Post-dural puncture headache (PDPH) due to excessive cerebrospinal fluid (CSF) leakage is a well-known complication of lumbar puncture. Although various factors, especially the type of spinal needle, have been demonstrated to be associated with PDPH, the clinical implications of CSF leakage detected on magnetic resonance myelography (MRM) images remain unclear. The objective of this case–control study was to evaluate the association between radiologically visualized CSF leakage and PDPH.Clinical data including patients’ age and gender, types of spinal needle, duration of bed rest, interval between lumbar puncture procedures and MRM studies, and incidence of PDPH were compared between patients who were radiologically-positive and -negative for CSF leakage.Of the 22 patients with definite CSF leakage on MRM images, most were asymptomatic . The remaining three patients, who were suffering from PDPH, only complained of headaches and were treated conservatively. In a review of patients’ clinical data, there were no significant differences in any parameter including the incidence of PDPH between the 22 patients who were radiologically-positive for CSF leakage and the 31 radiologically-negative patients.The significance of radiologically visualized CSF leakage should not be overestimated, as most such incidents are not associated with PDPH and do not require any treatment. Lumbar puncture is generally performed in daily medical practice to measure the pressure in the subarachnoid space, to obtain cerebrospinal fluid (CSF) samples for analysis, to inject contrast medium for myelography, or to induce spinal anesthesia. However, puncturing the dura has the potential to lead to excessive CSF leakage, and CSF hypovolemia subsequent to excessive CSF leakage can lead to post-dural puncture headache (PDPH), which has been regarded as a complication of lumbar puncture for over a century . Its cliRecently, neuroimaging techniques including radioisotope cisternography (RICG) and magnetic resonance imaging (MRI) have enabled the visualization of postpuncture CSF leakage in the epidural space ,5. ConsiThis was a retrospective study evaluating the incidence and clinical characteristics of postpuncture CSF leakage using data obtained at a single medical center, and was approved by the Ethics Committee for Clinical Research of Nagoya City University Graduate School of Medical Sciences, which waived the requirement for informed consent. The privacy of the patients was completely protected. Between January 2009 and March 2012, 329 lumbar punctures were performed to obtain samples for CSF analysis in 251 patients with various neurological disorders  at the Department of Neurology. Of these 329 examinations, 270 were excluded because no subsequent magnetic resonance myelography (MRM) study was performed within 14 days of the lumbar puncture. There were two reasons why MRI examinations were not performed in these patients. The one was that their primary illness did not require lumbar MRI examinations , and the other was that the examination and admission schedules did not allow to perform MRI examinations after lumbar punctures. As a result, 59 examinations involving 53 patients who underwent subsequent thoracolumbar or lumbar MRM studies were included in this study. The lumbar puncture procedures were mainly performed using 21-gauge (G) Quincke spinal needles at the L3/4 or L4/5 intervertebral level. PDPH was diagnosed according to the previously published diagnostic criteria [The thoracolumbar or lumbar MRM studies were performed on a 1.5-T imager  using a synergy spine phased-array coil. The 2D MRM sequence was performed using the following parameters: turbo spin echo (TSE) sequence; repetition time/echo time, 8000 ms/1000 ms; field of view, 350–500 mm; matrix, 512 × 142; slice thickness, 40–50 mm; section orientation, coronal; and TSE factor, 256. Basically, this sequence was utilized as a localizer scan in our institution. Postpuncture CSF leakage was diagnosed according to the previously reported imaging findings by the consensus of two experienced physicians (K.S. and M.N.), who were blinded to the patients’ clinical information [t test was used for comparisons of age distributions. Fisher’s exact test was used for comparisons of the gender distribution and PDPH incidence between the two patient populations. Pearson’s chi-squared test was used for comparisons of the spinal needle gauge and puncture level. The Mann–Whitney U test was used to compare the duration of bed rest and the interval between the lumbar puncture and MRM study. Differences were considered significant when p < 0.05.Statistical analyses were carried out using the SPSS 11.0 statistical software program . The unpaired The characteristics of the patients are summarized in Table In 37 CSF leakage-negative MRM studies, five patients suffered from PDPH 14%, 5/37). In a review of the patients’ clinical data including the incidence of PDPH, their basic characteristics and the details of puncture procedures, no significant differences were detected in any parameter between the radiologically CSF leakage-positive and -negative patients , MRM may be regarded as the first-line examination in the diagnosis of CSF leakages. Its high contrast resolution contributes to detect indirect findings such as epidural-paraspinal fluid collections, especially small amounts of leakages along bone structures -11. FurtAlthough the association between PDPH and CSF abnormalities  is not disputed, the exact pathophysiology of PDPH remains unclear. PDPH is considered to be caused by the hole left in the dura after the lumbar puncture needle has been withdrawn, which can allow CSF leakage from the subarachnoid space . Among tIt is worth noting that the incidence of PDPH did not differ significantly between the radiologically CSF leakage-positive and -negative patients in our study. On the surface, these findings do not seem to support the hypothesis that CSF leakage through dural holes causes PDPH. However, several previous studies have indicated that the volume of CSF lost via leakage and CSF hypotension are not associated with PDPH and have also questioned the dural hole hypothesis ,19. In aA simple older explanatory model for PDPH is that the reduction in intracranial pressure induced by persistent CSF leakage causes traction between pain-sensitive structures such as meningeal membranes, blood vessels, and nerves ,20. On tA number of limitations of the present study need to be addressed. The main limitation is the relatively small study population. The lack of available clinical data due to the study’s retrospective nature is also problematic point. These made it difficult to evaluate various factors that affect the incidence of PDPH, such as the number of lumbar punctures, body mass index, and the patients’ medical histories. Furthermore, the incidence of CSF leakages on MRM might also be affected by the number of lumbar punctures. Another limitation is our use of the 2D MRM sequence; i.e., the lower resolution made this study qualitative rather than quantitative evaluation. To perform the precise measurement of CSF leakage, it is necessary to use the 3D MRM sequence, which achieves higher spatial resolution. Additionally, the interval between lumbar punctures and MRM may be too long in some cases. Therefore, there is a chance of resorption of CSF leak in radiologically-negative patients. In spite of these limitations, the fact remains that most of the patients with definite CSF leakage after lumbar puncture with the relatively large 21G Quincke spinal needle were asymptomatic.In this study, most radiologically visualized CSF leakages are not associated with PDPH and do not require any treatment. It is important that we gain a better understanding of this asymptomatic and incidental phenomenon in order to avoid misdiagnosis and overtreatment.The Authors declare that there is no conflict of interests.KS drafted the manuscript. KS and MN performed the neuroimaging analysis. NM, KO and TY participated in the design of the study and evaluated the clinical information. MS and YO performed the statistical analysis. SK helped to check the imaging protocol. YS participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2253/13/35/prepub


G-CSF treatment had no effect on body weight in LETO or on food intake in either OLETF or LETO. Body fat in G-CSF/OLETF was more reduced than in saline/OLETF . Energy expenditure was higher in G-CSF/OLETF from 4 weeks after the treatments than in saline/OLETF. Serum levels of cholesterol, triglyceride, interleukin-6 and tumor necrosis factor-α were lower in G-CSF/OLETF than in saline/OLETF. Uncoupling protein-1 (UCP-1) expression in brown adipose tissue (BAT) was higher in G-CSF/OLETF than in saline/OLETF, but was unaffected in LETO. Immunofluorescence staining and PCR results revealed that G-CSF receptors were expressed in BAT. In vitro experiments using brown adipocyte primary culture revealed that G-CSF enhanced UCP-1 expression from mature brown adipocytes via p38 mitogen-activated protein kinase pathway. In conclusion, G-CSF treatment reduced body weight and increased energy expenditure in a diabetic model, and enhanced UCP-1 expression and decreased inflammatory cytokine levels may be associated with the effects of G-CSF treatment.Granulocyte-colony stimulating factor (G-CSF) has molecular structures and intracellular signaling pathways that are similar to those of leptin and ciliary neurotropic factor (CNTF). It also has immune-modulatory properties. Given that leptin and CNTF play important roles in energy homeostasis and that obesity is an inflammatory condition in adipose tissue, we hypothesized that G-CSF could also play a role in energy homeostasis. We treated 12 38-week-old male Otsuka-Long-Evans-Tokushima fatty rats  and 12 age-matched male Long-Evans-Tokushima rats  with 200 µg/day G-CSF or saline for 5 consecutive days. Body weight reduction was greater in G-CSF-treated OLETF (G-CSF/OLETF) than saline-treated OLETF  following 8 weeks of treatment (−6.9±1.6% vs. −3.1±2.2%,  Granulocyte-colony stimulating factor (G-CSF) is a cytokine found in vertebrates that primarily acts as a regulator in the formation and development of neutrophils Obesity is a disorder associated with an imbalance between food intake and energy expenditure. Appetite and energy expenditure are primarily governed by hypothalamic mediators such as neuropeptide Y (NPY), agouti-related peptide (AgRP), pro-opiomelanocortin (POMC) and cocaine-amphetamine regulated transcript (CART) ad libitum with standard rodent chow .This study was performed according to the ARRIVE guidelines for animal research The experimental protocol is shown schematically in β, IL-6 and TNFα were measured using a Milliplex Analyzer Luminex 200 System  with a Rat Cardiovascular Disease Panel kit.Blood samples were taken from the tail vein after 8 hours of fasting. Sera were obtained by centrifugation and stored at −70°C. Serum glucose, total cholesterol, triglyceride and free fatty acid (FFA) levels were measured using an Olympus AU400 auto analyzer . Insulin, IL-1The lean body and fat composition of the animals was measured by DEXA using a Hologic QDR 4500 device  with an internal adapted collimator for small animal measurements (Hologic QDR Software for Windows XP version). The scan field was adjustable to a maximum of 36 cm×18 