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Endocrinology, doi:10.1210/en.2003-1376
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Endocrinology Vol. 145, No. 4 2035-2045
Copyright © 2004 by The Endocrine Society

Differential Modulation of Energy Balance by Leptin, Ciliary Neurotrophic Factor, and Leukemia Inhibitory Factor Gene Delivery: Microarray Deoxyribonucleic Acid-Chip Analysis of Gene Expression

V. Prima, M. Tennant, O. S. Gorbatyuk, N. Muzyczka, P. J. Scarpace and S. Zolotukhin

Department of Molecular Genetics and Microbiology (V.P., M.T., O.S.G., N.M., S.Z.), Powell Gene Therapy Center, and Department of Pharmacology and Therapeutics (P.J.S.), University of Florida, Gainesville, Florida 32610

Address all correspondence and requests for reprints to: Sergei Zolotukhin, P.O. Box 100266, Powel Gene Therapy Center, J. Hillis Miller Health Science Center, University of Florida, Gainesville, Florida 32610-0266. E-mail: sergei{at}ufl.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Most obese animal models, whether associated with genetic, diet-induced, or age-related obesity, display pronounced leptin resistance, rendering leptin supplement therapy ineffective in treating obesity. Ciliary neurotrophic factor (CNTF) has been recently used to invoke leptin-like signaling pathways, thereby circumventing leptin resistance. In the current study, we characterize immediate and long-term molecular events in the hypothalamus of rats exposed to the sustained ectopic expression of leptin, CNTF, or leukemia inhibitory factor, another neurocytokine of IL-6 family, all delivered centrally via a viral vector. The respective transgene-encoded ligands induced similar but not identical metabolic responses as assessed by the reduction in body weight gain and changes in food intake. To define molecular mechanisms of weight-reducing and anorexigenic action of cytokines, we have analyzed the gene expression profiles of 1300 brain-specific genes in the hypothalami of normal rats subjected to the prolonged cytokine action for 10 wk. We present evidence that constitutive expression of cytokines in the brain induces changes in gene expression characteristic of chronic inflammation leading to either temporal weight reduction (CNTF) or severe cachexia (leukemia inhibitory factor). Our results convey a cautionary note regarding potential use of the tested cytokines in therapeutic applications.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GROWING EVIDENCE SUGGESTS that a prolonged exposure of the hypothalamus to exogenously administered leptin may result in the development of central leptin resistance. Indeed, chronic central infusion in obese AY mice (1) and in chow-fed male Sprague Dawley rats (2) results in attenuation of the leptin-induced anorexia, indicative of a leptin-induced hypothalamic leptin resistance. Supporting this hypothesis are the results from studies using recombinant adeno-associated virus (rAAV) vector-mediated leptin gene delivery into the brain that generated sustained elevation of central leptin (3, 4). In these studies, not only did the anorexic response to leptin attenuate, but also the energy expenditure response waned over time after chronic leptin delivery in both young and older rats. Furthermore, the leptin-induced leptin-resistant rats were completely unresponsive when challenged by central injection of a suprapharmacological dose of leptin, thus confirming their full hypothalamic leptin-resistant state (5, 6).

Recently, an alternative strategy using ciliary neurotrophic factor (CNTF) has emerged for the treatment of obesity associated with leptin resistance. Indeed, acute treatment with CNTF reduced obesity-related phenotypes in ob/ob and db/db mice, which lack functional leptin and leptin receptor, respectively (7). Consistent with this finding, Lambert et al. (8) showed that CNTF can activate hypothalamic leptin-like pathways even in diet-induced obese (DIO) models unresponsive to leptin. Consequently, recombinant human variant of CNTF has recently been tested in clinical trials for weight loss in obese adult subjects (9).

Leukemia inhibitory factor (LIF) is another member of the IL-6 cytokine family, mediating signaling through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway similar to leptin (10). LIF, like CNTF, is a potent cachexia-inducing agent (11), and the respective receptor subunits CNTFR{alpha}, LIF-R, and gp130 are expressed in hypothalamic nuclei involved in energy metabolism (7). Effective dose of LIF is two orders of magnitude lower than CNTF, suggesting much higher biological potency for LIF (12).

There are continuing discussions and experimental efforts to evaluate LIF for the potential clinical use, mostly for the peripheral nerve injuries treatment (13, 14, 15, 16, 17, 18), including clinical trials (19). Notably, a report has recently been published suggesting using LIF gene therapy as a treatment for obesity (20). Because peripherally administered LIF readily enters the brain and spinal cord by a saturable transport system across the blood-brain barrier (21), the long-term (LT) effect of the cytokine ought to be investigated in a more systematic and thorough manner.

rAAV-mediated leptin gene delivery has been demonstrated to be an effective method to provide sustained physiological levels of the hormone in rodent brain (6). Because neurocytokines LIF and CNTF induce pathways similar to leptin while bypassing the leptin resistance barrier, we decided to evaluate the LT consequences of the elevated levels of cytokines using rAAV-mediated delivery approach. The objective of the current investigation, therefore, was to analyze a global gene expression in the hypothalamus of rats exposed to sustained expression of leptin, CNTF, or LIF, with hopes of gaining new information on the mechanisms of the weight-reducing actions of neurocytokines in the brain and evaluating the safety of such treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Age-matched Sprague Dawley male rats weighing 250–300 g were obtained from Harlan Sprague Dawley (Indianapolis, IN). Upon arrival, rats were examined and remained in quarantine for 1 wk. Animals were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals. Rats were housed individually with a 12-h light, 12-h dark cycle (0700–1900 h) and fed standard Teklad rodent chow (Harlan Industries, Indianapolis, IN) and water ad libitum.

