The chiropractic profession has been well established in Denmark for several decades with state authorization, partial reimbursement by the state and a formal academic education. Biennial systematic data collections among all chiropractors and clinics have been performed since 2010 in order to provide exact information on the profession to The Danish Chiropractic Association (DCA). It is the aim of this study to outline the major characteristics and developments of the chiropractic profession in Denmark to make this information accessible to other stakeholders, domestic as well as foreign.Using contact information from the DCA, two questionnaires were distributed electronically to all individual members of the association actively working as chiropractors and all clinics respectively in 2010, 2012 and 2014. The questions asked were developed for this specific survey.Response rates varied between 59 and 78 % for the clinic questionnaires and 75 to 86 % for the individual questionnaires. Almost half the Danish chiropractors were educated in Denmark and a small majority was female. The average Danish chiropractor of 2014 was 44 years old, graduated 17 years earlier, and worked full time in a primary care clinic with at least one colleague. Half the chiropractors spent more than 20 h a year on continued professional development. Danish chiropractic clinics had a median of 3 treatment rooms, most had digital X-ray equipment, around 6 out of 10 had exercise facilities, and 1 out of 4 employed a physiotherapist. Three out of 4 clinics employed a secretary, too. The average duration of a consultation was 40 min for a new patient and 13 min for a follow-up consultation. Virtually all Danish chiropractors working in the primary sector made use of manipulation as one of their treatment modalities.This is the first study to describe the state and latest development of the chiropractic profession in Denmark using repeated surveys. Displaying various characteristics of both clinics and individual chiropractors, the image emerging is one of a stable profession where rapid or drastic changes are not taking place over short intervals of time. The first chiropractor in Denmark started practising in 1920. Since then a profession has emerged and it has been an acknowledged and accepted part of the official Danish health care system for several decades by now. Although only to a limited degree, partial reimbursement of expenses for chiropractic care was acceded to in 1978 by the Danish government, and in 1992 a state authorization for practicing as a chiropractor was introduced as the final step towards full recognition of the profession .A 5-year academic chiropractic education was introduced in 1994 at what is now called The University of Southern Denmark (SDU) in Odense, and since 1999 when the first students completed their education approximately 300 Danish chiropractors (by 2014) have joined the profession after graduating at SDU largely supplanting United Kingdom and USA as the most common place of education [The Danish Chiropractors’ Association (DCA) was established in 1925. Since then, it has been the sole chiropractic professional organisation in Denmark except for a two-year period in the late 1940s. Affiliation to the association is a requirement for receiving reimbursement from the national health insurance because the DCA negotiates the agreement with the Danish health authorities (The Regions’ Board for Wages and Tariffs) on behalf of the profession. Negotiations take place every three years and includes agreements on payment to chiropractors, regulations related to the profession, and which developmental initiatives the profession should take on. State reimbursement amounts to approximately 20 % of the treatment fee.The association organizes clinic owners, chiropractic employees in both the primary and the secondary sector, researchers and university lecturers with a chiropractic background, and retired chiropractors as non-active members.Patients either self-refer to chiropractors or are advised about chiropractic care by a general practitioner. There is no formal referral from general practice and patient costs are the same whether self-referring or being advised to seek chiropractic care by a general practitioner.In 1987, the DCA established The Foundation for Chiropractic Research and Post Graduate Education in collaboration with The Regions’ Board for Wages and Tariffs (RBWT). Among the objectives of the foundation is to provide financially support for research projects, international stipends, and quality development projects within the field of clinical biomechanics as well as supporting continued professional development. One third of the foundation’s yearly income is provided by RBWT while two thirds is drawn directly from the state reimbursements to the clinics.In 2010 a desire to investigate the major characteristics of the now fairly well established profession and to initiate an on-going monitoring of its further development caused the launching of the first systematic data collection on the chiropractic profession in Denmark. The main purpose of the initiative was to provide comprehensive information to The Danish Chiropractors’ Association to help the organisation in its effort to refine and develop the profession even further. However, we regard this information to be relevant to chiropractors, healthcare professionals, officials, and chiropractic associations in other countries too.n = 49 [n = 44 [n = 80 [n = 113 [Over the past decade, similar descriptive reports on the chiropractic profession have been issued from a number of our neighbouring countries, mainly displaying characteristics of the individual country at a specific time. A majority of these deal with chiropractic communities somewhat smaller than the Danish profession and presents comparatively few responders , and on[n = 113 . FinallyThe primary objective of this report is to describe the chiropractic profession in Denmark in 2014, tentatively outlining developments since 2009 when possible. However, due to the short intervals between the surveys, some data will only be reported for 2014.Though it is not a main objective of this study to present an exhaustive comparison between countries, in cases where conspicuous similarities or differences call for attention details from previously published reports will be taken into account in the discussion.Between 2010 and 2014, three cross-sectional data collections were carried out among Danish chiropractors using contact information obtained from the DCA. Two separate questionnaires were distributed electronically in each survey: one directed at all individual chiropractors who were registered members of the DCA and actively working as chiropractors at the time of initiating the data collection, and one addressed to each separate physical clinic. Hence one owner from each clinic was required to answer both questionnaires.Designing the project, data collection and analysis were anchored at the Nordic Institute of Chiropractic and Clinical Biomechanics (NIKKB). Mails with a short introduction to the survey signed by a senior researcher at NIKKB and a link to the questionnaire itself were distributed via the licensed online survey database SurveyXact . AnalysiYears since education, Physical size of the clinic, Number of treatment rooms, No. of chiropractors and all questions concerning the duration of consultations were collected as continuous variables. Answer categories to the other questions appear from the tables. Average age was calculated on the basis of 10-year age groups.The questions asked were developed for this specific survey and were not previously validated. According to Danish legislation stipulated in Act on Research Ethics Review of Health Research Projects, section 14, subsection 1–2, approval from The Health Research Ethics Committee System in Denmark was not required as the questionnaires do not ask for personal information and all data were collected anonymously [The response rates for the surveys are shown in Table Assessment of representativeness could be based on the actual regional distribution of the clinics in 2014 known from the DCA mailing list. A comparison with responses to the clinic questionnaires that same year  respondents were female representing a slight increase compared to the earlier surveys  in the 2014 survey.2 which is roughly the same as in the previous years . In 2014 the respondents were given the opportunity to state administrational tasks or management as their primary function too and 9 individuals (2 %) did so.In terms of each chiropractor’s primary job only (not reported in the tables) as opposed to employment in general, 11 individuals (2 %) did clinical work at a hospital in the secondary sector, another 11 were engaged by a health insurance company, and 16 chiropractors (4 %) were employed in research and/or teaching in 2014 . Teaching formed the largest single job function among this group and was done by 35 % in 2014 (34 % in 2012 and 38 % in 2010).Most Danish chiropractors worked full time or more Table . Lack ofOwners of a clinic tended to work more than their employees. Figures from 2014 showed that 40 % of the clinic owners reported to work more than 37 h a week in total versus 12 % of the chiropractors in the primary sector who were not self-employed. The proportion of owners working 25–29 h and less than 25 h weekly was 10 and 5 % respectively, whereas the corresponding percentages for employees were 15 and 12 %.Data on the effort dedicated to continued professional development (CPD) by each respondent was only available from the two latest surveys Table . Half thChiropractors working in a clinic, whether that may be as their primary or secondary job, naturally spent the vast majority of their working capacity on patient consultations. An average of 29 h weekly was dedicated by these chiropractors to the treatment of patients in 2014, and a little less than 4 h and 2 h were spent on administration and communication with other parties of the Danish health care sector respectively.The duration of the various kinds of consultations seems remarkably constant Table .Table 6WVirtually all Danish chiropractors working in the primary sector make use of manipulation while trigger point treatment and other soft tissue techniques are offered by 85-90 % and exercise instructions by more than 80 %. The Danish surveys did not ask about the proportion of patients the various treatment modalities were given to. The average Danish chiropractor of 2014 is 44 years old, graduated 17 years ago and works full time in a primary care clinic with at least one colleague. Danish chiropractic clinics have a median of 3 treatment rooms, most have digital X-ray equipment, around 6 out of 10 have exercise facilities, and 1 out of 4 employs a physiotherapist. The described profile of the Danish chiropractic profession did not undergo large changes from 2010 to 2014.A response rate above 75 % as seen in five out of the six questionnaires is considered satisfactory, and matches or exceeds comparable studies from our neighbouring countries –7, 9–11.With contact information made available by the only chiropractic association in Denmark, we were able to contact practically all Danish chiropractors. However, we do not know if non-responders to the surveys may represent a group of clinicians with a certain profile.Negatively affecting the response rate for the clinic questionnaire may be that owners were required to answer both questionnaires, perhaps mistakenly leading them to believe the questionnaire to be already answered when they got to the second one. A similar effect may have been caused by the fact that the data source was physical clinics which implied that a clinic working from several addresses received several questionnaires probably even using the same email address. However, the results, especially from the 2012- and 2014-surveys, can be viewed as confidently reflecting the actual state of the Danish chiropractic profession.Distinguishing the Danish profession most clearly from the Norwegian, Swiss, Belgian, Dutch or Finnish profession is the very high proportion of female practitioners of 56 % compared to 20–36 % in the said countries. With 43 % female chiropractors, the United Kingdom is the only country relatively close to the Danish gender distribution .The American survey displays a female proportion changing from 13 % in 1991 to 22 % in 2009 . A certaIn terms of average age the Danish chiropractor seems to be growing older too. From 2010 to 2014 the proportion of chiropractors still in their twenties has decreased from 12 to 8 % while the 61–70 years age group has increased from 5 to 8 %. At the same time the surveys display a decrease in the proportion of responders with a maximum of 10 years since the date of their graduation from 42 % in 2010 over 35 % in 2012 to just 28 % in 2014, but due to a comparatively high number of missing values in the latest survey on this specific issue we are unable to ascertain to what extent the apparent change may be caused by selection bias.Significant changes in the size or characteristic of the average Danish chiropractic clinic cannot be detected over the past four years.Clinics with 3–5 treatment rooms comprise nearly half the total number of clinics in 2014 which may represent a certain tendency towards medium sized clinics.The prevalence of diagnostic imaging facilities forms the most distinct development in relation to the physical appearance of the clinic. The proportion of clinics with digital X-ray facilities rose from 57 % in 2010 over 63 % in 2012 to 70 % in 2014. Even more significant is the decrease in the prevalence of analogue X-ray equipment in the same period of time from 25 % over 15 to 5 %. Considered together with the increase in the number of clinics reporting to have no such facilities at all from 9 % over 13 to 17 % we see that whereas a growing number of the clinics acquire digital imaging facilities of their own not all the discarded analogue equipment is substituted by digital facilities. Some clinics simply choose to refer their patients to somewhere else for imaging.Though a tendency towards