A regulatable adenovector system for GDNF and GFP delivery in the rat hippocampus.

Spatial memory performance declines in both normal aging and Alzheimer's disease. This cognitive deficit is related to hippocampus dysfunction. Gene therapy using neurotrophic factors like Glial cell line-derived neurotrophic factor (GDNF) emerges as a promising approach to ameliorate age-related cognitive deficits. We constructed a two vector regulatable system (2VRS) which consists of a recombinant adenoviral vector (RAd) harboring a Tet-Off bidirectional promoter flanked by GDNF and Green Fluorescent Protein (GFP) genes. A second adenovector, RAd-tTA, constitutively expresses the regulatory protein tTA. When cells are cotransduced by the 2VRS, tTA activates the bidirectional promoter and both transgenes are expressed. In the presence of the antibiotic doxycycline (DOX) transgene expression is silenced. We tested the 2VRS in CHO-K1 cells where we observed a dose-dependent GFP expression that was completely inhibited by DOX (1 mg/ml). The 2VRS injected in the hippocampal CA1 region transduced both neurons and astrocytes and was efficiently inhibited by DOX added to the drinking water. In order to assess GDNF biological activity we injected 2VRS and its Control (CTRL) vector in the hypothalamus and monitored body weight for one month. The results showed that GDNF retards weight recovery 6 days more than CTRL. In conclusion, our 2VRS demonstrated optimal GFP expression and showed a bioactive effect of transgenic GDNF in the brain.

Partial Reprogramming As An Emerging Strategy for Safe Induced Cell Generation and Rejuvenation.

BACKGROUND: Conventional cell reprogramming involves converting a somatic cell line into induced pluripotent stem cells (iPSC), which subsequently can be re-differentiated to specific somatic cell types. Alternatively, partial cell reprogramming converts somatic cells into other somatic cell types by transient expression of pluripotency genes thus generating intermediates that retain their original cell identity, but are responsive to appropriate cocktails of specific differentiation factors. Additionally, biological rejuvenation by partial cell reprogramming is an emerging avenue of research. OBJECTIVE: Here, we will briefly review the emerging information pointing to partial reprogramming as a suitable strategy to achieve cell reprogramming and rejuvenation, bypassing cell dedifferentiation. METHODS: In this context, regulatable pluripotency gene expression systems are the most widely used at present to implement partial cell reprogramming. For instance, we have constructed a regulatable bidirectional adenovector expressing Green Fluorescent Protein and oct4, sox2, klf4 and c-myc genes (known as the Yamanaka genes or OSKM). RESULTS: Partial cell reprogramming has been used to reprogram fibroblasts to cardiomyocytes, neural progenitors and neural stem cells. Rejuvenation by cyclic partial reprogramming has been achieved both in vivo and in cell culture using transgenic mice and cells expressing the OSKM genes, respectively, controlled by a regulatable promoter. CONCLUSION: Partial reprogramming emerges as a powerful tool for the genesis of iPSC-free induced somatic cells of therapeutic value and for the implementation of in vitro and in vivo rejuvenation keeping cell type identity unchanged.

Umbilical Cord Cell Therapy Improves Spatial Memory in Aging Rats.

There is a growing interest in the potential of adult stem cells for implementing regenerative medicine in the brain. We assessed the effect of intracerebroventricular (icv) administration of human umbilical cord perivascular cells (HUCPVCs) on spatial memory of senile (27 mo) female rats, using intact senile counterparts as controls. Approximately one third of the animals were injected in the lateral ventricles with a suspension containing 4.8 X 105 HUCPVC in 8 µl per side. The other third received 4.8 X 105 transgenic HUCPVC overexpressing Insulin-like growth factor-1 (IGF-1) and the last third of the rats received no treatment. Spatial memory performance was evaluated using a modified version of the Barnes maze test. In order to evaluate learning ability as well as spatial memory retention, we assessed the time spent (permanence) by animals in goal sector 1 (GS1) and 3 (GS3) when the escape box was removed. Fluorescence microscopy revealed the prescence of Dil-labeled HUCPVC in coronal sections of treated brains. The HUCPVC were located in close contact with the ependymal cells with only a few labeled cells migrating into the brain parenchyma. After treatment with naïve or IGF-1 transgenic HUCPVC, permanence in GS1 and GS3 increased significantly whereas there were no changes in the intact animals. We conclude that HUCPVC injected icv are effective to improve some components of spatial memory in senile rats. The ready accessibility of HUCPVC constitutes a significant incentive to continue the exploration of their therapeutic potential on neurodegenerative diseases.

