A systematic review of oral health outcomes produced by dental teams incorporating midlevel providers.

BACKGROUND: The authors conducted a systematic review on this research question: "In populations where nondentists conduct diagnostic, treatment planning, and/or irreversible/ surgical dental procedures, is there a change in disease increment, untreated dental disease, and/or cost-effectiveness of dental care?" METHODS: The authors searched 12 electronic databases for articles published through February 2012 and hand searched relevant articles. They assessed the risk of bias of included studies and extracted data. RESULTS: The authors screened 7,701 citations, resulting in 18 observational studies that met the inclusion criteria. They judged 13 of the studies to be at high risk of bias, five at moderate risk and one at low risk. The authors found no data regarding cost effectiveness, irreversible diagnostic procedures or diseases other than caries. CONCLUSIONS: The authors concluded that the quality of the evidence was poor. They found that in select groups in which participants received irreversible dental treatment from teams that included midlevel providers, caries increment, caries severity or both decreased across time; however, there was no difference in caries increment, caries severity or both compared with those in populations in which dentists provided all irreversible treatment. In select groups in which participants had received irreversible dental treatment from teams that included midlevel providers, there was a decrease in untreated caries across time and a decrease in untreated caries compared with that in populations in which dentists provided all treatment. CLINICAL IMPLICATIONS: Generalizability of results to populations other than those studied is limited owing to the age of some of the studies, as well as to clinical and methodological heterogeneity; consequently, the conclusions should be viewed with caution.

Evidence-based clinical recommendations regarding screening for oral squamous cell carcinomas.

BACKGROUND: This article presents evidence-based clinical recommendations developed by a panel convened by the American Dental Association Council on Scientific Affairs. This report addresses the potential benefits and potential risks of screening for oral squamous cell carcinomas and the use of adjunctive screening aids to visualize and detect potentially malignant and malignant oral lesions. TYPES OF STUDIES REVIEWED: The panel members conducted a systematic search of MEDLINE, identifying 332 systematic reviews and 1,499 recent clinical studies. They selected 5 systematic reviews and 4 clinical studies to use as a basis for developing recommendations. RESULTS: The panel concluded that screening by means of visual and tactile examination to detect potentially malignant and malignant lesions may result in detection of oral cancers at early stages of development, but that there is insufficient evidence to determine if screening alters disease-specific mortality in asymptomatic people seeking dental care. CLINICAL IMPLICATIONS: The panel suggested that clinicians remain alert for signs of potentially malignant lesions or early-stage cancers while performing routine visual and tactile examinations in all patients, but particularly in those who use tobacco or who consume alcohol heavily. Additional research regarding oral cancer screening and the use of adjuncts is needed.

Evidence-based clinical recommendations on the prescription of dietary fluoride supplements for caries prevention: a report of the American Dental Association Council on Scientific Affairs.

BACKGROUND: This article presents evidence-based clinical recommendations for the prescription of dietary fluoride supplements. The recommendations were developed by an expert panel convened by the American Dental Association (ADA) Council on Scientific Affairs (CSA). The panel addressed the following questions: when and for whom should fluoride supplements be prescribed, and what should be the recommended dosage schedule for dietary fluoride supplements? TYPES OF STUDIES REVIEWED: A panel of experts convened by the ADA CSA, in collaboration with staff of the ADA Center for Evidence-based Dentistry, conducted a MEDLINE search to identify publications that addressed the research questions: systematic reviews as well as clinical studies published since the systematic reviews were conducted (June 1, 2006). RESULTS: The panel concluded that dietary fluoride supplements should be prescribed only for children who are at high risk of developing caries and whose primary source of drinking water is deficient in fluoride. CLINICAL IMPLICATIONS: These recommendations are a resource for practitioners to consider in the clinical decision-making process. As part of the evidence-based approach to care, these clinical recommendations should be integrated with the practitioner's professional judgment and the patient's needs and preferences. Providers should carefully monitor the patient's adherence to the fluoride dosing schedule to maximize the potential therapeutic benefit.

