Polymer-mediated flocculation of transient CHO cultures as a simple, high throughput method to facilitate antibody discovery.

Most biopharmaceutical drugs, especially monoclonal antibodies (mAbs), bispecific antibodies (BsAbs) and Fc-fusion proteins, are expressed using Chinese Hamster Ovary (CHO) cell lines. CHO cells typically yield high product titers and high product quality. Unfortunately, CHO cell lines also generate high molecular weight (HMW) aggregates of the desired product during cell culture along with CHO host cell protein (HCP) and CHO DNA.

Transient and stable CHO expression, purification and characterization of novel hetero-dimeric bispecific IgG antibodies.

IgG bispecific antibodies (BsAbs) represent one of the preferred formats for bispecific antibody therapeutics due to their native-like IgG properties and their monovalent binding to each target. Most reported studies utilized transient expression in HEK293 cells to produce BsAbs. However, the expression of biotherapeutic molecules using stable CHO cell lines is commonly used for biopharmaceutical manufacturing.

Transcriptional and post-transcriptional targeting for enhanced transient gene expression in CHO cells.

Objective: To develop a simple approach to increase titers of transient gene expression in CHO cells without relying on host cell line engineering as recent reports suggest that for PEI-mediated transfections, under optimized conditions, DNA delivery into cells and nuclei is not the limiting factor.

Results: N, N-Dimethyl acetamide (DMA) was utilized to enhance transcription. To target post-transcriptional events, we evaluated the co-expression of various genes involved in the unfolded protein response, namely XBP1S, ATF4, CHOP and HSPA5.

Cariprazine exerts antimanic properties and interferes with dopamine D2 receptor β-arrestin interactions.

Activation of dopamine D2 receptors (D2R) modulates G protein/cAMP-dependent signaling and also engages Akt-GSK-3 signaling through D2R/β-arrestin 2 scaffolding of Akt and PP2A. This G protein-independent pathway may be important in mediating the antimanic effects of mood stabilizers and antipsychotics. The mood stabilizer lithium influences behavior and Akt/GSK-3 signaling in mice and many antipsychotics have been shown to more potently antagonize the activity of the β-arrestin-2 pathway relative to the G protein-dependent pathway.

A high cell density transient transfection system for therapeutic protein expression based on a CHO GS-knockout cell line: process development and product quality assessment.

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the volumetric productivity of TGE has improved significantly over the past decade, most methods involve extensive cell line engineering and plasmid vector optimization in addition to long fed batch cultures lasting up to 21 days. Our colleagues have recently reported the development of a CHO K1SV GS-KO host cell line.

High-throughput mAb expression and purification platform based on transient CHO.

A high-cell-density transient transfection system was recently developed in our laboratory based on a CHO-GS-KO cell line. This method yields monoclonal antibody titers up to 350 mg/L from a simple 7-day process, in volumes ranging from 2 mL to 2 L. By performing transfections in 24-deep-well plates, a large number of mAbs can be expressed simultaneously.

The Transcriptional Response of Neurotrophins and Their Tyrosine Kinase Receptors in Lumbar Sensorimotor Circuits to Spinal Cord Contusion is Affected by Injury Severity and Survival Time.

Traumatic spinal cord injury (SCI) results in changes to the anatomical, neurochemical, and physiological properties of cells in the central and peripheral nervous system. Neurotrophins, acting by binding to their cognate Trk receptors on target cell membranes, contribute to modulation of anatomical, neurochemical, and physiological properties of neurons in sensorimotor circuits in both the intact and injured spinal cord. Neurotrophin signaling is associated with many post-SCI changes including maladaptive plasticity leading to pain and autonomic dysreflexia, but also therapeutic approaches such as training-induced locomotor improvement.

Skin incision induces expression of axonal regeneration-related genes in adult rat spinal sensory neurons.

Unlabelled: Skin incision and nerve injury both induce painful conditions. Incisional and postsurgical pain is believed to arise primarily from inflammation of tissue and the subsequent sensitization of peripheral and central neurons. The role of axonal regeneration-related processes in development of pain has only been considered when there has been injury to the peripheral nerve itself, even though tissue damage likely induces injury of resident axons.

Effect of ouabain on sodium pump alpha-isoform expression in an animal model of mania.

While the pathophysiologic mechanisms of bipolar illness are unknown, a dysregulation of electrolytes, particularly intracellular sodium (Na) and calcium (Ca), are thought to contribute to the illness. Ouabain, a potent Na pump inhibitor, administered intracerebroventricularly (ICV), has been used previously to model mania. The current study evaluates the effect of ICV ouabain on Na pump isoform expression in rat brain.

Positron emission tomography with fluorodeoxyglucose-F18 in an animal model of mania.

Intracerebroventricular (ICV) administration of ouabain to young adult rats has been suggested to model human bipolar mania. In the human condition, mania and bipolar depression are both associated with reductions in frontal cerebral metabolism. We utilized [(18)F]-fluorodeoxyglucose [(18)FDG] positron emission tomography (PET) to visualize glucose uptake in animals receiving ICV ouabain.

Distribution of muscarinic receptors and acetylcholinesterase in the rat heart.

Experiments were performed to determine the degree of overlap in the distribution of muscarinic receptors and cholinergic innervation of the rat heart. Localization of muscarinic receptors was determined by autoradiography with [3H]quinuclidinyl benzilate. Adjacent sections were stained for acetylcholinesterase to determine innervation.

Expression of asialo GM1 on a subset of adult murine thymocytes: histological localization and demonstration that the asialo GM1-positive subset contains both the functionally mature and the proliferating thymocyte subpopulations.

A small population (10 to 14%) of adult murine thymocytes expresses the glycolipid asialo GM1 (aGM1). Flow cytometric analysis of the aGM1+ cells present in thymus demonstrates the expression of a mature or medullary phenotype by 50% of the aGM1+ cells. Analysis of cytotoxic T lymphocyte precursor activity, proliferative capacity, and IL 2 production displayed by aGM1+ and aGM1- thymocyte fractions isolated by cell sorting indicates that these functional compartments of the thymus are contained within the aGM1+ subset.

Innervation of guinea pig heart by neurons sensitive to capsaicin.

To determine the origin of non-vagal afferent fibers innervating the heart of guinea pigs, capsaicin was injected into the ventricular myocardium to induce depletion of substance P (SP). The lower cervical, upper thoracic and lumbar spinal ganglia, as well as the left atrium and base of ventricles, were assayed for SP depletion by using the enzyme-linked immunosorbent assay (ELISA) and immunohistochemical procedures. Capsaicin affected spinal ganglia from the 3 regions differently.

Evaluation of simultaneous teaching of extremities in gross anatomy program.

A gross anatomy program was designed to expose medical students to all areas of the body but shortened the dissection time on the extremities by having half the class dissect either the upper or lower extremity and then study the opposite extremity already dissected by other classmates. The program has been used for six years and was evaluated via an analysis of covariance by comparing the intramural examination performance on both the dissected and undissected extremities. There was no statistical difference in the students' performances regardless of the extremity dissected.

Human lung tryptase. Purification and characterization.

Human lung tryptase, a mast cell-derived trypsin-like serine protease, has been isolated from whole human lung tissue obtained at autopsy. Increased yields from this purification process have allowed extensive characterization of the enzyme. One of the critical steps in the purification scheme is the use of a linear heparin gradient to elute active material from cellulose phosphate.

Cross-sectional anatomy: preparation of teaching specimens.

Forty cross sections of a male cadaver were prepared for a Continuing Medical Education Seminar and have been used in teaching programs for the past 3 years. The sections are easy and inexpensive to construct and can be maintained with a minimal amount of effort.

Quantitation of sulfhydryl groups on erythrocytes in polycythemia vera.

The number of sulfhydryl groups on the surface of intact erythrocytes from patients with polycythemia vera is significantly less (34%) than those from control subjects. Sulfhydryl groups were measured indirectly by reversibly blocking -SH groups with the thiol reagent, 6,6'-dithiodinicotinic acid, which forms stable mixed disulfides on the surface of erythrocytes. Glutathione was used to break the disulfides and release thione into the supernatant.

Canine kidney cells. II. Neutral lipids, phospholipids and fatty acids of Madin-Darby canine kidney cells in suspension and monolayer culture.

Differences in lipid composition and the effects of monolayer and suspension culture were studied in Madin-Darby canine kidney cells and their plasma membranes isolated by the Warren fluorescein-mercuric acetate technique. Unesterified cholesterol was the major neutral lipid extracted from the whole cells and plasma membranes of cultures except for suspension-grown whole cells which had triglyceride as their major neutral lipid. Triglyceride increased about 7- to 20-fold and glycerol ethers increased about 3- to 20-fold in suspension grown whole cells and their plasma membranes compared to those from monolayer cultures.

Canine kidney cells: I. Neutral lipids, phospholipids and fatty acids of SV40 transformed canine kidney cells in suspension and monolayer culture.

Differences in lipid composition and the effects of monolayer and suspension culture on SV40 virus transformed canine kidney cells and their plasma membranes were studied. Plasma membranes from monolayer and suspension cultures of SV40 transformed canine kidney cells were isolated by the Warren fluorescein-mercuric acetate technique. Free cholesterol was the major neutral lipid extracted from the whole cells and plasma membranes of all these cultures.