When we measured the OHCCSF index that controls the effects of blood–brain barrier disruption on the level of OHC in the CSF, the 27-OHCCSF index was associated with disability – 0.181, 0.620; p = 0.002), while the 24-OHCCSF index  and 25-OHCCSF index  were associated with the number of white blood cells in the CSF of NMO patients. Our results imply that OHCs in the CNS could play a role in the pathogenesis of NMO.Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system (CNS). Hydroxycholesterols (OHCs), metabolites of CNS cholesterol, are involved in diverse cellular responses to inflammation and demyelination, and may also be involved in the pathogenesis of NMO. We aimed to develop a sensitive and reliable method for the quantitative analysis of three major OHCs , and to evaluate their concentration in the cerebrospinal fluid (CSF) and serum of patients with NMO. The levels of the three OHCs in the serum and CSF were measured using liquid chromatography-silver ion coordination ionspray tandem mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry with picolinyl ester derivatization, respectively. The linear range was 5–250 ng/mL for 24S- and 27-OHC, and 0.5–25 ng/mL for 25-OHC in serum, and was 0.1–5 ng/mL for 24S- and 27-OHC, and 0.03–1 ng/mL for 25-OHC in CSF. Precision and accuracy were 0.5%–14.7% and 92.5%–109.7%, respectively, in serum, and were 0.8%–7.7% and 94.5%–119.2%, respectively, in CSF. Extraction recovery was 82.7%–90.7% in serum and 68.4%–105.0% in CSF. When analyzed in 26 NMO patients and 23 control patients, the 25-OHC (0.54 ± 0.96 ng/mL vs. 0.09 ± 0.04 ng/mL,  Neuromyelitis optica (NMO) is considered to be the first inflammatory demyelinating disease of the central nervous system (CNS) caused by an identified autoantibody ,2. In thCholesterol is a major component of the CNS, undergoes side chain oxidizations, and is metabolized as some types of OHCs. These OHCs can modulate sex hormone receptors that prevent inflammation and/or demyelination of the brain , upregulThe rapid and accurate quantitative determination of 24S-, 25- and 27-OHC in biological fluids such as serum and cerebrospinal fluid (CSF) is highly challenging due to their low concentration. For example, in the brain, the ratio of cholesterol to OHCs varies from 500:1 to 1000:1 . MoreoveOver several years a variety of methods for the quantitative analysis of OHCs in biological fluids have been reported –22. The +CIS/MS/MS method was investigated to minimize the difficulties in derivatization and to enhance the ionization efficiency. Based on the LC-MS/MS method with Ag+CIS ionization, we performed simultaneous quantification of 24S-, 25- and 27-OHC in serum (OHCserum). However, this method was limited for the analysis of OHC in the CSF (OHCCSF) because OHCCSF is present in only a trace amount compared with OHCserum , 25-hydroxycholesterol , 27-hydroxycholesterol , 24(R/S)-hydroxycholesterol-d6 , 25-hydroxycholesterol-d6  and 27-hydroxycholesterol-d6  were obtained from Avanti Polar Lipids . 2-methyl-6-nitrobenzoic anhydride, 4-dimethylaminopyridine, picolinic acid, pyridine and trimethylamine were purchased from Sigma Aldrich Co. . Phosphoric acid and potassium hydroxide were provided by Junsei . Distilled water was purified using a Milli-Q purification system . High performance liquid chromatography (HPLC)-grade methanol, ethanol, hexane and 2-propanol were purchased from Burdick & Jackson .This study included 26 NMO patients who had positive AQP4-Ab tests, met the revised diagnostic criteria for NMO or NMO spectrum disorders ,31, visiThe control group comprised 23 age- and sex-matched patients who had neurological diseases other than inflammatory or degenerative CNS diseases , did not receive steroid treatment, and gave written consent. The basal characteristics and CSF findings of included subjects are described in CSF index, which can control the effect of the BBB disruption on the level of OHCCSF, using a previous formula with minor modifications + at m/z 507.In our preliminary studies, the measurement of OHC concentration by GC-MS using trimethylsilyl derivatization was attempted. However, the intensity of the TMS derivatized OHCs and the interference of unwanted by-products in the GC-MS system was unsatisfactory. Therefore, an initial effort was made to establish a sensitive and selective method using LC-ESI/MS/MS. Although the conventional ESI is generally used, it is not the best ionization method for OHCs because of its poor ionization efficiency. To overcome this ionization problem, a new method for the enhancement of ionization efficiency using silver ion coordination ionspray was developed. The use of silver ion coordination for the analysis of OHC in serum provided an excellent chromatographic peak shape and sufficient intensity for quantitative analysis. The silver ion is a soft Lewis acid, and carbon–carbon double bonds in unsaturated compounds are soft Lewis bases and serve as likely sites for π complex formation which is charged complex . Bayer ek et al.  suggeste+CIS conditions, this method was limited to analysis of the OHCs in serum, because the concentration of OHCs in the CSF is approximately 10-fold lower. To combat this, a highly sensitive analytical method for the analysis of OHCs in CSF was developed using LC-ESI/MS/MS with PE + (m/z 146) ion was also present. Therefore, the [M+2 picolinic acid+Na]+ (m/z 635) and [M+picolinic acid+Na]+ (m/z 512) ions were selected as a monitoring ion pair (Q1/Q3) for OHC derivatives . The linearity of all the calibration curves was higher than 0.99. Intra- and inter-day precision for the target analytes were between 0.5% and 14.7%, and the accuracy ranged from 92.5% to 119.2%. CSF), which are appropriate for such a technique. Coefficients of variation for values obtained at the four concentrations were lower than 10% (data not shown), which meant that the OHC concentration over the range analyzed did not affect recovery in CSF or serum. Mean recovery data are shown in The validation results for the quantification of three OHCs are summarized in Tables Matrix effects can significantly affect the ionization of the analyte by causing a reduction of the MS/MS response. Therefore, the ion chromatograms of the pooled serum and CSF were repeatedly examined to investigate potential interference. No interference and ion suppression were observed in the human serum and CSF samples at the retention times of the OHCs. In addition, no carryover effect was observed during multiple injections of serum and CSF samples when the instrument was run in batch mode.CSF  and 27-OHCCSF , compared with controls. However, levels of 24S-OHCCSF , 24S-OHCSerum , 25-OHCSerum , and 27-OHCSerum  did not differ significantly between the groups. The ratio of 27-OHC to 24S-OHC (27-OHCCSF/24S-OHCCSF ratio) in the CSF, which could represent either the degree of BBB disruption [p < 0.001) (Patients with NMO had significantly higher levels of 25-OHCsruption  or incre< 0.001) .CSF of OHCserum with the disability of NMO patients at their acute attack. Univariate linear regression analysis revealed that, among these OHCs, only the level of 27-OHCCSF was significantly associated with disability during acute attack (EDSS)  (We assessed the association of OHC= 0.009) .Qalb) was conducted. Multivariate regression analysis revealed that only 27-OHCCSF, but not Qalb, which represents BBB disruption, was significantly associated with the EDSS of NMO patients . The 27-OHCCSF index were associated with disability , while the 24-OHCCSF index  and 25-OHCCSF index  were associated with WBCCSF in NMO patients  massive microgliosis [CSF; 2) damaged astrocyte in NMO patients could cause altered de novo synthesis of cholesterol in the CNS [CSF in humans by increasing the diffusion of 27-OHCserum into the CNS [CSF index  . In prevogliosis  in NMO pogliosis , could l the CNS , which i the CNS . The dis the CNS , which c the CNS . HoweverSF index , suggestCSF is increased  in NMO patients  of our control patients did not have any CNS disease. Moreover, in our sub-group analysis comparing NMO patients (n = 26) with controls without CNS involvement (n = 20), the NMO patients still showed significantly higher levels of 25-OHCCSF and 27-OHCCSF than these controls without CNS involvements (data not shown). Therefore, we could conclude that the difference in the level of OHCCSF in our NMO and controls was due to the increased level of OHCCSF in NMO, rather than the decreased levels of OHCCSF in controls.The level of OHC can be altered by a number of degenerative or inflammatory disease in the CNS , 62, 64.CSF levels were increased in NMO patients, and were associated with disability during acute attack and/or number of the inflammatory cells in their CSF, no causal relationship was demonstrated since this study involved human subjects. Further studies with experimental NMO models are needed to determine the causal relationship between OHC concentration and NMO. Third, though it seems to be reasonable to consider that the increased 27-OHCCSF in our NMO patients was independent of BBB disruption, we cannot completely rule out the possibility that this disruption of BBB could have facilitate a minor degree of penetration of OHCserum into the CNS. Fourth, we did not assess the level of other inflammatory parameters, such as cytokines or CSF glial fibrillary acidic protein [CSF were increased in NMO patients compared to controls and associated with disability or CNS inflammation, for the following reasons; 1) all samples (NMO and controls) underwent same storage condition, 2) our storage condition was in accordant with the international biobank consent protocol [There are several limitations to this study. First, the number of samples was relatively small. Second, while OHC protein  nor asseprotocol , and 3) protocol .CSF were increased during their acute attack of NMO patients. Moreover, 27-OHCCSF was associated with the degree of disability, while 24-OHCCSF and 25-OHCCSF were associated with the number of inflammatory cells in these patients. These results imply that OHCs in the CNS might play a role in the pathogenesis of NMO, and may therefore be a potential treatment target.In this study, two highly sensitive and selective analytical methods for the simultaneous quantitation of 24S-, 25-, and 27-OHC levels in human serum and CSF were developed. This is the first reported study to simultaneously quantify 24S-, 25-, and 27-OHC levels in serum and CSF in the human subject by LC-MS/MS. The levels of 25- and 27-OHC


