In vitro and in vivo studies on a group of chalcones find promising results as potential drugs against fascioliasis.

About a third of the world population is infected by helminth parasites implicated in foodborne trematodiasis. Fascioliasis is a worldwide disease caused by trematodes of the genus Fasciola spp. It generates huge economic losses to the agri-food industry and is currently considered an emerging zoonosis by the World Health Organization (WHO). The only available treatment relies on anthelmintic drugs, being triclabendazole (TCBZ) the drug of choice to control human infections. The emergence of TCBZ resistance in several countries and the lack of an effective vaccine to prevent infection highlights the need to develop new drugs to control this parasitosis. We have previously identified a group of benzochalcones as inhibitors of cathepsins, which have fasciolicidal activity in vitro and are potential new drugs for the control of fascioliasis. We selected the four most active compounds of this group to perform further preclinical studies. The compound's stability was determined against a liver microsomal enzyme fraction, obtaining half-lives of 34-169 min and low intrinsic clearance values (<13 µL/min/mg), as desirable for potential new drugs. None of the compounds were mutagenic or genotoxic and no in vitro cytotoxic effects were seen. Compounds C31 and C34 showed the highest selectivity index against liver fluke cathepsins when compared to human cathepsin L. They were selected for in vivo efficacy studies observing a protective effect, similar to TCBZ, in a mouse model of infection. Our findings strongly encourage us to continue the drug development pipeline for these molecules.

Vascular inflammation and impaired reverse cholesterol transport and lipid metabolism in obese children and adolescents.

BACKGROUND AND AIMS: Childhood obesity is associated to complications such as insulin resistance and dyslipidemia. High density lipoproteins (HDL) constitute the only lipoprotein fraction with ateroprotective properties. The aim of the present study was to analyze inflammatory markers, carbohydrate metabolism, lipid profile and HDL functionality in obese children and adolescents compared to healthy controls. METHODS AND RESULTS: Twenty obese children and adolescents (Body mass index z score >3.0) (9-15 years old) and 20 age and sex similar controls were included in the study. Triglyceride (TG), total cholesterol (TC), HDL-C, LDL-C, apolipoproteins (apo) A-I and B, glucose and insulin levels were quantified. Lipid indexes and HOMA-IR were calculated. Cholesterol efflux (CEC), lipoprotein associated phospholipase A2 (Lp-PLA2), lecithin-cholesterol acyl transferase (LCAT) and cholesteryl ester transfer protein, plus paraoxonase and arylesterase (ARE) activities were evaluated. Obese children and adolescents showed significantly higher TG [69 (45-95) vs 96 (76-121); p < 0.05], non-HDL-C [99 ± 34 vs 128 ± 26; p < 0.01], TC/HDL-C [2.8 ± 0.6 vs 4.7 ± 1.5; p < 0.01], TG/HDL-C [1.1 (1.0-1.8) vs 2,2 (1.4-3.2); p < 0.01], and HOMA-IR [1.5 (1.1-1.9) vs. 2.6 (2.0-4.5); p < 0.01] values, plus Lp-PLA2 activity [8.3 ± 1.9 vs 7.1 ± 1.7 umol/ml.h; p < 0,05] in addition to lower HDL-C [57 ± 10 vs 39 ± 9; p < 0.01], apo A-I [143 ± 25 vs 125 ± 19; p < 0.05], and CEC [6.4 (5.1-6.8) vs. 7.8 (5.7-9.5); p < 0.01] plus LCAT [12.6 ± 3.3 vs 18.7 ± 2.6; p < 0.05] and ARE [96 ± 19 vs. 110 ± 19; p < 0.05] activities. Lp-PLA2 activity correlated with LDL-C (r = 0.72,p < 0.01), non-HDL-C (r = 0.76,p < 0.01), and apo B (r = 0.60,p < 0.01). LCAT activity correlated with triglycerides (r = -0.78,p < 0.01), HDL-C (r = 0.64,p < 0.01), and apo A-I (r = 0.62, p < 0.05). ARE activity correlated with HDL-C (r = 0.32,p < 0.05) and apoA-I (r = 0.43,p < 0.01). CEC was negatively associated with BMI z-score (r = -0.36,p < 0.05), and triglycerides (r = -0.28,p < 0.05), and positively with LCAT activity (r = 0.65,p < 0.05). In multivariate analysis, BMI z-score was the only parameter significantly associated to CEC (r2 = 0.43, beta = -0.38, p < 0.05). CONCLUSION: The obese group showed alterations in carbohydrate and lipid metabolism, which were associated to the presence of vascular specific inflammation and impairment of HDL atheroprotective capacity. These children and adolescents would present qualitative alterations in their lipoproteins which would determine higher risk of suffering premature cardiovascular disease.

