MEFV mutation analysis of familial Mediterranean fever in Japan.

BACKGROUND: Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent attacks of fever with serosal inflammation. FMF gene (MEFV) mutations have been identified primarily in patients from Mediterranean populations. Although several clinical cases have been reported in Japan, there have been few reports to date on mutation analysis. We studied FMF patients and their relatives to examine the clinical and genetic features of this disease in the Japanese population. METHODS: Twelve Japanese FMF patients who met the Tel Hashomer criteria and a total of 17 relatives from 5 of 10 families underwent molecular genetic studies to detect MEFV mutations. The characteristics of these Japanese FMF patients and geno-phenotypical correlations were examined. RESULTS: Almost all of our patients had been suffering for a long time from fever of unknown origin and one patient also had systemic amyloidosis. In our 12 FMF patients, we detected the substitutions E84K, L110P, E148Q, R761H and M694I. We also newly diagnosed 2 relatives as having FMF based on clinical symptoms and the existence of FMF mutations. One patient was homozygous for E148Q, the patient with systemic amyloidosis was a homozygote for M694I and 4 patients from 3 families were compound heterozygotes for E148Q and M694I. Three patients in one family were compound heterozygotes for E148Q, L110P and M694I. There were 3 patients who were heterozygous for E84K, L110P-E148Q or M694I and had no other nucleotide changes in the exons of MEFV. On the other hand, 2 relatives who had never experienced symptoms of FMF were homozygous for L110P-E148Q as well as compound heterozygous for E148Q/E148Q-R761H. E148Q and M694I were the most frequently detected substitutions in our study. CONCLUSIONS: MEFV mutations occur in Japanese FMF patients though FMF is rare in Japan. The identification of MEFV mutations could be a reliable diagnostic test for FMF. The results of genetic analyses on 14 Japanese FMF patients in this study revealed that E148Q and M694I are frequent alleles.

Screening of genes responsible for differentiation of mouse mesenchymal stromal cells by DNA micro-array analysis of C3H10T1/2 and C3H10T1/2-derived cell lines.

BACKGROUND: The molecular mechanisms underlying the biologic effects or differentiation of mesenchymal stromal cells (MSC) have not been clarified. Screening for genes differentially expressed at different stages is an important step in determining these molecular mechanisms. METHODS: In this study, we analyzed the gene expression profiles of C3H10T1/2 (10T1/2) cells and two sublines, A54 (pre-adipocyte) and M1601 (myoblast), as a model of MSC and downstream committed progenitors. RESULTS: We found up-regulated expression of delta-like-1 (Dlk), Wnt-5a and IL-1 receptor-like-1 (ST2) in 10T1/2 cells; stem cell factor (SCF) and stromal derived factor-1 (SDF-1) in A54 cells; and cardiac muscle-specific gene in M1601 cells. Overexpression of Dlk in A54 cells did not induce any effects on their differentiation into adipocytes. After differentiation into adipocytes, A54 cells reduced the expression of SCF, SDF-1 and Ang-1 as well as the ability to support the formation of a cobblestone appearance. DISCUSSION: The results suggest that these three lines hae different gene profiles and are a useful system for analyzing the differentiation and function of MSC and progenitor cells.

Sak serine-threonine kinase acts as an effector of Tec tyrosine kinase.

The murine sak gene encodes a putative serine-threonine kinase which is homologous to the members of the Plk/Polo family. Although Sak protein is presumed to be involved in cell growth mechanism, efforts have failed to demonstrate its kinase activity. Little has been, therefore, elucidated how Sak is regulated and how Sak contributes to cell proliferation. Tec is a cytoplasmic protein-tyrosine kinase (PTK) which becomes activated by the stimulation of cytokine receptors, lymphocyte surface antigens, heterotrimeric G protein-linked receptors, and integrins. To clarify the in vivo function of Tec, we have tried to isolate the second messengers of Tec by using the yeast two-hybrid screening. One of such Tec-binding proteins turned out to be Sak. In human kidney 293 cells, Sak became tyrosine-phosphorylated by Tec, and the serine-threonine kinase activity of Sak was detected only under the presence of Tec, suggesting Sak to be an effector molecule of Tec. In addition, Tec activity efficiently protects Sak from the "PEST" sequence-dependent proteolysis. Internal deletion of the PEST sequences led to the stabilization of Sak proteins, and expression of these mutants acted suppressive to cell growth. Our data collectively supports a novel role of Sak acting in the PTK-mediated signaling pathway.

