Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction.

Head and neck cancer patients treated with irradiation often present irreversible salivary gland hypofunction for which no conventional treatment exists. We recently showed that recombinant neurturin, a neurotrophic factor, improves epithelial regeneration of mouse salivary glands in ex vivo culture after irradiation by reducing apoptosis of parasympathetic neurons. Parasympathetic innervation is essential to maintain progenitor cells during gland development and for regeneration of adult glands. Here, we investigated whether a neurturin-expressing adenovirus could be used for gene therapy in vivo to protect parasympathetic neurons and prevent gland hypofunction after irradiation. First, ex vivo fetal salivary gland culture was used to compare the neurturin adenovirus with recombinant neurturin, showing they both improve growth after irradiation by reducing neuronal apoptosis and increasing innervation. Then, the neurturin adenovirus was delivered to mouse salivary glands in vivo, 24 hr before irradiation, and compared with a control adenovirus. The control-treated glands have ∼50% reduction in salivary flow 60 days post-irradiation, whereas neurturin-treated glands have similar flow to nonirradiated glands. Further, markers of parasympathetic function, including vesicular acetylcholine transporter, decreased with irradiation, but not with neurturin treatment. Our findings suggest that in vivo neurturin gene therapy prior to irradiation protects parasympathetic function and prevents irradiation-induced hypofunction.

Gene Therapy of Salivary Diseases.

For many years, our research group worked to develop gene transfer approaches for salivary gland disorders that lacked effective conventional therapy. The purpose of this chapter is to describe and update key methods used in this process. As described in our original chapter from the 2010 volume, we focus on one clinical condition, irradiation-induced salivary hypofunction, and address the choice of transgene and vector to be used, the construction of recombinant viral vectors, how vector delivery is accomplished, and methods for assessing vector function in vitro and in an appropriate animal model.

Radiation-Induced Microvascular Injury as a Mechanism of Salivary Gland Hypofunction and Potential Target for Radioprotectors.

Radiation therapy is commonly used to treat patients with head and neck squamous cell carcinoma (HNSCC). One of the major side effects of radiotherapy is injury to the salivary glands (SG), which is thought to be mediated by microvascular dysfunction leading to permanent xerostomia. The goal of this study was to elucidate the mechanism of radiation-induced microvasculature damage and its impact on SG function. We measured bovine aortic endothelial cell (BAEC) apoptosis and ceramide production in response to 5 Gy irradiation, either alone or with reactive oxygen species (ROS) scavengers. We then investigated the effect of a single 15 Gy radiation dose on murine SG function. BAECs exposed to 5 Gy underwent apoptosis with increased ceramide production, both prevented by ROS scavengers. Among the 15 Gy irradiated mice, there was considerable weight loss, alopecia and SG hypofunction manifested by reduced saliva production and lower lysozyme levels. All of these effects, except for the lysozyme levels, were prevented by pretreatment with ROS scavengers. Microvessel density was significantly lower in the SG of irradiated mice compared to the control group, and this effect was significantly attenuated by pretreatment with Tempol. This study demonstrates that radiation-induced SG hypofunction is to a large extent mediated by microvascular dysfunction involving ceramide and ROS generation. These findings strongly suggest that ROS scavengers may serve as potential radioprotectors of SG function in patients undergoing radiotherapy for HNSCC.

IP3R deficit underlies loss of salivary fluid secretion in Sjögren's Syndrome.

The autoimmune exocrinopathy, Sjögren's syndrome (SS), is associated with secretory defects in patients, including individuals with mild lymphocytic infiltration and minimal glandular damage. The mechanism(s) underlying the secretory dysfunction is not known. We have used minor salivary gland biopsies from SS patients and healthy individuals to assess acinar cell function in morphologically intact glandular areas. We report that agonist-regulated intracellular Ca(2+) release, critically required for Ca(2+) entry and fluid secretion, is defective in acini from SS patients. Importantly, these acini displayed reduction in IP3R2 and IP3R3, but not AQP5 or STIM1. Similar decreases in IP3R and carbachol (CCh)-stimulated [Ca(2+)]i elevation were detected in acinar cells from lymphotoxin-alpha (LTα) transgenic (TG) mice, a model for (SS). Treatment of salivary glands from healthy individuals with LT α, a cytokine linked to disease progression in SS and IL14α mice, reduced Ca(2+) signaling. Together, our findings reveal novel IP3R deficits in acinar cells that underlie secretory dysfunction in SS patients.

