Dominant-negative effects of a novel mutation in the filamin myopathy.

BACKGROUND: Filamin myopathy is associated with mutations in the filamin C gene (FLNC) and is a myofibrillar myopathy characterized by focal myofibrillar destruction and cytoplasmic aggregates containing several Z-disk-related proteins. METHODS: This study investigated 6 Japanese patients with dominantly inherited myofibrillar myopathy manifested by adult-onset, slow and progressive muscle weakness and atrophy in the distal extremities. RESULTS: The abundantly expressed proteins in the affected muscles were identified as filamin C by nano liquid chromatography-tandem mass spectrometry. A genetic analysis of FLNC identified a heterozygous c.8107delG mutation that was localized to the dimerization domain of filamin C. A biochemical crosslinking analysis of bacterially expressed recombinant wild-type and mutant filamin C fragments demonstrated that the mutant monomer disturbed the proper dimerization of the wild-type filamin dimer, resulting in formation of a heterotrimer with the wild-type filamin dimer. The expression study in C2C12 myoblasts showed that the mutant filamin fragments formed cytoplasmic aggregates with endogenous wild-type filamin C. CONCLUSIONS: This study provides evidence for the dominant-negative effects of the FLNC mutation. These effects may be mutation-specific and likely result in the variation in the clinical phenotypes seen in patients with filamin myopathy.

Suppression of prostaglandin synthesis by arachidonic acid or eicosapentaenoic acid in a macrophage-like cell line, RAW 264.7, treated with LPS.

In a mouse macrophage-like cell line, RAW 264.7, increasing the arachidonic acid (AA) content, by culturing cells with AA, caused profound AA release, irrespective of the lipopolysaccharide (LPS)-treatment, while lowering the AA content, by culturing cells with eicosapentaenoic acid (EPA), decreased it compared with the level in non-modified control cells. However, the release of prostaglandin D2 (PGD2), which had been generated from AA in response to LPS-treatment, was significantly decreased in both AA- and EPA-treated cells. Furthermore, although the amount of PG endoperoxide synthase-2 (PGHS-2) increased following LPS-treatment in all cases, both AA- and EPA-treatment caused a reduction of PGHS activity in LPS-treated cell lysates. Also, the addition of EPA or preincubation with AA in an in vitro PGHS assay system involving an LPS-treated, un-modified macrophage lysate, resulted in rapid inhibition of PGHS activity. These results suggest that both AA- and EPA-treatment inhibit PGD2 synthesis by inactivating PGHS-2 without affecting induction of the protein, and that the increase or decrease in AA content following AA- or EPA-treatment correlates simply with the level of AA release but not with that of PGD2 formation.

[Autopsy case of intravascular lymphomatosis with pancreatic carcinoma].

We report an autopsy case of intravascular lymphomatosis (IVL) that arose after radiation therapy and chemotherapy for an inoperable pancreatic carcinoma. A 66-year-old man who suffered from diabetes mellitus and pancreatic carcinoma presented with aggressive progression of consciousness disturbance and high fever. The laboratory findings disclosed marked thrombocytopenia, hypercalcemia, and elevated serum PTH-related peptide. The patient soon died of ventricular fibrillation due to uncontrollable hypercalcemia. Postmortem examination with immunohistochemical analysis revealed a well-differentiated tubular adenocarcinoma in the pancreatic body as well as an accumulation of neoplastic B-lymphocytes in small vessels throughout the body without systemic lymphadenopathy. To our knowledge, double neoplasms including IVL are extremely rare.

[A case of metastatic liposarcoma originating in the retroperitoneum successfully treated with combination chemotherapy].

We reported a 36-year-old woman with metastatic liposarcoma originating in the retroperitoneum, which responded well to adjuvant chemotherapy. The primary tumor was removed by surgery. Two months later, the patient developed metastasis to the brain, and to the lung four months later. Metastatic liposarcomas to the brain generally are extremely rare. The patient was treated with combination chemotherapy using cyclophosphamide, vincristine, adriamycin, and dacarbazine (CYVADIC). After she was examined, the former two drugs were alternated with vindesine and ifosfamide, and another regimen with cisplatin and etoposide was given after a three-week interval. As a result, both of the metastases totally disappeared. No recurrent lesion has been noted for two years. Although the role of chemotherapy for liposarcoma has not been well defined and little data support its use in an adjuvant setting, this combination chemotherapy seemed to be effective for advanced liposarcoma.

