Dipeptide-catalysed Michael reaction under physiological conditions: Examination of potential bioorthogonality.

Reactions for drug synthesis under cell-like conditions or even inside living cells can potentially be used e.g., to minimize toxic side effects, to maximize bioactive compound efficacy and/or to address drug delivery problems. Those reactions should be bioorthogonal to enable the generation of drug-like compounds with sufficiently good yields. In the known bioorthogonal Michael reactions, using thiols and phosphines as nucleophiles (e.g., in CS and CP bond formation reactions) is very common. No bioorthogonal Michael addition with a carbon nucleophile is known yet. Therefore, the development of such a reaction might be interesting for future drug discovery research. In this work, the metal-free Michael addition between cyclohexanone and various trans-ß-nitrostyrenes (CC bond formation reaction), catalysed by a dipeptide salt H-Pro-Phe-O-Na+, was investigated for the first time in the presence of glutathione (GSH) and in phosphate-buffered saline (PBS). We demonstrated that with electron-withdrawing substituents on the aromatic ring and in ß-position of the trans-ß-nitrostyrene yields up to 64% can be obtained under physiological conditions, indicating a potential bioorthogonality of the studied Michael reaction. In addition, the selected Michael products demonstrated activity against human ovarian cancer cells A2780. This study opens up a new vista for forming bioactive compounds via CC bond formation Michael reactions under physiological (cell-like) conditions.

Coexistence of ETV6-RUNX1 and MLL aberration among pediatric acute lymphoblastic leukemia: case reports and a literature review.

Background: It is known that ETV6-RUNX1 is usually related to favorable prognosis, but MLL aberration has been associated with poor prognosis among pediatric acute lymphoblastic leukemia (ALL). However, the outcome of coexistence of ETV6-RUNX1 and MLL aberration in pediatric ALL patients is unknown. Herein, we report 4 cases of the coexistence of ETV6-RUNX1 and MLL-partial tandem duplications (MLL-PTD) in pediatric ALL patients and show the favorable outcome, which was never reported before. Case Description: The frequency of coexistence of ETV6-RUNX1 and MLL aberration at our children's medical center was calculated as 0.98% (4/410). All of them were ETV6/RUNX1-positive cases that exhibited MLL-PTD, and the 10-year event-free survival (EFS) and overall survival (OS) were both 75%. With the following keywords of "ETV6-RUNX1", "MLL", "children" and "acute lymphoblastic leukemia", a literature search of coexistence of ETV6-RUNX1 and MLL aberration was conducted in the database of PubMed, and 4 articles were retrieved finally, involving 16 cases of children. Among the 16 cases of pediatric ALL, the age ranged from 2 to 7 years old, including 9 males and 7 females and the white blood cell (WBC) count was (2.66-68.6)×109/L. In terms of fusion genes, they all had positive ETV6/RUNX1. Among them, MLL deletion was exhibited among 8 ETV6/RUNX1-positive patients, and 2 cases of der(21) duplication. MLL allelic deletions were shown among the remaining ETV6/RUNX1-positive patients. All patients showed a favorable outcome. Conclusions: The results of our analysis primarily provide compelling evidence that cases with an MLL-PTD or other types of MLL aberration are in fact a distinct subentry among ETV6-RUNX1 B-cell ALL (B-ALL).

Outcome and prognostic factors of CBF pediatric AML patients with t(8;21) differ from patients with inv(16).

PURPOSE: To explore the outcome and prognostic factors between inv(16) and t(8;21) disrupt core binding factor (CBF) in acute myeloid leukemia (AML). METHODS: The clinical characteristic, probability of achieving complete remission (CR), overall survival (OS) and cumulative incidence of relapse (CIR) were compared between inv(16) and (8;21). RESULTS: The CR rate was 95.2%, 10-year OS was 84.4% and CIR was 29.4%. Subgroup analysis showed that patients with t(8;21) had significant lower 10-year OS and CIR than patients with inv(16). Unexpectedly, there was a trend for pediatric AML receiving five courses cytarabine to have a lower CIR than four courses cytarabine (19.8% vs 29.3%, P = 0.06). Among the cohort of no-gemtuzumab ozogamicin(GO) treatment, inv (16) patients showed a similar 10-year OS (78.9% vs 83.5%; P = 0.69) and an inferior outcome on 10-year CIR (58.6% vs 28.9%, P = 0.01) than those patients with t(8;21). In contrast, inv (16) and t(8;21) patients receiving GO treatment had comparable OS (OS: 90.5% vs. 86.5%, P = 0.66) as well as CIR (40.4% vs. 21.4%, P = 0.13). CONCLUSION: Our data demonstrated that more cumulative cytarabine exposure could improve the outcome of childhood patients with t(8;21), while GO treatment was beneficial to the pediatric patients with inv(16).

