Phospholipase Cε insufficiency causes ascending aortic aneurysm and dissection.

Phospholipase Cε (PLCε) is a phospholipase C isoform with a wide range of physiological functions. It has been implicated in aortic valve disorders, but its role in frequently associated aortic disease remains unclear. To determine the role of PLCε in thoracic aortic aneurysm and dissection (TAAD) we used PLCε-deficient mice, which develop aortic valve insufficiency and exhibit aortic dilation of the ascending thoracic aorta and arch without histopathological evidence of injury. Fourteen days of infusion of Plce1+/+ and Plce1-/- mice with angiotensin II (ANG II), which induces aortic dilation and dissection, led to sudden death secondary to ascending aortic dissection in 43% of Plce1-/- versus 5% of Plce1+/+ mice (P < 0.05). Medial degeneration and TAAD were detected in 80% of Plce1-/- compared with 10% of Plce1+/+ mice (P < 0.05) after 4 days of ANG II. Treatment with ANG II markedly increased PLCε expression within the ascending aortic adventitia. Total RNA sequencing demonstrated marked upregulation of inflammatory and fibrotic pathways mediated by interleukin-1ß, interleukin-6, and tumor necrosis factor-α. In silico analysis of whole exome sequences of 258 patients with type A dissection identified 5 patients with nonsynonymous PLCE1 variants. Our data suggest that PLCε deficiency plays a role in the development of TAAD and aortic insufficiency.NEW & NOTEWORTHY We describe a novel phenotype by which PLCε deficiency predisposes to aortic valve insufficiency and ascending aortic aneurysm, dissection, and sudden death in the setting of ANG II-mediated hypertension. We demonstrate PLCE1 variants in patients with type A aortic dissection and aortic insufficiency, suggesting that PLCE1 may also play a role in human aortic disease. This finding is of very high significance because it has not been previously demonstrated that PLCε directly mediates aortic dissection.

Intestinal Inflammation Reversibly Alters the Microbiota to Drive Susceptibility to Clostridioides difficile Colonization in a Mouse Model of Colitis.

Susceptibility to Clostridioides difficile infection (CDI) typically follows the administration of antibiotics. Patients with inflammatory bowel disease (IBD) have increased incidence of CDI, even in the absence of antibiotic treatment. However, the mechanisms underlying this susceptibility are not well understood. To explore the intersection between CDI and IBD, we recently described a mouse model where colitis triggered by the murine gut bacterium, Helicobacter hepaticus, in IL-10-/- mice led to susceptibility to C. difficile colonization without antibiotic administration. The current work disentangles the relative contributions of inflammation and gut microbiota in colonization resistance to C. difficile in this model. We show that inflammation drives changes in microbiota composition, which leads to CDI susceptibility. Decreasing inflammation with an anti-p40 monoclonal antibody promotes a shift of the microbiota back toward a colonization-resistant state. Transferring microbiota from susceptible and resistant mice to germfree animals transfers the susceptibility phenotype, supporting the primacy of the microbiota in colonization resistance. These findings shine light on the complex interactions between the host, microbiota, and C. difficile in the context of intestinal inflammation, and may form a basis for the development of strategies to prevent or treat CDI in IBD patients. IMPORTANCE Patients with inflammatory bowel disease (IBD) have an increased risk of developing C. difficile infection (CDI), even in the absence of antibiotic treatment. Yet, mechanisms regulating C. difficile colonization in IBD patients remain unclear. Here, we use an antibiotic-independent mouse model to demonstrate that intestinal inflammation alters microbiota composition to permit C. difficile colonization in mice with colitis. Notably, treating inflammation with an anti-p40 monoclonal antibody, a clinically relevant IBD therapeutic, restores microbiota-mediated colonization resistance to the pathogen. Through microbiota transfer experiments in germfree mice, we confirm that the microbiota shaped in the setting of IBD is the primary driver of susceptibility to C. diffiicile colonization. Collectively, our findings provide insight into CDI pathogenesis in the context of intestinal inflammation, which may inform methods to manage infection in IBD patients. More broadly, this work advances our understanding of mechanisms by which the host-microbiota interface modulates colonization resistance to C. difficile.

The Gut Bacterial Community Potentiates Clostridioides difficile Infection Severity.

