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Acquired aplastic anemia (AA) is a bone marrow failure disorder characterized by pancytopenia, and immunosuppressive therapy (IST) is the optional first-line management. Several studies identified the influencing factors on IST response; however, there are still a considerable number of patients suffering from poor prognoses. In this study, we enrolled 61 AA patients aged ≤ 40 years old, and whole-exome sequencing (WES) found unexpected high FANC heterozygous germline mutations (28/61, 45.9%). Patients with FANC mutations have a significantly lower absolute reticulocyte count and CD34+ % in the bone marrow and also lower 3-, 6-, and 9-month IST response than that without mutation, which were 0% vs. 25% (P = 0.017), 26.3% vs. 42.1% (P = 0.495), and 29.4% vs. 72.2% (P = 0.011), especially in anti-thymocyte globulin combined with the cyclosporin A (ATG + CsA) group, which were 0% vs.33.4% (P = 0.143), 25% vs.83.3% (P = 0.103), and 25% vs. 100% (P = 0.003), respectively. The event-free survival in the FANCwt group was also better than that in the FANCmut group (P = 0.016) and also showed in patients who received ATG + CsA treatment (P = 0.045). In addition, all the adverse effects of FANC germline mutation were not significant in stem cell–transplanted group. Our result indicated that the WES-based detection of FANC heterozygous germline mutations may have a great meaning in predicting IST response of acquired AA. This study was registered at chictr.org.cn (# ChiCTR2100054992).

Resibufogenin (RBG) is an active ingredient of toad venom that also has antitumor potential. The present study aimed to investigate the role of RBG in multiple myeloma (MM) and the underlying action mechanism involving the PI3K/Akt signaling pathway. A human MM cell line, RPMI8226, was treated with RBG and/or insulin-like growth factor 1 (IGF-1; an activator of the PI3K/AKT signaling pathway). Cell viability and apoptosis were detected using Cell Counting Kit-8 and flow cytometry, respectively. Cell migration and invasion were detected using a Transwell assay. In addition, the epithelial-mesenchymal transition (EMT)-associated proteins (E-cadherin, N-cadherin and Vimentin) and the PI3K/AKT pathway-associated proteins [AKT, phosphorylated (p)-AKT, PI3K and p-PI3K] were measured using western blotting. RBG inhibited the viability, migration and invasion, and promoted the apoptosis of RPMI8226 cells in a dose-dependent manner. RBG at concentrations of 4 and 8 µM upregulated E-cadherin, and downregulated N-cadherin and Vimentin in RPMI8226 cells. RBG also decreased the protein expression of p-AKT and p-PI3K in a dose-dependent manner. In addition, the intervention of IGF-1 weakened the inhibitory effects of RBG on the malignant characteristics of MM cells. RBG-induced inhibition of EMT and the PI3K/AKT pathway were also weakened by IGF-1 treatment. In conclusion, RBG inhibited viability, migration, invasion and EMT, and promoted the apoptosis of MM cells by blocking the PI3K/AKT signaling pathway.

Between 2015–2018, we collected approximately 2000 water column measurements of methane (CH4) and nitrous oxide (N2O) concentrations in the North American Arctic Ocean during summer and early fall. We also obtained 25 measurements of CH4 and N2O concentrations in rivers along the Northwest Passage and Ellesmere Island in mid-summer 2017–2019. Our results show that N2O is generated in the highly productive Bering and Chukchi Seas and transported northeastward, producing a persistent subsurface N2O peak in the Beaufort Sea. The Chukchi and Beaufort Sea sediments are a significant source of CH4 to the water column. These sedimentary sources and associated water column consumption display significant spatial gradients and interannual variability. CH4 isotope data demonstrate the importance of CH4 oxidation across the study region. We find that rivers are not a significant source of CH4 or N2O to the Arctic Ocean at the time of year sampled. The estimated annual sea-air flux across the study region (2.3 million km2) had a median (first quartile, third quartile) of 0.009 (0.002, 0.023) Tg CH4 y-1 and −0.003 (−0.013, 0.010) Tg N y-1. These results suggest that the North American Arctic Ocean currently plays a negligible role in global CH4 and N2O budgets. Our expansive dataset, with observations at many repeat stations, provides a synopsis of present-day Arctic CH4 and N2O distributions and their range of variability, as well as a benchmark against which future climate-dependent changes can be evaluated.

