Matrix Metalloproteinase 1 as a Marker of Tonsil-Derived Mesenchymal Stem Cells to Assess Bone Marrow Cell Migration

BACKGROUND@#To achieve optimal bone marrow engraftment during bone marrow transplantation, migration of donor bone marrow cells (BMCs) toward the recipient’s bone marrow is critical. Despite the enhanced engraftment of BMCs by co-administration of mesenchymal stem cells (MSCs), the efficiency can be variable depending on MSC donor. The purpose of this study is to examine the functional heterogeneity of tonsil-derived MSCs (TMSCs) and to identify a marker to evaluate efficacy for the enhancement of BMC migration. @*METHODS@#To examine the donor-to-donor variation of TMSCs in potentiating BMC migration, we isolated TMSCs from 25 independent donors. Transcriptome of TMSCs and proteome of conditioned medium derived from TMSC were analyzed. @*RESULTS@#Enhanced BMC migration by conditioned medium derived from TMSCs was variable depending on TMSC donor. The TMSCs derived from 25 donors showed distinct expression profiles compared with other cells, including fibroblasts, adipose-derived MSCs and bone marrow–derived MSCs. TMSCs were distributed in two categories: high- and low-efficacy groups for potentiating BMC migration. Transcriptome analysis of TMSCs and proteome profiles of conditioned medium derived from TMSCs revealed higher expression and secretion of matrix metalloproteinase (MMP) 1 in the high-efficacy group. MMP1 knockdown in TMSCs abrogated the supportive efficacy of conditioned medium derived from TMSC cultures in BMC migration. @*CONCLUSION@#These data suggest that secreted MMP1 can be used as a marker to evaluate the efficacy of TMSCs in enhancing BMC migration. Furthermore, the strategy of analyzing transcriptomes and proteomes of the MSCs may be useful to set the standard for donor variation.

Promotion of Platelet Production by Co-Transplantation of Mesenchymal Stem Cells in Bone Marrow Transplantation

BACKGROUND@#Therapeutic strategies that can promote platelet production are in demand to enhance clinical outcomes of bone marrow transplantation (BMT). Our research group has studied human tonsil-derived mesenchymal stem cells (TMSCs) and their effectiveness in promoting bone marrow (BM) engraftment. Here, we analyzed the effects of T-MSCs on platelet production and hemostasis. @*METHODS@#Donor BM cells (BMCs) were isolated from C57BL/6 mice and transplanted with or without T-MSCs to BALB/c recipient mice. Mice were sacrificed and blood cells were counted using an Auto Hematology Analyzer. Femur sections were stained with CD41 antibody to analyze megakaryocytes in the BM. Growth factor secretion from MSCs was analyzed using the Quantibody Array. Effects of T-MSC conditioned medium (CM) on megakaryopoiesis were investigated using the MegaCult assay. In a mouse model of BMT, T-MSC CM was injected with or without anti-placental growth factor (a-PlGF) blocking antibody, and blood cell numbers and coagulation were analyzed. @*RESULTS@#T-MSC co-transplantation increased percent survival of BMT mice. Platelet numbers were significantly lower in the BMC-only group, whereas T-MSC co-transplantation restored circulating platelets to levels similar to those of the control group. Significantly reduced numbers of CD41 ? megakaryocytes in Bu-Cy and BMC groups were increased by T-MSC co-transplantation. PlGF secretion from T-MSCs were detected and enhanced megakaryopoiesis, platelet production, and coagulation by T-MCS CM were disrupted in the presence of the a-PlGF blocking antibody. @*CONCLUSION@#We demonstrated the effectiveness of T-MSC co-transplantation in promoting platelet production and coagulation after BMT. These findings highlight the potential therapeutic relevance of T-MSCs for preventing thrombocytopenia after BMT.

