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Objective: To establish an accurate and selective UPLC-MS/MS) method for the determination of afatinib in rat plas-ma. Methods: Protein precipitating by acetonitrile was used to prepare the samples. A CORTECS BEH C18column ( 50 mm × 2. 1 mm, 1. 6 μm) was used to separate the analytes at 40℃. The mobile phase consisted of acetonitrile and water (0. 1% formic acid) with the flow rate of 0. 4 ml·min-1. The analytes were quantified by multiple reaction monitoring ( MRM) mode with positive electrospray ionization, while the target fragment ions were m/z 486. 19→112. 1 for afatinib and m/z 557. 3→112. 15 for neratinib (IS). Results: The calibration curve obtained good linearity for afatinib within the range of 1–200 ng·ml-1(r=0. 998 1), and the LLOQ in rat plasma was 1. 0 ng/ml. The intra-and inter-day precisions were both≤9. 51% . The recovery of afatinib from plasma was above 77. 1% . After intragastric administration and intravenous administration of afatinib in rats, the t1/2was 7. 19 h and 2. 69 h, Cmax was 97. 78 ng·ml-1and 123. 37 ng·ml-1,and AUC(0-∞)was 1 505. 4 ng·ml-1·h and 405. 55 ng·ml-1·h, respectively. Con-clusion: The validated method can be applied in the pharmacokinetic study of afatinib at the intragastric and intravenous dosage of 10 and 2 mg·kg-1, respectively.
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<p><b>OBJECTIVE</b>To analyze clinical manifestations and genetic mutation in a child with severe short stature and other malformations.</p><p><b>METHODS</b>The child has undergone history taking and physical examination. Genome DNA was extracted from peripheral blood samples of the proband and her family members. Candidate genes were captured with Agilent SureSelect and sequenced on an Illumina platform. Suspected mutation was verified by Sanger sequencing.</p><p><b>RESULTS</b>The patient, a six-year-and-10-month old girl, presented with non-symmetrical short stature, dysmorphism, abnormalities of limbs and spine, amblyopia of left eye, and cataract of right eye, in addition with frequent respiratory infection and micturition. Laboratory testing suggested 25-hydroxy vitamin D deficiency (18.9 ng/mL). Spine X-ray showed multiple malformations with centrums. Her mother also featured short stature (138 cm). Her aunt had short stature (130 cm) and limb-length discrepancy. Her little brother was 2.5 years old, and his height was 81 cm (-3.4 SD). Exome sequencing revealed a heterozygous mutation c.184C to T (p.Arg62Trp) in the proband and her mother. The same mutation was not found in her father and brother.</p><p><b>CONCLUSION</b>The patient was diagnosed with X-linked chondrodysplasia punctata 2. Mutation of the EBP gene probably underlied the disease in this family.</p>
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Objective To study the clinical and pathological features of histiocytic necmtizing lym-phadenitis( HNL) in children. Methods The clinical data and histological findings of 38 cases of HNL admitted in our hospital from June 2000 to May 2015 were reviewed. Results Most of the patients were school-age children with male-femal ratio of 1. 4: 1. The main clinical features were lymphadenopathy (100%),fever(68. 24%),leucocytopenia(52. 63%),rising of lymphocytes percentage(84. 21%). All of the lymph node excisional biopsy met the criterion of HNL. Some cases spontaneously relieved and some cases were treated with NSAID,glucocorticoid or immunoglobulin and benefited significantly. There was no recurrence. Conclusion The clinical situation is not specific. The diagnosis is established by lymph node ex-cisional biopsy. HNL is benign and self-limited disease. The effect of management using glucocorticoid, NSAID and immunoglobulin is remarkable. Long term follow-up is necessary.
