Long-Term (Postnatal Day 70) Outcome and Safety of Intratracheal Transplantation of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells in Neonatal Hyperoxic Lung Injury

PURPOSE: This study was performed to evaluate the long-term effects and safety of intratracheal (IT) transplantation of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in neonatal hyperoxic lung injury at postnatal day (P)70 in a rat model. MATERIALS AND METHODS: Newborn Sprague Dawley rat pups were subjected to 14 days of hyperoxia (90% oxygen) within 10 hours after birth and allowed to recover at room air until sacrificed at P70. In the transplantation groups, hUCB-MSCs (5x10(5)) were administered intratracheally at P5. At P70, various organs including the heart, lung, liver, and spleen were histologically examined, and the harvested lungs were assessed for morphometric analyses of alveolarization. ED-1, von Willebrand factor, and human-specific nuclear mitotic apparatus protein (NuMA) staining in the lungs and the hematologic profile of blood were evaluated. RESULTS: Impaired alveolar and vascular growth, which evidenced by an increased mean linear intercept and decreased amount of von Willebrand factor, respectively, and the hyperoxia-induced inflammatory responses, as evidenced by inflammatory foci and ED-1 positive alveolar macrophages, were attenuated in the P70 rat lungs by IT transplantation of hUCB-MSCs. Although rare, donor cells with human specific NuMA staining were persistently present in the P70 rat lungs. There were no gross or microscopic abnormal findings in the heart, liver, or spleen, related to the MSCs transplantation. CONCLUSION: The protective and beneficial effects of IT transplantation of hUCB-MSCs in neonatal hyperoxic lung injuries were sustained for a prolonged recovery period without any long-term adverse effects up to P70.

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.

Screening and Identification of Metallo-beta-Lactamase Gene in Clinical Isolates of Imipenem-Resistant Pseudomonas Aeruginosa / 대한진단검사의학회지

BACKGROUND: The therapeutic difficulty due to wide-spread emergence of multiply resistant strains is a major problem in Pseudomonas aeruginosa infection. Carbapenem-resistant P. aeruginosa strains are being isolated with increasing frequency. Clinical isolates of P. aeruginosa with transferable imipen-em resistance due to production of metallo-beta-lactamase (MBL) have been reported. This study was performed to determine the usefulness of the imipenem-EDTA disk test to detect MBL, to examine the prevalence of MBL in a tertiary care hospital in Korea. METHODS: One hundred sixteen P. aeruginosa isolates with reduced susceptibilities to imipenem were collected during the period of 2000-2003 in the Samsung Medical Center. Imipenem-resistant P. aeruginosa isolates were examined for MBL production by imipenem-EDTA disk tests. To detect of blaIMP-1 , blaVIM-1, and blaVIM-2 genes, polymerase chain reactions (PCR) were performed and the positive isolates were confirmed by sequencing. RESULTS: Among 116 clinical isolates of P. aeruginosa, 20 isolates (17.2%) were positive for the imipenem-EDTA disk tests. Nineteen isolates (16.4%) carried VIM-2. Accoroding to PCR results, the sensitivity, specificity, and test efficiency of the imipenem-EDTA disk tests were 89%, 97%, and 96%, respectively. CONCLUSIONS: The imipenem-EDTA disk test is sensitive and specific for detecting VIM producer. VIM-2 may be an important MBL in P. aeruginosa in tertiary care hospitals the Korea. The spread of MBL genes could compromise the future usefulness of carbapenem for the treatment of gram-neg-ative bacilli infections.

