Adolescent ; Adult ; Blood Pressure ; Gravity Suits ; Heart Function Tests ; Humans ; Male ; Positive-Pressure Respiration ; Young Adult

Objective To evaluate the feasibility of monitoring the gene expression of VEGF165 via the diglycylcysteine (GGC) reporter gene system by reporter probe of 99Tcm-GH. Methods DNA fragments encoding GGC binding motifs were prepared by PCR and positioned at the C end of VEGF165 gene after the linearization of pcDNA3-VEGF165 plasmid. A replication-defective adenovirus vector Ad5-VEGF165GGC motif-internal ribosomal entry site(IRES) -enhanced green fluorescent protein (EGFP) (Ad5-VIE)was constructed, with a cytomegalovirus (CMV) early promoter driving the expression of VEGF165 gene,GGC motif and EGFP, under the aid of an IRFS. A replication-defective adenovirus carrying the Ad5-EGFP was used as the control. Mensenchymal stem cells (MSC) were infected with the recombinant adenovirus at a multiplicity of infection (MOI) from 0 to 100 infectious units (0,10,25,50,100). The cellular uptake of 99Tcm-GH in infected MSC were then studied at 30, 60, 90 and 120 min. VEGF165 was detected by quantitative reverse transcriptase real-time PCR (RT-PCR), Western-blot, and immunohistochemisty. EGFP was observed by RT-PCR and fluorescence microscopy. The correlation analysis was studied between the cellular uptake of 99Tcm-GH and the expression of VEGF165. SPSS 13.0 was applied for statistical analysis. Independent samples t-test, q-test and Pearson correlation coefficient were used. Results After infected with different viral titer of Ad-VIE, the cellular uptake of 99Tcm-GH increased with the increasing virus titer(r2 =0.86, P ＜0.05), with the peak rate (7.94 ±0.75) % at MOI = 100. In time-dependent uptake study, the cellular uptake rates increased rapidly with the time extension, and the highest uptake occurred at 120 min with the peak uptake rate (7.72 ±0.22)%. The uptake rates of 99Tcm-GH in Ad5-VIE-infected cells were significantly higher than those of Ad5 -EGFP-transfected cells at all time points (t = 15.10- 54.92, all P ＜0.05). The VEGF165 and EGFP mRNA levels increased with increasing virus titer, and the VEGF165 mRNA correlated well with the EGFP mRNA(r2 = 0. 99, P ＜ 0.05). After infected with different MOI of Ad5-VIE, good relationship was found between the cellular uptake of 99Tcm-GH and the expression of VEGF165protein in MSC(r2 =0.90, P ＜0.05). Inmunohistochemisty showed VEGF165 protein expressed obviously at Ad5-VIE-infected MSC, and the EGFP was observed by fluorescence microscopy. Conclusions The cellular uptake of 99Tcm-GH in Ad5-VIE-infected MSC are well correlated with the expression of VEGF165 in vitro. The expression of therapeutic gene VEGF165 can be monitored by the GGC peptide expression.

Child, Preschool ; Chromosomes, Human, Pair 22 ; Chromosomes, Human, Pair 8 ; Chromosomes, Human, Pair 9 ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; Translocation, Genetic

OBJECTIVETo analyze the genetic relations of Shigella isolated from Shenzhen in 2001-2006 and develop primary molecular subtyping surveillance network of Shigella.

METHODSChromosomal DNAs from 55 isolated in agarose were digested with the restriction enzyme Xba I, and then were analyzed by pulsed-field gel electrophoresis. Pulsed-field gel electrophoresis (PFGE) patterns were clustered using BioNumerics software.

RESULTSAll 41 distinctive PFGE patterns were identified among 55 strains. 32 strains belonged to one cluster. Differences were observed in other strains.

CONCLUSIONBoth genetic-related clones and non-related clones of Shigella existed in Shenzhen. The development of PFGE molecular subtyping surveillance network would contribute to the active surveillance, outbreak investigation and source tracking for Shigellosis.

Bacterial Typing Techniques ; China ; Electrophoresis, Gel, Pulsed-Field ; methods ; Feces ; microbiology ; Humans ; Shigella ; classification ; isolation & purification

OBJECTIVETo investigate clinical and molecule genetics features of four Ph-positive leukemia patients characterized by pericentric inv(9)(p22q34) with the der(9)t(9;22)(q34;q11).

METHODSCytogenetic analysis was carried out on bone marrow directly or after short-period culture. R banding was used for karyotype analysis. BCR/ABL fusion gene was detected with interphase fluorescence in situ hybridization (FISH), and chromosome painting was carried out using specific probes. RT-PCR was used to detect BCR/ABL chimeric transcripts.

