OBJECTIVETo establish a novel bioartificial liver (BAL) consisting of spheroids of porcine hepatocytes in a hollow-fiber bioreactor, and to perform an in vitro study on its metabolic effects on the serum from severe hepatitis B patients.

METHODSHepatocytes were isolated from pup pigs and cultured as aggregate spheroids through rotation and vibration. Phase-contrast microscopy, transmission electron microscopy, and scanning electron microscopy were used for morphological detection of hepatocyte spheroids. The hepatocyte spheroids were then transferred into the shell of a polysulfone hollow-fiber bioreactor, creating a novel BAL. Diluted serum samples of severe hepatitis B patients were circulated for 3 hours each into the bioreactor, by using an extracorporeal circulatory system. Every half hour, including both before and after perfusion, serum samples were collected to assay total bilirubin (TBIL), total protein (TP), albumin (ALB), and globulin (GLB) concentrations in order to judge the metabolic effects of this novel BAL.

RESULTSMost hepatocytes had formed spheroids with high viability after 24 hours in culture. After 3 hours of perfusion, when compared with the control group, the serum concentration of TBIL in the treatment group decreased significantly (P < 0.01), but the serum concentrations of TP and ALB increased significantly (P < 0.05).

CONCLUSIONSHepatocytes can be conveniently cultured as aggregate spheroids through a rotation and vibration method. The novel BAL is efficient in removing bilirubin from the serum of severe hepatitis B patients, and in supplying the serum with ALB. Thus, the BAL might provide effective therapy for patients with severe hepatitis B.

Bilirubin ; blood ; Blood Proteins ; analysis ; Hepatitis B ; blood ; Humans ; In Vitro Techniques ; Liver, Artificial ; Serum Albumin ; analysis ; Serum Globulins ; analysis

OBJECTIVETo screen out serum differential proteins between vinyl chloride monomer (VCM)-exposed workers and healthy controls by proteomics and analyze the functions of differential proteins, and to provide a basis for elucidating the pathogenesis of diseases caused by VCM exposure and searching for the protein biomarkers.

METHODSFasting venous blood was collected from 125 VCM-exposed workers and 40 healthy controls according to accumulated exposure doses. Proteins were precipitated by acetone precipitation. These proteins were identified by 2D-nano LC-ESI-TOF/MS and quantified by isobaric tags for relative and absolute quantitation. The functions of differential proteins were analyzed by gene ontology.

RESULTSA total of 596 proteins were identified, including 194 quantified proteins. There were 21 differential proteins according to the screening criteria (19 upregulated proteins and 2 downregulated proteins), including complement, apolipoprotein, and glycoprotein. The functions of these differential proteins were binding, enzyme regulator activity, catalytic activity, and transporter activity, and they were involved in the biological processes including immune system process and response to stimulus.

CONCLUSIONThe complement, apolipoprotein, and glycoprotein identified in the proteomics may be related to liver injury caused by VCM exposure, and they could be used as candidate protein biomarkers of diseases caused by VCM exposure.

Biomarkers ; blood ; Blood Proteins ; analysis ; Humans ; Liver ; injuries ; Occupational Exposure ; Proteins ; metabolism ; Proteomics ; Vinyl Chloride ; toxicity

OBJECTIVETo investigate the biuret reagent to detect proteins in the application, the impact of different test conditions for test results.

METHODSThe biuret method to select three different instruments, reagents, calibrators are arranged in combination to form 27 sets of detection systems, each detection system is a combination of 5 serum samples for testing, 5 measured values obtained, the selection process normality good a serum for the study to determine the mean value of all AST after culling outliers obtained in order to calculate the various detection systems use a combination of biuret reagent to detect proteins bias.

RESULTSThe use of different detection equipment to detect proteins biuret reagent bias, homogeneity of variance (P = 0.467), the difference was not statistically significant (F = 1.688, P = 0.421). different detection reagents using biuret reagent to detect proteins bias, homogeneity of variance (P = 0.574), a statistically significant difference (F = 5.784, P = 0.011). different calibrators use biuret reagent to detect proteins bias, homogeneity of variance (P = 0.467), the difference was statistically significant (F = 5.289, P = 0.000).

CONCLUSIONBiuret reagent in the detection of protein applications, impact detection reagents and calibrators will test result, during the test than when it is necessary to detect deviation detection reagents and calibrators due to be considered.