Increased Cholesteryl Ester Transfer Protein and Lipoprotein-Associated Phospholipase A2 Activities in Children and Adolescents Presenting High Triglyceride/High-Density Lipoprotein Cholesterol (TG/HDL-C) Ratio.

OBJECTIVE: To explore the association between Triglyceride/High-density lipoprotein cholesterol (TG/HDL-C) index and these enzymes and proteins in a pediatric population. METHODS: Children and adolescents (7-14 y old) were recruited (n = 150) and anthropometric data were registered. Glucose, TG, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), HDL-C plus cholesteryl ester transfer protein (CETP), lipoprotein-associated phospholipase A2 (Lp-PLA2) and paraoxonase 1 (PON1) activities were determined. RESULTS: Twenty-five individuals presented TG/HDL-C ratio ≥ 3.0. These individuals exhibited higher TG [164 (126-186) vs. 65 (48-72) mg/dL; p < 0.01] CETP [250 (232-263) vs. 223 (193-237)% mL/min; p < 0.01] and Lp-PLA2 (4.5 ± 1.9 vs. 3.5 ± 1.3; p < 0.05) plus lower HDL-C [41 (37-49) vs. 52 (48-62) mg/dL; p < 0.01] compared to an age-matched group with TG/HDL-C < 3.0. TG/HDL-C ratio was associated to CETP (p < 0.01) and Lp-PLA2 (p < 0.05). Multiple lineal regression analyses showed TG/HDL-C index as an independent predictor of CETP (r2 = 0.29; beta = 0.49; p < 0.01) and Lp-PLA2 (r2 = 0.21; beta = 0.32; p < 0.05) activities. CONCLUSION: Children and adolescents with TG/HDL-C ≥ 3.0 presented a more atherogenic lipid profile and higher CETP and Lp-PLA2 activities, which would indicate alterations in lipoprotein metabolism and quality.

Thalamic tumors in children: case series from our institution and literature review.

PURPOSE: To describe a case series of children with thalamic tumors treated at our institution in a 5-year period. METHOD: A retrospective and observational study was performed. The records of 15 patients between 2013 and 2018 were analyzed. RESULTS: From 2013 to 2018, 15 patients were treated at our institution. The male to female index was 1.5, and the median age was 8.9 (IQR 4.75-13). Seven (46%) tumors were left-sided, seven (46%) were right-sided, and one (6%) was bilateral. All patients were symptomatic at the time of treatment. Motor deficit was the most common form of presentation (73%). Gross-total resection was performed in two (13.3%) patients, subtotal resection was performed in two other patients (13.3%), and for the rest of the patients (73.3%), the chosen surgical approach was a stereotactic-guided biopsy. The average of procedures was 3.4, mostly related to the treatment for hydrocephalus. Twelve patients (80%) received treatment for hydrocephalus. Nine patients (75%) were treated with ventriculoperitoneal shunts, and four patients (33%) underwent endoscopic third ventriculostomy. High-grade tumors predominated. Grade IV tumors were diagnosed in six patients (40%), followed by grade III in four patients (26.6%), grade II in three (20%) patients, and grade I in two (13.3%) patients. Chemotherapy was given in 93% of the cases, being temozolomide, the most used drug. CONCLUSION: The clinical and surgical approaches for thalamic tumors in children have changed over time. At our institution, the lesser invasive surgical procedures are now being used more frequently.

Effect of Roux-en-Y Gastric Bypass on Lipoprotein Metabolism and Markers of HDL Functionality in Morbid Obese Patients.

