Numerous reports have documented bacteria in the placental membranes and basal plate decidua in the absence of immunopathology using histologic techniques. Similarly, independent metagenomic ...characterizations have identified an altered taxonomic makeup in association with spontaneous preterm birth. Here we sought to corroborate these findings by localizing presumptive intact bacteria using molecular histology within the placental microanatomy.
Here we examined for microbes in term and preterm gestations using a signal-amplified 16S universal in situ hybridization probe set for bacterial rRNA, alongside traditional histologic methods of Warthin–Starry and Gram stains, as well as clinical culture methodologies. We further sought to differentiate accompanying 16S gene sequencing taxonomic profiles from germ-free (gnotobiotic) mouse and extraction and amplicon contamination controls.
Placentas were collected from a total of 53 subjects, composed of term labored (n = 4) and unlabored cesarean deliveries (n = 22) and preterm vaginal (n = 18) and cesarean deliveries (n = 8); a placenta from a single subject with clinical and histologic evident choriomanionitis was employed as a positive control (n = 1). The preterm cohort included spontaneous preterm birth with (n = 6) and without (n = 10) preterm premature rupture of membranes, as well as medically indicated preterm births (n = 10). Placental microbes were visualized using an in situ hybridization probe set designed against highly conserved regions of the bacterial 16S ribosome, which produces an amplified stable signal using branched DNA probes. Extracted bacterial nucleic acids from these same samples were subjected to 16S rRNA metagenomic sequencing (Illumina, V4) for course taxonomic analysis, alongside environmental and kit contaminant controls. A subset of unlabored, cesarean-delivered term pregnancies were also assessed with clinical culture for readily cultivatable pathogenic microbes.
Molecular in situ hybridization of bacterial rRNA enabled visualization and localization of low-abundance microbes after systematic high-power scanning. Despite the absence of clinical or histologic chorioamnionitis in 52 of 53 subjects, instances of 16S rRNA signal were confidently observed in 13 of 16 spontaneous preterm birth placentas, which was not significantly different from term unlabored cesarean specimens (18 of 22; P > .05). 16S rRNA signal was largely localized to the villous parenchyma and/or syncytiotrophoblast, and less commonly the chorion and the maternal intervillous space. In all term and unlabored cesarean deliveries, visualization of evident placental microbes by in situ hybridization occurred in the absence of clinical or histologic detection using conventional clinical cultivation, hematoxylin–eosin, and Gram staining. In 1 subject, appreciable villous bacteria localized to an infarction, where 16S microbial detection was confirmed by Warthin–Starry stain. In all instances, parallel sample principle coordinate analysis using Bray-Cutis distances of 16S rRNA gene sequencing data demonstrated consistent taxonomic distinction from all negative or potential contamination controls (P = .024, PERMANOVA). Classification from contaminant filtered data identified a distinct taxonomic makeup among term and preterm cohorts when compared with contaminant controls (false discovery rate <0.05).
Presumptively intact placental microbes are visualized as low-abundance, low-biomass and sparse populations within the placenta regardless of gestational age and mode of delivery. Their taxonomic makeup is distinct from contamination controls. These findings further support several previously published findings, including our own, which have used metagenomics to characterize low-abundance and low-biomass microbial communities in the placenta.
Abstract Background and purpose Type 1 diabetes mellitus (T1DM) is a pro-inflammatory state with increased toll-like receptor (TLR) activity. Inflammation is crucial in diabetic nephropathy (DN). We ...tested the effect of global deficiency of TLR4 on renal inflammation, fibrosis and podocytopathy using control (C) and streptozotocin (STZ) induced diabetic wildtype (WT) and TLR4-knockout (TLR4KO) mice. Methods Following STZ treatment, mice were euthanized at 17 weeks and plasma and kidneys collected. Results Compared to C, STZ-WT mice had significantly increased macrophage and TLR4 immunostaining in kidney, significant increases in MyD88, Interferon Regulatory Factor-3, NFKappaB activity, TNF-Alpha, IL-6, and MCP-1; all these were significantly decreased in the STZ-TLR4KO compared to STZ-WT mice. Compared to C, there were significant increases in fibrosis markers (collagen 4, and transforming growth factor-beta) in STZ-WT which were significantly decreased in the STZ-TLR4KO versus STZ-WT. Podocyte numbers and podocin were decreased in the STZ-WT versus C and increased in the STZ-TLR4KO mice. Conclusion Global genetic deficiency of TLR4 also ameliorates renal inflammation, fibrosis and podocytopathy and could be important in DN.
