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Trichinella spiralis: Effect of thymus factor X on apoptosis and necrosis in mice
Experimental Parasitology 123 (2009) 128–133
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Trichinella spiralis: Effect of thymus factor X on apoptosis and necrosis in mice J. Piekarska a,*, A. Michalski b, M. Szczypka c, B. Obmin´ska-Mrukowicz c a b c
Department of Internal and Parasitic Diseases, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Pl. Grunwaldzki 47, Poland Department of Mathematics, Wroclaw University of Environmental and Life Sciences, ul. Grunwaldzka 53, Poland Department of Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, ul. Norwida 31, Poland
a r t i c l e
i n f o
Article history: Received 11 January 2009 Received in revised form 1 June 2009 Accepted 15 June 2009 Available online 23 June 2009 Keywords: Trichinella spiralis Apoptosis Necrosis Calf thymus extract Mice
a b s t r a c t The aim of the study was to determine the effect of thymus factor X (TFX-Jelfa) on the percentage of apoptotic and necrotic lymphocytes in the spleen, mesenteric lymph nodes, and muscle tissue of mice infected with 200 larvae of Trichinella spiralis. TFX was administered subcutaneously at a dose of 15 mg/kg. On days 7, 14, 21, 28, 35, 42, and 60 after infection, apoptotic and necrotic cells were detected by flow cytometry after staining with the Annexin V-Fluos Staining Kit. TFX increased the percentage of apoptotic lymphocytes in the spleen, mesenteric lymph nodes, and muscle tissue of mice infected with T. spiralis. The effect of TFX on the percentage of necrotic lymphocytes was weaker and less clear. Parasite load was lower in infected mice treated with TFX than in the untreated control mice. The effect of TFX on the host immune response and the survival of parasite larvae was therefore probably affected by the extent of inflammatory infiltrates, and not by the percentage of lymphocytes undergoing apoptosis. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The host response to the strong antigenic stimulus of invasion by Trichinella spiralis involves the activation of various populations of lymphocytes. These react to the specific antigens encountered during each phase of the disease (Grencis et al., 1991). During the course of infection, CD4+ and CD8+ T lymphocytes are found in both intestinal and muscle tissue infiltrates. This indicates that these cells are involved in the regulation of the immune response during both the intestinal and the muscular phases of the disease (Karman´ska et al., 1995). The mechanism by which adult parasites are expelled from the small intestine is a complicated process mediated by the immune system. Th2 cells and mucosal mast cells play a key role (Finkelman et al., 1997; Faulkner et al., 1997; Urban et al., 2000; Grencis et al., 1993). During the intestinal phase, the response is mediated by CD4+ Th2 lymphocytes and the cytokines produced by them, including IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. Th1 lymphocytes play a smaller role (Grencis et al., 1991). In the lymphatic system, both Th1 and Th2 cells are actively involved in the host immune response (Kelly et al., 1991; Scott and Kaufmann, 1991). In the spleen, Th1 lymphocytes are more important. In the mesenteric lymph nodes, on the other hand, Th2 cells are more important. CD4+ Th2 lymphocytes produced in the mesenteric lymph nodes are involved in the host response to many intestinal nematodes, including T. spiralis (Grencis et al., 1991).
* Corresponding author. E-mail address: [email protected] (J. Piekarska). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.06.009
Apoptosis plays a key role in the pathogenesis of many parasitic diseases. Excretory/secretory antigens can induce apoptosis in various host organs. This indicates that they are a key element of the defense mechanism of the parasite (O’Connell and Rogan, 2000; Kuroda et al., 2002). Invasion by T. spiralis induces apoptosis in some cells in inflammatory infiltrates in the lamina propria of the small intestine and also in striated muscle tissue. Apoptotic cells in inflammatory infiltrates are significantly more numerous during the muscular phase than during the intestinal phase of the disease (Karman´ska et al., 2000). None of the studies carried out on animals infected with T. spiralis to date have focused on the proportions of apoptotic or necrotic cells in lymphatic organs. Apoptosis and necrosis are two types of cell death that can take place at the same time. Preliminary studies on mice infected with T. spiralis have indicated that the parasite attempts to protect itself from the host immune response from the earliest stages of infection. Apoptosis was observed primarily in lymphocytes. The aim of the present study was to determine the effect of thymus factor X (TFX-Jelfa), a natural stimulator of lymphocyte function, on the percentage of apoptotic and necrotic lymphocytes in the spleen, mesenteric lymph nodes, and muscle tissue of mice infected with T. spiralis. TFX-Jelfa is an aqueous extract of juvenile calf thymuses purified using a method patented by Jelfa Pharmaceuticals. The method involves fractionation with ammonium sulfate, desalting through a G-25 porosity molecular sieve, and ion exchange chromatography. The isolated protein is very large on polyacrylamide gel electrophoresis. TFX consists of polypeptide chains that are free of
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carbohydrate and lipid residues. The polypeptides are resistant to DNase and RNase (Czarnecki and Jaskólski, 1978). In experiments on nude or thymectomized mice, TFX induced the expression of markers and differentiation in T lymphocytes. Other affects include improvement or modulation of thymomimetic properties of T lymphocytes, such as response to mitogens, production of IL-2 and IFN-c, and suppressive activity (Da˛browski and Da˛browska-Bernstein, 1990). When TFX is administered to mice before restraint stress or a high dose of hydrocortisone, immunosuppression successfully counteracts the effects of acute stress or glucocorticoids (Obmin´ska-Domoradzka, 1997; Obmin´ska-Domoradzka et al., 2002).
2. Materials and methods 2.1. Experimental animals The experiment was carried out using male CFW mice between 8- and 10-weeks-old and weighing between 20 and 22 g. The mice were orally infected with 200 T. spiralis larvae and maintained under identical conditions. All procedures were carried out in strict accordance with all laws pertaining to the ethical treatment of animals prevailing in Poland. 2.2. Parasitological material The strain of T. spiralis used was T1(ISS1820). This strain was isolated in Poland and identified at the Istituto Superiore di Sanita, Rome, Italy. The larvae used in the infection were recovered from the muscle tissue of mice that had been infected two to three months earlier. The parasites were released from the muscle tissue by digesting with a 1% pepsin/HCl solution at 37 °C. 2.3. Treatment protocol Thymus factor X (TFX-Jelfa, Series 010204) was dissolved in saline immediately before use and was administered subcutaneously at a dose of 15 mg/kg. The first dose was administered on the third day before the mice were infected with T. spiralis. The dose was repeated at 24 h intervals until the thirteenth day after infection. In all, 17 doses were administered. The experiment was carried out using 134 mice divided into three groups: Group I: 46 mice infected with T. spiralis and treated with TFX; Group II: 42 uninfected mice treated with TFX; and Control group: 46 mice infected with T. spiralis and not treated with TFX.
