Diabetic cardiomyopathy (CMP) is a common and disabling disease in diabetic patients, however no effective treatments have been developed. Although granulocyte-colony stimulating factor (G-CSF) improves heart function in myocardial infarction, its effect on non-ischemic CMP such as diabetic CMP is unknown. In the present study, we investigated the effects of G-CSF on diabetic CMP in a rat model of type II diabetes.Twenty 7-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF: a rat model of diabetes) rats and 10 male Long-Evans Tokushima Otsuka  rats were used. All of the LETO and 8 OLETF rats were fed on tap water while the rest were fed on sucrose-containing water. After 10 weeks, saline or recombinant human G-CSF (100 μg/kg/day) was injected intraperitoneally for 5 days. Blood levels of glucose, total cholesterol and triglyceride, and Doppler echocardiograms for diastolic dysfunction were obtained just before and 4 weeks after the saline or G-CSF treatment. Light microscopy, electron microscopy (EM) and immunohistochemistry for transforming growth factor-β were employed to examine myocardial histology 4 weeks after the saline or G-CSF treatment.Diastolic dysfunction developed at 17 weeks  in the OLETF rats whether or not they were fed sucrose water, but were more severe in those fed sucrose water. Four weeks after saline or G-CSF treatment, diastolic function had recovered in the G-CSF-treated group regardless of sucrose water feeding, and perivascular and/or interstitial fibrosis in the G-CSF-treated group had decreased significantly. TGF-β immunoreactivity in the interstitial and perivascular tissue was also reduced in the G-CSF-treated group, and EM studies revealed less severe disruption of myofilaments and mitochondrial cristae, and decreased collagen deposition.G-CSF can ameliorate cardiac diastolic dysfunction and morphological damage, especially fibrosis of the myocardium, in OLETF rats with diabetic CMP. Diabetic cardiomyopathy (CMP) is defined as cardiac failure when possible etiologies such as alcohol, hypertension, and coronary and structural heart disease have been excluded in diabetic patients . In a prRecent studies have shown that granulocyte-colony stimulating factor (G-CSF) reduces the size of infarcts, induces myocardial regeneration and is responsible for recovery of heart function in myocardial infarction (MI) -17. BaseThis work was performed in compliance with the ARRIVE guidelines on animal research . We usedThe numbers of rats in each group of LETO and OLETF rats and a schema of the experimental protocol are given in Figure Body weight, fasting blood glucose (FBG), total cholesterol (TC) and triglyceride (TG), heart rate (HR), systolic blood pressure (SBP) were measured, and Doppler echocardiography performed, at two times: first at the age of 17 weeks (just before treated) and second at the age of 22 weeks . After the rats were killed, myocardial histology was examined with light microscopy, immunohistochemistry for transforming growth factor (TGF)-β and electron microscopy (EM).SBP was measured by the tail-cuff method . Blood tests were measured in tail capillary blood after 8 hours of fasting.Each rat was anesthetized with 50 mg/kg ketamine and 5.8 mg/kg xylazine HCl, and the left side of the chest was shaved to gain a clear image. Doppler echocardiography was performed by a single sonographer. Serial echocardiographic examinations  were performed with the rats in the left lateral decubitus position. The transmitral flow velocity profile was determined by positioning a sample volume at the tip of the mitral valve on the apical four chamber view. The Doppler beam was set with < 15° of the incident angle to flow direction identified on color Doppler image. Angle correction was performed by a semiautomated system installed in the machine. The peak velocity (E), deceleration time (DT) of the early diastolic filling wave and early mitral annulus velocity (E') were measured. DT was obtained by extrapolating the initial slope of early diastolic filling wave deceleration to the baseline. E' was measured at the septal portion of the mitral annulus in an apical four chamber view, using tissue Doppler technique with a Nyquist limit of 15 cm/s. The transmitral inflow pattern was recorded on a strip chart at 100 mm/s sweep speed with simultaneous 3-lead electrocardiography for offline analysis. All measurements represented the mean of 5 consecutive cardiac cycles, and heart rate was calculated on the basis of the strip chart of Doppler echocardiography.Rats were anesthetized with 50 mg/kg ketamine and 5.8 mg/kg xylazine HCl. The hearts were excised and separated into 2 halves along the anterior longitudinal middle line. One half of the heart was fixed with formalin solution, embedded in paraffin and cut into sections 4 μm thick for H&E, Masson's trichrome staining and immunostaining for the TGF-β receptor . The other half of each heart was frozen in liquid nitrogen and stored at 280°C for electron microscopic examination. Interstitial fibrosis and perivascular fibrosis were measured, using Image Pro plus 6.0 imaging software  and the results were expressed as percentages.For electron microscopic examination, a portion of the left ventricle was cut into 1 mm fragments and fixed in 2.5% glutaraldehyde  for 4 hours and in 1% buffered sodium tetroxide for 1 hour, and then embedded in Epon 812. Survey sections were cut at 1 μm and stained with toluidine blue. Ultrathin sections were cut in from this block and studied under a Hitachi H-7600S electron microscope at 80 KV.t-tests (for single comparisons) or one-way ANOVA (for multiple comparisons), and Post Hoc multiple comparisons were made with Tukey's test  or Tamhane's T2 test . p values of less than 0.05 were considered significant.All values are means ± standard deviation (SD). Statistical differences were determined with Statistical Package for the Social Sciences (SPSS) 18.0 software . Data were analyzed by Table E and E' were lower and E/E' was higher in the two OLETF groups than in the LETO (-) rats, showing that diastolic dysfunction had developed in the OLETF rats. Moreover the OLETF (+) rats had lower E' and higher E/E' than the OLETF (-) rats; thus diastolic dysfunction was more pronounced in the sucrose-fed OLETF rats. In that, diabetic CMP models using OLETF rats were comparatively well made when compared with age-matched LETO rats group, using diastolic function parameters by Doppler echocardiography.Doppler echocardiography was performed four weeks after the series of intraperitoneal injections of G-CSF or normal saline. In the tap water groups, E' showed a higher trend p = 0.062) and E/E' was lower (p < 0.05) in the G-CSF treated (OLETF (-/+)) rats than the saline-treated (OLETF (-/-)) rats. This shows that G-CSF had a protective effect on diastolic function. Furthermore, there were no significant differences of E, E' and E/E' between OLETF (-/+) and LETO (-), indicating that G-CSF increased diastolic function to a level similar to that in the control rats. (Table  and E/E'A more clear-cut set of findings was obtained in the two groups of OLETF rats fed on sucrose-containing water. The differences between the OLETF rats injected with G-CSF (OLETF (+/+)) and those injected with normal saline (OLETF (+/-)) were more prominent, with E' higher and E/E' lower in the OLETF (+/+) compared with the OLETF (+/-) rats. Moreover, E' and E/E' did not differ between the OLETF (+/+) and LETO (-) rats, indicating that G-CSF treatment also improved diastolic function to a level comparable with that in normal rats. Table The changes in E, E' and E/E' of OLETF rats fed on sucrose-containing water and then injected with saline of G-CSF are shown in Table Masson trichrome staining revealed less perivascular and interstitial fibrosis in the OLETF rats treated with G-CSF than in those treated with saline. In the Image-pro analysis, the extent of fibrosis was greater in the OLETF groups than in the LETO group, and significantly lower in the G-CSF- treated group than the saline-treated group. In subgroup analysis, both groups of OLETF rats treated with G-CSF displayed less fibrosis than those treated with saline. Figure Similarly only a low level of interstitial immunostaining for TFG-β was observed in the OLETF (-/-) rats, and a complete absence of staining in the OLETF (-/+) rats. Likewise, the level of TGF-β immunoreactivity in interstitial and perivascular tissue was significantly lower in the OLETF (+/+) rats than in the OLETF (+/-) rats. Figure Electron microscopy revealed more pronounced heterogeneous structural alterations in the myocardial tissue of the OLETF (+/-) rats than in that of the OLETF (+/+) rats. Ultrastructurally the OLETF (+/-) myocardium showed the presence of intracellular edema, deposition of collagen fibers in the pericapillary region, swelling and disrupted mitochondrial cristae, and damaged myofilaments and intracellular junctions, as well as increased accumulation of lipid droplets and lysosomes in the cardiac cells, In contrast, collagen deposition was absent from the OLETF (+/+) myocardium and there were fewer abnormal mitochondria. Moreover there was less evidence of disruption of the cristae of enlarged mitochondria, or of damaged myofilaments and intracellular junctions. Figure In the diabetic heart, the functional alterations are preceded by a variety of structural, molecular and cellular changes, many of which are present in asymptomatic diabetic individuals and experimental models of diabetes. Moreover, the undermining of myocardial and vascular integrity appears to begin during the pre-diabetic stage . In a reAnother study reported that in an MI model, hyperglycemia had a beneficial effect on delaying ischemic-reperfusion injury and left ventricular dysfunction, however hyperglycemia did not decrease mortality rate , and diaIn this study we evaluated the functional and histological effect of G-CSF on relatively early diabetic CMP. We observed no substantial differences in blood levels of FBG, TC and TG between G-CSF-treated and saline-treated OLETF rats, suggesting that G-CSF does not significantly affect glucose and lipid metabolism.In diabetic CMP, diastolic dysfunction is well on the way even before symptoms appear in the patient. Therefore, we evaluated E, E', DT and E/E' using Doppler echocardiography and tissue Doppler to assess the degree of diastolic dysfunction. We found that diastolic dysfunction was minimal or completely recovered in OLETF rats treated with G-CSF.The beneficial effects of G-CSF were shown in the histologic findings as well. We found that perivascular and interstitial fibrosis progressed more slowly or even showed improvement in G-CSF treated OLETF rat groups. Also, the extent of TGF-β immunoreactivity in the interstitial and perivascular tissue was significantly decreased in G-CSF treated OLETF rat groups. EM studies revealed decreased collagen deposition and lesser number of abnormal mitochondria, damaged myofilaments and intracellular junctions were also noted in G-CSF treated OLETF rats. Our results indicate that the protective effects of G-CSF are seen not only in relatively advanced diabetic CMP but also in its early stages.Although various animal models have been used to investigate diabetes-related changes in the myocardium including interstitial fibrosis, cardiomyocyte loss, impaired energy utilization, small vessel disease and neuropathy, the pathophysiology of diabetic cardiomyopathy remains to be fully elucidated . The preG-CSF has various functions such as induction of proliferation, survival and differentiation of hematopoietic cells and mobilization of bone marrow cells -27. It hHarada et al. have discussed the possible molecular mechanisms of G-CSF action in preventing left ventricular remodeling after MI. They found that G-CSF acted directly on a G-CSF receptor expressed on cardiomyocytes and activated the Jak/Stat pathway. G-CSF also induced antiapoptotic proteins and inhibited apoptotic death of cardiomyocytes . Baldo MIn the present study, we found in the immunohistochemical findings that G-CSF inhibited presentation of TGF-β and progression of fibrosis. Such findings can enhance our understanding of the effects of G-CSF on myocardium, though further investigation is required to establish the exact mechanism of the protective effect of G-CSF in diabetic CMP.This study has two main limitations. First the number of subjects was small. Second, data on optimal G-CSF regimens such as dose, timing, frequency and duration are limited. In order to overcome these limitations, a preclinical study of the effects of G-CSF on diabetic CMP should be carried out with a larger number of subjects, and the G-CSF regimen should be optimized. Elucidating the major mechanism underlying the improvement of heart function by G-CSF treatment will require much more investigation.In the clinical setting, patients with diabetic CMP have increased in number, and moreover end-stage dilated CMP is the reality. Despite the increasing burden of diabetic CMP, the development of new treatment modalities has received little attention, and treatment is still largely based on conventional approaches such as strict glycemic control, management of symptoms and the use of renin-angiotensin-aldosterone system inhibitors and beta blockers -12. In tIn this study we evaluated the functional and histological effect of G-CSF on relatively early diabetic CMP. We found that diastolic dysfunction was minimal or completely recovered in OLETF rats treated with G-CSF. The beneficial effects of G-CSF were shown in the histologic findings as well. Perivascular and interstitial fibrosis progressed more slowly or even showed improvement in G-CSF treated OLETF rat groups. Also, the extent of TGF-β immunoreactivity in the interstitial and perivascular tissue was significantly decreased in G-CSF treated OLETF rat groups. EM studies revealed decreased collagen deposition and lesser number of abnormal mitochondria, damaged myofilaments and intracellular junctions were also noted in G-CSF treated OLETF rats. Our results indicate that the protective effects of G-CSF are seen diabetic. Therefore, G-CSF can ameliorate cardiac diastolic dysfunction and morphological damage, especially fibrosis of the myocardium, in OLETF rats with diabetic CMP. The present study provides the first evidence of the beneficial effects of G-CSF on diabetic CMP in OLETF rats.CMP: cardiomyopathy; DM: diabetes mellitus; G-CSF: granulocyte-colony stimulating factor; OLETF: Otsuka Long-Evans Tokushima Fatty; LETO: Long-Evans Tokushima Otsuka; FBG: fasting blood glucose; TC: total cholesterol; TG: triglyceride; HR: heart rate; SBP: systolic blood pressure; TGF: transforming growth factor; EM: electron microscopy; E: peak velocity of the early diastolic filling wave; DT: deceleration time; E': early mitral annulus velocity; SD: standard deviation; SPSS: Statistical Package for the Social SciencesThe authors declare that they have no competing interests.YHL designed, coordinated the experiments, performed the echocardiographic measurements, and prepared the manuscript. JHJ participated in the immunohistochemistry and helped with the echocardiography measurements. KSJ coordinated and studied the pathology findings. YSS and CHF helped with the echocardiography measurements. BIS participated in the electron microscopy. JS helped to draft the manuscript. JHK and HKL participated in the design and data discussion. KSK conceived the study and participated in the design and coordination. All authors have read and approved the final manuscript.


