Lymphoid activity was studied in spleen and lymph node cells from Trypanosoma cruzi-infected mice. Blast transformation in each lymphocyte class was assessed by dual parameter analysis for size and surface markers by both FACS and conventional immunofluorescence, while proliferative activity was measured by tritiated thymidine uptake, autoradiography, and analysis of DNA content in single cells. Acute infection results in rapid blast transformation and proliferative activity of all three lymphocyte classes (Ig+, L3T4+, and Lyt 2+). At 2 weeks of infection most cells in these organs are enlarged and more than half are dividing. By 2 and 6 months after infection (chronic phase of resistant strains), large numbers of activated B lymphocytes and, to a lesser extent, of Lyt 2+ T cells are still detected. Similar results were obtained in C57BL/6 (resistant) and C3H/HeJ (susceptible) mouse strains. The implications of this massive polyclonal lymphocyte response to the parasite for the physiopathology of acute and chronic infection are discussed.
Abstract. A new gene in bacteriophage X is described. The product of this gene cro prevents expression of immunity and regulates the expression of those genes to the left of the immunity region. cro-mutants have been isolated and characterized.Introduction. The Ci gene of bacteriophage lambda, which codes for the repressor,' is located between the two operons controlled by the repressor itself2-5 and is transcribed from the 1 strand of the DNA, as is the N operon.4 Occurrence of a mechanism controlling the expression of the CI repressor has been evidenced by the study of lysogens in which prophage induction does not affect the growth of the host: These carry derivatives of the N-defective thermoinducible (C1,57) prophage X, with an additional mutation blocking phage DNA
Addition of Fab fragments from rabbit antiserum to surface antigen F9 to 2-cell stage mouse embryos in culture does not alter cleavage; however, the addition prevents the formation of compact morulae and blastocysts. A similar effect is observed when Fab fragments are added to already compact 8-cell stage or even older morulae, but disappears at the beginning of blastocoel formation. This effect is reversible: uncompact 30-cell embryos washed free of Fab become compact in a few hours, produce blastocysts, and upon reimplantation into pseudopregnant mothers can produce mice. Development is not altered by divalent anti-F9 antibodies, by Fab fragments from sera directed against other embryo surface antigens, or by succinyl concanavalin A. There are reasons to assume that the cell surface plays an important role in the development of the embryo (1). We have investigated this point by examining the effect of several antisera reacting with surface structures of embryonic cells on the in vitro development of the mouse preimplantation embryo (2). Among these sera, one which proved effective in blocking morula compaction and blastocyst formation without inhibiting cleavage was directed against the nullipotent, embryonal carcinoma (EC) line F9. This line carries the "F9 antigen," which has been shown to be expressed on EC cells, germ line cells, and preimplantation embryos (3, 4). MATERIALS AND METHODSMice. The following stocks were used: 129/Sv, an inbred subline of the original 129 strain; C57BI/6 X CBA, an F1 hybrid of C57BI/6 and CBA. All mice were produced at the Institut Pasteur.Cells. Two cultured cell lines were used: F9-41, a nullipotent EC cell (5); PYS-2, a parietal yolk sac cell (6) devoid of F9 antigen.Mouse Anti-F9 Serum was produced in syngeneic male 129/Sv mice by hyperimmunization with irradiated F9-41 cells (3). Before use anti-F9 serum was absorbed with PYS-2 cells (vol/vol, serum dilution 1:5 in Hank's medium containing 4% -heat-inactivated, gamma globulin-free, fetal calf serum, 1 hr at 40). The absorbed serum retained most of the original activity against F9 cells.Rabbit Anti-F9 Serum. Rabbits were immunized with 5 X 107 F9-41 cells in complete Freund's adjuvant. After 1 month the animals received four booster injections at 2-week intervals with 5 X 107 cells each. Serum was collected and heat inactivated 1 week after the last injection. The cytotoxic titer of the unabsorbed serum against F9-41 cells was 1/6000. Rabbit anti-F9 was massively absorbed against lymphocytes (twice, vol/vol, serum The absorbed conjugates were sampled and stored frozen at -80°. They were used at a 1/25 dilution.IgG and Fab Preparation. The immunoglobulin (IgG) fraction of an unabsorbed rabbit anti-F9 serum was isolated by precipitation with 40% saturated ammonium sulfate, pH 7.0, followed by a chromatography on Whatman DEAE-cellulose (DE52) according to Levy and Sober (7). For Fab preparation, the method of Porter (8) was used. IgG (100 mg) was digested with 1 mg of papain (Worthington). Undigested IgG was separ...
