Systemic lupus erythematosus (SLE) is associated with major gastrointestinal complications due to radiotherapy. A patient with active SLE and grade 4 nephropathy presented with inoperable advanced cancer of the cervix which proved to be contraindicated for chemotherapy. The patient was treated with intensity-modulated radiotherapy technique (IMRT). The patient, however, did not experience severe radiotherapy-related complications as expected with conventional techniques of radiotherapy. The tolerance of SLE patients to radiotherapy can thus be achieved by proper delivery of radiation and the sparing of normal tissues by IMRT although further confirmatory studies are required. Severe gastrointestinal tract (GIT) complications induced by radiotherapy (RT) have been well documented in patients with systemic lupus erythematosus (SLE). SLE is a chronic, inflammatory, multi-system disease characterized by remissions and exacerbations. Its pathogenesis shows multiple, immunological aberrations characterized by polyclonal B-cell activation associated with abnormal expression of cytokines.2The use of RT in SLE patients has been considered to be a relative contraindication by many oncologists due to these patients' intolerance to radiation-induced side effects.–5 In SLEIntensity-modulated radiotherapy (IMRT) is a new radiotherapy technique that uses an inverse planning algorithm and allows radiation oncologists to be able to decide the exact dose of radiation to be delivered to the tumor and normal tissues.A 32 year-old lady known to have SLE for eight years, and suffering from hypertension and grade IV lupus nephritis , presented with irregular vaginal bleeding and dysparaunia. Pelvic examination showed a bulky endocervical mass extending to the parametrium, but not reaching the pelvic wall, with an upper third vaginal wall extension. Biopsy revealed a high-grade squamous cell carcinoma. Thoracic, abdominal, and pelvic computed tomography (CT) and magnetic resonance imaging (MRI) confirmed the extension of the tumor to the lower uterine segment, which was associated with enlargement of the internal iliac pelvic lymph nodes without any evidence of metastasis. In addition, a bone scan was performed and showed no bony metastasis. Based on this work-up, the patient was diagnosed with stage IIB cervical cancer.Her renal function was normal as proved by a renogram. Antinuclear antibody titer was 320, antiDNA antibody titer was 361, and other immunological study results were normal. Chemotherapy was contraindicated due to lupus nephropathy and surgery was not performed due to the extension and size of the tumor. Based on the contraindication to have neither surgery nor chemotherapy, the decision was made to treat her with RT. She received local 45 Gray (Gy) pelvic irradiation in 25 fractions (fx) over five weeks, 1.8 Gy/fx, with the IMRT technique. The radiation dose to the bowel was calculated to be as low as possible (see below).45Gy  < 1%, V40 Gy of 40%, and V30 Gy of 77%.The target volume and bowel were delineated with the aid of CT with contrast and fused MRI images as shown in 45 Gy = 0.4%, V40 Gy = 7.6%, and V20Gy = 22%.It is evident from the DVH that the small bowel dose ranges from 1 to 105.7% with a mean dose of 22.6%. The differential doses were Vst week, ii) diarrhea which is the most worrisome expected side effect that started in the fourth week (after administering 36 Gy) and that was not more than four motions/day over the baseline (once daily). The rectal discomfort during defecation (G1 proctitis) started to appear in week 5 as shown in The acute GIT side effects were graded and recorded using the common toxicity criteria modified by the National Cancer Institute of Canada (CTC-NCIC). The gastIt is challenging to use radiotherapy in SLE patients due to severe acute and late side effects. In this In their review of the Michigan University experience, Lin and his colleagues showed that 86 patients had collagen vascular diseases and concluded that radiotherapy is associated with moderate side effects, but they were nevertheless not prohibitive for its use. FurthermThis report reveals that decreasing the radiation dose to the normal tissues by using IMRT is associated with mild side effects. However, more data are needed to reach conclusions in the use of radiation therapy in patients with active connective tissue disease, including SLE.


One strategy available to public health officials during a pandemic is physician recommendations for isolation of infected individuals. This study was undertaken during the height of the Australian pandemic (H1N1) 2009 outbreak to measure self-reported willingness to comply with physician recommendations to stay home for seven days, and to compare responses for the current strain of pandemic influenza, avian influenza, seasonal influenza, and the common cold.Data were collected as part of the Queensland Social Survey (QSS) 2009, which consisted of a standardized introduction, 37 demographic questions, and research questions incorporated through a cost-sharing arrangement. Four questions related to respondents' anticipated compliance with a physician's advice to stay home if they had a common cold, seasonal influenza, pandemic (H1N1) 2009 influenza or avian influenza were incorporated into QSS 2009, with responses recorded using a balanced Likert scale ranging from "very unlikely" to "very likely." Discordance between responses for different diseases was analysed using McNemar's test. Associations between demographic variables and anticipated compliance were analysed using Pearson's chi-square or chi-square for linear-by-linear association, and confirmed using multivariate logistic regression; p < 0.05 was used to establish statistical significance.Self-reported anticipated compliance increased from 59.9% for the common cold to 71.3% for seasonal influenza (p < .001), and to 95.0% for pandemic (H1N1) 2009 influenza and 94.7% for avian influenza . Anticipated compliance did not differ for pandemic (H1N1) 2009 and avian influenza (p = 0.815). Age and sex were both associated with anticipated compliance in the setting of seasonal influenza and the common cold. Notably, 27.1% of health and community service workers would not comply with physician advice to stay home for seasonal influenza.Ninety-five percent of people report they would comply with a physicians' advice to stay home for seven days if they are diagnosed with pandemic (H1N1) 2009 or avian influenza, but only 71% can be expected to comply in the setting of seasonal influenza and fewer still can be expected to comply if they are diagnosed with a common cold. Sub-populations that might be worthwhile targets for public health messages aimed at increasing the rate of self-imposed isolation for seasonal influenza include males, younger people, and healthcare workers. In late March 2009 an outbreak of a new strain of influenza A H1N1), swine-origin influenza virus (S-OIV) or "swine flu," was reported in North America N1, swine. The firAt the time the Australian cases peaked, there was no approved vaccine for pandemic (H1N1) 2009 virus; traditional public health measures were critical to containing the outbreak. One strategy available to public health officials is physician recommendations for self-imposed isolation of infected individuals; specifically, to stay home for at least seven days. Such public health measures, however, only work if patients are willing to comply -7. This Data for this study were collected as part of the Queensland Social Survey (QSS) 2009. QSS is an annual state-wide survey conducted by the Population Research Laboratory (PRL) in CQUniversity Australia's Institute for Health and Social Science Research. Through a cost-sharing arrangement, QSS enables researchers and policy-makers to incorporate questions into the survey.Queensland is the second largest Australian state by land area, and the third most populous state. QSS uses a computer-assisted telephone interviewing (CATI) system and trained interviewers to randomly sample households across Queensland, including metropolitan Brisbane (South East Queensland) and the rest of the state (Other Queensland). To ensure equal representation of males and females, households are randomly pre-determined to provide a male or female respondent; if a person of that sex is not available then the household is not included in the survey.QSS 2009 consisted of a standardized introduction, specific questions incorporated by researchers and the University, and 37 demographic questions. The questions were pilot tested by trained interviewers in 92 randomly-selected households, with modifications to the questions guided by both responses from the pilot study subjects and feedback from the interviewers. Final interviewing was conducted between 20 July 2009 and 19 August 2009, between the hours of 10:30 am to 2:30 pm and 4:30 pm to 8:30 pm on weekdays, and between the hours of 11:00 am and 4:00 pm on weekends.Four questions related to respondents' anticipated compliance with a physician's advice to stay home if they had a viral respiratory illness were incorporated into QSS 2009. The four questions were:• If you had a common cold and your doctor recommended that you stay home for at least seven days so as not to infect anyone else, how likely are you to do so?