The Relationship between the Expressions of CD33 and CD13 and the Prognosis of Patients with Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To investigate the expressions of CD33 and CD13 in newly diagnosed multiple myeloma (MM) patients and its relationship with prognosis.@*METHODS@#It was retrospectively observed that the expression of CD33 and CD13 in 121 MM patients who were newly diagnosed from January 2014 to January 2020, and the relationship between the expressions of CD33 and CD13 and patients prognosis was analyzed.@*RESULTS@#Among the 121 newly diagnosed MM patients, there were 30 patients (24.8%) in the CD33+ group and 12 patients (9.9%) in the CD13+ group. Kaplan-Meier analysis showed that, compared with the CD33- group, the progression-free survival (PFS) time and overall survival (OS) time were significantly shortened in MM patients in CD33+ group (PFS 17.5 vs 23 months, P=0.000; OS 18.5 vs 25 months, P=0.000); and the PFS time and OS time of MM patients in the CD13+ group were also significantly shortened than those in CD13- group (PFS 21 vs 22 months, P=0.012; OS 25 vs 26 months, P=0.006). Cox regression analysis showed that CD33 and CD13 were independent adverse prognostic factors in MM patients (CD33: P=0.000;CD13: P=0.003).@*CONCLUSION@#CD33 and CD13 are prognostic risk factors in patients with MM.

Interaction of Bombyx mori aminopeptidase N and cadherin-like protein with Bacillus thuringiensis Cry1Ac toxin / 生物工程学报

Bacillus thuringiensis (Bt) produces Cry toxins that are widely used as insecticides in agriculture and forestry. Receptors are important to elucidate the mode of interaction with Cry toxins and toxicity in lepidopteran insects. Here, we purified the Cry toxin from Bt and identified this toxin by flight mass spectrometry as Cry1Ac, and then recombinantly expressed aminopeptidase N (BmAPN6) and repeat domains of cadherin-like protein (CaLP) of B. mori. Using co-immunoprecipitation (co-IP), Far-Western blotting, and enzyme-linked immunosorbent assays (ELISAs), we identified the interaction between Cry1Ac and BmAPN6. Furthermore, analysis of the cytotoxic activity of Cry1Ac toxin in Sf9 cells showed that BmAPN6 directly interacted with Cry1Ac toxin to induce morphological aberrations and cell lysis. We also used co-IP, Far-Western blotting and ELISAs to analyze the interactions of Cry1Ac with three binding sites corresponding to cadherin repeat (CR) 7 CR11, and CR12 of CaLP. Notably, the three repeat domains were essential Cry1Ac binding components in CaLP. These results indicated that BmAPN6 and CaLP served as a functional receptor involved in Bt Cry1Ac toxin pathogenicity. These findings represent an important advancement in our understanding of the mechanisms of Cry1Ac toxicity and provide promising candidate targets for gene editing to enhance resistance to pathogens and increase the economic value of B. mori.

Mechanisms for effect of osthole on inhibiting the growth and invasion of bladder cancer cells / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of osthole on epidermal growth factor receptor tyrosine kinase (EGFR-TPK), matrix-metalloproteinase-2 (MMP-2), aminopeptidase N (APN) in bladder cancer cell and the underlying mechanism.
@*METHODS@#The T24 cell lines were cultured. The inhibitory effects of osthole on EGFR-TPK, APN and MMP-2 were evaluated by spectrophotometric and MTT assay. The caspase-3 activity and the expression COX-2 and VEGF in T24 were examined. The activity of NF-κB was determined by electrophoretic mobility shift assay.
@*RESULTS@#The half inhibition concentrations (IC50) of osthole on EGFR-TPK, APN and MMP-2 were (45.33±3.98), (28.21±3.23) and (8.11±0.54) µmol/L, respectively. The growth inhibitory rates for T24 cells were increased in a dose-dependent manner (P<0.05). The caspase-3 activities were significantly increased in T24 cells in the osthole group compared with control group, while the expression of angiogenesis related-protein COX-2, VEGF, and NF-κB in T24 cells were decreased.
@*CONCLUSION@#Through the inhibitory effect on EGFR-TPK, APN and MMP-2, osthole can decrease COX-2, VEGF and NF-κB expression while increase the activity of caspase-3, eventually blocking the growth and invasion of bladder cancer cell.

