Sex-Related Effect of Aging in Gingival Gamma-Delta T Cells.

Aging affects the number and function of gamma-delta (γδ) T cells in a tissue-specific manner, modifying the risk for inflammatory disease. These aging-related γδT-cell variations in gingival tissues that could increase the risk for inflammation and periodontal disease remain unknown. Here we sought to identify quantitative and qualitative variations in gingival γδT cells associated with aging that could have an impact in oral immunoinflammatory responses. For this, gingival tissues from young (4 mo) and aged (24 mo) male and female mice were collected and analyzed by flow cytometry. Cell suspensions were stimulated and stained with eFluor450 (cell viability), anti-CD45 (hematopoietic cells), anti-CD3 (lymphocytes), anti-TCRγδ (γδT cells), anti-IL-15rα (cell proliferation), and anti-Notch-3 (senescence marker). Detection of intracellular cytokines IL-17A and interferon γ (IFNγ) was performed. Gingival expression of specific γ- and δ-chains and cytokines was evaluated by quantitative reverse transcription polymerase chain reaction. A significantly higher number of IL-17A-producing γδT cells and IL-17A expression levels were observed in gingival tissues from aged females but not males. Similarly, the number of gingival Notch-3+ γδT cells increased with aging only in females. IL-15rα was not detected in gingival γδT cells. Chains γ1, 2, 4, 5, 6, and 7 as well as δ1, 2, 4, and 6 were detected. Detection levels of all γ chains except γ1 as well as δ1 and δ2 changed with aging in males, females, or both. Interestingly, number of IL-17A-producing conventional T cells similarly increased with aging only in females. Both sexes showed increased IFNγ+ conventional T-cell numbers with aging; however, it reached significance only in females. In conclusion, the number of gingival IL-17A-producing γδT cells and IL-17A expression increase naturally with aging specifically in females. This sexual dimorphism in gingival γδT and conventional Th17 cell numbers and phenotypes suggests distinct aging-related mechanisms of periodontitis in males and females.

Dynamic expression of JC virus in urine and its relationship to serostatus.

BACKGROUND: There is limited information regarding the daily shedding of JC virus (JCV) in urine and its correlation with serum JCV antibody levels. METHODS: The dynamic expression of JCV in urine and its correlation with JCV antibody status in patients receiving disease modifying therapy for multiple sclerosis were examined in a longitudinal case-control study. JCV antibody index levels were determined using a two-step ELISA (Stratify). JCV shedding in urine samples was determined by quantitative PCR during two 30-day study periods separated by intervals of at least 6 months. RESULTS: Of 42 study subjects (57% female; ages 22-56, average age 39.6 years), 27 (64.3%) were JCV antibody positive (index >0.40) at initial urine collection. Twelve seropositive subjects (44.4%) had detectable JCV in their urine with values ranging from 290 to 5.08 × 108 copies/mL. Daily viral shedding in these patients remained fairly constant throughout the study. Urinary JCV shedding was not detected in any JCV antibody index negative or indeterminate subject. In JCV urinary shedders, the average JCV antibody index was 2.69 (range 1.67-3.57). The average anti-JCV antibody index for the remaining JCV seropositive individuals without viral urinary shedding was 1.35 (range 0.46-3.91). CONCLUSION: MS patients displayed a consistent pattern of JCV shedding over days and months in which higher levels of viruria appeared to have driven higher levels of JCV antibody index. The findings provide additional insights into the dynamic expression of JCV and host response; however, studies in larger populations and of longer duration will be needed to determine their significance to the development of progressive multifocal leukoencephalopathy (PML).

Rapid assessment of salivary MMP-8 and periodontal disease using lateral flow immunoassay.

OBJECTIVE: The objective of this study was to determine the efficacy of a novel point-of-care immunoflow device (POCID) for detecting matrix metalloproteinase (MMP)-8 concentrations in oral fluids in comparison with a gold standard laboratory-based immunoassay. METHODS: Oral rinse fluid and whole expectorated saliva samples were collected from 41 participants clinically classified as periodontally healthy or diseased. Samples were analyzed for MMP-8 by Luminex immunoassay and POCID. Photographed POCID results were assessed by optical scan and visually by two examiners. Data were analyzed by Pearson's correlation and receiver-operating characteristics. RESULTS: MMP-8 was readily detected by the POCID, and concentrations correlated well with Luminex for both saliva and rinse fluids (r = 0.57-0.93). Thresholds that distinguished periodontitis from health were delineated from both the optical scans and visual reads of the POCID (sensitivity: 0.7-0.9, specificity: 0.5-0.7; P < 0.05). CONCLUSIONS: Performance of this POCID for detecting MMP-8 in oral rinse fluid or saliva was excellent. These findings help demonstrate the utility of salivary biomarkers for distinguishing periodontal disease from health using a rapid point-of-care approach.