Biuret ; chemistry ; Blood Proteins ; analysis ; Calibration ; Indicators and Reagents ; chemistry

OBJECTIVENephrolithiasis is one of the most common disorders of the urinary tract. The aim of this study was to examine a possible relationship between DNase I/II activity and E3 SUMO-protein ligase NSE2 in the sera of nephrolithiasis patients to evaluate the possibility of a new biomarker for evaluating kidney damage.

METHODSSixty nephrolithiasis patients and 50 control patients were enrolled in a case-control study. Their blood urea, creatinine, protein levels and DNase I/II activity levels were measured by spectrometry. Serum NSMCE2 levels were measured by ELISA. Blood was collected from patients of the government health clinics in Kuantan-Pahang and fulfilled the inclusion criteria.

RESULTSThe result indicated that mean levels of sera NSMCE2 have a significantly increase (P<0.01) in patients compared to control group. Compared with control subjects, activities and specific activities of serum DNase I and II were significantly elevated in nephrolithiasis patients (P$lt;0.01).

CONCLUSIONThis study suggests that an increase in serum concentrations of DNase I/II and E3 SUMO-protein ligase NSE2 level can be used as indicators for the diagnosis of kidney injury in patients with nephrolithiasis.

Adult ; Blood Proteins ; analysis ; Case-Control Studies ; Creatinine ; blood ; Deoxyribonuclease I ; blood ; Endodeoxyribonucleases ; blood ; Hemoglobins ; analysis ; Humans ; Ligases ; blood ; Malaysia ; Middle Aged ; Nephrolithiasis ; blood ; enzymology ; Urea ; blood

With the development of bio-technological drugs, drug immunogenicity evaluation has become key factor of clarifying safety and efficacy of these drugs. It has become the focus to establish a stable and reliable evaluation system. Due to the advantages such as continuous real-time monitoring, surface plasmon resonance (SPR) technology has been widely used in bio-technological drugs immunogenicity assessments. Our study applied this technology to detect anti-drug antibody (ADA) of a recombinant human anti-rabies monoclonal antibody NM57 in the sera of 48 volunteers admitted in phase I clinical trials. This method could satisfy the basic requirements of detection of ADA.
Antibodies, Anti-Idiotypic ; analysis ; blood ; immunology ; Antibodies, Monoclonal ; analysis ; blood ; immunology ; Antibodies, Viral ; analysis ; blood ; immunology ; Humans ; Rabies virus ; immunology ; Recombinant Proteins ; analysis ; blood ; immunology ; Surface Plasmon Resonance

BACKGROUND: Becton Dickinson (BD) Vacutainer tubes are the most widely used vacuum system for collection of blood in clinical laboratories. We compared the performance of two new tubes, Sekisui INSEPACK tube and Green Cross Green Vac-Tube, with the existing BD Vacutainer tubes for 49 common analytes. METHODS: A total of 20 apparently healthy volunteers were recruited for this study. For rountine chemistry and thyroid function tests, we compared the results of two new vacutainer tubes and BD Vacutainer tubes with those of BD glass tubes at t =0 hr by student paired t test. Hematology and coagulation test results of the two new vacutainer tubes were compared with those of BD Vacutainer tubes at t =0 hr. To study the stability of each analyte, results at t =24 +/- 2 hr, t =72 +/- 2 hr, and t =168 +/- 2 hr were compared with those at t =0 hr for each tube. RESULTS: Although paired t test analysis revealed statistically significant differences between two tested tubes and existing BD Vacutainer tube in several tests (total protein, total bilirubin, alkaline phosphatase, glucose, uric acid, calcium, inorganic phosphorus, direct bilirubin, triglyceride, HDL-cholesterol, LDL-cholesterol, lactate dehydrogenase, triiodothyronine, and thyroxine), these differences were not considered clinically significant. Stability of two new vacuum tubes for each analyte was similar to that of the BD Vacutainer tube. CONCLUSIONS: Sekisui INSEPACK tube and Green Cross Green-Vac Tube showed a satisfactory analytical performance compared with existing BD Vacutainer tubes. We conclude that these two new plastic vacutainer tubes are acceptable for the commonly ordered laboratory tests.
Blood Cells/chemistry ; Blood Proteins/analysis ; Blood Specimen Collection/*instrumentation ; Equipment and Supplies/standards ; Female ; Humans ; Male

BACKGROUNDProgrammed cell death 5 (PDCD5) is a novel apoptotic regulatory gene that promotes apoptosis in various tumor cells. Studies have shown that PDCD5 accelerates the apoptosis of synoviocytes in vitro, implying a potential role in rheumatoid arthritis (RA) pathogenesis. This study examined the expression of PDCD5 in serum and synovial fluid of RA patients, its effect on the expression of inflammatory cytokine, interleukin-17 (IL-17), and the assessment of disease activity in RA.