Effect of downregulation the expression of HDAC1 on cells differentiation of HL-60 cells / 药学学报

This study is to investigate the effect of downregulation histone deacetylases 1 (HDAC1) gene by the technology of RNA interference on the differentiation of HL-60 cells line. The optimal segment targeting HDAC1 gene was designed and transfected into HL-60 cells by Lipofectamine 2000. The HDAC1 mRNA and protein level were detected by RT-PCR and Western blotting. The morphologic change of HL-60 cells was detected by an optical microscope with Wright-Giemsa. Cell differentiation was tested by NBT reduction assay. Expression of CD13, CD33 and CD14 was measured by flow cytometry. The results indicated that HDAC1 mRNA and protein were markedly suppressed by the siRNA targeting HDAC1 in a concentration-dependent manner. HDAC1 siRNA promoted cell differentiation. HL-60 cells became more mature in morphology after transfected to HDAC1 siRNA at a concentration of 30-60 nmol x L(-1) for 24 h. NBT reduction ability of HDAC1 siRNA with 30 nmol x L(-1) was 0.31 +/- 0.09, compared with negative control (0.20 +/- 0.02) (t = -3.1, P < 0.01), and with 60 nmol x L(-1) was 0.25 +/- 0.02 in comparison with negative control (0.21 +/- 0.04) (t = -2.12, P < 0.05). But it has no change in HDAC1 siRNA > or = 120 nmol x L(-1). After transfection with 60 nmol x L(-1) HDAC1 siRNA to HL-60 cells, the expression of CD13 was (96.50 +/- 0.70)% in compared to siRNA-NC (3.39 +/- 0.68) % (t = 164.9, P < 0.000 5), CD33 was (66.73 +/- 0.50) % in compared to siRNA-NC (96.80 +/- 1.70) % (t = 43.4, P < 0.000 5). CD14 was (0.53 +/- 0.00) % by comparison with siRNA-NC (0.49 +/- 0.02) % (t = -0.97, P > 0.1). HDAC1 siRNA promoted cell differentiation in indicated concentration. HDAC1 might be one of the targets of gene therapy for leukemia.

Advance in studies on NGR peptide modified liposome and its anti-tumor performance / 中国中药杂志

Aspargine-glycine-arginine (NGR)-containing peptides are targeted peptides which can be integrated with CD13 receptors on tumor vascular endothelial cells. NGR peptides are connected to liposomes to obtain NGR peptide-modified liposomes. By intravenous injection of these liposomes, NGR peptides can be combined with CD13 receptors on tumor vascular endothelial cells, position liposomes in tumor tissues, and concentrate drug in liposomes in tumor, so as to enhance the antitumor effect. The article starts with NGR peptides, summarizes definition of NGR, NGR peptide-modified liposomes, strengths and weaknesses of NGR peptide-modified liposomes in antitumor and the latest study orientation of NGR peptide-modified liposomes, and looks into the future of studies on NGR peptide-modified liposomes.

Inhibition of tumor cell invasion and induction of apoptosis by ubenimex / 药学学报

This study is to investigate the effects of ubenimex on tumor cell invasion and apoptosis, dose relationship and mechanism. Immunofluorescence staining was performed to detect the expression of CD13 in HT-1080 cells. MTT assay was used to analyze the effect of ubenimex on cell proliferation. Annexin V-EGFP/PI was used to detect apoptotic cells by flow cytometry. Cell cycle was analyzed using flow cytometry. Ala-pNA was used as substrate to evaluate the effect of ubenimex on the aminopeptidase activity. Transwell assay was used to analyze the effect of ubenimex on cell invasion and migration ability. Western blotting was used to detect the expression level of CD13. MMP activity was analyzed using gelatin zymography. The results showed that ubenimex at high concentration inhibited the proliferation of HT-1080 cells (IC50: 3.8 mg x mL(-1)), and induced cell apoptosis. Cell cycle was blocked at G1 phase. Ubenimex at low concentration inhibited the aminopeptidase activity of HT-1080 cells (IC50: 8.3 microg x mL(-1)) and inhibited cell invasion, but it had no effects on the cell migration and proliferation. Ubenimex had no effects on CD13 expression and MMP activity. In conclusion, ubenimex at low concentration can inhibit the invasion ability of tumor cells by directly inhibiting the aminopeptidase activity; ubenimex at high concentration can inhibit the proliferation of tumor cells and induce cell apoptosis by a CD13-independent pathway.

