The development and validation of methods for evaluating the immune system in preweaning piglets

The preweaning piglet has been found to be a valuable research model for testing ingredients used in infant formula. As part of the safety assessment, the neonates' immune system is an important component that has to be evaluated. In this study three concurrent strategies were developed to assess immune system status. The methods included (1) immunophenotying to assess circulating innate immune cell populations, (2) monitoring of circulating cytokines, particularly in response to a positive control agent, and (3) monitoring of localized gastrointestinal tissue cytokines using immunohistochemistry (IHC), particularly in response to a positive control agent. All assays were validated using white papers and regulatory guidance within a GLP environment. To validate the assays precision, accuracy and sample stability were evaluated as needed using a fit for purpose approach. In addition animals were treated with proinflammtory substances to detect a positive versus negative signal. In conclusion, these three methods were confirmed to be robust assays to evaluate the immune system and GIT-specific immune responses of preweaning piglets.     

Low-density lipoprotein apheresis in children with familial hypercholesterolemia: follow-up to 21 years

Twenty-seven patients (14 girls, 13 boys) affected by familial hypercholesterolemia who had begun low-density lipoprotein (LDL) apheresis treatment before the age of 15 were studied. The median age at diagnosis was 4 years and the blood LDL cholesterol level was 704 +/- 163 mg/dL. Screening was performed for homozygous or double heterozygous mutations of the LDL cholesterol receptor gene and mutations were found in 24 of the patients. The mean age at the beginning of treatment was 8.5 years and the mean length of follow up was 12.6 years. The two main procedures used were direct adsorption of lipoproteins and dextran sulfate cellulose adsorption. Nine patients experienced anaphylactic reactions due to bradykinin and six had to have their treatment changed. The LDL cholesterol level before the session was lowered by 45 +/- 11% of the value at diagnosis. The LDL cholesterol reduction in a session was 72 +/- 10%. Tendinous xanthomas disappeared or diminished dramatically in 62% of the children. In 22 patients no cardiovascular event occurred during LDL apheresis treatment. Three had angina pectoris; two others had surgical management of aortic stenosis, but no clinical manifestations. Seven children had normal cardiovascular pictures while on treatment. Eleven had abnormalities of the aortic root or coronary arteries, which in six cases had appeared before treatment; the other five children did not undergo prior cardiac evaluation. In five children the abnormalities appeared during treatment. Based on these data, LDL-apheresis can be recommended for the treatment of homozygous familial hypercholesterolemia, even in young children, with good efficiency on biological parameters, cutaneous lesions and cardiovascular events.     

Monitoring recovery from diaphragm paralysis with ultrasound

BACKGROUND: Diaphragmatic paralysis is an uncommon, yet underdiagnosed cause of dyspnea. Data regarding the time course and potential for recovery has come from a few small case series. The methods that have been traditionally employed to diagnose diaphragmatic weakness or paralysis are either invasive or limited in sensitivity and specificity. A new technique utilizing two-dimensional, B-mode ultrasound (US) measurements of diaphragm muscle thickening during inspiration (Deltatdi%) has been validated in the diagnosis of diaphragm paralysis (DP). The purpose of this study was to assess whether serial US evaluation might be utilized to monitor the potential recovery of diaphragm function. METHODS: Twenty-one consecutive patients with clinically suspected DP were referred to the pulmonary physiology laboratory. Sixteen patients were found to have DP by US (unilateral, 10 patients; bilateral, 6 patients). Subjects were followed up for up to 60 months. On initial and subsequent visits, Deltatdi% was measured by US. Additional measurements included upright and supine vital capacity (VC), maximal inspiratory pressure (Pimax), and maximal expiratory pressure. RESULTS: Eleven of 16 patients functionally recovered from DP. The mean (+/- SD) recovery time was 14.9 +/- 6.1 months. No diaphragm thickening was noted in those patients who did not recover. Positive correlations were found between improvement in Deltatdi% and interval changes in VC, Pimax, and end-expiratory measurements of diaphragm thickness. CONCLUSIONS: US may be used to assess for potential functional recovery from diaphragm weakness or DP. As in previous series, recovery occurs in a substantial number of individuals, but recovery time may be prolonged.     

