Design and interpretation of clinical research studies in oral medicine: a brief review.

The objective of this short review is to help researchers improve the designs of their clinical studies. Also included is a discussion of the level of evidence provided by the various clinical research study designs.

Oral and dental phenotype of dyskeratosis congenita.

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome that is characterized by lacey reticular hyperpigmentation of the skin, dystrophic nails, mucous membrane leukoplakia and pancytopenia. Diagnosis may be delayed until clinical signs are apparent. Severe pancytopenia frequently causes early mortality of DC patients, who have an increased risk of developing oropharyngeal squamous cell carcinoma. Several case reports have described oral changes in DC, which include oral leukoplakia, increased dental caries, hypodontia, thin enamel structure, aggressive periodontitis, intraoral brown pigmentation, tooth loss, taurodontism and blunted roots. We determined the prevalence of these previously reported findings in a cohort of 17 patients with DC and 23 family members. The most common oral changes in DC patients were oral leukoplakia (65% of the entire DC population), decreased root/crown ratio (75% with sufficient tooth development) and mild taurodontism (57% with sufficient tooth development). From the clinical perspective, a diagnosis of DC or other inherited bone marrow failure syndrome should be considered in young persons with oral leukoplakia, particularly those with no history of smoking. Multiple permanent teeth with decreased root/crown ratios further suggest DC.

Oral graft-versus-host disease.

OBJECTIVE: Graft-versus-host disease (GVHD) is a leading cause of morbidity and mortality in patients receiving hematopoietic cell transplant. It is estimated that 40-70% of engrafted patients surviving the initial transplant eventually develop chronic GVHD (cGVHD), which can persist for months to years and require long-term management from multiple disciplines. This review describes the oral component of this transplant complication. DESIGN: The search related to GVHD patho-biology, salivary gland disease after hematopoietic cell transplant and treatments for oral GVHD encompassed literature from 1966 through 2008. Searches were limited to the MEDLINE/PubMed database and English language literature in peer-reviewed journals. RESULTS: Our understanding of the patho-biology of oral cGVHD is based on studies of other affected tissues. It is difficult to determine the prevalence and incidence of salivary gland disease after transplant because there is no universally accepted case definition. In general, clinical trials for treatment of oral cGVHD have been too small to make strong recommendations for use in clinical practice. CONCLUSIONS: Larger well-designed clinical studies are needed to understand the patho-biology of oral cGVHD and determine best treatments for this disease.

Identification of parotid salivary biomarkers in Sjogren's syndrome by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and two-dimensional difference gel electrophoresis.

OBJECTIVES: To identify the most significant salivary biomarkers in Sjögren's syndrome (SS) using proteomic methods. METHODS: Parotid saliva from 20 non-SS subjects and 41 primary SS patients was analysed. Protein expression profiles for each sample were generated by surface-enhanced laser desorption/ionization time-of-flight-mass spectrometry (SELDI-TOF-MS). Mean peak intensities of SS patients and non-SS subjects were compared by univariate analyses. Samples pooled by diagnosis (SS and non-SS) and labelled with different Cy dyes were compared by two-dimensional difference gel electrophoresis (2D-DIGE). Two protein levels that were most significantly different by SELDI-TOF-MS and 2D-DIGE were validated by enzyme-linked immunosorbent assay in individual samples. RESULTS: SELDI-TOF-MS of 10-200 kDa peaks revealed eight peaks with >2-fold changes in the SS group that differed from non-SS at P < 0.005. Peaks of 11.8, 12.0, 14.3, 80.6 and 83.7 kDa were increased, while 17.3, 25.4, and 35.4 kDa peaks were decreased in SS samples. 2D-DIGE identified significant increases of beta-2-microglobulin, lactoferrin, immunoglobulin (Ig) kappa light chain, polymeric Ig receptor, lysozyme C and cystatin C in all stages of SS. Two presumed proline-rich proteins, amylase and carbonic anhydrase VI, were reduced in the patient group. Three of these ten biomarkers have not been associated previously with SS. CONCLUSIONS: The salivary proteomic profile of SS is a mixture of increased inflammatory proteins and decreased acinar proteins when compared with non-SS. Future studies will test the ability of these biomarker levels, alone and in combination, to diagnose the salivary component of SS.

