In-home evaluation of efficacy and titration of a mandibular advancement device for obstructive sleep apnea There is increasing evidence that mandibular advancement devices -LRB- MADs -RRB- can be an effective treatment for some patients with obstructive sleep apnea , a highly prevalent chronic disease . In this study , the objectives were to objectively assess the effectiveness of MAD therapy using a limited channel recorder , and to develop a model for identifying patients who may be appropriate for MAD therapy as the initial treatment option . Thirty patients were prospectively recruited and studied at two independent dentist offices and the participants ' homes . Subjects wore the ARES Unicorder for two nights before insertion of the MAD , and again when the dentist determined that the patient had reached the titration endpoint . Self-reported measures of depression , sleepiness , and quality of life were obtained pre - and posttreatment . The reviewer was blinded to the study status while the physiological signals were being visually inspected . Significant reductions in the apnea/hypopnea index -LRB- AHI -RRB- , hypoxemia measures , and snoring level were observed posttreatment . Twenty-seven of the 30 -LRB- 90 % -RRB- patients had a posttreatment AHI -LRB- using a 4 % desaturation for hypopneas -RRB- below a clinical cut-off of 10 . All but one patient -LRB- 97 % -RRB- exhibited at least a 50 % decrease in AHI or had a posttreatment AHI ≤ 10 . Significant differences in body mass index , weight , and neck circumference in patients with posttreatment AHIs above and below a clinical cut-off of five were identified . The linear regression used to predict the posttreatment AHI using pretreatment data resulted in an R2 of 0.68 . The model correctly predicted two patients who were unable to obtain a posttreatment AHI of 10 or less . This study was designed to demonstrate two models of collaboration between a dental sleep medicine specialist and a sleep medicine physician in the monitoring of a patient treated with a MAD . The outcome data suggest that the limited channel recording system can be used as an alternative to laboratory polysomnography to reduce the cost of MAD treatment , and to improve the quality and consistency of posttreatment patient care . Introduction Obstructive sleep apnea -LRB- OSA -RRB- is the most common disorder observed in the practice of sleep medicine and is responsible for more mortality and morbidity than any other sleep disorder -LSB- 1 -RSB- . Although characterized over 40 years ago -LSB- 2 , 3 -RSB- , OSA has only recently gained recognition as one of the world 's most prevalent , underdiagnosed disorders -LSB- 4 , 5 -RSB- . Due to associated morbidity , OSA has been identified as a major public health concern -LSB- 6 -RSB- . The disorder is characterized by frequent loud snoring and recurrent failures to breathe adequately during sleep -LRB- termed apneas or hypopneas -RRB- , usually as a result of collapse of the upper airway . Mandibular advancement devices -LRB- MADs -RRB- are being increasingly recognized as a treatment alternative to continuous positive airway pressure -LRB- CPAP -RRB- , particularly for patients with mild to moderate OSA -LSB- 7 -- 12 -RSB- . MADs are designed to protrude the mandible and thus the tongue and epiglottis during sleep , preventing airway occlusion -LSB- 13 -RSB- . Comparisons of MAD to CPAP have revealed that although MADs are less efficacious than CPAP in reducing the RDI they are , on average , used more frequently , preferred by more patients and more readily accepted than CPAP -LSB- 14 -RSB- . Recent studies demonstrated that both MADs and CPAP can reduce the 24-h diastolic blood pressure by an average of 3 mmHg after 4 weeks of treatment -LSB- 15 -- 17 -RSB- . Similarly to CPAP , self-reported improvements in quality of life and decrease in sleepiness are reported in the majority of patients -LSB- 14 -RSB- . The primary goal of this study was to use a self-applied limited channel recorder to objectively assess the effectiveness of MAD therapy when the dentist determined the patient had reached the titration endpoint . A second goal was to determine factors which might predict successful treatment outcomes and provide a more refined method for identifying patients who may be appropriate for MAD therapy as the initial treatment option . Finally , we wanted to demonstrate two potential models of collaboration between the dentist and sleep medicine physician in monitoring MAD treatment outcomes . Materials and methods Eleven females and 19 males were recruited from two dental practices before treatment with an oral appliance and enrolled in the study . Twenty-five of the patients had failed CPAP therapy . After obtaining an informed consent -LRB- approved by the BioMed IRB , San Diego , CA , USA -RRB- patients completed a two-night pretreatment in-home study with the Apnea Risk Evaluation System -LRB- ARES TM -RRB- Unicorder TM -LRB- Advanced Brain Monitoring , Carlsbad , CA , USA -RRB- . This pretreatment recording was conducted between the time the dental impressions were taken and when the TAP II Mandibular Advancement Device -LRB- MAD -RRB- -LRB- Airway Management , Dallas , TX , USA -RRB- was fabricated and ready for insertion . The TAP is a custom-made oral appliance with separate upper and lower appliances joined by a titration or advancement mechanism on the upper and a transverse bar or socket on the lower . The titration mechanism uses a hook to engage the bar or socket on the lower once each device is placed in the mouth . A jackscrew controls the position of the hook and thus the amount of protrusion . The patient can self-titrate the device using a removable hex key which engages the screw . On the day of insertion of the MAD , patients completed the Beck depression index , Epworth sleepiness scale , and the Flemons ' quality of life questionnaire . At the insertion appointment , both study sites attempted to achieve a starting MAD titration position whereby the patient could just hook the lower tray with the upper tray using active protrusion with both trays in place . In the rare occasion that this level of advancement was not tolerated by the patient , the starting protrusion was reduced . Patients were instructed to begin adjusting the MAD in one-half turn increments as soon as it was tolerable , until a cessation in snoring or the symptoms had resolved . As a result , the titration endpoint was determined by the dentist based on the patient 's self-report . At the follow-up appointment , which was typically scheduled 3 to 4 weeks subsequent to the MAD insertion , the assessment questionnaires were completed again and the ARES Unicorder study was repeated . Twenty-seven of the 30 patients reached their endpoint within 34 days . The other three patients completed their endpoint in 40 , 61 , and 75 days ; the delay in reaching the endpoint was due to patient illness unrelated to this study . In between the time that treatment was initiated and the titration endpoint , each patient maintained a daily journal that recorded the time the appliance was inserted each night , and the time it was removed in the morning . The pre - and posttreatment studies were conducted with the ARES Unicorder . From a single site on the forehead , the wireless recorder measures oxygen saturation , pulse rate , airflow , respiratory effort , snoring levels , head movement , and head position -LSB- 18 -RSB- -LRB- Fig. 1 -RRB- . Reflectance oximetry is used to obtain the SpO2 and pulse rate signals . Respiratory effort is derived from the measurement of changes in forehead venous pressure acquired using a combination of