Herpes simplex virus bronchopneumonia in a non-immunocompromized individual.

We report a rare case of herpes simplex virus (HSV) bronchopneumonia in an otherwise healthy middle-aged individual. Bronchoscopy indicated scattered white-coated lesions in the bronchial mucosa. The diagnosis was established following immunohistopathological staining for HSV of specimens obtained by bronchial biopsy. This case suggests that HSV could be a pathological agent of not only oral and genital mucosal lesions but also lower respiratory tract infection.

Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction.

Myelodysplastic syndrome (MDS) is a slowly progressing hematologic malignancy associated with a poor outcome. Despite the relatively high incidence of MDS in the elderly, differentiation of MDS from de novo acute myeloid leukemia (AML) still remains problematic. Identification of genes expressed in an MDS-specific manner would allow the molecular diagnosis of MDS. Toward this goal, AC133 surface marker-positive hematopoietic stem cell (HSC)-like fractions have been collected from a variety of leukemias in a large-scale and long-term genomics project, referred to as "Blast Bank," and transcriptome of these purified blasts from the patients with MDS were then compared with those from AML through the use of oligonucleotide microarrays. A number of genes were shown to be expressed in a disease-specific manner either to MDS or AML. Among the former found was the gene encoding the protein Delta-like (Dlk) that is distantly related to the Delta-Notch family of signaling proteins. Because overexpression of Dlk may play a role in the pathogenesis of MDS, the disease specificity of Dlk expression was tested by a quantitative "real-time" polymerase chain reaction analysis. Examination of the Blast Bank samples from 22 patients with MDS, 31 with AML, and 8 with chronic myeloid leukemia confirmed the highly selective expression of the Dlk gene in the individuals with MDS. Dlk could be the first candidate molecule to differentiate MDS from AML. The proposal is made that microarray analysis with the Blast Bank samples is an efficient approach to extract transcriptome data of clinical relevance for a wide range of hematologic disorders.

IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-gamma production by NK cells.

The aim of this study was to examine the contribution of IL-18 in host defense against infection caused by Cryptococcus neoformans in mice with defective IL-12 production. Experiments were conducted in mice with a targeted disruption of the gene for IL-12p40 subunit (IL-12p40-/- mice). In these mice, host resistance was impaired, as shown by increased number of organisms in both lungs and brains, compared with control mice. Serum IFN-gamma was still detected in these mice at a considerable level (20-30% of that in control mice). The host resistance was moderately impaired in IL-12p40-/- mice compared with IFN-gamma-/- mice. Neutralizing anti-IFN-gamma mAb further increased the lung burdens of organisms. In addition, treatment with neutralizing anti-IL-18 Ab almost completely abrogated the production of IFN-gamma and also impaired the host resistance. Host resistance in IL-12p40-/- IL-18-/- mice was more profoundly impaired than in IL-12p40-/- mice. Administration of IL-12 as well as IL-18 increased the serum levels of IFN-gamma and significantly restored the reduced host resistance. Spleen cells obtained from infected IL-12p40-/- mice did not produce any IFN-gamma upon restimulation with the same organisms, while those from infected and IL-12-treated mice produced IFN-gamma. In contrast, IL-18 did not show such effect. Finally, depletion of NK cells by anti-asialo GM1 Ab mostly abrogated the residual production of IFN-gamma in IL-12p40-/- mice. Our results indicate that IL-18 contributes to host resistance to cryptococcal infection through the induction of IFN-gamma production by NK cells, but not through the development of Th1 cells, under the condition in which IL-12 synthesis is deficient.

