Ikaros is a mutational target for lymphomagenesis in Mlh1-deficient mice.

Deficiencies in DNA mismatch repair (MMR) result in replication errors within key tumor suppressor genes or oncogenes, and cause hereditary nonpolyposis colorectal cancer (HNPCC). Hematological malignancy with microsatellite instability is also associated with defective MMR, but little is known about the target genes for MMR. Here we identified Ikaros, a master transcription factor of lymphoid lineage commitment and differentiation, as a mutational target in spontaneous and radiation-induced T-cell lymphomas in Mlh1-deficient mice. Three quarters of lymphomas lacked Ikaros protein expression, which resulted from a frameshift mutation that created a stop codon. Mononucleotide repeat sequences at 1029-1034(C)6 and 1567-1572(G)6 in Ikaros were mutational hot spots with a one-base deletion occurring with a frequency of 45 and 50%, respectively. Point mutations and splicing alterations were also observed. In total, 85% of the lymphomas showed aberrations in Ikaros. The characteristic of Mlh1-deficient lymphomas is harboring of multiple mutations simultaneously in the same tumor, displaying a combination of two frameshift mutations at different repeats, frameshift and point mutations, and/or deletion mutations. This is the first report of Ikaros mutations coupled with Mlh1 deficiency in lymphomagenesis.

Up regulated expression of tumour necrosis factor {alpha} converting enzyme in peripheral monocytes of patients with early systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is accompanied by abnormalities in humoral and cellular immune systems. OBJECTIVE: To determine the genes specifically expressed in the immune system in SSc by analysis of the gene expression profile of peripheral blood mononuclear cells (PBMC) from patients with SSc, including those treated with haematopoietic stem cell transplantation (HSCT). Additionally, to investigate the clinical significance of the up regulation of tumour necrosis factor alpha (TNFalpha) converting enzyme (TACE). METHODS: PBMC from patients with SSc (n = 23) and other autoimmune diseases (systemic lupus erythematosus (SLE, n = 16), rheumatoid arthritis (RA, n = 29)), and from disease-free controls (n = 36) were examined. Complementary DNA arrays were used to evaluate gene expression of PBMC, in combination with real time quantitative polymerase chain reactions. TACE protein expression in PBMC was examined by fluorescence activated cell sorter (FACS). RESULTS: In patients with SSc 118 genes were down regulated after HSCT. Subsequent comparative analysis of SSc without HSCT and healthy controls indicated SSc-specific up regulation for three genes: monocyte chemoattractant protein-3 (p = 0.0015), macrophage inflammatory protein 3alpha (p = 0.0339), and TACE (p = 0.0251). In the FACS analysis, TACE protein was mainly expressed on CD14(+) monocytes both in patients with SSc and controls. TACE expression on CD14(+) cells was significantly increased in patients with early SSc (p = 0.0096), but not in those with chronic SSc, SLE, or RA. TACE protein levels in SSc monocytes correlated with the intracellular CD68 levels (p = 0.0016). CONCLUSIONS: Up regulation of TACE expression was a unique profile in early SSc, and may affect the function of TNFalpha and other immunoregulatory molecules.

Cerebral imaging by magnetic resonance imaging and single photon emission computed tomography in systemic lupus erythematosus with central nervous system involvement.

OBJECTIVE: To assess the significance of magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT) abnormalities in patients with systemic lupus erythematosus (SLE). METHODS: Forty-four patients with SLE were retrospectively analysed. Patients were classified into three groups [1 and 2: patients with central nervous system (CNS) manifestations before and after starting high-dose steroid therapy, respectively; 3: patients without CNS manifestations. MRI was performed in all 44 patients and SPECT in 31. RESULTS: Abnormal findings in MRI were found in 19 patients. MRI abnormalities were significantly more frequent in patients with CNS manifestations than in those without [71 vs 17%, odds ratio (OR) 11.9, confidence interval (CI) 2.8-49.9, P=0.0003]. After the initiation of steroid therapy, patients with CNS manifestations also had an increased frequency of abnormal MRI. No correlation was found between SPECT findings and CNS manifestations. However, patients with CNS manifestations after starting steroids showed a markedly increased frequency of abnormal MRI and SPECT compared with those without CNS manifestations (80 vs 7%; OR 56, CI 4.4-719, P=0.0003). The positive predictive value of abnormality in both techniques in developing CNS manifestations after starting steroids was 89%. CONCLUSION: MRI findings correlated with CNS manifestations in SLE. Where there is a high suspicion of CNS involvement, the combination of MRI and SPECT may be useful in predicting CNS manifestations after starting steroid therapy.