photoplethysmography and changes in surface pressure of the forehead reflectance oximetry sensor . Airflow is obtained via a cannula and nasal pressure transducer . A calibrated acoustic microphone is used to acquire quantified snoring levels -LRB- dB -RRB- . Actigraphy is used to measure head movement and derive head position . The recorder was designed to be affixed by the patient , and provide alerts during the study if poor quality airflow or SpO2 is detected so the device could be adjusted . Fig. 1Patient wearing a unicorder The description and validation of this device in 284 valid comparisons of the in-laboratory simultaneous PSG and ARES and 187 valid comparisons of the in-laboratory PSG with a separate two nights unattended self-applied ARES Unicorder has been previously published -LSB- 18 -RSB- . Using a diagnostic AHI cutoff of > 10 , the concurrent in-lab comparison yielded a sensitivity of 97.4 , a specificity of 85.6 , a positive predictive value of 93.6 , and a negative predictive value of 93.9 . The in-home comparison sensitivity , specificity , positive predictive value , and negative predictive value were 91.5 , 85.7 , 91.5 , and 85.7 , respectively . The failure rate was 2 % . In an independent validation of 40 subjects -LRB- 13 healthy controls and 27 patients with symptoms of EDS and/or snoring -RRB- using a clinical cut-off of 15 , the sensitivity of the ARES compared to concurrent PSG was 100 % -LRB- 25/25 patients -RRB- and specificity 67 % -LRB- 10/15 subjects -RRB- . The sensitivity for two-nights of ARES in-home compared to the PSG was 96 % ; specificity was 80 % . The failure rate was 5 % -LSB- 19 -RSB- . Automated scoring algorithms were applied off-line to detect sleep disordered breathing . The AHI was computed using a time-in-bed measure based on recording time with acceptable signal quality minus periods when the patient was upright or presumed to be awake based on actigraphy . Apneas , based on a 10-s cessation of airflow detected by the automated algorithms , were included in all apnea -- hypopnea indexes -LRB- AHI -RRB- . Hypopnea events required a 50 % reduction and recovery in airflow and were further stratified based on the depth of desaturation . The AHI-4 % criteria required a minimum 3.5 % reduction in SpO2 and at least a 1.0 % recovery . Hypopneas included in the AHI-3 % and AHI-1 % criteria required SpO2 desaturation and resaturation using stepped thresholds . For the AHI-3 % , if the SpO2 at the point of maximum saturation before the event was greater than or equal to 95 % then a 2.2 % reduction and a 2.2 % recovery in SpO2 was required . For maximum saturations of between 95 -- 93 % the required SpO2 change was a 2.5 % reduction and 2.5 % recovery ; between 93 -- 91 % a 3.0 % reduction and 2.7 % recovery ; between 91 -- 88 % a 3.5 % reduction and 3.0 % recovery ; and below 88 % a 4.0 % reduction and 3.5 % recovery . For the AHI-1 % , if the point of maximum saturation before the event was greater than 93 % , then a 1.0 % reduction and 1.0 % recovery was required ; and for events with a starting SpO2 between 93 -- 91 % , a 1.2 % reduction and 1.2 % recovery was required . For an AHI-1 % event to be called , the change in flow and desaturation needed to be associated with a behavioral arousal -LRB- i.e. , an abrupt change in pulse rate , snoring sound or a head movement -RRB- . After the automated scoring was applied , the full disclosure recordings were visually inspected by a sleep medicine physician to confirm the accuracy of the automated scoring , and to reclassify as central and/or exclude autodetected events if necessary . At the time of the review , the clinician was blinded to the study status -LRB- i.e. , pre - or posttreatment -RRB- . The physiological data , including AHI values , percent time with SpO2 below 90 , 85 , and 80 % , and percentage time snoring greater than 30 , 40 , 50 , and 60 dB were then calculated . Responses to the Beck depression index and Flemons ' quality of life questionnaire were tallied in accordance with published methods -LSB- 20 , 21 -RSB- . Paired t tests were used to identify significant changes in the pre - and posttreatment physiological data and questionnaire responses . To identify anthropomorphic factors that may impact MAD treatment outcomes , patients were stratified into two groups . Group 1 included all patients with a posttreatment AHI-4 % ≤ 5 -LRB- n = 18 -RRB- . The balance of patients with an AHI-4 % > 5 were assigned to group 2 -LRB- n = 12 -RRB- . Paired t tests were used to identify significant group differences . To develop and validate the prediction of the posttreatment AHI using pretreatment data , patients were paired and assigned into either the model development or cross validation group based on similarities in the pre - and post-4 % AHI and 1 % AHI . Correlation analysis was used to identify anthropomorphic variables and measures of obstructive breathing before treatment which might be useful in estimating the posttreatment 4 % AHI -LRB- post-T 4 % -RRB- . Variables with significant correlations were then used in a linear regression to derive predicted posttreatment values -LRB- predict AHI -RRB- . Results Overall effects of MAD treatment The mean ± SD and minimum pre - and posttreatment valid recording times were 9.6 ± 3.6 and 3.5 h ; and 10.3 ± 2.4 and 4.6 h , respectively.Paired t tests revealed significant changes -LRB- p < 0.001 -RRB- in all pre - vs posttreatment sleep disordered breathing measures , including : apnea -- hypopnea index with a 4 % -LRB- AHI-4 % -RRB- and 1 % -LRB- AHI-1 % -RRB- desaturation -LRB- Fig. 2 -RRB- . Fig. 2Mean + SE changes in pre - and posttreatment AHI-4 % and AHI-1 % A decrease in hypoxemia measures pre - and posttreatment were also observed including the percentage of time below 90 % SpO2 -LRB- T % < 90 % -RRB- -LRB- p < 0.05 -RRB- and the mean percentage change in SpO2 across all AHI events -LRB- % Dip -RRB- -LRB- p < 0.001 -RRB- -LRB- see Fig. 3 -RRB- . Fig. 3Mean + SE changes in pre - and posttreatment percentage of time the SpO2 was < 90 % , and the percentage change in SpO2 resulting from sleep disordered breathingSignificant reduction in the average snoring levels at 30 and 40 dB across the valid recording time were also observed -LRB- p < 0.001 -RRB- -LRB- Fig. 4 -RRB- . Fig. 4Mean + SE changes in pre - and posttreatment snoring loudness above 30 and 40 dBBased on conventional outcome measures , the MAD therapy was highly efficacious . Twenty-seven of the 30 -LRB- 90 % -RRB- patients had a posttreatment AHI-4 % < 10 -LRB- Fig. 5a -RRB- . All but one patient -LRB- 97 % -RRB- exhibited at least a 50 % decrease in AHI-4 % or had a posttreatment AHI-4 % ≤ 10 . Fig. 5Individual changes in pre - and posttreatment sleep disordered breathing using : a AHI-4 % desaturation and b AHI-1 % desaturationUsing an alternative measure of AHI-1 % -LRB- used to identify residual sleep disordered breathing/upper airway resistance -RRB- 21 of the 30 patients -LRB- 70 % -RRB- had a posttreatment AHI-1 % ≤ 15 -LRB- Fig. 5b -RRB- . Eighty-seven percent -LRB- 26 of 30 -RRB- of the patients exhibited at least a 50 % decrease in AHI-1 % or had a posttreatment AHI-1 % ≤ 15 . Paired t tests applied to the pre - and posttreatment scores revealed statistically significant differences for Beck depression index , Flemons ' QOL , Epworth sleepiness score -LRB- all at the p ≤ 0.001 level -RRB- -LRB- Fig. 6 -RRB- . Seventy-six percent -LRB- 23/30 -RRB- of the patients showed a decrease in Epworth score ; 60 % had an Epworth score reduction of three or more . Seventy-three percent -LRB- 22/30 -RRB- of the patients reported a posttreatment reduction in depression ; 53 % had a