Using recordings from a five-pin surface sensor array, a template-based surface electromyogram (sEMG) decomposition system has been developed to identify single motor unit discharge properties. However, the reliability of such template based decomposition results has not been thoroughly examined except by the developers. The focus of this study was to assess the validity of the motor unit decomposition technique, using EMG recordings from the first dorsal interosseous muscle of able-bodied human subjects.Two tests were utilized. In the first test, a spike triggered averaging (STA) analysis was used to derive motor unit action potential (MUAP) parameters. We examined these STA derived MUAP shapes after firing times were perturbed by added timing noise. In the second test, a cross-correlation analysis was performed between the sEMG signal and MUAP trains constructed using STA estimates and their firing times.In the first test, we found that MUAP shape features deteriorated significantly when rather small (0.6-2 ms) timing errors were added, affirming that the decomposed firing times are presumptively valid. The results of the second test reveal that the cross-correlation index between the EMG and MUAP trains increased monotonically up to 0.71 when the identified MUs were progressively added to reconstructed MUAP trains; however, this increment disappeared when the firing times or the MUAP templates were shifted randomly.Based on our STA selection criteria, our results suggest that the firing times and estimated MUAP shapes for each MU generated by the decomposition algorithms are presumptively valid. Single motor unit (MU) firing properties provide valuable information about the neuromuscular system and are widely used in physiology , motor cIn earlier studies, the accuracy of the decomposition results has been assessed using a reconstruct-and-test approach by De Luca and colleagues ,15. SpecTo address these concerns, the objective of this study was to employ two sets of tests to examine and assess the validity of decomposition results of the sEMG signal recorded from the first dorsal interosseous (FDI) muscle. We devised indirect methods of assessment, because the recorded signal consisted of a large number of motor units with a significant amount of superposition.In the first set of tests, a spike triggered averaging (STA) technique was used to reconstruct the action potential shape -20. ThisThe STA MUAP estimate was used for further analysis to derive decomposition related parameters. Two STA features were extracted: (1) the correlation between the STA estimates and the decomposition templates, and (2) the waveform stability of the STA estimates. Gaussian noise was then added to perturb the timing of the originally decomposed firings in the STA analysis. We hypothesized that features derived from the STA analysis would show substantial changes  if the decomposed firing times are largely correct. Conversely, if the firing times are already erroneous from the decomposition, we would expect little changes in the STA features.The second set of assessments involves a cross-correlation analysis between the raw EMG signal and the MUAP trains constructed using the STA estimates. Reliably decomposed MUs were selected and MUAP trains were constructed using the firing times and the STA estimates. The MUAP trains from different MUs were combined linearly, after which the combined MUAP trains were correlated with the recorded raw EMG signal. We hypothesized that the cross-correlation would increase with the number of MUAP trains added together, if the firing times were correct and the STA estimates represent the true MUAPs. Additionally, if the firing times or the MUAP templates were perturbed , we would expect low cross-correlations.Using these tests we were able to validate the decomposition results of the sEMG recorded from the FDI muscle. The outcome of our first assessment showed that the MUAP shape features of the STA estimates were lost even if a small (0.5-2 ms) amount of timing noise was added to the firing times. The second assessment, the cross-correlation index between the raw EMG and MUAP trains increased monotonically with an increasing number of MUs added to the simulated MUAP train. This incremental change was absent when the firing times or the MUAP templates were shifted randomly. The results of our analysis provide support for the validity of surface EMG decomposition systems, and also provide a potential tool for routine assessment of the output of general MU decomposition algorithms for surface EMG.Eight right hand-dominant neurologically intact individuals  volunteered to participate in this study. The EMG activity of the FDI muscle was recorded during isometric abduction of the right index finger. The participants gave informed consent via protocols approved by the Institutional Review Board under the Office for the Protection of Human Subjects at Northwestern University.nd metacarpal and the long axis of the forearm creating a neutral MCP joint angle. The proximal phalanx of the index finger was cast and fixed to a ring-mount interface attached to a six degrees-of-freedom load cell . The recorded forces from the abduction/adduction direction were low pass filtered (cutoff =200 Hz) and digitized at a sampling frequency of 1 kHz. The subjects were instructed to produce required abduction forces while minimizing the off-axis forces.Participants were seated upright in a Biodex chair with their upper arm resting on a support. To standardize hand position and to minimize contributions of unrecorded muscles, the forearm was immobilized in a brace and placed in a ring mount interface attached to an elbow rest. The forearm was placed in full pronation and the wrist was held neutral with respect to flexion/extension. The little, ring and middle fingers were extended away from the index finger and strapped to the support surface. The thumb was secured at an approximately 60 degree angle to the index finger. The index finger was placed in line with the 2The subject’s skin was prepared by cleaning the superficial layers with adhesive tape, and the skin was then cleaned with alcohol pads to ensure proper electric contact and low baseline noise. sEMG was recorded from the FDI Figure A using aFor the first trial of the experimental session, subjects were asked to perform maximal voluntary contractions (MVCs) for 3 s. This maximum contraction was repeated 3 times in total, with 1 min rest between trials. The largest value of the 3 trials was designated as the MVC. The rest of the session consisted of a series of isometric voluntary contractions during which the subject was asked to follow trapezoid force trajectories displayed on a computer screen, each at a different varying percentage of the MVC. The force output in one exemplar trial is shown in Figure In order to clarify the relation between the decomposed MUAP templates and the STA derived MUAP shapes, a brief summary of template formation and matching used in the decomposition algorithm follows.The decomposition algorithm consists of two stages. The first stage involves MUAP template creation, matching, and updating . In the The second stage identifies MUAPs within complex superpositions in the EMG signal. Multiple MUAP templates are iterated through a discrimination analysis to calculate the combination of MUAP templates that best matches the signal shape while also satisfying the criteria that the coefficient of variation of the inter-spike intervals (ISIs) of these MUs is at a minimum. It is worth noting that not all the MUAPs embedded in the EMG signal are identifiable, especially for those small MUAPs that are blurred by background noise and extensive superposition. The decomposition system output consists of the MUAPs that were most reliably identified, and the algorithm is not residual based. Thus the residual signal varies according to the recording circumstances, including experimental protocol. Detailed information for the decomposition algorithm is described in .Spike triggered averaging (STA) analysis aligns segments of EMG signals based on MU firing events. This method provides the ability to attenuate non-time-locked activities from the complex interference pattern and extract action potentials related exclusively to the triggering events. In this study, STA analyses were performed to assess the stability of the MUAP that include:1) The correlation between the STA and the decomposed action potential templates provided by the decomposition algorithm. A comparison of the STA and the decomposed templates is shown in Figure 2) The variability in the STA derived MUAP peak-peak amplitude across epochs/segments of the EMG signal. The stability of the MUAP peak-peak amplitude, instead of the entire MUAP waveform, was assessed, because the MUAP amplitude provides key information about the physiological properties of the MU .The STA analysis of sEMG involved three stages of processing.First, the STA was performed on the individual four sEMG channels. The firing times identified from the decomposition system were used as triggers for the STA calculation. This resulted in one representative waveform estimate for each algorithm identified MU in each channel Figure C. The tiSecond, two separate STA features were calculated to determine which MUs would be retained for EMG reconstruction. These features were designed to assess the stability of the waveform over the trial duration.For the first feature, the STA estimated MUAP was calculated based on a window (EMG segment) length of 4 s, and the MUAP variation across segments was then calculated. This window yielded approximately 50–100 firing events in each window, and the window was shifted over the sEMG signal, using a step size of 0.5 s. The number of steps ranged from 24 to 36 depending on the force level and recruitment threshold of individual MUs. The coefficient of variation  of the peak-peak (P-P) amplitude of the MUAP templates, for each step change, was calculated as a measure of the stability of the waveform average. The P-P amplitude was the difference between the maximal positive and negative peaks of the MUAP.For the second feature, the MUAP estimates derived using the STA method were compared with the decomposition based templates (a 20 ms segment). The maximum linear correlation coefficient between the STA estimate  and the decomposition estimated templates was computed as a second measure of the reliability of the waveform average. The STA derived template was shifted 10 ms backward and forward one data sample at a time  relative to the decomposition template to find the maximum correlation coefficient. The decomposition estimated templates are derived via a weighted average of the various templates used to represent each MU; the STA estimates were calculated directly from the raw EMG data. A high correlation between these two estimates signifies the reliability of the STA waveform average.Third, the calculated features of the STA estimates were used as criteria to select MUs. Specifically, the MUs with CV of P-P amplitude < 0.2 and a correlation coefficient > 0.7 were selected for later calculation. The average CV and average correlation coefficient across the 4 channels was calculated provided that the 4 channels give rise to similar values of these features.If the action potentials are correctly identified, a disturbance of the timing of firings should significantly change the STA waveform, because it is calculated from the identified firing times for each MU. Conversely, if the firing times are already