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Objective To preliminarily discuss the feasibility of geolocation inference of forensic individual origin by soil metagenomic analysis. Methods The 33 soil samples from Heilongjiang, Qinghai and Tibet were collected, total bacterial DNA in the samples were extracted, and universal primers were used to amplify the V3 and V4 hypervariable region of bacterial 16S rDNA. The region was sequenced by high-throughput sequencing （HTS） with the MiSeq sequencer. Bioinformatics analysis such as species composition and sample comparison was performed on sequencing data. The richness index and diversity index were calculated based on operational taxonomic unit （OTU） results. Results A total of 2 720 149 sequences were generated by sequencing. Those sequences were clustered into 114 848 OTUs. The Chao1 indexes of soil microorganisms in Heilongjiang, Qinghai, and Tibet were 797.45, 745.11 and 535.98, respectively, and Shannon indexes were 6.46, 6.36 and 6.25, respectively. The number of bacterial species and the community diversity in the soil from high to low were Heilongjiang > Qinghai > Tibet. The composition of soil bacteria in three provinces at various classification levels were obtained, the dominant genuses in Heilongjiang were Chthoniobacteraceae DA101 and an unannotated genus of Thermogemmatisporaceae; the dominant genuses in Qinghai were an unannotated genus of Cytophagaceae and an unannotated genus of Nocardioidaceae; the dominant genuses in Tibet were an unannotated genus of Comamonadaceae and Verrucomicrobiaceae Luteolibacter. The results of principal co-ordinates analysis demonstrated that, according to the weighted UniFrac analysis, the three principle components represented 56.36% of the total variable, and according to the unweighted UniFrac analysis, the three principle components represented 34.81% of the total variable. The samples from the same province could be clustered together, and the species and content of soil microorganisms from different provinces were significantly different. Conclusion Based on the metagenomic analysis method, soil samples from different regions can be effectively distinguished, which has potential application value in geolocation inference of forensic individual origin in the future.
Subject(s)
Bacteria/genetics , High-Throughput Nucleotide Sequencing , RNA, Ribosomal, 16S/genetics , Soil , Soil MicrobiologyABSTRACT
BACKGROUND@#Medicines for the treatment of 2019-novel coronavirus (2019-nCoV) infections are urgently needed. However, drug screening using live 2019-nCoV requires high-level biosafety facilities, which imposes an obstacle for those institutions without such facilities or 2019-nCoV. This study aims to repurpose the clinically approved drugs for the treatment of coronavirus disease 2019 (COVID-19) in a 2019-nCoV-related coronavirus model.@*METHODS@#A 2019-nCoV-related pangolin coronavirus GX_P2V/pangolin/2017/Guangxi was described. Whether GX_P2V uses angiotensin-converting enzyme 2 (ACE2) as the cell receptor was investigated by using small interfering RNA (siRNA)-mediated silencing of ACE2. The pangolin coronavirus model was used to identify drug candidates for treating 2019-nCoV infection. Two libraries of 2406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on Vero E6 cells by GX_P2V infection. The anti-viral activities and anti-viral mechanisms of potential drugs were further investigated. Viral yields of RNAs and infectious particles were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and plaque assay, respectively.@*RESULTS@#The spike protein of coronavirus GX_P2V shares 92.2% amino acid identity with that of 2019-nCoV isolate Wuhan-hu-1, and uses ACE2 as the receptor for infection just like 2019-nCoV. Three drugs, including cepharanthine (CEP), selamectin, and mefloquine hydrochloride, exhibited complete inhibition of cytopathic effects in cell culture at 10 μmol/L. CEP demonstrated the most potent inhibition of GX_P2V infection, with a concentration for 50% of maximal effect [EC50] of 0.98 μmol/L. The viral RNA yield in cells treated with 10 μmol/L CEP was 15,393-fold lower than in cells without CEP treatment ([6.48 ± 0.02] × 10vs. 1.00 ± 0.12, t = 150.38, P < 0.001) at 72 h post-infection (p.i.). Plaque assays found no production of live viruses in media containing 10 μmol/L CEP at 48 h p.i. Furthermore, we found CEP had potent anti-viral activities against both viral entry (0.46 ± 0.12, vs.1.00 ± 0.37, t = 2.42, P < 0.05) and viral replication ([6.18 ± 0.95] × 10vs. 1.00 ± 0.43, t = 3.98, P < 0.05).@*CONCLUSIONS@#Our pangolin coronavirus GX_P2V is a workable model for 2019-nCoV research. CEP, selamectin, and mefloquine hydrochloride are potential drugs for treating 2019-nCoV infection. Our results strongly suggest that CEP is a wide-spectrum inhibitor of pan-betacoronavirus, and further study of CEP for treatment of 2019-nCoV infection is warranted.
