Larval Taenia crassiceps secretes a protein with characteristics of murine interferon-gamma.

Previous studies have shown that excretory/secretory products of larval Taenia crassiceps have immunomodulatory activities. We report here that one of these products, termed p66, possesses activities that mimic some characteristics of murine interferon-gamma (IFN-gamma). Purified p66 cross-reacts with anti-murine IFN-gamma on immunoblots and increases concanavalin-A-induced splenic T-cell proliferative responses of normal and chronically infected mice. It has also been shown that p66 induces enhanced IFN-gamma and interleukin-10 (IL-10) production in cells from infected or normal mice. Adherent and non-adherent peritoneal exudates cells were stimulated with p66 and showed that the adherent cells were induced to produce IL-10 and that the non-adherent cells were induced to produce IFN-gamma. p66 was shown as well to upregulate nitric oxide production in macrophages and two T-cell lines, IEL and YAC-1, were shown to be stimulated to produce IFN-gamma and IL-10, respectively, by p66. Although the significance of p66 in immunoregulation is not known, we show here that this molecule mimics characteristics of IFN-gamma.

Suppressed cytotoxic T lymphocyte responses in experimental cysticercosis.

Mitogen-induced T cell responses are suppressed in mice infected with larvae of Taenia crassiceps. The effects of experimental infection on specific T cell responses, however, have not been examined. In the present study, we demonstrate that larval-infected mice exhibit suppressed ability to develop anti-virus specific cytotoxic T lymphocyte (CTL) responses while maintaining apparently normal natural killer (NK) cell responsiveness.

Essential role of signal transducer and activator of transcription (Stat)5a but not Stat5b for Flt3-dependent signaling.

The receptor tyrosine kinase Flt3 plays an important role in proliferation and survival of hematopoietic stem and progenitor cells. Although some post-receptor signaling events of Flt3 have been characterized, the involvement of the Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway in Flt3 signaling has not been thoroughly evaluated. To this aim, we examined whether Flt3 activates the Jak/Stat pathway in Baf3/Flt3 cells, a line stably expressing human Flt3 receptor. Stat5a, but not Stats 1-4, 5b, or 6, was potently activated by Flt3 ligand (FL) stimulation. Interestingly, FL did not activate any Jaks. Activation of Stat5a required the kinase activity of Flt3. A selective role for Stat5a in the proliferative response of primary hematopoietic progenitor cells to FL was documented, as FL did not act on progenitors from marrows of Stat5a(-/-) mice, but did stimulate/costimulate proliferation of these cells from Stat5a(+/+), Stat5b(-/-), and Stat5b(+/+) mice. Thus, Stat5a is essential for at least certain effects of FL. Moreover, our data confirm that Stat5a and Stat5b are not redundant, but rather are at least partially distinctive in their function.

Parasite-secreted products regulate the host response to larval Taenia crassiceps.

Parasite-induced immunosuppression is believed to play a significant role in the pathology of cysticercosis, a disease caused by the larval stage of cestode parasites. The biochemical basis for immunoregulation by Taenia crassiceps in experimental cysicercosis is unknown. In order to determine whether or not excretory/secretory (E/S) products from the parasite have the ability to regulate host immune function, the activity of these products was examined. Excretory/secretory products from larvae early in the infection were found to suppress T cell proliferative responses in vitro as well as the production of IFN-gamma and IL-4. In contrast, E/S products secreted from larvae harvested late in infection were not suppressive. Electrophoretic analysis of E/S products revealed both qualitative and quantitative differences in the pattern of proteins produced by larvae taken from an early infection versus those taken from a chronic infection. The viability of parasites taken from an early infection was greatly reduced compared to those taken from chronically infected mice, suggesting a change in the nature of the host immune response to the parasite during the course of the infection. The proliferative activity and cytokine profiles of host immune cells were examined. Both mesenteric lymph node cells and peritoneal exudate cells were found to produce high levels of both IFN-gamma and IL-4, consistent with the high levels of these cytokines in sera of chronically infected animals. Chronic infection with Taenia crassiceps therefore is characterized by high levels of production of both Th1 and Th2 cytokines by host cells.