Trigeminal Inflammatory Compression (TIC) injury induces chronic facial pain and susceptibility to anxiety-related behaviors.

Our laboratory previously developed a novel neuropathic and inflammatory facial pain model for mice referred to as the Trigeminal Inflammatory Compression (TIC) model. Rather than inducing whole nerve ischemia and neuronal loss, this injury induces only slight peripheral nerve demyelination triggering long-term mechanical allodynia and cold hypersensitivity on the ipsilateral whisker pad. The aim of the present study is to further characterize the phenotype of the TIC injury model using specific behavioral assays (i.e. light-dark box, open field exploratory activity, and elevated plus maze) to explore pain- and anxiety-like behaviors associated with this model. Our findings determined that the TIC injury produces hypersensitivity 100% of the time after surgery that persists at least 21 weeks post injury (until the animals are euthanized). Three receptive field sensitivity pattern variations in mice with TIC injury are specified. Animals with TIC injury begin displaying anxiety-like behavior in the light-dark box preference and open field exploratory tests at week eight post injury as compared to sham and naïve animals. Panic anxiety-like behavior was shown in the elevated plus maze in mice with TIC injury if the test was preceded with acoustic startle. Thus, in addition to mechanical and cold hypersensitivity, the present study identified significant anxiety-like behaviors in mice with TIC injury resembling the clinical symptomatology and psychosocial impairments of patients with chronic facial pain. Overall, the TIC injury model's chronicity, reproducibility, and reliability in producing pain- and anxiety-like behaviors demonstrate its usefulness as a chronic neuropathic facial pain model.

Antibacterial effects of blackberry extract target periodontopathogens.

BACKGROUND AND OBJECTIVE: Antimicrobial agents provide valuable adjunctive therapy for the prevention and the control of oral diseases. Limitations in their prolonged use have stimulated the search for new, naturally occurring agents with more specific activity and fewer adverse effects. Here we sought to determine the antibacterial properties of blackberry extract (BBE) in vitro against oral bacterial commensals and periodontopathogens. MATERIAL AND METHODS: The effects of whole and fractionated BBE on the metabolism of 10 different oral bacteria were evaluated using the colorimetric water-soluble tetrazolium-1 assay. The bactericidal effects of whole BBE against Fusobacterium nucleatum were determined by quantitating the numbers of colony-forming units (CFUs). Cytotoxicity was determined in oral epithelial (OKF6) cells. RESULTS: BBE at 350-1400 µg/mL reduced the metabolic activity of Porphyromonas gingivalis, F. nucleatum and Streptococcus mutans. The reduced metabolic activity observed for F. nucleatum corresponded to a reduction in the numbers of CFUs following exposure to BBE for as little as 1 h, indicative of its bactericidal properties. An anthocyanin-enriched fraction of BBE reduced the metabolic activity of F. nucleatum, but not of P. gingivalis or S. mutans, suggesting the contribution of species-specific agents in the whole BBE. Oral epithelial cell viability was not reduced following exposure to whole BBE (2.24-1400 µg/mL) for ≤ 6 h. CONCLUSION: BBE alters the metabolic activity of oral periodontopathogens while demonstrating a minimal effect on commensals. The specific antibacterial properties of BBE shown in this study, along with its previously demonstrated anti-inflammatory and antiviral properties, make this natural extract a promising target as an adjunct for prevention and/or complementary therapy of periodontal infections.

Salivary levels of Epstein-Barr virus DNA correlate with subgingival levels, not severity of periodontitis.

OBJECTIVE: The aim of this study was to determine the presence and quantity of human cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNA in the saliva of patients with periodontitis, and investigate the correlation between these factors. METHODS: Presence and amounts of viral DNA in saliva and subgingival plaque samples, from healthy and disease sites, of 65 adults diagnosed with chronic periodontitis were determined using quantitative real-time polymerase chain reaction. RESULTS: Epstein-Barr virus DNA was detected in saliva of 81.5% (53/65) of patients at a median concentration of 4325 copies ml(-1). CMV DNA was detected in saliva of one individual (1.5%) at low copy number. Patients who had EBV in saliva were 10 times more likely to have EBV in subgingival plaque than patients lacking EBV in saliva (odds ratio = 10.1, 95% confidence interval = 2.6-39.5; P = 0.0009). EBV DNA burden in saliva positively correlated with the amounts detected in plaque and with amounts detected in increasing number of affected sites (P < 0.0001). EBV DNA presence and quantity in saliva did not correlate with increasing severity of disease as measured by periodontal indices. CONCLUSIONS: Epstein-Barr virus DNA presence and burden in saliva are associated with its presence and burden in subgingival plaque, but presence and burden in saliva does not correlate with periodontal disease severity.

