Effects of charge balance and hydrophobicity of the surface of cytochrome c on the distribution behaviour in an ionic liquid/buffer biphasic system.

Factors contributing to the different distribution behaviour of cytochrome c were investigated in a biphasic tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate and potassium phosphate buffer system, which shows a lower critical solution temperature. To change charge balance and hydrophobicity of cytochrome c, surface modification with a few modifier molecules was applied. Surface charge and hydrophobicity affected the distribution behavior of chemically modified cytochrome c in the tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate and potassium phosphate buffer biphasic system. The distribution ratio into tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate decreased with decreasing isoelectric point of cytochrome c. Furthermore, cytochrome c possessing a low isoelectric point showed different distribution ratio depending on surface hydrophobicity. Taken together, these findings indicate that isoelectric point and surface hydrophobicity of cytochrome c are important factors controlling the distribution behavior in temperature sensitive biphasic systems.

Apheresis education and certification for nurses.

Hematopoietic stem cell transplantation requires a wide-range of expertise and procedural competence, in which nurses play important roles throughout, including stem cell mobilization and collection by apheresis. Little is published about post-licensure nursing education in apheresis, which suggests that it proceeds at the discretion of individual institutions, supplemented with practical training by equipment manufacturers. Information can be obtained on a small number of apheresis training courses for nurses in Australia, Canada, Indonesia, Italy, The Netherlands, Sweden and Turkey, and on nurse certification systems in Turkey and the United States. There seems to be no certification officially linked to institutional accreditation or medical insurance reimbursement related to apheresis, except in Turkey. Because apheresis is associated with various adverse events, including citrate toxicity and vasovagal reactions, the Japan Society of Transfusion Medicine and Cell Therapy, in cooperation with 3 other speciality societies, started a "Qualified Apheresis Nurse" certification in 2010, when the Japan Marrow Donor Program officially added circulating stem cell collection to bone marrow harvest from unrelated donors as a source of hematopoietic stem cells. Questionnaire surveys, collected when nurses must renew or surrender their 5-year certification, show that our system matches nurses' learning desire and can be an objective and motive of their learning, thus leading to safer and more effective apheresis practice. We dare to imagine that an internationally standardized curriculum might emerge, to which we would contribute, and from which we would learn.

Predictors of vasovagal reactions during preoperative autologous blood donation: a single-institution analysis.

Studies examining risk factors associated with vasovagal reactions (VVRs) during autologous blood donations, especially in younger subjects, have been limited. The aim of the present study was to define risk factors for VVRs during preoperative autologous blood donation in patients, including those younger than 18 years old. We retrospectively analyzed 4192 autologous, preoperative blood donations between 2007 and 2015 at Okayama University Hospital. Eighty-seven (2.08%) of the patients experienced VVRs. VVRs occurred approximately three times as often in patients 0-17 years old (16/320, 5.0%) than in patients 18 years and older (71/3872, 1.8%). In particular, VVRs occurred more frequently in those 10-13 years old, and decreased with older age (P = 0.006). In a univariate analysis, younger age, lower body mass index, lower systolic blood pressure, lower body weight, lower total blood volume, female gender, first-time collection, and higher heart rate were associated with a higher incidence of VVRs. In a multivariate analysis, lower systolic blood pressure (P < 0.001), higher heart rate (P = 0.007), and first-time collection (P = 0.015), remained independent predictors of VVRs. These results emphasize the need for careful attention during blood collection.

Allogeneic hematopoietic stem cell transplantation for advanced extranodal natural killer/T-cell lymphoma, nasal type.

The prognosis for patients with advanced or refractory extranodal natural killer (NK)/T-cell lymphoma, nasal type (ENKL) is extremely poor. Thus, allogeneic stem cell transplantation (allo-HSCT) should be considered for this disease. However, reports of allo-HSCT for ENKL are limited because of the rarity of the disease. Here, we describe the clinical course of 12 cases of advanced and refractory ENKL treated with allo-HSCT, including five cases with cord blood transplant. With a median follow-up of 13 months (range, 1-168 months), seven patients are alive in remission, five have died, and one treatment-related death occurred. All patients with disease progression at transplant died of disease progression, whereas seven of eight patients with a complete or partial response are long-term survivors. Allo-HSCT is a feasible and promising consolidation therapy for advanced and relapsed ENKL. The disease status before allo-HSCT is well associated with general outcome, and thus induction treatment is very important for this disease.

Prediction of number of apheresis procedures necessary in healthy donors to attain minimally required peripheral blood CD34+ cells.

