Viral-vectored boosting of OmcB- or CPAF-specific T-cell responses fail to enhance protection from Chlamydia muridarum in infection-immune mice and elicits a non-protective CD8-dominant response in naïve mice.

A vaccine is needed to combat the Chlamydia epidemic. Replication-deficient viral vectors are safe and induce antigen-specific T-cell memory. We tested the ability of intramuscular immunization with modified vaccinia Ankara (MVA) virus or chimpanzee adenovirus (ChAd) expressing chlamydial outer membrane protein (OmcB) or the secreted protein, chlamydial protease-like activating factor (CPAF), to enhance T-cell immunity and protection in mice previously infected with plasmid-deficient Chlamydia muridarum CM972 and elicit protection in naïve mice. MVA.OmcB or MVA.CPAF increased antigen-specific T cells in CM972-immune mice ∼150 and 50-fold, respectively, but failed to improve bacterial clearance. ChAd.OmcB/MVA.OmcB prime-boost immunization of naïve mice elicited a cluster of differentiation (CD) 8-dominant T-cell response dominated by cluster of differentiation (CD)8 T cells that failed to protect. ChAd.CPAF/ChAd.CPAF prime-boost also induced a CD8-dominant response with a marginal reduction in burden. Challenge of ChAd.CPAF-immunized mice genetically deficient in CD4 or CD8 T cells showed that protection was entirely CD4-dependent. CD4-deficient mice had prolonged infection, whereas CD8-deficient mice had higher frequencies of CPAF-specific CD4 T cells, earlier clearance, and reduced burden than wild-type controls. These data reinforce the essential nature of the CD4 T-cell response in protection from chlamydial genital infection in mice and the need for vaccine platforms that drive CD4-dominant responses.

Anti-CD22 and anti-CD79B antibody drug conjugates are active in different molecular diffuse large B-cell lymphoma subtypes.

Antibody drug conjugates (ADCs), in which cytotoxic drugs are linked to antibodies targeting antigens on tumor cells, represent promising novel agents for the treatment of malignant lymphomas. Pinatuzumab vedotin is an anti-CD22 ADC and polatuzumab vedotin an anti-CD79B ADC that are both linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE). In the present study, we analyzed the activity of these agents in different molecular subtypes of diffuse large B-cell lymphoma (DLBCL) both in vitro and in early clinical trials. Both anti-CD22-MMAE and anti-CD79B-MMAE were highly active and induced cell death in the vast majority of activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL cell lines. Similarly, both agents induced cytotoxicity in models with and without mutations in the signaling molecule CD79B. In line with these observations, relapsed and refractory DLBCL patients of both subtypes responded to these agents. Importantly, a strong correlation between CD22 and CD79B expression in vitro and in vivo was not detectable, indicating that patients should not be excluded from anti-CD22-MMAE or anti-CD79B-MMAE treatment because of low target expression. In summary, these studies suggest that pinatuzumab vedotin and polatuzumab vedotin are active agents for the treatment of patients with different subtypes of DLBCL.

Anti-CD22-MCC-DM1: an antibody-drug conjugate with a stable linker for the treatment of non-Hodgkin's lymphoma.

Antibody-drug conjugates (ADCs) are potent cytotoxic drugs linked to antibodies through chemical linkers, and allow specific targeting of drugs to neoplastic cells. The expression of CD22 is limited to B-cells, and we show that CD22 is expressed on the vast majority of non-Hodgkin's lymphomas (NHLs). An ideal target for an ADC for the treatment of NHL would have limited expression outside the B-cell compartment and be highly effective against NHL. We generated an ADC consisting of a humanized anti-CD22 antibody conjugated to the anti-mitotic agent maytansine with a stable linker (anti-CD22-MCC-DM1). Anti-CD22-MCC-DM1 was broadly effective in in vitro killing assays on NHL B-cell lines. We did not find a strong correlation between in vitro potency and CD22 surface expression, internalization of ADC or sensitivity to free drug. We show that anti-CD22-MCC-DM1 was capable of inducing complete tumor regression in NHL xenograft mouse models. Further, anti-CD22-MCC-DM1 was well tolerated in cynomolgus monkeys and substantially decreased circulating B-cells as well as follicle size and germinal center formation in lymphoid organs. These results suggest that anti-CD22-MCC-DM1 has an efficacy, safety and pharmacodynamic profile that support its use as a treatment for NHL.