In Alzheimer’s disease (AD) patients, apopoliprotein (APOE) polymorphism is the main genetic factor associated with more aggressive clinical course. However, the interaction between cerebrospinal fluid (CSF) tau protein levels and APOE genotype has been scarcely investigated. A possible key mechanism invokes the dysfunction of synaptic plasticity. We investigated how CSF tau interacts with APOE genotype in AD patients. We firstly explored whether CSF tau levels and APOE genotype influence disease progression and long-term potentiation (LTP)-like cortical plasticity as measured by transcranial magnetic stimulation (TMS) in AD patients. Then, we incubated normal human astrocytes (NHAs) with CSF collected from sub-groups of AD patients to determine whether APOE genotype and CSF biomarkers influence astrocytes survival. LTP-like cortical plasticity differed between AD patients with apolipoprotein E4 (APOE4) and apolipoprotein E3 (APOE3) genotype. Higher CSF tau levels were associated with more impaired LTP-like cortical plasticity and faster disease progression in AD patients with APOE4 but not APOE3 genotype. Apoptotic activity was higher when cells were incubated with CSF from AD patients with APOE4 and high tau levels. CSF tau is detrimental on cortical plasticity, disease progression and astrocyte survival only when associated with APOE4 genotype. This is relevant for new therapeutic approaches targeting tau. The accumulation of Aβ begins before the onset of cognitive symptoms and it is followed by the spreading of tau pathology that has been related to the progression of cognitive decline5. The course of AD is also influenced by genetic factors such as the APOE genotype6. Having one or two copies of APOE E4 allele is associated with increased risk of developing AD7 and with a more aggressive disease course8. Although there is some evidence that APOE4 genotype may be associated with more pronounced tau pathology10, it is still poorly understood how in AD patients APOE genotype may interact with underlying tau pathology.Alzheimer’s disease (AD) is pathologically characterized by senile plaques, composed of extracellular aggregates of amyloid-β (Aβ) protein, and neurofibrillary tangles formed by hyperphosphorylated tau proteintrait d’union could be the dysfunction of cortical plasticity, which is strictly linked to AD pathology11. This is especially relevant since long-term potentiation (LTP) is considered a main neurophysiological correlate of learning and memory12. Notably, APOE regulates these mechanisms in an isoform dependent manner13 and tau pathology has a detrimental effect on synaptic plasticity14. Emerging evidence is promoting a novel framework in which astrocytes are powerful regulators of formation and refinement of synaptic plasticity15. Astrocytes are essential for the Aβ-induced tau phosphorylation in neurons16 and conversely tau protein plays a central role in inducing astrocytes apoptosis17. Astrocytes functions could critically influence brain plasticity and consequently neuronal survival in AD18.A possible 21. Indeed, an altered LTP-like cortical plasticity is associated with a more-severe cognitive decline19 and with higher CSF tau levels20. However, it is still unknown if CSF tau levels interacts with cortical plasticity and with disease severity depending on the APOE genotype. Therefore, in a first series of in vivo experiments performed in AD patients, we explored the influence of CSF tau levels on LTP-like cortical plasticity and on clinical progression, according to the APOE genotype. We hypothesized that if APOE4 genotype is associated with more pronounced CSF tau pathology10, then we would expect in these patients an LTP-like cortical plasticity impairment and a more aggressive disease course, whose might be dependent on the CSF tau levels.Accordingly, we recently identified the disruption of LTP-like cortical plasticity as a core feature of AD by using transcranial magnetic stimulation (TMS)23. Recent works showed that APOE isoforms influence the progression of AD by impairing astrocytes structure and function24 and tau protein is thought to play a central role in inducing astrocytes apoptosis25. Therefore, in a second series of in vitro experiments we incubated normal human astrocytes (NHAs)26 with CSF sampled from the same AD enrolled for the TMS experiments in order to verify the effects of CSF tau on astrocytes survival. We hypothesized that higher CSF tau levels would be more detrimental on astrocyte survival especially when associated with APOE4 genotype.We also aimed at investigating the effects of CSF tau on synaptic impairment by exploring the influence of CSF tau on astrocytes survivalp = 0.38; p-tau: 89.7 ± 47.3 vs 80.7 ± 46.0, p = 0.54).Forty-one consecutive patients were recruited at the memory clinic of the University Hospital Tor Vergata, admitted for complaining memory symptoms Fig. . APOE4 ap = 0.75). Follow-up evaluation, with MMSE scores performed at 6, 12 and 18 months, revealed that APOE4 progress faster than APOE3 AD patients as shown by ANOVA  = 3.2782, p = 0.024)  and showed a further step of decline between the 12 months and the 18 months follow-up (p < 0.00001). On the other hand, in APOE3 AD patients MMSE scores became significantly lower respect to baseline only after 18 months (p = 0.001).During follow up AD patients in both APOE groups were all treated with standard acetylcholinesterase inhibitor (AchEI) therapy (rivastigmine/donepezil). The two groups did not differ in the number of patients undergoing different AchEI treatment  Fig. . Post hop = 0.007) and APOE4 (p = 0.001) AD patients compared to healthy subjects (HS) . Baseline mean motor evoked potentials (MEPs) amplitude did not differ between APOE4, APOE3 AD patients and HS . ANOVA analysis performed on the intermittent-theta burst stimulation (iTBS) protocol showed a significant GROUP main factor  = 6.08; p = 0.01) and GROUP x TIME interaction  = 2.498; p = 0.013), but not for TIME  = 1.99; p = 0.09) main factor. Post hoc analyses with Bonferroni correction showed that AD patients with APOE3 and APOE4 genotype had less LTP in comparison to healthy controls at 10, 15, 20 and 25 minutes (p < 0.0001 for both comparisons) and that AD patients with APOE3 genotype showed a more pronounced tendency towards long term depression (LTD) in comparison to AD patients with APOE4 genotype at 10 and 25 minutes (both p < 0.05) .The TMS procedures were well tolerated in all subjects. The mean resting motor threshold (RMT) to TMS did not different between APOE3 and APOE4 AD groups, but was lower in both APOE3 (05) Fig. .p = 0.005, r = −0.61; APOE3: p = 0.55, r = −0.13)    in the APOE4 but not in the APOE3 group (APOE4: 13) Fig. . Similar29) Fig. , but not03) Fig. .Figure 3p = 0.04, r = −0.45; APOE3: p = 0.33, r = −0.22)    only in the APOE4 but not in the APOE3 group (APOE4: 22) Fig. . On the 10) Fig.  and Aβ1-21) Fig. .Figure 419. In fact, the mean individual response to the iTBS correlated with delta MMSE scores at 18 months in both APOE3 and APOE4 AD patients . However, CSF t-tau levels had a strong influence on the relationship between cortical plasticity and cognitive decline depending on the APOE genotype. In this analysis, AD patients were stratified according to CSF t-tau levels into high CSF t-tau levels and low CSF t-tau levels, using as cut-off value the median of CSF tau levels of our entire sample (680 pg/ml). We found that APOE4 patients with high CSF t-tau levels showed a response to the iTBS protocol towards LTD, while conversely, APOE4 patients with low CSF t-tau showed a tendency to form LTP , and APOE genotype, resulting in 4 groups (each one consisting of four subjects), namely APOE3 low t-tau levels (LTE3), APOE3 high t-tau levels (HTE3), APOE4 low t-tau levels (LTE4) and APOE4 high t-tau levels (HTE4). The four resulting groups were matched for sex, age, MMSE at the baseline and disease onset ; conversely, at 7 days HTE4 sample showed significantly lower viability when compared to LTE4 and control samples . Hence, the results of this experiment show that the coexistence of high CSF levels of t-tau and APOE4 genotype is able to influence astrocytes viability; conversely CSF t-tau levels do not induce any change when associated to APOE3 genotype.To assess the effect of CSF incubation on physiological status of astrocytes, we analyzed cell viability using 7-amino actinomycin D (7-AAD), a membrane impermeant dye that is generally excluded from viable cells. A general time-dependant decrease in NHAs viability was detected during long-term CSF incubation. We found no differences among all CSF treated samples at 24 hours and 5 days Fig. ; conver28. Several studies suggest that activation of caspases and cleavage of cellular proteins may contribute to astrocyte injury and damage in the AD patient brain29. Thus, to evaluate the effect of CSF incubation on apoptosis in NHAs, we employed two approaches to detect early apoptosis by measuring the activity of caspase 3/7 and annexin V/7AAD after 24 hours, 5 days and 7 days of incubation. We found that activated caspase 3/7 levels were similar among groups until day 7 , when we observed higher activated caspase level in the HTE4 respect to LTE4, APOE3 and control groups . Similar results were obtained from Annexin V assay . In fact, after 24 hours and 5 days of incubation we did not observe any variation in apoptotic levels while astrocytes treated with HTE4 for 7 days showed the highest percentage of early apoptotic cells, if compared to all other groups . No differences were observed between LTE3 and HTE3 , suggesting that t-tau levels might be the factor responsible for cell apoptosis only in NHAs treated with CSF from patients with APOE4 isoform.Cell viability loss is often linked to apoptosis and the activation of caspases, a milestone in the path of programmed cell death, leads to proteolytic cleavage of several neuronal proteins in ADy 7 Fig. , when wsay Fig. . In facTE3 Fig. , suggesWe provide novel evidence that the presence of APOE polymorphisms imply different mechanisms of CSF tau-related dysfunction in AD patients. Our work reveals that high CSF tau levels are associated with impaired cortical plasticity and disease progression only in AD patients