CLAG combined with total body irradiation as intensive conditioning chemotherapy prior to allogeneic hematopoietic stem cell transplantation in patients with refractory or relapsed acute myeloid leukemia.

Refractory or relapsed acute myeloid leukemia (R/R AML) remains the major challenge of AML treatment. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only valid option to achieve cure, but the prognosis is still dismal. We conducted a retrospective analysis for the feasibility of CLAG regimens (cladribine, cytarabine, and granulocyte colony-stimulating factor) combined with total body irradiation (TBI) as new intensive conditioning chemotherapy prior to HSCT in R/R AML. A total of 70 patients, including 21 primary refractory and 49 relapsed AML, were analyzed. Forty-nine (70%) patients had extramedullary diseases, and 54 (77%) patients received haploidentical transplantation. Except for one who died before white blood cell engraftment, all of the 69 evaluable patients achieved measurable residual disease (MRD) negative complete remission. The 3-year overall survival (OS) and relapse-free survival (RFS) rates were 46.0% (95% confidence interval [CI], 33.5-57.7%) and 38.5% (95%CI, 26.8-50.0%). The 1-year cumulative incidences of relapse and non-relapse mortality (NRM) were 38.6% (95%CI, 27.3-49.3%) and 11.6% (95%CI: 5.4-20.3%), respectively. The presence of chronic graft-versus-host disease (cGVHD) showed a trend to be associated with a lower risk of relapse (P = 0.054) and extramedullary diseases with a higher risk of NRM (P = 0.074). Multivariate analyses identified low leukemia burden pre-HSCT (defined as bone marrow blasts ≤ 50%) and cGVHD as independent factors associated with favorable OS and RFS. In conclusion, intensive conditioning with CLAG regimens plus TBI may be an effective and well-tolerated choice for R/R AML patients undergoing allo-HSCT.

Cord Haploidentical Non-In Vitro T Cell Depletion Allogeneic Hematopoietic Stem Cell Transplantation Reduces Relapse of Refractory Acute Leukemia.

Whether a graft-versus-graft (GVG) response in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) is associated with an enhanced graft-versus-leukemia (GVL) effect remains highly controversial. Furthermore, it is unknown if the GVG response overwhelms the impact of refractory acute leukemia. We aimed to compare the characteristics and therapeutic outcomes between patients undergoing a modified haploidentical cord blood (cord-haplo) HSCT protocol (n = 97) and those undergoing haploidentical HSCT (n = 42) for refractory acute leukemia. A reliable and stable predominant haploidentical donor chimerism was established. The 2-year relapse rate was more favorable in patients undergoing cord-haplo HSCT than in those undergoing haploidentical HSCT (25.9% versus 53.2%; P = .007), as was progression-free survival (PFS; 35.5% versus 17.9%; P = .049). Meanwhile, nonrelapse mortality at 2 years was not significantly different (38.0% versus 24.6%; P = .367). We also found that a higher number of mutual haploidentical donor-mismatched antigens, a concept similar to HLA mismatching, was associated with better disease control. Multivariate analysis identified cord-haplo HSCT as an independent significant predictor of reduced relapse (hazard ratio [HR], .44; P = .028) and improved PFS (HR, .58; P = .033), as was chronic graft-versus-host disease (GVHD) (relapse: HR, .42; P = .013; PFS: HR, .63: P = .052). However, the incidences of neutrophil and platelet engraftment, GVHD, and virus reactivation were comparable in the 2 groups. This study demonstrates that cord-haplo HSCT significantly enhances the GVL effect and improves PFS, providing a reliable and efficient therapeutic platform for patients with refractory acute leukemia.

FLT3 inhibitors as maintenance therapy post allogeneic hematopoietic stem cell transplantation in acute myeloid leukemia patients with FLT3 mutations: A meta-analysis.

