Alex G. Peniche

Pregnancy enhances the innate immune response in experimental cutaneous leishmaniasis through hormone-modulated nitric oxide production

The maintenance of host defense during pregnancy may depend on heightened innate immunity. We evaluated the immune response of pregnant hamsters during early infection with Leishmania (Viannia) panamensis, a cause of American cutaneous leishmaniasis. At 7 days post‐infection, pregnant animals showed a lower parasite burden compared with nonpregnant controls at the cutaneous infection site (P=0.0098) and draining lymph node (P=0.02). Resident peritoneal macrophages and neutrophils from pregnant animals had enhanced Leishmania killing capacity compared with nonpregnant controls (P=0.018 each). This enhanced resistance during pregnancy was associated with increased expression of inducible NO synthase (iNOS) mRNA in lymph node cells (P=0.02) and higher NO production by neutrophils (P=0.0001). Macrophages from nonpregnant hamsters infected with L. panamensis released high amounts of NO upon estrogen exposure (P=0.05), and addition of the iNOS inhibitor L‐N6‐(1‐iminoethyl) lysine blocked the induction of NO production (P=0.02). Infected, nonpregnant females treated with estrogen showed a higher percentage of cells producing NO at the infection site than controls (P=0.001), which correlated with lower parasite burdens (P=0.036). Cultured macrophages or neutrophils from estrogen‐treated hamsters showed significantly increased NO production and Leishmania killing compared with untreated controls. iNOS was identified as the likely source of estrogen‐induced NO in primed and naïve macrophages, as increased transcription was evident by real‐time PCR. Thus, the innate defense against Leishmania infection is heightened during pregnancy, at least in part as a result of estrogen‐mediated up‐regulation of iNOS expression and NO production.

Recent insights into Clostridium difficile pathogenesis.

Purpose of review Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis in the healthcare setting. An emerging consensus suggests that CDI is caused by pathogenic toxin production, gut microbial dysbiosis and altered host inflammatory responses. The aim of this review is to summarize and highlight recent advances focused on CDI pathogenic mechanisms. Recent findings Potential paradigm shifts relating to the mechanisms of toxin action and inhibition have recently been reported, with new insights into spore germination and surface protein function also gaining traction. Multiomic analysis of microbiome dysbiosis has identified important CDI-associated microbial community shifts that may form the basis of future targeted bacteriotherapy, and functional metabolite biomarkers that require further characterization. Classical innate and adaptive immunity against CDI is rapidly being delineated, with novel innate S-nitrosylation signals also being identified. Summary 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.

Clinical, Parasitologic, and Immunologic Evolution in Dogs Experimentally Infected with Sand Fly-Derived Leishmania chagasi Promastigotes

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-gamma (IFN-gamma) 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.

Congenital Transmission of Experimental Leishmaniasis in a Hamster Model

Little information is available on transplacental transmission of Leishmania spp. We determined the frequency and impact of congenital infection caused by Leishmania panamensis or L. donovani in experimentally infected hamsters. A polymerase chain reaction showed that congenital transmission occurred in 25.8% (24 of 93) of offspring born to L. panamensis-infected hamsters and 14.6% (11 of 75) offspring born to L. donovani-infected hamsters. Mortality during lactation was higher in offspring born to L. panamensis-infected hamsters and offspring born to L. donovani-infected hamsters than controls, and lymphoproliferation to Leishmania was more frequent in offspring born to L. panamensis-infected hamsters (17.4%, 11 of 63) than in offspring born to L. donovani-infected hamsters (8.5%, 3 of 35). After weaning, only offspring born to L. donovani-infected hamsters had lower weight gain (P < 0.001) and hematocrit levels (P = 0.0045) than controls. Challenge of offspring born to L. panamensis-infected hamsters with L. panamensis showed no differences in lesion evolution, and offspring born to L. donovani-infected hamsters were more susceptible to L. donovani challenge than controls. Consequently, prenatal exposure of hamsters to L. donovani significantly increased the mortality risk and susceptibility to secondary homologous infection.

The malnutrition-related increase in early visceralization of Leishmania donovani is associated with a reduced number of lymph node phagocytes and altered conduit system flow.

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.

New Role for FDA-Approved Drugs in Combating Antibiotic-Resistant Bacteria

ABSTRACT 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.

