Biomedical subjects

Antonis Voutetakis

Publications and source records attributed to Antonis Voutetakis.

14 recordsLinked to original sources

PubMed2006

Effect of serotype 5 adenoviral and serotype 2 adeno- associated viral vector-mediated gene transfer to salivary glands on the composition of saliva.

Key to the development of a useful clinical therapy is the minimization of side effects. Routine safety testing, however, does not provide information about the physiological status of many potentially useful gene transfer target sites. In this study, we evaluated the longitudinal effects of intrasalivary duct delivery of recombinant serotype 5 adenoviral (rAd5; 10(9)-10(10) particles/gland in rats) and recombinant serotype 2 adeno-associated viral (rAAV2; 10(8)-10(9) particles/gland in mice) vectors on salivary composition. Both vectors led to modest, transient alterations in several salivary components that thereafter returned to normal. The changes suggested two initial specific consequences of rAd5 and rAAV2 vector administration: (1) a modest breach of the mucosal barrier in the targeted glands, indicated by elevations in salivary albumin, total protein, and Na+ levels, and (2) an innate host response, indicated by transient elevations in either salivary lactoferrin and IgA levels (rAd5) or mucin (rAAV2). These studies are consistent with the notion that administration of modest doses of rAd5 and rAAV2 vectors to salivary glands for a therapeutic purpose can be accomplished without severe or permanent injury to the target tissue, or compromise to its essential exocrine physiological function.

Adenoviridae↗

PubMed2005

Transfer of the AQP1 cDNA for the correction of radiation-induced salivary hypofunction.

The treatment of most patients with head and neck cancer includes ionizing radiation (IR). Salivary glands in the IR field suffer significant and irreversible damage, leading to considerable morbidity. Previously, we reported that adenoviral (Ad)-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to rat [C. Delporte, B.C. O'Connell, X. He, H.E. Lancaster, A.C. O'Connell, P. Agre, B.J. Baum, Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands. Proc. Natl. Acad. Sci. U S A. 94 (1997) 3268-3273] and miniature pig [Z. Shan, J. Li, C. Zheng, X. Liu, Z. Fan, C. Zhang, C.M. Goldsmith, R.B. Wellner, B.J Baum, S. Wang. Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands. Mol. Ther. 11 (2005) 444-451] salivary glands approximately 16 weeks following IR resulted in a dose-dependent increase in salivary flow to > or =80% control levels on day 3. A control Ad vector was without any significant effect on salivary flow. Additionally, after administration of Ad vectors to salivary glands, no significant lasting effects were observed in multiple measured clinical chemistry and hematology values. Taken together, the findings show that localized delivery of AdhAQP1 to IR-damaged salivary glands is useful in transiently increasing salivary secretion in both small and large animal models, without significant general adverse events. Based on these results, we are developing a clinical trial to test if the hAQP1 cDNA transfer strategy will be clinically effective in restoring salivary flow in patients with IR-induced parotid hypofunction.

Animals↗

PubMed2005

The Smad6-histone deacetylase 3 complex silences the transcriptional activity of the glucocorticoid receptor: potential clinical implications.

Glucocorticoids play pivotal roles in the maintenance of homeostasis but, when dysregulated, may also have deleterious effects. Smad6, one of the transforming growth factor beta (TGFbeta) family downstream transcription factors, interacts with the N-terminal domain of the glucocorticoid receptor (GR) through its Mad homology 2 domain and suppresses GR-mediated transcriptional activity in vitro. Adenovirus-mediated Smad6 overexpression inhibits glucocorticoid action in rat liver in vivo, preventing dexamethasone-induced elevation of blood glucose levels and hepatic mRNA expression of phosphoenolpyruvate carboxykinase, a well known rate-limiting enzyme of liver gluconeogenesis. Smad6 suppresses GR-induced transactivation by attracting histone deacetylase 3 to DNA-bound GR and by antagonizing acetylation of histone H3 and H4 induced by p160 histone acetyltransferase. These results indicate that Smad6 regulates glucocorticoid actions as a corepressor of the GR. From our results and known cross-talks between glucocorticoids and TGFbeta family molecules, it appears that the anti-glucocorticoid actions of Smad6 may contribute to the neuroprotective, anticatabolic and pro-wound healing properties of the TGFbeta family of proteins.

Acetylation↗

PubMed2005

Immune responses following salivary gland administration of recombinant adeno-associated virus serotype 2 vectors.

