Immunity in Mammals to Helminths of the Circulatory and Lymphatic Systems, Emphasizing Nonhuman Primate Models and Their Comparison to Human Infections and Rodent Models

The host—parasite relationship of intravascular helminthsin mammalian hosts is complex, consisting of several interrelatedaspects. These organisms are often very long lived in thesehosts that possess highly evolved immune systems, which impliesthe existence of mechanisms in these complex parasites for evadingthe immune response. On the other hand, mammalian hosts appearto be very successful in limiting their worm burdens, mainlyby preventing reinfection. Thus, a balance between host andparasite is often achieved. This balance extends to the immunopathologicalconsequences of the infections as well. Especially in the schistosomes,where relatively more is known about immunopathology, it isbecoming increasingly apparent that immunopathological reactionsto the eggs are multifunctional and paradoxical: they causedisease, they limit disease, and they even serve the schistosomeby making possible the escape of its eggs, laid deep withinthe tissues, from the host's body to propagate the life cycle.The comparative approach, using different species of schistosomesand filarids in different mammalian hosts, has proved to bevaluable for understanding the immunological basis of thesecomplex relationships. The knowledge gained from these modelsystems is already being vigorously applied to the human diseases,two of the great scourges of the tropics, schistosomiasis andlymphatic filariasis, to the end of their eradication from theearth by mass immunization.     

Establishment and Validation of SSCLIP Scoring System to Estimate Survival in Hepatocellular Carcinoma Patients Who Received Curative Liver Resection

Background and Aims

There is no prognostic model that is reliable and practical for patients who have received curative liver resection (CLR) for hepatocellular carcinoma (HCC). This study aimed to establish and validate a Surgery-Specific Cancer of the Liver Italian Program (SSCLIP) scoring system for those patients.

Methods

668 eligible patients who underwent CLR for HCC from five separate tertiary hospitals were selected. The SSCLIP was constructed from a training cohort by adding independent predictors that were identified by Cox proportional hazards regression analyses to the original Cancer of the Liver Italian Program (CLIP). The prognostic performance of the SSCLIP at 12 and 36-months was compared with data from existing models. The patient survival distributions at different risk levels of the SSCLIP were also assessed.

Results

Four independent predictors were added to construct the SSCLIP, including age (HR = 1.075, 95%CI: 1.019–1.135, P = 0.009), albumin (HR = 0.804, 95%CI: 0.681–0.950, P = 0.011), prothrombin time activity (HR = 0.856, 95%CI: 0.751–0.975, P = 0.020) and microvascular invasion (HR = 19.852, 95%CI: 2.203–178.917, P = 0.008). In both training and validation cohorts, 12-month and 36-month prognostic performance of the SSCLIP were significantly better than those of the original CLIP, model of end-stage liver disease-based CLIP, Okuda and Child-Turcotte-Pugh score (all P < 0.05). The stratification of risk levels of the SSCLIP showed an enhanced ability to differentiate patients with different outcomes.

Conclusions

A novel SSCLIP to predict survival of HCC patients who received CLR based on objective parameters may provide a refined, useful prognosis algorithm.     

NIKEI: A New Inexpensive and Non-Invasive Scoring System to Exclude Advanced Fibrosis in Patients with NAFLD

Aims

To develop, validate and compare a non-invasive fibrosis scoring system for non-alcoholic fatty liver disease (NAFLD) derived from routinely obtained clinical and biochemical parameters.

Methods

267 consecutive patients with biopsy proven fatty liver or non-alcoholic steatohepatitis were randomly assigned to the estimation (2/3) or validation (1/3) group to develop a model for the prediction of advanced fibrosis. Univariate statistics were performed to compare patients with and without advanced fibrosis, and following a multivariate logistic regression analysis a new scoring system was constructed. This non-invasive Koeln-Essen-index (NIKEI) was validated and compared to the FIB-4 index by calculating the area under the receiver operating characteristic curve (AUC). We evaluated a stepwise combination of both scoring systems for the precise prediction of advanced fibrosis. To set in contrast, we additionally tested the diagnostic accuracy of the AST/ALT ratio, BARD score and the NAFLD fibrosis score in our cohort.

