ALBERT

Description: Objective: We assessed the impact of high-resolution genotypic results of human leukocyte antigen (HLA) for all major class I and II loci between donors and recipients in the outcome of unrelated hematopoietic stem cell transplantation (HSCT). Method: Between 1999 and 2005, high-resolution genotyping for HLA-A, -B, -C and -DRB1 was performed for 23 unrelated HSCT. All the patients were typed as HLA identical by serologic technique and then they were also typed HLA identical by high resolution technique. Unrelated bone marrow transplantation using DNA-based high resolution HLA compatibilities were considered in the analyses of clinical outcomes such as hematopoietic engraftment, acute GVHD, and survival. And then, we compared with patients who received related HSCT (same institute, same duration) and also unrelated HSCT data from IBMTR. Results: Median follow up duration was 9 months (1–51). Fifteen patients were male and 8 were female. Median age was 22 years (range 6–52). Median time from diagnosis to transplantation was 7 months (range 4–63). Eight patients of acute myeloid leukemia (AML), 6 of chronic myeloid leukemia (CML, 2 of 6 were in blast crisis), 4 of acute lymphoid leukemia (ALL), 3 of severe aplastic anemia and each case of juvenile myelomonocytic leukemia and myelodysplastic syndrome were enrolled. Median value of total nucleated cell and CD34 positive cell count was 3.51 (1.06–20.7) x 108/kg and 4.88 (1.33–46.9) x 106/kg, respectively. The conditioning regimen and prophylaxis for graft versus host disease (GVHD) were not different from conventional HSCT except one case of non-myeloablative transplantation. Median value of granulocytic (absolute granulocyte count 〉 500/mm3) and platelet (〉 20,000/mm3) engraftment were D + 16, D + 17, respectively. Grade II acute GVHD developed in 4 patients (2 patients subsequently proceded to chronic GVHD). Treatment related mortality was 2 out of 23 patients (8.7%). Median value of overall survival duration was 30 months. For AML patients, 3-year survival rate was 72.9 %. Conclusions: Our survival data for unrelated HSCT based on high resolution genotyped HLA matching was superior to unrelated HSCT. Although the sample size is small, the survival data of AML patients (CR1 at transplant) was superior to the survivals of related HSCT, as well as that of unrelated HSCT. The findings were that transplantation using unrelated donors selected by high-resolution genotype identity improves the transplantation outcomes similar degree to the result of the related HSCT.

Description: BACKGROUND: Helicobacter pylori has clearly been implicated in the pathogenesis of gastric and duodenal ulcers, gastritis, and gastric malignancy. Remarkably, eradication of H. pylori from the gastric mucosa has been associated with improvement of systemic disease, including Sjögren’s syndrome, rheumatoid arthritis, autoimmune thyroid disease, and immune thrombocytopenic purpura (ITP). PURPOSE: To investigate the relationship between Helicobacter pylori infection and the clinical features of idiopathic thrombocytopenic purpura (ITP), and to examine the effects of H. pylori eradication on platelet counts. METHOD: A 13C urea breath test (UBT) for H. pylori infection was performed in a 25 consecutive patients with ITP at Ajou University School of Medicine, Suwon, Korea. Patients who tested positive for H. pylori received standard eradication therapy if their platelet count was 〈 50 x 109/L. RESULTS: H. pylori infection was detected in 18 patients (72%) and eradication therapy was successfully administered to all infected patients. H. pylori infection was not associated with dyspepsia or other clinical or laboratory features. Platelet responses were observed in 6 (33%) of these patients, which lasted for more than 4 months in 4 patients. Platelet associated antibody and anti platelet antibody were negative to all patients. CONCLUSION: H. pylori eradication may improve the platelet counts in some of adults (33%) in whom the ITP is of recent onset.