BACKGROUND: Gene transfer to salivary glands (SGs) can be accomplished in a minimally invasive manner, resulting in stable, long-term secretion of the transgene product. Therefore, SGs provide a novel target site for several potentially useful clinical gene therapeutics applications. Previous studies have indicated that intravenous, intramuscular and intranasal administration of recombinant adeno-associated virus serotype 2 (rAAV2) vectors induce host immune responses. There are no reported studies on immune responsiveness of rAAV2 vector administration to SGs. MATERIAL AND METHODS: Vectors were administered by retrograde infusion to the SGs of Balb/c mice in various combinations. Thereafter, transgene expression was determined, and evaluations of host innate and adaptive immune responsiveness performed over a 56-day period. RESULTS: Histological examination of SGs from vector-treated mice showed no significant changes in appearance from controls, including the frequency of activated macrophage detection. There were also no differences in salivary flow rates among experimental groups. In vitro stimulation of splenocytes from mice administered rAAV2 showed elevated interferon-gamma levels in culture media. Significant titers of neutralizing antibodies to rAAV2 were detected in serum of mice following rAAV2 vector administration. While SGs could be transduced with low doses of vector it was not possible to repeat the administration and detect transduction with the same serotype at low doses. However, repeat administration was possible with an alternative serotype (rAAV4). CONCLUSIONS: Following a single administration of rAAV2 vectors to SGs there is no significant innate immune response. However, rAAV2 vector administration to SGs results in both cellular and humoral immune responses. The latter may interfere with the efficacy of repeated rAAV2 vector administration.

Animals↗

PubMed2005

Spontaneous pregnancy and birth of a normal female from a woman with Turner syndrome and elevated gonadotropins.

OBJECTIVE: To provide data for pregnancies in girls with Turner syndrome. Only 5%-10% of TS girls undergo spontaneous puberty and have menses. Spontaneous pregnancy occurs in 2%-7% of TS girls and is associated with a high rate of miscarriages, stillbirths, malformations, and chromosomal aberrations. Besides fetal problems, pregnancy in TS girls is of high risk for the mothers as well. DESIGN: Case report. SETTING: Academic unit. PATIENT(S): One patient, now aged 28 years, was referred for short stature at age 13 years after an operation for coarctation of the aorta. The karyotype was 88% 45X, 5% 46XX, 5% 47XXX, 2% XO+Fr. The gonadotropin values at presentation and on follow-up ranged from high normal to high levels. Pubertal development and menses occurred spontaneously. At the age of 20 and 21 years, dominant follicles of 14 and 17 mm, respectively, were found on sonography. She conceived spontaneously at the age of 26 years and had an uneventful pregnancy, giving birth to a normal girl with normal chromosome constitution and birth weight of 2,800 g. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Pregnancy outcome. RESULT(S): Successful spontaneous pregnancy. CONCLUSION(S): Bearing in mind the serious problems of fertility and pregnancy outcome encountered in TS girls, we considered such a rare escape from the expected course of biological events to be worth reporting.

Adult↗

PubMed2005

Partial redirection of transgenic human growth hormone secretion from rat salivary glands.

Regulated secretory pathway proteins, when delivered as transgenes to salivary glands, are secreted predominantly into saliva. This is not useful for those proteins whose therapeutic function is required systemically, for example, human growth hormone (hGH). One strategy to improve the efficiency of hGH secretion into the bloodstream involves manipulation of existing sorting signals. The C terminus of hGH is highly conserved and contains a domain similar to the regulated pathway sorting domain of pro-opiomelanocortin (POMC). We hypothesized that, similar to POMC, mutation of this domain would divert hGH secretion from the regulated to the constitutive pathway, which in salivary glands leads to the bloodstream. Several mutations were made in the C terminus of the hGH cDNA and tested in vitro. One biologically active mutant containing E174A and E186A substitutions, and with an included C-terminal extension, was studied in greater detail. Compared with wild-type hGH, we found that this mutant hGH accumulated in the Golgi/trans-Golgi network and showed increased basal secretion in AtT20 cells, a model endocrine cell line. Importantly, in vivo, the mutant hGH displayed a relative increase in the proportion of constitutive pathway secretion seen from rat salivary glands, with a significantly lower saliva-versus-serum secretion ratio (p=0.03). Although this mutant is unlikely to be therapeutically beneficial, these results suggest that the final destination of a transgenic secretory protein may be controlled by reengineering its sorting determinants.

Adenoviridae↗

PubMed2005

Salivary glands as a potential gene transfer target for gene therapeutics of some monogenetic endocrine disorders.

