APTT critical value establishment in four different reagent/instrument systems based on single factor deficiencies.

INTRODUCTION: There are significant differences in the activated partial thromboplastin time (APTT) critical values reported in different studies, most of which does not make recommendations for any specific clear detection systems. The International Council for Standardization in Hematology (ICSH) recommends that APTT critical values be established based on the reagent type, coagulation factor sensitivity and heparin response. The objective of this study was to establish APTT critical values by using different reagents and based on single coagulation factor deficiencies. METHODS: The APTT values were determined in commercial endogenous coagulation factor-deficient plasma at concentrations of 1 IU/dL, 2 IU/dL, 5 IU/dL, 10 IU/dL, 20 IU/dL, and 30 IU/dL by using four assay systems. The retrospective collection of data from patients who lacked factor VIII (FVIII), FIX, or FXI alone was performed. Receiver operating characteristic (ROC) curves were constructed to assess the diagnostic accuracy of APTT for identifying patients with an endogenous coagulation factor activity < 5 IU/dL. RESULTS: The APTT values in the plasma samples with the same concentrations of endogenous coagulation factors were significantly different among the four assay systems (P < 0.001). The suggested critical values of APTT were 40.0 s for Sysmex CS5100 (Actin FSL), 58.0 s for Sysmex CS5100 (Actin), 51.8 s for STA-R Evolution (STA-PTTA), and 64.8 s for ACL TOP 700 (HemosIL SynthasIL). On the basis of the ROC curve, the optimal threshold values for APTT (STA-PTTA) were 55.8 s in patients with a simple deficiency of FVIII (sensitivity = 100%, specificity = 85.7%, area under the ROC curve (AUC) = 0.982), 54.3 s in patients with a simple deficiency of FIX (sensitivity = 100%, specificity = 92.9%, AUC = 0.986), and 71.7 s in patients with a simple deficiency of FXI (sensitivity = 100%, specificity = 94.1%, AUC = 0.992), which were closer (difference of 0.6-2.5 s) to the cutoff points for commercial plasma at equal factor levels. CONCLUSIONS: APTT critical values need to be established for different reagents based on the presence of a single coagulation factor deficiency.

A novel antimicrobial peptide derived from human BPIFA1 protein protects against Candida albicans infection.

Bactericidal/permeability-increasing fold containing family A, member 1 (BPIFA1) is an innate immunity defense protein. Our previous studies proved its antibacterial and antiviral effects, but its role in fungi remains unknown. The study aimed to identify antifungal peptides (AFP) derived from BPIFA1, and three antimicrobial peptides (AMP1-3) were designed. The antifungal effects were proved by growth inhibition assay. AMP3 activity was confirmed by germ tube growth experiment and XTT assay. Its effects on cell wall and membrane of Candida albicans were assessed by tannic acid and Annexin V-FITC/PI double staining, respectively. Additionally, scanning electron microscope (SEM) and transmission electron microscopy (TEM) were used for morphological and ultrastructural observation. The expression of ALS1, EAP1, and SUN41 was tested by qPCR. Ultimately, three AMPs could fight against C. albicans in vitro, and AMP3 was highly effective. It functioned by destroying the integrity of cell wall and normal structure of cell membrane. It also inhibited biofilm formation of C. albicans. In addition, AMP3 down-regulated the expression of ALS1, EAP1, and SUN41, those are known to be involved in virulence of C. albicans. Altogether, the study reported successful development of a novel AFP, which could be used as a new strategy for antifungal therapy.

Non-canonical Raf-1/p70S6K signalling in non-small-cell lung cancer.

