BackgroundNew drugs are needed to treat visceral leishmaniasis (VL) because the current therapies are toxic, expensive, and parasite resistance may weaken drug efficacy. We established a novel ex vivo splenic explant culture system from hamsters infected with luciferase-transfected Leishmania donovani to screen chemical compounds for anti-leishmanial activity.Methodology/Principal FindingsThis model has advantages over in vitro systems in that it: 1) includes the whole cellular population involved in the host-parasite interaction; 2) is initiated at a stage of infection when the immunosuppressive mechanisms that lead to progressive VL are evident; 3) involves the intracellular form of Leishmania; 4) supports parasite replication that can be easily quantified by detection of parasite-expressed luciferase; 5) is adaptable to a high-throughput screening format; and 6) can be used to identify compounds that have both direct and indirect anti-parasitic activity. The assay showed excellent discrimination between positive (amphotericin B) and negative (vehicle) controls with a Z' Factor >0.8. A duplicate screen of 4 chemical libraries containing 4,035 compounds identified 202 hits (5.0%) with a Z score of <–1.96 (p<0.05). Eighty-four (2.1%) of the hits were classified as lead compounds based on the in vitro therapeutic index (ratio of the compound concentration causing 50% cytotoxicity in the HepG2 cell line to the concentration that caused 50% reduction in the parasite load). Sixty-nine (82%) of the lead compounds were previously unknown to have anti-leishmanial activity. The most frequently identified lead compounds were classified as quinoline-containing compounds (14%), alkaloids (10%), aromatics (11%), terpenes (8%), phenothiazines (7%) and furans (5%).Conclusions/SignificanceThe ex vivo splenic explant model provides a powerful approach to identify new compounds active against L. donovani within the pathophysiologic environment of the infected spleen. Further in vivo evaluation and chemical optimization of these lead compounds may generate new candidates for preclinical studies of treatment for VL.

Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. infection (CDI) represents an important example where the rational design of next-generation probiotics is being actively pursued to prevent disease recurrence. Because intrinsic resistance to clinically relevant antibiotics used to treat CDI (vancomycin, metronidazole, and fidaxomicin) is a desired trait in such probiotic species, we screened several bacteria and identified to be a promising candidate for adjunct therapy. Human-derived bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the gene locus were potent reuterin producers, with 17938 inhibiting growth at a level on par with the level of growth inhibition by vancomycin. Targeted mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of with glycerol was effective against colonization in complex human fecal microbial communities, whereas treatment with either glycerol or alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with elicited changes in the composition and function of the human microbial community that preferentially targets outgrowth and toxicity, a finding consistent with glycerol fermentation and reuterin production. Antimicrobial resistance has thus been successfully exploited in the natural design of human microbiome evasion of , and this method may provide a prototypic precursor-directed probiotic approach. Antibiotic resistance and substrate bioavailability may therefore represent critical new determinants of probiotic efficacy in clinical trials.

h Antibiotic resistance in medically relevant bacterial pathogens, coupled with a paucity of novel antimicrobial discoveries, represents a pressing global crisis. Traditional drug discovery is an inefficient and costly process; however, systematic screening of Food and Drug Administration (FDA)-approved therapeutics for other indications in humans offers a rapid alternative approach. In this study, we screened a library of 780 FDA-approved drugs to identify molecules that rendered RAW 264.7 murine macrophages resistant to cytotoxicity induced by the highly virulent Yersinia pestis CO92 strain. Of these compounds, we identified 94 not classified as antibiotics as being effective at preventing Y. pestis-induced cytotoxicity. A total of 17 prioritized drugs, based on efficacy in in vitro screens, were chosen for further evaluation in a murine model of pneumonic plague to delineate if in vitro efficacy could be translated in vivo. Three drugs, doxapram (DXP), amoxapine (AXPN), and trifluoperazine (TFP), increased animal survivability despite not exhibiting any direct bacteriostatic or bactericidal effect on Y. pestis and having no modulating effect on crucial Y. pestis virulence factors. These findings suggested that DXP, AXPN, and TFP may modulate host cell pathways necessary for disease pathogenesis. Finally, to further assess the broad applicability of drugs identified from in vitro screens, the therapeutic potential of TFP, the most efficacious drug in vivo, was evaluated in murine models of Salmonella enterica serovar Typhimurium and Clostridium difficile infections. In both models, TFP treatment resulted in increased survivability of infected animals. Taken together, these results demonstrate the broad applicability and potential use of nonantibiotic FDA-approved drugs to combat respiratory and gastrointestinal bacterial pathogens.