bigger clinics cannot be ascertained based on physical size, more clinics find themselves in need of secretaries or a receptionist. Co-habitation or sharing facilities with other kinds of health practitioners such as masseurs or physiotherapists is widespread and more than half the clinics have formally employed health practitioners other than chiropractors. This proportion has increased from 52 % in 2010 to 56 % in 2014.Comparison with other countries in this respect must be looked upon with some reservation due to the considerable variation in the way related questions are posed. The Swiss survey from 2009 has 10 % of its respondents reporting to “practice in a multidisciplinary office” . Only unDespite the lack of homogeneity of the data, however, it seems safe to conclude that the co-habitation is far more developed in Denmark compared to most other countries, although further information on this subject as well as on the actual level of interdisciplinary collaboration is desirable.Looking at the average duration of a patient consultation, a remarkably stable pattern is revealed. Neither the first consultation with a new patient nor the follow-up consultations display any variation in length at all over the three surveys (40 min and 13 min respectively). In 2014 85 % of the chiropractors allotted 16–45 min to a new patient and approximately the same proportion allotted 6–15 min to follow-up consultations suggesting that Danish chiropractors have reached a rather firm concept of their individual treatment procedure.The average duration of consultations does not differ very much across countries. In The Netherlands and Belgium an appointment with a new patient takes on average 41 and 36 min respectively, and follow-up consultations are done in 15 min in both countries . A compaA similar pattern with comparatively long consultations is also seen in the other Nordic countries although different time categories make a one-on-one comparison impossible. In Norway 80 % dedicates 30–49 min to a new patient and 60 % see a follow-up patient for 15–20 min [This is the first study to describe the state and latest development of the chiropractic profession in Denmark using several consecutive surveys. Displaying various characteristics of both clinics and individual chiropractors, the image emerging is one of a stable profession where rapid or drastic changes are not taking place over short intervals of time.The typical Danish chiropractor works in primary care, she is in her mid-forties with some 17 years of working experience. About half the profession graduated from University of Southern Denmark. The average clinic has 3–5 treatment rooms, is equipped with digital X-ray facilities and offers exercise facilities too. It employs at least one secretary, more than one chiropractor and, unlike clinics in our neighbouring countries, very often other health practitioners too. The average duration of the consultations, however, resembles that of other countries in the North-Western Europe, and it has been quite stable across all three surveys on 40 min for a new patient and 13 min for a follow-up consultation. Virtually all Danish chiropractors use manipulation as one of their treatment modalities.


Herbal and nutritional supplements are more and more popular in the western population. One of them is an extract of an exotic plant, named Tribulus terrestris (TT). TT is a component of several supplements that are available over-the-counter and widely recommended, generally as enhancers of human vitality. TT is touted as a testosterone booster and remedy for impaired erectile function; therefore, it is targeted at physically active men, including male athletes. Based on the scientific literature describing the results of clinical trials, this review attempted to verify information on marketing TT with particular reference to the needs of athletes. It was found that there are few reliable data on the usefulness of TT in competitive sport. In humans, a TT extract used alone without additional components does not improve androgenic status or physical performance among athletes. The results of a few studies have showed that the combination of TT with other pharmacological components increases testosterone levels, but it was not discovered which components of the mixture contributed to that effect. TT contains several organic compounds including alkaloids and steroidal glycosides, of which pharmacological action in humans is not completely explained. One anti-doping study reported an incident with a TT supplement contaminated by a banned steroid. Toxicological studies regarding TT have been carried out on animals only, however, one accidental poisoning of a man was described. The Australian Institute of Sport does not recommend athletes’ usage of TT. So far, the published data concerning TT do not provide strong evidence for either usefulness or safe usage in sport. Tribulus terrestris (TT), which was used by traditional, ancient medicine in Greece, China and India (Ayurvedic medicine). It was recommended as a remedy for infertility, impotence, erectile dysfunction, and low libido. Since the early 1980s its extract has also been an attractive product of unconventional medicine in Western countries as a testosterone booster, an enhancer of libido and a adaptogenic aid for healthy and physically active men. Concurrently, physical activity in leisure time is widely recommended by WHO for prevention of lifestyle diseases, good performance and wellbeing; therefore, proper diet and the use of nutritional supplements, including herbs, can also be effectively advertised. TT as a testosterone enhancer would be attractive also for competitive athletes, as an alternative of the use of forbidden enhancing drugs such as androgenic-anabolic steroids. No wonder that recently TT promotion has been targeted in a high extent to athletes. An additional incentive and facilitation for the use is that the extract of TT is provided by the pharmaceutical market as an over-the-counter supplement.Nowadays, modern pharmacology is based mostly on synthetic chemical compounds. However, traditional herbal medicine which utilizes centuries-old experience, still plays an important and complementary role in enhancement of human health. The pharmaceutical market advertises and provides several products, so-called nutritional and dietary supplements, which are not considered as drugs; therefore, their quality and biomedical efficacy are not required to be strictly controlled by clinical trials. Nevertheless, herbal extracts containing biologically active chemical compounds serve sometimes as components of those supplements. The use of herbal extracts or nutritional supplements containing those components is becoming more and more attractive and popular among the Western population due to extensive marketing activities. However, there is a need to confirm the recommended usefulness and possible unknown side effects when using herbs, especially those that contain biologically active organic chemicals. It may be achieved due to advanced and trustworthy scientific methodologies of clinical trials, which allow one to verify the former attitudes and beliefs referring to the effectiveness of the action of herbal preparations. One of the many exotic herbal plants recommended for the use is Taking into consideration an increasing interest in the extract of TT as a supplement improving human vitality, the purpose of this review was to present current knowledge of the effects of the use of TT on androgenic status and physical performance in healthy, physically active men, with special focus on the benefits and risks for human health.The plant grows mainly in Africa, Asia, Australia and Europe. It has seeds with three spines and yellow flowers, and grows up to a metre high. For a long time it was believed that besides the special biological properties which make this plant attractive as an enhancer of human vitality, all the components of the plant, roots, seeds, fruit and leaves, are useful in cases of kidney stones, high cholesterol, hypertension and as a diuretic. Currently, TT is mainly advertised as a testosterone booster, that may be a way to improve androgenic status in case of male hypogonadism and may enhance performance in athletes. The majority of these assumptions are provided by the Internet and other media which aim to encourage the use of TT. Usually, TT extracts are offered by the pharmaceutical market as a separate product, or as a component of various nutritional supplements, which are offered for healthy, physically active adults, athletes and generally for those who want to maintain their health and well-being. However, in spite of alleged benefits, potential users should get to know, and take into consideration, the results of clinical trials, which are often not clear, and some of them are not fully credible because of methodological shortcomings. In fact, the effectiveness of several herbal supplements examined in a controlled experimental setting is often significantly lower compared to common opinions of the users, especially those of higher susceptibility to suggestions. All those doubts as to the effectiveness of TT have to be verified. The present review focused on currently available scientific and trustworthy information about potential advantages and side effects resulting from the persistent taking of TT, and a discussion of the rationale of TT usage for revitalization by improving androgenic status in athletes. Scientific literature referring to this topic is limited, and the results of existing studies do not present clear conclusions.Tribulus terrestris revealed contents of many chemical compounds, among which the best known are steroidal glycosides (saponins) and alkaloids. Presumably, so far, not all chemical compounds have been identified in the plant, since researchers are still discovering new substances of a high molecular weight which belong to the family of steroidal saponins, glycosides, alkaloids and flavonoids. The majority of those compounds have not been identified earlier by chemists; therefore, their new specific names come from the name of the plant   . The quarosin D) .First scientific reports about beneficial effects resulting from the use of TT were provided by researchers of the Bulgarian Pharmaceutical Group. They stated that TT extracts increased libido, blood testosterone and spermatogenesis, and improved the male sexual function. To date, there is a lack of reliable data concerning the usefulness and safe usage of TT in sport. However, TT is widely touted among athletes and used by them. Ergogenic (anabolic) properties are attributed to this plant, since it supposedly elevates the blood testosterone level and stimulates skeletal muscles hypertrophy. TT was especially promoted in Bulgaria, where it was used from the 1970s as the preparation named Tribestan . It was Indeed, that was a time when Bulgarian weightlifters were the world champions, until more accurate anti-doping controls revealed the use of anabolic-androgenic steroids in these athletes. Hence, until the absence of steroidal doping will be confirmed by urine analysis, good sport results in weightlifting should be taken with caution. Despite discovered doping cases among Bulgarian weightlifters during the Olympic Games  they didTribulus terrestris supplementation  and so-called pro-hormones that are weak androgens, which are precursors of stronger endogenous androgens such as testosterone and dihydrotestosterone (DHT). Such contamination is not usually described in the list of ingredients; however, on the market there also exist products with a full list of components, including compounds prohibited in sport. Moreover, some researchers undertake studies to examine the biological action of those compositions, i.e. TT plus AAS. In the search for appropriate androgens taken by athletes, entation , and praposition . Howeverposition . The menposition . Obviousposition . Despiteposition . That riposition .Studies on effects of TT on blood androgens and a sexual disposition were conducted mainly among animals, and the results were contradictory. As to effects on the libido level, the experiments carried out in castrated rats showed that an extract of TT administered orally (5 mg/kg) possesses aphrodisiac activity , and theA few studies with a TT extract carried out among humans also provided divergent results. Since for the pharmaceutical market, which provides TT extracts, the main targets are athletes and healthy, physically active adults, the whole investigative efforts are aimed at confirming such biomedical properties of TT that are expected by the potential users. As mentioned, athletes seek performance enhancers, i.e. supplements of ergogenic properties, to improve training tolerance, and increase muscle mass and physical strength or endurance. Since development of muscle mass and strength depends, in part, on androgenic status, competitive athletes seek substances that are able to increase their endogenous circulating testosterone, while guaranteeing a negative anti-doping test. So far, trustworthy studies on expected TT properties are still insufficient. Studies on TT toxicity have been conducted only among animals. Effects of the use of nutritional supplements in sports are widely studied. The results of numerous investigations referring to the biological action, duration of supplementation and recommended doses are currently published in the scientific literature , and thiConsidering a relatively low number of conducted studies on the influence of TT on athletes, especially with regard to the effect on performance and androgenic status, one should stress the lack of evidence to show an enhancing effect of the use of TT on expected biomedical properties in humans. Contradictory results of aforementioned studies suggest that marketing opinions about TT as a testosterone enhancer are unsubstantiated. Therefore, there are athletes who use it in order to improve their performance. It is caused by intensive advertising encouraging TT usage, which may only result in a temporary placebo effect. For that reason, further clinical trials should be carried out in the future.


