Objective To analyze clinical diagnosis and management of 5 patients with actinomycete keratitis.Design Retro- spective case series.Participants 5 patients (5 eyes) with actinomycete keratitis.Methods The clinical features and microbiologic da- ta of 5 culture-proven cases of actinomycete keratitis recorded between October 2004 to March 2006 were analyzed.Main Outcome Measures clinical characteristics,isolations identification,drug susceptibility test and treatments.Results All patients were males and farmers.Of the 5 cases presented in this study,4 cases were followed by minor trauma as a predominant risk factor,and were pre- sented by a chronic progressive corneal ulcer with a wreath pattern of infiltrate.The diagnosis of all cases was based on laboratory in- vestigations,by which 4 cases of nocardia and one case of streptomyce were identified.A variable drug sensitivities were presented in nocardia isolates,which including TMP-SMZ,amicasin,gentamicin and fluorine-quinolones.Conclusions Nocardia keratitis is mainly followed by a minor trauma.It is identified predominantly by laboratory investigations.Tropical and systemically sensitive biotic are the initial choice,while debridement and amnionic transplantation could be an effective alternative.

OBJECTIVETo explore the killing effect of the mutant herpes simplex virus thymidine kinase (HSV-sr39tk) and its wild-type (HSV-tk) mediated by lentiviral vector on T lymphocytes in vitro and compare T cell survival rate after GCV or ACV treatment.

METHODSThe three-plasmid lentiviral vector system including packaging plasmid DeltaNRF, envelope plasmid VSV-G and vector plasmid (pTK151 + HSV-sr39tk or pTK151 + HSV-tk) were cotransfected into human embryonic kidney 293T cells using modified calcium phosphate precipitation methods. The packaged virus was harvested 72 h later. The survival of T cells expressing HSV-sr39tk or HSV-tk was measured by MTT assay after 4 day-culture against a gradient of GCV or ACV concentrations.

RESULTSThe three plasmids were effectively cotransfected and a high titre of lentivirus was obtained (2 x 10(6) IU/ml). 39tk(+) T cell survival rates declined promptly when the prodrug GCV/ACV concentrations increased from 0 micromol/L to 10 micromol/L. The T cell survival rates in GCV group declined from (96.04 +/- 3.23)% to (36.76 +/- 4.38)% while in ACV group from (97.31 +/- 4.61)% to (43.75 +/- 8.99)%. However, when GCV/ACV concentrations were more than 10 micromol/L, further decline of 39tk(+) T cell survival rates became unobvious. The growth rate of 39tk(+) T cell exposed to GCV or ACV was obviously lower than that in un-transfected T cells (P < 0.05). Tk(+) T cells were sensitive to GCV (P < 0.05), but not to ACV (P > 0.05). There was a significant difference in killing effects between 39tk(+) T cell + GCV group and tk(+) T cell + GCV group (P < 0.05).

CONCLUSIONThe lentiviral vectors containing HSV-sr39tk gene could infect T lymphocytes effectively and stably without affecting the proliferation of the transduced cell. In contrast to HSV-tk gene, T cells infected HSV-sr39tk were more sensitive not only to GCV but also to ACV.

Acyclovir ; pharmacology ; Animals ; Cell Survival ; drug effects ; Cells, Cultured ; Ganciclovir ; pharmacology ; Genetic Vectors ; Lentivirus ; genetics ; Mice ; Mice, Inbred C57BL ; Plasmids ; genetics ; T-Lymphocytes ; cytology ; drug effects ; Thymidine Kinase ; genetics ; Transfection

OBJECTIVETo construct a recombinant lentiviral vector (pXZ208-BDDhFVIII) mediating B-domain-deleted human coagulation factor VIII (BDDhFVIII) gene and investigate its expression in HLF, Chang-Liver and MSC cells.

METHODSBDDhFVIII gene fragment was separated by endonuclease digestion and was cloned into the multiple cloning sites of pXZ208 to construct a recombinant lentiviral vector pXZ208-BDDhFVIII. Viral particles were prepared by means of three-plasmid cotransfection of 293T package cells by calcium phosphate precipitation. After infection, the coagulant activity of human FVIII in the culture medium of 293T, HLF, Chang-Liver and MSC cells was assayed by one-stage method. The gene transduction efficiency was assayed by flow cytometry (FCM). Furthermore, PCR was performed to test the integration of BDDhFVIII.