cm and spatial resolution was approximately 1 mm. All animals were anesthetized with Ketalar® (75 mg/kg) and Rompun® (5–10 mg/kg) before the measurement. After anesthesia, the animals were positioned ventrally on a reference film with all limbs extended.2 and VCO2 were measured in all animals for 6 hours at the end of every week using an indirect calorimeter . Energy expenditure was calculated from the following formula provided by the manufacturer:VO2/VO2)×VO2.Energy expenditure  = 3.815+1.232×.2.Brown preadipocytes were obtained from the interscapular BAT of postnatal (1–2 days after birth) Sprague-Dawley rats. Isolation and culture of brown adipocytes were performed, as described previously Brown preadipocytes were induced to differentiate to mature brown adipocytes 2 days after confluence (day 0), as described previously Oil red-O  staining was used to identify the lipid droplets of mature brown adipocytes on day 0 and 4 days after differentiation (day 4), as described previously Immunofluorescence staining for G-CSFR was performed to locate G-CSFR in BAT and on the surface of brown adipocytes. The interscapular BAT was obtained from a 20 week-old male OLETF rat. The BAT sections were blocked with 0.1% BSA containing 10% normal goat serum (NGS)  for 1 hour, and were incubated for 90 minutes with mouse monoclonal anti-G-CSFR primary antibodies . The sections were washed and then incubated with fluorescein isothiocyanate-conjugated secondary antibody  for 60 minutes. Brown adipocytes were fixed with 4% paraformaldehyde. After washed, the cells were exposed to 0.3% TritonX-100 for 10 minutes at room temperature to be permeable. Then, the cells were blocked with 0.1% BSA containing 10% NGS  for 1 hour. The cell were then, incubated for 2 hours with a monoclonal anti-G-CSFR antibody as the primary antibody . After washed again, the cells were incubated with fluorescein isothiocyanate-conjugated secondary antibody  for 60 minutes. Images were obtained on an ECLIPSE 80i microscope equipped with an iAi progressive scan camera  and CytoVision system software .Mature brown adipocytes were exposed to a maintenance media containing G-CSF for 30 minutes on day 4. The optimal G-CSF concentration was determined over multiple attempts by elevating the G-CSF concentration in log scale. In some experiments, mature brown adipocytes were pretreated with 10 µM p38 MAPK inhibitor  for 1 hour before G-CSF treatment.Hypothalami were obtained to perform quantitative PCR (Q-PCR) for NPY, POMC, CART and AgRP, RT-PCR for G-CSFR and Q-PCR for UCP-1 were performed in BAT, and cultured brown adipocytes were obtained on day 0 and day 4 for RT-PCR of G-CSFR, UCP-1, PR domain containing 16 (PRDM16), peroxisome proliferator-activated receptor-γ (PPARγ) and PPAR-γ co-activator 1-α (PGC-1α). RNA was extracted using Qiazol reagent . The primers used in the analyses are shown in RNA samples (1 µg) were reverse-transcribed with Moloney murine leukemia virus reverse transcriptase . PCR mixtures contained 1 µl cDNA, 2 µl 10X PCR buffer, 2 µl dNTP, 0.2 µL Taq DNA polymerase  and 0.4 µl 25 pmol/µl of sense and anti-sense primers . We performed PCR using a C1000TM Thermal Cycler  under the following conditions: initial denaturation at 95°C for 5 minutes, 30 amplification cycles  and final extension at 72°C for 5 minutes. The products were visualized on 2% agarose gels stained with ethidium bromide using Gel-Doc 2000 . For quantification of UCP-1 expression levels in brown adipocytes, gels were scanned, and pixel intensity for each band was determined using the Image J software  and normalized to the intensity of GAPDH.RNA samples (3 µg) were reverse-transcribed as above. Expression was quantified using a Light Cycler® 480 System  with a FastStart DNA Master SYBR Green I kit . We performed PCR using the following steps: incubation for 10 minutes at 95°C followed by 45 cycles of 10 sec at 95°C, 10 sec at 60°C and 8 s at 72°C and a final dissociation curve step at 65°C for 15 sec. The crossing point of each PCR was automatically determined by the LightCycler® program. PCR for all samples was run in duplicate. The transcript levels were normalized against those of GAPDH.BAT was obtained from all of the animals for Western blotting for UCP-1. Mature brown adipocytes on day 4 were used to assess p38 MAPK activity by Western blotting. The samples were homogenized in ice-cold homogenization buffer . We transferred 50 µg proteins into a sample buffer that was then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . After blocked in 5% skim milk solution for 60 minutes, the membrane was incubated with primary antibody for UCP-1 , followed by incubation with HRP-conjugated anti-rabbit antibody , as the secondary antibody. For p38 MAPK, we transferred 80 µg proteins. After the cells were blocked with 5% BSA for 60 minutes, the membrane was incubated with primary antibody for phosphorylated and total p38 MAPK , followed by incubation with the secondary antibody. GAPDH was used as a protein loading control. Positive protein bands were visualized using an ECL kit . The results were quantified with an image analyzer .p-value <0.05 was considered statistically significant. All statistical analyses were performed using SPSS statistics 20.0 for Windows .The results are presented as the mean ± standard error of the mean. Mann-Whitney U-tests were performed to compare the changes in body weights and food intakes of the G-CSF-treated groups and the saline-treated groups each week after the treatments and to compare the changes in body composition, biochemical measurements and UCP-1 expression of the G-CSF-treated groups and the saline-treated groups. Kruskal-Wallis tests were used to compare body weights pre-treatment and at 8 weeks after G-CSF or saline treatment and the expression of hypothalamic appetite mediators in all 4 groups. A The body weight of the OLETF rats peaked at 22 weeks of age, then gradually decreased and reached a similar value to that of the LETO rats at 34 weeks of age. The body weight of the LETO rats peaked at 34 weeks of age and then decreased at a rate similar to that of the OLETF rats . Pre-treThe differences in the percentage of body fat mass in the G-CSF/OLETF were greater than those in the saline/OLETF. In contrast, the differences in lean body mass percentages were not significantly different between the G-CSF-treated groups and the saline-treated groups in both OLETF and LETO rats .Energy expenditures in the OLETF rats did not change significantly for 3 weeks after the treatments. However, 4, 5 and 7 weeks after the treatments, energy expenditures were significantly higher in the G-CSF/OLETF than in the saline/OLETF. Energy expenditures in the G-CSF/LETO and saline/LETO were not different during the experimental period .p = 0.037; IL-6 64.3±8.7 pg/ml vs. 3.9±2.5 pg/ml, p = 0.003; TNFα 5.1±1.0 pg/ml vs. 0.0±0.0 pg/ml, p = 0.002). Serum levels of IL-6 and TNFα in the G-CSF/OLETF rats were lower than those in the saline/OLETF, whereas serum levels of IL-1β were not different in the G-CSF/OLETF and saline/OLETF. There was no difference between serum levels of the inflammatory cytokines in the G-CSF/LETO and those in the saline/LETO  and not observed at 6 days after differentiation . G-CSFR G-CSF is a member of the class-1 cytokine superfamily, many other members of which are known to play a role in energy homeostasis, and its intracellular signal pathways are similar to many cytokines that have known anti-obesity effects It has been reported that the body weights of OLETF rats peak approximately 30 weeks of age and then gradually decrease The effect of G-CSF treatment was not observed in the LETO rats. Appetites were not affected by G-CSF treatment in the OLETF or LETO rats; however, the reductions of body weight and fat mass, the decreases in serum levels of cholesterol and triglyceride, the improvement in systemic inflammation and increases in energy expenditure and UCP-1 expression all appeared only in the G-CSF/OLETF rats. Although the body weights of both the OLETF and LETO rats were similar at the time of the treatments, the OLETF rats are a spontaneous animal model for type 2 diabetes with obesity, while the LETO rats are healthy controls. Therefore, the effects of G-CSF appear to be associated with metabolic and immunologic alterations that have occurred in a diabetic phenotype. The principle function of BAT is to regulate energy expenditure via non-shivering thermogenesis; UCP-1, a mitochondrial membrane protein, plays a key role The potential mechanisms for G-CSF treatment effects could be associated with enhanced UCP-1 expression in BAT. It is well-known that the hypothalamus regulates BAT non-shivering thermogenesis via sympathetic innervations We demonstrated that the BAT of OLETF rats expressed G-CSFRs. The intracellular signaling pathway of G-CSF includes JAK/STAT and p38 MAPK Obesity is a low-grade systemic inflammatory condition mediated by cytokines such as TNFα and IL-6 The major limitation of our study is that the detailed mechanisms of the body weight reduction effect of G-CSF were not sufficiently explored. In vitro experiments with brown adipocytes may partly resolve the question of the enhanced UCP-1 expression in BAT. However, the influence of the immune-modulatory function of G-CSF on BAT still requires further histological and molecular investigation. Long-lasting effects of G-CSF on body weight and energy expenditure are also difficult to explain from our data. G-CSF serum levels significantly decreased only several hours after intra-peritoneal G-CSF injection In conclusion, G-CSF has anti-obesity effects in an animal model with diabetes and obesity. Treatment with G-CSF reduces body weight and fat components, improves lipid abnormalities and ameliorates systemic inflammation without altering appetite. The anti-obesity effect of G-CSF is not associated with improved insulin resistance or glycemic control but is associated with increased energy expenditure and enhanced UCP-1 expression in BAT in a diabetic animal model. The effects of G-CSF treatment on UCP-1 expression in BAT could be mediated by G-CSFR and p38 MAPK pathway or immune-modulatory function of G-CSF. Further experiments are needed to illuminate in detail the mechanisms of the effects of G-CSF on body weight reduction.Table S1Body weight before the treatments of G-CSF or saline.(XLSX)Click here for additional data file.Table S2Blood glucose level before the treatments of G-CSF or saline.(XLSX)Click here for additional data file.Table S3Body weight changes after the treatments of G-CSF and saline.(XLSX)Click here for additional data file.Table S4Weekly average food intakes after the treatments of G-CSF or saline.(XLSX)Click here for additional data file.Table S5Changes in body composition after the treatments of G-CSF and saline.(XLSX)Click here for additional data file.Table S6Energy expenditure changes after the treatment G-CSF or saline.(XLSX)Click here for additional data file.Table S7Serum inflammatory cytokines after the treatment of G-CSF and saline.(XLSX)Click here for additional data file.Table S8Quantitative PCR for appetite mediators in hypothalamus after the treatments of G-CSF and saline.(XLSX)Click here for additional data file.Table S9UCP-1 expression in brown adipose tissue after the treatments of G-CSF and saline.(XLSX)Click here for additional data file.


