Efficacy, Safety, and Population Pharmacokinetics of Eltrombopag in Children with Different Severities of Aplastic Anemia.

Eltrombopag was approved as a first-line treatment for patients older than 2 years old with severe aplastic anemia (SAA). However, data on eltrombopag in children with different types of aplastic anemia (AA), especially non-severe AA (NSAA), are limited. We performed a prospective, single-arm, and observational study to investigate eltrombopag's efficacy, safety, and pharmacokinetics in children with NSAA, SAA, and very severe AA (VSAA). The efficacy and safety were assessed every 3 months. The population pharmacokinetic (PPK) model was used to depict the pharmacokinetic profile of eltrombopag. Twenty-three AA children with an average age of 7.9 (range of 3.0-14.0) years were enrolled. The response (complete and partial response) rate was 12.5%, 50.0%, and 100.0% after 3, 6, and 12 months in patients with NSAA. For patients with SAA and VSAA, these response rates were 46.7%, 61.5%, and 87.5%. Hepatotoxicity occurred in one patient. Fifty-three blood samples were used to build the PPK model. Body weight was the only covariate for apparent clearance (CL/F) and volume of distribution. The allele-T carrier of adenosine triphosphate-binding cassette transporter G2 was found to increase eltrombopag's clearance. However, when normalized by weight, the clearance between the wild-type and variant showed no statistical difference. In patients with response, children with NSAA exhibited lower area under the curve from time zero to infinity, higher CL/F, and higher weight-adjusted CL/F than those with SAA or VSAA. However, the differences were not statistically significant. The results may support further individualized treatment of eltrombopag in children with AA.

Individualized Use of 6-Mercaptopurine in Chinese Children with ALL: A Multicenter Randomized Controlled Trial.

Continuous 6-mercaptopurine (6-MP) dose titration is necessary because of its narrow therapeutic index and frequently encountered dose-limiting hematopoietic toxicity. However, evidence-based guidelines for gene-based 6-MP dosing have not been established for Chinese children with acute lymphoblastic leukemia (ALL). This multicenter, randomized, open-label, active-controlled clinical trial randomly assigned Chinese children with low- or intermediate-risk ALL in a 1:1 ratio to receive TPMT-NUDT15 gene-based dosing of 6-MP (N = 44, 10 to 50 mg/m2 /day) or standard dosing (N = 44, 50 mg/m2 /day) during maintenance therapy. The primary end point was the incidence of 6-MP myelosuppression in both groups. Secondary end points included frequencies of 6-MP hepatotoxicity, duration of myelosuppression and leukopenia, event-free survival, and steady-state concentrations of active metabolites (6-thioguaninenucleotides and 6-methylmercaptopurine nucleotides) in erythrocytes. A 2.2-fold decrease in myelosuppression, the primary end point, was observed in the gene-based-dose group using ~ 50% of the standard initial 6-MP dose (odds ratio, 0.26, 95% confidence interval, 0.11 to 0.64, P = 0.003). Patients in the gene-based-dose group had a significantly lower risk of developing thiopurine-induced myelosuppression and leukopenia (P = 0.015 and P = 0.022, respectively). No significant differences were observed in the secondary end points of the incidence of hepatotoxicity and steady-state concentrations of active metabolites in erythrocytes between the two groups. TPMT- and NUDT15-based dosing of 6-MP will significantly contribute toward further reducing the incidence of leukopenia in Chinese children with ALL. This trial is registered at www.clinicaltrial.gov as #NCT04228393.

A simple and accurate LC­MS/MS method for monitoring cyclosporin A that is suitable for high throughput analysis.

With time, the number of samples in clinical laboratories from therapeutic drug monitoring has increased. Existing analytical methods for blood cyclosporin A (CSA) monitoring, such as high-performance liquid chromatography (HPLC) and immunoassays, have limitations including cross-reactivity, time consumption, and the complicated procedures involved. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has long been considered the reference standard owing to its high accuracy, specificity, and sensitivity. However, large numbers of blood samples, multi-step preparation procedures, and longer analytical times (2.5-20 min) are required as a consequence of the different technical strategies, to ensure good analytical performance and routine quality assurance. A stable, reliable, and high throughput detection method will save personnel time and reduce laboratory costs. Therefore, a high throughput and simple LC-MS/MS method was developed and validated for the detection of whole-blood CSA with CSA-d12 as the internal standard in the present study. Whole blood samples were prepared through a modified one-step protein precipitation method. A C18 column (50x2.1 mm, 2.7 µm) with a mobile phase flow rate of 0.5 ml/min was used for chromatographic separation with a total running time of 4.3 min to avoid the matrix effect. To protect the mass spectrometer, only part of the sample after LC separation was allowed to enter the mass spectrum, using two HPLC systems coupled to one mass spectrometry. In this way, throughput was improved with detection of two samples possible within 4.3 min using a shorter analytical time for each sample of 2.15 min. This modified LC-MS/MS method showed excellent analytical performance and demonstrated less matrix effect and a wide linear range. The design of multi-LC systems coupled with one mass spectrometry may play a notable role in the improvement of daily detection throughput, speeding up LC-MS/MS, and allowing it to be an integral part of continuous diagnostics in the near future.