Accumulating studies have defined a role for the intestinal microbiota in modulation of host behavior. Research using gnotobiotic mice emphasizes that early microbial colonization with a complex ...microbiota (conventionalization) can rescue some of the behavioral abnormalities observed in mice that grow to adulthood completely devoid of bacteria (germ-free mice). However, the human infant and adult microbiomes vary greatly, and effects of the neonatal microbiome on neurodevelopment are currently not well understood. Microbe-mediated modulation of neural circuit patterning in the brain during neurodevelopment may have significant long-term implications that we are only beginning to appreciate. Modulation of the host central nervous system by the early-life microbiota is predicted to have pervasive and lasting effects on brain function and behavior. We sought to replicate this early microbe-host interaction by colonizing gnotobiotic mice at the neonatal stage with a simplified model of the human infant gut microbiota. This model consortium consisted of four "infant-type" Bifidobacterium species known to be commensal members of the human infant microbiota present in high abundance during postnatal development. Germ-free mice and mice neonatally-colonized with a complex, conventional murine microbiota were used for comparison. Motor and non-motor behaviors of the mice were tested at 6-7 weeks of age, and colonization patterns were characterized by 16S ribosomal RNA gene sequencing. Adult germ-free mice were observed to have abnormal memory, sociability, anxiety-like behaviors, and motor performance. Conventionalization at the neonatal stage rescued these behavioral abnormalities, and mice colonized with Bifidobacterium spp. also exhibited important behavioral differences relative to the germ-free controls. The ability of Bifidobacterium spp. to improve the recognition memory of both male and female germ-free mice was a prominent finding. Together, these data demonstrate that the early-life gut microbiome, and human "infant-type" Bifidobacterium species, affect adult behavior in a strongly sex-dependent manner, and can selectively recapitulate the results observed when mice are colonized with a complex microbiota.
Probiotics and commensal intestinal microbes suppress mammalian cytokine production and intestinal inflammation in various experimental model systems. Limited information exists regarding potential ...mechanisms of probiotic-mediated immunomodulation in vivo. In this report, we demonstrate that specific probiotic strains of Lactobacillus reuteri suppress intestinal inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse colitis model. Only strains that possess the hdc gene cluster, including the histidine decarboxylase and histidine-histamine antiporter genes, can suppress colitis and mucosal cytokine (interleukin-6 IL-6 and IL-1β in the colon) gene expression. Suppression of acute colitis in mice was documented by diminished weight loss, colonic injury, serum amyloid A (SAA) protein concentrations, and reduced uptake of (18)Ffluorodeoxyglucose ((18)FFDG) in the colon by positron emission tomography (PET). The ability of probiotic L. reuteri to suppress colitis depends on the presence of a bacterial histidine decarboxylase gene(s) in the intestinal microbiome, consumption of a histidine-containing diet, and signaling via the histamine H2 receptor (H2R). Collectively, luminal conversion of l-histidine to histamine by hdc(+) L. reuteri activates H2R, and H2R signaling results in suppression of acute inflammation within the mouse colon.
Probiotics are microorganisms that when administered in adequate amounts confer beneficial effects on the host. Supplementation with probiotic strains was shown to suppress intestinal inflammation in patients with inflammatory bowel disease and in rodent colitis models. However, the mechanisms of probiosis are not clear. Our current studies suggest that supplementation with hdc(+) L. reuteri, which can convert l-histidine to histamine in the gut, resulted in suppression of colonic inflammation. These findings link luminal conversion of dietary components (amino acid metabolism) by gut microbes and probiotic-mediated suppression of colonic inflammation. The effective combination of diet, gut bacteria, and host receptor-mediated signaling may result in opportunities for therapeutic microbiology and provide clues for discovery and development of next-generation probiotics.
