It has been claimed that osteopathic manipulative treatment (OMT) is able to enhance the immune response of individuals. In particular, it has been reported that OMT has the capability to increase antibody titers, enhance the efficacy of vaccination, and upregulate the numbers of circulating leukocytes. Recently, it has been shown in human patients suffering chronic low back pain, that OMT is able to modify the levels of cytokines such as IL-6 and TNF-α in blood upon repeated treatment. Further, experimental animal models show that lymphatic pump techniques can induce a transient increase of cytokines in the lymphatic circulation. Taking into account all these data, we decided to investigate in healthy individuals the capacity of OMT to induce a rapid modification of the levels of cytokines and leukocytes in circulation. Human volunteers were subjected to a mixture of lymphatic and thoracic OMT, and shortly after the levels of several cytokines were evaluated by protein array technology and ELISA multiplex analysis, while the profile and activation status of circulating leukocytes was extensively evaluated by multicolor flow cytometry. In addition, the levels of nitric oxide and C-reactive protein (CRP) in plasma were determined. In this study, our results show that OMT was not able to induce a rapid modification in the levels of plasma nitrites or CRP or in the proportion or activation status of central memory, effector memory or naïve CD4 and CD8 T cells. A significant decrease in the proportion of a subpopulation of blood dendritic cells was detected in OMT patients. Significant differences were also detected in the levels of immune molecules such as IL-8, MCP-1, MIP-1α and most notably, G-CSF. Thus, OMT is able to induce a rapid change in the immunological profile of particular circulating cytokines and leukocytes. Osteopathic medicine is based on the premise that the primary role of the physician is to facilitate the body's inherent ability to heal itself. Osteopathic physicians view diseases as a disruption of the normal interactions of anatomy, physiology and behavior. One unique aspect of osteopathic medicine is treatment by manually applied procedures, often referred to as manipulative therapies. These therapies have been successfully used by osteopathic physicians for more than a hundred years in order to treat dysfunctions of the neuromusculoskeletal, lymphatic, or vascular tissue. As described in Foundations for osteopathic medicine et al in 1998 et alOf the few published studies that have examined changes in leukocyte counts following osteopathic lymphatic techniques Streptococcus pneumoniae colony-forming units in the lungs of rats with acute pneumonia et al. on human patients suffering chronic low back pain showed that repeated OMT treatment was able to modify the levels of TNFα in circulation In more recent years, exciting studies on the effect of OMT on immune parameters have been performed. Firstly, three publications from the Hodge lab have shown that abdominal lymphatic pump can significantly modify the leukocyte population in lymphatic circulation Herewith, in this pilot study, we decided to investigate rapid responses on several human circulating leukocyte populations and cytokines. To this end, human volunteers were subjected to a combination of lymphatic, splenic and hepatic pump osteopathic treatments and blood samples were collected at different times pre and post-OMT. Then, the proportion of different leukocyte populations was determined by hematological techniques combined with thorough flow cytometry analysis. Further, the presence of several chemokines, cytokines and growth factors were determined in our samples by means of antibody array technology and multiplex ELISA analysis.This study was approved by the Ohio University Institutional Review Board Ethics (protocol 09F007) Committee and informed consent was obtained from all volunteers. The research process, including the collection and storage of blood, isolation of immune cells and determination of cytokine levels in plasma samples, was explained in detail to every participant. Every participant provided written informed consent.Healthy volunteers were enrolled for these studies following an institutional review board approved human subjects' blood collection protocol as described above. Selection of volunteers was with the help of the Clinical Research Unit of the Heritage College of Osteopathic Medicine, Ohio University. As described in other studies As previously described for similar studies For the first series of studies, 21 participants were recruited. In this group, two of the participants withdrew due to problems with recovering blood during the second or third venipuncture. Ten participants fell in the OMT group and 10 in the sham group. For the second group of studies a total of 36 participants were recruited. In this group, three participants withdrew before the treatment or after due to problems with recovering blood during the second or third venipuncture. Sixteen participants fell in the OMT group and 17 in the sham group.et al. (1998), the time length of OMT on each participant was 7 minutes As previously described in detail by Jackson For the sham treatment group, we used light touch to the similar region as described previously For these studies we perform a simple random sampling. For each group of 10 participants, the clinician prepared 5 closed papers with OMT and 5 with sham labels in a closed box. At the day of the treatment, and after the participants signing the consent forms, the clinician drew one paper in order to determine to which group the participant was allocated. The participants were not informed whether they were subjected to OMT or sham treatment.All outcome analyses were performed in a blinded fashion. The researchers in charge of performing the hematological and immunological techniques did not receive any information regarding whether the samples belong to an OMT or sham-treated participant until the stage of statistical analysis, and did not witness the protocol treatments.Blood was drawn by research nurses from OMT and sham-treated participants at −1 h (baseline), 5 and 30 min after treatment ; or at −1 h, 30 min and 1 h after treatment . Two 8-ml VACUTAINER CPT (Cell Preparation Tube) and one 8-ml VACUTAINER PPT (Plasma Preparation Tube) , containing citrate as the anti-coagulant, were collected from each subject. Samples were treated according to the manufacturer's instruction in order to obtain plasma for nitrite determinations and mononuclear cells for different studies.Total and differential leukocyte numbers were determined in an automatic cell counter in triplicate . To confirm differential leukocyte counts, peripheral blood smears were made and stained with Wright Giemsa stain for each specimen. Two hundred white blood cells were counted at a 1000× magnification. Then, differences in the numbers and types of leukocytes among samples recovered from OMT and sham treated individuals were determined.+MHC-II (HLA-DR)+ and CD19(HIB19)+ cells. Monocytes were detected by means of CD14 (M5E2) staining; and different monocyte subpopulations identified by their expression of CD16 (CB16). DCs were identified by staining together with CD11c  or CD123 . Different myeloid DC populations were identified by their expression of CD1c (L161), CD16 (CB16) and CD141 . The activation status of circulating APCs  was defined based on the levels of expression of HLA-DR, CD80 (2D10.4) or CD86 (IT2.2). All antibodies except where indicated were acquired from eBioscience .In order to analyze the phenotype of circulating leukocytes from OMT and sham treated participants, mononuclear cells were stained with specific fluorochrome-labeled antibodies and analyzed by flow cytometry. All samples were stained with CD45 (2D1) antibody  a pan leukocyte marker. In order to define T lymphocyte subsets, CD3 (HIT3a), and CD8 (HIT8a) antibodies were used. Further staining with CD45RA and CCR7 (3D12) helped to identify naïve and memory lymphocytes within the T cell population. CD56 (B159) was used to detect NK cells, and NKT cells were defined as cells co-expressing CD56 and CD3. The activation status of T cells, NK cells and NKT cells was defined due to the levels of expression of the early activation antigen CD69 (FN50). B cells were defined as CD45Presence of cytokines, chemokine and growth factors in plasma samples obtained from OMT and sham participants was analyzed using a cytokine antibody array  following the manufacturer's instructions. Briefly, samples were seeded on glass microarrays and upon following incubation and washing procedures fluorescence signal intensities were measured in an Agilent microarray B-scanner . Once the fluorescent intensity data was obtained, the background was subtracted and the values normalized to the provided positive control signals before proceeding to analysis. Then, comparison of signal intensities for antigen-specific antibody spots was used to determine the relative differences in expression levels of each analyte between samples. As per manufacturer's instructions, any ≥1.5-fold increase or >0.65-fold decrease in signal intensity for a single analyte between samples was considered a measurable and significant difference in expression. This assay, as followed, is not able to assess absolute or relative concentrations of different analytes in the same sample. Plasma samples (pretreatment and 30 min post-treatment) from OMT and control subjects were compared for changing levels of 40 different cytokines and chemokines associated with inflammatory responses.In another set of experiments, plasma samples obtained from OMT and sham participants  were analyzed for the presence of chemokines and cytokines by using a MILLIPLEX MAP Human Cytokine/Chemokine Panel  according to manufacturer's specifications. The chemokines and cytokines chosen for analysis were Eotaxin, G-CSF, IL-1α, IL-1β, GM-CSF, IL-2, IL-8, IL-4, IL-6, IL-10, MCP-1, and MIP-1α.The levels of C-reactive protein (CRP) in plasma samples were determined by using a human CRP ELISA kit  following the manufacturer's instructions.Nitrate levels in plasma samples, a measure of nitric oxide metabolism, were determined by using the Nitrate/Nitrite Fluorometric Assay Kit  following the manufacturer's instructions. Briefly, fluorescent emissions were evaluated in a Synergy HT microplate reader , using a 360 excitation filter and a 420 emission filter.For this pilot project, multiple comparisons we performed ANOVA analysis with post-analysis comparisons by the Tukey-Kramer multiple comparisons test. When samples did not follow normal distribution, non-parametric statistics were used for analysis. When comparing the levels of a particular molecule within the OMT or sham group at different time-points, we used paired-sample statistics analysis. A value of p<0.05 was considered significant. Data are expressed as mean ± SD. Data was analyzed using Graph Pad Instat software .de novo synthesis” of particular immune factors, but rather an effect of changes in the secretion rates of preformed or constitutively produced molecules. Likewise, differences in the percentages of activated immune cells would surely reflect mobilization of populations rather than synthesis of markers of activation during this period of time. It is important to highlight that no adverse effects were reported due to OMT in any of the participants of these studies.The focus of our research was to analyze rapid responses to OMT. Thus, samples were obtained for analysis within an hour of treatment. This has the benefit of “homogenizing” the volunteer behavior, since