Subject(s)
Humans , Betacoronavirus , Genetics , Cell Line , Clinical Laboratory Techniques , Coronavirus Infections , Diagnosis , Drug Therapy , Drug Approval , Pandemics , Pneumonia, Viral , Diagnosis , Drug Therapy , RNA, Small Interfering , Genetics , Real-Time Polymerase Chain Reaction , Viral LoadABSTRACT
Background@#Medicines for the treatment of 2019-novel coronavirus (2019-nCoV) infections are urgently needed. However, drug screening using live 2019-nCoV requires high-level biosafety facilities, which imposes an obstacle for those without such facilities or 2019-novel coronavirus (2019-nCoV). This study aims to repurpose the clinically approved drugs for the treatment of coronavirus disease 2019 (COVID-19) in a 2019-nCoV related coronavirus model.@*Methods@#A 2019-nCoV related pangolin coronavirus GX_P2V/pangolin/2017/ Guangxi was described. Whether GX_P2X uses angiotensin-converting enzyme 2 (ACE2) as the cell receptor was investigated by using small interfering RNA (siRNA) -mediated silencing of ACE2. The pangolin coronavirus model was used to identify drug candidates for treating 2019-nCoV infection. Two libraries of 2406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on Vero E6 cells by GX_P2X infection. The antiviral activities and antiviral mechanisms of potential drugs were further investigated. Viral yields of RNAs and infectious particles were quantified by quantitative real-time polymerase chain reaction (qRT-PCR) and plaque assay, respectively.@*Results@#The spike protein of coronavirus GX_P2V shares 92.2% amino acid identity with that of 2019-nCoV isolate Wuhan-hu-1, and uses ACE2 as the receptor for infection just like 2019-nCoV. Three drugs-cepharanthine (CEP), selamectin and mefloquine hydrochloride exhibited complete inhibition of cytopathic effects in cell culture at 10 μmol/L. CEP demonstrated the most potent inhibition of GX_P2V infection, with a concentration for 50% of maximal effect [EC50] of 0.98 μmol/L. The viral RNA yield in cells treated with 10 μmol/L CEP was 15,393-fold lower than in cells without CEP treatment ([6.48±0.02]×10-4 vs. 1.00±0.12, t=150.38, P<0.001) at 72 h post-infection (p.i.). Plaque assays found no production of live viruses in media containing 10 μmol/L CEP at 48 h p.i. Furthermore, we found CEP has potent antiviral activities against both viral entry (1.00±0.37 vs. 0.46±0.12, t=2.42, P<0.05) and viral replication (1.00±0.43 vs. [6.18±0.95]×10-4, t=3.98, P<0.05).@*Conclusions@#Our pangolin coronavirus GX_P2V is a workable model for 2019-nCoV research. CEP, selamectin and mefloquine hydrochloride are potential drugs for treating 2019-nCoV infection. Our results strongly suggest that CEP is a wide-spectrum inhibitor of pan-betacoronavirus, and clinical trial of CEP for treatment of 2019-nCoV infection is warranted.
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Objective: To investigate the clinical value of ambulatory electrocardiogram and the neutrophils to lymphocytes ratio (NLR) in the diagnosis on Kawasaki disease combined with changes of coronary artery for children. Methods: 80 children with Kawasaki disease were enrolled in the research. According to the combined situation of coronary artery disease, all of children with combined coronary artery disease were divided in observation group (48 cases) and the children without combined coronary artery disease were divided in control group (32 cases). All of children received the examinations of dynamic electrocardiogram, routine electrocardiogram, ultrasound cardiogram scan and blood routine detection within 15d of occurring disease. And the amounts of neutrophil and lymphocyte, and change of dynamic electrocardiogram of all of children were recorded and analyzed. And then the amounts of neutrophil and lymphocyte and the NLR of the two groups were further compared. Results: The sinus tachycardia, atrioventricular block, abnormal average heart rate and the proportions of total abnormal number of observation group were significantly higher than that of control group, and the difference of total number of ambulatory electrocardiographic abnormality between the two groups were significant (x2=20.112, P<0.05). The results of ambulatory electrocardiogram for Kawasaki disease combined with abnormal coronary artery were significantly higher than that of control group (x2=17.778, P<0.05). Compared with control group, the coronary artery ectasia was more larger, the amount of neutrophils would be more greater in observation group, and the difference between the two groups was significant (t=-3.464, P<0.05). Besides, the difference of NLR between patients with mild ectasia of observation group and patients of control group was significantly (t=2.976, P<0.05). Conclusion: Dynamic electrocardiogram and the NLR have important significance in the diagnosis for children with Kawasaki disease combined with the change of coronary artery.
