The aim of this analysis was, however, to identify the influencing factors on high catecholamine levels Various potential risk factors that could impact the humoral stress response before induction of anaesthesia were recorded in 84 males undergoing coronary aortic bypass surgery, and were entered into a stepwise linear regression analysis. The plasma level of norepinephrine measured immediately after radial artery canulation was chosen as a surrogate marker for the humoral stress response, and it was used as the dependent variable in the regression model. Accordingly, the mean arterial blood pressure, heart rate and the calculated pressure-rate product were taken as parameters of the hemodynamic situation.-1) on the morning of surgery was the only significant predictor (p = 0.004) of the high variation in preoperative norepinephrine plasma levels. This intervention decreased norepinephrine levels by more than 40% compared to no clonidine administration, from 1.26 to 0.75 nmol·l-1. There was no evidence for dose-responsiveness of clonidine. All other potential predictors were removed from the model as insignificant (p > 0.05). The use of beta-blocker, ace-inhibitors, ejection fraction, and body mass index were significant determinants for the hemodynamic situation  of the patient during the pre-induction period.Stepwise regression analysis revealed that the oral administration of low-dose clonidine (mean dose 1.75 μg·kgThe oral administration of clonidine is the only significant predictor for the observed variation of norepinephrine levels during the preoperative period. Lack of significant dose responsiveness suggests that even a low dose of the drug can attenuate the preoperative stress response and thus is recommended in cardiovascular high risk patients. Even ve drugs ,6-19.However, little attention has been paid to the stress response of cardiac high risk patients when entering the operating area, during initiation of routine monitoring, and finally during awake venous and arterial canulation. Especially the latter procedure can cause significant discomfort for patients even when performed under local anaesthesia . In seveAfter Ethics Committee approval was obtained, patients gave their written and informed consent. Eighty-four consecutive male patients undergoing CABG-surgery were enrolled into this observational study. The only exclusion criterion was emergency operation.-1 and the same-day coefficient of variation for norepinephrine measurements determined by repeated measures of a standardized probe was 3%.Due to the observational character of the study, drugs administered preoperatively (including clonidine) were given at the discretion of the anaesthetist performing the preoperative examination. Each patient received an oral premedication with clorazepate 20 mg in the evening before surgery and in the morning of surgery. In about half of the patients, benzodiazepine premedication was combined with clonidine 75–300 μg. Patients were maintained on their regular cardiac and antihypertensive medication up to the day of surgery but all inhibitors of platelet aggregation were discontinued 3–7 day preoperatively. After arrival at the operating theatre an i.v. line was initiated and 500 ml hydroxyethyl-starch  was infused. A 12-lead channel ECG with an automatic ST-segment analysis, oxygen saturation and invasive blood pressure monitoring were connected to the patients. A radial artery catheter was then inserted after local anaesthesia with 1 ml mepivacaine 1%. Heart rate (HR) and mean arterial blood pressure (MAP) were recorded every 15 seconds online using a Laptop computer connected to a Solar 9500 monitor . HR and MAP were multiplied to receive the pressure-rate product (PRP). These variables were used as a measure of the hemodynamic stress response. To determine humoral stress response, an arterial blood sample for the measurement of norepinephrine plasma level was taken immediately after placing the radial artery catheter (this measurement was the main outcome of the study) and 5, 15, and 60 minutes after endotracheal intubation was performed  and sufentanil (10 μg.kg-1.h-1). After loss of consciousness propofol was reduced to 3 mg·kg-1.h-1 and sufentanil to 1.5 μg·kg-1.h-1. Endotracheal intubation was performed after administration of pancuronium bromide 0.1 mg·kg-1.General anaesthesia was standardized. After administration of midazolam 0.05 mg·kgTo allow comprehensive analysis of potential factors associated with a reduced stress response, the following data were recorded prospectively:• Age• Bodyweight• Height• Body mass index (BMI)• Clorazepate dose per kilogram bodyweight• Clonidine (yes – no)• Clonidine per kilogram bodyweight• Time from morning premedication until observational period• Inhibitors of the angiotensine converting enzyme system (ace-inhibitors)• Beta-blocking drugs• Calcium antagonists• Angiotensin-2 receptor inhibitors• Left ventricular ejection fraction (EF)• Number of affected vessels2 of 0.3 and higher, attributed to 14 independent variables using an F-test with a significance level of 0.05. All potential relevant factors were subjected to a stepwise linear regression analysis using a backward technique. In each step the least significant factor was eliminated when F was lower than 3.96. The quality of the regression model was judged using the Durbin-Watson statistic , and by checking if the standardized residuals are normally distributed. All calculations were performed using SPSS 11.0 for Windows. All continuous data are presented as mean and standard deviation when normally distributed and as median (25th–75th percentile) when normal distribution had to be rejected using the Kolmogorov-Smirnov-test.A power analysis had revealed that 80 patients provide a power of more than 95% to detect an R-1) on the morning of surgery was the only significant predictor (p = 0.004) of the high variation in preoperative norepinephrine plasma levels. This intervention decreased norepinephrine levels by 40% compared to no clonidine administration (from 1.26 to 0.75 nmol·l-1). In this analysis, the dichotomous variable (clonidine administration: yes-no) was a better predictor for the norepinephrine levels than variables including the clonidine dose (absolute dose or dose per body weight), indicating that our data provide no evidence for a strong dose-responsiveness of clonidine in this context.Stepwise regression analysis revealed that the single administration of low-dose clonidine (mean dose 1.75 μg·kgAll other of the investigated factors (see methods) were removed from the regression model as not significant. The two factors that were eliminated with a p < 0.1 during the last but one and during the final step were body mass index (removed in step 11 with a p = 0.064) and age (removed in step 12 with a p = 0.076). Both factors were associated with increased norepinephrine levels.The overall quality of the regression model was excellent. The Durbin-Watson coefficient was 2.04 (very near to the optimum of 2.0) and the standardized residuals were normally distributed.For mean arterial blood pressure, heart rate and the calculated pressure rate product, however, preoperative clonidine administration was not an influencing factor.For the mean arterial pressure (MAP), a higher ejection fraction (EF) was a statistically significant predictor (p = 0.024). Each 10% increase of EF was associated with a 2.7 mmHg higher MAP. Administration of an ace-inhibitor was the second predictor in the final model of MAP (p = 0.03). These patients had a 7.5 mmHg lower MAP than patients without ace-inhibitors.-2 (p = 0.001).For heart rate (HR) there were three significant predictors that remained in the model. Administration of beta-blockers and ace-inhibitors were both associated with a decreased HR (p = 0.004). Each of them decreased HR between 6–7 beats per minute (bpm). Additionally, a higher BMI was associated with a 1.3 bpm higher HR per kg·mSince the PRP is the product of HR and MAP, it is not surprising that similar variables contributed to its prediction. These were the administration of beta-blockers (p = 0.017) and ace-inhibitors (p = 0.004), each of them reducing the PRP, whereas the EF was associated with an increase in PRP (p = 0.014).No patient had signs of cardiac ischemia on arrival at the operating theatre until induction of anaesthesia (defined as ST-T change > 0.1 mV in any ECG lead). There were no major adverse events during the entire induction period and surgery.2-adrenergic receptor agonist clonidine acts by decreasing central sympathetic nervous system activity in all hyperadrenergic situations. In addition to its sedative, anxiolytic, analgesic and antihypertensive properties [The αoperties ,22 it haoperties ,24 and toperties ,26.In many investigations attention has been drawn to the stressful stimulus of endotracheal intubation ,4,6-19, Thus, it was the major aim of this observational study to identify factors that might contribute either to increased humoral stress or that might help to attenuate this response.Our results show, that a single application of low dose oral clonidine was the only factor that was associated with significantly decreased norepinephrine levels on arrival at the pre-induction area. The question that arises from this observation is, if this is simply an association  or if clonidine premedication is the cause for lower norepinephrine levels. In our opinion the latter is the case. Firstly, there were no differences considering any other variables between those patients who had received clonidine and those who had not (see table -1) was low compared to all other trials. Data concerning the appropriate dose of clonidine to attenuate the stress response to intubation vary considerably between 0.625 and 10 μg·kg-1. For example, one trial demonstrated that clonidine 0.625 and 1.25 μg·kg-1 i.v. were sufficient to attenuate pressure response to laryngoscopy and intubation [-1 clonidine i.v. was equally effective as placebo, and only 4 and 6 μg·kg-1 significantly attenuated hemodynamic and adrenergic reactions in an equal manner. It could also be shown that 4 or 6 μg·kg-1 were necessary to reduce norepinephrine levels before induction of anaesthesia, however 2 μg·kg-1 where not sufficient in this setting [However, it is interesting to notice that the mean dose administered to our patients . Furthermore, a post-hoc comparison between the patients receiving either 75 or 150μg clonidine did not show relevant differences (p = 0.91 using the Mann-Whitney U-test).Higher age and higher body mass index showed a non-significant tendency to increase the catecholamine concentration. No other of the investigated factors  had statistically significant impact on norepinephrine levels.An explorative post-hoc analysis of the impact of clonidine premedication (none versus any dose) and clonidine dose on norepinephrine levels during the entire induction period proves the results of the main analysis. There was a pronounced reduction of norepinephrine plasma levels after induction of general anaesthesia with lower values in the clonidine-group. However, a statistically significant interaction term p = 0.012) suggests that the fall of norepinephrine levels are more marked in the untreated group and thus mainly caused by induction of general anaesthesia rather than effects of clonidine (figure 2 suggestThis observational trial demonstrates that patients undergoing coronary artery bypass graft surgery have a great variation of norepinephrine levels when entering the operating theatre. We could identify oral clonidine premedication as the only predictor for increased humoral stress response. There was no strong evidence for a dose dependency, indicating that even small doses, like 75–150 μg attenuate the humoral stress response before coronary artery bypass graft surgery. Clonidine did not have a negative impact on hemodynamic parameters.None declared.AMM processed the data and wrote the manuscript.GG conceived the study, collected the clinical data and participated in its design.US collected the clinical data.MK designed the study and collected the clinical data.HAA performed the laboratory investigations.HW participated in the conception of the study.LHJE designed the study, performed the statistical analysis and extensively revised the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