during that time they were not allowed to eat food or drink and were monitored to by our research team. On the other hand, due to the short period of time after OMT, we do not expected to observe changes due to “de novo by the liver, very important changes in the levels of this molecule are usually observed after 4 h of stimulation. Nevertheless, taking into account some reports that indicate a small but significant increase of CRP at very early time points after stimulation In order to investigate the effect of OMT on the level of circulating metabolites and leukocytes, healthy volunteers were subjected to a mixture of lymphatic and hepatic pump treatments while a sham group received a “light touch” treatment. In order to determine the basal levels of circulating metabolites and leukocytes, 1 h prior treatment blood was extracted from the volunteers. To evaluate immediate response to OMT, blood was drawn at 5 and 30 min post-treatment. It has been previously reported that OMT can induce production of nitric oxide (NO) In order to further investigate the rapid response of the immune system to OMT, we evaluated the levels of various chemokines, cytokines and growth factors in the plasma of OMT and sham treated subjects using antibody array technology . Plasma samples (pretreatment and 30 min post-treatment) from 4 OMT and 4 control subjects were compared for changing levels of 40 different factors associated with inflammatory responses. Expression levels of four cytokines  were significantly increased (≥1.5× increase at 30 minutes vs. pretreatment level) only in OMT subjects (4 of 4 OMT subjects vs. 0 of 4 control subjects) Table 1.Taking into account this qualitative modification in the levels of some circulating cytokines without affecting leukocyte populations, we decided to change our time points for blood collection after OMT, selecting 30 min and 60 min post-treatment. We hypothesize that this would encompass the possibility of a modification in leukocyte levels in response to changes in the cytokine profile. As shown in + DC subpopulation was present at significantly lower levels in the PBMC fraction recovered from the OMT group. The significance of this observation is very important. DCs are the most powerful APCs present in the body, and as such have the capability to stimulate specific T cell responses +CD11c+HLA-DR+ population has been described as a subset of DCs involved in inflammatory responses, since they are characterized by high expression of TNFα (and low levels of IL-10) upon stimulation We then performed an exhaustive flow cytometry analysis of PBMCs recovered from OMT and sham-treated groups 1 h post-treatment. hi monocytes between OMT and sham groups , no differences in the activation levels of B cells, DCs or plasmacytoid DCs were observed in our samples upon OMT  were evaluated. Long-term effects involving de novo synthesis of immune molecules could be surely observed due to the limited time lapse between treatment and sampling. Even under these stringent conditions we were able to detect some modification in immune parameters in our studied population. This argues for future studies investigating the effect of OMT at later time points in order to observe effects on the synthesis of circulating immune molecules or the upregulation of activation markers in immune cells.In closing, our data show for the first time that OMT exerts a modification in the distribution of a particular blood DC population. This was possible due to thorough flow cytometry analysis and multiplex technology. This could help interpret data indicating that OMT can help fight infections, or can increase the efficacy of vaccinations. Furthermore, this argues for extended immunological studies aiming to determine that OMT might help some conventional anti-infectious therapies. In this context, combination of OMT with conventional therapeutic treatments has the potential of lowering healthcare costs which will result in a great benefit for both the patient and the healthcare system. Finally, this study focused in changes induced by OMT in which little or no effect from File S1Patient consent form regarding the studies described herewith. Each participant read and signed the consent form document.(DOC)Click here for additional data file.


We retrospectively reviewed magnetic resonance (MR) images of 96 patients with diagnosis of rectal cancer to evaluate tumour stage (T stage), involvement of mesorectal fascia (MRF), and nodal metastasis (N stage).Our gold standard was histopathology.All studies were performed with 1.5-T MR system  by using a phased-array coil. Our population was subdivided into two groups: the first one, formed by patients at T1-T2-T3, N0, M0 stage, whose underwent MR before surgery; the second group included patients at Tx N1 M0 and T3-T4 Nx M0 stage, whose underwent preoperative MR before neoadjuvant chemoradiation therapy and again 4-6 wks after the end of the treatment for the re-staging of disease.Our gold standard was histopathology.MR showed 81% overall agreement with histological findings for T and N stage prediction; for T stage, this rate increased up to 95% for pts of group I (48/96), while for group II (48/96) it decreased to 75%.Preoperative MR prediction of histologically involved MRF resulted very accurate  also after chemoradiation .Phased-array MRI was able to clearly estimate the entire mesorectal fat and surrounding pelvic structures resulting the ideal technique for local preoperative rectal cancer staging. Rectal cancer is one of the most common tumour in Europe and in the United States  ,3.At the present, the experts agreed that total mesorectal excision (TME) is the surgical approach of choice for rectal cancer, because it is able to reduce the local recurrence rate to less than 10% , improviDue to this, preoperative staging techniques for rectal cancer should also distinguish patients with high risk of local recurrence and involvement of MRF, who might benefit from preoperative radiation therapy, from those patients at low risk, with the tumour located far from MRF, who might go directly to surgery. Then, it is always necessary to pay attention to the main risk factor for local recurrence that are: incomplete resection, nodal disease and distal tumour.The aim of this report is to show that crucial role of phased-array MRI as technique of choice to preoperative locally stage rectal cancer, since it is the only technique able to accurately predict T and N stage and to depict the involvement of MRF.Between June 2005 - December 2008 we retrospectively reviewed 96 patients with histological diagnosis of rectal cancer, performed by endoscopical evaluation. This study population was subdivided into two groups: the first group was characterized by patients at T1-T2-T3 N0 M0 stage, whose underwent just a single MR examination before surgery. T3 stage patients, belonging to this group, despite the locally advanced disease, did not underwent neoadjuvant chemoradiation therapy, due to serious controindications to the treatment itself . The second group included patients at Tx N1 M0 and T3-T4 Nx M0 stage of disease, whose underwent MR staging, then received neoadjuvant chemoradiation therapy and finally performed a second MR for the re-staging of disease.The lesions were considered as T1 stage tumours when clearly limited to submucosa Figure  and as TT3 stage tumours were considered those lesions with a growth through all wall layers and extended into the perirectal fat tissue Figure  while T4All MR findings were compared with histological results.MR imaging was performed with a 1.5- Siemens Medical System, Erlangen, Germany T MR imager  using a pelvic phased-array surface RF coil, necessary to reduce patient discomfort and to obtain higher spatial resolution images providing a full evaluation of rectal wall layers with the additional advantage of a large field of view.All subjects were positioned in a supine, feet-first position with the lower edge of the coil placed at least 10 cm below the symphysis pubis in order to ensure adequate signal from the lower rectum and anorectal junction.In our experience there was no need to use the endorectal coil because despite it may produce very high signal, its primary limitation is its small volume of sensitivity that is an insufficient anatomic detail from surrounding tissues to adequately stage rectal cancer. In fact, the area that can be imaged with endorectal coil amounts to a total distance of one coil diameter away from it that results in a very rapid drop in signal intensity beyond the immediate vicinity of the coil itself. Moreover, as with any other endoluminal technique, stenosis and stricturing, pain and discomfort, bowel wall motion, low lesions, lesions in the upper rectum and coil migration are all factors that hamper image acquisition.No purgative bowel preparation or enemas, air-insufflation and contrast agents were administered. The use of intravenous antiperistaltic agents was not necessary to diminish movement in the small bowel since only the tumours of the lower two-thirds of the rectum were studied.Multiplanar localizing images were obtained to select transverse, coronal and sagittal images with a T2- weighted fast spin-echo (FSE) sequence . These images were obtained with a 24-cm field of view and 4-mm-thick sections with no intersection gap. The sagittal images obtained were used to plan large field of view T2- weighted axial sections of the whole pelvis, from the iliac crest to the symphisis pubis . While the second series was being acquired, we again use the sagittal T2-weighted images obtained to plan to plan high-resolution T2-weighted thin-section axial images  through the rectal tumour and adjacent perirectal tissues. These images were performed perpendicularly to the long-axis of the tumour and rectal wall. The images were obtained by using a 16-cm field of view, a 3-mm section thickness and no intersection gap.T1-weighted imaging (with/without contrast agent) was not performed, as it contributed any additional staging information to the analysis. No fat suppression was used to better delineate the tumour against the perirectal fat.Some patients ( < 5%), either due to co-existing medical conditions or claustrophobia, found the scan impossible to tolerate. In these cases some of the sequences were omitted. Of all sequences the oblique high-resolution scans are the most important so the sagittal views is the only sequence which may be shortened by modifying the parameters and the large field of view. In these cases, axial T2-weighted images should be performed last and may be omitted if the patient is in considerable discomfort.Pre-operative neo-adjuvant therapy has been shown to improve overall survival in patient with rectal carcinoma  especialThe challenge is to identify pre-operatively patients with high risk of local recurrence performing TME surgery alone and hence identify who may benefit from preoperative chemo-radiation.In our study we used high-resolution phased-array coil MRI to allow better selection of patients who could have required pre-operative neo-adjuvant therapy from those who could have be safely treated with surgery alone. 