The amount of irreversible injury on renal allograft biopsy predicts function, but little is known about the early evolution of this damage. In a single-center cohort, we examined the relationship between donor-, recipient-, and transplantation-associated factors and change in a morphometric index of chronic damage (ICD) between protocol biopsies performed at implantation and at 2–3 months. We then investigated whether early delta ICD predicted subsequent biochemical outcomes. We found little evidence to support differences between the study group, who had undergone serial biopsies, and a contemporaneous control group, who had not. In allografts with serial biopsies (n = 162), there was an increase in ICD between implantation  and 2–3 months post-transplant . Donation from younger or live donors was independently associated with smaller early post-transplant increases in ICD. There was no evidence for a difference in delta ICD between donation after cardiac death vs. donation after brain death, nor association with length of cold ischemia. After adjustment for GFR at the time of the second biopsy, delta ICD after three months did not predict allograft function at one yr. These findings suggest that graft damage develops shortly after transplantation and reflects donor factors, but does not predict future biochemical outcomes. Improvements in the outcomes of kidney transplantation have mainly been confined to prevention of early graft loss. Over the last 20 yr, the slopes of allograft survival curves from one yr post-transplant have remained almost parallel The total amount of irreversible histological damage on protocol biopsies at early time points, as estimated by Banff criteria for chronic damage Although previous studies have examined clinical predictors of chronic damage at a single time point or over many years, little is understood about the factors surrounding transplantation that are associated with the early evolution of such damage, that is, the relationship between clinical variables and changes in measures of chronic damage during the first post-transplant months. This is an issue of particular importance in kidneys from “extended criteria” donors (ECD) or donation following cardiac death (DCD) as these allografts are likely to have significant levels of pre-existing chronic damage in the former case and suffer increased warm ischemic injury in the latter. The use of a quantitative approach to assess damage, made possible by the use of digital analysis systems, has been explored in an investigation of recipients of live donor transplants Using protocol biopsies performed as a part of routine clinical care, the aim of this study was to examine donor and recipient factors associated with changes in a morphometric measure of chronic damage over the first three post-transplant months. We then investigated whether the early change in this same measure was a useful predictor of graft function at one yr.In this cohort study, adult recipients of solitary renal allografts under the care of the Royal Free London NHS Foundation Trust (RFLNHSFT), transplanted between February 1, 2008, and October 31, 2010, underwent protocol biopsies as part of their routine clinical care. The unit policy is for protocol biopsies to be performed at three time points: 30 min following allograft implantation (PB0), 6–12 wk post-transplantation (PB1), and one yr post-transplantation. Only data from the first two are described here, as uptake of the one-yr biopsy is low  and likely to be heavily influenced by clinical factors; that is, those patients with a well-functioning graft are likely to forego this biopsy. All patients undergoing biopsy gave informed consent as to the clinical risks and benefits of the program. The following were considered contraindication to protocol biopsy: high bleeding risk, pregnancy, and indicated biopsy within the last 14 d.The immunosuppressive protocol was unchanged for the duration of this study. Induction therapy consisted of basiliximab (days 0 and 4), methylprednisolone, tacrolimus, and mycophenolate mofetil. Maintenance therapy (commenced five d prior to the transplant in recipients of live donor kidneys) consisted of (i) tacrolimus adjusted to maintain a trough level of 9–12 μg/L for the first three months and then 6–9 μg/L (except in cases of delayed graft function where target trough levels are 6–9 μg/L until graft function is established); (ii) mycophenolate mofetil 1 g bd reduced to 750 mg bd after one month; (iii) prednisolone 20 mg daily for one wk and 10 mg daily for one wk followed by steroid withdrawal. Acute cellular rejection was treated with pulsed methylprednisolone and continuation of oral steroids with the addition of antithymocyte globulin (ATG) in resistant cases. Acute vascular rejection was treated with the addition of plasma exchange where there was an identifiable donor-specific antibody, plus rituximab in resistant cases. Patients underwent a preemptive CMV surveillance strategy , receiving antiviral therapy only where routine samples revealed CMV antigen levels >1250 copies/mL.Protocol biopsies were performed by senior clinicians, under direct vision at implantation or live ultrasound guidance post-transplantation. Samples were obtained using either 16-gauge needles (at implantation) or 18-gauge biopsy needles (post-transplant) and were examined using a dissecting microscope at the bedside to confirm adequate sampling. Biopsies were formalin-fixed, paraffin-embedded, and serially sectioned before staining using conventional techniques. The morphometric measure, called the index of chronic damage (ICD), was determined in sections from PB0 to PB1  as previously described Donor data were retrieved from NHS Blood and Transplant (NHSBT). NHSBT definitions were used for mismatch level t-test (Wilcoxon rank-sum test for nonparametric variables). Then, in a sensitivity analysis, we examined whether inclusion in the biopsy group was associated with differences in eGFR at eight wks or cumulative incidence of rejection over the follow-up period, after matching using a propensity score based on donor and recipient characteristics.As not all patients transplanted in the study period had undergone serial protocol biopsies, we examined whether there were differences between the group who had undergone such biopsies and the group who had not using the chi-squared test  and We went on to examine the associations between ICD and clinical variables. ICD scores were positively skewed at all time points so to examine predictors of ICD0, and change in ICD  scores was divided into equal frequency tertiles and quartiles, respectively. Univariable associations between clinical variables and ICD0 or delta ICD were examined using analysis of variance (ANOVA)  and chi-squared test . The change in ICD between paired samples was examined using the Wilcoxon rank-sum test.For multivariate analysis, we did not group ECD and DBD donors separately, firstly to avoid small cell numbers and also so we could examine clinical factors that would lead to the definition of a donor as ECD  in both DBD and DCD groups. To identify independent predictors of ICD0 and delta ICD, we used a logistic regression models and included exposure variables where we hypothesized an impact on ICD. For baseline chronic damage, these were donor type , age, sex, ethnic group, donor serum creatinine>133 μMol/L, ICH, diabetes mellitus (DM), and cold ischemic time . For change in chronic damage, these were as above plus recipient factors: mismatch level, age, sex, ethnic group, previous transplantation, primary disease, DM, BK nephropathy, and rejection. Adjustment was made for the timing of PB1, but otherwise variables were retained in the models only where there was an improvement in model fit as demonstrated by a reduction in the −2 log-likelihood ratio. We also performed a restricted analysis excluding donors dying from ICH. Following fitting of the ordinal logistic model, the proportional odds assumption was tested for each of the variables in the model individually. Where this was violated, as in the model of ICD0, a multinomial logistic approach was used, and relative risk ratios were reported for each category. Predicted odds ratios or relative risk ratios were compared with observed values for both multivariable models. A further sensitivity analysis examining change in chronic damage was carried out using a linear regression model with log ICD1 adjusted for log ICD0, and although model fit was poor, the main conclusions, as identified using the logistical modeling approach, were unchanged.To investigate whether delta ICD might predict future outcome, we then examined whether this same measurement (as an explanatory variable) was associated with biochemical measures of kidney function. First, we examined the relationship between delta ICD quartile and quartile of GFR at biopsy. Then, to test the usefulness of the measure as a predictor of future function, we examined the association between delta ICD quartile and the change in eGFR between the time of PB1 and one-yr post-transplant (eGFR at day 365 after adjustment for eGFR at day 56).Summary statistics are presented as means ± standard deviation . Statistical significance was accepted as p < 0.05. This was a descriptive study, and therefore, no power calculations were performed. Data were analyzed in Stata 11 .Two hundred and sixty seven patients under follow-up at the renal transplant unit RFLNHSFT underwent 269 kidney-alone transplants between February 1, 2008, and October 31, 2010. Six patients were transplanted elsewhere, so no ICD0 was available. A further 17 allografts were excluded from further analysis due to nephrectomy (n = 8), loss to follow-up (n = 2), recipient death (n = 5), and contraindication to protocol biopsy (n = 2). Either ICD0 (n = 45) or ICD1 (n = 61) or both were not available on a further 81 allografts due to patient refusal (n = 5), multiple recent indicated biopsies (n = 2), or undocumented reasons. Delta ICD was available for 162 allografts, and along with three allografts with biopsies that were technically inadequate, this group (n = 165) was compared with those who did not undergo both protocol biopsies (n = 81). The “6–12 wks” post-transplant protocol biopsies were performed a median of 56 d (IQR 48–72) following transplantation. There was a higher prevalence of female donors, and there were an increased number of indicated biopsies in the group who did not undergo serial biopsies, but there were no other differences observed between the groups , median ICD0 was 2% (IQR 0–7). On univariable analysis, donor age, transplant type, and donor ethnic group were associated with tertile of ICD0 Table . HoweverOverall , median ICD1 was 8% (IQR: 4–15). Restricting the analysis to those subjects for whom delta ICD was available, there was a small but highly significant increase between biopsies . Box and whisker plots of ICD0 and ICD1 stratified by donor type are shown in Fig. Univariable associations between clinical variables and quartile of delta ICD are presented in Table Donor age and donor type were the only variables independently associated with quartile of delta ICD on multivariable analysis using an ordinal logistic regression model Table . IncreasThere was a strong association between delta ICD and eGFR at day 56 Table . There wIn this study, we describe the clinical associations of early changes in chronic damage on protocol biopsies performed as a part of a routine clinical care. Although several other studies have examined the prevalence of chronic damage in routine biopsy samples, to our knowledge, this is the first one to examine early change in ICD outside a clinical trial, in a cohort including transplants from donors after cardiac and brain death, as well as live donors.As with all studies of this type, a major concern is whether patients undergoing protocol biopsies are representative of the cohort of transplant patients as a