Establishment and characterization of lung adenocarcinoma cell line XLA-07 / 中华病理学杂志

<p><b>OBJECTIVE</b>To establish and characterize a lung adenocarcinoma cell line from a female patient in Xuanwei, Yunnan province.</p><p><b>METHODS</b>Surgical specimen of the lung adenocarcinoma was obtained and cultured immediately in RPMI 1640 medium with 10% fetal bovine serum and 10(5) U/L penicillin and 100 mg/L streptomycin. When stable proliferation of the cells was achieved after over 40 passages in culture, the biological features of the cell line were investigated by cell morphology, karyotyping, protein marker expression [cytokeratins (CKs), epithelial membrane antigen (EMA) and CD proteins], growth kinetics, cell cycle phase distribution, mitotic index, colony formation in soft agar, cell invasion and tumorigenicity in Balb/c nude mice.</p><p><b>RESULTS</b>The established cell line was stably cultured for over 80 passages during a one-year period as an anchorage-dependent monolayer of short spindle, polygonal to epithelioid cells under phase contrast microscope. Microglandular cavities and disordered microfilaments were observed under transmission electron microscope. The growth curve presented in an "S" shape with the cell population doubled every 46.7 hours. The mitotic index was 1.5% and the colony formation rate was 8.3%. The cell cycle distribution included 76.9% in G(0)/G(1), 15.1% in S and 8.0% in G(2)/M. The cell line displayed a hypotriploid karyotype with a mode of 66 chromosomes and a median of 64 chromosomes. The cells expressed CK7, CK8, CK (Pan) and EMA by immunohistochemistry. A high level of cell surface expression of CD13 and CD59 was evident by flow cytometry. The cells were able to penetrate Matrigel in vitro but failed to form a stable xenograft in nude mice.</p><p><b>CONCLUSION</b>A new human lung adenocarcinoma cell line, designated as XLA-07, is successfully established from a Xuanwei lung cancer patient.</p>

influence of Bcl-2 and myeloid antigen expression on response to therapy in childhood acute lymphoblastic leukemia

The possible prognostic significance of the expression of a variety of markers has been investigated in acute lymphoblastic leukemia [ALL]. In the present study we investigated the prognostic significance of CD13 and CD33 myeloid antigens [MY] aberrantly expressed on the blasts of ALL patients and Bcl-2 anti-apoptotic molecule expression in childhood ALL. Aberrant expression of MY occurred in 8.8% of cases. Variant levels of Bcl-2 were expressed in patients [44.2 +/- 25.5%], with more than 20% positivity for Bcl-2 in 64.7% of patients. Bcl-2[+] patients survived 959 +/- 242 days compared to 1059 + 230 days for Bcl-2 patients [P=0.2]. Corresponding data for complete remission duration was 682 +/- 170 and 716 +/- 173 days [P=0.3], respectively, indicating no significant association between survival and complete remission duration of patients with expression of the Bcl-2 molecule. Analysis of clinical response according to MY expression, however, showed significant association with survival and complete remission duration. MY[+] patients had shorter complete remission duration [383 +/- 58 days] and survival [473 +/- 68 days] than MY[-] patients [complete remission duration, 724 +/- 144 days; survival, 1045 +/- 186 days; P<0.001]. Expression of Bcl-2 along with MY was not associated with a significant decrease in survival or complete remission duration. Results of this study indicated that expression of MY was a poor prognostic factor in childhood ALL. Bcl-2 expression in MY[+] patients could not influence the response to therapy

