Tanshinol inhibits trophoblast cell migration and invasion by regulating Gadd45a in preeclampsia.

OBJECTIVE: Tanshinol is an active constituent of Salvia miltiorrhiza that possesses anti-inflammatory, antioxidant, and antibacterial activities. Therefore, this study attempted to detect whether it has a role in the treatment of preeclampsia (PE). METHODS: In this study, we explored the effect of tanshinol on the development of PE at the cellular level. The effect of tanshinol on cell proliferation was measured by colony formation and EdU assays. The migration, invasion, and in vitro angiogenesis of HTR-8/SVneo cells were detected by wound-healing, transwell, and tube formation assays, respectively. In addition, a PE cell model was established by overexpression of Gadd45a, and this cell model was assessed with the optimal concentration of tanshinol. RESULTS: The results show that tanshinol enhanced proliferation, migration, invasion, and tube formation of HTR-8/SVneo cells in vitro. Furthermore, the reduction in proliferation, migration, invasion, and tube formation of cells by Gadd45a overexpression was partially reversed by tanshinol treatment. Tanshinol also inhibited the apoptosis of HTR-8/SVneo cells transfected with Gadd45a. CONCLUSIONS: In summary, tanshinol promoted proliferation, migration, invasion, and tube formation and inhibited the apoptosis of HTR-8/SVneo cells. It may be a novel therapeutic compound to attenuate the development of PE.

Traditional Chinese medicine has maintained the health of people in Asia for thousands of years and is increasingly used worldwide. Tanshinol has been found to be useful in the treatment and prevention of many diseases. Through experiments, we found that tanshinol is a novel therapeutic compound that promotes the proliferation, migration, invasion and tubular formation of HTR-8/SVneo cells. In addition, tanshinol also inhibited the apoptosis rate of preeclampsia cell models. Follow-up experiments will further validate the results of this study.

Safety and Acceptability of Three Ablation Treatments for High-Grade Cervical Precancer: Early Data From a Randomized Noninferiority Clinical Trial.

PURPOSE: This ongoing trial is comparing the efficacy and safety of three ablation treatments for cervical intraepithelial neoplasia grade 2 or higher. Here, we present early data regarding pain, side effects, and acceptability of CO2 gas-based cryotherapy (CO2), nongas cryotherapy, and thermal ablation (TA). Efficacy results are expected to become available in late 2023. MATERIALS AND METHODS: This noninferiority randomized trial is taking place in El Salvador, China, and Colombia. Patients are 1,152 eligible women with biopsy-confirmed cervical intraepithelial neoplasia grade 2 or higher who will receive one of three ablation treatments. Pain is measured before, during, and after treatment with a visual analog scale (1-10). Side effects and acceptability are assessed at 6 weeks. RESULTS: To date, 1,024 of 1,152 (89%) women were randomly assigned to treatment. The median pain level was higher during TA (4, IQR = 4) than CO2 (2, IQR = 4) or nongas cryotherapy (2, IQR = 4) (P < .01, range: 0-10). The most common post-treatment symptom was watery discharge, reported by 97.9% of women, and it lasted longer in the CO2 group than the other two treatments (in days, median [IQR]: CO2 = 20[20], nongas cryotherapy = 15[10], TA = 18[15], P < .01). Bleeding was reported more frequently in women treated with TA (27.6%) than CO2 (17.5) or nongas cryotherapy (18.7%) (P < .01). The majority of patients reported being very satisfied with the treatment they received at 6 weeks (91%) and again at 12 months post-treatment (97%). CONCLUSION: Despite differences in pain and side effects across ablation treatments, all were safe and highly acceptable to patients. In addition to efficacy, considerations such as cost and portability may be more significant in choosing a treatment method.

Evaluation of Liquid Versus Dry Specimen Transport With a Newly Validated Isothermal Amplification High-Risk Human Papillomavirus Assay.

