A Role for P-Glycoprotein in Clearance of Alzheimer Amyloid ß -Peptide from the Brain.

Most data indicates that Alzheimer's disease involves an accumulation of amyloid ß - peptide (Aß) in the CNS and that sporadic cases arise from a deficiency in Aß clearance. Considerable attention has been given to mechanisms by which Aß might be transported between the brain and blood, and evidence suggests that p-glycoprotein, also known as the multi-drug resistance (MDR) protein (product of the ABCB1 gene), plays a role in Aß transport across the blood-brain barrier (BBB). We tested this possibility through two approaches: First, wild-type and MDR1A-knockout mice were compared after intravenous injection of [(125)I]-labeled Aß; after 60 min, homogenates of brain parenchyma were subjected to γ-counting of TCA-precipitable material, and histological sections of brain were subjected to autoradiography. Second, MDR1Aknockout mice were crossed with Tg2576 APP transgenic mice, a line that routinely accumulates Aß in the brain; SDS and formic acid extracts of brain homogenates were assessed for Aß levels by ELISA. Each of these approaches yielded data indicating that Aß accumulates to a greater degree in mice lacking MDR1A. These findings confirm other reports linking p-glycoprotein to Aß clearance across the BBB and have important implications for Alzheimer's disease genetics, pharmacology, and epidemiology.

Effects of administration of two growth hormone-releasing hormone plasmids to gilts on sow and litter performance for the subsequent three gestations.

OBJECTIVE: To determine whether a novel optimized plasmid carrying the porcine growth hormone-releasing hormone (GHRH) wild-type cDNA administered at a lower dose was as effective at eliciting physiologic responses as a commercial GHRH plasmid approved for use in Australia. ANIMALS: 134 gilts. PROCEDURES: Estrus was synchronized and gilts were bred. Pregnant gilts were assigned to 2 treatment groups (40 gilts/group) or 1 untreated control group (24 gilts). Gilts in one of the treatment groups received the commercial GHRH plasmid, whereas gilts in the other treatment group received a novel optimized GHRH plasmid; both plasmids were administered IM in the right hind limb, which was followed by electroporation. Sow and litter performance were monitored for the 3 gestations after treatment. RESULTS: A significant increase in insulin-like growth factor-I concentrations, decrease in perinatal mortality rate, increase in the number of pigs born alive, and increase in the weight and number of pigs weaned were detected for both groups receiving the GHRH-expressing plasmids, compared with values for the control group. Additionally, there was a significant decrease in sow attrition in GHRH-treated females, compared with attrition in the control group, during the 3 gestations after treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Both of the GHRH plasmids provided significant benefits for sow performance and baby pig survivability for pregnant and lactating sows and their offspring during the 3 gestations after treatment, compared with results for untreated control gilts. Use of a novel optimized plasmid reduced the effective plasmid dose in these large mammals.

Regulation of small ubiquitin-like modifier-1, nuclear receptor coreceptor, histone deacetylase 3, and peroxisome proliferator-activated receptor-γ in human adipose tissue.

BACKGROUND: This study investigated the regulation of peroxisome proliferator-activated receptor-γ (PPARγ), the histone deacetylase 3 (HDAC3)-nuclear receptor coreceptor (NCoR) complex (a corepressor of transcription used by PPARγ), and small ubiquitin-like modifier-1 (SUMO-1) (a posttranslational modifier of PPARγ) in human adipose tissue and both adipocyte and macrophage cell lines. The objective was to determine whether there were alterations in the human adipose tissue gene expression levels of PPARγ, HDAC3, NCoR, and SUMO-1 associated either with obesity or with treatment of impaired glucose tolerance (IGT) subjects with insulin-sensitizing medications. METHODS: We obtained subcutaneous adipose tissue biopsies from 86 subjects with a wide range of body mass index (BMI) and insulin sensitivity (S(I)). Additionally, adipose tissue biopsies were obtained from a randomized subgroup of IGT subjects before and after 10 weeks of treatment with either pioglitazone or metformin. RESULTS: The adipose mRNA levels of PPARγ, NCoR, HDAC3, and SUMO-1 correlated strongly with each other (P<0.0001); however, SUMO-1, NCoR, and HDAC3 gene expression were not significantly associated with BMI or S(I). Pioglitazone increased SUMO-1 expression by 23% (P<0.002) in adipose tissue and an adipocyte cell line (P<0.05), but not in macrophages. Small interfering RNA (siRNA)-mediated knockdown of SUMO-1 decreased PPARγ, HDAC3, and NCoR in THP-1 cells and increased tumor necrosis factor-α (TNF-α) induction in response to lipopolysaccharide (LPS). CONCLUSIONS: These results suggest that the coordinate regulation of SUMO-1, PPARγ1/2, HDAC3, and NCoR may be more tightly controlled in macrophages than in adipocytes in human adipose and that these modulators of PPARγ activity may be particularly important in the negative regulation of macrophage-mediated adipose inflammation by pioglitazone.

