Diagnostic value of TWEAK for predicting active lupus nephritis in patients with systemic lupus erythematosus: a systematic review and meta-analysis.

PURPOSE: Accumulative studies showed that tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) was up-regulated in the blood and urine from patients diagnosed with lupus nephritis (LN) and that it might be used as a novel biomarker for active LN. This meta-analysis aimed to determine the diagnostic value of TWEAK in active LN. METHODS: We searched the Cochrane Library, Embase, PubMed, Springer, Wanfang and CNKI databases for articles published up to 20 August 2020. The diagnostic capacity of TWEAK for active LN was assessed using pooled sensitivity and specificity, positive and negative likelihood ratios (PLR and NLR), diagnostic odds ratio (DOR), and area under the receiver operating characteristic curve (AUC). Quality assessment and publication bias were also evaluated. STATA 11.0 and Meta-Disc 1.4 were used to perform these analyses. RESULTS: Nine cross-sectional studies were included in this meta-analysis. The overall pooled sensitivity of TWEAK for the diagnosis of active LN was 0.69 (95% CI, 0.63-0.75), and specificity was 0.77 (95% CI, 0.71-0.82). The overall pooled PLR and NLR were 3.31 (95% CI, 2.05-5.35) and 0.38 (95% CI, 0.26-0.55), respectively, with a DOR of 10.89 (95% CI, 6.73-17.63) and AUC (SE) of 0.8276 (0.0289). Deeks' funnel plot revealed that the publication bias was insignificant in the study (p = .32). CONCLUSIONS: Our results suggest that TWEAK might be a potential biomarker for patients with active LN. Future cross-sectional and longitudinal studies are needed to confirm its diagnostic value, as well as to establish more definite cutoff for active LN.

[Clinical and genetic analyses of juvenile myelomonocytic leukemia].

OBJECTIVE: To study the clinical and genetic features of juvenile myelomonocytic leukemia (JMML) and the association between genotype and prognosis. Methods The clinical data of 15 children who were diagnosed with JMML were collected. Next-generation sequencing was used to detect common gene mutations of JMML. RESULTS: The male/female ratio was 6.5:1, and the age of onset was 19 months (range 2-67 months). Of the 15 children, 11 (73%) experienced disease onset before the age of 4 years, with abdominal distension and pyrexia as initial symptoms. All children had hepatosplenomegaly and superficial lymphadenectasis, with a number of peripheral blood mononuclear cells of >1.0×109/L and a percentage of juvenile cells of 1%-7% in peripheral blood smear. The percentage of bone marrow blasts + juvenile cells was <20%, and the percentage of monoblasts + promonocytes was 1%-10%. Of the 15 children, 10 (67%) had a higher level of hemoglobin F than the normal level at the corresponding age, with the highest level of 62.5%. All 15 children had the absence of Philadelphia chromosome, and one child had chromosome 7 deletion. All 15 children had a negative result of BCR/ABL fusion gene detection. PTPN11 gene mutation was found in 5 children (33%), NF1 mutation in 4 children (27%), CBL mutation in 3 children (20%), and RAS mutation in 3 children (20%). No children received regular chemotherapy, and one child underwent hematopoietic stem cell transplantation. The median follow-up time of 15 children was 18 months (range 1-48 months). Among the 15 children, 8 died (among whom 4 had PTPN11 gene mutation, 3 had NF1 mutation, and 1 had RAS mutation) and 7 survived. The children with PTPN11 mutation had the worst prognosis and the highest mortality rate, and those with CBL or NRAS mutation had a relatively good prognosis. The level of hemoglobin F was negatively correlated with survival time (rs=-7.21, P=0.002). CONCLUSIONS: In children with JMML, the type of gene mutation is associated with prognosis. The children with PTPN11 mutation often have a poor prognosis, and those with CBL or NRAS mutation have a relatively good prognosis.

Multicenter randomized trial of arsenic trioxide and Realgar-Indigo naturalis formula in pediatric patients with acute promyelocytic leukemia: Interim results of the SCCLG-APL clinical study.