Supramolecular Recognition of Cytidine Phosphate in Nucleotides and RNA Sequences.

Supramolecular recognition of nucleotides would enable manipulating crucial biochemical pathways like transcription and translation directly and with high precision. Therefore, it offers great promise in medicinal applications, not least in treating cancer or viral infections. This work presents a universal supramolecular approach to target nucleoside phosphates in nucleotides and RNA. The artificial active site in new receptors simultaneously realizes several binding and sensing mechanisms: encapsulation of a nucleobase via dispersion and hydrogen bonding interactions, recognition of the phosphate residue, and a self-reporting feature-"turn-on" fluorescence. Key to the high selectivity is the conscious separation of phosphate- and nucleobase-binding sites by introducing specific spacers in the receptor structure. We have tuned the spacers to achieve high binding affinity and selectivity for cytidine 5' triphosphate coupled to a record 60-fold fluorescence enhancement. The resulting structures are also the first functional models of poly(rC)-binding protein coordinating specifically to C-rich RNA oligomers, e.g., the 5'-AUCCC(C/U) sequence present in poliovirus type 1 and the human transcriptome. The receptors bind to RNA in human ovarian cells A2780, causing strong cytotoxicity at 800 nM. The performance, self-reporting property, and tunability of our approach open up a promising and unique avenue for sequence-specific RNA binding in cells by using low-molecular-weight artificial receptors.

Chidamide as maintenance after chemotherapy or hematopoietic stem cell transplantation in 27 children with T-cell lymphoblastic leukemia: A real-world prospective study.

Background: The long-term overall survival of children with T-cell acute lymphoblastic leukemia (T-ALL) is limited to approximately 80-85% because of a high incidence of relapse after achieving remission with intensive chemotherapy and hematopoietic stem cell transplantation (HSCT). Novel treatment strategies inducing long-term remission are needed to improve the outcome. Histone deacetylase inhibitors (HDACis) have been reported to be effective in a series of T-ALL cases. Preclinical studies suggested that T-ALL cells are sensitive to Chidamide, which is a selective HDACi. Methods: This preliminary clinical study evaluated the efficacy and safety of Chidamide in combination with chemotherapy or post-HSCT for children with T-ALL at a dose of 0.5 mg/kg weight of patient twice per week for at least 6 months. Results: In total, 27 children with a mean age of 7.88 years were included. The high-risk proportion was 66.7%. After a median follow-up period of 37.8 months (9.5-67.9 months), the overall survival and event-free survival in the patients treated with Chidamide were 94.1 and 95.2%, respectively. All patients except two maintained persistent remission with <0.01% blast cells in minimal residual disease. Conclusion: The combination therapy with Chidamide in a case series of T-ALL shows the promising clinical efficacy and good safety in children. Clinical trial registration: https://www.chictr.org.cn/, identifier ChiCTR2000030357.

Prognostic value and outcome for acute lymphocytic leukemia in children with MLL rearrangement: a case-control study.

