Susceptibility of patients receiving chemotherapy for haematological malignancies to scabies.

BACKGROUND: Scabies is a contagious dermatosis. The risk factors for its transmission remain unclear. A scabies outbreak, involving patients who were receiving chemotherapy for haematological malignancies, occurred at our hospital. METHODS: The outbreak population was analysed to determine whether the incidence of scabies was higher among contact patients receiving chemotherapy for haematological malignancies. RESULTS: A patient with crusted scabies was the index case, and 18 of 78 contact healthcare workers (HCWs) and 22 of 135 contact patients were diagnosed with classical scabies. Ten of 17 contact patients with haematological malignancies and 12 of 118 contact patients with other diseases were infected with scabies. The incidence rate was significantly higher among the patients with haematological malignancies (P<0.001). The patients with haematological malignancies had a significantly lower mean minimum neutrophil count than those with other diseases (1159/µL vs 3761/µL, P=0.0012). Most haematological patients did not require special nursing assistance, suggesting that the higher incidence of scabies among these patients resulted from their immunodeficiency rather than greater skin-to-skin contact with infected HCWs. CONCLUSION: Our study suggests that patients receiving chemotherapy for haematological malignancies are more susceptible to scabies than patients with other diseases, and require stricter protection.

Aberrant expression of RasGRP1 cooperates with gain-of-function NOTCH1 mutations in T-cell leukemogenesis.

Ras guanyl nucleotide-releasing proteins (RasGRPs) are activators of Ras. Previous studies have indicated the possible involvement of RasGRP1 and RasGRP4 in leukemogenesis. Here, the predominant role of RasGRP1 in T-cell leukemogenesis is clarified. Notably, increased expression of RasGRP1, but not RasGRP4, was frequently observed in human T-cell malignancies. In a mouse bone marrow transplantation model, RasGRP1 exclusively induced T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) after a shorter latency when compared with RasGRP4. Accordingly, Ba/F3 cells transduced with RasGRP1 survived longer under growth factor withdrawal or phorbol ester stimulation than those transduced with RasGRP4, presumably due to the efficient activation of Ras. Intriguingly, NOTCH1 mutations resulting in a gain of function were found in 77% of the RasGRP1-mediated mouse T-ALL samples. In addition, gain-of-function NOTCH1 mutation was found in human T-cell malignancy with elevated expression of RasGRP1. Importantly, RasGRP1 and NOTCH1 signaling cooperated in the progression of T-ALL in the murine model. The leukemogenic advantage of RasGRP1 over RasGRP4 was attenuated by the disruption of a protein kinase C phosphorylation site (RasGRP1(Thr184)) not present on RasGRP4. In conclusion, cooperation between aberrant expression of RasGRP1, a strong activator of Ras, and secondary gain-of-function mutations of NOTCH1 have an important role in T-cell leukemogenesis.

Eµ/miR-125b transgenic mice develop lethal B-cell malignancies.

MicroRNA-125b-1 (miR-125b-1) is a target of a chromosomal translocation t(11;14)(q24;q32) recurrently found in human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). This translocation results in overexpression of miR-125b controlled by immunoglobulin heavy chain gene (IGH) regulatory elements. In addition, we found that six out of twenty-one BCP-ALL patients without t(11;14)(q24;q32) showed overexpression of miR-125b. Interestingly, four out of nine patients with BCR/ABL-positive BCP-ALL and one patient with B-cell lymphoid crisis that had progressed from chronic myelogenous leukemia overexpressed miR-125b. To examine the role of the deregulated expression of miR-125b in the development of B-cell tumor in vivo, we generated transgenic mice mimicking the t(11;14)(q24;q32) (Eµ/miR-125b-TG mice). Eµ/miR-125b-TG mice overexpressed miR-125b driven by IGH enhancer and promoter and developed IgM-negative or IgM-positive lethal B-cell malignancies with clonal proliferation. B cells obtained from the Eµ/miR-125b-TG mice were resistant to apoptosis induced by serum starvation. We identified Trp53inp1, a pro-apoptotic gene induced by cell stress, as a novel target gene of miR-125b in hematopoietic cells in vitro and in vivo. Our results provide direct evidence that miR-125b has important roles in the tumorigenesis of precursor B cells.

