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HIV progression is characterized by immune activation and microbial translocation. One factor that may be contributing to HIV progression could be a dysbiotic microbiome. We therefore hypothesized that the GI mucosal microbiome is altered in HIV patients and this alteration correlates with immune activation in HIV. 121 specimens were collected from 21 HIV positive and 22 control human subjects during colonoscopy. The composition of the lower gastrointestinal tract mucosal and luminal bacterial microbiome was characterized using 16S rDNA pyrosequencing and was correlated to clinical parameters as well as immune activation and circulating bacterial products in HIV patients on ART. The composition of the HIV microbiome was significantly different than that of controls; it was less diverse in the right colon and terminal ileum, and was characterized by loss of bacterial taxa that are typically considered commensals. In HIV samples, there was a gain of some pathogenic bacterial taxa. This is the first report characterizing the terminal ileal and colonic mucosal microbiome in HIV patients with next generation sequencing. Limitations include use of HIV-infected subjects on HAART therapy.

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic immune receptor sensing viral RNA. It triggers the release of type I interferons (IFN) and proinflammatory cytokines inducing an adaptive cellular immune response. We investigated the therapeutic potential of systemic RIG-I activation by short 5′-triphosphate-modified RNA (ppp-RNA) for the treatment of acute myeloid leukemia (AML) in the syngeneic murine C1498 AML tumor model. ppp-RNA treatment significantly reduced tumor burden, delayed disease onset and led to complete remission including immunological memory formation in a substantial proportion of animals. Therapy-induced tumor rejection was dependent on CD4+ and CD8+ T cells, but not on NK or B cells, and relied on intact IFN and mitochondrial antiviral signaling protein (MAVS) signaling in the host. Interestingly, ppp-RNA treatment induced programmed death ligand 1 (PD-L1) expression on AML cells and established therapeutic sensitivity to anti-PD-1 checkpoint blockade in vivo. In immune-reconstituted humanized mice, ppp-RNA treatment reduced the number of patient-derived xenografted (PDX) AML cells in blood and bone marrow while concomitantly enhancing CD3+ T cell counts in the respective tissues. Due to its ability to establish a state of full remission and immunological memory, our findings show that ppp-RNA treatment is a promising strategy for the immunotherapy of AML.

Replication competent vesicular stomatitis virus (VSV) is the basis of a vaccine against Ebola and VSV strains are developed as oncolytic viruses. Both functions depend on the ability of VSV to induce adequate amounts of interferon-α/β. It is therefore important to understand how VSV triggers interferon responses. VSV activates innate immunity via retinoic acid-inducible gene I (RIG-I), a sensor for viral RNA. Our results show that VSV needs to replicate for a robust interferon response. Analysis of RIG-I-associated RNA identified a copy-back defective-interfering (DI) genome and full-length viral genomes as main trigger of RIG-I. VSV stocks depleted of DI genomes lost most of their interferon-stimulating activity. The remaining full-length genome and leader-N-read-through sequences, however, still triggered RIG-I. Awareness for DI genomes as trigger of innate immune responses will help to standardize DI genome content and to purposefully deplete or use DI genomes as natural adjuvants in VSV-based therapeutics.

BackgroundPancreatic ductal adenocarcinoma (PDAC) remains largely refractory to chimeric antigen receptor (CAR)-T cell therapy. Insufficient T cell infiltration, a highly immunosuppressive microenvironment, and antigen loss pose major challenges for CAR-T cell therapy.MethodsWe investigated therapeutic synergies of synthetic 5′-triphosphate RNA (3p-RNA), an agonist of the cytoplasmic double-stranded RNA sensor Retinoic Acid Inducible Gene I (RIG-I), and CAR-T cell therapy using syngeneic and human xenograft PDAC models. Tumor growth, chemokine secretion, immune-cell composition, CAR-T persistence, and endogenous T cell responses were assessed by flow cytometry, multiplex cytokine arrays, Enzyme-linked Immunospot (ELISpot), and vaccination-challenge.Results3p-RNA provoked rapid type I interferon accompanied with chemokine ligand CCL5 and CXCL9/10/11 secretion, creating chemokine gradients that recruited chemokine receptor CCR5+/CXCR3+ CAR-T cells into tumors. RIG-I activation enhanced CAR-T cell proliferation, activity, and CAR-T persistence. Combination therapy eradicated established tumors in 60%–70% of mice, whereas either monotherapy was largely ineffective. Cured animals rejected CAR antigen-negative tumor cell rechallenge, demonstrating antigen-spreading and endogenous T cell responses.ConclusionsIntratumoral RIG-I priming reprograms the PDAC microenvironment, transforming a non-responsive cancer into a CAR-T-permissive one, supporting durable, poly-antigenic immunity. These findings position 3p-RNA as a rapid, clinically tractable co-therapy to extend CAR-T efficacy to solid tumors.