Herpesvirus quiescence in neuronal cells IV: virus activation induced by pituitary adenylate cyclase-activating polypeptide (PACAP) involves the protein kinase A pathway.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally occurring peptide found in the central nervous system that plays a role in somatosensory processing and activation of protein kinase A (PKA) and protein kinase C (PKC). Because activation of PKA or PKC results in reactivation of HSV-1 from latently infected embryonic neuronal cells, PACAP was used to evaluate HSV-1 activation from quiescently infected (QIF)-PC12 cells. Our studies demonstrate that physiologically relevant concentrations of PACAP38 and PACAP27 induce HSV-1 activation from QIF-PC12 cell cultures in a dose-dependent fashion. PACAP-induced activation of virus was significantly impaired by the PKA-inhibitor, H-89 (20 microM), whereas treatment with the PKC-inhibitor, GF109203X (1 microM), was without affect. Additionally, direct activation of PKA with cAMP analogs, 8-(4-chlorophenylthio)- and dibutyryl-cAMP, only partially mimicked the effect of PACAP on virus activation. Taken together, PACAP induced HSV-1 activation from QIF-PC12 cells involves the PKA and possibly cAMP-independent pathways. This report is the first to demonstrate that PACAP induces HSV-1 activation from a quiescent state and that this in vitro cell model is useful for studying early inductive events that lead to virus production from quiescence.

Herpesvirus quiescence in neuronal cells: antiviral conditions not required to establish and maintain HSV-2 quiescence.

We previously described a novel in vitro model of a non-productive herpes simplex virus type 1 (HSV-1) infection in neurally differentiated (ND)-PC12 cells that allows for inducible virus replication upon forskolin and heat stress (HS) treatment. In this research, we further characterized the model with respect to HSV-2 strain 333. We found that: (i) ND-PC12 cells are non-permissive to HSV-2 replication; (ii) HSV-2 can establish a quiescent infection, like HSV-1, in ND-PC12 cells with the transient use of acycloguanosine (ACV); however unlike HSV-1, anti-viral conditions are not obligatory to establish and maintain a quiescent state; (iii) the quiescent state is maintained in the presence of Vero cell cocultivation indicating that such cultures are free of infectious virus; and (iv) a high percentage of quiescently infected (QIF)-PC12 cell cultures (80 - 100%) produce HSV-2 in response to forskolin and HS (43 degrees C, 3 h) treatment for as long as 4 weeks post infection. These findings indicate that ND-PC12 cells can harbor HSV-2 in a cryptic and non-productive state that is reversible. This model has appealing features for studying gene expression during the establishment, maintenance and reactivation phases of the HSV-2 quiescent state in cell culture.

Heat stress activates production of herpes simplex virus type 1 from quiescently infected neurally differentiated PC12 cells.

We have previously described a novel in vitro model of a non-productive herpes simplex virus type 1 (HSV-1) infection in neurally differentiated (Nd)-PC12 cells that allows for inducible virus replication upon forskolin treatment. In this study, we further characterized the quiescent state of infection and examined the ability of heat stress (HS) to induce virus from this non-productive state. These studies demonstrated that (i) the quiescent state is characterized by the absence of cell-associated virus, capsids, and viral antigens; (ii) HS (43 degrees C, 3 h) efficiently activated virus from quiescently infected Nd-PC12 (QIF-PC12) cells; (iii) the rate of virus production was significantly greater following HS than forskolin treatment, and the rates of both were dependent on MOI; (iv) forskolin and HS appeared to affect pathways of viral activation from a quiescent state as they did not enhance viral growth in Nd-PC12 cells; (v) viral alpha4 gene and host HSP72 gene transcription were rapidly induced in QIF-PC12 as soon as 3 h post-HS initiation; (vi) induction of the viral alpha27 gene followed that of representative beta and gamma genes, U(L)30 and U(L)18, respectively, and (vii) HS induced asynchronous HSV-1 replication from QIF-PC12 cells with 1:400 to 1:22000 positive foci detected as rapid as 24 h post-induction when established at MOIs of 30 and 3, respectively. These findings provide evidence that alpha4 may be involved in the switch from quiescence to productive infection. Furthermore, this model has the potential to advance our understanding of how HS initiates the HSV-1 productive cycle from a cryptic viral genome.

Establishment of a quiescent herpes simplex virus type 1 infection in neurally-differentiated PC12 cells.