The significance of prior mechanical therapy for changes of periodontal status achieved by local delivery of a doxycyclinehyclate containing gel.

AIM: The purpose of this retrospective analysis was to examine what effect, if any, the time elapsed between an individual's last episode of scaling and root planing and subsequent treatment with a sustained-release doxycycline hyclate gel (DH) alone or scaling and root planing alone (SRP) would have upon adult periodontitis. METHODS: A total of 207 subjects were included in the DH group and 210 patients in the SRP group. Periodontitis was defined as those sites which presented with pocket depths > or =5 mm and exhibited bleeding upon probing. In both DH and SRP groups, 3 sub-groups of subjects were identified according to their last episode of scaling and root planing prior to the study baseline: within 2 to 6 months, >6 but < or =12 months, and one or more times in their life but not within the last 12 months. Each study site was treated twice over a 9-month study period, once at baseline and again at 4 months. Data from the study sites at 4, 6, and 9 months were then evaluated for changes in probing depth, clinical attachment level, and bleeding upon probing. RESULTS: At the 9-month evaluation, all sub-groups in the DH and SRP treatment arms presented with improvement in the measured clinical parameters, as compared to baseline. No significant differences were observed in the measured periodontal indices among the study sites between the three sub-groups for either treatment. CONCLUSIONS: It is concluded that the time interval since the last episode of scaling and root planing had no observable effect on the results achieved when treating periodontitis sites with locally delivered doxycycline hyclate alone or scaling and root planing alone. The treatment of periodontitis sites with locally delivered doxycycline hyclate resulted in clinical improvement comparable to scaling and root planing irrespective of the patient's prophylaxis frequency.

Subantimicrobial dose doxycycline as an adjunct to scaling and root planing: post-treatment effects.

BACKGROUND/OBJECTIVE: Subantimicrobial dose doxycycline (SDD 20 mg bid) plus scaling and root planing (SRP) significantly improved clinical attachment level (CAL) and reduced probing depth (PD) compared with placebo plus SRP in a double-blind, placebo-controlled, multicenter study of patients with adult periodontitis (AP). In a study conducted as a follow-up, the post-treatment effects of SDD were assessed in patients who completed the SRP study. METHODS: The SRP study was a 9-month, active-treatment study and the follow-up was a 3-month, no-treatment study. In the SRP study, tooth sites in qualifying quadrants were scaled and root planed and patients were randomized to receive twice daily SDD 20 mg or placebo. In the follow-up, patients received no study drug; investigators and patients remained blinded to the previous treatment group assignments. Efficacy measures included the change in CAL and PD from baseline values determined at the start of the SRP study in tooth sites stratified by baseline PD (i.e., 0-3 mm, 4-6 mm, > or =7 mm). Safety was evaluated using adverse event data and the results of clinical laboratory tests, oral pathology examinations, and microbiological assessments. RESULTS: Within each disease stratum, the incremental improvements in PD and CAL demonstrated in the SDD group over 9 months of active treatment were maintained through 3 additional months of no treatment. Treatment cessation did not result in an accelerated regression of periodontal health. No differences in the incidence of adverse events (including those related to infection) or laboratory or microbiological parameters were noted between the SDD group and the placebo group. CONCLUSIONS: The administration of SDD 20 mg bid for a period of up to 9 months is not associated with rebound effects or delayed or negative after-effects for a 3-month period after cessation of therapy.

Kaposi's sarcoma-associated herpesvirus K-bZIP protein is phosphorylated by cyclin-dependent kinases.

