Human leukemias with mutated FLT3 kinase are synergistically sensitive to FLT3 and Hsp90 inhibitors: the key role of the STAT5 signal transduction pathway.

17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of the molecular chaperone heat shock protein 90, results in cell type-specific inhibition of proliferation of leukemic cells. GTP14564 is a tyrosine kinase inhibitor actively against FLT3. The current study evaluated the single and combined effects of 17-AAG and GTP14564, and the role of FLT3 in their inhibitory effects. The importance of FLT3 mutations was demonstrated using small interfering RNA (siRNA) targeted to FLT3. Similar to FLT3 siRNA, GTP14564 inhibited FLT3 internal tandem duplication (ITD) cells (MV4;11) and FLT3 amplified wild-type cells (SEMK2-M1), but not wild-type FLT3 cells (RS4;11). However, when RS4;11 cells were stimulated with FLT3-ligand, phosphorylation of STAT5 and GTP14564 inhibition were observed. Responses to GTP14564 in all cell types were directly related to the level of STAT5 phosphorylation in the cells. We observed synergistic effects of combined 17-AAG and GTP14564 in cell lines with FLT3-ITD and amplified wild-type FLT3. Combined treatment with 17-AAG and GTP14564 reduced the levels of p-FLT3 and p-STAT5, enhanced G0/G1 arrest and apoptosis in FLT3-ITD and amplified wild-type FLT3. The combination of 17-AAG with FLT3 kinase inhibitors can enhance targeted therapy in leukemias with FLT3 mutations, such as MLL fusion gene leukemias.

Matrix metalloproteinase-2 is involved in A549 cell migration on laminin-10/11.

We have reported that laminin-10/11 strongly promotes migration of A549 human lung carcinoma cells by activating the alpha3beta1 integrin-dependent signaling pathway. To elucidate the mechanism involved, we investigated whether matrix metalloproteinases (MMPs) are involved in cell migration on laminin-10/11. Here, we demonstrate that laminin-10/11, but not fibronectin which does not greatly promote A549 cell movement, stimulated MMP-2 secretion approximately 3-fold. The cell migration-promoting activity of laminin-10/11 was down-regulated by an MMP inhibitor. In addition, cell motility was significantly increased when cells adhered to a mixture of fibronectin and laminin-10/11 with a concomitant decrease of focal contacts, compared with those adhering to fibronectin alone. The enhanced cell migration was partially suppressed by the MMP inhibitor. Furthermore, an anti-alpha3 integrin, but not an anti-alpha5 integrin, antibody induced the activated form of MMP-2. These data suggest that MMP-2 may play an important role in A549 cell migration on laminin-10/11 through an alpha3beta1 integrin-dependent pathway.

Secondary sclerosing cholangitis and portal hypertension after O157 enterocolitis: Extremely rare complications of hemolytic uremic syndrome.

The authors experienced an extremely rare case of secondary sclerosing cholangitis and portal hypertension developed as late complications of hemolytic uremic syndrome (HUS) owing to Escherichia coli O157:H7 in a 2-year-old boy. HUS after E coli O157 infection is the most frequent cause of acute renal failure in childhood and occasionally is accompanied by extrarenal complications such as encephalopathy, cardiomyopathy, ischemic colitis, and pancreatitis. Rarely, late colonic stenosis may develop secondary to the ischemic damage. Sclerosing cholangitis and subsequent cirrhosis with portal hypertension are very uncommon as late complications of HUS. To our knowledge, such a case has not been previously reported in the literature. J Pediatr Surg 36:1838-1840.

Molecular detection of metastatic retinoblastoma cells by reverse transcription polymerase reaction for interphotoreceptor retinoid-binding protein mRNA.

BACKGROUND: In the current study, the authors report a 4 year old girl with disseminated retinoblastoma. To find sensitive and specific molecular markers for detection of retinoblastoma cells in blood and marrow, the authors evaluated three photoreceptor-associated gene transcripts by using reverse transcription polymerase chain reaction (RT-PCR). METHOD: Samples of bone marrow and blood were obtained from healthy donors and the patient. RT-PCR was performed to detect the cone alpha'-subunit of cGMP phosphodiesterase (cone alpha'-PDE), the rod beta-subunit of cGMP (rod beta-PDE), and the interphotoreceptor retinoid-binding protein (IRBP) gene transcript in RNA extracted from the samples. RESULTS: While no expression of rod beta-PDE or IRBP was detected in any of the normal samples, expression of cone alpha'-PDE was detected in two out of seven normal marrow samples. Expression of rod beta-PDE was not detected in the patient samples. Expression of IRBP was detected in the patient samples obtained from iliac bone marrow before intensive chemotherapy but not thereafter. CONCLUSION: RT-PCR for IRBP was a useful method for detecting metastatic retinoblastoma cells as well as for evaluating the therapeutic effects of treatment in this particular case.

