Excessive intragastric alcohol administration exacerbates hepatic encephalopathy and provokes neuronal cell death in male rats with chronic liver disease.

Hepatic encephalopathy (HE) is defined as decline in neurological function during chronic liver disease (CLD). Alcohol is a major etiological factor in the pathogenesis of fibrosis/cirrhosis and has also been documented to directly impact the brain. However, the role of alcohol in the development of HE in CLD remains unclear. Here, we investigated the impact of excessive alcohol administration on neurological deterioration in rats with CLD. Starting day 7 post-BDL surgery, rats were administered alcohol twice daily (51% v/v ethanol, 3 g/kg, via gavage) for 4 weeks. Motor coordination was assessed weekly using rotarod and anxiety-like behavior was evaluated with open field and elevated plus maze at 5 weeks. Upon sacrifice, brains were collected for western blot and immunohistochemical analyses to investigate neuronal integrity and oxidative stress status. Alcohol worsened motor coordination performance and increased anxiety-like behavior in BDL rats. Impairments were associated with decreased neuronal markers of NeuN and SMI311, increased apoptotic markers of cleaved/pro-caspase-3 and Bax/Bcl2, increased necroptosis markers of pRIP3 and pMLKL, decreased total antioxidant capacity (TAC), and increased 4-hydroxynonenal (4-HNE)modified proteins in the cerebellum of BDL-alcohol rats when compared to respective controls. Immunofluorescence confirmed the colocalization of cleaved caspase-3 and pMLKL in the granular neurons of the cerebellum of BDL-alcohol rats. Excessive alcohol consumption exacerbates HE which leads to associated apoptotic and necroptotic neuronal loss in the cerebellum of BDL-alcohol rats. Additionally, higher levels of 4-HNE and decreased TAC in the cerebellum of BDL-alcohol rats suggest oxidative stress is the triggering factor of apoptotic and necroptotic neuronal loss/injury.

Increased cross-presentation by dendritic cells and enhanced anti-tumour therapy using the Arp2/3 inhibitor CK666.

BACKGROUND: Dendritic cell (DC) vaccines for cancer therapy offer the possibility to let the patient's own immune system kill cancer cells. However, DC vaccines have shown less efficacy than expected due to failure to induce cancer cell killing and by activating T regulatory cells. METHODS: We tested if inhibition of signalling via WASp and Arp2/3 using the small molecule CK666 would enhance DC-mediated killing of tumour cells in vitro and in vivo. RESULTS: Using CK666 during the ex vivo phase of antigen processing of ovalbumin (OVA), murine and human DCs showed decreased phagosomal acidification, indicating activation of the cross-presentation pathway. When compared to untreated DCs, DCs treated with CK666 during uptake and processing of OVA-induced increased proliferation of OVA-specific CD8+ OT-I T cells in vitro and in vivo. Using the aggressive B16-mOVA melanoma tumour model, we show that mice injected with CK666-treated DCs and OVA-specific CD8+ OT-I T cells showed higher rejection of B16 melanoma cells when compared to mice receiving non-treated DCs. This resulted in the prolonged survival of tumour-bearing mice receiving CK666-treated DCs. Moreover, combining CK666-treated DCs with the checkpoint inhibitor anti-PD1 further prolonged survival. CONCLUSION: Our data suggest that the small molecule inhibitor CK666 is a good candidate to enhance DC cross-presentation for cancer therapy.

Overactive WASp in X-linked neutropenia leads to aberrant B-cell division and accelerated plasma cell generation.

BACKGROUND: B-cell affinity maturation in germinal center relies on regulated actin dynamics for cell migration and cell-to-cell communication. Activating mutations in the cytoskeletal regulator Wiskott-Aldrich syndrome protein (WASp) cause X-linked neutropenia (XLN) with reduced serum level of IgA. OBJECTIVE: We investigated the role of B cells in XLN pathogenesis. METHODS: We examined B cells from 6 XLN patients, 2 of whom had novel R268W and S271F mutations in WASp. By using immunized XLN mouse models that carry the corresponding patient mutations, WASp L272P or WASp I296T, we examined the B-cell response. RESULTS: XLN patients had normal naive B cells and plasmablasts, but reduced IgA+ B cells and memory B cells, and poor B-cell proliferation. On immunization, XLN mice had a 2-fold reduction in germinal center B cells in spleen, but with increased generation of plasmablasts and plasma cells. In vitro, XLN B cells showed reduced immunoglobulin class switching and aberrant cell division as well as increased production of immunoglobulin-switched plasma cells. CONCLUSIONS: Overactive WASp predisposes B cells for premature differentiation into plasma cells at the expense of cell proliferation and immunoglobulin class switching.

