Impact of Ivermectin on the Gut Microbial Ecosystem.

Ivermectin is a an anti-helminthic that is critical globally for both human and veterinary care. To the best of our knowledge, information available regarding the influence of ivermectin (IVM) on the gut microbiota has only been collected from diseased donors, who were treated with IVM alone or in combination with other medicines. Results thus obtained were influenced by multiple elements beyond IVM, such as disease, and other medical treatments. The research presented here investigated the impact of IVM on the gut microbial structure established in a Triple-SHIME® (simulator of the human intestinal microbial ecosystem), using fecal material from three healthy adults. The microbial communities were grown using three different culture media: standard SHIME media and SHIME media with either soluble or insoluble fiber added (control, SF, ISF). IVM introduced minor and temporary changes to the gut microbial community in terms of composition and metabolite production, as revealed by 16S rRNA amplicon sequencing analysis, flow cytometry, and GC-MS. Thus, it was concluded that IVM is not expected to induce dysbiosis or yield adverse effects if administered to healthy adults. In addition, the donor's starting community influences the relationship between IVM and the gut microbiome, and the soluble fiber component in feed could protect the gut microbiota from IVM; an increase in short-chain fatty acid production was predicted by PICRUSt2 and detected with IVM treatment.

2-N-Arylthiazole inhibitors of Mycobacterium tuberculosis.

To develop agents for the treatment of infections caused by Mycobacterium tuberculosis, a novel phenotypic screen was undertaken that identified a series of 2-N-aryl thiazole-based inhibitors of intracellular Mycobacterium tuberculosis. Analogs were optimized to improve potency against an attenuated BSL2 H37Ra laboratory strain cultivated in human macrophage cells in vitro. The insertion of a carboxylic acid functionality resulted in compounds that retained potency and greatly improved microsomal stability. However, the strong potency trends we observed in the attenuated H37Ra strain were inconsistent with the potency observed for virulent strains in vitro and in vivo.

A Nanofiltration Decision Tool for Potable Reuse: A New Rejection Model for Recalcitrant CECs.

Effluent samples from three water resource recovery facilities (WRRFs) were individually characterized for presence and concentration of 94 different chemicals of emerging concern (CECs), using analytical methods with reporting limits in the low-parts-per-trillion. Following CEC analysis, each sample was subjected to dead-end, pressurized membrane filtration with either a nanofiltration (NF) or reverse osmosis (RO) membrane. The majority of the measured CECs were rejected by both membranes by 1-log removal (90%) or greater. However, nine of the 94 CECs had average rejection rates by the NF membrane less than 90%. A multilevel, multivariable model was developed to predict the probable rejection coefficients of CECs with the studied NF membrane. The resulting Quantitative Molecular Properties Model (QMPM) predicted the NF rejection of CECs based on size, ionic charge, and hydrophobicity. The model parameters that successfully predicted NF rejection in bench testing were log (Kow/Kaw) and the polar surface area of the CEC molecule.

Avulsion of the Distal Sealing Stent of a Zenith Aortouni-iliac Stent-Graft.

PURPOSE: To describe a life-threatening complication during endovascular aneurysm repair that was caused by a series of errors. CASE REPORT: A 64-year-old man received a Zenith aortouni-iliac stent-graft for a 61-mm saccular aortic aneurysm. During retrieval of the delivery system, the sheath became dislodged from the common femoral artery. Reintroduction caused the top cap to disengage from the pusher rod because the pin vice had not been tightened as per the instructions for use. Subsequent traction was applied but the delivery system could not be withdrawn from the sheath, thus it was decided to remove the delivery system and sheath en masse. On removal, the distal sealing stent was found deformed and wedged between the top cap and the sheath. Perforation of the common iliac artery by the avulsed stent was treated with iliac limb extension before contralateral iliac plug and femorofemoral bypass graft were performed as planned. The patient made an uneventful recovery. CONCLUSION: Trapping of the distal sealing stent between the top cap and the pusher rod of the Zenith graft is possible and can be prevented by securing the pin vice prior to retrieval. Description of this complication may prevent similar occurrences in future.

Immune Response to Marburg Virus Angola Infection in Nonhuman Primates.

