Perfusion method for bone marrow cell collection in poor mobilizer lymphoma patient.

We previously described a unique procedure for the collection of bone marrow cells (BMCs) using a perfusion method (PM). In cynomolgus monkeys, this method resulted in lower contamination with T cells (<10%). Here, we performed PM on a poor mobilizer lymphoma patient. We confirmed the safety of the intra-bone marrow injection of saline to collect the BMCs. The collected BMCs showed minimal contamination with T cells (<15%) and red blood cells (RBCs) (<4%) from the peripheral blood. It took a total of only 30 min to collect the BMCs. Moreover, transfusion of RBCs was unnecessary. There were no relevant post-operative side effects except for self-limiting pain at the sites of collection, and the patient was able to walk around the hospital after the operation.

Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a severe and incurable autoimmune disease characterized by chronic activation of plasmacytoid dendritic cells (pDCs) and production of autoantibodies against nuclear self-antigens by hyperreactive B cells. Neutrophils are also implicated in disease pathogenesis; however, the mechanisms involved are unknown. Here, we identified in the sera of SLE patients immunogenic complexes composed of neutrophil-derived antimicrobial peptides and self-DNA. These complexes were produced by activated neutrophils in the form of web-like structures known as neutrophil extracellular traps (NETs) and efficiently triggered innate pDC activation via Toll-like receptor 9 (TLR9). SLE patients were found to develop autoantibodies to both the self-DNA and antimicrobial peptides in NETs, indicating that these complexes could also serve as autoantigens to trigger B cell activation. Circulating neutrophils from SLE patients released more NETs than those from healthy donors; this was further stimulated by the antimicrobial autoantibodies, suggesting a mechanism for the chronic release of immunogenic complexes in SLE. Our data establish a link between neutrophils, pDC activation, and autoimmunity in SLE, providing new potential targets for the treatment of this devastating disease.

CXCL8 enhances the angiogenic activity of umbilical cord blood-derived outgrowth endothelial cells in vitro.

OECs (outgrowth endothelial cells), also known as late-EPCs (late-endothelial progenitor cells), have a high proliferation potential in addition to in vitro tube formation capability. In ischaemic animal models, injected OECs were integrated into regenerating blood vessels and improved neovascularization. Previous reports have demonstrated the expression of CXCL8 to be up-regulated in ischaemic tissues. It has also been documented that CXCL8 stimulates the angiogenic activity of mature ECs (endothelial cells). Therefore, it has been suggested that CXCL8 plays an important role in neovascularization in ischaemic tissues. However, it is still uncertain whether CXCL8 also stimulates the angiogenic activity of OECs. This study evaluated the effects of CXCL8 on the angiogenic activity of OECs in vitro. OECs were isolated from human UCB (umbilical cord blood)-derived mononuclear cells. Phenotypes of the OECs were assessed by flow cytometry, immunostaining, and real-time RT (reverse transcription)-PCR. The effects of CXCL8 on OECs were investigated by transwell migration assay and capillary tube formation assay on Matrigel. The OEC clones isolated from UCB expressed OEC phenotypes. In addition, CXCL8 receptors (CXCR1 and CXCR2) were expressed on these OEC clones. CXCL8 significantly stimulated the transwell migration and capillary tube formation of OECs. Neutralizing antibody against CXCR2, but not CXCR1, abolished a transwell migration of OECs induced by CXCL8, suggesting the involvement of CXCL8/CXCR2 axis in transwell migration. These results demonstrate that CXCL8 stimulates the angiogenic activity of UCB-derived OECs in vitro.

Epstein-barr virus in diffuse large B-Cell lymphoma in immunocompetent patients in Japan is as low as in Western Countries.

