Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

OBJECTIVES: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). METHODS: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). RESULTS: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. CONCLUSIONS: These recommendations are an important step to achieve high quality ANA testing.

Distinct shear-induced Ca2+ signaling in the left and right atrial myocytes: Role of P2 receptor context.

Atrial myocytes are continuously exposed to shear stress during cardiac cycles. Previous reports have shown that shear stress induces two different types of global Ca2+ signaling in atrial myocytes-longitudinal Ca2+ waves (L-waves) and action potential-involved transverse waves (T-waves), and suggested an underlying role of the autocrine activation of P2 receptors. We explored the correlations between ATP release and Ca2+ wave generation in atrial myocytes and investigated why the cells develop two Ca2+-wave types during the same shear force. We examined whether ATP release correlates with different shear-stress (~16 dyn/cm2)-mediated Ca2+ signaling by simultaneous measurement of local Ca2+ and ATP release in individual atrial myocytes using two-dimensional confocal imaging and sniffer patch techniques, respectively. Functional P2X7-receptor-expressing HEK293 cells were established as sniffer cells, which generated currents in real time in response to ATP released from a closely positioned atrial myocyte. Both shear-stress-induced L- and T-waves were preceded by sniffer currents with no difference in the current magnitude. Left atrial (LA) myocytes had two- to three-fold larger sniffer currents than right atrial (RA) cells, as was confirmed by ATP chemiluminescence assay. Shear-stress-induced ATP release was eliminated by connexin (Cx) 43 hemichannel inhibition using La3+, Gap19, or knock-down of Cx43 expression. The level of phosphorylated Cx43 at Ser386 (p-Cx43Ser368), but not total Cx43, was higher in LA versus RA myocytes. Most LA cells (~70%) developed L-waves, whereas most RA myocytes (~80%) presented T-waves. Shear-stress-induced T-waves were completely removed by inhibition of P2X4R, which were most abundant in rat atrial cells. Expression of P2X4R was higher in RA than LA myocytes, whereas expression of P2Y1R, the mediator of L-waves, was higher in LA than RA myocytes. ATP release mainly triggers L-waves in LA myocytes and T-waves in RA myocytes under the same shear force, partly because of the differential expression of P2Y1R and P2X4R between LA and RA myocytes. Higher ATP release in LA myocytes under shear stress may not contribute to determination of the wave pattern.

Evaluation of neutrophil extracellular traps as the circulating marker for patients with acute coronary syndrome and acute ischemic stroke.

INTRODUCTION: Neutrophil extracellular traps (NETs) are known to be induced by various factors. In this study, we tried to identify circulating levels of NETs in patients with acute coronary syndrome (ACS) and acute ischemic stroke (AIS) and to confirm its suitability as a new circulating marker in their detection. METHODS: We prospectively enrolled 95 patients with a diagnosis of ACS (N = 37) or AIS (N = 58) in Dong-A University Hospital, Busan, Korea. The control group was selected from healthy adults (N = 25) who visited the hospital for health screening. Circulating levels of NETs were evaluated by measuring plasma concentrations of double-stranded DNA (dsDNA) and DNA-histone complex. RESULTS: The concentrations of dsDNA were statistically higher in patients with ACS or AIS than those in the control group (both P < .001). In the univariable and multivariable analyses, statistically significant risk factors were troponin I (TnI) level and dsDNA concentration in the ACS group (P = .046 and P = .015, respectively) and only dsDNA concentration in the AIS group (P = .002). In the receiver operating characteristic curve analyses, the area under the curve values for TnI level and dsDNA concentration in the ACS group were 0.878 and 0.968, respectively, and the value for dsDNA concentration in the AIS group was 0.859. CONCLUSIONS: In this study, it was confirmed that the circulating level of NETs was increased in patients with ACS and AIS at initial presentation. Findings in this study show that NETs could be used as a new circulating marker for the initial diagnosis of ACS or AIS.

Association of neutrophil-to-lymphocyte ratio and natural killer cell activity revealed by measurement of interferon-gamma levels in a healthy population.

