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Taenia taeniaeformis: Fate and proliferation of mucosal cells during gastric hyperplasia in larvae infected rats
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Experimental Parasitology 118 (2008) 576–582 www.elsevier.com/locate/yexpr
Taenia taeniaeformis: Fate and proliferation of mucosal cells during gastric hyperplasia in larvae infected rats J.T. Lagapa a b
a,b,c,*
, Y. Oku b, N. Nonaka b, M. Kamiya
c
Department of Medicine, Surgery and Zootechnics, College of Veterinary Medicine, Central Mindanao University, Musuan, Bukidnon 8710, Philippines Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan c Laboratory of Environmental Zoology, Department of Biosphere and Environmental Sciences, Faculty of Environmental Systems, Rakuno Gakuen University, Ebetsu 069-8501, Japan Received 17 April 2007; received in revised form 19 October 2007; accepted 10 December 2007 Available online 28 January 2008
Abstract Fate and proliferation of gastric mucosal cells during hyperplasia of Taenia taeniaeformis eggs inoculated Wistar rats were investigated using PCNA immunohistochemistry, BrdU labeling and other histopathologic staining techniques. Results revealed marked cell proliferation in gastric corpus and antral mucosa of infected rats as evidenced by increased lengths of proliferative zones and indices of BrdU labeling. The gastropathy in corpus was characterized by massive accumulation of precursors, neck and intermediate cells following significant decreases in numbers of parietal and zymogenic cells. Gastropathy in antrum was described with significant increases in precursors and mucous cells. Our results suggested that T. taeniaeformis-induced gastric hyperplasia was initiated by depletion of parietal cells presumably due to the cestode’s ES products. As a result, there was inhibition of zymogenic cell differentiation due to the disruption of normal development pathways of gastric mucosal lineages. These sequences of events were considered to cause the increase in cell proliferation and accumulation of intermediate cells resulting to the hyperplastic lesions. Ó 2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Taenia taeniaeformis; Cestode; Cell proliferation; Gastric mucosa; Hyperplasia; AB–PAS, alcian blue–periodic acidSchiff reaction; BSA, bovine serum albumin; BrdU, bromodeoxyuridine; ES, excretory–secretory; HIER, heat-induced epitope retrieval; HPI, hours postinoculation; MXL, modified Maxwell staining; NBF, neutral buffered formalin; PBS, phosphate buffered solution; PCNA, proliferating cell nuclear antigen; SD, standard deviation; TGFa, transforming growth factor–alpha; WPI, weeks post-inoculation
1. Introduction Parasites in general produces pathological changes in organs or tissues of hosts where they are located, however, Taenia taeniaeformis larvae infection in the liver of rats induced hyperplasia in the gastrointestinal mucosa (Cook and Williams, 1981). Previous studies showed that the development of lesions is T cell independent (Abella et al., 1997a; Lagapa et al., 2002a) and is not a by product of increase gastrin secretions in the stomach (Konno et al., *
Corresponding author. Address: Laboratory of Environmental Zoology, Department of Biosphere and Environmental Sciences, Faculty of Environmental Systems, Rakuno Gakuen University, Ebetsu 069-8501, Japan. Fax: +81 11 3884909. E-mail address: [email protected] (J.T. Lagapa). 0014-4894/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.12.004
1999b). This phenomenon was suggested to be due to larval ES products that induced the pathological effects remote from predilection site of the parasite (Lagapa et al., 2002b). Hyperplastic gastropathy in rats due to T. taeniaeformis larvae infection have been described grossly by increases in sizes and weights of the stomachs. Gastric mucosa was characterized as heavily thickened with excessive mucous production. Histologically, mucous cells positive to AB and/or PAS were causing several fold increases on height of gastric units (Blaies and Williams 1983; Rikihisa et al., 1984; Abella et al., 1997a; Konno et al., 1999a). Increased accumulation of cells similar to immature surface mucous cells was observed and significant decreases in number of parietal and zymogenic cells are associated with concomitant increase of mucous cells (Lagapa et al., 2002a). Isolated gas-
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tric mucosa from infected rats had an increased 3H-thymidine incorporation in organ culture suggesting the cause as stimulation of growth and major differentiation of stem cells to neutral mucus producing cells (Rikihisa and Lin, 1984). Present study aimed to outline the cellular fate and proliferation involved in T. taeniaeformis-induced gastropathy. Further understanding of these events could provide insights regarding the pathogenic mechanisms of this phenomenon and related gastropathies. To our knowledge, this is the first report on fate and proliferation of gastric mucosal cells during a remote parasitic infection. 2. Materials and methods 2.1. Animals Thirty male Wistar rats 5 weeks old (CLEA, Japan) were used in this study. The rats were divided into five groups with four inoculated rats (orally dosed with approximately 4000 T. taeniaeformis eggs) and two uninoculated rats (as control) per group. A group was sacrificed at 6 WPI. The other four groups were used for time course labeling of BrdU on 9 WPI. 2.2. Parasites T. taeniaeformis eggs were collected from proglottids expelled by cats infected with a rat strain that has originally been isolated from Norways rats (Rattus norvegicus) in Sapporo, Japan, and maintained in our laboratory using cats and rats (Konno et al., 1999b). Proglottids were stored at 4 °C for at most one month in physiologic saline with penicillin and streptomycin. Eggs were extracted by mincing, crushing and repeated washing of the gravid tapeworm segments on a wire mesh (Abella et al., 1997b). Egg viability was determined by counting the number of oncospheres per number of eggs in a 10 ll aliquot, after hatching using sodium hypochlorite solution (Kanto Chem. Co. Inc., Japan).
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and allowed to boil for 10 min, cooled and washed with PBS two times. Anti-PCNA was diluted 1:10 with 1% BSA in 0.1 mM PBS and incubated for 30 min. Immunostained slides were determined for the degree of PCNA staining by measuring the length between the highest and lowest located PCNA-positive cells, which corresponded as the proliferative zone (Fox et al., 1996). 2.5. BrdU immunohistochemistry Rats were intraperitoneally injected with BrdU (Sigma) at a dose of 80 mg/kg body weight from a freshly diluted solution of 10 mg/ml in PBS. Groups of injected control and inoculated rats were sacrificed on 2, 6, 24 and 96 h after BrdU injection. Tissues fixed in 10% NBF were used for immunohistochemical detection of BrdU incorporation using a commercial BrdU staining kit (Zymed, CA, USA). Sections were processed according to the manufacturer’s suggested procedure except for some modified methods mentioned in PCNA immunohistochemistry. Biotinylated mouse anti-BrdU was diluted 1:5 with 1% BSA in 0.1 mM PBS and incubated with tissues for 30 min. Labeling index was determined by counting the number of BrdU-positive cells and expressing the result as a percentage ratio of the total number of cells per gastric and mucous units (Fox et al., 1996). 2.6. Gastric mucosal cell fate and hyperplasia Sections from corpus mucosa were stained using Alcian blue–periodic acid-Schiff reaction (AB–PAS) and modified Maxwell method as described by Martinelli et al. (1996). Counting of the total number of cells per corpus and mucous units and identification of the major type of cells were made from AB–PAS and modified Maxwell stained sections based on the description of Karam (1998) and Konno et al. (1999a). As described by Martinelli et al. (1996), zymogenic cells stain red-purple while parietal cells give a blue-green color and mucous neck cells yellow in the modified Maxwell method.
