at the largest diameter measured on angiogram. Results Forty-one aneurysms (45.6%) were large or giant, 41 (45.6%) were small, four (4.4%) were recurrent side-wall aneurysms and four (4.4%) were blood blister–like aneurysms. In total, 87 PFSs were placed in 80 patients with 90 aneurysms. In six patients, coexisting proximal stenosis of parent artery was also covered with PFS without balloon angioplasty. Adjunct coils were placed in 31 aneurysms (34%). One patient died of intracerebral hematoma after thrombolysis. There was one intrastent occlusion at six-month follow-up without any symptoms. The morbidity and the mortality rate is 0% and 1.3% (95% confidence interval (CI), 0%–3.7%). Control angiography was available in 74 (92.5%) patients with 83 aneurysms, and the aneurysm occlusion rate was 98.8% (95% CI, 96.5%–100%) in 6 to 12 months. Conclusion For wide-necked saccular, large/giant, blood blister–like aneurysms and recurrent side-wall aneurysms, PFS is a valid and safe treatment option.

Simple Summary The clinical effectiveness of Bacillus Calmette-Guérin (BCG) is limited to patients with early stages of bladder cancer (BlCa) and its effects are often transient. To understand the mechanisms limiting the effectiveness of BCG, we evaluated its impact on the human BlCa tumor microenvironment (TME) and the feasibility of its pharmacologic modulation. We observed that BCG non-selectively induces both CTL-attracting chemokines and Treg/MDSC attractants and suppressive factors in human BlCa tissue explants, in a mechanism involving NFκB-induced PGE2 synthesis and EP4 signaling. In contrast to non-selective impact of NFκB blockade on BCG-induced inflammation, the PGE2 antagonism selectively enhanced the BCG-driven production of CTL attractants but eliminated the induction of Treg/MDSC attractants and suppressive factors, enhancing the CTL migration but reducing Treg attraction to BCG-treated BlCa. Since intratumoral CTL accumulation predicts long term patient outcomes and the effectiveness of cancer immunotherapies, our data indicates the feasibility of targeting the PGE2-chemokine interplay to enhance the therapeutic effects of BCG. Abstract Bacillus Calmette-Guérin (BCG) is commonly used in the immunotherapy of bladder cancer (BlCa) but its effectiveness is limited to only a fraction of patients. To identify the factors that regulate the response of human BlCa tumor microenvironment (TME) to BCG, we used the ex vivo whole-tissue explant model. The levels of COX2 in the BCG-activated explants closely correlated with the local production of Treg- and MDSCS attractants and suppressive factors, while the baseline COX2 levels did not have predictive value. Accordingly, we observed that BCG induced high levels of MDSC- and Treg-attracting chemokines (CCL22, CXCL8, CXCL12) and suppressive factors (IDO1, IL-10, NOS2). These undesirable effects were associated with the nuclear translocation of phosphorylated NFκB, induction of COX2, the key enzyme controlling PGE2 synthesis, and elevation of a PGE2 receptor, EP4. While NFκB blockade suppressed both the desirable and undesirable components of BCG-driven inflammation, the inhibitors of PGE2 synthesis (Celecoxib or Indomethacin) or signaling (EP4-selective blocker, ARY-007), selectively eliminated the induction of MDSC/Treg attractants and immunosuppressive factors but enhanced the production of CTL attractants, CCL5, CXCL9 and CXCL10. PGE2 blockade allowed for the selectively enhanced migration of CTLs to the BCG-treated BlCa samples and eliminated the enhanced migration of Tregs. Since the balance between the CTLs and suppressive cells in the TME predicts the outcomes in patients with BlCa and other diseases, our data help to elucidate the mechanisms which limit the effectiveness of BCG therapies and identify new targets to enhance their therapeutic effects.

