BACKGROUND: The red macroalgae Asparagopsis is an effective methanogenesis inhibitor due to the presence of halogenated methane (CH4) analogues, primarily bromoform (CHBr3). This study aimed to investigate the degradation process of CHBr3 from A. taxiformis in the rumen and whether this process is diet-dependent. An in vitro batch culture system was used according to a 2 × 2 factorial design, assessing two A. taxiformis inclusion rates [0 (CTL) and 2% DM diet (AT)] and two diets [high-concentrate (HC) and high-forage diet (HF)]. Incubations lasted for 72 h and samples of headspace and fermentation liquid were taken at 0, 0.5, 1, 3, 6, 8, 12, 16, 24, 48 and 72 h to assess the pattern of degradation of CHBr3 into dibromomethane (CH2Br2) and fermentation parameters. Additionally, an in vitro experiment with pure cultures of seven methanogens strains (Methanobrevibacter smithii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, Methanosarcina barkeri, Methanobrevibacter millerae, Methanothermobacter wolfei and Methanobacterium mobile) was conducted to test the effects of increasing concentrations of CHBr3 (0.4, 2, 10 and 50 µmol/L). RESULTS: The addition of AT significantly decreased CH4 production (P = 0.002) and the acetate:propionate ratio (P = 0.003) during a 72-h incubation. The concentrations of CHBr3 showed a rapid decrease with nearly 90% degraded within the first 3 h of incubation. On the contrary, CH2Br2 concentration quickly increased during the first 6 h and then gradually decreased towards the end of the incubation. Neither CHBr3 degradation nor CH2Br2 synthesis were affected by the type of diet used as substrate, suggesting that the fermentation rate is not a driving factor involved in CHBr3 degradation. The in vitro culture of methanogens showed a dose-response effect of CHBr3 by inhibiting the growth of M. smithii, M. ruminantium, M. stadtmanae, M. barkeri, M. millerae, M. wolfei, and M. mobile. CONCLUSIONS: The present work demonstrated that CHBr3 from A. taxiformis is quickly degraded to CH2Br2 in the rumen and that the fermentation rate promoted by different diets is not a driving factor involved in CHBr3 degradation.

VCAM-1 and ICAM-1, receptors for leukocyte integrins, are recruited to cell-cell contact sites on the apical membrane of activated endothelial cells. In this study, we show that this recruitment is independent of ligand engagement, actin cytoskeleton anchorage, and heterodimer formation. Instead, VCAM-1 and ICAM-1 are recruited by inclusion within specialized preformed tetraspanin-enriched microdomains, which act as endothelial adhesive platforms (EAPs). Using advanced analytical fluorescence techniques, we have characterized the diffusion properties at the single-molecule level, nanoscale organization, and specific intradomain molecular interactions of EAPs in living primary endothelial cells. This study provides compelling evidence for the existence of EAPs as physical entities at the plasma membrane, distinct from lipid rafts. Scanning electron microscopy of immunogold-labeled samples treated with a specific tetraspanin-blocking peptide identify nanoclustering of VCAM-1 and ICAM-1 within EAPs as a novel mechanism for supramolecular organization that regulates the leukocyte integrin-binding capacity of both endothelial receptors during extravasation.

PURPOSE: The capability of ultrahigh dose rate FLASH radiation therapy to generate the FLASH effect has opened the possibility to enhance the therapeutic index of radiation therapy. The contribution of the immune response has frequently been hypothesized to account for a certain fraction of the antitumor efficacy and tumor kill of FLASH but has yet to be rigorously evaluated. METHODS AND MATERIALS: To investigate the immune response as a potentially important mechanism of the antitumor effect of FLASH, various murine tumor models were grafted either subcutaneously or orthotopically into immunocompetent mice or in moderately and severely immunocompromised mice. Mice were locally irradiated with single dose (20 Gy) or hypofractionated regimens (3 × 8 or 2 × 6 Gy) using FLASH (≥2000 Gy/s) and conventional (CONV) dose rates (0.1 Gy/s), with/without anti-CTLA-4. Tumor growth was monitored over time and immune profiling performed. RESULTS: FLASH and CONV 20 Gy were isoeffective in delaying tumor growth in immunocompetent and moderately immunodeficient hosts and increased tumor doubling time to >14 days versus >7 days in control animals. Similar observations were obtained with a hypofractionated scheme, regardless of the microenvironment (subcutaneous flank vs ortho lungs). Interestingly, in profoundly immunocompromised mice, 20 Gy FLASH retained antitumor activity and significantly increased tumor doubling time to >14 days versus >8 days in control animals, suggesting a possible antitumor mechanism independent of the immune response. Analysis of the tumor microenvironment showed similar immune profiles after both irradiation modalities with significant decrease of lymphoid cells by ∼40% and a corresponding increase of myeloid cells. In addition, FLASH and CONV did not increase transforming growth factor-β1 levels in tumors compared with unirradiated control animals. Furthermore, when a complete and long-lasting antitumor response was obtained (>140 days), both modalities of irradiation were able to generate a long-term immunologic memory response. CONCLUSIONS: The present results clearly document that the tumor responses across multiple immunocompetent and immunodeficient mouse models are largely dose rate independent and simultaneously contradict a major role of the immune response in the antitumor efficacy of FLASH. Therefore, our study indicates that FLASH is as potent as CONV in modulating antitumor immune response and can be used as an immunomodulatory agent.

