Background. The beneficial effects of testosterone treatment (TT) are debated. Methods. Double-blinded, placebo-controlled study of six months TT (gel) in 54 men aged 60–78 with bioavailable testosterone (BioT) <7.3 nmol/L and waist >94 cm randomized to TT , placebo (n = 18), or strength training (ST) (n = 16) for 24 weeks. Moreover, the ST group was randomized to TT (n = 7) or placebo (n = 9) after 12 weeks. Outcomes. Chemokines  and lean body mass (LBM), total, central, extremity, visceral, and subcutaneous (SAT) fat mass established by DXA and MRI. Results. From 0 to 24 weeks, MIF and SAT decreased during ST + placebo versus placebo, whereas BioT and LBM were unchanged. TT decreased fat mass  and increased BioT and LBM versus placebo. MIF levels increased during TT versus ST + placebo. ST + TT decreased fat mass  and increased BioT and LBM versus placebo. From 12 to 24 weeks, MCP-1 levels increased during TT versus placebo and MCP-1 levels decreased during ST + placebo versus placebo. Conclusion. ST + placebo was associated with decreased MIF levels suggesting decreased inflammatory activity. TT may be associated with increased inflammatory activity. This trial is registered with ClinicalTrials.gov NCT00700024. Testosterone replacement therapy is indicated in severe hypogonadism, whereas the indication for testosterone therapy (TT) in aging hypogonadal men without hypothalamic, pituitary, or testicular disease is debated , 2. In pAdiposity is a major component of the metabolic syndrome and an independent risk factor for the development of type 2 diabetes and cardiovascular disease . Human aAging men with low normal testosterone levels are often characterized by decreased LBM, decreased muscle strength, and relative inactivity . PhysicaIn the present study, we investigated the effects of TT on chemokine levels in aging men with low normal testosterone levels  and incrα reductase inhibitors, morphine, or oral glucocorticoid steroids. The study was a single center, randomized, placebo-controlled, six-month study to assess the effect of testosterone gel and strength training (ST) on body composition, components of the metabolic syndrome, and quality of life in men with low normal bioavailable testosterone (BioT) levels and increased body fat. The inclusion criteria for participation in the study were age 60–78 years, BioT <7.3 nmol/L, and waist circumference >94 cm. The exclusion criteria were hematocrit >50%, prostate cancer or a prostate specific antigen >3 ng/dL, previous or ongoing malignant disease, severe ischemic heart or respiratory disease, disability, diabetes mellitus, alcohol or drug abuse, abnormal routine blood samples , and treatment with 5The sample size of the study was determined by the effect of TT on LBM and has been described previously . LBM wasn = 20), placebo (n = 18), or engage in strength training (n = 16) as previously described [n = 7, and ST + placebo, n = 9) . All participants gave written informed consent. Exclusion criteria were hematocrit >50%, prostate cancer or a prostate specific antigen (PSA) >3 ng/dL, previous or ongoing malignant disease, severe ischemic heart or respiratory disease, disability, diabetes mellitus, alcohol or drug abuse, abnormal routine blood samples , and treatment with 5α reductase inhibitors, morphine, or oral glucocorticoid steroids. Four patients had previous apoplexia cerebri, and three patients had ischemic heart disease. None of the study participants were restricted during daily living activities. A total of 42% of participants were on antihypertensive drugs, 29% were on cholesterol-lowering drugs, 10% were on inhalation steroids, 6% were on antidepressants, and 4% were treated for enlarged prostate. The distribution of concomitant medication in the treatment arms was equal. No cholesterol-lowering drugs or other medications were introduced during the study. Participants received 5–10 g gel/50–100 mg testosterone  or 5–10 g gel/placebo. The randomization list, medicine labeling, and randomization and code break envelopes were generated by Ipsen Scandinavia  to ensure double blinding. Compliance was monitored by participant self-reporting at each visit. The study outcomes were evaluated at baseline and after three and six months' intervention.Subjects were randomly assigned to receive testosterone  . TreatmeThe training protocol has been described recently . In brieα, and MIF assays had intra-assay coefficient of variation (CV) of 8.1% (n = 12), 7.1% (n = 12), and 4.6% (n = 12), respectively. The samples used for the chemokine analyses had not previously been thawed.Plasma levels of the investigated chemokines were assessed using a specific human enzyme-linked immunosorbent assay (ELISA) method . MCP-1, macrophage inflammatory protein- (MIP-) 1http://www.issam.ch/freetesto.htm. During calculations, we used the assumption that albumin concentration in participants was 4.3 g/L. A single measurement of testosterone was performed to determine eligibility. BioT levels were all below 7.3 nmol/L at reevaluation after three weeks on placebo treatment (n = 18). The normal range and 95% confidence interval for BioT were 7.3 nmol/L (7.0–7.5 nmol/L) [Testosterone was measured after an overnight fast between 8 and 9 AM. Serum total testosterone was measured by liquid chromatography tandem mass spectrometry after ether extraction. For testosterone measurements, the intra-assay coefficient of variation was 10% for total testosterone >0.2 nmol/L and 30% in the range between 0.1 and 0.2 nmol/L. SHBG was measured by autoDELFIA assay, and BioT was calculated according to the formulas of Vermeulen et al. , http://ment n = . The norTFM, central fat mass (CFM), lower extremity fat mass (LEFM), and LBM were measured by DXA using a Hologic Discovery device . The CV was 0.8% for TFM and 0.6% for LBM, respectively.Magnetic resonance imaging was performed as previously reported . In brieU tests. The effect of placebo, TT, ST + placebo, and ST + TT was analyzed by comparing delta (Δ) values of hormonal and metabolic variables using the Mann-Whitney U tests. Δ-values were calculated as post treatment level minus pretreatment level of each analyzed variable. In this way, Δ-values were positive if the measured variable increased during intervention. Δchemokine levels were correlated with Δ-values of hormonal and metabolic variables using the Spearman nonparametric correlation tests. Pretreatment differences between patients in the testosterone and placebo group were tested using the Mann-Whitney P values < 0.05. were considered significant. Data are given as median and interquartile range.All statistics were performed using SPSS 17.0  for calculations and Baseline data are shown in α levels and VAT were unchanged during all interventions at 3 and 6 months, VAT data not shown.Changes in chemokines, BioT, and body composition during 3 and 6 months' intervention are shown in TT alone had no significant effects on muscle strength, whereas ST significantly increased muscle strength independent of the addition of testosterone . Circulating MIF levels decreased during ST + placebo and were significantly different from changes in MIF during placebo and changes during TT, but not from changes in MIF during ST + TT . TT increased BioT and LBM versus placebo, and fat mass  decreased. ST + placebo decreased fat mass  versus placebo without significantly affecting BioT and LBM.ST + TT was followed by similar BioT and LBM compared to TT alone. The beneficial effects of ST + TT on fat mass measures were comparable to ST + placebo.Chemokine levels were unchanged during the first three months of intervention . ST + placebo versus placebo and versus ST + TT was followed by decreased MCP-1 levels .r = −0.83, P < 0.05) and ΔLEfat . ΔMIP was significantly associated with Δfat , ΔCFM , and ΔLEfat .ΔMIF was significantly associated with Δfat (No significant associations were found between Δchemokine levels and Δfat mass measures during TT or during ST + placebo.In the present study, MIF levels increased during TT, whereas ST + placebo was associated with decreased MIF and MCP-1 levels in ageing men with low normal BioT and increased waist circumference. Our findings suggest opposing effects of TT and ST on inflammatory status. Given the on-going debate on testosterone therapy in aging hypogonadal men without pituitary or testicular disease, it is important to clarify possible favorable or unfavorable effects of testosterone therapy . Our finWe are not aware of previous studies that evaluated the long-term effect of randomized TT on chemokine levels in ageing male study populations. Six-month DHEAS treatment in women with adrenal insufficiency did not affect MCP-1 levels despite increased LBM , but fatIn the present study, ST was associated with reduced MIF and MCP-1 levels suggesting decreased inflammatory activity. These findings support previous reports in which lifestyle intervention with diet and/or increased physical activity decreased circulating chemokine levels, reduced infiltration of macrophages into the AT, and improved whole-body inflammation . We founIn the present study, resistance training and not aerobic training was included in the intervention. A recent study in patients with type 2 diabetes found that aerobic training was associated with similar weight loss but had more beneficial effects on inflammatory markers than ST . Future Change in LBM was our main study outcome . We founIn conclusion, the findings of the present study support that TT and ST have similar and positive effects on fat mass; however, ST is superior to TT regarding chemokine levels, inflammatory status, and body strength. Further studies on cardiovascular end points are needed to determine the long-term effects of TT alone and in combination with lifestyle intervention in ageing men with low normal BioT.