2.3. Histopathology 2.7. Analysis Stomachs were harvested and divided into two equal parts, one was fixed with 10% NBF and the other with 40% formaldehyde. Tissues were routinely processed for paraffin embedding and cut appropriate to the techniques conducted. 2.4. PCNA immunohistochemistry Gastric tissues from rats sacrificed on 6 and 9 WPI fixed in 10% NBF were used for PCNA immunostaining. A PCNA commercial staining kit (Zymed, CA, USA) was used and procedure was done according to the manufacturer’s recommendation except for modifications mentioned hereafter. Peroxidase quenching solution and blocking reagent was incubated with the tissues for 20 min each. Slides were subjected with HIER method by submerging into 0.01 M citrate buffer (pH 6.0), heated
Assessment of number of immunopositive and major type of cells was done from three to five sections per rat in 5–10 complete or at least 75% longitudinally cut corpus and mucous units. Statistical calculation of BrdU means between hours post-injection was conducted using analysis of variance and differences between means of control and treated groups were computed by the unpaired Student’s t-test. P values < 0.05 were considered significant. 3. Results 3.1. Gastric corpus hyperplasia At 6 WPI, lengths of pit regions (topmost immature cell in isthmus to tip of corpus unit) in gastric mucosa of
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infected rats were increased compared to control rats. Differences, however, in the total lengths of corpus units were not statistically significant. Neutral mucous AB–PAS-positive and Maxwell stained pyronin cells were slightly increased in mucosa of infected rats. Microscopically, these cells resembled the immature cells of isthmus region. Some of these cells were classified as either pre-pit or pre-neck mucous cells. Parietal and zymogenic cell counts were significantly decreased. Maxwell staining intensity of remaining parietal cell population was reduced. Infected rats sacrificed at 9 WPI were observed with severe gastric corpus hyperplasia (Fig. 1a–c). Maxwell stained pyronin pit cells occupied the top most part of the mucosa
(Fig. 1d). Majority of the cells at the middle region were neutral mucus-magenta cells and yellowish pigments in AB– PAS and MXL staining, respectively (Fig. 1e). Basal parts of hyperplastic mucosa were occupied with mucous neck cells being periodic acid-Schiff- and Maxwell staining alcian yellow-positive (Fig. 1c and f). These cells invaded the normal location of zymogenic cells. Parietal and zymogenic cells were rarely found in the hyperplastic mucosa. There were abundant AB–PAS- and Maxwell staining-yellow-positive cells in lower parts of the hyperplastic mucosa identified as intermediate cells. Mean total number of cells per gastric unit in infected rats increased fivefold [785–912; ave: 837.2 ± 36.1 (SD)] more than the control mucosa [152–182; ave:
Fig. 1. Hyperplastic mucosa of infected rats. (a) Approximately from the upper third part of a hyperplastic corpus stained by AB–PAS showed significant increase of AB–PAS-positive pre-pit cells (light red), pit cells (red) and intermediate cells (reddish blue); (b) from the lower third part of a hyperplastic corpus with an increasing number of intermediate cells (reddish blue) and neck cells (magenta); (c) basal part of a hyperplastic corpus occupied by intermediate cells (reddish blue) and neck cells (magenta) with the absence of zymogenic cells; (d) uppermost part of hyperplastic corpus stained with Maxwell showed the presence of cystic cavities and pit cells with red pigments; (e) middle region of a Maxwell stained hyperplastic corpus invaded by neck and intermediate cells (yellow and yellow green); (f) lower third of hyperplastic corpus occupied mostly by neck cells (yellow green); (g) uppermost part of a hyperplastic antrum with increased number of mucous cells (reddish blue); (h) the middle part of a hyperplastic antrum filled with undifferentiated cells (pink); (i) Maxwell stained middle part of hyperplastic antrum with undifferentiated cells (blue with red pigmentation and yellow surfaces); (j) basal hyperplastic antrum by AB–PAS staining with gland cells (blue) and increased number of mucous cells (reddish blue). Bars: 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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164.1 ± 11.4 (SD)]. Abundant cystic cavities were seen with mucus secretions inside. 3.2. Gastric antral hyperplasia Antral mucosa of infected rats appeared without visible lesions the same with that of the control rats at 6 WPI. At 9 WPI, antral mucosa of inoculated rats was hyperplastic with lengths increased four- to fivefold as compared to the control rats. The upper two third of mucous units were invaded by PAS-positive mucous cells with some AB–PAS-positive and Maxwell yellow stained cells at surfaces of the units (Fig. 1g–i). Lower third part of mucous units was heavily occupied with immature and precursor cells (Fig. 1j). Cystic cavities were also observed. 3.3. PCNA staining in corpus and antral region Immunostaining using PCNA was done in mucosa of control and infected rats at 6 and 9 WPI. Increased proliferative zones included the whole length of corpus units (Fig. 2a), while PCNA-positive cells in control mucosa were located in the isthmus regions (Fig. 2b). These cells were identified as immature and precursor cells. Mean lengths of proliferative zones were greater in corpus mucosa of infected rats than the control at both 6 and 9 WPI (P < 0.0001; Fig. 3). In antral mucosa, PCNA-labeled cells were found in isthmus region (Fig. 2c) but these cells occupied the whole length of the hyperplastic antral mucosa at 9 WPI (Fig. 2d). No difference in mean length of proliferative zones was observed between control and infected rats at 6 WPI, whereas at 9 WPI proliferative zones of antral mucosa of infected rats were greater than the control (Fig. 3, P < 0.0001). Labeled cells by PCNA were ranging from immature and precursor cells to mucous neck and intermediate cells. 3.4. BrdU labeling in corpus region At 2 HPI of BrdU, labeled cells of mucosa in control rats were located at isthmus region (Fig. 4a). In hyperplastic mucosa of infected rats these were found scattered throughout the lengths of corpus units (Fig. 4b and c). Labeling indices in mucosa of infected rats were higher compared to the control (P < 0.0001; Fig. 5a). Serial histopathologic sections revealed that BrdU-labeled cells were immature and precursor cells. The BrdU labeling indices at 6 HPI, in both the control and infected rats were increased. A remarkable increase was observed in the labeling index of hyperplastic mucosa of infected rats (P < 0.0001, Fig. 5a). Most cells in control mucosa were located at isthmus region, classified as immature cells and few cells have migrated to the neck region as pre-neck cells. BrdU-labeled cells in hyperplastic mucosa were found throughout the gastric units. These cells were mixtures of immature, pre-pit and pre-neck cells.