The K8 locus in Kaposi's sarcoma-associated herpesvirus (KSHV) is syntenic with the Epstein-Barr virus (EBV) BZLF (Z) locus and expresses three alternatively spliced transcripts. The fully spliced transcript encodes K-bZIP, the KSHV homologue of the EBV immediate-early transcriptional transactivator Z. Here we show that despite the presence of alternatively spliced transcripts, the protein from the fully spliced RNA, K-bZIP, is the principal product detectable in KSHV-infected B cells. The protein is detected only in lytically infected cells and is localized to the nucleus. We further characterized K-bZIP by determining its phosphorylation status. Phosphoamino acid analysis revealed phosphorylation on serine and threonine. Analysis of the sites of K-bZIP phosphorylation by tandem mass spectrometry revealed that K-bZIP was phosphorylated on Thr 111 and Ser 167. These phosphorylation sites are contained within cyclin-dependent kinase (CDK) recognition sites with the consensus sequence (S/T)PXR, suggesting that K-bZIP could be phosphorylated by CDKs. We tested this hypothesis using an in vitro kinase reaction performed in whole-cell extracts that resemble in vivo conditions more closely than standard in vitro kinase reactions. We found that the three CDK-cyclin complexes we tested phosphorylated K-bZIP but not the control ORF 73 protein, which contains four (S/T)PXR sites. Ectopic expression of K-bZIP cannot reactivate KSHV from latency, and single and double mutants of K-bZIP in which alanines replaced the phosphorylated serine and/or threonine also failed to induce lytic replication. These studies indicate that K-bZIP is a substrate for CDKs and should inform further functional analyses of the protein.

An economic evaluation of a chlorhexidine chip for treating chronic periodontitis: the CHIP (chlorhexidine in periodontitis) study.

BACKGROUND: The authors previously suggested that an adjunctive, controlled-release chlorhexidine, or CHX, chip may reduce periodontal surgical needs at little additional cost. This article presents an economic analysis of the CHX chip in general dental practice. METHODS: In a one-year prospective clinical trial, 484 chronic periodontitis patients in 52 general practices across the United States were treated with either scaling and root planing, or SRP, plus any therapy prescribed by treating, unblinded dentists; or SRP plus other therapy as above but including the CHX chip. Economic data were collected from bills, case report forms and 12-month treatment recommendations from blinded periodontist evaluators. RESULTS: Total dental charges were higher for SRP + CHX chip patients vs. SRP patients when CHX chip costs were included (P = .027) but lower when CHX chip costs were excluded (P = .012). About one-half of the CHX chip acquisition cost was offset by savings in other charges. SRP + CHX chip patients were about 50 percent less likely to undergo surgical procedures than were SRP patients (P = .021). At the end of the trial, periodontist evaluators recommended similar additional procedures for both groups: SRP, about 46 percent; maintenance, about 37 percent; surgery, 56 percent for SRP alone and 63 percent for SRP + CHX chip. CONCLUSIONS: Adjunctive CHX chip use for general-practice patients with periodontitis increased costs but reduced surgeries over one year. At study's end, periodontists recommended similar additional surgical treatment for both groups. CLINICAL IMPLICATIONS: In general practice, routine use of the CHX chip suggests that costs will be partially offset by reduced surgery over at least one year.

Treatment with subantimicrobial dose doxycycline improves the efficacy of scaling and root planing in patients with adult periodontitis.

BACKGROUND: In a previous study, subantimicrobial dose doxycycline (SDD) significantly improved clinical parameters associated with periodontal health in patients with adult periodontitis (AP) when used as an adjunct to a maintenance schedule of supragingival scaling and dental prophylaxis. In this double-blind, placebo-controlled, parallel-group, multicenter study, the efficacy and safety of SDD were evaluated in conjunction with scaling and root planing (SRP) in patients with AP. METHODS: Patients (n = 190) received SRP at the baseline visit and were randomized to receive either SDD 20 mg bid or placebo bid for 9 months. Efficacy parameters included the per-patient mean changes in clinical attachment level (CAL) and probing depth (PD) from baseline, the per-patient percentages of tooth sites with attachment loss (AL) > or = 2 mm and > or = 3 mm from baseline, and the per-patient percentage of tooth sites with bleeding on probing. Prior to analysis, tooth sites were stratified by the degree of disease severity evident at baseline RESULTS: In tooth sites with mild to moderate disease and severe disease (n = 183, intent-to-treat population), improvements in CAL and PD were significantly greater with adjunctive SDD than with adjunctive placebo at 3, 6, and 9 months (all P <0.05). In tooth sites with severe disease, the per-patient percentage of sites with AL > or = 2 mm from baseline to month 9 was significantly lower with adjunctive SDD than with adjunctive placebo (P<0.05). Improvements in clinical outcomes occurred without detrimental shifts in the normal periodontal flora or the acquisition of doxycycline resistance or multiantibiotic resistance. SDD was well tolerated, with a low incidence of discontinuations due to adverse events. CONCLUSIONS: The adjunctive use of SDD with SRP is more effective than SRP alone and may represent a new approach in the long-term management of AP.