carrying APOE4 but not APOE3 genotype. In parallel, we also found that CSF tau levels influence apoptosis in NHAs when incubated with CSF collected from AD patients with APOE4 but not APOE3 genotype. Taken together these findings reveal that CSF tau levels are linked to cortical plasticity, cognitive decline and astrocytes survival only when associated with APOE4 genotype.32.In our study, APOE3 and APOE4 AD patients did not initially differ in terms of neuropsychological symptoms and degree of functional status, thus making them indistinguishable from a clinical point of view, at least at the time of their first visit to the memory clinic. Indeed, our follow up evaluations revealed that the two groups show divergent clinical progression, since APOE4 group worsen faster than APOE3, accordingly to previous evidence20. Interestingly, APOE3 patients showed an evident tendency to form paradoxical LTD instead of the expected LTP-like cortical plasticity in comparison to APOE4 patients. This different profile of LTP-like cortical plasticity dysfunction can be explained according to the extensive experimental works in animal models of AD revealing that APOE polymorphism has a profound influence on disease-related synaptic dysfunction15. These studies showed that APOE selectively impairs glutamatergic neurotransmission and LTP by reducing NMDA and AMPA receptor functions33. In particular it was found that LTP is more impaired in APOE2 and APOE3 in comparison to APOE4 mice35. This is compatible with our findings, showing a relative less impaired LTP in APOE4 in comparison with APOE3 AD patients. It is possible that such differences could involve complex interactions with glutamatergic transmission36, although the exact mechanisms remain to be clarified in further studies.Both APOE4 and APOE3 AD patients showed, as expected, an impairment of LTP-like cortical plasticity22. However, we found a striking difference when considering the interaction between disease progression and CSF tau levels according to the APOE genotype. Only in APOE4 AD patients there is a strong association between cognitive decline, impaired cortical plasticity and CSF t-tau values, suggesting a relative susceptibility to APOE4 patients to the amount of tau-related pathological burden. On the other hand, this phenomenon is completely absent in APOE3 AD patients, which are indeed characterized by a strong correlation between LTP-like cortical plasticity dysfunction and cognitive decline, which is totally independent from CSF tau levels. Therefore, our data show a clear dependency on underlying CSF tau levels that is exclusive for APOE4 genotype. This finding is clinically relevant especially in light of recent findings showing that AD patients with very high levels of CSF tau and phosphorylated tau at threonine 181 (p-tau181) exhibit worse clinical outcomes over time, including faster progression of cognitive deficits and a higher mortality5. In addition, they are also compatible with a recent work showing that CSF tau and p-tau181 levels, but not Aβ42 levels, predict subsequent rates of whole-brain and regional atrophy in AD over the next three years37. Other recent clinical evidence also supports this result: for instance, a recent longitudinal study of CSF biomarkers found that APOE4 genotype influences dramatically markers of neuronal injury inducing a constant increase of CSF t-tau and p-tau181 levels36, and higher CSF tau levels were specifically correlated with increased tau in the temporal lobe, a region involved in memory processing38. Indeed, there is an increased risk of progression from mild cognitive impairment to AD with elevated CSF t-tau and p-tau and with the presence of the APOE4 genotype, but not with decreased CSF Aβ42 levels39. Moreover, APOE4 may accelerate onset of dementia and neuronal degeneration by differentially impairing the maintenance of synaptic strength and reducing glutamate signaling proteins, even in the absence of overexpression of APP and Aβ APOE439.In our sample, faster cognitive decline was associated with more impaired LTP-like cortical plasticity, a condition observed in both APOE3 and APOE4 group, confirming previous observations that an altered cortical plasticity could be involved in fastened clinical worsening40. In particular, the caspase-3 inhibits the production of ROS and is necessary for the efficient execution of apoptosis, while it is necessary for the effector caspase-7 cell detachment by apoptosis41. Several studies suggest that the plaques, tangles, and the activation of caspases share a common path and that induced caspase cleavage of tau plays an important role in the development of the disease neurofibrillary tangle42. Since caspase-3 activity can be also induced independently of apoptosis43 and apoptosis is not necessarily caspase-dependent44, we performed also the annexin V/7AAD assay in order to confirm the data on apoptosis deriving from caspase 3/7 activity. In fact, annexin V is a 35–36 kDa Ca2+-dependent phospholipid-binding protein with high affinity for membrane phosphatidylserine (PS) that binds to PS exposed on cell surface of apoptotic cells45.The current neurophysiological and clinical findings were also paralleled by similar interactions between CSF tau levels and APOE genotype when examining astrocytes vulnerability. Notably, the CSF samples used as incubation medium for the astrocytes cultures were collected from the same AD patients in whom we assessed cortical plasticity. We found that NHAs were less viable and more susceptible to apoptosis when incubated with CSF obtained from APOE4 patients with high levels of CSF tau but not from APOE3 ones. We also found a similar phenomenon when evaluating the activation of caspases, a milestone in the path programmed of cell death known as apoptosis, occurring in AD and leads to proteolytic cleavage of several neuronal proteins50. Moroever astrocytes apoptosis has been reported in several experimental47 and clinical neuropathological conditions including AD49 thereby reinforcing the notion that damage of astrocytes can contribute to disease progression.This is relevant since a growing body of evidence shows that alterations in astrocytic functions have potentially catastrophic consequences for neurons51. In alternative, APOE4 and its C-terminal-truncated fragments could regulate directly tau phosphorylation and the formation of neurofibrillary tangle-like inclusions in the intracellular space of neurons52. At this regard, studies investigating the relationship between apoE and the microtubule-stabilizing protein tau showed that APOE3 binds tightly to tau, whereas APOE4 does not. These data provided the basis for hypothesizing a role for APOE in microtubule stabilization. While binding of APOE3 to tau is proposed to stabilize microtubules and inhibit its hyperphosphorylation, binding deficiency of APOE4 would allow for tau self-association, hyperphosphorylation, and subsequent formation of paired helical filaments, the building blocks of neurofibrillary tangles in AD53. This experimental evidence could account for our current findings. In fact, while CSF collected from APOE3 AD patients with different tau values did not influenced astrocytes survival, the samples collected from AD APOE4 patients affected astrocytes viability and apoptosis depending on the CSF tau levels. Therefore, our data show that only APOE4 may influence astrocyte survival and functionality depending on CSF tau levels. Given the established role of astrocytes in controlling the complex synaptic machinery at the basis of the mechanisms of LTP, we propose that the interaction between altered synaptic plasticity, CSF tau levels and APOE genotype could explain why AD patients with APOE4 allele tend to have more impaired cortical plasticity and eventually a more aggressive course of disease. On the other hand, the different pathophysiological mechanisms linked to the presence of APOE3 allele remain to be elucidated.Regarding the possible mechanisms underlying the current findings we can only speculate about possible sites of interaction between CSF tau and APOE. One possibility is that CSF tau combines with the APOE receptors in the extracellular space, indirectly modulating the intracellular transduction signaling pathways that regulate tau kinases activityin vitro experiments are needed to better identify the interaction between Aβ42, t-tau and p-tau and astrocytes survival, to define the metabolic pathways involved in these mechanisms. Moreover additional work is required to better establish the pathophysiological role of CSF tau oligomers in relation to APOE polymorphism.A limitation of the current study is that we cannot exclude that other interactions could have taken place involving other molecules apart from tau in the experiments with astrocytes cultures. Therefore, further In conclusion, here we used a novel translational model to study the mechanisms of cortical synaptic plasticity impairment occurring in AD. Our findings suggest that: i) CSF tau drives cortical plasticity impairment and cognitive decline only in the presence of APOE4 genotype, while APOE3 patients seem to be characterized by other pathological mechanisms; ii) APOE4 patients CSF tau levels can influence astrocytes survival, independently from Aβ levels. Thus, our data point to the existence of independent pathophysiological pathways mediating cognitive decline in AD patients according to the APOE polymorphism. These findings could be relevant to specifically address new diagnostic and therapeutic approaches targeting tau, in order to select AD patients according to specific clinical and biological characteristics.). Patients fulfilled the clinical criteria of dementia as defined by the DSM-IV and probable or possible AD according to the criteria of the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s disease and Related Disorders Association54. Disease duration was calculated using standardized semi structured questionnaire55. Neurophysiological examinations were performed at the Santa Lucia Foundation within 30 days from CSF sampling. Patients included in this study did not receive drugs that could have modulated cerebral cortex excitability such as Acetylcholinesterase inhibitors (AchEI), L-Dopa or dopamine-agonists antidepressants or any other neuroactive drugs  in the 90 days prior to TMS evaluation. Exclusion criteria were the