Effect of Single and Double Administration of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Following Focal Cerebral Ischemia in Rats

Stem cell therapies are administered during the acute phase of stroke to preserve the penumbral tissues from ischemic injury. However, the effect of repeated cell therapy during the acute phase remains unclear. In this study, we investigated and compared the functional outcome of single (two days post-injury) and repeated (two and nine days post-injury) treatment with human umbilical cord derived mesenchymal stem cells (hUCB-MSCs) after middle cerebral artery occlusion (MCAO). The rotarod and limb placement tests were utilized to investigate functional outcomes, while infarct volume and tissue damage were measured by immunofluorescent staining for neovascularization, neurogenesis, apoptosis, and inflammation in the penumbral zones. We observed notable motor dysfunction and a significant decrease in infarcted brain volume, as well as increases in neurons and vessels in both single and repeated hUCB-MSC treatments compared to the control group. Interestingly, repeated administration of hUCB-MSCs was not found to elicit additional or synergistic improvements over monotherapy. This study suggests that a clearer understanding of the therapeutic window after stroke will facilitate the development of more efficient treatment protocols in the clinical application of stem cell therapy.

The Effect of Donor-Dependent Administration of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells following Focal Cerebral Ischemia in Rats

Stroke is an ischemic disease caused by clotted vessel-induced cell damage. It is characterized by high morbidity and mortality and is typically treated with a tissue plasminogen activator (tPA). However, this therapy is limited by temporal constraints. Recently, several studies have focused on cell therapy as an alternative treatment. Most researches have used fixed donor cell administration, and hence, the effect of donor-dependent cell administration is unknown. In this study, we administered 3 types of donor-derived human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) in the ischemic boundary zone of the ischemic stroke rat model. We then performed functional and pathological characterization using rotarod, the limb placement test, and immunofluorescent staining. We observed a significant decrease in neuron number, and notable stroke-like motor dysfunction, as assessed by the rotarod test (~40% decrease in time) and the limb placement test (4.5 point increase) in control rats with ischemic stroke. The neurobehavioral performance of the rats with ischemic stroke that were treated with hUCB-MSCs was significantly better than that of rats in the vehicle-injected control group. Regardless of which donor cells were used, hUCB-MSC transplantation resulted in an accumulation of neuronal progenitor cells, and angiogenic and tissue repair factors in the ischemic boundary zone. The neurogenic and angiogenic profiles of the 3 types of hUCB-MSCs were very similar. Our results suggest that intraparenchymal administration of hUCB-MSCs results in significant therapeutic effects in the ischemic brain regardless of the type of donor.

The Effect of Umbilical Cord Blood Derived Mesenchymal Stem Cells in Monocrotaline-induced Pulmonary Artery Hypertension Rats

Pulmonary arterial hypertension (PAH) causes right ventricular failure due to a gradual increase in pulmonary vascular resistance. The purposes of this study were to confirm the engraftment of human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) placed in the correct place in the lung and research on changes of hemodynamics, pulmonary pathology, immunomodulation and several gene expressions in monocrotaline (MCT)-induced PAH rat models after hUCB-MSCs transfusion. The rats were grouped as follows: the control (C) group; the M group (MCT 60 mg/kg); the U group (hUCB-MSCs transfusion). They received transfusions via the external jugular vein a week after MCT injection. The mean right ventricular pressure (RVP) was significantly reduced in the U group after the 2 week. The indicators of RV hypertrophy were significantly reduced in the U group at week 4. Reduced medial wall thickness in the pulmonary arteriole was noted in the U group at week 4. Reduced number of intra-acinar muscular pulmonary arteries was observed in the U group after 2 week. Protein expressions such as endothelin (ET)-1, endothelin receptor A (ERA), endothelial nitric oxide synthase (eNOS) and matrix metalloproteinase (MMP)-2 significantly decreased at week 4. The decreased levels of ERA, eNOS and MMP-2 immunoreactivity were noted by immnohistochemical staining. After hUCB-MSCs were administered, there were the improvement of RVH and mean RVP. Reductions in several protein expressions and immunomodulation were also detected. It is suggested that hUCB-MSCs may be a promising therapeutic option for PAH.

