A novel pyrosequencing strategy for RHD zygosity for predicting risk of hemolytic disease of the fetus and newborn.

OBJECTIVE: The aim of this study was the development of an accurate and quantitative pyrosequence (PSQ) method for paternal RHD zygosity detection to help risk management of hemolytic disease of the fetus and newborn (HDFN). METHODS: Blood samples from 96 individuals were genotyped for RHD zygosity using pyrosequencing assay. To validate the accuracy of pyrosequencing results, all the samples were then detected by the mismatch polymerase chain reaction with sequence-specific primers (PCR-SSP) method and Sanger DNA sequencing. Serological tests were performed to assess RhD phenotypes. RESULTS: Serological results revealed that 36 cases were RhD-positive and 60 cases were RhD-negative. The concordance rate between pyrosequencing assay and mismatch PCR-SSP assay was 94.8% (91/96). There were 5 discordant results between pyrosequencing and the mismatch PCR-SSP assay. Sanger sequencing confirmed that the pyrosequencing assay correctly assigned zygosity for the 5 samples. CONCLUSION: This DNA pyrosequencing method accurately detect RHD zygosity and will help risk management of pregnancies that are at risk of HDFN.

Creatinine accelerates APAP-induced liver damage by increasing oxidative stress through ROS/JNK signaling pathway.

Serum creatinine is an endogenous biomarker to estimate glomerular filtration rate (GFR) and is commonly used to assess renal function in clinical practice. Acetaminophen (APAP), the most available analgesic and antipyretic medication, is recommended as the drug of choice for pain control in patients with renal diseases. However, an overdose of APAP can lead to severe acute liver injury, which is also the most common cause of acute liver failure in western countries. The role of creatinine in APAP-induced liver injury is unclear and should be further explored. Herein, clinical data on patients with drug-induced liver injury revealed that the creatinine concentration between 82-442 µmol/L for female and 98-442 µmol/L for male is positively correlated with alanine aminotransferase (ALT), aspartate aminotransferase (AST). While there was no correlation between creatinine and ALT and AST when creatinine concentration is over 442 µmol/L. In addition, mice were administrated with creatinine intraperitoneally for 1 week before APAP injection to investigated the pathophysiological role of creatinine in APAP-induced acute liver injury. The results showed that creatinine administration aggravated hepatic necrosis and elevated serum lactate dehydrogenase (LDH) and ALT levels in mice upon APAP injection. The mechanism study demonstrated that creatinine could increase the production of reactive oxygen activation (ROS) and the activation of c-Jun N-terminal kinase (JNK). Furthermore, the liver injury was alleviated and the difference between APAP-treated mice and APAP combined with creatinine-treated mice was blunted after using specific ROS and JNK inhibitors. Significantly, creatinine stimulation aggravates APAP-induced cell death in HepaRG cells with the same mechanism. In summary, this study proposed that creatinine is closely related with liver function of drug-induced liver injury and exacerbates APAP-induced hepatocyte death by promoting ROS production and JNK activation, thus providing new insight into the usage of APAP in patients with kidney problems.

Immune microenvironment-related gene mapping predicts immunochemotherapy response and prognosis in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin's lymphoma (NHL). The R-CHOP immunochemotherapy regimen is the first-line treatment option for DLBCL patients and has greatly improved the prognosis of DLBCL, making it a curable disease. However, drug resistance or relapse is the main challenge for current DLBCL treatment. Studies have shown that the tumor microenvironment plays an important role in the onset, development, and responsiveness to drugs in DLBCL. Here, we used the CIBERSORT algorithm to resolve the composition of the immune microenvironment of 471 DLBCL patients from the GEO database. We found that activated memory CD4+ T cells and γδ T cells were significantly associated with immunochemotherapy response. Weighted gene co-expression networks (WGCNA) were constructed using differentially expressed genes from immunochemotherapy responders and non-responders. The module most associated with these two types of T cells was defined as hub module. Enrichment analysis of the hub module showed that baseline immune status was significantly stronger in responders than in non-responders. A protein-protein interaction (PPI) network was constructed for hub module to identify hub genes. After survival analysis, five prognosis-related genes (CD3G, CD3D, GNB4, FCHO2, GPR183) were identified and all these genes were significantly negatively associated with PD1. Using our own patient cohort, we validated the efficacy of CD3G and CD3D in predicting immunochemotherapy response. Our study showed that CD3G, CD3D, GNB4, FCHO2, and GPR183 are involved in the regulation of the immune microenvironment of DLBCL. They can be used as biomarkers for predicting immunochemotherapy response and potential therapeutic targets in DLBCL.

Identification of Circular RNAs Circ_0005008 and Circ_0005198 in Plasma as Novel Biomarkers for New-Onset Rheumatoid Arthritis.