Intravenous arsenic trioxide (ATO) has been adopted as the first-line treatment for acute promyelocytic leukemia (APL). Another arsenic compound named the Realgar-Indigo naturalis formula (RIF), an oral traditional Chinese medicine containing As4 S4 , has been shown to be highly effective in treating adult APL. In the treatment of pediatric APL, the safety and efficacy of RIF remains to be confirmed. This randomized, multicenter, and noninferiority trial was conducted to determine whether intravenous ATO can be substituted by oral RIF in the treatment of pediatric APL. From September 2011 to January 2017, among 92 patients who were 16 years old or younger with newly diagnosed PML-RARa positive APL, 82 met eligible criteria and were randomly assigned to ATO (n = 42) or RIF (n = 40) group. The remaining 10 patients did not fulfilled eligible criteria because five did not accept randomization, four died and one had hemiplegia prior to arsenic randomization due to intracranial hemorrhage or cerebral thrombosis. Induction and consolidation treatment contained ATO or RIF, all-trans-retinoic acid and low intensity chemotherapy. End points included event-free survival (EFS), adverse events and hospital days. After a median 3-year follow-up, the estimated 5-year EFS was 100% in both groups, and adverse events were mild. However, patients in the RIF group had significantly less hospital stay than those in the ATO group. This interim analysis shows that oral RIF is as effective and safe as intravenous ATO for the treatment of pediatric APL, with the advantage of reducing hospital stay. Final trial analysis will reveal mature outcome data.

[Effects of minimal residual disease level on day 33 of remission induction and IKZF1 genotype on the survival of children with B-lineage acute lymphoblastic leukemia].

OBJECTIVE: To study the effects of minimal residual disease (MRD) level on day 33 of remission induction and IKZF1 genotype on the survival of children with B-lineage acute lymphoblastic leukemia (B-ALL). METHODS: A total of 152 children with newly-diagnosed B-ALL who had complete remission after the first cycle of the chemotherapy and had complete follow-up information were enrolled in this study. According to the MRD detection by flow cytometry on day 33 of remission induction, they were divided into three groups: standard-risk (SR) group (MRD <10-4; n=60), intermediate-risk (IR) group (10-4≤ MRD <10-2; n=55), and high-risk (HR) group (MRD ≥10-2; n=37). Nested RT-PCR was used to determine the IKZF1 genotype of all children before chemotherapy. The effects of MRD level on day 33 of remission induction and IKZF1 genotype on the recurrence-free survival (RFS) of children with B-ALL were analyzed. RESULTS: There were 7 common IKZF1 subtypes in all the 152 children with B-ALL: IK1, IK2/3, IK4, IK6, IK8, IK9, and IK10. Of the 152 children, 130 had functional subtypes of IKZF1 and 22 had non-functional subtypes of IKZF1. During the follow-up period, relapse occurred in 26 (17%) children, and the recurrence rate was highest in the HR group (P<0.05). However, there was no significant difference in the recurrence rate between the SR group and the IR group (P>0.05). The cumulative recurrence rate of the children with non-functional subtypes of IKZF1 was significantly higher than that of those with functional types of IKZF1 (P<0.01). The predicted 5-year RFS rates in the SR, IR, and HR groups were (94.2±2.9)%, (86.7±3.8)%, and (56.2±4.5)% respectively (P<0.05). The 5-year RFS rate of the children with functional subtypes of IKZF1 was significantly higher than that of those with non-functional subtypes of IKZF1 (P<0.01). There was no significant difference in the predicted 5-year RFS rate between the children with functional subtypes of IKZF1 and those with non-functional subtypes of IKZF1 in the SR group (P>0.05). However, the predicted 5-year RFS rate of the children with functional subtypes of IKZF1 was significantly higher than that of those with non-functional subtypes of IKZF1 in the IR group and the HR group (P<0.05). CONCLUSIONS: B-ALL children with non-functional subtypes of IKZF1 have a high recurrence rate, and the recurrence rate will be even higher in B-ALL children with non-functional subtypes of IKZF1 and MRD ≥10-4 on day 33 of chemotherapy.

[Effect of glucocorticoid on dendritic cells in children with chronic immune thrombocytopenia].