Hydroxyethyl starch is an alternative washing solution for peripheral bloodstem cells products

Cryopreservation of hematopoietic stem cells (HSC) involves slow rate cooling in the presence of a cryoprotectant (DMSO) to avoid the damaging effects of intracellular ice formation. The infusion of DMSO with the thawed product has been related to adverse events. Reduction of DMSO content by washing the HSCs after thawing has been suggested as a method to avoid infusion-related side-effects. Albumin-dextran washing methods have proved useful in thawing HSC products. Dextran40 shortages prompted us to search for suitable alternatives. We report the results of a comparative study of the use of hydroxyethyl starch (HES) as an alternative to dextran40 for washing thawed HSCs products. A total of 10 HSC bags cryopreserved with 10 % DMSO were used. We conducted a paired study; one of the bags was thawed and washed with our standard washing solution (Dextran 40) and the paired bag with HES solution with a final HES and Human Serum Albumin (HSA) concentration of 2.4 % and 4.2 % respectively. Each final product was tested immediately after washing (sample 0’) and after 90 min (sample 90’) for total nucleated cells (TNC) recovery, acridine orange viability, viable CD34+ enumeration, and clonogenicity. No significant difference was found for any of the cell counts, viability tests, cell recovery, or potency. We can state that the washing solution based on 2.4 % HES and 4.2 % HSA is equivalent to that used in our routine practice. Therefore, we could use the solution with HES, paying special attention to the renal function of the recipient.     

不同浓度葡聚糖硫酸钠对小鼠炎症性肠病模型建立及其致病相关免疫因子表达的影响

目的 探讨饮用不同浓度的葡聚糖硫酸钠(dextran sulfate sodium, DSS)对于建立小鼠炎症性肠病(inflammatory bowel disease, IBD)模型及其致病相关免疫因子表达的影响。方法 雄性C57BL/6J小鼠随机分为对照组和不同浓度的DSS饮用组(3%、5%、7%)。观察小鼠的大便性状,体重和生存时间。饮用后的第6天处死各组小鼠,观察结肠大体形态并评分;取病变处进行石蜡包埋病理切片,苏木素伊红染色并进行病理组织学评分;定量PCR检测各组脾细胞免疫因子表达情况。结果 饮用3%、5%、7%浓度DSS的小鼠在第6天均有不同程度溃疡形成,成模率随着DSS浓度提升而增加,但是小鼠死亡率也相应增加。定量PCR结果表明促炎因子(TNF-α、IFN-γ和IL-17A)的表达水平与DSS浓度成正相关,而抑炎因子(IL-4和IL-10)以及调节性T细胞相关的转录因子Foxp3的表达水平与DSS浓度成负相关关系。结论 给予小鼠5%浓度的DSS溶液饮用有助于高效经济地建立小鼠IBD模型,为进一步研究IBD的发病机理、生物学特性、干预因素等打下了重要基础。     

Application of PK/PD Modeling and Simulation to Dosing Regimen Optimization of High-Dose Human Regular U-500 Insulin

Background:Pharmacokinetic/pharmacodynamic (PK/PD) studies of human regular U-500 insulin (U-500R) at high doses commonly used in clinical practice (>100 units) have not been performed. The current analysis applied PK/PD modeling/simulation to fit the data and simulate single-dose and steady-state PK/PD of U-500R high-dose regimens.Method:Data from 3 single-dose euglycemic clamp studies in healthy obese and normal-weight patients, and normal-weight patients with type 1 diabetes were used to build the model. The model was sequential (PK inputs fed into PD component). PK was described using a 1-compartment model with first-order absorption and elimination. The model estimated separate absorption rate constants for U-500R and human regular U-100 insulin. The PD component used an effect compartment model, parameterized in terms of maximum pharmacologic effect (E_max) and concentration to achieve 50% of E_max.Results:The model described the data well. Steady-state PK for once-daily (QD), twice-daily (BID), or thrice-daily (TID) administration appeared to be reached 24 hours after the first dose. At steady-state, QD dosing showed the greatest fluctuations in PK/PD. BID dosing showed a gradual increase in insulin action with each dose and a fairly stable basal insulin effect. For TID dosing, activity was maintained throughout the dosing interval.Conclusions:PK/PD modeling/simulation of high U-500R doses supports BID or TID administration with an extended duration of activity relative to QD. TID dosing may provide slightly better full-day insulin effect. Additional PK/PD studies and randomized controlled trials of U-500R are needed to validate model predictions in patients with insulin-resistant diabetes requiring high-dose insulin.     