BACKGROUND: While a method of assaying natural killer (NK) cell activity by measuring the amount of interferon (IFN)-γ released from NK cells has been proposed, no data are available about the factors that influence IFN-γ levels related to NK cell activity. NLR has recently been reported to be a predictor of several diseases. In the present study, we investigated the pre-analytical variables for NK cell activity using measurements of IFN-γ and the relationship between NLR and NK cell activity. METHODS: The NK cell activity was assessed with the measurement of IFN-γ after stimulation with an NK cell-specific stimulant (NK Vue™ , ATgen, Sungnam, Korea). One hundred and six adult volunteers were recruited and analysis of their complete blood count data and serum C-reactive protein was done. Blood sample from 59 of the participants was also used for analysis of lymphocyte subpopulations. RESULT: Natural killer cell activity varied widely (range, 44.2-1775.6 pg/mL). NK cell activity was higher in females than in males (P = 0.014). NK cell activity decreased with increasing NLR (P = 0.004, r = -0.32) but NK cell activity showed no significant association with NK cell count or other lymphocyte subpopulations. NK cell activity levels according to CRP quartile were significantly different (P = 0.025). CONCLUSION: We have observed that NK cell activity when assessed by IFN-γ level measurement was negatively correlating with NLR. This result can be helpful in interpreting or predicting NK cell activity in the clinical environment.

Early prediction of severity in acute ischemic stroke and transient ischemic attack using platelet parameters and neutrophil-to-lymphocyte ratio.

BACKGROUND: It is still not easy to predict severity promptly in patients with acute ischemic stroke (AIS) and transient ischemic attack (TIA). We investigated that platelet parameters or combinations of them could be a useful tool for early prediction of severity of AIS and TIA at admission and after 3 months. METHODS: We prospectively recruited 104 patients newly diagnosed with AIS and TIA. We investigated their neutrophil-to-lymphocyte ratio (NLR) and platelet parameters. According to the Modified Rankin Scale scores, the patients were divided into two groups. RESULTS: In receiver operating characteristic (ROC) curve analyses, mean platelet volume (MPV), NLR/platelet count (PLT), MPV/PLT, MPV*NLR, and MPV*NLR/PLT showed statistically significant results in both at admission and after 3 months. Values of area under ROC curves for those tests at admission were 0.646, 0.697, 0.664, 0.708, and 0.722, respectively. Also, values after 3 months were 0.591, 0.661, 0.638, 0.662, and 0.689, respectively. CONCLUSION: MPV*NLR/PLT could be used as a relatively good tool for predicting severity at the time of admission and after 3 months than other parameters or combinations of them. Further studies have to be carried out to investigate the best parameter for predicting the severity of AIS and TIA.

Histological Evidence of Intrinsic Muscle Degeneration in Compression Ulnar Neuropathy.

PURPOSE: We investigated the histological characteristics of the tissues in the intrinsic hand muscles of patients with compressive ulnar neuropathy (CUN) to determine how the compromised nerve influences the target muscle. METHODS: In total, 83 patients underwent surgery for CUN in our institution between March 2015 and August 2017. Of these, 45 patients who met our inclusion/exclusion criteria were initially included in this study. Basic demographic data including age, sex, hand dominance, duration of symptoms, bone mineral density, and clinical stage were evaluated. During the ulnar nerve decompression surgery, a biopsy of the ipsilateral adductor pollicis was performed. Using the biopsy samples, we analyzed the tissue composition and degree of degeneration, and investigated the association with demographic factors and clinical status. RESULTS: The ratio of muscle/connective tissue/fat was 62.62 ± 8.27%/23.44 ± 4.10%/14.16 ± 6.68% in the affected muscle. The ratio was different than that of samples taken from control groups. In samples containing muscle fibers, although the total fat content remained low, fat was consistently concentrated at the fascicular borders, between fascicles (perifascicular fat, 62.3 ± 18.2% of fascicles), and within fascicles (intrafascicular fat, 35.6 ± 11.8% of fascicles). The proportion of centrally nucleated muscle fibers was also significantly elevated (5.58 ± 4.25%; P < 0.001) compared with that of both controls (1.09 ± 1.26%) and the clinical threshold for abnormal central nuclei (3%). Preoperative scores assessed using Gabel and Amadio criteria were positively correlated with the muscle composition (r = 0.89, P < 0.001). In addition, the clinical status was negatively correlated with the degree of fat accumulation and the proportion of centrally nucleated muscle fibers (r = -0.94, P < 0.001, r = -0.82, P < 0.001). CONCLUSIONS: We demonstrated that target muscle in CUN underwent degeneration, which was potentially exacerbated by inflammation, and that the degree of degeneration was correlated with the patient's clinical status. Histologically, reversible recovery of the hand muscles may be possible if decompression of the ulnar nerve is performed at earlier stages.