Mesenchymal stem cell therapy improves spatial memory and hippocampal structure in aging rats.

There is a growing interest in the potential of mesenchymal stem cells (MSCs) for implementing regenerative medicine in the brain as they have shown neurogenic and immunomodulatory activities. We assessed the effect of intracerebroventricular (icv) administration of human bone marrow-derived MSCs (hBM-MSCs) on spatial memory and hippocampal morphology of senile (27 months) female rats, using 3-months-old counterparts as young controls. Half of the animals were injected in the lateral ventricles (LV) with a suspension containing 5 × 105hBM-MSCs in 8 µl per side. The other half received no treatment (senile controls). Spatial memory performance was assessed with a modified version of the Barnes maze test. We employed one probe trial, one day after training in order to evaluate learning ability as well as spatial memory retention. Neuroblast (DCX) and microglial (Iba-1 immunoreactive) markers were also immunohistochemically quantitated in the animals by means of an unbiased stereological approach. In addition, hippocampal presynaptic protein expression was assessed by immunoblotting analysis. After treatment, the senile MSC-treated group showed a significant improvement in spatial memory accuracy and extended permanence in a one- and 3-hole goal sectors as compared with senile controls. The MSC treatment increased the number of neuroblasts in the hippocampal dentate gyrus, reduced the number of reactive microglial cells, and restored presynaptic protein levels as compared to senile controls. We conclude that icv injected hBM-MSCs are effective in improving spatial memory in senile rats and that the strategy improves some functional and morphologic brain features typically altered in aging rats.

Regulatable adenovector harboring the GFP and Yamanaka genes for implementing regenerative medicine in the brain.

Biological rejuvenation by partial cell reprogramming is an emerging avenue of research. In this context, regulatable pluripotency gene expression systems are the most widely used at present. We have constructed a regulatable bidirectional adenovector expressing the humanized green fluorescent protein (GFP) and oct4, sox2, klf4, and c-myc genes (known as the Yamanaka genes or OSKM). The OSKM genes are arranged as a bicistronic tandem (hSTEMCCA tandem), which is under the control of a Tet-Off bidirectional promoter that also controls the expression of the gFP gene. Separately, a constitutive cassette expresses the regulatory protein tTA. Vector DNA was transfected in HEK293 Cre cells, which were additionally infected with the helper adenovector H14, unable to package its DNA due to the Cre recombinase produced by the HEK293 Cre cells. The newly generated vector was expanded by six iterated coinfections of the above cells which were lysed at the end of the process and the adenovector purified by ultracentrifugation in a CsCl gradient. The titer of the initial preparation was 1.2 × 1012 physical viral particles/ml. As expected, GFP fluorescence in vector-transduced rat fibroblast cultures declined with the dose of doxycycline (DOX) present in the medium. Immunocytochemical analysis of transduced cells confirmed the expression of the four Yamanaka genes. Additionally, 3 days after vector injection in the hypothalamus of rats, a significant level of fluorescence was observed in the region. Addition of 2 mg/ml DOX to the drinking water reduced the GFP expression. This adenovector constitutes a promising tool for implementing nonintegrative partial cell reprogramming.

IGF-1 Gene Therapy as a Potentially Useful Therapy for Spontaneous Prolactinomas in Senile Rats.

BACKGROUND: Insulin-like Growth Factor1 (IGF1) is a powerful neuroprotective molecule. We have previously shown that short-term hypothalamic IGF1 gene therapy restores tuberoinfundibular dopaminergic neuron function in aging female rats. OBJECTIVE: Our aim was to implement long-term IGF-I gene therapy in pituitary prolactinomas in senile female rats. METHODS: Here, we assessed the long-term effect of IGF1 gene therapy in the hypothalamus of young (4 mo.) and aging (24 mo.) female rats carrying spontaneous pituitary prolactinomas. We constructed and injected a Helper-Dependent (HD) adenovector expressing the gene for rat IGF1 or the reporter red fluorescent protein DsRed. Ninety-one days post vector injection, all rats were sacrificed and their brains and pituitaries fixed. Serum prolactin (PRL), Estrogen (E2) and progesterone (P4), as well as hypothalamic IGF1 content, were measured by RIA. Anterior pituitaries were immunostained with an anti-rat PRL antibody and submitted to morphometric analysis. RESULTS: DsRed expression in the Mediobasal Hypothalamus (MBH) was strong after the treatment in the DsRed group while IGF1 content in the MBH was higher in the IGF1 group. The IGF1 treatment affected neither pituitary weight nor PRL, E2 or P4 serum levels in the young rats. In the old rats, IGF1 gene therapy reduced gland weight as compared with intact counterparts and tended to reduce PRL levels as compared with intact counterparts. The treatment significantly rescued the phenotype of the lactotropic cell population in the senile adenomas. CONCLUSION: We conclude that long-term hypothalamic IGF1 gene therapy is effective to rescue spontaneous prolactinomas in aging female rats.

