Co-active receptor tyrosine kinases mitigate the effect of FGFR inhibitors in FGFR1-amplified lung cancers with low FGFR1 protein expression.

Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR inhibitor. These data reveal that biomarker-directed trials for FGFR1-amplified SCC require assessment of FGFR1 protein expression and uncover novel therapeutic strategies for FGFR1-amplified SCC with low FGFR1 protein expression.

Cyclic compressive loading on 3D tissue of human synovial fibroblasts upregulates prostaglandin E2 via COX-2 production without IL-1ß and TNF-α.

OBJECTIVE: Excessive mechanical stress on synovial joints causes osteoarthritis (OA) and results in the production of prostaglandin E2 (PGE2), a key molecule in arthritis, by synovial fibroblasts. However, the relationship between arthritis-related molecules and mechanical stress is still unclear. The purpose of this study was to examine the synovial fibroblast response to cyclic mechanical stress using an in vitro osteoarthritis model. METHOD: Human synovial fibroblasts were cultured on collagen scaffolds to produce three-dimensional constructs. A cyclic compressive loading of 40 kPa at 0.5 Hz was applied to the constructs, with or without the administration of a cyclooxygenase-2 (COX-2) selective inhibitor or dexamethasone, and then the concentrations of PGE2, interleukin-1ß (IL-1ß), tumour necrosis factor-α (TNF-α), IL-6, IL-8 and COX-2 were measured. RESULTS: The concentrations of PGE2, IL-6 and IL-8 in the loaded samples were significantly higher than those of unloaded samples; however, the concentrations of IL-1ß and TNF-α were the same as the unloaded samples. After the administration of a COX-2 selective inhibitor, the increased concentration of PGE2 by cyclic compressive loading was impeded, but the concentrations of IL-6 and IL-8 remained high. With dexamethasone, upregulation of PGE2, IL-6 and IL-8 was suppressed. CONCLUSION: These results could be useful in revealing the molecular mechanism of mechanical stress in vivo for a better understanding of the pathology and therapy of OA. Cite this article: Bone Joint Res 2014;3:280-8.

Clinical and prognostic significance of cytokine receptor expression in adult acute lymphoblastic leukemia: interleukin-2 receptor alpha-chain predicts a poor prognosis.

We quantitatively assessed the expression of cytokine receptors (interleukin-2 receptor (IL-2R), IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, granulocyte-macrophage colony-stimulating factor R (GM-CSFR), G-CSFR, c-fms, c-mpl, c-kit and FLT3) in cells from 211 adults with acute lymphoblastic leukemia (ALL) by flow cytometry and determined their prevalence and clinical significance. Although all cytokine receptors were expressed to various degrees, the levels of IL-3R alpha-chain (IL-3Ralpha), IL-2Ralpha, IL-2Rbeta, IL-7Ralpha, common-Rgamma(gammac), c-mpl, c-kit and FLT3 exhibited a wide spectrum > or =2000 sites/cell. Among them, IL-3Ralpha, IL-2Ralpha and FLT3 were highly expressed in B-lineage ALL, whereas IL-7Ralpha, gammac and c-kit predominated in T-lineage ALL. Higher levels of IL-3Ralpha, IL-2Ralpha, c-kit and FLT3 correlated with the expression of CD13/33. Increased IL-2Ralpha levels related to the presence of Philadelphia chromosome (Ph), leukocytosis and shorter event-free survival (EFS). C-kit preferred in male. Elevated FLT3 levels correlated with age > or =60 years. Multivariate analysis in B-lineage ALL revealed only IL-2Ralpha (P=0.028) and Ph (P=0.020) as independent factors for EFS. These findings suggest that several cytokine receptors associated with certain cellular and clinical features, but IL-2Ralpha solely had a prognostic value and should be considered as a major prognostic factor for adult ALL that is comparable with Ph.

Involvement of LEU13 in interferon-induced refractoriness of human RSa cells to cell killing by X rays.

