Undiminished regulatory T cells in the thymus of patients with myasthenia gravis.

OBJECTIVE: The thymus has been implicated as a possible site of origin that triggers autoimmunity in myasthenia gravis (MG). Although several groups have suggested that the decrease in the number of regulatory T (Treg) cells contributes to the onset of MG, the exact role of Treg cells in MG remains unclear. To address this point, we examined the number and distribution of Treg cells in a large number of patients with MG. METHODS: Immunohistofluorescence analysis of Foxp3 along with CD4 and CD8 was performed in thymic sections of MG (+) (n = 24) and MG (-) patients (n = 27). Circulating CD4(+)CD25(+) cells in the peripheral blood of patients with MG (n = 15) and age-matched healthy subjects (n = 15) were also analyzed. RESULTS: Foxp3(+)CD4(+)CD8(-) cells were predominantly found in the thymic medulla and their number declined with age. There was no significant difference in the number or the distribution of Foxp3(+)CD4(+)CD8(-) cells in the thymus between MG (+) and MG (-) patients. The number of circulating CD4(+)CD25(+) cells in the peripheral blood of patients with MG was not significantly altered compared to that in healthy subjects. CONCLUSION: The cellularity of Treg cells in the thymus and circulation is not diminished in patients with myasthenia gravis.

Transgenic over-expression of MafK suppresses T cell proliferation and function in vivo.

BACKGROUND: The small Maf proteins regulate gene transcription from Maf recognition elements (MARE). These proteins do not contain a canonical transactivation domain. Depending upon the ratio of small Maf proteins to their partner proteins, which either possess a transactivation domain or not, transcription can be switched on or off. RESULTS: Transgenic mice were generated which over-express the small Maf family member MafK, specifically in the T cell lineage. It was our expectation that the high level of MafK would shift the balance to the formation of MafK homodimer and thereby repress MARE-dependent transcription. The transgenic mice had a shortened life span because of Pneumocystis carinii pneumonia and displayed a decrease in thymocytes and lower IL-2 and IL-4 mRNA expression levels. Analyses by electrophoretic gel mobility shift assay revealed that over-expressed MafK could interact with the proximal AP-1 sequence of IL-2 and the MARE in the IL-4 promoter region. CONCLUSION: These results indicate that when over-expressed, MafK binds to a MARE-like sequence and represses MARE-dependent transcription. Consequently, T cell proliferation and cytokine secretion are affected. The MafK homodimer serves as an important molecular probe for evaluating the role played by cis-acting MAREs in the proliferation and function of T cells.

Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells.

The resetting of a somatic epigenotype to a totipotential state has been demonstrated by successful animal cloning, via transplantation of somatic nuclei into enucleated oocytes. We have established an experimental system, which reproduces the nuclear reprogramming of somatic cells in vitro by fusing adult thymocytes with embryonic stem (ES) cells. Analysis of the lymphoid-cell-specific V-(D)-J DNA rearrangement of the T cell receptor and immunoglobin genes shows that the ES cells have hybridized with differentiated cells. In these ES cell hybrids, the inactivated X chromosome derived from a female thymocyte adopts some characteristics of an active X chromosome, including early replication timing and unstable Xist transcription. We also found that an Oct4-GFP transgene, which is normally repressed in thymocytes, is reactivated 48 hr after cell fusion. The pluripotency of the ES-thymocyte hybrid cells is shown in vivo, since they contribute to all three primary germ layers of chimeric embryos. The somatic DNA methylation pattern of the imprinted H19 and Igf2r genes is maintained in these hybrids, unlike hybrids between ES and EG (embryonic germ) cells in which the differential methylation is erased. Thus, ES cells have the capacity to reset certain aspects of the epigenotype of somatic cells to those of ES cells.

Stat3 in thymic epithelial cells is essential for postnatal maintenance of thymic architecture and thymocyte survival.

This study describes abnormalities of the thymus in mice in which the Stat3 gene has been specifically disrupted behind the keratin 5 promoter. In these mice, virtually all of the thymic epithelial cells (TEC) were deficient for Stat3 activation. Adult mutant mice developed severe thymic hypoplasia, which included alterations in the cortical TEC architecture that coincided with the loss of thymocytes. Even during the asymptomatic period of preadolescence, these mice exhibited a higher susceptibility of the thymus to suboptimal doses of dexamethasone or gamma-irradiation, while their thymocytes per se were no more sensitive than controls. These results indicate that Stat3 in TEC plays an essential role in maintaining thymic architecture and thymocyte survival.

