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A group of mountain climbers, caught in the dark, fights to survive their descent; An American band finds more than they bargained for in Mexico while scouting remote locations for a photo shoot; A young student's exploration into the origins of a mysterious song leads him on a winding, dangerous path through the US's deep south; A group of kids scaring each other with ghost stories discovers alarming consequences. The Best Horror of the Year showcases the previous year's best offerings in horror short fiction. This edition includes award-winning and critically acclaimed authors Mark Morris, Kaaron Warren, John Langan, Carole Johnstone, Brian Hodge, and others. For more than three decades, award-winning editor and anthologist Ellen Datlow has had her finger on the pulse of the latest and most terrifying in horror writing. Night Shade Books is proud to present the tenth volume in this annual series, a new collection of stories to keep you up at night.

Little is known about the repertoire of cellular factors involved in the replication of pathogenic alphaviruses. To uncover molecular regulators of alphavirus infection, and to identify candidate drug targets, we performed a high-content imaging-based siRNA screen. We revealed an actin-remodeling pathway involving Rac1, PIP5K1- α, and Arp3, as essential for infection by pathogenic alphaviruses. Infection causes cellular actin rearrangements into large bundles of actin filaments termed actin foci. Actin foci are generated late in infection concomitantly with alphavirus envelope (E2) expression and are dependent on the activities of Rac1 and Arp3. E2 associates with actin in alphavirus-infected cells and co-localizes with Rac1-PIP5K1-α along actin filaments in the context of actin foci. Finally, Rac1, Arp3, and actin polymerization inhibitors interfere with E2 trafficking from the trans-Golgi network to the cell surface, suggesting a plausible model in which transport of E2 to the cell surface is mediated via Rac1- and Arp3-dependent actin remodeling.

In addition to complications of acute diseases, chronic viral infections are linked to both malignancies and autoimmune disorders. Lack of adequate treatment options for Epstein-Barr virus (EBV), Human T-lymphotropic virus type 1 (HTLV-1), and human papillomavirus (HPV) remains. The NexImmune Artificial Immune Modulation (AIM) nanoparticle platform can be used to direct T cell responses by mimicking the dendritic cell function. In one application, AIM nanoparticles are used ex vivo to enrich and expand (E+E) rare populations of multi-antigen-specific CD8 + T cells for use of these cells as an AIM adoptive cell therapy. This study has demonstrated using E+E CD8 + T cells, the functional relevance of targeting EBV, HTLV-1, and HPV. Expanded T cells consist primarily of effector memory, central memory, and self-renewing stem-like memory T cells directed at selected viral antigen peptides presented by the AIM nanoparticle. T cells expanded against either EBV- or HPV-antigens were highly polyfunctional and displayed substantial in vitro cytotoxic activity against cell lines expressing the respective antigens. Our initial work was in the context of exploring T cells expanded from healthy donors and restricted to human leukocyte antigen (HLA)-A*02:01 serotype. AIM Adoptive Cell Therapies (ACT) are also being developed for other HLA class I serotypes. AIM adoptive cell therapies of autologous or allogeneic T cells specific to antigens associated with acute myeloid leukemia and multiple myeloma are currently in the clinic. The utility and flexibility of the AIM nanoparticle platform will be expanded as we advance the second application, an AIM injectable off-the-shelf nanoparticle, which targets multiple antigen-specific T cell populations to either activate, tolerize, or destroy these targeted CD8 + T cells directly in vivo, leaving non-target cells alone. The AIM injectable platform offers the potential to develop new multi-antigen specific therapies for treating infectious diseases, cancer, and autoimmune diseases.