Construction and packaging of rAAV vectors
rAAV vectors designed for the study are shown in Fig. 1Go. Construction of the rAAV vectors encoding human secreted high-affinity mutant DH-CNTF (sDH-CNTF) cDNA and rat leptin cDNA were described previously (6, 22). Human LIF cDNA was cloned by PCR-mediated protocol from LIF-expressing melanoma cell line G361 (23). Vectors were packaged, purified, concentrated, and titered as previously described (24). The titers of rAAVs used in separate experiments of this study were as follows: rAAV-GFP (green fluorescent protein), 1.3 x 1011; rAAV-leptin, 2.9 x 1011; rAAV-LIF, 2.3 x 1011; rAAV-CNTF, 0.7 x 1011 infectious particles/ml. The average ratio of physical to infectious particles was 50. A mini-Ad helper plasmid pDG (25) was used to produce rAAV vectors with no detectable adenovirus or wild-type AAV contamination. rAAV vectors purified using iodixanol gradient/heparin-affinity chromatography were more than 99% pure, as judged by the polyacrylamide silver-stained gel electrophoresis.



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  		FIG. 1. Diagram of rAAV vectors. The construction of pTR-GFP negative control vector was described earlier (58 ). pTR-ObW contains two AAV2 terminal repeat sequences (TR); the rat leptin cDNA is driven by chicken ß-actin promoter linked to cytomegalovirus (CMV) enhancer (CBA) (59 ); the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is placed downstream of to enhance the expression of the transgene (60 ). The construction of pTR-sDH-CNTF was described earlier (22 ). The dicistronic transcription unit in pTR-hLIF mediates a coordinate expression of human LIF and GFP reporter gene via poliovirus type 1 internal ribosome entry site (IRES) (58 ). IVS, Intervening sequence; pA, polyA; TK, thymidine kinase promoter; neo, neomycin resistance gene.

 
rAAV administration
A single dose of vector (5 x 108 infectious particles/rat) was administered by intracerebroventricular injection into the third cerebral ventricle. Rats were anesthetized with xylazine, 8 mg/kg sc and 5 min later, 90 mg/kg ketamine ip. After a surgical plane of anesthesia has been reached, animals were placed into a stereotaxic frame, and a small incision (1.5 cm) was made over the midline of the skull to expose the landmarks of the cranium (Bregma and Lamda). The following coordinates were used for injection into the third cerebroventricle: 1.3 mm posterior to Bregma and 9.4 mm ventral from the skull surface on the midline (medial fissure), with the nose bar set at 3.3 mm below the ear bars (below zero) and the canula set at 20 degrees posterior from vertical. A small hole was drilled through the skull and a 23-gauge stainless steel guide canula was lowered to the third cerebroventricle. This was followed by an injection canula attached to a 10-µl syringe. We injected 5 µl of viral particles dissolved in Ringer’s solution at approximately 0.5 µl/min. Animals received the rAAV-leptin, rAAV-LIF, rAAV-CNTF, or the control virus encoding GFP.

Oligonucleotide microarray hybridization
Total RNA from hypothalamic tissue was isolated using Trizol reagent (Invitrogen, Carlsbad, CA), pooled from four to six animals for each experiment, and cleaned with RNeasy columns (QIAGEN, Valencia, CA). cDNA synthesis followed by biotin-labeled cRNA synthesis was completed according to the Affymetrix technical manual (Affymetrix, Santa Clara, CA). Biotinilated cRNA (5 µg per array) was hybridized to Affymetrix RN-U34 microarrays containing oligonucleotide probe sets representing approximately 1300 neurobiology-relevant rat genes. Hybridization, washing, and staining of RN-U34 microarrays were carried out according to the Affymetrix technical manual in an Affymetrix hybridization oven and fluidics station. The arrays were scanned using a Hewlett Packard confocal laser scanner and visualized using Affymetrix GeneChip 3.1 software.

Data analysis
The Affymetrix Rat Neurobiology U34 Array contained about 1300 neuronal-specific genes. Raw data were initially normalized using Affymetrix software. Noise filtering and correction for outliers and saturation effects with subsequent statistical analysis of hybridization data were performed by Probe Profiler software through the custom service of Corimbia (Berkeley, CA). Negative cross-hybridization values were filtered out using GeneSpring 4.2 software (Silicon Genetics, Redwood City, CA). The remaining 1040 genes were subjected to clustering analysis using GeneSpring 4.2. Hierarchical clustering of the data were performed using standard correlation as a measure of gene expression similarity with the following parameter settings: the 50th percentile of all measurements was used as a positive control for each sample; each measurement for each gene was divided by this synthetic positive control, assuming that this was at least 10. The bottom 10th percentile was used as a test for correct background subtraction. This was never less than the negative of the synthetic positive control. The measurement for each gene in each sample was divided by the corresponding value in a control sample [rAAV-GFP-injected rats, short-term (ST); or rAAV-GFP-injected rats, LT], assuming that it was at least 0.01. Genes represented on Affymetrix RN-U34 microarrays as expressed sequence tags were identified using GenBank UniGene System.

Northern blot hybridization
Total RNA from hypothalamic tissue was pooled from four to six animals for each experiment. Five micrograms of total RNA were separated by denaturing hot-agarose electrophoresis (26) and transferred by capillary blot to nylon membranes using standard protocols (27). Blots were prehybridized at 42 C with UltraHyb solution (Ambion, Austin, TX), and hybridized with 1 x 106 cpm/ml of [32P] deoxy-CTP-labeled DNA probe generated by random-primed labeling. Filters were washed to high stringency and exposed to x-ray film or scanned with Storm 850 PhosphorImager (Amersham Biosciences, Piscataway, NJ). Probes for specific mRNAs were either PCR-generated based on published sequences or isolated as expressed sequence tag fragments from sequence-verified rat cDNA clones (ResGen, Huntsville, AL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of body weight (BW) and food intake (FI) by rAAV vectors encoding cytokines
Two groups of rats were injected with each vector: ST cohort and LT cohort (Fig. 2Go). The initial physiological response in LT group was identical with ST cohort and, therefore, is not shown in Fig. 2Go, B and D. This experimental setup allowed us to determine changes in gene expression immediately after the onset of the phenotypic changes (judged by significant loss of BW, ST group), and during the chronic phase (10 wk post injection, LT group). All experimental groups experienced an initial loss in BW due to the surgical procedure of vector administration. After this period, beginning at d 2, the control rats (rAAV-GFP) steadily gained weight over the remainder of the study.