Rat pheochromocytoma (PC12) cells differentiated with nerve growth factor (Nd-PC12) were used to investigate the establishment of a non-productive herpes simplex virus type 1 (HSV-1) infection that is reversible. The results of this work are as follows: (i) Nd-PC12 cultures could be maintained as long term (>7 weeks) non-dividing cultures only when plated on collagen-coated dishes in the absence of serum; (ii) Infection of Nd-PC12 with HSV-1 strains KOS and 17 in the transient presence of acycloguanosine (ACV) resulted in all cultures free of detectable levels of infectious virus at the time of ACV removal and ACV was not needed to maintain the non-productive quiescent state in the subsequent 8 weeks; (iii) These persistently infected and quiescent (QIF)-PC12 cultures demonstrated both spontaneous and forskolin-inducible virus production, at low (5%) and high frequencies (92-100%), respectively during the first 2 weeks post-ACV withdrawal. (iv) In contrast to other in vitro models, HSV-1 failed to reactivate following removal of nerve growth factor. (v) A high percentage of QIF-PC12 cultures (50-100%) produced virus in response to forskolin treatment as long as 7 weeks post-ACV withdrawal. (vi) Expression of HSV-1 productive genes (i.e. alpha0, alpha4, alpha27, UL30 and UL18) dropped precipitously in the presence of ACV and remained undetectable or continued to decline following its removal, whereas the levels of LAT and the host gene G3PDH remained relatively constant throughout the 31 day study period as measured by RT-PCR. These results indicate that QIF-PC12 cells offer a novel, neuronal cell culture system that may enhance our ability to study HSV-1 reactivation from a cryptic, latent-like, non-productive state in the absence of replication inhibitors.

Molecular aspects of herpes simplex virus I latency, reactivation, and recurrence.

The application of molecular biology in the study of the pathogenesis of herpes simplex virus type 1 (HSV-1) has led to significant advances in our understanding of mechanisms that regulate virus behavior in sensory neurons and epithelial tissue. Such study has provided insight into the relationship of host and viral factors that regulate latency, reactivation, and recurrent disease. This review attempts to distill decades of information involving human, animal, and cell culture studies of HSV-1 with the goal of correlating molecular events with the clinical and laboratory behavior of the virus during latency, reactivation, and recurrent disease. The purpose of such an attempt is to acquaint the clinician/scientist with the current thinking in the field, and to provide key references upon which current opinions rest.

Antigenic variation in Neisseria gonorrhoeae: production of multiple lipooligosaccharides.

Individual cells of Neisseria gonorrhoeae may express a single lipooligosaccharide (LOS) component on their cell surfaces, or they may simultaneously express multiple LOS structures. Strain FA19 expresses LOS components that react with monoclonal antibodies (MAbs) 2-1-L8 and 1B2. The genetic locus responsible for this phenotype in FA19 was identified by isolating a clone that is able to impart the ability to simultaneously express both LOS molecules to strain 1291, a strain expressing only the MAb 1B2-reactive LOS. This clone, pCLB1, was characterized, and the gene responsible for the expression of both LOS components was determined to be lsi2. DNA sequence analysis of lsi2(Fa19) indicates that there are several differences between the DNA sequences of lsi2(FA19) and lsi2(1291). The region responsible for the LOS-specific phenotype change in lsi2(FA19) was identified by deletion and transformation analysis, mapping to a polyguanine tract within lsi2 where lsi2(FA19) possesses a +2 frameshift relative to lsi2(1291). The polyguanine tract in lsi2(FA19) was modified by site-directed mutagenesis to change the sequence to GGGAGGTGGCGGA to prevent frameshifting during DNA replication, transcription, and/or translation. Transformants of strain 1291 containing this DNA sequence express a single MAb 2-1-L8-reactive LOS component, the same phenotype exhibited by lsi2-defective strains. These data indicate that FA19 is able to generate a small amount of functional Lsi2 protein via transcriptional and/or translational frameshifting, and this limited amount of protein allows for the expression of MAb 1B2-reactive LOS molecules.

Genetic basis of Neisseria gonorrhoeae lipooligosaccharide antigenic variation.

Neisseria gonorrhoeae lipooligosaccharide (LOS) undergoes antigenic variation at a high rate, and this variation can be monitored by changes in a strain's ability to bind LOS-specific monoclonal antibodies. We report here the cloning and identification of a gene, lsi-2, that can mediate this variation. The DNA sequence of lsi-2 has been determined for N. gonorrhoeae 1291, a strain that expresses a high-molecular-mass LOS, and a derivative of this strain, RS132L, that produces a truncated LOS. In the parental strain, lsi-2 contains a string of 12 guanines in the middle of its coding sequence. In cells that had antigenically varied to produce a truncated LOS, the number of guanines in lsi-2 was altered. Site-specific deletions were constructed to verify that expression of a 3.6-kDa LOS is due to alterations in lsi-2.

