Recent data have suggested that patient admission during intensive care unit (ICU) morning bedside rounds is associated with less favorable outcome. We undertook the present study to explore the association between morning round-time ICU admissions and hospital mortality in a large Canadian health region.A multi-center retrospective cohort study was performed at five hospitals in Edmonton, Canada, between July 2002 and December 2009. Round-time ICU admission was defined as occurring between 8 and 11:59 a.m. Multivariable logistic regression analysis was used to explore the association between round-time admission and outcome.P < 0.004) and from the ward compared with the emergency department (ED) or operating theater . Round-time admissions were more often medical than surgical , had more comorbid illness  and higher APACHE II score , and were more likely to have a primary diagnosis of respiratory failure  or sepsis . Crude ICU mortality  and hospital mortality  were higher for round-time compared with non-round-time admissions. In multi-variable analysis, round-time admission was associated with increased ICU mortality  but was not significantly associated with hospital mortality . In the subgroup admitted from the ED, round-time admission showed significantly higher ICU mortality  and a trend for higher hospital mortality .Of 18,857 unique ICU admissions, 2,055 (10.9%) occurred during round time. Round-time admissions were more frequent in community hospitals compared with tertiary hospitals (12.0% vs. 10.5%; odds ratio [OR] 1.16; 95% CI, 1.05-1.29, Approximately 1 in 10 patients is admitted during morning rounds. These patients are more commonly admitted from the ward and are burdened by comorbidities, are non-operative, and have higher illness severity. These patients admitted during morning rounds have higher observed ICU mortality but no difference in hospital mortality. Bedside clinical rounds are an integral component for both patient care and medical education. Intensivists in academic medical centers and many metropolitan/community-based hospitals often must balance evidence-based and high-quality patient care with appropriate supervision of trainees, including medical students, residents, and fellows.The acuity of illness in the first few hours after patient admission to the intensive care unit (ICU) is most often used to prognosticate the clinical course on the basis of the premise that these patients are most physiologically unstable during this period . This reRecently, in a retrospective cohort study, Afessa and colleagues  found anAccordingly, to further explore the hypothesis that ICU admission during morning rounds portends risk of worse clinical outcome, we performed a multi-center cohort study at five teaching hospitals in a large Canadian health region. The objective was to investigate the effect of round-time ICU admissions on ICU and hospital mortality in critically ill patients.The study was approved by the Health Research Ethics Board at the University of Alberta prior to commencement. The requirement for individual informed consent was waived. The reporting of this study follows the STROBE  guideline .This was a multi-center retrospective observational cohort study performed at five teaching-affiliated hospitals in Edmonton, Canada, between 1 July 2002 and 31 December 2009. Edmonton has an estimated population of 730,000 and a metropolitan population of 1,034,945. The three metropolitan/community hospitals are the Grey Nuns Community Hospital (GNH) (eight beds and one daytime/one night-time intensivist), Misericordia Community Hospital (six beds and one daytime/one night-time intensivist), and Sturgeon General Hospital (six beds and one daytime/one night-time intensivist). These three hospitals all have mixed medical/surgical ICUs. The GNH is the regional referral center for all major vascular surgery. The two tertiary hospitals are the Royal Alexandria Hospital (RAH) (22 beds and two daytime intensivist teams and one night-time intensivist) and University of Alberta Hospital (UAH) (30 beds and three daytime intensivist teams and one night-time intensivist). Both of these tertiary hospitals have mixed medical/surgical/trauma ICUs. The RAH is a level II trauma center, and the UAH is a level I trauma center and the regional referral center for all non-cardiac solid-organ transplantation. Bedside morning rounds in each ICU were attended to by one or more consultant intensivists, residents, and/or bedside clinicians and by a multi-disciplinary team consisting of registered nurses, respiratory therapists, pharmacists, dieticians, and physiotherapists. Critical care fellows rotate between the ICUs, and the majority of their rotations occur at the two tertiary hospital sites.All consecutive adult (at least 18 years old) patients admitted to one of the five hospitals' participating ICUs were eligible. If a patient had multiple ICU admissions during a single hospitalization, only the first admission was included for analysis. Patients discharged from the ICU less than 24 hours after admission and those with missing data on vital status were excluded.Round-time admission was defined as an ICU admission occurring between 8 and 11:59 a.m., corresponding to the routine time during which multi-disciplinary and teaching bedside clinical rounds are performed at all participating ICUs. Non-round-time admission was defined as an ICU admission occurring between noon and 7:59 a.m. ICU admission source was classified as emergency department (ED), operating room/emergency post-operative status, operating room/elective post-operative status, transfer from other institutions, or in-hospital ward transfer. Severity of illness was defined according to the Acute Physiology and Chronic Health Evaluation II (APACHE II) score . HepaticWe used an ICU-specific clinical/administrative database maintained by the regional Division of Critical Care Medicine, termed the Minimal Data Set database, which routinely captures demographic, diagnostic, clinical, physiologic, and outcome data for all ICU admissions to the five participating hospitals. We extracted data on demographics, ICU admission source/time, post-operative status, comorbidities, admission diagnoses, necessity for invasive mechanical ventilation, APACHE II score, ICU and hospital duration of stay, and ICU and hospital mortality.t test. Non-normally distributed continuous data are reported as medians and interquartile ranges and were compared by Mann-Whitney U test. Categorical variables were compared by using the chi-squared test. Separate customized multi-variable logistic regression models with ICU or hospital mortality as dependent variable and round-time admission as an independent variable were created and were adjusted for demographics, comorbidity, APACHE II score, use of mechanical ventilation, surgical status, admission source, and primary diagnostic category, study year, and center. Data are reported as odds ratios (ORs) with 95% confidence intervals (CIs). Data were evaluated for multi-colinearity. Model calibration and fit were assessed by the area under the receiver operating characteristic curve (AUR ROC) and the Pearson goodness-of-fit (GoF) test, respectively. All statistical analyses were two-sided, and a P value of less than 0.05 was considered significant. Statistical analyses were conducted by using Intercooled Stata Release 10 .The primary exposure of interest was time of ICU admission (round-time versus non-round-time). The primary outcome measure was hospital mortality. The secondary outcome measures were ICU mortality and ICU and hospital lengths of stay. Continuous normally or near-normally distributed data are reported as means with standard deviations and compared by Student P = 0.004).There were 24,829 ICU admissions during the study period. After exclusion of 1,732 (7.0%) because of repeat admissions, 3,841 (15.5%) for an ICU stay of less than 24 hours, and 399 (1.6%) because of missing data on vital status, the study cohort consisted of 18,857 unique ICU admissions (75.9%). Of these, 2,055 (10.9%) occurred during round time, from 8 to 11:59 a.m., and 16,802 (89.1%) during non-round time, from noon to 7:59 p.m. The two tertiary hospitals accounted for the majority of admissions (73.4%) during the study period (Table P < 0.0001) (Table P < 0.0001). Round-time patients also had greater comorbid illness , and those with any comorbidity were more likely to have a higher number of conditions . Specifically, round-time patients had higher prevalences of hematologic malignancies (P = 0.005), chronic kidney disease (P = 0.04), and immunosuppression (P = 0.002) when compared with non-round-time admissions. Acuity of illness was higher for round-time admissions compared with non-round-time . Round-time admissions were more likely to have primary diagnoses of respiratory failure  and sepsis  and were less likely to have diagnoses of cardiovascular  or gastrointestinal  failure in comparison with non-round-time admissions, respectively.Patients admitted at round time were more likely to be admitted from the hospital ward when compared with the ED or operating theater  and hospital mortality  were significantly greater for ICU admissions occurring during round time compared with non-round time  but was not significantly associated with hospital mortality  (Tables P = 0.035).After covariate adjustment, round-time admission remained associated with higher odds of ICU death  Tables  and 5. TP = 0.009) when compared with the tertiary ICU subgroup  and a trend toward higher hospital death  for round-time admission compared with non-round-time admission  and hospital death  relative to other years for round-time compared with non-round-time admission  and were characterized by a higher burden of comorbid illness and greater illness severity. Likewise, we found that the distribution of admission diagnoses differed; patients arriving during morning rounds were more likely to have respiratory failure and septic diagnoses. Although we showed that patients admitted during morning rounds had higher crude ICU and hospital mortality, the association with ICU mortality remained after adjustment in multi-variable analysis; however, there was no significant association with hospital mortality. Finally, in additional sensitivity analyses, we found a higher mortality associated with admissions occurring during morning rounds for those occurring in community hospitals and for patients referred from the ED.A number of observational studies have evaluated the association between time of ICU admission and hospital mortality and have shown inconsistent findings -17. In dP < 0.001). The investigators were also able to show significant declines in mortality for round-time admissions over the course of the study (21.3% in 1994/1995 to 13.6% in 2006/2007) and an association with modifications to the ICU organizational model .To date, only one large retrospective study has investigated an association between ICU admission during morning rounds and mortality: Afessa and colleagues  examinedWe believe that our data extend the findings of this study, in particular by enabling the evaluation of multiple hospital ICUs, including community-based teaching hospitals, and more extensive covariate adjustment. Our data suggest that ICU admission during morning rounds portends an increased risk for ICU death, and while no statistical difference for in-hospital mortality, certainly fails to suggest that admission during this time leads to improved clinical outcomes, when theoretically all available staff  are present in the ICU.P = 0.004). This was furthermore associated with an adjusted ICU mortality that was 44% higher and a non-significant increase in hospital mortality, despite comparable patient characteristics and illness severity.We believe that the observed differences between community and tertiary hospital ICUs with respect to morning round-time admissions are novel. We observed the proportion of total ICU admissions during round time to be higher in the community as compared with those of tertiary hospital ICUs  and greater hospital mortality .In addition, we found that ICU admission from the ED during morning rounds was associated with greater ICU mortality and a trend for greater hospital mortality. This generates speculation about the influence of system-level factors associated with delays in transfer of critically ill patients from the ED, such as ICU capacity/bed availability, organizational structure, and/or personnel/staffing models, whereby selected patients may be 'held' in the ED during 'after hours' until an ICU bed is available the following morning. In a large observational study using the Project IMPACT database, Chalfin and colleagues  found thOur study has several limitations. First, although our study is large and multi-centric and represents all ICUs in a large Canadian health region, it is a retrospective analysis of prospectively collected data and is observational and thus is potentially predisposed to bias and residual confounding. However, the evaluation of health services, such as how the timing of admission to the ICU impacts patient course and outcomes, cannot feasibly be evaluated in a clinical trial setting. Accordingly, we believe that there is value in replicating observational studies of this nature to assess for consistency and generalizability across studies. Second, despite our adjustment of those variables available, we recognize that we may have omitted additional variables of prognostic importance. Third, we estimated that the routine time during which morning bedside rounds occur at all sites was between 8 and 11:59 a.m. and further recognize that this may have varied at times between and within sites during the study period and depended on additional unrecorded intangible factors.We believe that these observations suggest that system-level factors  in addition to patient-level factors  can have an important modifying effect on the outcome for critically ill patients. However, although our study adds to our current understanding of these patient-system interactions, further confirmatory studies that focus on the relative timing of admission in community/metropolitan hospitals, the impact of delayed transfers from the ED to the ICU, and their interaction with ICU organizational structure and capacity are needed.In summary, patients admitted during morning rounds have important differences in baseline characteristics, disposition, and illness severity in comparison with those admitted during other periods of the day. Moreover, in comparison with the latter, the former have a significantly higher ICU mortality but have similar hospital mortality. Importantly, ICU admission during morning rounds was associated with higher mortality in community hospitals and in patients referred from the ED. These data argue for a need to critically evaluate the patient-system factors associated with ICU admission, such as those that occur during morning bedside rounds and that have the potential to impact patient outcome.• Approximately 1 in 10 intensive care unit (ICU) admissions occurs during morning bedside clinical rounds.• Patients admitted during morning rounds had higher likelihoods to be transferred from the ward and to be medical (non-operative) and were characterized by a higher burden of comorbid illness and greater illness severity.• Patients admitted during morning rounds had higher crude and adjusted ICU mortality.• There was no significant association between morning rounds and hospital mortality after adjustment in multi-variable analysis.• We found a higher mortality associated with admissions occurring during morning rounds for those occurring in community hospitals and for patients referred from the emergency department.APACHE II: Acute Physiology and Chronic Health Evaluation II; AUR ROC: area under the receiver operating characteristic curve; CI: confidence interval; ED: emergency department; GNH: Grey Nuns Community Hospital; GoF: goodness-of-fit; ICU: intensive care unit; OR: odds ratio; RAH: Royal Alexandria Hospital; UAH: University of Alberta Hospital.The authors declare that they have no competing interests.IS participated in the design of the study, performed statistical analysis, and drafted the manuscript. CK and RG participated in the design of the study and helped draft the manuscript. SB conceived the study, participated in its design and coordination, performed statistical analysis, and helped to draft the manuscript. All authors read and approved the final manuscript.Sensitivity Analysis. Multiple variable logistic regression analysis showing the association of ICU death with round-time/non-round-time admission, APACHE II score, age, burden of comorbidities, mechanical ventilation at admission, source of admission, study year and admission diagnosis stratified by study site.Click here for fileSensitivity Analysis. Multiple variable logistic regression analysis showing the association of hospital death with round-time/non-round-time admission, APACHE II score, age, burden of comorbidities, mechanical ventilation at admission, source of admission, study year and admission diagnosis stratified by study site.Click here for fileSensitivity Analysis. Multiple variable logistic regression analysis showing the association of ICU and hospital death with round-time/non-round-time admission, APACHE II score, age, burden of comorbidities, mechanical ventilation at admission, study year, study site and admission diagnosis among patients referred from the Emergency Department.Click here for fileSensitivity Analysis. Multiple variable logistic regression analysis showing the association of ICU and hospital death with round-time/non-round-time admission, APACHE II score, age, burden of comorbidities, mechanical ventilation at admission, source of admission, study site and admission diagnosis among patients admitted during years 2006/2007.Click here for file


Apolipoprotein (Apo) levels are considered more reliable than plasma lipoprotein levels for predicting coronary artery disease (CAD). However, a unanimous Apo marker for CAD has not been identified. In the Chin-Shan Community Cardiovascular Cohort (CCCC), we sought to identify a common Apo marker for predicting CAD in the general population.We examined the cross-sectional association between Apo markers and CAD in the CCCC from 1990 to 2001. Among 3,602 subjects, 90 had angiographically proven CAD (>50% stenosis in ≥1 vessel), and 200 did not have CAD. These subjects were divided into the following 4 groups for analysis: normolipidemic , hypertriglyceridemic , hypercholesterolemic , and hyperlipidemic .P=0.0018), the hypertriglyceridemic group (P=0.0001), the hypercholesterolemic group (P=0.0001), and the hyperlipidemic group (P=0.0001). Overall, a high HDL-ApoCIII/VLDL-ApoCIII ratio was observed in all CAD patients, including those with a normal lipid profile. In multivariate analyses, the HDL-ApoCIII/VLDL-ApoCIII ratio was the strongest predictor for CAD among all lipid factors investigated .Compatible with findings in other populations, our results showed that CAD patients in the CCCC had higher ApoB and lower high-density lipoprotein (HDL) cholesterol and ApoAI concentrations than non-CAD subjects, but the differences were not significant in all groups. Plasma concentrations of ApoE and lipoprotein (a) were not consistently correlated with CAD. In contrast, the ratio of HDL-ApoCIII to very-low-density lipoprotein (VLDL)-ApoCIII was the only universal determinant for CAD in the normolipidemic group (A high HDL-ApoCIII to VLDL-ApoCIII ratio is a better marker for predicting CAD than are the conventional lipid markers or ApoAI and ApoB. High HDL-ApoCIII and low VLDL-ApoCIII values in CAD, irrespective of lipid variations, suggest that ApoCIII is markedly transported from VLDL to HDL in this disease. Measurement of plasma ApoCIII may improve CAD prediction in the general population. Dyslipidemia, which includes various lipid and lipoprotein abnormalities, is closely related to coronary artery disease (CAD) . ConventClinical and pathobiologic studies including the Apolipoprotein-related Mortality Risk study have shown that a high ApoB/ApoAI ratio is related to increased incidence of myocardial infarction and stroke ,5. RecenTo date, an Apo marker that is consistently associated with CAD in the general population has not been described. To identify such a marker and risk factors for CAD, we performed a cross-sectional study in the community-based Chin-Shan Community Cardiovascular Cohort (CCCC) and analyzed the correlation between CAD and various lipid and Apo abnormalities.All patients provided informed consent, and the study protocol conforms to the Declaration of Helsinki guidelines. The institutional review board of the National Taiwan University Hospital approved this study . This isAmong the CCCC study individuals, 90 (72 men and 18 women) with CAD were identified. CAD was defined as angiographically proven stenosis (>50%) of at least 1 major epicardial coronary artery. The non-CAD group comprised 200 subjects (96 men and 104 women) who were CCCC study individuals referred to the National Taiwan University Hospital for cardiac examinations and showed no clinical, electrocardiographic, or angiographic manifestations of CAD. All subjects underwent