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To improve the in vitro dissolution and in vivo absorption as well as the bioavailability after oral administration by increasing the solubility with the formation of solid dispersion remains a great challenge for the oral dosage form design of poorly water-soluble drugs. Compared with the other pharmaceutical techniques in improving the solubility for poorly water-soluble drugs, priorities are usually given to solid dispersion for its manufacturing convenience. Following the characteristics introduction, we were focused this review on the novel carriers and advanced techniques used for preparing solid dispersions. Amphiphilic polymers used as novel solid dispersion carriers are Solutol HS 15, Soluplus and poly [MPC-co-BMA]. Inorganic materials like magnesium aluminum metasilicat, mesoporous silica microparticle and mesoporous magnesium carbonate are introduced together with the advanced solid dispersing techniques such as supercritical fluid technology, high speed electro-spinning and microenvironmental pH modified technology.
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Objective To investigate the value of ultrasound-guided liver biopsy for infantile hepatitis syndrome regarding diagnosis,treatment and prognosis.Methods Fifty children with infantile hepatitis syndrome hospitalized in Guiyang Maternal and Child Hospital during Aug.2010 to May 2012 were involved in this study.Ultrasoundguided liver biopsies were performed to evaluate the inflammation grade and fibrosis stage.Immunohistochemical staining was used for pathogen diagnosis.The clinical outcomes were followed-up.Results Thirty-four cases (68％)were CMV infection,6 cases(12％) were vanishing bile duct syndrome,4 cases(8％) were chronic intrauterine infection,4 cases(8％) were congenital anomaly of bilirubin metabolism,and 2 cases (4％) were obstructive cholangitis.All 50 cases showed mild inflammation at portal area(G1-G2 grade).All 50 cases exhibited liver fibrosis.Sixteen cases were S1 stage,20 cases were S2 stage,8 cases were S3 stage and 6 cases were S4 stage.Pathogen analysis:all 50 cases showed intrahepatic cholestasis:38 cases were diffuse cholestasis,and 12 cases were moderate cholestasis.Treatment:all cases were treated using 2-week heteropathy; antivirus was used for CMV infected cases,thus 39 cases were finally cured,9 cases were relieved,and 2 cases were ineffective.Conclusion Liver biopsy is valuable for diagnosis,treatment and prognosis infantile hepatitis syndrome.
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<p><b>BACKGROUND</b>Acute lung injury (ALI) is a common syndrome associated with high morbidity and mortality in emergency medicine. Cell apoptosis plays a key role in the pathogenesis of ALI. Hydrogen sulfide (H(2)S) plays a protective role during acute lung injury. We designed this study to examine the role of H(2)S in the lung alveolar epithelial cell apoptosis in rats with ALI.</p><p><b>METHODS</b>Sixty-nine male Sprague Dawley rats were used. ALI was induced by intra-tail vein injection of oleic acid (OA). NaHS solution was injected intraperitonally 30 minutes before OA injection as the NaHS pretreatment group. Single sodium hydrosulfide pretreatment group and control group were designed. Index of quantitative assessment (IQA), wet/dry weight (W/D) ratio and the percentage of polymorphonuclear leukocyte (PMN) cells in the bronchoalveolar lavage fluid (BALF) were determined. H(2)S level in lung tissue was measured by a sensitive sulphur electrode. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Fas protein was measured by immunohistochemical staining.</p><p><b>RESULTS</b>The level of endogenous H(2)S in lung tissue decreased with the development of ALI induced by OA injection. Apoptosis and Fas protein in alveolar epithelial cells increased in the ALI of rats but NaHS lessened apoptosis and Fas protein expression in alveolar epithelial cells of rats with ALI.</p><p><b>CONCLUSION</b>Endogenous H(2)S protects rats from oleic acid-induced ALI, probably by inhibiting cell apoptosis.</p>
Subject(s)
Animals , Male , Rats , Acute Lung Injury , Drug Therapy , Metabolism , Apoptosis , Physiology , Epithelial Cells , Hydrogen Sulfide , Metabolism , In Situ Nick-End Labeling , Oleic Acid , Toxicity , Rats, Sprague-Dawley , Sulfides , Pharmacology , Therapeutic UsesABSTRACT
Wnt signaling has been shown to inhibit adipogenic differentiation while inducing the osteogenic pathway of bone marrow stromal cells (BMSC). Patients with aplastic anemia (AA) often show excess fat accumulation in the bone marrow, possibly due to overactivation of the adipogenic pathway. Therefore, an activator of the Wnt signaling may alleviate the symptoms by enhancing the inhibition on the differentiation of BMSC towards adipocytes. To judge this hypothesis, the therapeutic effects of Wnt signaling activator lithium chloride (LiCl) combined with the currently used immunosuppressor cyclosporine A (CsA) on mice with AA in vivo was investigated. Mouse model with AA was established and the disease was confirmed by increased fat cell counts and decreased hematopoietic cell counts in the bone marrow of these animals. These mice treated with CsA 50 mg/(kg·d) alone or together with LiCl 20 mg/(kg·d), once daily for 5 d, then at day 14, 21 and 28 after establishment of mouse model with AA, the treatment effects were observed, including peripheral blood cells, bone marrow mononuclear cells (BMMNC) count and bone marrow biopsy examination in each group. The results showed that compared with the AA group, Hb content, WBC and BMMNC counts of CsA group and the combination group significantly increased. HE staining of bone marrow biopsy sample showed that the fat cells were significantly reduced in the bone marrow cavity (P < 0.05). Compared with the CsA group, Hb content, WBC and BMMNC counts of the combination group significantly increased (P < 0.05); HE staining of bone marrow biopsy sample showed that fat cells were reduced, the hematopoiesis of bone marrow was close to the normal. It is concluded that Wnt signal activator (LiCl) combined with CsA displayed a better treatment effect on AA in mouse models than the effect of using CsA only. They can promote the hematopoietic function of bone marrow, which may correlate with inhibiting differentiation of bone marrow mesenchymal stem cells into the fat cells by Wnt signaling.