Checkpoint molecules such as programmed death 1 (PD-1) dampen excessive T cell activation to preserve immune homeostasis. PD-1-specific monoclonal antibodies have revolutionized cancer therapy, as they reverse tumour-induced T cell exhaustion and restore CTL activity. Based on this success, deciphering underlying mechanisms of PD-1-mediated immune functions has become an important field of immunological research. Initially described for T cells, there is emerging evidence of unconventional PD-1 expression by myeloid as well as tumor cells, yet, with cell-intrinsic functions in various animal tumor models. Here, we describe positive PD-1 antibody staining of various murine immune and tumour cells that is, unlike for T cells, not the PD-1 receptor and restricted to cells with low forward scatter characteristics. Based on flow cytometry and various approaches, including two established murine anti-PD-1 antibody clones, CRISPR/Cas9 genome editing and confocal imaging, we describe a staining pattern assigned to a nuclear antigen cross-reacting with anti-PD-1 monoclonal antibodies. Lack of PD-1 expression was further underlined by the analysis of PD-1 expression from B16-F10-derived 3D cultures and ex vivo tumours. Thus, our data provide multiple lines of evidence that PD-1 expression by non-T cells is unlikely to be the case and, taking recent data of PD-1 tumour cell-intrinsic functions into account, suggest that other antibody-mediated pathways might apply.

BackgroundThe tumor microenvironment (TME) combines features of regulatory cytokines and immune cell populations to evade the recognition by the immune system. Myeloid-derived suppressor cells (MDSC) comprise populations of immature myeloid cells in tumor-bearing hosts with a highly immunosuppressive capacity. We could previously identify RIG-I-like helicases (RLH) as targets for the immunotherapy of pancreatic cancer inducing immunogenic tumor cell death and type I interferons (IFN) as key mediators linking innate with adaptive immunity.MethodsMice with orthotopically implanted KrasG12D p53fl/R172H Ptf1a-Cre (KPC) pancreatic tumors were treated intravenously with the RLH ligand polyinosinic-polycytidylic acid (poly(I:C)), and the immune cell environment in tumor and spleen was characterized. A comprehensive analysis of the suppressive capacity as well as the whole transcriptomic profile of isolated MDSC subsets was performed. Antigen presentation capability of MDSC from mice with ovalbumin (OVA)-expressing tumors was investigated in T cell proliferation assays. The role of IFN in MDSC function was investigated in Ifnar1 −/− mice.ResultsMDSC were strongly induced in orthotopic KPC-derived pancreatic cancer, and frequencies of MDSC subsets correlated with tumor weight and G-CSF serum levels, whereas other immune cell populations decreased. Administration of the RLH-ligand induced a IFN-driven immune response, with increased activation of T cells and dendritic cells (DC), and a reduced suppressive capacity of both polymorphonuclear (PMN)-MDSC and monocytic (M)-MDSC fractions. Whole transcriptomic analysis confirmed an IFN-driven gene signature of MDSC, a switch from a M2/G2- towards a M1/G1-polarized phenotype, and the induction of genes involved in the antigen presentation machinery. Nevertheless, MDSC failed to present tumor antigen to T cells. Interestingly, we found MDSC with reduced suppressive function in Ifnar1-deficient hosts; however, there was a common flaw in immune cell activation, which was reflected by defective immune cell activation and tumor control.ConclusionsWe provide evidence that the treatment with immunostimulatory RNA reprograms the TME of pancreatic cancer by reducing the suppressive activity of MDSC, polarizing myeloid cells into a M1-like state and recruiting DC. We postulate that tumor cell-targeting combination strategies may benefit from RLH-based TME remodeling. In addition, we provide novel insights into the dual role of IFN signaling in MDSC’s suppressive function and provide evidence that host-intrinsic IFN signaling may be critical for MDSC to gain suppressive function during tumor development.

Bowel preparations (BPs) taken before colonoscopy may introduce a confounding effect on the results of gastrointestinal microbiota studies. This study aimed to determine the effect of bowel preparation on the mucosa-associated and luminal colonic microbiota in healthy subjects (HC) and inflammatory bowel disease (IBD) patients. Biopsy samples (n=36) and fecal samples (n=30) were collected from 10 HC and 8 IBD subjects pre- and post-BP. 16S rRNA gene was pyrosequenced using 454 Titanium protocols. We compared the differences between the pre- and post-BP samples (i.e., comparisons-across-bowel-prep); we examined the effect of BP on the expected separation of the mucosal vs. the luminal compartments (i.e., comparisons-across-compartments). Last, we compared the baseline differences between the HC vs. IBD groups (a secondary analysis), and examined whether the differences between the HC vs. IBD changed after BP. In comparisons-across-bowel-prep, the Shannon's index (SI) decreased only in the biopsy samples of IBD subjects post-BP (P=0.025) and phylogenetic diversity-whole tree (PD-WT) metric decreased in biopsy samples of HC subjects post-BP (P=0.021). In secondary comparisons, the subtle differences between the fecal samples of the HC vs. IBD groups, in terms of evenness and the SI, were not apparent post-BP. In terms of β-diversity, in comparisons-across-bowel-prep, the proportion of shared operational taxonomic units (OTUs) in pre- and post-BP samples was low (~30%) and unweighted Unifrac distances between pre- and post-BP specimens ranged from 0.52 to 0.66. HC biopsies were affected more than IBD biopsies with BP (P=0.004). In comparisons-across-compartments, the proportion of shared OTUs between biopsy and fecal samples increased and Unifrac distances decreased post-BP in IBD subjects, reducing the differences between the mucosal and luminal compartments of the gut microbiota. Interindividual differences in Unifrac distances were preserved even with BP effects, although the effects were greater on weighted Unifrac distances. Bacteroidetes and its subtypes increased post-BP in both the luminal and mucosal compartments. Bowel preparations affect the composition and diversity of the fecal and luminal microbiota in the short term, introducing potential bias into experiments examining the gut microbiota. The magnitude of the effect of BP is not greater than that of interindividual variation. Both the luminal and mucosal compartments of the gut microbiota get affected, and samples from controls and IBD subjects may get affected differently. Studies of the colonic microbiota should take into account the direction and the magnitude of the change introduced by BP during the design stage of the experiments, and consider sample sizes so that potential bias is minimized.