Results

Age, AST, AST/ALT ratio, and total bilirubin were identified as significant predictors of advanced fibrosis and used to construct the NIKEI with an AUC of 0.968 [0.937; 0.998] compared to 0.929 [0.869; 0.989] for the FIB-4 index. The absence of advanced fibrosis could be confirmed with excellent accuracy (99–100%). The positive predictive value of the FIB-4 index was higher (100% vs. 60%), however, the false negative rate was also high (33%). With a stepwise combination of both indices 82%–84% of biopsies would have been avoidable without a single misclassification. The AUROC for AST/ALT ratio, the NAFLD fibrosis score, and the BARD score were 0.81 (95% CI, 0.72–0.90), 0.96 (95% CI 0.92–0.99), and 0.67 (95% CI 0.55–0.78), respectively.

Conclusion

The NIKEI can reliably exclude advanced fibrosis in subjects with NAFLD. In combination with the FIB-4 index misclassification with inadequate clinical management can be avoided while the need for liver biopsies can be reduced.     

Human Liver Infection in a Dish: Easy-To-Build 3D Liver Models for Studying Microbial Infection

Human liver infection is a major cause of death worldwide, but fundamental studies on infectious diseases affecting humans have been hampered by the lack of robust experimental models that accurately reproduce pathogen-host interactions in an environment relevant for the human disease. In the case of liver infection, one consequence of this absence of relevant models is a lack of understanding of how pathogens cross the sinusoidal endothelial barrier and parenchyma. To fill that gap we elaborated human 3D liver in vitro models, composed of human liver sinusoidal endothelial cells (LSEC) and Huh-7 hepatoma cells as hepatocyte model, layered in a structure mimicking the hepatic sinusoid, which enable studies of key features of early steps of hepatic infection. Built with established cell lines and scaffold, these models provide a reproducible and easy-to-build cell culture approach of reduced complexity compared to animal models, while preserving higher physiological relevance compared to standard 2D systems. For proof-of-principle we challenged the models with two hepatotropic pathogens: the parasitic amoeba Entamoeba histolytica and hepatitis B virus (HBV). We constructed four distinct setups dedicated to investigating specific aspects of hepatic invasion: 1) pathogen 3D migration towards hepatocytes, 2) hepatocyte barrier crossing, 3) LSEC and subsequent hepatocyte crossing, and 4) quantification of human hepatic virus replication (HBV). Our methods comprise automated quantification of E. histolytica migration and hepatic cells layer crossing in the 3D liver models. Moreover, replication of HBV virus occurs in our virus infection 3D liver model, indicating that routine in vitro assays using HBV or others viruses can be performed in this easy-to-build but more physiological hepatic environment. These results illustrate that our new 3D liver infection models are simple but effective, enabling new investigations on infectious disease mechanisms. The better understanding of these mechanisms in a human-relevant environment could aid the discovery of drugs against pathogenic liver infection.     

Establishment and Validation of GV-SAPS II Scoring System for Non-Diabetic Critically Ill Patients

MethodsTraining and validation cohorts were exacted from the Multiparameter Intelligent Monitoring in Intensive Care database III version 1.3 (MIMIC-III v1.3). The GV-SAPS II score was constructed by Cox proportional hazard regression analysis and compared with the original SAPS II, Sepsis-related Organ Failure Assessment Score (SOFA) and Elixhauser scoring systems using area under the curve of the receiver operator characteristic (auROC) curve.Results4,895 and 5,048 eligible individuals were included in the training and validation cohorts, respectively. The GV-SAPS II score was established with four independent risk factors, including hyperglycemia, hypoglycemia, standard deviation of blood glucose levels (Glu_SD), and SAPS II score. In the validation cohort, the auROC values of the new scoring system were 0.824 (95% CI: 0.813–0.834, P< 0.001) and 0.738 (95% CI: 0.725–0.750, P< 0.001), respectively for 30 days and 9 months, which were significantly higher than other models used in our study (all P < 0.001). Moreover, Kaplan-Meier plots demonstrated significantly worse outcomes in higher GV-SAPS II score groups both for 30-day and 9-month mortality endpoints (all P< 0.001).ConclusionsWe established and validated a modified prognostic scoring system that integrated glucose variability for non-diabetic critically ill patients, named GV-SAPS II. It demonstrated a superior prognostic capability and may be an optimal scoring system for prognostic evaluation in this patient group.     

Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test interventions at various stages of the disease process. These animal models are particularly difficult to implement in immature rodents, a classic model organism for laboratory study of these disorders. Recording continuous EEG in young animal models of seizures and other neurological disorders presents a technical challenge due to the small physical size of young rodents and their dependence on the dam prior to weaning. Therefore, there is not only a clear need for improving pre-clinical research that will better identify those therapies suitable for translation to the clinic but also a need for new devices capable of recording continuous EEG in immature rodents. Here, we describe the technology behind and demonstrate the use of a novel miniature telemetry system, specifically engineered for use in immature rats or mice, which is also effective for use in adult animals.     

啮齿动物螺杆菌多重PCR检测方法的建立

目的建立一种可同时检测肝、胆汁、啮齿类三种螺杆菌的多重PCR方法。方法根据已公布的肝、胆汁、啮齿类三种螺杆菌16SrRNA基因序列设计三对特异性引物进行多重PCR并对反应条件进行优化。结果三对引物能分别扩增出特异性的417 bp、364 bp、324 bp目的条带。最佳退火温度为52℃,镁离子浓度为2.0mmol/L,dNTP浓度为200μmol/L,引物浓度为0.625μmol/L。在此条件下多重PCR同时检测的肝、胆汁、啮齿类三种螺杆菌敏感度均为10 fg。结论本实验建立的多重PCR是一种敏感、特异、高效的方法,为同时检测啮齿动物中肝、胆汁、啮齿类三种螺杆菌奠定了良好的基础。     

无饲养层培养人胚胎干细胞方法的建立

人胚胎干细胞(human embryonic stem cell,hES细胞)是当前医学研究的热点之一．然而hES细胞培养条件苛刻,通常需要采用鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEFs)饲养层来维持其未分化状态,成为目前hES细胞研究的瓶颈之一、本实验成功地将hES细胞接种在细胞外基质包被的六孔板上培养,传代20次后细胞仍然保持良好的未分化状态,各种hES细胞生物学特性(如表面标志物SSEA-3、SSEA-4、TRA-1-60和TRA-1-8l,OCT-4,碱性磷酸酶及体内外分化潜能等)均无改变;其冻存、复苏效果与生长在饲养层上的hES细胞无明显差异．因此,该无饲养层培养体系可以用于培养hES细胞,并为hES细胞转基因研究及大规模培养打下良好的基础．     

人类疾病模型小鼠的标准化维持方法研究

目的研究人类疾病模型小鼠的可遗传生物学特性,建立品系的标准化维持方法。方法选用FVB TgN、MRL Mpj和SAMP1Ka三个疾病模型小鼠品系作为代表性实验对象,通过测定生长曲线、RAPD同工、酶电泳、行为学试验等方法,找出三个品系相对于各自对照品系的不同特征,并建立了标准化检测指标作为维持方法的依据。结果MRL Mpj的生长曲线相对于对照品系有明显的统计学差异;FVB TgN、MRL Mpj、SAMP1Ka等三个品系的RAPD图谱在阳性引物及扩增出的条带方面均不一致;同工酶电泳的结果表明不同品系的个体之间表型不完全相同;行为学试验更从多方面直观地显示了各品系的不同特征。结论人类疾病模型小鼠除了用常规检测方法外,还应建立各品系在生长曲线、RAPD、同工酶电泳、行为学试验等方面的特殊检测方法,及时检测模型小鼠品系的独特性状并作为保种依据,以避免遗传漂变的发生。     

Establishment of a Reverse Genetics System for Studying Human Bocavirus in Human Airway Epithelia

Human bocavirus 1 (HBoV1) has been identified as one of the etiological agents of wheezing in young children with acute respiratory-tract infections. In this study, we have obtained the sequence of a full-length HBoV1 genome (including both termini) using viral DNA extracted from a nasopharyngeal aspirate of an infected patient, cloned the full-length HBoV1 genome, and demonstrated DNA replication, encapsidation of the ssDNA genome, and release of the HBoV1 virions from human embryonic kidney 293 cells. The HBoV1 virions generated from this cell line-based production system exhibits a typical icosahedral structure of approximately 26 nm in diameter, and is capable of productively infecting polarized primary human airway epithelia (HAE) from the apical surface. Infected HAE showed hallmarks of lung airway-tract injury, including disruption of the tight junction barrier, loss of cilia and epithelial cell hypertrophy. Notably, polarized HAE cultured from an immortalized airway epithelial cell line, CuFi-8 (originally derived from a cystic fibrosis patient), also supported productive infection of HBoV1. Thus, we have established a reverse genetics system and generated the first cell line-based culture system for the study of HBoV1 infection, which will significantly advance the study of HBoV1 replication and pathogenesis.     