Mediation by the protein-tyrosine kinase Tec of signaling between the B cell antigen receptor and Dok-1.

A variety of growth factor receptors induce the tyrosine phosphorylation of a nonreceptor protein-tyrosine kinase Tec as well as that of a Tec-binding protein of 62 kDa. Given the similarity in properties between this 62-kDa protein and p62(Dok-1), the possibility that these two proteins are identical was investigated. Overexpression of a constitutively active form of Tec in a pro-B cell line induced the hyperphosphorylation of endogenous Dok-1. Tec also associated with Dok-1 in a phosphorylation-dependent manner in 293 cells. Tec mediated marked phosphorylation of Dok-1 both in vivo and in vitro, and this effect required both the Tec homology and Src homology 2 domains of Tec in addition to its kinase activity. Expression of Dok-1 in 293 cells induced inhibition of Ras activity, suggesting that Dok-1 is a negative regulator of Ras. In the immature B cell line Ramos, cross-linking of the B cell antigen receptor (BCR) resulted in tyrosine phosphorylation of Dok-1, and this effect was markedly inhibited by expression of dominant negative mutants of Tec. Furthermore, overexpression of Dok-1 inhibited activation of the c-fos promoter induced by stimulation of the BCR. These results suggest that Tec is an important mediator of signaling from the BCR to Dok-1.

Reduced host resistance and Th1 response to Cryptococcus neoformans in interleukin-18 deficient mice.

Using interleukin (IL)-18 deficient (IL-18(-/-)) mice, we examined the role of IL-18 in the host resistance and Th1 response against infection with Cryptococcus neoformans. Fungal clearance in the lung was reduced in IL-18(-/-) mice, although there was no significant change in the level of dissemination to the brain. The DTH response, as determined by footpad swelling, was also diminished in IL-18(-/-) mice compared to control wild-type (WT) mice. The levels of IL-12 and interferon (IFN)-gamma in the sera were significantly lower in IL-18(-/-) mice than in WT mice. Spleen cells from infected WT mice produced a high level of IFN-gamma upon stimulation with the microbe, while only a low level of IFN-gamma production was detected in spleen cells from infected IL-18(-/-) mice. Administration of IL-18 almost completely restored the reduced response in IL-18(-/-) mice, while IL-12 showed a marginal effect. These results demonstrated the important role of IL-18 in the resistance and Th1 response of mice to C. neoformans by potentiating the production of IFN-gamma.

Chemokine synthesis and cellular inflammatory changes in lungs of mice bearing p40tax of human T-lymphotropic virus type 1.

To elucidate the pathogenic mechanisms of human T-lymphotropic virus type 1 (HTLV-1)-associated lung inflammation, we conducted a histopathological and molecular analysis study using transgenic mice bearing pX region of this virus. In these mice, accumulations of inflammatory cells consisting mainly of lymphocytes were present in peribronchiolar and perivascular areas and alveolar septa, while control littermate mice did not show such changes. In situ hybridization showed that the anatomic distribution of p40tax mRNA was similar to that of inflammatory cells, typically in peribronchiolar areas and to a lesser extent in perivascular and alveolar septa. Inflammatory cytokines, including IL-1beta, tumour necrosis factor-alpha and interferon-gamma, and several chemokines, such as monocyte chemotactic protein-1 (MCP-1), RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha) and IP-10, were detected in lungs of transgenic mice but not control mice. Semiquantitative analysis using reverse transcription-polymerase chain reaction showed a significant correlation between MCP-1 mRNA expression and p40tax mRNA, but not with other chemokines. The gene expression of the above chemokines, with the exception of MIP-1alpha, correlated with the severity of histopathological changes in the lung. Considered together, our results suggested that p40tax synthesis may be involved in the development of lung lesions caused by HTLV-1 through the induction of local production of chemokines.