Signaling Pathway for Endothelin-1- and Phenylephrine-Induced cAMP Response Element Binding Protein Activation in Rat Ventricular Myocytes: Role of Inositol 1,4,5-Trisphosphate Receptors and CaMKII.

BACKGROUND/AIMS: Endothelin-1 (ET-1) and the α₁-adrenoceptor agonist phenylephrine (PE) activate cAMP response element binding protein (CREB), a transcription factor implicated in cardiac hypertrophy. The signaling pathway involved in CREB activation by these hypertrophic stimuli is poorly understood. We examined signaling pathways for ET-1- or PE-induced cardiac CREB activation. METHODS: Western blotting was performed with pharmacological and genetic interventions in rat ventricular myocytes. RESULTS: ET-1 and PE increased CREB phosphorylation, which was inhibited by blockade of phospholipase C, the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway, protein kinase C (PKC) or Ca2+-calmodulin-dependent protein kinase II (CaMKII). Intracellular Ca2+ buffering decreased ET-1- and PE-induced CREB phosphorylation by ≥80%. Sarcoplasmic reticulum Ca2+ pump inhibitor, inositol 1,4,5-trisphosphate receptor (IP₃R) blockers, or type 2 IP₃R (IP₃R2) knock-out abolished ET-1- or PE-induced CREB phosphorylation. ET-1 and PE increased phosphorylation of CaMKII and ERK1/2, which was eliminated by IP₃R blockade/knock-out or PKC inhibition. Activation of CaMKII, but not ERK1/2, by these agonists was sensitive to Ca2+ buffering or to Gö6976, the inhibitor of Ca2+-dependent PKC and protein kinase D (PKD). CONCLUSION: CREB phosphorylation by ET-1 and PE may be mainly mediated by IP₃R2/Ca2+-PKC-PKD-CaMKII signaling with a minor contribution by ERK1/2, linked to IP₃R2 and Ca2+-independent PKC, in ventricular myocytes.

The clinical severity score of chronic actinic dermatitis correlates with in vivo photoallergic reactions and the immunologic parameters related to a shift towards Th2 immunity from the Th2/Th1 balanced status in patients with chronic actinic dermatitis.

BACKGROUND: Chronic actinic dermatitis (CAD) has a more complicated pathogenetic basis than others. The clinical grading system and its correlations with the clinical and immunological parameters still remained to be investigated to define the nature of CAD in a more detailed manner. OBJECTIVES: We investigated correlations of the clinical severity score of CAD (CSS-CAD) with the clinical and immunological parameters. METHODS: We evaluated 72 patients with CAD and classified them into three groups according to the CSS-CAD. We analysed total IgE level, peripheral blood (PB) eosinophil count, the ratio of Th2cell (CCR4 + CD4 +) percentage over Th1 cell (CXCR3 + CD4 +) percentage (Th2/Th1) and/or the sum of suppressor/cytotoxic T cells. RESULTS: The total IgE levels and the percentage of PB eosinophils were higher in the severer group than other groups. A shift towards Th2 from Th2/Th1 balanced status may be affected by total counts of suppressor T cells, and the patient with higher Th2/Th1 ratio than balanced status had the more proportion in the severer CSS-CAD group than other groups. CONCLUSION: The CSS-CAD correlates with total IgE level, PB eosinophil count and a shift towards Th2 immunity from Th2/Th1. So we suggest the Th1/Th2 dysbalance may be affected by the CSS-CAD.

p62/sequestosome-1 as a severity-reflecting plasma biomarker in Charcot-Marie-Tooth disease type 1A.