Subject(s)
Animals , Female , Mice , Anemia, Aplastic , Drug Therapy , Metabolism , Bone Marrow Examination , Cyclosporine , Therapeutic Uses , Disease Models, Animal , Drug Therapy, Combination , Lithium Chloride , Therapeutic Uses , Mice, Inbred BALB C , Wnt Signaling PathwayABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of Gli1 gene silencing by RNA interference (RNAi) on proliferation of K562 cells and its mechanisms.</p><p><b>METHODS</b>The small interference RNA (siRNA) was synthesized in vitro. K562 cells were transfected with Gli1 siRNA by the way of lipofection (lipofectamine 2000). Non-specific siRNA transfected cells were used as control. Transfection efficiencies of different siRNA concentrations were detected by flow cytometry and the best siRNA concentration was selected. The silencing effect of siRNA was demonstrated by real time PCR and Westem blot analysis. Cell proliferation was measured by MTT method, cell cycle by PI assay, c-myc and p21 mRNA level was detected by real time PCR analysis.</p><p><b>RESULTS</b>Transfection efficiency of siRNA was increased in a dose-dependent manner when siRNA concentration was below 200 pmol, and the highest transfection efficiency reached (80.11 ± 5.63)%. Both the mRNA and protein level of Gli1 was down-regulated in Gli1 specific siRNA group, the mRNA level was (52.60 ± 3.57)% of that of control group after 24 h (t = 20.33, P < 0.01) and the protein level was (79.31 ± 5.58)% of that of control group after 48 h (t = 6.54, P < 0.01). The cell proliferation rate in Gli1 siRNA group was (94.41 ± 3.58)% (t = 2.40, P = 0.05) and (90.22 ± 3.34)% (t = 4.37, P < 0.01) of that of control group after 24 h and 48 h, respectively. G(2)/M cell cycle arrest was observed, the mRNA level of c-myc was down-regulated while p21 was up-regulated in Gli1 siRNA group after 24 h and 48 h (P < 0.05).</p><p><b>CONCLUSIONS</b>Targeted silencing of Gli1 gene by RNAi inhibits the proliferation of K562 cells, which acts through the down-regulation of c-myc and up-regulation of p21 expression.</p>
Subject(s)
Humans , Cell Proliferation , Gene Silencing , K562 Cells , RNA Interference , RNA, Messenger , Genetics , RNA, Small Interfering , Genetics , Transcription Factors , Genetics , Transfection , Zinc Finger Protein GLI1ABSTRACT
This study was purposed to investigate the relation of the Notch signaling pathway to senescence of murine bone marrow stromal cells in vitro. Intracellular domain of Notch 1 (ICN) was transfected into cultured murine bone marrow stromal cells by lipofectamine transfection. After transfection for three days the proliferation of transfected cells was measured by MTT, cell cycle distribution was analyzed by flow cytometry. The percentage of senescence associated beta-galactosidase (SA-beta-Gal) positive cells were measured by cytochemical method, and the expression rates of P53 and p21Cip1/Waf1 at gene and protein levels were analyzed by RT-PCR and Western blot respectively. The results showed that after transfection for 3 days the proliferation of murine bone marrow stromal cells was inhibited with induction of G1 arrest, the percentage of SA-beta-gal positive cells increased and the p53 and p21Cip1/Waf1 mRNA and protein expression levels were upregulated. It is concluded that the activated Notch signaling can induce premature senescence of bone marrow stromal cells through the p53-p21Cip1/Waf1 pathway.
Subject(s)
Animals , Mice , Bone Marrow Cells , Cell Biology , Metabolism , Cellular Senescence , Cyclin-Dependent Kinase Inhibitor p21 , Metabolism , Receptor, Notch1 , Metabolism , Signal Transduction , Stromal Cells , Cell Biology , Metabolism , Tumor Suppressor Protein p53 , MetabolismABSTRACT
The aim of the present paper is to better understand the mechanism of hematopoietic development through studying the biological characteristics of hematopoietic progenitor cells at different stages of development. Firstly, the c-kit expression levels of the mononuclear cells from murine embryonic aorta-gonad-mesonephros (AGM) region at embryonic day (E)10.5 and E11.5, fetal liver (FL) at E12.5, E14.5, E16.5, E18 and bone marrow (BM) were assayed with fluorescence activated cell sorting (FACS). Secondly, hematopoietic progenitor cells derived from AGM at E10.5, FL at E14.5 and BM were isolated by using c-kit microbeads. Isolated c-kit(+) population cells from AGM, FL and BM were then co-cultured with E14.5 FL-derived stromal cells in transwell co-culture system in vitro. After 3, 7, 10 days of co-culture, numerous floating cells were generated. The floating cells generated in transwell inserts were collected for FACS cell count, migration activity detection and colony forming unit (CFU) formation assay. The results showed that the c-kit was highly expressed in E10.5 AGM, with the percentage of c-kit(+) cells declining during AGM development. c-kit expression was highly expressed again in E12.5 FL, declining along with the progressive development of the FL region. Co-cultured with FL-derived stromal cells, E10.5 AGM-derived c-kit(+) cells produced the highest number of hematopoietic cells, while BM-derived c-kit(+) cells produced the lowest number of hematopoietic cells. Compared with E10.5 AGM-derived c-kit(+) cells, E14.5 FL- and BM- derived c-kit(+) cells inclined to differentiate after 7 to 10 days of culture in vitro. E10.5 AGM and E14.5 FL-derived c-kit(+) cells exhibited a higher migration activity than BM-derived c-kit(+) cells. Moreover, E10.5 AGM-derived c-kit(+) cells showed a higher ability to form mixed colony-forming unit (CFU-Mix) colony. In conclusion, compared with FL- and BM-derived c-kit(+) cells, E10.5 AGM-derived c-kit(+) hematopoietic progenitor cells exhibit better proliferation, migration potential, and have a higher ability to maintain the undifferentiation state in vitro, providing an insight into their clinical manipulation.
Subject(s)
Animals , Mice , Aorta , Embryology , Coculture Techniques , Colony-Forming Units Assay , Gonads , Embryology , Hematopoiesis , Hematopoietic Stem Cells , Cell Biology , Mesonephros , Embryology , Proto-Oncogene Proteins c-kit , Metabolism , Stromal Cells , Cell BiologyABSTRACT
<p><b>OBJECTIVE</b>To establish a mouse model for the study of pathophysiologic mechanism and treatment of bone marrow failure (BMF).</p><p><b>METHODS</b>Balb/c mice (recipient) were irradiated 5.0 Gy by gamma rays of (60)Co, and then infused 5 x 10(6) lymph node (LN) cells from DBA/2 mice (donor) in 4 hours. Pancytopenia was monitored by cell counting, bone marrow damage was assessed by histological staining and mononuclear cell counting. Serum IFN-gamma concentration was measured by ELISA. The proportion of Treg in spleen was detected by flow cytometry.</p><p><b>RESULTS</b>Irradiation and infusion of LN cells led to rapid development of severe pancytopenia and BM hypoplasia, which reached the most severity at d14. The pancytopenia remained at d28 and displayed no signs of recovery. The bone marrow was full of adipose cells with scarcity of hematopoietic cells at d14 and persisted at least for 28 days, being similar to the feature of aplastic anemia. Serum IFN-gamma concentration was 6.3 fold increased \[(170.0 +/- 17.0) vs (27.7 +/- 7.1) pg/ml\] at d6. Tregs were decreased after infusion, and then increased \[(3.38 +/- 0.52)%\] and recovered to normal \[(4.04 +/- 0.44)%\] at d21. The expression level of the specific transcription factor Foxp3 was similar to normal.</p><p><b>CONCLUSION</b>The MHC antigen of Balb/c mice is identical to that of DBA/2 mice, but their minor antigen differs. 5.0 Gy irradiation and then 5 x 10(6) lymphocyte infusion can induce BMF similar to the features of aplastic anemia.</p>
Subject(s)
