Early prediction of instability of Chinese hamster ovary cell lines expressing recombinant antibodies and antibody-fusion proteins.

One of the most important criteria for the successful manufacture of a therapeutic protein (e.g., an antibody) is to develop a mammalian cell line that maintains stability of production. Problems with process yield, lack of effective use of costly resources, and a possible delay in obtaining regulatory approval of the product may ensue otherwise. Therefore the stability of expression in a number of Chinese hamster ovary (CHO) derived production cell lines that were isolated using the glutamine synthetase (GS) selection system was investigated by defining a culture as unstable if the titer (which is a measure of productivity) of a cell line expressing an antibody or antibody-fusion protein declined by 20-30% or more as it underwent 55 population doublings. Using this criterion, a significant proportion of the GS-selected CHO production cell lines were observed to be unstable. Reduced antibody titers correlated with the gradual appearance of a secondary, less productive population of cells as detected with flow cytometric analysis of intracellular antibody content. Where tested, it was observed that the secondary population arose spontaneously from the parental population following multiple passages, which suggested inherent clonal instability. Moreover, the frequency of unstable clones decreased significantly if the host cell line from which the candidate production cell lines were derived was apoptotic-resistant. This data suggested that unstable cell lines were more prone to apoptosis, which was confirmed by the fact that unstable cell lines had higher levels of Annexin V and caspase 3 activities. This knowledge has been used to develop screening protocols that identify unstable CHO production cell lines at an early stage of the cell line development process, potentially reducing the cost of biotherapeutic development.

Respiratory issues in beef and pork production: recommendations from an expert panel.

This paper summarizes "Respiratory Issues in Confined Feeding Operations," a panel discussion at the Agricultural Safety and Health Council of America/National Institute for Occupational Safety and Health conference, "Be Safe, Be Profitable: Protecting Workers in Agriculture," Dallas/Fort Worth, Texas, January 27-28, 2010. Occupational exposure to confined animal feeding operations is associated with cough, wheezing, and shortness of breath. Published data shows that 20% to 40% of hog confinement workers experience such symptoms, although most are able to continue working in this industry. Endotoxin is one component of hog barn dust that is associated with respiratory disease in workers. Endotoxin levels on cattle feedlots can also be in the range linked with occupational lung disease. The cattle industry has not yet prepared guidance documents for producers, in part because much less is known about the prevalence of lung disease in its workers. However, the pork industry provides information for pork producers on reducing their respiratory health risks through a multifaceted approach, including the use of respirators. Some jobs cannot be done safely without respiratory protection, such as entering manure pits. It is less clear for other jobs when respirators should be worn. Use of respiratory protection should be considered but not mandated for all persons working in close proximity to livestock in dusty conditions. A respiratory protection program may also serve as a cost effective biosecurity measure to protect animals from human pathogens such as influenza virus. Proper design and management of barn ventilation systems is critical for maintaining temperature and humidity levels for optimal animal growth; as well as decreasing the level of gases and respirable dusts. The pork and the cattle industries support occupational health and safety; however, the governmental guidance and recommendations for such programs are limited for the agricultural industries as a whole. The industries should lead the way in the effort to improve respiratory protection for workers. Overall, a team approach that includes input from managers, workers, and veterinarians is important for the reduction of respiratory hazards on livestock farms.

Expression of anti-apoptosis genes alters lactate metabolism of Chinese Hamster Ovary cells in culture.

In an effort to develop robust Chinese Hamster Ovary host cell lines, a variety of anti-apoptotic genes were over-expressed, either singly or in combination, followed by screening of transfectants for improved cell growth, extended longevity, reduced caspase 3/7 activity, and enhanced mitochondrial membrane potential (MMP). Two particular cell lines, one containing two anti-apoptotic genes, E1B-19K and Aven (EA167), and another containing three, E1B-19K, Aven, and a mutant of XIAP (EAX197), exhibited a reduction in caspase 3 activity of at least 60% and a 170% enhancement in mitochondrial membrane potential compared to controls when treated with staurosporine. In batch cell growth experiments, the peak viable cell densities and viabilities were higher resulting in a 186% increase in integrated viable cell densities. Analyses of metabolite utilization and formation of waste products indicated that the apoptotic resistant cell lines depleted all the lactate when grown in commercially available CD-CHO medium while significant levels (>1.8 g/L) accumulated in the host cell lines. When the lactate level was replenished daily in the apoptotic resistant cell lines, the cell lines consumed lactate and the culture longevity was extended up to four additional days compared to control cell lines. Furthermore, the anti-apoptosis cell lines also accumulated lower levels of ammonia. The ability of the apoptotic resistant cell lines to consume lactate was exploited by cultivating them in a "high" glucose medium containing 15 g/L (60 mM glucose) in which apoptotic resistant cell lines exhibited lower maximum lactate (1.8 g/L) compared to control cell lines which accumulated concentrations of lactate (2.2 g/L) that appeared to be deleterious for growth. The shaker flask titer of a therapeutic antibody product expressed in an apoptotic resistant cell line in "high" glucose medium reached 690 mg/L compared to 390 mg/L for a cell line derived from a control host cell line. These results represent to our knowledge the first example in the literature in which manipulation of the apoptosis pathway has altered the nutrient consumption profile of mammalian cells in culture; findings that underscore the interdependence of the apoptotic cellular machinery and metabolism and provide greater flexibility to mammalian bioreactor process development.