The Effect of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells in a Collagenase-Induced Intracerebral Hemorrhage Rat Model

Intracerebral hemorrhage (ICH) is one of the devastating types of stroke. Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have potential benefits in recovery from brain damage following ICH. This study aimed to identify the beneficial effects of hUCB-MSCs and investigate whether they have anti-inflammatory effects on the ICH brain via neurotrophic factors or cytokines. hUCB-MSCs were transplanted into a collagenase-induced ICH rat model. At 2, 9, 16, and 30 days after ICH, rotarod and limb placement tests were performed to measure behavioral outcomes. ICH rats were sacrificed to evaluate the volume of lesion using H&E staining. Immunostaining was performed to investigate neurogenesis, angiogenesis, and anti-apoptosis at 4 weeks after transplantation. Inflammatory factors (TNF-alpha, COX-2, microglia, and neutrophils) were analyzed by immunofluorescence staining, RT-PCR, and Western blot at 3 days after transplantation. hUCB-MSCs were associated with neurological benefits and reduction in lesion volume. The hUCB-MSCs-treated group tended to reveal high levels of neurogenesis, angiogenesis, and anti-apoptosis (significant for angiogenesis). The expression levels of inflammatory factors tended to be reduced in the hUCB-MSCs-treated group compared with the controls. Our study suggests that hUCB-MSCs may improve neurological outcomes and modulate inflammation-associated immune cells and cytokines in ICH-induced inflammatory responses.

Microarray analysis after umbilical cord blood derived mesenchymal stem cells injection in monocrotaline-induced pulmonary artery hypertension rats / 대한해부학회지

Pulmonary arterial hypertension (PAH) is associated with structural alterations of lung vasculature. PAH is still a devastating disease needing an aggressive therapeutic approach. Despite the therapeutic potential of human umbilical cord mesenchymal stem cells (MSCs), the molecular parameters to define the stemness remain largely unknown. Using high-density oligonucleotide microarrays, the differential gene expression profiles between a fraction of mononuclear cells of human umbilical cord blood (UCB) and its MSC subpopulation were obtained. Of particular interest was a subset of 46 genes preferentially expressed at 7-fold or higher in the group treated with human UCB-MSCs. This subset contained numerous genes involved in the inflammatory response, immune response, lipid metabolism, cell adhesion, cell migration, cell differentiation, apoptosis, cell growth, transport, cell proliferation, transcription, and signal transduction. Our results provide a foundation for a more reproducible and reliable quality control using genotypic analysis for the definition of human UCB-MSCs. Therefore, our results will provide a basis for studies on molecular mechanisms controlling the core properties of human MSCs.

Effect of Aging on the Pluripotential Capacity of Human Bone Marrow Derived Mesenchymal Stem Cells / 대한정형외과학회잡지

PURPOSE: The aim of study was to compare the differentiation capacity of mesenchymal stem cells (MSCs) obtained from human bone marrow (BM) according to the age of the donors. MATERIALS AND METHODS: MSCs were isolated from the BM of young (n=16, 12.5+/-5.8 years) and elder (n=4, 48.5+/-7.2 years) patients with the consent of them. We analyzed the cell morphology and the cell surface markers of the MSCs. In addition, we assessed the cell senescence with serial cultures from both age groups. Cell pluripotentiality was analyzed by osteogenic, chondrogenic, and adipogenic induction media. We performed RT-PCR, a measurement of expression of alkaline phosphatase, and staining with von Kossa, safranin O, and oil red O stain. RESULTS: All of the MSC samples tested, irrespective of the age of the donors, MSCs were all successfully isolated from twenty bone marrows. However, the number of cells of from the young donors was five times greater than that of the elderly donors. Senescence was observed over 10 passages in both age groups. The immunophenotypes of both age groups showed similar patterns. MSCs obtained from young and older donors showed the potential to differentiate into osteogenic, chondrogenic, and adipogenic lineages with no difference for both age groups. CONCLUSION: Our study supports that age does not influence the pluripotential capacity of human BM derived MSCs.