Sporadic Creutzfeldt-Jakob-disease (sCJD) is a fatal neurodegenerative condition that escapes detection until autopsy. Recently, brain iron dyshomeostasis accompanied by increased transferrin (Tf) was reported in sCJD cases. The consequence of this abnormality on cerebrospinal-fluid (CSF) levels of Tf is uncertain. We evaluated the accuracy of CSF Tf, a ‘new’ biomarker, as a pre-mortem diagnostic test for sCJD when used alone or in combination with the ‘current’ biomarker total-tau (T-tau). Levels of total-Tf (T-Tf), isoforms of Tf (Tf-1 and Tf-β2), and iron saturation of Tf were quantified in CSF collected 0.3–36 months before death (duration) from 99 autopsy confirmed sCJD (CJD+) and 75 confirmed cases of dementia of non-CJD origin (CJD-). Diagnostic accuracy was estimated by non-parametric tests, logistic regression, and receiver operating characteristic (ROC) analysis. Area under the ROC curve (AUC), sensitivity, specificity, positive and negative predictive values (PV), and likelihood ratios (LR) of each biomarker and biomarker combination were calculated. We report that relative to CJD-, CJD+ cases had lower median CSF T-Tf  and higher T-tau (11530 vs. 1266) values. AUC was 0.90 , 0.85–0.94) for T-Tf, and 0.93  for T-Tf combined with T-tau. With cut-offs defined to achieve a sensitivity of ∼85%, T-Tf identified CJD+ cases with a specificity of 71.6% , positive LR of 3.0 , negative LR of 0.2 , and accuracy of 80.1%. The effect of patient age and duration was insignificant. T-Tf combined with T-tau identified CJD+ with improved specificity of 87.5% , positive LR of 6.8 , negative LR of 0.2 , positive-PV of 91.0%, negative-PV of 80.0%, and accuracy of 86.2%. Thus, CSF T-Tf, a new biomarker, when combined with the current biomarker T-tau, is a reliable pre-mortem diagnostic test for sCJD. C) to a β-sheet rich pathogenic conformation called PrP-scrapie (PrPSc) Sc in the brain tissue obtained by biopsy or at autopsy Sporadic Creutzfeldt-Jakob disease is a uniformly fatal prion disorder of humans that results from refolding of the normal prion protein  variable false positives since 14-3-3 and T-tau are elevated in several other dementias besides sCJD One such potential biomarker is Tf that is increased in sCJD and prion disease affected animal brains Human tissue and CSF samples were obtained from the National Prion Disease Pathology Surveillance Center (NPDPSC) at Case Western Reserve University, Ohio. All samples are from deceased subjects, and personal information is limited to age, sex, symptoms, neuropathology, and classification of prion disease. The use of these samples has been granted waiver of informed consent by the Case Western Reserve University Institutional Review Board since the protocol meets criteria for exemption under Federal regulations 45 CFR 46.101 (b).To avoid sample bias, pre-mortem CSF from autopsy-confirmed cases of sCJD (CJD+) and dementia of non-CJD origin (CJD-) received between 2006 and 2008 were included in this study. The samples consisted of 99 CJD+ and 75 CJD- cases, amounting to a total of 174 cases. Specific diagnosis of CJD- cases included Alzheimer's Disease (AD) (17), encephalitis (3), meningoencephalitis (2), cortical dysplasia (1), cortical angiopathy (1), angiitis with microinfarcts (1), infarct (1), cerebral vasculopathy (1), Lewy body dementia (1), Lewy body variant of Alzheimer's disease (1), anoxic and Wernicke's encephalopathies (1), meningeal carcinomatosis (1), Alexander disease (1), leukoencephalopathy (1), hippocampal sclerosis (1), frontotemporal dementia (1), coccidioidomycosis meningoencephalitis (1), CNS lymphoma (1), chronic meningoencephalitis (1), epilepsy or encephalopathy (1), glioma (1), perivenous encephalomyelitis (1), CNS lymphoma (1), military metastases (1), leptomeningeal lymphomatosis/leukemia (1), mitochondrial encephalopathy (1), lymphoma (1), and non-CJD dementia of uncertain diagnosis (29). The age at autopsy ranged from 37–85 years for CJD+, and 47–84 years for CJD- cases. All CSF samples were stored at −80°C until use. Human brain tissue from the frontal cortex of CJD+ and age-matched cases of dementia (CJD−) was obtained from the NPDPSC and stored at −80°C (brain tissue and CSF samples are from different cases). Tissue homogenates (10%) were prepared in lysis buffer and analyzed by Western blotting following standard protocols.59Fe followed by fractionation on native gradient gels and autoradiography.Equal volume of CSF from CJD+ and CJD- cases was fractionated by SDS-PAGE and proteins transferred to PVDF membranes were probed with anti-transferrin antibody  followed by HRP-conjugated secondary antibody and visualization of reactive bands with ECL (Amersham). Several procedural and statistical precautions were taken to reduce error in comparing multiple samples. Procedurally, a similar protocol was followed for all Western blots, including exposure times. In addition, strips of PVDF membrane representing specific molecular weights were exposed to X-ray film simultaneously in large cassettes to obtain similar exposure. Quantification of immunoreactive bands was performed with UN-SCAN-IT software  using three exposures from a single membrane showing exponential increase in intensity. Statistically, results from different Western blots were analyzed simultaneously in logistic regression by treating these as clustered observations. The statistical software Stata allows estimation of regression coefficients after controlling for clustering to produce unbiased standard errors. Two Stata procedures were used to do this: the ‘vce cluster’ option and svy option. Each logistic regression model was first tested without adjustment for clustering and then tested with each of the 2 clustering options. Both had the effect, in general, of increasing standard errors and p-values for the coefficient estimates as expected. However, the significance of the coefficients of the biomarkers did not change; all their coefficients remained significant at the p<.05 level. T-Tf (ELISA) was estimated with Human Transferrin ELISA kit  following the manufacturer's instructions. T-tau was determined by Human Tau  ELISA Kit  as directed by the manufacturer. Iron saturation of CSF Tf was determined by radiolabeling with 59Fe-Tf). Of these, T-Tf, Tf-1, and Tf-β2 offered greater promise, and were analyzed further either alone or in combination with T-tau, a current biomarker that is quantifiable and is superior to other CSF biomarkers used for the diagnosis of sCJD The following new biomarkers were evaluated for their diagnostic accuracy: total transferrin by Western blot (T-Tf (WB)) and ELISA (T-Tf (ELISA)) techniques, Tf-1, Tf-β2, and iron saturation of Tf , which is also nonparametric. Finally, logistic regression, which makes no assumptions regarding the distributional properties of data  A combination of univariate and multivariable statistical methods were used to evaluate the relative diagnostic accuracy of individual biomarkers. Since the biomarkers were expected to evidence skewed distributions, nonparametric statistical procedures were used as these are subject to fewer distributional assumptions and provide less biased estimators when data are non-normal. For relative analysis of individual biomarkers, the nonparametric Mann-Whitney Using logistic regression results, an analytic expression for the risk of CJD was derived for each individual biomarker. Area under the receiver operating characteristic (ROC) curve and the Aikake Information Criterion (AIC) were obtained. A ROC curve graphically shows the trade-offs between sensitivity and specificity for different cut-offs used to discriminate between positive and negative cases . The area under the ROC curve (AUC) can be understood as an estimate of the probability that the biomarker being tested correctly ranks a CJD+ case higher than a CJD- case and, therefore, indexes the discriminating power of the biomarker. The AIC is used to compare different logistic regression models for different biomarkers . A model with a relatively lower AIC is considered superior due to its better fit to data and its parsimony. For each model, estimates of specificity and positive and negative likelihood ratios (LR) were obtained given a baseline sensitivity of 85%, which has precedence in the literature as a reasonable cut-off level for biomarker comparison of Alzheimer's disease (AD) To identify an optimal combination of biomarkers, all two way combinations of new and current biomarkers were also entered into a logistic regression model with controls for age, duration, and clustered data (as above). Combining new and current biomarker T-tau was intended to take advantage of the differential pathogenic mechanisms represented by these biomarkers. We focused on two-way combinations for reasons of practicality. However, a specific combination involving T-Tf and 14-3-3 could not be evaluated because the readouts for 14-3-3 are not precisely quantifiable, and this biomarker has demonstrated relatively low specificity in previous studies U test.All models were compared using three criteria: AUC, specificity, and AIC Evaluation of CSF Tf by Western blot (WB) reveals two distinct bands representing Tf-1 and Tf-β2, the latter representing deglycosylated Tf specific to the brain and CSF .Quantitative comparison shows a decrease in CSF T-Tf (WB) by 49%  and an increase in brain T-Tf (WB) by 39%  in CJD+ relative to CJD- cases  B, C. CoComparison of individual subunits of Tf shows a decrease in CSF Tf-1 by 35%  and Tf-β2 by 61%  in CJD+ relative to CJD- samples  C, E, an59FeCl3 with decreasing concentrations of unlabeled FeCl3 and fractionating 59Fe-Tf on a native gel for quantification , Tf-1, and Tf-β2 are more sensitive differentiators of the CJD+ vs. CJD- groups in comparison to the current biomarker T-tau , but not in CJD- cases . None of the new biomarkers showed a significant correlation with duration in the CJD+ or CJD- group. (Correlation of T-Tf (WB), Tf-1, and Tf-β2 in CJD+ group ρ = −0.08, p = 0.41; ρ = −0.02, p = 0.89; ρ = 0.11, p = 0.29, respectively, and in CJD- group ρ = 0.06, p = 0.62; ρ = 0.01, p = 0.96; ρ = 0.06, p = 0.64, respectively). These results indicate that T-tau changes as sCJD progresses, while the new biomarkers remain fairly stable in CJD+ and CJD- cases. None of the biomarkers showed any correlation with age.For comparative analysis of new and the current biomarker T-tau, logistic regression analysis was performed. Only T-Tf (WB), not T-Tf (ELISA) results are considered for reasons mentioned above. When individual biomarkers are tested for their diagnostic accuracy, the new biomarkers T-Tf (WB), Tf-1, and Tf-β2 are superior to the current biomarker T-tau . T-Tf W.Among the new group of biomarkers, T-Tf (WB) has by far the highest diagnostic accuracy relative to other individual biomarkers. Thus, T-Tf (WB) yields an AUC of 0.90 that is significantly higher than corresponding values for other biomarkers with the exception of Tf-1. The AIC associated with the logistic regression model using T-Tf (WB) is lowest across all other models. Finally, the specificity of T-Tf (WB) is significantly higher than that of T-Tau and equivalent to that of Tf-1 and Tf-β2. However, T-Tf (ELISA) does not have the same superior characteristics: the model has a lower AUC (0.81), higher AIC (1.06), and poorer specificity of 64.1% for reasons mentioned above.Past research shows that relative to any single biomarker, using two biomarkers in combination generally enhances the prediction of individuals likely to suffer from AD or sCJD This report describes the accuracy of new biomarkers CSF T-Tf (WB) including its two isoforms Tf-1 and Tf-β2 in identifying CJD+ from dementia of other causes. Using rigorous statistical modeling and analysis of new and currently used biomarker T-tau, we demonstrate that each of the new biomarkers is superior to T-tau, and the combination of T-Tf (WB) and T-tau is more accurate in identifying sCJD than the currently used combination of 14-3-3 and T-tau. Below, we discuss distinctive features of the methodology used for comparing new and current biomarkers, possible reasons for high specificity of new biomarkers when used singly and significant improvement in diagnostic accuracy when combined with T-tau, and limitations of our study leading to directed avenues in the identification of accurate CSF biomarker(s) for sCJD.A rigorous statistical approach was used to examine the significance (nonparametric M–W test), quantitative comparison using uniform criteria (logistic regression), and sensitivity-specificity interdependence (ROC) of different biomarkers. In contrast to specific cut-off values for individual biomarkers, a preset sensitivity of 85% was applied to all biomarkers to provide a common quantitative baseline for comparison Sc-protein complexes mitigates the signal for iron deficiency, reducing Tf secretion However, instead of the compensatory increase in CSF Tf in response to brain iron deficiency as observed in cases of Restless Leg Syndrome Regardless of the underlying cause, the difference in CSF Tf between CJD+ and CJD- cases is noted much before end-stage disease, providing a useful pre-mortem diagnostic biomarker for sCJD. When combined with the surrogate biomarker T-tau, the diagnostic accuracy of T-Tf (WB) and T-tau improves significantly, and is superior to the reported accuracy of 14-3-3 and T-Tau combination Contrary to the norm, a specific cut-off value for T-Tf or T-tau was not identified to calculate the specificity and sensitivity of these biomarkers. For T-Tf we thought it pre-mature to decide on such a value due to the limited sample size. For T-tau we did not use the conventional cut-off of 1200 or 1300 pg/ml for three reasons: 1) a consistent parameter was essential for comparing T-Tf and T-tau singly or in combination, 2) majority of CJD- samples were collected in the last month before death when T-tau is likely to be released into the CSF from damaged neurons, and 3) inclusion of conditions such as AD and brain inflammation that are associated with increased levels of T-tau in the CSF In addition to its superior sensitivity and specificity, CSF Tf by itself and in combination with T-tau offers several additional advantages as a biomarker for sCJD: 1) CSF T-Tf reflects prion disease associated brain iron imbalance and is therefore likely to be more specific In conclusion, reduced levels of CSF Tf reinforce previous reports indicating the association of brain iron dyshomeostasis with prion disease pathology, and offer promise as a pre-mortem diagnostic test for sCJD. Although alteration of CSF Tf in sCJD cases has been described previously Figure S1Tf is increased in the brain tissue of CJD+ cases. Original immunoblot of brain Tf from CJD+ and CJD- cases.(TIF)Click here for additional data file.Figure S2Iron saturation of CSF Tf is comparable in CJD+ and CJD- cases. (A) Competition of 59FeCl3 with graded concentrations of unlabeled FeCl3 for binding to CSF Tf from a CJD- case. (B) Standard curve demonstrating iron saturation of CSF Tf from a CJD- case. (C) Level of 59Fe-Tf in the CSF of CJD+ and CJD- cases is similar. (D & E) Difference between iron saturation of CSF Tf from CJD+, CJD-, and AD cases is not significant.(TIF)Click here for additional data file.