PURPOSE: To evaluate the prognostic factors and outcome for acute lymphoblastic leukemia (ALL) in children with MLL rearrangement (MLL-r). METHODS: A total of 124 pediatric patients who were diagnosed with ALL were classified into two groups based on the MLL-r status by using a retrospective case-control study method from June 2008 to June 2020. RESULTS: The prevalence of MLL-r positive in the whole cohort was 4.9%. The complete remission (CR) rate on Day 33 in the MLL-r positive group was not statistically different from the negative group (96.8% vs 97.8%, P = 0.736). Multivariate analysis showed that T-cell, white blood cell counts (WBC) ≥ 50 × 109/L, MLL-AF4, and D15 minimal residual disease (MRD) positive were independent risk factors affecting the prognosis of MLL-r positive children. Stem cell transplantation (SCT) was a favorable independent prognostic factor affecting event-free survival (EFS) in MLL-r positive patients (P = 0.027), and there was a trend toward an independent prognostic effect on overall survival (OS) (P = 0.065). The 10-year predicted EFS for patients with MLL-AF4, MLL-PTD, MLL-ENL, other MLL partner genes, and MLL-r negative cases were 46.67 ± 28.61%, 85.71 ± 22.37%, 75 ± 32.41%, 75 ± 32.41%, and 77.33 ± 10.81%, respectively (P = 0.048). The 10-year predicted OS were 46.67 ± 28.61%, 85.71 ± 22.37%, 75 ± 32.41%, 75 ± 32.41%, and 85.2 ± 9.77%, respectively (P = 0.049). The 124 patients with ALL were followed up and eventually 5 (4%) cases relapsed, with a median relapse time of 3.9 years. CONCLUSION: Patients with MLL-r positive ALL have moderate remission rates, but are prone to relapse with low overall survival. The outcome of MLL-r positive ALL was closely related to the partner genes, and clinical attention should be paid to screening for MLL partner genes and combining them with other prognostic factors for accurate risk stratification.

Impact of posttransplant cyclophosphamide on the outcome of patients undergoing unrelated single-unit umbilical cord blood transplantation for pediatric acute leukemia.

BACKGROUND: Umbilical cord blood transplantation (UCBT) from unrelated donors is one of the successful treatments for acute leukemia in childhood. The most frequent side effect of UCBT is peri-engraftment syndrome (PES), which is directly associated with the greater prevalence of acute and chronic graft-versus-host-disease (aGvHD and cGvHD). In haploidentical stem cell transplantation, posttransplant cyclophosphamide (PTCY) has been demonstrated to be an effective method against GvHD. However, the effects of PTCY as a GvHD prophylactic in UCBT had not been investigated. This study aimed to evaluate the effects of PTCY on the outcomes of UCBT for pediatric acute leukemia. METHODS: This retrospective study included 52 children with acute leukemia who underwent unrelated single-unit UCBT after myeloablative conditioning regimens. The results from the PTCY and non-PTCY groups were compared. RESULTS: The incidence of transplantation-related mortality in non-PTCY and PTCY were 5% and 10% (p = 0.525), respectively. The incidence of relapse in non-PTCY and PTCY were 5% and 23% (p = 0.095), respectively. Second complete remission status (CR2) was an independent risk factor for relapse-free survival (hazard ratio = 9.782, p = 0.001). The odds ratio for sepsis or bacteremia incidence was significantly greater in the PTCY group (9.524, p = 0.017). PTCY group had increased rates of cytomegalovirus activity and fungal infection. The incidence of PES, aGvHD, cGvHD, and hemorrhagic cystitis in the PTCY group was lower than that in the non-PTCY group, although it was not significantly different. Additionally, higher doses of PTCY (29 mg/kg and 40 mg/kg) were associated with lower incidences of aGvHD and severe GvHD (65% and 29%, respectively) than lower doses (93% and 57%, respectively). Engraftment time and graft failure incidence were similar across groups. CONCLUSION: The results support the safety and efficiency of PTCY as part of PES controlling and GvHD prophylaxis in single-unit UCBT for children with acute leukemia. A PTCY dosage of 29 mg/kg to 40 mg/kg appears to be more effective in GvHD prophylaxis for UCBT patients.

Anticancer Aminoferrocene Derivatives Inducing Production of Mitochondrial Reactive Oxygen Species.

Elevated levels of reactive oxygen species (ROS) and deficient mitochondria are two weak points of cancer cells. Their simultaneous targeting is a valid therapeutic strategy to design highly potent anticancer drugs. The remaining challenge is to limit the drug effects to cancer cells without affecting normal ones. We have previously developed three aminoferrocene (AF)-based derivatives, which are activated in the presence of elevated levels of ROS present in cancer cells with formation of electron-rich compounds able to generate ROS and reduce mitochondrial membrane potential (MMP). All of them exhibit important drawbacks including either low efficacy or high unspecific toxicity that prevents their application in vivo up to date. Herein we describe unusual AF-derivatives lacking these drawbacks. These compounds act via an alternative mechanism: they are chemically stable in the presence of ROS, generate mitochondrial ROS in cancer cells, but not normal cells and exhibit anticancer effect in vivo.

