Assessing the Respect of Children's Rights in Pediatric Hospitals.

Background and Objectives: In 1989, the United Nations (UN) General Assembly adopted the United Nations Convention on the Rights of the Child (UNCRC), with a considerable number of the Articles of the Convention being related to the health status of children. Therefore, adhering to and assessing the implementation of the rights of children during hospitalization is a very important step towards child protection. Herein, we attempt to highlight the depth of knowledge of employees working in children's hospitals with regard to children's rights as well as the degree of adherence to the UNCRC with respect to hospitalized children. Material and Methods: The target group included all healthcare professionals working in the various general pediatric clinics of the three Children's Hospitals of the Athens metropolitan area in Greece. We conducted a cross-sectional study, with data collection carried out in February and March 2020, using a structured questionnaire consisting of 46 questions which was handed out to all personnel. For the analysis, we used the IBM SPSS 21.0. Results: A total of 251 individuals participated in the study (physicians 20%, nurses 72%, and other employees 8%). A total of 54.5% of health professionals did not know what the UNCRC is, and 59.6% of them were not even aware that their hospital had rules and a bioethical committee related to clinical research involving children. Lack of awareness or trust of health professionals is also observed for other procedures or supervisory measures such as abuse protocols, complaint control, admission control, etc. With regard to the health system, there are shortcomings or weaknesses in (a) procedures followed with regard to respect for gender and privacy, (b) information on basic services provided by pediatric hospitals (such as recreation, education and free meals during hospitalization), (c) the logistical infrastructure (such as recreational facilities and facilities for the disabled), (d) the possibility of recording complaints, and (e) hospitalizations that were not necessary. A difference emerged concerning the nurses' responses between the three hospitals, with nurses participating in relevant seminars held in one of the hospitals being significantly more informed. Conclusions: The majority of healthcare personnel seem unaware of basic principles with respect to children's rights during hospitalization as well as relevant procedures and supervisory measures. Moreover, obvious weaknesses of the health system exist with respect to procedures, services, infrastructure, and complaint recording. There is a need for improved education of health professionals with respect to the implementation of children's rights in pediatric hospitals.

Congenital Hypopituitarism: Various Genes, Various Phenotypes.

The ontogenesis and development of the pituitary gland is a highly complex process that depends on a cascade of transcription factors and signaling molecules. Spontaneous mutations and transgenic murine models have demonstrated a role for many of these factors, including HESX1, PROP1, PIT1, LHX3, LHX4, SOX2, SOX3, OTX2, PAX6, FGFR1, SHH, GLI2, and FGF8 in the etiology of congenital hypopituitarism. Genetic mutations in any of these factors can lead to congenital hypopituitarism, which is characterized by the deficiency in one or more pituitary hormones. The phenotype can be highly variable, consisting of isolated hypopituitarism or more complex disorders. The same phenotype can be attributed to different gene mutations; while a given gene mutation can induce different phenotypes. This review highlights the genetic variations that lead to congenital hypopituitarism and their associated defects. The overall incidence of mutations in known transcription factors in patients with hypopituitarism is low; therefore many gene mutations or even gene- epigenetic interactions have to be unraveled in the future to explain the vast majority of still unclear cases of congenital hypopituitarism.

Pituitary stalk interruption syndrome: cause, clinical manifestations, diagnosis, and management.

PURPOSE OF REVIEW: Pituitary stalk interruption syndrome (PSIS) is characterized by a thin or absent pituitary stalk, hypoplasia of the adenohypophysis, and ectopic neurohypophysis. PSIS manifestations include a wide spectrum of clinical phenotypes and pituitary hormone deficiencies of variable degree and timing of onset. In this review, recent advances with respect to the cause of PSIS, clinical characteristics leading to earlier diagnosis, and management are outlined. RECENT FINDINGS: Diagnosis of PSIS is often delayed probably because clinical findings such as neonatal hypoglycemia, cholestasis, and/or micropenis as well as decreasing growth velocity are not appropriately and timely validated. Recently, molecular defects in various genes have been associated with PSIS albeit in a small number of cases. These findings suggest that PSIS belongs to the spectrum of holoprosencephaly-related defects. Phenotype-genotype discordance and the existence of asymptomatic carriers of a given molecular aberration indicate that penetrance may be modified favorably or unfavorably by the presence of other genetic and/or environmental factors. SUMMARY: PSIS constitutes an antenatal anatomical defect. Neonatal hypoglycemia, cholestasis, and/or micropenis with or without growth deficit should raise the possibility of combined pituitary hormone deficiency, a life-threatening condition in cases of coexisting cortisol deficiency. It is important to search for molecular defects in all PSIS cases, as precise identification of the cause is a prerequisite for genetic counseling.

