The Rising Challenge of Training Physician-Scientists: Recommendations From a Canadian National Consensus Conference.

Physician-scientists are individuals who actively participate in patient care, have undergone additional research training, and devote the majority of their time to research. Physician-scientists are traditionally the primary catalysts in bridging the translational gap-that is, the failure to link fundamental new knowledge in the pathobiology of disease with advances in health care and health policy in a timely manner. However, there has been a shift away from training physician-scientists, and financial support for the physician-scientist is diminishing globally, causing the translational gap to grow. Given its socialized health care system and cultural and geographic diversity, Canada can serve as a unique case study in understanding how to address this phenomenon as a national priority. To this end, a Canadian national consensus conference was convened to develop recommendations for training programs and early-career supports for physician-scientists. Five recommendations were generated: (1) Establish an independent, national council whose mandate is to provide pan-Canadian oversight of physician-scientist training programs; (2) develop capacity for funding and mentorship support for physician-scientists; (3) develop coherent networks across a broad range of clinician-scientists, including physician-scientists, to reflect the unique cultural and geographic diversity of Canada and to reflect the interdisciplinarity of health research; (4) ensure that medical school curricula integrate, as a core curriculum feature, an understanding of the scientific basis of health care, including research methodologies; and (5) ensure that the funding of the physician-scientist trainee is viewed as portable and distinct from the operational funding provided to the training program itself.

Fed-batch bioreactor performance and cell line stability evaluation of the artificial chromosome expression technology expressing an IgG1 in Chinese hamster ovary cells.

The artificial chromosome expression (ACE) technology system uses an engineered artificial chromosome containing multiple site-specific recombination acceptor sites for the rapid and efficient construction of stable cell lines. The construction of Chinese hamster ovary(CHO) cell lines expressing an IgG1 monoclonal antibody (MAb) using the ACE system has been previously described (Kennard et al., Biotechnol Bioeng. 2009;104:540-553). To further demonstrate the manufacturing feasibility of the ACE system, four CHO cell lines expressing the human IgG1 MAb 4A1 were evaluated in batch and fed-batch shake flasks and in a 2-L fed-batch bioreactor. The batch shake flasks achieved titers between 0.7 and 1.1 g/L, whereas the fed-batch shake flask process improved titers to 2.5­3.0 g/L. The lead 4A1 ACE cell line achieved titers of 4.0 g/L with an average specific productivity of 40 pg/(cell day) when cultured in a non optimized 2-L fed-batch bioreactor using a completely chemically defined process. Generational stability characterization of the lead 4A1-expressing cell line demonstrated that the cell line was stable for up to 75 days in culture. Product quality attributes of the 4A1 MAb produced by the ACE system during the stability evaluation period were unchanged and also comparable to existing expression technologies such as the CHO-dhfr system. The results of this evaluation demonstrate that a clonal, stable MAb-expressing CHO cell line can be produced using ACE technology that performs competitively using a chemically defined fed-batch bioreactor process with comparable product quality attributes to cell lines generated by existing technologies.

The generation of stable, high MAb expressing CHO cell lines based on the artificial chromosome expression (ACE) technology.

The manufacture of recombinant proteins at industrially relevant levels requires technologies that can engineer stable, high expressing cell lines rapidly, reproducibly and with relative ease. Commonly used methods incorporate transfection of mammalian cell lines with plasmid DNA containing the gene of interest. Identifying stable high expressing transfectants is normally laborious and time consuming. To improve this process, the ACE System has been developed based on pre-engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for the targeted transfection of single or multiple genes and eliminates the need for random integration into native host chromosomes. To illustrate the utility of the ACE System in generating stable, high expressing cell lines, CHO based candidate cell lines were generated to express a human monoclonal IgG1 antibody. Candidate cell lines were generated in under 6 months and expressed over 1 g/L and with specific productivities of up to 45 pg/cell/day under non-fed, non-optimized shake flask conditions. These candidate cell lines were shown to have stable expression of the monoclonal antibody for up to 70 days of continuous culture. The results of this study demonstrate that clonal, stable monoclonal antibody expressing CHO based cell lines can be generated by the ACE System rapidly and perform competitively with those cell lines generated by existing technologies. The ACE System, therefore, provides an attractive and practical alternative to conventional methods of cell line generation.

Auditioning of CHO host cell lines using the artificial chromosome expression (ACE) technology.