According to the World Health Organization, implementing mobile health (mHealth) technologies can increase access to quality health services worldwide. mHealth apps for smartphones, also known as health apps, are a central component of mHealth, and they are already used in diverse medical contexts. To benefit from health apps, potential users need specific skills that enable them to use such apps in a responsible and constructive manner. This study aimed to evaluate the effectiveness of the free and widely used web-based intervention, The APPocalypse?. Besides providing knowledge about health apps, the web-based intervention was designed to promote digital health and media literacy by teaching skills that enable users to distinguish between trustworthy and less trustworthy health apps. It was hypothesized that after completing the web-based intervention, participants' knowledge in the domain of health apps, their digital health literacy, and their media literacy would be higher than it was before completing the web-based intervention. The study was divided into 3 parts. During part 1, participants (n=365; 181 female, 181 male, and 3 diverse; mean age 17.74, SD 1.391 years) provided demographic information and answered the pre- and postmeasurements. The measurements included questionnaires about participants' knowledge in the domain of health apps, digital health literacy, and media literacy. During part 2, participants had 1 week to complete the web-based intervention. During part 3, participants answered the pre- and postmeasurements again. Furthermore, they answered educational quality and user experience questionnaires. Bayesian paired samples 2-tailed t tests were conducted to test the hypotheses. Overall, the results support the hypotheses. After completing the web-based intervention, participants demonstrated more elaborate knowledge in the domain of health apps. Specifically, they displayed higher competencies in the domains of subjective (Bayes factor [BF ]=1.475×10 ; effect size δ=-1.327) and objective health app knowledge (BF =8.162×10 ; effect size δ=-1.350). Furthermore, participants demonstrated higher digital health literacy. Specifically, they displayed higher competencies in the domains of information appraisal (BF =3.413×10 ; effect size δ=-0.870), information searching (BF =3.324×10 ; effect size δ=-0.604), evaluating reliability (BF =3.081×10 ; effect size δ=-0.766), and determining relevance (BF =3.451×10 ; effect size δ=-0.618). Regarding media literacy, the results were mixed. Participants displayed higher competencies in the domain of technology literacy beliefs (BF =1.533×10 ; effect size δ=-0.570). In the domain of technology control beliefs, their competencies did not seem to improve (BF =0.109; effect size δ=-0.058). In comparison to relevant benchmarks, the web-based intervention offers exceptional educational quality and a superior user experience. The free web-based intervention The APPocalypse? might promote the constructive use of health apps, digital health literacy, and media literacy. Therefore, it may contribute to achieving the health-related United Nations Sustainable Development Goals.

Following publication of the original article [1], the authors have reported that Fig. 2 and Additional file 1: Figure S1, S2 partially show red scripts.Following publication of the original article [1], the authors have reported that Fig. 2 and Additional file 1: Figure S1, S2 partially show red scripts.

When searching for health information, many people use the internet as their first source of information. In online health forums, for example, users post their questions and exchange health advice. In recent years, information givers from various professions have begun to use positive language (indicated by the frequent use of positively valenced adjectives) to communicate their information and persuade their audiences. The goal of the current study was to answer the following research questions: (1) How does positive language, in comparison to neutral language, influence the trustworthiness of a person arguing in an online health forum and the credibility of their health claims; (2) How does working for a university, compared to working for a lobbying organization, influence the trustworthiness of a person arguing in an online health forum and the credibility of their health claims; and (3) Do the two factors of language style and professional affiliation interact with each other to influence trustworthiness and credibility judgments? In a 2 × 2 between-subject experiment, 242 participants read a post from an online health forum and subsequently rated the trustworthiness of the forum post author and the credibility of their information. Within the post, the professional affiliation (scientist vs lobbyist) and language style (neutral vs positive) of the forum post author was varied. When the forum post author used a positive language style, they were perceived as less trustworthy (high Machiavellianism [P<.001; η =.076], low Integrity [P=.001; η =.045], and low Benevolence [P=.02; η =.025]) and their information was perceived as less credible (low Message Credibility [P=.001; η =.045]). The professional affiliation of the forum post author did not influence their trustworthiness or the credibility of their information. When searching for health information, information seekers evaluate the language style of forum posts to decide whether forum post authors are trustworthy and their information is credible.