In addition, brain tissue is equipped with the synthetic machinery for melatonin production and the astrocytes and glial cells have been proven to produce melatonin. These two sources of melatonin may be responsible for its high levels in CNS. The physiological significance of the high levels of melatonin in CNS presumably is to protect neurons and glia from oxidative stress. Melatonin as a potent antioxidant has been reported to be a neuroprotector in animals and in clinical studies. It seems that long term melatonin administration which elevates CSF melatonin concentrations will retard the progression of neurodegenerative disorders, for example, Alzheimer disease. Levels of melatonin in mammalian circulation are well documented; however, its levels in tissues and other body fluids are yet only poorly established. It is obvious that melatonin concentrations in cerebrospinal fluid (CSF) of mammals including humans are substantially higher than those in the peripheral circulation. Evidence indicates that melatonin produced in pineal gland is directly released into third ventricle  Its isolation and structural identification by Lerner et al.  have beeet al. , defendiet al. , balanciet al. , 58, 67 et al. , 64, 67.In addition to the pineal gland, melatonin is found in a large number of extrapineal tissues and organs which also have the capacity to biosynthesize the indoleamine. Cells and organs that produce melatonin include astrocytes, glial cells, lymphocytes, retinal cells, gut, testes, ovary, placenta, skin, etc . Among tRecent studies have also documented that a variety of food stuffs, e.g., vegetables, cereals, fruits, nuts, seeds, grapes, red wine and beer contain considerable amounts of melatonin , 38, 45.in vivo. For example, the gut produces several hundred-fold more melatonin than the pineal gland generates [In recent decades, it was commonly accepted that the physiological levels of melatonin in the blood represent the levels throughout the body. The blood values range from several pg/ml during the day to more than 50 pg/ml at its nighttime peak. These levels, however, do not reflect the real concentrations of melatonin in tissues or in other body fluids. These values significantly underestimate the levels of melatonin enerates ; this meenerates ; this isenerates , ovarianenerates  and amnienerates  and cereenerates , 71. Thein vitro conditions [While the pathway for melatonin synthesis is well documented, there is some controversy concerning the rate limiting enzyme in its production. The essential enzymes in the melatonin pathway are arylalkylamine N-acetyltrans-ferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT). The AANAT is often portrayed as the rate limiting enzyme but this may not be the case under all circumstances  and HIOMnditions .1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), a unique melatonin metabolite, have been measured in the CSF of patients with meningitis [On other hand, extremely high levels of Nningitis . The levningitis  and mighningitis . Due to As an effective antioxidant and neuroprotector , 12, 39 1).The human pineal gland is located near the center of the brain and is surrounded with basal cistern, ventricles, choroid fissure and choroid plexus  plus mass spectrum (MS) technologies are more specific and accurate than radioimmunoassay or ELISA. Complicating the measurement is that two forms of melatonin, i.e., free and protein- bound melatonin are present in the CSF. The amounts of bound melatonin are several-fold greater than the free melatonin in CSF . Route Hvia the lumbar puncture, which is often the case, melatonin levels would be expected to be significantly lower than CSF collected from the ventricles of the brain. The site of CSF collection is also important when estimating melatonin concentrations. As mentioned previously, there is a melatonin concentration gradient in CSF throughout the third ventricle. When CSF is obtained 1.The time of CSF collection is another important factor impacting CSF melatonin levels. Melatonin levels in CSF, as in the blood, exhibit a circadian rhythm with a peak at night and basal levels during the day . For mosvia lumbar punctured was collected at night, the patient was in light at the time. Under these conditions, melatonin levels are either at their basal values or in the process of degradation. In addition, most of these studies only measured the free melatonin present in the CSF. Based on the findings reported by Rizzo et al. [As indicated in this table, the majority of these studies are performed during the day or post mortem. Even in the single case where CSF o et al. , the freMelatonin is a pleiotropic molecule that plays several important roles in CNS including circadian rhythm regulation , sleep p.), superoxide anion radical (O2.-) hydrogen peroxide (H2O2), nitric oxide (NO.) and peroxynitrite anion (ONOO-) [In the current review, we address the neuroprotective effects of melatonin. These actions of melatonin are in part based on the discovery that melatonin is a potent endogenous free radical scavenger and antioxidant . It is w (ONOO-) , but als (ONOO-) , 50. In  (ONOO-) ; this is (ONOO-) .The potential associations of physiological levels of melatonin in CSF and neurodegenerative disease, e.g, Alzheimer disease, have been reported. Several clinical investigations have shown that melatonin concentrations in CSF of Alzheimer patients are several-fold lower than those in age-matched non-Alzheimer control subjects , 61, 72.Several small scale and non-double-blinded clinical trials have tested the treatment effect of melatonin as a powerful antioxidant in Alzheimer disease. Some promising results have been obtained from these clinical studies. These studies show that the oral administration of 6-9 mg melatonin at bed time significantly retards the progression of the disease, reduces symptoms such as sundowning and modifies the sleep pattern of the patients , 16, 36.In addition to the human studies, the protective effects of melatonin on brain damage caused by a variety of processes have been intensively investigated in animal studies. Melatonin administration or pineal grafts into the brain significantly reduced the infarct volume in the rat brain induced by the middle cerebral artery ischemia/reperfusion , 14. In An obvious advantage of melatonin in neurodegenerative diseases is its ready permeability into the CNS. Melatonin as a lipophilic and hydrophilic molecule  passes tEvaluation of the melatonin levels in tissues and other body fluids based on the blood melatonin concentrations appears to be inadequate since the distribution of melatonin in the body is not homogenous. Several studies have shown that levels of melatonin in CSF are much higher than those in the blood. Evidence indicates that melatonin originating from the pineal gland and melatonin synthesized by brain tissue both contribute to the high level of melatonin in CSF. The major source of melatonin in CSF seems to come from the direct release of melatonin from the pineal gland into the pineal recess of the third ventricle. This leads to a melatonin concentration gradient in CSF as the fluid flows through the ventricular system including the aqueduct, fourth ventricle, and subarachnoid space. Melatonin in the CSF is speculated to protect the surrounding brain structures from oxidative and nitrosative stress. A low level of melatonin in CSF may relate to the etiology of neurodegenerative diseases which have elevated oxidative stress, e.g., Alzheimer disease. Decreased melatonin levels in CSF have been observed in patients with this neurodegenerative condition. Long term melatonin supplementation may retard the progress of some neurodegenerative diseases. This conclusion is based on a variety of animal studies and several small scale clinical investigations.


