BACKGROUNDInteractions of tumor cells with the microenvironment were deemed to promote the tumor invasion and metastasis. CXC chemokine receptor 4 (CXCR4) and extracellular matrix metalloproteinase inducer (EMMPRIN) had reported to participate in this process. However the roles of bone marrow microenvironment in leukemic infiltration were not well investigated.

METHODSA co-culture system between SHI-1 cells and bone marrow stromal cells (BMSCs) is used to simulate the interactions of leukemic cells with their microenvironment. The trans-matrigel invasion was used to detect the capability of SHI-1 cells invasion. The BMSCs and SHI-1 cells were mixed in a ratio of 1:10 and added to the millicell chamber coated with matrigel. Either the co-culture supernatant or the functional blocking peptide of CXCR4 and EMMPRIN were added to the trans-matrigel invasion system. The expressions of EMMPRIN in SHI-1 cells and BMSCs were detected by RT-PCR. The changes of the expression of matrix metalloproteinase-2, 9 (MMP-2, MMP-9), tissue inhibitor of metalloproteinase 2 (TIMP-2), and CXCR4 mRNA in SHI-1 cells were determined by real-time PCR. The concentration of stromal cell derived factor 1 (SDF-1) in serum free supernatant was measured by ELISA.

RESULTSBoth SHI-1 cells and BMSCs express EMMPRIN. SHI-1 cells could hardly invade the matrigel membrane; the coculture supernatant did not induce the invasion of SHI-1 cells. When contacting directly with BMSCs, SHI-1 cells invaded to the lower chamber of millicell were significantly increased. The functional blocking peptide of CXCR4 and EMMPRIN could significantly inhibit the invasion triggered by BMSCs. When co-culturing with BMSCs, the expression of CXCR4, MMP-2, MMP-9 and TIMP-2 mRNA in SHI-1 cells were significantly elevated in company with a significantly higher level of SDF-1 in the co-cultured serum-free supernatant.

CONCLUSIONThe interactions of leukemic cells and BMSCs play important roles in leukemic cell infiltration.

Basigin ; physiology ; Cell Communication ; Cell Line, Tumor ; Coculture Techniques ; Humans ; Leukemia, Monocytic, Acute ; pathology ; Mesenchymal Stromal Cells ; physiology ; Neoplasm Invasiveness ; Receptors, CXCR4 ; physiology

OBJECTIVETo investigate the influence of tissue inhibitor of metalloproteinase 2 (TIMP-2) on the infiltrative patterns of human monocytic leukemic cell line SHI-1 in nude mice.

METHODS1) 1 x 10(7) TIMP-2 gene transduced SHI-1 (SHI-1-TIMP-2) and SHI-1 transduced MSCV gene (SHI-1-MSCV) cells were inoculated via tail vein into 6-week nude mice, which pretreated by splenectomy, cytoxan intraperitoneal injection, and sublethal irradiation(referred as SCI nude mice). 30 days after inoculation, half of the mice were sacrificed, and the infiltration patterns were investigated by histological exam and human CD45 immunohistochemistry, other mice were observed for survival time. 2) Leukemic cells inoculated subcutaneously into the axillary area of mice without any pre-treatment. On day 23 and 30, mice were sacrificed to measure the volume of neoplasm. TIMP-2 protein expression and the micro vein density were detected by immunohistochemistry.

RESULTSIn SCI nude mice inoculated via caudal vein with SHI-1-TIMP-2 cells, the survival time was shorter and infiltration (including in central nervous system) was higher than that in those inoculated with SHI-1-MSCV cells. However, in inoculated subcutaneously group, the neoplasm though grew rapidly at first, over expression of TIMP-2 limited the tumor growth and angiogenesis.

CONCLUSIONThe functions of TIMP-2 are diversity; the role of TIMP-2 in tumor infiltration and metastasis was worthy of further investigation.

Animals ; Cell Line, Tumor ; DNA, Complementary ; genetics ; Genetic Vectors ; Humans ; Leukemia, Experimental ; genetics ; pathology ; Leukemic Infiltration ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Tissue Inhibitor of Metalloproteinase-2 ; genetics ; Transfection