IGF-I Gene Therapy in Aging Rats Modulates Hippocampal Genes Relevant to Memory Function.

In rats, learning and memory performance decline during normal aging, which makes this rodent species a suitable model to evaluate therapeutic strategies. In aging rats, insulin-like growth factor-I (IGF-I), is known to significantly improve spatial memory accuracy as compared to control counterparts. A constellation of gene expression changes underlie the hippocampal phenotype of aging but no studies on the effects of IGF-I on the hippocampal transcriptome of old rodents have been documented. Here, we assessed the effects of IGF-I gene therapy on spatial memory performance in old female rats and compared them with changes in the hippocampal transcriptome. In the Barnes maze test, experimental rats showed a significantly higher exploratory frequency of the goal hole than controls. Hippocampal RNA-sequencing showed that 219 genes are differentially expressed in 28-month-old rats intracerebroventricularly injected with an adenovector expressing rat IGF-I as compared with placebo adenovector-injected counterparts. From the differentially expressed genes, 81 were down and 138 upregulated. From those genes, a list of functionally relevant genes, concerning hippocampal IGF-I expression, synaptic plasticity as well as neuronal function was identified. Our results provide an initial glimpse at the molecular mechanisms underlying the neuroprotective actions of IGF-I in the aging brain.

Rejuvenating Effect of Long-Term Insulin-Like Growth Factor-I Gene Therapy in the Hypothalamus of Aged Rats with Dopaminergic Dysfunction.

The aging female rat constitutes an interesting model of spontaneous and progressive age-related dopaminergic dysfunction as it allows assessing new therapeutic strategies for Parkinson's disease. Insulin-like growth factor I (IGF-I) is emerging as a powerful neuroprotective molecule, strongly induced in the central nervous system after different insults. We constructed a helper-dependent recombinant adenoviral vector (HDRAd-IGFI) harboring the gene for rat IGF-I. This was used to implement long-term IGF-I gene therapy in the hypothalamus of aged female rats, which display hypothalamic dopaminergic (DA) dysfunction and, as a consequence, chronic hyperprolactinemia. Rejuvenating long-term IGF-I gene therapy was implemented in young (3 months) and aged (24 months) female rats, which received a single intrahypothalamic injection of 4 × 109 viral particles of either HD-RAd-IGFI or HD-RAd-DsRed (control vector) and were sacrificed 119 days postinjection. In the young animals, neither vector modified serum prolactin (PRL) levels, but in the RAd-IGFI-injected aged rats a nearly full reversion of their hyperprolactinemic status was recorded. Morphometric analysis revealed a significant increase in the total number of tyrosine hydroxylase (TH)-positive cells in the hypothalamus of experimental compared with control aged animals (5874 ± 486 and 3390 ± 498, respectively). Our results indicate that IGF-I gene therapy in aged female rats is highly effective in rejuvenating the hypothalamic DA neuron groups.

A new adenovector system for implementing thymulin gene therapy for inflammatory disorders.