Cotransplanted Bone Marrow Derived Mesenchymal Stem Cells (MSC) Enhanced Engraftment of Hematopoietic Stem Cells in a MSC-dose Dependent Manner in NOD/SCID Mice

Transplantation of marrow-derived mesenchymal stem cells (MSCs), expanded by culture in addition to whole bone marrow, has been shown to enhance engraftment of human hematopoietic stem cells (HSCs). Our hypothesis was that there might be an optimum ratio range that could enhance engraftment. We examined the percent donor chimerism according to the ratio of HSCs to MSCs in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We tested a series of ratios of co-transplanted CD34+-selected bone marrow cells, and marrow-derived MSCs into sublethally irradiated NOD/SCID mice. In all experiments, 1 x 10(5) bone marrow derived human CD34+ cells were administered to each mouse and human MSCs from different donors were infused concomitantly. We repeated the procedure three times and evaluated engraftment with flow cytometry four weeks after each transplantation. Serial ratios of HSCs to MSCs were 1:0, 1:1, 1:2 and 1:4, in the first experiment, 1:0, 1:1, 1:2, 1:4 and 1:8 in the second and 1:0, 1:1, 1:4, 1:8 and 1:16 in the third. Cotransplantation of HSCs and MSCs enhanced engraftment as the dose of MSCs increased. Our results suggest that the optimal ratio of HSCs and MSCs for cotransplantation might be in the range of 1:8-1:16; whereas, an excessive dose of MSCs might decrease engraftment efficiency.

Cotransplanted Bone Marrow Derived Mesenchymal Stem Cells (MSC) Enhanced Engraftment of Hematopoietic Stem Cells in a MSC-dose Dependent Manner in NOD/SCID Mice

Transplantation of marrow-derived mesenchymal stem cells (MSCs), expanded by culture in addition to whole bone marrow, has been shown to enhance engraftment of human hematopoietic stem cells (HSCs). Our hypothesis was that there might be an optimum ratio range that could enhance engraftment. We examined the percent donor chimerism according to the ratio of HSCs to MSCs in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We tested a series of ratios of co-transplanted CD34+-selected bone marrow cells, and marrow-derived MSCs into sublethally irradiated NOD/SCID mice. In all experiments, 1 x 10(5) bone marrow derived human CD34+ cells were administered to each mouse and human MSCs from different donors were infused concomitantly. We repeated the procedure three times and evaluated engraftment with flow cytometry four weeks after each transplantation. Serial ratios of HSCs to MSCs were 1:0, 1:1, 1:2 and 1:4, in the first experiment, 1:0, 1:1, 1:2, 1:4 and 1:8 in the second and 1:0, 1:1, 1:4, 1:8 and 1:16 in the third. Cotransplantation of HSCs and MSCs enhanced engraftment as the dose of MSCs increased. Our results suggest that the optimal ratio of HSCs and MSCs for cotransplantation might be in the range of 1:8-1:16; whereas, an excessive dose of MSCs might decrease engraftment efficiency.

Maternal and Neonatal Predictors of Hematopoietic Potential of Umbilical Cord Blood Units / 대한혈액학회지

BACKGROUND: Umbilical cord blood (UCB) is a useful source for hematopoietic stem cells especially when HLA-matched donors are unavailable. UCB banking is expensive, however, because only a small fraction of cryopreserved units is used for transplantation. In order to make the criteria for selecting optimal UCB units at the time of collection, we examined the effects of maternal and neonatal factors on the laboratory parameters of hematopoietic potential of UCB units. METHODS: Total number of 1,692 UCB units processed in our UCB bank was analyzed to determine the effects of maternal and neonatal factors such as maternal age, birth weight, gestational duration, and sex of baby on the laboratory parameters of hematopoietic potential including the volume of UCB units and the numbers of total nucleated cells (TNC), mononuclear cells (MNC), and CD34+cells. RESULTS: Bigger babies yielded larger volumes of UCB, more TNC, MNC and CD34+cells. Babies of longer gestational duration also yielded higher numbers of TNC and MNC, but the number of CD34+cells was declined. No significant effect by maternal age was noticed on the volume of UCB, TNC, MNC, or CD34+cells. CONCLUSION: Optimal results would be obtained by selecting babies with weight equal to or more than 3,350g and gestational duration from 38 weeks through 39.7 weeks. These data may be used as important criteria for selecting UCB units for banking and cost-effective bank operation.

