The aim of this study was to establish a stable subline of K562 cells expressing the HLA-A(*)1101 protein, which was expected to provide target cells for characterizing the HLA-I restrictive antigen specific cytotoxic T lymphocyte (CTL) effects against chronic myeloid leukemia (CML). The HLA-A(*)1101 protein encoding gene was amplified from peripheral blood mononuclear cell (PBMNC) of CML patient by RT-PCR; the 2A peptide linker (D-V-E-X-N-P-G-P) gene was linked to the 3'terminal of the HLA-A(*)1101 gene by recombinant PCR, then the recombinant was cloned into the pEGFP-N3 plasmid which contains an enhanced green fluorescent protein gene, and the eukaryotic recombinant expression vector containing HLA-A(*)1101-T2A-EGFP transcription box was constructed; the pEGFP-N3 vector and recombinant vector was separately electroporated into K562 cells. The expression of GFP was monitored by fluorescence microscopy, finally stably transfected sublines of K562 cells containing HLA-A(*)1101 gene, and of K562 containing pEGFP-N3 vector were obtained by G418 selection; the transcriptional or translational expression of HLA-A(*)1101 gene was detected with RT-PCR and flow cytometry respectively. The results indicated that the eukaryotic expression vector HLA-A(*)1101-T2A-EGFP plasmid was successfully constructed; after G418 selection for 2 months, two sublines of K562 cells (HLA-A(*)1101(+)K562, pEGFP-N3(+)K562) expressing GFP were constructed. The expression of HLA-A*A1101 gene could be determined in HLA-A(*)1101(+)K562 cell line by RT-PCR, while the pEGFP-N3(+)K562 cells could not express HLA-A*A1101 gene. HLA-A(*)1101 protein and GFP double positive HLA-A(*)1101(+)K562 cells were up to 88.5%, which was obviously higher than pEGFP-N3(+)K562 cells (0.698%) by flow cytometric analysis. It is concluded that a simple and effective method to select HLA-A(*)1101(+)K562 cells has been established and a subline of K562 cell expressing HLA-A(*)1101 protein on its cell membrane was successfully constructed, which provides the tool cells for further studying the specific cellular immunity against-CML.
Genetic Vectors ; HLA-A11 Antigen ; genetics ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; Leukocytes, Mononuclear ; Plasmids ; Transfection

OBJECTIVE: To analyze the indications of the therapies for rheumatoid arthritis (RA) with neural network model analysis. METHODS: Three hundred and ninety-seven patients were included in the clinical trial from 9 clinical centers. They were randomly divided into Western medicine (WM) treated group, 194 cases; and traditional Chinese herbal medicine (CM) treated group, 203 cases. A complete physical examination and 18 common clinical manifestations were prepared before the randomization and after the treatment. The WM therapy included voltaren extended action tablet, methotrexate and sulfasalazine. The CM therapy included Glucosidorum Tripterygii Totorum Tablet and syndrome differentiation treatment. The American College of Rheumatology 20 (ACR20) was taken as efficacy evaluation. All data were analyzed on SAS 8.2 statistical package. The relationships between each variable and efficacy were analyzed, and the variables with P<0.2 were included for the data mining analysis with neural network model. All data were classified into training set (75%) and verification set (25%) for further verification on the data-mining model. RESULTS: Eighteen variables in CM and 24 variables in WM were included in the data-mining model. In CM, morning stiffness, swollen joint number, peripheral immunoglobulin M (IgM) level, tenderness joint number, tenderness, rheumatoid factor (RF), C-reactive protein (CRP) and joint pain were positively related to the efficacy, and disease duration and more urination at night negatively related to the efficacy. In WM, erythrocyte sedimentation rate (ESR), weak waist, white fur in tongue, joint pain, joint stiffness and swollen joint were positively related to the efficacy, and yellow fur in tongue, red tongue, white blood negatively related to the efficacy. In the analysis with the neural network model in the patients of verification set, the predictive response rates of 20% patients would be 100% and 90% in the treatment with CM and WM, respectively. CONCLUSION: Neural network model analysis, based on the full clinical trial data with collection of both traditional Chinese medicine and modern medicine diagnostic information, shows a good predictive role for the information in the efficacy in rheumatoid arthritis.