Animals , Female , Male , Mice , Anemia, Aplastic , Allergy and Immunology , Pathology , Bone Marrow , Pathology , Disease Models, Animal , Gamma Rays , Interferon-gamma , Allergy and Immunology , Lymphocyte Transfusion , Mice, Inbred BALB C , T-Lymphocytes, Regulatory , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To explore the mechanism of Wnt and Notch pathway involved modulating time and spatial restricted hematopoiesis.</p><p><b>METHODS</b>Murine hematopoietic stem and progenitor cells (HSPCs) were isolated from bone marrow (BM) by using c-kit microbeads. E10.5 aorta-gonad-mesonephros (AGM), E12.5, E14.5, E16.5 fetal liver (FL) and adult BM derived stromal cells (StroCs) were isolated and co-cultured with c-kit(+)HSPCs. The floating cells in co-culture system were sorted and counted by FACS. Gene expressions of Wnt and Notch pathway were detected by quantitative PCR and protein expressions by immunostaining.</p><p><b>RESULTS</b>Co-culturing HSPCs with AGM and FL-derived StroCs resulted in an expansion of c-kit(+)population from 0.4 x 10(5)/well to (19.2 +/- 3.2) x 10(5)/well and (26.8 +/- 5.4) x 10(5)/well, respectively, being greater than that with BM-derived StroCs (P < 0.05). The percentage of c-kit(+)cells detected in AGM- and BM- derived StroCs culture system was (75.2 +/- 7.1)%, (74.1 +/- 6.2)% respectively, being higher than FL- derived StroCs culture system (63.4 +/- 5.3)% (P < 0.05). Wnt and Notch pathway genes expression varied at different phases of hematopoiesis. Wnt was highly expressed in AGM and FL derived StroCs, and, Notch did in AGM and BM derived StroCs.</p><p><b>CONCLUSION</b>Wnt and Notch pathway are important modulators in regulating time and spatial restricted hematopoiesis.</p>
Subject(s)
Animals , Humans , Aorta , Cell Biology , Coculture Techniques , Hematopoiesis , Hematopoietic Stem Cells , Cell Biology , Mesonephros , Cell Biology , Stromal CellsABSTRACT
<p><b>OBJECTIVE</b>To explore the activation and proliferation of specific T cells induced by artificial antigen-presenting cells (aAPCs) simulated dendritic cells (DCs) and to observed the effect of these T cells on leukemic cell killing.</p><p><b>METHODS</b>aAPCs were developed by coating a human leukocyte antigen-immunoglobulin fusion protein ( HLA-lg), which was connected each one of the four CML28 antigen epitopes (DLMSSTKGL, DLMSSTKGL, ALFCGVACA, VLTFALDSV), and CD28-specific antibody, to magnet-beads CML cell specific peptides (CML28) served as target peptides. Bone marrow (BM) or peripheral blood (PB) mononuclear cells (MNCs) were isolated from HLA-A2 healthy volunteers, and co-cultured with aAPCs. Specific T lymphocyte were detected by flow cytometry. The fresh acute leukemic cells were used as target cells. The specific T cells incubated with leukemic cells for 4 h at ratios of 5:1, 10:1, 20:1, 40:1, 80: 1, respectively. The effect of leukemic cells killing was detected by lactate dehydrogenase release test.</p><p><b>RESULTS</b>The average ratio of CML-28 specific T lymphocyte in control group was (2.2 +/- 0.4)% and in experimental groups (DLMSSTKGL, DLMSSTKGL, ALFCGVACA, VLTFALDSV) were (13.5 +/- 1.6)%, (15.2 +/- 1.5)%, (14.7 +/- 1.8)% and (34.3 +/- 3.5)%, respectively, being significantly higher than that in control group (P < 0.01). Induction efficiencies of acute leukemic cells killing were significantly enhanced by increase of effector cells. The cytotoxic activity of specific T lymphocyte in one experimental group (VLTFALDSV) was much higher than that in other three experimental group (P < 0.05).</p><p><b>CONCLUSION</b>This "prime and expand" regimen should be an alternative method for large scale amplification of rare tumor-specific CTLs and aAPCs might be a useful tool for leukemia immunotherapy.</p>
Subject(s)
Adolescent , Adult , Female , Humans , Male , Middle Aged , Young Adult , Antigen-Presenting Cells , Allergy and Immunology , Cell Proliferation , Cells, Cultured , Coculture Techniques , Cytotoxicity, Immunologic , Leukemia , Allergy and Immunology , Pathology , Lymphocyte Activation , T-Lymphocytes , Cell Biology , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To establish a sensitive and effective method for detection of immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangement,and to explore its role in diagnosis and differential diagnosis of lymphoproliferative disorders.</p><p><b>METHODS</b>Fifty-eight lymphoid tissue samples from 54 patients with lymphoproliferations were evaluated by the novel BIOMED-2 multiplex polymerase chain reaction (PCR) for antigen receptor genes rearrangement.</p><p><b>RESULTS</b>Multiplex PCR demonstrated monoclonal Ig/TCR gene rearrangements in 22 of 25 (88.0%) B-cell malignancies and 8 of 15 (53.3%) T-cell malignancies. Among 17 benign lymphoproliferations confirmed histopathologically, polyclonal rearrangements were detected in 14 cases (82.4%). In total, the clonality analysis and the final clinico-histopathological diagnosis were concordant in 77.2%. Combination detection of Iglambda and TCR delta gene rearrangements did not increase the detection rate of monoclonal rearrangement of Ig/TCR, but might help to the detection of Iglambda+ or TCR delta+ lymphomas.</p><p><b>CONCLUSION</b>The novel BIOMED-2 multiplex PCR strategy is a rapid, reliable and sensitive approach to detecting clonality in suspected lymphoproliferations, especially in atypical cases.</p>