IL-6 is a proinflammatory cytokine reported to play an important role in the induction of cerebral vasospasm after subarachnoid hemorrhage (SAH). Suppressor of cytokine signaling 3 (SOCS3) is known to act as an inhibitor of signal transduction of IL-6. However, there have been no reports on the expression of SOCS3 in cerebrospinal fluid (CSF) after SAH.The concentration of IL-6 was measured serially up until day 10, in CSF of eight patients with SAH. CSF samples obtained from patients suffering from an unruptured aneurysm were used as controls. The expression of SOCS3 in CSF was further examined by immunoprecipitation methods. Concentrations of IL-6 in CSF increased immediately after the onset of SAH and remained chronically elevated over control values. SOCS3 was significantly expressed in CSF on days 1 to 3 after SAH.Our findings suggest that SOCS3 regulates IL-6 signaling as an antagonist in CSF, immediately following SAH. As the expression of SOCS3 decreases after day 5, IL-6 signals might then be more easily transmitted, presumably resulting in cerebral vasospasm.The online version of this article (doi:10.1186/s12974-014-0142-2) contains supplementary material, which is available to authorized users. The traThe present study was therefore undertaken to clarify the time-course of expression of SOCS3 in CSF after SAH. Our study demonstrates that SOCS3 is significantly expressed in CSF on days 1 to 3 after onset of SAH.Eight patients who underwent surgical obliteration of cerebral aneurysms at Aichi Medical University Hospital within one day after onset of SAH were enrolled in this study. The patients ranged in age from 41 to 66 years (mean 53 years); 3 were men and 5 were women. Initial clinical status was classified according to Hunt and Kosnik grade; four patients were grade II and four were grade III. The amount of blood in the subarachnoid space was defined by Fisher’s criteria; six patients were group 3 and two were group 4. Locations of all aneurysms were in the anterior circulation. After aneurysms were clipped, a cisternal catheter was inserted into the chiasmatic or prepontine cistern and postoperative CSF samples were collected serially with a drainage tube over the following ten days. Cerebral angiography or magnetic resonance angiography revealed focal cerebral vasospasm in some cases. No patients suffered from clinically symptomatic vasospasm or developed infection. Clinical outcomes were assed using the Glasgow Outcome Scale; six patients were classified as good recovery and two classified as moderate disability due to initial brain damages. As a control, CSF samples were obtained from six patients (mean 59 years) undergoing neck clipping for unruptured cerebral aneurysm. All samples were immediately centrifuged and supernatant fluids were stored at −80°C until analysis. The Ethics Committee of Aichi Medical University approved this clinical experiment.IL-6 was measured using an enzyme immuno-assay . The limit of detection of this assay was 0.7 pg/ml.CSF samples after SAH in five patients were used for further study. CSF (500 μl) and SOCS3 polyclonal antibodies  were incubated with gentle shaking for 1 hour at 4°C. After this procedure, protein G Sepharose  was added and incubated overnight at 4°C. The immunocomplexes were then washed with PBS and boiled in 50 μl of Laemmli sample buffer for 10 minutes.All sample buffers were centrifuged and supernatant samples were subjected to 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes under wet conditions and incubated with primary monoclonal antibodies against SOCS3  at a dilution of 1:1,000 overnight at 4°C. After washing, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase  at a dilution of 1:3,000 for 30 minutes at room temperature. Reactions were developed with electrochemiluminescence (ECL) . Bands were scanned with image analyzer LAS-1000  and intensities were quantitated using the NIH IMAGE program.P < 0.05 level.Data are represented as box-and-whisker plots indicating the median, the upper and lower quartile, the largest and the lowest value in the data set. Data are expressed as the mean ± SD values. Significant differences between the groups were assessed by one-way analysis of variance (ANOVA) followed by the Bonferroni/Dunn for multiple comparisons. Statistical significance was concluded at the CSF concentrations of IL-6 were remarkably high in the acute stage  - more than one thousand times the control CSF value (11.4 ± 0.9 pg/ml) -and remained chronically elevated (Figure SOCS3 has been previously identified as an inhibitor of the JAK/STAT signaling pathway activated by IL-6. We examined the expression of SOCS3 in the CSF by immunoprecipitation methods. As shown in Figure In this study we demonstrated that concentrations of IL-6 in CSF increase immediately after the onset of SAH and are maintained for a week after the event. In addition, a significant increase in SOCS3 in CSF was confirmed on days 1 to 3 by immunoprecipitation.et al. argued that IL-6 is a reliable early marker for predicting vasospasm on days 4 and 5 before clinical onset [Significant increases in IL-6 levels in CSF after SAH have been previously reported ,7-9, whial onset . Moreoveal onset , resultiin vivo [The SOCS protein is one of the key factors involved in the negative regulation of JAK/STAT signaling . SOCS gein vivo . In respin vivo ,14. Takein vivo ,16. The The present investigation has, for the first time, clarified chronological expressions of SOCS3 in CSF after SAH. Of particular note is our finding that SOCS3 is significantly expressed in CSF on days 1 to 3 after onset of SAH, suggesting that SOCS3 may inhibit IL-6 signal transduction immediately after SAH. Further studies using SOCS3 as an antagonist of IL-6 should be conducted to more precisely clarify the mechanisms of IL-6 regulation after SAH.The study was conducted with the approval of the Aichi Medical University Ethics Committee.


Hematopoietic chimerism is known to promote donor-specific organ allograft tolerance; however, clinical translation has been impeded by the requirement for toxic immunosuppression and large doses of donor bone marrow (BM) cells. Here, we investigated in mice whether durable chimerism might be enhanced by pre-treatment of the recipient with liposomal clodronate, a macrophage depleting agent, with the goal of vacating BM niches for preferential reoccupation by donor hematopoietic stem cells (HSC). We found that liposomal clodronate pretreatment of C57BL/6 mice permitted establishment of durable hematopoietic chimerism when the mice were given a low dose of donor BM cells and transient immunosuppression. Moreover, clodronate pre-treatment increased durable donor-specific BALB/c skin allograft tolerance. These results provide proof-of-principle that clodronate is effective at sparing the number of donor BM cells required to achieve durable hematopoietic chimerism and donor-specific skin allograft tolerance and justify further development of a tolerance protocol based on this principle. Clinical application of vascularized composite allograft (VCA) transplantation has been limited and is controversial because of the high risk of toxicity from life-long immunosuppression relative to the benefit of acquiring a non-vital organ12326Despite significant breakthroughs in the field, attaining durable mixed hematopoietic chimerism with clinically feasible doses of donor BM generally requires aggressive recipient conditioning. Such regimens are thought to provide a competitive advantage to the infused donor BM cells, especially HSCs, by damaging (or destroying) endogenous stem cells, but are generally associated with significant morbidity and mortality10HSC activity in the niche is tightly controlled; however, under certain circumstances, they can be induced to egress from the BM and enter into the peripheral circulation, a process termed “mobilization”, which is still poorly understood11Previously, we have attempted unsuccessfully to enhance mixed chimerism induction and donor-specific tolerance by mobilizing recipient HSCs with AMD3100 and/or G-CSF before allogeneic BM infusionMale C57BL/6, BALB/c and CBA/Ca mice 8 to 12 weeks old (body weight range: 26 ~ 28 gram) were obtained from The Jackson Laboratory  and were maintained in our specific pathogen-free animal facility. All care and handling of animals was carried out in accordance with guidelines provided in the Guide for the Care and Use of Laboratory Animals published by the U.S. Department of Health and Human Services. All studies were reviewed and approved by the Animal Care and Use Committee of the NIAID, NIH.7 or 5 × 107, which are respectively below and above the threshold for establishing hematopoietic chimerism in our system without BM conditioning using radiation or cytotoxic agents. The immunosuppression protocol used in all experiments involved three consecutive 1 mg injections  of each of the following monoclonal antibodies: rat anti-mouse CD4 , anti-CD40 ligand (MR1) and anti-mouse CD8 , all from BioXcell . In addition, two doses of 12 mg/kg of rapamycin  were administered i.p. at days 6 and 30 post-transplantationAllogeneic BM transplantation was modified from a protocol described elsewhereWhere indicated, skin transplantation was performed as previously described by grafting full thickness tail skin measuring 1 × 1 cm on the two lateral flanks, one for the BALB/c donor type and the other for the CBA/Ca third partyin vivo macrophage depletion was modified from that described previouslyOur protocol of d FITC or PB (SF1-1.1), CD4-APC-Cy7 or -PB (GK1.5), CD8-APC or -PE (53-6.7), CD19-APC-Cy7 (6D5), CD11b-FITC or eFluor 450 (M1/70), F4/80-APC or -PB (BM8). The recipient Vβ repertoire was screened with the following antibodies from BD Pharmingen : anti-Vβ5.1-5.2-PE (MR9-4), anti-Vβ6-PE (RR4-7), and anti-Vβ11-PE (RR3-15) in association with anti-CD4 and anti-CD8. All data were collected using an LSRII flow cytometer  and analyzed with FlowJo X software .Leukocytes were purified from the blood, thymus, spleen, lymph node, BM, liver and lung from mice that had received BM transplantation, then were washed after red blood cell lysis and incubated for 20 min with Fc blocking antibody  followed by the relevant antibody combinations (1:50 dilution) and FACS staining buffer for 30 min at 4 °C. Mixed chimerism was assessed in multiple lineages after staining cells with the following antibodies obtained from BioLegend or eBioscience : H-2Kd-FITC . Finally, nuclei in the samples were counterstained with 4’, 6-diamidino-2-phenylindole  at RT for 40 minutes. All images were acquired directly under 10× NA 0.75 objectives using confocal light scanning microscopy on Zeiss LSM 510 and LSM 710 confocal systems . 