The Impact of the ENDORSE Digital Weight Management Program on the Metabolic Profile of Children and Adolescents with Overweight and Obesity and on Food Parenting Practices.

Childhood obesity is a serious public health problem worldwide. The ENDORSE platform is an innovative software ecosystem based on Artificial Intelligence which consists of mobile applications for parents and health professionals, activity trackers, and mobile games for children. This study explores the impact of the ENDORSE platform on metabolic parameters associated with pediatric obesity and on the food parenting practices of the participating mothers. Therefore, the metabolic parameters of the 45 children (mean age: 10.42 years, 53% girls, 58% pubertal, mean baseline BMI z-score 2.83) who completed the ENDORSE study were evaluated. The Comprehensive Feeding Practices Questionnaire was used for the assessment of food parenting practices. Furthermore, regression analysis was used to investigate possible associations between BMI z-score changes and changes in metabolic parameters and food parenting practices. Overall, there was a statistically significant reduction in glycated hemoglobin (mean change = -0.10, p = 0.013), SGOT (mean change = -1.84, p = 0.011), and SGPT (mean change = -2.95, p = 0.022). Emotional feeding/food as reward decreased (mean change -0.21, p = 0.007) and healthy eating guidance increased (mean change = 0.11, p = 0.051). Linear regression analysis revealed that BMI z-score change had a robust and significant correlation with important metabolic parameters: HOMA-IR change (beta coefficient = 3.60, p-value = 0.046), SGPT change (beta coefficient = 11.90, p-value = 0.037), and cortisol change (beta coefficient = 9.96, p-value = 0.008). Furthermore, healthy eating guidance change had a robust negative relationship with BMI z-score change (beta coefficient = -0.29, p-value = 0.007). Conclusions: The Endorse digital weight management program improved several metabolic parameters and food parenting practices.

The ENDORSE Feasibility Study: Exploring the Use of M-Health, Artificial Intelligence and Serious Games for the Management of Childhood Obesity.

Childhood obesity constitutes a major risk factor for future adverse health conditions. Multicomponent parent-child interventions are considered effective in controlling weight. Τhe ENDORSE platform utilizes m-health technologies, Artificial Intelligence (AI), and serious games (SG) toward the creation of an innovative software ecosystem connecting healthcare professionals, children, and their parents in order to deliver coordinated services to combat childhood obesity. It consists of activity trackers, a mobile SG for children, and mobile apps for parents and healthcare professionals. The heterogeneous dataset gathered through the interaction of the end-users with the platform composes the unique user profile. Part of it feeds an AI-based model that enables personalized messages. A feasibility pilot trial was conducted involving 50 overweight and obese children (mean age 10.5 years, 52% girls, 58% pubertal, median baseline BMI z-score 2.85) in a 3-month intervention. Adherence was measured by means of frequency of usage based on the data records. Overall, a clinically and statistically significant BMI z-score reduction was achieved (mean BMI z-score reduction -0.21 ± 0.26, p-value < 0.001). A statistically significant correlation was revealed between the level of activity tracker usage and the improvement of BMI z-score (-0.355, p = 0.017), highlighting the potential of the ENDORSE platform.

Severe hyperinsulinemia, decreased GLUT3 and GLUT4 expression, and increased retinol binding protein 4 in a patient with chronic graft-versus-host disease post bone marrow transplantation.