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  		FIG. 2. Effect of centrally injected rAAV encoding cytokines on BW and FI. A and B, Change in BW in ST and LT experiments, respectively; open circles, rats injected with rAAV-GFP (n = 7 for ST, n = 8 for LT group); open triangles, rats injected with rAAV-leptin (n = 5 for ST, n = 7 for LT group); open rectangles, rats injected with rAAV-LIF (n = 5 for ST, n = 6 for LT group); open diamonds, rats injected with rAAV-CNTF (n = 5 for ST, n = 8 for LT group). C and D, Change in FI in ST and LT experiments, respectively. The initial response to the treatment in LT experiment was similar to the ST; therefore, the data in panels B and D are shown starting from d 5 post injection.

 
The respective ligands encoded by leptin, CNTF, or LIF transgenes induced similar but not identical physiological responses as assessed by the reduction in BW gain and changes in FI. rAAV-leptin: The response to this vector was recorded as early as d 3 post injection, judged by the significant reduction in FI, followed by the respective decline in BW (Fig. 2Go, A and C). The anorexigenic affect of this vector persisted until the end of the experiment, whereas BW continued to decline until d 18 followed by the period of slow weight gain (Fig. 2Go, B and D). At the end of the study, rAAV-leptin LT cohort gained on average 30% less weight compared with the rAAV-GFP control group. rAAV-LIF: Although the anorectic response to LIF vector became significant only at d 7, the initial reduction in BW gain mirrored rAAV-leptin ST group. Subsequently, FI was similar in both LIF and leptin groups, yet LIF animals experienced more severe weight loss with some animals losing up to 41% of the initial weight. Some of the animals in the LIF group displayed apparent signs of stress and depression experiencing cachexia-like symptoms characterized by dehydration and overall reduction of physical activity (grooming, responsiveness). Because of the severe cachexia and wasting, one animal died at d 27 post injection; the other was euthanized at d 50. rAAV-CNTF: The response to this cytokine was moderate; the initial anorexigenic effect attenuated at about d 20 post injection. After initial gain, CNTF group maintained BW relatively constant until d 25 before drawing level with GFP control group at the end of the experiment.

DNA microarray analysis of hypothalamic gene expression
We have documented two distinct phases in the physiological response to transgenes: 1) onset of phenotypic changes of BW loss concomitant with statistically significant reduction in FI corresponding to the acute phase of cytokine action; 2) sustained maintenance of the lean phenotype (chronic phase). Respectively, separate cohorts of rats were used to analyze changes in gene expression profiles at ST and LT time points. Gene expression levels in animals injected with rAAV-GFP were used as hybridization control references.

Of about 1300 genes represented on the Affymetrix RN U34 microarray, 1040 passed two sequential filters of significance (applied by Affymetrix Microarray Suite and Probe Profiler algorithms) and were used for subsequent clustering analysis using GeneSpring 4.2 software. First, we applied hierarchical clustering to the filtered data to visualize patterns of gene expression globally (Fig. 3AGo). In this dendrogram, samples or genes having similar effects on the gene expression pattern are clustered together, the nearness of any two branches measured on the basis of the standard correlation between the expressions profiles of the two genes. At the sample axis, the dendrogram grouped rAAV-leptin cohorts (both ST and LT) separately from rAAV-GFP control groups. Curiously, both rAAV-LIF and rAAV-CNTF groups were clustered in one node at ST time point, as well as at LT time point reflecting the functional similarity of the two cytokines and justifying the selected time course of the experiment. We then applied additional filter to select only those genes whose mRNA levels changed more than 2-fold in either direction. The hierarchical clustering of such filtered database of 350 genes produced similar pairs of experimental groups (Fig. 3BGo).



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  		FIG. 3. Hierarchical clustering of gene expression data. From the Rat Neurobiology U34, 1040 neuronal-specific genes were clustered in two dimensions, according to their gene expression, using the hierarchical clustering algorithm (GeneSpring 4.2). A, Clustering of all genes vs. all experimental groups; B, clustering of all genes filtered with 2-fold threshold vs. all experimental groups. Both databases were initially normalized using ProbeProfiler algorithm, filtered for positive expression values, and clustered using the parameters settings indicated in Data analysis. A single column represents each gene; each sample (ST, LT, vector administered) is represented by a single row. Up-regulated genes (compared with GFP controls) are shown in different shades of red color; down-regulated genes are shown in blue.

 
A complete catalog of genes exhibiting 2-fold (or higher) change threshold is published as supplemental data Tables 1–4 on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org. These genes were grouped to demonstrate a pairwise/threesome similarity between modes of action of the products of three transgenes (leptin, LIF, and CNTF). Venn diagrams shown in Fig. 4Go illustrate the graphic representation of this analysis. By the number of genes modulated, rAAV-LIF appeared to introduce the most of perturbation, whereas rAVV-leptin was the least intrusive (LIF>CNTF>leptin). All genes affected by each treatment (depicted by one full circle in the Venn diagram) were assigned to several functional categories (Table 1Go). At ST, a considerable fraction of up-regulated genes were genes related to stress and inflammation, whereas the down-regulated included mostly genes involved in intracellular signal transduction and ion transport. The total number of chronically (LT) modulated genes decreased compared with the ST phase. The up-regulated genes included members of neurotransmission and signal transduction gene families, whereas the down-regulated at the LT phase were mostly genes of stress- and immune-response systems, and those involved in cell proliferation. In addition, we have compiled a list of the arbitrarily selected genes (Table 2GoGo) that displayed a dramatic change in the expression values and/or were known to be involved in energy metabolism, inflammatory response, signal transduction, and neurotransmission (for the full listing, please see supplemental data published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Finally, we have selected the short list of stimulatory and inhibitory genes that mediate feeding behavior and grouped them under the respective headings to illustrate the dynamics of the hypothalamic network of neurotransmitters directly involved in energy metabolism (Fig. 5BGo). We have observed down-regulation in expression of most of the orexigenic genes and reciprocal up-regulation of anorexigenic ones, whereas some genes [leptin receptor isoform b (Ob-Rb), CRH, oxytocin, cholecystokinin] displaying the reversal trend at ST vs. LT time points.