The severity of Clostridioides difficile infections (CDI) has increased over the last few decades. Patient age, white blood cell count, and creatinine levels as well as C. difficile ribotype and toxin genes have been associated with disease severity. However, it is unclear whether specific members of the gut microbiota are associated with variations in disease severity. The gut microbiota is known to interact with C. difficile during infection. Perturbations to the gut microbiota are necessary for C. difficile to colonize the gut. The gut microbiota can inhibit C. difficile colonization through bile acid metabolism, nutrient consumption, and bacteriocin production. Here, we sought to demonstrate that members of the gut bacterial communities can also contribute to disease severity. We derived diverse gut communities by colonizing germfree mice with different human fecal communities. The mice were then infected with a single C. difficile ribotype 027 clinical isolate, which resulted in moribundity and histopathologic differences. The variation in severity was associated with the human fecal community that the mice received. Generally, bacterial populations with pathogenic potential, such as Enterococcus, Helicobacter, and Klebsiella, were associated with more-severe outcomes. Bacterial groups associated with fiber degradation and bile acid metabolism, such as Anaerotignum, Blautia, Lactonifactor, and Monoglobus, were associated with less-severe outcomes. These data indicate that, in addition to the host and C. difficile subtype, populations of gut bacteria can influence CDI disease severity. IMPORTANCE Clostridioides difficile colonization can be asymptomatic or develop into an infection ranging in severity from mild diarrhea to toxic megacolon, sepsis, and death. Models that predict severity and guide treatment decisions are based on clinical factors and C. difficile characteristics. Although the gut microbiome plays a role in protecting against CDI, its effect on CDI disease severity is unclear and has not been incorporated into disease severity models. We demonstrated that variation in the microbiome of mice colonized with human feces yielded a range of disease outcomes. These results revealed groups of bacteria associated with both severe and mild C. difficile infection outcomes. Gut bacterial community data from patients with CDI could improve our ability to identify patients at risk of developing more severe disease and improve interventions that target C. difficile and the gut bacteria to reduce host damage.

Molecular genetic evaluation of pediatric renovascular hypertension due to renal artery stenosis and abdominal aortic coarctation in neurofibromatosis type 1.

The etiology of renal artery stenosis (RAS) and abdominal aortic coarctation (AAC) causing the midaortic syndrome (MAS), often resulting in renovascular hypertension (RVH), remains ill-defined. Neurofibromatosis type 1 (NF-1) is frequently observed in children with RVH. Consecutive pediatric patients (N = 102) presenting with RVH secondary to RAS with and without concurrent AAC were prospectively enrolled in a clinical data base, and blood, saliva and operative tissue, when available, were collected. Among the 102 children, 13 were having a concurrent clinical diagnosis of NF-1 (12.5%). Whole exome sequencing was performed for germline variant detection, and RNA-Seq analysis of NF1, MAPK pathway genes and MCP1 levels were undertaken in five NF-1 stenotic renal arteries, as well as control renal and mesenteric arteries from children with no known vasculopathy or NF-1. In 11 unrelated children with sequencing data, 11 NF1 genetic variants were identified, of which 10 had not been reported in gnomAD. Histologic analysis of NF-1 RAS specimens consistently revealed intimal thickening, disruption of the internal elastic lamina and medial thinning. Analysis of transcript expression in arterial lesions documented an approximately 5-fold reduction in NF1 expression, confirming heterozygosity, MAPK pathway activation and increased MCP1 expression. In summary, NF-1-related RVH in children is rare but often severe and progressive and, as such, important to recognize. It is associated with histologic and molecular features consistent with an aggressive adverse vascular remodeling process. Further research is necessary to define the mechanisms underlying these findings.

An Osmotic Laxative Renders Mice Susceptible to Prolonged Clostridioides difficile Colonization and Hinders Clearance.