Development of mammalian production cell lines expressing CNTO736, a glucagon like peptide-1-MIMETIBODY: factors that influence productivity and product quality.

In an attempt to develop a high producing mammalian cell line expressing CNTO736, a Glucagon like peptide-1-antibody fusion protein (also known as a Glucagon like peptide-1 MIMETIBODY), we have noted that the N-terminal GLP-1 portion of the MIMETIBODY was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. Therefore, a number of parameters that had an effect on productivity as well as product quality were examined. Results suggest that the choice of the host cell line had a significant effect on the overall product quality. Product expressed in mouse myeloma host cell lines had a lesser degree of proteolytic degradation and variability in O-linked glycosylation as compared to that expressed in CHO host cell lines. The choice of a specific CHOK1SV derived clone also had an effect on the product quality. In general, molecules that exhibited minimal N-terminal clipping had increased level of O-linked glycosylation in the linker region, giving credence to the hypothesis that O-linked glycosylation acts to protect against proteolytic degradation. Moreover, products with reduced potential for N-terminal clipping had longer in vivo serum half-life. These findings suggest that early monitoring of product quality should be an essential part of production cell line development and therefore, has been incorporated in our process of cell line development for this class of molecules.

Transforming care teams to provide the best possible patient-centered, collaborative care.

Patient experience of care is now a crucial parameter in assessing the quality of healthcare delivered in the United States. Continuity, patient-driven access to care, and being "known" by a provider or practice, particularly for patients with chronic diseases, have been shown to enhance patient satisfaction with care and health outcomes. Healthcare systems are challenged to effectively meet the wants and needs of patients by tailoring interventions based on each person's unique set factors-his or her strengths, preferences, and personal and social context. Creating care teams, a coordinated multidisciplinary group of healthcare professionals, enables a practice to take advantage of the skill sets represented and redesign care delivery with the patient and community as the focal point. This article describes the attributes of highly functioning care teams, how to measure them, and guidance on creating them. A case example illustrates how these ideas work in practice.

A self-instructional model to teach systems-based practice and practice-based learning and improvement.

BACKGROUND: When mandated as resident competencies in 1999, systems-based practice (SBP) and practice-based learning and improvement (PBLI) were new concepts to many. OBJECTIVE: To describe and evaluate a 4-week clinical elective (Achieving Competence Today-ACT) to teach residents SBP and PBLI. DESIGN: ACT consisted of a four-week active learning course and follow-up teaching experience, guided and supported by web-based materials. The curriculum included readings, scheduled activities, work products including an improvement project, and weekly meetings with a non-expert preceptor. The evaluation used a before-after cross-comparison of ACT residents and their peers. PARTICIPANTS: Seventy-eight residents and 42 faculty in 18 US Internal Medicine residency programs participated between 2003 and 2005. RESULTS AND MAIN MEASUREMENTS: All residents and faculty preceptors responded to a knowledge test, survey of attitudes, and self-assessment of competency to do 15 tasks related to SBP/PBLI. All measures were normalized to a 100-point scale. Each program's principal investigator (PI) identified aspects of ACT that were most and least effective in enhancing resident learning. ACT residents' gains in knowledge (4.4 on a 100-point scale) and self-assessed competency (11.3) were greater than controls' (-1.9, -8.0), but changes in attitudes were not significantly different. Faculty preceptors' knowledge scores did not change, but their attitudes became more positive (15.8). PIs found a ready-to-use curriculum effective (rated 8.5 on a 10-point scale). CONCLUSIONS: ACT increased residents' knowledge and self-assessment of their own competency and raised faculty's assessment of the importance of residents' learning SBP/PBLI. Faculty content expertise is not required for residents to learn SBP/PBLI.

Reforming the primary care physician payment system: eliminating E & M codes and creating the financial incentives for an "advanced medical home".

The problem faced by primary care physicians is that they can only maintain or increase their (inflation adjusted) incomes by increasing the volume of visits and associated services. The fundamental flaw in a fee-for-service system is that only paying for individual services creates incentives for more services. This article offers a very different approach to paying primary care physicians that will result in both significantly higher incomes for these underpaid professionals together with incentives for creating a medical home.

Educating interprofessional learners for quality, safety and systems improvement.

Most health professionals in training, as well as those in practice, lack the knowledge and skills they need to play an effective role in systems improvement. Until very recently, these competencies were not included in formal (or informal) educational curricula. Interprofessional collaboration - another core competency needed for successful systems improvement - is also inadequately taught and learned. Achieving Competence Today (ACT) was designed as a new model for interprofessional education for quality, safety and health systems improvement. The core of ACT is a four-module active learning course during which learners from different disciplines work together to develop a Quality Improvement Project to address a quality or safety problem in their own practice system. In this paper we describe the ACT program and curriculum model, discuss our strategies for maximizing ACT's interprofessional potential, and make recommendations for the future.