Background We describe use of a canine model to evaluate physiological effects and neuroprotective strategies in the setting of cerebral ischemia and endovascular neurosurgery training. Methods We performed transfemoral digital subtraction cerebral and cervical angiography on eight anesthetized dogs. Angiographic images of cerebral arteries were obtained following cannulation of the femoral artery. Cerebral ischemia models were made after angiography. Results The canine cerebral vasculature exhibited extensive tortuosity of the carotid and vertebral arteries. Conversely, the bilateral anterior spinal arteries were easily catheterized using microcatheters and microguidewires. The basilar artery and its branches were facilely cannulable. Circle of Willis continuity sans hypoplasia or aplasia of its constitutive segments was appreciated in all animals. The middle cerebral arteries could be easily accessed via the posterior communicating arteries. We generated an empirically evaluable therapeutically interventional experimental animal model of cerebral ischemia by occluding the middle cerebral artery using small coils for a duration between 15 and 60 min. Conclusion Unique amenability of the canine intracranial vasculature to selective and microcatheter cannulation renders experimentally induced cerebral, cerebellar, and brainstem via occlusion of the supratentorial and infratentorial arteries a simple matter. The neural vasculature irrigating the canine cerebrum, brainstem, and cerebellum may consequently prove useful in helping young and nascent endovascular neurosurgeons in developing and refining their skills of microcatheter navigation and manipulation and deployment of therapeutic devices to achieve effective occlusion of aneurysms, arteriovenous malformations, arteriovenous fistulas, and neoplasms of the intracranial cavity.

Gradients of soluble molecules coordinate cellular communication in a diverse range of multicellular systems. Chemokine-driven chemotaxis is a key orchestrator of cell movement during organ development, immune response and cancer progression. Chemotaxis assays capable of examining cell responses to different chemokines in the context of various extracellular matrices will be crucial to characterize directed cell motion in conditions which mimic whole tissue conditions. Here, a microfluidic device which can generate different chemokine patterns in flow-free gradient chambers while controlling surface extracellular matrix (ECM) to study chemotaxis either at the population level or at the single cell level with high resolution imaging is presented. The device is produced by combining additive manufacturing (AM) and soft lithography. Generation of concentration gradients in the device were simulated and experimentally validated. Then, stable gradients were applied to modulate chemotaxis and chemokinetic response of Jurkat cells as a model for T lymphocyte motility. Live imaging of the gradient chambers allowed to track and quantify Jurkat cell migration patterns. Using this system, it has been found that the strength of the chemotactic response of Jurkat cells to CXCL12 gradient was reduced by increasing surface fibronectin in a dose-dependent manner. The chemotaxis of the Jurkat cells was also found to be governed not only by the CXCL12 gradient but also by the average CXCL12 concentration. Distinct migratory behaviors in response to chemokine gradients in different contexts may be physiologically relevant for shaping the host immune response and may serve to optimize the targeting and accumulation of immune cells to the inflammation site. Our approach demonstrates the feasibility of using a flow-free gradient chamber for evaluating cross-regulation of cell motility by multiple factors in different biologic processes.

Constructing tissue engineered vascular grafts (TEVG) is of great significance for cardiovascular research. However, most of the fabrication techniques are unable to construct TEVG with a bifurcated and curved structure. This paper presents multilayered biodegradable TEVGs with a curved structure and multi-branches. The technique combined 3D printed molds and casting hydrogel and sacrificial material to create vessel-mimicking constructs with customizable structural parameters. Compared with other fabrication methods, the proposed technique can create more native-like 3D geometries. The diameter and wall thickness of the fabricated constructs can be independently controlled, providing a feasible approach for TEVG construction. Enzymatically-crosslinked gelatin was used as the material of the constructs. The mechanical properties and thermostability of the constructs were evaluated. Fluid-structure interaction simulations were conducted to examine the displacement of the construct&rsquo, s wall when blood flows through it. Human umbilical vein endothelial cells (HUVECs) were seeded on the inner channel of the constructs and cultured for 72 h. The cell morphology was assessed. The results showed that the proposed technique had good application potentials, and will hopefully provide a novel technological approach for constructing integrated vasculature for tissue engineering.

