valuation of the Collection Efficiency of the Granulocyte in Leukapheresis Using 10% Pentastarch

Background :To collect high concentration of granulocytes for transfusion to neutropenic cancer patients with infections, we investigated the effect of G-CSF or dexamethasone as granulocyte mobilizers and 10% pentastarch (PS) as the sedimentation agent in granulocyte collection by leukapheresis. Subsequently, the therapeutic effect of the granulocyte transfusions was assessed. Method :Forty five leukapheresis were performed with CS-3000Plus (Baxter, Deerfield, IL, USA) using 10% pentastarch. The donors were classified into three groups according to their premedication drugs and the interface detector offset; group 1 used dexamethasone with offset 15 (n=16), group 2 used dexamethasone with offset 33 (n=19), and group 3 used G-CSF with offset 33 (n=10). We compared total collected granulocyte counts and granulocyte collection efficiency (GCE). Result :The mean counts of total granulocytes collected and GCE were as follows; 0.9±0.5 × 1010 and 31.6±14.3% in group 1, 1.3±0.6 × 1010 and 39.0±14.2% in group 2, and 1.6±0.9 × 1010 and 63.9±32.2% in group 3, respectively. The counts of granulocytes collected in group 3 was significantly higher than that in group 1 (P Conclusion :G-CSF indicates greater potency than dexamethasone although its high cost is limitation of routine use as mobilizing agents and PS was an excellent red cell sedimenting agent in granulocyte collection. Large volume granulocyte transfusions allow high therapeutic effects in neutropenic patients with marrows of sufficient regenerating capacity.ope

ELISpot for measuring human immune responses to vaccines

The enzyme-linked immunosorbent spot (ELISpot) assay is one of the most commonly used methods to measure antigen-specific T cells in both mice and humans. Some of the primary reasons for the popularity of the method are that ELISpot is highly quantitative, can measure a broad range of magnitudes of response and is capable of assessing critical cellular immune-related activities such as IFN-γ secretion and granzyme B release. Furthermore, ELISpot is adaptable not only to the evaluation of a variety of T-cell functions, but also to B cells and innate immune cells. It is no wonder that ELISpot has evolved from a research tool to a clinical assay. Recent Phase I and II studies of cancer vaccines, tested in a variety of malignancies, have suggested that ELISpot may be a useful biomarker assay to predict clinical benefit after therapeutic immune modulation. This article will discuss the most common applications of ELISpot, overview the efforts that have been undertaken to standardize the assay and apply the method in the analysis of human clinical trials, and describe some important steps in the process of developing a clinical-grade ELISpotope

Clinical Significance of Anti-HSP 70 Antibody in the Patients with Systemic Lupus Erythematosus

Background :Heat shock proteins (HSPs), or stress proteins, are immunodominant antigens of many microorganisms. In this study, we have detected the anti-HSP 70 antibody and tried to explain the role of the antibody with respect to the pathogenesis of SLE. Furthermore, we have attempted to find out the possibility to link the presence of the autoantibody with the monitoring and diagnosis of systemic lupus erythematosus (SLE). Method :A total of 80 samples from 55 SLE patients were screened for the presence of anti-HSP 70 antibodies. Simultaneously 59 healthy people were tested as a control group. The anti-HSP 70 antibodies were measured by enzyme-linked immunosorbent assay (ELISA) and confirmed by western blot in anti-HSP 70 antibody ELISA positive samples. The activity of disease state was confirmed by the patients’medical record and systemic lupus activity measure (SLAM). Result :The mean optical density (O.D.450) of ELISA in healthy controls and SLE patients were 0.15±0.18 (mean±S.D.) and 0.13±0.14. The correlation of SLAM Score and ELISA O.D. was r2 = 0.19, P = 0.014. And, the mean O.D. value of ELISA was 0.18±0.02 and 0.11±0.01 before and after treatment (P < 0.05). We compared samples with SLAM Score. The O.D. of anti-HSP 70 ELISA in these patients were 0.20±0.02 and 0.08±0.002 before and after treatment respectively (n=10, mean±S.D., P < 0.01). Conclusion :Anti-HSP 70 antibody was not a clinically useful diagnostic marker in SLE patients. However, the titer of anti-HSP 70 antibody can be used for the monitoring of the therapeutic effectiveness in these patients.ope