Culture of human cells with human interferon alpha and beta (IFNA and IFNB) results in increased resistance of the cells to cell killing by X rays. To identify candidate genes responsible for the IFN-induced X-ray resistance, we searched for genes whose expression levels are increased in human RSa cells treated with IFNA, using an mRNA differential display method and Northern blotting analysis. RSa cells, which showed increased survival (assayed by colony formation) after X irradiation when they were treated with IFNA prior to irradiation, showed increased expression levels of LEU13 (IFITM1) mRNA after IFNA treatment alone. In contrast, IF(r) and F-IF(r) cells, both of which are derived from RSa cells, showed increased X-ray resistance and high constitutive LEU13 mRNA expression levels compared to the parental RSa cells. Furthermore, the IFNA-induced resistance of RSa cells to killing by X rays was suppressed by antisense oligonucleotides for LEU13 mRNA. LEU13, a leukocyte surface protein, was previously reported to mediate the actions of IFN such as inhibition of cell proliferation. The present results suggest a novel role of LEU13 different from that in the inhibition of cell proliferation, involved in IFNA-induced refractoriness of RSa cells to X rays.

Characteristics of t(8;21) acute myeloid leukemia (AML) with additional chromosomal abnormality: concomitant trisomy 4 may constitute a distinctive subtype of t(8;21) AML.

t(8;21)(q22;q22) is the most frequently observed karyotypic abnormality associated with acute myeloid leukemia (AML), especially in FAB M2. Clinically, this type of AML often shows eosinophilia and has a high complete remission rate with conventional chemotherapy. t(8;21) AML is also frequently associated with additional karyotypic aberrations, such as a loss of the sex chromosome; however, it is unclear whether these aberrations change the biological and clinical characteristics of t(8;21) AML. To investigate this issue, 94 patients with t(8;21) AML were categorized according to their additional karyotypic aberrations, which were detected in more than three cases, and then morphologic features, phenotypes, expression of cytokine receptors, and clinical features were compared to t(8;21) AML without other additional aberrant karyotypes. t(8;21) AML with loss of the sex chromosome and abnormality of chromosome 9 were found in 27 cases (29.3%) and 10 cases (10.6%), respectively; however, no differences were observed from the t(8;21) AML without other additional karyotypes in terms of morphological and phenotypic features. There was also no significant difference in the clinical outcome among these three groups. On the other hand, trisomy 4 was found in three cases (3.2%) and these cells showed low expressions of CD19 (P=0.06) and IL-7 receptor (P=0.05), and high expressions of CD33 (P=0.13), CD18 (P=0.03), and CD56 (P=0.03) when compared to t(8;21) AML without additional karyotypes. Moreover, all three t(8;21) AML cases with trisomy 4 did not show eosinophilia in their bone marrow and died within 2.4 years. These observations suggest that additional karyotypic aberration, t(8;21) with trisomy 4 is rare, but it may constitute a distinctive subtype of t(8;21) AML.

Modification of delayed radiation-induced reactions of duodenal vessels and mast cells in old rats.

Pronounced ultrastructural changes in vessels and mast cells were observed in duodenal lamina propria of Wistar rats 1 year after single whole-body gamma-irradiation in a dose of 7.5 Gy. Inhibition of adrenocortical function with methopyrone reduced structural damage and improved animal survival.

Non-DNA-binding Ikaros isoform gene expressed in adult B-precursor acute lymphoblastic leukemia.

Ikaros, a zinc finger transcription factor, is essential for lymphoid development. Mutant mice expressing dominant-negative Ikaros gene (Ikaros) isoforms develop an aggressive form of lymphoid malignancies. We examined the expression of Ikaros isoforms in 11 leukemic cell lines and adult acute lymphoblastic leukemia cells from 36 patients with B-precursor acute lymphoblastic leukemia (pre-B ALL) and nine with T-precursor acute lymphoblastic leukemia (pre-T ALL), using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. In one pre-B ALL cell line, INC cells, and primary leukemic cells from 16 patients with pre-B ALL, we found the predominant expression of a non-DNA-binding Ikaros isoform, Ik-6. However, Ik-6 was not detected in pre-T ALL cells. All of pre-B ALL cells expressing Ik-6 were CD10(+), whereas CD10(-) pre-B ALL cells did not express Ik-6. The expression of Ik-6 was not related to karyotype abnormalities such as t(9;22) and t(4;11). Proteins from the cells that expressed Ik-6 alone failed to bind to the Ikaros protein-specific binding sequence in DNA. Ikaros proteins lacking the DNA binding sequences were detected in the cytoplasm but not in the nucleus of the cells. When INC and primary pre-B ALL cells that express Ik-6 alone were irradiated and cultured in the absence of serum, these cells produced functional Ikaros isoforms, Ik-1 and Ik-2. Purified CD19(+) CD10(-) and CD19(+) CD10(+) cells from normal human bone marrow did not express Ik-6. The predominant expression of Ik-6, which is the result of post-transcription dysregulation, is characteristic of adult pre-B ALL, especially CD10(+) pre-B ALL.