A pyruvate kinase deficiency child with novel PK-LR gene mutations was successfully cured by matched unrelated donor peripheral blood stem cell transplantation.

BACKGROUND: Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by a PK-LR gene mutation. Allogeneic hematopoietic cell transplantation (HCT) is an effective cure for PKD. However, the experience of applying HCT in PKD is limited. METHODS: We present a child with novel PK-LR gene mutations who was successfully cured by matched unrelated donor peripheral blood stem cell transplantation (MUD-PBSCT). RESULTS: A 4-year-old, male patient suffered severe hemolytic anemia and jaundice 5 h after birth. Gene sequencing showed that the pyruvate kinase-liver and RBC (PK-LR) gene had a nonsense mutation in exon 5: c.602G>A (p.W201X), and large deletions in exons 3-9. Both of them were novel pathogenic mutations of the PK-LR gene. After transplantation, the hemoglobin level became normal and the nonsense mutation was undetectable. Grade Ⅳ acute graft-versus-host disease (aGVHD) and extensive chronic graft-versus-host disease (cGVHD) occurred in the patient. However, the GVHD was controlled effectively. The patient is alive and has good quality of life 22 months post-transplant, but has mild oral lichen planus-like lesion. CONCLUSION: Gene sequencing contributes to the diagnosis of PKD. HCT is an effective method for curing PKD, but we should explore how to reduce severe GVHD.

An Endoplasmic Reticulum Specific Pro-amplifier of Reactive Oxygen Species in Cancer Cells.

The folding and export of proteins and hydrolysis of unfolded proteins are disbalanced in the endoplasmic reticulum (ER) of cancer cells, leading to so-called ER stress. Agents further augmenting this effect are used as anticancer drugs including clinically approved proteasome inhibitors bortezomib and carfilzomib. However, these drugs can affect normal cells, which also rely strongly on ER functions, leading, for example, to accumulation of reactive oxygen species (ROS). To address this problem, we have developed ER-targeted prodrugs activated only in cancer cells in the presence of elevated ROS amounts. These compounds are conjugates of cholic acid with N-alkylaminoferrocene-based prodrugs. We confirmed their accumulation in the ER of cancer cells, their anticancer efficacy, and cancer cell specificity. These prodrugs induce ER stress, attenuate mitochondrial membrane potential, and generate mitochondrial ROS leading to cell death via necrosis. We also demonstrated that the new prodrugs are activated in vivo in Nemeth-Kellner lymphoma (NK/Ly) murine model.

Reduced intensity of early intensification does not increase the risk of relapse in children with standard risk acute lymphoblastic leukemia - a multi-centric clinical study of GD-2008-ALL protocol.

BACKGROUND: The prognosis of childhood acute lymphoblastic leukemia (ALL) is optimistic with a 5-year event-free survival (EFS) rate of 70-85%. However, the major causes of mortality are chemotherapy toxicity, infection and relapse. The Guangdong (GD)-2008-ALL collaborative protocol was carried out to study the effect of reduced intensity on treatment related mortality (TRM) based on Berlin-Frankfurt-Münster (BFM) 2002 backbone treatment. The study was designed to elucidate whether the reduced intensity is effective and safe for children with ALL. METHODS: The clinical data were obtained from February 28, 2008 to June 30, 2016. A total of 1765 childhood ALL cases from 9 medical centers were collected and data were retrospectively analyzed. Patients were stratified into 3 groups according to bone marrow morphology, prednisone response, age, genotype, and karyotype information: standard risk (SR), intermediate risk (IR) and high risk (HR). For SR group, daunorubicin was decreased in induction IA while duration was reduced in Induction Ib (2 weeks in place of 4 weeks). Doses for CAM were same in all risk groups - SR patients received one CAM, others got two CAMs. RESULTS: The 5-year and 8-year overall survival (OS), event-free survival (EFS) and cumulative incidence of relapse (CIR) were 83.5±0.9% and 83.1±1.0%, 71.9±1.1% and 70.9±1.2%, and 19.5±1.0% and 20.5±1.1%, respectively. The 2-year treatment-related mortality (TRM) was 5.2±0.5%. The 5-year and 8-year OS were 90.7±1.4% and 89.6±1.6% in the SR group, while the 5-year and 8-year EFS were 81.5±1.8% and 80.0±2.0%. In the SR group, 74 (15.2%) patients measured minimal residual disease (MRD) on Day 15 and Day 33 of induction therapy. Among them, 7 patients (9.46%) were MRD positive (≥ 0.01%) on Day 33. The incidence of relapse in the MRD Day 33 positive group (n=7) was 28.6%, while in the MRD Day 33 negative group (n=67) was 7.5% (p=0.129). CONCLUSIONS: The results of GD-2008-ALL protocol are outstanding for reducing TRM in childhood ALL in China with excellent long term EFS. This protocol provided the evidence for further reducing intensity of induction therapy in the SR group according to the risk stratification. MRD levels on Day 15 and Day 33 are appropriate indexes for stratification.

Prognostic Value and Outcome for ETV6/RUNX1-Positive Pediatric Acute Lymphoblastic Leukemia: A Report From the South China Children's Leukemia Group.

PURPOSE: To analyzed the outcome of ETV6/RUNX1-positive pediatric acute B lymphoblastic leukemia (B-ALL) with the aim of identifying prognostic value. METHOD: A total of 2,530 pediatric patients who were diagnosed with B-ALL were classified into two groups based on the ETV6/RUNX1 status by using a retrospective cohort study method from February 28, 2008, to June 30, 2020, at 22 participating ALL centers. RESULTS: In total, 461 (18.2%) cases were ETV6/RUNX1-positive. The proportion of patients with risk factors (age <1 year or ≥10 years, WB≥50×109/L) in ETV6/RUNX1-positive group was significantly lower than that in negative group (P<0.001), while the proportion of patients with good early response (good response to prednisone, D15 MRD < 0.1%, and D33 MRD < 0.01%) in ETV6/RUNX1-positive group was higher than that in the negative group (P<0.001, 0.788 and 0.004, respectively). Multivariate analysis of 2,530 patients found that age <1 or ≥10 years, SCCLG-ALL-2016 protocol, and MLL were independent predictor of outcome but not ETV6/RUNX1. The EFS and OS of the ETV6/RUNX1-positive group were significantly higher than those of the negative group (3-year EFS: 90.11 ± 4.21% vs 82 ± 2.36%, P<0.0001, 3-year OS: 91.99 ± 3.92% vs 88.79 ± 1.87%, P=0.017). Subgroup analysis showed that chemotherapy protocol, age, prednisone response, and D15 MRD were important factors affecting the prognosis of ETV6/RUNX1-positive children. CONCLUSIONS: ETV6/RUNX1-positive pediatric ALL showed an excellent outcome but lack of independent prognostic significance in South China. However, for older patients who have the ETV6/RUNX1 fusion and slow response to therapy, to opt for more intensive treatment.

Intracellular Amplifiers of Reactive Oxygen Species Affecting Mitochondria as Radiosensitizers.

Radiotherapy (RT) efficacy can be improved by using radiosensitizers, i.e., drugs enhancing the effect of ionizing radiation (IR). One of the side effects of RT includes damage of normal tissue in close proximity to the treated tumor. This problem can be solved by applying cancer specific radiosensitizers. N-Alkylaminoferrocene-based (NAAF) prodrugs produce reactive oxygen species (ROS) in cancer cells, but not in normal cells. Therefore, they can potentially act as cancer specific radiosensitizers. However, early NAAF prodrugs did not exhibit this property. Since functional mitochondria are important for RT resistance, we assumed that NAAF prodrugs affecting mitochondria in parallel with increasing intracellular ROS can potentially exhibit synergy with RT. We applied sequential Cu+-catalyzed alkyne-azide cycloadditions (CuAAC) to obtain a series of NAAF derivatives with the goal of improving anticancer efficacies over already existing compounds. One of the obtained prodrugs (2c) exhibited high anticancer activity with IC50 values in the range of 5-7.1 µM in human ovarian carcinoma, Burkitt's lymphoma, pancreatic carcinoma and T-cell leukemia cells retained moderate water solubility and showed cancer specificity. 2c strongly affects mitochondria of cancer cells, leading to the amplification of mitochondrial and total ROS production and thus causing cell death via necrosis and apoptosis. We observed that 2c acts as a radiosensitizer in human head and neck squamous carcinoma cells. This is the first demonstration of a synergy between the radiotherapy and NAAF-based ROS amplifiers.