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then suspended in the same buffer to a final density of 1 106 cells/ml. Cell viability, as determined by trypan blue exclusion, was greater then 96%. 2.5. Measurement of apoptosis and necrosis Apoptotic and necrotic lymphocytes were detected by flow cytometry after staining with the Annexin V-Fluos Staining Kit (Roche Diagnostics) in accordance with the procedure recommended by the manufacturer. The method depends on the selective binding of Annexin V to phosphatidylserine residues on the cell surface of apoptotic cells, and the staining by propidium iodide of cells that have lost membrane integrity. Cells were analyzed with a FACS Calibur flow cytometer (Becton-Dickinson Biosciences). Data were analyzed using the Cell Quest 3.1f software package. 2.6. Parasite load in muscle tissue Four additional mice each from Group I and the control group were sacrificed on day 60 after infection. Parasite load in the muscle tissue was counted after digesting with a 1% pepsin/HCl solution at 37 °C. 2.7. Statistical analysis The experiment was carried out using a complete two-factor design, with the first factor representing whether TFX was administered or not, and the second factor representing the number of days after infection. In order to determine the effect of TFX, the experiment was also carried out on uninfected mice using a onefactor design, with that factor representing the number of days after infection. All data were analyzed using appropriate ANOVA methods (Rao, 1973). Before analysis, values for the proportion of apoptotic and necrotic lymphocytes were transformed using the following logit function (Curtiss, 1943):
y ¼ logðy=ð1 yÞÞ This was done so that the transformed values would conform to a distribution that approximates a normal distribution. The transformation also stabilizes variances in the groups determined by the respective levels of each factor and by the interactions between them, which is an important assumption in hypothesis testing using parametric ANOVA models (Levene, 1960; Brown, 1998). Numerical calculations were carried out using the STATISTICA ver. 8.0 software package, supplemented with procedures developed by the authors.
3. Results 2.4. Isolation of lymphocytes 3.1. Apoptotic lymphocytes On days 7, 14, 21, 28, 35, 42, and 60 after infection, six mice from each group were sacrificed using halothane (Narcotan, Leciva). The spleen, mesenteric lymph nodes and masseter muscles were excised from each mouse and transferred to sterile phospho-buffered saline at 4 °C. The tissue samples were then separately passed through a nylon cell strainer. One part of each filtrate was layered on top of three parts of Ficoll 400/Uropolinum 75% (density 1.071) and centrifuged for 15 min at 4 °C at 1000g. The fraction that gravitated to the interphase was then collected (Fandrich et al., 1999). For spleen samples, this fraction was treated with 0.84% NH4Cl in order to disrupt erythrocytes. All fractions were then washed twice with phospho-buffered saline containing 1% bovine serum albumin (Sigma) at 4 °C. The lymphocytes were
The percentage of apoptotic lymphocytes in the spleen in Group I was approximately the same as the control group on days 7 and 14, significantly lower on day 21, and significantly higher for the rest of the experiment [with a maximum of 32.9% at 35 days after infection (p < 0.0002)]. The percentage in Group I was approximately the same as in Group II throughout the experiment except on day 21, when it was significantly higher (Fig. 1A and B). The percentage of apoptotic lymphocytes in mesenteric lymph nodes in Group I was lower than in the control group on day 7, and significantly higher for the rest of the experiment (p < 0.0002). The highest percentage reached 55.7% on 35 days after infection. The percentage in Group I was approximately the
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Fig. 1. Percentage of apoptotic lymphocytes in the spleen of mice: (A) infected with T. spiralis and treated with TFX (Group I) and in infected mice not treated with TFX (Control), (B) infected with T. spiralis and treated with TFX (Group I) and in uninfected mice treated with TFX (Group II). The vertical bars represent 95% confidence intervals.
same as in Group II on days 7, 14, and 21, significantly lower on day 28, and significantly higher for the rest of the experiment (Fig. 2A and B). The percentage of apoptotic lymphocytes in muscle tissue in Group I was approximately the same as in the control group on days 7 and 14, and significantly higher for the rest of the experiment, with a maximum of 50.6% at 35 days after infection (p < 0.0015). The percentage in Group I was significantly higher than in Group II throughout the whole experiment (Fig. 3A and B). 3.2. Necrotic lymphocytes The percentage of necrotic lymphocytes in the spleen in Group I was significantly lower than in the control group on day 7, and approximately the same as in the control group for the rest of the experiment. The percentage in Group I was approximately the same as in Group II throughout the whole experiment (Fig. 4A and B). The percentage of necrotic lymphocytes in mesenteric lymph nodes in Group I was approximately the same as in the control
group on days 7 and 14, and significantly lower than in the control group for the rest of the experiment. The percentage in Group I was approximately the same as in the control group throughout the experiment, except on days 35 and 42, when it was significantly lower (Fig. 5A and B). The percentage of necrotic lymphocytes in muscle tissue in Group I was approximately the same as in the control group on day 7, significantly lower on days 14 and 21 (p < 0.0002), approximately the same on days 28 and 35, significantly lower on day 42, and significantly higher on day 60 (p < 0.005). The percentage in Group I was significantly higher than in Group II throughout the whole experiment, except on days 21 and 42, when it was slightly lower (Fig. 6A and B). 3.3. Parasite load The number of muscle larvae per mouse at 60 days after infection was 17,400 ± 2000 for Group I, and 23,500 ± 5400 for the control group. The difference was significant at p < 0.01.
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Fig. 2. Percentage of apoptotic lymphocytes in mesenteric lymph nodes of mice: (A) infected with T. spiralis and treated with TFX (Group I) and in infected mice not treated with TFX (Control), (B) infected with T. spiralis and treated with TFX (Group I) and in uninfected mice treated with TFX (Group II). The vertical bars represent 95% confidence intervals.
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0.0
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Fig. 3. Percentage of apoptotic lymphocytes in muscle tissue of mice: (A) infected with T. spiralis and treated with TFX (Group I) and in infected mice not treated with TFX (Control), (B) infected with T. spiralis and treated with TFX (Group I) and uninfected mice treated with TFX (Group II). The vertical bars represent 95% confidence intervals.
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Fig. 4. Percentage of necrotic lymphocytes in the spleen of mice: (A) infected with T. spiralis and treated with TFX (Group I) and in infected mice not treated with TFX (Control), (B) uninfected mice treated with TFX (Group II). The vertical bars represent 95% confidence intervals.
4. Discussion In the present study, TFX affected the levels of both apoptosis and necrosis in lymphocytes of mice infected with T. spiralis. The effect was dependent on the drug itself, on the amount of time that had lapsed since the mice were infected, and on the interaction between these two factors. Apoptosis in T and B lymphocytes is involved in all fundamental processes in the immune system. It also plays a role in the establishment of immunological memory and in the development of central and peripheral tolerance. Parasites have developed several mechanisms to evade the action of the host immune response. One of these is modulation of the process of apoptosis in the host. The size and activity of particular cell populations can be regulated by antigens produced by the parasite (Krammer et al., 1994). TFX is a biologically active polypeptide that affects the activity of thymus hormones, thereby restoring equilibrium and normalizing physiological function in the lymphatic system. It acts mainly on T lymphocytes. TFX also stimulates phagocytosis and accelerates the elimination of infectious agents.