Objective Sperm screening is an essential step in IVF procedures. The swim-up method, an assay on sperm motility, is used clinically to select the ideal sperm for subsequent manipulation. However, additional parameters, including acrosome reaction capability, chemotaxis, and thermotaxis are also important indicators of mammalian sperm health. To monitor both sperm motility and chemotaxis simultaneously during sperm screening, we designed and constructed a microdevice comprising a straight channel connected with a bi-branch channel that mimics the mammalian female reproductive tract. Methods The width and length of the straight channel were optimized to select the motile sperm. Cumulus cells were selectively cultured in the bi-branch channel to generate a chemoattractant-forming chemical gradient. Sperm chemotaxis was represented by the ratio of the sperm swimming towards different branches. Results The percentage of motile sperm was improved from ( 58. 5 ± 3. 8 ) % to ( 82. 6±2.9)% by a straight channel 7 mm in length and 1 mm in width. About 10% of sperm were found chemotactically responsive in our experiment, which is consistent with previous studies. Conclusion The combined evaluation of both sperm motility and chemotaxis was achieved for the first time, and the motile and chemotactically responsive sperm can be easily enriched on a lab-on-a-chip device to improve IVF outcome.

OBJECTIVETo evaluate the prevalence and distribution of C-kit, NPM1 and FLT3 gene mutations in patients with acute myeloid leukemia (AML), and to analyze the relationship between the gene mutations and their prognosis.

METHODSMutations in exon 8 and 17 of C-kit gene, exon 12 of NPM1 gene, exon 20 of FLT3-TKD gene, and exon 14/15 of FLT3-ITD gene were detected by direct sequencing. Clinical data was collected and followed up if the patient had accepted treatment in our hospital.

RESULTSAmong the 656 AML patients, mutations in C-kit exon 8 were found in 6 patients (0.9%), C-kit exon 17 in 33 (5.0%), NPM1 in 169 (25.8%), FLT3-TKD in 46 (7.1%), and FLT3-ITD in 178 (27.1%). Six subtypes of mutations were detected in C-kit exon 8, 8 in C-kit exon 17, 11 in FLT3-TKD, 15 in NPM1, of which 5 were not reported before. C-kit exon 17 mutations were more frequently detected in patients with t(8;21) and exon 8 in patients with inv(16) cytogenetic abnormality. No other gene mutations except FLT3 were detected in M(3) patients. NPM1 and ITD mutations were often detected in individuals with normal cytogenetics or M(5) and M(1) of FAB classification, and accompanied with high white blood cell counts in peripheral blood, high blast counts in bone marrow and low CD34 expression. The older the patients were when diagnosed, the more gene mutations and the higher white blood cell count were detected. More mutations were found in individuals with normal karyotype than that with other karyotypes. It appeared that FLT3-ITD was significantly associated with shorter overall survival (OS) (P = 0.004), NPM1 was not significantly associated with OS, but NPM1(+)/ITD(-) patients had the longest OS.

CONCLUSIONSOur results showed that the mutation types and amounts had particular distribution in MICM subtypes, and were associated with white blood cell counts in peripheral blood, blast counts in bone marrow and prognosis. Especially for patients with normal karyotype, the genetic mutations could be new molecule marker.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; genetics ; DNA Mutational Analysis ; Female ; Humans ; Karyotyping ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Male ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics ; Prognosis ; Proto-Oncogene Proteins c-kit ; genetics ; Young Adult ; fms-Like Tyrosine Kinase 3 ; genetics

OBJECTIVETo investigate the expression of AML1/ETO9a isoform in the acute myeloid leukemia (AML)-M2 patients.

METHODSExpressions of AML1/ETO fusion gene and AML1/ETO9a isoform were detected by using reverse transcriptase-polymerase chain reaction (RT-PCR) in leukemia patients, MDS patients, leukemia cell lines and healthy subjects. Karyotype was studied by R-banding technique.

RESULTIn 30 newly diagnosed AML-M2 patients 15 were found to express AML1/ETO9a isoform, while the rest including 20 AML-M2CR, 18 other subtypes of AML, 5 chronic myelogenous leukemia (CML), 3 myelodysplastic syndromes (MDS), 3 leukemia cell lines (NB4, KG-1, K562) and 5 healthy subjects were AML1/ETO9a negative. Among the 15 AML/ETO9a isoform expressing cases, 13 were demonstrated t(8;21) translocation and AML1/ETO expression.

CONCLUSIONIsoform AML1/ETO9a was correlated to AML/M2, and it may promote the development of leukemia in combination with the AML1/ETO fusion gene.

Adolescent ; Adult ; Aged ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Female ; Gene Expression ; Humans ; Karyotyping ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; metabolism ; Protein Isoforms ; genetics ; metabolism ; RUNX1 Translocation Partner 1 Protein

OBJECTIVETo analyze chlorogenic acids contained in Lonicera japonica water extracts, and to investigate the regularity of changes in chlorogenic acids during the water extraction process.