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Objective To study the clinical and pathological features of histiocytic necmtizing lym-phadenitis( HNL) in children. Methods The clinical data and histological findings of 38 cases of HNL admitted in our hospital from June 2000 to May 2015 were reviewed. Results Most of the patients were school-age children with male-femal ratio of 1. 4: 1. The main clinical features were lymphadenopathy (100%),fever(68. 24%),leucocytopenia(52. 63%),rising of lymphocytes percentage(84. 21%). All of the lymph node excisional biopsy met the criterion of HNL. Some cases spontaneously relieved and some cases were treated with NSAID,glucocorticoid or immunoglobulin and benefited significantly. There was no recurrence. Conclusion The clinical situation is not specific. The diagnosis is established by lymph node ex-cisional biopsy. HNL is benign and self-limited disease. The effect of management using glucocorticoid, NSAID and immunoglobulin is remarkable. Long term follow-up is necessary.
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Objective@#To analyze and summarize the clinical and molecular characteristics of the patients with multiple congenital anomalies- hypotonia-seizures syndrome 1 (MCAHS 1).@*Method@#Clinical data and test results were collected from a patient who was diagnosed with confirmed genetic basis of MCAHS 1 in Shanghai Children′s Medical Center since December 2015. The patient and his parents were examined by the next generation sequencing (NGS) technology using peripheral blood genomic DNA, and the relevant mutations identified by NGS were verified with Sanger sequencing. Related literature was searched from PubMed and Embase databases (from their establishment to January 2017) by using "PIGN gene" as a keyword, the retrieved articles were further reviewed for the clinical manifestations, results and prognosis of PIGN related variants.@*Result@#A nearly 4-month-old Chinese boy was presented with epilepsy, hypotonia, developmental delay, accompanied by nearly normal laboratory test results. The NGS analysis revealed a compound heterozygous variations in the PIGN gene, included a known splice site mutation (c.963G>A) which was inherited from his father, and a novel nonsense mutation (c.2773A>T, p.Lys925*) which was inherited from his mother. Nine associated articles were retrieved. Including our patient, a total of 22 cases were identified as the PIGN variants. The most common clinical manifestations were developmental delay, hypotonia, and epilepsy.Missense varients were most frequently found. Prognosis was poor. Eight cases died, while survived cased suffered from refractory epilepsy, profound mental retardation, muscle weakness, etc.@*Conclusion@#MCAHS1 is characterized by epilepsy, severe developmental delay, hypotonia, and may be accompanied by multiple malformations of other systems. Homozygous or compound heterozygous variants in PIGN gene are the cause of the disease.
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Objective To investigate the characteristics and essential points of diagnosis and treatment of double trisomy 47,XXX/48,XXX,+8 combined Behcet disease, a rare inherited immunodeficiency disorder. Methods The clinical manifestations, karyotype analysis and gene test results of the patients were analyzed, and relevant literatures were reviewed. Results A 11-year-old girl presented repeated fever for more than 6 years, accompanied with recurrent genital herpes infection and oral apthosis, was clinically diagnosed with Behcet disease. Cytogentic and molecular karyotyping on peripheral lymphocytes demonstrated 47,XXX[12]/48,XXX,+8[18]. Conclusions Conventional karyotype analysis and chromosomal microarray analysis have a complementary role in the diagnosis of the disease. We conclude that patients with constitutional trisomy 8 and those with trisomy 8 confined to the bone marrow are both at increased risk of developing features of Behcet disease. The mechanism may relate to increased gene dosage of candidate genes for Behcet's disease on chromosome 8.
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Objective To investigate the effects of low dose of extracellular histone on endothelial cells in infectious diseases such as sepsis. Methods The endothelial cells were treated with 10 μg/mL recombinant human histone H3/H4 complex in replacement of calf thymus histones (CTH) for various periods of time, and the morphology changes and the viability of the endothelial cells were recorded. In addition, flow cytometry was applied to identify the characteristics of endothelial cells and enzyme-linked immunosorbnent assay (ELISA) was used to detect the extracellular histones level in endothelial cells culture. Results The low dose of CTH could continuously induce endothelial cells death, cell morphological changes and function loss, which was reproduced by 10 μg/mL recombinant histone H3/H4 complex. Results of histones quantitation showed that histone can cause a series of intracellular reactions in a short period of time. Conclusions It is showed that 10 μg/mL H3/H4 can induce the toxicity in infectious disease and this level of the dose is a lower than those used in previous studies and more close to the pathological conditions.