A Novel Method to Differentiate Tonsil-Derived Mesenchymal Stem Cells In Vitro into Estrogen-Secreting Cells

BACKGROUND@#The advantages of tonsil-derived mesenchymal stem cells (TMSCs) over other mesenchymal stem cells (MSCs) include higher proliferation rates, various differentiation potentials, efficient immune-modulating capacity, and ease of obtainment. Specifically, TMSCs have been shown to differentiate into the endodermal lineage. Estrogen deficiency is a major cause of postmenopausal osteoporosis and is associated with higher incidences of ischemic heart disease and cerebrovascular attacks during the postmenopausal period. Therefore, stem cell-derived, estrogen-secreting cells might be used for estrogen deficiency. @*METHODS@#Here, we developed a novel method that utilizes retinoic acid, insulin-like growth factor-1, basic fibroblast growth factor, and dexamethasone to evaluate the differentiating potential of TMSCs into estrogen-secreting cells. The efficacy of the novel differentiating method for generation of estrogen-secreting cells was also evaluated with bone marrow- and adipose tissue-derived MSCs. @*RESULTS@#Incubating TMSCs in differentiating media induced the gene expression of cytochrome P450 19A1 (CYP19A1), which plays a key role in estrogen biosynthesis, and increased 17b-estradiol secretion upon testosterone addition. Furthermore, CYP11A1, CYP17A1, and 3b-hydroxysteroid dehydrogenase type-1 gene expression levels were significantly increased in TMSCs. In bone marrow-derived and adipose tissue-derived MSCs, this differentiation method also induced the gene expression of CYP19A1, but not CYP17A1, suggesting TMSCs are a superior source for estrogen secretion. @*CONCLUSION@#These results imply that TMSCs can differentiate into functional estrogen-secreting cells, thus providing a novel, alternative cell therapy for estrogen deficiency.

A Novel Method to Differentiate Tonsil-Derived Mesenchymal Stem Cells In Vitro into Estrogen-Secreting Cells

BACKGROUND@#The advantages of tonsil-derived mesenchymal stem cells (TMSCs) over other mesenchymal stem cells (MSCs) include higher proliferation rates, various differentiation potentials, efficient immune-modulating capacity, and ease of obtainment. Specifically, TMSCs have been shown to differentiate into the endodermal lineage. Estrogen deficiency is a major cause of postmenopausal osteoporosis and is associated with higher incidences of ischemic heart disease and cerebrovascular attacks during the postmenopausal period. Therefore, stem cell-derived, estrogen-secreting cells might be used for estrogen deficiency. @*METHODS@#Here, we developed a novel method that utilizes retinoic acid, insulin-like growth factor-1, basic fibroblast growth factor, and dexamethasone to evaluate the differentiating potential of TMSCs into estrogen-secreting cells. The efficacy of the novel differentiating method for generation of estrogen-secreting cells was also evaluated with bone marrow- and adipose tissue-derived MSCs. @*RESULTS@#Incubating TMSCs in differentiating media induced the gene expression of cytochrome P450 19A1 (CYP19A1), which plays a key role in estrogen biosynthesis, and increased 17b-estradiol secretion upon testosterone addition. Furthermore, CYP11A1, CYP17A1, and 3b-hydroxysteroid dehydrogenase type-1 gene expression levels were significantly increased in TMSCs. In bone marrow-derived and adipose tissue-derived MSCs, this differentiation method also induced the gene expression of CYP19A1, but not CYP17A1, suggesting TMSCs are a superior source for estrogen secretion. @*CONCLUSION@#These results imply that TMSCs can differentiate into functional estrogen-secreting cells, thus providing a novel, alternative cell therapy for estrogen deficiency.

Conditioned Medium from Tonsil-Derived Mesenchymal Stem Cells Relieves CCl₄-Induced Liver Fibrosis in Mice

BACKGROUND: The liver is an organ with remarkable regenerative capacity; however, once chronic fibrosis occurs, liver failure follows, with high mortality and morbidity rates. Continuous exposure to proinflammatory stimuli exaggerates the pathological process of liver failure; therefore, immune modulation is a potential strategy to treat liver fibrosis. Mesenchymal stem cells (MSCs) with tissue regenerative and immunomodulatory potential may support the development of therapeutics for liver fibrosis. METHODS: Here, we induced hepatic injury in mice by injecting carbon tetrachloride (CCl₄) and investigated the therapeutic potential of conditionedmedium from tonsil-derivedMSCs (T-MSCCM). In parallel, we used recombinant human IL-1Ra,which, as we have previously shown, is secreted exclusively from T-MSCs and resolves the fibrogenic activation of myoblasts. Hepatic inflammation and fibrosis were determined by histological analyses using H&E and Picro-Sirius Red staining. RESULTS: The results demonstrated that T-MSC CM treatment significantly reduced inflammation as well as fibrosis in the CCl₄-injured mouse liver. IL-1Ra injection showed effects similar to T-MSC CM treatment, suggesting that T-MSC CM may exert anti-inflammatory and anti-fibrotic effects via the endogenous production of IL-1Ra. The expression of genes involved in fibrosis was evaluated, and the results showed significant induction of alpha-1 type I collagen, transforming growth factor beta, and tissue inhibitor of metalloproteases 1 upon CCl₄ injection, whereas treatment with T-MSC CM or IL-1Ra downregulated their expression. CONCLUSION: Taken together, these data support the therapeutic potential of T-MSC CM and/or IL-1Ra for the alleviation of liver fibrosis, as well as in treating diseases involving organ fibrosis.

