Multiscale model for forecasting Sabin 2 vaccine virus household and community transmission.

Since the global withdrawal of Sabin 2 oral poliovirus vaccine (OPV) from routine immunization, the Global Polio Eradication Initiative (GPEI) has reported multiple circulating vaccine-derived poliovirus type 2 (cVDPV2) outbreaks. Here, we generated an agent-based, mechanistic model designed to assess OPV-related vaccine virus transmission risk in populations with heterogeneous immunity, demography, and social mixing patterns. To showcase the utility of our model, we present a simulation of mOPV2-related Sabin 2 transmission in rural Matlab, Bangladesh based on stool samples collected from infants and their household contacts during an mOPV2 clinical trial. Sabin 2 transmission following the mOPV2 clinical trial was replicated by specifying multiple, heterogeneous contact rates based on household and community membership. Once calibrated, the model generated Matlab-specific insights regarding poliovirus transmission following an accidental point importation or mass vaccination event. We also show that assuming homogeneous contact rates (mass action), as is common of poliovirus forecast models, does not accurately represent the clinical trial and risks overestimating forecasted poliovirus outbreak probability. Our study identifies household and community structure as an important source of transmission heterogeneity when assessing OPV-related transmission risk and provides a calibratable framework for expanding these analyses to other populations. Trial Registration: ClinicalTrials.gov This trial is registered with clinicaltrials.gov, NCT02477046.

Megasphaera in the Stool Microbiota Is Negatively Associated With Diarrheal Cryptosporidiosis.

BACKGROUND: The protozoan parasites in the Cryptosporidium genus cause both acute diarrheal disease and subclinical (ie, nondiarrheal) disease. It is unclear if the microbiota can influence the manifestation of diarrhea during a Cryptosporidium infection. METHODS: To characterize the role of the gut microbiota in diarrheal cryptosporidiosis, the microbiome composition of both diarrheal and surveillance Cryptosporidium-positive fecal samples from 72 infants was evaluated using 16S ribosomal RNA gene sequencing. Additionally, the microbiome composition prior to infection was examined to test whether a preexisting microbiome profile could influence the Cryptosporidium infection phenotype. RESULTS: Fecal microbiome composition was associated with diarrheal symptoms at 2 timepoints. Megasphaera was significantly less abundant in diarrheal samples compared with subclinical samples at the time of Cryptosporidium detection (log2 [fold change] = -4.3; P = 10-10) and prior to infection (log2 [fold change] = -2.0; P = 10-4); this assigned sequence variant was detected in 8 children who had diarrhea and 30 children without diarrhea. Random forest classification also identified Megasphaera abundance in the pre- and postexposure microbiota as predictive of a subclinical infection. CONCLUSIONS: Microbiome composition broadly, and specifically low Megasphaera abundance, was associated with diarrheal symptoms prior to and at the time of Cryptosporidium detection. This observation suggests that the gut microenvironment may play a role in determining the severity of a Cryptosporidium infection. Clinical Trials Registration. NCT02764918.

Fluorescent indomethacin-dansyl conjugates utilize the membrane-binding domain of cyclooxygenase-2 to block the opening to the active site.

Many indomethacin amides and esters are cyclooxygenase-2 (COX-2)-selective inhibitors, providing a framework for the design of COX-2-targeted imaging and cancer chemotherapeutic agents. Although previous studies have suggested that the amide or ester moiety of these inhibitors binds in the lobby region, a spacious alcove within the enzyme's membrane-binding domain, structural details have been lacking. Here, we present observations on the crystal complexes of COX-2 with two indomethacin-dansyl conjugates (compounds 1 and 2) at 2.22-Å resolution. Both compounds are COX-2-selective inhibitors with IC50 values of 0.76 and 0.17 µm, respectively. Our results confirmed that the dansyl moiety is localized in and establishes hydrophobic interactions and several hydrogen bonds with the lobby of the membrane-binding domain. We noted that in both crystal structures, the linker tethering indomethacin to the dansyl moiety passes through the constriction at the mouth of the COX-2 active site, resulting in displacement and disorder of Arg-120, located at the opening to the active site. Both compounds exhibited higher inhibitory potency against a COX-2 R120A variant than against the WT enzyme. Inhibition kinetics of compound 2 were similar to those of the indomethacin parent compound against WT COX-2, and the R120A substitution reduced the time dependence of COX inhibition. These results provide a structural basis for the further design and optimization of conjugated COX reagents for imaging of malignant or inflammatory tissues containing high COX-2 levels.

Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis.

The disease severity of Entamoeba histolytica infection ranges from asymptomatic to life-threatening. Recent human and animal data implicate the gut microbiome as a modifier of E. histolytica virulence. Here we have explored the association of the microbiome with susceptibility to amebiasis in infants and in the mouse model of amebic colitis. Dysbiosis occurred symptomatic E. histolytica infection in children, as evidenced by a lower Shannon diversity index of the gut microbiota. To test if dysbiosis was a cause of susceptibility, wild type C57BL/6 mice (which are innately resistant to E. histiolytica infection) were treated with antibiotics prior to cecal challenge with E. histolytica. Compared with untreated mice, antibiotic pre-treated mice had more severe colitis and delayed clearance of E. histolytica. Gut IL-25 and mucus protein Muc2, both shown to provide innate immunity in the mouse model of amebic colitis, were lower in antibiotic pre-treated mice. Moreover, dysbiotic mice had fewer cecal neutrophils and myeloperoxidase activity. Paradoxically, the neutrophil chemoattractant chemokines CXCL1 and CXCL2, as well as IL-1ß, were higher in the colon of mice with antibiotic-induced dysbiosis. Neutrophils from antibiotic pre-treated mice had diminished surface expression of the chemokine receptor CXCR2, potentially explaining their inability to migrate to the site of infection. Blockade of CXCR2 increased susceptibility of control non-antibiotic treated mice to amebiasis. In conclusion, dysbiosis increased the severity of amebic colitis due to decreased neutrophil recruitment to the gut, which was due in part to decreased surface expression on neutrophils of CXCR2.

Detection of carcinogen-induced bladder cancer by fluorocoxib A.

BACKGROUND: Conventional cystoscopy can detect advanced stages of bladder cancer; however, it has limitations to detect bladder cancer at the early stages. Fluorocoxib A, a rhodamine-conjugated analog of indomethacin, is a novel fluorescent imaging agent that selectively targets cyclooxygenase-2 (COX-2)-expressing cancers. METHODS: In this study, we have used a carcinogen N-butyl-N-4-hydroxybutyl nitrosamine (BBN)-induced bladder cancer immunocompetent mouse B6D2F1 model that resembles human high-grade invasive urothelial carcinoma. We evaluated the ability of fluorocoxib A to detect the progression of carcinogen-induced bladder cancer in mice. Fluorocoxib A uptake by bladder tumors was detected ex vivo using IVIS optical imaging system and Cox-2 expression was confirmed by immunohistochemistry and western blotting analysis. After ex vivo imaging, the progression of bladder carcinogenesis from normal urothelium to hyperplasia, carcinoma-in-situ and carcinoma with increased Ki67 and decreased uroplakin-1A expression was confirmed by histology and immunohistochemistry analysis. RESULTS: The specific uptake of fluorocoxib A correlated with increased Cox-2 expression in progressing bladder cancer. In conclusion, fluorocoxib A detected the progression of bladder carcinogenesis in a mouse model with selective uptake in Cox-2-expressing bladder hyperplasia, CIS and carcinoma by 4- and 8-fold, respectively, as compared to normal bladder urothelium, where no fluorocoxib A was detected. CONCLUSIONS: Fluorocoxib A is a targeted optical imaging agent that could be applied for the detection of Cox-2 expressing human bladder cancer.

Identification of Etiology-Specific Diarrhea Associated With Linear Growth Faltering in Bangladeshi Infants.

