Up to 50 % of HIV-infected persons in sub-Saharan Africa are lost from care between HIV diagnosis and antiretroviral therapy (ART) initiation. Structural barriers, including cost of transportation to clinic and poor communication systems, are major contributors.We conducted a prospective, pragmatic, before-and-after clinical trial to evaluate a combination mobile health and transportation reimbursement intervention to improve care at a publicly operated HIV clinic in Uganda. Patients undergoing CD4 count testing were enrolled, and clinicians selected a result threshold that would prompt early return for ART initiation or further care. Participants enrolled in the pre-intervention period (January – August 2012) served as a control group. Participants in the intervention period (September 2012 – November 2013) were randomized to receive daily short message service (SMS) messages for up to seven days in one of three formats: 1) messages reporting an abnormal result directly, 2) personal identification number-protected messages reporting an abnormal result, or 3) messages reading “ABCDEFG” to confidentially convey an abnormal result. Participants returning within seven days of their first message received transportation reimbursements (about $6USD). Our primary outcomes of interest were time to return to clinic and time to ART initiation.P < 0.001); and median time to ART initiation was 47 days (IQR 11–75) versus 12 days (IQR 5–19), (P < 0.001). In multivariable models, participants in the intervention period had earlier return to clinic  and earlier time to ART initiation . All three randomized message formats improved time to return to clinic and time to ART initiation .There were 45 participants in the pre-intervention period and 138 participants in the intervention period  with low CD4 count results. Median time to clinic return was 33 days (IQR 11–49) in the pre-intervention period and 6 days (IQR 3–16) in the intervention period  contains supplementary material, which is available to authorized users. Despite expanded access to antiretroviral therapy (ART) , programStructural barriers to care in resource-limited settings, including transportation costs and absence of communication between providers and patients, are major contributors to poor linkage –14. CommScalable interventions that mitigate structural barriers to clinical care in resource-limited settings are urgently needed. Mobile health  applications hold promise in this area by leveraging existing cellular phone infrastructure to improve patient-provider communication and prioritize care delivery for those most in need. Cellular phone coverage in sub-Saharan Africa increased from 5 to 70 % of the population during the past decade, while personal subscriptions increased from 16 to 380 million . While sStudy participants were enrolled from a publicly operated President’s Emergency Plan for HIV/AIDS Relief (PEPFAR)-supported HIV clinic at the Mbarara Regional Referral Hospital in Mbarara, Uganda. Eligibility criteria included: a) current enrollment at the adult HIV clinic, b) self-reported access to a cellular phone, whether personal or shared, c) residence in a district immediately surrounding the clinic , and d) undergoing a CD4+ T-lymphocyte (CD4) test determined by the ordering clinician to be of critical importance . Clinicians selected the abnormal CD4 result threshold on the day the laboratory test was ordered. The threshold was meant to specify a result below which an early return to clinic would be requested for clinical evaluation. Potential participants could be ART-naïve or on ART (with concern for treatment failure). We did not include viral load testing as an inclusion criterion because it is not available free of charge at the clinic and was rarely performed outside of research activities.The intervention was developed based on a conceptual framework developed through pre-study mixed-methods research. Pre-intervention interviews with patients and providers at the clinic revealed two major barriers to clinic return after abnormal test results at this site: 1) lack of communication between staff and patients outside of clinical visits, which limited patient decision-making capacity , and 2) We initially considered a fully randomized control trial, including a control group without a notification SMS message. We decided against this for two reasons. First, in a preliminary acceptability study at the clinic, we found that 100 % of surveyed participants desired cellular phone communication about their laboratory test results . Second,During the intervention period, clinicians used eligibility criteria identical to those used during the pre-intervention period, including selection of an abnormal CD4 count threshold to trigger the SMS and transportation reimbursement intervention. We discussed message design with study clinicians during the intervention design phase. They requested an individualized determination of abnormal test results over a standardized method, largely to allow enrollment of patients already on therapy who were considered to be at risk for treatment failure. Similarly, clinicians scheduled the next clinic appointment prior to study enrollment during both study periods. Eligible participants completed written informed consent to receive health messages on their phone, completed a baseline structured survey to collect SMS scheduling preferences, and were given a brief instructional session to describe possible messages and select a personal identification number (PIN) to open messages.Those with a CD4 test result above the clinician-specified threshold  received a single SMS message stating that their test result was within the normal range and that they should return to clinic as scheduled. For participants with a CD4 test result below the stated threshold , study staff used the randomization module within the Research Electronic Data Capture (REDCap ) study dMessages were scheduled and initiated with CommCare , a web-based mobile health application. Participants could receive up to seven daily messages at preferred days of the week and times of day  that were selected by participants during the baseline survey. On the date and time of each scheduled message, CommCare sent a web-based automated prompt with the content of the message to a Ugandan-based telecommunications company , which relayed an automated SMS to the indicated phone number. The messages cost $0.02 each to send, and were paid for by the study. Participants in the intervention period with a normal laboratory result received a single message at the next preferred date and time that was indicated on their enrollment scheduling form. Participants in the intervention group received a single abnormal result message daily, up to a maximum of seven days after the abnormal result. Participants who returned to clinic within seven days of the first abnormal result message received a transportation reimbursement stipend of 15,000 Ugandan shillings . Participants with normal laboratory results or those presenting after seven days from the first message were not eligible for transportation reimbursement. There were no blinding procedures as part of this study.For both study groups, we recorded the date of clinic return after an SMS message and, for the ART-naïve participants, the date of ART initiation. To assess receipt and comprehension of the messages, research assistants called participants in the intervention period who did not return to clinic within 14 days (after determination of primary outcome success or failure). We performed home-based tracking for participants with abnormal laboratory results who did not return to clinic by 28 days.In our initial protocol, our prespecified primary outcome of interest was return to clinic within 7 days of an abnormal test result notification. However, we noted during study planning that the time from test result to first SMS transmission varied by participant. For example, if a participant selected only a single day to receive messages  and the laboratory result returned on the following calendar day (Tuesday), the maximum time period from laboratory result to receipt of the first message was 7 days, which corresponded to a maximum period of 14 days from test result to clinic return to achieve the desired outcome. To ensure unbiased outcome assessment between study arms, we changed our primary outcome to: return to clinic within 14 days of when the laboratory result was received back at the clinic from the laboratory. The study was powered to detect a difference in return time for participants with abnormal results. Whereas the study was initially powered based on assumptions about clinic return with 7 days, we updated our sample size estimates with the 14-day outcome using data from the pre-intervention period. During the pre-intervention period, approximately 30 % of 45 participants with abnormal laboratory results returned to clinic within 14 days. We planned to enroll 45 participants with abnormal laboratory results in each intervention arm in order to have 80 % power to detect a doubling in return rate  between the pre-intervention period and each of the intervention groups.We conducted survival analyses and fit Cox proportional hazards regression models to estimate differences between study arms in: 1) time to return to clinic after an abnormal result and 2) time to initiation of ART among ART-naïve participants. Our primary predictors of interest were message type (comparing each of the three message formats with the control group) and study period (comparing the control period with the intervention period). Models were adjusted for known predictors of clinic retention in care based on prior studies, including age, gender, district of residence, education, and CD4 result . ParticiThe study was reviewed and approved by the ethical review committees of the Mbarara University of Science and Technology (Reference Number: 1/7), Partners Healthcare (Partners Reference Number: 2011P001538), and the Ugandan National Council of Science and Technology (Reference Number: IS 83). The trial and study protocol were registered prior to study procedures at clinicaltrials.gov (NCT01579214).Of 554 participants screened, 21 (6 %) were excluded due to invalid laboratory results (n = 21). An additional 2 % (n = 12) were excluded from the intervention period due to prior participation in the control period  in the pre-intervention period and 67 % (93/138) in the intervention period ; and median time to ART initiation was 8 (P = 0.002), 15 (P = 0.016), and 15 days (P < 0.001). All comparisons between the pre-intervention period and each individual message format remained statistically significant in multivariable analyses adjusted for sociodemographic and clinical factors (Table P = 0.09), the proportion that returned before their scheduled visits (P =0.96), or the proportion that initiated ART within 14 days (P = 0.32). Lastly, among those with normal results, a greater proportion who received an SMS message returned within 7 days of their scheduled return date  and targeted end users, during application design, evaluation, and implementation . ImportaOur study is subject to a number of limitations. First, we implemented an unblinded, non-randomized study design, and found differences in the study periods in participant age and proportion naïve to ART. While the differences in time to clinic return and time to ART initiation could be explained by unmeasured confounders or temporal changes in clinic outcomes, we believe this is unlikely because 1) our results were independent of the most likely confounding variables, including immune status, gender, and education, 2) our effect sizes were large and demonstrated across all randomized subgroups, and 3) the intervention period immediately followed the control period and no other changes in clinical protocols took place during either period. Second, our study was implemented within a research setting. Effectiveness studies should be pursued to evaluate its impact on a larger, real-world scale. In its current format, our intervention requires a single phone and/or Internet connection, a staff member to process and enter results, and the cost of a transportation stipend ($6 per patient in our case). These inputs are comparable to the cost, infrastructure, and human resources requirements for standard point-of-care CD4 tests, which require similar resources and cost approximately $10 per sample. While the greatest threat to sustainability of our intervention is the cost of the transportation reimbursement, it is likely that this could be a one-time cost for most patients. Data from our site and others has demonstrated that economic restoration is rapid and largely complete during the first year of ART therapy, suggesting the potential to decrease reliance on economic support early after initiation of care , 40. WhiIn summary, we found significant improvements in time to clinic return and ART initiation after abnormal laboratory results in southwestern Uganda with an SMS-based combination intervention including laboratory result notifications and transportation reimbursements. The benefit of the intervention was seen in three different SMS formats with varying degrees of clarity and confidentiality. Future investigations should evaluate the scalability of similar interventions. Successful reproducibility would add a promising strategy to improving HIV care in sub-Saharan Africa.