Autophagy is a self-degradation system for recycling to maintain homeostasis. p62/sequestosome-1 (p62) is an autophagy receptor that accumulates in neuroglia in neurodegenerative diseases. The objective of this study was to determine the elevation of plasma p62 protein levels in patients with Charcot-Marie-Tooth disease 1A (CMT1A) for its clinical usefulness to assess disease severity. We collected blood samples from 69 CMT1A patients and 59 healthy controls. Plasma concentrations of p62 were analyzed by ELISA, and we compared them with Charcot-Marie-Tooth neuropathy score version 2 (CMTNSv2). A mouse CMT1A model (C22) was employed to determine the source and mechanism of plasma p62 elevation. Plasma p62 was detected in healthy controls with median value of 1978 pg/ml, and the levels were significantly higher in CMT1A (2465 pg/ml, p < 0.001). The elevated plasma p62 levels were correlated with CMTNSv2 (r = 0.621, p < 0.0001), motor nerve conduction velocity (r = - 0.490, p < 0.0001) and disease duration (r = 0.364, p < 0.01). In C22 model, increased p62 expression was observed not only in pathologic Schwann cells but also in plasma. Our findings indicate that plasma p62 measurement could be a valuable tool for evaluating CMT1A severity and Schwann cell pathology.

Characterization of novel HLA-A*24:608N allele discovered in Koreans.

A novel null HLA-A*24 allele, HLA-A*24:608N, was identified in five Korean subjects including three from a family and two separate individuals. This study was performed to discern its immunological function in transplantation settings. Because this null variant had deletions of approximately 12 k base pairs from intron 3 to 3' end of the HLA-A gene, low resolution HLA typing and amplicon-based next generation sequencing (NGS) typing methods had failed to assign it. Hybrid capture-based NGS method confirmed that this novel variant had a large deletion. T-lymphocyte crossmatching by complement-dependent lymphocytotoxicity and flow cytometry with a serum consisting anti-HLA-A24 antibody revealed negative results, implying that an individual with this allele would not carry a functioning A24 antigen. These findings highlight the importance of identifying a null HLA allele by employing appropriate molecular method and providing expected crossmatching outcomes in a real-world transplantation setting.

Current Status of Flow Cytometric Immunophenotyping of Hematolymphoid Neoplasms in Korea.

Background: Flow cytometric immunophenotyping of hematolymphoid neoplasms (FCI-HLN) is essential for diagnosis, classification, and minimal residual disease (MRD) monitoring. FCI-HLN is typically performed using in-house protocols, raising the need for standardization. Therefore, we surveyed the current status of FCI-HLN in Korea to obtain fundamental data for quality improvement and standardization. Methods: Eight university hospitals actively conducting FCI-HLN participated in our survey. We analyzed responses to a questionnaire that included inquiries regarding test items, reagent antibodies (RAs), fluorophores, sample amounts (SAs), reagent antibody amounts (RAAs), acquisition cell number (ACN), isotype control (IC) usage, positive/negative criteria, and reporting. Results: Most hospitals used acute HLN, chronic HLN, plasma cell neoplasm (PCN), and MRD panels. The numbers of RAs were heterogeneous, with a maximum of 32, 26, 12, 14, and 10 antibodies used for acute HLN, chronic HLN, PCN, ALL-MRD, and multiple myeloma-MRD, respectively. The number of fluorophores ranged from 4 to 10. RAs, SAs, RAAs, and ACN were diverse. Most hospitals used a positive criterion of 20%, whereas one used 10% for acute and chronic HLN panels. Five hospitals used ICs for the negative criterion. Positive/negative assignments, percentages, and general opinions were commonly reported. In MRD reporting, the limit of detection and lower limit of quantification were included. Conclusions: This is the first comprehensive study on the current status of FCI-HLN in Korea, confirming the high heterogeneity and complexity of FCI-HLN practices. Standardization of FCI-HLN is urgently needed. The findings provide a reference for establishing standard FCI-HLN guidelines.

Choice of ABO Group for Blood Component Transfusion in ABO-Incompatible Solid Organ Transplantation: A Questionnaire Survey in Korea and Guideline Proposal.