whole. Therefore, we compared clinical variables in the group undergoing both protocol biopsies with the group undergoing only one or neither. We could only detect differences in donor sex and number of indicated biopsies. The higher number of indicated biopsies in the group not undergoing protocol biopsies may reflect causation  or reverse causation . Reassuringly rates of biopsy proven rejection were no different, suggesting that our analysis of change in ICD is not restricted to a selected group of patients in this context. The reasons underlying lower numbers of female donors in the protocol biopsy group are less clear, but it could be that there are differences in transplant course in recipients of kidneys from female donors Donor age was independently associated with ICD0 underlining the well-recognized importance of this variable in considering the suitability of organs for transplantation. Furthermore, DCD kidneys had a higher baseline ICD independent of age, which may reflect a difference in the underlying clinical characteristics of donors of kidneys of these types accepted at our institution. No differences in baseline ICD were observed between kidneys from live donors and DBD after adjustment for donor age. Others have reported associations between early changes in fibrosis and diabetes, rejection, and kidneys from male donors Younger donor age and live donation were independently associated with reduced odds of higher delta ICD. As age is known risk factor for failure to recover from acute kidney injury Overall, there was a small, but highly significant increase in ICD between biopsies. This increase in chronic damage in our cohort appears consistent with what has been reported in other studies , 9. ThisDespite differences in ICD at implantation, there was no evidence for a difference in delta ICD between DBD and DCD after adjustment for donor age, suggesting that although kidneys from DCD in our cohort may have higher levels of chronic damage prior to transplantation, the post-transplant progression of this damage is similar between these two donor types. We also found no evidence that either CIT (within the range of CIT seen in deceased donors) or early acute rejection was associated with delta ICD. It is impossible to confidently separate the impact of CIT from donor type in a study of this design, but these data suggest that there may be non-ischemic mediators of the association between live donors and lower delta ICD. The attenuation of the differences between live and DBD donors after exclusion of donors dying from ICH may implicate this cause of donor death in aggravated transplantation-associated allograft injury and is consistent with the worse medium-term outcomes observed in kidneys transplanted from such donors We examined associations between delta ICD (as the explanatory variable) and eGFR, both at the time of second biopsy and at one yr. Unsurprisingly, delta ICD was associated with eGFR at both time points. These finding are similar to those reported by Servais and colleagues Almost 16% of serial protocol biopsies demonstrated a negative delta ICD. Areas of chronic damage are not thought to recover, so these values are unlikely to represent true changes. A similar finding has been reported elsewhere In addition to those mentioned above, our study has several other weaknesses. Although we examined, and found little evidence for, differences between the group undergoing protocol biopsies and those who did not, we cannot exclude the possibility that we have examined a selected group of patients. All our patients were from a single center, and therefore, our conclusions may not be generalizable. Furthermore, we did not examine our biopsies for inflammation in scarred areas, a finding that has been reported to be a useful predictor of adverse graft outcomes In summary, this study demonstrated an early, small, but measurable increase in irreversible damage in allograft kidneys over the first three months following transplantation. This increase in chronic damage was associated with donor age. Furthermore, transplantation from a live donor was associated with a smaller increase in ICD. The routine clinical value of measurement of chronic damage is unclear due to both the potential for sampling variation and the observation that the early increase in histological damage appears to reflect past insults, but not future course as judged by biochemical change in function over the following nine months.


Therefore, the mechanism of GM-CSF-producing CD4+ T cell differentiation and the role of GM-CSF in the development of autoimmune and inflammatory diseases are gaining increasing attention. This review summarizes the latest knowledge of GM-CSF and its relationship with autoimmune and inflammatory diseases. The potential therapies targeting GM-CSF as well as their possible side effects have also been addressed in this review.Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor, which stimulates the proliferation of granulocytes and macrophages from bone marrow precursor cells. In autoimmune and inflammatory diseases, Th17 cells have been considered as strong inducers of tissue inflammation. However, recent evidence indicates that GM-CSF has prominent proinflammatory functions and that this growth factor (not IL-17) is critical for the pathogenicity of CD4 Granulocyte macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor which was originally recognized as a stimulator for the proliferation of granulocytes and macrophages from bone marrow precursor cells . It has β and IL-23 in mice [β and IL-12 in humans [α and IL-1, and in macrophage/monocytes it is stimulated by toll like receptors (TLRs) [γ [GM-CSF is produced by a variety of cells. Major sources of GM-CSF include activated T and B cells, monocytes/macrophages, endothelial cells, fibroblasts, and other sources such as neutrophils, eosinophils, epithelial cells, mesothelial cells, chondrocytes, Paneth cells, and tumor cells –7. The p in mice , IL-1β an humans , and alsn humans . In fibrs (TLRs) . In lymps (TLRs) , 12. HowTLRs) [γ , IL-4 [1TLRs) [γ , IL-10 [TLRs) [γ , and alsTLRs) [γ , 17 or gTLRs) [γ .α-subunit which binds GM-CSF with low affinity and a signaling βc-subunit which is shared with the IL-3 and IL-5 receptors [βc-subunit constitutively associates with Janus kinase 2 (JAK2) and is tyrosine phosphorylated by it resulting in an assembly of dodecameric signaling complex and initiation of signaling [β-chain residues: Ser585 and Tyr577 [β-chain, which leads to activation of the PI-3 kinase pathway and results in myeloid cell survival. At high concentrations of GM-CSF, as at the site of inflammation, signaling via Ser585 is extinguished and signaling occurs exclusively via Tyr577 residue, which activates the Jak2/STAT5 pathway, Ras/mitogen-activated protein kinase pathway and PI-3 kinase pathway, resulting in cell survival, proliferation, and activation [The GM-CSF receptor is expressed on myeloid cells and on some nonhaemopoietic cells such as endothelial cells but not on T cells , 20. Theeceptors . The βc-ignaling . The effd Tyr577 . At low tivation –26.GM-CSF stimulates proliferation and activation of macrophages, monocytes, neutrophils, eosinophils, dendritic cells, and microglia , 27. Howγ receptors via PU.1 [β [GM-CSF stimulates the terminal differentiation of macrophages and the acquisition of normal immune functions via the transcription factor PU.1 . GM-CSF via PU.1 –34, 43. via PU.1 . GM-CSF  PU.1 [β , 45, 46, PU.1 [β , 47, 48  PU.1 [β . On the  PU.1 [β . GM-CSF  PU.1 [β , pulmona PU.1 [β , and sev PU.1 [β .+CD11b+ DCs [+ DCs [ in vitro [ in vivo. It was reported that the number of moDCs was increased in GM-CSF transgenic mice [κB1-dependent GM-CSF production in CD4 T cells was reported to be required for the generation of moDCs in inflammatory arthritis and antigen-induced peritonitis mouse models. The number of moDCs was markedly reduced in draining lymph nodes from GM-CSF−/− mice with inflammatory arthritis or in the spleen of mice reconstituted with NF-κB1−/− CD4 T cells in acute peritonitis, demonstrating that GM-CSF contributes to the differentiation of these cells during inflammation in vivo [ in vitro and in vivo, there may be another GM-CSF-independent pathway for the development of moDCs [GM-CSF positively regulates the development of migratory CD10311b+ DCs  but negas [+ DCs . GM-CSF in vitro . Howevernic mice . Further in vivo . On the  in vivo , 56. Theof moDCs , or prodof moDCs .In mature neutrophils, GM-CSF upregulates the expression of the integrin CD11b, which increases cellular adhesion and tissue entry . GM-CSF Among B cells, the innate-like B1 B cells reside predominantly in serosal cavities such as the pleural or peritoneal cavity. In response to microbial infection, B1a B cells (a subset of B1 B cells) recognize bacteria via direct TLR-dependent pathogen recognition and differentiate into innate response activator (IRA) B cells, which produce GM-CSF and also express the GM-CSF receptor , 60. GM-+ T cells is essential in inflammatory mouse models such as experimental autoimmune encephalomyelitis (EAE), arthritis models such as collagen-induced arthritis (CIA) or SKG-arthritis, interstitial lung disease in SKG mice (SKG-ILD), peritonitis, or myocarditis [Although GM-CSF is widely expressed in both stromal and hematopoietic cells, recent murine studies suggest that GM-CSF from CD4carditis , 62–64. carditis . Moreoveγ, and GM-CSF and are identified by the coexpression of T-bet, RORγt, and the chemokine receptors CXCR3 and CCR6 [γt [Th17 cells have been shown to be strong inducers of tissue inflammation and autoimmune diseases. However, a number of studies determined that IL-17 inhibition does not prevent but rather only ameliorates the development of EAE , 68, 69,and CCR6 –78. HumaCCR6 [γt , 79, 80.CCR6 [γt  and the CCR6 [γt , 82.β induce the production of GM-CSF in T cells whereas IL-12 suppresses its expression [β renders Th17 cells sensitive to IL-12 and both IL-1β and IL-12 promote the differentiation of Th1/17 cells [β from antigen presenting cells (APCs) [In mice, IL-23 and IL-1pression , 77, 83.17 cells , 84, 85 s (APCs) . This res (APCs) .+ T cells is not regulated by T-bet [γt expression in CD4+ T cells results in GM-CSF production, indicating that GM-CSF production in Th1/17 cells is induced by RORγt [γt-deficient CD4+ T cells can produce GM-CSF, indicating the existence of additional pathways to induce GM-CSF production in CD4+ T cells [Studies show that GM-CSF expression in CD4by T-bet , 4 and R T cells .+ T cells with low or undetectable expression of T-bet, GATA-3, RORγt transcripts. These cells represent a new distinct subset of Th cells, namely, Th-GM [+CCR4+CXCR3−CCR6− Th cells [Recently, it was reported that IL-2- or IL-7-activated STAT5 promotes the generation of GM-CSF-producing CD4y, Th-GM , 64. In Th cells . The conTh cells , 87, whiTh cells –90, was Th cells . These cTh cells , 64. It  in vitro culture [Th1 cells are another source of GM-CSF . It was  culture .+ cells and GM-CSF+ cells in the nasal mucosa of patients with allergic rhinitis [+ T cells and their contribution to the development of autoimmune and inflammatory diseases.Th2 cells also produce GM-CSF , 67. Altrhinitis , there i+ T cells play a critical role in the development of MS and experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of MS. It has been widely believed that Th17 cells are the main encephalitogenic population in autoimmune inflammation [Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system and is pathologically characterized by demyelination and subsequent axonal degeneration. Past studies have shown that CD4ammation ; howeverammation . On the ammation , 95. Theammation  and elevammation , 97.α [+ T cells induce the differentiation of neurotoxic M1-like phenotype of microglia [β, IL-6, and TNFα, which contribute to myelin sheath damage, via upregulation of TLR and CD14 expression [Recent findings show that GM-CSF is secreted by CNS-infiltrating helper T cells and is essential for encephalitogenicity in EAE , 4, 64. α –102. Furicroglia  and uprepression .