METHODSPDCD5 and IL-17 levels in serum and synovial fluid from 18 patients with RA and 22 patients with osteoarthritis (OA) were detected using enzyme-linked immunosorbent assay (ELISA). Concentrations of serum PDCD5 in 40 healthy people were also detected as controls. As disease activity indices, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), and X-ray grading scale were also evaluated.

RESULTSSerum and synovial fluid PDCD5 levels in RA patients were significantly higher than those in OA and healthy controls. Serum PDCD5 level was inversely correlated to CRP and ESR, and was significantly higher in the RF negative group than in the positive group. PDCD5 level was also negatively correlated with IL-17 levels both in serum and synovial fluid of RA patients. However, differences in synovial fluid PDCD5 level from RA patients at different Larsen stages were not detectable.

CONCLUSIONSPDCD5 affects RA pathogenesis. Insufficient apoptosis of fibroblast-like synoviocytes and inflammatory cells in RA could increase the expression of PDCD5 protein. As PDCD5 levels correlated negatively with disease activity indices and IL-17 level, PDCD5 could become a target in the diagnosis and treatment of RA.

Aged ; Apoptosis ; Apoptosis Regulatory Proteins ; analysis ; blood ; physiology ; Arthritis, Rheumatoid ; etiology ; Blood Sedimentation ; C-Reactive Protein ; analysis ; Female ; Humans ; Interleukin-17 ; analysis ; blood ; physiology ; Male ; Middle Aged ; Neoplasm Proteins ; analysis ; blood ; physiology ; Synovial Fluid ; chemistry

OBJECTIVETo investigate the relationship between the expression of murine double minute 2 (MDM2) oncogene and non-Hodgkin lymphoma (NHL) in childhood.

METHODSThirty-one cases of NHL were enrolled in this study as patient group and 8 cases of lymphadenitis as control group. (1) Immunohistochemistry ultrasensitive S-P assay was used to detect the expression of MDM2 protein in pathological tissues in all cases. Positive cells were dyed yellow or brown in nuclei. MDM2 positive cell was defined as >/= 10% of the tumor cells were positive, which was overexpression of MDM2 protein. (2) RT-PCR (reverse transcription-polymerase chain reaction) was performed to value the overexpression of MDM2 mRNA in the pathological tissues and mononuclear cells in peripheral blood. While the ratio of MDM2/beta-actin was >16% was defined as overexpression of MDM2 mRNA.

RESULTS(1) Rates of overexpression of MDM2 protein and MDM2 mRNA were 64.5% and 61.3%, respectively, which were significantly different as compared to that of control group (P < 0.05 and P < 0.01, respectively). (2) The relationship analysis among subgroups in the experiment group showed that the overexpression of MDM2 protein did not correlate with classifications of working formulation, cellular origin, sex, clinical stage and involved extranodal sites (P > 0.05), but significantly correlated with classifications of B status and the increased serum LDH level (P < 0.05). It was shown that the overexpression of MDM2 mRNA did not correlate with classifications of working formulation, cellular origin, sex and clinical stage (P > 0.05), significantly correlated with B status (P < 0.05), and was remarkably significantly correlated with the involved extranodal sites and the increased serum LDH level (P < 0.01). (3) It was demonstrated that the overexpression of MDM2 mRNA in the pathological tissues was similar to the overexpression of MDM2 protein in the pathological tissues and MDM2 mRNA in peripheral blood (P > 0.05, kappa = 0.655 and 0.571), and the overexpression of MDM2 protein in the pathological tissues was similar to that of MDM2 mRNA in peripheral blood (P > 0.05, kappa = 0.609).