A modified LC-MS/MS method for the detection of whole blood tacrolimus and its clinical value in Chinese kidney transplant patients.

Background: For patients who treated with tacrolimus after kidney transplant, therapeutic drug monitoring is essential to improve their prognosis. However, previous detection methods have limitations, such as the overestimation and unacceptable bias in the immunoassays. Precision medicine has been challenged. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is recognized as the gold standard due to its accuracy and specificity, but lack of throughput and complex process limits its clinical application. Therefore, an accurate, simple and high throughput method for tacrolimus monitoring is needed for clinical practice. Methods: A modified LC-MS/MS method was introduced and validated. Whole blood samples were prepared by a one-step protein precipitation method. Chromatographic separation was achieved using a Phenomenex Kinetex 2.6 µm XB-C18 2.1 × 50 mm column with a total run time of 3.5 min to avoid matrix effect. An electrospray ionization source (ESI) was used in positive ion multiple reaction monitoring (MRM) mode for mass spectrometric detection. In order to protect the mass spectrometer, only part of the sample after LC separation was allowed to enter the mass spectrum, through a two HPLC systems coupled one mass spectrometry design. In this way, the instrument throughput is also improved and realizing the detection of 2 samples within 3.5 min and carried out a shorter analyzing time for each sample of 1.75 min. Additionally, we calculated tacrolimus-intrapatient variant (Tac-IPV) based on this modified method and assessed the prognostic value of Tac-IPV in Chinese kidney transplant patients. Results: The LC-MS/MS was modified by streamlining the procedure and increasing the throughput. The method proved to be accurate and reproducible with all performance parameters suitably meeting the clinical requirements over a calibration ranged from 0.37 to 42.90 ng/mL. Parameters such as linearity, limit of quantification (LoQ) and dilution integrity were validated with a clinical reportable range from 0.37 to 343.20 ng/mL, which was particularly useful for high drug concentrations patients (rare but very serious). Both cross-contamination and matrix effects were negligible. Clinical data of 83 patients showed that Tac-IPV was associated with poor kidney transplant outcome in Chinese (Hazard Ratio (HR) = 3.96, 4.75; 95% Cl: 1.10-14.21, 1.23-18.36; P < 0.05). Conclusions: This modified LC-MS/MS method possessed high throughput and simple sample preparation, allowing it to meet daily clinical needs. At the same time, Tac-IPV based on this modified LC-MS/MS had excellent prognostic value in kidney transplantation. These advantages have great significance for the individualized treatment of Chinese kidney transplant patients and broad application of Tac-IPV.

Integrated Medical Care and the Continuous 4C Nursing Model to Improve Nursing Quality and Clinical Treatment of Patients with Acute Stroke: Based on a Retrospective Case-Control Study.

Objective: This research paper is based on a retrospective case-control study for exploring the effects of medical nursing integration and the continuous 4C nursing model to improve the clinical treatment and nursing quality of patients with acute stroke. Method: For this purpose, a total of 313 patients with acute stroke, treated in our hospital from February 2020 to April 2021, were enrolled. They were divided into control and study groups with an even number of patients. The control group received integrated medical care number (N = 156), while the study group received integrated medical care and a continuous 4C nursing model (N = 157). In integrated medical care, the general data, self-nursing ability, degree of neurological impairment, Fugl-Meyer Assessment (FMA) score, Barthel index score, and quality of life score were compared between the two groups. Result: The self-nursing concept, self-nursing responsibility, self-nursing skills, health knowledge, and total score of the patients in the study group were higher than those in the control group (P < 0.05). The neurological function scores of the study group were lower than those of the control group at 1, 3, and 6 months after discharge (P < 0.05). The scores of the study group were higher than those of the control group at 1, 3, and 6 months after discharge (P < 0.05). The Barthel index score of the study group was higher than that of the control group at 1, 3, and 6 months after discharge. The scores of physical function, psychological function, social function, and health self-cognition in the study group were lower than those in the control group (P < 0.05). Conclusion: The application of integrated medical care and the continuous 4C nursing model for patients with acute stroke is beneficial to enhance the degree of neurological impairment of stroke patients, improve activities of daily life and motor function, and facilitate patients' quality of life. It is helpful to strengthen the attitude and feeling of cooperation between doctors and nurses, promote cooperation between doctors and nurses, reduce the defects of nursing work, heighten the quality of nursing, and achieve the requirement and goal of effectively promoting high-quality nursing.

Prognostic implications of PPL expression in ovarian cancer.

Periplakin (PPL) is a main member in plakin family, which plays important role in cellular adhesion complexes supporting and cytoskeletal integrity supplying. PPL was reported to be a potential biomarker candidate for several types of cancers. However, the biological functions and underlying mechanisms of PPL in ovarian cancer (OV) remain unclear. In the present study, we used GEPIA 2, Human Protein Atlas, Oncomine, LinkedOmics, Kaplan-Meier Plotter, STRING, CytoHubba plug-in and TIMER to determine the associations among PPL expression, prognosis, and immune cell infiltration in OV. RT-qPCR and IHC analysis were conducted to validated the role of PPL in an independent OV cohort. Compared with the normal ovary tissues, the levels of PPL mRNA and protein expression were both obviously higher in OV tumors from multiple datasets (P < 0.05), and a poor survival was observed to be strongly correlated with high PPL expression (P < 0.05). Moreover, the results were further validated by RT-qPCR and IHC analysis in an independent OV cohort. A gene-clinical nomogram was constructed, including PPL mRNA expression and clinical factors in TCGA. Functional network analysis suggested that PPL participates in the important pathways like Wnt signaling pathway, MAPK signaling pathway. Ten hub genes (LAMC2, PXN, LAMA3, LAMB3, LAMA5, ITGA3, TLN1, ACTN4, ACTN1, and ITGB4) were identified to be positively associated with PPL. Furthermore, PPL expression was negatively correlated with infiltrating levels of CD4+ T cell, macrophages, neutrophils, and dendritic cells. In conclusion, PPL may be an unfavorable prognostic biomarker candidate in OV, which was also correlated with immune infiltrating and function in immunotherapy response.

Population Pharmacokinetics and Safety of Dasatinib in Chinese Children with Core-Binding Factor Acute Myeloid Leukemia.

BACKGROUND: Dasatinib, an orally administered Src-family kinase inhibitor, is combined with the standard chemotherapeutic regimen to enhance antineoplastic activity against core-binding factor acute myeloid leukemia (CBF-AML) in adults; however, limited data are available for use in children. In the present study, we studied the pharmacokinetics and safety of dasatinib in children. METHODS: Dasatinib (60 or 80 mg/m2 once daily) was administered to 20 children with CBF-AML. Blood samples were collected and drug concentrations were quantified by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Population pharmacokinetic analysis and Monte-Carlo simulations were performed using NONMEM software, and safety analyses were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 4.0 (NCT03844360). RESULTS: Twenty pediatric patients (3.3-14.4 years of age) were included, and a total of 40 dasatinib concentrations were available for population pharmacokinetic analysis. The mean (standard deviation) of the estimated area under the concentration-time curve extrapolated to steady state (AUCss) of dasatinib 60 and 80 mg/m2 was 366.1 (146.6) ng·h/mL and 425.3 (150.7) ng·h/mL, respectively. The majority of adverse events were grade 1/2 in severity, including thrombocytopenia, rash, and pain in the extremities. The estimated cumulative incidence of complete remission and complete molecular response were 95.0% and 75.5%, respectively. CONCLUSIONS: The population pharmacokinetics of orally administered dasatinib were evaluated in pediatric CBF-AML patients. The AUCss of dasatinib (80 mg/m2) in CBF-AML pediatric patients was similar to those of dasatinib (100 mg) in adult patients. Dasatinib is well-tolerated in pediatric patients with CBF-AML.

ZNF76 predicts prognosis and response to platinum chemotherapy in human ovarian cancer.