The role of the school nurse in special schools for pupils with severe learning difficulties.

The work of two paediatric nurses working full-time in special schools was monitored over a full school year. Most of their time was spent on routine tasks with small numbers of pupils who required enteral feeding and suctioning. They were also responsible for administering medications to around 1 in 6 of the pupils. Both nurses had an involvement in staff training and health promotion classes; more so in one school than the other. In two similar schools which did not have a nurse, the routine tasks were done mainly by teachers or assistants with support from visiting community nurses. Health promotion formed part of the school curriculum. The presence of the nurses in schools was valued by school staff, parents and other health and social care professionals. The implications of these findings are discussed in the context of the British Government's aspirations for school nurses to play a key role in reducing health inequalities.

Primary care medicine in crisis: toward reconstruction and renewal.

Primary care is in crisis. Despite its proud history and theoretical advantages, the field has failed to hold its own among medical specialties. While the rest of medicine promises technology and sophistication, the basic model of primary care has changed little over the past half-century. Why has the transition from general practice to today's primary care been so difficult? Many of the causes of this struggle may lie within primary care itself, ranging from failure to articulate to the public (and insurers and policymakers) what value it, and it alone, can offer, to taking on an ever-broadening set of roles and responsibilities while all too often falling short of its promises. Perhaps most important, in the emerging health care system, the lack of a discrete definition of primary care has allowed managed care organizations and payers, among others, to define the role of primary care to suit their own interests. In response to a changing marketplace, political uncertainty, and shifting consumer expectations, primary care will need to reconstruct itself. The reconstruction will not be easy. Nevertheless, a process should begin that moves the field in the right direction. Building on its unique abilities, primary care can emerge as a redefined product that is attractive to patients, payers, and primary care practitioners alike.

Obesity in people with intellectual disabilities: the impact of nurse-led health screenings and health promotion activities.

BACKGROUND: Obesity appears to be more common among people with intellectual disabilities, with few studies focusing on achieving weight reduction. AIM: Firstly, to follow-up people identified as overweight and obese following special health screening clinics and to determine the actions taken. Secondly, to evaluate the impact of health promotion classes on participants' weight loss. METHODS: A clinic led by two learning disability nurses was held for all people aged 10 years and over (n = 464) who attended special services within the area of one Health and Social Services Trust in Northern Ireland. In a second study, the nurses organized health promotion classes for 20 people over a 6- or 8-week period. FINDINGS: The health screen identified 64% of adults and 26% of 10-19-year olds as being overweight or obese. Moreover, those aged 40-49 years who were obese had significantly higher levels of blood pressure. However, information obtained from a follow-up questionnaire sent after 3 months suggested that of the 122 people identified for weight reduction, action had been taken for only 34% of them and only three were reported to have lost weight. The health promotion classes, however, led to a significant reduction in weight and body mass index scores. CONCLUSIONS: Health screening per se has limited impact on reducing obesity levels in this client group. Rather, health personnel such as general practitioners, nurses and health promotion staff need to work in partnership with service staff, carers and people with intellectual disabilities to create more active lifestyles.

Going solo: making the leap.

Frustrated by current practice and convinced of a better way, the author left his salaried position and opened a solo practice with no staff. Because his overhead costs are extremely low, the author is able to see fewer patients per day and create more meaningful interactions. By offering unfettered access, the author finds that his patients trust him more and actually call him less.

Metabolic Control of Avocado Fruit Growth (Isoprenoid Growth Regulators and the Reaction Catalyzed by 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase).

The effect of isoprenoid growth regulators on avocado (Persea americana Mill. cv Hass) fruit growth and mesocarp 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity was investigated during the course of fruit ontogeny. Both normal and small-fruit phenotypes were used to probe the interaction between the end products of isoprenoid biosynthesis and the activity of HMGR in the metabolic control of avocado fruit growth. Kinetic analysis of the changes in both cell number and size revealed that growth was limited by cell number in phenotypically small fruit. In small fruit a 70% reduction in microsomal HMGR activity was associated with an increased mesocarp abscisic acid (ABA) concentration. Application of mevastatin, a competitive inhibitor of HMGR, reduced the growth of normal fruit and increased mesocarp ABA concentration. These effects were reversed by co-treatment of fruit with mevalonic acid lactone, isopentenyladenine, or N-(2-chloro-4-pyridyl)-N-phenylurea, but were not significantly affected by either gibberellic acid or stigmasterol. However, stigmasterol appeared to partially restore fruit growth when co-injected with mevastatin in either phase II or III of fruit growth. In vivo application of ABA reduced fruit growth and mesocarp HMGR activity and accelerated fruit abscission, effects that were reversed by co-treatment with isopentenyladenine. Together, these observations indicate that ABA accumulation down-regulates mesocarp HMGR activity and fruit growth, and that in situ cytokinin biosynthesis modulates these effects during phase I of fruit ontogeny, whereas both cytokinins and sterols seem to perform this function during the later phases.