OBJECTIVE: The aim of the study was to determine whether the proportion of positive high-risk human papillomavirus (HR-HPV) tests in endocervical specimens transported dry differs from paired specimens transported in liquid media. METHODS: Five hundred women aged of 30 to 55 years were recruited, Shanxi Bethune Hospital, China. Two samples were collected from the endocervix per patient, one placed into empty vial, the other into a liquid transport solution. All samples were analyzed by AmpFire HR-HPV assay. RESULTS: Total 1,000 samples collected from 500 patients were analyzed by the AmpFire HR-HPV assay. The total invalid rate was 0.2% (2/1,000). The proportion of endocervical samples testing positive for HR-HPV transported dry (42.2%, 210/498 [95% CI = 37.8%-46.6%]) was similar to the proportion of paired endocervical samples testing positive transported in liquid media (40.4%, 201/498 [95% CI = 36.0%-44.8%], p = .18 [McNemar test]). That the 2 transport methods are likely measuring the same positive (and negative) specimens is suggested by the finding that κ value for the correlation of positive HR-HPV in endocervical specimens transported dry with those transported in liquid media was 0.86 (95% CI = 0.81-0.90). CONCLUSIONS: Endocervical specimens transported dry have similar proportion of positive HR-HPV tests as those transported in liquid media. Dry brush transport of endocervical samples paired with the special characteristics of AmpFire HR-HPV may become an important addition to population based cervical cancer screening.

Pregnancy in a woman with untreated bladder exstrophy: a case report.

OBJECTIVE: To report the management of urinary tract obstruction and infection in a pregnant woman with unrepaired bladder exstrophy. CASE REPORT: A 27-year-old pregnant woman with unrepaired bladder exstrophy was referred to our hospital with a complaint of bilateral flank pain in the second trimester. After two-dimensional abdominal ultrasound, magnetic resonance imaging and a urine analysis, she was diagnosed with an upper urinary tract infection due to ureteral obstruction secondary to unrepaired congenital bladder exstrophy and an intrauterine pregnancy. J-tube insertion was performed after locating the ureteral orifices and antibiotics were administered. Symptoms rapidly resolved. She delivered a normal male infant by caesarean section at 34 weeks of gestation. CONCLUSION: Standard urological management of the ureteral obstruction in pregnancy was successful in this extreme case of unrepaired bladder exstrophy associated with an intrauterine pregnancy. The perinatal outcome was good.

A novel ocular delivery of brinzolamide based on gellan gum: in vitro and in vivo evaluation.

BACKGROUND: The aim of the study was to develop a sustained ocular delivery of brinzolamide (BLZ) based on gellan gum. METHODS: The formulations were characterized for clarity, gelling capacity, rheological studies, pH, drug content, and in vitro drug-release behavior. In vivo rabbit eye irritation test was conducted to evaluate irritation of the BLZ gel drug-delivery system. The prepared BLZ formulations were then investigated in vivo and compared with commercially available BLZ eyedrops with regard to pharmacodynamics. RESULTS: The results showed that the optimum concentration of gellan gum was 0.25% w/v; the prepared liquid was converted into a flowing gel after the addition of simulated tear fluid. In vitro release profiles showed that the release of BLZ from the in situ gel exhibited sustained characteristics. Draize test results showed that BLZ in situ gels did not stimulate signs of eye tissue activity and were less irritating than BLZ solutions and commercial Azopt. CONCLUSION: The results of pharmacodynamics implied that the novel preparation of BLZ in situ gel effectively prolonged the intraocular pressure-lowering effect after administration.

Publisher Correction: Gene-based Therapy in a Mouse Model of Blue Cone Monochromacy.

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Gene-based Therapy in a Mouse Model of Blue Cone Monochromacy.

Cones are responsible for daylight, central, high acuity and color vision. Three proteins found in human cones, i.e. long-wavelength (L)-, middle-wavelength (M)-, and short-wavelength sensitive (S)-opsins, are responsible for red, green and blue color recognition, respectively. Human blue cone monochromacy (BCM) is characterized by functional loss of both L- and M-cone opsins due to mutations in the OPN1LW/OPN1MW gene cluster on the X chromosome. BCM patients, who rely on their vision from only S-cones and rods, suffer severely reduced visual acuity and impaired color vision. Recent studies show that there is sufficient cone structure remaining in the central fovea of BCM patients to consider AAV-mediated gene augmentation therapy. In contrast, mouse retina has only two opsins, S-opsin and M-opsin, but no L-opsin. We generated an M-opsin knockout mouse (Opn1mw -/-) expressing only S-opsin as a model for human BCM. We show that recombinant M-opsin delivered by AAV5 vectors rescues M-cone function in Opn1mw -/- mice. We also show that AAV delivered M-opsin localizes in the dorsal cone outer segments, and co-localizes with S-opsin in the ventral retina. Our study demonstrates that cones without M-opsin remain viable and respond to gene augmentation therapy, thereby providing proof-of-concept for cone function restoration in BCM patients.