Background: Autoimmune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) can be observed in Waldenström Macroglobulinemia (WM). The cause of AIHA should be carefully distinguished during the disease management. Case Presentation: A 63-year-old female WM patient complicated with thrombocytopenia, who was admitted to our department with a complaint of abdominal pain. After first half of bortezomib/dexamethasone/rituximab (BRD) chemotherapy, her platelet level recovered, but subsequently decreased to extremely low level (around 1-2×109/L), and the patient suffered from platelet transfusion refractoriness. During the management of refractory thrombocytopenia, the patient developed severe hemolytic anemia, and further tests confirmed warm AIHA. FcγRIIα polymorphism test showed that the patient had FcγRIIα-131RH, which implied that the AIHA may not be WM-related. Given the effects of ibrutinib in controlling WM, ibrutinib single treatment was started, which quickly corrected the thrombocytopenia within five days, but not hemolysis. With a relatively safe platelet level, eltrombopag was stopped, and the hemolysis alleviated within three days after eltrombopag withdrawal. Conclusion: This is the first report on eltrombopag-induced AIHA, and we should always keep in mind of the drug induced hemolysis even in disorders with autoimmune background.

Patients with advanced stage of squamous cell carcinoma of esophagus have a poor prognosis with a lethal outcome. In order to explore the feasibility and effectiveness of dendritic cell (DC)-based immunotherapy for squamous cell carcinoma of esophagus, we performed a phase I/II clinical trial of monocyte-derived dendritic cells (moDCs) pulsed with SART1 peptide in seven patients with advanced stage of this disease. Although the feasibility of this therapy was definite, the effectiveness was not clearly confirmed in advanced stage of squamous cell carcinoma of esophagus. However, in vitro study revealed that moDCs generated for this therapy possessed a potent ability of inducing SART1 peptide-specific cytotoxic T lymphocytes (CTLs). In addition, these moDCs were demonstrated to be able to produce exosomes with an antigen presenting ability for inducing SART1 peptide-specific CTLs. ELISPOT assay using cryopreserved patient's lymphocytes demonstrated that IFN-γ ELISPOTs were increased after four times of SART1 peptide-pulsed moDC vaccinations compared with before the vaccination in a patient. The present study demonstrated that moDCs prepared from advanced stage of squamous cell carcinoma of esophagus possess a good immune function and in vivo immune responses (detected by ELISPOT assay) were evoked by the infusion of these moDCs. These findings suggest that DC-based immunotherapy could be one of the modalities applicable for squamous cell carcinoma of esophagus.

To understand the risks associated with aplastic anemia (AA) in 4 cities of Zhejiang Province, China, with special focus on the joint contributions of multiple risks. Based on an Electronic Data Capture (EDC), a case control study was carried out. Data regarding socio-demographic, diseases history, living habits, and exposures to toxic substances, etc., were collected through survey questionnaires. t Test, chi-square test, or non-parametric rank sum test, and univariate and multivariate Logistic regression analysis were conducted to analyze data. The univariate logistic regression analysis results indicated that among all study participants (n = 1802), AA was associated with over 30 risks, in terms of their individual behaviors, daily and environmental exposures, diseases history, and family history. Multivariate logistic regression analysis further confirmed that the independent risks related to AA included presence of chemical factory within 3 km of living residence (odds ratio [OR] = 8.73, 95% CI: 1.42–53.74, P = .019), living in a newly decorated house/apartment (OR = 25.37, 95% CI: 4.44–144.81, P