Hyperinsulinemia with or without DM2 is a frequent long-term sequela of BMT, especially following cGvHD. In this report, an extensive evaluation of a patient with cGvHD is described: glucose and insulin during OGTT, markers of inflammation, adiponectin and RBP4, body composition analysis, and the kinetics of GLUT3 and GLUT4 in circulating monocytes were evaluated. Hyperinsulinemia, associated with partial lipodystrophy, elevated RBP4, low adiponectin levels, and decreased expression of GLUT3 and GLUT4 were detected. The defects disclosed in this particular patient possibly explain, at least in part, the mechanisms underlying insulin resistance in patients undergoing BMT. It is not clear whether insulin resistance was caused by the drugs, the process itself, or the residual damage to the muscles and/or adipose tissue.

Non-inferiority of liquid thyroxine in comparison to tablets formulation in the treatment of children with congenital hypothyroidism.

OBJECTIVES: The aim of the current prospective randomized control study was to assess efficacy, safety, and non-inferiority of a new liquid L-thyroxine formulation dissolved in glycerol and water (T4® drops, produced by a Greek pharmaceutical Company, Uni-Pharma, Athens, Greece) in comparison to the standard Tablets form (T4® tablets, Uni-Pharma, Athens, Greece) in the substitutive treatment of children with congenital hypothyroidism (CH). METHODS: Thirty-nine children with CH, aged 3-12 years old, were enrolled in the study, after parental Informed Consent has been obtained, while three patients were lost from follow-up. At baseline, all participants had normal thyroid-stimulating hormone (TSH) and Free T4 values. Patients were randomly subdivided according to the assigned treatment in Group A (n=17)-Tablet Form and Group B (n=19)-Liquid Form. TSH and Free T4 levels were evaluated at 0, 2, 4, and 6 months. RESULTS: TSH values showed a statistically significant difference (p=0.017) between groups only at six months (Group A having higher TSH levels than Group B, albeit within the normal range), while Free T4 levels had no statistical difference throughout the six month study period and were always within the normal range. Moreover, dose adjustments were more frequent in Group A (p=0.038) during the six months. Liquid L-thyroxine substitutive treatment exhibited no statistically significant adverse effects in comparison to the widely used tablets. CONCLUSIONS: Levothyroxine (LT4) as liquid solution formulation is safe and noninferior to the widely used L-thyroxine Tablets, with less need for dose adjustment, and can therefore be safely used in the treatment of children with CH.

Pituitary stalk interruption syndrome.

Pituitary stalk interruption syndrome (PSIS) is a distinct developmental defect of the pituitary gland identified by magnetic resonance imaging and characterized by a thin, interrupted, attenuated or absent pituitary stalk, hypoplasia or aplasia of the adenohypophysis, and an ectopic posterior pituitary. The precise etiology of PSIS still remains elusive or incompletely confirmed in most cases. Adverse perinatal events, including breech delivery and hypoxia, were initially proposed as the underlying mechanism affecting the hypothalamic-pituitary axis. Nevertheless, recent findings have uncovered a wide variety of PSIS-associated molecular defects in genes involved in pituitary development, holoprosencephaly (HPE), neural development, and other important cellular processes such as cilia function. The application of whole exome sequencing (WES) in relatively large cohorts has identified an expanded pool of potential candidate genes, mostly related to the Wnt, Notch, and sonic hedgehog signaling pathways that regulate pituitary growth and development during embryogenesis. Importantly, WES has revealed coexisting pathogenic variants in a significant number of patients; therefore, pointing to a multigenic origin and inheritance pattern of PSIS. The disorder is characterized by inter- and intrafamilial variability and incomplete or variable penetrance. Overall, PSIS is currently viewed as a mild form of an expanded HPE spectrum. The wide and complex clinical manifestations include evolving pituitary hormone deficiencies (with variable timing of onset and progression) and extrapituitary malformations. Severe and life-threatening symptomatology is observed in a subset of patients with complete pituitary hormone deficiency during the neonatal period. Nevertheless, most patients are referred later in childhood for growth retardation. Prompt and appropriate hormone substitution therapy constitutes the cornerstone of treatment. Further studies are needed to uncover the etiopathogenesis of PSIS.

Molecular Analysis of the CYP11B2 Gene in 62 Patients with Hypoaldosteronism Due to Aldosterone Synthase Deficiency.

