Genetic analysis of basophil function in vivo.

Contributions by basophils to allergic and helminth immunity remain incompletely defined. Using sensitive interleukin 4 (Il4) reporter alleles, we demonstrate here that basophil IL-4 production occurs by a CD4(+) T cell-dependent process restricted to the peripheral tissues affected. We genetically marked and achieved specific deletion of basophils and found that basophils did not mediate T helper type 2 (T(H)2) priming in vivo. Two-photon imaging confirmed that basophils did not interact with antigen-specific T cells in lymph nodes but engaged in prolonged serial interactions with T cells in lung tissues. Although targeted deletion of IL-4 and IL-13 in either CD4(+) T cells or basophils had a minimal effect on worm clearance, deletion from both lineages demonstrated a nonredundant role for basophil cytokines in primary helminth immunity.

Human schistosomiasis in Nigeria: present status, diagnosis, chemotherapy, and herbal medicines.

Schistosomiasis is a neglected tropical disease caused by a parasitic, trematode blood fluke of the genus Schistosoma. With 20 million people infected, mostly due to Schistosoma haematobium, Nigeria has the highest burden of schistosomiasis in the world. We review the status of human schistosomiasis in Nigeria regarding its distribution, prevalence, diagnosis, prevention, orthodox and traditional treatments, as well as snail control strategies. Of the country's 36 states, the highest disease prevalence is found in Lagos State, but at a geo-political zonal level, the northwest is the most endemic. The predominantly used diagnostic techniques are based on microscopy. Other methods such as antibody-based serological assays and DNA detection methods are rarely employed. Possible biomarkers of disease have been identified in fecal and blood samples from patients. With respect to preventive chemotherapy, mass drug administration with praziquantel as well as individual studies with artemisinin or albendazole have been reported in 11 out of the 36 states with cure rates between 51.1 and 100%. Also, Nigerian medicinal plants have been traditionally used as anti-schistosomal agents or molluscicides, of which Tetrapleura tetraptera (Oshosho, aridan, Aidan fruit), Carica papaya (Gwanda, Ìbépe, Pawpaw), Borreria verticillata (Karya garma, Irawo-ile, African borreria), and Calliandra portoricensis (Tude, Oga, corpse awakener) are most common in the scientific literature. We conclude that the high endemicity of the disease in Nigeria is associated with the limited application of various diagnostic tools and preventive chemotherapy efforts as well as poor knowledge, attitudes, and practices (KAP). Nonetheless, the country could serve as a scientific base in the discovery of biomarkers, as well as novel plant-derived schistosomicides and molluscicides.

Structure-Based Identification of Naphthoquinones and Derivatives as Novel Inhibitors of Main Protease Mpro and Papain-like Protease PLpro of SARS-CoV-2.

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In this study, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 µM and 9.0 µM. In addition, three compounds inhibited PLpro with IC50 ranging from 1.9 µM to 3.3 µM. To verify the specificity of Mpro and PLpro inhibitors, our experiments included an assessment of common causes of false positives such as aggregation, high compound fluorescence, and inhibition by enzyme oxidation. Altogether, we confirmed novel classes of specific Mpro and PLpro inhibitors. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

Computer-aided design of 1,4-naphthoquinone-based inhibitors targeting cruzain and rhodesain cysteine proteases.

