Introduction: Coronary artery bypass surgery (CABG) is associated with ischemia-reperfusion injury and tissue damage. CoQ10 as an antioxidant has an important role and may have cardio-protective effects after myocardial dysfunction and CABG. We aimed to evaluate whether CoQ10 has a myocardial cardio protective impact on cardiac biomarkers after CABG.Methods: In this double-blind study, 80 patients with coronary artery disease (CAD) who underwent CABG surgery were divided into intervention and control groups and received Q10 supplement or placebo, respectively. The surgical characteristics of the patients in the two groups were similar. The intervention group received 150 mg of Q10 supplement per day for 7 days before the surgery. The control group received placebo capsule. After operation the inter- and intra-group blood levels of CK-MB and troponin, before and after supplementation and 12 hours after the CABG, and postoperative outcomes such as intensive care unit (ICU) stay and hospital stay were compared.Results: In this study, 40 subjects were located in each group. The participation rate was 97.5% and men and women accounted for 52.5% and 47.5% respectively. The mean age of the subjects was 58.17 ± 8.55. The two groups were not significantly different in terms of basic variables. Within-group comparison showed a significant increase in the level of troponin enzymes over time (P < 0.001) and CK-MB (P < 0.001). However, between-group comparison showed no significant difference between the two groups in terms of CK-MB (P = 0.384) and troponin (P = 0.115). In the end, no interaction was observed between the intervention and time on CK-MB (P = 0.095) and troponin (P = 0.198) variables.Conclusion: Q10 supplementation 7 days before surgery was not effective in reducing CK-MB and troponin after CABG. Reduced glutathione levels lead to the formation of ROS, oxidative stress, and increase intracellular calcium levels.4 Dysrhythmia after the surgery, as an indicator visible from oxidant stress during operation is considered, and can provide a basis for the comparison of strategies in protecting the heart during cardiac surgery.5 CK-MB and Troponin as a marker of myocardial infarction are used to measure these biomarkers to assess patients and controls in terms of infarction after the surgery.6 According to previous studies, a low level of troponin after the surgery is predictive of success in short and long-term. For this reason, decreases in the levels of these biomarkers could be useful.7 Peak plasma levels of troponin in the study of Cummins B, and others, was expressed 14 hours after the onset of acute myocardial infarction (AMI). For this reason, we also measured troponin in the first 12 hours after the surgery.8 It appears that during CABG, total antioxidant capacity is significantly reduced. Coenzyme Q10, as an antioxidant can reduce free radicals and may play an effective role in reducing cardiac enzymes and complications after the surgery.11 The main objective of this study was to determine the effect of Q10 supplements on the level of CK-MB and troponin levels after elective coronary artery bypass graft.Coronary artery bypass surgery (CABG) surgery is a high-risk procedure. Evidence indicates that cardiopulmonary bypass (CPB) leads to inflammatory response that may be responsible for some of the major complications after open-heart surgery.This was a double-blind randomized clinical trial implemented in Shahid Rajai Heart Center in 2013 through a parallel design.12Considering the type of analysis, effect size = 0.25, power = 0.80, α = 0.05 and the fact that the two groups were studied the total number of required samples was determined 82 using the software G-power.All patients who qualified elective CABG surgery based on angiography were included in the study. Also, patients who have experience of warfarin use, antioxidant intake, hybrid surgery, creatinine greater than 1.5 and advance heart failure were excluded from the present study.At first, 100 patients candidate for CABG surgery admitted to hospital were selected with a daily visit to the different wards of Shahid Rajai hospital and according to the study inclusion and exclusion criteria, 88 of them were selected and given a full explanation about the goals of the study and were asked to offer informed consent. Six people refused to consent after hearing explanations and were excluded from the study . FinallyThe considered intervention was a supplement called Q10 which was a vitamin-like enzyme and can reduce the CK-MB and troponin enzymes. This supplement was made in 50-mg capsules by the Elixir Pharmaceuticals, Inc. Eighty-two boxes were used in this study and 41 of which had Label A and