RESULTSThe infection rates of HLF, Chang-Liver and MSC were (74.52 +/- 7.57)%, (27.24 +/- 6.53)% and (42.34 +/- 5.84)% respectively. The activities of FVIII in supernatants of HLF, Chang-Liver and MSC were (54.1 +/- 5.6)%, (22.5 +/- 2.9)% and (12.5 +/- 2.7)% respectively. BDDhFVIII gene integration was detected in all the infected cells.

CONCLUSIONThe recombinant lentiviral vector pXZ208-BDDhFVIII was successfully constructed and efficiently integrated into target cells to express human FVIII activity in vitro.

Cell Line ; Factor VIII ; biosynthesis ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Plasmids ; Transfection

Objective To investigate the distribution characteristics of methicillin-resistant Staphylococcus aureus (MRSA)in a children's hospital,and provide basis for the prevention and control of MRSA infection in children. Methods Children who admitted to a children's hospital from 2011 to 2015 were analyzed retrospectively,clinical data of children,isolation of pathogens,types of specimens,and healthcare-associated infection(HAI)status were analyzed.Results From 2011 to 2015,a total of 911 children isolated Staphylococcus aureus (SA,1108 positive specimens),494 of whom isolated MRSA (599 positive specimens),54.23% of children isolated MRSA(isolation rate of specimens was 54.06%);there was no significant difference in the isolation rate of MRSA between children of different genders(P > 0.05);isolation rate of MRSA in different age groups was statistically significant(P <0.05).Isolation rates of MRSA from blood,puncture fluid,secretion,and pus were 68.97%,66.00%,55.81%, and 54.47% respectively.Isolation rate of SA and MRSA increased from 0.61% and 21.74% in 2011 to 1.40%and 75.59% in 2015 respectively,difference were both significant(both P <0.05).Incidence of SA and MRSA in-creased from 0.198% in 2011 to 2.697% and 2.119% in 2015 respectively,both showed an upward trend year by year(both P <0.05).Conclusion Isolation rate of MRSA and incidence of HAI in this children's hospital increased year by year,it is necessary to intensify management,use antimicrobial agents scientifically and rationally,timelyperform disinfection and isolation,so as to curb the emergence and spread of MRSA in hospital settings.

OBJECTIVETo construct a three plasmids lentiviral vector containing canine coagulation factor IX (cFIX) gene with ubiquinone promoter (PUB) and observe the expression of cFIX gene.

METHODSLentivirus was generated by transient three-plasmid transfection, namely, the VSV-G envelope expression cassette, the delta NRF packaging plasmid and the PTK 164 plasmid. Viral particles were used to infect the target cell, third passage mesenchymal stem cells (MSCs) and 293T cell respectively at MOI 3: 1. The cFIX activity was detected in cultured cells with one-stage clotting assay.

RESULTSThe MSCs were obtained in vitro. The lentivirus infected MSCs and 293T cells all expressed the active factor IX with the activity of (26.30 +/- 2.10)% and (19.70 +/- 1.53)%, respectively, which are significantly higher than that of control (1.00 +/- 0.05)%.

CONCLUSIONSThe lentiviral vector of three plasmids with ubiquinone promoter (PUB) was constructed and can transfect the MSCs and 293T cells.

Animals ; Bone Marrow Cells ; metabolism ; Cells, Cultured ; Dogs ; Factor IX ; genetics ; metabolism ; Genetic Vectors ; Hemophilia B ; genetics ; metabolism ; Humans ; Lentivirus ; genetics ; Plasmids ; genetics ; Transfection

BACKGROUNDThe B7/CD28 pathway provides critical costimulatory signals for complete T cell activation, and members of this pathway have served as useful targets for immunotherapeutic strategies. In this study, we investigated the RNA interference (RNAi) effect induced by small interfering RNA (siRNA) targeting CD28 mRNA on human lymphocytes and its specificity.

METHODSAccording to CD28 gene sequence, we designed and synthysized three different siRNAs (siRNA-1, siRNA-2, siRNA-3) containing 21 bases using Silencertrade mark siRNA construction kit. These siRNAs were transfected into freshly isolated human lymphocytes with Lipofectamine 2000 reagent. At 24-hour, 48-hour and 72-hour post transfection, these cells were collected and analyzed. The changes of surface expression of CD28 gene were detected by flow cytometry, and the changes of CD28 mRNA levels were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The cell viability of transfected lymphocytes was determined by methyl thiazolyl tetrazolium (MTT) assay and trypan blue dye exclusion assay.