The fourth "Melanoma Bridge Meeting" took place in Naples, December 3-6th, 2014. The four topics discussed at this meeting were: Molecular and Immunological Advances, Combination Therapies, News in Immunotherapy, and Tumor Microenvironment and Biomarkers. Until recently systemic therapy for metastatic melanoma patients was ineffective, but recent advances in tumor biology and immunology have led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS). New therapies, such as mitogen-activated protein kinase (MAPK) pathway inhibitors as well as other signaling pathway inhibitors, are being tested in patients with metastatic melanoma either as monotherapy or in combination, and all have yielded promising results. These include inhibitors of receptor tyrosine kinases (BRAF, MEK, and VEGFR), the phosphatidylinositol 3 kinase (PI3K) pathway [PI3K, AKT, mammalian target of rapamycin (mTOR)], activators of apoptotic pathway, and the cell cycle inhibitors (CDK4/6). Various locoregional interventions including radiotherapy and surgery are still valid approaches in treatment of advanced melanoma that can be integrated with novel therapies. Intrinsic, adaptive and acquired resistance occur with targeted therapy such as BRAF inhibitors, where most responses are short-lived. Given that the reactivation of the MAPK pathway through several distinct mechanisms is responsible for the majority of acquired resistance, it is logical to combine BRAF inhibitors with inhibitors of targets downstream in the MAPK pathway. For example, combination of BRAF/MEK inhibitors (e.g., dabrafenib/trametinib) have been demonstrated to improve survival compared to monotherapy. Application of novel technologies such sequencing have proven useful as a tool for identification of MAPK pathway-alternative resistance mechanism and designing other combinatorial therapies such as those between BRAF and AKT inhibitors. Improved survival rates have also been observed with immune-targeted therapy for patients with metastatic melanoma. Immune-modulating antibodies came to the forefront with anti-CTLA-4, programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) pathway blocking antibodies that result in durable responses in a subset of melanoma patients. Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors and other approaches such as adoptive cell transfer demonstrate clinical benefit in patients with melanoma as well. These agents are being studied in combination with targeted therapies in attempt to produce longer-term responses than those more typically seen with targeted therapy. Other combinations with cytotoxic chemotherapy and inhibitors of angiogenesis are changing the evolving landscape of therapeutic options and are being evaluated to prevent or delay resistance and to further improve survival rates for this patient population. This meeting's specific focus was on advances in combination of targeted therapy and immunotherapy. Both combination targeted therapy approaches and different immunotherapies were discussed. Similarly to the previous meetings, the importance of biomarkers for clinical application as markers for diagnosis, prognosis and prediction of treatment response was an integral part of the meeting. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage. Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma.

Human foragers are obligately group-living, and their high dependence on mutual aid is believed to have characterized our species' social evolution. It was therefore a central adaptive problem for our ancestors to avoid damaging the willingness of other group members to render them assistance. Cognitively, this requires a predictive map of the degree to which others would devalue the individual based on each of various possible acts. With such a map, an individual can avoid socially costly behaviors by anticipating how much audience devaluation a potential action (e.g., stealing) would cause and weigh this against the action's direct payoff (e.g., acquiring). The shame system manifests all of the functional properties required to solve this adaptive problem, with the aversive intensity of shame encoding the social cost. Previous data from three Western(ized) societies indicated that the shame evoked when the individual anticipates committing various acts closely tracks the magnitude of devaluation expressed by audiences in response to those acts. Here we report data supporting the broader claim that shame is a basic part of human biology. We conducted an experiment among 899 participants in 15 small-scale communities scattered around the world. Despite widely varying languages, cultures, and subsistence modes, shame in each community closely tracked the devaluation of local audiences (mean r = +0.84). The fact that the same pattern is encountered in such mutually remote communities suggests that shame's match to audience devaluation is a design feature crafted by selection and not a product of cultural contact or convergent cultural evolution.

Whereas preclinical investigations and clinical studies have established that CD8⁺ T cells can profoundly affect cancer progression, the underlying mechanisms are still elusive. Challenging the prevalent view that the beneficial effect of CD8⁺ T cells in cancer is solely attributable to their cytotoxic activity, several reports have indicated that the ability of CD8⁺ T cells to promote tumor regression is dependent on their cytokine secretion profile and their ability to self-renew. Evidence has also shown that the tumor microenvironment can disarm CD8⁺ T cell immunity, leading to the emergence of dysfunctional CD8⁺ T cells. The existence of different types of CD8⁺ T cells in cancer calls for a more precise definition of the CD8⁺ T cell immune phenotypes in cancer and the abandonment of the generic terms "pro-tumor" and "antitumor." Based on recent studies investigating the functions of CD8⁺ T cells in cancer, we here propose some guidelines to precisely define the functional states of CD8⁺ T cells in cancer.

Abstract Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer.