This study was aimed to prepare the polyclonal antibody against the soluble proliferation-inducing ligand (sAPRIL) antigen and to investigate its effects in suppressing sAPRIL mediated lymphocyte proliferation. Mutated recombinant sAPRIL protein, which lacks biological activity but maintains immunogenicity, was used as antigen to immunize humanized SCID mice. Sera were obtained at 6 weeks after immunization. Indirect ELISA and Western blot were used to detect the antibody titer and specificity. The inhibition of polyclonal antibodies on Raji and Jurkat cell proliferation stimulated by sAPRIL was assessed by the MTT assay. The results showed that the mutant of sAPRIL could induce the production of polyclonal antibodies against human sAPRIL. Western blot and indirect ELISA analyses indicated that the anti-serum had higher specificity with a titer of 1:640. Functional analysis revealed that these polyclonal antibodies significantly inhibited the proliferation of Raji and Jurkat cell stimulated by sAPRIL (p < 0.05). It is concluded the polyclonal antibody against human sAPRIL is successfully prepared, which can inhibit the proliferation of Raji and Jurkat cells stimulated by sAPRIL in vitro.
Animals ; Antibodies ; genetics ; immunology ; pharmacology ; Antibody Specificity ; immunology ; Cell Proliferation ; drug effects ; Cloning, Molecular ; Humans ; Immune Sera ; analysis ; immunology ; Jurkat Cells ; Mice ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; genetics ; immunology

OBJECTIVETo identify the incidence of PAX5 deletion in childhood B-lineage acute lymphoblastic leukemia (B-ALL) without reproducible chromosomal abnormalities and to investigate the association between PAX5 abnormalities and prognosis of ALL.

METHODSMultiplex ligation-dependent probe amplification was used to determine the copy numbers of PAX5 gene in children newly diagnosed with B-ALL without reproducible chromosomal abnormalities between April 2008 and April 2013 and controls (children with non-hematologic diseases or tumors). The patients were classifiied into deletion group and non-deletion group based on the presence of PAX5 deletion.

RESULTSEighteen (21%) out of 86 children with B-ALL had PAX5 deletion. The deletion group had a significantly higher total white blood cell count at diagnosis than the non-deletion group (P=0.001). The Kaplan-Meier analysis demonstrated that the deletion group had a significantly lower disease-free survival (DFS) rate than the non-deletion group (0.69±0.12 vs 0.90±0.04; P=0.017), but there was no significant difference in the overall survival rate between the two groups (P=0.128). The Cox analysis showed that PAX5 deletion was a risk factor for DFS (P=0.03).

CONCLUSIONSPAX5 deletion is an independent risk factor for DFS in B-ALL children without reproducible chromosomal abnormalities.

Acute Disease ; Adolescent ; Cell Lineage ; Child ; Child, Preschool ; Chromosome Aberrations ; Disease-Free Survival ; Female ; Gene Deletion ; Humans ; Infant ; Male ; PAX5 Transcription Factor ; genetics ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; mortality

OBJECTIVETo study the clinical and laboratory characteristics of cases with warts, hypogammaglobulinemia, infections and myelokathexis (WHIM) syndrome.

METHODAn 11-year-old boy was diagnosed as WHIM syndrome and CXCR4 gene mutation analysis was performed.

RESULTSince 3 years of age, the patient had recurrent fever and persistent cough. Since 6 years of age, he had warts on his fingers, the warts increased gradually. His complete blood count showed: white blood cell (WBC) 0.65×10(9)/L, neutrophil 0.15×10(9)/L, hemoglobin 116 g/L, platelet 200×10(9)/L, reticulocyte 0.62%. Results of serum biochemical tests: total protein (TP) 72.2 g/L (reference value 60 - 80 g/L), albumin 20.4 g/L (reference value 20 - 35 g/L), gammaglobulin 20.4 g/L (reference value 20 - 35 g/L). IgG 5.56 g/L (reference value 7.51 - 15.6 g/L), IgA 0.48 g/L (reference value 0.82 - 4.53 g/L), IgM 0.29 g/L (reference value 0.46 - 3.04 g/L). Peripheral blood lymphocyte subsets: CD3(+)T lymphocyte 43.6% (reference value 64.01% - 75.95%), CD19(+)B lymphocyte 1.00% (reference value 9.02% - 14.1%). Bone marrow smears showed that many of the neutrophils had a reactive appearance, with cytoplasmic vacuolation. Most neutrophils had hypersegmentation with four or five nuclear lobules. In some cells, the filaments connecting the nuclear lobes were long. CXCR4 mutation was detected.