CSF-ERK1/2 was significantly increased in the AD group as compared to stable MCI patients and the ND group. Western blot analysis of a pooled cerebrospinal fluid sample revealed that both isoforms, ERK1 and ERK2, and low amounts of doubly phosphorylated ERK2 were detectable. As a predictive diagnostic AD biomarker, CSF-ERK1/2 was inferior to tTau, pTau, and Aβ42.Cerebrospinal fluid (CSF) samples from 33 patients with Alzheimer dementia (AD), 21 patients with mild cognitive impairment who converted to AD during followup (MCI-AD), 25 patients with stable mild cognitive impairment (MCI-stable), and 16 nondemented subjects (ND) were analyzed with a chemiluminescence immunoassay to assess the levels of the mitogen-activated protein kinase ERK1/2 . The results were evaluated in relation to total Tau (tTau), phosphorylated Tau (pTau), and beta-amyloid 42 peptide (A The increase in total Tau is believed to reflect the intensity of the neuronal degeneration, while pTau may indicate the phosphorylation state of the Tau protein  consisting of nondemented subjects (ND). An early event in the development of the neurofibrillary changes in Alzheimer's disease is the pathological hyperphosphorylation of Tau . The undAll study procedures were approved by the respective local ethics committees, and the patients or their legal caregivers gave their informed consent. At baseline, a comprehensive medical history was taken, and the patients underwent thorough physical, neurological, and psychiatric examination. For each patient, a computed tomography or an MRI scan was recorded. Alzheimer dementia (AD) was diagnosed according to the NINCDS-ADRDA criteria . Mild con = 33); (2) patients who fulfilled the Petersen criteria for MCI [n = 21; average time to conversion 30 months; range: 12–59 months); (3) patients with MCI who remained stable during the follow-up period ; (4) nondemented individuals referring to the centers for symptoms or other neurological diseases who underwent lumbar puncture for diagnostic reasons  (ND n = 16). The ND group included 4 individuals (“controls”) who were examined for symptoms but were later found to be neurologically healthy and one person who fulfilled the MCI diagnosis at baseline but was later found out to be cognitively healthy. The clinical diagnoses were made blinded to the biomarker results. For the assessment of cognitive dysfunction's severity, the Mini-Mental Status Examination (MMSE) was used [The subjects included in this study were categorized into four groups according to clinical and neuropsychological criteria: (1) patients with Alzheimer dementia  and Kuopio, Finland (n = 50). CSF was taken by lumbar puncture and processed according to standardized procedures as described elsewhere [μL (prior to centrifugation) were excluded from the analysis.The CSF samples were collected in Perugia, Italy . The measurements were performed in the Clinica Neurologica, University of Perugia, Italy and in the Department of Neurology, University of Kuopio, Finland. The tTau, pTau, and Aβ42 data were collected in the context of other studies, and these biomarker results have been published before, at least in parts [β42 data were reevaluated in relation to the current ERK1/2 measurements.CSF biomarkers were determined by ELISA method  was initially spiked into a pooled human CSF sample at a concentration of 480 ng/mL (“CSF-stabilized ERK2 stock solution”). Previous observations had indicated that the recombinant-activated kinase was relatively stable under these conditions and could be stored this way in aliquots at −80°C. Starting from this ERK2 stock solution in CSF, serial dilutions (250–16000-fold) were prepared in 1x assay buffer (see above) and measured in triplicates against 1x assay buffer. The standard curve for each assay plate was calculated with the MSD Discovery Workbench 3.0 software. The lower limit of detection (LLOD) was defined as 3 standard deviations (SD) above the blank signal (mean of 3 blanks for each plate). For 9 out of the 95 CSF samples studied here, the ERK1/2 measurements (means of the duplicate reads) were below the corresponding LLOD of the respective assay plate. As a means to include them in the statistical comparison between the diagnostic groups, these samples are reported here with an arbitrary value of 39 pg/mL corresponding to a CSF concentration just below the mean LLOD. When analyzing the correlations between ERK1/2 and age or classical CSF biomarkers, the 9 ERK1/2 measurements below LLOD were excluded. ERK1/2 in human CSF was measured with the monoplex total ERK1/2 whole cell lysate 96-well electrochemiluminescence assay  essentially as described previously . For meaP < 0.05 was regarded as statistically significant. The actual P levels are given for descriptive purposes.Statistical tests were performed with GraphPad Prism 5.01 software . Nonparametric tests  were applied in most cases, since D'Agostino-Pearson omnibus K2 test had indicated that some of the analytes did not follow normal distribution. A level of μL was achieved with Vivaspin spin concentrators  according to the manufacturer's protocol. Rehydration solution was added to the concentrated sample to a final volume of 2 mL, and the proteins were separated according to their isoelectric points (pI) on a 3100 off-gel fractionator (Agilent). The isoelectric focusing was performed at a maximum current of 50 μA for 20.000 Vhrs on 13 cm long pH 3–10 IPG strips . From the resulting 12 fractions, those covering pH ~6.5–~8.5 were pooled. This way, a substantial reduction of albumin was achieved, facilitating the further analysis. The proteins in the prefractionated sample were precipitated with trichloroacetic acid and desoxycholate. Briefly, the sample was mixed with sodium desoxycholate to reach a final concentration of 0.02%. Following 30 min incubation on ice, trichloroacetic acid was added to a final concentration of 15%. After an additional hour on ice, the sample was centrifuged at 16.000 g and 4°C for 30 min. Finally, the pellet was washed twice with ice cold acetone and air dried. For SDS-PAGE, the pellet was dissolved in 60 μL of sample buffer  SDS, 10% (v/v) Glycerol; 100 mM DTT; 0.005% (w/v) bromophenol blue) for 10 min at 95°C with agitation. To facilitate the subsequent Western blot analysis, pooled CSF was prefractionated by isoelectric focusing. Ten individual CSF samples from AD- and non-AD patients were combined. 3.25 mL of the resulting CSF pool was mixed with a 10x protease inhibitor cocktail and a 10x phosphatase inhibitor cocktail  to yield 1x inhibitor concentrations. Buffer exchange into “off-gel rehydration solution”  DTT, 0.5% (v/v) Pharmalyte) and concentration of the sample to a final volume of ~100 2 for 60 min with 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol [Advance blocking agent  in PBS/0.075% Tween-20 (PBS-T) for 45 min and incubated overnight at 4°C with antibodies diluted in blocking solution. The following antibodies were used: rabbit anti-ERK2 (C-14) sc-154 (Santa Cruz Biotechnology) (working dilution 100 ng/mL); rabbit anti-ERK1/MAPK3 (RnD Systems) (working dilution 250 ng/mL); monoclonal anti-MAP Kinase, activated (clone MAPK-YT) (Sigma) (working dilution 1 : 1000). The next day the membranes were washed with PBS-T and incubated for 1 h at room temperature with horseradish-peroxidase-coupled secondary antibodies. After washing with PBS-T, the blots were developed with ECL-plus chemiluminescent substrate according to the manufacturer's instructions  and visualized with an Intas Imager .The proteins were separated on 12% T/2.7% C SDS-polyacrylamide gels  and blotβ42is shown in P = 0.0999). The CSF levels (rank sums) of tTau and pTau were significantly higher in the AD- and the MCI-AD groups than in the MCI-stable and ND groups . Aβ42 was significantly lower in AD- and MCI-AD patients than in MCI-stable and ND subjects . A summary of the demographical data of the 4 diagnostic groups under investigation, as well as mean MMSE scores and median levels of the CSF biomarkers tTau, pTau, and An = 86; the measurements with mean calculated ERK1/2 levels below the LLOD were excluded).The ERK1/2 concentrations in the CSF samples were quantified with the total ERK1/2 monoplex electrochemiluminescence assay. The standard curves were calculated from measurements of serial dilutions in assay buffer of a “CSF-stabilized” ERK2 stock solution . The lower limit of detection of the assay (LLOD) was defined as 3 standard deviations (SD) above the blank signal. For the 4 assay plates employed here, the LLODs were calculated as 31.1 pg/mL, 31.4 pg/mL, 40.5 pg/mL, and 31.7 pg/mL, respectively (mean LLOD ± SD: 33.7 ± 4.6 pg/mL). Taking into account the 1.17-fold dilution of the CSF samples resulting from addition of an assay buffer concentrate prior to the measurements, the mean LLOD of the assay corresponded to an ERK1/2 concentration of 39.4 pg/mL in undiluted CSF. In total, for this study, 95 CSF samples were included in the analysis. With regard to the raw signal counts, the coefficient of variation (CV) between the duplicate reads was on average 2.4% ±2 (mean ± SD). The maximum CV between the respective duplicate reads  observed in the whole sample was 10.3%, indicating an acceptable technical assay performance. It should be noted, however, that the imprecision regarding the individually calculated CSF-ERK1/2 concentrations for the duplicates was higher  and the stable MCI cohort   below the respective assay plate LLOD are reported here with an artificially set value of 39 pg/mL. This corresponds to a CSF concentration just below the mean LLOD (see above). As a group, the AD patients showed significantly higher CSF-ERK1/2 concentrations than the nondemented individuals (on test) . The medβ42. The CSF concentrations of pTau and tTau were strongly correlated with each other (Spearman test)  . A signian test) , while sβ42 are known to show a substantial degree of variation between different centers [β42 in the samples from both sites when no classification into diagnostic groups was applied, and in the AD group from Kuopio. ERK1/2 and tTau as well as ERK1/2 and pTau were significantly correlated with each other in the samples from Perugia when all subjects were considered, but not in any of the diagnostic groups per se .The measurements of the CSF biomarkers tTau, pTau, and A centers . When wen = 29, MCI-AD: n = 21, MCI-stable: n = 25, ND: n = 9), MMSE scores as a measure for cognitive functions were available. 