In a murine model of moderate childhood malnutrition we found that polynutrient deficiency led to a 4–5-fold increase in early visceralization of L. donovani (3 days post-infection) following cutaneous infection and a 16-fold decrease in lymph node barrier function (p<0.04 for all). To begin to understand the mechanistic basis for this malnutrition-related parasite dissemination we analyzed the cellularity, architecture, and function of the skin-draining lymph node. There was no difference in the localization of multiple cell populations in the lymph node of polynutrient deficient (PND) mice, but there was reduced cellularity with fewer CD11c+dendritic cells (DCs), fibroblastic reticular cells (FRCs), MOMA-2+ macrophages, and CD169+ subcapsular sinus macrophage (p<0.05 for all) compared to the well-nourished (WN) mice. The parasites were equally co-localized with DCs associated with the lymph node conduit network in the WN and PND mice, and were found in the high endothelial venule into which the conduits drain. When a fluorescent low molecular weight (10 kD) dextran was delivered in the skin, there was greater efflux of the marker from the lymph node conduit system to the spleens of PND mice (p<0.04), indicating that flow through the conduit system was altered. There was no evidence of disruption of the conduit or subcapsular sinus architecture, indicating that the movement of parasites into the subcortical conduit region was due to an active process and not from passive movement through a leaking barrier. These results indicate that the impaired capacity of the lymph node to act as a barrier to dissemination of L. donovani infection is associated with a reduced number of lymph node phagocytes, which most likely leads to reduced capture of parasites as they transit through the sinuses and conduit system.

Background & Aims Regenerating islet-derived protein (REG3γ), an antimicrobial peptide, typically expressed by intestinal epithelial cells (IEC), plays crucial roles in intestinal homeostasis and controlling gut microbiota. However, the mechanisms that regulate IEC expression of REG3γ are still largely unclear. In this study, we investigated whether and how interleukin (IL) 33, an alarmin produced by IEC in response to injury, regulates REG3γ expression in IEC, thus contributing to intestinal homeostasis. Methods IEC were isolated from wild-type and IL33 -/- mice to determine expression of REG3γ and other antimicrobial peptides by quantitative real-time polymerase chain reaction and Western blot. IEC cell lines were used for mechanistic studies. 16S rRNA pyrosequencing analysis was used for measuring gut microbiota. Citrobacter rodentium was used for enteric infections. Results The expression of REG3γ, but not β-defensins, in IECs of IL33 -/- mice was significantly lower than wild-type mice. IL33 treatment induced IEC expression of REG3γ in both mice and human cell lines. Mechanistically, IL33 activated STAT3, mTOR, and ERK1/2 in IEC. Inhibition of these pathways abrogated IL33-induction of REG3γ. IL33 -/- mice demonstrated higher bacteria loads and altered microbiota composition. IL33 did not directly inhibit bacterial growth, but promoted wild-type, not REG3γKO, IECs to kill bacteria in vitro . Consistently, C rodentium infection induced IEC IL33 expression, and IL33 -/- mice demonstrated an impaired bacterial clearance with C rodentium infection. Conclusions Our study demonstrated that IL33, which is produced by IEC in response to injury and inflammatory stimulation, in turn promotes IEC expression of REG3γ, and controls the gut microbiota of the host.

Studies in patients and animal disease models are shedding new light on the critical roles of the microbiota, metabolome and host responses in primary and recurrent CDI. An improved understanding of the CDI disease pathogenesis will provide the basis for developing new therapies for treating disease and preventing recurrence.

e Leishmaniasis is a vector-borne zoonotic infection affecting people in tropical and subtropical regions of the world. Current treatments for cutaneous leishmaniasis are difficult to administer, toxic, expensive, and limited in effectiveness and availability. Here we describe the development and application of a medium-throughput screening approach to identify new drug candidates for cutaneous leishmaniasis using an ex vivo lymph node explant culture (ELEC) derived from the draining lymph nodes of Leishmania major-infected mice. The ELEC supported intracellular amastigote proliferation and contained lymph node cell populations (and their secreted products) that enabled the testing of compounds within a system that mimicked the immunopathological environment of the infected host, which is known to profoundly influence parasite replication, killing, and drug efficacy. The activity of known antileishmanial drugs in the ELEC system was similar to the activity measured in peritoneal macrophages infected in vitro with L. major. Using the ELEC system, we screened a collection of 334 compounds, some of which we had demonstrated previously to be active against L. donovani, and identified 119 hits, 85% of which were confirmed to be active by determination of the 50% effective concentration (EC 50 ). We found 24 compounds (7%) that had an in vitro therapeutic index (IVTI; 50% cytotoxic/effective concentration [CC 50 ]/EC 50 ) > 100; 19 of the compounds had an EC 50 below 1 M. According to PubChem searchs, 17 of those compounds had not previously been reported to be active against Leishmania. We expect that this novel method will help to accelerate discovery of new drug candidates for treatment of cutaneous leishmaniasis.

Abstract. Experimental infection of dogs with Leishmania infantum has yielded heterogeneous clinical, parasitologic, and immunologic results. We studied dogs infected with 10 5 or 10 4 sand fly-derived promastigotes delivered by the intradermal (ID) or intravenous (IV) routes. Total mortality over 1 year post-infection reached 23.8%. The mortality and proportion of sustained polysymptomatic dogs was highest in the IV-10 5 group. The early appearance of polysymptoms was associated with an increased risk of progression to death. Dissemination of the parasite to lymph nodes was faster, and the subsequent infectivity to sand flies higher, in the IV compared with ID-infected dogs. Parasite-specific IgG1 or IgG2 production was similar among the groups, but higher interferon-γ (IFN-γ) and interleukin-10 (IL-10) expression was associated with polysymptomatic dogs. On the basis of the data obtained from this study, a sample size analysis using different endpoints for future vaccine trials is described.