incorrectly identified, disturbed firing times should have little effect on the STA features .To test this hypothesis, different levels of Gaussian noise (each generated independently) were added to the identified firing times of each MU such that each individual firing time is shifted randomly. Specifically, the noise had a zero mean and a standard deviation (SD) that was scaled with the SD of the ISIs of MU firings. The scaling of the SD ranged from 0 to 0.6 with an increment of 0.1. The exemplar MU spike trains  are shown in Figure When the interference sEMG patterns are decomposed into constituent MUAP trains, each MUAP train contains key information about the EMG signal, provided that the firing times are identified correctly, and the estimated MUAP templates represent the true MUAP shape. Therefore, one would expect measurable correlation between the interference EMG signal and MUAP train derived from the decomposed MUs and firing times, and this correlation should become stronger when more MUAP trains are combined together. Conversely, if the identified firing times or the estimated MUAPs did not match the true firings and the true MUAP shapes of the MUs that constitute the EMG signal, we would expect a low correlation between the EMG signal and the combined MUAP trains of multiple MUs.We performed a cross-correlation analysis to test this hypothesis. This analysis consists of several steps:1) MUs with stable STA derived MUAP shapes were selected based on the STA features. Specifically, the MUAPs with CV of P-P amplitude < 0.2 and a correlation coefficient > 0.7 were selected for later calculation.2) MUAP trains were constructed using the firing times of the selected MUs and the STA derived MUAPs.one at a time and the cross-correlation between the combined MUAP trains and the raw EMG was calculated. The MUAP trains were combined in two separate ways (from the first to the last recruited MUs and from the last to the first recruited MUs) to examine the order effect on the cross-correlation.3) MUAP trains of different MUs were combined SD = 0.1 * SD of ISIs) was added to the identified firing times to disturb the timing. We expect a low cross-correlation, if the original firing times were correctly identified.4) To further validate the identified firing times, Gaussian noise (a zero mean and 5) To further validate the estimated MUAP shapes, instead of using the original STA MUAP template, we randomly assigned a template from other MUs and constructed the MUAP trains. We expect a reduced cross-correlation between the random MUAP train and the EMG signal, if the original STA templates were estimated accurately.In summary, we examined 4 ways of combining the MUAP trains when performing the cross-correlation with the raw EMG signal:1) Using original firing times and original STA templates and combining MUAP trains from the first to the last recruited MUs ).2) Using original firing times and original STA templates and combining MUAP trains from the last to the first recruited MUs ).noisy firing times and original STA templates.3) Construct MUAP trains using randomly shuffled STA templates.4) Construct MUAP trains using original firing times and SD = 0.1 * SD of ISIs) was added to the firing times. When different amounts of firing noise was added  that already had low correlations between decomposition and STA estimates using the original firings.SD = 0.1 * SD of ISIs) was added. The different noise levels  for all 8 trials of all the 8 subjects.A paired SD = 0.1 * SD of ISIs) was added to the firing times. Similar to the correlation results, when different amount of firing noise was added . The average CV was calculated across different noise level conditions. Because CV values are lower bounded by 0, a logarithmic transformation was performed prior to the t-test. As shown in Table p < 0.05) for the 8 trials of all the 8 subjects.The CV of the MUAP P-P amplitude of a second exemplar trial at 50% MVC is shown in Figure circles) increased almost linearly from 0.1 up to 0.66. When the MUAP trains were combined from late to early recruited MUs , the cross-correlations (open squares) also increased monotonically from 0.2 up to 0.66. Because the later recruited MUs tends to be larger units, as expected from the size principle, [squares) had initially high values and rose at a faster rate than the open circles, but eventually plateaued at a value of 0.66. The different trials from 20% to 50% MVC showed similar cross-correlation function results.The cross-correlation function values between the raw EMG signal and MUAP trains constructed using STA MUAP estimates are shown in Figure inciple, , and theSD of noise = 0.1 * SD of ISIs), the cross-correlations (triangles) were below 0.2 even after all the MUAP trains were combined. Similarly, when the MUAP templates were randomly shuffled between the various identified MUs during the construction of MUAP trains, the cross-correlations (crosses) fluctuated between −0.2 to 0.2.In contrast, when the MUAP train was constructed using noisy firing times (circles and squares) increased from 0.06 up to 0.6 when the MUAP trains were constructed using the original firing times and STA estimated templates. When the firing times (triangles) or the action potential templates (crosses) were disturbed, the cross-correlations were below 0.3. As for subject 2, the initial increase of cross-correlation in the shuffled template condition is likely due to the fact that some MUAPs shared similar shapes in the particular electrode configuration (position and orientation over the FDI muscle). As a result, the shuffling of MU templates has only a moderate effect on the changes in correlation.The second subject Figure B demonstp < 0.05). Post-hoc pairwise multiple comparisons with Bonferroni’s correction method were then performed to examine the changes between individual conditions. Specifically, the correlation value was significantly higher when MUAP trains were added based on original firings than based on noisy firings and shuffled MUs (p < 0.05). When 5 MUAPs were added, the correlation value was significantly higher when MUAP trains were added from large to small than from small to large (p < 0.05).To quantify these observations across these four conditions of all the 8 subjects, the cross-correlation at each condition was summarized when the first 5 MUAP trains had been added and was compared with the cross-correlation when all the MUAP trains had been added  technique was used to extract the action potential features from the raw recorded EMG utilizing the individual MU event times output from the decomposition algorithm. Subsequently the MU timings were altered and the STA analysis was repeated. The results show that the MUAP shape features are lost i.e., the correlation between the STA derived template and the decomposition template declines and the coefficient of variation of the STA estimated template increases, even for a small amount of added timing error. To our surprise, the change in the STA features was not sensitive to the amount of added timing noise. Meanwhile, there are also MUs that show minimal change in STA features when different amount of noise is added, possibly due to inaccurate firing identification or excessive superposition.In the second set of tests, a cross-correlation analysis was performed between the recorded EMG signal and MUAP trains constructed using the STA derived MUAP estimates combined with the algorithm derived MU timings. The results reveal that the cross-correlation index between the raw EMG and constructed MUAP trains increases monotonically when more MUs are added to the MUAP trains; however, this increment is absent when the firing times or the MUAP templates were altered, which suggests that the firing times and estimated MUAPs are valid.We did not expect that a small shift of the spike timing by 0.6-2 ms) could substantially change the properties of the STA derived MUAP estimates. Such a high sensitivity of the STA estimates to the timing errors can arise from the particular characteristics of the recorded action potentials. Specifically, the diameter of the recording electrodes is 0.5 mm and the inter-electrode distance is 3.6 mm (center to corner electrodes) or 5 mm (corner to corner electrodes). This electrode design gives rise to sharp action potential waveforms that are broadly comparable to some recorded intramuscular EMG signals; namely the peak-peak duration of the action potential in FDI muscle is approximately 2–3 ms , the cross-correlation was initially small and increased in a smaller rate than the condition where the MUs were added in a reverse order , likely because large MUs contain more correlated information with the raw EMG signal. These results suggest that the decomposed firing times and MUAP templates are reliable. Our results are further confirmed by shifting the firing times by a small amount (approximately 0.6-2 ms) and by randomly shuffling the MUAP templates between MUs during the construction of MUAP trains. The cross-correlations in these perturbed condition are considerably lower, suggesting that the perturbed MUAP trains do not represent the raw EMG signal any more.The cross-correlation calculated based on the original firings and templates tends to plateau around 0.6 – 0.7, which is lower than 1. This effect can arise from several factors. First, only the MUs that passed the selection criteria were used to construct the MUAP trains, and the excluded MUs may still contain information of the EMG signal. Second, during the decomposition, not all the MUs embedded in the raw EMG are identifiable by the algorithm , especiaThe series of tests used in this study affirm the general validity of the decomposed MUs; however, the accuracy of the timing for a particular firing event of a MU train is not assessed. In MU studies, the mean firing rate or the average ISI of a time segment is typically the variable of interest . The STAOne way to assess the accuracy of individual firing events is the two-source method ,17,24. IOur STA analysis detects spurious firings  and position errors  in the decomposition outputs. In contrast, a spike omission will not influence the STA estimate, because that action potential is missed and will not be taken into account during the averaging calculation. Therefore, the frequency of spike omission in a MUAP train is not directly assessed here. However, the spurious firings are as frequent as spike omissions for a particular MUAP train ,17. In aThis study used two sets of analyses based on STA estimates to evaluate the overall validity of a sEMG decomposition method. Our results reveal that the identified firing times and estimated MUAPs are reliable in sEMG signals recorded from FDI muscles at low to moderate force contraction levels. Additionally, the STA analysis provides a tool to detect possible identification errors and select reliably decomposed MUs from MU decomposition algorithms in general.EMG: Electromyogram; sEMG: Surface electromyogram; STA: Spike triggered averaging; MU: Motor unit; MUAP: Motor unit action potential; FDI: First dorsal interosseous; MVC: Maximum voluntary contraction; P-P: Peak-peak; CV: Coefficient of variation; SD: Standard deviation; ISI: Inter-spike intervals; ANOVA: Analysis of variance.The authors declared that they have no competing interests.All the authors have made substantial contributions to conception and design, acquisition, analysis and interpretation of data; have been involved in drafting and revising the manuscript; and have given final approval of the version to be published.