Chronic graft-versus-host disease in children and adolescents after bone marrow transplantation from HLA-matched donors.

We analyzed 98 pediatric patients who underwent bone marrow transplantation (BMT) from serologically HLA-matched related donors (RD) or unrelated donors (UD) at our institute to clarify the actual status of chronic graft-versus-host disease (cGVHD). There were 36 evaluable cases of RD-BMT and 35 of UD-BMT. cGVHD was observed in 8 RD-BMT cases (22.2%) and in 23 UD-BMT cases (65.7%). In the RD-BMT cases, the limited and extensive types of cGVHD were observed in 4 cases each, whereas in the UD-BMT cases, the limited type was seen in 11 cases and the extensive type in 12. Prior acute GVHD was observed in 6 RD-BMT cases and in 18 UD-BMT cases. Two RD-BMT patients with extensive type cGVHD died of relapse and cytomegalovirus infection, and 4 UD-BMT patients died because of bronchiolitis obliterans, fungal infection, liver failure, and multiple organ failure, respectively. The incidence of cGVHD in these pediatric patients was as high as that in adult patients when UD-BMT was performed. Some UD-BMT patients required long-term immunosuppressive therapy after BMT. These findings suggest that cGVHD is a serious problem in pediatric UD-BMT. Therefore, intensive prophylaxis and treatment of GVHD must always be performed after UD-BMT.

Cutaneous lymphocyte antigen is expressed on memory/effector B cells in the peripheral blood and monocytoid B cells in the lymphoid tissues.

Cutaneous lymphocyte antigen (CLA) is expressed on a subpopulation of human memory T cells and is involved in the primary step of their skin homing. T cells and some B cells in the peripheral blood express CLA, but the pathophysiologic roles of CLA(+) B cells have not yet been clarified. We examined the relationships among CLA expression in B cells and immunoglobulin heavy chain subtype, the localization of CLA(+) B cells in the peripheral lymphoid tissues, and their functional binding to E-selectin. CLA was expressed on class-switched, memory B cells in the peripheral blood and tonsils as revealed by flow cytometry. Immunohistochemical staining of the lymph nodes with various types of inflammation or reactive hyperplasia showed CLA on the monocytoid B cells, which correspond to memory cells. The functional study revealed that CLA on B cells bound to E-selectin transfectants. E-selectin was detected on some of the high endothelial venules in the monocytoid B-cell-rich lymph nodes. These findings suggest that CLA is also expressed on a subset of memory/effector B cells, in addition to a subset of memory T cells. Such B cells were located in the lymph nodes or tonsils and rarely in chronic dermatitis. Therefore, CLA seems to be related to memory/effector B-cell trafficking to the lymph nodes or tonsils. According to the multistep theory, mechanisms involved in the second or third step might be different between CLA(+) B and T cells.

CD95 ligand is expressed in Reed-Sternberg cells of Hodgkin's disease.

Reed-Sternberg (RS) cells and their mononuclear variants, Hodgkin's (H) cells, are considered to be the neoplastic cells of Hodgkin's disease (HD). The cellular origin of H-RS cells remains the subject of considerable controversy, although most recent papers have claimed that H-RS cells are of B cell origin. Recently, however, it has been reported that some H-RS cells express granzyme B, as observed in cytotoxic T cells and/or natural killer cells, which also express CD95 ligand (FasL/APO-1L). In the present study, the expression of CD95L and granzyme B in H-RS cells of HD was investigated. CD95L was detected in H-RS cells in five of nine HD cases (one case of lymphocyte-rich classical HD, two of these cases of nodular sclerosis type, and two of four cases of mixed cellularity type). All three examined HD cell lines expressed CD95L in the cytoplasm, although cell surface expression was seen only in L428 cells. Three HD cases expressed both CD95L and granzyme B. It was concluded that CD95L is frequently expressed in H-RS cells, which is one of their notable characteristics; albeit it seems to be irrespective of cell lineage.

Expression of Fas ligand mRNA in germinal centres of the human tonsil.

