Adverse pregnancy outcomes in Toxoplasma gondii seropositive Hispanic women.

AIMS: Chronic Toxoplasma gondii infection is not thought to affect pregnancy or birth outcomes, but there are few prospective studies. The study aims were T. gondii immunoglobulin G measurement and relationship of chronic T. gondii infection with gestational age at birth and adverse pregnancy outcomes in 690 Hispanic women in Tampa, Florida. METHODS: Hispanic women, born either in the United States or in Latin America or the Caribbean had a venous blood sample drawn to measure T. gondii IgG and T. gondii serotype at the first prenatal visit, along with collection of demographic and health-related measures. Seropositive and seronegative women were followed throughout their pregnancy. Gestational age, infant weights, and adverse pregnancy outcomes (miscarriages, preterm births) were compared in the two groups. RESULTS: There were 740 women of self-reported Hispanic ethnicity screened and enrolled in Tampa, Florida, with 690 having birth data extracted from the electronic health record (538 T. gondii negative and 152 T. gondii seropositive). T. gondii seropositivity was 22.4% and the majority (83%) had high avidity titers, indicating chronic infection. Compared to T. gondii seronegative Hispanic women, seroseropositive women had more smaller for gestational age infants and higher prevalences of miscarriages and preterm birth. CONCLUSION: This is one of the largest longitudinal cohort studies of women with chronic T. gondii infection followed through pregnancy. There was a higher percentages of adverse pregnancy outcomes in this group compared to T. gondii seronegative controls. The mechanism for this is unknown and warrants reexamination of the dogma that chronic T. gondii infection in pregnant women has no significant clinical consequences.

Miniaturized Cultivation of Microbiota for Antimalarial Drug Discovery.

The ongoing search for effective antiplasmodial agents remains essential in the fight against malaria worldwide. Emerging parasitic drug resistance places an urgent need to explore chemotherapies with novel structures and mechanisms of action. Natural products have historically provided effective antimalarial drug scaffolds. In an effort to search nature's chemical potential for antiplasmodial agents, unconventionally sourced organisms coupled with innovative cultivation techniques were utilized. Approximately 60,000 niche microbes from various habitats (slow-growing terrestrial fungi, Antarctic microbes, and mangrove endophytes) were cultivated on a small-scale, extracted, and used in high-throughput screening to determine antimalarial activity. About 1% of crude extracts were considered active and 6% partially active (≥ 67% inhibition at 5 and 50 µg/mL, respectively). Active extracts (685) were cultivated on a large-scale, fractionated, and screened for both antimalarial activity and cytotoxicity. High interest fractions (397) with an IC50 < 1.11 µg/mL were identified and subjected to chromatographic separation for compound characterization and dereplication. Identifying active compounds with nanomolar antimalarial activity coupled with a selectivity index tenfold higher was accomplished with two of the 52 compounds isolated. This microscale, high-throughput screening project for antiplasmodial agents is discussed in the context of current natural product drug discovery efforts.

Bastimolide A, a Potent Antimalarial Polyhydroxy Macrolide from the Marine Cyanobacterium Okeania hirsuta.

Bastimolide A (1), a polyhydroxy macrolide with a 40-membered ring, was isolated from a new genus of the tropical marine cyanobacterium Okeania hirsuta. This novel macrolide was defined by spectroscopy and chemical reactions to possess one 1,3-diol, one 1,3,5-triol, six 1,5-diols, and one tert-butyl group; however, the relationships of these moieties to one another were obscured by a highly degenerate (1)H NMR spectrum. Its complete structure and absolute configuration were therefore unambiguously determined by X-ray diffraction analysis of the nona-p-nitrobenzoate derivative (1d). Pure bastimolide A (1) showed potent antimalarial activity against four resistant strains of Plasmodium falciparum with IC50 values between 80 and 270 nM, although with some toxicity to the control Vero cells (IC50 = 2.1 µM), and thus represents a potentially promising lead for antimalarial drug discovery. Moreover, rigorous establishment of its molecular arrangement gives fresh insight into the structures and biosynthesis of cyanobacterial polyhydroxymacrolides.

Depression in pregnant Hispanic women: Risk factors, pregnancy outcomes and plasma cytokines.

