Abnormal behaviors and developmental disorder of hippocampus in zinc finger protein 521 (ZFP521) mutant mice.

Zinc finger protein 521 (ZFP521) regulates a number of cellular processes in a wide range of tissues, such as osteoblast formation and adipose commitment and differentiation. In the field of neurobiology, it is reported to be an essential factor for transition of epiblast stem cells into neural progenitors in vitro. However, the role of ZFP521 in the brain in vivo still remains elusive. To elucidate the role of ZFP521 in the mouse brain, we generated mice lacking exon 4 of the ZFP521 gene. The birth ratio of our ZFP521Δ/Δ mice was consistent with Mendel's laws. Although ZFP521Δ/Δ pups had no apparent defect in the body and were indistinguishable from ZFP521+/+ and ZFP521+/Δ littermates at the time of birth, ZFP521Δ/Δ mice displayed significant weight reduction as they grew, and most of them died before 10 weeks of age. They displayed abnormal behavior, such as hyper-locomotion, lower anxiety and impaired learning, which correspond to the symptoms of schizophrenia. The border of the granular cell layer of the dentate gyrus in the hippocampus of the mice was indistinct and granular neurons were reduced in number. Furthermore, Sox1-positive neural progenitor cells in the dentate gyrus and cerebellum were significantly reduced in number. Taken together, these findings indicate that ZFP521 directly or indirectly affects the formation of the neuronal cell layers of the dentate gyrus in the hippocampus, and thus ZFP521Δ/Δ mice displayed schizophrenia-relevant symptoms. ZFP521Δ/Δ mice may be a useful research tool as an animal model of schizophrenia.

A new four-way variant t(5;17;15;20)(q33;q12;q22;q11.2) in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by t(15;17)(q22;q21), which results in the fusion of the promyelocytic leukemia (PML) gene at 15q22 with the retinoic acid alpha-receptor (RARA) at 17q21. We report a patient with APL carrying a new complex variant translocation (5;17;15;20). Spectral karyotyping analysis of bone marrow cells revealed t(5;17;15;20)(q33;q12;q22;q11.2). Fluorescence in situ hybridization with a PML/RARA dual-color DNA probe showed a single fusion signal, and RT-PCR analysis showed PML/RARA fusion transcripts. Complete remission was attained with a course of conventional chemotherapy with all-trans retinoic acid (ATRA). To our knowledge, this is the first report of a four-way translocation of 5q33 and 20q11 involvement in APL.

The role of zinc finger protein 521/early hematopoietic zinc finger protein in erythroid cell differentiation.

ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in erythroid cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL cell lines as compared with control cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates erythroid cell differentiation through direct binding with GATA-1.

Cooperative role of the membrane-proximal and -distal residues of the integrin beta3 cytoplasmic domain in regulation of talin-mediated alpha IIb beta3 activation.

Integrin cytoplasmic tails regulate integrin activation that is required for high affinity binding with ligands. The interaction of the integrin beta subunit tail with a cytoplasmic protein, talin, largely contributes to integrin activation. Here we report the cooperative interaction of the beta3 membrane-proximal and -distal residues in regulation of talin-mediated alpha IIb beta3 activation. Because a chimeric integrin, alpha IIb beta3/beta1, in which the beta3 tail was replaced with the beta1 tail was constitutively active, we searched for the residues responsible for integrin activation among the residues that differed between the beta3 and beta1 tails. Single amino acid substitutions of Ile-719 and Glu-749 in the beta3 membrane-proximal and -distal regions, respectively, with the corresponding beta1 residues or alanine rendered alphaIIbbeta3 constitutively active. The I719M/E749S double mutant had the same ligand binding activity as alpha IIb beta3/beta1. These beta3 mutations also induced alphaVbeta3 activation. Conversely, substitution of Met-719 or Ser-749 in the beta1 tail with the corresponding beta3 tail residue (M719I or S749E) inhibited alpha IIb beta3/beta1 activation, and the M719I/S749E double mutant inhibited ligand binding to a level comparable with that of the wild-type alpha IIb beta3. Knock down of talin by short hairpin RNA inhibited the I719M- and E749S-induced alpha IIb beta3 activation. These results suggest that the beta3 membrane-proximal and -distal residues cooperatively regulate talin-mediated alpha IIb beta3 activation.

Absence of chlamydial infection in Japanese patients with ocular adnexal lymphoma of mucosa-associated lymphoid tissue.