Advances in salivary gland gene therapy - oral and systemic implications.

INTRODUCTION: Much research demonstrates the feasibility and efficacy of gene transfer to salivary glands. Recently, the first clinical trial targeting a salivary gland was completed, yielding positive safety and efficacy results. AREAS COVERED: There are two major disorders affecting salivary glands: radiation damage following treatment for head and neck cancers and Sjögren's syndrome (SS). Salivary gland gene transfer has also been employed in preclinical studies using transgenic secretory proteins for exocrine (upper gastrointestinal tract) and endocrine (systemic) applications. EXPERT OPINION: Salivary gland gene transfer is safe and can be beneficial in humans. Applications to treat and prevent radiation damage show considerable promise. A first-in-human clinical trial for the former was recently successfully completed. Studies on SS suffer from an inadequate understanding of its etiology. Proof of concept in animal models has been shown for exocrine and endocrine disorders. Currently, the most promising exocrine application is for the management of obesity. Endocrine applications are limited, as it is currently impossible to predict if systemically required transgenic proteins will be efficiently secreted into the bloodstream. This results from not understanding how secretory proteins are sorted. Future studies will likely employ ultrasound-assisted and pseudotyped adeno-associated viral vector-mediated gene transfer.

Recovery of radiation-induced dry eye and corneal damage by pretreatment with adenoviral vector-mediated transfer of erythropoietin to the salivary glands in mice.

Therapeutic doses of radiation (RTx) causes dry eye syndrome (DES), dry mouth, and as in other sicca syndromes, they are incurable. The aims of this work are as follows: (a) to evaluate a mouse model of DES induced by clinically relevant doses of radiation, and (b) to evaluate the protective effect of erythropoietin (Epo) in preventing DES. C3H female mice were subjected to five sessions of RTx, with or without pre-RTx retroductal administration of the AdLTR2EF1a-hEPO (AdEpo) vector in the salivary glands (SG), and compared with naïve controls at Day 10 (10d) (8 Gy fractions) and 56 days (56d) (6 Gy fractions) after RTx treatment. Mice were tested for changes in lacrimal glands (LG), tear secretion (phenol red thread), weight, hematocrit (Hct), and markers of inflammation, as well as microvessels and oxidative damage. Tear secretion was reduced in both RTx groups, compared to controls, by 10d. This was also seen at 56d in RTx but not AdEpo+RTx group. Hct was significantly higher in all AdEpo+RTx mice at 10d and 56d. Corneal epithelium was significantly thinner at 10d in the RTx group compared with AdEpo+RTx or the control mice. There was a significant reduction at 10d in vascular endothelial growth factor (VEGF)-R2 in LG in the RTx group that was prevented in the AdEpo+RTx group. In conclusion, RTx is able to induce DES in mice. AdEpo administration protected corneal epithelia and resulted in some recovery of LG function, supporting the value of further studies using gene therapy for extraglandular diseases.

Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction.

