AIMS:Diffuse large B-cell lymphoma (DLBCL) usually proliferates effacing lymph follicles. In occasional cases, tumour cells show an interfollicular pattern of proliferation preserving lymph follicles. The aim was to analyse clinicopathological findings in DLBCL showing an interfollicular pattern of proliferation to determine whether this type of lymphoma is a distinct entity of DLBCL.METHODS AND RESULTS:Clinicopathological findings in 12 cases of DLBCL showing an interfollicular pattern of proliferation [interfollicular group (IF)] were examined and compared with those in 30 cases of DLBCL with ordinary morphology [control group (CG)]. IF showed a significantly lower lactate dehydrogenase level and International Prognostic Index scores than CG (P = 0.023 and P < 0.01, respectively). The frequency of localized disease, clinical stage 1 and 2, in IF was higher than that in CG (P = 0.016). A morphologically polymorphous pattern of proliferation was found in seven of 12 cases (58.3%) in IF, which was higher than that in CG, five (16.7%) of 30 cases (P < 0.01). Clonality analysis with the polymerase chain reaction method revealed that all 11 IF cases examined showed a monoclonal pattern. Immunohistochemically, the majority (11 of 12) of IF cases showed a non-germinal centre B-cell phenotype and the frequency was higher than that in CG (P = 0.021).CONCLUSION:Diffuse large B-cell lymphoma with an interfollicular pattern of proliferation shows distinct clinical and pathological findings from ordinary DLBCL.

We initially conducted a multicenter, randomized trial (n=43), and subsequently a questionnaire study (n=209) of participating hospitals, to evaluate whether infused fresh frozen plasma (FFP) could prevent the occurrence of hepatic veno-occlusive disease (VOD) after stem cell transplantation (SCT). Forty-three patients were divided into two groups: 23 receiving FFP infusions and 20 not receiving it. VOD developed in three patients not receiving FFP. Plasma von Willebrand factor (VWF) antigen levels were lower at days 0, 7 and 28 after SCT in patients receiving FFP than in those not receiving it, whereas plasma ADAMTS13 activity (ADAMTS13:AC) did not differ between them. Plasma VWF multimer (VWFM) was demonstrated to be defective in the high∼intermediate VWFM during the early post-SCT phase, but there was a significant increase in high VWFM just before VOD onset. This suggests that a relative enzyme-to-substrate (ADAMTS13/high-VWFM) imbalance is involved in the pathogenesis of VOD. To strengthen this hypothesis, the incidence of VOD was apparently lower in patients receiving FFP infusions than in those not receiving it (0/23 vs 3/20) in the randomized trial. Further, the results combined with the subsequent questionnaire study (0/36 vs 11/173) clearly showed the incidence to be statistically significant (0/59 vs 14/193, P=0.033).

The complete remission (CR) rate in acute leukemia (AL) has been improved due to progressive chemotherapy. However, relapse frequently occurs as minimal residual disease (MRD). To eradicate MRD, immunotherapy using autologous cytotoxic T-lymphocytes has been available. Leukemia cells (LC) of the patients were stored frozen before chemotherapy. Furthermore, after achieving CR, an EBV-transformed lymphoblastoid B-cell line (LCL) was established in each case. To induce Cytotoxic T-lymphocytes (CTL), co-culture system was carried out in two types. One is co-culture of peripheral blood mononuclear cells (PBMCs) and original leukemia cells (LC), the other is co-culture of PBMCs and the patient’s own LCL. After co-culture for 5 days, cytotoxic activity was examined by FACS with PKH-26 staining. Interestingly, cytotoxicity against not only autologous LC but the patient’s own LCL was observed in almost cases. This phenomenon is called cross-killing. Moreover, regulatory T-cells (Treg) were detected in the effecter phase determined with FACS (CD4+ CD25+) and FOXP3 mRNA by RT-PCR. Enhanced cytotoxicity was observed in inverse relation to a low proportion of Treg. These results suggested that more cytotoxicity may be induced by deletion of the Treg population. In conclusion, cell therapy as an immunotherapy should be useful tool to improve the prognosis, and if stocked LC is not available, CTL activity must be induced by co-culture with the patient’s own LCL.