METHODAgilent Zorbax SB-C18 analytical column (4.6 mm x 250 mm, 5 microm) was eluted with 0.1% formic acid (A)-acetonitrile (B) as the mobile phase at a flow rate of 0.5 mL x min(-1). The detection wavelength was 327 nm and the column temperature was 30 degrees C. Negative MS(n) mode was adopted in mass spectrum for ananlyzing the 22 samples of L. japonica water extracts.

RESULTCaffeic acid and six organic acids were accurately identified from the water extracts. During the extraction, the contents of chlorogenic acid and 3,5-dicaffeoylquinic acid became stable or gradually decreased after reaching the highest value. The contents of other components had long been increasing, but with a decreasing rate of change.

CONCLUSIONThis study provides basis for improving the production process of traditional Chinese medicine preparations containing L. japonica.

Chlorogenic Acid ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Lonicera ; chemistry ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization ; methods ; Tandem Mass Spectrometry ; methods

OBJECTIVETo find out the distributed characteristics of KIR2DL1 alleles frequencies and the recognition HLA-C ligand in the Chinese Han population.

METHODSThe 111 patients and 116 donors from CMDP were performed the KIR2DL1 high-resolution typing and KIR genotyping using sequence-based testing (SBT) and PCR-SSP methods.

RESULTSA total of 224 individuals with KIR2DL1 locus was predominantly observed and accounted for 98.68% (224/227). There were 3 different KIR2DL1 alleles, including KIR2DL1*00302, *00201 and *00401 alleles polymorphism. The most common phenotype observed were KIR2DL1*00302 (84.82%, 380/448), KIR2DL1*00201 (12.05%, 54/448) and KIR2DL1*00401(3.13%,14/448), present at allele genotype frequencies of 61.04%,6.22% and 1.58% respectively. The allele homozygotic types of KIR2DL1*00302 and KIR2DL1*00302 were the most frequent in 6 KIR2DL1 allele by high resolution typing. The allele heterozygous types of KIR2DL1*00302 and KIR2DL1*00401 presented statistically different in haplotypes A/A and B/x (P=0.001), and KIR2DL1*00401 lacked of all A/A haplotype. The KIR2DL1*00302 and KIR2DL1*00201 allele had significant positive associations between different KIR pairs of KIR2DS1, KIR2DL3, KIR2DS4 and KIR3DL1/S1 using linkage disequilibrium analysis (P<0.01), respectively. In the receptor-ligand of KIR/HLA model after allo-HSCT, KIR2DL1*00302 alleles correlated with their HLA-C2 group ligands. KIR2DL1*00302 and HLA-C*06:02 was the most common combination ligand model, but KIR2DL1*00302 and HLA-C*01:02 was the most frequent mismatch ligand model with the development of NK cell-induced alloreactivity, meanwhile there was statistically significant difference of frequency distribution (P<0.05).

CONCLUSIONThe KIR2DL1*00302 was the most frequent allele in Chinese Han population. The KIR2DL1 high resolution typing would be beneficial for predicting donor NK cells all activity after hematopoietic stem cell transplantation and selecting suitable donors.

Alleles ; Asian Continental Ancestry Group ; genetics ; Gene Frequency ; Genotype ; HLA-C Antigens ; genetics ; Haplotypes ; Histocompatibility Testing ; Humans ; Ligands ; Polymorphism, Genetic ; Receptors, KIR2DL1 ; genetics

OBJECTIVETo identify the distribution and differentiation of ABL kinase domain mutation in the Chinese Han nationality imatinib resistant chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+)ALL).

METHODSBone marrow or peripheral blood samples of 112 imatinib resistant CML patients and 21 Ph(+)ALL patients were obtained from the first affiliated hospital of Soochow university according to local law. Total RNA was extracted from the mononuclear cells using a TRIzol reagent. ABL kinase domain (KD) mutation was detected by direct sequencing.

RESULTSOf the 112 imatinib resistant CML patients, 54.46%(61 cases) had ABL KD mutation. Twenty-three mutants were identified in 20 amino acid sites and 23.21% (26 cases) ABL KD mutations were in P-loop region. ABL KD mutations were also detected in 71.43% (15 cases) imatinib resistant Ph(+)ALL patients, with 10 mutations in 8 amino acid sites. The most frequent mutation was T315I (28.57%), followed by E255K/V (19.05%) and Y253F/H (14.29%). The frequency of T315I was much higher in imatinib resistant Ph(+) ALL than that in imatinib resistant CML (P = 0.001). Ph(+)ALL with additional chromosomal aberrations also had a higher rate of ABL KD mutation than that of CML (P = 0.010). Ph(+)ALL gained ABL KD mutation faster than CML (P < 0.010).