Akt inhibits the orphan nuclear receptor Nur77 and T-cell apoptosis.

Akt is a common mediator of cell survival in a variety of circumstances. Although some candidate Akt targets have been described, the function of Akt is not fully understood, particularly because of the cell type- and context-dependent apoptosis regulation. In this study, we demonstrate that one of the mechanisms by which Akt antagonizes apoptosis involves the inhibition of Nur77, a transcription factor implicated in T-cell receptor-mediated apoptosis. It has been suggested that Akt phosphorylates Nur77 directly, but whether Akt suppresses biological functions of Nur77 remains unknown. We found that Akt inhibited the DNA binding activity of Nur77 and stimulated its association with 14-3-3 in a phosphorylation site-dependent manner. Moreover, we found that expression of Akt suppressed Nur77-induced apoptosis in fibroblasts and activation-induced cell death of T-cell hybridomas. The inhibition of Nur77 by Akt suggests a mechanism that explains how T-cell receptor activation can promote survival in some instances even when Nur77 is induced. Collectively, these results may suggest that Akt is a negative regulator of Nur77 in T-cell apoptosis.

Annexin II overexpression is correlated with poor prognosis in human gastric carcinoma.

Annexins belong to a family of the calcium-dependent phospholipid binding proteins. They are also substrates of receptor tyrosine kinases. Overexpression of Annexin II, which has been reported in various carcinomas, is thought to be associated with cell proliferation, differentiation and cell-cell adhesion in the pathogenesis of carcinoma, but the functions of Annexins have not been fully elucidated. In this study, we investigated the role of Annexin II (p36) and its relationship with c-erbB-2 overexpression in gastric carcinoma. We studied Annexin II expression using Western blot analysis in 8 human gastric carcinoma cell lines and expression of Annexin II and c-erbB-2 using, immunohistochemistry in 153 primary gastric carcinomas. Western blot revealed that Annexin II was expressed in 8 human gastric carcinoma cell lines. It was more strongly expressed in the cell membrane than in the cytoplasm of tumor cells in primary gastric carcinoma tissues. Thirty-three percent of all cases were immunopositive for Annexin II, overexpression of which was more frequent in differentiated type (p = 0.0009), lymph node, metastasis (p = 0.0147) and venous invasion (p = 0.0092). Annexin II and c-erbB-2 overexpression were significantly correlated p = 0.0002) and patients with Annexin II had poorer prognoses (p = 0.0066). Multivariate analysis showed that immunopositivity of both Annexin II and c-erbB-2 was an independent and poor prognostic factor (p = 0.0037). In conclusion, Annexin II was overexpressed in advanced gastric carcinomas and it could contribute to the progression of gastric carcinoma.

Genetic modulation of immature T lymphocytes and its application.

T lymphocytes are the cells that play an essential role in regulating immune responses. The thymus is the organ in which T lymphocytes are generated. Our laboratory has investigated molecular signals that determine cell fate during T lymphocyte development in the thymus. To this goal, we have devised a technique in which one may efficiently introduce foreign genes into immature T lymphocytes. The somatic gene-transfer into developing T lymphocytes are likely useful to restore various immunodeficiencies and to establish immune tolerance to any introduced genes. The genetically engineered immune tolerance may be applied to reduce allergies and autoimmune diseases, as well as to sustain gene therapies by allowing prolonged survival of therapeutic vectors.

Synchronous deletion of Mtv-superantigen-reactive thymocytes in the CD3(medium/high) CD4(+)CD8(+) subset.

Multiple model systems have demonstrated that negatively selected thymocytes can be deleted during the immature CD4(+)CD8(+) CD3(low) stage after high affinity interaction of T-cell receptors (TCRs) with antigen:major histocompatibility complex (MHC) complexes. Superantigens (SAGs) derived from endogenous mammary tumour viruses (Mtv) induce negative selection of Mtv-SAG-reactive thymocytes regardless of which peptide antigen is presented by MHC molecules. In this study, the timing of deletion of multiple subsets of Mtv-SAG-reactive CD4(+)CD8(+) thymocytes was investigated by a 4 colour flow cytometry in SJL x CBA/J cross-bred mice. Deletion of V beta 3(+), V beta 5(+), V beta 11(+), and V beta 17(+) Mtv-SAG-reactive thymocytes was found to occur synchronously in the most mature CD3(medium) and early CD3(high) subsets of CD4(+)CD8(+) thymocytes, in contrast with reports showing that the deletion of Mtv-SAG-reactive thymocytes can occur at different stages in particular model systems.