Rapid amplification of immunoglobulin heavy chain switch (IGHS) translocation breakpoints using long-distance inverse PCR.

Molecular cloning of immunoglobulin heavy chain (IGH) translocation breakpoints identifies genes of biological importance in the development of normal and malignant B cells. Long-distance inverse PCR (LDI-PCR) was first applied to amplification of IGH gene translocations targeted to the joining (IGHJ) regions. We report here successful amplification of the breakpoint of IGH translocations targeted to switch (IGHS) regions by LDI-PCR. To detect IGHS translocations, Southern blot assays using 5' and 3' switch probes were performed. Illegitimate Smu rearrangements were amplified from the 5' end (5'Smu LDI-PCR) from the alternative derivative chromosome, and those of Sgamma or Salpha were amplified from the 3' end (3'Sgamma or 3'alpha LDI-PCR) from the derivative chromosome 14. Using a combination of these methods, we have succeeded in amplifying IGHS translocation breakpoints involving FGFR3/MMSET on 4p16, BCL6 on 3q27, MYC on 8q24, IRTA1 on 1q21 and PAX5 on 9p13 as well as BCL11A on 2p13 and CCND3 on 6p21. The combination of LDI-PCR for IGHJ and IGHS allows rapid molecular cloning of almost all IGH gene translocation breakpoints.

Expression of a gene for Mn-peroxidase from Coriolus versicolor in transgenic tobacco generates potential tools for phytoremediation.

In efforts aimed at the detoxification of contaminated areas, plants have many advantages over bacteria and fungi. We are attempting to enhance the environmental decontamination functions of plants by transferring relevant genes from microorganisms. When the gene for Mn-peroxidase (MnP) from Coriolus versicolor was expressed in transgenic tobacco plants, one line (designated fMnP21) expressed MnP activity at levels 54-fold higher than in control lines. When undamaged roots of transgenic plants were applied to liquid medium supplemented with 250 microM pentachlorophenol (PCP), the decrease in the level of PCP in fMnP21 (86% reduction) was about 2-fold higher than that in control lines (38% reduction). Expression of the gene for MnP in the transgenic plants had no obvious negative effects on their vegetative and sexual growth. Our system should contribute to the development of novel methods for the removal of hazardous chemicals from contaminated environments using transgenic plants.

Lack of somatic hypermutation of IG V(H) genes in lymphoid malignancies with t(2;14)(p13;q32) translocation involving the BCL11A gene.

The t(2;14)(p13;q32.3) involving the BCL11A and IGH genes is a rare but recurrent chromosomal aberration in B-cell malignancies. Hitherto, juxtaposition of BCL11A and IGH has only been described in B-cell chronic lymphocytic leukemia (B-CLL) and immunocytoma. As subgroups of B-CLL can be distinguished by the pattern of somatic mutation of immunoglobulin variable (V) genes we investigated four lymphomas with IGH/BCL11A involvement for IGH hypermutation. Clonal V(H) gene rearrangements were amplified; in all four cases, sequencing of the amplificates revealed the rearranged V(H) genes to lack somatic mutations. These results suggest that t(2;14)(p13;q32.3) is associated with a subset of B-CLL/immunocytoma characterized by non-mutated IG genes deriving from pre-germinal center B cells. As the translocations in both informative cases are targeted to the switch regions of the IGG2 gene, which is mainly used in T cell-independent immune responses, these translocations presumably occurred in activated B cells in the course of T cell-independent immune responses outside the germinal center.

The BCL11 gene family: involvement of BCL11A in lymphoid malignancies.