The purpose of this study was to investigate the effects of a low-load gluteal warm-up protocol on countermovement and squat jump performance. Research by  Improving an athlete’s explosiveness and power capabilities is a central objective of many training programs. The type of a warm-up undertaken prior to the performance of explosive movements, such as jumping or sprinting, can have an effect on the power output during these dynamic exercises. Research has been conducted to investigate the effect of warm-up protocols on subsequent explosive exercise performance with the aim of optimizing dynamic performance . These pA large number of studies have investigated the acute effects of performing heavy resistance exercises as part of a warm-up protocol. Many of these have supported the efficacy of performing heavy loads  of the back squat exercise in order to enhance subsequent jump performance . DespiteThe gluteal muscles play a major role in running and jumping activities, and studies investigating lower limb muscular activity during running and jumping have found the gluteal muscles to be vigorously activated . The hipIn light of the existing research the present study sought to further investigate the effectiveness of the low-load gluteal protocol employed by Eleven track and field athletes  formed the subject base for this study . Seven of the athletes specialized in sprint events , one in the high jump, one long jump, one pole vault and one in the heptathlon. All subjects had previously competed at the national level in their event, with one subject having also competed internationally. The athletes were proficient in the technique of the CMJ and SJ, with both exercises having formed a part of their regular training for a minimum of 2 years. The subjects were injury-free and were participating in pre-season training at the time of the study. Approval from the University of Limerick Ethics Committee was received prior to recruitment. Subjects were informed of the experimental risks and signed an informed consent form and a Physical Activity Readiness Questionnaire before the investigation.The experiment involved three testing sessions at equal intervals over a three-week period. For reliability reasons, and to control for circadian variation, each subject completed all three testing sessions on the same day of the week and at the same time . Each teTesting session one was a familiarization session in which the subjects completed the aforementioned warm-up, followed by a number of CMJ and SJ trials until the experimenter was satisfied with their technique. For the CMJ the subjects were instructed to start from a straight leg position with hands placed on the hips, squat down to a self-selected depth before exploding upwards in an attempt to gain maximum height. They were instructed that this should be a smooth action with no pause in the crouch position. For the squat jump subjects were instructed to start by squatting to a 90° angle at the knee and hold that position until given the cue to jump by the experimenter, at which point they were to explode forcefully upwards and jump for maximum height. No further dip or counter movement was permitted as only an upward motion constitutes a correct SJ. In both jumping exercises subjects were instructed to keep both legs extended while in the air and land back on the force platform before bending their knees. The force-time trace for each SJ was examined to ensure that no dip occurred at the beginning of the contraction as this would indicate a counter movement. To conclude the familiarization session the subjects were asked to perform the low-load gluteal warm-up protocol and instructed on the correct technique for each of the seven exercises. The present study replicated the low-load gluteal warm-up protocol employed by In the second testing session the subjects completed the general warm-up followed by recording of three baseline CMJs. The baseline jumps were separated with ninety seconds rest. The subjects then performed the gluteal warm-up followed by a further five CMJs, one at each of the pre-determined rest intervals . Subjects then completed a cool-down consisting of light jogging and static stretching of each of the major lower body muscle groups. Testing session three followed the same procedure as session two except the CMJs were replaced by SJs. A timeline of testing sessions 2 and 3 is illustrated in All jumps were performed on an AMTI force platform (AMTI OR6-5), mounted flush with the surrounding laboratory floor. Data were collected with the system sampling at 1000 Hz. Force-time traces were real-time displayed and saved with the use of computer software  for further analysis. Reliability of this particular force platform had previously been established . A pilotThe dependent variables for the CMJ were height jumped and peak ground reaction force (GRF). Peak GRF was calculated from the force-time CMJ traces. Jump height was derived from the flight time score, obtained via inspection of the force-time CMJ traces. The SJ force platform data were used to calculate jump height, peak GRF, time to peak GRF, max RFD, time to max RFD, starting strength, change in force in first 100 ms and speed strength index. The details of how each variable was derived are detailed in All statistical analyses were conducted using IBM SPSS Statistics (Release 20.0.0). The differences between the average of the three baseline scores for each dependent variable and the scores after each recovery interval were evaluated individually using a repeated-measure GLM ANOVA. The ANOVA had 1 within-subjects factor, namely Condition, with 2 levels . This analysis was performed on all recorded variables in both the CMJs and SJs.d (1988) were also obtained for each variable found to be significantly different from baseline using the following formula:1 = mean of group 1; M2 = mean of group 2; σpooled = pooled standard deviation), 2 min , 4 min  and 6 min  rest. Similar to the CMJ, the analysis of height jumped for the SJ showed a significant decrease in performance after 30 s , 2 min , 4 min  and 6 min  rest, as shown in The results for FT and peak GRF are presented in d=0.238, small). The mean CMJ peak GRF increased at the 2, 4 and 6 min interval, before decreasing again after 8 min, however the GLM ANOVA did not report these differences as significant. Mean SJ peak GRF scores increased from baseline at all rest intervals, excluding 4 min, however the GLM ANOVA did not report these differences to be significant (p>0.05).The peak GRF results for the CMJ and the SJ are presented in d=0.498, small). Max RFD also increased at all other rest intervals, however these increases were not deemed significant (p>0.05). Time to peak GRF and time to peak RFD showed an improvement in both variables at the 8 min rest interval, with a significant 43% reduction in the time taken to reach peak GRF  and 65.7% reduction in the time to max RFD . The statistical analysis also showed a significant 63.4% increase in starting strength for the 8 min rest interval  and a significant increase of 49.1% in the change in force in the first 100 ms at this recovery interval . Finally, statistical analysis of the speed strength results also showed a significant 43.6% increase after 8 min rest .The results pertaining to the SJ fast force production variables are presented in The results of this investigation provide insight into the effects of a low-load gluteal warm-up on explosive jump performance. The height jumped results for both the CMJ and SJ showed a significant reduction compared to baseline scores at the 30 s, 2 min, 4 min and 6 min rest intervals, suggesting that the warm-up protocol elicited fatigue rather than performance enhancement in the athletes. In order to understand the true effects of the low-load gluteal warm-up routine on explosive jump performance, a number of other important variables were also examined. While height jumped is the outcome measure of jump performance, other variables recorded in this study provide insight into the jumping process and the generation of impulse. Significant improvements were seen in many of the explosive SJ variables. Seven of the eight variables recorded during SJ performance showed improvement at the 8 min rest interval, with analysis finding five of these improvements to be statistically significant. These improvements in explosive ability may be of great interest to those participating in sports where rapid force production is required.As shown in the results section height jumped in both the CMJ and SJ decreased significantly after 30 s, 2 min, 4 min and 6 min of rest. A decrease in height jumped was also evident after 8 min, however this decrease was of a small magnitude (CMJ: 2.3% lower and SJ: 3.5% lower) and was not deemed significant by statistical analysis. The height jumped variable in both jumping exercises demonstrated a trend towards recovery to baseline values as the rest interval increased. Previous research has found squat warm-up protocols to have a detrimental effect on height jumped in CMJs performed immediately after the resistance exercise , time to max RFD (65.7%), starting strength (63.4%), change of force in first 100 ms of contraction (49.1%) and speed strength (43.6%) all showing significant positive improvement at the 8 min rest interval. Max RFD was improved at every post warm-up rest interval, however statistical analysis deemed only the improvement after 2 min rest as significant.RFD parameters have important functional significance in fast and forceful muscle contraction . It takeThe results of the current study suggest that a low-load gluteal warm-up is effective in enhancing fast force production variables related to SJ and CMJ performance. The results are applicable to those participating in sports where explosive force production is necessary to optimize performance, for example sprinting and jumping. No improvement, however, is evident for CMJ or SJ height jumped post the gluteal warm-up indicating that such a protocol may be inappropriate for sports where maximum height is the key contributor to performance.The warm-up employed in this study required little equipment and thus can be easily incorporated into pre-training and competition routines. Large and statistically significant improvements in performance were recorded at the 8 min interval across a number of measures of fast force production. Therefore, in order to enhance dynamic performance in rapid movement activities a gluteal warm-up could be performed 8 min prior to commencement of the event. For comparative reasons, future research should include additional warm-up intervention groups within the research design.


