Inflammasome components Asc and caspase-1 mediate biomaterial-induced inflammation and foreign body response

Implantation of biomaterials and devices into soft tissues leads to the development of the foreign body response (FBR), which can interfere with implant function and eventually lead to failure. The FBR consists of overlapping acute and persistent inflammatory phases coupled with collagenous encapsulation and currently there are no therapeutic options. Initiation of the FBR involves macrophage activation, proceeding to giant cell formation, fibroblast activation, and collagen matrix deposition. Despite the recognition of this sequence of events, the molecular pathways required for the FBR have not been elucidated. We have identified that the acute inflammatory response to biomaterials requires nucleotide-binding domain and leucine-rich repeat-containing 3 (Nlrp3), apoptosis-associated speck-like protein containing CARD (Asc), and caspase-1, as well as plasma membrane cholesterol, and Syk signaling. Full development of the FBR is dependent on Asc and caspase-1, but not Nlrp3. The common antiinflammatory drug aspirin can reduce inflammasome activation and significantly reduce the FBR. Taken together, these findings expand the role of the inflammasome from one of sensing damage associated molecular patterns (DAMPs) to sensing all particulate matter irrespective of size. In addition, implication of the inflammasome in biomaterial recognition identifies key pathways, which can be targeted to limit the FBR.     

Comparison of HIV- and EIAV-based vectors on their efficiency in transducing murine and human hematopoietic repopulating cells.

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.     

Characteristics of murine histidinaemia and its potential for genetic manipulation.

BACKGROUND: Histidinaemia is an autosomal recessive disorder affecting the hepatic enzyme histidine ammonia lyase (histidase) resulting in elevated plasma and urinary histidine and is prototypic of a series of hepatic cytosolic enzyme defects. AIMS: To characterise the physiology of murine histidinaemia with respect to histidine excretion and catabolism, and explore the potential for manipulating cellular and whole body histidase metabolism by gene transfer. MATERIALS AND METHODS: We studied his/his mice which have a G to A substitution in the gene encoding histidase, using both in vitro transduction of isolated hepatocytes by lipofection with wild-type histidase cDNA, and in vivo transduction of whole liver using a retroviral construct. RESULTS AND CONCLUSION: Histidase cDNA expression restored histidase activity in vivo and in vitro towards normal levels, demonstrated both at the cellular level and by whole body metabolic studies, establishing the potential of this model for the development of new gene therapeutic approaches.     

The role of food restriction on CCl4-induced cirrhosis model in rats.

Effects of food restriction on susceptibility to the toxic effect of some chemicals are controversial. In order to identify an exposure model that could maximize cirrhosis and minimize mortality rate, this study aimed to evaluate the effect of food restriction on tetrachloride carbon (CCl(4))-induced cirrhosis model in rats. Fifty-three male Wistar rats received CCl(4) 0.25 ml/kg weekly intragastrically once a week. Thirty-three had 44% food restriction (group 1); 10 rats had 25% food restriction (group 2); and 10 rats received ad libitum food (group 3). After 10 weeks, the animals were sacrificed and liver sections were collected for histology. Of the 53 animals enrolled for the study, 22 (41.5%) died before completing 10-week CCl(4). Mortality rate was significantly higher in group 1 compared to other groups (p<0.05). Cirrhosis was significantly more prevalent in group 1 than in group 3 (p<0.01), but without significant difference between groups 1 and 2 (p=0.624). We concluded that food restriction is an important issue to be considered when establishing a CCl(4)-induced cirrhosis model in rats. Moreover, there is an ideal range of food intake that predisposes to liver damage without increasing mortality leading to a more effective model.     

Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice.

Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases by stable vector insertion into the patients' chromosomes. However, three cases of T cell lymphoproliferative disease have been identified almost 3 years after retrovirus gene therapy for X-linked severe combined immune deficiency. In two of these cases vector insertion into the LMO2 locus was implicated in leukemogenesis, demonstrating that a more profound understanding is required of the genetic and molecular effects imposed on the host by vector integration or transgene expression. In vivo models to test for retro- and lentiviral vector safety prior to clinical application are therefore needed. Here we present a high incidence of lentiviral vector-associated tumorigenesis following in utero and neonatal gene transfer in mice. This system may provide a highly sensitive model to investigate integrating vector safety prior to clinical application.     

A methodology for the automated creation of fuzzy expert systems for ischaemic and arrhythmic beat classification based on a set of rules obtained by a decision tree

OBJECTIVE: In the current work we propose a methodology for the automated creation of fuzzy expert systems, applied in ischaemic and arrhythmic beat classification. METHODS: The proposed methodology automatically creates a fuzzy expert system from an initial training dataset. The approach consists of three stages: (a) extraction of a crisp set of rules from a decision tree induced from the training dataset, (b) transformation of the crisp set of rules into a fuzzy model and (c) optimization of the fuzzy model's parameters using global optimization. MATERIAL: The above methodology is employed in order to create fuzzy expert systems for ischaemic and arrhythmic beat classification in ECG recordings. The fuzzy expert system for ischaemic beat detection is evaluated in a cardiac beat dataset that was constructed using recordings from the European Society of Cardiology ST-T database. The arrhythmic beat classification fuzzy expert system is evaluated using the MIT-BIH arrhythmia database. RESULTS: The fuzzy expert system for ischaemic beat classification reported 91% sensitivity and 92% specificity. The arrhythmic beat classification fuzzy expert system reported 96% average sensitivity and 99% average specificity for all categories. CONCLUSION: The proposed methodology provides high accuracy and the ability to interpret the decisions made. The fuzzy expert systems for ischaemic and arrhythmic beat classification compare well with previously reported results, indicating that they could be part of an overall clinical system for ECG analysis and diagnosis.     

Restoration of LDL receptor function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1

Familial hypercholesterolemia is an autosomal dominant disorder with a gene-dosage effect that is usually caused by mutations in the LDL receptor gene that disrupt normal clearance of LDL. In the homozygous form, it results in a distinctive clinical phenotype, characterized by inherited hypercholesterolemia, cholesterol deposition in tendons, and severe premature coronary disease. We described previously two families with autosomal recessive hypercholesterolemia that is not due to mutations in the LDL receptor gene but is characterized by defective LDL receptor-dependent internalization and degradation of LDL by transformed lymphocytes from the patients. We mapped the defective gene to chromosome 1p36 and now show that the disorder in these and a third English family is due to novel mutations in ARH1, a newly identified gene encoding an adaptor-like protein. Cultured skin fibroblasts from affected individuals exhibit normal LDL receptor activity, but their monocyte-derived macrophages are similar to transformed lymphocytes, being unable to internalize and degrade LDL. Retroviral expression of normal human ARH1 restores LDL receptor internalization in transformed lymphocytes from an affected individual, as demonstrated by uptake and degradation of (125)I-labeled LDL and confocal microscopy of cells labeled with anti-LDL-receptor Ab.     

Widespread and efficient marker gene expression in the airway epithelia of fetal sheep after minimally invasive tracheal application of recombinant adenovirus in utero

Cystic fibrosis is a common lethal genetic disease caused by functional absence of the cystic fibrosis transmembrane conductance regulator (CFTR). Although a candidate disease for in utero gene therapy, demonstration of potentially therapeutic levels of transgene expression in the fetal airways after minimally invasive gene delivery is a mandatory prerequisite before application of this approach in humans can be considered. We report here on the delivery of a beta-galactosidase expressing adenovirus directly to the airways of fetal sheep in utero using ultrasound-guided percutaneous injection of the trachea in the fetal chest. Injection of adenoviral particles to the fetal airways was not associated with mortality and resulted in low-level expression in the peripheral airways. However, complexation of the virus with DEAE dextran, which confers a positive charge to the virus, and pretreatment of the airways with Na-caprate, which opens tight junctions, increased transgene expression, and a combination of these two enhancers resulted in widespread and efficient gene transfer of the fetal trachea and bronchial tree. Using a percutaneous ultrasound-guided injection technique, we have clearly demonstrated proof of principle for substantial transgene delivery to the fetal airways providing levels of gene expression that could be relevant for a therapeutic application of CFTR expressing vectors.     

Highly efficient EIAV-mediated in utero gene transfer and expression in the major muscle groups affected by Duchenne muscular dystrophy

Gene therapy for Duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. In addition, the prenatal onset of disease complicates postnatal gene therapy. We have therefore proposed a fetal approach to overcome these barriers. We have applied beta-galactosidase expressing equine infectious anaemia virus (EIAV) lentiviruses pseudotyped with VSV-G by single or combined injection via different routes to the MF1 mouse fetus on day 15 of gestation and describe substantial gene delivery to the musculature. Highly efficient gene transfer to skeletal muscles, including the diaphragm and intercostal muscles, as well as to cardiac myocytes was observed and gene expression persisted for at least 15 months after administration of this integrating vector. These findings support the concept of in utero gene delivery for therapeutic and long-term prevention/correction of muscular dystrophies and pave the way for a future application in the clinic.