BACKGROUND: Allogeneic peripheral blood stem cell (PBSC) transplantation is widely performed as a curative therapy for hematopoietic malignancies. Donors for PBSC harvest (PBSCH) are usually healthy subjects and undergo granulocyte-colony-stimulating factor treatment and apheresis procedures. A considerable proportion of donors experience poor mobilization, necessitating additional harvesting or marrow collection or remobilization. Although some characteristics have been reported to correlate with poor mobilization, they may not be taken into account in selecting PBSC donors. To protect healthy donors, it is preferable to predict the number of apheresis procedures needed for PBSCH before the procedure is initiated. STUDY DESIGN AND METHODS: A retrospective cohort study of 83 subjects was conducted, using statistical models to predict the probability of obtaining a sufficient number of CD34+ cells (>or=2.0 x 10(6)/kg) in the first to the third apheresis procedures and the probability of failure to obtain sufficient cells within three apheresis sessions. This study explored potential candidate factors in an ordinal probit regression analysis. RESULTS: Significant factors predicting successful PBSCH were donor age, donor sex, and body weight difference between donor and recipient. The predictive model showed good agreement with the observed number of apheresis sessions. Simulation tables are presented with this model. CONCLUSION: The statistical model developed to predict the number of apheresis procedures for PBSCH may be useful for planning PBSCH in clinical practice.

Efficacy and feasibility of IDEA therapy for refractory or relapsed non-Hodgkin's lymphoma.

BACKGROUND: The effects of a novel salvage regimen, IDEA (ifosfamide, cytosine arabinoside, etoposide and dexamethasone), which does not include anthracycline or platinum, in patients with non-Hodgkin's lymphoma (NHL) were examined. PATIENTS AND METHODS: Thirty-four patients with refractory or relapsed NHL were treated with IDEA. RESULTS: The overall remission and complete remission rates were 67.6% and 35.3%, respectively. The toxicity of IDEA was tolerable. With a median follow-up of 14 months, one-year overall survival (OS) and progression-free survival rates were 75.1% and 43.7%, respectively. Adequate numbers of CD34(+) cells were obtained in 77.8% of the patients assigned to receive autologous peripheral blood stem cell (PBSC) transplantation. High-dose chemotherapy with autologous PBSC transplantation was carried out in 14 patients; their 3-year OS was 75.0%, with a median follow-up of 38 months. CONCLUSION: IDEA is an effective second-line chemotherapy regimen for NHL patients and has an excellent PBSC-mobilizing effect.

Clinical outcomes of unrelated donor umbilical cord blood transplantation for 30 adults with hematological malignancies.

BACKGROUND: Umbilical cord blood transplantation (CBT) has increasingly been used as a therapeutic option for adult patients for whom allogeneic stem-cell transplantation is not indicated, due to the availability of cord blood. However, myeloablative conditioning regimens are associated with significant mortality, and high relapse rates in reduced-intensity regimens may result in a poor rate of disease-free survival for those with advanced stages of hematological malignancies. Therefore, it remains unknown whether CBT is a truly effective option for such adults with high-risk disease, as well as for those with standard-risk disease. PATIENTS AND METHODS: Thirty adult patients with a median age of 45 years (range: 16-67) with standard or high-risk disease underwent CBT from unrelated donors at Okayama University Hospital between October 2002 and May 2007. Twenty-one patients had diseases classified as high-risk for transplantation. The median number of nucleated cells in infused cord blood was 2.65 x 10(7)/kg (range: 1.73-4.87). RESULTS: Twenty-three patients achieved neutrophil engraftment at a median time of 22 days (range: 13-42) after CBT. The cumulative incidence of grade II to IV acute graft-versus-host disease (GVHD) was 53.6% . Out of the 30 patients, 11 were alive and disease-free at a median time of 446 days (range: 124-1153) after CBT. The cumulative 1-year overall survival in patients with standard-risk or high-risk disease was 63.5% and 15.4%, respectively (p=0.01). CONCLUSION: Although from a retrospective study, these results suggest that unrelated donor CBT could be safe and effective for adult patients with standard-risk disease who cannot find a suitable HLA-matched volunteer marrow or peripheral blood donor.

[The biological characteristics of peripheral blood CD123+ myeloid dendritic cell].