Low-level laser irradiation in the visible as well as infrared range is applied to skin for treatment of various diseases. Here we summarize and discuss effects of laser irradiation on mast cells that leads to degranulation of the cells. This process may contribute to initial steps in the final medical effects. We suggest that activation of TRPV channels in the mast cells forms a basis for the underlying mechanisms and that released ATP and histamine may be putative mediators for therapeutic effects. Heat prammation ,8,9. In ammation . The masammation . These rammation ,14. In fammation , and we In the following we will review and discuss effects of laser irradiation in the visible, infra-red (IR) as well as the ultra-violet (UV) range on ion channels of the TRPV family; effects of NIR have not been investigated. Since several of the medical effects, including wound healing  and antiXenopus oocyte with heterologously expressed TRPV1. The TRPV1-mediated current exhibited in solution with 2 mM Ca2+ a reversal potential of about −30 mV; a similar value of −20 mV has been reported for oocytes with solution containing 0.5 mM Ca2+ i) indeed increased in response to irradiation by red laser light of 657 nm  (2+]i was independent on extracellular Ca2+. With the same irradiation parameters that stimulate [Ca2+]i increase an elevation of extracellular ATP could be detected (2+]i was demonstrated to trigger exocytotic release of ATP i , but al2+-permeable ion channels, their activation may contribute to the laser-induced increase in intracellular Ca2+ that triggers degranulation and endocytotic release of ATP. Such light-induced mechanism may contribute to the basis of the medical effects of LLLT. This hypothesis still needs confirmation in animal tests and clinical trials.We have shown in this review that laser irradiation in the visible and IR as well as UV range can modulate the function and expression of TRPV ion channels, and in particular TRPV1, TRPV2, and TRPV4. This may form the basis for effect of LLLT. As Ca


Ambystoma mexicanum) fibula to regenerate across segment defects of different size in the absence of intervention or after implant of a unique 8-braid pig small intestine submucosa (SIS) scaffold, with or without incorporated growth factor combinations or tissue protein extract. Fractures and defects of 10% and 20% of the total limb length regenerated well without any intervention, but 40% and 50% defects failed to regenerate after either simple removal of bone or implanting SIS scaffold alone. By contrast, scaffold soaked in the growth factor combination BMP-4/HGF or in protein extract of intact limb tissue promoted partial or extensive induction of cartilage and bone across 50% segment defects in 30%-33% of cases. These results show that BMP-4/HGF and intact tissue protein extract can promote the events required to induce cartilage and bone formation across a segment defect larger than critical size and that the long bones of axolotl limbs are an inexpensive model to screen soluble factors and natural and synthetic scaffolds for their efficacy in stimulating this process.We tested the ability of the axolotl ( Mammalian bones fail to regenerate across gaps that exceed the critical size defect (CSD), defined as the smallest defect that cannot be bridged by regenerated skeletal tissue over the life of the animal . CSDs inOrthopedic regenerative medicine seeks new strategies aimed at regeneration across CSDs in both intramembranous and endochondral bones. These strategies have included implanting osteoinductive and osteoconductive scaffolds, with or without osteogenic cells and/or growth factors or growth factor genes , 6–11. WXenopus hind limb.Most studies on segment defects in endochondral bones have been carried out on mice, rats, rabbits and sheep. Amphibians have been used extensively for studies on the regeneration of amputated limbs, and to a lesser extent for experiments on regeneration of individual limb skeletal elements in unamputated limbs , 14. ExtAmbystoma mexicanum (axolotl)   has beenng bones , 22. AxoMultiple bone morphogenetic proteins (BMPs) have been implicated in skeletal development and regeneration . We havehttp://www.cytoscape.org/) to select the proteins most commonly identified as significant. The topological properties evaluated were: bottleneck nodes, maximal cliques (MCC), eccentricity, and maximum connected component (MNC).The information extracted by BioMAP was normalized using the protein and gene names from the UniProt database. The Human Protein Reference Database (HPRD) was then used to identify growth factors and transcription factors from this gene/protein list. These growth factors and transcription factors were used to determine the predominant pathways and networks of protein interaction in cartilage regeneration, using MetaCore (GeneGO Inc). These were further analyzed using four topological parameters of the CytoHubba plugin  in CytosEleven growth factors emerged from this analysis: These were FGF-2, PDGF-A, PDGF-B, PDGF-D, EGF, HGF, TGF-β2, TGF-β3, Follistatin, VEGF-A, and Lefty-2. Nine of these factors have been implicated experimentally in fracture repair, nine in bone regeneration, and seven in cartilage regeneration . TGF-β2 Xenopus counterparts  of 55% (EGF) to 99% (BMP-4). Five different combinations of BMP-4 and the other selected growth factors were tested for their ability to promote regeneration across a 50% segment defect  and white (dd mutant) axolotls obtained as small larvae from the Ambystoma Stock Center at the University of Kentucky and raised to a length of 12–15 cm . Both types are widely used in limb regeneration studies and are not known to differ in any respect related to regeneration. They were maintained in individual containers with daily changes of artificial pond water (10% Holtfreter solution) and fed salmon chow daily . Axolotls (unfed for 24 hrs prior to surgery) were anesthetized by immersion in 0.1% (w/v) of MS-222  in 10% Holtfreter solution buffered with bicarbonate to pH 7.3. The age of the animals used was approximately the same, but because of individual differences in individual growth rates, the limbs were not uniform in size from animal to animal.A simple cut with microscissors through the mid-diaphysis was used to create a fracture of the fibula. To create a segment defect, the length of the fibula was first measured externally with calipers. A longitudinal cut was made with microscissors along the posterior aspect of the hind limb zeugopodium and the muscle pushed to one side to expose the fibula. A length of bone equivalent to a defect of 10%, 20%, 40% or 50% of the total fibula length was then excised from the middle of the bone. The average length of the fibula was 7 mm. The average length of the defects was thus approximately 0.7 mm, 1.4 mm, 2.8 mm, and 3.5 mm, respectively. Wounds were closed with two sutures of #6 silk thread . The animals were kept immersed in 0.01% MS-222 to control pain during a recovery period of two hours and then returned to artificial pond water. The wounds healed within one week without other treatment. Control limbs  were harvested at intervals of one to six months, and fixed for one week in 10% formalin.All animal care and experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of laboratory animals of the National Institutes of Health. The protocol was approved by the School of Science Institutional Animal Care and Use Committee of Indiana University-Purdue University Indianapolis (Protocol SC 226R).Defects of 50% were used to test growth factor combinations and protein extracts. SIS scaffolds loaded with growth factor combinations, blastema extract, or tissue extract were inserted into 50% defects immediately after removal of the bone segment from the fibula and the wounds closed with two #6 silk sutures. Controls consisted of limbs in which the fibular defect received no treatment or was implanted with scaffold soaked overnight at 4°C in 0.8x aPBS. Growth factor-treated and tissue extract-treated limbs were harvested at intervals of two to six months, and fixed for one week in 10% formalin.An eight-strand biodegradable braid of SIS, a generous gift from Cook Biotech , was used to deliver combinations of growth factors, blastema extract, and tissue extract to 50% segment defects. The braid was half the diameter of the fibula and hydrated to the diameter of the fibula when soaked in growth factor or extract solution prior to implantation into a segment defect. The braid was cut into pieces approximating the length of the segment defect that were then sterilized for 30 min with UV light. For each set of implants, five sterile pieces were immersed together overnight at 4°C in 50 μl of sterile growth factor or tissue extract solution contained in the inverted cap of a 1 ml Eppendorf tube. The cap in turn was placed in a small culture dish floored with sterile moist paper to maintain humidity and prevent changes in concentration of the solutions.Kinetics of protein release was analyzed for BMP-4. Three scaffolds at a time were soaked in 50 μl of BMP-4 solution (10 ng/μl) at 4°C overnight. Each scaffold was then serially transferred to new Eppendorf tubes containing 50 μl of 0.8x aPBS and allowed to release for 2 hr, 4hr, 8hr, 1 day, 2 days, and 3 days at room temperature. The concentration of BMP-4 per 50 μl of BMP-4 released into the 50 μl of aPBS at each time point was measured using a human BMP-4 Quantikine ELISA kit .All of the fixed limbs for each time point were first imaged by X-ray  and then by microcomputed tomography (micro-CT), using a high-resolution desktop imaging system . The micro-CT scans included the region from the distal end of the femur to the proximal tarsus and were obtained using an X-ray source set at 60kV and 167 μA over an angular range of 180°  with a 17-μm pixel size. NRecon Reconstruction software was used to construct cross-sectional images with up to 8000 x 8000 pixels, which were then used to reconstruct a volumetric 3-D image. These techniques image ossified tissue, but do not detect cartilage formation. Bone volume per total volume (BV/TV) was calculated at the defective