While previous authors have emphasized the importance of integrating and reinforcing evidence-based medicine (EBM) skills in residency, there are few published examples of such curricula. We designed an EBM curriculum to train family practice interns in essential EBM skills for information mastery using clinical questions generated by the family practice inpatient service. We sought to evaluate the impact of this curriculum on interns, residents, and faculty.Interns (n = 13) were asked to self-assess their level of confidence in basic EBM skills before and after their 2-week EBM rotation. Residents (n = 21) and faculty (n = 12) were asked to assess how often the answers provided by the EBM intern to the inpatient service changed medical care. In addition, residents were asked to report how often they used their EBM skills and how often EBM concepts and tools were used in teaching by senior residents and faculty. Faculty were asked if the EBM curriculum had increased their use of EBM in practice and in teaching.Interns significantly increased their confidence over the course of the rotation. Residents and faculty felt that the answers provided by the EBM intern provided useful information and led to changes in patient care. Faculty reported incorporating EBM into their teaching (92%) and practice (75%). Residents reported applying the EBM skills they learned to patient care (86%) and that these skills were reinforced in the teaching they received outside of the rotation (81%). All residents and 11 of 12 faculty felt that the EBM curriculum had improved patient care.To our knowledge, this is the first published EBM curriculum using an individual block rotation format. As such, it may provide an alternative model for teaching and incorporating EBM into a residency program. Evidence-based medicine (EBM) strives to provide a systematic approach to integrating the best research evidence with clinician expertise and patient preferences to provide better patient care . While tWhile there have been several studies of residency EBM curricula ,9-11, noThe UCSF family practice residency program is based at San Francisco General Hospital (SFGH), a large county hospital serving the urban poor. The program runs a busy family practice inpatient service (at SFGH) as well as the Family Health Center, an outpatient clinic that includes both continuity practices and acute care services. Within this setting, we formulated 3 primary goals for our EBM curriculum: (1) to teach interns basic EBM concepts and skills; (2) to disseminate and reinforce EBM skills to second and third year residents and faculty; and (3) to apply EBM to the care of patients. This study was approved by the UCSF Committee on Human Research.The EBM curriculum for UCSF family practice residents began, in its current form, summer of 2001. The core of the curriculum is a 2-week, individual EBM rotation for interns. In contrast to the usual format of multiple lectures or learning modules scattered throughout residency, the two-week individual block format provides for a concentrated time in which to learn EBM. It also allows tailoring of the rotation to fit the residents' backgrounds and interests. During each of the two weeks, residents have 3 half-day clinics, with the remainder of the time available for EBM. The EBM portion of the rotation fulfills the ACGME requirements for resident research and scholarly activity.patient/problem, intervention, comparison, and outcome). The entire process generally takes 5 to 10 minutes and includes modeling of how to formulate an appropriate 'answerable' question by senior residents and the inpatient attending faculty. The question(s) generated are then used by the librarian Co-director as material for a tutorial on developing search strategies and using high-quality web-based EBM resources. The emphasis of the tutorial is to introduce the intern to the concept of information mastery [The rotation begins with a meeting with the EBM faculty Director (DT) during which the structure and goals of the rotation are communicated, the intern's knowledge and experience related to EBM are assessed by review of his or her experience and by a pre-rotation test of EBM skills and knowledge (described below). Interns receive reference materials ,12 and a mastery , and lea mastery .During the first week, the intern completes a web-based EBM tutorial  which coDuring the initial 3 months of the curriculum, interns completed a written test of their EBM skills and knowledge before and immediately following the rotation. The tests were adapted, with permission, from a similar instrument developed by Sean Schafer, MD and Katie Ramos, PhD at the University of California Fresno Family Practice Residency Program . Scores As others have noted, progress in the use of EBM depends on the availability of information support services to resident and faculty at the point of patient care . In conjWe estimate that training 13 interns per year requires approximately 70 hours of librarian time and 200 hours of the faculty Director's time. In the event that the medical librarian Co-Director (JH) is not available, the faculty Director (DT) provides coverage for this portion of the rotation. Absence of the faculty Director for one or two days can usually be covered by schedule modifications and communication by telephone and e-mail. An extended absence of the faculty Director requires another faculty member assuming supervisory responsibility.We evaluated our EBM curriculum with respect to our 3 primary goals using pre- and post-rotation questionnaires (completed by each EBM intern) and by a survey of family practice residents and faculty.The EBM intern questionnaires consisted of pre- and post-rotation self-assessments of confidence in EBM knowledge and skills . Self-confidence in EBM knowledge and skills was assessed by asking the intern to rate his or her level of comfort from 1 = very uncomfortable to 5 = very comfortable for (1) MEDLINE searching to answer a clinical question; (2) use of Web-based EBM resources to answer a clinical question; and (3) use of EBM principles to critically evaluate articles. At the end of the rotation interns were asked to identify the most useful and least useful aspects of the rotation and what could make the rotation better. In addition, residents completed and returned to the Residency Coordinator a standard evaluation of the rotation.We evaluated the dissemination and reinforcement of EBM skills and knowledge to residents and faculty  and the application of EBM to patient care  by surveying residents  and faculty. These surveys were distributed and collected by a program assistant. Surveys were labeled with a code number for each resident and faculty and results were reported in aggregate to provide anonymity. The resident survey asked "How frequently have you continued to apply EBM concepts and tools from the rotation to answer clinical questions?" and "How frequently have EBM concepts and tools been reinforced via teaching by faculty or senior residents?" For both questions, response options were 1 = never, 2 = seldom (less than once per month on average); 3 = occasionally (1 to 3 times per month on average); 4 = often (1 or 2 times a week on average); and 5 = frequently (3 or more times per week on average). For faculty and residents who had had a clinical question answered by the EBM intern on the inpatient service were asked. "How often did the EBM answer provide useful information?" and "How often did the answer change your management of a patient?" For both questions, response options were 1 = less than 25% of the time; 2 = 25% to 75% of the time; and 3 = more than 75% of the time. Finally, all faculty and residents were asked if they believed that "the presence of the EBM rotation improves the quality of patient care within the family practice residency program."During the period from July 1, 2001 to September 30, 2003, 30 EBM interns presented 30 journal clubs and generated 74 CATs in response to questions from the inpatient service. Journal club presentations and CATS are electronically archived and make accessible via the residency website . InternsSurveys to evaluate goals 2 and 3 were completed by 21 of 25 residents (response rate of 84%) and 12 of 13 faculty (response rate or 92%). As seen in Table As shown in Table While the above findings support the acceptance and perceived utility of our EBM program, they do not provide a formal measure of its effectiveness. Changes in resident knowledge, skills and confident were measured over a short period of time. The frequency of reinforcement of EBM and the impact of EBM on clinical care was by resident and faculty report and may have been biased. No attempt was made to observe changes in physician behaviors or patient outcomes. There were no measures of EBM use prior to the introduction of the EBM rotation and no comparison group was available. It is therefore not possible to objectively determine to what degree current levels of awareness and utilization of EBM are the result of the rotation. It is also not possible to separate out the effects of the 2-week EBM rotation from the adjunct changes of establishing a medical information website or promoting the use of PDAs, except to the extent that the questions asked specifically about the 2-week EBM rotation.Our block EBM rotation differs substantially from the approaches reported in previous studies ,11,16 inInterns reported greater confidence in their search skills after the two week rotation. Two randomized controlled trials and a controlled before-after study have demonstrated benefits of training in electronic search skills -19. InteOne study that evaluated the impact of a 1-month pilot program to use EBM methods on an inpatient service, reported results roughly similar to ours . Our curPrevious studies have found only small increases in residents' knowledge and skills from journal clubs alone, leading to the suggestion that journal clubs should be used as a component of EBM training rather then being 'stand alone' activities -8. WhileOther investigators have reported educational interventions aimed at improving faculty knowledge and skills in medical informatics and EBM. One study reported increases in faculty self-rating of EBM skills following an intervention consisting of 2 half-day workshops and substantial amount of individual mentoring . We founOur EBM curriculum, based on an individual 2-week EBM rotation in the first year, appears to be successful in increasing resident's EBM skills and confidence. In addition, resident and faculty both perceive EBM as being incorporated and reinforced beyond the rotation, and that the presence of EBM in the residency improves the quality of patient care. We hope that our experience provides a useful model for teaching and integrating EBM into a busy, resource-limited, family practice residency.The authors declare that they have no competing interests.All authors participated in the development of the curriculum. DT conceived of and conducted the evaluation. JH and PSS reviewed the survey instrument. JH and PSS reviewed and suggested changes to the paper. JH drafted the subsection describing the teaching the use of electronic. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