CD4+ T Cell Tolerance to Tissue-Restricted Self Antigens Is Mediated by Antigen-Specific Regulatory T Cells Rather Than Deletion

Deletion of self-antigen-specific T cells during thymic development provides protection from autoimmunity. However, it is unclear how efficiently this occurs for tissue-restricted self antigens, or how immune tolerance is maintained for self-antigen-specific T cells that routinely escape deletion. Here we show that endogenous CD4+ T cells with specificity for a set of tissue-restricted self antigens were not deleted at all. For pancreatic self antigen, this resulted in an absence of steady-state tolerance, while for the lung and intestine, tolerance was maintained by the enhanced presence of thymically-derived antigen-specific Foxp3+ regulatory T (Treg) cells. Unlike deletional tolerance, Treg cell-mediated tolerance was broken by successive antigen challenges. These findings reveal that for some tissue-restricted self antigens, tolerance relies entirely on nondeletional mechanisms that are less durable than T cell deletion. This might explain why autoimmunity is often tissue-specific, and it offers a rationale for cancer vaccine strategies targeting tissue-restricted tumor antigens.ope

High-mobility group box-1 contributes tumor angiogenesis under interleukin-8 mediation during gastric cancer progression

Many soluble factors are involved in tumor angiogenesis. Thus, it is valuable to identify novel soluble factors for effective control of tumor angiogenesis in gastric cancer (GC). We investigated the role of extracellular high-mobility group box-1 (HMGB1) and its associated soluble factors in the tumor angiogenesis of GC. Clinically, we measured serum levels of HMGB1 and GC-associated cytokines/chemokines using GC serum samples (n = 120), and calculated microvessel density (MVD) by CD34 immunostaining using human GC tissues (n = 27). Then we analyzed the correlation of serum HMGB1 levels with MVD or that with cytokine/chemokine levels by linear regression. As in vitro angiogenesis assay for HMGB1, HUVEC migration and capillary tube formation assay were carried out using different histological types of human GC cells (N87 and KATOIII). CD34-positive microvessels were detected from early GC, but MVD increased according to GC stages, and were closely correlated with serum HMGB1 levels (R = 0.608, P = 0.01). The HUVECs cultured in conditioned media derived from rhHMGB1-treated or HMGB1-TF GC cells showed remarkably enhanced migration and tube formation activities. These effects were abrogated by anti-HMGB1 antibody or HMGB1 siRNA in both N87 and KATOIII cells (all P < 0.05). Among tested cytokines/chemokines, interleukin-8 (IL-8) was the most remarkable cytokine correlated with serum HMGB1 (P < 0.001), and enhanced HUVEC migration and tube formation activities by rhHMGB1 or HMGB1-TF were significantly reversed by IL-8 inhibition. These results indicate overexpressed HMGB1 contributes to tumor angiogenesis through IL-8 mediation, and combined targeting of HMGB1 and IL-8 can control tumor angiogenesis in GC.ope

Annual Report on External Quality Assessment in Therapeutic Drug Monitoring and Drug of Abuse in Korea (2007)

Two trials of external quality assessment for Therapeutic Drug Monitoring (TDM) subcommittee of Korean Association of Quality Assurance for Clinical Laboratory (KAQACL) were performed in 2007. Number of participating laboratories were increased to 109, by 5.63% increase comparing with the previous year. Response rates reached 98.7% for both trials slightly lower than that of the previous year. Two kinds of control materials were requested to be tested in each trial so that each institution could know the possible systematic error. In both trials, 20 test items were responded at least from one laboratory. The average drug item was 6.3 per institution, which was decreased slightly from 6.8 in recent 5 years. The most common test items were valproic acid, digoxin, carbamazepine, theophylline, phenytoin, and phenobarbital which were peformed in more than 55% of the participating laboratories, followed by cyclosporine, vancomycin, lithium, tacrolimus, methotrexate, amikacin, gentamicin, salicylate, tobramycin, acetaminophen, primidone, free phenytoin, and amitryptyline. The inter-laboratory coefficients of variations showed simliar tendency comparing with those of the previous years. We started the proficiency test for drug of abuse from 2007 and got the response from 13 and 17 laboratories in the 1st and 2nd trial, respectively. Average drug items were 3.4 for the 2nd trial. We found the relatively good performances as we got the correct answers from all laboratories except 4 for each one mistake. In conclusion, the TDM external quality assessment of 2007 showed grossly similar pattern comparing with those of previous year and drug of abuse proficiency testing showed a relatively good performance.ope