RESULTSOne patient with acute myeloid leukemia (AML) presented three clones, which included one with a normal karyotype, one with t(9;22)(q34;q11), and one with inv(9)(p22q34) involving the der(9)t(9;22) and additional t(8;12)(q12;p11). The inv(9)(p22q34) has always co-occurred with der(9)t(9;22)(q34;q11) accompanied by der(22)t(9;22)(q34;q11) in all metaphases from the three patients with chronic myeloid leukemia (CML). B3a2 transcript was detected in all patients by RT-PCR. Inv(9)(p22q34) was found in both CML and AML, and was associated with poor prognosis.

CONCLUSIONInv(9)(p22q34) is a novel, rare, but recurrent secondary chromosomal abnormality for Ph-positive leukemia. Leukemia with der(9)t(9;22) and inv(9)(p22q34) has unique clinical and laboratory characteristics.

Adult ; Chromosome Inversion ; Chromosomes, Human, Pair 22 ; Chromosomes, Human, Pair 9 ; Female ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; genetics ; Leukemia, Myeloid, Acute ; genetics ; Male ; Middle Aged ; Translocation, Genetic

OBJECTIVETo perform immunophenotyping and molecular genetic analysis for diffuse large B-cell lymphoma (DLBCL), and to explore their correlation and implication for prognosis.

METHODSImmunohistochemical streptavidin peroxidase (SP) method was used to determine the expression of CD10, BCL6 and MUM1 in 59 cases of DLBCL. A Hans algorithm was used to classify DLBCL into germinal center B-cell (GCB) and non-GCB subtypes. Interphase fluorescence in situ hybridization (FISH) assay was performed on paraffin-embedded lymphoma tissue sections to detect translocations and amplifications of BCL6, BCL2 and MYC genes with dual-color break-apart BCL6 probe, dual-color dual-fusion IgH/ BCL2 probe and dual-color break-apart MYC probe, respectively.

RESULTSIn the 59 cases of DLBCL, 28.8% (17/59) belonged to GCB subtype, and 71.2% (42/59) belonged to non-GCB subtype. The incidences of BCL6, BCL2 and MYC gene translocations were 24.1% (14/58), 1.7% (1/59) and 5.3% (3/57), respectively. The incidences of BCL6, BCL2 and MYC gene amplifications were 17.2% (10/58), 22.0% (13/59) and 21.1% (12/57), respectively. BCL6 amplification was not correlated with BCL6 translocation (P=0.424), but was correlated with amplifications of BCL2 and MYC (C=0.405 and 0.403, respectively, P <0.01). The incidence of BCL6 translocation in GCB type was higher than that in non-GCB type, and amplifications of BCL6, BCL2 or MYC were more frequently encountered in non-GCB type, though no statistical significance was detected (P=0.089 and 0.106, respectively). By univariate analysis, immunophenotyping and international prognostic index (IPI) exerted a significant effect on overall survival (OS) (P=0.047 and 0.001, respectively), but to which BCL6 translocation and amplification of the 3 genes were not related (P=0.150 and 0.444, respectively). By multivariate analysis, IPI score was the only independent prognostic factor for OS (RR =3.843, P=0.017).

CONCLUSIONThe GCB subtype of DLBCL is less common in the patient cohort. Common genetic aberrations have included BCL6 translocation and BCL6, BCL2 and MYC amplifications. Amplification of the 3 genes is strongly correlated with each other, and the incidence of BCL2 translocation is low. Immunophenotyping only has minor significance for the prognosis. Genetic aberrations cannot predict the clinical outcome of DLBCL.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; DNA-Binding Proteins ; genetics ; Female ; Genes, bcl-2 ; Genes, myc ; Humans ; Immunophenotyping ; In Situ Hybridization, Fluorescence ; Lymphoma, Large B-Cell, Diffuse ; genetics ; immunology ; Male ; Middle Aged ; Proto-Oncogene Proteins c-bcl-6

OBJECTIVETo report a case of myelodysplastic syndrome(MDS) with t(1;18)(p31;p11).

METHODSChromosome specimens were prepared by short-term culture of bone marrow cells. Karyotype analysis was made by R banding technique. Chromosome painting was performed using whole chromosome probes 1 and 18.

RESULTSConventional karyotype analysis revealed t(1;18)(p31;p11) in this patient. Chromosome painting analysis confirmed this result.

CONCLUSIONThe translocation of (1;18) was an unusual recurrent chromosome change and was reported on MDS for the first time.