Circulating soluble CD4 directly prevents host resistance and delayed-type hypersensitivity response to Cryptococcus neoformans in mice.

In the present study, we examined the effect of soluble CD4 (sCD4) on host resistance and delayed-type hypersensitivity (DTH) response to Cryptococcus neoformans using a novel mutant mouse that exhibits a defect in the expression of membrane-bound CD4 but secretes high levels of sCD4 in the serum. In these mice, host resistance to this pathogen was impaired as indicated by an increased number of live pathogens in the lung. To elucidate the mechanism of immunodeficiency, three different sets of experiments were conducted. First, administration of anti-CD4 mAb restored the attenuated host defense. Second, in CD4 gene-disrupted (CD4KO) mice, host resistance was not attenuated compared to control mice. Third, implantation of sCD4 gene-transfected myeloma cells rendered the CD4KO mice susceptible to this infection, while similar treatment with mock-transfected cells did not show such an effect. These results indicated that immunodeficiency in the mutant mice was attributed to the circulating sCD4 rather than to the lack of CD4+ T cells. In addition, DTH response to C. neoformans evaluated by footpad swelling was reduced in the mutant mice compared to that in the control, and the reduced response was restored by the administration of anti-CD4 mAb. Finally, serum levels of IFN-gamma, IL-12 and IL-18 in the mutant mice were significantly reduced, while there was no difference in Th2 cytokines, such as IL-4 and IL-10. Considered collectively, our results demonstrated that sCD4 could directly prevent host resistance and DTH response to C. neoformans through interference with the production of Th1-type cytokines.

Molecular cloning of a docking protein, BRDG1, that acts downstream of the Tec tyrosine kinase.

Tec, Btk, Itk, Bmx, and Txk constitute the Tec family of protein tyrosine kinases (PTKs), a family with the distinct feature of containing a pleckstrin homology (PH) domain. Tec acts in signaling pathways triggered by the B cell antigen receptor (BCR), cytokine receptors, integrins, and receptor-type PTKs. Although upstream regulators of Tec family kinases are relatively well characterized, little is known of the downstream effectors of these enzymes. The yeast two-hybrid system has identified several proteins that interact with the kinase domain of Tec, one of which is now revealed to be a previously unknown docking protein termed BRDG1 (BCR downstream signaling 1). BRDG1 contains a proline-rich motif, a PH domain, and multiple tyrosine residues that are potential target sites for Src homology 2 domains. In 293 cells expressing recombinant BRDG1 and various PTKs, Tec and Pyk2, but not Btk, Bmx, Lyn, Syk, or c-Abl, induced marked phosphorylation of BRDG1 on tyrosine residues. BRDG1 was also phosphorylated by Tec directly in vitro. Efficient phosphorylation of BRDG1 by Tec required the PH and SH2 domains as well as the kinase domain of the latter. Furthermore, BRDG1 was shown to participate in a positive feedback loop by increasing the activity of Tec. BRDG1 transcripts are abundant in the human B cell line Ramos, and the endogenous protein underwent tyrosine phosphorylation in response to BCR stimulation. BRDG1 thus appears to function as a docking protein acting downstream of Tec in BCR signaling.

Induction of lymphocytic inflammatory changes in lung interstitium by human T lymphotropic virus type I.

Human T lymphotropic virus type I (HTLV-I) is the etiological agent of adult T cell leukemia, and reports suggest that several other clinical conditions are associated with HTLV-I infection, including myelopathy and inflammatory pulmonary diseases. However, the clinical entity of HTLV-I-associated lung disease remains unsubstantiated more than 10 years after its description. In the present study, we conducted a histopathological analysis of lung tissues of transgenic mice that expressed gene segments of HTLV-I p40(tax) regions. The aim of the study was to examine the relationship between expression of viral components and development of lung disorders. In these mice, inflammatory changes with infiltration of lymphocytes in peribronchial and perivascular areas and in alveolar septa developed at 11 wk of age and increased in incidence during the observation period (26 wk). There was a significant correlation between the pulmonary pathological changes and the level of expression of p40(tax) mRNA in the lungs. Our results provided for the first time strong evidence of a direct relationship between HTLV-I and development of bronchopulmonary infection.