Macrophage migration inhibitory factor promoter polymorphisms (-794CATT5-7 ) as potential biomarker for early-stage cervical cancer.

AIM: The objective was to investigate the correlation between macrophage migration inhibitory factor (MIF) promoter polymorphisms (-794CATT5-7 ) and early-stage cervical cancer (ESCC) and to identify a potential biomarker for ESCC. METHODS: A hospital-based case-control study was performed. The case group contained 250 patients with histologically confirmed ESCC. The control group included 147 healthy women. Polymerase chain reaction-single strand conformation polymorphism was used to genotype polymorphisms of MIF promoter -794CATT5-7 . Enzyme-linked immunosorbent assay was applied to detect serum concentration of MIF. RESULTS: The genotype distribution and allele frequency of MIF-794CATT in the ESCC group were significantly different from those in the control group (P < 0.05). The 7-CATT repeat carriers were significantly higher in the ESCC group than those in the control group (P < 0.05). The 7-CATT repeat carriers (5/7, 6/7, and 7/7) were associated with ESCC and increased risks of cervical cancer (odds ratio = 3.5, 3.0, and 5.6; 95% confidence interval = 1.2-10.5, 1.2-7.9, and 1.3-25.3, respectively). Serum concentration of MIF was significantly higher in the ESCC group than in the control group (P < 0.05), and it was significantly higher in 7-CATT carriers than in non-7-CATT carriers (P < 0.05). Neither polymorphisms of MIF-794 nor serum MIF were associated with lymph node metastases and differentiation (P > 0.05). CONCLUSION: MIF promoter polymorphisms (-794CATT) were correlated with ESCC; and 7-CATT might play a role in ESCC. It could be a potential biomarker for ESCC.

[Hematopoietic reconstitution after transplantation of uncontrolled-rate cryopreservation autologous peripheral blood hematopoietic stem cells using -80 °C mechanical freezer].

OBJECTIVE: This study was to identify the efficacy of -80°C cryopreservated peripheral blood hemato-poietic stem cell (PBHSC) transplantation for hematopoietic reanstitution in patients. METHODS: The efficacy of 104 patients underwent autologous peripheral blood hematopoietic stem cell transplantation using uncontrolled-rate freezing and storage at -80°C was evaluated. RESULTS: This cryopreservation method could effectively cryopreserve peripheral blood stem cells. Out of 104 patients only 2 patients died, other patients got hematologic reconstition satisfactorily, the median engrafement times of neutrophils and platelet were 12 and 14 days respectively, the activity of cells after rehabilitation was 94%, the mean recovery rates of CD34(+) cells and mononuclear cells (MNC) were 86% and 80.3% respectively. There were no significant influences on engrafement time in sex, chemotherapy circles and radiotherapy. The engrafement of leukocytes associated with amount of CD34(+) cells. CONCLUSION: This simple uncontrolled-rate freezing PBHSC at -80°C is safe, effective and economic, and can meet clinical needs. As compared with the classical cryopreservation, there were no significant differences in hematopoietic reconstitution. Therefore, this method worth to popularize and apply in clinic.

[Association of XRCC1 genetic polymorphism with susceptibility to non-Hodgkin's lymphoma].

The purpose of this study was to explore the association between X-ray repair cross-complementing group 1 (XRCC1)gene polymorphism and non-Hodgkin's lymphoma risk. A total of 282 non-Hodgkin's lymphoma (NHL) patients and 231 normal controls were used to investigate the effect of three XRCC1 gene polymorphisms (rs25487, rs25489, rs1799782) on susceptibility to non-Hodgkin's lymphoma. Genotyping was performed by using SNaPshot method. All statistical analyses were done with R software. Genotype and allele frequencies of XRCC1 were compared between the patients and controls by using the chi-square test. Crude and adjusted odd ratios and 95% confidence intervals were calculated by using logistic regression on the basis of genetic different models. For four kinds of NHL, subgroup analyses were also conducted. Combined genotype analyses of the three XRCC1 polymorphisms were also done by using logistic regression. The results showed that the variant genotype frequency was not significantly different between the controls and NHL or NHL subtype cases. Combined genotype analyses of XRCC1 399-280-194 results showed that the combined genotype was not associated with risk of NHL overall, but the VT-WT-WT combined genotype was associated with the decreased risk of T-NHL (OR: 0.21; 95%CI (0.06-0.8); P = 0.022), and the WT-VT-WT combined genotype was associated with the increased risk of FL(OR:15.23; 95%CI (1.69-137.39); P = 0.015). It is concluded that any studied polymorphism (rs25487, rs25489, rs1799782) alone was not shown to be rela-ted with the risk of NHL or each histologic subtype of NHL. The combined genotype with mutation of three SNP of XRCC1 was not related to the risk of NHL. However, further large-scale studies would be needed to confirm the association of decreased or increased risk for T-NHL and FL with the risk 3 combined SNP mutants of XRCC1 polymorphism.