Chagas disease and Human African Trypanosomiasis (HAT) are caused by Trypanosoma cruzi and T. brucei parasites, respectively. Cruzain (CRZ) and Rhodesain (RhD) are cysteine proteases that share 70% of identity and play vital functions in these parasites. These macromolecules represent promising targets for designing new inhibitors. In this context, 26 CRZ and 5 RhD 3D-structures were evaluated by molecular redocking to identify the most accurate one to be utilized as a target. Posteriorly, a virtual screening of a library containing 120 small natural and nature-based compounds was performed on both of them. In total, 14 naphthoquinone-based analogs were identified, synthesized, and biologically evaluated. In total, five compounds were active against RhD, being three of them also active on CRZ. A derivative of 1,4-naphthoquinonepyridin-2-ylsulfonamide was found to be the most active molecule, exhibiting IC50 values of 6.3 and 1.8 µM for CRZ and RhD, respectively. Dynamic simulations at 100 ns demonstrated good stability and do not alter the targets' structures. MM-PBSA calculations revealed that it presents a higher affinity for RhD (-25.3 Kcal mol-1) than CRZ, in which van der Waals interactions were more relevant. A mechanistic hypothesis (via C3-Michael-addition reaction) involving a covalent mode of inhibition for this compound towards RhD was investigated by covalent molecular docking and DFT B3LYP/6-31 + G* calculations, exhibiting a low activation energy (ΔG‡) and providing a stable product (ΔG), with values of 7.78 and - 39.72 Kcal mol-1, respectively; similar to data found in the literature. Nevertheless, a reversibility assay by dilution revealed that JN-11 is a time-dependent and reversible inhibitor. Finally, this study applies modern computer-aided techniques to identify promising inhibitors from a well-known chemical class of natural products. Then, this work could inspire other future studies in the field, being useful for designing potent naphthoquinones as RhD inhibitors.

The Antifungal Drug Isavuconazole Is both Amebicidal and Cysticidal against Acanthamoeba castellanii.

Current treatments for Acanthamoeba keratitis rely on a combination of chlorhexidine gluconate, propamidine isethionate, and polyhexamethylene biguanide. These disinfectants are nonspecific and inherently toxic, which limits their effectiveness. Furthermore, in 10% of cases, recurrent infection ensues due to the difficulty in killing both trophozoites and double-walled cysts. Therefore, development of efficient, safe, and target-specific drugs which are capable of preventing recurrent Acanthamoeba infection is a critical unmet need for averting blindness. Since both trophozoites and cysts contain specific sets of membrane sterols, we hypothesized that antifungal drugs targeting sterol 14-demethylase (CYP51), known as conazoles, would have deleterious effects on A. castellanii trophozoites and cysts. To test this hypothesis, we first performed a systematic screen of the FDA-approved conazoles against A. castellanii trophozoites using a bioluminescence-based viability assay adapted and optimized for Acanthamoeba The most potent drugs were then evaluated against cysts. Isavuconazole and posaconazole demonstrated low nanomolar potency against trophozoites of three clinical strains of A. castellanii Furthermore, isavuconazole killed trophozoites within 24 h and suppressed excystment of preformed Acanthamoeba cysts into trophozoites. The rapid action of isavuconazole was also evident from the morphological changes at nanomolar drug concentrations causing rounding of trophozoites within 24 h of exposure. Given that isavuconazole has an excellent safety profile, is well tolerated in humans, and blocks A. castellanii excystation, this opens an opportunity for the cost-effective repurposing of isavuconazole for the treatment of primary and recurring Acanthamoeba keratitis.

Herbal melanin inhibits colorectal cancer cell proliferation by altering redox balance, inducing apoptosis, and modulating MAPK signaling.

BACKGROUND: Colorectal carcinoma is one of the most deadly cancers that requests effective and safe chemotherapy. Evaluation of natural product-based anticancer drugs as adjuvant treatment with fewer side effects is largely unexplored research fields. Herbal melanin (HM) is an extract of the seed coats of Nigella sativa that modulates an inflammatory response through toll-like receptor 4 (TLR4). This TLR4 receptor is also involved in the modulation of apoptosis. We therefore explored the anticancer potential of HM and specifically its effect on the molecular mechanisms underlying adenocarcinoma and metastatic colorectal cancer (mCRC) cell death in vitro. METHODS: Cell viability was evaluated using the MTT assay. Cellular reactive oxygen species (ROS), glutathione levels, and apoptotic status were assessed using fluorometric and colorimetric detection methods. HM-induced apoptotic and other signaling pathways were investigated using Western blot technology and mitochondrial transition pore assay kit. TLR4 receptor downregulation and blockade were performed using siRNA technology and neutralizing antibody, respectively. RESULTS: Our results showed that HM inhibited the proliferation of the colorectal adenocarcinoma HT29 and mCRC SW620 cell lines. Furthermore, HM enhanced ROS production and decreased glutathione levels. HM-induced apoptosis was associated with mitochondrial outer membrane permeability and cytochrome c release, inhibition of the Bcl2 family proteins, and activation of caspase-3/-7. In addition, HM modulated MAPK pathways by activating the JNK pathway and by inhibiting ERK phosphorylation. TLR4 receptor downregulation enhanced HM-induced apoptosis while TLR4 receptor blockade partially alleviated HM-inhibited ERK phosphorylation. CONCLUSION: Altogether, these findings indicate that HM exerts pro-apoptotic effects and inhibits MAPK pathway through TLR4 in mCRC and colorectal adenocarcinoma cells, suggesting HM as a promising natural-based drug for the treatment of colorectal cancer.

