Biomedical subjects

Amir S Khan

Publications and source records attributed to Amir S Khan.

12 recordsLinked to original sources

PubMed2006

Transplacental transfer of a growth hormone-releasing hormone peptide from mother to fetus in the rat.

Previous studies showed that when growth hormone-releasing hormone (GHRH) was administered to either pregnant rats or pigs as a plasmid-mediated therapy, pituitary weight, somatotroph and lactotroph numbers, and postnatal growth rate of the offspring increased. To determine if these responses resulted from direct effects of GHRH on the fetus or were secondary to effects incurred in the mother, we studied in the rat the transplacental transfer of a GHRH analog (HV-GHRH) to the fetus from the maternal circulation. For the in vivo study, HV-GHRH was labeled with 125I and purified by reverse-phase high-performance liquid chromatography (HPLC). At 18 days of gestation, pregnant dams were administered a priming intravenous dose followed by a constant infusion of the labeled peptide. Approximately 2 days later, intact [125I]-HV-GHRH was isolated from the fetal liver, stomach contents, and brain. The amounts of tracer were positively correlated with those present in the corresponding dam's plasma. These data suggest that a GHRH analog of nonplacental origin, even at physiologic concentrations, can cross the placenta and, therefore, has the potential to influence fetal pituitary development directly.

Animals↗

PubMed2006

Plasmid-based expression technology using growth hormone releasing hormone: a novel method for physiologically stimulating long-term growth hormone secretion.

Novel DNA-based technologies were recently introduced for various purposes, such as screening of targets identified from genomic projects, shuffled molecules for vaccination, or to direct the in vivo production of hormones and other peptides for therapeutic or preventative applications. We have used a plasmid-based technology to deliver growth hormone releasing hormone (GHRH) to various animal species for screening, toxicology and therapy. A single intramuscular injection of a low dose of plasmid followed by electroporation can ensure that the target species will produce physiological levels of GHRH for extended periods of time, which would replace costly, frequent injections of the recombinant hormone and improve the quality of life and compliance of patients. This therapeutic modality is of particular importance in circumstances requiring long-term administration of small molecules with naturally short half-life (e.g. treatment of anemia and cachexia associated with renal failure, cancer or other chronic disability). A similar technique was used to create, test and validate protease-resistant analogs of GHRH with significantly longer half-life. Analysis of the characteristics of each of the plasmid components and tissue-specific transcription factors and the choice of target tissue is imperative when designing plasmids for therapeutic applications. Using the species-specific sequences of GHRH or other molecule along with the appropriate choice of plasmid backbone and expression cassette components can result in long and steady expression of the transgene product.

Amino Acid Sequence↗

PubMed2006

Electroporation-enhanced nonviral gene transfer for the prevention or treatment of immunological, endocrine and neoplastic diseases.

Nonviral gene transfer is markedly enhanced by the application of in vivo electroporation (also denoted electro-gene transfer or electrokinetic enhancement). This approach is safe and can be used to deliver nucleic acid fragments, oligonucleotides, siRNA, and plasmids to a wide variety of tissues, such as skeletal muscle, skin and liver. In this review, we address the principles of electroporation and demonstrate its effectiveness in disease models. Electroporation has been shown to be equally applicable to small and large animals (rodents, dogs, pigs, other farm animals and primates), and this addresses one of the major problems in gene therapy, that of scalability to humans. Gene transfer can be optimized and tissue injury minimized by the selection of appropriate electrical parameters. We and others have applied this approach in preclinical autoimmune and/or inflammatory diseases to deliver either cytokines, anti-inflammatory agents or immunoregulatory molecules. Electroporation is also effective for the intratumoral delivery of therapeutic vectors. It strongly boost DNA vaccination against infectious agents (e.g., hepatitis B virus, human immunodeficiency virus-1) or tumor antigens (e.g., HER-2/neu, carcinoembryonic antigen). In addition, we found that electroporation-enhanced DNA vaccination against islet-cell antigens ameliorated autoimmune diabetes. One of the most likely future applications, however, may be in intramuscular gene transfer for systemic delivery of either endocrine hormones (e.g., growth hormone releasing hormone and leptin), hematopoietic factors (e.g., erythropoietin, GM-CSF), antibodies, enzymes, or numerous other protein drugs. In vivo electroporation has been performed in humans, and it seems likely it could be applied clinically for nonviral gene therapy.

Animals↗

PubMed2005

Growth hormone releasing hormone plasmid supplementation, a potential treatment for cancer cachexia, does not increase tumor growth in nude mice.