Thymulin is a thymic peptide possessing anti-inflammatory effects. In order to manipulate thymulin expression in gene therapy studies, we built a bidirectional regulatable two-vector Tet-Off system and the corresponding control system. The experimental two-vector system, ETV, consists of a recombinant adenovector (RAd) harboring an expression cassette centered on a Tet-Off bidirectional promoter flanked by a synthetic gene for thymulin and the gene for humanized Green Fluorescent Protein (hGFP). The second adenovector of this system, RAd-tTA, constitutively expresses the regulatory protein tTA. When cells are co-transduced by the two adenovector components, tTA activates the bidirectional promoter and both transgenes are expressed. In the presence of the antibiotic doxycycline (DOX) transgene expression is deactivated. The control two-vector system, termed CTV, is similar to ETV but only expresses hGFP. In CHO-K1, BHK, and C2C12 cells, ETV and CTV induced a dose-dependent hGFP expression. In CHO-K1 cells, transgene expression was almost completely inhibited by DOX (1mg/ml). After intracerebroventricular injection of ETV in rats, thymulin levels increased significantly in the cerebrospinal fluid and there was high hGFP expression in the ependymal cell layer. When injected intramuscularly the ETV system induced a progressive increase in serum thymulin levels, which were inhibited when DOX was added to the drinking water. We conclude that our regulatable two-adenovector system is an effective molecular tool for implementing short and long-term anti-inflammatory thymulin gene therapy in animal models of acute or chronic inflammation.

Identification of a conserved gene signature associated with an exacerbated inflammatory environment in the hippocampus of aging rats.

There have been a few descriptive studies in aged rodents about transcriptome changes in the hippocampus, most of them in males. Here, we assessed the age changes in spatial memory performance and hippocampal morphology in female rats and compared those changes with changes in the hippocampal transcriptome. Old rats displayed significant deficits in spatial memory. In both age groups, hole exploration frequency showed a clear peak at hole 0 (escape hole), but the amplitude of the peak was significantly higher in the young than in the old animals. In the hippocampus, there was a dramatic reduction in neurogenesis, whereas reactive microglial infiltrates revealed an inflammatory hippocampal state in the senile rats. Hippocampal RNA-sequencing showed that 210 genes are differentially expressed in the senile rats, most of them being downregulated. Our RNA-Seq data showed that various genes involved in the immune response, including TYROBP, CD11b, C3, CD18, CD4, and CD74, are overexpressed in the hippocampus of aged female rats. Enrichment analysis showed that the pathways overrepresented in the senile rats matched those of an exacerbated inflammatory environment, reinforcing our morphologic findings. After correlating our results with public data of human and mouse hippocampal gene expression, we found an 11-gene signature of overexpressed genes related to inflammatory processes that was conserved across species. We conclude that age-related hippocampal deficits in female rats share commonalities between human and rodents. Interestingly, the 11-gene signature that we identified may represent a cluster of immune and regulatory genes that are deregulated in the hippocampus and possibly other brain regions during aging as well as in some neurodegenerative diseases and low-grade brain tumors. Our study further supports neuroinflammation as a promising target to treat cognitive dysfunction in old individuals and some brain tumors. © 2017 Wiley Periodicals, Inc.

Insulin-like growth factor-I gene therapy increases hippocampal neurogenesis, astrocyte branching and improves spatial memory in female aging rats.

In rats, learning and memory performance decline during aging, which makes this rodent species a suitable model to evaluate therapeutic strategies of potential value for correcting age-related cognitive deficits. Some of these strategies involve neurotrophic factors like insulin-like growth factor-I (IGF-I), a powerful neuroprotective molecule in the brain. Here, we implemented 18-day long intracerebroventricular (ICV) IGF-I gene therapy in 28 months old Sprague-Dawley female rats, and assessed spatial memory performance in the Barnes maze. We also studied hippocampal morphology using an unbiased stereological approach. Adenovectors expressing the gene for rat IGF-I or the reporter DsRed were used. Cerebrospinal fluid (CSF) samples were taken and IGF-I levels determined by radioimmunoassay. At the end of the study, IGF-I levels in the CSF were significantly higher in the experimental group than in the DsRed controls. After treatment, the IGF-I group showed a significant improvement in spatial memory accuracy as compared with DsRed counterparts. In the dentate gyrus (DG) of the hippocampus, the IGF-I group showed a higher number of immature neurons than the DsRed controls. The treatment increased hippocampal astrocyte branching and reduced their number in the hippocampal stratum radiatum. We conclude that the ependymal route is an effective approach to increase CSF levels of IGF-I and that this strategy improves the accuracy of spatial memory in aging rats. The favorable effect of the treatment on DG neurogenesis and astrocyte branching in the stratum radiatum may contribute to improving memory performance in aging rats.

A Rat Treated with Mesenchymal Stem Cells Lives to 44 Months of Age.