Background: Maternal depression is considered a major contributor to morbidity and mortality in pregnancy. A population at risk are U.S. born or immigrant Hispanic women, and few prenatal depression or immune studies have focused on this population. Objective: The research questions for the study were 1) What are the occurrences, risk factors and outcomes associated with depression in Hispanic pregnant women in the United States and 2) What are the associations of plasma immune cytokines and prenatal depression in this population. Study design: Women of self-reported Hispanic ethnicity were born in the continental United States or foreign-born. Screening of potential participants (n = 690) at a first prenatal clinic visit consisted of antibody testing for Toxoplasma gondii antibodies in a larger grant, and only the women with antibody levels below the cutoff for T. gondii positivity (N = 536) were included in the present study. All participants completed a health and demographic questionnaire, the Edinburgh Postpartum Depression (EPDS) scale, the Perceived Stress Scale (PSS), and the Medical Outcomes Study Social Support (MOS) scale. We surveyed electronic health records (EHR) for risk factors and adverse pregnancy outcomes in the sample. We further measured physical and mental health and seven plasma immune cytokines at four study visits during pregnancy in a longitudinal subsample (N = 128). Results: The frequency of EPDS scores of 10 (depression risk) or above was 18.6 % at the time of enrollment. Socioeconomic factors such as less education, greater unemployment, and U.S. born nativity were associated with greater depression risk, but these relationships became insignificant when we corrected for false discovery rate. Depression scores were not associated with adverse birth and pregnancy outcomes. The inflammatory cytokine TNF-α was significantly higher across pregnancy in women with depression risk (p < 0.03). Other inflammatory cytokines were higher in depressed women, but only at one time point in mid-pregnancy. Conclusions: Prenatal depression occurs in early pregnancy and then declines in Hispanic women. The frequency of depression and stress were higher in U.S. born compared to immigrant Hispanic women. There was an elevation in plasma levels of TNF-α through the pregnancy in depressed women, and elevations in other cytokines, at midpregnancy. The adverse pregnancy outcomes, including preterm delivery, known to be associated with prenatal depression were not present in this cohort.

Optimization of diastereomeric dihydropyridines as antimalarials.

The increase in research funding for the development of antimalarials since 2000 has led to a surge of new chemotypes with potent antimalarial activity. High-throughput screens have delivered several thousand new active compounds in several hundred series, including the 4,7-diphenyl-1,4,5,6,7,8-hexahydroquinolines, hereafter termed dihydropyridines (DHPs). We optimized the DHPs for antimalarial activity. Structure-activity relationship studies focusing on the 2-, 3-, 4-, 6-, and 7-positions of the DHP core led to the identification of compounds potent (EC50 < 10 nM) against all strains of P. falciparum tested, including the drug-resistant parasite strains K1, W2, and TM90-C2B. Evaluation of efficacy of several compounds in vivo identified two compounds that reduced parasitemia by >75 % in mice 6 days post-exposure following a single 50 mg/kg oral dose. Resistance acquisition experiments with a selected dihydropyridine led to the identification of a single mutation conveying resistance in the gene encoding for Plasmodium falciparum multi-drug resistance protein 1 (PfMDR1). The same dihydropyridine possessed transmission blocking activity. The DHPs have the potential for the development of novel antimalarial drug candidates.

4(1H)-Quinolones with liver stage activity against Plasmodium berghei.

With the exception of primaquine, tafenoquine, and atovaquone, there are very few antimalarials that target liver stage parasites. In this study, a transgenic Plasmodium berghei parasite (1052Cl1; PbGFP-Luc(con)) that expresses luciferase was used to assess the anti-liver stage parasite activity of ICI 56,780, a 7-(2-phenoxyethoxy)-4(1H)-quinolone (PEQ), as well as two 3-phenyl-4(1H)-quinolones (P4Q), P4Q-146 and P4Q-158, by using bioluminescent imaging (BLI). Results showed that all of the compounds were active against liver stage parasites; however, ICI 56,780 and P4Q-158 were the most active, with low nanomolar activity in vitro and causal prophylactic activity in vivo. This potent activity makes these compounds ideal candidates for advancement as novel antimalarials.

Screening mangrove endophytic fungi for antimalarial natural products.

