Bone marrow mesenchymal stem cell-derived exosomes miR-202-5p inhibited pyroptosis to alleviate lung ischemic-reperfusion injury by targeting CMPK2.

Bone mesenchymal stem cell-derived exosome (BMSC-exosome) is a potential candidate for lung ischemia-reperfusion injury (LIRI) treatment. This study aims to investigate the anti-pyroptosis effect of BMSC-exosomes in LIRI. The LIRI cell model was established by hypoxia/reoxygenation (H/R) treatment. Interleukin (IL)-1ß and IL-18 levels were examined by enzyme-linked immunosorbent assay. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. Lactate dehydrogenase (LDH) release was examined using a LDH assay kit. The interaction between microRNA (miR)-202-5p and cytidine monophosphate kinase 2 (CMPK2) was analyzed using dual-luciferase reporter assay and RNA immunoprecipitation. BMSC-exosomes promoted cell viability and suppressed pyroptosis in H/R-treated mouse lung epithelial. miR-202-5p was enriched in BMSC-exosomes, and exosomal miR-202-5p inhibition upregulated pyroptosis-associated proteins, including cleaved N-terminal Gasdermin D, nucleotide-binding domain-like receptor family member pyrin domain-containing protein 3, and Caspase1. Meanwhile, miR-202-5p suppressed CMPK2 expression by directly targeting CMPK2. Expectedly, CMPK2 knockdown reversed the promoting effect of exosomal miR-202-5p inhibition on pyroptosis in LIRI. Therefore, BMSC-derived exosome miR-202-5p repressed pyroptosis to inhibit LIRI progression by targeting CMPK2.

Comparison of two dosages of rabbit antithymocyte globulin (r-ATG) in treating children with severe aplastic anemia.

In this study, efficacy and safety of two different dosages of rabbit antithymocyte globulin (r-ATG) combined with cyclosporine (CsA) in treating children with severe aplastic anemia (SAA) were compared. The clinical data of 122 SAA children treated by r-ATG/CsA between Jan 2005 and Jan 2017 at Sun Yat-sen Memorial Hospital of Sun Yat-sen University were retrospectively analyzed. The r-ATG dose of 55 cases was 2.5mg/(kg·d, group 1), and in the other 67 cases it was 3.5 mg/(kg·d, group 2). r-ATG was continuously administered for 5 days. In the 3rd and 6th month after treatment, the efficacy rate of group 2 was higher than that of group 1 (45.5% vs 26.4%, P=0.032; 54.5% vs 35.8%, P=0.042). In the 9th and 12th month after treatment, the efficacy rates of both groups were similar (71.2% vs 54.9%, P=0.077; 75.9% vs 68.6%, P=0.399). The incidence rates of serum diseases (74.5% vs 79.1%, P=0.551), short-term infection rates (76.4% vs 62.7%, P=0.105), early mortality (3.6% vs 1.5%, P=0.447), and 3-year overall survival rates (89.5% vs 90.1%, P=0.932) of both groups showed no significant differences. The r-ATG/CSA therapy was safe and effective towards SAA. The final efficacies and safety of the two r-ATG dosages were equal. However, the follow-up period in this study was relatively short, so the intergroup comparison of the long-term complications and survival rates needed to be further followed up.

[Comparison of Curative Effect between Fu Fang Huang Dai Pian and Arsenic Trioxide in Treatment of 45 Patients with Acute Promyelocytic Leukaemia].