• If you had the regular flu, but not swine or bird flu, and your doctor recommended that you stay home for at least seven days so as not to infect anyone else, how likely are you to do so?• If you had the swine flu and your doctor recommended that you stay home for at least seven days so as not to infect anyone else, how likely are you to do so?• If you had the avian or bird flu and your doctor recommended that you stay home for at least seven days so as not to infect anyone else, how likely are you to do so?Responses were recorded using a 4-point Likert scale ranging from "very unlikely" to "very likely." Responses were subsequently dichotomized as "yes" (very likely or likely) and "no" (very unlikely or unlikely) and cross-tabulated in a 2 × 2 table. Because the data are essentially repeated measures of likelihood to comply under different circumstances, discordance between responses for the different diseases was analysed using McNemar's test. Bivariate associations between relevant demographic variables and anticipated compliance were analysed using chi-square or Fisher's exact test; where demographic variables were recorded as ordinal data, analyses utilizing chi-square for linear-by-linear association were conducted to identify any significant trend effects. Subsequently, multivariate logistic regression was conducted to identify covariates and interaction effects, and to adjust for confounding. Each variable was entered into or removed from the logistic regression model using both forward and backward methods to identify significant covariates, the remaining variables were then individually entered into the model to identify potential confounders. The final model included significant covariates, potential confounders and significant interaction effects. For all analyses, p < 0.05 was used to establish statistical significance; for the multivariate analysis, adjusted odds ratios (AOR) and their 95% confidence intervals (CI) are reported.a priori estimated sampling error at the 95% confidence level was ± 2.9% overall, ± 3.6% for the South East Queensland sub-sample, and ± 5.1% for the Other Queensland sub-sample.QSS 2009 had a target sample size of 1,200 subjects, with 800 subjects from South East Queensland and 400 from Other Queensland; thus the QSS 2009 was approved by the Human Ethics Review Panel at CQUniversity (H09/06-037) and the incorporation of the influenza-related questions was approved by the Human Research Ethics Committee at James Cook University (H3456).QSS 2009 contacted or attempted to contact 3,112 households; 1,536 subjects declined participation, 142 households could not be contacted, and 129 were otherwise ineligible. Thus, the final sample for QSS 2009 included 1,292 respondents; 860 from South East Queensland and 432 from Other Queensland for an overall response rate of 41.5%. The sample was nearly equally divided between males and females (50.2% vs. 49.8%). Younger people (aged 18 - 34 years) were under-represented in the sample and older people (aged > 55 years) were over-represented in the sample, otherwise the demographics of the participants reasonably approximated that of the general population[Responses to the four questions concerning anticipated compliance with a physician's advice to stay home are shown in Table Bivariate associations between demographic variables and anticipated compliance with a physician's advice to stay home for the four viral diseases are shown in Additional file In multivariate analysis, only sex and age remained significantly associated with anticipated compliance, and there was no interaction effect between these two variables.  2009 influenza, and the same level of compliance could be expected in the setting of avian influenza. These findings are similar to those that have been previously reported; our study adds data in the context of an actual, rather than hypothetical, pandemic.Prior to the current pandemic, Eastwood et al read a brief description of a pandemic influenza outbreak analogous to the 1918 Spanish flu to Australian telephone survey participants, and found 97.5% of respondents would stay home for seven to ten days if they were told they might have had contact with the disease  reportedFrom a public health planning perspective, the more useful data from this study may be that regarding the level of compliance with stay at home advice that can be anticipated for seasonal influenza, and the relative lack of compliance that can be expected for the common cold. Seasonal influenza is a more common disease, each year leading to approximately 18,000 hospitalizations and costing around $115 million in Australia; the burden in the United States is much greater with the direct costs of influenza-related medical care exceeding $10 billion . Yet, thThis study did find some significant associations between demographic characteristics and likelihood to comply with stay at home advice for seasonal influenza that might be useful for targeting public health efforts to increase compliance. Males were less likely to report anticipated compliance with stay home advice for both a common cold and seasonal influenza, and this is consistent with other studies from Australia ,9. MalesIncreasing age was associated with increased anticipated compliance with stay at home advice for both the common cold and seasonal influenza, while increasing education and income were associated with decreased anticipated compliance for both diseases. Although the associations for education and income did not withstand multivariate analysis, the finding is consistent with previous work and both variables were retained as potential confounders in the final logistic regression model. Like males, wealthier and better educated people tend to view themselves as less susceptible to influenza, while older people tend to view themselves as more susceptible . Many inEmployed respondents were less likely than unemployed respondents to report anticipated compliance with stay home advice for both a common cold and seasonal influenza. This association, also, did not withstand multivariate analysis, but it is an intuitive finding. Even in the setting of pandemic influenza, many people would have to forgo income in order to stay home . For exaA particularly novel and important finding of this study was that more than one-quarter of health and community service workers reported they would not comply with a physician's advice to stay home if they had seasonal influenza. This may represent a misplaced sense of duty. Previous research has demonstrated that most healthcare workers (HCWs) would not abandon their responsibilities during an influenza pandemic,,17 but iThis study was limited in that it relied upon a telephone survey to collect data, but telephone surveys have been previously used to gather information regarding public perceptions of risk and willingness to comply with containment strategies for influenza,,7,9-12 aNinety-five percent of people report they would comply with a physicians' advice to stay home for seven days if they are diagnosed with pandemic (H1N1) 2009 or avian influenza, but only 71% can be expected to comply with the same advice in the setting of seasonal influenza and fewer still (60%) can be expected to stay home if they are diagnosed with a common cold. Sub-populations that might be worthwhile targets for public health messages aimed at increasing the rate of self-imposed isolation for seasonal influenza include males and younger people. Notably, more than one-quarter of health and community service workers report that they are unlikely to comply with stay home advice for seasonal influenza; thus they too may be an appropriate  target for influenza-related public health campaigns.CATI: computer-assisted telephone interviewing; PRL: Population Research Laboratory; QSS: Queensland Social Survey; RR: relative risk; SARS: severe acute respiratory syndrome; S-OIV: swine origin influenza virus.The authors declare that they have no competing interests.LHB, PA, PAL and RS participated in the development of the research question and the influenza-related questionnaire items for inclusion in QSS 2009. LHB conducted the primary analysis. LHB, PA, PAL and RS participated in the interpretation of the data and the initial drafting of 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/1471-2458/10/138/prepubTable S1 - Bivariate associations between demographic variables and anticipated compliance with physician's advice to stay home for seven days for common cold and three strains of influenza. A table showing the bivariate associations between demographic variables and anticipated compliance.Click here for fileTable S2 - Final models and results of the multivariate logistic regression. A table showing the final models, coefficients, and adjusted odds ratios for the logistic regressions predicting anticipated compliance for the common cold and seasonal influenza.Click here for file


Colorectal cancer (CRC) is considered a complex disease, and thus the majority of the genetic susceptibility is thought to lie in the form of low-penetrance variants following a polygenic model of inheritance. Candidate-gene studies have so far been one of the basic approaches taken to identify these susceptibility variants. The consistent involvement of some signaling routes in carcinogenesis provided support for pathway-based studies as a natural strategy to select genes that could potentially harbour new susceptibility loci.We selected two main carcinogenesis-related pathways: Wnt and BMP, in order to screen the implicated genes for new risk variants. We then conducted a case-control association study in 933 CRC cases and 969 controls based on coding and regulatory SNPs. We also included rs4444235 and rs9929218, which did not fulfill our selection criteria but belonged to two genes in the BMP pathway and had consistently been linked to CRC in previous studies. Neither allelic, nor genotypic or haplotypic analyses showed any signs of association between the 37 screened variants and CRC risk. Adjustments for sex and age, and stratified analysis between sporadic and control groups did not yield any positive results either.Despite the relevance of both pathways in the pathogenesis of the disease, and the fact that this is indeed the first study that considers these pathways as a candidate-gene selection approach, our study does not present any evidence of the presence of low-penetrance variants for the selected markers in any of the considered genes in our cohort. Colorectal cancer (CRC) is one of the main forms of cancer, being the second most frequent neoplasm in both sexes and one of the most important morbidity causes in the western world Candidate-gene studies have been one of the most commonly used tools in the screening for new variants affecting CRC risk. Gene selection in these studies is mainly based on the functional implications of a possible association, and thus genes selected have either been chosen because of the previous presence of other high/low risk alleles This latter approach has also allowed, together with the development of high-throughput technologies, the implementation of new hypothesis-free approaches (in opposition with hypothesis-based candidate-gene approaches), covering the majority of the genome (genome-wide association studies or GWAS). This implementation has successfully led to the identification of some new susceptibility loci We hence aimed to find such susceptibility variants through a candidate-gene approach screening a selected number of variants within two cellular pathways that have consistently been linked to CRC tumorogenesis: the Wnt and the BMP signaling pathways APC and familial adenomatous polyposis APC are found in almost 80% of the sporadic colorectal cancers, and Wnt signaling activation is involved in the best part of sporadic colorectal carcinomas SMAD4 and BMPR1A, are responsible for juvenile polyposis syndrome, another hereditary CRC condition cis-regulatory sequences (5′ and 3′ unstranslated regions) were selected to analyse their relationship with CRC susceptibility.The Wnt pathway contains genes that have for long been known to be responsible of some hereditary CRC syndromes, such as AXIN1, HDAC9, BMP4, DACT1, CDH3, CDH1, BTRC, and APC), , . Stratifnd APC), .Thus, our strategy has not managed to detect any new susceptibility loci for CRC risk.Pathway-based expectations have proved to be quite discouraging in the literature as well, for strong candidate pathways, such as DNA-repair ones, surprisingly failed too in identifying any new risk variants BMP4 and CDH1 gene regions with the disease Still, in light of the recent discoveries that followed up the analysis of genome-wide data, both Wnt and BMP have earned a renewed fame. The susceptibility locus found on 8q24 (rs6983267) has been linked to an enhanced Wnt signaling through its interaction with TCF4 APC, CCND1, CDH1 and TCF7) had already been screened for risk alleles APC (rs459552). This missense change is widely documented in the literature, with some studies defending it as neutral (this study and others)Even though this is actually the first association study that considers the pathways as a whole for gene selection, some of the genes included in our analysis , and 1.24 for MAFs down to 0.2 (78% of the SNPs), assuming a log-additive model and α = 0.05, we were unable to detect any positive associations suggesting the presence of any new CRC susceptibility variants. Nevertheless, it is quite remarkable that, albeit our failure to replicate the associations for the a priori good candidates to be directly associated with the disease. This indeed may constitute a limitation in the study, for most of the genetic variation within the loci was not investigated. Thus, we believe further efforts should be made to screen a wider variety of loci within these pathways, specially considering the previous positive associations described so far for both Wnt and BMP-related genes.Despite our negative results, we must consider that we did not whatsoever comprehensively cover all possible low-penetrance variants within the selected genes. This is mainly due to the fact that our strategy was purely functional, selecting the variants that were Pondering the potential odds ratios of the variants described so far , we assume larger cohorts may be required to detect such subtle effects. On the other hand, when considering candidate-gene approaches, it would also be useful to meta-analyse previous studies and pull the information across of them altogether in the search of evidences of potential new pathways linked to the pathogenesis of the disease.de-novo histologically confirmed diagnosis of colorectal adenocarcinoma and who attended 11 community hospitals across Spain between November 2006 and December 2007. Patients in whom CRC developed in the context of familial adenomatous polyposis or inflammatory bowel disease, and cases where patients or family refused to participate in the study were excluded. Demographic, clinical and tumour-related characteristics of probands, as well as a detailed family history were obtained using a pre-established questionnaire, and registered in a single database. Of these, 592 (63%) were male and 341 (37%) female. Median age for cases was 73 (range 26–95), whereas mean was 71(SD±10.7). Hospital-based controls were recruited together with cases and were confirmed to have no cancer or prior history of neoplasm, and no family history of CRC. All controls were randomly selected and matched with cases for sex and age (±5 years) in a 1∶1 ratio. Both cases and controls were of European ancestry and from Spain.Subjects were 933 CRC patients and 969 controls that belonged to the EPICOLON project, a prospective, multicentre, population-based epidemiology survey studying the incidence and features of familial and sporadic CRC in the Spanish population The study was approved by the “Comité Ético de Investigación Clínica de Galicia”, and each of the institutional review boards of the hospitals where samples were collected . All samples were obtained with written informed consent reviewed by the ethical board of the corresponding hospital.DNA was obtained from frozen peripheral blood; extraction was performed in a CHEMAGEN robot  in accordance with the manufacturer's instructions, at the Galician Public Fundation of Genomic Medicine in Santiago de Compostela. Cases and controls were extracted in mixed batches to avoid any kind of bias.Both Wnt and BMP pathways were initially selected after the findings of Nishanian et al. cis (5′or 3′ UTR ends), as defined by the FESD web browser SNP selection criteria only considered functional markers with minor allele frequencies above 0.05 and at least two independent validation criteria as established in dbSNP BMP4 and CDH1, respectively, that have been recently reported to be associated with the disease BMP4, and 0.02 for those in CHD1) (rs4444235 and rs9929218 are two variants lying in the near-by and intronic regions of in CHD1) , we deciGenotyping was performed with the MassARRAY  technology at the Santiago de Compostela node of the Spanish Genotyping Center. Calling of genotypes was done with Sequenom Typer v4.0 software using all the data from the study simultaneously.2 test (1df). All p-values obtained were ≥0.05, thereby excluding the possibility of genotyping artifacts (data not shown). Population stratification was assessed with Structure v2.2 smartpcaQuality control was performed, first by excluding both SNPs and samples with genotype success rates below 95%, with the help of the Genotyping Data Filter (GDF) Association tests were performed by chi-squared tests for every single SNP and haplotypes where possible with both Haploview v4.0 Figure S1Haplotype structure and analysis for the 8 genes for which more than one SNP was genotyped. The table shows association values for each SNP generated by Haploview.(3.40 MB TIF)Click here for additional data file.Figure S2Principal component analysis plot for the first vs. second component, comparing our case and control populations.(0.96 MB TIF)Click here for additional data file.Note S1(0.03 MB DOC)Click here for additional data file.


Yttrium-90 chelated by the tetraphosphonate EDTMP achieved a high uptake in bone and a rapid clearance from all soft tissues compared with 90Y nitrilotriacetate, citrate and acetate. The biological half-life of 90Y in the bone was greater than 72 h, but the quantity, and therefore dose, could be reduced by 50% using repeated, non-toxic chelation therapy with the calcium salt of DTPA. This treatment should be able to supplement current treatments for leukaemia where the dose of external beam radiation is associated with considerable morbidity.