48/96 patients underwent 5 weeks of pre-operative chemo-radiation therapy using standard dose radiation (45 Gy in 25 daily fractions) concurrently with systemically effective chemotherapy.Pre-operative MRI restaging was planned to take place 4-6 weeks after completion of neo-adjuvant therapy. Surgery was performed within 2 weeks following this pre-operative MRI restaging (6-8 weeks after completion of neo-adjuvant therapy).Good pathological reporting of rectal carcinoma is essential if we are to achieve the optimum possible results for patients with this tumour. There is nowadays good evidence that survival differs dramatically between surgeons and that the adoption of TME can improve local recurrence rates and survival -11.After TME, each resected specimen was sent to the pathologist unopened and fresh . Not opening the specimen facilitates comparison with MRI which means an important quality control assessment for the radiologist. The specimen was photographed, prior and after inking, to allow audit of the quality of surgery. For a good resection the mesorectum should be smooth with no violation of the fat, good bulk to the mesorectum anteriorly and posteriorly and the distal margin should appear adequate with no coning near the tumour. No defect should be more than very superficial or 5 mm deep. Then the specimen was opened down to just above the tumour, but not through the tumour which was kept untouched to preserve assessment of the anterior aspect where CRM or peritoneal involvement may be seen. Anterior and posterior non-peritonealised surfaces were painted with ink.After the resection surfaces were inked the specimen was fixed in formalin for a minimum of 3 days (72 hours). Good fixation allows thinner slices to be taken and thus a better assessment of tumour spread. The segment of the fixed specimen containing the tumour was then sectioned transversely at 5 mm intervals from 2 cm below to 2 cm above the tumour itself, producing slices that corresponded precisely to the MR sections.Finally each slice was photographed and whole-mount histopathologic slices were cut and stained with hematoxylin-eosin. The extent of local tumor spread in each histopathologic slice was then assessed according to the TNM staging.The local lymph nodes were identified and embedded, as should all the lymph nodes above and below the tumour.The circumferential margin was considered involved if the tumour extended to within 1 mm of the circumferential excision margin.The whole MR examination time was about 20 minutes, depending on the length of the tumour, and it was always well tolerated. Each MR image was interpreted by two experienced readers independently. The tumour, the layers of the rectal wall, the perirectal tissues and the mesorectal fascia were always well visualized in all patients (n = 96).The zonal anatomy of the rectal wall was best appreciated on thin-section axial images where the three different layers may be easily recognized: the inner hypointense and hyperintense layers respectively represent mucosa and submucosa, while the hypointense inner and outer layers represent the muscolaris propria characterized by the inner circular muscle and the outer longitudinal muscle; the external hyperintense layer represents perirectal fat tissue Figure .Even very thin hypointense structures such as the mesorectal fascia was always identified independent of the body habitus of the patient, owing to the high contrast between the hypointense fascia and the hyperintense fat tissue in and outside the mesorectum.Rectal carcinoma appeared as a higher signal intensity structure than rectal wall in all T2-weighted sequences so the infiltration was well appreciated thanks to the contrast between high-intermediate signal intensity of tumour and low signal intensity of surrounding muscle tissues. The fat tissue remained high in signal intensity and in this way, the tumour contrasted well also with the surrounding fat tissue Figure .Moreover, the major anatomic structures such as the levator ani muscle, the puborectal muscle, the internal and external anal sphincters, and the anal canal were easily evaluated Figure .In the first group of pts MR demonstrated high accuracy in the prediction of correct T stage achieving an agreement rate with histology of 95%: in 2 cases, MRI overstaged attributing a T3 stage rather than a T2 Table . Also foMRI with phased-array coil showed high accuracy in prediction of a tumour-involved MRF (MRF+) with a sensibility and a specificity of 100% Table  Figure .Preoperative chemo-radiotherapy was administered to patients belonging to group II with locally advanced rectal cancer and then treated with TME. Locally advanced rectal cancer was defined as tumour extension through the bowel wall, based on clinical and MR evaluation, without associated distant metastases.In this second group population of patients submitted to neoadjuvant therapy MRI correctly assigned tumour stage (T) in 36/48 cases with 75% of agreement with the histological results. In 12 cases MRI overstaged: 2 patients were staged as T2 rather than T1; 8 cases were staged as T3 rather than T2; 2 lesions were classified as T4 but resulted T3 at histology (Table MRI showed a sensibility of 100% and a specificity of 67% in prediction of a tumour-involved MRF (MRF+) as shown in table For nodal metastasis (N stage) MRI showed 92% accuracy with 2 cases of overstaging ( N1 stage rather than N0) and in 1 case MRI did not reveal any nodal involvement whereas histology reported N1 Table .Moreover, in this second group of patients phased-array MRI assessed also the treatment outcome. In fact, 22/48 cases resulted downstaged: 2 rectal cancer staged as T4 at preoperative MR became T3 lesions after chemoradiation; 14 cases staged as T3 have became T2 Figure ; 4 case Our study demonstrated the high accuracy of preoperative MRI in the prediction of correct T stage; the agreement with histopatholgy was about 95% and it correlated with the data reported in the most of the studies published in literature (65%-100%) -16.For T staging, results of accuracy of phased-array coil have been demonstrated to be similar to those obtained with an endorectal coil, but with the advantage that also nodal staging and a detailed imaging of all anatomical structures above the pelvic floor is possible.Moreover, there is no patient discomfort because no insertion of any rectal coil is needed: the patient have merely just to lie on the MR table and his/her comfort is further enhanced in our technique by using the feet-first supine position, resulting in fewer problems with claustrophobia.MRI demonstrated high performance also to assign the correct tumour stage of small lesions confined to rectal wall: T1 (16/20) and T2 (18/28).The most frequent diagnostic error caused by MRI consisted in correctly differentiated T2 from early T3 lesions: this overstaging was often caused by the presence of desmoplastic reaction within the peritumoral tissues that made difficult the MR differentiation between perirectal fat spiculation, caused by fibrosis alone from those containing viable tumour cells.The major problems occurred when MRI was performed after neoadjuvant therapy because the agreement rate with histhological results comes down from 95 to 75% due to post-radiation oedema, inflammation, fibrosis and necrosis that made difficult a correct diagnosis.As far as concern the prediction of MRF tumour invasion, MRI was extremely accurate achieving a sensibility and a specificity of 100% in the first group. Also for this aspect our results were in accordance with those published in literature, but decreased after neoadjuvant therapy (II group) .A recent Dutch study introduced a profile very similar to our study as far as concerns the population of patients that performed neoadjuvant therapy (48/96); our data were indeed comparable with those : in both cases the lowest values of specificity came from the overstaging. In the most of patients when disease was overstaged at MRI, a diffuse hypointense tissue infiltrating MRF, corresponded to sterilized areas of fibrosis at histological analysis. Indeed postchemoradiation MRI was not able to discriminate between the fibrotic spiculations in the perirectal fat and spiculations with contextually malignant cells at histological analysis .The performances of MRI in the local staging of rectal cancer disease confirm the possibility of this technique to discriminate patients candidated to preoperative neoadjuvant therapy especially considering the high performances of MRI in defining MRF involvement. A recent trial reported that, in selected patients, with FMR+ at the moment of the diagnosis, the use of the preoperative radiation therapy is mandatory because reduces the recurrence rate from 8.2% to 2.4% to 2 years .In our experience, radiologist can be sure that the MRF will be free, only when an intact rectal wall is detected.In this respect, the present T-staging system have its shortcomings because it does not discriminate between cancers with a wide FMR and cancers with a close or involved FMR. Although most of those tumours are classified as stage T3, they have a different risk of local recurrence. In fact today it's unanimously agreed that the distance of the cancer from the CRM, represented by the MRF, is a more powerful predictor for the local recurrence rate, much more significant than the T stage. In particular, if the distance of the tumour from MRF is ≥ 5 mm, radiologist may consider the fascia uninvolved; if the distance is 2-5 mm the involvement is considered border-line; if it is ≤ 1 mm, MRF is involved.As regard N stage, identification of nodal disease is still a diagnostic problem for the radiologist. Despite the identification of lymph nodes as small as 2-3 mm on high-spatial-resolution images, reliable detection of nodal metastasis presently is not possible. The radiologist assessment of nodal involvement generally relies on morphologic criteria such as the size and shape of the node. The problem with morphologic imaging, however, is that with enlarged nodes is difficult to distinguish between reactive and metastatic nodes, and with small nodes micrometastases are easily missed. An additional problem in rectal cancer is the high frequency of micrometastases in normal-sized nodes. In fact, there is currently no consensus about lymph node size vis-à-vis nodal involvement: some authors report any detectable lymph node, whereas others report only those lymph nodes that are larger than a given size  .In our study nodal evaluation had been estimated with dimensional cut-off of 10 mm achieving an agreement rate with histology of 96% that was comparable with the values of literature but it is obvious that also our approach has been arbitrary.However, results of early anatomic studies showed that over half of the metastatic nodes were within 3 cm of the primary tumour and were smaller than 5 mm in size -25; thesOur experience has confirmed that MR with phased-array coil is the technique of choice in the preoperative local staging of rectal cancer since it helps to estimate in a detailed and non-invasive way the entire mesorectal fat and all the surrounding pelvic structures, MRF included; moreover, it allows to identify the different risk group of patients helping to decide about the treatment and its outcome (downstaging with complete or incomplete response), and finally to optimize a complete excision allowing to those patients with an advantaced rectal cancer to benefit from a sphincter-preserving surgery.The authors declare that they have no competing interests.SG: carried out the study and drafted the manuscript. PB: performed surgery. MC: performed anatomopathological evaluation. EF: participated in the design of the study. SF: participated in the anatomopathological evaluation. EC: participated in the anatomopathological evaluation. DC: coordinated the study. CB: coordinated the study and participate in its design. All authors read and approved the final manuscript