Corticosteroids are highly effective anti-inflammatory or immunosuppressive drugs used commonly to treat human systemic lupus erythematosus (SLE). All-trans-retinoic acid (ATRA), which belongs to a class of retinoids that exert immunomodulatory and anti-inflammatory functions, can also suppress the development of lupus nephritis in an animal model. However, both agents can inflict serious adverse effects. Here, we have asked whether ATRA can serve as a steroid-sparing drug in the treatment of lupus nephritis. To examine the efficacy of combining predonisolone (PSL) with ATRA, we treated intraperitoneally New Zealand black/white F-1 (NZB/W F-1) mice with PSL, ATRA or both agents. Survival rate and proteinuria were determined once a month. Cytokine and anti-DNA antibody production were determined by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Renal histopathology was observed by haematoxylin and periodic acid Schiff (PAS), immunoperoxidase and immunohistochemical assay. Survival rate and proteinuria were improved in all experimental groups, and were much improved in the mice receiving the combination of ATRA and PSL (P < 0.05). A single administration of ATRA reduced the Th1 [interleukin (IL)-2, interferon (IFN)-gamma and IL-12], and a Th2 (IL-4) cytokine level, as effectively as administration of PSL. ATRA also suppressed the expression of inducible nitric oxide synthetase (iNOS) and monocyte chemoattractant protein-1 (MCP-1) in the kidney. The combination of PSL and ATRA significantly reduced IgG2 (especially IgG2b)-specific anti-DNA antibody levels in comparison with administration of either agent alone. These data suggest that ATRA might have the potential to act as a new therapeutic and steroid-sparing drug against lupus nephritis.

Objective: Some patients who had carried out long-term continuous ambulatory peritoneal dialysis discontinued the treatment because of progressive peritoneal fibrosis. It has been previously reported that transforming growth factor-β1 (TGF-β1) is one of the factors that induces peritoneal fibrosis. Also, hepatocyte growth factor (HGF) plays a role in the prevention of fibrosis and in inhibiting TGF-β1 production. In this study, we examined the effects of HGF on peritoneal fibrosis by TGF-β1 induced by high concentrations of D-glucose. Design: We transfected a full-length human HGF cDNA in an expression vector into human peritoneal mesothelial cells (HPMCs) using the calcium phosphate method. Transfected HPMCs were cultured with high concentrations of D-glucose solution and co-cultured with fibroblasts using a transwell system. Cell proliferation was determined using the Tetra Color One method. TGF-β1 and HGF protein were measured by enzyme-linked immunosorbent assay. Results: In addition to recombinant HGF, the growth inhibition of HPMCs by high concentration D-glucose or TGF-β1 was significant. By transfecting HGF cDNA into HPMCs, growth inhibition by high concentration D-glucose was completely restored. Furthermore, the production of TGF-β1 was also significantly decreased. Conclusion: These results suggested that exogenous HGF could possibly prevent peritoneal fibrosis.

We previously reported that all-trans retinoic acid (ATRA) inhibits growth in HTLV-1-positive T-cell lines and fresh cells from patients with adult T-cell leukemia. However, the mechanism of this inhibition is not clear. In the present study, we observed that NF-κB transcriptional activity as well as cell growth decreased significantly in HTLV-1-positive T-cell lines in the presence of ATRA. Furthermore, we observed that ATRA reduced HTLV-1 proviral DNA, HTLV-1 genes (gag, tax or pol mRNA) using the real time quantitative polymerase chain reaction. SIL-2R was reduced by ATRA in both protein level (culture supernantant) and mRNA level in HTLV-1-positive T-cell lines. Interestingly, ATRA significantly inhibited RT activity similar to azidothimidine (AZT) in HTLV-1-positive T-cell lines. Moreover, AZT was inhibitory of proviral DNA but not NF-kB transcriptional activity and sIL-2R on HTLV-1, however ATRA was inhibitory of NF-kB, proviral DNA and sIL-2R on HTLV-1. These results suggested that the decrease of sIL-2R induced by ATRA may be caused by the actions of a NF-kB inhibitor acting on the NF-kB/sIL-2R signal pathway. These results suggested that ATRA could have two roles, as a NF-kB inhibitor and as a RT inhibitor.

CD34 + progenitors from MDS patients are unresponsive to SDF-1, despite high levels of SDF-1 in bone marrow plasma