3D tiled-images comprising the entire sternum fossae volume were collected for assessment of the number and distribution of donor and recipient cells.Immunofluorescence labeling and confocal imaging was performed using whole mount preparations of the sternum as previously describedt-tests (two-tailed) with Prism 6 (GraphPad Software) and are presented as the mean ± SEM of summary data .Data were analyzed using unpaired parametric The use of liposomal clodronate in mice, especially frequent and large intravenous doses, is known to be toxic and even fatal in a subset of animals+ F4/80+ macrophages in the BM compared to control liposome-injected animals on the day after the last injection , consistent with previously published studiesUsing the same dosing schedule specified above, we found that injection of 300 μl of liposomal clodronate resulted in a 50% reduction of CD11bOn the day after the last liposomal clodronate or control liposome injection of C57BL/6 recipient mice, we infused allogeneic BALB/c donor BM to test whether mixed chimerism induction could be enhanced under transient immunosuppression conditions  . InfusioC57BL/6 mice receiving 20 million BALB/c donor BM cells  showed clear evidence of durable mixed hematopoietic chimerism when they were pre-treated with four every other day injections of 200 μl of liposomal clodronate before receiving the transplant, in contrast to mice receiving the same doses of control liposomes and BM cells .Skin allotransplantation with donor-type (BALB/c) and third-party (CBA/Ca) allografts was performed next in these chimeric mice to determine whether donor-specific skin allograft tolerance could be established in association with clodronate-dependent hematopoietic chimerism. Skin allotransplantation was first performed  90 days after the BM transplant, a time-point when all the recipient mice had been off immunosuppression for at least two months. As expected, all third-party CBA/Ca primary allografts were rapidly rejected (fewer than 20 days) after skin transplantation in all groups tested. The BALB/c donor-type primary allografts were also rapidly rejected in mice receiving 20 million donor BM cells without liposomal clodronate pretreatment. In contrast, prolonged donor-specific primary skin allograft survival was observed in mice pretreated with liposomal clodronate. In particular, in liposomal clodronate-pretreated mice, rejection was markedly delayed in 3 out of 7 animals; in the other 4 mice, no evidence of rejection was observed for up to 90 days post-primary skin allotransplantation , at whic7 donor BM cells (cells) without liposomal clodronate pretreatment , whereas all third party CBA/Ca secondary skin allografts were rejected in fewer than 20 days. Rejection of primary and secondary donor-type allografts in the other two mice occurred 20 and 30 days after the time of secondary skin transplantation .Mice in the low dose clodronate group (200 μL/injection) were sacrificed at 180 days after BM infusion  to check the status of mixed chimerism in immune organs  as well as in liver, lung and kidney. Only background levels of hematopoietic chimerism were observed in mice that had received 20 million donor BM cells without liposomal clodronate pretreatment . In cont+ F4/80+ macrophages, CD4+ T lymphocytes, and CD49b+ NK cells  and peripheral deletion of donor-reactive T cells from the recipiento groups . A similA possible mechanism underlying our finding is that depletion of macrophages in the lungs decreased the passive entrapment of infused donor BM cells (first-pass effect), thus increasing the probability of BM niche engraftment. To test this, we performed BM infusion after administering liposomal clodronate via the intranasal route, a classical approach to selectively deplete lung macrophagesIn the present study, we have demonstrated that the macrophage-depleting agent clodronate promotes durable mixed hematopoietic chimerism and donor-specific skin allograft tolerance in mice. It has been known since the 1940’s that a state of mixed hematopoietic chimerism in allotransplant recipients relative to the genetic background of the donor may be associated with donor-specific tolerance8Several studies have demonstrated that monocyte/macrophage depletion may promote mobilization of HSCs from BM to peripheral blood and spleen in mice1725in vitro-prepared, polyclonal recipient-type regulatory T cells (Tregs) leads to engraftment of clinically feasible doses of fully allogeneic BM without cytoreductive recipient conditioningex vivo is complicated and expensive and may not scale as a community-based treatment option.A number of other mixed chimerism approaches have been investigated trying to transplant clinically realistic doses of BM without irradiation or cytotoxic drugsOur study has several important limitations. First, although it did deplete macrophages in the BM and in other organs, we do not know exactly how clodronate is working. Second, clodronate was toxic and resulted in ~30% mortality in recipient mice, mainly due to cardiac complications (infarction and hemorrhage). We took advantage of the surviving mice, which represented the majority, to perform this proof-of-principle study; however, a role for one or more of the toxic side-effects of clodronate at the doses we used cannot be excluded with regard to promoting chimerism. Third, our data indicate that immunosuppression, although transient, is still needed to induce a state of mixed hematopoietic chimerism and possibly to prevent graft-versus-host disease (GVHD).In conclusion, we have developed proof-of-principle that pretreatment of recipient mice with the macrophage-depleting agent liposomal clodronate can enhance induction of mixed hematopoietic chimerism and promote robust donor-specific skin allograft tolerance. In order to be translated to the clinic, further optimization of the protocol will be necessary, at the level of both safety and efficacy, or instead by identifying another less toxic macrophage-depleting agent able to enhance chimerism and tolerance.How to cite this article: Li, Z. et al. The Macrophage-depleting Agent Clodronate Promotes Durable Hematopoietic Chimerism and Donor-specific Skin Allograft Tolerance in Mice. Sci. Rep.6, 22143; doi: 10.1038/srep22143 (2016).


Over the past years, the essential role of vitamin B6 in brain development and functioning has been recognized and genetic metabolic disorders resulting in functional vitamin B6 deficiency have been identified. However, data on B6 vitamers in children are scarce.B6 vitamer concentrations in simultaneously sampled plasma and cerebrospinal fluid (CSF) of 70 children with intellectual disability were determined by ultra performance liquid chromatography-tandem mass spectrometry. For ethical reasons, CSF samples could not be obtained from healthy children. The influence of sex, age, epilepsy and treatment with anti-epileptic drugs, were investigated.The B6 vitamer composition of plasma  > pyridoxic acid > pyridoxal (PL)) differed from that of CSF (PL > PLP > pyridoxic acid > pyridoxamine). Strong correlations were found for B6 vitamers in and between plasma and CSF. Treatment with anti-epileptic drugs resulted in decreased concentrations of PL and PLP in CSF.We provide concentrations of all B6 vitamers in plasma and CSF of children with intellectual disability (±epilepsy), which can be used in the investigation of known and novel disorders associated with vitamin B6 metabolism as well as in monitoring of the biochemical effects of treatment with vitamin B6. Fig. 1). Pyridoxal phosphate (PLP) is the active B6 vitamer, which is produced from its precursor vitamers by phosphorylation  and oxidation (of PN phosphate and PM phosphate), through the actions of pyridoxal kinase and pyridox(am)ine phosphate oxidase (PNPO), respectively. For transport across the cell membrane, a vitamin B6-specific phosphatase enzyme is indispensable , the aldehyde pyridoxal (PL), the amine pyridoxamine (PM) and their phosphate-esterified forms . PyridoxOver the past years, the essential role of vitamin B6 in normal brain development and functioning has been recognized. Specific genetic metabolic disorders resulting in a functional deficiency of vitamin B6 have been identified as an underlying cause of vitamin B6-responsive epilepsy in children. Most patients suffering from PNPO OMIM #610090) [0090 [1] Optimal treatment strategies for PNPO and antiquitin deficiencies are not known. Whereas antiquitin deficiency can be treated with both PN and PLP, PNPO deficiency usually needs PLP treatment, although patients responsive to PN have been recently reported ,10. A suIn literature, data on B6 vitamers in children are scarce –22. ReceIn this study, we determined concentrations of all B6 vitamers in plasma and CSF of children and we investigated the influence of sex and age. Given the fact that analysis of B6 vitamers is of particular importance in patients suffering from epilepsy, we also studied the influence of epilepsy and treatment with anti-epileptic drugs (AEDs). Our approach to simultaneously sample plasma and CSF allows presentation of the relationship between the vitamers in both body fluids.S1 Table). Plasma was obtained by venous sampling (4 mL in heparin tube) and subsequent centrifugation . For most children, CSF was also obtained. This was done on the same day, by lumbar puncture (fraction IV) [For this cross-sectional study, we used remnant plasma and CSF samples that were taken for diagnostic purposes. Plasma and/or CSF were sampled from 70 children (1–18 years of age) visiting the Sylvia Tóth Center  for diagnostic evaluation of intellectual disability (tion IV) . An addiAfter withdrawal, samples had been protected from light and stored at −80°C until analysis. The longest time to analysis was 6 years and 3 months; B6 vitamers are known to be stable during storage at −80°C ,27 for aStatistical analyses were performed using SPSS 20.0 (IBM Corporation). Because none of the B6 vitamers in plasma and CSF, nor their unstandardized residuals, showed a normal distribution, nonparametric tests (Mann-Whitney U (comparison of two groups) and Mann-Whitney U with Bonferroni correction (comparison of more than two groups)) were applied to study differences in B6 vitamer concentrations. Spearman’s rho (ρ) was used to describe correlations.Written parental informed consent was obtained for the use of all remnant samples. Approval by the Medical Ethics Committee of the UMC Utrecht was obtained.Concentrations of PL, PLP, PM and PA were determined in both plasma and CSF. Since PMP in plasma is highly instable, quantification was not possible . PMP in Three children with extreme B6 vitamer concentration(s) in plasma and/or CSF were excluded . In one Therefore, concentrations of PM (in CSF), PL, PLP and PA (in plasma and CSF) of 67 children were studied in more detail. In two children, plasma and CSF were not withdrawn on the same day, but 21 and 22 days apart. Relative B6 vitamer concentrations did not differ from those in the other 65 children.Table 1 displays median B6 vitamer concentrations in plasma and CSF of children. The most abundant vitamer in plasma was PLP (median concentration 33.9 (range 20.5–151) nmol/L), whereas in CSF the concentration of PL (28.2 (16.1–55.7) nmol/L) was the highest.Fig. 2A). In CSF, concentrations of PA and PL were most strongly correlated .Table 2, ratios and correlations are shown for PL, PLP and PA between CSF and plasma. The strongest correlation was found for PL .In Median concentrations of PL in CSF were lower in boys than in girls (24.3 (16.1–55.7) versus 33.1 (21.1–45.9) nmol/L, p = 0.014) . ConcentConcentrations of PL and PLP in CSF marginally decreased from one to 18 years of age . B6 vitamer concentrations in plasma did not correlate with age (data not shown).Of all 67 children, 37 did not have epilepsy nor used any AEDs, 11 did have epilepsy for which they did not use any AEDs and 19 had epilepsy and used one or more AEDs . B6 vitan = 35) were compared with B6 vitamer concentrations in CSF of the original set of (non-)epileptic children not using any AEDs, since in our dataset there was no influence of epilepsy itself   . The oriTable 3; Fig. 3). In addition, median CSF concentrations of PL as well as PM were lower compared to children not using any AEDs , concentrations of PL were below the lower limit of the concentration range observed in children not using any AEDs, whereas PLP in CSF was decreased in seven children. Three of these children showed decreased concentrations of both PL and PLP in CSF. In addition, three children with a decreased concentration of PL in CSF also showed a decreased concentration of PLP in plasma.n = 16), valproate combined with at least one other AED (n = 14) and monotherapies and/or combinations of other AEDs (n = 21); data not shown).Concentrations of PL, PLP and PM in CSF did not differ between types of AED therapy  was excluded from the original dataset , 5-hydroxyindoleacetic acid (5-HIAA), 3-O-methyl-DOPA and 5-methyltetrahydrofolate (5-MTHF) in CSF. It therefore is very likely that, when being the cause of the clinical picture in these children, genetic metabolic disorders associated with a functional deficiency of vitamin B6 are picked up by these investigations. To our knowledge, none of the children in our dataset suffered from one of these defects, although we cannot exclude the possibility of an undiagnosed neurometabolic disorder subtly influencing B6 vitamer concentrations. So although we used plasma and CSF of children with intellectual disability, we are confident that our approach provides the best possible reflection of healthy vitamin B6 homeostasis in children and that it enables us to obtain further insight in B6 vitamers and their interrelationships.In children, like in adults , the B6 ALPL gene (rs1256335) was associated