To try to improve the outcome of our TKAs, we started to use the CKS prosthesis. However, in a retrospective analysis this design tended to give worse results. We therefore conducted a randomized, controlled trial comparing this CKS prosthesis and our standard PFC prosthesis. Because many randomized studies between different TKA concepts generally fail to show superiority of a particular design, we hypothesized that these seemingly similar designs would not lead to any difference in clinical outcome. 82 patients (90 knees) were randomly allocated to one or other prosthesis, and 39 CKS prostheses and 38 PFC prostheses could be followed for mean 5.6 years. No patients were lost to follow-up. At each follow-up, patients were evaluated clinically and radiographically, and the KSS, WOMAC, VAS patient satisfaction scores and VAS for pain were recorded. With total Knee Society score (KSS) as primary endpoint, there was a difference in favor of the PFC group at final follow-up (p = 0.04). Whereas there was one revision in the PFC group, there were 6 revisions in the CKS group (p = 0.1). The survival analysis with any reoperation as endpoint showed better survival in the PFC group (97% (95% CI: 92–100) for the PFC group vs. 79% (95% CI: 66–92) for the CKS group) (p = 0.02). Our hypothesis that there would be no difference in clinical outcome was rejected in this study. The PFC system showed excellent results that were comparable to those in previous reports. The CKS design had differences that had considerable negative consequences clinically. The relatively poor results have discouraged us from using this design. Although current results of total knee arthroplasty (TKA) are relatively good, there is still room for improvement. There is constant research and development, with a view to obtaining longer survival rates , a betteWe started to use the CKS prosthesis , based on previous research at our institution showing that the natural patella groove does not have an isolated lateral orientation . In contMany randomized studies of TKAs with different bearings , cruciatWe designed a randomized, controlled trial with 2 posterior cruciate ligament (PCL) retaining total knee designs. The study protocol was approved by the institutional review board at our hospital and it was carried out in line with the Helsinki Declaration. The study was registered in the ClinicalTrials.gov Protocol Registration System (Identifier: NCT 00228137). All patients who were scheduled to undergo primary total knee arthroplasty because of osteoarthritis or rheumatoid arthritis at the Radboud University Nijmegen Medical Centre were considered for inclusion and were enrolled prospectively. Exclusion criteria were dementia, hemophilia, juvenile rheumatoid arthritis, and ligament insufficiency that needed a posterior-stabilized or otherwise more constrained type of design. Between November 2002 and December 2004, 87 consecutive patients (95 knees) were assessed for eligibility. 5 patients (5 knees) were excluded before randomization: 2 patients refused to participate, 2 patients had hemophilia, and 1 patient had dementia.After written informed consent had been obtained, the knees were randomly allocated to 2 groups. 45 knees received a press-fit condylar prosthesis  and 45 knees received a continuum knee system prosthesis . Computer-generated randomization with stratification for age, co-morbidity, and flexion contracture was performed by an independent observer to allocate the patients in equal numbers to the 2 groups.Both cemented designs are PCL-retaining and have a fixed polyethelene (PE) insert on a tibial tray with central keel. The femoral and tibial components are made of the same material . In contrast to the lateral orientation of the patellar groove in the PFC prosthesis, the trochlea of the CKS prosthesis is deeper and has a neutral direction. The femoral component of the PFC has a fixation peg in both condyles, whereas the CKS design uses one central peg. Furthermore, the CKS prosthesis has a different surface texture of the femoral component. Additionally, the PE insert of the CKS design has a more prominent and sharp posterior edge compared to the PFC design .Identical surgical techniques were used in the groups according to the manuals of the designers. 6 surgeons were involved in the study. All procedures were performed by an experienced knee surgeon or under the direct supervision of one. A pneumatic tourniquet was used for all patients. A medial parapatellar capsular incision was used. No patellas were resurfaced. All implants were cemented after pulsed lavage, drying, and pressurization of the cement . Continuous passive motion was started on the second postoperative day. Thereafter, active range-of-motion exercises and walking were started under the supervision of a physiotherapist.Routine follow-up evaluation was scheduled at postoperative intervals of 3 months, 6 months, 1 year, and annually thereafter. Preoperative and postoperative review data were recorded by a physician assistant who was blinded regarding patient allocation. At each follow-up visit, we took anteroposterior, lateral, and skyline patellar radiographs, which were evaluated according to the guidelines of the Knee Society . The priA sample size estimation showed that 37 knees per group would be required to detect a clinically relevant difference of 10 points with a standard deviation of 15 points in the total KSS, with an alpha of 0.05 and a power of 80%. Intergroup differences were determined using Student's t-test for continuous variables and the Pearson chi-square test or Fisher's exact test for categorical variables. Survival analyses were performed using the Kaplan-Meier method and compared using log-rank tests. Survival estimates are presented with 95% confidence intervals (CIs). For all data sets, differences were considered statistically significant at p-values < 0.05.After randomization, 5 patients were excluded because a posterior-stabilized design was needed after routinely sacrificing the PCL in cases with a flexion contracture of 25 degrees or more (1 in the CKS group and 4 in the PFC group). Because bilateral involvement might cause bias, 8 other knees were excluded (5 in the CKS group and 3 in the PFC group). No patients were lost to follow-up, but 12 relatively elderly patients died of unrelated causes. These patients were analyzed according to the latest available follow-up. Consequently, we analyzed 39 knees with a CKS prosthesis and 38 knees with a PFC prosthesis , with a With total KSS as primary endpoint, there was a difference between groups in favor of the PFC group at final follow-up (p = 0.04) . EvaluatThere was 1 revision in the PFC group; a thicker polyethylene insert was placed for instability. In contrast, there were 6 revisions in the CKS group: in 5 patients, the CKS prosthesis was removed because of poor function and pain and 1 patient was treated with arthrolysis and secondary resurfacing of the patella. During the removal of the prostheses, it appeared that all femoral components of the failed CKS group were easy to remove, leaving an intact cement layer on the bones—indicating inadequate fixation between prosthesis and cement. Cultures were positive in 2 of the CKS revisions. 8-year survival analysis with revision for any reason as endpoint showed 97% (95% CI: 92–100) survival for the PFC group and 84% (72–96) survival for the CKS group (p = 0.05) . The sur2 other patients in the CKS group were reoperated. 1 patient developed postoperative arthrofibrosis and was manipulated under anesthesia, but the knee remained stiff with 20° fixed flexion deformity and 70° of flexion. 1 patient was treated with open debridement followed by antibiotics for 6 months because of a culture-proven deep infection. 4 years later, there were no signs of infection and the knee functioned well. 2 patients (1 in each group) developed a hematoma, both of which were treated conservatively. There were no thromboembolic complications. The 8-year survival analysis with any reoperation as endpoint showed a difference between the PFC group and the CKS group (97% (92–100) for PFC and 79% (CI: 66–92) for CKS; p = 0.02) . The surAnalysis of the radiographs at final follow-up showed a radiolucency smaller than 2 mm in one zone under the tibial component in 2 cases in the PFC group and in 3 cases in the CKS group. These radiolucent lines were already present in the direct postoperative radiographs and no radiolucent line was progressive; none of these 5 cases were classified as radiographic loosening. The skyline patellar radiographs did not show (sub)luxation of the patella in the PFC group or in the CKS group.Our hypothesis that there would be no difference in clinical outcome between the PFC prosthesis and the CKS prosthesis was rejected. With total KSS as primary endpoint, and for survival with any reoperation as endpoint, the CKS group showed a worse result. With our standard prosthesis, the PFC, we found an excellent survival rate of 97% for any revision after 8 years. Other authors have recently reported similar 10-year survival rates for the PFC prosthesis: 97% survival for aseptic loosening  and 97% The excellent clinical scores of the PFC prosthesis do not leave a lot of room for improvement, which is probably why recent RCTs have failed to show a superior design . Subtle In addition, we have to realize that the outcome after TKA not only depends on the type of implant; The question remains as to why we found such a difference between the PFC and the CKS prostheses, as the designs appear to be quite similar. Concerning the articular part of the prosthesis, the most prominent difference is the orientation of the patella groove. Although patellofemoral complaints are one of the complications after TKA, with the highest incidence (1–24%) and an important reason for revision surgery , it seemAn important issue is the observation of bad fixation strength of the prosthetic components to the bone. It appeared that all femoral components of the failed CKS group were easy to remove. Only 2 revisions could be attributed to positive bacterial cultures; the other 3 revisions were defined as aseptic loosening after 13, 16, and 51 months. This high rate of aseptic loosening is uncommon, especially at this early stage . MoreoveWe therefore wondered what the reason could be for a weak cement-metal interface of the CKS components. We analyzed the differences in the backside of both designs. The femoral component of the PFC has fixation pegs in both condyles, whereas the CKS design uses only 1 central peg. 2 pegs might enhance the fixation relative to 1 central peg, but to our knowledge this has not been described in the literature. We also analyzed the surface roughness of the femoral components and found that the CKS components had a lower surface roughness value than the PFC design . As shown by Another difference between the designs concerns the PE insert. The CKS design uses a tibial insert with a more prominent and sharp posterior edge compared to the PFC design. This sharp edge may come in contact with the PCL, leading to damage and subsequent PCL laxity. In a previous study comparing the CKS and the PFC prostheses, kinematic analysis supported the suspicion that the CKS design has larger AP translations than the PFC design . AlthougOur study had some limitations. First, a relatively high number of patients (12) died before final follow-up. Even so, all the patients were analyzed with the latest available  follow-up. Including these patients, the mean follow-up was 5.6 years. Furthermore, no patients were lost to follow-up. Another possible limitation is the potential bias from there being 6 different surgeons involved in this study. However, since we are a teaching hospital all procedures were performed by—or under direct supervision of—an experienced knee surgeon and none of the reoperated cases had originally been operated by a surgeon with low volume.One strength of our study was the randomization process with stratification for age, flexion contracture, and co-morbidity. Consequently, patient demographics and the baseline clinical status of both groups were similar. Thus, we are convinced that the differences in clinical function and survival between the groups were caused by the differences in design between the CKS and the PFC prostheses. Our study was not designed to determine the reason for the worse results of the CKS design. We believe that the reason may have been multi-factorial, and a combination of low fixation strength and possible PCL insufficiency. Initially, we thought that the CKS system was very similar to the PFC system, but the large differences in clinical outcome were evident and discouraged us from further use of the CKS system.