Childhood diarrhea in low-resource settings has been variably linked to linear growth shortfalls. However, the association between etiology-specific diarrhea and growth has not been comprehensively evaluated. We tested diarrheal stools collected from the Performance of Rotavirus and Oral Polio Vaccines in Developing Countries study from 2011 to 2013 in Dhaka, Bangladesh, by quantitative polymerase chain reaction for a broad range of enteropathogens to characterize diarrhea etiology and examine the association between etiology-specific diarrhea and linear growth and systemic inflammation. Pathogen-specific burdens of diarrhea were determined using attributable fractions. Linear regression was used to examine associations of pathogen-specific diarrhea with length-for-age z scores (LAZ) and serum C-reactive protein. There was no relationship between all-cause diarrhea and length at 12 months (change in 12-month LAZ per episode, -0.01, 95% confidence interval (CI): -0.06, 0.03). However, Cryptosporidium (change in 12-month LAZ per attributable episode, -0.23, 95% CI: -0.50, 0.03), Campylobacter jejuni/coli (change of -0.16, 95% CI: -0.32, -0.01), and Shigella/enteroinvasive Escherichia coli diarrhea (change of -0.12, 95% CI: -0.26, 0.03) were associated with linear growth deficits. Diarrhea attributable to C. jejuni/coli and Shigella/enteroinvasive E. coli were associated with elevated C-reactive protein. The association between diarrhea and linear growth appears to be pathogen-specific, reinforcing the need for pathogen-specific interventions.

Kinetics of poliovirus shedding following oral vaccination as measured by quantitative reverse transcription-PCR versus culture.

Amid polio eradication efforts, detection of oral polio vaccine (OPV) virus in stool samples can provide information about rates of mucosal immunity and allow estimation of the poliovirus reservoir. We developed a multiplex one-step quantitative reverse transcription-PCR (qRT-PCR) assay for detection of OPV Sabin strains 1, 2, and 3 directly in stool samples with an external control to normalize samples for viral quantity and compared its performance with that of viral culture. We applied the assay to samples from infants in Dhaka, Bangladesh, after the administration of trivalent OPV (tOPV) at weeks 14 and 52 of life (on days 0 [pre-OPV], +4, +11, +18, and +25 relative to vaccination). When 1,350 stool samples were tested, the sensitivity and specificity of the quantitative PCR (qPCR) assay were 89 and 91% compared with culture. A quantitative relationship between culture(+)/qPCR(+) and culture(-)/qPCR(+) stool samples was observed. The kinetics of shedding revealed by qPCR and culture were similar. qPCR quantitative cutoffs based on the day +11 or +18 stool samples could be used to identify the culture-positive shedders, as well as the long-duration or high-frequency shedders. Interestingly, qPCR revealed that a small minority (7%) of infants contributed the vast majority (93 to 100%) of the total estimated viral excretion across all subtypes at each time point. This qPCR assay for OPV can simply and quantitatively detect all three Sabin strains directly in stool samples to approximate shedding both qualitatively and quantitatively.

Risk assessment of four toxic heavy metals in terrestrial and aquatic ecosystems around BSCIC tannery industrial estate of Savar, Dhaka, Bangladesh.

The shifting of tannery industries from Hazaribagh to Savar adjacent to Dhaleshwari River might have inevitable adverse impacts, especially the heavy metal contamination of both terrestrial and aquatic ecosystems. The study was carried out to investigate the concentrations of four heavy metals in the soil, water, and plant samples collected from around Dhaleshwari River adjacent to the Bangladesh Small & Cottage Industries Corporation Tannery Industrial Estate, Dhaka. This study revealed that average concentrations of cadmium and chromium in soil exceeded the maximum permissible limit of World Health Organization (1996) and average concentrations of lead, cadmium, and chromium in water exceeded the maximum permissible limit of World Health Organization (2011) and Environmental Conservation Rules (1997). The average concentrations of lead, cadmium, and chromium in the roots and shoots of both Eichhornia crassipes and Cynodon dactylon exceeded the maximum permissible limit of Food and Agriculture Organization/ World Health Organization (2016). Ecological risk assessment using indices model for soil pollution indicated that soil is mostly contaminated with cadmium and chromium which can pose strong ecological risk Health risk assessment using indices model for water pollution revealed the high degree of contamination and unacceptable non-carcinogenic risk and carcinogenic risk for adults as well as children through ingestion of water. Average bioconcentration factor and bioaccumulation factor were higher in Eichhornia crassipes than Cynodon dactylon for lead, cadmium, and chromium. Average translocation factor was also higher in Eichhornia crassipes for all the metals except cadmium. It is suggested remedial and mitigation measures be instituted to control environmental degradation of the newly established Tannery Industrial Estate.

