Polarized secretion of transgene products from salivary glands in vivo.

Previously (Kagami et al. Hum. Gene Ther. 1996;7:2177-2184) we have shown that salivary glands are able to secrete a transgene-encoded protein into serum as well as saliva. This result and other published data suggest that salivary glands may be a useful target site for vectors encoding therapeutic proteins for systemic delivery. The aim of the present study was to assess in vivo if transgene-encoded secretory proteins follow distinct, polarized sorting pathways as has been shown to occur "classically" in cell biological studies in vitro. Four first-generation, E1-, type 5 recombinant adenoviruses were used to deliver different transgenes to a rat submandibular cell line in vitro or to rat submandibular glands in vivo. Subsequently, the secretory distribution of the encoded proteins was determined. Luciferase, which has no signal peptide, served as a cell-associated, negative control and was used to correct for any nonspecific secretory protein release from cells. The three remaining transgene products tested, human tissue kallikrein (hK1), human growth hormone (hGH), and human alpha1-antitrypsin (halpha1AT), were predominantly secreted (>96%) in vitro. Most importantly, in vivo, after a parasympathomimetic secretory stimulus, both hK1 and hGH were secreted primarily in an exocrine manner into saliva. Conversely, halpha1AT was predominantly secreted into the bloodstream, i.e., in an endocrine manner. The aggregate results are consistent with the recognition of signals encoded within the transgenes that result in specific patterns of polarized protein secretion from rat submandibular gland cells in vivo.

Construction and in vivo efficacy of a replication-deficient recombinant adenovirus encoding murine growth hormone.

We have constructed a recombinant, replication-deficient, first-generation adenovirus-encoding mouse GH (mGH), AdCMVmGH. This virus directed mGH production from an epithelial cell line in vitro in a dose-dependent manner. When injected into the quadriceps muscle or submandibular ducts of mGH-deficient Snell dwarf mice, AdCMVmGH resulted in the production of significantly elevated serum mGH levels. Furthermore, after i.m. injection, dwarf mice increased in weight by 8% over 4 days and close to 100% by 30 days. When AdCMVmGH was administered to 3- to 4-week-old rats by i.v. injection to assess general metabolic responses, serum mGH, insulin-like growth factor 1, triglycerides and cholesterol levels were significantly elevated. AdCMVmGH should be a valuable experimental tool for the controlled, directed expression of mGH in preclinical mouse model studies.

Salivary gland transplantation: a canine model.

Impaired salivary function with resultant severe dryness of the mouth, or xerostomia, may occur in association with a variety of systemic disorders or therapies. No adequate treatment exists for this debilitating condition, which impedes normal oral function, in particular alimentation and phonation. This study explores the feasibility of salivary gland autotransplantation, using a canine model. A salivary gland with its duct and surrounding blood vessels still attached was excised and reimplanted in the dog's thigh by anastomosing the graft's blood vessels to the femoral artery and vein. The duct was sutured to an artificial orifice cut in the thigh's skin, from which the saliva was collected. Salivary secretion was induced by a single intravenous bolus of pilocarpine (5 mg). Preoperative (normal) salivation was measured by collecting saliva from the gland in situ. Periodic functional studies showed normal saliva production during the first month after grafting, after which the salivary flow was reduced by 35% over the next 2 months. This reduction was interpreted as a sign of disuse atrophy resulting from the lack of autonomic innervation. To overcome this impediment, oral pilocarpine (5 mg/day) was administered to the recipient dog, after which normal levels of saliva were excreted through the graft during the 3-month follow-up period. The quality of the graft saliva was assessed by its protein and electrolyte levels, which showed close to normal values.

Parotid salivary gland dysfunction in chronic graft-versus-host disease (cGVHD): a longitudinal study in a mouse model.