Ovarian cancer (OV) is the most lethal gynecologic malignancy. One major reason of the high mortality of the disease is due to platinum-based chemotherapy resistance. Increasing evidence reveal the important biological functions and clinical significance of zinc finger proteins (ZNFs) in OV. In the present study, the relationship between the zinc finger protein 76 (ZNF76) and clinical outcome and platinum resistance in patients with OV was explored. We further analyzed ZNF76 expression via multiple gene expression databases and identified its functional networks using cBioPortal. RT-qPCR and IHC assay shown that the ZNF76 mRNA and protein expression were significantly lower in OV tumor than that in normal ovary tissues. A strong relationship between ZNF76 expression and platinum resistance was determined in patients with OV. The low expression of ZNF76 was associated with worse survival in OV. Multivariable analysis showed that the low expression of ZNF76 was an independent factor predicting poor outcome in OV. The prognosis value of ZNF76 in pan-cancer was validated from multiple cohorts using the PrognoScan database and GEPIA 2. A gene-clinical nomogram was constructed by multivariate cox regression analysis, combined with clinical characterization and ZNF76 expression in TCGA. Functional network analysis suggested that ZNF76 was involved in several biology progressions which associated with OV. Ten hub genes (CDC5L, DHX16, SNRPC, LSM2, CUL7, PFDN6, VARS, HSD17B8, PPIL1, and RGL2) were identified as positively associated with the expression of ZNF76 in OV. In conclusion, ZNF76 may serve as a promising prognostic-related biomarker and predict the response to platinum in OV patients.

Population Pharmacokinetics and Safety of Oral Tetra-Arsenic Tetra-Sulfide Formula in Pediatric Acute Promyelocytic Leukemia.

BACKGROUND: An oral tetra-arsenic tetra-sulfide (AS4S4) formula has been recommended as an outpatient post-remission treatment for Chinese adults with acute promyelocytic leukemia (APL) but limited data are available for children. In this exploratory study, we aimed to evaluate the pharmacokinetics and safety of the AS4S4 formula in children. METHODS: Eleven newly diagnosed and one relapsed pediatric patient (4-14 years of age) treated with the AS4S4 formula were included. Blood samples were collected from 12 children, and drug concentrations were quantified by ICP-MS. Population pharmacokinetic analysis and Monte-Carlo simulation were performed using NONMEM software. Toxic effects were graded according to the NCI-CTCAE, Version 3. RESULTS: A total of 107 arsenic concentrations (0.1-75.0 µg L-1) were used for population pharmacokinetic analysis. The median (range) of estimated weight-normalized CL and volume distribution at steady-state were 45.26 (35.63-82.18) L h-1 kg-1 and 230.37 (85.96-495.68) L kg-1, respectively. No patients discontinued AS4S4 treatment owing to adverse events, and there were no drug-related adverse events over grades 3-4. All newly diagnosed APL patients were in MCR with a median follow-up of 28 months (range, 23 to 37 months). Both the estimated 3-year EFS and OS rates were 100%. CONCLUSION: The pharmacokinetics and safety oral AS4S4 formula was evaluated for the first time in pediatric APL. The pharmacokinetic assessment demonstrated that the dosing regimen of 60 mg/kg/d TID resulted in a higher steady-state through concentration in children than that which was achieved in adults. The results of this study indicate that the AS4S4 formula is safe in newly diagnosed pediatric APL patients.

[Clinical effect of two different regimens in treatment of acute lymphoblastic leukemia children with bone marrow recurrence].

OBJECTIVE: To study the short-term effect of two different re-induction regimens in the treatment of acute lymphoblastic leukemia (ALL) children with bone marrow recurrence. METHODS: A retrospective analysis was performed for 57 ALL children with bone marrow recurrence. According to their treatment regimen, they were divided into two groups: VMDP (vincristine + mitoxantrone + dexamethasone + PEG-asparaginase; n=42) and VIDP (vincristine + idarubicin + dexamethasone + PEG-asparaginase; n=15). The two groups were compared in terms of complete response rate and incidence rate of adverse reactions. RESULTS: There was no significant difference in complete response rate between the VMDP and VIDP groups (74% vs 73%, P>0.05). All children experienced grade ≥3 hematological adverse events. The VMDP group had a significantly lower chemotherapy-related mortality rate than the VIDP group (P<0.05). There was no significant difference in the incidence rate of infection between the two groups (P>0.05). CONCLUSIONS: For ALL children with bone marrow recurrence, both re-induction regimens can achieve a relatively high complete response rate, and VMDP regimen has a lower chemotherapy-related mortality rate and can thus be used as an option for re-induction in ALL children with bone marrow recurrence.

The Legionella pneumophila effector WipA disrupts host F-actin polymerisation by hijacking phosphotyrosine signalling.

