[Effects of Platelet-Rich Plasma on Macrophage Phenotype and Its Influencing Factors].

OBJECTIVE: To evaluate the effects of platelet-rich plasma (PRP) supernatants with different activation methods and storage time on human monocyte-derived macrophages phenotype and explore the possible mechanism. METHODS: Human monocyte-derived macrophages were cultured in vitro with PRP or activated PRP supernatants activated with different activators. The expression of marker molecules on the surface of macrophages was detected by flow cytometry, and the concentration of growth factors in PRP supernatants was detected by ELISA. RESULTS: After 24 h of coculture, the expression level of CD86 in macrophages stimulated by PRP supernatant (thrombin and Cacl2 activated) was significantly higher than that by PRP group (P<0.05), and the expression of CD163 in macrophages was increased by Cacl2 activated PRP supernatant. Compared with different activator groups, the expression of CD163 in macrophages of Cacl2 activated group was significantly higher than that of thrombin and ADP groups (P<0.05). ELISA results showed that the concentrations of FGF (P<0.001) and EGF (P<0.05) in the supernatant of PRP stored at -80 ℃ for more than 20 months and 10-20 months were significantly higher than those in the group stored at less than 10 months after Cacl2 activation, and the expressions of CD86 (P<0.01), CD163 (P<0.001) and CD206 (P<0.001) in macrophages cocultured with the supernatant of the two groups were significantly increased. CONCLUSION: PRP activated by different activators has different effects on the phenotype of macrophages. Meanwhile, the storage time will also affect the growth factor concentration and effect of PRP.

[Preliminary Study on Screening and Identification of Lewis a Antigen Mimic Epitope in Alpaca Phage Display Nanobody Library].

OBJECTIVE: To establish a new method for synthesizing Lewis blood group antigens, that is, the mimotopes of Lewis blood group antigens were screened by using an alpaca phage display nanobody library. METHODS: We selected mimotopes of the Lewis a (lea) antigen by affinity panning of an alpaca phage display nanobody library using a monoclonal anti-lea antibody. Enzyme-linked immunosorbent assay (ELISA) was used to test the affinity of the positive clones for the monoclonal anti-lea antibody, and the high-affinity positive clones were selected for sequencing and synthesis. Finally, the sensitivity, specificity and reactivity of the synthesized lea mimotope in clinical samples were verified by ELISA. RESULTS: A total of 96 phage clones were randomly selected, and 24 were positive. Fourteen positive clones with the highest affinity were selected for sequencing. The result showed that there were 5 different sequences, among which 3 sequences with the highest frequency, largest difference and highest affinity were selected for expression and synthesis. The sensitivity and specificity of lea mimic antigen by ELISA showed that, the minimum detection limit of gel microcolumn assay (GMA) and ELISA method were 25 times different, and the lea mimic antigen had no cross reacted with the other five unrelated monoclonal antibodies(P<0.001). Finally, 30 clinical plasma samples were analyzed. The mean absorbance of the 15 positive plasma samples was significantly higher than that of the 15 negative plasma samples (P=0.02). However, the positive signal values of the clinical samples were much lower than those of the monoclonal antibodies. CONCLUSION: A new method of screening lea mimic antigen by using alpaca phage nanoantibody library has been established, which is expected to realize the screening of lea mimotopes, thus realizing the application of high-sensitivity detection methods such as ELISA and chemiluminescence in blood group antibody identification.

[Thalassemia Gene Detection Results and Application Value of Hematological Indexes Among Pregnant Women in Xindu District of Chengdu City].

OBJECTIVE: To analyze the genotype characteristics of α- and ß-thalassemia and the diagnostic value of hematological indexes in pregnant women in Xindu District of Chengdu. METHODS: The blood routine parameters(MCV) <80 fl and (or) (MCH) <27 pg and hemoglobin electrophoresis were used to screen the pregnant women, PCR-reverse dot blot hybridization(PCR-RDB) technique was used to detect the common α- and ß-thalassemia gene types in the primary screening positive population. The husbands of the diagnosed pregnant women were recalled for gene testing, and the highly suspected patients were checked by gene sequencing. RESULTS: Among the 7 049 pregnant women, 1 740(24.68%) cases were positive for primary screening. 180 patients were diagnosed as thalassemia gene positive, among them, 94 cases (52.22%) of α-thalassemia were detected and six genotypes were found, in which --SEA /αα genotype was the highest (58 cases, 61.70%); 82 cases (45.56%) of ß-thalassemia were detected and ten genotypes were found while CD41-42/N and CD17/N genotypes were the most common; there were 4 cases(2.22%) with α combined with ß-thalassemia. Through clinical follow-up survey, there were 4 couples with the same type of thalassemia, one of them was induced labor after diagnosis of hemoglobin H disease. Receiver operating curve (ROC curve) was used to analyze the diagnostic value of hematological parameters in thalassemia positive pregnant women. The results showed that AUC(HBA2)

[Changes in Metabolism and Function of Platelets Stored at Different Temperature].

