Project description:The helminth Acanthocheilonema viteae serves as model organism for research on parasitic filarial nematodes. Total RNA secreted or excreted by 1500 adult female and male A. viteae over 3 weeks was isolated from culture media previously processed by differential ultracentrifugation, and subjected to miRNA sequencing.
Project description:microRNAs (miRNAs) are an abundant class of non-coding RNA species with important regulatory roles in gene expression at the posttranscriptional level. The helminth Acanthocheilonema viteae serves as model organism for research on parasitic filarial nematodes. Total RNA secreted or excreted in vitro by 1500 adult female and male A. viteae over 3 weeks was isolated from culture media previously processed by differential ultracentrifugation. miRNA sequencing revealed the presence of 360 unique miRNA candidates released by adult A. viteae in vitro. Among them, 74 high-confidence unique miRNAs, as well as several potential novel miRNA candidates were discovered. A large proportion of the sequenced miRNA candidates appeared differentially expressed between the male and female samples based on normalized copy count. The presence of extracellular vesicles, often rich in miRNAs, could not be confirmed unambiguously by transmission electron microscopy.
Project description:Filariae are vector-borne parasitic nematodes that are endemic worldwide, in tropical and subtropical regions. Important human filariae spp. include Onchocerca volvulus, Wuchereria bancrofti and Brugia spp., and Loa loa and Mansonella spp. causing onchocerciasis (river blindness), lymphatic filariasis (lymphedema and hydrocele), loiasis (eye worm), and mansonelliasis, respectively. It is estimated that over 1 billion individuals live in endemic regions where filarial diseases are a public health concern contributing to significant disability adjusted life years (DALYs). Thus, efforts to control and eliminate filarial diseases were already launched by the WHO in the 1970s, especially against lymphatic filariasis and onchocerciasis, and are mainly based on mass drug administration (MDA) of microfilaricidal drugs (ivermectin, diethylcarbamazine, albendazole) to filarial endemic areas accompanied with vector control strategies with the goal to reduce the transmission. With the United Nations Sustainable Development Goals (SDGs), it was decided to eliminate transmission of onchocerciasis and stop lymphatic filariasis as a public health problem by 2030. It was also requested that novel drugs and treatment strategies be developed. Mouse models provide an important platform for anti-filarial drug research in a preclinical setting. This review presents an overview about the Litomosoides sigmodontis and Acanthocheilonema viteae filarial mouse models and their role in immunological research as well as preclinical studies about novel anti-filarial drugs and treatment strategies.
Project description:Chitinases of pathogens have been proposed as potential targets of vaccines or specific inhibitors. We studied the genomic organization, transcript levels, developmental expression, and biological function of chitinases in the rodent filarial nematode Acanthocheilonema viteae, a model organism for human-pathogenic filarial worms. Characterization of nine genomic clones from an A. viteae phage library and Southern blot experiments revealed the existence of three different chitinase genes, two of which could theoretically yield functional transcripts. The deduced proteins of these genes had the common modular organization of family 18 chitinases. Northern blot experiments and rapid amplification of cDNA ends-PCR with adult worms and larval stages showed that only one gene is expressed, with high variation in transcript levels, as determined by real-time PCR. Chitinase transcript levels were lowest in the late male stage 4 larva (L4) and peaked in the stage 3 larva (L3), which was corroborated by Western blotting. RNA interference (RNAi) experiments showed that treatment of L3 and adult female worms with double-stranded RNA of chitinase inhibited molting of L3 worms and hatching of microfilariae. RNAi also led to the death of 50% of female worms, revealing the essential role of chitinase in the life cycle of filarial nematodes.
Project description:Cystatins are reversible, tightly binding inhibitors of cysteine proteases. Filarial cystatins have been ascribed immunomodulatory properties and have been implicated in protective immunity. To continue exploration of this potential, here we have determined the sequence, structure and genomic organization of the cystatin gene locus of A. viteae. The gene is composed of 4 exons separated by 3 introns and spans approximately 2 kb of genomic DNA. The upstream genomic sequence contains transcriptional factor binding sites such as AP-1 and NF-Y, an inverted CCAAT sequence, and a TATA box. To investigate sites of cystatin expression, Caenorhabditis elegans worms were transformed by microinjection with the putative promoter region and the first exon of the A. viteae cystatin gene fused to the reporter GFP. In transgenic worms fluorescence was observed in the pharyngeal and rectal gland cells suggesting that cystatin is secreted. Additionally, A. viteae cystatin was expressed in C. elegans to explore its potential as an expression system for filarial genes.