CONTEXT: Isolated congenital hypoaldosteronism presents in early infancy with symptoms including vomiting, severe dehydration, salt wasting, and failure to thrive. The main causes of this rare autosomal recessive disorder is pathogenic variants of the CYP11B2 gene leading to aldosterone synthase deficiency. OBJECTIVE: To investigate the presence of CYP11B2 pathogenic variants in a cohort of patients with a clinical, biochemical, and hormonal profile suggestive of aldosterone synthase deficiency. DESIGN: Clinical and molecular study. SETTING: Tertiary academic Children's Hospital, Center for Rare Pediatric Endocrine Diseases. PATIENTS AND METHODS: Sixty-two patients (56 unrelated patients and 6 siblings), with hypoaldosteronism and their parents, underwent CYP11B2 gene sequencing after its selective amplification against the highly homologous CYP11B1 gene. In silico analysis of the identified novel variants was carried out to evaluate protein stability and potential pathogenicity. RESULTS: CYP11B2 gene sequencing revealed that 62 patients carried a total of 12 different pathogenic CYP11B2 gene variants, 6 of which are novel. Importantly, 96% of the 56 patients carried the previously reported p.T185I variant either in homozygosity or in compound heterozygosity with another variant. The 6 novel variants detected were: p.M1I, p.V129M, p.R141Q, p.A165T, p.R448C, and the donor splice site variant of intron 8, c.1398 + 1G > A. CONCLUSION: Molecular diagnosis was achieved in 62 patients with aldosterone synthase deficiency, the largest cohort thus far reported. Six novel genetic variants were identified as possibly pathogenic, extending the spectrum of reported molecular defects of the CYP11B2 gene.

Long-term transduction of miniature pig parotid glands using serotype 2 adeno-associated viral vectors.

BACKGROUND: Previously, using an adenoviral vector, we showed that miniature pigs could provide a valuable and affordable large animal model for pre-clinical gene therapy studies to correct parotid gland radiation damage. However, adenoviral vectors lead to short-term transgene expression and, ideally, a more stable correction is required. In the present study, we examined the suitability of using a serotype 2 adeno-associated viral (AAV2) vector to mediate more stable gene transfer in the parotid glands of these animals. METHODS: Heparan sulfate proteoglycan was detected by immunohistochemistry. beta-galactosidase expression was determined histochemically. An AAV2 vector encoding human erythropoietin (hEpo) was administered via Stensen's duct. Salivary and serum hEpo levels were measured using an enzyme-linked immunosorbent assay. Serum chemistry and hematological analyses were performed and serum antibodies to hEpo were measured throughout the study. Vector distribution was determined by a quantitative polymerase chain reaction. RESULTS: Transgene expression was vector dose-dependent, with high levels of hEpo being detected for up to 32 weeks (i.e. the longest time studied). hEpo reached maximal levels during weeks 4-8, but declined to approximately 25% of these values by week 32. Haematocrits were elevated from week 2. Transduced animals exhibited low serum anti-hEpo antibodies (1 : 8-1 : 16). Vector biodistribution at animal sacrifice revealed that most copies were in the targeted parotid gland, with few being detected elsewhere. No consistent adverse changes in serum chemistry or hematology parameters were seen. CONCLUSIONS: AAV2 vectors mediate extended gene transfer to miniature pig parotid glands and should be useful for testing pre-clinical gene therapy strategies aiming to correct salivary gland radiation damage.

Adeno-associated virus type 12 (AAV12): a novel AAV serotype with sialic acid- and heparan sulfate proteoglycan-independent transduction activity.

Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy. Recent isolations of novel AAV serotypes have led to significant advances by broadening the tropism and increasing the efficiency of gene transfer to the desired target cell. However, a major concern that remains is the strong preexisting immune responses to several vectors. In this paper, we describe the isolation and characterization of AAV12, an AAV serotype with unique biological and immunological properties. In contrast to those of all other reported AAVs, AAV12 cell attachment and transduction do not require cell surface sialic acids or heparan sulfate proteoglycans. Furthermore, rAAV12 is resistant to neutralization by circulating antibodies from human serum. The feasibility of rAAV12 as a vector was demonstrated in a mouse model in which muscle and salivary glands were transduced. These characteristics make rAAV12 an interesting candidate for gene transfer applications.

Carney Complex.