with plasma concentrations of PLP [Interestingly, the correlation between PLP and PL in plasma is different for children (this study) than it is for adults . Plasma s of PLP .n = 11), we cannot fully exclude a possible effect of epilepsy on B6 vitamer concentrations in plasma and CSF. We did find lower concentrations of PL and PLP as well as PM in CSF of children with AED therapy. This implicates that AED-treated children are at risk of a deficiency of vitamin B6, which might have adverse effects on their brain development and functioning.Our study demonstrates that B6 vitamer concentrations in CSF are not influenced by epilepsy. This finding is supported by Footitt et al , althougB6 vitamer concentrations in plasma were not influenced by epilepsy nor by AEDs. However, three children with AED therapy showed a decreased concentration of PLP in plasma besides a decreased concentration of PL in CSF. In literature, several AEDs have been associated with decreased concentrations of PLP in plasma, for children ,33, as wn = 6), which probably does not affect vitamin B6 metabolism, and children without a diagnosis (n = 7). In the latter group, extensive metabolic investigations, like mentioned above, did not result in a diagnosis. In none of these children, PNPO and antiquitin deficiency were ruled out by genetic analyses. However, the complex clinical phenotypes were not suggestive of these defects. The children were not treated with anti-tuberculosis medication or antibiotic drugs at the time of plasma and CSF sampling.The group of AED-treated children with low PLP and/or PL concentrations in CSF consisted of children (aged 1.3–16.9 years) with a genetic diagnosis . Our data suggest a tight regulation of B6 vitamers in and between plasma and CSF of children. Treatment with anti-epileptic drugs may result in decreased concentrations of several B6 vitamers in CSF. Quantification of vitamin B6 in body fluids is useful in the investigation of known and novel disorders associated with vitamin B6 metabolism and will teach us about the biochemical effects of treatment with vitamin B6.S1 Table(DOCX)Click here for additional data file.


We analyzed the influence of prior exercise designed to reduce predominantly muscle glycogen in either type I or II fibers on pacing and performance during a 4-km cycling time trial (TT). After preliminary and familiarization trials, in a randomized, repeated-measures crossover design, ten amateur cyclists performed: 1) an exercise designed to reduce glycogen of type I muscle fibers, followed by a 4-km TT (EX-FIB I); 2) an exercise designed to reduce glycogen of type II muscle fibers, followed by a 4-km TT (EX-FIB II) and; 3) a 4-km TT, without the prior exercise (CONT). The muscle-glycogen-reducing exercise in both EX-FIB I and EX-FIB II was performed in the evening, ∼12 h before the 4-km TT. Performance time was increased and power output (PO) was reduced in EX-FIB I (432.8±8.3 s and 204.9±10.9 W) and EX-FIB II (428.7±6.7 s and 207.5±9.1 W) compared to CONT , without a difference between EX-FIB I and EX-FIB II (P>0.05). The PO was lower in EX-FIB I than in CONT at the beginning and middle of the trial (P<0.05). The mean aerobic contribution during EX-FIB I was also significantly lower than in CONT (P<0.05), but there was no difference between CONT and EX-FIB II or between EX-FIB I and EX-FIB II (P>0.05). The integrated electromyography was unchanged between conditions (P>0.05). Performance may have been impaired in EX-FIB I due a more conservative pacing at the beginning and middle, which was associated with a reduced aerobic contribution. In turn, the PO profile adopted in EX-FIB II was also reduced throughout the trial, but the impairment in performance may be attributed to a reduced glycolytic contribution (i.e. reduced lactate accumulation). To date, the effects of reduced endogenous carbohydrate (CHO) stores on performance during a time trial (TT) has received limited attention While it seems clear that pre-exercise muscle glycogen availability affects the pacing strategy, it is not known if fiber-selective glycogen depletion will have different effects on pacing strategy. Due to the high-intensity at the beginning of a middle-distance cycling TT An interesting and practical manner to test the effects of fiber-selective glycogen depletion on pacing strategy would be to use validated protocols to reduce selectively the muscle glycogen content of type I or II fibers Therefore, the aim of this study was to analyze the influence of prior selective muscle glycogen-lowering exercise, designed to predominantly reduce the muscle glycogen content of either type I or II fibers, on pacing strategy and performance during a 4-km cycling TT. We hypothesized that the exercise protocol designed to predominantly lower the muscle glycogen content of type I fibers would impair PO during the midpoint of the TT, while the exercise protocol designed to predominantly lower the muscle glycogen content of type II fibers would impair PO at the beginning and during the last part of the TT. In order to provide some insights about muscle recruitment This study was approved by the Ethics Committee of the Federal University of Alagoas.2peak) 4.1±0.1 L·min−1 (55.3±2.6 mL·kg−1·min−1), peak PO (PPO) 253.5±8.6 W] took part in this study. The athletes trained regularly (4–5 days per week) for more than 60 minutes per day and cycled more than 200 km per week. The sample size required was estimated, using mean power output (i.e. performance) as the main outcome, from the equation n = 8e2/d2, as suggested by Hopkins n, e, and d denote predicted sample size, coefficient of variation, and the magnitude of the treatment effect, respectively. Coefficient of variation was assumed to be 1.9% Ten amateur, competitive cyclists  were measured through enzymatic colorimetric reactions in a spectrophotometer .To obtain the EMG signal, the skin of the vastus lateralis muscle of the right leg was shaved, abraded, and cleansed with isopropyl alcohol. Then, bipolar surface silver-silver chloride electrodes , at a 20-mm interelectrode distance, were placed over the muscle belly and aligned to the muscle fiber direction. Before the TT, four 5-s submaximal isometric contractions of the knee  were performed as a warm-up aer and Pan, respectively) were estimated from calculations using the metabolic power and gross mechanical efficiency, in accordance with Hettinga et al. met was calculated from the following equation 2 is the oxygen uptake in L·min−1 and RER is the respiratory exchange ratio. During the warm-up for each experimental trial, the gross mechanical efficiency was determined using the following equation:warm-up is the PO in the warm-up . As gross mechanical efficiency was not different across the conditions, the values were averaged for subsequent analyses. The Paer during the TT was calculated by the equation:aer is the aerobic mechanical power output. An RER equal to 1.00 was assumed during the TT to calculate Pmetan was calculated from the following equation:an is the anaerobic mechanical power output, PO is the power output and Paer is the aerobic power output.The PO corresponding to aerobic and anaerobic contribution . To allow magnitude-based inferences about practical effects of prior low or high-intensity exercises on 4-km cycling time-trial performance, the uncertainty in the effects were expressed as likelihoods that the true value of the effects represent substantial changes  Data distribution was verified by Shapiro-Wilk’s test. Means of the performance time, PO, PThe maximal voluntary contraction carried out immediately before each TT was not significantly different between the three experimental conditions , indicating that there was no influence  from the exercise protocol performed in the evening before the TT.aer, Pan, and iEMG are shown in aer in the EX-FIB I was significantly lower than in the CONT (P<0.05), but there was no difference between CONT and EX-FIB II or between EX-FIB I and EX-FIB II (P>0.05). Mean Pan and iEMG were similar for all conditions (P>0.05).The means for performance time, PO, PThere were significant main effects for condition (P<0.05), distance (P<0.05), and interaction effects (P<0.05) for PO . In the aer and Pan every 200 m are shown in aer (P<0.05), but there was no significant interaction (P>0.05). In all conditions, the Paer increased exponentially and stabilized after the first 800 m (P<0.05), but was systematically lower during EX-FIB I than in the control (P<0.05).The results concerning the Pan only for distance (P<0.05), but no condition (P>0.05) or interaction effect (P>0.05). In general, the Pan in the first 600 m and last 200 m was higher than the other intervals for all conditions  and supra VO2peak exercise  is impaired if athletes start the trial after a muscle-glycogen-lowering exercise protocol A lower mean PO and a longer time to completion of the TT was observed in EX-FIB I and EX-FIB II compared with CONT. Qualitative inferences indicated that these changes were ‘very likely’ harmful and ‘likely’ when EX-FIB I and EX-FIB II were compared to CON, respectively, suggesting that the impairments in performance were meaningful. Using time-to-exhaustion tests, it has previously been reported that performance during high-intensity exercise  of the TT, when the PO increased again in the last part, i.e. a U-shaped curve. This pacing model has previously been shown in studies investigating pacing during a TT of similar distance and duration aer and Pan for EX-FIB II, but Paer was systematically reduced during EX-FIB I , suggesting that selective muscle glycogen depletion may not produce a reduction in the anaerobic metabolism of the same magnitude as when both types of fibers are depleted concomitantly The Post-TT [La] was significantly lower in EX-FIB II than in the CONT condition. An interaction effect indicates that the increase in [La] with exercise was less pronounced during EX-FIB II than during the CONT or EX-FIB I. A previous study has suggested that an exercise protocol designed to reduce glycogen from both type I and II muscle fibers reduces the anaerobic capacity by close to 20% during a single high-intensity (175 to 400 W) square-wave exercise The iEMG of the vastus lateralis did not parallel the changes in PO throughout the trial. Specifically, iEMG remained unaltered throughout the trial . We [33]Finally, we found that although the overall TT performance was impaired in both reduced muscle glycogen conditions, force production  prior to the TT was similar between the three conditions. These results highlight the task-specific nature of fatigue and indicate that the mechanism(s) that dictate task failure during a TT may not necessarily be the same as those influencing pre-exercise maximal voluntary contraction performances. In other words, the specific mechanism responsible for performance reduction may be exercise task dependent A possible limitation of the present study is that we did not use a gold-standard technique  to quantify the muscle glycogen content. However, the muscle biopsy technique is invasive and could have interfered directly with the athletes’ pacing and overall performance. Furthermore, the effectiveness of the selective muscle glycogen-lowering exercises used in this study is well established The results of the present study suggest that prior, prolonged, low-intensity exercise (designed to reduce glycogen of type I muscle fibers) was associated with impairment in PO at the beginning and middle of a 4-km TT, and this was accompanied by a reduction in the aerobic contribution. On the other hand, the prior, short, high-intensity exercise (designed to reduce glycogen of type II muscle fibers) impaired the PO and reduced the plasma [La] accumulation. Irrespective of the inclusion of either a prior prolonged, low-intensity exercise or short, high-intensity exercise, an impairment in overall performance of a middle-distance cycling TT was evident, but the pacing adopted, as well as energy system alterations, occurred in a specific way. To the best of our knowledge, this is the first study demonstrating that the distribution of energy expenditure during a middle-distance cycling TT is dependent of the intensity and duration of the exercise carried out in the evening before.