BACKGROUND: Acute myeloid leukemia (AML) patients with a Fms-like tyrosine kinase 3 (FLT3) mutation have a high incidence of relapse despite allogeneic hematopoietic stem cell transplantation (allo-HSCT) and a subsequent poor prognosis. FLT3 inhibitors (FLT3i) have been suggested to reduce the post-transplant relapse risk in recent studies. As more evidence is accumulated, we perform the present meta-analysis to assess the efficacy and safety of FLT3i as post-transplant maintenance therapy in AML patients. METHODS: Literature search was performed in public databases from inception to December 31, 2021. Overall survival (OS), relapse-free survival (RFS), cumulative incidence of relapse (CIR), non-relapse mortality (NRM), graft-versus-host disease (GVHD) and adverse events were compared between FLT3i and control groups. Pooled hazard ratio (HR) or relative risk (RR) with corresponding 95% confidence interval (CI) were calculated. RESULTS: We identified 12 eligible studies with 2282 FLT3-mutated AML patients who had received HSCT. There was no between-study heterogeneity and a fix-effect model was used. Post-transplant FLT3i maintenance significantly prolonged OS (HR = 0.41, 95%CI: 0.32-0.52, p < 0.001) and RFS (HR = 0.39, 95%CI 0.31-0.50, p < 0.001), and reduced CIR (HR = 0.31, 95%CI 0.20-0.46, p < 0.001) as compared with control. There were no significant risk differences in NRM (RR = 0.69, 95%CI 0.41-1.17, p = 0.169), acute GVHD (RR = 1.17, 95%CI 0.93-1.47, p = 0.175), chronic GVHD (RR = 1.31, 95%CI 0.91-1.39, p = 0.276) and grade ≥3 adverse events between both groups, except for skin toxicity (RR = 5.86, 95%CI 1.34-25.57, p = 0.019). CONCLUSION: Post-transplant FLT3i maintenance can improve survival and reduce relapse in FLT3-mutated AML patients and is tolerable.

[Four Cases of Wernicke's Encephalopathy Following Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To make through introduction of Wernicke's encephalopathy (WE) following hematopoietic stem cell transplantation (HSCT) in terms of clinical characteristics, diagnostic process and treatment. METHODS: The clinical charactaristics, diagnostic and therapeutic process and prognostic follow-up in 4 patients diagnosed of WE following HSCT between January 2016 to January 2017 at Department of Hematology, Chinese Aerospace Center Hospital were retrospectively analyzed. RESULTS: Four patients included 2 ALL and 2 AML, and 3 males and 1 female, their age ranged from 8 to 20 years old. 4 patients accouted for about 3% of all petients who received HSCT at that time. Typical triad syndrome consisting of ocular motility disorders, ataxia, global confusion was seen in only 1 patient. However, confusion and heterophthongia as onset of this complication were seen in all patients. Cerebral computed tomograph scan was universally unremarkable and useless. Cerebral MRI scan disclosed that typical involvement including thalamus, fourth ventricle, third ventricle, middle cerebral aqueduct was seen in 3, while untypical site including mamillary body was in the remaining 1 patient. All received vitamin B1 supplement therapy by intramuscular injection at a dose of 100 mg each day. Initial response was observed at 2, unknown, 3, 4 days after treatment and all obtained complete remission within 2 weeks without any event of relapse after median follow-up period of 8 (7-12) months. CONCLUSION: Any recipient of HSCT with clinical signs or symptoms of central nervous system should receive vitamin B1 supplementary therapy immediately to decrease risk of mortality of WE even if the diagnosis of WE is uncertain.

[Clinical Analysis of Treating EBV Infection Accompanied with GVHD after Allon-HSCT by EBV-Specific Cytotoxic T Lymphocytes].