Why Funding for Neglected Tropical Diseases Should Be a Global Priority.

Neglected tropical diseases affect >1 billion of the world's poorest persons. Control programs range from near-elimination (dracunculiasis) to increasing prevalence (dengue and cutaneous leishmaniasis). These are some of the most cost-effective public health interventions and should be a global priority.

Identification of cysteine protease inhibitors as new drug leads against Naegleria fowleri.

Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. PAM occurs principally in healthy children of less than 13 years old with a history of recent exposure to warm fresh water. While as yet not a reportable disease, the Centers for Disease Control and Prevention (CDC) documents a total of 143 cases in the United States. Only four patients have survived. Infection results from water containing N. fowleri entering the nose, followed by migration of the amebae to the brain. Within the brain, N. fowleri infection results in extensive necrosis, leading to death in 3-7 days. Mortality among patients with PAM is greater than 95%. The drugs of choice in treating PAM are the antifungal amphotericin B, and the antileishmanial, miltefosine. However neither drug is FDA-approved for this indication and the use of amphotericin B is associated with severe adverse effects. Moreover, very few patients treated with amphotericin B have survived PAM. Therefore, development of new, safe and effective drugs is a critical unmet need to avert future deaths of children. The molecular mechanisms underlying the pathogenesis of PAM are poorly understood but it is known that cysteine proteases of N. fowleri play a role in the progression of PAM. We therefore assessed the in vitro activity of the synthetic vinyl sulfone cysteine protease inhibitor, K11777, and 33 analogs with valine, phenylalanine or pyridylalanine at P2 position, against cysteine protease activity in the lysate of N. fowleri. Inhibitors with phenylalanine or pyridylalanine at P2 position were particularly effective in inhibiting the cysteine protease activity of N. fowleri cell lysate with IC50 ranging between 3 nM and 6.6 µM. Three of the 34 inhibitors also showed inhibitory activity against N. fowleri in a cell viability assay and were 1.6- to 2.5-fold more potent than the standard of care drug miltefosine. Our study provides the first evidence of the activity of synthetic, small molecule cysteine protease inhibitors against N. fowleri.

Impact of Single Dose Praziquantel Treatment on Schistosoma haematobium Infection among School Children in an Endemic Nigerian Community.

Schistosomiasis is prevalent in Nigeria, and the foremost pathogen is Schistosoma haematobium, which affects about 29 million people. Single dose of the drug praziquantel is often recommended for treatment but the efficacy has not been documented in certain regions. Therefore, this study was designed to assess the impact of single dose praziquantel treatment on S. haematobium infection among school children in an endemic community of South-Western Nigeria. Urine samples were collected from 434 school children and 10 ml was filtered through Nucleopore filter paper before examination for egg outputs by microscopy. The prevalence was 24.9% at pre-treatment. There was no statistically significant difference for the prevalence of infection between males (14.7%) and females (10.2%), although the mean egg count for the females (9.87) was significantly more (P < 0.05) than the males (6.06). At 6 and 12 months post-treatment there was 74.4% and 86.4% reduction in the mean egg count, respectively. Interestingly, an increased prevalence of infection from 2.1% at 6 months to 7.7% at 12 months post-treatment was observed, nonetheless the mean egg count was reduced to 0.27 at 12th month from 1.98 at 6 months post-treatment. Resurgence in the prevalence rate between 6 and 12 months post-treatment with praziquantel is herein reported and the need for a follow-up treatment in endemic areas for adequate impact on schistosomiasis control is discussed.

Phase I Clinical Trial Results of Auranofin, a Novel Antiparasitic Agent.