OBJECTIVE: To evaluate the correlations between clinical symptoms and treatment efficacy in patients with rheumatoid arthritis (RA). METHODS: Four hundred and thirteen patients were included in the clinical trial from 9 clinical centers. They were randomly divided into Western medicine-treated group with 204 cases and Chinese herbal drug-treated group with 209 cases. Eighteen clinical symptoms were evaluated before and after treatment. The Western medicine therapy included voltaren extended release tablets, methotrexate and sulfasalazine. The Chinese herbal drug therapy included glucosidorum Tripterygii totorum tablets and Yishen Juanbi Tablets combined with treatment based on syndrome differentiation. The American College of Rheumatology 20 (ACR20) was used as efficacy evaluation criteria. RESULTS: In the Chinese herbal drug-treated group, clinical symptoms such as arthralgia and tenderness of joints were positively correlated with the efficacy after 12-week treatment, while frequent urination at night was negatively correlated. In the same group, tenderness of joints and fever were positively correlated with the efficacy after 24-week treatment, while deep-colored and turbid urine was negatively correlated. In the Western medicine-treated group, tenderness of joints and thirst were positively correlated with the efficacy after 12-week treatment, while vertigo was negatively correlated. And in the same group, tenderness of joints was positively correlated with the efficacy after 24-week treatment, while heaviness of limbs was negatively correlated to the efficacy. The statistical results showed that the treatment efficacy was improved when the correlated symptoms were included in the indications. CONCLUSION: The treatment efficacy of RA is correlated with some symptoms, so further studies should proceed on these correlations in order to achieve better treatment outcome.

HLA-A* 2402 is one of the most frequently encountered HLA-A alleles in East Asian populations. In order to study the CD8+ T cell responses in Chinese populations, we have described the generation and functional test of HLA-A* 2402 tetramer loaded with HCMV pp65(341-349) peptide (QYDPVAALF, QYD). The cDNA of HLA-A* 2402 heavy chain was cloned by RT-PCR from one of the donors. DNA fragment encoding the ectodomain of HLA-A* 2402 heavy chain fused at its carboxyl-terminal a BirA substrate peptide (BSP) was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type gene of HLA-A* 2402-BSP was not expressed in Escherichia coli (E. coli), while mutant HLA-A* 2402-BSP gene with optimized codons was overexpressed as inclusion bodies in E. coli. Furthermore, the soluble HLA-A* 2402-QYD monomers were generated by in vitro refolding of washed inclusion bodies in the presence of beta2-microglobulin and QYD peptide. The tetramer was subsequently formed by mixing HLA-A* 2402-QYD monomers with streptavidin-PE at a molar ratio of 4:1. Flow cytometry analysis indicated that this tetramer possessed binding activity with specific CTL from HLA-A24+ donors and the frequencies of tetramer-binding CTL were 0.09% - 0.37% within total CD8+ T cells. This tetrameric agent provides a powerful tool to explore the secrets of CTL responses against HCMV antigens in HLA-A* 2402 individuals.
Amino Acid Sequence ; CD8-Positive T-Lymphocytes ; cytology ; metabolism ; Carbon-Nitrogen Ligases ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Escherichia coli Proteins ; metabolism ; Flow Cytometry ; Gene Expression ; HLA-A Antigens ; chemistry ; genetics ; metabolism ; HLA-A24 Antigen ; Humans ; Oligopeptides ; genetics ; metabolism ; Phosphoproteins ; chemistry ; genetics ; metabolism ; Protein Multimerization ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism ; Repressor Proteins ; metabolism ; Substrate Specificity ; T-Lymphocytes, Cytotoxic ; cytology ; metabolism ; Viral Matrix Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo investigate the clinical results of the proximal femoral nail antirotation (PFNA) system in the treatment of unstable intertrochanteric femoral fractures.

METHODSFrom September 2006 to September 2009, 90 patients (40 males and 50 females, ranged in age from 64 to 95 years with an average of 73.2 years with unstable intertrochanteric femoral fractures were surgically treated with PFNA. Fifty patients had the fractures in the right hip, and 40 patients had the fractures in the left hip. The fractures were classified according to the AO classification: 11 patients were type A2.1, 21 patients were type A2.2,25 patients were type A2.3 9 patients were type A3.1,6 patients were A3.2 and 18 patients were A3.3. The patients underwent surgery within a mean of 3.2 days(ranged,2 to 20.1 days) from injury. The mean hospital stay was 12.8 days(ranged,7 to 24 days). Closed reduction was achieved in all the patients. Harris hip score were used for the evaluation of clinical effects.