OBJECTIVE: To investigate the efficiency and safety of treating Epstein-Barr virus (EBV) infection of acute graft versus host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) by EBV specific cytotoxic T lymphocytes (EBV-CTL). METHODS: The Clinical characteristics, therapeutic efficacy and safety of 12 patients with EBV infection treated by EBV-CTL infusion after allo-HSCT in Department of Hemahlogy of Aero Space Center Hospital between Jan 2015 and May 2017 were analyzed retrospectioely. RESULTS: Our of 12 cases received EBV-CTL infusion after transplantation, 9 did not received Rituximab therapy due to the active infection, 4 cases including 3 received Ritaximab progressed into posttransplantation lymphoroliferetive disease (PTLD). The median time of EBV infection was 47 (22-71) days, median time of antivirus therapy before tramplantation was 10 (8-33) days, median time of first CTL infusion was 59(34-86) days after transplatation. The 43 cases-time CTL infusion was performed smoothly, no related harmful evnts occoured, no progression of GVHD was observed. After the first course of infusion, complete remission (CR), Partial remssion (PR) and no remssion (NR) were obtained in 9, 1 and 2 patients respectively, the relapse was observed in 4 patients who then received the socond course of infusion and all reached CR, the patient in PR did not reathed CR finally and died of GVGD at 5 months after transpplantation . Only 1 out of 2 cases of NR obtained CR, another 1 still was in NR, and died of transplantation related infection at 5 months after transplantation. 4 cases of PTLD were all cared. CONCLUSION: Preliminary results of this study suggest that EBV-CTL infusion is safe for the EBV infection combined with acute GVHD after all-HSCT. However, a further larger scale clinical studies are needed to prove the efficiency.

Salvaged allogeneic hematopoietic stem cell transplantation for pediatric chemotherapy refractory acute leukemia.

There is an ongoing debate concerning the performance of salvaged allogeneic hematopoietic stem cell transplantation (allo-HSCT) in pediatric patients with acute refractory leukemia, in whom the prognosis is quite dismal. Few studies have ever been conducted on this subject. This may be partly due to missed opportunities by majority of the patients in such situations. To investigate the feasibility, evaluate the efficiency, and identify the prognostic factors of allo-HSCT in this sub-setting, the authors performed a single institution-based retrospective analysis. A total of 44 patients, of whom 28 had acute myeloid leukemia (AML), 13 had acute lymphocytic leukemia (ALL), and 3 had mixed phenotype leukemia (MPL), were enrolled in this study. With a median follow-up of 19 months, the estimated 2-year overall survival (OS) and progression free survival (PFS) were 34.3% (95% CI, 17.9-51.4%) and 33.6% (95% CI, 18.0-50.1%), respectively. The estimated 2-year incidence rates of relapse and non-relapse mortality (NRM) were 43.8% (95% CI 26.4-60.0%) and 19.6% (95% CI 9.1-32.9%), respectively. The estimated 100-day cumulative incidence of acute graft versus host disease (aGvHD) was 43.6% (95% CI 28.7-57.5%), and the 1-year cumulative incidence of chronic GvHD (cGvHD) was 45.5% (95% CI 30.5-59.3%). Compared with the previous studies, the multivariate analysis in this study additionally identified that female donors and cGvHD were associated with lower relapse and better PFS and OS. Male recipients, age younger than 10 years, a diagnosis of ALL, and the intermediate-adverse cytogenetic risk group were associated with increased relapse. On the contrary, extramedullary disease (EMD) and aGvHD were only linked to worse PFS. These data suggested that although only one-third of the patients would obtain PFS over 2 years, salvaged allo-HSCT is still the most reliable and best therapeutic strategy for refractory pediatric acute leukemia. If probable, choosing a female donor, better management of aGvHD, and induction of cGvHD promotes patient survival.

A new intensive conditioning regimen for allogeneic hematopoietic stem cell transplantation in patients with refractory or relapsed acute myeloid leukemia.