Subject(s)
Female , Humans , Male , Gene Rearrangement, B-Lymphocyte, Light Chain , Gene Rearrangement, T-Lymphocyte , Lymphoproliferative Disorders , Diagnosis , Genetics , Polymerase Chain Reaction , Methods , Sensitivity and SpecificityABSTRACT
<p><b>OBJECTIVE</b>To explore the characteristics of CpG islands methylation at promoter region of HOX A gene cluster in leukemia cells before and after all-trans retinoic acid (ATRA) treatment.</p><p><b>METHODS</b>Eleven human leukemia cell lines, bone marrow cells from leukemia patients before and after therapy and white blood cells from normal subjects were collected. HL-60 and K562 cells were treated by 2-deoxy-5-azacytidine (DAC) or ATRA respectively. Bisulfite modified DNA of these cells were amplified with PCR and quantitatively analyzed by pyrosequencing for methylation of CpG islands.</p><p><b>RESULTS</b>In normal cells, CpGs at all loci of HOX A cluster were unmethylated. In HOX A4, A6, A7, A9, A10 and A11, many CpG sites were methylated (>20%) or hypermethylated (>50%) in leukemia cell lines. Percentages of methylated CpGs were higher in T-cell leukemia (71.4%) and B-cell leukemia (85.7%) than in others. For individual CpGs methylations there were HOX A4 in all leukemia cells, HOX A6 and HOX A7 in most of the leukemia samples and HOX A10 and HOX A11 in K562 and HL-60 cells (38%-86%). HOX A9 CpGs showed hypomethylation in most of myeloid leukemia cells, whereas HOX A11 CpGs were hypermethylated in B-cell leukemia (>50%). Methylation levels of HOX A4 and A6 in AML and ALL patients after complete remission were decreased obviously, and so did HOX A6 and A9 in CML patients. Methylation levels of HOX A4, A6 and A10 in HL-60 cells and of HOX A6 in K562 cells were reduced by ATRA treatment.</p><p><b>CONCLUSIONS</b>In all leukemia cell lines, aberrant methylation of CpGs was observed at promoter regions of 6 HOX A cluster genes, and some of these genes showed leukemia-type-specific hypermethylation. CpGs methylation of some HOX A genes in leukemia cell lines, especially in HL-60 cells, were down-regulated by ATRA.</p>
Subject(s)
Humans , Cell Line, Tumor , CpG Islands , Genetics , DNA Methylation , Homeodomain Proteins , Genetics , Leukemia , Genetics , Multigene Family , Promoter Regions, Genetic , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To observe the efficacy and safety of amphotericin B for treatment of invasive fungal infections (IFI) in patients with hematologic diseases.</p><p><b>METHODS</b>121 patients were given amphotericin B 5 -50 mg/d for 5 - 101 d with a median of 19 d.</p><p><b>RESULTS</b>The clinical efficacy rate was 67.3%, and fungal elimination rate 66.7%. The adverse events included rigor and fever, hypokalaemia, hepatic damage, nephrotoxicity, nausea and vomiting, phlebitis and teeter.</p><p><b>CONCLUSION</b>Amphotericin B is still a high-efficiency drug in the treatment of IFI, although it has many side effects. With monitoring of hepatic and renal function, it is still a relatively safe and effective drug.</p>
Subject(s)
Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Amphotericin B , Therapeutic Uses , Antifungal Agents , Therapeutic Uses , Mycoses , Drug Therapy , Treatment OutcomeABSTRACT
This study was aimed to investigate the effect of artificial antigen-presenting cells (aAPCs) on inducing activation and proliferation of specific T-lymphocytes through stimulating biological function of dendritic cells with aAPCs in vitro. The specific antigen of chronic myeloid leukemia CML-28 was screened as objective antigen peptide by using magnetic microbeads as vector; the CML-28 epitope sequence (Vltfaldsv) was obtained by antigen epitope prediction software; this epitope was coupled with MHC molecule and used as first signal molecule, the B7-1 molecule was used as second signal molecule; these 2 molecules simultaneously were loaded onto surface of magnetic microbeads so as to contract aAPC complex. The bone marrow mononuclear cells were derived from HLA-A2(+) healthy bone marrow donors, CD8(+) T lymphocytes were screened and co-cultured with aAPC complex. During culture the 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) was added and proliferation of T-lymphocytes was detected by CPSE and proliferation level of specific T lymphocytes was detected by flow cytometry. The results showed that the proliferation level of CML-28 specific T lymphocytes obviously increased in experimental group, average level was 17.34 +/- 0.65%, while average level in control was 2.25 +/- 0.43%, there was significant difference between them (p < 0.01). It is concluded that the aAPC complex can mimic human APCs in vitro, and stimulate activation and proliferation of CD8(+) specific T lymphocytes.