PURPOSE: Morbid obesity represents the most severe form of obesity and surgical intervention would be its only successful treatment. Bariatric surgery could generate modifications in carbohydrate metabolism and in lipid profile plus lipoprotein-associated proteins and enzymes, such as lipoprotein-associated phoslipase A2 (Lp-PLA2), cholesteryl ester transfer protein (CETP), and paraoxonase (PON) 1. The aim of the present study was to analyze changes in inflammation markers, carbohydrate metabolism, and lipid parameters in patients who underwent bariatric surgery. METHODS: Thirty-seven patients with morbid obesity were recruited. Evaluations were performed before (T0) and 1 (T1) and 6 (T2) months after surgery. Glucose, insulin, high-sensitivity C-reactive protein (hsCRP), triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol, apolipoproteins (apo) A-I, and B plus Interleukin 1ß and 6 levels in addition to CETP, Lp-PLA2, and PON 1 activities were determined. RESULTS: Body mass index decreased at T1 and T2 (p < 0.01). An improvement in all markers of insulin resistance (p < 0.05) was observed at T1. hsCRP levels diminished at T2 (p < 0.05). Triglyceride levels decreased at T1 and T2 (p < 0.05). HDL-C and apo A-I showed a decrease at T1 which was completely reversed at T2 (p < 0.05). Lp-PLA2 activity increased at T1, which was reversed at T2 (p < 0.05), and CETP activity was diminished at T2 (p < 0.05). PON and ARE activities decreased at T1 and partially recovered at T2 (p < 0.05). CONCLUSIONS: These results would be indicative of a favorable effect of bariatric surgery on markers of carbohydrate metabolism and cardiovascular disease lipid risk factors.

Leukocyte telomere length is associated with iron overload in male adults with hereditary hemochromatosis.

BACKGROUND: Hereditary hemochromatosis (HH) is a primary iron overload (IO) condition. Absolute telomere length (ATL) is a marker of cellular aging and DNA damage associated with chronic diseases and mortality. AIM: To evaluate the relationship between ATL and IO in patients with HH. METHODS: Cross-sectional study including 25 patients with HH: 8 with IO and 17 without IO (ferritin < 300 ng/ml) and 25 healthy controls. Inclusion criteria were: age > 18 years, male sex and HH diagnosis. Patients with diabetes or other endocrine and autoimmune diseases were excluded. ATL was measured by real-time PCR. RESULTS: HH patients with IO were older (P<0.001) and showed higher ferritin concentration (P<0.001). Patients with HH, disregarding the iron status, showed higher glucose and body mass index (BMI) than controls (both P<0.01). ATL was shorter in patients with IO than controls [with IO: 8 (6-14), without IO: 13 (9-20), and controls: 19 (15-25) kilobase pairs, P<0.01]; with a linear trend within groups (P for trend <0.01). Differences in ATL remained statistically significant after adjusting by age, BMI and glucose (P<0.05). DISCUSSION: Patients with IO featured shorter ATL while patients without IO showed only mild alterations vs. controls. Screening for IO is encouraged to prevent iron-associated cellular damage and early telomere attrition.

Drug Targets: Screening for Small Molecules that Inhibit Fasciola hepatica Enzymes.

The in vitro screening of small molecules for enzymatic inhibition provides an efficient means of finding new compounds for developing drug candidates. This strategy has the advantage of being rapid and inexpensive to perform. Enzymes are suitable targets for screening when simple methods to obtain them and measure their activities are available and there is evidence of their essential role in the parasite's life cycle. Here, we describe the screening of small molecules as inhibitors of two Fasciola hepatica enzyme targets (cathepsin L and triose phosphate isomerase), an initial step to find new potential compounds for drug development strategies.

Comparison of different systems of ultrasound (US) risk stratification for malignancy in elderly patients with thyroid nodules. Real world experience.