A Disintegrin and Metalloproteinase 8 as a Potential Blood Biomarker for Early Diagnosis of Gastric Cancer

PURPOSE: We aimed to evaluate the clinical significance of a disintegrin and metalloproteinase 8 (ADAM 8) as a potential blood biomarker for gastric cancer (GC). MATERIALS AND METHODS: Blood ADAM 8 was measured by ELISA. Cytokines/chemokines [interleukin-23 (IL-23), stromal cell-derived factor 1α/CXC chemokine ligand 12 (SDF-1α/CXCL12), interleukin-8 (IL-8), and soluble CD40 ligand (sCD40L)] were measured by chemiluminescent immunoassay. They were compared among five groups; normal/gastritis, high-risk, early GC (EGC), advanced GC (AGC) without distant metastasis, and AGC with distant metastasis by one-way analysis of variance in both training (n=80) and validation dataset (n=241). Clinicopathological features of GC and GC-associated cytokines were evaluated for their correlations with blood ADAM 8. To evaluate the diagnostic accuracy to predict GC, receiver operating characteristic (ROC) curve and logistic regression were used. RESULTS: Blood ADAM 8 significantly increased along GC carcinogenesis in both training (ANOVA, p<0.001) and validation dataset (p<0.001). It was significantly higher in EGC compared to high-risk (post-hoc Bonferroni, p=0.041) and normal (p<0.001). It was also higher in AGC compared with high-risk (p<0.001) and normal (p<0.001) groups. However, no significant difference was found between cancer groups. Blood ADAM 8 was correlated with N-stage (Spearman's correlation, γs=0.320, p=0.011), but not with T-stage or M-stage. Pearson's correlations showed blood ADAM 8 was closely correlated with pre-inflammatory cytokines, IL-23 (p=0.036) and SDF-1α/CXCL12 (p=0.037); however, it was not correlated with pro-angiogenic cytokine IL-8 (p=0.313), and sCD40L (p=0.702). ROC curve and logistic regression demonstrated that blood ADAM 8 showed higher diagnostic accuracy (sensitivity, 73.7%; specificity, 86.2%) than CEA (sensitivity, 23.1%; specificity, 91.4%). Combination of ADAM 8 and CEA further increased the diagnostic accuracy to predict GC (sensitivity, 81.8%; specificity, 84.0%). CONCLUSION: Blood ADAM 8 is a promising biomarker for early detection of GC.ope

Clinical significance and usefulness of soluble heparin binding-epidermal growth factor in gastric cancer

AIM: To evaluate the clinical usefulness of soluble heparin-binding epidermal growth factor (sHB-EGF) as a serum biomarker for gastric cancer (GC). METHODS: Serum sHB-EGF levels were measured by a commercially available human HB-EGF ELISA Kit and compared among 60 normal controls, 30 high-risk patients, 37 early gastric cancer (EGC), and 30 advanced gastric cancer (AGC) through ANOVA test. Correlations between serum sHB-EGF and clinicopathological features of GC were analyzed through Spearman's correlation. The diagnostic performance of serum sHB-EGF for GC was evaluated through receiver operating characteristic (ROC) curve and logistic regression analysis. RESULTS: Serum sHB-EGF levels were significantly higher in AGC group (314.4±127.5 pg/mL) than EGC (165.3±123.2 pg/mL), high-risk (98.7±67.3 pg/mL), and control (94.7±83.6 pg/mL) groups (post-hoc Bonferroni, all P<0.001), respectively. Serum sHB-EGF levels were also significantly higher in EGC group than high-risk (P=0.049) and control (P=0.006) groups. Clinicopathologically, serum sHB-EGF levels closely correlated with depth of invasion (T-stage, γs=0.669, P<0.001), lymph node metastasis (N-stage, γs=0.407, P=0.001), and distant metastasis (M-stage, γs=0.261, P=0.030). ROC curve and logistic regression analysis demonstrated a remarkable diagnostic potential of serum sHB-EGF. CONCLUSION: Serum sHB-EGF is closely correlated with advanced stage GC and can be a promising serological biomarker for GC.ope

