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

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

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.

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 report a rare variant of t(15;17), ins(17;15)(q21;q14q22) in an acute promyelocytic leukemia (APL) patient and the results of cytogenetic and molecular genetic studies.

METHODSChromosomes were prepared after 24 hours culture of bone marrow cells and peripheral blood cells. R-banding technique was used to analyze karyotypes. Chromosome painting analysis was performed using whole chromosome paints for chromosomes 15 and 17. PML-RAR alpha and RAR alpha-PML fusion transcripts were detected by reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSKaryotypic analysis using both specimens from bone marrow and peripheral blood leukemic cells revealed 15q- and 17q+. Chromosome painting analysis confirmed that the karyotypic abnormality was ins(17;15). PML-RAR alpha fusion transcript (S type) was detected by RT-PCR, while RAR alpha-PML fusion transcript was not detected.

CONCLUSIONChromosome painting and RT-PCR are reliable methods for characterization of the insertion involving chromosomes 15 and 17 in APL patients.

Adult ; Chromosome Painting ; methods ; Chromosomes, Human, Pair 15 ; genetics ; Chromosomes, Human, Pair 17 ; genetics ; Humans ; Leukemia, Promyelocytic, Acute ; diagnosis ; genetics ; Male ; Neoplasm Proteins ; genetics ; Oncogene Proteins, Fusion ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription, Genetic ; genetics ; Translocation, Genetic

OBJECTIVETo explore the value of dual-color fluorescence in situ hybridization (D-FISH) in the detection of inv(16) in acute myeloid leukemia (AML).

METHODSEleven AML patients were investigated by D-FISH with two-color break apart probe for MYH11 labeled directly by fluorescein isocyanate (FITC) and a Texas Red. The results were associated or compared with those of cell morphology, cytogenetics, single color fluorescence in situ hybridization (FISH) and reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSFour cases (M4Eo three cases, M2a one case) had inv(16), of which one had trisomy 22 in addition to inv(16), while the other seven cases had no inv(16), of which, five cases (M4Eo three cases, M4 two cases)had a normal karyotype, one (M2a) had 5p+ and trisomy 22, one (M4Eo) had a translocation t(9;22) on G-banded karyotypic analysis. All 11 cases of AML were positive for the rearrangement of inv(16) detected by D-FISH. The average positive cell rate for these 11 AML patients was 93.45% (range 86.6%-98.7%). Of them, four had a minimal deletion of 16p13 in addition to inv(16). The results of D-FISH coincided with those of RT-PCR or single color FISH.

CONCLUSIOND-FISH is a powerful tool for the detection of inv(16) due to its sensitivity and specificity. For raising the detecting rate of inv(16), it is necessary to screen inv(16) rearrangement by D-FISH in all M4- and M2-AML cases or the cases with trisomy 22, no matter whether they are accompanied by bone marrow eosinophilia.

Chromosome Inversion ; Chromosomes, Human, Pair 16 ; genetics ; Female ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Leukemia, Myeloid, Acute ; genetics ; Male ; Reverse Transcriptase Polymerase Chain Reaction

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 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

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 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