Immune destruction of larval taenia crassiceps in mice.

Immune destruction of larval Taenia crassiceps was examined by first injecting BALB/cJ mice subcutaneously with larval buds and 30 to 60 days later challenging the mice with larvae injected into the peritoneal cavity. The larvae injected intraperitoneally (i.p.) secondarily are killed by host cells that completely encase the larvae in a thick sheath. The peritoneal exudate cells and the cytokines they produced were characterized by flow cytometry, enzyme-linked immunosorbent assays (ELISAs), and reverse transcription PCR (RT-PCR). No changes in percentage of CD4(+) T cells, CD8(+) T cells, B1 cells, or macrophages were detected in the peritoneal cavities of mice that were killing larvae compared to mice with a primary 7-day infection i.p. Both RT-PCR and ELISA demonstrated a decrease in cytokines including gamma interferon (IFN-gamma), interleukin-4 (IL-4), and IL-10 in mice that were killing the larvae compared to control mice infected for 30 to 60 days i.p. alone, although there was little difference compared to mice infected for 7 days i.p. alone. Serum cytokine levels in mice that were killing the larvae showed a decrease in IFN-gamma and IL-4, an increase in IL-10 when compared to mice infected for 30 to 60 days i.p. alone, and increases in all cytokines compared to mice infected for 7 days i.p. alone. Inhibition of nitric oxide production did not significantly affect the number or the viability of larvae in the peritoneal cavity of mice that were killing larvae during secondary infection.

gamma delta T cells do not play a major role in controlling infection in experimental cysticercosis.

Protective immunity against larval Taenia crassiceps has been shown to rely on T cells; however, the roles of the specific subsets of T cells during infection are not known. To investigate a possible role for gamma delta T cells, this study investigated larval infection in delta-chain knock-out C57BL/6 (deltaKO) and wild-type C57BL/6 mice. It was found that deltaKO mice and C57BL/6 mice were equally susceptible to infection suggesting gamma delta T cells do not play a major role in protective immunity. Cytokine production by concanavalin A (ConA)-stimulated spleen cells from infected deltaKO mice and C57BL/6 mice were determined. All infected mice demonstrated an increased IL-10 production suggesting a Th1-inhibitory function. Cells from infected deltaKO mice and C57BL/6 mice did not show increases in IL-4 production. Heavily-infected C57BL/6 mice showed a decrease in IFN-gamma production compared to deltaKO mice. These observations suggest that an increase in IL-10 production best correlates with a non-protective immune response. To make comparisons between in vitro cytokine production and systemic immune responses, cytokine levels in serum were determined. C57BL/6 mice and deltaKO mice showed increases in serum levels of IL-4 and IFN-gamma at 52 days post-infection. The systemic immune response of these mice, therefore, is a mixed Th1/Th2-type response and gamma delta T cells are apparently not responsible for the systemic increases in these cytokines.

The systemic immune response of BALB/c mice infected with larval Taenia crassiceps is a mixed Th1/Th2-type response.

The subsets of lymphocytes and cytokines regulating the site-specific immune response in experimental cysticercosis (Taenia crassiceps) are not known. This study investigated the cells present at the site of infection (PECs) using flow cytometry and measured the cytokines produced by these cells through 50 days of infection. The results showed an expansion of B220+CD5+, B220+CD5-, alpha beta TCR+CD4+ and CD8+ cells coincident with a transient increase in IL-10 production. After the initial increase, the percentage of B220+CD5- and helper T cells decreased with a concomitant decrease in IL-10 production. CD8+ T cells continued to increase throughout infection and gamma delta TCR+ cells increased after 10 days of infection. PECs demonstrated an increased IFN-gamma and IL-4 production throughout infection when stimulated with larval antigens. Because a Th2-type polarization has been shown for spleen cells from infected BALB/c mice, cytokine profiles of spleen cells and PECs in response to ConA and larval antigens were compared. ConA and antigen-specific stimulation of spleen cells from 50-day-infected mice produced increased amounts of IL-10 while PECs showed a decreased IL-10 production suggesting that anatomically distinct lymphoid populations produce different cytokines and promote different types of responses. Surprisingly, late in infection the levels of IL-4 and IFN-gamma in serum increased substantially (460-fold and 100-fold, respectively). The systemic immune response of BALB/c mice during experimental cysticercosis, therefore, is a mixed Th1/Th2-type response.