Humoral responses are essential for the protective efficacy of most Ebola virus (EBOV) candidate vaccines; however, the in vivo development of protective anti-EBOV B-cell responses is poorly defined. Here, by using the virus-like particle (VLP) as a model antigen, we demonstrate that humoral responses are generated through follicular B-cell and T-cell-dependent mechanisms in a mouse model of EBOV infection. In addition, we show that the inclusion of the clinical-grade dsRNA adjuvant known as poly-ICLC in VLP vaccinations both augments and sustains germinal center B-cell reactions, antigen-specific B-cell frequencies and anti-EBOV serum titers. Finally, we used mice that were deficient in either B-cells or T-cell-dependent antibody production to distinguish the contributing roles of EBOV humoral responses. We demonstrate that while anti-EBOV antibody responses promote protection, VLP-vaccinated mice can survive EBOV infection in the absence of detectable anti-EBOV antibodies. Moreover, we found that adjuvant signaling could circumvent the complete requirement for B-cell immunity in protection against EBOV. Collectively, these studies may prove valuable for the characterization and future development of additional EBOV vaccine candidates.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovial joints. Inflammation, new blood vessel formation (angiogenesis) and bone resorption (osteoclastogenesis) are three key processes involved in the joint damage and deformities of arthritis. Various gut microbiota-derived metabolites are implicated in RA pathogenesis. However, there is barely any information about the impact of two such metabolites, indole-3-aldehyde (IAld) and indole-3-acetic acid (I3AA), on arthritis-related processes. We conducted a comparative analysis of IAld and I3AA using established cell-based models to understand how they might influence RA pathogenesis. Although structurally similar, the bioactivities of these two metabolites were profoundly different. IAld but not I3AA, inhibited the expression of pro-inflammatory cytokines (IL-1β and IL-6) in RAW 264.7 (RAW) cells stimulated with heat-killed M. tuberculosis sonicate (Mtb) and lipopolysaccharide (LPS). IAld also exhibited pro-angiogenic activity and pro-osteoclastogenic activity. In contrast, I3AA exhibited anti-angiogenic activity on endothelial cell tube formation but had no effect on osteoclastogenesis. Both IAld and I3AA have been proposed as aryl hydrocarbon receptor (AhR) agonists. Use of CH-223191, an inhibitor of the AhR, suppressed the anti-angiogenic activity of I3AA but failed to mitigate the effects of IAld. Further investigation of the anti-inflammatory activities of IAld and I3AA in LPS-treated RAW cells indicated that inhibition of MyD88-dependent activation of NF-κB and MAPK pathways was not likely involved. Our results suggest that the relative bioavailability of these indole derivatives may differentially impact RA progression and possibly other diseases that share similar cellular processes.

Rheumatoid arthritis (RA) is a chronic, debilitating illness characterized by painful swelling of the joints, inflammation of the synovial lining of the joints, and damage to cartilage and bone. Several anti-inflammatory and disease-modifying drugs are available for RA therapy. However, the prolonged use of these drugs is associated with severe side effects. Furthermore, these drugs are effective only in a proportion of RA patients. Hence, there is a need to search for new therapeutic agents that are effective yet safe. Interestingly, a variety of herbs and other natural products offer a vast resource for such anti-arthritic agents. We discuss here the basic features of RA pathogenesis; the commonly used animal models of RA; the mainstream drugs used for RA; the use of well-characterized natural products possessing anti-arthritic activity; the application of nanoparticles for efficient delivery of such products; and the interplay between dietary products and the host microbiome for maintenance of health and disease induction. We believe that with several advances in the past decade in the characterization and functional studies of natural products, the stage is set for widespread clinical testing and/or use of these products for the treatment of RA and other diseases.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the synovial tissue that can lead to joint damage and deformities. Inflammation, new blood vessel formation (angiogenesis), and bone resorption (osteoclastogenesis) are three key processes of the pathophysiology of RA. ‘Dysbiosis’ of the gut microbiota is implicated in RA pathogenesis because it can cause an imbalance in the microbial metabolites that regulate host health and disease. However, there is little information about the impact of two such indole derivatives, indole-3-aldehyde (IAld) and indole-3-acetic acid (I3AA), on arthritis-related processes. Using established cell-based models and the adjuvant-induced arthritis animal model, we conducted a comparative analysis of IAld and I3AA to understand how these metabolites might impact RA pathogenesis. To our surprise, despite their structural similarities, the bioactivities of these two metabolites were profoundly different. IAld, but not I3AA, altered the expression of genes encoding arthritis-associated cytokines (IL-1β, IL-6, VEGF) in RAW 264.7 (murine macrophage) cells stimulated with heat-killed M. tuberculosis. Further investigation of this anti-inflammatory activity of IAld suggested that inhibition of the MyD88-dependentactivation of NF-κB and MAPK pathways was unlikely to be involved. IAld also exhibited pro-osteoclastogenic and pro-angiogenic activity. In contrast, I3AA exhibited only anti-angiogenic activity. Both IAld and I3AA are proposed agonists of the aryl hydrocarbon receptor (AhR). However, AhR inhibitor CH-223191 suppressed the anti-angiogenic activity of I3AA, but failed to mitigate any of the effects of IAld. There is a cross-talk between the AhR and Nrf2 pathways, and some plant-derived phytochemicals are multifunctional ligands of both pathways. Our findings show that IAld, unlike I3AA, can suppress the Nrf2-dependent antioxidant response of macrophages to an Nrf2 agonist, but that IAld also potentially reduces intracellular ROS levels during osteoclast differentiation. Furthermore, oral administration of IAld to rats resulted in the reduction of arthritis severity compared to the arthritic control group. Taken together, our findings suggest that the relative bioavailability of these microbial indole derivatives has the potential to influence their immunomodulatory effects in healthy individuals as well as patients with RA.

