Intracranial hemorrhage in large vessel occlusion patients receiving endovascular thrombectomy with or without intravenous alteplase: a secondary analysis of the DIRECT-MT trial.

BACKGROUND: Alteplase before thrombectomy for patients with large vessel occlusion stroke raises concerns regarding an increased risk of intracranial hemorrhage (ICH), but the details of this relationship are not well understood. METHODS: This was a secondary analysis of the DIRECT-MT trial. ICH and its subtypes were independently reviewed and classified according to the Heidelberg Bleeding Classification. The effects of alteplase before thrombectomy on ICH and ICH subtypes occurrence were evaluated using logistic regression. Clinical and imaging characteristics that may modify these effects were exploratorily tested. RESULTS: Among 591 patients, any ICH occurred in 254 (43.0%), including hemorrhagic infarction type 1 in 12 (2.1%), hemorrhagic infarction type 2 in 127 (21.7%), parenchymal hematoma type 1 in 34 (5.8%), parenchymal hematoma type 2 in 50 (8.6%), and other hemorrhage types (3a-3c) in 24 (4.1%). Similar ICH frequencies were observed with combined alteplase and thrombectomy versus thrombectomy only (134/292 (45.9%) vs 120/299 (40.1%); OR 1.27, 95% CI 0.91 to 1.75, P=0.16), but patients treated with alteplase had a higher parenchymal hematoma rate (51/287 (17.8%) vs 33/297 (11.1%); OR 1.75, 95% CI 1.08 to 2.85, P=0.024). In the adjusted model, difference in parenchymal hematoma occurrence between groups remained significant (adjusted OR 1.71, 95% CI 1.00 to 2.92, P=0.049). Patients with history of diabetes (Pinteraction=0.048), hypertension (Pinteraction=0.02), antiplatelet therapy (Pinteraction=0.02), anticoagulation therapy (Pinteraction=0.04), and statin administration (Pinteraction=0.02) harbored a higher ICH rate when they received combination therapy. CONCLUSIONS: Our data showed that in the DIRECT-MT trial, alteplase did not increase overall ICH for large vessel occlusion patients treated with thrombectomy, but it increased the parenchymal hematoma rate.

Effect of stroke etiology on endovascular thrombectomy with or without intravenous alteplase: a subgroup analysis of DIRECT-MT.

BACKGROUND: Stroke etiology might influence the clinical outcomes in patients with large vessel occlusion receiving endovascular treatment (EVT) with or without thrombolysis. OBJECTIVE: To examine whether stroke etiology resulted in different efficacy and safety in patients treated with EVT-alone or EVT preceded by intravenous alteplase (combined therapy). METHODS: We assessed the efficacy and safety of treatment strategy based on prespecified stroke etiology, cardioembolism (CE), large-artery atherosclerosis (LAA), and undetermined cause (UC) for patients enrolled in the DIRECT-MT trial. The primary outcome was the modified Rankin Scale (mRS) score at 90 days. Multivariate ordinal logistic regression analysis was used to calculate the adjusted common OR for a shift of better mRS score for EVT-alone versus combined therapy. A term was entered to test for interaction. RESULTS: In this study, 656 patients were grouped into three prespecified stroke etiologic subgroups. The adjusted common ORs for improvement in the 90-day ordinal mRS score with EVT-alone were 1.2 (95% CI 0.8 to 1.8) for CE, 1.6 (95% CI 0.8 to 3.3) for LAA, and 0.8 (95% CI 0.5 to 1.3) for UC. Compared with CE, EVT-alone was more likely to result in an mRS score of 0-1 (pinteraction=0.047) and extended Thrombolysis in Cerebral Infarction ≥2b (pinteraction=0.041) in the LAA group. The differences in mortality and symptomatic intracranial hemorrhage within 90 days were not significant between the subgroups (p＞0.05). CONCLUSIONS: The results did not support the hypothesis that a specific treatment strategy based on stroke etiology should be used for patients with large vessel occlusion (NCT03469206).

Diphenyleneiodonium chloride synergizes with diazoxide to enhance protection against amyloid ß induced neurotoxicity.