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  		FIG. 4. Venn diagrams demonstrating the relationship between genes modulated in the hypothalami of rats in response to treatment with rAAV-leptin, rAAV-LIF, or rAAV-CNTF (threshold of 2-fold). A and B, ST experiment, up-, or down-regulated genes, respectively; C and D, LT experiment, up-, or down-regulated genes, respectively. Red circles indicate genes affected by rAAV-CNTF; green, by rAAV-leptin; blue, by rAAV-LIF. Sectors’ color codes correspond to codes in the gene supplemental data Tables 1–4. The numbers, displayed within the intersections of the circles indicate the identical genes induced/repressed by two (or three) vectors. The number at the bottom right corner of each panel indicates the rest of the genes analyzed from the Rat Neurobiology U34 microchip.

 

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  		TABLE 1. Functional classification of genes modulated in hypothalami of rats in response to treatment with rAAV-leptin, rAAV-LIF, or rAAV-CNTF (2-fold threshold)

 

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  		TABLE 2. Partial list of hypothalamic genes modulated in response to sustained expression of rAAV vectors encoding leptin, LIF, or CNTF in ST or LT experiment1

 

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  		TABLE 2A. Continued

 


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  		FIG. 5. Neuronal pathways in rat brain activated by leptin, CNTF, and LIF. A, Interactions of orexigenic (AGRP/NPY) and anorexigenic [POMC/CART (cocaine- and amphetamine-regulated transcript)] first-order neurons in the arcuate nucleus. The stimulatory (+), or inhibitory (-) actions of neurocytokines, or hormone leptin activate the respective subpopulations of arcuate neurons (see panel B), connected to second-order neurons in PVN and LHA, coordinating centers for energy metabolism. These two distinct arcuate neuronal populations send widespread projections that follow seemingly parallel routes and terminate on common postsynaptic targets (panel B) that mediate autonomic and neuroendocrine (PVN) as well as behavior (LHA) components of the physiological effects of leptin and cytokines. B, Longitudinal view of the rat brain illustrating neuroanatomical model of leptin, CNTF or LIF actions. Key brain regions implicated in adiposity signaling and regulation of FI are shown as follows: the arcuate nucleus (ARC), the paraventricular nucleus (PVN), the lateral hypothalamic area (LHA), the median eminence (ME), the dorsal vagal complex (DVC). Open arrows indicate catabolic pathways; filled arrows, anabolic pathways. Hypothalamic genes known to be involved in feeding behavior and modulated in response to the sustained expression of rAAV vectors are grouped under the respective headings. Upward filled arrowheads indicate the up-regulation of the transcription in ST, or chronic LT experiment; downward open arrowheads indicate the down-regulation of transcription. CCK, Cholecystokinin; SSTR, somatostatin receptor.

 
Validation of DNA microarray analysis
To perform independent validation of the microarray hybridization data, Northern blot hybridization was employed to analyze the expression of a subset of genes (Fig. 6Go). The results of this assay were in general agreement with the microarray data for the selected genes. Hybridization with human LIF and CNTF probes resulted in strong signals at both ST and LT time points that reflected the expression of the respective rAAV-mediated transgenes. Because of the mismatches between human and rat sequences, the expression of these transgenes was not detected by the DNA microarray analysis. However, the hybridization with rat leptin probe was consistent for both methods in detecting virus-encoded rat leptin mRNA. These data support the notion of the constitutive expression of transgenes for the duration of the experiment. We have also included proopiomelanocortin (POMC) gene into the Northern assay because the respective oligo set was not included in the list of genes of RN_U34 microarray. Curiously, POMC was down-regulated for all effector transgenes compared with the control GFP vector.



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  		FIG. 6. Northern blot validation of selected genes from Rat Neurobiology U34 microarray. Each lane contains 5 µg of total RNA isolated from the hypothalami of rats, pools of four to six animals, injected with the respective vector (indicated along the top margin). Each panel was hybridized to the probe indicated along the left margin. The bottom panel shows ethidium bromide-stained 28S rRNA. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; SOCS, suppressor of cytokine signaling.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We conducted microarray DNA-chip analysis of gene expression in the hypothalamus induced by constitutive expression of cytokine transgenes delivered centrally via rAAV vectors. After the period of postsurgical recovery, we documented two distinct phases of metabolic response. The first, acute phase was characterized by significant reduction in FI and BW. The duration of this stage as well as the dynamics of BW/FI change were different for each of the transgenes used. Whereas rAAV-CNTF-injected animals experienced mild anorexia and moderate loss of BW, LIF, and leptin induced more profound changes that lasted for the duration of the experiment. During the second, chronic phase anorexia and BW losses in CNTF group gradually attenuated and were no longer significant at the end of the experiment. On the contrary, both LIF and leptin groups had significant anorexia and BW loss. These physiological readouts were in contrast with the hierarchical clustering of experimental groups that combined CNTF and LIF cohorts in one node. Clustering analysis shows the similarity of the mode of action of these two neurocytokines, whereas difference in physiological responses between CNTF and LIF apparently reflects the higher potency (12) and stability (28) of the latter.

Hypothalamic targets of leptin
Within the hypothalamus, leptin receptor is expressed in the arcuate, paraventricular, supraoptic, ventromedial, and dorsomedial nuclei and lateral hypothalamic area, as revealed by in situ hybridization (29). However, there is strong evidence pointing at the hypothalamic arcuate nucleus as a main target of leptin in the brain, where it has been identified to interact with both POMC and agouti-related protein (AGRP)/neuropeptide Y (NPY) cells (Fig. 5Go). Leptin treatment has been shown to block a decrease of POMC mRNA expression normally observed in fasted rats (30). High supraphysiologic doses of leptin administered to normal or obese mice had been demonstrated to increase expression of POMC and decrease AGRP and NPY mRNAs. However, Ahima et al. (30) have shown that leptin infusion, which increased plasma leptin in ad libitum-fed rats to moderate levels observed with mild obesity, suppressed NPY mRNA below that of the fed state but did not affect the expression of POMC. As we have shown earlier, constitutive expression of rAAV-mediated leptin in brain resulted in moderately increased cerebrospinal fluid levels of leptin within the physiological range (4, 6). Under these conditions, we anticipated detecting little or no change in POMC expression levels, whereas in fact, POMC was down-regulated at both ST and LT time points (Fig. 6Go).