The major virulence determinant of Legionella pneumophila is the type IVB secretion system (T4BSS), which delivers approximately 330 effector proteins into the host cell to modulate various cellular processes. However, the functions of most effector proteins remain unclear. WipA, an effector, was the first phosphotyrosine phosphatase of Legionella with unknown function. In this study, we found that WipA induced relatively strong growth defects in yeast in a phosphatase activity-dependent manner. Phosphoproteomics data showed that WipA was likely involved into endocytosis, FcγR-mediated phagocytosis, tight junction, and regulation of actin cytoskeleton pathways. Western blotting further confirmed WipA dephosphorylates several proteins associated with actin polymerisation, such as p-N-WASP, p-ARP3, p-ACK1, and p-NCK1. Thus, we hypothesised that WipA targets N-WASP/ARP2/3 complex signalling pathway, leading to disturbance of actin polymerisation. Indeed, we demonstrated that WipA inhibits host F-actin polymerisation by reducing the G-actin to F-actin transition during L. penumophila infection. Furthermore, the intracellular proliferation of wipA/legK2 double mutant was significantly impaired at the late stage of infection, although the absence of WipA does not confer any further effect on actin polymerisation to the legK2 mutant. Collectively, this study provides unique insights into the WipA-mediated regulation of host actin polymerisation and assists us to elucidate the pathogenic mechanisms of L. pnuemophila infection.

[Mitoxantrone-cytarabine-etoposide induction therapy in children with acute myeloid leukemia: a single-center study of complications and clinical outcomes].

OBJECTIVE: To investigate the complications and clinical outcome of children with acute myeloid leukemia (AML) undergoing mitoxantrone-cytarabine-etoposide (MAE) induction therapy. METHODS: A total of 170 children with AML were given MAE induction therapy, and the complications and remission rate were analyzed after treatment. RESULTS: The male/female ratio was 1.33:1 and the mean age was 7.4 years (range 1-15 years). Leukocyte count at diagnosis was 29.52×109/L [range (0.77-351)×109/L]. Of all children, 2 had M0-AML, 24 had M2-AML, 2 had M4-AML, 48 had M5-AML, 3 had M6-AML, 7 had M7-AML, 69 had AML with t(8;21)(q22;q22), and 15 had AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22). The most common complication was infection (158/170, 92.9%). Among these 158 patients, 22 (13.9%) had agranulocytosis with pyrexia (with no definite focus of infection), and 136 (86.1%) had definite focus of infection (including bloodstream infection). Other complications included non-infectious diarrhea, bleeding, and drug-induced hepatitis. Treatment-related mortality was observed in 10 children, among whom 8 had severe infection, 1 had multiple organ failure, and 1 had respiratory failure. Remission rate was evaluated for 156 children and the results showed a complete remission rate of 85.3%, a partial remission rate of 4.5%, and a non-remission rate of 10.3%. CONCLUSIONS: Induction therapy with the MAE regimen helps to achieve a good remission rate in children with AML after one course of treatment. Infection is the main complication and a major cause of treatment-related mortality.

Erratum: Publisher Correction: Cytological, physiological, and transcriptomic analyses of golden leaf coloration in Ginkgo biloba L.

[This corrects the article DOI: 10.1038/s41438-018-0015-4.].

Cytological, physiological, and transcriptomic analyses of golden leaf coloration in Ginkgo biloba L.

Ginkgo biloba is grown worldwide as an ornamental plant for its golden leaf color. However, the regulatory mechanism of leaf coloration in G. biloba remains unclear. Here, we compared G. biloba gold-colored mutant leaves and normal green leaves in cytological, physiological and transcriptomic terms. We found that chloroplasts of the mutant were fewer and smaller, and exhibited ruptured thylakoid membranes, indistinct stromal lamellae and irregularly arranged vesicles. Physiological experiments also showed that the mutant had a lower chlorophyll, lower flavonoid and higher carotenoid contents (especially lutein). We further used transcriptomic sequencing to identify 116 differentially expressed genes (DEGs) and 46 transcription factors (TFs) involved in chloroplast development, chlorophyll metabolism, pigment biosynthesis and photosynthesis. Among these, the chlorophyll biosynthesis-related PPO showed down-regulation, while chlorophyll degradation-related NYC/NOL had up-regulated expression in mutant leaves. Z-ISO, ZDS, and LCYE, which are involved in carotenoid biosynthesis were up-regulated. Quantitative real-time PCR (RT-qPCR) further confirmed the altered expression levels of these genes at three stages. The alteration of PPO and NYC/NOL gene expression might affect chlorophyll biosynthesis and promote degradation of chlorophyll b to chlorophyll a, while the up-regulated genes Z-ISO, ZDS and LCYE enhanced carotenoid accumulation. Consequently, changes in the ratio of carotenoids to chlorophylls were the main factors driving the golden leaf coloration in the mutant G. biloba.