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OBJECTIVE:To establish the method for pharmacokinetic study of luteolin and cynaroside in rats and to determine pharmacokinetic parameters. METHODS:16 SD rats were randomly divided into luteolin group (sublingual iv,1.34 mg/kg) and cynaroside group(sublingual iv,0.64 mg/kg). 0.5 ml blood were collected before administration and 0,15,30 min and 1,2,3,4,6, 8,12,24,48 h after administration respectively to prepare plasma. UPLC-TQ-MS was adopted to determine plasma concentration, and pharmacokinetic parameters were calculated. A CORTECSTM UPLC? C18(100 mm×2.1 mm,1.6 μm)column was used with mobile phase consisted of acetonitrile-water (containing 0.1% formic acid) at a flow rate of 0.4 ml/min,the column temperature was set at 40 ℃,and quercetin was used as internal standard. RESULTS:The linear range of luteolin and cynaroside were 2.5-500 ng/ml (r=0.998 2) and 10-2 500 ng/ml (r=0.993 5). The lowest quantitation limits were 1 and 2.5 ng/ml,and extraction were 70.75%-87.72% and 75.40%-91.18%(n=6);RSD of inter-day and intra-day were all lower than 10%(n=3). Pharmacokinetic parameters as t1/2 were (1.88 ± 0.32) and (1.57 ± 0.08) h;CL were (0.77 ± 0.18) and (0.06 ± 0.01) L/(h·kg);AUC0-6 h were (189.60±40.04)and(1 093.14±187.36)ng·h/ml;AUC0-∞ were(195.18±38.37)and(1 097.11±188.07)ng·h/ml. CONCLU-SIONS:The method can be used for pharmacokinetic study of luteolin and cynaroside in rats,and the pharmacokinetics of them in rats are in line with two-compartment model.
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Infective endocarditis(IE) is a kind of endocardial infection which cardiac intima is affected by some pathogens.Intravascular catheter,implantable artificial device and surgery are the common reasons for IE,congenital heart diseases and cardiac surgery are the main causes for IE in children.The incidence of infective endocarditis is high and case fatality rate is as high as 20％.The dominant pathogen of IE is gram-positive bacteria.Modified Duke standard is the gold standard in the diagnosis of IE,some other imaging evidence and new laboratory tests and biomarkers for diagnosis of IE have improved the sensitivity of the diagnostic criteria.Good oral hygiene and regular dental check are the most effective prevention measures.The idea ofendocarditis management team in the newest guidelines on IE requires muhidisciplinary experts to participate in diagnosis and treatment of IE.Individualized treatment is the best way to improve the patients survival rate.
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Objective:To establish an ultra performance liquid chromatography-tandem quadrupole mass spectrometry method for the determination of warfarin and its metabolite 7-hydroxywarfarin in human plasma. Methods: An ACQUITY UPLC? BEH C18 (50 mm × 2. 1 mm, 1. 7 μm) column was used as the stationary phase at 40℃. The mobile phase consisted of acetonitrile and water (con-taining 0. 1% formic acid) with gradient elution at a flow rate of 0. 4 ml·min-1 . Warfarin-d5 was used as the internal standard. The analytes were detected on a triple-quadrupole mass spectrometer equipped with an ESI interface in a positive mode. Results:The reten-tion time of warfarin and 7-hydroxywarfarin was 1. 8 min and 1. 5 min, respectively. Excellent linear calibration curve of warfarin and 7-hydroxywarfarin was obtained within the concentration range of 25-2 000 ng · ml-1 ( r =0. 999 3 ) and 5-500 ng · ml-1 ( r =0. 999 6), respectively. The lower limit of quantification of warfarin and 7-hydroxywarfarin was 5 ng·ml-1 and 2. 5 ng·ml-1 with the average recovery of 96. 9%-105. 3% and 97. 1% -103. 3%, respectively. The intra-and inter-day standard deviations were both less than 10%. Conclusion: The method is accurate and simple, and suitable for the determination of warfarin and its metabolite 7-hydroxywarfarin in human plasma.