University students in Sweden routinely receive proactive mail-based alcohol Internet interventions sent from student health services. This intervention provides personalized normative feedback on alcohol consumption with suggestions on how to decrease drinking. Earlier feasibility trials by our group and others have examined effectiveness in simple parallel-groups designs.To evaluate the effectiveness of electronic screening and brief intervention, using a randomized controlled trial design that takes account of baseline assessment reactivity (and other possible effects of the research process) due to the similarity between the intervention and assessment content. The design of the study allowed for exploration of the magnitude of the assessment effects per se.This trial used a dismantling design and randomly assigned 5227 students to 3 groups: (1) routine practice assessment and feedback, (2) assessment-only without feedback, and (3) neither assessment nor feedback. At baseline all participants were blinded to study participation, with no contact being made with group 3. We approached students 2 months later to participate in a cross-sectional alcohol survey. All interventions were fully automated and did not have any human involvement. All data used in the analysis were based on self-assessment using questionnaires. The participants were unaware that they were participating in a trial and thus were also blinded to which group they were randomly assigned.Overall, 44.69% (n = 2336) of those targeted for study completed follow-up. Attrition was similar in groups 1  and 2  and lower in group 3 . Intention-to-treat analyses among all participants regardless of their baseline drinking status revealed no differences between groups in all alcohol parameters at the 2-month follow-up. Per-protocol analyses of groups 1 and 2 among those who accepted the email intervention (36.2% of the students who were offered the intervention in group 1 and 37.3% of the students in group2 ) and who were risky drinkers at baseline (60.7% follow-up rate in group 1 and 63.5% in group 2) suggested possible small beneficial effects on weekly consumption attributable to feedback.This approach to outcome evaluation is highly conservative, and small benefits may follow the actual uptake of feedback intervention in students who are risky drinkers, the precise target group.International Standard Randomized Controlled Trial Number (ISRCTN): 24735383; http://www.controlled-trials.com/ISRCTN24735383 (Archived by WebCite at http://www.webcitation.org/6Awq7gjXG) University entrance is associated with increased levels of heavy drinking. Alcohol is often used to promote social integration despite the potential negative consequences of excessive drinking among students. A survey of more than 18,000 students aged 17 to 30 years in 21 countries found that substantial numbers of them used alcohol, ranging from 29% of men and 6% of women in South Africa to 95% of men and 93% of women in Ireland [Several studies have assessed the effectiveness of various forms of Internet-based screening, combined with brief interventions, to change drinking behaviors, as shown for example by Boon et al  and SaitThe use of e-SBI has to a great extent emerged as an efficient approach to reaching large numbers of adolescents as a result of high levels of Internet use among young people. Advantages of the use of Internet-delivered interventions compared with face-to-face consultations include greater reach and implementation, higher consistency of intervention content, and closer matching of intervention to patient characteristics and recommended guidelines ,8. The pMany earlier studies of e-SBI among university students have focused on the effectiveness of a normative feedback on students’ drinking behaviors -15. A meMore research is needed on many aspects of e-SBI in student populations. The majority of the research has been conducted in the United States. Many previous studies have required respondents to participate in e-SBIs taking place in controlled settings, rather than allowing students to access e-SBIs using their own computers ,18. OnlyAnother area where more knowledge is needed concerns whether the assessment or screening of alcohol consumption per se reduces drinking -26. SeveAlcohol screening involves answering a series of questions and is thus a form of brief assessment. A recently published systematic review of randomized evaluations of assessment effects in brief intervention found somewhat equivocal evidence of assessment reactivity when studies with adults, some of which identified no brief intervention effects, were included . When atThis study addressed several of the identified gaps in previous research. The overall aim of this study was to evaluate the effectiveness of e-SBI, using an randomized controlled trial design that takes account of baseline assessment reactivity (and other possible effects of the research process) due to the similarity between the intervention and assessment content. The design of the study allowed exploration of the magnitude of the assessment effects per se. More specifically, the study tested 4 main hypotheses as follows:1. Alcohol Use Disorders Identification Test (AUDIT) scores in the group that was assessed at baseline and received feedback (group 1) will be lower than in the group that was not contacted at baseline (group 3), providing a test of the effects of universal e-SBI provision in an unselected population of university students.2. AUDIT scores in the baseline assessment-only group (group 2) will be lower than in the no-contact baseline group (group 3), providing a test of the effects of assessment-only in an unselected population of university students.3. AUDIT scores among risky drinkers in group 1 will be lower than in group 2, providing a test of the effects of adding feedback to assessment-only among risky drinkers who participated at baseline.4. AUDIT scores in group 1 as a whole will be lower than in group 2 as a whole, providing a test of the effects of adding feedback to assessment-only in an unselected population of university studentsThe study was performed at Linköping University in the mid to southern part of Sweden among all 5227 freshmen entering the university. Every student at the university has a personal official university email address that is obligatory for the student to use, with all official mail delivered through this address. The e-SBI was performed by the individual students on personal computers at a location of their preference, usually at home.The study was performed as a three-arm parallel-groups trial in which routine provision of e-SBI (group 1) was compared with the same without feedback  and no-contact control (group 3) study conditions. Groups 1 and 2 completed identical assessments, the sole difference between them being that group 1 was provided with normative feedback as usual, whereas participants in group 2 were simply thanked for their participation and given a link to a commonly used website concerning alcohol information with no normative feedback. Group 3 was contacted only after 2 months, at which time both groups 1 and 2 also completed outcome data collection .The study was designed to protect the control group as much as possible from the possible effects of research participation . Thus, pWe collected email addresses from the official university register in three separate data files. All participants had a 1 in 3 probability of allocation to any particular study condition. Randomization was computerized and did not use any strata or blocks. Each participant was given a random number between 0.0 and 1.0 with 5 decimals. For instance, participant A might be given number 0.12345 and participant B might get 0.54321. The list of participants was then sorted in descending order by this number, and the list was cut into 3 equal parts .At baseline the students received an email from the student health care service with a short greeting welcoming them to the university followed by an invitation to perform the Internet alcohol intervention. All three groups were sent an email 2 months later by the Swedish principal investigator (PB). This mail made no reference to the previous email from the student health care services and comprised an invitation to participate in an online alcohol research survey, with inclusion in a lottery draw for 100 cinema tickets as a small incentive to increase participation. The alcohol study outcomes were derived from the 10-item AUDIT questionnaire  in this Groups 1 and 2 were unaware that they were participating in a research study when they responded to the initial emails. Both groups were led to expect that these emails were provided as routine practice by the student health care center to help students think about their drinking. All three groups were unaware they were participating in an intervention study and that they had been randomly assigned to a study group. At follow-up, no explanation of the true nature of the study was given to students. Instead they were invited to participate in a seemingly unrelated student alcohol survey. As all study procedures were automated, the research team had no direct contact with study participants. The use of blinding and deception in this trial raised ethical issues. All students were subsequently offered the opportunity to provide feedback on their alcohol consumption at the time of the follow-up. Ethical approval for the study was given by the regional ethics committee in Linköping, Sweden (reference number: 2010/291-31).The study was based on an email-based Internet alcohol intervention (e-SBI) that has been developed by the Lifestyle Intervention Research group at Linköping University ,19. The After students received the welcoming email from the student health care service, they were sent an invitation to perform the Internet alcohol intervention by clicking on a hyperlink to the test. Two reminders were sent 1 week apart to those who had not answered and thereafter the link was closed. The link could be used only once in order to ensure that each student performed the test only once. The test included questions about average consumption day by day in a typical week during the last 3 month, frequency of binge drinking, highest blood alcohol concentration in the last 3 months , negative consequences related to alcohol, views on how much other students and peers drink, and questions about motivation to change. A demo version of the computerized test can be viewed at demo.livsstilstest.nu.Group 1 students then got immediate feedback consisting of 3 statements summarizing their weekly consumption, their frequency of heavy episodic drinking, and