Individualized, heterologous chimpanzee adenovirus and self-amplifying mRNA neoantigen vaccine for advanced metastatic solid tumors: phase 1 trial interim results.

Checkpoint inhibitor (CPI) therapies provide limited benefit to patients with tumors of low immune reactivity. T cell-inducing vaccines hold promise to exert long-lasting disease control in combination with CPI therapy. Safety, tolerability and recommended phase 2 dose (RP2D) of an individualized, heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA (samRNA)-based neoantigen vaccine in combination with nivolumab and ipilimumab were assessed as primary endpoints in an ongoing phase 1/2 study in patients with advanced metastatic solid tumors (NCT03639714). The individualized vaccine regimen was safe and well tolerated, with no dose-limiting toxicities. Treatment-related adverse events (TRAEs) >10% included pyrexia, fatigue, musculoskeletal and injection site pain and diarrhea. Serious TRAEs included one count each of pyrexia, duodenitis, increased transaminases and hyperthyroidism. The RP2D was 1012 viral particles (VP) ChAd68 and 30 µg samRNA. Secondary endpoints included immunogenicity, feasibility of manufacturing and overall survival (OS). Vaccine manufacturing was feasible, with vaccination inducing long-lasting neoantigen-specific CD8 T cell responses. Several patients with microsatellite-stable colorectal cancer (MSS-CRC) had improved OS. Exploratory biomarker analyses showed decreased circulating tumor DNA (ctDNA) in patients with prolonged OS. Although small study size limits statistical and translational analyses, the increased OS observed in MSS-CRC warrants further exploration in larger randomized studies.

Microbiota and adipocyte mitochondrial damage in type 2 diabetes are linked by Mmp12+ macrophages.

Microbiota contribute to the induction of type 2 diabetes by high-fat/high-sugar (HFHS) diet, but which organs/pathways are impacted by microbiota remain unknown. Using multiorgan network and transkingdom analyses, we found that microbiota-dependent impairment of OXPHOS/mitochondria in white adipose tissue (WAT) plays a primary role in regulating systemic glucose metabolism. The follow-up analysis established that Mmp12+ macrophages link microbiota-dependent inflammation and OXPHOS damage in WAT. Moreover, the molecular signature of Mmp12+ macrophages in WAT was associated with insulin resistance in obese patients. Next, we tested the functional effects of MMP12 and found that Mmp12 genetic deficiency or MMP12 inhibition improved glucose metabolism in conventional, but not in germ-free mice. MMP12 treatment induced insulin resistance in adipocytes. TLR2-ligands present in Oscillibacter valericigenes bacteria, which are expanded by HFHS, induce Mmp12 in WAT macrophages in a MYD88-ATF3-dependent manner. Thus, HFHS induces Mmp12+ macrophages and MMP12, representing a microbiota-dependent bridge between inflammation and mitochondrial damage in WAT and causing insulin resistance.

Transkingdom interactions between Lactobacilli and hepatic mitochondria attenuate western diet-induced diabetes.

Western diet (WD) is one of the major culprits of metabolic disease including type 2 diabetes (T2D) with gut microbiota playing an important role in modulating effects of the diet. Herein, we use a data-driven approach (Transkingdom Network analysis) to model host-microbiome interactions under WD to infer which members of microbiota contribute to the altered host metabolism. Interrogation of this network pointed to taxa with potential beneficial or harmful effects on host's metabolism. We then validate the functional role of the predicted bacteria in regulating metabolism and show that they act via different host pathways. Our gene expression and electron microscopy studies show that two species from Lactobacillus genus act upon mitochondria in the liver leading to the improvement of lipid metabolism. Metabolomics analyses revealed that reduced glutathione may mediate these effects. Our study identifies potential probiotic strains for T2D and provides important insights into mechanisms of their action.

CVID enteropathy is characterized by exceeding low mucosal IgA levels and interferon-driven inflammation possibly related to the presence of a pathobiont.

Common variable immunodeficiency (CVID), the most common symptomatic primary antibody deficiency, is accompanied in some patients by a duodenal inflammation and malabsorption syndrome known as CVID enteropathy (E-CVID).The goal of this study was to investigate the immunological abnormalities in CVID patients that lead to enteropathy as well as the contribution of intestinal microbiota to this process.We found that, in contrast to noE-CVID patients (without enteropathy), E-CVID patients have exceedingly low levels of IgA in duodenal tissues. In addition, using transkingdom network analysis of the duodenal microbiome, we identified Acinetobacter baumannii as a candidate pathobiont in E-CVID. Finally, we found that E-CVID patients exhibit a pronounced activation of immune genes and down-regulation of epithelial lipid metabolism genes. We conclude that in the virtual absence of mucosal IgA, pathobionts such as A. baumannii, may induce inflammation that re-directs intestinal molecular pathways from lipid metabolism to immune processes responsible for enteropathy.