The number of ABO-incompatible solid organ transplantations (ABOi SOTs) has markedly increased worldwide since the early 2000s. We investigated the choice of ABO group for blood component transfusion in ABOi SOT. We conducted a survey by e-mailing a questionnaire to blood bank specialists at 77 major hospitals in Korea, among whom 34 responded to the survey. In major ABOi SOT, for red blood cells (RBCs), the recipient's type (70.6%) was the most common choice, followed by group O (29.4%); for platelets, group AB (50.0%) was the most common choice, followed by the donor type (38.2%); for plasma, group AB (55.9%) was the most common choice, followed by the donor type (32.4%). In bidirectional ABOi SOT, for RBCs, the recipient's type (55.9%) was the most common choice, followed by group O (44.1%); for platelets and plasma, group AB was the most common choice (94.1% and 97.1%, respectively). The policies for transfusion in ABOi SOT were diverse. We suggest a guideline on the choice of ABO group for transfusion in ABOi SOT to secure patient health and enable an efficient use of blood components.

Clinical significance of preoperative serum vascular endothelial growth factor, interleukin-6, and C-reactive protein level in colorectal cancer.

BACKGROUND: Angiogenesis is a multistep process in which many growth factors and cytokines have an essential role. Vascular endothelial growth factor (VEGF) is a potent angiogenic agent that acts as a specific mitogen for vascular endothelial cells through specific cell surface receptors. The interleukin-6 (IL-6) pathway is another mechanism linking angiogenesis to malignancy. C-reactive protein (CRP), a representative marker for inflammation, is known for its association with disease progression in many cancer types. The aim of this study was to determine preoperative serum levels of VEGF, IL-6, and CRP in colorectal carcinoma, and to correlate them with disease status and prognosis. METHODS: A 132 of 143 patients who underwent curative resection for colorectal cancer were enrolled in this study. 11 patients with resection margin positive were excluded. Factors considered in analysis of the relationship between VEGF, IL-6, and CRP and histological findings. Patient prognosis was investigated. Serum levels of VEGF and IL-6 were assessed using Enzyme-Linked Immuno-Sorbent Assay (ELISA), and CRP was measured using immunoturbidimetry. RESULTS: Median follow-up duration was 18.53 months (range 0.73-43.17 months) and median age of the patients was 62 years (range, 26-83 years). Mean and median levels of VEGF and CRP in colorectal cancer were significantly higher than in the normal control group; 608 vs. 334 pg/mL and 528 (range 122-3242) vs. 312 (range 16-1121) (p < 0.001); 1.05 mg/dL vs. 0.43 mg/dL and 0.22 (range 0.00-18.40) vs. 0.07 (range 0.02-6.94) (p = 0.002), respectively. However mean and median level of IL-6 in patients were not significantly higher than in control; 14.33 pg/mL vs. 5.65 pg/mL and 6.00 (range 1.02-139.17) vs. 5.30 (4.50-13.78) (p = 0.327). Although IL-6 and CRP levels were not correlated with other pathological findings, VEGF level was significantly correlated with tumor size (p = 0.012) and CEA (p = 0.038). When we established the cutoff value for VEGF (825 pg/mL), IL-6 (8.09 pg/mL), and CRP (0.51 mg/dL) by Receiver Operating Characteristic (ROC) curve, we noted that high VEGF levels tended to reduce overall survival (p = 0.053), but not significantly. However, IL-6 and CRP demonstrated no significance with regard to disease free survival (p = 0.531, p = 0.701, respectively) and overall survival (p = 0.563, p = 0.572, respectively). Multivariate analysis showed that VEGF (p = 0.032), CEA (p = 0.012), lymph node metastasis (p = 0.002), and TNM stage (p = 0.025) were independently associated with overall survival. CONCLUSIONS: Preoperative serum VEGF and CRP level increased in colorectal cancer patients. High VEGF level has been proposed as a poor prognostic factor for overall survival in patients with colorectal cancer.

Bloodless cancer treatment results of patients who do not want blood transfusion: single center experience of 77 cases.

