In Vitro and in Vivo Study of a Photostable Quinone Compound with Enhanced Therapeutic Efficacy against Chagas Disease.

Chagas disease, a neglected tropical disease caused by the protozoan Trypanosoma cruzi poses a significant health challenge in rural areas of Latin America. The current pharmacological options exhibit notable side effects, demand prolonged administration, and display limited efficacy. Consequently, there is an urgent need to develop drugs that are safe and clinically effective. Previously, we identified a quinone compound (designated as compound 2) with potent antiprotozoal activity, based on the chemical structure of komaroviquinone, a natural product renowned for its antitrypanosomal effects. However, compound 2 was demonstrated considerably unstable to light. In this study, we elucidated the structure of the light-induced degradation products of compound 2 and probed the correlation between the quinone ring's substituents and its susceptibility to light. Our findings led to the discovery of quinones with significantly enhanced light stability, some of which exhibiting antitrypanosomal activity. The most promising compound was evaluated for drug efficacy in a mouse model of Chagas disease, revealing where a notable reduction in blood parasitemia.

Differential cardiomyocyte transcriptomic remodeling during in vitro Trypanosoma cruzi infection using laboratory strains provides implications on pathogenic host responses.

BACKGROUND: Chagas disease can lead to life-threatening cardiac manifestations. Regional factors, including genetic characteristics of circulating Trypanosoma cruzi (T. cruzi), have attracted attention as likely determinants of Chagas disease phenotypic expression and Chagas cardiomyopathy (CCM) progression. Our objective was to elucidate the differential transcriptomic signatures of cardiomyocytes resulting from infection with genetically discrete T. cruzi strains and explore their relationships with CCM pathogenesis and progression. METHODS: HL-1 rodent cardiomyocytes were infected with T. cruzi trypomastigotes of the Colombian, Y, or Tulahuen strain. RNA was serially isolated post-infection for microarray analysis. Enrichment analyses of differentially expressed genes (fold-change ≥ 2 or ≤ 0.5) highlighted over-represented biological pathways. Intracellular levels of reactive oxygen species (ROS) were compared between T. cruzi-infected and non-infected HL-1 cardiomyocytes. RESULTS: We found that oxidative stress-related gene ontology terms (GO terms), 'Hypertrophy model', 'Apoptosis', and 'MAPK signaling' pathways (all with P < 0.01) were upregulated. 'Glutathione and one-carbon metabolism' pathway, and 'Cellular nitrogen compound metabolic process' GO term (all with P < 0.001) were upregulated exclusively in the cardiomyocytes infected with the Colombian/Y strains. Mean intracellular levels of ROS were significantly higher in the T. cruzi-infected cardiomyocytes compared to the non-infected (P < 0.0001). CONCLUSIONS: The upregulation of oxidative stress-related and hypertrophic pathways constitutes the universal hallmarks of the cardiomyocyte response elicited by T. cruzi infection. Nitrogen metabolism upregulation and glutathione metabolism imbalance may implicate a relationship between nitrosative stress and poor oxygen radicals scavenging in the unique pathophysiology of Chagas cardiomyopathy.

Re-emerging threat of Trypanosoma cruzi vector transmission in El Salvador, update from 2018 to 2020.

BACKGROUND: Since the late twentieth century, Chagas disease gained global attention to suppress the vector burden as a main control strategy in endemic countries. In Central America, multi-national initiative successfully achieved significant reduction in the estimated disease prevalence as well as elimination of the region's principal vector species at the time in 2012. While the last decade has witnessed significant changes in ecosystem-such as urbanization and replacement of the main vector species-that can possibly affect the vector's habitation and residual transmission, the up-to-date vector burden in the region has not been evaluated thoroughly due to the cessation of active vector surveillance. The aim of this study was to update the risk of vector-borne Trypanosoma cruzi infection in El Salvador, the top Chagas disease-endemic country in Central America. METHODS: A nationwide vector survey was conducted in the domestic environment of El Salvador from September 2018 to November 2020. The selection of the houses for inspection was based on expert purposeful sampling. Infection for T. cruzi was examined by microscopic observation of the insects' feces, followed by a species confirmation using PCR. The data were analyzed using R software version 4.1.3. Proportion estimates with 95% confidence intervals were inferred using the Jeffrey's method provided under the epiR package. RESULTS: A total of 1529 Triatoma dimidiata was captured from 107 houses (infestation rate, 34.4%; 107/311) in all the fourteen departments of the country visited within the period; prevalence of T. cruzi infection was as high as 10% (153/1529). In the country, domestic T. dimidiata infestation was distributed ubiquitously, while T. cruzi infection rates varied across the departments. Five out of fourteen departments showed higher infection rates than the average, suggesting sporadic high-risk areas in the country. CONCLUSIONS: Our comprehensive study revealed substantial T. cruzi infection of T. dimidiata across the country, indicating potential active transmission of the disease. Therefore, strengthened surveillance for both vector and human infection is required to truly eliminate the risk of T. cruzi transmission in Central America.