Beck depression index score reduction of three or more . Eighty-seven percent of the patients reported an increase in the Flemons ' QOL index ; 60 % showed in improvement score of one or greater . All patients reported some level of subjective improvement on at least one of these subjective measures . Fig. 6Pre - and posttreatment changes in Beck depression index , Epworth sleepiness scale , and Flemons ' quality of life indexThe correlation between the posttreatment percentage time snoring above 30 dB and the AHI-4 % was computed because snoring is a principal measure used to assess the MAD titration endpoint . The results in Fig. 7 suggest that snoring is a good predictor of outcome when objectively measured . Fig. 7Regression plot predicting posttreatment AHI-4 % based on the posttreatment percentage of time the snoring loudness was above 30 dBDaily self-reported compliance for the MAD revealed that out of a total of 693 nights in bed recorded across all subjects , only 20 nights were reported as not wearing the MAD . The mean usage per night was 7.34 ± 1.3 h. Three of the 30 patients had incomplete paperwork due to either confusion on the part of the patient or problems in collecting the paperwork at the study sites . Although all three patients said that they had worn the MAD for the duration of the study , there was no written record available that allowed the investigators to assess compliance/usage . Factors that affect treatment outcomes Analysis of variance was used to compare demographic and anthropomorphic measures of patients who had a posttreatment AHI-4 % ≤ 5 and those who did not respond optimally to treatment -LRB- Table 1 -RRB- . Significant differences in the two groups were observed in the body mass index -LRB- p < 0.01 -RRB- , weight -LRB- p < 0.01 -RRB- , and neck circumference -LRB- p < 0.02 -RRB- . Table 1Characteristics of group that was treated optimally and the group that was not treated optionally with a MAD -LRB- mean + SE -RRB- Treated optimally post-T AHI-4 % < 5Did not treat optimally post-T AHI-4 % > 5GenderFemales74Males118Age -LRB- years -RRB- 48.3 + 2.550.9 + 3.4 BMI -LRB- kg/m -RRB- ** 27.6 + 0.833.1 + 2.1 Weight -LRB- kg -RRB- ** 84.4 + 3.0100.2 + 5.0 Neck circumference -LRB- cm -RRB- * 40.0 + 0.743.2 + 1.0 * p < 0.05 ** p < 0.01 Prediction of successful treatment outcome Correlation analysis was applied to the entire data set -LRB- n = 30 -RRB- identify pretreatment variables that had a significant relationship with the posttreatment AHI-4 % -LRB- see Table 2 -RRB- . Table 2Correlations between posttreatment AHI-4 % and pretreatment measuresPearson r -LRB- p < 0.01 -RRB- Pearson r -LRB- p < 0.05 -RRB- Pretreatment 4 % AHIr = 0.53 Snoring > 30 dBr = 0.44 Pretreatment 3 % AHIr = 0.44 Neck circumferencer = 0.45 Pretreatment 1 % AHIr = 0.54 Body mass indexr = 0.45 Snoring > 40 dBr = 0.52 Weightr = 0.45 % Time < 90 % SpO2r = 0.56 Various combinations of variables were submitted to linear regression using the model development data set in an effort to predict the posttreatment AHI-4 % . Limiting the number of variables to four -LRB- due to the small sample size -RRB- , the variables AHI-4 % , AHI-3 % , AHI-1 % , and percent time snoring > 30 dB resulted in an R2 of 0.68 . The coefficients derived from the linear regression were applied to the model development and cross-validation data sets.Twenty-seven of the 30 patients had predict-AHI suggesting a successful outcome would be attained using a clinical cutoff post-T 4 % AHI ≤ 10 -LRB- Fig. 8 -RRB- . Two of the three patients were accurately predicted as unable to achieve a post-T AHI ≤ 10 . The Bland -- Altman plots comparing the predicted-AHI and posttreatment AHI -LRB- Fig. 9 -RRB- suggest a reasonable fit of the data given the small sample size used for the model development and cross validation data sets . Fig. 8Comparison of pretreatment , posttreatment , and predicted posttreatment AHI-4 % for a the model development , and b the cross validation groupsFig . 9Bland -- Altman plot of the difference between the predicted posttreatment AHI-4 % vs the actual posttreatment AHI for a the model development and b the cross validation groups Discussion Consecutive patients who were referred to the dentist for a MAD were provided the opportunity to enroll in the study . The only exclusion criteria applied were ages less than 18 or older than 70 years . The inclusion of 25 subjects who had previously failed CPAP therapy contributed to the wide OSA severity range : nine of the subjects -LRB- 30 % -RRB- were considered to have severe OSA -LRB- i.e. , AHI-4 % ≥ 30 -RRB- and an additional 30 % were considered to have moderate OSA -LRB- AHI-4 % > 15 and < 30 -RRB- . Seventy-seven percent of the patients -LRB- 23/30 -RRB- had at AHI-1 % > 25 , a clinical cut-off which was considered at least moderate . In this study , a treatment efficacy rate for the MAD was 90 % using an AHI-4 % clinical cut-off of 10 , and 97 % when a 50 % reduction in AHI was included in determining a successful outcome . Eighty-seven percent of the patients showed improvement using an AHI-1 % with a clinical cut-off of 15 or a 50 % reduction in AHI . Eighty percent of the patients -LRB- 24/30 -RRB- had a posttreatment AHI-1 % ≤ 16 . A recent evidence-based review summarized results from 87 published studies -LRB- over 2,000 patients evaluated -RRB- including 15 Sackett Level I and Level II randomized controlled trials -LSB- 14 -RSB- . Using a definition of treatment success that required achieving a posttreatment respiratory disturbance index below 10 , success was achieved on average across all studies for 52 % of patients treated with oral appliances . When the more liberal criteria of a 50 % reduction in RDI were applied to assess outcome , 65 % of patients were shown to have a positive result with MAD . In an attempt to compare these previous finding to this data set , a clinical cut-off of 10 was applied to the AHI-1 % . The differences between the AHI-1 % and RDI include a 50 % reduction in flow vs 30 % ; a 1 % desaturation vs no desaturation ; arousal indicators based on changes in snoring , pulse rate , and head movement vs cortical arousals ; and the use of time-in-bed vs total-sleep-time . Using this alternative clinical cut-off , 33 % of the patients had a successful outcome . When a 50 % reduction in AHI-1 % was included , 77 % had a successful outcome . Results are more consistent with the previous research . The findings in this study were consistent with previous reports which found self-reported improvements in quality of life and decrease in sleepiness in the majority of patients -LSB- 14 -RSB- . No effort was made in this study to obtain objective measures of improvement . To date , only four studies have utilized objective measures of alertness or neurocognitive function to assess treatment outcomes with MAD therapy , with mixed results -LSB- 22 -- 25 -RSB- . Further investigations need to be conducted to assess the effectiveness of MAD therapy in ameliorating cognitive impairments in OSA . The strong correlation -LRB- r = 0.63 -RRB- between posttreatment % time snoring > 30 dB and the posttreatment AHI-4 % suggests that the current clinical practice of relying on residual snoring to asses MAD titration is valid . It is interesting to note that there was no apparent correlation between snoring levels and pretreatment AHI values . Given the discrepancy between the self-reported and objectively measured snoring at the titration endpoint , it appears that the bed-partner or patient 's observation may not provide the most accurate means for titrating a MAD . Several studies have reported increased efficacy for MADs in patients with