Regulation of expression of T cell gamma chain, L3T4 and Ly-2 messages in Abelson/Moloney virus-transformed T cell lines.

Recent results have suggested that T cells may exist in two distinct pathways, one expressing alpha and beta chain of the T cell receptor genes with either or both of the cell surface markers CD4 and CD8, while the other is negative for these cell surface markers and expresses the T cell-specific gamma chain genes. The relationship between these two pathways is not known. In this study, we have examined a series of either Abelson virus or Moloney virus-derived T cell lines for their expression of these T cell receptor and cell surface marker genes. Results indicate that the Abelson T cell lines do not express the cell surface markers CD4 and CD8, but express relatively high levels of gamma chain transcripts. After culture of these cell lines with the phorbol ester phorbol myristate acetate and interleukin 2, a down-regulation of these gamma chain transcripts can be observed. More interestingly, we found that the Moloney virus-derived T cell lines, which express the cell surface markers CD4 and/CD8, contain high levels of alpha and beta chain T cell receptor transcripts but little or no gamma transcripts even though they have rearranged these latter genes. The gamma transcripts, however, can be induced to high levels after culture with phorbol myristate acetate and interleukin 2. In the process, the cell surface markers CD4 and CD8 and their transcripts were dramatically down-regulated resulting in cells with high levels of gamma chain transcripts and a CD4-CD8- phenotype. The regulation of expression of these genes is reversible. Taken together, these results indicate that the T cell receptor gamma chain genes and those of the cell surface markers CD4 CD8 can be regulated in vitro by external factors and it opens up the possibility of studying the regulatory sequences associated with these genes.

Liver parenchymal cell proliferation during secondary induction with estradiol-17 beta in Xenopus.

We have previously demonstrated that injection of adult male frogs with estradiol-17 beta causes extensive proliferation of liver parenchymal cells together with the induction of vitellogenin (R. J. Spolski, W. Schneider, and L. J. Wangh (1985) Dev. Biol. 108, 332-340). In addition, purified parenchymal cells placed in culture synthesize DNA in an estrogen-dependent manner (B. S. Aprison, L. Martin-Morris, R. J. Spolski, and L. J. Wangh (1986) In Vitro 22, 457-464). We now describe conditions under which secondary exposure to estradiol-17 beta, either in vivo or in vitro, can lead to further DNA synthesis and cell division. The extent of this proliferation depends upon both the magnitude of the primary dose and the length of time elapse before secondary stimulation. A hormone dose of 0.5 mg, which causes little cell proliferation initially, allows for maximal secondary proliferation in response to 2.0 mg, while a maximal primary dose of 2.0 mg substantially inhibits further division in response to a secondary treatment with the same hormone dose. Cell culture experiments demonstrate that the failure of liver cells, in maximally stimulated males, to synthesize DNA in response to estrogen is not irreversible. But, cell crowding in culture does restrict DNA synthesis. The restrictions seen in vivo may therefore be due to structural features of the intact tissue rather than to terminal differentiation at the genetic level. These results are discussed with regard to our understanding of hormone-dependent differentiation in the frog liver system.

A human T cell-specific cDNA clone (YT16) encodes a protein with extensive homology to a family of protein-tyrosine kinases.