In preliminary studies on mice infected with T. spiralis, TFX reduced the intensity of both the intestinal and muscular phases of the disease (Kocie˛cka et al., 1989; Piekarska 2003). In both phases, TFX increased the extent of inflammatory infiltrates. In both the lamina propria of the small intestine and in striated muscle, TFX stimulated the mobilization of eosinophils and CD4+ T lymphocytes. TFX affected the dynamics of mastocytes during the intestinal phase, and of macrophages during the muscular phase (Piekarska, 2004). TFX also stimulated B lymphocytes in muscle tissue. In the spleen and mesenteric lymph nodes, TFX mobilized CD4+ T lymphocytes. During the second month after infection, it also increased the proportion of CD8+ T lymphocytes (Obmin´skaMrukowicz et al., 2002). In various studies conducted over the last few years, apoptosis was found to play a key role in the regulation of the host response to invasion by helminths. During the course of chronic schistosomiasis, for example, antigens produced by the parasite induced apoptosis in populations of both CD4+ and CD8+ T lymphocytes (Lundy et al., 2001; Rutitzky et al., 2003). Excretory/secretory (ES) antigens of Taenia crassiceps and cysteine protease excreted
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(B) -2.5
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Fig. 5. Percentage of necrotic lymphocytes in mesenteric lymph nodes of mice: (A) infected with T. spiralis and treated with TFX (Group I) and in infected mice not treated with TFX (Control), (B) uninfected mice treated with TFX (Group II). The vertical bars represent 95% confidence intervals.
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Fig. 6. Percentage of necrotic lymphocytes in muscle tissue of mice: (A) infected with T. spiralis and treated with TFX (Group I) and in infected mice not treated with TFX (Control), (B) uninfected mice treated with TFX (Group II). The vertical bars represent 95% confidence intervals.
by Taenia solium induced apoptosis of CD4+ and CD19+ splenocytes in infected mice (Lopez-Briones et al., 2003; O’Connell and Rogan, 2000; Tato et al., 2004). ES antigens of Taenia crassiceps larvae suppressed the proliferation of T lymphocytes and the production of IFN-c and IL-4 in the early phases of infection (Spolski et al., 2000). In mice infected with T. spiralis, immunosuppression is caused by disturbances to both the humoral and the cellular host responses (Ljungström and Ferrante, 1984). This is caused by disturbances in the function of T lymphocytes during the intestinal phase of the disease (Luft and Kim, 1988). The elimination of adult parasites from the intestines begins with a rise in the number of CD8+ T lymphocytes and a drop in the number of CD4+ lymphocytes in the lamina propria (Boz´ic´ et al., 2000). CD4+ T lymphocytes also play a role in the induction of intestinal muscle contraction (Vallance et al., 1999). CD4+ T cells also regulate the intensity of the invasive process. ES antigens produced by the pathogen induce apoptosis in the CD4+ T lymphocytes. This may therefore be the mechanism by
which the parasite suppresses the immune system of the host in order to ensure its continued survival. In the present study, TFX increased the percentage of apoptotic lymphocytes in the spleen, mesenteric lymph nodes and muscle tissue of mice infected with T. spiralis. The effect of TFX on the percentage of necrotic lymphocytes was weaker and less clear. TFX had no significant effect on the percentage of necrotic lymphocytes in the spleen, and generally reduced the percentage of necrotic lymphocytes in mesenteric lymph nodes and muscle tissue. The only exception was on day 60 in muscle tissue, when the percentage of necrotic lymphocytes was higher than in the control. In both infected and uninfected mice, TFX similarly modified the kinetics of apoptotic cells in the spleen, but not in the mesenteric lymph nodes. Similarities in the kinetics of necrotic cells were also observed in the spleen and mesenteric lymph nodes in infected and uninfected mice (Groups I and II), but not in the muscle tissue.
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Apoptosis proceeded differently in lymphatic tissue and in muscle tissue. This indicates that the immune response was different in these tissues. TFX had a similar effect on apoptosis in infected and uninfected mice, which indicates that TFX stimulated lymphocytes in both the spleen and lymph nodes. Parasite load was significantly lower in infected mice treated with TFX than in the untreated control mice, in spite of the increased level of apoptosis. In earlier studies, the immune response of the host during the course of trichinellosis was modulated using phytohemagglutinin-P (PHA-P). Although the percentage of apoptotic cells was higher in the treated mice, this had no effect on the survival of parasite larvae in muscle tissue (Piekarska et al., 2009). In earlier experiments, the Tunel method was used to study cells in inflammatory infiltrates in muscle tissue of mice infected with T. spiralis. TFX had no affect on the proportion of apoptotic cells in the muscle tissue. On the other hand, when the mice were treated with either PHA-P or TFX, inflammatory infiltrates in muscle tissue were more extensive, and the number of inflammatory cells inside the cysts containing the larvae was higher (Karman´ska et al., 2001; Piekarska, 2004). The effect of TFX on the host immune response and the survival of parasite larvae is therefore probably affected by the extent of inflammatory infiltrates, and not by the percentage of lymphocytes undergoing apoptosis. Acknowledgments This study was funded by the Polish State Committee for Scientific Research, Ministry of Science and Higher Education (2 PO6K 01727). References Brown, K.S., 1998. Analysis of variance. In: Armitage, P., Colton, T. (Eds.), Encyclopedia of Biostatistics, vol. 1. John Wiley and Sons, Chichester, pp. 146– 161. ´ Bozic´, F., Marinculic´, A., Durakovic´, E., 2000. Analysis of intestinal intra-epithelial lymphocyte populations in experimental Trichinella spiralis infection of mice. Folia Parasitologica 47, 55–59. Curtiss, J.H., 1943. On transformations used in the analysis of variance. The Annals of Mathematical Statistics 14, 107–122. Czarnecki, J., Jaskólski, B., 1978. Preparation TFX-Polfa Jelenia Góra. Archivum Immunologiae et Therapiae Experimentalis 26, 475–482. Da˛browski, M., Da˛browska-Bernstein, B., 1990. Thymus influence on differentiation and functional maturation of T lymphocytes. In: Da˛browski, M. (Ed.), Immunoregulatory Role of Thymus. CRC Press, Boca Raton, FL, USA, pp. 45–94. Fandrich, F., Zhu, X., Schroder, J., Dresske, B., Henne-Bruns, D., Oswald, H., Zavazava, N., 1999. Different in vivo tolerogenicity of MHC class I peptides. Journal of Leukocyte Biology 65, 16–27. Faulkner, H., Humphreys, N., Renauld, J.C., Van Snick, J., Grencis, R., 1997. Interleukin-9 involved in host protective immunity to intestinal nematode infection. European Journal of Immunology 27, 2536–2540. Finkelman, F.D., Shea-Donohue, T., Goldhill, J., Sullivan, C.A., Morris, S.C., Madden, K.B., Gause, W.C., Urban, J.F., 1997. Cytokine regulation of host defense against parasitic gastrointestinal nematodes: lessons from studies with rodent models. Annual Review of Immunology 15, 505–533. Grencis, R.K., Hultner, L., Else, K.J., 1991. Host protective immunity to Trichinella spiralis in mice. activation of Th cell subsets and lymphokine secretion in mice expressing different response phenotypes. Immunology 74, 329–332. Grencis, R.K., Else, K.J., Huntley, J.F., Nishikawa, S.I., 1993. The in vivo role of stem cell factor (c-kit ligand) on mastocytosis and host protective immunity to the intestinal nematode Trichinella spiralis in mice. Parasite Immunology 15, 55–59. Karman´ska, K., Houszka, M., Mis´ta, D., Stefaniak, E., 1995. CD4+ and CD8+ cells during infection with Trichinella spiralis in mice. Acta Parasitologica 40, 53–57. Karman´ska, K., Houszka, M., Piekarska, J., 2000. The phenomenon of apoptosis in the course of experimental trichinellosis in mice. Wiadomos´ci Parazytologiczne 46, 111–115.