Antibiotics are a major risk factor for Clostridioides difficile infections (CDIs) because of their impact on the microbiota. However, nonantibiotic medications such as the ubiquitous osmotic laxative polyethylene glycol 3350 (PEG 3350) also alter the microbiota. Clinicians also hypothesize that PEG helps clear C. difficile. But whether PEG impacts CDI susceptibility and clearance is unclear. To examine how PEG impacts susceptibility, we treated C57BL/6 mice with 5-day and 1-day doses of 15% PEG in the drinking water and then challenged the mice with C. difficile 630. We used clindamycin-treated mice as a control because they consistently clear C. difficile within 10 days postchallenge. PEG treatment alone was sufficient to render mice susceptible, and 5-day PEG-treated mice remained colonized for up to 30 days postchallenge. In contrast, 1-day PEG-treated mice were transiently colonized, clearing C. difficile within 7 days postchallenge. To examine how PEG treatment impacts clearance, we administered a 1-day PEG treatment to clindamycin-treated, C. difficile-challenged mice. Administering PEG to mice after C. difficile challenge prolonged colonization up to 30 days postchallenge. When we trained a random forest model with community data from 5 days postchallenge, we were able to predict which mice would exhibit prolonged colonization (area under the receiver operating characteristic curve [AUROC] = 0.90). Examining the dynamics of these bacterial populations during the postchallenge period revealed patterns in the relative abundances of Bacteroides, Enterobacteriaceae, Porphyromonadaceae, Lachnospiraceae, and Akkermansia that were associated with prolonged C. difficile colonization in PEG-treated mice. Thus, the osmotic laxative PEG rendered mice susceptible to C. difficile colonization and hindered clearance. IMPORTANCE Diarrheal samples from patients taking laxatives are typically rejected for Clostridioides difficile testing. However, there are similarities between the bacterial communities from people with diarrhea and those with C. difficile infections (CDIs), including lower diversity than the communities from healthy patients. This observation led us to hypothesize that diarrhea may be an indicator of C. difficile susceptibility. We explored how osmotic laxatives disrupt the microbiota's colonization resistance to C. difficile by administering a laxative to mice either before or after C. difficile challenge. Our findings suggest that osmotic laxatives disrupt colonization resistance to C. difficile and prevent clearance among mice already colonized with C. difficile. Considering that most hospitals recommend not performing C. difficile testing on patients taking laxatives, and laxatives are prescribed prior to administering fecal microbiota transplants via colonoscopy to patients with recurrent CDIs, further studies are needed to evaluate if laxatives impact microbiota colonization resistance in humans.

Intestinal Inflammation and Altered Gut Microbiota Associated with Inflammatory Bowel Disease Render Mice Susceptible to Clostridioides difficile Colonization and Infection.

Clostridioides difficile is a noteworthy pathogen in patients with inflammatory bowel disease (IBD). Patients with IBD who develop concurrent C. difficile infection (CDI) experience increased morbidity and mortality. IBD is associated with intestinal inflammation and alterations of the gut microbiota, both of which can diminish colonization resistance to C. difficile. Here, we describe the development of a mouse model to explore the role that IBD-induced changes of the gut microbiome play in susceptibility to C. difficile. Helicobacter hepaticus, a normal member of the mouse gut microbiota, triggers pathological inflammation in the distal intestine akin to human IBD in mice that lack intact interleukin 10 (IL-10) signaling. We demonstrate that mice with H. hepaticus-induced IBD were susceptible to C. difficile colonization in the absence of other perturbations, such as antibiotic treatment. Concomitant IBD and CDI were associated with significantly worse disease than observed in animals with colitis alone. Development of IBD resulted in a distinct intestinal microbiota community compared to that of non-IBD controls. Inflammation played a critical role in the susceptibility of animals with IBD to C. difficile colonization, as mice colonized with an isogenic mutant of H. hepaticus that triggers an attenuated intestinal inflammation maintained full colonization resistance. These studies with a novel mouse model of IBD and CDI emphasize the importance of host responses and alterations of the gut microbiota in susceptibility to C. difficile colonization and infection in the setting of IBD. IMPORTANCE The incidence of C. difficile infection (CDI) has increased significantly among patients with IBD, independently of antibiotic use, yet the relationship between IBD and increased risk for CDI remains to be understood. Our study sought to describe and utilize an antibiotic-independent mouse model to specifically explore the relationship between the IBD-associated gut and susceptibility to C. difficile colonization and CDI development. We demonstrate that the development of IBD is sufficient to render mice susceptible to C. difficile colonization and results in significantly worse disease than IBD alone. Furthermore, this model requires IBD-induced inflammation to overcome colonization resistance to C. difficile. This model recapitulates human IBD and CDI comorbidity and will aid in developing new clinical approaches to predict, diagnose, and treat C. difficile infection in the IBD population.

A Multi-Mineral Intervention to Modulate Colonic Mucosal Protein Profile: Results from a 90-Day Trial in Human Subjects.