measurement of plasma lipid levels ≥3 times; none received lipid-lowering agents within 12 weeks before blood sampling. Cardiovascular risk factors were documented.g at 4°C for 15 min. Fresh plasma samples were used for lipid and Apo analysis and for lipoprotein preparations. VLDL (density <1.006 kg/L) and HDL (density = 1.063–1.21 kg/L) fractions were isolated by sequential ultracentrifugation by using a Beckman 50.4 Ti Rotor Assembly  [Venous blood was collected from subjects after overnight fasting. Plasma from each sample was obtained by centrifuging at 300 × CA, USA) . The comTC and TG concentrations were measured enzymatically in an Eppendorf Epose 5060 autoanalyzer  by using commercial kits  . LipoproP-value <0.05 was considered statistically significant.The natural logarithm of Lp(a) levels was used to normalize their right-skewed distribution. After forced entry of Apo covariables, multivariate logistic regression analysis was used to determine adjusted odds ratios (ORs) and 95% confidence intervals (CIs). Receiver operating characteristic (ROC) curve analysis was performed to compare the ability of different lipid factors to discriminate between subjects with CAD and those without CAD. All analyses were performed by using SAS, version 6.11 . A P=0.233). Mean lipid and Apo values for CAD and non-CAD groups are shown in Table The mean age of CAD patients and non-CAD subjects was 57.4±10.9 years and 53.4±10.7 years, respectively , hypertriglyceridemic , hypercholesterolemic HC; TC, ≥200 mg/dL), and hyperlipidemic . Although plasma TG levels in the NL, HTG, and HC groups were higher in CAD patients than in non-CAD subjects, the differences were not significant. However, TG levels in the HLP group were significantly lower in CAD patients than in non-CAD patients  levels were significantly higher in the NL group of CAD patients than of non-CAD subjects. In addition, plasma Lp(a) levels were significantly lower in the HC group of CAD patients than of non-CAD subjects. No significant difference in plasma Lp(a) levels was observed between CAD patients and non-CAD subjects in the HTG and HLP groups Table . Plasma P=0.002–0.0001; Table Because ApoCIII and ApoE are transferable between VLDL and HDL, they may play different roles in CAD development ; therefoP <0.001).To assess the association between CAD and various lipid and Apo parameters, we performed multivariate logistic regression analyses by forcing entry of HDL-ApoCIII, VLDL-ApoCIII, and other covariables that were significantly different between groups in the univariate analysis. Results of the analyses Table  showed tIn our study, HDL-ApoCIII was positively associated with the presence of CAD, and VLDL-ApoCIII was inversely associated with the presence of CAD. We further identified the HDL-ApoCIII/VLDL-ApoCIII ratio to be a predictor of CAD. Adjustments for age and sex increased the ratio’s predictive potential. To our knowledge, we are the first to report that the ApoCIII/VLDL-ApoCIII ratio is a unanimous predictor of CAD, irrespective of lipidemic phenotype. We demonstrate that, regardless of TG and TC levels, a high HDL-ApoCIII/VLDL-ApoCIII ratio suggests the presence of CAD, and a low HDL-ApoCIII/VLDL-ApoCIII ratio suggests the absence of CAD Figure . CompareThe Cholesterol and Recurrent Events trial showed that plasma VLDL-ApoCIII concentration and hypertriglyceridemia are positively associated with the incidence of cardiovascular disease . In diabAlthough LDL-C and HDL-C are the recommended lipid variables in the international guidelines for the treatment of CAD , we haveIn our study, the HDL-ApoCIII/VLDL-ApoCIII ratio was the strongest predictor of CAD in the general population and represents an accurate measure of atherogenic HDL-ApoCIII and VLDL-ApoCIII particles rather than a measure of lipids levels per particle. This ratio may help to identify individuals at risk of angiographically significant CAD, irrespective of lipid concentrations, and it may also be useful in clinical practice for routinely assessing CAD risk in the general population and for making decisions about diet and pharmacologic treatment. Importantly, the assays for analyzing the HDL-ApoCIII/VLDL-ApoCIII ratio are automated, accurate, and inexpensive, making this parameter applicable for routine use in the clinical setting.Notably, numerous results of CCCC-based studies are in accordance with findings in other ethnic populations ,16,27. TBecause of the lack of longitudinal assessment of biochemical markers in our study, we could not account for the effects of serial changes in the HDL-ApoCIII/VLDL-ApoCIII ratio that may be associated with the incidence of cardiovascular events. In addition, we did not obtain complete information regarding the study subjects’ medical therapy, preventing us from analyzing the influence of medications on ApoCIII concentration. Finally, the lipid cutoff value defined in our study has not been tested in an independent data set or prospectively validated.The HDL-ApoCIII/VLDL-ApoCIII ratio is a better predictor of CAD risk than are the conventional lipid factors, even though levels of plasma lipids ApoAI and ApoB remain important predictors for CAD. In addition to its potential clinical importance, high levels of ApoCIII-containing HDL may represent a class of dysfunctional HDL—a topic that has gained much attention in atherogenesis research ,30.Apo: Apolipoprotein; AUC: Area under the curve; CAD: Coronary artery disease; CCCC: Chin-Shan Community Cardiovascular Cohort; CI: Confidence interval; HC: Hypercholesterolemic; HDL-C: High-density lipoprotein cholesterol; HLP: Hyperlipidemic; HTG: Hypertriglyceridemic; LDL-C: Low-density lipoprotein cholesterol; Lp(a): Lipoprotein (a); NL: Normolipidemic; OR: Odds ratio; ROC: Receiver operating characteristic; TC: Total cholesterol; TG: Triglyceride; VLDL: Very-low-density lipoprotein.The authors declare that they have no competing interests.All authors made substantial contributions to the conception and design of the study and manuscript, contributing to the drafting of the article or revising it critically for important contents, and all gave final approval of the version to be published.Lipid and apolipoprotein parameters (stratified by sex) in non-CAD and CAD subjects.Click here for fileCorrelation of lipid and apolipoprotein parameters with CAD (stratified by sex) in various lipidemic groups.Click here for fileCorrelation of VLDL- and HDL-associated ApoE and ApoCIII with CAD (stratified by sex) in various lipidemic groups.Click here for file


Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis, which is the most common neoplastic disease of cattle. BLV infects cattle worldwide, imposing a severe economic impact on the dairy cattle industry. Recently, we developed a new quantitative real-time polymerase chain reaction (PCR) method using Coordination of Common Motifs (CoCoMo) primers to measure the proviral load of known and novel BLV variants in BLV-infected animals. Indeed, the assay was highly effective in detecting BLV in cattle from a range of international locations. This assay enabled us to demonstrate that proviral load correlates not only with BLV infection capacity as assessed by syncytium formation, but also with BLV disease progression. In this study, we compared the sensitivity of our BLV-CoCoMo-qPCR method for detecting BLV proviruses with the sensitivities of two real-time PCR systems, and also determined the differences of proviral load with serotests.et al. and the commercial TaKaRa cycleave PCR system. The BLV copy number determined by BLV-CoCoMo-qPCR was only partially correlated with the positive rate for anti-BLV antibody as determined by the enzyme-linked immunosorbent assay, passive hemagglutination reaction, or agar gel immunodiffusion. This result indicates that, although serotests are widely used for the diagnosis of BLV infection, it is difficult to detect BLV infection with confidence by using serological tests alone. Two cattle were experimentally infected with BLV. The kinetics of the provirus did not precisely correlate with the change in anti-BLV antibody production. Moreover, both reactions were different in cattle that carried different bovine leukocyte antigen (BoLA)-DRB3 genotypes.BLV-CoCoMo-qPCR was found to be highly sensitive when compared with the real-time PCR-based TaqMan MGB assay developed by Lew Our results suggest that the quantitative measurement of proviral load by BLV-CoCoMo-qPCR is useful tool for evaluating the progression of BLV-induced disease. BLV-CoCoMo-qPCR allows us to monitor the spread of BLV infection in different viewpoint compared with classical serotest. Bovine leukemia virus (BLV) is associated with enzootic bovine leucosis (EBL) , which iBLV is closely related to human T-cell leukemia virus types 1 and 2 (HTLV-1 and −2), which are associated with adult T-cell leukemia (ATL) and with the chronic neurological disorder tropical spastic paraparesis/HTLV-1-associated myelopathy -7. By coThe above findings suggest that the BLV provirus remains integrated in cellular genomes ,10, eventax region, which is present at only one copy per provirus, and encodes a transactivator protein Tax. Lew et al./[(absorbance of antigen existence and positive control added well)-(absorbance of antigen absence and positive control added well)]. The dilution ratio of PHA indicates the observed limit point of hemagglutination.BoLA-DRB3 alleles were typed by the PCR-sequence based typing (SBT) method [BoLA-DRB3 exon 2 was amplified by the DRB3FRW and DRB3REV primer set by single-step PCR, and the nucleotide sequences were subsequently determined. Sequence data were analyzed by ASSIGN 400 ATF software , and both BoLA-DRB3 alleles of the cattle were determined.) method . In brieA comparison of the sensitivity and the reproducibility of BLV-CoCoMo-qPCR to those of other TaqMan real-time PCR methods for the detection of the BLV provirus.et al.