葡聚糖硫酸钠诱导小鼠实验性结肠炎的临床和组织病理学特征研究

目的研究葡聚糖硫酸钠(DSS)诱导小鼠实验性结肠炎的临床和病理特征,以便于指导选择合适的溃疡性结肠炎(ulcerative colitis,UC)小鼠模型。方法予3%DSS溶液喂饲野生型C57BL/6成年小鼠诱导急性结肠炎模型,分别于第0天、第3天、第5天、第7天和第10天麻醉处死小鼠,取结肠观察不同时期结肠病理学特征,造模过程中连续观察并记录小鼠体质量、大便和死亡情况。结果小鼠造模第3天开始出现粪便潜血,第5天开始出现血便,第10天开始出现死亡小鼠,死亡率为30%。DSS诱导小鼠急性结肠炎模型疾病活动指数第3天后与饮用纯净水的小鼠比较明显增高(P0.05)。小鼠结肠炎造模第3天黏膜上皮细胞开始逐渐丧失,第7~10天最为严重;隐窝结构的紊乱从第3天开始发生,第7天出现固有层塌陷;炎性细胞浸润从第3天开始数量逐渐增加,第10天最为严重。DSS诱导小鼠急性结肠炎模型结肠组织病理评分第5天后与饮用纯净水的小鼠比较明显增高(P0.05)。结论 3%DSS诱导野生型C57BL/6成年小鼠急性结肠炎模型可用于UC的实验研究,第3天出现明显的炎症表现,第7天模型较理想,小鼠死亡少。     

葡聚糖包裹的超顺磁性Fe3O4纳米颗粒用于MRI造影剂的研究

目的探讨葡聚糖包裹的超顺磁性Fe3O4纳米颗粒（dextran-coated superparamagnetic iron oxides nanoparticles,dextran／SPIONP）作为间质注射的MRI造影剂的可行性。方法应用界面共沉淀法,制备dex—tran／SPIONP,用透射电镜、能谱分析、X射线衍射、红外光谱测试、热重分析等方法对dextran／SPIONP的大小、磁性能等进行表征,并研究葡聚糖对SHONP的影响。然后,将dextran／SPIONP稀释成不同剂量,兔舌粘膜下间质注射后的不同时间段,切取前哨淋巴结,行核磁共振波谱分析,确定dextrart／SPIONP的最适剂量和最佳检测时间。结果dextran／SPIONP的平均直径为6～9nm,其表征与经典的共沉淀法制备的颗粒一致,具有超顺磁性,葡聚糖是影响SPIONP饱和磁化强度的主要因素。dextran／SPIONP用于间质注射的最适剂量为含铁量20μmol,最佳检测时间为注射后2Ah。结论用界面共沉淀法制备的dextran／SPIONP是一种适用于间质注射的MRI造影剂。     

Preparation of immunomagnetic iron-dextran nanopartides and application in rapid isolation of E.coli O157:H7 from foods

AIM: To prepare a kind of magnetic iron-dextran nanopartides that was coated with anti-E.coli O157:H7 IgG, analyze its application conditions, and try to use it to isolate E.coli O157:H7 from foods. METHODS: Magnetic iron-dextran nanopartides were prepared by the reaction of a mixture of ferric and ferrous ions with dextran polymers under alkaline conditions. The particles were coated with antiserum against E.coli O157: H7 by the periodate oxidation-borohydride reduction procedure. The oxidation time, amount of antibody coating the particles, amount of nanoparticles, incubation time and isolation time were varied to determine their effects on recovery of the organisms. Finally, the optimum conditions for isolating E.coli O157:H7 from food samples were established. RESULTS: E.coli O157:H7 can be isolated from samples within 15 min with the sensitivity of 101 CFU/mL or even less. In the presence of 108 CFU/mL of other organisms, the sensitivity is 101-102 CFU/mL. Nonspecific binding of other bacteria to the particles was not observed. Two and a half hours of enrichment is enough for the particles to detect the target from the food samples inoculated with 1 CFU/g. CONCLUSION: Isolation of target bacteria by immuno magnetic nanoparticles is an efficient method with high sensitivity and specificity. The technique is so simple that it can be operated in lab and field even by untrained personnel.