We examined synergistic effects of inhibiting reactive oxygen species generated from the mitochondria and from nicotinamide adenine dinucleotide phosphate oxidase on neurotoxicity. Primary hippocampal neurons were exposed to amyloid ß, and the cells were treated with diazoxide or/and diphenyleneiodonium chloride. We found that the cell viability was decreased significantly after exposure to amyloid ß for 72 h with higer reactive oxygen species and malondialdehyde levels, higher caspase-3 and cleaved caspase-3 levels and lower B-cell lymphoma 2 (Bcl-2) level. Both diazoxide and diphenyleneiodonium increased cell viability by inhibiting the increase in reactive oxygen species and caspase-3 activity as well as the decrease in Bcl-2 induced by amyloid ß. The combination of diazoxide and diphenyleneiodonium exhibited better protective effects compared to a single treatment. In conclusion, the activation of a mitochondrial potassium channel in combination with the inhibitor of nicotinamide adenine dinucleotide phosphate oxidase exhibit synergistic protective effects against amyloid ß neurotoxicity.

Activation of PPARγ mediates icaritin-induced cell cycle arrest and apoptosis in glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is the most prevalent primary malignancy of the brain. This study was designed to investigate whether icaritin exerts anti-neoplastic activity against GBM in vitro. MATERIALS AND METHODS: Cell Counting Kit-8 (CCK-8) assay was utilized to examine the viability of GBM cells. The apoptotic cell population was measured by flow cytometry analysis. Cell cycle distribution was detected by flow cytometry as well. Western blot analysis was performed to examine the level of biomarker proteins in GBM cells. Levels of PPARγ mRNA and protein were detected by qPCR and western blot analysis, respectively. To examine the role of PPARγ in the anti-neoplastic activity of icaritin, PPARγ antagonist GW9662 or PPARγ siRNA was used. The activity of PPARγ was determined by DNA binding and luciferase assays. RESULTS: Our findings revealed that icaritin markedly suppresses cell growth in a dose-dependent and time-dependent fashion. The cell population at the G0/G1 phase of the cell cycle was significantly increased following icaritin treatment. Meanwhile, icaritin promoted apoptotic cell death in T98G and U87MG cells. Further investigation showed upregulation of PPARγ played a key role in the anti-neoplastic activities of icaritin. Moreover, our result demonstrated activation of AMPK signaling by icaritin mediated the modulatory effect of icaritin on PPARγ. CONCLUSION: Our results suggest the PPARγ may mediate anti-neoplastic activities against GBM.

Three-dimensional structure discrepancy between HLA alleles for effective prediction of aGVHD severity and optimal selection of recipient-donor pairs: a proof-of-concept study.

The optimal selection of recipient-donor pair and accurate prediction of acute graft-versus-host disease (aGVHD) severity are always the two most crucial works in allogeneic hematopoietic stem cell transplantation (allo-HSCT), which currently rests mostly with HLA compatibility, the most polymorphic loci in the human genome, in clinic. Thus, there is an urgent need for a rapid and reliable quantitative system for optimal recipient-donor pairs selection and accurate prediction of aGVHD severity prior to allo-HSCT. For these reasons, we have developed a new selection/prediction system for optimal recipient-donor selection and effective prediction of aGVHD severity based on HLA three-dimensional (3D) structure modeling (HLA-TDSM) discrepancy, and applied this system in a pilot randomized clinical allo-HSCT study. The 37 patient-donor pairs in the study were typed at low- and high-resolution levels for HLA-A/-B/-DRB1/-DQB1 loci. HLA-TDSM system covering the 10000 alleles in HLA class I and II consists of the revised local and coordinate root-mean-square deviation (RMSD) values for each locus. Its accuracy and reliability were confirmed using stably transfected Hmy2.CIR-HLA-B cells, TCR Vß gene scan, and antigen-specific alloreactive cytotoxic lymphocytes. Based on the preliminary results, we theoretically defined all HLA acceptable versus unacceptable mismatched alleles. More importantly, HLA-TDSM enabled a successful retrospective verification and prospective prediction for aGVHD severity in a pilot randomized clinical allo-HSCT study of 32 recipient-donor transplant pairs. There was a strong direct correlation between single/total revised RMSD and aGVHD severity (92% in retrospective group vs 95% in prospective group). These results seem to be closely related to the 3D structure discrepancy of mismatched HLA-alleles, but not the number or loci of mismatched HLA-alleles. Our data first provide the proof-of-concept that HLA-TDSM is essential for optimal selection of recipient-donor pairs and effective prediction of aGVHD severity before allo-HSCT.

