A Size-Exclusion Chromatography Method for Analysis of Clostridium difficile Vaccine Toxins.

High-performance size-exclusion chromatography (HPSEC or SEC) is a method that can be applied to measure size distribution of proteins, including aggregates, monomers, and fragments. In the biopharmaceutical industry the quantitation of aggregates contained in biotherapeutics and protein-based vaccines is critical given the potential impact on safety, immunogenicity, and efficacy. Hence, aggregation analysis of therapeutic proteins or protein-based vaccine products is almost always a requirement of regulatory agencies. SEC, also referred to as gel-filtration chromatography, separates molecules by size through a porous resin stationary phase. Under isocratic flow small molecules are retained on the column longer than large molecules. Here we describe the use of this SEC technique to characterize aggregation levels for four different protein antigens for a Clostridium difficile vaccine.

Neuronal PINCH is regulated by TNF-α and is required for neurite extension.

During HIV infection of the CNS, neurons are damaged by viral proteins, such as Tat and gp120, or by inflammatory factors, such as TNF-α, that are released from infected and/or activated glial cells. Host responses to this damage may include the induction of survival or repair mechanisms. In this context, previous studies report robust expression of a protein called particularly interesting new cysteine histidine-rich protein (PINCH), in the neurons of HIV patients' brains, compared with nearly undetectable levels in HIV-negative individuals (Rearden et al., J Neurosci Res 86:2535-2542, 2008), suggesting PINCH's involvement in neuronal signaling during HIV infection of the brain. To address potential triggers for PINCH induction in HIV patients' brains, an in vitro system mimicking some aspects of HIV infection of the CNS was utilized. We investigated neuronal PINCH expression, subcellular distribution, and biological consequences of PINCH sequestration upon challenge with Tat, gp120, and TNF-α. Our results indicate that in neurons, TNF-α stimulation increases PINCH expression and changes its subcellular localization. Furthermore, PINCH mobility is required to maintain neurite extension upon challenge with TNF-α. PINCH may function as a neuron-specific host-mediated response to challenge by HIV-related factors in the CNS.

Estrogen receptor beta-mediated nuclear interaction between IRS-1 and Rad51 inhibits homologous recombination directed DNA repair in medulloblastoma.

In medulloblastomas, which are highly malignant cerebellar tumors of the childhood genotoxic treatments such as cisplatin or gamma-irradiation are frequently associated with DNA damage, which often associates with unfaithful DNA repair, selection of new adaptations and possibly tumor recurrences. Therefore, better understanding of molecular mechanisms which control DNA repair fidelity upon DNA damage is a critical task. Here we demonstrate for the first time that estrogen receptor beta (ERbeta) can contribute to the development of genomic instability in medulloblastomas. Specifically, ERbeta was found highly expressed and active in mouse and human medulloblastoma cell lines. Nuclear ERbeta was also present in human medulloblastoma clinical samples. Expression of ERbeta coincided with nuclear translocation of insulin receptor substrate 1 (IRS-1), which was previously reported to interfere with the faithful component of DNA repair when translocated to the nucleus. We demonstrated that ERbeta and IRS-1 bind each other, and the interaction involves C-terminal domain of IRS-1 (aa 931-1233). Following cisplatin-induced DNA damage, nuclear IRS-1 localized at the sites of damaged DNA, and interacted with Rad51--an enzymatic component of homologous recombination directed DNA repair (HRR). In medulloblastoma cells, engineered to express HRR-DNA reporter plasmid, ER antagonist, ICI 182,780, or IRS mutant (931-1233) significantly increased DNA repair fidelity. These data strongly suggest that both molecular and pharmacological interventions are capable of preventing ERbeta-mediated IRS-1 nuclear translocation, which in turn improves DNA repair fidelity and possibly counteracts accumulation of malignant mutations in actively growing medulloblastomas.

Activation of PPARalpha inhibits IGF-I-mediated growth and survival responses in medulloblastoma cell lines.

Recent studies suggest a potential role of lipid lowering drugs, fibrates and statins, in anticancer treatment. One candidate for tumor chemoprevention is fenofibrate, which is a potent agonist of peroxisome proliferator activated receptor alpha (PPARalpha). Our results demonstrate elevated expression of PPARalpha in the nuclei of neoplatic cells in 12 out of 13 cases of medulloblastoma, and of PPARgamma in six out of 13 cases. Further analysis demonstrated that aggressive mouse medulloblastoma cells, BsB8, express PPARalpha in the absence PPARgamma, and human medulloblastoma cells, D384 and Daoy, express both PPARalpha and PPARgamma. Mouse and human cells responded to fenofibrate by a significant increase of PPAR-mediated transcriptional activity, and by a gradual accumulation of cells in G1 and G2/M phase of the cell cycle, leading to the inhibition of cell proliferation and elevated apoptosis. Preincubation of BsB8 cells with fenofibrate attenuated IGF-I-induced IRS-1, Akt, ERKs and GSK3beta phosphorylation, and inhibited clonogenic growth. In Daoy and D384 cells, fenofibrate also inhibited IGF-I-mediated growth responses, and simultaneous delivery of fenofibrate with low dose of the IGF-IR inhibitor, NVP-AEW541, completely abolished their clonogenic growth and survival. These results indicate a strong supportive role of fenofibrate in chemoprevention against IGF-I-induced growth responses in medulloblastoma.