their highest blood alcohol concentration during the last 3 months, comparing the respondent’s drinking patterns against the safe drinking limits established by the Swedish National Institute of Public Health. Immediately after this followed comprehensive normative feedback with information describing the participant’s alcohol use compared with that of Swedish university students and, if applicable, personalized advice concerning the need for reducing any unhealthy levels or pattern of consumption. The student viewed the feedback on screen and could print it out. In addition the student received an email with a pdf file of the feedback.In the baseline assessment, used in the per-protocol analysis, we calculated the average weekly consumption, in a typical week, as the number of standard glasses day by day. Frequency of heavy episodic drinking was assessed as at most once a month, 2–3 times a month, 1–2 times a week, or 3 or more times a week. Risky drinking at baseline was defined as either having risky weekly consumption  or engaged in heavy episodic drinking  2 times a month or more often.In the intention-to-treat analysis we assessed risky drinking at follow-up with the AUDIT questionnaire , asking t test. The absolute change in weekly alcohol consumption  between baseline and follow-up was compared between groups with multilevel linear regression . The relative change in weekly alcohol consumption between baseline and follow-up was compared between groups by Mann-Whitney U test. All tests were performed 2-sided at P < .05. The statistical analyses were performed using SPSS version 19  and Stata version 12 .We examined differences in proportions between groups with chi-squared tests. Weekly consumption of alcohol, total AUDIT score, AUDIT problem and dependence scores were logarithmically transformed  and compared between groups by Student P < .001, χ22 = 54.6). The proportion who were risky drinkers at baseline was similar in groups 1 and 2 (around 56%) as well as in all groups at follow-up (around 50%) (The response rate to the baseline survey was similar (approximately 37%) in groups 1 and 2. At follow-up, however, attrition was higher in groups 1 and 2 than in group 3 . Thus, tund 50%) .The sociodemographic characteristics of the participants in the follow-up from all three groups did not differ with regard to sex, age, and university section see . The intThe per-protocol analyses of groups 1 and 2 assessed outcomes among those participants with risky drinking at baseline who also answered the follow-up survey. Thus, 201 (57.4%) participants of the 377 who provided follow-up data from group 1 also provided data from the baseline survey, and similarly 207 (54.9%) of 421 from group 2 were included in the per-protocol analysis. There were no significant differences between groups 1 and 2 in the sociodemographic characteristics of the participants in the baseline survey. Weekly alcohol consumption at baseline was similar in both group with a mean consumption of 135.9 g/week for group 1 and 133.4 g/week for group 2 (median 120 g/week in both groups). Frequency of heavy episodic drinking did not differ significantly between the two groups at baseline.P = .03); the absolute change in this outcome was not statistically significant. There were no other statistically significant differences between groups 1 and 2. The majority of risky drinkers in both groups reported drinking in a nonrisky way at follow-up.This study found no between-group differences, indicating an absence of evidence of assessment or feedback benefit in the intention-to-treat analyses among unselected university students. There was one between-group difference among those who were risky drinkers at study entry in the per-protocol analysis. This potentially provides some very modest evidence of benefit attributable to receiving feedback in addition to assessment, although this should be interpreted in the context of the wider study finding of no effects.There are three principal obstacles to making more definitive statements attesting to evidence of intervention ineffectiveness in the present study. First, the differential attrition between group 3 and groups 1 and 2 suggests problems of nonequivalence between the groups and thus bias in direct comparisons. Second, the present study was undertaken to prepare for a subsequent large trial and was thus not originally designed to produce such conclusions. Lastly, the approach taken to outcomes evaluation in which populations are randomized and compared regardless of their need for intervention should be carefully considered. Each of these issues shall be addressed in some detail.Because of the randomized nature of this study, it can be inferred that differential attrition was caused by the earlier involvement of groups 1 and 2 than of group3 with the study. The earlier invitation to participate in the alcohol e-SBI was apparently not sufficiently different from the later alcohol survey, or the mere fact of a second alcohol-related email invitation may have interfered with the likelihood of accepting the invitation. Whatever the precise mechanism, the main implication is clear: selection bias is possible, if not likely, and outcomes for group 3 cannot be validly assumed to be directly comparable with those for groups 1 and 2 in relation to intervention effectiveness. Because of the lack of contact with group 3, the basis for making comparisons to evaluate this possibility is limited. This is restricted to the unlikelihood of data being collected at follow-up having been altered differentially between groups by the passage of time. Here there is evidence of a slight tendency for a greater proportion of male participants, and the presence of other unmeasured confounders cannot be ruled out. It should be noted that this issue of differential attrition particularly complicates randomized comparisons involving group 3 and is less of a problem for comparisons restricted to groups 1 and 2 only. A stronger conclusion can thus be drawn in relation to whether feedback added to the effects of assessment-only, and there is little evidence here that it did, notwithstanding one statistically significant difference among the per-protocol analysis outcomes.The second reason for caution relates to the highly naturalistic Internet study context and the need to confront the difficult methodological challenges that this implies during the course of a long-term research program. Attrition has been a major source of difficulty in previous work in developing e-SBI in Linköping and other Swedish universities .It is also a significant problem in the conduct of online trials in other populations as underscored by Eysenbach  and otheThe third main reason for caution in drawing conclusions from the present study relates to our intention-to-treat approach to outcomes evaluation, which was highly conservative. The intervention comprised an automated email providing a means of accessing a website in an unselected population with an elevated prevalence of hazardous and harmful drinking. Thus, the intervention was delivered more widely than was necessary, as we only wished to intervene with risky drinkers. The intervention could be defined more narrowly as being delivered to those who accessed the website, with the email merely being the means of recruitment. Even if this definition is applied, the intervention would still have been accessed by students whose drinking was not risky and who would thus not have been deemed to merit individual targeting for intervention. More narrowly still, outcome evaluation could have been restricted to those whose drinking was found to be risky. The overarching problem is that a greater number of people were randomly assigned than would have been targeted for intervention. The primary rationale for proceeding in this way was that assessing eligibility, baseline data collection, and intervention delivery were all quickly integrated in 1 brief online session. We shaped our research design pragmatically around the real-world intervention opportunity, matching the research study to routine practice as it is delivered, rather than interfering with it for research purposes, which would have introduced external validity problems. This approach takes advantage of an opportunity to avoid any research participation effects that may be associated with screening and other aspects of study entry, in much the same way as cluster randomized trials can be used for this purpose. The obvious major disadvantage of this approach is that it biases hypothesis testing toward the null and thus is highly conservative. Thinking about outcomes evaluation needs to take account of these issues.There then arises the question of the consistency of study findings with the existing literature. Put simply, there are no existing studies against which to compare our intention-to-treat findings, as none have used no-contact control groups. The per-protocol comparisons more closely reflect existing studies, and smaller between-group differences are observed. Thus, for both internal and external validity purposes, our test of the third hypothesis is particularly important. Comparisons with the existing literature also need to take account of the highly naturalistic study context. If our results are confirmed in further studies, they have important implications when considering the effectiveness of online alcohol interventions.Our unusual study design confers many limitations, as well as strengths, some of which have already been considered. We used the AUDIT as an efficient summary measure of alcohol consumption and whether it may be hazardous or harmful. Although the AUDIT has been validated in online student contexts , this do


Protein residue-residue contact prediction is important for protein model generation and model evaluation. Here we develop a conformation ensemble approach to improve residue-residue contact prediction. We collect a number of structural models stemming from a variety of methods and implementations. The various models capture slightly different conformations and contain complementary information which can be pooled together to capture recurrent, and therefore more likely, residue-residue contacts.L/5 medium range contacts and. 36 for the top L/5 long range contacts for CASP8 targets (L being the target domain length). When applied to targets from CASP9, the accuracies of the top L/5 medium and long range contact predictions were. 34 and. 30 respectively.We applied our conformation ensemble approach to free modeling targets from both CASP8 and CASP9. Given a diverse ensemble of models, the method is able to achieve accuracies of. 48 for the top When operating on a moderately diverse ensemble of models, the conformation ensemble approach is an effective means to identify medium and long range residue-residue contacts. An immediate benefit of the method is that when tied with a scoring scheme, it can be used to successfully rank models. In part, this is due to the inadequacy of current de novo sampling techniques which are incapable of guiding the folding process through such a vast conformational space , T0531 [6-63], T0534 , T0534 [81-256], T0537 [65-350], T0537 [351-381], T0544 [1-135], T0547 [343-421], T0547 [554-609], T0550 [178-339], T0553 [3-65], T0553 [66-136], T0555 [12-145], T0561 , T0571 [197-331], T0578 , T0581 [27-131], T0604 [11-94], T0604 [292-496], T0608 [29-117], T0618 [6-175], T0621 [2-170], T0624 [5-73], T0629 [50-208], T0637 [1-135] and T0639 [3-126]. All the targets along with their corresponding domain definitions and experimental structures are available on the CASP websites . It