Anthelmintic Activity and Cytotoxic Effects of Compounds Isolated from the Fruits of Ozoroa insignis Del. (Anacardiaceae).

Ozoroa insignis Del. is an ethnobotanical plant widely used in traditional medicine for various ailments, including schistosomiasis, tapeworm, and hookworm infections. From the so far not investigated fruits of Ozoroa insignis, the anthelmintic principles could be isolated through bioassay-guided isolation using Caenorhabditis elegans and identified by NMR spectroscopic analysis and mass spectrometric studies. Isolated 6-[8(Z)-pentadecenyl] anacardic (1), 6-[10(Z)-heptadecenyl] anacardic acid (2), and 3-[7(Z)-pentadecenyl] phenol (3) were evaluated against the 5 parasitic organisms Schistosoma mansoni (adult and newly transformed schistosomula), Strongyloides ratti, Heligmosomoides polygyrus, Necator americanus, and Ancylostoma ceylanicum, which mainly infect humans and other mammals. Compounds 1-3 showed good activity against Schistosoma mansoni, with compound 1 showing the best activity against newly transformed schistosomula with 50% activity at 1µM. The isolated compounds were also evaluated for their cytotoxic properties against PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma) cell lines, whereby compounds 2 and 3 showed antiproliferative activity in both cancer cell lines, while compound 1 exhibited antiproliferative activity only on PC-3 cells. With an IC50 value of 43.2 µM, compound 3 was found to be the most active of the 3 investigated compounds.

Synthetic hookworm-derived peptides are potent modulators of primary human immune cell function that protect against experimental colitis in vivo.

The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% of the world's population. Current treatments available for autoimmune diseases are inadequate, expensive, and tend to focus on symptom management rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease and other gastrointestinal autoimmune inflammatory conditions. As an alternative and better controlled approach to live infection, we have identified and characterized two peptides, Acan1 and Nak1, from the excretory/secretory component of parasitic hookworms for their therapeutic activity on experimental colitis. We synthesized Acan1 and Nak1 peptides from the Ancylostoma caninum and Necator americanus hookworms and assessed their structures and protective properties in human cell-based assays and in a mouse model of acute colitis. Acan1 and Nak1 displayed anticolitic properties via significantly reducing weight loss and colon atrophy, edema, ulceration, and necrosis in 2,4,6-trinitrobenzene sulfonic acid-exposed mice. These hookworm peptides prevented mucosal loss of goblet cells and preserved intestinal architecture. Acan1 upregulated genes responsible for the repair and restitution of ulcerated epithelium, whereas Nak1 downregulated genes responsible for epithelial cell migration and apoptotic cell signaling within the colon. These peptides were nontoxic and displayed key immunomodulatory functions in human peripheral blood mononuclear cells by suppressing CD4+ T cell proliferation and inhibiting IL-2 and TNF production. We conclude that Acan1 and Nak1 warrant further development as therapeutics for the treatment of autoimmunity, particularly gastrointestinal inflammatory conditions.

Geohelmintiasis, estado nutricional y antropometría de niños de 3 a 6 años del Centro Escolar de la ciudad de Guayaquil, Ecuador / Geohelminthiasis, nutritional status and anthropometry of children from 3 to 6 years old from the School Center of the city of Guayaquil, Ecuador

Las geohelmintiasis son nematodos que afectan al hombre a través de la ingesta de huevos parasitarios, afectando el estado nutricional antropométrico de la población infantil, motivo por el cual se planteó como objetivo determinar incidencia de las helmintiasis transmitidas por el suelo en el estado nutricional antropométrico de niños de 3 a 6 años en un centro escolar Guayaquil en Ecuador, durante el último semestre del 2018. Se aplicó la metodología descriptiva de corte transversal; donde se incluyeron 92 preescolares de 3 a 6 años. A cada niño(a) se le hizo su estudio antropométrico nutricional, unido a un estudio coprológico realizado a 45 niños. Se obtuvo el Índice de Masa Corporal (IMC) con el cual se determinó el estado nutricional (IMC/Edad/Sexo), usando los percentiles del Ministerio de Salud Pública de Ecuador. Se revisaron los IMC de los 35 niños que presentaron geohelminiasis quienes evidenciaron una ligera disminución en la ganancia de los valores de las variables antropométricas. Se encontró predominio del sexo femenino (54,35%), la edad fue 3,78 años, peso: 15,21 Kg y talla: 100,23 cm2, el IMC fue 16,62 Kg/cm2, sin diferencias entre el sexo; la especie de mayor ocurrencia fue la Trichuris. trichiura 43,47% (niñas 10/23) y 66,67% (niños 8/12). Se concluye que 29,34% de los preescolares estudiados están malnutridos, presentan geohelmintiasis, por ello es recomendable realizar intervenciones dirigidas a representantes, estudiantes y docentes, para atender paralelamente geohelmintiasis y nutrición para garantizar la calidad de vida(AU)

