Targeting the arginine metabolic brake enhances immunotherapy for leukaemia.

Therapeutic approaches which aim to target Acute Myeloid Leukaemia through enhancement of patients' immune responses have demonstrated limited efficacy to date, despite encouraging preclinical data. Examination of AML patients treated with azacitidine (AZA) and vorinostat (VOR) in a Phase II trial, demonstrated an increase in the expression of Cancer-Testis Antigens (MAGE, RAGE, LAGE, SSX2 and TRAG3) on blasts and that these can be recognised by circulating antigen-specific T cells. Although the T cells have the potential to be activated by these unmasked antigens, the low arginine microenvironment created by AML blast Arginase II activity acts a metabolic brake leading to T cell exhaustion. T cells exhibit impaired proliferation, reduced IFN-γ release and PD-1 up-regulation in response to antigen stimulation under low arginine conditions. Inhibition of arginine metabolism enhanced the proliferation and cytotoxicity of anti-NY-ESO T cells against AZA/VOR treated AML blasts, and can boost anti-CD33 Chimeric Antigen Receptor-T cell cytotoxicity. Therefore, measurement of plasma arginine concentrations in combination with therapeutic targeting of arginase activity in AML blasts could be a key adjunct to immunotherapy.

Merkel cell polyomavirus small T antigen mediates microtubule destabilization to promote cell motility and migration.

UNLABELLED: Merkel cell carcinoma (MCC) is an aggressive skin cancer of neuroendocrine origin with a high propensity for recurrence and metastasis. Merkel cell polyomavirus (MCPyV) causes the majority of MCC cases due to the expression of the MCPyV small and large tumor antigens (ST and LT, respectively). Although a number of molecular mechanisms have been attributed to MCPyV tumor antigen-mediated cellular transformation or replication, to date, no studies have investigated any potential link between MCPyV T antigen expression and the highly metastatic nature of MCC. Here we use a quantitative proteomic approach to show that MCPyV ST promotes differential expression of cellular proteins implicated in microtubule-associated cytoskeletal organization and dynamics. Intriguingly, we demonstrate that MCPyV ST expression promotes microtubule destabilization, leading to a motile and migratory phenotype. We further highlight the essential role of the microtubule-associated protein stathmin in MCPyV ST-mediated microtubule destabilization and cell motility and implicate the cellular phosphatase catalytic subunit protein phosphatase 4C (PP4C) in the regulation of this process. These findings suggest a possible molecular mechanism for the highly metastatic phenotype associated with MCC. IMPORTANCE: Merkel cell polyomavirus (MCPyV) causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer with a high metastatic potential. However, the molecular mechanisms leading to virally induced cancer development have yet to be fully elucidated. In particular, no studies have investigated any potential link between the virus and the highly metastatic nature of MCC. We demonstrate that the MCPyV small tumor antigen (ST) promotes the destabilization of the host cell microtubule network, which leads to a more motile and migratory cell phenotype. We further show that MCPyV ST induces this process by regulating the phosphorylation status of the cellular microtubule-associated protein stathmin by its known association with the cellular phosphatase catalytic subunit PP4C. These findings highlight stathmin as a possible biomarker of MCC and as a target for novel antitumoral therapies.

The golden age of bio-logging: how animal-borne sensors are advancing the frontiers of ecology.

Great leaps forward in scientific understanding are often spurred by innovations in technology. The explosion of miniature sensors that are driving the boom in consumer electronics, such as smart phones, gaming platforms, and wearable fitness devices, are now becoming available to ecologists for remotely monitoring the activities of wild animals. While half a century ago researchers were attaching balloons to the backs of seals to measure their movement, today ecologists have access to an arsenal of sensors that can continuously measure most aspects of an animal's state (e.g., location, behavior, caloric expenditure, interactions with other animals) and external environment (e.g., temperature, salinity, depth). This technology is advancing our ability to study animal ecology by allowing researchers to (1) answer questions about the physiology, behavior, and ecology of wild animals in situ that would have previously been limited to tests on model organisms in highly controlled settings, (2) study cryptic or wide-ranging animals that have previously evaded investigation, and (3) develop and test entirely new theories. Here we explore how ecologists are using these tools to answer new questions about the physiological performance, energetics, foraging, migration, habitat selection, and sociality of wild animals, as well as collect data on the environments in which they live.

An analysis of the function and expression of D6 on lymphatic endothelial cells.