According to previous reports, the frequency of Epstein-Barr virus (EBV) positivity in diffuse large B-cell lymphoma is higher in East Asia (approximately 9%) than in Western countries. The presence of the EBV genome was examined in diffuse large B-cell lymphoma patients registered with the Osaka Lymphoma Study Group (OLSG) in Osaka, Japan, situated in East Asia. The EBV-positive rate was examined with in situ hybridization (ISH) in 484 immunocompetent diffuse large B-cell lymphoma patients registered with OLSG. The male-to-female ratio was 1.29, with ages ranging from 16 to 95 (median, 68) years. ISH with EBV-encoded small RNAs (EBER) probes revealed positive signals in the nuclei of tumor cells: the frequency of positively stained cells among all tumor cells was almost none in 458 cases, 5-10% in 5, 10-20% in 5, 20-50% in 11, and >50% in 5. When the frequency was >20% or >50%, the EBV-positive rate in the present series (3.3% or 1.0%) was rather similar to that reported in Western cases. Careful evaluation of patient backgrounds, including age distribution, type of lymphomas, exclusion of immunocompromised patients, and establishment of definite criteria for EBV positivity (>20%, >50%, or almost all tumor cells) are essential in comparing geographical differences.

Development of a high-resolution purification method for precise functional characterization of primitive human cord blood-derived CD34-negative SCID-repopulating cells.

OBJECTIVE: We have successfully identified human cord blood (CB)-derived CD34-negative (CD34(-)) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphomyeloid repopulating ability using the intrabone marrow injection method. In our previous study, a limiting dilution analysis demonstrated the frequency of CD34(-) SRCs in CB-derived 13lineage-negative (Lin(-)) CD34(-) cells to be approximately 1/25,000. In this study, we intended to develop a high-resolution purification method to obtain highly purified CD34(-) SRCs. MATERIALS AND METHODS: The pooled CB-derived Lin(-) cells were stained with 13 reported Lin monoclonal antibodies (mAbs) and 5 more Lin mAb, against CD11b, CD33, CD66c, CD45RA, and CD127. Then 18Lin(-)CD34(high), 18Lin(-)CD34(-), and 13Lin(-)CD34(high)CD38(-) cells were sorted by fluorescence-activated cell sorting. Stem cell characteristics of these three fractions of cells were analyzed by in vitro cultures and in vivo repopulation assays for evaluation of this new purification method. RESULTS: A limiting dilution analysis demonstrated the frequency of CD34(-) SRCs in these 18Lin(-)CD34(-) cells to be approximately 1/1,000, which is associated with a seeding efficiency 25 times greater than the previous method. All primary recipient nonobese diabetic/Shi-scid/IL-2Rγc(null) mice that received transplants of only two CD34(-) SRCs were highly engrafted with human lymphomyeloid cells at 24 weeks after primary transplantation and showed secondary multilineage repopulating abilities. CONCLUSIONS: We succeeded to highly purify the CD34(-) SRCs using 18Lin mAbs and the intrabone marrow injection technique. This newly developed high-resolution purification method is indispensable to precisely characterize a distinct class of primitive human CB-derived CD34(-) hematopoietic stem cells.

Dic (17;20) (p11;q11) preceded MLL gene amplification in a patient with de novo mixed-lineage leukemia.

We report a case of acute mixed-lineage leukemia, as seen in a 65 year-old female with MLL gene amplification and biallelic loss of wild type p53 gene. The diagnosis was based on the findings that her bone marrow (BM) blasts expressed cytoplasmic CD3 (cyCD3), B-lineage antigens and myeloid antigens accompanied by clonal rearrangements of IgH gene. The BM blasts consisted of small-sized peroxidase-negative blasts (97%) and large-sized peroxidase-positive blasts (3%). The BM blasts showed a complex "karyotype," including dic(17;20) (p11;q11), -5 and add (11q23). Add (11q23) abnormality was found in sideline karyotypes as well as the stemline abnormality of dic(17;20) (p11;q11). For the p53 gene, which is located at 17p13, fluorescence in situ hybridization analysis showed the loss of one of two p53 alleles. Furthermore, polymerase chain reaction-single-strand conformation polymorphism and following nucleotide sequencing showed that the p53 gene was mutated at codon 215, leading to an amino acid substitution from Ser to Arg. For the MLL gene, southern blot analysis showed that the MLL gene locus was amplified but not rearranged at its breakpoint cluster region, which is usually rearranged in balanced translocations with many partner genes. These findings suggest that MLL gene amplification may in this case be based on the genetic instability caused by the preceding biallelic loss of the wild type p53 gene.