Eltrombopag as first-line treatment for thrombocytopenia among paediatric patients after allogeneic haematopoietic stem cell transplantation.

AIMS: The purpose of this study is to examine the safety and efficacy of eltrombopag as first-line treatment for thrombocytopenia among paediatric patients after haematopoietic stem cell transplantation (HSCT). METHODS: Forty-three childhood patients with thrombocytopenia after HSCT who received eltrombopag were retrospectively analysed. RESULT: Eltrombopag was began at the median of 27 days after HSCT and lasted for 24 days. Thirty-five children responded to eltrombopag therapy, and the cumulative platelet recovery rate was 88.9%. The cumulative incidence of platelet recovery was lower (83.9 vs 100%; P = .035) in patients with decreased numbers of megakaryocytes before starting eltrombopag than in those with normal. Factors associated with a significantly elevated response to eltrobopag from univariate analysis were donor type. Results from the multiple regression analysis found that weight (hazard ratio [HR] = 0.7, 95% confidence interval [CI] 0.5-0.9, P = .022), platelet engraftment time (HR = 1.0, 95%CI 1.0-1.0, P = .012) and bone marrow megakaryocytes (HR = 8.0, 95%CI 1.5-43.3, P = .016) before starting eltrombopag were the independent risk factors. Based on Youden's index algorithm in the receiver-operating characteristic curve, the optimal cut-off value of the maintenance dose of eltrombopag in predicting nonresponders was 4 mg/kg. The area under the receiver-operating characteristic curve was 0.923 with sensitivity of 97.8%, specificity of 87.9%, positive predictive value of 72.3%, and negative predictive value of 92%. None of the paediatric patients stopped using eltrombopag due to side effect or intolerability. CONCLUSION: Eltrombopag is effective and safe in paediatric patients with thrombocytopenia after HSCT. The number of megakaryocytes in bone marrow before eltrombopag treatment may serve as a predictor of the response to eltrombopag. We recommend that the maintenance dose of eltrombopag should not exceed 4 mg/kg/d.

Prediction, management, and prognosis of mixed chimerism after hematopoietic stem cell transplantation in transfusion-dependent pediatric thalassemia patients.

BACKGROUND: Early-onset mixed chimerism (MC) with a high proportion of residual host cells is considered a signal of graft rejection in patients undergoing allogenic hematopoietic stem cell transplantation for transfusion-dependent thalassemia. In order to prevent graft rejection and minimize the risk of treatment-related graft-versus-host disease (GVHD), we established a hierarchical management system based on chimerism analysis. METHOD: This retrospective study provides a comprehensive review of the characteristics, interventions, and outcomes of the 38 patients who developed MC after transplantation among the 144 pediatric thalassemia patients between July 2007 and January 2019 at our center. RESULTS: A sibling donor, a blood type-matched donor, conditioning regimens without fludarabine, and transplants containing <10 × 108 total nucleated cells/kg were identified to be associated with the development of MC. Among the 38 patients developing MC, only four patients rejected the grafts. The response rate to donor lymphocyte infusion (DLI, only for patients receiving sibling donor transplantation) and cytokine immunomodulation without DLI was 70.6% and 42.9%, respectively. Patients that developed GVHD after DLI or cytokine therapy had a more significant increase in donor cell chimerism (16%, range 0%-35%) than those without (8.5%, range -21% to 40%, P = .049). However, even when treatment-related GVHD was included, patients with MC had a lower cumulative incidence of total acute GVHD than patients with complete donor chimerism (29.2% vs 48.0%, P = .030). CONCLUSIONS: Interventions based on chimerism analysis were effective in preventing graft rejection and did not increase treatment-related GVHD in thalassemia patients with MC.