CONCLUSIONWHIM syndrome is a rare immunodeficiency disorder with an autosomal-dominant pattern of inheritance. The disease is less progressive, and may accompany the patients' whole life.

Agranulocytosis ; genetics ; pathology ; Amino Acid Sequence ; Child ; Humans ; Immunoglobulins ; blood ; Immunohistochemistry ; Immunologic Deficiency Syndromes ; genetics ; pathology ; Leukocyte Count ; Male ; Mutation ; Receptors, CXCR4 ; genetics ; Warts ; genetics ; pathology

OBJECTIVETo study the efficacy and safety of Chinese Childhood Leukemia Group ALL 2008 (CCLG-ALL2008) protocol combined with tyrosine kinase inhibitor (TKI, imatinib) for the treatment of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) in children.

METHODSThe clinical data of 53 patients aged less than 15 years when first diagnosed with Ph+ ALL between October 2008 and December 2013 were retrospectively analyzed. The patients were assigned to two groups: HR (n=26) and HR+TKI (n=27). The HR group was treated with CCLG-ALL2008 protocol (for high-risk patients). The HR+TKI group was treated with imatinib in combination with CCLG-ALL2008 protocol (for high-risk patients).

RESULTSThe complete remission rate and chemotherapy induction-related mortality rate in the TKI+HR and HR groups were 100% vs 75% and 0 vs 15%, respectively. The 3-year event-free survival (EFS) rate in the HR group was (6±5)%; the 5-year EFS rate of the TKI+HR group was (52±11)%. Compared with the HR group, the TKI+HR group had no increase in the toxic responses to chemotherapy and had a decrease in the infection rate during the induction period.

CONCLUSIONSApplication of imatinib significantly improves the clinical efficacy in children with Ph+ ALL and has good safety.

Adolescent ; Antineoplastic Agents ; therapeutic use ; Child ; Child, Preschool ; Female ; Humans ; Imatinib Mesylate ; adverse effects ; therapeutic use ; Male ; Philadelphia Chromosome ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; genetics ; mortality ; Protein Kinase Inhibitors ; therapeutic use

OBJECTIVETo investigate the application of multiplex ligation-dependent probe amplification (MLPA) in the detection of copy number variations (CNVs) in pediatric ETV6/RUNX1-positive acute lymphoblastic leukemia (ALL), to compare this method with conventional karyotype analysis and fluorescence in situ hybridization (FISH), and to evaluate the value of MLPA.

METHODSThe clinical data of 95 children with ETV6/RUNX1-positive ALL who were treated from January 2006 to November 2012 were analyzed retrospectively, including clinical features, results of karyotype analysis, and results of FISH. CNVs were detected with MLPA.

RESULTSCNVs were detected in 73 (77%), and the median number of CNVs was 1 (range 0-6). The CNVs of EBF1, CDKN2A/2B, PAX5, ETV6, RB1, and BTG1 were detected in more than 10% of all the patients. The changes in the chromosome segments carrying the genes with CNVs detected by MLPA were not detected by conventional karyotype analysis. The coincidence rate between the CNVs in ETV6 gene detected by FISH and those detected by MLPA was 66%.

CONCLUSIONSMLPA is an efficient and convenient method to detect CNVs in children with ETV6/RUNX1-positive ALL.