Ocular adnexal extranodal marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (ocular adnexal MALT lymphoma) are predominately low-grade, small B-cell types and may be caused by several putative inflammatory agents. Twenty-three ocular adnexal MALT lymphoma cases, the monoclonality of which was confirmed by examination of immunoglobulin heavy chain gene rearrangement and/or cell surface antigens, were analyzed for evidence of several causative factors. A serologic evaluation of our series of patients showed no evidence of infection by Epstein-Barr virus, hepatitis C virus, or Chlamydophila psittaci. Two patients tested positive for serum antibodies for autoimmunity, and another 2 patients tested positive for antibodies against Chlamydia trachomatis. Polymerase chain reaction analysis did not reveal the presence of the chlamydial 16S ribosomal RNA (rRNA) gene or the 16S-23S spacer rRNA gene. These results indicate that the inflammatory agents in our series of ocular adnexal MALT lymphomas are still unknown and that some types of chlamydial infections are not associated with orbital adnexal MALT lymphoma in southern regions of Japan.

Estimation of the initial dose setting of vancomycin therapy with use of cystatin C as a new marker of renal function.

In recent years, it has been suggested that the glomerular filtration rate (GFR) can be predicted on the basis of serum cystatin C concentrations and that this measurement is more sensitive than serum creatinine concentration as a marker of renal function. In this study, to investigate the clinical utility of the initial dose setting of vancomycin by the population mean method with use of serum cystatin C as a marker of renal function, we compared the correlations between measured vancomycin concentrations and predicted vancomycin concentrations based on serum cystatin C or serum creatinine concentrations in elderly (>/=65 years old) and nonelderly (<65 years old) patients. An analysis of prediction accuracy (bias) and precision was evaluated by calculating the mean prediction error (ME), the mean absolute error (MAE), and the root mean squared prediction error (RMSE). For nonelderly patients (n = 50), there was no significant difference in the MAE based on the use of serum creatinine or serum cystatin C concentration. However, for elderly patients (n = 105), the MAE based on serum cystatin C concentration was significantly better than that based on serum creatinine level. These results suggest that serum cystatin C is a good marker of renal function in comparison with serum creatinine for dose setting of vancomycin, especially in an elderly population.

Ubp43 regulates BCR-ABL leukemogenesis via the type 1 interferon receptor signaling.

Interferon (IFN) signaling induces the expression of interferon-responsive genes and leads to the activation of pathways that are involved in the innate immune response. Ubp43 is an ISG15-specific isopeptidase, the expression of which is activated by IFN. Ubp43 knock-out mice are hypersensitive to IFN-alpha/beta and have enhanced resistance to lethal viral and bacterial infections. Here we show that in addition to protection against foreign pathogens, Ubp43 deficiency increases the resistance to oncogenic transformation by BCR-ABL. BCR-ABL viral transduction/transplantation of wild-type bone marrow cells results in the rapid development of a chronic myeloid leukemia (CML)-like myeloproliferative disease; in contrast, a significantly increased latency of disease development is observed following BCR-ABL viral transduction/transplantation of Ubp43-deficient bone marrow cells. This resistance to leukemic development is dependent on type 1 IFN (IFN-alpha/beta) signaling in Ubp43-deficient cells. Increased levels of type 1 IFN are also detected in the serum of CML mice. These results suggest that inhibition of Ubp43-negative effect on IFN signaling can potentiate the response to increased endogenous IFN levels in innate immune responses against cancer development, indicating that pharmacological inhibition of Ubp43 may be of benefit in cancers and others diseases in which interferon is currently prescribed.

[Esophageal stricture following complete remission after chemotherapy for malignant lymphoma in an elderly patient].

A 75-year-old woman was given a diagnosis of malignant lymphoma (non-Hodgkin, diffuse large B cell type, stage IIA) at our hospital on August 2003. She received six courses of rituximab-based chemotherapy (R-CHOP regimen) and then she achieved complete remission. On August 16, 2004, she was readmitted in our hospital for difficulty in swallowing. Upper gastrointestinal endoscopy reveled esophageal stricture and an ulcerative lesion on the esophageal mucosa. The X-ray examination of the upper gastrointestinal tract reveled a severe esophageal stricture with niches and hiatus hernia. No malignancy was seen on CT scanning, gallium radioisotope scanning and histological examination of biopsy specimens with the upper gastrointestinal endoscopy. The physical examination showed gibbosity, and MR imaging showed multiple compression spined fractures. Finally, we diagnosed benign esophageal stricture with reflux esophagitis. She underwent laparoscopic partial esophagectomy in September 21, 2004, and the postoperative course was satisfactory. The pathological findings showed benign esophageal stricture caused by esophagitis. We report here a case of esophageal stricture following complete remission after chemotherapy for malignant lymphoma in an elderly patient.