[We compared the sensitivity and the reproducibility of the BLV-CoCoMo-qPCR method with those of two real-time PCR systems for BLV provirus detection: the TaqMan MGB assay developed by Lew et al., and theet al. , but BLV-CoCoMo-qPCR (100%) and TaKaRa cycleave PCR (66.7%) resulted in high and moderate detection rates. Together, these results indicate that, at low proviral loads of pBLV-IF, the sensitivity of BLV-CoCoMo-qPCR is better than those of the TaqMan MGB assay developed by Lew et al. and the TaKaRa cycleave PCR assay.To determine the sensitivity, we performed a 2-fold dilution of pBLV-IF2 = 0.93744), the copy number determined by TaKaRa cycleve PCR was moderately reproducible (R2 = 0.85754), and the copy number detected by the TaqMan MGB assay developed by Lew et al. was the least reproducible (R2 = 0.39447). These results clearly demonstrated that this assay has good reproducibility.Next, we evaluated the reproducibility of the copy numbers obtained by the three methods Figure . At low Thus, it appeared that BLV-CoCoMo-qPCR was the most suitable method for estimating BLV proviral load.We compared the sensitivity of BLV-CoCoMo-qPCR with those of nested PCR and conventional serological techniques, including AGID, PHA, and ELISA, using 370 clinical samples from two farms in Japan Table .A total of 39 out of 370 cattle were negative for BLV provirus and anti-BLV antibody, as determined by the four methods. A total of 150 out of 370 cattle were negative for BLV provirus, as determined by both BLV-CoCoMo-qPCR and nested PCR. However, some animals that were negative for proviral load, as determined by BLV-CoCoMo-qPCR, were positive in the serological tests. For example, 75 of 160 samples (46.9%) were positive by PHA, 25 of 163 samples (15.3%) were positive by AGID, and 94 of 151 samples (62.3%) were positive by ELISA.5 cells, indicating that the positive rate for nested PCR in animals correlated well with the level of proviral load determined by BLV-CoCoMo-qPCR. Moreover, some animals that were positive for proviral load, as determined by BLV-CoCoMo-qPCR were positive in the serological tests: 127 of 199 samples (63.8%) were positive by PHA, 181 of 207 samples (87.4%) were positive by AGID, and 152 of 154 samples (98.7%) were positive by ELISA.Furthermore, a total of 56 out of 370 cattle were positive for BLV provirus and anti-BLV antibody, as determined by all five methods. A total of 161 out of 370 cattle were positive for BLV provirus, as determined by both BLV-CoCoMo-qPCR and nested PCR. By contrast, a total of 22 out of 183 cattle (12.0%) were positive for BLV provirus by BLV-CoCoMo-qPCR but were negative in the nested PCR assay. However, the positive rate for nested PCR was 100% in animals with proviral loads of >1,500 copies per 105 cells . By contrast, 22 cattle were negative by nested PCR but were positive by BLV-CoCoMo-qPCR, with proviral loads ranging from 0 to 1,233 copies per 105 cells (mean 20 copy). A total of 202 samples were positive for anti-BLV antibody as determined by PHA, with copy numbers ranging from 0 to 42,015 copies per 105 cells . By contrast, 157 cattle were negative for anti-BLV antibody as determined by PHA but were positive as determined by BLV-CoCoMo-qPCR, with proviral loads ranging from 0 to 52,680 copies per 105 cells . A total of 206 samples were positive for anti-BLV antibody by AGID, with copy numbers ranging from 0 to 52,680 copies per 105 cells . By contrast, 164 cattle were negative for anti-BLV antibody by AGID but positive by BLV-CoCoMo-qPCR, with proviral loads ranging from 0 to 32,909 copies per 105cells (mean 693 copies). A total of 246 samples were positive for anti-BLV antibody by ELISA, with copy numbers ranging from 0 to 32,909 copies per 105 cells . By contrast, 59 cattle were negative for anti-BLV antibody by ELISA but positive by BLV-CoCoMo-qPCR, with proviral loads ranging from 0 to 5 copies per 105 cells (mean 0.1 copies). Moreover, Figure Figure 5 per 105 cells, as evaluated by BLV-CoCoMo-qPCR . Thus, the positive rate for the nested PCR in animals correlated well with the level of proviral load determined by BLV-CoCoMo-qPCR.We next calculated the positive rate for the nested PCR method in animals with BLV proviral copy numbers of 0 to 10R Figure B. The prBoLA class II genes are responsible for the outcomes of infectious diseases such as neosporosis, Lone Star tick, clinical mastitis, and enzootic bovine leukosis [BoLA-DRB3 alleles by the PCR-SBT method. SK576 carried alleles DRB3*0101/1201, and SK577 carried alleles DRB3*14011/1201. Our results showed an inconsistency between the proviral load as evaluated by BLV-CoCoMo-qPCR and the detection of BLV infection by serological tests. To investigate the reasons for these different results, two cattle were experimentally infected with BLV, and the anti-BLV antibody titer in the serum and proviral load were examined Figure . Polymorleukosis -30. Ther5 cells increased gradually throughout the experimental period. The ELISA S/P value started to increase gradually at 5 weeks, and the value remained high for the experimental period. Interestingly, the PHA titer was lower at the high viral load stage and was higher at the low viral load stage.We evaluated the number of BLV-infected cells in the two cattle by BLV-CoCoMo-qPCR from 1 to 10 weeks after experimental infection with BLV, and compared the production of anti-BLV antibodies by PHA and ELISA. In Figure et al. and the commercial TaKaRa cycleave PCR kit), using an infectious full-length molecular clone of BLV, pBLV-IF [Recently, we developed the BLV-CoCoMo-qPCR system to detect various BLV strains with broad geographical origins. Proviral load determined in this manner was found to correlate not only with BLV infection capacity as assessed by syncytium formation, but also with BLV-induced disease progression. The analyses described here show that the BLV-CoCoMo-qPCR method is a useful tool for evaluating BLV infection status. Conventional serological techniques, including AGID, PHA, and ELISA, are commonly used to diagnose BLV infection in Japan. Especially, the AGID test is currently a “golden standard” for determining BLV infection in Japan. We demonstrated that animals that were BLV-positive, as determined by the serological test , were negative for proviral load, as determined by BLV-CoCoMo-qPCR   The TaqMan probe was used to improve the sensitivity and specificity and to counter any drawbacks associated with high degeneracy. Importantly, the sequence of the BLV TaqMan probe, located between positions corresponding to two of the CoCoMo primers, was completely conserved among the 52 BLV variants. 3) A preliminary experiment demonstrated that the primer annealing regions of the TaqMan MGB assay developed by Lew et al. were variable in 10% of the 78 pol sequences selected from GenBank . This finding indicates that it is difficult to detect all of these variants. By contrast, use of degenerate primers allows for the detection of BLV sequence variants, including those that arise from mutations. Indeed, we previously demonstrated that this method can detect various BLV strains of broad geographical origins, including Japan, Peru, Bolivia, Chile, and the U.S.A. [An advantage of the BLV-CoCoMo-qPCR method is that it uses degenerate primers designed from 52 individual BLV LTR sequences identified from 356 BLV sequences in GenBank. It also uses the CoCoMo algorithm that was developed specifically for the detection of multiple viral species . It is pR) Table , as folle U.S.A. .BoLA-DRB3 genotypes. This experiment may help direct future research into examining whether or not progression of BLV-induced diseases correlates with not only viral propagation, but also with host factors that are associated with the immune response. We previously reported that, in sheep experimentally infected with BLV, quantitative and/or qualitative aspects of the immunoresponse, production of neutralizing antibodies against BLV, and elimination of BLV depended on the particular allelic forms of the MHC class II molecules expressed by an individual and, in particular, on certain polymorphic amino acid residues in class II molecules [In the present study, we found that the propagation of and immunoresponses to BLV were different in two cattle that carried different olecules ,38. Addi+-B cells [Using BLV-CoCoMo-qPCR, we previously found an increase in the proviral load during disease progression . This re-B cells , it is eet al. and the commercial TaKaRa cycleave PCR system. We observed that numerous cattle that were negative for anti-BLV antibody by each serotest were positive for the provirus as determined by BLV-CoCoMo-qPCR. By contrast, numerous animals that were BLV-positive by the serological test showed a negative proviral load by BLV-CoCoMo-qPCR. This result was confirmed by the finding that the kinetics of the proviral load did not quite correlate with changes in anti-BLV antibody production in two cattle experimentally infected with BLV. This result indicates that it is difficult to detect BLV infection by using the serological test alone. Collectively, these results suggest that the quantitative measurement of proviral load by BLV-CoCoMo-qPCR is a useful for monitoring the spread of BLV.Recently, we developed the BLV-CoCoMo-qPCR system to detect various BLV strains with broad geographical origins. The BLV-CoCoMo-qPCR method is a useful tool for evaluating the progression of BLV-induced disease. In this study, BLV-CoCoMo-qPCR was found to be highly sensitive when compared with the real-time PCR–based TaqMan MGB assay developed by Lew ATL: Adult T-cell leukemia; AGID: Agarose Gel Immuno-diffusion; BLV: Bovine leukemia virus; BoLA: Bovine leukocyte antigen; CoCoMo: Coordination of Common Motifs; EBL: Enzootic bovine leucosis; ELISA: Enzyme-linked immunosorbent assay; FAM: 5'-carboxyfluorescein; HTLV: Human T-leukemia virus; LTR: Long terminal repeat; MGB: Minor groove binder; PBMC: Peripheral blood mononuclear cell; PCR: Polymerase chain reaction; PHA: Passive Hemagglutination antigen; PL: Persistent lymphocytosis; qPCR: Quantitative real-time PCR.Non-financial competing interests.MJ participated in real time-PCR and nested PCR, analyzed data and helped to draft of manuscript. ST participated in the experimental design, analyzed date and helped to draft the manuscript. HM experimented of real-time PCR. JK carried out experimentally infection with BLV of cattle. NK and TM experimented of AGID and ELISA. TO and TN experimented of AGID and ELISA. YA conceived the study, participated in experiments, participated in experimental design, coordinated experiments, and drafted the manuscript. All authors read and approved the final manuscript.Mayuko Jimba (Ph.D.): Ph.D. student of The University of Tokyo and Junior Research Associate (JRA) in RIKEN. Present position is Postdoctoral Research of RIKEN. Shin-nosuke Takeshima (Ph.D.): ASI researcher of RIKEN and Associate professor of The University of Tokyo. Hironobe Murakami : Visiting researcher of RIKEN and postdoctoral researcher of Japan Foundation for AIDS Prevention, Junko Kohara : Hokkaido Animal Research Center. Naohiko Kobayashi : Gifu prefectual livestoch research institute. Tamako Matsuhashi (Ph.D.): Gifu prefectual livestoch research institute. Takashi Ohmori : Nippon institute for biological science. Tetsuo Nunoya : Nippon institute for biological science. Yoko Aida : Unit leader of RIKEN and Professor of The University of Tokyo.