Chondrogenic Differentiation of Mesenchymal Stem Cells from Human Umbilical Cord Blood / 대한정형외과학회잡지

PURPOSE: The aim of this study was to demonstrate the existence of circulating mesenchymal stem cells (MSC) in the human umbilical cord blood (hUCB) and to evaluate the chondrogenic differentiation potential of hUCB-derived MSC in vitro. MATERIALS AND METHODS: Fifty hUCB harvests were cultured in media supplemented with 10% fetal bovine serum. The adherent fibroblast-like cells were characterized by immunophenotyping and induced to differentiate into chondrocytes in the pellet culture with and without BMP-6. This study performed RTPCR of the chondrogenic markers, Safranin-O stain and type II collagen immunohistochemical stain. RESULTS: The mononuclear cells isolated from hUCB formed adherent colonies with an attached wellspread fibroblast-like morphology. The cells positively expressed the MSC-related antigens, but did not express the hematopoietic, HLA-DR, endothelial, or osteoclast antigens and could be induced to differentiate into chondrocytes under proper stimulation. BMP-6 increased the size of the pellet and the mRNA levels for aggrecan, type II collagen and type IX collagen and enhanced the levels of proteoglycan synthesis during chondrogenic differentiation. CONCLUSION: The homogenous fibroblast-like cells developed in cultures from hUCB with chondrogenic differentiation potential were considered to be MSC. Furthermore, it was found that BMP-6 enhanced chondrogenic differentiation of the hUCB-derived MSC in the pellet culture.

Isolation, Characterization and Tri-lineage Differentiation of Mesenchymal Stem Cells from Human Umbilical Cord Blood / 대한정형외과학회잡지

PURPOSE: Human umbilical cord blood (hUCB) is well known for a good source of hematopoietic stem cells (HSCs). However, the presence of mesenchymal stem cells (MSCs) in hUCB is still not well approved by many authors. We hereby report the isolation and characterization of MSCs from hUCB, as well as their differentiation into osteogenic, chondrogenic and adipogenic lineages. MATERIALS AND METHODS: Mononuclear cells were isolated from each hUCB harvest (n=411) by density gradient centrifugation, and suspended in -minimum essential medium supplemented with 10% fetal bovine serum (FBS). The cell population was expanded by successive sub-cultivation under the same condition. The cell population that showed more than 1, 000-fold expansion at fifth to eighth passage was inspected with known surface antigens of MSCs and other cell lineage. The isolated MSCs were cultured in osteogenic, chondrogenic, and adipogenic condition to identify their potential to differentiate into different mesenchymal cell lineage. RESULTS: Ninety five out of 411 hUCB units (23.1%) generated the MSC-like cell population during initial cultivation. Nine cell populations (2.2%) showed more than 1, 000-fold expansion capacity at fifth to eighth passage. These cells positively expressed all known MSC-related antigens. They did not express any of myeloid, endothelial, or histo-compatibility antigens. All of the MSCs isolated showed the potential to differentiate into osteogenic, chondrogenic, and adipogenic lineages. CONCLUSION: Our study supports that hUCB does contain MSCs, which can be differentiated into different cell lineages. We believe hUCB will be a good source of MSCs with the advantage of availability and relative abundance. We think hUCB should not be considered as a medical waste, and it will serve as a good source of cells for tissue engineering and cellular therapy in the future.

Osteogenic Differentiation of Mesenchymal Stem/Progenitor Cells Developed in Cultures from Human Umbilical Cord Blood / 대한정형외과연구학회지

PURPOSE: To demonstrate the existence in human umbilical cord blood (hUCB) of circulating mesenchymal stem/progenitor cells(MSPC), the adherent cells developed in cultures from hUCB were characterized and induced to differentiate into osteoblasts. MATERIALS AND METHODS: Fifty hUCB harvests were cultured in the media supplemented with 10% fetal bovine serum. Homogeneously adherent fibroblast-like cells obtained during successive subcultivation were characterized by immunophenotyping analysis and induced to differentiate into osteoblasts. Reverse transcription-polymerase chain reaction of osteogenic markers, alkaline phosphatase (ALP) stain, and von Kossa stain were performed. RESULTS: The adherent fibroblast-like cells developed in cultures from hUCB positively expressed the MSPCrelated antigens, but, did not express the hematopoietic, HLA-DR, osteoclast, or endothelial antigens. These cells were well proliferated during successive subcultivation. Under osteogenic condition, these cells showed increased levels of osteogenic mRNAs and strong positivity in ALP and von Kossa stains at 4th week. CONCLUSION: The homogeneous fibroblast-like cells developed in cultures from hUCB were considered to be MSPC. Morphological and immunophenotypical characteristics of these cells were very similar to those of bone marrow-derived MSPC, and well differentiated into osteoblasts.