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Karman´ska, K., Piekarska, J., Widyma, A., 2001. Attempts of modification of apoptosis in the course of experimental trichinellosis in mice. Wiadomos´ci Parazytologiczne 47, 211–216. Kelly, E.A.B., Enderson, S.C., Hauda, K.M., Wassom, D.L., 1991. IFN-gamma and IL-5 producing cells compartmentalize to different lymphoid organs in Trichinella spiralis-infected mice. Journal of Immunology 147, 306–311. Kocie˛cka, W., Rauhut, W., Gustowska, L., 1989. The effect of thymus factor X (TFX Polfa) on Trichinella spiralis muscle invasion in mice. Parasitological and histological study. Wiadomos´ci Parazytologiczne 35, 413–421. Krammer, P.H., Behrmann, I., Daniel, P., Dhein, J., Debatin, K.M., 1994. Regulation of apoptosis in the immune system. Current Opinion of Immunology 6, 279–289. Kuroda, A., Uchikawa, R., Matsuda, S., Hamada, M., Tegoshi, T., Arizono, N., 2002. Upregulation of Fas (CD95) and induction of apoptosis in intestinal epithelial cells by nematode-derived molecules. Infection and Immunity 70, 4002–4008. Levene, H., 1960. Robust tests for equality of variances. In: Olkin, I., Ghurye, S.G., Hoeffding, W., Madow, W.G., Mann, H.B. (Eds.), Contributions to Probability and Statistics: Essays in Honor of Harold Hotteling. Stanford University Press, pp. 278–292. Ljungström, I., Ferrante, A., 1984. The in vivo and in vitro immunodepression by Trichinella spiralis, a preliminary report. In: Kim, C.W. (Ed.), Trichinellosis, vol. 6. State University of New York Press, New York, pp. 63–70. Lopez-Briones, S., Sciutto, E., Ventura, J.L., Zentella, A., Fragoso, G., 2003. CD4+ and CD19+ splenocytes undergo apoptosis during an experimental murine infection with Taenia crassiceps. Parasitology Research 2, 157–163. Luft, B.J., Kim, Ch.W., 1988. T-cell dysfunction during acute murine trichinellosis. In: Tanner, C.E., Martinez-Fernandez, A.R., Bolas-Fernandez, F. (Eds.), Trichinellosis, vol. 7. Consejo Superiore de Investigaciones Cientificas Press, Madrid, pp. 153– 158. Lundy, S.K., Lerman, S.P., Boros, D.L., 2001. Soluble egg antigen-simulated T helper lymphocyte apoptosis and evidence for cell death mediated by FasL+ T and B cells during murine Schistosoma mansoni infection. Infection and Immunity 69, 271–280. O’Connell, K.M., Rogan, M.T., 2000. Apoptosis in human Jurkat T cell after culture with live Taenia crassiceps cysticerci in vitro. Parasitology 120, 649–655. Obmin´ska-Domoradzka, B., 1997. The effect of DTC on mitogen-induced proliferation of thymocytes in restrained mice. Comparison with calf thymus extract. International Journal of Immunopharmacology 19, 235–241. Obmin´ska-Domoradzka, B., Szczypka, M., De˛bowy, J., 2002. Effects of thymomimetic drugs and zinc supplementation on the cellular immune response in hydrocortisone-suppressed mice. Journal of Veterinary Medicine B 49, 469–475. Obmin´ska-Mrukowicz, B., Piekarska, J., Szczypka, M., Widyma, A., Połozowski, A., 2002. Modulatory effects of thymus extract on the subset of T lymphocytes in Trichinella spiralis-infected mice. Polish Journal of Veterinary Sciences 5, 243– 249. Piekarska, J., 2003. Influence of selected immunotrophic compounds on T (CD4+, CD8+) and B-lymphocytes during the course of experimental trichinellosis in mice. Veterinary Medicine 59, 277–368 (in Polish). Piekarska, J., 2004. Influence of the select immunotrophic compounds on mast cells, eosinophiles and macrophages in the course of the experimental trichinellosis in mice. Veterinary Medicine 60, 561–672 (in Polish). Piekarska, J., Szczypka, M., Obmin´ska-Mrukowicz, B., Gorczykowski, M., 2009. Effect of phytohaemagglutinin-P on apoptosis and necrosis in Trichinella spiralis infected mice. Veterinary Parasitology 159, 240–244. Rao, C.R., 1973. Linear Statistical Inferences and its Application, second ed. Wiley, New York. Rutitzky, L.I., Mirkin, G.A., Stadecker, M.J., 2003. Apoptosis by neglect of CD4+ Th cells in granulomas: a novel effector mechanism involved in the control of egginduced immunopathology in murine schistosomiasis. Journal of Immunology 171, 1859–1867. Scott, P., Kaufmann, S.H.E., 1991. The role of T-cell subset and cytokines in the regulation of infection. Immunology Today 12, 346–348. Spolski, R.J., Corson, J., Thomas, P.G., Kuhn, R.E., 2000. Parasite-secreted products regulates the host response to larval Taenia crassiceps. Parasite Immunology 22, 297–305. Tato, P., Fernandez, A.M., Solano, S., Borgonio, V., Garrido, E., Sepulveda, J., Molinari, J.L., 2004. A cysteine protease from Taenia solium metacestodes induces apoptosis in human CD4+ T-cells. Parasitology Research 92, 197–204. Urban, J.F., Schopf, L., Morris, S.C., Orekhova, T., Madden, K.B., Betts, K.J., Gamble, H.R., Byrd, C., Donaldson, D., Else, K., Finkelman, F.D., 2000. Stat6 signaling promotes protective immunity against Trichinella spiralis through a mast celland T cell-dependent mechanism. Journal of Immunology 164, 2046–2052. Vallance, B.A., Galeazzi, F., Collins, S.M., Snider, D., 1999. CD4 T cells and major histocompatibility complex class II expression influence worm expulsion and increased intestinal muscle contraction during Trichinella spiralis infection. Infection and Immunity 67, 6090–6097.
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Trichinella spiralis: Effect of thymus factor X on apoptosis and necrosis in mice J. Piekarska a,*, A. Michalski b, M. Szczypka c, B. Obmin´ska-Mrukowicz c a b c
Department of Internal and Parasitic Diseases, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Pl. Grunwaldzki 47, Poland Department of Mathematics, Wroclaw University of Environmental and Life Sciences, ul. Grunwaldzka 53, Poland Department of Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, ul. Norwida 31, Poland
a r t i c l e
i n f o
Article history: Received 11 January 2009 Received in revised form 1 June 2009 Accepted 15 June 2009 Available online 23 June 2009 Keywords: Trichinella spiralis Apoptosis Necrosis Calf thymus extract Mice
a b s t r a c t The aim of the study was to determine the effect of thymus factor X (TFX-Jelfa) on the percentage of apoptotic and necrotic lymphocytes in the spleen, mesenteric lymph nodes, and muscle tissue of mice infected with 200 larvae of Trichinella spiralis. TFX was administered subcutaneously at a dose of 15 mg/kg. On days 7, 14, 21, 28, 35, 42, and 60 after infection, apoptotic and necrotic cells were detected by flow cytometry after staining with the Annexin V-Fluos Staining Kit. TFX increased the percentage of apoptotic lymphocytes in the spleen, mesenteric lymph nodes, and muscle tissue of mice infected with T. spiralis. The effect of TFX on the percentage of necrotic lymphocytes was weaker and less clear. Parasite load was lower in infected mice treated with TFX than in the untreated control mice. The effect of TFX on the host immune response and the survival of parasite larvae was therefore probably affected by the extent of inflammatory infiltrates, and not by the percentage of lymphocytes undergoing apoptosis. Ó 2009 Elsevier Inc. All rights reserved.
1. Introduction The host response to the strong antigenic stimulus of invasion by Trichinella spiralis involves the activation of various populations of lymphocytes. These react to the specific antigens encountered during each phase of the disease (Grencis et al., 1991). During the course of infection, CD4+ and CD8+ T lymphocytes are found in both intestinal and muscle tissue infiltrates. This indicates that these cells are involved in the regulation of the immune response during both the intestinal and the muscular phases of the disease (Karman´ska et al., 1995). The mechanism by which adult parasites are expelled from the small intestine is a complicated process mediated by the immune system. Th2 cells and mucosal mast cells play a key role (Finkelman et al., 1997; Faulkner et al., 1997; Urban et al., 2000; Grencis et al., 1993). During the intestinal phase, the response is mediated by CD4+ Th2 lymphocytes and the cytokines produced by them, including IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13. Th1 lymphocytes play a smaller role (Grencis et al., 1991). In the lymphatic system, both Th1 and Th2 cells are actively involved in the host immune response (Kelly et al., 1991; Scott and Kaufmann, 1991). In the spleen, Th1 lymphocytes are more important. In the mesenteric lymph nodes, on the other hand, Th2 cells are more important. CD4+ Th2 lymphocytes produced in the mesenteric lymph nodes are involved in the host response to many intestinal nematodes, including T. spiralis (Grencis et al., 1991).
* Corresponding author. E-mail address: [email protected] (J. Piekarska). 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.06.009
Apoptosis plays a key role in the pathogenesis of many parasitic diseases. Excretory/secretory antigens can induce apoptosis in various host organs. This indicates that they are a key element of the defense mechanism of the parasite (O’Connell and Rogan, 2000; Kuroda et al., 2002). Invasion by T. spiralis induces apoptosis in some cells in inflammatory infiltrates in the lamina propria of the small intestine and also in striated muscle tissue. Apoptotic cells in inflammatory infiltrates are significantly more numerous during the muscular phase than during the intestinal phase of the disease (Karman´ska et al., 2000). None of the studies carried out on animals infected with T. spiralis to date have focused on the proportions of apoptotic or necrotic cells in lymphatic organs. Apoptosis and necrosis are two types of cell death that can take place at the same time. Preliminary studies on mice infected with T. spiralis have indicated that the parasite attempts to protect itself from the host immune response from the earliest stages of infection. Apoptosis was observed primarily in lymphocytes. The aim of the present study was to determine the effect of thymus factor X (TFX-Jelfa), a natural stimulator of lymphocyte function, on the percentage of apoptotic and necrotic lymphocytes in the spleen, mesenteric lymph nodes, and muscle tissue of mice infected with T. spiralis. TFX-Jelfa is an aqueous extract of juvenile calf thymuses purified using a method patented by Jelfa Pharmaceuticals. The method involves fractionation with ammonium sulfate, desalting through a G-25 porosity molecular sieve, and ion exchange chromatography. The isolated protein is very large on polyacrylamide gel electrophoresis. TFX consists of polypeptide chains that are free of
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carbohydrate and lipid residues. The polypeptides are resistant to DNase and RNase (Czarnecki and Jaskólski, 1978). In experiments on nude or thymectomized mice, TFX induced the expression of markers and differentiation in T lymphocytes. Other affects include improvement or modulation of thymomimetic properties of T lymphocytes, such as response to mitogens, production of IL-2 and IFN-c, and suppressive activity (Da˛browski and Da˛browska-Bernstein, 1990). When TFX is administered to mice before restraint stress or a high dose of hydrocortisone, immunosuppression successfully counteracts the effects of acute stress or glucocorticoids (Obmin´ska-Domoradzka, 1997; Obmin´ska-Domoradzka et al., 2002).
2. Materials and methods 2.1. Experimental animals The experiment was carried out using male CFW mice between 8- and 10-weeks-old and weighing between 20 and 22 g. The mice were orally infected with 200 T. spiralis larvae and maintained under identical conditions. All procedures were carried out in strict accordance with all laws pertaining to the ethical treatment of animals prevailing in Poland. 2.2. Parasitological material The strain of T. spiralis used was T1(ISS1820). This strain was isolated in Poland and identified at the Istituto Superiore di Sanita, Rome, Italy. The larvae used in the infection were recovered from the muscle tissue of mice that had been infected two to three months earlier. The parasites were released from the muscle tissue by digesting with a 1% pepsin/HCl solution at 37 °C. 2.3. Treatment protocol Thymus factor X (TFX-Jelfa, Series 010204) was dissolved in saline immediately before use and was administered subcutaneously at a dose of 15 mg/kg. The first dose was administered on the third day before the mice were infected with T. spiralis. The dose was repeated at 24 h intervals until the thirteenth day after infection. In all, 17 doses were administered. The experiment was carried out using 134 mice divided into three groups: Group I: 46 mice infected with T. spiralis and treated with TFX; Group II: 42 uninfected mice treated with TFX; and Control group: 46 mice infected with T. spiralis and not treated with TFX.