ClpP-deletion impairs the virulence of Legionella pneumophila and the optimal translocation of effector proteins.

BACKGROUND: The opportunistic bacterial pathogen Legionella pneumophila uses substrate effectors of Dot/Icm type IVB secretion system (T4BSS) to accomplish survival and replication in amoebae cells and mammalian alveolar macrophages. During the conversion between its highly resistant, infectious dormant form and vigorously growing, uninfectious replicative form, L. pneumophila utilizes a complicated regulatory network in which proteolysis may play a significant role. As a highly conserved core protease, ClpP is involved in various cellular processes as well as virulence in bacteria, and has been proved to be required for the expression of transmission traits and cell division of L. pneumophila. RESULTS: The clpP-deficient L. pneumophila strain failed to replicate and was digested in the first 3 h post-infection in mammalian cells J774A.1. Further investigation demonstrates that the clpP deficient mutant strain was unable to escape the endosome-lysosomal pathway in host cells. We also found that the clpP deficient mutant strain still expresses T4BSS components, induces contact-dependent cytotoxicity and translocate effector proteins RalF and LegK2, indicating that its T4BSS was overall functional. Interestingly, we further found that the translocation of several effector proteins is significantly reduced without ClpP. CONCLUSIONS: The data indicate that ClpP plays an important role in regulating the virulence and effector translocation of Legionella pneumophila.

Identification of endonuclease domain-containing 1 gene in Japanese flounder Paralichthys olivaceus.

The mRNA level of the endonuclease domain-containing 1 gene (Jf_ENDOD1) in Japanese flounder Paralichthys olivaceus kidney was significantly increased after injection of formalin-killed bacteria cells (FKC) in the previous microarray study. ENDOD1 is a member of the DNA/RNA non-specific nucleases family, and its role in fish immunity has not been reported. The open reading frame of Jf_ENDOD1 cDNA was 912 bp, encoding 303 amino acids. The first 27 amino acids were predicted to be a signal peptide and the mature Jf_ENDOD1 was calculated as 32 kDa. The amino acid sequence of Jf_ENDOD1 showed 76% identity to that of large yellow croaker Larimichthys crocea. Transcripts of Jf_ENDOD1 were marginally detected in all sampled tissues from healthy fish, while they were significantly detected in brain, kidney, spleen and intestine at 6 h post FKC injection. Jf_ENDOD1 recombinant protein produced in Escherichia coli showed DNase activity. Furthermore, to evaluate the DNase activities in vivo, total proteins from Japanese flounder kidney and spleen were extracted at 12, 24 and 72 h post Edwardsiella tarda FKC injection. The DNase activity of extracted protein was higher in treated fish than in untreated fish. Since the mRNA levels were significantly up-regulated after the FKC treatment, Jf_ENDOD1 might be responsible for the activities.

[Immunotherapeutic Effect of Dermatophagoides pteronyssinus Group 1 Allergen T Cell Epitope Peptide Against Allergic Asthma in Mice].