Organised colorectal cancer screening is likely to be cost-effective, but cost-effectiveness results alone may not help policy makers to make decisions about programme feasibility or service providers to plan programme delivery. For these purposes, estimates of the impact on the health services of actually introducing screening in the target population would be helpful. However, these types of analyses are rarely reported. As an illustration of such an approach, we estimated annual health service resource requirements and health outcomes over the first decade of a population-based colorectal cancer screening programme in Ireland.A Markov state-transition model of colorectal neoplasia natural history was used. Three core screening scenarios were considered: (a) flexible sigmoidoscopy (FSIG) once at age 60, (b) biennial guaiac-based faecal occult blood tests (gFOBT) at 55–74 years, and (c) biennial faecal immunochemical tests (FIT) at 55–74 years. Three alternative FIT roll-out scenarios were also investigated relating to age-restricted screening (55–64 years) and staggered age-based roll-out across the 55–74 age group. Parameter estimates were derived from literature review, existing screening programmes, and expert opinion. Results were expressed in relation to the 2008 population .FIT-based screening would deliver the greatest health benefits, averting 164 colorectal cancer cases and 272 deaths in year 10 of the programme. Capacity would be required for 11,095-14,820 diagnostic and surveillance colonoscopies annually, compared to 381–1,053 with FSIG-based, and 967–1,300 with gFOBT-based, screening. With FIT, in year 10, these colonoscopies would result in 62 hospital admissions for abdominal bleeding, 27 bowel perforations and one death. Resource requirements for pathology, diagnostic radiology, radiotherapy and colorectal resection were highest for FIT. Estimates depended on screening uptake. Alternative FIT roll-out scenarios had lower resource requirements.While FIT-based screening would quite quickly generate attractive health outcomes, it has heavy resource requirements. These could impact on the feasibility of a programme based on this screening modality. Staggered age-based roll-out would allow time to increase endoscopy capacity to meet programme requirements. Resource modelling of this type complements conventional cost-effectiveness analyses and can help inform policy making and service planning. Various international organisations have recommended screening for colorectal cancer ,2. WhileCost-effectiveness is only one of the issues which needs to be considered when taking decisions on screening implementation. Long-established criteria from the World Health Organisation (WHO) recommend also considering the safety of the screening test and associated diagnostic investigations, the acceptability of the test, and availability of diagnosis and treatment facilities . The chastatus quo of no screening. The cost per QALY gained was €589 for FSIG, €1,696 for FIT and €4,428 for gFOBT [We conducted a cost-effectiveness analysis of three strategies for colorectal cancer screening in Ireland: biennial gFOBT at ages 55–74, biennial FIT at ages 55–74 and once-only FSIG at age 60. All three options were considered to be highly cost-effective compared to the In this paper we describe how we used a Markov state-transition model to estimate health service resource requirements and health outcomes, relative to the actual population of Ireland, over the first decade of a population-based colorectal cancer screening programme. These analyses were done with a view to aiding decision-making and service planning, and intended to complement the cost-effectiveness analyses.The country of Ireland is a republic comprising the largest part of the island of Ireland (the remainder of the island is Northern Ireland). The country has a population of 4.4 million, 700,800 of whom are aged 55–74. The healthcare system involves a mixture of public and private provision. Cancer screening programmes are provided free at the point of delivery, so the costs of provision fall on the healthcare payer, in this instance the Health Service Executive (HSE).This analysis was part of a health technology assessment (HTA) of population-based colorectal cancer screening in Ireland. The commissioners, the Health Information & Quality Authority, established an Expert Group to scope the HTA, and recommend which screening modalities and age-groups should be evaluated . These dSince FIT-based screening was considered the optimal option in terms of cost-effectiveness, health service requirements associated with alternative scenarios for implementing FIT-screening were also considered. The two main alternative implementation approaches involve either restrictions by area or by age. The former is considered unacceptable by the National Cancer Screening Service (NCSS) in Ireland, so only age-based implementation strategies were considered. The first variant involved restricting screening to the 55–64 age-group (i.e. a maximum of five screening rounds). The second and third variants involved staggered roll-out of biennial screening in the 55–74 age-group. “Medium implementation” involved inviting those aged 55 and 65 in year one; those aged 55, 57, 65 and 67 in year two; those aged 55, 57, 59, 65, 67 and 69 in year three; and so on until the full age range would be included in year five. “Slow implementation” involved inviting those aged 55 in year one; those aged 55 and 57 in year two, those aged 55, 57 and 59 in year three; and so on until year ten when the full age-range would be included. These scenarios were not intended to represent the only or preferred options, but rather were chosen to illustrate the health service impact of different possible approaches to implementation.“Conventional” analysis of a Markov model - for the purposes of cost-effectiveness analysis - follows a single notional cohort, usually comprising 100,000 individuals, over their lifetime accumulating costs and benefits as the cohort ages. The approach adopted for this analysis was similar to that taken in a budget impact analysis . We consResources associated with screening were assumed to be provided by the HSE and, within that, the NCSS. The health service resources estimated included those associated with: screening ; and diagnosis and treatment of screen-detected lesions (up to and including the point of surgery) and surveillance of individuals with screen-detected adenomas  and colorectal resection). The health outcomes considered were the numbers of individuals who were diagnosed with screen-detected adenomas and cancers and the numbers who sustained complications .We used a Markov state-transition model which is described in detail elsewhere ,12. BrieTo populate the model, comprehensive literature reviews were undertaken. These were augmented with data and results from population-based screening programmes and pilot programmes. When relevant data was unavailable, values were based on expert clinical opinion. Table Base-case estimates of screening uptake and colonoscopy compliance came from the UK pilot colorectal cancer screening programmes and the UK FSIG trial ,35,38. GSeveral parameters, including natural history transition probabilities, could not be empirically observed and were obtained by calibration: This involved fitting the model to colorectal cancer incidence (by stage) and mortality data for Ireland. Further details of the approach and the transition probabilities are provided elsewhere ,50. The The primary analysis concerned absolute screening impact . One-way sensitivity analyses explored the impact of varying (a) screening uptake, (b) screening test sensitivity and (c) screening test specificity. Values for lower and higher uptake were based on experience in other countries ,37,39,40The secondary analysis assessed the relative impact of screening. For each core screening scenario, we estimated the additional resources needed for diagnosis and treatment, and the population-level health gains, compared to those required or generated under a policy of no screening.The model predicted that, in year one of a programme based on gFOBT or FIT, 357,812 individuals would be sent test kits, and 189,640 completed kits would be returned for laboratory processing  would require pathological analysis, compared to 1,599  using FSIG, and 1,004 (695 and 309) using gFOBT. Resources would be required to conduct 774 resections of screen-detected cancers in year one with FIT screening, compared to 280 with gFOBT and 59 with FSIG. In year 10, these numbers would rise to 635, 307 and 71 for FIT, gFOBT and FSIG screening, respectively.Numbers of diagnostic colonoscopies and screen-detected cancers estimated from the sensitivity analyses are shown in Additional file Additional file Compared to a policy of no screening, screening by gFOBT, FIT and FSIG all had potential to reduce deaths from colorectal cancer in the population within a decade of implementation. This reduction was evident from the second year, and was greatest for FIT-based screening Figure . FIT-basThe resource modelling described in this paper could be viewed as a particular type of budget impact analysis, with costs expressed in terms of resources and harms rather than monetary units. As in budget impact analyses , we focuThe few previous analyses of the health service impact of colorectal cancer screening ,52,53 alIn this study, we found that a programme based on FIT the option considered most cost-effective  – would If not prepared for, increases in colonoscopy demand can increase waiting times . StudiesStrategies that might be used to help address endoscopy capacity and other resource challenges include restricting screening to a narrower age-range, and/or gradually phasing in screening across the full age-range. Although restricting FIT-based screening to those aged 55–64 would reduce resource requirements by around 40%, our cost-effectiveness analyses concluded that, when fully implemented this strategy would be slightly less desirable than screening the 55–74 age group . This suSafety is a key consideration in the implementation of any screening programme . The chaA screening test must be acceptable to the population  and one Since it is impossible to be entirely certain what uptake might be attained for any screening test in any population, base-case values were chosen to be conservative. For the faecal tests, the base-case estimate of 53% was based on the second round of the UK pilot programmes ,35. AlthSince we conducted our analysis evidence has accrued that uptake may be higher with FIT than gFOBT , althougAlthough parameter values were based on review of the best available evidence, as with all modelling studies, some uncertainty remains. Even for gFOBT, which has been shown to reduce mortality in several trials , performWe assumed, for gFOBT and FIT, that sensitivity and specificity remained constant across all screening rounds. Recent evidence suggests that the ability of gFOBT-based programmes to identify cancers might decline with repeated screenings . The samWe evaluated a strategy that combined gFOBT with reflex FIT, instead of the more conventional approach of reflex gFOBT. This was because second-line FIT has been shown to limit the number of referrals to colonoscopy ,73. FindWe did not estimate resources required for adjuvant chemotherapy in screen-detected colon cancers, or for follow-up after resection  since responsibility for providing these would most likely fall outwith the screening programme/NCSS. Facilities would, however, need to be provided elsewhere in the health services, so this distinction is somewhat arbitrary.This study illustrates how resource modelling complements cost-effectiveness analyses and can help inform health service decision making and planning. Our results show that the introduction of FIT-based screening would quite quickly generate attractive health outcomes – in terms of reduced numbers of colorectal cancer cases and deaths – but that it has heavy resource requirements. These heavy requirements could impact on the feasibility of any programme based on this screening modality. Staggered age-based roll-out of FIT-based screening would allow time to increase endoscopy capacity to meet requirements. Overall out findings suggest that there are likely to be many challenges ahead in delivering a high-quality, safe, and acceptable screening programme to the population of Ireland.CT: Computed tomography; FIT: Faecal immunochemical test; FSIG: Flexible sigmoidoscopy; gFOBT: guaiac-based Faecal Occult Blood; HSE: Health Service Executive; LYS: Life years saved; MRI: Magnetic resonance imaging; NCSS: National Cancer Screening Service; PET: Positron emission tomography; QALY: Quality adjusted life year; UK: United Kingdom; WHO: World Health Organisation.None of the authors have any conflicts of interest to declare.All authors were involved in the design of the study, interpretation of data and commenting critically on the manuscript. In addition, LS co-ordinated the study, drafted the initial version of the manuscript, and undertook systematic reviews together with LT, AOC and CU. LT, CU and MB collated cost data. PT, JC and SW developed and modified the Markov model. CW provided mathematical and statistical input. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/13/105/prepubSimplified diagram of Markov states in natural history mode.Click here for fileSensitivity analysis: estimated numbers of diagnostic colonoscopies required and screen-detected cancers, for years one and 10, by screening scenario, as test sensitivity, test specificity and screening uptake vary around base-case values.Click here for file1.Scenario analysis: estimated screening-related resource use and health outcomes by year for alternative FIT implementation options: biennial FIT at 55-64 years, biennial FIT at 55-74 years (medium roll-out), and biennial FIT at 55-74 years (slow roll-out)Click here for file