Mutant p53 gain of function mediates cancer immune escape that is counteracted by APR-246.

BACKGROUND: p53 mutants contribute to the chronic inflammatory tumour microenvironment (TME). In this study, we address the mechanism of how p53 mutants lead to chronic inflammation in tumours and how to transform it to restore cancer immune surveillance. METHODS: Our analysis of RNA-seq data from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) project revealed that mutant p53 (mtp53) cancers correlated with chronic inflammation. We used cell-based assays and a mouse model to discover a novel gain of function of mtp53 and the effect of the mtp53 reactivating compound APR-246 on the anti-tumour immune response. RESULTS: We found that tumour samples from patients with breast carcinoma carrying mtp53 showed elevated Interferon (IFN) signalling, Tumour Inflammation Signature (TIS) score and infiltration of CD8+ T cells compared to wild type p53 (wtp53) tumours. We showed that the expression of IFN and immune checkpoints were elevated in tumour cells in a mtp53-dependent manner, suggesting a novel gain of function. Restoration of wt function to mtp53 by APR-246 induced the expression of endogenous retroviruses, IFN signalling and repressed immune checkpoints. Moreover, APR-246 promoted CD4+ T cells infiltration and IFN signalling and prevented CD8+ T cells exhaustion within the TME in vivo. CONCLUSIONS: Breast carcinomas with mtp53 displayed enhanced inflammation. APR-246 boosted the interferon response or represses immune checkpoints in p53 mutant tumour cells, and restores cancer immune surveillance in vivo.

Bile-duct ligation renders the brain susceptible to hypotension-induced neuronal degeneration: Implications of ammonia.

Hepatic encephalopathy (HE) is a debilitating neurological complication of cirrhosis. By definition, HE is considered a reversible disorder, and therefore HE should resolve following liver transplantation (LT). However, persisting neurological complications are observed in as many as 47% of LT recipients. LT is an invasive surgical procedure accompanied by various perioperative factors such as blood loss and hypotension which could influence outcomes post-LT. We hypothesize that minimal HE (MHE) renders the brain frail and susceptible to hypotension-induced neuronal cell death. Six-week bile duct-ligated (BDL) rats with MHE and respective SHAM-controls were used. Several degrees of hypotension (mean arterial pressure of 30, 60 and 90 mm Hg) were induced via blood withdrawal from the femoral artery and maintained for 120 min. Brains were collected for neuronal cell count and apoptotic analysis. In a separate group, BDL rats were treated for MHE with the ammonia-lowering strategy ornithine phenylacetate (OP; MNK-6105), administered orally (1 g/kg) for 3 weeks before induction of hypotension. Hypotension 30 and 60 mm Hg (not 90 mm Hg) significantly decreased neuronal marker expression (NeuN) and cresyl violet staining in the frontal cortex compared to respective hypotensive SHAM-operated controls as well as non-hypotensive BDL rats. Neuronal degeneration was associated with an increase in cleaved caspase-3, suggesting the mechanism of cell death was apoptotic. OP treatment attenuated hyperammonaemia, improved anxiety and activity, and protected the brain against hypotension-induced neuronal cell death. Our findings demonstrate that rats with chronic liver disease and MHE are more susceptible to hypotension-induced neuronal cell degeneration. This highlights MHE at the time of LT is a risk factor for poor neurological outcome post-transplant and that treating for MHE pre-LT might reduce this risk.

Genetically engineered E. coli Nissle attenuates hyperammonemia and prevents memory impairment in bile-duct ligated rats.