OBJECTIVE: To investigate the clinical efficacy of Fu Fan Huang Dai Pian(RIF) and arsenic trioxide (ATO) regimens for treatment of children with acute promyelocytic leukemia (APL) and to explore the risk factors affecting the prognosis of patients. METHODS: The clinical data of 45 newly diagnosed APL children admitted in our hospital from January 2004 to May 2017 were analyzed retrospectively. Among 45 APL children, 25 children were treated by chemotherapetic regimen including RIF (RIF group), another 20 children were treated by chemotherapeutic regimen including ATO (ATO group). The follow-up was performed in all APL children. The prognosis and incidence of side reactions from drugs in 2 groups were compared, and the high risk factors affecting the prognosis of patients were analyzed. RESULTS: The median follow-up time was 49.8% months. In RIF group, no early death occured in 25 APL children; 5 cases did not achieve complete remission (CR) after induction therapy, CR rate was 88%. Out of 25 cases 2 caes relapsed, 3 cases died, 20 cases maintained contined CR (CCR), 2 cases failed to be followed-up. In ATO group, 2 cases suffered from early death, 5 cases did not achieve CR after induction therapy, CR rate was 90%, 2 caese relapsed and died, 15 cases maintained CCR, the follow-up failed in 1 caes. The 5 year- OS and EFS rate in all the patients were predicted as (82.2±6.2)% and (76.4±6.6)% respectively. The OS and EFS rate in RIF group were (86.1±7.4)% and (78.4±8.6)% respectively, which were significantly different from OS and EFS rate (76.4%±10.6%) and (74.0%±10.1%) respectively in ATO group (all P>0.05). As for the side reaction from drug, except for the cardiac damage (P<0.05), incidence of other side reactions was not significantly different between 2 groups (P>0.05). In addition, the 5 year-OS and EFS rates in APL children with CNSL were significantly lower than those in APL children without CNSL (all P<0.05), the 5 year OS and EFS rate in APL children did not reache M1 and with high risk were significantly lower than those in APL children reached M1 after induction therapy and with low and standerd risk (P<0.05 and P<0.05); the 5 year-OS and EFS rates did not correlate with age and sex. CONCLUSION: The Fu Fang Huang Dai Pian shows the therapeutic efficacy on APL children same as ATO, moreover, no obvious enhancement in incidence of side reactions is observed, therefore, the Fu Fang Huang Dai Pian is effective and safe for treatment of APL children. The CNSL, poor respond to treatment, high risk in clinical stratification are high risk factors affecting prognosis of patients.

Correlation analysis of severe aplastic anemia immunosuppressive therapy and human leukocyte antigen alleles in pediatric patients.

The aim of the present study was to investigate the correlation between the efficacy of immunosuppressive therapy (IST) in children with severe aplastic anemia (SAA) and human leukocyte antigen (HLA) alleles. The polymerase chain reaction-sequence based typing high-resolution genotyping method was used to profile the HLA alleles of 115 SAA cases that were treated with rabbit-antithymocyte globulin (r-ATG) + cyclosporine (CsA) immunosuppressive therapy and 222 normal control subjects. The aim was to compare the frequency distribution of HLA alleles among the IST-effective group, the IST-ineffective group and the healthy control group. The results showed that the gene frequencies (GFs) of HLA-B*15:02, B*40:02, B*48:01, DRB1*09:01, C*01:02, C*03:04, DQB1*03:03 and DQB1*06:02 in the IST-effective group were significantly higher compared with those in the healthy control group, with a statistically significant difference. The GFs of HLA-B*15:11, B*38:01, B*39:05, DRB1*15:01, C*01:02 and C*08:22 in the IST-ineffective group were significantly increased compared with those in the healthy control group, with a statistically significant difference. The gene frequency of HLA-A*29:01 in the IST-effective group was significantly reduced compared with that in the IST-ineffective group, and the difference was statistically significant. In summary, IST efficacy in children with SAA that express the HLA-B*15:02, B*40:02, B*48:01, DRB1*09:01, C*01:02, C*03:04, DQB1*03:03 and DQB1*06:02 alleles may be superior, while the efficacy may be mitigated in children with SAA who express HLA-A*29:01, B*15:11, B*38:01, B*39:05, DRB1*15:01, C*01:02, C*08:22 alleles.

[Comparison between two different dose of r-ATG combined with CsA for treating children with severe aplastic anemia].

This study was purposed to compare the efficacy and safety of two different doses of rabbit anti-thymocyte globulin (r-ATG) combined with cyclosporine (CsA) for treating children with severe aplastic anemia (SAA). From January 2005 to July 2010, a total of 95 children with SAA accepted intensive immunosuppressive therapy (IIST) in our department, out of them 55 cases were treated with r-ATG 2.5 mg/(kg·d) for 5 days in combination with CsA (group I) and other 40 cases were treated with r-ATG 3.5 mg/(kg·d) for 5 days in combination with CsA (group II). The responsive rate, adverse reactions, early mortality, relapse and clonal disease were analyzed retrospectively and results between the two groups were compared. Out of 95 patients 43 were boys and 52 were girls, their ages were from 1 to 16 years. The sex, age, severity and course of the disease were comparable between the two groups. The results showed that after treating for 3 and 6 months, the response of patients in group II was higher than that of patients in group I (50% vs 32.1%, P = 0.08 and 65% vs 45.3%, P = 0.059), at 9 and 12 months the response rate of patients in group II and group I did not show significant difference (70.0% vs 71.1%,P = 0.904 and 82.5% vs 80.8%,P = 0.832); at 12 months of treatment, the complete response rate of patients in group II was significantly higher than that of patients in group I (40.0% vs 23.1%,P = 0.08); at 3, 6, 9 months of treatment, the complete response rate of 2 groups showed no obvious difference. The incidence of serum disease, early infection and early mortality did not show statistical difference between two groups. There was no statistical difference in 2 year overall survival rate of two groups. In group I 39 patients were followed-up for more than 2 years, among them 3 patients relapsed, 1 patient died and 1 patient was diagnosed as acute monocytic leukemia (M5). In group II 15 patients were followed up for more than 2 years, there were no relapse, death and clonal disease. It is concluded that the r-ATG combined with CsA is an effective and safe therapeutic regimen for the SAA children. The effect of r-ATG 3.5 mg/(kg·d) is better than the 2.5 mg/(kg·d). The early safety is comparable between the two groups. However, the long-term effect, complications and survival rate need longer follow-up study to evaluate.

