Reduced oral wound healing in the NOD mouse model for type 1 autoimmune diabetes and its reversal by epidermal growth factor supplementation.

Using the NOD mouse, a model for type 1 diabetes, we examined how reduced concentrations of epidermal growth factor (EGF) in the saliva, after onset of type 1 diabetes, affect oral wound healing. Diabetic NOD/LtJ mice on insulin therapy, prediabetic NOD/LtJ, and age- and sex-matched BALB/cJ mice were given a cutaneous tongue punch and allowed to undergo normal healing. With diabetes onset and a reduction in saliva-derived growth factor levels, the rate of tongue wound healing was reduced compared with nondiabetic NOD/LtJ and healthy BALB/cJ mice. Addition of exogenous EGF to the drinking water did not accelerate the rate of healing in BALB/cJ or prediabetic NOD/LtJ; however, diabetic NOD/LtJ mice exhibited accelerated wound healing similar to healthy mice. These results demonstrate that loss of growth factors from saliva is associated with profoundly reduced oral wound healing, suggesting that therapeutic treatment with topical delivery may be beneficial to patients with type 1 diabetes and oral wound complications.

Differential absorption and distribution of epidermal growth factor and insulin-like growth factor in diabetic NOD mice.

Previous studies have shown that absorption of growth factors occurs through the gastrointestinal tract and the oral cavity. The non-obese diabetic (NOD) mouse, a model for spontaneous development of type 1 insulin-dependent diabetes (IDDM), was evaluated for the absorption and systemic distribution of growth factors. Radiolabeled epidermal growth factor (EGF) and insulin-like growth factor, type I (IGF-I), were administered by gavage into the stomach or by lozenge into the sublingual vasculature of either diabetic or nondiabetic mice. After a time-dependent uptake, the levels of absorption and distribution through the tissues were measured. A similar time course of EGF absorption following gavage administration was determined for NOD and C57BL/6 mice, with a maximum tissue distribution by 30-min post infusion. Diabetic NOD mice showed similar levels of IGF uptake and tissue distribution compared with nondiabetic NOD and normal healthy C57BL/6 mice, whether administered by gavage or sublingual lozenge. On the other hand, gavage uptake and tissue distribution of EGF was significantly higher in diabetic mice when compared to sublingual administration in nondiabetic NOD or C57BL/6 healthy control mice. These findings suggest that the overall potential uptake and distribution of saliva-derived growth factors in systemic wound-healing processes is retained with diabetes onset, and may offer a new avenue to treating this complication of diabetes.

Salivary EGF levels reduced in diabetic patients.

Oral problems such as periodontitis are recognized major complications associated with diabetes. Salivary derived growth factors, including epidermal growth factor (EGF), are thought to play a role in helping maintain levels of oral health, promoting wound healing, and maintaining mucosal integrity. In the present study, salivary levels of EGF in diabetic vs. healthy control patients was evaluated. Twenty-one diabetic patients participated in this study. Age, race, sex and smoking histories were matched with 21 systematically healthy nondiabetic patients. Three milliliters of unstimulated resting whole saliva was collected from each patient at 6 h intervals up to 42 h and whole saliva protein concentrations were determined for each sample. EGF concentrations for each sample were quantitated spectrophometrically utilizing an immunoassay. Diabetic patients had greater salivary protein concentrations over 42 h of collection with a mean of 1.502+/-0.09 vs. 1.242+/-0.05 mg/ml for healthy control patients. The EGF concentration was significantly lower (p<0.05) for the diabetic patients compared to control patients, whether expressed relative to 1 ml volume of saliva (873.43+/-106.5 vs. 1101.09+/-116.8 pg/ml) or 1 mg whole saliva protein (629.18+/-92.6 vs. 931.20+/-124.6 pg/mg saliva protein). This study suggests that reduced levels of salivary EGF in diabetic patients may contribute to the development of oral and systemic complications of diabetes, which may have future clinical applications.

Aberrant proteolytic digestion of biglycan and decorin by saliva and exocrine gland lysates from the NOD mouse model for autoimmune exocrinopathy.