Investigating the impact of antibiotic-induced dysbiosis on protection from Clostridium difficile colitis by mouse colonic innate lymphoid cells.

Innate lymphoid cells (ILCs) play a critical role in maintaining intestinal health in homeostatic and diseased conditions. During Clostridium difficile infection (CDI), IL-33 activates ILC2 to protect from colonic damage and mortality. The function of IL-33 and ILC is tightly regulated by the intestinal microbiota. We set out to determine the impact of antibiotic-induced disruption of the microbiome on ILC function. Our goal was to understand antibiotic-induced changes in ILC function on susceptibility to C. difficile colitis in a mouse model. We utilized high-throughput single-cell RNAseq to investigate the phenotypic features of colonic ILC at baseline, after antibiotic administration with or without IL-33 treatment. We identified a heterogeneous landscape of colonic ILCs with gene signatures of inflammatory, anti-inflammatory, migratory, progenitor, plastic, and antigen-presenting ILCs. Antibiotic treatment decreased ILC2 while coordinately increasing ILC1 and ILC3 phenotypes. Notably, Ifng+, Ccl5+, and Il23r+ ILC increased after antibiotics. IL-33 treatment counteracted the antibiotic effect by downregulating ILC1 and ILC3 and activating ILC2. In addition, IL-33 treatment markedly induced the expression of type 2 genes, including Areg and Il5. Finally, we identified amphiregulin, produced by ILC2, as protective during C. difficile infection. Together, our data expand our understanding of how antibiotics induce susceptibility to C. difficile colitis through their impact on ILC subsets and function.IMPORTANCEClostridium difficile infection (CDI) accounts for around 500,000 symptomatic cases and over 20,000 deaths annually in the United States alone. A major risk factor of CDI is antibiotic-induced dysbiosis of the gut. Microbiota-regulated IL-33 and innate lymphoid cells (ILCs) are important in determining the outcomes of C. difficile infection. Understanding how antibiotic and IL-33 treatment alter the phenotype of colon ILCs is important to identify potential therapeutics. Here, we performed single-cell RNAseq of mouse colon ILCs collected at baseline, after antibiotic treatment, and after IL-33 treatment. We identified heterogeneous subpopulations of all three ILC subtypes in the mouse colon. Our analysis revealed several potential pathways of antibiotic-mediated increased susceptibility to intestinal infection. Our discovery that Areg is abundantly expressed by ILCs, and the protection of mice from CDI by amphiregulin treatment, suggests that the amphiregulin-epidermal growth factor receptor pathway is a potential therapeutic target for treating intestinal colitis.

Design, synthesis, and structure-activity relationship studies of fluorescent inhibitors of cycloxygenase-2 as targeted optical imaging agents.

Cycloxygenase-2 (COX-2) is an attractive target for molecular imaging because it is an inducible enzyme that is expressed in response to inflammatory and proliferative stimuli. Recently, we reported that conjugation of indomethacin with carboxy-X-rhodamine dyes results in the formation of effective, targeted, optical imaging agents able to detect COX-2 in inflammatory tissues and premalignant and malignant tumors (Uddin et al. Cancer Res. 2010, 70, 3618-3627). The present paper summarizes the details of the structure-activity relationship (SAR) studies performed for lead optimization of these dyes. A wide range of fluorescent conjugates were designed and synthesized, and each of them was tested for the ability to selectively inhibit COX-2 as the purified protein and in human cancer cells. The SAR study revealed that indomethacin conjugates are the best COX-2-targeted agents compared to the other carboxylic acid-containing nonsteroidal anti-inflammatory drugs (NSAIDs) or COX-2-selective inhibitors (COXIBs). An n-butyldiamide linker is optimal for tethering bulky fluorescent functionalities onto the NSAID or COXIB cores. The activity of conjugates also depends on the size, shape, and electronic properties of the organic fluorophores. These reagents are taken up by COX-2-expressing cells in culture, and the uptake is blocked by pretreatment with a COX inhibitor. In in vivo settings, these reagents become highly enriched in COX-2-expressing tumors compared to surrounding normal tissue, and they accumulate selectively in COX-2-expressing tumors as compared with COX-2-negative tumors grown in mice. Thus, COX-2-targeted fluorescent inhibitors are useful for preclinical and clinical detection of lesions containing elevated levels of COX-2.