Synthesis and Evaluation of Quinone Derivatives for Activity against Trypanosome cruzi.

A series of quinone derivatives with a variety of side chains were synthesized. These synthetic quinone compounds were evaluated for in vitro antitrypanosomal activity against trypomastigotes and amastigotes of Trypanosoma cruzi, the causative agent of Chagas disease. Measurement of solubility of quinones and their ability to permeate cell membranes were assessed to address their possible use as oral drugs. Some synthesized compounds exhibited potent antitrypanosomal activity. However, most compounds with a promising activity showed poor solubility that did not seem suitable for oral usage. Meanwhile, compound 5a, an N-tert-butoxycarbonylpiperidine derivative, exhibited good antitrypanosomal activity, ability to permeate membranes, and good solubility.

Variation of urinary follicle-stimulating hormone level after menopause†:†From the results of Japan Nurses' Health Study.

The change in follicle-stimulating hormone (FSH) during the menopausal transition and associations of FSH with various diseases have been assessed by using blood samples. We examined cross-sectionally the variation of FSH levels, associations of estrone and estradiol with FSH, and associations of BMI with these hormones by using urinary samples from peri- and postmenopausal women in Japan. Of 4472 participants in the Urinary Isoflavone Concentration Survey of the Japan Nurses' Health Study, we analyzed urinary levels of estrone, estradiol and FSH in 547 women aged from 45 to 54 years. Urinary FSH levels varied widely in postmenopausal women and the pattern of change in urinary FSH levels seems to be similar to that in blood FSH levels in previous studies. There were no significant differences in age, body mass index (BMI), estradiol, estrone and estradiol/estrone ratio among three groups according to the tertile of FSH. In postmenopausal women, there were significant associations of BMI with levels of estrone and estradiol, but there was no significant association of BMI with FSH. Studies using urinary samples will allow us to establish a study project as a large-scale population-based study to determine associations between FSH and various diseases after menopause. J. Med. Invest. 66 : 297-302, August, 2019.

Association between FSH, E1, and E2 levels in urine and serum in premenopausal and postmenopausal women.

OBJECTIVE: We aimed to establish correlations for the levels of follicle-stimulating hormone (FSH), estrone (E1) and estradiol (E2) between urine and serum in premenopausal and postmenopausal women using immunoassays. METHODS: In this study of 92 women (61 postmenopausal, 31 premenopausal), both urine and blood specimens were collected on the same day and stored at 4 °C for analysis by chemiluminescent immunoassay, radioimmunoassay and/or electrochemiluminescent immunoassay. RESULTS: There were correlations in the levels of FSH, E1 and E2 between urine and serum in both postmenopausal (r = 0.96 for FSH, r = 0.91 for E1, r = 0.80 for E2) and premenopausal (r = 0.98 for FSH, r = 0.92 for E1, r = 0.90 for E2) women. It is indicated that the correlations were stronger in the premenopausal group compared with the postmenopausal group, especially for FSH. CONCLUSION: The levels of FSH, E1 and E2 in urine correlated with those in the serum in premenopausal and postmenopausal women. Urine samples could be used instead of serum samples to measure hormone levels, which would reduce the difficulty of conducting large survey studies.

