Objective To establish a rapid and convenient assay to screen for emerging ganciclovir(GCV) resistant mutations in the UL97 gene of human cytomegalovirus (HCMV) from transplant recipients. Methods Thirteen spefcific oligonucleotide probes were designed according to the target sequence encoding HCMV-UL97 gene. A nested polymerase chain reaction (PCR) amplifying UL97 gene was employed. Line probes assay (LiPA) was configured to detect relevant non-synonymous mutations at codon 460,520,594,595,603, and 607. In parallel, nested-PCR amplicons were subsequently sequenced directly. Results Four of 16 heart and lung transplant recipients had been infected with HCMV resistant strains during GCV therapy. The mutant codons of UL97 gene were at codon 520、595 and 603. All amplicons detected by LiPA were fully concordant with the nucleotide sequenced.Conclusions Long-term prophylaxis and treatment with GCV in heart and lung transplant recipients may develop HCMV resistant strains. HCMV-LiPA proves to be an alternative method for the resistance genotype analysis of HCMV.

Objective To investigate the biological effects of exosomes secreted by KYSE410 cells on migration and invasion of KYSE410,KYSE510,YES2 cells and the possible mechanisms underlying the phenotype change.Methods The exosomes were isolated from the conditional supernatant of esophageal cancer cell line KYSE410 by ultracentrifugation.The morphology of exosomes was observed by transmission electron microscopy (TEM).Western blotting was used to detect the protein markers of exosomes.The uptaken of fluorescence-labeled KYSE410 exosomes by KYSE410,KYSE510 and YES2 was also recorded under confocal microscopy.Migration and invasion ability of the three esophageal carcinoma cell lines and the effects of exosomes from KYSE410 on migration and invasion of KYSE410,KYSE510 and YES2 cells were analyzed by Transwell chamber,respectively.The alteration of Wnt/β-catenin and PI3K/Akt pathway-related proteins were detected by Western blotting.Results The membrane structure of KYSE410 derived exosomes could be observed with its diameter ranged between 30-100nm.The invasion and migration ability of three esophageal cancer cells are KYSE410＞ KYSE510＞ YES2.KYSE410 exosomes promoted the migration and invasion of KYSE410,KYSE510 and YES2 cells.Conclusions Concentrated exosomes derived from the highly migratory and invasive esophageal cancer cell line KYSE410 promoted the migration and invasion potentials of itself and esophageal cancer cell lines KYSE510 and YES2,which possibly exerted the effects by activating Wnt/β-catenin and PI3K/Akt signaling pathways.

To investigate the cardioprotective effect of formononetin (FMN) on no-reflow (NR) after myocardial ischemia-reperfusion and its molecular mechanism based on integrated pharmacology and experimental verification, firstly, human breast cancer MCF-7 cells and myocardial NR rats were used to confirm the estrogenic activity and the effect of alleviating NR of FMN, respectively. Male SD rats were divided into Sham, NR, FMN (20 mg·kg^-1) and sodium nitroprusside (SNP, 5.0 mg·kg^-1) groups, which were administered once a day for one week, the experiment was approved by the Ethics Committee of Tianjin University of Traditional Chinese Medicine (TCM-LAEC2019095). The pharmacological analysis and in vivo study of NR rats were integrated to reveal the mechanism of FMN improving NR. The results showed that FMN had estrogenic effect and reduced NR by improving cardiac structure and function, reducing NR, ischemic myocardial area and pathological injury of cardiomyocytes. Integrated pharmacology predicts that the mechanism of FMN improving NR is mainly related to phosphatidyinositol-3-kinase-protein kinase B (PI3K-Akt) signal pathway. Phytoestrogens play a role in cardiovascular protection mainly by activating G protein-coupled estrogen receptor (GPER). GPER is also an important regulator in the upstream of PI3K-Akt signaling pathway. This study found that FMN can significantly activate GPER, p-PI3K, p-Akt and phospho-endothelial nitric oxide synthase (p-eNOS). It has good binding ability with GPER and eNOS protein. In this study, through the integration of pharmacology and experimental evaluation, it is revealed that FMN activates PI3K/Akt/eNOS signal pathway by activating GPER, thus significantly improving NR.