Treatment combining RU486 and Ad5IL-12 vector attenuates the growth of experimentally formed prostate tumors and induces changes in the sentinel lymph nodes of mice.

BACKGROUND: Tumor immune responses are first generated and metastases often begin in tumor sentinel lymph nodes (TSLN). Therefore, it is important to promote tumor immunity within this microenvironment. Mifepristone (RU486) treatment can interfere with cortisol signaling that can lead to suppression of tumor immunity. Here, we assessed whether treatment with RU486 in conjunction with an intratumor injection of Ad5IL-12 vector (a recombinant adenovirus expressing IL-12) could impact the TSLN microenvironment and prostate cancer progression. METHODS: The human PC3, LNCaP or murine TRAMP-C1 prostate cancer cell lines were used to generate subcutaneous tumors in NOD.scid and C57BL/6 mice, respectively. Adjuvant effects of RU486 were looked for in combination therapy with intratumor injections (IT) of Ad5IL-12 vector in comparison to PBS, DL70-3 vector, DL70-3 + RU486, RU486 and Ad5IL-12 vector treatment controls. Changes in tumor growth, cell cytotoxic activity and populations of CD4+/FoxP3+ T regulatory cells (Treg) in the TSLN were evaluated. RESULTS: Treatment of human PC3 prostate xenograft or TRAMP-C1 tumors with combination Ad5IL-12 vector and RU486 produced significantly better therapeutic efficacy in comparison to controls. In addition, we found that combination therapy increased the capacity of TSLN lymphocytes to produce Granzyme B in response to tumor cell targets. Finally, combination therapy tended towards decreases of CD4+/FoxP3+ T regulatory cell populations to be found in the TSLN. CONCLUSION: Inclusion of RU486 may serve as a useful adjuvant when combined with proinflammatory tumor killing agents by enhancement of the immune response and alteration of the TSLN microenvironment.

Evidence-based clinical recommendations regarding screening for oral squamous cell carcinomas.

BACKGROUND: This article presents evidence-based clinical recommendations developed by a panel convened by the American Dental Association Council on Scientific Affairs. This report addresses the potential benefits and potential risks of screening for oral squamous cell carcinomas and the use of adjunctive screening aids to visualize and detect potentially malignant and malignant oral lesions. TYPES OF STUDIES REVIEWED: The panel members conducted a systematic search of MEDLINE, identifying 332 systematic reviews and 1,499 recent clinical studies. They selected five systematic reviews and four clinical studies to use as a basis for developing recommendations. RESULTS: The panel concluded that screening by means of visual and tactile examination to detect potentially malignant and malignant lesions may result in detection of oral cancers at early stages of development, but that there is insufficient evidence to determine if screening alters disease-specific mortality in asymptomatic people seeking dental care. CLINICAL IMPLICATIONS: The panel suggested that clinicians remain alert for signs of potentially malignant lesions or early-stage cancers while performing routine visual and tactile examinations in all patients, but particularly in those who use tobacco or who consume alcohol heavily. Additional research regarding oral cancer screening and the use of adjuncts is needed.

Managing clinical risk: right person, right care, right time.

Dentists and the dental health care industry have a renewed interest in clinical risk assessment, because they offer the potential to identify a patient's clinical needs for oral health care more specifically, to maximize prevention by early intervention, and to educate patients to become more informed consumers of oral health care and direct resources where they are most needed and can produce the greatest value. To realize this potential, risk assessment must be applied appropriately, and its indirect ramifications for access to care should be considered. Several ideas for the appropriate application of risk assessment are discussed and the ramifications for access to care are explored.

Attenuation of the glucocorticoid response during Ad5IL-12 adenovirus vector treatment enhances natural killer cell-mediated killing of MHC class I-negative LNCaP prostate tumors.