This study proposes the implementation of a Multiple-Network Poroelastic Theory (MPET) model coupled with finite-volume computational fluid dynamics for the purpose of studying, in detail, the effects of obstructing CSF transport within an anatomically accurate cerebral environment. The MPET representation allows the investigation of fluid transport between CSF, brain parenchyma and cerebral blood, in an integral and comprehensive manner. A key novelty in the model is the amalgamation of anatomically accurate choroid plexuses with their feeding arteries and a simple relationship relaxing the constraint of a unique permeability for the CSF compartment. This was done in order to account for the Aquaporin-4-mediated swelling characteristics. The aim of this varying permeability compartment was to bring to light a feedback mechanism that could counteract the effects of ventricular dilation and subsequent elevations of CSF pressure through the efflux of excess CSF into the blood system. This model is used to demonstrate the impact of aqueductal stenosis and fourth ventricle outlet obstruction (FVOO). The implications of treating such a clinical condition with the aid of endoscopic third (ETV) and endoscopic fourth (EFV) ventriculostomy are considered. We observed peak CSF velocities in the aqueduct of the order of 15.6 cm/s in the healthy case, 45.4 cm/s and 72.8 cm/s for the mild and severe cases respectively. The application of ETV reduced the aqueductal velocity to levels around 16–17 cm/s. Ventricular displacement, CSF pressure, wall shear stress (WSS) and pressure difference between lateral and fourth ventricles (ΔP) increased with applied stenosis, and subsequently dropped to nominal levels with the application of ETV. The greatest reversal of the effects of atresia come by opting for ETV rather than the more complicated procedure of EFV. Hydrocephalus (HCP) can be succinctly described as the abnormal accumulation  of CSF within the brain ml, where only 25 ml are segregated to the ventricles and the remainder occupies the cranial and subarachnoid spaces + clearance, such as nonmyelinated axons and the nodes of Ranvier In this work we also consider the effects of including aquaporin-4 (AQP4) within our mathematical framework. It is the most predominant aquaporin in the brain, and is located on the external and internal glial limiting membranes, the basolateral membrane of ependymal cells CSF is secreted continuously; however, findings indicate strong circadian variations to its production thought to be due to the finely regulated responsibilities of the autonomic nervous system CSF absorption is more complex than the existing classical theory just outlined. As opposed to the highly orchestrated transport process of CSF secretion, CSF absorption occurs via other outflow mechanisms, such as along the sheaths of major blood vessels and cranial nerves Endoscopic third ventriculostomy (ETV) was initially restricted to patients with triventricular HCP, where a bulging translucent floor of the third ventricle floor was evident and with a patient age threshold of greater than two years A special case of atresia of the foramina of the fourth ventricle was witnessed by Gianetti et al. For relevant overviews on CSF hydrodynamics and their applications, the reader may refer to the work of Dutta-Roy et al. The aim of the paper is to justify a means by which derivative pathologies of HCP  can be alleviated via two surgical means, namely endoscopic third and fourth ventriculostomy. A computational comparison of the two techniques is presented using a novel application of Multiple-Network Poroelastic Theory (MPET) to investigate cerebral fluid transport. A detailed investigation of multiscalar, spatio-temporal transport of fluid between the cerebral blood, cerebrospinal fluid (CSF) and brain parenchyma is conducted. Specifically, the MPET model of the cerebral tissue is coupled with a three-dimensional representation of the CSF flow patterns arising from the source of production (CP) within a patient-derived cerebroventricular system.3. These were then interpolated to 0.5 mm isotropic voxel size volume data which was used as an input for further processing.Imaging was performed on a 1.5T GE Signa system  and a T2 weighted imaging sequence was used for obtaining the brain anatomy data . Images www.blender.org). The geometry can be seen in Solution Method and post processing section.The acquired voxels were manually segmented for the ventricular system using Amira  and the raw segmented geometry from this process was converted to a Stereo Lithography (STL) file. In order to preserve key anatomical features such as the Sylvian aqueduct, subsequent smoothing of the STL file was done using the open-source modelling software, Blender  and (2).a), lower pressure arteriole/capillary network (c), extracellular/CSF network (e) and finally a venous network (v).The MPET theory will revolve around the quadruple-MPET model. This means letting In the ensuing discussion, a spherical representation of the brain geometry will be utilised pμ, pκ and pα, where p = a,e,c,v (described above) represent the viscosity, permeability and Biot parameters of the interstitial fluid networks. The parameters a in equations 1 and The parenchyma of an adult brain is represented as a spherical shell. The outer radius of this shell is given by Assuming a linear stress-strain relationship and isotropic permeability, the effective stress is represented by Hooke’s law, and is inverted for stress and subsequently represented as a function of displacement with the aid of Einstein notation to calculate divergence:G is the shear modulus and is equivalent to E is the Young’s modulus and v is the Poisson ratio. An isotropic permeability would imply u, ∇(∇⋅u), ∇⋅b and From the above, Additional assumptions include the notion that HCP has a long time scale for development, of the order of days, weeks or even years (if one considers the chronic case). It is therefore evident that an assumption of a quasi-steady system is not unrealistic. It should be noted that the MPET model does not take pulsatile effects into account.An insight into the specifics of continuity and inter-compartment transport for the system is also important. It is necessary to restrict the transfer of CSF between fluid networks, and inQ, outQ and accCVQ represent the flux entering, leaving and accumulating in the control volume. The rest of the flux transfer terms have been explained in the earlier parts of this chapter, and are driven by a hydrostatic pressure gradient.From equations 4a–4d, it is noted that More segregated assessments on the transfer include: i) Transfer of flux occurs between the arterial and capillary network on an on-going basis, hence s and t. The values of the Biot parameters, permeabilities and transfer coefficients are given in 10 permutations. The details are outlined in Tully & Ventikos In equations 5a–5e, the transfer between networks is via a hydrostatic pressure gradient, hence The following relationship was proposed to alleviate the constraint of a unique permeability for the CSF compartment in order to account for AQP4′s swelling characteristics:ek and eµ have already been described, eP is the CSF pressure, refP is a reference pressure and finally fA is an amplification factor, and here it has a value of unity. The permeability was made to vary between the interval: eP. refP was chosen to possess a value refP = 1 kPa. Due to insufficient experimental data, AQP4 expression is not accounted for in From Finally, we define a useful parameter, namely the increment of fluid content, ζ, in terms of strain, ε as:K, the bulk modulus, is given by K, λ and G are given in In As discussed earlier, the shell responsible for outlining the skull lies at The blood pressures in the arterial and venous compartments are assigned physical values:c) at the skull and the resulting accumulation of CSF into the venous network ultimately results in a pressure rise, hence:In addition to the above boundary conditions, there is a restraint on the flow both into and out of the capillary network , and more specifically, it represents the amount of CSF produced by the porous choroid plexuses:The final boundary condition involves the conservation of mass of fluid within the ventricular system  and pressure difference between isolated points in the lateral ventricle and fourth ventricle  possibilities taken into consideration. Firstly, the atresia of the bilateral foramina of Luschka was investigated, along with corresponding values for peak velocity in the aqueduct, Magendie and central canal. In addition to this, the WSS values at the surface of the aqueduct were investigated along with the previously defined pressure difference ΔP, in order to determine the extent of ventriculomegaly under FVOO.Secondly the sole atresia of the foramen of Magendie was investigated along with corresponding values for peak velocity in the aqueduct, (average of the right and left) bilateral Luschka and central canal. Once again the WSS values at the surface of the aqueduct along with the pressure difference are recorded. Finally the occlusion of both of the aforementioned outlets was investigated along with same additional comparators previously outlined.Atresia was simulated as a blocked outlet, whilst the application of EFV was to apply a concentric hole within the blocked outlet under consideration. As in the case for ETV, a boundary condition of 0 Pa was assigned.The governing multicompartmental poroelastic equations are solved with an implicit second-order central finite differences scheme on the midpoints and forward/backward Euler used on the boundary nodes. The quasi-steady time discretization  is performed via a first-order Euler approach.Flow through the multidimensional ventricles is solved using the multiphysics software CFD-ACE+  which is based on the finite volume approach, along with central spatial differencing, algebraic multigrid scheme and the SIMPLEC pressure–velocity coupling. The coupling between the poroelastic solver and the flow solver is achieved through appropriate CFD-ACE+ user-defined subroutines.Mesh generation see  for the The base discretisation for the spherically symmetric MPET model involved 81 nodes, as this yielded results within a 5% band from what we considered as the fully converged solution: full grid independence was achieved at 221 nodes. Similarly, adequate discretization of the 3D ventricular domain is required in order to acquire fully resolved results. Similar grid independence analysis resulted in meshes of over 2 million cells for that domain.The coupling between the poroelastic solver and the flow solver is achieved through the use of CFD-ACE+s’ user-defined subroutines. After an initial assumption of the aqueductal flow, the pressures at the skull and inside the ventricles are passed to the user-defined subroutines as pressure boundary conditions (after each time step of the poroelastic solver). Following the Finite Volume solution of the ventricular and therefore aqueductal flow, the mass flux is calculated in the user-defined subroutines and passed to the poroelastic solver for the next iteration.CFD-VIEW  is used as the post processing tool to analyze the flow physics see . This tod, for the open, mild and severe cases is given in The application of ETV substantially reduced the level of ventricular displacement to 0.70 mm for the mild case and around 2.25 mm for the severe case. This is a fair reduction in both cases, but especially for the mildly stenosed case, as its ventricular displacement level has been reduced to a level resembling the case of the open aqueduct. All of the aforementioned ventricular displacement values are taken at The CSF pressure variation to aqueductal stenosis shown in 2, whilst for the mild and severe cases of stenosis this increased to around 5.8 and 12.2 N/m2 respectively. The WSS was alleviated by the application of ETV , whilst EFV reduces it only marginally to 16.7 Pa. The peak velocities in the central canal, bilateral foramina and aqueduct are reduced to a greater extent with the application of ETV as opposed to EFV. Applying ETV and EFV reduced the ventricular displacement to just under 1.22 mm and 0.77 mm respectively. CSF Pressure increases to 1044.12 Pa for the ETV case, whilst for EFV it increases to 1060.56 Pa.The situation is similar for the occluded Magendie. The peak velocity through the aqueduct increases from 15.6 cm/s in the patent aqueduct case  to 16.6 2. The pressure difference remains more or less constant, decreasing by just 2.1 Pa when compared to the open case. The ventricular displacement increases to approximately 1.25 mm, whilst the CSF pressure decreases to 1042.95 Pa.The final scenario involves the occlusion of all fourth ventricle exits except the central canal. The peak aqueduct velocity decreases slightly from 15.6 cm/s to 15.0 cm/s, whilst through the central canal it increases drastically from 2 cm/s to 35.5 cm/s. In addition, the WSS possesses a modest value of 0.63 N/mApplying ETV, we see a reduced peak velocity through the aqueduct to a greater extent than EFV (which increased compared to the combined obstruction of Luschka and Magendie); however EFV reduced the velocity in the central