CD7, CD4 and myeloid antigen-positive acute lymphoblastic leukemia.

We report a case of acute lymphoblastic leukemia (ALL, FAB-L2) with unique cellular characteristics. Leukemic cells were negative for various cytochemical stainings except acid phosphatase. Immunophenotypic studies revealed CD7+, CD4+, CD8-, CD2+, CD3-, CD13+, CD25+, CD33+ and CD34+. The immunoglobulin heavy chain and T-cell receptor beta, gamma and delta chain genes were germline configurations. This patient had a karyotype of complex abnormalities involving Nos. 5 and 7. Leukemic cells showed a prominent response to interleukin-3, granulocyte macrophage colony stimulating factor (GM-CSF) and G-CSF with partial granulocytic differentiation in a colony assay. A binding assay confirmed the presence of both high and low affinity receptors for GM-CSF. These findings suggest that CD7+CD4+CD8-CD3- putative T-cell precursors may retain the capacity to differentiate to myeloid lineage.

Biologic characteristics of acute leukemia after myelodysplastic syndrome.

Ten patients with acute leukemia after primary myelodysplastic syndrome (MDS-AL) were examined to clarify the biologic nature of the leukemic cells in comparison with that of de novo acute myelocytic leukemia (AML). The morphologic and cytochemical features of the leukemic cells from all these patients corresponded well to those of de novo AML, and they were diagnosed with MDS-AML. Phenotypically, the frequent expression of the lymphocyte activation antigens, CD25 and CD30, was characteristic in MDS-AML. The in vitro response of MDS-AML cells to various growth factors was similar to that of de novo AML cells. Transforming growth factor beta 1 (TGF beta 1) suppressed growth factor-dependent colony formation by normal bone marrow cells, MDS bone marrow cells, and de novo AML cells, but did not inhibit colony formation by MDS-AML cells. The number of TGF beta 1 high-affinity binding sites of MDS-AML samples (< 5 to 47 sites/cell) was markedly lower than that in de novo AML samples (120 to 221 sites/cell). Our results indicate that the reduced TGF beta 1 may represent disregulation of the proliferation system in MDS-AML cells. This is thought to occur during the MDS phase, and may be related to the poorer response shown to conventional chemotherapy of AML.

Interleukin-2 receptor alpha chain on acute myelocytic leukemia cells is involved in cell-to-cell interactions.

Leukemic cells from 21 to 197 adult patients with de novo acute myelocytic leukemia (AML) were positive for IL-2R alpha chain (IL-2R alpha), whereas IL-2R beta chain (IL-2R beta), which is responsible for IL-2 signal transduction, was not found on leukemic cells from any of these cases tested. The expression of IL-2R alpha was closely associated with that of adhesion molecules CD4, CD11b and CD22, and endopeptidase CD10. None of the IL-2R alpha (+) AML cells responded to recombinant human IL-2. These data suggest that IL-2R alpha on AML cells may not be involved in cellular proliferation as one of growth factor receptors but may have a role in the control of cell-to-cell interactions.

Structural alterations of the p53 gene in human leukemias.

We have investigated alterations of the p53 gene in human leukemias by polymerase chain reaction-mediated restriction fragment length polymorphism analysis. This method detects the codon 72 polymorphism of the p53 gene, allowing identification of loss of heterozygosity (LOH) of the p53 gene. In this study, at least two specimens were obtained from each patient to compare the allele status at different points of clinical course. Of 21 cases examined 7 were heterozygous at this polymorphic site and were evaluable for LOH study. Only one patient of Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL) lost the heterozygosity in the specimen of her last relapse. Leukemic cells from her repeated relapses including the last one revealed to have the clonally rearranged immunoglobulin H chain gene of the same size, indicating that alteration of the p53 gene in this patient might account for the lethal relapse as a clonal evolution event. Northern-blot analysis of the p53 gene showed that one case of CD7(+) ALL had altered p53 mRNA. Overall, it is demonstrated that alterations of the p53 gene might be involved in the pathogenesis or progression of at least some human leukemias, although the alterations in leukemias seemed to be not as frequent as in solid tumors.