77 out of these with calculated CSF-ERK1/2 concentrations above the LLOD were subjected to correlation analysis. In the whole sample, CSF-ERK1/2 showed a statistically significant negative Spearman correlation with the MMSE scores  were calculated. To provide additional information regarding the ERK1/2 isoform(s) contributing to the signals detected by the ERK1/2 electrochemiluminescence assay, a pooled CSF sample was prefractionated by off-gel isoelectric focusing to facilitate the subsequent Western blot analysis. The fractions covering the pH range from ~6.5 to ~8.5 were combined and separated by SDS-polyacrylamide-gel electrophoresis followed by Western blotting. Probing with a panel of anti-ERK antibodies revealed the presence of ERK1, ERK2, and doubly phosphorylated ERK2 in the pooled human CSF . Visual β42, as is obvious from the ROC curves shown in In the present study, we have confirmed our previous finding of measurable levels of the MAP kinase ERK1/2 in human CSF , and we β42, tTau and pTau, in CSF were significantly altered at baseline in those MCI patients who later converted to AD . This was not the case for CSF-ERK1/2. In the AD group, but not in the MCI-AD patients, CSF-ERK1/2 was statistically significantly elevated as compared to the ND and MCI-stable groups. Therefore, it appears that the increase in CSF-ERK1/2 may possibly occur at a later disease stage than those of tTau and pTau. In the whole sample, CSF-ERK1/2 was significantly correlated with the CSF concentrations of tTau and pTau and negatively correlated with Aβ42. A correlation between ERK1/2 and tTau had also been observed in our previous study in a small set of CSF samples from patients with different neuropsychiatric disorders [r = 0.404 as compared to r = 0.933 for the correlation between pTau and tTau), and the association between the two did not reach statistical significance in any of the diagnostic groups per se. To gain more insight whether the increase in CSF-ERK1/2 in AD represents simply another marker of neuronal destruction in addition to Tau, or if possibly different cell populations or distinct underlying molecular mechanisms are reflected, it might be interesting to measure CSF-ERK1/2 levels in patients with other non-AD neurodegenerative disorders. In a recent study, a statistically significant correlation between the CSF levels of tTau and ERK1/2 was not observed in patients with Creutzfeldt-Jakob disease [β42. One can therefore speculate that in the long run, CSF-ERK1/2 may turn out to be of interest as a biomarker sensitive to disease progression at the MCI stage. Whether or not the ApoE genotype has an impact on the CSF-ERK1/2 levels has to remain open at this stage since a larger sample size will be required for reasonable statistical evaluation. In line with published biomarker studies , 22, in isorders . A simpl disease . InteresThe mitogen-activated protein/extracellular signal-regulated kinases ERK1 and ERK2 are ubiquitous components of cellular signal transduction pathways regulating diverse downstream processes, including proliferation, differentiation, survival, and apoptosis , 25. In β42. Although our findings need to be confirmed and extended in future studies, the observations suggest that the increase in ERK-1/2 levels may occur at a later stage during disease development or progression than the changes of tTau, pTau, and Aβ42 in CSF.Taken together, we report here a statistically significant elevation of CSF-ERK1/2 in AD patients as compared to ND and MCI-stable subjects. As a predictive diagnostic tool, CSF-ERK1/2 turned out to be inferior to the “classical” CSF biomarkers tTau, pTau, and Aβ42 and the ages of the subjects included in the 4 diagnostic groups under investigation were analyzed by Kruskal-Wallis test followed by Dunn's multiple comparison test . The CSF concentrations of tTau and pTau were significantly higher in the AD and MCI-AD patients than in the MCIstable and ND groups. CSF Aβ42 was significantly lower in AD and MCI-AD than in MCIstable and ND. Significant group differences in the median or mean ages were not observed with Kruskal-Wallis and ANOVA tests.The CSF levels of the classical biomarkers tTau, pTau, and Aβ42 were inversely correlated in the samples from both sides when no classification into diagnostic groups was applied. Furthermore, a statistically significant negative association between ERK1/2 and Aβ42 was observed in the AD group from Kuopio. The CSF-levels of tTau and of pTau were significantly correlated with CSF-ERK1/2 in the Perugia sample when no classification into diagnostic groups was applied.To complement the analysis for correlations between CSF-ERK1/2 and classical CSF biomarkers in the whole sample and to address the possibility of center effects, additional separate correlation analyses were done for each of the two centers involved. The results are summarized in Table S1. CSF-ERK1/2 and AClick here for additional data file.


ABCD1 gene disrupting the metabolism of very-long-chain fatty acids. The most serious form of ALD, cerebral adrenoleukodystrophy (cALD), causes neuroinflammation and demyelination. Neuroimaging in cALD shows inflammatory changes and indicates blood-brain-barrier (BBB) disruption. We hypothesize that disruption may occur through the degradation of the extracellular matrix defining the BBB by matrix metalloproteinases (MMPs). MMPs have not been evaluated in the setting of cALD.X-linked adrenoleukodystrophy results from mutations in the 2 = 0.34, 0.20, 0.55 respectively). Levels of TIMP1 and total protein in CSF significantly correlated with pre-transplant neurologic functional scores (R2 = 0.22 and 0.48 respectively), and levels of MMP10 and total protein in CSF significantly correlated with one-year post-transplant functional scores (R2 = 0.38 and 0.69). There was a significant elevation of MMP9 levels in plasma compared to control, but did not correlate with the MRI or neurologic function scores.We used a multiplex assay to correlate the concentration of MMPs in cerebrospinal fluid and plasma to the severity of brain inflammation as determined by the ALD MRI (Loes) score and the neurologic function score. There were significant elevations of MMP2, MMP9, MMP10, TIMP1, and total protein in the CSF of boys with cALD compared to controls. Levels of MMP10, TIMP1, and total protein in CSF showed significant correlation [p<0.05 for each with pre-transplant MRI Loes Loes scores (RMMPs were found to be elevated in the CSF of boys with cALD and may mechanistically contribute to the breakdown of the blood-brain-barrier. MMP concentrations directly correlate to radiographic and clinical neurologic severity. Interestingly, increased total protein levels showed superior correlation to MRI score and neurologic function score before and at one year after transplant. ABCD1, the gene that encodes for a peroxisomal transporter of very long chain fatty acid (VLCFA) and subsequently disrupts their metabolism and results in the accumulation of these compounds in all tissues. X-linked adrenoleukodystrophy (ALD) is a neurometabolic disease that results from mutations in ABCD1 mutation, there is currently no reliable way to predict who will develop neuroinflammation or when neuroinflammation may occur. There are very few therapeutic options once neuroinflammation occurs. Treatment with Lorenzo’s oil (glycerol trioleate and glycerol trierucate) combined with a VLCFA-low diet normalizes the concentration of VLCFA and may delay neurologic disease in those predestined to develop it, but it does not slow the progress of neurological symptoms once they have begun Cerebral ALD is characterized by central inflammatory demyelination A defining feature found on the MRIs of boys with cALD is the enhancement around the apparent lesion upon infusion of gadolinium indicating an ongoing pathological and neuroinflammatory process. While this is true in any neuroinflammatory disorder, the garland of enhancement of a posterior white matter lesion in a boy is nearly pathognomic of ALD In preparation for HCT, we have collected CSF and plasma samples from boys with cALD prior to their undergoing transplant and have previously reported elevated cytokine levels that correlated to the ALD MRI score This study and the use of all bodily fluids were approved by the Committee on the Use of Human Subjects in Research at the University of Minnesota. Informed written consent was obtained for all patient samples from the parents or guardians on behalf of the child participants. Patient written assent was also obtained if patients were greater than 8 years of age.n = 19) were patients undergoing intrathecal chemotherapy as treatment for a prior diagnosis of acute lymphoblastic leukemia (ALL) and were at least 3 months into maintenance therapy and without CSF leukemia. These patients were deemed to be the most suitable control group because the risk of obtaining CSF from “healthy” controls was too great. Use of these patients as controls has been previously published Samples were taken from patients with cALD (n = 20) 2 to 6 months prior to HSCT at the University of Minnesota. Controls  for a panel of human MMPs including MMP-1, −2, −3, −8, −9, −10, −13, TIMP-1, and TIMP-2 . Each sample was run in duplicate and an average value was calculated to determine the MMP or TIMP concentration using standard curves generated with the relevant recombinant human proteins provided with the commercial kits. Means for the cALD and control groups were calculated and subjected to a two-tailed Student’s t-test to determine a p-value. A linear regression analysis of MMPs and TIMPs of interest and MRI Loes score or neurologic function score was performed using GraphPad Prism software (version 5.0d).We performed a comprehensive examination of MMP, TIMP, and total protein levels in the CSF of boys with cALD prior to HSCT (n = 20) versus control patients (n = 19). Our approach was to use a multiplex ELISA for a panel of human MMPs including MMP-1, −2, −3, −8, −9, −10, −13, TIMP-1, and TIMP-2. Initial analysis of the data showed no significant differences between our cALD and control groups for: MMP1, MMP3, MMP8, MMP13 or TIMP2 levels in the CSF (data not shown). The MMP family members for which we found differences are reported below and were subjected to a more detailed analysis and clinical correlation.The level of total protein in the CSF of boys with cALD was significantly elevated (p<0.0001) as indicated in We also performed a comprehensive examination of MMP/TIMP family members in the plasma of boys with cALD that showed a significant elevation in MMP9 levels  as shown in 2 = 0.34) and CSF TIMP1  as shown in 2 = 0.55).The ALD MRI (Loes) score is an MRI-based quantification of the amount of cerebral involvement; it is based on a point system derived from location, extent of disease, and the presence of focal and/or global atrophy that is present in boys with cALD 2 = 0.22; and p<0.001, R2 = 0.48, respectively). With regard to transplant outcomes, post-transplant neurologic functional score correlated significantly with CSF levels of MMP10 and CSF total protein levels . None of the other MMPs and TIMPs in our panel correlated significantly to pre-transplant or post-transplant functional scores.The neurologic function score quantifies the gross neurological dysfunction through assessment of patients in the following areas: vision, hearing, speech, gait, and other areas such as fine motor skills and activities of daily living as shown in This study provides the first evidence of elevated MMPs and TIMPs in the CSF of boys with cALD. We report that there are significant elevations of CSF total protein, MMP2, MMP9, MMP10, TIMP1 and plasma MMP9 in boys with cALD at the time of transplant compared to controls. The levels of MMP10 and TIMP1 in CSF correlated significantly with the pre-transplant MRI scores. The levels of TIMP1 and CSF total protein also correlated significantly with the pre-transplant neurologic functional scores, and the levels of MMP10 and CSF total protein correlated significantly with the 1-year