Chronic graft-versus-host disease (cGVHD) is an autoimmune-like phenomenon resulting in morbidity and mortality following allogeneic bone marrow transplantation (BMT). Major salivary gland dysfunction and hyposalivation is one of the prevalent manifestations of cGVHD. We have used the B10.D2 to Balb/C cGVHD mice model in order to assess major salivary gland function in cGVHD, evaluating sialometric, sialochemical and histopathological parameters for almost 3 months. As cGVHD is a chronic debilitating disease it is of vast importance to evaluate these parameters on a prolonged longitudinal basis. We observed significant reduction in parotid salivary flow rate and disturbance in the salivary dynamic function in cGVHD mice in comparison to the normal and syngeneic transplanted controls. On days 18, 25, 46, 56 and 88 the mean flow rates of the cGVHD group were 37.4 +/- 4.4 microl/30 min, 40.5 +/- 4.6 microl/30 min, 32.5 +/- 2.3 microl/30 min, 22.2 +/- 3.2 microl/30 min and 14.8 +/- 3.8 microl/30 min, respectively, values which were lower than those of the syngeneic transplanted controls group by 42% (P < 0.04), 32% (P < 0.03), 44% (P < 0.01), 49% (P < 0.01) and 64% (P < 0.01), respectively. These changes in flow rates were paralleled by changes in the biochemical composition of the saliva. Moreover, the reduction in flow rates correlated with the degree of salivary gland destruction observed in the pathological slides. An inverse correlation was observed between the mean parotid salivary flow rate and the degree of fibrosis observed in the histopathological evaluation of the cGVHD mice (P < 0.01). Maximal flow rate 34.8 +/- 4.6 microl/30 min was observed when no fibrosis was observed while in mice with maximal fibrosis flow rates were minimal. This may point to the pathological mechanism leading to the major salivary gland dysfunction and hyposalivation observed in cGVHD. Thus, it may broaden our knowledge and provide the scientific background for designing better therapeutic strategies for this complication. Bone Marrow Transplantation (2000).

Major salivary gland dysfunction in patients with hematological malignancies receiving interleukin-2-based immunotherapy post-autologous blood stem cell transplantation (ABSCT).

lnterleukin-2 (IL-2) is known to cause xerostomia and skin manifestations similar to graft-versus-host disease (GVHD). We therefore evaluated major salivary gland function in patients with hematological malignancies treated with IL-2 and interferon-alpha (IFN-alpha) after ABSCT. Eleven patients (seven male, four female) of median age 40 (24-47) were evaluated, seven with non-Hodgkin lymphoma (NHL); one with Hodgkin's disease (HD) and three with acute myelogenous leukemia (AML). Parotid and submandibular salivary gland function was assessed before, during and after IL-2/IFN-alpha administration by evaluation of the salivary flow rate and the composition of secreted saliva. Significant reductions in both the resting and stimulated parotid and submandibular salivary flow rates were observed during IL-2/IFN-alpha immunotherapy compared with the pre- and post-therapy values (P < 0.01), while no hyposalivation was observed in the control patients who underwent ABSCT and did not received IL-2. Sialochemical evaluation revealed a significant increase in potassium concentration (24.4+/-0.6 mEq/l to 28.9+/-1.4 mEq/l) and a significant decrease in sodium concentration (6.7+/-2.1 mEq/l to 3.3+/-1.0 mEq/l) (P < 0.05) in the stimulated parotid gland saliva secreted during IL-2/IFN-alpha administration. Salivary protein concentrations were not altered by the IL-2/IFN-alpha immunotherapy. Similar changes were previously observed in mice and humans with chronic GVHD. We conclude that IL-2 immunotherapy induces major salivary gland dysfunction in humans, similar to our previous observations in patients with chronic GVHD, which may indicate similar pathophysiologic mechanisms.

Major salivary gland dysfunction in human acute and chronic graft-versus-host disease (GVHD).