Many malignancies of mature B cells are characterized by chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32.3 and result in deregulated expression of the translocated oncogene. t(2;14)(p13;q32.3) is a rare event in B-cell malignancies. In contrast, gains and amplifications of the same region of chromosome 2p13 have been reported in 20% of extranodal B-cell non-Hodgkin lymphomas (B-NHL), in follicular and mediastinal B-NHL, and in Hodgkin disease (HD). It has been suggested that REL, an NF-kappaB gene family member, mapping within the amplified region, is the pathologic target. However, by molecular cloning of t(2;14)(p13;q32.3) from 3 cases of aggressive B-cell chronic lymphocytic leukemia (CLL)/immunocytoma, this study has shown clustered breakpoints on chromosome 2p13 immediately upstream of a CpG island located about 300 kb telomeric of REL. This CpG island was associated with a Krüppel zinc finger gene (BCL11A), which is normally expressed at high levels only in fetal brain and in germinal center B-cells. There were 3 major RNA isoforms of BCL11A, differing in the number of carboxy-terminal zinc fingers. All 3 RNA isoforms were deregulated as a consequence of t(2;14)(p13;q32.3). BCL11A was highly conserved, being 95% identical to mouse, chicken, and Xenopus homologues. BCL11A was also highly homologous to another gene (BCL11B) on chromosome 14q32.1. BCL11A coamplified with REL in B-NHL cases and HD lymphoma cell lines with gains and amplifications of 2p13, suggesting that BCL11A may be involved in lymphoid malignancies through either chromosomal translocation or amplification.

Cyclin D3 is a target gene of t(6;14)(p21.1;q32.3) of mature B-cell malignancies.

Chromosomal translocation t(6;14)(p21.1;q32.3) has been reported as a rare but recurrent event not only in myeloma and plasma cell leukemia but also in diffuse large B-cell non-Hodgkin lymphoma (B-NHL) (diffuse large B-cell lymphoma [DLBCL]) and splenic lymphoma with villous lymphocytes (SLVL); however, the nature of the target gene(s) has not been determined. This study identified t(6;14)(p21.1;q32.3) in 3 cases of transformed extranodal marginal zone B-NHL, in 1 case of SLVL, and in 1 case of a low-grade B-cell lymphoproliferative disorder. In a sixth case, a CD5(+) DLBCL, the translocation was identified by molecular cloning in the absence of cytogenetically detectable change. Two chromosomal translocation breakpoints were cloned by using long-distance inverse polymerase chain reaction methods. Comparison with the genomic sequence for chromosome 6p21.1 showed breakpoints approximately 59 and 73.5 kilobases 5' of the cyclin D3 (CCND3) gene with no other identifiable transcribed sequences in the intervening region. Although Southern blotting with derived genomic 6p21.1 probes failed to detect other rearrangements, fluorescent in situ hybridization assays, using BAC (bacterial artificial chromosome) clones spanning and flanking the CCND3 locus, along with probes for IGH confirmed localization of 6p21.1 breakpoints within the same region, as well as fusion of the CCND3 and IGH loci. Furthermore, in all cases, high-level expression of CCND3 was demonstrated at RNA and/or protein levels by Northern and Western blotting and by immunohistochemistry. These data implicate CCND3 as a dominant oncogene in the pathogenesis and transformation in several histologic subtypes of mature B-cell malignancies with t(6;14)(p21.1;q32.3) and suggest that CCND3 overexpression seen in about 10% of DLBCL cases may have a genetic basis.

Detection of translocations affecting the BCL6 locus in B cell non-Hodgkin's lymphoma by interphase fluorescence in situ hybridization.

Structural alterations in 3q27 affecting the BCL6 locus are among the most frequent changes in B-NHL. The aim of the present study was to establish an interphase-FISH assay for the detection of all diverse BCL6 translocations in B-NHL. Two different approaches were tested, one using a PAC-clone spanning the major breakpoint region (MBR) of BCL6 (span-assay), and another using two BAC clones flanking the MBR (flank-assay). Interphase FISH with the span-assay detected the various BCL6 translocations in seven B-NHL cell lines. The dual-color flank-assay was evaluated in two laboratories independently: in normal controls, the cutoff level for false-positive signals was 2.6%, whereas the cutoff level for false-negatives in the seven cell lines was 7.5%. To test the feasibility of the FISH strategies, 30 samples from patients with B-NHL with cytogenetic abnormalities of 3q27 were evaluated with both assays. In 21 cases, the span-assay indicated a BCL6 rearrangement. In 18 of the 21 cases, the dual-color flank-assay confirmed the translocation including 12 different partner chromosomal loci. The three false-positive cases detected with the span-assay showed trisomy of chromosome 3 by cytogenetic analyses, and they were correctly classified as non-rearranged with the flank-assay. In summary, our FISH strategy using two differently labeled flanking BCL6 BAC probes provides a robust, sensitive, and reproducible method for the detection of common and uncommon abnormalities of BCL6 gene in interphase nuclei. The routine application of this assay to patients with B-NHL will allow the assessment of the diagnostic and prognostic significance of BCL6 rearrangements.