Fig. 2. PCNA immunohistochemistry. (a) proliferative zones (PZ) extending from base to tip of hyperplastic mucosa in corpus of infected rats; (b) proliferative zones in corpus mucosa of control concentrated on isthmus region; (c) proliferative zones in antral mucosa of control located in isthmus at basal part; (d) hyperplastic mucosa of antrum with proliferative zones including the whole lengths of mucous units. Bars: a and b = 200 lm; c and d = 100 lm.
In infected rats, BrdU labeling index at 24 HPI dropped when compared to the 6 HPI (P < 0.0001; Fig. 5a). Mean labeling index of infected rats, however, was still significantly higher if paired with the control rats (P < 0.0002). Some of the labeled cells in control rats were located in pit region as mucous pit cells while most cells were found in neck region as pre-neck or mucous neck cells. Labeled cells in hyperplastic mucosa were scattered throughout
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Fig. 3. Mean lengths of proliferative zones in gastric mucosa of control and infected rats at 6 and 9 weeks post-infection. Control (open columns); infected (closed columns). Asterisk (*) means P < 0.05. Bars = SD.
the corpus units, as immature, pre-neck or mucous neck cells and pre-pit or pit cells. The BrdU labeling index decreased in infected rats at 96 HPI from 24 HPI (P = 0.0001; Fig. 5a). Control rats apparently have decreased labeling index (Fig. 4d) but not statistically different from 24 HPI. Labeled cells in mucosa of control rats were identified as mostly pit and neck mucous cells. In infected rats, most cells of the hyperplastic mucosa were described as neck and intermediate mucous cells (Fig. 4e and f). Mean labeling index of the hyperplastic mucosa at 96 HPI was greater than the control rats (P = 0.0060). 3.5. BrdU labeling in antral region Labeling indices in antral mucosa of infected rats were higher than control rats at 2 HPI (P < 0.0001; Fig. 5b). Labeled cells in control mucosa were located in the basal parts of mucosa (Fig. 4g) while they were found in the whole length of mucous units in infected rats (Fig. 4h). At 6 HPI, no significant changes were observed in labeling indices of both mucosas. Labeling index of hyperplastic mucosa decreased at 24 HPI, while lesser changes were observed in mucosa of control rats except the location of labeled cells in middle part of antral mucosa. Hyperplastic antral mucosa (Fig. 4i) of inoculated rats further decreased its labeling index at 96 HPI while lesser BrdU-labeled cells were observed in mucosa of control rats (Fig. 4j). 4. Discussion The study determined the rate of epithelial cell proliferation by time course labeling with BrdU and PCNA immunohistochemical staining. The time course of BrdU labeling reflected the stimulation of DNA synthesis and subsequent cell replication within the mucosa (Ohning and Guth, 1995)
whereas PCNA labeling indicated level of cell mitosis in S, G2 and M phases. Fates of major gastric cell types in the hyperplastic gastric mucosa were examined using various staining techniques. Cell proliferations are restricted to isthmus regions in gastric mucosa. Granule-free cells are most proliferative in isthmus regions, although, pre-pit, pre-neck and their precursor cells have also some potential for mitosis (Karam, 1998). In this study, however, regions of the hyperplastic mucosa of inoculated rats were not recognizable because of numerous cystic cavities. Furthermore, epithelial cells were not found in their normal locations. This abnormality displaced the isthmus region and the whole length of corpus and mucous units was transformed as proliferative zones. These were demonstrated by the presence of BrdU and PCNA-labeled cells throughout the lengths of hyperplastic mucosa. Hyperplasia of gastric corpus epithelium was observed primarily as the result of a remarkable increase in the rate of cell proliferation and accumulation of intermediate precursor cells. Significant increase in lengths of proliferative zones at 6 WPI even without marked mucosal hyperplastic lesions yet, suggested initiation of hyperproliferative activity at this point. The dramatic increase in labeling indices of BrdU and proliferative zone lengths at 9 WPI created severe hyperplastic lesions in mucosa of infected rats. These events suggested obvious association between excessive cell proliferation and gastric mucosal hyperplasia. It was showed in SCID mice that larval ES products have stimulated hyperproliferation leading to hyperplastic changes (Lagapa et al., 2002b). Phenomenal elevation in labeling indices of BrdU within 6 h indicated that the specific inducing factor must have tremendous effect on the mitotic activity of gastric cell poll either directly or indirectly. Karam and Leblond (1993) reported that labeled cells in mice 6 h after injection with 3H-thymidine injection were granule-free cells
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Fig. 5. Time course BrdU labeling indices (LI) of gastric mucosa between control and infected rats during 9 WPI at 2, 6, 24 and 96 h. (a) corpus mucosa; (b) antral mucosa. Control (open circles); infected rats (closed circles). Asterisk (*) means P < 0.05. Bars = SD.
Fig. 4. Bromodeoxyuridine (BrdU) immunohistochemistry of gastric mucosa at 9 weeks post-infection. (a) control corpus mucosa at 2 h post-injection (HPI) of BrdU; (b and c) hyperplastic corpus mucosa at 2 HPI; (d) control corpus mucosa at 96 HPI; (e and f) hyperplastic corpus mucosa at 96 HPI; (g) control antral mucosa at 2 HPI; (h) hyperplastic antral mucosa at 2 HPI; (i) hyperplastic antral mucosa at 96 HPI; (j) control antral mucosa at 96 HPI. Bars: 50 lm.
with pre-pit and pre-neck mucous cells. In this study, however, hyperplastic mucosa was composed already of mature pit and neck mucous cells indicating an unusually rapid differentiation of cells.