Health risks linked to obesity and the difficulty most have in achieving weight loss underscore the importance of identifying dietary factors that contribute to successful weight loss.2 and without chronic illness enrolled in 2004 in a randomized trial evaluating behavioral treatments for long-term weight loss. Subjects completed a 62-item food frequency questionnaire at baseline and at 6, 12, and 18 months.This study examined the association between change in dietary energy density and weight loss over time. Subjects were 213 men and women with BMI of 30–39 kg/m2) of those in the quartile representing greatest decrease in energy density at 18 months compared to those in the quartile with the least was -1.95 (p = 0.006). The association was especially strong in the first six months (estimated β = -1.43), the period with greatest weight loss (mean change in BMI = -2.50 kg/m2 from 0–6 months vs. 0.23 kg/m2 from 12–18 months) and the greatest contrast with respect to change in energy density.Pearson correlations between BMI and energy density  at baseline were not significantly different from zero . In a longitudinal analysis, change in energy density was strongly related to change in BMI. The estimated β for change in BMI (kg/mDecreased energy density predicted weight loss in this 18 month weight loss study. These findings may have important implications for individual dietary advice and public health policies targeting weight control in the general population Further30 kg/m2)-3. Givencancers) -6, identper se was investigated as a predictor of weight change, subjects with the greatest decrease in energy density achieved the greatest weight loss[Dietary energy density, or an individual's average daily energy intake divided by the grams of food consumed per day, is a factor that could help explain the broader trends in obesity prevalence and that could also serve as a potentially important point of focus in efforts to achieve weight loss. At the population level, dietary energy density is associated with energy intake and weight status in a limited number of cross-sectional studies -10. At tight loss.We conducted a longitudinal analysis of data collected from a randomized weight loss trial. It was our primary aim in these analyses to determine if a change in dietary energy density would result in weight loss over 18 months of follow-up, a follow-up period that extended beyond what has been reported in the small number of prior studies of energy density and weight loss. We had the secondary aim of analyzing the data at intermediate stages of follow up in order to explore the dynamics of the relationship between changes in energy density and weight loss and to provide insight into potential mechanistic explanations for the associations we observed.Subjects: Participants in the study were 100 men and 113 women recruited in 2004 and 2005 to participate in the Lose It Forever (LIFE) Study, a randomized clinical trial designed to compare the efficacy of prescribing different behavior combinations over time during therapy as a means of achieving and then maintaining weight loss.2, be free from serious medical conditions or other consideration that would contraindicate treatment , agree to be randomized to either of two treatment groups, and provide their informed consent to participate. The study protocol was approved by the Institutional Review Boards at both the University of Minnesota and University of Washington.Participants were recruited by general mass media advertising and specialized advertising targeting men and minorities. To be eligible for the study, subjects had to be at least 18 years of age, have a body mass index (BMI) between 30 and 39 kg/mSince the primary endpoint for this analysis was weight loss at 18 months of follow-up, we included only those subjects who had weight measurements at the 18 month clinic visit (see below) in the final analyses as well as in the cross-sectional analyses at baseline and in the analyses of intermediate time points. Doing so resulted in an analytic sample of 155 subjects (73% of those randomized). Including all subjects for the baseline cross-sectional analyses or including subjects with weight measures at the 6 or 12 month clinic visits but not the 18 month visit did not produce results materially different from those using only the subjects who had weight measures at 18 month completion point (data not shown).LIFE Study Intervention: The present analysis uses the LIFE Study data in an observational manner, though the larger study used an experimental design as described here. Following enrollment, participants were randomized to one of two treatment groups. The treatment in both groups was conducted in small groups of 11 to 21 individuals at the Epidemiology Clinical Research Center at the University of Minnesota. The format of both treatment groups was comprised of a presentation by treatment staff of recommendations for changes in diet and physical activity behaviors necessary for successfully controlling weight, interactive discussion of these behavioral goals and strategies to achieve them, and homework assignments to be completed between sessions to reinforce learning.per se was not the focus of any of the weight loss units for either treatment arm. Encouragement to eat lower energy foods was a recurring theme, however, and individuals who act on this encouragement will often eat a lower energy density diet.Content for the Standard Behavioral Treatment (SBT) group was closely modeled after prior work of the investigatorsand closeOutcome and Covariate Measures: The primary outcome was change in BMI. Study personnel used standardized procedures to measure subjects: Weight measurements were repeated, in light clothing without shoes, on an electronic scale during assessment sessions  at baseline and at 6, 12, and 18 months of follow-up. Height was measured at the baseline assessment visit using a wall mounted stadiometer. LIFE Study personnel called participants prior to each six-month outcome assessment session to encourage attendance. In the event of a no-show at the assessment session, study personnel made multiple reschedule attempts.Demographic characteristics of age, gender, and ethnicity were assessed at baseline. The Paffenbarger Activity Questionnaire, a well known assessment instrument with established validity and reliability -22, was i.e., without coaching from study personnel, although study personnel did review the FFQs for completeness accuracy when participants submitted them). Detailed descriptions of this FFQ and its validity have appeared elsewhere [Dietary Assessment: Subjects completed the 62-item Block Food Frequency Questionnaire (FFQ) to assess usual intake in the previous 6 months. Subjects completed the questionnaires individually and in a self-administered manner (lsewhere -25. Softvs. SBT) since treatment group was related to change in BMI (data not shown) and to change in energy density  into a single observational analysis. We used PROC CORR in the SAS statistical software package (version 8.2) to identify cross-sectional Pearson correlation coefficients among baseline values for BMI, energy consumed, and energy density of the diet. We used PROC GLM to generate beta estimates for change in BMI across quartiles of energy density change. We categorized subjects according to quartiles of change in energy density. In this way quartile 1 (Q1) contained subjects with a large decrease in energy density from baseline, and they had large negative values for change in energy density. Thus if we use Q4  as the reference category, comparing Q1 to Q4 will compare those with large decreases in energy density to those with no change or even an increase in energy density. All beta estimates were derived from multivariable-adjusted models controlling for age, ethnicity, gender, change in physical activity level from baseline (based on Paffenbarger score), and treatment group . By contrast, we did observe a positive correlation between energy density and total energy consumed. This association was weak if we included any liquid foods in the definition of energy density, but was relatively strong  if we considered only solid foods.We observed no correlation between energy density and BMI at baseline in a cross sectional analysis Table . This revs. Q4 (no change or slight increase in energy density) was -1.95 kg/m2 (p = 0.006), which is roughly equivalent to 5 or 6 kg Looking at different phases of follow-up, we observed that the estimated β was strongest in the first 6 months of the intervention period  and grew noticeably smaller as time went forward such that by the 12 month to 18 month period the association was essentially null .In a longitudinal analysis, the subjects who had the greatest decrease in energy density from baseline to 18 months had significantly decreased BMI compared to those who had the least change in energy density Table . The mulvs. Q4) during the different phases of follow-up represented much different levels of change in both the energy density and BMI variables during the different time periods. During the first six months of follow-up, Q1 represented a mean decrease in energy density of 1.39 kcal/g while Q4 represented a mean decrease of only 0.05 kcal/g. By contrast, in the 12–18 month period, Q1 represented a decrease in mean energy density of just 0.05 kcal/g while Q4 represented an increase of 0.38 kcal/g. Thus the difference between Q1 and Q4 was more than 3 times greater during the 0–6 month period than it was in the 12–18 month period. The absolute decrease in energy density in Q1 was more than 20 times greater during the early period of follow-up compared to the 12–18 month period.That the relationship between change in energy density and weight change would decline over time may at first appear puzzling, but it is important to note that the comparison that formed the basis of the β estimates  to those who reported no change or a slight increase in energy density (Q4) (data not shown).We also compared grams of food consumed and energy consumed at each clinic visit by quartile of energy density change between 0 and 18 months of follow-up Table . Resultsi.e., weight) of food they consumed but did have notable changes in energy intake. These observations are consistent with what Rolls and colleagues have observed in short-term feeding studies where study subjects consuming diets differing in energy density ate a constant volume of food but the low energy-density diet subjects consumed significantly fewer calories[In this 18 month weight loss intervention study, we observed that subjects with the greatest degree of change in energy density did not change the amount , but no one had a BMI above 39 kg/m2. By restricting the range of values for BMI in this way, we may have obscured the cross-sectional association most previous investigators have observed.Some of our findings were somewhat unexpected, however. For example, despite prior studies consistently showing an association between energy density of the diet and BMI -10, we fi.e., -1.39 kcal/g for those in the quartile with the greatest decline in energy density from 0–6 months compared to -0.05 kcal/g for those showing the greatest decline in energy density from 12–18 months) and in relative difference  suggest that this apparently changing association may simply be a function of the greater range in exposure levels early in the follow-up period compared to later.A second unexpected finding was that the association between change in energy density and change in weight or BMI was different at different phases of follow-up. During periods of active weight loss, that weight loss was strongly correlated with decreased energy density, but later in follow-up, the association was markedly attenuated. Our evaluation of the magnitude of change in energy density during each of these time periods showed that large changes in energy density, both in absolute magnitude , is worth noting. This result suggest that the variation in energy consumption that comes from changes in energy density is primarily a function of solid foods, which should not be surprising as liquid foods as a rule are low in energy density[That the association of energy density with weight loss and energy intake was strongest if we defined it only in terms of solid food consumed  would be required for effective weight loss. Alternatively, the low energy density diets reported in the quartile showing the least change in energy density over the course of the study might be erroneous and as such reflect poor dietary self awareness. Given that all subjects had starting BMIs of between 30 and 39 kg/m2 and thus had similar degrees of energy imbalance, it is perhaps not unreasonable to consider that those reporting the lowest energy density diets were in fact underestimating the amount of high energy density foods consumed relative to the rest of the study population. Thus reporting a low energy density diet at baseline may be an indicator of poor behavioral compliance  among subjects who do not recognize the energy density of their diets at the outset. These explanations are strictly hypothetical, however, and require further investigation in future studies before any conclusions are possible.An interesting secondary observation in this study was the poor weight loss outcome in subjects starting with low energy density diets at baseline. There are several possible explanations for this result. The first is simply that subjects eating high energy density diets had substantial room for improvement on that front, and when they did reduce the energy density of their diets, their energy intake dropped in turn. For subjects with low energy density at baseline, however, no additional improvement of this type was possible. For the latter group of subjects, reducing energy intake could not happen through changes in energy density of the diet, and alternative strategies (over reporting of "good" habits (eating low energy density foods) in those who were successful at weight loss. While we cannot exclude this possibility, the reverse scenario  seems much more likely. That we found a positive result despite the limitations of the FFQ is notable.The use of a food frequency questionnaire to assess energy density of diet might be a point of criticism for this study as most investigations of energy density have been short term feeding studies with tightly regulated (and measured) food intakes. There is no question that food frequency questionnaires measure diet with error, so much so that some have suggested dispensing with them altogether. But thiper se could have different effects in populations with different ethnic or educational profiles. Nonetheless, some caution is warranted in interpreting these results.A final limitation of the study may be in the demographic makeup of the study population. Results from this mostly white, generally well-educated population (in excess of 97% had more than a high school education) may not be generalizable to the broader public. Certainly, it is reasonable to consider if achieving these changes in dietary energy density would be similarly practical in different populations, but it is not immediately clear how change in energy density In summary, decreases in energy density predicted weight loss in an 18 month observational analysis. Changes in energy density predicted weight loss most strongly in the first 6 months of follow-up. Diminished predictive value of energy density later in follow-up may be due to smaller changes in energy density during this period making for a less sensitive analysis. Volume of food consumed did not change with decreasing energy density suggesting energy intake went down, but curiously, controlling for energy intake did not eliminate the effect of changing energy density on weight loss.The authors declare that they have no competing interests.AF participated in the design of the study, designed the analysis, interpreted the data, and drafted the manuscript. NM performed the data analysis and assisted in drafting the manuscript. MJ and EAF assisted in the design of the study and supervised the operations of the weight loss intervention activities. SLL, PSL, EMW, and AH directed the intervention activities. RLL, and RWJ designed the study, provided overall study management, and assisted in the drafting of the manuscript. All authors read and approved the final manuscript.


