Monitoring minimal residual disease by qualitative detection of immunoglobulin H/T cell receptor γ by using multiplex polymerase chain reaction in children with acute lymphocytic leukemia / 中华实用儿科临床杂志

Objective To explore the prognostic significance in monitoring minimal residual disease (MRD) in childhood acute lymphoblastic leukemia (ALL) by a simple method,and to detect cloned immunoglobulin H (IgH) and T cell receptor γ (TCRγ) gene rearrangements by using multiplex polymerase chain reaction (PCR) and automated fragment analysis.Methods Bone marrow samples were collected from 86 newly diagnosed cases of childhood ALL at the Department of Pediatrics,the First Affiliated Hospital of Sun Yat-Sen University,from May of 2009 to August of 2013.IgH and TCRγ gene rearrangements were amplified by qualitative multiplex PCR.The clonality of PCR production was analyzed by GENEMAPPERID software.Only those carried monoclonal IgH/TCRγ on diagnosis were arranged to monitor MRD.Detectable monoclonal IgH/TCRγby the end of induction was defined as MRD positive.All patients were treated with GD2008 ALL protocol.Clinical data of all newly-diagnosed ALL patients in the corresponding period were reviewed.The final follow-up on May 31,2014.Survival rates and event free survival (EFS) curves were estimated by the Kaplan-Meier,and compared by using the log-rank test.Results The percent age of 94.2 (81/86 cases) patients was at least 1 marker positive.Subsequent MRD was monitored in 79 cases.The median follow-up time was 20 months (9-61 months).By the end of induction,20 cases were MRD positive and 59 cases were M RD negative,and the 3-year EFS were 56.4％ ± 14.7％ and 94.0％ ± 3.4％ (x2 =8.563,P =0.003),respectively.According to the traditional prognostic stratification criteria,MRD was detected 65 cases in the non-high risk group:23 cases in standard risk group and 42 cases in intermediate risk group,and the difference of 3-year EFS had no statistical significance (95.3％ ±4.7％ vs 76.6％ ±9.0％,x2 =0.934,P =0.334).While using MRD by the end of induction as a risk stratification criterion,there was a statistical significant difference compared with the 3-year EFS for MRD-negative (n =52) group and MRD-positive (n =13) group (93.1％ ± 3.8％ and 59.5％ ± 16.2％,x2 =7.128,P =0.008).Conclusions It is a simple but feasible method to monitor MRD in childhood ALL by using this qualitative multiplex PCR with automated fragment analysis for monoclonal IgH/TCRγ gene rearrangements.MRD by the end of induction can be used as a more accurate risk stratification criterion than the traditional one.It is worth of further research.

Functional role of protein kinase D1 in Aspergillus fumigatus-induced activation of nuclear factor-κB signal pathway and transcription / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the functional role of protein kinase D1 (PKD1) in the activation of nuclear factor-κB (NF-κB) signal pathway and NF-κB transcription mediated by Aspergillus fumigatus.</p><p><b>METHODS</b>A549 cells and HEK293 cells were transfected with green fluorescence protein (GFP) or GFP-PKD1 followed by treatment with 1×10(5) CFU/ml Aspergillus fumigatus conidia for different time lengths. The phosphorylation levels of PKD1, IκB and p65 (pS276) in the transfected cells were measured by Western blotting. A549 cells were transfected with GFP-PKD1 or siRNA-PKD1, and the phosphorylation of IκB and p65 (pS276) was examined. Finally, NF-κB-luc and renilla luciferase reporter pRL-SV40 were cotransfected into GFP- or GFP-PKD1-transfected A549 cells before exposure of the cells to Aspergillus fumigatus conidia for 24 h, and NF-κB transcriptional activity in the cells was determined using dual-luciferase reporter assay.</p><p><b>RESULTS</b>Overexpression of PKD1 significantly increased Aspergillus fumigatus conidia-stimulated phosphorylation of PKD1, IκB and p65 (pS276), whereas PKD1 knockdown by siRNA-PKD1 suppressed IκB and p65 (pS276) phosphorylation. Dual luciferase assay demonstrated that PKD1 overexpression markedly enhanced Aspergillus fumigatus-induced NF-κB transcription in A549 cells.</p><p><b>CONCLUSION</b>PKD1 may contribute to the activation of NF-κB signal pathway and NF-κB transcription induced by Aspergillus fumigatus.</p>