Growth hormone releasing hormone (GHRH) is known to have multiple anabolic effects and immune-stimulatory effects. Previous studies suggest that treatment with anabolic hormones also has the potential to mitigate the deleterious effects of cancer cachexia in animals. We studied the effects of plasmid-mediated GHRH supplementation on tumor growth and the role of antitumor immune cells with two different human tumor cell lines, NCI-H358 human bronchioalveolar carcinoma and MDA-MB-468 human breast adenocarcinoma, subcutaneously implanted in nude mice. GHRH supplementation by delivery of human GHRH from a muscle-specific GHRH expression plasmid did not increase tumor progression in tumor-bearing nude mice. Male animals implanted with the NCI-H358 tumor cell line and treated with the GHRH-expressing plasmid exhibited a 40% decrease in the size of the tumors (P<.02), a 48% increase in white blood cells (P<.025) and a 300% increase in monocyte count (P<.0001), as well as an increase in the frequency of activated CD3+ and CD4+ cells in the tumors, compared to tumors of control animals. No adverse effects were observed in animals that received the GHRH-plasmid treatment. The present study shows that physiological stimulation of the GHRH-GH-IGF-I axis in mice with cancer does not promote tumor growth and may provide a viable treatment for cancer cachexia in humans.

Adenocarcinoma↗

PubMed2005

Highly efficient constant-current electroporation increases in vivo plasmid expression.

Electroporation has been demonstrated as an effective technique for enhancing the delivery of plasmids coding for DNA vaccines and therapeutic proteins into skeletal muscle. Nevertheless, constant-voltage techniques do not take into account the resistance of the tissue and result in tissue damage, inflammation, and loss of plasmid expression. In the present study, we have used a software-driven constant-current electroporator to deliver plasmids to mice and small and large pigs. The voltage, amperage, and resistance of the tissue during pulses were recorded and analyzed. Optimal conditions of electroporation were identified in both species, and found to be highly dependent on the individual tissue resistance. Six- to 10-week-old pigs had higher muscle resistance compared to 1- to 2-year-old pigs, but both values were four to five times lower than the resistance of the mouse muscle. In mice, optimum amperage, pulse length, and lag time between plasmid injection and electroporation were identified to be 0.1 Amps, 20 msec and 0 sec. The electroporation pulse pattern among the electrodes also affected plasmid expression. These results indicate that age- and tissue-specific resistance, pulse pattern, and other variables associated with the electroporation need to be optimized for each separate species to achieve maximum plasmid expression.

Age Factors↗

PubMed2005

Plasmid-based growth hormone-releasing hormone supplementation and its applications.

A single dose of a plasmid expressing growth hormone-releasing hormone (GHRH) has been safely used in a number of animal species and applications to physiologically increase growth hormone and insulin-like growth factor-I for over a year. An array of constructs encoding for analogs of, or species-specific, GHRH has been tested to treat anemia and cachexia associated with cancer and its treatment, and renal failure, as well as to increase immune surveillance and animal welfare. The positive results obtained with plasmid-based GHRH in companion and farm animals may be translated to a number of human applications.

Anemia↗

PubMed2003

Maternal GHRH plasmid administration changes pituitary cell lineage and improves progeny growth of pigs.

Previous studies from our laboratory have demonstrated that administration of a myogenic plasmid that encodes a protease-resistant growth hormone-releasing hormone (HV-GHRH) to pregnant rat dams augmented long-term growth in first-generation progeny. In the present study, gilts were injected intra-muscularly at day 85 of gestation with 0, 0.1, 0.5, 1, or 5 mg of the HV-GHRH-expressing plasmid and were then electroporated. Piglets were weighed and bled periodically from birth to 100 kg. Piglets from gilts treated with 1 and 5 mg of HV-GHRH plasmid were larger at birth and weaning compared with controls. These two groups reached 100 kg 9 days earlier than the other groups. GHRH levels were increased at birth in piglets from treated gilts. IGF-I levels were significantly increased in the 5-mg group beginning at 21 days of age compared with controls. Pituitaries from the 5-mg group contained a significantly increased number of somatotrophs and lactotrophs from birth to 100 kg. This study confirms that enhanced maternal GHRH production results in intergenerational growth augmentation and that the magnitude of the response is dose dependent. The similarity of the response across species suggests that the effect is likely exerted as a fundamental component of gestational and developmental physiology.

Animals↗

PubMed2003

Effects of plasmid-mediated growth hormone-releasing hormone supplementation on LL-2 adenocarcinoma in mice.