The progression of autoimmune diseases is affected by the differential expression of circular RNAs (circRNAs). However, in the plasma from rheumatoid arthritis (RA), circRNAs have an uncertain role. Herein, microarray analysis was used to determine the plasma expression profile of circRNAs from new-onset patients with RA and healthy controls (HCs). CircRNA expression was verified using quantitative real-time reverse transcription PCR. The correlation between clinical variables and circRNA expression was assessed using Spearman's correlation test. The diagnostic value of plasma circRNAs was evaluated using receiver operating characteristic (ROC) curves. Circ_0005008 and circ_0005198 were confirmed to be elevated significantly in plasma samples from new-onset patients with RA compared with those from HCs and from patients with systemic lupus erythematosus. Among these new-onset patients with RA, we found that the levels of circ_0005008 and circ_0005198 correlated positively with the severity of disease, including the rheumatoid factor, C-reactive protein, the erythrocyte sedimentation rate, and the disease activity score in 28 joints (DAS28). However, their expression levels did not correlate with anti-cyclic citrullinated peptide antibodies. Analysis using ROC curves implied that circ_0005008 and circ_0005198 have significant value in the diagnosis of RA. In addition, we found that compared with that in osteoarthritis fibroblast-like synoviocytes (OA-FLSs), circ_0005198 expression was enhanced in RA-FLSs and correlated positively with DAS28. The level of the miRNA target of circ_0005198, miR-4778-3p, was identified as significantly decreased in RA-FLSs, and the expression levels of circ_0005198 and miR-4778-3p correlated significantly and negatively. The results suggested that in new-onset patients with RA, plasma circ_0005008 and circ_0005198 levels are associated with disease activity and represent possible RA biomarkers.

Preliminary study on the function of TMEM50A and its correlation with the RH genes.

OBJECTIVES: The purpose of this study was to investigate the association and impact of TMEM50A on RH genes activity and function. BACKGROUND: SMP1 is located on chromosome 1p36.11 in the RH gene locus, between the RHD and RHCE gene, where its position may be linked to RH haplotypes and contribute to selective pressures regarding certain RH haplotypes. TMEM50A is encoded by the SMP1 located in the intergenic region of RH, its influence on the function of the RH genes remains unclear. METHODS: The expression of TMEM50A was regulated by transfection of plasmid and siRNA in K562 cell model. Western blot and real-time PCR were used to detect possible expression changes in the RH. The ammonium transport function of cells was monitored using pH-sensitive dye, while transcriptome sequencing was used to predict the potential function of TMEM50A. RESULTS: The overexpression of TMEM50A significantly up-regulated RHCE gene activity (63.56%). The inhibition of TMEM50A resulted in significantly decreased RHCE (41.82%) and RHD expression (27.35%). Compared to control group, there was no significant change in the NH4 + transport function of cells in the overexpressed TMEM50A group. Transcriptome analysis showed that TMEM50A not only affected the transcription of target gene through splicing activities, but also played a role in the development of embryonic nervous system. CONCLUSIONS: TMEM50A may regulate the expression of RH gene by affecting the stability of RH mRNA through splicing function. It speculates that TMEM50A may play an important role in the development of embryonic nervous system.

Changes in Coagulation and Fibrinolysis in Post-Cesarean Section Parturients Treated With Low Molecular Weight Heparin.

BACKGROUND: Cesarean section is an independent risk factor for Venous thromboembolism (VTE). Low molecular weight heparin (LMWH) is extensively used for VTE prophylaxis after cesarean section. In this study, the effects of LMWH on coagulation and fibrinolysis after cesarean section and its clinical value were explored by studying the changes in laboratory indicators. METHODS: Antepartum and postpartum peripheral blood of 44 pregnant women who underwent vaginal delivery and 44 pregnant women who underwent cesarean section treated per routine with LMWH thromboprophylaxis on the first day post-operatively were collected for the following tests: D-dimer; thrombotic markers such as thrombomodulin (TM), thrombin-antithrombin complex (TAT), α2-plasmin inhibitor-plasmin complex (PIC), and tissue plasminogen activator inhibitor complex (t-PAIC); thromboelastography. RESULTS: Compared to the antepartum levels, PIC increased, TM, TAT, and t-PAIC decreased significantly in the parturients after a spontaneous vaginal delivery. Compared to the antepartum levels, parturients routinely treated with LMWH after cesarean section had higher PIC levels and lower D-dimer, TAT, and t-PAIC levels. Compared with parturients after vaginal delivery, parturients treated with LMWH after cesarean section had higher levels of TM, R, and MA, while there was no significant differences in the levels of D-dimer, TAT, PIC, t-PAIC, K, angle, LY30, and CI. CONCLUSION: The coagulation and fibrinolytic systems in gravidas and parturients are in a high level of dynamic equilibrium. The levels of coagulation and fibrinolytic system activation were similar in parturients who were routinely treated with LMWH after cesarean section compared with parturients after a spontaneous vaginal delivery.

[Angelica Polysaccharide Resists Platelets Apoptosis Induced by LY294002].