Characteristics of T-cell lineage immunophenotype in 95 patients with acute lymphoid leukemia / 中国实验血液学杂志

The objective of this study was to investigate the immunophenotype of T-lineage acute lymphoid leukemia (T-ALL) and to find valuable immunologic markers in T-ALL diagnosis and therapy. Four-color multiparametric flow cytometry(FCM) with CD45/SSC gating was used for immunophenotyping of 95 patients with newly diagnosed T-ALL. The results demonstrated that T-ALL occurred more frequently in males younger than 30 years of age and was usually accompanied by a high WBC count and tumor mass at diagnosis. Univariate analysis showed an influence on achievement of CR1 for age (< 30 years) but not for WBC count and tumor mass. According to WHO (2008) classification of tumors of haematopoietic and lymphoid tissues, 87 patients with confirmed subtype included 27 cases of Pro-T-ALL (31.0%), 31 cases of Pre-T-ALL (35.6%), 23 cases of cortical-T-ALL (26.4%), 6 cases of medullary-T-ALL (6.9%). CD34 expression in Pro-T-ALL was significantly higher than that of Pre-T-ALL (p = 0.001). After the first chemotherapy, the complete remission rate in Pro-T-ALL was statistically lower than that of Pre-T-ALL. Besides, the complete remission rate of immature T-ALL (including Pro-T-ALL and Pre-T-ALL) was also significantly lower than that in mature T-ALL (including cortical-T-ALL and medullary-T-ALL). Myeloid antigen (CD13, CD33) expression was associated with T-ALL subtype and treatment effect. While 66.7% of CD13(+) patients belonged to Pre-T-ALL, most (60.0%) of CD33(+) patients were classified into Pro-T-ALL; CD13 expression had no effect on CR1 rate whereas CD33(+) patients had worse treatment effect compared with CD33(-) groups (p = 0.001). Notably, the expression of CD117 reached up to 26.7% and the positive cases were primarily distributed in pro-T-TAll and pre-T-ALL. It is found that CD117 expression in CD34(-) group was homogeneous and CD117 expression level was less than 10% in 73.2% patients, but CD117 expression level in CD34(+) group was not homogenous, in which group the CD117 expression levels < 10%, 10% - 20% and > 20% were 44.2%, 17.3% and 38.5% respectively. As compared with CD34(-) group, the proportion of patients with CD117 expression levels < 10%, > 20% in CD34(+) group was higher, and there was significant difference between these 2 group. It is concluded that immunophenotype has great value in T-ALL diagnosis, classification as well as treatment. Flow cytometry provides access to find valuable immunologic markers for T-ALL biological research.

Effect of APN/CD13 on bestatin enhancing all-trans-retinoic acid-inducing differentiation in NB4 cells / 中国实验血液学杂志

This study was purposed to investigate the effect of aminopeptidase N/CD13 on bestatin enhancing all-trans-retinoic acid (ATRA)-inducing differentiation in NB4 cells. The nitroblue-tetrazolium (NBT) reduction assay was performed to determine the differentiation of NB4 cells, MR2 cells and primary APL blasts. The expression of P38 MAPK protein and the phosphorylation of P38 MAPK protein in NB4, MR2 and K562 cells were detected by Western blot. The results showed that pre-incubation with 5 µg/ml WM-15 blocked the enhancement effect of bestatin on differentiation of NB4 cells induced by ATRA. 5 µg/ml CD13 antibody WM-15 partly blocked the inhibition of bestatin on the phosphorylation of P38 MAPK in NB4 cells. 100 µg/ml bestatin inhibited the phosphorylation of P38 MAPK in NB4 cells and MR2 cells in a time-dependent manner. 100 µg/ml bestatin had no effect on the phosphorylation of P38 MAPK in K562 cells with low level of CD13. Bestatin could not restore the sensitivity to ATRA in ATRA-resistant primary APL blasts and MR2 cells. It is concluded that aminopeptidase N/CD13 inhibitor bestatin may enhance the differentiation-inducing activity of ATRA through inhibiting the phosphorylation of P38 MAPK in NB4 cells mediated by the cell surface APN/CD13.