RESULTSThe mean operation time was 36.8 min (ranged, 23 to 110 min) and the mean blood loss was 150 mi (ranged, 100 to 500 ml). The mean follow-up period was 12 months (ranged, 6 to 24 months). All the patients had fracture union. Sixty-nine patients got excellent reduction, 14 good and 7 bad. The mean collodiaphysial angle was 135.60 (ranged, 1260 to 1470). Postoperative complications included secondary varus in 2 patients,calcification at the tip of the greater trochanter in 5 patients, medial thigh pain in 7 patients,and screw cut-out in 1 petient. Ten patients had femoral shortness (mean 9.3 mm,ranging from 8 to 14 mm). The mean Harris hip score was (80.5 +/- 9.8). According to Harris hip scores evaluation system, 26 patients reached an excellent result, 37 good, 18 poor and 9 bad.

CONCLUSIONDue to advantages of high union rate, short operation time, and early postoperative mobilization, PFNA osteosynthesis is an idea method for surgical treatment of unstable intertrochanteric femoral fractures.

Aged ; Aged, 80 and over ; Bone Nails ; Female ; Fracture Fixation, Internal ; methods ; Hip Fractures ; diagnostic imaging ; surgery ; Humans ; Male ; Middle Aged ; Radiography

This study reports the preparation and identification of soluble programmed death-1 (PD-1) ligand-1 (sPD-L1) and its antibodies of mouse origin. Immobilized metal ion affinity chromatography was used to perform on-column refolding with simultaneous purification of denatured sPD-L1, and soluble sPD-L1 with purity of 95% was obtained. The purified sPD-L1 was verified by immunoblotting using a commercial goat-anti-human PD-L1 antibody. An ELISA-based assay showed that it also had high binding activity for its cognate receptor PD-1. Furthermore, mouse anti-sPD-L1 antiserum of high titer was raised using the purified sPD-L1 as an immunogen, and the specific IgG antibodies were purified using sPD-L1-HiTrap affinity chromatography. In addition, a sensitive sandwich ELISA was established using the purified IgG antibodies together with the commercial goat antibodies. In conclusion, the preparation of soluble sPD-Ll and its antibodies provide the basis for detection of the potential anti-PD-L1 antibodies and soluble PD-L1 in humans as well as for further investigation of its in vivo bioactivities and characterization of its potential receptors.
Animals ; Antibodies ; immunology ; isolation & purification ; Antigens, CD ; genetics ; immunology ; metabolism ; B7-H1 Antigen ; Chromatography, Affinity ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Female ; Humans ; Immune Sera ; immunology ; Immunoblotting ; Mice ; Mice, Inbred C57BL ; Recombinant Proteins ; immunology ; isolation & purification ; metabolism ; Solubility

BACKGROUND:As a routine method after lumbar spine surgery,a drainage tube is convenient for postoperative bleeding drainage and management,and there is still no consensus on the choice of postoperative removal time for short-segment lumbar spine surgery with less risk. OBJECTIVE:To explore the effect of different drainage times on early clinical efficacy after short-segment lumbar fusion. METHODS:A prospective randomized controlled study was performed on 220 patients in the Affiliated Hospital of Southwest Medical University who underwent posterior lumbar interbody fusion for lumbar degenerative diseases from March 2017 to April 2021.According to the different drainage times,the patients were randomly divided into removal on the second day after operation(group A),removal on the third day after operation(group B),and removal after the observation method 24-hour drainage volume<30 mL(group C).The perioperative indicators and follow-up results of the three groups of patients were observed and compared. RESULTS AND CONCLUSION:(1)Because 7 patients were lost to follow-up,2 patients were excluded,and 211 patients were finally included(72 patients in group A,71 patients in group B,and 68 patients in group C).(2)The average drainage time of group C was 2.91 days.The postoperative drainage volume in group A was significantly less than that in groups B and C,and the difference was statistically significant(P<0.05).On day 3 after operation,the hematocrit value of group C was lower than that of group A and group B,and the difference was statistically significant(P<0.05).Postoperative activity time and hospital stay in group A were shorter than those in groups B and C,and the difference was statistically significant(P<0.05).(3)Four patients in group A,two patients in group B and three patients in group C received an allogeneic blood transfusion.There was no significant difference among the groups(P>0.05).(4)In terms of postoperative complications,there were no statistical differences in postoperative wound leakage and surgical site infection in all three groups(P>0.05).(5)All patients were followed up for more than 12 months.Visual analog scale score and Oswestry dysfunction index of the three groups of patients before discharge and at the last follow-up were significantly improved compared with those before surgery(P<0.05).There was no statistical significance among the groups(P>0.05).(6)It is indicated that the removal of the drainage tube on the second day after a posterior lumbar fusion can effectively reduce the time to get out of bed and hospital stay,without increasing the postoperative blood loss and the risk of complications.