Expression of endothelial cell-associated molecules in AML cells.

Recently, it has been clarified that interaction between hematopoietic cells and endothelial cells is important in normal hematopoiesis and leukemogenesis. In this study, we examined the relationship between AML cells and endothelial cells by analyzing the expression profile of angiogenic factors, angiopoietin-1 (Ang-1), Ang-2, Tie-2 (a receptor for angiopoietins) and vascular endothelial growth factor (VEGF). Our results demonstrated that CD7(+)AML expressed Ang-2 mRNA frequently and integrin-family adhesion molecules (CD11c and CD18) intensively, suggesting the close correlation with endothelial cells. On the other hand, in t(8;21) AML cells, expression of Ang-2 was infrequent and expression of integrin-family adhesion molecules (CD11b, CD11c and CD18) was weak, suggesting the sparse association with endothelial cells. As for CD7(+)AML cells, despite the frequent and intense expression of endothelial cell-associated molecules (such as Ang-2, CD11c and CD18), intensity of Tie-2 expression was quite low (P < 0.05). Ang-2 expressed in CD7(+)AML cells is not considered to act in an autocrine fashion, but to work on endothelial cells to "feed" leukemic cells. Although Ang-2 is recognized as a natural antagonist for Tie-2, our data presented here suggested the alternative role of Ang-2 in the relationship between endothelial cells and leukemia cells, at least in a subset of leukemia such as CD7(+)AML. These results were supported by the study using AML cell lines, KG-1 (CD7 negative) and its subline KG-1a (CD7 positive); KG-1 had mRNA expression profile of Ang-1(+)Ang-2(-)Tie-2(+), while KG-1a showed Ang-1(+)Ang-2(+)Tie-2(-). These difference in the expression profile of angiogenic factors between CD7(+)AML and t(8;21)AML may explain the characteristic morphological features of these leukemias (CD7(+)AML as blastic type and t(8;21)AML as differentiative type).

Hypermutable change of human UV(r)-1 cells by p53 overexpression.

The p53 protein has been reported to regulate cellular responses to genetic stress such as far-ultraviolet light (UV), protecting human cells from mutation. Levels of p53 protein in hypermutable RSa cells were found here to increase soon after UV irradiation, while those in UV(r)-1 cells, a hypomutable variant of RSa cells, showed a delayed increase. Three cell lines overexpressing wild-type p53 in UV(r)-1 cells exhibited higher sensitivity to UV mutagenicity than did control U-V-7 cells transfected with vector alone, assessed using the ouabain-resistance phenotypic mutation test and identification of K-ras codon 12 base substitution mutation. On the other hand, U-V-7 cells showed UV-induced elevation of antipain-sensitive protease activity, but p53 transfectants did not. Moreover, antipain treatment to U-V-7 cells was increased susceptibility to UV mutagenicity. Thus, p53 protein overproduction may sensitize human cells, at least those tested, to UV mutagenicity, in association with inhibition of protease activity.

Porcine growth hormone and LongR(3)IGF-I can improve recovery from surgery-induced weight loss in guinea pigs.