post-transplant neurologic functional score.MMP9 is a fairly well studied metalloproteinase, and increases in MMP9 have been shown in the CSF of patients with both viral and bacterial meningitis Elevated levels of MMP2 and MMP10 were also observed in boys with cALD, and MMP2 has been shown to be significantly higher in the brain following traumatic or viral brain injury TIMP1 has been shown to be elevated in the CSF of patients with both viral and bacterial meningitis MMP9 was elevated in the plasma of boys with cALD, as well as in the CSF. Elevation of serum MMP9 has been identified in other neurodegenerative disease such as MS where it was found to be elevated in patients with relapsing-remitting MS and other inflammatory neurological diseases as compared to patients with non-inflammatory neurological diseases. It was suggested that the elevated MMP9 serum level may influence the elevations of MMP9 that is observed in the CSF Microglia activation is known to occur in cALD and has been suggested to be partially responsible for the inflammation that causes cALD The increases of MMPs we have found in the CSF of boys with cALD could have several origins including: plasma, choroid plexus, or infiltrating hematopoietic cells, though it likely that all three sources play an intertwined role. Additionally, all the patients in our study had abnormal MRIs at the time of sample collection which begs the question whether elevations in MMP family members occurs before, or as a result of, the damaged BBB. This will be difficult to determine without the prospective sampling of CSF in boys prior to development of cALD. The role of increased MMP2 and MMP9 is an important observation as these MMPs are central to MMP family activation It has been previously shown that CSF total protein levels in boys with cALD are elevated We have first identified elevated levels of MMPs and TIMP1 in the CSF of boys with cALD. MMP and TIMP1 levels were directly associated with the severity of neurodegeneration and probably contribute to the disruption in the BBB seen in boys with cALD. This opens the possibility that inhibition of MMPs may provide a method for reducing breakdown of the BBB and neuroinflammation. Finally, CSF total protein level was elevated in boys with cALD, and showed superior correlation to the pre-transplant Loes score as well as the pre- and post- transplant neurologic function score. Although it contains a milieu of different proteins, CSF total protein may be, in fact, the best indicator of disease severity and outcome after HSCT in boys with cALD.


Macrophage Colony Stimulating Factor (CSF-1) controls the survival, differentiation and proliferation of cells of the mononuclear phagocyte system. A second ligand for the CSF-1R, Interleukin 34 (IL-34), has been described, but its physiological role is not yet known. The domestic pig provides an alternative to traditional rodent models for evaluating potential therapeutic applications of CSF-1R agonists and antagonists. To enable such studies, we cloned and expressed active pig CSF-1. To provide a bioassay, pig CSF-1R was expressed in the factor-dependent Ba/F3 cell line. On this transfected cell line, recombinant porcine CSF-1 and human CSF-1 had identical activity. Mouse CSF-1 does not interact with the human CSF-1 receptor but was active on pig. By contrast, porcine CSF-1 was active on mouse, human, cat and dog cells. IL-34 was previously shown to be species-specific, with mouse and human proteins demonstrating limited cross-species activity. The pig CSF-1R was equally responsive to both mouse and human IL-34. Based upon the published crystal structures of CSF-1/CSF-1R and IL34/CSF-1R complexes, we discuss the molecular basis for the species specificity. Garceau et al. op/op mice and tl/tl rats) and born with congenital osteopetrosis, reduced tissue macrophage populations and multiple developmental abnormalities op/op mice weight defects Mice and rats with a homozygous mutation in their CSF-1 gene are deficient in biologically active CSF-1 , has been identified in humans and mice Molecular tools targeting either CSF-1 or CSF-1R have proved very useful, allowing investigation of the myeloid population (macrophages/monocytes). Antibodies against the CSF-1R developed for the mouse and human ligands (CD115) are commonly used as markers to enable purification of blood monocytes in mice and humans Much of our knowledge of CSF-1 biology derives from studies of rodents. We have reviewed elsewhere the evidence of a role for macrophages, and CSF-1, in the growth hormone/insulin like growth factor-1 axis 22.1l-glutamine , 100 μg/ml streptomycin, and 100 U/ml penicillin . Untransfected Ba/F3 cells were maintained in medium containing 10% conditioned medium from X63 Ag8-653 myeloma cells carrying an expression vector for IL-3 4 U/ml rh-CSF-1 . HEK293T cells  were cultured in DMEM  supplemented with 10% HI-FCS (Sigma), 2 mM l-glutamine (Invitrogen), 100 μg/ml streptomycin (Invitrogen), 100 U/ml penicillin and 0.1 mM nonessential amino acids (Invitrogen). All cell lines and primary bone marrow cells were incubated at 37 °C with 5% CO2.The Ba/F3 cell line, transfected Ba/F3 cells and primary bone marrow cells were cultured in RPMI 1640 medium  containing 10% HI-FCS (Sigma), 2 mM 2.2All procedures performed on these animals were in accordance with national regulations and established guidelines and were reviewed and approved by the Institutional Animal Care and Use Committee or Ethical Review Panel. Spleen, liver and mesenteric lymph node samples were collected from a healthy, 3-year-old male, Large White × Landrace pig, euthanised by intramuscular injection of ketamine followed by captive bolt. Total RNA was prepared using Qiagen RNeasy kit  according to manufacturer’s instructions, including a DNase digestion step. Porcine specific cDNA was produced using 1ug of total RNA and reversed transcribed using ImPromII . Successful cDNA production without genomic DNA contamination was demonstrated using porcine HPRT primers 2.3For mammalian expression of porcine CSF-1 and CSF-1R, PCR primer pairs  were des2 (CSF-1R) or 1 mM MgCl2 (CSF-1) using an initial cycle of 94 °C for 2 min (3 min for CSF-1R), followed by 30 cycles (35 for CSF-1R) of 94 °C for 30 s, 60 °C for 30 s (56 °C for CSF-1R), 72 °C for 3 min and one cycle of 72 °C for 10 min. PCR products were gel-purified using QIAquick gel extraction kit (Qiagen) and cloned in frame with V5-His C-terminal tag of pEF6/V5-His expression construct using TOPO cloning kit (Invitrogen). DNA sequencing was performed by DNA Sequencing & Services  using Applied Biosystems Big-Dye Ver 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer.Amplification was achieved using porcine cDNA and expand high fidelity enzyme  with 3 mM MgClEscherichia coli and synthesized by Blue Heron Biotechnologies . The sequence was engineered with a BspHI restriction site at the 5′ end and an EcoRI restriction site at the 3′ end and cloned into the expression plasmid pET-28(b) using the complimentary restriction sites NcoI and EcoRI. The resulting plasmid, pTLW53, was transformed into MAX Efficiency® DH5α™ Chemically Competent E. coli according to the manufacturer’s protocol . A kanamycin resistant transformant was selected and the plasmid sequenced to verify the error-free ORF. The pTLW53 plasmid was isolated via QIAprep® spin miniprep kit  according to the manufacturer’s recommendations and transformed into One Shot® BL21 Star™ Chemically Competent E. coli .For bacterial expression of porcine CSF-1, the sequence corresponding to the active fragment of porcine CSF-1 (Ser36–Arg189) was codon optimised for expression in 50 broth of pTLW53/One Shot® BL21 Star™ E. coli incubating at 37 °C with 225 rpm shaking was refreshed 1:10 into 1 l of TB/Kan50 broth into baffled, vented 2L flasks. Protein expression was induced with 0.5 mM IPTG, final concentration, with incubation conditions continued at 37 °C and 300 rpm shaking. After 2 h induction, the culture was centrifuged and the E. coli pellet was stored at −80 °C.An overnight TB/KanE. coli cell culture were suspended in five volumes of lysis buffer  and lysis was completed by passing the suspension through a Microfluidizer. Lysate was centrifuged and insoluble pellets were washed in 1% Trition X-100, and 5 mM EDTA. Inclusion body pellets were suspended in DEAE buffer , and mixed at room temperature for 60 min. Following clarification, the soluble protein was loaded onto a DEAE Sepharose column and eluted with a gradient of 0–150 mM NaCl in buffer containing 8 M Urea. Protein fractions containing pCSF-1 were pooled and diluted slowly into two parts redox buffer . Protein was dialysed against redox buffer overnight and dialysis buffer exchanged to contain 0.5 mM reduced glutathione and 1 mM oxidised glutathione. Refolded pCSF-1 dimer was loaded onto a Q Sepharose column equilibrated with 50 mM Tris pH 8.5, 5 mM EDTA. Protein was eluted with a 10 BV gradient of 0–250 mM NaCl. The pooled pCSF-1 was dialysed against PBS and sterile filtered prior to use. Protein concentration was calculated by UV absorbance at 280 nm.Frozen cell pellets from 2.46 Ba/F3 cells were transfected by electroporation  with 10 μg DNA (pEF6_pCSF-1R or empty pEF6 DNA), or no DNA, and selected with 30 μg/ml blasticidin (Invitrogen) and 10% IL-3 for 6 days prior to further selection with 30 μg/ml blasticidin and 104 Units/ml of rh-CSF-1. For generation of stable pEF6_pCSF-1 HEK293T cells, 0.8 × 106 cells/well of a six well plate were plated with antibiotic-free DMEM for 24 h, followed by transfection with 4 μg DNA , or no DNA, using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. Selection of stable cells was achieved by the addition of 10 μg/ml blasticidin (Invitrogen) after 48 h.For generation of stable Ba/F3 cells expressing porcine CSF-1R, 5 × 102.56 cells in 2% SDS 10 mM Tris buffer and boiling for 10 min at 100 °C. 100 μl of centrifuged supernatant from stably transfected HEK293T cell cultures of pEF6_pCSF-1, and empty pEF6 was also prepared. Protein concentration was determined using DC protein assay  with 10 μg of protein mixed with Laemmli buffer (Invitrogen) and 5 mM DTT. Samples were run on a 4–12% gradient precast SDS–PAGE gel (Bio-Rad) and transferred onto polyvinylidene difluoride membrane, as per manufacturer’s directions (Bio-Rad). The membrane was blocked with 5% skimmed milk powder in TBS-Tween 20 at 4 °C overnight, prior to being washed and probed with 1:5000 dilution of mouse anti-v5 tag antibody  and 1:5000 dilution of anti-mouse IgG HRP conjugated antibody  and detected using enhanced chemiluminescence (ECL) reagents . β-Actin  was used at 1:200 dilution as a loading control.Whole cell lysate was prepared by lysing 0.5 × 102.66 cells were pelleted and re-suspended in 4 ml of complete RPMI containing supernatant from empty pEF6 or pEF6_pCSF-1 transfected HEK293T cells . Cells were plated into 60 mm bacteriological plates and incubated for 10 days at 37 °C with 5% CO2.Pig bone marrow cells were obtained by flushing the bone marrow from five caudal ribs with 20 ml of complete RPMI with 5 mM EDTA using a bone marrow biopsy/aspirate needle . For each condition, 0.25 × 106 cells were cultured in 60 mm bacteriological plates with 4 ml RPMI, supplemented with either 104 Units/ml rh-CSF-1, or 300 ng/ml porcine CSF-1 and incubated for 12 days at 37 °C with 5% CO2.With owners consent, 0.5 ml of surplus feline bone marrow from clinical diagnostic investigation of a 2 year old, neutered male, DSH cat was obtained from a bone marrow aspirate of the left humerus using a bone marrow biopsy/aspirate needle  and placed in EDTA. Canine bone marrow was collected post-mortem with owner’s consent from a 3-year-old male entire Staffordshire Bull Terrier dog which was euthanised for behavioural reasons. Bone marrow was flushed from the left femur with 20 ml of RPMI and 5 mM EDTA using a bone marrow biopsy/aspirate needle . For both feline and canine bone marrow cultures, 10 × 104 Units/ml rh-CSF-1 for 5 days at 37 °C with 5% CO2.Mouse bone marrow cells were flushed from the femurs of an 8-week-old male BALB/c mouse with 10 ml complete RPMI and a 27 g needle, and plated in 100 mm bacteriological plates with complete RPMI supplemented with 102.74 Units/ml rh-CSF-1 or 10% IL-3 conditioned medium prior to MTT assay. 