Description: BACKGROUND Extranodal NK/T-cell lymphoma is notable for its unique behavior; Many patients die before 1 year from the diagnosis. However, the patients who survive the first year from the diagnosis live for longer period. Known prognostic factors were insufficient to explain this unique pattern. The objective of this study was to investigate clinicopathological features according to survival outcome(OS

Description: The risk of factor VIII (FVIII) inhibitor development increases by the disease severity, presence of family history and patient-related environment. Recently, FVIII genotypes and genes related to immune response were found to be decisive risk factors for inhibitor development. To identify the differentially expressed genes (DEGs) for developing inhibitory antibodies in hemophilia A, we analyzed the gene expression profiles between inhibitor and non-inhibitor by microarray technique. The results show that the 384 genes were up-regulated and 161 genes were down-regulated in inhibitor patients as compared with non-inhibitor patients. The 545 of DEGs were classified by the functional gene grouping using Panther classification method. Of interest was the finding that the expression levels of immunity and signal transduction related genes were significantly modified in inhibitor patients. For 7 genes, validation of these bead-array data was carried out by semi-quantitative RT-PCR. Finally, we performed Real Time PCR analysis for the significantly changed DEG450. As a result, expression of DEG450 was significantly down-regulated in inhibitor patients. We demonstrate that inhibitor development is closely related to the expression levels of immune-related genes and that these genes can be used as biomarker genes for prediction of inhibitor development

Description: Mesenchymal stem cells (MSCs) are a highly promising source of adult stem cells for purposes of cell therapy and tissue repair in the field of regenerative medicine. Although the most studied and accessible source of MSC is the bone marrow, the clinical use of bone marrow-derived MSCs (BMSCs) has presented problems, including pain, morbidity, and low cell number upon harvest. For those reasons, we isolated, cultured, and characterized MSCs from a number of tissues; including wharton’s jelly, cord blood, and adipose tissues that were discarded routinely in the past, and evaluated the usefulness of these MSCs compared to BMSCs. Proliferation ability of Wharton’s jelly-derived MSCs (WJ-MSCs), Cord blood-derived MSCs (CB-MSCs), or adipose tissue-derived MSCs (ASCs) was lost at passage 8–10 (22–27 population doubling), passage 7–10, or passage 7–12 (45–50 population doubling), respectively. WJ-MSCs, CB-MSCs, and ASCs expressed CD73, CD90, and CD105, CD90, CD105, and CD166, and CD44, CD73, CD90, and CD166, respectively, were absent for CD14, CD31, and CD45, and differentiated into osteoblast, adipocyte, and chondrogenic lineages under appropriate culture condition. In this study, like BMSCs, WJ-MSCs, CB-MSCs, and ASCs expressed similar cell surface antigens, were able to differentiate into mesenchymal lineages, and possessed highly proliferation potential. Therefore, MSCs isolated from wharton’s jelly, cord blood, and adipose tissue may become useful alternative sources of MSCs to cell therapy and tissue repair in the field of regenerative medicine.

Description: The best prognostic predictor for acute leukemia is known to be the finding of genetic abnormalities of leukemic cells. Methods for detecting the genetic abnormalities include chromosomal studies for karyotyping, FISH(Fluorescence in situ hybridization) and RT-PCR. However, each methods have limitations i.e. low sensitivity in karyotyping, uncertainty of molecular probe to be used in FISH or RT PCR methods. Multiplex RT-PCR (MRT-PCR) allows simultaneous detection of 28 fusion genes, more than 80 breakpoints and splice variants associated with leukemia. Therefore, this method can be used for detection of molecular abnormality in fresh unknown leukemic cases, as well as for molecular remission in follow-up cases. The aim was to demonstrate whether MRT-PCR system might be successfully used to screen a large number of patients with acute leukemia and compare the result with that of chromosome studies. Frozen bone marrow cells from 78 patients, who were diagnosed with acute leukemia at Ajou university hospital between September 1994 and February 2004, were used for MRT-PCR. In all samples with a known conventional cytogenetic results, we performed MRT-PCR and to compared with conventional cytogenetic study regarding the concordance rate and analyzed discordant cases regarding their types. 78 samples(40 male and 38 female patients) were analyzed, and there were 59 AML patients and 19 ALL patients. We successfully obtained the mRNA from all frozen samples. In 21 cases with gene abnormalities by chromosome studies most of them (15/21) showed the same abnormalities with MRT-PCR. In 57 patients with normal karyotype by cytogenetic technique, we identified 18 translocations of clinical significance by MRT-PCR method. In 18 discordant cases, there were 4 cases with t(15;17), 4 cases with t(8;21), 3 cases with t(9;22), 2 cases with t(11;19) and 4 others [t(9;11),t(9;9),t(3;11),inv(16)]. Conventional cytogenetics detected 10 cases of good prognostic gene abnormalities [t(15;17),t(8;21),inv(16)], but MRT-PCR method detected 10 additional cases of good prognostic gene abnormalities which might change the treatment paln. The twenty good prognostic cases with MRT-PCR showed better overall survival than others. (median f/u = 10.6 month, p=0.0443) There were 69% concordance rate between cytogenetic technique and MRT-PCR. Furthermore clinically significant translocations were detected by MRT-PCR in 18 of 57 normal karyotype patients, indicating improved sensitivity and prognostic value with MRT-PCR. Further investigations are needed to ascertain the usefulness of MRT-PCR for the screening tool of leukemic gene abnormalities.