+ T cell-derived GM-CSF was shown to be vital for the development of EAE [Studies also show that GM-CSF is required for recruitment of peripheral myeloid cells that contribute to blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) disruption and demyelization into the CNS , 105. Ast of EAE .+ T cells initially activate microglia and induce production of proinflammatory cytokines, which contribute to myelin sheath damage [Taken together, CNS-infiltrating CD4h damage . This inh damage , leadingThese reports indicate that GM-CSF plays a central role in MS and indicate that the inhibition of GM-CSF will be a useful therapeutic strategy for MS. MOR103, a fully human monoclonal antibody that binds human GM-CSF, is currently being tested in a phase Ib trial for MS  Table 1Table 1. γ but not IL-17 [Rheumatoid arthritis (RA) is a systemic chronic autoimmune disease characterized by persistent and erosive inflammatory polyarthritis. Recent studies indicate that GM-CSF plays a central role in the pathogenesis of RA as in MS, by activating or promoting differentiation and survival of macrophages and neutrophils , 110. Thot IL-17 , indicatot IL-17 , 116.The contribution of GM-CSF in the development of arthritis was also reported in several mouse models of arthritis. In the collagen-induced arthritis (CIA) model, GM-CSF deficient mice failed to develop arthritis , and theβ or IL-6 from macrophages in a dose dependent manner [+ T cells [In SKG mice, another model of autoimmune arthritis, GM-CSF, upregulated proinflammatory cytokine production such as IL-1t manner , 120. Th T cells . The pro T cells , indicatα antibody, is currently being developed and a phase II study in RA patients reported significant efficacy with no serious adverse events such as pulmonary alveolar proteinosis [Mavrilimumab, a fully human anti-GM-CSF receptor teinosis . In thisteinosis .ClinicalTrials.gov.MOR103, which is also being tested for MS, has shown preliminary evidence of efficacy in a phase Ib/IIa trial for patients with active RA . SubjectPulmonary alveolar proteinosis (PAP) is a rare syndrome characterized by the accumulation of surfactant in pulmonary alveoli resulting in varying degrees of respiratory insufficiency and myeloid cell dysfunction leading to increased risk of infection , 124. SePulmonary surfactant is tightly regulated by balanced production, secretion, reuptake, and catabolism within alveoli. GM-CSF regulates surfactant catabolism in alveolar macrophages via PU.1 but does not regulate surfactant endocytosis or uptake and catabolism of surfactant by alveolar epithelial cells type II (AEC-II) , 127. GMβ produced by damaged epithelial cells. In fact, anti-inflammatory therapies have little benefit in IPF [α-induced endothelin-1 (ET-1) upregulates GM-CSF production from airway smooth muscle cells (ASMCs) [The mechanism of pulmonary fibrosis in interstitial lung disease (ILD) has been studied using Idiopathic pulmonary fibrosis (IPF) or a bleomycin-induced mouse model of pulmonary fibrosis. Pulmonary fibrosis in IPF is considered to be inflammation-independent and mainly initiated by TGF-t in IPF –135. Howt in IPF –138 or Tt in IPF , 140, ha (ASMCs)  and that (ASMCs) , 143. GM (ASMCs) , 145. In (ASMCs) .+ T cells, and CD11b+Gr1+ neutrophils with fibrosis [β production by macrophages, which in turn enhanced differentiation of IL-17A and/or GM-CSF-producing T cells and infiltration of neutrophils into the lungs [α antibody and MOR103, a fully human monoclonal anti-GM-CSF antibody, are undergoing clinical trials in RA patients [In contrast with idiopathic pulmonary fibrosis (IPF), connective tissue disease-associated ILD (CTD-ILD) is often characterized by a clearer response to immunosuppression, indicating that autoimmune/inflammatory mechanisms play a more significant and central role in the pathogenesis of CTD-ILD , 149. SKfibrosis . Naive She lungs . Neutralhe lungs . Importahe lungs , suggestpatients , 122. FuRecent developments suggest that the development of Crohn's disease (CD) is caused by a mucosal innate immunodeficiency with a variety of genetic defects  and a dyThese findings indicate that administration of GM-CSF could be beneficial for the treatment for CD patients. Initial reports indicated that the administration of GM-CSF in CD patients with moderate to severe disease activity had a high rate of clinical response and remission with minimal adverse effects , 166. MoGM-CSF also was reported to take part in Th2 response in allergic airway inflammation via activation of DCs –171. In GM-CSF plays pivotal roles not only in maintaining immune homeostasis but also in exacerbating inflammatory reactions. Recent findings indicate that GM-CSF inhibition will be an attractive therapeutic strategy for many autoimmune and inflammatory diseases. Studies also indicate that it is necessary to monitor possible side effects such as PAP or CD, although GM-CSF inhibition has no demonstrated serious adverse reactions so far, which is indicative of its wide therapeutic index , 174. Fu


The magnitude and consequences of prescription opioid (PO) misuse and harms (including rising demand for PO disorder treatment) in Canada have been well-documented. Despite a limited evidence-base for PO dependence treatment, opioid maintenance therapy (OMT) - mostly by means of methadone maintenance treatment (MMT) - has become the de facto first-line treatment for PO-disorders. For example in the most populous province of Ontario, some 50,000 patients - large proportions of them young adults - are enrolled in MMT, resulting in a MMT-rate that is 3–4 times higher than that of the United States. MMT in Ontario has widely proliferated towards a quasi-treatment industry within a system context of the public fee-payer offering generous incentives for community-based MMT providers. Contrary to the proliferation of MMT, there has been no commensurate increase in availability of alternative , and less intrusive and/or costly, treatments which may provide therapeutic benefits at least for sub-sets of PO-dependent patients. Given the extensive PO-dependence burden combined with its distinct socio-demographic and clinical profile , an evidence-based ‘stepped-care’ model for PO dependence treatment ought to be developed in Canada where MMT constitutes one, but likely a last resort or option, for treatment. Other, less intrusive treatment options as well as the best mix of treatment options should be systematically investigated and implemented. This case study has relevance and implications for evidence-based treatment also for the increasing number of other jurisdictions where PO misuse and disorders have been rising. By now, the detrimental extent and consequences of the ‘epidemic’ of prescription opioid (PO) misuse and harms in North America are well documented. Despite recent ‘plateauing’ effects, some 4.2 % of adults report non-medical PO use (NMPOU) and some 17,000 PO-related poisoning fatalities occur annually in the United States –7. AmpleA related but commonly overlooked phenomenon concerns the realities of treatment for PO-related disorders. A recent report in the Lancet (2012) concluded that “research into the treatment for [PO] addiction has been chronically neglected. As a result, the evidence base that informs best practice is thin […] The ‘standard treatment’ for [PO] dependence is evolving, and [there is no] single current standard at this time” . Yet, cuThe above data reflect that OMT - mostly with methadone but some suboxone-based in exceptional cases - has proliferated as the de facto first-line treatment for PO-related disorders in Ontario. This is despite the fact that OMT is designed as long-term - in many cases for life - pharmacotherapy for most patients . The preWhile the pharmaceutical industry’s corporate greed and tactics have been popularly blamed - and legally punished - for the PO abuse epidemic e.g., , 33), ec, ec33]),Allow us to be perfectly clear: Our position is not ‘anti’-OMT for PO-disorders. In fact, several of the present authors have actively argued for the expansion of OMT availability in Canada when this was still a highly restricted and scarce treatment for the treatment of opioid disorders not so long ago , 40. We Unquestionably, there are a large number of individuals suffering from PO disorders in Canada who require treatment. While OMT undoubtedly brings therapeutic benefits to many opioid-dependent people, and is the best available therapuetic choice for a large sub-group of patients with PO disorder it also implies the continued exposure of patients to potential correlated adverse effects  of chronic opioid intake - risks that should be minimized especially with young and non-severely dependent patients –50. LongIn summary, we urge policy-makers at relevant levels in Canada to both facilitate the development of an evidence-base - building on existent and facilitate the generation of new data required - for effective non-OMT options towards a comprehensive overall continuum-of-care for PO disorder treatment with OMT as a ‘last resort’; related, we urge the correction of the predominant economic parameters in opioid disorder treatment that seem to have unduly influenced the recent excessive expansions of OMT in Ontario, towards a more and overall public health oriented approach to treatment and care. As the burdensome issue of PO misuse and disorders is increasing in other national system contexts , 55, the


During brain development the neural stem cells are regulated by both intrinsic and extrinsic sources. One site of origin of extrinsic regulation is the developing choroid plexuses, primely situated inside the cerebral ventricles. The choroid plexuses are very active in terms of both secretion and barrier function as soon as they appear during development and control the production and contents of cerebrospinal fluid (CSF). This suggests that regulated secretion of signaling molecules from the choroid plexuses into CSF can regulate neural stem cell behavior (as they are in direct contact with CSF) and thereby neurogenesis and brain development. Here, choroid plexus development, particularly with regards to molecular regulation and specification, is reviewed. This is followed by a review and discussion of the role of the developing choroid plexuses in brain development. In particular, recent evidence suggests a region-specific reciprocal regulation between choroid plexuses and the neural stem cells. This is accomplished by site-specific secretion of signaling molecules from the different choroid plexuses into CSF, as well as brain region specific competence of the neural stem cells to respond to the signaling molecules present in CSF. In conclusion, although in its infancy, the field of choroid plexus regulation of neurogenesis has already and will likely continue to shed new light on our understanding of the control and fine-tuning of overall brain development. Neurogenesis, both in the adult and during development, occurs in a specialized environment established by both the neurogenic  and non-neurogenic cells within the neurogenic area, and from cells and compartments in direct contact with the niche  lining the ventricle . The processes of the NSCs span the entire thickness of the cortical wall and their nuclei move up and down in the ventricular zone (VZ) during the cell cycle  but they stay in contact with the ventricle via the apical process and the cilium responsive to elements present in CSF through-out the cell cycle. The NSCs divide and give rise to the next cells in the differentiation cascade, the transient amplifying cells, TAPs (yellow cells) or neurons (green cells). The TAPs are a heterogeneous population and vary in their morphology, location and abundance  are derived from plasma and are transferred across the choroid plexus epithelium by a subset