Synthesis and photooxidation of oligodeoxynucleotides containing 5-dimethylaminocytosine as an efficient hole-trapping site in the positive-charge transfer through DNA duplexes.

We have designed and synthesized DNA duplexes containing 5-dimethylaminocytosine ((DMA)C) to investigate the effects of C(5)-substituted cytosine bases on the transfer and trapping of positive charge (holes) in DNA duplexes. Fluorescence quenching experiments revealed that a (DMA)C base is more readily one-electron oxidized into a radical cation intermediate as compared with other natural nucleobases. Upon photoirradiation of the duplexes containing (DMA)C, the photosensitizer-injected hole migrated through the DNA bases and was trapped efficiently at the (DMA)C sites, where an enhanced oxidative strand cleavage occurred by hot piperidine treatment. The (DMA)C radical cation formed by hole transfer may undergo specific hydration and subsequent addition of molecular oxygen, thereby leading to its decomposition followed by a predominant strand cleavage at the (DMA)C site. This remarkable property suggests that the modified cytosine (DMA)C can function as an efficient hole-trapping site in the positive-charge transfer in DNA duplexes.

[Fatal hepatic failure due to AL amyloidosis in a patient with multiple myeloma].

Although about 10 to 15% of patients with multiple myeloma (MM) develop AL amyloidosis, liver-restricted fatal amyloidosis is rare. We encountered such an MM patient. A 73-year-old female without a history of carpal tunnel syndrome was diagnosed with IgG-κ MM (Stage I by Durie & Salmon) in January, 2005. Because MM was exacerbated to Stage III in May, 2007, VAD (vincristine, adriamycin, dexamethasone) chemotherapy was performed with minor response, despite 3 courses of this regimen. Three courses of salvage chemotherapy (cyclophosphamide+melphalan; CM) were then performed with near partial response. In March, 2008, just before the 4th cycle of CM chemotherapy, she was slightly icteric with elevated biliary tract enzymes; therefore, treatment was switched to oral cyclophosphamide and prednisolone. At this time, she did not have macroglossia, skin eruption, gastrointestinal dysfunction, or bleeding. Echocardiography was also non-specific. One month later, she developed a marked bleeding tendency and leg edema. Laboratory tests showed a severe deterioration in liver function. In the middle of May, 2008, she progressed to hepatic coma and died of intracranial hemorrhage several days later. Autopsy showed that the liver was almost substituted by AL amyloid substance.

[Exacerbation of autoimmune neutropenia to agranulocytosis in association with severe autoimmune thrombocytopenia and hemolytic anemia in a patient with Sjögren's syndrome].

A 73-year-old woman with Sjögren's syndrome and autoimmune neutropenia (AIN) associated with large granular lymphocytosis of the polyclonal T cell type, demonstrated autoimmune thrombocytopenia (AIT) at diagnosis of sigmoid colon cancer. Ten months later, both AIN and AIT had exacerbated to agranulocytosis and severe thrombocytopenia below 10×10(9)/L, respectively. There were no dysplastic features of bone marrow hematopoietic cells. Furthermore, an in vitro assay of hematopoietic progenitors showed normal granuloid and erythroid colony formation. Although we serially treated her with prednisolone (oral), filgrastim, intravenous high-dose immunoglobulin infusion, cyclophosphamide (oral), danazol, cyclosporine A (oral), and rituximab, number of neutrophils and platelets elevated only temporarily. During the course of agranulocytosis and severe thrombocytopenia, the patient also developed autoimmune hemolytic anemia (AIHA). She died of pneumonia 5 months after the onset of agranulocytosis. This case is very unique and novel in terms of autoimmune phenomena simultaneously directed to granulocytes, platelets, and red blood cells under the background of Sjögren's syndrome.

Hepatosplenic alphabeta T cell lymphoma.