Gene therapy has the potential to counter problems that still hamper standard HIV antiretroviral therapy, such as toxicity, patient adherence and the development of resistance. RNA interference can suppress HIV replication as a gene therapeutic via expressed short hairpin RNAs (shRNAs). It is now clear that multiple shRNAs will likely be required to suppress infection and prevent the emergence of resistant virus.status quo.We have developed the first biologically relevant stochastic model in which multiple shRNAs are introduced into CD34+ hematopoietic stem cells. This model has been used to track the production of gene-containing CD4+ T cells, the degree of HIV infection, and the development of HIV resistance in lymphoid tissue for 13 years. In this model, we found that at least four active shRNAs were required to suppress HIV infection/replication effectively and prevent the development of resistance. The inhibition of incoming virus was shown to be critical for effective treatment. The low potential for resistance development that we found is largely due to a pool of replicating wild-type HIV that is maintained in non-gene containing CD4+ T cells. This wild-type HIV effectively out-competes emerging viral strains, maintaining the viral The presence of a group of cells that lack the gene therapeutic and is available for infection by wild-type virus appears to mitigate the development of resistance observed with systemic antiretroviral therapy. Human Immunodeficiency Virus type 1 (HIV-1) is a positive strand RNA retrovirus that can cause Acquired Immunodeficiency Syndrome (AIDS) resulting in destruction of the immune system. HIV infection is currently treated with Highly Active Anti-Retroviral Therapy (HAART), a combination treatment of 3 or more drugs that significantly reduces viral replication and disease progression [et. al. [et. al. [RNA interference (RNAi) is a relatively recently discovered mechanism of gene suppression that has received considerable attention for its potential use in gene therapy strategies for HIV (for Reviews see -4). RNAiet. al.  and ter [et. al. , our gro[et. al. .et. al. unpublished data). Our analysis indicates that at least 6 highly conserved shRNAs are required to ensure that 100% of Clade B patients will have complete homology to at least 4 of these shRNAs.While shRNA is known to be an effective tool to regulate gene expression, the efficacy of single shRNAs in treating HIV infection is limited due to the rapid development of resistance in the target region -12. ManyGene therapy is an emerging technology that has demonstrated clinical efficacy and biological effect in treating diseases such as severe combined immune deficiencies  ,14 and ctat/vpr specific anti-HIV ribozyme (OZ1) in autologous HSC [Mathematical modelling of gene therapy has been limited and has mostly considered the average response over time of frequent and predictable events such as CD4+ T cell numbers and HIV viral load -20. Despgous HSC . Over 10et al. [et al. [In that phase II study , there wet al.  investig [et al. . It is iThe model was developed to determine i) how many shRNAs and ii) their level of inhibition (when delivered to HSC as a gene therapeutic), are required to prevent virological escape. The stochastic model incorporated a 3-dimensional space to represent lymphoid tissue where transmission of HIV is high, and tracked the survival and expansion of individual cells and the evolution of viral sequences in the shRNA targeted region. Using conservative assumptions, we found that combinations of 4 or more shRNAs can stabilize infection at a low level, as long as the shRNAs act prior to integration of pro-viral DNA. Escape mutants did not emerge due to a pool of wild-type (wt) virus replicating in UNTx cells. This wt virus effectively out-competes all emerging mutated strains of reduced fitness. This indicates that gene therapy delivered to HSC can suppress viral load, and can forestall the development of resistance due to a sizeable proportion of cells that do not contain the gene therapeutic. This produces a situation very different to systemic HAART where the drugs are distributed at varying concentrations across all target cells.3  CD4+ T cells and was followed for 5,000 days, with data collected every 12 hours. Each shRNA was assumed to inhibit both incoming virus prior to integration (Class I) and nascent viral transcripts produced from integrated proviral DNA (Class II); see Methods for a more complete model description. CD34+ HSC were assumed to have been transduced with the gene therapy ex vivo and returned to the patient to engraft and to continuously give rise to a supply of gene-containing CD4+ progeny T cells through the thymus [The model was designed to monitor the impact of HSC-delivered gene therapy, in which a combination of non-overlapping shRNAs were expressed, on the development of resistance in a 3-dimensional cube representing lymphoid tissue. The cube contained 70e thymus . A propo-5 mutations per HIV RNA nucleotide per round of replication [Each of the scenarios in Table lication . With thIn the absence of gene therapy, the proportion of infected cells increased rapidly and completely saturated the tissue in less than 500 days Figure . A propoThe first gene therapy scenarios that we modeled compared the expression of 2 S3), 4 (S2) and 6 S1) independent shRNAs  When 2 shRNAs provided inadequate protection, the resistance profile indicated that > 99% of replication was wt and occurred in approximately equal amounts in the UNTx 38%) and Tx (36%) compartments  conservation amongst different viral variants and ii) experimentally determined suppressive activity. We have previously identified suitable anti-HIV shRNAs that are both highly active (> 75% efficacy) and whose target sequence is highly conserved. We used the model to determine if a reduction in shRNA efficacy is likely to affect overall infection or resistance profiles, assuming shRNAs can target both incoming and nascent viral transcripts -26. We sThe reduction in efficacy from 80% (S1) to 60% S4) led to a slight increase in the number of infected cells after 5000 days  to 1% (S5) increased the number of infected cells from 35 to 42% after 5000 days  was chosen to ensure i) that low frequency events could be meaningfully quantified, including the evolution of randomly mutated strains occurring in the absence of gene therapy and ii) consistency of results. As a validation of the model, it is relevant that variations in assumptions between scenarios produced quantitative results in the expected ordering of percentage resistant mutations and the variation over the 10 simulations for each scenario were small Table . Neverthdividual  suggest Not only is HIV established in short-lived activated CD4+ T cells, but it also infects resting CD4+ T cells, monocytes and macrophages and creates a latent pool. These other cell types can produce virus over lengthy life-spans and latently infected cells in particular exhibit the history of infection evolution within the individual. They are also strongly implicated in re-establishing high viral levels after the cessation of antiretroviral therapy. Hence a realistic model of HIV infection should duplicate i) infection not being in all target cells, ii) infection being maintained even at low levels, iii) and long-lived infected cells stopping eradication of virus even when infection is reduced to very low levels. Our model was designed to replicate these properties and the results presented show that it achieves these goals.The inclusion of long-lived infected cells in the model was necessary in achieving these properties, as is expected in vivo as well. If the model only included short-lived infected cells then infection in the absence of the gene therapy either swamped the entire population or was completely extinguished. Moreover the addition of the gene therapeutic also either completely extinguished the infection, or established itself in all cells due to the high turnover with extensive infection. None of these situations duplicated what is expected to occur in practice and so models consisting solely of short-lived cells were discarded. It is interesting that the inclusion of a long-lived infected cell component allowed a better model of HIV infection both in the presence and absence of gene therapy. Although long-lived infected cells play an important role in vivo their half-lives have been estimated to be between 2 weeks and 44 months ,37, and The model assumed that every infected cell that died was replaced by a new cell from the thymus or by one of its neighbors regardless of its phenotype, and if selective pressure is high, this results in Tx cells quickly becoming the dominant population. While this assumption and outcome are conservative, its consequence is that the selection pressure for resistant virus was even greater than would be expected. Further, it was assumed that each shRNA inhibited virus by 80% compared with wt, and that the effect of each additional shRNA was multiplicative irrespective of the presence of other shRNAs. For example, 6 shRNAs exhibited a 99.994% efficacy. However, multiple short interfering RNAs (siRNAs) and shRNAs may compete with each other and with host miRNAs for access to the RNAi machinery and therefore may not inhibit their targets as effectively as if they were expressed independently at