[Feasibility of Preheating at 41 ℃ to Correct Red Blood Cell Parameters in the Presence of High-titer Cold Agglutinins].

Objective To explore the feasibility of preheating in 41 ℃ water bath for 30 minutes to correct the red blood cell parameters in the specimens containing high-titer cold agglutinins(CAs). Methods Two specimens containing high-titer CAs were selected during work,and the parameters of complete blood count at room temperature or after preheating in 37 ℃ or 41 ℃ water bath were compared.The smears were stained,and the distribution of red blood cells was observed with a microscope.Further,74 specimens without CAs were collected for complete blood count,and then the test results at room temperature and after preheating at 41 ℃ were compared. Results At room temperature,the specimens containing high-titer CAs showed significantly reduced red blood cell count(RBC)and hematocrit(HCT),abnormally increased mean corpuscular hemoglobin(MCH)and mean cell hemoglobin concentration(MCHC),abnormal percents of hemoglobin(HGB)and RBC,and aggregation of a large number of red blood cells.After being preheated at 37 ℃ for a certain time,the specimens demonstrated obviously improved parameters while still aggregation of a small number of red blood cells.After being preheated at 41 ℃ for 30 minutes,the specimens showed significantly increased RBC,normal HCT,MCH,and MCHC,and evenly distributed red blood cells.The 74 specimens without CAs showed the comparability was ≥80% between room temperature and preheating at 41 ℃ for 30 minutes or 60 minutes. Conclusion We can preheat the specimens containing high-titer CAs in a water bath at 41 ℃ to obtain accurate red blood cell parameters.

Acute diffuse alveolar haemorrhage accompanied by gastrointestinal bleeding in a patient with serious systemic lupus erythematosus: A case report.

Systemic lupus erythematosus (SLE) is an autoimmune disease that affects many organs, but multisystem dysfunction is rare. Here, we report a case of a 29-year-old woman who was initially diagnosed with SLE complications including lupus nephritis, lupus encephalopathy, renal hypertension, thrombocytopenia, anaemia and hyperkalaemia. She recovered following treatment with high dose methylprednisolone, intravenous immunoglobulin (IVIG) and continuous renal replacement therapy (CRRT). However, a few days after hospital discharge, she developed gastrointestinal bleeding. Although intensive treatment was administered, the patient deteriorated rapidly and had a progressive decline in oxygen saturation followed by diffuse alveolar haemorrhage and acute left heart failure. Inotropic therapy, mechanical ventilation, blood transfusion, CRRT, antibiotics, intravenous glucocorticoids and other support therapies were initiated and gradually the patient's vital signs stabilized and haemoptysis subsided. This case report emphasises that complications of SLE can occur at any stage of the disease, especially in patients with active SLE. Therefore, it is important for clinicians to be aware of the rare presentations of SLE and its complex management. For multisystem dysfunction, early intensive treatment with high dose corticosteroids and cyclophosphamide is advocated.

Quality specifications of routine clinical chemistry methods based on sigma metrics in performance evaluation.

INTRODUCTION: Sigma metrics were applied to evaluate the performance of 20 routine chemistry assays, and individual quality control criteria were established based on the sigma values of different assays. METHODS: Precisions were expressed as the average coefficient variations (CVs) of long-term two-level chemistry controls. The biases of the 20 assays were obtained from the results of trueness programs organized by National Center for Clinical Laboratories (NCCL, China) in 2016. Four different allowable total error (TEa) targets were chosen from biological variation (minimum, desirable, optimal), Clinical Laboratory Improvements Amendments (CLIA, US), Analytical Quality Specification for Routine Analytes in Clinical Chemistry (WS/T 403-2012, China) and the National Cholesterol Education Program (NECP). RESULTS: The sigma values from different TEa targets varied. The TEa targets for ALT, AMY, Ca, CHOL, CK, Crea, GGT, K, LDH, Mg, Na, TG, TP, UA and Urea were chosen from WS/T 403-2012; the targets for ALP, AST and GLU were chosen from CLIA; the target for K was chosen from desirable biological variation; and the targets for HDL and LDL were chosen from the NECP. Individual quality criteria were established based on different sigma values. CONCLUSIONS: Sigma metrics are an optimal tool to evaluate the performance of different assays. An assay with a high value could use a simple internal quality control rule, while an assay with a low value should be monitored strictly.

Establishment of reference intervals for serum thyroid-stimulating hormone, free and total thyroxine, and free and total triiodothyronine for the Beckman Coulter DxI-800 analyzers by indirect method using data obtained from Chinese population in Zhejiang Province, China.

BACKGROUND: In order to establish suitable reference intervals of thyroid-stimulating hormone (TSH), free (unbound) T4 (FT4), free triiodothyronine (FT3), total thyroxine (T4), and total triiodothyronine (T3) for the patients collected in Zhejiang, China, an indirect method was developed using the data from the people presented for routine health check-up. METHODS: Fifteen thousand nine hundred and fifty-six person's results were reviewed. Box-Cox or Case Rank was used to transform the data to normal distribution. Tukey and Box-Plot methods were used to exclude the outliers. Nonparametric method was used to establish the reference intervals following the EP28-A3c guideline. Pearson correlation was used to evaluate the correlation between hormone levels and age, while Mann-Whitney U test was employed for quantification of concentration differences on the people who are younger and older than 50 years old. RESULTS: Reference intervals were 0.66-4.95 mIU/L (TSH), 8.97-14.71 pmol/L (FT4), 3.75-5.81 pmol/L (FT3), 73.45-138.93 nmol/L (total T4), and 1.24-2.18 nmol/L (total T3) in male; conversely, reference intervals for female were 0.72-5.84 mIU/L (TSH), 8.62-14.35 pmol/L (FT4), 3.59-5.56 pmol/L (FT3), 73.45-138.93 nmol/L (total T4), and 1.20-2.10 nmol/L (total T3). FT4, FT3, and total T3 levels in male and FT4 level in female had an inverse correlation with age. Total T4 and TSH levels in female were directly correlated. Significant differences in these hormones were also found between younger and older than 50 years old except FT3 in female. CONCLUSIONS: Indirect method can be applied for establishment of reference intervals for TSH, FT4, FT3, total T4, and total T3. The reference intervals are narrower than those previously established. Age factor should also be considered.

Secondary B-cell lymphoma diagnosed by fine-needle aspiration cytology and flow cytometry following penile carcinoma: A case report.

The number of studies reporting lymphoma as a secondary tumor has gradually increased. However, few studies have reported that occurrence of lymphoma as a secondary tumor following treatment for penile carcinoma, particularly cases in which the lymphoma was diagnosed by fine-needle aspiration cytology and flow cytometry. The present study reports the case of a 62-year-old male patient who was troubled with frequent urination and repeated chest tightness for 5 years. The diagnosis upon admission was penile carcinoma. Two months subsequent to the tumor removal surgery, enlarged lymph nodes were extracted from the patient using fine-needle biopsy, to be analyzed using light microscopy and flow cytometry. Smear results indicated a large number of abnormal cells scattered in the right axillary lymph node. Flow cytometry immunophenotyping of fine-needle aspiration samples indicated the increased expression of cluster of differentiation (CD)79a, CD19, CD20, CD38, κ chain and human leukocyte antigen-DR, which supported a diagnosis of B-cell lymphoma. Thus, the patient was diagnosed with B-cell lymphoma based on the results of the fine-needle aspiration biopsy and flow cytometry. The method of diagnosis and causes of therapy-related leukemia are discussed in the present report.

N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1) suppresses the proliferation of PANC-1 pancreatic cancer cells via apoptosis and G2/M cell cycle arrest.

Pancreatic cancer is one of the human gastrointestinal malignancies with a high mortality and poor prognosis. Approximately eighty percent of patients are diagnosed with unresectable or metastatic disease. Thus, development of novel chemicals in the treatment of pancreatic cancer is imperative. This study aimed to investigate the anticancer effects of N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1), a new tetrazine derivative, on the PANC-1 pancreatic cancer cell line and clarify the underlying molecular mechanism. Using an MTT assay, we found that ZGDHu-1 significantly suppressed the proliferation of PANC-1 cells in a time- and dose-dependent manner. Moreover, according to the morphological and flow cytometric analysis, the results indicated that ZGDHu-1 induced PANC-1 cell apoptosis and G2/M cell cycle arrest in a dose-dependent manner. In the western blot analysis, expression of the pro-apoptotic Bax gene was upregulated while the anti-apoptotic Bcl-2 gene was downregulated following treatment with ZGDHu-1. ZGDHu-1 also activated pro-caspase-3 and PARP and increased the expression of NF-κB inhibitor IκB. Furthermore, the expression levels of G2/M regulatory molecules such as cyclin B1 and cdc2 were decreased while that of Chk1 was increased. These results suggested that ZGDHu-1 suppressed the proliferation of pancreatic cancer cells, rendering it a potential therapeutic drug for the treatment of pancreatic cancer.

ZGDHu-1 induces G2/M phase arrest and apoptosis in Kasumi-1 cells.

The present study examined the effects of N,N'­di­(m­methylphenyi)­3, 6­dimethyl­1, 4­dihydro­1,2,4,5­tetrazine­1,4­dicarboamide (ZGDHu­1), a novel oxazine derivative, in Kasumi­1 cells. Following incubation with various concentrations of ZGDHu­1, fluorescence­activated cell sorting (FACS) was used in order to detect changes in mitochondrial membrane permeability in Kasumi­1 cells. Western blot analysis was performed in order to analyze the expression of nuclear factor­κB, inhibitor of κB and AML1/ETO. In addition FACS was used to analyze leukemia cell cycles and the expression levels of cyclin, cyclin­dependent kinases and cyclin­dependent kinase inhibitors in G2/M phase were determined using FACS and western blot analysis. The upregulation of reactive oxygen species production and mitochondrial membrane permeability was ascribed to apoptosis. The growth of Kasumi­1 cells was inhibited through the downregulation of nuclear factor­κB, degradation of AML1/ETO fusion protein and cell cycle arrest at the G2/M phase. This study documented that G2/M regulatory molecules, including cyclin B1, cell division control (cdc)2 and cdc25c were downregulated and checkpoint kinase 1 (CHK1), p53, p27, phospho­cdc25c, phospho­CHK1 and phospho­p53 were upregulated following treatment with ZGDHu­1. In the present study, pretreatment with CHIR­124, a selective CHK1 inhibitor, abrogated G2/M arrest via ZGDHu­1. These results demonstrated the anti­tumor activity of ZGDHu­1, which may therefore a potential target for further investigation and may be useful for the treatment of patients with t(8;21) acute myeloid leukemia.

Synthesis and antitumor activity of feruloyl and caffeoyl derivatives.

We developed two efficient protocols for the synthesis of feruloyl and caffeoyl derivatives from commercial vanillin and veratraldehyde. Pharmacological activities were assessed against a panel of human cancer cell lines in vitro. Most synthesized compounds demonstrated attractive cytotoxicity. Several new compounds demonstrated significant antiproliferative and cytotoxic activities against HeLa and Bewo tumor cell lines. In particular, 5-nitro caffeic adamantyl ester showed broad spectrum of tumor inhibition in 10 cell lines, and reduced tumor weight by 36.7% in vivo when administered at a dose of 40 mg kg(-1).

[Screening and detection of patients with macroprolactinemia by application of polyethylene glycol precipitation method].

OBJECTIVE: To establish a polyethylene glycol (PEG6000) precipitation method for screening macroprolactinemia in patients with high serum prolactin (PRL). METHODS: PEG6000 precipitation method was used to remove macroprolactin (MPRL) molecules in serum of PRL-elevated patients. The effect of PEG6000 precipitating serum MPRL was determined by Sephacryl S-100HR chromatography plus chemiluminescent immunoassay and SDS-PAGE plus Western Blot assay. The PEG6000 precipitation plus chemiluminescent immunoassay was applied to screen serum samples of PRL-elevated patients for macroprolactinemia. The clinical manifestations of patients with true-hyperprolactinemia, hyperprolactinemia/macroprolactinemia or true-macroprolactinemia were analyzed and compared. RESULTS: After precipitation with PEG6000, MPRL peak or hybridization signal in the serum samples was markedly decreased, while the big or small prolactin (BPRL or SPRL) levels were not affected. In 1538 PRL-elevated patients, 16.1% (247/1538) were detectable for macroprolactinemia, while the 83.9% (1291/1538) were identified as true-hyperprolactinemia. In 247 samples of macroprolactinemia, 93.5% (231/247) were determined as true-macroprolactinemia, while 6.5% (16/247) were identified as hyperprolactinemia plus macroprolactinemia. In 508 true-hyperprolactinemia patients, menoxenia, menolipsis/menostasia, dysgenesia or hypophysoma were manifested in 438 (86.2%), which were also manifested in 85.7% (6/7) of hyperprolactinemia/macroprolactinemia patients. However, only 11 cases in 71 true-macroprolactinemia patients (15.5%) presented above clinical diseases. CONCLUSION: There is a certain proportion of true-macroprolactinemia (pseudo-hyperprolactinemia) in serum PRL-elevated patients. The PEG6000 precipitation method established in this study can efficiently distinguish true-hyperprolactinemia from pseudo-hyperprolactinemia in patients.

[Macrocalin A induces apoptosis of multiple myeloma U266 cells through inhibiting the proteasome].

This study was purposed to investigate the inhibitory effect of macrocalin A (MA) on proteasome of multiple myeloma U266 cells in vitro and molecular mechanism of MA-inducing apoptosis. U266 cells in vitro were incubated with different concentrations (2, 4, 8 µg/mL) of MA, the Hochest staining and Annexin-V/PI double staining were used to detect the apoptosis of U266 cells. The expressions of protein ß1, ß1i, ß2, ß2i, ß5, ß5i, ubiquitous, 19S subunit S6', and BAD,BCL-2, FAS, FAS-L,MAPK, PARP, Pro-caspase 3, cleaved-caspase 3 were detected by Western blot technique. The results showed that along with time prolonging and dose increasing of MA, the small and compact fluorescent particles were observed in cytoplasm and nucleus of U266 cells stained with Hoechst 33258, the Annexin V(+)/PI(-) cells and the total apoptosis cells (Annexin V(+)/PI(-) and Annexin V(+)/PI(+)) increased. MA could elevate the ubiquitylation level in U266 cells, suppress the expression of ß1i,ß2, ß5i and 19S subunit S6', meanwhile the expression of BCJ-2, MAPK, PARP and pro-caspase 3 were down-regulated along with increasing of drug concentrations, but the expressions of BAD, FAS, FAS-L cleaved-caspase 3 were enhanced. It is concluded that MA can inhibit the effect of proteasome, and the mitochondrial pathway and death receptor pathway may play important roles in apoptosis of U266 cells induced by MA.

ZGDHu-1 promotes apoptosis of chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is a low-grade lymphoid malignancy incurable with conventional modalities of chemotherapy. We aimed to examine the proapoptotic effects of a novel proteasome inhibitor, N,N'-di-(m-methylphenyi)-3,6- dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1), on CLL cells. B lymphocytes were isolated from CLL patients and normal healthy controls, and treated with various concentrations of ZGDHu-1 for different days. CLL cell viability was detected by MTT assay. The apoptosis, mitochondrial membrane potential (∆ψm) and reactive oxidative species (ROS) were examined by flow cytometry. The expression of caspase-3 and Bcl-2/Bax ratio was detected by western blotting. ZGDHu-1 significantly reduced the viability of CLL cells and induced apoptosis in comparison to the control cells (both P<0.05). Normal peripheral B cells were resistant to the apoptosis-inducing effects of ZGDHu-1. Apoptosis induced by ZGDHu-1 was accompanied with generation of ROS, loss of ∆ψm, downregulation of Bcl-2 and increase of caspase-3 cleavage. Results of this study indicate that ZGDHu-1 is a promising specific treatment for CLL in the clinic.

[Effect of early enteral nutrition supplemented with glutamine on postoperative intestinal mucosal barrier function in patients with gastric carcinoma].

OBJECTIVE: To investigate the effect of early enteral nutrition (EEN) supplemented with glutamine on postoperative intestinal mucosal barrier function of patients with gastric carcinoma. METHODS: Eighty patients with gastric carcinoma who underwent intraoperative peritoneal hyperthermic chemotherapy(IPHC) were randomized into two groups: EEN+glutamine (EEN+Gln) group(n=40) and EEN group(n=40). Intestinal mucosal barrier function was evaluated by serum diamine oxidase (DAO), ratio of lactulose to mannitol(L/M), endotoxin lipopolysaccharides(LPS), and tumor necrosis factor-α(TNF-α) at 1 day before operation, 1 day, 7 days, 12 days after operation. Time to first flatus and tolerance to EEN were recorded as well. RESULTS: There were no significant differences in the two groups in demographics(all P>0.05). Two cases(5%) in the EEN+Gln group and 1 case (2.5%) in the EEN group could not tolerate well(P>0.05). On postoperative day 1, there were no differences in serum DAO, L/M ratio, LPS, TNF-α between the two groups (P>0.05). On postoperative day 7, all the parameters for mucosal barrier function were significantly lower in the EEN+Gln group. On postoperative day 12, the urinary L/M and DAO, LPS, and TNF-α were still significantly lower in the EEN+Gln group, however, urinary L/M was comparable between the two groups. There were no differences between the two groups in the time to first flatus (P>0.05). CONCLUSION: The immunologic tolerance of enteral nutrition supplemented with glutamine is favorable, which provides protective effect on intestinal mucosal barrier in patients with gastric carcinoma undergoing IPHC.

[The value of plasma pro-gastrin-releasing peptide, cytokeratin 19-fragments and carcinoembryonic antigen in patients with lung cancer].

OBJECTIVE: To evaluate the value of plasma ProGRP, CYFRA 21-1 and CEA in patients with lung cancer. METHODS: The levels of plasma ProGRP, CYFRA 21-1 and CEA were detected in 85 healthy control, 49 benign lung diseases and 143 lung neoplasms. The levels of ProGRP in the patients with SCLC was monitored. RESULTS: The level of plasma ProGRP in SCLC (M 179.1 ng/ml) was significantly higher than adenocarcinoma (M 35.3 ng/ml), squamous-cell carcinoma (M 33.3 ng/ml), healthy control (M 35.6 ng/m) and benign lung diseases (M 33.3 ng/m), P < 0.001. The sensitivity and specificity for diagnosing SCLC by ProGRP were 60.6% and 95.0% respectively. In the effective treatment group, ProGRP reduced 45.9%, in the progression group, ProGRP increased 103.1%, P < 0.05. The level of CEA in the metastatic adenocarcinoma (M 10.22 ng/ml) was significantly higher than non-metastatic adenocarcinoma (M 3.85 ng/ml) and squamous cell carcinoma (M 2.56 ng/ml) (P < 0.01). CONCLUSION: The plasma ProGRP is a good indicator for diagnosing and evaluating cure effect in SCLC; the high expression of CEA is related to the metastatic adenocarcinoma.

[Apoptosis of human lung carcinoma cell line EBC-1 induced by N, N'-di-(m-methylphenyl)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide and its molecular mechanism].

OBJECTIVE: To study whether N, N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1) inhibits proliferation and induces apoptosis in human lung carcinoma cell line EBC-1 cells and its molecular mechanism. METHODS: Different concentrations of ZGDHu-1 and different times of culture were used to treat EBC-1 cells in vitro. The inhibition of proliferation was measured by BrdU-ELISA. Cell apoptosis was detected by Annexin V/PI staining and cellular DNA fragmentation ELISA. Phosphorylated p38MAPK and STAT3 were examined by flow cytometry. The protein expressions of bcl-2, bax, p53, Fas, and caspase-3 were detected by Western blot analysis. RESULTS: ZGDHu-1 inhibited EBC-1 cell proliferation within a certain range of treating times and does, with a 24 h IC(50) of (295 ± 25) ng/ml, 48 h of (112 ± 8) ng/ml and 72 h of (23 ± 2) ng/ml. The EBC-1 cell apoptosis was confirmed by Annexin V/PI labeling and cellular DNA fragmentation ELISA in a dose-related manner. When EBC-1 cells were treated with 50, 200, and 500 ng/ml ZGDHu-1 for 48 h, the expression rates of phosphor-p38MAPK protein were 67.4%, 88.2%, 91.1%, respectively, and that of the control was 10.6%. That of STAT3 protein were 56.5%, 43.6% and 34.6%, respectively, and that of the control was 89.1%. The expression of bax, p53 and Fas protein was significantly increased, that of bcl-2 was not changed, and that of caspase-3 was significantly decreased by the ZGDHu-1 treatment. CONCLUSION: ZGDHu-1 can inhibit proliferation and induce apoptosis in EBC-1 cells. The mitochondrial pathway mediated by Fas may be one of its mechanisms. The apoptosis of EBC-1 cells may associate with up-regulation of phosphor-p38MAPK and down-regulation of phosphor-STAT3 in the cells.

[Effect of macrocalyxin A on HL-60 cells proliferation, differentiation and apoptosis].

OBJECTIVE: To study the effect of macrocalyxin A (MA) on proliferation, differentiation and apoptosis in HL-60 cells and explore its possible mechanisms. METHODS: Different concentration of MA were used to treat HL-60 cells. Proliferation inhibition was analyzed by Trypan blue staining and MTT assay, cell apoptosis by cell morphology, DNA content, cell cycle analysis, Annexin-V/PI and Hoechst 33258 fluorescence staining. The differentiation of HL-60 cells was evaluated by cell morphology, NBT tests and expression of CD11b, CD13, CD14. The expressions of bcl-2, bax, Fas, P53, mitochondrial transmembrane-potential (DeltaPsim) and mitochondrial membrane protein were analyzed by flow cytometry. RESULTS: MA could inhibit HL-60 cells proliferation capacity in a time-and dose-effect, with a 24 h IC50 value of 8.76 microg/ml, 48 h of 7.17 microg/ml and 72 h of 7.14 microg/ml. The HL-60 cells apoptosis was confirmed by cell morphology, sub-G1 phase and Annexin-V/PI labeling in a time and dose dependent manner. The more mature HL-60 cells were a with higher positivity of NBT and expressions of CD11b than those cultured without MA. The expression of bax was increased, while bcl-2, P53, Fas were unchanged on the MA treatment. MA could increase the expression of mitochondrial membrane protein in a dose-dependent manner while the DeltaPsim was reduced. CONCLUSION: MA can inhibit proliferation and induce differentiation and apoptosis of the HL-60 cells. The mechanism may be related with up-regulating bax, opening the mitochondrial permeability transition pore and reducing DeltaPsim.

[Effects of anti-heparanase antibody on the growth and invasion of HCCLM6 human hepatocellular carcinoma cells].

OBJECTIVE: To investigate the effects of the self-developed anti-heparanase polypeptide antibodies on growth and invasion of human hepatocellular carcinoma HCCLM6 cells. METHODS: Using MTT, flow cytometry, plate clone formation, transwell invasion and heparan degrading enzyme assay, the growth and invasion changes of human hepatocellular carcinoma HCCLM6 cells by co-culture with each of three self-developed rabbit anti-heparanase polyclonal antibodies were detected. RESULTS: Compared with normal rabbit IgG, in the presence of each anti-heparanase polypeptide antibody, the growth, cell cycle and clone formation remained unchanged, and under the P1 or P2 anti-heparanase polypeptide antibody (with final concentration 100 microg/ml), the cell invasiveness was inhibited by 52.5% and 36.6%, respectively, and the heparanase activity was inhibited by 42.9% and 39.1%, respectively. CONCLUSION: The P1 and P2 anti-heparanase polypeptide antibodies can effectively inhibit the invasion ability and heparanase activity of liver cancer HCCLM6 cells. However, All the three antibodies have no effects on its growth, cell cycle and clone formation.

Screening and identification of novel B cell epitopes in human heparanase and their anti-invasion property for hepatocellular carcinoma.