Antibiotic-Induced Alterations in Gut Microbiota Are Associated with Changes in Glucose Metabolism in Healthy Mice.

The gut microbiome plays an important role in health and disease. Antibiotics are known to alter gut microbiota, yet their effects on glucose tolerance in lean, normoglycemic mice have not been widely investigated. In this study, we aimed to explore mechanisms by which treatment of lean mice with antibiotics (ampicillin, metronidazole, neomycin, vancomycin, or their cocktail) influences the microbiome and glucose metabolism. Specifically, we sought to: (i) study the effects on body weight, fasting glucose, glucose tolerance, and fasting insulin, (ii) examine the changes in expression of key genes of the bile acid and glucose metabolic pathways in the liver and ileum, (iii) identify the shifts in the cecal microbiota, and (iv) infer interactions between gene expression, microbiome, and the metabolic parameters. Treatment with individual or a cocktail of antibiotics reduced fasting glucose but did not affect body weight. Glucose tolerance changed upon treatment with cocktail, ampicillin, or vancomycin as indicated by reduced area under the curve of the glucose tolerance test. Antibiotic treatment changed gene expression in the ileum and liver, and shifted the alpha and beta diversities of gut microbiota. Network analyses revealed associations between Akkermansia muciniphila with fasting glucose and liver farsenoid X receptor (Fxr) in the top ranked host-microbial interactions, suggesting possible mechanisms by which this bacterium can mediate systemic changes in glucose metabolism. We observed Bacteroides uniformis to be positively and negatively correlated with hepatic Fxr and Glucose 6-phosphatase, respectively. Overall, our transkingdom network approach is a useful hypothesis generating strategy that offers insights into mechanisms by which antibiotics can regulate glucose tolerance in non-obese healthy animals. Experimental validation of our predicted microbe-phenotype interactions can help identify mechanisms by which antibiotics affect host phenotypes and gut microbiota.

Arsenic exposure and intestinal microbiota in children from Sirajdikhan, Bangladesh.

BACKGROUND: Arsenic has antimicrobial properties at high doses yet few studies have examined its effect on gut microbiota. This warrants investigation since arsenic exposure increases the risk of many diseases in which gut microbiota have been shown to play a role. We examined the association between arsenic exposure from drinking water and the composition of intestinal microbiota in children exposed to low and high arsenic levels during prenatal development and early life. RESULTS: 16S rRNA gene sequencing revealed that children with high arsenic exposure had a higher abundance of Proteobacteria in their stool compared to matched controls with low arsenic exposure. Furthermore, whole metagenome shotgun sequencing identified 332 bacterial SEED functions that were enriched in the high exposure group. A separate model showed that these genes, which included genes involved in virulence and multidrug resistance, were positively correlated with arsenic concentration within the group of children in the high arsenic group. We performed reference free genome assembly, and identified strains of E.coli as contributors to the arsenic enriched SEED functions. Further genome annotation of the E.coli genome revealed two strains containing two different arsenic resistance operons that are not present in the gut microbiome of a recently described European human cohort (Metagenomics of the Human Intestinal Tract, MetaHIT). We then performed quantification by qPCR of two arsenic resistant genes (ArsB, ArsC). We observed that the expression of these two operons was higher among the children with high arsenic exposure compared to matched controls. CONCLUSIONS: This preliminary study indicates that arsenic exposure early in life was associated with altered gut microbiota in Bangladeshi children. The enrichment of E.coli arsenic resistance genes in the high exposure group provides an insight into the possible mechanisms of how this toxic compound could affect gut microbiota.

Akkermansia muciniphila mediates negative effects of IFNγ on glucose metabolism.

Cross-talk between the gut microbiota and the host immune system regulates host metabolism, and its dysregulation can cause metabolic disease. Here, we show that the gut microbe Akkermansia muciniphila can mediate negative effects of IFNγ on glucose tolerance. In IFNγ-deficient mice, A. muciniphila is significantly increased and restoration of IFNγ levels reduces A. muciniphila abundance. We further show that IFNγ-knockout mice whose microbiota does not contain A. muciniphila do not show improvement in glucose tolerance and adding back A. muciniphila promoted enhanced glucose tolerance. We go on to identify Irgm1 as an IFNγ-regulated gene in the mouse ileum that controls gut A. muciniphila levels. A. muciniphila is also linked to IFNγ-regulated gene expression in the intestine and glucose parameters in humans, suggesting that this trialogue between IFNγ, A. muciniphila and glucose tolerance might be an evolutionally conserved mechanism regulating metabolic health in mice and humans.