The remarkable loss of parietal cells in the corpus units of infected rats at early stages corroborated with a previous study that parietal cells were significantly ablated even in slight hyperplastic lesions (Lagapa et al., 2002a). Further, parietal cell dysfunction was proposed as the key event leading to loss of mature zymogenic cells resulting to mucous cell hyperplasia in abomasal nematodosis (Simpson, 2000). Nomura et al., 2005 established that parietal cells secrete a number of growth factors that influence the differentiation of other gastric lineages. The accompanying loss of zymogenic cells was likely a consequence of the interruption of the normal development pathway in the gastric mucosa that followed after destruction of parietal cells (Marshall et al., 2005). Parietal cells were observed to play a central role in the regulation of mucosal proliferation during gastric inflammations (Beales, 2004). Thus, we hypoth-
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esized that the loss of parietal cells brought chaos in the gastric mucosal lineage differentiations that caused the increase proliferation of gastric cells and accumulation of immature and undifferentiated cells. Accumulation of intermediate forms of cells suggested also a cell dynamic attempting to replace the loss of parietal and zymogenic cells. As previously reported by Fujita and Kaneko (1994), mucous neck cells were converted to zymogenic cells via intermediate cells. Reports also noted that during gastric ulcer healing, parietal cells are not formed directly from dividing cells, but were recruited from existing precursor cells (Helander and Poorkhalkali, 2004). Based on the above claims and our present findings, we extrapolated a proposition that cessation of normal developmental pathway to zymogenic cells was the cause of the massive accumulation of intermediate cells. Hyperproliferation and accumulation of immature cells with decreased number of parietal and zymogenic cells were noted also in murine autoimmune gastritis (Franic et al., 2004). A critical feature in this gastropathy was the occurrence of corpus hyperplasia prior to antral lesions. The absence of increased proliferative zone at 6 WPI by PCNA staining in the antrum implied that epithelial hyperplasia was an aftermath of the corpus gastropathy. From this sequential development of the lesion from corpus to antrum, a basis for a strong argument that factors from larval ES products could have first evoked its effects on the corpus mucosa as shown also during a parabiotic transfer experiment (Cook and Williams, 1981). Thus, our result indicated that T. taeniaeformis larvaeinduced gastropathy might be associated with the effect of larval ES products on the depletion of parietal cells. Our finding could be summarized to have started with parietal cell loss followed by the blocking of zymogenic cell differentiation. The loss of control in cell differentiation caused the accumulation of intermediate cells. Marked depletion of the major cells of corpus mucosa initiated accompanying increase of stem cell proliferation that enhanced further accumulation of intermediate mucous cells. Antral lesions emerged later with mucous cell hyperproliferation of which cause is still unclear. References Abella, J.A., Oku, Y., Konno, K., Altamirano, Z., Nonaka, N., Kamiya, M., 1997a. Concomitant onset of hypergastrinemia, intragastric alkalinity and gastric hyperplasia, and numbers of antral G cells in Taenia taeniaeformis-infected rats. Parasitology International 46, 97– 104. Abella, J.A., Oku, Y., Nonaka, N., Ito, M., Kamiya, M., 1997b. Role of host immune response in the occurrence of gastropathy in rats infected with larval Taenia taeniaeformis. Journal of Veterinary Medical Science 59, 1039–1043. Beales, I.L., 2004. Gastrin and interleukin-1 beta stimulate growth factor secretion from cultured rabbit gastric parietal cells. Life Science 75, 2983–2995.
Blaies, D.M., Williams, J.F., 1983. Taenia taeniaeformis: gastrointestinal hyperplasia in chronically infected rats. Experimental Parasitology 55, 197–206. Cook, R.W., Williams, J.F., 1981. Pathology of Taenia taeniaeformis infection in the rat: gastrointestinal changes. Journal of Comparative Pathology 91, 205–217. Fox, J.G., Li, X., Cahill, R.J., Andrutis, K., Rustgi, A.K., Odze, R., Wang, T.C., 1996. Hypertrophic gastropathy in Helicobacter felisinfected wild-type C57BL/6 mice and p53 hemizygous transgenic mice. Gastroenterology 110, 155–166. Franic, T.V., Judd, L.M., Nguyen, N.V., Samuelson, L.C., Loveland, L.L., Giraud, A.S., Gleeson, P.A., van Driel, I.R., 2004. Growth factors associated with gastric mucosal hypertrophy in autoimmune gastritis. American Journal of Physiology 287, G910–G918. Fujita, K., Kaneko, K., 1994. Immunohistochemical examination of the relationship between two types of mucous neck cells and the intermediate cells in the rat fundic gland. Okajimas Folia Anatomica Japonica 71, 137–142. Helander, H.F., Poorkhalkali, N., 2004. Parietal cell density during gastric ulcer healing in the rat. Scandinavian Journal of Gastroenterology 39, 20–26. Karam, S.M., 1998. Cell lineage relationship in the stomach of normal and genetically manipulated mice. Brazilian Journal of Medicine and Biological Research 31, 271–279. Karam, S.M., Leblond, C.P., 1993. Dynamics of epithelial cells in the corpus of the mouse stomach III. Inward migration of neck cells followed by progressive transformation into zymogenic cells. The Anatomical Record 236, 297–313. Konno, K., Abella, J.A., Oku, Y., Nonaka, N., Kamiya, M., 1999a. Histopathology and physiopathology of gastric mucous hyperplasia in rats heavily infected with Taenia taeniaeformis. Journal of Veterinary Medical Science 61, 317–324. Konno, K., Oku, Y., Nonaka, N., Kamiya, M., 1999b. Hyperplasia of gastric mucosa in donor rats orally infected with Taenia taeniaeformis eggs and in recipient rats surgically implanted with the larvae in the abdominal cavity. Parasitology Research 85, 431–436. Lagapa, J.T., Konno, K., Oku, Y., Nonaka, N., Ito, M., Kamiya, M., 2002a. Gastric hyperplasia and parietal cell loss in Taenia taeniaeformis inoculated immunodeficient mice. Parasitology International 51, 81–89. Lagapa, J.T., Oku, Y., Nonaka, N., Ito, M., Kamiya, M., 2002b. Taenia taeniaeformis larval product induces gastric mucosal hyperplasia in SCID mice. Japanese Journal of Veterinary Research 49, 273–285. Marshall, A.C., Alderuccio, F., Murphy, K., Toh, B.H., 2005. Mechanisms of gastric mucosal cell loss in autoimmune gastritis. International Reviews of Immunology 24, 123–134. Martinelli, T.M., van Driel, I.R., Alderuccio, F., Gleeson, P.A., Toh, B.H., 1996. Analysis of mononuclear cell infiltrate and cytokine production in murine autoimmune gastritis. Gastroenterology 110, 1791–1802. Nomura, S., Yamaguchi, H., Ogawa, M., Wang, T.C., Lee, J.R., Goldenring, J.R., 2005. Alterations in gastric mucosal lineages induced by acute oxyntic atrophy in wild-type and gastrin-deficient mice. American Journal of Physiology 288, G362–G375. Ohning, G.V., Guth, P.H., 1995. Time course of mucosal cell proliferation following acute aspirin injury in rat stomach. Digestive Disease Science 40, 1351–1356. Rikihisa, Y., Letonja, T., Pratt, N., Lin, Y.C., 1984. Taenia taeniaeformis: characterization of larval metabolic products and growth of host gastric cells in vitro. Experimental Parasitology 58, 230–238. Rikihisa, Y., Lin, Y.C., 1984. Taenia taeniaeformis: increased cell growth and neutral mucus production in the gastric mucosa of the rat with a larval infection. Experimental Parasitology 58, 147–155. Simpson, H.V., 2000. Pathophysiology of abomasal parasitism: is the host or parasite responsible? The Veterinary Journal 160, 177–190.