region in each animal.After X-ray and micro-CT imaging, half of the samples for each time point were stained in 0.25% methylene blue for cartilage matrix, or double-stained in methylene blue and alizarin red for both cartilage and bone matrix. The other half of the fixed specimens was decalcified in Calciclear  for three weeks at room temperature, embedded in paraffin (Fisher Scientific), sectioned at 10 μm, and stained with Ehrlich’s hematoxylin and eosin (Fisher Scientific). Some slides were stained with Gomori’s Trichrome stain  after being re-fixed overnight in Bouin’s solution after the re-hydration step. Imaging was done on a Leica model DM2000 light microscope equipped with a camera .Two fluorochromes were used to measure early bone regeneration in untreated 10% vs. 50% defects. On the day of surgery, a 1% calcein solution  was injected subcutaneously into the proximal one-third of the hind limb zeugopodium at a dose of 15μg/gm body wt. This fluorochrome fluoresces green. One week after the surgery, a solution of 3% alizarin complexone red was injected at the same site at a dose of 30 μg/gm body wt. This fluorochrome fluoresces red. The limbs were harvested at 3 weeks post-surgery and fixed in 10% neutral formalin solution for one week. The soft tissues were removed with jeweler’s forceps and the fibular bone on either side of the defect was imaged whole with Leica fluorescent optics for red and green fluorescence . Two color images were merged using Image J software and the extent of bone regeneration was assessed by measuring the length of red color extending from green color on the anterior, middle and posterior sides of the bone.X-ray imaging proved to be of little use, but micro-CT scans showed that ossification had taken place in the defects . Fig 3 iThe process of regeneration appeared to be very similar in fractures and 10% segment defects, which were bridged within 2–3 weeks with cartilage that ossified earlier than in 20% defects. Examination of sections at three weeks post-operation indicated that in the 20% defects, the ossification centers arose not in the center of the cartilage bridge, as in the fetal development of endochondral bones, but in the cartilage regenerating from each end of the fibula, suggesting that regeneration took place from both ends of the cut bone toward the center. Flattened chondrocytes, hypertrophying chondrocytes, and regions of osteogenesis with osteoblasts and osteoclasts were observed extending from the ends of the bone in these sections. prior to insertion into the defect. Trichrome and H&E staining of sections were used to monitor degradation of the scaffold and the tissue filling the defect space. After three months there was no visible skeletal regeneration in any of the defects and they were filled with disorganized connective tissue and regenerated muscle . These rFifty percent defects were implanted with 8-braid SIS soaked in growth factor, blastema extract or tissue extract solutions. This defect size was chosen to provide a regenerative challenge well beyond the CSD. We noted that upon examination of whole mounts and sections that the bone at the cut ends of the fibula in many cases had undergone substantial regression, making the segment defect closer to 70%.The BMP-4/HGF combination yielded two cases of partial regeneration  and fourWhole limb tissue extract also was effective at inducing regeneration. Of 18 limbs, two had partial regeneration and four had significant regeneration. Figs None of the other growth factor combinations, or blastema protein extract, evoked any regeneration by three months post-implantation.More than half of the 10% and 20% segment defects showed signs of regeneration two months after operation and 80%-88% of these defects were bridged by three months post-implant as evidenced by micro CT, whole mount staining for cartilage and bone, and histological examination of stained tissue sections. Cartilage developed first and was replaced by bone. Regeneration appeared to take place by extension from one or both ends of the cut bone. Extension from one end continued to the other end, whereas extensions from both ends met in the middle to bridge the gap. These extensions were often at first irregular in appearance but eventually became more regular as endochondral ossification replaced the cartilage template. The impression from histological examination was that the cells providing the chondrocytes for regeneration came from the periosteum at the cut ends of the bone. The bone volume in the middle of the newly regenerated bone of the 10% and 20% segment defects was more than 50% of the total regenerated bone volume.In contrast to the 10–20% segment defects, the cut ends of the fibula in 40% and 50% defects were capped off and no regeneration took place across the gaps. These defects were filled in by regenerated muscle and connective tissue that lacked organization. The regenerated bone volume was very low in the capped off ends of the 40% and 50% segment defects. Fluorochrome staining showed clearly that regeneration of calcified skeletal tissue took place in the 10% defects within three weeks, whereas none was initiated in the 50% defects. We also observed that the cut ends of the bone in 40% and 50% defects often underwent regression such that the defect space became larger than that created by removing the bone segment. It is possible that such regression sufficient to cause regenerative failure also occurred in the small number of 10% and 20% control defects that did not regenerate by two months. Hutchison et al  and Satode novo without the extensive processing done to create the version of SIS that we used and thus may have retained significant amounts of growth factors, such as TGF-β, FGF-2, and VEGF  founThe ability of BMP-4/HGF to support regeneration in 50% segment defects is not surprising. BMPs are known to be expressed early in axolotl limb regeneration –43 and tin vitro release kinetics assay, in vivo the growth factors migth have suffered substantial inactivation thus reducing their effectiveness to promote regeneration. Since clinical use ultimately demands a one-step surgical protocol that simultaneously removes damaged bone and implants regeneration-promoting factors, research directed design and testing of slow release or timed release scaffolds is of prime importance.Why we did not see cartilage and bone induced in all the combinations that contained BMP-4 and HGF is not known. The ratios of growth factor concentrations in combinations other than BMP-4/HGF might have been suboptimal, there may have been functional interference in these combinations that canceled out positive effects, or the growth factors needed to be released in a temporal sequence to be effective. We can also ask why the incidence of regeneration was not higher. One possibility is that there may be obligatory pre-regeneration events that take place in the interval between bone removal and initiation of the regenerative response by growth factors. Even tough BMP-4 release remained high for at least three days in our In the treated 50% defects, one case  was noteIn BMP-4/HGF-treated 50% defects, as in untreated 10% and 20% defects, the regenerating cartilage appears to grow from either or both cut ends of the fibula, suggesting either a periosteal or cartilage origin. Cartilage contributes only a small percentage of the blastema cells that regenerate cartilage in an amputated axolotl limb, most regenerated chondrocytes arising by the transdifferentiation of dermal fibroblasts . In mammProtein extract of whole limb tissues was a somewhat more effective promoter of skeletal tissue regeneration than BMP-4/HGF across 50% gaps in the fibula, whereas protein extract of regeneration blastemas had no effect. This result suggests that limb tissue extract contains factors in addition to BMPs and HGF, or simply more optimum concentrations of BMP-s and HGF that initiate the molecular cascade leading to cartilage regeneration and ossification. Pekarinen et al  and TollXenopus hind limb. They used a biocompatible 1,6 hexanedioldiacrylate (HDDA) scaffold loaded with BMP-4 and VEGF to induce bridging of the CSD by a cartilage template, which was followed by the beginning of osteogenesis in the mid-region of the cartilage. The scaffold did not act as an osteoinductive substrate, but rather to deliver the growth factors over the whole length of the CSD, and was pushed to one side of the regenerating cartilage. Untreated CSDs formed only fibrous scar tissue. A disadvantage of this model was that the HDDA scaffold was not biodegradable.Feng et al  demonstrPrrx-1. Prrx-1 is a transcription factor that plays a role in limb development and fibroblast migration [Prrx-1. This result suggested that the nerve and wound epidermis provided signals that induced histolysis and dedifferentiation to produce blastema cells, similar to what happens in the amputated limb.Satoh et al  found thigration , 60 and In our experiments, wound epidermis may have been formed by sufficient injury to the limb to induce the regeneration of a supernumerary foot in one case Figs  and 16. We have established that the axolotl (and most likely other urodele species) can serve as an inexpensive and surgically amenable model to screen different combinations of factors for their ability to promote regeneration of cartilage and bone across a CSD. The model has established that a combination of BMP-4 and HGF, as well as whole limb issue extract is effective in evoking regeneration across gaps of 50% or greater in the axolotl fibula. This model will likely also be amenable to the screening of different scaffold types to optimize the osteoconductive and osteoinductive characteristics of biomaterials and architectures for delivery of cartilage cascade-initiating and sustaining factors that promote cartilage regeneration across defects of critical size or greater in long bones. The model may also be informative about bone regeneration under conditions of weightlessness, such as are encountered on long space flights, since limb and skeletal regeneration in axolotls takes place in a buoyant environment that mimics reduced gravity.