Fas ligand (FasL), a cell surface molecule belonging to the tumour necrosis family, induces apoptosis through its receptor, Fas antigen (Fas). Germinal centre B cells strongly express Fas, but the role of the Fas-FasL system in B-cell selection in the germinal centre remains unclear. In the present study, FasL mRNA in the tonsils was examined by RNA in situ hybridization. FasL mRNA was detected in the lymphocytes of both germinal centres and interfollicular areas, but much more intensively in the former. The distribution of cells strongly expressing FasL mRNA in the germinal centres was quite similar to that of CD45RO-positive T cells. Immunohistochemically, however, most of the germinal centre cells were positive for FasL. Flow cytometric analysis demonstrated that FasL-positive cells of the tonsils included not only CD3-positive/CD45RO-positive T cells, but also CD19-positive B cells. This finding therefore suggests either that germinal centre B cells can produce FasL, although the level of mRNA was equivocal, or that the soluble form of FasL may be released from FasL-positive T cells in the germinal centres and then bind to Fas-positive germinal centre B cells. Thus, the Fas-FasL system may participate in the positive selection of B cells.

[The role of anti L-asparaginase antibody in childhood acute lymphoblastic leukemia].

The function of IgG antibody with regard to L-asparaginase (L-asp) was investigated in vivo. Blood samples were collected before, during and after IV administration of L-asp (6,000 U/sqm for 10 days) in 18 children with acute lymphoblastic leukemia (ALL) previously treated with L-asp. Using enzyme-linked immunosorbent assay (ELISA), serum levels of L-asp and anti-L-asp IgG antibody were measured simultaneously. In 11 cases, the level of anti-L-asp IgG antibody increased prior to, but decreased to within the normal range after drug administration whereas the level of serum L-asp increased after drug administration. In 5 cases, the level of anti-L-asp IgG antibody increased as the level of serum L-asp decreased after drug administration. In contrast, in the 13 cases with no increase in anti-L-asp IgG antibody during L-asp administration, the serum L-asp level was stable. These data indicate that anti-L-asp IgG antibodies play an important role in the immunoclearance of L-asp. We would like to continue to carefully follow patients showing high titers of anti-L-asp IgG antibody during the administration of L-asp.

The Fas antigen is detected on immature B cells and the representative cell lines show Fas-mediated apoptosis.

The expression and functions of Fas antigen, a major regulator of apoptosis, in T-cell selection have been intensively investigated, but little is known about its expression i immature B cells which are also selected in the bone marrow, and plasma cells which are at the terminal stage of B-cell differentiation and are designed to die. We examined bone marrow cells and found Fas antigen on these cells at low levels. Next we analysed Fas expression and susceptibility to anti-Fas antibody-mediated apoptosis on B-cell lines representing various stages of differentiation. We also examined the expression of Bcl-2 and Bax on these lines, which were intimately related to apoptosis. Fas antigen was almost negative on pre-pre-B cell lines and was detected on pre-B-cell lines at low levels. All plasma cell lines expressed Fas at a low to moderate level. Some cell lines with peripheral B-cell phenotype expressed Fas antigen. Except for the Burkitt cell lines and one plasma cell line, susceptibility to Fas-mediated apoptosis depended on Fas expression. Bcl-protein was detected on all but one cell line and Bax was detected on 15/23 lines, but neither were related to cellular differentiation or Fas expression.

Clonal analysis by polymerase chain reaction of B-cell lymphoma with late relapse: a report of five cases.

BACKGROUND: Little is known about the clonal heterogeneity of non-Hodgkin's lymphoma between presentation and relapse, although several such reports have been published on acute lymphoblastic leukemia. METHODS: We examined five patients with B-cell lymphoma who relapsed more than 5 years after initial presentation. Formalin fixed, paraffin embedded tissues were analyzed for clonal immunoglobulin heavy chain gene rearrangement by the polymerase chain reaction (PCR) and sequencing of the PCR products. Four specimens retained the original histologic type, but one showed histologic transformation from diffuse large cell lymphoma to follicular small cleaved cell lymphoma. RESULTS: Although the size of the PCR products looked identical on the gel between presentation and relapse in all patients, only three of the four specimens that retained the original type had identical gene rearrangements at both presentation and relapse. One of these four and the fifth specimen showed novel gene rearrangements. CONCLUSIONS: This study suggests that late relapse lymphoma may present as a new clone. Sequencing of the PCR products is important in the evaluation of clonal heterogeneity.

Ligation of HLA class II molecules promotes sensitivity to CD95 (Fas antigen, APO-1)-mediated apoptosis.