We previously reported that portable gait rhythmogram(PGR) equipped with accelerometers to identify gait-induced accelerations enable us to quantitative gait analysis in Parkinson's disease (PD). We objected to reveal the quantitative gait differences between idiopathic normal pressure hydrocephalus (iNPH), PD and normal controls (NC) using PGR.The study subjects were 7 patients with iNPH , 12 patients with de novo PD who were not medicated with any kinds of anti-parkinsonian drug  We also studied 17 NC .24hours continuous recordings were performed twice before and after CSF drainage in iNPH patients with a trunk-mounted PGR. In PD and NC, 24hours continuous recordings were performed once at arbitrary point.We calculated the “amount of overall movements per 24 hrs” from all motion-induced acceleration as an index of hypokinesia, “gait cycle and cadence” from the gait signals as indexes of step length, and “gait acceleration range” as an index of step power.Comparing with NC and PD, the average data obtained from 6 CSF drainage responded iNPH patients showed lower “amount of overall movements per 24 hrs”. Faster “gait cycle” and higher “cadence” which indicate narrower step lengths, and lower “gait acceleration range” which indicate ineffective floor reaction forces in iNPH patients were also detected. Improvements in gait following spinal CSF drainage were observed using PGR.In representative case of CSF drainage responded patients, gait defects improved after CSF drainage, and the improvement lasted after LP shunt was performed. Same tendency were observed in the average result of responded patients, though it was not statistical significant.Whereas, no alteration was observed after CSF drainage in an un-responded subject.When considering percent changes of parameters, positive correlations were found between “amount of overall movements” and points of FAB, between “cadence” and points of MMSE, between “cadence” and 10m walk time. Negative correlation was found in “amount of overall movements” and 10m walk time, between “gait acceleration range” and points of FAB.PGR revealed narrower and more monotonous step length in iNPH patients than PD and NC. And it also detected gait improvements following CSF drainage in the CSF drainage responded patients. Thus, PGR can analyze long durational gait in daily life and provide additional quantitative data on former measurements of CSF drainage easily and usefully.


After death, a series of changes occur naturally in the human body in a fairly regular pattern. These postmortem changes are detectable on postmortem CT scans (PMCT) and may be useful in estimating the postmortem interval (PMI). The purpose of our study is to correlate the PMCT radiodensities of the cerebrospinal fluid (CSF) and vitreous humor (VH) to the PMI.Three patient groups were included: group A consisted of 5 donated cadavers, group B, 100 in-hospital deceased patients, and group C, 12 out-of-hospital forensic cadavers. Group A were scanned every hour for a maximum of 36 h postmortem, and the tympanic temperature was measured prior to each scan. Groups B and C were scanned once after death (PMI range 0.2–63.8 h). Radiodensities of the VH and CSF were measured in Hounsfield units. Correlation between density and PMI was determined using linear regression and the influence of temperature was assessed by a multivariate regression model. Results from group A were validated in groups B and C.r2 CSF, 0.65). PMI overruled the influence of temperature (r = 0.99 and p = 0.000). Groups B and C showed more diversity, with CSF and VH radiodensities below the mean regression line of Group A. The formula of this upper limit indicated the maximum PMI and was correct for >95 % of the cadavers.Group A showed increasing radiodensity of the CSF and VH over time (The results of group A showed a significant correlation between CSF radiodensity and PMI. The radiodensities in groups B and C were higher than in group A, therefore the maximum PMI can be estimated with the upper 95 % confidence interval of the correlation line of group A. Estimating the time of death is an important part of forensic pathology with the postmortem interval (PMI) being defined as the period of time that has elapsed since a person has died. In a medico-legal investigation of death, an accurate estimation of the PMI can be of great importance. It may aid in identification of the victim and, in cases of a violent death, limit the number of suspects and help to validate or reject an alibi and verify witnesses’ statements . Also, iSeveral techniques can be used if the time of death is unknown. In the immediate hours after death, medical examiners and pathologists usually ascertain the PMI utilizing early postmortem changes such as algor mortis (the decrease in body temperature after death), livor mortis (postmortem lividity or hypostasis), and rigor mortis  . These tFor around a decade, postmortem computed tomography (PMCT) and postmortem magnetic resonance imaging (PMMRI) have been introduced into clinical and forensic postmortem investigations, to visualize disease and cause of death and to serve as a virtual guide for autopsy , 13. It The correlation between PMI and radiodensities of CSF and VH was determined in 3 distinct groups of cadavers, with Group A as the standard and Groups B and C validation groups. In all groups PMCT included head, cervical spine and a thoracic-abdominal series. For this study cadavers were scanned in a natural, supine position with arms next to or on the torso, using the scan parameters as shown in Table Individuals who had donated their bodies for scientific research after their death were prospectively included for PMCT scanning from January to March 2014. Ethical approval was waived as these individuals had already consented to any scientific procedure. This is also in accordance with Dutch law. Date of birth, sex, cause, and time of death were provided by the undertaker and the cadavers quickly transported, without a cooling period, to a dedicated CT suite. Immediately after arrival the cadavers were scanned using a 16-slice scanner , every hour for up to a maximum of 36 h postmortem. During this time the cadavers were not moved and prior to each scan, tympanic temperature, using a Basetech LT-80 Digital thermometer, was measured in both ears and the mean calculated. The temperature within the CT suite was 22 to 25 °C with an air humidity varying from 24 to 57 % (TFA Thermo-/hygrometer).Patients who had died in our university hospital or in our emergency room and had had a cerebral PMCT scan, were prospectively included in this group from July 2012 to January 2015. Exclusion criteria were: intracranial intervention or hemorrhage for the CSF measurements and after ocular donation for the VH measurements. Relatives were required to give consent for the PMCT and the local ethical committee waived any ethical approval for study purposes. Basic patient data, including time and assumed cause of death, were obtained from the digital patient files. The PMCT was performed as soon as possible after death and whenever possible, prior to an eventual autopsy. If immediate scanning was not possible, the cadaver was stored in the mortuary at 4 °C. The scanners used were, Siemens Somatom Sensation 16, a Siemens Sensation 64  or an Aquilion ONE . Temperatures of these cadavers were not measured with the scan suite temperature and humidity standardized according to the hospital settings.Out-of-hospital cadavers from a forensic institute were retrospectively included if the head anatomy was intact . Patient data included the cause and the assumed or estimated time of death, based on forensic investigations. The cadaver was transported to the CT suite after forensic investigation of the crime scene and having being stored in the mortuary. The PMCT was performed with the body in a natural supine position, using an Aquilion 64 . The temperature of the cadavers was not measured in the CT suite. During the scan procedure, room temperature and humidity were standardized according to the institutes’ settings.Radiodensity in Hounsfield units (HU) of the VH and CSF were measured by two researchers. Circular regions of interest (ROI) automatically calculated the mean using IMPAX . The radiodensity of the CSF was determined by the mean of 4 measurements with a maximum fitting ROI positioned in the anterior and posterior horns of the left and right ventricles, excluding the choroid plexus or visible beam hardening artifacts of plexus calcifications. The radiodensity of the VH was determined by the mean value of the left and right VH, excluding the lenses .The correlation between the PMI and postmortem CSF and VH density measurements were evaluated using linear regression. In Group A, a mixed model was used to predict the PMI, in hours, with the fixed effect being the mean radiodensity. A random intercept and slope was calculated per cadaver to manage the multiple observations per individual. The fixed effect regression line is the average regression line of the donated cadavers. Using the fixed effect regression line, a prediction of the maximum PMI was calculated to validate this model for Groups B and C and was compared to the actual PMI. Results are shown in scatterplots .Correlations of the temperature and the PMI to the radiodensity of CSF and VH were analyzed in multivariate regression models. The inter- and intra-observer reliability were calculated with interclass correlation (ICC) and 95 % confidence interval (CI), using a random sample of 15 cases in group B [Five donated cadavers, 100 in-hospital deceased patients and 12 out-of-hospital forensic cadavers were included in this study. Basic patient data are shown in Table This group included 4 males (80 %) and 1 female (20 %) donated cadavers, with an age range from 68 to 90 years (mean 78.8 years). The PMI of the first scan varied from 6 to 10 h, with a mean PMI starting at 7.6 h. Measurements of CSF and VH radiodensity on the PMCTs were performed in all cases. Tympanic temperature was measured in only 4 of the 5 cadavers due to a technical failure.The in-hospital cadavers consisted of 62 males (62 %) and 38 females (38 %). Their age ranged from 0 to 91 years  and the PMI at PMCT ranged from 0.18 to 63.8 h . VH density measurements were performed in 99 cadavers as 1 cadaver was excluded due to a cornea donation prior to the scan. Another cadaver had an ocular prosthesis of the left eye so only the right eye was included. Density measurements of the CSF were performed in 98 of the 100 cadavers as one cadaver was excluded due to intracranial hemorrhage and in another cadaver, measurements were not possible as the ventricles had collapsed.The forensic cadaver group comprised 9 males (75 %) and 3 females (25 %) with an age range of 29–84 years . Four individuals had died after gunshot wounds to the thoracic-abdominal region, but without any intracranial wounds. Two individuals died of asphyxia , two other individuals died after blunt trauma and one after a fall from height. Three individuals were found with an unknown cause of death and after forensic investigations, cause of death was determined to be due to natural causes . Measurements of CSF and VH radiodensity were performed in all cases.The radiodensity of CSF on the PMCT of group A, showed an increase over time for all 5 cadavers, with radiodensities of 5.7–8.1 HU on the first scan at a PMI of 6–10 h, followed by an almost linear increase over time to a radiodensity of 10–12 HU at 36 h.r2) for the CSF was 0.65, which indicated a fairly accurate application of the model  of cerebrospinal fluid (CSF) and vitreous humor (VH) increase with the increasing postmortem interval (PMI).A linear correlation is found between the CSF radiodensity and the PMI.CSF radiodensity is a predictor of the PMI; and overrules body temperature in a multivariate regression model.We present a formula to estimate the PMI using the radiodensity of CSF.Postmortem computed tomography may be more than anatomical information; it may possibly be a helpful modality to estimate the PMI.