Adolescent ; Child ; Child, Preschool ; Core Binding Factor Alpha 2 Subunit ; analysis ; DNA Copy Number Variations ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Infant ; Male ; Multiplex Polymerase Chain Reaction ; methods ; Oncogene Proteins, Fusion ; analysis ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics

OBJECTIVETo identify IKZF1 gene copy number abnormalities in BCR/ABL-negative B-lineage acute lymphoblastic leukemia (B-ALL) in children, and to investigate the association between such abnormalities and prognosis.

METHODSMultiplex ligation-dependent probe amplification (MLPA) was applied to detect IKZF1 gene copy number abnormalities in 180 children diagnosed with BCR/ABL-negative B-ALL. These children were classified into IKZF1 deletion group and IKZF1 normal group according to the presence or absence of IKZF1 gene deletion. The association between IKZF1 copy number abnormalities and prognosis of children with BCR/ABL-negative B-ALL was analyzed retrospectively.

RESULTSAmong 180 children, 27 (15.0%) had IKZF1 deletion; among the 27 children, 4 had complete deletions of 8 exons of IKZF1 gene, 17 had deletion of exon 1, 3 had deletions of exons 4-7, and 3 children had deletions of exons 2-7. Compared with those in the IKZF1 normal group, children in the IKZF1 deletion group had higher white blood cell (WBC) count and percentage of individuals with high risk of minimal residual disease at the first visit. IKZF1 deletions often occurred in BCR/ABL-negative children with no special fusion gene abnormalities. They were frequently accompanied by abnormalities in chromosomes 11, 8, 5, 7, and 21. The analysis with Kaplan-Meier method showed that disease-free survival (DFS) in the IKZF1 deletion group was significantly lower than that in the IKZF1 normal group (0.740 ± 0.096 vs 0.905 ± 0.034; P=0.002). Cox analysis showed that after exclusion of sex, age, initial WBC count, cerebrospinal fluid state at the first visit, prednisone response, and chromosome karyotype, IKZF1 deletion still affected the children's DFS (P<0.05).

CONCLUSIONSSome children with BCR/ABL-negative B-ALL have IKZF1 deletion, and IKZF1 deletion is an independent risk factor for DFS in children with BCR/ABL-negative B-ALL.

Adolescent ; Child ; Child, Preschool ; Female ; Fusion Proteins, bcr-abl ; analysis ; Gene Dosage ; Humans ; Ikaros Transcription Factor ; genetics ; Infant ; Male ; Multiplex Polymerase Chain Reaction ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; mortality ; Prognosis

OBJECTIVETo evaluate the protective efficacy of plague subunit vaccine, BALB/c mice, guinea pigs and rabbits were used in this study.

METHODSGroups of mice (10 per group), guinea pigs (14 per group) and rabbits (6 per group) were immunized with F1 + rV270 vaccine, EV76 vaccine and alum adjuvant by intramuscular route, respectively. Serum antibody titres of mice, guinea pigs and rabbits were determined by ELISA and the immunized animals were challenged with 10(6) CFU of Y. pestis strain 141 at the 8th week after the primary immunization.