The mechanisms by which CC chemokine receptor (CCR)7 ligands are selectively presented on lymphatic endothelium in the presence of inflammatory chemokines are poorly understood. The chemokine-scavenging receptor D6 is expressed on lymphatic endothelial cells (LEC) and contributes to selective presentation of CCR7 ligands by suppressing inflammatory chemokine binding to LEC surfaces. As well as preventing inappropriate inflammatory cell attachment to LECs, D6 is specifically involved in regulating the ability of LEC to discriminate between mature and immature dendritic cells (DCs). D6 overexpression reduces immature DC (iDC) adhesion to LECs, whereas D6 knockdown increases adhesion of iDCs that displace mature DCs. LEC D6 expression is regulated by growth factors, cytokines, and tumor microenvironments. In particular, interleukin-6 and interferon-γ are potent inducers, indicating a preferential role for D6 in inflamed contexts. Expression of the viral interleukin-6 homolog from Kaposi sarcoma-associated herpesvirus is also sufficient to induce significant D6 upregulation both in vitro and in vivo, and Kaposi sarcoma and primary effusion lymphoma cells demonstrate high levels of D6 expression. We therefore propose that D6, which is upregulated in both inflammatory and tumor contexts, is an essential regulator of inflammatory leukocyte interactions with LECs and is required for immature/mature DC discrimination by LECs.

An interaction between KSHV ORF57 and UIF provides mRNA-adaptor redundancy in herpesvirus intronless mRNA export.

The hTREX complex mediates cellular bulk mRNA nuclear export by recruiting the nuclear export factor, TAP, via a direct interaction with the export adaptor, Aly. Intriguingly however, depletion of Aly only leads to a modest reduction in cellular mRNA nuclear export, suggesting the existence of additional mRNA nuclear export adaptor proteins. In order to efficiently export Kaposi's sarcoma-associated herpesvirus (KSHV) intronless mRNAs from the nucleus, the KSHV ORF57 protein recruits hTREX onto viral intronless mRNAs allowing access to the TAP-mediated export pathway. Similarly however, depletion of Aly only leads to a modest reduction in the nuclear export of KSHV intronless mRNAs. Herein, we identify a novel interaction between ORF57 and the cellular protein, UIF. We provide the first evidence that the ORF57-UIF interaction enables the recruitment of hTREX and TAP to KSHV intronless mRNAs in Aly-depleted cells. Strikingly, depletion of both Aly and UIF inhibits the formation of an ORF57-mediated nuclear export competent ribonucleoprotein particle and consequently prevents ORF57-mediated mRNA nuclear export and KSHV protein production. Importantly, these findings highlight that redundancy exists in the eukaryotic system for certain hTREX components involved in the mRNA nuclear export of intronless KSHV mRNAs.

Complete response to neoadjuvant chemoradiotherapy in rectal cancer is associated with RAS/AKT mutations and high tumour mutational burden.

BACKGROUND: Pathological complete response (pathCR) in rectal cancer is beneficial, as up to 75% of patients do not experience regrowth of the primary tumour, but it is poorly understood. We hypothesised that the changes seen in the pre-treatment biopsies of pathCR but not seen in residual tumour after chemoradiotherapy were the determinants of responsiveness. METHODS: Two groups of patients with either complete response (pathCR group, N = 24) or no response (poor response group, N = 24) were retrieved. Pre-treatment biopsies of cancers from these patients underwent high read depth amplicon sequencing for a targeted panel, exome sequencing, methylation profiling and immunohistochemistry for DNA repair pathway proteins. RESULTS: Twenty four patients who underwent pathCR and twenty-four who underwent poor response underwent molecular characterisation. Patients in the pathCR group had significantly higher tumour mutational burden and neoantigen load, frequent copy number alterations but fewer structural variants and enrichment for driver mutations in the PI3K/AKT/mTOR signalling pathway. There were no significant differences in tumour heterogeneity as measured by MATH score. Methylation analysis demonstrated enrichment for hypomethyation in the PI3K/AKT/mTOR signalling pathway. DISCUSSION: The phenomenon of pathCR in rectal cancer may be related to immunovisibility caused by a high tumour mutational burden phenotype. Potential therapy resistance mechanisms involve the PI3K/AKT/mTOR signalling pathway, but tumour heterogeneity does not seem to play a role in resistance.

Combination Lenalidomide and Azacitidine: A Novel Salvage Therapy in Patients Who Relapse After Allogeneic Stem-Cell Transplantation for Acute Myeloid Leukemia.