Attention-deficit/hyperactivity disorder (ADHD) is a common neuro-psychiatric problem, affecting 7-9% of children. Pharmacological interventions are widely used with behavioral treatments in ADHD. Still, the origin of ADHD is unclear, limiting pharmacological effectiveness and making adverse effects common. The use of complementary and alternative medicine (CAM) has increased, especially for developmental and behavioral disorders, such as ADHD. CAM is used by 60-65% of parents of children with ADHD to relieve ADHD-associated symptoms and to avoid the side effects of conventional medication. Acupuncture has been widely used to treat patients with ADHD, but the available evidence of its effectiveness is insufficient. Our aim was to evaluate the effectiveness and safety of acupuncture in patients  with ADHD (any subtype) compared to the waitlist control.This study is a waitlist controlled open trial. We used a computer generated randomization scheme. This randomised, controlled trial had two parallel arms . Each arm consisted of 40 participants. The acupuncture group received acupuncture treatment two times per week for a total of 12 sessions over 6 weeks. Post-treatment follow-up was performed 3 weeks later to complement the 12 acupuncture sessions. Participants in the waitlist group did not receive acupuncture treatments during the first six weeks but were only required to be assessed. After 6 weeks, the same treatments given to the acupuncture group were provided to the waitlist group. The primary outcome of this trial included differences in Korean version of ADHD-Rating Scale (K-ADHD-RS) before randomization, 3 weeks and 6 weeks after randomization, and 3 weeks after completing the treatment.Subjective measurements, like K-ADHD-RS, are commonly used in ADHD. Although these measurements have adequate reliability and validity, lack of objective assessment in ADHD may lead to some disputes, like parental placebo effects. More objective measurements, like Computerized Neurocognitive function Test (CNT) in this study, are needed in ADHD trials. Furthermore, this trial will provide evidence for the effectiveness of acupuncture as a treatment for ADHD.Clinical Research Information Service (CRiS) KCT0000019 Attention-deficit/hyperactivity disorder (ADHD) is a common problem, affecting 7-9% of children. The defining features of ADHD are inattention, hyperactivity, and impulsivity . There iThe widespread use of stimulant medications for ADHD, such as methylphenidate (MPD) and dex-amphetamine, is thought to enhance short-term behavioral, academic, and social functioning . HoweverThe use of CAM therapies has increased, especially for developmental and behavioral disorders such as ADHD ,13,16-18Acupuncture can change brain activity -35. A brThere are a large number of studies of the clinical efficacy of acupuncture in ADHD published globally, especially in China, but not all demonstrate a beneficial effect on ADHD ,23,24,36The present study aimed to evaluate the effectiveness of acupuncture in patients with ADHD (any subtype) compared to waitlist control and to evaluate the safety of acupuncture in patients with ADHD (any subtype).A 6-week, randomised, waitlist controlled trial of acupuncture for the treatment of ADHD in children/adolescents is being conducted at Kyung-Hee Oriental Medical center, Seoul, Korea. All screening appointments and study visits occur in the neuropsychiatry clinic for outpatients. This study was ethically approved by the Kyung-Hee Korean Medical Center Institutional Review Board, and was registered with the Clinical Research Information Service (CRiS) (KCT0000019) as a waitlist controlled, randomised clinical trial. This study had two parallel arms (acupuncture and waitlist group) with a 1:1 patient allocation ratio (40 participants per arm).The providers went through 6 years of regular Korean medical college education and each was licensed a Korean Medical Doctor (KMD) degree. All the KMDs have undergone acupuncture safety education. These practitioners were chosen on the basis of their expertise in the field, their certified status and agreement to abide by the study protocols. Every practitioner is an apprentice doctor of "Department of Neuropsychiatry, College of Korean medicine, Kyung-Hee University" during this trial period.Parents were initially screened by phone, and their children were included in the study if the inclusion criteria were met. The inclusion criteria includes being between 7-18 years of age, having a diagnosis of ADHD (any subtype) according to Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria and having an informed verbal and written consent from parents and participants. It did not matter whichever intervention  was used, if there were not any change of ADHD treatments/symptoms for the past 2 weeks. Exclusion criteria are a diagnosis of a developmental disorder, past history of epilepsy, the presence of another neurotic disorder, pregnancy, and any changes in medications during the course of the study. During waitlist period, a CGI-I score ≥6, is a cue for the participant to stop waiting and to drop-out.DSM-IV ADHD diagnostic criteria ,37 were The primary outcomes for the study were changes in ADHD symptoms from baseline to week 6 (or from week 6-12) as measured by the K-ADHD-RS . The K-AThe Clinical Global Impression (CGI) rating scale is also a commonly used measure of symptom severity, treatment response, and the efficacy of treatments in treatment studies of patients with mental disorders . The CliThe Korean version of Child Behavior Checklist (K-CBCL) was used at week 3 and 6 . CBCL has been proposed as a non-ideological measure to characterize comorbidity in individuals with ADHD ,43. The The Frankfurt Attention Inventory (FAIR)  measurin© Sample size calculations are performed to determine the number of participants needed to detect effect sizes similar to those that have been reported in recent ADHD medication trials . This sthttp://www.ebombit.com and happiend.tistory.com. Short, text-only advertisements were also placed on several ADHD parents' internet community sites for free. As part of the informed consent forms, the parents of the participants were advised that they were free to withdraw their child/children from the study at any stage without any consequences.Recruitment for the study commenced in July 2010 and the first participant was enrolled in July 2010. Participant recruitment is ongoing. Each participant is qualified and enrolled into the study separately, and has their own start date. The study end-point goal is December 2011. Paid advertisements were placed in free-newspapers with metropolitan-wide circulations. Paid advertising bills were also inserted between the pages of the morning newspaper. Websites advertisements were placed on several blogs for free, including Group A (acupuncture group) continued with their existing program and the acupuncture protocol was added to their regimen. Group W (waitlist group) continued with their existing program and the waitlist protocol was added to their regimen. The participants in group A attended a clinical facility for the first 6 weeks and received 12 treatments (two times per week) for approximately 20 minutes. During this first 6-week period, group W consisted of children who continued with their existing ADHD treatment regimen as prescribed by their pediatrician, clinical psychologist and/or medical doctor. Then, the participants in group W were scheduled for another 12 treatments over the next 6 weeks .Acupuncture originated in ancient East Asia approximately 2,500 years ago and is a key component of traditional Korean medicine or traditional Chinese medicine. Acupuncture techniques involve insertion of needles and the stimulation of the needles may involve manual, electrical, heat, laser or other forms of stimulation .http://www.dbneedle.com; Seoul, Korea) are used. This treatment is conducted two times per week for 6 weeks. There are no co-interventions without a life-style education for their parents. An education for parents is conducted during visits in weeks 3 and 6.For every treatment visit, participants are treated with 13 acupuncture points  . After the waiting endpoint, participants will similarly be given acupuncture treatment for 6 weeks without an additional life-style education for parents.One of the advantages of acupuncture is that the incidence of adverse effects is substantially lower than that of many drugs used for the same conditions . In a reThe trial protocol was reviewed by the Kyung-Hee Korean Medical Center Institutional Review Board. We prepared two types of consent forms, one for participants who are 7-13-years-of-age (explained in easier words) and another for those who are 14-18-years-of-age. For 7-13-year-old participants, a "comprehension check form" is used to confirm their full understanding. All children participants make their own decisions about participation in a clinical trial. All parents of the participants are issued with duplicate copies of the informed consent forms, with the original copies kept by the hospital. The informed consent form conveys the following information: names of the chief investigator and supervisors; the name of the hospital and the department conducting the research, a brief description of the control and treatment groups, the duration of the study, the questionnaires used, the Ethics approval, the right to withdraw their child at any time, and the fact that the results of the study will be disseminated via conferences and publications while maintaining the child's privacy.2 for categorical variables. Every analysis conducted using the SPSS software (SPSS 12.0KO for Windows©), and performed among two randomised  groups and their stratified  sub-groups. The primary analysis will compare outcomes for each primary and secondary outcome measure among the groups to estimate treatment effect with 95% confidence interval (CI). An intention-to-treat (ITT) analysis will be undertaken of the 3-week and 6-week (respectively 9-week and 12-week) data from the K-ADHD-RS, due to some participants who commence the study but do not complete.Tests of potential differences across study groups on demographic and clinical history variables were performed by means of analysis of covariance (ANCOVA) for continuous measures and Pearson χWe have a presented the design and the protocol for the randomised controlled trial of acupuncture for childhood ADHD. The participants of this study will have acupuncture added to their existing medical regimen  or no previous therapy at all, and will be monitored for changes in their outcomes  in the short-term (3 and 6 weeks) and during a 9 week follow-up. Completion of this trial will help provide the answer to the question "Does acupuncture have a role to play in the management of children with ADHD?" Results of this trial will be disseminated at the completion of the study.This is the first acupuncture trial protocol study in ADHD. Case studies of acupuncture effects for ADHD already exist, especially in China, but trial protocols are not precisely described. Our protocol will help to make better quality acupuncture studies for ADHD. Furthermore, we tried to gather objective measurements using CNT40. Subjective measurements, like K-ADHD-RS in this study, are commonly used in ADHD and have adequate reliability and validity. But lack of objective measurements in ADHD may lead to some disputes like parental placebo effects.In traditional Korean medical theories, the insufficiency of yin, results in an excess of yang. The imbalanced condition of the whole body leads to impaired mental symptoms like attention deficit, hyperactivity and impulsivity. Thirteen acupuncture points have been chosen in this context. The acupuncture points located on the head region takes a main role for controlling the spirit. Qi of five viscera and six bowels belong to the head region. For balance of the whole mind and body, acupuncture points in the head region are essential. Baihui (GV20), Sishencong (EX-HN1) are representative points for the head region. Quchi (LI11) takes a role in the elimination of excessive yang and Sanyinjiao (SP6) supplements insufficient yin. It yields a better mental balance. Hegu (LI4), Taichong (LR3) assists in harmonious circulating qi and blood.One limitation of this study involves the waitlist for the controlled group. Many researchers commonly prefer placebo controlled designs to the waitlist ones in randomised control trials. Placebo effects of acupuncture were actively studied in many articles and sham acupuncture is a representative method for an acupuncture placebo tool. But there is a lack of consensus on the most valid approach to establishing an acupuncture placebo. So, important aspects of the placebo acupuncture control need to be considered. "Inert" is the main topic in placebo acupuncture control. Placebo acupuncture strategies mainly consist of superficial  insertion, non-acupuncture insertion points insertion, and sham acupuncture. But each approach has some defects. First, a superficial  insertion is not a real placebo method because it induces peripheral sensory stimulation. It does not seem possible to insert acupuncture needles without any sensory stimulation ,65. In fNot separating assessor from randomization is an additional limitation. Every measurement was assessed by the person who conducted the screening phase, so as not to be blinded from randomization. Although we tried to maintain an objective standpoint, we could not exclude observer bias, which all researchers brought to their work and which helped to determine their method of research and their observations. To minimize this bias, we involved objective ADHD measurement tools, like CNT40 and FAIR.Finally, a paper case record form (CRF) was used in this study. These days, electronic case record form (e-CRF) system, like the Clinical Data Interchange Standards Consortium (CDISC) e-CRF, is become preferred because of its improved quality and efficiency in trial study. In July 2010, the Korea CDISC Coordination Committee (K3C) started making standard e-CRF forms, but we used paper CRF instead, due to insufficient e-CRF infrastructure. Paper CRF may lead to data loss and input errors. The Kyung-Hee Korean Medical Center Institutional Review Board strictly supervised data storage and management. We also reexamined the inputted data.Subjective measurements, like K-ADHD-RS, are commonly used in ADHD, and have the best evidence. Although these measurements have adequate reliability and validity, lack of objective assessment in ADHD may lead to some disputes, like parental placebo effects. More objective measurements, like the presently used CNT40, have to be validated in ADHD trials. Furthermore, as an evidence-based treatment in ADHD, acupuncture has little evidences compared with conventional pharmacological or psychosocial treatment. This study will give some evidence for the effectiveness of acupuncture as an add-on treatment in treating children with ADHD.ADHD: Attention Deficit Hyperactivity Disorder; CAM: Complementary and Alternative Medicine; DMPFC: Dorsomedial Prefrontal Cortex; FDG-PET/CT: Fluorodeoxyglucose Positron Emission Tomography combined Computed Tomography; K-ADHD-RS: Korean version of ADHD-Rating Scale; CRiS: Clinical Research Information Service; MPD: methylphenidate; KMD: Korean Medical Doctor; CGI: Clinical Global Impression rating scales; CGI-S: Clinical Global Impression severity scale; CGI-I: Clinical Global Impression improvement scale; K-CBCL: Korean version of Child Behavior Checklist; FAIR: The Frankfurt Attention Inventory; MV: marker value; PV: performance value; QV: quality value; CV: continuity value; CNT: Computerized Neurocognitive function Test; CPT: Continuous Performance Test; CPM: Colored Progressive Matrices; SPM: Standard Progressive Matrices; K-CPRS: Korean version of Conners' parent Rating scale; K-IOWA: Korean version of IOWA-Conners Rating scale; group A: acupuncture group; group W: waitlist group; SD: Standard Deviation; GV: Governor Vessel; EX-HN: Extra Points of Head and Neck; LI: Large Intestine Meridian; SP: Spleen Meridian; LR: Liver Meridian; B-cun: proportional bone cun; WHO: World Health Organization; CI: confidence interval; ITT: Intention-To-Treat; CRF: Case Record Form; e-CRF: Electronic Case Record Form; CDISC: Clinical Data Interchange Standards Consortium; K3C: Korea CDISC Coordination.The authors declare that they have no competing interests.SHC conceived the research project. SHC, SSH designed the study. SSH designed and tailored the protocol for the ADHD cohort. All authors contributed to the writing of the manuscript. All authors read and approved the final manuscript.