In the present study, porcine growth hormone (pGH) and LongR(3)IGF-I (LR(3)IGF-I), a potent analogue of IGF-I, were infused continuously into 430-g guinea pigs for 7 days, either alone or in combination, to examine whether pGH can counteract the reduction in circulating IGF-I concentrations induced by LR(3)IGF-I administration. The pGH and LR(3)IGF-I were infused at rates of 400 microg/day (0.93 mg/kg/day) and 120 microg/day (0.28 mg/kg/day), respectively, by miniosmotic pumps. The same doses were infused in the combination treatment. During the first day of treatment, animals lost between 2 and 3% of body weight. Cumulative body weight gains as a percentage of initial body weight were significantly (P < 0.001) increased relative to vehicle-treated controls by the LR(3)IGF-I, pGH, and combination treatments when effects were analyzed across the whole 7-day treatment period. The increased weight gains relative to controls were largely made on day 2, but these gains were not associated with increased water or feed intakes, indicating that pGH and LR(3)IGF-I improved feed conversion efficiency. LR(3)IGF-I alone or in combination with pGH significantly increased the fractional weight of kidneys at the end of the 7-day treatment period, whereas LR(3)IGF-I alone increased the fractional weight of spleens. Concentrations of IGF-I in serum collected after 7 days of treatment were decreased by LR(3)IGF-I, but this decrease was not ameliorated by coinfusion with pGH. GH alone did not have any effects on IGF-I concentration. This study suggests that pGH does not have a strong influence on circulating IGF-I concentrations in the guinea pig. We have also demonstrated that pGH and LR(3)IGF-I are capable of enhancing the recovery of body weight lost in response to surgery in the guinea pig.

Expression pattern of hybrid phenotype in adult acute lymphoblastic leukemia.

We examined the expression of hybrid phenotype in 236 adults with acute lymphoblastic leukemia (ALL; 188 B-lineage ALL and 48 T-lineage ALL). In B-lineage ALL, myeloid antigen (mAg) CD15 was concentrated in CD10-CD20- cases (49%); CD13 (42%); and CD33 (43%) in CD10+CD20- cases. This trend had no correlation with the presence of Ph1 or t(4;11) chromosomal abnormality. T-cell antigen CD2, CD4, and CD7 was seen in four, four, and two cases, respectively, and CD4+ and CD7+ cases commonly expressed CD13 and/or CD33 (CD13/CD33). In T-lineage ALL, expression of mAg, CD11b (47%), CD13 (38%), CD15 (28%), and CD33 (51%) was restricted to CD3- cases. B-cell antigen CD19 was found in two cases with CD7 solely as T-cell antigen, and these cases possessed CD13/CD33. CD21 was detected in three cases with CD3. In whole ALL, CD13/CD33 was associated closely with the presence of stem-cell antigen CD34, and in T-lineage ALL, CD13/CD33 had a significant correlation with additional stem-cell features, such as HLA-DR, multidrug resistance 1 (MDR1) and c-kit gene expression. Our results suggest that immature ALL cells frequently express B+M+, T+M+, and occasionally B+T+M+ phenotype; that B+T+M- phenotype is extremely rare; and that mAg expression in B-lineage ALL is complicated as compared to T-lineage ALL.

Increased and decreased expression of CD69 and CD23, respectively, in gravity-stressed lymphocytes.

BACKGROUND: Recent studies have shown that gravity-changing stress modulates expression levels of cell surface molecules on human lymphocytes. However, previous in vitro microgravity studies have been performed with lymphocytes treated with mitogenic agents. HYPOTHESIS: The aim of the study was to test if exposure of cells to gravity-changing stress alone alters the expression levels of cell surface molecules. Specifically, we examined whether the expression of activation markers is altered after exposure of lymphocytes to combinations of microgravity and hypergravity. METHODS: We used free-fall in parabolic flight for human subjects and a drop-shaft to expose peripheral blood mononuclear cells (PBMC) to gravity-changing stress. After such exposure, PBMC were isolated, and expression levels of CD69, CD23 and CD38 were estimated using three-color flow cytometry. RESULTS: Increased percentages of CD69-positive cells were observed with PBMC from 3 of 4 volunteers who undertook 10 parabolic flights. Exposure of blood to gravity-changing stress in the drop-shaft increased both ratios of CD69-positive cells and levels of CD69 expression on T and B cells. In contrast, the percentages of CD23-positive B cells was decreased. However, gravity-changing stress was not always followed by significant alteration in CD38 expression. CONCLUSIONS: Our findings suggest that CD69 and CD23 might be useful markers that are up- and down-regulated, respectively, after exposure of lymphocytes to gravity-changing stress.

Structural Modification Influences the Characteristics of Langmuir Monolayers from Aromatic Carboxylic Acids.