Salivary glands (SGs) exhibit several important features which are also common to endocrine glands: self-containment due to a surrounding capsule, highly efficient protein production and the ability to secrete proteins into the bloodstream. We have hypothesized that SGs are potentially useful as gene transfer targets for the correction of inherited monogenetic endocrine disorders. In the present communication, we extend our studies and attempt to test our hypothesis by comparing the efficacy of two commonly used viral vectors and the resulting serum and salivary distribution of transgene encoded hormones. A low dose (5 x10(9) particles) of either an adenoviral serotype 5 (Ad5) vector encoding the human erythropoietin (hEPO) cDNA or an adeno-associated virus sero-type 2 (AAV2) vector encoding either the hEPO or human growth hormone (hGH) cDNA was administered to individual submandibular SGs of Balb/c mice. Serum and salivary hEPO and hGH levels were determined at defined time points. Two additional recombinant viruses encoding enhanced green fluorescence protein (GFP) were also used (AdGFP and AAVGFP) in order to perform immunohistochemical analyses of transgenic protein localization in SG sections post-administration. AAV2 vectors led to stable gene transfer unlike the results with the Ad5 vectors. Indeed, in one mouse we observed hEPO production for a period of two years after administration of AAVhEPO to SGs. hEPO, which is a constitutive pathway secretory protein, was readily secreted into the bloodstream from the SGs, yielding therapeutically adequate serum levels. Conversely, hGH, a regulated secretory pathway protein, was preferentially secreted into saliva. SGs may be an attractive candidate target tissue for gene therapeutics of some monogenetic endocrine deficiency disorders. At present, AAV2 vectors seem particularly useful for such applications, and transgenes encoding constitutive secretory pathway hormones are more suitable for this application with SGs than those encoding regulated secretory pathway hormones.

Adenoviridae↗

PubMed2004

Reengineered salivary glands are stable endogenous bioreactors for systemic gene therapeutics.

The use of critical-for-life organs (e.g., liver or lung) for systemic gene therapeutics can lead to serious safety concerns. To circumvent such issues, we have considered salivary glands (SGs) as an alternative gene therapeutics target tissue. Given the high secretory abilities of SGs, we hypothesized that administration of low doses of recombinant adeno-associated virus (AAV) vectors would allow for therapeutic levels of transgene-encoded secretory proteins in the bloodstream. We administered 10(9) particles of an AAV vector encoding human erythropoietin (hEPO) directly to individual mouse submandibular SGs. Serum hEPO reached maximum levels 8-12 weeks after gene delivery and remained relatively stable for 54 weeks (longest time studied). Hematocrit levels were similarly increased. Moreover, these effects proved to be vector dose-dependent, and even a dosage as low as 10(8) particles per animal led to significant increases in hEPO and hematocrit levels. Vector DNA was detected only within the targeted SGs, and levels of AAV copies within SGs were highly correlated with serum hEPO levels (r = 0.98). These results show that SGs appear to be promising targets with potential clinical applicability for systemic gene therapeutics.

Animals↗

PubMed2004

Utilizing endocrine secretory pathways in salivary glands for systemic gene therapeutics.

Mammalian salivary glands are commonly used models of exocrine secretion. However, there is substantial experimental evidence showing the physiological existence of endocrine secretory pathways in these tissues. The use of gene transfer technology in vivo has allowed the unambiguous demonstration of these endocrine pathways. We and others have exploited such findings and evaluated salivary glands as possible target tissues for systemic applications of gene therapeutics. Salivary glands present numerous advantages for this purpose, including being well encapsulated, which limits extra-glandular vector dissemination, and having the luminal membranes of almost all parenchymal cells accessible via intraoral delivery of vectors through the main excretory ducts. Existing studies suggest that clinical benefits will result from salivary gland targeted systemic gene therapeutics.

Animals↗

PubMed2004

Ovulation induction and successful pregnancy outcome in two patients with Prop1 gene mutations.

OBJECTIVE: To describe ovulation induction and pregnancy outcome in a unique model of genetically determined combined pituitary hormone deficiency (CPHD), with respect to the necessity for GH substitution therapy. DESIGN: Case report. SETTING: Academic units. PATIENT(S): Two patients with childhood onset of CPHD (GH, PRL, TSH, LH, FSH) caused by a genetic defect (GA296del mutation) of the Prop1 gene. MAIN OUTCOME MEASURE(S): Ovulation, pregnancy outcome, and fetal growth. RESULT(S): Successful pregnancy outcome and delivery of normal, full-term newborns were achieved in both patients with the use of gonadotropins and L-T(4). Growth hormone supplementation was not necessary. No lactation was observed. CONCLUSION(S): Patients with Prop1 gene mutations constitute a unique model for studying the role of GH and PRL in ovulation, pregnancy, and fetal growth. Our data indicate that for women with CPHD, ovulation and pregnancy are possible with a classic regimen for hypogonadotropic hypogonadism, without the need for GH substitution therapy.

Adolescent↗

PubMed2004

Salivary glands: novel target sites for gene therapeutics.