Csf1r null and op/opCsf1 mice were isolated and sectioned for IHC identification of enterocytes, enteroendocrine cells, goblet cells and proliferating cells. Both −/−Csf1r and op/opCsf1 mice were found to have colon defects in enterocytes and enteroendocrine cell fate, with excessive goblet cell staining and reduced cell proliferation. In addition, the gene expression profiles of the cell cycle genes, cyclinD1, c-myc, c-fos, and c-myb were suppressed in −/−Csf1r colonic crypt, compared with those of WT mice and the expression of the stem cell marker gene Lgr5 was markedly reduced. However, analysis of the proliferative responses of immortalized mouse colon epithelial cells  indicated that CSF-1R is not a major regulator of colonocyte proliferation and that its effects on proliferation are indirect. In an examination of the acute inflammatory response, Csf1r+/− male mice were protected from the adverse affects of DSS-induced colitis compared with WT mice, while Csf1r+/− female mice were significantly less protected. These data indicate that CSF-1R signaling plays an important role in colon homeostasis and stem cell gene expression but that the receptor exacerbates the response to inflammatory challenge in male mice.The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) directly regulates the development of Paneth cells (PC) and influences proliferation and cell fate in the small intestine (SI). In the present study, we have examined the role of CSF-1 and the CSF-1R in the large intestine, which lacks PC, in the steady state and in response to acute inflammation induced by dextran sulfate sodium (DSS). As previously shown in mouse, immunohistochemical (IHC) analysis of CSF-1R expression showed that the receptor is baso-laterally expressed on epithelial cells of human colonic crypts, indicating that this expression pattern is shared between species. Colons from  Colonic and small intestinal (SI) crypts share a range of architectural and molecular features and for convenience are often discussed interchangeably. Each generates the three cell lineages; the enterocytes, which secrete hydrolases and absorb nutrients, We have recently shown that the cytokine colony stimulating factor receptor (CSF-1R) is required for PC development and that CSF-1R-deficient mice possess deficits in SI enterocytes and enteroendocrine cells and excess goblet cell production, as well as substantively reduced crypt proliferative capacity and stem cell niche maintenance In vivo CSF-1 regulation has been investigated in the CSF-1-deficient op/oposteopetrotic (Csf1) mutant mouse, Csf1 gene −/−Csf1r mice with a targeted deletion of the only cellular receptor for CSF-1 in vitro and that CSF-1R is expressed on the basal lateral surface of mouse colonic crypts Csf1r gene is protective in male mice. Collectively, these studies support a central role for CSF-1R signaling in gastrointestinal homeostasis and disease.We have previously reported the baso-lateral presence of CSF-1R on isolated mouse colonic crypts −/−Csf1r and op/opCsf1 mutant mice and matching wild type (WT) littermate controls were excised from cecum to anus. Three mice of each genotype were evaluated and both mutants were found to have significantly shorter colons (p>0.01), commensurate with their reduced overall size . Staining of sections for Chromogranin A, to evaluate the number of enteroendocrine cells for the whole colon, revealed that both mutant mice had fewer enteroendocrine cells than WT (p<0.01)   which showed that the mutant crypts had fewer cells that had progressed to these later phases of the cell cycle  . Images of these sections , phospho-ERK1/2 induction  and c-myb, c-myc, cyclinD1, Ets-2 and c-jun gene expression . Cytokine antibody arrays confirmed the presence of CSF-1 in PMUE, but also demonstrated the presence of several other factors . However, attempts to replicate the level of growth stimulation with either purified recombinant CSF-1, or purified IL-34, which also activates the CSF-1R ). Although these experiments were carried out with cell lines, the lack of a strong proliferative response to CSF-1R ligands raised the possibility that the in vivo requirement of the CSF-1R for epithelial cell proliferation is indirect. As recent studies have shown that PC, the CSF-1R-responsive cells of the SI Figure S2F).To further explore the role of the CSF-1R signaling in colonic epithelial cells, we examined the response of cells of the immortalized, colonic epithelial cell lines YAMC −/−Csf1r FVB/NJ mice do not normally survive beyond 1 month of age, we queried whether loss of one Csf1r allele in +/−Csf1 mice was sufficient. WT (+/+Csf1) and +/−Csf1 mice were provided with water containing 2% w/v DSS ad libitum for 8 days and evaluated for inflammatory responses. While the weights of male WT male mice at day 8 had decreased significantly by ∼20%   . To further assess the effects of DSS treatment, we monitored the Whole Animal Disease Activity Index data not shown). The most profound differences between the WT and +/−Csf1r male mice were noted at day 8 . Others have also reported that male mice are more susceptible to DSS-induced colitis, but not specifically in FVB/n mice mdr1a mutant males As pre-treatment of mice with neutralizing anti-CSF-1 antibodies protects them from DSS-induced colitis +/−Csf1r male mice failed to reveal significant differences in SI crypt or villi morphology, cell number or intestinal length . However, DSS treatment had pronounced effects on the colon as assessed using the modified criteria of Dieleman et al (1998) +/−Csf1r male mice. However, +/−Csf1r mice have significantly less crypt damage throughout the colon compared to WT mice  . .In contrast to the localization of CSF-1R+ cells (PC) to the crypt base of the SI, in both the mouse Csf1r gene in the mouse intestinal epithelium and found a reduction in PC, Lgr5 expression, cell proliferation and altered cell fate that phenocopied those observed in mice with the global Csf1r deletion . As PCs appear to be the only CSF-1R-expressing cells of the SI epithelium, these results are consistent with an aberrant regulation by PC of the proliferation and fate of epithelial cells resulting from disruption of the direct, local regulation of PCs by CSF-1. Thus it appears that it is through its action in PCs that the CSF-1R plays such a critical role in the SI. In colon, despite the lack of PCs, we have shown that, as in the SI, CSF-1R deficiency results in decreased cell proliferation, reduction in the expression of cell cycle genes and altered cell fate. Most importantly, as in SI, we have found that the expression of the mRNA for Lgr5, the most compelling stem cell molecular marker in the GI tract to date, is markedly reduced in the colonic crypts of −/−Csf1r compared with WT mice. Our failure to demonstrate robust CSF-1 stimulation of the proliferation of cells of two colonic epithelial cell lines, compared with their robust response to R spondin, suggests that, as in SI, epithelial cell proliferation is not directly regulated through the CSF-1R, despite the broader expression of the CSF-1R in colonic crypts. Thus these experiments demonstrate a critical role for the CSF-1R in the development of the colonic epithelium that does not appear to be a direct effect of regulation of the proliferation and differentiation of epithelial cells. In colon, it is possible that the CSF-1R is required for the development of cells with a function analogous to that of intestinal stem cell supporting PCs.Previously we showed that expression of the cell surface CSF-1 isoform could completely rescue the CSF-1-deficient phenotype within the SI and that the CSF-1-reporter expressing cells were localized in the crypts adjacent to PC, suggesting that regulation of PC by CSF-1 was local, if not juxtacrine Csf1r+/− female mice were minimally affected by the same DSS treatment regime. Interestingly the same gender difference has been reported for colitis in man Csf1r+/− mice, heterozygous loss of PPRAγ gene in female mice exacerbated DSS-induced colitis +/−Csf1r mice are indistinguishable from WT litter-mates when unchallenged, but protected when stressed indicates the importance of Csf1r gene dosage in DSS-induced colitis. Humans with hypomorphic mutations that dampen CSF-1R signaling might be protected from IBD and, as has been suggested by others CSF-1 has been implicated in a range of inflammatory contexts CSF-1 is the primary regulator of monocyte proliferation and differentiation Normal human mucosa was obtained from a surgical resection for colon cancer as tissue adjacent, but separate from the tumor, with institutional human ethics approval. Crypts were released as described elsewhere Csf1op/+−/Csf1r+R software. c-Myb was visualized using Mab1.1 and processed as described previously Post-natal day 14 mouse pups were perfused at atmospheric pressure with periodate-lysine-2% paraformaldehyde-0.05% glutaraldehyde, pH 7.4 (PLPG) ad libitum for 8 days and observed for inflammatory responses as well as changes in body weight. Clinical symptoms were assessed based on the modified criteria reported by Marshall et al., 2007 et al (1998) Female and male FVB/n mice aged from 8–10 weeks were fed water with 2% w/v DSS gapdh.Crypt epithelium was prepared and its purity confirmed as described YAMC R Cytokine antibody Array 3 was used to assess the presence of CSF-1 and other factors present in PMUE according to manufacturer instructions.RayBioData were analyzed using GraphPad Prism5 statistical package where p<0.05 was considered to be statistically significant.Figure S1Alcian blue (AB) staining shows increased acidic mucin production in −/−Csf1r and op/opCsf1 colonic epithelium. Similar to, but more extensive than observed with PAS staining (−/−Csf1r colonic epithelium. Bar = 50 µm.(PDF)Click here for additional data file.Figure S2PMUE-stimulation of immortalized colonic epithelial cells YAMC cells following serum starvation. (A) Five×104 YAMC cells were cultured in fetal calf serum-free media with and without increasing amounts of PMUE (a source of CSF-1) prior to assessment of viable cell numbers by MTT assay at day 5. Upper panel: Representative images of wells incubated without or with the indicated concentrations of PMUE. Lower panel: Quantitation of data from multiple plates . (B) Time course of Immorto-5 (IM-5) cells Erk1/2 phosphorylation status in response to PMUE. Cytosolic fractions of cells grown with fetal calf serum (FCS), or serum-starved, or serum-starved and then incubated with 5 µl/ml PMUE for the indicated times were subjected to SDS-PAGE and western blotted for phospho-ERK 1/2 and total ERK 1/2. (C) Cytokine antibody arrays show that CSF-1 is the predominant, but not the only growth factor/cytokine in PMUE (red ellipse). The next most abundant factors identified were IGFBP-3 and MIP-2 (black rectangles). (D–E) IM-5  cells show robust proliferation by MTT assay in response to PMUE but and slight stimulation by purified the CSF-1R ligands, CSF-1 or IL-34. (F) Immorto-5 cells respond strongly to R-spondin in the presence or absence of CSF-1, while Wnt3a has no stimulatory effect alone or in combination with R-spondin or CSF-1, .(PDF)Click here for additional data file.Figure S3Cell cycle and immediate gene expression induction in colonic epithelial cells following PMUE stimulation. IM-5 cells were cultured in the presence of FCS (+FCS), or serum-starved (-FCS), or serum-starved and incubated with 5 µl/ml PMUE for the indicated times prior to extraction of RNA for analysis of gene expression by qRT-PCR. Results show induction of immediate early genes (ets-2 & c-Jun) and cell cycle genes  following PMUE stimulation, .(PDF)Click here for additional data file.Figure S4Absence of obvious differences between male FVB/NJ +/−Csf1r and WT small intestines following DSS-induced colitis. As it has been reported that an increase in villus height and crypt depth may occur in response to DSS-induced colitis (bottom left panel) or villi (bottom middle panel) between WT and +/−Csf1r mice was observed. In addition, no significant differences in intestinal length between the DSS-treated WT and +/−Csf1r mice were detected in small intestines or colons (bottom right panel). Images are representative H&E stained section of small intestine, (Bar = 100 µm).(PDF)Click here for additional data file.Figure S5CD24a and R-spondin mRNA expression are not significantly altered in −/−Csf1r colonic crypts. (A) Absence of Csf1r mRNA in Csf1r−/− crypts. No significant change in CD24a mRNA (B) or R-spondin-1 mRNA (C) expression in −/−Csf1r compared with WT crypts was observed, .(PDF)Click here for additional data file.