Effects of Hand Training During the Aftereffect Period of Low-Frequency rTMS in Subacute Stroke Patients

OBJECTIVE: To investigate the effects of hand training using low-frequency repetitive transcranial magnetic stimulation (rTMS) within the aftereffect period on hand function in patients with subacute stroke. METHODS: The subacute stroke patients with hand weaknesses were divided randomly into two groups. Patients in the intervention group underwent hand training within the aftereffect period, that is, immediately after receiving low-frequency rTMS treatment. Patients in the control group underwent hand training 2 hours after the low-frequency rTMS treatment. A manual function test (MFT) for ‘grasp and pinch’ and ‘hand activities’; a manual muscle test (MMT) for ‘grasp’, ‘release’, and ‘abductor pollicis brevis (APB)’; and the Modified Ashworth Scale for finger flexion were performed and measured before and immediately after combined therapy as well as 2 weeks after combined therapy. RESULTS: Thirty-two patients with hand weakness were enrolled in this study. The intervention group patients showed more improvements in grasp MMT and MMT APB tested immediately after combined therapy. However, the changes in all measurements were not significantly different between the two groups 2 weeks after the combined therapy. In both groups, hand functions improved significantly immediately after combined therapy and 2 weeks after combined therapy. CONCLUSION: Hand training immediately after low-frequency rTMS showed more rapid improvement in the motor power of hands than hand training conducted 2 hours after low-frequency rTMS. Our results suggest that conducting hand training immediately after low-frequency rTMS could be an improved useful therapeutic option in subacute stroke patients.

Effect of Caregiver Driven Robot-Assisted In-Ward Training in Subacute Stroke Patients: A Case Series

OBJECTIVE: To evaluate the effect of caregiver driven robot-assisted in-ward training in subacute stroke patients. METHODS: A retrospective evaluation was performed for patients treated with caregiver driven robot-assisted in-ward training to retain gait function from June 2014 and December 2016. All patients received more than 2 weeks of caregiver driven robot-assisted in-ward training after undergoing conventional programs. The robot was used as a sitting device, a standing frame, or a high-walker depending on functional status of the patient. Patients were evaluated before and after robot training. Patient records were assessed by Korean version of Modified Barthel Index (K-MBI), Functional Independence Measure (FIM), and Functional Ambulation Category (FAC). RESULTS: Initially, patients used the robot as a sitting device (n=6), a standing frame (n=7), or a partial body-weight support high-walker (n=2). As patient functions were improved, usage level of the robot was changed to the next level. At the end of the treatment, the robot was used as a sitting device (n=1), a standing frame (n=6), or high-walker (n=8). Scores of K-MBI (Δ17.47±10.72) and FIM (Δ19.80±12.34) were improved in all patients. CONCLUSION: Patients' usage level of the robot and functional scores were improved. Therefore, performing additional caregiver driven robot-assisted in-ward training is feasible and beneficial for subacute stroke patients.

Mosaic Turner syndrome associated with schizophrenia

Turner syndrome is a sex-chromosome disorder; occurring in 1 in 2,500 female births. There are sporadic few case reports of concomitant Turner syndrome with schizophrenia worldwide. Most Turner females had a 45,X monosomy, whereas the majority of comorbidity between Turner syndrome and schizophrenia had a mosaic karyotype (45,X/46,XX). We present a case of a 21-year-old woman with Turner syndrome, mosaic karyotype (45,X/46,XX), showing mental retardation, hypothyroidism, and schizophrenia. HOPA gene within Xq13 is related to mental retardation, hypothyroidism, and schizophrenia. Our case may be a potential clue which supports the hypothesis for involvement of genes on X chromosome in development of schizophrenia. Further studies including comorbid cases reports are need in order to discern the cause of schizophrenia in patients having Turner syndrome.

