The PCNA is a ring-like protein that provides the DNA polymerase the processivity for DNA replication. It is believed that the gene sequence and functions of PCNA are remarkably conserved among eukaryotes. PCNA has been reported in several cell types in mammalian tissues and PCNA-positivity has been reported from a number of different organs in fish[2, 3, 24, 25]. The aquatic environment is continuously exposed to a range of organic chemicals and there is growing concern in how each of these compounds affects the molecular and cellular mechanisms within the intestinal tract. An increase in expression of PCNA is widely accepted as a marker of proliferation associated with the development of neoplastic tissue[3, 24, 27, 28]. Teleost fish have, therefore, become a popular model for use in cancer studies, where there is growing interest on the concurrent detection of PCNA, tumour protein p53 and apoptosis[23, 29, 30].
The stimulation or inhibition of normal cell proliferation, therefore, serves as an early indication of potential abnormality within the intestinal tract, making these an appropriate model for study in toxicity bioassays[1, 31]. New intestinal epithelial cells are continuously produced by stem cells in the crypts which subsequently migrate along the crypt-villi axis. An increase in PCNA labelling, therefore, signals marked increases in the rate of cellular division. A number of experimental studies have examined the cellular localisation of PCNA within the intestines of fish exposed to a model toxicant and through the dietary administration of compounds[25, 32, 33], but no information exists regarding the expression of PCNA in infected fish tissues. The acanthocephalan D. truttae, in addition to its armed proboscis, bears trunk spines that facilitate its attachment within the intestinal villi of its fish host. These spines, during the process of attachment, inflict damage to the intestinal folds, causing destruction of the villi epithelium resulting in the development of necrotic tissue. The immunohistochemical results demonstrate that the levels of acanthocephalan infection observed in the current study (i.e. 5–320 individuals fish-1) effect a significant increase in the number of PCNA-positive cells in the intestinal villi that are close to the sites of parasite attachment. The increase in PCNA-positive epithelial cells were observed within the villi crypts that were either occupied by or close to intestinal helminths, when compared to the lower numbers seen in uninfected fish or in villi that were situated at least 0.7 cm away from the site of helminth activity.
The extent of intestinal damage inflicted by acanthocephalans is related to the intensity of infection and the degree to which the parasite penetrate the host tissues[35, 36]. Parasitic infection of the alimentary canal can have detrimental effects on digestive function[10, 37], with many species of intestinal helminth inducing an inflammatory response at the site of attachment[21, 38]. In fish, the innate defences in response to helminth infection are associated with inflammatory reactions[10, 18, 39]. The innate immunity of teleosts, involves a range of cell types, which commonly include MCs[13, 40, 41]. In perciform fish, it has been reported that the mast cells contain histamine, which can regulate the fish’s inflammatory responses. Moreover, MCs degranulation has been shown to promote intestinal contraction in Sparus aurata L. and in Oncorhynchus mykiss (Walbaum)[42, 43].
Although Roberts et al. introduced the term eosinophilic granule cell, there has been a tendency in recent years to use the term mast cell as these cells have functional and morphological similarities to [mammalian] MCs[45, 46]. MCs are present in most species of teleost and are found in a variety of tissues, including the gastrointestinal tract, skin and gills[45–47]. MCs are motile[19, 48] and until recently, their origin was unclear but it is now known that in mammals their precursors are from pluripotent bone marrow-derived stem cells circulating in the blood and lymphatic fluid. In fish, it is most likely that MCs differentiate in the haematopoietic organs and reach their target tissues via the circulatory system as immature cells, as has been reported for mammalian MCs.
Evidence of MCs migration has been documented in the gills of rainbow trout, O. mykiss, exposed to bacteria and in the intestine of fish infected with tapeworms. In all vertebrates, MCs may be strategically positioned at perivascular sites to regulate inflammatory responses[42, 49]. This places them in a unique position to encounter invading organisms and to orchestrate a response. In the current study, and in particular within the intestines of infected fish, numerous MCs were seen in close contact with capillaries and the outer layer of the endothelia as well as within the lumen of the blood vessels. MCs play an important role in responding to inflammation; their number increases in allergic reactions and as a consequence of helminth infection[39, 53–55]. The close association of MCs with the endothelial cells of capillaries and their presence within gill capillaries suggests that they may migrate across the endothelium[47, 52, 54]. Nonetheless, the intra-tissue migratory nature of MCs has been observed in the gills and intestine of fish[48, 56, 57]. In addition, the occurrence of MCs throughout the loose connective tissue of the gill arch, suggests that there is a resident population of these cells[47, 56]. In the current study, numerous MCs were observed within the connective tissue, and both on the outside and within capillaries in the sub-mucosal layers of infected brown trout intestines. Based on a considerable body of descriptive data, it is reasonable to presume that fish may have two populations of MCs, a circulating and a resident population, and the presence of parasites may induce recruitment of MCs to the site(s) of infection[18, 20]. Accordingly, acute MC activation is a feature of many types of tissue injury; experimental studies have demonstrated that pathogen products can activate MCs.
In humans, PCNA-positive MCs have been observed in the nasal sub-epithelial and lamina propria layers in patients suffering allergic rhinitis. A proliferation of mature MCs has also been reported in the nasal mucosa of those suffering allergies[60–62]. In the current study, a high number of PCNA-positive cells were seen in the sub-mucosal layer, immediately around the proboscis of D. truttae; most of these were MCs with some fibroblasts. The co-occurrence of fibres-fibroblasts and MCs has been described from a range of fish species including O. mykiss (see Flaño et al. 1996), coho salmon, Oncorhynchus kisutch (Walbaum) (see) and minnows, Phoxinus phoxinus L. (see). It has been suggested that MCs have the potential to directly influence fibroblasts and/or indirectly influence other cells, leading to a profibrotic response. Several lines of evidence suggest that MCs are involved in the fibrotic process and in tissue remodelling[66, 67]. In the process of attachment, the proboscis of D. truttae penetrates deep into the sub-mucosal layer and destroys the architecture of the host’s intestinal wall; the recruitment and proliferation of numerous MCs around the site of proboscis insertion may serve also to repair and remodel damaged intestinal tissue.