Experimental Parasitology 118 (2008) 576–582 www.elsevier.com/locate/yexpr
Taenia taeniaeformis: Fate and proliferation of mucosal cells during gastric hyperplasia in larvae infected rats J.T. Lagapa a b
a,b,c,*
, Y. Oku b, N. Nonaka b, M. Kamiya
c
Department of Medicine, Surgery and Zootechnics, College of Veterinary Medicine, Central Mindanao University, Musuan, Bukidnon 8710, Philippines Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan c Laboratory of Environmental Zoology, Department of Biosphere and Environmental Sciences, Faculty of Environmental Systems, Rakuno Gakuen University, Ebetsu 069-8501, Japan Received 17 April 2007; received in revised form 19 October 2007; accepted 10 December 2007 Available online 28 January 2008
Abstract Fate and proliferation of gastric mucosal cells during hyperplasia of Taenia taeniaeformis eggs inoculated Wistar rats were investigated using PCNA immunohistochemistry, BrdU labeling and other histopathologic staining techniques. Results revealed marked cell proliferation in gastric corpus and antral mucosa of infected rats as evidenced by increased lengths of proliferative zones and indices of BrdU labeling. The gastropathy in corpus was characterized by massive accumulation of precursors, neck and intermediate cells following significant decreases in numbers of parietal and zymogenic cells. Gastropathy in antrum was described with significant increases in precursors and mucous cells. Our results suggested that T. taeniaeformis-induced gastric hyperplasia was initiated by depletion of parietal cells presumably due to the cestode’s ES products. As a result, there was inhibition of zymogenic cell differentiation due to the disruption of normal development pathways of gastric mucosal lineages. These sequences of events were considered to cause the increase in cell proliferation and accumulation of intermediate cells resulting to the hyperplastic lesions. Ó 2007 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Taenia taeniaeformis; Cestode; Cell proliferation; Gastric mucosa; Hyperplasia; AB–PAS, alcian blue–periodic acidSchiff reaction; BSA, bovine serum albumin; BrdU, bromodeoxyuridine; ES, excretory–secretory; HIER, heat-induced epitope retrieval; HPI, hours postinoculation; MXL, modified Maxwell staining; NBF, neutral buffered formalin; PBS, phosphate buffered solution; PCNA, proliferating cell nuclear antigen; SD, standard deviation; TGFa, transforming growth factor–alpha; WPI, weeks post-inoculation
1. Introduction Parasites in general produces pathological changes in organs or tissues of hosts where they are located, however, Taenia taeniaeformis larvae infection in the liver of rats induced hyperplasia in the gastrointestinal mucosa (Cook and Williams, 1981). Previous studies showed that the development of lesions is T cell independent (Abella et al., 1997a; Lagapa et al., 2002a) and is not a by product of increase gastrin secretions in the stomach (Konno et al., *
Corresponding author. Address: Laboratory of Environmental Zoology, Department of Biosphere and Environmental Sciences, Faculty of Environmental Systems, Rakuno Gakuen University, Ebetsu 069-8501, Japan. Fax: +81 11 3884909. E-mail address: [email protected] (J.T. Lagapa). 0014-4894/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2007.12.004
1999b). This phenomenon was suggested to be due to larval ES products that induced the pathological effects remote from predilection site of the parasite (Lagapa et al., 2002b). Hyperplastic gastropathy in rats due to T. taeniaeformis larvae infection have been described grossly by increases in sizes and weights of the stomachs. Gastric mucosa was characterized as heavily thickened with excessive mucous production. Histologically, mucous cells positive to AB and/or PAS were causing several fold increases on height of gastric units (Blaies and Williams 1983; Rikihisa et al., 1984; Abella et al., 1997a; Konno et al., 1999a). Increased accumulation of cells similar to immature surface mucous cells was observed and significant decreases in number of parietal and zymogenic cells are associated with concomitant increase of mucous cells (Lagapa et al., 2002a). Isolated gas-
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tric mucosa from infected rats had an increased 3H-thymidine incorporation in organ culture suggesting the cause as stimulation of growth and major differentiation of stem cells to neutral mucus producing cells (Rikihisa and Lin, 1984). Present study aimed to outline the cellular fate and proliferation involved in T. taeniaeformis-induced gastropathy. Further understanding of these events could provide insights regarding the pathogenic mechanisms of this phenomenon and related gastropathies. To our knowledge, this is the first report on fate and proliferation of gastric mucosal cells during a remote parasitic infection. 2. Materials and methods 2.1. Animals Thirty male Wistar rats 5 weeks old (CLEA, Japan) were used in this study. The rats were divided into five groups with four inoculated rats (orally dosed with approximately 4000 T. taeniaeformis eggs) and two uninoculated rats (as control) per group. A group was sacrificed at 6 WPI. The other four groups were used for time course labeling of BrdU on 9 WPI. 2.2. Parasites T. taeniaeformis eggs were collected from proglottids expelled by cats infected with a rat strain that has originally been isolated from Norways rats (Rattus norvegicus) in Sapporo, Japan, and maintained in our laboratory using cats and rats (Konno et al., 1999b). Proglottids were stored at 4 °C for at most one month in physiologic saline with penicillin and streptomycin. Eggs were extracted by mincing, crushing and repeated washing of the gravid tapeworm segments on a wire mesh (Abella et al., 1997b). Egg viability was determined by counting the number of oncospheres per number of eggs in a 10 ll aliquot, after hatching using sodium hypochlorite solution (Kanto Chem. Co. Inc., Japan).
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and allowed to boil for 10 min, cooled and washed with PBS two times. Anti-PCNA was diluted 1:10 with 1% BSA in 0.1 mM PBS and incubated for 30 min. Immunostained slides were determined for the degree of PCNA staining by measuring the length between the highest and lowest located PCNA-positive cells, which corresponded as the proliferative zone (Fox et al., 1996). 2.5. BrdU immunohistochemistry Rats were intraperitoneally injected with BrdU (Sigma) at a dose of 80 mg/kg body weight from a freshly diluted solution of 10 mg/ml in PBS. Groups of injected control and inoculated rats were sacrificed on 2, 6, 24 and 96 h after BrdU injection. Tissues fixed in 10% NBF were used for immunohistochemical detection of BrdU incorporation using a commercial BrdU staining kit (Zymed, CA, USA). Sections were processed according to the manufacturer’s suggested procedure except for some modified methods mentioned in PCNA immunohistochemistry. Biotinylated mouse anti-BrdU was diluted 1:5 with 1% BSA in 0.1 mM PBS and incubated with tissues for 30 min. Labeling index was determined by counting the number of BrdU-positive cells and expressing the result as a percentage ratio of the total number of cells per gastric and mucous units (Fox et al., 1996). 2.6. Gastric mucosal cell fate and hyperplasia Sections from corpus mucosa were stained using Alcian blue–periodic acid-Schiff reaction (AB–PAS) and modified Maxwell method as described by Martinelli et al. (1996). Counting of the total number of cells per corpus and mucous units and identification of the major type of cells were made from AB–PAS and modified Maxwell stained sections based on the description of Karam (1998) and Konno et al. (1999a). As described by Martinelli et al. (1996), zymogenic cells stain red-purple while parietal cells give a blue-green color and mucous neck cells yellow in the modified Maxwell method.