To explore the efficacy, and safety of the intensive conditioning regimen consisting of cladribine, cytarabine (Ara-C), and granulocyte colony-stimulating factor (G-CSF) plus modified busulfan (Bu) combined with cytoxan (Cy) (BuCy), prior to allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with refractory, or relapsed acute myeloid leukemia (R/R AML).Thirty-Six R/R AML patients scheduled to receive allo-HSCT were consecutively, enrolled in this prospective study, and treated using intensive conditioning regimen consisting of CLAG plus modified BuCy. Median follow-up duration was 11.25 (range 0.5 - 21.0) months and the last follow up date was August 15, 2017.All patients (100%) achieved white blood cell (WBC) recovery within a median time of 16.00 (13.25 - 18.00) days, and 34 of them (94%) attained platelet (PLT) recovery within a median time of 13.50 (9.25 - 19.75) days. Incidence of acute graft-versus-host disease (aGVHD) was 50.00%, with median time of 71.50 (41.00 - 401.25) days. Three patients developed Grade I; nine, Grade II; 5, Grade III; and 1, Grade IV aGVHD. The incidence of chronic GVHD (cGVHD) was 44.40%, with median time of 255.00 (120.00 - 390.00) days. Four patients developed limited cGVHD, and 12, extensive cGVHD. One-year accumulating leukemia free survival (LFS), and overall survival (OS) rates between 52.9 ±â€Š8.8% to 69.4 ±â€Š7.7%, respectively. Eighteen (50%) patients were infected with cytomegalovirus; 2 (5.6%), with Epstein-Barr virus (EBV), 7 (19.4%), with hemorrhagic cystitis; 13 (36.1%), with bacteria; and 8 (22.2%), with fungus.Intensive conditioning regimen of CLAG plus modified BuCy for allo-HSCT may be effective and well-tolerated in R/R AML patients.

TCR spectratyping revealed T lymphocytes associated with graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Clonal expansion of T cells after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been observed, but their characteristics remain to be fully elucidated. We report here that CD8(+) T cells were the dominant T lymphocytes seen and T-cell repertoire diversity decreased dramatically during the first 3 months after allo-HSCT. Patients with GVHD grade II - IV had significantly lower T-cell repertoire diversity compared with non-GVHD patients. TCR beta variable gene (TCRBV) subfamily 8, 5.1, 5.2, 4, and 13 were the five most frequently expanded subfamilies among these patients. Among the 49 over-expanded clones identified, clonotype "TCR3-5" and "TCR18-5" were isolated from four patients with HLA-A2 allele and skin GVHD. Their frequencies correlated well with skin symptoms (i.e. rash). Moreover, they were detected in donors but not detected in recipients before transplantation. Lastly, three common TCRBV CDR3 motifs shared by T cells related with GVHD were discovered: TGDS, GLAG, and GGG. These findings suggest that TCR spectratyping is helpful for revealing GVHD-related T cells and may have utility in early diagnosis.

[Real-time PCR array for simultaneous detection of 37 kinds of fusion genes in leukemia].

OBJECTIVE: To develop a real-time PCR array for simultaneous quantitative detection of translocations/chromosomal aberrations in patients with leukemia, and to investigate the feasibility and utility thereof. METHODS: By construction and optimization a set of specific primes (totally 82 primers), an array containing 66 parallel PCR reactions was developed. That array was used on the specimens of bone marrow or peripheral blood from 31 patients with leukemia to detect simultaneously 37 fusion genes and 4 proto-oncogene activations often occurring in patients with leukemia. Eva Green fluorescent dye method was chosen in the protocol. Relative quantification was performed by Ct analysis and the result was expressed as the ratio of the target gene versus the internal control gene (ABL). Six patients with chronic myelocytic leukemia (CML) among the 31 cases underwent prior to and after treatment so as to study the expression changes of fusion genes and/or proto-oncogene. RESULTS: The established PCR array showed high efficiency of amplification and good sensibility (232 copies/microl) in the fusion gene detected. The standard curve had a satisfying linear range (10(2) approximately 10(8) copies/microl), showing a good reproducibility. Fourteen fusion genes, including PML/RARalpha, PLZF/RARalpha, BCR/ABL, MLL/AF1, MLL/AF6, MLL/AF10, AML/Eto, CBFbeta/MYH11, TLS/ERG, TEL/AML1, MOZ/CBP, MLL/hCDCrel, LAF4/MLLT2, and FIP1L1/PDGFRalpha, and activation of all 4 proto-oncogenes were found in the 31 samples. In one patient, 5 fusion genes and activation of 2 proto-oncogenes were observed. Such results were compared with those of RT-nested PCR in 28 samples. The comparison showed that this array was a bit less sensitive than RT-nested PCR, however, without significant difference between them (P = 0.009). The expression of BCR/ABL fusion gene, WT1 gene, and EVI1 gene decreased after treatment in the 6 CML patients, which was in accordance with the clinical features. CONCLUSION: The PCR array newly-established successfully detects various leukemia related fusion genes and proto-oncogene activation. It is useful in molecule diagnosis and monitoring minimal residual disease in leukemia, and therapeutic effect monitoring.

