Ocular Inflammatory Response to Intravitreal Injection of Adeno-Associated Virus Vector: Relative Contribution of Genome and Capsid.

Both subretinal dosing and intravitreal (IVT) dosing of adeno-associated virus (AAV) in higher species induce mild and transient inflammatory responses that increase with dose. Foreign protein and foreign DNA are known inducers of inflammation, which is also true in the immune-privileged ocular environment. We explored which component(s) of AAV vectors, viral capsid, or viral DNA drive inflammatory responses. Recombinant AAV with three tyrosine to phenylalanine substitutions in the capsid of AAV serotype 2 (rAAV2tYF), and with a generic ubiquitous promoter (cytomegalovirus [CMV]) controlling the expression of humanized green fluorescent protein (hGFP), was processed to enrich for AAV capsids containing genome (full capsids), capsids without genome (empty capsids), and residual material. Nonhuman primate eyes were injected by IVT in both eyes. During in-life, ocular inflammation and development of neutralizing antibodies (NAb) were measured. Following termination, lymph node immunophenotyping was performed, vitreous was processed for cytokine and RNAseq analyses, and ocular sections were assessed for transgene expression (by in situ hybridization) and histopathology. IVT dosing of AAV vectors transiently raised cellular inflammation in the aqueous and induced a more sustained inflammation in the vitreous. Lowering the total capsid dose by removing empty AAV capsids reduced inflammation and improved viral transduction. IVT dosing of AAV induced systemic NAb to AAV irrespective of the vector preparation. Similarly, lymph node immunophenotyping revealed identical profiles irrespective of viral preparation used for dosing. Immune cells in the vitreous were identified based on RNAseq analysis. Three months postdose, cytokine levels were low, indicative of minimal levels of inflammation in agreement with histopathological assessment of the retina.

Coordinated Regulation of the EIIMan and fruRKI Operons of Streptococcus mutans by Global and Fructose-Specific Pathways.

The glucose/mannose-phosphotransferase system (PTS) permease EIIMan encoded by manLMN in the dental caries pathogen Streptococcus mutans has a dominant influence on sugar-specific, CcpA-independent catabolite repression (CR). Mutations in manL affect energy metabolism and virulence-associated traits, including biofilm formation, acid tolerance, and competence. Using promoter::reporter fusions, expression of the manLMN and the fruRKI operons, encoding a transcriptional regulator, a fructose-1-phosphate kinase and a fructose-PTS permease EIIFru, respectively, was monitored in response to carbohydrate source and in mutants lacking CcpA, FruR, and components of EIIMan Expression of genes for EIIMan and EIIFru was directly regulated by CcpA and CR, as evinced by in vivo and in vitro methods. Unexpectedly, not only was the fruRKI operon negatively regulated by FruR, but also so was manLMN Carbohydrate transport by EIIMan had a negative influence on expression of manLMN but not fruRKI In agreement with the proposed role of FruR in regulating these PTS operons, loss of fruR or fruK substantially altered growth on a number of carbohydrates, including fructose. RNA deep sequencing revealed profound changes in gene regulation caused by deletion of fruK or fruR Collectively, these findings demonstrate intimate interconnection of the regulation of two major PTS permeases in S. mutans and reveal novel and important contributions of fructose metabolism to global regulation of gene expression.IMPORTANCE The ability of Streptococcus mutans and other streptococcal pathogens to survive and cause human diseases is directly dependent upon their capacity to metabolize a variety of carbohydrates, including glucose and fructose. Our research reveals that metabolism of fructose has broad influences on the regulation of utilization of glucose and other sugars, and mutants with changes in certain genes involved in fructose metabolism display profoundly different abilities to grow and express virulence-related traits. Mutants lacking the FruR regulator or a particular phosphofructokinase, FruK, display changes in expression of a large number of genes encoding transcriptional regulators, enzymes required for energy metabolism, biofilm development, biosynthetic and degradative processes, and tolerance of a spectrum of environmental stressors. Since fructose is a major component of the modern human diet, the results have substantial significance in the context of oral health and the development of dental caries.

A new arginine-based dental adhesive system: formulation, mechanical and anti-caries properties.