contained 50 mg Q10 supplement capsules and 41 other capsules had Label B and contained similar and empty capsules as placebo which were made by the same company. After determining patients and arranging blocks, boxes were given to patients and given that the study was double blinded, patient and the nurse who was the responsible for the hospital ward were unaware from the contents of the boxes and capsules.For the intervention group, Q10 supplement was daily given to patients as 50-mg capsules for seven days and the control group was given capsules as placebo with identical shape and without any drug content (only a similar coating).Standard intravenous anesthesia  was done. Similar standard surgical techniques was done in all cases. Sternotomy was performed from the middle part of the sternum. In addition, grafting one of internal thoracic arteries with 1-5 peripheral veins from lower limbs was considered in each case. CPB was prepared and an anstomosised with proximal area with moderate hypothermia. After surgery, patients were transferred to the intensive care unit (ICU) and separated from mechanical ventilation with the following criteria: Hemodynamic stability, ambient temperature over 36°C, cooperation, lack of major bleeding and acceptable arterial blood gases.® Troponin I Ultra kit is made in France and the MB kit Audit Diagnostics facility in Cork, Ireland), blood samples of all the patients were measured in terms of these enzymes in three occasions before beginning supplements and seven days after supplementation and twelve hours after surgery and were compared on this basis. All patients were transferred to ICU after surgery.The variables of age, gender, average graft time, average time of cross clamp and graft and pre-operation ejection fraction were measured as basic variables. Venous blood samples were taken from the antecubital without tourniquet for cardiac enzyme levels. For the measurement of outcome variables such as CK-MB and Troponin  for the categorical variables. Significance level was set at 0.05.Data were analyzed using SPSS version 16. To study the normality of data distribution, Kolmogorov-Smirnov test was used. For comparing baseline data, P=0.0836). Variable distribution of blood pressure (P=0.115), sex (P=0.179) and diabetes (P=0.799) were not significantly different between the two groups. Besides, the variables bypass time (P=0.667), cross clamp time (P=0.621), Pre-operation (ejection fraction) (P=0.269) and graft (P=0.179) were not significantly different between the two groups . Forty-two patients were male (52.5%) and 38 patients (47.5%) were female. Comparing baseline variables between the two groups of intervention and control showed no significant difference of variable age between the two groups (o groups .P<0.001). Bonferroni test results used for pair-wise comparison showed that CK-MB variable at the second time is less than the first time (P<0.001). Moreover, at the third time it is more than the first time (P<0.001) and at the third time it is more than the second time (P<0.001) and all of these cases were significant (P<0.001). Bonferroni test results showed that troponin variable at the second time is more than the first time (P<0.001), at the third time is more than the second time (P<0.001) and at the third time is more than the second time (P<0.001), which all were significant (Comparing the average variable CK-MB in three time measurements showed that the average variable CK-MB decreased over time and then increased so that it was (21.04 ± 8.33) at the first time, (18.89 ± 6.71) at the second time, and (32.19 ± 20.83) at the third time and the three values were significantly different   .P=0.095) and troponin (P=0.198) variables do not exist  has harmful effects on the heart15 which may be prevented by antioxidant effects caused by supplements,16 reactive oxygen intermediates can cause imbalance and excessive intracellular calcium and these states show themselves with increasing levels of cardiac enzymes of CK-MB and troponin.17There is a lot of information, which suggests that protecting heart during CABG has important long-term effects after surgery.20 However, trying to identify a threshold and the upper and lower limits that predict patients’ prognosis is faced with failure.21Interventional studies of coronary artery showed that the peak level of CK-MB not only is along with higher mortality, but with a higher risk of events after surgery leading to further cost of treatment in these patients.22 It is surely clear that the risk of side effects increases with any increase in CK-MB. Therefore, measurement of cardiac enzymes following coronary intervention is strongly recommended.23 and the reduced