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Objective To study the clinical characteristics, treatment and prognosis of infective endocarditis in children. Methords Clinical data from 83 patients of infective endocarditis admitted from 1998 to 2012 were retrospectively analyzed. Results In a total of 83 patients, there were 53 males and 30 females, and the average age was 6.8±4.6 years. The main clinical characteristics were fever (77.1%) and mild to moderate anemia (71.1%). The C-reaction protein (67.5%), erythrocyte sedimen-tation rate (60.2%), and white blood cell (47.0%) were elevated. Twenty (24.1%) patients had embolism. Blood culture was pos-itive in 56 (67.5%) cases with bacteria mainly being Gram-positive and Streptococcus and Staphylococcus accounted for 89.3%. Vancomycin and other sensitive antibiotics were effective. Neoplasm was detected in 68 cases (82%) by transthoracic echocar-diograerphy. Fifty-ifve (66.2%) patients underwent cardio surgery. Seven patients (8.4%) died. Conclusion In recent years, the distribution of pathogenic bacteria in infective endocarditis had changed. Streptococcus mitis and Staphylococcus aureus has become a major pathogens and need to be treated by vancomycin and other sensitive antibiotics. The detection rate of neoplasm is higher by echocardiography.
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With the inappropriate use of antibiotics, the situation of bacterial resistance is more and more severe. The emergence of multidrug-resistant (MDR) bacteria has made it difficult to cure the infections in clinical. For treatment the infections caused by MDR and reducucton of the generation of resistant bacteria, researchers are actively studying on the non-antibiotic substances for antibacterial activity. In this paper, the advances in those with conifrmed effects such as phage therapy, metal/chelation therapy, immunization therapy, photodynamic therapy, and nitric oxide (NO)-based therapies, small molecule inhibitors, antimicrobial peptides and Chinese herb were reviewed.
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Objective:To establish an ultra performance liquid chromatography-tandem quadruple mass spectrometry ( UPLC-MS/MS) method to determine CYP2C9 activity in vitro. Methods:An ACQUITY UPLC? BEH C18 (100 mm × 2. 1 mm, 1. 7 μm) column was used as the stationary phase at 30℃. The mobile phase consisted of acetonitrile-water ( containing 0. 1% formic acid and 0. 5%ammonia water) (40∶60, v/v). The flow rate was 0. 2 ml·min-1. Chlorpropamide was used as the internal standard. The MS condi-tions were as follows:ESI with positive ion detection mode. Self-prepared CYP2C9?1, ?2, ?3 and ?13 protein were incubated with tolbutamide at 37℃ and 800μl ethyl acetate was added to stop the reaction. After centrifuged at 10 000g, the organic layer was then dried using nitrogen, the residue was re-dissolved in 200μl mobile phase and determined by UPLC-MS/MS. Results: The reten-tion time of 4-hydroxytolbutamide was 1. 21 min. An excellent linear calibration curve of 4-hydroxytolbutamide was obtained within the concentration range of 0. 05-5 ng·μl-1(r=0. 999 8). The lower limit of quantification of 4-hydroxytolbutamide was 0. 01 ng·μl-1 with the average recovery of 99. 3%-100. 3%. The intra- and inter-day RSDs were all less than 5%. There was no interference from the endogenous substances existing in the incubation system. The catalytic activity of the variants CYP2C9?2,?3 and?13 after tol-butamide was incubated with CYP2C9?1,?2,?3 and?13 was 47. 3%, 11% and 0. 3% of wild type CYP2C9?1. Conclusion:The method is simple and stable, and suitable for the fast evaluation of cytochrome CYP2C9 activity in vitro and relevant studies on the inhibitors.