maximal levels ,38-42. FOur own experience, as well as that of others, in gene therapy delivered to HSC and/or directly to CD4+ T cells, indicates that an anti-HIV gene therapy will not lead directly to the development of an entire CD4+ T cell population containing the therapeutic gene(s). It will likely be contained within a minority of these cells. Hence gene therapy provides a very different scenario to systemic antiretroviral therapy where every cell is bathed in some concentration of drugs. Our model was also designed to duplicate this situation where there should always be a sizeable proportion of target cells that do not contain the gene therapeutic. Given that this would present a situation very different to systemic therapy our model was designed specifically with this in mind, and it also achieves this goal.et al. [The model was designed to follow cells, their survival and their replacement longitudinally through multiple rounds of possible infection. Unlike the model of Leonard , it alloet al.  studied et al. -51.In summary, resistance to gene therapy appears to differ from that for antiretroviral therapy. Although HSC gene therapy aims to establish a large protected population of target CD4+ T cells, monocytes and macrophages with the gene therapeutic, there will always be a proportion of UNTx target cells, particularly in the absence of complete bone marrow ablation. Thus there will be two distinct populations of target cells; UNTx cells which have no selective pressure driving the evolution of virus, and Tx cells which have the inhibitory pressure of gene therapy limiting viral replication. This differs from systemic antiretroviral therapy where cells contain a continuous distribution of the inhibitory effects of therapy and therefore provide a spectrum of selection from the outset Figure . Thus, iFailure of gene therapy will occur if there is sufficient replication of virus within Tx cells and if the fitness of the resistant virus remains competitive with wild-type in UNTx cells. This situation is exemplified by cases where the gene therapeutic is suboptimal, allowing preferential expansion and eventual outgrowth of virus within Tx cells. This is shown by scenarios where the gene therapeutic contains 2 shRNA and generates outgrowth of virus regardless of the fitness of resistant virus. However, the modelling shows that viruses containing mutations that confer as little as a 5% and 1% loss in fitness are rapidly outcompeted by the wt in the UNTx cell compartment. This predicts that resistant viruses harbouring a reduction in fitness will not survive in the presence of an adequate gene therapy. The dynamics of virus outgrowth will be determined by i) efficacy of the gene therapeutic inhibiting wt replication in Tx cells, ii) fitness of resistant virus allowing it to replicate in UNTx cells in competition with wt; and iii) the relative sizes of the Tx and UNTx compartments. It will thus be important to develop a gene therapeutic to which resistant viruses have significantly reduced fitness, and that provides maximum inhibition by targeting incoming virus prior to integration. As it is presently unclear whether anti-HIV shRNAs alone can achieve this, multiple shRNA gene therapies may need to be combined with a Class I inhibitor to allow Tx cells to survive and mitigate the effects of HIV.et al. [This stochastic model of gene therapy for HIV incorporated the introduction of multiple anti-HIV shRNA into HSC cells which then differentiated into gene-containing progeny CD4+ T cells (referred to as Tx in the model). This model generated a longitudinal description of the expansion of both Tx cells, and infected cells, while monitoring the development of resistance. While the model incorporates some features similar to the model developed by Leonard et al. , it modeCD4+ T cells were assumed organised on the grid points of a 70 × 70 × 70 3-dimensional lattice in order to roughly duplicate the 3-dimensional layout of CD4+ T cells in a portion of lymphoid tissue. Initially all cells were uninfected and untransduced. The time step in these calculations was taken to be 12 hours.Infection was established on this lattice by randomly choosing cells with a probability of 0.05 to become infected with wild-type virus. This initial infection pre-transduction was run for 200 time steps to generate viral diversity. Within each time step of this initial phase as well as the later post-transduction phase, cells died, were replaced by new cells from the HSC or through cellular division of a neighbouring cell, or were infected by neighbour cells. Each of these components is described in detail below.Transduction with the gene therapy was assumed to establish a proportion of HSC that produced this same proportion of new CD4+ T cells. However, initially no cells on the lattice were transduced. Starting with the distribution of infection established in the initial phase, the model was run over 10,000 time steps, resulting in simulations over 5,000 days.At the final time point, the proportion of cells on the lattice that were infected and/or transduced was calculated. Furthermore we determined the proportion of cells that were totally resistant to 1, 2, 3, ... of the shRNA components. Means and standard deviations over 10 simulations for each scenario are reported.HSC were assumed transduced with a set number of shRNA. Simulations were performed where was either 2, 4 or 6 in separate experiments. Each shRNA target site was assumed to be 19 nucleotides (nt) long. Model parameters and their values are summarised in Table Upon infection a cell was assigned a lifespan from a Poisson distribution with a mean of 4 time-steps (2 days ). Those (eff = 0). A mutation in the remaining regions provided partial resistance (eff = 0.5). Any 2 mutations among the 19 nt rendered the shRNA completely ineffective.Each viral genome was described in terms of the presence or absence (1 or 0) of mutations in each of the 19 nucleotide positions for each of the shRNA. A mutation in the 7 central nucleotides resulted in complete resistance to that particular shRNA N shRNA with no resistance mutations, viral production was scaled down by a factor of N(1-eff). If the infected cell contained a partial or fully resistant mutation to a single shRNA, then the efficacy of the shRNA to which the virus sequence contained the mutation was reduced from 80% to 50% or 0% respectively. Hence a cell infected with a viral strain totally resistant to all shRNA would have the same viral productivity, modulo fitness, as an untransduced cell. Viral production was multiplied by a fitness factor if the viral genome was not wild-type.Each infected cell produced virions at a rate dependent on the sequence of its integrated viral genome and whether it was transduced or not. An untransduced infected cell produced virions at a standardised rate of 1. For an infected transduced cell containing 2), and so forth.It was assumed that all shRNAs inhibited viral production (Class II) but depending on the scenario simulated could also inhibit incoming viral transcripts (Class I) at the same rate . This inIf an uninfected cell possessed infected neighbours then the chance it became infected per time step was determined by the viral production of these cells, the transduction status of the uninfected cell, the viral sequence of the infecting virions, and chance. Viral production from neighbouring infected cells was calculated as described above.P. inf. The resulting value was taken to be the mean of a Poisson distribution. The random number generated from this distribution and for the uninfected cell determined the number of virions that could infect the cell. Multiple infecting virions allowed the possibility of recombination and further viral diversity. The infecting virions were then randomly chosen over all neighbours relative to their viral productivity. The sequences of each of these infecting virions were used as the basis for mutation and recombination as described below.1. If the uninfected cell was untransduced then viral production over all neighbouring cells was summed, and multiplied by a scaling factor 2. If the uninfected cell was transduced, and if the shRNA were allowed to inhibit infection and act as a Class 1 gene therapy, then the viral productivity of each infected neighbour was modified by the inhibition by the shRNA in the uninfected cell relative to the particular viral sequence. The efficacy of inhibition was assumed equivalent to the efficacy of viral production as described above. This calculation took into account the viral sequence of each of the infected neighbours. The calculations then proceeded as above. The choice of the infecting virions was also based on this modified viral production for each cell.-5 mutations/nt/reverse transcription (P.mut) in accord with the estimated error rate for the HIV-1 reverse transcriptase (RT) [Due to the lack of a proof-reading mechanism, HIV replication  is error prone and characterized by a high mutation rate. HIV sequence variation was randomly incorporated at a rate of 3.4 × 10ase (RT) . If a taase (RT) , furtherThe authors declare that they have no competing interests.TLA, GLM, JMM and DJB conceived and designed the model. JMM wrote and developed the mathematical model. All authors participated in data analysis and interpretation. TLA, GJM and JMM prepared the manuscript.