OBJECTIVE: To investigate the change in dendritic cells (DCs) in children with chronic immune thrombocytopenia (cITP) and the effect of glucocorticoid on DCs in children with cITP. METHODS: Fifteen children with cITP and 20 healthy controls were included in the study. Flow cytometry was used to measure the DC subsets count in the 15 children with cITP before and after glucocorticoid treatment as well as the corresponding values in the 20 healthy controls. The DCs derived from peripheral blood monocytes in children with cITP were cultured in vitro and collected, and their immunophenotypes were determined by flow cytometry. RESULTS: Before glucocorticoid treatment, the children with cITP showed no notable change in the absolute count of myeloid DCs (mDCs) but showed decreased absolute count of plasmacytoid DCs (pDCs) and increased mDC/pDC ratio compared with the healthy controls (P<0.05). After glucocorticoid treatment, the children with cITP demonstrated increased absolute count of pDCs and decreased absolute count of mDCs and mDC/pDC ratio compared with before treatment (P<0.05). Before glucocorticoid treatment, the children with cITP had significantly higher positive rates of HLA-DR, CD80, CD83 and CD86 on peripheral blood DCs than the healthy controls (P<0.01). All the positive rates were significantly decreased after glucocorticoid treatment (P<0.01), so that there was no significant difference from the healthy controls (P>0.05). CONCLUSIONS: Disproportion and functional disturbance of DC subsets is associated with the pathogenesis of cITP in children. Glucocorticoid can strengthen the immunosuppression of DCs in children with cITP, which may contribute to the effectiveness of glucocorticoid as a treatment.

Inhibition of Human Sulfotransferases by Phthalate Monoesters.

Objective: This study aimed to investigate the inhibition of human important phase II metabolic enzyme sulfotransferases (SULTs) by phthalate monoesters, which are important metabolites of phthalate esters (PAEs). Method: Recombinant SULT-catalyzed metabolism of p-nitrophenol (PNP) was employed as the probe reactions of SULTs to investigate the inhibition of 8 kinds of phthalate monoesters towards SULT isoforms. An in vitro incubation system was utilized for preliminary screening, and 100 µM of phthalate monoesters was used. Inhibition kinetics were carried out to determine the inhibition of SULTs by phthalate monoesters. Result: Multiple phthalate monoesters have been demonstrated to exert strong inhibition potential towards SULT1A1, SULT1B1, and SULT1E1, and no significant inhibition of phthalate monoesters towards SULT1A3 was found. The activity of SULT1A1 was strongly inhibited by mono-hexyl phthalate (MHP), mono-octyl phthalate (MOP), mono-benzyl phthalate (MBZP), and mono-ethylhexyl phthalate (MEHP). Monobutyl phthalate (MBP), MHP, MOP, mono-cyclohexyl phthalate (MCHP), and MEHP significantly inhibited the activity of SULT1B1. MHP, MOP, and MEHP significantly inhibited the activity of SULT1E1. MOP was chosen as the representative phthalate monoester to determine the inhibition kinetic parameters (Ki) towards SULT1B1 and SULT1E1. The inhibition kinetic parameters (Ki) were calculated to be 2.23 µM for MOP-SULT1B1 and 5.54 µM for MOP-SULT1E1. In silico docking method was utilized to understand the inhibition mechanism of SULT1B1 by phthalate monoesters. Conclusions: All these information will be beneficial for understanding the risk of phthalate monoester exposure from a new perspective.

Prognostic significance of CNSL at diagnosis of childhood B-cell acute lymphoblastic leukemia: A report from the South China Children's Leukemia Group.

Objectives: The prognostic significance of acute lymphoblastic leukemia (ALL) patients with central nervous system leukemia (CNSL) at diagnosis is controversial. We aimed to determine the impact of CNSL at diagnosis on the clinical outcomes of childhood B-cell ALL in the South China Children's Leukemia Group (SCCLG). Methods: A total of 1,872 childhood patients were recruited for the study between October 2016 and July 2021. The diagnosis of CNSL depends on primary cytological examination of cerebrospinal fluid, clinical manifestations, and imaging manifestations. Patients with CNSL at diagnosis received two additional courses of intrathecal triple injections during induction. Results: The frequency of CNLS at the diagnosis of B-cell ALL was 3.6%. Patients with CNSL at diagnosis had a significantly higher mean presenting leukocyte count (P = 0.002) and poorer treatment response (P <0.05) compared with non-CNSL patients. Moreover, CNSL status was associated with worse 3-year event-free survival (P = 0.030) and a higher risk of 3-year cumulative incidence of relapse (P = 0.008), while no impact was observed on 3-year overall survival (P = 0.837). Multivariate analysis revealed that CNSL status at diagnosis was an independent predictor with a higher cumulative incidence of relapse (hazard ratio = 2.809, P = 0.016). Conclusion: CNSL status remains an adverse prognostic factor in childhood B-cell ALL, indicating that additional augmentation of CNS-directed therapy is warranted for patients with CNSL at diagnosis.