BACKGROUND: The 2005 outbreak of Marburg virus (MARV) infection in Angola was the most lethal MARV infection outbreak in history, with a case-fatality rate (90%) similar to that for Zaire ebolavirus (EBOV) infection. However, very little is known about the pathogenicity of MARV Angola, as few studies have been conducted to date. Therefore, the immune response was examined in MARV Angola-infected nonhuman primates. METHODS: Cynomolgus macaques were infected with MARV Angola and monitored for survival. The effect of MARV Angola on the immune system was examined by immunophenotyping whole-blood and by analyzing cytokine and chemokine levels in plasma and spleen specimens, using flow cytometry. RESULTS: The prominent clinical findings were rapid onset of disease and death (mean time after infection, 6.7 days), fever, depression, anorexia, petechial rash, and lymphopenia. Specifically, T, B, and natural killer cells were severely depleted in the blood by day 6. The typical cytokine storm was present, with levels of interferon γ, tumor necrosis factor, interleukin 6, and CCL2 rising in the blood early during infection. CONCLUSIONS: MARV Angola displayed the same virulence and disease pathology as EBOV. MARV Angola appears to cause a more rapid onset and severe outcome of infection than other MARV strains.

The efflux pump inhibitor timcodar improves the potency of antimycobacterial agents.

Previous studies indicated that inhibition of efflux pumps augments tuberculosis therapy. In this study, we used timcodar (formerly VX-853) to determine if this efflux pump inhibitor could increase the potency of antituberculosis (anti-TB) drugs against Mycobacterium tuberculosis in in vitro and in vivo combination studies. When used alone, timcodar weakly inhibited M. tuberculosis growth in broth culture (MIC, 19 µg/ml); however, it demonstrated synergism in drug combination studies with rifampin, bedaquiline, and clofazimine but not with other anti-TB agents. When M. tuberculosis was cultured in host macrophage cells, timcodar had about a 10-fold increase (50% inhibitory concentration, 1.9 µg/ml) in the growth inhibition of M. tuberculosis and demonstrated synergy with rifampin, moxifloxacin, and bedaquiline. In a mouse model of tuberculosis lung infection, timcodar potentiated the efficacies of rifampin and isoniazid, conferring 1.0 and 0.4 log10 reductions in bacterial burden in lung, respectively, compared to the efficacy of each drug alone. Furthermore, timcodar reduced the likelihood of a relapse infection when evaluated in a mouse model of long-term, chronic infection with treatment with a combination of rifampin, isoniazid, and timcodar. Although timcodar had no effect on the pharmacokinetics of rifampin in plasma and lung, it did increase the plasma exposure of bedaquiline. These data suggest that the antimycobacterial drug-potentiating activity of timcodar is complex and drug dependent and involves both bacterial and host-targeted mechanisms. Further study of the improvement of the potency of antimycobacterial drugs and drug candidates when used in combination with timcodar is warranted.

Novel Ranking System for Identifying Efficacious Anti-Influenza Virus PB2 Inhibitors.

Through antigenic drift and shifts, influenza virus infections continue to be an annual cause of morbidity in healthy populations and of death among elderly and at-risk patients. The emergence of highly pathogenic avian influenza viruses such as H5N1 and H7N9 and the rapid spread of the swine-origin H1N1 influenza virus in 2009 demonstrate the continued need for effective therapeutic agents for influenza. While several neuraminidase inhibitors have been developed for the treatment of influenza virus infections, these have shown a limited window for treatment initiation, and resistant variants have been noted in the population. In addition, an older class of antiviral drugs for influenza, the adamantanes, are no longer recommended for treatment due to widespread resistance. There remains a need for new influenza therapeutic agents with improved efficacy as well as an expanded window for the initiation of treatment. Azaindole compounds targeting the influenza A virus PB2 protein and demonstrating excellent in vitro and in vivo properties have been identified. To evaluate the in vivo efficacy of these PB2 inhibitors, we utilized a mouse influenza A virus infection model. In addition to traditional endpoints, i.e., death, morbidity, and body weight loss, we measured lung function using whole-body plethysmography, and we used these data to develop a composite efficacy score that takes compound exposure into account. This model allowed the rapid identification and ranking of molecules relative to each other and to oseltamivir. The ability to identify compounds with enhanced preclinical properties provides an opportunity to develop more-effective treatments for influenza in patients.

A novel inhibitor of gyrase B is a potent drug candidate for treatment of tuberculosis and nontuberculosis mycobacterial infections.