Human herpesvirus 6 impairs differentiation of monocytes to dendritic cells.

OBJECTIVE: Monocyte-derived dendritic cells (DCs) play important roles in the immune response against infections and malignancies. Human herpesvirus 6 (HHV-6) infects monocytes and is reactivated in immunodeficient patients. To clarify the mechanisms of HHV-6-induced immunodeficiency, we investigated the effect of HHV-6 infection on differentiation of monocytes to DCs. METHODS: Monocytes were inoculated with or without HHV-6 and then allowed to differentiate to myeloid DCs in culture medium containing granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. The expression of cell surface molecules on DCs and the capacity of the DCs for antigen capture were examined by flow cytometric analysis. Alteration of antigen-presenting capacity induced by HHV-6 infection was examined. RESULTS: The morphology of HHV-6-infected monocyte-derived DCs was distinctly different from that of the DCs derived from mock-infected monocytes. Although expression levels of DC-associated surface antigens, including CD80, CD83, and CD86, were significantly higher on HHV-6-infected monocyte-derived DCs than on DCs derived from mock-infected monocytes, antigen-presenting capacity was significantly lower in the former group. Addition of culture supernatant of HHV-6-infected monocytes resulted in suppression of the T-lymphocyte proliferative response, and anti-IL-10 neutralizing antibody partly inhibited this suppressive effect. The antigen-presenting capacity of DCs generated from a patient with severe HHV-6 reactivation was significantly lower than that of DCs generated from the same patient in the recovery phase. CONCLUSIONS: HHV-6 infection induces immunodeficiency via impaired differentiation of DCs. These results present a new concept for the pathogenesis of HHV-6-induced immunodeficiency.

Leukemoid reaction in association with bone marrow necrosis due to metastatic prostate cancer.

An 80-year-old man presented to the internist with fever, fatigue and leukocytosis up to 66.8 x 10(3)/microl. Although a chronic myelogenous leukemia was initially suspected, he was diagnosed as metastatic bone marrow tumor with bone marrow necrosis from primary prostate cancer on the basis of the clinical and pathological findings. The serum concentrations of IL-6 and TNF-alpha were mildly elevated to 65.0 pg/ml and, 54.0 pg/ml respectively. It is probable that these humoral factors were partially responsible for the leukemoid reaction although other factors induced by the bone marrow necrosis with bone marrow metastasis of prostate cancer are also likely involved.

Secondary amyloidosis and eosinophilia in a patient with uterine leiomyosarcoma.

We report a rare case demonstrating the relationship between secondary amyloidosis and uterine leiomyosarcoma. A 59-year-old female with high fever was referred to our hospital. Laboratory tests revealed increased white blood cells, eosinophilia and an accelerated erythrocyte sedimentation rate. Endoscopic examination of the stomach and colon revealed amyloid deposits in their mucosa. The patient showed no symptoms that suggested amyloidosis. No other organs or tissues were surveyed for amyloid deposition. Ga scintigraphy, computed tomography and magnetic resonance imaging suggested necrotic infectious leiomyoma of the uterus, which was considered to be the cause of the fever. The patient underwent total hysterectomy. The histological diagnosis of the mass revealed a low-grade uterine leiomyosarcoma with necrosis. Amyloid deposits in the gastric mucosa disappeared 1 year after the operation. In this case, amyloid deposition was detected by endoscopic biopsy before clinical manifestations. The deposition was reversible and was successfully treated. Thus, it is logical and useful to undertake endoscopic mucosa biopsy to check for amyloid deposition in patients with systemic inflammation, whose serum amyloid A protein level has been high for several months. In addition, peripheral eosinophilia was also detected in this case. Although eosinophilia associated with malignant tumor has been recognized, it is an uncommon occurrence.

Novel RUNX1-PRDM16 fusion transcripts in a patient with acute myeloid leukemia showing t(1;21)(p36;q22).