Description: Abstract 4450 Acquired hemophilia is a rare bleeding disorder caused by autoantibodies directed against the coagulation factor VIII (FVIII). Usually acquired hemophilia is found in the elderly person, autoimmune disorder or women in the postpartum. Incontrast to alloantibody inhibitors of hereditary hemophilia, acquired inhibitor is presumably due to a spontaneous autoimmune disorder in which patients with previously normal hemostasis develop autoantibodies against FVIII. However, the precise cause of the antibody formation remains obscure and the possibility of abnormal FVIII protein derived from the FVIII gene mutation might have caused the antibody formation was investigated by direct FVIII gene sequencing method. Blood samples were obtained from a 72 year old male who initially presented with a large hematoma in the Rt. Shoulder, and had a prolonged aPTT (46 sec), FVIII level of 14%, and VIII inhibitor titer 2∼5 BU/ml. Direct sequencing of FVIII was performed via amplification for all 26 exons, including the 5′-UTR and 3′-UTR region, using the 35 synthesized primers. Sequencing was conducted using BigDye Terminator cycle sequencing kit on an ABI 3730XL DNA analyzer (Applied Biosystems, Foster City, CA). Sequence variations were analyzed with CLC main workbench program (CLC, Aarhus. Denmark). Firstly, we analyzed translational region of FVIII gene. But we couldn't find any sequence variation. Then we analyzed the sequence of untranslationed region, 5′-UTR and 3′-UTR. As a result, we identified gene variation of substitution to c.8889G〉A in the 3′-UTR. We speculate that this variation may have affected the RNA processing or mRNA stability. Thus we need to study about the relationship between this sequence variation and FVIII function. Also, we will investigate whether this gene variation is functional mutation. The cause of acquired hemophilia remains obscure. Variations in FVIII molecules including in untranslatable 5′- or 3′-region should be studied further to delineate the cause of antibodies in acquired HA. We expect that the accumulation of sequence variation can help to understand the cause of acquired hemophilia through this study. Disclosures: No relevant conflicts of interest to declare.

Description: Background: The aim of this study was to examine the differentiation potentials and characteristics of adipose tissue-derived stem cells (ASCs) in other to evaluate for the ASCs to be used as alternative cell sources of mesenchymal stem cells. Methods: ASCs were isolated from lipo-aspirated adipose tissues by treatment of collagenase A and cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM). To know the characteristics of ASCs, the expression of cell surface antigens was analyzed by flow cytometry, and the proliferation potentials were estimated by colony forming abilities or capacities of population doubling. The differentiation potentials into adipocytes or osteoblasts were confirmed by accumulation of neutral lipid vacuoles stained with Oil-red O and expression of alkaline phosphatases. Results: When the nucleated cells were isolated by collagenase treatment after lipo-aspiration, the mean cell yield was about 3.1 X 106 or 1.2 X 106 cells per gram of lipo-aspirate (n=8) processed at 3 or 21 hours, respectively. But cells processed at 21 hours were able to form more colonies than those at 3 hours. Flowcytometric analysis showed that Adipose tissue-derived stem cells (ASCs) have a marker expression that is similar to that of bone marrow stromal cells (BMSCs). ASCs expressed CD44, CD73, CD90, and CD105 and were absent for CD14, CD31 and CD45 expression. When primary cells were plated at 50 or 1000 cells/cm2 on 6-well plates, cumulative population doublings were about 50 times until passage 7 or 13 (approximately 130 days), respectively, and ASCs expanded to 1018 cells. ASCs were multipotent, differentiating to the adipocyte and osteoblast lineages. ASCs did not provoke in vitro alloreactivity of incompatable lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogen. Conclusion: Our results suggested that ASCs have highly proliferative potentials, multiple differentiation potentials, and immunosuppressive properties like BMSCs. Therefore, ASCs-based reconstructive therapy could employ allogeneic cells and because of their immunosuppressive properties, ASCs could be an alternative source of BMSCs to treat allogeneic conflicts.