Tumor cells can evolve to evade immune responses by down-modulating surface MHC class I expression and become refractory to T cell-directed immunotherapy. We employed a strategy to bypass this escape mechanism using a recombinant adenovirus vector expressing interleukin-12 (Ad5IL-12) to target natural killer (NK) cell-mediated killing of human prostate tumors in NOD.scid mice. Fluorescence-activated cell sorting analysis revealed that LNCaP tumor cells bear negligible levels of MHC class I molecules; yet, they express MICA/B molecules, ligands for the NKG2D receptors found on NK cells. Transduction of LNCaP cells with the Ad5IL-12 vector prevented tumor formation in NOD.scid mice, indicating that NK cells alone can conduct tumor immunosurveillance and mediate protection. Intratumor injection of the Ad5IL-12 vector to established LNCaP tumors in NOD.scid mice resulted in a significant delay of tumor growth mediated by NK cell killing activity. The dependency of NK cells in this protective response was shown by the complete loss of Ad5IL-12 therapeutic efficacy on LNCaP tumors established in NOD.Cg-Rag1(tm1Mom)Prf1(tm1Sdz) congenic mice, which are devoid of NK cell activity. More pronounced attenuation of tumor growth and enhanced NK killing activity was observed when pharmacologic adrenalectomy with mitotane was done in combination with Ad5IL-12 vector treatment. The Ad5IL-12 vector treatment also induced killing of MICA/B-negative MHC class I-positive PC3 tumors formed in NOD.scid mice. Together, these results indicate that a targeted NK cell response could provide a generic approach for cancer immunotherapy, and that enhancing the NK cell response via control of cortisol levels may provide an additional therapeutic avenue in cancer.

Comparative evaluation of two severe acute respiratory syndrome (SARS) vaccine candidates in mice challenged with SARS coronavirus.

Two different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated by beta-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection.

A convenient plasmid system for construction of helper-dependent adenoviral vectors and its application for analysis of the breast-cancer-specific mammaglobin promoter.

BACKGROUND: Helper-dependent (HD) adenovirus (Ad) vectors, deleted of all viral coding sequences, have a higher cloning capacity, improved performance of tissue-specific promoters, and reduced toxicity in animals relative to first-generation Ad vectors, making these vectors promising tools for gene transfer in vitro and in vivo. However, the large size of HDAd precursor plasmids renders them relatively difficult to manipulate due to the paucity of unique restriction enzyme sites suitable for transgene insertion and to the size constraints imposed by the viral packaging machinery. METHODS: We have constructed a series of HDAd precursor plasmids that allows cassette insertion at a unique site in the vector backbone. We have tested whether these vector backbones will support the tissue-specificity of inserted expression cassettes in a study of the activity of the potentially breast-cancer-specific mammaglobin promoter and enhancer. RESULTS: We report here the generation of a series of HDAd precursor plasmids, both with and without an additional reporter expression cassette, that were designed to accommodate a wide range in size of inserted DNA. The system was validated for transcriptional targeting studies by demonstrating the tissue-specificity and activity of the mammaglobin promoter rescued using this precursor system. In addition, we have extended our previous studies on the mammaglobin promoter by demonstrating that two copies of the mammaglobin enhancer fused to the minimal promoter surpassed the activity of the single enhancer/promoter by at least 10-fold in breast cancer cells while maintaining only minimal expression in normal cells both in vitro and in a mouse tumor model. CONCLUSIONS: This versatile plasmid system simplifies the construction of HDAd vectors and was valuable in demonstrating the targeting potential of the mammaglobin promoter for breast cancer gene therapy.

Correction of chromosomal mutation and random integration in embryonic stem cells with helper-dependent adenoviral vectors.