BACKGROUND: Previous research has demonstrated that dermal tissue has mesenchymal stem cells, which have a possibility of autologous transplantation. If the mesenchymal stem cells derived from the skin differentiate into lymphocytes under a certain condition, the immune system disease can be solved generally.OBJECTIVE: To investigate the possibility of differentiation of human skin-derived mesenchymal stem cells into lymphocytes. METHODS: Surface marker expression was detected in the 14th passage human skin-derived mesenchymal stem cells using flow cytometry. Transdifferentiation medium of human skin-derived mesenchymal stem cells consisted of human lymphocyte supernatant and fresh human skin-derived mesenchymal stem cells based on the ratio of 7:3. Inverted microscope was employed to observe morphological changes, and flow cytometry was used to detect surface marker expression in the lymphocytes at 1-8 days after induction. Self-marker expression of human skin-derived mesenchymal stem cells was then detected at 3,6, and 9 days after induction.RESULTS AND CONCLUSION: Human skin-derived mesenchymal stem cells stably expressed self-specific marker CD73, Vimentin and so on, but did not express specific markers of hematopoietic system, I.e., CD34, CD45 and so on, lowly expressed HLA-I, but did not express HLA-DR at all. At 3 days after induction, the cell volume significantly increased, cell proliferation rate was significantly lower than before induction, and a lot of cystic-like particles with strong refraction were observed in or between cells. The CD45 lymphocyte expression was not significantly changed, but CD3, CD19, CD16, CD4, and CD8 expression rates of human skin-derived mesenchymal stem cells were linearly increased at 1-4 days after induction and stabilized at 5-8 days after induction. In addition, CD37, CD34, Vimentin, and HLA-DR expressions were not changed at 3, 6, and 9 days after induction, but HLA-I expression rate was gradually increased with the prolongation time of induction. This suggested that human skin-derived mesenchymal stem cells can differentiate into lymphocyte and potentially participate in repairing immune system injury.

OBJECTIVETo study the characteristics histologic and cytologic features and clinical usefulness of plasma cell myeloma (PCM) subtyping according to WHO PCM classification.

METHODSBone marrow biopsy plastic-embedded sections were stained with H-G-E and Gomori's stains, and bone marrow aspirate smears were stained with Wright's stain. The clinicopathologic findings were then analyzed.

RESULTSOf the 131 cases with PCM, three types of growth patterns were noted: interstitial (21 cases, 16.0%), nodular (46 cases, 35.1%) and packed (64 cases, 48.9%). Besides, there were three cytologic subtypes: mature plasma cell type (43 cases, 32.8%), immature (81 cases, 61.8%) and pleomorphic (7 cases, 5.3%) types. The age of patients with mature plasma cell type was significantly higher than that of immature type (P = 0.005); and the number of tumour cells in bone marrow smears was significantly higher than that of immature type (P = 0.003). The numbers of WBC and platelets in peripheral blood were also significantly higher than that of pleomorphic type (P = 0.024, P = 0.002, respectively). On the other hand, the number of platelets in peripheral blood of immature type was significantly higher than that of pleomorphic type (P = 0.019). Marrow fibrosis was more frequently observed in immature type than in mature plasma cell type (P = 0.000). The incidence of marrow fibrosis and osteolytic lesions was higher in high risk group than in low risk group (P = 0.000, P = 0.023 respectively). Twenty-one cases (56.8%) of the 37 cases treated with MP or MP and M2 chemotherapeutic regimens showed good response. However, there was no significant difference in treatment response and survival between different subtypes.

CONCLUSIONSEach subtype of PCM carries different clinicopathologic features in some aspects. The classification carries important value in pathologic diagnosis and probably in predicting prognosis.

Adult ; Aged ; Aged, 80 and over ; Biopsy ; Bone Marrow Examination ; Female ; Humans ; Immunophenotyping ; Male ; Middle Aged ; Multiple Myeloma ; classification ; immunology ; pathology ; Prognosis

The aim of this study was to investigate the clinical characteristics and prognosis of acute erythroleukemia (AEL, AML-M6). The clinical features and results of morphologic, immunophenotypic, cytogenetic and molecular biologic detections were retrospectively analyzed in 13 cases of AEL from 305 acute leukemia patients hospitalized between October 2007 and October 2012. The results showed that the expression of erythroid and non-erythroid cells increased at the same time. The myeloid antigens mainly expressed CD13/CD33/CD117/CD34, while the erythroid antigens expressed Gly and CD71. The karyotypic detection indicated that there were 1 case with normal karyotype, 3 cases with simple karyotypic abnormality and 2 cases with complex karyotypic abnormality, the other cases were not detected. The molecular biological detection found that the poor prognosis gene existed in 5 cases [38.5% (5/13)], including 3 cases with MLL-MLL fusion gene, 1 case with MLL mutation, and 1 cases with NRAS gene mutation, the abnormal genes were not detected in remainder 8 cases. After chemotherapy with decitabine, the complete remission (CR) rate achieved 53.5% (7/13), partial remission (DR) rate achieved 15.4% (2/13). Finally, 8 patients received allo-HSCT, the median overall survival (OS) was 20.7 months, 3 year survival rate was 79%, 3 year disease-free survival rate was 78%. It is concluded that the acute erythroleukemia is a rare subtype of AML, which is transformed from MDS and has harmful genes and poor prognosis. Allo-HSCT and treatment with decitabine may enhance the survival rate of AEL.
Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Female ; Humans ; Karyotyping ; Leukemia, Erythroblastic, Acute ; diagnosis ; genetics ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Survival Rate ; Young Adult

OBJECTIVETo investigate whether the p38 mitogen-activated protein kinase (MAPK) signaling pathway mediates advanced oxidation protein products (AOPPs)-induced epithelial-to-mesenchymal transition (EMT) in tubular cells.