Geohelminthiasis are nematodes that affect man through the ingestion of parasitic eggs, affecting the anthropometric nutritional status of the infant population, which is why the objective was to determine the incidence of soil-transmitted helminthiasis in the anthropometric nutritional status in children aged 3 to 6 years of a school in Ecuador, during the last semester of 2018. A cross-sectional descriptive methodology was applied; where 92 preschoolers from 3 to 6 years old were included. Each child was given his or her nutritional anthropometric study, together with a coprological study of 45 children. There was obtained the Body Mass Index (BMI) with which there decided the nutritional state (BMI / Age / Sex), using the centiles of the Department of Public health of Ecuador. The BMI of the 35 children with geohelminiasis were reviewed and showed a slight decrease in the gain of anthropometric variables. The prevalence of female sex was found (54,35%), the age was 3,78 years, weight: 15,21 Kg and height: 100,23 cm2, the BMI was 16,62 Kg/cm2, with no differences between sex; the species of greatest occurrence was Trichuris trichiura 43,47% (girls 10/23) and 66,67% (boys 8/12). It is concluded that 29.34% of the preschools studied are malnourished, have geohelminthiasis, so it is advisable to make interventions aimed at representatives, students and teachers, to attend parallel geohelminthiasis and nutrition to ensure quality of life(AU)

Diferencias morfológicas relevantes para la identificación específica de larvas de uncinarias y Strongyloides stercoralis / Relevant morphological differences for the specific identification of larvae of hookworms and Strongyloides stercoralis

Objetivos: Describir las características morfológicas diferenciales más relevantes de los estadios larvarios de uncinarias (Ancylostoma duodenale y Necator americanus), y S. stercoralis, para su identificación específica en laboratorio clínico convencional. Material y métodos: Estudio observacional durante julio del 2015 a agosto del 2016. Se realizaron cultivos en placa de agar de muestras fecales positivas por microscopía a larvas de nematodos o huevos de uncinaria. Las larvas desarrolladas en cultivo fueron descritas morfológicamente mediante claves, se identificaron las características morfológicas diferenciales más relevantes y se ilustraron mediante microfotografías. Resultados: Las características morfológicas más relevantes para la diferenciación específica fueron la longitud de la cavidad bucal y la prominencia del poro genital en el estadio rabditiforme; y la forma del cuerpo, el extremo terminal de la cola y el diámetro del extremo anterior del intestino en relación al esófago en el estadio filariforme. Conclusiones: Las uncinarias A. duodenale y N. americanus, y S. stercoralis pueden identificarse específicamente por microscopía óptica mediante el análisis morfológico de sus estadíos larvarios desarrollados en cultivo. Debido al requerimiento de equipos e insumos de uso común en laboratorio de microbiología, se recomienda esta metodología para uso en laboratorios clínicos convencionales. (AU)

Objectives: To determine the most relevant morphologic differences of larval stages of hookworms (Ancylostoma duodenale y Necator americanus) and S. stercoralis for their specific identification in a conventional laboratory. Methods: An observational study was carried-out between July and August 2016. Positive fecal samples at microscopy to nematode larvae or to eggs of hookworms were cultured in agar plate. Larvae that developed in culture were described morphologically using passwords. We identified the most relevant differential morphologic features and illustrated them using microphotography. Results: The most relevant morphologic features that allow differentiating these nematodes were the longitude of the oral cavity and the prominence of the genital primordium for the rabditiform larva; the shape of the body, the terminal end of the tail and the diameter of the anterior extreme of the intestine in relation to the esophagus for the filariform larvae. Conclusions: Hookworms and S. stercoralis can be specifically identified by optic microscopic examination of their larval stages developed in culture. We recommend this methodology for conventional microbiology laboratories due to the requirement of simple equipment. (AU)

The Use of Microwave-Assisted Solid-Phase Peptide Synthesis and Click Chemistry for the Synthesis of Vaccine Candidates Against Hookworm Infection.