The Military Orthopedic Trauma Registry: The potential of a specialty specific process improvement tool.

BACKGROUND: The Military Orthopaedic Trauma Registry (MOTR) was designed to replicate the Department of Defense Trauma Registry's (DoDTR's) role as pillar for data-driven management of extremity war wounds. The MOTR continuously undergoes quality assurance checks to optimize the registry data for future quality improvement efforts. We conducted a quality assurance survey of MOTR entrants to determine if a simple MOTR data pull could provide robust orthopedic-specific information toward the question of causes for late amputation. METHODS: Forty-five entrants into the DoDTR with late transtibial amputation were sequentially abstracted into MOTR by MOTR data abstractors. The MOTR record was then examined by an independent reviewer for three data fields pertaining to the events leading up to the late amputation: injury before limb amputation, complications before and after amputation, and complication or other factor directly contributing to the decision for amputation. RESULTS: Thirty-nine subjects had at least one fracture of the tibial diaphysis, tibial pilon, calcaneus, or multiple foot fractures. Twenty-nine fractures were described as open injuries for which 27 included a Gustilo and Anderson classification in the available data fields. Complications could be identified along the treatment course for 43 of the 45 entrants specific to the amputated limb. A directly contributing factor to late amputation was identified in 36 (80%) of the subjects. Infection, either alone or associated with fracture nonunion, was a contributing factor in 46% of late amputations. Wound infection was the most common complication both before and after the amputation. CONCLUSION: The MOTR, using a simple data extraction from a few registry fields, can provide a robust amount of information that can direct process and care improvement for severely injured limbs by providing the level of detail pertinent to an orthopedic surgeon. LEVEL OF EVIDENCE: Prognostic/epidemiological study, level IV.

Military Orthopaedic Trauma Registry: Quality Data Now Available.

The Military Orthopaedic Trauma Registry (MOTR) orginally began as part of the Department of Defense Trauma Registry (DoDTR) and became a live registry in 2013. As a quality improvement process, this study examined MOTR data for 20 female amputees compared with DoDTR data. The DoDTR provided diagnosis and procedure codes as a list but no details. The MOTR provided additional data, including specific limb, fracture classifications, and associated injuries per limb. The MOTR allowed for construction of a treatment time line for each limb, including number and timing of debridements, antibiotics, and implant types. Orthopaedic-specific complications were also coded more frequently in the MOTR and clearly identified with a specific injury and treatment. During initial quality control checks, the MOTR provides a greater volume and granularity of detail for orthopaedic-specific injury and treatment information, indicating that the MOTR is on track to provide a valuable repository for data-driven orthopaedic management of combat injury.

Investigating a holobiont: Microbiota perturbations and transkingdom networks.

The scientific community has recently come to appreciate that, rather than existing as independent organisms, multicellular hosts and their microbiota comprise a complex evolving superorganism or metaorganism, termed a holobiont. This point of view leads to a re-evaluation of our understanding of different physiological processes and diseases. In this paper we focus on experimental and computational approaches which, when combined in one study, allowed us to dissect mechanisms (traditionally named host-microbiota interactions) regulating holobiont physiology. Specifically, we discuss several approaches for microbiota perturbation, such as use of antibiotics and germ-free animals, including advantages and potential caveats of their usage. We briefly review computational approaches to characterize the microbiota and, more importantly, methods to infer specific components of microbiota (such as microbes or their genes) affecting host functions. One such approach called transkingdom network analysis has been recently developed and applied in our study. (1) Finally, we also discuss common methods used to validate the computational predictions of host-microbiota interactions using in vitro and in vivo experimental systems.

Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation.

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation.

Uncovering effects of antibiotics on the host and microbiota using transkingdom gene networks.