Directing the local or systemic expression of therapeutic proteins is a potentially important clinical application of gene transfer technology. Gene-based therapeutics theoretically offer many advantages over protein therapeutics. Numerous tissues have been evaluated for this purpose in animal models, most commonly the liver and skeletal muscle. Based on pre-clinical studies, we suggest that salivary glands are a valuable, yet under-appreciated, target tissue for both systemic and upper gastrointestinal tract gene therapeutic applications.

Animals↗

PubMed2004

Pituitary magnetic resonance imaging in 15 patients with Prop1 gene mutations: pituitary enlargement may originate from the intermediate lobe.

Pituitary morphology in patients with Prop1 gene mutations varies. Most patients demonstrate a normal or small pituitary gland. Occasionally, pituitary enlargement of undetermined origin has also been detected. In the present study we use long-term magnetic resonance imaging findings to characterize the morphological abnormalities of the pituitary gland in 15 patients (aged 2.5-45 yr) with combined pituitary hormone deficiency caused by Prop1 gene mutations (GA296del/GA296del in seven, GA296del/A150del in two, A150del/A150del in five, and GA296del/R73H in one patient) and attempt to uncover the origin and nature of the pituitary enlargement. Small pituitary gland was detected in seven patients (25.2 +/- 14.4 yr of age), normal pituitary size in three patients (10.2 +/- 5.8 yr of age), and pituitary enlargement in five patients (6.5 +/- 2.7 yr of age). The pituitary enlargement consisted of a nonenhancing mass lesion interposed between the normally enhancing anterior lobe and the neurohypophysis. The pituitary stalk was displaced anteriorly, whereas the neurohypophysis was orthotopic, displaying a normal signal. Spontaneous regression of the mass lesion with normalization of the pituitary stalk position was observed in three patients. Our data indicate that although a small pituitary gland is usually observed in older subjects, a significant number of young patients with Prop1 gene mutations demonstrate pituitary enlargement with subsequent regression. The distinct magnetic resonance imaging characteristics of the pituitary enlargement in our patients in conjunction with pertinent data from Prop1-deficient mice suggest that the mass causing the pituitary enlargement most likely originates from the intermediate lobe.

Adolescent↗

PubMed2004

Prolonged jaundice and hypothyroidism as the presenting symptoms in a neonate with a novel Prop1 gene mutation (Q83X).

OBJECTIVE: To identify the genetic defect in a neonate presented with prolonged jaundice and central hypothyroidism. DESIGN: Central hypothyroidism was detected in a neonate initially examined for prolonged jaundice, and levothyroxine therapy was initiated. Direct sequencing of the Prop1 gene was carried out and pituitary function and morphology were evaluated using hormonal testing and magnetic resonance imaging (MRI) respectively. METHODS: Dynamic hormonal testing was carried out using established methodologies. Hormones were determined by RIA or chemiluminescence immunoassays. Genomic analysis of the Prop1 gene was performed by direct sequencing. MRI protocol: sagittal spin echo T2-weighted scans 2500/90 (TR/TE), plain and contrast-enhanced sagittal and coronal spin echo T1-weighted scans 500/20 (TR/TE). RESULTS: Low thyroid hormones (coupled with lack of TSH rise), low GH, normal cortisol and normal prolactin values were detected. Direct sequencing revealed the presence of two mutations in the Prop1 gene: GA296del and Q83X. The Q83X was further confirmed by PvuII restriction digestion and represented a novel Prop1 gene mutation, which was not detected in 100 controls tested. Pituitary enlargement was detected, with respect to normal-for-age controls. CONCLUSIONS: (i) The Q83X mutation extends the spectrum of Prop1 gene mutations; (ii) central hypothyroidism in a neonate might constitute the initial sign of Prop1 gene defect; (iii) the patient is the youngest individual with Prop1 gene defect and pituitary enlargement presented to date; and (iv) early detection of Prop1 gene mutations facilitates genetic counseling and ensures prompt management of the anticipated hormonal insufficiencies.

Codon, Nonsense↗

PubMed2003

Advances in vector-mediated gene transfer.

Clinical applications of gene transfer technology initially targeted the treatment of inherited monogenetic disorders and cancers refractory to conventional therapies. Today, gene transfer approaches are being developed for most tissues and for multiple disorders including those affecting quality of life. The focus herein is eventual application of gene transfer technology for the management of organ-directed autoimmunity. A specific example is presented: Sjögren's syndrome and localized salivary gland gene transfer. The status of relevant pre-clinical gene transfer studies is reviewed, with an emphasis on use of adenoviral and adeno-associated viral vectors. Current limitations of effective organ-directed gene transfer are also discussed.

Adenoviridae↗