Coexistence of two different O demethylation systems in lignin metabolism by Sphingomonas paucimobilis SYK-6: cloning and sequencing of the lignin biphenyl-specific O-demethylase (LigX) gene.

Sphingomonas paucimobilis SYK-6 can grow on several dimeric model compounds of lignin as a carbon and energy source. It has O demethylation systems on three kinds of substrates: 5, 5'-dehydrodivanillic acid (DDVA), syringate, and vanillate. We previously reported the cloning of a gene involved in the tetrahydrofolate-dependent O demethylation of syringate and vanillate. In the study reported here, we cloned the gene responsible for DDVA O demethylation. Using nitrosoguanidine mutagenesis, a mutant strain, NT-1, which could not degrade DDVA but could degrade syringate and vanillate, was isolated and was used to clone the gene responsible for the O demethylation of DDVA by complementation. Sequencing analysis showed an open reading frame (designated ligX) of 1,266 bp in this fragment. The deduced amino acid sequence of LigX had similarity to class I type oxygenases. LigX was involved in O demethylation activity on DDVA but not on vanillate and syringate. DDVA O demethylation activity in S. paucimobilis SYK-6 cell extracts was inhibited by addition of the LigX polyclonal antiserum. Thus, LigX is an essential enzyme for DDVA O demethylation in SYK-6. S. paucimobilis SYK-6 has two O demethylation systems: one is an oxygenative demethylase system, and the other is a tetrahydrofolate-dependent methyltransferase system.

A plasma cell leukemia patient showing bialleic 14q translocations: t(2;14) and t(11;14).

We report here an IgG/lambda-type plasma cell leukemia patient showing bialleic 14q32 translocations. All immunoglobulins were suppressed in this patient, but a small amount of monoclonal IgG was detected by immunoelectrophoresis. Two cells of six peripheral blood mononuclear cells showed 46,XY,t(2;14)(q11;q32), i(8)(q10), t(11;14)(q13;q32), del(12)(q13.1) by karyotypic analysis. We confirmed the juxtaposition of IgH and PRAD1/Cyclin D1 genes by fluorescent in situ hybridization and overexpression of the PRAD1/Cyclin D1 gene, but Southern analysis showed no bcl-1 rearrangement. We analyzed the t(2;14)(q11;q32) using DNA fragments derived from childhood B-chronic lymphocytic leukemia cases bearing t(2;14)(p13;q32). Southern and Northern analyses demonstrated no alteration of these genes, indicating that this t(2;14) was different from that of childhood B-chronic lymphocytic leukemia. At the IgH loci, Southern analysis showed two rearranged bands and one germ-line band of JH. Cmicro was deleted on one rearranged allele but remained on the other, suggesting that the chromosome translocation occurred after productive class switch recombination on the Cmicro deleted allele.

Establishment and characterization of a CD95 (Fas/Apo-1)-negative myeloma cell line.