2 × 104 cells/well (Ba/F3 cells and Ba/F3 transfectants), or 5 × 104 cells/well (mouse BMM) of a 96 well plate were plated in triplicate or quadruplicate and appropriate treatment  rhIL-34 (R&D Systems 5265), or rmIL-34 (R&D Systems 5195), or supernatant from pEF6_pCSF-1 were added to make a total volume of 100 μl per well. Cells were incubated for 48 h at 37 °C with 5% CO2. For Ba/F3 cells, 10 μl of MTT (Sigma Aldrich M5655) stock solution (5 mg/ml) was added directly to each well to achieve a final concentration of 0.5 mg/ml and incubated at 37 °C for 3 h prior to solubilisation overnight. For adherent mouse BMM cells, culture medium was replaced with 50 μl of 1 mg/ml MTT solution and incubated for 1 h at 37 °C. MTT solution was removed and tetrazolium salt solubilised with 100 μl of solubilisation agent  followed by incubation at 37 °C with 5% CO2 for 10 min. Plates were read at 570 nm with reference wavelength of 405 nm.Stable Ba/F3 cells expressing porcine CSF-1R were maintained in culture with complete RPMI supplemented with either 102.850 for rh-CSF-1 and bacterially produced porcine CSF-1 in the Ba/F3pCSF-1R MTT cell viability assay were calculated from dose responses performed in triplicate or quadruplicate. Data was analysed using GraphPad Prism software using nonlinear regression curve fit. The R2 (coefficient of determination) values for all curve fits were >0.90.The EC2.9http://bmm.cancerresearchuk.org/3djigsaw/) using structure-based alignments (performed by Domain Fishing). 3D models of human IL-34-CSF-1R (PDB 4DKD), mouse IL-34-CSF-1R (PDB 4EXP), and mouse CSF-1 (PDB 3EJJ) were obtained and viewed in FirstGlance in Jmol (http://firstglance.jmol.org). Human and pig CSF-1 were generated using 3D-Jigsaw with the mouse CSF-1 structure as template (3EJJ) and pig CSF-1R was generated using human CSF-1R as template (4DKD chain X). Contact amino acids between IL-34, CSF-1 and CSF-1R were identified using recently published data 3D models in PDB format were generated with 3D-Jigsaw  and a 950 amino acid mature chain . The 520 amino acid extracellular segment contains the predicted ligand binding domains (D1–D5) and a 25 amino acid hydrophobic transmembrane region. The 430 amino acid intracellular region contains the tyrosine kinase domain and ATP binding region required for catalytic activity of the receptor upon binding of CSF-1 or IL-34 to the extracellular domain. Comparison between species of CSF-1R sequences demonstrates that porcine CSF-1R shares 80% homology with human CSF-1R and 72% homology with the mouse CSF-1R amino acid sequence, while human and mouse CSF-1R share 75% amino acid homology . In part3.2in vitro studies, the pEF6_CSF-1 expression plasmid encoding porcine CSF-1 was transfected into HEK293T cells using Lipofectamine 2000. Secreted CSF-1 in the HEK293T supernatant was successfully detected using Western blotting for the V5 epitope tag encoded by the expression plasmid. The CSF-1 protein has a predicted weight of 27 KDa. Under non-reducing conditions, two bands of approximately 37 KDa and 50 KDa were detected, whereas two smaller bands (20 and 25 KDa) were detected in the presence of dithiothreitol (DTT)  A. These 3.3To confirm biological activity of secreted porcine pEF6_CSF-1, we stably transfected the IL-3 dependant Ba/F3 cell line Western blot analysis of these cells based upon detection of the V5 epitope tag demonstrated successful expression of porcine CSF-1R. Prior selection of these cells with rh-CSF-1 and/or IL-3 altered the levels of receptor expression B. Growth3.4Using an MTT cell viability assay, we developed and optimised a bioassay for assessing the biological activity of secreted CSF-1 by HEK293T transfection with porcine pEF6_CSF-1, and rh-CSF-1. The bioassay was optimised for cell number/well, and culture conditions  prior to assay, but these variables did not make a substantive difference to sensitivity. Although IL-3 was equivalent to rh-CSF-1 in the bioassay in terms of cellular viability and proliferative response of the Ba/F3 cells expressing CSF-1R, rh-CSF-1 was used as the standard culture growth factor to maintain selection pressure for CSF-1R expression. Using the MTT cell viability assay, the presence of 10% conditioned medium from the IL-3-expressing cell line allowed survival of both parent Ba/F3 and Ba/F3 cells expressing porcine CSF-1R. The supernatant from transfected HEK293T with porcine pEF6_pCSF-1 also maintained the viability of the Ba/F3 cells expressing porcine CSF-1R A.Bone marrow-derived macrophages (BMDMs) grown in CSF-1 have been used extensively in studies of mouse macrophage biology 3.5E. coli. The advantage of an E. coli expression system is not only the high yield which enables both preclinical and structural studies, but also the possibility of introducing defined mutations to support structure–function analysis. To enable such studies of the porcine protein, and to further optimise expression, a synthetic codon-optimised cDNA encoding the active amino acids was generated as described in Section 50 of the bacterially produced porcine CSF-1 and rh-CSF-1 on Ba/F3 cells expressing porcine CSF-1R was essentially identical (29 ng/ml and 34 ng/ml respectively) (The recombinant human CSF-1 used here and in previous studies on pig marrow ctively) D.3.64 Units/ml rh-CSF-1 or increasing concentrations of HEK pEF6_pCSF-1 supernatant. There was a dose-dependent increase in BMM survival with increasing concentrations of HEK pEF6_pCSF-1 supernatant  (Previous studies suggest that CSF-1 and IL-34 bind different parts of the CSF-1R 0 ng/ml) B. This f4We have produced a stable Ba/F3 cell line expressing porcine CSF-1R which can be utilised in a cell viability bioassay to assess biological activity of CSF-1. Numerous receptors and oncogenes have been tested in Ba/F3 cells for their ability to generate factor-independent, or novel factor-dependent growth P388D1Although transfected Ba/F3 cells grew well in CSF-1 and provided a useful assay, they nevertheless generally proliferated less rapidly than in IL-3. Ba/F3 cells were originally identified as pro-B cells that express surface Ig and lack endogenous CSF-1R N/DS ratios, noted by Garceau et al. We have used the recombinant proteins and receptor-expressing cells to further dissect the species-specificity of CSF-1 and IL-34. Porcine CSF-1 was able to activate porcine, mouse, feline and canine CSF-1 receptors, a property shared by recombinant human CSF-1 We have demonstrated that mouse CSF-1 can bind and activate the porcine CSF-1R. Porcine CSF-1 can also stimulate the mouse, dog and cat receptors. Conversely, mouse CSF-1 can activate porcine CSF-1R but not human CSF-1R. As with IL-34 binding, there must be a difference between all three species of CSF-1R and between mouse and human CSF-1. There are 19 contact amino acids between mouse CSF-1 and CSF-1R op/op), have been previously well studied Mice with no detectable biologically-active serum CSF-1 (


The mixed chimerism approach induces donor-specific tolerance in both pre-clinical models and clinical pilot trials. However, chronic rejection of heart allografts and acute rejection of skin allografts were observed in some chimeric animals despite persistent hematopoietic chimerism and tolerance toward donor antigens in vitro. We tested whether additional cell therapy with regulatory T cells (Tregs) is able to induce full immunologic tolerance and prevent chronic rejection.We recently developed a murine “Treg bone marrow (BM) transplantation (BMT) protocol” that is devoid of cytoreductive recipient pre-treatment. The protocol consists of a moderate dose of fully mismatched allogeneic donor BM under costimulation blockade, together with polyclonal recipient Tregs and rapamycin. Control groups received BMT under non-myeloablative irradiation and costimulation blockade without Treg therapy. Multilineage chimerism was followed by flow cytometry, and tolerance was assessed by donor-specific skin and heart allografts.Durable multilineage chimerism and long-term donor skin and heart allograft survival were successfully achieved with both protocols. Notably, histologic examination of heart allografts at the end of follow-up revealed that chronic rejection is prevented only in chimeras induced with the Treg protocol.In a mouse model of mixed chimerism, additional Treg treatment at the time of BMT prevents chronic rejection of heart allografts. As the Treg-chimerism protocol also obviates the need for cytoreductive recipient treatment it improves both efficacy and safety over previous non-myeloablative mixed chimerism regimens. These results may significantly impact the development of protocols for tolerance induction in cardiac transplantation. Deliberate induction of donor-specific tolerance is a major goal in transplantation medicine as it would eliminate the need for chronic immunosuppression and the risk of chronic rejection. The mixed chimerism approach was shown to be a successful tolerance strategy in experimental animal modelsIntensive research has led to the development of experimental BMT protocols devoid of global T-cell depletion.In experimental models, especially mice, skin grafts are considered to be the most stringent test for tolerance and skin graft survival is usually followed for >100 days, which is commonly considered to indicate permanent survival. Nevertheless, although skin grafts may remain macroscopically intact for >100 days, and histologic examination does not reveal major changes in skin architecture, the risk of chronic rejection cannot be fully assessed as skin grafts are only secondarily vascularized with endothelium derived from the recipient. Thus, permanent survival of skin allografts in mixed chimeras does not allow for a reliable conclusion as to whether full donor-specific tolerance capable of preventing chronic rejection of solid-organ allografts has been established. This is of particular interest, as some studies demonstrated chronic rejection of primarily vascularized heart grafts in skin graft–tolerant recipients.In this study we examined the potency of Tregs in preventing chronic rejection of primarily vascularized heart allografts in a murine BMT model without cytotoxic recipient