PURPOSE: To comparatively assess the performance of three sonographic classification systems, American Thyroid Association (ATA), the American College of Radiology Thyroid Imaging Reporting and Data System (ACR TI-RADS), and American Association of Clinical Endocrinologists (AACE)/American College of Endocrinology (ACE)/Associazione Medici Endocrinologi (AME) in identifying malignant nodules in an elderly population. METHODS: Cross-sectional study of patients referred for fine needle aspiration biopsy in an academic center for the elderly. One nodule/patient was considered. Nodules classified Bethesda V/VI were considered malignant. Receiver operating characteristics (ROC) curves were established and compared to evaluate diagnostic performance. Malignancy among biopsies below the size cutoff for each ultrasound classification was also compared. RESULTS: One thousand, eight hundred sixty-seven patients (92% females); median (Q1-Q3), age 71 (67-76) years, were studied showing 82.8% benign (Bethesda II) and 2.6% malignant cytology. The three classifications correctly identified malignancy (P < 0.01). Nonetheless, in the ATA and AACE/ACE/AME 16 and 2 malignant nodules, respectively, were unclassifiable. Including unclassified malignant nodules (n = 1234, malignant = 50), comparison of the ROC curves showed lower performance of ATA [area under the curve (AUC) = ATA (0.49) vs. ACR TI-RADS (0.62), p = 0.008 and ATA vs. AACE/ACE/AME (0.59), p = 0.022]. Proportion of below size cutoff biopsies for ATA, ACR TI-RADS, and AACE/ACE/AME was different [16, 42, and 29% (all p < 0.001)], but no differences in malignancy rate were observed in these nodules. CONCLUSION: The present study is the first to validate in elderly patients these classifications showing that AACE/ACE/AME and ACR TI-RADS can predict thyroid malignancy more accurately than the ATA when unclassifiable malignant nodules are considered. Moreover, in this aged segment of the population, the use of ACR TI-RADS avoided more invasive procedures.

Novel and selective inactivators of Triosephosphate isomerase with anti-trematode activity.

Trematode infections such as schistosomiasis and fascioliasis cause significant morbidity in an estimated 250 million people worldwide and the associated agricultural losses are estimated at more than US$ 6 billion per year. Current chemotherapy is limited. Triosephosphate isomerase (TIM), an enzyme of the glycolytic pathway, has emerged as a useful drug target in many parasites, including Fasciola hepatica TIM (FhTIM). We identified 21 novel compounds that selectively inhibit this enzyme. Using microscale thermophoresis we explored the interaction between target and compounds and identified a potent interaction between the sulfonyl-1,2,4-thiadiazole (compound 187) and FhTIM, which showed an IC50 of 5 µM and a Kd of 66 nM. In only 4 hours, this compound killed the juvenile form of F. hepatica with an IC50 of 3 µM, better than the reference drug triclabendazole (TCZ). Interestingly, we discovered in vitro inhibition of FhTIM by TCZ, with an IC50 of 7 µM suggesting a previously uncharacterized role of FhTIM in the mechanism of action of this drug. Compound 187 was also active against various developmental stages of Schistosoma mansoni. The low toxicity in vitro in different cell types and lack of acute toxicity in mice was demonstrated for this compound, as was demonstrated the efficacy of 187 in vivo in F. hepatica infected mice. Finally, we obtained the first crystal structure of FhTIM at 1.9 Å resolution which allows us using docking to suggest a mechanism of interaction between compound 187 and TIM. In conclusion, we describe a promising drug candidate to control neglected trematode infections in human and animal health.

Cathepsin L Inhibitors with Activity against the Liver Fluke Identified From a Focus Library of Quinoxaline 1,4-di-N-Oxide Derivatives.

Infections caused by Fasciola species are widely distributed in cattle and sheep causing significant economic losses, and are emerging as human zoonosis with increasing reports of human cases, especially in children in endemic areas. The current treatment is chemotherapeutic, triclabendazole being the drug of preference since it is active against all parasite stages. Due to the emergence of resistance in several countries, the discovery of new chemical entities with fasciolicidal activity is urgently needed. In our continuous search for new fasciolicide compounds, we identified and characterized six quinoxaline 1,4-di-N-oxide derivatives from our in-house library. We selected them from a screening of novel inhibitors against FhCL1 and FhCL3 proteases, two essential enzymes secreted by juvenile and adult flukes. We report compounds C7, C17, C18, C19, C23, and C24 with an IC50 of less than 10 µM in at least one cathepsin. We studied their binding kinetics in vitro and their enzyme-ligand interactions in silico by molecular docking and molecular dynamic (MD) simulations. These compounds readily kill newly excysted juveniles in vitro and have low cytotoxicity in a Hep-G2 cell line and bovine spermatozoa. Our findings are valuable for the development of new chemotherapeutic approaches against fascioliasis, and other pathologies involving cysteine proteases.