Carney complex is a rare, autosomal dominant, multiple endocrine neoplasia and lentiginosis syndrome, caused in most patients by defects in the PRKAR1A gene, which encodes the regulatory subunit type 1α of protein kinase A. Inactivating defects of PRKAR1A lead to aberrant cyclic-AMP-protein kinase A signaling. Patients may develop multiple skin abnormalities and a variety of endocrine and non-endocrine tumors. Endocrine manifestations include primary pigmented nodular adrenocortical disease, that may cause Cushing syndrome, growth-hormone secreting pituitary adenoma or pituitary somatotropic hyperplasia which can result in acromegaly, as well as gonadal and thyroid tumors. Non-endocrine tumors associated with Carney complex include myxomas of the heart, breast, and other sites, psamommatous melanotic schwannomas, breast ductal adenomas, osteochondromyxomas, and a predisposition to a number of malignancies from adrenal to pancreatic and liver cancer.

SGPL1 Deficiency: A Rare Cause of Primary Adrenal Insufficiency.

CONTEXT: Multiple autosomal recessive genes have been etiologically linked to primary adrenal insufficiency (PAI). Recently, sphingosine-1-phosphate lyase 1 (SGPL1) gene mutations were recognized as a cause of steroid-resistant nephrotic syndrome type 14 (NPHS14), a sphingolipidosis with multisystemic manifestations, including PAI. OBJECTIVE: To check if SGPL1 mutations are involved in the pathogenesis of PAI in patients who do not exhibit nephrotic syndrome. METHODS: Sequencing of the SGPL1 gene in 21 patients with familial glucocorticoid disease or triple A syndrome. RESULTS: We identified two missense SGPL1 variants in four patients, two of whom were first cousins. We describe in detail the proband, a boy born to Saudi Arabian consanguineous parents with a homozygous c.665G>A, p.R222Q SGPL1 variant. The patient presented with hypoglycemia and seizures at age 2 years and was ultimately diagnosed with PAI (isolated glucocorticoid deficiency). Brain MRI showed abnormalities in the basal ganglia consistent with a degenerative process albeit the patient had no neurologic symptoms. CONCLUSIONS: New genetic causes of PAI continue to be identified. We suggest that screening for SGPL1 mutations should not be reserved only for patients with nephrotic syndrome but may also include patients with PAI who lack other clinical manifestations of NPHS14 because, in certain cases, kidney disease and accompanying features might develop. Timely diagnosis of this specific sphingolipidosis while the kidneys still function normally can lead to prompt initiation of therapy and improve outcome.

The Dilemma of Sex of Rearing: A Case of a 45,X/46,XY Neonate with Hydrocolpos.

BACKGROUND: A rare disorder of sex development is 45,X/46,XY mosaicism, which is phenotypically very heterogenous, ranging from normal male (or female) to that of genital ambiguity of varying degrees. CASE: We report a case of a neonate with 45,X/46,XY mosaicism and hydrocolpos, and we point out the dilemma and the difficulty in gender assignment. SUMMARY AND CONCLUSION: Gender assignment of cases with frank genital ambiguity is often difficult to be determined, because several factors have to be taken into consideration, such as genital appearance, anticipated urological and sexual function, capacity for future fertility, gonadal malignancy risk, and psychosocial factors. A multidisciplinary approach is definitely needed in the management of such cases.

Adrenal Steroids in Female Hypothyroid Neonates: Unraveling an Association Between Thyroid Hormones and Adrenal Remodeling.