level of cardiac enzymes is along with improved surgical outcomes in the short-term and long-term,26 although 0.1 ng/mL troponin was used as the cutoff. Even low levels may cause myocardial injury.27 In this study, 0.1 ng troponins were considered as high levels (positive troponin). After CBP, troponin and CKMB levels begin to rise after six hours and two hours respectively,28 so we used both markers.According to the definition of ACC, cardiac biomarkers level (CK-MB and troponin I) is considered as more than 3 times the upper limit of normal as myocardial infarction and the range between one and three times is considered as myocardial injury.29 these mechanisms minimize cellular damage and a decrease in cardiac enzymes can be experienced. According to previous studies,32 the used dose was determined as 150 mg per day for seven days. On the other hand, according to the study, it is found that a daily dose more than 300 mg creates negative effects such as increased levels of serum LDH.33 that’s why some studies that have used excessive doses of supplements have not reported a positive effect in patients.CoQ10 plays an important role in stabilizing the membrane while helping replenish ATP storage inside the cell.30 so that Foroughinia et al study showed omega-3 positive impact in reducing the levels of cardiac enzymes.34 Makhija et al showed that preoperative use of Q10 can lower levels of cardiac enzymes.11 Other studies have shown that Q10 supplement is effective on troponin and CK-MB enzymes after surgery, even when supplement is administered only once.32 Pepping noted in his study that it takes at least four days to achieve therapeutic level.35 That is why we raised supplementation period to seven days in this study. In a review study by Bulatao and Banez,31 it was shown that oral supplement Q10 in spite of providing lower surgery indices including less drainage, less hospital stay and less need for inotropic had no effect on cardiac biomarkers level. Increasing the number of grafts is directly correlated with higher levels of the enzyme. Jang et al showed in his study that increasing troponin serum levels is associated with the number of involved arteries that underwent PCI.36 In our study, a statistically significant difference was not observed in number of grafts. Previous studies have indicated a strong correlation between the number of grafts and graft type with increased levels of cardiac enzymes as observed in one of the studies37 that internal mammary artery  graft was along with less increase in cardiac enzyme levels. Age and gender were also evaluated as a risk factor for elevating troponin serum levels. But no significant statistical difference was observed between the two groups, age and sex with troponin. Wiviott et al in 2004 compared the effects of gender on designed cardiac enzyme levels. It was observed that the level of CK-MB and troponin is higher in men, while inflammatory marker following AMI and angina is higher in women.38Various studies have indicated the effect of antioxidants in reducing cardiac enzymes level39 In explaining why the supplement could not reduce the enzyme levels, items such as long time surgery can be cited. Because in this study, the time of surgery was more than 73 minutes, while in other studies40 it has been for about 49 minutes and it is obvious that whatever this period is more, the cardiac enzymes CK-MB and troponin will be higher.41 That is why the present study has not been able to be successful in reducing the level of cardiac enzymes.In this study from among 80 cases of intervention and control at baseline, 71.3% had negative troponin (troponin larger than 0.1) and 28.7% had positive troponin and after surgery, total troponin level was 25% percnt negative and 75% positive. Iner and intra group comparisons showed that Q10 supplements could not have a significant effect in reducing the levels of troponin. Although in previous studies, using some supplements such as omega-3 could have a positive effect on the number of positive troponins.43After surgery, the incidence of arrhythmia in the intervention group who received CoQ10 supplement was significantly lower compared with the control group. However, hospital stay and ICU stay were not significantly different between the two groups. Although in some studies, the effect of supplement was seen to reduce the length of stay in the ICU and reduced drainage.According to the results obtained from a randomized double-blinded study, the prescription of Q10 supplements was not effective unlike the designers’ original idea in reducing cardiac enzyme (CK-MB) and troponin after CABG.The results of the present study were based on a research on a small population