New drugs to treat drug-resistant tuberculosis are urgently needed. Extensively drug-resistant and probably the totally drug-resistant tuberculosis strains are resistant to fluoroquinolones like moxifloxacin, which target gyrase A, and most people infected with these strains die within a year. In this study, we found that a novel aminobenzimidazole, VXc-486, which targets gyrase B, potently inhibits multiple drug-sensitive isolates and drug-resistant isolates of Mycobacterium tuberculosis in vitro (MICs of 0.03 to 0.30 µg/ml and 0.08 to 5.48 µg/ml, respectively) and reduces mycobacterial burdens in lungs of infected mice in vivo. VXc-486 is active against drug-resistant isolates, has bactericidal activity, and kills intracellular and dormant M. tuberculosis bacteria in a low-oxygen environment. Furthermore, we found that VXc-486 inhibits the growth of multiple strains of Mycobacterium abscessus, Mycobacterium avium complex, and Mycobacterium kansasii (MICs of 0.1 to 2.0 µg/ml), as well as that of several strains of Nocardia spp. (MICs of 0.1 to 1.0 µg/ml). We made a direct comparison of the parent compound VXc-486 and a phosphate prodrug of VXc-486 and showed that the prodrug of VXc-486 had more potent killing of M. tuberculosis than did VXc-486 in vivo. In combination with other antimycobacterial drugs, the prodrug of VXc-486 sterilized M. tuberculosis infection when combined with rifapentine-pyrazinamide and bedaquiline-pyrazinamide in a relapse infection study in mice. Furthermore, the prodrug of VXc-486 appeared to perform at least as well as the gyrase A inhibitor moxifloxacin. These findings warrant further development of the prodrug of VXc-486 for the treatment of tuberculosis and nontuberculosis mycobacterial infections.

Preclinical activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase PB2 subunit.

VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m(7)GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a KD (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection.

Prepublication data sharing.

Rapid release of prepublication data has served the field of genomics well. Attendees at a workshop in Toronto recommend extending the practice to other biological data sets.

Computational fluid dynamic analysis of the effect of morphologic features on distraction forces in fenestrated stent grafts.

OBJECTIVE: Secure fixation of endovascular stent grafts is essential for successful endovascular aneurysm repair. Hemodynamic distraction forces are generated by blood pressure and blood flow and act against fixation force to encourage migration that may eventually lead to late stent graft failure. The aim of this in silico study was to determine which morphologic features were associated with greater distraction force. METHODS: Computer models of 54 in situ fenestrated stent grafts were constructed from postoperative computed tomography scans by use of image processing software. Computational fluid dynamic analysis was then performed by use of a commercial finite volume solver with boundary conditions representative of peak systole. Distraction force results were obtained for each component of the stent graft. Distraction force was correlated with lumen cross-sectional area (XSA) at the inlet and outlet of components and was compared between groups of components, depending on the magnitude of four predefined angles within the aortoiliac territory that we describe in detail. RESULTS: Median total resultant distraction force (RDF) acting on the fenestrated proximal bodies was 4.8 N (1.3-15.7 N); bifurcated distal bodies, 5.6N (1.0-8.0 N); and limb extensions, 1.7 N (0.6-8.4N). Inlet XSA exhibited strong, positive correlation with total RDF in proximal body and distal body components (Spearman correlation coefficient ρ, 0.883 and 0.802, respectively). Outlet XSA exhibited a similarly strong, positive correlation with total RDF in limb extension components (ρ, 0.822). Outlet angulation ≥ 45 degrees was associated with greater total RDF in the limb extension components only (P = .004). CONCLUSIONS: For a given blood pressure, XSA was the most important morphologic determinant of total RDF. Angulation within the aorta was not large enough to influence this, whereas iliac angulation affecting outlet angulation of limb extension components was associated with significantly greater total RDF.

Type IIIb endoleak is an important cause of failure following endovascular aneurysm repair.

PURPOSE: To present confirmed cases of type IIIb endoleak in second and third-generation stent-grafts used for endovascular aneurysm repair (EVAR). CASE REPORTS: Four patients developed type IIIb endoleak caused by fabric tears between 4 and 13 years following their initial EVAR. Three patients presented with rupture and one with aneurysm expansion of unknown cause. In each case, the type IIIb endoleak was confirmed at open surgery after imaging proved non-diagnostic. Only one patient survived. Had the cause for the expansion or ruptures been found prior to open reintervention, relining of the stent-graft may have been possible. CONCLUSION: Type IIIb endoleak remains difficult to diagnose. Avoidance of the high mortality associated with open secondary intervention requires a high degree of suspicion and it should be considered in any post-EVAR aneurysm expansion without an obvious cause.

Supplementation with soluble or insoluble rice-bran fibers increases short-chain fatty acid producing bacteria in the gut microbiota in vitro.