[HLA antigen compatibility between patients with hematologic diseases and their parents].

This study was aimed to investigate and analyze the HLA antigen compatibility between patients with hematologic diseases and their parents so as to provide basis for selecting the suitable donors in haploidentical hematopoietic stem cell transplantation. The HLA low resolution for 174 families was typed and analyzed by using PCR-SSP. The results showed that 52.30% of patients with hematologic diseases possessed father and/or mother with HLA matching over haploidentity, 10.92% patients were over 8/10 matched with their father and/or mother. 11.49% were over semi-matched with both their father and mother. The rate of 6/10 matched pairs (28.16%), 7/10 matched pairs (16.1%) and 8/10 matched pairs (8.62%) were all beyond 5%; 9/10 (2.3%) and 10/10 matched pairs (1.15%) were all below 5%. It is concluded that with the matching degree increasing between two generations, HLA matching rate is decreasing. Over 50% and 10% patients were over HLA semi-matched and 8/10 matched with their father and/or mother, respectively. This high matching rate offered a big chance for success of haploidentical HSCT. Patients are more likely over semi-matched with their father and/or mother when they have high frequency and strong linkage HLA disequilibrium. High frequency and strong linkage disequilibrium in populations are main reason, and population concentrating and isolated living may be another reason for this phenomenon.

Targeting photoreceptors via intravitreal delivery using novel, capsid-mutated AAV vectors.

Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY-F+T-V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY-F+T-V) -hGRK1-GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.

The human rhodopsin kinase promoter in an AAV5 vector confers rod- and cone-specific expression in the primate retina.

Adeno-associated virus (AAV) has proven an effective gene delivery vehicle for the treatment of retinal disease. Ongoing clinical trials using a serotype 2 AAV vector to express RPE65 in the retinal pigment epithelium have proven safe and effective. While many proof-of-concept studies in animal models of retinal disease have suggested that gene transfer to the neural retina will also be effective, a photoreceptor-targeting AAV vector has yet to be used in the clinic, principally because a vector that efficiently but exclusively targets all primate photoreceptors has yet to be demonstrated. Here, we evaluate a serotype 5 AAV vector containing the human rhodopsin kinase (hGRK1) promoter for its ability to target transgene expression to rod and cone photoreceptors when delivered subretinally in a nonhuman primate (NHP). In vivo fluorescent fundus imaging confirmed that AAV5-hGRK1-mediated green fluorescent protein (GFP) expression was restricted to the injection blebs of treated eyes. Optical coherence tomography (OCT) revealed a lack of gross pathology after injection. Neutralizing antibodies against AAV5 were undetectable in post-injection serum samples from subjects receiving uncomplicated subretinal injections (i.e., no hemorrhage). Immunohistochemistry of retinal sections confirmed hGRK1 was active in, and specific for, both rods and cones of NHP retina. Biodistribution studies revealed minimal spread of vector genomes to peripheral tissues. These results suggest that AAV5-hGRK1 is a safe and effective AAV serotype/promoter combination for targeting therapeutic transgene expression protein to rods and cones in a clinical setting.

[Clinical analysis for 3 cases of HLA-matched between father and son and 1 case of post-hematopoietic stem cell transplant efficacy].

Getting a HLA-matched donor is a key factor for successful hematopoietic stem cell transplantation. People are almost semi-matched with their parents, while a person HLA-matched with his/her father or mother was rarely seen, if so, usually whose father and mother are genetically related. HLA-low resolution for patients and their relatives were performed using PCR-SSP technique and three patients were found HLA-matched with their father in these results. One of them accepted hematopoietic stem cell transplantation using his HLA-matched father as his donor. The results showed that the chimerism was detected as stable complete donor chimerism, fusing gene of MLL-ENL was detected all negatively in the post-transplant period. This case got well hematopoietic reconstruction and GVHD didn't occur, so far he has survived for two years in health conditioning. It is concluded that people HLA-matched with his/her father or mother can be found when there is one identical haplotype of high frequency and strong linkage disequilibrium between father and mother. This case is valuable for hematopoietic stem cell transplantation development.