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then suspended in the same buffer to a final density of 1 106 cells/ml. Cell viability, as determined by trypan blue exclusion, was greater then 96%. 2.5. Measurement of apoptosis and necrosis Apoptotic and necrotic lymphocytes were detected by flow cytometry after staining with the Annexin V-Fluos Staining Kit (Roche Diagnostics) in accordance with the procedure recommended by the manufacturer. The method depends on the selective binding of Annexin V to phosphatidylserine residues on the cell surface of apoptotic cells, and the staining by propidium iodide of cells that have lost membrane integrity. Cells were analyzed with a FACS Calibur flow cytometer (Becton-Dickinson Biosciences). Data were analyzed using the Cell Quest 3.1f software package. 2.6. Parasite load in muscle tissue Four additional mice each from Group I and the control group were sacrificed on day 60 after infection. Parasite load in the muscle tissue was counted after digesting with a 1% pepsin/HCl solution at 37 °C. 2.7. Statistical analysis The experiment was carried out using a complete two-factor design, with the first factor representing whether TFX was administered or not, and the second factor representing the number of days after infection. In order to determine the effect of TFX, the experiment was also carried out on uninfected mice using a onefactor design, with that factor representing the number of days after infection. All data were analyzed using appropriate ANOVA methods (Rao, 1973). Before analysis, values for the proportion of apoptotic and necrotic lymphocytes were transformed using the following logit function (Curtiss, 1943):
y ¼ logðy=ð1 yÞÞ This was done so that the transformed values would conform to a distribution that approximates a normal distribution. The transformation also stabilizes variances in the groups determined by the respective levels of each factor and by the interactions between them, which is an important assumption in hypothesis testing using parametric ANOVA models (Levene, 1960; Brown, 1998). Numerical calculations were carried out using the STATISTICA ver. 8.0 software package, supplemented with procedures developed by the authors.
3. Results 2.4. Isolation of lymphocytes 3.1. Apoptotic lymphocytes On days 7, 14, 21, 28, 35, 42, and 60 after infection, six mice from each group were sacrificed using halothane (Narcotan, Leciva). The spleen, mesenteric lymph nodes and masseter muscles were excised from each mouse and transferred to sterile phospho-buffered saline at 4 °C. The tissue samples were then separately passed through a nylon cell strainer. One part of each filtrate was layered on top of three parts of Ficoll 400/Uropolinum 75% (density 1.071) and centrifuged for 15 min at 4 °C at 1000g. The fraction that gravitated to the interphase was then collected (Fandrich et al., 1999). For spleen samples, this fraction was treated with 0.84% NH4Cl in order to disrupt erythrocytes. All fractions were then washed twice with phospho-buffered saline containing 1% bovine serum albumin (Sigma) at 4 °C. The lymphocytes were
The percentage of apoptotic lymphocytes in the spleen in Group I was approximately the same as the control group on days 7 and 14, significantly lower on day 21, and significantly higher for the rest of the experiment [with a maximum of 32.9% at 35 days after infection (p < 0.0002)]. The percentage in Group I was approximately the same as in Group II throughout the experiment except on day 21, when it was significantly higher (Fig. 1A and B). The percentage of apoptotic lymphocytes in mesenteric lymph nodes in Group I was lower than in the control group on day 7, and significantly higher for the rest of the experiment (p < 0.0002). The highest percentage reached 55.7% on 35 days after infection. The percentage in Group I was approximately the
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Fig. 1. Percentage of apoptotic lymphocytes in the spleen of mice: (A) infected with T. spiralis and treated with TFX (Group I) and in infected mice not treated with TFX (Control), (B) infected with T. spiralis and treated with TFX (Group I) and in uninfected mice treated with TFX (Group II). The vertical bars represent 95% confidence intervals.
same as in Group II on days 7, 14, and 21, significantly lower on day 28, and significantly higher for the rest of the experiment (Fig. 2A and B). The percentage of apoptotic lymphocytes in muscle tissue in Group I was approximately the same as in the control group on days 7 and 14, and significantly higher for the rest of the experiment, with a maximum of 50.6% at 35 days after infection (p < 0.0015). The percentage in Group I was significantly higher than in Group II throughout the whole experiment (Fig. 3A and B). 3.2. Necrotic lymphocytes The percentage of necrotic lymphocytes in the spleen in Group I was significantly lower than in the control group on day 7, and approximately the same as in the control group for the rest of the experiment. The percentage in Group I was approximately the same as in Group II throughout the whole experiment (Fig. 4A and B). The percentage of necrotic lymphocytes in mesenteric lymph nodes in Group I was approximately the same as in the control
group on days 7 and 14, and significantly lower than in the control group for the rest of the experiment. The percentage in Group I was approximately the same as in the control group throughout the experiment, except on days 35 and 42, when it was significantly lower (Fig. 5A and B). The percentage of necrotic lymphocytes in muscle tissue in Group I was approximately the same as in the control group on day 7, significantly lower on days 14 and 21 (p < 0.0002), approximately the same on days 28 and 35, significantly lower on day 42, and significantly higher on day 60 (p < 0.005). The percentage in Group I was significantly higher than in Group II throughout the whole experiment, except on days 21 and 42, when it was slightly lower (Fig. 6A and B). 3.3. Parasite load The number of muscle larvae per mouse at 60 days after infection was 17,400 ± 2000 for Group I, and 23,500 ± 5400 for the control group. The difference was significant at p < 0.01.
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Fig. 2. Percentage of apoptotic lymphocytes in mesenteric lymph nodes of mice: (A) infected with T. spiralis and treated with TFX (Group I) and in infected mice not treated with TFX (Control), (B) infected with T. spiralis and treated with TFX (Group I) and in uninfected mice treated with TFX (Group II). The vertical bars represent 95% confidence intervals.
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0.0
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Fig. 4. Percentage of necrotic lymphocytes in the spleen of mice: (A) infected with T. spiralis and treated with TFX (Group I) and in infected mice not treated with TFX (Control), (B) uninfected mice treated with TFX (Group II). The vertical bars represent 95% confidence intervals.
4. Discussion In the present study, TFX affected the levels of both apoptosis and necrosis in lymphocytes of mice infected with T. spiralis. The effect was dependent on the drug itself, on the amount of time that had lapsed since the mice were infected, and on the interaction between these two factors. Apoptosis in T and B lymphocytes is involved in all fundamental processes in the immune system. It also plays a role in the establishment of immunological memory and in the development of central and peripheral tolerance. Parasites have developed several mechanisms to evade the action of the host immune response. One of these is modulation of the process of apoptosis in the host. The size and activity of particular cell populations can be regulated by antigens produced by the parasite (Krammer et al., 1994). TFX is a biologically active polypeptide that affects the activity of thymus hormones, thereby restoring equilibrium and normalizing physiological function in the lymphatic system. It acts mainly on T lymphocytes. TFX also stimulates phagocytosis and accelerates the elimination of infectious agents.