of patients undergoing coronary bypass surgery that may be the main reason for not observing the effect of supplementation in reducing cardiac enzyme levels. Besides, the short time of supplementation or inadequate dose of Q10 may have a significant role in the lack of impact of reducing troponin. Moreover the limitations of this study include lack of body antioxidant levels during the period mentioned, in fact, our blindfold, just looked at the effect of antioxidant supplements That’s why more studies are needed in this regard.We would like to offer our appreciation and thanks to Tehran Rajaee heart hospital nurses and doctors who have supported us.After obtaining the ethics code of Tehran University of Medical Sciences (25555-161-01-93) and registration at Iranian Registry of Clinical Trials (IRCT ID: 2013122815957N1) was conducted according to the Helsinki protocol..None


In the supratentorial space, the volume of CSF was 3375.2 cm3 and the volume of brain parenchyma was 80.3 cm3. In the infratentorial space, the volume of CSF was 101.3 cm3 and the volume of the brain parenchyma was 88.7 cm3. In the supratentorial space, there was severe malacia of almost all brain parenchyma with no visible remnants of the choroid plexuses. Infratentorial structures of the brainstem and cerebellum were hypoplastic but completely developed. Since our patient had no choroid plexuses in the supratentorial space and no obstruction between dural sinuses and CSF, development of hydrocephalus and macrocephaly cannot be explained by the classic hypothesis of CSF physiology with secretion, unidirectional circulation, and absorption as its basic postulates. However, the origin and turnover of the enormous amount of intracranial CSF volume, at least 10-fold larger than normal, and the mechanisms of macroencephaly development could be elucidated by the new hypothesis of CSF physiology recently published by our research team.The aim of this study was to perform for the first time the intracranial volumetric analysis of cerebrospinal fluid (CSF) and brain parenchyma in the supratentorial and infratentorial space in a 30-year-old female patient with hydranencephaly and macrocephaly. A head scan performed using a 3T magnetic resonance was followed by manual segmentation of the brain parenchyma and CSF on T2 coronal brain sections. The volume of CSF and brain parenchyma was measured separately for the supratentorial and infratentorial space. The total volume of the intracranial space was 3645.5 cm Hydranencephaly is a difficult and rare developmental disorder that occurs in fewer than 1 of 10 000 newborn children, most likely due to bilateral occlusion of the internal carotid artery between 8 and 12 weeks of pregnancy . It is oOur patient, aged 30 years, is one of the oldest living patients with hydranencephaly. The clinical picture is dominated by permanent vegetative state with spastic tetraparesis and the morphological picture by extreme macrocephaly. Although in many patients with hydranencephaly detailed qualitative descriptions of intracranial changes have been made, to our knowledge no volumetric analysis of the cerebrospinal fluid and residual parenchyma has been performed separately for the supratentorial and infratentorial spaces. The aim of this article was to quantify the volume reduction of the parenchyma and volume increase of cerebrospinal fluid (CSF) in the supratentorial and infratentorial spaces and to discuss the developmental mechanism of hypertensive hydrocephalus and macrocephaly with respect to dynamic changes of head circumference.We report on the case of a 30-year-old woman with hydranencephaly, in permanent vegetative state, with severe spastic tetraparesis and emphasized macrocephaly. Immediately after birth she had a somewhat larger head circumference but without manifest neurological symptoms. The head circumference was rapidly increasing during the following months, so that at 2.5 months of age it was 45 cm (standard deviation [SD] = 3.43), at 5.5 months 49 cm (SD = 4.52), at 6 months 53 cm (SD = 6.45), at 9 months 57 cm (SD = 8.40), and at the time of the study 75 cm (SD = 14.1).Signs of spastic tetraparesis started to appear from the 5th month of age and progressed to such a degree that the patient needed continuous medical care. Menstrual cycles are regular but the secondary sex characteristics are incompletely developed. A written informed consent of the legal guardian was obtained before MR exam.Magnetic resonance imaging (MRI) was performed using the 3T MR scanner , with a 12-channel Tx/Rc head coil, the only one that has a sufficient diameter to perform head scan in a patient with severe macrocephaly. After the head scan, a coronal T2 sequence  