AXIN1-related CSRNP1 mRNA expression and its transcriptional regulation in TGF-β1-induced tumor cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate AXIN1-related CSRNP1 gene expression and the mechanism of its transcriptional regulation in TGF-β1-induced tumor cells.</p><p><b>METHODS</b>Human lung carcinoma A549 cells or human prostate cancer PC3 cells were treated with TGF-β1 at different doses (0, 20, 40, and 80 ng/ml) or at 20 ng/ml for 0, 8, 12, or 24 h, and the dose and time effect of TGF-β1 on CSRNP1 mRNA expression in the tumor cells were evaluated with real-time RT-PCR. A549 cells were also treated with TGF-β1 and cycloheximide to clarify whether CSRNP1 expression induced by TGF-β1 required de novo protein synthesis. A549 cells transfected with pcDNA3.1, flag-SMAD3, or flag-SMAD3-mu, after serum starvation, were treated with or without TGF-β1 (20 ng/mL) for 24 h, and the overexpression of wild-type SMAD3 and dominant negative SMAD3-mu mutant were confirmed by Western blotting. The effect of SMAD3 or SMAD3-mu overexpression on CSRNP1 mRNA expression was also measured by real-time RT-PCR.</p><p><b>RESULTS</b>In both A549 and PC3 cells, TGF-β1 dose- and time-dependently stimulated CSRNP1 expression, which required de novo protein synthesis in A549 cells. Overexpression of wild-type SMAD3 significantly increased the expression of CSRNP1 mRNA induced by TGF-β1, while overexpression of dominant negative SMAD3 mutant remarkably reduced CSRNP1 mRNA expression in response to TGF-β1 in A549 cells.</p><p><b>CONCLUSION</b>TGF-β1 may contribute to CSRNP1 expression through SMAD3 activation and downstream signaling in tumor cells.</p>

Maintenance therapy with dose-adjusted 6-mercaptopurine in 15 cases of idiopathic pulmonary hemosiderosis / 中国综合临床

Objective To review the diagnosis of idiopathic pulmonary hemosiderosis ( IPH),and to evaluate the efficacy of maintenance therapy with dose-adjusted 6-mercaptopurine (6MP) in IPH children. Methods The diagnosis of IPH was confirmed by in-patient examination and at least 1 year follow-up to exclude secondary causes of pulmonary hemorrhage. Fifteen children met the criteria of IPH and were enrolled. The age at diagnosis was 2-13 years ( median 7 years). Prednisone was administered at 2 mg/( kg·d) for 4 weeks in acute phase of the disease followed by taper. 6MP was also started at 60 mg/( m2·d) simultaneously and continued for 3 years. Results The diagnosis was delayed in most children, which was due to the lack of initial classical manifestation of the disease. The time between the onset of symptoms and diagnosis ranged from 2 weeks to 108 months ( median 8 months) . All the patients exhibited response to the initial treatment and prednisone was successfully tapered off. Only 1 of 8 patients with relative leucopenia (3 × 109/L -6 × 109/L) on 6MP maintenance recurred while 5 of 7 others recurred (P < 0. 05) during median 6-year (range 2. 5 - 9. 5 years) follow-up. Of the latter 5 patients who recurred,4 remained recurrence-free after adjusting the dose of 6MP upwards to keep relative leucopenia. Conclusions Diagnostic delayed is still a main problem in pediatric IPH. Most IPH children in our group tolerated maintenance treatment with 6MP and achieved long-term remission, and these suggested growth retardation on long-term steroids therapy could be avoided. Because of interindividual difference in 6MP metabolism, adjusting the dose of 6MP may be necessary for treatment of IPH children and avoid under-treatment or overtreatment in some children,and thus improve the prognosis. White blood count could be a simple and useful indicator to predict clinical response in most IPH children on 6MP.