AAbstractObjective:To compare and analyze the metabolic and functional changes in platelets stored at 4 ℃ and ones stored at 22 ℃ with agitation so as to provide an experimental basis for the cryopreservation technology of platelets. METHODS: Samples were collected from platelets stored at 4 ℃ in 2, 4, 6, 11, 15 and 21 days, and from ones stored at 22 ℃ with agitation during the same days, the metabolism indicators and thromboelastogram (TEM) were analysed. RESULTS: In metabolism, there were no significant changes of pH, GLU，PCO2, PCO2 and MPV levels of platelets stored at 4 ℃ for <6 days (P＞0.05), However, the Plt count decreased, the PDW and LDH level incrseased (P＜0.05). At the same time, only MPV had no changes of platelets stored at 22 ℃ during above-mentioned same days (P＞0.05), while the pH, PCO2, GLU, Plt all decreased, and PO2, LDH, PDW incrseased (P＜0.05). There were significant changes about the pH value, PO2, Plt, MPV, LDH, GLU levels between the two kinds of stored platelets during the same storing period (P＜0.05). The pH value and MPV of platelets stored at 4 ℃ were obviously lower than ones stored at 22 ℃, while GLU, PO2, LDH and Plt levels showed reverse changes (P＜0.05). Meanwhile, the PCO2 of platelets stored at 4 ℃ not could be detected and the Plt count reduced rapidly from d15. In function, the MA level of platelets stored at 4 ℃ was slower than that of platelets stored at 22 ℃, that is, the MA level of platelets stored at 4 ℃ were higher than that of platelets stored at 22 ℃ during the same storeing period (P＜0.05). CONCLUSION: Platelets stored at 4 ℃ have much slower metabolism than ones stored at 22 ℃, and the aggregation is stronger of platelets stored at 4 ℃ than that of ones at 22 ℃ during the same conservation period.

[Effect of red blood cell storage duration on the clinical effect of exchange transfusion and internal environment in neonates with hyperbilirubinemia].

OBJECTIVE: To study the effect of red blood cell (RBC) storage duration on the clinical effect of exchange transfusion (ET) and internal environment in neonates with hyperbilirubinemia. METHODS: A retrospective analysis was performed for the clinical data of 135 neonates with hyperbilirubinemia who received ET between January 2015 and August 2018. According to RBC storage duration, the neonates were divided into short-term storage group (RBCs were stored for ≤7 days) with 56 neonates and long-term storage group (RBCs were stored for >7 days) with 79 neonates. The two groups were compared in terms of serum total bilirubin (TBIL) level and the rate of TBIL reduction at 0 and 12 hours after ET, as well as the duration of continued phototherapy and rate of repeated ET. Routine blood test parameters, electrolytes, blood glucose, and blood gas parameters were measured before ET and at 0 hour after ET. RESULTS: At 0 hour after ET, there were no significant differences in the TBIL level and the rate of TBIL reduction between the two groups (P>0.05). At 12 hours after ET, the long-term storage group had a significantly higher TBIL level and a significantly lower rate of TBIL reduction than the short-term storage group (P<0.01). The long-term storage group had a significantly longer duration of continued phototherapy after ET than the short-term storage group (P<0.05). Compared with the short-term storage group, the long-term storage group had significantly higher incidence rates of ET-related complications, including hyponatremia, hyperkalemia, and metabolic acidosis (P<0.05). CONCLUSIONS: The use of RBCs with a storage duration of >7 days in ET for neonates with hyperbilirubinemia does not affect the immediate effect of ET, but these neonates tend to have a poor outcome after continued phototherapy and high risk of hyponatremia, hyperkalemia, and metabolic acidosis.

Platelet proteomics in diagnostic differentiation of primary immune thrombocytopenia using SELDI-TOF-MS.

BACKGROUND: Primary immune thrombocytopenic purpura (pITP) is defined as isolated autoimmune thrombocytopenia with idiopathic low platelet count, normal bone marrow, and unexplained causes of thrombocytopenia. Currently there is no definite criterion for ITP diagnosis. METHODS: We conducted proteomic screen of patients with pITP, secondary immune thrombocytopenia (sITP), and healthy controls using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). The proteomic profiles were obtained from platelet lysate samples of 82 healthy adult controls, 64 pITP, and 70 sITP patients, from which we screened marker proteins with significant differences, and constructed a diagnosis model using the artificial neural network (ANN) technique. RESULTS: We identified 6 marker proteins in the platelet lysates of pITP patients. This diagnosis method differentiated pITP patients from sITP effectively with a sensitivity of 96.9% (31/32), a specificity of 71.0% (54/76), and the area under the ROC curve of 0.864 in the training set, and a sensitivity of 87.5% (28/32), a specificity of 69.7% (53/76), and a positive predictive value of 75.0% (81/108) in the test set. CONCLUSION: The artificial neural network model based on platelet protein profiling established a potential pITP diagnosis platform.

Identification of Streptococcus pneumoniae genes specifically induced in mouse lung tissues.

