Packaging cells for lentiviral vectors generated using the cumate and coumermycin gene induction systems and nanowell single-cell cloning.

Lentiviral vectors (LVs) are important for cell therapy because of their capacity to stably modify the genome after integration. This study describes a novel and relatively simple approach to generate packaging cells and producer clones for self-inactivating (SIN) LVs pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). A novel gene regulation system, based on the combination of the cumate and coumermycin induction systems, was developed to ensure tight control for the expression of cytotoxic packaging elements.

Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors.

To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow.

Development of suspension adapted Vero cell culture process technology for production of viral vaccines.

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated.

Investigating Patients' Perception of Microvascular Free Toe Flap for Reconstruction of Amputated Thumbs: A Guide for Surgeons during Informed Consent.

Background:  After thumb amputations, restoration of function and aesthetic can be accomplished with microvascular free toe flaps. However, many patients in clinical practice do not choose this reconstruction despite positive reported outcomes. This study aims to determine patients' perceptions with respect to free toe flaps to improve areas of informed consent.

Concomitant use of corticosteroids and immune checkpoint inhibitors in patients with hematologic or solid neoplasms: A systematic review.

Purpose: Clinical trials studying immune checkpoint inhibitors exclude patients on corticosteroids, due to the hypothesis that corticosteroids may antagonize immunotherapy. We performed a systematic review of the literature looking at the clinical outcomes of cancer patients treated with immune checkpoint inhibitors and concomitant corticosteroids.

Methods: The following databases were searched for relevant studies: MEDLINE, Embase Classic+Embase, BIOSIS Previews, the Cochrane Database of Systematic Reviews, the CENTRAL Registry of Controlled Trials, Web of Science and Scopus.

Long-term Outcomes of Induction Carboplatin and Gemcitabine Followed by Concurrent Radiotherapy With Low-dose Paclitaxel and Gemcitabine for Stage III Non-small-cell Lung Cancer.

Background: Standard treatment for unresectable stage III non-small-cell lung cancer (NSCLC) is concurrent chemo-radiation (CRT). A regimen of induction carboplatin and gemcitabine followed by CRT was developed at the McGill University Health Centre to prevent delays in treatment initiation. We report the long-term outcomes with this regimen based on a pooled analysis of both protocol patients from a phase II study and nonprotocol patients.

Establishment and validation of new complementing cells for production of E1-deleted adenovirus vectors in serum-free suspension culture.

E1-deleted adenovirus vectors (AdV) are important gene transfer vehicles for gene therapy and vaccination. Amplification of AdV must take place in cells that express the adenovirus E1A and E1B genes. Sequence homology between AdV and the E1 genes integrated within the complementing cells should be minimal to reduce the odds of generating replication-competent adenovirus (RCA).

Fenretinide corrects the imbalance between omega-6 to omega-3 polyunsaturated fatty acids and inhibits macrophage inflammatory mediators via the ERK pathway.

We previously identified Fragile X-related protein 1 (FXR1) as an RNA-binding protein involved in the post-transcriptional control of TNF and other cytokines in macrophages. Macrophages derived from FXR1-KO mice overexpress several inflammatory cytokines including TNF. Recently, we showed that fenretinide (4HPR) is able to inhibit several inflammatory cytokines in the lungs of cystic fibrosis mice, which also have abnormal immune responses.

Cystic fibrosis fatty acid imbalance is linked to ceramide deficiency and corrected by fenretinide.

Patients with cystic fibrosis (CF) and Cftr-knockout mice (CF mice) display an imbalance in fatty acids, with high arachidonic acid (AA) and low docosahexaenoic acid (DHA) concentrations. Our recent studies demonstrated defects in another class of lipids, ceramides, in patients with CF and in CF mice. This study investigates the relationship between ceramide, AA, DHA, and the correction of lipid imbalances in CF mice after treatment with fenretinide.

New adenovirus vectors for protein production and gene transfer.

Based on two new adenovirus expression cassettes, we have constructed a series of Ad transfer vectors for the overexpression of one or two genes either in a dicistronic configuration or with separate expression cassettes. Inclusion of the green or blue fluorescent protein in the vectors accelerates the generation of adenovirus recombinants and facilitates the functional characterization of genes both in vitro and in vivo by allowing easy quantification of gene transfer and expression. With our optimized tetracycline-regulated promoter (TR5) we have generated recombinant adenoviruses expressing proteins, that are either cytotoxic or which interfere with adenovirus replication, at levels of 10-15% of total cell protein.

Novel pharmaceutical approaches for treating patients with cystic fibrosis.

Before the cloning of the CFTR gene in 1989, there were relatively few treatment options for the many phenotypes associated with cystic fibrosis (CF). The advancement of research in areas such as immunology, molecular biology and pharmacology have provided new insights into the mechanism and evolution of CF. More than 40 systematic clinical trials evaluating new therapies for CF are presently registered with the NIH.

Fenretinide prevents the development of osteoporosis in Cftr-KO mice.

Background: The most recently described phenotype associated with Cystic Fibrosis (CF) is reduced bone mineral density which results in osteopenia and osteoporosis. The etiology of the early onset of osteoporosis in CF patients has remained to be established. It has been suggested that inadequate nutritional absorption of essential fatty acids may play a role in the altered bone metabolism.

Fenretinide corrects newly found ceramide deficiency in cystic fibrosis.