[Predictable recurrence by regular monitoring minimal residual disease with flow cytometry in the patients with both AML and ALL: a single-center study of 163 cases].

OBJECTIVE: To study the predictable value of monitoring minimal residual disease (MRD) regularly by flow cytometry (FCM) in patients with acute leukemia (AL) in the first complete remission (CR(1)). METHODS: From April 2005 to July 2009, AL patients who had got CR(1) after chemotherapy were regularly monitored for MRD in bone marrow by FCM to relapse or to July 2010 in Beijing Daopei Hospital (not including those received stem cell transplantation). The special antibody combinations were employed for each patient according to aberrant expression of leukemia cells. MRD(+) was defined as the aberrant cells more than 0.01%. The probability of continuous CR (CCR) was calculated by Kaplan-Meier formula, and the statistical difference between two CCR probabilities was evaluated by log-rank test. RESULTS: A total of 163 AL patients in CR(1) were monitored to relapse or to July 2010. Among 89 AML patients referred to our hospital within 1 year after diagnosis, 30 cases were in MRD(+) and 59 cases MRD(-) till 12 months following chemotherapy, 3/30 patients in MRD(+) and 47/59 remained in CCR to July 2010. The probability of CCR at 24, 36 months was 13%, 13%in MRD(+) group, 94%, 78% in MRD(-) group respectively, the difference between them was statistically significant (P < 0.01). Among 35 ALL referred to our hospital within 5 months after diagnosis, 13 cases were MRD(+) and 22 cases MRD(-) till 5 months following chemotherapy, 0/13 patients in MRD(+) and 20/22 patients in MRD(-) remained in CCR to July 2010. The probability of CCR at 24, 36 months was 0% in MRD(+) group, 96%, 96% in MRD(-) group respectively, the difference between them was statistically significant (P < 0.01). Over the time point above, all patients with MRD(+) or their MRD from negative to positive relapsed finally, and most patients with MRD(-) remained CCR to July 2010. CONCLUSION: It had a clinical prognostic value to monitor MRD regularly by FCM in the patients with AL after CR(1).

An improved design of PCR primers for detection of human T cell receptor beta chain repertoire.

Analysis of T cell receptor beta variable region (TCRBV) gene rearrangement is useful for clonal assessment of abnormal T cell proliferations in various diseases. However, most primer panels previously used can only amplify the third complementarity-determining region. Following IMGT database we established a panel of primers, which can amplify entire sequences of all functional TCRBV families. To confirm the usefulness of this panel of primers, we detected different TCRBV repertoires. In 15 healthy donors, most of the BV families were expressed and appeared as a Gaussian distribution. 13 acute myeloid leukemia patients showed monoclonal or oligoclonal changes of BV15 family, and some of them also had monoclonal or oligoclonal expansion of BV2, BV4, BV6 or BV13 families. In one patient after allo-hematopoietic stem cell transplantation, monoclonal proliferation of BV10 family occurred during graft-versus-host disease. In conclusion, this panel of primers improves our abilities to analyze TCRBV repertoire changes in related diseases.

[Changes of T-cell clonality after induction-cultivation of peripheral T lymphocytes in adoptive immunotherapy for leukemias].