In preliminary studies on mice infected with T. spiralis, TFX reduced the intensity of both the intestinal and muscular phases of the disease (Kocie˛cka et al., 1989; Piekarska 2003). In both phases, TFX increased the extent of inflammatory infiltrates. In both the lamina propria of the small intestine and in striated muscle, TFX stimulated the mobilization of eosinophils and CD4+ T lymphocytes. TFX affected the dynamics of mastocytes during the intestinal phase, and of macrophages during the muscular phase (Piekarska, 2004). TFX also stimulated B lymphocytes in muscle tissue. In the spleen and mesenteric lymph nodes, TFX mobilized CD4+ T lymphocytes. During the second month after infection, it also increased the proportion of CD8+ T lymphocytes (Obmin´skaMrukowicz et al., 2002). In various studies conducted over the last few years, apoptosis was found to play a key role in the regulation of the host response to invasion by helminths. During the course of chronic schistosomiasis, for example, antigens produced by the parasite induced apoptosis in populations of both CD4+ and CD8+ T lymphocytes (Lundy et al., 2001; Rutitzky et al., 2003). Excretory/secretory (ES) antigens of Taenia crassiceps and cysteine protease excreted
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by Taenia solium induced apoptosis of CD4+ and CD19+ splenocytes in infected mice (Lopez-Briones et al., 2003; O’Connell and Rogan, 2000; Tato et al., 2004). ES antigens of Taenia crassiceps larvae suppressed the proliferation of T lymphocytes and the production of IFN-c and IL-4 in the early phases of infection (Spolski et al., 2000). In mice infected with T. spiralis, immunosuppression is caused by disturbances to both the humoral and the cellular host responses (Ljungström and Ferrante, 1984). This is caused by disturbances in the function of T lymphocytes during the intestinal phase of the disease (Luft and Kim, 1988). The elimination of adult parasites from the intestines begins with a rise in the number of CD8+ T lymphocytes and a drop in the number of CD4+ lymphocytes in the lamina propria (Boz´ic´ et al., 2000). CD4+ T lymphocytes also play a role in the induction of intestinal muscle contraction (Vallance et al., 1999). CD4+ T cells also regulate the intensity of the invasive process. ES antigens produced by the pathogen induce apoptosis in the CD4+ T lymphocytes. This may therefore be the mechanism by
which the parasite suppresses the immune system of the host in order to ensure its continued survival. In the present study, TFX increased the percentage of apoptotic lymphocytes in the spleen, mesenteric lymph nodes and muscle tissue of mice infected with T. spiralis. The effect of TFX on the percentage of necrotic lymphocytes was weaker and less clear. TFX had no significant effect on the percentage of necrotic lymphocytes in the spleen, and generally reduced the percentage of necrotic lymphocytes in mesenteric lymph nodes and muscle tissue. The only exception was on day 60 in muscle tissue, when the percentage of necrotic lymphocytes was higher than in the control. In both infected and uninfected mice, TFX similarly modified the kinetics of apoptotic cells in the spleen, but not in the mesenteric lymph nodes. Similarities in the kinetics of necrotic cells were also observed in the spleen and mesenteric lymph nodes in infected and uninfected mice (Groups I and II), but not in the muscle tissue.
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Apoptosis proceeded differently in lymphatic tissue and in muscle tissue. This indicates that the immune response was different in these tissues. TFX had a similar effect on apoptosis in infected and uninfected mice, which indicates that TFX stimulated lymphocytes in both the spleen and lymph nodes. Parasite load was significantly lower in infected mice treated with TFX than in the untreated control mice, in spite of the increased level of apoptosis. In earlier studies, the immune response of the host during the course of trichinellosis was modulated using phytohemagglutinin-P (PHA-P). Although the percentage of apoptotic cells was higher in the treated mice, this had no effect on the survival of parasite larvae in muscle tissue (Piekarska et al., 2009). In earlier experiments, the Tunel method was used to study cells in inflammatory infiltrates in muscle tissue of mice infected with T. spiralis. TFX had no affect on the proportion of apoptotic cells in the muscle tissue. On the other hand, when the mice were treated with either PHA-P or TFX, inflammatory infiltrates in muscle tissue were more extensive, and the number of inflammatory cells inside the cysts containing the larvae was higher (Karman´ska et al., 2001; Piekarska, 2004). The effect of TFX on the host immune response and the survival of parasite larvae is therefore probably affected by the extent of inflammatory infiltrates, and not by the percentage of lymphocytes undergoing apoptosis. Acknowledgments This study was funded by the Polish State Committee for Scientific Research, Ministry of Science and Higher Education (2 PO6K 01727). References Brown, K.S., 1998. Analysis of variance. In: Armitage, P., Colton, T. (Eds.), Encyclopedia of Biostatistics, vol. 1. John Wiley and Sons, Chichester, pp. 146– 161. ´ Bozic´, F., Marinculic´, A., Durakovic´, E., 2000. Analysis of intestinal intra-epithelial lymphocyte populations in experimental Trichinella spiralis infection of mice. Folia Parasitologica 47, 55–59. Curtiss, J.H., 1943. On transformations used in the analysis of variance. The Annals of Mathematical Statistics 14, 107–122. Czarnecki, J., Jaskólski, B., 1978. Preparation TFX-Polfa Jelenia Góra. Archivum Immunologiae et Therapiae Experimentalis 26, 475–482. Da˛browski, M., Da˛browska-Bernstein, B., 1990. Thymus influence on differentiation and functional maturation of T lymphocytes. In: Da˛browski, M. (Ed.), Immunoregulatory Role of Thymus. CRC Press, Boca Raton, FL, USA, pp. 45–94. Fandrich, F., Zhu, X., Schroder, J., Dresske, B., Henne-Bruns, D., Oswald, H., Zavazava, N., 1999. Different in vivo tolerogenicity of MHC class I peptides. Journal of Leukocyte Biology 65, 16–27. Faulkner, H., Humphreys, N., Renauld, J.C., Van Snick, J., Grencis, R., 1997. Interleukin-9 involved in host protective immunity to intestinal nematode infection. European Journal of Immunology 27, 2536–2540. Finkelman, F.D., Shea-Donohue, T., Goldhill, J., Sullivan, C.A., Morris, S.C., Madden, K.B., Gause, W.C., Urban, J.F., 1997. Cytokine regulation of host defense against parasitic gastrointestinal nematodes: lessons from studies with rodent models. Annual Review of Immunology 15, 505–533. Grencis, R.K., Hultner, L., Else, K.J., 1991. Host protective immunity to Trichinella spiralis in mice. activation of Th cell subsets and lymphokine secretion in mice expressing different response phenotypes. Immunology 74, 329–332. Grencis, R.K., Else, K.J., Huntley, J.F., Nishikawa, S.I., 1993. The in vivo role of stem cell factor (c-kit ligand) on mastocytosis and host protective immunity to the intestinal nematode Trichinella spiralis in mice. Parasite Immunology 15, 55–59. Karman´ska, K., Houszka, M., Mis´ta, D., Stefaniak, E., 1995. CD4+ and CD8+ cells during infection with Trichinella spiralis in mice. Acta Parasitologica 40, 53–57. Karman´ska, K., Houszka, M., Piekarska, J., 2000. The phenomenon of apoptosis in the course of experimental trichinellosis in mice. Wiadomos´ci Parazytologiczne 46, 111–115.