The general population is exposed to cadmium from food and smoking. Cadmium is a widely spread toxic pollutant that seems to be associated with cardiovascular diseases, although little is known if it contributes to the occurrence of atherosclerotic plaques and the process whereby plaques become vulnerable and are prone to rupture. We tested the hypotheses that cadmium exposure is associated not only with an increased subclinical burden of atherosclerotic plaques in different vascular territories and early signs of plaque vulnerability, but also with cadmium content and plaque-rupture in the clinical phase of the disease. Ultrasound technique was used to measure plaque prevalence and echogenicity in the carotid and femoral arteries in a population sample of women (n = 599) in whom blood cadmium was measured. In addition cadmium was measured in snap-frozen endarterectomies and whole blood obtained from patients who were referred to surgery because of symptomatic carotid plaques (n = 37). Sixteen endarterectomies were divided into three parts corresponding to different flow conditions and plaque vulnerability. In the population sample blood cadmium was associated with the number of vascular territories with plaques . The cadmium concentrations in symptomatic plaques were 50-fold higher in plaque tissue than in blood. Cadmium levels in blood and plaque correlated, also after adjustment for smoking and other cardiovascular risk factors (p<0.001). Compared with the other parts of the plaque, the cadmium content was double as high in the part where plaque rupture usually occurs. In conclusion, the results show that cadmium exposure is associated with the burden of subclinical atherosclerosis in middle-aged women with different degrees of glucose tolerance, and that the content of cadmium in symptomatic plaques in patients is related to that in blood, but much higher, and preferentially located in the part of plaque where rupture often occurs. The general population is exposed to cadmium from food and smoking. The major dietary sources of cadmium are rice, cereals, potatoes, and other root vegetables due to uptake of cadmium from the soil, which is contaminated with fertilizers with high cadmium content and fall-out from industrial emissions and other urban sources .Smoking is a major source of exposure as cadmium in tobacco smoke is effectively absorbed in the lungs . CadmiumThe prevalence of atherosclerotic plaques is closely associated with old age and most plaques never cause clinical disease . A key pB-mode ultrasound images of carotid atherosclerotic plaques with low echogenicity are associated with histological characteristics typical of vulnerable plaques and cerebrovascular clinical events –18. SuchAnother approach to explore symptomatic atherosclerotic lesions is to study endarterectomies of symptomatic carotid plaques. We, as well as others have previously shown that carotid plaques most often rupture in the upstream part, which is before the blood flow passes the maximum stenosis of the plaque –23. AlthIn order to better understand how cadmium may cause symptomatic atherosclerosis we hypothesized that cadmium exposure is associated not only with an increased subclinical burden of atherosclerotic plaques in different vascular territories and early signs of plaque vulnerability, but also with cadmium content and rupture of plaques in the late clinical phase. Accordingly our first aim was to examine if cadmium exposure is positively associated with the sub-clinical plaque-burden in the carotid and femoral arteries and the prevalence of low echogenicity plaques. The second, clinical aim was to examine if human symptomatic atherosclerotic plaques have higher concentration of cadmium than in blood, if cadmium levels in blood and carotid plaques correlate, and if the cadmium concentrations within symptomatic plaques are highest in the upstream part were plaque rupture most often takes place. In the present study we succeeded to test these hypotheses in a population-sample of women, and in a biobank of endarterectomies, representing patients who were operated on for symptomatic carotid atherosclerosis.The subclinical prevalence of plaque burden and echogenicity were examined in a previously described population-based sample , 28, 29.In the clinical substudy of symptomatic carotid plaques we used endarterectomies from the Göteborg Atheroma Study Group (GASG) biobank at the Sahlgrenska University Hospital  , 30. ThiBoth studies have been carried out in accordance with the Declaration of Helsinki and were approved by the Regional Ethical Review Board in Gothenburg and all participants gave written informed consent to participate.Smoking history included current, previous and never smoking. Pack years were calculated . Supine The ultrasound examination of the carotid and femoral arteries was performed with an ultrasound scanner equipped with a linear 8L5-MHz transducer . The examination of the carotid arteries for occurrence of plaques in the near and far wall was performed in accordance with previously published semi-automated methods and definitions , 32–34. The sample consisted of 37 frozen endarterectomies. Sixteen of those allowed full identification of the common, external and internal carotid artery. From a previous study based on MR angiography imaging and serial section of carotid endarterectomy we know that in two thirds of all cases, the maximum stenosis is located 1.5 mm below to 4.5 mm above the flow divider between the external and internal carotid artery . As showIn the remaining 21 carotid endarterectomies it was not technically possible to remove the plaques with a conserved recognizable anatomical structure and cadmium concentration was therefore measured in each endarterectomy without knowledge of which vascular section the plaque originated from.The dry weight of all plaque samples was determined after drying at 90°C for 24 h in a thermostat-controlled cupboard. When the samples had dried, 0.10 ml deionised water was added followed by addition of 0.10 ml with a mixture of concentrated perchloric acid and nitric acid (5:1). The samples were then digested at 110°C for 24 h and thereafter diluted to a volume of 5 ml with deionised water. The blood samples were diluted ten times with an alkaline solution according to Barany et al. . The conTwo reference materials were used for the blood analysis. For Seronorm Trace Elements Whole Blood L-1 , lot. 1103128, the result  obtained was 0.34±0.02 (n = 4) vs. recommended 0.32–0.40, and for Human Blood Reference Samples, lot. C-06-16  the result was 1.06±0.02 (n = 4) vs. recommended 1.1±0.13. All blood samples were prepared in duplicate and the method imprecision  was 3.1%Cadmium in whole blood in the 599 women was analysed with ICP-MS as described above, for further details see ) and from 0.010 to 0.29 μg/g dry weight (geometric mean 0.069 [5–95 percentiles 0.019–0.226]).The characteristics of the patients are shown in Blood concentrations of cadmium correlated with the cadmium content in the plaques, both by wet and dry weight . Smoking2 = 0.46). This partial correlation coefficient was 0.52 (p<0.001) after further adjustment for sex, age, statin treatment, prevalent hypertension or diabetes (R2 = 0.60). Similar results were obtained for cadmium content in plaque by dry weight and when smoking history was replaced by pack years of smoking (data not shown).In a multiple linear regression analysis, log cadmium in plaques/wet weight was associated with log blood cadmium  after adjustment for smoking history and apolipoprotein B/A-I ratio (RThe distribution of cadmium is shown in In the group who had never smoked (n = 5) the geometric mean (SE) of the cadmium concentration/g wet plaque weight was 0.014 (0.002) μg/g, to be compared with 0.022 (0.004) and 0.027 (0.006) μg/g in the previous (n = 18) and current (n = 13) smokers. The corresponding data for cadmium levels by dry weight were 0.045 (0.014), 0.071 (0.016), and 0.082 (0.019) μg/g, respectively. Similarly, the geometric means of blood cadmium levels were 0.26, 0.37 and 0.76 μg/L in the never, previous, and current smoking groups. Hence, in never-smokers there was a 50-fold increase in the concentration of cadmium in plaque tissue, compared with the blood level.In one never-smoking patient it was possible to examine the concentrations of cadmium within a carotid plaque. The distribution was exactly as in the total group with a high cadmium level in the upstream section (0.034 μg/g wet weight) and low levels in the stenosis and downstream sections .The results of the present study showed that increasing levels of blood cadmium were associated with increasing numbers of arterial territories with subclinical atherosclerotic plaques in the carotid and femoral arteries. This finding extends our previous observations of an association between cadmium exposure and carotid plaques and supports the concept of a pro-atherogenic effect , 29, 37.There are autopsy studies from Finland, the Netherlands and England in which cadmium concentrations were measured in different organs or in the aorta –27. TakeWe interpret our results as supporting the hypotheses that cadmium may have both pro-atherogenic effects and effects on the mechanisms leading to plaque rupture. Firstly, our results are in line with previous data showing that cadmium exposure enhances the atherosclerotic process in experimental models –12 and iFor initiation of atherosclerosis the passage of proatherogenic lipoproteins and monocytes through the endothelial barrier and retention in the intimal layer seem to be key mechanisms which lead to an inflammatory response and development of plaques . In suppper se.It is a limitation that this is a cross-sectional study and it is not possible to clarify causal mechanisms. Moreover, it is important to consider the confounding effect of smoking, which is a major source of cadmium exposure together with cadmium intake by food. However, previous large prospective epidemiological studies have shown that cadmium exposure is associated with future cardiovascular disease also in never-smokers, particularly in men –8. In thIn the subclinical study, a subgroup analysis of the never smokers showed that the 95% confidence interval of the beta-coefficient overlapped with that in the total group, indicating that with a larger study it would have been possible to demonstrate if there was an association between blood cadmium and the number of vascular territories with prevalent plaques. The study of subclinical plaques was performed in women and the results cannot be inferred to men, although the pro-atherogenic effects were shown in the sex with the smallest risk for cardiovascular disease.We conclude that our data extend the knowledge from epidemiological and experimental studies of cadmium as a risk factor for atherosclerotic diseases. We have shown that increasing cadmium exposure is associated with increased burden of subclinical atherosclerotic plaques and that symptomatic carotid plaques contain cadmium in levels much higher than those in blood, that cadmium concentrations in blood and plaques correlate and that within the plaques the cadmium concentration is highest in upstream section, where plaque vulnerability often is most pronounced.