The overall goal of this study was to determine whether Aquamin®, a calcium-, magnesium-, trace element-rich, red algae-derived natural product, would alter the expression of proteins involved in growth-regulation and differentiation in colon. Thirty healthy human subjects (at risk for colorectal cancer) were enrolled in a three-arm, 90-day interventional trial. Aquamin® was compared to calcium alone and placebo. Before and after the interventional period, colonic biopsies were obtained. Biopsies were evaluated by immunohistology for expression of Ki67 (proliferation marker) and for CK20 and p21 (differentiation markers). Tandem mass tag-mass spectrometry-based detection was used to assess levels of multiple proteins. As compared to placebo or calcium, Aquamin® reduced the level of Ki67 expression and slightly increased CK20 expression. Increased p21 expression was observed with both calcium and Aquamin®. In proteomic screen, Aquamin® treatment resulted in many more proteins being upregulated (including pro-apoptotic, cytokeratins, cell-cell adhesion molecules, and components of the basement membrane) or downregulated (proliferation and nucleic acid metabolism) than placebo. Calcium alone also altered the expression of many of the same proteins but not to the same extent as Aquamin®. We conclude that daily Aquamin® ingestion alters protein expression profile in the colon that could be beneficial to colonic health.

Protection from Lethal Clostridioides difficile Infection via Intraspecies Competition for Cogerminant.

Clostridioides difficile, a Gram-positive, spore-forming bacterium, is the primary cause of infectious nosocomial diarrhea. Antibiotics are a major risk factor for C. difficile infection (CDI), as they disrupt the gut microbial community, enabling increased germination of spores and growth of vegetative C. difficile To date, the only single-species bacterial preparation that has demonstrated efficacy in reducing recurrent CDI in humans is nontoxigenic C. difficile Using multiple infection models, we determined that precolonization with a less virulent strain is sufficient to protect from challenge with a lethal strain of C. difficile, surprisingly even in the absence of adaptive immunity. Additionally, we showed that protection is dependent on high levels of colonization by the less virulent strain and that it is mediated by exclusion of the invading strain. Our results suggest that reduction of amino acids, specifically glycine following colonization by the first strain of C. difficile, is sufficient to decrease germination of the second strain, thereby limiting colonization by the lethal strain.IMPORTANCE Antibiotic-associated colitis is often caused by infection with the bacterium Clostridioides difficile In this study, we found that reduction of the amino acid glycine by precolonization with a less virulent strain of C. difficile is sufficient to decrease germination of a second strain. This finding demonstrates that the axis of competition for nutrients can include multiple life stages. This work is important, as it is the first to identify a possible mechanism through which precolonization with C. difficile, a current clinical therapy, provides protection from reinfection. Furthermore, our work suggests that targeting nutrients utilized by all life stages could be an improved strategy for bacterial therapeutics that aim to restore colonization resistance in the gut.

N-Acetyl-L-cysteine and aminooxyacetic acid differentially modulate trichloroethylene reproductive toxicity via metabolism in Wistar rats.

Exposure to the industrial solvent trichloroethylene (TCE) has been associated with adverse pregnancy outcomes in humans and decreased fetal weight in rats. TCE kidney toxicity can occur through formation of reactive metabolites via its glutathione (GSH) conjugation metabolic pathway, largely unstudied in the context of pregnancy. To investigate the contribution of the GSH conjugation pathway and oxidative stress to TCE toxicity during pregnancy, we exposed rats orally to 480 mg TCE/kg/day from gestational day (GD) 6 to GD 16 with and without N-acetyl-L-cysteine (NAC) at 200 mg/kg/day or aminooxyacetic acid (AOAA) at 20 mg/kg/day as pre/co-treatments from GD 5-16. NAC is a reactive oxygen species scavenger that modifies the GSH conjugation pathway, and AOAA is an inhibitor of cysteine conjugate ß-lyase (CCBL) in the GSH conjugation pathway. TCE decreased fetal weight, and this was prevented by AOAA but not NAC pre/co-treatment to TCE. Although AOAA inhibited CCBL activity in maternal kidney, it did not inhibit CCBL activity in maternal liver and placenta, suggesting that AOAA prevention of TCE-induced decreased fetal weight was due to CCBL activity inhibition in the kidneys but not liver or placenta. Unexpectedly, NAC pre/co-treatment with TCE, relative to TCE treatment alone, altered placental morphology consistent with delayed developmental phenotype. Immunohistochemical staining revealed that the decidua basale, relative to basal and labyrinth zones, expressed the highest abundance of CCBL1, flavin-containing monooxygenase 3, and cleaved caspase-3. Together, the findings show the differential effects of NAC and AOAA on TCE-induced pregnancy outcomes are likely attributable to TCE metabolism modulation.

Magnetic resonance imaging of human neural stem cells in rodent and primate brain.