Mtb) to progressive hypoxia in vitro induces a dormant state characterized by reduced replication and metabolism analogous to that postulated for bacilli in LTBI. In vitro, Mtb has been shown to enter microaerophilic nonreplicating persistent (NRP) stage 1, if the dissolved oxygen content falls below 1 %. NRP stage 1 is characterized by thickening of the outer cell wall of Mtb and termination of DNA synthesis. If the oxygen content further drops to approximately 0.06 %, the bacilli enter NRP stage 2, which is accompanied by reduced susceptibility to standard anti-TB drugs but increased susceptibility to nitroimidazole drugs  (1-(2-1-(1H-methyl) ethoxy)-methyl-2-nitroimidazole (18F-RP-170). These tracers have been validated for detection of hypoxia in different cancer types. 18F-FMISO, the prototype 2-nitroimidazole, has been extensively studied, and other tracers have been developed to address shortfalls with this tracer, such as its stability, and slow clearance from background tissue causing modest signal to noise ratio. 18F-FAZA and 18F-FETNIM are more hydrophilic than 18F-MISO, thus should have better clearance than 18F-MISO. 18F-EF3 and 18F-EF5 are relatively more lipophilic than 18F-MISO but more stable and a study of the metabolites showed the tracers were unchanged.There are currently a number of 2-nitroimidazole compounds available. These include 18F-FMISO was able to detect anaerobic odontogenic infections with a sensitivity of 93 % and a specificity of 97 % [18F-FAZA has been evaluated in vitro and in vivo to assess alveolar echinococcosis (AE) infection caused by the larval from metacestode of Echinococcus multilocularis in rodents [18F-FAZA displayed a slightly elevated uptake in the Echinococcus multilocularis vesicles in the in vitro studies, but was not found useful in in vivo studies. This appears to be the first and only study up till now, in which 18F-FAZA has been used to evaluate infection. It is important to note that metronidazole which is a nitroimidazole has not been found to be effective against alveolar echinococcosis. Metronidazole is a prodrug that is activated under conditions of hypoxia. Metronidazole is an effective therapy for the parasites Entamoeba histolytica and Giardia lamblia. Both parasites are found in hypoxic habitats. Both parasites reside in the intestinal lumen, whereas Entamoeba histolytica is also present in abscesses. The hypoxic environment provides the right milieu for activation of the nitroimidazole drug. The metacestode of Echinococcus multilocularis is not subject to hypoxic conditions as it is transported through blood and settles in organs . It is no surprise, therefore, that metronidazole is neither used nor effective for the treatment of alveolar echinococcosis even though metronidazole has been shown to have different modes of action in different parasites [18F-FAZA involving Echinococcus multilocularis till now, the uptake was minimal and limited to the periphery of the AE lesions. In the same study, both 18F-FDG and 18F-FLT showed good uptake, indicating imaging of glycolytic and DNA proliferation of metacestode and that probably there was not enough hypoxia in the AE lesion to be imaged by a nitroimidazole.Although there have been reports of hypoxia playing a role in infectious disease, there are few studies involving hypoxia imaging in infection. Mtb bacteria 28 days after hypoxia was induced in an in vivo model of latent TB. This suggests that radiolabeled nitroimidazole PET/CT imaging may provide information not only on the presence of hypoxia, but also on the number of latent bacilli present in a granuloma [PET/CT imaging with radiolabeled nitroimidazoles could provide a unique opportunity to non-invasively study the hypoxic state of all granulomas within the body. In vitro evidence supports the potential use of hypoxia imaging in TB. Pimonidazole is a nitroimidazole used for immunohistochemical staining of hypoxic tissue. The intensity of pimonidazole staining was found to correlate directly with the number of encapsulated living ranuloma . The conranuloma .Imaging TB with radiolabeled nitroimidazole could potentially provide answers not only for the latent stage of TB, but may help in assessing risk of progression to reactivation based on the number of dormant or persistent bacilli. It may be instrumental in developing new antimicrobials that would need to be taken for only a short period of time, as these drugs would target the latent phenotype bacteria. Future research may provide new insights into the biology of the hypoxic TB granulomas, and thereby increase our understanding of the role of hypoxia in the latency and persistence stage. In the future, hypoxia imaging may also provide a stratification approach for individuals with LTBI and serve as an objective test in determining individuals with LTBI who would require treatment. Finally, hypoxia imaging may help with the development of new vaccines targeting patients with LTBI or persistent (MDR-TB) and may promote the development of new TB drugs targeting LTBI which will eventually lead to reducing the duration of current treatment. This would increase adherence and ultimately make the goal of eradication of TB by 2050 achievable.


In addition to the disruption of neural function below spinal cord injuries (SCI), there also can be changes in neuronal properties above and below the lesion site. The relevance of these changes is generally unclear, but they must be understood if we are to provide rational interventions. Pharmacological approaches to improving locomotor function have been studied extensively, but it is still unclear what constitutes an optimal approach. Here, we have used the lamprey to compare the modulatory effects of 5-HT and lesion-induced changes in cellular and synaptic properties in unlesioned and lesioned animals. While analyses typically focus on the sub-lesion spinal cord, we have also examined effects above the lesion to see if there are changes here that could potentially contribute to the functional recovery. Cellular and synaptic properties differed in unlesioned and lesioned spinal cords and above and below the lesion site. The cellular and synaptic modulatory effects of 5-HT also differed in lesioned and unlesioned animals, again in region-specific ways above and below the lesion site. A role for 5-HT in promoting recovery was suggested by the potential for improvement in locomotor activity when 5-HT was applied to poorly recovered animals, and by the consistent failure of animals to recover when they were incubated in PCPA to deplete 5-HT. However, PCPA did not affect swimming in animals that had already recovered, suggesting a difference in 5-HT effects after lesioning. These results show changes in 5-HT modulation and cellular and synaptic properties after recovery from a spinal cord transection. Importantly, effects are not confined to the sub-lesion spinal cord but also occur above the lesion site. This suggests that the changes may not simply reflect compensatory responses to the loss of descending inputs, but reflect the need for co-ordinated changes above and below the lesion site. The changes in modulatory effects should be considered in pharmacological approaches to functional recovery, as assumptions based on effects in the unlesioned spinal cord may not be justified. Spinal cord injury is associated with a loss of sensory and motor function below a lesion site due to damage to ascending and descending tracts and local circuitry, as well as disturbances of autonomic function. There is currently no effective intervention to overcome the effects of SPI  between 100–130 mm were obtained from commercial suppliers . All experiments were performed under license and conformed to the requirements of the UK Home Office Animals (Scientific Procedures) Act 1986. For spinal cord transections animals were anesthetized by immersion in MS-222  and a ~5 mm dorsal incision was made approximately 1 cm below the last gill to expose the spinal cord. The spinal cord was transected with iridectomy scissors and the incision site sealed with tissue glue (Vetbond). Following transection animals were kept at 20°C for 8–10 weeks : at this time most animals had recovered locomotor function  recordings were made from unlesioned animals and animals 8–10 weeks after lesioning. EMG recordings were made by inserting bipolar Teflon insulated electrodes (0.075 mm diameter) into the muscle under MS-222 anesthesia. The electrodes were inserted approximately 0.5 cm rostral and caudal to the lesion site. After recovery from anesthesia muscle activity was recorded as the animals swam in a plastic aquarium (29 × 23.5 × 5.5 cm). Swimming activity was initiated by lightly pinching the tail or head of the animal using serrated forceps: stimulation was given 30 s after the end of the previous swimming episode. A qualitative assessment of the functional recovery was made from video recordings based on the scale derived by  to the aquarium water: plateau effects on the episode length and coefficient of variation , aspects where significant effects of 5-HT were seen, occurred at a concentration of 200–400 μM -glucose; 2.0 mM HEPES; 0.5 mM L-glutamine, bubbled with O2 and adjusted to pH 7.4 with 1 M NaOH). The spinal cord was isolated from the notochord and pinned to a Sylgard lined chamber and superfused with lamprey Ringer at 10°C. Intracellular recordings were made from cells above and below the lesion site using an Axoclamp 2B amplifier (Molecular Devices). Motor neurons  and unidentified cells were examined in unlesioned animals and in lesioned animals above and below the lesion site. Given the relative size and number of different types of neurons, unidentified cells are likely to be motor neurons: as there were no obvious differences in motor neurons and unidentified cells they were grouped for analysis . Recordings were not made from different classes of interneurons as this analysis is not trivial even in this “simpler” system  in oxygenated lamprey Ringer at 4°C  spontaneous PSPs were instead measured over a 40 s period in each cell using the event detection feature in Clampfit.The properties of specific presynaptic inputs were examined by making paired intracellular recordings from postsynaptic motor neurons and Müller reticulospinal axons. These inputs were also used to examine the modulatory effects of 5-HT, but for this analysis cord stimulation-evoked responses were also used when reticulospinal axons that connected to the recorded postsynaptic cell could not be found. Cord-stimulation responses were evoked by stimulating the medial column that runs between the cell body layer (the region where locomotor network interneurons and motor neurons are located) and the midline of the spinal cord extracellularly using a glass suction electrode for 1 ms at 0.1 Hz for 50 s. The stimulation strength was adjusted to evoke what appeared to be a unitary EPSP, determined by the short rise time, longer decay time, and absence of multiple peaks . This was done in control and in the presence of 5-HT: the overlaid PSPs