Peptide nanofiber–CaCO3 composite microparticles as adjuvant-free oral vaccine delivery vehicles

To combat mucosal pathogens that cause gastrointestinal (GI) infections, local mucosal immunity is required which is best achieved through oral vaccination. Oral delivery of vaccines is also a safe and convenient alternative to injected vaccines due to its non-invasive nature and high compliance rate for all ages. However, the lack of effective and safe mucosal adjuvants, the selective permeability of the mucus barrier, and the harsh GI environment continue to pose a significant challenge for oral vaccine development. Microparticle-based strategies are attractive for oral vaccination due to their ability to efficiently penetrate the mucus barrier and have the added advantage of protecting the antigen in the harsh gastric environment. In this work, self-adjuvanting peptide nanofiber-CaCO3 composite microparticles were prepared and investigated for oral vaccine delivery. Compared to polymeric microparticles, inorganic CaCO3 microparticles have unique advantages due to the biocompatibility of CaCO3 as a natural mineral, mild preparation conditions, and its porous structure that is suitable for loading other materials. Particle size distribution, nanofiber loading efficiency, morphology, and degradation in simulated gastric fluid were characterized. The composite microparticles were efficient at penetrating the mucus barrier and were localized to immune inductive sites and elicited the production of mucosal antibody responses, particularly the protective IgA isotype following oral administration. The magnitude of the mucosal immune response was comparable to the gold-standard adjuvant cholera toxin B (CTB). Our results indicate that OVA-KFE8/CaCO3 composite microparticles are efficient self-adjuvanting oral vaccine delivery vehicles for induction of mucosal antibody responses.

Development of an Ex Vivo Lymph Node Explant Model for Identification of Novel Molecules Active against Leishmania major

ABSTRACT 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 (EC50). We found 24 compounds (7%) that had an in vitro therapeutic index (IVTI; 50% cytotoxic/effective concentration [CC50]/EC50) > 100; 19 of the compounds had an EC50 below 1 μM. 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.

Next-generation probiotics targeting Clostridium difficile through precursor-directed antimicrobial biosynthesis

ABSTRACT Integration of antibiotic and probiotic therapy has the potential to lessen the public health burden of antimicrobial-associated diseases. Clostridium difficile 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 Lactobacillus reuteri to be a promising candidate for adjunct therapy. Human-derived L. reuteri bacteria convert glycerol to the broad-spectrum antimicrobial compound reuterin. When supplemented with glycerol, strains carrying the pocR gene locus were potent reuterin producers, with L. reuteri 17938 inhibiting C. difficile growth at a level on par with the level of growth inhibition by vancomycin. Targeted pocR mutations and complementation studies identified reuterin to be the precursor-induced antimicrobial agent. Pathophysiological relevance was demonstrated when the codelivery of L. reuteri with glycerol was effective against C. difficile colonization in complex human fecal microbial communities, whereas treatment with either glycerol or L. reuteri alone was ineffective. A global unbiased microbiome and metabolomics analysis independently confirmed that glycerol precursor delivery with L. reuteri elicited changes in the composition and function of the human microbial community that preferentially targets C. difficile 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 C. difficile, 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.

Antileishmanial Activity of Disulfiram and Thiuram Disulfide Analogs in an Ex Vivo Model System Is Selectively Enhanced by the Addition of Divalent Metal Ions

ABSTRACT Current treatments for cutaneous and visceral leishmaniasis are toxic, expensive, difficult to administer, and limited in efficacy and availability. Disulfiram has primarily been used to treat alcoholism. More recently, it has shown some efficacy as therapy against protozoan pathogens and certain cancers, suggesting a wide range of biological activities. We used an ex vivo system to screen several thiuram disulfide compounds for antileishmanial activity. We found five compounds (compound identifier [CID] 7188, 5455, 95876, 12892, and 3117 [disulfiram]) with anti-Leishmania activity at nanomolar concentrations. We further evaluated these compounds with the addition of divalent metal salts based on studies that indicated these salts could potentiate the action of disulfiram. In addition, clinical studies suggested that zinc has some efficacy in treating cutaneous leishmaniasis. Several divalent metal salts were evaluated at 1 μM, which is lower than the normal levels of copper and zinc in plasma of healthy individuals. The leishmanicidal activity of disulfiram and CID 7188 were enhanced by several divalent metal salts at 1 μM. The in vitro therapeutic index (IVTI) of disulfiram and CID 7188 increased 12- and 2.3-fold, respectively, against L. major when combined with ZnCl2. The combination of disulfiram with ZnSO4 resulted in a 1.8-fold increase in IVTI against L. donovani. This novel combination of thiuram disulfides and divalent metal ions salts could have application as topical and/or oral therapies for treatment of cutaneous and visceral leishmaniasis.