TPX2 enhances the transcription factor activation of PXR and enhances the resistance of hepatocellular carcinoma cells to antitumor drugs.

The pregnane X receptor (PXR) is an important regulator of hepatocellular carcinoma cellular resistance to antitumor drugs. Activation of PXR was modulated by the co-regulators. The target protein for the Xenopus plus end-directed kinesin-like protein (Xklp2) known as TPX2 that was previously considered as a tubulin regulator, also functions as the regulator of some transcription factors and pro-oncogenes in human malignances. However, the actions of TPX2 on PXR and HCC cells are still unclear. In the present study, our results demonstrate that the high expression of endogenous mRNA level of TPX2 not only correlated with the poor prognosis of advanced HCC patients who received sorafenib treatment but also with expression of PXR's downstream genes, cyp3a4 and/or mdr-1. Results from luciferase and real-time polymerase chain reaction (qPCR) showed that TPX2 leads to enhancement of the transcription factor activation of PXR. Protein-protein interactions between PXR and TPX2 were identified using co-immunoprecipitation. Mechanically, overexpression of TPX2 led to enhancement of PXR recruitment to its downstream gene cyp3a4's promoter region (the PXRE region) or enhancer region (the XREM region). Treatment of HCC cells with paclitaxel, a microtubule promoter, led to enhancement of the effects of TPX2, whereas vincristine, a microtubule depolymerizing agent caused a decrease in TPX2-associated effects. TPX2 was found to cause acceleration of the metabolism or clearance of sorafenib, a typical tyrosine kinase inhibitor (TKI) in HCC cells and in turn led to the resistance to sorafenib by HCC cells. By establishing novel actions of TXP2 on PXR in HCC cells, the results indicate that TPX2 could be considered a promising therapeutic target to enhance HCC cells sensitivity to antitumor drugs.

MTBP enhances the activation of transcription factor ETS-1 and promotes the proliferation of hepatocellular carcinoma cells.

Increasing evidence indicates that the oncoprotein murine double minute (MDM2) binding protein (MTBP) can be considered a pro-oncogene of human malignancies; however, its function and mechanisms in hepatocellular carcinoma (HCC) are still not clear. In the present work, our results demonstrate that MTBP could function as a co-activator of transcription factor E26 transformation-specific sequence (ETS-1), which plays an important role in HCC cell proliferation and/or metastasis and promotes proliferation of HCC cells. Using luciferase and real-time polymerase chain reaction (qPCR) assays, MTBP was found to enhance the transcription factor activation of ETS-1. The results from chromatin co-immunoprecipitation showed that MTBP enhanced the recruitment of ETS-1 to its downstream gene's (mmp1's) promoter region with ETS-1 binding sites. In cellular and nude mice models, overexpression of MTBP was shown to promote the proliferation of MHCC97-L cells with low endogenous MTBP levels, whereas the knockdown of MTBP led to inhibition of the proliferation of MHCC97-H cells that possessed high endogenous levels of MTBP. The effect of MTBP on ETS-1 was confirmed in the clinical specimens; the expression of MTBP was positively correlated with the downstream genes of ETS-1, mmp3, mmp9, and uPA. Therefore, by establishing the role of MTBP as a novel co-activator of ETS-1, this work expands our knowledge of MTBP or ETS-1 and helps to provide new ideas concerning HCC-related research.

The Combination of AFP and "Up-To-Seven" Criteria May Be a Better Strategy for Liver Transplantation in Chinese Cirrhotic HCC Patients.