Subject(s)
Adolescent , Adult , Female , Humans , Male , Middle Aged , Young Adult , Antigen Presentation , Allergy and Immunology , Antigen-Presenting Cells , Allergy and Immunology , Metabolism , Antigens, Neoplasm , Allergy and Immunology , Biomimetics , Methods , CD8-Positive T-Lymphocytes , Allergy and Immunology , HLA-A2 Antigen , Allergy and Immunology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Allergy and Immunology , Lymphocyte Activation , Tissue DonorsABSTRACT
This study was aimed to explore fgfr3 gene expression in leukemic cells and its clinical significance. The expression levels of fgfr3 mRNA in 4 leukemic cell lines and bone marrow samples from 96 patients with leukemia and 14 controls were assayed by RT-PCR. 96 patients with leukemia included 36 cases of acute myeloid leukemia (AML), 29 cases of acute lymphoblastic leukemia (ALL), 31 cases of chronic myelogenous leukemia (CML). The results indicated that fgfr3 gene could be detected in both K562 and U937 cell lines, but not in HL-60 and SHI-1 cell lines. The positive expression rates of fgfr3 mRNA in AL and CML were 46.15% and 51.61% respectively, and were higher than that in control (14.29%, p < 0.05). The positive expression rates of fgfr3 mRNA in AML and ALL were 44.44% and 48.28% respectively, and were higher than that in control (p < 0.05). The increased level of fgfr3 mRNA had a positive correlation with high white blood cell count of > or = 20 x 10(9)/L (p < 0.05). Higher fgfr3 mRNA expression positively correlated with fuse genes of bcr/abl in ALL (r = 0.151, p < 0.05). The expression of fgfr3 gene in AL was not related with chromosome abnormality. In conclusion, the over expression of fgfr3 gene exists in AL and CML patients, it suggests that fgfr3 gene may be partially involved in leukemogenesis.
Subject(s)
Humans , Acute Disease , K562 Cells , Leukemia , Genetics , Metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , Receptor, Fibroblast Growth Factor, Type 3 , Genetics , Metabolism , U937 CellsABSTRACT
<p><b>BACKGROUND</b>CD4(+) T cells play a crucial role in the pathogenesis of aplastic anaemia. However, the mechanisms of over-proliferation, activation, infiltration of bone marrow and damage to haematopoietic cells of CD4(+) T cells in aplastic anaemia are unclear. Therefore, we screened differentially expressed genes of bone marrow CD4(+) T cells of aplastic anaemia patients and normal donors by suppressive subtractive hybridization to investigate the pathogenesis of aplastic anaemia.</p><p><b>METHODS</b>The bone marrow mononuclear cells of a first visit aplastic anaemia patient and a healthy donor of the same age and sex were isolated using lymphocyte separating medium by density gradient centrifugation. With the patients as "tester" and donor as "driver", their CD4(+) T cells were separated with magnetic bead sorting and a cDNA library established by suppressive subtractive hybridization. Then 15 of the resulting subtracted cDNA clones were randomly selected for DNA sequencing and homological analysis. With semiquantitative RT-PCR, bone marrow samples from 20 patients with aplastic anaemia and 20 healthy donors assessed the expression levels of differentially expressed genes from SSH library.</p><p><b>RESULTS</b>PCR detected 89 clones in the library containing an inserted fragment of 100 bp to 700 bp. Among 15 sequenced clones, 12 were known genes including 3 repeated genes. Compared with normal donors, there were 9/12 genes over-expressed in bone marrow CD4(+) T cells of patients with aplastic anaemia. The effects of these genes included protein synthesis, biology oxidation, signal transduction, proliferative regulation and cell migration. Not all these genes had been reported in the mechanisms of haematopoietic damage mediated by CD4(+) T cells in aplastic anaemia.</p><p><b>CONCLUSIONS</b>Screening and cloning genes, which regulate functions of CD4(+) T cells, are helpful in elucidating the mechanisms of over proliferation, activation, infiltrating bone marrow and damaging haematopoietic cells of CD4(+) T cells in aplastic anaemia.</p>