OBJECTIVE: To investigate the expression and clinical significance of PD-L1 and microRNA-138-5p in the peripheral blood mononuclear cells of patients with acute myeloid leukemia. METHODS: The SYBR GreenⅠreal-time PCR was used to detect the expression levels of PD-L1 mRNA and miR-138 in 20 cases of primary AML, 9 cases of relapsed/refractory AML and 8 cases of complete remission. The samples of peripheral blood of 20 healthy peoples were used as controls. RESULTS: The expression levels of PD-L1 in both the primary AML and the relapsed/refractory AML groups were significantly higher than those in the healthy control group (P<0.01), and the expression level of PD-L1 in the relapsed/refractory AML group was significantly higher than that in the primary AML group (P<0.01). The expression level of miR-138 in both the primary AML and the relapsed/refractory AML groups were significantly lower than that in the healthy control group (P<0.01). The 8 sampes out of 20 cases of primary AML patients achieved complete remission (CR) were collected and detected. The results showed that the expression level of miR-138 in the complete remission group was higher than that in the primary AML group (P<0.05), but the expression level of PD-L1 mRNA in the CR group was not significantly different from that in the primary AML group (P>0.05). and there was a negative correlation between PD-L1 mRNA and miR-138 in primary AML patients (P<0.05). CONCLUSION The expression of PD-L1 increases and the expression of miR-138 down-regulates in PBMNCs of AML patients, furthermore, both correlate each other.
B7-H1 Antigen ; genetics ; Humans ; Leukemia, Myeloid, Acute ; genetics ; Leukocytes, Mononuclear ; MicroRNAs ; genetics ; Remission Induction

OBJECTIVE: To analyze the changes in the gene expression profile of T cells in CML patients after TCRζ up-regulation expression, and to explore the molecular mechanism of T cell reactivation after transgenic up-regulation of TCRζ. METHODS: The peripheral blood mononuclear cells(PBMCs) from 3 newly untreated chronic-stage CML patients were collected, and the CD3 RESULTS: A total of 2248 differentially-expressed genes were obtained, including 553 up-regulated genes and 1695 down-regulated genes in experimental group as compared with those in control group (P<0.05) . The GO and KEGG enrichment analyses showed that differentially expressed genes involved in the biological processes related to T cell immune function, such as TCR signaling pathway, T cell proliferation and activation. Some of core genes involved in promoting the TCR signaling pathway, T cell proliferation, activation and apoptosis pathways were significantly up-regulated, while some core genes involved in inhibiting T cell activation were significantly down-regulated. CONCLUSION The molecular mechanism of the significantly improved T cell activation and proliferation ability in CML patients after TCRζ up-regulation may be related to the differential transcripts mediated signaling pathways of T cell activation, proliferation and apoptosis.
Humans ; Leukocytes, Mononuclear ; Lymphocyte Activation ; Receptors, Antigen, T-Cell/genetics* ; T-Lymphocytes ; Up-Regulation

This study investigated the choroplast genome sequence of wild Atractylodes lancea from Yuexi in Anhui province by high-throughput sequencing, followed by characterization of the genome structure, which laid a foundation for the species identification, analysis of genetic diversity, and resource conservation of A. lancea. To be specific, the total genomic DNA was extracted from the leaves of A. lancea with the improved CTAB method. The chloroplast genome of A. lancea was sequenced by the high-throughput sequencing technology, followed by assembling by metaSPAdes and annotation by CPGAVAS2. Bioiformatics methods were employed for the analysis of simple sequence repeats(SSRs), inverted repeat(IR) border, codon bias, and phylogeny. The results showed that the whole chloroplast genome of A. lancea was 153 178 bp, with an 84 226 bp large single copy(LSC) and a 18 658 bp small single copy(SSC) separated by a pair of IRs(25 147 bp). The genome had the GC content of 37.7% and 124 genes: 87 protein-coding genes, 8 rRNA genes, and 29 tRNA genes. It had 26 287 codons and encoded 20 amino acids. Phylogenetic analysis showed that Atractylodes species clustered into one clade and that A. lancea had close genetic relationship with A. koreana. This study established a method for sequencing the chloroplast genome of A. lancea and enriched the genetic resources of Compositae. The findings are expected to lay a foundation for species identification, analysis of genetic diversity, and resource conservation of A. lancea.
Phylogeny ; Atractylodes/genetics* ; Genome, Chloroplast ; Whole Genome Sequencing ; Microsatellite Repeats ; Lamiales