Adult ; Chromosomes, Human, Pair 1 ; Chromosomes, Human, Pair 18 ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Male ; Myelodysplastic Syndromes ; genetics ; Translocation, Genetic ; genetics

To explore the value of detection of PML/RARalpha gene rearrangement on bone marrow smears (BMS) by dual-color fluorescence in situ hybridization (D-FISH) for the diagnosis of acute promyelocytic leukemia (APL), the locus-specific probes for PML and RARalpha genes labeled directly and respectively by Spectrum Green and Spectrum Orange and the D-FISH technique were used to detect the PML/RARalpha gene rearrangement on BMS in 27 suspected APL patients. The results were compared with that of conventional cytogentics (CCG) and reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that out of 18 newly diagnosed patients 14 were found having t(15;17) translocation by CCG and PML/RARalpha gene rearrangement were confirmed by BMS-D-FISH and RT-PCR. Thus, their APL diagnosis was determined; out of 4 patients in whom t(15;17) translocation was not detected by CCG, one had positive BMS-D-FISH and RT-PCR results, thus, this case was considered as having a cryptic t(15;17) translocation, three had negative BMS-D-FISH and RT-PCR results, thus, they were diagnosed as having acute myeloid leukemia rather than APL. In 9 cases with remission, one case with partial remission was found having t(15;17) translocation by CCG, and he had positive BMS-D-FISH and RT-PCR results, the other 8 patients (6 cases with normal karyotype and 2 cases without CCG examination) displayed different BMS-D-FISH and RT-PCR results: negative in 6 cases, but positive in 2 cases. The 2 cases were believed that they survived with minimal residual disease (MRD). It is concluded that BMS-D-FISH is a sensitive and reliable method for the detection of PML/RARalpha rearrangement. It is helpful for diagnosing APL and monitoring its MRD, and especially fit to those patients presenting a cryptic translocation or with failed cytogenetics, lacking suitable material for RT-PCR, as well as needing retrospective study.
Adolescent ; Adult ; Bone Marrow Cells ; metabolism ; Female ; Gene Rearrangement ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Leukemia, Promyelocytic, Acute ; diagnosis ; genetics ; Male ; Middle Aged ; Oncogene Proteins, Fusion ; genetics ; Receptors, Retinoic Acid ; genetics ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity

OBJECTIVETo determine the frequency of the derivative 9 [der(9)] deletion among chronic myeloid leukemia (CML) patients with classic and variant Ph translocations, and assess the correlation between this deletion and clinical prognosis.

METHODSCytogenetic analysis of bone marrow cells was performed by direct method and/or 24 h culture method. RHG banding was used for karyotype analysis. Dual-color and dual-fusion DNA probe was used to perform FISH for investigating the deletion of der(9) in Ph+ CML patients.

RESULTSCytogenetics studies showed typical Ph translocation in 76/105 and variant Ph translocation in 29/105 cases. Interphase-FISH studies showed deletion of der(9) in 12 (15.8%) of 76 patients with classic Ph translocation and in 4 (13.7%) of 29 patients with variant translocation. The frequency of deletion was similar in classic and variant translocations (P > 0.05). When the deletion was seen in the patient, it was present in all the Ph+ metaphases and nuclei. In 3 patients there were mixed cell populations with either 5'-abl or 3'-bcr deletion and all the 3 patients had both 5'-abl and 3'-bcr deletion. The median survival time of patients with deletion was significantly shorter than those without deletion (34 months vs 76 months; P < 0.05).

CONCLUSIONDeletion of der(9) is seen in about 1/6 of Ph+ CML patients in our study on Chinese CML patients, Ph+ CML patients with the deletion have shorter median survival time than those without it, indicating that it is a poor prognostic index.

Adolescent ; Adult ; Aged ; Child ; Chromosome Deletion ; Chromosomes, Human, Pair 9 ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; diagnosis ; genetics ; mortality ; Male ; Middle Aged ; Philadelphia Chromosome ; Prognosis ; Survival Rate ; Translocation, Genetic ; Young Adult

OBJECTIVETo investigate the application of pulsed-field gel electrophoresis (PFGE) in food-borne outbreak.

METHODSPathogens were isolated and further characteristics identified by traditional methods. The strains isolated were carried out with molecular typing with using PFGE. PFGE was performed by Laboratory Directions for molecular subtyping of Salmonella by PFGE (CDC, USA) and the results of PFGE were analyzed by BioNumerics soft.

RESULTSTotally 14 Salmonella serotype Muenchen strains were isolated from 19 patients, 3 of 9 suspicious foods were positive for S. muenchen and 7 strains were isolated from 18 cooks. The biochemistry characterization and antimicrobial susceptibility of all the strains isolated were the same. 23 S. muenchen isolates were all shown indistinguishable by PFGE.

CONCLUSIONPFGE should play a key role in identifying the outbreak-associated isolates and distinguishing them from unrelated sporadic isolates. It might also demonstrate that the genetic fingerprints of serotype Muenchen isolates derived from patients were indistinguishable from those derived from drinks. PFGE might provide precise information on bacterial food-borne pathogens, promptly identify the source of infection, and effectively prevent from spreading. It should be one of the early warning method on controlling outbreak of the food-borne disease.

China ; epidemiology ; Disease Outbreaks ; Electrophoresis, Gel, Pulsed-Field ; methods ; Humans ; Microbial Sensitivity Tests ; Salmonella Food Poisoning ; epidemiology ; microbiology ; Salmonella enterica ; classification ; isolation & purification ; Serotyping