RESULTSThree siRNAs (siRNA-1, siRNA-2, siRNA-3) specifically targeting CD28 mRNA were successfully designed and constructed. Flow cytometry analysis showed that a decrease in CD28 expression was detectable at 24-hour post transfection. Different siRNA showed different inhibition effects on CD28 expression. At 48-hour post transfection, the degrees of reduction with siRNA-1, siRNA-2 and siRNA-3 were 22.10% +/- 1.63%, 73.50% +/- 1.02% and 42.90% +/- 0.89% respectively compared with the control (P < 0.001). Neither of the groups transfected only with siRNA or lipo showed marked reduction in CD28 expression (3.15% +/- 0.75% and 4.55% +/- 0.80%) (P > 0.05). Moreover, lymphocytes treated with siRNA-co showed no marked reduction in CD28 expression (5.07% +/- 0.96%) (P > 0.05). The results of semi-quantitative RT-PCR assay indicated CD28 mRNA level was inhibited after transfection of specific siRNAs. At least 4-fold of reduction in siRNA-2 group occurred at 48-hour post transfection compared with the control (P < 0.001). MTT assay and trypan blue dye exclusion assay demonstrated that the viable cell rations of transfected lymphocytes were significantly reduced in siRNA-1, siRNA-2 and siRNA-3 groups at 48-hour post transfection (P < 0.01). The control groups showed no marked reduction in cell viability (P > 0.05).

CONCLUSIONSThree different siRNAs were synthesized and transfected into lymphocytes. They could reduce the expression of CD28 and the CD28 mRNA level. siRNA-2 was the most efficient. The cell viability reduced correspondingly. Therefore, the silencing effect on CD28 mRNA induced by siRNA may contribute to costimulatory blockade. This result show that siRNA may be useful for further study on graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT).

Adolescent ; Adult ; CD28 Antigens ; genetics ; Cell Survival ; Cells, Cultured ; Flow Cytometry ; Gene Silencing ; Humans ; Lymphocytes ; metabolism ; RNA, Small Interfering ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction

The aim was to study minimal residual disease (MRD) in blood and bone marrow after complete remission of patients with acute myeloid leukemia (AML) and explore the role of MRD in detecting relapse of acute myeloid leukemia. The blood and bone marrow samples from 33 AML patients who had been in complete remission were determined for residual leukemic cells (RLC) with flow cytometry. The results showed that RLC in AML group of complete remission was higher than that of normal group both in blood by (4.7518 +/- 4.1537)% vs (0.4835 +/- 0.2005)% and bone marrow by (17.9082 +/- 20.4819)% vs (0.7285 +/- 0.2209)%, while the RLC in relapsed group was higher than that in non-relapsed group both in blood by (2.233 +/- 1.5923)% vs (10.2369 +/- 9.4714)% and bone marrow by (4.779 +/- 3.0336)% vs (38.0685 +/- 19.4295)%. In conclusion, early detection of leukemic residual cells with flow cytometry contributes to treatment of relapse in time.
Acute Disease ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow Cells ; pathology ; Female ; Flow Cytometry ; Humans ; Leukemia, Myeloid ; blood ; pathology ; therapy ; Male ; Middle Aged ; Neoplasm, Residual ; blood ; diagnosis ; Remission Induction

OBJECTIVETo establish strategies for preventing graft versus host disease (GVHD) and reducing treatment associated morbidity while preserving graft versus leukemia (GVL) effect in nonmyeloablative allogeneic bone marrow transplantation (allo-BMT), with or without donor lymphocyte infusion (DLI) after BMT.

METHODS3 x 10(7) bone marrow cells mixed with 1 x 10(7) spleen cells from the same BALB/c mouse were transplanted into the nonablative irradiated inbred 615 mouse which received a single subcutaneous injection of 1 x 10(6) L615 leukemia cells three days before. The experiments were designed as follows (ten mice in each group): myeloablative BMT control group (group A), nonmyeloablative conditioning without BMT group (group B), nonmyeloablative BMT group (group C), and nonmyeloablative BMT + DLI group (group D). GVL effects were assessed by survival time, white blood cell count and L615 cells in peripheral blood and histologic changes. GVHD was assessed by signs of weight loss, ruffled fur, diarrhea and histologic changes of skin, liver and small intestines. Chimerism was detected by cytogenetic analysis and PCR technique.