Salivary gland dysfunction is frequently observed in patients suffering from acute (a) and chronic (c) GVHD. We studied the influence of GVHD on the function of major salivary glands in 20 patients with GVHD (cGVHD, 15; aGVHD, 5). A subjective evaluation of salivary function was performed, in which the score ranged from 0-4 where a higher score indicated more oral dryness. Patients with aGVHD scored 4.0 while patients with cGVHD scored 2.1 (P < 0.01). In addition to this subjective evaluation, patient's salivary flow rates were measured and a reduction of 90% and 60% in aGVHD and cGVHD patients respectively, was observed as compared to controls (P < 0.01; P < 0.05). No hyposalivation was observed in patients who underwent bone marrow transplantation but did not develop GVHD as compared to normal individuals. A direct correlation was observed between the degree of hyposalivation and the severity of the GVHD. Hyposalivation was also documented by scintigraphy of the major salivary glands in the GVHD patients. Furthermore, hyposalivation was associated with extensive mucosal atrophy, erythema, tongue surface depapillation, lichenoid lesions of the buccal and labial mucosa as well as lupus-like lesions. Routine assessment of these glands in patients with GVHD could play a role in monitoring response to therapy.

Novel protection strategy against X-ray-induced damage to salivary glands.

Exposure of the major salivary glands to ionizing radiation often results in severe alterations in structure and function. The mechanism of these effects is still unknown, and no adequate prevention or treatment is yet available. The purpose of this study was to examine a mechanism based on the assumption that redox-active metal ions, which propagate the production of highly reactive free radicals, are responsible for the unique radiosensitivity of salivary glands. Zinc-desferrioxamine (Zn-DFO) was recently reported to be a very potent protector against the injuries induced by such metal ions in the vicinity of sensitive cellular targets. We chose to examine its protective potential against the damage to salivary glands induced by X rays. Head and neck irradiation (15 Gy) was delivered to rats 90 min after the intraperitoneal administration of 20 mg/kg Zn-DFO. This group was compared to two control groups, irradiated and nonirradiated. At 2 months after irradiation, both systemic and salivary parameters were analyzed. The results demonstrated that X irradiation induced a profound attenuation of body weight (30%) and a reduction of parotid gland saliva flow rate (74%), parotid gland weight (36%), submandibular gland/sublingual gland saliva flow rate (46%), and submandibular/sublingual gland weight (24%) (P < 0.01 for all parameters). The content of potassium in parotid gland saliva was increased by 46% (P < 0.01), while the protein content was unaltered. The increase in the potassium concentration of the saliva is considered to be another indication of salivary gland hypofunction. Administration of Zn-DFO prior to irradiation resulted in partial protection against radiation-induced injury to the parotid gland but not the submandibular gland. In the Zn-DFO-treated and irradiated group, the parotid gland saliva flow rate was reduced by 42%, the weight of the parotid gland was reduced by 13%, and the potassium concentration in the parotid gland saliva was increased by 21% (P < 0.05 for all parameters). These results give credence to the validity of the hypothesis which correlates radiation-induced damage of the salivary glands with the injurious role of intracellular redox-active metal ions. Furthermore, the results offer prospects in the clinical setting, as Zn-DFO is a modification of DFO, which is a clinically approved and widely used medication. Further examination of the clinical use of Zn-DFO is currently under way, focusing on its beneficial protective effect on healthy non-neoplastic tissue.

Irradiation-induced damage to the salivary glands: the role of redox-active iron and copper.

The mechanism of irradiation-induced hypofunction of the salivary glands is a process that is not fully understood. Here we examine the hypothesis that intracellular and redox-active ions of iron and copper, which are associated with the secretion granules, play a catalytic role in the irradiation-induced damage. Rats were subjected to head and neck irradiation (15 Gy X rays) and allowed to recover for 2 months. The function of the parotid and submandibular glands was then determined by pilocarpine-stimulated salivary secretion. A 45% decrease in the function of both glands was obtained when compared to nonirradiated controls. Treatment prior to irradiation (90 min) with cyclocytidine (200 mg/kg) led to a massive degranulation of the parotid gland and yielded nearly complete protection from radiation-induced damage. In contrast, pilocarpine stimulation prior to irradiation led to a marginal degranulation of the parotid gland and yielded only 13% protection. Neither agent caused degranulation of the submandibular gland mucous cells or yielded functional protection of this gland. Treatment with both agents yielded a marked increase in iron, copper and manganese levels in the parotid gland saliva. An analogous marked increase in the redox activity of iron and copper ions was recorded for the parotid saliva stimulated by pilocarpine and cyclocytidine. Pilocarpine-stimulated submandibular gland saliva contained metal levels similar to those of the parotid gland saliva. However, no redox activity and no increase in metal mobilization could be demonstrated in the submandibular gland saliva stimulated by both agents. The correlation between the patterns of gland degranulation, mobilization of redoxactive metals and the protection of gland function, for both parotid and submandibular glands, focuses attention on the catalytic roles played by transition metal ions in promoting free radical reactions, which likely participate in the process of injury to the tissue.