Stem cell transplantation therapies are currently under investigation for central nervous system disorders. Although preclinical models show benefit, clinical translation is somewhat limited by the absence of reliable noninvasive methods to confirm targeting and monitor transplanted cells in vivo. Here, we assess a novel magnetic resonance imaging (MRI) contrast agent derived from magnetotactic bacteria, magneto-endosymbionts (MEs), as a translatable methodology for in vivo tracking of stem cells after intracranial transplantation. We show that ME labeling provides robust MRI contrast without impairment of cell viability or other important therapeutic features. Labeled cells were visualized immediately post-transplantation and over time by serial MRI in nonhuman primate and mouse brain. Postmortem tissue analysis confirmed on-target grft location, and linear correlations were observed between MRI signal, cell engraftment, and tissue ME levels, suggesting that MEs may be useful for determining graft survival or rejection. Overall, these findings indicate that MEs are an effective tool for in vivo tracking and monitoring of cell transplantation therapies with potential relevance to many cellular therapy applications.

A Novel Recurrent COL5A1 Genetic Variant Is Associated With a Dysplasia-Associated Arterial Disease Exhibiting Dissections and Fibromuscular Dysplasia.

OBJECTIVE: While rare variants in the COL5A1 gene have been associated with classical Ehlers-Danlos syndrome and rarely with arterial dissections, recurrent variants in COL5A1 underlying a systemic arteriopathy have not been described. Monogenic forms of multifocal fibromuscular dysplasia (mFMD) have not been previously defined. Approach and Results: We studied 4 independent probands with the COL5A1 pathogenic variant c.1540G>A, p.(Gly514Ser) who presented with arterial aneurysms, dissections, tortuosity, and mFMD affecting multiple arteries. Arterial medial fibroplasia and smooth muscle cell disorganization were confirmed histologically. The COL5A1 c.1540G>A variant is predicted to be pathogenic in silico and absent in gnomAD. The c.1540G>A variant is on a shared 160.1 kb haplotype with 0.4% frequency in Europeans. Furthermore, exome sequencing data from a cohort of 264 individuals with mFMD were examined for COL5A1 variants. In this mFMD cohort, COL5A1 c.1540G>A and 6 additional relatively rare COL5A1 variants predicted to be deleterious in silico were identified and were associated with arterial dissections (P=0.005). CONCLUSIONS: COL5A1 c.1540G>A is the first recurring variant recognized to be associated with arterial dissections and mFMD. This variant presents with a phenotype reminiscent of vascular Ehlers-Danlos syndrome. A shared haplotype among probands supports the existence of a common founder. Relatively rare COL5A1 genetic variants predicted to be deleterious by in silico analysis were identified in ≈2.7% of mFMD cases, and as they were enriched in patients with arterial dissections, may act as disease modifiers. Molecular testing for COL5A1 should be considered in patients with a phenotype overlapping with vascular Ehlers-Danlos syndrome and mFMD.

Nanoparticle Digestion Simulator Reveals pH-Dependent Aggregation in the Gastrointestinal Tract.

Determining the physicochemical properties of ingested nanoparticles within the gastrointestinal tract (GIT) is critical for evaluating the impact of environmental exposure and potential for nanoparticle drug delivery. However, it is challenging to predict nanoparticle physicochemical properties at the point of intestinal absorption due to the changing chemical environments within the GIT. Herein, a dynamic nanoparticle digestion simulator (NDS) was constructed to examine nanoparticle evolution due to changing pH and salt concentrations in the stomach and upper intestine. This multicompartment, flow-through system simulates digestion by transferring gastrointestinal fluids and digestive secretions at physiologically relevant time scales and flow rates. Pronounced differences in aggregation and aggregate stability were observed with silver nanoparticles (citrate-coated) with an initial hydrodynamic diameter (Dh) of 24.6 ± 0.4 nm examined under fasted (pH 2) and fed (pH 5) gastric conditions using nanoparticle tracking analysis (NTA) for size distributions and transmission electron microscopy with energy dispersive X-ray spectroscopy (TEM-EDX) for morphology and elemental composition. Under fasted stomach conditions, particles aggregated to Dh = 130 ± 10 nm and remained as large aggregates in the upper intestinal compartments (duodenum and jejunum) ending with Dh = 110 ± 20 nm and a smaller mode at 59 ± 8 nm. In contrast, under fed conditions, nanoparticles aggregated to 60 ± 10 nm in the stomach, then disaggregated to individual nanoparticles (26 ± 2 nm) in the intestinal compartments. The NDS provides an analytical approach for studying nanoparticle physicochemical modifications within the GIT and the impacts of intentionally and unintentionally ingested nanoparticles.

Systemic Inflammatory Mediators Are Effective Biomarkers for Predicting Adverse Outcomes in Clostridioides difficile Infection.