BACKGROUND: This study aims to evaluate the clinical efficacy and prognosis of currently popular treatments for patients with transfusion-dependent non-severe aplastic anemia (TD-NSAA). METHODS: Medical records of patients with TD-NSAA were collected from three hospitals and divided into four groups based on their treatment. Treatment efficacy was evaluated at 6 months after treatment, with subgroup analysis based on patients' disease courses. Adverse events and 3-year survival status were also reviewed. RESULTS: A total of 175 patients' records were collected in this study. The analysis of treatment efficacy indicated that the most effective treatment was allo-hematopoietic stem cell transplant (HSCT) with the response rate of 84.62%, which was followed by Anti thymocyte globulin and ciclosporin A (ATG + CsA) (68.52%), CsA + androgen (26.92%), and androgen (0%), respectively. The subgroup analysis showed that for patients with less than 6-month disease courses, ATG + CsA treatment could achieve similar response rate as allo-HSCT (84.00%). Besides, ATG + CsA showed better efficacy than CsA + androgen in all stratified arms (P CONCLUSIONS: Therefore, we concluded that ATG+ CsA could achieve better efficacy to treat TS-NSAA than did CsA+ androgen. Disclosures No relevant conflicts of interest to declare.

The induction of donor T-cell anergy to recipient cells for reducing GVHD could be one way of expanding donor candidates for HLA-mismatched hematopoietic SCT. The present study was designed to clarify whether recipient cell-specific T-cell anergy could be induced by priming donor lymphocytes with recipient monocyte-derived DCs (mo-DCs) irradiated with ultraviolet-C (UV-C). By irradiation of mo-DCs with UV-C, the expression of DC-associated surface phenotypes such as CD83, CD80, CD86 and CD40 was reduced and the antigen-presenting ability of UV-C-irradiated mo-DCs was clearly decreased. By co-culturing normal donor 1 lymphocytes with UV-C-irradiated donor 2 immature mo-DCs, the response of the lymphocytes to donor 2 mature mo-DCs was markedly reduced as compared with that of the lymphocytes prestimulated with non-irradiated donor 2 immature mo-DCs or UV-C-irradiated mo-DCs derived from a different individual donor 3. The present study demonstrated that recipient cell-specific T-cell anergy could be induced by priming donor lymphocytes with UV-C-irradiated recipient immature mo-DCs in hematopoietic SCT. These data suggest the applicability of donor graft cells, which have been prestimulated with UV-C-irradiated recipient immature mo-DCs, for expanding donor candidates in HLA-mismatched hematopoietic SCT.

Experimental characterization of time-dependent properties for rectangular hollowcored, continuous fiber reinforced, commingled recycled plastic extruded forms under long-term (creep) flexural loading has been presented in this paper. Finite element based computer models have been developed to predict the effects of damage progression in such reinforced extruded plastic forms. In the long-term (creep) tests, reinforced and unreinforced extrusions with varying compositions were used as specimens. These extruded specimens were submerged in heated water and subjected to different loads. Experimental results indicate that fiber micro-buckling and fiber–matrix interface failure occur during the creep loading environment. The fiber micro-buckling occurs over time, compared with similar but dramatic damage that occurs in a short-period of time during short-term (static) loading, as discussed in Part I of this work. Experimental data also shows that these damage modes significantly reduce the long-term (life cycle) flexural properties, and the specimens with a coupling agent demonstrated much better performance. “Damage dependent” finite element models were developed using different material property types to represent the glass-fiber roving, fiber–matrix interface and plastic matrix respectively. Material nonlinearity of the plastic matrix has been incorporated along with stress-based failure criteria to account for fiber–matrix interfacial shear failure and local fiber micro-buckling. A user-defined material model has been incorporated into an industrial standard finite element software package to accommodate damage progression. The developed finite element based model(s) have correlated well with the long-term (creep) test results, and can provide a valuable tool in designing reinforced plastics for long-term loading conditions.

Experimental results on fiber micro-buckling of continuous glass-fiber reinforced hollow-cored recycled plastic extrusions under creep tests are introduced in the paper. The full size specimens with dimensions in 2.5×3.5×42 in. 3 were submerged in warm water at a temperature of 125 °F when they were under a four-point bending creep test. The results show that the micro-buckling of the embedded glass-fiber roving occurs along 90% the length of the specimen on the upper inner surface (compressive side) and mainly during the time between 5 and 100 h from the initial loading moment. The micro-buckling causes the steady-state apparent flexural modulus of the composite drop faster, and it also causes the plastic matrix local crackling which subsequently leads to the structural failure of the composite. The stress level has little effect on the steady-state creep rate. The results also show some evidence that the plastic matrix becomes more brittle when it is submerged in warm water for certain long time. From the results, it is indicated that the pattern or distribution of the micro-buckling is significantly different from that of short-term four-point bending test for the same composite materials, for which the fiber micro-buckling occurs locally only on the middle section of the specimen.