We conducted a screening campaign to investigate fungi as a source for new antimalarial compounds. A subset of our fungal collection comprising Chinese mangrove endophytes provided over 5000 lipophilic extracts. We developed an accelerated discovery program based on small-scale cultivation for crude extract screening and a high-throughput malaria assay. Criteria for hits were developed and high priority hits were subjected to scale-up cultivation. Extracts from large scale cultivation were fractionated and these fractions subjected to both in vitro malaria and cytotoxicity screening. Criteria for advancing fractions to purification were developed, including the introduction of a selectivity index and by dereplication of known metabolites. From the Chinese mangrove endophytes, four new compounds (14-16, 18) were isolated including a new dimeric tetrahydroxanthone, dicerandrol D (14), which was found to display the most favorable bioactivity profile.

Tryptophan metabolism and immune alterations in pregnant Hispanic women with chronic Toxoplasma gondii infection.

PROBLEM: Pregnancy markedly modifies women's metabolism and immune functions. We hypothesized that pregnancy might alter the immune and metabolic responses to chronic Toxoplasma gondii infection in pregnancy. METHOD OF STUDY: A population of 690 pregnant Hispanic women were screened for antibodies to T. gondii and 158 women were positive (23% positivity) with 83% showing high avidity indices. These seropositive women were followed through their pregnancies with four data collection time points and a postpartum collection at two clinics in Tampa, Florida. A T. gondii seronegative group (N = 128) was randomly selected to serve as a control group and measured along pregnancy in the same way. Serum levels of tryptophan, kynurenine, and their ratio, phenylalanine, tyrosine and their ratio, neopterin, and nitrite were measured through pregnancy and the postpartum. A plasma cytokine panel (IFN-γ, TNFα, IL-2, IL-10, IL-12, IL-6, IL-17) was analyzed in parallel. RESULTS: The major findings suggest that indoleamine 2,3-dioxygenase (IDO-1) was less activated in T. gondii seropositive pregnant Hispanic women with chronic infection. Evidence for IDO-1 suppression was that tryptophan catabolism was less pronounced and there were lower levels of multiple inflammatory cytokines including IFN-γ, which is the major inducer of IDO-1, and higher nitrite concentration, a surrogate marker for nitric oxide, an inhibitor of IDO. CONCLUSIONS: Latent T. gondii infection was associated with higher plasma tryptophan levels, and lower inflammatory cytokines across pregnancy, suggesting suppression of the IDO-1 enzyme, and possible T cell exhaustion during pregnancy.

Phenotypic and genotypic analysis of in vitro-selected artemisinin-resistant progeny of Plasmodium falciparum.

Emergence of artemisinin resistance in Cambodia highlights the importance of characterizing resistance to this class of drugs. Previously, intermediate levels of resistance in Plasmodium falciparum were generated in vitro for artelinic acid (AL) and artemisinin (QHS). Here we expanded on earlier selection efforts to produce levels of clinically relevant concentrations, and the resulting lines were characterized genotypically and phenotypically. Recrudescence assays determined the ability of resistant and parent lines to recover following exposure to clinically relevant levels of drugs. Interestingly, the parent clone (D6) tolerated up to 1,500 ng/ml QHS, but the resistant parasite, D6.QHS340×3, recovered following exposure to 2,400 ng/ml QHS. Resistant D6, W2, and TM91c235 parasites all exhibited elevated 50% inhibitory concentrations (IC(50)s) to multiple artemisinin drugs, with >3-fold resistance to QHS and AL; however, the degree of resistance obtained with standard methods was remarkably less than expected for parasite lines that recovered from 2,400-ng/ml drug pressure. A novel assay format with radiolabeled hypoxanthine demonstrated a greater degree of resistance in vitro than the standard SYBR green method. Analysis of merozoite number in resistant parasites found D6 and TM91c235 resistant progeny had significantly fewer merozoites than parent strains, whereas W2 resistant progeny had significantly more. Amplification of pfmdr1 increased proportionately to the increased drug levels tolerated by W2 and TM91c235, but not in resistant D6. In summary, we define the artemisinin resistance phenotype as a decrease in susceptibility to artemisinins along with the ability to recover from drug-induced dormancy following supraclinical concentrations of the drug.

Novel 4-aminoquinoline analogs highly active against the blood and sexual stages of Plasmodium in vivo and in vitro.