Impact of chemotherapy-related hyperglycemia on prognosis of child acute lymphocytic leukemia.

PURPOSE: To investigate the impact of hyperglycemia during inductive treatment on the prognosis of acute lymphocytic leukemia (ALL) in children. MATERIALS AND METHODS: Clinical data of 159 ALL childhood cases were reviewed. The patients were divided into the hyperglycemia group (fasting glucose≥126 mg/dl and/or random blood glucose≥200 mg/dl) and the euglycemia group according to the blood glucose values. The ?2 test was performed to compare the complete remission rates of the two groups, and Kaplan-Meier and log-rank tests were performed to compare the 5-year overall and relapse-free survival. RESULTS: The incidence of hyperglycemia in the age≥10-year-old group was higher than the younger-age group (P=0.009). Values in the interim- and high-risk groups were higher than the standard-risk group (P=0.028), while there was no significant difference between genders (P=0.056). The complete remission rates of the 2 groups demonstrated no significant difference (P=0.134), while the 5-year OS of the hyperglycemia group was lower than in the euglycemia group (83.8±6.0% vs 94.9±2.4%, P=0.014). The 5 -year RFS was significantly lower than the euglycemia group (62.9±8.7%) vs 80.2±9.1%, P<0.001). CONCLUSIONS: Children with age≥10 years old, and in the middle- and high-risk groups appear prone to complicating hyperglycemia during inductive chemotherapy, associated with lower 5-year OS and RFS.

[Hyperglycemia during chemotherapy influences the prognosis of children with acute lymphocytic leukemia].

This study was aimed to explore whether hyperglycemia during chemotherapy influences the prognosis of children with acute lymphocytic leukemia (ALL). The clinical medical records of all newly diagnosed patients with ALL at SUN Yat-Sen Memorial Hospital from June 2008 to May 2012 were analyzed retrospectively. The median time of follow-up for patients was 2.6 years (range 0.08 to 4.9 years). Patients were divided to hyperglycemia and euglycemia groups according to their blood glucose concentrations during chemotherapy which contains L-asp and dexamethasone. The variables between two groups were compared using χ(2) test, the RFS and OS among two groups were compared by use of Kaplan-Meier and Cox-proportional hazard analyses. The results showed that the hyperglycemia correlated with older age (43.33% vs 19.23%, P = 0.008) and high-risk disease at diagnosis (26.62% vs 4.76%, P = 0.017) , but did not associate with sex (P = 0.059). Patients with hyperglycemia had worse OS (94.2 ± 2.9% vs 83.1 ± 6.3%, P = 0.014) and more poor RFS (64.1 ± 8.9% vs 88.6 ± 3.8%, P < 0.001) at 5 years than their counterpart. It is concluded that the incidence rate of hyperglycemia during chemotherapy correlated with older age and high-risk disease in ALL children, and the patients with hyperglycemia during chemotherapy may have poorer prognosis.

[Expression of IGF-IR and IR in children with acute lymphoblastic leukemia].

