Regulation of BCL-6 gene expression in human myeloid/monocytoid leukemic cells.

We demonstrated in the present study that the BCL-6 transcripts were detectable not only in B cells, but also in circulating granulocytes and monocytes from normal individuals, and in human acute nonlymphocytic leukemia cells of certain subtypes (M3, M4, M5). Then, with an assumption that the BCL-6 gene expression may be related to the differentiation of myeloid cells, we analyzed the inducibility of BCL-6 gene expression along monocytic lineage differentiation in HL-60 and U-937 cells by treating them with 12-O-tetradecanoylphorbol-13-acetate (TPA). Although the expression of BCL-6 transcripts was very low or undetectable in untreated HL-60 or U-937 cells, treatment of these cells with TPA to induce monocytic differentiation resulted in an apparent increase of BCL-6 mRNA, suggesting that BCL-6 gene expression is not limited to B cells and it is closely associated with monocytic lineage differentiation. The BCL-6 transcripts in TPA-treated U-937 cells were superinduced by the treatment with cycloheximide (CHX) and the half-life of the BCL-6 mRNA was apparently prolonged when TPA-treated U-937 cells were exposed to CHX in the presence of actinomycin D (ACD). Furthermore, the nuclear run-on assay revealed that the BCL-6 transcription signals were enhanced by TPA treatment. These results suggest that the increase of BCL-6 mRNA in U-937 cells stimulated with TPA to induce monocytic lineage differentiation is mediated by both transcriptional and post-transcriptional regulation.

Cell-permeable ceramide inhibits the growth of B lymphoma Raji cells lacking TNF-alpha-receptors by inducing G0/G1 arrest but not apoptosis: a new model for dissecting cell-cycle arrest and apoptosis.

We examined the effects of a cell-permeable ceramide analog, C2-ceramide, on the growth of TNF-alpha-resistant B lymphoma Raji cells lacking TNF-alpha-receptors (TNF-R). C2-ceramide inhibited the clonal growth of not only TNF-alpha-sensitive myeloid leukemia cells (HL60 and U937) but also Raji cells. Following stimulation with C2-ceramide, HL60 and U937 cells showed apoptotic cell death, whereas Raji cells did not show a detectable level of apoptosis. However, a cell-cycle arrest in G0/G1 phase was observed in Raji cells after the treatment with C2-ceramide, which was accompanied by the dephosphorylation of retinoblastoma (RB) gene products and decreased expression of p53 proteins. Failure of C2-ceramide to induce apoptosis in Raji cells might be explained by the lack or low expression of apoptosis-inducing proteins by two lines of evidence: (1) Raji cells were resistant to apoptosis induced by ceramide even in the presence of transcription/translation inhibitors; (2) Bax protein expression was not detectable in Raji cells, although Bcl-2 protein expression in Raji cells was even less than that in HL60 and U937 cells. Moreover, protein kinase C (PKC), whose activation has been described to inhibit ceramide-induced apoptosis, inhibitor H-7 did not induce apoptotic cell death in Raji cells, suggesting that an imbalance between PKC and ceramide pathways is not the reason for the resistance of Raji cells against ceramide-induced apoptosis. Finally, ceramide-induced activation of nuclear factor kappaB (NF-kappaB) was observed in Raji cells as well as HL60 cells, indicating that activation of this molecule may not be specific for apoptosis. By using the present model, one can dissect cell-cycle arrest and apoptosis induced by ceramide.

The presence and longevity of peripherally expanded donor-derived TCRalphabeta+ mature T lymphocyte clones after allogeneic bone marrow transplantation for adult myeloid leukemias.

There are two major pathways for T-cell regeneration after allogeneic bone marrow transplantation; thymus-dependent T-cell differentiation of T-cell progenitors, and peripheral expansion of mature T cells in the graft. In order to learn to what extent the peripheral expansion of donor-derived mature T lymphocytes contributes to reconstitution of the TCRalphabeta+ T-cell repertoire after allogeneic bone marrow transplantation for adult myeloid leukemias, we pursued the fate of donor-derived T-cell clones using the amino-acid sequences of the complementarity-determining region 3 (CDR3) of the TCR-beta chain as a clonal marker. Clonal expansion of TCRalphabeta+ T lymphocytes with specific TCRBV subfamilies was identified in donor blood. Identical T-cell clones were not found in blood from recipients before transplantation. The donor-derived T-cell clones were identified in the circulating blood from recipients a few months after allogeneic bone marrow transplantation, and they remained in the blood for 18 months after transplant in two recipients, and for 56 months in one. These results suggest that the peripheral expansion of mature T lymphocytes in the graft makes a significant contribution to post-transplant T-cell regeneration during the early period of transplantation in humans, and that mature T cells can survive in recipients for several years. Further investigation will be required to explore which antigens drive the expansion of T-cell clones in donors and recipients, and the mechanisms of maintaining homeostatic balance between the thymus-dependent pathway and the peripheral expansion of mature T cells in post-transplant T-cell regeneration.