Background: Orthotopic liver transplantation (OLT) is a life-saving option for patients with hepatocellular carcinoma (HCC), but the expanded OLT criteria remain controversial. Objective: The study aimed to explore whether expanded OLT criteria can be applied to Chinese cirrhotic patients with HCC. Methods: This retrospective study analyzed risk factors for HCC recurrence and death and compared patients' tumor characteristics and outcomes in groups of Milan, "Up-to-seven," and Hangzhou criteria, and groups between met and unmet the combinative criteria of "Up-to-seven" and AFP of < 1000 ng/mL. Results: Among 153 patients who underwent OLT for HCC from January 2015 to February 2019 in 4 years of follow-up, 20 (13.1%) patients had HCC recurrence, and 11 (7.2%) had HCC-related death. Multivariate Cox regression analysis showed that preoperative alpha-fetoprotein (AFP) of > 1000 ng/mL (hazard ratio [HR]: 10.05, 95% confidence interval [CI]: 2.45-41.13, P = 0.001) was an independent risk factor for HCC recurrence and HCC-related death (HR: 6.63, 95%CI: 1.31-33.52, P = 0.022). Patients who did not meet Milan criteria but satisfied the "Up-to-seven" criteria had no differences in overall survival (OS) (P = 0.69) and disease-free survival (DFS) (P = 0.35) than patients who met the Milan criteria. The combination of "Up-to-seven" criteria and AFP of < 1000 ng/mL differed significantly (HR: 18.9; 95% CI: 4.0-89.2; P < 0.001). Patients with HCC who met the "Up-to-seven" criteria and AFP of < 1000 ng/mL (n = 121) had excellent survival with 4-year OS of 91.6% (P < 0.001) and DFS of 90.8% (P < 0.001), which is significantly better compared to the other group (n = 32) (OS of 67.5% and DFS of 46.5%) and patients who met the Milan criteria (n = 108, OS of 89.8%, DFS of 89.6%), allowing 28.9% (13/45) of patients who did not meet the Milan criteria to benefit from OLT. Conclusion: Chinese cirrhotic patients with HCC who met the combinative criteria of "Up-to-seven" and AFP of < 1000 ng/mL had better survival than those who met the Milan criteria, and these combinative criteria benefited more patients and may become a better option for OLT.

[Expression Analysis of miRNA Profiles in Apheresis Platelets during Storage].

OBJECTIVE: To study the expression profiles changes of miRNA in apheresis platelets after 1, 3 and 5 days of storage. METHODS: The apheresis platelets were collected from 20 volunteer blood donors. After mixing fully, the platelets were stored in a shaker with (22±2) ℃ horizontal oscillation. The samples were taken on the 1st, 3rd and 5th day, and used to sequence for miRNAs by DNA nanoball (DNB) sequencing technology, which were named as C_1, C_3 and C_5, respectively. The expression level of platelets miRNA was standardized by transcripts per kilobase million (TPM) algorithm. MiRNAs with P-value < 0.001 and the expression difference of more than two times were considered as significant difference between two groups. The expression of miRNAs was verified by real-time fluorescence quantitative PCR (RT-qPCR). RESULTS: By DNB sequencing, there were 688, 730, and 679 platelet miRNAs expressed in C_1, C_3 and C_5 group, respectively. Cluster analysis showed that the expression profile of miRNAs changed significantly. The expression level of the first 20 high abundance miRNAs was about 4/5 of the total amounts of expressed miRNAs in each group, which the top five miRNAs were miR-21-5p, miR-26a-5p, miR-199a-3p, miR-126-3p, and let-7f-5p. The correlation of high abundance platelet miRNAs among the three groups was high (R2=0.876, R2=0.979, R2=0.937, respectively) and the differences were not statistically significant (P>0.05). Compared with the differential expression of platelet miRNAs with more than 1 000 TPM in the C_3 and C_1 group, there were 6 differentially expressed miRNAs, including 3 up-regulated (miR-146a-5p, miR-379-5p, and miR-486-5p) and 3 down-regulated (miR-652-3p, miR-142-5p, and miR-7-5p). While in the C_5 and C_1 group, there were 4 differentially expressed miRNAs, including 2 up-regulated (miR-146a-5p and let-7b-5p) and 2 down-regulated (miR-30d-5p and miR-142-5p). Compared with the differentially expression of platelet miRNAs between 1-1 000 TPM in the C_3 and C_1 group, there were 133 differentially expressed miRNAs, in which 99 were up-regulated and 34 were down-regulated. While in the C_5 and C_1 group, there were 77 differentially expressed miRNAs, in which 31 were up-regulated and 46 were down-regulated. The six selected differentially expressed miRNAs verified by RT-qPCR were consistent with those of sequencing. CONCLUSION: The expression profiles of platelets miRNAs change significantly among 1, 3, and 5 d of storage in vitro.

MicroRNA expression profiles analysis of apheresis platelets treated with vitamin B2 and ultraviolet-B during storage.