Objective: To study the specific immunotherapeutic effect of Dermatophagoides pteronyssinus group 1 major allergen T-cell fusion epitope peptide vaccine TAT-IhC-DPTCE against allergic asthma. Methods: One hundred and twenty SPF-grade BALB/c mice were randomized into PBS group (group A), asthma group （group B）, and immune treatment groups respectively receiving intraperitoneal (i.p.) injections of ProDer p 1 allergen (group C), DPTCE （group D）, TAT-DPTCE (group E) or TAT-IhC-DPTCE (group F) （n=20 in each group）. In detail, PBS （group A） or allergen extract derived from Dermatophagoides pteronyssinus （groups B-F, 10 µg） was intraperitoneally injected on days 0, 7 and 14, and was continued by aerosol inhalation from day 21 for 7 consecutive days （0.5 µg/ml, once/day, 30 min each time）. The mice in groups C-F received i.p. injections of 100 µg/ml ProDer p 1, DPTCE, TAT-DPTCE and TAT-IhC-DPTCE respectively 30 min prior to inhalation challenge on days 25-27 as a specific immunotherapy, while those in groups A and B received 200 µl PBS. Twenty-four hours after the last inhalation challenge, all the mice were sacrificed. The lung histopathological changes were examined by HE staining. The levels of IFN-γ, IL-13, IL-10 and TGF-ß in the bronchoalveolar lavage fluid (BALF) was determined with ELISA, and eosinophils in the BALF were counted (n=20 mice in each group). The serum level of IgE, IgG1 and IgG2a in orbital blood was determined by ELISA（n=5 mice in each group）. Results: HE staining revealed increased BALF eosinophils and decreased pulmonary inflammation in group F compared with group B. The IFN-γ level in group F ［（298.75±26.09） pg/mlï¼½ was significantly higher than those in groups B［（158.71±20.89） pg/mlï¼½, C［（210.38±18.92） pg/mlï¼½, D ［（229.44±13.00） pg/mlï¼½ and E［（233.24±20.39） pg/mlï¼½ （all P<0.01）. Similar results were also found for IL-10 and TGF-ß, while the IL-13 levels in groups C ［（47.35±4.71） pg/mlï¼½, D ［（41.90±4.28） pg/mlï¼½, E［（41.05±6.50） pg/mlï¼½ and F［（18.53±5.67） pg/mlï¼½ were all significantly lower than that in group B ［（66.68±6.63） pg/ml］（all P<0.01）. The number of BALF eosinophils in group B ï¼»5.65±0.91ï¼½×105/mlï¼½ was significantly higher than that in group A ［（0.45±0.39）×105/mlï¼½ （P<0.01）, while the BALF eosinophils in groups C ［（4.00±0.59）×105/mlï¼½, D ［（3.39±0.63）×105/mlï¼½, E ［（3.24±0.69）×105/mlï¼½ and F ［（1.42±0.49）×105/mlï¼½ decreased after immune treatment (all P<0.01). ELISA results showed that the serum IgE level in group F ［（5.26±1.72） ng/mlï¼½ was significantly lower than those in group B ［（32.81±2.98） ng/mlï¼½ and the other 3 treatment groups [group C, （20.06±3.17） ng/ml; D, （17.06±3.18） ng/ml; E, （16.23±3.61） ng/mlï¼½. Similar results were also obtained for IgG1. In contrast, the serum IgG2a level in group F［（43.10±1.34） ng/mlï¼½ was significantly higher than those in group B［（12.61±1.87） ng/mlï¼½ and the other 3 treatment groups ï¼»group C, （23.37±2.67） ng/ml; D, （25.60±2.10） ng/ml; E, （25.91±1.33） ng/mlï¼½ （all P<0.01）. Conclusion: Immunotherapy with chimeric TAT-IhC-DPTCE can effectively ameliorate the allergic airway response and pulmonary inflammation in mice.

[Analysis of sensitization effect of chimeric allergen TAT-IhC-R8 derived from major allergen group 1 genes of dust mites].

OBJECTIVE: To explore the sensitization effect of allergen TAT-IhC-R8, derived from major allergen group 1 genes of dust mites. METHODS: Forty BALB/c mice were randomly divided into 4 groups, namely PBS group, ovalbumin (OVA) group, R8 group and TAT-IhC-R8 (TIR8) group, 10 mice each group. All the mice in OVA, R8 and TIR8 groups were treated with corresponding allergens (10 µg/ml) on the 0, 7th and 14th day by intraperitoneal injection and nebulized inhalation on day 21 with the concentration of 30 min/d for 7 days. The mice in PBS group were treated with PBS. Twenty-four hours after the last challenge, all the mice were sacrificed, their bronchoalveolar lavage fluids (BALFs) and sera were collected and their spleen cells were cultured. ELISA was performed to detect the levels of IFN-γ and IL-13 in BALFs and supernatants of cultured splenocytes (SCSs) of the mice, as well as the levels of allergen-specific IgE (sIgE), IgG, and IgG2 in their sera. The number of white blood cells and eosinophils in BALF were calculated. In addition, the airway inflammation and mucus secretion were analyzed by haematoxylin and eosin (H&E) staining. RESULTS: Compared with the PBS group, the lung inflammations of mice in the OVA, R8 and TIR8 groups were observed obviously, including inflammatory infiltration, bronchial epithelial cell breakage and falling off, as well as vasculitis. The numbers of the total white blood cells and eosinophils in BALF of mice in the TIR8 group were significantly more than those in the OVA and R8 groups (all P < 0.01). The IL-13 levels in BALFs and SCSs of mice in the TIR8 group were significantly higher than those in the OVA group and R8 group (all P < 0.01). However, the level of IFN-γ of mice in the TIR8 group was lower than those in the latter 2 groups (all P < 0.01). In addition, the levels of sera sIgE and IgG of mice from the TIR8 group were significantly higher than those in the OVA group and R8 group (all P < 0.01), but the level of IgG2a of the former was significantly lower than those of the latter groups (all P < 0.01). CONCLUSIONS: TAT-IhC-R8 can effectively stimulate lung inflammations of mice, and its sensitization effect is better than R8's.