Expression and biological function analysis of chicken aminopeptidase N / 生物工程学报

To clone and express the gene encoding chicken aminopeptidase N (chAPN), and analysis the biological function of chAPN expressed in Escherichia coli (E. coli). The chAPN gene was amplified by RT-PCR from the kidney cells of chicken embryo and then cloned into the prokaryotic expression vector pCOLD-TF. Recombinant expression plasmid of pCOLD-TF-chAPN was constructed and then transformed into the competent E. coli BL21(DE3) cells for expression under different conditions such as induction time and inductor concentrations. Purified soluble recombinant chAPN was obtained by Ni-NTA His Bind Resin affinity chromatography and identified by SDS-PAGE gel and Western blotting assay. Its biological function was detected by its reaction with Leu-PNA and Enzyme-Linked Immunosorbent Assay (ELISA). The results showed that the expression product of chAPN gene in E. coli was soluble. It was able to bind infectious bronchitis virus (IBV) dose-dependently. In conclusion, chAPN gene has been successfully cloned and expressed in E. coli, which will establish a basis for further research the enzymatic activity and antiviral function.

Immunophenotyping characteristics of adult patients with acute lymphoblastic leukemia in different ages / 中国实验血液学杂志

The purpose of this study was to investigate the immunophenotyping characteristics of adult acute lymphoblastic leukemia (ALL) patients in groups of different ages. Immunophenotyping was performed in 260 ALL patients by flow cytometry using a panel of monoclonal antibodies and CD45/SSC gating. The results indicated that (1) all the 82 cases of T-cell acute lymphoblastic leukemia (T-ALL) expressed CD7 (100%) while the positive rate of CD2 remarkably decreased with aging. The positive rate of CD2 in patients aged 14 to 18 years (adolescents) was 91.67%, which is significantly higher than that in cases aged 19 to 35 years (young adults) and > 35 years (older adults) (65.71% and 43.48% respectively, p < 0.05); the positive rate of CD34 and HLA-DR increased with aging, there was significant difference of the HLA-DR expression between the older adults group (39.13%) and the other two groups (4.17% in adolescents and 11.43% in young adults respectively (p < 0.05). Moreover, there were significant differences of the myeloid antigen (MyAg) and CD13 expression between the older adults and younger adults (p < 0.05). (2) As to adult B-cell acute lymphoblastic leukemia (B-ALL), the positive rates of CD19 and HLA-DR in 178 cases were 100%; the positive rate of CD33 in young adults was significant higher than that in adolescents (p < 0.05), the differences of the other marker expressions failed to reach statistical significance in adult B-ALL patients. It is concluded that the immunophenotypes of adult T-ALL are evidently heterogeneous in different ages, and expression with more aberrant phenotypes indicates poor prognostic significance in patients older than 35 years. There is no significant association of immunophenotypes with ages among different age groups of adult B-ALL.

A clinical and laboratory study on acute myeloid leukemia with t(6;9)(p23;q34) / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To explore the clinical and laboratory features of 6 cases of acute myeloid leukemia (AML) with t(6;9)(p23;q34).</p><p><b>METHODS</b>Chromosome preparation of bone marrow cells was performed with regular method. R-banding by heating using Giemsa banding technique (RHG) was used for karyotype analysis. The immunoprofile was studied by flow cytometry (FCM) using a panel of monoclonal antibodies. Chromosome painting was performed by using whole chromosome paint probes for chromosomes 6 and 9 in all the 6 cases. The expression of fusion gene DEK/CAN and FLT3-ITD mutation were analyzed by reverse transcription-PCR(RT-PCR).</p><p><b>RESULTS</b>The t(6;9)(p23;q34) was found in all the 6 cases including 4 cases of M2 and 2 cases of M4. Blast cells were positive for CD13 and CD33 in 6 patients, for HLA-DR in 4 patients, for CD34 and CD117 in 3 cases, for CD38 or CD15 each in 1 case, respectively. A reciprocal translocation between chromosome 6 and 9 was confirmed by chromosome painting technique in the 6 cases. The DEK/CAN fusion gene was found in all the 6 cases, FLT3-ITD mutation was detected in three of them. Follow-up showed that 3 patients died with a survival time of 3 months, 5 months and 6 months, respectively. The other three obtained complete remission and are still alive.</p><p><b>CONCLUSION</b>The t(6;9)(p23;q34) is a rare recurrent abnormity. AML with t(6;9)(p23;q34) has unique clinical and laboratory features and its prognosis is poor in most cases.</p>