was used for a manual segmentation of CSF and brain parenchyma in the supratentorial and infratentorial spaces using the Analyze 8.1 program , which measures the volume of the segmented structures in radiological sections .The analysis of the standard structural MR sequences showed macrocephaly with atypically shaped bones of the neurocranium and a particularly marked anterioposterior diameter. Almost all brain parenchyma of the supratentorial space was affected by malacic changes. A small part of the remaining parenchyma was located in the diencephalon area, the mediobasal part of the left temporal lobe, and the mediobasal part of the occipital lobes, with one small island of parenchyma on the right side, just below the frontal bone. There were no visible remains of the ventricular system or choroid plexuses.3), while the total amount of parenchyma was significantly reduced (169.0 cm3), which represents a significant deviation from the average   .3 of CSF, while the brain parenchyma was reduced to only 80.3 cm3. In the infratentorial area, the volume of the brainstem and cerebellum parenchyma was 88.7 cm3, with the CSF volume being 101.3 cm3.The greatest part of the increased intracranial volume was located in the supratentorial space with 3375.2 cmWe report on a patient with an extremely large intracranial space, which was primarily the result of an increase in supratentorial space. The supratentorial space was mainly filled with CSF, with only small areas of sustained parenchyma. These results comply with the assumed pathophysiological mechanism of hydranencephaly development due to impaired perfusion in the area supplied by the internal carotid arteries. A small island of sustained brain parenchyma below the frontal bone was primarily perfused by a branch of the right middle meningeal artery and was in no way connected with the rest of the brain parenchyma. Reports of marked macrocephaly are very common in patients with hydranencephaly, but there is no satisfactory pathophysiological explanation for this impressive clinical presentation.The generally accepted classic hypothesis of CSF pathophisiology has three basic postulates: active secretion of CSF predominantly in the choroid plexuses, unidirectional flow of CSF from the ventricular system to the subarachnoidal area, and resorption of CSF in the dural venous sinuses via arachnoid granulations -15. MacrIt is very hard to explain the developmental mechanism of hydrocephalus and macrocephaly in our patient using the basic postulates of the classic hypothesis. Since she had no choroid plexuses in the supratentorial space, the cause of intracranial hypertension and macrocephaly remains unclear. This is especially the case if we take into account that is strongly believed, even taken as a fact , that CSHowever, all this can be explained with the new hypothesis of CSF physiology, since it is based primarily on the hydrostatic and osmotic forces at the level of cerebral and spinal capillary network, which has more than 5000 times larger surface than the choroid plexuses ,26. CNS This new hypothesis ,26,28 enIn conclusion, our patient is one of the oldest living patients with hydranencephaly and to our knowledge in such patients no volumetric analysis of the CSF and residual parenchyma has ever been made separately for the supratentorial and infratentorial space. Furthermore, since our patient has no choroid plexuses in the supratentorial space and no obstruction between the dural sinuses and CSF, the development of hydrocephalus and macrocephaly cannot be explained by the classic hypothesis of CSF physiology. However, it can be explained by the new hypothesis of CSF physiology and development of hydrocephalus ,28-30. T


Polypeptide hormone Leptin suppresses inflammation in the heart muscle and protects heart from diseases. The purpose of this study is to evaluate the relationship between leptin and troponin serum levels with cardiac involvement in patients with major beta thalassemia.In this cross-sectional study, 70 children with major thalassemia were selected. Two ml blood was taken as sample from all children and after separating serum; the samples were maintained in -20°C temperature. Then, regarding cold chain conditions, the sample were sent to the Biochemistry Lab. Afterwards, leptin and troponin serum levels with the relevant kits and BMI were measured in all children, and information about age and gender was recorded. Collected data were analyzed with SPSS. The mean of leptin in girls and boys were 2.47 ± 3.13 and 0.96 ± 1.08 respectively which showed a significant difference . A significant correlation was also observed between BMI and leptin . Another significant association was found