Impact of cell culture process changes on endogenous retrovirus expression

Cell culture process changes (e.g., changes in scale, medium formulation, operational conditions) and cell line changes are common during the development life cycle of a therapeutic protein. To ensure that the impact of such process changes on product quality and safety is minimal, it is standard practice to compare critical product quality and safety attributes before and after the changes. One potential concern introduced by cell culture process improvements is the possibility of increased endogenous retrovirus expression to a level above the clearance capability of the subsequent purification process. To address this, retrovirus expression was measured in scaled down and full production scaled Chinese hamster ovary (CHO) cell cultures of four monoclonal antibodies and one recombinant protein before and after process changes. Two highly sensitive, quantitative (Q)-PCR-based assays were used to measure endogenous retroviruses. It is shown that cell culture process changes that primarily alter media components, nutrient feed volume, seed density, cell bank source (i.e., master cell bank vs. working cell bank), and vial size, or culture scale, singly or in combination, do not impact the rate of retrovirus expression to an extent greater than the variability of the Q-PCR assays (0.2-0.5 log(10)). Cell culture changes that significantly alter the metabolic state of the cells and/or rates of protein expression (e.g., pH and temperature shifts, NaButyrate addition) measurably impact the rate of retrovirus synthesis (up to 2 log(10)). The greatest degree of variation in endogenous retrovirus expression was observed between individual cell lines (up to 3 log(10)). These data support the practice of measuring endogenous retrovirus output for each new cell line introduced into manufacturing or after process changes that significantly increase product-specific productivity or alter the metabolic state, but suggest that reassessment of retrovirus expression after other process changes may be unnecessary.     

The effects of centrally administered apelin-13 on food intake, water intake and pituitary hormone release in rats

Apelin is the recently identified endogenous ligand for the G-protein-coupled receptor, APJ. Preproapelin and APJ mRNA are found in hypothalamic regions known to be important in the regulation of food and water intake, and pituitary hormone release. The effects of intracerebroventricular (ICV) administration of pyroglutamylated apelin-13 on food and water intake and pituitary hormone release in rats were investigated. Apelin-13 had little effect on food intake, but dose-dependently increased drinking behaviour and water intake at 1 h. Apelin-13 (10 nmol) increased water intake by up to sixfold compared to saline. Compared to saline control, apelin-13 (10 nmol) significantly increased plasma ACTH and corticosterone and decreased plasma prolactin, LH and FSH at 30 min. In vitro, apelin-13 stimulated the release of CRH and AVP from hypothalamic explants, but had no effect on NPY release. These results suggest that apelin may play an important role in the hypothalamic regulation of water intake and endocrine axes.     

A Trypanosoma brucei protein complex that binds G-overhangs and co-purifies with telomerase activity.

The chromosomal ends of Trypanosoma brucei, like those of most eukaryotes, contain conserved 5'-TTAGGG-3' repeated sequences and are maintained by the action of telomerase. Fractionated T. brucei cell extracts with telomerase activity were used as a source of potential regulatory factors or telomerase-associated components that might interact with T. brucei telomeres. Electrophoretic mobility shift assays and UV cross-linking were used to detect possible single-stranded telomeric protein.DNA complexes and to estimate the approximate size of the protein constituents. Three single-stranded telomeric protein.DNA complexes were observed. Complex C3 was highly specific for the G-strand telomeric repeat sequence and shares biochemical characteristics with G-rich, single-stranded telomeric binding proteins and with components of the telomerase holoenzyme described in yeast, ciliates, and humans. Susceptibility to RNase A or chemical nuclease (hydroxyl radical) pre-treatment showed that complex C3 was tightly associated with an RNA component. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to estimate the molecular mass of the peptides obtained by in-gel Lys-C digestion of low abundance C3-associated proteins. The molecular masses of the peptides showed no homologies with other proteins from trypanosomes or with any protein in the data bases screened.     

The Effects of Overfeeding on Spontaneous Physical Activity in Obesity Prone and Obesity Resistant Humans

Despite living in an environment that promotes weight gain in many individuals, some individuals maintain a thin phenotype while self‐reporting expending little or no effort to control their weight. When compared with obesity prone (OP) individuals, we wondered if obesity resistant (OR) individuals would have higher levels of spontaneous physical activity (SPA) or respond to short‐term overfeeding by increasing their level of SPA in a manner that could potentially limit future weight gain. SPA was measured in 55 subjects (23 OP and 32 OR) using a novel physical activity monitoring system (PAMS) that measured body position and movement while subjects were awake for 6 days, either in a controlled eucaloric condition or during 3 days of overfeeding (1.4× basal energy) and for the subsequent 3 days (ad libitum recovery period). Pedometers were also used before and during use of the PAMS to provide an independent measure of SPA. SPA was quantified by the PAMS as fraction of recording time spent lying, sitting, or in an upright posture. Accelerometry, measured while subjects were in an upright posture, was used to categorize time spent in different levels of movement (standing, walking slowly, quickly, etc.). There were no differences in SPA between groups when examined across all study periods (P > 0.05). However, 3 days following overfeeding, OP subjects significantly decreased the amount of time they spent walking (?2.0% of time, P = 0.03), whereas OR subjects maintained their walking (+0.2%, P > 0.05). The principle findings of this study are that increased levels of SPA either during eucaloric feeding or following short term overfeeding likely do not significantly contribute to obesity resistance although a decrease in SPA following overfeeding may contribute to future weight gain in individuals prone to obesity.     

Dendritic Cells Control CD4+CD25+ Treg Cell Suppressor Function In Vitro through Juxtacrine Delivery of IL-2

CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) restrict inflammatory responses to self and nonself. Aberrant Treg activity is pathologic: Insufficient Treg activity is implicated in autoimmunity, allergy, and graft-versus-host-disease; overabundant activity is implicated in chronic infection and cancer. Tregs require IL-2 for their expansion and acquisition/execution of suppressor function; however, because Tregs cannot produce IL-2, they depend on IL-2 from an exogenous source. Until now, that IL-2 source had not been established. We asked whether dendritic cells (DCs) could supply IL-2 to Tregs and, if so, what was required for that delivery. We used flow cytometry, IL-2 ELISPOT, RT-qPCR, and IL-2 promoter-driven reporter assays to measure intracytoplasmic IL-2, secreted protein, IL-2 message and IL-2 promoter activity in bone marrow-derived (BMDC) and splenic DCs. We examined conjugate formation between Tregs, conventional CD4⁺ cells, and IL-2-expressing DCs. We measured Treg levels of CD25, Foxp3, and suppressor function after co-culture with IL-2 sufficient and IL-2^−/− DCs. We generated IL-2-mCherry-expressing DCs and used epifluorescence microscopy and flow cytometry to track IL-2 transfer to Tregs and test requirements for transfer. Between 0.7 to 2.4% of DCs constitutively produced IL-2 and diverted IL-2 secretion to Tregs by preferentially forming conjugates with them. Uptake of DC IL-2 by Tregs required cell-cell contact and CD25. Tregs increased levels of CD25 and Foxp3 from baseline and showed greater suppressor function when co-cultured with IL-2-sufficient DCs, but not when co-cultured with IL-2^−/− DCs. Exogenous IL-2, added in excess of 500 U/ml to co-cultures with IL-2^−/− DCs, restored Treg suppressor function. These data support a model of juxtacrine delivery of IL-2 from DCs to Tregs and suggest that a subset of DCs modulates Treg function through controlled, spatial delivery of IL-2. Knowledge of how DCs regulate Tregs should be integrated into the design of interventions intended to alter Treg function.     

Overexpression of A(3) adenosine receptors decreases heart rate,preserves energetics,and protects ischemic hearts

To determine whether A(3) adenosine receptor (A(3)AR) signaling modulates myocardial function, energetics, and cardioprotection, hearts from wild-type and A(3)AR-overexpressor mice were subjected to 20-min ischemia and 40-min reperfusion while (31)P NMR spectra were acquired. Basal heart rate and left ventricular developed pressure (LVDP) were lower in A(3)AR-overexpressor hearts than wild-type hearts. Ischemic ATP depletion was delayed and postischemic recoveries of contractile function, ATP, and phosphocreatine were greater in A(3)AR-hearts. To determine the role of depressed heart rate and to confirm A(3)AR-specific signaling, hearts were paced at 480 beats/min with or without 60 nmol/l MRS-1220 (A(3)AR-specific inhibitor) and then subjected to ischemia-reperfusion. LVDP was similar in paced A(3)AR-overexpressor and paced wild-type hearts. Differences in ischemic ATP depletion and postischemic contractile and energetic dysfunction remained in paced A(3)AR-overexpressor hearts versus paced wild-type hearts but were abolished by MRS-1220. In summary, A(3)AR overexpression decreased basal heart rate and contractility, preserved ischemic ATP, and decreased postischemic dysfunction. Pacing abolished the decreased contractility but not the ATP preservation or cardioprotection. Therefore, A(3)AR overexpression results in cardioprotection via a specific A(3)AR effect, possibly involving preservation of ATP during ischemia.