should be noted that the ensemble prediction approach could be applied to hard template based modeling as well. In this study we limited ourselves to the free modeling targets as they are typically the type of target chosen when evaluating residue-residue contact prediction methods.The prediction targets used in our study were the protein domains classified as free modeling (FM) targets for CASP8 and CASP9. These are domains which did not have structural templates or the templates existed but were extremely difficult to detect . For CASβ atoms (Cα for glycine) in the experimental structure is less than 8Å. Long range contacts are defined as residues in contact whose separation in the sequence is greater than or equal to 24 residues. Medium range contacts are defined by interacting residues which are 12 to 23 residues apart in the sequence. These definitions were used in accordance with previous studies [For the purposes of our investigation two amino acid residues are said to be in contact if the distance between their C studies ,15,16 an studies -27,29.L/5 or L/10 predictions where L is the length of the protein in residues. If evaluating predictions over a domain, L can also be the length of the domain. Accuracy is defined as the number of correctly predicted residue-residue contacts divided by the total number of contact predictions considered. The recall is defined as the number of correctly predicted residue-residue contacts divided by the total number of true contacts. Additionally, we also calculated the number of contact predictions which were very close to a true contact. For this calculation, a prediction is considered correct if there is a true contact within ± δ residues for small values (ie 1 or 2) of δ.A common evaluation metric for residue-residue contact predictions is the accuracy of the top The starting point for our conformation ensemble contact predictor is a collection of structural models generated for a protein. This collection of structural models we define as the input ensemble. From each model in the input ensemble, the residue-residue contacts are extracted and then counted across all models. This list of contacts is then normalized so that all counts are between 0 and 1 and sorted according to frequency. At this point, the contacts can be filtered (ie restricted to a domain) and the most commonly occurring contacts are selected as the predicted contacts. The entire procedure is depicted by Figure The primary source of input ensembles was CASP. During the most recent CASP experiments, prediction groups were allowed to submit up to 5 tertiary structure predictions per target to the prediction center. The models for the groups which participated in the server category are available on the CASP website and provided us with a rich collection of ensembles for our prediction targets. On average there were 301 models in each ensemble.L/5 medium and long range contacts. This is a standard evaluation metric for contact predictors and has been used in recent CASP experiments [L/5 predictions when compared to small neighborhoods around true contacts. In this case, a prediction is counted as correct if it ± δ residues  from a true contact. Tables To establish an initial baseline for the effectiveness of our conformation ensemble approach, we first evaluated it on the free modeling targets from CASP8 and then tested it blindly during CASP9 as the MULTICOM human residue contact predictor. For the input ensemble, we used the tertiary structure predictions submitted by predictors in the server category. For each target domain, we calculated the precision (ie the percent of correct predictions) of top eriments -27. As ade novo 3D structure predictors. This assessment was conducted on the CASP9 free modeling targets. For contact predictors, we selected SVMcon [L/5). To rank the contacts, we applied our ensemble approach on the 5 models submitted by BAKER-ROSSETASERVER and Zhang-Server during the CASP9 experiment. This is to say that for each predictor, we took the 5 models submitted during the CASP experiment and used these models as the input ensemble. Contacts were extracted from models and ranked according to the procedure the same procedure as that outlined by Figure de novo tertiary structure predictors. This demonstrates that the ensemble-based contact prediction very likely can be used to improve de novo structure modeling.We also compared our conformation ensemble approach with existing predictors of residue-residue contacts and to contacts extracted from individual d SVMcon  - a methd SVMcon . To compAs the quality of the contact predictions depends on the quality of the models in the ensemble, we reevaluated our method on the CASP9 targets using filtered ensembles. This allowed us to assess the method's effectiveness in coping with poor quality models and verify that the method was not relying on a small number of good models to make quality predictions. Three filtering processes were applied. In the first approach, we used ModelEvaluator  to predide novo structure prediction.Given a diverse pool of models, the conformation ensemble approach performs better than existing contact prediction methods. The method is rather robust as well. Removing poor quality models or the best models from the starting ensembles does not significantly affect performance. In this work we did not directly address the usability of contacts predicted by our conformation ensemble approach to aid in tertiary structure prediction. It is a matter which we hope to explore further in a future investigation. Nevertheless, we are optimistic that the contacts will prove useful. This is due to the high accuracy of the contact predictions when evaluating them in neighborhoods of true contacts and also the clustered nature of the contacts' distribution. This is particularly true for short to medium length proteins and Figure L medium range contacts and all long range contacts. More specifically, we calculated the percentage of the predicted medium range contacts satisfied exactly, the percentage of predicted medium range contacts satisfied within 1 residue , the percentage of predicted long range contacts satisfied exactly and the percentage of predicted long range contacts satisfied within 1 residue. The sum of these percentages was calculated and used to rank the models.One application of our conformational ensemble approach which we demonstrate here is its usability and effectiveness in ranking models. It should be noted that use of predicted contacts to rank and select models has been studied previously and shown to be useful ,8. MotivOne measure of the effectiveness of a ranking scheme is loss. The loss for a target is defined as the difference in GDT-TS score ,32,33 beAs indicated in Table de novo tertiary structure predictors) all four key long range interactions are still present in the predicted contacts.A principle advantage of this approach is its ability to consolidate contact information across multiple models. Target T0618 is an excellent example. Several of the models submitted for target T0618 had misplaced some of the helical bundles. By pooling all of the models together into an ensemble and extracting the most common  long range contacts, four key long range interactions can be identified Figure . To checTo further evaluate the method's effectiveness in collecting and consolidating contacts across an ensemble of models, we clustered long range predicted contacts and then calculated the coverage of these clusters by each model in the ensemble. By doing so, we could determine if the conformation ensemble approach was pulling together more localized contacting clusters or if it was simply identifying a combination of interacting clusters which was already quite prevalent and represented in the individual models. To cluster predicted contacts, we grouped contacts based on their separation in sequence. If two contacts were within 4 residues in sequence they were placed in the same cluster. After the clusters were formed, the predicted contact closest to the average position  in sequence to all of the contacts in a cluster was selected as the representative contact for the cluster. This list of representative contacts was filtered and only those representative contacts that were within 4 residues of a true contact were retained. Then each model was checked and the coverage of the clusters calculated. A cluster was considered covered if there was a contact in the model within 4 residues of the cluster's representative contact. Table This ability to consolidate contact information across multiple models is a concept that several protein structure predictors could use as part of their own prediction pipeline. Clustering is widely used as a means to identify more probable structures from a pool of models. However, with clustering only similar models are capable of being clustered and contribute information. With the conformation ensemble approach, all models are able to contribute and help identity likely residue-residue interactions. One could easily envision an iterative approach in which a protein structure predictor could generate a diverse set of models, extract contact data and use it to generate more models. This would allow information about the conformation space to be passed from one round to the next via the likely contacts extracted from the models.A disadvantage of the method is its dependency on a diverse ensemble of mildly accurate 3D models. In order for the approach to work, the models generated need to be able to capture at least some local portion of the overall topology of the protein. If all of the models in the ensemble are of poor quality then the method does not perform very well.An additional consideration which must be taken into the account is the generation of the models. In practice, one would need to generate a varied ensemble of models before using the method. This could be done using a variety of protein structure prediction methods or variants of a few approaches. The time and computing resources needed to generate the models would depend on the methods used to produce the models. These decisions would affect the general practicality and usefulness of the method as a general residue-residue contact predictor. Yet, as we have demonstrated the method is applicable to ensembles of smaller sizes and still generates relatively accuracy predictions. The size of the ensemble and the sources of the models are choices which must be made when implementing a conformational ensemble predictor and inevitably affect the time needed to make contact predictions, the accuracy of those predictions and the method's ability to extract varied contact information across the models.In this work we have presented a conformation ensemble approach for predicting protein residue-residue contacts. The method draws contact data from an ensemble of models which capture slightly different conformations and contain complementary information. This information can be pooled together to capture recurrent, and therefore more likely, residue-residue contacts. We evaluated our approach on hard targets from CASP8 and CASP9 and found that it is capable of achieving state of the art performance for medium and long range residue-residue contact prediction. We have also demonstrated that the generated contact information coupled with a simple scoring scheme is capable of effectively ranking models.JE, JC conceived the project. JE, JC designed the experiment. JE implemented the method and carried out experiment. JE, JC, ZW analyzed the results. JE, JC wrote the manuscript. All authors read, edited and approved the manuscript.