Mevalonate kinase deficiency (MKD) has a wide spectrum and severity of clinical manifestation. Patients with mutations in MVK gene leading to complete lack of the enzyme, suffer from the most severe form of disease, also known as mevalonic acydosis, whereas defects with preserved, but insufficient enzyme activity present with autoinflammatory syndrome , also known as hiperIgD syndrome (HIDS). Both diagnosis and treatment of MKD is a great challenge for clinicians. Hematopoietic stem cell transplantation (HSCT) is the only available therapy that allows delivery to the tissues of the missing enzyme produced by healthy donor hematopoietic cells.We present a case of 4-year-old girl with genetically confirmed mevalonic kinase deficiency.She has suffered from recurrent fever, lymphadenopathy, hepatosplenomegaly, hepatitis, impaired growth since she was born. Constant mevalonic acyduria was the first key for diagnosis, then confirmed by genetical analysis of MVK and lack of MK enzyme activity in blood cells. The response to treatment with steroids and IL1-blocker was poor, with only partial resolution of autoinflammation and steroid-dependent adverse events. Due to worsening clinical condition, at the age of 2.5 years patient received bone marrow transplantation (BMT) from matched sibling donor. Due to decreasing donor chimerism, the girl required second transplantation from the same donor at the age of 3 years, followed by repeated donor lymphocyte infusions (DLI). Five months after last DLI the patient achieved stable full donor chimerism. At last follow up, 12 months after HSCT, the girl is in excellent clinical condition, without signs of autoinflammation for 7 last months, progress in psychomotorical and physical development and good immunological reconstitution was observed. Activity of mevalonate kinase is present after transplantation, although still below normal range and mevalonic acyduria is still observed. To assess further improvement of laboratory markers and its clinical consequences longer follow up of the patient is required.Presented case encourages to qualify patients with severe course of MKD to hematopoietic stem cells transplantation.None declared


The cerebrospinal fluid is secreted at the choroid plexus and occupies the cavities of the four ventricles, as well as the cranial and spinal sub-arachnoid space. The cerebrospinal fluid moves over the surfaces of the brain and spinal cord and is rapidly absorbed into the general circulation. The choroid plexus forms the blood-cerebrospinal fluid barrier, and this barrier is functionally distinct from the brain microvascular endothelium, which forms the blood-brain barrier. Virtually all non-cellular substances in blood distribute into cerebrospinal fluid, and drug entry into cerebrospinal fluid is not an index of drug transport across the blood-brain barrier. Drug injected into the cerebrospinal fluid rapidly moves into the blood via bulk flow, but penetrates into brain tissue poorly owing to the limitations of diffusion. Drug transport into cerebrospinal fluid vs. brain interstitial fluid requires knowledge of the relative expression of transporters at the choroid plexus  This idea emerged from studies with vital dyes, which were acidic such as trypan blue, and the acidic dyes did not penetrate the vasculature, which forms the blood-brain barrier (BBB). It was not until the 1930s, when studies with basic vital dyes, which do cross the vasculature, demonstrated the importance of the vascular route to brain. Nevertheless, the misconceptions of a hundred years ago are alive and well today. Virtually all neurologists, academic neuroscientists, and industry scientists believe that drug entry into CSF is a measure of drug penetration through the BBB. It is also widely believed that the trans-cranial delivery of drug to the CSF compartment is a viable solution to the brain drug delivery problem.One hundred years ago, the prevailing view was that nutrients in blood were transferred to brain not There are two barrier systems in brain: the BBB, which is formed by the brain microvascular endothelium, and the blood-CSF barrier, which is formed by the choroid plexus epithelium. In addition, the arachnoid epithelium forms a barrier around the brain creating the sub-arachnoid space. Drug transport from blood to CSF is regulated by the choroid plexus, or blood-CSF barrier. Drug transport from blood to interstitial fluid (ISF) is regulated by the brain microvascular endothelium, or BBB. In order to delineate the relative roles of the choroid plexus and the BBB in limiting drug transport in the brain, it is useful to consider the quantitative aspects of CSF flow within the 1200 g human brain.2 [There is approximately 140 mL of CSF in the human brain . In the 2 , which iin vivo, owing to active efflux transport processes at the brain microvasculature [Drug entry into the CSF compartment from blood should not be taken as an index of drug transport across the BBB. Drug transfer from blood to CSF occurs across the choroid plexus, which is leaky compared to the BBB. Virtually all small and large molecules in blood penetrate into CSF, at a rate inversely related to the molecular weight of the substance. The finding of drug entry into CSF is expected, and does not discriminate between those drugs that do, and do not, cross the BBB. This is because the choroid plexus, which regulates drug transfer into CSF from blood, and the brain capillary endothelium, which regulates drug transfer into ISF from blood, are comprised of completely different epithelial/endothelial barriers. The application of recent proteomics technology will enable the description of the different transporter expression profiles in the BBB and choroid plexus . The difculature .Drug that is injected into the CSF compartment is rapidly transported out of brain to the blood. Following the ICV injection of drug, it moves through the CSF flow tracks, and is absorbed into the peripheral bloodstream across the arachnoid villi to enter the general circulation. This is illustrated by barbiturate studies in the dog. The dose of barbiturate that causes anesthesia in the dog is the same, whether the drug is given by an intravenous or intra-cerebroventricular (ICV) route . The barWhen drug is injected into the CSF compartment, the drug exits this space relatively rapidly via convection and bulk flow through the CSF flow tracts. Conversely, the entry of drug into brain parenchyma from the CSF compartment is mediated by diffusion, a process that is much slower than CSF convection and bulk flow. Moreover, diffusion decreases exponentially with the distance. It takes four days for albumin to diffuse 5 mm through water . During Since the penetration of drug into brain decreases exponentially with the distance from the CSF surface , it is nThe CSF 'macrocirculation' involves production of the CSF within the ventricles, bulk flow through the subarachnoid space, and absorption into the arachnoid villi. However, along this route there is a CSF 'microcirculation' that originates from the pial surface of the brain . There iThe CSF macrocirculation functions as a closed hydraulic system, which provides a mechanical buffer between the skull and the brain, and relieves the effects of gravitational and accelerational stresses . WithoutDrug entry into CSF from blood is expected for all drugs, irrespective of whether the drug crosses the BBB. The finding of drug penetration into the CSF should not be taken as evidence that the drug crosses the BBB. Drug injected into the CSF compartment is rapidly exported to the blood with minimal penetration of brain parenchyma more than 1-2 mm from the ependymal surface of brain.The author is consultant to ArmaGen Technologies, Inc.Sole author and had read and approved the final version of the manuscript.


To determine the activity of pseudocholinesterase (PChE) in cerebrospinal fluid (CSF) and serum in children with solid central nervous system (CNS) tumor and to assess whether PChE activity could be a valid biomarker for solid CNS tumors in children.The study and control group included 30 children each. Children in the study group had a solid CNS tumor, while those from the control group had never suffered from any tumor diseases. CSF and serum samples were collected from all participants and PChE activity was determined using the Ellman’s spectrophotometric method. PChE activity in CSF was shown as a cerebrospinal fluid/serum ratio expressed in percentage, ie, PChE CSF/serum ratio. Receiver operating characteristic (ROC) curve was used to assess whether PChE activity can be used as a biomarker for identifying children with solid CNS tumors.P = 0.001). PChE CSF/serum ratio in the study group was 2.38% (interquartile range [IQR] 1.14-3.97) and 1.09% (IQR 0.95-1.45) in the control group. ROC curve analysis of PChE CSF/serum ratio resulted in an area under the curve (AUC) value of 0.76  and a cut-off of 1.09. Twenty five of 29 patients with elevated PChE CSF/serum ratio had a tumor, corresponding to a sensitivity of 83% and a specificity of 53%.Children with solid CNS tumor had significantly higher PChE activity in CSF and serum, as well as PChE CSF/serum ratio (PChE CSF/serum ratio may be used as a test or biomarker with good sensitivity for solid CNS tumors in children. Pediatric solid central nervous system (CNS) tumors are rarely detected at an early stage. The reasons for this include the tumor site, acute intracranial bleeding, unavailable imaging tests, inconsistent conduct of clinical diagnostic procedures, etc . CNS tumOne of the biological materials used for biomarker testing is cerebrospinal fluid (CSF) . CSF wasPChE is an enzyme (EC 3.1.1.8) synthesized in the liver and distributed to body tissue and fluids. Cholinesterases (acetylcholinesterase – AChE and PChE) activity changes in an abnormal tissue metabolism and/or they become part of the oncogenic process . CholinePChE activity in CSF in healthy people is rarely investigated, most often as a part of studies on specific diseases or anesthetics -13. LumbThe study was conducted at the Reference Center for Solid Tumors in Children of the Ministry of Health and Social Welfare in Croatia, Children’s Hospital Zagreb, Croatia, between October 1, 2007 and November 30, 2011. Ethical approval was received from the Ethics Committee of the Children’s Hospital Zagreb. Written consent was obtained for each child from their parents, who had been explained the study objectives and procedures verbally and in writing.The number of study participants was limited to 60 because we had only 60 laboratory kit tests. We had 30 patients with solid CNS tumor and 30 patients who met the criteria for the control group. When all the available laboratory kit tests had been used we finished the study. There was a total of 60 participants aged between 2 months and 16 years. The study group comprised of 30 children with solid CNS tumors. Tumors were classified according to the 2007 World Health Organization classification . We diviThe control group comprised 30 children who had never suffered from any tumor diseases. Eight children were hospitalized due to the management of febrile convulsion; after a neurotrauma without any sign of hemorrhaging into the CNS (7 children); for the replacement of the ventriculoperitoneal drain system as a result of growth in case of