Significance of magnetic resonance imaging in the diagnosis of nodular regenerative hyperplasia of the liver complicated with systemic lupus erythematosus: a case report and review of the literature.

Nodular regenerative hyperplasia of the liver (NRH), characterized by multiple hepatic nodules in the absence of fibrosis, is a rare but important complication of systemic lupus erythematosus (SLE) associated with non-cirrhotic portal hypertension. The diagnosis of NRH is based on the pathological examination, and radiological findings of NRH are poorly documented. We report a case of a 40-year-old woman with SLE complicated with NRH. Sixteen years after diagnosis of SLE, esophageal varices were incidentally found and diagnosis of portal hypertension due to NRH was made by magnetic resonance imaging (MRI) and confirmed by needle liver biopsy. Although MRI showed the lesions as significant nodules, neither computed tomography nor ultrasonography could demonstrate the nodules. However, serial MRI showed significant enlargement of the nodules for 2 years Because NRH may lead to portal hypertension with life-threatening variceral haemorrhage in patients with SLE, MRI is a useful, non-invasive examination to screen the patients for its presence and follow-up. We reviewed the literature regarding NRH in SLE and discuss the management of the affected patients.

Correlation between fatty liver and coronary risk factors: a population study of elderly men and women in Nagasaki, Japan.

The relation between fatty liver, detected by ultrasonography as a marker of visceral fat accumulation, and coronary risk factors was studied in 810 elderly men and 1,273 elderly women in Nagasaki, Japan from 1990 to 1992. The prevalence of fatty liver was 3.3% in the male and 3.8% in the female non-obese participants (BMI, body mass index < 26.0 kg/m2) and 21.6% in the male and 18.8% in the female obese participants (26.0 kg/m2 < or = BMI). Fatty liver was significantly (p < 0.01) related to hypercholesterolemia and hypertriglyceridemia in the men and to hypertension, hypercholesterolemia, low-HDL cholesterol, hypertriglyceridemia and diabetes mellitus or impaired glucose tolerance (DM+IGT) in the women independent of age, obesity, smoking and drinking. Non-obesity with fatty liver, rather than obesity with or without fatty liver, had the highest odds ratio for hypertension and low-HDL cholesterol in the men and for hypercholesterolemia, low-HDL cholesterol, hypertriglyceridemia and DM+IGT in the women. The prevalence of fatty liver is the same in elderly men and women, and fatty liver is an independent correlate of coronary risk factors in the elderly.

Stem cell factor protects c-kit+ human primary erythroid cells from apoptosis.

OBJECTIVE: It has been reported that stem cell factor (SCF) promotes cell survival in primary cultured human erythroid colony-forming cells (ECFC). Given the heterogeneous nature of ECFC, which may affect interpretation of the data, we purified c-kit+ ECFC and investigated the specificity and mechanisms of the anti-apoptotic effects of SCF on these cells. MATERIALS AND METHODS: Glycophorin A+ (GPA+) c-kit+ cells were purified from primary cultured ECFC derived from purified human CD34+ cells. The GPA+c-kit- and nonerythroid cells were generated from the same CD34+ cells. Apoptosis of ECFC was investigated in the absence or presence of SCF and erythropoietin (EPO) in serum-free medium. DNA fragmentation was measured with enzyme linked immunosorbent assay for oligonucleosome-sized DNA, gel electrophoresis, and annexin V labeling. Characterization of expanded cells and enriched cells was performed using multiparameter flow cytometry. For Akt assay, cells were lysed and the cleared lysates subjected to SDS-PAGE followed by Western blotting. RESULTS: In GPA+c-kit+ cells, deprivation of cytokine caused rapid DNA fragmentation within 4 hours that reached a maximum at 6 hours. This was partially but clearly prevented by SCF or EPO. In contrast, no significant DNA fragmentation was seen in GPA+c-kit- and nonerythroid cells within 24 hours. PP2, a specific Src family kinase inhibitor, but not its inactive analogue PP3, reversed the anti-apoptotic effects of SCF. PP2 also inhibited SCF-induced phosphorylation of Akt. CONCLUSION: These data indicate that SCF protects purified human GPA+c-kit+ cells from apoptosis and suggest that kit-mediated Src kinase activation is involved in Akt activation and cell survival.