New drugs to treat malaria must act rapidly and be highly potent against asexual blood stages, well tolerated, and affordable to residents of regions of endemicity. This was the case with chloroquine (CQ), a 4-aminoquinoline drug used for the prevention and treatment of malaria. However, since the 1960s, Plasmodium falciparum resistance to this drug has spread globally, and more recently, emerging resistance to CQ by Plasmodium vivax threatens the health of 70 to 320 million people annually. Despite the emergence of CQ resistance, synthetic quinoline derivatives remain validated leads for new drug discovery, especially if they are effective against CQ-resistant strains of malaria. In this study, we investigated the activities of two novel 4-aminoquinoline derivatives, TDR 58845, N(1)-(7-chloro-quinolin-4-yl)-2-methyl-propane-1,2-diamine, and TDR 58846, N(1)-(7-chloro-quinolin-4-yl)-2,N(2),N(2)-trimethylpropane-1,2-diamine and found them to be active against P. falciparum in vitro and Plasmodium berghei in vivo. The P. falciparum clones and isolates tested were susceptible to TDR 58845 and TDR 58846 (50% inhibitory concentrations [IC(50)s] ranging from 5.52 to 89.8 nM), including the CQ-resistant reference clone W2 and two multidrug-resistant parasites recently isolated from Thailand and Cambodia. Moreover, these 4-aminoquinolines were active against early and late P. falciparum gametocyte stages and cured BALB/c mice infected with P. berghei. TDR 58845 and TDR 58846 at 40 mg/kg were sufficient to cure mice, and total doses of 480 mg/kg of body weight were well tolerated. Our findings suggest these novel 4-aminoquinolines should be considered for development as potent antimalarials that can be used in combination to treat multidrug-resistant P. falciparum and P. vivax.

Epigenetic tailoring for the production of anti-infective cytosporones from the marine fungus Leucostoma persoonii.

Recent genomic studies have demonstrated that fungi can possess gene clusters encoding for the production of previously unobserved secondary metabolites. Activation of these attenuated or silenced genes to obtain either improved titers of known compounds or new ones altogether has been a subject of considerable interest. In our efforts to discover new chemotypes that are effective against infectious diseases, including malaria and methicillin-resistant Staphylococcus aureus (MRSA), we have isolated a strain of marine fungus, Leucostoma persoonii, that produces bioactive cytosporones. Epigenetic modifiers employed to activate secondary metabolite genes resulted in enhanced production of known cytosporones B (1, 360%), C (2, 580%) and E (3, 890%), as well as the production of the previously undescribed cytosporone R (4). Cytosporone E was the most bioactive, displaying an IC(90) of 13 µM toward Plasmodium falciparum, with A549 cytotoxicity IC(90) of 437 µM, representing a 90% inhibition therapeutic index (TI(90) = IC(90) A459/IC(90)P. falciparum) of 33. In addition, cytosporone E was active against MRSA with a minimal inhibitory concentration (MIC) of 72 µM and inhibition of MRSA biofilm at roughly half that value (minimum biofilm eradication counts, MBEC90, was found to be 39 µM).

CNS and antimalarial activity of synthetic meridianin and psammopemmin analogs.

The marine invertebrate-derived meridianin A, the originally proposed structure for psammopemmin A, and several related 3-pyrimidylindole analogs were synthesized and subsequently investigated for central nervous system, antimalarial, and cytotoxic activity. A Suzuki coupling of an indoleborate ester to the pyrimidine electrophile was utilized to form the natural product and derivatives thereof. The 3-pyrimidineindoles were found to prevent radioligand binding to several CNS receptors and transporters, most notably, serotonin receptors (<0.2 µM K(i) for 5HT(2B)). Two compounds also inhibited the human malaria parasite Plasmodium falciparum (IC(50) <50 µM). Only the natural product was cytotoxic toward A549 cells (IC(50)=15 µM).

A Pilot Study To Establish an In Vitro Model To Study Premature Intestinal Epithelium and Gut Microbiota Interactions.