Icaritin inhibits the invasion and epithelial-to-mesenchymal transition of glioblastoma cells by targeting EMMPRIN via PTEN/AKt/HIF-1α signalling.

Icaritin, a hydrolytic product of icariin from the Epimedium genus, exerts anti-tumour effects on a variety of tumour cell types, mainly by inhibiting cell proliferation and inducing apoptosis. However, little is known about the role of icaritin in cancer invasion and epithelial-to-mesenchymal transition (EMT). In the present study, the glioblastoma (GBM) cell line U87MG was used as a model to investigate the effects of icaritin on the invasion and EMT of cancer cells. The results showed that icaritin significantly inhibited the invasion and EMT of GBM cells by targeting extracellular matrix metalloproteinase (EMMPRIN). Furthermore, the findings strongly indicate that the modulatory effect of icaritin on EMMPRIN is mediated via the PTEN/Akt/HIF-1α signalling pathway. The data provide the first experimental evidence of the inhibitory effect of icaritin on cancer cell invasion and EMT, thus highlighting the potential of icaritin to be employed as a promising anti-cancer agent in the treatment of GBM.

Icaritin Sensitizes Human Glioblastoma Cells to TRAIL-Induced Apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been considered to be one of the most promising candidates in research on treatments for cancer, because it induces apoptosis in a wide variety of cancer cells but not in most normal human cell types. However, many cells including glioblastoma (GBM) cells are resistant to TRAIL-induced apoptosis, which limits the potential application of TRAIL in cancer therapy. Icaritin, a hydrolytic product of icariin from Epimedium Genus, has been identified as a potential therapeutic and preventive agent in renal cell carcinoma and breast cancer. In this study, we investigated whether Icaritin treatment could modulate TRAIL-induced apoptosis in GBM. The effect of icaritin on TRAIL sensitivity was assessed in human GBM U87 and U373 cells. The underlying regulatory cascades were approached by biochemical and pharmacological strategies. We found that nontoxic concentration of icaritin alone had no significant effect on the level of apoptosis, but a combination treatment of TRAIL and icaritin caused a significantly more profound apoptosis. The sensitization was accompanied by c-FLIP down-regulation and inhibition of NF-κB activity. Studies have further demonstrated that silencing NF-κB alone was sufficient to down-regulate c-FLIP expression and sensitized both tested cells to TRAIL-induced apoptosis. These data suggest that icaritin sensitizes TRAIL-induced tumor cell apoptosis via suppression of NF-κB-dependent c-FLIP expression, providing in vitro evidence supporting the notion that icaritin is a potential sensitizer of TRAIL in anticancer therapy against human GBM.

Amplified voltammetric characterization of cleavage of the biotinylated peptide by BACE1 and screening of BACE1 inhibitors.

Cleavage of amyloid precursor protein (APP) by the ß-site APP cleaving enzyme 1 (BACE1) is a key step in the formation of amyloid beta (Aß) peptide, the main component of amyloid plaques in Alzheimer's disease (AD). Suppression of BACE1 activity has thus become an efficient way for the treatment of AD. In this study, BACE1 in the absence or presence of BACE1 inhibitors was exposed to the biotinylated peptide substrate-modified electrode. This step was followed by the attachment of ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. Due to the blockage of the BACE1 activity by select inhibitors, well-defined voltammetric peaks of high signal intensity were obtained. However, featureless voltammogram was obtained upon initiating the cleavage reaction. The proposed method is simple, sensitive, and suitable for monitoring of BACE1 activity and screening of BACE1 inhibitors.

Direct quantification of microRNA at low picomolar level in sera of glioma patients using a competitive hybridization followed by amplified voltammetric detection.