A 32-year-old male with chronic hepatitis B was admitted to a hospital with cellulitis in the right leg in September 2006. Pancytopenia, hepatosplenomegaly, and systemic superficial lymph node swelling were noted, and he was referred to our hospital. He developed fever and liver dysfunction in June 2007 and underwent a splenectomy. His pancytopenia subsequently improved. A pathologic diagnosis of hepatosplenic alphabeta T cell lymphoma was made by examining spleen tissue and biopsy specimens of the liver and mesenteric lymph node. He had stage IVB disease because neoplastic T cells were noted in the bone marrow. The response of the lymphoma to conventional chemotherapy including the CHOP (cyclophosphamide, adriamycin, vincristine, prednisolone) and DeVIC (dexamethasone, etoposide, ifoshamide, carboplatin) regimens was poor and transient. A partial remission was obtained with an ESHAP (etoposide, cisplatin, cytarabine, methylprednisolone) regimen. Therefore, we planned a bone marrow transplantation (BMT) from an HLA-haploidentical sibling donor. He was moved to the Department of Hematology, Hyogo Medical College, to receive this BMT as part of a clinical trial. During the conditioning procedure for the transplantation, however, he died of septicemia. Since hepatosplenic alphabeta T cell lymphoma is very rare with only 23 reported cases to date, herein we report this case and discuss the therapeutic strategy.

[Successful treatment of chronic disseminated intravascular coagulation syndrome with continuous subcutaneous infusion of heparin using a mobile infusion pump: report of 2 cases].

We report here two patients with chronic disseminated intravascular coagulation (chronic DIC) secondary to aortic aneurysm, who were successfully treated with continuous subcutaneous infusion of heparin. The patients were 69- and 89-year-old males, who were admitted to our hospital because of thrombocytopenia and marked bleeding tendency. The underlying conditions were aortic dissection and aortic aneurysm, respectively. Coagulation test demonstrated that these patients had DIC, and a diagnosis of chronic DIC secondary to aortic aneurysm was made. Anti-coagulation treatment with oral camostat mesylate and daily subcutaneous infusion of heparin calcium was started. However, the treatment was insufficient to control chronic DIC, and these patients developed recurrent severe subcutaneous hemorrhages. Therefore, we attempted continuous subcutaneous infusion of heparin using a mobile infusion pump. This delivery of heparin markedly improved the coagulopathy, and the hemorrhagic episode disappeared with good compliance in the use of infusion equipment in these patients. Continuous subcutaneous infusion of heparin using a mobile infusion pump is effective and useful for long term treatment of chronic DIC by the outpatient department.

[Angioimmunoblastic T-cell lymphoma complicated by recurrent drug-induced agranulocytosis].

A 73-year-old man was hospitalized with fever, erythema, generalized superficial lymphadenopathy and marked neutropenia in July 2007. Hematologic examination demonstrated a white blood cell count of 1,400/microl with 0% neutrophils, and 18% abnormal lymphocytes. A bone marrow aspirate showed marked myeloid hypoplasia. A diagnosis of drug-induced agranulocytosis was made. Although neutrophil counts immediately returned to normal levels in response to filgrastim, fever, skin rash and systemic lymphadenopathy were all persistent. He also developed autoimmune hemolytic anemia and a second episode of agranulocytosis. The causative agent of the both episodes of agranulocytosis appeared to be acetaminophen. The histologic picture of a biopsied lymph node showed diffuse infiltration of polymorphous lymphoid cells with clear cytoplasm and proliferation of arborizing capillary vessels. Based on the histologic findings, PCR, and immunohistologic analyses, he was diagnosed with angioimmunoblastic T cell lymphoma (AILT) in leukemic state. The response of the lymphoma to conventional chemotherapy (CHOP and ESHAP) was poor. We next performed an immunomodulatory therapy using cyclosporine A to suppress cytokine production by neoplastic T cells. The treatment resulted in a partial remission of AILT including disappearance of circulating lymphoma cells. To our knowledge, this is the first published report of AILT complicated by drug-induced agranulocytosis.

Systemic lupus erythematosus complicated by cytomegalovirus-induced hemophagocytic syndrome and pneumonia.