Whether platelet (PLT) microRNA (miRNA) profiles are affected by pathogen reduction technology (PRT) using vitamin B2 and ultraviolet-B (VB2-PRT) remains unclear. Samples from VB2-PRT-treated (experimental group, E_) and untreated (control group, C_) apheresis PLTs were taken on days 1, 3 and 5 of storage, designated as E_1, E_3, E_5, C_1, C_3 and C_5, respectively. The miRNA expression profiles were assessed by DNA Nano Ball (DNB) sequencing technology, and verified by quantitive real-time fluorescence quantitative PCR (qRT-PCR). Compared with the expression profiles of PLT miRNAs, 3895 miRNAs were identified in the E_ groups while 4106 were in the C_ groups. There were 487 significant differentially expressed miRNAs in E_1 vs C_1 group, including 220 upregulated and 287 downregulated, such as miR-146a-5p and let-7b-5p. There were 908 significant differentially expressed miRNAs in E_3 vs C_3 group, including 297 upregulated and 611 downregulated, such as miR-142-5p and miR-7-5p. There were 229 significant differentially expressed miRNAs in E_5 vs C_5 group, including 80 upregulated and 149 downregulated, such as miR-3529-3p and miR-451a. These differentially expressed miRNAs had been suggested to have functional roles in energy homeostasis, cell communication, proliferation, migration and apoptosis. GO analysis showed a significant enrichmen in relevant biological process categories as receptor activity, signal transduction, cell transport, motility and chemotaxis. The significantly enriched KEGG pathway of predicted target genes was Glycosaminoglycan biosynthesis in E_ vs C_ groups. These new observation could provide insights on the understanding of change of miRNA profiles of PLT treated with VB2-PRT.

Association of Killer Cell Immunoglobulin-like Receptor Genotypes and Haplotypes in Dry Eye Disease Patients Treated with Restasis and Systane.

Purpose: whether the Killer immunoglobulin-like receptor (KIR) genotypes and haplotypes are associated with the improvement in dry eye disease (DED) patients treated with Restasis and Systane (RS) remain unclear.Methods: Polymerase chain reaction with sequence-specific primers (PCR-SSP) was used to analyze KIR genes in a Chinese Han population of 198 severe DED patients treated with RS.Results: The higher frequencies of KIR genotype M, AF, AJ and haplotype 2 and 8 (P = .001, P = .03, P = .004, P = .000 and P = .023, respectively) and the lower frequencies of genotype AG and haplotype 1 (P = .000 and P = .000, respectively) were observed in complete responders (CR) than those in null or partial responders (NPR) of DED patients treated by RS.Conclusions: The patients with KIR genotype M, AF and AJ might be of advantage to therapy with RS, which are useful for improving novel personalized precise therapy strategy in DED patients.

[Analysis of miRNA-326's action on its target gene BCL-XL].

OBJECTIVE: To analyze the action of miRNA-326 on its target gene BCL-XL and the molecular mechanism of platelet apoptosis regulated by miRNAs. METHODS: Dual-luciferase vectors containing respectively the wild-type and mutant 3'-untranslated region (3'UTR) fragments of the BCL-XL gene were constructed with firefly and renilla luciferases and transfected into 293T cells. Relative fluorescence intensities of the transfected cells were measured. RESULTS: Dual-luciferase reporter gene vectors for PsiCHECK- BCL-XL -3'UTR-WT (wild-type) and PsiCHECK- BCL-XL -3' UTR-MT (variant) were respectively constructed. Relative fluorescence intensities of the 293T cells co-transfected by miRNA-326 and PsiCHECK- BCL-XL -3'UTR-WT plasmid were significantly lower compared with the control group (co-transfected by a miRNA-326 negative sequence and PsiCHECK- BCL-XL -3' UTR-WT plasmid) ( P = 0.034). The relative fluorescence intensity was also significantly reduced in cells co-transfected by miRNA-326 and PsiCHECK- BCL-XL -3' UTR-WT plasmid compared with the mutant control group co-transfected by miRNA-326 and PsiCHECK- BCL-XL -3'UTR-MT plasmid (P = 0.022). CONCLUSION: miRNA-326 may participate in the regulation of platelet apoptosis by acting on the 3'-UTR of the BCL-XL gene.

A novel HLA-A allele, A*31:74, was identified by sequence-based typing in a Chinese potential donor.

A*31:74: one nucleotide change resulting in a new motif ACG at codon 29 in HLA-A.