following: patients with isolated cognitive deficits, patients with clinically manifest acute stroke in the last 6 months showing a Hachinsky scale score >4, and a radiological evidence of ischemic lesions, Aβ1-42 CSF values >600 pg/mL. After the neurophysiological assessment patients started treatment with rivastigmine patch (n = 26) or donepezil (n = 15) and were followed longitudinally with clinical assessments and MMSE at 6, 12 and 18 months. Twenty age, sex- and education-matched healthy subjects (HS) were recruited as controls. All participants or their legal guardian provided written informed consent after receiving an extensive description of the study. The study was performed according to the Declaration of Helsinki. The ethics committee of the Santa Lucia Foundation approved this protocol (Prot. CE/AG4/PROG.392-08).Forty-one consecutive patients recruited at the memory clinic of the University Hospital Tor Vergata, admitted for complaining symptoms. After the first visit to our Centre, all patients underwent for diagnostic purposes a complete clinical investigation in a period not superior to 60 days, including medical history, neurological examination, Mini-Mental State Examination (MMSE), a complete blood screening, neuropsychological assessment, magnetic resonance or CT imaging, and lumbar puncture for CSF analysis Table . Patien56. APOE genotyping was performed using a standard PCR. Two groups of AD patients were classified using the APOE genotype. APOE4 group consisted of fifteen E3/E4 and five E4/E4 allele carriers AD patients while APOE3 group consisted of twenty-one AD patients homozygous for E3 allele . Aβ1-42 levels were determined using a sandwich ELISA (Innotest® ß- amyloid (1–42), Innogenetics, Gent, Belgium), specifically constructed to measure Aβ-amyloid containing both the first and 42nd amino-acid, as previously describedin vivo experiments we investigated the role of APOE genotype on cortical plasticity by applying theta burst stimulation (TBS) protocols29 in a large sample of AD patients. TBS, a form of repetitive TMS has been put forward as a reliable tool to examine cortical plasticity in AD patients in analogy with hippocampal plasticity assessed in animal models of AD. We verified how APOE polymorphism eventually affected LTP-like cortical plasticity by interacting with CSF levels of total tau, p-tau181 and Aβ42. Motor evoked potentials (MEPs) were recorded from the right first dorsal interosseous muscle using 9 mm diameter, Ag–AgCl surface cup electrodes. A monophasic Magstim 200 device  was used to define the motor hot spot and to assess MEPs size using standard 70 mm figure-of-eight shaped coil. The motor hot-spot was defined as the location where TMS consistently produced the largest MEP size at 120% of resting motor threshold (RMT) in the target muscle57. A second coil was connected to a biphasic Super Rapid Magstim stimulator  to deliver TBS. In the intermittent-TBS (iTBS) protocol, a 2 s train of TBS was repeated twenty times, every 10 seconds for a total of 190 seconds (six-hundred pulses)58. The intensity of the test pulse was adjusted so that it evoked a MEP of about 1 mV peak-to-peak amplitude in each individual. Twenty MEPs were collected and averaged at baseline. Then, over the same hot-spot, twenty MEPs were recorded at 1–5, 6–10, 11–15, 16–20 and 21–25 minutes after TBS and averaged.In a first series of 2 flasks containing astrocyte basal medium (ABM™) at 37 °C in 5%CO2 humidified incubator, changing the growth medium the day after and every other day thereafter. The cells were sub-cultured when they were 70–80% confluent and containing many mitotic figures. New culture flasks were prepared by adding pre-warmed medium and the flasks were returned in the incubator. In a sterile field the medium was aspirated from flask and the cells were rinsed with 5 ml of room temperature HEPES-BSS and after a brief swirling were removed and replaced with 2 ml of trypsin/EDTA solution and placed the flask in the 37 °C incubator for 3 to 4 minutes for trypsinization. After detached, the cells suspension was quickly transferred to a sterile 15 ml centrifuge tube. The cell suspension was then centrifuged at 160 × g to 200 × g for 5 minutes at 2 °C to 8 °C. The supernatant was aspirated and the cell pellet was re-suspended in 2 ml of AGM™ medium (Lonza) and the cells were seeded at density of 5,000 cells/cm2 to a tissue culture flask or dish (pre-filled with pre-warmed medium).Lonza NHAs were purchased from Euroclone  and cultured according instructions. These experiments were performed at the University of Bologna. NHAs were seeded in 75 cm2). The day after medium was changed with 500 µl of fresh medium and 100 µl of CSF from AD patients with different tau levels was added to a final 1:6 dilution38. Using the same methods, cells were also incubated in CSF derived from healthy control (HC) subjects. In accordance with the institution’s ethical standards which prevent the recruitment of healthy people for CSF collection, the control group is composed of astrocytes treated with CSF collected from patients that received lumbar puncture and did not show any feature of neurological disorder.The cells were seeded into a 24 cell plates at the recommended density  were diluted 1:1 with the reagent and incubated at room temperature in the dark for 5 min before detection with Guava EasyCyte 5 Flow Cytometer (Millipore); at least 10,000 cells/sample were analyzed.Cell viability was assessed by Guava ViaCount Reagent (Millipore) that contains 7-amino-actinomycin D (7-AAD). Briefly, NHA (105 cells/sample) were harvested following 24 h, 5 and 7 days treatment with AD patients’ CSF, and incubated in Caspase Reagent Working Solution for 1 hour at 37 °C in a CO2 incubator. Then cells were washed twice and labeled with Caspase 7-ADD working solution for 10 min at room temperature. Astrocytes were analyzed on a Guava EasyCyte 5 flow cytometer (Millipore); at least 10,000 cells/sample were analyzed.Apoptosis was also assayed by measuring caspase-3/7 activity using Guava Caspase 3/7 FAM kit (Millipore) following manufacturer’s instructions. NHA (105 cells/sample) were harvested following 24 h, 5 and 7 days treatment with AD patients’ CSF and resuspended in 100 μl of complete medium; the cells were then stained with 100 μl annexin V-PE and 7-AAD for 20 min at room temperature in the dark, following manufacturer’s instructions, and analyzed on a Guava EasyCyte 5 flow cytometer (Millipore). Three populations of cells can be distinguished by this assay: viable cells (annexin V-PE and 7-AAD negative), early apoptotic cells (annexin V-PE positive and 7-AAD negative), and late stages apoptosis or necrotic cells (annexin V-PE and 7-AAD positive).To confirm data on NHA apoptosis obtained with Caspase 3/7 assay, phycoerythrin-conjugated annexin V (annexin V-PE) and 7-AAD  method was used to determine the percentage of viable, early-apoptotic and late apoptotic/necrotic cells by flow cytometry. Astrocytes , cognitive decline  and CSF biomarkers levels were investigated using Pearson’s correlation analysis or Spearman’s Rho analysis, as appropriate. For TMS experiments, two-way repeated measure ANOVAs were performed on MEPs amplitude expressed as percentage of change in comparison to baseline for iTBS protocol with TIME  as within subjects factors and GROUP  as between subjects factor. The Greenhouse-Geisser correction was used for non-spherical data. When a significant main effect was reached, paired t-test with Bonferroni correction was employed. A p value of <0.05 was considered to be significant.The analysis was performed using SPSS for Windows version 11.0. The assumptions and calculations at the bases of the sample size were 90% power to detect, using a two-sided alpha-level of 5%, a difference between APOE groups of at least 2.2 point in MMSE at 18 months. Data were presented as mean ± standard deviation (SD) or as mean ± standard deviation (SEM) as reported. Kruskal-Wallis analysis of variance was performed for analyzing the difference among 3 or more independent groups, followed by Mann-Whitney U test with Bonferroni correction when significant, χin vitro assays, every experimental group was composed by cells treated with CSF from four different patients. In viability and apoptosis measurements performed with Guava, each reading at least 10.000 cells/sample was analyzed in triplicate. The statistical analysis was performed using GraphPad Prism. A one-way ANOVA followed by a post-hocBonferroni multiple comparison test was used for parametric data; a Kruskal-Wallis test, followed by Dunn’s multiple comparisons test was used for non-parametric data. Values of p < 0.05 were considered as being significant.For


M. tuberculosis infection and we asked how GM-CSF neutralization might affect host response, both in immunocompetent and in immunocompromised TNFα-deficient mice. GM-CSF neutralizing antibodies, at a dose effectively preventing acute lung inflammation, did not affect M. tuberculosis bacterial burden, but increased the number of granuloma in wild-type mice. We next assessed whether GM-CSF neutralization might affect the control of M. tuberculosis by isoniazid/rifampicin chemotherapy. GM-CSF neutralization compromised the bacterial control under sub-optimal isoniazid/rifampicin treatment in TNFα-deficient mice, leading to exacerbated lung inflammation with necrotic granulomatous structures and high numbers of intracellular M. tuberculosis bacilli. In vitro, GM-CSF neutralization promoted M2 anti-inflammatory phenotype in M. bovis BCG infected macrophages, with reduced mycobactericidal NO production and higher intracellular M. bovis BCG burden. Thus, GM-CSF pathway overexpression during acute M. tuberculosis infection contributes to an efficient M1 response, and interfering with GM-CSF pathway in the course of infection may impair the host inflammatory response against M. tuberculosis.Host directed immunomodulation represents potential new adjuvant therapies in infectious diseases such as tuberculosis. Major cytokines like TNFα exert a multifold role in host control of mycobacterial infections. GM-CSF and its receptor are over-expressed during acute  New approaches, such as host-directed therapies (HDT) are being developed to improve the control of Mycobacterium tuberculosis infection4. Indeed, although one-third of the global population is considered