Hyperammonemia associated with chronic liver disease (CLD) is implicated in the pathogenesis of hepatic encephalopathy (HE). The gut is a major source of ammonia production that contributes to hyperammonemia in CLD and HE and remains the primary therapeutic target for lowering hyperammonemia. As an ammonia-lowering strategy, Escherichia coli Nissle 1917 bacterium was genetically modified to consume and convert ammonia to arginine (S-ARG). S-ARG was further modified to additionally synthesize butyrate (S-ARG + BUT). Both strains were evaluated in bile-duct ligated (BDL) rats; experimental model of CLD and HE. METHODS: One-week post-surgery, BDLs received non-modified EcN (EcN), S-ARG, S-ARG + BUT (3x1011 CFU/day) or vehicle until sacrifice at 3 or 5 weeks. Plasma (ammonia/pro-inflammatory/liver function), liver fibrosis (hydroxyproline), liver mRNA (pro-inflammatory/fibrogenic/anti-apoptotic) and colon mRNA (pro-inflammatory) biomarkers were measured post-sacrifice. Memory, motor-coordination, muscle-strength and locomotion were assessed at 5 weeks. RESULTS: In BDL-Veh rats, hyperammonemia developed at 3 and further increased at 5 weeks. This rise was prevented by S-ARG and S-ARG + BUT, whereas EcN was ineffective. Memory impairment was prevented only in S-ARG + BUT vs BDL-Veh. Systemic inflammation (IL-10/MCP-1/endotoxin) increased at 3 and 5 weeks in BDL-Veh. S-ARG + BUT attenuated inflammation at both timepoints (except 5-week endotoxin) vs BDL-Veh, whereas S-ARG only attenuated IP-10 and MCP-1 at 3 weeks. Circulating ALT/AST/ALP/GGT/albumin/bilirubin and gene expression of liver function markers (IL-10/IL-6/IL-1ß/TGF-ß/α-SMA/collagen-1α1/Bcl-2) were not normalized by either strain. Colonic mRNA (TNF-α/IL-1ß/occludin) markers were attenuated by synthetic strains at both timepoints vs BDL-Veh. CONCLUSION: S-ARG and S-ARG + BUT attenuated hyperammonemia, with S-ARG + BUT additional memory protection likely due to greater anti-inflammatory effect. These innovative strategies, particularly S-ARG + BUT, have potential to prevent HE.

An intronic deletion in megakaryoblastic leukemia 1 is associated with hyperproliferation of B cells in triplets with Hodgkin lymphoma.

Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor and together they regulate transcription of actin cytoskeleton genes. MKL1 is associated with hematologic malignancies and immunodeficiency, but its role in B cells is unexplored. Here we examined B cells from monozygotic triplets with an intronic deletion in MKL1, two of whom had been previously treated for Hodgkin lymphoma (HL). To investigate MKL1 and B-cell responses in the pathogenesis of HL, we generated Epstein-Barr virus-transformed lymphoblastoid cell lines from the triplets and two controls. While cells from the patients with treated HL had a phenotype close to that of the healthy controls, cells from the undiagnosed triplet had increased MKL1 mRNA, increased MKL1 protein, and elevated expression of MKL1-dependent genes. This profile was associated with elevated actin content, increased cell spreading, decreased expression of CD11a integrin molecules, and delayed aggregation. Moreover, cells from the undiagnosed triplet proliferated faster, displayed a higher proportion of cells with hyperploidy, and formed large tumors in vivo This phenotype was reversible by inhibiting MKL1 activity. Interestingly, cells from the triplet treated for HL in 1985 contained two subpopulations: one with high expression of CD11a that behaved like control cells and the other with low expression of CD11a that formed large tumors in vivo similar to cells from the undiagnosed triplet. This implies that pre-malignant cells had re-emerged a long time after treatment. Together, these data suggest that dysregulated MKL1 activity participates in B-cell transformation and the pathogenesis of HL.

Essential and toxic elements in human milk concentrate with human milk lyophilizate: A preclinical study.