Introduction: Studies have shown that a diet high in fiber and prebiotics has a positive impact on human health due largely to the fermentation of these compounds by the gut microbiota. One underutilized source of fiber may be rice bran, a waste product of rice processing that is used most frequently as an additive to livestock feed but may be a good source of fibers and other phenolic compounds as a human diet supplement. Previous studies focused on specific compounds extracted from rice bran showed that soluble fibers extracted from rice bran can improve glucose response and reduce weight gain in mouse models. However, less is known about changes in the human gut microbiota in response to regular rice bran consumption. Methods: In this study, we used a Simulator of the Human Intestinal Microbial Ecology (SHIME®) to cultivate the human gut microbiota of 3 different donors in conditions containing either soluble or insoluble fiber fractions from rice bran. Using 16S rRNA amplicon sequencing and targeted metabolomics via Gas Chromatography-Mass Spectrometry, we explored how gut microbial communities developed provided different supplemental fiber sources. Results: We found that insoluble and soluble fiber fractions increased short-chain fatty acid production, indicating that both fractions were fermented. However, there were differences in response between donors, for example the gut microbiota from donor 1 increased acetic acid production with both fiber types compared with control; whereas for donors 2 and 3, butanoic acid production increased with ISF and SF supplementation. Both soluble and insoluble rice bran fractions increased the abundance of Bifidobacterium and Lachnospiraceae taxa. Discussion: Overall, analysis of the effect of soluble and insoluble rice bran fractions on the human in vitro gut microbiota and the metabolites produced revealed individually variant responses to these prebiotics.

Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus.

The 1918 influenza pandemic was unusually severe, resulting in about 50 million deaths worldwide. The 1918 virus is also highly pathogenic in mice, and studies have identified a multigenic origin of this virulent phenotype in mice. However, these initial characterizations of the 1918 virus did not address the question of its pathogenic potential in primates. Here we demonstrate that the 1918 virus caused a highly pathogenic respiratory infection in a cynomolgus macaque model that culminated in acute respiratory distress and a fatal outcome. Furthermore, infected animals mounted an immune response, characterized by dysregulation of the antiviral response, that was insufficient for protection, indicating that atypical host innate immune responses may contribute to lethality. The ability of influenza viruses to modulate host immune responses, such as that demonstrated for the avian H5N1 influenza viruses, may be a feature shared by the virulent influenza viruses.

Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses.

Vaccines and therapies are urgently needed to address public health needs stemming from emerging pathogens and biological threat agents such as the filoviruses Ebola virus (EBOV) and Marburg virus (MARV). Here, we developed replication-competent vaccines against EBOV and MARV based on attenuated recombinant vesicular stomatitis virus vectors expressing either the EBOV glycoprotein or MARV glycoprotein. A single intramuscular injection of the EBOV or MARV vaccine elicited completely protective immune responses in nonhuman primates against lethal EBOV or MARV challenges. Notably, vaccine vector shedding was not detectable in the monkeys and none of the animals developed fever or other symptoms of illness associated with vaccination. The EBOV vaccine induced humoral and apparent cellular immune responses in all vaccinated monkeys, whereas the MARV vaccine induced a stronger humoral than cellular immune response. No evidence of EBOV or MARV replication was detected in any of the protected animals after challenge. Our data suggest that these vaccine candidates are safe and highly efficacious in a relevant animal model.

Single immunization with a monovalent vesicular stomatitis virus-based vaccine protects nonhuman primates against heterologous challenge with Bundibugyo ebolavirus.

The recombinant vesicular stomatitis virus (rVSV) vector-based monovalent vaccine platform expressing a filovirus glycoprotein has been demonstrated to provide protection from lethal challenge with Ebola (EBOV) and Marburg (MARV) viruses both prophylactically and after exposure. This platform provides protection between heterologous strains within a species; however, protection from lethal challenge between species has been largely unsuccessful. To determine whether the rVSV-EBOV vaccines have the potential to provide protection against a newly emerging, phylogenetically related species, cynomolgus macaques were vaccinated with an rVSV vaccine expressing either the glycoprotein of Zaire ebolavirus (ZEBOV) or Côte d'Ivoire ebolavirus (CIEBOV) and then challenged with Bundibugyo ebolavirus (BEBOV), which was recently proposed as a new EBOV species following an outbreak in Uganda in 2007. A single vaccination with the ZEBOV-specific vaccine provided cross-protection (75% survival) against subsequent BEBOV challenge, whereas vaccination with the CIEBOV-specific vaccine resulted in an outcome similar to mock-immunized animals (33% and 25% survival, respectively). This demonstrates that monovalent rVSV-based vaccines may be useful against a newly emerging species; however, heterologous protection across species remains challenging and may depend on enhancing the immune responses either through booster immunizations or through the inclusion of multiple immunogens.