Lung cancer is the leading cause of cancer-related death globally, with non-small-cell lung cancer (NSCLC) being the predominant subtype. Overall survival remains low for NSCLC patients, and novel targets are needed to improve outcome. Raf-1 is a key component of the Ras/Raf/MEK signalling pathway, but its role and downstream targets in NSCLC are not completely understood. Our previous study indicated a possible correlation between Raf-1 levels and ribosomal protein S6 kinase (p70S6K) function. In this study, we aimed to investigate whether p70S6K is a downstream target of Raf-1 in NSCLC. Raf-1 was silenced in NSCLC cell lines by using small hairpin RNA, and Raf-1 and p70S6K protein levels were measured via Western blot. p70S6K was then overexpressed following Raf-1 knock-down; then, cell proliferation, apoptosis and the cell cycle in NSCLC cell lines were examined. Tumour xenografts with NSCLC cells were then transplanted for in vivo study. Tumours were measured and weighed, and Raf-1 and p70S6K expression, cell proliferation and apoptosis were examined in tumour tissues by Western blot, Ki-67 staining and TUNEL staining, respectively. When Raf-1 was silenced, p70S6K protein levels were markedly decreased in the A549 and H1299 NSCLC cell lines. A significant decrease in NSCLC cell proliferation, a profound increase in apoptosis and cell cycle arrest were observed in vitro following Raf-1 knock-down. Overexpression of p70S6K after Raf-1 depletion effectively reversed these effects. Xenograft studies confirmed these results in vivo. In conclusion, Raf-1 targets p70S6K as its downstream effector to regulate NSCLC tumorigenicity, making Raf-1/p70S6K signalling a promising target for NSCLC treatment.

Establishing reference intervals for ALT, AST, UR, Cr, and UA in apparently healthy Chinese adolescents.

BACKGROUND: The current child-specific reference intervals (RIs) are inadequate or even unavailable for many analyses in China. Many of the RIs used in Chinese laboratories were derived from Chinese adult standards or from foreign studies. The aim of this study was to establish specific RIs for alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UR), creatinine (Cr) and uric acid (UA) for apparently healthy Chinese adolescents. METHODS: Overall, 1682 apparently healthy adolescents were enrolled. Serum ALT, AST, UR, Cr and UA were measured by an ARCHITECT C-8000 automated chemistry analyzer. The 2.5th and 97.5th percentile RIs were determined using non-parametric methods. RESULTS: The established reference intervals for ALT, AST, UR, CR and UA were 7.5-42.8 U/L, 12.8-40.2 U/L, 3.12-6.38 mmol/L, 42.7-91.2 µmol/L, and 180.2-409.6 µmol/L in boys and 6.5-32.8 U/L, 10.4-32.5 U/L, 3.05-6.47 mmol/L, 40.2-88.8 µmol/L and 176.5-394.0 µmol/L in girls, respectively. The median and upper and lower limits for the RIs of ALT, AST, Cr and UA were higher in boys than they were in girls (P < 0.05). CONCLUSION: RIs based on adult criteria are not applicable to adolescents. It was necessary to establish specific, accurate and suitable RIs for Chinese adolescents. We have established reference intervals of ALT, AST, UR, Cr and UA that are defined specifically for Chinese adolescents and are appropriate for universal use among Chinese laboratories.

Serum indoleamine 2,3-dioxygenase and kynurenine aminotransferase enzyme activity in patients with ischemic stroke.

We investigated the activation and pathophysiological roles of indoleamine 2,3-dioxygenase (IDO) and kynurenine aminotransferase (KAT) in patients with ischemic stroke. Patients were recruited from the acute stroke unit of a general hospital within 24 hours post-stroke. The immune transmission turbidity method was used to determine the concentration of serum high-sensitivity C-reactive protein (hsCRP), apolipoprotein A-1 and apolipoprotein B. The concentrations of triglyceride, cholesterol, high density lipoprotein (HDL), low density lipoprotein and non-esterified fatty acids were determined using an enzymatic method. Tryptophan (TRP), kynurenine (KYN) and kynurenine acid (KYNA) concentrations were determined by high performance liquid chromatography. The National Institutes of Health Stroke Scale (NIHSS) was used to assess the neurological deficits at admission and 3 weeks post-stroke. The IDO and KAT activity ratio were calculated by KYN/TRP and KYNA/KYN, respectively. The correlation between hsCRP and IDO, KAT and NIHSS score was also analyzed. A total of 81 patients with ischemic stroke and 35 normal controls were recruited. Lower TRP, KYNA, HDL and KAT activity ratio were found in the stroke group compared to the control group (p<0.05). The levels of hsCRP and IDO activity ratio were much higher in the stroke group than the control group (p<0.01). The IDO activity in patients with ischemic stroke showed a positive correlation with hsCRP (r=0.425, p=0.027). In addition, hsCRP and IDO levels were positively associated with the NIHSS score both at admission and 3 weeks post-stroke. These data suggest an inflammatory response characterized by up-regulated IDO activation in ischemic stroke, which might be closely relevant to its pathophysiology.