A new study adds to the mounting evidence implicating T cells as an important component of the inflammation in chronic obstructive pulmonary disease Chronic obstructive pulmonary disease (COPD) is a global epidemic of major proportions that is predicted to become the third most common cause of death and fifth most frequent cause of chronic disability by 2020. In developed countries it is mainly caused by cigarette smoking, but the reasons why only a proportion 10%–20%) of smokers develop progressive airflow limitation is currently unknown. The disease is characterized by a chronic inflammatory process predominantly in the small airways and lung parenchyma, with increased numbers of macrophages, neutrophils, and T lymphocytes [0%–20% of3 of lung and the extent of emphysema [+ (T helper) and CD8+ (suppressor/cytotoxic) T cells were increased in the airways and lung parenchyma of patients with COPD, with a predominance of CD8+ cells [+ cells, which are predominantly of the T helper 2 (Th2) pattern, with increased expression of interleukin (IL)-4, IL-5, and IL-13 (see Glossary), and which are associated with an increased number of eosinophils. In smokers who develop COPD there appears to be activation of adaptive immunity, with the infiltration of CD8+ and CD4+ cells in the alveolar walls and small airways and—in patients with the most severe disease—the presence of lymphoid follicles that contain a core of B lymphocytes surrounded by T cells [+ and, to an even greater extent, CD8+ cells.T lymphocytes were first reported to be increased in patients with COPD by Finkelstein and colleagues, who showed a correlation between the number of T lymphocytes/mm8+ cells ,5. This PLoS Medicine takes the story forward [+ and CD8+ cells appear to be fully activated, as they would be after being presented with antigens, and they show predominantly a T helper 1 (Th1)/cytotoxic T 1 (Tc1) pattern, with increased expression of interferon-γ (IFN-γ) and Th1 chemokines. This is consistent with the recent demonstration of increased expression of IL-12 in bronchial biopsies of patients with COPD and activation of the transcription factor STAT-4 in T cells, subsequent STAT-4 nuclear translocation, and IFN-γ gene induction, and thus a Th1 commitment in the T cells [The study by Grumelli et al. (2004) published in this issue of  forward . The CD4 T cells .+ cells [As well as producing the cytokines IL-2 and IFN-γ, Th1 and Tc1 cells also express the chemokine receptor CXCR3 and the ligands that activate this receptor, IFN-γ inducible protein 10 (IP-10, CXCL10), monokine induced by IFN-γ (CXCL9), and IFN-inducible T cell α chemoattractant (CXCL11). There is an increase in the expression of IP-10 in the airways of patients with COPD and an increase in the number of CXCR3+ cells . CXCR3 i+ cells and the severity of emphysema [It is likely that Th1 cells are the major source of IFN-γ in the lungs of patients with COPD and therefore drive and maintain the T cell response and promote an “immune inflammation” with neutrophils and macrophages. However, it is the role of Tc1 cells that is of particular interest, as these cells are cytotoxic to epithelial cells through the release of granzymes and perforins, which induce apoptosis. Increased concentrations of perforins have recently been reported in the sputum of patients with COPD . In suppmphysema .The T cell inflammatory response appears in mild COPD but increases markedly with disease severity. It is possible that the initial immune response becomes self-perpetuating because of endogenous autoantigens resulting from inflammatory and oxidative lung injury. There are also antigens in tobacco, but the inflammatory response appears to become independent of smoking status, and there is intense inflammation even in patients who stopped smoking many years previously , as seenCOPD is characterized by destruction of the lung parenchyma and loss of elastin due to elastolytic enzymes, such as neutrophil elastase and certain matrix metalloproteinases (MMPs). The predominant MMP in COPD appears to be MMP9, which is released in much larger amounts from alveolar macrophages of patients with COPD than from those of smokers without the disease . The stuThere are currently no treatments that reduce the relentless progression of COPD, and none that have significant anti-inflammatory effects. However the recognition that an adaptive immune T cell response, most likely driven by antigens, may play an important pathophysiological role in the pathogenesis of COPD has important therapeutic implications. It is possible that T cell inhibitory strategies, such as the use of immunosuppressants, might be effective, although side effects may be a problem, and there is particular concern about increasing the risk of bacterial infection. Another approach might be to block the trafficking of Th1 and Tc1 cells to the lungs by blocking CXCR3, and there is now a search for small-molecule inhibitors of these receptors. Inhibition of IFN-γ signaling might be another approach.The mounting evidence implicating T cells, and thus an adaptive immune response, as an important component of the inflammation in COPD is overwhelming. A better understanding of the immune mechanisms involved in COPD is important, since it might lead us to new and more effective therapeutic approaches to this important disease.+ (helper) T cell:CD4 T lymphocyte that enhances the inflammatory response+ (cytotoxic/suppressor) T cell:CD8 T lymphocyte that suppresses the inflammatory responseCXCR3: Chemokine receptor that is selectively activated by IP-10, monokine induced by IFN-γ, and IFN-inducible T cell chemoattractantCytotoxic (Tc1) cell: T cell that is characterized by secretion of INF-γGranzyme: Enzyme released by cytotoxic T cellsInterferon-γ inducible protein 10 (IP-10, CXCL10): Chemokine of 10 kDa that selectively activates CXCR3Interferon-inducible T cell γ chemoattractant (I-TAC, CXCL11): Chemokine that selectively activates CXCR3Interferon-γ (IFN-γ): Protein secreted by Th1 and Tc1 cellsInterleukin-4 (IL-4): Protein secreted by Th2 cells that is important in increasing IgE secretionInterleukin-5 (IL-5): Protein secreted by Th2 cells that is important for eosinophiliaInterleukin-12 (IL-12): Protein secreted by antigen-presenting cells that promotes differentiation of Th1 cellsInterleukin-13 (IL-13): Protein secreted by Th2 cells that is important for IgE secretionMatrix metalloproteinase (MMP): Proteolytic enzyme that degrades connective tissueMMP9, MMP12: MMPs that destroy elastin fibersMonokine induced by interferon-γ (MIG, CXCL9): Chemokine that selectively activates CXCR3Neutrophil elastase: Enzyme released from neutrophils that destroys elastin fibersPerforin: Protein released by cytotoxic T cells that induces apoptosisSTAT-4: Transcription factor specifically activated by IL-1T helper 1 (Th1) cell: T lymphocyte that is characterized by secretion of INF-γT helper (Th2) cell: T lymphocyte that is characterized by increased secretion of the cytokines IL-4, IL-5, and IL-13; characteristically increased in allergic inflammation


Chronic food restriction augments the rewarding effect of centrally administered psychostimulant drugs and this effect may involve a previously documented upregulation of D-1 dopamine receptor-mediated MAP kinase signaling in nucleus accumbens (NAc) and caudate-putamen (CPu). Psychostimulants are known to induce striatal glutamate release, and group I metabotropic glutamate receptors (mGluR) have been implicated in the cellular and behavioral responses to amphetamine. The purpose of the present study was to evaluate whether chronic food restriction increases striatal MAP kinase signaling in response to the group I mGluR agonist, DHPG.Western immunoblotting was used to demonstrate that intracerebroventricular (i.c.v.) injection of DHPG (500 nmol) produces greater activation of ERK1/2 and CREB in CPu and NAc of food-restricted as compared to ad libitum fed rats. Fos-immunostaining induced by DHPG was also stronger in CPu and NAc core of food-restricted relative to ad libitum fed rats. However, i.c.v. injection of saline-vehicle produced greater activation of ERK1/2 and CREB in CPu and NAc of food-restricted relative to ad libitum fed rats, and this difference was not seen when subjects received no i.c.v. injection prior to sacrifice. In addition, although DHPG activated Akt, there was no difference in Akt activation between feeding groups. To probe whether the augmented ERK1/2 and CREB activation in vehicle-injected food-restricted rats are mediated by one or more GluR types, effects of an NMDA antagonist (MK-801, 100 nmol), AMPA antagonist (DNQX, 10 nmol), and group I mGluR antagonist (AIDA, 100 nmol) were compared to saline-vehicle. Antagonist injections did not diminish activation of ERK1/2 or CREB.These results indicate that a group I mGluR agonist induces phosphorylation of Akt, ERK1/2 and CREB in both CPu and NAc. However, group I mGluR-mediated signaling may not be upregulated in food-restricted rats. Rather, a physiological response to "i.c.v. injection stress" is augmented by food restriction and appears to summate with effects of the group I mGluR agonist in activating ERK1/2 and CREB. While the augmented cellular response of food-restricted rats to i.c.v. injection treatment represents additional evidence of enhanced CNS responsiveness in these subjects, the functional significance and underlying mechanism(s) of this effect remain to be elucidated. Correspc-fos ,4. Changc-fos -7, (ii) tagonist , and  are of particular interest. Group I mGluRs are densely expressed in striatal medium spiny neurons  and acticascades . The grocascades , as do ccascades ,13. DHPGcascades . It is tcascades  and stimcascades  while becascades . A secon1,18 = 10.8, p < .005; NAc: F1,18 = 8.2, p < .01) and by DHPG injection . However, there was no interaction between feeding condition and drug treatment in either brain region . This analysis supports the impression .In both the caudate-putamen and nucleus accumbens ERK1/2 phosphorylation was increased by food restriction  or NAc . Food-restricted rats have elevated striatal BDNF levels which appear to be involved in an enhanced neuroprotective response to diverse insults . BDNF acThe present results indicate that a group I mGluR agonist activates Akt, ERK1/2 and CREB in both the CPu and NAc. Further, activation of ERK1/2, CREB, and c-Fos are stronger in food-restricted than in ad libitum fed rats. The augmented response is not attributed to increased Group I mGluR function but, instead, to an augmented response of food-restricted rats to the i.c.v. injection procedure. Some evidence casting doubt on attribution of the latter response to GluRs was obtained. It will be of interest to evaluate whether striatal MAPK signaling in food-restricted rats is generally augmented in response to stressors or whether this response is peculiar to i.c.v. infusion. In addition, it will be of interest to evaluate whether this physiological response is mediated by BDNF and its TrkB receptor.All experimental procedures were approved by the New York University School of Medicine Institutional Animal Care and Use Committee and were performed in accordance with the "Principles of Laboratory Animal Care" (NIH publication number 85-23, revised 1996).Subjects were male Sprague-Dawley rats (375–425 g) housed individually in plastic cages with free access to food and water except when food restriction conditions applied. Animals were maintained on a 12-h light/dark cycle, with lights on at 07:00 h. Rats were anesthetized with ketamine  and xylazine  and stereotaxically implanted with a 26-gauge guide cannula (Plastics One, Roanoke, VA USA) in the right lateral ventricle. The cannula was permanently affixed to the skull by flowing dental acrylic around it and four surrounding mounting screws. Patency of the guide cannula was maintained with an occlusion stylet. Several days after surgery, cannula placements were confirmed by demonstration of a vigorous and short latency (i.e. <60 s) drinking response to 50 ng of angiotensin II.ad libitum access to water. Once body weight had declined by 20–25% (approximately 15 days) daily food allotments were titrated for an additional week to maintain stable body weight. During the 3 weeks required for food-restricted rats to attain and stabilize at target body weights, all rats were habituated, on five occasions, to the handling and injection procedures to be employed on the terminal day of the experiment.Seven days following surgery, half the subjects were put on a food restriction regimen whereby a single 10 g meal of Purina (Gray Summit, MO USA) rat chow was delivered at approximately 17:00 h each day. Rats continued to have In Experiment 1, six food-restricted and six ad libitum fed rats received i.c.v injections of sterile 0.9% saline (5 μl). Six food-restricted and six ad libitum fed rats received i.c.v injections of the group I metabotropic glutamate receptor agonist, DHPG . An additional five food-restricted and four ad libitum fed rats received i.c.v. injections of DHPG and brains were processed for Fos-immunostaining. In Experiment 2, six unoperated food-restricted