2.3. Histopathology 2.7. Analysis Stomachs were harvested and divided into two equal parts, one was fixed with 10% NBF and the other with 40% formaldehyde. Tissues were routinely processed for paraffin embedding and cut appropriate to the techniques conducted. 2.4. PCNA immunohistochemistry Gastric tissues from rats sacrificed on 6 and 9 WPI fixed in 10% NBF were used for PCNA immunostaining. A PCNA commercial staining kit (Zymed, CA, USA) was used and procedure was done according to the manufacturer’s recommendation except for modifications mentioned hereafter. Peroxidase quenching solution and blocking reagent was incubated with the tissues for 20 min each. Slides were subjected with HIER method by submerging into 0.01 M citrate buffer (pH 6.0), heated
Assessment of number of immunopositive and major type of cells was done from three to five sections per rat in 5–10 complete or at least 75% longitudinally cut corpus and mucous units. Statistical calculation of BrdU means between hours post-injection was conducted using analysis of variance and differences between means of control and treated groups were computed by the unpaired Student’s t-test. P values < 0.05 were considered significant. 3. Results 3.1. Gastric corpus hyperplasia At 6 WPI, lengths of pit regions (topmost immature cell in isthmus to tip of corpus unit) in gastric mucosa of
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infected rats were increased compared to control rats. Differences, however, in the total lengths of corpus units were not statistically significant. Neutral mucous AB–PAS-positive and Maxwell stained pyronin cells were slightly increased in mucosa of infected rats. Microscopically, these cells resembled the immature cells of isthmus region. Some of these cells were classified as either pre-pit or pre-neck mucous cells. Parietal and zymogenic cell counts were significantly decreased. Maxwell staining intensity of remaining parietal cell population was reduced. Infected rats sacrificed at 9 WPI were observed with severe gastric corpus hyperplasia (Fig. 1a–c). Maxwell stained pyronin pit cells occupied the top most part of the mucosa
(Fig. 1d). Majority of the cells at the middle region were neutral mucus-magenta cells and yellowish pigments in AB– PAS and MXL staining, respectively (Fig. 1e). Basal parts of hyperplastic mucosa were occupied with mucous neck cells being periodic acid-Schiff- and Maxwell staining alcian yellow-positive (Fig. 1c and f). These cells invaded the normal location of zymogenic cells. Parietal and zymogenic cells were rarely found in the hyperplastic mucosa. There were abundant AB–PAS- and Maxwell staining-yellow-positive cells in lower parts of the hyperplastic mucosa identified as intermediate cells. Mean total number of cells per gastric unit in infected rats increased fivefold [785–912; ave: 837.2 ± 36.1 (SD)] more than the control mucosa [152–182; ave:
Fig. 1. Hyperplastic mucosa of infected rats. (a) Approximately from the upper third part of a hyperplastic corpus stained by AB–PAS showed significant increase of AB–PAS-positive pre-pit cells (light red), pit cells (red) and intermediate cells (reddish blue); (b) from the lower third part of a hyperplastic corpus with an increasing number of intermediate cells (reddish blue) and neck cells (magenta); (c) basal part of a hyperplastic corpus occupied by intermediate cells (reddish blue) and neck cells (magenta) with the absence of zymogenic cells; (d) uppermost part of hyperplastic corpus stained with Maxwell showed the presence of cystic cavities and pit cells with red pigments; (e) middle region of a Maxwell stained hyperplastic corpus invaded by neck and intermediate cells (yellow and yellow green); (f) lower third of hyperplastic corpus occupied mostly by neck cells (yellow green); (g) uppermost part of a hyperplastic antrum with increased number of mucous cells (reddish blue); (h) the middle part of a hyperplastic antrum filled with undifferentiated cells (pink); (i) Maxwell stained middle part of hyperplastic antrum with undifferentiated cells (blue with red pigmentation and yellow surfaces); (j) basal hyperplastic antrum by AB–PAS staining with gland cells (blue) and increased number of mucous cells (reddish blue). Bars: 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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164.1 ± 11.4 (SD)]. Abundant cystic cavities were seen with mucus secretions inside. 3.2. Gastric antral hyperplasia Antral mucosa of infected rats appeared without visible lesions the same with that of the control rats at 6 WPI. At 9 WPI, antral mucosa of inoculated rats was hyperplastic with lengths increased four- to fivefold as compared to the control rats. The upper two third of mucous units were invaded by PAS-positive mucous cells with some AB–PAS-positive and Maxwell yellow stained cells at surfaces of the units (Fig. 1g–i). Lower third part of mucous units was heavily occupied with immature and precursor cells (Fig. 1j). Cystic cavities were also observed. 3.3. PCNA staining in corpus and antral region Immunostaining using PCNA was done in mucosa of control and infected rats at 6 and 9 WPI. Increased proliferative zones included the whole length of corpus units (Fig. 2a), while PCNA-positive cells in control mucosa were located in the isthmus regions (Fig. 2b). These cells were identified as immature and precursor cells. Mean lengths of proliferative zones were greater in corpus mucosa of infected rats than the control at both 6 and 9 WPI (P < 0.0001; Fig. 3). In antral mucosa, PCNA-labeled cells were found in isthmus region (Fig. 2c) but these cells occupied the whole length of the hyperplastic antral mucosa at 9 WPI (Fig. 2d). No difference in mean length of proliferative zones was observed between control and infected rats at 6 WPI, whereas at 9 WPI proliferative zones of antral mucosa of infected rats were greater than the control (Fig. 3, P < 0.0001). Labeled cells by PCNA were ranging from immature and precursor cells to mucous neck and intermediate cells. 3.4. BrdU labeling in corpus region At 2 HPI of BrdU, labeled cells of mucosa in control rats were located at isthmus region (Fig. 4a). In hyperplastic mucosa of infected rats these were found scattered throughout the lengths of corpus units (Fig. 4b and c). Labeling indices in mucosa of infected rats were higher compared to the control (P < 0.0001; Fig. 5a). Serial histopathologic sections revealed that BrdU-labeled cells were immature and precursor cells. The BrdU labeling indices at 6 HPI, in both the control and infected rats were increased. A remarkable increase was observed in the labeling index of hyperplastic mucosa of infected rats (P < 0.0001, Fig. 5a). Most cells in control mucosa were located at isthmus region, classified as immature cells and few cells have migrated to the neck region as pre-neck cells. BrdU-labeled cells in hyperplastic mucosa were found throughout the gastric units. These cells were mixtures of immature, pre-pit and pre-neck cells.