METHODSHuman proximal tubular cells (HK-2 cells) exposed to AOPP-bovine serum albumin (BSA) were examined for expressions of p38 MAPK and phosphorylated p38 MAPK using Western blotting. Western blotting and quantitative RT-PCR were used to examine the protein and mRNA expressions of EMT markers E-cadherin and vimentin and endoplasmic reticulum stress marker glucose-regulated protein (GRP) 78 in cells treated with SB203580 (an inhibitor of the p38 MAPK signaling pathway) prior to AOPP exposure. The cells treated with AOPPs following pretreatment with salubrinal (an inhibitor of endoplasmic reticulum stress) were also examined for expressions of p38 MAPK and phosphorylated p38 MAPK.

RESULTSAOPP treatment induced the phosphorylation of p38 MAPK in HK-2 cells. AOPP-induced decrease in E-cadherin expression and overexpression of vimentin and GRP78 were partly inhibited by pretreatment of the cells with SB203580. Salubrina partly suppressed AOPP-induced phosphorylation of p38 MAPK in the cells.

CONCLUSIONp38 MAPK signaling pathway, which is regulated by endoplasmic reticulum stress, might mediate AOPP-induced EMT in HK-2 cells.

OBJECTIVEThe severe acute respiratory syndrome (SARS) is a highly contagious infection caused by a newly discovered strain of coronavirus (SARS-CoV). During the outbreak of SARS in the first half of 2003, children appeared to be less susceptible to the SARS coronavirus and pediatric patients presented with a less aggressive clinical course than adult patients did, demonstrating the traits which were rarely observed in other viral contagious disease. The present study aimed to preliminarily examine the presence of serum specific antibodies against severe acute respiratory syndrome (SARS)-associated coronavirus virus (SARS-CoV) in pediatric SARS patients and explore the possibility of subclinical infection in children/adults through close association with SARS cases.

METHODS(1) Clinicians and nurses visited families and collected general and epidemiological information about the subjects using a standard questionnaire and took serum specimens. (2) Specific antibodies against SARS-CoV were assayed with two methods, indirect immunofluorescence assay (IFA) for detecting IgG antibodies and enzyme linked immunosorbent assay (ELISA) for mixed antibodies. Serum specimens tested included those from 21 clinically confirmed pediatric SARS cases (aged from 8 months to 14 years, 11 male and 10 female) and their 23 parents who had close contact with the children, 36 adult patients in convalescence stage of SARS, 24 children (aged 1.5 to 14 years) and other 34 adults who had close contact with infected adults.

RESULTS(1) The positive rates of specific IgG and mixed antibodies against SARS-CoV were 38% (8/21) and 33% (7/21) in pediatric cases; whereas the rates were 75% (27/36) and 69% (25/36) in adult patients. (2) The proportion of the patients who had close contact to SARS patients was 7/8 among the antibody-positive group vs. 1/13 for the antibody-negative group (P < 0.05). (3) The IgG antibody emerged in one of 24 children, whose mother, a nurse, had suffered from SARS (4%). (4) Among 23 parents of children with SARS, one was positive for IgG and the mixed antibodies, whose grandson and husband suffered from SARS; The IgG antibody and the mixed antibodies were also positive in another adult who had close contact with adult SARS cases (3%).

CONCLUSIONS(1) SARS-CoV infection was confirmed by serological methods in 38.1% of clinically diagnosed pediatric SARS cases, which leads to the assumption that correct diagnosis of pediatric SARS requires more accurate and efficient ways, for example, screening for antigen or gene of SARS-CoV. (2) The proportion of the patients who had close contact to SARS patients among antibody-positive cases was higher than that in antibody-negative cases. (3) It is possible that subclinical SARS CoV infection exists in children and adults, although the rate of occurrence is low. The data of the present study did not confirm that SARS had subclinical infection among adults who had close contact to pediatric SARS cases.