To identify Streptococcus pneumoniae genes expressed specifically during infections, a selection system based on the in vivo expression technology (IVET) was established. galU, which is critical for capsular polysaccharide biosynthesis, and lacZY encoding beta-galactosidase were employed as dual reporter genes to screen in-vivo-induced (ivi) genes of S. pneumoniae. The galU-deficient mutant of S. pneumoniae is incapable of utilizing galactose, thus failing to synthesize capsular polysaccharide, and therefore loses its ability to survive in the host. A promoter-trap library was constructed in S. pneumoniae, which was used to infect BALB/c mice in an intranostril model. Those strains recovered from lung tissue of mice and exhibiting a white colony phenotype on tryptic soy agar containing X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) were collected and identificated. A total of 15 unique sequences were obtained through in vivo screening. The ivi genes of S. pneumoniae are involved in many processes, such as colonization and adherence, energy metabolism, nutrient substance transport, transcription regulation, DNA metabolism, and cell wall synthesis. There are some hypothetical proteins whose functions are not clear. This novel IVET is a useful tool for identifying ivi genes in S. pneumoniae.

Microarray analysis of microRNA expression in hepatocellular carcinoma and non-tumorous tissues without viral hepatitis.

BACKGROUND AND AIM: MicroRNAs (miRNAs) are non-coding RNA molecules of 21-24 nt that regulate the expression of target genes in a post-transcriptional manner. Evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation, and pathogenesis of human diseases including cancer. METHODS: We analyzed the miRNA expression profiles in 10 pairs of hepatocellular carcinoma (HCC) and adjacent non-tumorous tissue (NT) from 10 non-viral hepatitis patients, using a mammalian miRNA microarray containing whole human mature and precursor miRNA sequences. RESULTS: A total of 15 miRNAs exhibited higher expression in the HCC samples than that in the NT samples, and one miRNA demonstrated lower expression in the HCC samples than in the NT samples. A total of 18 miRNAs identified valid expression only in HCC samples, with six only in NT samples. The chip results were confirmed by Northern blot analysis. CONCLUSION: Our study may help clarify the molecular mechanisms involved in the pathogenesis of HCC, and miRNAs potentially serve as a novel diagnostic tool of HCC.

[Screen and identification of Streptocococcus pneumoniae genes specifically induced in host].

To identify in vivo-induced genes of S. pneumoniae and search new potential drug targets and vaccine candidates, a selection system was developed based on the in vivo expression technology (IVET). Promoter galU gene which is critical for the capsular polysaccharide biosynthesis and lacZY gene which encodes bea-galactosidase were employed as dual reporter genes. The galU-deficient mutant of S. pneumoniae is incapable of utilizing galactose and synthesizing capsular polysaccharide, therefore can't survival in the host. Firstly, the random pieces of S. pneumoniae chromosomal DNA (200-500 bp), obtained by partial Sau3Al restriction digestion, were subcloned into the Bgl II site of pEVP3-galU. Transformation by this plasmid library yielded promoter-trap library in S. pneumoniae. Then, the library was used to infect animals. Bacteria were harvested from lung tissue. White clones on TSA agar containing X-gal were used to reinfect animals. The sequence of infection and sorting was performed twice, 165 white clones harvested from the final round of infection were analyzed. A total of 15 unique sequences were obtained through in vivo screen. The bioinformatics analysis showed that these ivi genes involved in colonization and adherence, energy metabolism, nutrient substance transport, transcription regulation, DNA metabolism and cell wall synthesis. And there were some hypothetical proteins have unknown functions. Part of these genes may be related with virulence and can be used as vaccine candidates and drug targets.

The effect of transformation on the virulence of Streptococcus pneumoniae.

Although pneumococcus is one of the most frequently encountered opportunistic pathogen in the world, the mechanisms responsible for its infectiveness have not yet been fully understood. In this paper, we have attempted to characterize the effects of pneumococcal transformation on the pathogenesis of the organism. We constructed three transformation-deficient pneumococcal strains, which were designated as Nos. 1d, 2d, and 22d. The construction of these altered strains was achieved via the insertion of the inactivated gene, comE, to strains 1, 2 and 22. We then conducted a comparison between the virulence of the transformation-deficient strains and that of the wild-type strains, via an evaluation of the ability of each strain to adhere to endothelial cells, and also assessed psaA mRNA expression, and the survival of hosts after bacterial challenge. Compared to what was observed with the wild-type strains, our results indicated that the ability of all of the transformation-deficient strains to adhere to the ECV304 cells had been significantly reduced (p < 0.05), the expression of psaA mRNA was reduced significantly (p < 0.05) in strains 2d and 22d, and the median survival time of mice infected with strains 1d and 2d was increased significantly after intraperitoneal bacterial challenge (p < 0.05). The results of our study also clearly indicated that transformation exerts significant effects on the virulence characteristics of S. pneumoniae, although the degree to which this effect is noted appears to depend primarily on the genetic background of the bacteria.