Analyses of variable panoramic radiographic characteristics of maxillo-mandibular fibrous dysplasia in McCune-Albright syndrome.

OBJECTIVE: Fibrous dysplasia (FD) is a rare skeletal disease caused by activating GNAS1 gene mutations often found in association with the McCune-Albright syndrome (MAS). Multiple bones may be affected in FD, including maxilla and mandible. Patients with MAS have different endocrinopathies that can further influence bone metabolism. The purposes of this cross-sectional study are to characterize FD panoramic radiographic patterns, and to evaluate the effects of age, endocrinopathies and renal phosphate wasting on radiographic characteristics of maxillo-mandibular FD in MAS. SUBJECTS AND METHODS: Fifty-one consecutive MAS patients were screened and panoramic radiographs of 43 patients with craniofacial FD were evaluated and analyzed for FD involvement. Clinical chemistries were evaluated for associations between radiographic patterns and age, endocrinopathies or renal phosphate wasting using Fisher's Exact Test. RESULTS: Four types of radiographic changes were observed: ground glass (granular/condensed trabeculae), radiolucent (lytic), mixed radiolucent/radio-opaque (mixed density) or radio-opaque (sclerotic). Masking or displacement of the maxillary sinus (range: 77.8-86.4%) and mandibular canal (range: 55.6-75.0%) were prevalent in FD sites. Sixty-three percent of the MAS patients had multiple dysregulated endocrine/metabolic functions, the most common were hyperthyroidism, precocious puberty and renal phosphate wasting. There were no statistically significant associations between radiographic patterns and age, endocrinopathies or renal phosphate wasting. CONCLUSIONS: Maxillo-mandibular FD images in panoramic radiographs fall within a spectrum of four different patterns. Patients with facial asymmetry and any of these radiographic patterns should be promptly referred for further radiographic tests and endocrine evaluation if MAS is suspected.

Salivary gland delivery of pDNA-cationic lipoplexes elicits systemic immune responses.

OBJECTIVE: To test the ability of two cationic lipoplexes, Vaxfectin and GAP-DLRIE/DOPE, to facilitate transfection and elicit immune responses from plasmid DNAs (pDNAs) after retrograde instillation into salivary glands. METHODS: Two pDNA expression vectors encoding either the influenza NP protein or human growth hormone (hGH) were complexed with the cationic lipid transfection reagents, GAP-DLRIE/DOPE or Vaxfectin, and delivered to the submandibular glands of rats. Samples from rats receiving the influenza NP protein pDNA and cationic lipoplexes were analyzed for anti-influenza NP-specific IgG1, IgG2a, and IgG2b in serum, and IgA in saliva, by an enzyme- linked immunosorbent assay (ELISA). Cytotoxic T-cell lymphocyte (CTL) assays were also performed. Transgene protein expression (hGH) was determined from extracts of submandibular glands of rats receiving hGH lipoplexes. RESULTS: Serum antibodies (IgG) against the NP protein developed and were highest in all rats vaccinated with GAP-DLRIE/DOPE or Vaxfectin. The major serum IgG subclass stimulated by this route of immunization was IgG2b, followed by IgG2a. CTL assay results showed statistically significantly higher percentage killing in the Vaxfectin group than controls (P < 0.05). No rats developed IgA antibodies to NP protein in saliva. Animals receiving plasmid encoding hGH and either lipoplex expressed significantly higher amounts of hGH compared with those receiving the hGH plasmid and water. Although hGH expression was higher in the animals receiving pDNA/Vaxfectin (approximately 30-fold > pDNA/water), the difference with those receiving pDNA/GAP-DLRIE/DOPE (approximately 10-fold > pDNA/water) was not significant. CONCLUSIONS: Retrograde instillation of pDNA complexed with Vaxfectin into the salivary glands can stimulate cytotoxic and humoral responses to the influenza NP protein antigen. Optimization of the conditions required to stimulate humoral and secretory antibody formation may facilitate use of this tissue for specific clinical applications of pDNA immunization.

Oral manifestations in patients with aplastic anemia.