Under an NIH priority to identify new drugs to treat class B parasitic agents, we performed high-throughput screens, which identified the activity of auranofin (Ridaura) against Entamoeba histolytica and Giardia intestinalis, major causes of water- and foodborne outbreaks. Auranofin, an orally administered, gold (Au)-containing compound that was approved by the FDA in 1985 for treatment of rheumatoid arthritis, was effective in vitro and in vivo against E. histolytica and both metronidazole-sensitive and -resistant strains of Giardia We now report the results of an NIH-sponsored phase I trial to characterize the pharmacokinetics (PK) and safety of auranofin in healthy volunteers using modern techniques to measure gold levels. Subjects received orally 6 mg (p.o.) of auranofin daily, the recommended dose for rheumatoid arthritis, for 7 days and were followed for 126 days. Treatment-associated adverse events were reported by 47% of the subjects, but all were mild and resolved without treatment. The mean gold maximum concentration in plasma (Cmax) at day 7 was 0.312 µg/ml and the half-life (t1/2) 35 days, so steady-state blood levels would not be reached in short-term therapy. The highest concentration of gold, 13 µM (auranofin equivalent), or more than 25× the 50% inhibitory concentration (IC50) for E. histolytica and 4× that for Giardia, was in feces at 7 days. Modeling of higher doses (9 and 21 mg/day) was performed for systemic parasitic infections, and plasma gold levels of 0.4 to 1.0 µg/ml were reached after 14 days of treatment at 21 mg/day. This phase I trial supports the idea of the safety of auranofin and provides important PK data to support its potential use as a broad-spectrum antiparasitic drug. (This study has been registered at ClinicalTrials.gov under identifier NCT02089048.).

Mass Spectrometry-Based Chemical Cartography of a Cardiac Parasitic Infection.

Trypanosoma cruzi parasites are the causative agents of Chagas disease, a leading infectious form of heart failure whose pathogenesis is still not fully characterized. In this work, we applied untargeted liquid chromatography-tandem mass spectrometry to heart sections from T. cruzi-infected and uninfected mice. We combined molecular networking and three-dimensional modeling to generate chemical cartographical heart models. This approach revealed for the first time preferential parasite localization to the base of the heart and regiospecific distributions of nucleoside derivatives and eicosanoids, which we correlated to tissue-damaging immune responses. We further detected novel cardiac chemical signatures related to the severity and ultimate outcome of the infection. These signatures included differential representation of higher- vs lower-molecular-weight carnitine and phosphatidylcholine family members in specific cardiac regions of mice infected with lethal or nonlethal T. cruzi strains and doses. Overall, this work provides new insights into Chagas disease pathogenesis and presents an analytical chemistry approach that can be broadly applied to the study of host-microbe interactions.

Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease.

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

X-ray structures of thioredoxin and thioredoxin reductase from Entamoeba histolytica and prevailing hypothesis of the mechanism of Auranofin action.

The anti-arthritic gold-containing drug Auranofin is lethal to the protozoan intestinal parasite Entamoeba histolytica, the causative agent of human amebiasis, in both culture and animal models of the disease. A putative mechanism of Auranofin action proposes that monovalent gold, Au(I), released from the drug, can bind to the redox-active dithiol group of thioredoxin reductase (TrxR). Au(I) binding in the active site is expected to prevent electron transfer to the downstream substrate thioredoxin (Trx), thus interfering with redox homeostasis in the parasite. To clarify the molecular mechanism of Auranofin action in more detail, we determined a series of atomic resolution X-ray structures for E. histolytica thioredoxin (EhTrx) and thioredoxin reductase (EhTrxR), the latter with and without Auranofin. Only the disulfide-bonded form of the active site dithiol (Cys(140)-Cys(143)) was invariably observed in crystals of EhTrxR in spite of the addition of reductants in various crystallization trials, and no gold was found associated with these cysteines. Non-catalytic Cys(286) was identified as the only site of modification, but further mutagenesis studies using the C286Q mutant demonstrated that this site was not responsible for inhibition of EhTrxR by Auranofin. Interestingly, we obtained both of the catalytically-relevant conformations of this bacterial-like, low molecular weight TrxR in crystals without requiring an engineered disulfide linkage between Cys mutants of TrxR and Trx (as was originally done with Escherichia coli TrxR and Trx). We note that the -CXXC- catalytic motif, even if reduced, would likely not provide space sufficient to bind Au(I) by both cysteines of the dithiol group.