Arnold Chiari malformation (3 children) and Dandy Walker malformation (4 children); because of the congenital hydrocephalus (6 children); meningomyelocele (1 child); and syringomyelia (1 child). We chose these children because each child had normal neurological status or only chronic motor handicap; brain CT result showing only anatomical variations, and normal biochemical, cytological, and microbiological CSF test results. We did not choose random healthy children, since they do not have an indication for the lumbar puncture, which would interfere with the ethical principles.Samples of blood and CSF (both 0.5 µL) were collected at the same time, only once from each child. The samples were immediately centrifuged and separated from erythrocytes, and then stored at -70°C. We determined PChE activity using the Ellman’s spectrophotometric method with a Careside TM cholinesterase test system  using p-hydroxybenzoil choline as a substrate, manufactured by Vitros DT Slides .There was a difference in PChE activity between age groups, which was even more pronounced in girls. Moreover, previous measurements of PChE only in CSF showed high variability . This isCytological evaluation (complete cell count), microbiological , and biochemical analysis  was performed on CSF obtained by lumbar puncture. All drugs taken by the participants at the time of sampling were noted.P < 0.05. Receiver operator characteristic (ROC) curve analysis was used for each of three variables  to determine the optimal cut-off point that maximized the desired test properties. The optimal cut-off of the investigated parameters was calculated as the threshold value with the highest specificity and sensitivity. For the cut-off points, results are presented as sensitivity, specificity, and positive and negative predictive values (PPV and NPV). For each variable, area under the curve (AUC) of sensitivity plotted against 1-specificity is also reported with 95% confidence intervals (CI).All analyses were performed using the IBM SPSS statistical package version 20.0 . As PChE levels were found not to be normally distributed, results are reported as median and interquartile ranges (IQR). In a skeletal box-and-whisker plot, the whiskers are drawn from the quartiles to the extreme values of the group. Normality was evaluated with the Kolmogorov-Smirnov and the Shapiro-Wilk test. Group comparisons were performed using the nonparametric Mann-Whitney U tests for continuous data and Fisher exact test for categorical data. As a nonparametric measure of statistical dependance between two variables we used Spearman's rank correlation coefficient. The significance level was set at P = 0.795), but they did by age (P = 0.001). In the study group, there were 12 girls and 18 boys between the ages of 2.8 and 16 years . In the control group there were 16 boys and 14 girls between the ages of 2 months and 16 years .Groups did not significantly differ by sex  and a negative correlation in the control group . It was the same in the serum . PChE CSF/serum ratio showed a positive correlation with age in both study  and control group .PChE activity in CSF showed a positive correlation with age in the study group . PChE activity in the serum was higher in the study group, but not significantly (P = 0.056).In the control group, the median PChE activity in the serum was 6488 U/L IQR, 5202-7772) and in CSF 70 U/L  ,2. Media02-7772 aP = 0.001). In the control group, this correlation was also positive (r = 0.26) but not significant (P = 0.167).The results indicate high variability of PChE activity in CSF in the study group. In this group, we also found a positive correlation (r = 0.68) between PChE activity in the serum and PChE CSF/serum ratio , for CSF PChE 0.65 (95% CI 0.55-0.75), and for PChE CSF/serum ratio 0.76 (95% CI 0.63-0.88). ROC analysis suggested that the CSF PChE cut-off of 80 U/L (21/30 patients with tumors had PChE in CSF>80 U/L) could produce 70% sensitivity, 70% specificity, 70% PPV, and 70% NPV. When the cut-off value was further increased to 150 U/L, all 16 patients with PChE CSF levels >150 U/L were positive for solid tumor, corresponding to a specificity of 100%, sensitivity of 53%, PPV of 100%, and NPV of 68%. For PChE CSF/serum ratio, a cut-off of 1.09 was evaluated. Twenty-five of 29 patients with elevated PChE CSF/serum ratio were positive for tumor, corresponding to a sensitivity of 83%, specificity of 53%, PPV of 64%, and NPV of 76%. In order to maximize specificity, a cut-off of 2.70% was introduced, corresponding to a specificity of 100%, sensitivity of 50%, PPV of 100%, and NPV of 67% .All children in both groups had CSF cytological, biochemical, and microbiological test results within the reference range according their age. Their total protein and albumin values were also within the reference range. This confirmed that other clinical conditions affecting CSF were not present in these children.We registered all drugs taken by children in both groups at the time of sampling. In the control group, 21 children received no therapy and 9 received medication. Three children received ranitidine, 2 valproic acid, 1 child promazine sodium, 1 cefuroxime, 1 prednisone, and 1 phenobarbitone, nitrazepam, pantoprazole, vitamin D3, lactulose, and vigabatrin. In the study group, 18 children received no therapy. Seven children received ranitidine, 1 hydrocortisone, 1 acyclovir, 1 child fluconazole, 1 trimetoprim sulfometoxazole, and 1 radiotherapy.Our research was first to analyze PChE activity in CSF in children with solid CNS tumors and it showed that they had higher PChE activity in CSF than children who did not have tumors. Our aim was to find a relatively easily available biomarker for solid CNS tumors in children that would require less invasive methods. Tissue samples from the CNS are mainly unavailable, which is why we chose to test CSF, which is in direct contact with the brain structures. Around 528 proteins secreted by a tumor have been identified in CSF to date , many ofThere are several limitations of this study. Research was conducted in the Children’s Hospital Zagreb, which is the Reference Centre for Solid Tumors in Children of the Ministry of Health and Social Welfare in Croatia. Due to the size of Croatian population, collecting a larger number of examinees would require considerably more time. For this reason, different types of solid CNS tumors were included in the study. Also groups differed by age, although it is known that PChE activity is different in different age groups . ConsideParticipants in both groups received drugs that could have affected PChE activity. Antiepileptic drugs or sedatives are acting as AChE and PChE inductors in the serum ,19, whilThe variability of PChE activity in CSF was higher in children with tumors. We believe that this is a result of differences between tumor types, especially in relation to glioma tissue. Cavanagh et al showed that glioma tissue had high PChE concentration levels . In our


Minocycline is a tetracycline antibiotic that has been shown to attenuate central nervous system (CNS) lentivirus infection, immune activation, and brain injury in model systems. To initiate assessment of minocycline as an adjuvant therapy in human CNS HIV infection, we conducted an open-labelled pilot study of its effects on cerebrospinal fluid (CSF) and blood biomarkers of infection and immune responses in 7 viremic subjects not taking antiretroviral therapy.There were no discernable effects of minocycline on CSF or blood HIV-1 RNA, or biomarkers of immune activation and inflammation including: CSF and blood neopterin, CSF CCL2, CSF white blood cell count, and expression of cell-surface activation markers on CSF and blood T lymphocytes and monocytes.This pilot study of biological responses to minocycline suggests little potential for its use as adjunctive antiviral or immunomodulating therapy in chronic untreated HIV infection. Human immunodeficiency virus type one (HIV) infection of the central nervous system (CNS) is a nearly ubiquitous facet of systemic infection that begins early after exposure -6. This adjuvant modes of treatment to mitigate brain injury by attenuating immunopathology or interfering with downstream neurotoxic pathways. While a number of adjunctive therapies have been advocated or tested [These indirect mechanisms of injury have led to a search for r tested , none ofr tested -27 and ar tested -35. Thisr tested  or low-cr tested ,37. For r tested ,38, CSF r tested ,40 and Tr tested .Of 17 subjects screened over a period of 3 years (2006-2009), 6 were excluded because of low CSF HIV-1 RNA (N = 3) or unsuccessful lumbar punctures (N = 3). Three other subjects withdrew from the study without starting minocycline treatment. One subject enrolled in the study but stopped after 4 days due to a reaction to minocycline (nausea and vomiting) that resolved after stopping the drug. The remaining 7 subjects entered the study and were prescribed minocycline. Their baseline characteristics are shown in Table Figure This pilot study was undertaken to explore the use of minocycline as an adjuvant treatment for chronic HIV infection, particularly for attenuating the CNS components of immunoactivation and infection. It aimed to provide a preliminary view of the biological effects of minocycline on CNS HIV immune reactions and infection, and to obtain effect-size estimates for power calculations prior to planning a larger controlled trial. Our underlying mechanistic hypotheses centered on the proposed capacity of minocycline to attenuate CNS immune and systemic perturbations and their effects on CNS infection as revealed by changes in CSF and blood biomarkers. We hypothesized that attenuating these immunological effects would be reflected in reductions in CSF (and perhaps plasma) neopterin and CSF CCL2 concentrations, and in the expression of surface activation markers on T cells and monocytes. Additionally, we hypothesized that minocycline might also indirectly reduce CNS infection through its effects on various immune system-related mechanisms that contribute to the magnitude of CNS (and CSF) infection, including: CD4+ T cell traffic that brings both infected cells and uninfected targets into the CNS, and CD4+ T cell and macrophage activation that enhance viral replication in these cell types. Unfortunately, in this study, none of these effects were seen. Similarly, there were no changes in the other secondary endpoints, including CSF WBC counts, CSF:blood albumin ratios or the brief neurological performance battery, the QNPZ-4.in vitro and in in vivo animal models [in vitro [in vitro studies on human peripheral blood CD4+ T cells demonstrate that minocycline has anti-viral effects in CD4+ T cells and reduces cellular CD4+ T cell activation [Minocycline, a licensed tetracycline antibiotic, has been reported to have a number of properties that make it an attractive adjuvant therapy candidate. In various model systems, it has been shown to have anti-inflammatory effects ,44, incll models -52. Thesl models , and neul models ,52,54,55in vitro . Importain vitro . Recent tivation . Since aWhile it is possible that the CSF measurements were insensitive to salutary effects on the brain parenchyma, including the important perivascular environment, this does not seem likely. CSF neopterin is a marker of CNS macrophage activation  