supine positional OSA -LSB- 7 , 26 -RSB- ; however , the association has not been widely confirmed . This is due in part to the fact that the majority of studies on MAD therapy do not report the number of abnormal respiratory events by sleep position despite the recommendation of the ASDA Practice Parameters for separate calculations of supine and lateral RDIs -LSB- 27 , 28 -RSB- . Supine positionality did not appear to influence the treatment outcome in our study , possibly , because there were so few MAD treatment failures . This may be a result of the appliance which was used or that the small sample size was not representative of the population treated with oral appliances . A recent evidence-based review reported that median MAD treatment compliance across patients was 77 % after 1 year -LSB- 14 -RSB- . In this study , compliance was only monitored during the titration period which may have contributed to the favorable finding -LRB- i.e. , 97 % -RRB- . Other factors that may have influenced this outcome were a relatively small sample size and/or patient compensation being provided . Given that 80 % of the study participants were CPAP failures , the influence of prior CPAP use on MAD compliance should be explored . While an objective measure of MAD compliance would eliminate potential bias contributed by self-reported use , practical methods are not currently available . The suggested decrease in MAD efficacy with increasing body mass index -LRB- BMI -RRB- was confirmed -LSB- 27 , 28 -RSB- . Weight and neck circumference also appeared to influence the posttreatment apnea/hypopnea index . These variables make sense : the upper airway tends to be narrower in patients with more fatty tissue around the neck and the additional mass combines with gravity to contribute to greater collapsibility when sleeping supine . The successful treatment outcome of patients with severe sleep apnea suggests that a more quantitative approach should be investigated to identify candidates appropriate for a MAD therapy . The results from the predictive model , once fully validated , could provide the guidance needed for sleep medicine physicians to recommend an oral appliance as an initial treatment option for more severe patients . Alternatively , substantial differences between the predicted and actual posttreatment AHI could help dentists determine when a patient has not been fully titrated . Given the small sample size of the model development data set -LRB- n = 15 -RRB- , only four variables were included in the regression model even though the correlations presented in Table 2 suggested additional variables would be predictive . It is expected that the error between the predicted and actual posttreatment AHI can be reduced with larger data sets . In addition to expanding the database used for the predictive model , future investigations should be conducted to determine if the accuracy of the predictive model is influenced by the type of MAD . Dentists represent an important access point for identifying and treating patients with undiagnosed OSA . This study was designed to demonstrate two models of collaboration between a dental sleep medicine specialist and a sleep medicine physician . As recommended by the AASM , only patients with mild to moderate OSA were offered MAD therapy as the initial treatment choice in this study . Patients with severe sleep apnea were enrolled only after failure of CPAP therapy . In one model , the dentist referred the patient to the sleep medicine physician and his staff to obtain and review the pre - and posttreatment physiological data . In the second model , the dentist acquired the data and transmitted it to the sleep medicine physician for review . In both models , the physical and history was made available to the physician for interpretation of the data -LSB- 29 -RSB- . A follow-up PSG is generally not affordable in cases where it is not covered by an insurance company or health ministry ; sleep centers do not tend to offer less expensive level III studies as an alternative . This creates a situation whereby treatment outcomes are simply not assessed , or the dentist conducts the study independently , without the assistance or oversight of the sleep medicine professional . Left on their own , many dentists rely upon oximetry and other level IV devices to monitor treatment outcomes , in part because of their limited experience in interpreting the more sophisticated signals obtained by level III devices . Although several level III devices have automated scoring algorithms , the physiological data should still be reviewed by a trained professional . In two patients , the review of the full disclosure recording allowed recognition of complex sleep apnea -LRB- i.e. , central sleep apnea was revealed posttreatment after the obstructive breathing was resolved -RRB- suggesting the importance of having an experienced professional review the data . This study suggests an alternative approach for assessing MAD treatment outcomes that is based on a collaborative relationship between dentists and sleep medicine physician using a limited channel recording system .

O O O O O O O O B I I O O B I O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

sleep apnea
sleep disordered breathing
mandibular advancement device


Novel vectors expressing anti-apoptotic protein Bcl-2 to study cell death in Semliki Forest virus-infected cells Semliki Forest virus -LRB- SFV , Alphavirus -RRB- induce rapid shut down of host cell protein synthesis and apoptotic death of infected vertebrate cells . Data on alphavirus-induced apoptosis are controversial . In this study , the anti-apoptotic bcl-2 gene was placed under the control of duplicated subgenomic promoter or different internal ribosome entry sites -LRB- IRES -RRB- and expressed using a novel bicistronic SFV vector . The use of IRES containing vectors resulted in high-level Bcl-2 synthesis during the early stages of infection . Nevertheless , in infected BHK-21 cells translational shutdown was almost complete by 6 h post-infection , which was similar to infection with appropriate control vectors . These results indicate that very early and high-level bcl-2 expression did not have a protective effect against SFV induced shutdown of host cell translation . No apoptotic cells were detected at those time points for any SFV vectors . Furthermore , Bcl-2 expression did not protect BHK-21 or AT3-neo cells at later time points , and infection of BHK-21 or AT3-neo cells with SFV replicon vectors or with wild-type SFV4 did not lead to release of cytochrome c from mitochondria . Taken together , our data suggest that SFV induced death in BHK-21 or AT3-neo cells is not triggered by the intrinsic pathway of apoptosis . 