A Nomogram for Predicting Event-Free Survival in Childhood Acute Lymphoblastic Leukemia: A Multicenter Retrospective Study.

Objective: Even though childhood acute lymphoblastic leukemia (ALL) has an encouraging survival rate in recent years, some patients are still at risk of relapse or even death. Therefore, we aimed to construct a nomogram to predict event-free survival (EFS) in patients with ALL. Method: Children with newly diagnosed ALL between October 2016 and July 2021 from 18 hospitals participating in the South China children's leukemia Group (SCCLG) were recruited and randomly classified into two subsets in a 7:3 ratio (training set, n=1187; validation set, n=506). Least absolute shrinkage and selection operator (LASSO) and multivariate Cox regression analysis were adopted to screen independent prognostic factors. Then, a nomogram can be build based on these prognostic factors to predict 1-, 2-, and 3-year EFS. Concordance index (C-index), area under the curve (AUC), calibration curve, and decision curve analysis (DCA) were used to evaluate the performance and clinical utility of nomogram. Result: The parameters that predicted EFS were age at diagnosis, white blood cell at diagnosis, immunophenotype, ETV6-RUNX1/TEL-AML1 gene fusion, bone marrow remission at day 15, and minimal residual disease at day 15. The nomogram incorporated the six factors and provided C-index values of 0.811 [95% confidence interval (CI) = 0.792-0.830] and 0.797 (95% CI = 0.769-0.825) in the training and validation set, respectively. The calibration curve and AUC revealed that the nomogram had good ability to predict 1-, 2-, and 3-year EFS. DCA also indicated that our nomogram had good clinical utility. Kaplan-Meier analysis showed that EFS in the different risk groups stratified by the nomogram scores was significant differentiated. Conclusion: The nomogram for predicting EFS of children with ALL has good performance and clinical utility. The model could help clinical decision-making.

Molecular structure, expression and bioactivity characterization of TNF13B (BAFF) gene in mefugu, Takifugu obscurus.

B-cell activating factor of the TNF family (BAFF) induces B-cell survival, proliferation, immunoglobulin secretion and plays a role in enhancing immune responses. In the present study, a BAFF homolog has been identified in mefugu Takifugu obscurus, and its biological activities have been characterized. The mefugu BAFF (fBAFF) cDNA is 789 bp in length and translates into a 262-aa protein. The fBAFF genomic sequence consists of six exons and five introns, is approximately 1.8 kb in size. Amino acid sequence comparison indicated that fBAFF possessed the TNF signatures, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues, which were the typical characteristics of TNF gene in mammals and birds. The predicted three-dimensional (3D) structure of the fsBAFF monomer analyzed by comparative protein modeling revealed that it was very similar to its human counterpart. Real-time quantitative PCR (qPCR) analysis revealed that fBAFF was predominantly expressed in mefugu lymphoid tissue spleen. The SUMO-fsBAFF and GFP/fsBAFF efficiently expressed in Escherichia coli Rosetta (DE3) were confirmed by SDS-PAGE and Western blotting analysis. After purification, the GFP/fsBAFF fusion protein obtained similar fluorescence spectrum to those of GFP. Laser scanning confocal microscopy analysis showed GFP/fsBAFF could bind to its receptors. In vitro, the MTT assays and flow cytometric analysis indicated that SUMO-fsBAFF could promote the proliferation of mefugu splenocytes or mouse splenic B cells together with/without anti-mouse IgM. These findings indicate that SUMO-fsBAFF plays an important role in proliferation of mefugu B cells and has functional cross-reactivity among mefugu and other mammalians. Therefore, BAFF may be a potential immunologic factor for enhancing immunological efficacy in fish.

Production of bioactive γ-glutamyl transpeptidase in Escherichia coli using SUMO fusion partner and application of the recombinant enzyme to L-theanine synthesis.