No conventional therapy exists for salivary hypofunction in surviving head and neck cancer patients with Radiation Therapy Oncology Group late grade 2-3 toxicity. We conducted a phase I clinical trial to test the safety and biologic efficacy of serotype 5, adenoviral-mediated aquaporin-1 cDNA transfer to a single previously irradiated parotid gland in 11 subjects using an open label, single-dose, dose-escalation design (AdhAQP1 vector; four dose tiers from 4.8 × 10(7) to 5.8 × 10(9) vector particles per gland). Treated subjects were followed at scheduled intervals. Multiple safety parameters were measured and biologic efficacy was evaluated with measurements of parotid salivary flow rate. Symptoms were assessed with a visual analog scale. All subjects tolerated vector delivery and study procedures well over the 42-d study period reported. No deaths, serious adverse events, or dose-limiting toxicities occurred. Generally, few adverse events occurred, and all were considered mild or moderate. No consistent changes were found in any clinical chemistry and hematology parameters measured. Objective responses were seen in six subjects, all at doses <5.8 × 10(9) vector particles per gland. Five of these six subjects also experienced subjective improvement in xerostomia. AdhAQP1 vector delivery to a single parotid gland was safe and transfer of the hAQP1 cDNA increased parotid flow and relieved symptoms in a subset of subjects.

A novel hybrid adenoretroviral vector with more extensive E3 deletion extends transgene expression in submandibular glands.

Salivary glands are an attractive target for gene transfer. Salivary epithelial cells are considered to be highly differentiated and have low rates of cell division (~6 months), affording the opportunity to obtain relatively long-term transgene expression in the absence of genomic integration. Here, we report a novel modified hybrid adenoretroviral vector, which provides stable transgene expression in salivary epithelial cells in vivo for up to 6 months in the absence of genomic integration. This modified hybrid vector, Ad(ΔE1/3)LTR(2)EF1α-hEPO, encodes human erythropoietin (hEPO) and differs from a previously developed hybrid vector, AdLTR(2)EF1α-hEPO, by having more extensive E3 gene deletion. Following direct salivary gland gene transfer by retroductal cannulation, rats transduced with Ad(ΔE1/3)LTR(2)EF1α-hEPO had sustained, elevated serum hEPO levels and hematocrits for 6 months (length of experiment), as compared with ~2 months for animals administered the AdLTR(2)EF1α-hEPO vector. Immunohistochemistry demonstrated that this novel vector could transduce both acinar and ductal cells. Interestingly, the Ad(ΔE1/3)LTR(2)EF1α-hEPO vector evoked much weaker local (salivary gland) immune responses than seen after AdLTR(2)EF1α-hEPO vector delivery, which likely permits its significantly lengthened transgene expression in this tissue.

Pharmacological protection from radiation ± cisplatin-induced oral mucositis.

PURPOSE: To evaluate if two pharmacological agents, Tempol and D-methionine (D-met), are able to prevent oral mucositis in mice after exposure to ionizing radiation ± cisplatin. METHODS AND MATERIALS: Female C3H mice, ∼8 weeks old, were irradiated with five fractionated doses ± cisplatin to induce oral mucositis (lingual ulcers). Just before irradiation and chemotherapy, mice were treated, either alone or in combination, with different doses of Tempol (by intraperitoneal [ip] injection or topically, as an oral gel) and D-met (by gavage). Thereafter, mice were sacrificed and tongues were harvested and stained with a solution of Toluidine Blue. Ulcer size and tongue epithelial thickness were measured. RESULTS: Significant lingual ulcers resulted from 5 × 8 Gy radiation fractions, which were enhanced with cisplatin treatment. D-met provided stereospecific partial protection from lingual ulceration after radiation. Tempol, via both routes of administration, provided nearly complete protection from lingual ulceration. D-met plus a suboptimal ip dose of Tempol also provided complete protection. CONCLUSIONS: Two fairly simple pharmacological treatments were able to markedly reduce chemoradiation-induced oral mucositis in mice. This proof of concept study suggests that Tempol, alone or in combination with D-met, may be a useful and convenient way to prevent the severe oral mucositis that results from head-and-neck cancer therapy.

Prevention of radiation-induced salivary hypofunction following hKGF gene delivery to murine submandibular glands.