were averaged and the peak amplitude was measured. Putative monosynaptic reticulospinal-evoked inputs were examined by intracellular stimulation of reticulospinal axons in the medial column above the lesion site several segments rostral to the postsynaptic cell at 20 Hz for 1 s, followed by low frequency recovery test pulses . The amplitude of the initial EPSP and of EPSPs over the spike train was measured from the baseline preceding each EPSP to the peak of the EPSP. The trains were evoked at 20 s intervals. As there is no activity-dependent plasticity of the input at this frequency, the initial EPSP in the spike trains provided a measure of single low-frequency-evoked EPSPs. Putative monosynaptic connections were determined by the presence of reliable EPSPs that occurred with a consistent short latency  in response to presynaptic stimulation at 20 Hz  could fail the criteria for monosynapticity even though the connections were monosynaptic). Inputs were considered to be putatively polysynaptic if the inputs were unreliable  in the resting potential  or below the lesion site . With 10 μM 5-HT , but in the population as a whole there was still a mean hyperpolarization of −0.54 ± 0.39 mV, while above the lesion site it depolarized the membrane potential in 9 of 24 cells, which in this case gave a mean depolarization of 0.19 ± 0.76 mV . The RMP effect thus changed from a highly consistent hyperpolarization in unlesioned animals to a more variable effect where there was a mix of depolarization and hyperpolarization.In cells from unlesioned animals, 1 μM 5-HT non-significantly hyperpolarised the RMP , but it did significantly reduce the sAHP amplitude in each case (unlesioned (n = 11), above lesion (n = 10), lesioned below (n = 25), the reduction not differing significantly in the different conditions .There was no significant effects of 1 μM 5-HT on the input resistance, I-V relationship, excitability, or sAHP amplitude in unlesioned animals or lesioned animals above or below the lesion site (data not shown). For 10 μM 5-HT there was again no significant effect on the excitability or I-V relationship in cells from unlesioned animals or lesioned animals above or below the lesion site , and in lesioned animals above (n = 19) and below the lesion site (n = 20), the magnitude of the EPSP reduction not differing significantly between conditions . Similarly, 10 μM 5-HT also significantly reduced the amplitude of cord stimulation-evoked EPSPs in unlesioned animals  or below (n = 26) the lesion site  and in lesioned animals above the lesion site (n = 3): it is generally harder to find connections in lesioned animals, especially below the lesion site, which may be a reflection of the sparseness of regenerated inputs . In unlesioned animals and lesioned animals below the lesion site when all connections were averaged the EPSP depressed across the spike train , but not above (n = 15), the lesion site compared to unlesioned animals  was significantly increased in cells above and below the lesion site compared to cells from unlesioned animals Figures ; Table 1n = 1 of 17 in unlesioned animals, n = 3 of 6 lesioned animals, p < 0.05, Chi square). While the properties of spontaneous PSPs above the lesion site did not differ to unlesioned animals, there was additional evidence for an increase in excitatory connectivity: putative polysynaptic EPSPs were more common above the lesion site than in unlesioned animals . The IPSP value for spinal cord injured animals was significantly greater below than those in both the unlesioned and above lesion values, while the EPSP value was significantly greater above than in unlesioned animals  was calculated for each cell there was a significant reduction below the lesion site compared to unlesioned and above lesion cases, presumably reflecting the increase in inhibition  significant effects of 5-HT on the CV and episode length occurred at concentrations of between 200–300 μM , but control values varied and in animals with shorter cycle periods (<400 ms) the cycle period was increased , the swimming episode length was significantly reduced by 5-HT at higher concentrations , but the CV was again significantly reduced , 5-HT could improve swimming in poorly recovered animals (swimming score of 2). In these animals activity was absent below the lesion site but was evoked by 5-HT, and the activity above the lesion site became more regular . The failure of PCPA incubated animals to recover was not due to the acute effect of PCPA as animals were tested after being removed from PCPA for at least 2 weeks. However, in the five animals that recovered in the absence of PCPA, incubation in PCPA for 5 days 10 weeks after lesioning resulted in no disruption of locomotor activity  and lower levels of the swimming score .We have previously found significant differences in larval animals separated into those that recovered well or poorly  or poorly (n = 10), but above the lesion site excitability was significantly reduced with current steps from 1–2.5 nA in poorly recovered animals  than those that recovered well . Excitability did not differ to that in unlesioned animals for either those that recovered well  compared to those that recovered well  compared to those that recovered well (n = 17). The EPSPAmplitude × EPSPNumber was significantly greater above the lesion site in animals that recovered well than in those that recovered poorly  that gave a mean change of 0.31 ± 0.74 mV (n = 12), but in cells from poorly recovered there was a hyperpolarization in every cell that was significantly different to cells from animals that recovered well  effects, above the lesion site the only significant difference was that the sAHP was only significantly reduced in animals that recovered well Figure . Below tWhile several of the effects shown here are preliminary and require further analysis, the results show that there are changes in cellular and synaptic properties in the isolated lesioned compared to the isolated unlesioned spinal cord; that these changes differ above and below the lesion site; and that the modulatory effects of 5-HT are altered by lesioning, with effects again differing above and below the lesion site.There were several changes in functional properties in lesioned animals. Spontaneous synaptic and cord activity was increased above and below the lesion site compared to unlesioned animals. The reticulospinal axon-evoked EPSP half-width and the slow synaptic depolarization during trains of action potentials were also greater above and below the lesion site compared to unlesioned animals. However, the activity-dependent plasticity of reticulospinal inputs differed above and below the lesion site: depression occurred below that matched that seen in unlesioned animals, but above the lesion site the input facilitated which will make these connections functionally stronger. Facilitation is typically associated with a lower release probability and smaller initial EPSP amplitude , changes in EIN excitability , but they were not blocked by specific 5-HT2 antagonists: 5-HT3 and 5-HT4 agonists and antagonists were without effect . Exogenous and endogenous 5-HT slows the frequency of network activity in unlesioned animals  sAHP following an action potential or via activation of NMDA channels in the crossing (CC) inhibitory interneurons, which delays switching of activity between the two sides and thus increases the cycle period (e.g., Matsushima and Grillner, A major issue is to place the differences in 5-HT effects in lesioned animals into a functional context. While this lack of understanding is an obvious weakness, similar uncertainty exists over the effects of 5-HT in unlesioned animals despite several studies that examine these effects. 5-HT has numerous cellular and synaptic effects in unlesioned animals: it reduces somatosensory and reticulospinal-evoked EPSP amplitudes (Buchanan and Grillner, Pharmacological, electrical stimulation, and training effects modulate distinct functions in experimental and clinical situations (Courtine et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


With 14.234 diagnoses and over 4047 deaths reported in 2007, colorectal cancer (CRC) is the second most common cancer and second most common cause of cancer-related mortality in Australia. The direct treatment cost has recently been estimated to be around AU$1.2 billion for the year 2011, which corresponds to a four-fold increase, compared the cost reported in 2001. Excluding CRCs due to known rare genetic disorders, 20% to 25% of all CRCs occur in a familial aggregation setting due to genetic variants or shared environmental risk factors that are yet to be characterised. A targeted screening strategy addressed to this segment of the population is a potentially valuable tool for reducing the overall burden of CRC.We developed a Markov model to assess the cost-effectiveness of three screening strategies offered to people at increased risk due to a strong family history of CRC. The model simulated the evolution of a cohort of 10,000 individuals from age 50 to 90 years. We compared screening with biennial iFOBT, five-yearly colonoscopy and ten-yearly colonoscopy versus the current strategy of the Australian National Bowel Cancer Screening Programme (i.e. base case).Under the NBCSP scenario, 6,491 persons developed CRC with an average screening lifetime cost of AU$3,441 per person. In comparison, screening with biennial iFOBT, colonoscopy every ten years, and colonoscopy every five years reduced CRC incidence by 27%, 35% and 60%, and mortality by 15%, 26% and 46% respectively. All three screening strategies had a cost under AU$50,000 per life year gained, which is regarded as the upper limit of acceptable cost-effectiveness in the Australian health system. At AU$12,405 per life year gained and an average lifetime expectancy of 16.084 years, five-yearly colonoscopy screening was the most cost-effective strategy.The model demonstrates that intensive CRC screening strategies targeting people at increased risk would be cost-effective in the Australian context. Our findings provide evidence that substantial health benefits can be generated from risk-based CRC screening at a relatively modest incremental cost. With 14.234 diagnoses and over 4047 deaths reported in 2007, colorectal cancer (CRC) is the second most common cancer and second most common cause of cancer-related mortality in Australia[Approximately 10-15% of the population have a family history of CRC, which increases the disease personal risk by two-four fold  depending on the number of relatives affected, the degree of relationship of the affected relatives and the age of diagnosis. Even afThe focus of this study is people at increased risk of CRC because of a strong family history of CRC. Here, we define strong family history as people with one first-degree relative diagnosed before the age of 55 years, or with two first-degree relatives or one first- and one second-degree relative on the same side of the family diagnosed at any age.On average, these people have an estimated risk of developing