MicroRNAs (miRNAs), acting as oncogenes or tumor suppressors in humans, play a key role in regulating gene expression and are believed to be important for developing novel therapeutic treatments and clinical prognoses. Due to their short lengths (17-25 nucleotides) and extremely low concentrations (typically < picomolar) in biological samples, quantification of miRNAs has been challenging to conventional biochemical methods, such as Northern blotting, microarray, and quantitative polymerase chain reaction (qPCR). In this work, a biotinylated miRNA (biotin-miRNA) whose sequence is the same as that of a miRNA target is introduced into samples of interest and allowed to compete with the miRNA target for the oligonucleotide (ODN) probe preimmobilized onto an electrode. Voltammetric quantification of the miRNA target was accomplished after complexation of the biotin-miRNA with ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. The Fc oxidation current was found to be inversely proportional to the concentration of target miRNA between 10 fM and 2.0 pM. The method is highly reproducible (relative standard deviation (RSD) < 5%), regenerable (at least 8 regeneration/assay cycles without discernible signal decrease), and selective (with sequence specificity down to a single nucleotide mismatch). The low detection levels (10 fM or 0.1 attomoles of miRNA in a 10 µL solution) allow the direct quantification of miRNA-182, a marker correlated to the progression of glioma in patients, to be performed in serum samples without sample pretreatment and RNA extraction and enrichment. The concentration of miRNA-182 in glioma patients was found to be 3.1 times as high as that in healthy persons, a conclusion in excellent agreement with a separate qPCR measurement of the expression level. The obviations of the requirement of an internal reference in qPCR, simplicity, and cost-effectiveness are other additional advantages of this method for detection of nucleic acids in clinical samples.

Impact of incompatible killer cell immunoglobulin-like receptor and its ligand on the outcome of haploidentical bone marrow transplantation.

The purpose of study was to investigate the impact of killer immunoglobulin-like receptor (KIR) and its ligand on haploidentical bone marrow transplantation. 74 cases were analyzed for the distribution frequencies and characteristics of KIR and its ligand as well as the impact of KIR ligand for the haploidentical bone marrow transplantation in terms of the overall survival, disease-free survival (DFS), GVHD and relapse. The results showed that among the 19 KIR genotypes currently nominated KIR2DL1, KIR2DL4 and KIR3DL2-3 could be detected in all the cases. Other high frequency genotypes included KIR3DP1 (98.6%), KIR2DP1 (98.6%), KIR3DL1 (97.3%) and KIR2DL3 (97.3%). Inhibitory receptor genotypes were 1.37-fold of activating receptor genotypes. KIR2DL1, KIR3DL2, KIR3DL3 and KIR2DL4 were found in all haplotypes and at least one genotype of KIR2DL2 and/or KIR2DL3 existed in all haplotypes. Among the 14 genotypes found in the test, the HLA-Cw7 was the most popular (37.8%) and the group 2 (HLA-Cw1, 3, 7, 8, 13, 14) recognized by KIR2DL2/2DL3 counted for 43.2%. The incompatibility of KIR for 32 cases of haploidentical BMT was 43.8%, of which 9/14 were KIR2DL incompatible, 5/14 were KIR2DL2 or KIR3DL1 incompatible. Among the 46 cases of haploidentical BMT, 29 cases were HLA-Cw matched and 14 cases were mismatched. The completed mismatch ratio of HLA-Cw was 30.4% and the match ratio was 63.4%. The survival rate was higher for the 14 cases of KIR genotype compatible group than the 13 cases of KIR genotype incompatible group (p = 0.032). The disease-free survival was significantly higher for the 17 cases of mismatched KIR ligands (HLA-Cw) group than the matched group (p = 0.024). The survival rate was higher in GVHD group than that in non-GVHD group when the KIR ligand was missing. The acute and severe GVHD was related to the existence of activating receptor of KIR2DS1/2DS2. The incompatibility group was accompanied with frequent acute and severe GVHD and less relapse and vice versa for the compatibility group. One patient died after BMT among the 14 mismatched KIR ligand group suffering from myelogenous leukemia while 4 patients out of 12 patients died in the matched group. It is concluded that the haploidentical BMT is characterized by mismatch between donor and recipient and its immunological reactions also features by the incompatibility of KIR genotype and missing ligand. The missing ligand for the donor KIR has strong effect on the outcome of BMT and it means a lot to analyze the KIR genotype and its ligand for the selection of best donor and prognostic evaluation in haploidentical BMT.