We report here the isolation of a human T cell-specific cDNA clone (YT16) that encodes a protein with extensive homology to a family of transformation and proliferation-related protein-tyrosine kinases (PTK). The message size of YT16 is 2.2 kb and it encodes a protein of 507 amino acids with a mol. mass of 57,136 Daltons. The deduced protein sequence has an overall homology of about 50% to the viral oncogene products of src and yes. Around the PTK catalytic domain, the homologies increase to 70-80% between the deduced protein sequence of YT16 and those of the PTK viral oncogenes. Of special interest is the extensive homology between the deduced protein sequence of YT16 and that of a recently reported murine B and T lymphocyte-specific PTK, lskT. It is possible that YT16 either encodes the human homologue of this murine gene, or a protein with similar function(s) in T cells.

Estrogen-dependent DNA synthesis in cultures of Xenopus liver parenchymal cells.

We have recently shown that extensive proliferation of liver parenchymal cells takes place in adult male Xenopus frogs in response to estradiol-17 beta, which also induces synthesis and secretion of vitellogenin, the precursor of yolk proteins. We demonstrate here that liver parenchymal cells from adult male animals can be maintained for several weeks in a defined tissue culture medium containing added insulin, dexamethasone, and triiodothyronine. Under these conditions the cells do not divide, but can synthesize DNA. Maximum DNA synthesis occurs in cells that have achieved monolayer morphology under low plating densities. Estradiol-17 beta causes a dose-dependent increase in the number of cells synthesizing DNA, as well as inducing synthesis of vitellogenin. Estrogen-dependent, but not background, DNA synthesis is inhibited by the antiestrogen tamoxifen. These results imply that estradiol-17 beta acts directly on liver cells to initiate DNA replication, probably by interaction with a receptor protein and induction of specific gene transcription.

Estrogen-dependent DNA synthesis and parenchymal cell proliferation in the liver of adult male Xenopus frogs.

Estradiol-17 beta treatment of adult male Xenopus laevis induces liver parenchymal cells to synthesize DNA and proliferate. DNA synthesis begins 3 to 4 days after estrogen treatment and continues for approximately 10 days. Over this 2-week period, the total number of liver parenchymal cells increases fourfold, the wet weight of the liver remains constant, and there is a 50% reduction in cell volume. The elevated number of cells persists for several months and then returns to the control value. The extent of proliferation is hormone dose dependent. Pulse-chase experiments demonstrate that as a result of hormone treatment a minority of the parenchymal cells in the initial population enter the cell cycle, and via repeated divisions become the majority (79%) of the population by Day 14. The implications of this phenomenon for estrogen-induced liver cell differentiation and vitellogenin gene function are discussed.

Xenopus fibrinogen. Characterization of subunits and hormonal regulation of biosynthesis.

In this paper we describe the purification and characterization of Xenopus plasma fibrinogen and the hormonal factors which regulate synthesis and secretion of fibrinogen in liver parenchymal cells in primary culture. As in other vertebrate species, Xenopus fibrinogen is composed of three nonidentical polypeptide chains, A alpha, B beta, and gamma. In contrast to mammalian fibrinogens, the B beta chain of Xenopus fibrinogen has a higher apparent molecular weight than the A alpha chain. The gamma chain has the lowest molecular weight in the frog protein, as in that of other species. The relatively large size of the frog B beta chain results from the unusually large size of the NH2-terminal B fibrinopeptide, which is released by thrombin cleavage of fibrinogen. Hormonal regulation of fibrinogen biosynthesis was examined using a primary cell culture system. Purified Xenopus liver parenchymal cells, maintained for several weeks in a defined culture medium, gradually decrease the synthesis and secretion of fibrinogen. Sustained production of this protein is dependent upon the addition of a glucocorticoid, dexamethasone, to the culture medium. Fibrinogen production is suppressed if an estrogen, estradiol-17 beta, is added to the culture medium together with dexamethasone and triiodothyronine. The Xenopus system provides new insight into the structure of fibrinogen, the evolution of this protein, and the hormonal factors which regulate its synthesis.