CCL25 (also known as thymus-expressed chemokine) and CCL28 (also known as mucosae-associated epithelial chemokine) play important roles in mucosal immunity by recruiting IgA Ab-secreting cells (ASCs) into mucosal lamina propria. However, their exact roles in vivo still remain to be defined. In this study, we first demonstrated in mice that IgA ASCs in small intestine expressed CCR9, CCR10, and CXCR4 on the cell surface and migrated to their respective ligands CCL25, CCL28, and CXCL12 (also known as stromal cell-derived factor 1), whereas IgA ASCs in colon mainly expressed CCR10 and CXCR4 and migrated to CCL28 and CXCL12. Reciprocally, the epithelial cells of small intestine were immunologically positive for CCL25 and CCL28, whereas those of colon were positive for CCL28 and CXCL12. Furthermore, the venular endothelial cells in small intestine were positive for CCL25 and CCL28, whereas those in colon were positive for CCL28, suggesting their direct roles in extravasation of IgA ASCs. Consistently, in mice orally immunized with cholera toxin (CT), anti-CCL25 suppressed homing of CT-specific IgA ASCs into small intestine, whereas anti-CCL28 suppressed homing of CT-specific IgA ASCs into both small intestine and colon. Reciprocally, CT-specific ASCs and IgA titers in the blood were increased in mice treated with anti-CCL25 or anti-CCL28. Anti-CXCL12 had no such effects. Finally, both CCL25 and CCL28 were capable of enhancing alpha4 integrin-dependent adhesion of IgA ASCs to mucosal addressin cell adhesion molecule-1 and VCAM-1. Collectively, CCL25 and CCL28 play essential roles in intestinal homing of IgA ASCs primarily by mediating their extravasation into intestinal lamina propria.

Fractalkine (CX3CL1) and its receptor CX3CR1 play an important role in natural killer (NK) cell- and cytotoxic T cell-mediated endothelium damage. Here we describe the cytotoxicity of myelodysplastic syndrome (MDS)-derived T cell line, K2-MDS, through the fractalkine–CX3CR1 system. K2-MDS cells induced apoptosis against CD34+ cells from normal bone marrow (BM) in a direct cell contact manner. K2-MDS cells expressed perforin and granzyme B, but they lacked Fas ligand expression. A specific inhibitor for perforin, concanamycin A, blocked K2-MDS-dependent cytotoxicity. Furthermore, a CX3C-chemokine, fractalkine, was expressed in CD34+ cells, and its receptor, CX3CR1, was expressed on K2-MDS cells. The neutralizing monoclonal antibody (MoAb) for fractalkine and soluble fractalkine significantly inhibited K2-MDS-dependent cytotoxicity. K2-MDS cells also induced the cytotoxicity against human umbilical cord endothelial cells (HUVECs) expressing fractalkine. These data indicate that K2-MDS may be a perforin–granzyme-positive T cell line that exerts a cytotoxic effect on CD34+ cells mediated through the fractalkine–CX3CR1 system.

The frequent involvement of adult T-cell leukemia (ATL) cells in organs such as the skin and lymph nodes is observed in ATL. Epstein-Barr virus (EBV) infection has been observed in the clinical course of ATL, and ATL cells in all skin cases were strongly positive for EBV nuclear antigen (EBNA)-1 and latent membrane protein (LMP)-1 by immunohistochemical staining. Interestingly, ATL cells in all skin cases were positive for CD45R0 (UCHL1) and CD20 (L26), indicating that they have both T and B cells characterization. We suggested that HTLV-1 enhances the entry of EBV into endothelial cells, epithelial cells and EBV presents for an active role of HTLV-1 in the development of ATL. Here, we established two B-cell lines from peripheral blood of patients with ATL. EBV DNA, proviral DNA for HTLV-1 and Tax mRNA were detected in both B cell lines. On characterization of the cell lines, enhanced expression of intercellular adhesion molecule-1 or-3 (CD54, CD50), lymphocyte function-associated antigen-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) was observed compared to an EBV-infected B cell line, Raji. Moreover, EBV oncogenes such as EBV-encoded small RNA (EBER)-1, EBNA-1,-2 and LMP-2A were also detected. To investigate the role of the interaction of these viruses, we transfected EBV and/or HTLV-1 into a healthy donor's lymphocytes, Raji cells and a HTLV-1 negative T-cell line, Jurkat. Enhanced expression of adhesion molecules was observed in double transfectants (EBV and HTLV-1). Furthermore, we detected high production levels of interleukin-4 (IL-4) in those B-cell lines and transfectants. These results indicated co-expression of HTLV-1 and EBV may induce aggressive organ involvement by enhanced expression of adhesion molecules via IL-4 signaling. Furthermore, HTLV-1 could greatly enhance EBV infectivity and attribute to increased replication of EBV. EBV also could be a necessary cofactor for ATL development in immunodeficiency states. ATL patients who have been coinfected with EBV and HTLV-1 have a high risk of worse progress of disease. ATL patients have a poor treatment outcome, even with strong chemotherapy, especially who have severe skin involvements have a resistance for chemotherapy. Viewed from a different angle, our observations suggest that EBV infection may be used as one of bad prognostic factors and an identification of bad-risk patients who need to benefit from early high dose chemotherapy. Collectively, a reactivation of EBV infection and EBV oncogenes can provide an acceleration of organ involvement in ATL.These results can explain why EBV-associated ATL is more frequent and clinically more aggressive.