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Objective To investigate the pathogen of 21 infective endocarditis (IE) cases treated with operation in Shanghai Children's Medical Center from 2007 to 2010.Methods Blood culture,vegetation culture and vegetation PCR assay(target gene to the conserved region V3 in 16SrRNA gene) were detected in 21 IE patients; multiplex PCR amplification of staphylococci for methicillin-resistant staphylococcus was performed.Results Of 21 IE cases,20 cases were detected positive by vegetation PCR with the detection rate of 95.2％,12 IE cases were detected positive by blood culture with the detection rate of 57.1％,2 IE cases were detected positive by vegetation culture with the detection rate of 9.5％.The difference of the positive rates of the three methods was statistically significant (P ＜ 0.0001).The vegetation PCR of one case was actinobacillus actinomycetemcomitans,while the blood culture was haemolysis pasteurell which was inconsistent with the vegetation PCR result.Howerver,the PCR result of colony obtained by blood culture was consistent with vegetation PCR that was confirmed as actinobacillus actinomycetemcomitans.The endocardium PCR results of 11 IE cases were consistent with the results of blood culture.MecA gene was detected by multiplex PCR,which could identify methicillin-resistant staphylococcus quickly,sensitively and accurately and could also effectively identify methicillin resistant staphylococcus aureus,when coupled with femA gene detection,thus glycopeptides antibiotic could be prescribed promptly.All the 21 patients recovered and discharged without infection recurrence in the follow-up.Conclusion Universal primer V3 coupled with multiplex PCR can improve vegetation pathogen detection rate of IE patients and is minimally influenced by antibiotic therapy.Multiplex PCR can be applied for etiological diagnosis of IE patients with indication of surgery and negative blood culture or difficult diagnosis,contributing to post-surgery antibiotics selection and improvement of recovery rate of IE patients.
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Objective To assess the diagnostic potential of circulating galactomannan (GM),(1,3)-β-D-glucan (BG), and a combination of both biomarkers among high-risk pediatric patients.Methods Circulating GM antigen was detected by ELISA kits (Platelia~(TM) Aspergillus) and BG antigen by a turbidimetric kinetic method (GKT-5M Set Kinetic Fungus Detection Kit).Positive tests were defined by two consecutive values of GM index ≥0.5 or by a single value ≥0.8, and by BG detection ≥ 10 pg/ml.Results A total of 130 patients were enrolled.Two was identified with proven IFI, twenty probable IFI.Sensitivity, specificity were 81.8%, 82.4% for plasma BG detection, respectively; 75.0%, 94.4% for GM detection, respectively; 50.0%, 96.3% for combined GM and BG detection, respectively.Conclusions Both circulating GM and BG detections are available for most of the common pathogens and demonstrated desirable sensitivity and specificity among pediatric high-risk population.Both assays can be used as prospective screening tools.Combination of detections of both biomarkers would improve specificity for IA diagnosis.
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Objective To investigate pediatric infective endocarditis(IE) risk factors and the feature of pathogen.Methods We conducted a retrospective study of 46 cases who were diagnosed as IE from January 2000 to June 2007.Sixteen variables such as sex,primary disease,infectious site,complication,culture result and therapeutic measure were selected for logistic regression analysis.Results Forty-six IE patients accounted for 0.78‰ of all the hospitalized patients during the same period.Of 46 cases,40 had heart diseases.The incidence rate of left-sided endocarditis was higher than that of right-sided endocarditis.Twenty-four had positive blood culture result,one patient had positive culture from vegetation sample.The total positive culture rate was 54%.Gram-positive bacteria were found in 15 cases,and the most common organisms were α-hemolytic streptococcus.Gram-negative bacteria were in 7 cases and fungus infection was present in 3 cases.After antiboitic treatment,40 were cured,6 died.In the logistic regression model,the variables significantly associated with death included negative blood culture (OR=25.127,95%CI=1.110-363.236),hematological system disease (OR=28.620,95%CI=2.261-354.448) and multiple organ failure (OR=19.843,95%CI=0.786-221.754).Conclusion Streptococcus viridans is the most common pathogen in pediatric IE in our hospital.Higher incidence in the left heart than that in the right heart is found.Negative blood culture,hematological system disease and MOF are the risk factors in IE patients.