OBJECTIVE: The aim of the present study was to characterize the prevalence and risks of oral complications in aplastic anemia (AA). STUDY DESIGN: Approximately 79 patients with AA (age, 37 +/- 17 years) and 66 control patients with schizophrenia (age, 33 +/- 12 years) were examined. Records were reviewed for demographic, clinical, and radiographic information. Prior medical therapy, laboratory values, disease duration, and medical treatment response were noted for patients with AA. Odds ratios (OR) and 95% CI were calculated for oral manifestations in cases and in control subjects. Univariate analysis identified important variables for logistic regression. RESULTS: Patients with AA presented more frequently with oral petechiae (OR = 49; 95% CI, 2.9-825), gingival hyperplasia (OR = 27; 95% CI, 1.6-463.5), spontaneous gingival bleeding (OR = 27; 95% CI, 1.6-463.5), and herpetic lesions (OR = 27; 95% CI, 1.6-463.5). Prior cyclosporine use was associated with gingival hyperplasia (P =.0001). No other predictors for oral manifestations or treatment outcomes were found. CONCLUSIONS: Oral soft tissue changes and infections were more common in patients with AA. Prior cyclosporine use was predictive of the presence of gingival hyperplasia.

Salivary enhancement: current status and future therapies.

Saliva provides the principal protective milieu for teeth by modulating oral microbial ecosystems and reversing the initial phases of caries development. Patients with inadequate salivary function are at increased risk for dental decay. Therefore, it is likely that therapies that increase overall fluid output of these individuals will reverse early carious lesions. The most common causes of salivary dysfunction are medication usage, Sjögren's syndrome, and damage of salivary parenchyma during therapeutic irradiation. For patients with remaining functional acinar tissue, treatment with the parasypathomimetic secretogogues pilocarpine and Cevimeline may provide relief. However, these medications do not benefit all patients. The possibilities of using gene therapy and tissue engineering to develop treatments for those with severe salivary dysfunction are discussed.

Dental health and viridans streptococcal bacteremia in allogeneic hematopoietic stem cell transplant recipients.

Viridans streptococci were the most common cause of bacteremia in 61 consecutive myeloablative allogeneic hematopoietic stem cell transplant (HSCT) recipients, occurring in 19 of 31 bacteremic patients (61%) during the period of post-transplant neutropenia. Seven of the 19 had more than one viridans streptococcus in the same blood culture. Twenty isolates from 15 patients were Streptococcus mitis. Most viridans streptococci were resistant to norfloxacin, used routinely for prophylaxis. Comparison of the 19 patients with viridans streptococcal bacteremia with a contemporaneous group of 23 allogeneic HSCT recipients with fever and neutropenia but no identified focus of infection found that patients with viridans streptococcal bacteremia were more likely to have severe intraoral pathology while neutropenic (26% vs 0%) and slightly shorter interval between the last dental procedure and the onset of neutropenia (11 vs 14 days). Poor underlying dental health and the use of norfloxacin thus appear to predispose to viridans streptococcal bacteremia.

Adenoviral-mediated gene transfer to mouse salivary glands.

Adenoviral vectors effectively transfer genes to rat salivary glands. However, potent immune responses limit their use in vivo. Mice offer more opportunities than rats for the study of these immune processes. We first established conditions for infection of mouse salivary glands, with an adenoviral vector. The effects of time, viral dose, viral diluent buffer volume, and dexamethasone on expression of a transgene, luciferase, were determined by means of the recombinant vector AdCMVluc. Optimal luciferase expression was observed when the vector was suspended in 50 microL of buffer. This volume completely filled the gland parenchyma and slightly distended the capsule. Dexamethasone increased immediate transgene expression and reduced the acute inflammation one day following viral administration, but did not alter subsequent mononuclear inflammation or transgene expression 14 or 28 days later. An adenoviral vector encoding either anti-inflammatory cytokine IL-4 or IL-10 was co-administered with AdCMVluc to increase transgene expression at 14 and 28 days. While this strategy did not extend the duration of luciferase expression, co-administration of AdCMVIL-10 with AdCMVluc almost completely eliminated the chronic inflammatory infiltrate in the glands after 28 days. This study demonstrates that adenoviral-mediated gene transfer to mouse submandibular glands is possible by intraductal cannulation and that reduction of either the acute or chronic inflammatory infiltrates was insufficient to increase long-term transgene expression in this tissue.

Oral manifestations of primary immunological diseases.