A protein-based vaccine approach against hookworm infection has failed to deliver the expected outcome, due to a problem with an allergic response in the patient or difficulties in the proteins' production. This implication could be overcome by using a chemically synthesized peptide-based vaccine approach. This approach utilizes minimal pathogenic components that are necessary for the stimulation of the immune response without triggering adverse side effects. To boost the peptide's immunogenicity, a lipid core peptide (LCP) system can be utilized as a carrier molecule/immunostimulant. This chapter describes in detail the synthesizing of protected lipoamino acid, the self-adjuvanting moiety (LCP core), the peptide epitope, and the final vaccine candidate. The subunit peptide and the LCP core were synthesized using microwave-assisted solid-phase peptide synthesis (SPPS). Then the final hookworm vaccine construct was assembled using the copper-catalyzed azide-alkyne cycloaddition, or "click," reaction.

Immune polarization by hookworms: taking cues from T helper type 2, type 2 innate lymphoid cells and alternatively activated macrophages.

Cellular and molecular investigation of parasitic helminth infections has greatly accelerated the understanding of type 2 immune responses. However, there remains considerable debate regarding the specific leucocytes that kill parasites and whether these mechanisms are distinct from those responsible for tissue repair. Herein, we chronicle discoveries over the past decade highlighting current paradigms in type 2 immunity with a particular emphasis upon how CD4(+) T helper type 2 cells, type 2 innate lymphoid cells and alternatively activated macrophages coordinately control helminth-induced parasitism. Primarily, this review will draw from studies of the murine nematode parasite Nippostrongylus brasiliensis, which bears important similarities to the human hookworms Ancylostoma duodenale and Necator americanus. Given that one or more hookworm species currently infect millions of individuals across the globe, we propose that vaccine and/or pharmaceutical-based cure strategies targeting these affected human populations should incorporate the conceptual advances outlined herein.

Isolation, characterization, and anthelminthic activities of a novel dichapetalin and other constituents of Dichapetalum filicaule.

CONTEXT: Dichapetalum filicaule Breteler (Dichapetalaceae) is a rare species occurring only in Côte d'Ivoire and Ghana. Although research on several species of the genus has produced interesting bioactive compounds, particularly the Dichapetalins, a novel class of triterpenoids with antineoplastic properties, there is virtually no information on the ethnobotanical uses and chemical constituents of D. filicaule. OBJECTIVE: The phytochemical and anthelminthic activities of the constituents of D. filicaule were investigated. MATERIALS AND METHODS: Chemical constituents of the petroleum ether, chloroform-acetone, and methanol root extracts of D. filicaule were isolated by column chromatography and characterized by their physico-chemical properties, 1-D and 2-D NMR spectroscopy and mass spectrometry. In vitro anthelminthic activity of the extracts and compounds against the human hookworm, Necator americanus, Stiles 1902 (Nematoda: Ancylostomatidae) was determined within a concentration range of 2500-250 µg/ml using the Egg Hatch Inhibition (EHI) Assay. The hookworm species were identified using a published polymerase chain reaction (PCR) method. RESULTS: A new dichapetalin, dichapetalin X (1), together with the known dichapetalin A (2), pomolic acid (3), glycerol monostearate (4), D:A-friedooleanan-3ß-ol (5), and D:A-friedooleanan-3-one (6) were isolated. Compounds 1, 2, and 4 exhibited EHI with IC50 values of 523.2, 162.4, and 306.0 µg/ml, respectively, against the hookworm. The positive control albendazole gave an IC50 value of 93.27 µg/ml. DISCUSSION AND CONCLUSION: This is the first report of the phytochemical investigation of D. filicaule. The study has yielded a new dichapetalin and also demonstrated the potential anthelminthic properties of the constituents.

Expression, purification, and characterization of the Necator americanus aspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine.

Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.

Structure of glutathione S-transferase 1 from the major human hookworm parasite Necator americanus (Na-GST-1) in complex with glutathione.

Glutathione S-transferase 1 from Necator americanus (Na-GST-1) is a vaccine candidate for hookworm infection that has a high affinity for heme and metal porphyrins. As part of attempts to clarify the mechanism of heme detoxification by hookworm GSTs, co-crystallization and soaking studies of Na-GST-1 with the heme-like molecules protoporphyrin IX disodium salt, hematin and zinc protoporphyrin were undertaken. While these studies did not yield the structure of the complex of Na-GST-1 with any of these molecules, co-crystallization experiments resulted in the first structures of the complex of Na-GST-1 with the substrate glutathione. The structures of the complex of Na-GST-1 with glutathione were solved from pathological crystalline aggregates comprising more than one crystal form. These first structures of the complex of Na-GST-1 with the substrate glutathione were solved by molecular replacement from data collected with a sealed-tube home source using the previously reported apo structure as the search model.