PURPOSE: Salivary glands are significantly affected when head and neck cancer patients are treated by radiation. We evaluated the effect of human keratinocyte growth factor (hKGF) gene transfer to murine salivary glands on the prevention of radiation-induced salivary hypofunction. EXPERIMENTAL DESIGN: A hybrid serotype 5 adenoviral vector encoding hKGF (AdLTR(2)EF1α-hKGF) was constructed. Female C3H mice, 8 weeks old, were irradiated by single (15 Gy) or fractionated (6 Gy for 5 days) doses to induce salivary hypofunction. AdLTR(2)EF1α-hKGF or AdControl was administered (10(8) - 10(10) particles per gland) to both submandibular glands (SG) by retrograde ductal instillation before irradiation (IR). Salivary flow was measured following pilocarpine stimulation. Human KGF levels were measured by ELISA. SG cell proliferation was measured with bromodeoxyuridine labeling. Endothelial and progenitor or stem cells in SGs were measured by flow cytometry. The effect of SG hKGF production on squamous cell carcinoma (SCC VII) tumor growth was assessed. RESULTS: In 3 separate single-dose IR experiments, salivary flow rates of mice administered the AdLTR(2)EF1α-hKGF vector were not significantly different from nonirradiated control mice (P > 0.05). Similarly, in 3 separate fractionated IR experiments, the hKGF-expressing vector prevented salivary hypofunction dramatically. Transgenic hKGF protein was found at high levels in serum and SG extracts. AdLTR(2)EF1α-hKGF-treated mice showed increased cell proliferation and numbers of endothelial cells, compared with mice treated with AdControl. hKGF gene transfer had no effect on SCC VII tumor growth ± radiation. CONCLUSIONS: hKGF gene transfer prevents salivary hypofunction caused by either single or fractionated radiation dosing in mice. The findings suggest a potential clinical application.

Bone marrow-derived cells rescue salivary gland function in mice with head and neck irradiation.

Treatment for most patients with head and neck cancers includes ionizing radiation. A consequence of this treatment is irreversible damage to salivary glands (SGs), which is accompanied by a loss of fluid-secreting acinar-cells and a considerable decrease of saliva output. While there are currently no adequate conventional treatments for this condition, cell-based therapies are receiving increasing attention to regenerate SGs. In this study, we investigated whether bone marrow-derived cells (BMDCs) can differentiate into salivary epithelial cells and restore SG function in head and neck irradiated mice. BMDCs from male mice were transplanted into the tail-vein of 18Gy-irradiated female mice. Salivary output was increased in mice that received BMDCs transplantation at week 8 and 24 post-irradiation. At 24 weeks after irradiation (IR), harvested SGs (submandibular and parotid glands) of BMDC-treated mice had greater weights than those of non-treated mice. Histological analysis shows that SGs of treated mice demonstrated an increased level of tissue regenerative activity such as blood vessel formation and cell proliferation, while apoptotic activity was increased in non-transplanted mice. The expression of stem cell markers (Sca-1 or c-kit) was detected in BMDC-treated SGs. Finally, we detected an increased ratio of acinar-cell area and approximately 9% of Y-chromosome-positive (donor-derived) salivary epithelial cells in BMDC-treated mice. We propose here that cell therapy using BMDCs can rescue the functional damage of irradiated SGs by direct differentiation of donor BMDCs into salivary epithelial cells.

Effect of irradiation on microvascular endothelial cells of parotid glands in the miniature pig.