Intestinal microbiota has emerged as an important player in the health and disease of preterm infants. The interactions between intestinal flora and epithelium can lead to local injury and systemic diseases. A suitable in vitro cell model is needed to enhance our understanding of these interactions. In this study, we exposed fetal epithelial cell cultures (FHs-74 int cells, human, ATCC CCL 241) to sterile fecal filtrates derived from stool collected from preterm infants at <2 and at 3 to 4 weeks of age. We measured the cytokine levels from the culture media after 4, 24, and 48 h of exposure to the fecal filtrates. We analyzed the 16S rRNA V4 gene data of the fecal samples and transcriptome sequencing (RNA-seq) data from the fetal epithelial cells after 48 h of exposure to the same fecal filtrates. The results showed correlations between inflammatory responses (both cytokine levels and gene expression) and the Proteobacteria-to-Firmicutes ratio and between fecal bacterial genera and epithelial apoptosis-related genes. Our in vitro cell model can be further developed and applied to study how the epithelium responds to different microbial flora from preterm infants. Combining immature epithelial cells and preterm infant stool samples into one model allows us to investigate disease processes in preterm infants in a way that had not been previously reported. IMPORTANCE The gut bacterial flora influences the development of the immune system and long-term health outcomes in preterm infants. Studies of the mechanistic interactions between the gut bacteria and mucosal barrier are limited to clinical observations, animal models, and in vitro cell culture models for this vulnerable population. Most in vitro cell culture models of microbe-host interactions use single organisms or adult origin cell lines. Our study is innovative and significant in that we expose immature epithelial cells derived from fetal tissues to fecal filtrates from eight stool samples from four preterm infants to study the role of intestinal epithelial cells. In addition, we analyzed epithelial gene expression to examine multiple cellular processes simultaneously. This model can be developed into patient-derived two- or three-dimensional cell cultures exposed to their own fecal material to allow better prediction of patient physiological responses to support the growing field of precision medicine.

Norselic acids A-E, highly oxidized anti-infective steroids that deter mesograzer predation, from the Antarctic sponge Crella sp.

Five new steroids, norselic acids A-E (1-5), were isolated from the sponge Crella sp. collected in Antarctica. The planar structures of the norselic acids were established by extensive NMR spectroscopy and mass spectrometry studies, and the configuration of norselic acid A (1) was elucidated by X-ray crystallography. Norselic acid A displays antibiotic activity against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive S. aureus (MSSA), vancomycin-resistant Enterococcus faecium (VRE), and Candida albicans and reduces consumption of food pellets by sympatric mesograzers. Compounds 1-5 are also active against the Leishmania parasite at low micromolar levels.

Design and Synthesis of Orally Bioavailable Piperazine Substituted 4(1H)-Quinolones with Potent Antimalarial Activity: Structure-Activity and Structure-Property Relationship Studies.

Malaria deaths have been decreasing over the last 10-15 years, with global mortality rates having fallen by 47% since 2000. While the World Health Organization (WHO) recommends the use of artemisinin-based combination therapies (ACTs) to combat malaria, the emergence of artemisinin resistant strains underscores the need to develop new antimalarial drugs. Recent in vivo efficacy improvements of the historical antimalarial ICI 56,780 have been reported, however, with the poor solubility and rapid development of resistance, this compound requires further optimization. A series of piperazine-containing 4(1H)-quinolones with greatly enhanced solubility were developed utilizing structure-activity relationship (SAR) and structure-property relationship (SPR) studies. Furthermore, promising compounds were chosen for an in vivo scouting assay to narrow selection for testing in an in vivo Thompson test. Finally, two piperazine-containing 4(1H)-quinolones were curative in the conventional Thompson test and also displayed in vivo activity against the liver stages of the parasite.

Synthesis and Activity of a New Series of Antileishmanial Agents.

We have determined that tetrahydroindazoles such as 1 show potent activity against Leishmania donovani, the causative agent of leishmaniasis. While the Hsp90 activity and anticancer properties of 1 have previously been explored, we present here our efforts to optimize their activity against L. donovani via the synthesis of novel analogues designed to probe the hydrophobic pocket of the protozoan Hsp90 orthologue, specifically through the auspices of functionalization of an amine embedded into the scaffold.

ICI 56,780 Optimization: Structure-Activity Relationship Studies of 7-(2-Phenoxyethoxy)-4(1H)-quinolones with Antimalarial Activity.