OBJECTIVE: To investigate the anti-apoptotic effect of Angelica polysaccharide (APS) on cryopreservated platelets and its mechanism. METHODS: The platelets were divided into 4 group: control group(4 ℃ stored platelets),APS group (APS-treated platelets stored at 4 ℃), LY294002 group (LY294002-treated platelets stored at 4 ℃) and LY294002+APS group(LY294002+APS treated platelets stored at 4 ℃ ). The expression of platelet membrane glycoprotein CD41 and CD61, as well as the platelet apoptotic rate, Caspase 3 expression and mitochondrial membrane potential (MMP) were detected by flow cytometry; the anti-apoptotic mechanism of APS by PI3K /AKT signaling pathway was analyzed by Western blot assay. RESULTS: The apoptosis rate of platelets in LY294002 group obviously increased, the activity of CD41 and CD61 expression gradually decreased along with the enhancement of LY294002 concentrations (r=-0.953)； compared with control group, the apoptosis rate of platelets in LY294002 group was enhanced significantly(P<0.05)，while the apoptosis rate of platelets in LY294002+APS group significantly was reduced(P<0.05) as compare with LY294002 group, which suggest that APS has an anti-apoptotic effect on the cryopreserved platelets. APS decreased the expression of Caspase-3 and inhibited the reduction of mitochondrial membrane potential induced by LY294002, moreover, APS could increase the activation of PI3K /AKT pathway in Plt. CONCLUSION: APS has an anti-apoptotic effect on the cryopreserved platelets through activating the PI3K /AKT pathway, decreasing the expression of apoptosis protease Caspase-3 and inhibiting the reduction of MMP.

Intron 4 of the RH Gene in Han Chinese, Tibetan, and Mongol Populations.

AIMS: This study aimed to compare the intron 4 sequence of the RHD and RHCE genes from Han Chinese, Tibetans, and Mongols, and explore its polymorphisms. MATERIALS AND METHODS: To investigate the distinction in the RHD and RHCE intron 4, polymerase chain reaction (PCR) was performed by a set of primers: Intron4F and Intron4R. Primer Intron4F for a sequence located in exon 4 and primer Intron4R for a sequence located in exon 5, respectively. RHD and RHCE intron 4 of all the samples from 26 cases of random unrelated Hans (13 RhD-positive donors and 13 RhD-negative donors), 25 cases of random unrelated Tibetans (18 RhD-positive donors and 7 RhD-negative donors), and 4 cases of random unrelated Mongols (1 RhD-positive donor and 3 RhD-negative donors) were amplified with PCR. The PCR products were then sequenced. RESULTS: A 576-bp product was detected in all the Han, Tibetan, and Mongol RhD-positive donors, whereas a 1228-bp product was detected in RhD-negative donors. The sequences of RHCE gene intron 4 were identical to each other in all Han, Tibetan, and Mongol RhD-negative donors, including 335 bp of Alu element, with a whole length of 1078 bp. By contrast, a 426-bp product was detected in all Han, Tibetan, and Mongol RhD-positive donors. Compared with the RHCE gene, a 652-bp deletion was noted in the RHD gene of Chinese, including the whole Alu element. The results were similar to the findings of Caucasians, whereas the lengths of RHD gene deletion fragments of Japanese and French were 649 and 654 or 651 bp, respectively. CONCLUSIONS: The RHCE gene intron 4 of Han Chinese, Tibetans, and Mongols differs from the RHD gene intron 4 in the presence of a 652-bp fragment. The RHCE gene intron 4 in Chinese has its own structural characteristics and differs among various ethnicities and regions.

Anti-H can trigger apoptosis and down-regulate FUT1 expression in erythroid differentiated K562 cells without complement mediation.

The reason why delayed RBC engraftment and pure red cell aplasia (PRCA) develop only in some but not all recipients of major ABO-incompatible hematopoietic stem cell transplantation (HSCT) remains elusive and the underlying mechanisms are not fully understood. Understanding how incompatible erythroid blood group antibodies (Abs) interact with ABH antigens (Ags) of grafts, and investigating how to induce artificially accommodation of grafts are of obvious importance in transplantation immunology. The effects of anti-H on proliferation, apoptosis, and α-(1,2)-fucosyltransferase gene (FUT1) expression in erythroid differentiated K562 cells were analyzed by the MTT assay, Annexin V/PI staining, and quantitative RT-PCR method. The growth of erythroid differentiated K562 cells was significantly suppressed when anti-H dilution was ≤ 1:8 (P<0.001, as compared with 1:16). Under the complement-free culture conditions, the apoptotic ratio of erythroid differentiated K562 cells was significantly increased when anti-H dilution was ≤ 1:16 (P<0.05, as compared with 1:32). The apoptosis was not only closely associated with anti-H dilution (F=138.991, P<0.001), but also correlated with treated time (F=583.249, P<0.001), which indicated typical dose- and time-dependent effects. Under the complement-free culture conditions, the FUT1 mRNA expression level was also suppressed when anti-H dilution was ≤ 1:16 (P<0.05, as compared with 1:32), which also manifested in typical dose-dependent (F=130.356, P<0.001) and time-dependent (F=1432.00, P<0.001) effects. The results confirm that anti-H can trigger apoptosis and down-regulate FUT1 expression in erythroid differentiated K562 cells without complement mediation. The findings suggest that anti-H could accommodate grafts through triggering apoptosis and down-regulating Fut1 expression to reduce ABH antigens.