Although expression of CD95 (Fas/Apo-1) on myeloma cells has been reported, its significance is not clearly understood. We established a myeloma cell line, KHM-11ad (11ad), from a parental cell line, KHM-11, by collecting cells adhered to a plastic dish. KHM-11 cells have been reported to be positive for CD45 and CD95 (Fas/Apo1), and negative for a myelomonocytic antigen, CD13. Interestingly, CD95 was not detected in 11ad. Expression of CD45 was also significantly decreased in 11ad cells while expression of CD13 was detected in these cells. The growth rate of 11ad cells was 1.7 times lower than that of KHM-11 cells. Analysis of adhesion molecules showed that expression of VLA4 and CD44 was significantly suppressed in 11ad. The IC50 of melphalan (L-PAM) for 11ad cells was 50 times higher than that for KHM-11, indicating that 11ad is significantly refractory to L-PAM than KHM-11 cells. Induction of apoptosis by doxorubicin and cycloheximide was suppressed in 11ad cells compared with those in KHM-11 cells. Western blot analysis for Bcl-2 family of proteins showed that Bax was expressed at a 2.2 times lower level in 11ad cells than in KHM-11 cells while there was no difference in expression of Bcl-2, Bcl-Xs nor Bcl-XL. These results suggest that CD95-negative myeloma cells may have characteristics as follows: (1) slow proliferation; (2) low sensitivity to apoptosis; (3) low expression of VLA4, CD44 and Bax. Although these intraclonal variations were based on the findings of cell lines, these may reflect similar variations in vivo. The 11ad line may be a suitable model for analyzing intraclonal variation of myeloma cells.

Detection of inducible nitric oxide synthase (iNOS) mRNA by RT-PCR in ATL patients and HTLV-I infected cell lines: clinical features and apoptosis by NOS inhibitor.

Various tumors have been reported to express an inducible form of nitric oxide synthase (iNOS), and nitric oxide (NO) may affect the clinicopathological features of these tumors. Previously, Burkitt's lymphoma and Epstein-Barr virus (EBV)-infected cells were shown to express iNOS constitutively at a low level. We analyzed iNOS expression by the reverse transcriptase-polymerase reaction method (RT-PCR) in eight HTLV-I-infected cell lines (five were ATL-derived lines and there were in vitro transformed lines), nine ATL patients (three were chronic, two were acute, and four were lymphoma type), and an HTLV-I-negative T cell line (CEM). In four ATL derived and in all three in vitro transformed cell lines, iNOS was expressed constitutively, but it was not expressed in CEM cells. Four out of nine ATL patients also showed iNOS expression. The expression of iNOS was found in all subtypes of ATL. Three of four iNOS-positive patients had infiltration of ATL cells to organs such as skin, lung, or liver. In NOS inhibitor (NG-monomethyl-L-arginine: L-NMMA)-containing medium, an iNOS-positive ATL cell line (K3T) showed growth inhibition and DNA ladder. Although only a limited number of patients was analyzed, our results suggest that NO may be involved in the invasive character of ATL cells. The NOS inhibitor can induce apoptosis in an ATL cell line, as it does in EBV-infected cell lines.

Expression of PRAD1/cyclin D1 in plasma cell malignancy: incidence and prognostic aspects.

We analysed PRAD1/cyclin D1 expression in 20 patients with plasma cell malignancy by Northern analysis. 6/17 multiple myeloma patients and 3/3 plasma cell leukaemia patients showed PRAD1/cyclin D1 expression. This incidence appeared to be higher than the expected incidence based on previous studies. Southern analysis did not show rearrangement of the bcl-1 region. Although there was no statistical difference, the PRAD1/cyclin D1 negative group showed a 1-year survival of 81.8%, 3-year survival of 45.5% and 5-year survival of 22.7%, and those for the PRAD1/cyclin D1 positive group were 63.5%, 16.9% and 16.9%, respectively. Further study is required to determine whether PRAD1/cyclin D1 expression is a prognostic factor.

Regulation of the urea cycle enzyme genes in nitric oxide synthesis.