Tissue-specific activation of mitogen-activated protein kinases for expression of transthyretin by phenylalanine and its metabolite, phenylpyruvic acid

Phenylketonuria is an autosomal recessive disorder caused by a deficiency of phenylalanine hydroxylase. Transthyretin has been implicated as an indicator of nutritional status in phenylketonuria patients. In this study, we report that phenylalanine and its metabolite, phenylpyruvic acid, affect MAPK, changing transthyretin expression in a cell- and tissue-specific manner. Treatment of HepG2 cells with phenylalanine or phenylpyruvic acid decreased transcription of the TTR gene and decreased the transcriptional activity of the TTR promoter site, which was partly mediated through HNF4alpha. Decreased levels of p38 MAPK were detected in the liver of phenylketonuria-affected mice compared with wild-type mice. In contrast, treatment with phenylalanine increased transthyretin expression and induced ERK1/2 activation in PC-12 cells; ERK1/2 activation was also elevated in the brainstem of phenylketonuria-affected mice. These findings may explain between-tissue differences in gene expression, including Ttr gene expression, in the phenylketonuria mouse model.

Congenital Zinc Deficiency from Mutations of the SLC39A4 Gene as the Genetic Background of Acrodermatitis Enteropathica

Acrodermatitis enteropathica (AE) is an autosomal recessive disorder with the clinical triad of acral dermatitis, diarrhea and alopecia. AE is known to be caused by mutations of the SLC39A4 gene on the chromosome band 8q24.3, encoding the zinc transporter in human. An 8-month-old Korean boy presented with eczematous changes on the inguinal area and knees and was diagnosed with AE. Blood tests revealed a markedly decreased level of plasma zinc, and his symptoms improved on oral zinc replacement. To confirm the diagnosis of AE from congenital zinc deficiency, direct sequencing analysis of SLC39A4 was performed and revealed that he was compound heterozygous for a known missense mutation (Arg95Cys) and a novel splicing mutation in the donor site of intron 7 (c.1287+2T>C). Family study showed that his parents were heterozygous carriers of the mutations. To the best of our knowledge, this is the first report of genetically confirmed AE in Korea.

Protective Effect of Recombinant Adeno-Associated Virus 2/8-Mediated Gene Therapy from the Maternal Hyperphenylalaninemia in Offsprings of a Mouse Model of Phenylketonuria

Phenylketonuria (PKU) is an autosomal recessively inherited metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and the fetus of an uncontrolled pregnant female patient presents with maternal PKU syndrome. We have reported previously on the cognitive outcome of biochemical and phenotypic reversal of PKU in a mouse model, Pah(enu2), by the AAV serotype 2-mediated gene delivery of a human PAH transgene. However, the therapeutic efficacy had been limited to only male PKU mice. In this study, we generated a pseudotyped recombinant AAV2/8-hPAH vector and infused it into female PKU mice through the hepatic portal vein or tail vein. Two weeks after injection, complete fur color change to black was observed in female PKU, as in males. The PAH activity in the liver increased to 65-70% of the wild-type activity in female PKU mice and to 90% in male PKU mice. Plasma phenylalanine concentration in female PKU mice decreased to the normal value. In addition, the offsprings of the treated female PKU mice can rescue from the harmful effect of maternal hyperphenylalaninemia. These results indicate that recombinant AAV2/8-mediated gene therapy is a potential therapeutic strategy for PKU.