PURPOSE: Salvage options for patients who relapse after allogeneic stem-cell transplantation (allo-SCT) for acute myeloid leukemia (AML) and myelodysplasia (MDS) remain limited, and novel treatment strategies are required. Both lenalidomide (LEN) and azacitidine (AZA) possess significant antitumor activity effect in AML. Administration of LEN post-transplantation is associated with excessive rates of graft-versus-host disease (GVHD), but AZA has been shown to ameliorate GVHD in murine transplantation models. We therefore examined the tolerability and activity of combined LEN/AZA administration in post-transplantation relapse. PATIENTS AND METHODS: Twenty-nine patients who had relapsed after allo-SCT for AML (n = 24) or MDS (n = 5) were treated with sequential AZA (75 mg/m2 for 7 days) followed by escalating doses of LEN on days 10 to 30. Dose allocation and maximum tolerated dose (MTD) estimation were guided by a modified Bayesian continuous reassessment method (CRM). RESULTS: Sequential AZA and LEN therapy was well tolerated. The MTD of post-transplantation LEN, in combination with AZA, was determined as 25 mg daily. Three patients developed grade 2 to 4 GVHD. There was no GVHD-related mortality. Seven of 15 (47%) patients achieved a major clinical response after LEN/AZA therapy. CD8+ T cells demonstrated impaired interferon-γ/tumor necrosis factor-α production at relapse, which was not reversed during LEN/AZA administration. CONCLUSION: We conclude LEN can be administered safely post-allograft in conjunction with AZA, and this combination demonstrates clinical activity in relapsed AML/MDS without reversing biologic features of T-cell exhaustion. The use of a CRM model delivered improved efficiency in MTD assessment and provided additional flexibility. Combined LEN/AZA therapy represents a novel and active salvage therapy in patients who had relapsed post-allograft.

To migrate, stay put, or wander? Varied movement strategies in bald eagles (Haliaeetus leucocephalus).

BACKGROUND: Quantifying individual variability in movement behavior is critical to understanding population-level patterns in animals. Here, we explore intraspecific variation in movement strategies of bald eagles (Haliaeetus leucocephalus) in the north Pacific, where there is high spatiotemporal resource variability. We tracked 28 bald eagles (five immature, 23 adult) using GPS transmitters between May 2010 and January 2016. RESULTS: We found evidence of four movement strategies among bald eagles in southeastern Alaska and western Canada: breeding individuals that were largely sedentary and remained near nest sites year-round, non-breeding migratory individuals that made regular seasonal travel between northern summer and southern winter ranges, non-breeding localized individuals that displayed fidelity to foraging sites, and non-breeding nomadic individuals with irregular movement. On average, males traveled farther per day than females. Most nomadic individuals were immature, and all residential individuals (i.e. breeders and localized birds) were adults. CONCLUSIONS: Alternative movement strategies among north Pacific eagles are likely associated with the age and sex class, as well as breeding status, of an individual. Intraspecific variation in movement strategies within the population results in different space use patterns among contingents, which has important implications for conservation and management.

Environmental DNA from Residual Saliva for Efficient Noninvasive Genetic Monitoring of Brown Bears (Ursus arctos).

Noninvasive genetic sampling is an important tool in wildlife ecology and management, typically relying on hair snaring or scat sampling techniques, but hair snaring is labor and cost intensive, and scats yield relatively low quality DNA. New approaches utilizing environmental DNA (eDNA) may provide supplementary, cost-effective tools for noninvasive genetic sampling. We tested whether eDNA from residual saliva on partially-consumed Pacific salmon (Oncorhynchus spp.) carcasses might yield suitable DNA quality for noninvasive monitoring of brown bears (Ursus arctos). We compared the efficiency of monitoring brown bear populations using both fecal DNA and salivary eDNA collected from partially-consumed salmon carcasses in Southeast Alaska. We swabbed a range of tissue types from 156 partially-consumed salmon carcasses from a midseason run of lakeshore-spawning sockeye (O. nerka) and a late season run of stream-spawning chum (O. keta) salmon in 2014. We also swabbed a total of 272 scats from the same locations. Saliva swabs collected from the braincases of salmon had the best amplification rate, followed by swabs taken from individual bite holes. Saliva collected from salmon carcasses identified unique individuals more quickly and required much less labor to locate than scat samples. Salmon carcass swabbing is a promising method to aid in efficient and affordable monitoring of bear populations, and suggests that the swabbing of food remains or consumed baits from other animals may be an additional cost-effective and valuable tool in the study of the ecology and population biology of many elusive and/or wide-ranging species.

Differential use of salmon by vertebrate consumers: implications for conservation.

Salmon and other anadromous fish are consumed by vertebrates with distinct life history strategies to capitalize on this ephemeral pulse of resource availability. Depending on the timing of salmon arrival, this resource may be in surplus to the needs of vertebrate consumers if, for instance, their populations are limited by food availability during other times of year. However, the life history of some consumers enables more efficient exploitation of these ephemeral resources. Bears can deposit fat and then hibernate to avoid winter food scarcity, and highly mobile consumers such as eagles, gulls, and other birds can migrate to access asynchronous pulses of salmon availability. We used camera traps on pink, chum, and sockeye salmon spawning grounds with various run times and stream morphologies, and on individual salmon carcasses, to discern potentially different use patterns among consumers. Wildlife use of salmon was highly heterogeneous. Ravens were the only avian consumer that fed heavily on pink salmon in small streams. Eagles and gulls did not feed on early pink salmon runs in streams, and only moderately at early sockeye runs, but were the dominant consumers at late chum salmon runs, particularly on expansive river flats. Brown bears used all salmon resources far more than other terrestrial vertebrates. Notably, black bears were not observed on salmon spawning grounds despite being the most frequently observed vertebrate on roads and trails. From a conservation and management perspective, all salmon species and stream morphologies are used extensively by bears, but salmon spawning late in the year are disproportionately important to eagles and other highly mobile species that are seasonally limited by winter food availability.