Regulation of nuclear factor of activated T-cell family transcription factors during T-cell development in the thymus.

BACKGROUND: T lymphocytes undergo a series of developmental events in the thymus, and signaling through the T-cell antigen receptor is crucial in this differential program. The nuclear factor of activated T cells (NFATs) may be involved in transcriptional induction of cytokine genes and other immunoregulatory genes in T cells. OBJECTIVES: We have examined the distribution of 3 NFAT family members (NFAT1, NFATc, and NFATx) in human fetal thymocytes, by using semiquantitative RT-PCR and electrophoretic mobility shift assay. RESULTS: The messenger RNA of NFATx was expressed in all T-lymphocyte subsets tested, and expression was highest in CD4(+)CD8(+) thymocytes. Conversely, mRNA of NFAT1 was preferentially expressed in mature CD4(+) single-positive cells. NFATc mRNA was present at low levels in all subsets but was strongly induced by treatment with phorbol ester plus calcium ionophore. Using electrophoretic mobility shift assay, we observed stimulation-dependent NFAT-DNA binding in CD4(+)CD8(+) thymocytes, which was largely dependent on NFATx protein. This DNA-binding activity was inhibited by cyclosporin A, which indicated that NFATx nuclear translocation in CD4(+)CD8(+) thymocytes was regulated by calcineurin phosphatase. In contrast, NFAT1 and NFATc (and to some extent NFATx) were responsible for NFAT DNA binding in the CD4(+) cells. CONCLUSIONS: Expression of NFAT family members is differentially regulated during T-cell development, and NFATx may be involved in T-cell antigen receptor/calcineurin-dependent signaling in CD4(+)CD8(+) thymocytes.

Successive expression and activation of NFAT family members during thymocyte differentiation.

Differentiation of immature CD4(+)CD8(+) thymocytes to mature CD4(+) or CD8(+) T cells is induced by positive selection and appears to involve calcineurin-dependent activation of NFAT, a family of transcription factors. NFATx is predominantly expressed in CD4(+)CD8(+) thymocytes, whereas NFATp and NFATc are expressed at much lower levels in the thymus than in mature T cells. However, how or when each NFAT member is involved in the differentiation pathway is unclear. Using an in vitro model system where isolated CD4(+)CD8(+) thymocytes can survive and differentiate into semi-mature CD4-lineage T cells, we suggest that low calcineurin activity sustained for approximately 20 h is required for cell survival and differentiation. Accordingly, the DNA binding activity of NFAT slowly increased during the stimulation of 20 h to induce the differentiation. NFATx significantly contributed to the early rise, but the late increase was mostly due to NFATc activation. Meanwhile, the expression of NFATx mRNA decreased and that of NFATc mRNA increased. The DNA-binding activity of NFATp was detectable but low throughout the stimulation. NFATp became dominantly active after the semi-mature T cells differentiated into mature and activated CD4 T cells. These findings suggest that NFATx and NFATc successively play roles in T cell development.

Signalling into the T-cell nucleus: NFAT regulation.