Concentrated human milk (HM-concentrate) can be obtained from the simple and inexpensive method of donated breast milk direct lyophilization. A previous study reported that HM-concentrate contains the adequate amount of main macro- and micronutrients for use as a nutritional resource for preterm infants with very low birth weight admitted to neonatal intensive care units. However, further details need to be elucidated about HM-concentrate composition, particularly its content of essential and potentially toxic trace elements. Therefore, this study aimed to determine the concentration of essential and toxic elements in human milk considered baseline (HM-baseline) and HM-concentrate, as well as to quantify changes in concentration of these elements after the HM concentration process. The concentration of Aluminum, Arsenic, Cadmium, Chromium, Iron, Mercury, Manganese, Nickel, Lead, Selenium, Tin, and Thallium was analyzed by inductively coupled plasma-mass spectrometry (ICP-MS). Moreover, Bayesian linear mixed effect models were applied to estimate the mean difference between HM-baseline and HM-concentrate samples. After comparison (HM-concentrate versus HM-baseline), a significant increase in concentration was observed only for Manganese (0.80 µg/L; 95% CrI [0.16; 1.43]) and Selenium (6.74 µg/L; 95% CrI [4.66; 8.86]), while Lead concentration (-6.13 µg/L; 95% CrI [-8.63; -3.61]) decreased. This study provides latest and reliable information about HM composition. After milk concentration by lyophilization, there was a significant increase only in the essential elements Manganese and Selenium. The essential micronutrient content in HM-concentrate was similar or higher than that in preterm mothers' milk, which suggests it is viable for nutritional support of preterm infants. In addition, the low concentrations of potentially toxic elements in HM-concentrate indicates that it is safe for consumption by premature newborns.

An Investigation of PS-b-PEO Polymersomes for the Oral Treatment and Diagnosis of Hyperammonemia.

Ammonia-scavenging transmembrane pH-gradient poly(styrene)-b-poly(ethylene oxide) polymersomes are investigated for the oral treatment and diagnosis of hyperammonemia, a condition associated with serious neurologic complications in patients with liver disease as well as in infants with urea cycle disorders. While these polymersomes are highly stable in simulated intestinal fluids at extreme bile salt and osmolality conditions, they unexpectedly do not reduce plasmatic ammonia levels in cirrhotic rats after oral dosing. Incubation in dietary fiber hydrogels mimicking the colonic environment suggests that the vesicles are probably destabilized during the dehydration of the intestinal chyme. The findings question the relevance of commonly used simulated intestinal fluids for studying vesicular stability. With the encapsulation of a pH-sensitive dye in the polymersome core, the local pH increase upon ammonia influx could be exploited to assess the ammonia concentration in the plasma of healthy and cirrhotic rats as well as in other fluids. Due to its high sensitivity and selectivity, this polymersome-based assay could prove useful in the monitoring of hyperammonemic patients and in other applications such as drug screening tests.

Synthesis, Antioxidant Activity and Cytotoxicity of N-Functionalized Organotellurides.

The use of antioxidants is the most effective means to protect the organism against cellular damage caused by oxidative stress. In this context, organotellurides have been described as promising antioxidant agents for decades. Herein, a series of N-functionalized organotellurium compounds has been tested as antioxidant and presented remarkable activities by three different in vitro chemical assays. They were able to reduce DPPH radical with IC50 values ranging from 5.08 to 19.20 µg mL-1, and some of them also reduced ABTS+ radical and TPTZ-Fe3+ complex in ABTS+ and FRAP assays, respectively. Initial structure-activity relationship discloses that the nature of N-substituent strongly influenced both activity and cytotoxicity of the studied compounds. Furthermore, radical scavenging activities of N-functionalized organotellurides have been compared with those of their selenilated congeners, demonstrating that the presence of tellurium atom has an essential role in antioxidant activity.

The bile duct ligated rat: A relevant model to study muscle mass loss in cirrhosis.

Muscle mass loss and hepatic encephalopathy (complex neuropsychiatric disorder) are serious complications of chronic liver disease (cirrhosis) which impact negatively on clinical outcome and quality of life and increase mortality. Liver disease leads to hyperammonemia and ammonia toxicity is believed to play a major role in the pathogenesis of hepatic encephalopathy. However, the effects of ammonia are not brain-specific and therefore may also affect other organs and tissues including muscle. The precise pathophysiological mechanisms underlying muscle wasting in chronic liver disease remains to be elucidated. In the present study, we characterized body composition as well as muscle protein synthesis in cirrhotic rats with hepatic encephalopathy using the 6-week bile duct ligation (BDL) model which recapitulates the main features of cirrhosis. Compared to sham-operated control animals, BDL rats display significant decreased gain in body weight, altered body composition, decreased gastrocnemius muscle mass and circumference as well as altered muscle morphology. Muscle protein synthesis was also significantly reduced in BDL rats compared to control animals. These findings demonstrate that the 6-week BDL experimental rat is a relevant model to study liver disease-induced muscle mass loss.