Postnatally, engrafted donor skins and leukocytes were examined for their MHC expression by quantitative real-time PCR and flow cytometry. Engrafted donor skins upregulated their MHC class I related gene transcripts after short-term (1~2 weeks) or long-term (>1 month) engraftment. This biological phenomenon was simultaneously associated with upregulation of TAP1 gene transcripts, suggesting an important role of TAP1 in the regulation of MHC class I pathway. The surface MHC class I molecules of H-2Kb in engrafted donor leukocytes consistently showed overexpression. Conclusively, the induction of allograft tolerance involved biological modifications of donor transplants. The overexpression of MHC class I within engrafted transplants of tolerant mice might be used as the tolerance biomarkers for identifying a state of graft tolerance.The alterations in MHC class I expression play a crucial step in immune evasion of cancer or virus-infected cells. This study aimed to examine whether tolerized grafts modified MHC class I expression. FVB/N mice were rendered tolerant of C57BL/6 alloantigens by  Organ transplantation has been the standard practice for failed or failing organs. However, long-term graft survivals rely upon immunosuppressive therapies that lessen or prevent rejection episodes at the cost of direct organ toxicity, susceptibility to opportunistic infections, malignancy, and accelerated cardiovascular diseases , 2. Despq) mice were used as recipients, C57BL/6 (H-2b) as donors, and C3H (H-2k) as the source of third-party alloantigens. They were purchased at their age of 6–8 weeks and bred for this study with the approval of the Committee on Animal Research at Chang Gung Memorial Hospital. Recipient females were caged with males in the afternoon and checked for vaginal plugs the following morning. The day when the plug was observed was designated as day 0 of the pregnancy.FVB/N  and anti-FITC microbeads . T-cell-depleted BMCs contained CD3+ cells of <0.5% by flow cytometry.C57BL/6 bone marrow cells (BMCs) were harvested by flushing the tibias and femurs with phosphate buffer saline (PBS) using a 26-gauge needle, layered over NycoPrep 1.077A , and centrifuged at 600 g for 25 minutes. Light-density BMCs were then depleted of T-cells by anti-CD3μm glass micropipette with beveled tip was used to inject 5–10 × 106 T-cell-depleted BMCs in 5–10 μL of PBS into the peritoneal cavities of all fetuses at a litter via transuterine approach. Then, the abdomen of the pregnant mice was closed in two layers with 5–0 silk. After operation, all mice were housed in an undisturbed room without bedding changes until the pups were 1 week old. Pups were weaned at 3 weeks of age.Donor BMCs were freshly injected within 3 hours after preparation. Briefly, the uteri of gestational day 14 pregnant FVB/N mice were exposed through a vertical laparotomy. A 60 γII/FcγIII antibody (BioLegend) and then stained with anti-H-2Kq FITC (BioLegend) and anti-H-2Kb PE (BioLegend). Chimerism levels were determined by flow cytometry after gating out dead cells by high propidium iodide staining. A negative control consisted of anti-H-2Kq FITC and mouse IgG2a PE (BioLegend) to define background staining. Recipients with first month peripheral chimerism of >3% were collected to further validate tolerance by skin transplantation because they could be consistently rendered tolerant to donor skin [b PE were measured. Wild-type C57BL/6 mice were used as controls.Chimerism levels were examined at the age of 1 month old. Peripheral blood was sampled via tail veins and depleted of red cells using ACK lysing buffer. Cells were first incubated with anti-mouse Fcnor skin . As for Under anesthesia, the recipient's back was shaved by clippers and disinfected by beta-iodine solution. Graft beds with intact panniculus carnosus were created by the scissors . Skin trThe engrafted skin grafts were taken down from tolerant mice at indicated time points. Then, their cell constituents were homogenized by physical treatment using a tissue tearor in Trizol Reagent . mRNAs were extracted with RNeasy Mini Kit  according to the instruction manual. The purity of RNA samples was evaluated using absorption of light at 260 and 280 nm (A260/280) by a ND-2000 spectrophotometer . High-quality mRNA showed the A260/280 ratio of ≥1.97.First-strand cDNA were reversely transcribed from mRNA templates of each skin sample in equal amount, using MMLV High Performance Reverse Transcriptase Kit with an oligo(dT) primer . Then, MHC-I related target gene expression was quantified by Bio-RAD iQ5 real-time PCR detection system, using Maxima SYBR Green/ROX qPCR Master Mix (2X) . The specificity of real-time PCR reaction was further confirmed by melting curve analysis. Normalized values for target mRNA expression in each sample were calculated as the relative quantity of target gene divided by the relative quantity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All the gene primers were designed by online primer design tool of Primer 3 as shown in t-test between two independent or paired-sample groups. Differences were regarded as significant in case of P < 0.05.The equality of means was examined by Student's β2m, H-2Kα, H-2Dα, H-2Lα, H-2Aα, and H-2Aβ genes was determined by normalized fold expression relative to the mRNA quantity of GAPDH internal control gene. This survey for MHC gene expression revealed that engrafted donor skins significantly upregulated β2m, H-2Kα, H-2Dα, and H-2Lα as opposed to wild-type controls (We collected 13 mixed chimeras with first month donor cell (C57BL/6) levels of 6.05~28.18%. All were readily rendered tolerant to donor skin for at least 4 months . Engraftcontrols .We further examined the expression of mRNA that translates accessory proteins responsible for assembly and transport of MHC-I molecules. These chaperone-like proteins in MHC-I processing pathway include the transporter associated with antigen processing (TAP1 & TAP2), tapasin, calnexin, calreticulin, and ERp57. Engrafted donor skins showed significant upregulation of TAP1, but only marginal upregulation of TAP2 .β2m, H-2Kα, H-2Dα, and H-2Lα) and TAP1&2 gene expressions of skin grafts before and after their transplantation. The mice with skin tolerance were used as recipients for secondary skin transplantation after removal of prior engrafted skins. Both allogeneic (C57BL/6) and syngeneic (FVB/N) tail skins were first collected before their placement on tolerant mice. Following skin transplantation, the engrafted skin graft from the same donor tail was harvested at indicated time points (1~2 weeks and >1 month). Following mRNA extraction (A260/280 ratio ≥1.97) and reverse transcription, the expressions of target genes were measured in relation to those of an endogenous GAPDH reference gene by quantitative real-time PCR. Paired-sample t-test was used to compute the differences between values of the two normalized expression variables for each skin graft from the same donor before and after skin transplantation (pre-ST and post-ST). The expressions of β2m, H-2Kα, H-2Lα, and TAP1 were significantly increased in donor skins after short-term (1~2 weeks) and long-term (>1 month) engraftment. However, H-2Dα was only significantly upregulated in short-term engrafted donor skin .In the pursuit of tolerance biomarkers, researchers mostly focused on the recipients in preference to the donor grafts for examining various biological activities or products that sufficed to signify a tolerant state. However, local regulatory mechanisms might contribute to graft tolerance due to Treg activity specifically within tolerated grafts , 33. EviDiscovering a biomarker for transplantation tolerance is not necessarily equivalent to uncovering their causal relationship. However, our approach to the secondary donor skin acceptance in tolerant recipients suggested that MHC-I overexpression was the recipients' tolerizing effects on donor grafts. Within the donor grafts, inflammatory responses might upregulate their MHC-I and related genes. Graft inflammation usually resulted from alloimmune reactions. However, the absence of mononuclear cell infiltration reflected the lack of any ongoing inflammatory process within the engrafted donor skins. Thus, MHC-I upregulation should be specific to tolerance rather than the results of alloimmunity. We have known that the alterations in MHC-I expression play a crucial step in immune evasion of cancer or virus-infected cells through the regulation of adaptive T-cell cytotoxicity  and innaImmune tolerance to donor transplants involved biological modifications of MHC-I upregulation within engrafted donor grafts. This biological phenomenon has great potential to be a tolerance biomarker in the settings of transplantation. More importantly, it may pave the way to further mechanism studies for transplantation tolerance in the future.