A 58-year-old female with systemic lupus erythematosus (SLE) and anti-phospholipid syndrome (APS) was referred and admitted to our hospital because of fever and pancytopenia. She had been taking small dose of prednisolone and azathiprine since 2003 with stable SLE and APS. Two weeks before the admission, she developed fever and common cold-like symptoms and was admitted to a hospital. Antibiotics were ineffective and thrombocytopenia manifested. Under a diagnosis of progressive SLE, bolus methylprednisolone was administrated. However, she became pancytopenic and was transferred to our hospital. A bone marrow aspirate showed the hemophagocytosis and chest CT scanning revealed interstitial pneumonia. Cytomegalovirus (CMV) antigenemia test gave a positive result. A diagnosis of CMV-induced hemophagocytic syndrome (HPS) and CMV pneumonia was made. Gancyclovir resolved the pancytopenia, pneumonia, and fever. There have been only 3 reported cases of CMV-related HPS in collagen disease.

[Successful treatment with azathioprine for autoimmune thrombocytopenia developing after autologous peripheral blood stem cell transplantation].

A 60-year-old man with acute promyelocytic leukemia in complete remission had minimal residual disease (MRD). After two courses of arsenic trioxide treatment, the MRD disappeared. In October 2005, he received an unmanipulated autologous peripheral blood stem cell transplantation (autoPBSCT). Hematologic recovery was prompt; however, after day 21 following the autoPBSCT, platelet counts decreased to below 10 x 10(9)/l. A bone marrow aspirate showed an increased number of immature megakaryocytes, and platelet-associated IgG was elevated to 48.5 ng/10(7) platelets. A diagnosis of autoimmune thrombocytopenia was made. The combination of oral prednisolone (40 mg/day) and bolus immunoglobulin infusion (400 mg/kg, for 5 consecutive days) was ineffective. He was given azathioprine (50 mg/day, orally), and 10 days after the initiation of the treatment, the platelet counts gradually increased and recovered to over 50 x 10(9)/l on day 168, and 100 x 10(9)/l on day 364. To the best of our knowledge, successful treatment of ITP following auto PBSCT with azathioprine has not been previously reported.

Chronic eosinophilic leukemia with the FIP1L1-PDGFRalpha fusion gene in a patient with a history of combination chemotherapy.

Hypereosinophilic syndrome (HES) was diagnosed in December 2000 in a 43-year-old man on the basis of persistent eosinophilia (11.7 x 10(9)/L) and a normal karyotype of the bone marrow cells. He had developed intra-abdominal non-Hodgkin's lymphoma and in 1992 had received 3 courses of combination chemotherapy with doxorubicin (Adriamycin), cyclophosphamide, vincristine, methotrexate, bleomycin, and prednisolone. The patient was orally given prednisolone (10 mg/day) and cyclophosphamide (50 mg/day) as HES treatment without a subsequent improvement of the eosinophilia. In May 2003, anemia (hemoglobin, 7.9 g/dL) and thrombocytopenia (65 x 10(9)/L) manifested with progressive eosinophilia (21.0 x 10(9)/L) and a small number of blasts. The patient became febrile and was admitted in July 2003. Cytogenetic reexamination of the bone marrow cells disclosed the deletion of 4q12, indicating the presence of a fusion of the Fip1-like 1 (FIP1L1) gene to the plateletderived growth factor receptor alpha (PDGFRalpha) gene and consequently the clonal nature of his hematopoietic cells. DNA sequence analysis demonstrated that the breakpoints of the FIP1L1 and PDGFRalpha genes were present in exon 9 and exon 12, respectively. Treatment with imatinib mesylate (300 mg/day) promptly brought about complete remission. Although a number of similar eosinophilic cases have been reported, our patient may be the first such patient with a history of chemotherapy.

Multiple granulocytic sarcomas in essential thrombocythemia.

A 59-year-old woman was diagnosed with essential thrombocythemia in 1988 and had been treated with hydroxyurea, mitobronitol, busulfan, and ranimustine, in that order. Hepatosplenomegaly, low-grade fever, and body weight loss manifested, and a few blasts were noted in the peripheral blood studied in March 2002. A biopsied specimen of the bone marrow showed myelofibrosis but not a leukemia in August 2004. An abnormal karyotype with der(1; 13) appeared for the first time. She was treated with low-dose prednisolone. In January 2005, she experienced left hip joint pain, and magnetic resonance scanning showed a tumoral lesion in the femoral head. Histological diagnosis of the biopsied mass revealed that it was a granulocytic sarcoma, and radiotherapy was performed. In April 2005, bone scintigraphy showed multiple lesions. She became febrile and red blood cell transfusion-dependent with hepatosplenomegaly and a small number of circulating blasts. Intravenous cytarabine (low dose) and etoposide relieved the fever and hepatosplenomegaly; however, she developed a pathologic fracture of the right humerus. An additional karyotypic abnormality (7q22 deletion) was noted. She subsequently died of infection. Granulocytic sarcoma is very rare in essential thrombocythemia, and this patient may be the first reported case of essential thrombocythemia that developed multiple lesions and a pathologic fracture without transformation to overt leukemia.