BACKGROUND: Primary immunodeficiencies have many oral manifestations. The clinical presentation of these diseases demonstrates the roles of different immune cells for the maintenance of oral health. METHODS: The authors reviewed selected literature describing systemic and oral manifestations of the primary immunodeficiencies published between 1966 and 1999. RESULTS: The authors found that oral candidiasis and herpetic infections are seen frequently in patients with T-cell deficiencies, while patients with B-cell deficiencies are most susceptible to bacterial infections. Periodontitis and oral candidiasis are found in some, but not all, phagocyte deficiencies. CONCLUSIONS: These findings demonstrate that T cells, B cells and phagocytes all have roles in oral immunity. CLINICAL IMPLICATIONS: Acquired conditions that affect the immune system such as diabetes, alcoholism and acquired immunodeficiency syndrome, as well as certain medications, will affect oral defense mechanisms. The effects that acquired immunodeficiencies will have on oral health can be predicted from the oral manifestations of primary immunodeficiencies.

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.

Salivary gland cytokine expression in NOD and normal BALB/c mice.

The autoimmune diabetes-prone nonobese diabetic (NOD) mouse develops a chronic lymphocytic infiltration of endocrine and exocrine glands. The objectives of this study were to characterize the salivary immune infiltration and cytokine expression of NOD mice and compare these findings to those of normal BALB/c mice. A decline in salivary flow rates in NOD mice began between 8 and 12 weeks of age. At this same time lymphocytic foci are detectable in the salivary glands. Lymphocytic infiltration in the salivary glands of NOD mice increased with age and simultaneously salivary function declined. No lymphocytic infiltration was seen in BALB/c salivary tissues. Messenger RNA expression of several inflammatory cytokines, including interleukin-1beta (IL-1beta), IL-2, IL-10, interferon-gamma, and tumor necrosis factor-alpha was detected in the submandibular glands of both NOD and BALB/c mice by the reverse transcription polymerase chain reaction. IL-4 synthesis was also present in some tissues. Immunohistochemical analysis demonstrated the intense expression of inflammatory cytokines within lymphocytic infiltrates and epithelial cells of all NOD mice. Minimal expression of the same cytokines was detected only occasionally in BALB/c tissues stained in parallel. These results demonstrate cytokine expression in the salivary glands of normal mice and suggest that the overexpression of these inflammatory cytokines is likely involved in the development and progression of the organ-localized autoimmunity in the salivary glands of NOD mice.

Effect of clodronate on macrophage depletion and adenoviral-mediated transgene expression in salivary glands.

Expression of transgenes from adenoviral vectors is short-lived in salivary glands, in part because of immune responses to the virus and/or transgene product. Previous studies demonstrated that depletion of macrophages with multilamellar liposomes containing clodronate (Cl2MBP) increases adenoviral-mediated transgene expression in the liver. This technique was tested in salivary glands. Rats were treated with Cl2MBP-liposomes or control liposomes by femoral vein, intraperitoneal, or carotid artery injections. Thereafter, a recombinant adenovirus, AdCMVluciferase, was instilled intraductally in submandibular glands (SMGs), or delivered to the liver via femoral vein injection. Marked depletion (>94%) of liver macrophages and increased levels of luciferase activity in the liver (45-fold higher than controls) were present in animals receiving Cl2MBP-liposomes. In contrast, the same treatment never depleted more than 41% of SMG macrophages nor increased luciferase activity in SMGs, regardless of the route of administration. In conclusion, while macrophage depletion with Cl2MBP-liposomes is associated with markedly increased adenoviral-mediated transgene expression, this strategy was ineffective for salivary glands.

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.

Repetitive adenovirus administration to the parotid gland: role of immunological barriers and induction of oral tolerance.