Expression of PP2A B regulatory subunit beta isotype in rat testis.

We isolated a rat cDNA encoding part of the beta-isotype of the B regulatory subunit (BR beta) of protein phosphatase 2A (PP2A). The isolated cDNA encoded the region corresponding to amino acids positions 8(R) to 177(N) of human BR beta. The identities of the nucleotide and amino acid sequences of the rat and human BR beta s were 95.7% and 100%, respectively. The BR beta mRNA was specifically expressed in rat brain and testis, the lengths of mRNAs in these two organs being different. In the testis, the BR beta mRNA was first detected 40 days after birth, increasing gradually thereafter, and was expressed specifically in elongated spermatids, while mRNA of the alpha-isotype (BR alpha) was expressed equally in all spermatogenic cells. After meiosis, round spermatids change morphologically to elongated spermatids. BR beta may regulate the activity of the PP2A catalytic subunit in spermatids, and be involved in spermatogenic maturation, especially spermatid elongation.

Transmembrane signaling through CD80 (B7-1) induces growth arrest and cell spreading of human B lymphocytes accompanied by protein tyrosine phosphorylation.

We addressed the issue of the role for CD80 (B7-1) expressed on human B cells in transmembrane signaling. Cross-linking of CD80 on B lymphoma Raji cells induced tyrosine phosphorylation in 160-, 120-, 55-, 46- and 44-kDa proteins, which was inhibited by genistein. CD80-mediated signaling resulted in the inhibition of DNA replication of B cells and induced the changes in morphology like macrophages or fibroblasts. This cell spreading was inhibited by the pre-treatment of the cells with genistein. These results suggest that the CD80 antigen is involved in transmembrane outside-in signaling in B cells and its biological effects appear to be mediated by tyrosine kinases.

Hematopoietic cell transplantation-related nephropathy in Japan.

To clarify the incidence and characteristics of hematopoietic cell transplantation (HCT)-related nephropathy (HCT-N) in Japan, we sent questionnaire letters to 188 hematologic divisions of 91 hospitals and analyzed the responses. Of 2,136 Japanese hematopoietic cell transplant recipients, 51 patients (2.4%) had HCT-N. The early-onset (180 days after HCT) groups included 20, 16, and 15 patients, respectively. The early-onset group mainly consisted of patients with acute renal failure (ARF) and hemolytic uremic syndrome and/or thrombotic thrombocytopenic purpura. ARF was the dominant type in the middle-onset group. The main phenotype of the late-onset group was nephrotic syndrome, which correlated with chronic graft-versus-host disease (P=0.008). The total amounts of irradiation for patients with chronic renal failure and urinary abnormality were significantly greater than those for patients with ARF (P=0.004). The survival rate of the early-onset and middle-onset groups was 47.2%, whereas 87% of patients in the late-onset group survived (P=0.002). HCT-N is expected to become a serious and important problem in Japan because of the increasing number of HCTs from unrelated donors.

Hemoperitoneum due to acute cytomegalovirus infection in a patient receiving peritoneal dialysis.

A 27-year-old man receiving continuous ambulatory peritoneal dialysis (CAPD) developed high-grade fever, dyspnea, and hemoperitoneum 32 months after the start of CAPD. A chest computed tomograph showed fine reticular shadows in the bilateral lower lung fields. Cytomegalovirus (CMV) antigenemia were detected, and immunoglobulin (Ig) M and IgG antibodies for CMV were also positive. The absolute counts of helper T cells (478/microL) and the ratio of helper T cells/suppressor T cells (0.25) decreased, despite no evidence of hematologic or immunologic diseases, including human immunodeficiency virus (HIV) or human T cell lymphoma virus-1 (HTLV-1) infection, or the use of immunosuppressive drugs. All symptoms, including hemoperitoneum and the ratio of helper T cells/suppressor T cells, improved gradually and spontaneously. Acute and primary cytomegalovirus (CMV) infection induced hemoperitoneum in a patient who was receiving CAPD.