Re-engineering the functions of a terminally differentiated epithelial cell in vivo.

Because of their easy access, and important role in oral homeostasis, mammalian salivary glands provide a unique site for addressing key issues and problems in tissue engineering. This manuscript reviews studies by us in three major directions involving re-engineering functions of salivary epithelial cells. Using adenoviral-mediated gene transfer in vivo, we show approaches to i) repair damaged, hypofunctional glands and ii) redesign secretory functions to include endocrine as well as exocrine pathways. The third series of studies show our general approach to develop an artificial salivary gland for clinical situations in which all glandular tissue has been lost.

Evidence for contribution of effector organ cellular responses to the biphasic dynamics of heat acclimation.

The involvement of cellular processes in the biphasic dynamics of heat acclimation was studied. Key steps in the cholinergic signal transduction pathway for water secretion were measured in the submaxillary gland of acclimating [2-day short-term heat acclimation (STHA) and 30-day long-term heat acclimation (LTHA) at 34 degrees C] or acute heat-stressed (2 h at 40 degrees C) rats in vitro. Both the carbamylcholine (CCh)-induced maximal fractional rate and the total 86Rb+ efflux, reflecting K+ efflux and water transport, transiently decreased in STHA (P < 0.001). In LTHA, the total K+ efflux increased (P < 0.001), whereas the maximal fractional rate of efflux increased only slightly. During STHA, the density of the high-affinity binding site of the muscarinic receptors (MRs) increased by 50% and their affinity for the muscarinic antagonist [3H]-N-methylscopolamine decreased transiently by 87%. Basal cytosolic Ca2+ concentration ([Ca2+]i) decreased (P < 0.05), but the peak CCh-induced [Ca2+]i increase resembled the control values. In LTHA, MR density continued to increase (100%; P < 0.05), whereas affinity resumed control values. Basal and CCh-induced [Ca2+]i increases returned to control levels. We conclude that glandular cellular processes follow a biphasic pattern with major apparent changes attributable to events distal to the [Ca2+]i rise. This was further validated by employing heat stress, which produced qualitatively different effects on the MR profile with a decrease in 86Rb+ efflux comparable to STHA. Hence, although heat-induced changes in the proximal components of the signal transduction pathway may contribute to altered regulatory span, the predominant apparent cellular effect is on the distal part of the pathway.

Primary lymphoma of the parotid gland: a report of twelve cases with a review of the literature.

Primary extranodal lymphoma of the salivary gland is an extremely rare disease. In this report we describe twelve cases of primary lymphoma of the parotid gland seen at a single centre, and review the relevant literature. The 12 cases were treated in different departments and did not receive a uniform therapeutic approach. All three patients with Hodgkin's disease are still alive and two are in complete remission after initial radiotherapy. One of these cases developed stage 4 disease and had to receive combination chemotherapy subsequently. Of the 9 non-Hodgkin's lymphoma (NHL) patients, four had low grade NHL and 5 intermediate or high grade NHL. Of these, 2 died with disseminated disease. However, 6 are still alive and well from 1 to 5 years after therapy. These cases were treated with surgery alone, radiotherapy alone or combination chemotherapy with an anthracycline-bearing regimen. Consequently, we are unable to draw any conclusions relating the success of therapy in these cases, nor can we suggest therapeutic guidelines on the basis of this study alone. The treatment of parotid lymphoma is discussed briefly in the light of the available literature. In most cases, symptoms related to an enlarging mass in the parotid region, were evident. In the light of the above data, we suggest that, despite its rarity, lymphoma of the salivary gland should always be considered in the differential diagnosis of a parotid mass. No correlation between lymphoma and Sjogren's syndrome was noted in the present study.