of choroid plexus epithelial cells in a highly specific and regulated manner  and alterations in developmental neurogenesis  choroid plexus Figure . The choIn the adult, the choroid plexuses and CSF have several know functions such as: (i) protecting and regulating the internal environment of the brain via the blood-CSF barrier; (ii) secretion and modulation of CSF through the activity of the choroid plexus epithelial cells; and (iii) waste and metabolite removal, via the “CSF sink,” through the continuous production and then removal of CSF into peripheral circulation. During development two out of these three functions are already performed by the choroid plexuses; protection of the brain via the blood-CSF barrier and regulation of CSF composition via specific and regulated transfer and secretion (see more below). However, due to the lack of CSF reabsorption during embryogenesis, the removal of waste and metabolites does not occur . Ttr is known as the choroid plexus marker and is expressed in the mouse lateral ventricular choroid plexus from E11  choroid plexus has also been described  the choroid plexus was reduced in size but Cajal-Retzius cells were increased in number , resulting in a transient doubling of the size of the lateral ventricles  free passage of water and solutes between the cells has to be inhibited via the presence of tight-junctions , (ii) the capacity of water transport across the cells , and (iii) the presence of transporters and exchangers that can create an ionic or osmotic gradient that drives water across the epithelium. These criteria are of course all met in the adult choroid plexus via the presence of the blood-CSF barrier, the presence of the water channel AQP1 and the creation of ion gradients between blood and CSF via a multitude of ion transporters and exchangers  creates a pressure inside the cerebral ventricles. The requirement of this pressure for normal brain development was shown by a study in chick embryos (Stage18), where the intraventricular pressure was removed via insertion of hollow tubes into the ventricular system  or via changes in the secretion of molecules produced by the choroid plexuses themselves Figure . In the in vitro experiments. Here it was shown that isolated cortical cells can be maintained on embryonic CSF alone .The choroid plexuses appear whilst neurogenesis is occurring through-out the nervous system. This gives the potential for the choroid plexuses (via their modulation of CSF) to alter the behavior of the neural stem cells along the neuroaxis Figure , Box 1. With the advancement of region and time specific deletion techniques new strategies to isolate choroid plexuses involvement in neurogenesis are available. Along these lines, some recent work has demonstrated, via choroid plexus specific deletions, that molecules expressed in the choroid plexus have the capability to influence brain development at other sites. This was first achieved by deletion of Shh from the hindbrain choroid plexus (using the Wnt1-Cre), resulting in a more than 50% decrease in the proliferation of the neural stem cells in the nearby cerebellum and impaired GABAergic progenitor expansion  it not only significantly altered the size of the choroid plexus but also the relative expression of many secreted signaling molecules . We suggest that select tissues are prepared for certain signaling molecules, by the expression of not only receptors but also by the expression of modulators and inhibitors of the different signaling pathways. Thus, neurogenesis can be seen as a reciprocal process between the choroid plexus and the neural stem cells mediated via the CSF Figure .In conclusion, there is substantial evidence that there is indeed a role played by the choroid plexus-CSF-signaling axis in the modulation of developmental neurogenesis. However, much more remains to be discovered. The specific mechanisms by which the choroid plexuses modulate brain development both in terms of the choroid plexuses as whole organs as well as specific aspects of their functions  needs to be investigated. Further detailed studies of choroid plexus impact on different brain regions and developmental time-points would increase our understanding of the whole process of brain development. Understanding the role of the choroid plexus-CSF signaling axis and its impact on brain development can also lead to novel routes and mechanisms for treatment approaches for a multitude of developmental disorders and in the long-run also for application to adult neurodegeneration and brain injuries.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


APOE) ɛ4 and low cerebrospinal fluid (CSF) amyloid-β42 (Aβ42) levels are predictors for developing Alzheimer’s disease (AD). The results of several studies indicate an interaction between docosahexaenoic acid (DHA) consumption and cognitive outcomes by APOE genotype. Our objective in the present study was to examine whether APOE ɛ4 genotype and low CSF Aβ42 levels were associated with reduced delivery of DHA to CSF in the Alzheimer’s Disease Cooperative Study-sponsored DHA clinical trial.Apolipoprotein E (n = 15) or DHA (n = 29). Plasma and CSF DHA levels, CSF Aβ42, Tau, and phosphorylated Tau were measured at baseline and after the 18-month intervention. Participants were divided into tertiles based on baseline Aβ42 CSF levels. To assess DHA delivery across the blood-brain barrier, the ratio of CSF to plasma DHA levels was calculated.Phospholipid DHA was assayed in the plasma of 384 participants and CSF of 70 participants at baseline. Forty-four of the 70 participants completed the 18-month follow-up visit after allocation to placebo (p = 0.01) and lower CSF-to-plasma DHA ratios (p = 0.05) compared to the other tertiles. Baseline CSF Aβ42 levels were significantly lower in ɛ4 carriers than in ɛ4 noncarriers (p = 0.01). Participants carrying the ɛ4 allele (n = 25) demonstrated a less pronounced increase in CSF DHA level compared with noncarriers (n = 4), with a possible interaction effect between treatment and APOE genotype (p = 0.07).At baseline, there were no significant differences between CSF or plasma phospholipid DHA levels by CSF Aβ42 tertiles or ɛ4 status. After 18 months of DHA supplementation, participants at the lowest Aβ42 tertile had significantly lower CSF DHA levels (APOE ɛ4 allele and lower CSF Aβ42 levels were associated with less transport of DHA to CSF. Brain amyloid pathology may limit the delivery of DHA to the brain in AD.NCT00440050. Registered on 22 Feb 2007.Clinicaltrials.gov identifier:  APOE) ɛ4 has the strongest correlation with disease onset  [n = 818] [n = 286] [There is evidence in several studies other than the ADCS-sponsored DHA trial  that thepatients . In 2005patients  examinedcarriers . In cont = 1228] , Chicagon = 818] , and then = 286] ) suggestAPOE ε4 status before the onset of detectable brain amyloidosis and AD. These studies would then clarify whether cognitively healthy younger APOE ε4 carriers have a chronic defect in DHA brain delivery years before the onset of neurodegeneration, presenting with an opportunity for DHA supplementation to prevent or slow the progression of AD.In summary, these studies indicate an interaction between ɛ4 allele and DHA efficacy, with the ɛ4 allele potentially limiting the effectiveness of DHA supplementation on cognition later in the disease process. It would be important to assess the delivery of DHA to the brain by APOE genotypes and CSF Aβ42 peptide levels. Our main finding suggests decreased DHA delivery to CSF in participants with lower CSF Aβ42 peptide and in carriers of the APOE ε4 allele. These findings can help explain the lack of efficacy of DHA in participants with established AD. Future studies are needed to clarify if differences in DHA transport in participants carrying the APOE ε4 allele appear earlier in life, before the onset of cognitive decline.To our knowledge, this is the first study to demonstrate changes in CSF DHA levels in relation to APOE, apolipoprotein E; cPLA2, calcium-dependent phospholipase A2; CSF, cerebrospinal fluid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; MMSE, Mini Mental State Examination; n-3 PUFA, ω-3 polyunsaturated fatty acid; PLA2, phospholipase A2; p-Tau, phosphorylated Tau; Tg, transgenicAA, arachidonic acid; Aβ42, amyloid-β42; AD, Alzheimer’s disease; ADAS-cog, Alzheimer’s Disease Assessment Scale-Cognition; ADCS, Alzheimer’s Disease Cooperative Study;


Danio rerio) is an ideal vertebrate model to investigate the developmental molecular mechanism of organogenesis and regeneration. Recent innovation in genome editing technologies, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system, have allowed researchers to generate diverse genomic modifications in whole animals and in cultured cells. The CRISPR/Cas9 and TALEN techniques frequently induce DNA double-strand breaks (DSBs) at the targeted gene, resulting in frameshift-mediated gene disruption. As a useful application of genome editing technology, several groups have recently reported efficient site-specific integration of exogenous genes into targeted genomic loci. In this review, we provide an overview of TALEN- and CRISPR/Cas9-mediated site-specific integration of exogenous genes in zebrafish.The zebrafish ( Usintyrosine hydroxylase locus in four of 275 potential F0 zebrafish founders. Subsequently, Shin et al. reported the efficient TALEN-mediated knock-in of donor DNA templates by optimizing the length of the homology arms and the configuration of the donor DNA construct [sox2 locus, and 29 of 363 F0 founders produced sfGFP-positive F1 embryos. Furthermore, tandem dimeric Tomato (tdTomato) was integrated into the gfap locus, and five of 44 F0 founders produced tdTomato-positive F1 embryos [et al. reported the CRISPR/Cas9-mediated knock-in of a donor vector [albino mutant , which carries a nonsense mutation in the slc45a2 gene essential for melanin production. Because the alb mutant embryos exhibit a pale phenotype with pigment defects, the phenotypic rescue by the HR-mediated knock-in of a donor vector containing the wild-type slc45a2 gene is easily determined by the appearance of the pigmented cells. They demonstrated that the frequency of phenotypic rescue was increased using circular donor DNA containing CRISPR target sites. In the rescue experiment, three of 28 F0 founders produced pigmented embryos with the wild-type phenotype [Genome editing technologies (TALEN and CRISPR/Cas9) were applied to perform HR-mediated integration of donor DNA templates because the HR of the donor vector at the target site is largely enhanced by the production of site-specific DSBs. The eGFP reporter donor containing approximately 1 kb of homology arms was integrated at the target site using HR in zebrafish ; howeveronstruct . They usr vector . To examhenotype . Althouget al. reported the efficient CRISPR/Cas9-mediated knock-in of donor vectors at targeted sites in zebrafish [Recently, site-specific insertion via homology-independent repair mechanisms, presumably mediated by NHEJ, was developed. When the targeted genomic locus and the donor vector containing the ZFN or TALEN target sequences were simultaneously cleaved by ZFN or TALEN, the NHEJ-mediated insertion of donor DNA was observed in cultured cells at the target site ,39. One ebrafish . In thisebrafish . When thet al. improved the NHEJ-mediated knock-in of donor vectors for establishing transgenic zebrafish [evx2 gene. Furthermore, the introduction of the hsp promoter usually enhances the basal expression of the integrated reporter/driver gene. Immunostaining using an anti-Evx2 antibody revealed that the eGFP expression generally overlapped with Evx2 protein expression. However, the expression of the Evx2 protein was not detected in some of the eGFP-positive cells, presumably owing to the enhanced leaky expression of the hsp-dependent promoter activity. Using this strategy, the authors consistently established stable knock-in lines in four