In order to maximize recombinant protein expression in mammalian cells many factors need to be considered such as transfection method, vector construction, screening techniques and culture conditions. In addition, the host cell line can have a profound effect on the protein expression. However, auditioning or directly comparing host cell lines for optimal protein expression may be difficult since most transfection methods are based on random integration of the gene of interest into the host cell genome. Thus it is not possible to determine whether differences in expression between various host cell lines are due to the phenotype of the host cell itself or genetic factors such as gene copy number or gene location. To improve cell line generation, the ACE System was developed based on pre-engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for targeted transfection and has been effectively used to rapidly generate stable CHO cell lines expressing high levels of monoclonal antibody. A key feature of the ACE System is the ability to isolate and purify ACEs containing the gene(s) of interest and transfect the same ACEs into different host cell lines. This feature allows the direct auditioning of host cells since the host cells have been transfected with ACEs that contain the same number of gene copies in the same genetic environment. To investigate this audition feature, three CHO host cell lines (CHOK1SV, CHO-S and DG44) were transfected with the same ACE containing gene copies of a human monoclonal IgG1 antibody. Clonal cell lines were generated allowing a direct comparison of antibody expression and stability between the CHO host cells. Results showed that the CHOK1SV host cell line expressed antibody at levels of more than two to five times that for DG44 and CHO-S host cell lines, respectively. To confirm that the ACE itself was not responsible for the low antibody expression seen in the CHO-S based clones, the ACE was isolated and purified from these cells and transfected back into fresh CHOK1SV cells. The resulting expression of the antibody from the ACE newly transfected into CHOK1SV increased fivefold compared to its expression in CHO-S and confirmed that the differences in expression between the different CHO host cells was due to the cell phenotype rather than differences in gene copy number and/or location. These results demonstrate the utility of the ACE System in providing a rapid and direct technique for auditioning host cell lines for optimal recombinant protein expression.

The antimicrobial peptide polyphemusin localizes to the cytoplasm of Escherichia coli following treatment.

The horseshoe crab peptide polyphemusin I possesses high antimicrobial activity, but its mechanism of action is as yet not well defined. Using a biotin-labeled polyphemusin I analogue and confocal fluorescence microscopy, we showed that the peptide accumulates in the cytoplasm of wild-type Escherichia coli within 30 min after addition without causing substantial membrane damage.

Salmonella and Enteropathogenic Escherichia coli Interactions with Host Cells: Signaling Pathways.

The host-pathogen interaction involves a myriad of initiations and responses from both sides. Bacterial pathogens such as enteropathogenic Escherichia coli (EPEC) and Salmonella enterica have numerous virulence factors that interact with and alter signaling components of the host cell to initiate responses that are beneficial to pathogen survival and persistence. The study of Salmonella and EPEC infection reveals intricate connections between host signal transduction, cytoskeletal architecture, membrane trafficking, and cytokine gene expression. The emerging picture includes elements of molecular mimicry by bacterial effectors and bacterial subversion of typical host events, with the result that EPEC is able to survive and persist in an extracellular milieu, while Salmonella establishes an intracellular niche and is able to spread systemically throughout the host. This review focuses on recent advances in our understanding of the signaling events stemming from the host-pathogen interactions specific to Salmonella and EPEC.

Interaction and cellular localization of the human host defense peptide LL-37 with lung epithelial cells.

LL-37 is a human cationic host defense peptide that is an essential component of innate immunity. In addition to its modest antimicrobial activity, LL-37 affects the gene expression and behavior of effector cells involved in the innate immune response, although its mode of interaction with eukaryotic cells remains unclear. The interaction of LL-37 with epithelial cells was characterized in tissue culture by using biotinylated LL-37 and confocal microscopy. It was demonstrated that LL-37 was actively taken up into A549 epithelial cells and eventually localized to the perinuclear region. Specific inhibitors were used to demonstrate that the uptake process was not mediated by actin but required elements normally involved in endocytosis and that trafficking to the perinuclear region was dependent on microtubules. By using nonlinear regression analysis, it was revealed that A549 epithelial cells have two receptors for LL-37B, with high and low affinity for LL-37, respectively. These results indicate the mode of interaction of LL-37 with epithelial cells and further our understanding of its role in modulating the innate immune response.

Adenovirus infection: taking the back roads to viral entry.

Certain virus receptors are sequestered on the basolateral surface of polarized epithelial cells. A recent study has shown how adenovirus--and perhaps other viruses--are able to overcome this physical barrier.

SifA, a type III secreted effector of Salmonella typhimurium, directs Salmonella-induced filament (Sif) formation along microtubules.

A unique feature of Salmonella enterica serovar typhimurium (S. typhimurium) is its ability to enter into (invade) epithelial cells and elongate the vacuole it occupies into tubular structures called Salmonella-induced filaments (Sifs). This phenotype is dependent on SifA, a Salmonella virulence factor that requires the SPI-2-encoded type III secretion system for delivery into host cells. Previous attempts to study SifA and other type III secreted proteins have been limited by a lack of suitable reagents. We examined SifA function by expressing SifA with two internal hemagglutinin epitope tags. By employing subcellular fractionation techniques, we determined that translocated SifA was membrane associated in infected HeLa cells. Confocal microscopy revealed that SifA associated with the Salmonella vacuole and with Sifs. Our analysis also revealed that microtubules serve as a scaffold for Sifs, and that SifA colocalizes with microtubules at sites of interaction between lysosomal glycoprotein-containing vesicles and Sifs. Treatment with the microtubule inhibitor nocodazole blocked Sif formation but did not prevent SifA translocation into the Salmonella vacuole. While polymerized actin has been observed on Sifs, this phenotype was transient and did not play a role in promoting or maintaining Sif formation. Our findings demonstrate the essential role of microtubule dynamics in the formation of Sifs and the utility of this epitope tagging strategy for the study of bacterial type III secreted proteins.