Annual Report on External Quality Assessment in Therapeutic Drug

Two trials of external quality assessment for Therapeutic Drug Monitoring (TDM) Subcommittee of Korean Association of Quality Assurance for Clinical Laboratory were performed in 2006. The number of participating laboratories were increased to 104, by 9.5% increase comparing with the previous year. Response rates were 100.0% for both trials as the recent 4 years. Two kinds of control materials were requested simultaneously to be tested in each trial so that each institution could know the possible systematic error. In both trials, 20 test items were responded at least from one laboratory. The average drug item was 6.5 per institution, which was decreased slightly from 6.8 in recent 5 years. The most common test items were valproic acid, digoxin, phenytoin, carbamazepine, and theophylline which were peformed in more than 71% of participating laboratories. The response rate of phenobarbital were decreased to 61%, followed by cyclosporine, lithium, vancomycin, tacrolimus, methotrexate, amikacin, gentamycin, salicylate, tobramycin, acetaminophen, salicylate, primidone, free phenytoin, and amitryptyline, in decreasing order. The most widely used TDM analyzer was Abbott TDx/TDxFLx (38.9%), followed by Abbott AxSym (25.9%), and Roche Cobas Integra (20.5%). The inter-laboratory coefficients of variations were not greatly improved comparing with those of previous years. In conclusion, the TDM external quality assessment of 2006 was showed the similar patterns except the increase of the participating laboratories (n=104) with the slightly decreased usage of popular 6 items.ope

Clinical significance of elevated serum soluble CD40 ligand levels as a diagnostic and prognostic tumor marker for pancreatic ductal adenocarcinoma

BACKGROUND: CD40-CD40 ligand (CD40L) interaction is considered to contribute to the promotion of prothrombotic responses and production of angiogenesis-associated factor in addition to adaptive immune responses. Recently, the role of soluble CD40L (sCD40L) has gained interest in cancer, although its exact functions remain unknown. This study evaluated the clinical significance of sCD40L in patients with pancreatic ductal adenocarcinoma (PDAC) and validated its utility as a PDAC diagnostic and prognostic biomarker. METHODS: Serum sCD40L levels were measured by chemiluminescent immunoassay and compared among normal, chronic pancreatitis (CP, high-risk), and PDAC group in both training (n=25 per group) and independent validation (n=30, 30, and 55, respectively) datasets through one-way ANOVA test with the post-hoc Bonferroni method. To evaluate the diagnostic potential of serum sCD40L for PDAC, receiver operating characteristic (ROC) curves were generated and logistic regression analysis was conducted. To investigate the sCD40L-assoicated cytokines/chemokines in PDAC, cytokines/chemokines levels were analyzed by a MILLIPLEX MAP Human Cytokine/Chemokine Kit. To assess the prognostic potentials of sCD40L, Kaplan-Meier survival curve and Cox proportional-hazards regression analysis were applied. RESULTS: Serum sCD40L levels were significantly higher in PDAC group compared with non-cancer groups in both training (p35,000 ng/ml) had a poorer prognosis than those with low-serum sCD40L (log-rank, p=0.015). Multivariate Cox regression analysis yielded a hazard ratio of 2.509 (95% CI, 1.038-6.067, p=0.041) for mortality in the high-serum sCD40L group. CONCLUSIONS: Serum sCD40L is correlated with immunosuppression and angiogenesis in PDAC carcinogenesis/progression, and is a promising diagnostic and prognostic biomarker for PDAC superior to CA19-9 and CEA.ope