This study was aimed to investigate the mRNA expression levels of insulin-like growth factor-I receptor (IGF-IR) and insulin receptor (IR) in peripheral blood lymphocytes of childhood ALL and their relationship with clinical features. The expression levels of IR and IGF-IR in peripheral blood lymphocytes of ALL children and healthy children were analysed. Peripheral blood samples were obtained from 43 ALL patients and 14 healthy children. The mRNA expression of IGF-IR and IR was measured by RT-PCR. The results showed that IGF-IR was widely expressed on peripheral blood lymphocytes of ALL children and healthy controls, mRNA level of IGF-IR significantly increased in ALL children of newly diagnosed, relapsed and hyperglycemia groups (P = 0.000, P = 0.002 and P = 0.05)as compared with the normal control group. mRNA level of IGF-IR in CR group was significantly lower than that in newly diagnosed and relapsed groups (P = 0.000 and P = 0.018); mRNA level of IR in newly diagnosed and relapsed groups were significantly lower than that in the normal control group (P = 0.001 and P = 0.018). mRNA level of IR in CR group was significantly higher than that in newly diagnosed and relapsed groups (P = 0.000 and P = 0.001); mRNA levels of IGF-IR and IR correlated with the clinical stages of the disease, while they had no association with the sex, age and white blood cell counts at new diagnosis. It is concluded that IGF-IR positively correlates with the stages of the disease, indicating that IGF-IR plays a leading role in the proliferation of tumor cells, and its increased expression levels may reflect the malignant proliferation of ALL cells.

[Effects of insulin on expression of insulin receptor and insulin-like growth factor-1 receptor and proliferation in Reh cells].

This study was aimed to explore the effects of insulin on expression of insulin receptor (IR) and insulin-like growth factor-I receptor (IGF-IR) in Reh cells and promoting effect on proliferation of Reh cells. The proliferation of Reh cells were evaluated by CCK-8 assay. The expression levels of IR and IGF-IR mRNA in Reh cells at different times were detected by real-time quantitative polymerase chain reaction. The results showed that insulin promoted the proliferation of Reh cells in dose- and time-dependent manners. Compared with the control group, insulin promoted the proliferation of Reh cells obviously (P < 0.05). When Reh cells were treated with insulin 10(-9) mol/L for 24, 48 and 72 h, the relative quantity of IR expression (2(-ΔCt1)/2(-ΔCt2)) was 2.2520 ± 0.7431, 1.9956 ± 0.9692 and 3.9766 ± 1.3189, respectively, the relative quantity of IGF-IR expression was 1.0803 ± 0.2238, 1.6026 ± 0.6158 and 3.1013 ± 0.1008, respectively, compared with the control group. The expression levels of IR and IGF-IR mRNA in Reh cells treated with insulin were obviously increased compared with the control group. It is concluded that insulin promotes the proliferation of Reh cells. The high expression levels of IR and IGF-IR may closely related with the growth of leukemia cells.

[Analysis of PTPN11 mutation in children leukemia and its clinical significance].

This study was aimed to explore the frequency of PTPN11 mutation in children with leukemia and its clinical significance. Genomic DNAs were extracted from peripheral leukocytes of 131 patients with leukemia, including 101 cases of ALL, 26 cases of AML, 3 cases of CML and 1 case of juvenil myelomonocytic leukemia (JMML). The sequences of PTPN11 exons 3, 8, 13 were amplified by polymerase chain reaction (PCR), and the clinical characteristics of positive patients were analyzed. The results indicated that the PTPN11 mutation was found in 10 cases (9.9%) from newly diagnosed 101 cases of ALL. Grouping the newly diagnosed ALL children by various clinical features, it was found that the PTPN11 mutation did not show associations with sex, age, white blood cell (WBC) count, prednisone test sensitivity, clinical risk and disease recurrences at the first visit (P > 0.05). PTPN11 mutations were found in 2 cases out of 26 AML patients, including one AML-M(2) and one AML-M(4). No PTPN11 mutation in 3 CML patients was found. Exon 13 mutation of PTPN11 gene was found in 1 case of JMML. It is concluded that the E76 of exon 3 is the hot spot of PTPN11 mutation in children leukemia. The novel G503E (1508G > A) mutation is detected in one JMML patient. The PTPN11 mutation does not associate with the sex, age, WBC count, prednisone sensitive test and early recurrence.

[Relationship between polymorphism of HLA-A, -B, -DRB1 alleles and susceptibility of children to acquired aplastic anemia].