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, function as inhibitors of cellular and molecular components involved in type I interferon production.

OBJECTIVE: Statins, which are used as cholesterol-lowering agents, have pleiotropic immunomodulatory properties. Although beneficial effects of statins have been reported in autoimmune diseases, the mechanisms of these immunomodulatory effects are still poorly understood. Type I interferons (IFNs) and plasmacytoid dendritic cells (PDCs) represent key molecular and cellular pathogenic components in autoimmune diseases such as systemic lupus erythematosus (SLE). Therefore, PDCs may be a specific target of statins in therapeutic strategies against SLE. This study was undertaken to investigate the immunomodulatory mechanisms of statins that target the IFN response in PDCs. METHODS: We isolated human blood PDCs by flow cytometry and examined the effects of simvastatin and pitavastatin on PDC activation, IFNalpha production, and intracellular signaling. RESULTS: Statins inhibited IFNalpha production profoundly and tumor necrosis factor alpha production modestly in human PDCs in response to Toll-like receptor ligands. The inhibitory effect on IFNalpha production was reversed by geranylgeranyl pyrophosphate and was mimicked by either geranylgeranyl transferase inhibitor or Rho kinase inhibitor, suggesting that statins exert their inhibitory actions through geranylgeranylated Rho inactivation. Statins inhibited the expression of phosphorylated p38 MAPK and Akt, and the inhibitory effect on the IFN response was through the prevention of nuclear translocation of IFN regulatory factor 7. In addition, statins had an inhibitory effect on both IFNalpha production by PDCs from SLE patients and SLE serum-induced IFNalpha production. CONCLUSION: Our findings suggest a specific role of statins in controlling type I IFN production and a therapeutic potential in IFN-related autoimmune diseases such as SLE.

Thymic stromal lymphopoietin plays an adjuvant role in BCG-mediated CD8(+) cytotoxic T cell responses through dendritic cell activation.

Although Bacillus Calmette-Guérin (BCG) has historically emerged as a potent adjuvant in cancer immunization through dendritic cell (DC) activation, the efficacy of its antitumor effect has been limited. Therefore, the strategy of adjuvant therapy using BCG needs to be improved by adding enhancers. Here we found that thymic stromal lymphopoietin (TSLP) acts as an enhancer for the BCG-mediated antitumor effect. While BCG-stimulated DCs induced CD8(+) T cell production of IFN-gamma without strong cell expansion, TSLP-stimulated DCs induced robust CD8(+) T cell expansion without high quantities of IFN-gamma production. Notably, DCs stimulated with both BCG and TSLP induced robust expansion of CD8(+) T cells that produced a large amount of IFN-gamma with a potent cytolytic activity related to granzyme B expression. Our data suggest that TSLP is a good adjuvant to enhance the BCG-mediated cytotoxic T cell effect through DC activation, and provide a functional basis for a novel strategy for antitumor immune-based therapy.

Inhibitor of IkappaB kinase activity, BAY 11-7082, interferes with interferon regulatory factor 7 nuclear translocation and type I interferon production by plasmacytoid dendritic cells.