that increases with disease severity and is especially elevated in HIVE/HAD ,38. Thistransitory infection [autonomous or compartmentalized infection, is characteristically detected as a minor contributor to CSF HIV levels in neuroasymptomatic chronic infection, but predominates in more advanced infection, particularly HIV encephalitis (HIVE) [CSF HIV-1 RNA levels reflect more than one cellular source, with the relative contributions differing depending on the stage of systemic and CNS infection and disease evolution -66. Shornfection ,37 and infection . A secons (HIVE) .Minocycline might attenuate both types of infection by its effects on T cell and monocyte-macrophage activation. In the case of transitory infection, T cell activation is critical to support HIV replication and also promotes T cell traffic that carries infected and uninfected target CD4+ T cells into the meninges and perivascular spaces. Hence, if minocycline alters these T cell properties it might reduce this type of CSF infection. Similarly, activation is likely important for macrophages in sustaining infection and also, perhaps, in their entry into the CNS, including into the perivascular spaces, meninges and parenchyma. Minocycline might, therefore, reduce this type of autonomous infection. However, we detected no evidence of reduced CSF infection, although in the subjects studied with relatively preserved blood CD4+ T cell counts, the major CSF viral population likely originated from transitory type infection, although this was not directly examined in these subjects.Our methods of examining the hypothesized actions of minocycline should have been adequate to detect a substantial immunological or virological effect of minocycline. Possible reasons as to why there were no discernable effects similar to those in the SIV-infected pigtailed macaques may have included species differences. Perhaps more likely were differences in the disease targets. The SIV model differs from our subjects in the relatively short disease duration and the presence of frank lentivirus encephalitis . Our stuhttp://clinicaltrials.gov/ct2/show/NCT00361257), and these issues should be addressed by that study.The study also did not assess the more direct neuroprotective properties of minocycline. With one exception, our subjects were largely neuroasymptomatic, and we performed only brief quantitative neurological performance testing (QNPZ-4) on four measures. The small improvement noted in this measure, which was not statistically significant, might have related to practice effect. However, if the observed improvement was indeed real, then a study with 20-25 subjects in each of two treatment arms (minocycline and placebo) would be needed for an 80% power to detect the difference found here at 8 weeks. An AIDS Clinical Trials Group study is studying whether minocycline might improve performance in cognitively impaired HIV-infected subjects  in CSF HIV-1 RNA at 8 weeks was indeed a 'real' finding, then it would require more than 100 subjects in each of the two arms (minocycline and placebo) to have an 80% power to detect this difference between the groups, a difference with likely little clinical meaning. In the case of CSF neopterin, there was no statistically significant reduction, but if the slight increase at 8 weeks (0.033 nmol/L) was inverted and actually a reduction, it would take 500 subject in each group to detect this difference. Thus, the effects of minocycline on infection and immune activation appeared too weak to justify a study of the requisite size, particularly when viewed in comparison to the potent effects of combination antiretroviral on these variables .instead of combination antiretroviral drugs. However, given the reported in vitro and SIV effects of this tetracycline [In conclusion, this small pilot study suggests that any effects of minocycline on CNS HIV infection and immune activation were not sufficient to impact chronic HIV in the absence of antiretroviral treatment. Therefore, there seems little justification or indeed ethical basis for treating chronic HIV infection with minocycline acycline , there sThis study was approved by the University of California San Francisco Committee on Human Research and conducted according to the principles expressed in the Declaration of Helsinki. Informed written consent was obtained from all subjects. The study was registered with ClinicalTrials.gov (number: NCT01064752).This was an open-labelled, uncontrolled, pilot study examining the effects of 100 mg of minocycline taken orally twice daily for 8 weeks. Subject entry criteria included: ≥18 years of age; chronic HIV infection with plasma and CSF HIV-1 RNA concentrations >1,000 copies/mL; not taking antiretroviral therapy (either naïve to therapy or >6 weeks off treatment with no plans to start during the period of study); predicted medication adherence; blood CD4+ T cell counts >100 cells/μl; no previous adverse reaction to tetracyclines; no tetracycline treatment for the past 6 months; no contraindications to lumbar puncture (LP); no active opportunistic infection or neurological disease confounding evaluations; ADC stage <1 ; no concThe primary outcome measures were the change from baseline during treatment in CSF HIV-1 RNA and CSF neopterin concentrations as indices of CNS infection and immunoactivation . Change HIV-1 RNA was measured in cell-free CSF and plasma by the Roche Amplicor HIV-1 Monitor assay . Neopterin concentrations in cell-free CSF and plasma were measured in batch by ELISA according to the manufacturer's instructions . Blood CD4+ and CD8+ T cell counts were performed in the San Francisco General Hospital (SFGH) Clinical Laboratories using standard flow cytometric methods. CCL2 was measured in cell-free CSF by ELISA . Other measurements performed in the SFGH Clinical Laboratories using routine clinical methods included CSF and blood albumin , CSFCSF and blood CD4+ and CD8+ T cell activation were assessed by the percent of these cells in fresh specimens co-expressing surface CD38 and HLA-DR by multiparameter flow cytometry as previously described . Blood mChanges from baseline to follow-up test intervals were analysed by Kruskal-Wallis test with Dunn's post hoc comparison of individual intervals and additionally from baseline through week 8 using repeated measures ANOVA with Dunnet's post hoc comparison. All P values were two-sided with values <0.05 considered significant. Statistical analyses used Prism 5  while power calculations used StatMate 2.00 (GraphPad Software Inc).Dr. Price has received funding from Merck to support an investigator-initiated research study and an honorarium from Abbott for a conference presentation. The other authors have no competing interests.ELH examined study participants, performed lumbar punctures, and assisted with the analysis of the data and preparation of the manuscript. SSS examined study participants, performed lumbar punctures, and assisted in design of the study and reviewed the manuscript. EL served as the patient study coordinator, aided in the design of the study, performed the quantitative neurological performance testing and managed the data. DF performed assays of CSF and plasma neopterin. ES designed the flow cytometry assays, directed the SFGH Clinical Immunology Laboratory that performed the flow cytometry assays and CSF CCL2 ELISA assays, and analysed and interpreted flow cytometry data. RWP designed and oversaw the study, examined study participants, performed lumbar punctures, analysed and interpreted the data, and participated in preparation of the manuscript. All authors read and approved of the final manuscript.


CSF glucose 5th percentile values ranged from 1.8 to 2.9 mmol/L and 95th percentile values from 3.8 to 5.6 mmol/L. CSF/plasma glucose ratio 5th percentile values ranged from 0.41 to 0.53 and 95th percentile values from 0.82 to 1.19. CSF lactate 5th percentile values ranged from 0.88 to 1.41 mmol/L and 95th percentile values from 2.00 to 2.71 mmol/L. Reference ranges for all three parameters were widest in neonates and narrowest in toddlers, with lower and upper limits increasing with age. These reference values allow a reliable interpretation of CSF results in everyday clinical practice. Furthermore, hypoglycemia was associated with an increased CSF/plasma glucose ratio, whereas hyperglycemia did not affect the CSF/plasma glucose ratio.Cerebrospinal fluid (CSF) analysis is an important tool in the diagnostic work-up of many neurological disorders, but reference ranges for CSF glucose, CSF/plasma glucose ratio and CSF lactate based on studies with large numbers of CSF samples are not available. Our aim was to define age-specific reference values. In 1993 The Nijmegen Observational CSF Study was started. Results of all CSF samples that were analyzed between 1993 and 2008 at our laboratory were systematically collected and stored in our computerized database. After exclusion of CSF samples with an unknown or elevated erythrocyte count, an elevated leucocyte count, elevated concentrations of bilirubin, free hemoglobin, or total protein 9,036 CSF samples were further studied for CSF glucose , CSF/plasma glucose ratio  and CSF lactate values . CSF glucose, CSF/plasma glucose ratio and CSF lactate were age-, but not sex dependent. Age-specific reference ranges were defined as 5–95 Cerebrospinal fluid (CSF) analysis is an important tool in the diagnostic work-up of neurological disorders like infection or inflammation of the central nervous system, intracranial hemorrhage and inherited metabolic disorders. It is therefore remarkable that little consensus exists amongst different laboratories about the reference ranges for CSF glucose and lactate We performed an extensive literature search using PubMed for studies on the subject of reference values for CSF glucose, CSF/plasma glucose ratio and CSF lactate by using MeSH terms . Furthermore, we studied the relevant references mentioned in the articles.www.neurochemistry.nl) were systematically collected and stored in our computerized database. This study was approved by the local Medical Ethical Committee, METC Arnhem – Nijmegen. Since laboratory data had routinely been stored in the hospital database and were anonymously processed for the purpose of this study, the METC waived the need for patient informed consent. CSF samples that were analyzed between September 1993 and December 2008  were included. To define reference values for glucose and lactate in CSF obtained by lumbar puncture we have excluded (1) all CSF samples from patients from the neurosurgical ward or intensive care units (where CSF is often obtained from ventricular shunts); and CSF samples with (2) an erythrocyte count ≥200/µL or unknown erythrocyte count; (3) a bilirubin concentration ≥0.5 µmol/L and/or a free hemoglobin concentration ≥0.25 µmol/L; (4) an elevated leucocyte count; or (5) an elevated total protein concentration  and CSF lactate  concentrations if only the first CSF sample of each patient was included.th percentile ranges were determined after exclusion of (1) CSF samples from patients with hypoglycaemia (defined as plasma glucose <3.0 mmol/L), hyperglycemia (defined as plasma glucose >7.8 mmol/L) or unknown plasma glucose level at the moment of lumbar puncture  CSFLumbar punctures were performed in everyday clinical practice, thus on any time of the day, using standard procedures, and preferably after a ‘fasting’ period of at least 4–6 hours. CSF was collected in polypropylene tubes and was immediately transported after withdrawal to the adjacent laboratory. The CSF samples were analyzed within two hours after sample collection. After cell count, CSF was centrifuged at 860 g at room-temperature and glucose, lactate, total protein and blood pigments concentrations were analyzed the same day.CSF glucose was measured by the hexokinase method The CSF lactate concentration was determined by the enzymatic conversion of NAD into NADH, measured at 340 nm, in the presence of lactate dehydrogenase. CSF lactate was measured linear until 13.32 mM with a LOD of 0.020 mM, cients of variation (CV) <1.2% and a recovery of 98–100%.Total protein concentration in CSF was determined with the Lowry reaction and absorbance is measured at 720 nm. Lactate, glucose and total protein were analyzed with an automated analyzer . The laboratory participates in external quality control programs of Instand  for the quality control of the glucose, lactate and total protein analyses. Results are available upon request. CSF erythrocytes and leucocytes count were determined manually in a Fuchs-Rosenthal counting chamber. Assay methods were completely validated if the method was altered during the inclusion period (e.g. change in detection device) and were evaluated according to the QC-system (ISO 15189).The allowed time frame for parallel sampling of plasma and CSF was 30 minutes. Glucose was measured in plasma by the hexokinase method th data-observed percentile ranges. 