The amino acid L-theanine (γ-glutamylethylamide) has potential important applications in the food and pharmaceutical industries and increased demand for this compound is expected. It is the major "umami" (good taste) component of tea and its favorable physiological effects on mammals have been reported. An enzymatic method for the synthesis of L-theanine involving recombinant Escherichia coli γ-glutamyltranspeptidase (GGT) has been developed. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of recombinant Escherichia coli γ-GGT. In order to obtain γ-GGT with high theanine-forming activity, safety, and low cost for food and pharmaceutics industry, M9 (consisting of glycerol and inorganic salts) and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blot analysis. The fusion protein was purified to 90% purity by nickel-nitrilotriacetic acid (Ni-NTA) resin chromatography with a yield of 115 mg per liter fermentation culture. After the SUMO/γ-GGT fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni-NTA column. Finally, about 62 mg recombinant γ-GGT was obtained from 1 l fermentation culture with no less than 95% purity. The recombinant γ-GGT showed great transpeptidase activity, with 1500 U of purified recombinant γ-GGT in a 1-l reaction system, a biosynthesis yield of 41 g of L-theanine was detected by paper chromatography or high pressure liquid chromatography (HPLC). Thus, the application of SUMO technology to the expression and purification of γ-GGT potentially could be employed for the industrial production of L-theanine.

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.

[Clinical Significance of CRLF2 High Expression in Bone Marrow Mononuclear Cells from Children with Acute Lymphoblastic Leukemia].

OBJECTIVE: To detect the expression of CRLF2 in bone marrow mononuclear cells from children with newly diagnosed acute lymphoblastic leukemia(ALL) and to explore its clinical significance in pediatric ALL. METHODS: A total of 218 children with newly diagnosed ALL who achieveal the complete remission and had the complete follow-up information were selected, and the expression level of CRLF2 in bone marrow mononuclear cells of these children was detected by real-time fluorescent quantitative PCR, and the significance of CRLF2 expression level in clinical prognosis of ALL children was analyzed by using statistical method. RESULTS: 28 cases in 218 children with complete data showed high expression of CRLF2. The cumulative recurrence rate in the CRLF2 high expression group was significantly higher than that in the low expression group (53.6% vs 12.6%) (P＜0.01). The predicted 5-year recurrence-free survival rate (RFS) of ALL children with CRLF2 high expression was significantly higher than that of low expression group (P＜0.01). There was no significant difference in the predicted 5-year RFS between ALL children with CRLF2 low and high expression in the standard-risk(SR) group (P＞0.05). The predicted 5-year RFS of ALL children with CRLF2 low expression was higher than that of ALL children with CRLF2 high expression in the intermediate-risk (IR) and high-risk (HR) groups. (P＜0.05). Cox analysis showed that CRLF2 high expression is an independent risk factor for the relapse of children with ALL. CONCLUSION: The recurrence rate of pediatric ALL with CRLF2 high expression is high, and CRLF2 high expression is an important prognostic factor for high risk of relapse in ALL children with IR and HR. It is necessary to use CRLF2 expression as an indicator of risk stratification in pediatric ALL.

[Effect of Icaritin on Proliferation and Apoptosis of THP-1 Cell and Its Mechanism].

OBJECTIVE: To investigate the effect of icaritin on the proliferation and apoptosis of THP-1 cells and its mechanism. METHODS: After treated with various concentrations of icaritin, cell proliferation was detected by MTS method, and apoptosis was measured with flow cytometry and Hoechst 33258 staining. Expression of BCL-2, BAX and Caspase-3 protein in THP-1 cell was detected by Western blot. RESULTS: After treatment with various concentrations (4-32 µmol/L) of icaritin for 24, 48, 72 h, the inhibition rate of cell growth significantly increased (P<0.05) in time-dose dependent manner(r=0.946); and the apoptotic rate of cells significantly increased (P<0.05) in time-dose dependent manner(r= 0.924). The expression of BCL-2 protein at 48 h decreased significantly in icaritin-treated group, compared with that in control group (P<0.05), while the expression of BAX and Caspase3 protein at 48 h increased significantly in icaritin-treated group, compared with that in control group (P<0.05). CONCLUSION: Icaritin can inhibit proliferation and induce apoptosis of THP-1 in vitro, Icaritin may induce apoptosis in THP-1 cells through the mitochondrial pathway.

cDNA cloning, recombinant expression and bioactivity of Père David's deer BAFF.