Human Telomerase Reverse Transcriptase (hTERT): A Target Molecule for the Treatment of Cisplatin-resistant Tumors / 대한진단검사의학회지

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme that is required for telomerase activity (TA) and cancer progression. Telomerase inhibition or inactivation increases cellular sensitivity to UV irradiation, DNA-damaging agents, the tyrosine kinase inhibitor, imatinib, and pharmacological inhibitors, such as BIBR1532. hTERT is associated with apoptosis. Some patients show drug-resistance during anti-cancer drug treatment and the cancer cell acquire anti-apoptotic mechanism. Therefore, we attempted to study correlation between hTERT and drug-resistance. METHODS: To study the correlation between protein level and activity of hTERT and drug-resistance, Western blotting and telomerase repeat amplification protocol (TRAP) assays were performed. To investigate whether hTERT contributes to drug resistance in tumor cells, we transiently decreased hTERT levels using small interfering RNA (siRNA) in T24/R2 cells. RESULTS: hTERT knockdown increased Bax translocation into the mitochondria and cytochrome C release into the cytosol. Caspase inhibitors, especially Z-VAD-FMK, rescued this phenomenon, suggesting that the stability or expression of hTERT might be regulated by caspase activity. CONCLUSIONS: These data suggest that hTERT might be a target molecule for drug-resistant tumor therapy.

PHEX Gene Mutations and Genotype-Phenotype Analysis of Korean Patients with Hypophosphatemic Rickets

X-linked hypophosphatemic rickets (XLH) results from mutations in the PHEX gene. Mutational analysis of the PHEX gene in 15 unrelated Korean patients with hypophosphatemic rickets revealed eight mutations, including five novel mutations, in nine patients: two nonsense mutations, two missense mutations, one insertion, and three splicing acceptor/donor site mutations. Of these, c.64G>T, c.1699C>T, c.466_467 insAC, c.1174-1G>A, and c.1768+5G>A were novel mutations. To analyze the correlation between genotype and phenotype, phenotypes were compared between groups with and without a mutation, in terms of mutation location, mutation type, and sex. Skeletal disease tended to be more severe in the group with a mutation in the C-terminal half of the PHEX gene, but no genotype-phenotype correlation was detected in other comparisons. Further extensive studies of the PHEX gene mutations and analyses of the genotype-phenotype relationships are required to understand PHEX function and the pathogenesis of XLH.

Upregulation of Reactive Oxygen Species and Resistance to Fas-mediated Apoptosis in Cervical Cancer Cell Lines Transfected with the IL-18 Receptor / 대한진단검사의학회지

BACKGROUND: The fas (CD95/Apo-1)/Fas ligand (FasL) system is reported to be involved in the suppression and stimulation of immune responses, and the reactive oxygen species (ROS) play a key role in the mechanism for resisting Fas-induced apoptosis of tumor cells. In this work, we investigated the effect of endogenous interleukin (IL)-18 on the regulation of immune related factors such as Fas/Fas ligand and intercellular adhesion molecules (ICAM), and of the ROS level in IL-18 receptor (IL-18R) transfected C-33A cells. METHODS: The cervical cancer cell line C-33A was transfected with IL-18R (C-33A/IL-18R). For the detection of pro-inflammatory cytokines in C-33A/IL-18R, reverse-transcriptase (RT) polymerase chain reaction (PCR), in situ enzyme-linked immunosorbent assay (ELISA), Western blot, and Northern blot analyses were performed. The level of p53 was determined by Western blot. Intracellular ROS, ICAM-1, FasL, and apoptosis in C-33A/IL-18R were measured by flow cytometry. RESULTS: In situ ELISA and RT-PCR showed that, among pro-inflammatory cytokines, IL-18 was induced in C-33A/IL-18R whereas there appeared no induction of the IL-1alpha, IL-1beta, tumor necrosis factor (TNF)-alpha, and IL-6. IL-18R transfection showed a slight enhancement of the Fas via upregulation of intracellular ROS and IL-18 in C-33A cells whereas there was no effect on the expression of p53, ICAM-1 and FasL. However, treatment with the agonistic anti-Fas antibody showed that the enhanced surface Fas was not functional or was not enough to induce apoptosis and the C-33A/IL-18R cells escaped still resistant to Fas-mediated apoptosis. CONCLUSIONS: IL-18R transfection induced IL-18 expression and enhanced ROS and Fas expression in C-33A cells. These results show that C-33A/IL-18R cells escaped from immunuosurveillance by failure to express ICAM-1 adhesion molecules and Fas ligand, and are resistant to Fas-mediated apoptosis.