The effect of leptin on the expression of other neuropeptides is also mediated indirectly through the hypothalamic neuronal projections of the arcuate POMC and AGRP/NPY neurons, which are well positioned to coordinate the activity of the hypothalamo-pituitary-adrenal, gonadal, thyroid and somatotroph axes (31, 32, 33). For example, both NPY/AGRP and melanocortinergic terminals have been demonstrated to converge on neurosecretory parvocellular neurons expressing CRH and TSH-releasing hormone (34, 35, 36, 37, 38, 39). Moreover, magnocellular neurosecretory cells, which release oxytocin and vasopressin in the posterior pituitary, are under direct influence of the arcuate nucleus (40, 41, 42). Supporting this notion, our data indicate that rAAV leptin modulates expression of wide variety of orexigenic and anorexigenic peptides (Table 2GoGo). For example, early acute phase (ST) was characterized by the dramatic induction of CRH that apparently increased pituitary ACTH release and subsequent secretion of glucocorticoids by the adrenal gland (hypothalamo-pituitary-adrenal axis) (43). The following LT decline of hypothalamic CRH expression reflects negative feedback regulation by high concentrations of stress-induced circulating glucocorticoids (44).

The initial (ST) anorexigenic effect at all conditions tested was mediated by dramatic down-regulation of NPY. At the ST time point, repression of NPY enhances sympathetic stimulation of brown adipose tissue (45), inducing thermogenesis via up-regulation of uncoupling protein 1. The subsequent LT maintenance of the lean phenotype in the case of rAAV-leptin could be attributed in part to the combination of chronically reduced NPY expression (orexigenic peptide) and increased cholecystokinin expression (anorexigenic peptide) (46).

Central action of neurocytokines CNTF and LIF
The rationale of using CNTF as a leptin substitute was based upon an assumption that both effectors produce similar effects on energy homeostasis, while apparently employing distinct pathways (Fig. 5AGo). For example, unlike leptin, peripheral administration of DH-CNTF significantly reduced FI and BW of obese mice lacking melanocortin receptor MC4-R, indicating that the melanocortin pathway is not required for the anorectic effect of CNTF (47). Similarly, CNTF injections produced anorexigenic effects in a DIO rodent model resistant to leptin (7, 8). Because CNTF is under investigation in clinical trials, it is essential to study a LT effect of its action in brain where it exerts anorexigenic effect. As a second effector, we also tested another member of the IL-6 family of neurocytokines (10, 48), LIF, characterized by higher potency and stability.

Absolute comparisons of cytokines are difficult because equal doses of two different ligands do not produce equivalent effects on the target variables. Indeed, constitutive expression of the CNTF and LIF transgenes produced different LT physiological effects. Whereas in the rAAV-CNTF group, the anorexigenic and weight-reducing effects attenuated at 5 and 10 wk, respectively, the rAAV-LIF group experienced a dramatic weight loss with attending cachexia that resulted in the deaths of two rats (in two independent studies). Despite these obvious differences, the clustering analysis revealed a remarkable similarity in gene expression profiles of both cytokines, grouping the two together in the same nodes at ST and LT time points and separately from leptin groups. This is not surprising considering the fact that both cytokines share identical signal-transducing receptor domains and initiate the JAK-STAT3 signaling cascade (10). The difference in the physiological readouts has apparently resulted from the abundance and/or brain distribution of the respective receptor complexes LIFR/gp130 (LIF) vs. LIFR/gp130/CNTFR{alpha} (CNTF) (10, 39). The potency/stability of the ligand is also a factor enhancing the biological effect of LIF.

Earlier, Lambert et al. (8) have documented the reduction in body fat in rats injected daily with recombinant CNTF (Axokine) for 26 d. Upon cessation of the treatment, animals demonstrated a lack of rebound in FI for 1 wk, apparently contributing to reduction in BW set point. In the current study, rats were subjected to the constitutive central action of the DH-CNTF transgene that was similar in expression levels at both time points tested (Fig. 6Go). Despite the constant supply of the cytokine, its weight-reducing effect attenuated and rAAV-CNTF animals experienced the gradual rebound of BW by wk 10. This effect of decreasing sensitivity to CNTF is comparable with the gradual development of leptin resistance documented earlier in rats injected with rAAV-leptin (3, 5, 6). Notably, human subjects in a clinical study, treated with recombinant human variant CNTF demonstrated a weight-gaining trend at 1 yr upon termination of the treatment (9).

The format of the current report does not allow us to conduct an extensive analysis of a complex network of hypothalamic genes modulated in response to continuous action of cytokine transgenes. Instead, we focus our attention on genes known to be markers of cellular injury and vulnerability in the nervous system. Despite profound physiological effect, rAAV-leptin modulated relatively few genes at ST, and even fewer at LT time points (Table 1Go). On the contrary, the number of genes perturbed by LIF and CNTF, as well as the amplitude of change, was considerably higher at both time points. In addition to genes involved in energy metabolism, the cytokines initiated a cascade of changes in stress and immune response-related genes (Table 2GoGo). In the central nervous system, cytokines act as immunoregulators and neuromodulators maintaining anorectic, pyrogenic, and somnogenic neurological responses of an organism at norm or during disease (48). Normally, the cellular expression of cytokines in the central nervous system is tightly regulated and maintained at low levels. In the current experiment, the constitutive expression of the cytokine transgenes in the hypothalamus initiated a cascade of downstream changes in stress-related genes such as hsp27, or hsp70. Induction of these heat-shock proteins (hsp) has protective action against thermal or ischemic stress (49, 50, 51), or brain injury (52). Induction of hsp27 mediated antiapoptotic effects in neural cells (49, 53, 54). Consistent with our data, strong induction of hsp27 in hypothalamus was shown to accompany anorectic response to the central infusion of CNTF and FGF-1 but not leptin (55). Similarly, food deprivation has been shown to induce hsp27 and hsp70 in hypothalamus, presumably as a mechanism of adjustment to the caloric restriction stress (56).