Effects of Pubertal Status and Inflammation on the Use of Ferritin to Define Iron Deficiency in Children With Overweight or Obesity.

BACKGROUND AND AIMS: A worldwide increase in childhood overweight (OW) and obesity (OB) has been reported. OB is an inflammatory state which affects iron metabolism and the sensibility of the tests to detect iron deficiency (ID). Our aim was to evaluate the adequacy of current ferritin cut-offs to define ID in children with OW/OB. METHODS: This cross-sectional study included 152 children (54% girls) aged (median [Q1-Q3]) 11 (8-13) years with OW/OB. Complete blood count and iron metabolism were evaluated. Low ferritin, transferrin saturation (TSat), and anemia were defined by age- and sex-specific cut-offs recommended by National Guidelines. Iron intake was assessed in a subgroup (n = 80) by a 24-hour dietary recall. Analyses were made according to pubertal development and ferritin tertiles. RESULTS: The overall prevalence of low ferritin, TSat, and anemia was 2.6%, 23.8%, and 5.2%, respectively. Among pre-pubertal children (n = 87), the frequency of low TSat rose across ferritin tertiles (P < .05), whereas it decreased among pubertal children (n = 65; P < .005). Cases of anemia among pre-pubertal children were found in the highest ferritin tertile, whereas 4/6 anemia cases in pubertal children were found in the lowest ferritin tertile (<39 µg/L). Pubertal children within the lowest ferritin tertile + low TSat (n = 11) showed lower hemoglobin (-9%; P < .005) and hematocrit (-8%, P < .01) than those in the same tertile + normal TSat (n = 16). The overall prevalence of children with ferritin < 39 µg/L + low TSat was 9.2%. CONCLUSIONS: Higher ferritin cut-off values are required to define ID in children with OW/OB. Such cut-off remains to be validated in larger, multi-ethnic cohorts of children with OW/OB.

Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L.

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket) of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.

Identification of Chalcones as Fasciola hepatica Cathepsin L Inhibitors Using a Comprehensive Experimental and Computational Approach.

BACKGROUND: Increased reports of human infections have led fasciolosis, a widespread disease of cattle and sheep caused by the liver flukes Fasciola hepatica and Fasciola gigantica, to be considered an emerging zoonotic disease. Chemotherapy is the main control measure available, and triclabendazole is the preferred drug since is effective against both juvenile and mature parasites. However, resistance to triclabendazole has been reported in several countries urging the search of new chemical entities and target molecules to control fluke infections. METHODOLOGY/PRINCIPLE FINDINGS: We searched a library of forty flavonoid derivatives for inhibitors of key stage specific Fasciola hepatica cysteine proteases (FhCL3 and FhCL1). Chalcones substituted with phenyl and naphtyl groups emerged as good cathepsin L inhibitors, interacting more frequently with two putative binding sites within the active site cleft of the enzymes. One of the compounds, C34, tightly bounds to juvenile specific FhCL3 with an IC50 of 5.6 µM. We demonstrated that C34 is a slow-reversible inhibitor that interacts with the Cys-His catalytic dyad and key S2 and S3 pocket residues, determinants of the substrate specificity of this family of cysteine proteases. Interestingly, C34 induces a reduction in NEJ ability to migrate through the gut wall and a loss of motility phenotype that leads to NEJ death within a week in vitro, while it is not cytotoxic to bovine cells. CONCLUSIONS/SIGNIFICANCE: Up to date there are no reports of in vitro screening for non-peptidic inhibitors of Fasciola hepatica cathepsins, while in general these are considered as the best strategy for in vivo inhibition. We have identified chalcones as novel inhibitors of the two main Cathepsins secreted by juvenile and adult liver flukes. Interestingly, one compound (C34) is highly active towards the juvenile enzyme reducing larval ability to penetrate the gut wall and decreasing NEJ´s viability in vitro. These findings open new avenues for the development of novel agents to control fluke infection and possibly other helminthic diseases.