This study was purposed to analyze the changes of T-cell clonality after induction of peripheral T lymphocytes by autogenous DC and cytokines in the preparation of adoptive immunotherapy for leukemias. The bone marrow and peripheral blood from 21 leukemia patients at remission stage after treatment and subjected to adoptive immunotherapy were collected. Their DCs and T-cells were stimulated with cytokines and then were mixed to activate T-cells. T-cell receptor beta variable region (TCRBV) families were amplified by RT-PCR, and genescan method and sequencing of the PCR products were used to observe the clonality changes of T-cells before and after the induction and cultivation of T-cells. The flow cytometry was used to identify CD3(+), CD4(+), CD8(+), CD3(+)CD56(+) and CD4(+)CD25str(+)FOXP3(+) cells to disclose the ratio change of cytotoxic T lymphocytes (CTL), helper T-cells, regulatory T-cells and NK T-cells before and after induction and cultivation of T-cells. The results showed that in the 21 patients, most of the 24 TCRBV families presented as oligoclonal distribution on genescan, several families were not expressed, and only a few families remained polyclonal. TCRBV24 was found to be oligoclonal in all of the 21 patients. DNA sequence analysis of TCRBV24 revealed a common motif of VAG in CDR3 in 3 cases and a common motif of GGG in CDR3 in 2 cases. In patient 5, both TCRBV 24 and TCRBV8 contained the same motif of GGG in CDR3. The identical motif in these patients may suggest that these T-cells recognize the same antigen. The peripheral lymphocytes demonstrated recovery of clonal profile on genescan from oligoclonal profile and absence of several families before the induction and cultivation to typical polyclonal profile in all TCRBV families after the induction by DC and cytokines for 13 days. After the induction and cultivation, the number of lymphocytes increased to 3.38 +/- 1.20 times. CD3(+), CD4(+), CD8(+), CD3(+)CD56(+) and CD4(+)CD25str(+)FOX P3(+) cells were 71.1 +/- 11.8%, 26.7 +/- 11.4%, 35.7 +/- 12.9%, 3.1 +/- 1.6% and 0.12 +/- 0.1% respectively before the induction and cultivation, and changed to 95.4 +/- 3.2% (p < 0.01), 27.0 +/- 13.1% (p > 0.01), 55.5 +/- 13.8% (p < 0.01), 9.8 +/- 6.1% (p < 0.01) and 0.22 +/- 0.18% (p < 0.01) respectively after the induction and cultivation. It is concluded that the major action of this induction and cultivation method on T-lymphocytes in vitro is the promotion of CTL and NK T-cell proliferation. In leukemic patients at the remission stage, the TCRBV profile is characterized by the oligoclonal proliferation of T-lymphocytes. Several proliferated clones may have the same motif in CDR3, suggesting the recognition of the same antigen by these lymphocyte clones. Cytokine induction and co-culture with autogenous DCs can stimulate the T-lymphocytes to recover their immunocompetence as manifested by the polyclonal profile and the proliferation of CTL and NK-T cells.

[T cells recognizing EBV-epitopes arose in co-culture of peripheral blood mononuclear cells from EB-infected patients and dendritic cells loaded with LMP2-mixed peptides].

The latent membrane protein 2 (LMP2) is a kind of protein expressed by EBV-infected cells. This study was aimed to investigate whether the stimulation of peripheral blood mononuclear cells with peptides induces EBV-specific cytotoxic T lymphocytes (CTL). The peptides were mixture of LMP2 protein and available for people with different HLA types. Peripheral blood sample was collected from a patient with EBV-associated hemophagocytic syndrome. The mononuclear cells were isolated and cultured to obtain dendritic cells (DCs). Immature DCs were pulsed with MIX-LMP2 and added with different maturation-promoting factors. The auto-T lymphocytes were stimulated weekly with the harvested mature DCs loaded with MIX-LMP2, and totally for two times. Part of isolated lymphocytes was cultured without any stimulation as control. T-cell receptor (TCR) beta spectratyping was used to analyze the distribution of different T cell subgroups before and after culture. The phenotype of T lymphocytes was determined by flow cytometry. The IFN-gamma assay was used to estimate specific cytoxic activity of the cultured T cells. The results showed that the distribution of TCRbeta was changed according to analysis of TCR spectratypes. From the distribution of gene families of TCRbeta, the T lymphocytes were oligoclonal before culture, but shifted to a polyclonal after culture in vitro like the normalization of TCR diversity, suggesting the subgroups of lymphocyte could return to normal. The percentage of CD3+, CD3+CD8+ CD3+ CD45RA- CD45 RO+ on T lymphocytes from freshly isolated mononuclear cells were 70.73%, 42.99%, 27.56% respectively. After being stimulated twice with DC loaded with MIX-LMP2, they further increased to 95.17%, 52.54% and 81.41%. The percentages of CD3-CD56+ NK cells and CD4+CD35+ FOXP3+ regulation T cells seldom changed, from 2.12%, 0.03% to 2.35%, 0.02% respectively. The increase of CD3+CD45RA-CD45RO+ cells obviously indicated that most naive T cells could be activated. ELISA for IFN-gamma showed that when DCs loaded with LMP2 peptide were used as target cells, IFN-gamma level secreted by the T cells stimulated with LMP2 peptide-pulsed DCs was 805+/-16 pg/ml and 1729+/-49 pg/ml, the IFN-gamma level secreted by T cells stimulated twice with LMP2 peptide-pulsed DCs was 956+/-23 pg/ml and 2325+/-58 pg/ml respectively at effector-target ratios of 10:1 and 10:2. They were both significantly higher than that secreted by T cells without any stimulation (441+/-27 pg/m and 557+/-19 pg/ml) (p<0.05). But DCs unpulsed with LMP2 peptide were used as target cells, there were no significant differences between the T cells stimulated with LMP2 peptide-pulsed DCs and the T cells without stimulation (p>0.05). It is concluded that the antigen specific T cells recognizing EBV epitopes can be obtained by using DCs pulsed with MIX-LMP2 peptide in vitro, meanwhile the distribution of T cell subgroups can be changed and normalized.