[Prediction and identification of T-cell epitopes in major group 3 allergen derived from Dermatophagoides farina].

OBJECTIVE: To predict and identify T cell epitopes of major group 3 allergen derived from Dermatophagoidesfarina (Der f 3). METHODS: The T cell epitopes of Der f 3 were analyzed through the sequence analysis by using the bioinformatics online tools. The five predicted peptides of T-cell epitopes were artificially synthesized. The spleen lymphocytes were co-cultured with the five T cell epitopes by using the modified MTT method and the levels of IL-2, IFN-γ, IL-4 and IL-5 in the supernatant of the cultures were detected by ELISA. RESULTS: Five T cell epitopes of Der f 3 were predicted and three of which could promote the proliferation of the mouse spleen lymphocytes. The secretions of IL-2 and IFN-γ were significantly induced and the secretions of IL-4 and IL-5 were significantly decreased by three of five prediction epitopes of Der f 3: 37GDCPYQISLQSSSHFCGG54, 98IYQHENYDSMTIDNDVALIKLKTPMT123 and 164SELQRVDIDVVSREQCDQLYS184. CONCLUSION: Three T cell epitopes of Der f 3 have been initially identified, which lays the foundation of the diagnosis and treatment of asthma.

[Effect of immunotherapy with recombinant allergen group 3 from dermatophagoides farinae in asthma mice].

OBJECTIVE: To explore the effect of specific immunotherapy with major 3 group recombinant allergen rDer f 3 of Dermatophagoides farinae in murine asthma model. METHODS: Forty BALB/c mice were randomly divided into 4 groups, namely PBS group (negative control), ovalbumin(0VA) group (positive control), rDerf3 allergen sensitization group (asthma group), and rDerf3 specific immunotherapy group(SIT group). The mice in asthma group and SIT group were injected intraperitoneally with purified rDer f 3 protein on days 0, 7 and 14, respectively, and rDer f 3 solution was inhalated from day 21 for 7 days. During the 25th-27th day, mice in SIT group were injected subcutaneously with 100 jg rDer f 3 allergen for 30 min before nasal inhalation. Mice in groups of PBS and OVA were treated with PBS and OVA, respectively. Twenty-four hours after the final challenge, all mice were sacrificed, the bronchoalveolar lavage fluid (BALF) was collected and the total number of white blood cells and the number of eosinophils were recorded. The levels of IL-5 and IFN-gamma in BALF and supernatant of cultured splenocytes were detected by ELISA, as well as the serum levels of specific IgE and IgG2, antibodies. Lung tissues were stained with haematoxylin and eosin for histological analysis. RESULTS: Compared with the asthma group, the rDer f 3-induced lung inflammation was significantly alleviated in SIT group. The total number of white blood cells [(7.03 +/- 1.38) x 10(8)/ml] in SIT group was considerably lower than that of OVA group [(22.11 +/- 3.70) x 10(8)/ml] and asthma group [(22.75 +/- 3.24) x 10(8)/ml] (P<0.01). The change trend of eosinophil leukocytes was similar with that of white blood cells. IL-5 levels in BALF [(108.20 +/- 11.02) pg/ml] and splenocyte culture supernatant [(98.34 +/- 13.06) pg/ml] in SIT group were significantly lower than that of OVA group [(182.04 +/- 13.94) pg/ml, (208.26 +/- 10.63) pg/ml] and asthma group [(195.33 +/- 15.33) pg/mL, (179.54 +/- 13.65) pg/ml] (P<0.01). Whereas, the level of IFN-gamma in BALF [(107.98 +/- 12.64) pg/ml] and supernatant of cultured splenocytes [(105.51 +/- 1.62) pg/ml] in SIT group was significantly higher than those of OVA group and asthma group (P<0.01). Compared with OVA group [(26.87 +/- 4.30) IU/ml] and asthma group [(35.25 +/- 8.84) IU/ml], a lower level of allergen-specific IgE [(9.12 +/- 3.78) IU/ml] and higher level of allergen-specific IgG2, [(38.52 +/- 6.33) microg/ml] were observed in SIT group (P<0.01). CONCLUSION: rDer f 3 allergen can reverse allergen-induced airway and lung inflammation in murine asthma model.