Clostridioides difficile infection (CDI) can result in severe disease and death, with no accurate models that allow for early prediction of adverse outcomes. To address this need, we sought to develop serum-based biomarker models to predict CDI outcomes. We prospectively collected sera ≤48 h after diagnosis of CDI in two cohorts. Biomarkers were measured with a custom multiplex bead array assay. Patients were classified using IDSA severity criteria and the development of disease-related complications (DRCs), which were defined as ICU admission, colectomy, and/or death attributed to CDI. Unadjusted and adjusted models were built using logistic and elastic net modeling. The best model for severity included procalcitonin (PCT) and hepatocyte growth factor (HGF) with an area (AUC) under the receiver operating characteristic (ROC) curve of 0.74 (95% confidence interval, 0.67 to 0.81). The best model for 30-day mortality included interleukin-8 (IL-8), PCT, CXCL-5, IP-10, and IL-2Rα with an AUC of 0.89 (0.84 to 0.95). The best model for DRCs included IL-8, procalcitonin, HGF, and IL-2Rα with an AUC of 0.84 (0.73 to 0.94). To validate our models, we employed experimental infection of mice with C. difficile Antibiotic-treated mice were challenged with C. difficile and a similar panel of serum biomarkers was measured. Applying each model to the mouse cohort of severe and nonsevere CDI revealed AUCs of 0.59 (0.44 to 0.74), 0.96 (0.90 to 1.0), and 0.89 (0.81 to 0.97). In both human and murine CDI, models based on serum biomarkers predicted adverse CDI outcomes. Our results support the use of serum-based biomarker panels to inform Clostridioides difficile infection treatment.IMPORTANCE Each year in the United States, Clostridioides difficile causes nearly 500,000 gastrointestinal infections that range from mild diarrhea to severe colitis and death. The ability to identify patients at increased risk for severe disease or mortality at the time of diagnosis of C. difficile infection (CDI) would allow clinicians to effectively allocate disease modifying therapies. In this study, we developed models consisting of only a small number of serum biomarkers that are capable of predicting both 30-day all-cause mortality and adverse outcomes of patients at time of CDI diagnosis. We were able to validate these models through experimental mouse infection. This provides evidence that the biomarkers reflect the underlying pathophysiology and that our mouse model of CDI reflects the pathogenesis of human infection. Predictive models can not only assist clinicians in identifying patients at risk for severe CDI but also be utilized for targeted enrollment in clinical trials aimed at reduction of adverse outcomes from severe CDI.

Aging Dampens the Intestinal Innate Immune Response during Severe Clostridioides difficile Infection and Is Associated with Altered Cytokine Levels and Granulocyte Mobilization.

Clostridioides (formerly Clostridium) difficile is the most common cause of hospital-acquired infection, and advanced age is a risk factor for C. difficile infection. Disruption of the intestinal microbiota and immune responses contribute to host susceptibility and severity of C. difficile infection. However, the specific impact of aging on immune responses during C. difficile infection remains to be well described. This study explores the effect of age on cellular and cytokine immune responses during C. difficile infection. Young mice (2 to 3 months old) and aged mice (22 to 28 months old) were rendered susceptible to C. difficile infection with the antibiotic cefoperazone and then infected with C. difficile strains with varied disease-causing potentials. We observe that the host age and the infecting C. difficile strain influenced the severity of disease associated with infection. Tissue-specific CD45+ immune cell responses occurred at the time of peak disease severity in the ceca and colons of all mice infected with a high-virulence strain of C. difficile; however, significant deficits in intestinal neutrophils and eosinophils were detected in aged mice, with a corresponding decrease in circulating CXCL1, an important neutrophil recruiter and activator. Interestingly, this lack of intestinal granulocyte response in aged mice during severe C. difficile infection was accompanied by a simultaneous increase in circulating white blood cells, granulocytes, and interleukin 17A (IL-17A). These findings demonstrate that age-related alterations in neutrophils and eosinophils and systemic cytokine and chemokine responses are associated with severe C. difficile infection and support a key role for intestinal eosinophils in mitigating C. difficile-mediated disease severity.

A Calcium-Rich Multimineral Intervention to Modulate Colonic Microbial Communities and Metabolomic Profiles in Humans: Results from a 90-Day Trial.