Because leptin, LIF, and CNTF are proinflammatory cytokines (48), their constitutive expression in hypothalamus initiated a typical neuroinflammatory response characterized by up-regulation of acute phase and immediate-early response genes (Table 2GoGo), with anorexia being one of the neurological symptoms. Subsequent up-regulation of antiinflammatory cytokines, such as IL-10 and, most notably, IL-13 signifies a negative feedback to ameliorate the condition perceived by the organism as chronic inflammation.

Earlier we have demonstrated a development of leptin-induced hypothalamic leptin resistance that was associated in part with the reduction in Ob-Rb expression (3, 5). In the current experiment, the rAAV-leptin LT group demonstrated a similar reduction in the expression levels of Ob-Rb, not detected in LIF, or CNTF groups (Table 2GoGo). This finding supports the hypothesis that the down-regulation of Ob-Rb expression contributes causally to central leptin resistance, and it argues against potential therapeutic application of leptin either a recombinant, or vector-derived for the treatment of DIO. Similarly, none of neurocytokines tested in the current research, appear to fulfill the requirements of sustained (CNTF) and safe (LIF) therapy. Because the effect of CNTF started wearing off at about 30 d post vector delivery, higher doses of the cytokine may be required to achieve persistent weight-reducing effect. In experimental as well as in a clinical setting, however, high doses of CNTF induce side effects as reported earlier (9, 22). Our data related to LIF treatment support this notion indicating that a potent JAK/STAT inducer initiates pleiotropic processes in the brain of a treated animal that, depending on the individual response, might trigger untoward or even fatal outcome. In primate model, for example, injections of recombinant human LIF that reduced sc fat, also resulted in severe splenomegaly and trombocytosis, associated with acute or chronic inflammation (57).

In summary, we have tested a weight-reducing effect of sustained central expression of leptin, CNTF, or LIF. Using DNA microarray analysis of gene expression in the hypothalamus, we present evidence that constitutive expression of cytokines in the brain evokes a state of perceived chronic inflammation leading to either temporal weight reduction (CNTF) or severe cachexia (LIF). Our results convey a cautionary note regarding potential use of the tested cytokines in therapeutic applications.


   Footnotes
 
Abbreviations: AGRP, Agouti-related protein; BW, body weight; CNTF, ciliary neurotrophic factor; DIO, diet-induced obese; FI, food intake; GFP, green fluorescent protein; hsp, heat-shock protein; JAK, Janus kinase; LIF, leukemia inhibitory factor; LT, long-term; NPY, neuropeptide Y; Ob-Rb, leptin receptor isoform b; POMC, proopiomelanocortin; rAAV, recombinant adeno-associated virus; sDH, human secreted high-affinity mutant DH-CNTF; ST, short-term; STAT, signal transducer and activator of transcription.

Received October 14, 2003.