This study was designed to measure the effects of plasmid growth hormone-releasing hormone (GHRH) supplementation on LL-2 (Lewis lung adenocarcinoma) tumor-bearing immunocompetent mice. Male and female mice (n = 20/group/experiment) received 2.5 x 10(6) LL-2 cells in the left flank. One day later, we injected the mice intramuscularly with 20 micro g of a myogenic plasmid, pSP-hGHRH or pSP-betagal, as a control. Mean serum IGF-I was significantly higher in treated animals versus controls (P < 0.05). Male and female mice constitutively expressing GHRH exhibited a decline in tumor growth rate relative to controls (20% for males, P < 0.03, and 11% for females, P < 0.13). Histopathological analysis revealed that treated animals were less likely to develop lung metastases than controls (11%) and had no alternate-organ metastases. The number of metastases/lung was reduced by 57% in female mice with GHRH treatment (P < 0.006). When tumor size exceeded 8% of body weight, GHRH-treated mice showed normal urea, creatinine, and kidney volume, while controls displayed signs of renal insufficiency. This study provides evidence that with plasmid-mediated GHRH supplementation in tumor-bearing mice, tumor growth rate is not increased but is actually attenuated.

Animals↗

PubMed2003

Optimization of electroporation parameters for the intramuscular delivery of plasmids in pigs.

Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimum conditions are not well defined. Using a plasmid with a muscle-specific secreted embryonic alkaline phosphatase (SEAP) gene, we have optimized the electroporation conditions in a large mammal (pig). Parameters tested included electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. Electric pulses, between 0.4 and 0.6 Amp constant current, applied 80 sec after the injection of 0.5 mg SEAP-expressing plasmid in a total volume of 2 mL produced the highest levels of expression. Further testing demonstrated that electroporation of a nondelineated injection site reduces the levels of SEAP expression. These results demonstrate that electroporation parameters such as amperage, lag time, and the number of pulses are able to regulate the levels of reporter gene expression in pigs.

Alkaline Phosphatase↗

PubMed2002

Growth hormone, insulin-like growth factor-1 and the aging cardiovascular system.

There is a large body of evidence that biological aging is related to a series of long-term catabolic processes resulting in decreased function and structural integrity of several physiological systems, among which is the cardiovascular system. These changes in the aging phenotype are correlated with a decline in the amplitude of pulsatile growth hormone secretion and the resulting decrease in plasma levels of its anabolic mediator, insulin like growth factor-1 (IGF-1). The relationship between growth hormone and biological aging is supported by studies demonstrating that growth hormone administration to old animals and humans raises plasma IGF-1 and results in increases in skeletal muscle and lean body mass, a decrease in adiposity, increased immune function, improvements in learning and memory, and increases in cardiovascular function. Since growth hormone and IGF-1 exert potent effects on the heart and vasculature, the relationship between age-related changes in cardiovascular function and the decline in growth hormone levels with age have become of interest. Among the age-related changes in the cardiovascular system are decreases in myocyte number, accumulation of fibrosis and collagen, decreases in stress-induced cardiac function through deterioration of the myocardial conduction system and beta-adrenergic receptor function, decreases in exercise capacity, vessel rarefaction, decreased arterial compliance and endothelial dysfunction leading to alterations in blood flow. Growth hormone has been found to exert potent effects on cardiovascular function in young animals and reverses many of the deficits in cardiovascular function in aged animals and humans. Nevertheless, it has been difficult to separate the effects of growth hormone deficiency from age-related diseases and associated pathologies. The development of novel animal models and additional research are required in order to elucidate the specific effects of growth hormone deficiency and assess its contribution to cardiovascular impairments and biological aging.

Aged↗

PubMed2002

Electrical enhancement of formulated plasmid delivery in animals.

Electroporation has been shown to significantly increase plasmid transfer to the skeletal muscle, but this procedure is also implicated in muscle damage. We are reporting a highly efficient in vivo transfer of a plasmid formulated with poly-(L-glutamate) (PLG) into murine, canine and porcine muscle fibers using electric pulses of low field intensity. In mice and pigs, the use of secreted embryonic alkaline phosphatase (SEAP) as the indicator gene caused increased PLG expression by 2-3 fold compared to naked plasmid; while delivery of a PLG-plasmid formulation to dogs showed a 10-fold increase in serum SEAP levels compared to plasmid alone. Muscle lesions were reduced by the protective PLG. Thus, PLG may constitute a useful adjuvant for increased expression and reduced muscle trauma to plasmid DNA delivered by electroporation.

Alkaline Phosphatase↗

PubMed2002

Nonhereditary enhancement of progeny growth.

The im electroporated injection of a protease-resistant GH-releasing hormone cDNA into rat dams at 16 d gestation resulted in enhanced long-term growth of the F(1) offspring. The offspring were significantly heavier by 2 wk of age, and the difference was sustained to 10 wk of age. Consistent with their augmented growth, the plasma IGF-I concentration of the F(1) progeny was increased significantly. The pituitary gland of the offspring was significantly heavier and contained an increased number of somatotrophs and PRL-secreting cells, which is indicative of modification of cell lineage differentiation. These unique findings demonstrate that enhanced GH-releasing hormone expression in pregnant dams can result in intergenerational growth promotion by altering development of the pituitary gland in the offspring.

Adrenal Glands↗