RESULTSThe survival time of group A, B, C and D was (20.3 +/- 13.4), (15.9 +/- 1.1), (21.6 +/- 1.7) and (37.8 +/- 2.0) days, respectively, being no significant difference between group A and group C (P > 0.05). The survival time of group C was longer than that of group B (P < 0.01). And among group B, C and D, group D had the longest survival time (P < 0.01). GVHD signs and histologic changes were observed in 60% of control group mice at + 14 day, but none of group C and group D. 40% of mice in group A died of treatment associated morbidity within two weeks, but none in group C and group D. Allogeneic chimerism was kept in group A, but excluded gradually in group C.

CONCLUSIONGVL effect seems preserved in nonmyeloablative BMT mice, but weaker than that in myeloablative BMT mice. GVL effect seems to be enhanced by DLI after nonmyeloablative BMT. GVHD and transplantation associated morbidity seems to be reduced in nonmyeloablative BMT.

Animals ; Bone Marrow Transplantation ; immunology ; methods ; Combined Modality Therapy ; Female ; Graft vs Host Disease ; prevention & control ; Graft vs Leukemia Effect ; Leukemia, Experimental ; therapy ; Leukemia, Lymphoid ; therapy ; Lymphocyte Transfusion ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Transplantation Conditioning ; methods ; Transplantation, Heterologous

This study was aimed to detect the methylation status of FHIT gene promoter region in the DNA from plasma of patients with myelodysplastic syndrome (MDS), and to investigate the demethylating effect of decitabine. Methylation-specific PCR method was used to detect the methylation status of FHIT gene promoter region in the DNA from plasma of 4 patients with MDS before and after treatment with decitabine plus semis CAG therapy (among them, 1 case of newly diagnosed MDS, 3 cases progressed into acute leukemia). The results indicated that 3 cases were found to have an increased methylation in the promoter region. After treatment with decitabine plus semis CAG, increased methylation was reversed in 2 cases. In 4 cases, 2 cases displayed clinical response. It is concluded that FHIT gene hypermethylation is associated with MDS pathogenesis. Decitabine has demethylating effect on the FHIT gene hypermethylation of plasma from MDS patients. Detecting the methylation status of FHIT gene in DNA from plasma may play a role in MDS auxiliary diagnosis or prognosis.
Acid Anhydride Hydrolases ; genetics ; Adult ; Aged ; Azacitidine ; analogs & derivatives ; therapeutic use ; DNA ; blood ; DNA Methylation ; Female ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; blood ; drug therapy ; Neoplasm Proteins ; genetics ; Promoter Regions, Genetic

OBJECTIVETo study the association between the polymorphism of human monoamine oxidase type A (MAO-A) gene and Parkinson's disease(PD).

METHODSFnu4HI restriction fragment length polymorphism(RFLP) and PCR-RFLP were used to detect the mutation of MAO-A gene. The frequencies of alleles and genotypes at the MAO-A Fnu4HI locus on the X chromosome in different PD group were compared with those of the control group.

RESULTSIt was found that the frequencies of G allele in the patients with PD and controls were 0.613 and 0.527 respectively, P=0.039 "the frequencies of TT genotype were 0.303 and 0.415(P=0.014), and the frequencies of GG genotype were 0.564 and 0.451 respectively(P=0.021). When the patients were divided into two groups by age-onset, significant difference in the allelic and genotypic frequencies was observed only between early-onset PD group and control group. And when the PD patients were grouped by sex, significant difference was observed only between male PD group and male control group (the frequencies of G allele being 0.669 and 0.500 respectively, P=0.005).

CONCLUSIONThis study revealed significant differences between PD group and control group in allelic and genotypic frequencies. The findings supported the hypothesis about an association between MAO-A gene and PD, suggesting that age at onset of PD and gender predisposition might be related to the putative association, and Fnu4HI SNP be a risk factor for PD.

Alleles ; Asian Continental Ancestry Group ; Deoxyribonucleases, Type II Site-Specific ; analysis ; genetics ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Monoamine Oxidase ; genetics ; Parkinson Disease ; genetics ; Polymorphism, Genetic ; genetics ; Polymorphism, Restriction Fragment Length