[A recombination event occurring between HLA-A and -A loci from father's HLA haplotype chromosome].

This study was aimed investigate the recombination event occurring between HLA-A and-A loci discovered from father's HLA haplotype chromosome in a family. Peripheral blood samples were collected from a family. HLA class I (-A, -B, and -Cw) and II (-DRB1 and -DQB1) alleles were amplified and typed by both low and high resolution PCR with sequence-specific primers (PCR-SSP) and sequence-based typing (SBT). The results showed that 2 haplotypes of the patient were A(*)3001-B(*)1302-DRB1(*)0701 and A(*)3001-B(*)5601-DRB1(*)1454 respectively, those of her father were A(*)3001-B(*)1302-DRB1*0701 and A(*)1101-B(*)5601-DRB1(*)1454. Family analysis demonstrated that the patient's A(*)3001-B(*)1302-DRB1(*)0701 came from her mother and A(*)1101-B(*)5601-DRB1(*)1454 came from her father, but the A of patient was A(*)3001 and B, DR were the same to her father. This showed that the chromosome exchange and recombination event of father's 2 haplotypes occurring between HLA-A and -A loci at meiosis. And recombinate haploid chromosome was completely inherited to his daughter 1. HLA typing and Paternity testing demonstrated that father was the natural father, and the recombination event occurring between HLA-A and -A loci of the daughter 1 with father's HLA haplotype chromosome. It is concluded that the HLA-A/A of father's HLA haplotype chromosome recombination event occurring between HLA-A an-A loci has been found in a family in China, which helps further study on the mechanisms of HLA recombination.

Strand-specific PCR of UV radiation-damaged genomic DNA revealed an essential role of DNA-PKcs in the transcription-coupled repair.

BACKGROUND: In eukaryotic cells, there are two sub-pathways of nucleotide excision repair (NER), the global genome (gg) NER and the transcription-coupled repair (TCR). TCR can preferentially remove the bulky DNA lesions located at the transcribed strand of a transcriptional active gene more rapidly than those at the untranscribed strand or overall genomic DNA. This strand-specific repair in a suitable restriction fragment is usually determined by alkaline gel electrophoresis followed by Southern blotting transfer and hybridization with an indirect end-labeled single-stranded probe. Here we describe a new method of TCR assay based on strand-specific-PCR (SS-PCR). Using this method, we have investigated the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related protein kinases (PIKK) family, in the TCR pathway of UV-induced DNA damage. RESULTS: Although depletion of DNA-PKcs sensitized HeLa cells to UV radiation, it did not affect the ggNER efficiency of UV-induced cyclobutane pyrimidine dimers (CPD) damage. We postulated that DNA-PKcs may involve in the TCR process. To test this hypothesis, we have firstly developed a novel method of TCR assay based on the strand-specific PCR technology with a set of smart primers, which allows the strand-specific amplification of a restricted gene fragment of UV radiation-damaged genomic DNA in mammalian cells. Using this new method, we confirmed that siRNA-mediated downregulation of Cockayne syndrome B resulted in a deficiency of TCR of the UV-damaged dihydrofolate reductase (DHFR) gene. In addition, DMSO-induced silencing of the c-myc gene led to a decreased TCR efficiency of UV radiation-damaged c-myc gene in HL60 cells. On the basis of the above methodology verification, we found that the depletion of DNA-PKcs mediated by siRNA significantly decreased the TCR capacity of repairing the UV-induced CPDs damage in DHFR gene in HeLa cells, indicating that DNA-PKcs may also be involved in the TCR pathway of DNA damage repair. By means of immunoprecipitation and MALDI-TOF-Mass spectrometric analysis, we have revealed the interaction of DNA-PKcs and cyclin T2, which is a subunit of the human transcription elongation factor (P-TEFb). While the P-TEFb complex can phosphorylate the serine 2 of the carboxyl-terminal domain (CTD) of RNA polymerase II and promote transcription elongation. CONCLUSION: A new method of TCR assay was developed based the strand-specific-PCR (SS-PCR). Our data suggest that DNA-PKcs plays a role in the TCR pathway of UV-damaged DNA. One possible mechanistic hypothesis is that DNA-PKcs may function through associating with CyclinT2/CDK9 (P-TEFb) to modulate the activity of RNA Pol II, which has already been identified as a key molecule recognizing and initializing TCR.