OBJECTIVE: The protein components of the extracellular matrix (ECM) are responsible for driving tissue morphogenesis, the development of differentiated function, and the sequestration of biologically active molecules such as growth factors in close proximity to tissue and organ cells. Recent reports indicate that saliva and exocrine tissue lysates from Sjögren's syndrome patients and the non-obese diabetic (NOD) mouse model for autoimmune exocrinopathy demonstrate elevated levels of specific enzymes that degrade the ECM, especially the matrix metalloproteinases (MMPs). To determine if elevated levels of MMPs could be important in exocrine tissue destruction, we examined proteolytic activity against two ECM proteoglycans, decorin and biglycan. METHODS: Purified recombinant human core protein for decorin or biglycan was incubated with saliva or gland lysates from either control BALB/c or NOD mice. Degraded proteoglycan products were estimated by Western blotting analysis using anti-decorin or anti-biglycan monospecific polyclonal antibodies. The levels of TGF beta protein were measured by ELISA. RESULTS: Proteolytic activity for decorin and biglycan was not observed in the saliva and salivary gland lysates collected from C57BL/6 or BALB/c mice used as normal controls. In contrast, both proteoglycans were degraded by saliva and exocrine gland lysates from NOD mice and the congenic partner strains NOD-scid and NOD.B10.H2b. This proteolytic activity for proteoglycans was inhibited by the MMP inhibitors, EDTA, GM6001 and 1,10-phenanthroline. Protein steady state levels for TGF beta were increased in the saliva and gland lysates from 20-week old NOD strains, as compared to BALB/c mice and NOD treated with the MMP inhibitor GM6001. With the inhibition of MMP activity, TGF beta levels declined in saliva and gland lysates. CONCLUSION: Proteolytic degradation of the ECM molecules decorin and biglycan is elevated in the exocrine tissues of the NOD mouse model for Sjögren's syndrome. Furthermore, the proteolysis of small leucine-rich proteoglycans correlates with the presence of elevated levels of TGF beta in gland lysates and saliva.

Elevated levels of human salivary epidermal growth factor after oral and juxtaoral surgery.

PURPOSE: Saliva provides a natural reservoir of growth factors whose purposes have remained elusive. Animal studies suggest that saliva-derived growth factors play a role in systemic and oral wound healing. In the current study, salivary concentrations of epidermal growth factor (EGF) were monitored in patients before and after oral and juxtaoral surgery. PATIENTS AND METHODS: Whole resting saliva was collected from a group of patients with parotid gland tumors requiring surgical resection. Another group of patients a history of periodontal disease requiring surgical intervention also provided whole salivary samples. Healthy age- and sex-matched persons served as controls. RESULTS: Salivary EGF levels were elevated in both groups of patients within 24 hours after surgery. In the periodontitis patients, a second smaller peak was assayed noted between 36 and 48 hours. After this, EGF concentrations returned to levels comparable to healthy controls in both experimental groups. CONCLUSIONS: Although the local cells have the ability to synthesize and secrete growth factors at a site of injury, these results suggest that surgery stimulates increased synthesis and secretion of growth factors in the saliva as well. This increased level of saliva-derived growth factor may also aid in promoting wound healing.

Excessive synthesis of matrix metalloproteinases in exocrine tissues of NOD mouse models for Sjögren's syndrome.

OBJECTIVE: Matrix metalloproteinases (MMP) and their substrates, components of the extracellular matrix, regulate environmental signals for cellular differentiation and tissue function. Changes in the levels of these enzymes may influence cell survival as well as pathology involving ectopic apoptosis. Using the non-obese diabetic (NOD) mouse model for Sjögren's syndrome, we evaluated the synthesis and expression of MMP in the exocrine target tissues of autoimmunity. METHODS: NOD, immunodeficient NOD-scid, and nondiabetic NOD.B10.H2b mice were evaluated for MMP activity in their saliva and exocrine gland lysates by gelatin zymography and reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, changes in protein content of saliva and gland lysates were determined by specific Western blot and by enzymatic activity of amylase and cysteine proteases. Mice continuously treated with the MMP inhibitor GM6001 were evaluated from 7 to 20 weeks of age for the contribution of MMP activity to development of these hallmark biochemical markers of Sjogren's syndrome-like disease of NOD mice. RESULTS: Gelatin zymography of whole saliva and gland lysates indicated the presence of increased proteolytic activity, corresponding to proteins with a molecular mass ranging from 50 to 95 kDa, in the saliva of older (> 20 weeks of age) NOD mice as well as NOD.B10.H2b and NOD-scid mice compared to BALB/c controls. Elevated steady state levels of mRNA transcripts for the gelatinases MMP-2 and MMP-9 were detected in total RNA extracted from parotid and submandibular glands by RT-PCR. Despite prophylactic injection of the broad spectrum MMP inhibitor GM6001 into mice beginning at 7 weeks of age and continuing to 20 weeks, development of the autoimmune exocrinopathy was neither stopped nor retarded. CONCLUSION: These observations suggest that excessive MMP activity is associated with autoimmune Sjögren's syndrome-like disease in NOD mice. However, a possible contribution by increased MMP activity in initiation and progression of this autoimmune disease is yet to be elucidated.