endogenous loci  [More recently, Kimura ebrafish . They prvglut2a) . Importavglut2a) . The CRItyrosinase locus, the NHEJ-mediated knock-in of the donor vector in the forward direction was detected in 53% of the injected F0 embryos using PCR, while the addition of 10–40 bp homology arms in the donor vector increased its efficiency to 85%. Genomic sequence analysis revealed that the MMEJ-mediated knock-in with precise targeted integration was 60%–77% of analyzed knock-in events. Therefore, the introduction of short homology arms promotes precise targeted integration of exogenous genes. In order to eliminate the undesired integration of the backbone vector sequences, we designed an improved donor vector that configures the eGFP and polyA signal between two bait sequences in the case of the keratin type 1 c19e/krtt1c19e locus (It has been recently shown that DSBs can be repaired by MMEJ that uses the existing microhomology sequences (five to 25 bases) around the broken DNA ends . Both Dr9e locus . We obseGenetic manipulation in whole organisms plays an important role in basic, applied and medical sciences. Recent advances in genome editing technologies  have enabled researchers to make precise genomic modifications that were previously difficult or impossible in various organisms including humans. In this review, we summarized the targeted integration of exogenous genes using genome editing technologies mediated by DNA repair mechanisms, such as NHEJ, MMEJ and HR. Because integration directions become random and various indel mutations occur at the junction in the process of NHEJ-mediated knock-in, we recommend that MMEJ-mediated knock-in technology is suitable for the precise targeted integration of donor DNA templates. Furthermore, the construction of a donor vector for MMEJ-mediated knock-in is much easier than that of HR-mediated knock-in. Therefore, we predict that the MMEJ-mediated targeted integration could be widely applied in various organisms. Importantly, genome editing technologies can be used to precisely model human genetic disorders in animals including zebrafish, which could lead to novel findings with respect to the molecular mechanisms of human diseases. Furthermore, these technologies make it easier to correct serious genetic disorders in affected patients and in induced pluripotent stem (iPS) cells derived from patients. Further innovation of genome editing technologies will accelerate various applications in basic biology, biotechnology and medicine, including potential therapeutic strategies for human genetic disorders.


The aim of this study was to determine whether the improvement of cerebrospinal fluid (CSF) flow dynamics by CSF shunting, can suppress the oligomerization of amyloid β-peptide (Aβ), by measuring the levels of Alzheimer’s disease (AD)-related proteins in the CSF before and after lumboperitoneal shunting. Lumbar CSF from 32 patients with idiopathic normal pressure hydrocephalus (iNPH) (samples were obtained before and 1 year after shunting), 15 patients with AD, and 12 normal controls was analyzed for AD-related proteins and APLP1-derived Aβ-like peptides (APL1β) (a surrogate marker for Aβ). We found that before shunting, individuals with iNPH had significantly lower levels of soluble amyloid precursor proteins (sAPP) and Aβ38 compared to patients with AD and normal controls. We divided the patients with iNPH into patients with favorable  and unfavorable  outcomes. Compared to the unfavorable outcome group, the favorable outcome group showed significant increases in Aβ38, 40, 42, and phosphorylated-tau levels after shunting. In contrast, there were no significant changes in the levels of APL1β25, 27, and 28 after shunting. After shunting, we observed positive correlations between sAPPα and sAPPβ, Aβ38 and 42, and APL1β25 and 28, with shifts from sAPPβ to sAPPα, from APL1β28 to 25, and from Aβ42 to 38 in all patients with iNPH. Our results suggest that Aβ production remained unchanged by the shunt procedure because the levels of sAPP and APL1β were unchanged. Moreover, the shift of Aβ from oligomer to monomer due to the shift of Aβ42 (easy to aggregate) to Aβ38 (difficult to aggregate), and the improvement of interstitial-fluid flow, could lead to increased Aβ levels in the CSF. Our findings suggest that the shunting procedure can delay intracerebral deposition of Aβ in patients with iNPH. Alzheimer’s disease (AD) is a neurodegenerative disorder and the most common form of dementia. In the brains of patients with AD, a toxic oligomeric species of the amyloid β-peptide (Aβ) induces synaptic degeneration and neuronal death. The amyloid cascade hypothesis posits that polymerization of Aβ and subsequent accumulation of this toxic species is the principal cause of AD pathogenesis , 2.Another type of dementia, the normal pressure hydrocephalus (NPH), has been linked to the reduction in compliance between the cerebrospinal compartments and disruption in cerebrospinal fluid (CSF) outflow-absorption . IdiopatInterestingly, some studies have reported cases of comorbid iNPH and AD , 10, witAmyloid precursor protein (APP) plays a significant role in AD pathogenesis since its cleavage by the proteolytic enzymes, β- and γ-secretase, generates the various types of Aβ peptides. Of these, Aβ42 is a major component of senile plaques in patients with AD. APLP1-derived Aβ-like peptides (APL1β), homologues of APP, are similar to soluble APP (sAPP) β in its primary sequence and function . HoweverWe hypothesized that the promotion of CSF production and drainage by CSF shunt surgery may suppress the oligomerization of Aβ and result in increased Aβ clearance. In the current study, in order to determine the effects of CSF shunting on the amyloid cascade, we measured the levels of AD-related proteins in the CSF of patients with iNPH, before and after the lumboperitoneal shunting (LPS) surgery. We also investigated the influence of oligomerization of Aβ by comparing the levels of Aβ42 in the CSF, and determining the changes in the production of Aβ42, as estimated by the levels of APL1β28.LPS was performed on 32 patients with iNPH, which included 23 men and 9 women aged 73.7 ± 6.8 years (mean ± SD), between 2007 and 2012. Diagnostic criteria were symptoms and signs of iNPH in accordance with the Japanese guidelines for iNPH , mini meFifteen patients with AD, 11 men and 4 women, aged 71.5 ± 10.6 years (mean ± SD), were recruited in this study. AD was diagnosed using standard clinical criteria , 22.Finally, 12 normal controls (NCs), 3 men and 9 women, aged 67.1 ± 11.0 years (mean ± SD), were recruited in this study. The NC group had no history of dementia and did not show any signs of other psychiatric illnesses. All patients with AD and NCs consented to lumbar punctures at the Juntendo University Hospital.Lumbar puncture was performed in the L3-L4 or L4-L5 interspace before LPS. CSF was sampled through direct lumbar puncture before LPS, and through a puncture of the reservoir 1 year after LPS. All CSF samples were centrifuged at 3,000 rpm for 10 min at 4°C to remove cells and debris. Samples were aliquoted and stored in polypropylene tubes at −80°C until biochemical analyses. Levels of the CSF biomarkers, tau and phosphorylated tau  were determined using standardized, commercially available ELISA kits (Innotest hTau-Ag and Innotest Phosphotau (181P), Innogenetics, Ghent, Belgium). Levels of sAPPα and sAPPβ; Aβ38, 40, 42, and 43; and APL1β25, 27, and 28 were measured using specific ELISA kits obtained from Immuno Biological Laboratories .t-test, was considered significant.Non-parametric statistical methods were used in all analyses. The Wilcoxon signed-ranks test was used for within-group comparisons of mRS, iNPHGS, MMSE, FAB, and TMT-A scores, while the Mann-Whitney U test was used for comparisons between groups. The Spearman rank correlation coefficient (r) was used to estimate associations between variables. Statistical analyses were performed with IBM SPSS Version 18.0  for Windows, and p < 0.05, determined with a In 72% of the iNPH cases (23/32), the median mRS score improved by 1 point after LPS . AdditioComparisons of CSF measurements indicated that the levels of sAPP, sAPPα, sAPPβ, and Aβ38 were not significantly different between the AD and NC groups .However, their levels were significantly lower in patients with iNPH before LPS than in patients with AD and NCs. Aβ42 level and Aβ42/p-tau ratio were significantly lower in patients with AD than in individuals with NC and iNPH, before LPS. The CSF levels of tau and p-tau were significantly higher in patients with AD than in NCs and patients with iNPH before LPS.We found positive correlations between Aβ38 levels and APL1β25 , APL1β27 , and APL1β28  levels in iNPH before LPS . AdditioImportantly, we found that in patients with iNPH, levels of Aβ38, 40, and 42, as well as tau and p-tau were higher after LPS than before LPS ; Fig. 3.Levels of sAPPα and sAPPβ showed a positive correlation before and after shunting, with a minor shift from sAPPβ to sAPPα, although not significant. Similarly, APL1β25 and APL1β28, and Aβ38 and Aβ42 also showed a significant positive correlation, with a shift from APL1β28 to APL1β25 and from Aβ42 to Aβ38, respectively, after shunting .Consistent with previous reports, we determined that the patients with iNPH showed significantly lower concentrations of sAPPα and sAPPβ in the CSF compared to NCs, suggesting that these could be potential biomarkers for iNPH , 23, 24.In the current study, no significant difference was observed in Aβ42 level between patients with iNPH and NCs in the CSF. On the other hand, Aβ42 level was found to be significantly decreased in patients with AD. This is in partial conflict with previous studies that have frequently reported decreases in Aβ42 levels in the CSF of patients with AD as well as iNPH, compared to NCs , 24, 26.In the current study, we found that sAPP, sAPPα, sAPPβ, and APL1β25, 27, and 28 levels did not change after LPS. In contrast, levels of Aβ38, 40, and 42 significantly increased after LPS. We also speculated that the shunting would affect α- and β-secretase activities, but found no significant changes in sAPPβ, sAPPα, and sAPPα/β level after LPS, suggesting no effect of shunting on α- or β-secretase activity. Interestingly, we found no change in APL1β28 before and after LPS suggesting that Aβ42 production did not significantly change after LPS. Furthermore, the postoperative shifts from APL1β28 to APL1β25 and from Aβ42 to Aβ38 suggest that the shunting procedure caused a change in γ-secretase activity . More spIn the present study, levels of tau and p-tau significantly increased after LPS in the favorable outcome group. Consistent with our findings, increases in tau and p-tau have been reported in lumbar CSF collected after shunting , 34, 35.To our knowledge, the present study is the first to compare levels of AD-related proteins in the lumbar CSF before and after shunting in patients with iNPH. Importantly, CSF collection during operation through a ventricular puncture may cause contamination of the destructed cerebral parenchyma, thus causing errors in AD-related protein concentrations. It is better to collect lumbar CSF than ventricular CSF before and after operation, because of reduced error and stability of AD-related protein levels in the lumbar method , 16. MorOur results indirectly show a shift of Aβ42 from oligomer to monomer, and a change in γ-secretase activity due to the improvement in CSF turnover by shunting. Moreover, this procedure could induce increases in CSF levels of Aβ38 and Aβ42 and may delay intracerebral deposition of Aβ, if the drainage system from the interstitial space into the CSF is not completely damaged. It should be noted that a limitation of this study was that the number of patients with iNPH was too small for sufficiently thorough evaluation. Therefore, future studies with increased the number of enrolled subjects are required to confirm our results.