Major royal jelly proteins (MRJPs) have been used as endogenous biomarkers for honey authentication. However, no study on quantification of MRJPs in honey authentication was reported. In order to develop a new honey authenticity method based on quantitative analysis of MRJPs in honey, trypsin-digested peptides from MRJPs were examined by mass spectrometry (MS). Three peptides, YNGVPSSLNVISK, TLQMIAGMK, and LTVAGESFTVK were selected as specific markers for MRJP1, MRJP 2 and MRJP 3(MRJP1∼3), respectively. An ultra performance liquid chromatography-triple quadrupole MS (UPLC-TQMS) method used for quantification of MRJPs in honey was developed and validated. A database on the MRJP1∼3 contents of honey established by quantification of 70 authentic samples showed a potential application in honey authenticity identification. The analysis results of 10 commercial honey samples by UPLC-TQMS with reference to the MRJP1∼3 database of authentic honey were compared and confirmed by widely used isotope ratio mass spectrometry, a AOAC Official Method. In conclusion, the results showed our method, a proteomic-based UPLC-TQMS method, has a high accuracy, sensitivity and specificity for honey authenticity.

Bacillus Calmette-Guérin (BCG) is commonly used in the immunotherapy of bladder cancer (BlCa) but its effectiveness is limited to only a fraction of patients. To identify the factors that regulate the response of human BlCa tumor microenvironment (TME) to BCG, we used the ex vivo whole-tissue explant model. The levels of COX2 in the BCG-activated explants closely correlated with the local production of Treg- and MDSCS attractants and suppressive factors, while the baseline COX2 levels did not have predictive value. Accordingly, we observed that BCG induced high levels of MDSC- and Treg-attracting chemokines (CCL22, CXCL8, CXCL12) and suppressive factors (IDO1, IL-10, NOS2). These undesirable effects were associated with the nuclear translocation of phosphorylated NFκB, induction of COX2, the key enzyme controlling PGE2 synthesis, and elevation of a PGE2 receptor, EP4. While NFκB blockade suppressed both the desirable and undesirable components of BCG-driven inflammation, the inhibitors of PGE2 synthesis (Celecoxib or Indomethacin) or signaling (EP4-selective blocker, ARY-007), selectively eliminated the induction of MDSC/Treg attractants and immunosuppressive factors but enhanced the production of CTL attractants, CCL5, CXCL9 and CXCL10. PGE2 blockade allowed for the selectively enhanced migration of CTLs to the BCG-treated BlCa samples and eliminated the enhanced migration of Tregs. Since the balance between the CTLs and suppressive cells in the TME predicts the outcomes in patients with BlCa and other diseases, our data help to elucidate the mechanisms which limit the effectiveness of BCG therapies and identify new targets to enhance their therapeutic effects.

