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 Department of Internal and Parasitic Diseases, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Pl. Grunwaldzki 47, Poland b Department of Mathematics, Wroclaw University of Environmental and Life Sciences, ul. Grunwaldzka 53, Poland c Department of Biochemistry, Pharmacology and Toxicology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, ul. Norwida 31, Poland article info 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 abstract 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 in- volved in the host immune response (Kelly et al., 1991; Scott and Kaufmann, 1991). In the spleen, Th1 lymphocytes are more impor- tant. In the mesenteric lymph nodes, on the other hand, Th2 cells are more important. CD4 + Th2 lymphocytes produced in the mes- enteric lymph nodes are involved in the host response to many intestinal nematodes, including T. spiralis (Grencis et al., 1991). Apoptosis plays a key role in the pathogenesis of many parasitic diseases. Excretory/secretory antigens can induce apoptosis in var- ious 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 inflam- matory infiltrates in the lamina propria of the small intestine and also in striated muscle tissue. Apoptotic cells in inflammatory infil- trates 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. Preli- minary studies on mice infected with T. spiralis have indicated that the parasite attempts to protect itself from the host immune re- sponse from the earliest stages of infection. Apoptosis was ob- served primarily in lymphocytes. The aim of the present study was to determine the effect of thy- mus factor X (TFX-Jelfa), a natural stimulator of lymphocyte func- tion, on the percentage of apoptotic and necrotic lymphocytes in the spleen, mesenteric lymph nodes, and muscle tissue of mice in- fected with T. spiralis. TFX-Jelfa is an aqueous extract of juvenile calf thymuses puri- fied 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 electro- phoresis. TFX consists of polypeptide chains that are free of 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.06.009 * Corresponding author. E-mail address: piekarsk@wp.pl (J. Piekarska). Experimental Parasitology 123 (2009) 128–133 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr