PKCζ in prostate cancer cells represses the recruitment and M2 polarization of macrophages in the prostate cancer microenvironment.

Tumor-associated macrophages are key regulators of the complex interplay between tumor and tumor microenvironment. M2 Macrophages, one type of tumor-associated macrophages, are involved in prostate cancer growth and progression. Protein kinase C zeta has been shown to suppress prostate cancer cell growth, invasion, and metastasis as a tumor suppressor; however, its role in chemotaxis and activation of tumor-associated macrophages remains unclear. Here, we investigated the role of protein kinase C zeta of prostate cancer cells in regulation of macrophage chemotaxis and M2 phenotype activation. Immunohistochemistry was performed to analyze the expression of protein kinase C zeta and the number of CD206+ M2 macrophages in human prostate tissue. Macrophage chemotaxis and polarization were examined using Transwell migration assays and a co-culture system. Quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were used to detect M2 markers, protein kinase C zeta, interleukin-4, and interleukin-10 expression. We found the expression of protein kinase C zeta increased in prostate cancer tissues, especially in the early stage, and was negatively associated with tumor grade and the number of CD206+ macrophages. Inhibition of protein kinase C zeta expression in prostate cancer cells promoted chemotaxis of peripheral macrophages and acquisition of M2 phenotypic features. These results were further supported by the finding that silencing of endogenous protein kinase C zeta promoted the expression of prostate cancer cell-derived interleukin-4 and interleukin-10. These results suggest that protein kinase C zeta plays an important role in reducing infiltration of tumor-associated macrophages and activation of a pro-tumor M2 phenotype, which may constitute an important mechanism by which protein kinase C zeta represses cancer progression.

Positivity of both plasma Epstein-Barr virus DNA and serum Epstein-Barr virus capsid specific immunoglobulin A is a better prognostic biomarker for nasopharyngeal carcinoma.

BACKGROUND: Positivity of plasma Epstein-Barr virus (EBV)-DNA or serum virus capsid antigen-specific IgA (VCA-IgA) is a biomarker for the prognosis of nasopharyngeal carcinoma (NPC). The objective of this study was to determine the value of positivity for plasma EBV-DNA and/or VCA-IgA in predicting the survival of patients with NPC. METHODS: Plasma EBV-DNA and serum VCA-IgA in 506 NPC patients in this retrospective study were detected by quantitative real time polymerase chain reaction and enzyme-linked immunoabsorbent assay, respectively. The value of positivity for EBV-DNA and/or VCA-IgA in predicting the survival of patients with NPC was analyzed. RESULTS: Patients with positivity for both EBV-DNA and VCA-IgA had significantly shorter periods of relapse free survival (RFS) and overall survival (OS) than those with positive single measure or negative for both measures, and patients with positive single measure had significantly shorter periods of RFS and OS than those with negative for both. Multivariate analysis indicated that the positivity for EBV-DNA and/or VCA-IgA were significant risk factors for shorter periods of RFS and OS. CONCLUSION: These data indicated that positivity for both EBV-DNA and VCA-IgA was a better biomarker for the prognosis of patients with NPC. Our findings may provide new references for clinical practice.

[Clinical diagnostic value of procalcitonin detection in local infection and sepsis].

OBJECTIVE: To evaluate the value of procalcitonin (PCT) detection in the diagnosis of local infection and sepsis. METHODS: PCT, C-reactive protein (CRP), white blood cell count (WBC), neutrophil ratio (neu%) and lymphocyte ratio (lym%) were measured in patients with negative or positive blood culture test. The receiver operating characteristic (ROC) curves were constructed for PCT CRP, WBC, neu%, lym%, and the diagnostic model using SPSS software. Based on the binary logistic regression model, the predictors or probabilities were obtained and applied to establish the empirical and binormal model of the ROC curves to compare the area under the curve (AUC). RESULTS: A highly significant difference in PCT concentrations was noted between the two groups (chi(2)=52.52, P<0.001), and the diagnostic criteria at <2 of the ROC curves resulted in the greatest Youden index with a sensitivity of 63.3% and specificity of 86.8%. The AUC of PCT, CRP, WBC, neu% and lym% were 0.700, 0.765, 0.636, 0.618 and 0.648, respectively; the combined predicted ROC AUC was 0.776. The maximum Youden index was acquired at the optimal cutoff point of 0.566 with a diagnosis sensitivity and specificity of 63.8% and 84.7%, respectively. CONCLUSIONS: The PCT level is a valuable predictor for a rapid and reliable early diagnosis of sepsis. The diagnostic model based on the laboratory parameters, using the combined predictors of PCT, CRP and lym%, can be a useful means for predicting early-onset sepsis.

Detection and evaluation of antibodies against neutrophil-activating protein of Helicobacter pylori in patients with gastric cancer.

AIM: To detect and evaluate the antibodies against Helicobacter pylori (H pylori) neutrophil-activating protein (HP-NAP) in patients with gastric cancer and other gastroduodenal diseases. METHODS: Recombinant HP-NAP was prepared from a prokaryotic expression system in Escherichia coli. Serum positivity and level of HP-NAP-specific antibodies in sera from 43 patients with gastric cancer, 28 with chronic gastritis, 28 with peptic ulcer, and 89 healthy controls were measured by rHP-NAP-based ELISA. rHP-NAP-stimulated production of interleukin-8 (IL-8) and growth-related oncogene (GRO(alpha)) cytokines in the culture supernatant of SGC7901 gastric epithelial cells was also detected. RESULTS: The serum positivity and mean absorbance value of HP-NAP-specific antibodies in the gastric cancer group (97.7% and 1.01 +/- 0.24) were significantly higher than those in the chronic gastritis group (85.7% and 0.89 +/- 0.14, P < 0.005) and healthy control group (27.7% and 0.65 +/- 0.18, P < 0.001). The sensitivity and specificity of ELISA for the detection of HP-NAP-specific antibodies were 95.5% and 91.5%, respectively. HP-NAP could slightly up-regulate IL-8 production in gastric epithelial cell lines but had no effect on GRO(alpha) production. CONCLUSION: Infection with virulent H pylori strains secreting HP-NAP is associated with severe gastroduodenal diseases, and HP-NAP may play a role in the development of gastric carcinoma. rHP-NAP-based ELISA can be used as a new method to detect H pylori infection. The direct effect of HP-NAP on gastric epithelial cells may be limited, but HP-NAP may contribute to inflammatory response or carcinogenesis by activating neutrophils.

[RelB silencing in mouse bone-marrow derived dendritic cells mediated by lentiviral vector].

AIM: To silence RelB gene in mouse bone-marrow derived dendritic cells (DC) utilizing lentiviral vector, a novel tolerogenic dendritic cell with a relatively low expression level RelB was constructed and a new way to treat and prevent autoimmune diseases was explored. METHODS: Interferential targeting sequence R5 of RelB in mice was designed, synthesized and cloned into lentiviral vectors. Together with viral packaging materials were co-cultured in 293FT cell line to package lentiviral vector. Supernatant fluids were harvested, then virus titer detected. Mouse bone marrow derived DCs were infected by lentivirus particle. RelB gene expression level was detected by RT-PCR and immunofluorescence staining and analyzed by software of geo pro. There are three experiment control groups including immature DC, mature DC and DC infected by a negative independent control of T6. RESULTS: A similar RelB expression was detected by RT-PCR and immunofluorescence staining assay between DC infected virus R5 and immature DC, but was lower than that of mature DC. Significant difference in statistics P < 0.05. A similar RelB expression was detected by RT-PCR and immunofluorescence staining approaches between DC infected virus T6 and mature DC, but was higher than that of immature DC. Significant difference in statistics P < 0.05. CONCLUSION: RelB gene expressed by mouse bone marrow derived DC was silenced by Lentivirus vector effectively. The lentivirus vector with a low immunogenicity can be used to immunotherapy in vivo and overcome difficult transfection problem of primary DC. A new viral vector of DC immunotherapy can be obtained.

[Inhibition of tissue factor expression in endothelial cells by lentivirus mediated shRNA].

OBJECTIVE: To construct the recombinant lentivirus RNAi vector, and to determine whether the lentivirus mediated short hairpin RNA (shRNA) can inhibit the tissue factor (TF) expression in endothelial cells. METHODS: Two short hairpin RNAs targeting to human TF were cloned into pENTRTM/U6 plasmid to obtain an entry clone, and the positive clones were verified by sequencing. A recombination reaction was performed between the pENTR/U6 entry construction and pLenti6/BLOCKiTTM-DEST vector, and then the positive clones were confirmed by sequencing. The 293FT cell line was transfected by the above recombined plasmid and lentivirus packing materials, the culture supernatant was harvested, and the virus titer was determined. RT-PCR and ELISA were used to observe the inhibition of TF gene expression after the lentivirus transduction in human umbilical vein endothelial cells. RESULTS: The shRNA sequences targeting to human TF were cloned into the vectors, and an entry clone and an expression clone were constructed successfully, which were proved by sequence determination. Viral particles were packaged in the 293FT cell line, all virus stocks were collected, and the transfection titer was 5*10(5)/transduced unit. RT-PCR and enzyme linked immunosorbent assay demonstrated that the lentivirus stocks could suppress the TF expression in endothelial cells remarkably. CONCLUSION: Lentivirus RNAi vectors containing human TF gene are successfully constructed, and lentivirus mediated shRNA can inhibit the TF expression in endothelial cells, which may provide a highly effective method for the prevention and treatment of thrombo-embolic diseases.

[Relationship between polymorphisms of interleukin 10 promoter and serum levels of lipoprotein in the Chinese Han population].

OBJECTIVE: To study the relationship between polymorphisms of interleukin 10 (IL10QX) promoter and serum levels of lipoprotein in the healthy Chinese Han population. METHODS: PCR restriction fragment length polymorphism assay was used to detect the distribution of genotypes of IL10 -592,-819,-1082 in 200 healthy Chinese Han subjects. Serum levels of total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and very low-density lipoprotein (VLDL) in all subjects were measured to analyze the relationship with the polymorphisms of IL10 promoter. RESULTS: Comparing with AA genotype, the group with GA genotype at IL10 promoter -1082 position had a significant elevation of serum HDL-C level [(1.514+/-0.501) mmol/L vs. (1.261+/-0.346) mmol/L, t=-2.225, P=0.028] and a lower serum TG level[(1.701+/-1.836) mmol/L vs. (0.981+/-0.314) mmol/L,Z=-2.096,P=0.036]. The TG, TC, HDL-C, LDL-C and VLDL levels did not show any statistically significant differences among different genotypes (CC, AA, CA) of the IL10 -592, as well as the genotypes (TT, TA, AA) ofIL10 -819 (P>0.05). CONCLUSION: The results suggest that in the Chinese Han population, the polymorphism at position -1082 in the promoter region of IL10 gene may be associated with the serum HDL-C level and TG level.

[Relationship between surface molecules and RelB gene expression in DC2.4 cell line].

OBJECTIVE: To explore the relationship between the expression of the nuclear factor-kappaB transcription factor RelB gene and the surface molecules in DC2.4 cell line. METHODS: DC2.4 cell line was cultured in complete RPMI 1640 medium, whose morphology was observed with optical microscope and the intracellular structures with transmission electron microscope. Flow cytometry was performed to analyze the surface markers of the cells, including MHC-II, CD86 and CD40, and RelB mRNA expression was detected by RT-PCR. RESULTS: Under optical microscope, the cells appeared irregular in shape with obvious dendritic cell processes, and electron microscopy revealed homogenous fat drops and phagocytic vesicles in the cytoplasm. Flow cytometry demonstrated low expression levels of MHC-II and CD40, but high level of CD86 molecules. Low-level expression of RelB mRNA was detected by RT-PCR, resembling its expression level in bone marrow-derived DC with immature phenotype. CONCLUSION: DC2.4 is a mouse bone marrow dendritic cell line with strong phagocytic capacity, and the low expression of both RelB gene and surface CD40 molecules suggests an immature dendritic cell line.

[Heart-type fatty acid-binding protein detection for early diagnosis of acute coronary syndrome].

OBJECTIVE: To estimate the reliability of heart-type fatty acid-binding protein (H-FABP) for identifying acute coronary syndrome (ACS) in the early stage of chest pain onset. METHODS: This investigation was conducted based on a small population consisting of 40 healthy individuals, 19 established AMI patients and 20 unstable angina pectoris (UAP) patients. Serum H-FABP concentrations were measured in these subjects by sandwich ELISA, and receiver operating characteristics (ROC) curves for H-FABP for diagnosing AMI and UAP against normal subjects were then generated respectively. The areas under curve (AUCs) were calculated, and 0.5 was defined as the critical value of AUC to evaluate the diagnostic ability. RESULTS: The concentrations of H-FABP in healthy individuals, AMI patients and UAP patients were 1.29+/-0.64, 24.45+/-32.40 and 1.95+/-3.11 ng/ml, respectively; AUC (AMI) and AUC UAP were 0.978 (95%CI: 0.948-1.000) and 0.503 (95% CI: 0.334-0.671) respectively, and the former was significantly greater than 0.5. CONCLUSIONS: In the early stage of chest pain onset H-FABP detection is sufficient in distinguishing AMI patients from healthy individuals, but not capable of distinguishing UAP patients from healthy individuals. H-FABP may be used as a diagnostic biochemical marker in the early stage of AMI.

Antisense epidermal growth factor receptor (EGFR) transfection down-regulates EGFR expression and suppresses the malignant phenotype of human nasopharyngeal carcinoma CNE-2 cell line.

OBJECTIVE: To determine whether epidermal growth factor receptor (EGFR) expression contributes to tumor growth of poorly differentiated human nasopharyngeal carcinoma CNE-2 cell lines. METHODS: An expression vector containing a N-terminal fragment (1.35 kb) of human EGFR in the antisense orientation was transfected into CNE-2 cell lines via lipofectamine. The established clones resistant to G418 were isolated and characterized, and the tumor-inhibiting effect of antisense EGFR expression was evaluated in terms of tumor growth and metastasis at different time after subcutaneous inoculation into nude mice. RESULTS: Down-regulated EGFR expression in the cells with antisense vector transfection was demonstrated by ligand binding assay. The growth rate and the ability to grow in soft agarose of these antisense transfectants were also reduced. After inoculation into nude mice, EGFR antisense transfectants showed a longer latency period, slower tumor growth and lower metastatic rates to the lymph nodes and lung in comparison with the parental cells. CONCLUSIONS: These results suggest that these EGFR antisense cDNA-transfected CNE-2 cells are of value to further delineate the role of EGFR in the development and progression of nasopharyngeal carcinoma.

Effect of anti-tumor necrosis factor-alpha monoclonal antibody in alleviating renal ischemia-reperfusion injury.

OBJECTIVE: To investigate the effect of anti-tumor necrosis factor-alpha monoclonal antibody (anti-TNF-alpha mAb) in alleviating renal ischemia-reperfusion injury. METHODS: Fifty normal male Sprague-Dawley rats were randomly divided into 3 groups, namely group A that was subjected to ischemia-reperfusion injury with intravenous administration of anti-TNF-alpha mAb (0.1mg/kg.b.w.) 5 min before reperfusion (treatment group), group B with the same injury followed by saline administration in the same manner (control group), and group C with only anesthetization and leparotomy but not ischemia (sham operation group). Routine assays were performed for testing the levels of blood creatine (Cr), blood urea nitrogen (BUN), plasma TNF-alpha and the cell apoptosis. Ultrastructure of the kidney was also observed. RESULTS: Renal ischemia-reperfusion resulted in significant increase of the levels of Cr, BUN and TNF-alpha in the plasma (P<0.01), but these effects were offset by administration of anti-TNF-alpha mAb (P<0.01). In group B, widespread pathological changes and cell apoptosis were observed in the renal tissue following renal ischemia-reperfusion injury, while similar changes were scarcely visible in group A due to the protective effect of intravenous administration of anti-TNF-alpha mAb 5 min before reperfusion. CONCLUSION: Renal ischemia-reperfusion injury can be alleviated by anti-TNF-alpha mAb treatment.

Evaluation of the clinical applicability of bromcresol purple method for measuring serum albumin.

OBJECTIVE: To evaluate the clinical applicability of bromcresol purple (BCP) method in serum albumin measurement, and to investigate the possibility to render the results thus obtained in agreement with those by traditional bromcresol green (BCG) method. METHODS: The assessment of BCP method was conducted in comparison with BCG method by measuring their linearity ranges, coefficients of variation (CV) in batch and between batches, recovery rates, and resistance to interference by certain normal clinical factors. RESULTS: The linearity of BCP method was 10 to 80 g/L, CV was 0.95% in batch and 3.03% between batches, with an average recovery rate of 99.4%, showing no obvious interference by 252 micromol/L bilirubin, 4% Trilitid, or by 4 g/L hemoglobin separately. Close correlation was observed between the measurement results by the 2 methods (y=1.08x+5.98). The adult reference range was 34-49 g/L by BCP method, which was 6 g/L lower, on average, than that by traditional BCG method. CONCLUSION: BCP method is more specific than BCG method in serum albumin measurement, and the results of the former can be easily and accurately converted so as to be comparable with the results by the latter method.

Study of direct lung injury by seawater in canine models.

OBJECTIVE: To study the mechanism of direct lung injury by seawater and explore its possible management. METHODS: To exclude the interference of hypoxia and acidosis during the study of seawater-induced direct lung injury, 18 normal hybrid dogs were randomly assigned into group A (with all lung lobes perfused with seawater), group R (with the right lung lobe perfused with seawater) and group D (with the diaphragmatic lobe of lung perfused with seawater), with 6 dogs in each group. The changes in blood gas dynamics, blood gas acid-base status and electrolytes, along with the histological changes in the lung tissues were comparatively analyzed between the 3 groups. Bronchial microscope was employed to observe the continuous changes in the bronchioles before and after seawater perfusion in group D, and the concentration of the bronchoalveolar fluid and blood LDH-L and ALP levels were tested. RESULTS: The values of PaO(2), PaCO(2), pH, actual bicarbonate (AB), base excess (BE), tidal volume, and respiration rate in groups A and R were significantly different from those in group D (P < 0.01), and in groups A and R, the above measurements at every stage after seawater perfusion were significantly different from those before perfusion (P < 0.01). In group D, however, blood gas dynamics, blood gas acid-base status and electrolytes changed little after seawater perfusion (P > 0.05). In all the groups, obvious lung tissue injuries were observed under optical microscope after seawater perfusion. Observation with electron microscope revealed injuries to type II alveolar epithelial cells, broadened respiratory mucosa, and platelet adherence. Bronchial microscope in group D presented the bronchus filled with bronchoalveolar fluid, and blood LDH-L and ALP levels kept rising significantly (P < 0.01). Within 4 h after seawater perfusion, no pathological changes were seen in the lung tissues without direct contact with seawater. CONCLUSIONS: Seawater inspiration and retention in the lungs may lead to severe direct lung injury, and is the primary factor responsible for acute lung injury after drowning in the sea.