HLA-DRB1*09 is associated with increased incidence of cytomegalovirus infection and disease after allogeneic hematopoietic stem cell transplantation.

Cytomegalovirus (CMV) infection is a major complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT); however, we have little information on the clinical association of various human leukocyte antigen (HLA) alleles with CMV infection. We reviewed medical records of 60 patients who underwent allo-HSCT. The effect of the 7 most frequent HLA alleles on the incidence of CMV infection and disease was analyzed, including HLA-A*02, A*11, A*24, B*13, B*40(60), DRB1*15, and DRB1*09. All the patients were monitored for CMV infection at least once weekly within 3 months. CMV infection was found in 38 (63.3%) patients on a median of day 36 (range: 16-89). Diagnosis of CMV disease was established in 6 (10.0%) patients, comprising pneumonia (n = 2), enterocolitis (n = 2), and hemorrhagic cystitis (n = 2). CMV disease was successfully treated using ganciclovir or foscarnet combined with immune globulins in 4 patients. The other 2 patients died without improvement of CMV disease. In multivariate analysis, grade II-IV acute graft-versus-host disease (aGVHD), CMV seronegative donors, and HLA-DRB1*09 were associated with increased incidence of CMV infection and disease after allo-HSCT. We suggest that more cautions should be taken to prevent CMV infection in patients with HLA-DRB1*09 after allo-HSCT.

Identification of disease-associated proteins by proteomic approach in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic systemic inflammatory disorder of the axial skeleton and shows significant inherited susceptibility. Auto-immune responses have been traditionally considered as a putative pathogenetic event in AS. However, no consistent self-antigen has been identified to responsible for the disorders in AS to this day. In this study, serum protein profiles of AS patients and healthy controls from a large Chinese AS family were investigated by two dimensional electrophoresis analysis. A group of four highly expressed protein spots was observed in all AS patients' profiles and subsequently identified as isoforms of haptoglobin precursor (pre-Hp) by ESI-Q-TOF MS/MS. Increased expression of haptoglobin precursor were also observed in sera of sporadic AS patients. Moreover, bioinformatics analysis revealed epitopes derived from haptoglobin precursor with high affinity binding to HLA-B( *)2705, a primary subtype associated with AS. These results indicate that pre-Hp may be involved in the pathogenesis of AS.

[Expression of PRAME gene in acute leukemia and its clinical significance].