Accepted for publication December 26, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Halaas JL, Boozer C, Blair-West J, Fidahusein N, Denton DA, Friedman JM 1997 Physiological response to long-term peripheral and central leptin infusion in lean and obese mice. Proc Natl Acad Sci USA 94:8878–8883[Abstract/Free Full Text]
  2. Sahu A 2002 Resistance to the satiety action of leptin following chronic central leptin infusion is associated with the development of leptin resistance in neuropeptide Y neurones. J Neuroendocrinol 14:796–804[CrossRef][Medline]
  3. Wilsey J, Zolotukhin S, Prima V, Scarpace PJ 2003 Central leptin gene therapy fails to overcome leptin resistance associated with diet-induced obesity. Am J Physiol Regul Integr Comp Physiol 285:R1011–R1020
  4. Scarpace PJ, Matheny M, Zhang Y, Tumer N, Frase CD, Shek EW, Hong B, Prima V, Zolotukhin S 2002 Central leptin gene delivery evokes persistent leptin signal transduction in young and aged-obese rats but physiological responses become attenuated over time in aged-obese rats. Neuropharmacology 42:548–561[CrossRef][Medline]
  5. Scarpace PJ, Matheny M, Zolotukhin S, Tumer N, Zhang Y 2003 Leptin-induced leptin resistant rats exhibit enhanced responses to the melanocortin agonist MT II. Neuropharmacology 45:211–219[CrossRef][Medline]
  6. Scarpace PJ, Matheny M, Zhang Y, Shek EW, Prima V, Zolotukhin S, Tumer N 2002 Leptin-induced leptin resistance reveals separate roles for the anorexic and thermogenic responses in weight maintenance. Endocrinology 143:3026–3035[Abstract/Free Full Text]
  7. Gloaguen I, Costa P, Demartis A, Lazzaro D, Di Marco A, Graziani R, Paonessa G, Chen F, Rosenblum CI, Van der Ploeg LH, Cortese R, Ciliberto G, Laufer R 1997 Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance. Proc Natl Acad Sci USA 94:6456–6461[Abstract/Free Full Text]
  8. Lambert PD, Anderson KD, Sleeman MW, Wong V, Tan J, Hijarunguru A, Corcoran TL, Murray JD, Thabet KE, Yancopoulos GD, Wiegand SJ 2001 Ciliary neurotrophic factor activates leptin-like pathways and reduces body fat, without cachexia or rebound weight gain, even in leptin-resistant obesity. Proc Natl Acad Sci USA 98:4652–4657[Abstract/Free Full Text]
  9. Ettinger MP, Littlejohn TW, Schwartz SL, Weiss SR, McIlwain HH, Heymsfield SB, Bray GA, Roberts WG, Heyman ER, Stambler N, Heshka S, Vicary C, Guler HP 2003 Recombinant variant of ciliary neurotrophic factor for weight loss in obese adults: a randomized, dose-ranging study. JAMA 289:1826–1832[Abstract/Free Full Text]
  10. Turnley AM, Bartlett PF 2000 Cytokines that signal through the leukemia inhibitory factor receptor-ß complex in the nervous system. J Neurochem 74:889–899[CrossRef][Medline]
  11. Metcalf D, Gearing DP 1989 Fatal syndrome in mice engrafted with cells producing high levels of the leukemia inhibitory factor. Proc Natl Acad Sci USA 86:5948–5952[Abstract/Free Full Text]
  12. Ikeda K, Iwasaki Y, Shiojima T, Kinoshita M 1996 Neuroprotective effect of various cytokines on developing spinal motoneurons following axotomy. J Neurol Sci 135:109–113[CrossRef][Medline]
  13. Kurek JB, Radford AJ, Crump DE, Bower JJ, Feeney SJ, Austin L, Byrne E 1998 LIF (AM424), a promising growth factor for the treatment of ALS. J Neurol Sci 160(Suppl 1):S106–S113
  14. McKay Hart A, Wiberg M, Terenghi G 2003 Exogenous leukaemia inhibitory factor enhances nerve regeneration after late secondary repair using a bioartificial nerve conduit. Br J Plast Surg 56:444–450[CrossRef][Medline]
  15. Zou Y, Takano H, Mizukami M, Akazawa H, Qin Y, Toko H, Sakamoto M, Minamino T, Nagai T, Komuro I 2003 Leukemia inhibitory factor enhances survival of cardiomyocytes and induces regeneration of myocardium after myocardial infarction. Circulation 108:748–753[Abstract/Free Full Text]
  16. Feeney SJ, Austin L, Bennett TM, Kurek JB, Jean-Francois MJ, Muldoon C, Byrne E 2003 The effect of leukaemia inhibitory factor on SOD1 G93A murine amyotrophic lateral sclerosis. Cytokine 23:108–118[CrossRef][Medline]
  17. Azari MF, Lopes EC, Stubna C, Turner BJ, Zang D, Nicola NA, Kurek JB, Cheema SS 2003 Behavioural and anatomical effects of systemically administered leukemia inhibitory factor in the SOD1(G93A G1H) mouse model of familial amyotrophic lateral sclerosis. Brain Res 982:92–97[CrossRef][Medline]
  18. Laura M, Gregson NA, Curmi Y, Hughes RA 2002 Efficacy of leukemia inhibitory factor in experimental autoimmune neuritis. J Neuroimmunol 133:56–59[CrossRef][Medline]
  19. Kurek J 2000 AM424: history of a novel drug candidate. Clin Exp Pharmacol Physiol 27:553–557[CrossRef][Medline]
  20. Beretta E, Dhillon H, Kalra PS, Kalra SP 2002 Central LIF gene therapy suppresses food intake, body weight, serum leptin and insulin for extended periods. Peptides 23:975–984[CrossRef][Medline]
  21. Pan W, Kastin AJ, Brennan JM 2000 Saturable entry of leukemia inhibitory factor from blood to the central nervous system. J Neuroimmunol 106:172–180[CrossRef][Medline]
  22. Bok D, Yasumura D, Matthes MT, Ruiz A, Duncan JL, Chappelow AV, Zolotukhin S, Hauswirth W, LaVail MM 2002 Effects of adeno-associated virus-vectored ciliary neurotrophic factor on retinal structure and function in mice with a P216L rds/peripherin mutation. Exp Eye Res 74:719–735[CrossRef][Medline]
  23. Mori M, Yamaguchi K, Honda S, Nagasaki K, Ueda M, Abe O, Abe K 1991 Cancer cachexia syndrome developed in nude mice bearing melanoma cells producing leukemia-inhibitory factor. Cancer Res 51:6656–6659[Abstract/Free Full Text]
  24. Zolotukhin S, Byrne BJ, Mason E, Zolotukhin I, Potter M, Chesnut K, Summerford C, Samulski RJ, Muzyczka N 1999 Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 6:973–985[CrossRef][Medline]
  25. Grimm D, Kern A, Rittner K, Kleinschmidt JA 1998 Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum Gene Ther 9:2745–2760[Medline]
  26. Almeida TA, Perez JA, Pinto FM 2000 Size-fractionation of RNA by hot agarose electrophoresis. Biotechniques 28:414–416[Medline]
  27. Sambrook J, Fritsch EF, Maniatis T 2001 Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press
  28. Austin L, Bower JJ, Kurek JB, Muldoon CM 1997 Controlled release of leukaemia inhibitory factor (LIF) to tissues. Growth Factors 15:61–68[Medline]
  29. Meister B, Hakansson ML 2001 Leptin receptors in hypothalamus and circumventricular organs. Clin Exp Pharmacol Physiol 28:610–617[CrossRef][Medline]
  30. Ahima RS, Kelly J, Elmquist JK, Flier JS 1999 Distinct physiologic and neuronal responses to decreased leptin and mild hyperleptinemia. Endocrinology 140:4923–4931[Abstract/Free Full Text]