Remission of the nephrotic syndrome in a patient with renal amyloidosis due to rheumatoid arthritis treated with prednisolone and methotrexate.

A 46-year-old woman developed nephrotic syndrome secondary to rheumatoid arthritis (RA). A renal biopsy showed deposition of amyloid fibrils in the subendothelial space of the glomerular capillary walls. After treatment with prednisolone (PSL, 40 mg/day), the levels of C-reactive protein (CRP) and serum amyloid A decreased to within normal limits for 2 weeks. However, the nephrotic syndrome persisted for 6 months after the therapy. To maintain the suppression of disease activity and to reduce PSL, methotrexate (5 mg/week) was added. The nephrotic syndrome resolved gradually, and the level of serum albumin returned to normal. Although renal prognosis of patients with nephrotic syndrome due to amyloidosis caused by RA has been considered poor, adequate and long-term treatment of RA with antiinflammatory drugs, including PSL and methotrexate, is useful for patients with secondary amyloidosis complicated by RA.

Endocapillary proliferative glomerulonephritis in a patient with parvovirus B19 infection.

A 45-year-old woman developed acute nephritic syndrome after erythema infectiosum. Laboratory data on admission showed decreased serum C3, C4, and CH50 levels and the presence of both immunoglobulin (Ig) M and IgG antibodies to human parvovirus B19 (HPV). A renal biopsy showed diffuse endocapillary proliferative glomerulonephritis. Immunofluorescence microscopy indicated 2+ granular staining for IgG, IgM, and C3 over the mesangial area and along glomerular capillary walls. HPV antigen was also detected in glomeruli by immunohistochemistry. Electron microscopy showed electron-dense deposits in the subendothelial space and the paramesangial area. These findings suggest that immune complex-type glomerulonephritis is caused by glomerular deposition of HPV antigen-antibody complexes in some patients with HPV infection.

Disappearance of nodular mesangial lesions in a patient with light chain nephropathy after long-term chemotherapy.

A 64-year-old man developed multiple myeloma (kappa light chain type), nephrotic syndrome, and renal insufficiency in 1993. A renal biopsy showed typical histological findings of light chain nephropathy: nodular glomerulosclerosis with deposition of kappa light chains in the mesangial area and subendothelial space of the glomerular capillary walls. Long-term intermittent MEVP chemotherapy (melphalan, 4 mg/d for 4 days; cyclophosphamide, 100 mg/d for 4 days; vincristine, 1 mg/d; prednisolone, 40 mg/d for 4 days) diminished proteinuria and improved renal function. In April 1999, a follow-up biopsy showed remarkable diminution of nodular lesions and disappearance of kappa light chain deposits. Although the prognosis of light chain nephropathy has been considered poor, long-term successful chemotherapy can clear light chain deposits and restore renal function.

Collagenofibrotic glomerulopathy: a systemic disease.

Collagenofibrotic glomerulopathy is a recently discovered entity that is characterized by massive accumulation of spiraled and frayed collagen fibrils in mesangial and subendothelial areas, and elevated serum levels of procollagen III peptide. We report the autopsy of a patient who received continuous ambulatory peritoneal dialysis (CAPD) therapy for 7 years. Autopsy disclosed that massive accumulation of peculiar collagen fibers was found not only in the kidney, but also in many organs including spleen, liver, myocardium, and thyroid gland. Although the possibility remains that CAPD for 7 years might change or aggravate the deposition of abnormal collagen, the current case suggests a possibility that collagenofibrotic glomerulopathy is a systemic disorder with abnormal metabolism of type III collagen.

Extensive intraglomerular thrombi of monoclonal IgM-kappa in a patient with malignant lymphoma.

We describe an 80-year-old man who developed malignant lymphoma (ML) complicated by extensive intraglomerular thrombi of immunoglobulin M (IgM)-kappa monoclonal immunoglobulin. The clinical picture was characterized by nephrotic syndrome and systemic lymphadenopathy. Laboratory examination showed mild anemia and a small amount of monoclonal IgM-kappa in the blood. The histopathologic findings and surface immunoglobulin analysis of the lymph node biopsy specimen were consistent with CD5-positive diffuse large B-cell (type, IgM-kappa) lymphoma. The subsequent renal biopsy showed a massive deposition of amorphous material in the glomerular capillary lumens, subendothelial areas, and mesangium. Nodular glomerulosclerosis was not found. An immunofluorescent study showed that the deposits consisted of IgM-kappa monoclonal immunoglobulin. Ultrastructurally, the deposits were composed of granular electron-dense material. Chemotherapy was effective for both the ML and nephrotic syndrome, and the patient's urine analysis results returned to normal. The histopathologic manifestations of this case are rare, and the pathogenesis of these glomerular lesions was obviously associated with ML.