133
Karman´ska, K., Piekarska, J., Widyma, A., 2001. Attempts of modification of apoptosis in the course of experimental trichinellosis in mice. Wiadomos´ci Parazytologiczne 47, 211–216. Kelly, E.A.B., Enderson, S.C., Hauda, K.M., Wassom, D.L., 1991. IFN-gamma and IL-5 producing cells compartmentalize to different lymphoid organs in Trichinella spiralis-infected mice. Journal of Immunology 147, 306–311. Kocie˛cka, W., Rauhut, W., Gustowska, L., 1989. The effect of thymus factor X (TFX Polfa) on Trichinella spiralis muscle invasion in mice. Parasitological and histological study. Wiadomos´ci Parazytologiczne 35, 413–421. Krammer, P.H., Behrmann, I., Daniel, P., Dhein, J., Debatin, K.M., 1994. Regulation of apoptosis in the immune system. Current Opinion of Immunology 6, 279–289. Kuroda, A., Uchikawa, R., Matsuda, S., Hamada, M., Tegoshi, T., Arizono, N., 2002. Upregulation of Fas (CD95) and induction of apoptosis in intestinal epithelial cells by nematode-derived molecules. Infection and Immunity 70, 4002–4008. Levene, H., 1960. Robust tests for equality of variances. In: Olkin, I., Ghurye, S.G., Hoeffding, W., Madow, W.G., Mann, H.B. (Eds.), Contributions to Probability and Statistics: Essays in Honor of Harold Hotteling. Stanford University Press, pp. 278–292. Ljungström, I., Ferrante, A., 1984. The in vivo and in vitro immunodepression by Trichinella spiralis, a preliminary report. In: Kim, C.W. (Ed.), Trichinellosis, vol. 6. State University of New York Press, New York, pp. 63–70. Lopez-Briones, S., Sciutto, E., Ventura, J.L., Zentella, A., Fragoso, G., 2003. CD4+ and CD19+ splenocytes undergo apoptosis during an experimental murine infection with Taenia crassiceps. Parasitology Research 2, 157–163. Luft, B.J., Kim, Ch.W., 1988. T-cell dysfunction during acute murine trichinellosis. In: Tanner, C.E., Martinez-Fernandez, A.R., Bolas-Fernandez, F. (Eds.), Trichinellosis, vol. 7. Consejo Superiore de Investigaciones Cientificas Press, Madrid, pp. 153– 158. Lundy, S.K., Lerman, S.P., Boros, D.L., 2001. Soluble egg antigen-simulated T helper lymphocyte apoptosis and evidence for cell death mediated by FasL+ T and B cells during murine Schistosoma mansoni infection. Infection and Immunity 69, 271–280. O’Connell, K.M., Rogan, M.T., 2000. Apoptosis in human Jurkat T cell after culture with live Taenia crassiceps cysticerci in vitro. Parasitology 120, 649–655. Obmin´ska-Domoradzka, B., 1997. The effect of DTC on mitogen-induced proliferation of thymocytes in restrained mice. Comparison with calf thymus extract. International Journal of Immunopharmacology 19, 235–241. Obmin´ska-Domoradzka, B., Szczypka, M., De˛bowy, J., 2002. Effects of thymomimetic drugs and zinc supplementation on the cellular immune response in hydrocortisone-suppressed mice. Journal of Veterinary Medicine B 49, 469–475. Obmin´ska-Mrukowicz, B., Piekarska, J., Szczypka, M., Widyma, A., Połozowski, A., 2002. Modulatory effects of thymus extract on the subset of T lymphocytes in Trichinella spiralis-infected mice. Polish Journal of Veterinary Sciences 5, 243– 249. Piekarska, J., 2003. Influence of selected immunotrophic compounds on T (CD4+, CD8+) and B-lymphocytes during the course of experimental trichinellosis in mice. Veterinary Medicine 59, 277–368 (in Polish). Piekarska, J., 2004. Influence of the select immunotrophic compounds on mast cells, eosinophiles and macrophages in the course of the experimental trichinellosis in mice. Veterinary Medicine 60, 561–672 (in Polish). Piekarska, J., Szczypka, M., Obmin´ska-Mrukowicz, B., Gorczykowski, M., 2009. Effect of phytohaemagglutinin-P on apoptosis and necrosis in Trichinella spiralis infected mice. Veterinary Parasitology 159, 240–244. Rao, C.R., 1973. Linear Statistical Inferences and its Application, second ed. Wiley, New York. Rutitzky, L.I., Mirkin, G.A., Stadecker, M.J., 2003. Apoptosis by neglect of CD4+ Th cells in granulomas: a novel effector mechanism involved in the control of egginduced immunopathology in murine schistosomiasis. Journal of Immunology 171, 1859–1867. Scott, P., Kaufmann, S.H.E., 1991. The role of T-cell subset and cytokines in the regulation of infection. Immunology Today 12, 346–348. Spolski, R.J., Corson, J., Thomas, P.G., Kuhn, R.E., 2000. Parasite-secreted products regulates the host response to larval Taenia crassiceps. Parasite Immunology 22, 297–305. Tato, P., Fernandez, A.M., Solano, S., Borgonio, V., Garrido, E., Sepulveda, J., Molinari, J.L., 2004. A cysteine protease from Taenia solium metacestodes induces apoptosis in human CD4+ T-cells. Parasitology Research 92, 197–204. Urban, J.F., Schopf, L., Morris, S.C., Orekhova, T., Madden, K.B., Betts, K.J., Gamble, H.R., Byrd, C., Donaldson, D., Else, K., Finkelman, F.D., 2000. Stat6 signaling promotes protective immunity against Trichinella spiralis through a mast celland T cell-dependent mechanism. Journal of Immunology 164, 2046–2052. Vallance, B.A., Galeazzi, F., Collins, S.M., Snider, D., 1999. CD4 T cells and major histocompatibility complex class II expression influence worm expulsion and increased intestinal muscle contraction during Trichinella spiralis infection. Infection and Immunity 67, 6090–6097.