The Association of HLA-DRB1 and DQB1 Alleles and a Study of Anticardiolipin Antibody and Anti-beta2 Glycoprotein I Antibody in Korean SLE Patients

BACKGROUND: Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by diverse clinical manifestations and autoantibody production, which is known to be strongly influenced by genetic factors. Previous studies have revealed the associations of SLE with HLA class II alleles and antiphospholipid antibody system (anticardiolipin antibody (aCL) and anti-beta2 glycoprotein I antibody (anti-beta2 GPI)). Therefore, we studied the associations of HLA class II alleles with SLE and antiphospholipid antibody system. METHODS: The genotyping for HLA-DRB1 and DQB1 alleles were performed in 61 SLE patients and 100 controls by the polymerase chain reaction (PCR)-sequence specific oligonucleotide probe method. ELISA tests for aCL and anti-beta2 GPI were performed in 39 of the 61 SLE patients. The results were evaluated statistically by Chi-square test. RESULTS: The frequencies of the HLA-DRB1*15 and DQB1*06 in SLE patients were significantly higher than those in controls. HLA-DRB1*12 was significantly lower in SLE patients than controls. Nine of 39 patients were positive for aCL (IgG) and three were positive for aCL (IgM). One of 39 patients were positive for anti-beta2 GPI (IgG) and none of them positive for anti-beta2 GPI (IgM). Association of aCL with HLA class II alleles was not observed in our study. CONCLUSION: According to our results, it was found that HLA-DRB1*15 and DQB1*06 were associated with genetic susceptiblility and DRB1*12 was associated with resistance to SLE in Korean population. No Association of aCL with HLA class II alleles was observed and the positive rate for anti-beta2 GPI was very low.

NRAMP1 gene polymorphisms in patients with rheumatoid arthritis in Koreans

Natural resistance-associated macrophage protein 1 (Nramp1) is a genetic locus associated with innate resistance or susceptibility of murine hosts to infection with intracellular pathogens such as Salmonella, Leishmania and Mycobacterium. The human homologue of the Nramp1 gene, designated NRAMP1, has been investigated as a candidate gene for genetic susceptibility to autoimmune diseases as well as infections. This study tries to determine whether NRAMP1 polymorphisms are associated with susceptibility to rheumatoid arthritis in Koreans. The nine NRAMP1 polymorphisms (1 microsatellite, 1 variation in 3' UTR, 5 silent substitution, 2 amino acid substitution) were typed by PCR-RFLP in 74 patients with rheumatoid arthritis (RA) and 53 healthy controls in Koreans. The distribution of allele and genotype frequencies were compared between patients and controls. Three NRAMP1 polymorphisms (823C/T, D543N and 1729+55del4) were significantly associated with RA. In addition, there were significant differences in the genotype frequencies for 823C/T, D543N and 1729+ 55del4 polymorphisms between RA patients and controls. Genotypes of A/A homozygote for D543N and TGTG deletion homozygote for 1729+55del4 were only detected in the patient group. These data indicate that genetic polymorphisms of NRAMP1 might be associated with the susceptibility to rheumatoid arthritis in Koreans.

Evaluation of In-house Lymphocyte Panel of 72 Wells for the Identification of HLA Antibody Specificity / 대한임상병리학회지

BACKGROUND: To detect anti-human leukocyte antigen(HLA) class I alloantibodies in patients awaiting solid organ transplantation, panel reactive antibody(PRA) test using complement-dependent lymphocytotoxicity(CDC) has been used. The enough size of lymphocyte panel in PRA test enables the identification of HLA antibody specificities. So we made lymphocyte panel of 72 wells to evaluate the usefulness comparing with 36 wells screening panel. METHODS: A total of 55 sera(positive 20, negative 25, quality control materials provided by "International Cell Exchange" program of UCLA Tissue Typing Laboratory 10), which had been tested for PRA using 36 wells screening panel, were re-tested using newly produced 72 wells lymphocyte panel. RESULTS: The results of the 25 negative sera were same except one serum, which might be due to non-specific reaction. The %PRA values of the 20 positive sera using 36 wells screening panel were distributed into 1-10%(n=4), 10-50%(n=9), 50-80%(n=5), and 80-100%(n=2). Using lymphocyte panel of 72 wells, %PRA values of 20 positive sera showed no difference(p=0.61) from that of 36 wells and we could not identify the specificity of HLA antibodies for the 10 sera, which previously had not been identified with 36 wells screening panel. But we additionally or newly identified the specificity of HLA antibodies in 4 positive sera and 2 quality control materials. CONCLUSION: Identification of HLA antibodies was not much improved using a PRA test with 72 lymphocyte panel and therefore 36 lymphocyte panel is considered to be enough to screen the HLA antibodies. However the increase of the size of lymphocyte panel is expected to resolve the difficulty, caused by linkage disequilibrium, for the identification of HLA antibody specificity.