[Serological and molecular genetic analysis of a family with B(A) blood type].

OBJECTIVE: To analyze the blood type of a family with B(A) blood type. METHODS: The serological blood type of the family was determined by routine tube method. Exons 6 and 7 of the ABO gene were amplified by PCR and subjected to Sanger sequencing. RESULTS: Serological testing of the proband and her elder son showed a discrepancy which was initially identified as B(A) subtype. Her husband and second son were identified as blood type O. Sequencing of the proband and her elder son has identified an O02 allele and a 640A>G mutation compared with the B101 gene. Her husband and second son possessed the same genotype of O01/O02. CONCLUSION: The 640A>G mutation of ABO gene probably underlies the B(A)04 subtype.

Association of KIR Genotypes and Haplotypes in HBeAg-positive Chronic Hepatitis B Patients Treated with Entecavir.

BACKGROUND: A large proportion of patients with chronic hepatitis B (CHB) in China do not respond to entecavir (ETV) treatment. It remains unclear whether the Killer immunoglobulin-like receptor (KIR) genotypes and haplotypes were associated with the advantage of seroconversion in phepatitis B e-Antigen (HBeAg) positive CHB patients treated with ETV. METHODS: Polymerase chain reaction with sequence-specific primers (PCR-SSP) was used to analyze KIR genes in a Chinese Han population of 198 ETV-treated HBeAg-positive CHB patients and 200 healthy controls. Of the 198 patients, 59 were complete response group (CRG) and 139 were null or partial response group (NPRG) to the treatment with ETV. RESULTS: The frequencies of KIR genotype M, and haplotype 8 were significantly higher(P = 0.017, OR = 2.497，95%CI = 5.39-1.16 and P = 0.034, OR = 1.905，95%CI = 3.48-1.04, respectively), while the frequencies of genotype AH and haplotype 5 were significantly lower (P = 0.039, OR = 0.504, 95%CI = 0.97-0.26 and P = 0.031, OR = 0.601, 95%CI = 0.96-0.38, respectively) in HBeAg-positive CHB patient group than those in healthy group. Of note, the frequencies of KIR genotype AF and haplotype 1 were significantly higher (P = 0.022, OR = 2.860, 95%CI = 7.24-1.13 and P = 0.001, OR = 3.261, 95%CI = 6.47-1.64, respectively), while the frequencies of genotype AH and haplotype 5 were significantly lower (P = 0.038, OR = 0.338, 95%CI = 0.98-0.12 and P = 0.004, OR = 0.354, 95%CI = 0.73-0.17, respectively) in NPRG than those in CRG. CONCLUSIONS: The patients with KIR genotype AF and haplotype 1 might be negative, while genotype AH and haplotype 5 might be of advantage to the therapy with ETV, which are useful for improving novel personalized precise therapy strategy in HBeAg-positive CHB patients.

Association between KIR Genes and Efficacy of Treatment of HBeAg-Positive Chronic Hepatitis B Patients with Entecavir.

Entecavir (ETV) is commonly used to treat chronic hepatitis B (CHB) in China. However, certain percentages of e-Antigen (HBeAg) positive CHB patients do not respond to ETV therapy. OBJECTIVE: To investigate whether the killer immunoglobulin-like receptor (KIR) genes were associated with seroconversion in HBeAg positive CHB responder patients treated with ETV. METHODS: Polymerase chain reaction with sequence-specific primers (PCR-SSP) method was performed to genotype KIR genes in 200 healthy controls and 198 HBeAg-positive CHB patients which 59 were defined as the complete response group (CRG) to the treatment with ETV and 139 were defined as null or partial response group (NPRG). RESULTS: The frequencies of KIR2DS2 and KIR2DS3 were significantly higher (P=0.030, OR=1.57，95%CI=2.36-1.05 and P=0.018, OR=1.773，95%CI=2.77-1.13, respectively), while, the frequencies of KIR2DL3, KIR2DS1 and KIR3DS1 were significantly lower (P=0.038, OR=0.525, 95%CI=0.96-0.29,and P=0.031, OR=0.640, 95%CI =0.95-0.43, and P=0.035, OR=0.641, 95%CI=0.96-0.43, respectively) in HBeAg-positive CHB patients than those in healthy controls. The frequency of KIR2DS3 gene was significantly higher in NPRG than that in CRG (P=0.018, OR=0.402, 95%CI=0.83-0.20). The frequencies of KIR2DL3 and KIR3DS1 genes were significantly higher in CRG than those in NPRG (P=0.019, OR=3.625, 95%CI=10.83-1.21 and P=0.041, OR=1.949, 95%CI=3.65-1.04, respectively). CONCLUSION: Patients with KIR2DS3 might have negative responses to anti-HBV therapy with ETV and patients with KIR2DL3 and KIR3DS1 might have advantage in the therapy with ETV.