B cell activating factor (BAFF), a member of the TNF family, is a critical cytokine for the survival, proliferation, maturation, and differentiation of B cells. In the present study, Père David's deer BAFF (miBAFF) was amplified from Elaphurus davidianus using RT-PCR. This is the first BAFF cloned from a member of Cervidae family. The open reading frame (ORF) of the miBAFF cDNA consists of 843 bases that encode a 280-amino acid protein bearing typical TNF homology domain. Sequence alignment shows that miBAFF shares 39.3%-97% sequence homology with the BAFF sequences of other mammals. Comparative protein modeling predicted that the 3D structure of the soluble mature portion of miBAFF (misBAFF) is very similar to that of human BAFF (hsBAFF). Recombinant misBAFF fused to a SUMO-tag was efficiently expressed in Escherichia coli BL21 (DE3) cells. The protein molecular weight of ~36 KDa was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In vitro, purified misBAFF was shown to promote the survival and proliferation of Père David's deer peripheral blood lymphocytes and mouse B cells. These results indicate that miBAFF plays an important role in the survival/proliferation of mouse B cells and, shows highly conserved evolutionarily, leading to functional cross-reactivity that exists between mouse and Père David's deer BAFF.

Molecular cloning and partial functional characterization of a proliferation inducing ligand (APRIL) in zebrafish (Danio rerio).

Here we describe the identification of a Danio rerio homologue of a proliferation-inducing ligand (APRIL) of the TNF family (designated zAPRIL). Sequence analysis showed that the open reading frame of zAPRIL consists of 600 bases encoding a protein of 199-amino acids. Recombinant soluble APRIL (zsAPRIL) was constructed consisting of fluorescence-enhanced green fluorescent protein (EGFP) and cloned into a pET28a vector. SDS-PAGE and western blotting analysis indicated a high-level expression of soluble EGFP/zsAPRIL protein in Escherichia coli BL21 (DE3). Observation by confocal microscopy demonstrated that EGFP/zsAPRIL could successfully bind to the surface receptors of zebrafish lymphocytes. In vitro survival analysis revealed that purified EGFP/zsAPRIL was able to promote the survival of zebrafish lymphocytes in a dose-dependent manner. The biological role of APRIL does not seem to be restricted to proliferation induction. Zebrafish may could served as a model organism for further study of APRIL.

cDNA cloning and recombinant expression of a proliferation inducing ligand (APRIL) from Père David's deer.

In the present study, the cDNA of Père David's deer APRIL (miAPRIL) was successfully amplified, and nucleotide sequence analysis showed that the open reading frame (ORF) of miAPRIL consists of 753 bases encoding 250-amino acids. Phylogenetic analysis exhibited high homology with the even-toed ungulates (artiodactyla). The extracellular soluble domain of the miAPRIL (misAPRIL) fragment was cloned into the expression vector pET43.1a and transformed into Escherichia coli BL21 (DE3) for recombinant expression. SDS-PAGE and western blotting analysis indicated that misAPRIL protein expression was successfully achieved. Indirect immunofluorescence staining demonstrated that misAPRIL has the ability to bind to the surface of Père David's deer lymphocytes. Flow cytometry analysis revealed that misAPRIL was able to enhance lymphocytes survival in a dose-dependent manner.

Molecular structure, bioinformatics analysis, expression and bioactivity of BAFF (TNF13B) in dog (Canis familiaris).

B cell activating factor (BAFF), belonging to the tumor necrosis factor (TNF) family, plays an important role in B cell survival, proliferation and differentiation. In the present study, we amplified the cDNA of dog (Canis familiaris) BAFF (designated doBAFF) from spleen by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) strategies. The open reading frame (ORF) of doBAFF covers 879 bp encoding 292 amino acids, with a 152-aa mature peptide. The soluble mature part of doBAFF (dosBAFF) shares 91.5%, 72.1%, 96.7%, 94.0% and 91.5% identity with the human, mouse, cattle, pig and rabbit counterparts, respectively. The predicted three-dimensional (3D) structural analysis of dosBAFF analyzed by "comparative protein modeling" revealed that it was very similar to its human counterpart. Real-time quantitative PCR analysis revealed that doBAFF was mainly expressed in spleen. Two fusion proteins SUMO-dosBAFF and GFP/dosBAFF were efficiently expressed in Escherichia coli BL21 (DE3) and purified using metal chelate affinity chromatography (Ni-NTA). Laser scanning confocal microscopy analysis showed that GFP/dosBAFF could bind to the mouse splenic B-cell. In vitro, SUMO-dosBAFF and GFP/dosBAFF were able to promote the survival/proliferation of dog lymphocytes or mouse splenic B cells with/without anti-IgM. Therefore, BAFF may be a potential immunologic factor for enhancing immunological efficacy in dog.