Performance Analysis of the Beckman Coulter GEN-S Automated Hematology Analyzer / 대한임상병리학회지

BACKGROUND: The complete blood cell count (CBC) and leukocyte differential counts are useful tools for making a diagnosis, treating and monitoring a disease. This study evaluated the perfor-mance of Beckman-Coulter's new model, the Coulter GEN-S system (Beckman Coulter Corpora-tion, Miami, USA; GEN-S), and compared it with the Sysmex NE-8000 (Sysmex Corporation, Kobe, Japan; NE-8000) and Sysmex R-3000 (Sysmex Corporation, Kobe, Japan; R-3000). METHODS: One hundred and four blood samples and 120 blood samples were randomly chosen for a comparison analysis from various in-patients and healthy persons, who visited Samsung med-ical center, respectively. The GEN-S system was evaluated according to the linearity and how well it compared with the NE-8000 in terms of the CBC, and the efficiency of the leukocyte suspect flags. RESULTS: The GEN-S showed that the determination coefficients (R(2)) of the WBC, RBC, platelet count and hemoglobin level were more than 0.94 (P 0.95 (P < 0.001) when compared with the NE-8000. In addition, the correlations of the leukocyte differential counts for neutrophils, eosinophils, and lymphocytes were good, but those for monocytes and basophils were poor. The sensitivity, specificity and positive predictive value of the leukocyte suspect flags of the GEN-S system were 89%, 73%, and 43%, respectively. CONCLUSIONS: These results demonstrate the comparable performance of the GEN-S system in clinical laboratories. However, a separate microscopic differential count is required due to the low positive predictive value for the leukocyte suspect flags.

Evaluation of Serologic Marker Tests for Hepatitis B Viral Infection Using the Automated Immunoassay System ARCHITECT i2000 / 대한임상병리학회지

BACKGROUND: The prevalence of Hepatitis B virus (HBV) in Korea is still higher than that of devel-oped countries. Recently, the automated chemiluminescent microparticle immunoassay analyzer ARCHITECT i2000 (Abbott Laboratories, Abbott Park, IL USA) was introduced in Korea and we evaluated performance of the tests for serological markers for HBV infection. METHODS: We analyzed precision, agreement, sensitivity, specificity and throughput of the HBs antigen, anti-HBs and anti-HBc as well as linearity and compared with the AxSYM (Abbott Labora-tories, Abbott Park, IL USA) for anti-HBs. Precision, linearity and comparison were performed on the basis of the National Committee for Clinical Laboratory Standards guidelines. Random patients 'sera were used for this study. RESULTS: The coefficients of variations of precision were below 5% for anti-HBs and anti-HBc (total) except for the HBs antigen. The agreements, sensitivities and specificities for serologic mark-ers were more than 90%. The linearity and comparison for anti-HBs were statistically significant (P < 0.001). The throughput of ARCHITECT i2000 was 110 tests/hours and that was 2.8 times faster than that of the AxSYM. CONCLUSIONS: These results suggest that ARCHITECT i2000 can provide rapid and effective results for serologic markers for HBV infection. However, each laboratory should decide the utiliza-tion of this analyzer on the basis of volume of samples, other items tested concurrently, and the inter-face of existing facilities etc.

Surface Immunoglobulin Light Chain-Positive Acute Lymphoblastic Leukemia of FAB L1 Type: Case Report / 대한혈액학회지

ymphoblastic leukemia (ALL) of B-cell lineage can be classified using the French- American-British (FAB) classification as L1, L2 and L3 type. L1 and L2 ALLs express terminal deoxynucleotidyl transferase (TdT) and are surface immunoglobulin (sIg)-negative. SIg expression in adults with L1 or L2 ALL is extremely rare. We report a case of L1 ALL with positive sIg. A 39-year-old woman had suffered from fever and abdominal pain for 15 days. Her initial complete blood cell counts were WBC 1.3x109/L, hemoglobin 8.8g/dL and platelet 59.0x109/L. Blast cells on blood were counted up to 24% and showed typical FAB L1 morphology on bone marrow. Immunophenotyping was performed and showed expression of CD5, CD19, CD20, HLA-DR, TdT and sIglamda. Karyotype was 46,XX,der (8;9) (q10;q10),+der (8;9) (q10;q10),t (9;22) (q34;q11.2)[3]/47, idem,+der (22)t (9;22)[5]/46,XX[12]. The case was finally diagnosed as the sIg positive ALL, L1. Chemotherapy consisting of cytoxan, daunorubicin, vincristine, L-asparaginase, prednisolone and intrathecal methotrexate was initiated. The patient had been in complete remission for 12 months. Twelve months later, blasts were detected in cerebrospinal fluid. The patient received intrathecal methotrexate and radiation therapy. Thereafter six months later, blasts were observed on peripheral blood. Bone marrow examination showed diffuse infiltration by blasts with L2 morphology and loss of previously positive sIg. At that time, she had given up the treatment. Although several cases of sIg positive B cell ALL, L1 or L2 have been reported, we could hardly find same case of ours in Korean.