RESULTSThe immunized mice, guinea pigs or rabbits with subunit vaccine developed anti-F1 IgG titre of 41 587.3 +/- 2.1, 11 543.7 +/- 2.1 or 522.4 +/- 22.4 and elicited statistical anti-F1 IgG titre difference among them (F = 17.58, P < 0.01). The immunized mice, guinea pigs or rabbits with subunit vaccine had anti-rV270 IgG titre of 15 748.7 +/- 1.6, 12.6 +/- 1.4 or 1648.0 +/- 5.0 and induced statistical anti-rV270 IgG titre difference among them (F value was 16.34, P < 0.01). There was significant anti-F1 IgG titre difference among mice, guinea pigs and rabbits immunized with EV76 vaccine that developed anti-F1 IgG titre of 913.4 +/- 4.5, 937.0 +/- 2.0 or 342.0 +/- 12.0 (F = 23.67, P < 0.01), whereas the immunized mice, guinea pigs and rabbits with EV76 vaccine developed anti-rV270 IgG titre of 12.0 +/- 1.0, 447.0 +/- 10.0, 40.0 +/- 11.0 and there was no anti-rV270 IgG titre difference between them (F = 2.20, P = 0.1314). The immunized mice with subunit vaccine developed significantly higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 30.57 and 19.04, respectively, P < 0.01), and there were no anti-F1 IgG titre differences between the immunized guinea pigs and rabbits (q = 0.04, P = 0.8485). The immunized mice with subunit vaccine developed significantly higher anti-rV270 IgG titres than immunized guinea pigs and rabbits (q value was 27.10 and 19.49, respectively, P < 0.01), and there were no anti-rV270 IgG titre differences between the immunized guinea pigs and rabbits with the subunit vaccine (q = 0.25, P = 0.6187). The immunized mice with EV76 elicited higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 40.67 and 29.10, respectively, P < 0.01), whereas there was no difference of F1 IgG titer between immunized guinea pigs and rabbits (q = 0.06, P = 0.8098). The immunized mice, guinea pigs and rabbits with subunit vaccine provided 100% (10/10), 86% (12/14) and 100% (5/5) protection against 10(6) CFU Y. pestis of challenge, respectively. The immunized mice, guinea pigs and rabbits with EV76 vaccine gave 100% (6/6), 93% (13/14) and 100% (6/6) protection against 10(6) CFU Y. pestis of challenge respectively.

CONCLUSIONBALB/c mice is the best small animal model for valuation of protective efficacy of plague subunit vaccine. The guinea pigs showed a high individual variation for this purpose. The rabbits can be used as an alternative model for evaluating plague subunit vaccine.

Animals ; Antibodies, Bacterial ; blood ; Dose-Response Relationship, Immunologic ; Female ; Guinea Pigs ; Immunization ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Models, Animal ; Plague ; prevention & control ; Plague Vaccine ; immunology ; Rabbits ; Vaccines, Subunit ; immunology

OBJECTIVETo study the clinical features of children with relapsed acute lymphoblastic leukemia (ALL) treated with the CCLG-ALL2008 protocol.

METHODSThe data of 591 children who were newly diagnosed with ALL and were treated with the CCLG-ALL 2008 protocol between April 2008 and June 2013 were collected, and the clinical features of 80 children with relapsed ALL were retrospectively analyzed.

RESULTSAfter treatment with the CCLG-ALL2008 protocol, the recurrence rate in the standard-risk, intermediate-risk and the high-risk groups were 7.0%, 10.7% and 28.7% respectively (P<0.05). The recurrence rate in patients with TEL/AML1-positive ALL was 8.0%, and the 5-year overall survival (OS) of the relapsed patients was 37.04%. The recurrence rates in patients with MLL-positive and BCR/ABL-positive ALL were 35.0% and 24.2% respectively, and none of the relapsed patients had long-term survival. The recurrence mainly occurred at the very early stage (53%), and none of patients with recurrence at the very early stage had long-term survival. The recurrence occurred at early stage and late stage accounted for 34% and 14% respectively, and the 5-year OS rates of patients with recurrence at early stage and late stage were 11.44% and 60.00% respectively. The sites of recurrence were mainly bone marrow alone (83%), and the 5-year OS of patients with recurrence at bone marrow alone was 9.23%. The recurrence in bone marrow and outside bone marrow accounted for 11%, and the 5-year OS of patients with recurrence in both bone marrow and outside bone marrow was 25.00%. The recurrence only outside bone marrow accounted for 6%, and the 5-year OS of patients with recurrence only outside bone marrow was 100%. The recurrence rate in patients with T-cell ALL was 9.5%, and none of the relapsed patients had long-term survival. The recurrence rate in patients with B-cell ALL was 14.3%, and the 5-year OS of the relapsed patients was 15.52%.

CONCLUSIONSAfter treatment with the CCLG-ALL2008 protocol, a relatively high recurrence rate is observed in children with high-risk ALL. Positive MLL and positive BCR/ABL are high-risk factors for recurrence. The recurrence rate is not significantly correlated with immunophenotype. A very low survival rate is seen in children whose recurrence have the following features: at early stage, only in bone marrow, T-cell ALL, and abnormal BCR/ABL and MLL.