Diminution of the AML1 transcription factor function causes differential effects on the fates of CD4 and CD8 single-positive T cells.

In the thymic cortex, T lymphocytes are positively selected to survive and committed either to the CD4 single-positive (SP) or the CD8 SP lineage. The SP cells then pass through a step of maturation in the medulla and are delivered to peripheral lymphoid tissues. We examined the role of AML1, the gene encoding a transcription factor, in the above processes by using the transgenic mice expressing a dominant interfering form of AML1 as well as mice targeted heterozygously for AML1. One phenotypic change seen in the AML1-diminished mice was the reduction in the numbers of both CD4 SP and CD8 SP thymocytes, reflecting the partial impairment of the transition from the double-positive to SP stage. In addition, distinct from the above abnormality, perturbed were several aspects of SP cells, including the maturation of SP thymocytes, the recent thymic emigration, and the proliferative responsiveness of peripheral T cells to TCR stimulation. Interestingly, the AML1 diminution caused inhibitory and enhancing effects on the CD4 SP and CD8 SP cells, respectively. These differential effects are most likely related to the reduction in the peripheral CD4 SP/CD8 SP ratio observed in the AML1-diminished mice. The AML1 transcription factor thus maintains the homeostasis of each SP subset by functioning at the later stages of T lymphocyte differentiation.

Detection of spatial localization of Hst-1/Fgf-4 gene expression in brain and testis from adult mice.

HST-1, a member of the fibroblast growth factor (FGF) family (FGF-4), has been shown to be a signaling molecule whose expression is essential for embryonic development. However, HST-1/FGF-4 expression has not been detected or reported in adult tissues so far analysed. To investigate whether there is a possible role of HST-1/FGF-4 in adult stage, we have carried out a highly sensitive RT-PCR analysis of Hst-1/Fgf-4 gene expression in adult mice tissues. Results show Hst-1/Fgf-4 gene expression in the nervous system, intestines, and testis of normal adult mice. In situ hybridization technique was used to localize Hst-1/Fgf-4 gene expression in the cerebellum and testis from 10-week-old mice. Cell type-specific gene expression was detected: Purkinje cells in the cerebellum and Sertoli cells in testis. These findings suggest that the Hst-1/Fgf-4 gene also plays an important role in adult tissues, and may offer insights into the biological significance of HST-1/FGF-4 in cerebellar and testicular functions.

Inhibition of liver metastases and tumor cell invasion in spontaneous liver metastasis model (LMFS) by sodium D-glucaro-delta-lactam (ND2001).

We examined the inhibitory effect of sodium D-glucaro-delta-lactam (sodium 5-amino-5-deoxy-D-glucosaccharic acid-delta-lactam: ND2001) upon liver metastases of the LMFS tumor. A permanent cell line, LMFS, was established from a spontaneously occurring murine retroperitoneal tumor of BALB/c mouse origin, and after a subcutaneous injection, the LMFS cells proliferated at the inoculation site (100% take) with liver metastases. ND2001 had little effect on the cell growth, cell cycle and phagokinesis of the LMFS cells in vitro. However, when the invasive activity was measured by the Boydem chamber method, the number of LMFS cells was reduced, with inhibition rates of 98.0%. After the LMFS cells treated with ND2001 in vitro, the numbers of hepatic metastases of subcutaneous inoculation of treated cells were reduced dose-dependently, and those of intravenous inoculation were not found by microscopical study. When the LMFS tumor-bearing mice were treated with ND2001 (0, 30, 100 mg/kg/d) from day 1, ND2001 (30 mg/kg) inhibited the liver metastases with a rate of 56.4%, and when given from day 15, ND2001 (100 mg/kg) inhibited with a rate of 47.5%. But ND2001 showed neither cytocidal nor anti-tumor activity. Combination therapy of primary tumor resection and ND2001 administration revealed that preoperative use of ND2001 was more effective in preventing liver metastases. These results suggested that ND2001 might have a potential use as an anti-metastatic agent for operative patients without metastasis.

A role for pref-1 and HES-1 in thymocyte development.