The effect of locally delivered controlled-release doxycycline or scaling and root planing on periodontal maintenance patients over 9 months.

BACKGROUND: This research report evaluates clinical changes resulting from local delivery of doxycycline hyclate (DH) or traditional scaling and root planing (SRP) in a group of patients undergoing supportive periodontal therapy (SPT). METHODS: In all, 141 patients received either DH (67) or SRP (74) treatment in sites > or =5 mm on one-half of their dentition at baseline and month 4. RESULTS: Clinical results were determined at month 9. Baseline mean probing depth recordings were similar between the two groups (DH = 5.9 mm; SRP = 5.9 mm). Mean month 9 results showed similar clinical results for attachment level gain (DH 0.7 mm; SRP 0.8 mm) and probing depth reduction (DH 1.3 mm; SRP 1.1 mm). Percentage of sites showing > or =2 mm attachment level gain at month 9 was 24.7% in the DH group and 21.2% in the SRP group. Thirty-nine percent (39%) of DH sites and 38% of SRP sites showed > or =2 mm probing depth reduction. When treated sides of the dentition were compared to untreated sides, DH showed a difference in disease activity (> or =2 mm attachment loss) from 19.3% (untreated) to 7.2% (treated); and SRP from 14.3% (untreated) to 8.1% (treated). CONCLUSIONS: Results show that both DH without concomitant mechanical instrumentation and SRP were equally effective as SPT in this patient group over the 9-month study period.

In situ localization and characterization of active proteases in chronically inflamed and healthy human gingival tissues.

BACKGROUND: Studies have indicated an important role for host-derived proteases in the pathogenesis of periodontal disease. The objectives of this study were: 1) to develop an assay measuring protease activity in situ and 2) to localize and characterize the enzymatic activity in intact inflamed and healthy gingiva. METHODS: Gingival specimens were prepared and over-laid with a quenched fluorescent substrate. Protease activity was visualized by fluorescence microscopy and correlated with histologic features. RESULTS: In inflamed tissues, enzymatic activity was detected mainly in the connective tissue (predominantly matrix metalloproteases) and, to some extent, in the epithelium (predominantly serine proteases). In contrast, clinically healthy tissues failed to exhibit significant amounts of protease activity. Quantitative and qualitative characteristics of protease activity in intact tissues were found to be pH dependent. CONCLUSIONS: The method described here enabled assessment of active proteases in intact tissues where cell-cell and cell-matrix interactions had been maintained. Our results indicate that there are substantial differences in the distribution of specific proteases between clinically healthy and inflamed periodontal tissues.

Effects of smoking on local delivery of controlled-release doxycycline as compared to scaling and root planing.

This paper examines the effects of smoking on the treatment outcomes of two nonsurgical therapies: (1) scaling and root planing alone (SRP) or (2) controlled-release of subgingivally delivered doxycycline hyclate in a polylactic acid based polymer gel. Subjects from 2 9-month multicenter studies were classified as nonsmokers (never smoked: 100 subjects), former smokers (137 subjects), and current smokers (> or = 10 cigarettes/day: 121 subjects). Clinical parameters were analyzed for treated sites with baseline probing depths > or = 5 mm and for a subset of treated sites with baseline probing depths of > or = 7 mm. Clinical parameters (plaque levels, clinical attachment levels, pocket depths, and bleeding on probing) were analyzed at baseline, 4, 6, and 9 months. In the doxycycline treated group in general, there were neither marked significant differences in clinical attachment gain nor differences in probing depth reduction among the 3 smoking groups. On the other hand, in the scaling and root planing treated group in general, there were significant differences in clinical attachment gain and pocket depth reduction, with non-smokers responding better than former smokers and current smokers at 6 and 9 months. These differences in clinical response between scaling and root planing alone versus controlled-release of locally-delivered doxycycline hyclate among these 3 smoking groups are discussed in relation to treatment implications for smokers.