Neuroblastoma Arginase Activity Creates an Immunosuppressive Microenvironment That Impairs Autologous and Engineered Immunity.

Neuroblastoma is the most common extracranial solid tumor of childhood, and survival remains poor for patients with advanced disease. Novel immune therapies are currently in development, but clinical outcomes have not matched preclinical results. Here, we describe key mechanisms in which neuroblastoma inhibits the immune response. We show that murine and human neuroblastoma tumor cells suppress T-cell proliferation through increased arginase activity. Arginase II is the predominant isoform expressed and creates an arginine-deplete local and systemic microenvironment. Neuroblastoma arginase activity results in inhibition of myeloid cell activation and suppression of bone marrow CD34(+) progenitor proliferation. Finally, we demonstrate that the arginase activity of neuroblastoma impairs NY-ESO-1-specific T-cell receptor and GD2-specific chimeric antigen receptor-engineered T-cell proliferation and cytotoxicity. High arginase II expression correlates with poor survival for patients with neuroblastoma. The results support the hypothesis that neuroblastoma creates an arginase-dependent immunosuppressive microenvironment in both the tumor and blood that leads to impaired immunosurveillance and suboptimal efficacy of immunotherapeutic approaches.

Inflammatory cell distribution in primary merkel cell carcinoma.

Merkel cell carcinoma (MCC) is an aggressive poorly differentiated neuroendocrine cutaneous carcinoma associated with older age, immunodeficiency and Merkel cell polyomavirus (MCPyV) integrated within malignant cells. The presence of intra-tumoural CD8+ lymphocytes reportedly predicts better MCC-specific survival. In this study, the distribution of inflammatory cells and properties of CD8+ T lymphocytes within 20 primary MCC specimens were characterised using immunohistochemistry and multicolour immunofluorescent staining coupled to confocal microscopy. CD8+ cells and CD68+ macrophages were identified in 19/20 primary MCC. CD20+ B cells were present in 5/10, CD4+ cells in 10/10 and FoxP3+ cells in 7/10 specimens. Only two specimens had almost no inflammatory cells. Within specimens, inflammatory cells followed the same patchy distribution, focused at the edge of sheets and nodules and, in some cases, more intense in trabecular areas. CD8+ cells were outside vessels on the edge of tumour. Those few within malignant sheets typically lined up in fine septa not contacting MCC cells expressing MCPyV large T antigen. The homeostatic chemokine CXCL12 was expressed outside malignant nodules whereas its receptor CXCR4 was identified within tumour but not on CD8+ cells. CD8+ cells lacked CXCR3 and granzyme B expression irrespective of location within stroma versus malignant nodules or of the intensity of the intra-tumoural infiltrate. In summary, diverse inflammatory cells were organised around the margin of malignant deposits suggesting response to aberrant signaling, but were unable to penetrate the tumour microenvironment itself to enable an immune response against malignant cells or their polyomavirus.

Raising money for scientific research through crowdfunding.

In this article we discuss the utility of crowdfunding from the perspective of individual scientists or laboratory groups looking to fund research. We address some of the main factors determining the success of crowdfunding campaigns, and compare this approach with the use of traditional funding sources.

Infection and transmission dynamics of rKSHV.219 in primary endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the aetiologic agent of Kaposi's sarcoma (KS), a tumour of endothelial cell origin. The study of KS development was aided by the generation of a recombinant GFP (latent)/RFP (lytic)-expressing KSHV (rKSHV.219) by Vieira and O'Hearn (2004). In this study the first data characterising primary endothelial cell infection and transmission with this virus is presented. Infection was predominantly latent and the percentage of GFP-positive cells increased over time. Neither horizontal transmission of infection, nor cellular proliferation, explained this increase. Analysis of latency-associated nuclear antigen (LANA-1) expression revealed that a threshold level of infection was required for GFP expression early post infection. At later time points GFP correlated more closely with LANA-1 expression, likely due to the accumulation of GFP over time. This study provides methodological guidance for the use of rKSHV.21. In addition, it highlights potential problems associated with the use of fluorescent proteins as markers of viral infection.