rats and six unoperated ad libitum fed rats were prepared and habituated as above and received no unusual handling or drug injection on the terminal day of the experiment. In the second part of Experiment 2, twenty four food-restricted rats with lateral ventricular cannulas were prepared and habituated as in Experiment 1. On the terminal day of the experiment, six were injected i.c.v. with saline vehicle (5.0 μl), six were injected with the AMPA glutamate receptor antagonist, DNQX , six were injected with the NMDA glutamate receptor antagonist, MK-801 , and six were injected with the group I mGluR antagonist, AIDA . Antagonist doses were chosen on the basis of prior findings e.g. ,26,28] a,28 a26,22 followed by decapitation. The twelve unoperated/uninjected rats were simply removed from home cages and exposed to CO2 followed by decapitation. Brains were rapidly removed and immediately frozen in powdered dry ice. Five hundred-micrometer sections were cut using an IEC Minotome cryostat, and CPu and NAc were micropunched, under an Olympus dissecting microscope, from a series of 8 consecutive frozen sections. The tissue was then homogenized in 10 volumes of 50 mM Tris-HCl, pH 7.5 containing 50 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 40 mM β-glycerophosphate, 50 mM NaF and 5 mM Na4P2O7, 1% Tx-100, 0.5 μM okadaic acid, 0.5% sodium deoxycholate and 0.1% SDS, followed by centrifugation and protein determination using BCA reagent kit as described by the manufacturer (Pierce) Supernatants were mixed with 5 × SDS-PAGE sample buffer, boiled for 5 min, cooled on ice and kept at -80°C until future use.Prior studies have indicated that MAP kinase activation is transient and the optimal time-point to study phosphorylation of ERK 1/2 after physiological or pharmacological treatment is 15–20 min . TherefoProtein (10–30 μg per lane) was separated by electrophoresis on precast 10% polyacrylamide gels (Cambrex, East Rutherford, NJ, USA). Precision Plus protein standard molecular weight markers (Bio-Rad, Hercules, CA, USA) were also loaded to assure complete electrophoretic transfer and to estimate the size of bands of interest. The gels were transferred to nitrocellulose membrane (Osmonics) for 2 h, with a constant voltage of 100 volts. Membranes were blocked for 1 hr at room temperature with blocking buffer, 5% non fat dry milk in 50 mM Tris-HCl, pH 7.5 containing 150 mM NaCl and 0.1% Tween 20 (TBS-T), then probed overnight at 4°C using primary monoclonal antibodies for phospho-(Thr202/Tyr204)-p44/42 ERK1/2 , or polyclonal antibodies for phospho-Akt (Ser 473) , and phospho (Ser 133) CREB . Total levels of ERK1/2, Akt and CREB were detected on the same blots using anti-rabbit p42/44 ERK1/2 antibody 1:2000, , anti-rabbit total Akt , or anti-rabbit CREB antibody . After detection of phosphorylated ERK1/2, phospho-Akt and CREB blots were stripped with 25 mM Glycine, pH 2.0 containing 1% SDS for 30 min at room temperature, washed six times in TBS-T buffer, blocked in blocking buffer for 1 h and then incubated overnight at 4°C in total ERK1/2, total Akt or CREB antibody. After probing with primary antibodies and washing with TBS-T buffer (3 × 5 min), membranes were incubated with 1:2000 dilution horseradish peroxidase conjugated anti-mouse or 1: 2000 dilution anti-rabbit IgG . Proteins were visualized using a chemiluminescense ECL kit (Pierce). Densitometric analysis of the bands was performed using NIH image software. Phospho-p42/44 MAPK, phospho Akt and phospho CREB values were normalized to total p42/44 MAPK, Akt and CREB values respectively.Ninety min after DHPG injection rats were anesthetized with sodium pentobarbital (50.0 mg/kg,i.p.) and transcardially perfused with isotonic phosphate buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Brains were then removed and maintained in 20% sucrose at 4°C for 48 h. Forty μm sections were cut on an IEC Minotome cryostat and collected in a cryoprotective solution. Fos immunostaining was carried out using a rabbit polyclonal c-Fos antiserum  and the avidin-biotin peroxidase complex .Sections were washed in 1% sodium borohydride followed by PBS and incubated for 2 hrs in 4% normal goat serum plus 1% BSA in PBS containing 0.2% Triton X-100 (Sigma-Aldrich) to block nonspecific binding. This was followed by incubation, overnight, with rabbit polyclonal c-fos antiserum (1:5000 dilution). Following several PBS washes, sections were incubated with a secondary antiserum (Vector, Burlingame, CA) for 60 min and subsequently reacted with avidin-biotin complex (ABC) (Vector). The peroxidase reaction was visualized with a chromogen solution containing 100 mM nickel sulfate, 125 mM sodium acetate, 10 mM imidazole, 0.03% diaminobenzidine (DAB), and 0.01% hydrogen peroxide at pH 6.5. Sections were then mounted on chrome-alum coated slides, dehydrated, and coverslipped.Objective counting of c-Fos positive cells in CPu at coronal levels +1.7 and -0.3 mm, and NAc core and shell at coronal level +1.5 mm in relation to bregma  was accoFor each Western blot, film exposure time was set as needed to visualize distinct bands in the control samples of each experiment. Immunoblots were analyzed using NIH imaging software. For each blot, relative phospho-protein levels were calculated from the ratio of optical density of the phosphorylated protein/total protein to correct for small differences in protein loading. In addition, tubulin levels were analyzed in several representative gels and no differences were observed between treatment groups. Results were expressed by comparison to the normalized control, which in Experiment 1 was defined as the ad libitum fed group injected with vehicle. In the first part of Experiment 2, unoperated/uninjected ad libitum fed rats served as control. In the second part of Experiment 2, food-restricted rats injected with i.c.v. saline vehicle served as control. Differences between treatment conditions in Experiment 1 were analyzed by two-way analysis of variance . Differences between treatment conditions in the first and second parts of Experiment 2 were analyzed by student's t-test and one-way ANOVA, respectively.YP conducted the majority of immunoblotting experiments plus the immunohistochemistry experiment, contributed to experimental design and assisted in manuscript preparation. YB provided technical supervision of immunoblotting experiments and assisted in manuscript preparation. KC contributed to design of the study, assisted in all experiments, and wrote the final draft of the manuscript.


The role of amino acids as substrates for protein synthesis is well documented. However, a function for amino acids in modulating the signal transduction pathways that regulate mRNA translation has only recently been described. Interesting, some of the signaling pathways regulated by amino acids overlap with those classically associated with the cellular response to hormones such as insulin and insulin-like growth factors. The focus of this review is on the signaling pathways regulated by amino acids, with a particular emphasis on the branched-chain amino acid leucine, and the steps in mRNA translation controlled by the signaling pathways. The response of translation initiation to a change in amino acid and/or hormone availability can be general, i.e., affecting the translation of most if not all mRNAs, and/or specific, i.e., affecting the translation of a single class or subset of mRNAs. Both the general and specific responses can be mediated through regulation of either the met-tRNAi and/or mRNA binding steps. The specific response may also involve an additional regulatory site, i.e., the phosphorylation status of ribosomal protein rpS6, one of the proteins composing the 40S ribosomal subunit. Learning how the cell recognizes a sufficiency of amino acids is presently the objective of intense research. Present evidence, however, suggests multiple recognition sites and multiple signaling pathways. Below, we summarize our current knowledge of the signaling pathways known to respond to changes in amino acid availability. In addition, the translation initiation factors and mRNA structural elements that are involved in changes in both global and specific modulation of mRNA translation are discussed.Recent advances in biomedical research reveal a key role for amino acids as nutritional signals in the regulation of a number of cellular processes. Studies employing a variety of cell types and different tissues demonstrate that one such process affected is the regulation of gene expression through modulation of the translation of messenger RNA (mRNA). The studies show that cells recognize changes in amino acid availability and generate alterations in signal transduction pathways that are also regulated by hormones and growth factors. The cells then respond to the integrated signaling input by either upregulating or downregulating translation initiation, i.e., the process during which initiator methionyl-tRNA  and internal ribosome entry sites (IRESs). Phosphorylation of eIF2α is mediated by any of four known eIF2α kinases in mammalian cells: the mammalian ortholog of the yeast general control non-derepressing kinase-2 (mGCN2), the heme-regulated inhibitor (HRI), the protein kinase dsRNA-activated (PKR), and the PKR-like endoplasmic reticulum kinase [PERK, reviewed in ] and livresponse . Howeverresponse . A recenresponse . Thus, iHRI was first identified in rabbit reticulocytes and shown to be activated in response to hyperoxia and iron and heme deficiency . SubsequPKR is a ubiquitously expressed serine-threonine protein kinase that is activated by double-stranded RNA and is induced by interferon [reviewed in ]. PKR isi essentially halts cell growth and triggers apoptosis [In addition to changes in eIF2α phosphorylation, eIF2B activity can be altered through changes in expression of the catalytic ε-subunit. Knockdown of the catalytic ε-subunit using RNApoptosis . In contpoptosis . BecauseThe guanine nucleotide exchange activity of eIF2B may also be subject to regulation through phosphorylation of its ε-subunit. In vitro, at least four kinases phosphorylate eIF2Bε including casein kinases (CK)-I and -II, glycogen synthase kinase (GSK)-3, and DYRK. Phosphorylation of eIF2Bε by either CK-I  or CK-IIThe protein kinase mTOR is a common intermediate in both nutrient and hormone signal transduction pathways Fig. . SignaliThe activity of mTOR toward downstream targets such as 4E-BP1 and S6K1 is controlled in part through the interaction of mTOR with the regulatory proteins raptor and GβL. Evidence linking raptor with nutrient signaling through mTOR is provided by studies wherein raptor expression was downregulated using siRNA ,34. In sThe most proximal upstream protein that has been identified in the mTOR signaling pathway is the Ras homolog enriched in brain (Rheb). Rheb is a small G protein that enhances phosphorylation of S6K1, rpS6, and 4E-BP1 in an mTOR-dependent fashion when overexpressed [reviewed in ,36]. Morrsk [The mechanism(s) involved in the regulation of TSC2 GAP activity are poorly understood, but likely involve phosphorylation of the protein by multiple upstream protein kinases. For example, TSC2 has been shown to be directly phosphorylated by AKT on multiple serine and threonine residues and phosphorylation by AKT represses the inhibitory action of the TSC1/TSC2 complex on signaling through mTOR to 4E-BP1 and S6K1 -53. Likersk . These s7GTP cap, and binding domains for specific regulatory proteins (e.g. the iron-responsive element in the ferritin mRNA). Each of these elements serves to modulate the translation of a subset of mRNAs in response to various stimuli. For example, uORF elements repress the translation of most mRNAs under normal growth conditions. The translation of mRNAs bearing multiple uORFs is paradoxically enhanced in response to phosphorylation of the α-subunit of eIF2, an event that is associated with repressed translation of most mRNAs. The mechanism through which eIF2α phosphorylation enhances the translation of mRNAs containing multiple uORFs is complex and involves inhibition of the guanine nucleotide exchange activity of a second translation initiation factor, eIF2B, by phosphorylated eIF2. Examples of enhanced translation of mRNAs containing