Mechanisms of allogeneic stimulation induced tumor necrosis factor-alpha (TNF-alpha) production.

We investigated the mechanisms of allogeneic stimulation induced TNF-alpha production in vitro by using human peripheral blood mononuclear cells (PBMC) and Daudi lymphoblastoid B-cells. PBMC produced TNF-alpha in response to mitomycin C-treated or paraformaldehyde-fixed Daudi cells, reaching a peak level after 4-6 h of culture. Monocytes were identified as the major source of TNF-alpha produced during allogeneic cell interaction. The second potent producer of TNF-alpha was E-rosette non-forming natural killer cells. Purified T-cells did not produce significant levels of TNF-alpha, even in the presence of IL-1 and IL-6. Interleukin-4 (IL-4) down-regulated TNF-alpha production by monocytes, but in contrast interferon-gamma (IFN-gamma) moderately enhanced TNF-alpha production. Our results indicate that monocytes are mainly responsible for the production of TNF-alpha in response to allogeneic stimulation, and T-cells modulate monocyte function by their soluble factors.

Evidence for the in vivo production of interleukin-3 in lethally irradiated mice transplanted with syngeneic murine splenocytes.

We wish to demonstrate new evidence for the in vivo production of interleukin-3 (IL-3). Syngeneic murine splenocytes were transplanted into lethally irradiated mice. The spleen cells in these transplant mice spontaneously produced IL-3 in cultures, and IL-3-like activity was detected in the serum. Chronological changes of the Thy-1 antigen expression on the bone marrow cells, and the number of myeloid progenitors in the bone marrow from post-transplant mice correlated with the amount of IL-3 produced in vivo. These results suggest that IL-3 may be produced by activated T cells during the syngeneic mixed lymphocyte reaction induced in vivo.

Mesangiolytic glomerulonephritis associated with Takatsuki's syndrome: an analysis of five renal biopsy specimens.

Renal biopsy specimens from five patients with Takatsuki's syndrome were studied. Proteinuria and hematuria were modestly positive in four cases and three cases, respectively. One case demonstrated early azotemia, while four cases revealed normal serum creatinine levels. All cases displayed glomerular enlargement and lobulation, mesangial cell proliferation, and double contour structure, simulating membranoproliferative glomerulonephritis. It is noteworthy that mesangial loosening was also detected in the renal biopsy specimens of this study. This change was present diffuse-globally in the well-developed cases and focal-globally or segmentally in the developing cases. In addition, various morphologic expressions were observed, ie, microaneurysms congested with RBCs, tentatively termed mesangial hyaline thrombi, nodule-like lesions, and tiny to small-sized mesangiolytic lesions. Since all these features could be explained by low-grade mesangiolysis, the renal alteration associated with this syndrome was termed mesangiolytic glomerulonephritis. Humoral factor(s) also toxic to the mesangium were implicated.

Reconstitution of gammadelta T cell repertoire diversity after human allogeneic hematopoietic cell transplantation and the role of peripheral expansion of mature T cell population in the graft.

We have examined the reconstitution of gammadelta T cell repertoire diversity after human allogeneic hematopoietic cell transplantation using a polymerase chain reaction (PCR)-based complementarity-determining region (CDR) 3 size spectratyping and DNA sequencing. The CDR3 complexity in the variable region of the T cell receptor (TCR)-delta chain was different amongst the individuals studied. Furthermore, CDR3 size distribution patterns of allogeneic hematopoietic cell transplant recipients were almost completely recovered by a few months after transplantation. In some patients, clonal predominance of the TCRDV1+ T cells became evident during the period after transplantation. In one particular donor/recipient pair, clonal predominance of TCRDV1+ T cells was already present in blood lymphocytes of the donor, and was also observed in the recipient after transplantation. Using this donor/recipient pair, we have questioned whether gammadelta T cell regeneration occurs via the peripheral expansion of mature T cells in the graft. In the donor lymphocytes, two expanding gammadelta T cell clones, which were demonstrated by CDR3 sequences of the TCR-delta chain, were recognized. These two clones were identified in the T cells from the recipient post transplant, but not before transplantation. One of the two clones was still detectable 1(1/2) years after the transplant procedure. These results strongly suggest that peripheral expansion of mature T cells in the graft is the principal pathway of gammadelta T cell regeneration after allogeneic hematopoietic cell transplantation in adults.