Comparison of HLA-Cw Typing by Serology and PCR-SSP in the Korean Population / 대한임상병리학회지

BACKGROUND: Using the classical serological methods, the HLA-Cw typing resulted in a high frequency of Cw blank because of the lack of suitable antisera coupled with low cell surface expression. Recent data on association between graft-versus-host disease and serologically undetectable HLA-Cw mismatches in bone marrow transplantation (BMT) facilitate the investigations into the biological role of HLA-Cw and more reliable HLA-Cw typing. METHODS: We performed the HLA-Cw DNA typing using PCR-SSP technique with sequence-specific primers of 22 pairs in 150 Koreans (79 organ transplant recipients, 71 healthy potential donors). These results were compared with those of serological HLA-Cw typing, which had been performed by complement-dependent microlymphocytotoxicity technique using Terasaki Tissue typing tray. RESULTs: Comparison between serological and PCR-SSP typing revealed a discrepancy rate of 24.0% (36/150). The majority of total discrepancies (29/36, 80.6%) were due to antigens that were not detected serologically and these antigens consisted of mainly Cw*12 and Cw*15. In five cases, no Cw allele was detected by DNA typing, whereas serological typing showed antigens and different antigen assignments between two methods were found in three cases. Of 66 individuals typed serologically with one blank, 66.6% (44 cases) were confirmed to be homozygous, whereas an additional Cw allele was found in remaining 22 cases using the PCR-SSP technique. In the case of the serologically undetectable HLA-Cw blank antigens, the gene frequencies of Cw*12 and Cw*15 were 6.4% and 2.8%, respectively. CONCLUSIONS: These results indicated that serological typing is insufficient for accurate HLA-Cw typing. DNA typing using PCR-SSP technique appears a reliable and practical method for accurate HLA-Cw typing which can contribute to the evaluation of the biological role of the HLA-Cw in transplantation.

The Frequency and Characteristics of the Platelet Refractoriness and Alloimmunization in Platelet Concentrate Multiple-Transfused Thrombocytopenic Patients / 대한혈액학회지

BACKGROUND: Understanding the cause for platelet refractoriness in a given patient is the critical step in determining the strategy for optimum management. The aim of this study was to establish the causes and frequency of platelet refractoriness as well as the incidence of anti- HLA antibodies and anti-platelet specific antibodies in multiple transfused thrombocytopenic patients. METHODS: Our study was based on 58 patients requiring platelet transfusions on at least three consecutive occasions from September 1997 to December 1997 in our hospital. The platelet refractoriness was defined as 18-24 hour post-transfusion corrected count increment (CCI) of less than 5,000. Enzyme immunosorbent assay (EIA), panel reactive antibody test (PRA) and Modified antigen capture ELISA (MACE) were applied for the detection of alloimmunization. RESULTS: Thirty-nine patients had episodes of refractoriness (67%). In 39 patients, poor response was seen in 38 patients (97%) with presence of non-immune factors known to be associated with platelet refractoriness. The total rate of alloimmunization was 31%, accounting for fourteen patients (24%) who had HLA antibodies, and four patients (7%) who had platelet specific antibodies. No patient had platelet-specific antibodies in addition to HLA antibodies. From our results, alloimmunization has shown a statistically meaningful relationship with CCI and the use of leukoreduction filtered blood components. CONCLUSION: Our data suggest that immune mechanisms are not the predominant cause of platelet refractoriness and HLA antibodies are produced separately with platelet-specific antibodies, as well as more frequently in alloimmunization.

Comparison between Daunorubicin and Idarubicin in Acute Myelocytic Leukemia Induction Therapy and the Analysis of Prognostic Factors / 대한혈액학회지