Adolescent ; Adult ; Antibodies, Viral ; blood ; immunology ; Child ; Child, Preschool ; Enzyme-Linked Immunosorbent Assay ; Female ; Fluorescent Antibody Technique, Indirect ; Humans ; Immunoglobulin G ; blood ; immunology ; Infant ; Male ; SARS Virus ; immunology ; Severe Acute Respiratory Syndrome ; blood ; epidemiology ; immunology

This study was aimed to investigate the incidence of thrombosis in patients with primary thrombocytosis (PT) and its correlation with function changes of platelets, and to explore the effect of thromboxane A2 (TXA2) inhibitor-ozagrel sodium on platelet activity and its efficacy for prevention and treatment of thrombosis. The CD62P and PAC-1 levels on platelet surface were detected by flow cytometry; the levels of TXB2 (metabolic product of TXA2) and 6-keto-PGFIalpha (metabolic product of prostacyclin) were detected by FLISA. The function change of platelets and its correlation with thrombosis were observed and compared in PT patients with and without thrombosis. The results indicated that the TXB2, PAC-1 and CD62P level, and TXB2/6-keto-PGF1alpha ratio in PT patients with thrombosis were higher than those in PT patients without thrombosis before treatment with ozagrel sodium (p<0.01). After treatment with ozagrel sodium, the function indexes of platelets such as CD62P, PAC-1, TXB2 and TXB2/6-keto-PGF1alpha except 6-keto-PGF1alpha in PT patients with and without thrombosis decreased obviously (p<0.01), but there was no significant difference in TXB2, 6-keto-PGF1alpha and TXB2/6-keto-PGF1alpha levels between PT patients with and without thrombosis except CD62P and PAC-1. It is concluded that the multi-index of platelets in PT patients with thrombosis are higher than that in PT patients without thrombosis, the activation of platelet function is a high risk factor for thrombosis of PT patients. The ozagrel sodium can obviously reduce the platelet activation, decrease the production of TXA2 and ameliorate the TXB2/6-keto-PGF1alpha ratio. The ozagrel sodium not only possesses therapeutic effect, but also preventive efficacy for thrombosis.
Adolescent ; Adult ; Aged ; Female ; Fibrinolytic Agents ; therapeutic use ; Humans ; Male ; Methacrylates ; therapeutic use ; Middle Aged ; Thrombocythemia, Essential ; complications ; drug therapy ; Thrombosis ; complications ; drug therapy ; Young Adult

BACKGROUNDSevoflurane preconditioning (SP) has been shown to invoke potent myocardial protection in animal studies and clinical trials. However, the mechanisms underlying SP are complex and not yet well understood. We investigated the hypothesis that the cardioprotection afforded by SP is mediated via the Wnt/glycogen synthase kinase 3β (GSK3β)/β-catenin signaling pathway.

METHODSTwo models were established: a Langendorff perfused rat heart model and the H9C2 cell hypoxia/reoxygenation model. Both rats and H9C2 cells were randomly divided into 6 groups as follows: S group, ischemia-reperfusion (I/R) group, DMSO group, IWP group, SP group, and SP + IWP group. Hemodynamic parameters, lactate dehydrogenase (LDH) activity in coronary effluent and cell culture supernatant, and the infarct size were measured to evaluate myocardial ischemia-reperfusion injuries. To determine the activity of Wnt/GSK3β/β-catenin signaling pathway, the expressions of Wnt3a, phospho-GSK3β, and β-catenin were measured by Western blotting.

RESULTSSP improved cardiac function recovery, reduced infarct size (18 ± 2% in the SP group compared with 35 ± 4% in the I/R group; P < 0.05), decreased LDH activity in coronary effluent, and culture supernatant. IWP-2, an inhibitor of Wnt, abolished the cardioprotection by SP. In addition, Western blotting analysis demonstrated that the expressions of Wnt3a, phospho-GSK3β, and β-catenin significantly (P < 0.05) increased in the I/R group, compared with the S group; and compared to I/R group, SP significantly (P < 0.05) increased Wnt3a, phospho-GSK3β, and β-catenin expressions. Pretreatment with IWP-2 significantly (P < 0.05) abolished SP-induced Wnt/GSK3β/β-catenin signaling activation.

CONCLUSIONSThe results showed for thefirst time that cardioprotection afforded by SP may be mediated partly via the Wnt/GSK3β/β-catenin signaling pathway.

Animals ; Cell Hypoxia ; drug effects ; Cell Line ; Glycogen Synthase Kinase 3 ; genetics ; metabolism ; Glycogen Synthase Kinase 3 beta ; Hemodynamics ; drug effects ; Male ; Methyl Ethers ; therapeutic use ; Myocardial Reperfusion Injury ; drug therapy ; prevention & control ; Rats ; Rats, Wistar ; Wnt Signaling Pathway ; drug effects ; genetics ; beta Catenin ; genetics ; metabolism