A New Variant Translocation t(8;16)(q22;p13.3) in a Case of Acute Myeloid Leukemia-M2 / 대한혈액학회지

No abstract available.

Correlation between Progenitor Cell Dose and the Rate of Engraftment in Autologous Peripheral Blood Stem Cell Transplantation and Allogeneic Bone Marrow Transplantation / 대한수혈학회지

BACKGROUND: Bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT) following high dose chemotherapy has been an important therapeutic option for patients with hematologic malignancies or some solid tumors. The number of progenitor cells in the collection products has been used to determine the optimum time to stop the collections and to predict the hematopoietic engraftment after transplantation. In this study, we investigated the relationship between end-product cell counts measured by different methods and the influence of the infused cell dose on the engraftment rate. METHODS: Twenty five patients receiving autologous PBSCT and 25 patients receiving allogeneic BMT were studied. The number of total nucleated cells (TNC), of mononuclear cells (MNC), of CD34+ cells, and of CFU-GM (colony-forming unit-granulocyte monocyte) colonies were measured in each collection product. The number of days required to achieve an absolute neutrophil count (ANC) of 0.5x109/L with TNC count of 1.0x109/L and platelet count of 20x109/L without transfusions was taken as an arbitrary measure of the engraftment rate. RESLUTS: A close correlation between CD34+ cells/kg and CFU-GM/kg was observed in both collection products (p<0.05). However, MNC/kg also showed significant correlations with CD34+ cells/kg and CFU-GM/kg in allogeneic bone marrow collection products (p<0.05). The CFU-GM amount in the PBSC products was greater than that in the bone marrow collection products (p<0.05). Time to engraftment was a median of 14 (range 9-50) days in autologous PBSCT group, but 29 (range 17-57) days in allogeneic BMT group. In autologous PBSCT, infused CD34+ cells/kg and CFU-GM/kg correlated significantly with ANC recovery (p<0.05). CONCLUSIONS: The number of CD34+ cells was correlated with that of CFU-GM in the collection products, and the infused cell doses showed positive relation to the engraftment rate in autologous PBSCT. These findings suggest that measurement of CD34+ cell counts alone would be a sufficient parameter to predict the engraftment rate in autologous PBSCT.

CyCD3+MPO- Biphenotypic Leukemia With Unusual Presentation: A Case Report / 대한혈액학회지

No abstract available.

Peripheral Lymphocyte Subsets in Patients with Pulmonary and Extrapulmonary Tuberculosis / 대한임상병리학회지