Characterization of t(3;6)(q27;p21) breakpoints in B-cell non-Hodgkin's lymphoma and construction of the histone H4/BCL6 fusion gene, leading to altered expression of Bcl-6.

A recurrent translocation, t(3;6)(q27;p21), in non-Hodgkin's lymphoma results in fusion of BCL6 with a particular histone H4 gene on 6p21. We cloned five H4/BCL6 junctions from both der(3) and der(6) chromosomes. The breakpoints on H4 were distributed within the single exon or close to the terminal palindrome, and those on BCL6 were localized within or close to the translocation hypercluster. Deletions or duplications of variable numbers of nucleotides were identified at the junctions. A total of eight single nucleotide alterations were introduced into the translocation/mutation cluster of BCL6, whereas four single nucleotide substitutions were identified within a 360-bp region of H4. Thus, the somatic hypermutation mechanism is likely to target H4, resulting in a predisposition to the development of translocation with BCL6. Lymphoma cells carrying H4/BCL6 produced fusion transcripts containing both H4 and BCL6 messages; however, the cells expressed only moderate levels of BCL6 mRNA. We constructed expression plasmids that mimicked the H4/BCL6 fusion gene and transiently introduced them into COS-7 cells. H4/BCL6-transfected cells expressed markedly higher levels of Bcl-6 protein than cells transfected with a plasmid carrying BCL6 driven by its normal promoter and displayed bright nuclear staining with a characteristic punctate pattern with an anti-Bcl-6 antibody. Deletion analyses revealed that the high-level Bcl-6 expression was promoted by the H4 regulatory sequences. The levels of expression of activating transcription factor 3, prefoldin 4, and retinoblastoma-binding protein 7 significantly increased in accordance with that of BCL6, suggesting that Bcl-6 may act as a transcriptional activator. Our study suggested that t(3;6)(q27;p21) leads to BCL6 overexpression; however, the high-level BCL6 expression may not be required to maintain the malignant phenotype of lymphoma cells.

The gene for interleukin-21 receptor is the partner of BCL6 in t(3;16)(q27;p11), which is recurrently observed in diffuse large B-cell lymphoma.

BCL6 translocation affecting the chromosomal band 3q27 can involve a number of non-immunoglobulin (non-IG) gene loci as partners. We report here that the gene for interleukin-21 receptor (IL-21R) is the partner of BCL6 in t(3;16)(q27;p11) translocation. The two breakpoints on 16p11 of a lymphoma cell line YM and case no. 1012 with diffuse large B-cell lymphoma, both of which carried t(3;16), were localized within the 27-kb intron 1 of IL-21R. As a result of t(3;16), the promoter region of IL-21R was substituted for the regulatory sequences of BCL6 in the same transcriptional orientation. Reverse transcriptase-mediated polymerase chain reaction revealed chimeric mRNA consisting of two non-coding exons 1a/1b of IL-21R and coding exons of BCL6 in both lymphoma cells. Fluorescence in situ chromosomal hybridization of YM metaphase cells revealed fusion signals that contained both the BCL6 and IL-21R sequences on the der(3)t(3;16) chromosome. IL-21R was actively transcribed in YM cells, while BCL6 that was under the control of the IL-21R promoter was only moderately expressed at the mRNA and protein level. We constructed expression plasmid of BCL6 that followed the promoter sequences of IL-21R. COS-7 cells transiently transfected with the plasmid expressed high level Bcl-6 protein and displayed nuclear staining with a characteristic punctate pattern by immunofluorescence, indicating that expression of BCL6 can be enhanced by t(3;16). This study added to the list of non-IG partners of BCL6 translocations a new class of gene, i.e. cytokine receptor gene, the expression of which is closely associated with lymphoid cells.