95% Confidence Intervals were based on Bootstrap Percentiles (based on 1000 bootstrap samples).Statistical analyses were performed using SPSS version 20.0 . Patients were divided into age groups which are commonly used in daily clinical practice  . We inclReference ranges for CSF glucose, CSF/plasma glucose ratio and CSF lactate based on studies including large numbers of CSF samples were not available. Most studies had been performed in newborn or young infants and were based on a small number of patients (i.e. <350 patients) and only a few had been performed in patients without an explicit neurological disorder. We have summarized the results of our literature study in th percentile values until the age of 6 months (1.7–2.0 mmol/L) . CSF glucose 95th percentile values were highest in neonates (5.6 mmol/L) and lowest in patients aged 3–6 months (3.9 mmol/L) and 3–4 years (3.8 mmol/l). Above the age of 4 years a gradual increase in the CSF glucose 95th percentile was seen (from 4.0 to 6.1 mmol/L). After exclusion of CSF samples from patients with an abnormal or unknown plasma glucose level at the moment of lumbar puncture, the CSF glucose 5th percentile value did not show a change of >10% of the original value. CSF glucose 95th percentile values, however, did change more than 10% in patients aged 4–8 weeks, 3–12 months and ≥60 years after the application of these exclusion criteria. Numbers were unaffected in the other age groups.CSF glucose concentrations were measured in 8,871 out of 9,036 (98%) CSF samples. CSF glucose ranged from 1.0 to 11.9 mmol/L . CSF glucose concentrations were age-dependent with remarkably low 5 mmol/L) . After tPlasma glucose was measured in 4,516 out of 9,036 (50%) paired samples and ranged from 0.80 to 40.4 mmol/L . An elevated plasma glucose (>7.8 mmol/L) was found in 338 samples (7.5%). Plasma glucose was elevated in 13.5% of the samples from patients older than 50 years of age, compared to 3.7% in samples from patients younger than 50 years of age. A lowered plasma glucose (defined as <3.0 mmol/L) was found in 24 samples (0.5%) of patients aged 0–78 years .th percentile values were seen below the age of 6 months (0.36–0.42) and in children aged 3–4 years (0.43) compared to neighboring age groups. Above the age of 4 years the CSF/plasma glucose 5th percentile values gradually increased from 0.44 to 0.46 until the age of 40 years, with a continuous decrease afterwards to 0.35. CSF/plasma glucose ratio 95th percentiles were high in patients below the age of 12 months (1.03–1.38).The CSF/plasma glucose ratio is described in th percentiles were >10% lower compared to the original value in patients below the age of 4 weeks, whereas >10% higher 5th percentile values were found in patients older than 50 years of age. Numbers were unaffected in the other age groups.After exclusion of CSF samples from patients with abnormal (defined as >7.8 mmol/L or <3.0 mmol/L) or unknown plasma glucose level, CSF/plasma glucose ratio 95th percentile concentrations were, like CSF glucose, remarkably low in (young) children (0.86–1.14 mmol/L) and increased with age. CSF lactate 95th percentile values were particularly high in neonates (3.4 mmol/L) and low in children aged 6 months to 10 years (2.0–2.1 mmol/L). After the age of 18 years a gradual increase of the 95th percentile value was seen from 2.2 to 2.7 mmol/L. Exclusion of CSF samples with a CSF lactate value of >3000 µmol/L did not lead to a change of >10% of the original value of the 5th of 95th percentile value, except in patients under the age of 4 weeks and in patients aged 3–6 months.CSF lactate concentrations were measured in 7,614 out of 9,036 (84%) CSF samples. CSF lactate ranged from 0.16 to 12.5 mmol/L  CSF lactate showed a clear age-dependent pattern . CSF lacth and 95th percentile ranges for CSF glucose , CSF/plasma glucose ratio  and CSF lactate  values did not change >10% compared to the original value). Age-specific reference values were therefore based on the 5th and 95th percentile ranges of all CSF samples   . ReferenA linear relation between plasma glucose and CSF glucose was found . HypoglyOur study allowed for the first time to define age-specific reference values for CSF glucose, CSF lactate and CSF/plasma glucose for children and adults based on a large number of CSF samples. Reference ranges did not differ between males and females, which is in line with previous findings Since it seems impossible to obtain CSF from healthy volunteers  under standardized conditions (e.g. after fasting), we included CSF samples from our computerized database of patients with a medical indication for a lumbar puncture, which reflects the daily clinical practice. Because of the large number of samples it was not feasible to obtain clinical information from the patients. In order to exclude as many as possible CSF samples of patients with overt neurological disorders, we have therefore used strict exclusion criteria based on other CSF parameters . Furtherth percentile values ranging from 1.7 to 2.9 mmol/L and 95th percentile values from 4.5 to 5.6 mmol/L. Age-dependency of CSF glucose had been described before in children th percentile values were remarkably low until the age of 6 months (1.7–2.0 mmol/L), which is in line with previous studies th percentile value in our study was especially high in neonates (5.6 mmol/L), which is higher than previously described (3.5–4.0 mmol/L) th percentile values and high 95th percentile values in neonates in our study might be related to the high frequency of hypoglycemia and hyperglycemia in neonates, especially in preterm infants and neonates with a low birth weight th percentile values in infants aged 4 to 8 weeks and 3 to 12 months increased more than 10% compared to the original value. This was due to a skewed distribution most likely caused by the low number of CSF samples in these subgroups after exclusion of samples with abnormal or unknown CSF glucose . In a study comparing the CSF/blood glucose ratio from patients with diabetes mellitus and noninflammatory neurological conditions with patients with bacterial meningitis, the CSF glucose ranged from 2.78 to 25 mmol/L in patients with diabetes, with CSF/blood glucose ratio ranging from 0.20–3.46 CSF glucose 5–95th percentile values were seen (0.36–0.44), whereas 95th percentiles were high in patients younger than 12 months (1.05–1.20). A high CSF/plasma glucose ratio in newborns is thought to be related to the higher cerebral blood flow in newborns in combination with a smaller brain that utilizes less glucose The reference value for CSF/plasma glucose ratio is typically about 0.6 and is generally set at 0.5–0.8 A linear correlation was found between plasma glucose (range 2.2–23.3 mmol/L) and CSF glucose (range 1.2–11.0 mmol/L), with CSF glucose values exceeding 8 mmol/L in patients with hyperglycemia. Whereas hypoglycemia was associated with an increase of the CSF/plasma glucose ratio, hyperglycemia barely affected the CSF/plasma glucose ratio . This fith percentile values around 1.95 mmol/L. Higher 95th percentiles for children under the age of 6 months were found in the present study (2.01–2.64 mmol/L). Furthermore, we observed increasing 95th percentile values above the age of 40 years. The tendency for CSF lactate to increase in adulthood (after the age of 54 years) has been described before in a study on cisternal CSF specimens The reference range for CSF lactate is generally set at 1.2–2.1 mmol/L, but ranges from 0.6 to 3.1 mmol/L are used as well  [1]. We th percentile range of the reference range. We have therefore excluded CSF samples with a CSF lactate concentration >3000 µmol/L in second instance. This, however, did not change the 5–95th percentile value more than10% compared to the original value, except for >10% lower 95th percentile value in patients younger than 4 weeks and patients aged 3–6 months. For these patients we have therefore based the reference range for CSF lactate on the CSF samples with CSF lactate <3000 µmol/L , a neurometabolic disorder caused by defective glucose transport into brain. With the exception of rare cases In conclusion, we have defined age-specific reference values for CSF glucose, CSF/plasma glucose ratio and CSF lactate. They allow a reliable interpretation of CSF results in everyday clinical practice.Table S1N  – total number of CSF samples. N – number of CSF samples with measured CSF glucose concentration. 95% CI – 95% Confidence Interval based on Bootstrap Percentiles (based on 1000 bootstrap samples). Numbers and CSF glucose concentrations between brackets represent the results after exclusion of CSF samples with hypoglycemia (blood glucose <3.0 mmol/L), hyperglycemia (blood glucose >7.8 mmol/L), or unknown blood glucose at the moment of lumbar puncture .(DOC)Click here for additional data file.Table S2N  – total number of CSF samples. N – number of CSF samples with measured CSF glucose concentration. 95% CI – 95% Confidence Interval based on Bootstrap Percentiles (based on 1000 bootstrap samples).*) Sample size too small for bootstrapping. Numbers and CSF/blood glucose values between brackets represent the results after exclusion of CSF samples with hypoglycemia (blood glucose <3.0 mmol/L), hyperglycemia (blood glucose >7.8 mmol/L), or unknown blood glucose at the moment of lumbar puncture .(DOC)Click here for additional data file.Table S3N  – total number of CSF samples. N – number of CSF samples with measured CSF glucose concentration. 95% CI – 95% Confidence Interval based on Bootstrap Percentiles (based on 1000 bootstrap samples). Numbers and CSF lactate concentrations between brackets represent the results after exclusion of CSF samples with CSF lactate >3000 µmol/L .(DOC)Click here for additional data file.