uORFs concomitant with eIF2α phosphorylation include the induction of the transcription factors ATF4 in mouse embryo fibroblasts deprived of amino acids (5) and CD36 in response to hyperglycemia (6) and the induction of the cationic amino acid transporter (CAT-1) in response to deprivation of amino acids [Changes in translation initiation can manifest as either altered translation of most or all mRNAs  or as altered translation of mRNAs encoding specific proteins. The mRNAs that encode proteins whose expression are specifically regulated through changes in mRNA translation  typically have one or more structural elements within the 5'-untranslated region (5'-UTR) that mediate translational control. Examples of such elements include multiple upstream open reading frames (uORF), internal ribosome entry sites (IRES), highly structured 5'-UTRs, terminal oligopyrimidine (TOP) tracts immediately downstream of the 5'-mno acids  or glucono acids . HoweverA second 5'-UTR structure that allows preferential translation when eIF2α is phosphorylated is an IRES. An IRES allows the ribosome to bind to an internal site in the 5'-UTR and bypass the normal route of association with the mRNA, i.e. binding to the 5'-cap structure [reviewed in ,65]. TheAlthough eIF2α phosphorylation is one mechanism for enhancing the translation of mRNAs containing an IRES element(s), it is not unique. For example, during apoptosis or infection by certain types of viruses, eIF4G is cleaved. The normal function of eIF4G is to assemble the translation initiation factors eIF4A and eIF4E and the poly(A) binding protein into a complex that mediates the binding of mRNA to the 40S ribosomal subunit. Cleavage of eIF4G during apoptosis or viral infection separates the binding domain for PABP and the mRNA cap binding protein, eIF4E, from the domains that bind eIF4A and allow ribosome attachment (referred to as the middle fragment of eIF4G or M-FAG). A recent study reported that M-FAG generated in etoposide-treated cells M-FAG promotes the preferential translation of certain, but not all, IRES-containing mRNAs including Apaf-1 and death-associated protein (DAP)-5 . MoreoveAnother structural element within the 5'-UTR of some mRNAs that is involved in selective mRNA translation is an oligopyrimidine tract, referred to as a TOP sequence, immediately downstream of the 5'-cap structure ,69. MessMost mRNAs that are efficiently translated, e.g. GAPDH and β-actin, have 5'-UTRs that are short (<200 nt), have a low content of G and C residues, and are relatively unstructured . In contSecondary structure may also function as a cis-acting regulatory element through the binding of specific trans-acting factors. A well-characterized example of such regulation is the modulation of ferritin and δ-aminolevulinate (ALA) synthase mRNA translation in response to changes in iron availability . Both thNutrients, and in particular certain amino acids, play important roles in the control of gene expression through their ability to modulate the initiation phase of mRNA translation. All essential amino acids have the potential to globally regulate mRNA translation through the eIF2α kinase mGCN2. In addition, changes in eIF2α phosphorylation can selectively modulate the translation of mRNAs encoding particular proteins if the 5'UTR of the mRNA contains uORFs and/or IRES elements. Selective control of mRNA translation can also occur through changes in signaling through mTOR. Activation of S6K1 by mTOR leads to phosphorylation of rpS6 and eIF4B which is thought to promote preferential translation of TOP mRNAs and mRNAs with highly structured 5'-UTRs, respectively. In addition, mTOR phosphorylates the eIF4E binding proteins leading to enhanced assembly of the eIF4F complex. In combination with eIF4B phosphorylation, enhanced eIF4F assembly leads to preferential translation of mRNAs with highly structured 5'-UTRs. Although other amino acids have been shown to increase signaling through mTOR, leucine is arguably the most potent of the amino acids in activating the pathway.Both authors contributed equally to the writing of this manuscript.None declared.


In this paper I will describe some of the sentinel events in Aboriginal and Torres Strait Islander health policy and strategy during 2003 and the early part of 2004. This will involve discussion on the:• National Strategic Framework in Aboriginal and Torres Strait Islander Health• National Strategic Framework for Aboriginal and Torres Strait Islander Peoples Mental Health and Social and Emotional Well Being 2004–2009• National Aboriginal and Torres Strait Islander Health Performance Framework• The roll-out of the Primary Health Care Access Program• The National Aboriginal and Torres Strait Islander Social Survey and the National Indigenous Health SurveyThese developments are consistent with a policy agenda that has evolved, in general terms, since the release of the National Aboriginal Health Strategy in 1989. However, I will also consider significant developments in the broader context for Aboriginal and Torres Strait Islander affairs, particularly the decision made in early 2004 by the Howard government to abolish the Aboriginal and Torres Strait Islander Commission (ATSIC). While the key events and developments that are reported in this paper elaborate on an agenda that has been developing for more than a decade, the decision to abolish ATSIC is likely to have a revolutionary impact on the future development of Aboriginal health strategy. Following the lead of the National Aboriginal Health Strategy (NAHS) , nationa• Framework Agreements in Aboriginal and Torres Strait Islander Health ;• Joint Planning Forums .st of July 2003, the "National Strategic Framework for Aboriginal and Torres Strait Islander Health" . Agreement has also been recently brokered that details strategies for Indigenous social and emotional well-being, which is one of the Key Result Areas for the "National Framework". Significant progress has also been made in the development of a national performance management framework for Aboriginal and Torres Strait Islander health that aligns with the "National Framework".The NAHS has been the guiding framework for action in this field since it was endorsed in 1989. Consequently, it was significant that the Australian Health Ministers Conference endorsed its successor on the 31The agenda in Aboriginal and Torres Strait Islander health strategy that was adopted by the health portfolio post 1995 had focussed on reform priorities focussed on the development of :• The capacity of primary health services to respond to Aboriginal and Torres Strait Islander health need (with a particular focus on financing and workforce);• disease and risk strategies that aimed to improve Aboriginal and Torres Strait Islander health outcomes;• the evidence base for policy and practice in this sector .With respect to primary care capacity, the roll-out of the Primary Health Care Access Program (PHCAP) continues to be one of the central planks of this agenda and I will provide an overview of recent progress. Significant progress has also been made over the last couple of years in the development of the Australian Bureau of Statistics Indigenous Survey program, which promises to enhance the information available to assist decision-making within the sector. I will provide a report on the recent developments in the roll-out of this program.While the recent developments in Aboriginal and Torres Strait Islander health policy and strategy represent an evolution of a health reform agenda that has been developing for more than a decade, the abolition of ATSIC points to a much more revolutionary change in the broader institutional and programmatic context for Aboriginal affairs. ATSIC had play a critical role in integrating Australian government programs in Indigenous affairs and providing an institutional structure that facilitated Aboriginal and Torres Strait Islander input into policy and program development. ATSIC, for instance, continued to play a role in health strategy following the transfer of specific health program responsibilities in 1995. It retained, for instance, responsibility for the delivery of environmental health service. A memorandum of understanding was developed between the Department of Health and Human Services and ATSIC to support collaboration between the sectors . ConsequThe National Aboriginal and Torres Strait Islander Health Council (NATSIHC) oversaw the development of the "National Framework". However, this process was stalled by political conflict between the key stakeholders. In December 2000, Council members representing the National Aboriginal Community Controlled Health Organisation (NACCHO) resigned in protest over a consultation draft of the 'National Framework'. NACCHO, which is the peak body representing the Aboriginal community controlled health sector, was concerned with :The way the Draft is written distances Aboriginal and Torres Strait Islander people, undermines the concept of Aboriginal community control of primary health care service delivery and diminished structures which NACCHO believe are still useful. The document's tone and language is wrong in a number of ways...Following further negotiation, NACCHO withdrew its resignation, and the NATISHC was reconstituted with revised membership and Terms of Reference . DespiteThe agreed "National Framework" consists of two documents:• The "National Strategic Framework for Aboriginal and Torres Strait Islander Health – Framework for action by Governments", which sets out a five- to ten-year reform agenda in 9 key result areas .• The "National Strategic Framework for Aboriginal and Torres Strait Islander Health – Context", which outlines the rationale for the Framework and its context .There are nine Key Result Areas set out in the Framework including :• Community controlled primary health care: building community capacity so that individuals and communities can better address and manage their own health needs.• Health system delivery framework: improving the responsiveness of the mainstream health system to Indigenous Australians and developing stronger partnerships between mainstream and Indigenous-specific services.• A competent health workforce: improving the training, supply, recruitment and retention of appropriately skilled health professionals, health service managers and policy officers in both mainstream and Indigenous-specific health services.• Emotional and social well-being: improving outcomes with respect to mental health, suicide, family violence, substance misuse and male health .• Environmental health: improving the delivery of safe housing, water, sewerage and waste disposal.• Wider strategies that impact on health: undertaking action in portfolios outside the health sector and implementing health gain strategies in the areas of education, employment transport, food and nutrition, custodial health, aged and disability services, recreation and exercise.• Data, research and evidence: aiming to improve the quality of information about how well the health sector is meeting the needs of Indigenous Australians.• Resources and finance: aiming to provide an optimal level of resources for Indigenous health commensurate with levels of need, costs of delivering services and community capacity to deliver health outcomes.