[Renal insufficiency caused by leukemic cell infiltration in Sézary syndrome].

A 77-year-old woman was admitted to our hospital because of pneumonia and heart failure in July 2002. She had been diagnosed as having with Sézary syndrome in 1993, and had been treated with a combination of prednisolone, methotrexate, and cyclosporin A with subsequent stable disease but persistent generalized erythroderma. On admission, the white blood count was 23.9 X 10(9)/L with 28% Sézary cells, and serum creatinine levels were within normal limits. One month after admission, the pneumonia and heart failure improved remarkably with antibiotics and diuretics. However, at the same time, her renal function deteriorated with increasingly high serum creatinine levels. She died of anuria in September, 2002. An autopsy showed marked perivascular and peritubular infiltration of abnormal lymphocytes with degenerative nephrotubuli in the kidneys. This patient may be the first reported case of Sézary syndrome with renal failure caused by leukemic infiltration.

Sugar beet (Beta vulgaris L.).

Creating transgenic plants is invaluable for the genetic analysis of sugar beet and will be increasingly important as sugar beet genomic technologies progress. A protocol for Agrobacterium-mediated transformation of sugar beet is described in this chapter. Our protocol is optimized for a sugar beet genotype that performs exceptionally well in tissue culture, including the steps of dedifferentiation, callus proliferation, and regeneration. Because of the infrequent occurrence of such a genotype in sugar beet populations, our protocol includes an in vitro propagation method for germplasm preservation. The starting materials for transgenic experiments are aseptic shoots grown from surface-sterilized seed balls. Callus is induced from leaf explants and subsequently infected with Agrobacterium. Plantlets are regenerated from transgenic callus and vernalized for flowering, if necessary. The efficiency of transformation was quite high; in our laboratory, the culture of only ten leaf explants, on average, generated one transgenic plant.

[Severe hyponatremia with consciousness disturbance caused by hydroxyurea in a patient with chronic myeloid leukemia].

A 79-year-old man was admitted because of consciousness disturbance on August 9, 2002. He had been diagnosed as having chronic myeloid leukemia in 1999, and since then, he had continued to take hydroxyurea (1500 mg/day) orally. On admission, his serum sodium concentration was as low as 119 mEq/L, while urinary sodium excretion was high. Based on the blood picture and lack of hepatosplenomegaly, we considered that the leukemia was still in the chronic phase. Because of normal blood level of the antidiuretic hormone (ADH) concentration and sufficient urine volume, the syndrome of inappropriate ADH secretion (SIADH) was unlikely, and sodium-losing nephropathy was suspected. After discontinuation of hydroxyurea, the urinary sodium excretion decreased and the patient's consciousness became clear concomitantly with improvement in the serum Na level. This patient appears to be the first case of hyponatremia caused by hydroxyurea.

[Pleural involvement in the course of chronic myelomonocytic leukemia and the development of multiple colonic perforation due to leukemic infiltration in the acute leukemia phase].

A 68-year-old man with chronic myelomonocytic leukemia (CMML) was initially treated with hydroxyurea with subsequent stable disease. In the time course, he developed bilateral pleuritis accompanied by leukocytosis and spiking fever. Cytologic analysis of the pleural effusion revealed abundant mature granulocytes and monocytes. He was treated with intravenous or oral etoposide with consequent resolution of the pleuritis, indicating the pleural involvement of CMML. Three months later, he developed hepatomegaly and became febrile. One month thereafter, the CMML transformed to acute myeloid leukemia, and the patient developed massive bloody stools associated with epigastric pain and leukocytosis. A gastrofiberscopic examination showed multiple bleeding gastric ulcers. The bleeding ulcers were treated with the clipping procedure; however, the bloody stools continued. Although intravenous etoposide was effective for the leukocytosis and hepatomegaly, the treatment did not improve the bloody stools. The patient finally died of panperitonitis. The autopsy showed multiple ulcers of the transverse colon, some of which were perforated. Microscopically, the ulcerated areas were densely infiltrated with leukemic cells predominantly consisting of immature monocytes and granulocytes. This patient may be the first reported case of CMML complicated by colonic perforation due to leukemic infiltration.