1 Introduction Semliki Forest virus -LRB- SFV -RRB- is a positive-stranded RNA virus in Alphavirus genus -LRB- family Togaviridae -RRB- , a widely distributed group of human and animal pathogens -LRB- Strauss and Strauss , 1994 -RRB- . SFV genomic RNA -LRB- so-called 42S RNA -RRB- is approximately 11.5 kb long and encodes four non-structural proteins designated nsP1 -- 4 ; these are involved in viral RNA synthesis . The remaining proteins form the virus capsid and envelope and are not essential for virus replication . After virus entry , the 42S RNA is translated into a large non-structural polyprotein , which is processed to form an early and subsequently a late replicase complex -LRB- Strauss and Strauss , 1994 -RRB- . The early replicase mediates synthesis of the negative-stranded RNA complementary to the genomic 42S RNA . Minus strands are used by the late replicase as templates for the synthesis of new positive strand 42S RNA , and for transcription of subgenomic mRNAs encoding the structural proteins . The structural genes of SFV are not required for replication and can be removed or replaced with a polylinker and/or with foreign gene sequences . This property forms the basis of the SFV-based replicon vector systems -LRB- Liljestrom and Garoff , 1991 ; Smerdou and Liljestrom , 1999 -RRB- . SFV-based replicon vectors mediate high-level expression of heterologous proteins . However , as with virus , vectors cause shutdown of cellular biosynthesis and induce apoptotic death -LRB- Glasgow et al. , 1997 , 1998 ; Scallan et al. , 1997 -RRB- . This precludes long-term foreign gene expression , and several attempts to reduce the cytotoxicity of alphavirus vectors have been made -LRB- Fazakerley et al. , 2002 ; Lundstrom et al. , 2003 , 2001 ; Perri et al. , 2000 -RRB- . The anti-apoptotic gene bcl-2 is an antagonist of the intrinsic mitochondrial pathway of apoptosis -LRB- for reviews see Ashe and Berry , 2003 ; Cory and Adams , 2002 ; Tsujimoto and Shimizu , 2000 -RRB- . Bcl-2 can prevent release of cytochrome c from mitochondria , thus , precluding the apoptotic cascade -LRB- Kluck et al. , 1997 ; Yang et al. , 1997 -RRB- . Bcl-2 can block apoptosis induced by several viruses , including influenza virus and reovirus -LRB- Nencioni et al. , 2003 ; Rodgers et al. , 1997 -RRB- . Existing data on Bcl-2 in SFV - or Sindbis virus-induced apoptosis are contradictory . On one hand it has been shown that alphavirus-induced apoptosis of baby hamster kidney -LRB- BHK -RRB- cells , Chinese hamster ovary cells , rat insulinoma cells and rat prostatic adenocarcinoma -LRB- AT3 -RRB- cells can be prevented by over-expression of Bcl-2 -LRB- Levine et al. , 1993 ; Lundstrom et al. , 1997 ; Mastrangelo et al. , 2000 ; Scallan et al. , 1997 -RRB- . Similarly , a Sindbis virus expressing Bcl-2 produces reduced encephalitis in infected mice -LRB- Levine et al. , 1996 -RRB- . That Bcl-2 expression can block apoptosis , suggests involvement of intrinsic pathway of apoptosis . In contrast , other studies using rat embryo fibroblasts and monocyte cell lines overexpressing Bcl-2 failed to detect a protective effect against alphavirus-induced apoptosis -LRB- Grandgirard et al. , 1998 ; Murphy et al. , 2001 -RRB- . The aim of this study was to determine whether expression of anti-apoptotic Bcl-2 directly from SFV-based replicon vectors in BHK-21 cells could be used to prolong co-expression of marker proteins from a bicistronic SFV replicon . Using the SFV1 vector system -LRB- Liljestrom and Garoff , 1991 -RRB- , the bcl-2 gene was placed either under the control of a duplicated SFV subgenomic promoter or an internal ribosome entry site -LRB- IRES -RRB- . It is possible that expression of Bcl-2 from the subgenomic promoter occurs too late to prevent cell death . Expression from an IRES element within the genomic RNA should be more rapid . We tested two different IRES elements , the Encephalomyocarditis virus IRES -LRB- EMCV-IRES -RRB- and the crucifer-infecting tobamovirus IRES -LRB- CR-IRES -RRB- . The latter is a 148-nt element , which precedes the CR coat protein gene and displays IRES activity across all kingdoms -LRB- Dorokhov et al. , 2002 -RRB- . Using this novel approach we demonstrate that early Bcl-2 expression does not protect SFV-infected BHK-21 cells from alphavirus-induced translational shutdown or cell death . Moreover , our results indicate that SFV-induced cell death in BHK-21 cells does not involve the release of cytochrome c from mitochondria , and most likely does not occur by the apoptotic intrinsic pathway . 2 Materials and methods 2.1 Plasmid construction The BamHI-XmaI multicloning site of the pSFV1 replicon -LRB- Liljestrom and Garoff , 1991 -RRB- was replaced with a BamHI , ApaI , ClaI , AvrII , NruI , NsiI and XmaI multicloning site ; the resulting construct was designated as pSFV-PL . The spliced sequences encoding the mouse Bcl-2 alpha protein -LRB- locus AAA37282 -RRB- , the EMCV-IRES -LRB- pIRES2-EGFP ; BD Clontech -RRB- and the 148 bp CR-IRES -LRB- Ivanov et al. , 1997 -RRB- were amplified by PCR , cloned and verified by sequence analysis . Each IRES was fused to the Bcl-2 coding sequence and cloned into NsiI-XmaI digested pSFV-PL vector ; obtained constructs were designated as pSFV-EMCV-bcl2 and pSFV-CR-bcl2 . To create constructs expressing Bcl-2 protein from the duplicated subgenomic promoter , the IRES from pSFV-EMCV-bcl2 was replaced by an oligonucleotide duplex representing the minimal SFV subgenomic promoter -LRB- Hertz and Huang , 1992 -RRB- ; the resulting construct was designated pSFV-PR-bcl2 . The d1EGFP reporter gene -LRB- BD Clontech -RRB- was amplified by PCR , sequenced and cloned into pSFV-PL , pSFV-EMCV-bcl2 , pSFV-CR-bcl2 and pSFV-PR-bcl2 vectors treated with ClaI-NsiI . Resulting constructs were designated as pSFV-PL-d1EGFP , pSFV-d1EGFP-EMCV-bcl2 , pSFV-d1EGFP-CR-bcl2 and pSFV-d1EGFP-PR-bcl2 , respectively -LRB- Fig. 1 -RRB- . Sequences and primers are available upon request . To construct SFV replicons expressing mutated chromoprotein HcRed -LRB- from the reef coral Heteractis crispa -RRB- , HcRed was PCR amplified -LRB- from pHcRed1-N1 ; BD Clontech -RRB- , cloned and sequenced . The sequence encoding Bcl-2 from pSFV-d1EGFP-EMCV-bcl2 , pSFV-d1EGFP-CR-bcl2 and pSFV-d1EGFP-PR-bcl2 was replaced with HcRed to give constructs pSFV-d1EGFP-EMCV-HcRed , pSFV-d1EGFP-CR-HcRed and pSFV-d1EGFP-PR-HcRed -LRB- Fig. 1 -RRB- . To obtain constructs used for viability analysis under puromycin selection , the sequence encoding d1EGFP from pSFV-PL-d1EGFP , pSFV-d1EGFP-EMCV-bcl2 , pSFV-d1EGFP-CR-bcl2 and pSFV-d1EGFP-PR-bcl2 was replaced by that of puromycin acetyltransferase -LRB- Pac -RRB- , and constructs were designated pSFV-PL-Pac , pSFV-Pac-EMCV-bcl2 , pSFV-Pac-CR-bcl2 and pSFV-Pac-PR-bcl2 . To generate infectious RNA , constructs were linearised by SpeI digestion and in vitro transcription was carried out as previously described -LRB- Karlsson and Liljestrom , 2003 -RRB- . 2.2 Cells and viruses BHK-21 cells were grown in Glasgow 's Minimal Essential Medium containing 5 % foetal calf serum , 0.3 % tryptose phosphate broth , 0.1 U/ml penicillin and 0.1 μg/ml streptomycin . AT3-neo and AT3-bcl2 cells were grown in Roswell Park Memorial Institute-1640 medium containing 10 % foetal calf serum , 0.1 U/ml penicillin and 0.1 μg/ml streptomycin . All cells were grown at 37 °C in a 5 % CO2 atmosphere . SFV4 was derived from the infectious cDNA clone pSP6-SFV4 -LRB- Liljestrom et al. , 1991 -RRB- . 2.3 Transfection and collection of virus-like particles -LRB- VLPs -RRB- BHK-21 cells were co-transfected with equal amounts of vector and helper RNA -LRB- Liljestrom and Garoff , 1991 -RRB- . Helper RNA encodes the structural proteins under subgenomic promoter . Transfected cells were grown at 28 °C for 72 h and the VLPs collected , concentrated , purified and titrated as described by Karlsson and Liljestrom -LRB- 2003 -RRB- . Although the replicase encoded by the replicon vector will amplify both RNAs , helper RNA is not packed into VLPs due to a missing packaging signal . To determine if any replication-proficient viruses were formed due to the recombination between replicon vector RNA and helper RNA , batches of VLPs were tested as described by Smerdou and Liljestrom -LRB- 1999 -RRB- . All infections with VLPs were carried out in BHK-21 cells at a multiplicity of infections 10 -LRB- moi = 10 -RRB- for 1 h at 37 °C . 