Purpose of Study: Dendritic Cells (DC) maturing in the conditions of acute/early versus late/chronic inflammation (such as alpha-type-1-polarized DCs [αDC1s] and “standard” nonpolarized DCs; sDCs) deliver differential “signal 3” to resting T cells, resulting in their different induction of effector functions in tumor-specific CD8+ T cells. Here, using single-cell mRNAseq (scRNAseq), a unique technology allowing for unbiased analysis of gene expression (GEX), we evaluated the GEX patterns and cellular heterogeneity of CD8+ T cells expanded by cancer-cell loaded αDC1s or sDCs. Procedure: Freshly isolated autologous (compared to DCs) CD8+ T cells were in vitro sensitized for 1 week, using either αDC1s loaded with UV/gamma-radiation-killed ovarian cancer (OvCa) cell lines OVCAR or SKOV3, or sDCs loaded with the same antigens. αDC1s and sDCs not loaded with exogenous antigen were used as controls. The expanded CD8+ T cells were harvested, captured at the single-cell level and immobilized in a vertical flow array chip (VFAC), which contain 100-microchambers packed with 105 beads immobilizing 1010 oligo(dT) probes with unique cell-ID, UMI, and PCR-tags. Each cell was lysed and its mRNA was individually tagged with unique cell ID sequences prior to library preparation and targeted scRNAseq using a custom 94-gene inflammation RNA-seq panel was performed. Post-clustering (Scanpy), unsupervised cell-type identification (louvain), and downstream analyses were performed on GEX data to determine impact of αDC1s versus sDCs (loaded with OVCAR or SKOV3 versus not loaded with antigens) on the effector and memory pathways of differentiation of the expanded T cells and their unique functional status. Results: PCA and unsupervised clustering revealed sample groups with distinct phenotypic signatures associated with loaded versus unloaded aDC1s, with differential GEX subclusters identified for the αDC1- activated T cells (independently on DC loading with either OVCAR or SKOV3 cells) to include at least two populations with differential expression of genes typical of central memory, effector memory and effector cells (killer molecules and chemokine receptors typical of both lymphoid and peripheral homing potential). In sharp contrast, sDC1 activated T cell showed a more uniform gene distribution patterns lacking the effector T cell component. These differences correlated with the differential ability of αDC1- and sDC-activated T cells to respond to migrate to different chemokines and to recognize and kill cancer cells. Conclusion: scRNAseq was able to identify distinct single-cell T-cell phenotypes caused by induction of αDC1s or sDCs (loaded with OVCAR or SKOV3), consistent with the differential ability of these two DC types to induce effector, effector memory and central memory CD8+ T cells in response to different tumor targets and with their differential ability to modulate peripheral tissue homing patterns. Citation Format: Bowen Dong, Sarabjot Pabla, Vincent Giamo, Weijian Jiang, Kiyomi Taniguchi, Sean T. Glenn, Masataka Shirai, Pawel Kalinski. Distinct pathways of DC-induced CD8+T cell differentiation revealed by single-cell mRNA sequencing analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 968.

Heat stress (HS) causes serious physiological dysfunction associated with cardiovascular diseases. Curcumin (CUR) may increase animal survival and lifespan under HS. However, its effects and mechanism on mammal are underexplored. The goal of this study was to examine the protective effect of CUR on the cardiac health of mice exposed to HS. Mice were divided into six groups (n=8 per group): no-heat treatment (NHT), heat treatment (HT), aspirin, CUR 50 mg/kg/day, CUR 100 mg/kg/day and CUR 200 mg/kg/day. After administration for 4 weeks, except for NHT, other groups were exposed once to HS at 41°C for 20 min. After HS treatment, the physiological-related indexes of blood pressure, rectal temperature and heart rate were measured. Serum biochemical indexes and the levels of cardiac troponin I (cTn-I) in serum and angiotensin II (Ang II) in cardiomyocytes were analyzed. Furthermore, the mRNA and proteins levels of angiotensin receptor 1 (AT1), 78-kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP) and B-cell lymphoma 2 (Bcl-2) were measured. Our results indicated that CUR supplementation could alleviate HS-induced physiological disorders and the increasing of cTn-I and Ang II. The expression of AT1 gene in HT group was significantly higher than that of CUR groups, indicating the cardioprotective effects of CUR. Moreover, the levels of GRP78 and CHOP proteins in the HT group were significantly higher than those of CUR groups, indicating that CUR supplementation reversed the endoplasmic reticulum HS-mediated apoptosis. In summary, CUR supplementation alleviates physiological stress and cardiac damage caused by HS.