This study assessed the mucosal and systemic immune responses following repetitive adenoviral vector instillation to the parotid glands. Also, we investigated the feasibility of oral tolerance induction as a rational strategy to overcome the immunological reactions. The replication-deficient recombinant adenovirus vector AdCMVCAT was instilled into rat parotid glands. Chloramphenicol acetyltransferase (CAT) activity in the parotid was observed after a first or second AdCMVCAT infection, but not after a third vector administration. ELISA assays showed increased anti-adenovirus immunoglobulin G (IgG) and IgM in serum, and also anti-adenovirus IgA in gland extracts and saliva after virus administration. The results of in vivo neutralization experiments demonstrated that salivary IgA and IgM prevented reinfection of the parotids with adenoviral vectors. Subsequently, studies were conducted to induce tolerance to adenovirus by peroral feedings of ultraviolet (UV)-inactivated virus before gene administration to the parotid glands. Between 3 and 13 doses of virus were fed to rats. Final parotid gene expression was dependent on the number of viral feedings and the amount fed. Tolerized animals showed prolonged and heightened gene expression in the salivary glands compared to control animals and displayed gene expression even after three administrations of vector. Mononuclear cells from the spleens of these animals showed reduced proliferation following adenovirus stimulation. This same cell population was depleted of CD8+ T cells and found to produce less interferon-gamma (IFN-gamma) after virus challenge. This profile indicates the down regulation of Th1 cell-mediated responses. These results indicate that oral tolerance induction is a potentially useful adjunct to virus-based gene therapy.

Safety of salivary gland-administered replication-deficient recombinant adenovirus in rats.

We have examined the safety of a replication-deficient recombinant adenovirus administered at a single, high dose intraductally to rat submandibular glands or systemically via the femoral vein. The virus used directed the synthesis of human aquaporin-1, a water channel protein, and is termed AdhAQP1. Comparisons were made 1 and 9 days post-infection with animals administered either a similar virus encoding no transgene or the viral suspension buffer. Animals were specifically not given anti-inflammatory drugs to impede the well-known immunopathologic response to recombinant adenoviral administration. Serum chemistries and hematological parameters were monitored. Rats were subjected to complete gross necropsy and selected tissues were evaluated by histopathology. Most clinical chemistry and hematology values were within normal ranges; however, evidence of inflammation (e.g., elevated lactic dehydrogenase, total leukocyte count) was seen. Gross pathology was normal, as was histopathology, excepting rare focal areas of necrosis. The results show that intrasalivary gland or intravenous AdhAQP1 administration leads to low levels of toxicity in rats.

Collagen VI regulates normal and transformed mesenchymal cell proliferation in vitro.

Suggestions exist that, in addition to traditional growth factors, the extracellular matrix (ECM) of a cell can regulate its proliferation. This hypothesis was investigated with normal and transformed fibroblasts because they exhibit specific intracellular responses after adherence to ECM and produce large quantities of ECM proteins. Although cells cultured on different ECM proteins grew more rapidly than those on plastic, adherence and cell growth on an individual ECM protein were not correlated. To test if ECM can stimulate cell growth, soluble ECM proteins were given to cells after plating. In this culture system only collagen VI (CVI), at a concentration of 20 microg/ml in medium, increased 3T3 cell number to 402% of control by 72 h. Similar increases of human fibroblasts and HT 1080 cell numbers were noted. DNA synthesis of all three cell types increased 24 h after addition of soluble CVI. A mixture of CVI single chains, yielded by reduction and alkylation, was not stimulatory. However, this mixture efficiently inhibited the DNA synthesis induced by native CVI. Antibody inhibition studies showed that the region of CVI stimulating proliferation differs from the site bound by the integrin receptor alpha2beta1, which mediates cell adhesion to immobilized CVI. Heparin inhibited a portion of CVI-induced proliferation. These data demonstrate that CVI can stimulate mesenchymal cell growth via a pathway that is independent of the integrin alpha2beta1 and that the stimulatory region appears to be within the native helical portion of the collagen.

Anti-salivary antibodies in primary Sjögren's syndrome.

Earlier studies have described an antibody that recognized salivary ductal epithelium in sera from 15-50% of patients with primary Sjögren's syndrome; however, the specific salivary antigen in those studies was not identified. The present study further investigated this unknown salivary antigen. Twenty-nine of 31 patients (94%) with primary Sjögren's syndrome demonstrated IgG antinuclear antibodies that bound to an epithelial cell line with ductal characteristics derived from a human salivary gland. Seventy-seven percent of these patients had serum antibodies that bound to ductal cells of normal human parotid tissue after formalin fixation. Western blots of cell extracts, immunofluorescence, and adsorption studies indicated that SS-A/Ro and SS-B/La were the antigens recognized in the salivary cell line. The pattern of fluorescence seen when anti-SS-B/La bound to normal parotid tissue was identical to the fluorescence pattern of the anti-salivary ductal antibodies described in earlier literature.