Two multi-center studies evaluating locally delivered doxycycline hyclate, placebo control, oral hygiene, and scaling and root planing in the treatment of periodontitis.

BACKGROUND: The clinical efficacy and safety of doxycycline hyclate (8.5% w/w) delivered subgingivally in a biodegradable polymer (DH) was compared to placebo control (VC), oral hygiene (OH), and scaling and root planing (SRP) in 2 multi-center studies. METHODS: Each study entered 411 patients who demonstrated moderate to severe periodontitis. Patients had 2 or more quadrants each with a minimum of 4 qualifying pockets > or =5 mm that bled on probing. At least 2 of the pockets were > or =7 mm. Treatment with DH, VC, OH, or SRP was provided at baseline and again at month 4. Clinical parameters were recorded monthly. RESULTS: DH and SRP resulted in nearly identical clinical changes over time in both studies. Mean 9 month clinical attachment level gain (ALG) was 0.8 mm for the DH group and 0.7 mm for the SRP group in Study 1, and 0.8 mm (DH) and 0.9 mm (SRP) in Study 2. Mean probing depth (PD) reduction was 1.1 mm for the DH group and 0.9 mm for the SRP group in Study 1 and 1.3 mm for both groups in Study 2. Frequency distributions showed an ALG > or =2 mm in 29% of DH sites versus 27% of SRP sites in Study 1 and 31% of DH sites versus 34% of SRP sites in Study 2. PD reductions > or =2 mm were seen in 32% of DH sites versus 31% of SRP sites in Study 1 and 41% of DH sites versus 43% of SRP sites in Study 2. Comparisons between DH, VC, and OH treatment groups showed DH treatment to be statistically superior to VC and OH. Safety data demonstrated a benign safety profile with use of the DH product. CONCLUSIONS: Results of this trial demonstrate that treatment of periodontitis with subgingivally delivered doxycycline in a biodegradable polymer is equally effective as scaling and root planing and superior in effect to placebo control and oral hygiene in reducing the clinical signs of adult periodontitis over a 9-month period. This represents positive changes resulting from the use of subgingivally applied doxycycline as scaling and root planing was not limited regarding time of the procedure or use of local anesthesia.

Effects of prognosis, perceived benefit, and decision style on decision making and critical care on decision making in critical care.

OBJECTIVE: To assess the effects of prognostic estimates, perceived benefit of treatment, and practice style on decision-making in critical care. DESIGN: Randomized assignment of subjects to either of two versions of a questionnaire designed to elicit treatment decisions for six intensive care unit cases based on actual patients. One version offered optimistic survival forecasts; the other, pessimistic forecasts. SUBJECTS: A random sample of 120 clinicians obtained from the Canadian Critical Care Society was contacted by mail. One version of the questionnaire was randomly assigned and mailed to each. Thirty-four replies, 17 for each version (response rate, 28%), were received and analyzed. MEASUREMENTS AND MAIN RESULTS: A list of treatment/management options was developed for each case, in three categories: recommended, questionable, and unacceptable. Subjects were also able to list new options that they would order that were not on the list. The dependent variables were the number of actions ordered in each category and the total for each case. Perceived benefit was measured by comparing subjective estimates of the probability of survival with the optimistic/pessimistic forecast given in the case. Practice style was assessed by correlating the total number of actions ordered across all possible pairs of cases. There were no significant differences between the two questionnaires on actions ordered either by category or by amount per category. Perceived benefit did not appear to be an important factor in decision-making. However, statistically significant correlations provide evidence for practice style in intensive care unit decision-making on an interventionist/noninterventionist dimension. CONCLUSIONS: There is no evidence that erroneous or biased prognostic estimates affect intensive care unit treatment choices. Neither the principle of maximizing expected utility nor the Rule of Rescue appear to affect these decisions systematically, but practice style does.

Dental implant outcomes in postmenopausal women undergoing hormone replacement.