pre-conditioning.H-2b), BALB/c  and C3H/HeNCrl  mice were purchased from Charles River Laboratories . The animals were housed under specific pathogen-free conditions and were used at 6 to 12 weeks of age. All experiments were approved by the local review board of the Medical University of Vienna and were performed in accordance with national and international guidelines of laboratory animal care. All mice received humane care in compliance with the “Principles of Laboratory Animal Care,” formulated by the National Society for Medical Research, and The Guide for the Care and Use of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health .Female C57BL/6  and costimulation blockade with cytotoxic T-lymphocyte–associated antigen 4 immunoglobulin  and anti-CD154 monoclonal antibodies . Groups of mice received additional Treg treatment (described in what follows) and a short course of rapamycin .Groups of age-matched B6 recipients received approximately 2 × 10+ cells were isolated from B6 spleen and lymph nodes by magnetic bead sorting , and cultivated in plates coated with 10 µg/ml anti-CD3 (145-2C11), 1 µg/ml anti-CD28 (37.51) (BD Pharmingen) in the presence of 100 U/ml interleukin-2  and 5 ng/ml recombinant human transforming growth factor-beta  for 5 days. At the end of culture, the Treg-enriched cell population was used for therapeutic administration without additional sorting steps. FoxP3 expression was usually >80%. Then 3 × 106 cells were injected intravenously per mouse.Induced Tregs (iTregs) were generated as described previously.Cervical heterotopic heart transplantation was performed 6 to 8 weeks after BMT with a modified cuff technique for revascularization, as described previously.Full-thickness tail skin from donor (BALB/c) and fully mismatched third-party (C3H) mice was grafted 4 to 6 weeks after BMT and visually inspected thereafter at short intervals. Grafts were considered to be rejected when <10% remained viable.Recipient serum harvested >3 months post-BMT was heat-inactivated and incubated with recipient-type, donor-type and third-party-type thymocytes . Binding of serum IgG antibodies to thymocytes was analyzed by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse IgG1 and IgG2a/2b (BD Pharmingen).5 responder splenocytes were incubated in triplicate with 4 × 105 irradiated (at 30 Gy) stimulator cells of either B6 (recipient), BALB/c (donor) or C3H (third party) origin or with medium only. After 72 hours of incubation, cells were pulsed with [3H]-thymidine  for 18 hours. Incorporated radioactivity was measured using scintillation fluid in a β-counter. Stimulation indices (SIs) were calculated in relation to medium controls. Results represent averaged data from each group.Mixed lymphocyte reactions (MLRs) were performed as described in detail previously.+  cells among specific leukocyte lineages, as described previously.Multicolor flow-cytometric analysis of multilineage macrochimerism and Vβ-subunit expression were performed as described previously.Four-micron sections were cut from paraffin-embedded tissue fixed in 4.5% formalin (with buffered pH 7.5), stained with hematoxylin–eosin (HE), Giemsa–elastica van Gieson (EvG) stain according to standard protocols, and analyzed by an experienced clinical pathologist in a blinded fashion. Heart allografts were scored according to the International Society for Heart and Lung Transplantation (ISHLT) 2005 guidelines for cellular rejection .t-test with unequal variances was used to compare percentages of Vβ-family–positive cells, chimerism levels and SI values between groups. Fisher’s exact test was used to compare chimerism rates between groups and rejection scores. Skin and heart allograft survival were calculated according to the Kaplan–Meier product limit method and compared between groups using the log-rank test. p < 0.05 was considered statistically significant.A 2-sided Student's +CD25+ Tregs [nTregs] and TGF-β induction [iTregs]In previous work we reported that therapeutic administration of polyclonal recipient Tregs enhances BM engraftment and therefore allows reduction of recipient pre-conditioning.+ cells after 5 days in vitro culture was usually around 80% (6 cells per recipient (which corresponds to ~2.4 × 106 FoxP3+ CD4 cells/mouse [120 × 106 Tregs/kg]). Tregs were administered simultaneously with allogeneic BM to allow activation of alloreactive Tregs, potentially enhancing their suppressor activity . Although this protocol reliably induced long-term chimerism, the chimerism levels were substantially lower than in the group employing 3-Gy TBI   and rapamycin  led to the engraftment of a conventional number of BALB/c BM cells in otherwise untreated wild-type B6 recipients  A. Notablost-BMT) B and C. To assess donor-specific tolerance, skin transplants were performed 4 to 8 weeks post-BMT. All chimeras induced by Treg treatment and the majority of chimeras pre-treated with 3-Gy TBI accepted donor skin for the length of follow-up, whereas unirradiated control mice without Tregs rejected donor skin A. Third-p = 0.010). Fragmented elastic fibers could also be demonstrated in the myoelastic tissue of cardiac vessels of 3-Gy chimeras (but not Treg chimeras or syngeneic controls) using EvG staining. Allograft vasculopathy found in 3-Gy chimeras included interstitial hemorrhage, intravascular macrophages, capillary fragmentation, mixed inflammatory infiltrates, endothelial cell pyknosis, karyorrhexis and marked edema, indicating moderate to severe signs of chronic rejection.Cellular rejection was significantly more frequent in chimeras induced by irradiation than in those induced by Tregs  A. To ass+ and Vβ5+ T cells allow assessment of donor-reactive T cells in the strain combination used in the present studies (Vβ8 serves a specificity control). As reported previously,Clonal deletion is one of the main tolerance mechanisms in mixed chimerism models.p = 0.0014]; Vβ5: 3 Gy 0.43% vs Tregs 1.34% [p = 0.0140])   , whereas deletion in Treg-treated chimeras was evident but significantly less substantial (Vβ11: 3 Gy 0.84% vs Tregs 1.76% [0.0140]) C. Deleti0.0140]) D (Vβ11: 0.0001]) D. Furthe0.0012]) E.Thus, deletional mechanisms seem to be less important in chimeras induced by Treg treatment than in chimeras induced by non-myeloablative irradiation.In this study we have demonstrated that therapeutic Treg treatment as part of a BMT protocol to induce chimerism efficiently prevents chronic rejection of murine heart allografts. Thus, additional Treg treatment not only obviates the need for cytotoxic recipient treatment , but also improves outcome after cardiac transplantation in a murine mixed chimerism model.The mixed chimerism approach has been shown to potently induce robust donor-specific tolerance in both animals and humans.Herein we have shown that polyclonal recipient Tregs can substitute cytotoxic recipient irradiation to facilitate BM engraftment and induce chimerism. Moreover, we demonstrated that this Treg-based protocol is superior to protocols based on irradiation in the prevention of chronic rejection of heart allografts. Notably, levels of chimerism induced with mild TBI are significantly higher than in BMT recipients receiving Treg therapy instead. However, there is no clear evidence indicating which level of chimerism is needed to induce tolerance. Besides, it is unclear whether higher levels of chimerism per se are more likely to induce full tolerance. In the clinical setting, it was shown that tolerance toward renal allografts can be achieved with transient chimerism.We also found that the therapeutic use of polyclonal recipient Tregs in a non-cytotoxic BMT model induced full immunologic tolerance toward skin and heart allografts. Transplantation of skin/heart allografts for testing donor-specific tolerance was performed with some delay after BMT in order to avoid non-specific immunosuppressive effects that could result from the drugs used for recipient conditioning . Notably, both in vitro and in vivo donor-specific tolerance are established with this regimen immediately after BMT (unpublished data from our laboratory and).Importantly, chimeras uniformly lacked detectable levels of donor-specific antibiodies, demonstrating humoral tolerance in these mice. This is of clinical relevance as one clinical pilot trial of chimerism showed that operationally tolerant patients developed donor-specific antibodies, with some even showing complement depositions in the graft.27As mentioned previously, immunologic tolerance depends on several complementary mechanisms, including intrathymic and peripheral deletion of alloreactive T cells. In this study, we demonstrated the presence of central tolerance by intrathymic deletion as well as peripheral deletion in both BMT conditioning regimens. Notably, deletion of donor-reactive T cells in Treg-induced chimeras was less pronounced and incomplete even 7 months post-BMT, suggesting other mechanisms to be more important in this model. The presence of donor-reactive T cells, despite the superior histologic outcome of cardiac allografts, suggests that dependence of active regulatory tolerance mechanisms rather than complete clonal deletion may be favorable for induction of full tolerance. This is in contrast to studies in a graft-versus-host-disease model where the administration of double-negative Tregs was shown to mediate clonal deletion of T and natural killer cells.This new tolerance approach shows remarkable potency in the establishment of donor-specific tolerance including the prevention of chronic rejection and the potential for clinical translation. Nevertheless, implementation of Treg therapy into the clinical setting remains challenging. Although initial trials using Treg therapy in clinical hematopoietic stem cell transplantation seem promising, there are still many questions to be answered. Safety concerns include a potentially non-specific immunosuppressive effect of Treg therapy that may lead to an increased risk of infections or malignancies. In addition, lineage instability of transferred Tregs could cause them to lose their suppressor phenotype and become effector T cells eventually.The use of young mice raised in a protected environment is a limitation of the present study due to avoidance of heterologous immunity and immunosenescence, which are known to impede translation of murine mixed chimerism protocols in the clinic. Strategies to overcome these hurdles have been reported, however,In conclusion, we have demonstrated that Tregs can be used to enhance BM engraftment, rendering cytotoxic recipient treatment unnecessary, and can protect cardiac allografts from chronic rejection. These results underline the clinical potential of therapeutic Treg treatment in induction of BMT-based hematopoietic chimerism and subsequent prevention of acute and chronic allograft rejection. We believe these results are relevant in the development of new protocols for tolerance induction in cardiac transplantation.The authors have no conflicts of interest to disclose.This work was supported by a research award from the International Society for Heart and Lung Transplantation (Research Fellowship award to N.P.) and the Austrian Science Fund (FWF TRP151-B19 to T.W.).We thank Dr A. Zuckermann for critical reading of the manuscript.