There is a growing interest in the potential of mesenchymal stem cells (MSCs) for implementing regenerative medicine. We assessed the effect of intravenous administration of human bone marrow-derived MSC on the life span of a single Sprague-Dawley female rat. The treatment was started when the rat was 6 months old and the cells were administered every 2 weeks afterward. The treatment did not induce any obvious changes in body growth or behavior and the rat showed the typical age changes for this strain, except that, unlike intact counterparts, the animal did not develop mammary tumors or pituitary gland hyperplasia. The more remarkable effect of the treatment was on life span, which was 44 months compared with an average of 36 months for intact laboratory rats. We conclude that despite the low N value, it is likely that the MSC treatment was responsible for the exceptionally long survival of the rat. The potential rewards of confirming the present findings warrant further studies involving higher N values.

Physiology and therapeutic potential of the thymic peptide thymulin.

Thymulin is a thymic hormone exclusively produced by the epithelial cells of the thymus. After its discovery and initial characterization in the '70s, it was demonstrated that the production and secretion of thymulin are strongly influenced by the neuro-endocrine system. Conversely, a growing body of evidence, to be reviewed here, suggests that thymulin is a hypophysiotropic peptide. Additionally, a substantial body of information pointing to thymulin and a synthetic analog as anti-inflammatory and analgesic peptides in the central nervous system brain and other organs will be also reviewed. In recent years, a synthetic DNA sequence encoding a biologically active analog of thymulin, metFTS, was constructed and cloned in a number of adenovectors. These include bidirectional regulatable Tet-Off vector systems that simultaneously express metFTS and green fluorescent protein and that can be down-regulated reversibly by the addition of the antibiotic doxycycline. A number of recent studies indicate that gene therapy for thymulin may be an effective therapeutic strategy to prevent some of the hormonal and reproductive abnormalities that typically appear in congenitally athymic (nude) mice, used as a suitable model of neuroendocrine and reproductive aging. Summing up, this article briefly reviews the publications on the physiology of the thymulin-neuroendocrine axis and the anti-inflammatory properties of the molecule and its analog. The availability of novel biotechnological tools should boost basic studies on the molecular biology of thymulin and should also allow an assessment of the potential of gene therapy to restore circulating thymulin levels in thymodeficient animal models and eventually, in humans.

Regenerative Medicine for the Aging Brain.

In the central nervous system, cholinergic and dopaminergic (DA) neurons are among the cells most susceptible to the deleterious effects of age. Thus, the basal forebrain cholinergic system is known to undergo moderate neurodegenerative changes during normal aging as well as severe atrophy in Alzheimer's disease (AD). Parkinson's disease (PD), a degeneration of nigro-striatal DA neurons is the most conspicuous reflection of the vulnerability of DA neurons to age. In this context, cell reprogramming offers novel therapeutic possibilities for the treatment of these devastating diseases. In effect, the generation of induced pluripotent stem cells (iPSCs) from somatic cells demonstrated that adult mammalian cells can be reprogrammed to a pluripotent state by the overexpression of a few embryonic transcription factors (TF). This discovery fundamentally widened the research horizon in the fields of disease modeling and regenerative medicine. Although it is possible to re-differentiate iPSCs to specific somatic cell types, the tumorigenic potential of contaminating iPSCs that failed to differentiate, increases the risk for clinical application of somatic cells generated by this procedure. Therefore, reprogramming approaches that bypass the pluripotent stem cell state are being explored. A method called lineage reprogramming has been recently documented. It consists of the direct conversion of one adult cell type into another by transgenic expression of multiple lineage-specific TF or microRNAs. Another approach, termed direct reprogramming, features several advantages such as the use of universal TF system and the ability to generate a rejuvenated multipotent progenitor cell population, able to differentiate into specific cell types in response to a specific differentiation factors. These novel approaches offer a new promise for the treatment of pathologies associated with the loss of specific cell types as for instance, nigral DA neurons (in PD) or basal forebrain cholinergic neurons in the early stages of AD. The above topics are reviewed here.

Hypothalamic IGF-I gene therapy prolongs estrous cyclicity and protects ovarian structure in middle-aged female rats.