To determine if hormone supplementation reduces the risk of failure for osseointegration of dental implants in postmenopausal women, the treatment outcomes of 116 women older than age 50, treated with 450 endosseous dental implants, were analyzed in this retrospective study. The findings indicated that hormone replacement therapy may not be linked with improved outcomes of endosseous dental implant treatment in postmenopausal women. Smoking, however, appears to significantly increase the implant failure rate in the group observed in this study.

Biodegradation and biocompatibility of a guided tissue regeneration barrier membrane formed from a liquid polymer material.

Biodegradable barrier films were made by coagulating a solution of poly(DL-lactide) in N-methyl-2-pyrrolidone on porous polyethylene pads wetted with saline solution. The semisolid films were cut into 10 x 10 mm barriers and implanted subcutaneously in rabbits. At monthly intervals, the polymer implant sites were compared histologically to those implanted with USP negative control plastic. The polymer films were retrieved from the surrounding tissue, dried, weighed, and the changes in molecular weight determined using gel permeation chromatography. The molecular weight of the polymer decreased at a relatively constant rate over 5 months; however, no significant mass loss occurred until 5 months postimplantation. Also, no distinct histological differences were noted between the polymer barrier and the control plastic sites until 6 months when histiocytes and multinucleated giant cells showed a modest increase around fragmented polymer films. Similar barrier films also were fitted over naturally occurring buccal dehiscence defects in beagle dogs and the tissue sites compared histologically at 6 months to sham-operated control sites. New bone and dense connective tissues closely approximated segments of the remaining polymer and demonstrated the biocompatibility of the biodegradable films. Histomorphometric analyses of treated sites compared to sham controls showed that the polymer barrier is effective in promoting bone and cementum regeneration in periodontal defects in dogs.

Neuralgic amyotrophy: A rare cause of bilateral diaphragmatic paralysis.

Neuralgic amyotrophy, also known as brachial neuritis, is a well described clinical entity. Diaphragmatic dysfunction, as a result of phrenic nerve root involvement (cervical roots 3 to 5), is an uncommon, but increasingly recognized association. The case of a previously healthy 61-year-old woman who, after a prodrome of neck and shoulder discomfort, presented with severe orthopnea is described. Pulmonary function and electrophysiological studies led to a diagnosis of bilateral diaphragmatic paralysis. The patient's clinical course and the exclusion of other nerve entrapment syndromes and neurological disorders strongly favoured the diagnosis of neuralgic amyotrophy.

The role of local drug delivery in the management of periodontal diseases: a comprehensive review.

This review article evaluates the role of local drug delivery systems in the management of periodontal diseases. The efficacy of several local delivery devices (i.e., tetracycline fibers, metronidazole and minocycline gels, chlorhexidine chips, and doxycycline polymer) which are either commercially available in the United States or abroad, or are currently under consideration for Food and Drug Administration (FDA) approval are discussed. The drug delivery systems are assessed with regard to their functional characteristics, effectiveness as a monotherapy, as compared to scaling and root planing, and ability to enhance conventional therapy. Furthermore, controversies associated with local delivery are addressed (e.g., induction of bacterial resistant strains, the efficacy of systemic versus local drug delivery, and whether local drug delivery should function as an alternative or as an adjunct to conventional treatment).

Controlled local delivery of antimicrobials in the treatment of periodontitis.

Five local delivery systems with five different antimicrobial agents have been discussed. All are capable of delivering high concentrations of their antimicrobial to the site of the periodontal infection. Although only one system, tetracycline fiber, is available in United States, two other systems, chlorhexidine chip and doxycycline polymer, may be available in the near future. Two other systems, metronidazole gel and minocycline ointment, are available in other countries. Data from pertinent studies were presented as were techniques for using the various systems. Indications for the use of the products were also discussed. In selecting the appropriate delivery system, the clinician has to weigh the efficacy of the products, ease of use, availability, and cost. Although local delivery systems do not replace existing periodontal therapies, they do have a place in the treatment of periodontitis and offer the dentist additional methods to aid in the control of periodontal diseases.

Hepatitis delta virus RNA editing is highly specific for the amber/W site and is suppressed by hepatitis delta antigen.

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.