Numerical computation of pulsatile hemodynamics and diagnostic concern of coronary bifurcated artery flow for Newtonian and non-Newtonian fluid.

Atherosclerotic with the high occurrence of plaque formation due to stenosis has attracted wide attention among researchers. The left coronary artery has been studied in two-dimensional and in three-dimensional (3D) bifurcation as the models of blood flow through Newtonian and non-Newtonian fluids to better understand the physical mechanism. The computational Fluid Dynamics (CFD) technique is incorporated in COMSOL Multiphysics and then it is justified by satisfactory validation. It is found that the Newtonian model shows larger recirculation zones than non-Newtonian does. The present study also focuses on the evaluations of the lesion of diagnostic and the coefficient of pressure drop assessments on the basis of the diagnostic parameter's critical values affected by the rheological model. Nevertheless, the leading concentration of the subsisting investigation works is confined to the change of importance factor (IFc) affected by arterial blockage. But the IFc of non-Newtonian fluid for 3D left coronary artery bifurcation model decreases with increasing bifurcation angle and the time-averaged inlet pressure is the least for smaller bifurcation angles. The current research further concentrates that the flow separation length reduces with developing bifurcation angle in bifurcated geometry. It is significant to mention that non-Newtonian blood flow model incorporating hemodynamic and diagnostic parameters has great impacts on instantaneous flow systems.

Development of Pluoronic nanoparticles of fluorocoxib A for endoscopic fluorescence imaging of colonic adenomas.

Significance: Current white light colonoscopy suffers from many limitations that allow 22% to 32% of preneoplastic lesions to remain undetected. This high number of false negatives contributes to the appearance of interval malignancies, defined as neoplasms diagnosed between screening colonoscopies at a rate of 2% to 6%. Aim: The shortcomings of today's white light-based colorectal cancer screening are addressed by colonoscopic fluorescence imaging of preneoplastic lesions using targeted fluorescent agents to enhance contrast between the lesion and the surrounding normal colonic epithelium. Approach: We describe the development of Pluronic® nanoparticles of fluorocoxib A (FA), a fluorescent cyclooxygenase-2 (COX-2) inhibitor that enables targeted imaging of inflammation and cancer in numerous animal models, for endoscopic florescence imaging of colonic adenomas. Results: We formulated FA, a fluorescent COX-2 inhibitor, or fluorocoxib negative control (FNC), a nontargeted fluorophore and a negative control for FA, in micellar nanoparticles of FDA approved Pluronic tri-block co-polymer using a bulk solvent evaporation method. This afforded FA-loaded micellar nanoparticles (FA-NPs) or FNC-loaded micellar nanoparticles (FNC-NPs) with the hydrodynamic diameters ( D h ) of 45.7 ± 2.5 nm and 44.9 ± 3.8 nm and the zeta potentials ( ζ ) of - 1.47 ± 0.3 mV and - 1.64 ± 0.5 mV , respectively. We intravenously injected B6;129 mice bearing colonic adenomas induced by azoxymethane and dextran-sodium sulfate with FA-loaded Pluronic nanoparticles (FA-NPs). The diffusion-mediated local FA release and its binding to COX-2 enzyme allowed for clear detection of adenomas with high signal-to-noise ratios. The COX-2 targeted delivery and tumor retention were validated by negligible tumor fluorescence detected upon colonoscopic imaging of adenoma-bearing mice injected with Pluronic nanoparticles of FNC or of animals predosed with the COX-2 inhibitor, celecoxib, followed by intravenous dosing of FA-NPs. Conclusions: These results demonstrate that the formulation of FA in Pluronic nanoparticles overcomes a significant hurdle to its clinical development for early detection of colorectal neoplasms by fluorescence endoscopy.

Ferroptosis Inhibitors Suppress Prostaglandin Synthesis in Lipopolysaccharide-Stimulated Macrophages.