• Accountability: both to communities and to governments for the delivery and effectiveness of health services.The "National Framework" was endorsed as a plan to guide all Australian governments in a coordinated, collaborative and multi-sectoral approach to achieving Aboriginal and Torres Strait Islander health gain over the next decade. It does not have a specific funding program attached to its implementation, although arguably, the roll-out of the Primary Health Care Access Program (described later) will provide additional capacity to the implementation of the "National Framework". It is also possible that the National Framework will guide the allocation of any new resources made available through the joint planning processes established under the Framework Agreements.To further these ends, it is significant that the "National Framework" was endorsed through each government's cabinet process, providing a whole-of-government commitment to its implementation in each State and Territory and at the Commonwealth level. Each jurisdiction is developing its implementation plan against which it will report annually on progress and outcomes in health portfolios and biennially on whole of government progress. The plans will identify the specific strategies and timeframes for each action area. The National Aboriginal and Torres Strait Islander Health Council will develop a plan for an independent mid-term and final evaluation.The "Social and Emotional Well Being Framework (SEWB Framework)"  is basedThe nine guiding principles for the "SEWB Framework" were been extracted from "Ways Forward" , an earlIn 2003 the Social Health Reference Group (SHRG) (established to oversee its development) conducted extensive consultations on a draft framework document. Since then the 'SEWB Framework' has endorsed by the NATSIHC and the National Mental Health Working Group in November 2003, and the Standing Committee on Aboriginal and Torres Strait Islander Health in December 2003. It was anticipated that the final 'SEWB Framework' document would be endorsed out of session by the Australian Health Ministers Advisory Council by the middle of 2004.The development of the Aboriginal and Torres Strait Islander Health Performance Framework has built on the foundations of earlier work which has established the key elements of this framework, including the:• national performance indicators in Aboriginal and Torres Strait Islander health for the Australian Health Ministers Advisory Council ;• service activity reporting for Aboriginal community controlled health services ;• Australian government health portfolio indicators ; and• the reporting against key indicators of Aboriginal and Torres Strait Islander disadvantage for the Council of Australian Governments .It is intended that the Aboriginal and Torres Strait Islander Health Performance Framework will both integrate those government performance reporting processes that have already been developed; streamline reporting processes in Indigenous health and, ensure the strategic management of policy relevant and quality information in published reports  . As star• mapping the relationship between the Key Result areas of the 'National Framework' and key domains of health performance (effectiveness, safety, responsiveness etc); and• identifying priorities for data development and improvement based on priorities of the 'National Framework'.The Primary Health Care Access Program (PHCAP) was introduced in the 1999–2000 Federal Budget to improve access to primary health care for Aboriginal and Torres Strait Islander people. PHCAP achieves this by funding increased primary health care provision, such as additional general practitioners, nurses, Aboriginal Health Workers, and through preventive and health promotional activities, such as diabetes education and management. Funds are also used for supports to service provision such as capital works and equipment. The program also aims to work with existing health services to ensure they are responsive to the needs of Aboriginal and Torres Strait Islander people.On average across Australia, PHCAP aims to bring the level of Commonwealth funding for Indigenous primary health care to three times the average MBS usage for all Australians. The key objectives of PHCAP are :• Increased availability of appropriate primary health care services where they are currently inadequate;• Local health systems that better meet the needs of Aboriginal and Torres Strait Islander people; and• Individuals and communities that are empowered to take greater responsibility for their own health.Services can be provided through a mix of arrangements, including Indigenous specific, mainstream or a combination of these. Funding can also be used to support mechanisms to assist service providers to deliver better services and enable individuals and communities to become more involved in improving their health.Up until March 2004, new and additional services have been funded in Central Australia (5 regions), Queensland (3 regions) and South Australia (4 regions) through PHCAP, as well as continued funding of services provided through the former Aboriginal Coordinated Care Trials .During 2003 a more streamlined approach to the management of PHCAP rollout was developed ,16, resuFrom these proposals, $11.8 million in funding was approved on 14 March 2004 for:• additional health professional and support staff, for example, over 20 more health professional positions in the Kimberley region of WA;• capital works for health clinic upgrades and the construction of staff housing in remote communities;• minor capital purchases such as medical equipment; and• one-off health promotional activities and health board support and training.Longer term strategies around enhancement of local service systems, to ensure they are more accessible for Aboriginal and Torres Strait Islander people, and ensuring the commitment by state/territory governments to at least maintain their funding commitments, will continue to be pursued. While the PHCAP program has provided a significant injection of resources into what is generally considered an inadequately funded primary heath care system, the amount made available through this program still does not meet its programmatic benchmarks and targets .One of the development priorities established by the heath portfolio when it took responsibility for the administration of the Aboriginal health program was to develop the evidence base to support policy reform and the development of health service capacity .The National Health Survey, undertaken by the Australian Bureau of Statistics with funding support from the Australian Department of Health and Ageing, collects information about the health status, use of health services and facilities, socio-economic status and health-related aspects of the lifestyle of Australians. The Indigenous component of this survey aims to benchmark information on a range of health issues and enable comparisons between the health characteristics of Indigenous and non-Indigenous Australians and to allow trends in the health of Indigenous Australians to be monitored over time.The Indigenous Health Survey that was run in 2001, collected data from approximately 3,500 individuals which was reportable at the national level . In 2004In parallel with the health survey program the Australian Bureau of Statistics collected data for the 2002 National Aboriginal and Torres Strait Islander Social Survey from August 2002 to April 2003 . It is pOn 20 April 2004, the Prime Minister, John Howard and the Minister for Aboriginal Affairs, Senator Amanda Vanstone, announced the intention of the Australian government to abolish the ATSIC.ATSIC had been established in 1989 when the program responsibilities of the Commonwealth Department of Aboriginal Affairs and the Aboriginal Development Corporation were merged into a structure that enable the regional allocation of resources through elected regional councils. The board of Commissioners, elected by ATSIC regional councils, was responsible for national policy development and the oversight of national programs. At the Commonwealth level, ATSIC had the lead agency responsible for the administration of a range of programs such as: community development and employment (CDEP); housing and infrastructure; cultural heritage, broadcasting services; legal services; native title, land rights and the Indigenous land fund, etc.The agency also played a critical role in co-ordinating and integrating the Aboriginal strategy across the different government program areas. ATSIC and the health portfolio had collaborated in the implementation of health infrastructure priority projects . The MemATSIC played a pivotal institutional role in the development of 'whole of government' strategies across the Australian government. It was in effect the only institutional mechanism  that enabled this. This was until the Council of Australian Governments (COAG) resolved (in 2000 and 2002) to trial, in up to 10 regions across the country, innovative administrative arrangements, developed in partnership with Indigenous communities, which aimed to provide "more flexible programs and services based on priorities agreed with communities" [From its first term in 1996, the Howard Coalition government had a conflictual relationship with the Commission. However, government confidence in the ATSIC Board deteriorated significantly under the chairmanship of Geoff Clark (first elected chairperson in 1999) to the extent that the Minister for Indigenous Affairs suspended him on the ground of misbehaviour (under section 40 of the ATSIC Act 1989) . A revieThe Federal cabinet, nevertheless, resolved to a more radical agenda than outlined in the review findings, and announced its intention to abolish ATSIC, its regional councils and the mainstreaming of the administration of all the programs for which ATSIC had been responsible. It is proposed that the elected advisory structures will be replaced by a government appointed national council. It is also proposed that Indigenous specific program dollars will be quarantined and a whole of government approach is to be developed for the delivery of Indigenous specific funding. The key elements of this reform package have been positioned within the broader context of a government commitment to reforms aimed at producing "'joined up' government and the 'seamless' delivery of programmes" . This ne• Ministerial Taskforce: which would operate as a cabinet committee, provide collaborative leadership at a government level and set strategic directions.• Secretaries group: which would support Ministerial decision-making, coordinate across government agencies, and oversight annual reporting.• National Indigenous Council: in which the Minister would appointment Indigenous leaders in health, education, employment, law and justice to provide advice and monitor performance.The proposed mechanisms and structures that would be established to deliver this 'joined-up' framework including regional partnership agreements and community shared responsibility agreements . It is also proposed to establish Indigenous co-ordination centres which will provide a single shopfront in regional and remote Australia for indigenous specific programs, lead the negotiation of regional partnerships an shared responsibility agreements but maintain line responsibility to mainstream departments.The impact of this radical reform agenda to national Aboriginal health strategy is difficult to predict. One the one hand the actual changes to the administration of the health program is insignificant . A mainstream department has administered this program since 1995. One the other hand, the implementation of this reform agenda could have potentially very significant consequences for the development of inter-sectoral strategies in Indigenous health. This depends on the success in implementing the new mechanisms, and on their effectiveness. Furthermore, the political consequences of this radical agenda on the relationship between the Australian government and Aboriginal and Torres Strait Islander peoples have yet to really become clear. Specific partnership arrangements that have been developed within the health sector are tenuous – as is evident by the politics in the development of the "National Framework". These partnerships are critical to successful implementation of strategy in Indigenous health. Consequently, deterioration in the broader relationship between Indigenous Australians and the Australian government may have significant negative consequences for the partnership processes specific to the health sector. Even though 2003 was a year in which policy and strategy in Indigenous health made no or new radical departures, it was a year of considerable tumult in relations between the Australian government and Indigenous peoples. The ramifications of this are only now beginning to unfold.ATSIC Aboriginal and Torres Strait Islander CommissionNAHS National Aboriginal Health StrategyNATSICH National Aboriginal and Torres Strait Islander Health CouncilNACCHO National Aboriginal Community Controlled Health OrganisationPHCAP Primary Health Care Access ProgramThe authors declare that they have no competing interests.