BACKGROUND: T-cell mediated cellular immunity has been suggested as an important mechanism in mycobacterial infection. Also, it is known that there is a imbalance between helper and suppressor T cells in the pathogenesis of tuberculosis in human. This study was designed to evaluate the changes of lymphocyte subsets in patients with pulmonary and extrapulmonary tuberculosis. METHODS: Lymphocyte subset analysis was performed on 53 pulmonary tuberculosis and 21 extrapulmonary tuberculosis patients. The proportion of total T (CD3+), B (CD19+), helper T (H, CD3+CD4+) and suppressor T (S, CD3+CD8+) cells, natural killer (NK, CD16+CD56+) cells and activated T (CD3+HLA-DR+) cells were analyzed using SimultestTM (Becton-Dickinson, California, USA) by FACSortTM (Becton-Dickinson, California, USA) and each absolute cell counts and helper T/suppressor T (H/S) ratio were calculated. RESULTS: In pulmonary and extrapulmonary tuberculosis patients groups, there were no significant changes in percentage and absolute cell counts of lymphocyte subset compared to control group. But H/S ratio was significantly decreased in both groups and the H/S ratio in extapulmonary tuberculosis was lower than that in pulmonary tuberculosis (1.06+/-0.44 vs. 1.64+/-0.97). CONCLUSION: Decreased or reversed H/S ratio reflect the role of cell mediated immune response in patients with tuberculosis, expecially in the spreading of pulmonary tuberculosis. Lymphocyte subset test seems to be helpful for access the different clinical forms of tuberculosis, pulmonary and extapulmonary tuberculosis.

CMV Infection in AIDS Patients Depends on the Decrease of CD4+ T Cell Count eauth

Cytomegalovirus is the most common cause of life-threatening viral infection in HIV-infected patients. This study was done prospectively to investigate the incidence of CMV infection according to the decrease of CD4+ T cell count (CD4+) in Korean AIDS patients. Thirty-nine HIV-infected patients diagnosed before 1994 were followed for regular immunological monitoring. We have used urine shell vial method for the CMV detection from 1994 and have also checked clinical findings. Positive urine culture rate definitely depended on the CD4+ as follows; 45%, 22%, 17%, 11% and 0%, CD4+ >50, 50-100, 100-200, 200-500 and <500, respectively. Except culture positive 2 patients with CD4+ of 200~300/ul, all eight culture positive patients with CD4+ less than 200/ul showed CMV related diseases on or before urine culture. But, we could not get a positive culture for a late AIDS patient with vision loss. With ganciclovir therapy, all culture results were at least negative just after or on late of first 14 days-ganciclovir infusion-course. These data suggest that the incidence of CMV disease in Korean AIDS patients is very high, and early diagnosis and treatment for CMV diseases is required for the prevention of life threatening results.

Sequence Analysis of C2-V3 Region of Human Immunodeficiency Virus Type 1 gp120 and its Correlation with Clinical Significance : the Effect of Long-Term Intake of Korean Red Ginseng on env Gene Variation

Human immunodeficiency virus type 1 (HIV-1) phenotype plays an important role in the pathogenesis of AIDS. The presence of syncytium-inducing (SI) HIV-1 isolates in infected persons is associated with a rapid decline of CD4+T cells (CD4+), rapid disease progression, and reduced survival time after AIDS diagnosis. We have reported the effects of Korean red ginseng (KRG) on HIV-1 infected patients. To investigate whether KRG affects HIV-1 at gene level and there is a correlation between genotype and decline of CD4+, the C2-V3 region of env gene from 65 HIV-1 isolates were cloned and sequenced. Distributions of subtype were subtype B 57 (88%), subtype A 4 (6%), subtype C 2 (3%), subtype G 1 (2%), and subtype H 1 (2%). The prevalences of SI according to the number of CD4+ are as follows; 40% (6/15) in CD4+ 200/ul. Seventy-five percent (6/8) of SI were detected in rapid progressor with the decline of CD4+ over 60/ul per year. The correlation between SI genotype and the detection of immune complex dissociated (ICD) p24 antigen was significant (p<0.001). In the 40 patients followed-up over 60 months by CD4+, there was significant correlation between annual decrease of CD4+ and duration of KRG intake (R=-0.380, p<0.01), whereas no correlation between CD4+ and zidovudine (ZDV) was observed. The intrapatient variation of amino acid level showed significant inverse correlation with the months of KRG intake (R=-0.47, p<0.01). These results suggest that the determination of genotype by C2- V3 sequencing may be used for the evaluation of prognosis of AIDS patient, and long-term intake of KRG may prevent or delay the progression from NSI to SI.