Adolescent ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; mortality ; Prognosis ; Recurrence ; Retrospective Studies

OBJECTIVETo study the long-term efficacy of CAMSBDH-ALL chemotherapy protocol for the treatment of childhood acute lymphoblastic leukemia (ALL).

METHODSThree hundred and eighteen children who were newly diagnosed with ALL between January 1999 and December 2007 were enrolled in this study. Among the 318 children, 83 children who hospitalized before December 2002 were treated with CAMSBDH-ALL99 protocol, including 48 patients of standard risk and 35 patients of high risk. The patients (n=235; 131 in standard risk and 104 in high risk) who hospitalized after December 2002 were treated with CAMSBDH-ALL03 protocol. Patients in the CAMSBDH-ALL99 protocol group were treated with conventional chemotherapy. CAMSBDH-ALL03 protocol was modified based on the CAMSBDH-ALL99 protocol.

RESULTSThe long-term overall survival (OS) and event-free-survival (EFS) in the CAMSBDH-ALL03 group was significantly higher than in the CAMSBDH-ALL99 (P<0.01). The long-term OS and EFS of standard risk and high risk patients in the CAMSBDH-ALL03 protocol group were significantly higher than in the CAMSBDH-ALL99 protocol group (P<0.01). The CAMSBDH-ALL03 protocol group showed a significantly lower recurrence rate (28.9%) than in the CAMSBDH-ALL99 protocol group (50.6%) (P<0.05). The mortality rate in the CAMSBDH-ALL03 protocol group was 28.5% vs 56.6% in the CAMSBDH-ALL99 protocol group (P<0.05).

CONCLUSIONSThe therapeutic effect of the CAMSBDH-ALL03 protocol is supior to the CAMSBDH-ALL99 protocol group for childhood ALL, with a higher long-term survival rate.

Adolescent ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Child ; Child, Preschool ; Clinical Protocols ; Female ; Humans ; Infant ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; mortality ; Recurrence

OBJECTIVE: To study the association of platelet level at diagnosis with prognosis in children with acute lymphoblastic leukemia (ALL). METHODS: A total of 892 children with ALL who underwent chemotherapy with the CCLG-ALL 2008 regimen were enrolled. According to the platelet count at diagnosis, these children were divided into normal platelet count group (platelet count ≥100×109/L; n=263) and thrombocytopenia group (platelet count <100×10/L; n=629). The thrombocytopenia group was further divided into (50- <100)×10/L (n=243), (20- <50)×10/L (n=263), and <20×10/L (n=123) subgroups. The association of clinical features (sex, age, immunophenotype, and molecular biology) with event-free survival (EFS) and overall survival (OS) was analyzed. RESULTS: Compared with the thrombocytopenia group, the normal platelet count group had significantly lower positive rate of MLL gene rearrangement and recurrence rate (P<0.05), as well as a significantly higher 10-year EFS rate (P<0.05). There was no significant difference in 10-year OS between the two groups (P>0.05). The normal platelet count group still had a significantly higher 10-year EFS rate than the thrombocytopenia group after the children with MLL gene rearrangement were excluded (P<0.05), and there was still no significant difference in 10-year OS between the two groups (P>0.05). The <20×10/L subgroup had significantly lower 10-year EFS and OS rates than the normal platelet count group, the (50- <100)×10/L subgroup, and the (20- <50)×10/L subgroup (P<0.05). After the children with MLL gene rearrangement were excluded, the <20×10/L subgroup still had significantly lower 10-year EFS and OS rates than the normal platelet count group, the (50-<100)×10/L subgroup, and the (20- <50)×10/L subgroup (P<0.05). CONCLUSIONS ALL children with MLL gene rearrangement often have the clinical manifestation of thrombocytopenia. Platelet level at diagnosis is associated with the prognosis of ALL children. The children with normal platelet count have a low recurrence rate and good prognosis, and those with a platelet count of <20×10/L have the worst prognosis.
Child ; Disease-Free Survival ; Humans ; Immunophenotyping ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Prognosis ; Recurrence