to be infected by M. tuberculosis, only 5% to 10% will develop active disease. This indicates that a healthy host immune system effectively controls the infection, although mycobacteria remain present for years in a very slow or non-replicating dormant state5. Host immune responses involved in controlling M. tuberculosis infection include innate and adaptive immune responses, with multiple key players including macrophages, T cells, and mediators such as tumor necrosis factor alpha (TNFα), IFNγ, IL-1, IL-12p40, reactive oxygen and nitrogen intermediates10. In front of multidrug resistant M. tuberculosis strains, allying host immunomodulation with chemotherapy could help to control the pathogen and limit dissemination12. TNFα is essential for granuloma formation and maintenance, and experimental host directed adjunctive therapies combining TNFα neutralization with chemotherapy reported enhanced M. tuberculosis bacterial clearance15. However, neutralizing TNF is a risky measure to take in M. tuberculosis infected individuals. Here we hypothesized that GM-CSF immunomodulation could contribute to host defense against M. tuberculosis.Tuberculosis (TB) remains a major global health problem with the emergence and spreading of new drug resistant strains, including in ‘developed’ countries16. Beside its role in homeostasis, GM-CSF is considered as a key mediator in tissue inflammation, notably in chronic obstructive pulmonary disease (COPD), allergy and asthma. Neutralization of GM-CSF or its receptors are in development for indications including cancer or severe inflammatory diseases such as rheumatoid arthritis18. However, the role of GM-CSF during bacterial infections like TB is poorly understood. GM-CSF is induced after in vitro M. tuberculosis infection of THP-1 cells via PI3-K/MEK1/p38 MAPK signaling pathway19. Additionally, GM-CSF contributes to proinflammatory macrophage M1 polarization20. Indeed, GM-CSF induces monocyte and macrophage production of IL-6, IL-8, G-CSF, M-CSF, TNFα and IL-1β, although less potently than LPS20. On the other hand, GM-CSF seemed to control TNFα and IFNγ expression, since lung cells from GM-CSF deficient mice produced less TNFα and IFNγ after exposure to M. tuberculosis culture filtrate proteins than wild-type cells21.Indeed, a new, pivotal role of GM-CSF at the T cell - myeloid cell interface has recently emerged, and targeting the crosstalk between T cells and myeloid cells through GM-CSF seems promising in immunopathology and chronic tissue inflammationM. tuberculosis infection since GM-CSF deficient mice exhibited granuloma disruption22. However, while GM-CSF is essential for maintaining the ability of pulmonary alveolar macrophages to clear surfactant lipids and proteins from the lung surface, as evidenced in GM-CSF deficient mice23, it is uncertain whether GM-CSF has a direct effect on granuloma formation. Specific GM-CSF expression either in lung epithelial cells or in the hematopoietic compartment partially restored the control of the M. tuberculosis infection by GM-CSF deficient mice24.It was proposed that GM-CSF controls granuloma formation in M. tuberculosis infection in vivo and we tested how neutralizing pharmacologically GM-CSF host response might affect lung inflammation and granuloma formation, and interfere with the action of antibiotic treatment in M. tuberculosis infection. GM-CSF neutralizing antibodies that efficiently prevented sterile lung inflammation had no significant effect on bacterial burden and host response to M. tuberculosis infection in wild-type mice, while promoting granuloma formation. We then tested GM-CSF neutralization under sub-optimal antibiotic chemotherapy in TNFα-deficient mice. We propose that GM-CSF overexpression during acute M. tuberculosis infection contributes to an efficient M1 response, and neutralizing GM-CSF might be detrimental to the host inflammatory response to M. tuberculosis infection.Here, we show that GM-CSF and its receptor are overexpressed in the lung during acute in vivo after inflammatory challenge or M. tuberculosis infection. Indeed, GM-CSF levels are increased in both bronchoalveolar lavage fluid (BAL) and lungs 24 h after LPS exposure  in the lungs 28 days post- infection . Administration of anti GM-CSF antibodies (200 µg per mouse i.p.) strongly reduced neutrophil recruitment in the bronchoalveolar lavage  or IgG2b isotype control were administered (200 µg i.p.) on day 14 and 21 after infection with M. tuberculosis H37Rv (1000 ± 200 CFU/mouse i.n.). After a transient decrease in bodyweight following anti GM-CSF MAB administration, the susceptible TNFα−/− mice showed a strong deterioration of their general status and had to be killed by day 26 ± 2, while WT mice were less affected  and rifampicin (RIF) bi-therapy sufficient to induce a limited protection in TNFα−/− mice against M. tuberculosis infection, in order to appreciate a potential adjuvant effect of the immunotherapy. A low dose of 1 mg/L of both INH and RIF (INH/RIF) given from day 14 to 35 post-infection in drinking water was totally ineffective, with a drastic body weight loss , GM-CSF neutralization did not impair bacterial control, nor pulmonary IL12p40 and IFNγ levels. Lung integrity was preserved under the 20 mg/L antibiotics dose, and anti GM-CSF antibody treatment did not induce morphological differences 32 days post-infection, although macroscopically larger nodules were observed. Thus, in susceptible TNFα−/− mice GM-CSF neutralization compromised the bacterial control by sub-optimal antibiotic treatment, leading to exacerbated lung inflammation, necrosis and intracellular bacterial load, while it did not affect host response under more effective antibacterial chemotherapy.TNFαion Fig.  and the ion Fig.  and baction Fig. , as compp40 Fig.  and IFNγp40 Fig.  were notice Fig. , as compice Fig. . Co-admions Fig. . Very inrol Fig. . We thenrol Fig. . When us20, and we thus asked how GM-CSF neutralization might impact M1/M2 polarization in vitro. Macrophages express GM-CSF after in vitro M. bovis BCG infection33. To appreciate the effects of GM-CSF neutralization on macrophage polarization and mycobacterial control in vitro, bone marrow-derived macrophages were stimulated with LPS or M. bovis BCG in the presence of anti-GM-CSF antibody or IgG2b isotype control. GM-CSF neutralization led to an increase in CD11b+F4/80+CD206+ M2 macrophage population and in CD206+ MFI values, as compared to isotype control, after 24 hour stimulation with LPS or M. bovis BCG , the upregulation of M1 related genes Nos2 and Il12b was reduced in the lung after GM-CSF neutralization  and rifampicin (RIF) bi-therapy partially contained the infection in terms of CFU titer per lung, number of M. tuberculosis clusters and the number of M. tuberculosis per cluster, and reduced necrosis in the lung tissue. GM-CSF neutralization actually compromised the bacterial control by sub-optimal INH/RIF treatment in TNFα−/− mice, leading to extended necrotic granulomatous lesions. However, under a higher dose chemotherapy that effectively reduced pulmonary bacterial load, the anti GM-CSF treatment did not compromise bacterial control. There was a transient body weight loss after GM-CSF neutralization in infected mice. Cachexia may result from systemic ‘cytokine storm’ as seen during a systemic inflammatory response syndrome. Indeed body weight loss did not seem an effect of the anti-GM-CSF MAB administration itself but occurred in conjunction with M. tuberculosis infection, as there was no body weight loss in the sterile LPS-induced lung inflammation model, nor in infected mice under INH/RIF antibiotics chemotherapy.Adjuvant immunotherapies under discussion for tuberculosisM. tuberculosis infection is still a matter of debate. Indeed, M1 macrophages seem to play a key role in bacterial killing and granuloma formation while M2 macrophages were found in non-granulomatous lung tissues, inhibiting M1 macrophage effects37. Therefore, in tuberculosis a strong regulation of M1/M2 macrophages balance is essential for the host defense. GM-CSF was recently recognized to induce M1 polarization, culminating in monocyte and macrophage IL-6, IL-8, G-CSF, M-CSF, TNFα and IL-1β production20. Here, we report that macrophages produce GM-CSF after M. bovis BCG infection and hypothesized that GM-CSF may contribute to the host response to mycobacteria infection by favoring macrophage M1 polarization. We show that in vitro GM-CSF neutralization induced a shift towards M2 macrophages after M. bovis BCG infection. Indeed, blocking GM-CSF triggered the overproduction of anti-inflammatory IL-10, while TNFα and IL-12, pro-inflammatory cytokines which play a key role for the host-response to tuberculosis, were reduced. Although GM-CSF increased the phagocytic capacity of alveolar macrophages in vitro35, we show here that anti GM-CSF treatment increased the intracellular bacterial load in macrophages in vitro but also in vivo. Thus, we propose that the anti-inflammatory milieu induced by GM-CSF neutralization impairs the ability of macrophages to eradicate mycobacteria. Indeed, while M2-related markers such as Arg1 and Ym1 genes or CD206 were overexpressed after GM-CSF neutralization, M. bovis BCG-induced M1 marker Nos2 was reduced, leading to a deficient production of nitric oxide in macrophages infected in vitro in the presence of anti-GM-CSF antibodies.Macrophage polarization during M. tuberculosis. Although, in vivo GM-CSF MAB treatment reduced M1 polarization after LPS-induced acute lung inflammation, GM-CSF neutralization did not influence the M1/M2 balance in the context of M. tuberculosis infection. Indeed, several in vivo factors such as extracellular matrix composition, cell maturation as well as cell adhesion could greatly influence macrophages polarization20. During M. tuberculosis infection, high levels of Th1-derived IFNγ, but also TNFα and IL-12 produced by macrophages, dendritic cells and neutrophils contribute to a complex milieu favoring M1 macrophage polarization32. Thus, although GM-CSF played a prominent role for M1 macrophage polarization in an acute LPS-induced lung inflammation model, GM-CSF was not essential for M1 polarization during M. tuberculosis infection. Indeed, the complex M1 milieu was not affected by GM-CSF neutralization, with no M1/M2 switch, indicating that the main pathway inducing M1 macrophage polarization after