Validation of the Accuracy of Self-Reported ABO Blood Types in the Japan Nurses' Health Study

Background: The associations between ABO blood type and risk of diseases including cancer have been reported from epidemiological studies. Self-reporting is one of the most widely used methods of collecting the ABO blood type information. Verifying the accuracy of self-reporting is important to consider measurement errors. We aimed to conduct validation of self-reported ABO blood types in the Japan Nurses' Health Study (JNHS), which is a large prospective cohort study. Methods: The concordance rate between self-reported and serologically or genetically inferred ABO blood groups was investigated for a subsample of 41 subjects from the Gunma Nurses' Health Study, which was a pilot cohort study that preceded the JNHS. The presence of antibodies to A or B antigens in serum (serological test) and allele types of the ABO gene (genotyping test) were determined by using frozen blood samples that were preserved for approximately 7 years. ABO blood types were determined from these tests and compared with self-reported data. Results: All of the nurses reported that their ABO blood groups were concordant with those determined by a serological and/or genotyping test. Self-reported ABO blood types of 35 of 38 (92.1%) participants were consistent with the results from serological typing, while the answers of three participants were not. In these three participants, ABO genotypes that were inferred from genotyping of three single nucleotide polymorphisms in ABO loci perfectly matched with their self-reported ABO types, and all of these were O-type. Conclusions: Japanese health professionals report their blood type with a high degree of accuracy. Special attention should be paid to the O-type group in serological analysis of blood samples that have been preserved for several years in longitudinal studies.

The association of urinary estrogen levels with urinary isoflavone levels: Difference between premenopausal women and postmenopausal women.

Results of studies on the associations of soy food intake with urinary estrogen levels in premenopausal women and in postmenopausal women have been inconsistent. We examined the associations of urinary isoflavone levels as well as soy food intake with estrone (E1) and estradiol (E2) in pre- and postmenopausal women. In addition, we compared the levels of isoflavones, E1 and E2 across current hormone users such as those receiving hormone replacement therapy and those using oral contraceptives and non-users among both pre- and postmenopausal women. Urinary levels of isoflavones, E1 and E2 in 498 women (36 hormone users and 462 non-users) were analyzed. Premenopausal women with a higher frequency of soy food intake had higher urinary isoflavone levels, but there were no significant associations between E1 and E2 levels and urinary isoflavone levels. Levels of E1 and E2 in hormone users were significantly lower than those in hormone non-users among premenopausal women, but levels of E1 and E2 in hormone users were significantly higher than those in hormone non-users among postmenopausal women. Postmenopausal women with a higher frequency of soy food intake had higher urinary isoflavone levels, and postmenopausal women with high urinary isoflavone levels had significantly higher E1 and E2 levels. In conclusion, the associations of urinary isoflavone levels with urinary estrogen levels differed with menopausal status. Urinary levels of E1 and E2 were high in postmenopausal women with high urinary isoflavone levels but not in premenopausal women with high urinary isoflavone levels.

Optimal cut-off value for equol-producing status in women: The Japan Nurses' Health Study urinary isoflavone concentration survey.

Equol is one of the most active soy isoflavones. When the association between soy food intake in daily life and health outcomes is examined in epidemiological studies, it is important to define the equol-producing status of each individual. However, few studies have assessed equol-producing status without a soy challenge test. To determine a robust cutoff criterion for equol producer classification in observational studies, we conducted a urinary isoflavone concentration survey in daily life among women. Furthermore, we examined the association between eating habits regarding soy foods and equol-producing status. A total of 4,412 participants were included in the analyses. Urinary isoflavones were analyzed using a high-performance liquid chromatography method. We examined the distribution of the log10 equol/daidzein ratios, finding a mixture of two normal distributions, corresponding to equol producer and non-producer subpopulations. Applying a finite mixture model, we estimated the means, standard deviations, and mixing proportions of these two distributions. The estimation was carried out using the SAS NLIN procedure. The optimal cutoff point for the log10 equol/daidzein ratio in the study population was determined to be -1.42, according to the estimated parameters of the mixture distribution. Based on this criterion, 1,830 (41.5%) of the participants were identified as equol producers. Compared with non-consumers of soy foods, consumers of soy foods had significantly higher odds of being equol producers. Using log10-transformed equol/daidzein ratios ≥ -1.42 to define equol producers among Japanese women is reasonable and suitable for determining equol-producing status in epidemiological studies. We found that soy food eating habits were associated with equol-producing status. Further investigation is required to evaluate associations between equol-producing status in daily life and health outcomes. The results of this study suggest the best cutoff point to use in the definition of equol-producing status in daily life.

Inhibition of autolysosome formation in host autophagy by Trypanosoma cruzi infection.