The experimental results of fiber local micro-buckling and partial failure due to the fiber micro buckling of extruded continuous glass fiber reinforced hollow-cored recycled plastic lumber under four point bending are presented in the paper. The test specimens are commercially produced E-glass roving reinforced recycled HDPE extrusions (2.5″ by 3.5″ hollow-cored cross section and 42″ in length) with/without coupling agent and different color pigment. The test results show that the fiber micro buckling causes a significant drop in strength of the extruded lumber and subsequently causes the matrix cracking that will lead to the total structural failure. It becomes a concern for the continuous fiber reinforced plastic beams used in construction fields because most of them are under bending loads. The micro buckling occurs on the compressive half of the hollow beam when it undergoes bending loads. The critical compressive stress is reasonably close to the predictions of Rosen’s model if considering a factor of 0.63 as proposed by Hahn and Williams (1986). For the specimens without the additives and coupling agent, the results show no sign of fiber micro buckling though their strength is much lower than those with the additives and coupling agent. In addition, it is shown that the fiber micro buckling initiates the matrix longitudinal as well as latitudinal cracking.

The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34+ hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34+ progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34+ hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7--11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-alpha. By the two-step culture, total nucleated cells were increased 208+/-66 (+/-SD, n=13), or 94+/-29 (n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34+ cells at the time of starting culture. Out of the total nucleated cells, 23 +/-10.4% of cells in CB cell culture and 25 +/-5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a+ cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34+ cells using SCF, GM-CSF and TNF-alpha. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a+ DC generated from CD34+ cells, and the phenotypes and functions of these CD1a+ DC could be enhanced efficiently by treatment with a calcium ionophore agent.

The aim of this study was to determine the association of the microRNA-146a (miR-146a) polymorphism with the risk of diffuse large B cell lymphoma (DLBCL). The genotyping of miR-146a rs2910164 polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results showed that CC genotype and C alleles distribution in the DLBCL patient was significantly higher than that of the controls (p = 0.01 and p = 0.01, respectively). No significant differences were found between the two subgroups when stratified by clinical characteristics including sexual, age at admission, performance status, pathological type, Ann Arbor stage, LDH and β2-MG value. The miR-146a expression was detected by the Taqman real-time PCR. The result showed that the miR-146a expression was notably upregulated in DLBCL patients when compared with controls (p = 0.02). In addition, the miR-146a expression of CC genotypes subgroup was drastically downregulated than that of GC/GG genotype subgroup in DLBCL patients (p = 0.0003), suggesting that this polymorphism can functionally affect the expression of miR-146a. In conclusion, it was shown that the miR-146a rs2910164 polymorphism is associated with the risk of DLBCL in the Chinese Han population.

Hollow-cored structural profiles have been processed using blends of polyethylene with continuous glass-fiber roving reinforcement through an offset extrusion process. These profiles have been produced with and without coupling agents. The intent has been to achieve high bending stiffness (El) by locating the reinforcing fibers in the mid-plane of the extrusion walls. However, the process was not optimally tuned which results in the fibers being nearer the inner surfaces of extrusion walls. Four-point bending and compression tests have been performed on the hollow-cored beams according to ASTM D6109 and ASTM D61 12. The nonlinear elastic nature of the unreinforced plastic blends has been quantified by performing tensile tests according to ASTM D638. A finite element-based computational model has been developed through the assembly of "repeating cells" representing individual fiber rovings and neighboring plastic. Comparing experimental and computational results demonstrates the validity of the model in predicting bending stiffness (El). Furthermore, simple multi-material beam models are shown to over-predict bending stiffness. The experiments also provide evidence suggesting shear failure at fiber-matrix interfaces.