INTRODUCTION: Plasmacytoid dendritic cells (pDCs) play not only a central role in the antiviral immune response in innate host defense, but also a pathogenic role in the development of the autoimmune process by their ability to produce robust amounts of type I interferons (IFNs), through sensing nucleic acids by toll-like receptor (TLR) 7 and 9. Thus, control of dysregulated pDC activation and type I IFN production provide an alternative treatment strategy for autoimmune diseases in which type I IFNs are elevated, such as systemic lupus erythematosus (SLE). Here we focused on IkappaB kinase inhibitor BAY 11-7082 (BAY11) and investigated its immunomodulatory effects in targeting the IFN response on pDCs. METHODS: We isolated human blood pDCs by flow cytometry and examined the function of BAY11 on pDCs in response to TLR ligands, with regards to pDC activation, such as IFN-alpha production and nuclear translocation of interferon regulatory factor 7 (IRF7) in vitro. Additionally, we cultured healthy peripheral blood mononuclear cells (PBMCs) with serum from SLE patients in the presence or absence of BAY11, and then examined the inhibitory function of BAY11 on SLE serum-induced IFN-alpha production. We also examined its inhibitory effect in vivo using mice pretreated with BAY11 intraperitonealy, followed by intravenous injection of TLR7 ligand poly U. RESULTS: Here we identified that BAY11 has the ability to inhibit nuclear translocation of IRF7 and IFN-alpha production in human pDCs. BAY11, although showing the ability to also interfere with tumor necrosis factor (TNF)-alpha production, more strongly inhibited IFN-alpha production than TNF-alpha production by pDCs, in response to TLR ligands. We also found that BAY11 inhibited both in vitro IFN-alpha production by human PBMCs induced by the SLE serum and the in vivo serum IFN-alpha level induced by injecting mice with poly U. CONCLUSIONS: These findings suggest that BAY11 has the therapeutic potential to attenuate the IFN environment by regulating pDC function and provide a novel foundation for the development of an effective immunotherapeutic strategy against autoimmune disorders such as SLE.

Diffuse large B-cell lymphoma in the spinal epidural space: A study of the Osaka Lymphoma Study Group.

Diffuse large B-cell lymphoma (DLBCL) involving spinal epidural space (SEDLBCL) is relatively rare, constituting 1.8% of DLBCLs in Osaka, Japan. The aim of this study was to analyze SEDLBCL cases for their clinical and histopathologic findings, including an association with Epstein-Barr virus (EBV) and immunohistochemical characteristics. We analyzed the clinicopathologic findings of 27 SEDLBCL cases. They consisted of 16 males and 11 females, their age ranging from 37-86 years (median 64 years). Eight patients had stage I disease, 3 had stage II, 5 had stage III, and 11 had stage IV. Based on the staining pattern for anti-CD10, bcl-6, and MUM-1, the cases were categorized into 17 cases of the germinal center B-cell (GCB) type and nine of the non-GCB type. There was a 4%-positive rate for EBV in the tumor cells. When compared to nodal DLBCL, the frequency of patients with a high performance status (PS) is higher in SEDLBCL. Compared to the DLBCL of the central nervous system (CNS), the frequency of cases with high stage, 2 or more extranodal lesions, high international prognostic index (IPI), and GCB-type is higher in SEDLBCL. There were no significant differences in the histologic features between SEDLBCL and nodal/CNS DLBCL. Univariate analysis revealed that advanced stage was an unfavorable factor for overall survival (P=0.060). SEDLBCL is different from nodal and CNS DLBCL, but an association with EBV is unlikely in every group.

Polymorphous lymphoproliferative disorder: a clinicopathological analysis.

Lymphoproliferative disorder (LPD) with polymorphous composition of proliferation (polymorphous LPD), containing large lymphoid cells together with small lymphocytes, plasma cells, macrophages, and/or eosinophils, is found in individuals with immunodeficiency conditions. Clinicopathological findings in 19 cases of polymorphous LPD registered with the Osaka Lymphoma Study Group, Osaka, Japan, were analyzed; they represented 0.4% of the registered cases. In six cases, there was a history of rheumatoid arthritis; five of them had received immunosuppressive agents. There were no acquired immunodeficiency syndrome cases or organ transplant recipients. Southern blotting and/or polymerase chain reaction (PCR)-based clonality analysis revealed monoclonal B cell and T cell proliferation in eight and six cases (B- and T-LPD), respectively, and polyclonality in one. In B-LPD, there was polymorphous proliferation, containing large B-lymphoid cells, while medium-to-large T lymphoid cells with occasional eosinophilic infiltration were seen in T-LPD. Epstein-Barr virus (EBV) was detected in three of eight B-LPD, four of six T-LPD, and one of one polyclonal LPD. The prognosis was not favorable; the 3-year overall survival rate was 49.7 +/- 17.3%. Thus, polymorphous LPD is relatively rare in Japan and is a heterogeneous disease with monoclonal proliferation of B or T cells; additionally, it is occasionally EBV-associated, and behaves as an aggressive lymphoma.