This paper reports on a study investigating the epidemiology of sporadic cryptosporidiosis in the North West of England and Wales using a Geographical Information System (GIS) to map location of residence of cases. Some 747 reports of cases were made to CDSC North West of which 649 reports were suitable for analysis. Cases were plotted on the maps of water supply zone and water quality area boundaries, provided by the two main water utilities.It was notable that there were major spatial variations in attack rate across the North West and Wales. The most dramatic example was the large difference between the Greater Manchester conurbation with many reports and Liverpool with none. Given the distribution of previously detected waterborne outbreaks in the region it was initially thought that drinking water source may be an explanation. However, an analysis of the distribution of cases in the Greater Manchester area showed no correlation with any of five water supplies that serve the conurbation.Our study has shown a dramatic variation in the incidence of laboratory confirmed cryptosporidiosis within two regions of the United Kingdom. Further analysis has not been able to prove drinking water as a likely explanation of this variation which so far remains unexplained. Cryptosporidium. Most infections in the UK are due either to C. parvum (previously known as C. parvum genotype 2 or bovine strain) or C. hominis (previously known as C. parvum genotype 1 or human strain) [Cryptosporidium spp. are protozoan parasites. In otherwise healthy individuals they tend to cause a self-limiting form of gastroenteritis which can last for several days and, sometimes, weeks. In patients with certain forms of immune deficiency, most notable the Acquired Immune Deficiency Syndrome (AIDS), the infection can cause a severe and prolonged diarrhoeal illness which, prior to the widespread use of at highly effective antiretroviral therapy was often fatal [Cryptosporidiosis is infection with species of the genus Cryptosporidiosis has now become the most commonly identified protozoal cause of gastroenteritis in the United Kingdom. Most of the epidemiological data to-date has been related to reports of outbreaks. Between the years 1983 to 1997 there were 80 outbreaks cryptosporidiosis in England and Wales affecting 4649 individuals . OutbreaThe epidemiology of sporadic (non-outbreak-related) cases is largely unknown. Of three large case-control studies reported in the past few years only one found an association with drinking mains water -9. The sThe question remains what proportion of sporadic cryptosporidiosis infections may be due to the consumption of mains drinking water. This paper reports a study using GIS to investigate the epidemiology of sporadic cryptosporidiosis and particularly address the issues of whether sporadic cases are also associated with consumption of drinking water.Table It can be seen that there is substantial spatial variation in the distribution of reported cases. In part, this variation can be explained by variation in population density. However, much of the variation is unexplained. For example, reports from Liverpool are very uncommon, whilst reports from Greater Manchester are very common.It was decided to investigate the excess case reporting from Greater Manchester in further detail to look for any possible association with water supplies. Water to the Greater Manchester area comes from five main water treatment works; Lostock (derived from Thirlmere in the Lake District and chlorinated but not filtered), Woodgate Hill , Arnfield-Godley , Buckton Castle  and Wybersley .In order to determine whether there was any relationship between attack rate and water supply, all water supply zones in the North West that received any water from one or more of these five supplies were identified Figure . For eacAs already mentioned, care should be taken in the interpretation of this analysis. It is notable that the proportion of reports that could not be allocated a correct postcode varied from one health authority to another to some extent. Also variation in attack rate between water supply zones or water quality areas was as likely to be due to differences in population size as to differences in reported cases. This was most obvious in zones/areas with relatively small population sizes where random effects could have a particularly important affect. However, there are a number of obvious features.The most obvious is the large number of cases from the Greater Manchester conurbation. This covered the Bury and Rochdale, Manchester, Salford, Stockport, West Pennine, and Wigan and Bolton Health Authorities. This excess of cases in Manchester is even more remarkable when compared with the virtual absence of cases from the Liverpool conurbation . The reason for the excess of cases in Greater Manchester is unclear. Although different reporting habits could play a part, we doubt that it could explain more than a small part of the difference. Reporting practices are not that greatly different across the North West . A sero-Cryptosporidium [An alternate explanation could be that the increase represents different water supplies. Salford, and Wigan and Bolton Health Authorities get much of their water supply from Thirlmere, a supply known to be prone to contamination by poridium , none ofporidium . NeverthA further explanation could be that the Manchester population experience other risk factors more commonly than the Liverpool population. Possible explanations include contact with contact with animals, visiting swimming pools and overseas travel. We do not have access to data to show whether or not people from Manchester are more exposed to these factors than people from Liverpool. However, Manchester is closer to a major National Park, The Peak District. If people from Manchester use their proximity to the Peak District to spend more time in the countryside and so are more likely to come into contact with farm animals, this could explain the difference. This would be an interesting hypothesis to test in a further study.In addition to Greater Manchester, there are also areas of increased reporting from North Wales and from North West Lancashire. These hotspots also remain unexplained. North West Lancashire, however, receives much of its water from Thirlmere and a water source cannot be excluded. However, many cases were reported from the Fylde peninsula which only receives a small proportion of its water from Thirlmere.et al. [The use of GIS to study the spatial distribution of cases has been useful in identifying geographical variation, but not necessarily for identifying the reasons for this variation. However, initial analysis does not support the hypothesis that differences in drinking water source is the major reason for this variation. We agree with Dangendorf et al.  that GISConsultants in Communicable Disease Control in the North West Region of England and in Wales were asked to forward details of cryptosporidium cases upon notification from the laboratory. A data collection form was completed for each case, giving the following details: name, address, postcode, date of birth, GP name, GP address and date of notification. The form was faxed or e-mailed to Communicable Disease Surveillance Centre (CDSC) – North West as soon as possible.Enhanced surveillance for the North West of England and Wales were set up separately, North West England in mid December 2000 and Wales in February 2001. Both ran until February 2002. To check for accuracy, the data were audited every 2 months. Each CCDC was sent a list of the cases they had notified to CDSC North West in the preceding 2 months. Any cases that had not been notified were forwarded to CDSC.The first stage in the geographical analysis was to check the 747 records for possible duplicate records. These were selected on the criteria of 2 individuals with identical names, dates of birth and postcodes being present in the database. Given that a postcode contains on average only 15 addresses the chances of these being legitimate is highly unlikely. Through this procedure 10 records were deleted from the database. Consequently 737 cases of cryptosporidiosis were identified during the period of enhanced surveillance.The next step was to assign a grid reference to each postcode and this was achieved using the Royal Mail Postcode Address File. Eighty eight records were excluded as either, an incomplete postcode was entered into the cryptosporidiosis database or a match could not be found in the postcode address file. Therefore, in total the database was reduced to 649 cryptosporidiosis cases. These were plotted as points against a backdrop of the water supply zones for the two main water utilities. The water supply zone and water quality area boundaries were provided by the two main water utilities (United Utilities and Welsh Water). A "water supply zone" is an area designated by a water undertaker providing water to the residences of not more than 50,000 people. In general the source is consistent across a particular zone.Using the GIS each case was also assigned its corresponding water supply zone and the number of cases in each WSZ was divided by the population, based upon data supplied by the two water utilities, to produce the attack rate maps. The analysis was undertaken in ArcGIS 8.1 using point in polygon techniques .PRH was lead on study design, did the statistical analyses and co-wrote the paper.SH co-designed the study and co-wrote the paper and undertook most of the data collection.QS co-designed the study and co-wrote the paper.SW co-designed the study and co-wrote the paper.IL undertook the geographical analyses and co-wrote the paper.KO co-designed the study, co-wrote the paper and obtained data on the water distribution.RC co-designed the study and co-wrote the paper.