PURPOSE: To evaluate the effect of irradiation on microvascular endothelial cells in miniature pig parotid glands. METHODS AND MATERIALS: A single 25-Gy dose of irradiation (IR) was delivered to parotid glands of 6 miniature pigs. Three other animals served as non-IR controls. Local blood flow rate in glands was measured pre- and post-IR with an ultrasonic Doppler analyzer. Samples of parotid gland tissue were taken at 4 h, 24 h, 1 week, and 2 weeks after IR for microvascular density (MVD) analysis and sphingomyelinase (SMase) assay. Histopathology and immunohistochemical staining (anti-CD31 and anti-AQP1) were used to assess morphological changes. MVD was determined by calculating the number of CD31- or AQP1-stained cells per field. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay was used to detect apoptotic cells. The activity of acid and neutral Mg(2+)-dependent SMase (ASMase and NSMase, respectively) was also assayed. RESULTS: Local parotid gland blood flow rate decreased rapidly at 4 h post-IR and remained below control levels throughout the 14-day observation period. Parotid MVD also declined from 4 to 24 hours and remained below control levels thereafter. The activity levels of ASMase and NSMase in parotid glands increased rapidly from 4 to 24 h post-IR and then declined gradually. The frequency of detecting apoptotic nuclei in the glands followed similar kinetics. CONCLUSIONS: Single-dose IR led to a significant reduction of MVD and local blood flow rate, indicating marked damage to microvascular endothelial cells in miniature pig parotid glands. The significant and rapid increases of ASMase and NSMase activity levels may be important in this IR-induced damage.

Gene therapy of salivary diseases.

For many years, our laboratory has been developing gene transfer approaches for salivary gland disorders that currently lack effective therapy. The purpose of this chapter is to describe key methods used in this developmental process. Specifically, we focus on one clinical condition, irradiation-induced salivary hypofunction, and address the choice of transgene and vector to be used, the construction of recombinant viral vectors, how vector delivery is accomplished, and methods for assessing vector function in vitro and in an appropriate animal model.

Systemic delivery of bioactive glucagon-like peptide 1 after adenoviral-mediated gene transfer in the murine salivary gland.

An adenoviral (Ad) vector that expresses bioactive glucagon-like peptide 1 (GLP-1) was generated, and its effectiveness at modulating glucose homeostasis was evaluated after transduction of murine salivary glands. The construct was engineered with the signal sequence of mouse GH to direct the peptide into the secretory pathway, followed by a furin cleavage site and the GLP-1(7-37) sequence encoding an Ala to Gly substitution at position 8 to achieve resistance to degradation. When expressed in Neuro2A and COS7 cells, an active form of GLP-1 was specifically detected by RIA in the conditioned medium of transduced cells, showed resistance to degradation by dipeptidyl-peptidase IV, and induced the secretion of insulin from NIT1 pancreatic beta-cells in vitro. In vivo studies demonstrated that healthy mice transduced with Ad-GLP-1 in both submandibular glands had serum GLP-1 levels approximately 3 times higher than mice transduced with the control Ad-luciferase vector. In fasted animals, serum glucose levels were similar between Ad-GLP-1 and Ad-luciferase transduced mice in keeping with GLP-1's glucose-dependent action. However, when challenged with glucose, Ad-GLP-1 transduced mice cleared the glucose significantly faster than control mice. In an animal model of diabetes induced by alloxan, progression of hyperglycemia was significantly attenuated in mice given the Ad-GLP-1 vector compared with control mice. These studies demonstrate that the bioactive peptide hormone, GLP-1, normally secreted from endocrine cells in the gut through the regulated secretory pathway, can be engineered for secretion into the circulatory system from exocrine cells of the salivary gland to affect glucose homeostasis.

Radioprotectors and mitigators of radiation-induced normal tissue injury.

Radiation is used in the treatment of a broad range of malignancies. Exposure of normal tissue to radiation may result in both acute and chronic toxicities that can result in an inability to deliver the intended therapy, a range of symptoms, and a decrease in quality of life. Radioprotectors are compounds that are designed to reduce the damage in normal tissues caused by radiation. These compounds are often antioxidants and must be present before or at the time of radiation for effectiveness. Other agents, termed mitigators, may be used to minimize toxicity even after radiation has been delivered. Herein, we review agents in clinical use or in development as radioprotectors and mitigators of radiation-induced normal tissue injury. Few agents are approved for clinical use, but many new compounds show promising results in preclinical testing.

Development of a gene transfer-based treatment for radiation-induced salivary hypofunction.