Efficacy and tolerability of reduced-dose 21-day cycle rituximab and cyclophosphamide, doxorubicin, vincristine and prednisolone therapy for elderly patients with diffuse large B-cell lymphoma.

Rituximab combined with cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) is regarded as the first-line treatment for elderly patients with diffuse large B-cell lymphoma (DLBCL), but it is often necessary to reduce the dose or prolong the intervals between doses. In our center, dose reduction is performed with elderly patients with DLBCL on an individual basis: for patients in their 70s and 80s, the initial CHOP dose is reduced to 70% and 50%, respectively, and the subsequent doses are adjusted so that the patients receive, at 21-day intervals, the highest dose they can tolerate (reduced-dose R-CHOP21). To verify this practice, a retrospective analysis was performed. Between January 2004 and January 2011, 109 ≥ 70-year-old patients with DLBCL received reduced-dose R-CHOP21 with curative intent. The 2-year overall survival rates of the 70-79- and ≥ 80-year-old patients were 75.2% and 64.6%, respectively. Of 35 deaths, 20 were due to disease progression and five were related to treatment toxicity. Multivariate analysis revealed that an age of 75-79 years and an age of 80 years or older were associated with shorter survival. Given that many patients had poor performance status and comorbidities, reduced-dose R-CHOP21 may provide a reasonable balance between efficacy and tolerability for elderly patients with DLBCL.

Impact of occult bone marrow involvement on the outcome of rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone therapy for diffuse large B-cell lymphoma.

We assessed the prognostic impact of occult bone marrow involvement, determined by flow cytometry and/or polymerase chain reaction, in a population of 117 consecutive patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL) treated with rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). Twenty-four (20.5%) had morphologically diagnosed and 16 (13.7%) had occult marrow involvement, and 77 (65.8%) had no marrow involvement. Although the pretreatment characteristics of the negative or occult marrow involvement group were similar, severe hematological toxicity after R-CHOP was more frequent in the occult marrow involvement group. Progression-free survival (PFS; p = 0.015) and overall survival (OS; p = 0.035) for the occult marrow involvement group were significantly shorter than those for the negative group, and were comparable to those of the morphologic marrow involvement group, independent of the International Prognostic Index score for PFS. Occult bone marrow involvement predicts severe hematological toxicity and negatively impacts on the PFS and OS of R-CHOP therapy.

Unusual and typical features of a novel restorer-of-fertility gene of sugar beet (Beta vulgaris L.).

Male gametogenesis in plants can be impaired by an incompatibility between nuclear and mitochondrial genomes, termed cytoplasmic male sterility (CMS). A sterilizing factor resides in mitochondria, whereas a nuclear factor, Restorer-of-fertility (Rf), restores male fertility. Although a majority of plant Rf genes are thought to encode a family of RNA-binding proteins called pentatrico-peptide repeat (PPR) proteins, we isolated a novel type of Rf from sugar beet. Two BACs and one cosmid clone that constituted a 383-kbp contig covering the sugar beet Rf1 locus were sequenced. Of 41 genes borne by the contig, quadruplicated genes were found to be associated with specific transcripts in Rf1 flower buds. The quadruplicated genes encoded a protein resembling OMA1, a protein known from yeast and mammals to be involved in mitochondrial protein quality control. Construction of transgenic plants revealed that one of the four genes (bvORF20) was capable of restoring partial pollen fertility to CMS sugar beet; the level of restoration was comparable to that evaluated by a crossing experiment. However, the other genes lacked such a capability. A GFP-fusion experiment showed that bvORF20 encoded a mitochondrial protein. The corresponding gene was cloned from rf1rf1 sugar beet and sequenced, and a solitary gene that was similar but not identical to bvORF20 was found. Genetic features exhibited by sugar beet Rf1, such as gene clustering and copy-number variation between Rf1 and rf, were reminiscent of PPR-type Rf, suggesting that a common evolutionary mechanism(s) operates on plant Rfs irrespective of the translation product.