ST estimates, Nei's DA distance and phylogenetic relationships.Approximately 800,000 primarily feral dogs live on the small island of Bali. To analyze the genetic diversity in this population, forty samples were collected at random from dogs in the Denpasar, Bali region and tested using 31 polymorphic microsatellites. Australian dingoes and 28 American Kennel Club breeds were compared to the Bali Street Dog (BSD) for allelic diversity, heterozygosities, F-statistics, GST of 0.088 and also with high bootstrap support to the Australian dingo and Akita in the phylogenetic analysis.The BSD proved to be the most heterogeneous, exhibiting 239 of the 366 total alleles observed across all groups and breeds and had an observed heterozygosity of 0.692. Thirteen private alleles were observed in the BSD with an additional three alleles observed only in the BSD and the Australian dingo. The BSD was related most closely to the Chow Chow with a FThis preliminary study into the diversity and relationship of the BSD to other domestic and feral dog populations shows the BSD to be highly heterogeneous and related to populations of East Asian origin. These results indicate that a viable and diverse population of dogs existed on the island of Bali prior to its geographic isolation approximately 12,000 years ago and has been little influenced by domesticated European dogs since that time. Bali, a province of the Republic of Indonesia, is an island just 87 km from north to south and 142 km from east to west and home to more than 2.9 million people [More than 90% of the residents of Bali are Hindu  with mytAs a result of overpopulation, many BSDs suffer from chronic skin diseases, internal parasites, parvo- and distemper-virus infections, and malnutrition. In an effort to reduce the dog population and to care for their medical needs, the Bali Street Dog Foundation (Yayasan Yudisthira Swarga) was founded in 1998 . They prThe BSD population is of interest for both its genetic diversity and historical relationships. It is also a population that has bred more or less randomly for thousands of years with limited genetic influx, due mainly to geographic barriers and a strict rabies control program in effect since 1926. The present study is concerned with the genetic diversity of this unique canine population and its relationship to other canine subpopulations in Asia and throughout the world. Data presented herein was derived from the DNA testing of 40 BSD samples from the Denpasar city region of Bali with 31 polymorphic microsatellite loci. The genetic diversity of the BSD was compared to that of the Australian dingo and 28 American Kennel Club (AKC) breeds.T values greater than 0.700. Average HS was 0.577 for the 30 subpopulations, with all but three loci having HS values greater than 0.500. The HS and HT values were closest for C23.123 and farthest for C22.279 and C10.404. HWE analysis revealed that all but one locus had at least one population out of equilibrium for the 30 populations sampled. C01.424, C31.646 and CPH16 had 7 populations out of HWE and AHT130 did not have any populations with p values below 0.05. The level of locus diversity attributable to subpopulation structure was evaluated with two statistics – RST and FST. Both statistics gave similar average values at 0.230 and 0.236 respectively. However, RST ranged from 0.098 to 0.486 while FST ranged from 0.179 to 0.328.Analysis of locus diversity across all 30 subpopulations revealed that the number of observed alleles ranged from six to 20 with a total of 366 for all loci Table . OverallE) and observed (HO) heterozygosities  and a 28.4% higher HE than the average AKC breed (0.573). HO was also highest in the BSD at 0.692, versus 0.426 in the Australian dingo and 0.563 in the average AKC breed.Overall, the BSD was the most genetically diverse population surveyed here, displaying 239 total alleles out of the 366 seen in all 30 subpopulations or 65.3% of the total observed alleles .IS estimates were calculated to assess the level of inbreeding for each subpopulation . Appearing at the highest frequency was allele 201 of locus CPH08 in the BSD, Australian dingo and Chow Chow at frequencies of 21.3%, 10% and 20%, respectively. Further, the BSD, Australian dingo, Chow Chow and Akita share allele 113 of locus C22.279 at frequencies of 23.8%, 67.5%, 15% and 5%, respectively. Results for locus PEZ08 demonstrate a lack of influence of European alleles where a high frequency of deviations from n+4 alleles were observed in the AKC breeds sampled yet no deviation from n+4 alleles was observed in the BSD or the Australian dingo.ST analysis was performed for each locus between the BSD and the two closest subpopulations: the Australian dingo and Chow Chow  and the Australian dingo (DA = 0.242, FST = 0.126), and least closely with the Airedale Terrier (DA = 0.454, FST = 0.258).Further distance analysis was performed for all 31 loci between all 30 subpopulations using both Nei's DA distance and pairwise Fes Table . Across Genetic distance relationships amongst the five Asian subpopulations were further explored using neighbor-joining dendograms with four non-Asian subpopulations for comparison Fig. . The BSDO was high in three feral dog subpopulations of Korea, Sakhalin and Taiwan, ranging from 0.539 in the Taiwanese to 0.717 in the Korean dogs. Given that the loci used in that study had an average allele number of 7.75, these values are similar to the HO of 0.692 observed in the BSD. Wilton et al. [O of 0.387 using microsatellites with an average allele number of 6.93, similar to the HO of 0.426 for the Australian dingoes reported herein with an average allele number of 11.8.Microsatellites have been previously used to assess genetic diversity and relationships in feral dog subpopulations ,7. Kim eIS. Even after adjusting for sample size, the BSD maintains their status as the most heterogeneous population in the study. Unlike the Australian dingo which exhibits a much lower level of diversity, the BSD findings suggest either a large founding population on Bali and/or a consistent genetic influx since the geographic isolation of ~12,000 years ago. This data also supports that the BSD appears to approximate a randomly breeding population with little selection pressure.Given the size of the island of Bali, it is extraordinary that 800,000 feral dogs can thrive and maintain such high levels of genetic diversity. Of all the subpopulations surveyed here, the BSD has the highest number of observed alleles, the highest heterozygosity, the fewest number of loci out of Hardy-Weinberg equilibrium and the lowest FE, such as the Boxer with a HE of 0.320, breeds like the Jack Russell Terrier have a high HE of 0.713 and overall their HE is higher than that of the dingo. Of first note, the selection of the dogs that contribute to a breed composition mostly occurs prior to official breed recognition primarily by genetic drift due to geographic isolation and selection for particular working or physical characteristics. After official breed recognition future breeding choices are based primarily on the availability of sires and dams that approximate the breed standard. As a result, there is a founding population that proceeds to breed mostly by convenience. Also, many breed standards have changed considerably over the years resulting in retention of a certain level of diversity within each breed, some breeds retaining much more than others. Finally, dogs comprising the comparison AKC breeds were sampled from across the United States, removing any geographical bias of the genotypes observed and slightly elevating the heterozygosities.When comparing the heterogeneity of the BSD to that observed within the AKC breeds some caveats should be addressed. One may initially expect long established, well-defined dog breeds to be much less heterogeneous than reported here. While some breeds do have a low HT is 0.577. The average values for the 11 subpopulations surveyed in the Kim et al [The average allelic diversity of the loci used in the present study was 11.8 alleles per locus, versus 7.75 in the Kim et al work . Howeverim et al  work werST and FST values were nearly identical across all subpopulations and all loci, indicating that approximately 23% of the differences observed in allele frequencies can be attributed to differences between subpopulations. FST provides an unbiased estimate of genetic drift between subpopulations by comparing alleles identical by state. RST takes advantage of the stepwise mutation model, which assumes that mutations most often occur as whole repeat unit losses or gains from the original allele size. As a result, the number of mutations provides an estimate of time from divergence. It is interesting, therefore, to compare RST and FST values by locus. Eighteen of the 31 loci studied have an RST to FST ratio greater than 1.1  over that of other study groups. Blood samples from the Australian dingo were taken from captive animals in Australia. Australian dingoes were known to be unrelated by at least one generation.Dogs from 28 American Kennel Club (AKC) breeds, equally representing the AKC group designations, were sampled with buccal swabs for a previous study . Twenty Thirty-one of the 100 microsatellites multiplexed into 12 PCRs by the Veterinary Genetics Laboratory  had beenForward primers were synthesized and dye labeled with either Fam, Hex or Vic, or Tamra or Ned (Applied Biosystems, Inc. (ABI), Foster City, CA). Reverse primers were synthesized by Operon (Alameda, CA). Primer sequences and concentrations for all markers are available as BSD and AKC breed DNA was derived from buccal cells harvested from the inside of the cheek with nylon bristle cytology brushes . Samples were collected by owners or field volunteers and submitted directly to the laboratory. DNA was extracted by heating a single swab for 10 min at 95°C in 400 μl 50 mM NaOH and then neutralized with 140 μl 1 M Tris-HCl, pH 8.0. Australian dingo DNA was extracted from blood using a standard sodium hydroxide digest.2, 200 μM of each dNTP (Hoffmann-La Roche Inc, Nutley, NJ), 0.6 unit AmpliTaq (ABI), and 2% DMSO (Sigma) then covered with 15 ul Chill-out™ Liquid Wax  to prevent evaporation. One of five thermal cycler programs was used for each primer mix ranging from 56° to 64° degrees for the annealing temperature. All PCR work was done in polycarbonate 96-well v-bottom microtiter plates  on MJ Research PTC-100 thermal cyclers . Protocols are also available in A 2 μl aliquot of extract was used in each PCR which equates to approximately 50 ng DNA. All markers and DNAs were amplified with a PCR reagent mix of 1X PCR buffer (ABI), 4.17 mM MgClOne μl aliquots of PCR product were mixed with 2 μl Fluorescent Ladder (CXR) 60–400 (Promega 400) or Internal Lane Standard 600 (Promega 600) (Promega, Madison, WI) fluorescent size standard, denatured on MJ Research PTC-100 thermal cyclers for three minutes at 95°C, then held at 5°C or placed on ice for at least one minute before gel loading. Two μl aliquots were then loaded onto a 6% denaturing polyacrylamide gel and run on an ABI 377 Automated Sequencer using ABI 10" × 7 1/8" short plates (12 cm). Gels were run at 1.10 kV (constant) voltage, 60.0 mA current, 200 W power, 51°C and 40.0 mW (constant) laser power for up to 2 hours when using Promega 400, and up to 3 hours using Promega 600. DNA fragment analysis was performed with in-house designed STRand software , which rO) were determined by direct counting for each of the 30 subpopulations. Hardy-Weinberg equilibrium (HWE) tests were performed using Genepop version 3.4 [ST estimates and subpopulation expected heterozygosities (HE) for the 30 breeds or dog groups were performed using Genepop version 3.4 [IS estimates (inbreeding coefficient of each subpopulation) for each allele following Weir and Cockerham [Allelic diversity and observed heterozygosities  and its associated parameters, HS (average heterozygosity among subpopulations) and GST (coefficient of genetic differentiation), were calculated across all loci using the public domain software, DISPAN [ST [ST [Gene diversity or total population heterozygosity (H, DISPAN . Two addSPAN [ST  and RST  [ST [ST ,28 were Allele frequencies were used to compute a matrix of genetic distances , which wBSD: Bali Street DogIS, FST RST, GST: F-statistics indicesFS, HT, HE, HO: Heterozygosity indicesHHWE: Hardy-Weinberg equilibriumA: Number of allelesNAES: American Eskimo DogAS: Australian ShepherdAT: Airedale TerrierBCO: Border CollieBLT: Bull TerrierBMD: Bernese Mountain DogBS: Brittany SpanielBT: Belgian TervurenBU: BulldogBX: BoxerBZ: BorzoiChow: Chow ChowDingo: Australian dingoDP: Doberman PinscherGH: GreyhoundMBT: Miniature Bull TerrierPG: PugRR: Rhodesian RidgebackGR: Golden RetrieverJRT: Jack Russell TerrierKE: KeeshondLR: Labrador RetrieverNE: Norwegian ElkhoundPG: PugPM: PomeranianPPN: PapillonPWC: Pembroke Welsh CorgiRR: Rhodesian RidgebackWM: WeimaranerYT: Yorkshire TerrierDNI performed the majority of data acquisition and analysis, wrote first draft of the manuscript and prepared the final draft for submission. ALS performed the majority of sample processing, assisted in data acquisition and the writing of the subsequent drafts of the manuscript as well as final draft preparation. SG sampled the dogs tested, provided background for the manuscript and assisted in the final draft preparation. ANW provided the Australian dingo data for comparison and assisted in the subsequent drafts of the manuscript. NCP directed the research and assisted in the writing of the manuscript. All authors read and approved the final manuscript."MS 2784776144318916 Supplement1.xls" and contains the primer sequences, expected size range, primer concentration used and annealing temperatures used. In a separate sheet within the same file the protocols for each PCR reaction are listed.Click here for file"MS 2784776144318916 Supplement2.xls" and contains the individual genotype data for each animal used to derive the statistics and phylogenetic results presented herein.Click here for file