Histone deacetylase inhibitors induce reactivation of herpes simplex virus type 1 in a latency-associated transcript-independent manner in neuronal cells.

Histone acetylation is implicated in the regulation of herpes simplex virus type 1 (HSV-1) latency. However, the role of histone acetylation in HSV-1 reactivation is less clear. In this study, the well-established model system, quiescently infected, neuronally differentiated PC12 (QIF-PC12) cells, was used to address the participation of histone acetylation in HSV-1 reactivation. In this model, sodium butyrate and trichostatin A (TSA), two histone deacetylase inhibitors, stimulated production of infectious HSV-1 progeny from a quiescent state. To identify viral genes responsive to TSA, the authors analyzed representative alpha, beta, and gamma viral genes using quantitative real-time polymerase chain reaction. Only the latency-associated transcript (LAT) accumulated in response to TSA treatment, under culture conditions that restricted virus replication and spread. This led the authors to evaluate the importance of LAT expression on TSA-induced reactivation. In QIF-PC12 cells, the LAT deletion mutant virus dLAT2903 reactivated equivalently with its wild-type parental strain (McKrae) after TSA treatment, as well as forskolin and heat stress treatment. Both viruses also reactivated equivalently from latently infected trigeminal ganglia explants from rabbits. In contrast, there was a marked reduction in the recovery of dLAT2903, as compared to wild-type virus, from the eyes of latently infected rabbits following epinephrine iontophoresis. These combined in vitro, ex vivo, and in vivo data suggest that LAT is not required for reactivation from latently infected neuronal cells per se, but may enhance processes that allow for the arrival of virus at, or close to, the site of original inoculation (i.e., recrudescence).

Multiple astrocyte responses to lysophosphatidic acids.

Lysophosphatidic acid (LPA) and LPA receptors are enriched in the brain. Moreover, the levels of these receptors and ligand are modulated during brain development and injury, respectively, suggesting multiple roles for LPA in the brain. In cultured astrocytes and glioma-derived cells, LPA increases intracellular calcium concentrations and causes morphological changes. LPA also induces glioma cell migration. In normal astrocytes, LPA stimulates reactive oxygen species synthesis, activation of multiple protein kinases and expression of c-fos and c-jun. It is noteworthy that LPA-induced astrocyte responses vary as a function of the specific brain region of origin of the astrocytes. This may be one factor in the finding of LPA-stimulated proliferation in some, but not all, astrocyte studies. The species and/or developmental stage also differed in many of the astrocyte proliferation analyses. Micromolar LPA is required to elicit some astrocyte responses, including the stimulation of cytokine expression and inhibition of glutamate uptake. These events could significantly impact on survival of injured neurons and micromolar LPA concentrations are likely in diverse brain pathologies. There are important aspects of astrocyte LPA responses still to be fully evaluated, including functions in development and activation, synergy between LPA and other biomediators, and astrocyte interactions with other cells.

Poly(ethylene glycol) (PEG) conjugated arginine deiminase: effects of PEG formulations on its pharmacological properties.

Some tumors, such as melanomas and hepatocellular carcinomas, have a unique nutritional requirement for arginine. Thus, enzymatic degradation of extracellular arginine is one possible means for inhibiting these tumors. Arginine deiminase is an arginine degrading enzyme (ADI) that has been studied as an anti-cancer enzyme. However, ADI has a short serum half-life and, as a microbial enzyme, is highly immunogenic. Formulation of other therapeutic proteins with poly(ethylene glycol) (PEG) has overcome these problems. Here, ADI-PEGs were synthesized using PEGs of varying size, structure (linear or branched chain) and linker chemistries. All ADI-PEGs retained approximately 50% of enzyme activity when PEG was covalently attached to approximately 40% of the primary amines irrespective of the PEG molecular weight or attachment chemistry used. However, it was observed that, as the PEG size increases to 20 kDa, there was a corresponding increase in the pharmacokinetic (pK) and pharmacodynamic (pD) properties of the formulation. Variation in PEG linker or structure, or the use of PEGs >20,000 mw, did not affect the pK or pD. As has been shown with other therapeutic proteins, repeated injection of ADI-PEG into experimental animals resulted in significantly lower titers of antibodies against this protein than unmodified ADI. These data suggest that formulation of ADI with PEG of 20,000 mw results is the optimal method for formulating this promising therapeutic agent.