This study was aimed to detect the expression levels of preferentially expressed antigen of melanoma (PRAME) gene in acute leukemia (AL) and to evaluate the clinical significance of PRAME gene. The quantitative detection method was established by SYBR Green I real-time quantitative RT-PCR, then PRAME mRNA was measured by this method in 55 cases of acute leukemia, out of which 43 cases were acute myeloid leukemia (AML), 9 cases were acute lymphocytic leukemia (ALL) and other types leukemia were 3 cases. In addition, expression of PRAME gene was also analyzed in 7 cases of non-malignant hematological diseases and 8 healthy volunteers. K562 cell line was used as a positive control. The results showed that the expression of PRAME gene was found in 35 cases of acute leukemia, the positive percentage was 64%. No expression could be detected in any of the non-malignant hematological diseases and healthy volunteers. In 35 PRAME positive cases, 28 cases were AML, which mainly belonged to M3, M4 and M2 subtypes, and 5 cases was ALL. In 31 fusion gene positive cases, 23 cases were PRAME positive, and in 24 fusion gene negative cases 12 cases were PRAME positive. No significant relationship was found between PRAME expression level and clinical characteristics (age, sex, WBC count, blast cells in BM). The expression of PRAME gene decreased or disappeared in 6 patients achieving complete remission (CR). It is concluded that the PRAME gene expresses in 64% AML patients, which mainly belonged to M3, M4 and M2 subtypes, no expression could be detected in any of the non-malignant hematological diseases and healthy volunteers. There is remarkable difference in the level of PRAME transcript of the 35 cases and the expression of PRAME gene decreases or disappears when the patients achieved complete remission. These results suggest that PRAME expression in acute leukemia may be a useful marker to detect the minimal resi-dual disease (MRD) and to determine the response to therapy in AL patients.

[Characteristics of T cell receptors recognizing antiphospholipid syndrome associated antigens].

To understand the characteristics of T cell receptors recognizing antiphospholipid syndrome associated antigen, the characteristics of T cells were analyzed using T cell receptor beta variable region (TCRbetaV) gene spectrotyping in a case of antiphospholipid syndrome (APS). The results indicated that in the case of APS there were 2 dominant T cell clones. The TCRbetaVs sequences of the 2 T cell clones showed the TCRbetaVs belonged to 8 and 23 gene families respectively. The peptides of third complementarity-determining regions (CDR3) in the TCRbetaVs were CASSLLVAGGPRAYNEQFFGPG and CASSLAGFGQPQHFGDG. Comparing the motifs in CDR3 with another autoimmune disease, the motif YNEQFFGPG in TCRbetaV8 and motif QHFGDG in TCRbetaV23 were identical with that of idiopathic thrombocytopenic purpura and systemic lupus erythematosus reported before. In conclusion, some T cell clones proliferating in these autoimmune diseases may recognize the same antigens.

[Construction of recombinant vector expressing ALAS2 gene in X-linked sideroblastic anemia].

X-linked sideroblastic anemia (XLSA) is caused by mutations of erythroid-specific 5-aminolevulinate synthetase (ALAS2) gene. In this study a eukaryotic expression vector of ALAS2 was constructed and transfected into eukaryotic cells to observe the expression of ALAS2 gene. The full length cDNA of ALAS2 gene was inserted into plasmid pDs-red2-N1, named pDs-red2-N1/ALAS2. Then, the vector was transfected into K562 cells via electroporation. At 48 hours after transfection, total RNA from K562 cells was extracted, expressions of ALAS2 gene and protein with red fluorescence in the K562 cells were detected by RT-PCR and flow cytometry, respectively. The vector was also transfected into COS 7 cells via liposome. Both mRNA and protein expression in COS7 cells were detected by RT-PCR and fluorescence microscopy. The result showed that after the pDs-red2-N1/ALAS2 eukaryotic expression vector was digested by KpnI and BamHI, two fragments of 4 700 bp and 1 764 bp were displayed by electrophoresis on agarose gel. Sequence method confirmed that the sequence was correct. RT-PCR amplified the total RNA extracted from the transfected K562 and COS7 cells, and could find mRNA of ALAS2 gene that can't be found in K562 and COS7 cells usually. The expressions of both fluorescein and ALAS2 were significantly increased. The percentage of positive cells reached about 19.2% and 10.7%, respectively. ALAS2 expression lasted for 10 days in COS7 cells and the peak was at the third day. It is concluded that the eukaryotic expression vector of ALAS2 gene is successfully constructed; K562 and COS7 cells transfected with the vector via electroporation and liposome can express ALAS2 protein. So, the vector has the potential in gene replacement and can be used for patients with XLSA in future.