The aim has been to give researchers who are presenting experimental research at the main meeting, the opportunity to present their work in an informal atmosphere with time for feedback and useful discussion from the audience which usually amounts to 20 - 40 people.This year was a milestone since it was the 10Behavioral and neuroimaging correlates of white matter injury in experimental hydrocephalus”. He said that white matter tracts are known to be affected by ventriculomegaly and can be probed using diffusion tensor imaging (DTI), but not enough data have been accumulated to determine if this particular marker has reliable predictive power. He and colleagues have hypothesized that DTI and CSF pulsatility could serve as biomarkers to predict disease severity in experimental communicating hydrocephalus. This was investigated using their novel adult rat model of kaolin-induced communicating hydrocephalus, characterized by CSF flow impairments in the basal cisterns, marked changes in ventricular size, and variations in disease severity. Preliminary data has suggested that DTI changes in the corpus callosum and internal capsule were predictive of poor motor and cognitive outcomes, but that CSF pulsatility was not predictive. Ongoing analyses will correlate these non-invasive neuroimaging changes with specific cytopathology.The first speaker was Pat McAllister (University of Utah), who spoke on “Validation of diffusion tensor imaging as a biomarker for neonatal hydrocephalus”. He described an ongoing project designed to validate diffusion tensor imaging (DTI) as a biomarker to identify injury to the developing brain as a consequence of hydrocephalus. They propose to use DTI at 7 Tesla in an animal (rat) model of neonatal hydrocephalus and to compare results with outcome measures using histological examination of brain tissues and behavioral tests. The data obtained is expected to help to further understanding of pediatric hydrocephalus at the radiographic, pathologic and behavioral level.The second speaker was Weihong Yuan  on ““Tethered Cord – our first long term results in pigs”. This year they presented their first long term survival results on six pigs that underwent either sham surgery (2) or kaolin-induced experimental tethered cord (4). At the point of sacrifice (3 months), magnetic resonance imaging showed what seemed to be an increased length and thinning of the medulla of the induced tethered cord animals when compared to sham. This study supports their view that the model is useful and development of the tethered cord is possible. However, the main goal, to achieve clinical symptoms of tethered cord syndrome, has yet to be accomplished.The third talk was by Dorte Clemmensen and Mikkel Mylius Rasmussen  on “Can the Power of Zebrafish genetics be exploited to study hydrocephaly and CNS fluid balance?” He discussed their joint efforts that include investigating whether a zebrafish model might help understand the role of factors produced by the choroid plexus and ependyma in hydrocephalus. After describing the many advantages and the significant disadvantages of a fish model, he emphasized that zebrafish have been extensively exploited by developmental biologists to understand the genetic basis for disease and that the results are now widely recognized as relevant to man, within limitations. Zebrafish can be used to selectively knockdown different genes in vivo including one they are studying called ECRG4 that produces augurin in the choroid plexus epithelium and ependyma. Knockdown ECRG4 produces a gene-dependant specific hydrocephalus-like phenotype that developmental biologists call CNS "edema". Because of its low cost, amenability to high throughput experimental analyses and its proven utility as a model for human diseases, the group propose that it should be considered as a complementary -- albeit not stand alone -- alternative to other animal models of hydrocephalus. If so, they believe it might be possible to ask very basic mechanistic, yet more physiological questions that cannot be addressed using either cells in culture or mammalian models of CNS injury and inflammation.After a coffee break, Andrew Baird (UCSD) spoke on behalf of a small network of five research groups that collaborate in hydrocephalus research between San Diego, Providence (Brown University) and the UK (University of Birmingham), on “Brain biomechanics during acute obstructive hydrocephalus in live rats”. He described how biomechanically, viscoelastic, nonlinear brain can accumulate strain with cyclic loading, in young more than in mature animals. In the normal state, intracranial pressure is maintained by vascular pulsation, and pulsating stress is mitigated by CSF oscillatory exits. In the case of CSF obstruction, due to Pascal’s principles, pulsating stress transmits undiminished to all parts of the enclosed fluid, resulting in a large multiplication of applied forces. CSF is a Newtonian (non compressible) fluid, the brain is viscoelastic, enabling accumulate strain (deformation) with cyclic, pulsating stress. Pulsating stress shrinks brain parenchyma (reducing extracellular space) leads to ventricular enlargement and hydrocephalus.The next speaker was Alexander Shulyakov (University of Manitoba) who spoke on “A study of human choroid plexus mRNA reveals disease-related changes in gene expression”. To do this human lateral ventricle choroid plexuses from advanced Alzheimer's disease patients, healthy aged controls, and diseased controls  were obtained at autopsy. RNA was extracted and amplified, and human Affymetrix 48K gene arrays were utilized to investigate neurodegenerative disease-related changes in choroid plexus gene expression. The gene sets of greatest significance could be separated out into four experimental groups, revealing differences in choroid plexus gene expression when comparing the AD samples with both the normal and diseased control groups.This talk was followed by Miles Miller (Brown University) who spoke on “Pulsatility and cerebral blood flow in hydrocephalus”. They described how the movement of fluid through the cranial and spinal spaces appears to play a physiological role in cerebral compliance and therefore in blood flow. They suggest that the relationship between CSF flow and pressure pulsatility in altering cerebral blood flow is not clearly understood. In order to study the possible effect of cranial pulsatility on cerebral blood flow, they have developed a novel method and device to directly control CSF pulse amplitude via a cardiac-gated, oscillating bladder which dynamically modulates the CSF space, either accommodating or opposing entering systolic waves of blood. In an experimental model of hydrocephalus, it was possible to either augment or reduce intracranial pressure wave amplitude without affecting systemic conditions. Preliminary findings from this study suggest a relationship between CSF and CBF that may be involved in the underlying pathophysiology of hydrocephalus.The final talk of the morning was by Mark Luciano and Steve Dombrowski (Cleveland Clinic) who spoke on Overall, this was a very interesting session with lively presentations of novel research. Throughout the morning there were many discussions among presenters and audience participants that we hope were beneficial to all present.


The pressure gradient (PSG) and flow velocities in the left coronary artery were measured and compared in the left coronary models with and without presence of plaques during cardiac cycle. Our results showed that the highest PSG was observed in stenotic regions caused by the plaques. Low flow velocity areas were found at postplaque locations in the left circumflex, LAD, and bifurcation. WSS at the stenotic locations was similar between the non-Newtonian and Newtonian models although some more details were observed with non-Newtonian model. There is a direct correlation between coronary plaques and subsequent hemodynamic changes, based on the simulation of plaques in the realistic coronary models.This study was to investigate the hemodynamic effect of simulated plaques in left coronary artery models, which were generated from a sample patient's data. Plaques were simulated and placed at the left main stem and the left anterior descending (LAD) to produce at least 60% coronary stenosis. Computational fluid dynamics analysis was performed to simulate realistic physiological conditions that reflect the  Coronary artery disease (CAD) is the leading cause of death in advanced countries. The most common cause of CAD is atherosclerosis which is caused by the presence of plaques on the artery wall, resulting in the lumen stenosis. Plaques have been particularly associated with blood clots and compromise blood flow to the myocardium. This occurs when the coronary plaques suddenly rupture; if a clot cannot be treated in time, then the heart muscle will be impaired due to ischemic changes, leading to myocardial ischemia or infarction or, more severely, necrosis . Therefoin vivo will enable risk stratification for the entire coronary circulation [The natural history of coronary plaque is dependent not only on the formation and progression of atherosclerosis, but also on the vascular remodelling response. If the local wall shear stress is low, a proliferative plaque will form. Local inflammatory response will stimulate the formation of so-called “vulnerable plaque” which is prone to rupture with superimposed thrombus formation. The vast majority of these inflamed high-risk plaques cannot be detected by anatomic and myocardial perfusion imaging. Since the progression and development of vulnerable plaque is associated with low wall shear stress and the presence of expansive remodelling, measurement of these characteristics culation .The wall shear stress (WSS), wall pressure, and blood flow changes in the human body cannot be measured directly on blood vessels, whereas computational fluid dynamics (CFD) can provide alternative ways to diagnose CAD . The WSSin vivo simulation of coronary artery flow changes [CFD allows for efficient and accurate computations of hemodynamic features of both normal and abnormal situations in the cardiovascular system,  changes –6. CFD i changes , 8. In cA sample patient suspected of CAD who underwent multislice CT angiography was selected, and the patient's volume CT data was used to generate a 3D coronary model. The original CT data was saved in digital imaging and communication in medicine (DICOM) format and then transferred to a workstation equipped with Analyze 7.0  for image after-processing and segmentation. Three-dimensional (3D) volume data was postprocessed and segmented using a semiautomatic method with a CT number thresholding technique , 10, andThe actual plaques and degree of lumen stenosis on coronary artery wall were simulated at the left main stem (LMS) and the left anterior descending (LAD), as these artery branches are the common locations where plaques tend to form and induce myocardial ischemic changes , 11. TheThe surface of LCA model with and without plaques  was prepin vivo conditions, realistic physiological boundary conditions were applied for 3D numerical analysis. The transient simulation was performed using accurate hemodynamic rheological and material properties, as described in a previous study [3 and blood viscosity of 0.0035 Pa s [In order to ensure that our analysis reflects the realistic simulation of us study .  