Effects of rosiglitazone on the growth and lymphangiogenesis of human gastric cancer transplanted in nude mice.

Gastric cancer mainly metastasizes via lymphatic vessels. Thus, it is critical to identify efficacious chemopreventive agents for lymphangiogenesis. The present study was undertaken to explore the effects of rosiglitazone (ROSI) on the growth and lymphangiogenesis of human gastric cancer. We established a model of gastric cancer by subcutaneously inoculating the human gastric cancer cell line SGC-7901 into nude mice. Mice were randomly divided into 4 groups and each group received a different agent by oral gavage. The control group received normal saline and treatment groups received different doses of ROSI once every 2 days. The growth of the tumor in vivo was assessed by measuring tumor volume. After 42 days, the mice were sacrificed and the tumors were removed. H&E staining was used to observe the histomorphological features; immunohistochemistry staining for lymphatic vessel density (LVD) was used to evaluate tumor lymphangiogenesis, RT-PCR was performed to determine the mRNA expression of vascular endothelial growth factor C (VEGF-C) and VEGF receptor-3 (VEGFR-3), and western blotting was used to detect the protein expression of VEGF-C and VEGFR-3. Compared with the control group, all treatment groups had smaller tumor volume and higher tumor growth inhibitory rate every day. The number of typical tumor cells in the control group was higher compared to that in the treatment groups, and the highest level of LVD was found in the control group. Furthermore, both the expression of VEGF-C and VEGFR-3 mRNA and proteins in the control group were significantly higher compared to those in the treatment groups. Markedly, these changes were correlated in a dose-dependent manner with ROSI. These results demonstrated that, through simultaneously blocking the expression of VEGF-C and VEGFR-3, ROSI suppresses lymphangiogenesis. This may represent a powerful therapeutic approach for controlling gastric cancer cell growth and metastasis.

Survivin up-regulates the expression of breast cancer resistance protein (BCRP) through attenuating the suppression of p53 on NF-κB expression in MCF-7/5-FU cells.

Both breast cancer resistance protein (BCRP, ABCG2) and apoptosis-related molecules are involved in the development of multidrug resistance (MDR) in cancer cells. However, the association of BCRP with apoptosis-related molecules in the development of MDR is unknown. In this study, we investigated the changes in expression levels of BCRP, Survivin, p53, Bcl-2, Bcl-xL or Bax in cultured MCF-7 and MCF-7/5-FU cells, and explored whether these changes affected the expressions of BCRP. Our data showed that the protein and mRNA expression levels of BCRP, Survivin and Bcl-2 were significantly higher in MCF-7/5-FU cells than in MCF-7 cells, while p53 expression lower in MCF-7/5-FU cells than in MCF-7 cells. Knockdown of Survivin or Bcl-2 in MCF-7/5-FU cells and overexpression of Survivin in MCF-7 cells revealed that Survivin had significant association with BCRP expression. Luciferase reporter gene assays showed that Survivin up-regulated BCRP expression at transcriptional level and this response was mediated through NF-κB(p50) pathway. However, may be due to the physical interaction between p53 and Survivin, p53 directly affected Survivin-regulated BCRP expressions. Interestingly, we found that Survivin would suppress p53 expression. Furthermore, our data revealed that Survivin itself had no apparent effect on NF-κB(p50) and BCRP expression when knockdown of p53 in MCF-7 cells; whereas p53 exerted significant inhibitory action on these when knockdown of Survivin. In conclusion, through down regulation of p53 expression, Survivin attenuates the suppressing effect of p53 on NF-κB(p50) expression and then enhances BCRP expression. This may represent a novel strategy for reversal of BCRP drug transporter activity to modulate MDR in cancer cells.