Combination Effects of Various Beta-Lactam Antibiotics with Vancomycin or Teicoplanin against Staphylococcus aureus with Reduced Susceptibility to Vancomycin / 대한진단검사의학회지

BACKGROUND: Combination effects of various beta-lactam antibiotics with vancomycin or teicoplanin against methicillin-resistant Staphylococcus aureus (MRSA) that were detected as pos-sible hetero-vancomycin resistant Staphylococcus aureus (hetero-VRSA) by the Mu-3 agar method, were evaluated. METHODS: Twenty-four strains of MRSA (possible hetero-VRSA) from 22 inpatients of Dankook University Hospital from July through November 1998, were subjected to the study. Minimum inhibitory concentrations (MICs) of antibiotics, alone or in combination, were tested with the agar dilution method and the fractional inhibitory concentration (FIC) indices were calculated to estimate the combination effects. RESULTS: Six strains of 24 MRSA were estimated as hetero-VRSA by population analysis. The aver-age FIC index of imipenem (I), flomoxef (F), cephalothin (C), cefpirome (E) in combination with van-comycin (V) and teicoplanin (T) were 0.584 for I-V, 0.200 for I-T, 0.747 for F-V, 0.230 for F-T, 0.633 for C-V, 0.374 for C-T, 0.773 for E-V, and 0.386 for E-T, respectively. The presence of synergy and addi-tivity in beta-Lactams were observed as 5.3% (16/304) and 90.1% (274/304) for the combination of van-comycin with I, F, C, or E, respectively, and 29.3% (164/560) and 69.8% (391/560) for the combina-tion of teicoplanin with I, F, C, or E, respectively. CONCLUSIONS: We concluded that the selected beta-lactam antibiotics with vancomycin or teicoplanin showed effective against possible hetero-VRSA, as the combination effects were syner-gistic or additive with the average of the FIC index and the frequency of synergy and additivity in this study.

Quantitative Determination of Plasma and Urine Paraquat Concentrations using Spectroscopic Method / 대한진단검사의학회지

BACKGROUND: It is well known that plasma paraquat concentration is one of the most important prognostic indicators for paraquat poisoning. Quantitative analyses of paraquat, however, are not generally used in clinical laboratories. In this work, we evaluated the second-derivative spectroscop-ic method for quantitation of paraquat in plasma and urine, and investigated the clinical significance in patients with paraquat poisoning. METHODS: Linearity, precision, interferences, and comparison with high-performance liquid chro-matography (HPLC) were evaluated in 20 paraquat-poisoning cases using the UV-160 A recording spectrophotometer. The relationship of plasma and urine paraquat concentrations with the clinical outcomes was also studied. RESULTS: The within-run and between-day coefficients of variation (CV) for groups of low and high levels were less than 5%. The derivative amplitude was linearly related to paraquat concentra-tion through the range from 0.5 to 10 ng/mL. The correlation coefficient (r) between spectrophotom-etry and HPLC was 0.992. The accuracy for predicting the outcome for patients based on plasma paraquat concentration was 84.6%. The urine paraquat levels on admission were more than 10 ng/ mL in all of the 9 non-survivors group and in 5 out of 11 of the survivors group. The eliminating rates for plasma and urine paraquat concentrations by extracorporeal procedures were not statistically different between the two groups. CONCLUSIONS: Second-derivative spectroscopic methods for quantitation of paraquat showed an acceptable performance and suitable procedure for clinical laboratory use and it was thought to be seful in assessing the severity and in predicting the prognosis for paraquat poisoning.

A Correlation of Prothrombin Time in Citrated Specimens and EDTA Specimens Examined by Owren Method / 대한진단검사의학회지

BACKGROUND: Sodium citrate has been used as a coagulation test because factor V and VIII are more stable in a citrated specimen. Ethylenediaminetetraacetic acid (EDTA) has been used for the hematologic test because blood cells are preserved better in the EDTA specimen. Both sodium cit-rate and EDTA have the same principle of anticoagulation. They bind free plasma calcium to pre-vent clotting. This study was performed to see if we could substitute EDTA for sodium citrate in pro-thrombin time. METHODS: Blood samples from 133 patients who underwent the prothrombin time test in our hos-pital and 36 healthy controls were used. Each sample was anticoagulated with both sodium citrate and EDTA. We examined the prothrombin time with citrated specimen and EDTA specimen using the Owren reagent; Nycotest PT and also the Quick reagent; and the IL Test PT-Fibrinogen HS. RESULTS: The mean prothrombin time with EDTA specimen was longer than that with the citrated specimen by the Quick method but was shorter using the Owren method. In the Owren method, there was a significant correlation between mean prothrombin time with the citrated and EDTA spec-imen but not using the Quick method. CONCLUSIONS: The prothrombin time with the citrated and EDTA specimens correlated with each other by the Owren method. This correlation could make it possible to replace sodium citrate with EDTA.