Sclerocornea in a patient with van den Ende-Gupta syndrome homozygous for a SCARF2 microdeletion.

Van den Ende-Gupta Syndrome (VDEGS) is an autosomal recessive disorder characterized by blepharophimosis, distinctive nose, hypoplastic maxilla, and skeletal abnormalities. Using homozygosity mapping in four VDEGS patients from three consanguineous families, Anastacio et al. [Anastacio et al. (2010); Am J Hum Genet 87:553-559] identified homozygous mutations in SCARF2, located at 22q11.2. Bedeschi et al. [2010] described a VDEGS patient with sclerocornea and cataracts with compound heterozygosity for the common 22q11.2 microdeletion and a hemizygous SCARF2 mutation. Because sclerocornea had been described in DiGeorge-velo-cardio-facial syndrome but not in VDEGS, they suggested that the ocular abnormalities were caused by the 22q11.2 microdeletion. We report on a 23-year-old male who presented with bilateral sclerocornea and the VDGEGS phenotype who was subsequently found to be homozygous for a 17 bp deletion in exon 4 of SCARF2. The occurrence of bilateral sclerocornea in our patient together with that of Bedeschi et al., suggests that the full VDEGS phenotype may include sclerocornea resulting from homozygosity or compound heterozygosity for loss of function variants in SCARF2.

Natural killer cells drive 4-1BBL positive uveal melanoma towards EMT and metastatic disease.

BACKGROUND: Inflammation in the eye is often associated with aggravated ocular diseases such as uveal melanoma (UM). Poor prognosis of UM is generally associated with high potential of metastatic liver dissemination. A strong driver of metastatic dissemination is the activation of the epithelial-mesenchymal transition (EMT) regulating transcription factor ZEB1, and high expression of ZEB1 is associated with aggressiveness of UM. While ZEB1 expression can be also associated with immune tolerance, the underlying drivers of ZEB1 activation remain unclear. METHODS: Transcriptomic, in vitro, ex vivo, and in vivo analyses were used to investigate the impact on clinical prognosis of immune infiltration in the ocular tumor microenvironment. A metastatic liver dissemination model of was developed to address the role of natural killer (NK) cells in driving the migration of UM. RESULTS: In a pan-cancer TCGA analysis, natural killer (NK) cells were associated with worse overall survival in uveal melanoma and more abundant in high-risk monosomy 3 tumors. Furthermore, uveal melanoma expressed high levels of the tumor necrosis factor superfamily member 4-1BB ligand, particularly in tumors with monosomy 3 and BAP1 mutations. Tumors expressing 4-1BB ligand induced CD73 expression on NK cells accompanied with the ability to promote tumor dissemination. Through ligation of 4-1BB, NK cells induced the expression of the ZEB1 transcription factor, leading to the formation of liver metastasis of uveal melanoma. CONCLUSIONS: Taken together, the present study demonstrates a role of NK cells in the aggravation of uveal melanoma towards metastatic disease.

Tumor-associated NK cells drive MDSC-mediated tumor immune tolerance through the IL-6/STAT3 axis.

Apart from their killer identity, natural killer (NK) cells have integral roles in shaping the tumor microenvironment. Through immune gene deconvolution, the present study revealed an interplay between NK cells and myeloid-derived suppressor cells (MDSCs) in nonresponders of immune checkpoint therapy. Given that the mechanisms governing the outcome of NK cell-to-myeloid cell interactions remain largely unknown, we sought to investigate the cross-talk between NK cells and suppressive myeloid cells. Upon contact with tumor-experienced NK cells, monocytes and neutrophils displayed increased expression of MDSC-related suppressive factors along with increased capacities to suppress T cells. These changes were accompanied by impaired antigen presentation by monocytes and increased ER stress response by neutrophils. In a cohort of patients with sarcoma and breast cancer, the production of interleukin-6 (IL-6) by tumor-infiltrating NK cells correlated with S100A8/9 and arginase-1 expression by MDSCs. At the same time, NK cell-derived IL-6 was associated with tumors with higher major histocompatibility complex class I expression, which we further validated with b2m-knockout (KO) tumor mice models. Similarly in syngeneic wild-type and IL-6 KO mouse models, we then demonstrated that the accumulation of MDSCs was influenced by the presence of such regulatory NK cells. Inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) axis alleviated suppression of T cell responses, resulting in reduced tumor growth and metastatic dissemination. Together, these results characterize a critical NK cell-mediated mechanism that drives the development of MDSCs during tumor immune escape.