Lack of acute effects of methotrexate on rat parotid salivary gland function.

Like other cytotoxic drugs, methotrexate (MTX) produces adverse reactions in oral tissues. Parotid gland function was examined, in vivo and in vitro, 18 h after MTX administration (15 mg/kg, i.p.). No salivary effects could be detected consistently. In addition, the integrity of the oral mucosa remained intact. Thus, at this dose, MTX does not exert an acute cytotoxic effect either on a rapidly replicating oral tissue like the mucosa, or on tissue with a slow turnover rate like the parotid.

A novel and non-invasive method for the removal of salivary gland stones.

A non-invasive method for salivary gland stone disintegration by shock waves is proposed. An in vitro experiment, in which a large sialolith was subjected to shock waves produced by a Dornier lithotriptor, demonstrated the complete destruction of the stone. The advantages of the method and the need for modification of the existing equipment is discussed.

Hyperostosis corticalis generalisata: surgical management and long-term follow-up of one patient.

A 24-year-old woman presented for treatment of her distorted facial appearance. She showed marked widening of the face and skull, which had first become noticeable in childhood. Significant thickening of the cortical bone was seen radiographically throughout the skeleton. Routine laboratory and endocrinological tests showed normal results. These findings, together with a family history of bone disorder, led to the diagnosis of hyperostosis corticalis generalisata. The lower border of the mandible was resected, resulting in improved facial appearance. During the 8-year follow-up, no changes were seen with regard to the mandible.

Thyroid papillary carcinoma presenting as jaw and parotid gland metastases.

A slowly progressing colloidal goiter of 20 years standing was excised in a 67-year old woman. Two years later, a metastatic tumor inducing facial swelling was diagnosed radiologically in the left parotid gland and left ascending mandibular ramus. The incisional biopsy confirmed papillary carcinoma of thyroid origin. The patient refused treatment. Six months later, she returned to the clinic and presented with advanced destructive changes of the involved structures. This rare case of unrestricted malignant growth may shed light upon the sequence and mode of metastatic invasion.

The use of monoclonal anti-CEA antibody immunohistochemistry in detecting the origin of oral cavity metastasis.

A 75-year-old woman presented with a painless swelling in the right mandibular retromolar area and numbness of the left lower lip. Radiographic examination of the mandible demonstrated an osteolytic lesion of the ascending ramus. Biopsy revealed adenocarcinoma of obscure origin. Staining of the specimen with a monoclonal antibody specific to colon carcinoma revealed its origin. On subsequent examinations, a primary tumor in the rectosigmoid region with extensive lung, liver and skeletal metastases were diagnosed. This unusual case of colonorectal carcinoma, presenting as a metastatic lesion of the mandible, was readily diagnosed by a novel immunohistochemical technique that utilizes highly specific monoclonal antibodies.

The diagnostic value of computed tomography in temporomandibular joint synovial chondromatosis.

Synovial chondromatosis of the temporomandibular joint was diagnosed and location of dens particles was assessed by means of computed tomography. Based upon the data collected, the joint was operated conservatively and most of the calcifications were removed leaving the patient symptom free. The importance of an accurate preoperative diagnosis of synovial chondromatosis by use of CT scan is stressed in view of numerous unnecessary condylectomies and parotidectomies reported in the literature.

The mouth is a gateway to the body: gene therapy in 21st-century dental practice.

Gene therapy may become an integral tool in dental practice early in the 21st century. It and other biological therapies are expected to be applied to oral diseases and disorders during the midpractice lifetime of today's dental students. If the applications of oral gene transfer are expanded to systemic diseases, oral health care providers in the future could routinely be "gene therapists" with therapeutic targets well outside the oral cavity.