The nuclear factor of activated T cells (NFAT) plays an important role in T-cell biology. Activation of T cells results in the rapid calcineurin-dependent translocation of NFAT transcription factors from the cytoplasm to the nucleus. This translocation process coupled to the subsequent active maintenance of NFAT in the nucleus compartment is critical for the induction of expression of several genes encoding cytokines and membrane proteins that modulate immune responses. The molecular cloning of the NFAT family of transcription factors has facilitated rapid progress in the understanding of the signalling mechanisms that control the activity of NFAT.

Distinct NFAT family proteins are involved in the nuclear NFAT-DNA binding complexes from human thymocyte subsets.

The nuclear factor of activated T cells (NFAT) is involved in the transcriptional induction of cytokine and other immunoregulatory genes during an immune response. Among four distinct NFAT family members identified to date, mRNAs of NFAT1, NFATc, and NFATx are expressed in the thymus. Here, we report the distribution of these three NFAT family members in human fetal thymocyte subsets and in peripheral mature T cells. We show that NFATx mRNA was expressed in all T lymphocyte subsets tested and was highest in CD4+CD8+ double positive (DP) thymocytes. Conversely, NFAT1 mRNA was preferentially expressed in the mature CD4+ single positive (SP) populations. NFATc mRNA was present at low levels in all subsets but strongly induced upon treatment with phorbol ester and calcium ionophore. Interestingly, we detected NFAT-DNA binding complexes in DP thymocytes, albeit at lower levels than in CD4 SP cells. Corresponding to the mRNA expression, we observed that NFATx was responsible for the NFAT-DNA binding in DP thymocytes. Moreover, this DNA binding was inhibited by cyclosporin A, indicating that NFATx nuclear translocation was regulated by the calcineurin phosphatase in DP thymocytes. For the CD4 SP populations, NFAT1 and NFATc, and to some extent NFATx, were responsible for the NFAT-DNA binding complexes. These results indicate that NFAT family members are differentially regulated during the development of T cells, and that NFATx may play a distinct role in calcineurin-dependent signaling in DP thymocytes.

Molecular cloning and functional characterization of murine cDNA encoding transcription factor NFATc.

Transcription factors of the NFAT (nuclear factor of activated T cells) family play important roles in immune and inflammatory responses by regulating the expression of genes encoding cytokines and immunoregulatory proteins. Here we describe cloning and characterization of full-length cDNA encoding murine (m) NFATc which predicts that the protein has all the conserved structural motifs of NFAT family members, including the rel homology domain, the NFAT homology domain and the nuclear translocation signals. mNFATc complexed with AP-1 bound specifically to the murine IL-2 NFAT recognition sequence and activated transcription from the co-transfected IL-2 promoter in COS-7 cells. Northern blot analysis showed that the cDNA probe hybridized with a 4.5 kb transcript which is highly inducible in murine T cells. By Northern and in situ hybridization, mNFATc transcript was detected from the early stage of development. In the mouse embryo, mNFATc transcript was strongly expressed in thymus, lung and submandibular gland and weakly in skeletal muscle and heart suggesting that mNFATc may have a role both in embryogenesis and in mature T cells.

[Cytokine gene expression in Th1 and Th2: its molecular mechanism and clinical implication].

Activation of helper T-cells mediated by the T-cell receptor induces a series of biochemical events. Among them, both the activation of PKC/Ras- and CaM/CN-mediated pathways play a central role in the signal transduction of cytokine gene induction. Cytokines produced by non-transformed Th1 and Th2 clones were classified into three groups, based on their signal requirement patterns for their expression. Closer examination using various stimulation conditions suggested that the balance between the activities of the two signaling pathways contributes to cytokine expression. Th1 and Th2 effector functions and their development are attributable to their coordinated and differential expression of cytokines. Clarification of the mechanisms of Th1/Th2 differentiation should lead to rational strategies for manipulating pathological immune responses.

Calcineurin-dependent nuclear translocation of a murine transcription factor NFATx: molecular cloning and functional characterization.