Screening and identification of CD13-binding peptides with phage display peptide library / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen and identify the peptides that specifically bind to CD13 on monocytes.</p><p><b>METHODS</b>The phages capable of specific binding to CD13 were screened in the phage-displayed 12-peptide library. The affinity of the selected phages with CD13 was verified with enzyme-linked immunosorbent assay (ELISA). The sequences of the peptides bound to the phages were deduced according to the phage DNA sequences, and the functional peptides aligned using the BLASTP on the Website NCBI were synthesized. To analyze the biological function of the screened peptides, the location of the peptides bound to THP-1 cells was detected using immunofluorescence assay. The blocking effect of WM15 on the peptide binding to THP-1 cells was assessed by immunofluorescence assay.</p><p><b>RESULTS</b>The phages that specifically bound to CD13 were effectively enriched to approach saturation after 4 rounds of panning. The recovery rate in the fourth round was 30 times that in the first round. Twenty selected phages were verified by ELISA, and the signals of 10 phages were higher than the control. The sequences of the peptides P9 and P7 showed 83% and 100% identity with those of human cytomegalovirus (HCMV) UL38 and UL105, respectively. The peptides bound to the cell membrane of THP-1 cells as shown by immunofluorescence assay. The binding of the peptides P9 and P7 to THP-1 cells was blocked by CD13-specific monoclonal antibody WM15 at different levels.</p><p><b>CONCLUSION</b>Two peptides (P7 and P9) that can specifically bind to CD13 have been screened successfully, and these two peptides show specific binding to CD13 on the membrane of THP-1 cells.</p>

Immunophenotype characteristics and prognosis of acute leukemia patients with cross expressing lymphoid and myeloid lineage associated antigens / 中国实验血液学杂志

The aim of study was to investigate the immunophenotype characteristics and prognosis of acute leukemia patients with cross-expressing lymphoid and myeloid lineage-associated antigens. The immunophenotypes of leukemic cells were examined by using flow cytometry. All patients were classified into several groups according to FAB subtypes and immunophenotyping. The cross-expressed antigens analyzed for AML included CD2, CD7, CD19, CD56 and other co-expressed lymphoid antigens. The myeloid antigens analyzed for ALL included CD13 and co-expressed CD13/CD33. ALL and AML patients without expression of cross-expressing antigens were selected as control. Complete remission (CR) ratio and relapse-free survival (RFS) of patients in all groups were compared. The results indicated that among 161 patients analyzed, 91 cases of AML with cross-expressing lymphoid and myeloid antigens included that 24 cases of AML expressed lymphoid surface marker-CD7, namely CD7(+) AML, 14 cases of AML only expressed lymphoid surface marker-CD19, namely CD19(+) AML, 8 cases of AML expressed lymphoid surface marker-CD2 (including CD2/CD19 co-expressed), namely CD2(+) AML, 10 cases of AML expressed lymphoid surface marker-CD56 (including CD56/CD19 or CD56/CD2 co-expressed), namely CD56(+) AML, 16 cases of AML expressed two or more lymphoid surface markers, namely Ly ≥ 2(+) AML, 9 cases of ALL expressed myeloid surface markers CD13, namely CD13(+) ALL, 10 cases of ALL expressed myeloid surface markers CD13 and CD33, namely CD13/CD33(+) ALL. 29 cases of ALL did not expressed myeloid surface markers, namely My(-) ALL, and 41 case of AML did not expressed lymphoid surface markers, namely Ly(-) AML. CR ratio and RFS of Ly ≥ 2(+) AML patients were lower than those of Ly(-) AML patients. RFS of CD56(+) AML patients was lower, but CR ratio had no significant difference, when compared with Ly(-) AML patients. CR ratio and RFS of other AML patients with cross-expressing antigens had no significant difference when compared with Ly(-) AML patients. CR ratio and RFS of CD13(+) ALL and CD13/CD33(+) ALL patients had no significant difference when compared with My(-) ALL patients. It is concluded that the importance of cross-expressing antigens for prognosis of patients should be analyzed concretely. CD56(+) AML and Ly ≥ 2(+) AML have bad prognosis, while other cross-expressed lymphoid and myeloid lineage-associated antigens have no impact on prognosis of acute leukemia patients.