  31. Ahima RS, Saper CB, Flier JS, Elmquist JK 2000 Leptin regulation of neuroendocrine systems. Front Neuroendocrinol 21:263–307[CrossRef][Medline]
  32. Broberger C, Hokfelt T 2001 Hypothalamic and vagal neuropeptide circuitries regulating food intake. Physiol Behav 74:669–682[CrossRef][Medline]
  33. Elmquist JK 2001 Hypothalamic pathways underlying the endocrine, autonomic, and behavioral effects of leptin. Physiol Behav 74:703–708[CrossRef][Medline]
  34. Toni R, Jackson IM, Lechan RM 1990 Neuropeptide-Y-immunoreactive innervation of thyrotropin-releasing hormone-synthesizing neurons in the rat hypothalamic paraventricular nucleus. Endocrinology 126:2444–2453[Abstract/Free Full Text]
  35. Broberger C, Visser TJ, Kuhar MJ, Hokfelt T 1999 Neuropeptide Y innervation and neuropeptide-Y-Y1-receptor-expressing neurons in the paraventricular hypothalamic nucleus of the mouse. Neuroendocrinology 70:295–305[CrossRef][Medline]
  36. Diano S, Naftolin F, Goglia F, Horvath TL 1998 Segregation of the intra- and extrahypothalamic neuropeptide Y and catecholaminergic inputs on paraventricular neurons, including those producing thyrotropin-releasing hormone. Regul Pept 75–76:117–126
  37. Fekete C, Legradi G, Mihaly E, Tatro JB, Rand WM, Lechan RM 2000 {alpha}-Melanocyte stimulating hormone prevents fasting-induced suppression of corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular nucleus. Neurosci Lett 289:152–156[CrossRef][Medline]
  38. Legradi G, Lechan RM 1998 The arcuate nucleus is the major source for neuropeptide Y-innervation of thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus. Endocrinology 139:3262–3270[Abstract/Free Full Text]
  39. Li C, Chen P, Smith MS 2000 Corticotropin releasing hormone neurons in the paraventricular nucleus are direct targets for neuropeptide Y neurons in the arcuate nucleus: an anterograde tracing study. Brain Res 854:122–129[CrossRef][Medline]
  40. Kagotani Y, Tsuruo Y, Hisano S, Daikoku S, Chihara K 1989 Synaptic regulation of paraventricular arginine vasopressin-containing neurons by neuropeptide Y-containing monoaminergic neurons in rats. Electron-microscopic triple labeling. Cell Tissue Res 257:269–278[CrossRef][Medline]
  41. Piekut DT 1985 Relationship of ACTH1–39-immunostained fibers and magnocellular neurons in the paraventricular nucleus of rat hypothalamus. Peptides 6:883–890[CrossRef][Medline]
  42. Sawchenko PE, Pfeiffer SW 1988 Ultrastructural localization of neuropeptide Y and galanin immunoreactivity in the paraventricular nucleus of the hypothalamus in the rat. Brain Res 474:231–245[CrossRef][Medline]
  43. Morimoto I, Yamamoto S, Kai K, Fujihira T, Morita E, Eto S 2000 Centrally administered murine-leptin stimulates the hypothalamus-pituitary-adrenal axis through arginine-vasopressin. Neuroendocrinology 71:366–374[CrossRef][Medline]
  44. Tanimura SM, Watts AG 1998 Corticosterone can facilitate as well as inhibit corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular nucleus. Endocrinology 139:3830–3836[Abstract/Free Full Text]
  45. Billington CJ, Briggs JE, Grace M, Levine AS 1991 Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism. Am J Physiol 260:R321–R327
  46. Barrachina MD, Martinez V, Wang L, Wei JY, Tache Y 1997 Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice. Proc Natl Acad Sci USA 94:10455–10460[Abstract/Free Full Text]
  47. Marsh DJ, Hollopeter G, Huszar D, Laufer R, Yagaloff KA, Fisher SL, Burn P, Palmiter RD 1999 Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides. Nat Genet 21:119–122[CrossRef][Medline]
  48. Turrin NP, Plata-Salaman CR 2000 Cytokine-cytokine interactions and the brain. Brain Res Bull 51:3–9[CrossRef][Medline]
  49. Wagstaff MJ, Collaco-Moraes Y, Smith J, de Belleroche JS, Coffin RS, Latchman DS 1999 Protection of neuronal cells from apoptosis by Hsp27 delivered with a herpes simplex virus-based vector. J Biol Chem 274:5061–5069[Abstract/Free Full Text]
  50. David JC, Landry J, Grongnet JF 2000 Perinatal expression of heat-shock protein 27 in brain regions and nonneural tissues of the piglet. J Mol Neurosci 15:109–120[CrossRef][Medline]
  51. Lee JE, Yenari MA, Sun GH, Xu L, Emond MR, Cheng D, Steinberg GK, Giffard RG 2001 Differential neuroprotection from human heat shock protein 70 overexpression in in vitro and in vivo models of ischemia and ischemia-like conditions. Exp Neurol 170:129–139[CrossRef][Medline]
  52. Harada S, Kamiya K, Masago A, Iwata A, Yamada K 1997 Subarachnoid hemorrhage induces c-fos, c-jun and hsp70 mRNA expression in rat brain. Neuroreport 8:3399–3404[Medline]
  53. Mehlen P, Schulze-Osthoff K, Arrigo AP 1996 Small stress proteins as novel regulators of apoptosis. Heat shock protein 27 blocks Fas/APO-1- and staurosporine-induced cell death. J Biol Chem 271:16510–16514[Abstract/Free Full Text]
  54. Akbar MT, Wells DJ, Latchman DS, de Belleroche J 2001 Heat shock protein 27 shows a distinctive widespread spatial and temporal pattern of induction in CNS glial and neuronal cells compared to heat shock protein 70 and caspase 3 following kainate administration. Brain Res Mol Brain Res 93:148–163[Medline]
  55. Suzuki S, Li AJ, Ishisaki A, Hou X, Hasegawa M, Fukumura M, Akaike T, Imamura T 2001 Feeding suppression by fibroblast growth factor-1 is accompanied by selective induction of heat shock protein 27 in hypothalamic astrocytes. Eur J Neurosci 13:2299–2308[CrossRef][Medline]
  56. Aly KB, Pipkin JL, Hinson WG, Feuers RJ, Duffy PH, Lyn-Cook L, Hart RW 1994 Chronic caloric restriction induces stress proteins in the hypothalamus of rats. Mech Ageing Dev 76:11–23[CrossRef][Medline]
  57. Akiyama Y, Kajimura N, Matsuzaki J, Kikuchi Y, Imai N, Tanigawa M, Yamaguchi K 1997 In vivo effect of recombinant human leukemia inhibitory factor in primates. Jpn J Cancer Res 88:578–583[CrossRef][Medline]
  58. Zolotukhin S, Potter M, Hauswirth WW, Guy J, Muzyczka N 1996 A "humanized" green fluorescent protein cDNA adapted for high-level expression in mammalian cells. J Virol 70:4646–4654[Abstract]
  59. Miyazaki J, Takaki S, Araki K, Tashiro F, Tominaga A, Takatsu K, Yamamura K 1989 Expression vector system based on the chicken ß-actin promoter directs efficient production of interleukin-5. Gene 79:269–277[CrossRef][Medline]
  60. Loeb JE, Cordier WS, Harris ME, Weitzman MD, Hope TJ 1999 Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: implications for gene therapy. Hum Gene Ther 10:2295–2305[CrossRef][Medline]



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