The molecular organization of purely aromatic, polyphenyl carboxylic acids, as Langmuir monolayers at the air/water interface, has been investigated by means of surface pressure and electric surface potential measurements upon film compression. The monolayer characteristics of the basic compound, a symmetrical triphenylbenzene (5'-phenyl-m-terphenyl) ring with a carboxylic group at the 4 position (namely 5'-phenyl-1,1' : 3',1"-terphenyl-4-carboxylic acid), are compared with those of its derivatives containing hydrophilic (nitro) or hydrophobic (phenyl) substituents. The nature of the substituent as well as its position (2' or 4') has a profound influence on the monolayer properties. The results are discussed in view of molecular orientation deduced from values of effective dipole moments. Copyright 2001 Academic Press.

Mutagenicity of bisphenol A and its suppression by interferon-alpha in human RSa cells.

Bisphenol A is used as a monomer in the production of polycarbonate plastic products. The widespread use of bisphenol A has raised concerns about its effects in humans. Since there is little information on the mutagenic potential of the chemical, the mutagenicity of bisphenol A was tested using human RSa cells, which has been utilized for identification of novel mutagens. In genomic DNA from cells treated with bisphenol A at concentrations ranging from 1x10(-7) to 1x10(-5)M, base substitution mutations at K-ras codon 12 were detected using PCR and differential dot-blot hybridization with mutant probes. Mutations were also detected using the method of peptide nucleic acid (PNA)-mediated PCR clamping. The latter method enabled us to detect the mutation in bisphenol A-treated cells at a dose (1x10(-8)M) equivalent to that typically found in the environment. Induction of ouabain-resistant (Oua(R)) phenotypic mutation was also found in cells treated with 1x10(-7) and 1x10(-5)M of bisphenol A. The induction of K-ras codon 12 mutations and Oua(R) mutations was suppressed by pretreating RSa cells with human interferon (HuIFN)-alpha prior to bisphenol A treatment. The cells treated with bisphenol A at the concentration of 1x10(-6)M elicited unscheduled DNA synthesis (UDS). These findings suggested that bisphenol A has mutagenicity in RSa cells as well as mutagens that have been tested in these cells, and furthermore, that a combination of the PNA-mediated PCR clamping method with the human RSa cell line may be used as an assay system for screening the mutagenic chemicals at very low doses.

Additional t(11;17)(q23;q21) in a patient with Philadelphia-positive mixed lineage antigen-expressing leukemia.

We describe very uncommon phenotypic and cytogenetic findings in a 40-year-old female with blast phase of Philadelphia chromosome (Ph)-positive CML. In addition to the t(9;22)(q34;q11) that was detected in all metaphases, a t(11;17)(q23;q21) was identified in 15 of 20 metaphases. Reverse transcription-polymerase chain reaction showed the major and minor bcr/abl fusion transcripts in the cells from a bone marrow (BM) sample. Fluorescence in situ hybridization (FISH) analysis also showed that fusion signals of the bcr and abl probes were found in 95% of blastic cells and in 64% of neutrophils. MLL gene rearrangement was also detected in some blastic cells but not in neutrophils by FISH analysis. Phenotypically, blastic cells expressed mixed lineage antigens such as CD34, CD33, CD13, CD19, CD7, and CD41. Immunogenotypically, some population of BM cells showed monoclonal rearrangements of immunoglobulin heavy chain and T-cell receptor gamma chain genes by Southern blot analysis. Clinical course was aggressive, and therapy was poorly tolerated. Such findings seem to support an association between Ph and an abnormality of 11q23 with poor prognosis, and suggest that the expression of both abnormal genes may be related to this mixed lineage antigen-expressing leukemia.

Analysis of clonal relationship using single-cell polymerase chain reaction in a patient with concomitant mantle cell lymphoma and multiple myeloma.

We report a case of concomitant mantle cell lymphoma (MCL) and multiple myeloma (MM) in which we investigated the possibility of a clonal relationship. A 76-year-old man was diagnosed with MCL [immunoglobulin (Ig)M,D-kappa; stage IVB] and MM (IgG-kappa; stage I). Ig heavy chain (IgH) gene complementarity-determining region 3 in DNA from both the MCL tumor and from single MM cells from bone marrow smears was amplified to investigate whether there was a clonal relationship between MCL and MM. Sequence analysis revealed no clonal relationship between MCL and MM in our patient.

Aggressive neoplastic plasma cell growth with MLL gene rearrangement after high-dose therapy with autologous stem cell support for multiple myeloma.