Simultaneous determination of phenylalanine and tyrosine in peripheral capillary blood by HPLC with ultraviolet detection.

OBJECTIVES: Phenylalanine (Phe) and tyrosine (Tyr) are the most reliable indicators for the diagnosis of phenylketonuria (PKU). The purpose of this study is to establish a simple and rapid method for the determination of Phe and Tyr in peripheral capillary blood from newborns and children by high performance liquid chromatography with ultraviolet detection (HPLC-UV). METHODS: Peripheral blood specimens were taken from newborns or children by heel stick or skin puncture on the fingers. Blood samples were deproteinized by equal volume of 5% perchloric acid. Amino acid separation was carried out on a Hypersil C8 column. The mobile phase containing 5% acetonitrile (v/v) was run at a flow rate of 1.5 mL/min. Phe and Tyr were determined in the ultraviolet detector at 210 nm. RESULTS: The determination was within 10.0 min. The linear range was from 6.0 to 1512.0 µmol/L for Phe and 5.5 to 1250.0 µmol/L for Tyr. Although Phe and Tyr levels from peripheral blood samples were relatively lower than those from serum or plasma, the data showed a good agreement between them. CONCLUSION: The method we developed is simple, rapid and convenient. It is useful for screening and diagnosis of PKU in newborns and children.

[Simultaneous determination of kynurenine and kynurenic acid in serum by high performance liquid chromatography-fluorescence detection].

A method of high performance liquid chromatography-fluorescence detection (HPLC-FLD) for the simultaneous determination of kynurenine (Kyn) and kynurenic acid (KYNA) in serum was developed. A 20 microL of the supernatant of a serum sample deproteinized by 5% perchloric acid solution was separated on a Hypersil C8 column (300 mm x 6.0 mm, 10 microm) with an isocratic elution of 0.25 mol/L zinc acetate-50 mmol/L acetate containing 3% (v/v) acetonitrile. The fluorescence excitation and emission wavelengths were 365 nm and 480 nm at the beginning of the run; and 10 min later, the excitation and emission wavelengths were changed to 344 nm and 404 nm, respectively. The retention times of Kyn and KYNA were 8.1 min and 13.0 min, respectively. The linearities of the assay were from 98 to 19,600 nmol/L for Kyn and from 2.62 to 1047 nmol/L for KYNA; the detection limits were 50 nmol/L for Kyn and 0.11 nmol/L for KYNA. The average recoveries were 94.88% for Kyn, and 102.72% for KYNA. The within-day and between-day relative standard deviations (RSD) were 3.87% and 3.94% for Kyn; and 3.79% and 4.71% for KYNA, respectively. The results indicated phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), 5-hydroxytryptamine (5-HT), and creatinine (Cr) had no interfering effects to the determination of Kyn and KYNA. Kyn and KYNA concentrations in the sera of 71 health people were (1.40 +/- 0.34) micromol/L and (24.22 +/- 8.67) nmol/L, respectively. The method is simple, rapid, accurate, convenient, and suitable for routine analysis.

More rapid and sensitive method for simultaneous determination of tryptophan and kynurenic acid by HPLC.

OBJECTIVE: To describe a simple, rapid, and sensitive HPLC method for simultaneous determination of TRP and KYNA in human serum. DESIGN AND METHOD: Samples were deproteinized by perchloric acid. KYNA was detected at 344 nm of excitation wavelength and 404 nm of emission wavelength, TRP was detected at 254 nm and 404 nm, with a total run time of 13 min per sample. RESULTS: Standard curves of 0.49 micromol/L to 196 micromol/L of TRP were linear. Inter-day and intra-day coefficient of variations were 3.31% and 4.14%, respectively. Average recovery was 104.43%. Detection limit was 0.001 micromol/L. The linearity of the assay was maintained from 1.5 nmol/L to 2093 nmol/L of KYNA. Inter-day and intra-day CVS were 3.20% and 4.27%, respectively. Average recovery was 101.19%. Detection limit was 0.05 nmol/L. CONCLUSION: The developed HPLC method is simple, convenient and can be applied in the diagnosis of related diseases.