Detection of Hepatitis B Virus X Antigen and Anti-X Antibody in Sera of HBV Infected Patients by ELISA using Recombinant X Proteins Expressed in E. coli and Anti-HBx Monoclonal Antibodies / 대한임상병리학회지

BACKGROUND: The X gene is the smallest coding region of the hepatitis B virus (HBV) genome. Several studies reported that X gene-encoded protein may be related to viral replication, and possibly used as a new marker indicative of HBV infection. However, its practical application as a diagnostic reagent remains limited. In this study, we developed anti-X monoclonal antibodies using recombinant hepatitis B virus X (HBx) proteins and investigated the humoral immune responses against HBx in sera of HBV-infected patients by enzyme-linked immunosorbent assay (ELISA). METHODS: Sera of 47 HBV-associated patients and 12 normal controls were studied. Using recombinant HBx expressed in Escherichia coli, seven clones of monoclonal anti-HBx antibodies were developed. The binding site and activity of each monoclonal antibody were determined by ELISA and Western blot analysis, and antibodies that gave the best signals in both assays were selected for the detection of HBx antigen. An ELISA to detect anti-X was also constructed by using recombinant HBx proteins. RESULTS: Clinical samples from patients with liver cirrhosis and hepatocellular carcinoma (HCC) were more than 60% positive for anti-HBx antibody. The positive rate of X antigen in patients with liver cirrhosis and HCC was 27% and 33%, respectively. None of acute hepatitis patients and chronic asymptomatic carriers were positive for HBx antigen or anti-X antibody. The present ELISA system detected circulating HBx with a dynamic range from 5 to 1000 ng per milliliter and the specificity of the assay was also acceptable. The analysis of binding site and activity of monoclonal antibodies performed by ELISA were in agreement with Western blotting results. CONCLUSIONS: ELISA using recombinant HBx and monoclonal antibodies showed good sensitivity and corresponded well with immunoblotting results. For the clinical application of this assay, however, further study is needed on the relationship between HBx and the progression of the disease.

Evaluation of ES-300 for the Detection of Anti-HCV Antibody / 대한임상병리학회지

BACKGROUND: A fully automated enzyme-immunoassay (EIA) analyzer, Enzymun System, ES-300 (Boehringer Mannheim, Germany) uses streptavidin technology and performs single test or panels of up to 12 tests per run. We evaluated the results of ES-300 for anti-HCV by comparing the results with microplate-EIA, radioimmunoassay (RIA), and confirmatory test. METHODS: Total 79 sera (51 positive, 24 negative, 4 indeterminate results confirmed by Lucky HCD Confirm) were analysed. ES-300 with Enzymun-Test(R) Anti-HCV (Boehringer Mannheim, Germany) and microplate-EIA (Green Cross Center Innotest HCV 3.0(R)) were used. Fifty one sera were examined additionally by 2nd-generation RIA method, NANBDINE 125C(General Biologicals Corp., R.O.C.). And all results were compared to the results of Lucky HCD Confirm. RESULTS: The overall concordance rate of ES-300 and Innotest(R) was 72/79 (91.1%). The results of Lucky HCD Confirm on seven discrepant samples were five negative and two indeterminate. The results of ES-300 and NANBDINE 125C showed concordance rate of 90.2%. The sensitivity and specificity of ES-300 with regard to Lucky HCD Confirm were 94.5%, and 87.5%, respectively, and that of Innotest(R) were 98.2% and 66.7%, respectively. Clear distinction of positive and negative results by signal/cut off ratio was available in both EIAs. The positive predictive values of ES-300 and Innotest(R) were 94.5%, and 87.1%, respectively. CONCLUSIONS: ES-300 showed relatively good results in sensitivity and positive predictive value with regard to confirmatory test. In EIA-positive persons, however, follow-up study would be necessary for reliable evaluation of HCV infection.