canal (4.6 cm/s as opposed to 5.6 cm/s). Finally, the pressure difference assumes a larger value due to ETV (∼6 Pa) as opposed to its competing neurosurgical technique (which raises it to ∼18 Pa). Further investigation in the vicinity of the ETV outlet was required to justify the lower than expected pressure difference between lateral and fourth ventricle. The velocity magnitude of CSF near the lower region , and the occlusion of individual or a combination of outlets of the fourth ventricle. The physical comparators used to describe the effects of this induced HCP include ventricular displacement, CSF pressure, peak velocities, pressure difference between lateral and fourth ventricle and wall shear stress. These comparators are also used to give an indication of the alleviating effects of the two surgical procedures proposed.It is evident from the results garnered in this study that the central canal does not simply act as a pathway for CSF flow. Gupta et al. A noticeable difference is seen during the cases of FVOO, where the peak velocity in the central canal exceeded 35 cm/s during the combined occlusion of the bilateral foramina and the foramen of Magendie. The application of both ETV and EFV reduced the impact of the occlusions on the central canal for nearly all cases; however, ETV did so with greater success, as can be seen from the results in Likewise, applying ETV at the floor of the third ventricle reduces both the ventricular displacement and CSF compartmental pressure to manageable levels. As the aqueduct tries to maintain the laminar CSF flow (350< Re <700) through the third and fourth ventricle by assisting in repelling the core of central fluid from the aqueductal wall effects One could appreciate that such a form of pronounced atresia could initiate secondary pathologies to FVOO, such as Syringomyelia. Indeed, recent developments indicate an increase in the expression of AQP4 in conjunction with the level of central canal occlusion and subsequent dilation P was reduced further, wall shear stress as well as ventricular displacement. CSF pressure was reduced below the levels expected from an individual with an open aqueduct. This fortifies the view of Mohanty et al. What was interesting is that although the management of the FVOO seems to be commensurate, it must be noted that ETV performed better in comparison, reducing: the average peak velocity in the aqueduct for the three cases of atresia, the pressure difference ΔA simplified boundary condition of 2000 Pa for the arteries was utilised for conservation of computational resources. Unlike previous work, the amount of CSF produced in the lateral, third and fourth ventricles was assumed identical, and not confined to a ratio in terms of total CSF produced. However, there is general agreement that there is no literature outlining the exact proportions of CSF production in the various choroid plexus sites ek) was relaxed in this study, and given way to a fluctuating adaptation represented by e <PrefP, then the permeability increases, whilst the converse is true for e >PrefP. Strictly speaking, AQP9 is also found in astrocytes and may possess a synergy in aiding AQP4 The unique permeability of the CSF compartment . It should be noted that the aforementioned velocity mappings were not used on any volunteers suffering from hydrocephalic symptoms, and it was stressed that the effect of aging on the CSF flow categorisations could be ignored One of the main reasons for considering detailed CSF flow categorisations is that areas of interest such as CSF flow diversion techniques  for treating obstructive HCP can only benefit from more detailed qualitative (and quantitative where applicable) CSF flow observations and categorisations. For instance, CSF flow dynamics in known to be influenced by the position of the interthalamic adhesion see , and it P drops back to levels similar to pre-stenosis states. The change in pressure is heavily influenced by very high degrees of stenosis, as expected from the governing MPET formulation. During FVOO however, we see a seemingly constant pressure difference of around 20 Pa present. Treating individual cases of obstructed Luschka or Magendie with ETV reduces ΔP to levels below those witnessed by an unobstructed configuration. This could prove to induce adverse therapeutic effects for alleviating the symptoms of HCP, and would prove an interesting scenario to validate experimentally.Levine The pressure difference between lateral and fourth ventricle indicates an area that requires further investigation. This is especially important for FVOO cases treated with ETV. Our results showed lower than expected levels of WSS and peak aqueductal velocity. Once applied, ETV also manages to increase the CSF velocity in the vicinity of the outlet by an average of The limitations surrounding the proposed MPET model include the use of a linear stress-strain relationship in light of evidently large strains. A spherically symmetric geometry which could theoretically prohibit the interpretation of the order of dilation amongst the various ventricular cavities is also an area of improvement. The simplification to quasi-stationary equations assumes altered pulsatility in the environment and does not play a role in hindering the investigation of properties over the time scale of HCP. In order to more accurately investigate their role, the more general transient equations must be solved. A constant isotropic permeability is also assumed, barring the possible effects of investigations of the diminishing of cerebral blood flow. The use of constant material parameters, such as elastic properties of the parenchyma, compartmental porosity, permeability and transfer coefficients is an additional limitation.Methods section. Current MRI techniques utilizing single point ramped imaging with T1 enhancement have been used to assess the porosity of sandstone The quadruple MPET model introduces an additional ten parameters that are unknown; these are the Biot parameters and permeabilities of the additional blood networks, and the transfer coefficients between the fluid networks. As far as the authors are aware, no suitable experimental data exist and approximations are made through an extensive parametric search detailed in Tully & Ventikos in vivo; however, they are yet to establish consistent and agreeable values. Evidence of this is that the reported numbers differ by orders of magnitude Promising studies using magnetic resonance elastography (MRE) calculate the shear stiffness of cerebral tissue in vivo pressure measurements are not commonly obtained with current technological means, and so this virtual boundary condition was chosen for simplicity, since the subarachnoid space was not taken into consideration.The inclusion of the choroid plexuses in the region of the lateral, third and fourth ventricles, along with a pressure inlet boundary condition of 2 kPa (15 mmHg) at these locations substantially increased the magnitude of the peak CSF velocity flowing through ventricular cavities (with the most pronounced effect being in the aqueduct). This should be expected as this is the first time that this level of detail is attached to the inlet boundary conditions of the regionally accurate choroid plexuses, and in addition, at all CSF producing sites  of the ventricular system. The level of porosity and permeability at the surface of the choroid plexuses plays an important role in ultimately determining the egressing CSF velocities; however, there is an evident lack of experimental data surrounding these values. The value of 2 kPa for the aforementioned boundary conditions arises from generally accepted pressure values in the relevant conflated feeding arteries. Work conducted by Bammer et al. There is currently no clinical literature that allows for a comparison of the MPET model, with all the sites of CSF production present to be compared. It would also be taxing to suggest at this point in time, that a comparison of the ETV and EFV procedures on artificially induced aqueductal stenosis and FVOO be made with clinical data. To the authors’ knowledge, no MRI obtained velocities exist in these circumstances for such a comparison to be made. Shunting on the other hand, would have proven easier to compare to, however, there is an exhaustive range of literature using shunting as opposed to ETV as a method of alleviating the symptoms of aqueductal stenosis, and none for EFV alleviating the symptoms of FVOO.This study presents the first assessment of the impact of aqueductal stenosis and fourth ventricle outlet obstruction along with the applications of ETV and EFV, on an anatomically accurate representation of the cerebroventricular system. A simple relationship relaxing the constraint of a unique permeability for the CSF compartment in order to account for the Aquaporin-4 swelling characteristics has also been incorporated into this MPET framework which can allow for the intrinsic investigation of multiscalar, spatio-temporal transport of fluid between cerebral blood, CSF and the brain parenchyma.The findings in this study support the appeal of ETV in cases of mild and severe aqueductal stenosis, as the metrics of comparison used in this work show an appreciable reversal of the effects of stenosis. FVOO is a new case taken under consideration, and the comparison of ETV and EFV on the different exit sites of the fourth ventricle proves to be more intricate. The results from this study outline that the greatest reversal of the effects of all three forms of atresia come by opting for EFV rather than ETV. However, our ETV findings indicate levels of aqueductal velocity and pressure difference between lateral and fourth ventricles to be low in comparison to the normal case. A full understanding of these effects will require a detailed estimate of ETV outlet boundary conditions, with the use of 3D PC-MRI in order to assess the complex CSF dynamics in the region. The interthalamic adhesion has the potential to alter the CSF dynamics in the third ventricle, and so this investigation should preferably be done simultaneously.Other methods that could influence the CSF flow such as shunt placement, can be easily incorporated into this current framework. The addition of the aquaporin swelling characteristics into this detailed model allows the initiation of a formal discussion on how these channels influence CSF transport. Pharmaceutical regulation of AQP’s may also be incorporated into such an extendable mathematical framework.


Expanding the criteria for deceased organ donors increases the risk of delayed graft function (DGF) and complicates kidney transplant outcome. We studied whether donor neutrophil gelatinase-associated lipocalin (NGAL), a novel biomarker for acute kidney injury, could predict DGF after transplantation.We included 99 consecutive, deceased donors and their 176 kidney recipients. For NGAL detection, donor serum and urine samples were collected before the donor operation. The samples were analyzed using a commercial enzyme-linked immunosorbent assay kit (serum) and the ARCHITECT method (urine).P = 0.028), and 1-year graft survival was worse . High U-NGAL concentration was also associated with significantly more histological changes in the donor kidney biopsies than the low U-NGAL concentration. In a multivariate analysis, U-NGAL, expanded criteria donor status and eGFR emerged as independent risk factors for prolonged DGF. U-NGAL concentration failed to predict DGF on the basis of receiver operating characteristic curve analysis.Mean donor serum NGAL (S-NGAL) concentration was 218 ng/mL  145.1) and mean donor urine NGAL (U-NGAL) concentration was 18 ng/mL . Donor S-NGAL and U-NGAL concentrations correlated directly with donor plasma creatinine levels and indirectly with estimated glomerular filtration rate (eGFR) calculated using the modification of diet in renal disease equation for glomerular filtration rate. In transplantations with high (greater than the mean) donor U-NGAL concentrations, prolonged DGF lasting longer than 14 days occurred more often than in transplantations with low (less than the mean) U-NGAL concentration (23% vs. 11%, This first report on S-NGAL and U-NGAL levels in deceased donors shows that donor U-NGAL, but not donor S-NGAL, measurements give added value when evaluating the suitability of a potential deceased kidney donor. P = 0.001; unpublished data, Helsinki University Hospital, Division of Transplantation, Kyllönen L and Salmela K).Deceased kidney donors are expected to have healthy kidneys which will function well in the recipient after transplantation. However, a considerable number of kidney transplantations from deceased donors are complicated by delayed graft function (DGF). There is no consensus on the ultimate effect of short DGF, lasting less than one week, on graft survival; however, when the duration of allograft dysfunction becomes prolonged, the negative effect on kidney graft survival becomes evident ,2. The cThe quality of donor kidneys has a clear impact on long-term kidney allograft outcomes -7. VarioNeutrophil gelatinase-associated lipocalin (NGAL) is a new marker for acute kidney injury (AKI) which has been studied after cardiac surgery, liver transplantation and contrast media administration, as