Polymorphisms of tumor necrosis factor alpha in Middle Eastern population with colorectal cancer.

Tumor necrosis factor-alpha (TNF-α) contributes in inflammation and has been implicated in the development of colorectal cancer (CRC). Single nucleotide polymorphisms (SNPs) in TNF-α promoter could affect the risk of CRC by regulating TNF-α production. This is the first study to investigate TNF-α SNPs in a Middle Eastern population. In this study, we examined three SNPs in TNF-α for association with CRC. One hundred CRC patients and 100 controls were genotyped for TNF-α -308, -238, and -857 using TaqMan allelic discrimination assay. The TNF-α -238 (G/A) genotype was significantly associated with high risk of CRC (p = 0.003552). The distribution of three genotypes of -238 G/A was significantly different between the controls and CRC patients even after Bonferroni's correction. The AA genotype of -238 G/A SNP was observed at considerably higher proportion (13 %) in CRCs compared to controls (1 %). Additionally, similar to genotypes, the allelic frequencies of -238 G/A were significantly different between the CRC cases and controls (odds ratios (OR) = 7.647, χ (2) = 18.50, p = 0.00002). The genotype frequencies of -308 and -857 were not notably different between the cases and controls. TNF-α -238A may be useful as a screening marker to identify individuals prior to their acquiring CRC in the Saudi population although, further validations in larger cohorts are needed.

Recognition of the role of Natural Products as drugs to treat neglected tropical diseases by the 2015 Nobel prize in physiology or medicine.

The 2015 Nobel Prize in Physiology or Medicine recognised the advances made in treating neglected tropical diseases, using drugs whose origins lie in natural products.

Synthesis of a sugar-based thiosemicarbazone series and structure-activity relationship versus the parasite cysteine proteases rhodesain, cruzain, and Schistosoma mansoni cathepsin B1.

The pressing need for better drugs against Chagas disease, African sleeping sickness, and schistosomiasis motivates the search for inhibitors of cruzain, rhodesain, and Schistosoma mansoni CB1 (SmCB1), the major cysteine proteases from Trypanosoma cruzi, Trypanosoma brucei, and S. mansoni, respectively. Thiosemicarbazones and heterocyclic analogues have been shown to be both antitrypanocidal and inhibitory against parasite cysteine proteases. A series of compounds was synthesized and evaluated against cruzain, rhodesain, and SmCB1 through biochemical assays to determine their potency and structure-activity relationships (SAR). This approach led to the discovery of 6 rhodesain, 4 cruzain, and 5 SmCB1 inhibitors with 50% inhibitory concentrations (IC50s) of ≤ 10 µM. Among the compounds tested, the thiosemicarbazone derivative of peracetylated galactoside (compound 4i) was discovered to be a potent rhodesain inhibitor (IC50 = 1.2 ± 1.0 µM). The impact of a range of modifications was determined; removal of thiosemicarbazone or its replacement by semicarbazone resulted in virtually inactive compounds, and modifications in the sugar also diminished potency. Compounds were also evaluated in vitro against the parasites T. cruzi, T. brucei, and S. mansoni, revealing active compounds among this series.

Global identification of peptidase specificity by multiplex substrate profiling.

We developed a simple and rapid multiplex substrate-profiling method to reveal the substrate specificity of any endo- or exopeptidase using liquid chromatography-tandem mass spectrometry sequencing. We generated a physicochemically diverse library of peptides by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated information on prime and nonprime sites as well as on substrate specificity that matched or expanded upon known substrate motifs. This method biochemically confirmed the activity of the klassevirus 3C protein responsible for polypeptide processing and allowed granzyme B substrates to be ranked by enzymatic turnover efficiency using label-free quantitation of precursor-ion abundance. Additionally, the proteolytic secretions from schistosome parasitic flatworm larvae and a pancreatic cancer cell line were deconvoluted in a subtractive strategy using class-specific peptidase inhibitors.