Grb10/GrbIR as an in vivo substrate of Tec tyrosine kinase.

BACKGROUND: Tec is a member of the recently emerging subfamily among nonreceptor protein-tyrosine kinases (PTKs). Although many members of this family have been shown to be involved in a wide range of cytokine-mediated signalling systems, the molecular mechanism by which they exert in vivo effects remains obscure. To gain insights into the downstream pathways of Tec, we here looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid screening. RESULTS: One of TIPs turned out to be Grb10/GrbIR, which carries one pleckstrin homology domain and one Src homology 2 domain. Grb10/GrbIR was known to bind receptor PTKs in a ligand-dependent fashion, but not to be phosphorylated on tyrosine residues. In a transient expression system in human kidney 293 cells, however, Grb10/GrbIR becomes profoundly tyrosine-phosphorylated by Tec, but not by Syk, Jak2 or insulin receptor. We also reveal that expression of Grb10/GrbIR suppresses the cytokine-driven and Tec-driven activation of the c-fos promoter. CONCLUSION: Our results indicate a novel role of Grb10/GrbIR as an effector molecule to a subset of nonreceptor PTKs.

[Retrospective analysis of elderly patients > or = 60 years of age with acute leukemia].

A retrospective analysis was performed on 76 consecutive elderly patients with acute leukemia aged 60 years or more (48 men, 28 women). Forty patients were 60-69 years old, 28 were 70-79 years old and 8 were > or = 80 years old. There were 55 patients with acute myelogenous leukemia (AML), 13 acute lymphoblastic leukemia (ALL) and 8 AML from myelodysplastic syndrome (MDS/AML). Patients were treated with the JALSG protocol, CAG regimen, or low-dose Ara-C regimen for AML, and DVP/M-CHOP protocol for ALL. The complete remission (CR) rates were 52.7% (29 of 55) in AML, 61.5% (8 of 13) in ALL, and 0% in MDS/AML. The median CR durations were 226, 85, 0 days, and the median survivals were 204, 177, 99 days, respectively. CR rates were 65.3% for the JALSG protocol, 62.5% for the CAG regimen and 25.0% for low-dose Ara-C regimen. According to age, CR was obtained 62.5% in patients aged 60-69 years and 33.3% in patients over 70 years old. Our results indicated that patients aged 60-69 years should be treated with intensive chemotherapy.

Tec and Jak2 kinases cooperate to mediate cytokine-driven activation of c-fos transcription.

Although transcriptional activation of the c-fos proto-oncogene plays an intrinsic role in the mechanism of blood cell growth, it is still obscure how protein-tyrosine kinases (PTKs) regulate the cytokine-driven c-fos activation pathway. We present here that Tec PTK is tyrosine-phosphorylated and activated by granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation in a human GM-CSF-dependent cell line. Moreover, we could show that introduction of Tec into mouse BA/F3-hGMRalphabeta cells can profoundly activate the c-fos promoter in response to GM-CSF or to interleukin-3 (IL-3). In contrast, introduction of a kinase-deleted Tec could suppress cytokine-driven c-fos activation, indicating that Tec is directly involved in the regulation of c-fos transcription. Interestingly, strong activation by Tec of the c-fos promoter was blocked by the co-expression of dominant negative Jak2. The molecular interaction between Tec and Jak2 was then investigated both in mammalian and insect cell systems, revealing that they can not only bind to each other, but either of the two can phosphorylate the other. Thus, Tec and Jak2 can "cross-talk" in a complexed way to mediate cytokine-driven c-fos activation.

SOCS-1/JAB/SSI-1 can bind to and suppress Tec protein-tyrosine kinase.