Chronic circadian dysfunction increases the risk of non-alcoholic fatty liver disease (NAFLD)-related hepatocellular carcinoma (HCC), but the underlying mechanisms and direct relevance to human HCC ...have not been established. In this study, we aimed to determine whether chronic circadian dysregulation can drive NAFLD-related carcinogenesis from human hepatocytes and human HCC progression.
Chronic jet lag of mice with humanized livers induces spontaneous NAFLD-related HCCs from human hepatocytes. The clinical relevance of this model was analysed by biomarker, pathological/histological, genetic, RNA sequencing, metabolomic, and integrated bioinformatic analyses.
Circadian dysfunction induces glucose intolerance, NAFLD-associated human HCCs, and human HCC metastasis independent of diet in a humanized mouse model. The deregulated transcriptomes in necrotic-inflammatory humanized livers and HCCs bear a striking resemblance to those of human non-alcoholic steatohepatitis (NASH), cirrhosis, and HCC. Stable circadian entrainment of hosts rhythmically paces NASH and HCC transcriptomes to decrease HCC incidence and prevent HCC metastasis. Circadian disruption directly reprogrammes NASH and HCC transcriptomes to drive a rapid progression from hepatocarcinogenesis to HCC metastasis. Human hepatocyte and tumour transcripts are clearly distinguishable from mouse transcripts in non-parenchymal cells and tumour stroma, and display dynamic changes in metabolism, inflammation, angiogenesis, and oncogenic signalling in NASH, progressing to hepatocyte malignant transformation and immunosuppressive tumour stroma in HCCs. Metabolomic analysis defines specific bile acids as prognostic biomarkers that change dynamically during hepatocarcinogenesis and in response to circadian disruption at all disease stages.
Chronic circadian dysfunction is independently carcinogenic to human hepatocytes. Mice with humanized livers provide a powerful preclinical model for studying the impact of the necrotic-inflammatory liver environment and neuroendocrine circadian dysfunction on hepatocarcinogenesis and anti-HCC therapy.
Human epidemiological studies have linked chronic circadian dysfunction to increased hepatocellular carcinoma (HCC) risk, but direct evidence that circadian dysfunction is a human carcinogen has not been established. Here we show that circadian dysfunction induces non-alcoholic steatohepatitis (NASH)-related carcinogenesis from human hepatocytes in a murine humanized liver model, following the same molecular and pathologic pathways observed in human patients. The gene expression signatures of humanized HCC transcriptomes from circadian-disrupted mice closely match those of human HCC with the poorest prognostic outcomes, while those from stably circadian entrained mice match those from human HCC with the best prognostic outcomes. Our studies establish a new model for defining the mechanism of NASH-related HCC and highlight the importance of circadian biology in HCC prevention and treatment.
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•Circadian dysfunction promotes NAFLD-induced carcinogenesis in human hepatocytes.•Chronic jet lag shifts NASH and HCC transcriptomes to activate hallmarks of cancer.•Circadian dysfunction reprograms transcriptomes in HCC to stimulate tumour metastasis.•Circadian disruption shifts prognostic biomarkers to interfere with HCC diagnosis.•Circadian homeostasis is strongly relevant to future precision oncology.
Programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) are immunomodulatory molecules overexpressed in lymphomas and are promising immunotherapy targets for hematologic ...malignancies. However, studies of PD-1/PD-L1 overexpression and their clinical significance in aggressive pediatric non-Hodgkin lymphomas (NHLs) are limited. We assessed PD-1/PD-L1 overexpression using immunohistochemistry in 68 aggressive pediatric NHLs: ALK-positive anaplastic large cell lymphoma (ALK+ ALCL, n=8), Burkitt lymphoma (BL, n=27), and large B-cell lymphoma (LBCL) de novo LBCL (n=22) and diffuse LBCL arising as monomorphic post-transplant lymphoproliferative disorder (PTLD-DLBCL, n=11). In LBCL, correlations between PD-L1 overexpression and Epstein-Barr virus (EBV) status, cell of origin, stage, nodal status, overall survival (OS), and event-free survival (EFS) were examined. The genetic mechanisms of PD-L1 overexpression were investigated using targeted nextgeneration sequencing (NGS) and cytogenetic data. All ALK+ ALCL samples, 50.0% of de novo LBCLs (11/22), 72.7% of PTLD-DLBCLs (8/11), and no BLs overexpressed PD-L1. Overexpressed PD-L1 correlated with EBV-positivity (p=0.033) in LBCLs and lower EFS in de novo LBCL (p=0.017). NGS of select LBCLs revealed distinct somatic mutations and an ultrahypermutated PTLD-DLBCL. Most cases with 9p24.1 copy gains overexpressed PD-L1 although some cases had no discernible genetic drivers of PD-L1 overexpression. Overexpressed PD-L1 is common in pediatric LBCL, associated with EBV-positivity and 9p24.1 gains, and may have prognostic significance in de novo LBCL. Furthermore, diverse molecular mechanisms for PD-L1 overexpression in aggressive pediatric NHL can occur. Thus, additional studies exploring the therapeutic and prognostic significance and molecular mechanisms of PD-L1 overexpression in aggressive pediatric NHLs are warranted.
Fanconi anemia (FA) is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Bone marrow transplantation is currently the only curative therapy for ...the hematopoietic complications of this disorder. However, long-term morbidity and mortality remain very high, and new therapeutics are badly needed. Here we show that the widely used diabetes drug metformin improves hematopoiesis and delays tumor formation in Fancd2−/− mice. Metformin is the first compound reported to improve both of these FA phenotypes. Importantly, the beneficial effects are specific to FA mice and are not seen in the wild-type controls. In this preclinical model of FA, metformin outperformed the current standard of care, oxymetholone, by improving peripheral blood counts in Fancd2−/− mice significantly faster. Metformin increased the size of the hematopoietic stem cell compartment and enhanced quiescence in hematopoietic stem and progenitor cells. In tumor-prone Fancd2−/−Trp53+/− mice, metformin delayed the onset of tumors and significantly extended the tumor-free survival time. In addition, we found that metformin and the structurally related compound aminoguanidine reduced DNA damage and ameliorated spontaneous chromosome breakage and radials in human FA patient–derived cells. Our results also indicate that aldehyde detoxification might be one of the mechanisms by which metformin reduces DNA damage in FA cells.
•The widely used diabetes drug metformin improves hematopoiesis and delays tumor formation in a preclinical murine model of FA.•Metformin reduces DNA damage in human FA patient–derived cells.
ABSTRACT
Although active duty women (ADW) represent over 17% of the total U.S. Armed Forces, there are few evidence-based guidelines to promote the health of women who serve in contemporary military ...roles. The existing body of evidence does not support guideline development because much of the evidence is not generalizable to ADW. The authors of this commentary recently conducted seven scoping reviews of the literature relevant to ADW’s health and healthcare. While completing the literature reviews, it was noted that a substantial proportion of military studies either do not include ADW as research participants or fail to examine findings according to sex and/or active duty status. The authors of this commentary outline a rigorous, step-by-step approach to research design in which ADW are accounted for at every stage of the process. Furthermore, this team of authors identifies opportunities for key stakeholders to provide oversight of the research process to ensure rigorous methodology that includes ADW. Implementing these strategies is critical to building the evidence on which to support the health and healthcare of ADW, who represent a substantial and growing component of the U.S. Military. Optimizing the health of this population is critical to maintaining the strength and readiness of our U.S. Armed Forces.