2.4 Metabolic labelling BHK-21 cells in 35 mm diameter plates were infected with SFV VLPs as described above . Infected cells were washed twice with PBS , once with methionine and cysteine free Dulbecco 's Modified Eagle Medium followed by 30 min labelling with 50 μCi/ml of -LSB- 35S -RSB- methionine and -LSB- 35S -RSB- cysteine -LRB- RedivuePRO-MIX , Amersham Biosciences -RRB- . After labelling , cells were washed with PBS , lysed in Laemmli buffer and analyzed by SDS-PAGE . Gels were dried under vacuum and exposed to film . 2.5 Immunoblot analysis BHK-21 cells were infected and samples collected as described above . After SDS-PAGE , proteins were transferred to a nitrocellulose membrane , probed with rabbit polyclonal antisera against SFV nsP1 , EGFP -LRB- in-house -RRB- , HcRed -LRB- Clontech -RRB- , with a mouse monoclonal antibody against Bcl-2 -LRB- Santa Cruz Biotechnology , Inc. -RRB- or with a mouse monoclonal antibody against beta-actin -LRB- C4 -RRB- -LRB- Cruz Biotechnology , Inc. -RRB- and visualized by ECL immunoblot detection kit -LRB- Amersham Life Science -RRB- . 2.6 Analysis of bicistronic SFV vector cytotoxicity in BHK-21 cells Cytotoxicity of SFV vectors was analyzed as described by Garmashova et al. , 2006 . Replicon RNA was obtained from pSFV-PL-Pac , pSFV-Pac-EMCV-bcl2 , pSFV-Pac-CR-bcl2 and pSFV-Pac-PR-bcl2 . 106 BHK-21 cells were electroporated with 5 μg of RNA . Cells were seeded into wells -LRB- growth area 2.0 cm2 per well ; Cellstar , Greiner bio-one plates -RRB- and selected with puromycin -LRB- 10 μg/ml -RRB- from 6 h post-transfection . Viable adherent cells were determined at 6 , 24 , 48 and 72 h post-transfection using Trypan blue -LRB- Flow Laboratories -RRB- . The viability of infected cells was also analyzed by WST-1 assay -LRB- Roche -RRB- . The assay is based on the reduction of WST-1 to a water-soluble formazan dye by viable cells . BHK-21 cells were seeded in 96-well plates -LRB- 7 × 103 cells/well -RRB- , grown for 18 h and infected with VLPs containing recombinant replicons at moi = 10 . Control cells were mock-infected . Infected cells were analyzed 6 , 24 or 48 h post-infection -LRB- p.i. -RRB- by adding 10 μl of WST-1 to each well , followed by incubating the plate for 1 h and measuring the change in color intensity at 450 nm in a microplate reader . 2.7 Immunofluorescence microscopy Cells were grown on cover slips and infected for selected times , mock-infected cells were used as controls . Cells were washed with PBS , fixed with 4 % paraformaldehyde for 10 min at room temperature and permeabilized with cold methanol for 7 min at − 20 °C . Cells were then washed with PBS , blocked in the 3 % BSA-PBS and incubated for 1 h with primary antibody -LRB- mouse anti-cytochrome c monoclonal antibody -LRB- BD Pharmingen -RRB- or rabbit polyclonal antibody against SFV nsP1 -RRB- . Then the cells were washed again with PBS and incubated with a AlexaFluor 568 -LRB- Invitrogen -RRB- or Cy3 conjugated secondary antibody for 1 h , washed three times with PBS and air-dried . Staurosporine -LRB- final concentration 0.5 μM -RRB- was added to mock-infected cells 1 -- 2 h before fixing to induce release of cytochrome c from mitochondria . Samples were analyzed on an Olympus U-RFL-TX microscope or a Bio-Rad MRC-1024 confocal microscope . 2.8 Analysis of the viability of AT3 cells infected with SFV VLPs 3 × 106 AT3-neo or AT3-bcl2 cells were infected with VLPs in serum-free RPMI1640 media supplemented with 0.2 % BSA . The amount of VLPs used for infections corresponded to a moi = 20 for BHK-21 cells . At 12 h p.i. , EGFP positive cells were separated using a BD FACSAria cell sorter ; EGFP-positive cells were seeded in 24-well plates . Viable cells were determined at 24 and 48 h p.i. using Trypan blue -LRB- Flow Laboratories -RRB- . 3 Results 3.1 Expression of d1EGFP and Bcl-2 using bicistronic SFV vectors To study the expression of foreign proteins using the bicistronic replicons , BHK-21 cells were infected with SFV-d1EGFP-CR-bcl2 , SFV-d1EGFP-EMCV-bcl2 and SFV-d1EGFP-PR-bcl2 VLPs and in addition , with monocistronic SFV-PL-d1EGFP VLPs . Samples were collected at 2 , 4 , 6 , 8 , 12 and 24 h p.i. and analyzed by immunobloting . Expression of SFV nsP1 was generally detectable by 2 h p.i. and increased up to 8 -- 12 h p.i. -LRB- Fig. 2a -- e -RRB- . The amount of nsP1 was approximately equal for all bicistronic vectors -LRB- Fig. 2b -- d -RRB- . Expression of d1EGFP was also detectable by 2-4 h post-infection -LRB- Fig. 2a -- d -RRB- and was highest in SFV-PL-d1EGFP infected cells -LRB- Fig. 2a -RRB- . Major differences were observed with Bcl-2 expression , which was detected on immunoblots as two bands -LRB- Fig. 2b -- d -RRB- . Bcl-2 expression was strongest and earliest -LRB- 2 h p.i. -RRB- in SFV-d1EGFP-EMCV-bcl2 infected cells -LRB- Fig. 2c -RRB- . In cells infected with SFV-d1EGFP-CR-bcl2 -LRB- Fig. 2b -RRB- , expression of Bcl-2 was detected 4 h p.i. Expression of Bcl-2 was also found in cells infected with SFV-d1EGFP-PR-bcl2 -LRB- Fig. 2d -RRB- although levels were lower compared to IRES containing constructs . To assess the ability of the bicistronic vectors to express proteins in general , Bcl-2 sequences were replaced with non-cytotoxic HcRed . The resulting replicons SFV-d1EGFP-EMCV-HcRed , SFV-d1EGFP-CR-HcRed and pSFV-d1EGFP-PR-HcRed were used to infect BHK-21 cells . There was no significant difference in the expression of nsP1 and d1EGFP proteins between bicistronic vectors also expressing HcRed or Bcl-2 -LRB- Fig. 2e for SFV-d1EGFP-EMCV-HcRed , data not shown for SFV-d1EGFP-CR-HcRed and pSFV-d1EGFP-PR-HcRed -RRB- . Expression of HcRed was strongest for SFV-d1EGFP-EMCV-HcRed -LRB- detectable by Western blotting by 4 h p.i. ; Fig. 2e -RRB- . This is in agreement with previous observations suggesting that the highest expression levels of the second target proteins are achieved with SFV-d1EGFP-EMCV vectors . The apparent delay in detection of protein expression -LRB- 2 h p.i. for Bcl-2 versus 4 h p.i. for HcRed -RRB- is most likely due to the quality of the HcRed antibody -LRB- Clontech -RRB- . Thus , vectors containing IRES elements expressed Bcl-2 earlier and to higher levels . Presence of a duplicated subgenomic promoter or IRES element following the Bcl-2 encoding sequence did not have any major effect on the time-course of infection , however , differences in expression levels of the first marker protein , d1EGFP , were observed . 3.2 Effects of infections by recombinant SFV VLPs on host cell protein synthesis Metabolic labelling was used to study the effects of infection by SFV VLPs on host cell protein synthesis . Infected BHK-21 cells were pulse labelled at 2 , 4 , 6 , 8 , 12 and 24 h p.i. . In contrast to mock-infected cells , where protein synthesis is ongoing -LRB- Fig. 3A -RRB- , translation in infected cells was rapidly inhibited -LRB- Fig. 3B -- E -RRB- . Shutdown of host protein synthesis was detected as early as 4 h p.i. -LRB- Fig. 3B -- E -RRB- . High-level and continuous expression of Bcl-2 was visible in cells infected with SFV-d1EGFP-EMCV-bcl2 -LRB- Fig. 3D -RRB- . In cells infected with other bicistronic vectors , expression of Bcl-2 could only be detected by Western blot -LRB- Fig. 2b -- d -RRB- . These results indicate that high-level , continuous expression of Bcl-2 from a bicistronic replicon vector does not protect against shut-off of host cell protein synthesis . 