Comparison of the overall treatment outcome of two protocols for childhood acute promyelocytic leukemia / 白血病·淋巴瘤

Objective For further improving the prognosis of childhood acute promyelocytic leukemia (APL) in China,the treatment efficacies, outcomes and costs of protocols for childhood APL between in developed countries and in our hospital were compared. Methods 30 cases aged ＜15 years were diagnosed according to the FAB classification and detection of PML-RARα rearrangement. From December 1999 to September 2004,sixteen eligible children were treated with an intensive in-house protocol including high-dose Ara-C and anthracycline for post remission treatment. From September 2004 to January 2008,14 cases enrolled were treated with a less intensive protocol modified from the PETHEMA LPA99. Results The 3.5 years EFS was 37.5 % (s-x=0.121) for total 16 patients on in-house protocol. Six patients (37.5 %) abandoned treatment,2 died of intracranial hemorrhage at diagnosis (6.3 %) and sepsis in remission (6.3 %),respectively,and 2 relapsed (12.5 %). The 14 cases treated with modified PETHEMA had a 3.5 years EFS of 79.6 % (s-x=0.136). One died of intracranial hemorrhage at diagnosis (7.1 %) and 1 relapsed (7.1 %). Patients on modified PETHEMA had a significantly higher EFS (P=0.012),lower frequency of sepsis during treatment (7.7 % vs 77.8 %; P=0.0015) and lower hospitalization cost (median,RMB 35 200 vs 150 000; P ＜0.0001) than those on in-house protocol. Conclusion Treatment with the less intensive protocol based on the PETHEMA LPA99 study for childhood APL successively reduced complication of chemotherapy and reduced hospitalization cost without increasing relapses, which led to decreases in treatment-related toxicity and treatment abandonment rate,thus improving overall outcome.

Clinic features and me chanism of scald-like skin damage induced by high-dose methotrexate / 中国医师进修杂志

Objective To study the clinic features and the mechanism of skin damage induced by high-dose methotrexate(HD-MTX).Method Children treated with HD-MTX were enrolled in a retrospective study in which the incidence and presentations of skin damage were concluded.Mechanism of the damage was studied base on skin pathology of an animal model.Result Skin damage presented in 3～9(median 5) days after HD-MTX and usually followed by bullation and exfoliation.Twelve cases,more severe than the others,accompanied with fever and symptoms of other organs especially the respiratory tract and the intestine.Skin damage was reduced when large dose rehydration used before,during and after HD-MTX.Conclusion HD-MTX may cause scald-like skin damage directly by its prolonged toxic effect on epidermal cells.Besides extended tetrahydrofolic acid rescue and supportive treatments,anti infection is the most important in dealing this condition.

Improvement of leptin and insulin sensitivity due to early nutritional intervention in rats born with intrauterine growth retardation / 中华内分泌代谢杂志

Objective To look for an appropriate dietary pattern of early nutritional intervention, which does not only meet the need of catch-up growth but also avoids or reduces the incidence of insulin resistance (IR) in adulthood of rats born with intrauterine growth retardation (IUGR). Methods The model of IUGR in rats was established by maternal nutrition restriction. Sixty newborn female rats with IUGR were randomly divided into 5 groups: (1) IUGR control group fed with common diet. (2) IUGR high-carbohydrate diet group. (3) IUGR high-fat diet group. (4) IUGR high-protein diet group. (5) IUGR low-protein diet group. The IUGR newborn rats were breast-fed for 3 weeks, while the mother rats were fed with the above different diets with the same caloric amount. Twelve normal newborn female rats served as a normal control group and were fed with common diet. All newborn rats were fed routine diet starting from the 4th week of experiment. The body weight, perirenal fat weight and the serum leptin, blood glucose, insulin concentration were measured and the insulin sensitive index (ISI) were calculated at the 4th week and the 12th week of life. Results The IUGR rats fed with high-protein diet showed a catch-up growth without the increase of perirenal fat at the 4th week, a normal level of perirenal fat, leptin and ISI at the 12th week compared with the normal control group, and did not show IR. The groups fed with high carbohydrate diet and high fat diet also showed a catch-up growth, but did the same as the IUGR control group in other aspects, they all showed increased perirenal fat, higher levels of leptin, lower ISI and IR at adulthood. The group fed with low protein diet did not show IR, but kept a small body size with increased perirenal fats. The serum leptin level of rats was positively correlated to body weight at the 4th week of life and was positively correlated to the weight of perirenal fat and negatively correlated to ISI at the 12th week of life. Conclusion High protein diet is an appropriate early nutritional intervention for rats with IUGR. The serum leptin level at the 12th week of life seems to be an index of IR in adult rats with IUGR.