Differences in vector-genome processing and illegitimate integration of non-integrating lentiviral vectors.

A variety of mutations in lentiviral vector expression systems have been shown to generate a non-integrating phenotype. We studied a novel 12 base-pair U3-long terminal repeats (LTR) integrase (IN) attachment site deletion (U3-LTR att site) mutant and found similar physical titers to the previously reported IN catalytic core mutant IN/D116N. Both mutations led to a greater than two log reduction in vector integration; with IN/D116N providing lower illegitimate integration frequency, whereas the U3-LTR att site mutant provided a higher level of transgene expression. The improved expression of the U3-LTR att site mutant could not be explained solely based on an observed modest increase in integration frequency. In evaluating processing, we noted significant differences in unintegrated vector forms, with the U3-LTR att site mutant leading to a predominance of 1-LTR circles. The mutations also differed in the manner of illegitimate integration. The U3-LTR att site mutant vector demonstrated IN-mediated integration at the intact U5-LTR att site and non-IN-mediated integration at the mutated U3-LTR att site. Finally, we combined a variety of mutations and modifications and assessed transgene expression and integration frequency to show that combining modifications can improve the potential clinical utility of non-integrating lentiviral vectors.

Electromagnetic field effects: changes in protein phosphorylation in the Jurkat E6.1 cell line.

This study is aimed at expanding the role of electromagnetic field (EMF) therapy for treatment of inflammatory diseases and obtaining new information on the biophysical mechanism of action of weak EMFs. The mechanism of action of EMFs on biological systems is a question that has yet to be answered. Several models have been proposed to explain the coupling of low frequency fields to biological systems, although no consensus has been reached as to which most adequately portrays the true mechanism. Protein phosphorylation is a major cellular metabolic regulator. As such, it has the potential to be a valuable indicator of the impact of EMFs on cellular metabolism. Using a well-controlled EMF exposure system, we examined the regulatory role of EMFs on low molecular weight protein phosphorylation in Jurkat E6.1 cells, a transformed human leukemic T cell line. Jurkat cells were grown to mid-log phase, preloaded with 32P and exposed to EMF (0.1 mT, 60 Hz) or sham for 30 minutes. Cell proteins were separated by SDS-polyacrylamide gel electrophoresis and incorporated radioactivity of low molecular weight proteins (18-23 kDa) was quantified by AMBIS data analysis. Three of five experiments showed no difference in protein phosphorylation in EMF exposed samples compared to controls, while two experiments revealed an EMF effect. We identified stathmin, an important T cell signaling phosphoprotein, as one of the low molecular weight proteins present in our Jurkat cell system. Stathmin expression as well as its phosphorylation was decreased in samples that were exposed to EMFs compared to controls. These data indicate that phosphorylation of individual proteins might be masked by the presence of numerous other proteins in whole cell lysate experiments. Further studies testing other low molecular weight T cell signaling molecules may validate this hypothesis.

Effect of a wound healing electromagnetic field on inflammatory cytokine gene expression in rats.

In earlier studies, we have shown that pulsed electromagnetic fields (PEMFs) induce programmed cell death in cultured T cells and that rats exposed in vivo to PEMFs have decreased T-cell proliferative capacity. These data led us to hypothesize that PEMFs might be used to control proliferation of inflammatory lymphocytes and therefore beneficially affect inflammatory diseases. Tendinitis is characterized by painful inflammation of the tendon. Inflammation is characterized by massive infiltration of T lymphocytes, neutrophils and macrophages into the damaged tissue. These inflammatory cells produce a variety of cytokines, which are the cellular regulators of inflammation. The current study tests whether in vivo PEMF effects are mediated via systemic cytokine production in rat tendinitis. Inflammation was chemically induced in female Harlan Sprague Dawley rats Achilles' tendons and a wound healing PEMF (Electrobiology, Inc.) was applied for 4 hours immediately following injury. Spleens from control and experimental animals were harvested 24 hours later and total RNA was extracted from the tissues. Gene expression was analyzed by reverse transcription of mRNA, and polymerase chain reaction amplification (RT-PCR) using primers specific for the cytokines IFN-gamma, IL-1 beta, IL-6, TNF-alpha, and TGF-beta, as well as for the control beta-actin. RT-PCR products were separated on 1.5% agarose gels and band intensities were normalized to beta-actin gene expression of the same sample. TGF-beta was the only cytokine produced at high levels in rats with tendinitis in comparison to the other cytokines. PEMFs did not show an effect on any cytokine expression in the spleens, 24 hours after induction of tendinitis. Further studies need to test if cumulative exposures of PEMFs are able to regulate inflammatory cytokine expression either at the site of inflammation or at the local lymph nodes.