[Identification of a Bw14 subtype and exploration for its molecular basis].

OBJECTIVE: To identify a rare subtype of the ABO blood group system and explore its molecular basis. METHODS: Based on a standard serological assay, ABO subtype and haplotype were analyzed through PCR amplification of the 7 exons and adjacent introns of the ABO gene and TA clone sequencing. RESULTS: Forward typing showed a B type, while reverse typing demonstrated an extremely weak anti-B on routine gel analysis, which indicated a forward and reverse typing discrepancy. Absorption-elution testing confirmed that there was no A antigen on the surface of patient's red blood cells. Sequencing of the ABO gene showed a G>A exchange at position 523 in exon 7, which resulted in a Val to Met substitution at codon 175. Clone sequencing of the amplificons of the ABO gene showed an ABO* Bw14/O01 heterozygote genotype. CONCLUSION: Molecular method is useful for the identification of ambiguous blood groups. A 523G>A substitution of the ABO gene resulting in a Bw14 subtype probably underlies the weak B phenotype noted in the patient.

The potential association of the transcription levels of the ABO gene with the disease phases in AML patients.

Patients with AML may show ABO blood typing discrepancy, and the expression levels of the ABO antigens may show some alterations with the disease progression. To better understand this phenomenon, the blood samples of 25 AML patients and 25 healthy blood donors were examined. The serological ABO blood types of the patients were determined in different AML stages, and gene sequencing was performed to identify the precise ABO genotypes. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out to detect the transcription levels of the antigens. The genotyping result showed that there were 4 patients with genotype A1O, 5 patients with B1O, and 16 patients with A1B1. RT-PCR results indicated that the transcription levels of the ABO gene in 76% (19/25) of the patients were significantly lower compared with those in controls (p <0.05). After therapy, 3/4 patients with A1O returned to normal A, 4/5 patients with B1O returned to normal B, and 10/16 patients with A1B1 returned to normal AB. The patients who achieved complete remission (CR) showed no difference of transcription levels of the ABO gene from those of controls. The data indicated that the transcription levels of the ABO gene changed with the disease progression, suggesting its potential role in the progression of AML disease.

[Effects of Apheresis Platelets Treated with Vitamin B2 Photochemical Technology on the Release of White Blood Cells- and Platelet-Derived Cytokines during Storage].

OBJECTIVE: To investigate whether vitamin B2 photochemical pathogen reduction technology(PRT) treatment may lead to increase white cell- and platelet- derived cytokines release from platelets during storage. METHODS: Sixty milliliters of leukodepleted apheresis platelets were collected from 20 healthy donors, then were divided into 2 parts: one part (30 ml) remained untreated to serve as control, while the other part was treated with vitamin B2-UVB photo-chemical technology as experimental group. During 7 d of storage under standard blood bank conditions, platelet coun-ting (PC), platelet distribution width (PDW), mean platelet volume (MPV), white cell-derived cytokines (IL-1ß, IL-2, IL-6, IL-8, TNF-α and IFN-γ) and platelet-derived cytokines (CCL3, CCL5, TGF-ß-1 and PF4), P-selectin and phosphatidyl serine (PS) were analyzed on day 1, 3, 5 and 7 of storage, respectively. RESULTS: No signi-ficant differences were observed on PC, PDW and MPV between the experimental and control groups, respectively. The higher levels of platelet-derived cytokines were detected and reached a plateau after 5-7 days of storage, and the cyto-kines showed significant increase in experimental group compared with the control group. PS expression increased signi-ficantly in experimental group as compared with control group on day 3, 5 and 7 of storage, respectively. The accumula-tion of P-selectin was significant higher in experimental group than that in control group on day 5 and 7 of storage (P<0.05). The white cell-derived cytokines were not elevated by PRT treatment during 7 days of storage. CONCLUSION: The PRT-treated platelets are the main source of released cytokines during storage of PRT treatment. The levels of platelet-derived cytokines reach a plateau after 5-7 days of storage, most likely due to accelerated platelet activation and apoptosis.