[Relationship between Single Nucleotide Polymorphisms of IL2RA, IL-10 Gene and Epstein-Barr Virus Associated Hemophagocytic Lymphohistiocytosisin in children].

OBJECTIVE: To investigate the correlation of single nucleotide polymorphisms (SNPs) of IL2RA and IL-10 gene with the pathogenesis of Epstein Barr Virus associated hemophagocytic lymphohistiocytosis (EBV-HLH) in children and the effect of correlated SNPs on the prognosis of children with EBV-HLH. METHODS: For EBV-HLH group (51 cases), EBV-associated infectious mononucleosis (EBV-IM) group (48 cases) and EBV-positive healthy children group (52 cases), the genotypes at rs2104286, rs12722489, rs11594656 of IL2RA gene and rs1800896, rs1800871 and rs1800872 of IL-10 gene were detected with the SNaPshot technique. The distribution differences of genotype frequency and allele frequency of each SNP in each group were analyzed, and the correlated SNPs were taken as the research object for survival analysis. RESULTS: the frequency of AA genotype at rs1800896 of IL-10 gene in EBV-HLH group was higher than that in IM group (58.8% vs 25.0%) and healthy control group (58.8% vs 26.9%). The frequency of A allele in EBV-HLH group was higher than that in IM group (74.5% vs 54.2%) and healthy control group (74.5% vs 57.7%). Similarly, the frequency of AA genotype at rs2104286 of IL2RA gene in EBV-HLH group was higher than that in both IM (54.9% vs 27.1%) and healthy control group (54.9% vs 25.0%). The frequency of A allele in EBV-HLH group was higher than that in both IM (70.6% vs 51.0%) and healthy control group (70.6% vs 46.2%). Kaplan-Meier survival curves of EBV-HLH children with different genotypes were not statistically significant. CONCLUSION: The polymorphism of rs1800896 in IL-10 gene and rs2104286 in IL2RA gene may be related with the incidence of EBV-HLH in children, and the AA genotype and A allele of the both sites may be the susceptible risk factors for EBV-HLH.

[Efficacy of BMMSCs on aGVHD and Its Correlation with SerumInflammatory Cytokines in Pediatric Patients with Severe Refractory Acute Graft-Versus-Host Disease].

OBJECTIVE: To investigate the efficacy of bone marrow mesenchymal stem cells (BMMSC) on children with refractory graft-versus-host disease (GVHD) and to judge the efficacy of BMMSC by dynamically monitoring the changes of cytokines in children with GVHD before and after infusion of BMMSC, so as to provide a theoretical basis for clarifying the mechanism of BMMSC. METHODS: 17 children with refractory aGVHD including 7 of grade II, 6 cases of grade III and 4 cases of grade IV after allo-HSCT were enrolled. All the children with aGVHD, who received routine immunosuppressive therapy, but the state of disease not improved, were treated with immunosuppressive drugs combined with BMMSC infusion. Study endpoints included safety of BMMSC infusion, response to BMMSC, and overall response of aGVHD. The serum levels of IL-2α, IL-6, IL-10, IL-8 and TNF-α in aGVHD patients were measured by chemiluminescence before infusion of BMMSCs and Day 7, Day 14 after infusion of BMMSCs. RESULTS: The cumulative median dose of BMMSCs was 5.5 (3.4-11.1) × 106/kg for average of 3.7 times, and the median time of 16.5 (4-95) days for the first infusion of MSCs. In 17 cases of refractory GVHD, 14 responded to treatment, whereas 3 patients failed. The total effective rate was 82.4% and no adverse reactions occurred. Of the 14 survived cases (82.4%), the median follow-up time was 944 (559-1245) days from the first infusion of MSCs. The levels of TNF-α in children with grade II, III and IV GVHD before treatment were 9.5±4.3 pg/ml, 16.3±10.9 pg/ml and 35.8±21.2 pg/ml respectively. The difference between grade II and IV, III and IV was statistically significant (P<0.05). Compared with the ineffective group of BMMSC infusion, the serum TNF-αlevel in the BMMSCs treatment effective group was 10.8±5.6 pg/ml vs 40.6±14.8 pg/ml (t=-3.901, P<0.05) before treatment. In the effective group of BMMSCs infusion, IL-10 20±17.4 pg/ml of day 14 was significantly higher than that 7.3±3.1 pg/ml before the treatment (t=-2.850, P<0.05), while , the serum levels of IL-2α, IL-6, IL-8, TNF-α were not statistically significantly different (P＞0.05). CONCLUSION: The infusion of BMMSC is safe and effective in the treatment of refractory GVHD in children. TNF-αlevel relates with the severity of GVHD. BMMSC may play an anti-GVHD role by up regulating the level of cytokine IL-10 in vivo.