between leptin and age . However, a significant inverse correlation between serum ferritin and age  was discovered. No correlation was found between leptin, troponin, and ferritin. Since Leptin increases with the advent of cardiac involvement and independent from troponin T, it can be a predictive marker of cardiac involvement in patients with major beta thalassemia. Leukemia causes a significant decrease in the Major Beta - thalassemia is the most common hereditary hemoglobinopathies. About 100000 babies are born with this problem around the world annually . Leptin This study aimed to assess the relationship between levels of leptin and troponin serums in patients with major thalassemia.In this cross-sectional  study, 70 children aged between 2 and 18 years were selected among children who had referred to Ali Ebne Abitaleb Hospital randomly to get a pack red cell for transfusion and, diagnosed by hemoglobin electrophoresis. The participants were not treated with any heart medication and had no kidney disease, diabetes, fever, systemic diseases filling out the consent by parents, those children were enrolled for current study. While fasting 2ml blood was drawn from each sample. Samples were centrifuged with 3000 rpm at 5 ° C for 10 minutes. Separated serums were held in a -20° C until the time of measuring leptin and troponin. Considering the cold chain, serums were transferred to the Biochemistry Laboratory of Zahedan University of Medical Sciences (ZaUMS). Then, 250 microns from serums were used to measure troponin and same process for measuring leptin in which was evaluated using ELISA kit. Data were collected and analyzed with SPSS 20. Descriptive statistics are shown by frequency tables and for inferential statistics, independent t-tests, ANOVA, Pearson correlation, and Chi-square were used. P < 0.05 was considered as significant level.In the present study, 70 children were selected among those who had referred to Ali Ebne abitaleb hospital in Zahedan and fulfilled the study criteria. The mean age of participants was 13.75± 5.91, 12.63 ±1.12 for boys and 14.70 ± 0.88 for girls. This study aimed to assess the relationship between levels of leptin and troponin serums in patients with major Beta thalassemia. The analysis showed a significant difference in the mean of leptin in girls and boys had but not for ferritin and troponin. Significant correlation between troponin and ferretine was not observed whereas leptin depicted a positive relationship with age. In a study by Smith CC on the role of adipose- kinesis in pathophysiology of the cardiovascular heart disease, the protective role of leptin was proposed . Ku IA, Our results showed that leptin increased with age and since from the age of 10, the cardiac involvement begins to increase, we then can conclude that there is a remarkable and positive association between leptin and cardiac involvement. Based on Noori, it can be concluded that leptin’s change is independent from troponin, which is related to the cardiac involvement and the age of cardiac involvement in patients with beta-thalassemia major is 10–15 years. According to the results of the present study, leptin may be used in children with major thalassemia as a selected Cardiac marker involvement screening.


The Marmarou model has fundamentally influenced the development of mathematical pressure-volume models of cerebrospinal fluid (CSF) dynamics. It is a non-linear ordinary differential equation which is solvable in closed-form. The solution provides valuable analytical insights into the nature of the circulatory dynamics of CSF. It would be useful to generalize the Marmarou model to a broader class of polynomials to expand its scope to other CSF and Hydrocephalus phenomena.The Marmarou model relating the temporal evolution of ICP in pressure-volume studies to infusions incorporates observed fluctuations in the ICP through a nonlinear ordinary differential equation (ODE) of second order whose structure is based on physical analogies between CSF dynamics and an electrical circuit. This ODE is extended from its quadratic structure to a general polynomial of order ‘n’.An algorithm to solve the general polynomial ODE is developed. The solution to the original Marmarou model emerges from this algorithm as a special case. The solution for the general polynomial ODE is obtained from the algorithm which is shown to be easily implementable.The general polynomial Marmarou model expands the domain of applicability of the original one, thereby providing insights into a broader class of CSF phenomena. From a clinical perspective, these insights have implications for risk management of the patient for a large class of CSF and Hydrocephalus phenomena.