Novel association of soluble intercellular adhesion molecule 1 and soluble P-selectin with the ABO blood group in a Chinese population.

Recent studies have reported that the ABO gene can affect circulating expression levels of soluble intercellular adhesion molecule 1 (sICAM-1) and soluble P-selectin (sP-selectin) in Caucasians. However, several factors may affect the association, including the distribution and variations of the ABO gene, ethnic diversity and the inflammatory response status. The aim of the present study was to investigate this issue in Asian subjects of various blood groups. A total of 800 blood samples were randomly selected from healthy blood donors. The ABO blood groups were examined using standard serological tests, and ABO genotypes of group A and group AB specimens were analyzed. Plasma concentrations of sICAM-1 and sP-selectin were detected by standard enzyme-linked immunosorbent assays. In healthy Chinese individuals, blood group A was detected to be significantly associated with lower circulating expression levels of sICAM-1 and sP-selectin, compared with group O. Individuals with ≥1 A1 allele had significantly lower expression levels of sICAM-1 and sP-selectin compared with all other ABO groups. The data indicate the significant association of ABO blood group antigens with sICAM-1 and sP-selectin expression levels in a healthy Chinese population, independent of the specific variations and distributions of ABO blood groups among ethnic populations. This result provides evidence for the previously unidentified role of ABO blood group antigens in the regulation of the inflammatory adhesion process. Accordingly, it can be proposed that ABO blood groups may require consideration when soluble adhesion molecules are identified as predictors for cardiovascular disease.

Possible association of killer cell immunoglobulin-like receptor genotypes and haplotypes with dry eye disease in a Han Chinese population.

PURPOSE: The objective of this study was to explore whether killer immunoglobulin-like receptor (KIR) genotypes and haplotypes are associated with dry eye disease (DED) in a Han Chinese population. METHODS: Polymerase chain reaction with sequence-specific primers (PCR-SSP) method was used to genotype KIR genes in 106 patients with DED and 220 healthy controls. RESULTS: Twenty-three KIR genotypes were observed in the DED patient and healthy control groups, ten of which had not been described previously. The genotype G and haplotype 4 were associated with increased risk of DED, and the odds ratio (OR) and 95% confidence interval (95% CI) were 2.58, 1.10-6.02 and 2.48, 1.31-4.69, respectively; while haplotype 2 appeared to have an inverse association with the disease (OR, 0.64; 95% CI, 0.44-0.92). Genotype B/B was also associated with increased risk of DED, and the OR and 95% CI were 2.35 and 1.09-5.10, respectively. KIR haplotypes A and B have distinctive centromeric (Cen) and telomeric (Tel) gene-content motifs, and Cen-B/B was associated with increased risk of DED (OR, 2.38; 95% CI, 1.03-5.49). However, all frequencies of these KIR genotypes and haplotypes were no longer statistically significant between the two groups after the Bonferroni correction was applied for multiple testing. CONCLUSIONS: There was a possible association between certain KIR genotypes and haplotypes with DED in a Han Chinese population. However, additional confirmation is required.

[Confirmation of 17 rare HLA alleles and prediction of their haplotypes].

OBJECTIVE: To confirm 17 rare HLA alleles detected during routine HLA typing and deduce their haplotypes. METHODS: Bi-allelic sequence-based typing and Luminex DNA PCR-SSOP assay were applied for the initial or repeat HLA typing, respectively. The rare HLA alleles were confirmed with mono-allelic sequence-based typing. Predicted haplotypes of the rare alleles were inferred based on the frequencies of HLA alleles and haplotypes in Han population. RESULTS: The authenticity of the total 17 rare HLA alleles was proven, and 18 predicted haplotypes associated with the rare alleles were recognized. A*11:12 and DRB1*13:19 were detected twice among unrelated individuals. CONCLUSION: Study of rare HLA alleles and predicted haplotype can provide useful information for donor searching and transplantation, and enrich polymorphisms of HLA in this population.

In vitro properties of apheresis platelet during extended storage in plasma treated with anandamide.