CRC between three and six times higher compared to the average risk population,8.A targeted screening strategy addressed to this segment of the population is a potentially effective approach to reduce the burden of CRC.Whilst three randomised controlled trials have demonstrated that screening with faecal occults blood test (FOBT)—followed with a diagnostic colonoscopy in case of positive test—is effective in reducing CRC incidence and mortality in the average risk population, no direct evidence exists to support the effectiveness of screening in people in this increased risk category. Based on expert opinion, current guidelines generally advise that persons with a strong family history of CRC should initiate screening at an earlier age or undergo more intensive screening compared to the average risk population-11. For Similar to the clinical effectiveness, the cost-effectiveness of CRC screening in the average-risk population, based on various strategies and using different screening modalities, has been established by several studies-18. PignThere is a strong rationale for designing risk-based CRC screening policies (i.e. intensiveness of screening based on risk) as this would, theoretically at least, permit a more efficient allocation of limited health care resources. HoweverThe main objective of this analysis was to provide an economic evaluation for people at increased risk of CRC, based on Australian data assessing the cost-effectiveness of biennial iFOBT, five-yearly colonoscopic screening starting at age 50 years, and ten-yearly colonoscopic screening starting at age 50 years to the current program of the National Bowel Cancer Screening programme of Australia (NBCSP) which currently consists of a one-off iFOBT screening offered at age 50, 55, 60 and 65 years (9) irrespective of family history.We developed a Markov microsimulation model to simulate the natural history of CRC and to evaluate costs and outcomes of screening. Figure Based on current understanding of the variability in the natural history of the disease, we created nine mutually exclusive, possible health states  and specified transition probabilities for movements between these health states. An individual can remain in the same health state or move to another health state according to the predetermined transition probabilities. Costs and health outcomes (including life expectancy) were assigned to each state and transition. The software TreeAge Pro was used to implement the model.The parameters used in the Markov model are presented in Table Age-specific incidence rates of small polyps, large polyps, adenomas and CRC at different stages were obtained from the health economics review conducted in 2008 by Bishop et al. for the Cancer Institute of New South Wales.To reflect a cohort of persons in the “moderately increased risk” category , we followed the approach adopted by Ladabaum et al. and RamsWe compared four alternative CRC screening strategies:1. Screening according to the current NBCSP programme. Under this screening scenario, which was used as the baseline strategy in the model, participants were invited to undertake CRC screening with an immunochemical iFOBT at age 50, 55, 60 and 65 years .2).2. iFOBT biennial screening (every two years) from age 50 years (iFOBT5).3. Colonoscopy screening every five years starting at age 50 years. This strategy reflects the current screening recommendation for people at moderately increased risk of CRC (COLO10).4. Colonoscopy screening every ten years starting at age 50 years (COLO2), a person invited to screen could “choose” to undertake (screeners) or to decline screening (non-screeners). We assigned screener status on a 40% probability based on the participation rate observed in the NBCSP reported in the latest monitoring report of the programme[In both iFOBT strategies  or negative tests  was determined by the participant’s probability of disease status (i.e. presence of adenoma or CRC) and the diagnostic accuracy of the test (i.e. sensitivity and specificity). Participants with negative tests were re-invited to screen according to the protocol of each iFOBT-based screening strategy. We assumed that all screeners with a positive iFOBT result were eligible for a follow-up diagnostic colonoscopy referred by a general practitioner. The compliance for the recommended diagnostic procedure was also based on the compliance observed in the NBCSP. If diagnostic colonoscopy confirmed the presence of a small adenoma <10 mm), a polypectomy was assumed to having been performed and the participant returned to the normal state in the natural history model. Subjects with large adenomas (≥10 mm) exited the screening programme after treatment and were followed up with five-yearly surveillance colonoscopy as recommended in the current NHMRC guidelines[ mm, a poFor colonoscopy-based screening strategies, a similar logic to that of iFOBT-based screening was applied, the only differences being the direct removal of any detected polyp and the use of colonoscopy values for sensitivity and specificity. In the base case model, level of screening uptake was determined based on a meta-analysis of eight studies reporting colonoscopy screening participation rates for people at increased risk of CRC due to family history. SubjectFor this analysis we adopted a third-party payer  perspective. Only direct costs, such as unit cost or annual costs related to screening and diagnostic tests, adenoma surveillance and CRC treatment were taken into account. Indirect costs related to production loss due to undergoing screening, colonoscopy or hospitalisation, were not considered in this analysis. Screening and diagnostic costs were based on the estimates reported by Bishop et al.. For iFOAnnual costs of treating CRC according to stage at diagnosis were derived from lifetime treatment costs: AU$34,337 for treating a Dukes’ A CRC, AU$53,487 for Dukes’ B, AU$79,924 for Dukes’ C and AU$71,156 for Dukes’ D, and represent the average cost to the Australian health system for an individual diagnosed with CRC over the course of their life. Here, t2, COLO10 and COLO5 screening strategies reduced the number of CRC cases by 27%, 35% and 60% and CRC mortality by 15%, 26% and 46% respectively. All three approaches resulted in more LYG and were more costly than Usual Care. Five-yearly colonoscopy screening starting at age 50 was the most costly option with a lifetime cost estimated to be AU$8,734 per person but also provided the highest LYG of all the screening strategies.Table 10 and COLO5 respectively. iFOBT2 was dominated by both screening approaches in the model  and the lowest number of colonoscopy procedures needed to save one life, COLO5 screening appeared as the most cost-effective strategy of the models compared.The incremental cost-effectiveness ratios of the different screening strategies compared to Usual Care are shown in Table 5 and COLO10 strategies were $9,108 and $7,835 per LYG respectively. The cost of both strategies were positively correlated with the level of screening uptake but increased by only 13% and 24% respectively when screening participation was set to 90%.Sensitivity analyses showed that above 20% participation rate the level of colonoscopy screening uptake was not a very influential parameter on the ICER Table . For exaEffects of varying the level of CRC risk (i.e. 3 and 6 fold risk increase compared to 4 fold increase used in the base-case model) associated with a strong family history on the different outcomes of the model are presented in Additional fileOur aim was to provide an insight into the cost-effectiveness of CRC screening for people at increased risk of the disease due to having a strong family history. We compared three different screening alternatives to the current NBCSP using Australian data on CRC incidence, adenoma distribution and cancer stage for invasive carcinoma. We chose the current NBCSP screening strategy as the base case scenario in our model instead of a no screening scenario as we consider this approach to be more realistic and more relevant to the Australian context where a population-based screening programme has been in place since 2006 and will be expended to cover all individuals aged 50 to 75 years by 2017.All three strategies had an ICER under $50,000 per LYG, which is regarded as the upper limit of acceptable cost-effectiveness in the Australian health system,31. An iLadabaum et al. conducted a cost-effectiveness analysis including only direct medical costs and using data from a screening pilot programme implemented in Aragón (Spain) targeting first-degree relatives of patients with CRC. The authors analysed the cost-effectiveness of CRC screening starting at age 40 years comparing colonoscopy every ten years and colonoscopy every five years, to no screening. Both screening strategies dominated the “no screening” option. Similar to our findings, five-yearly colonoscopic screening was found to be the most cost-effective strategy.In a study conducted in the United States, Ramsey and colleagues used a validated microsimulation model adoptingIn this study we attempted to conduct a similar analysis in the Australian context where an iFOBT-based screening programme has been in place since 2006. To our knowledge, this is the first analysis attempting to measure the economic aspect of different CRC screening strategies (including NHMRC recommendations) that target persons at “moderately increased risk” of CRC in the Australian context. It should be noted that this category does not include all individuals with a family history of CRC. Our model is particularly relevant to a segment of the population at a substantially increased risk of CRC—namely, individual whom CRC family history characteristics place them in the risk category 2 of the current Australian guidelines. PersonsThe modelling presented here has several limitations. For example the model assumed that family history of CRC was known for all participants and did not include administrative costs associated with the implementation of a family history assessment.While the model accounted for the higher incidence of polyps observed in people with family history of CRC as a cause of a higher CRC incidence, it did Our analysis was conducted from a third-party payer perspective and was limited to direct costs only. Indirect costs such as production loss due to CRC treatment need be taken into account for a more exhaustive economic assessment of a family history-based CRC screening programme. We did not take into account the costs involved with potential complications from screening tests, and the combination of screening and administrative costs. Also, our costs parameters rely heavily on estimates reported in Bishop et al. review, which was published in 2008. It is possible that some of those estimates might be out-of-date given the constant progress and changes in CRC treatment protocols.In conclusion, our results are consistent with findings from previous research and present informative preliminary estimates in the perspective of familial risk-based CRC screening strategies within the context of a national screening programme addressed to the average risk population. Given limited colonoscopy capacity and budget, there is an increasing number of investigators calling for a better inclusion of personal disease risk in the design of CRC screening policies. The rational of this approach being that more screening resources should be allocated to those who would benefit most from more screening while at the same time reducing the intensity of screening among those who have less to gain. Several risk prediction tools have been developed,37 and fThe authors have no competing interests to declare in relation to this manuscript.DA designed the study, carried out data collection, statistical analysis and writing of the manuscript. MJ participated in the design of the study, the interpretation of the statistical analysis and the writing of the manuscript. TL, AB and JH participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/261/prepubDescription of data: Results of cost-effectiveness analysis with three and six fold increased risk of colorectal cancer.Click here for file