Systemic and Local Drug Delivery for Treating Diseases of the Central Nervous System in Rodent Models

Thorough preclinical testing of central nervous system (CNS) therapeutics includes a consideration of routes of administration and agent biodistribution in assessing therapeutic efficacy. Between the two major classifications of administration, local vs. systemic, systemic delivery approaches are often preferred due to ease of administration. However, systemic delivery may result in suboptimal drug concentration being achieved in the CNS, and lead to erroneous conclusions regarding agent efficacy. Local drug delivery methods are more invasive, but may be necessary to achieve therapeutic CNS drug levels. Here, we demonstrate proper technique for three routes of systemic drug delivery: intravenous injection, intraperitoneal injection, and oral gavage. In addition, we show a method for local delivery to the brain: convection-enhanced delivery (CED). The use of fluorescently-labeled compounds is included for in vivo imaging and verification of proper drug administration. The methods are presented using murine models, but can easily be adapted for use in rats.     

Establishment of Magnetic Microparticles-Assisted Time-Resolved Fluoroimmunoassay for Determinating Biomarker Models in Human Serum

In order to early screen and detect suspected biomarkers from pathogens and the human body itself, tracers or reaction strategies that can act as signal enhancers have been proposed forth at purpose. In this paper, we discussed the applicability of magnetic microparticles-assisted time-resolved fluoroimmunoassay (MMPs-TRFIA) for sensitive determination of potential analytes. Hepatitis B e antigen, antibody to hepatitis B surface antigen and free triiodothyronine were used as biomarker models to explore the reliability of the method. By coupling with bioprobes, MMPs were used as immunoassay carriers to capture target molecules. Under optimal condition, assay performance, including accuracy, precision and specificity, was outstanding and demonstrated satisfactory. To further evaluate the performance of the MMPs-TRFIA in patients, a total of 728 serum samples from hospital were analyzed for three biomarkers in parallel with the proposed method and chemiluminescence immunoassay kit commercially available. Fairly good agreements are obtained between the two methods via data analysis. Not only that but the reliability of MMPs-TRFIA has also been illustrated by three different reaction models. It is confirmed that the novel method modified with MMPs has been established and showed great potential applications in both biological detection and clinical diagnosis, including big molecule protein and low molecular weight haptens.     

两种变应性接触性皮炎动物模型的建立及比较

目的比较两种动物作为变应性接触性皮炎（allergic contact dermatitis,ACD）模型各自的优势,为实际应用中恰当选择动物模型提供依据。方法利用二硝基氯苯（dinitrochlorobenzene,DNCB）作为致敏剂,以腹部致敏、背部激发的方法分别建立豚鼠（连续激发4次）和小鼠（1次激发）两种ACD动物模型,并以丙酮作为对照。激发后0～96h,对激发部位进行动态分级。激发后96h,H-E染色观察激发部位皮肤病理变化,并计算脾指数和胸腺指数。结果动态评分结果显示：豚鼠激发后72h红斑程度最强,临床分级以3级为主,并于72～96h保持不变;小鼠激发后24h红斑程度最强,临床分级以4级为主,48h后红斑程度减轻。病理结果显示：两种模型激发部位皮肤内均有大量炎症细胞浸润。脾指数和胸腺指数计算结果显示：两种动物模型的脾指数和胸腺指数均较对照组明显增加（P〈0．05）。结论通过上述方法分别成功建立了豚鼠和小鼠ACD动物模型。豚鼠红斑程度较弱,且出现较晚,持续时间较长;小鼠红斑程度较强,出现较早,持续时间较短。     

Comparison of Human Genomics and Genetic Models of Cancer to Identify Novel Therapeutic Targets

No abstract available.     

Comparison of Different Scoring Systems Based on Both Donor and Recipient Characteristics for Predicting Outcome after Living Donor Liver Transplantation

Background and Objectives

In order to provide a good match between donor and recipient in liver transplantation, four scoring systems [the product of donor age and Model for End-stage Liver Disease score (D-MELD), the score to predict survival outcomes following liver transplantation (SOFT), the balance of risk score (BAR), and the transplant risk index (TRI)] based on both donor and recipient parameters were designed. This study was conducted to evaluate the performance of the four scores in living donor liver transplantation (LDLT) and compare them with the MELD score.

Patients and Methods

The clinical data of 249 adult patients undergoing LDLT in our center were retrospectively evaluated. The area under the receiver operating characteristic curves (AUCs) of each score were calculated and compared at 1-, 3-, 6-month and 1-year after LDLT.