Autophagy has emerged as an essential component of the defense system against intracellular pathogens. We demonstrated that Trypanosoma cruzi, an intracellular protozoan parasite, was not eliminated by the host's autophagic machinery despite exposure to the host cell cytoplasm. Puncta of microtubule-associated protein 1 light chain 3 (LC3), an autophagy marker, and LC3-II, a lipidated form of LC3, were significantly increased after infection with T. cruzi, indicating that the parasite activated the early steps of host autophagy and induced autophagosome formation. However, autolysosomes were not observed in the infected cells. In addition, T. cruzi was not enwrapped by autophagosomes, suggesting that the parasite has mechanisms to allow it to evade autophagic capture. The results of this study indicate that host autophagy is incomplete following T. cruzi infection.

Host cytoplasmic processing bodies assembled by Trypanosoma cruzi during infection exert anti-parasitic activity.

Processing bodies (PBs) are cytoplasmic granules containing mRNAs and proteins involved in translation and degradation of mRNAs. PBs are constitutively present in cells and are induced to accumulate when external stressors including microbial infection are applied to cells, followed by a rapid translational arrest. We have examined the impact of Trypanosoma cruzi (T. cruzi, Tc) infection on host cytoplasmic PB assembly. Within 24h post-infection, we found the average number of PB foci per cell increased by more than 2-fold. Protein levels of PB components were unaltered during infection. These results indicated that Tc infection caused accumulation of PBs by changing the localization pattern of PB protein components. To elucidate the role of the accumulated PBs on Tc infection, we knocked down PBs using a siRNA specific for PB components EDC4 and Lsm14A, which are involved in mRNA decapping and translational repression, respectively. We observed that the inhibition of PB accumulation significantly enhanced the infectivity and growth of intracellular amastigotes. Depletion of PBs did not affect nitric oxide (NO) production during Tc infection, indicating that the growth promotion was not caused by modulation of NO-mediated killing of Tc. Our results suggest that the accumulated PBs partially contribute to anti-parasitic responses by manipulating the host's mRNA metabolism.

Synthesis and biological evaluation of quinones derived from natural product komaroviquinone as anti-Trypanosoma cruzi agents.

Current chemotherapy drugs for Chagas' disease are insufficient due to their limited efficacy; however, anti-trypanosomal agents have recently shown promise. As such, synthetic intermediates of komaroviquinone were evaluated for anti-trypanosomal activity. Based on the results, a series of novel quinone derivatives were screened for anti-trypanosomal activity and mammalian cytotoxicity. Several quinone derivatives displayed higher antiprotozoal activity against Trypanosoma cruzi trypomastigotes than the reference drug benznidazole, without concomitant toxicity toward the host cell.

Derivatives of Dictyostelium discoideum differentiation-inducing factor-3 suppress the activities of Trypanosoma cruzi in vitro and in vivo.

Chagas disease (human American trypanosomiasis), which is caused by the protozoan parasite Trypanosoma cruzi, is responsible for numerous deaths each year; however, established treatments for the disease are limited. Differentiation-inducing factor-1 (DIF-1) and DIF-3 are chlorinated alkylphenones originally found in the cellular slime mold Dictyostelium discoideum that have been shown to possess pharmacological activities. Here, we investigated the effects of DIF-3 derivatives on the infection rate and growth of T. cruzi by using an in vitro assay system utilizing host human fibrosarcoma HT1080 cells. Certain DIF-3 derivatives, such as butoxy-DIF-3 (Bu-DIF-3), at micro-molar levels strongly suppressed both the infection rate and growth of T. cruzi in HT1080 cells and exhibited little toxicity for HT1080 cells. For example, the IC50 of DIF-3 and Bu-DIF-3 versus the growth of T. cruzi in HT1080 cells were 3.95 and 0.72µM, respectively, and the LD50 of the two compounds versus HT1080 cells were both greater than 100µM. We also examined the effects of DIF-3 and Bu-DIF-3 on T. cruzi activity in C57BL/6 mice. Intraperitoneally administered Bu-DIF-3 (50mg/kg) significantly suppressed the number of trypomastigotes in blood with no apparent adverse effects. These results strongly suggest that DIF-3 derivatives could be new lead compounds in the development of anti-trypanosomiasis drugs.