In this study gene expression profiles were obtained from fresh human lung cancer tissue fragments cultured ex vivo under different oxygen concentrations in order to study responses to hypoxia in a model that mimics human lung cancer in vivo.Hypoxia-induced genes are potential targets in cancer therapy. Responses to hypoxia have been extensively studied ex vivo in normoxia or hypoxia in short-term culture. Viability, apoptosis rates and tissue hypoxia were assessed. Gene expression profiles were studied using Affymetrix GeneChip 1.0 ST microarrays.Non-small cell lung cancer (NSCLC) fragments from altogether 70 patients were maintained Apoptosis rates were comparable in normoxia and hypoxia despite different oxygenation levels, suggesting adaptation of tumor cells to hypoxia. Gene expression profiles in hypoxic compared to normoxic fragments largely overlapped with published hypoxia-signatures. While most of these genes were up-regulated by hypoxia also in NSCLC cell lines, membrane metallo-endopeptidase  expression was not increased in hypoxia in NSCLC cell lines, but in carcinoma-associated fibroblasts isolated from non-small cell lung cancers. High MME expression was significantly associated with poor overall survival in 342 NSCLC patients in a meta-analysis of published microarray datasets.ex vivo model allowed for the first time to analyze hypoxia-regulated gene expression in preserved human lung cancer tissue. Gene expression profiles in human hypoxic lung cancer tissue overlapped with hypoxia-signatures from cancer cell lines, however, the elastase MME was identified as a novel hypoxia-induced gene in lung cancer. Due to the lack of hypoxia effects on MME expression in NSCLC cell lines in contrast to carcinoma-associated fibroblasts, a direct up-regulation of stroma fibroblast MME expression under hypoxia might contribute to enhanced aggressiveness of hypoxic cancers.The novel  Ex vivo human cancer models based on the short-term culture of small tumor fragments or slices are suitable to study tumor responses within the natural in situ microenvironment, comprising a close contact between tumor cells and the accompanying stroma cells. Such models have been used e.g. for the study of drug effects in lung cancer ” yielded 84 results (status June 2011). Of these 84 datasets/series, 68 contained expression profiling data. Four of these series included expression data of a minimal number of 50 NSCLC patients treated by surgery with linked information on survival, GSE11969 [Next, we examined whether expression of the four hypoxia genes was associated with survival in patients with NSCLC. Due to the relatively short observation period in our patient cohort, we used large published microarray datasets containing gene expression data linked to clinical and prognostic information in NSCLC patients. The Gene Expression Omnibus  in a multivariate analysis with pathological tumor stage as stratification variable. The interaction between MME and histology (adenocarcinoma vs. non-adenocarcinoma) was statistically significant (P = 0.027). Thus survival analyses were performed in adenocarcinoma patients and non-adenocarcinoma patients separately  and series GSE14814 (P = 0.029), and in the combined cohort including 182 patients , the 3D-morphology is preserved, and inter-patient variability is taken into account by using material derived from different patients. The major limitation of our study is that the exact oxygen concentration could only be controlled on the surface of the fragments. Inside the tumor fragments there are supposed to be oxygen gradients, depending on the size and composition of the tissue fragment. The size of normoxic and hypoxic fragments did not differ in our study. In fact, using pimonidazole staining, fragments cultured in hypoxia were found to be entirely hypoxic, while only a core of hypoxia was found in fragments cultured in normoxia. In addition to pimonidazole, also other major hypoxia-markers were significantly increased in the hypoxic fragments, such as HIF-1α and CA IX. However, HIF-2α, which is known to be stabilized by hypoxia similarly to HIF-1α, was expressed only at low levels, both in normoxia and hypoxia, and was not elevated in hypoxic fragments. Different co-activators and different kinetics of activation under hypoxia [The model has several advantages compared to  hypoxia  might pl2 or normoxia for three days. This agrees with our previous study where we showed that hypoxia-induced adaptation and cisplatin-resistance are reversible in lung cancer cells and occur without hypoxia-induced cell death and selection [Apoptosis rates were comparable in NSCLC fragments cultured in 1% Oelection . In an aelection  identifielection ) with ouelection  were sigFour genes were significantly up-regulated by hypoxia in both adenocarcinoma and squamous cell carcinoma fragments in our setting. We confirmed the differential expression of the four overlapping hypoxia genes under hypoxia in an independent validation set using quantitative PCR (qPCR). Also the well-established hypoxia-responsive gene HK2, which phosphorylates glucose and thus contributes to the glycolytic flux in cancer cells, was significantly up-regulated by hypoxia in the fragments, both in the microarray analysis and by qPCR.The four hypoxia-genes identified in our study have been found to be up-regulated by hypoxia in several microarray studies, however these findings were not validated e.g. by qPCR -32. To tIn our study we found MME localized to neoplastic tumor cells, but also to stroma cells in fresh NSCLC tissue, which is in line with published data ,41. The in vitro, but only at very high concentrations and after long exposure [MME, which is identical to common acute leukemia antigen (CALLA), is a 90–110 kDa zinc binding cell surface peptidase, which cleaves small peptides, such as atrial natriuretic peptide, substance P, endothelin-1, and bombesin . It exposure . On the exposure . While texposure ,57. Elasexposure , which mIn order to analyze, whether levels of the common hypoxia-genes identified in our study are associated with overall survival in NSCLC patients we used all eligible studies deposited in one of the largest microarray depositories, the GEO database. We were able to show that MME expression is a highly significant, independent adverse prognostic factor in surgically treated lung adenocarcinoma patients in multivariate analysis involving tumor stage and MME status. No association was found in the subgroup of non-adenocarcinoma patients. The reason for the different results in the histological subgroups is unknown, however, lung adenocarcinomas have been shown to possess more elastin than squamous cell carcinomas . Since tTo the best of our knowledge, three other studies examined the association of MME expression and survival in lung cancer ,41,51. AAll of these studies were immunohistochemical studies, in contrast to our MME mRNA based survival analysis. Since MME may be excreted in exosomes , which hAdditional studies examined the expression of MME in combination with other factors and survival. In the study by Tokuhara et al.  132 NSCLex vivo model allowed for the first time to analyze hypoxia-regulated gene expression in preserved human lung cancer tissue. The study shows that gene expression profiles in human hypoxic lung cancer tissue overlap with hypoxia-signatures from cancer cell lines, however, MME was identified as a novel hypoxia-induced gene in lung cancer. Despite the advantages of ex vivo tissue culture, cell monolayers still appear to be the method of choice to study mechanisms of adaptation of individual cell types to hypoxia, since the oxygen concentration can be controlled only on the surface of such three-dimensional structures. Thus we analyzed expression of the hypoxia-regulated genes identified in the NSCLC fragments in different NSCLC cell lines and primary CAFs isolated from NSCLC tissue. We show that MME expression is up-regulated by hypoxia in CAFs, not in NSCLC cells. High global levels of MME mRNA in NSCLC tissue were shown in our study to predict poor survival. A direct effect of hypoxia on stromal fibroblast MME expression might thus contribute to enhanced aggressiveness of hypoxic cancers.The novel NSCLC: Non-small cell lung cancer; MME: Membrane metallo-endopeptidase; CALLA: Common acute leukemia antigen; HIF: Hypoxia-inducible factor; CA IX: Carbonic anhydrase IX; ACTB: β-actin; FDR: False discovery rate; PPP1R3C: Protein phosphatase 1 regulatory subunit 3C; KCTD11: Potassium channel tetramerisation domain containing 11; FAM115C: Family with sequence similarity 115 member C; HK2: Hexokinase 2; CAFs: Carcinoma-associated fibroblasts; HR: Hazard ratio; CI: Confidence interval.The authors declare that they have no competing interests.KL contributed to the study design and data interpretation, obtained funding for the project, carried out cell culture and pimonidazole experiments, performed RNA isolation and immunohistochemistry and prepared the manuscript. CW carried out experiments with NSCLC fragments. HHP and ES contributed to study design and data interpretation, contributed to immunohistochemistry analysis and revised the manuscript. JL and FM-SJ contributed to establishment of the fragment model, coordinated the isolation of fresh NSCLC fragments and revised the manuscript. NAH contributed to immunohistochemistry and revised the manuscript. BG and CG participated in the design of the project, performed the microarray analyses and participated in interpretation of the data. FQ contributed to the survival meta-analysis, performed the hierarchical cluster analysis, and revised the manuscript. PS provided donor lungs and revised the manuscript. SP provided clinical data of patients included in the meta-analysis and critically revised the manuscript. AH contributed to study design and project coordination and revised the manuscript. AO contributed to the study design and data interpretation. HO contributed to the study design and data interpretation, coordinated the project, obtained project funding and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/40/prepubSupplementary Methods. Table S1. Probesets identifying genes of interest. Table S2. TaqMan® Gene Expression Assays for qPCR.Click here for fileGenes down-regulated by hypoxia.Click here for fileP-values were calculated with pvclust based on 1000 bootstrap replications in 1426 selected genes with highest variability. In this software P-values greater than 95% are considered significant. Bootstrap P-values (bp) are shown in green. Asymptotically unbiased P-values (au) are shown in red. Cluster numbers (edge #) are shown in grey. The most similar pairs of arrays (shortest dendrogram branches) were the hypoxic and normoxic fragments from each patient. This close similarity was significant in 9 of 10 patients. Ad, adenocarcinoma; Sq, squamous cell carcinoma; No, normoxia; Hy, hypoxia.Hierarchical clustering of gene expression profiles including all genes. Click here for file