Parasitic infections and immune function: effect of helminth infections in a malaria endemic area.

According to the hygiene hypothesis, reduced exposure to infections could explain the rise of atopic diseases in high-income countries. Helminths are hypothesised to alter the host's immune response in order to avoid elimination and, as a consequence, also reduce the host responsiveness to potential allergens. To elucidate the effect of current helminth infections on immune responsiveness in humans, we measured cytokine production in a rural Ghanaian population in an area with multiple endemic parasites including malaria, intestinal helminths and protozoa. Multiplex real-time PCR in stool samples was used for the detection of four gastrointestinal helminths, of which only Necator americanus was commonly present. A similar assay was used to test for Giardia lamblia in stool samples and malaria infection in venous blood samples. Levels of the cytokines interleukin (IL)-10, tumour necrosis factor (TNF)-α, IL-17, IL-6, IL-13, and interferon (IFN)-γ were determined in whole-blood samples ex vivo-stimulated either with lipopolysaccharide (LPS) and zymosan (for innate cytokine production) or the T-cell mitogen phytohaemagglutinin (PHA). There were no significant differences in either innate or PHA-stimulated cytokine production dependent on current N. americanus infection. Plasmodium falciparum malarial infection was associated with a pro-inflammatory response indicated by increased innate production of TNF-α, IL-17 and IL-6. There was no clear pattern in cytokine responses dependent on G. lamblia-infection. In conclusion, in this rural Ghanaian population current N. americanus infections are not associated with altered immune function, while infection with P. falciparum is associated with pro-inflammatory innate immune responses.

Real-time PCR demonstrates Ancylostoma duodenale is a key factor in the etiology of severe anemia and iron deficiency in Malawian pre-school children.

BACKGROUND: Hookworm infections are an important cause of (severe) anemia and iron deficiency in children in the tropics. Type of hookworm species (Ancylostoma duodenale or Necator americanus) and infection load are considered associated with disease burden, although these parameters are rarely assessed due to limitations of currently used diagnostic methods. Using multiplex real-time PCR, we evaluated hookworm species-specific prevalence, infection load and their contribution towards severe anemia and iron deficiency in pre-school children in Malawi. METHODOLOGY AND FINDINGS: A. duodenale and N. americanus DNA loads were determined in 830 fecal samples of pre-school children participating in a case control study investigating severe anemia. Using multiplex real-time PCR, hookworm infections were found in 34.1% of the severely anemic cases and in 27.0% of the non-severely anemic controls (p<0.05) whereas a 5.6% hookworm prevalence was detected by microscopy. Prevalence of A. duodenale and N. americanus was 26.1% and 4.9% respectively. Moderate and high load A. duodenale infections were positively associated with severe anemia (adjusted odds ratio: 2.49 (95%CI 1.16-5.33) and 9.04 (95%CI 2.52-32.47) respectively). Iron deficiency (assessed through bone marrow examination) was positively associated with intensity of A. duodenale infection (adjusted odds ratio: 3.63 (95%CI 1.18-11.20); 16.98 (95%CI 3.88-74.35) and 44.91 (95%CI 5.23-385.77) for low, moderate and high load respectively). CONCLUSIONS/SIGNIFICANCE: This is the first report assessing the association of hookworm load and species differentiation with severe anemia and bone marrow iron deficiency. By revealing a much higher than expected prevalence of A. duodenale and its significant and load-dependent association with severe anemia and iron deficiency in pre-school children in Malawi, we demonstrated the need for quantitative and species-specific screening of hookworm infections. Multiplex real-time PCR is a powerful diagnostic tool for public health research to combat (severe) anemia and iron deficiency in children living in resource poor settings.

Acquired hookworm immunity in the golden hamster (Mesocricetus auratus) elicited by living Necator americanus third-stage infective larvae.