OBJECTIVE: Despite widespread use of antibiotics for the treatment of life-threatening infections and for research on the role of commensal microbiota, our understanding of their effects on the host is still very limited. DESIGN: Using a popular mouse model of microbiota depletion by a cocktail of antibiotics, we analysed the effects of antibiotics by combining intestinal transcriptome together with metagenomic analysis of the gut microbiota. In order to identify specific microbes and microbial genes that influence the host phenotype in antibiotic-treated mice, we developed and applied analysis of the transkingdom network. RESULTS: We found that most antibiotic-induced alterations in the gut can be explained by three factors: depletion of the microbiota; direct effects of antibiotics on host tissues and the effects of remaining antibiotic-resistant microbes. Normal microbiota depletion mostly led to downregulation of different aspects of immunity. The two other factors (antibiotic direct effects on host tissues and antibiotic-resistant microbes) primarily inhibited mitochondrial gene expression and amounts of active mitochondria, increasing epithelial cell death. By reconstructing and analysing the transkingdom network, we discovered that these toxic effects were mediated by virulence/quorum sensing in antibiotic-resistant bacteria, a finding further validated using in vitro experiments. CONCLUSIONS: In addition to revealing mechanisms of antibiotic-induced alterations, this study also describes a new bioinformatics approach that predicts microbial components that regulate host functions and establishes a comprehensive resource on what, why and how antibiotics affect the gut in a widely used mouse model of microbiota depletion by antibiotics.

Dicer regulates differentiation and viability during mouse pancreatic cancer initiation.

miRNA levels are altered in pancreatic ductal adenocarcinoma (PDA), the most common and lethal pancreatic malignancy, and intact miRNA processing is essential for lineage specification during pancreatic development. However, the role of miRNA processing in PDA has not been explored. Here we study the role of miRNA biogenesis in PDA development by deleting the miRNA processing enzyme Dicer in a PDA mouse model driven by oncogenic Kras. We find that loss of Dicer accelerates Kras driven acinar dedifferentiation and acinar to ductal metaplasia (ADM), a process that has been shown to precede and promote the specification of PDA precursors. However, unconstrained ADM also displays high levels of apoptosis. Dicer loss does not accelerate development of Kras driven PDA precursors or PDA, but surprisingly, we observe that mouse PDA can develop without Dicer, although at the expense of proliferative capacity. Our data suggest that intact miRNA processing is involved in both constraining pro-tumorigenic changes in pancreatic differentiation as well as maintaining viability during PDA initiation.

Bridging immunity and lipid metabolism by gut microbiota.

The human gut is a unique organ in which hundreds of different microbial species find their habitat and in which different host physiologic functions, such as digestion, nutrition, and immunity, coexist. Although all these players were studied separately for decades, recently, there has been an explosion of studies demonstrating the essential role for interactions between these components in gut function. Furthermore, new systems biology methods provide essential tools to study this complex system as a whole and to identify key elements that define the crosstalk between the gut microbiota, immunity, and metabolism. This review is devoted to several human diseases resulting from the disruption in this crosstalk, including immunodeficiency-associated and environmental enteropathies, celiac disease, inflammatory bowel disease, and obesity. We describe findings in experimental models of these diseases and in germ-free animals that help us understand the mechanisms and test new therapeutic strategies. We also discuss current challenges that the field is facing and propose that a new generation of antibiotics, prebiotics, and probiotics coupled with novel, systems biology-driven diagnostics will provide the basis for future personalized therapy.

Numb regulates acinar cell dedifferentiation and survival during pancreatic damage and acinar-to-ductal metaplasia.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDA) is a leading cause of cancer-related death. Through the process of acinar-to-ductal metaplasia (ADM), pancreatic acinar cells give rise to pancreatic intraepithelial neoplasia (PanIN), the most common precursor of PDA. However, even when Kras is activated in a majority of acinar cells, ADM and subsequent development of PanINs is inefficient in the absence of additional stresses. Numb regulates cell junctions, integrins, and the activity of embryonic signaling pathways; therefore, we investigated its effects on acinar cell dedifferentiation, regeneration, and metaplasia. METHODS: We used mouse models of pancreatic regeneration and PDA as well as mice with loss-of-function alleles of Numb (p48Cre/p48Cre(ER);Numb(f/f) and p48Cre/p48Cre(ER);Kras(G12D);Numb(f/f) mice) to study the roles of Numb in pancreatic regeneration and ADM. RESULTS: Loss of Numb resulted in premature dedifferentiation of acinar cells in response to injury due to administration of the cholecystokinin analogue cerulein and interfered with acinar cell regeneration. Numb was found to regulate multiple signaling pathways in acinar cells during cerulein-induced pancreatitis. Disruption of Numb accelerated and destabilized ADM in the context of oncogenic Kras (in p48Cre;Kras(G12D);Numb(f/f) and p48Cre(ER);Kras(G12D);Numb(f/f) mice). CONCLUSIONS: Numb is an important regulator of acinar cell differentiation and viability during metaplasia. In mice with pancreatitis or pancreatic injury, elimination of Numb causes dedifferentiated acinar cells to undergo apoptosis, and this is not mitigated by oncogenic Kras.