Publication bias jeopardizes evidence-based medicine, mainly through biased literature syntheses. Publication bias may also affect laboratory animal research, but evidence is scarce.To assess the opinion of laboratory animal researchers on the magnitude, drivers, consequences and potential solutions for publication bias. And to explore the impact of size of the animals used, seniority of the respondent, working in a for-profit organization and type of research  on those opinions.Internet-based survey.All animal laboratories in The Netherlands.Laboratory animal researchers.Median (interquartile ranges) strengths of beliefs on 5 and 10-point scales .Overall, 454 researchers participated. They considered publication bias a problem in animal research (7 (5 to 8)) and thought that about 50% (32–70) of animal experiments are published. Employees (n = 21) of for-profit organizations estimated that 10% (5 to 50) are published. Lack of statistical significance (4 (4 to 5)), technical problems (4 (3 to 4)), supervisors (4 (3 to 5)) and peer reviewers (4 (3 to 5)) were considered important reasons for non-publication . Respondents thought that mandatory publication of study protocols and results, or the reasons why no results were obtained, may increase scientific progress but expected increased bureaucracy. These opinions did not depend on size of the animal used, seniority of the respondent or type of research.Non-publication of “negative” results appears to be prevalent in laboratory animal research. If statistical significance is indeed a main driver of publication, the collective literature on animal experimentation will be biased. This will impede the performance of valid literature syntheses. Effective, yet efficient systems should be explored to counteract selective reporting of laboratory animal research. This is not just an issue of publication bias [.]. Scientific experiments are designed to test for evidence in favour of a particular experimental hypothesis and to abandon it if insufficient evidence is acquired.”Publication bias jeopardizes evidence-based medicine through biased literature syntheses of clinical studies. We approached respondents via a two-step procedure: (i) the Dutch professional association of animal welfare officers, and (ii) all animal welfare officers. In March 2011, we sent a standard letter of invitation to participate by email to all animal welfare officers in the Netherlands via one liaison person (ML) who had access to them through their professional association. Since the animal welfare officers have address lists of all researchers that (had) performed LAR in their institutes, they were asked to send the invitation letter to all their animal researchers. The invitation letters  contained the link to the internet-based survey and explained that confidentiality was guaranteed for the respondent and their institute. In the months prior to the survey’s launch, two authors (GtR and LH) had secured informal commitment to the survey from animal welfare officers in ten institutes.The survey  addressewww.surveymonkey.com) and STATA software (version 10.1). Mann-Whitney and Kruskal-Wallis tests (in combination with Tukey’s post-hoc test) were used to assess statistical significance . We applied the Bonferroni correction to adjust for multiple testing.We used Surveymonkey software  received the invitation letter and an internet link for the survey. We estimate that between 2,000 and 3,500 laboratory animal researchers received the invitation to participate. Between 17 March and 30 July 2011, 474 (between 14–24%) laboratory animal researchers returned the survey. Fifty-one respondents did not fill in the survey completely. Of these, we excluded 20 because of absence of (at least) their background data. This left 454 participants for the analysis. The variation in the number of respondents across Publication bias is an important problem in laboratory animal research (LAR) according to laboratory animal researchers. We estimate that only fifty percent of LAR is published, but it may be far less in for-profit organizations given that their employees estimated that only ten percent of LAR gets published overall, including their own. Lack of statistical significance, technical problems, the opinions of supervisors and peer reviewers were considered important drivers of non-publication. Respondents thought that mandatory publication of study protocols, research results or the reasons why results could not be obtained may accelerate scientific progress.To our knowledge, this is the first survey among laboratory animal researchers focusing on publication bias. This survey has several limitations. First, we estimate the response rate to this survey to be between 14 and 24 percent. We do not know to which extent the results are representative for the Dutch LAR community, let alone for the wider LAR community. The number of laboratory animal researchers in The Netherlands is unknown. We were unable to obtain exact information from the institutes on to the number of E-mail addresses to which the survey had been sent. Another difficulty is that such address lists may not always be fully up to date. In particular, researchers who retire or change jobs may be listed in error. Second, the survey was restricted to one country. Third, only few researchers in for-profit organizations participated. Fourth, our results are reminiscent of the joke about surveys on driving ability in which 90% of respondents think that they belong to the group of people whose driving abilities are above-average. Likewise, it seems somewhat paradoxical that our respondents estimate the publication rate of their own work as much higher  than the overall rate. Another explanation may be that the 50% rate mentioned in the introduction to the survey acted as an anchor that made respondents estimate the overall rates as too low. That would imply that a non-publication rate of 20% is closer to the truth. This issue is related to the next one. Fifth, our study investigated researchers’ opinions, which may not reflect the true rate(s) of non-publication. Sixth, due to the large number of statistical significance tests (n = 121), application of the Bonferroni correction for multiple testing  implies that only p-values below 0.0004 should be considered statistically significant  for ethics approval, IACUCs may play a crucial role in the registration of all LAR and prevention of publication bias. A system ensuring periodic follow-up of each experiment’s fate would reinforce such registration. It may be challenging to build a watertight system that simultaneously minimizes bureaucracy. Application of modern information technology may be crucial. One option to prevent that study results have an effect on the editorial decision is to initially submit manuscripts without any results. Statistical significance testing is probably a main driver of non-publication. This is especially unfortunate given the widespread errors involved in understanding the meaning of p-values.What are the implications for further research? As we have learnt from randomized trials in humans, follow-up of cohorts of study protocols may help us understand the magnitude and the causes of publication bias in LAR, which in turn may affect the research community’s motivation to deal with it. In the meantime, more research into statistical correction of publication bias seems useful.Table S1Statistically significant differences between medians are highlighted with an asterisk (*). An asterisk in the column that corresponds to the first level of one of the four variables , refers to the difference between the medians of the first and second level. An asterisk in the second column refers to the difference between the second and third level. An asterisk in the third column refers to the difference between the first and the third level.(DOCX)Click here for additional data file.Table S2All numbers are medians after bootstrapping the analysis 200 times. † Bootstrapped quantile regression on the median, simultaneously adjusting for all four stratification variables, that were modeled as dummy variables. CI denotes confidence interval. The intercept of the fully adjusted model, that is, the estimate for the median proportion of papers published of not-for profit researchers working with small animals, having co-authored between 0 and 5 papers, and working on both fundamental and pre-clinical topics was 50 percent (95% 43.5–56.5).(DOCX)Click here for additional data file.Table S3All numbers are medians after bootstrapping the analysis 200 times. † Bootstrapped quantile regression on the median, simultaneously adjusting for all four stratification variables, that were modeled as dummy variables. CI denotes confidence interval. The intercept of the fully adjusted model, that is, the estimate for the median proportion of papers published of not-for profit researchers working with small animals, having co-authored between 6 and 20 papers, and working on both fundamental and pre-clinical topics was 80 percent (95% 73.9–86.1). § The group that (co-)authored 0–5 studies was excluded from this row because very junior investigators very often had either zero or 100 percent of their papers published.(DOCX)Click here for additional data file.Appendix S1(DOC)Click here for additional data file.Appendix S2(PDF)Click here for additional data file.