Successful vidarabine therapy for adenovirus type 11-associated acute hemorrhagic cystitis after allogeneic bone marrow transplantation.

A 36-year-old man underwent an allogeneic BMT for acute lymphoblastic leukemia. Eighteen days later the patient developed sudden onset of painful, gross hematuria due to adenovirus type 11 infection that did not respond to conservative therapy. There was an increase in serum creatinine levels and delayed recovery of the platelet count, associated with hemophagocytosis. After obtaining informed consent, vidarabine, which has been shown to be active against adenovirus in vitro, was started. The patient's symptoms improved within a few days of vidarabine therapy and urine cultures for adenovirus became negative. No serious adverse effects were observed. Vidarabine may be one therapeutic option in life-threatening adenovirus infection.

Autoantibodies against 70-kDa heat shock proteins (HSP70) in allogeneic bone marrow transplant recipients.

We report that autoantibodies against the 70-kDa heat shock protein family (HSP70) were detected in allogeneic bone marrow transplant recipients. Antibodies to HSP70 family proteins were detected in three out of 14 recipients of an allogeneic marrow graft but in none of the seven patients receiving autologous peripheral blood stem cell transplant (PBSCT). Immunoblotting analysis combined with two-dimensional SDS-PAGE revealed that these patients had antibodies to a constitutive 73-kDa/pI 5.5 heat shock protein (HSP73) and to a stress-inducible 72-kDa/pI 5.6 protein (HSP72). This is the first report, to our knowledge, describing the presence of autoantibody against HSP73 in allogeneic marrow transplant recipients. Our results may provide additional insight into the etiology and the pathophysiology of allogeneic transplant-related disorders.

Distinct TCRAV and TCRBV repertoire and CDR3 sequence of T lymphocytes clonally expanded in blood and GVHD lesions after human allogeneic bone marrow transplantation.

Acute graft-versus-host disease (GVHD) is a disorder involving the skin, gut and liver that is caused by mismatches of major and/or minor histocompatibility antigens between the HLA-identical donor and recipient. If T lymphocytes infiltrating GVHD lesions recognize antigens expressed in these organs, T cell clones should expand in inflammatory tissues. We previously reported that recipients of allogeneic bone marrow grafts have clonally expanded TCRalphabeta(+) T lymphocytes soon after transplantation, which leads to a skew of TCR repertoires. To establish whether or not the same antigens cause clonal expansion of T lymphocytes in both blood and GVHD tissues, we examined the usage of TCR alpha and beta chain variable regions (TCRAV and TCRBV) and determined the complementarity-determining region 3 (CDR3) of T lymphocytes clonally expanded in circulating blood and GVHD lesions. We found that the repertoires and CDR3 diversity of TCRAV and TCRBV differed between the GVHD lesions and circulating blood, suggesting the selective recruitment of antigen-specific T cells into GVHD tissues. We also found that the usage of TCRAV and TCRBV by the clonally expanded T lymphocytes and their CDR3 sequences differed between the GVHD tissues and blood. These results suggest that the antigen specificity of TCRalphabeta(+) T lymphocytes clonally expanded in blood and GVHD lesions is different.

Extensive clonal expansion of T lymphocytes causes contracted diversity of complementarity-determining region 3 and skewed T cell receptor repertoires after allogeneic hematopoietic cell transplantation.

We previously described skewed repertoires of the T cell receptor-beta chain variable region (TCRBV) and the TCR-alpha chain variable region (TCRAV) soon after allogeneic hematopoietic cell transplantation. To determine the characteristics of skewed TCRBV after transplantation, we examined the clonality of T lymphocytes carrying skewed TCRBV subfamilies and determined the CDR3 sequences of expanded T cell clones. In all 11 recipients examined, TCR repertoires were skewed, with an increase of certain TCRBV subfamilies that differed among individuals. In nine of 11 patients, clonal/oligoclonal T cell expansion was observed, although the expanded T cells were not necessarily oligoclonal. The extent of expansion after transplantation appeared to predict clonality. The arginine (R)-X-X-glycine (G) sequence was identified in clonally expanded T cells from four of five recipients examined, and glutamic acid (E), aspartic acid (D) and alanine (A) were frequently inserted between R and G. These results suggest that T lymphocyte expansion may result from the response to antigens widely existing in humans, and that the extensive clonal expansion of a limited number of T cells leads to contracted CDR3 diversity and post-transplant skewed TCR repertoires.