BACKGROUND: In China apheresis platelets (PLTs) are stored in plasma for only 5 days, resulting in PLT inventory pressures. Anandamide (ANA) was reported to be a potential agent to inhibit PLT apoptosis. The aim of this study was to evaluate the characteristics of extended storage PLTs in plasma treated with ANA in vitro. METHODS: Apheresis PLTs (n = 20) were prepared in plasma treated with ANA, and stored at 22 °C for up to 11 days. On day 1, 3, 5, 7, 9 and 11, PLTs were tested for PLT count, mean PLT volume (MPV), PLT distribution width (PDW), pH, pCO(2), pO(2), hypotonic shock response (HSR), phosphatidylserine (PS) exposure and soluble P-selectin content. RESULTS: PLTs stored in plasma with/without ANA didn't show significant differences during the first 5 days of storage. From the 7(th) day on, PLTs stored in plasma with ANA displayed significantly lower PS expression, soluble P-selectin content and higher HSR scores than those stored in plasma without ANA (P <0.05), respectively. CONCLUSION: The extended storage of PLTs in plasma treated with 0.5 µmol/l ANA showed better characteristics of the PLTs, compared with the control group, which was suggested to potentially alleviate the PLT storage lesion.

[Mechanism investigation of platelet apoptosis inhibition by N-Arachidonoylethanolamine].

OBJECTIVE: To investigate the mechanism of N- Arachidonoylethanolamine (ANA) on inhibiting platelets (PLT) apoptosis under standard blood bank storage conditions. METHODS: Samples taken from collected apheresis PLT by the Amicus instrument were split into three parts. An aliquot of 0.5 µmol/L ANA were added to one part of storage PLT as the ANA group; an aliquot of 0.5 µmol/L ANA and 1 µmol/L SR141716 was added to the another part as the ANA + SR141716 group; and the third part without ANA and SR141716 as the control group. These samples were stored on a flat-bed shaker at (22 ± 2) °C for 7 days. The expression of phosphatidyl serine (PS) positive, phospho (p)-Akt, Akt, p-Bad, Bad, caspase-3, caspase-9, cytochrome C (Cyt-C) and BCL-XL interaction with Bak were detected. RESULTS: The rate of PLT PS positive in ANA group decreased significantly than that in control group[ (8.29 ± 1.44) % vs (14.24 ± 2.47) %, P<0.05]. The release of Cyt-C from mitochondria to cytosol in ANA group decreased significantly compared with control group[ (3.29 ± 1.44) % vs (15.24 ± 3.40) %, P<0.05]. Also the expressions of p-Akt and p-Bad in ANA group increased significantly than those in control group[ (71.33 ± 10.26) % vs (35.00 ± 6.00) %, P<0.05; (39.00 ± 9.64) % vs (10.33 ± 1.53) %, P<0.05, respectively]. Higher amounts of Bak protein were co-precipitated with BCL-XL in ANA group than that in control group (about 2.6 fold, P<0.05). The expressions of cleaved caspase- 9 and caspase- 3 in ANA group decreased significantly than those in control group[ (9.63 ± 1.47) % vs (23.24 ± 2.47) %, P<0.05; (6.30 ± 1.40) % vs (13.20 ± 2.50) %, P<0.05, respectively]. There were no significantly changes between ANA+SR141716 and control groups (P>0.05). CONCLUSION: ANA protected PLTs from apoptosis as a result of inhibiting the release of Cyt-C from mitochondria to cytosol by modifying the expressions of apoptosis-relative proteins.

[Identification of a novel HLA allele, HLA-DRB1*03:80, by sequencing-based typing].

This study was aimed to identify a novel HLA-DRB1 allele from a Chinese potential hemopoietic stem cell donor of Northeast China. A rare HLA-DRB1 allele was initially detected by Luminex PCR-SSO typing, then the sample was sequenced by sequence-based typing (SBT) and the alignments of sample's alleles was identified by single allele-specific sequencing strategy. The results revealed the existence of a new allele which differs from the closest matching allele DRB1*03:06 by a single nucleotide substitution at position 239, where C→G in exon 2, resulting in an amino acid exchange from Thr to Arg at codon 51. It is concluded that a novel allele has been confirmed and its name DRB1*03:80 is officially assigned by the WHO Nomenclature Committee in February 2012.