Single intraperitoneal injections of dimethylnitrosamine (DMN) in concentrations of up to 16 mg/g body weight, failed to have any lethal effect in newts. This treatment also failed to induce tumours in newts maintained for one year after injection.Six or 7 injections of 16 mg/g body weight of DMN, administered over a period of 3-4 weeks, gave rise to a short-term lethal effect due to liver necrosis and in the long term to liver tumours. The tumours induced were of similar type to nitrosamineinduced tumours in rats, one being an anaplastic liver tumour and the other nodular hepatic cell tumours.Attempts to maintain the tumours by grafting failed, probably due to homograft rejection.


Muscle functional magnetic resonance imaging (mfMRI) measures transverse relaxation time (T2), and allows for determination of the spatial pattern of muscle activation. The purposes of this pilot study were to examine whether MRI-derived T2 or side-to-side differences in T2 (asymmetries) differ in low back muscles between subjects with acute low back pain (LBP) compared to asymptomatic controls, and to determine if a single osteopathic manipulative treatment (OMT) session alters these T2 properties immediately and 48-hours after treatment.Subjects with non-specific acute LBP  and asymptomatic controls (n = 9/group) underwent an MRI, and subsequently the LBP subjects received OMT and then underwent another MRI. The LBP subjects reported back for an additional MRI 48-hours following their initial visit. T2 and T2 asymmetry were calculated from regions of interest for the psoas, quadratus lumborum (QL), multifidus, and iliocostalis lumborum/longissimus thoracis (IL/LT) muscles.r = 0.75, p = 0.02).No differences were observed between the groups when T2 was averaged for the left and right side muscles. However, the QL displayed a significantly greater T2 asymmetry in LBP subjects when compared to controls . The psoas muscle also displayed a relatively large, albeit non-significant, mean difference . In the subjects with LBP, psoas T2 asymmetry was significantly reduced immediately following OMT , and the change in LBP immediately following OMT was correlated with the change in psoas T2 asymmetry (Collectively, this pilot work demonstrates the feasibility of mfMRI for quantification and localization of muscle abnormalities in patients with acute low back pain. Additionally, this pilot work provides insight into the mechanistic actions of OMT during acute LBP, as it suggests that it may attenuate muscle activity asymmetries of some of the intrinsic low back muscles. Low back pain (LBP) is one of the most common reasons for seeking medical care, and in a given year 12-15% of the United States population will visit their physician with a complaint of back pain [Numerous studies have utilized electromyography (EMG), mainly surface EMG, to examine whether patients with LBP exhibit side-to-side differences (asymmetry) in their muscle activation patterns under resting  position. The majority of these studies have not observed differences in paraspinal muscle EMG asymmetry between patients with LBP compared to controls -6, althoIn addition to characterizing differences in paravertebral muscle activity between controls and patients with LBP, it is important to determine the effects of clinically relevant interventions on muscle activity. Manual therapies, such as spinal manipulation, mobilization and massage therapy, are used by a variety of health practitioners to treat acute LBP . OsteopaNine study participants exhibiting non-specific acute LBP and nine asymptomatic controls participated in the study. The control subjects were matched to the acute LBP subjects for age (range: 27-69 years), sex (7 women and 2 men for each group) and body mass (range: 50-86 kg) (matched within 5 years and 10 kg). Acute, rather than chronic, LBP subjects were chosen for study because it was felt that they would be more likely to respond to a single manipulative treatment than subjects with more established dysfunctions. To qualify for study inclusion the LBP subjects had to report a current acute episode of LBP that had begun within the preceding 3 weeks. When subjects inquired about the study or were referred to us we made every attempt to enroll them in the study as soon as possible. However, in many instances it required several days beyond this initial inquiry to arrange and schedule the MRI's, and on average the time from LBP onset to the first study day was 15.4 ± 2.0 days. None of the subjects reported a history of chronic LBP. Subjects were excluded if they exhibited radicular symptoms deficits , if they had any bowel or bladder symptoms, had a history of spinal injuries, or had any contraindications to magnetic resonance imaging . Study participants were also excluded if they were less than 18 or greater than 70 years of age or weighed more than 136 kg. The LBP subjects completed an Oswestry Low Back Pain Disability Index before the experiment (mean score = 12.17 ± 3.43 (out of 50)), and rated their LBP on a 0-10 visual analog scale (mean score = 3.02 ± 2.81 cm). LBP subjects were recruited from print advertisements in the local media. The Ohio University and O'Bleness Memorial Hospital institutional review boards approved the experimental protocol, and all subjects provided written informed consent before testing.The study design is illustrated in figure Subjects were assessed in the standing, seated, and supine positions to evaluate for the presence of somatic dysfunction in the thoracic, lumbar, sacral, or pelvic regions. This involved a palpatory screening assessment for alterations in tissue texture change and alterations in normal regional motion, followed by more detailed palpatory diagnostic procedures designed to localize the specific dysfunctional spinal segment or segments in each of the subjects. These palpatory procedures utilized normal landmark identification in the named regions and motion testing at a segmental level to determine the extent and severity of motion restriction along with increases in tissue hypertonicity and/or tenderness to palpation.Standard spin-echo magnetic resonance images of the lumbar spine were obtained using a 1.5-T superconducting magnet  Figure . These pAfter scanning, the images were transferred to a computer for calculation of muscle T2 using the ImageJ software . Muscle T2 was calculated on a pixel-by-pixel basis and averaged over 5-6 slices. Special care was taken to ensure the same slices were used for analysis for all of the MRI scans. The T2 values were calculated from regions of interest within the following muscles: psoas, quadratus lumborum (QL), multifidus, and the iliocostalis lumborum/longissimus thoracis (IL/LT) . When defining regions of interest, non-muscular tissue  was avoided. The dependent variables calculated from the MRI data were T2 and T2 asymmetry. T2 was calculated by averaging the left and right side values for each individual muscle, whereas T2 asymmetry is the percent difference in T2 between the sides. The coefficient of variations for the measurement error of T2 in our laboratory of manually selecting a region of interest from the respective muscles of the same image on different days in 17 subjects ranged between 1-11% . It should be noted that exercise-induced increases in T2 appear to persist for ~30-min . The MRIOsteopathic manipulation was performed by one of two osteopathic physicians (SW or DE) who have over 15-years of clinical experience each performing osteopathic manipulation on a daily basis. Combinations of osteopathic manipulative techniques were utilized. The techniques were the ones most commonly used in the treatment of LBP, and were limited to those listed in the Glossary of Osteopathic Terminology . The fol2, which represents the proportion of total variation attributable to the factor, partialing out other factors from the total non-error variation) are reported as an additional statistical parameter to aid in interpretation of the findings. Due to the pilot nature of this study we have denoted instances where significance was not reached but modest effect sizes were observed (eta2 ≥ 0.15). The SPSS statistical package  was used for data analysis. Data are presented as means+SEM, unless otherwise stated.A one-way analysis of variance (ANOVA) was performed to evaluate group differences (LBP versus control) in T2 and T2 asymmetry for each muscle. In instances where we observed significant differences in T2 asymmetry LBP subjects and controls we performed additional ANOVA's to determine whether the sides with the lower and higher T2 were different between groups. A repeated measures ANOVA was performed to evaluate changes in muscle T2 asymmetry, pain, and Oswestry score over time . In instances where we observed significant differences in T2 asymmetry over time, additional ANOVA's were performed to determine if there were directional changes over time in the lower and higher T2 sides. Pearson's correlation coefficients were calculated to examine the relationship between T2 asymmetries and pain in the LBP group. For all analyses, a pre-set alpha level of significance equal to 0.05 was required for statistical significance, and significant main effects were followed up with a Sidak post hoc test. As stated before, this was a pilot project, and thus with its relatively