Chronic and persistent lung infections cause the majority of morbidity and mortality in patients with cystic fibrosis (CF). Galactosyl ceramide has been previously shown to be involved in Pseudomonas internalization. Therefore, we assessed ceramide levels in the plasma of patients with CF and compared them to healthy volunteers using high-performance liquid chromatography followed by mass spectrometry.

High-level recombinant protein production in CHO cells using an adenoviral vector and the cumate gene-switch.

To facilitate and accelerate the production of eukaryotic proteins with correct post-translational modifications, we have developed a protein production system based on the transduction of Chinese hamster ovary (CHO) cells using adenovirus vectors (AdVs). We have engineered a CHO cell line (CHO-cTA) that stably expresses the transactivator (cTA) of our newly developed cumate gene-switch transcription system. This cell line is adapted to suspension culture and can grow in serum-free and protein-free medium.

The cumate gene-switch: a system for regulated expression in mammalian cells.

Background: A number of expression systems have been developed where transgene expression can be regulated. They all have specific characteristics making them more suitable for certain applications than for others. Since some applications require the regulation of several genes, there is a need for a variety of independent yet compatible systems.

Cystic fibrosis mouse models.

Animal models of cystic fibrosis (CF) are powerful tools that enable the study of the mechanisms and complexities of human disease. Murine models have several intrinsic advantages compared with other animal models, including lower cost, maintenance, and rapid reproduction rate. Mice can be easily genetically manipulated by making transgenic or knockout mice, or by backcrossing to well-defined inbred strains in a reasonably short period of time.

Distinct pattern of lung gene expression in the Cftr-KO mice developing spontaneous lung disease compared with their littermate controls.

Cystic fibrosis (CF) is caused by a defect in the CF transmembrane conductance regulator (CFTR) protein that functions as a chloride channel. Dysfunction of the CFTR protein results in salty sweat, pancreatic insufficiency, intestinal obstruction, male infertility, and severe pulmonary disease. Most of the morbidity and mortality of CF patients results from pulmonary complications.

Cystic fibrosis lung disease following infection with Pseudomonas aeruginosa in Cftr knockout mice using novel non-invasive direct pulmonary infection technique.

To better understand the mechanism of lung infection with Pseudomonas aeruginosa (P. aeruginosa), many techniques have been developed in order to establish lung infection in rodents. A model of chronic lung infection, using tracheotomy to inoculate the bacteria, has been extensively used in the cystic fibrosis (CF) mouse model of lung infection.

Modulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa.

Background: The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the alpha, beta and gamma subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase.

Methods: Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains.

The R1 subunit of herpes simplex virus ribonucleotide reductase has chaperone-like activity similar to Hsp27.

HSV-2 R1, the R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, protects cells against apoptosis. Here, we report the presence in HSV-2 R1 of a stretch exhibiting similarity to the alpha-crystallin domain of the small heat shock proteins, a domain known to be important for oligomerization and cytoprotective activities of these proteins. Also, the HSV-2 R1 protein, which forms multimeric structures in the absence of nucleotide, displayed chaperone ability as good as Hsp27 in a thermal denaturation assay using citrate synthase.

Influence of gender and interleukin-10 deficiency on the inflammatory response during lung infection with Pseudomonas aeruginosa in mice.

Cystic fibrosis females have a worse prognosis compared to male patients. Furthermore, cystic fibrosis patients infected with Pseudomonas aeruginosa have been shown to have dysregulated cytokine profiles, as higher levels of tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-8, and lower levels of IL-10 are found in the bronchoalveolar lavage fluid compared to healthy controls. The present study was aimed at investigating the importance of gender and IL-10 in the susceptibility of C57BL/6 mice to pulmonary infection with Pseudomonas aeruginosa.

The R1 subunit of herpes simplex virus ribonucleotide reductase protects cells against apoptosis at, or upstream of, caspase-8 activation.

The R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, which in addition to its C-terminal reductase domain possesses a unique N-terminal domain of about 400 amino acids, is thought to have an additional, as yet unknown, function. Here, we report that the full-length HSV-2 R1 has an anti-apoptotic function able to protect cells against death triggered by expression of R1(Delta2-357), an HSV-2 R1 subunit with its first 357 amino acids deleted. We further substantiate the R1 anti-apoptotic activity by showing that its accumulation at low level could completely block apoptosis induced by TNF-receptor family triggering.

Genotypic characterization of macrolide-resistant strains of Streptococcus pneumoniae isolated in Quebec, Canada, and in vitro activity of ABT-773 and telithromycin.

Increasing resistance of Streptococcus pneumoniae to macrolides represents a challenge for clinicians. New ketolides have an enhanced activity against macrolide-resistant strains. Four hundred and seventy-four strains of S.

A noninvasive cell-based assay for monitoring proteolytic activity within a specific subcellular compartment.

A noninvasive cell-based assay has been developed to monitor the proteolytic activity of cathepsin L within a specific subcellular compartment, the lysosome. The green fluorescent protein (GFP) of Aequorea victoria was selected as a substrate. Targeting to lysosomes was achieved by fusing GFP to preprocathepsin L, which also ensures colocalization of the enzyme and the substrate.

Development and evaluation of a PCR method for detection of the Clostridium difficile toxin B gene in stool specimens.

A PCR assay detecting Clostridium difficile toxin B gene in stool specimens was compared to the cytotoxicity assay as the reference standard for the diagnosis of C. difficile antibiotic-associated diarrhea (CDAD). Overall, 118 stool samples were tested.