The aim of this study was to identify the relationship between susceptibility of children to acquired aplastic anemia (AA) and HLA-A, -B, -DRB1 alleles. 80 children with AA were enrolled in this study. Among of them, 34 patients collected from tissue typing test centers of Nanfang Hospital; 46 patients were diagnosed at Department of Pediatrics of Sun Yat-Sen Memorial Hospital. In these patients, 48 were males, 32 were females, and with average age 8.1 years old, 6 cases were non-severe AA (nSAA), 74 case were severe AA (SAA). The healthy control group consisted of 109 donors who were from the same area. All the patients and healthy controls were of Han Chinese, and all were unrelated individuals. The polymerase chain reaction sequence specific primers (PCR-SSP) was used to analyze the polymorphism of HLA-A, -B and -DRB1 alleles. Pearson Chi-square or continuity correction or two-sided Fisher's exact test were used. The results showed that the genotype frequency of HLA-B*48:01 and DRB1*09:01 were significantly higher in children with AA as compared with healthy controls (P < 0.05). The genotype frequency of HLA-B*51:01, DRB1*03:01 and DRB1*11:01 were significantly lower in children with AA as compared with healthy controls (P < 0.05). Besides, the results also demonstrated that the genotype frequencies of HLA-B*48:01 and DRB1*09:01 were significantly higher in SAA as compared with controls, the genotype frequencies of B*51:01, DRB1*03:01 and DRB1*11:01 were significantly lower in SAA, as compared with controls. In conclusion, HLA-B*48:01 and DRB1*09:01 are related with children AA, and may be susceptible alleles to the development of children AA. Besides, the expression of HLA-B*51:01, DRB1*03:01 and DRB1*11:01 are low in children with AA, whether they are relative protection alleles of children needs to be further studied.

[Expression of perforin in cord blood NK cells after IL-2/IL-15 stimulation and its relation with cytotoxicity].

This study was aimed to investigate the expression level of perforin in cord blood NK cells and the relation of perforin expression after IL-2, IL-15 stimulation to cytotoxicity of NK cells. NK cells were isolated from cord blood MNC by depleting CD3(+) cells and then enriching CD56(+) cells using immunomagnetic separation (CD3 and CD56 cell isolation kit, autoMACS, miltenyi). The purity was analysed by flow cytometry. According to the different combination of cytokines, there were two groups: group A (IL-2) and group B (IL-2 + IL-15). The cytotoxicity and perforin expression rate of fresh and different cultured CB-NK cells against K562/Jurkat cell lines were estimated by LDH release test (cytotoxic 96 non-radioactive cytotoxicity assay). The results showed that the purity of NK cells after separation was more than 90%. The cytotoxicity towards both tumor lines in group B was higher than that in group A (p < 0.05), and cytotoxicity in group A was higher than that of fresh NK cells (p < 0.05). Perforin expression rate of group A (84.55%) was higher than that of fresh NK cells (67.21%) (p < 0.05), and there was no significant difference between group A and B (84.55% versus 87.22%) Cytotoxic activity of CB-NK cells was positively correlated with perforin expression rate (r = 0.886, p < 0.05). It is concluded that IL-2 can enhance cytotoxicity of CB/BM-NK cells by increasing the perforin expression.

[Cytotoxic effect of IL-2/IL-15 stimulated cord blood derived NK cells on K562/Jurkat cell lines].

The aim of this study was to explore the cytotoxicity of fresh cord blood(CB) NK cells and the influence of IL-12 and IL-15 on activity of the NK cells killing K562 and Jurkat cells lines. The NK cells were isolated from cord blood by depleting CD3(+) cells and then enriching CD56(+) cells using sorting with immunomagnetic beads. The experiment was divided into 3 groups: group A (fresh CB-NK cells without cytokines), group B (CB-NK cells cultured by IL-2) and group C (CB-NK cells cultured by IL-2 and IL-15). The purity of NK cells was determined by flow cytometry; the cytotoxity of fresh and different cytokine-treated CB-NK cells on K562 and Jurkat cell lines was detected by LDH release test. The results showed that the purity of NK cells before and after sorting was 14.88 ± 9.2% and 92.39 ± 0.8% respectively. After culture for 3 days, NK-forming colony amounts in group B and group C were 148.60 ± 13.0 and 831.80 ± 23.0 respectively, the comparison between group B and group C showed the significant difference (p < 0.05). The cytotoxicities of NK cells in group A, B and C on K562 and Jurkat cell lines were 27.76 ± 8.8%, 61.90 ± 9.1% and 87.62 ± 3.7%; 29.32 ± 2.5%, 69.43 ± 4.4% and 92.95 ± 3.2% respectively, the difference was significant (p < 0.05). It is concluded that the fresh isolated CB-NK cells show low cytotoxic activity. After stimulated with IL-2 or IL-2 plus IL15, cytotoxicity of CB-NK cells increases obviously, the effect of IL-2 plus IL-15 is much better than IL-2 alone for promoting the growth and enhancing the cytotoxicity of CB-NK cells.