Phenotypical characteristics of acute myelocytic leukemia associated with the t(8;21)(q22;q22) chromosomal abnormality: frequent expression of immature B-cell antigen CD19 together with stem cell antigen CD34.

Twenty-three acute myelocytic leukemia (AML) patients with t(8;21) chromosomal abnormality, all classified as M2 (French-American-British [FAB] classification), were investigated. Blastic cells from all patients were positive for the stem cell-associated antigens, CD34 and HLA-DR, and the immature myeloid antigens, CD13 and CD33. The nonblastic leukemic cells expressed the more mature myeloid antigens, CD11b and CD15, with loss of the immature phenotype. The incidence of positivities for the stem cell-associated antigens, CD34 and HLA-DR, in t(8;21) AML cells was significantly higher in comparison with those in other AML showing granulocytic differentiation (M2 or M3). AML cells with t(8;21) also showed some phenotypic abnormalities. Frequent expression of CD19 was found in the blastic population of t(8;21) AML (18 of 23 cases) without other B-cell antigens and Ig gene rearrangements. CD19 expression was confirmed by immunocytochemistry and Northern blotting. The CD19+ blastic cells coexpressed both CD34 and HLA-DR. In addition, CD33+ cells among the blastic fraction in t(8;21) AML cells were fewer in number than in those of M2 or M3 AML without t(8;21). Our findings indicate that leukemic blasts of t(8;21) AML commonly express CD19 while preserving the stem cell-associated antigens, and differentiate into the granulocytic pathway with discordant maturation such as low CD33 expression.

Diagnostic and clinical importance of interleukin-2 receptor alpha chain expression on non-T-cell acute leukaemia cells.

The expression of interleukin-2 receptors (IL-2R) was examined in 328 adult patients with non-T-cell (non-T) acute leukaemia and blast crisis of chronic myelocytic leukaemia (CML.BC) using two monoclonal antibodies, anti-Tac for IL-2R alpha chain (IL-2R alpha) and Mik beta 1 for IL-2R beta chain (IL-2R beta). Leukaemic cells in the following cases were positive for anti-Tac; 28/192 of acute myelocytic leukaemia (AML), 24/44 CML-BC, 4/28 CD19(+)CD10(-) acute lymphoblastic leukaemia (ALL), and 20/64 common ALL (c-ALL). IL-2R beta was not detected on leukaemic cells of any case examined. Eleven of IL-2R alpha(+) AML were derived from myelodysplastic syndrome. None of the IL-2R alpha positive leukaemic cells responded to exogenous recombinant human IL-2 (rhIL-2) in culture. In addition, IL-2R alpha expression on non-T leukaemic cells was closely correlated with coexpressing different lineage markers and the presence of the Philadelphia abnormality. Marked increase of serum soluble IL-2R alpha was demonstrated in the IL-2R alpha(+) patients examined. Clinically, the IL-2R alpha(+) patients showed significantly lower response to chemotherapy and poorer prognosis than IL-2R alpha(-) patients. Our results clearly indicate the diagnostic importance of IL-2R alpha expression in non-T acute leukaemia with a close relation to the particular cellular characteristics and the prognosis.

Establishment of a human acute myeloid leukemia cell line (Kasumi-1) with 8;21 chromosome translocation.

A novel leukemic cell line with an 8;21 chromosome translocation, designated as Kasumi-1, was established from the peripheral blood of a 7-year-old boy suffering from acute myeloid leukemia (AML). The Kasumi-1 cells were positive for myeloperoxidase showing a morphology of myeloid maturation. The response in proliferation assay was observed in the culture with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), and granulocytemacrophage CSF (GM-CSF), but not with IL-1 or IL-5. Neither granulocytic nor eosinophilic maturation was observed in the liquid culture by the addition of dimethyl sulfoxide, G-CSF, or IL-5, respectively. In contrast, induction of macrophagelike cells was seen by the addition of phorbol ester. This is the first report of a human AML cell line with t(8;21) that has characteristics of myeloid and macrophage lineages. The cell line could be a useful tool for elucidating the pathophysiology of AML with t(8;21).

[Syncytial variant of nodular sclerosing Hodgkin's disease expressing T-cell antigens].