Porcine aminopeptidase N is a functional receptor for the PEDV coronavirus / 病毒学报

Porcine epidemic diarrhea virus (PEDV) causes lethal diarrhea in piglets that leads to great economic losses in East Asia. It was reported that aminopeptidase N (APN) was the receptor for Transmissible gastroenteritis virus (TGEV), Human coronavirus 229E (HCoV-229E) and Feline coronavirus (FeCoV) which all belonged to group I coronavirus including PEDV. It was also confirmed previously that porcine aminopeptidase N (pAPN) could bind to PEDV, and anti-pAPN antibodies could inhibit the combination. To investigate whether pAPN was a receptor for PEDV, we transfected MDCK cells with porcine aminopeptidase (pAPN) cDNA and this enabled non-susceptible cells to support PEDV replication and serial viral propagation. Moreover, the infection was blocked by antibodies against pAPN, implying the critical role of pAPN during virus entry. In addition, immunofluorescence assays for detection of pAPN and PEDV antigens, together with neutralization assays using antibodies against pAPN, further confirmed the correlation between pAPN expression and viral replication in pAPN-transfected MDCK cells. These results indicated that pAPN is a functional receptor for PEDV.

Angiogenesis following three-dimensional culture of isolated human endometrial stromal cells

Endometriosis is the presence of endometrial tissue outside of the uterine cavity and is the most common gynecologic disorder in women of reproductive age. We have preliminary evidence that in the presence of a 3-dimensional [3-D] fibrin matrix, human endometrial glands, stroma, and neovascularization can develop in vitro, mimicking the earliest stages of endometriosis. The aim of the present study was to determine if angiogenesis can be developed in a 3-D culture of human stromal cells in vitro. This was an in vitro study of human endometrial biopsies in 3-D culture of fibrin matrix and conducted at a university affiliated infertility center. Biopsies were taken from ten normal ovulating women undergoing infertility treatment. The samples obtained from fundus of the uterine cavity were minced, stromal cells isolated and placed in a 3-D fibrin matrix culture system. Degree of proliferation of stromal cells, invasion of the fibrin matrix, gland formation, vessel sprouting and immunohistochemical characterization of cellular components were recorded. Three-dimensional culture of human stromal cells formed sheets of cells in the fibrin matrix. By 3-4 weeks, endothelial cell branching was observed and rudimentary capillary-like structures formed and endothelial cells confirmed by CD31 immunostaining. These data show that stromal cells from endometrial explants can proliferate and invade a fibrin matrix in vitro generating new vessels. This procedure represents a controlled, quantifiable model for the study of angiogenesis during the menstrual cycle, and in conditions such as endometriosis and cancer

Clinical study on prognosis of acute leukemia subtypes Ly + AML and My + ALL / 中国实验血液学杂志