Elevated salivary EGF levels stimulated by periodontal surgery.

Previous studies have shown epidermal growth factor (EGF) to be involved in oral wound healing as well as gastric cytoprotection. EGF functions with hormone-like properties to stimulate epithelial cells by binding to the EGF receptor which ultimately leads to proliferation via signal transduction mechanisms. Salivary glands are a major source of EGF The purpose of this study was to determine if intra-oral wounding by periodontal surgery stimulated increased salivary EGF levels. Salivary EGF levels were determined for 12 systemically healthy individuals (ages 27 to 70 years old) presurgically and postsurgically at 6, 12, 18, 24, 30, 36, and 42 hours and 2 and 6 weeks. Three ml of unstimulated whole saliva was obtained at each time point to allow immunoassay quantitation. Age and gender matched unoperated patients served as controls. Salivary samples were incubated with monoclonal and polyclonal EGF antibodies in these "sandwich" enzyme immunoassays. Quantitation was obtained by spectrometric analysis at OD 450 nm after addition of 200 microl of colorimetric substrate. Mean EGF levels ranged from 2441 pg/ml presurgically to 3349 pg/ml at 18 hours postsurgically and 1207 pg/ml at 6 weeks postsurgically. Repeated measures analysis of variance indicated statistically significant differences in 18 hours postsurgical salivary EGF levels when compared to controls and to postsurgical levels from 36 hours forward (P < 0.01). A second smaller rise in EGF was detected at 30 hours. These results suggest a transient increase in salivary EGF levels in response to intra-oral wounding.

Treatment of 3rd molar-induced periodontal defects with guided tissue regeneration.

Recent reports provide evidence of increased attachment levels when using guided tissue regeneration (GTR) techniques for the treatment of periodontal defects. Periodontal defects frequently occur at the distal aspect of mandibular 2nd molars which are next to mesioangular impacted 3rd molars that have oral communication. The purpose of this study was to determine whether the use of GTR can enhance probing attachment levels (PALs) following extraction of mesioangular impacted third molars. 12 patients with bilateral soft tissue impacted mandibular 3rd molars entered this split mouth study. After extractions, the previously exposed distal root surface of the 2nd molars were debrided. The defects on the randomly selected experimental sites were covered with expanded polytetraflouro-ethylene (e-PTFE) membrane and the tissue was replaced to cover the membrane. Membranes were removed after 6 weeks. Control sites were treated identically except no membrane was placed. GI, P1I, PD, PAL and BOP records were obtained at 0, 3 and 6 months. The use of barrier material did not provide statistically-significant differences in PAL when comparing experimental versus control sites. Nevertheless, PAL gain was consistently greater at 3 and 6 months when GTR techniques were used in sites with deep impactions.

Extracellular-matrix gene expression during mouse submandibular gland development.

Early morphogenesis of mouse submandibular glands begins on late day 11 of fetal development when the epithelium begins to bud from the surrounding mandibular mesenchyme. Using total RNA collected from fetal BALB/c submandibular glands, steady-state levels of mRNA expression for extracellular matrix molecules were measured using quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR). By comparing the PCR amplification products of both the cellular mRNA and a synthetic template, pMATRIX, it was possible to measure the direct expression of collagens alpha2(I), alpha1(III), alpha1(IV), fibronectin, laminin B2, elastin and lysyl oxidase genes. There was an observed trend for an increasing concentration of collagen alpha2(I), collagen alpha1(III) and lysyl oxidase mRNA molecules per cell on day 16 of development. The relative abundance of elastin mRNA was detectable only on day 16. Fibronectin and laminin B2 were more constitutively present but had their highest copy number per cell on day 16. The presence of extracellular-matrix protein was confirmed by immunohistochemistry using day-16 fetal glands and adult glands. With the construction of the pMATRIX supertemplate and the advent of quantitative, competitive RT-PCR technology, it has been possible to measure small changes in the steady-state concentrations for extracellular-matrix mRNA during salivary gland development.

Gastrointestinal absorption of insulin-like growth factor in the mouse in the absence of salivary insulin-like growth factor binding protein.