For gene therapy of inherited diseases, targeted integration/gene repair through homologous recombination (HR) between exogenous and chromosomal DNA would be an ideal strategy to avoid potentially serious problems of random integration such as cellular transformation and gene silencing. Efficient sequence-specific modification of chromosomes by HR would also advance both biological studies and therapeutic applications of a variety of stem cells. Toward these goals, we developed an improved strategy of adenoviral vector (AdV)-mediated HR and examined its ability to correct an insertional mutation in the hypoxanthine phosphoribosyl transferase (Hprt) locus in male mouse ES cells. The efficiency of HR was compared between four types of AdVs that contained various lengths of homologies at the Hprt locus and with various multiplicities of infections. The frequency of HR with helper-dependent AdVs (HD AdVs) with an 18.6-kb homology reached 0.2% per transduced cell at a multiplicity of infection of 10 genomes per cell. Detection of random integration at DNA levels by PCR revealed extremely high efficiency of 5% per cell. We also isolated and characterized chromosomal sites where HD AdVs integrated in a random manner. In contrast to retroviral, lentiviral, and adeno-associated viral vectors, which tend to integrate into genes, the integration sites of AdV was distributed randomly inside and outside genes. These findings suggest that HR mediated by HD AdVs is efficient and relatively safe and might be a new viable option for ex vivo gene therapy as well as a tool for chromosomal manipulation of a variety of stem cells.

Use of a murine secreted alkaline phosphatase as a non-immunogenic reporter gene in mice.

BACKGROUND: The development of any vector system as a gene delivery system requires its optimization in vitro and in vivo. Preliminary studies frequently involve the use of a reporter gene, which allows for the rapid and simple assay of vector function through monitoring expression levels of the reporter gene. However, evaluation of vector efficacy can be compromised by immune responses directed against immunogenic reporter proteins. METHODS: We have cloned a murine secreted alkaline phosphatase (mSEAP), and explored its use as a reporter gene in the context of an early region 1 (E1)-deleted adenovirus (Ad) vector. Studies involved characterization of gene expression in vitro and in vivo, and immunological responses after gene delivery to mice. RESULTS: In tissue culture, we show that mSEAP is easily measured quantitatively using a sensitive, commercially available chemiluminescent assay, or visualized directly using histological staining. The level of transgene expression from AdmSEAP was similar to that observed for an Ad vector encoding the human placental secreted alkaline phosphatase (hSEAP). After intravenous administration in mice, AdmSEAP continued to express at high levels for the duration of the experiment (1 month), whereas expression from AdhSEAP declined to background levels over the course of the experiment. Although cytotoxic T-lymphocytes were not detected against either the murine or human SEAP proteins in mice, antibodies were readily detected against the human protein. No antibodies were detected to mSEAP. CONCLUSIONS: Taken together, these data illustrate that mSEAP is a sensitive, non-immunogenic reporter gene for preclinical mouse studies.

Life-long systemic protection in mice vaccinated with L. major and adenovirus IL-12 vector requires active infection, macrophages and intact lymph nodes.

Immunization with soluble leishmanial antigen (SLA) in IFA plus Ad5IL-12 vector induced protection confined to the immunized footpad in BALB/c mice. However, animals that controlled a primary infection with a Leishmania major challenge in the same immunized footpad, became resistant to subsequent contralateral rechallenges due to expansion of IFN-gamma secreting cells. This systemic immunity could be disrupted either by macrophage depletion during immunization or by lymphadenectomy after challenge. We show that this procedure does not interfere with tissue-compartmentalized protection, since lymphadenectomized and splenectomized animals were resistant to rechallenges performed in the immunized footpads. Our results indicate that SLA-Ad5IL-12 vector priming requires macrophages to generate systemic protection. Furthermore, a previously undescribed lymphoid organ-independent, protective immune response is contained within the tissue microenvironment of the immunized/challenged footpad. These results have important implications for vaccine design against leishmanial and mycobacterial infections and diseases caused by intracellular pathogens.

Heterogeneity of the immune response to adenovirus-mediated factor VIII gene therapy in different inbred hemophilic mouse strains.