Members of the nuclear factor of activated T cells (NFAT) are involved in the induction of a number of cytokine genes. We report here cDNA cloning and chromosomal localization of a murine homologue of human NFATx, designated as mNFATx1, and its splicing variants mNFATx2 and m delta NFATx. Northern blot analysis showed mNFATx1 to be predominantly expressed in the thymus. mNFATx1, but not m delta NFATx, produced in COS-7 cells, bound to all NFAT-binding sites of the interleukin (IL)-2 and IL-4 promoters tested. Immunofluorescence assay showed that both mNFATx1 and m delta NFATx introduced into COS-7 cells localized predominantly to the cytoplasm, but did translocate to the nucleus, either by cotransfection with an active form of calcineurin or wild-type calcineurin followed by stimulation with calcium ionophore. Translocation of mNFATx1 correlated well with activation of the murine IL-2 promoter; mNFATx1 translocated under conditions described above, in combination with phorbol 12-myristate 13-acetate, activated the transiently transfected murine IL-2 promoter. Thus, nuclear-translocated mNFATx1 is involved in activation of the IL-2 promoter. These results provide the first evidence for the requirement of calcineurin in the control of mNFATx imported from the cytoplasm to the nucleus and implies that mNFATx may possibly be a substrate of calcineurin in vivo.

Serum levels of soluble Fas/APO-1 (CD95) and its molecular structure in patients with systemic lupus erythematosus (SLE) and other autoimmune diseases.

There are two major forms of the Fas molecule, membranous Fas and soluble Fas (sFas). To clarify the clinical significance of sFas in autoimmune diseases, we designed a sandwich ELISA to determine serum concentrations of sFas and its molecular structure, and we then analysed the correlation between levels of sFas and laboratory findings in patients with SLE and other autoimmune diseases. The levels of serum sFas were significantly higher in SLE patients than in subjects with other autoimmune diseases and in healthy donors, and the frequency of a positive serum sFas was much greater in SLE patients with high SLE disease activity index scores than in those with low scores. In addition, sFas-positive SLE patients showed a significant difference in various laboratory parameters from sFas-negative SLE patients. Serial measurements of serum sFas levels in SLE patients with active disease revealed that the elevated level of sFas dramatically decreased with improvement in clinical and laboratory findings, following corticosteroid therapy. We propose that the serum level of sFas can serve as an appropriate marker for evaluating SLE disease activity. Serum sFas is heterogeneous with respect to molecular structure, thus several mechanisms are involved in the generation of sFas.

[A case of melorheostosis with linear sclerodermatous skin changes].

A 69-years old Japanese woman complained of pain in the left elbow joint and thickened skin over the left upper limb. The pain had been present for 20 years, and the thickened area of the skin gradually enlarged during this period. Her left elbow joint showed some limitation of motion. There was no record of any similar condition in her family history. Radiographs of the left limb showed cortical hyperostosis extending from the middle of the left humerus to the distal end of the radius. Radiographs of the other limbs were normal. A technetium 99m-methylene diphosphonate bone scintigraphy revealed increased uptake in the areas of radiographic hyperostosis. The diagnosis of melorheostosis was made. Skin biopsy of thickened area was performed. The epidermis was normal, and proliferation of normal-appearing collagen fibers into the subcutaneous fat was noted. No inflammatory changes were found. The cause of sclerodermatous skin changes was thought to be not by linear scleroderma but by melorheostosis. In cases of linear sclerodermatous changes, melorheostosis as its origin should be considered.

[The association of the disease activity of rheumatoid factor positive vasculitis and the level of rheumatoid factor].

Rheumatoid factor (RF), an autoantibody against the Fc portion of denatured IgG, has long been recognized as an important biologic marker not only for rheumatoid arthritis but also for other auto-immune diseases. In this study, we measured the level of serum RF in four patients with RF positive systemic vasculitis using laser nephelometry. Three patients were diagnosed as polyarteritis nodosa and the other patient was diagnosed as systemic vasculitis without the finding of typical necrotizing vasculitis from biopsies. In the result, we found that the level of RF paralleled the disease activity in these cases. When active phase of the disease, the level of RF showed very high, and after the treatment combined with plasmapheresis, corticosteroid and immunosuppressive agent, the level of RF decreased in accordance with CRP, ESR and clinical features. These suggested that RF was the disease specific marker for RF positive vasculitis and beneficial informations for proper diagnosis and better treatment could be provided by measurement of the level of RF in patients with RF positive systemic vasculitis.