Expression of killer cell inhibitor receptors on immunocompetent cells with relation to graft-versus-host disease after hematopoietic stem cell transplantation.

The study was aimed at the exploration of relationship between T cells expressing killer cell inhibitor receptors (KIR, CD158 and CD94) and graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation. The expression rates of CD158a, CD158b and CD94 on T cells and NK cell were detected by flow cytometry and donor/recipient HLA-Cw was analyzed using PCR after peripheral blood stem cell transplantation (PBSCT) and umbilical cord blood transplantation (UCBT). After both PBSCT and UCBT, the rates of CD3(+)CD158a(+) and CD3(+)CD158b(+) T cells increased, especially the rate of CD8(+)CD158b(+) T cells. In both acute and chronic GVHD groups, the rate of CD3(+)CD158b(+) T cells increased, especially in acute GVHD. The CD94 mainly expressed on CD3(+)CD8(+) T cells. The percentage of the expression of CD94 on CD4(+) and CD8(+) cells after UCBT and PBSCT increased significantly. The expression of KIR in GVHD (early stage of transplantation) increased but the expression of KIR in chronic GVHD (advanced stage of transplantation) decreased. Five patients who HLA-Cw matched had no severe GVHD. In four patients who underwent allo-PBSCT and UCBT from related HLA-matched donors, only 2 patients had no aGVHD. Four patients underwent transplantation from unrelated HLA-matched donors had GVHD. These observations suggested that there is some relationship between GVHD and KIR expression on T cells. CD158b might be an inhibitory molecule of T cell activated at early stage after transplantation. Understanding the mechanism of GVHD with the expression of KIR on T cells, especially those binding the HLA-Cw might shed light on the establishment of the specific immunotolerance for the prevention of GVHD. To pay attention to HLA-Cw typing is very important to reduce GVHD and increase GVL effect in related or unrelated HLA-matched transplantation.

[Selection of donor in mismatched hematopoietic stem cell transplantation by CREG, residue match and HLA three-dimensional structure].

After search at hematopoietic stem cell banks for transplant donors, there may be several donors matched with given standards. To determine the most appropriate donor for a specific patient, the potential donors were analyzed and compared by three methods. The first is cross-reactive group (CREG) antigens, which defined as the public antigens that shared specific serological reaction patterns. The second is residue match theory, which concerned the three residues oriented upward toward the T-cell receptor. The third is comparing HLA three-dimensional structure models. The results of the three methods were not completely accorded in our case. However, some less matched donors could be excluded from the candidates and the range of selection was further reduced. It is concluded that combined application of three methods would contribute in selecting donor for hematopoietic stem cell transplantation in clinics.

[A New Criterion for Donor and Recipient Selection in Hematopoietic Stem Cell Transplantation - the Matching of Three-Dimensional Structure of HLA Molecular Modeling]

The purpose of the research is to provide a new standard for matching of HLA three-dimensional structure, and summarize the major permissible mismatch and immunogenic mismatch antigens. The molecular modeling method was used to create HLA molecular structures by Swiss Model Server, and the comparison of the differences among the alleles was done by SPDV software with the function of iterative magic fit. The results were recorded by relative mean square deviation (RMSD, nm). The differences among alleles were scattered below 0.06 nm for HLA-A and -B molecules, and below 0.03 nm for HLA-DRB1 molecules. On the basis of the statistical analysis, when RMSD is greater than 0.04 nm for -A and -B molecules and 0.02 nm for -DRB1 molecules, the difference is meaningful and can be related with graft versus host disease. When RMSD is lower than 0.02 nm for -A and -B molecules and 0.01 nm for -DRB1 molecules, the difference is decided unmeaningful. From the data, the permissible mismatch and immunogenic mismatch alleles within HLA-A, HLA-B and HLA-DRB1 molecules were summarized. Three-dimensional structure matching is a new area in the transplantation field, much research should be done in the future.