A case of syncytial variant of nodular sclerosing Hodgkin's disease in a 34-year-old woman is reported. Histologically, numerous Reed-Sternberg (RS) cells and their variants were seen in sheets. They were positive for CD30, CD15, CD25, and HLA-DR antigens. In addition, they expressed CD45 antigen and T-cell antigens (CD2, CD4). Molecular genetic analysis showed that this case had a germ line configuration for T-cell receptor (TCR) beta chain, TCR gamma chain, and immunoglobulin heavy chain genes. The percentage of the neoplastic cells in the specimen identified by CD30 positive cells was about 20% which was far above sensitivity of the molecular genetic analysis. Such analysis is reliable as to judgement of the results. Thus, these findings suggest that the RS cells and their variants in the present case are not derived from mature T cell in spite of their T-cell antigens expression.

[Transient large granular lymphocytosis associated with pulmonary tuberculosis: a case report].

We report a case of a 61-year-old woman with large granular lymphocytosis associated with pulmonary tuberculosis. She was admitted to our hospital because of high fever, anemia and splenomegaly. On admission, the leukocyte count was 6,890/microliters with 52% of large granular lymphocytes. Immunophenotypical analysis of the increased cells showed following results; CD2+, CD3-, CD16+, CD57+. These cells had natural killer (NK) activity. Molecular genetical analysis showed these cells had germline configuration of the T cell receptor beta chain genes. About four months after admission, chest X-P revealed multiple mass shadow and the diagnosis of pulmonary tuberculosis was made by the examination of gastric juice. Anti-tuberculosis therapy was started, and soon after clinical symptom and pancytopenia were improved. For about one year, anti-tuberculosis therapy was continued, and now hematological abnormality is not found. We considered that this case was reactive large granular lymphocytosis of NK cells to lung tuberculosis.

[Nasal lymphoma of natural killer cell origin].

We report a case of nasal lymphoma with characteristics of natural killer (NH) cells. A 44-year-old man was admitted to our hospital because of right nasal obstruction. Physical examination revealed a tumor in the right nasal cavity and swelling of the right cervical lymph nodes. Histopathological examination showed diffuse medium sized cell lymphoma. The neoplastic cells expressed CD2, NKH-1, HLA-DR and leukocyte common antigen, but lacked other T-cell, B-cell and myeloid markers. They were in germline configuration for immunoglobulin heavy chain and T-cell receptor genes by southern blot analysis. These findings suggest that they are derived from NK cell.

Acanthocephalan, probably Bolbosoma, from the peritoneal cavity of man in Japan.

An immature female acanthocephalan in a tumor on the serosa over the ileum of a 16-year-old boy in Kagoshima, Japan, was identified as probably a species of Bolbosoma. This is the second such case to be reported.

Tumors of Sprague-Dawley rats induced by long-term feeding of phenacetin.

Carcinogenicity of phenacetin was tested using Sprague-Dawley rats. Two groups of animals containing 50 males and 50 females per group were fed respectively with 2.5% and 1.25% phenacetin diet for 18 months and fed thereafter with basal diet for 6 months. Control animals containing 65 males and 65 females were fed with basal diet for 24 months. Animals surviving more than 24 months were regarded as effective animals and killed. Rats that died of tumor development within 24 months were also regarded effective animals. Every organ from the killed and dead animals was fixed in 10% formaldehyde solution and examined histopathologically. Effective number of rats was 27 males and 27 females in 2.5% phenacetin feeding group, and 22 males and 25 females in 1.25% phenacetin feeding group. In control group, 19 males and 25 females were effective. Neoplasms including spontaneous tumors were detected in 26 out of 27 males (96.3%) and 21 out of 27 females (77.8%) of 2.5% phenacetin feeding group, and in 20 out of 22 males (90.9%) and 19 out of 25 females (76.0%) of 1.25% phenacetin feeding group. In control group, 1 out of 19 males (5.3%) and 6 out of 25 females (24.0%) showed spontaneous tumor development. Histopathologically, carcinomas of the nasal cavity, such as adenocarcinoma, squamous cell carcinoma, and transitional cell carcinoma, and the urinary passage, as renal cell carcinoma of the kidney pelvis, and transitional cell carcinoma of the urinary bladder, were most conspicuous, suggesting the target organs of phenacetin carcinogenesis. Males showed higher tumor incidence compared to females. The higher the concentration of phenacetin given, higher incidence of tumors was observed.