The t(1;21)(p36;q22) is a recurrent chromosome abnormality associated with therapy-related acute myeloid leukemia (AML). Although involvement of RUNX1 has been detected by fluorescence in situ hybridization analysis, the partner gene has not been reported previously. We identified a novel RUNX1 partner gene, MDS1/EVI1-like-gene 1 (PRDM16), in an AML patient with t(1;21). Alternative splicing of the fusion gene generates five different fusion transcripts. In two of them, the PRDM16 reading frame is maintained in the fusion with RUNX1, suggesting that the RUNX1-PRDM16 gene fusion results in the production of a protein that is highly homologous to the RUNX1-MDS1/EVI1 chimeric protein. It is suggested that PRDM16 and MDS1/EVI1 share a common molecular mechanism for the leukemogenesis of RUNX1-associated leukemia. Characterization of the RUNX1-PRDM16 fusion protein and comparison with the RUNX1-MDS1/EVI1 protein will facilitate the understanding of the mechanisms underlying RUNX1-associated leukemia.

Differential regulation of perforin expression in human CD4+ and CD8+ cytotoxic T lymphocytes.

OBJECTIVE: Because perforin is an essential cytolytic mediator of cytotoxic T lymphocytes (CTLs), it is important to understand the regulatory mechanisms of perforin expression in CTLs. In the present study, we investigated the relationship between cytotoxic activity, perforin expression, and cell-activated status of CD4(+) and CD8(+) CTLs. METHODS: Herpes simplex virus-specific CD4(+) CTL clones and Epstein-Barr virus-specific CD8(+) CTL clones were established, and their cytotoxic activities were examined in both the activated and resting phases. Perforin mRNA expression was examined by reverse transcriptase polymerase chain reaction quantitatively. Transcriptional regulation of perforin was examined by electrophoretic mobility shift assay. RESULTS: The degrees of cytotoxic activity of CD8(+) CTLs did not differ significantly between the two phases; however, CD4(+) CTLs in the activated phase appeared to be significantly more cytotoxic than those in the resting phase. Similarly, expression levels of perforin mRNA in activated and resting CD8(+) CTLs did not differ significantly, but activated CD4(+) CTLs appeared to express perforin more abundantly than resting CD4(+) CTLs. In addition, it appeared that binding of STAT5 to the perforin gene promoter was increased in activated CD4(+) CTLs compared to resting CD4(+) CTLs; however, there was no significant detectable difference of STAT5 binding activity to the perforin gene promoter between activated and resting CD8(+) CTLs. CONCLUSIONS: The present study has revealed a difference in the control of perforin expression between CD4(+) and CD8(+) CTLs; that is, perforin is expressed constitutively in memory CD8(+) CTLs, but is dependent on cell activation in memory CD4(+) CTLs.

Direct recognition and lysis of leukemia cells by WT1-specific CD4+ T lymphocytes in an HLA class II-restricted manner.

Wilms tumor gene 1 product (WT1) has been recognized as an attractive target antigen of immunotherapy for various malignancies including leukemia. Because tumor-associated antigen-specific CD4+ T lymphocytes undoubtedly play an important role in the induction of an antitumor immune response, we attempted to generate WT1-specific CD4+ T lymphocytes in vitro and examined their antileukemia functions. A CD4+ T-cell line, designated NIK-1, which proliferated and produced Th1 cytokines specifically in response to stimulation with the WT1-derived peptide, WT1(337-347) LSHLQMHSRKH, in an HLA-DP5-restricted manner was established. NIK-1 exhibited cytotoxicity against HLA-DP5-positive, WT1-expressing leukemia cells but did not lyse HLA-DP5-negative, WT1-expressing leukemia cells or HLA-DP5-positive, WT1-negative cells. NIK-1 did not inhibit colony formation by normal bone marrow cells of HLA-DP5-positive individuals. This is the first report to describe WT1-specific and HLA class II-restricted CD4+ T lymphocytes possessing direct cytotoxic activity against leukemia cells.

Expression of SOCS3 mRNA in bone marrow cells from CML patients associated with cytogenetic response to IFN-alpha.

Previously, we have demonstrated that constitutive expression of suppressor of cytokine signaling-3 (SOCS3) affects the sensitivity of chronic myelogenous leukemia (CML) cell lines to interferon-alpha (IFN-alpha). In the present study, we analyzed the expression of SOCS3 mRNA in bone marrow cells from patients with CML at diagnosis, with the aid of real-time polymerase chain reaction. SOCS3 mRNA expression in bone marrow cells from CML patients who responded well to IFN-alpha therapy was significantly lower than that in cells from healthy volunteers and patients who were resistant to IFN-alpha therapy. Methylation of SOCS3 promoter was absent in bone marrow cells from all CML patients examined. These results indicate that the expression of SOCS3 mRNA is inversely associated with the sensitivity to IFN-alpha both in vitro and in vivo and that differences in SOCS3 mRNA expression are not due to the methylation status of SOCS3 promoters.