BACKGROUND: The development of anti-factor VIII (FVIII) antibodies (inhibitors) is a critical concern when considering gene therapy as a potential treatment modality for hemophilia A. We used a hemophilia A mouse model bred on different genetic backgrounds to explore genetically controlled differences in the immune response to FVIII gene therapy. METHODS: C57BL/6 FVIII knockout (C57-FVIIIKO) mice were bred with normal BALB/c (BAL) mice, to generate a recombinant congenic BAL-FVIIIKO model of hemophilia A. Early generation adenoviral (Ad) vectors containing the canine FVIII B-domain-deleted transgene under the control of either the CMV promoter or a tissue-restricted (TR) promoter were administered to C57-FVIIIKO, C57xBAL(F1)-FVIIIKO crosses, and BAL-FVIIIKO mice. FVIII expression, inhibitor development, inflammation, and vector-mediated toxicity were assessed. RESULTS: In response to administration of Ad-CMV-cFVIII, C57-FVIIIKO mice attain 3-fold higher levels of FVIII expression than BAL-FVIIIKO. All strains injected with Ad-CMV-FVIII displayed FVIII expression lasting only 2 weeks, with associated inhibitor development. C57-FVIII-KO mice that received Ad-TR-FVIII expressed FVIII for 12 months post-injection, whereas FVIII expression was limited to 1 week in C57xBAL(F1)-FVIIIKO and BAL-FVIIIKO mice. This loss of expression was associated with anti-FVIII inhibitor development. BAL-FVIIIKO mice showed increased hepatotoxicity with alanine aminotransferase levels reaching 4-fold higher levels than C57-FVIIIKO mice. However, C57-FVIIIKO mice initiate a more rapid and effective cell-mediated clearance of virally transduced cells than BAL-FVIIIKO, as evidenced by real-time PCR analysis of transduced tissues. Overall, strain-dependent differences in the immune response to FVIII gene delivery were only noted in the adaptive response, and not in the innate response. CONCLUSIONS: Our results indicate that the genetic background of the murine model of hemophilia A influences FVIII expression levels, the development of anti-FVIII inhibitors, clearance of transduced cells, and the severity of vector-mediated hepatotoxicity.

The human SCGB2A2 (mammaglobin-1) promoter/enhancer in a helper-dependent adenovirus vector directs high levels of transgene expression in mammary carcinoma cells but not in normal nonmammary cells.

Expression of secretoglobin family 2A member 2 (SCGB2A2, also known as mammaglobin-1) has been detected in a high percentage of primary and metastatic breast tumors, to a lesser extent in normal breast, but not in other normal tissues. Plasmid transfection studies in our lab and others, however, were unable to identify the genetic elements regulating this specificity. Here we demonstrate that a 25-kb DNA fragment derived from the human SCGB2A2 gene upstream of the protein coding sequence was highly active and preferentially expressed in breast cancer cells when introduced via a helper-dependent adenoviral (HDAd) vector. HDAd delivery was selected for its high cloning capacity, its high efficiency of gene transfer, and the absence of cis-acting viral sequences that can potentially interfere with specificity of the inserted promoters. A series of vectors with deletions in the 25-kb fragment was constructed to identify important regulatory regions of the SCGB2A2 promoter. We have determined that elements controlling the specificity of expression reside within the first 345 bp upstream of the coding sequence. In addition, we identified a strong enhancer several kilobases upstream of this minimal promoter. We suggest that the SCGB2A2 promoter/enhancer should be particularly advantageous for gene therapy protocols involving oncolytic viruses or toxic gene transfer via adenovectors to mammary tumors.

Functional rescue of vitamin C synthesis deficiency in human cells using adenoviral-based expression of murine l-gulono-gamma-lactone oxidase.

