Assessing the long-term stability of anti-drug antibodies in method validation: what is the added value?

The stability of analytes in biological matrices is a critical parameter assessed under bioanalytical method validations. Assessing the stability of anti-drug antibodies (ADA) is, however, not evident given the polyclonal antibody response and the practicality of obtaining reference material from human subjects. Moreover, the same argument that different test articles exhibit different stabilities does not translate to polyclonal antibodies, for which there is a body of literature supporting the stability of human antibodies in undiluted human biological matrix for several years. Herein, the current recommendations from industry leaders and health authorities is summarized, and the literature supporting the stability of ADAs is described to provide a consolidated reference for bioanalytical scientists submitting ADA method validations to regulatory agencies.

Gold nanoparticles induce nuclear damage in breast cancer cells, which is further amplified by hyperthermia.

Gold nanoparticles have emerged as promising tools for cancer research and therapy, where they can promote thermal killing. The molecular mechanisms underlying these events are not fully understood. The geometry and size of gold nanoparticles can determine the severity of cellular damage. Therefore, small and big gold nanospheres as well as gold nanoflowers were evaluated side-by-side. To obtain quantitative data at the subcellular and molecular level, we assessed how gold nanoparticles, either alone or in combination with mild hyperthermia, altered the physiology of cultured human breast cancer cells. Our analyses focused on the nucleus, because this organelle is essential for cell survival. We showed that all the examined gold nanoparticles associated with nuclei. However, their biological effects were quantitatively different. Thus, depending on the shape and size, gold nanoparticles changed multiple nuclear parameters. They redistributed stress-sensitive regulators of nuclear biology, altered the nuclear morphology, reorganized nuclear laminae and envelopes, and inhibited nucleolar functions. In particular, gold nanoparticles reduced the de novo biosynthesis of RNA in nucleoli, the subnuclear compartments that produce ribosomes. While small gold nanospheres and nanoflowers, but not big gold nanospheres, damaged the nucleus at normal growth temperature, several of these defects were further exacerbated by mild hyperthermia. Taken together, the toxicity of gold nanoparticles correlated with changes in nuclear organization and function. These results emphasize that the cell nucleus is a prominent target for gold nanoparticles of different morphologies. Moreover, we demonstrated that RNA synthesis in nucleoli provides quantitative information on nuclear damage and cancer cell survival.

Modulation of inflammatory signaling and cytokine release from microglia by celastrol incorporated into dendrimer nanocarriers.

AIM: This study investigates the capacity of a potent anti-inflammatory nanomedicine, celastrol, incorporated into poly(amidoamine) dendrimers, to inhibit endotoxin-mediated signaling in microglia. MATERIALS & METHODS: Celastrol was incorporated into amino (Cel/G4-NH(2)) and hydroxyl (Cel/G4-OH) terminus poly(amidoamine) (G4) dendrimers. Cell viability, release of nitric oxide, IL-6, TNF-α and activation of MAPK (e.g., p38 and JNK) and NF-κB were assessed in endotoxin (i.e., lipopolysaccharide) stimulated microglial cells. RESULTS: G4-OH and G4-NH(2) increased celastrol aqueous solubility by seven- and 12-fold, respectively. G4-OH and Cel/G4-OH suppressed lipopolysaccharide-mediated release of proinflammatory mediators, such as nitric oxide and IL-6, but not TNF-α, without reducing microglial cell viability, while Cel/G4-NH(2) potentiated cytotoxicity and cytokine release. Blockade of proinflammatory signaling was accompanied by attenuation of p38 MAPK activation. CONCLUSION: This study supports the potential use of poly(amidoamine) dendrimers for effective anti-inflammatory therapy in the chronically inflamed CNS.

A novel mouse model of Alzheimer's disease with chronic estrogen deficiency leads to glial cell activation and hypertrophy.

The role of estrogens in Alzheimer's disease (AD) involving ß-amyloid (Aß) generation and plaque formation was mostly tested in ovariectomized mice with or without APP mutations. The aim of the present study was to explore the abnormalities of neural cells in a novel mouse model of AD with chronic estrogen deficiency. These chimeric mice exhibit a total FSH-R knockout (FORKO) and carry two transgenes, one expressing the ß-amyloid precursor protein (APPsw, Swedish mutation) and the other expressing presenilin-1 lacking exon 9 (PS1Δ9). The most prominent changes in the cerebral cortex and hippocampus of these hypoestrogenic mice were marked hypertrophy of both cortical neurons and astrocytes and an increased number of activated microglia. There were no significant differences in the number of Aß plaques although they appeared less compacted and larger than those in APPsw/PS1Δ9 control mice. Similar glia abnormalities were obtained in wild-type primary cortical neural cultures treated with letrozole, an aromatase inhibitor. The concordance of results from APPsw/PS1Δ9 mice with or without FSH-R deletion and those with letrozole treatment in vitro (with and without Aß treatment) of primary cortical/hippocampal cultures suggests the usefulness of these models to explore molecular mechanisms involved in microglia and astrocyte activation in hypoestrogenic states in the central nervous system.