Aquamin is a calcium-, magnesium-, and multiple trace element-rich natural product with colon polyp prevention efficacy based on preclinical studies. The goal of this study was to determine the effects of Aquamin on colonic microbial community and attendant metabolomic profile. Thirty healthy human participants were enrolled in a 90-day trial in which Aquamin (delivering 800 mg of calcium per day) was compared with calcium alone or placebo. Before and after the intervention, colonic biopsies and stool specimens were obtained. All 30 participants completed the study without serious adverse event or change in liver and renal function markers. Compared with pretreatment values, intervention with Aquamin led to a reduction in total bacterial DNA (P = 0.0001) and a shift in the microbial community measured by thetaYC (θYC; P = 0.0087). Treatment with calcium also produced a decline in total bacteria, but smaller than seen with Aquamin, whereas no reduction was observed with placebo in the colon. In parallel with microbial changes, a reduction in total bile acid levels (P = 0.0375) and a slight increase in the level of the short-chain fatty acid (SCFA) acetate in stool specimens (P < 0.0001) from Aquamin-treated participants were noted. No change in bile acids or SCFAs was observed with calcium or placebo. We conclude that Aquamin is safe and tolerable in healthy human participants and may produce beneficial alterations in the colonic microbial community and the attendant metabolomic profile. Because the number of participants was small, the findings should be considered preliminary.

Outbreak of Murine Infection with Clostridium difficile Associated with the Administration of a Pre- and Perinatal Methyl Donor Diet.

Between October 2016 and June 2017, a C57BL/6J mouse colony that was undergoing a pre- and perinatal methyl donor supplementation diet intervention to study the impact of parental nutrition on offspring susceptibility to disease was found to suffer from an epizootic of unexpected deaths. Necropsy revealed the presence of severe colitis, and further investigation linked these outbreak deaths to a Clostridium difficile strain of ribotype 027 that we term 16N203. C. difficile infection (CDI) is associated with antibiotic use in humans. Current murine models of CDI rely on antibiotic pretreatment to establish clinical phenotypes. In this report, the C. difficile outbreak occurs in F1 mice linked to alterations in the parental diet. The diagnosis of CDI in the affected mice was confirmed by cecal/colonic histopathology, the presence of C. difficile bacteria in fecal/colonic culture, and detection of C. difficile toxins. F1 mice from parents fed the methyl supplementation diet also had significantly reduced survival (P < 0.0001) compared with F1 mice from parents fed the control diet. When we tested the 16N203 outbreak strain in an established mouse model of antibiotic-induced CDI, we confirmed that this strain is pathogenic. Our serendipitous observations from this spontaneous outbreak of C. difficile in association with a pre- and perinatal methyl donor diet suggest the important role that diet may play in host defense and CDI risk factors.IMPORTANCEClostridium difficile infection (CDI) has become the leading cause of infectious diarrhea in hospitals worldwide, owing its preeminence to the emergence of hyperendemic strains, such as ribotype 027 (RT027). A major CDI risk factor is antibiotic exposure, which alters gut microbiota, resulting in the loss of colonization resistance. Current murine models of CDI also depend on pretreatment of animals with antibiotics to establish disease. The outbreak that we report here is unique in that the CDI occurred in mice with no antibiotic exposure and is associated with a pre- and perinatal methyl supplementation donor diet intervention study. Our investigation subsequently reveals that the outbreak strain that we term 16N203 is an RT027 strain, and this isolated strain is also pathogenic in an established murine model of CDI (with antibiotics). Our report of this spontaneous outbreak offers additional insight into the importance of environmental factors, such as diet, and CDI susceptibility.

An Early Investigation into Possible Alternatives to Stapled Hysterotomy in Open Fetal Surgery.

PURPOSE: We hypothesized that surgical energy could be used to create hysterotomies in open fetal surgery. STUDY DESIGN: Initial studies compared the LigaSure Impact and Harmonic ACE + 7 Shears in the efficiency of hysterotomy and thermal damage. Pregnant ewes at an estimated gestational age (EGA) of 116 to 120 days (term = 145; n = 7) underwent hysterotomy using either device. Hysterotomy edges were resected, and thermal injury extent was determined by histopathological assessment. Upon determining a superior device, subsequent studies compared this to the AutoSuture Premium Poly CS*-57 Stapler in uterine healing. Pregnant ewes (n = 6) at an EGA of 87 to 93 days underwent 6-cm hysterotomy in each gravid horn with either the stapler (n = 5) or Harmonic (n = 5) followed by closure and animal recovery. After 37 to 42 days, uterine healing was assessed by evaluating tensile strength and histopathology. RESULTS: Thermal damage was more extensive with the LigaSure (n = 11 hysterotomies) than with the Harmonic (n = 11; 5.6 ± 1 vs. 3.1 ± 0.6 mm; p < 0.0001);therefore, the Harmonic was selected for healing studies. Gross scar appearance and tensile strength were the same between the Harmonic and stapler. The stapler caused more fibrosis (4/7 samples with "moderate" fibrosis vs. 0/8 with the Harmonic; p = 0.02). CONCLUSION: The Harmonic ACE + 7 caused less thermal injury than the LigaSure Impact and performed similar to the CS*-57 Stapler in uterine healing with continued gestation.