Necrostatin-1 blocks ferroptosis via an unknown mechanism and necroptosis through inhibition of receptor-interacting protein kinase-1 (RIP1). We report that necrostatin-1 suppresses cyclooxygenase-2-dependent prostaglandin biosynthesis in lipopolysaccharide-treated RAW264.7 macrophages (IC50 ∼ 100 µM). This activity is shared by necrostatin-1i (IC50 ∼ 50 µM), which lacks RIP1 inhibitory activity, but not the RIP1 inhibitors necrostatin-1s or deschloronecrostatin-1s. Furthermore, we show that the potent ferroptosis inhibitors and related compounds ferrostatin-1, phenoxazine, phenothiazine, and 10-methylphenothiazine strongly inhibit cellular prostaglandin biosynthesis with IC50's in the range of 30 nM to 3.5 µM. None of the compounds inhibit lipopolysaccharide-mediated cyclooxygenase-2 protein induction. In the presence of activating hydroperoxides, the necrostatins and ferroptosis inhibitors range from low potency inhibition to stimulation of in vitro cyclooxygenase-2 activity; however, inhibitory potency is increased under conditions of low peroxide tone. The ferroptosis inhibitors are highly effective reducing substrates for cyclooxygenase-2's peroxidase activity, suggesting that they act by suppressing hydroperoxide-mediated activation of the cyclooxygenase active site. In contrast, for the necrostatins, cellular prostaglandin synthesis inhibition does not correlate with peroxidase-reducing activity but rather with the presence of a thiohydantoin substituent, which conveys the ability to reduce the endoperoxide intermediate prostaglandin H2 to prostaglandin F2α in vitro. This finding suggests that necrostatin-1 blocks cellular prostaglandin synthesis and ferroptosis via a redox mechanism distinct from action as a one-electron donor. The results indicate that a wide range of compounds derived from redox-active chemical scaffolds can block cellular prostaglandin biosynthesis.

Clostridioides difficile Binary Toxin Binding Component Increases Virulence in a Hamster Model.

Background: Clostridioides difficile is the leading cause of hospital-acquired gastrointestinal infection, in part due to the existence of binary toxin (CDT)-expressing hypervirulent strains. Although the effects of the CDT holotoxin on disease pathogenesis have been previously studied, we sought to investigate the role of the individual components of CDT during in vivo infection. Methods: To determine the contribution of the separate components of CDT during infection, we developed strains of C difficile expressing either CDTa or CDTb individually. We then infected both mice and hamsters with these novel mutant strains and monitored them for development of severe illness. Results: Although expression of CDTb without CDTa did not induce significant disease in a mouse model of C difficile infection, we found that complementation of a CDT-deficient C difficile strain with CDTb alone restored virulence in a hamster model of C difficile infection. Conclusions: Overall, this study demonstrates that the binding component of C difficile binary toxin, CDTb, contributes to virulence in a hamster model of infection.

Secondary bile acids function through the vitamin D receptor in myeloid progenitors to promote myelopoiesis.

Metabolic products of the microbiota can alter hematopoiesis. However, the contribution and site of action of bile acids is poorly understood. Here, we demonstrate that the secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA), increase bone marrow myelopoiesis. Treatment of bone marrow cells with DCA and LCA preferentially expanded immunophenotypic and functional colony-forming unit-granulocyte and macrophage (CFU-GM) granulocyte-monocyte progenitors (GMPs). DCA treatment of sorted hematopoietic stem and progenitor cells (HSPCs) increased CFU-GMs, indicating that direct exposure of HSPCs to DCA sufficed to increase GMPs. The vitamin D receptor (VDR) was required for the DCA-induced increase in CFU-GMs and GMPs. Single-cell RNA sequencing revealed that DCA significantly upregulated genes associated with myeloid differentiation and proliferation in GMPs. The action of DCA on HSPCs to expand GMPs in a VDR-dependent manner suggests microbiome-host interactions could directly affect bone marrow hematopoiesis and potentially the severity of infectious and inflammatory disease.

The IL-33-ILC2 pathway protects from amebic colitis.