Objectives. Some animal studies showed that granulocyte-colony stimulating factor (G-CSF) provides beneficial environment for bone healing. It has been well documented that endothelial cells and osteoblasts play critical roles in multiple phases of bone healing. However, the biological effects of G-CSF on these cells remain controversial. This study aimed to investigate the influence of G-CSF at various concentrations on endothelial cells and osteoblasts. Materials and Methods. Human umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) were treated with G-CSF at 1000, 100, 10, and 0 ng/mL, respectively. The capacity of cell proliferation, migration, and tube formation of HUVECs was evaluated at 72, 8, and 6 hours after treatment, respectively. The capacity of proliferation, differentiation, and mineralization of hOBs was evaluated at 24 hours, 72 hours, and 21 days after treatment, respectively. Results. HUVECs treated with 100 and 1000 ng/mL G-CSF showed a significantly higher value comparing with controls in migration assay ; the group treated with 1000 ng/mL G-CSF showed a significantly lower value on tube formation. No significant difference was detected in groups of hOBs. Conclusions. G-CSF showed favorable effects only on the migration of HUVECs, and no direct influence was found on hOBs. Angiogenesis and osteogenesis are two interdependent activities which play crucial roles during bone development and regeneration . AngiogeDuring skeletal development and fracture repair, the coordination of multiple events, such as migration, differentiation, and activation of multiple cell types and tissues, is required. Endothelial cells and osteoblasts are two of the major cell types involved in the process of bone regeneration. The endothelial cells make up the inner surface of the microvascular or blood vessels which supply the surrounding cells with oxygen and nutrients and remove waste products. The development of a microvasculature and microcirculation is critical for the homeostasis and regeneration of living bone, without which the tissue would simply degenerate and die . Inadequ in vivo  cell proliferation assay was performed. All samples were run in triplicate in 48-well plates. Briefly, HUVECs were plated at 2 × 10out cell , 10.4 cells/well with ECM and incubated overnight at 37°C with 5% CO2. After incubation, cells were then starved for 24 hours by low serum (0.5% FBS). Then monolayers were horizontally scratched using a P100 pipette tip to create the wound. Cells were washed with phosphate-buffered saline to remove the debris. ECM alone or ECM with rhG-CSF at 10 ng/mL, 100 ng/mL, or 1000 ng/mL was added to allow for wound healing. Immediately after the change of medium, three randomly selected views along the scraped line were photographed on each well at 50x magnifications. After 8 hours of incubation, another set of images was taken using the same method.To assess the effects of rhG-CSF on the migration ability of HUVECs, wound healing assay was conducted as described by Lam et al. . All samImage analysis for signs of migration was performed using Leica Qwin Image Processing & Analysis Software V.2.6 . The increment of migration was calculated by subtracting the length of scraped line at 8 hours from that of 0 hours. A reduction in the scraped area indicates a sign of migration. in vitro tube formation on growth factor reduced Matrigel matrix . Matrigel solution was thawed overnight at 4°C, and all plasticware was precooled at −20°C. Resuspended HUVECs in ECM alone or ECM with rhG-CSF at 10 ng/mL, 100 ng/mL, or 1000 ng/mL were plated (1.5 × 104/well) on growth factor reduced Matrigel (60 μL/well) in a 96-well tissue culture-treated plate, as suggested by Arnaoutova et al.  alone, or calcification medium with rhG-CSF added at concentrations of 10 ng/mL, 100 ng/mL, or 1000 ng/mL. The medium in each group was replaced every 4 days. Cells cultured in calcification medium have been used as a basal control. After an incubation period of 21 days, the mineralized matrix was stained for calcium by Alizarin-red (ARS) staining. The cultures were fixed by covering with 4% paraformaldehyde and incubated at room temperature for 15 minutes. After rinsing with distilled water 3 times, ARS solution was added and incubated at room temperature for 20 minutes. The excess dye was removed by washing 4 times with deionized water. Then, 1 mL of water was added to each well to prevent the cells from drying, and the images were acquired. To quantify the ARS staining, 10% acetic acid was added to each well and incubated for 30 minutes. Then, the cells and acetic acid were vortexed for 30 seconds and heated to 85°C for 10 minutes. After cooling, the slurry was centrifuged at 20,000 g for 15 minutes, and the supernatant was collected. The standard/sample was added to a transparent-bottom 96-well plate, and optical density was measured at 405 nm with spectrophotometry.Mineralization of the extracellular matrix was used as a late marker ofp < 0.05.Normality and homogeneity of variance were checked. One-way ANOVA was performed to determine the levels of significant differences with Bonferroni post hoc tests (SPSS V.20). All statistical tests adopted significance level of All three treatment groups showed a slightly lower proliferation rate than the control group . No signp < 0.001 and p < 0.01, resp.).The effect of rhG-CSF on cell migration of HUVECs was determined by wound healing assay . At 8 hop < 0.01), indicating compromised ability of tube formation.The processes of angiogenesis typically consist of proliferation and alignment to form tubular structures . To testhOBs treated with 1000 ng/mL rhG-CSF showed similar proliferation rate as the control group, while the other two groups showed slightly lower rates . No signSeventy-two hours after treatment, the 10 and 100 ng/mL rhG-CSF groups showed slightly higher value of ALP when compared to the control group, while the value in the group of 1000 ng/mL rhG-CSF group was lower . No statA quantitative ARS method was performed to detect calcium compounds deposited in the extracellular matrix as a result of mineralization. After 21-day culture in the presence of osteogenic supplements, no obvious difference of staining was observed between groups. The rhG-CSF groups showed slightly higher absorbance than the control group, with no statistical difference detected between rhG-CSF treated groups and the control group .G-CSF has been widely used to mobilize CD34+ HSCs and to increase circulating granulocytes in patients receiving bone marrow transplantation or chemotherapy . It is r in vitro studies have shown that G-CSF (100 ng/mL) had direct stimulatory actions on mature vascular endothelial cells, by showing positive effect on cell migration, proliferation in culture, and capillary-like tube formation on Matrigel culture in vitro [ in vitro study, Tura et al. reported that G-CSF at 100 ng/mL exhibited an inhibitory effect on tube formation in HVUECs [μg/kg per day, for 7 days) [Early in the 1990s,in vitro . A recenin vitro . Besidesin vitro . An animin vitro . These f 7 days) .To clarify the effect of different dosages of G-CSF on endothelial cells and osteoblasts, the present study evaluated the influence of G-CSF on HUVECs and hOBs of three concentrations. in vitro [The angiogenic process of endothelial cells involves cell proliferation, migration, alignment, and tube formation . In earlin vitro . Howeverin vitro , 21. In in vitro . As it iin vitro .Based on the differentiation of endothelial cells, tube formation assay replicates many steps in angiogenic process, including cell adhesion, migration, protease activity, alignment, and tube formation . It has μg/kg per day, for 7 days) induced osteoblasts apoptosis and inhibited osteoblasts differentiation in a mouse model [μg/rabbit) mobilized more osteoblasts into the defect area and resulted in enhanced osteogenesis [The role of osteoblast in bone tissue regeneration is characterized by three sequential stages: proliferation, differentiation, and mineralization of the extracellular matrix, which indicates the endpoint of osteoblast phenotypic expression . To evalse model . Howeverogenesis . Bone reIn conclusion, these findings indicated that when applied at 100 ng/mL in HUVECs, G-CSF significantly stimulated the cell migration, which was a crucial process during angiogenesis. However, G-CSF showed little direct effect on hOBs regardless of the concentration. Further investigation is needed to evaluate a detailed mechanism, such as how high concentration of G-CSF inhibits the tube formation of HUVECs and how hOBs is stimulated by G-CSF.