The excessive accumulation of adipose tissue in the obese state is linked to an altered secretion profile of adipocytes, chronic low-grade inflammation and metabolic complications. RBP4 has been implicated in these alterations, especially insulin resistance. The aim of the present study was to determine if a local inflammatory micro-environment in adipose tissue regulates RBP4 expression and secretion.in vitro model for adipose inflammation. Adipocytes were exposed to recombinant TNF-α, IL-1β, IL-6 or IL-8. In addition, coexpression of IL-1β and RBP4 was measured in adipose tissue samples from 18 healthy females. RBP4 expression was studied by quantitative PCR and ELISA.Human SGBS and primary adipocytes cultured with conditioned media from human THP-1 macrophages were used as an RBP4 mRNA expression and secretion was significantly reduced upon incubation with macrophage-conditioned media in SGBS adipocytes and human primary adipocytes. Out of several factors studied we identified IL-1β as a new factor regulating RBP4. IL-1β significantly downregulated RBP4 mRNA and secretion in a time- and dose-dependent manner. IL-1β mediated its inhibitory effects on RBP4 expression via IL-1 receptor and NF-κB, as incubation with the IL-1 receptor blocking antibody and the NF-κB inhibitors CAPE and SC-514 reversed its effect. Most interestingly, RBP4 mRNA was negatively correlated with IL-1β mRNA in subcutaneous adipose tissue.Adipose tissue inflammation as found in the obese state might lead to a downregulation in local RBP4 levels. IL-1β was identified as a major factor contributing to the decrease in RBP4. The increase in circulating RBP4 that often precedes the development of systemic insulin resistance is most likely unrelated to inflammatory processes in adipose tissue. Adipose tissue has a major role in the regulation of systemic metabolism by secreting numerous factors, collectively called adipokines that act both locally and on distant tissues RBP4 is a lypocalin protein transporting retinol in the circulation in vitro model of inflamed adipose tissue, i.e. SGBS adipocytes cultured with conditioned media from human THP-1 macrophages. We furthermore aimed at identifying single factors regulating the expression of RBP4.The aim of the present study was to determine if an inflammatory micro-environment in adipose tissue might influence the production of RBP4 in human adipocytes. To this end, we used an 2) undergoing plastic surgery. Patients with malignancies were excluded from the study. Three adipose tissue samples from non-obese women  were used for the preparation of primary preadipocytes. All procedures in experimental subjects were performed in accordance with the Declaration of Helsinki guidelines and were approved by the ethics committee of the University of Ulm. Written informed consent was obtained from all subjects.Subcutaneous adipose tissue was collected from 18 healthy women  were cultured in RPMI medium containing non-essential amino acids, L-glutamine, sodium pyruvate, antibiotics and 10% fetal bovine serum. Differentiation into macrophages was induced by 125 ng/ml phorbol myristate acetate for 48 hours. MacCM was collected after additional 48 hours of incubation in serum-free basal medium containing 0.5% BSA and cleared by centrifugation. MacCM were pooled from 5 independently performed experiments and then used for experiments. Cytokine concentrations in MacCM were determined by Bio-Plex system  and a commercially availably ELISA kit  and were as follows: TNF-α (39 ng/ml), IL-1β (390 pg/ml), IL-6 (2.1 ng/ml), IL-8 (55 ng/ml) and RBP4 (below detection limit).Adipocytes were used for experiments on day 9 of adipogenic differentiation. For dose response experiments cells were washed in PBS and then treated in serum-free DMEM/F12 medium supplemented with 10 µg/ml iron-poor transferrin, 10 nM insulin, 200 pM thyroid hormone, 0.1 µM cortisol with in increasing doses of MacCM  or respective doses of vehicle (RPMI +0.5% BSA) for 48 hours. For kinetics, cells were treated for 6, 24, 48, and 72 hours with either 10% MacCM or 10% vehicle.Cells were also treated with TNF-α (100 ng/ml), IL-1β , IL-6 (50 ng/ml), and IL-8 (50 ng/ml) for 48 hours. To determine time-dependent effects SGBS adipocytes were treated with 5 ng/ml IL-1β for 6, 24, 48 and 72 hours.To determine mechanisms of IL-1β action, adipocytes were treated with IL-1 receptor type 1 neutralizing antibody (IL-1R1 Ab) (100 µg/ml) or NF-κB inhibitors caffeic acid phenethyl ester (CAPE) (1 µg/ml) or SC-514 (100 µM) with or without IL-1β (0.5 ng/ml).5′-GGC CGT GAT GGC AGA GAT-3′; adiponectin-rev: 5′-CCT TCA GCC CGG GTA CT-3′; peroxisome proliferator-activated receptor gamma (PPARγ)-fw: 5′-GAT CCA GTG GTT GCA GAT TAC AA-3′; PPARγ-rev: 5′-GAG GGA GTT GGA AGG CTC TTC-3′; CCAAT-enhancer-binding protein alpha (C/EBPα)-fw: 5′-GAC CCT CAG CCT TGT TTG TAC TGT ATG CC-3′; C/EBPα-rev: 5′-TTT GGA AAG CTT GTC ATA ACT CCG GTC CC-3′; CCAAT-enhancer-binding protein beta (C/EBPβ)-fw: 5′-CCG CCC GTG GTG TTA TTT AAA GAA GAA ACG TC-3′; C/EBPβ-rev: 5′-GCC CGT AGG AAC ATC TTT AAG CGA TTA CTC AG-3′. Data was analyzed by a comparative 2−ΔΔCT method Total RNA was isolated from adipocytes or adipose tissue using RNeasy Lipid Tissue Kit  following manufacturers instructions. cDNA was reversely transcribed using Superscript II reverse transcriptase . Gene expression was determined by qPCR . Intron-spanning primers were RBP4-fw: 5′-TAG CCT CCT TTC TCC AGA AAG GAA ATG ATG AC-3′, RBP4-rev: 5′-GGT TTC TTT CTG ATC TGC CAT CGC AGT AAC C-3′; succinate dehydrogenase (SDHA)-fw: 5′-CAT GCT GCC GTG TTC CGT GTG GG-3′; SDHA-rev: 5′-GGA CAG GGT GTG CTT CTT CCA GTG CTC C-3′; adiponectin-fw: Medium supernatants were collected and stored at −80°C until further analysis. RBP4 levels were determined by ELISA , according to the manufacturers instructions. Sensitivity of the test was 0.9 µg/l and inter- and intra-assay confidence values (CV) were 5 and 9.8% respectively.The number of morphologically differentiated adipocytes was determined by direct microscopic counting using a net micrometer. Three independent experiments performed in triplicate were analyzed.In vitro differentiated SGBS adipocytes were incubated in insulin-free medium supplemented with 10% MacCM for 48 h. Cellular triglyceride contents were measured using a commercially available triglyceride assay kit .In vitro differentiated SGBS adipocytes were incubated in insulin-free medium supplemented with 10% MacCM for 48 h. Cell-free supernatants were harvested by centrifugation and glycerol content was determined using the Free Glycerol Assay Kit  following the manufacturer’s protocol.t-test or ANOVA and post hoc Dunnetts test were used for comparison between groups. Statistical analysis was performed by GraphPad Prism version 5.0 . P-value <0.05 was considered statistically significant.Data from 3 or more independent experiments were expressed as mean ± SEM. When applicable, Student’s To determine the effect of an inflammatory micro-environment on the expression of RBP4 in human fat cells, SGBS adipocytes were exposed to macrophage-conditioned media (MacCM) obtained from human THP-1 macrophages. Morphologically, we observed the previously described delipidation most likely caused by lipolysis RBP4 mRNA expression was decreased by MacCM in a dose-dependant manner . AlreadyStarting at a concentration of 20%, the corresponding vehicle control also showed an inhibitory effect on RBP4 mRNA expression and secretion most likely due to dilution of the adipogenic basal medium and an increase in BSA concentration.ex vivo differentiated adipocytes obtained from three different donors. Comparable to SGBS adipocytes, we detected a downregulation of RBP4 mRNA expression  to study the dose-dependent effects of this cytokine on RBP4 production. With 0.05 ng/ml IL-1β we detected a downregulation to 69±7% of the vehicle after 48 hours and reached a maximal effect at 5 ng/ml . Concentt donors . The inht donors .We next aimed to identify the signaling mechanisms by which IL-1β mediates its effects on RBP4 production. First, we used an antagonistic antibody to block the function of the IL-1R1 . The IL-in vivo. We therefore studied the mRNA expression of RBP4 and markers of adipose tissue inflammation in subcutaneous adipose tissue from 18 healthy women. As expected from previous reports, we detected a positive association of RBP4  and also the macrophage marker CD68 with BMI . The mRNA expression of IL-1β was however not associated with BMI (R = −0.268) and expression of CD68 (R = −0.121). Most interestingly however and in line with our in vitro data, we detected a negative correlation between mRNA expression of RBP4 and IL-1β  (We next wanted to explore if IL-1β might be a putative regulator of RBP4 expression  p<0.05) . This daExcess adipose tissue accumulation is linked to the accumulation of inflammatory cells within adipose tissue, which is causally associated with the change in the secretion pattern of human adipocytes In several mouse models as well as in humans, Graham et al. showed that a systemic increase in RBP4 precedes the onset of insulin resistance The knowledge on the hormonal regulation of RBP4 is still scarce. Rosiglitazone and adiponectin were identified as positive regulators, while TNF-α was described as a potent inhibitor of RBP4 production in human primary adipocytes Additional, to date non-identified, macrophage-derived factors must however play a role in the described downregulation of RBP4. It is likely that a combination of factors has a more potent effect than single factors, since MacCM was more potent in downregulating RBP4 than single identified factors.It was shown that MacCM in addition to promoting insulin resistance inhibits adipogenic differentiation in murine 3T3-L1 adipocytes and human adipocytes in vitro data, a negative correlation between RBP4 and IL-1β mRNA expression was determined in subcutaneous adipose tissue from these women  and the NF-κB pathway In conclusion, the expression of an adipokine implicated in the development of whole body insulin resistance, RBP4, is downregulated by an inflammatory micro-environment in adipose tissue. In addition to the previously described effect of TNF-α, we determined IL-1β as an additional pro-inflammatory cytokine inhibiting RBP4 expression and secretion in human adipocytes. These results are corroborated by the finding of a negative correlation between RBP4 and IL-1β expression in the human adipose tissue explants.Altogether our results implicate adipose tissue inflammation and specifically IL-1β in the regulation of RBP4 expression in human adipose tissue. The increase in circulating RBP4 that often precedes the development of systemic insulin resistance is most likely unrelated to these processes.