Serum Interleukin-10 Levels in Rheumatoid Arthritis Patients / 대한류마티스학회지

OBJECTIVE: To investigate whether the serum levels of IL-10 in patients with rheumatoid arthritis are different from those of normal controls and SLE patients and to find out any correlation with disease activity parameters of rheumatoid arthritis. METHODS: Sera from 20 healthy normal persons, 16 rheumatoid arthritis patients and 16 patients with systemic lupus erythematosus were collected and measured for IL-10 and IL-6. Various disease activity parameters were measured in RA patients. RESULTS: The serum level of IL-10 in RA patients was significantly elevated compared to normal controls but lower than those of SLE patients. In RA patients there was no definite correlation between the disease activity parameters and serum IL-10 levels. Despite significant improvements in terms of various disease activity parameters, there was no significant change of serum IL-10 levels after treatment in RA patients. In seropositive RA patients, positive correlation was found between serun IL-10 and rheumatoid factor levels. CONCLUSION: Our findings show that the serum IL-10 levels in patietns with RA are elevated compared to normal controls but lower than those of SLE patients. There was no correlation between serum IL-10 levels and disease acivity parameters of RA.

Filtration Effects of Leukocyte Removal Filter for Platelet Concentrates / 대한수혈학회지

Some variables can influence the efficiency of leukocyte removal filter for red cell concentrates. We performed this study to evaluate whether the filtration efficiency for platelet concentrates(PCs) can be influenced by bedside filter(PXL8K, Pall Corporation, USA) used, platelet storage time and transient stopping of platelet agitation, and to measure the amount of cytokines released from donor leukocytes in PCs during the storage time. In addition, we examined the effect of leukocyte reduction by filtration before the storage of PCs on the subsequent generation of cytokines. Any of the above mentioned variables did not make significant differences. From day 1 to day 5, there were significant increases in IL-1 beta (<1 vs. 8.6 pg/mL, p<0.05), IL-8 (<10 vs. 455.3 pg/mL, p<0.05) and TNF-alpha (<4 vs. 16.6 pg/mL, p<0.05) in unflltered PCs, whereas no increased revels of cytokines were found in the cases of prestorage filtration.

Acute leukemias with unusual immunophenotypes

Over a two-year period, immunophenotypic patterns of 266 acute leukemia cases were analyzed using a panel of tests including TdT, SmIg and 9 surface antigens by the immunofluorescence stains for the assessment of the incidence and grade of phenotypic ambiguity (lineage infidelity) and the possible clinical significance of unusual immunophenotypes. Immunophenotypes were classified into four groups according to the degree of ectopic antigen expression. We classified as Group A (91.7%, 244 of 266 cases) those expressing conventional pattern without ectopic antigen. Group B (3.0%, 8 of 266 cases) was defined to have at least two lineage specific markers and single ectopic antigen. Such a "low grade deviation" did not prevent a definite immunodiagnosis. Group C (4.2%, 11 of 266 cases) revealed a promiscuous coexpression of markers related to different lineages, including two cases (0.8%, 2 cases) of biphenotypic leukemia. Group D (1.1%, 3 cases) included unclassifiable immunophenotypes with no antigen or HLA-DR only expression. Both patients with biphenotypic leukemia and one patient with unclassifiable immunophenotypes failed to respond to induction chemotherapy, suggesting a poor prognosis in these patients. The incidence of acute myelogenous leukemia (AML) cases with one or more ectopic surface antigens was 10 (8.1%) of the 124 AML cases. Ectopic antigen expression was seen in 5 (4%) of the 125 B-lineage acute lymphoblastic leukemia (ALL) cases and 3 (25%) of the 12 T-ALL cases. It is concluded that nearly 95% of cases of acute leukemia cases can be diagnosed accurately with immunophenotyping alone including patients with a mild degree of deviation from expected antigenic patterns.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunologic marker analysis in acute myeloid leukemia / 대한혈액학회지

No abstract available.