Presence of cytotoxic CD8+ T cells (CTL) in tumor microenvironments (TME) is critical for the effectiveness of immune therapies and patients' outcome, whereas regulatory T(reg) cells promote cancer progression. Immune adjuvants, including double-stranded (ds)RNAs, which signal via Toll-like receptor-3 (TLR3) and helicase (RIG-I/MDA5) pathways, all induce intratumoral production of CTL-attractants, but also Treg attractants and suppressive factors, raising the question of whether induction of these opposing groups of immune mediators can be separated. Here, we use human tumor explant cultures and cell culture models to show that the (ds) RNA Sendai Virus (SeV), poly-I:C, and rintatolimod (poly-I:C12U) all activate the TLR3 pathway involving TRAF3 and IRF3, and induce IFNα, ISG-60, and CXCL10 to promote CTL chemotaxis to ex vivo–treated tumors. However, in contrast with SeV and poly I:C, rintatolimod did not activate the MAVS/helicase pathway, thus avoiding NFκB– and TNFα-dependent induction of COX2, COX2/PGE2-dependent induction of IDO, IL10, CCL22, and CXCL12, and eliminating Treg attraction. Induction of CTL-attractants by either poly I:C or rintatolimod was further enhanced by exogenous IFNα (enhancer of TLR3 expression), whereas COX2 inhibition enhanced the response to poly-I:C only. Our data identify the helicase/NFκB/TNFα/COX2 axis as the key suppressive pathway of dsRNA signaling in human TME and suggest that selective targeting of TLR3 or elimination of NFκB/TNFα/COX2-driven suppression may allow for selective enhancement of type-1 immunity. Significance: This study characterizes two different poly-I:C-induced signaling pathways in their induction of immunostimulatory and suppressive factors and suggests improved ways to reprogram the TME to enhance the antitumor efficacy of immunotherapies. Cancer Res; 78(15); 4292–302. ©2018 AACR.

Two recanalization stroke cases are presented, one led to harmful brain hemorrhage and the other beneficial effect and patient recovered smoothly. Discussed the potential mechanisms behind beneficial recanalization and reperfusion after recanalization.

Recanalization and reperfusion injury in ischemic stroke patients casts shadows on current stroke management. The potential mechanisms of reperfusion injury remain obscure and need further investigation. This article summarized several clinically important mechanisms and especially discussed a potential role of blood pressure control in reperfusion injury.

Sulphur mustard (2,2'-dichloroethyl sulfide; SM) is a vesicant chemical warfare agent whose mechanism of acute or chronic action is not known with any certainty and to date there is no effective antidote. SM accumulation in adipose tissue (AT) has been originally verified in our previous study. To evaluate the biological effect caused by the presence of abundant SM in adipocyte and assess the biological role of AT in SM poisoning, in vitro and in vivo experiments were performed. High content analysis revealed multi-cytotoxicity in SM exposed cells in a time and dose dependent manner, and adipocytes showed a relative moderate damage compared with non-adipocytes. Cell co-culture model was established and revealed the adverse effect of SM-exposed adipocyte supernatant on the growth of co-cultured cells. The pathological changes in AT from 10mg/kg SM percutaneously exposed rats were checked and inflammation phenomena were observed. The mRNA and protein levels of inflammation-related adipokines secreted from AT in rats exposed to 1, 3 and 10mg/kg doses of SM were determined by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays. The expressions of proinflammatory and anti-inflammatory adipokines together promoted the inflammation development in the body. The positive correlations between AT and serum adipokine levels were explored, which demonstrated a substantial role of AT in systemic inflammation responding to SM exposure. Thus, AT is not only a target of SM but also a modulator in the SM toxicity.

This study aimed to explore the association between GPIa, COX-2 gene polymorphisms and aspirin resistance in the ischemic stroke patients from the southern part of Jiangsu province.In all, 97 patients with acute ischemic stroke were enrolled in the study. GPIa gene polymorphism at 807CT (rsl126643) locus and COX-2 gene polymorphism at -765GC (rs20417) locus were genotyped by PCR pyrosequencing technology. Patients were divided into the aspirin sensitivity (AS) group and aspirin resistance (AR) group according to the platelet aggregation rate. The relationship between the two gene polymorphisms and aspirin resistance was investigated and analyzed.The distribution of the genotype (CC, CT, TT, CT + TT, and CC) and the frequency of allele T of GPIa gene at 807CT locus were significantly different in AS and AR groups in female patients (P .05). Logistic regression analysis showed that the genotype of CT+TT at 807CT locus was significantly correlated with AR after adjustment for relative factors (P = .047, OR = 4.856, 95% CI: 1.020-23.108). There were no significant differences in the genotype distribution and allele frequency of the COX-2 gene -765GC site between two groups (P .05).GPIa gene polymorphism at 807CT locus was associated with AR in Chinese Han females, and the expression of allele T increased the incidence of AR. The gene polymorphism of COX-2 gene at -765GC locus was not significantly correlated with AR.