CD95 (Fas antigen/APO-1) is up-regulated in activated lymphocytes, and monoclonal antibody (mAb) to CD95 induces apoptosis. HLA class II molecules play a key role in antigen presentation, ligation of which induces signal transduction. We examined 18 lymphoid cell lines (15 B cell and 3 T cell lines) to investigate the effects of ligation of HLA class II molecules on CD95-mediated apoptosis. All of the five immature B cell lines were sensitive to anti-CD95 mAb, and ligation of HLA class II molecules promoted CD95-mediated apoptosis. In seven B-blastoid cell lines, two Burkitt lines were resistant to anti-CD95 mAb in spite of high expression of CD95. In three of five non-Burkitt B-blastoid lines, CD95-mediated apoptosis was augmented by treatment with anti-HLA class II mAb, while the other two lines lacking CD95 were resistant to anti-CD95 mAb. Three plasmacytic cell lines showed CD95-mediated apoptosis, but enhancement by anti-HLA class I mAb was slight in one cell line and was not observed in the other two lines. Out of three HLA class II antigen-positive T cell lines, CD95-mediated apoptosis was observed to some degree in one call line but was not promoted by the treatment with anti-HLA class II mAb, and the other two cell lines were resistant to anti-CD95 mAb. Ligation of HLA class II molecules did not alter CD95 expression in the five cell lines examined, except Su-DHL-4 originated from a follicular lymphoma, which showed slight up-regulation. Taken together, ligation of HLA class II molecules apparently promotes CD95-mediated apoptosis in immature B cells and non-Burkitt B blasts. These findings highlight the role of HLA class II molecules in CD95-mediated apoptosis, which may facilitate rapid clearance of functionally useless cells from the immune system and might be involved in negative selection of B cells.

Homotypic cell aggregation via conformational change of CD44 molecule induced by anti-CD44 monoclonal antibodies.

The homotypic cell aggregation of leukocytes is an unique adhesive event which is caused by cellular activation. Anti-CD44 monoclonal antibody (mAb) induces homotypic cell aggregation of hematopoietic cell lines expressing CD44, but the mechanism of homotypic cell aggregation is poorly understood. We used four mAbs against CD44: TL-1 which was newly developed and seemed to react with a non-hyaluronate binding site, OS/37 and BU52 which recognized a hyaluronate binding site, and Hermes-3 which recognized a non-hyaluronate binding site. TL-1 treatment induced strong homotypic cell aggregation in several types of cell lines including a B cell line from a patient with leukocyte adhesion deficiency syndromes (LAD) and normal peripheral blood lymphocytes (PBL). OS/37 and BU52 also induced weak homotypic cell aggregation. None of these anti-CD44 mAbs-induced homotypic cell aggregations was blocked by antibodies against LFA-1, ICAM-1, VLA-4, or L-selectin. Interestingly, the TL-1-induced homotypic cell aggregation was blocked by Hermes-3 or OS/37, but not by BU52. BU52-induced homotypic cell aggregation was blocked by Hermes-3 or OS/37, but not by TL-1. OS/37-induced homotypic cell aggregation was blocked by Hermes-3, TL-1 or BU52. The blocking experiments with anti-metabolic agents revealed that the induced homotypic cell aggregation was energy-dependent and associated with intracytoplasmic actin filaments. This homotypic cell aggregation did not require de novo protein synthesis, because it was not affected by pretreatment with either cycloheximide or actinomycin D. FACS analysis revealed that TL-1 binding did not affect the intensity of expression of the CD44 molecule on the cell surface.

[Occurrence of follicular small cleaved cell lymphoma following chemotherapy for diffuse large cell lymphoma].

A 59-year-old man was admitted in July, 1985 because of generalized lymphadenopathy and hypercalcemia. The biopsy specimen of the left inguinal lymph node showed diffuse large cell lymphoma. He achieved a complete remission (CR) in July, 1986 by CHOP therapy. In January, 1992, he visited the outpatient clinic with splenomegaly, and swelling of the left axillary and left supraclavicular lymph nodes. The biopsy specimen of the axillary lymph node showed follicular small cleaved cell lymphoma. Because the spleen and the lymph nodes regressed after the biopsy, he has been followed up without any specific treatment for lymphoma. Histologic change from low-grade lymphoma into more aggressive histologic pattern is well documented. However the converse phenomenon, occurrence of low-grade lymphoma after intermediate-, or high-grade lymphoma has rarely been reported. Histological examination is mandatory when a patient with diffuse large cell lymphoma is suspected to have relapsing disease after the long-term CR.