CONCLUSIONChinese imatinib resistant CML and Ph(+)ALL patients had different characteristics in ABL KD mutation. The rate of ABL KD mutation in Ph(+)ALL with additional chromosomal aberrations was much higher than that of CML with additional chromosomal aberrations.

Adolescent ; Adult ; Aged ; Asian Continental Ancestry Group ; genetics ; Benzamides ; pharmacology ; Chromosome Aberrations ; Drug Resistance, Neoplasm ; genetics ; Female ; Humans ; Imatinib Mesylate ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Middle Aged ; Mutation ; Philadelphia Chromosome ; Piperazines ; pharmacology ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Protein-Tyrosine Kinases ; genetics ; Proto-Oncogene Proteins c-abl ; genetics ; Pyrimidines ; pharmacology ; Young Adult

OBJECTIVETo explore the incidence of dementia among elderly people in Xi'an and its related risk factors.

METHODSSubjects that had been studied on the prevalence of dementia were follow-up, and the incidence of dementia, Alzheimer disease (AD) , and vascular dementia (VD) were counted by person-years. The clinical diagnosis on dementia,AD and VD were based upon the 3rd Edition of Diagnostic and Statistical of Manual of Mental Disorder, Revised version (DSM-III-R) and NINCDS-ADRDA criteria.

RESULTS2197 subjects of non-dementia being identified in 1998, were re-surveyed in 2001. Out of them, 47 new cases of dementia including 37 cases of AD and 8 cases of VD were identified, with an annual incidence rates of dementia, AD and VD as 0.68%, 0.54% and 0.12% among those of 55 years and over and 0.89%, 0.69% and 0.17% in 65 years and over, respectively. Analysis from single factor logistic regression showed that age and education but not gender were closely related to the occurrence of AD. On the contrary, age, hypertension and stroke were closely related to the occurrence of VD.

CONCLUSIONThe incidence of dementia in the "Xi' an cohort" was similar to that being reported from other countries. AD and VD were an age-related diseases but education seemed to have had great protective effect while lack of formal education served as risk factor to AD.

Age Distribution ; Aged ; Aged, 80 and over ; China ; epidemiology ; Dementia ; classification ; epidemiology ; Educational Status ; Female ; Follow-Up Studies ; Humans ; Logistic Models ; Male ; Middle Aged ; Multivariate Analysis ; Risk Factors ; Sex Distribution

OBJECTIVETo detect the frequencies of KIR2DS4 alleles in Chinese Han population and to study the impact of KR2DS4 alleles on clinical outcomes of HLA identical unrelated allo-HSCT.

METHODSA Sequence-Based Testing (SBT) and TOPO TA cloning system for identifying and distinguishing alleles of the KIR2DS4 gene were established. A total of 150 Chinese-Han individuals, including 75 leukemia patients who received allo-HSCT and their HLA high-resolution typing identical unrelated donors (URD) were entered this study. The patients underwent transplantation for CML (n = 24), AML (n = 19), ALL (n = 29) and other malignancies (n = 3).

RESULTSThe majority (139) of the 150 samples (92.7%) were positive for KIR2DS4. Sequencing of the whole length coding region of this gene identified four of the 12 known KIR2DS4 alleles, KIR2DS4*00101, *003, *004, and *007. 2DS4*00101 was the most frequent, being found in 109 of the 139 individuals (78.4%). The ratio of deleted to non-deleted versions of KIR2DS4 was approximately 1:2. Three novel KIR2DS4 alleles were identified. Transplantations from KIR haplotype B/x donors showed significantly higher overall survival rates than those from KIR haplotype A/A donors [RR 3.1 (95%CI 1.1 - 8.6), P = 0.007]. There was a lower overall survival rates in recipients when their donors carried two 2DS4 full-length allele (2DS4*001) than those carried less (0 or 1) 2DS4*001 allele (P = 0.031). In the haplotype A/A group, a higher risk of acute GVHD (aGVHD) [RR 9.0 (95%CI 1.2 - 66.9), P = 0.01], especially grade III-IV aGVHD (P = 0.006), was seen when the donor was homozygous for the full-length KIR2DS4*00101 allele.

CONCLUSIONThe development and application of the described SBT 2DS4 allele typing method highlights the diverse nature of the KIR gene family and displays the existence of KIR polymorphism that remains uncharacterized. Our findings suggest that KIR typing for 2DS4 be beneficial for selecting suitable donors.

Alleles ; Graft vs Host Disease ; genetics ; Haplotypes ; Hematopoietic Stem Cell Transplantation ; Humans ; Receptors, KIR ; genetics