An Expandable Mechanopharmaceutical Device (2): Drug Induced Granulomas Maximize the Cargo Sequestering Capacity of Macrophages in the Liver.

PURPOSE: Drug-induced liver injuries (DILI) comprise a significant proportion of adverse drug reactions leading to hospitalizations and death. One frequent DILI is granulomatous inflammation from exposure to harmful metabolites that activate inflammatory pathways of immune cells of the liver, which may act as a barrier to isolate the irritating stimulus and limit tissue damage. METHODS: Paralleling the accumulation of CFZ precipitates in the liver, granulomatous inflammation was studied to gain insight into its effect on liver structure and function. A structural analog that does not precipitate within macrophages was also studied using micro-analytical approaches. Depleting macrophages was used to inhibit granuloma formation and assess its effect on drug bioaccumulation and toxicity. RESULTS: Granuloma-associated macrophages showed a distinct phenotype, differentiating them from non-granuloma macrophages. Granulomas were induced by insoluble CFZ cargo, but not by the more soluble analog, pointing to precipitation being a factor driving granulomatous inflammation. Granuloma-associated macrophages showed increased activation of lysosomal master-regulator transcription factor EB (TFEB). Inhibiting granuloma formation increased hepatic necrosis and systemic toxicity in CFZ-treated animals. CONCLUSIONS: Granuloma-associated macrophages are a specialized cell population equipped to actively sequester and stabilize cytotoxic chemotherapeutic agents. Thus, drug-induced granulomas may function as drug sequestering "organoids" -an induced, specialized sub-compartment- to limit tissue damage.

Indomethacin increases severity of Clostridium difficile infection in mouse model.

AIM: To evaluate the effect on the nonsteroidal anti-inflammatory drug indomethacin on Clostridium difficile infection (CDI) severity. MATERIALS & METHODS: Indomethacin was administered in two different mouse models of antibiotic-associated CDI in two different facilities, using a low and high dose of indomethacin. RESULTS: Indomethacin administration caused weight loss, increased the signs of severe infection and worsened histopathological damage, leading to 100% mortality during CDI. Indomethacin-treated, antibiotic-exposed mice infected with C. difficile had enhanced intestinal inflammation with increased expression of KC, IL-1ß and IL-22 compared with infected mice unexposed to indomethacin. CONCLUSION: These results demonstrate a negative impact of nonsteroidal anti-inflammatory drugs on antibiotic-associated CDI in mice and suggest that targeting the synthesis or signaling of prostaglandins might be an approach to ameliorating the severity of CDI.

Effects of hydroxyurea on CNV induction in the mouse germline.

Copy number variants (CNVs) are important in genome variation and genetic disease, with new mutations arising frequently in the germline and somatic cells. Replication stress caused by aphidicolin and hydroxyurea (HU) is a potent inducer of de novo CNVs in cultured mammalian cells. HU is used extensively for long-term management of sickle cell disease. Here, we examined the effects of HU treatment on germline CNVs in vivo in male mice to explore whether replication stress can act as a CNV mutagen in germline mitotic divisions as in cultured cells and whether this would support a concern for increased CNV mutations in offspring of men treated with HU. Several trials of HU administration were performed by oral gavage and subcutaneous pump, with CNVs characterized in C57BL/6 x C3H/HeJ hybrid mouse offspring by microarray and mate-pair sequencing. HU had a short half-life of ~14 min and a narrow dose window over which studies could be performed while maintaining fertility. Tissue histopathology and reticulocyte micronucleus assays verified that doses had a substantial tissue and genetic toxicity. CNVs were readily detected in offspring that originated in both paternal and maternal mouse strains, as de novo and inherited events. However, HU did not increase CNV formation above baseline levels. These results reveal a high rate of CNV mutagenesis in the mouse germline but do not support the hypothesis that HU would increase CNV formation during mammalian spermatogenesis, perhaps due to highly toxic effects on sperm development or experimental variables related to HU pharmacology in mice. Environ. Mol. Mutagen. 59:698-714, 2018. © 2018 Wiley Periodicals, Inc.