The aim of the study is to demonstrate and understand the acquired immunity in golden hamsters (Mesocricetus auratus) elicited by primary Necator americanus infective third-stage larvae (L3) infection. Hamsters infected with 150 L3 for 1, 2, 3, 6 and 10 weeks, were challenged with the same number of L3 and sacrificed 25 days post challenge. The primarily infected hamsters exhibited 99-100% protection against subsequent L3 challenge compared to un-infected naive hamsters. The acquired immunity was developed as early as 1 week post L3 infection and lasted up to 10 weeks. Similar protective immunity was obtained in hamsters infected with N. americanus L3 and then treated orally with a single of 100mg/kg albendazole, followed by challenge with N. americanus L3 4 and 8 weeks post-treatment. The infected hamsters exhibited a rise in IgG antibodies against L3 and juvenile adult worm antigens. Histological examination showed that challenging L3 were trapped in the skin of primarily infected hamsters and surrounded or infiltrated by different inflammatory cells. The trapped L3 were damaged and dead followed by the formation of granulomas encasing dead worms. The results demonstrate that hamsters primarily infected with N. americanus L3 develop acquired immunity against re-infection.

Suppression of inflammatory immune responses in celiac disease by experimental hookworm infection.

We present immunological data from two clinical trials where the effect of experimental human hookworm (Necator americanus) infection on the pathology of celiac disease was evaluated. We found that basal production of Interferon- (IFN-)γ and Interleukin- (IL-)17A from duodenal biopsy culture was suppressed in hookworm-infected participants compared to uninfected controls. Increased levels of CD4+CD25+Foxp3+ cells in the circulation and mucosa are associated with active celiac disease. We show that this accumulation also occurs during a short-term (1 week) oral gluten challenge, and that hookworm infection suppressed the increase of circulating CD4+CD25+Foxp3+ cells during this challenge period. When duodenal biopsies from hookworm-infected participants were restimulated with the immunodominant gliadin peptide QE65, robust production of IL-2, IFN-γ and IL-17A was detected, even prior to gluten challenge while participants were strictly adhering to a gluten-free diet. Intriguingly, IL-5 was produced only after hookworm infection in response to QE65. Thus we hypothesise that hookworm-induced TH2 and IL-10 cross-regulation of the TH1/TH17 inflammatory response may be responsible for the suppression of these responses during experimental hookworm infection.

Structure of a two-CAP-domain protein from the human hookworm parasite Necator americanus.

Major proteins secreted by the infective larval stage hookworms upon host entry include Ancylostoma secreted proteins (ASPs), which are characterized by one or two CAP (cysteine-rich secretory protein/antigen 5/pathogenesis related-1) domains. The CAP domain has been reported in diverse phylogenetically unrelated proteins, but has no confirmed function. The first structure of a two-CAP-domain protein, Na-ASP-1, from the major human hookworm parasite Necator americanus was refined to a resolution limit of 2.2 Å. The structure was solved by molecular replacement (MR) using Na-ASP-2, a one-CAP-domain ASP, as the search model. The correct MR solution could only be obtained by truncating the polyalanine model of Na-ASP-2 and removing several loops. The structure reveals two CAP domains linked by an extended loop. Overall, the carboxyl-terminal CAP domain is more similar to Na-ASP-2 than to the amino-terminal CAP domain. A large central cavity extends from the amino-terminal CAP domain to the carboxyl-terminal CAP domain, encompassing the putative CAP-binding cavity. The putative CAP-binding cavity is a characteristic cavity in the carboxyl-terminal CAP domain that contains a His and Glu pair. These residues are conserved in all single-CAP-domain proteins, but are absent in the amino-terminal CAP domain. The conserved His residues are oriented such that they appear to be capable of directly coordinating a zinc ion as observed for CAP proteins from reptile venoms. This first structure of a two-CAP-domain ASP can serve as a template for homology modeling of other two-CAP-domain proteins.

A case of severe anemia by Necator americanus infection in Korea.

This report describes clinical and parasitological findings of an 82-yr-old female patient who lived in a local rural village and suffered from severe chronic anemia for several years. She was transferred to the National Police Hospital in Seoul for management of severe dyspnea and dizziness. At admission, she showed symptoms or signs of severe anemia. Gastroduodenoscopy observed hyperemic mucosa of the duodenum and discovered numerous moving roundworms on the mucosa. Endoscopy isolated seven of them, which were identified as Necator americanus by characteristic morphology of cutting plates in the buccal cavity. The patient was treated with albendazole and supportive measures for anemia, and her physical condition much improved. This case suggests the possibility that hookworm N. americanus is still transmitted in a remote local mountainous area in Korea.