Innate lymphoid cells (ILCs) contribute to host defence and tissue repair but can induce immunopathology. Recent work has revealed tissue-specific roles for ILCs; however, the question of how a small population has large effects on immune homeostasis remains unclear. We identify two mechanisms that ILC3s utilise to exert their effects within intestinal tissue. ILC-driven colitis depends on production of granulocyte macrophage-colony stimulating factor (GM-CSF), which recruits and maintains intestinal inflammatory monocytes. ILCs present in the intestine also enter and exit cryptopatches in a highly dynamic process. During colitis, ILC3s mobilize from cryptopatches, a process that can be inhibited by blocking GM-CSF, and mobilization precedes inflammatory foci elsewhere in the tissue. Together these data identify the IL-23R/GM-CSF axis within ILC3 as a key control point in the accumulation of innate effector cells in the intestine and in the spatio-temporal dynamics of ILCs in the intestinal inflammatory response.DOI:http://dx.doi.org/10.7554/eLife.10066.001 Crohn’s disease and ulcerative colitis are diseases in which the body’s own immune system causes inflammation of the large intestine. These autoimmune diseases can be severely debilitating and difficult to treat. However an improved understanding of the factors that contribute to the intestinal inflammation may lead to new and effective treatments.Immune cells called innate lymphoid cells were discovered recently, and shown quickly to play a role in host defense, tissue repair and inflammation regulation. Several groups of innate lymphoid cells are now known; each group is characterized by the genes that control the cell’s development and the small proteins  that the cells release. One group of innate lymphoid cells, the ILC3s, are generally found in the intestinal tract, albeit in small numbers. Given that innate lymphoid cells are known to manage inflammatory responses, it is possible that ILC3s contribute to intestinal inflammation. However, it remains unclear how such a small population of cells could so dramatically inflame the gut.Pearson et al. now reveal two mechanisms that these innate lymphoid cells use to amplify the inflammatory response and exacerbate intestinal inflammation. First, in both mice and humans, ILC3s were found to be a key source of a cytokine called GM-CSF, which recruits additional immune cells that further promote intestinal inflammation. Secondly, while ILC3s were traditionally regarded as immobile immune cells, Pearson et al. discovered that these cells can move within the intestinal tissue and mobilize from their starting points within this tissue if they are activated. These two mechanisms could explain how ILC3s can trigger inflammation that occurs throughout the gut.The experiments suggest that blocking production of the GM-CSF cytokine or altering ILC3 movement or activity may help reduce intestinal inflammation. However, the use of GM-CSF blocking drugs to protect against colitis and similar conditions could be problematic, because GM-CSF also plays an important protective role in the intestines. Nevertheless, clinical trials are underway to investigate the use of anti-GM-CSF drugs to treat other inflammatory conditions (such as rheumatoid arthritis). These studies could offer insight into whether these drugs provide relief to trial participants who suffer from intestinal inflammation as well.DOI:http://dx.doi.org/10.7554/eLife.10066.002 Innate lymphoid cells (ILCs) are a recently defined family of evolutionarily ancient cells involved in many facets of host defence. As with conventional Th1, Th2 and Th17 T cells, ILCs can be functionally classified based on the expression of transcription factors and associated signature cytokines . ILC1 arThe multi-functional roles of ILCs in disease development and pathogenesis , as well+ RORγt+ CD4- NKp46- ILCs in the development of innate intestinal inflammation in Rag-/- mice following Helicobacter hepaticus infection or αCD40 stimulation  cells expressed GM-CSF . The majof T-bet .10.7554/We next investigated the cytokine milieu