Nitric oxide (NO) is synthesized from arginine by nitric-oxide synthase (NOS), and citrulline that is generated can be recycled to arginine by argininosuccinate synthase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS) and expression of the inducible isoform of NOS (iNOS), AS and AL was analysed. In RNA blot analysis, iNOS mRNA was induced by LPS in the lung, heart, liver and spleen, and less strongly in the skeletal muscle and testis. AS and AL mRNAs were induced in the lung and spleen. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2-5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6-12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high up to 24 h. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS and AL were co-induced in rat tissues and cells, citrulline-arginine recycling seems to be important in NO synthesis under the conditions of stimulation. Arginine is a common substrate of NOS and arginase. Rat peritoneal macrophages were cultured in the presence of LPS and expression of iNOS and livertype arginase (arginase I) was analysed. mRNAs for iNOS and arginase I were induced by LPS in a dose-dependent manner. iNOS mRNA appeared 2 h after LPS treatment and increased up to a near-maximum at 8-12 h. On the other hand, arginase I mRNA began to increase after 4 h with a lag time and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and arginase I proteins were also induced. Induction of iNOS and arginase I mRNAs were also observed in LPS-injected rats in vivo. Thus, arginase I appears to have an important role in downregulating NO synthesis in murine macrophages by decreasing the availability of arginine. A cDNA for human arginase II, an arginase isozyme, was isolated. A polypeptide of 354 amino acid residues including the putative NH2-terminal presequence for mitochondrial import was predicted. It was 59% identical with arginase I. mRNA for human arginase II was present in the kidney and other tissues but was not detected in the liver. Arginase II mRNA was co-induced with iNOS mRNA in murine macrophage-like RAW 264.7 cells by LPS. This induction was enhanced by dexamethasone and dibutyrul cAMP, and was prevented by interferon-gamma. These results indicate that NO synthesis is regulated by arginine-synthesizing and -degrading enzymes in a complicated manner.

Cloning and sequencing of the Sphingomonas (Pseudomonas) paucimobilis gene essential for the O demethylation of vanillate and syringate.

Sphingomonas (Pseudomonas) paucimobilis SYK-6 is able to grow on 5,5'-dehydrodivanillic acid (DDVA), syringate, vanillate, and other dimeric model compounds of lignin as a sole carbon source. Nitrosoguanidine mutagenesis of S. paucimobilis SYK-6 was performed, and two mutants with altered DDVA degradation pathways were isolated. The mutant strain NT-1 could not degrade DDVA, but could degrade syringate, vanillate, and 2,2',3'-trihydroxy-3-methoxy-5,5'-dicarboxybiphenyl (OH-DDVA). Strain DC-49 could slowly assimilate DDVA, but could degrade neither vanillate nor syringate, although it could degrade protocatechuate and 3-O-methylgallate. A complementing DNA fragment of strain DC-49 was isolated from the cosmid library of strain SYK-6. The minimum DNA fragment complementing DC-49 was determined to be the 1.8-kbp insert of pKEX2.0. Sequencing analysis showed an open reading frame of 1,671 bp in this fragment, and a similarity search indicated that the deduced amino acid sequence of this open reading frame had significant similarity (60%) to the formyltetrahydrofolate synthetase of Clostridium thermoaceticum.

Multiple myeloma associated with serum amino acid disturbance and high output cardiac failure.

We experienced a plasma cell leukemia (PCL) patient complicated with high output cardiac failure (HOCF), proved as his elevated cardiac index and pulmonary artery wedge pressure and decreased systemic vascular resistance index in a hemodynamic study. We found no possible causes of HOCF. Interestingly, HOCF was improved as PCL responded to intensive chemotherapy. On the other hand, he showed consciousness disturbance, and had frequent attacks of generalized seizure. His electroencephalogram showed slow waves, and a spike and wave complex. Hyperammonemia and abnormal amino acid distribution were also found. This abnormal serum amino acid distribution, especially elevated glycine level, was different from that seen in chronic liver failure, and he had no hepatic disease. After intensive chemotherapy, the serum ammonia level and glycine level decreased. In this patient, PCL seemed to be responsible for HOCF, hyperammonemia, and abnormal amino acid distribution. We experienced two more cases of multiple myeloma (MM) with HOCF, hyperammonemia, abnormal serum amino acid distribution, and consciousness disturbance of unknown origin. Those two cases showed slow waves in the electroencephalogram. Improvement was seen in their HOCF, hyperammonemia, and abnormal amino acid levels after chemotherapy. The possibility of MM as a cause of HOCF is discussed.