Secondary caries at the margins of composite restorations has been attributed to adhesive failure and consequent accumulation of cariogenic biofilms. OBJECTIVES: To develop and evaluate an etch-and-rinse adhesive system containing arginine for sustainable release and recharge without affecting its mechanical properties. Arginine metabolism by oral bacteria generates ammonia, which neutralizes glycolytic acids and creates a neutral environmental pH that is less favorable to the growth of caries pathogens, thus reducing the caries risk at the tooth-composite interface. METHODS: Experimental adhesives were formulated with methacrylate monomers and arginine at 5%, 7%, and 10% or no arginine (control). Adhesives were tested for: (i) mechanical properties of true stress (FS and UTS), modulus of elasticity (E), degree of conversion (DC), Knoop hardness number (KHN) and dentin microtensile bond strength (µ-TBS), (ii) arginine release and recharge, and (iii) antibacterial activities. Data was analyzed by t-test, one-way ANOVA and Tukey's tests. RESULTS: FS and UTS results showed no statistically significant differences between the 7% arginine-adhesive and control, while the results for E, DC, KHN and µ-TBS showed no difference among all groups. The 7% arginine-adhesive showed a high release rate of arginine (75.0µmol/cm2) at 2h, and a more sustainable, controlled release rate (up to 0.2µmol/cm2) at 30days. CONCLUSIONS: Incorporation of 7% arginine did not affect the physical and mechanical properties of the adhesive. Arginine was released from the adhesive at a rate and concentration that exhibited antibacterial effects, regardless of shifts in biofilm conditions such as sugar availability and pH. CLINICAL SIGNIFICANCE: Secondary caries is recognized as the main reason for failure of dental restorations. The development of an arginine-based adhesive system has the potential to dramatically reduce the incidence and severity of secondary caries in adhesive restorations in a very economical fashion.

Amino Sugars Enhance the Competitiveness of Beneficial Commensals with Streptococcus mutans through Multiple Mechanisms.

UNLABELLED: Biochemical and genetic aspects of the metabolism of the amino sugars N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) by commensal oral streptococci and the effects of these sugars on interspecies competition with the dental caries pathogen Streptococcus mutans were explored. Multiple S. mutans wild-type isolates displayed long lag phases when transferred from glucose-containing medium to medium with GlcNAc as the primary carbohydrate source, but commensal streptococci did not. Competition in liquid coculture or dual-species biofilms between S. mutans and Streptococcus gordonii showed that S. gordonii was particularly dominant when the primary carbohydrate was GlcN or GlcNAc. Transcriptional and enzymatic assays showed that the catabolic pathway for GlcNAc was less highly induced in S. mutans than in S. gordonii Exposure to H2O2, which is produced by S. gordonii and antagonizes the growth of S. mutans, led to reduced mRNA levels of nagA and nagB in S. mutans When the gene for the transcriptional regulatory NagR was deleted in S. gordonii, the strain produced constitutively high levels of nagA (GlcNAc-6-P deacetylase), nagB (GlcN-6-P deaminase), and glmS (GlcN-6-P synthase) mRNA. Similar to NagR of S. mutans (NagRSm), the S. gordonii NagR protein (NagRSg) could bind to consensus binding sites (dre) in the nagA, nagB, and glmS promoter regions of S. gordonii Notably, NagRSg binding was inhibited by GlcN-6-P, but G-6-P had no effect, unlike for NagRSm This study expands the understanding of amino sugar metabolism and NagR-dependent gene regulation in streptococci and highlights the potential for therapeutic applications of amino sugars to prevent dental caries. IMPORTANCE: Amino sugars are abundant in the biosphere, so the relative efficiency of particular bacteria in a given microbiota to metabolize these sources of carbon and nitrogen might have a profound impact on the ecology of the community. Our investigation reveals that several oral commensal bacteria have a much greater capacity to utilize amino sugars than the dental pathogen Streptococcus mutans and that the ability of the model commensal Streptococcus gordonii to compete against S. mutans is substantively enhanced by the presence of amino sugars commonly found in the oral cavity. The mechanisms underlying the greater capacity and competitive enhancements of the commensal are shown to depend on how the genes for the catabolic enzymes are regulated, the role of the allosteric modulators affecting such regulation, and the ability of amino sugars to enhance certain activities of the commensal that are antagonistic to S. mutans.