Based on previous observations of the presence of both insulin-like growth factors I and II (IGF-I and IGF-II) in murine saliva (kerr et al., Biochem Pharmacol 49: 1521-1531, 1995), the saliva from BALB/c and Non-obese diabetic (NOD) mice was examined for the presence of insulin-like growth factor binding proteins (IGFBPs). Using a western-blot type ligand binding assay with 125I-labeled IGF-I, a series of binding proteins with molecular masses (M), between 25 and 45 kDa were detected in the sera, but not saliva, from both BALB/c and diabetic NOD mice. In the diabetic NOD mice, there were detectable changes in the concentrations of several of the IGFBPs relative to BALB/c mice. Using specific antibody to the binding proteins, one of these was identified as IGFBP-2. Gavage administration of [125I]IGFI indicated substantial uptake from the gastrointestinal tract and significant tissue distribution. There was an increase in serum concentrations of radiolabeled IGF-I in diabetic NOD mice over that in BALB/c mice but less recovered from most of the tissues. Intact 125I-labeled IGF-I was extracted and purified from various tissues, following gavage, and shown to retain biological activity. Thus, the uptake of biologically active IGFs from saliva would appear to take place independently of specific binding proteins.

Detection of alterations in the levels of neuropeptides and salivary gland responses in the non-obese diabetic mouse model for autoimmune sialoadenitis.

The salivary glands of non-obese diabetic (NOD) mice and BALB/c controls were evaluated for the stimulatory effects of the following neuropeptides; substance P (SP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY). Injection of either of the three neuropeptides in combination with the muscarinic-cholinergic agonist pilocarpine increased saliva flow rates in BALB/c mice while there was no observable augmentation to flow rates in pre-diabetic or diabetic NOD mice. Small increases in protein content of the stimulated saliva were observed in the BALB/c group of animals with the injection of any of the above neuropeptides in combination with pilocarpine. In pre-diabetic NOD animals, only VIP and NPY increased the protein content-ratio above pilocarpine alone. Radioimmunoassay determination of neuropeptide concentrations in the submandibular and parotid glands revealed reduced levels of SP with diabetes onset as compared with pre-diabetic NOD or BALB/c mice. The levels of NPY were similar between BALB/c and NOD animals except in the pre-diabetic parotid gland where NPY concentrations were 1.3-fold greater. On the other hand, VIP concentrations were substantially reduced in the submandibular gland of NOD mice, while in the parotid gland neuropeptide levels were evaluated 3.8-fold relative to BALB/c controls. Immunohistochemical staining of the parotid and submandibular glands for SP revealed primarily ductal cell staining which was reduced with diabetes onset in NOD animals. These findings further define the sialoadenitis observed in NOD mice to be due, in part, to a general loss of neurotransmitter responsiveness on the part of salivary gland cells.

Oral diseases, mycology and periodontal microbiology of HIV-1-infected women.

HIV-1 infection is increasing more rapidly among heterosexual women. Relatively limited information is available on HIV-related oral pathoses in these individuals. To gain insight into the type and occurrence of oral lesions in this population, 25 HIV-1 infected women including asymptomatic, symptomatic and AIDS patients were examined clinically and sampled for detection of oral yeast and characterization of their subgingival microbial flora. Sixty percent of the subjects were African-American, with 80% infected via heterosexual contact. Oral candidiasis was the most common nonperiodontal oral lesion, observed in 44% of the patients. Oral yeast was cultured from all women with candidiasis and 76% of the total subjects. Oral hairy leukoplakia was clinically diagnosed in 16% of the subjects. Clinically mild to moderate gingivitis and periodontitis were observed in 84% and 52% of the patients, respectively. Candidiasis and the presence of cultivable yeast were observed in patients with low, intermediate, and high CD4+ T lymphocyte numbers. Plaque samples were collected from each subject and enumerated by predominant cultivable methods, selective media and microscopy. No differences were detected in the microflora associated with seropositive women with existing periodontitis relative to those without periodontitis or to seronegative women with periodontitis. Candidiasis was the most notable oral clinical manifestation in the HIV-1-infected women and may be a useful clinical indicator of early immune dysfunction mediated by HIV-1.

Saliva and growth factors: the fountain of youth resides in us all.

The predominant focus of research dealing with saliva revolves around the role in the maintenance of oral health through a number of physiological and biological properties of constituent proteins. An ever-expanding literature exists indicating that the salivary glands additionally synthesize, and secrete into saliva, a wide range of growth factors. Animal studies with epidermal growth factor have provided evidence for a role in both oral and systemic health, through the promotion of wound healing rates. Thus, the ability to manipulate their rates of synthesis and absorption from saliva holds the potential to enhance tissue regeneration and homeostasis.