well as in intensive care unit (ICU) patients (in heterogeneous patient groups and in patients with septic vs. nonseptic AKI), in unselected patients who present to the emergency department and in critically ill multiple trauma patients -22. So fPlasma creatinine level is known to be a poor early detector of AKI. Thus, a simple laboratory test revealing AKI early on would be useful for clinicians taking care of potential donors in ICU when evaluating the quality of their kidneys. In this prospective study, we wanted to examine (1) the levels of serum NGAL (S-NGAL) and U-NGAL in deceased kidney donors, (2) whether donor S-NGAL and/or U-NGAL could be used as predictors of DGF and especially (3) prolonged DGF after kidney transplantation.The present study was performed at Helsinki University Hospital, which provides organ transplant service for Finland, which has a population of 5.2 million. For this study, we prospectively enrolled 99 consecutive, deceased, heartbeating donors and their 176 adult kidney recipients between August 2007 and December 2008. The study protocol was approved by the Helsinki University Hospital Ethics Committee and the hospital's Department of Surgery. Written informed consent was obtained from the recipients before enrollment.Altogether 198 kidneys were obtained from the 99 donors. One kidney was not transplanted because of a vascular lesion. Twenty-one kidneys were not included in the study: six were used for pediatric recipients, two were used for recipients who underwent combined kidney and liver transplantation and one was used for a combined kidney and lung transplantation. Nine kidneys were shipped to the other Nordic countries according to the Scandiatransplant exchange rules. Three patients did not consent to participate in the study. The recipients of the remaining 176 kidneys were included in this study.et al. [Donor clinical history data were obtained from the hospital records. The following variables were gathered: age, gender, history of hypertension, need for cardiopulmonary resuscitation, need for intracranial surgery, use of vasopressor support, use of antidiuretic hormone (ADH), plasma creatinine level, length of hospital stay before brain death diagnosis, cause of death and multiorgan or kidney-only donation. Estimated glomerular filtration rate (eGFR) was calculated using the modification of diet in renal disease equation for glomerular filtration rate (MDRD equation)  in 96 adet al. , which iin situ perfusion was initiated. The University of Wisconsin solution was used for in situ perfusion and cold storage preservation of the kidneys. A biopsy for histological evaluation was taken from the donor kidney before the initiation of in situ perfusion. The biopsies were examined later and scored using the Banff 97 criteria [Intravenous steroids were given to all donors before undergoing the organ retrieval operation, and they were given mannitol before criteria  and the criteria  to quantRecipient clinical data were obtained from the patients' hospital records and the Finnish Kidney Transplant Registry database. Plasma creatinine concentration was recorded daily after transplantation during the recipient's stay in the transplant unit, then at 3 months and 1 year after transplantation. eGFR was calculated using the MDRD equation  at 3 monet al. [n = 106), short DGF lasting less than 14 days (n = 43) and prolonged DGF lasting 14 days or longer (n = 27).The primary recipient outcome variable was onset of graft function after transplantation. DGF was defined as described by Halloran et al. : oliguriSerum samples for NGAL analyses were taken for logistical reasons in the donor hospital simultaneously with blood samples for HLA determination. The serum sample was drawn before the diagnosis of brain death in 36 cases  and after that in 63 cases . The serum samples were taken before steroid administration in 77 donors and after that in 22 donors. Urine samples were taken by the transplant team at the beginning of donor surgery. Thus all donors had already received the steroids before their urine samples were taken. All samples were immediately centrifuged at 2,500 rpm at 4°C for 10 minutes, and after that the serum and urine supernatant were divided into tubes and frozen at -70°C. No additives were used.n = 2) or incorrectly processed (n = 2) sampling.The S-NGAL assays were performed using a commercial enzyme-linked immunosorbent assay (ELISA) kit  as recommended by the manufacturer. The measurements were performed in duplicate and blinded to sample sources and clinical outcomes. Serum samples were available for NGAL analyses from 95 donors. In four cases, S-NGAL levels could not be analyzed because of inadequate (n = 1) or incorrectly processed (n = 3) sampling.The U-NGAL assays were performed using a standardized clinical platform  as previously described . Urine sn = 38; U-NGAL ≥18 ng/mL, n = 26) and a low NGAL group .We divided the donors using the mean NGAL concentrations as cutoffs into a high NGAL group  were used. Factors which were significantly different between the DGF and EGF groups in the univariate analyses, as well as for the other clinically relevant factors in this respect, were included in the multivariate analyses. The factors in the multivariate analyses consisted of categorical variables and the covariates of continuous variables. The parametric correlations were assessed using the Pearson correlation coefficient, and the nonparametric correlations were assessed using the Spearman correlation coefficient. Receiver operating characteristic curve (ROC) analysis was performed to assess the potential of NGAL to predict DGF. Positive and negative predictive values were calculated using Bayes' formula. A P value < 0.05 was considered significant.SPSS version 18.0 software  was used for statistical analyses. All analyzed variables were tested for distribution. Student's P = 0.001). Acute rejection occurred in 10 (5.7%) of 176 transplantations at a mean of 16.8 days after transplantation .Table R2 = 0.35, P = 0.001) and inversely with donor eGFRs .The mean donor S-NGAL concentration was 212 ng/mL . Donor S-NGAL concentrations correlated directly with donor plasma creatinine levels  and inversely with donor eGFRs . Donor U-NGAL concentrations correlated directly with donor S-NGAL concentrations .The mean donor U-NGAL concentration was 18 ng/mL . Donor U-NGAL concentrations correlated directly with donor plasma creatinine levels . Donor S-NGAL and U-NGAL levels did not correlate with donor age  and were not affected by gender, history of hypertension, use of vasopressors, length of hospital stay, need for cardiopulmonary resuscitation or intracranial surgery before brain death, ECD or standard criteria donor status, and multiorgan or kidney-only donation. In addition, there were no significant differences between donor S-NGAL levels in samples taken before or after brain death or before or after steroid administration  and U-NGAL  levels compared to those not treated with ADH  showed normal histology. The mean CADI score of the biopsies was 0.72, ranging from 0 to 5 Figure . OverallP = 0.002). Mean donor S-NGAL did not differ significantly between the prolonged DGF , short DGF  and EGF  (P = NS) groups. There were no significant differences in mean donor S-NGAL and U-NGAL levels in the DGF (including both short and prolonged DGF)  and EGF  groups. High donor U-NGAL level was associated with more prolonged DGF and worse 1-year graft survival compared to low donor U-NGAL level  compared to those with short DGF  or EGF   in addition to donor S-NGAL, U-NGAL, eGFR and plasma creatinine levels.per se. Donor U-NGAL, ECD status and eGFR emerged as independent risk factors for prolonged DGF (Table None of the included factors appeared to be a significant risk factor for DGF GF Table . ROC anaGF Table  resultedGF Table  resultedP = NS).A pair kidney analysis was possible in 77 donors (154 kidneys). In 28 of 77 cases both donated kidneys had EGF, in 13 of 77 cases both kidneys had DGF and in 36 of 77 cases one of the kidneys had DGF and the other had EGF. If one kidney had DGF, the other was not at increased risk for DGF  in donor biopsies, but the predictive power of U-NGAL with regard to the onset of graft function was weak. Donor S-NGAL levels did not have predictive power with regard to the onset of graft function after transplantation. Donor U-NGAL level, but not S-NGAL level, is useful when evaluating a potential deceased organ donor.• Deceased donor U-NGAL concentration is useful when evaluating the kidneys of potential organ donor candidates in the ICU.• The mean S-NGAL concentration (determined by ELISA) in deceased donors was 212 ng/mL, corresponding to the previously reported levels in critically ill patients.• The mean U-NGAL concentration (determined by the ARCHITECT method) was 18 ng/mL, which was well within the range of healthy controls.• Deceased donor U-NGAL concentration is more specific than S-NGAL concentration to kidney injury.• High deceased donor U-NGAL concentration was associated with prolonged oliguria after kidney transplantation and chronic histological changes in donor kidney biopsies, but it was a poor predictor of DGF in individual cases.ADH: antidiuretic hormone; AKI: acute kidney injury; CADI: Chronic Allograft Damage Index; DGF: delayed graft function; ECD: expanded criteria donor; eGFR: estimated glomerular filtration rate; EGF: early graft function; MDRD: modification of diet in renal disease equation for glomerular filtration rate; NGAL: neutrophil gelatinase-associated lipocalin; S-NGAL: serum neutrophil gelatinase-associated lipocalin; SD: standard deviation; U-NGAL: urine neutrophil gelatinase-associated lipocalin.One author of this manuscript has conflicts of interest to disclose. MEH was sponsored by Abbott Diagnostics to the American Transplant Congress in 2010, where an oral presentation of this study was presented. She also received an honorarium for presenting the recipient U-NGAL data  in a meeMEH collected data, carried out S-NGAL analyses, analyzed data and wrote the paper. LEK designed study, analyzed data and wrote the paper. KAI carried out U-NGAL analyses. MLTL and JM designed study. KTS designed study and wrote the paper. All authors read and approved the final manuscript.Donor serum and urine neutrophil gelatinase-associated lipocalin (U-NGAL) and donor parameters. The additional data file shows the association between donor parameters and serum NGAL and U-NGAL concentrations.Click here for file


We also show that high expression of the mannose receptor (CD206), a marker of alternative activation, is a distinct feature of GM-CSF MΦ. Changes of the chromatin structure carried out by chromatin modification enzymes (CME) have been shown to regulate myeloid differentiation. We analyzed the expression patterns of CME during MΦ polarization and show that M1 up-regulate the histone methyltransferase MLL and demethylase KDM6B, while resting and M2 MΦ were characterized by DNA methyltransferases and histone deacetylases. We demonstrate that MLL regulates CXCL10 expression and that this effect could be abrogated using a MLL-Menin inhibitor. Taken together we describe the distinct phenotypic differences of GM-CSF or M-CSF MΦ and demonstrate that MΦ polarization is regulated by specific epigenetic mechanisms. In addition, we describe a novel role for MLL as marker for classical activation. Our findings provide new insights into MΦ polarization that could be helpful to distinguish MΦ activation states.Macrophages (MΦ) play an essential role in innate immune responses and can either display a pro-inflammatory, classically activated phenotype (M1) or undergo an alternative activation program (M2) promoting immune regulation. M-CSF is used to differentiate monocytes into MΦ and IFN-γ or IL-4+IL-13 to further polarize these cells towards M1 or M2, respectively. Recently, differentiation using only GM-CSF or M-CSF has been described to induce a M1- or M2-like phenotype, respectively. In this study, we combined both approaches by differentiating human MΦ in GM-CSF or M-CSF followed by polarization with either IFN-γ or IL-4+IL-13. We describe the phenotypic differences between CD14 Macrophages (MΦ) are tissue resident cells of myeloid origin that are found in various organs where they contribute to immune responses by the killing of pathogens and the secretion of pro-inflammatory cytokines -3. They in vitro to induce a M1 phenotype, while IL-4 and IL-13 induce M2 MΦ. GM-CSF and M-CSF, growth factors traditionally used to differentiate monocytes into dendritic cells or MΦ, were recently found to alone promote a M1- and M2-like phenotype, respectively [IFN-γ alone or in combination with LPS is commonly used ectively -8. Thus,Hematopoietic differentiation involves epigenetic modifications that are responsible for specific changes in the chromatin structure enabling or inhibiting the transcription of genes . Using mhi CD163hi CD206int cells that express FOLR2 and GM-CSF MΦ as CD14lo CD163lo CD206hi. In addition, we demonstrate that polarization with either IFN-γ or IL-4+IL-13 induces a distinct pattern of gene expression that is independent of the growth factors. Importantly, the mannose receptor CD206, a well-described marker