AIM: To investigate the biological characteristics of CD123(+)DC derived from cord blood monocytes in myeloid culture system. METHODS: The monocytes isolated from peripheral blood were cultured with GM-CSF and IL-4 to induce DC for 7 days, and the related phenotype of DC was analyzed by flow cytometery. CD123(+)DC were purified with magnetic cell sorting separation technique. The morphological properties of CD123(+)DC was observed under confocal laser-scanning microscope and scan electron microscope. The concentration of IL-12 secreted by DC was analyzed by ELISA. The function of endocytosis and antigen presentation was evaluated by uptaking FITC-dextran and allogeneic mixed T lymphocyte reaction (MLR). RESULTS: FACS analysis showed the high expression of CD86 and CD11C, moderate expression of CD1a and CD123, low levels of CD83 on DC after induction by GM-CSF and IL-4 for 7 days, while the expression of CD304 was rather low. Well-distributed CD123 and CD11C were detected on DC. Purified CD123(+)DC showed typical morphology of immature DC with similar short cytoplasmic projections as CD123(-)DC. CD123(+)DC secreted lower level of IL-12, and exhibited more significant endocytosis and less antigen presentation function than that of CD123(-)DC(P<0.05). CONCLUSION: CD123(+)DC induced with GM-CSF and IL-4 might be more early phase of immature myeloid DC along with the DC differentiation, with special biological characteristics.

The effect of adding rituximab to CHOP-based therapy on clinical outcomes for Japanese patients with diffuse large B-cell lymphoma: a propensity score matching analysis.

We conducted a retrospective analysis to evaluate the impact on clinical outcomes of adding rituximab to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) treatment for diffuse large B-cell lymphoma (DLBCL) patients in Japan. A propensity score method was used to compensate for the non-randomized study design. From January 2000 to December 2004, 378 patients who were newly diagnosed with DLBCL at 13 institutes were enrolled: 123 in the rituximab plus CHOP-based chemotherapy (R+) group, and 255 in the CHOP-based chemotherapy only (R-) group. The complete response rate was significantly higher in the R+ group than in the R- group (77.7 vs. 69.4%, P < 0.001). The progression-free survival (PFS) at 2 years was 62.4% in the R+ group and 57.0% in the R- group. The 2-year overall survival (OS) was 76.9% for the R+ group and 70.5% for the R- group. A multivariate analysis revealed that the addition of rituximab was a strong independent prognostic factor for PFS (hazard ratio 0.64, 95% CI 0.43-0.96, P = 0.031). A subgroup analysis revealed that R+ particularly benefited younger patients (hazard ratio 0.25, 95% CI 0.08-0.75, P = 0.013). IPI also showed significant impact for PFS (hazard ratio 1.82, 95% CI 1.55-2.14 for one score increase, P < 0.001) as well as OS (hazard ratio 2.10, 95% CI 1.71-2.57, P < 0.001). In summary, the addition of rituximab to CHOP-based chemotherapy results in better outcomes for Japanese DLBCL patients, particularly younger patients.

Use of micafungin versus fluconazole for antifungal prophylaxis in neutropenic patients receiving hematopoietic stem cell transplantation.

A prospective randomized clinical trial assessed the efficacy and tolerance of micafungin compared with that of standard fluconazole treatment in patients undergoing hematopoietic stem cell transplantation (HSCT). Adult patients (n = 106) were randomly assigned to receive prophylaxis with either micafungin 150 mg (n = 52), or fluconazole 400 mg (n = 52). Success was defined as the absence of suspected, proven, or probable invasive fungal infection (IFI) through the end of therapy and the absence of proven or probable IFI through the end of the 4-week period following treatment. The overall efficacy of micafungin was comparable to that of fluconazole (94 vs. 88%; difference 6.0%; 95% confidence interval, -5.4 to +17.4%; P = 0.295). A total of 2 (4.0%) of 50 patients in the micafungin arm and 6 (12.0%) of 50 patients in the fluconazole arm received empirical antifungal therapy (P = 0.06). Micafungin treatment did not result in increasing adverse effects and had a safe profile as fluconazole in neutropenic patients. This randomized trial indicates that the efficacy and tolerance of micafungin 150 mg was comparable to that of fluconazole 400 mg, suggesting that micafungin at 150 mg daily represents a valuable new treatment option for antifungal prophylaxis in HSCT recipients.

Identification of CD123+ myeloid dendritic cells as an early-stage immature subset with strong tumoristatic potential.