Chagas' disease, caused by Trypanosoma cruzi, is an excellent model for autoimmune disease induced by an infectious agent. Transfer of T cells, directed against crossreactive antigens of T. cruzi and nervous tissue, have been shown to reproduce pathology found in chronic Chagas' disease. We used recombinant DNA technology to characterize one of these crossreactive antigens (Fl-160). We have cloned DNA from T. cruzi, which expresses a protein corresponding to a 160-kD protein found on the surface of the trypanosome, overlying the flagellum. This clone hybridizes to a 4.5-kb poly(A)+ RNA that is distributed in a differentiation-specific manner, suggesting expression of this protein is transcriptionally controlled. Antibodies to this protein crossreact with a 48-kD mammalian nervous tissue protein found in sciatic nerve, brain, and myenteric plexi of gut. The myenteric plexi are destroyed by inflammatory infiltrates in Chagas' disease, leading to the characteristic megaesophagus and megacolon Chagas' disease pathology. Thus, this antigen is a candidate antigen for autoimmune mimicry leading to nervous tissue pathology.
The tissue and cellular distribution and regulation ofthe chromatin protein HI' has been examined in developing and adult mouse and in rat. The protein appears in specific cell types of solid tissues only when the cells have terminated their maturation. This was found for brain, retina, striated and cardiac muscle, and liver. In tissues that depend on hormones for their function and maintenance, the expression of HI' is dependent on the continued presence of the specific maintenance hormone. In regenerating rat liver the amount of HI0 decreases to one-third after the onset of DNA synthesis. The possible role of HI0 is discussed in light of these results.Proteins with properties similar or identical to those of the very lysine rich chromosomal protein Hi°have been described in several mammalian tissues and in tissue culture cells (1-16). The protein isolated from ox or mouse liver has been shown to consist of two subfractions that have identical molecular weights (25,000) but differ slightly in charge (2, 3). The two subfractions appear to have similar or identical amino acid compositions and peptide patterns. Recently it has been shown that Hi0 has extensive sequence homology with histone H5 from goose erythrocytes (4).Hi0 is of interest for several reasons. Its level in different tissues varies widely and it is predominantly found in tissues exhibiting little cellular proliferation (1,6,7,9,15,17). In earlier work, using indirect immunofluorescence with monospecific antibodies, we showed that Hi0 was present in many but not all fully mature postmitotic cells, and therefore that its distribution in different cells of the adult mouse is heterogeneous (15). These data suggest that Hi0 may play an important role in cell regulation.The concept that Hi0 is involved in developmental regulation is strengthened by the observations that in rat pancreas, liver, and spleen the protein appears after birth once cell proliferation in the organs has ceased (7,8,17). It has also been shown to decrease in regenerating rat pancreas and liver (5, 18).We report here studies on the regulation of Hi0 in developing mouse and in adult mouse and rat. The presence of Hi0 was shown to be correlated with cell maturation during development and liver regeneration. It was also found to be hormonally regulated in several glands in the adult. The results suggest that Hi0 may play a role in the maintenance of the mature differentiated state of some cells.MATERIALS AND METHODS Animals. Strain 129 male mice were used unless otherwise indicated. Fisher or Wistar male rats (both normal and hypophysectomized) were purchased from Iffa Credo (Paris, France). Hormones were injected either intraperitoneally or subcutaneously as described in the text or figure legends.Biochemical Methods. Lysine-rich histones were extracted from whole tissues with 5% perchloric acid as described (3). Proteins were analyzed by electrophoresis in polyacrylamide gels as described (10). After staining with Coomassie brilliant blue the gels were scanned at 590 nm and ...
Acute infection of mice with Trypanosoma cruzi results in severe immunodepression and the appearance of autoimmune symptoms. In vitro, concanavalin A-stimulated T cells from spleens of infected animals could neither produce nor respond to interleukin 2. Interleukin 2 production was not restored by addition of exogenous interleukin 1, and proliferative response to concanavalin A was not restored by exogenous interleukin 2. A population of Thy-i-negative cells in the spleen of infected animals was shown to suppress the concanavalin A proliferative response and, to a lesser extent, the production of interteukin 2. These and other symptoms of T. cruzi-infected mice are similar to the immune dysfunction of autoimmune Ipr/lpr mice. These findings are discussed in relationship to the pathology of Chagas disease.Trypanosoma cruzi causes Chagas disease in man. The disease is characterized by a variable acute phase during which trypomastigotes circulate, followed by a lifelong chronic phase in which parasites are not detected in circulation but rather appear to be sequestered in the tissues (1). The parasites persist in the chronically infected individuals in spite of the presence of high levels of circulating anti-T. cruzi antibodies. The mouse has been used as a laboratory model for Chagas disease because injection of parasites gives rise to both acute and chronic infections.Severe perturbations of the host immune system accompany T. cruzi infections in the mouse (1-4). On one hand, autoantibodies directed to various host antigens are induced. On the other hand, severe immunodepression has been reported to occur during the acute phase of the infection, and cellular responses to mitogens, parasite antigens, or irrelevant antigens are depressed. These results suggest that the parasites perturb the host immune system at the level of the T cells. We have studied this possibility by examining T-cell proliferation and the production of T-cell regulatory molecules in T. cruzi-infected mice.Interleukin 2 (IL-2) appears to play a key role in the regulation of cellular immune responses (5, 6). It is produced by T lymphocytes in the presence of interleukin 1 (IL-1) and mitogen or antigen, and it sustains the proliferation of helper and effector T lymphocytes and thus amplifies the effector phase of the immune response. Because of this central role of IL-2 in regulating the immune response, we have examined its production and activity in T. cruzi-infected mice.We report here that acute infection with T. cruzi results in a severe depression of IL-2 production which parallels the depressed proliferative response to the mitogen concanavalin A (Con A). Furthermore, Thy-l-spleen cells from infected animals are able to suppress the proliferative response to Con A and, to a lesser extent, IL-2 production by spleen cells from normal animals. MATERIALS AND METHODSMice and Infection. All experiments were performed with pools of three to eight C3H/HeJ Pas male mice (Pasteur Institute, Paris). Mice were 6-8 weeks of age at the time of...