Electroporation of plasmid DNA to swine muscle.

For plasmid-mediated gene therapy applications, a major limitation to scale up from rodents to large animals is the low expression level of injected plasmid DNA. The electroporation technique, which results in the passage of foreign material through the cell membrane, is one method that has been shown to be effective at improving local plasmid uptake and consequently, expression levels. Previous studies have determined that optimized electroporation parameters (such as electric field intensity, number of pulses, lag time between plasmid injections and electroporations, and optimal plasmid formulation conditions) are dependent on the target muscle type and individual species. Here, we provide a detailed protocol to optimize conditions for the successful intramuscular electroporation of plasmid DNA to swine, a large animal model. Our results suggest that the technique is safe and effective for veterinary applications. Furthermore, these results provide evidence for the feasibility of upcoming human applications.

Effects of maternal plasmid GHRH treatment on offspring growth.

To differentiate prenatal effects of plasmid growth hormone-releasing hormone (GHRH) treatment from maternal effects mediated by lactation on long-term growth of offspring, a cross-fostering study was designed. Pregnant sows (n=12) were untreated (n=6) or received either a Wt-GHRH (n=2) or HV-GHRH (n=4) plasmid. At birth, half of each litter was cross-fostered (treated to controls and controls to treated only). Piglets from plasmid-injected sows were heavier at birth (HV-GHRH, 1.65+/-0.07kg; Wt-GHRH, 1.46+/-0.05kg vs. Controls, 1.27+/-0.03kg; P>or=0.001) and at weaning (Wt-GHRH, 6.01+/-0.21kg and HV-GHRH, 6.34+/-0.15kg vs. Controls, 5.37+/-0.14kg; P>or=0.02, respectively). Control piglets cross-fostered to plasmid-injected sows grew faster to weaning (Wt-GHRH, 5.61+/-0.15kg and HV-GHRH, 5.70+/-0.29kg vs. Controls, 5.08+/-0.22kg; P>0.05, respectively). Piglets from plasmid-injected sows that suckled on control sows were larger than control piglets on control sows (Wt-GHRH, 5.93+/-0.20kg and HV-GHRH, 6.2+/-0.19kg vs. Controls, 5.08+/-0.22kg; P>0.05, respectively), but smaller than their littermates left on their treated mothers. The observed improvements were maintained until the end of the study when the offspring were 170-day-old. The results suggest that the improved growth of offspring of GHRH plasmid-treated sows pre-weaning is attributable to improved maternal performance, while after weaning the effects on the pituitary component are relevant.

Electroporation for the delivery of DNA-based vaccines and immunotherapeutics: current clinical developments.

Electroporation (EP) has been used in basic research for the past 25 years to aid in the transfer of DNA into cells in vitro. EP in vivo enhances transfer of DNA vaccines and therapeutic plasmids to the skin, muscle, tumors, and other tissues resulting in high levels of expression, often with serological and clinical benefits. The recent interest in nonviral gene transfer as treatment options for a vast array of conditions has resulted in the refinement and optimization of EP technology. Current research has revealed that EP can be successfully used in many species, including humans. Clinical trials are currently under way. Herein, the transition of EP from basic science to clinical trials will be discussed.

Gene therapy by electroporation for the treatment of chronic renal failure in companion animals.

BACKGROUND: Growth hormone-releasing hormone (GHRH) plasmid-based therapy for the treatment of chronic renal failure and its complications was examined. Companion dogs (13.1+/-0.8 years, 29.4+/-5.01 kg) and cats (13.2+/-0.9 years, 8.5+/-0.37 kg) received a single 0.4 mg or 0.1 mg species-specific plasmid injection, respectively, intramuscularly followed by electroporation, and analyzed up to 75 days post-treatment; controls underwent electroporation without plasmid administration. RESULTS: Plasmid-treated animals showed an increase in body weight (dogs 22.5% and cats 3.2%) compared to control animals, and displayed improved quality of life parameters including significant increases in appetite, activity, mentation and exercise tolerance levels. Insulin-like growth factor I (IGF-I, the downstream effector of GHRH) levels were increased in the plasmid treated animals. Hematological parameters were also significantly improved. Protein metabolism changes were observed suggesting a shift from a catabolic to an anabolic state in the treated animals. Blood urea nitrogen and creatinine did not show any significant changes suggesting maintenance of kidney function whereas the control animal's renal function deteriorated. Treated animals survived longer than control animals with 70% of dogs and 80% of cats surviving until study day 75. Only 17% and 40% of the control dogs and cats, respectively, survived to day 75. CONCLUSION: Improved quality of life, survival and general well-being indicate that further investigation is warranted, and show the potential of a plasmid-based therapy by electroporation in preventing and managing complications of renal insufficiency.