Diffuse large B-cell lymphoma with a high number of epithelioid histiocytes (lymphoepithelioid B-cell lymphoma): a study of Osaka Lymphoma Study Group.

The aim of this study was to clarify whether diffuse large B-cell lymphoma (DLBCL) with a high number of epithelioid histiocytes (DLBCL-EH) could have distinctive clinicopathological characteristics. Clinicopathological findings in 22 cases with DLBCL-EH and, as a control, 96 cases with ordinary type of DLBCL were analyzed. There were ten men and 12 women with ages ranging from 38 to 91 (median, 64) years. The primary site was lymph node in 16 cases, extranodal organs in three, and unknown in three. Stage of disease was I in five cases, II in three, III in nine, and IV in five. Histologically, there was a diffuse proliferation of large lymphoid cells admixed with numerous clusters of epithelioid histiocytes sprinkling throughout the lesions. Immunohistochemically, the large lymphoid cells were CD20(+), CD15(-), and CD3(-) and positive for CD10, bcl-6, and MUM1 in nine (41%), eight (36%), and 12 (55%) of 22 cases, respectively. Epstein-Barr virus positive rate was higher in DLBCL-EH (23.8%) than that in ordinary DLBCL (4.5%; P<0.05). Clonality analysis revealed monoclonal bands in all of the examined 20 cases with DLBCL-EH. Multivariate analysis revealed the prominent epithelioid reaction to be an independent factor for favorable prognosis. These findings suggest that DLBCL-EH could be a specific morphological variant of DLBCL associated with a better prognosis.

Synergistic effect of interleukin-6 and endoplasmic reticulum stress inducers on the high level of ABCG2 expression in plasma cells.

ABCG2 is a transporter preferentially expressed in a primitive subpopulation of cells and recently reported as a surviving factor for trophoblasts. To date, manner of ABCG2 expression in lymphoid tissues is not known. Immunohistochemically, strong ABCG2 expression was found in a small proportion of plasma cells mainly located in the interfollicular space of lymphoid tissues. The number of ABCG2-high plasma cells increased in interleukin-6- (IL-6) rich lesions, such as Castleman's disease of plasma cell type. Plasma cells are subjected to endoplasmic reticulum (ER) stress when excess proteins are synthesized, and IL-6 stimulates protein synthesis. Therefore, the effect of IL-6 and ER stress on ABCG2 expression in plasma cells was examined. The expression level of ABCG2 increased by treatment with either IL-6 or ER stress inducers, and further increased with both. The promoter analysis revealed that the effect of IL-6 and ER stress inducers was mediated through the site overlapping XBP-1 and HIF-1 binding sequences. Knocked-down of ABCG2 by siRNA or ABCG2 inhibitor reduced plasma cell viability under ER stress. These suggest that ABCG2 is a surviving factor for plasma cells. To our knowledge, this is the first study reporting the effect of ER stress on ABCG2 expression.

Change of CD20 Expression in Diffuse Large B-Cell Lymphoma Treated with Rituximab, an Anti-CD20 Monoclonal Antibody: A Study of the Osaka Lymphoma Study Group.

Change of CD20 expression was examined in cases of diffuse large B-cell lymphoma (DLBCL). CD20 expression after treatment with anti-CD20 antibody (rituximab, Rx) for DLBCL was examined in 23 cases who received serial biopsy by immunohistochemistry (IHC) and flow cytometry (FCM). CD20- by IHC and/or FCM was defined as CD20-. Four cases were CD20- at initial biopsy but became CD20+ after chemotherapy with Rx (CH-R) (group A). Recurrent tumors in three group A cases became resistant to CH-R. Initial and recurrent tumors were CD20+ before and after CH-R in 17 cases (group B). Tumors before CH-R were CD20- in two cases (group C) and continued to be CD20- in one and turned CD20+ in the other with survival time after the relapse of 8 and 23 months, respectively. Evaluation of CD20 expression with immunohistochemical and flow cytometric methods is used for the prediction of responsiveness of relapsed DLBCL for CH-R.