BackgroundNexImmune is developing novel, antigen-specific immunotherapies to meet serious unmet clinical needs. Here we report on the development of adoptive cell therapies (ACT) for virally driven malignancies. The proprietary Artificial Immune Modulation (AIM™) platform mimics natural T cell biology to target, activate and expand antigen-specific CD8+ T cells. EBV, HPV, and HTLV are estimated to contribute to 6-7% of global cancer cases. The targeted antigens are implicated in multiple malignancies including B cell lymphomas (EBV), adult T cell leukemia/lymphoma (HLTV1) as well as multiple HPV related malignancies such as oropharyngeal, cervical, and anal cancers.MethodsUsing the paramagnetic AIM nanoparticle as an artificial antigen presenting cell (aAPC), in two-weeks T cells were enriched and expanded from HLA-A*02:01 donor apheresis material against immunodominant antigens of EBV, HTLV, and HPV. The memory phenotype of these cells was determined by CD45RA, CD62L, and CD95 expression. Antigen specific killing was observed on HLA-A2+ cell lines and polyfunctional activity characterized by an intracellular cytokine staining assay.ResultsGreater than 90% of the total resulting CD8+ T cells display a phenotype of effector, central, or long-lived stem-like memory. From 8 independent healthy donor clinical scale manufacturing runs of NEXI-003, a pentavalent specific AIM ACT against the E6 and E7 antigens of both HPV-16 and HPV-18, as well as the tumor-associated antigen Survivin, 0.28E9 to 3.79E9 cells were generated. These cells showed dual HPV-16+ and HPV-18+ cancer antigen specificity and cytotoxic activity against HLA-A2+ cell lines, without significant cytotoxic activity against autologous PBMCs. With respect to EBV, using HLA-A2+ cell lines we further show antigen specific killing directed at LMP2, BRLF1, BMLF1, EBNA3, and LMP1 from a single healthy donor AIM ACT selected on 6 EBV antigens. In addition, these expanded memory T cells demonstrate a high degree of polyfunctional activity upon stimulation.ConclusionsNEXI-003 is an immunotherapy for HPV cancers in HLA*A02:01 patients that recently received IND clearance and it is anticipated to begin clinical trials in 2022. In addition to B cell lymphomas, B cells that are present in the plaques of multiple sclerosis patients have been found to express EBV antigens. Therefore, by directing a multivalent EBV T cell response there is the potential to treat other EBV-associated diseases using one AIM ACT product. Results reported here, will support the expansion of the AIM platform modalities for use in the treatment of virally driven malignancies, as well as potential virally associated autoimmune and infection diseases.

Humoral responses are essential for the protective efficacy of most Ebola virus (EBOV) candidate vaccines; however, the in vivo development of protective anti-EBOV B-cell responses is poorly defined. Here, by using the virus-like particle (VLP) as a model antigen, we demonstrate that humoral responses are generated through follicular B-cell and T-cell-dependent mechanisms in a mouse model of EBOV infection. In addition, we show that the inclusion of the clinical-grade dsRNA adjuvant known as poly-ICLC in VLP vaccinations both augments and sustains germinal center B-cell reactions, antigen-specific B-cell frequencies and anti-EBOV serum titers. Finally, we used mice that were deficient in either B-cells or T-cell-dependent antibody production to distinguish the contributing roles of EBOV humoral responses. We demonstrate that while anti-EBOV antibody responses promote protection, VLP-vaccinated mice can survive EBOV infection in the absence of detectable anti-EBOV antibodies. Moreover, we found that adjuvant signaling could circumvent the complete requirement for B-cell immunity in protection against EBOV. Collectively, these studies may prove valuable for the characterization and future development of additional EBOV vaccine candidates.

Humoral responses are essential for the protective efficacy of most Ebola virus (EBOV) candidate vaccines; however, the in vivo development of protective anti-EBOV B-cell responses is poorly defined. Here, by using the virus-like particle (VLP) as a model antigen, we demonstrate that humoral responses are generated through follicular B-cell and T-cell-dependent mechanisms in a mouse model of EBOV infection. In addition, we show that the inclusion of the clinical-grade dsRNA adjuvant known as poly-ICLC in VLP vaccinations both augments and sustains germinal center B-cell reactions, antigen-specific B-cell frequencies and anti-EBOV serum titers. Finally, we used mice that were deficient in either B-cells or T-cell-dependent antibody production to distinguish the contributing roles of EBOV humoral responses. We demonstrate that while anti-EBOV antibody responses promote protection, VLP-vaccinated mice can survive EBOV infection in the absence of detectable anti-EBOV antibodies. Moreover, we found that adjuvant signaling could circumvent the complete requirement for B-cell immunity in protection against EBOV. Collectively, these studies may prove valuable for the characterization and future development of additional EBOV vaccine candidates.