CONTEXT: The adrenal gland undergoes substantial remodeling during the neonatal period, an essential developmental process that remains incompletely understood. With respect to control over the remodeling process and, specifically, the role of thyroid hormones (THs), no human studies have been published. The effects of both hypo- and hyperthyroidism have only been evaluated in adults, focusing on the mature adrenal. Recent studies have identified expression of the TH receptor ß1 in the mouse adrenal X-zone and have demonstrated that TH administration could alter the postnatal adrenal remodeling process. OBJECTIVE: To address whether THs influence adrenal steroid profiles and adrenal remodeling during the neonatal period. METHODS: We compared the adrenal steroid profile of a naturally occurring prototype, female neonates with severe congenital hypothyroidism (CH) (n = 22, upon diagnosis of CH), with that of euthyroid neonates (n = 20). RESULTS: Significantly higher levels of adrenal steroids (17-OH-progesterone, dehydroepiandrosterone sulfate, Δ4-androstenedione, and testosterone) were measured in neonates with severe CH compared with euthyroid neonates and returned to within normal range after euthyroid state had been established on l-thyroxine replacement therapy, whereas cortisol levels did not differ. TSH values in the CH group were positively correlated with circulating adrenal steroids, whereas free T4 levels were negatively correlated with circulating adrenal steroids. CONCLUSIONS: The hormonal profile of female neonates with severe CH suggests a more active adrenal fetal zone compared with control subjects. These data indirectly associate THs with the adrenal remodeling and maturation process in humans. Based on our results, we suggest that severe hypothyroidism decelerates the involution of the adrenal fetal zone that normally occurs postnatally.

The genetic etiology in cerebral palsy mimics: The results from a Greek tertiary care center.

OBJECTIVE: Non-progressive genetic disorders may present with motor dysfunction resembling cerebral palsy (CP). Such patients are often characterized as CP mimics. The purpose of this work was to delineate the clinical manifestations and molecular findings of CP mimic patients, with the ultimate goal to offer specific disease-modifying therapy and genetic counseling. METHODS: Retrospective study of 47 patients diagnosed with CP and no acquired etiology. Chart review of clinical, neuroradiological, biochemical and molecular data was performed. RESULTS: 31,91% of patients manifested with features resembling dyskinetic CP, 19,14% spastic CP, 10,63% ataxic CP and 38,30% mixed CP. In 23 patients molecular diagnosis was reached and included 5 hereditary spastic paraplegia genes (SPG) in spastic CP mimics; HPRT1, TH, QDPR, DDC in dystonic CP mimics; ADCY5 and NIKX2-1 in choreic CP mimics; CANA1A in ataxic CP mimics; and SPG, PDHA1, NIKX2-1, AT, SLC2A1 and SPR in mixed CP mimics. In 14 patients, the etiological diagnosis led to specific treatment. CONCLUSIONS: CP mimics show a number of features that differ from classic CP and can be used as diagnostic clues, including presence of mixed motor features, minor dysmorphic features, oculogyric movements, multiple features of autonomic dysfunction, and acquired microcephaly. A more stringent use of the concept of CP focused on acquired lesions during the perinatal and infancy periods, and excluding disorders that could be of genetic origin, could contribute to a purer use of the term. Identification of a specific genetic cause for CP mimics may in certain cases lead to etiologic treatment.

Adeno-associated virus serotype 2-mediated gene transfer to the parotid glands of nonhuman primates.

Salivary glands (SGs) are promising gene transfer targets with potential clinical applicability. Previous experiments in rodents using recombinant serotype 2 adeno-associated viral (rAAV2) vectors have demonstrated relatively stable transgene-encoded protein levels after SG gene transfer. In the present study, we examine direct SG administration of rAAV2 vectors encoding rhesus macaque erythropoietin (RhEPO) to the parotid glands of nonhuman primates using two different doses (n = 3 per group; 1 x 10(10) or 3 x 10(11) particles/gland, respectively). Gene transfer had no negative effects on general macaque physiology (e.g., weight, complete blood count, and serum chemistry). Macaques were euthanized 6 months after vector administration and complete necropsy and pathology assessments were performed, revealing no vector-related pathological lesions in any of the examined organs. In the high-dose group, RhEPO expression increased quickly (i.e., by week 1) and levels remained relatively stable both in serum and saliva until the end of the study. Serum-to-saliva ratios of RhEPO revealed secretion of the transgene product into the bloodstream, but not to the extent previously observed in mice. Furthermore, the kinetic results were not predicted by those observed in murine SGs. With respect to viral biodistribution, at necropsy vector was found overwhelmingly in the targeted parotid gland ( approximately 100 times more than levels in other tissues, most of which were similar to tissue levels in nontreated animals). We conclude that administration of modest doses of rAAV2 vectors to SGs for therapeutic purposes can be accomplished without significant or permanent injury to the targeted gland or to distant organs of nonhuman primates.

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.

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.

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.