Adult T-cell leukemia (ATL) is a leukemia of mature peripheralT lymphocytes associated with human T-lymphotropic virustype I (HTLV-I). We previously reported that all-transretinoic acid (ATRA) inhibited cell growth and inducedapoptosis in HTLV-I (þ) T-cell lines as well as fresh ATL cellsobtained from ATL patients.

Autoimmune disease in Fas-deficient MRL-Fas(lpr) mice is dependent on infiltrating autoreactive leukocytes and autoantibodies, and IFN-gamma plays an important role in the pathogenesis. As IL-18 is capable of inducing IFN-gamma production in T cells, we hypothesized that signaling through IL-18R is involved in the pathogenesis. To investigate the impact of IL-18 in this autoimmune disease, we generated an MRL-Fas(lpr) strain deficient in IL-18Ralpha. Compared with the wild-type strain, IL-18Ralpha-deficient MRL-Fas(lpr) mice survived longer and showed a significant reduction in renal pathology, including glomerular IgG deposits, proteinuria, and serum anti-DNA Abs. Intrarenal transcripts encoding IFN-gamma, TNF-alpha, IL-12, and IL-10, which have been linked to nephritis, were all markedly reduced. Skin lesions, lymphadenopathy, and lung pathology characteristic of the MRL-Fas(lpr) mouse disease were diminished in IL-18Ralpha-deficient MRL-Fas(lpr) mice. Thus, we conclude that IL-18Ralpha signaling is critical to the pathogenesis of autoimmune disease in MRL-Fas(lpr) mice.

Classical Hodgkin's disease (HD) is characterized by rare neoplastic Hodgkin and Reed-Sternberg (H-RS) cells within abundant reactive cellular backgrounds. In most cases, H-RS cells originate from the B-cell lineage, but their immunophenotypes are unusual. Here we newly found frequent expression of chemokine receptors CXCR6 and CCR10 and their respective ligands CXCL16 and CCL28 in HD-derived cell lines. CCR10 is known to be selectively expressed by plasma cells, whereas CCL28 attracts eosinophils via CCR3 and plasma cells via CCR10 and CCR3. Therefore, we examined their expression in HD tissues by immunohistochemistry. We found that H-RS cells in 15 of 19 cases were positive for CCL28. Among them, seven cases were also positive for CCR10, suggesting a potential autocrine effect. In situ hybridization confirmed the expression of CCL28 mRNA in H-RS cells. The CCL28 positivity in H-RS cells did not significantly correlate with that of LMP-1, CCL17, CCL22, or CCL11. However, it significantly correlated with the background accumulation of eosinophils, plasma cells, and CCR10+ cells. Thus, the production of CCL28 by H-RS cells may play a major role in tissue accumulation of eosinophils and/or plasma cells in classical HD. The frequent expression of CCR10 in H-RS cells themselves also supports their close relationship to plasma cells.

: To determine and directly compare the clinical course of white and Asian patients with paroxysmal nocturnal hemoglobinuria (PNH), data were collected for epidemiologic analysis on 176 patients from Duke University and 209 patients from Japan. White patients were younger with significantly more classical symptoms of PNH including thrombosis, hemoglobinuria, and infection, while Asian patients were older with more marrow aplasia. The mean fraction of CD59-negative polymorphonuclear cells (PMN) at initial analysis was higher among Duke patients than Japanese patients. In both cohorts, however, a larger PNH clone was associated with classical PNH symptoms, while a smaller PNH clone was associated with marrow aplasia. Thrombosis was significantly more prevalent in white patients than Asian patients, and was associated with a significantly higher proportion of CD59-negative PMN. For individual patients, CD59-negative populations varied considerably over time, but a decreasing PNH clone portended hematopoietic failure. Survival analysis revealed a similar death rate in each group, although causes of death were different and significantly more Duke patients died from thrombosis. Japanese patients had a longer mean survival time (32.1 yr vs. 19.4 yr), although Kaplan-Meier survival curves were not significantly different. Poor survival in both groups was associated with age over 50 years, severe leukopenia/neutropenia at diagnosis, and severe infection as a complication; additionally, thrombosis at diagnosis or follow-up for Duke patients and renal failure for Japanese patients were poor prognostic factors. These data identify important differences between white and Asian patients with PNH. Identification of prognostic factors will help the design of prospective clinical trials for PNH.