Aberrant activation of the transcription factor GLI1, a central effector of the Hedgehog (HH) pathway, is associated with several malignancies, including pancreatic ductal adenocarcinoma (PDAC), one of most deadly human cancers. GLI1 has been described as an oncogene in PDAC, making it a promising target for drug therapy. Surprisingly, clinical trials targeting HH/GLI1 axis in advanced PDAC were unsuccessful, leaving investigators questioning the mechanism behind these failures. Recent evidence suggests the loss of GLI1 in the later stages of PDAC may actually accelerate disease. This indicates GLI1 may play a dual role in PDAC, acting as an oncogene in the early stages of disease and a tumor-suppressor in the late stages. The protein GLI1, originally isolated in 1987 due to high levels of amplification in malignant glioma  defined a key role for GLI1 on PDAC initiation through the modulation of the activity of fibroblasts .While dysregulation of HH-GLI1 signaling has been shown to play an important role in PDAC formation, several studies have demonstrated that GLI1 expression can be activated through HH-independent mechanisms in PDAC, particularly in the epithelial compartment   signaling, a cascade aberrantly activated in the majority of PDACs, has been demonstrated to play a critical role in HH/GLI1-regulated tumor-initiating pancreatic cancer cells . This suThe concept that HH/GLI1 signaling might be required for PDAC growth, hence a suitable therapeutic target, has been first validated in a genetically engineered mouse model of pancreatic cancer that combines expression of oncogenic Kras with mutation of the tumor suppressor p53, the KPC mouse , in PDAC cells. Lowered expression of GLI1 in PDAC cells lead to a loss of CDH1 expression and promotion of EMT. The transition from epithelial to motile mesenchymal cells is thought to be a critical event for metastasis of carcinomas. Decreased expression of CDH1 is associated with increased metastasis and invasion, while increased expression is associated with lower tumor malignancy (Keratin 19 (KRT19) and adherens junctions componentsEVA1 andPTPRM, leading to increased cell motility. This indicates that as PDAC progresses, lower GLI1 levels may actually prime tumor cells towards an EMT program, which would be associated with metastasis and advanced stages of the disease.In contrast to the current paradigm for GLI1 expression and tumor progression, one study found GLI1 expression may actually decrease cell motility in advanced PDAC  in the Olive’s experiments, which may have not accurately detected disease progression following HH inhibition. This indicates that as PDAC progresses, the initial positive effects of HH inhibition may be eliminated as GLI1 levels decrease.in vivo. As previously discussed, one potential mechanism for lowered GLI1 expression in advanced PDAC may be due to activation of the kinase DYRK1B, which inhibits GLI1 expression through expression of the repressor GLI3 . This manuscript highlights the dual role of GLI1 during pancreatic carcinogenesis, acting as an oncogene in the early stages of disease and as a tumor-suppressor in the late stages. Recent evidence suggests the loss of GLI1 in the later stages of PDAC might accelerate disease progression. This might explain why Smoothened (SMO) inhibitors have been successful for treating basal cell carcinoma and medulloblastoma, but do not appear to have the same effect in metastatic PDAC. Moreover, this article summarizes recent data on the integration of GLI1 with other signaling pathways, suggesting that GLI1 is not only regulated by the upstream Hedgehog signaling in a SMO-dependent manner, but also by other oncogenic inputs, such as KRAS, TGF-beta and EGFR signaling.Recent experimental data suggest that lower GLI1 levels associate with PDAC progression, whereas increasing GLI1 levels could drive terminal differentiation and decreased PDAC tumorigenicity. What is missing in this review is a consideration about the factors that might contribute to decrease GLI1 levels and activity during PDAC progression. I would also suggest to mention in the Discussion that the dual role of GLI1 is so far limited to PDAC, as the evidence is lacking in other cancer types.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Recent experimental data suggest that lower GLI1 levels associate with PDAC progression, whereas increasing GLI1 levels could drive terminal differentiation and decreased PDAC tumorigenicity. What is missing in this review is a consideration about the factors that might contribute to decrease GLI1 levels and activity during PDAC progression.Response: We added additional considerations regarding other factors that may drive down GLI1 levels during PDAC progression to the “Discussion” section. Mechanisms included are activation of DYRK1B kinase, which promotes a shift from autocrine to paracrine signaling, which may lead to decreased GLI1 expression. Also, decreased HH signaling in advanced PDAC may allow for increased expression of GLI1 repressors.I would also suggest to mention in the Discussion that the dual role of GLI1 is so far limited to PDAC, as the evidence is lacking in other cancer types.Response: The following statement was added to the final paragraph of the Discussion section “Further studies are needed to fully understand the role of GLI1 in PDAC carcinogenesis. While there is evidence for a dual role of GLI1 in PDAC, this phenomenon has yet to be linked with other cancer types.” The review is timely and addresses a very important problem in pancreatic cancer. The Smoothened inhibitors that work well for other tumor types have not only failed to stop the progression, but instead promote aggressive behavior in pancreatic cancer. The authors make a good case of balancing the evidence that suggests that there is HH-independent upregulation of GLI1 in the epithelial cells and a Hh-dependent upregulation in fibroblasts. Moreover, they nicely discuss how GLI1 is necessary for PanIN formation and then restrains further cancer progression. What is lacking in the review is a consideration of potential non-canonical effects of the Smo inhibitors. It is known that Smo induces cytoskeletal changes in fibroblasts, for instance, and that it can regulate glucose uptake in other cell types. Perhaps modulation of GLI1 is a bystander effect confusing the results. And the GLI1 knockout animals only partly acknowledge this interpretation, since some effects can be cell-type specific and opposing, as discussed in the review.A minor criticism is the following: the introduction erroneously says that GLI1 is conserved from Drosophila to humans. However, GLI3 is the closest homolog in sequence and function to Drosophila Ci. It seems that the authors meant the GLI family is conserved.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. What is lacking in the review is a consideration of potential non-canonical effects of the Smo inhibitors. It is known that Smo induces cytoskeletal changes in fibroblasts, for instance, and that it can regulate glucose uptake in other cell types. Perhaps modulation of GLI1 is a bystander effect confusing the results. And the GLI1 knockout animals only partly acknowledge this interpretation, since some effects can be cell-type specific and opposing, as discussed in the review.Response:  Additional considerations were added concerning the use of SMO inhibitors and downstream GLI-independent effects to the Discussion section.A minor criticism is the following: the introduction erroneously says that GLI1 is conserved from Drosophila to humans. However, GLI3 is the closest homolog in sequence and function to Drosophila Ci. It seems that the authors meant the GLI family is conserved.Response: This statement was corrected to “the GLI family of transcription factors is highly conserved and is required for developmental response via transcriptional regulation of target genes” in the Introduction. The statement regarding Drosophila to humans was removed. et al. provide us with a timely review regarding the current knowledge about the role of the transcription factor GLI1 in pancreatic carcinoma. There is an important focus on the dual activity of GLI1 during pancreatic carcinogenesis depending on the stage of disease progression, as evidenced in most recent works in this field, both clinical and experimental, that are nicely summarized in this review.In this article HogensonAnother important aspect of this review consists in integrating GLI1 as a transcription factor that is not solely regulated by Hedgehog signaling downstream of SMO but also by other pro-tumorigenic pathways, such as TGF-beta and KRAS signaling.Overall, I believe that this manuscript is very focused and contains valuable information for the broader readership, with up-to-date citation of the most relevant and recent literature in the field.A figure summarizing the role of GLI1 downstream of the various pathways described to modulate its expression/activity would be helpful.On page 3, one reads "TGFβ induces the expression of GLI1 through Smad3 and LET-dependent up regulation of GLI2". "LET-dependent" should be replaced by beta-catenin/LEF-TCF-dependent or something similar.A couple of minor points may be corrected or improved:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. A figure summarizing the role of GLI1 downstream of the various pathways described to modulate its expression/activity would be helpful.Response: An additional figure was added to the review  to describe the various pathways that modulate GLI1 expression as discussed in the review. Figure 1 from the first version of this article was changed to Figure 2.On page 3, one reads "TGFβ induces the expression of GLI1 through Smad3 and LET-dependent up regulation of GLI2". "LET-dependent" should be replaced by beta-catenin/LEF-TCF-dependent or something similar. Response:  “LET-dependent” was corrected to “β-catenin/LEF-TCF-dependent” on page 3.