3.3 Survival of cells infected with Bcl-2 expressing bicistronic replicons It has been shown that similar to wild-type virus , alphavirus based replicon vectors induce apoptotic death of infected vertebrate cells -LRB- Glasgow et al. , 1997 , 1998 ; Scallan et al. , 1997 -RRB- and that over-expression of Bcl-2 can protect cells against alphavirus-induced apoptosis -LRB- Lundstrom et al. , 1997 ; Scallan et al. , 1997 -RRB- . To study the effect of Bcl-2 expression from bicistronic vectors on cell death , BHK-21 cells were transfected with SFV-PL-Pac , SFV-Pac-EMCV-bcl2 , SFV-Pac-CR-bcl2 and SFV-Pac-PR-bcl2 replicons or mock-transfected , and puromycin selection was applied 6 h post-transfection . Almost all mock-transfected cells died within 24 h of adding puromycin -LRB- Fig. 4a -RRB- . The transfected cells survived longer and the percentage of viable adherent cells was found to be similar at the selected time points -LRB- Fig. 4a -RRB- . Most of the transfected cells were dead 3 days post-transfection regardless of the replicon . Thus , our results demonstrate that high-level expression of Bcl-2 does not protect BHK-21 cells against SFV induced death . Similar results were also obtained when Annexin-V PE conjugate or propidium iodide were used to label apoptotic and necrotic cells -LRB- data not shown -RRB- . The WST-1 assay , used to analyze cell viability , measures mitochondrial activity in cells . Results obtained with this assay were similar to cell survival experiments : expression of Bcl-2 from bicistronic vectors -LRB- SFV-d1EGFP-CR-bcl2 , SFV-d1EGFP-EMCV-bcl2 or SFV-d1EGFP-PR-bcl2 -RRB- did not provide any protective effect -LRB- Fig. 4b -RRB- . In fact , viability of cells infected with bicistronic replicons at 48 h p.i. was reduced , compared to cells infected with SFV-PL-d1EGFP -LRB- Fig. 4b -RRB- . Infection with SFV-d1EGFP-EMCV-bcl2 caused the most important reduction in cell viability , which is in accordance with our previous data . -LRB- Fig. 4a -RRB- . This tendency was also seen when cells infected with SFV-d1EGFP-EMCV-HcRed , SFV-d1EGFP-CR-HcRed and SFV-d1EGFP-PR-HcRed VLPs were analyzed by WST-1 assay -LRB- data not shown -RRB- , indicating that not Bcl-2 expression , but rather the second gene expression unit -LRB- especially EMCV-IRES -RRB- is responsible for this effect . Taken together , our results suggest that Bcl-2 expression does not have a detectable effect on the viability of SFV-infected BHK-21 cells . 3.4 SFV infection does not induce release of cytochrome c from mitochondria The anti-apoptotic effect of Bcl-2 is connected to its ability to prevent the release of cytochrome c from mitochondria -LRB- Kluck et al. , 1997 ; Yang et al. , 1997 -RRB- . Apoptosis induced by death receptors or by ER stress bypasses this mitochondrial pathway and is , therefore , relatively insensitive to protection by Bcl-2 -LRB- Scaffidi et al. , 1998 -RRB- . The finding that high-levels of Bcl-2 expression did not have a protective effect against SFV induced death suggests that apoptosis in SFV-infected BHK-21 cells may not involve the mitochondrial pathway . To determine whether the mitochondrial pathway is activated in SFV-infected BHK cells , localization of cytochrome c was visualized by immunofluorescence in BHK-21 cells infected with the SFV-PL-d1EGFP 4 and 24 h p.i. Localization of cytochrome c in mock-infected BHK-21 cells was mitochondrial as evidenced by dotty localization -LRB- Fig. 5A -RRB- . In cells , treated with the non-selective protein kinase inhibitor staurosporine , cytochrome c staining was diffuse , demonstrating its release from mitochondria -LRB- Fig. 5B -RRB- . In cells infected with SFV-PL-d1EGFP , the cytochrome c staining maintained a granular pattern 4 h p.i. -LRB- Fig. 5C and D -RRB- , indicating that when shut-off of cellular gene expression starts cytochrome c was not released from mitochondria . Furthermore , the cytochrome c localization pattern was unchanged even at 24 h p.i. -LRB- Fig. 5E and F -RRB- . We conclude that induction of cell death in SFV replicon-infected BHK-21 cells does not involve the release of mitochondrial cytochrome c . This result is consistent with the absence of a protective effect of Bcl-2 . SFV replicons lack the coding sequences for structural proteins . To analyze the role of the structural proteins in the release of cytochrome c from mitochondria , BHK-21 cells were infected with SFV4 -LRB- moi = 1 -RRB- and localization of cytochrome c was determined at 4 , 12 and 24 h p.i. By 4 h p.i. infected cells did not release cytochrome c -LRB- Fig. 6A and B -RRB- . At 12 and 24 h p.i. detection of cytochrome c became more difficult due to extensive cytopathic effects . This includes cell rounding and re-localization of mitochondria into the perinuclear region -LRB- Fig. 6C -- F -RRB- ; all these effects were more extensive than in SFV-PL-d1EGFP infected cells -LRB- Fig. 5 -RRB- . Nevertheless , the localization pattern of cytochrome c remained dotty indicating that its mitochondrial localization was preserved -LRB- Fig. 6C -- F -RRB- . Taken together , these results indicate that for both replicon vector and SFV4 infection , BHK-21 cells do not release cytochrome c from the mitochondria . 3.5 Bcl-2 expression does not protect SFV-infected AT3-neo cells against cell death To analyze whether the absence of cytochrome c release and lack of the protection against SFV induced cell death are specific to BHK-21 cells , two lines of AT3 cells , AT3-neo -LRB- Cepko et al. , 1984 -RRB- and AT3-Bcl2 -LRB- Levine et al. , 1993 -RRB- cells were also characterized . Firstly , AT3-neo cells were infected with SFV4 and cytochrome c distribution was determined . Infected AT3-neo cells showed extensive virus-induced cytopathic effects but as with BHK-21 cells no detectable release of cytochrome c -LRB- Fig. 7 -RRB- . Similarly , no detectable release of cytochrome c was observed in AT3-neo or AT3-bcl2 cells infected with SFV-d1EGFP-EMCV-bcl2 -LRB- Fig. 8A -- D and I -- L -RRB- or SFV-PL-d1EGFP VLPs -LRB- Fig. 8E -- H and M -- P -RRB- . Therefore , we conclude that the inability of SFV to induce cytochrome c release from mitochondria was not a BHK-21 cell line-restricted phenomenon . Secondly , the viability of the AT3-neo and AT3-bcl2 cells , infected with SFV VLPs was analyzed . It should be noted that we were unable to obtain highly efficient infection of the AT3-neo and especially the AT3-bcl2 cells by use of the SFV VLPs . Typically , up to 20 % of AT3-neo and 10 % of AT3-bcl2 cells were infected when the amount of VLPs , corresponding to moi = 20 in BHK-21 cells , was used for infection ; using larger amounts of VLPs did not result in higher percentages of infected cells . Thus , we conclude that AT3 cells are not as susceptible for SFV infection as BHK-21 cells and sorting of EGFP positive -LRB- i.e. infected -RRB- cells before the viability analysis was required . This revealed that , in the case of AT3-neo cells , expression of Bcl-2 by any SFV replicon vector did not provide any protective effect and almost all infected cells were dead by 48 h p.i. -LRB- Fig. 9a -RRB- . Thus , the results were highly similar to those obtained in BHK-21 cells . In contrast , we found that almost all AT3-bcl2 cells , infected with SFV VLPs , were viable at 24 h p.i. and over the next 24 h numbers of viable cells rapidly increased in all samples . This indicates efficient cell division -LRB- Fig. 9b -RRB- . Since cell sorting ruled out the possibility of non-infected cells being present in analyzed samples , we concluded that AT3-bcl2 cells , infected with SFV replicons were either able to recover from infection or , alternatively , established a persistent infection . Our observation that surviving AT3-bcl2 cells rapidly lost EGFP fluorescence favours the first option . Expression of Bcl-2 by SFV replicons had little or no effect on survival and subsequent division of infected AT3-bcl2 cells . All results taken together we conclude that not bcl-2 expression as such but some other property of AT3-bcl2 cells is likely responsible for this phenomenon . 