Multiple ammonia-induced episodes of hepatic encephalopathy provoke neuronal cell loss in bile-duct ligated rats.

Background & Aims: Hepatic encephalopathy (HE) is defined as a reversible syndrome and therefore should resolve following liver transplantation (LT). However, neurological complications have been reported in up to 47% of LT recipients, which have been documented to be associated with a history of overt HE pre-LT. We hypothesise that multiple episodes of HE lead to permanent cell injury and exacerbate neurological dysfunction. Our goal was to evaluate the impact of cumulative HE episodes on neurological status and brain integrity in rats with chronic liver disease. Methods: Episodes of overt HE (loss of righting reflex) were induced following injection of ammonium acetate in bile duct ligation (BDL) rats (BDL-Ammonia) every 4 days starting at week 3 post-BDL. Neurobehaviour was evaluated after the last episode. Upon sacrifice, plasma ammonia, systemic oxidative stress, and inflammation markers were assessed. Neuronal markers including neuron-specific nuclear antigen and SMI311 (anti-neurofilament marker) and apoptotic markers (cleaved caspase-3, Bax, and Bcl2) were measured. Total antioxidant capacity, oxidative stress marker (4-hydroxynonenal), and proinflammatory cytokines (tumour necrosis factor-alpha and interleukin-1ß) were measured in brain (hippocampus, frontal cortex, and cerebellum). Proteomic analysis was conducted in the hippocampus. Results: In hippocampus of BDL-Ammonia rats, cleaved caspase-3 and Bax/Bcl2 ratio were significantly increased, whereas NeuN and SMI311 were significantly decreased compared with BDL-Vehicle rats. Higher levels of oxidative stress-induced post-translational modified proteins were found in hippocampus of BDL-Ammonia group which were associated with a lower total antioxidant capacity. Conclusions: Ammonia-induced episodes of overt HE caused neuronal cell injury/death in BDL rats. These results suggest that multiple bouts of HE can be detrimental on the integrity of the brain, translating to irreversibility and hence neurological complications post-LT. Impact and implications: Hepatic encephalopathy (HE) is defined as a reversible neuropsychiatric syndrome resolving following liver transplantation (LT); however, ∼47% of patients demonstrate neurological impairments after LT, which are associated with a previous history of overt HE pre-LT. Our study indicates that multiple episodes of overt HE can cause permanent neuronal damage which may lead to neurological complications after LT. Nevertheless, preventing the occurrence of overt HE episodes is critical for reducing the risk of irreversible neuronal injury in patients with cirrhosis.

Exposure of volunteers to microgravity by dry immersion bed over 21 days results in gene expression changes and adaptation of T cells.

The next steps of deep space exploration are manned missions to Moon and Mars. For safe space missions for crew members, it is important to understand the impact of space flight on the immune system. We studied the effects of 21 days dry immersion (DI) exposure on the transcriptomes of T cells isolated from blood samples of eight healthy volunteers. Samples were collected 7 days before DI, at day 7, 14, and 21 during DI, and 7 days after DI. RNA sequencing of CD3+ T cells revealed transcriptional alterations across all time points, with most changes occurring 14 days after DI exposure. At day 21, T cells showed evidence of adaptation with a transcriptional profile resembling that of 7 days before DI. At 7 days after DI, T cells again changed their transcriptional profile. These data suggest that T cells adapt by rewiring their transcriptomes in response to simulated weightlessness and that remodeling cues persist when reexposed to normal gravity.

The WASp L272P gain-of-function mutation alters dendritic cell coordination of actin dynamics for migration and adhesion.