Rupture of the gallbladder in a patient with acquired factor VIII inhibitors and systemic lupus erythematosus.

A 54-year-old woman with a 21-year history of systemic lupus erythematosus (SLE) was admitted to the Matsuyama Red Cross Hospital due to subcutaneous and gingival hemorrhaging. She was diagnosed with acquired factor VIII inhibitors based on a prolonged activated partial-thromboplastin time (APTT) and factor VIII inhibitors. Steroid pulse and factor VIII plasma concentrate were administered to her, not long after which she was transferred to Ehime University Hospital due to gallbladder hematoma. Although her APTT and factor VIII activity were improved after treatment with human factor VIII, she died of multiple organ failure. The autopsy demonstrated a ruptured gallbladder.

Existence of leukemic clones resistant to both imatinib mesylate and rituximab before drug therapies in a patient with Philadelphia chromosome-positive acute lymphocytic leukemia.

Imatinib mesylate and rituximab are molecularly targeted drugs against the BCR-ABL fusion protein and the CD20 antigen, respectively. Although these drugs have excellent anticancer effects, a major concern is drug resistance. We have investigated the case of a patient with Philadelphia chromosome-positive and CD20+ acute lymphocytic leukemia who acquired resistance to imatinib and rituximab. Imatinib therapy resulted in prompt cytogenetic remission, but resistance developed shortly thereafter. Sequencing of the kinase domain of the ABL gene and allele-specific polymerase chain reaction analysis revealed a point mutation resulting in an E255V substitution that was present before the therapy. After the patient received mild chemotherapy followed by rituximab administration, hematologic and cytogenetic remission was sustained for 5.5 months. The recurrent leukemic cells after the rituximab therapy showed not only the E255V mutation in the ABL gene but also loss of the CD20 antigen due to impaired transcription of the CD20 gene. The results of 2-color flow cytometry analysis showed that a small population of CD20(-) leukemic cells existed before the imatinib therapy. These results suggest that leukemic subclones carrying a genetic perturbation of the targeted molecules for both imatinib and rituximab were present before the therapies. The preexistence of primary resistant clones suggests the inability of combination therapy with 2 molecularly targeted drugs to overcome drug resistance in leukemia.

[Double B-cell malignancies with simultaneous onset].

We encountered a case of a 59-year-old female who simultaneously contracted a non-Hodgkin lymphoma (NHL) and a plasma cell neoplasm. The patient consulted her physician about her abdominal tumor and anemia in March 1999. She was diagnosed as having NHL (follicular center lymphoma, grade I, stage IIA) after an open tumor biopsy, and treated by cycles of CHOP chemotherapy which resulted in complete remission. However, the patient's abdominal tumor appeared again in March 2000 and she was hospitalized at the Ehime University Hospital. A tumor biopsy was performed laparoscopically at that time. Follicular lymphoma (with positive LCA, L-26, and bcl-2 immuno-staining) with the development of retroperitoneal fibrosis was diagnosed again. When a bone marrow puncture was performed because of a condition of monoclonal gammopathy which had continued for two years, a smoldering myeloma was additionally diagnosed. This diagnosis was made after the presence of IgG-lambda M protein when the marrow showed an increase in the number of plasma cells. In a Southern blot analysis which studied the abdominal tumor and the bone marrow cells, each B-cell tumor had a different IgH gene rearrangement pattern. Therefore, this case was diagnosed as an example of the simultaneous existence of two different B-cell tumors. Double cancers in hematological malignancies are very rare and this was thought to be an interesting case.

Hypocellular acute promyelocytic leukemia with a tetraploid clone characterized by two t(15;17).

We describe a case of hypocellular acute promyelocytic leukemia with a tetraploid clone characterized by two t(15;17). The large leukemia cells had a bizarre nuclear configuration and multiple Auer rods. A bone marrow biopsy specimen revealed a markedly hypocellular marrow (<10% cellularity) in the absence of myelofibrosis. Myelodysplastic features were not detected. Chromosome analysis of marrow cells revealed a karyotype of 92,XXYY,del(2)(q?),t(15;17)(q22;q21)x2. Interphase fluorescence in situ hybridization revealed that the marrow cells were composed of a tetraploid clone carrying double t(15;17) and normal diploid cells. The leukemia responded well to all-trans retinoic acid. We think that the tetraploidy could be caused by endoreduplication or endomitosis of the diploid clone with single t(15;17). The unique karyotype largely contributed to the cell morphology and marrow hypoplasia, while it may not have affected on the prognosis of the acute promyelocytic leukemia.