M. tuberculosis in vivo infection is not GM-CSF-dependent but most likely IFNγ produced by Th1, NK or NKT cells40.We next hypothesized that GM-CSF overexpression during acute infection contributes to an efficient M1 response against 41. In healthy individuals, anti-GM-CSF autoantibodies present at low levels may neutralize GM-CSF and therefore regulate GM-CSF mediated inflammation. In contrast, high levels of anti-GM-CSF autoantibodies have been associated with pulmonary alveolar proteinosis, and disseminated Crytococcus and Nocardia infections41. Higher levels of anti-GM-CSF autoantibodies, together with anti-IFNγ antibodies have been reported in patients with pulmonary disease due to non-tuberculous mycobacteria (NTM), relative to healthy controls, questioning whether these anti-cytokine autoantibodies might be a predisposing factor for pulmonary NTM disease42.Anti-cytokine autoantibodies present at steady-state may cause susceptibility to infections18. Our data, using anti GM-CSF antibodies in a tuberculosis infectious model may be relevant for the neutralization of GM-CSF pathway in the clinic. Indeed, we show here that murine monoclonal antibodies to GM-CSF induce a modification of granuloma integrity in the lung of immunocompromised mice infected by M. tuberculosis. The data raise concerns about the effect that such GM-CSF neutralizing therapies may have on latent tuberculosis infection in patients suffering from chronic inflammatory diseases. Indeed, recent mavrilimumab long-term safety and efficacy phase IIb studies in rheumatoid arthritis patients identified upper respiratory tract infection, including 2 cases of active pulmonary tuberculosis infection (0.22/100 PY) in patients with no active or latent tuberculosis at screening44. Thus, neutralization or adjuvant host-directed therapies using antibodies to a major cytokine such as GM-CSF might compromise the balance of host responses to mycobacterial infection and host immune system should be modulated with care in future immunotherapies.Several antibodies neutralizing GM-CSF or its receptor are in development for indications such as severe inflammatory diseases, chronic myeloid leukemia or cancer, either humanized IgG1 (leuzilumab) or fully human IgG . Mavrilimumab, a human monoclonal antibody targeting GM-CSFRα, has been tested in subjects with rheumatoid arthritis−/−)45 were bred in our specific pathogen free animal facility at CNRS . For experiments, adult (8–12 week old) animals were kept in ventilated cages or for the infectious protocols in biological isolation safety cabinet glove boxes in a biohazard animal unit. The infected mice were monitored every day for clinical status, weighted twice weekly and were sacrificed in accordance with ethical guidelines whenever reaching an endpoint such as 20% bodyweight loss. This study was carried out in accordance with the recommendations of the French Government’s animal experiment regulations and the protocol was approved by the “Ethics Committee for Animal Experimentation of CNRS Campus Orleans” (CCO) under number CLE CCO 2015-1085 and CLE CCO 2015-1071.C57BL/6 (B6) WT mice and TNFα deficient mice  on the indicated days. IgG1  and IgG2b 47 were used as isotype controls, respectively, as indicated.Mouse anti-mouse GM-CSF monoclonal antibodies (IgG1 Clone B2.6 or IgG2b Clone A7.39), obtained after immunization as describedEscherichia coli  diluted in saline (1 µg per mouse) or vehicle alone was applied by nasal or intra-tracheal instillation in a volume of 40 µL under light ketamine (100 mg/kg)-xylasine (10 mg/kg) anaesthesia.LPS from Bronchoalveolar lavage (BAL) fluid was collected by canulating the trachea and washing the lungs 4 times with 0.5 mL of ice-cold PBS. After centrifugation at 1850 rpm for 10 min at 4 °C, the supernatant of the first lavage was stored at −80 °C for cytokine analysis. Cell pellets were recovered, pooled and counted. For differential counts, cell cytospins were stained with Diff-Quik Staining  and twice 200 cells scored.M. tuberculosis H37Rv kept frozen at −80 °C were thawed, diluted in sterile saline containing 0.05% Tween 20 and clumping disrupted by 50 repeated aspirations through 18, 20, 26 and 27 gauge needles . Pulmonary infection with M. tuberculosis H37Rv was performed by delivering 1000 ± 200 CFU/mouse into the nasal cavities under ketamine-xylasine anesthesia as above. The bacterial load in the lungs was determined on day 1 post infection on control mice. Isoniazid (INH) and Rifampicin (RIF) were administered at the indicated concentration (1 to 100 mg/L) dissolved in drinking water from day 14 to 35 post-infection. For quantifying bacterial burden, lungs were weighted, defined aliquots homogenized in phosphate-buffered saline , and tenfold serial dilutions prepared in 0.05% Tween 20 containing 0.9% NaCl were plated in duplicates onto Middlebrook 7H11 (Difco) agar plates containing 10% OADC. After 3 weeks incubation at 37 °C colonies were enumerated and results expressed as log10 CFU per organ.Aliquots of 2 per lung section. Data are represented as percentage of granulomatous lesions corresponding to % area of lesions in mm2/total lung tissue in mm2. The number of bacterial clusters was counted in the lung lesions (0.5 mm²) and the average number of bacilli per cluster estimated in the same surface area of granulomatous lesions. Analyses were performed by two independent observers including a trained pathologist.Lung tissues were fixed in 4% buffered formaldehyde overnight, paraffin-embedded, and 3 µm sections were stained with haematoxylin and eosin (HE). Lung cellular infiltration, emphysema and necrosis were quantified using a semi-quantitative score with increasing severity of changes (0–5). The area surface of pulmonary granulomatous lesions were quantified on digitalized sections using a Zeiss Axio Scan Z1 Zeiss microscope and the ZEN software. Granulomatous lesions were determined in two lobes per mouse comprising a lung surface area of 25 to 35 mm48. Bone marrow derived macrophages (BMDM) were then harvested with cold PBS (Dulbecco), and 106 and 105 cells per well dispensed into 48 and 96 well plates (Corning® and Costar® Multiple Well Plates), respectively. Cells were incubated with 1 µg/mL of anti GM-CSF (Clone A7.39) or IgG2b isotype control  and stimulated with M. bovis BCG (MOI:2), M. bovis BCG-GFP (MOI:4) or LPS 100 ng/mL , as indicated.Bone marrow cells harvested from femur of C57/BL6 wild-type mice were cultured for 10 days in Dulbecco’s modified Eagle’s medium supplemented with 2 mM L-Glutamine, 25 mM HEPES, 20% heat-inactivated horse serum, 30% L929 supernatant containing macrophage-colony stimulating factor (M-CSF), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco)®), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco), 10% decomplemented FCS and 500 µL/plate of J558 supernatants containing GM-CSF49. BMDC were then harvested and stimulated with M. bovis BCG (MOI:2) or 100 ng/mL LPS, as above.For bone marrow derived dendritic cells (BMDC), bone marrow cells were cultured for 10 days in Roswell Park Memorial Institute RPMI medium supplemented with 25 mM Hepes, 1 mM sodium pyruvate, 40 µg/ml gentamycin, 50 µmol/L 2-mercaptoethanol, 10 mM L-Glutamine, 0.2% vitamins , F4/80-V450 (clone BM8) and CD206-APC (clone MR5D3) were from BD Biosciences. Stained cells were washed twice as above, fixed with 1% paraformaldehyde  and analyzed with a CANTO II analyser (Becton Dickinson). Data were processed with FlowJo software .After 24 hours of stimulation, cells were washed twice in CaTNFα, IL-1α, IL-1β, IL-12p40, GM-CSF, CXCL1, IFNγ, MMP9, TIMP1 and MPO protein concentration in the BAL fluid, lung homogenate or cell culture supernatants were quantified by enzyme-linked immunosorbent assay  following the manufacturer’s instructions. Lung homogenates were centrifuged , the supernatants sterilized by centrifugation through 0.45 µm and 0.22 µm filters , were immediately frozen on dry ice and stored at −80 °C until determination.M. bovis BCG-GFP (MOI:4) and with anti GM-CSF antibodies  or IgG2b isotype control (1 µg/mL). After 24 hours, the slides were washed twice in PBS, fixed in PFA 4% for 15 min, and coverslips mounted using Fluoromount®. The cells were observed using a confocal microscope LSM510 Meta (Carl Zeiss SAS) with a Plan-Apochromat 63x/1.4 OIL objective. Images were processed using LSM510 Meta software and ImageJ.Macrophages adhered on glass slides for 18 h at 37 °C were stimulated with 50 whole transcriptome microarray analysis of mouse lungs 4 weeks post-M tuberculosis infection in WT and TNFα−/− mice publicly available in ArrayExpress (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-5218, www.nature.com/scientificreports/Scientific Reports|6:36923| 10.1038/srep36923. For quantitative PCR determination of mRNA expression, total RNA was isolated from lung or cells using TRI-Reagent (Sigma) and RNA integrity and quality controlled using Agilent RNA 6000 Nano kit®. Reverse transcription was performed with SuperScript®III Kit (Invitrogen) and cDNA was subjected to quantitative real-time PCR using primers for Nos2, Arg1, Mrc1, Ym1 and Socs3 (Qiagen) and GoTaq® qPCR-Master Mix (Promega). Gapdh expression (Qiagen) was used for normalization. Raw data were analyzed using the comparative analysis of relative expression, using ΔΔCt methods51.The current study included microarray data of Csf2rb, Csfrb2 and Csf2 genes present in a previously reportedin vivo groups were analysed by means of one-way non-parametric ANOVA test  and values of p ≤ 0.05 were considered significant. Two-tailed, non-parametric Mann-Whitney test was used for analysing in vitro results. Grubb’s test was used to exclude significant outliers.Statistical significance was determined with GraphPad Prism . Differences between multiple The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Figures S1 to S3