BACKGROUND: The aim of this study was to screen and identify novel B cell epitopes within the human heparanase protein and to investigate the impact of self-developed anti-heparanase polypeptide antibodies on growth and invasion of HCCLM6 human hepatocellular carcinoma cells in vitro. METHODS: The flexible regions of secondary structure and the B cell epitopes of the human heparanase amino acid sequence were predicted by DNAStar and Bcepred software.The multiple antigenic peptides (MAP) of the epitopes were synthesized in eight-branched form. Rabbits were immunized with the eight-branched MAPs mixed with the universal T-helper epitope human IL-1beta peptide (VQGEESNDK, amino acid 163-171). The immunogenicity of the synthesized peptides was evaluated by ELISA, western blot and immunohistochemistry. The impact of the self-developed rabbit anti-heparanase polyclonal antibodies on growth and invasion ability of HCCMLM6 cells were analyzed in a cell culture model. The cells were first treated with one of the three antibodies, respectively, and then measured by using MTT, flow cytometry, plate clone formation, invasion assay and heparan sulfate degrading enzyme assay. RESULTS: The three amino acid sequences 1-15 (MAP1), 279-293 (MAP2), and 175-189 (MAP3) in the large subunit of the human heparanase protein were predicted as its most potential epitopes. ELISA, western blot and immunohistochemistry analysis showed that all three MAPs were capable to induce high titer of serum antibodies. Antibodies induced by MAP1 and MAP2 were high specific. Furthermore, anti-MAP2 antibodies showed the strongest avidity towards liver cancer tissues. Under the treatment with the three anti-heparanase antibodies, respectively, the growth, cell cycle and clone formation of the cells remained unchanged when compared with a treatment with normal rabbit IgG. However, an inhibition of cell invasiveness and heparanase activity could be detected under the treatment with anti-MAP1- or anti-MAP2-antibody (with a terminal concentration of 100 mug/ml). The cell invasiveness was decreased by 54 and 38%, respectively, the heparanase activity by 43 and 39%, respectively. CONCLUSION: The multiple antigenic peptides MAP1 (AC 1-15) and MAP2 (AC 279-293) may be the dominant B cell epitopes in the human heparanase protein. The induced polypeptide antibodies can effectively inhibit the heparanase activity of HCCLM6 liver cancer cells and therefore influence their invasion ability, which provides a theoretic basis for the development of anti-heparanase antibodies and their clinical use as vaccine.

Serum thymidine kinase 1 reflects the progression of pre-malignant and malignant tumors during therapy.

This study evaluated the clinical utility of serum thymidine kinase 1 (STK1) in following the progression of pre-malignancies and malignancies and in monitoring the response of common carcinomas to therapy within a routine clinical setting. The STK1 concentration levels of patients with malignancies (n=224), pre-malignancies (n=10), non-tumor/non-proliferating diseases (systemic lupus erythematosus, SLE) (n=53), benign tumors (n=20) and healthy volunteers (n=761) were determined by enhanced chemoluminescence dot blot assay. Prior to treatment, STK1 levels in the pre-malignant group alone (3.1±2.3) or in the pre-malignant and malignant groups together (2.3±1.9) were significantly higher than in the benign (1.4±0.8), SLE (1.1±0.8) or healthy volunteer (0.6±0.4) groups (p<0.01). According to ROC analysis, the STK1 assay provided a high degree of discrimination between STK1-positive pre-malignant (0.978) or pre-malignant + malignant (0.941) patients and STK1-negative healthy individuals. After varying treatments (surgery, chemotherapy, X-ray), STK1 levels increased by 40-50% during the first month, then decreased back to normal values or even lower. Following treatment, STK1 levels were significantly increased in squamous cell carcinoma (SCC) as compared to adenocarcinoma (AC) patients. In other types of malignancies, STK1 levels decreased from as early as the first month. The STK1 levels of relapsed treated patients were significantly higher (50-60%) than those of mid/long-term treated patients. In conclusion, the STK1 assay discriminated between patients with malignancies and healthy individuals very well, and is therefore potentially useful for a broad range of clinical applications. For example, it could be used for the evaluation of early tumor progression or of tumor progression during therapy in routine clinical settings, as well as for the screening of healthy individuals.

[Evaluation and clinical significance of HBV large protein (LHBs) in diagnosis of hepatitis B].

OBJECTIVE: To explore the significance of HBV large protein (LHBs) in diagnosis of hepatitis B, we detected the LHBs, HBV DNA, PreS1 and other hepatitis B viral markers (HBV M) in the serum of patients infected with HBV. METHODS: HBV DNA was quantitatively detected using RT-PCR, LHBs, PreS1 and HBV M were analyzed by ELISA in totally 385 serum samples. RESULTS: There was a significant difference between the positive rate of LHBs (86.97%) and PreS1 (49.5%) in the 307 serum positive for HBV DNA (P less than 0.05). There was a correlation between the levels of LHBs and the logarithm of HBV DNA (r=0.935). In the serum specimens of patients negative for HBeAg, there was no significant difference between the positive rate of LHBs (76.92%) and the HBV DNA (67.95%), but the positive rate of PreS1 (45.73%) was lower than that of LHBs or HBV DNA. CONCLUSION: There was a close correlation between the copies of HBV DNA and the levels of LHBs, both the positive rate and the coincidence rate of LBHs and HBV DNA were higher than those of PreS1. LHBs can reflect the replication status of HBV.