Chromatin In an attempt to understand the role of chromosomal proteins we have begun a study of erythroid differentiation using murine erythroleukemia cells transformed by Friend virus (FL cells) (5,6). FL cells offer a useful model system for the study of the role of chromosomal proteins because at least the terminal events of erythroid differentiation can be provoked in vitro. FL cells contain little or no hemoglobin or globin mRNA and do not display such characteristic erythroid antigens as glycophorin or spectrin. Addition of various compounds induces a terminal erythroid differentiation in these cells (7)(8)(9)(10)(11). This differentiation, which takes place over a period of 3-5 days, is characterized by the accumulation of globin mRNA and hemoglobin (12,13) and by the appearance of glycophorin and spectrin (ref. 14; H. Eisen, unpublished data). During the induction period the cells become smaller, globin RNA and protein synthesis is shut off, and, after two or three divisions, cell division ceases (15-17). Peterson and McConkey (18) compared the proteins of differentiating and nondifferentiating FL cells and observed some changes in the chromosomal proteins. We report here the preliminary characterization of a major chromatin protein that appears after induction of FL cells.MATERIALS AND METHODS Cells and Culture Methods. FL cells of clone F4N were obtained from W. Ostertag (19,20). The clone MA-1 was derived from the DBA/2 mastocytoma P815 obtained from P. Vassalli. The isolation and characterization of resistant cell lines F4+ and F4N+2 will be described elsewhere (H. Eisen and W. Ostertag, unpublished data). All cells were grown in suspension culture in minimal essential medium (21) Chromatin Purification. Chromatin was purified by a modification of the method of Bonner et al. (23). Cells (50 to 100 X 106) were harvested by centrifugation at 800 X g for 10 min, washed twice with phosphate-buffered saline, and disrupted by homogenization in hypotonic buffer (24) containing 1 mM phenylmethylsulfonylfluoride. Nuclei were sedimented at 1000 X g and washed once with the same buffer. The washed nuclei were lysed in 10 mM Tris-HCI (pH 8)/25 mM EDTA/ 0.5% Triton X-100/1 mM phenylmethylsulfonylfluoride and homogenized. After two washes with 5 mM Tris-HCI, pH 8, chromatin was sedimented through a solution of 65% sucrose in 5 mM Tris for 70 min. at 80,000 X g. The chromatin pellet was washed twice with 10 mM TrisMHCI (pH 7.4)/1 mM EDTA and resuspended in the same buffer either by sonication (three times for 15 sec in a MSE sonicator) or by a Sorvall omnimixer (90 sec at 25,000 rpm). Chromatin was quantitated on the basis of its DNA content by measuring its absorbance at 260 nm.Polyacrylamide Gel Electrophoresis. Chromatin proteins were analyzed on slab gels (25) by a modification of Laemmli's discontinuous buffer system (26,27). The running gel contained 15% acrylamide and 0.087% N',N-methylene bisacrylamide. The gels were stained in 0.1% Coomassie brilliant blue/10% acetic acid/50% methanol, destained...
Myeloproliferative virus, derived from Moloney sarcoma virus, causes erythroleukemia and myeloid leukemia in adult mice. This virus is also capable of fibroblast transfornation in vitro. The virus consists of two separable biological entities which have been cloned. The helper virus component caused no visible changes in adult mice, whereas the defective virus induced both spleen focus formation and a large increase in erythroid precursor cells but retained the sarcoma virus property of transforming fibroblasts in vitro. Thus, myeloproliferative virus is the first murine sarcoma virus which induces erythroleukemia in adult animals.
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