4 Discussion Previously published data on mechanisms of alphavirus-induced cell death are controversial -LRB- Glasgow et al. , 1997 ; Griffin , 2005 ; Li and Stollar , 2004 -RRB- . On the one hand , induction of apoptotic cell death via the death receptor pathway has been suggested -LRB- Li and Stollar , 2004 ; Nava et al. , 1998 -RRB- ; on the other hand , it has been shown that over-expression of the anti-apoptotic Bcl-2 can protect against alphavirus-induced apoptosis and pathogenesis -LRB- Levine et al. , 1993 ; Lundstrom et al. , 1997 ; Mastrangelo et al. , 2000 ; Scallan et al. , 1997 -RRB- . However , in these studies different alphaviruses , strains , cell lines and experimental systems were used . It is possible that mechanisms , by which cell death is induced may differ between viruses , cells etc. . In this study , shut-off of cellular translation and cell death in SFV-infected BHK-21 cells were studied using a novel bicistronic replicon vector system . This avoids transient or stable expression of Bcl-2 in host cells . In transiently transfected cells , cell metabolism can be seriously altered by the transfection procedure -LRB- Lepik et al. , 2003 -RRB- . Stable expression of Bcl-2 , known to have oncogenic properties -LRB- Reed , 1994 ; Reed et al. , 1991 -RRB- , may lead to adaptation of the cell , which in turn might affect virus replication and the outcome of infection . It is also important to mention that both transient and stable cellular expression are mediated by cellular RNA polymerase II and are , therefore , subjected to the alphavirus-induced transcriptional shutdown , which starts early in infection . In this study , Bcl-2 was expressed using novel vectors containing IRES elements . The use of IRESs has significant advantages over other systems . The first important advantage , as shown by our results -LRB- Fig. 2 -RRB- , is that IRES-mediated expression of Bcl-2 can take place directly from incoming genomic RNA . In contrast , expression from the subgenomic promoter requires replication and is activated later on in infection . Early expression of Bcl-2 may be crucial since it is not known when and how cell death is induced . It is possible that expression of Bcl-2 from the subgenomic promoter is too late to protect from cell death . Another important advantage of IRES-mediated expression is the level of expression , which is significantly higher than from the minimal subgenomic promoter -LRB- Fig. 2 -RRB- . It is also important to mention that at least for the EMCV-IRES , synthesis of Bcl-2 was resistant to inhibition of cellular translation -LRB- Fig. 3D -RRB- . Detection of Bcl-2 protein by immunoblotting in BHK-21 cells infected with VLPs of SFV1 vectors harboring bcl-2 revealed a lower molecular band , which increased in intensity proportional to Bcl-2 expression . This is presumably protein expressed from an internal initiation site -LRB- Tsujimoto and Croce , 1986 -RRB- . The truncated protein could also be the product of Bcl-2 cleavage by endogenous caspase-3 , resulting in curtailed Bcl-2 with proapoptotic activity -LRB- Cheng et al. , 1997 ; Kirsch et al. , 1999 -RRB- . The latter localizes to mitochondria and causes the release of cytochrome c , thus , promoting further caspase activation as a part of a positive feedback loop -LRB- Kirsch et al. , 1999 -RRB- . It has been shown that alphaviruses induce apoptosis in Bcl-2-overexpressing cells by caspase-mediated proteolytic inactivation of Bcl-2 -LRB- Grandgirard et al. , 1998 -RRB- . Viral capsid protein was suggested to trigger activation of the cell death machinery . However , in this study neither release of cytochrome c nor increase in the proportion of truncated product due to feedback loop action was observed . Infection of cells with VLPs eliminated the possibility that capsid protein alone triggers apoptosis . Simultaneous expression of two target proteins was achieved with all the bicistronic SFV expression vectors constructed in this study . The expression level of d1EGFP was slightly reduced for bicistronic vectors , compared to the monocistronic SFV-PL-d1EGFP , independent of the nature of the second gene -LRB- bcl-2 or HcRed -RRB- -LRB- Fig. 2 -RRB- . Interference between the native subgenomic promoter and the duplicated promoter or IRES element may have influenced expression from the first promoter . It is also possible that presence of the IRES elements had some effect on replication . However , infectivity of bicistronic vectors was not altered . Taken together , these results indicate that different IRES elements can be used to construct novel and efficient bicistronic SFV vectors . In this study , we show that early and high-level of Bcl-2 expression , achieved with bicistronic SFV replicon vectors , did not have a detectable effect on host protein synthesis shut-off -LRB- Fig. 3 -RRB- or cell death -LRB- Fig. 4 -RRB- . This can not be attributed to the delayed expression of bcl-2 since its high expression levels were observed at early time points -LRB- Fig. 2 -RRB- . These findings are coherent with our data suggesting that infection of BHK-21 cells with SFV replicons or with SFV4 does not cause the release of cytochrome c from mitochondria -LRB- Figs. 5 and 6 -RRB- . This effect was not restricted to BHK-21 cells since similar results were also obtained for AT3-neo -LRB- Figs. 7 and 8 -RRB- and AT3-bcl2 -LRB- Fig. 8 -RRB- cells . The release of cytochrome c in SFV-infected BHK-21 or AT3-neo cells did not even occur at late time points . This explains the lack of protective effect by Bcl-2 expression against cell death in infected cells , as observed by us . At the same time , the mechanism of cell death in infected cells remains unknown . We can rule out that binding of virions to the cellular receptor and/or virus internalisation have crucial roles , since induction of cell death does not depend on whether the cells are infected with virus or transfected by infectious transcripts . It seems most likely that cell death is induced by some non-mitochondrial pathway . This is consistent with previous data , which suggested that apoptosis in alphavirus infected cells uses the death receptor pathway -LRB- Nava et al. , 1998 ; Li and Stollar , 2004 -RRB- . However , our findings do not exclude the possibility that cell death in SFV-infected BHK-21 cells may be triggered by the ER pathway . A delay of cell death in SFV-infected AT3 cell line expressing bcl-2 has been reported -LRB- Scallan et al. , 1997 -RRB- . Our results confirm that the AT3-bcl2 cell line is remarkably resistant to infection by SFV VLPs . However , our data is more coherent with the hypothesis that these cells are not just delaying cell death but are also capable to recover from SFV infection . The finding that Bcl-2 expression by SFV replicons did not have any effect on the survival of AT3-neo cells suggests that resistance of the AT3-bcl2 cells to the SFV infection most likely represents an indirect effect of Bcl-2 . The Bcl-2 gene is potent oncogene and its constitutive expression significantly changes cell cycle and gene expression . It is indeed evident from data presented in Fig. 9 that AT3-bcl2 cells grow much more rapidly than AT3-neo cells . It could be hypothesized that constitutive expression of Bcl-2 in AT3-bcl2 cells may have resulted in changes protecting cells from virus infection . The most likely candidates for roles of these factors may be the components of the innate immune system and/or host factor -LRB- s -RRB- like rat zinc-finger antiviral protein , which provides resistance against alphavirus infection -LRB- Bick et al. , 2003 -RRB- .

O O O O O O O O B I O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

alphaviruses
semliki forest virus
cell death
at3