Results

The BAR at 1-, 3-, 6-month and 1-year after LDLT and the D-MELD and TRI at 1-, 3- and 6-month after LDLT showed acceptable performances in the prediction of survival (AUC>0.6), while the SOFT showed poor discrimination at 6-month after LDLT (AUC = 0.569). In addition, the D-MELD and BAR displayed positive correlations with the length of ICU stay (D-MELD, p = 0.025; BAR, p = 0.022). The SOFT was correlated with the time of mechanical ventilation (p = 0.022).

Conclusion

The D-MELD, BAR and TRI provided acceptable performance in predicting survival after LDLT. However, even though these scoring systems were based on both donor and recipient parameters, only the BAR provided better performance than the MELD in predicting 1-year survival after LDLT.     

Predicting Invasive Plants: Prospects for a General Screening System Based on Current Regional Models

Screening systems for predicting invasive plants have been independently developed for the non-indigenous floras of North America, the South African fynbos, and Australia. To evaluate the performance of these screening systems outside the regions for which they were developed, we tested them for the non-indigenous flora of the Hawaiian Islands. When known invasive plant species in the Hawaiian Islands were evaluated using the North American and Australian systems, 82% and 93% of the species were predicted to be invasive, respectively, and the remainder were classified as requiring further study. The South African fynbos system correctly predicted only 60% of the invasive species in the Hawaiian Islands. All three screening systems correctly classified a majority of the non-invaders as non-invasive. The Australian system has several advantages over the other systems, including the highest level of correct identification of invaders (>90%), ability to evaluate non-woody plants, and ability to evaluate a species even when the answers to some questions are unknown. Nevertheless, with the Australian system, a large fraction of species known not to be invasive were recommended for further study before importing, so there remains room for improvement in identifying non-invasive species. Based on our results for the Hawaiian Islands and a previous evaluation in New Zealand, the Australian system appears to be a promising template for building a globally applicable system for screening out invasive plant introductions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Checklist and Scoring System for the Assessment of Soft Tissue Preservation in CT Examinations of Human Mummies

The purpose of this study was to develop a checklist for standardized assessment of soft tissue preservation in human mummies based on whole-body computed tomography examinations, and to add a scoring system to facilitate quantitative comparison of mummies. Computed tomography examinations of 23 mummies from the Capuchin Catacombs of Palermo, Sicily (17 adults, 6 children; 17 anthropogenically and 6 naturally mummified) and 7 mummies from the crypt of the Dominican Church of the Holy Spirit of Vilnius, Lithuania (5 adults, 2 children; all naturally mummified) were used to develop the checklist following previously published guidelines. The scoring system was developed by assigning equal scores for checkpoints with equivalent quality. The checklist was evaluated by intra- and inter-observer reliability. The finalized checklist was applied to compare the groups of anthropogenically and naturally mummified bodies. The finalized checklist contains 97 checkpoints and was divided into two main categories, “A. Soft Tissues of Head and Musculoskeletal System” and “B. Organs and Organ Systems”, each including various subcategories. The complete checklist had an intra-observer reliability of 98% and an inter-observer reliability of 93%. Statistical comparison revealed significantly higher values in anthropogenically compared to naturally mummified bodies for the total score and for three subcategories. In conclusion, the developed checklist allows for a standardized assessment and documentation of soft tissue preservation in whole-body computed tomography examinations of human mummies. The scoring system facilitates a quantitative comparison of the soft tissue preservation status between single mummies or mummy collections.     

SW1990和Capan-2红色荧光标记胰腺癌移植肿瘤模型的建立和比较

目的建立稳定表达红色荧光蛋白基因的人胰腺癌细胞系,为体内监测肿瘤的早期生长及抗肿瘤药物的药效评价建立一种新的肿瘤动物模型。方法以Lipofectamine 2000介导chickenβ-actin-RFP-NEO转染人胰腺癌细胞SW1990和Capan-2,经梯度浓度G418筛选获得稳定表达红色荧光蛋白的细胞克隆并扩大培养。BALB/cA-nu裸鼠皮下接种1×106个发光细胞使其成瘤,活体荧光成像系统观察肿瘤的生长情况。结果获得了稳定表达RFP的两种不同的人胰腺癌细胞株,将其接种到裸鼠体内可成瘤,利用活体成像系统观察了肿瘤的生长动态过程,并且SW1990肿瘤细胞的生长速度较Capan-2细胞快。结论用红色荧光蛋白标记的人胰腺癌细胞建立的裸鼠肿瘤模型为胰腺癌的研究和相关药物筛选提供了可进行荧光影像活体、动态分析的动物模型。