Though malaria mortality rates are down 48% globally since 2000, reported occurrences of resistance against current therapeutics threaten to reverse that progress. Recently, antimalarials that were once considered unsuitable therapeutic agents have been revisited to improve physicochemical properties and efficacy required for selection as a drug candidate. One such compound is 4(1H)-quinolone ICI 56,780, which is known to be a causal prophylactic that also displays blood schizonticidal activity against P. berghei. Rapid induction of parasite resistance, however, stalled its further development. We have completed a full structure-activity relationship study on 4(1H)-quinolones, focusing on the reduction of cross-resistance with atovaquone for activity against the clinical isolates W2 and TM90-C2B, as well as the improvement of microsomal stability. These studies revealed several frontrunner compounds with superb in vivo antimalarial activity. The best compounds were found to be curative with all mice surviving a Plasmodium berghei infection after 30 days.

Orally bioavailable 6-chloro-7-methoxy-4(1H)-quinolones efficacious against multiple stages of Plasmodium.

The continued proliferation of malaria throughout temperate and tropical regions of the world has promoted a push for more efficacious treatments to combat the disease. Unfortunately, more recent remedies such as artemisinin combination therapies have been rendered less effective due to developing parasite resistance, and new drugs are required that target the parasite in the liver to support the disease elimination efforts. Research was initiated to revisit antimalarials developed in the 1940s and 1960s that were deemed unsuitable for use as therapeutic agents as a result of poor understanding of both physicochemical properties and parasitology. Structure-activity and structure-property relationship studies were conducted to generate a set of compounds with the general 6-chloro-7-methoxy-2-methyl-4(1H)-quinolone scaffold which were substituted at the 3-position with a variety of phenyl moieties possessing various properties. Extensive physicochemical evaluation of the quinolone series was carried out to downselect the most promising 4(1H)-quinolones, 7, 62, 66, and 67, which possessed low-nanomolar EC50 values against W2 and TM90-C2B as well as improved microsomal stability. Additionally, in vivo Thompson test results using Plasmodium berghei in mice showed that these 4(1H)-quinolones were efficacious for the reduction of parasitemia at >99% after 6 days.

Quinolone-3-diarylethers: a new class of antimalarial drug.

The goal for developing new antimalarial drugs is to find a molecule that can target multiple stages of the parasite's life cycle, thus impacting prevention, treatment, and transmission of the disease. The 4(1H)-quinolone-3-diarylethers are selective potent inhibitors of the parasite's mitochondrial cytochrome bc1 complex. These compounds are highly active against the human malaria parasites Plasmodium falciparum and Plasmodium vivax. They target both the liver and blood stages of the parasite as well as the forms that are crucial for disease transmission, that is, the gametocytes, the zygote, the ookinete, and the oocyst. Selected as a preclinical candidate, ELQ-300 has good oral bioavailability at efficacious doses in mice, is metabolically stable, and is highly active in blocking transmission in rodent models of malaria. Given its predicted low dose in patients and its predicted long half-life, ELQ-300 has potential as a new drug for the treatment, prevention, and, ultimately, eradication of human malaria.

Optimization of 1,2,3,4-tetrahydroacridin-9(10H)-ones as antimalarials utilizing structure-activity and structure-property relationships.

Antimalarial activity of 1,2,3,4-tetrahydroacridin-9(10H)-ones (THAs) has been known since the 1940s and has garnered more attention with the development of the acridinedione floxacrine (1) in the 1970s and analogues thereof such as WR 243251 (2a) in the 1990s. These compounds failed just prior to clinical development because of suboptimal activity, poor solubility, and rapid induction of parasite resistance. Moreover, detailed structure-activity relationship (SAR) studies of the THA core scaffold were lacking and SPR studies were nonexistent. To improve upon initial findings, several series of 1,2,3,4-tetrahydroacridin-9(10H)-ones were synthesized and tested in a systematic fashion, examining each compound for antimalarial activity, solubility, and permeability. Furthermore, a select set of compounds was chosen for microsomal stability testing to identify physicochemical liabilities of the THA scaffold. Several potent compounds (EC(50) < 100 nM) were identified to be active against the clinically relevant isolates W2 and TM90-C2B while possessing good physicochemical properties and little to no cross-resistance.