CD123 has been identified as a specific surface marker for plasmacytoid dendritic cells (PDCs). However, CD123 has recently been shown to be expressed on freshly isolated or in vitro generated myeloid dendritic cells (MDCs). In this article, we investigated whether the expression of CD123 on monocyte-derived MDCs was related to their function, especially to tumor-inhibiting potential. MDCs were induced from cord blood CD14+ monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7 days, and then CD123+ cells were isolated by positive immunomagnetic cell selection. We observed that CD123+ cells lost monocyte CD14 expression, acquired immature myeloid dendritic cell phenotype and morphology. They exerted more significant endocytosis and less antigen-presenting function than CD123(-)MDCs which are often referred to as typical MDCs. Meanwhile, CD123+ MDCs exhibited more significant tumor-inhibiting activity toward hematological tumor cell lines of U937 and Jurkat even at a low effector:target ratio. CD123+ MDCs expressed higher level of cytoplasmic TNF-alpha-related apoptosis-inducing ligand (TRAIL), but no detectable surface TRAIL and very little soluble TRAIL. Pretreatment with recombinant human TRAIL receptor 2:Fc fusion protein significantly reduced the tumor-inhibiting effect of CD123+ MDCs, but not of CD123(-) MDCs. Overall, our data demonstrated that CD123+ MDCs were an early-stage immature DC subset, with a significant tumor-inhibiting activity partially via involvement of enhanced cytoplasmic TRAIL.

Increased incidence of interstitial pneumonia by CHOP combined with rituximab.

Several authors have reported interstitial pneumonia (IP) during rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy, while others have encountered Pneumocystis jirovecii pneumonia during rituximab-combined bi-weekly CHOP. Herein, we report that 13 of 90 (14%) patients developed IP during R-CHOP therapy, compared with none of 105 patients treated with CHOP alone as a historical control. There were no differences in baseline data between patients undergoing the two therapies. Among R-CHOP-treated patients, serum beta-D-glucan was increased in 8 of 12 (75%) IP patients compared with none of 30 non-IP patients examined. In five IP patients who underwent sputum evaluation, two were positive for P. jirovecii by the polymerase chain reaction and another two were positive for Candida albicans. No other organisms were detected as causative pathogens. Treatment with steroids, sulfamethoxazole-trimethoprim (ST), and antifungals was effective. Our results suggest that R-CHOP raises the incidence of IP, possibly through increasing the susceptibility to P. jirovecii and fungal infection. The need for prophylactic antifungals and ST during R-CHOP should be evaluated by randomized controlled trials.

Rapid hematopoietic progenitor mobilization by sulfated colominic acid.

Hematopoietic progenitor cells (HPCs) can be mobilized from bone marrow (BM) to the blood by G-CSF. In this process, CXCR4 and CD26 play critical roles. Sulfated colominic acid (SCA) inhibits HIV entry, the step which requires CXCR4 and CD26 as co-receptors. Thus, we hypothesized that SCA would modulate HPC trafficking. We first found that SCA mobilized HPCs rapidly via CD26-independent mechanism. In vitro progenitor migration toward chemokine SDF-1 was significantly enhanced by SCA, and it was completely abrogated by CXCR4 inhibition. This likely originated from the inhibition of CXCR4 down-regulation after interaction with SDF-1. Serum SDF-1 level increased after SCA injection, whereas no change was observed in BM and bone. These results suggest that SCA induces HPC mobilization by modulating CXCR4 function resulting in attraction toward increased SDF-1 in the circulation. Furthermore, we confirmed an additive effect with G-CSF in mobilization. SCA may provide an efficacy in clinical mobilization.

Predominant infiltration of monocytes in chronic graft-versus-host disease.

Pathogenesis of chronic graft-versus-host disease (cGVHD) is largely unknown. It is important to determine the responsible cell types and the factors that play roles to recruit these cells into sites of disease. We examined whether monocytes and chemokine fractalkine/receptor CX3CR1 axis might be involved. We found that the absolute number of CX3CR1+ monocytes in the blood was significantly decreased in patients with severe cGVHD. Immunohistochemical staining revealed the extensive infiltration of CD14+ cells as well as strong expression of fractalkine in the cGVHD skin. The number of infiltrated CD14+ cells on the margin of fractalkine+ epidermis was larger in cGVHD skin compared to that of acute graft-versus-host disease, whereas no difference was observed in CD3+ T cells. These results suggest that CX3CR1+ monocytes may be recruited from the circulation to the fractalkine+ epidermis in cGVHD, and highlight these cells and this chemokine/receptor axis as additional targets for cGVHD therapy.

Loss or down-regulation of HLA class I expression at the allelic level in freshly isolated leukemic blasts.