Echo) and left atrial enlargement (LAEEcho) on echocardiography. 78 subjects who underwent echocardiography were included in this study. C-statistics  of the four biomarkers for predicting LVHEcho were 0.84 (0.72–0.92), 0.84 (0.73–0.92), 0.75 (0.63–0.85) and 0.62 (0.49–0.74); for LAEEcho 0.74 (0.6–0.85), 0.78 (0.66–0.88), 0.55 (0.42–0.67) and 0.68 (0.62–0.85); and composite SHD 0.79 (0.66–0.88), 0.87 (0.75–0.94), 0.62 (0.49–0.73) and 0.74 (0.62–0.84) respectively. Optimal cut points for SHD were >1.2 ng/L, >1.6 ng/L, >8 ng/L and >18 pmol/L respectively. These results advocate the potential role of hs-troponins as screening tools for structural heart disease with theranostic implications.The conventional use of high-sensitivity troponins (hs-troponins) is for diagnosing myocardial infarction however they also have a role in chronic disease management. This pilot study assessed the relationship of hs-troponins with echocardiographic markers of left ventricular hypertrophy (LVH) and structural heart disease (SHD). Patients undergoing computer gomography (CT) coronary angiogram for low-intermediate risk chest pain and healthy volunteers were recruited. Hs-troponins Singulex I, Abbott I and Roche T and N-terminal pro-brain natriuretic peptide (NT-proBNP) were evaluated in relation to SHD parameters including left ventricular hypertrophy (LVH Troponins assays have revolutionised the diagnosis of myocardial necrosis and infarction . HoweverTroponins have both diagnostic and prognostic capacity and are independently associated with the development of hypertension, heart failure, cardiovascular events, stroke and total mortality ,13. WithThe results of these studies demonstrate an emerging role of troponin as a longitudinal biomarker of cardiovascular risk and treatment response. However, many of these population studies and lack coronary or LV imaging information on patients. There are few studies where multimodal imaging has been applied alongside high sensitivity troponin testing. Whilst it is accepted that not all troponin assays are equivalent there have also been few studies comparing the utility of multiple contemporary troponin assays in parallel with multimodal imaging. We therefore undertook a pilot study employing two contemporary troponin assays and a research assay, using single molecule detection, in a group of patients undergoing computed tomography (CT) coronary angiography, echocardiography and multi-omic analysis.The myHealth e-Body study comprised of 200 patients undergoing CT coronary angiogram for low-intermediate risk chest pain. This interim analysis focuses on troponins and imaging markers of structural heart disease and reports on 78 patients from this study, including healthy volunteers who did not undergo CT. Institutional ethics approval was attained prior to the commencement of the study . All subjects gave written informed consent. Clinical characteristics collected included demographics, past medical history, 5-min electrocardiogram, a coronary artery and atrial fibrillation genetic risk score, metabolomics, echocardiography, CT coronary angiography and troponin assays and long-term outcomes including atrial fibrillation, cardiovascular events and mortality. Healthy volunteers did not undergo CT coronary angiography.Echo) defined as an interventricular septal thickness ≥11 mm and left atrial enlargement (LAEEcho) defined as ≥34 mls/m2, indexed to body surface area (BSA). Left ventricular global longitudinal strain and an echocardiographic calcium score was also applied but is described in another paper. Echocardiography was performed by a single Level III trained cardiologist, using a Philips CX50 machine . Echocardiographic markers of structural heart disease examined included left ventricular hypertrophy  was acquired using a 320-slice Aquillon ONE CT scanner . Oral and intravenous Metoprolol were used according to institutional protocol to achieve a scanning heart rate of <65 beats per minute. Obstructive coronary artery disease was defined as the presence of coronary stenosis of diameter loss >50%. Vitrea automated software was used for LV mass calculation, with LVHut point . g, for 5-min plasma was stored at −80 degrees centigrade before being shipped on dry ice to core lab facilities for testing. The fourth generation hs-troponin assays used included Singulex Erenna SMC troponin I , Abbott ArchitectSTAT troponin I  and Roche Elecsys troponin T . N-terminal pro-brain natriuretic peptide (NT-proBNP) was measured using a Roche Elecsys assay on a Cobas 6000 analyser . Singulex troponin concentrations <0.19 ng/L, Abbott troponin I < 0.2 ng/L and Roche troponin T <0.3 ng/L were reported as absolute values corresponding to the lower cut-off for detection.Blood was collected and after centrifugation at 3000 t-test for continuous variables and Spearman coefficient ) for correlations. Receiver-operative characteristics curve (ROC) analysis was used to assess performance of troponin assays at predicting echocardiographic markers by c-statistic and identifying the optimal cut point . A Bland Altman plot was used to demonstrate differences between the Singulex and Abbott troponin assays. Medcalc software version 16.8.4  was used to analyse the data. All tests were two-tailed and p < 0.05 deemed statistically significant. Median  were used to present continuous and categorical variables respectively. Univariate analysis was performed using student p = 0.03), male  and hypertension  were significantly associated with higher high-sensitivity troponins (Singulex assay). Diagnostic quality echocardiographic data was available for 78 patients. Cardiac CT was available for 62 (79%) patients, though LV mass could not be calculated for two patients due to poor contrast opacification of the LV. Eleven (14%) subjects were healthy volunteers and underwent an echocardiogram but not a CTCA. Measures of interventricular septal diameter (IVSd) were available in 75 (96%) of patients whereas echocardiographic LV mass could only be calculated in 64 (82%). Baseline characteristics for all patients are shown in 2/kg/m2. Correlation was strong between the two hs-troponin I assays, Spearman coefficient R2 = 0.87 (2 = 0.37/0.54 (Abbott/Singulex) and weakest between hs-troponin and NT-proBNP assays R2 = 0.3/0.31. Cardiac characteristics of the whole patient cohort are also found in 2 = 0.87  but weakp < 0.0001. NT-proBNP had a high sensitivity and specificity for the detection of moderate-severe LV systolic dysfunction as shown in LV systolic dysfunction (LVSD) was present in 8 (11%) patients, mean ± standard deviation (SD) ejection fraction 34 ± 6% versus 56 ± 3%, p < 0.0001, however IVSd and CT LV mass were less so r = 0.50, p < 0.0001. Although body mass index >25 or >30 m/kg2 was not associated with troponin concentration, body mass index (BMI) ≥ 27.4 m/kg2 predicted the presence of LVHEcho c-statistic 0.79, 95% confidence interval (CI) 0.68 to 0.89  and were superior to the Roche troponin I assay (c = 0.55–0.75), as well as NT-proBNP (c = 0.59–0.74)  b,c. The 2 predicted the presence of LVHEcho. Our study has also found that hs-troponins, particularly Singulex and Abbott assays, as a stand-alone tool, to be strongly associated with echocardiographic markers of structural heart disease. As previously described NT-proBNP had a higher sensitivity and specificity for LV systolic dysfunction, compared to hs-troponin. From this one could infer that NT-proBNP would serve as clinically useful when used in conjunction with hs-troponin or in a sequential manner when screening populations [In this study, we have shown that clinical factors such as age, gender and hypertension are strongly associated with troponin concentration, consistent with other data which has also shown an influence of physical activity, genetics and other factors including BMI on troponin . Althougulations . After eulations ,20,21,22ulations ,7. As onulations . Since Lulations ,19 and iulations  and stroulations ,25. In this small cohort, the number of patients with significant obstructive coronary disease was too limited to evaluate the relationship with hs-troponin. Whilst larger studies have shown a strong negative predictive value of Singulex troponin I (<1.53 ng/L) for the presence of obstructive coronary disease, causing ischemia, we could not demonstrate this finding due to the low numbers of patients in this study with obstructive coronary disease . In addiEcho/CT/LAEEcho as a stand-alone biomarker. In patients where white coat or mild hypertension is suspected hs-troponin could be used as a marker of end-organ damage, similar to the use of urine microalbumin creatinine ratio to detect hypertensive nephropathy.Our findings with those of others lend weight to the clinical applicability for ‘fourth generation’ hs-troponins. Apart from predicting cardiovascular events, failure and mortality, hs-troponins may be a useful screening tool for subclinical cardiac injury and dysfunction. This may provide a window of opportunity for earlier detection of LVH and act as a therapeutic guide. Two studies have shown that the addition of cardiac biomarkers, including hs-troponin with ECG and traditional cardiovascular risk factors, incrementally improves prediction of LVH ,26. Our Further research is required in large prospective studies to evaluate and determine the use of hs-troponins in cardiovascular screening. Multimarker studies are demonstrating that biomarkers such as troponin and BNP have utility in predicting coronary outcomes and stroke. Whether alterations in treatment, corresponding with a change in biomarker concentration then reflects a clinical positive clinical outcome remains uncertain, however one study did find decreased troponin concentrations correlating with reduced coronary events when treated with statins . LongituOur study has several limitations. The limited sample size of a pilot study meant not all significant associations could be found. It is an observational study which inevitably will contain bias. Note that patients included had low-intermediate risk chest pain so results may not be generalizable to asymptomatic healthy patients or with high-risk chest pain or established coronary artery disease and we did not have their final clinical diagnosis available to us at this point in time. Not all clinical characteristics were available for all patients, as these were collected as part of clinical care and their definitions were therefore open to clinician discretion. We also did not collect long-term outcomes to show the temporal adverse outcomes rates for this cohort.In conclusion, hs-troponins, particularly Singulex and Abbott assays, were strongly predictive of LVH and LAE on echocardiography. Our study and others and advocate for the potential role for troponins as a screening tool for structural heart disease and larger studies are required to assess this to guide clinical decision-making.