Application of fresh herbs is a kind of special forms of traditional Chinese medicine. In China, there is a long and rich experience in clinical application of fresh herbs. Many studies showed that the efficacy of fresh herbs was better than that of dried herbs, but the further study about the difference of their chemical composition, effective components and the overall material basis were few. In this paper, the ideas and methods to study on material basis of the fresh herbs by comparing the difference of the fresh and dry herbs in medicine chemical composition and pharmacological activity of effective components with modern advanced separation, analysis and screening technology under the "Constituent structure theory" were proposed. It was an effectual method for studying on the reasonable development of Chinese medicine and fresh herbs resources.

Although dendritic cells (DC) can be cultured from cord blood (CB) CD34+ progenitor cells, the generation of DC from CB monocytes has not been reported. In this paper, we explored the generation of DC from CB monocytes to establish the simplest way to obtain a substantial number of DC from CB. We isolated monocytes from CB mononuclear cells (CB-MNC) by the plastic adherence method. These adherent cells (monocyte-rich cells) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) or in serum-free X-VIVO 15 medium (SFM) for 7 days, both of which contained 100 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng/ml interleukin-4 (IL-4) with or without 10 ng/ml tumor necrosis factor-alpha (TNF-alpha) (added at day 5). In the presence of GM-CSF and IL-4, CB-adherent cells became nonadherent, acquired DC morphology, and showed increased expression of CD1a, CD80, CD86, and HLA-DR; they lost membrane CD14 and some cells with the expression of CD83 and CMRF-44 were generated. With the addition of TNF-alpha to these cultures and culturing for further 2 days, the proportion of CD83+ cells was elevated in both the FBS and SFM culture systems, compared with the culture without TNF-alpha. In the culture with TNF-alpha, cells expressing CD1a, CD80, CD86, HLA-DR, and HLA-DQ were markedly increased. TNF-alpha-treated cells were demonstrated to be stronger stimulators for proliferation of both allogeneic CB lymphocytes and PB lymphocytes than were cells not treated with TNF-alpha. The yield of CD83+ DC at day 7 of cultures was 4.9 +/- 1.1 x 10(5) or 3.0 +/- 0.5 x 10(5) per 1.2 x 10(7) CB-MNC plated initially when cultured in FBS or SFM, respectively. These results have shown that a substantial number of mature DC could be generated from CB-adherent cells even by serum-free culture. We then compared these CB-adherent cell-derived DC (CB-DC) with peripheral blood (PB)-adherent cell-derived DC (PB-DC) in cell-surface phenotype and function. We found day 7 CB-DC have lower expression of CD80, CD1a, CD83, and CMRF-44 than day 7 PB-DC, but CB-DC have a similar capacity to stimulate the proliferation of both allo-CB lymphocytes and PB lymphocytes, compared with PB-DC. CB-DC cultured with GM-CSF and IL-4 have almost identical capacity of phagocytosis to take up fluorescein isothiocyanate (FITC)-dextran and Lucifer yellow (LY), compared with PB-DC. In summary, our findings suggest CB adherent cells, when cultured with GM-CSF, IL-4, and TNF-alpha, are a potent source of functional DC. Thus, CB-DC as well as PB-DC may become valuable tools for immunotherapy.

SUMMARY In order to develop an effective immunotherapy for hematological malignancies, we investigated the applicability of class II transactivator (CIITA), which had been demonstrated to regulate the expression of MHC class II (MHC-II) by assembling the transcription factors of MHC-II molecules, for immunotherapy by potentiating the antigenicity of tumour cells by inducing MHC expression. First, 32 hematopoietic cell lines were analysed for the expression of HLA-DR, CIITA, RFX5 or HLA-ABC. Fourteen cell lines were positive and 18 were negative for HLA-DR. All the 14 HLA-DR positive cell lines were demonstrated to express CIITA mRNA by RT-PCR. On the other hand, in all the 18 HLA-DR negative cell lines, the expression of CIITA was not demonstrated. RFX5, which is one of the transcription factors of MHC-II, was expressed ubiquitously in all 32 cell lines. Three cell lines out of 23 hematopoietic cell lines examined were negative for HLA-ABC, and all three of these cell lines were negative for both HLA-DR and CIITA expression. Furthermore, CIITA cDNA was transfected into K562 cells, which were negative for HLA-ABC, -DR and -DQ, but positive for HLA-DP. The transfection rendered HLA-DR negative to positive and increased the expression level of HLA-DP, but HLA-DQ remained negative. In addition to HLA-DR, HLA-ABC was also induced to express by the transfection of CIITA gene. The present study demonstrated that the expression of HLA-DR in hematopoietic cells is regulated in subordination to CIITA and the expression of HLA-DR (and HLA-ABC in K562) is induced by transfection with the CIITA gene. These findings revealed the applicability of CIITA in potentiating antitumour immunity of HLA-DR negative tumour cells for immunotherapy of hematological malignancies. Copyright # 1999 John Wiley & Sons, Ltd.