The purpose of this study was to investigate the prognosis of acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), lymphoid antigen-positive acute myeloid leukemia (Ly + AML), myeloid antigen-positive acute leukemia (My + ALL) and biphenotypic acute leukemia (BAL). Immunophenotyping was performed on medullary specimens of 197 acute leukemia (AL) patients by using three-color flow cytometry analysis and CD45/SSC gating. The scoring systems proposed by EGIL was adopted to classify the AL patients into five groups: 43 of ALL, 53 of AML, 53 of My + ALL, 39 of Ly + AML and 9 of BAL patients. The results showed that in Ly + AML, CD7 was the most common (53.8%) as compared to other lymphoid markers, however, in My + ALL CD13 was the most common (47.2%) as compared to other myeloid markers. Compared with Ly + AML, My + ALL had higher incidences of enlargement of liver, spleen and lymphnodes significantly (P<0.05). As for the case numbers of WBC counts > 100 x 10(9)/L, the positive rate of CD34 and the complete remission rate there was no obvious difference between groups of Ly + AML and My + ALL (P>0.05). As for incidences of enlargement of liver, spleen and lymphnodes, the case numbers of WBC counts > 100 x 10(9)/L, the positive rate of CD34 and complete remission rate, no obvious difference was found between ALL and My + ALL (P>0.05). Compared with AML, Ly + AML had lower complete remission rate significantly (P<0.05). As for incidences of enlargement of liver, spleen and lymphnodes, the case numbers of WBC counts > 100 x 10(9)/L and the positive rate of CD34, no obvious difference was found between AML and Ly + AML (P>0.05). Compared with Ly + AML and My + ALL, BAL showed no significant difference in complete remission rate (P>0.05) because the number of BAL patients was too small. It is concluded that since Ly + AML has lymphoid markers, and the prognosis of Ly + AML is worse than AML, the clinical therapy for Ly + AML should contain both AML and ALL. Though My + ALL had myeloid markers, no significant difference was found between My + ALL and ALL, it might be supposed that their therapy could be the same.

Influence of Interferon alpha-2b on proliferation inhibition and apoptosis induction in HL-60 cells / 中国实验血液学杂志

To investigate the effects of interferon alpha-2b on proliferation and apoptosis in HL-60 cells, HL-60 cells were cultured in different concentrations of IFN alpha-2b. The morphologic changes were observed by Wright's and acridine orange (AO) and ethidium bromide (EB) staining respectively. Inhibition of proliferation was detected by MTT. Expression of CD13(+) was checked by indirect fluoroimmunoassay. The results showed that apoptosis rate of HL-60 cells assayed by the above-mentioned two methods was (51 +/- 2)% and (78 +/- 3)% respectively and OD(570) values of proliferation inhibited were 1.8 +/- 0.1 and 1.0 +/- 0.1 respectively when the concentrations of the IFN(alpha-2b) were 500 and 10,000 U/ml in culture for 48 hours. Morphology and count of CD13(+) cells were changed. CD13(+) cell expression rate was (62 +/- 2)% and (30 +/- 3)% respectively when the concentrations of the IFN(alpha-2b) were 500 and 10,000 U/ml in culture for 48 hours. It is concluded that IFN(alpha-2b) can enhance the apoptosis of HL-60 cells, inhibit their proliferation, promote their maturation and differentiation.

Differentiation phenotypes of k562 cells induced by exogenous wnt5a / 中国实验血液学杂志

This study was aimed to investigate the effect of exogenous Wnt5a on directional differentiation of K562 cells. Wnt5a and GFP condition mediums were prepared by recombinant adenoviral vector AdWnt5a and AdGFP transfecting CHO cells. K562 cells were treated with Wnt5a and the GFP condition mediums for 1 - 7 days as Wnt5a treated group and control group respectively. The morphological changes of K562 cells were observed by light microscope and electron microscope; the differentiation phenotypes of K562 cells were identified by the cytochemical staining of POX, PAS, alpha-NAE and immunocytochemistry of CD13, CD14, CD68, and the effect of Wnt5a on cell cycle distribution of K562 cells was detected by flow cytometry. The results showed that the morphology and ultrastructure of K562 cells treated by Wnt5a displayed differentiation mature feature; both POX and PAS staining showed higher positive ratio in Wnt5a treated group than that in control group; the alpha-NAE staining also was positive, but positive intensity in Wnt5a treated group could be inhibited up to 70% by NaF. The expressions of monocytic differentiation antigens of CD14, CD68 in Wnt5a treated group were higher than those in control group, but the expression differences of granulocytic differentiation antigen CD13 between Wnt5a treated group and control group were not significant. The cell cycle in treated group was blocked at G2 phase as compared with control group. It is concluded that exogenous Wnt5a can induce K562 cells to differentiate towards mature and K562 cells treated with Wnt5a displays features of differentiation towards monocytic lineage.