for alternative activation [In this current study we analyzed the expression of phenotypic markers in GM-CSF- and M-CSF-derived, IFN-γ- and IL-4+IL-13-polarized M1 and M2 MΦ. We define M-CSF MΦ as CD14Because of the importance of epigenetic changes during hematopoietic cell differentiation ,17, we a6 cells/ml in complete RPMI 1640 medium (Lonza) containing 10% FBS (Cell Generation), 100 U/ml penicillin, 100 µg/ml streptomycin (Mediatech), supplemented with either 50 ng/ml M-CSF or GM-CSF (R&D Systems). On day+3 fresh media containing the growth factors was added. On day+7 the cells were washed twice with PBS containing Ca2+ and Mg2+ (Lonza) and fresh complete RPMI (for resting MΦ), complete RPMI containing 100 ng/ml IFN-γ (for M1) or 10 ng/ml IL-4 and IL-13 (for M2)  was added for 24h-72h. In some experiments 40 μM of the MLL-Menin inhibitor MI-2-2 at a 1:500 dilution (kindly provided by J. Grembecka and T. Cierpicki) [The study was conducted in accordance with the principles expressed in the Declaration of Helsinki and approved by the Institutional Review Board of the University of Michigan (HUM00001441). Blood samples were obtained from healthy donors after written informed consent. Peripheral blood mononuclear cells (PBMC) were obtained by a standard Ficoll gradient . Monocytes were isolated from PBMC using anti-CD14 magnetic beads (Miltenyi Biotec) and cultured at a concentration of 0.5-1.0 x 10erpicki)  or vehicDNA/RNA Mini Kit (Qiagen). In experiments with RNA isolation only, Trizol® Reagent (Invitrogen) was used, followed by a column purification step with an additional on-column DNAse digestion . RNA and genomic DNA were quantified in a NanoDrop 1000 spectrometer (Thermo Scientific). Equal amounts of genomic DNA were analyzed for DNA methylation using the MethylFlashTM and Imprint® Methylated DNA Quantification Kits (Epigentek and Sigma-Aldrich) according to the manufacturers’ instructions. RNA and genomic DNA were isolated from adherent MΦ using the AllPrep TM cDNA Synthesis Kit (Bio-Rad). All primers were from AB Biosystems except for MRC1, which was custom made [Hs00609608_m1), CDH1 (Hs01013959_m1), CCR7 (Hs99999080_m1), CXCL10 (Hs00171042_m1), DNMT1 (Hs00154749_m1), DNMT3A (Hs01027166_m1), FOLR2 (Hs01044732_g1), IL10 (Hs00174086_m1), IL12B (Hs00233688_m1), KDM6B (Hs00389738_m1), MLL (Hs00610538_m1), RPS6KA5 (Hs00178054_m1), and SMYD3 (Hs00224208_m1). Quantitative real-time PCR was performed with human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Applied Biosystems) as housekeeping gene on a TaqMan 7500 sequence detection system (Applied Biosystems) and fold expression was normalized to resting MΦ using the ΔΔCt method. To compare the effect of growth factors, fold expression of GM-CSF MΦ was normalized to M-CSF MΦ. RNA was reverse-transcribed using random hexamer primers (Applied Biosystems) and M-MLV reverse transcriptase (Invitrogen) or iScripttom made . The fol2 First Strand kit (SA Bioscience). Expression of CME was analyzed using RT2 ProfilerTM PCR Array PAHS-085 according to the manufacturer’s instructions and the web-based RT2 ProfilerTM PCR Array Data Analysis software (SA Biosciences). Because of genomic DNA contamination in one sample, Ct values ≤ 31 cycles were considered for analysis and normalized to the housekeeping genes beta-2-microglobulin (B2M), ribosomal protein L13a (RPL13A) and GAPDH.MΦ were differentiated in RPMI 5% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 50 ng/ml M-CSF or GM-CSF for 7 days and polarized for 48h with either 100 ng/ml IFN-γ + 1 µg/ml LPS or 10 ng/ml IL-4 and IL-13. RNA was extracted and subjected to RNeasy MinElute cleanup (Qiagen). A total of 1 µg mRNA was reverse transcribed using the RTTM (Mediatech) and Fc receptors were blocked with human IgG (Sigma-Aldrich) for 30 min. Cells were stained for 25 min at 4°C. For intracellular staining, cells were fixed in 2% formaldehyde and then permeabilized using the Perm/WashTM buffer kit (BD Biosciences) followed by antibody incubation. The following antibodies were used: Biotinylated anti-CD14, FITC-conjugated anti-CD14, Pacific BlueTM-conjugated anti-CD64, FITC-conjugated anti-CD68, APC-conjugated anti-CD163, APC-conjugated anti-CD206, PE-conjugated anti-CXCL10 (Biolegend), PE-conjugated anti-CD206 (eBioscience), and Pacific OrangeTM-conjugated streptavidin (Invitrogen). For the detection of histone marks rabbit anti-trimethylated H3K4 and anti-trimethylated H3K27 (Millipore) were used, followed by secondary stain with DyLightTM 488-AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch). Flow cytometry data was collected on a BD Biosciences LSR II and analyzed using FlowJo software (Tree Star). MΦ were harvested using CellstripperTM Nuclear Extraction Kit I (Epigentek) and stored at -80° C until analyzed. Protein content was quantified using Bradford assay (Bio-Rad) and the H3K4-specific methyltransferase activity was determined in equalized amounts of nuclear protein using the EpiQuikTM Histone Methyltransferase Activity/Inhibition Assay Kit (H3-K4) (Epigentek) according to the manufacturer’s instructions. Nuclear extracts were prepared from MΦ 24h after polarization using EpiQuikMΦ were polarized for 48h and ChIP was performed as described . In brie® Gold Antifade Reagent with DAPI (Invitrogen). The following antibodies were used: mouse anti-MLLc, rabbit anti-trimethylated H3K4 (Millipore), goat anti-rabbit Alexa Fluor® 568, and anti-mouse Alexa Fluor® 488 (Invitrogen). Cytospin slides were fixed in 4% paraformaldehyde 4% sucrose for 20 min at RT. Cells were permeabilized with PBS 0.05% Tween 20 and Fc receptors were blocked with human FcR Blocking Reagent (Miltenyi Biotec) for 30 min at RT. The slides were stained with primary antibodies over night at 4°C followed by a secondary antibody stain for 1h at RT. The slides were mounted using ProLongData were analyzed using GraphPad prism software Version 6 (GraphPad). Mann-Whitney U test and Kruskal-Wallis one-way ANOVA followed by Dunn’s multiple comparison were applied for statistical analysis and p-values ≤ 0.05 were considered significant.Monocytes were differentiated into MΦ using either M-CSF or GM-CSF and subsequently polarized with IFN-γ (M1) or IL-4+IL-13 (M2). In our observations and as previously described , the majWe next assessed the expression of MΦ markers CD68, CD14, mannose receptor CD206, the haptoglobin-hemoglobin scavenger receptor CD163, and the FcγRI CD64, 24h after polarization with IFN-γ or IL-4+IL-13 on M-CSF and GM-CSF MΦ by flow cytometry . M-CSF aResting, M1 and M2 MΦ were compared for expression of CCR7, CXCL10, and IL12B genes as representative markers for M1 and the mannose receptor (MRC1), e-cadherin (CDH1), folate receptor beta (FOLR2), 15-lipoxygenase (ALOX15) and IL10 genes as representative markers for M2 activation ,21,24-26We also compared the resting, M1 and M2 gene profiles of M-CSF and GM-CSF MΦ differentiated in parallel from a single donor  +D. In liOverall, our results suggest that polarization with IFN-γ or IL-4+IL-13 leads to a similar gene expression pattern in both M-CSF and GM-CSF differentiated MΦ. In addition, GM-CSF MΦ displayed elevated levels of MRC1 mRNA, thus confirming the increased CD206 expression that we observed in flow cytometry.Epigenetic changes constitute the underlying mechanism for hematopoietic cell differentiation  and distOur results demonstrate that in general in both M-CSF and GM-CSF differentiated MΦ, polarization with IFN-γ leads to the up-regulation of MLL and KDM6B, while the CME pattern of IL-4+IL-13 treated MΦ does not significantly differ from resting MΦ.The histone demethylase KDM6B has been shown to regulate classical activation -29. HoweIn our PCR array, we initially found an up-regulation of DNMT1 and DNMT3A in M2 MΦ relative to M1 MΦ after 48h. But we did not observe significant changes in mRNA levels after 24h when we confirmed the array data using quantitative real-time PCR in 6 independent donors. However, this did not exclude a functional impact of DNMT enzymes on DNA methylation in M2 MΦ. In order to test for DNMT activity, we looked at changes in total DNA methylation. Genomic DNA from resting, M1 and M2 activated M-CSF and GM-CSF MΦ was isolated and tested for total DNA methylation . In contWith elevated MLL levels and a higher H3K4-specific methyltransferase activity in M1 MΦ, we next looked at the mark set by the enzyme. We used flow cytometry to assess the methylation status of H3K4 72h after polarization . We alsoWe next looked at MLL protein expression and activity by immunofluorescence . Both M1Our data implied that increased levels of total H3K4me3 accompany M1 activation in particular in M-CSF differentiated MΦ. To assess whether histone modifications also regulate MΦ polarization on a gene specific level in M1 and M2 MΦ, we analyzed the promoter regions of CXCL10, MRC1 and ALOX15 for changes in H3K4me3 and H3K27me3 using a ChIP approach . In lineNotably, the ALOX15 promoter in M2 was characterized by similar histone modifications with increased H3K4me3 and decreased H3K27me3. Thus, we re-evaluated our initial data from the PCR array  focusingInterestingly, the MRC1 promoter displayed a constitutively activated state in all MΦ indicated by increased H3K4me3 and low levels of H3K27me3 . This suThese data further support our previous findings and demonstrate that epigenetic mechanisms regulate the expression of polarization-specific genes in M1 and M2 MΦ. In line with our hypothesis that MLL is involved during classical activation of MΦ, we were able to show that CXCL10 displays elevated H3K4me3 at its promoter site. To verify the role of MLL in epigenetically regulating CXCL10 expression and promoting M1 polarization, we next used a recently described inhibitor of the MLL-Menin interaction, MI-2-2, to specifically target MLL function . Menin ihi IL-10lo phenotype, while M-CSF differentiation leads to IL12lo IL-10hi MΦ corresponding to an M2-like cell type [in vivo, monocytes simultaneously encounter growth factors and T cell-derived IFN-γ and/or IL-4+IL-13 depending on the prevailing tissue microenvironment. In order to better understand MΦ polarization, we combined growth factor treatments with cytokine-induced activation. In line with previous reports [hi CD163hi CD206int surface phenotype and express FOLR2. Conversely, GM-CSF MΦ were CD14low CD163low CD206hi with elevated IL-12B expression (Polarized MΦ are found during infections and tissue repair, where their role is fundamental in shaping the immune response towards inflammation or tolerance -4. Circuell type -8. Thus,ell type ,24 or poell type -8,25. It reports -34 we depression . IFN-γ lpression . Notablyin vitro experiments in which MΦ were differentiated under controlled conditions by adherence to a plastic surface, the markers described herein have been demonstrated to exert in vivo relevance in human diseases. CD163 and CD206 expression on tissue MΦ has been used to distinguish an alternative activation state from classical activation in many human diseases like asthma, obesity and atherosclerosis [in vitro findings [Although the phenotypic changes described in this manuscript are derived from clerosis -40. Inteclerosis . Taken tfindings -34 and sBut irrespective of their differences with regard to morphology, gene and surface receptor expression, M-CSF and GM-CSF MΦ appear to respond to IFN-γ or IL-4+IL-13 in a similar fashion with up- and down-regulation of common M1 and M2 genes , indicatEmbryonic development and cellular differentiation are accompanied by considerable changes in the chromatin structure including DNA methylation and histone modifications. These modifications regulate gene transcription and silencing and are carried out by distinct CME. Notably, hematopoietic development and monocyte to MΦ differentiation are governed by changes in the epigenome ,17. ThusThe role of KDM6B during MΦ polarization remains controversial. In contrast to murine MΦ, in which KDM6B mediates alternative activation ,12, we fUnexpectedly, alternative activation did not reveal a specific set of up-regulated CME in either M-CSF or GM-CSF MΦ, when compared to resting MΦ. When compared to classically activated MΦ, the M2 pattern of regulation resembled the one observed when M1 were compared to resting MΦ with preferential expression of DNMTs and HDACs. Selecting DNMT1 and DNMT3A as markers for M2, we found comparable expression levels and a similar amount of methylated DNA in M1 and M2 MΦ. However, at the promoter site of ALOX15 we also found an increase of H3K4me3 upon M2 polarization, indicative of methyltransferase activity. We hypothesize that this is due to SMYD3 activity, as we found that SMYD3, another H3K4-methyltransferase, is preferentially expressed in M2 MΦ. Recently, SMYD3 has been shown to regulate 15-Lipoxygenase expression  which fuNotably, the MRC1 promoter region displayed elevated H3K4me3 and low levels of H3K27me3 independent of polarization conditions. This indicates that MRC1 transcription is not regulated by polarization-induced changes in histone methylation. Interestingly, MRC1 expression also seems to be independent of c-MYC transcription factor binding that was shown to regulate M2 gene expression . Considehi CD163hi CD206int FOLR2-expressing M-CSF and CD14lo CD163lo CD206hi GM-CSF MΦ. We also show that M-CSF and GM-CSF MΦ reacted similarly upon activation with IFN-γ or IL-4+IL-13 with the induction of well described M1 and M2 marker genes, respectively. This indicates that the transcription program initiated by IFN-γ or IL-4+IL-13 is independent of the growth factors, suggesting that transcription factors might control the expression of marker genes [In conclusion, we describe the phenotypic differences between CD14er genes . Further


