Previously localization studies have been conducted

Previously, localization studies have been conducted using antibody-based detection of Pgp protein in tissues of Clade V nematodes. In transgenic C. elegans, Cel-Pgp-3 and Cel-Pgp-1 proteins localized to the apical membranes of the excretory and intestinal cells and the anterior pharynx (Broeks et al., 1995). In Haemonchus contortus, antibodies developed by immunization with synthesized peptides detected Hco-Pgp-2 in the pharynx, lateral nerve cords, deirids and mid-intestine (Godoy et al., 2015b). A monoclonal antibody targeting human Pgp bound to the cuticle, eggs, and intestinal cells of H. contortus (Riou et al., 2005). Fewer studies have assessed the localization of Pgp mRNA transcripts in situ. In H. contortus, in situ hybridization revealed that mRNA of Pgp-A (Hco-Pgp-2) was present in the worm gut, anterior to the pharyngeal intestinal junction, lateral cords, vas deferens and spicules, with no significant differences being observed in males or females (Smith and Prichard, 2002). In the present study, multiple nucleic az628 synthesis hybridization revealed significant levels of Peq-pgp-11 transcripts in the intestines of both sexes and in the reproductive tissue of the male and significant levels of Peq-pgp-16 mRNA transcripts in the intestines of male and female Parascaris. This technique has a significant advantage over worm dissection and qPCR, as tubular organs such as the testes that take significant skill to isolate without contamination. In addition, small organs such as nerve cords can be studied in the context of the surrounding tissues. Detection of Pgp transcripts in some cell types suggests that Pgps could prevent entry of ML into the body of the nematode. Pgp mRNA was visualized in the hypodermis and in the coelomyarian musculature. Since transcuticular diffusion of anthelmintics has been demonstrated in ascarids (Alvarez et al., 2001; Martin et al., 1992), anthelmintic efflux could be relevant at the level of the body wall. High levels of Pgp were also observed within intestinal cells. In mammals, the intestinal epithelium is the first line of defense and effluxes many foreign substances. There is a possibility that ingested anthelmintics are effluxed at the level of the intestine, thereby preventing their entry into the body of the nematode. Pgp transcripts were also visualized, albeit in lower numbers, in the lateral cords through which the excretory canal passes (Martin et al., 1992; Sanglas et al., 2009). It has been postulated that since nematodes lack a liver or kidneys, drug and xenobiotic clearance occurs through the excretory canal by P-glycoproteins on the surface of cell membranes (Broeks et al., 1995). In Ascaris, vacuolated cells of the lateral cords absorb ML from the peri-enteric fluid (Martin et al., 1992). Thus, the presence of Pgps in the lateral cords may enable drug efflux into the excretory canal causing a reduction in effective concentration of the drug at other active sites in the parasite. In mammals, nervous system Pgps exclude xenobiotics at the level of the blood brain barrier (de Boer et al., 2003). In the present study, Pgp mRNA was visualized in low numbers in the dorsal and ventral nerve cords. One binding site for MLs is in the outer monolayer of the plasma membrane of muscle cells and nerve-cords (Martin et al., 1992), therefore, efflux of anthelmintics could also be relevant at the level of the nerves bearing ligand-gated ion channels. Thus, although the mammalian body components are different structurally, the function of Pgp-mediated neuroprotection could be similar. Pgp mRNA was visualized in the reproductive tissues of both the male and female worms. Increased uterine expression of Peq-pgp-11 and Peq-pgp-16 has also been detected by PCR for Parascaris (Janssen et al., 2013). Uterine muscles are sites of action of for MLs and the presence of Pgps in the uterus and uterine muscles has been proposed (Godoy et al., 2015a; Prichard, 2001) However, the potential function of Pgps in the male reproductive organs such as the testis, vas deferens, and seminal vesicles are largely unknown.