[A case of mixed connective tissue disease with acute interstitial pneumonitis].

A 43-years-old woman was admitted to the Hokkaido University Hospital because of high fever, muscle weakness and dyspnea in May 1993. She had has muscle weakness of upper extremities since December 1992. She had developed swollen hand, polyarthralgia and Raynaud's phenomenon. High fever and severe dyspnea developed in May 1993. Chest roentogenogram was normal in April 1993. Physical examination showed Velcro rales in both lower lung fields. Her laboratory data showed increased muscle enzymes, high titers of anti-nuclear-antibody (1: 1280) and anti-RNP-antibody (index 199.4 (normal < 7)). Anti-DNA, anti-Sm and anti-Jo-1-antibodies were all negative. Blood gas analysis showed severe hypoxemia. Chest roentogenogram revealed diffuse bilateral interstitial infiltrates prominent in the bases. Diagnosis of mixed connective tissue disease with acute interstitial pneumonitis was made. She was treated with steroid pulse therapy (methylprednisolone 1 g x 3 days) followed by high dose oral prednisolone (60 mg/day), and diffuse interstitial infiltrates disappeared within one week. Prednisolone could be tapered to 17.5 mg/day without relapse. Acute interstitial pneumonitis is a rare complication of mixed connective tissue disease, but may be life threatening. In such cases, high dose steroid therapy should be started without delay.

Severe hepatic involvement without inflammatory changes in systemic lupus erythematosus: report of two cases and review of the literature.

Hepatic diseases in systemic lupus erythematosus (SLE) are not rare, but liver biopsies of those cases are usually reported as chronic hepatitis or steroid-induced steatosis. We describe two unusual patients with active SLE who displayed liver dysfunction without inflammatory changes or associated with drug administration. A liver biopsy in case 1 showed massive hepatic cell damage resulting in acute hepatic failure. In case 2, the liver specimen revealed diffuse fatty degeneration without symptoms specific to liver dysfunction. No inflammatory cell infiltrate was observed in the liver tissue of either patient. After steroid pulse therapy (case 1) and the administration of 60 mg/day of prednisolone (case 2), liver function improved in parallel with the stabilization of the other manifestations of SLE. No other causes for liver damage except for SLE were observed in either case. Therefore it is supposed that the liver impairments in these cases were one manifestation of SLE.

Up-regulated expression of Fas antigen (CD95) by peripheral naive and memory T cell subsets in patients with systemic lupus erythematosus (SLE): a possible mechanism for lymphopenia.

Fas antigen (CD95) is a membrane-associated molecule that mediates apoptotic cell death and may play a role in the induction and maintenance of T cell tolerance. To elucidate the involvement of Fas antigen in human autoimmune diseases, we analysed Fas antigen expression by peripheral T cells from patients with SLE and rheumatoid arthritis (RA), using three-colour flow cytometry. Both CD4+ and CD8+ T cells from SLE patients expressed Fas antigen in a higher density than did these cells from healthy donors and from RA patients. Enhancement of Fas antigen density was noted in Fas+CD45RO+ memory T cells from SLE patients. More remarkably, a significant expression of Fas antigen was observed in CD45RO- naive T cells from SLE patients. CD4+CD45RO- T cells from SLE patients co-expressed Fas antigen and early to intermediate activation antigens such as CD25 and CD71, and late activation antigen HLA-DR in only FashiCD4+ naive T cells. Such up-regulation of Fas antigen expression in SLE patients seems to be clinically meaningful, because mean fluorescence intensity (MFI) of Fas antigen on CD4+ T cell subsets inversely correlates with the absolute size of CD4+ T cell subsets in peripheral blood of SLE patients. These results suggest that T cells with increased Fas antigen expression may be highly susceptible to apoptotic cell death, in vivo. A putative mechanism for lymphopenia in SLE patients is discussed.