This study describes the effect of dendritic cells (DC) pulsed with tumor cell extracts on the induction and activation of autologous and HLA-identical sibling tumor cell specific cytotoxic T lymphocytes (CTL). The subjects of the study were patients with hematological malignancies (acute myeloid leukemia, acute lymphoblastic leukemia, and multiple myeloma), and stem cell transplant donors. Although lymphocytes primed with non-pulsed DC did not show definite cytotoxic activity against autologous or HLA-identical sibling patient tumor cells, both autologous and HLA-identical sibling CTL activated by DC pulsed with tumor cell extracts demonstrated markedly significant killing activity against tumor cells in the fashion of higher cytotoxicity with the increase of effector:target ratio (p

Dendritic cells (DC), the most potent 'professional' antigen-presenting cells, hold promise for improving the immunotherapy of cancer. In this study, we investigated the ability of normal donor DC pulsed ex vivo with 12 mer bcr-abl (b3a2) peptide to generate b3a2-specific autologous or HLA-identical sibling donor's cytotoxic T-lymphocytes (CTL). DC that were grown from normal peripheral blood adherent cells or purified DC precursors in the presence of GM-CSF and IL-4, were pulsed with b3a2-peptide then were induced to become mature and functional cells by the addition of TNF-alpha. These peptide-pulsed mature DC elicited a potent b3a2-specific CTL response in vitro. The b3a2-peptide pulsed DC-primed peripheral blood lymphocytes (PBL) displayed significantly higher cytotoxic activity compared with peptide non-pulsed DC-primed PBL against target cells, which are b3a2 positive marrow cells derived from HLA-identical sibling chronic myelogenous leukemia (CML) patient, or peptide-pulsed autologous macrophages (P < 0.001). In addition, the b3a2 peptide-pulsed DC-primed and non-pulsed DC-primed PBL showed no cytotoxic response against peptide non-pulsed autologous macrophages. These findings revealed that normal donor PBL pre-immunized with b3a2-peptide pulsed autologous DC could increase the graft-versus-leukemia effect without exaggerating graft-versus-host-disease. Both CD8+ and CD4+ T lymphocytes were shown to be involved in the effector cell populations. The b3a2 peptide-pulsed DC-primed T cells were significantly superior in their production of GM-CSF and TNF-alpha compared with peptide non-pulsed DC-primed T cells. These intriguing preclinical results imply the feasibility of developing b3a2 peptide-DC based protocol for in vitro sensitization of normal donor leukocytes before donor leukocyte transfusions for patients with CML, who relapsed after HLA-matched sibling bone marrow transplantation.