Genetic basis of pyocin resistance in Neisseria gonorrhoeae.

The genetic basis for pyocin resistance in Neisseria gonorrhoeae 1291d, 1291e, and FA5100 was determined by Southern blot and DNA sequence analyses. The genes defective in these strains are present as single copies in the gonococcal chromosome. The mutant regions of 1291d, 1291e, and FA5100 were amplified by the PCR. Sequence analysis of the mutant regions demonstrated that strain 1291d contains a 12-bp deletion that results in the loss of four amino acids in phosphoglucomutase, while strain 1291e contains a point mutation that results in the change of an uncharged glycine residue to a charged glutamic acid residue in the same protein. FA5100 contains a nonsense mutation in the gene encoding heptosyltransferase II. The gene previously described as lsi-1 was shown to complement an rfaF mutation in Salmonella typhimurium and has been renamed rfaF.

Use of xylE fusions to demonstrate that lsi-1, a Neisseria gonorrhoeae lipooligosaccharide biosynthetic gene, and lsi-3 are not transcriptionally linked.

The Neisseria gonorrhoeae lipooligosaccharide biosynthetic gene lsi-1 is preceded by a 281-bp non-protein-encoding sequence, lsi-3, that contains two pairs of inverted repeats. Gonococcal chromosomal lsi-xylE gene fusions were generated to measure the effect of the secondary structure on transcriptional attenuation. The data obtained indicate that lsi-3 and lsi-1 are not transcriptionally linked and therefore that lsi-3 is not involved in the regulation of lsi-1.

Analysis of the lsi region involved in lipooligosaccharide biosynthesis in Neisseria gonorrhoeae.

The genetic locus (lsi-1) responsible for the transformation of the lipooligosaccharide (LOS)-defective Neisseria gonorrhoeae mutant FA5100 to LOS expression was studied by deletion mutagenesis and sequence analysis. An open reading frame that was preceded by a leader sequence containing regions with the potential to form hairpin loops was identified. A perfect sigma 70 promoter consensus sequence was found upstream from this open reading frame. Promoter function was screened for functionality by using lac fusion cassettes and in vitro transcription-translation analysis. A frameshift mutation in the lsi-1 gene was constructed by site-directed mutagenesis and introduced into the chromosome of FA19, the LOS-expressing isogenic parent strain of FA5100. The mutant was characterized by Southern blotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting (immunoblotting) and found to be phenotypically identical to FA5100.

Characterization of a gyrB mutation responsible for low-level nalidixic acid resistance in Neisseria gonorrhoeae.

Nalidixic acid-resistant derivatives of Neisseria gonorrhoeae WR302 were identified and categorized into two classes on the basis of their susceptibilities to this antimicrobial agent. The MIC of nalidixic acid for the derivative strain MUG116 was fourfold greater than that for its isogenic parental strain WR302 (2 versus 0.5 micrograms/ml, respectively). MUG324 was significantly more resistant to nalidixic acid (greater than 64 micrograms/ml). The MICs of other antimicrobial agents known to interact with either the gyrA or gyrB gene products were determined. Although the nalidixic acid MIC for MUG116 increased, no significant increases in the MICs of other agents that interact with the gyrA gene product were seen. The MICs of all agents that interact with the gyrA gene product were significantly increased for MUG324. The gene that imparts low-level nalidixic acid resistance was cloned from strain MUG116. The DNA sequence of this gene was determined, and by comparing the deduced amino acid sequence with sequences of proteins in data bases, this protein was found to be approximately 70% homologous with the gyrB gene product of Escherichia coli.

Expression of cloned restriction and modification genes, hjaIRM from Hyphomonas jannaschiana in Escherichia coli.

A type-II RM system, HjaI, was identified in the marine bacterium, Hyphomonas jannaschiana. The ENase recognizes GATATC, and DNA fragments generated after cleavage with this enzyme contain blunt ends. A DNA fragment encoding these enzymes was cloned and expressed in Escherichia coli, although the level of expression of the cloned genes was low. DNA methylated by M.HjaI was not restricted by the Mcr or Mrr restriction systems of E. coli. Although H. jannaschiana is a marine bacterium isolated near the thermal vents on the floor of the Pacific Ocean, the biochemical properties of the ENase were similar to those of EcoRV, an isoschizomer isolated from E. coli.