Hybrids of a 9-anthracenyl moiety and fluorescein as chemodosimeters for the detection of singlet oxygen in live cells.

Singlet oxygen (1O2) plays an important role in human innate immune response, plant physiology and anticancer photodynamic therapy (PDT). Therefore, its monitoring by convenient and sensitive methods (e.g. by detecting a fluorescence signal) by using non-toxic reagents would be advantageous. Known fluorogenic 1O2-chemodosimeters can potentially consume reducing agents in cells leading to the generation of toxic side products that limit their applications. In this paper we report on a series of 9-anthracenyl-fluorescein hybrids, which do not require any reducing agents for their reaction with 1O2. The selected compound 8d at a very low concentration of 100 nM is able to detect 1O2 in live human promyelocytic leukemia HL-60 cells with over 35-fold fluorescence signal enhancement within only 20 min assay time. This chemodosimeter is not toxic to HL-60 cells at concentrations ≤1 µM (higher concentrations were not tested) even at long incubation times ≤48 h.

The early diagnostic value of serum galactomannan antigen test combined with chest computed tomography for invasive pulmonary aspergillosis in pediatric patients after hematopoietic stem cell transplantation.

OBJECTIVE: The purpose of our study was to evaluate the diagnostic value of serum galactomannan antigen (GM) testing combined with chest computed tomography (CT) of invasive pulmonary aspergillosis (IPA) in pediatric patients after hematopoietic stem cell transplantation. METHODS: A retrospective nested case-control study was conducted in the identifying IPA among pediatric patients. RESULTS: A total of 141 eligible pediatric recipients with febrile neutropenia were enrolled in this study. All patients in the cases were diagnosed with proven-probable IPA(PP-IPA), while only 9 patients in the controls. GM testing was positive in 38 pediatric recipients in the cases and nine recipients in the controls with sensitivity of 62.3%, specificity of 81.8%. Among all patients with IPA, 46 patients in the cases and 9 patients in the controls had typical features of CT imaging with sensitivity of 79.3%, specificity of 85.2%. For discrimination of participants' GM testing combined with CT evaluation, the AUC of the diagnostic model was 0.887 with PPV of 0.764, and NPV of 0.872. Sensitivity was 0.793, and specificity was 0.852 in IPA. CONCLUSION: The combination methods with serum GM and CT scan might be used as a valuable marker for early diagnosis of IPA in pediatric patients after HSCT.

Iron Metabolism and Oxidative Status in Patients with Hb H Disease.

To evaluate the iron metabolism and oxidative status in patients with Hb H disease, we investigated 43 patients with Hb H disease, including eight deletional Hb H disease patients and 35 nondeletional Hb H disease patients and 20 healthy controls. The levels of hematological parameters, serum ferritin, hepcidin, superoxide dismutase (SOD), malondialdehyde (MDA) and total antioxidant capacity (TAC), were examined. We found higher serum ferritin levels and lower hepcidin, MDA and TAC levels in Hb H disease patients than in controls. The hepcidin level in Hb H disease patients was positively correlated with MDA and TAC levels but not with serum ferritin and SOD levels. The patients with nondeletional Hb H disease showed higher serum ferritin and Hb H concentrations than those patients with deletional Hb H disease. However, no statistically significant differences in SOD, MDA and TAC levels were found in patients with deletional and nondeletional Hb H disease. Oxidative stress and antioxidant defense were related to hepcidin levels. Our study indicated that hepcidin might be an important parameter for monitoring the iron metabolism and oxidative status of Hb H disease patients.