small sample size rather large differences may not reach statistical significance, resulting in Type II errors. Therefore, effect sizes . Differences in T2 asymmetry were observed, however, between LBP subjects and controls . All of the LBP subjects displayed QL asymmetry values greater than that of the average control subject (> 15%), and in 8 of these 9 subjects the right side exhibited the higher T2 value. Further analysis indicated that, compared to controls, the LBP subjects exhibited a reduced T2 on the side with the lower T2 value , but that no difference existed for the side with the higher T2 value . The psoas muscle exhibited a modest effect size for greater T2 asymmetry in LBP subjects when compared to controls , although this difference failed to reach significance (p = 0.11). Six of the LBP subjects displayed psoas asymmetry values greater than that of the average control subject (> 9%), and in all of these LBP patients the higher T2 value was associated with the right side muscle. The multifidus muscle also exhibited a modest effect size for between-group differences with the LBP subjects exhibiting a lesser degree of T2 asymmetry in relation to controls , although this difference failed to reach significance (p = 0.11). The T2 asymmetry for the IL/LT was similar between LBP subjects and controls .No differences were observed between LBP subjects and controls when T2 was averaged for the left and right side muscles Figure  (Psoas: 2 = 0.39) , but it returned to baseline levels after 48-hours although a modest effect size was still observed . Further analysis indicated that the OMT resulted in an increase in T2 on the side with the lower initial T2 value immediately following OMT  that returned back to baseline levels after 48-hours . The side with the higher initial T2 value did not decrease significantly immediately following OMT  although a modest effect size was observed (eta2 = 0.51); however, 48-hours post-OMT this side was significantly reduced   , but it was slightly increased 48-hours later . Further analysis did not yield any information on the direction of effect, as neither the lower side nor the higher side exhibited significant changes following OMT . The QL and IL/LT muscles did not display significant changes in T2 asymmetry with OMT , although modest effect sizes for a reduction were observed   Figure . Psoas T2 = 0.61). Pain was significantly reduced immediately following OMT , and it remained reduced 48-hours later . The Oswestry Low Back Pain Disability Index score was also reduced 48-hours after treatment .With regards to patient perceptions of the effects of treatment, OMT resulted in a significant reduction in self-reported pain . Additionally, we observed that the change in pain immediately and 48-hours following OMT was correlated with the change in T2 asymmetry . No other significant correlations were observed between pain and T2 asymmetry in other muscles (p > 0.05). As stated above, for the psoas muscle six of the LBP subjects displayed asymmetry values greater than that of the average control subject (> 9%), and in all of these LBP patients the higher T2 value was associated with the right side muscle. Interestingly, in all six of these subjects the L4 or L5 vertebrae were rotated left. We did not observe any association between the side with the higher T2 asymmetry and self reported localization of LBP, as 3 subjects reported pain on the left side, 1 reported pain on both sides, 3 reported centralized pain, and 2 reported pain on the right side.We observed a significant correlation between pain  and psoas T2 asymmetry at baseline (before OMT) .Recently, the use of mfMRI has begun to extend beyond the scope of skeletal muscle and exercise physiology, and has been applied as a tool to examine pathologic muscle. For example, we have used it to examine muscle activity in spastic elbow flexor muscles following stroke , and othOur observation of asymmetrical activation patterns of the lumbar paraspinal muscles in patients with LBP is contrary to numerous studies approaching this research question using EMG -6. In coWe also observed that OMT decreased T2 asymmetry with the most notable reduction occurring in the psoas muscle. It has long been postulated that the mechanism(s) of OMT are related to an attenuation of the gain of the muscle spindle afferents that reduces reflexive contractile activity ,28. IndeIt could be argued that other characteristics of skeletal muscle, beyond the activity state of the muscle, could explain our T2-related findings. For example, the lumbar muscles of chronic LBP subjects exhibit muscle atrophy , and musThe findings from the present study raise several other pertinent questions as to the relative contribution of individual muscle activity asymmetries to the etiology of non-specific LBP and to the efficacy and mechanism of OMT in treating acute LBP. For example, we observed a modest effect size for differences in psoas T2 asymmetry and controls, a significant correlation between LBP and psoas T2 asymmetry, and that the subjects with high absolute T2 values on the right side exhibited L4 or L5 vertebrae that were rotated left. The latter finding is particularly interesting when one considers the functional anatomy and structural diagnosis of the lumbar spine. The segmental diagnosis of left rotation implies a relative tendency of a vertebral body to prefer rotation to the left and restriction of rotation to the right. The palpatory and motion testing reveals a relatively posterior transverse process on the left, suggesting an anterior transverse process on the right . Thus, it is possible that the right anterior displacement is due to the hyperactive musculotendinous unit pulling on the pulling on the transverse process.To fully address these questions, we must consider them within the context of the present study's limitations. With regards to the role of individual muscle asymmetry in contributing to LBP, we did observe that the QL exhibited greater T2 asymmetry than asymptomatic controls, and that the psoas muscle displayed a relatively large, albeit non-significant, mean difference. We must caution, however, against interpreting these findings as an indication that muscle activity asymmetry patterns in these given muscles causes or contributes to acute LBP as the observation of a difference between groups does not indicate causality. Thus, further work is needed to fully elucidate the extent and impact of muscle asymmetry patterns of the individual components of the lumbar muscles in individuals with acute LBP. In the present work our study population consisted mostly of subjects with mild, acute LBP , and it is suggested that subsequent work investigate muscle asymmetries in a cohort with more severe, acute LBP. Additionally, we should also reiterate that our study examined subjects with acute LBP, and that these findings should not be extrapolated to individuals with chronic LBP.We must also express caution when interpreting our data on the efficacy and mechanisms of OMT. While we did observe that LBP subjects reported an immediate reduction in pain with persistent effects two days following treatment and that the reduction in pain was associated with reduced T2 asymmetry, it should be noted that we did not have appropriate comparison groups to fully allow for the assessment of clinical efficacy or mechanisms of action . Additionally, the present study utilized a combination of OMT techniques, making it impossible to know which individual techniques had the primary effects, or if, indeed, a combination of techniques is necessary for the observed effects.This pilot study utilized muscle functional magnetic resonance imaging to examine if T2 asymmetries in the low back muscles, an index of side-to-side variation in muscle activation, differed between subjects with acute LBP and controls, and to determine if OMT altered T2 asymmetries. We observed that subjects with acute LBP had a greater T2 asymmetry in the quadratus lumborum. In the subjects with LBP, OMT resulted in an immediate reduction of T2 asymmetry in the psoas muscle, and this reduction in T2 asymmetry was associated with reduction in pain. Collectively, this pilot work shows the feasibility of employing muscle functional MRI for quantification and localization of muscle abnormalities in patients with acute low back pain. The implications for clinical research could be immense as the precise quantification of specific muscle abnormalities associated with LBP would allow for better evaluation of interventional treatments . Additionally, this pilot work provides insight into the mechanism(s) of action of OMT during acute LBP, as it suggests that OMT may function to attenuate muscle activity asymmetries of some of the intrinsic low back muscles.The authors declare that they have no competing interests.BC was responsible for overseeing the development and implementation of the MRI aspects of the study and drafted the manuscript. SW performed around half of the OMT sessions and structural examinations. RC coordinated subject recruitment and scheduling and performed the data analyses. DE performed around half of the OMT sessions and structural examinations. JH conceived of the study, and directed its overall design and coordination. All authors read and approved the final manuscript.