Figureus study  at the aus study  in Matlaus study . Appropr035 Pa s , 21. The035 Pa s . Blood w035 Pa s , 23. In 035 Pa s . A non-N035 Pa s , 25 whicμ∞ = 0.035, n∞ = 1.0, Δμ = 0.25, Δn = 0.45, a = 50, b = 3, c = 50, and d = 4. Generalized power law model fits experimental stress-strain measurements over the range of strain rates, −1 [−4 by approximately 100 iterations. The CFD solution was fully converged by approximately 8,000 time iterations per LCA model. The calculation time for each LCA model was approximately 2 hours. The configuration of this simulation is similar to previously published simulations [The Navier-Stokes equations were solved using the ANSYS CFX CFD package , on a Microsoft Windows 7 32-bit machine, 6 MB RAM with an Xeon W3505 2.53 GHz CPU. The CFD simulation was run for 80 timesteps, representing 1.0 second of pulsatile flow, (0.0125 seconds per timestep), with each timestep converged to a residual target of less than 1 × 10ulations , 15. Floulations , 27, whip is the pressure in the area of interest, u, v, and w are the Cartesian x, y, and z coordinates in the direction of blood flow velocity. The local PSG is calculated by taking the time derivative of the local pressure. Finally, the value of PSG oscillated in relation to the percentage of plaques in the coronary lumen [ry lumen .in vivo physiological conditions during the systolic and diastolic phases. Peak systolic velocity and pressure were reached at a time of 0.4 sec, and middiastolic phase was reached at a time of 0.7 sec during the cardiac cycles, respectively. The analysis demonstrates a strong relationship between hemodynamic change and plaques at the left coronary artery.The realistic left coronary artery models with plaques and without plaques were successfully performed with CFD analysis under 2 s2 to 345.71–629.64 kg/m2 s2, corresponding to the LMS, LAD, and LCx in the presence of plaques. In contrast, in the absence of plaques, measured PSG values were significantly lower than those measured with presence of plaques, and these values ranged from 61.79–118.57 kg/m2 s2 to 5–61.79 kg/m2 s2.Flow velocity increased significantly in the presence of plaques due to resultant lumen stenosis. Poststenotic recirculation was observed in the LMS and LAD according to the CFD analysis, at the locations where plaques were present, as shown in −1). However, the flow velocity increased in Sections C–E (velocity ranged from 23.96 to 30.50 mm s−1), at the location of plaques during the systolic peak. In addition, the flow pattern was affected by the presence of plaques, which started from Sections A–E as observed in the poststenotic region, during the middiastolic phase, with velocity increasing from 28.32 to 30.50 mm s−1.Flow velocity was visualised inside the LMS at Sections A–E, as shown in −1. Furthermore, a recirculation region was apparent at the postplaque locations in Sections I and J. The velocity increased slightly with measured values ranging from 17.43 to 23.96 mm s−1 as observed in the post-stenotic regions.−1 in both systolic peak and middle diastolic phases. Furthermore, velocity changes were not observed in Sections M–O as shown in the pre and post-stenotic regions with very similar flow patterns and velocity measured ranging from 0 to 21.79 mm s−1.Analysis of WSS was particularly focused at the stenotic locations with comparison of non-Newtonian and Newtonian fluid models. This study shows that coronary plaques produce a significant impact on the subsequent flow changes in the coronary artery, in addition to the local hemodynamic interference due to the presence of plaques. This is clinically important as further potential effects could result from the plaques' interference, leading to adverse effects on the coronary artery, such as lumen stenosis or worsening of atherosclerosis.2 s2.It is well known that plaques most commonly form in the coronary bifurcation and coronary angulation, and that this is an important factor that has been found to be related to the development of atherosclerosis, as confirmed by our and other studies , 29–31. −1. This is confirmed by our previous analysis [The presence of plaques in the coronary artery is responsible for obstructing blood flow to the myocardium, consequently affecting the flow velocity . Moreoveanalysis  showing analysis , 24. A nThere are some limitations in our study that should be addressed. Firstly, realistic left coronary models, both pre and post-stenotic, were assumed to have a rigid wall rather than elastic wall; therefore, the simulation does not fully reflect the realistic physiological situation as the coronary wall moves during cardiac cycles. Secondly, the assumption of a Newtonian blood model becomes important especially in low flow and low wall shear stress regions. Nevertheless, a previous study has shown that the assumption of a Newtonian model is reasonable in this configuration . ThirdlyIn conclusion, we studied the effect of simulated plaques in the realistic left coronary artery on hemodynamic changes at the locations of plaques, as well as pre and post-stenotic regions inside the coronary artery. There is a direct effect of plaques in the left coronary artery on hemodynamic changes such as recirculation flow, low flow velocity regions, wall shear stress, and wall pressure gradient, indicating the potential for plaques to rupture, causing atherosclerosis. Further studies focusing on the realistic plaque's effect on coronary side branches should be performed to verify our results.


Cystodiscus australis and Cystodiscus axonis are two species of myxosporean parasites infecting Australian frogs and tadpoles that have been recently recognised as important disease agents impacting amphibian conservation. Yet despite their importance to wildlife health, the mechanism of emergence for these parasites is unknown. We hypothesise that these parasites are capable of being accidentally translocated with their amphibian hosts in fresh produce  shipments into naïve environments and host populations.The spread of wildlife pathogens into new geographical ranges or populations is a conservation concern for endangered species. C. australis and C. axonis.We surveyed 33 Australian “Banana box” frogs from Sydney fruit markets during 2011 using faecal smears and multiplex species specific PCR on DNA isolated from frog faeces or using histopathology to demonstrate the presence of both Litoria gracilenta) was positive for C. australis and C. axonis in its faeces and continuously shed the parasites for eight months.One of the “Banana box” frogs, the Dainty green tree frog (Cystodiscus parasites and a non-invasive screening method to be used as a diagnostic test. In the future, vigilance and communication between wildlife managers/researchers and veterinarians will provide valuable information about these parasites, their host range and true distribution. This will aid risk management assessments for threatened populations within the range of Cystodiscus parasites and ultimately enhance conservation efforts.We present a possible mechanism for the emergence of  Cystodiscus australis and Cystodiscus axonis are two species of myxosporean parasites infecting Australian frogs and tadpoles that have been recently recognised as important disease agents impacting amphibian conservation [Cystodiscus is globally distributed and may have ecological implications outside of Australia. Eight species in three frog families  have been identified to be the hosts in New South Wales, Australia. Examination of archival specimens suggests that these parasites were not present in New South Wales prior to the mid 1960’s, but since then they have widely expanded their range and there is a significant prevalence of infection in several species of frogs [ervation -3. The gof frogs . How theBatrochochytrium dendrobatidis) via environmentally resilient spores and/or frog trade [Rhabdias pseudosphaerocephala was spread with the translocation of the Cane toad around the world [Haematoloechus floedae was introduced to Costa Rica with Rana catesbeiana [Thousands of frogs hitch rides every year in building materials, plants, and fresh food produce around Australia . As an eog trade ,8. Similhe world  and a diesbeiana .Cystodiscus shedding in the faeces. An infected frog was found and it was shown to shed both species of Cystodiscus during the entire study period, supporting a possible means of dissemination of these parasites across Australia.The aim of this study was to investigate if frogs translocated with fruit were a potential source of recently discovered emerging myxosporean parasites. To do this we surveyed frogs collected from Sydney fruit markets for evidence of Litoria infrafrenata (n = 9); Green tree frog, L. caerulea (n = 6); Desert tree frog, L. rubella (n = 15); Peron’s tree frog, L. peronii (n = 2) and Dainty tree frog L. gracilenta (n = 1). All animals were found in fruit containers at Sydney markets, collected by the Frog and Tadpole Society and examined at the Bird and Exotics Veterinary Clinic . All animals were kept isolated in the clinic for faecal sample collection, before being re-homed for captive care.In total 33 frogs were included in the study: White lipped tree frog, L. infrafrenata was examined for the presence of parasites. The animals died whilst in care for unknown reasons. Tissues including brain, liver, gallbladder, kidney, spleen and stomach were fixed in 10% buffered formalin and processed for histological sectioning and staining with H&E as described previously [Tissue from two adult frogs eviously .Cystodiscus spp. spores. During the parasitological examinations other suspected parasites were noted, including nematodes, ciliates and coccidian, but not reported in this study. Three smears were made for each sample and the number of Cystodiscus spp. spores counted and multiplied to enumerate the total number per faecal sample. Using the faecal weight the concentration was reported as spores per gram of faeces.Faecal samples were collected from all individually quarantined animals. Direct smear was used for both qualitative and quantitative examination. Faecal samples were weighed using balances with the precision of 0.001 g. Due to the small size of the faecal pellet (10–40 mg) no concentration techniques were employed. A tenth of the total volume of the faeces was mixed with 50 uL of Hartmann’s solution  and the sample viewed at 40x magnification (ISSCO BM-Lab microscope) and the entire slide observed for the presence of C. axonis and C. australis species specific PCR  targeting ITS rDNA according to Hartigan et al. [DNA was extracted from frozen or ethanol (70-100%) fixed faeces from adult frogs using the FastDNA Soil Kit Protocol with a Fast Prep-24 homogenisation system equipped with QuickPrep Adapter . The speed setting used was 6.0 for 40s, otherwise the manufacturer’s instructions were followed as described previously [L. gracilenta) adult was found to be shedding Cystodiscus spores in its faeces   × 8.7 (8–10) μm with 5–11 transverse ridges, and C. axonis, 14.1 (13–15.5) × 8.5 (8–10.5) μm with 5–12 transverse ridges and caudal filaments.Faeces from 31 frogs were examined, a single Dainty GREEN tree frog  that died spontaneously.No lesions  consistent with infection with either Cystodiscus parasites are associated with inflammation of nervous tissue and hepatic disease in a number of threatened and common frog species, yet the current distribution is still unknown [Cystodiscus parasites [Cystodiscus parasites. unknown . It was arasites . Their aarasites , with 90arasites . We testCystodiscus to New South Wales and potentially other parts of Australia. The Dainty GREEN tree frog has a range that extends from northern New South Wales all the way to the northern tip of the east coast of Australia, it is a new host record for both Cystodiscus species. Thus it would be found in the fruit and vegetable growing areas of Queensland and therefore it is likely that this frog originated from outside of New South Wales. Our study shows that both parasites are shed in significant numbers in this translocated frog and that shedding can occur for a minimum of 8 months and thus this translocated frog and possibly others could be an important source of environmental contamination at their final destination.Although only one infected frog was found in this small study set, its identification provides proof of concept that frogs from other geographic areas can disseminate both species of Litoria castanea) infected with Cystodiscus axonis[Cystodiscus spp. movement in translocated frogs. Myxospores of myxosporean species are known to be resilient [Cystodiscus spp. and potentially other pathogens.Baseline data is essential for correct quarantine and health screens of any individual. Detection of myxosporean parasites can be extremely important especially if the infected host is a critically endangered species as observed in Australia’s most threatened frog, the Yellow spotted bell frog (us axonis. This stesilient , so our Cystodiscus parasites in New South Wales and Southern Queensland, Australia with an analogous mechanism for Cystodiscus parasites in other parts of the world where fresh produce is translocated across a large geographic distance. Vigilance and communication between wildlife managers/researchers and veterinarians provides valuable information about these parasites, their host range and true distribution. These findings aid risk management assessments for threatened populations within the range of Cystodiscus spp. parasites and ultimately enhance conservation efforts. Moreover, it emphasizes how the detection of translocated frogs and the ultimate disposition of these frogs should be considered by regulatory agencies to minimize the risk that they will spread new pathogens to naive frog populations.We present a possible mechanism for the emergence of The authors declare that they have no competing interests.All authors contributed to this study. AH, LP, AR, DNP and JŠ designed the study. LP and AH performed the experiments and collected data. AH and LP analysed the data. AH and JŠ drafted the manuscript. All authors read and approved the final manuscript.