l-Gulono-gamma-lactone oxidase (GULO) is a critical enzyme present in most mammalian species that is required for the terminal step in vitamin C biosynthesis. Primates are absolutely dependent on exogenously supplied dietary vitamin C due to inactivation of the Gulo gene by mutation over 40 million years ago. In this study, we report the cloning and expression of the murine l-gulono-gamma-lactone oxidase cDNA and gene. The cDNA (2.3 kb) encodes an open reading frame of 440 amino acids that shows high homology to the rat l-gulono-gamma-lactone oxidase (>94%). The Gulo gene is 22 kb long and contains 12 exons. The 11 introns range in size from 479 to 5641 bp. Northern blot analysis revealed high expression of Gulo transcript in the liver. To investigate whether metabolic loss of vitamin C biosynthesis in human cells can be corrected by heterologous expression of GULO, we constructed a first-generation adenoviral vector expressing the murine GULO cDNA under the transcriptional control of the murine cytomegalovirus (MCMV) early promoter. Low rescue efficiency of Gulo-expressing adenoviral constructs and reduced viral growth in HEK293 cells were observed, suggesting that overexpression of Gulo may be inhibitory to cell growth. Placement of a removable stuffer fragment flanked by lox sites between the MCMV promoter and the Gulo gene resulted in efficient vector rescue and normal viral replication in parental HEK293 cells and high-level expression of Gulo in HEK293 cells expressing Cre recombinase. Cells infected with Gulo-expressing vectors overexpressed an FAD-containing protein that corresponded in size to that predicted for recombinant GULO protein and expressed a functional enzyme as measured by the conversion of l-gulono-gamma-lactone to ascorbic acid in cell-free extracts. The cloning of the murine Gulo cDNA and the construction of Gulo-expressing adenoviral vectors are vital steps toward determining the role of vitamin C in basic metabolism and in disease.

Five novel mutations in the lysosomal sialidase gene (NEU1) in type II sialidosis patients and assessment of their impact on enzyme activity and intracellular targeting using adenovirus-mediated expression.

Sialidosis is an autosomal recessive disease resulting from a deficiency of lysosomal sialidase. Type II sialidosis is a rare disease characterized clinically by hydrops fetalis, hepatosplenomegaly, and severe psychomotor retardation. Genomic DNA from four unrelated sialidosis patients was screened for mutations within the sialidase gene NEU1. Five novel mutations were identified. Four are missense and one is nonsense: c.674G>C (p.R225P), c.893C>T (p.A298V), c.3G>A (p.M1?), c.941C>G (p.R341G), and c.69G>A (p.W23X). We have used our findings and diagnostic tools to confirm the presence of a homozygous null allele in a neonate sibling. Recombinant adenoviruses expressing the mutant sialidase alleles in primary cell cultures were utilized to assess the impact of each mutation on enzyme activity and intracellular localization. None of the mutant alleles expressed significant enzymatic activity. The p.R341G mutation exerts its pathological effect by perturbing substrate binding, while the p.A298V and p.R225P mutations appear to impair the folding of the sialidase enzyme. Our findings point to mutation-sensitive amino acids involved in catalytic function or structural stability and indicate the potential utility of these mutations for molecular diagnosis of this rare disease.

Helper-dependent adenoviral vectors mediate therapeutic factor VIII expression for several months with minimal accompanying toxicity in a canine model of severe hemophilia A.

Two helper-dependent (HD) adenoviral vectors encoding a canine factor VIII B-domain-deleted transgene (cFVIII) were constructed and evaluated in 4 hemophilia A dogs. One vector was regulated by the cytomegalovirus (CMV) promoter (HD-CMV-cFVIII), while the other vector contained a tissue-restricted promoter comprised of the human FVIII proximal promoter with an upstream concatemer of 5 hepatocyte nuclear factor 1 binding sites (HD-HNF-cFVIII). We detected no toxicity at low dose (5 x 10(11) vp/kg), but at higher vector doses (> 1 x 10(12) vp/kg) transient hepatotoxicity and thrombocytopenia were observed. Low-level increases in FVIII activity were detected in all 3 HD-HNF-cFVIII-treated dogs, which corresponded with decreased whole blood clotting times. None of the animals receiving the HD-HNF-cFVIII vector developed FVIII inhibitors, and in 1 of the 3 animals, FVIII activity was sustained for over 6 months after treatment. One animal, which received the HD-CMV-cFVIII vector, achieved peak levels of FVIII above 19 000 mU/mL, but FVIII activity disappeared within 1 week, coincident with the development of a potent anti-canine FVIII antibody response. This study supports previous demonstrations of improved safety using HD gene transfer and suggests that these vectors can provide transient FVIII expression with minimal, acute toxicity in the absence of inhibitor formation.