Microglial response to gold nanoparticles.

Given the emergence of nanotherapeutics and nanodiagnostics as key tools in today's medicine, it has become of critical importance to define precisely the interactions of nanomaterials with biological systems and to characterize the resulting cellular response. We report here the interactions of microglia and neurons with gold nanoparticles (GNPs) of three morphologies, spheres, rods, and urchins, coated with poly(ethylene glycol) (PEG) or cetyl trimethylammonium bromide (CTAB). Microglia are the resident immune cells of the brain, primarily involved in surveillance, macrophagy, and production of cytokines and trophic factors. Analysis by dark-field microscopy and by two-photon-induced luminescence (TPL) indicates that the exposure of neural cells to GNPs resulted in (i) GNP internalization by both microglial cells and primary hippocampal neurons, as revealed by dark-field microscopy and by two-photon-induced luminescence (TPL), (ii) transient toll-like receptor 2 (TLR-2) up-regulation in the olfactory bulb, after intranasal administration in transgenic mice, in vivo, in real time, and (iii) differential up-regulation in vitro of TLR-2 together with interleukin 1 alpha (IL-1alpha), granulocyte macrophage colony-stimulating factor (GM-CSF) and nitric oxide (NO) in microglia. The study demonstrates that GNP morphology and surface chemistry strongly influence the microglial activation status and suggests that interactions between GNPs and microglia can be differentially regulated by tuning GNP nanogeometry.

The binding of pullulan modified cholesteryl nanogels to Abeta oligomers and their suppression of cytotoxicity.

Among various hydrogels able to form monodisperse and stable nanoparticles (20-30 nm) are those with pullulan-bearing cholesteryl moieties (CHP). These nanoparticles can interact with soluble proteins through hydrophobic bonding. The objectives of this study were to investigate whether CHP nanogels would interact with oligomeric forms of the 42 amino acid variant of beta-amyloid (Abeta(1-42)) and if the formation of CHP-Abeta(1-42) oligomer entities will reduce cytotoxicity of Abeta(1-42) in primary cortical cells and microglial (N9) cells. By employing fluorescent CHP analogs with different charges we provide evidence that, (i) both neutral and positively charged CHP nanoparticles interact with Abeta(1-42) monomers and oligomers, (ii) neutral CHP is non-toxic, but positively charged derivatives (CHPNH2) are toxic, particularly in primary cortical cultures, and (iii) binding of both monomeric and oligomeric Abeta(1-42) to CHP significantly reduces Abeta(1-42) toxicity in both the primary cortical and microglial cells. These results suggest that CHP nanogels could provide a valid complementary approach to antibody immunotherapy in neurological disorders characterized by the formation of soluble toxic aggregates, such as those in Alzheimer's disease (AD).

Molecular imaging of lipid peroxyl radicals in living cells with a BODIPY-alpha-tocopherol adduct.

An increasing number of reports discuss the role reactive oxygen species (ROS) have in cellular pathologies and cellular signaling processes. Critical to elucidating the underlying chemical mechanism behind these biological processes is the development of novel sensors and reporters with chemical sensitivity and, more importantly, molecular specificity, enabling the spatial and temporal monitoring of a specific ROS concentration in live cells. Here we report for the first time on the application of BODIPY-alpha-Tocopherol adduct (B-TOH), a novel lipophilic fluorescent antioxidant indicator, toward detection of peroxyl radicals in model lipid membranes and their imaging in the lipid membrane of live cells. Studies conducted in model lipid membranes show a 5-fold fluorescence enhancement upon reaction of liposome-embedded B-TOH with peroxyl radicals. The enhancement is independent of the solution pH and membrane composition. In studies in live cells performed under states of growth factor withdrawal and increased oxidative stress, a significant increase in B-TOH emission was also observed. Exogenous sources of free radicals were utilized herein, namely, N,N'-dimethyl-4,4'-bipyridinium dichloride (also known as methyl viologen or paraquat) and uncoated nonemissive CdTe nanoparticles, a source of Cd(2+). The recorded fluorescence intensity of B-TOH was proportional to the concentration of the dye and to the level of cellular oxidative stress. By employing fluorescent dyes such as Lysotracker and Nile Red, we demonstrate the formation of peroxyl radicals in subcellular locations in rat pheochromocytoma (PC12 cells) and in primary mouse hippocampal neural cells under oxidative stress conditions. Specifically, we observed peroxyl radicals in lysosomes. The assessment of the subcellular distribution of B-TOH in living cells deprived from growth factors and/or under oxidative stress may be useful in the future in determining subcellular sites of lipid peroxidation. In summary, results from this study underscore the potential of B-TOH as a sensitive and specific probe enabling the molecular imaging of peroxyl radicals in the lipid membranes of live cells.