A significant long-term side effect of radiation therapy for head and neck cancers is xerostomia, a dry mouth, due to salivary gland damage. Despite continuing efforts to eliminate this problem, many patients continue to suffer. This brief review describes our efforts to develop a gene transfer approach, employing the aquaporin-1 cDNA, to treat patients with existing radiation-induced salivary hypofunction. A Phase I/II clinical trial, using a recombinant adenoviral vector to mediate gene transfer, is currently underway.

Transient detection of E1-containing adenovirus in saliva after the delivery of a first-generation adenoviral vector to human parotid gland.

BACKGROUND: Radiation-induced salivary hypofunction is a common side-effect of treatment for head and neck cancers. Patients suffer significant morbidity and there is no suitable conventional therapy. We are conducting a Phase I clinical trial, using a first-generation serotype 5 adenoviral (Ad5) vector encoding human aquaporin-1 (AdhAQP1) to treat such patients. One week after the administration of AdhAQP1 to an enrolled, generally healthy patient, E1-containing adenovirus was detected in parotid saliva. METHODS: The real-time quantitative polymerase chain reaction (PCR) was used to measure the Ad5 E1 gene and AdhAQP1 in saliva and serum. PCR and sequencing were used to characterize viral/vector DNA extracted from saliva. The presence of infectious adenovirus was assessed by the inoculation of A549 cells with aliquots of saliva. Serum Ad5 neutralizing antibodies were measured by the inhibition of 293-cell transduction with an Ad5 vector encoding luciferase. Multiple clinical evaluations were performed. RESULTS: On day 7 after AdhAQP1 delivery, low levels of the Ad5 E1 gene were detected in parotid saliva (82 copies/microl). In addition, significant levels of AdhAQP1 were also detected (1.5 x 10(3) copies/microl). The patient was asymptomatic and subsequent analysis of parotid saliva samples prior to day 7 and after day 7 until day 42 was negative for both virus and vector. No virus or vector was detected in serum at any time. Detailed PCR analyses of DNA extracted from the day 7 parotid saliva sample suggested the absence of a recombination event, and no infectious virus was found. CONCLUSIONS: The patient most likely had a latent Ad5 infection in the targeted parotid gland that was activated after gene transfer and was without clinical consequence.

Prevention of radiation-induced oral mucositis after adenoviral vector-mediated transfer of the keratinocyte growth factor cDNA to mouse submandibular glands.

PURPOSE: The study aims to evaluate if human keratinocyte growth factor (hKGF), secreted after transduction of murine salivary glands with adenoviral vectors, can prevent oral mucositis resulting from radiation. EXPERIMENTAL DESIGN: Two serotype 5 adenoviral vectors encoding hKGF were constructed: AdEF1alpha-hKGF and AdLTR(2)EF1alpha-hKGF. Female C3H mice, 8 weeks old, were irradiated by single (22.5 Gy) or fractionated (5 x 8 Gy for 5 days) doses to induce oral mucositis (ulcers on tongue). One day before irradiation, the above viral vectors or an empty vector, Adcontrol, was given (10(10) particles per gland) to both submandibular glands by retrograde ductal instillation. Each experiment included five groups: no irradiation and irradiation (+/-Adcontrol, AdEF1alpha-hKGF, or AdLTR(2)EF1alpha-hKGF). Blood, saliva, submandibular glands, and tongue were collected on day 7 for single-dose studies or day 10 for fractionated dosing. hKGF levels were measured by ELISA. RESULTS: In three separate single-dose irradiation experiments, lingual ulcers were dramatically reduced after either KGF-expressing vector. Similarly, in two separate fractionated irradiation experiments, the hKGF-expressing vectors completely prevented ulcer formation. QPCR data indicated that approximately 10(7) to 10(8) particles of each vector remained in the targeted submandibular glands at the terminal time. Transgenic hKGF protein was found at high levels in saliva, serum, and submandibular gland extracts. CONCLUSIONS: hKGF gene transfer to salivary glands prevented radiation-induced oral mucositis in mice. This proof of concept study suggests that transgenic hKGF secreted from transduced salivary glands may be useful clinically to prevent oral mucositis caused by radiation.

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.