Fructooligosaccharides (FOS) differentially modifies the in vitro gut microbiota in an age-dependent manner.

Introduction: Fructooligosaccharides (FOS) are well-known carbohydrates that promote healthy gut microbiota and have been previously demonstrated to enhance levels of Bifidobacterium and Lactobacillus. Its bifidogenic properties are associated with positive health outcomes such as reduced obesity and anti-inflammatory properties, and, therefore, is in use as a prebiotic supplement to support healthy gut microbiota. However, the gut microbiota changes with age, which may lead to differential responses to treatments with prebiotics and other dietary supplements. Methods: To address this concern, we implemented a 24-h in vitro culturing method to determine whether FOS treatment in three different adult age groups would have a differential effect. The age groups of interest ranged from 25 to 70 years and were split into young adults, adults, and older adults for the purposes of this analysis. Metagenomics and short-chain fatty acid analysis were performed to determine changes in the structure and function of the microbial communities. Results: These analyses found that FOS created a bifidogenic response in all age groups, increased overall SCFA levels, decreased alpha diversity, and shifted the communities to be more similar in beta diversity metrics. However, the age groups differed in which taxa were most prevalent or most affected by FOS treatment. Discussion: Overall, the results of this study demonstrate the positive effects of FOS on the gut microbiome, and importantly, how age may play a role in the effectiveness of this prebiotic.

An in vitro analysis of how lactose modifies the gut microbiota structure and function of adults in a donor-independent manner.

Introduction: Following consumption of milk, lactose, a disaccharide of glucose and galactose, is hydrolyzed and absorbed in the upper gastrointestinal tract. However, hydrolysis and absorption are not always absolute, and some lactose will enter the colon where the gut microbiota is able to hydrolyze lactose and produce metabolic byproducts. Methods: Here, the impact of lactose on the gut microbiota of healthy adults was examined, using a short-term, in vitro strategy where fecal samples harvested from 18 donors were cultured anaerobically with and without lactose. The data were compiled to identify donor-independent responses to lactose treatment. Results and discussion: Metagenomic sequencing found that the addition of lactose decreased richness and evenness, while enhancing prevalence of the ß-galactosidase gene. Taxonomically, lactose treatment decreased relative abundance of Bacteroidaceae and increased lactic acid bacteria, Lactobacillaceae, Enterococcaceae, and Streptococcaceae, and the probiotic Bifidobacterium. This corresponded with an increased abundance of the lactate utilizers, Veillonellaceae. These structural changes coincided with increased total short-chain fatty acids (SCFAs), specifically acetate, and lactate. These results demonstrated that lactose could mediate the gut microbiota of healthy adults in a donor-independent manner, consistent with other described prebiotics, and provided insight into how dietary milk consumption may promote human health through modifications of the gut microbiome.

Characterization of Zaire ebolavirus glycoprotein-specific monoclonal antibodies.

Zaire ebolavirus (ZEBOV) can be transmitted by human-to-human contact and causes acute haemorrhagic fever with case fatality rates up to 90%. There are no effective therapeutic or prophylactic treatments available. The sole transmembrane glycoprotein (GP) is the key target for developing neutralizing antibodies. In this study, recombinant VSVΔG/ZEBOVGP was used to generate monoclonal antibodies (MAbs) against the ZEBOV GP. A total of 8 MAbs were produced using traditional hybridoma cell fusion technology, and then characterized by ELISA using ZEBOV VLPs, Western blotting, an immunofluorescence assay, and immunoprecipitation. All 8 MAbs worked in IFA and IP, suggesting that they are all conformational MAbs, however six of them recognized linearized epitopes by WB. ELISA results demonstrated that one MAb bound to a secreted GP (sGP 1-295aa); three bind to a part of the mucin domain (333-458aa); three MAbs recognized epitopes on the C-terminal domain of GP1 (296-501aa); and one bound to full length GP (VLPs/GP1,2 ΔTm). Using a mouse model these MAbs were evaluated for their therapeutic capacity during a lethal infection. All 8 MAb improved survival rates by 33%-100% against a high dose lethal challenge with mouse-adapted ZEBOV. This work has important implications for further development of vaccines and immunotherapies for ZEBOV infection.