Entamoeba histolytica is a pathogenic protozoan parasite that causes intestinal colitis, diarrhea, and in some cases, liver abscess. Through transcriptomics analysis, we observed that E. histolytica infection was associated with increased expression of IL-33 mRNA in both the human and murine colon. IL-33, the IL-1 family cytokine, is released after cell injury to alert the immune system of tissue damage. Treatment with recombinant IL-33 protected mice from amebic infection and intestinal tissue damage; moreover, blocking IL-33 signaling made mice more susceptible to amebiasis. IL-33 limited the recruitment of inflammatory immune cells and decreased the pro-inflammatory cytokine IL-6 in the cecum. Type 2 immune responses were upregulated by IL-33 treatment during amebic infection. Interestingly, administration of IL-33 protected RAG2-/- mice but not RAG2-/-γc-/- mice, demonstrating that IL-33-mediated protection required the presence of innate lymphoid cells (ILCs). IL-33 induced recruitment of ILC2 but not ILC1 and ILC3 in RAG2-/- mice. At baseline and after amebic infection, there was a significantly higher IL13+ILC2s in C57BL/J mice, which are naturally resistant to amebiasis, than CBA/J mice. Adoptive transfer of ILC2s to RAG2-/-γc-/- mice restored IL-33-mediated protection. These data reveal that the IL-33-ILC2 pathway is an important host defense mechanism against amebic colitis.

Discovery of a Redox-Activatable Chemical Probe for Detection of Cyclooxygenase-2 in Cells and Animals.

Cyclooxygenase-2 (COX-2) expression is up-regulated in inflammatory tissues and many premalignant and malignant tumors. Assessment of COX-2 protein in vivo, therefore, promises to be a powerful strategy to distinguish pathologic cells from normal cells in a complex disease setting. Herein, we report the first redox-activatable COX-2 probe, fluorocoxib Q (FQ), for in vivo molecular imaging of pathogenesis. FQ inhibits COX-2 selectively in purified enzyme and cell-based assays. FQ exhibits extremely low fluorescence and displays time- and concentration-dependent fluorescence enhancement upon exposure to a redox environment. FQ enters the cells freely and binds to the COX-2 enzyme. FQ exhibits high circulation half-life and metabolic stability sufficient for target site accumulation and demonstrates COX-2-targeted uptake and retention in cancer cells and pathologic tissues. Once taken up, it undergoes redox-mediated transformation into a fluorescent compound fluorocoxib Q-H that results in high signal-to-noise contrast and differentiates pathologic tissues from non-pathologic tissues for real-time in vivo imaging.

Host Protective Mechanisms to Intestinal Amebiasis.

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, an infection that manifests as colitis and, in some cases, liver abscess. A better understanding of host protective factors is key to developing an effective remedy. Recently, significant advances have been made in understanding the mechanisms of MUC2 production by goblet cells upon amebic infection, regulation of antimicrobial peptide production by Paneth cells, the interaction of commensal microbiota with immune stimulation, and host genetics in conferring protection from amebiasis. In addition to host pathways that may serve as potential therapeutic targets, significant progress has also been made with respect to development of a vaccine against amebiasis. Here, we aim to highlight the current understanding and knowledge gaps critically.

Optimizing a Multi-Component Intranasal Entamoeba Histolytica Vaccine Formulation Using a Design of Experiments Strategy.

Amebiasis is a neglected tropical disease caused by Entamoeba histolytica. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition.

Targeting diacylglycerol lipase reduces alcohol consumption in preclinical models.

Alcohol use disorder (AUD) is associated with substantial morbidity, mortality, and societal cost, and pharmacological treatment options for AUD are limited. The endogenous cannabinoid (eCB) signaling system is critically involved in reward processing and alcohol intake is positively correlated with release of the eCB ligand 2-Arachidonoylglycerol (2-AG) within reward neurocircuitry. Here we show that genetic and pharmacological inhibition of diacylglycerol lipase (DAGL), the rate limiting enzyme in the synthesis of 2-AG, reduces alcohol consumption in a variety of preclinical models ranging from a voluntary free-access model to aversion resistant-drinking and dependence-like drinking induced via chronic intermittent ethanol vapor exposure in mice. DAGL inhibition during either chronic alcohol consumption or protracted withdrawal was devoid of anxiogenic and depressive-like behavioral effects. Lastly, DAGL inhibition also prevented ethanol-induced suppression of GABAergic transmission onto midbrain dopamine neurons, providing mechanistic insight into how DAGL inhibition could affect alcohol reward. These data suggest reducing 2-AG signaling via inhibition of DAGL could represent an effective approach to reduce alcohol consumption across the spectrum of AUD severity.