PURPOSE: Certain patients may be unwilling to accept blood products for religious reasons. In this study, we have assessed the clinical cancer treatment outcomes of Jehovah's Witnesses (JW) cancer patients in order to identify the risks associated with their treatment, as well as their transfusion needs. METHODS: We analyzed 77 cases of histologically confirmed cancer patients (JW) from January 2001 to April 2008. RESULTS: The median age of the patients was 59 years (range, 8-83 years). The most common primary site was the stomach (20.8%), followed by the breast (14.3%), and colorectal region (11.7%). Operations were performed on 44 patients (89.8%). Changes in complete blood count profiles after operation were detected in the patients' hemoglobin (mean +/- SD; 12.7 +/- 2.1 g/dL to 10.6 +/- 2.3 g/dL, P < 0.001). Twenty-six patients received adjuvant chemotherapy. Among these, 21 (80.8%) completed their planned schedule. One hundred twenty-seven cycles of palliative intravenous chemotherapy were administered to 19 patients. Granulocyte-colony stimulating factor and erythropoietin were used in 45 and 20 cycles of treatment, respectively. Grade > or =III thrombocytopenia and anemia were noted in 3.9% and 2.4% of the patients. Three- and 5-year survival rates were 80% and 70%, respectively. The most frequent cause of death was disease progression rather than bleeding. CONCLUSIONS: Bloodless cancer operation and chemotherapy were not accompanied by serious complications. A few cases of palliative chemotherapy also required transfusions. A prospective cohort study group will need to be used to determine precisely the safety of bloodless cancer treatment and the efficacy of transfusion alternatives.

Toxicodendron vernicifluum Stokes extract inhibits solid tumor growth and lung metastasis of 4T1 murine mammary carcinoma cells in BALB/c mice.

Toxicodendron vernicifluum Stokes has long been used as a food supplement and traditional herbal medicine in East Asia. We applied a new extraction method to produce Toxicodendron vernicifluum Stokes extract (TVSE), that doesn't contain urushiol (an allergenic toxin) but dose have higher levels of some flavonoids such as fustin and fisetin. This study was conducted to investigate the anticancer effects of TVSE in an in vivo system. Fifty BALB/c mice were acclimated for one week and then injected with 4T1 murine mammary carcinoma cells in mammary fat pads. After 7 days, the mice were randomly divided into 5 groups, and orally administered with 0, 50, 100, 200 or 400 mg of TVSE/kg body weight (BW)/day for 20 days. TVSE reduced tumor volume and weight dose-dependently. The expression of Ki67 was significantly reduced and the number of TUNEL-positive apoptotic cells was significantly increased in the TVSE-treated group over 100 mg/kg BW/day. While tumor nodules were not found in the liver, but only in lungs, the number of tumor nodules was reduced in a dose-dependent manner in the TVSE treated groups compared to the control group. In breast tumors, expression of platelet endothelial cell adhesion molecule (PECAM-1) and vascular endothelial growth factor (VEGF) was reduced by TVSE treatment. TVSE treatment significantly suppressed mRNA expression in tumors of matrix metalloproteinase (MMP)-2, tissue inhibitor of metalloproteinase (TIMP)-1, urokinase-type plasminogen activator (uPA), intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 while increasing plasminogen activator inhibitor (PAI)-1. These results suggest that TVSE is potentially beneficial for the suppression of breast cancer growth and its-associated lung metastasis.

Rhus verniciflua Stokes extract suppresses migration and invasion in human gastric adenocarcinoma AGS cells.

BACKGROUND/OBJECTIVES: Many studies have suggested that Rhus verniciflua Stokes (RVS) and its extract are anticancer agents. However, RVS had limited use because it contains urushiol, an allergenic toxin. By improving an existing allergen-removal extraction method, we developed a new allergen-free Rhus verniciflua Stokes extract (RVSE) with higher flavonoid content. In this study, we examined whether RVSE inhibits the ability of AGS gastric cancer cells to migrate and invade. MATERIALS/METHODS: The flavonoids content of RVSE was analyzed by HPLC. The effects of RVSE on migration and invasion in AGS cells were analyzed by each assay kit. Matrix metalloproteinase (MMP)-9, plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) protein expression was analyzed by protein antibody array. The Phosphorylation of signal transducer and activator of transcription (STAT) 3 were assayed by Western blot analysis. RESULTS: RVSE treatment with 0-100 µg/mL dose-dependently reduced the ability of AGS cells to migrate and invade. Notably, treatment with RVSE strongly inhibited the expression of MMP-9 and uPA and the phosphorylation of STAT3. In contrast, RVSE treatment dramatically increased the expression of PAI-1. These results indicate that the inhibition of MMP-9 and uPA expression and STAT3 phosphorylation and the stimulation of PAI-1 expression contributed to the decreased migration and invasion of AGS cells treated with RVSE. CONCLUSIONS: These results suggest that RVSE may be used as a natural herbal agent to reduce gastric cancer metastasis.