The Australian chiropractic and osteopathic professions underwent a period of significant transformation between 1960 and 2000. This resulted in an improvement in the views held by the medical profession towards the two professions. However, a recent survey of Australian general practitioners (GPs) reported that a number of GPs still hold negative views towards chiropractors and osteopaths. This paper examines these views from the perspective of critical realism and explores the generative mechanisms that can influence the willingness of health practitioners to collaborate over patient care. A qualitative analysis of open-ended responses to a survey of 630 Australian GPs was conducted. Unfavourable attitudes of GPs towards chiropractors and osteopaths included perceived lack of safety, efficacy, and inadequacy of training, despite chiropractic's and osteopathy's reliance on the same evidence base and similar training to those of other manual therapy professions such as physiotherapy. These attitudes may be underpinned by the professional biases against chiropractic and osteopathy that continue to marginalise the professions within the Australian healthcare system. Continued investment in the research base for chiropractic and osteopathic practice is required, along with raising the awareness of GPs about the education and skills of chiropractors and osteopaths. While both chiropractic and osteopathy are formally recognised as allied health professions, their role within the Australian healthcare system has not been clearly delineated . This maBetween 1960 and 2000, the chiropractic and osteopathic professions in Australia underwent a period of significant transformation which included practitioner registration, the creation of codes of practice, and an upgrade in educational standards . The perThese changes brought a shift in attitude by sections of the medical profession in favour of chiropractic and osteopathy. Some GPs were willing to refer patients for chiropractic and osteopathic treatment in an attempt to collaborate over patient care –13. DespA recent survey of 630 Australian GPs about their views towards chiropractors and osteopaths reported that a number of GPs still hold negative views towards chiropractors and osteopaths . While tCritical realism, first described by Bhaskar , is a phAn assessment of free text responses from a cross-sectional study designed to investigate the current views of Australian GPs towards chiropractic and osteopathy was conducted from the perspective of this stratified ontology. The cross-sectional study targeted GPs currently working in Australian private practice and used an anonymous online survey to gather data. Prior to the survey, the questions were tested on a focus group of 19 experienced vocational trainers attending a regional GP training conference. The survey link was promoted at a national GP conference followed by publication on a national association website and electronic publications and advertorials on a national commercial website for Australian doctors. The response rate for the survey was 2.6%. While this rate limits the generalisability of the data, the themes generated by the qualitative analysis should still be considered trustworthy. Comparison between the demographics of respondents in the cross-sectional study and the relevant workforce data showed a reasonably good correlation for gender and location by state. However, a greater proportion of respondents were over 40 years of age with less than 10 years in practice compared to the national average making the current cohort somewhat different to the national profile  was used together with the “exact2x2” package [Statistical analyses were performed on the themes from the responses to the four questions involving free text responses; pairs of questions relating to chiropractic and osteopathy were analysed using McNemar's exact test for dependent binary data . A level package  for all All respondents consented to participate in the online survey. The study was approved by Macquarie University's Human Research Ethics Committee .One hundred and eighty-four (184) GPs responded to both the chiropractic and osteopathic questions. Five themes emerged from an analysis of these responses. The themes were often strongly expressed, as demonstrated by quotations from the free texts. Unfavourable responses towards chiropractic outnumbered those towards osteopathy with significant differences between the two professions reported below.p < 0.001). Typical comments included the following:I have had patients with serious detrimental outcomes following these “treatments.” I have seen patients firsthand that have had bad results at the hands of a chiropractor.Some of my patients have had vertebral artery dissections from chiropractic treatment. I am aware of patients who have died from this complication. The first theme to emerge in the free text responses was that chiropractic was not a safe practice. A total of 24 out of 184 (13.0%) respondents raised concerns about the potentially harmful side effects associated with chiropractic but not osteopathic intervention. Only 2 out of 184 (1.1%) respondents raised similar concerns about osteopathic but not chiropractic treatment while 2/184 (1.1%) raised concerns about both. McNemar's Test: estimated odds of concern about harm in osteopathic versus chiropractic were 0.08 .The second theme to emerge in the free text responses was a lack of evidence to support either chiropractic or osteopathy. Just over one fifth of respondents  referred to the lack of evidence for the efficacy of chiropractic intervention while almost the same proportion  raised a similar concern with respect to the efficacy of osteopathic intervention. Of these respondents, 21/184 were concerned with the efficacy of both practitioner types (11.4%). McNemar's Test: estimated odds of concern about efficacy in osteopathic versus chiropractic were 0.74  reported that chiropractic and osteopathy offered real benefits, especially when dealing with musculoskeletal issues, these benefits were thought to be largely dependent on a patient's belief in these professions . Typical comments included the following:p = 0.002).The third theme to emerge in the free text responses was the inadequacy of chiropractic and osteopathy education programs. Overall, respondents were apprehensive about the fitness of graduates with a Master's degree in either discipline to practise as primary contact health practitioners. A total of 48 out of 184 respondents (24.5%) raised the issue of credibility with regard to the education background of chiropractors, while 28 out of 184 (15.2%) raised the same concern about osteopathic education. Among these respondents, 23/184 (12.5%) raised concerns about both chiropractic and osteopathic education. McNemar's Test: estimated odds of concern about osteopathic versus chiropractic education were 0.23 . Typical comments included the following:Lack of trust in their ability to effectively manage a patient's pain and its aetiology.I keep getting stupid letters from chiropractors about my patients that indicate chiros are practising well beyond their scope of practice. For example, one chiro informed me he had done a thorough assessment and diagnosed the patient's problem as their “adrenal meridians” were out of alignment.Why would I? It uses a system of imaginary structures like the “innate” to treat real disease. It's make-believe.Included as part of this theme was the issue of variability in treatment approaches within the professions. A total of 19 out of 184 responses (10.3%) referred to an unwillingness to refer patients to chiropractors due to an inability to identify “good” from “bad” chiropractors. Only 7 out of 184 respondents (3.8%) raised similar concerns about osteopaths. Among these respondents, 2 were concerned about referring to either practitioner type. McNemar's Test: estimated odds of concern about referring to osteopath versus chiropractor were 0.29 . Typical comments included the following:There are also a number of chiropractors who openly advocate against vaccination, and I can't support this.Whilst there seems to be some evidence for spinal mobilisation in the short term treatment of back pain, it's difficult to know which practitioners will stick to the evidence and which will be hosting seminars on the evils of immunisation.I don't believe in the science behind chiro and I am very concerned about reported inappropriate treatments and advice people get from chiropractors, especially regarding childhood health and immunisation.The issue of chiropractors' and osteopaths' views on vaccination was also raised: 24 out of 184 respondents (13.0%) identified the antivaccination stance that some chiropractors uphold as problematic; 2 of these respondents (1.1%) had the same concern about osteopaths while none of the respondents were concerned about osteopaths but not chiropractors. McNemar's Test: estimated odds of concern about antivaccination policies for osteopaths versus chiropractors were 0 .The fourth theme to emerge in the free text responses was the issue of unprofessional practices. Just under one-third  of the responses referred to concerns about chiropractors making financially motivated decisions and their use of false claims about the benefits of chiropractic intervention in their advertising. Only 13 out of 184 responses (7.1%) raised similar concerns about osteopathy. Among these, 10 respondents were concerned about both osteopaths and chiropractors motivations. McNemar's Test: estimated odds of concern about osteopaths versus chiropractors were 0.06 . The fifth theme to emerge from the free text responses was that chiropractors and osteopaths were duplicating physiotherapy services. As physiotherapy was regarded as evidence-based and safe by many respondents, GPs could refer patients to physiotherapists for treatment of musculoskeletal conditions, thereby making chiropractic and osteopathic services redundant. Typical comments included the following:The findings of this study reveal that GPs' negative attitudes towards chiropractic and osteopathy were based on a perceived lack of safety and efficacy, perceived inadequacy of educational programs, questionable practices of some practitioners, and the failure of both professions to promote their contributions to healthcare. From the perspective of critical realism, these perceptions are located at the empirical level; that is, they are based on GPs' observations and experiences. The views of GPs towards chiropractors were generally less favourable than towards osteopaths. These negative views raise concerns for both professions as they have the potential to impact government policy and budget determinations.In critical realism, the “empirical” level does not tell the whole story; the “actual” ontological level comprises the unobservable as well as the observable and experiential . It inclWhile safety should always be a primary concern when considering an intervention, a recent systematic review on the use of nonpharmacological therapies for low back pain reported continuing support for the use of spinal manipulation in managing this common condition . Further where the education occurs but rather its adequacy in preparing primary contact health practitioners. This is based on their experience and observation . What appears to be hidden from the view of some GPs  is that all accredited chiropractic, osteopathy, and physiotherapy curricula in Australia are designed to produce primary contact health practitioners who have patients that can present with previously undiagnosed health conditions. The training ensures that graduates have the necessary knowledge and skills for appropriate evidence-based management of musculoskeletal conditions and referral procedures where applicable. Consistent with the proposed interpretation of the data, improving GPs' knowledge about the level and quality of training of chiropractors and osteopaths may be indicated. Of concern is the general lack of interest by GPs to learn more about chiropractic and osteopathic education: 68% of GPs reported a lack of interest in chiropractic education and 63% in osteopathic education [All chiropractic and osteopathic training programs in Australia are university-based . The comducation .Observed or experienced  questionable practices fall within the realm of a profession's Code of Conduct which, in Australia, is under the jurisdiction of the relevant professional regulatory body, in this case the Chiropractic and Osteopathy Boards of Australia. At the “actual” ontological level, both boards have revised their respective Codes of Conduct. However, the number of complaints about questionable practices and unsubstantiated claims of efficacy continues to rise , 27.The issue of relevance to the current Australian healthcare system continues to receive little attention from both the chiropractic and osteopathic professions in Australia. By not clearly defining their respective roles, we believe that both professions have failed to represent their respective contributions to the health of the Australian community. GPs often see the duplication of musculoskeletal services as confusing and want to simplify the message to their patients by referring to a single group, in this case physiotherapists. This is their experience and observation . Moreover, health policy makers could claim that a duplication of services leads to higher costs and recommend referring to one group on the basis of budgetary impact alone. Chiropractors and osteopaths argue that their professions are distinct from one another as well as being different from physiotherapy and other manual therapies . HoweverWhile there were significant differences in the views of GPs towards each profession, these differences may simply be a product of the size of the workforce: as of June 2016 there were approximately two and a half times more chiropractors than osteopaths in Australia  so there are more chances for GPs to interact with chiropractors than with osteopaths . This arA number of generative mechanisms emerged from the data analysis. These are the deep-seated mechanisms (causative factors and tendencies) that can influence our perceptions of the “actual” and the “empirical” and can include personal and professional values and institutional discrimination. In this analysis, some apparent professional biases were identified:(i) While it is understandable that GPs may not be aware that the minimum level of chiropractic and osteopathic training is at Master's degree level, nor be fully acquainted with the content of the curricula, the general lack of interest by GPs in learning more about chiropractic and osteopathic education could be considered alarming (68% of GPs reported a lack of interest in learning about chiropractic education and 63% in osteopathic education)  and rais(ii) Many GPs in the survey stated their preference for physiotherapy over chiropractic and osteopathy even though both professions use similar diagnostic and treatment techniques . A clear(iii) Attitudes and behaviours of members of the chiropractic and osteopathy professions may have contributed to the observed and experienced reality of GPs. Both the chiropractic and osteopathic professions need to take responsibility for instances of alleged unprofessional conduct. Certain chiropractors and osteopaths avow their unorthodoxy in the belief that their practice transcends the accepted constraints of evidence-based practice. This bias derives from a philosophical standpoint and is supported by anecdotal claims of clinical success. Such behaviour should not be supported as it represents an attempt to circumvent the scientific process.The views of Australian GPs towards chiropractors and osteopaths are somewhat different to other jurisdictions. In the US, osteopathy has been considered part of mainstream medicine since the 1960s, with osteopaths awarded full MD status in all 50 states. Chiropractic continues to gain recognition with an increasing number of chiropractors appointed under the Veterans Affairs health system. In Canada and the UK, chiropractors work alongside medical practitioners in public hospitals and multidisciplinary medical centres , while, There are a number of limitations associated with this qualitative analysis. Apart from the issue of sample size which has been addressed previously , the majNegative views towards chiropractic and osteopathy are evident among some Australian GPs. These views centre on five themes: perceived lack of safety and efficacy, perceived inadequacy of training as primary contact health practitioners, questionable practice motives of some practitioners, and relevance to the Australian healthcare system. These attitudes may be the result of ignorance and/or bias on the part of GPs, a situation that has the potential to perpetuate the marginalisation of chiropractors and osteopaths within the Australian healthcare system.