There is substantial evidence that age-related ovarian failure in rats is preceded by abnormal responsiveness of the neuroendocrine axis to estrogen positive feedback. Because IGF-I seems to act as a permissive factor for proper GnRH neuronal response to estrogen positive feedback and considering that the hypothalamic content of IGF-I declines in middle-aged (M-A) rats, we assessed the effectiveness of long-term IGF-I gene therapy in the mediobasal hypothalamus (MBH) of M-A female rats to extend regular cyclicity and preserve ovarian structure. We used 3 groups of M-A rats: 1 group of intact animals and 2 groups injected, at 36.2 weeks of age, in the MBH with either a bicistronic recombinant adeno-associated virus (rAAV) harboring the genes for IGF-I and the red fluorescent protein DsRed2, or a control rAAV expressing only DsRed2. Daily vaginal smears were taken throughout the study, which ended at 49.5 weeks of age. We measured serum levels of reproductive hormones and assessed ovarian histology at the end of the study. Although most of the rats injected with the IGF-I rAAV had, on the average, well-preserved estrous cyclicity as well as a generally normal ovarian histology, the intact and control rAAV groups showed a high percentage of acyclic rats at the end of the study and ovaries with numerous enlarged cysts and scarce corpora lutea. Serum LH was higher and hyperprolactinemia lower in the treated animals. These results suggest that overexpression of IGF-I in the MBH prolongs normal ovarian function in M-A female rats.

Role of thymulin on the somatotropic axis in vivo.

AIMS: There is clear evidence for the existence of a bi-directional thymus-somatotropic axis and several studies suggest that the thymic peptide thymulin may be involved in this communication. We undertook to assess the impact of serum thymulin immunoneutralization in C57BL/6 mice and that of neonatal thymulin gene therapy (NTGT) in nude mice on body weight (BW) gain and on the histomorphometric profile of the somatotrope population. MAIN METHODS: Immunoneutralization of thymulin was done from postnatal day 1 to 35 by i.p. injections of rabbit anti-thymulin serum (α-FTS) and normal rabbit serum (NRS) in controls. NTGT was implemented in nudes using an adenoviral vector expressing a synthetic gene for thymulin (RAd-FTS). On postnatal day 1, heterozygous (nu/+) and homozygous (nu/nu) pups received a single bilateral i.m. injection either RAd-FTS or RAd-GFP (a control vector expressing green fluorescent protein). BW gain was recorded and at the end of the study the pituitaries were immunostained for growth hormone (GH). Serum GH and thymulin were determined by radioimmunoassay and bioassay, respectively. KEY FINDINGS: Thymulin immunoneutralization induced a significant decrease in BW gain, serum GH and somatotrope cell density as well as an increase in somatotrope cell size. NTGT markedly increased BW gain, serum thymulin (P<0.01) and somatotrope cell and volume density in nu/nu mice. SIGNIFICANCE: Our results suggest that thymulin plays a relevant physiological role on the thymus-somatotropic axis in mice.

Neonatal thymulin gene therapy prevents ovarian dysgenesis and attenuates reproductive derangements in nude female mice.

Congenitally athymic (nude) female mice show severe ovarian dysgenesis after puberty, which seems to be consequential to a number of neuroendocrine derangements described in these mutants. Thus, considerable evidence suggests that thymulin, a thymic peptide, may be involved in thymus-pituitary communication. In order to clarify the relevance of thymulin for the maturation of the female reproductive system, we assessed at hypothalamic, pituitary, ovarian, and uterine level the preventive action of neonatal thymulin gene therapy (NTGT) on the changes that typically occur after puberty in congenitally athymic female mice. We injected (im) an adenoviral vector harboring a synthetic DNA sequence encoding a biologically active analog of thymulin, methionine-serum thymic factor, in newborn nude mice (which are thymulin deficient) and killed the animals at 70-71 d of age. NTGT in the athymic mice restored the serum thymulin levels. Morphometric analysis revealed that athymic nudes have reduced numbers of brain GnRH neurons and pituitary gonadotropic cells as compared with heterozygous controls. NTGT prevented these changes and also rescued the premature ovarian failure phenotype typically observed in athymic nude mice (marked reduction in the number of antral follicles and corpora lutea, increase in atretic follicles). Serum estrogen, but not progesterone, levels were low in athymic nudes, a reduction that was partially prevented by NTGT. Little to no morphological changes were observed in the endometrium of female nudes. The delay in the age of vaginal opening that occurs in athymic nudes was significantly prevented by NTGT. Our results suggest that thymulin plays a relevant physiologic role in the thymus-hypothalamo-pituitary-gonadal axis.

Magnetic field-assisted gene delivery: achievements and therapeutic potential.