Dendritic cells (DCs) devoid of the actin regulator Wiskott-Aldrich syndrome protein (WASp) show reduced directed migration and decreased formation of podosome adhesion structures. We examined DCs expressing a gain-of-function mutation in WASp, WASp L272P, identified in X-linked neutropenia patients. Analysis of WASp L272P DCs was compared to WASp-deficient DCs to examine how WASp activity influences DC migratory responses. In confined space, WASp-deficient DCs had increased migration speed whereas WASp L272P DCs had similar average speed but increased speed fluctuations, reduced displacement, and atypical rounded morphology, compared to wild-type (WT) DCs. Using an ear inflammation model and flow cytometry analysis, WT, WASp-deficient, and WASp L272P DCs were found to migrate in comparable numbers to the draining lymph nodes (LNs). However, histology analysis revealed that migratory DCs of WASp deficient and WASp L272P mice were mainly located in the collagenous capsule of the LN whereas WT DCs were located inside the LN. Analysis of ultrastructural features revealed that WASp L272P DCs had reduced cell area but formed larger podosome structures when compared to WT DCs. Together, our data suggest that WASp activity regulates DC migration and that loss-of-function and gain-of-function in WASp activity lead to different and phenotype-specific DC migratory behavior.

Pharmacologic Activation of p53 Triggers Viral Mimicry Response Thereby Abolishing Tumor Immune Evasion and Promoting Antitumor Immunity.

The repression of repetitive elements is an important facet of p53's function as a guardian of the genome. Paradoxically, we found that p53 activated by MDM2 inhibitors induced the expression of endogenous retroviruses (ERV) via increased occupancy on ERV promoters and inhibition of two major ERV repressors, histone demethylase LSD1 and DNA methyltransferase DNMT1. Double-stranded RNA stress caused by ERVs triggered type I/III interferon expression and antigen processing and presentation. Pharmacologic activation of p53 in vivo unleashed the IFN program, promoted T-cell infiltration, and significantly enhanced the efficacy of checkpoint therapy in an allograft tumor model. Furthermore, the MDM2 inhibitor ALRN-6924 induced a viral mimicry pathway and tumor inflammation signature genes in patients with melanoma. Our results identify ERV expression as the central mechanism whereby p53 induction overcomes tumor immune evasion and transforms tumor microenvironment to a favorable phenotype, providing a rationale for the synergy of MDM2 inhibitors and immunotherapy. SIGNIFICANCE: We found that p53 activated by MDM2 inhibitors induced the expression of ERVs, in part via epigenetic factors LSD1 and DNMT1. Induction of IFN response caused by ERV derepression upon p53-targeting therapies provides a possibility to overcome resistance to immune checkpoint blockade and potentially transform "cold" tumors into "hot." This article is highlighted in the In This Issue feature, p. 2945.

Perioperative coagulation profile in living liver donors as assessed by rotational thromboelastometry.

Transient postoperative coagulation abnormalities frequently occur in living liver donors, particularly after right liver resection. Usually, this coagulopathy is diagnosed by alterations in conventional coagulation tests (CCTs) such as the international normalized ratio (INR) of the prothrombin time. However, recent studies using other methods of coagulation monitoring have suggested that postoperative hypercoagulability may also occur in living donors. The rotational thromboelastometry (ROTEM) system is a coagulation monitor based on the viscoelastic properties of blood. The use of ROTEM for perioperative coagulation monitoring in the setting of living liver donors has not been reported. We evaluated the perioperative coagulation profile as assessed by ROTEM in 16 consecutive donors who underwent either right or left liver resection at our institution. ROTEM analysis and CCTs were performed at the baseline (before skin incision) and on postoperative days 1 and 3. According to the CCTs, hypocoagulability was revealed in all but 1 (left liver) donor. The INR was highest on postoperative day 1 [median = 1.67 (interquartile range = 1.37-1.8)]. In contrast, all donors had a normal coagulation profile as assessed by the ROTEM system throughout the study period. In conclusion, this study showed the disagreement between the CCTs and the ROTEM system, as no significant coagulation abnormalities could be revealed with the latter method during the study period. Further studies are needed to confirm the role of the ROTEM system as a perioperative coagulation monitor in this setting and ultimately its influence on the outcome of living donors.

Cytoskeletal regulation of dendritic cells: An intricate balance between migration and presentation for tumor therapy.

Dendritic cells (DCs) are the main players in many approaches for cancer therapy. The idea with DC tumor therapy is to promote activation of tumor infiltrating cytotoxic T cells that kill tumor cells. This requires that DCs take up tumor Ag and present peptides on MHC class I molecules in a process called cross-presentation. For this process to be efficient, DCs have to migrate to the tumor draining lymph node and there activate the machinery for cross-presentation. In this review, we will discuss recent progress in understanding the role of actin regulators for control of DC migration and Ag presentation. The potential to target actin regulators for better DC-based tumor therapy will also be discussed.