A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation.

Background: Measurement of natural killer (NK) cell function has important clinical utility in several diseases. Although the flow cytometry (FC)-based 4-h NK cytotoxicity assay using peripheral blood mononuclear cells (PBMCs) in the clinical laboratory has been used for this purpose, this assay requires large amounts of blood and a rapid PBMC isolation step. Here, we developed an FC-based overnight NK cytotoxicity assay using whole blood (WB), and applied it to patients with liver diseases. Methods: Peripheral blood of healthy volunteers (n = 28) and patients with liver diseases, including hepatocellular carcinoma (n = 19) and liver cirrhosis (n = 7), was analyzed for complete blood count, absolute NK cell count, and NK cell activity (NKA). NKA was evaluated in three assay types: an FC-based overnight WB NK cytotoxicity assay using carboxyfluorescein diacetate succinimidyl ester-labeled K562 cells in the presence of various cytokine combinations [including interleukin (IL)-2, IL-18, and IL-21], an FC-based 4-h PBMC NK cytotoxicity assay, and an FC-based CD107a degranulation assay using WB and PBMCs. Results: Optimal cytokine combinations for NK cell activation in WB were determined (IL-2/IL-18, IL-2/IL-21, and IL-2/IL-18/IL-21). A good correlation was observed between WB and PBMC NK cytotoxicity assays; absolute NK cell counts were better correlated with the WB NK cytotoxicity assay than with the PBMC NK cytotoxicity assay. This WB NK cytotoxicity assay showed that patients with liver diseases had significantly lower NK cytotoxicity than healthy volunteers, under stimulation with various cytokines (p < 0.001). Conclusion: The proposed FC-based overnight WB NK cytotoxicity assay correlates well with the conventional 4-h PBMC NK cytotoxicity assay, demonstrating future potential as a supportive assay for clinical laboratory research and observational studies.

Clinical significances of preoperative serum interleukin-6 and C-reactive protein level in operable gastric cancer.

BACKGROUND: The interleukin-6 (IL-6) pathway is one of the mechanisms that link inflammation and angiogenesis to malignancy. Because the C-reactive protein (CRP) is a representative marker for inflammation, CRP has recently been associated with the progression of disease in many cancer types. The principal objective of this study was to determine the preoperative serum levels of IL-6 and CRP in gastric carcinoma, and to correlate them with disease status and prognosis. METHODS: A total of 115 patients who underwent gastrectomy were enrolled in this study. Serum levels of IL-6 were assessed via Enzyme-Linked Immuno-Sorbent Assay (ELISA), and CRP was measured via immunoturbidimetry. Histological findings included tumor size, depth of tumor invasion, lymph node (LN) metastasis, and TNM stage (6th AJCC Stage Groupings: The staging systems; Primary tumor, regional LN, metastasis). RESULTS: Increases in cancer invasion and staging are generally associated with increases in preoperative serum IL-6 levels. IL-6 and CRP levels were correlated with invasion depth (P < 0.001, P = 0.001), LN metastasis (P < 0.001, P = 0.024) and TNM stage (P < 0.001, P < 0.001). The presence of peritoneal seeding metastasis is associated with IL-6 levels (P = 0.012). When we established the cutoff value for IL-6 level (6.77 pg/dL) by ROC curve, we noted significant differences in time to progression (TTP; P < 0.001) and overall survival (OS; P = 0.010). However, CRP evidenced no significance with regard to patients' TTP and OS levels. Serum IL-6 levels were correlated positively with CRP levels (r2 = 0.049, P = 0.018). CONCLUSION: Preoperative serum IL-6 and CRP levels might be markers of tumor invasion, LN metastasis, and TNM stage. Preoperative high IL-6 levels were proposed as a poor prognostic factor for disease recurrence and overall survival in patients with gastric cancers.