Infections remain a major cause of morbidity and mortality in transplant patients. Organ recipients are also susceptible to donor-derived pathogens and the majority of donor infections are easily treatable. Rarely, some pathogens have produced life-threatening complications by compromising the vascular anastomosis. In this case series we report loss of two kidney allografts secondary to vascular complications due to Candida albicans. Both recipients received grafts from a common donor, in whom Candida bacteremia in the donor was not apparent at the time of organ acceptance but became apparent on delayed cultures. Candida albicans, and Aspergillus have produced life-threatening complications in organ recipients by compromising the vascular anastomosis [Infections remain a major cause of morbidity and mortality in solid organ transplant recipients. In addition to nosocomial infections, transplant patients are also susceptible to a variety of donor-derived pathogens . Fortunastomosis , 3. CandCandida albicans. Liver and both kidneys were harvested without incident. We do not add any antibiotics to the preservative solution according to our department policy.The donor was a 21-year-old man, a victim of motor vehicle accident, underwent initial resuscitation and intubation at accident site, and hospitalized 4 days prior to declaration of brain death and multiorgan procurement. Routine lab tests were normal including blood and urine cultures. Sputum culture was positive for Candida albicans. With antifungal therapy, patient had protracted recovery.The 1st recipient was a 51-year-old Filipino-American female with end-stage renal disease (ESRD) secondary to focal segmental glomerulosclerosis. Patient was induced with thymoglobulin and steroids, maintained on tacrolimus and mycophenolate mofetil (MMF) (steroid free), and had good graft function. On postoperative day (POD) 17, he presented to the ER with hypotension and low hematocrit. A CT scan revealed a large perinephric hematoma and underwent emergent exploration. An arterial bleeder at the renal hilum was ligated with restoration of hemostasis. On POD29 the patient presented with acute abdomen and pulsatile mass across the lower abdomen. On exploration complete disruption of the arterial anastomosis with necrosis of the renal artery and adjacent external iliac artery was noted. Explant of the renal graft with resection of the necrotic segments of iliac artery and primary repair was done. Histological examination did not show any evidence of fungus, but a blood clot sent intraoperatively grew Candida albicans. Histopathology of explant is shown in The 2nd recipient was a 49-year-old Hispanic female with ESRD secondary to glomerulosclerosis. She received a similar induction, followed by tacrolimus and rapamycin maintenance (steroid-free), and had good graft function. She was admitted on POD88 with febrile neutropenia and painful mouth sores. Creatinine remained normal and blood and urine cultures were negative. Neutropenia responded quickly to Neupogen, fevers resolved, and patient was discharged 4 days later. In view of neutropenia and oral lesions, rapamycin was substituted with MMF. She was readmitted with fevers and elevated creatinine on POD112. Imaging studies performed are shown in In retrospect Candida bacteremia in the donor was not apparent at the time of organ acceptance but became apparent on delayed cultures, but not available for patient management. We could have investigated for potential Candida infection in the 2nd recipient soon after the first case to prevent such an outcome. Transplant teams should be vigilant and follow other organ recipients from the same donor when donor transmitted infection is a possibility.Donor-derived infections may occur at different phases of the transplantation: (a) from donor, especially from victims of polytrauma and in those who had prolonged intensive care unit stay; (b) during harvest, particularly multiorgan procurement, injury to gut during procurement, and harvesting kidneys from non-heart-beating donors; and (c) during organ preservation and graft implantation (contamination of preservation fluid) .Vascular Candidal infections typically present in 2 forms.Anastomotic leak or vessel rupture: it occurs early in the postoperative course (within 2 months post-transplant) and responsible for 2/3 of cases . Lack ofPseudoaneurysm: it occurs “late” after transplant (>2 months) . With apAnother important preventive measure may be routine culture of preservation media to detect contamination during organ procurement and/or graft back-table preparation. The rate of positive culture of preservation fluid ranged from 5 to 23% and fungi represented 2 to 10% of all positive cultures [C. albicans is a potentially life-threatening complication of renal transplantation leading to graft loss in most of the cases and utmost care is needed to prevent contamination at all phases of transplantation to prevent this complications. It is of utmost importance to report and follow up other organ recipients from the same donor if one encounters a suspected donor-related infection to prevent devastating complications.In conclusion, mycotic arteritis due to


To assess the functional donor site morbidity of the forearm free flap in patients surviving at least 2 years after ablative head and neck cancer surgery in a tertiary care centre.This study involved nine long-term survivors (2 year post-operative) who had forearm free flaps to reconstruct head and neck defects. All flaps were raised from the non-dominant arm. The non-donor side acted as a control for all patients. Objective measurements were as follows: grip, tip pinch and key pinch strength measured with dynamometers; flexion, extension, radial and ulnar deviation and pronation and supination range of motion at the wrist measured with goniometry; A timed manual dexterity task was performed with a grooved pegboard test, and sensation of the radial nerve was tested with Semmes Weinstein monofilaments. Subjective measurements included a validated patient questionnaire of hand function and opinions of scar appearance as well as a validated scar assessment from two different observers.Pronation at the wrist, manual dexterity and sensation were found to be significantly reduced in the donor side compared to the non-donor side. Inter-rater agreement between the two observers was found to be poor, except for an acceptable correlation between overall scar opinions. No correlations were found between any subjective or objective items or between the patient’s and the observers’ subjective evaluations.Donor site morbidity can be demonstrated with objective testing however this is accepted and well tolerated by head and neck cancer patients. The forearm flap is a reliable and versatile method to reconstruct various structures in the head and neck -3. It reMuch attention in the literature has addressed the success of the forearm flap for various head and neck defects -4. ThereSome previous studies have found statistically significant functional morbidity . In ordeCapital Health Research Ethics Board granted approval of this study. Nineteen individuals were identified as having a radial forearm free flap (RFFF) for head and neck reconstruction at least two years post cancer resection. Ten patients who met the inclusion criteria signed consent forms and were enrolled in the study, of which nine completed all aspects of the study Figure . Allen’sValidated patient and observer scar assessment questionnaires developed by van de Kar et al. ,20 were The observers were a medical student and an otolaryngology senior resident.A Jamar grip dynamometer  was used to measure grip strength. Tip pinch strength and key grip strength were measured with a pinch gauge . A stainless steel wrist goniometer was used to measure wrist flexion, extension, radial and ulnar deviation, and pronation and supination. Measurement protocols were taken from Solgaard et al. .A timed grooved pegboard test evaluated dexterity . Sensation was tested via Semmes Weinstein monofilaments applied to the radial nerve distribution over the scaphoid on the distal forearm.We analyzed the results of this study using a Student’s t test, Pearson’s Correlation, and Cohen’s Kappa was used to return a measurement of inter-rater agreement between observers. Statistical analysis was performed using Microsoft Excel and statistical significance was defined as < 0.05.Patient demographic information for those enrolled in the study are illustrated in Table Two patients had post-operative complications of the donor arm – one patient had a donor forearm fracture on post-operative day 2, the other patient had a stitch abscess. Only one patient reported difficulty in using the donor arm to perform household, outdoor, or work tasks. In patient reported aesthetics, 3 patients reported being bothered by the appearance of their scar and being uncomfortable wearing short sleeves in public. Three patients also reported the inability to wear a wristwatch on the donor arm, while four patients reported cold intolerance , extension (p = 0.31), radial p = 0.47) and ulnar deviation (p = 0.25), though the difference was not significant . Mean percentages of the un-operated arm were all over 94%. An expected 10-15% reduction in strength on the non-dominant side was not observed  and observer 2 (a senior otolaryngology resident). Inter-rater agreement was found to be poor throughout all categories of evaluation with Cohen’s Kappa values of 0.50, 0.26, 0, 0.46, and -0.07 for the first five questions respectively. However, use of Pearson’s Correlation found acceptable Pearson’s Correlation value of 0.78 for overall opinion of patient scars between the two observers. A weaker correlation was noted between the total scar assessment scores of observer 1 and total patient questionnaire scores with a Pearson’s Correlation value of 0.69.Although donor site morbidity was demonstrated objectively, this did not coincide with subjective patient reporting. This study attempted to address the lack of literature regarding radial forearm flap donor site morbidity in long-term head and neck cancer survivors. An unfortunate limitation of this study was a small sample size. Absence of non-donor arm evaluated as an internal control is cited as a limitation in previous studies . WithoutSensation over the anatomical snuffbox was significantly reduced compared to the un-operated arm, although there was no correlation with sensation as reported in the patients’ questionnaires. Dexterity of the donor arm as determined by the grooved pegboard test was also significantly less than the non-donor side. Due to the donor arm also being the non-dominant arm, it is expected that a portion of the time difference in dexterity will be due to the use of the dominant hand versus the non-dominant hand. Pre-operative donor hand dexterity testing to compare to post-operative donor hand dexterity was not completed and is a limitation of this study. Normative values for this age range have found a mean difference of 106% . HoweverPatient demographic information for those enrolled in the study are illustrated in Table The forearm flap remains a reliable and versatile option in head and neck reconstruction. However, objective and subjective concerns regarding the donor site post-operatively must be considered prior to surgery. This study has shown that while objective testing can demonstrate donor site morbidity, subjective testing reveals that overall patients are functionally satisfied and tolerate the donor site post-operatively.At the time of the research, the authors had no conflicts of interest to disclose.JO was responsible for study design, data collection, statistical analysis and manuscript preparation. PH was responsible for manuscript preparation. CB, JT, and RH were involved in study design, data collection, and interpretation of results. SMT is the corresponding author and was involved in study design, data collection, interpretation of results and manuscript preparation. All authors read and approved the final manuscript.Patient Scar Assessment Scale modified from scar assessment scale by van de Kar et al.[ar et al.,20.Click here for fileObserver Scar Assessment Scale from scar assessment scale by van de Kar et al.[ar et al.,20.Click here for file


The Basal Ganglia represent subcortical structures that have a crucial role in determining when a given motor program should be selected and called into action . The inpWe simultaneously recorded local field potentials (LFPs) in the cortex and striatum in order to determine how striatum processes cortical neuronal avalanches. Cortical neuronal avalanches represent activity clusters with a cluster size distribution that follows a power law with exponent -1.5 . To unde