BACKGROUND: Anthracycline is the most important chemotherapy drug of acute myelocytic leukemia (AML). It has been reported that idarubicin could have better complete remission (CR) rate than daunorubicin. However, it is not completely established concerning the effectiveness of idarubicin. There are many prognostic indicators of AML, however, many discrepancies still exist in prognostic indicators among each centers. METHODS: We analyzed initial CR rate of 39 AML patients treated with combination of cytarabine plus idarubicin or daunorubicin at Samsung Medical Center from April, 1995 to December, 1997. We subgrouped the patients according to age, sex, initial WBC count, status of initial CR, CD34, and chromosome. We analyzed the initial CR rate and long term survival of each subgroups. RESULTS: Initial CR rates of idarubicin and daunorubicin were 76.5% and 72.7%, respectively. The median survival days of 39 patients was 727+/-308.8 days. 1-year survival rate and 1-year event free survival rate were 64.2% and 59.6%, respectively. Patients who had failure of initial CR, old age (>60 years), and initial high WBC counts (>100,000/L) showed a statistically significant shorter survival in univariate analysis. However, we could not find the significant difference in the positivity of CD34 and chromosomal abnormalities. CONCLUSION: The effectiveness of idarubicin may be equivalent to that of daunorubicin. Failure of initial CR, old age, and high WBC counts were regarded as a prognostic risk factors of AML. However, a more definitive characterization of prognostic factors is warranted in further prospective study.

HLA Class I Typing for Umbilical Cord Blood / 대한임상병리학회지

BACKGROUND: Accurate HLA typing is important for umbilical cord blood (UCB) transplantation as well as bone marrow transplantation. However, HLA class I typing by standard microcytotoxicity method has been often unsatisfactory for UCB samples. This study was conducted to investigate the characteristics of cytotoxic reaction in HLA class I microlymphocytotoxicity testing for UCB. METHODS: We compared the strength index (SI) and the frequencies of HLA antigens with weak reaction between 87 UCB and 103 adult peripheral blood samples by analysis of reaction score in microlymphocytotoxicity typing using Terasaki Oriental HLA-ABC (72) Well Tray (One Lambda, USA). RESULTS: The mean SI of UCB samples in HLA class I typing was significantly lower than that of adult blood (78.5% vs. 96.8%). The SI of 100% among UCB and adult blood samples were 24.1% and 71.8%, respectively. Among HLA-A, B, C antigens, those showing significantly high frequencies of weak reaction in UCB were HLA-A31, B48, B51, B59, B61, Cw3 and Cw7. Cytotoxic reactions of Bw4 and Bw6 in UCB were significantly weaker than those in adult blood. CONCLUSIONS: The strategy of using a supplementary DNA typing method in selected cases with doubtful or unreliable results in serological typing would be effective for an accurate HLA class I typing of UCB samples.

Immunophenotypes and Proliferative Function of Lymphocytes in Umbilical Cord Blood / 대한임상병리학회지

BACKGROUND: The umbilical cord blood (UCB) is highlighted to be a alternative source of hematopoietic stem cells with a hope of resolving major problems of bone marrow transplantation, which are the lack of suitable HLA-matched donors and the occurrence of graft-versus-host disease (GVHD). In this study we have assessed the immunological potential of lymphocytes in UCB, which could mediate GVDH, by immunophenotypes and functional assays on 20 UCB and 19 adult peripheral blood as controls. METHODS: Immunophenotyping of lymphocytes were performed by one and two color flow cytometry (FACSort, Becton Dickinson, USA) using monoclonal antibodies (CD3, CD4, CD8, CD19, CD16/56, CD45RA, CD45RO, HLA-DR, HLA-DQ, HLA-DP, TCRalphabeta, TCRgammadelta). To assess proliferative responses, UCB lymphocytes were cultured in the presence of phytohemagglutinin (PHA) (5, 20, 100 ug/mL) and concanavalin A (ConA) (1, 5, 10 ug/mL). After 72 hour culture and 3H-thymidine pulsing, specific 3H-thymidine uptake was compared to control cells (without mitogen) by stimulation index (SI). RESULTS: In phenotypic analyses, the percentages of CD4, CD8, CD45RO, TCRgammadelta and HLA- DP positive cells were significantly lower in UCB compared with the adult controls, while the percentage of CD45RA positive cells was increased in UCB. In addition, the percentages of B cell (CD19), NK cell (CD16/56), HLA-DR, HLA-DQ and TCRalphabeta were comparable to those in adult controls. In mitogen stimulated culture, UCB lymphocytes showed a significant lower proliferative responses at 20, 100 ug/mL of PHA, and 5, 10 ug/mL of ConA concentrations. CONCLUSIONS: We have observed that UCB lymphocytes appeared to be phenotypically and functionally immature. Thus, UCB might be a attractive source for hematopoietic stem cells in that these UCB cells may have lower immunological potentials mediating GVHD after transplantation.