Molecular structure, phylogenetic analysis, tissue distribution, and function characterization of interferon-γ-inducible lysosomal thiol reductase (GILT) gene in sheep (Ovis aries).

Interferon-γ-inducible-lysosomal thiol reductase (GILT) plays a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction. In this study, a sheep cDNA, sGILT, encoding GILT protein was isolated from the spleen cDNA library of Ovis aries. It codes for a deduced protein of 244 amino acids with a putative molecular weight of 27.6 kDa, which has all the typical structural features of GILT protein including an active-site CXXC motif, a GILT signature sequence CQHGX(2)ECX(2)NX(4)C, and 5 conserved cysteines. Sequence comparison indicated the amino acid sequence of sGILT showed high identity to cow GILT (93.03%). Phylogenetic analysis showed that sGILT and cow GILT shared the greatest homology. The result of real-time PCR suggested that sGILT mRNA was expressed in a tissue-specific manner and obviously up-regulated in splenocytes and PBMCs after induction with lipopolysaccharide (LPS). Recombinant sGILT fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Its expression was confirmed by SDS-PAGE and Western blotting analysis. Thiol reductase activity was assessed using antibody as the substrate. These results suggest that sGILT has the activity of disulfide bond reduction and indicate that sheep also express a protein that has been found to maintain first line of innate immune defense at basal level.

Molecular structure and characterization of the cytokine TWEAK and its receptor Fn14 in bovine.

Full-length cDNA encoding TWEAK and Fn14 from bovine was isolated. We used bioinformatics to analyze the gene structure, function, evolutionary relationships, and predicted three-dimensional structure of proteins and binding sites. Real-time quantitative PCR (Q-PCR) analysis revealed that both TWEAK and Fn14 are constitutively expressed in various tissues in bovine. Bovine TWEAK and Fn14 interaction analysis by yeast two-hybrid. Our results suggest that the TWEAK-Fn14 pathway is evolutionarily highly conserved. It will be helpful for investigation on the biological role of the TWEAK/Fn14 system in this important animal model. Furthermore, it provides insight into the molecular evolution of the emerging TWEAK and Fn14 families.

Molecular structure, expression analysis and functional characterization of APRIL (TNFSF 13) in goat (Capra hircus).

A proliferation-inducing ligand (APRIL) is an important member of the tumor necrosis factor (TNF) superfamily. In the present study, a novel cDNA was isolated from the spleen of goat by RT-PCR and designated as goat APRIL (gAPRIL). The open reading frame (ORF) of this cDNA covered 753bp, encoding a protein of 250 amino acids. Sequence comparison showed that gAPRIL contains a predicted transmembrane domain, a putative furin protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF gene in mammals. The predicted three dimensional (3D) structure of soluble part of the gAPRIL (gsAPRIL) monomer analyzed by comparative protein modeling revealed that it is very similar to its counterparts. Real-time PCR analysis revealed that gAPRIL was constitutively expressed in various tissues. Recombinant gsAPRIL fused with NusA tag was efficiently produced in Escherichia coli BL21 (DE3) and then analyzed by the SDS-PAGE as well as western blot. Laser scanning confocal microscopy analysis showed gsAPRIL could bind to its receptors. In vitro, the MTT and flow cytometric methods revealed that purified gsAPRIL protein was not only able to promote survival/proliferation of goat splenocytes, but also able to stimulate survival/proliferation of mouse B cells. These results indicated that gAPRIL plays an important role in survival/proliferation of goat splenocytes and provided a basis for investigating its potential to be used as an immunoadjuvant for enhancing vaccine efficacy and as an immunotherapeutic in goats.