Fig. 2. PCNA immunohistochemistry. (a) proliferative zones (PZ) extending from base to tip of hyperplastic mucosa in corpus of infected rats; (b) proliferative zones in corpus mucosa of control concentrated on isthmus region; (c) proliferative zones in antral mucosa of control located in isthmus at basal part; (d) hyperplastic mucosa of antrum with proliferative zones including the whole lengths of mucous units. Bars: a and b = 200 lm; c and d = 100 lm.
In infected rats, BrdU labeling index at 24 HPI dropped when compared to the 6 HPI (P < 0.0001; Fig. 5a). Mean labeling index of infected rats, however, was still significantly higher if paired with the control rats (P < 0.0002). Some of the labeled cells in control rats were located in pit region as mucous pit cells while most cells were found in neck region as pre-neck or mucous neck cells. Labeled cells in hyperplastic mucosa were scattered throughout
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Fig. 3. Mean lengths of proliferative zones in gastric mucosa of control and infected rats at 6 and 9 weeks post-infection. Control (open columns); infected (closed columns). Asterisk (*) means P < 0.05. Bars = SD.
the corpus units, as immature, pre-neck or mucous neck cells and pre-pit or pit cells. The BrdU labeling index decreased in infected rats at 96 HPI from 24 HPI (P = 0.0001; Fig. 5a). Control rats apparently have decreased labeling index (Fig. 4d) but not statistically different from 24 HPI. Labeled cells in mucosa of control rats were identified as mostly pit and neck mucous cells. In infected rats, most cells of the hyperplastic mucosa were described as neck and intermediate mucous cells (Fig. 4e and f). Mean labeling index of the hyperplastic mucosa at 96 HPI was greater than the control rats (P = 0.0060). 3.5. BrdU labeling in antral region Labeling indices in antral mucosa of infected rats were higher than control rats at 2 HPI (P < 0.0001; Fig. 5b). Labeled cells in control mucosa were located in the basal parts of mucosa (Fig. 4g) while they were found in the whole length of mucous units in infected rats (Fig. 4h). At 6 HPI, no significant changes were observed in labeling indices of both mucosas. Labeling index of hyperplastic mucosa decreased at 24 HPI, while lesser changes were observed in mucosa of control rats except the location of labeled cells in middle part of antral mucosa. Hyperplastic antral mucosa (Fig. 4i) of inoculated rats further decreased its labeling index at 96 HPI while lesser BrdU-labeled cells were observed in mucosa of control rats (Fig. 4j). 4. Discussion The study determined the rate of epithelial cell proliferation by time course labeling with BrdU and PCNA immunohistochemical staining. The time course of BrdU labeling reflected the stimulation of DNA synthesis and subsequent cell replication within the mucosa (Ohning and Guth, 1995)
whereas PCNA labeling indicated level of cell mitosis in S, G2 and M phases. Fates of major gastric cell types in the hyperplastic gastric mucosa were examined using various staining techniques. Cell proliferations are restricted to isthmus regions in gastric mucosa. Granule-free cells are most proliferative in isthmus regions, although, pre-pit, pre-neck and their precursor cells have also some potential for mitosis (Karam, 1998). In this study, however, regions of the hyperplastic mucosa of inoculated rats were not recognizable because of numerous cystic cavities. Furthermore, epithelial cells were not found in their normal locations. This abnormality displaced the isthmus region and the whole length of corpus and mucous units was transformed as proliferative zones. These were demonstrated by the presence of BrdU and PCNA-labeled cells throughout the lengths of hyperplastic mucosa. Hyperplasia of gastric corpus epithelium was observed primarily as the result of a remarkable increase in the rate of cell proliferation and accumulation of intermediate precursor cells. Significant increase in lengths of proliferative zones at 6 WPI even without marked mucosal hyperplastic lesions yet, suggested initiation of hyperproliferative activity at this point. The dramatic increase in labeling indices of BrdU and proliferative zone lengths at 9 WPI created severe hyperplastic lesions in mucosa of infected rats. These events suggested obvious association between excessive cell proliferation and gastric mucosal hyperplasia. It was showed in SCID mice that larval ES products have stimulated hyperproliferation leading to hyperplastic changes (Lagapa et al., 2002b). Phenomenal elevation in labeling indices of BrdU within 6 h indicated that the specific inducing factor must have tremendous effect on the mitotic activity of gastric cell poll either directly or indirectly. Karam and Leblond (1993) reported that labeled cells in mice 6 h after injection with 3H-thymidine injection were granule-free cells
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Fig. 5. Time course BrdU labeling indices (LI) of gastric mucosa between control and infected rats during 9 WPI at 2, 6, 24 and 96 h. (a) corpus mucosa; (b) antral mucosa. Control (open circles); infected rats (closed circles). Asterisk (*) means P < 0.05. Bars = SD.
Fig. 4. Bromodeoxyuridine (BrdU) immunohistochemistry of gastric mucosa at 9 weeks post-infection. (a) control corpus mucosa at 2 h post-injection (HPI) of BrdU; (b and c) hyperplastic corpus mucosa at 2 HPI; (d) control corpus mucosa at 96 HPI; (e and f) hyperplastic corpus mucosa at 96 HPI; (g) control antral mucosa at 2 HPI; (h) hyperplastic antral mucosa at 2 HPI; (i) hyperplastic antral mucosa at 96 HPI; (j) control antral mucosa at 96 HPI. Bars: 50 lm.
with pre-pit and pre-neck mucous cells. In this study, however, hyperplastic mucosa was composed already of mature pit and neck mucous cells indicating an unusually rapid differentiation of cells.