CONCLUSIONS(1) The rate of MDM2 oncogene overexpression was quite high. (2) The overexpression of MDM2 protein in pathological tissues determined by using immunohistochemistry ultrasensitive S-P assay was similar to that of MDM2 mRNA in pathological tissues detected by using RT-PCR method. Both methods might be used to detect the overexpression of MDM2 oncogene in the cases of childhood NHL. (3) The overexpression of MDM2 oncogene related to the poor status and poor prognosis of patients with childhood NHL.

Biomarkers, Tumor ; analysis ; blood ; Child ; Humans ; Immunohistochemistry ; Lymphoma, Non-Hodgkin ; blood ; genetics ; metabolism ; Neoplasm Proteins ; blood ; genetics ; Oncogenes ; Proto-Oncogene Proteins c-mdm2 ; blood ; genetics ; metabolism ; RNA, Messenger

OBJECTIVETo evaluate two-dimensional gel electrophoresis (2DGE) and mass spectrometry in the studies of the serum proteins expressed in patients with BPH and those with high-grade prostatic intraepithelial neoplasm (HGPIN).

METHODSWe extracted serum proteins from BPH and HGPIN patients by 2DGE and cut the differentially expressed interesting protein spots from the gel. Then we digested the proteins, obtained the peptide mass fingerprint by mass spectrometry and identified the proteins through database retrieval.

RESULTSWe successfully achieved the 2DGE maps of the serum proteins from the BPH and HGPIN patients, obtained 1 421-1 532 protein spots from the 2D map of HGPIN and 1 466-1 778 from that of BPH. Based on peptide mass fingerprinting, 9 of the protein spots were identified. Serum amyloid A was found to be expressed in the HGPIN group, but weakly or not at all in the BPH.

CONCLUSIONProteomics can be applied to the study of the serum proteins in BPH and HGPIN patients. It can afford experimental evidence for the early diagnosis and development HGPIN, promote the search of functional and specific proteins of prostate diseases and shed new light on the network mechanisms of the problems.

Blood Proteins ; analysis ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Male ; Mass Spectrometry ; Prostatic Hyperplasia ; blood ; Prostatic Intraepithelial Neoplasia ; blood ; Prostatic Neoplasms ; blood ; Proteome ; analysis ; Proteomics ; methods

OBJECTIVETo screen and identify the serum specific protein markers of patients with gastric cancer by proteomics technology, and to provide more comprehensive serum protein fingerprint model for the early diagnosis of gastric cancer.

METHODSPreoperative and postoperative blood samples were collected from 60 gastric cancer patients. Mass spectrometry (SELDI-TOF-MS) technology was used to detect and screen serum specific proteins in gastric cancer patients(preoperative group, postoperative group, metastasis group), and the result was compared with normal control group. Gel electrophoresis(TRICINE SDS-OAGE) technology was applied in the separation and purification for those different protein. Matrix assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF) technology was used in the identification for the proteins following separation and purification.

RESULTMass spectrometry data of preoperative group and normal group resulted in 15 specific m/z peak(P<0.01). SVM screened by a combination of the highest index model Youden get m/z peak at 6 449.1 protein markers. The protein expression of preoperative group was significantly higher than that of normal group(2 299.3±2 029.3 vs. 509.5±168.3, P<0.01). Mass spectrometry data of preoperative group and postoperative group resulted in 6 specific m/z peak(P<0.01). SVM screened by a combination of the highest Youden index model indentified get m/z peak at 6 449.2 protein markers. The protein expression of preoperative group was significantly higher than that of postoperative group(1 247.9±685.0 vs. 476.5±157.8, P<0.01). Mass spectrometry data of preoperative group and metastasis group resulted in 12 specific m/z peak (P<0.01). SVM screened by a combination of the highest Youden index model indentified get m/z peak at 6 448.9 protein markers. The protein expression of metastasis group was higher than that of preoperative group(1 506.9±1 036.5 vs. 649.7±621.0). MALDI-TOF/TOF identified that the protein with m/z peak at 6 449 was Apo CIII(.

CONCLUSIONApo CIII( may be the specific serum protein marker of gastric cancer, which may provide a more comprehensive serum protein fingerprint model for the early diagnosis of gastric cancer and a new way for further research.

Biomarkers, Tumor ; blood ; Blood Proteins ; analysis ; Humans ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Stomach Neoplasms ; blood ; diagnosis