The induction of donor T cell anergy to recipient cells for reducing GVHD could be one way of expanding donor candidates for HLA-mismatched hematopoietic stem cell transplantation. The present study was designed to clarify whether recipient cell-specific T cell anergy could be induced by priming donor lymphocytes with recipient monocyte-derived dendritic cells (mo-DCs) irradiated with UV-C. By irradiation of mo-DCs with 100 J/m2 or more UV-C, the expression of DC-associated surface phenotypes such as CD1a, CD54, CD40, CD80, CD83 and CD86 was reduced in one day after irradiation and the effects of UV-C irradiation continued for at least 7 days. By irradiation of mo-DCs with 100 J/m2 or more UV-C, the antigen-presenting ability of both immature and mature mo-DCs, which was examined by 3H-thymidine incorporation assay, was clearly decreased at UV-C dose-dependent manner. Proliferation of CSFE-labeled lymphocytes by the stimulation with immature or mature Mo-DCs was suppressed by 300 J/m2 UV-C irradiation to immature or mature mo-DCs. The response of normal donor 1 lymphocytes, which had been co-cultured with 300–3,000 J/m2 UV-C-irradiated donor 2 immature mo-DCs for 7 days, against mature donor 2 mo-DCs in mixed leukocyte culture (MLC) for 7 days was markedly reduced, compared with the response of the donor 1 lymphocytes co-cultured with non-irradiated donor 2 mo-DCs or UV-C-irradiated mo-DCs derived from a different individual donor 3. CFSE-labeling analysis of donor 1 lymphocytes, which were co-cultured with 300 J/m2 UV-C irradiated donor 2 mo-DCs in the first MLC and then stimulated with donor 2 mature mo-DCs in the second MLC, showed that by stimulation with mature mo-DCs in the second MLC, the proliferation of donor 1 lymphocytes co-cultured with UV-C irradiated donor 2 mo-DCs in the first MLC was less than that of the lymphocytes co-cultured with non-irradiated mature mo-DCs. Flow cytometry analysis of the lymphocytes co-cultured with 300 J/m2 UV-C irradiated mo-DCs using surface CD4/CD25 and cytoplasmic Foxp3 monoclonal antibodies revealed that there was no increase of regulatory T cell population in the lymphocytes co-cultured with UV-C-irradiated immature mo-DCs, compared with the lymphocytes co-cultured with non-irradiated immature mo-DCs. Cell proliferation in allogeneic MLC consisting of lymphocytes as responder cells and mature mo-DCs as stimulator cells was not suppressed by the addition of the lymphocytes co-cultured with UV-C-irradiated immature mo-DCs. The present study demonstrated that recipient cell-specific T cell anergy could be induced by priming donor lymphocytes with UV-C-irradiated immature mo-DCs derived from a recipient and the T cell anergy was not associated with regulatory T cells. These data suggest the applicability of donor graft cells, which have been pre-stimulated with UV-C-irradiated recipient immature mo-DCs for expanding donor candidates for HLA-mismatched hematopoietic stem cell transplantation.

Objective: To explore the feasibility of Chinese herbal medicine in haploidentical related allogeneic bone marrow transplantation (Allo-BMT) without T-cell depletion for the treatment of leukemia. Methods: From 2003 to 2005, four patients with CML and four patients with AML received allo-BMT with grafts from 1 or 3 HLA antigen mismatched related donors. All patients were preconditioned with high dose Ara-c, cyclophosphamide (CY)and total body irradiation (TBI) or Busulfan (Bu). Donors were given G-CSF 250μg/d for seven days prior to marrow harvest. GVHD prevention regimen consisted of CSA, MTX, ATG, MMF and CD25-mab. Chinese herbal medicine was given orally to all patients after transplantation according to the theory (syndrome differentiation) of Traditional Chinese Medicine (TCM). One or 2 weeks after transplantation, TCM were given with the purpose to increase Qi and cultivate Yin, strengthen the function of Spleen and Stomach. At the time of bone marrow convalescence, about 1 month post transplantation, different TCM was prescribed for increasing Qi, cultivating Blood, strengthening the function of Kidney, and fasting distillation. Results: All patients were successfully engrafted. The median days of granulocyte >0.5×109/L and platelet >20×109/L were 12 (10–14) and 20(18–25) days, respectively. Seven patients developed grade I-IVGVHD, with two of them advanced to grade IV gut and liver GVHD and died of concurrent infection eventually. Two patients developed hemorrhagic cystitis. Six patients were alive in a disease-free situation after median following-up of 20 (6–36) months. Conclusion: The major histoincompatibility barriers in the haploidentical related donor/recipient allo-BMT could be successfully carried out with G-CSF stimulated donor bone marrow combined with GVHD preservation and Chinese Herbal Medicine. Our patients were treated with TCM to regulate immunity immediately after clearing out of their bong marrow; With this remedy method, we improved the curative effect of bone marrow transplant and this deserves further study with a well designed clinical trial.