[Application of spectral karyotyping to cytogenetic analysis in acute myeloid leukemia].

OBJECTIVE: To evaluate the value of spectral karyotyping (SKY) in cytogenetic analysis of acute myeloid leukemias (AML). METHODS: Nine AML patients were analyzed by R-banding and SKY. MLL, PML-RARalpha, AML1-ETO fusion genes were detected by dual fusion- fluorescence in situ hybridization (D-FISH). RESULTS: All 9 samples were successfully hybridized. SKY identified structural aberrations including 9q -, t(15;17) and ins(10;17) (q22;p11p12) ; and some numeral abnormalities. The results of SKY confirmed those of R-band karyotyping and D-FISH; with more accurate localization. CONCLUSION: SKY appears to be fairly stable, accurate and sensitive, for AML cytogenetic study.

[Allogeneic hematopoietic stem cell transplantation combined with imatinib for treatment of Philadelphia chromosome positive acute lymphoblastic leukemia].

The study was purposed to explore the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) combined with Imatinib for treatment of Philadelphia chromosome positive acute lymphoblastic leukemia (Ph(+)ALL) patients. From 2007 to 2008, 3 patients with Ph(+)ALL were treated with allogeneic hematopoietic stem cell transplantation and Imatinib, and the follow-up ended at Oct 21(st) 2009. 1 patient received HSCT from matched sibling donor and 2 patients from haploidentical related donors. All 3 patients achieved complete remission before transplantation and were treated with Imatinib for distinct time at different periods before and/or after transplantation. The level of bcr/abl mRNA was monitored using real-time PCR. The results showed that all 3 patients achieved stable engraftments without severe transplantation related complications. The level of bcr/abl mRNA declined and achieved zero level finally. In conclusion, the allo-HSCT combined with Imatinib is an effective therapy regimen for Ph(+)ALL patients.

[Killer immunoglobulin-like receptor gene genotype and expression pattern in NK-92MI and K562 cells].

AIM: To make clear the repertoire of Killer immunoglobulin-like receptor at the level of DNA and RNA in NK-92MI and K562 cells and compare KIRs gene genotype and expression patterns in both cells. METHODS: KIRs gene genotype, include KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, was analyzed by PCR. KIRs gene phenotype, include KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR2DS1, KIR2DS3, KIR2DS2/4 gene, was determined by RT-PCR and sequencing. RESULTS: KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5A, 2DS2, 2DS4*003-006, 2DS5, 3DL1, 3DL2 and 3DL3 gene were present in K562 cells, but all corresponding receptors were not expressed by K562 cells. KIR2DL1, 2DL2, 2DL3, 2DL4, 2DS2, 2DS4*001/002, 2DS4*003-006, 3DL1, 3DL2 and 3DL3 gene were present in NK-92MI cells, but only transcripts of KIR2DL1, KIR2DL3 and KIR2DS2/4 were detectable. CONCLUSION: KIR2DL1, 2DL2, 2DL3, 2DL4, 2DS2, 2DS4*003-006, 3DL1, 3DL2 and 3DL3 gene were present in both K562 and NK-92MI cells. All corresponding KIR receptors were not expressed by K562 cells and only partial KIRs by NK-92MI cells. The repertoire of KIRs were specific for K562 and NK-92MI cells.

[Effect of demethylation treatment on the expression of inhibitory receptor KIR gene in NK-92MI cell line].

The aim of this study was to analyze the promoter methylation patterns of inhibitory killer cell immunoglobulin-like receptor (KIR) which gene expression and the effect of demethylation treatment were studied, and to explore the possible regulation mechanism of inhibitory kir gene expression. The promoter methylation levels of kir2DL1 and kir2DL2/kir2DL3 in NK-92MI cell line were detected by bisulfite sequencing technique. Then NK-92MI cells were treated with 5-azacytidine to induce the demethylation of CpG islands. The levels of gene expression of kir were determined by RT-PCR. The results demonstrated that the methylation frequencies of CpG dinucleotides surrounding the promoter regions of kir2DL1 and kir2DL2/kir2DL3 genes were 25% to 88% and 5% to 80% respectively. DNA-demethylating treatment with 5-azacytidine resulted in re-expression of kir2DL1 gene and increased expressions of kir2DL1, kir2DL2 and kir2DL3 genes in NK-92MI cells. In conclusion, the promoter DNA methylation participates in the regulation of kir gene expression in NK-92MI cells.