Imidazoquinoline acts as immune adjuvant for functional alteration of thymic stromal lymphopoietin-mediated allergic T cell response.

Atopic dermatitis is a major allergic disease that develops through dysregulation of Th2-mediated inflammation. Although dendritic cells (DCs) have been thought to play a critical role in the upstream phase of the allergic cascade, conventional drugs such as steroids and chemical mediator antagonists target the effector cells or factors in allergic inflammation. Recently, it has been demonstrated that interaction between thymic stromal lymphopoietin (TSLP) and human DCs plays an essential role in evoking inflammatory Th2 responses in allergy through OX40 ligand expression on DCs. In this study, we provide evidence that R848, an imidazoquinoline compound, which is a TLR ligand and a strong Th1 response-inducing reagent, is a potent adjuvant for the alteration of the Th2-inducing potency of human DCs activated by TSLP (TSLP-DCs). R848 inhibited the inflammatory Th2-inducing capacity of TSLP-DCs and redirected them to possessing an IL-10 and IFN-gamma-producing regulatory Th1-inducing capacity. This functional alteration depended on both repression of OX40 ligand expression and induction of IL-12 production from DCs by the addition of R848. Additionally, R848 had the ability to inhibit the TSLP-mediated expansion and maintenance of the Th2 memory response. These findings suggest that imidazoquinoline may be a useful in the treatment of allergic diseases that are triggered by TSLP.

Mycobacterium bovis Bacillus Calmette-Guérin suppresses inflammatory Th2 responses by inducing functional alteration of TSLP-activated dendritic cells.

Allergic diseases such as atopic dermatitis and asthma develop as a consequence of dysregulated T(h)2 responses. Recently, it has been demonstrated that interaction between dendritic cells (DCs) and thymic stromal lymphopoietin (TSLP), an IL-7-like cytokine, is essential for evoking T(h)2 responses in allergy. In this study, we investigated whether Mycobacterium bovis Bacillus Calmette-Guérin (BCG), a strong T(h)1 response-inducing adjuvant, can alter the function of DCs activated by TSLP (TSLP-DCs). We demonstrated that BCG redirects TSLP-DCs away from inducing inflammatory T(h)2 cells that produce IL-4, IL-5, IL-13 and tumor necrosis factor (TNF)-alpha and toward regulatory T(h)1 cells that produce IFN-gamma and IL-10. We also demonstrated that this functional alteration of TSLP-DCs by BCG depended on both production of IL-12 from DCs and down-regulation of OX40 ligand, a member of the TNF family, on DCs. These findings suggest that BCG might be a useful adjuvant for the treatment of allergic diseases that are triggered by TSLP.

NK-cell intravascular lymphomatosis--a mini-review.

The majority of cases of intravascular lymphomatosis (IVL) is derived from B cells. However, IVL may also arise from T cells, or more rarely NK cells. The clinicopathological findings in six cases of NK-cell IVL (NK-IVL), including one new case, were summarised and compared with B-cell IVL (B-IVL) and T-cell IVL (T-IVL). Earlier onset of disease and female predominance were found in NK-IVL. NK-IVL was typically Epstein-Barr virus (EBV)-positive, whereas EBV was rarely detected in B-IVL. Cutaneous manifestations were common in NK-IVL with constant EBV infection. B-IVL showed a more favourable prognosis than T- or NK-IVL. Irrespective of immunophenotype, however, IVL showed a less favourable prognosis than ordinary lymphomas within the same immunophenotype. In summary, IVL of the B-, T- and NK-cell phenotypes is clinicopathologically distinct and shows similarities to their more common counterparts, i.e. diffuse large B-cell lymphoma, peripheral T-cell lymphoma, unspecified and extranodal NK/T-cell lymphoma, nasal type.