Inflammatory bowel disease (IBD) is a well-known risk factor for the development of colorectal cancer. Prior studies have demonstrated that microbial histamine can ameliorate intestinal inflammation ...in mice. We tested the hypothesis whether microbe-derived luminal histamine suppresses inflammation-associated colon cancer in Apc
mice. Mice were colonized with the human-derived Lactobacillus reuteri. Chronic inflammation was induced by repeated cycles of low-dose dextran sulfate sodium (DSS). Mice that were given histamine-producing L. reuteri via oral gavage developed fewer colonic tumors, despite the presence of a complex mouse gut microbiome. We further demonstrated that administration of a histamine H1-receptor (H1R) antagonist suppressed tumorigenesis, while administration of histamine H2-receptor (H2R) antagonist significantly increased both tumor number and size. The bimodal functions of histamine include protumorigenic effects through H1R and antitumorigenic effects via H2R, and these results were supported by gene expression profiling studies on tumor specimens of patients with colorectal cancer. Greater ratios of gene expression of H2R ( HRH2) vs. H1R ( HRH1) were correlated with improved overall survival outcomes in patients with colorectal cancer. Additionally, activation of H2R suppressed phosphorylation of mitogen-activated protein kinases (MAPKs) and inhibited chemokine gene expression induced by H1R activation in colorectal cancer cells. Moreover, the combination of a H1R antagonist and a H2R agonist yielded potent suppression of lipopolysaccharide-induced MAPK signaling in macrophages. Given the impact on intestinal epithelial and immune cells, simultaneous modulation of H1R and H2R signaling pathways may be a promising therapeutic target for the prevention and treatment of inflammation-associated colorectal cancer. NEW & NOTEWORTHY Histamine-producing Lactobacillus reuteri can suppress development of inflammation-associated colon cancer in an established mouse model. The net effects of histamine may depend on the relative activity of H1R and H2R signaling pathways in the intestinal mucosa. Our findings suggest that treatment with H1R or H2R antagonists could yield opposite effects. However, by harnessing the ability to block H1R signaling while stimulating H2R signaling, novel strategies for suppression of intestinal inflammation and colorectal neoplasia could be developed.
Risk of stroke-related morbidity and mortality increases significantly with age. Aging is associated with chronic, low-grade inflammation, which is thought to contribute to the poorer outcomes after ...stroke seen in the elderly. Histamine (HA) is a major molecular mediator of inflammation, and mast cells residing in the gut are a primary source of histamine.
Stroke was induced in male C57BL/6 J mice at 3 months (young) and 20 months (aged) of age. Role of histamine after stroke was examined using young (Yg) and aged (Ag) mice; mice underwent MCAO surgery and were euthanized at 6 h, 24 h, and 7 days post-ischemia; sham mice received the same surgery but no MCAO. In this work, we evaluated whether worsened outcomes after experimental stroke in aged mice were associated with age-related changes in mast cells, histamine levels, and histamine receptor expression in the gut, brain, and plasma.
We found increased numbers of mast cells in the gut and the brain with aging. Using the middle cerebral artery occlusion (MCAO) model of ischemic stroke, we demonstrate that stroke leads to increased numbers of gut mast cells and gut histamine receptor expression levels. These gut-centric changes are associated with elevated levels of HA and other pro-inflammatory cytokines including IL-6, G-CSF, TNF-α, and IFN-γ in the peripheral circulation. Our data also shows that post-stroke gut inflammation led to a significant reduction of mucin-producing goblet cells and a loss of gut barrier integrity. Lastly, gut inflammation after stroke is associated with changes in the composition of the gut microbiota as early as 24-h post-stroke.
An important theme emerging from our results is that acute inflammatory events following ischemic insults in the brain persist longer in the aged mice when compared to younger animals. Taken together, our findings implicate mast cell activation and histamine signaling as a part of peripheral inflammatory response after ischemic stroke, which are profound in aged animals. Interfering with histamine signaling orally might provide translational value to improve stroke outcome.
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