Numerous biologic approaches are being investigated as anti-cancer therapies in an attempt to induce tumor regression while circumventing the toxic side effects associated with standard chemo- or radiotherapies. Among these, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown particular promise in pre-clinical and early clinical trials, due to its preferential ability to induce apoptotic cell death in cancer cells and its minimal toxicity. One limitation of TRAIL use is the fact that many tumor types display an inherent resistance to TRAIL-induced apoptosis. To circumvent this problem, researchers have explored a number of strategies to optimize TRAIL delivery and to improve its efficacy via co-administration with other anti-cancer agents. In this review, we will focus on TRAIL-based gene therapy approaches for the treatment of malignancies. We will discuss the main viral vectors that are being used for TRAIL gene therapy and the strategies that are currently being attempted to improve the efficacy of TRAIL as an anti-cancer therapeutic. Despite the many significant medical breakthroughs that have been made in recent years, advanced cancers remain difficult to treat, and standard therapies such as chemotherapy and radiotherapy are often associated with substantial toxicities that can limit their use in the clinic. For these reasons, biologic therapies have been increasingly explored as alternative approaches due to their potential to specifically target tumor cells for eradication, while leaving untransformed cells intact. By restricting cell death to only malignant cells, many of the side effects associated with standard therapies can be potentially avoided.TNF-related apoptosis-inducing ligand (TRAIL) is a biologic therapy that has shown great promise in pre-clinical studies, due to its ability to preferentially induce apoptotic cell death in transformed cells. Apoptosis is a tightly regulated cellular process, and differs from necrosis in that it does not trigger inflammatory host responses. Apoptosis proceeds by two main pathways, referred to as intrinsic, which is controlled by interactions of pro-apoptotic and anti-apoptotic members of the B-cell leukemia/lymphoma 2 (Bcl-2) protein family , and extTherapeutic TRAIL use has been investigated in many different formats. Initial approaches employed systemic or local administration of recombinant TRAIL protein to ligate the TRAIL receptors on cancer cells and activate the extrinsic cell death machinery . As the 2.i.e., induction of normal cell/tissue death) was observed after systemic administration of TNF and FasL (or anti-Fas mAb), which put the utility of these molecules as anti-cancer therapeutics into question. In 1995, Wiley et al. reported the cloning of the full length TRAIL cDNA  , TRAIL r4.Upon ligation of TRAIL trimers to TRAIL-R1 or -R2 in humans, the extrinsic apoptotic pathway is activated via a series of intracellular changes that culminate in the apoptotic death of the TRAIL receptor-bearing cell . One of Cells expressing functional TRAIL receptors can be classified as either Type I or Type II, depending on their differential requirements for involvement of the intrinsic pathway in triggering apoptosis. Type I cells undergo apoptosis in response to extrinsic signals that lead to caspase 8 cleavage. Type II cells do not undergo apoptosis upon activation of the extrinsic pathway alone; they also require activation of the intrinsic apoptotic pathway and inactivation of intracellular proteins that inhibit caspase signaling cascades, such as X-linked Inhibitor of Apoptosis Protein (XIAP) . Initiat5.in vitro, whereas untransformed cells were resistant .-62.60-625.3.Sorafenib is a protein tyrosine kinase inhibitor approved for use in the treatment of renal cell carcinoma, and it has also received attention for its ability to sensitize tumor cells to TRAIL-mediated apoptosis by shifting the intracellular ratio of pro-apoptotic and anti-apoptotic molecules . This ca5.4.Histone deacetylase inhibitors (HDACi) are a new class of antitumor drugs that epigenetically alter gene expression through increased histone acetylation , which i6.i.e. immunization), and/or to introduce genes that encode cytotoxic proteins. There has also been a great deal of work performed in the gene therapy arena focused on improving the delivery of therapeutic genes by modifying viral vectors to increase their tropism for target cells, or by restricting transgene expression via incorporation of cell/tissue-specific promoters. Regardless of its intended use, one of the most important aspects of viral vector-based gene therapy is that this type of treatment should be capable of treating the disease in question while remaining benign to the host.Depending on the disease, gene therapy has become a viable therapeutic alternative for patients over the last decade because this technology can be used to restore proper gene expression in host cells, to express proteins with the intent of stimulating immunity , which is activated upon entry into the host cell and vital for transcription and function of the other early genes [E1A also renders Ad replication-deficient and allows space within the genome for transgene insertion. E3 deletion has no apparent effect on viral infectivity [E1A and E3 were deleted to create a replication-deficient vector that could accommodate larger transgene insertions [E2 region deleted to alleviate host inflammatory responses and potential toxicity [Ad vectors can be engineered to remain replication-sufficient, conditionally replication-sufficient, or replication-deficient in the host. First-generation Ad vectors contained a deletion of the early gene 1  require interaction between the viral fiber capsid protein with the coxsackievirus and adenovirus receptor (CAR), and the viral penton base binding to αntegrins -87. Thusntegrins . CAR is ntegrins . Cellulain vitro and in vivo [in vitro [in vitro and in vivo [v integrin expression, which enhances adenoviral infection and transgene expression [Modifications to the Ad vector can aid in overcoming cellular resistance. A modification to the viral fiber, via insertion of an Arg-Gly-Asp (RGD) integrin-binding motif, allows for CAR-independent binding interactions ,89. Fibe in vivo  and coloin vitro . Importain vitro . Similarin vitro ,94. An Apression .Site-specific cell targeting for directed transgene expression is another modification that can be made in Ad vectors. For example, targeting Ad-mediated transgene delivery to tumor cells has been achieved through expression of the transgene under the human telomerase reverse transcriptase (hTERT) promoter . Telomerin vitro and in vivo tumoricidal activity of Ad-TRAIL [We were the first to describe the Ad-TRAIL ,98. ThesAd-TRAIL ,91,99. OAd-TRAIL . Data frAd-TRAIL . In addi6.2.In addition to Ad vectors for gene therapy, AAV vectors have also been engineered and tested as gene transfer vectors. AAV is a small, nonenveloped, single stranded DNA virus . Unlike in vivo [However, AAV vectors are desirable for, and have been successfully used for, gene therapy due to their other unique features. For example, AAV has high tropism for many proliferating and quiescent cell types, and in human cells AAV is able to site-specifically integrate into chromosome 19. Despite chromosome integration, AAV transgene expression is not life-long in the host; however, expression does have the potential to persist for several years in vivo ,101,102.in vivo . Modificin vivo . Also, mt al. utilized AAV2/5 encoding TRAIL and showed increased tropism for the A549 cell line in vitro and in vivo, while histology and primary cell culture showed no detectable toxicity to normal tissues [In vivo experiments exhibited hepatocellular specific tumor cell death and tumor regression when treated with AAV-hTERT-TRAIL [et al. achieved hepatocellular-specific TRAIL expression by fusing the human insulin signal peptide to TRAIL (AAV-ISN-T) within the vector [The positive features of AAV have made it a popular vector option for TRAIL gene administration. In a model of colorectal cancer, AAV-encoded expression of TRAIL resulted in rapid transgene expression, followed by apoptosis of several tumor cell lines and either complete eradication of tumors or blunted progression of existing tumors . Similar tissues . Similar tissues ,108. Tum tissues . Treatme tissues ,110. In RT-TRAIL -111. Tise vector . Oral ade vector .6.3.in vitro, but was not as efficacious in vivo [With low cytoxicity in normal tissues and site-specific therapeutic effects, treatment with Ad- or AAV-vector encoded TRAIL in pre-clinical studies has provided strong evidence for future gene therapy of cancer patients. While TRAIL-encoding Ad and AAV have been the most popular vectors so far, other TRAIL-encoding vectors have been investigated. Lentiviral-mediated TRAIL expression was shown to be successful for specific induction of apoptosis in human lung cancer cells  in vivo . A genet in vivo , indicat7.In summary, the use of TRAIL-based gene therapy for cancer patients holds a great deal of promise. Known limitations of TRAIL, such as resistance to TRAIL-mediated killing, and cancer cell resistance to viral vector infection are being overcome in a variety of ways. Pre-clinical studies in mice have demonstrated the safety and efficacy of viral-based gene therapy using TRAIL, and phase I clinical trial results have demonstrated the safety of this approach. These advances pave the way for future clinical applications of TRAIL gene therapy as a treatment option for a variety of malignancies.