DNA vaccination and gene therapy: optimization and delivery for cancer therapy.

This review will focus on DNA vaccine approaches for the prevention or treatment of cancer and its complications. DNA vaccine therapies are a relatively novel method of cancer treatment with the goal to induce immunity against tumor-associated antigens. Both viral and nonviral vaccines have been tested in preclinical and clinical models with variable success. However, the development of new delivery methods, such as electroporation, as well as the use of agents that improve antigen uptake or presentation, and the optimization of the transgene sequences, are overcoming historical drawbacks. Efficacy and safety issues of the in vivo use of DNA-based vaccines, as well as data from preclinical and recent clinical studies, are discussed. Novel developments will improve clinical efficacy, with the potential for DNA vaccination to enter in to the arsenal of cancer therapies in the near future.

Effects of plasmid growth hormone-releasing hormone treatment during heat stress.

A gene therapy treatment with plasmid-based growth hormone-releasing hormone (GHRH) delivered by electroporation (EP) was investigated during heat stress; 32 primiparous cows received 2.5 mg of a GHRH-expressing myogenic plasmid (pSP-HV-GHRH), while 20 were designated as controls. Offspring of treated animals showed a reduction in mortality (47%; p < 0.02), and survival from birth to 260 days was dramatically improved (0% mortality vs. 21% in controls) along with an increase in weight gain (p < 0.05). Milk production was increased compared to controls with an average yield gain of 421 kg/cow (p = 0.028). Prolactin (PRL) levels were also significantly increased compared to controls (p < 0.05). The second pregnancy rate was improved by GHRH treatment (53.3% vs. 30.8%). This study shows that the use of plasmid-mediated therapy delivered by EP can maintain health status during periods of heat stress, important for both animals and potentially humans in hot, challenging climates.

Growth hormone-releasing hormone plasmid treatment by electroporation decreases offspring mortality over three pregnancies.

LifeTideSW5 is a growth hormone-releasing hormone (GHRH)-expressing plasmid delivered by intramuscular (IM) electroporation (EP), and the first therapeutic plasmid delivered by this physical method to be approved for use in food animals. Gestating sows (n = 997) were treated once with a single 5-mg GHRH-plasmid by EP or served as controls. Data on offspring from three parities subsequent to treatment were collected. No adverse effects related to treatment were noted. First parity post-treatment offspring from treated sows displayed a 2.93 kg (P < 0.0001) increase in carcass weight (CW), 1.0 mm (P < 0.0001) less back-fat (P2), and a 27.0 g CW/day (P < 0.0001) increase in rate of gain (ROG) compared with controls. An increase of 21.6% was recorded in the number of offspring surviving. In the second and third parities post-treatment, offspring from treated females displayed higher number of born alive and total born number, and lower stillborn rates. Third parity offspring from treated sows displayed a 1.6 kg advantage in CW (P < 0.05), 1.0 mm less P2 (P < 0.05), and a 10.0 g CW/day benefit in ROG. Furthermore, offspring from treated females had a 19.04% lower post-wean loss rate. Overall, plasmid GHRH administration decreased morbidity and mortality in treated females and their offspring over three consecutive pregnancies.

Double-blinded, Placebo-controlled plasmid GHRH trial for cancer-associated anemia in dogs.

The use of growth hormone releasing hormone (GHRH) plasmid-based therapy to treat companion dogs with spontaneous malignancies and anemia receiving a cancer-specific treatment was examined in a double-blinded, placebo-controlled trial. The dogs (age 10.5 +/- 2.5 years, weight 24.9 +/- 12.9 kg) received a single 0.35 mg dose of plasmid or placebo intramuscularly (i.m.), followed by electroporation (EP), and were analyzed for up to 120 days. The response rate was defined as > or = 5% increase above the nadir in the red blood cell (RBC), hemoglobin (Hb), and hematocrit (Ht) levels. Plasmid-treated dogs had at least a 7% increase in all three parameters. The initial response rates for the plasmid-treated dogs were 40.6 and 35.5%, respectively on days 40 and 60, which increased to 54.2% on day 90. Although the response rate reduced to 47.1% by day 120, it was still 22.1% higher than in the control dogs. Post-hoc analysis of the GHRH-treated group showed that responder dogs survived 84% longer, 178 +/- 26 days post-treatment, while nonresponders and controls survived for 95 +/- 16 and 97 +/- 31 days post-treatment, respectively. The quality of life, defined by 10 different parameters, dramatically improved with treatment. Overall, the possibility of a GHRH plasmid-based therapy for anemia in cancer-afflicted subjects is important enough to deserve further investigation.