The remarkable loss of parietal cells in the corpus units of infected rats at early stages corroborated with a previous study that parietal cells were significantly ablated even in slight hyperplastic lesions (Lagapa et al., 2002a). Further, parietal cell dysfunction was proposed as the key event leading to loss of mature zymogenic cells resulting to mucous cell hyperplasia in abomasal nematodosis (Simpson, 2000). Nomura et al., 2005 established that parietal cells secrete a number of growth factors that influence the differentiation of other gastric lineages. The accompanying loss of zymogenic cells was likely a consequence of the interruption of the normal development pathway in the gastric mucosa that followed after destruction of parietal cells (Marshall et al., 2005). Parietal cells were observed to play a central role in the regulation of mucosal proliferation during gastric inflammations (Beales, 2004). Thus, we hypoth-
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esized that the loss of parietal cells brought chaos in the gastric mucosal lineage differentiations that caused the increase proliferation of gastric cells and accumulation of immature and undifferentiated cells. Accumulation of intermediate forms of cells suggested also a cell dynamic attempting to replace the loss of parietal and zymogenic cells. As previously reported by Fujita and Kaneko (1994), mucous neck cells were converted to zymogenic cells via intermediate cells. Reports also noted that during gastric ulcer healing, parietal cells are not formed directly from dividing cells, but were recruited from existing precursor cells (Helander and Poorkhalkali, 2004). Based on the above claims and our present findings, we extrapolated a proposition that cessation of normal developmental pathway to zymogenic cells was the cause of the massive accumulation of intermediate cells. Hyperproliferation and accumulation of immature cells with decreased number of parietal and zymogenic cells were noted also in murine autoimmune gastritis (Franic et al., 2004). A critical feature in this gastropathy was the occurrence of corpus hyperplasia prior to antral lesions. The absence of increased proliferative zone at 6 WPI by PCNA staining in the antrum implied that epithelial hyperplasia was an aftermath of the corpus gastropathy. From this sequential development of the lesion from corpus to antrum, a basis for a strong argument that factors from larval ES products could have first evoked its effects on the corpus mucosa as shown also during a parabiotic transfer experiment (Cook and Williams, 1981). Thus, our result indicated that T. taeniaeformis larvaeinduced gastropathy might be associated with the effect of larval ES products on the depletion of parietal cells. Our finding could be summarized to have started with parietal cell loss followed by the blocking of zymogenic cell differentiation. The loss of control in cell differentiation caused the accumulation of intermediate cells. Marked depletion of the major cells of corpus mucosa initiated accompanying increase of stem cell proliferation that enhanced further accumulation of intermediate mucous cells. Antral lesions emerged later with mucous cell hyperproliferation of which cause is still unclear. References Abella, J.A., Oku, Y., Konno, K., Altamirano, Z., Nonaka, N., Kamiya, M., 1997a. Concomitant onset of hypergastrinemia, intragastric alkalinity and gastric hyperplasia, and numbers of antral G cells in Taenia taeniaeformis-infected rats. Parasitology International 46, 97– 104. Abella, J.A., Oku, Y., Nonaka, N., Ito, M., Kamiya, M., 1997b. Role of host immune response in the occurrence of gastropathy in rats infected with larval Taenia taeniaeformis. Journal of Veterinary Medical Science 59, 1039–1043. Beales, I.L., 2004. Gastrin and interleukin-1 beta stimulate growth factor secretion from cultured rabbit gastric parietal cells. Life Science 75, 2983–2995.
Blaies, D.M., Williams, J.F., 1983. Taenia taeniaeformis: gastrointestinal hyperplasia in chronically infected rats. Experimental Parasitology 55, 197–206. Cook, R.W., Williams, J.F., 1981. Pathology of Taenia taeniaeformis infection in the rat: gastrointestinal changes. Journal of Comparative Pathology 91, 205–217. Fox, J.G., Li, X., Cahill, R.J., Andrutis, K., Rustgi, A.K., Odze, R., Wang, T.C., 1996. Hypertrophic gastropathy in Helicobacter felisinfected wild-type C57BL/6 mice and p53 hemizygous transgenic mice. Gastroenterology 110, 155–166. Franic, T.V., Judd, L.M., Nguyen, N.V., Samuelson, L.C., Loveland, L.L., Giraud, A.S., Gleeson, P.A., van Driel, I.R., 2004. Growth factors associated with gastric mucosal hypertrophy in autoimmune gastritis. American Journal of Physiology 287, G910–G918. Fujita, K., Kaneko, K., 1994. Immunohistochemical examination of the relationship between two types of mucous neck cells and the intermediate cells in the rat fundic gland. Okajimas Folia Anatomica Japonica 71, 137–142. Helander, H.F., Poorkhalkali, N., 2004. Parietal cell density during gastric ulcer healing in the rat. Scandinavian Journal of Gastroenterology 39, 20–26. Karam, S.M., 1998. Cell lineage relationship in the stomach of normal and genetically manipulated mice. Brazilian Journal of Medicine and Biological Research 31, 271–279. Karam, S.M., Leblond, C.P., 1993. Dynamics of epithelial cells in the corpus of the mouse stomach III. Inward migration of neck cells followed by progressive transformation into zymogenic cells. The Anatomical Record 236, 297–313. Konno, K., Abella, J.A., Oku, Y., Nonaka, N., Kamiya, M., 1999a. Histopathology and physiopathology of gastric mucous hyperplasia in rats heavily infected with Taenia taeniaeformis. Journal of Veterinary Medical Science 61, 317–324. Konno, K., Oku, Y., Nonaka, N., Kamiya, M., 1999b. Hyperplasia of gastric mucosa in donor rats orally infected with Taenia taeniaeformis eggs and in recipient rats surgically implanted with the larvae in the abdominal cavity. Parasitology Research 85, 431–436. Lagapa, J.T., Konno, K., Oku, Y., Nonaka, N., Ito, M., Kamiya, M., 2002a. Gastric hyperplasia and parietal cell loss in Taenia taeniaeformis inoculated immunodeficient mice. Parasitology International 51, 81–89. Lagapa, J.T., Oku, Y., Nonaka, N., Ito, M., Kamiya, M., 2002b. Taenia taeniaeformis larval product induces gastric mucosal hyperplasia in SCID mice. Japanese Journal of Veterinary Research 49, 273–285. Marshall, A.C., Alderuccio, F., Murphy, K., Toh, B.H., 2005. Mechanisms of gastric mucosal cell loss in autoimmune gastritis. International Reviews of Immunology 24, 123–134. Martinelli, T.M., van Driel, I.R., Alderuccio, F., Gleeson, P.A., Toh, B.H., 1996. Analysis of mononuclear cell infiltrate and cytokine production in murine autoimmune gastritis. Gastroenterology 110, 1791–1802. Nomura, S., Yamaguchi, H., Ogawa, M., Wang, T.C., Lee, J.R., Goldenring, J.R., 2005. Alterations in gastric mucosal lineages induced by acute oxyntic atrophy in wild-type and gastrin-deficient mice. American Journal of Physiology 288, G362–G375. Ohning, G.V., Guth, P.H., 1995. Time course of mucosal cell proliferation following acute aspirin injury in rat stomach. Digestive Disease Science 40, 1351–1356. Rikihisa, Y., Letonja, T., Pratt, N., Lin, Y.C., 1984. Taenia taeniaeformis: characterization of larval metabolic products and growth of host gastric cells in vitro. Experimental Parasitology 58, 230–238. Rikihisa, Y., Lin, Y.C., 1984. Taenia taeniaeformis: increased cell growth and neutral mucus production in the gastric mucosa of the rat with a larval infection. Experimental Parasitology 58, 147–155. Simpson, H.V., 2000. Pathophysiology of abomasal parasitism: is the host or parasite responsible? The Veterinary Journal 160, 177–190.