T lymphocyte development requires a series of interactions between developing thymocytes and thymic epithelial (TE) cells. In this paper we show that TE cells in the developing thymus express Pref-1, a Delta-like cell-surface molecule. In fetal thymus organ cultures (FTOC), thymocyte cellularity was increased by the exogenous dimeric Pref-1 fusion protein, but was reduced by the soluble Pref-1 monomer or anti-Pref-1 Ab. Dimeric Pref-1 in FTOC also increased thymocyte expression of the HES-1 transcription factor. Thymocyte cellularity was increased in FTOC repopulated with immature thymocytes overexpressing HES-1, whereas FTOC from HES-1-deficient mice were hypocellular and unresponsive to the Pref-1 dimer. We detected no effects of either Pref-1 or HES-1 on developmental choice among thymocyte lineages. These results indicate that Pref-1 expressed by TE cells and HES-1 expressed by thymocytes are critically involved in supporting thymocyte cellularity.

Adenovirus-mediated transfer of HST-1/FGF-4 gene protects mice from lethal irradiation.

Intraperitoneal injection of a replication-deficient adenovirus containing the HST-1 (FGF-4) gene (Adex1HST-1) increased peripheral platelet counts in mice, and also effectively prevented experimentally induced thrombocytopenia. Here, we report the therapeutic potential of Adex1HST-1 on severely injured mice after exposure to otherwise lethal irradiation. Eighteen out of 20 mice that received Adex1HST-1 prior to gamma-irradiation (9 Gy) survived, while all the 20 mice with prior administration of control adenoviruses died after irradiation (P<0.0001). Hematological and histopathological analyses revealed that Adex1HST-1 acts as a potent protector against lethal irradiation, which causes injury of intestinal tract as well as myelosuppression in the bone marrow and spleen. These data demonstrate that the protective effects of administration of Adex1HST-1 against irradiation are superior to any other protective effects of cytokines against a lethal dose of irradiation, and that the pre-administration of Adex1HST-1 may be useful for lessening the side effects of currently used chemo- and radio-therapy against cancer.

Gamma interferon production is critical for protective immunity to infection with blood-stage Plasmodium berghei XAT but neither NO production nor NK cell activation is critical.

We have examined the roles of gamma interferon (IFN-gamma), nitric oxide (NO), and natural killer (NK) cells in the host resistance to infection with the blood-stage malarial parasite Plasmodium berghei XAT, an irradiation-induced attenuated variant of the lethal strain P. berghei NK65. Although the infection with P. berghei XAT enhanced NK cell lytic activity of splenocytes, depletion of NK1.1(+) cells caused by the treatment of mice with anti-NK1.1 antibody affected neither parasitemia nor IFN-gamma production by their splenocytes. The P. berghei XAT infection induced a large amount of NO production by splenocytes during the first peak of parasitemia, while P. berghei NK65 infection induced a small amount. Unexpectedly, however, mice deficient in inducible nitric oxide synthase (iNOS-/-) cleared P. berghei XAT after two peaks of parasitemia were observed, as occurred for wild-type control mice. Although the infected iNOS-/- mouse splenocytes did not produce a detectable level of NO, they produced an amount of IFN-gamma comparable to that produced by wild-type control mouse splenocytes, and treatment of these mice with neutralizing anti-IFN-gamma antibody led to the progression of parasitemia and fatal outcome. CD4(-/-) mice infected with P. berghei XAT could not clear the parasite, and all these mice died with apparently reduced IFN-gamma production. Furthermore, treatment with carrageenan increased the susceptibility of mice to P. berghei XAT infection. These results suggest that neither NO production nor NK cell activation is critical for the resistance to P. berghei XAT infection and that IFN-gamma plays an important role in the elimination of malarial parasites, possibly by the enhancement of phagocytic activity of macrophages.

Evaluation of cationic liposome suitable for gene transfer into pregnant animals.

Cationic liposome-mediated in vivo gene transfer represents a promising approach for somatic gene therapy. To assess the most suitable liposome for gene delivery into a wide range of organs and fetuses in mice, we have explored several types of cationic liposomes conjugated with plasmid DNA carrying the beta-galactosidase gene through intravenous injection into pregnant animals. Transduction efficiency was assessed by Southern blot analysis and expression of the transferred gene was evaluated by enzymatic demonstration of beta-galactosidase activity. Through the analysis of several types of recently synthesized cationic liposome/lipid formulations, DMRIE-C reagent, a liposome formulation of the cationic lipid DMRIE (1, 2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) and cholesterol in membrane-filtered water met our requirements. When the plasmid DNA/DMRIE-C complexes were administered intravenously into pregnant mice at day 11.5 post coitus (p.c.), transferred genes were observed in several organs in dams and were expressed. Furthermore, although the copy numbers transferred into embryos were low, we observed reporter gene expression in the progeny.