We report a case of a patient with IgA kappa multiple myeloma (MM) mobilized with etoposide and subsequently receiving high-dose melphalan (HDM) with stem cell support. She relapsed rapidly post transplantation. Southern blot and fluorescent in situ hybridization analysis showed MLL gene rearrangement in the myeloma cells, which was not detected in the sample at diagnosis or in the PBSC harvested with etoposide plus G-CSF. These observations suggest that clonal rearrangement of the MLL gene is caused by etoposide. Patients with MM undergoing HDM with stem cell rescue may be at an increased risk of not only secondary leukemia, but also secondary genetic abnormalities in myeloma cells, especially those receiving priming with etoposide for peripheral blood stem cell collection.

Protective immunity to Schistosoma japonicum infection depends on the balance of T helper cytokine responses in mice vaccinated with gamma-irradiated cercariae.

Although the strain difference in protection of mice to Schistosoma mansoni infection has been described, limited information is available in the case of Schistosoma japonicum. In the present study, we compared the protective immunity to S. japonicum infection and cytokine production in various strains of mice vaccinated with gamma-irradiated cercariae. A significant reduction in worm recovery was observed in male and female mice of DBA/2 at a 6-week interval between vaccination and a challenge infection, whereas vaccinated mice of C57BL/6, C57BL/10, (C57BL/6 x DBA/2) F1 (B6D2F1) and (C57BL/10 x DBA/2) F1 (B10D2F1) showed no detectable level of protection. No sex-linked difference in development of resistance was observed in any of the strains so far examined. Vaccination with gamma-irradiated cercariae twice with a 3-week interval also induced significant protection against a challenge infection in DBA/2 but not in BALB/c or C57BL/6 strains. Further studies demonstrated that spleen cells of vaccinated C57BL/6 mice produced lower levels of IFN-gamma compared to the cells of vaccinated BALB/c and DBA/2. On the other hand, production of IL-10 by spleen cells was relatively higher in BALB/c mice than in the other two strains. Macrophages that had been stimulated with spleen cell culture supernatants derived from vaccinated DBA/2 damaged schistosomula more effectively than cells stimulated with supernatants derived from the other strains. These results suggest that different levels of protection observed among strains of mice depend on the balance of cytokine responses which consequently activate or suppress macrophage-mediated damage to schistosomula.

Successful autologous peripheral blood stem cell transplantation for relapsed intravascular lymphomatosis.

We describe a case of relapsed intravascular lymphomatosis (IVL) successfully treated with autologous PBSCT. A 59-year-old Japanese female patient with IVL who had achieved CR after six courses of biweekly CHOP therapy developed lymphoma. She achieved a second CR after six courses of modified biweekly CHOP therapy, followed by autologous PBSCH and high-dose chemotherapy (CBDCA, VP-16, MCNU, CY) with PBSCT. There has not been any evidence of recurrence 48 months after PBSCT. Our case suggests that PBSC is acceptable as a source for stem cell rescue in IVL.

Characterization of the reversible nature of the reaction catalyzed by sphingolipid ceramide N-deacylase. A novel form of reverse hydrolysis reaction.

Sphingolipid ceramide N-deacylase catalyzes a reversible reaction in which the amide linkages of the ceramides of various sphingolipids are cleaved or synthesized. Hydrolysis of sphingolipids by the enzyme proceeded efficiently at acidic pH in the presence of high concentrations of detergents, whereas the reverse reaction tended to be favored at neutral pH with a decrease in the detergent concentration. Although the catalytic efficiency (V(max)/K(m)) of the hydrolysis and reverse reactions was changed mainly by the concentration of detergents in the reaction mixture, V(max) and K(m) for the reverse reaction were relatively higher than those for the forward reaction, irrespective of the detergent concentration. The reverse reaction proceeded most efficiently when the molar ratio of lyso-sphingolipids and fatty acids was fixed at 1 : 1-2, the yield of the reaction exceeding 70-80%. The reverse and exchange (transacylation) reactions did not require ATP, CoA, metal ions or addition of organic solvents. Studies using inhibitors and chemical modifiers of the enzyme protein suggested that both the hydrolysis and condensation reactions are catalyzed at the same catalytic domain. These results indicate that the reverse hydrolysis reaction of the enzyme is unique, being completely different from those of lipases, proteases and glycosidases reported to date.