Loss or down-regulation of human leukocyte antigen (HLA) class I expression has been demonstrated in a variety of solid tumors. To date, such altered HLA expression has not been studied extensively in freshly isolated leukemic blasts. If it occurs, leukemic cells could escape T-cell surveillance as a consequence. Genotypes of nine leukemic cell lines were determined using a polymerase chain reaction for HLA classes I and II. Cells were also examined for HLA beta2-microglobulin, and allele-specific HLA protein expression using flow cytometry. Next, 44 samples of freshly isolated leukemic blasts from 43 patients with malignant hematological diseases were examined for allele-specific HLA expression using flow cytometry. Microsatellite analysis was performed to determine heterozygosity in the HLA region on chromosome 6. Genotype analysis for HLA class I together with microsatellite analysis demonstrated loss of HLA haplotype in HL-60 cells. No loss of HLA haplotype was observed in 44 samples of freshly isolated leukemic blasts. As reported previously, flow cytometric analysis rarely demonstrated loss or down-regulation of HLA expression at initial diagnosis (3/39; 7.7%); however, this was evident in two of five cases in relapse (40.0%), which contrasts with previous reports. In one patient with acute leukemia, HLA-A2 cell surface expression was present at initial diagnosis, lost at relapse, and completely restored after 48 h of culture in the presence of interferon-gamma. These results suggest loss of allele-specific HLA expression may be involved in the pathogenesis of relapse in patients with leukemia. The findings should be valuable in designing new strategies for clinical immunotherapy.

Chronic lymphoproliferative disorder with regulatory T-cell phenotype.

We report a case of T-cell chronic lymphoproliferative disorder (CLPD) that shows neither features of T-cell prolymphocytic leukemia nor disease progression for more than 34 months. Flow cytometric analyses of the lymphocytes revealed high expression of CD4 and CD25. Up-regulation of Foxp3, a master regulatory gene for developmental differentiation of regulatory T cells (Treg), was confirmed at mRNA and protein levels. To our knowledge, this is the first case of extremely indolent CLPD with Treg phenotype.

Calcineurin inhibitor-induced irreversible neuropathic pain after allogeneic hematopoietic stem cell transplantation.

The calcineurin inhibitors (CIs) cyclosporine A and tacrolimus are essential for graft-versus-host disease prophylaxis but are associated with adverse effects, including neurotoxicity. We report a case of irreversible CI-induced neuropathic pain following allogeneic hematopoietic stem cell transplantation. The patient developed dysesthesia, electric shock-like pain, and severe itching followed by intractable analgesic-resistant pain in the lower extremities. There were no abnormal radiographic findings, and there was no improvement with a reduction of CI dosage or with administration of a calcium channel blocker. These clinical findings are similar to but inconsistent with CI-induced musculoskeletal pain syndromes previously reported in organ transplantation.

Serum cytokine concentrations and acute graft-versus-host disease after allogeneic peripheral blood stem cell transplantation: concurrent measurement of ten cytokines and their respective ratios using cytometric bead array.

Both inflammatory and anti-inflammatory cytokines have been reported to be associated with acute graft-versus-host disease (aGVHD). However, their role and possible mutual interactions during aGVHD are not well understood. Eight patients with aGVHD and eight without who had undergone allogeneic HLA-identical peripheral blood stem cell transplantation were studied. The patients had no other complications known to affect serum concentration of cytokines, including infection. Serum concentrations of IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF-alpha and IFN-gamma were concurrently measured by a new technique, the cytometric bead array (CBA). We found that serum concentrations of IL-5, IL-6 and IL-10 were significantly higher in patients with aGVHD than in patients without it. By ratiometric analysis, the ratios of IL-5/IL-2, IL-5/IL-4, IL-6/IL-4 in patients with aGVHD were increased compared to the patients with no evidence of aGVHD. ROC analysis demonstrated that the ratio of IL-5/IL-4 was the most sensitive parameter associated with aGVHD. The second best marker of aGVHD was increased IL-5 concentration. Thus, our results indicate that the ratio of a particular cytokine/cytokine could be a potential diagnostic marker for aGVHD, more sensitive that the serum level of a given cytokine. This observation is consistent with a cross-talk among some cytokines and their possible interactions via respective receptors on cytokine-producing cells; these interactions may play an important role in pathogenesis of aGVHD. Further studies including a large number of patients and concurrent measurement of a variety of cytokines are needed to fully assess the diagnostic value of the cytokine ratiometric analysis. The CBA methodology provides a convenient and useful tool in such studies.