Tec is the prototype of a recently emerging subfamily among nonreceptor type protein-tyrosine kinases and is known to become tyrosine-phosphorylated and activated by a wide range of cytokine stimulations in hematopoietic cells. Although Tec was recently shown to be involved in the cytokine-driven activation mechanism of c-fos transcription, it is yet obscure how Tec relays the signals from cell surface receptors to the nucleus. To identify signaling molecules acting downstream of Tec, we have looked for Tec-interacting proteins (TIPs) by using the yeast two-hybrid system. Here we report the identification and characterization of a novel protein, TIP3, which has been simultaneously identified by other groups as SOCS-1, JAB, or SSI-1. TIP3 carries one Src homology 2 domain with a sequence similarity to that of CIS. In 293 cells, TIP3 associates with Tec and suppresses its kinase activity. Interestingly, TIP3 can also down-regulate the activity of Jak2 but not that of Lyn. We propose that SOCS-1/JAB/SSI-1/TIP3 is a novel type of negative regulator to a subset of protein-tyrosine kinases.

Tec protein-tyrosine kinase is involved in the thrombopoietin/c-Mpl signaling pathway.

Thrombopoietin (TPO) is the ligand of the proto-oncogene product, c-Mpl, and supports the growth and maturation of megakaryocytic cells. Although much attention has been paid to the in vivo role of the TPO/c-Mpl system in the context of maintenance of circulating platelets, still little is understood about how activation of c-Mpl leads to mitogenesis and differentiation inside cells. Tec protein-tyrosine kinase (PTK) is the prototype of a recently emerging subfamily of nonreceptor type PTKs. Tec has been demonstrated to be activated by a wide range of cytokine stimulations. In this paper we show that TPO stimulation also rapidly enhances tyrosine-phosphorylation and activity of the Tec kinase in a human TPO-dependent cell line. In addition, the Vav protein, a blood cell-specific signaling molecule, is shown to be tyrosine-phosphorylated in response to TPO and to be constitutively associated with the Tec protein. From this evidence, we conclude that Tec is involved in the intracellular signaling system of TPO/c-Mpl.

[A case of pulmonary sarcoidosis mimicking relapse of pulmonary tuberculosis].

A 61-year-old woman was suspected of relapse of pulmonary tuberculosis. A chest X-ray film taken at a regular health check-up suggested relapse of pulmonary tuberculosis in a 61-year-old woman. Chest X-ray revealed irregular shadow with calcification in the upper lobe of the left lung and pulmonary tuberculosis was initially diagnosed a despite a negative reaction for acid-fast bacilli on examination of her sputum and bronchial lavage. Chest CT revealed thickened bronchi and blood vessels and nodules in the lung field. Transbronchial by biopsy failed to reveal caseating epithelioid cell granulomas transbronchial lung biopsy. Past history of facial palsy, uveitis, high plasma levels of angiotensin-converting enzyme (31.6IU/L), and abnormal HRCT levels. Bronchoalveolar lavage analysis revealed an increase in lymphocytes and a CD4/CD8 ratio of 8.67. Sarcoidosis was diagnosed after a liver biopsy. This appears that HRCT findings are useful in the diagnosis of pulmonary sarcoidosis.

Deletion of Src homology 3 domain results in constitutive activation of Tec protein-tyrosine kinase.

Tec protein-tyrosine kinase (PTK) is the prototype of a new subfamily of non-receptor type PTKs, and is abundantly expressed in hematopoietic tissues. We have revealed that Tec is inducibly tyrosine-phosphorylated and activated by stimulation with a wide range of cytokines. To get more insight into the signaling mechanism through Tec, we have generated a constitutively active form of Tec PTK. Deletion of the Src homology (SH) 3 domain gave rise to a hyperphosphorylated and activated Tec kinase (Tec deltaSH3). The activity of Tec deltaSH3 was confirmed in 293 cells, as well as in cytokine-dependent hematopoietic cells (BA/F3). Tec deltaSH3 should be a useful tool to study the in vivo substrates of Tec PTK.