The discovery in the early 2000's that magnetic nanoparticles (MNPs) complexed to nonviral or viral vectors can, in the presence of an external magnetic field, greatly enhance gene transfer into cells has raised much interest. This technique, called magnetofection, was initially developed mainly to improve gene transfer in cell cultures, a simpler and more easily controllable scenario than in vivo models. These studies provided evidence for some unique capabilities of magnetofection. Progressively, the interest in magnetofection expanded to its application in animal models and led to the association of this technique with another technology, magnetic drug targeting (MDT). This combination offers the possibility to develop more efficient and less invasive gene therapy strategies for a number of major pathologies like cancer, neurodegeneration and myocardial infarction. The goal of MDT is to concentrate MNPs functionalized with therapeutic drugs, in target areas of the body by means of properly focused external magnetic fields. The availability of stable, nontoxic MNP-gene vector complexes now offers the opportunity to develop magnetic gene targeting (MGT), a variant of MDT in which the gene coding for a therapeutic molecule, rather than the molecule itself, is delivered to a therapeutic target area in the body. This article will first outline the principle of magnetofection, subsequently describing the properties of the magnetic fields and MNPs used in this technique. Next, it will review the results achieved by magnetofection in cell cultures. Last, the potential of MGT for implementing minimally invasive gene therapy will be discussed.

Thymulin-based gene therapy and pituitary function in animal models of aging.

Thymulin is a thymic hormone exclusively produced by the thymic epithelial cells. After its discovery and initial characterization in the 1970s, it was demonstrated that thymulin production and secretion is strongly influenced by the neuroendocrine system. Conversely, a growing core of information, to be reviewed here, points to thymulin as a hypophysiotropic peptide. Additionally, thymulin was shown to possess anti-inflammatory and analgesic properties in the brain. In recent years, a synthetic DNA sequence coding for a biologically active analog of thymulin, metFTS, was constructed and cloned in different adenoviral vectors. These include bidirectional regulatable Tet-Off vector systems that simultaneously express metFTS and green fluorescent protein and that can be downregulated reversibly by the addition of the antibiotic doxycycline. A number of recent studies suggest that thymulin gene therapy may be a suitable therapeutic strategy to prevent some of the endocrine and reproductive alterations that typically appear in congenitally athymic (nude) mice, taken as a suitable model of neuroendocrine and reproductive aging. The present article briefly reviews the literature on the physiology of the thymulin-pituitary axis as well as on the new molecular tools available to exploit the therapeutic potential of thymulin.

Insulin-like growth factor-I gene delivery to astrocytes reduces their inflammatory response to lipopolysaccharide.

BACKGROUND: Insulin-like growth factor-I (IGF-I) exerts neuroprotective actions in the central nervous system that are mediated at least in part by control of activation of astrocytes. In this study we have assessed the efficacy of exogenous IGF-I and IGF-I gene therapy in reducing the inflammatory response of astrocytes from cerebral cortex. METHODS: An adenoviral vector harboring the rat IGF-I gene and a control adenoviral vector harboring a hybrid gene encoding the herpes simplex virus type 1 thymidine kinase fused to Aequorea victoria enhanced green fluorescent protein were used in this study. Primary astrocytes from mice cerebral cortex were incubated for 24 h or 72 h with vehicle, IGF-I, the IGF-I adenoviral vector, or control vector; and exposed to bacterial lipopolysaccharide to induce an inflammatory response. IGF-I levels were measured by radioimmunoassay. Levels of interleukin 6, tumor necrosis factor-α, interleukin-1ß and toll-like receptor 4 mRNA were assessed by quantitative real-time polymerase chain reaction. Levels of IGF-I receptor and IGF binding proteins 2 and 3 were assessed by western blotting. The subcellular distribution of nuclear factor κB (p65) was assessed by immunocytochemistry. Statistical significance was assessed by one way analysis of variance followed by the Bonferroni pot hoc test. RESULTS: IGF-I gene therapy increased IGF-I levels without affecting IGF-I receptors or IGF binding proteins. Exogenous IGF-I, and IGF-I gene therapy, decreased expression of toll-like receptor 4 and counteracted the lipopolysaccharide-induced inflammatory response of astrocytes. In addition, IGF-I gene therapy decreased lipopolysaccharide-induced translocation of nuclear factor κB (p65) to the cell nucleus. CONCLUSION: These findings demonstrate efficacy of exogenous IGF-I and of IGF-I gene therapy in reducing the inflammatory response of astrocytes. IGF-I gene therapy may represent a new approach to reduce inflammatory reactions in glial cells.