In vitro efficacy of corifungin against Acanthamoeba castellanii trophozoites and cysts.

Painful blinding keratitis and fatal granulomatous amebic encephalitis are caused by the free-living amebae Acanthamoeba spp. Several prescription eye medications are used to treat Acanthamoeba keratitis, but the infection can be difficult to control because of recurrence of infection. For the treatment of encephalitis, no single drug was found useful, and in spite of the use of a combination of multiple drugs, the mortality rate remains high. Therefore, efficient, novel drugs are urgently needed for the treatment of amebic keratitis and granulomatous amebic encephalitis. In this study, we identified corifungin, a water-soluble polyene macrolide, as amebicidal. In vitro, it was effective against both the trophozoites and the cysts. Transmission electron microscopy of Acanthamoeba castellanii incubated with corifungin showed the presence of swollen mitochondria, electron-dense granules, degeneration of cytoplasm architecture, and loss of nuclear chromatin structure. These changes were followed by lysis of amebae. Corifungin also induced the encystment process of A. castellanii. There were alterations in the cyst cell wall followed by lysis of the cysts. Corifungin is a promising therapeutic option for keratitis and granulomatous amebic encephalitis.

Hsp90 inhibitors as new leads to target parasitic diarrheal diseases.

Entamoeba histolytica and Giardia lamblia are anaerobic protozoan parasites that cause amebiasis and giardiasis, two of the most common diarrheal diseases worldwide. Current therapy relies on metronidazole, but resistance has been reported and the drug has significant adverse effects. Therefore, it is critical to search for effective, better-tolerated antiamebic and antigiardial drugs. We synthesized several examples of a recently reported class of Hsp90 inhibitors and evaluated these compounds as potential leads for antiparasitic chemotherapy. Several of these inhibitors showed strong in vitro activity against both E. histolytica and G. lamblia trophozoites. The inhibitors were rescreened to discriminate between amebicidal and giardicidal activity and general cytotoxicity toward a mammalian cell line. No mammalian cytotoxicity was found at >100 µM for 48 h for any of the inhibitors. To understand the mechanism of action, a competitive binding assay was performed using the fluorescent ATP analogue bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt) and recombinant E. histolytica Hsp90 preincubated in both the presence and absence of Hsp90 inhibitors. There was significant reduction in fluorescence compared to the level in the control, suggesting that E. histolytica Hsp90 is a selective target. The in vivo efficacy and safety of one Hsp90 inhibitor in a mouse model of amebic colitis and giardiasis was demonstrated by significant inhibition of parasite growth at a single oral dose of 5 mg/kg of body weight/day for 7 days and 10 mg/kg/day for 3 days. Considering the results for in vitro activity and in vivo efficacy, Hsp90 inhibitors represent a promising therapeutic option for amebiasis and giardiasis.

Upregulation of retinal dehydrogenase 2 in alternatively activated macrophages during retinoid-dependent type-2 immunity to helminth infection in mice.

Although the vitamin A metabolite retinoic acid (RA) plays a critical role in immune function, RA synthesis during infection is poorly understood. Here, we show that retinal dehydrogenases (Raldh), required for the synthesis of RA, are induced during a retinoid-dependent type-2 immune response elicited by Schistosoma mansoni infection, but not during a retinoid-independent anti-viral immune response. Vitamin A deficient mice have a selective defect in T(H)2 responses to S. mansoni, but retained normal LCMV specific T(H)1 responses. A combination of in situ imaging, intra-vital imaging, and sort purification revealed that alternatively activated macrophages (AAMφ) express high levels of Raldh2 during S. mansoni infection. IL-4 induces Raldh2 expression in bone marrow-derived macrophages in vitro and peritoneal macrophages in vivo. Finally, in vivo derived AAMφ have an enhanced capacity to induce Foxp3 expression in CD4+ cells through an RA dependent mechanism, especially in combination with TGF-ß. The regulation of Raldh enzymes during infection is pathogen specific and reflects differential requirements for RA during effector responses. Specifically, AAMφ are an inducible source of RA synthesis during helminth infections and T(H)2 responses that may be important in regulating immune responses.