that regulates GM-CSF production by ILCs in vivo. To determine whether IL-23 signalling induces GM-CSF, we cultured total colonic lamina propria cells overnight with IL-12 or IL-23 and measured GM-CSF production in the cultures. IL-12 had no effect on either the proportion of GM-CSF-expressing ILCs or the amount of GM-CSF per cell . By contIl23a mRNA expression within colonic tissue revealed a peak in expression at day 1 following anti-CD40 treatment that rapidly declined but was followed by increased expression of Csf2, the gene for GM-CSF, at day 3  structures  present within a cryptopatch  patients were recruited through the Oxford IBD cohort study, and samples were obtained from Oxford GI Biobank in collaboration with Oxford Radcliffe Biobank. Colon specimens were collected from patients undergoing surgery for severe disease. Macroscopically healthy sections from CRC patients were used as controls. For gene expression data, intestinal mucosal pinch biopsies were taken from patients undergoing routine endoscopy. From 20 patients with evidence of active intestinal inflammation, matched biopsies were collected from both inflamed tissue and regions of the intestine with no apparent inflammation. IBD blood samples were collected from patients attending the outpatient clinic and in some cases from patients undergoing surgery. Control blood samples were obtained from healthy donors. Ethical approval was obtained from the Oxfordshire Research Ethics Committee (Reference numbers: 11/YH/0020 and 09/H0606/5) and informed written consent was given by all study patients.A previously published study with a publically available dataset  was used to confirm mRNA observations of the Oxford cohort, using colonic mucosal biopses obtained at endoscopy from healthy controls or patients refractory to corticosteroids and/or immunosuppression. Gene expression data were generated using the Affymetrix Human Genome U133 Plus 2.0 array as described in .Rag1-/-, 129SvEvS6.Rag2-/- and C57BL/6 Rag1-/-Il23rgfp/+  strains used in this study were bred and maintained at the University of Oxford. C57BL/6 Rag1-/-Il15ra-/- and C57BL/6 Rag1-/- were bred and maintained at the National Institute for Medical Research. Experiments were conducted in accordance with local animal care committees (UK Scientific Procedures Act of 1986). Mice were routinely screened for the absence of pathogens, and were kept in individually ventilated cages with environmental enrichment.All mice were bred and maintained under specific pathogen-free conditions in accredited animal facilities. C57BL/6 g, 20 min). Lymphocytes were isolated from the 40%/30% interface.For cLP isolation, colon tissue was cut into 1cm pieces, and epithelial cells were removed by incubation (2x) in RPMI containing 5% heat-inactivated FCS and 5mM EDTA. Remaining tissue was incubated in RPMI containing 5% FCS, 15mM HEPES and 300U/ml of Collagenase VIII  to digest the remaining tissue. Cell populations were purified by 75%:40%:30% Percoll  gradient centrifugation . Following centrifugation, PBMCs were collected from the Ficoll-plasma interface and washed 3x in PBS prior to culture or flow cytometry.g, 30 min). Lymphocytes were isolated from the 60%/40% interface.Specimens were incubated in 1 mM DTT solution, followed by three washes in 0.75 mM EDTA solution. Tissues were then digested overnight in 0.1 mg/ml collagenase A solution . Cell populations were purified by 100%:60%:40%:30% Percoll  gradient centrifugation  and stored at -20oC until processed. Human and murine cLP cells were lysed in RLT buffer  containing ß-mercaptoethanol. RNA was isolated using the RNEasy kit (Qiagen) including a DNAse I digestion. Content and purity of RNA was measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific). cDNA was synthesised using the Superscript III reverse transcription kit  or the High Capacity cDNA kit . Quantitative real-time PCR for the candidate genes was performed using the Taqman system. cDNA samples were analysed in triplicate  using the CFX96 detection system  or the Viia7 system . Values were normalised to Hprt expression, and analysed using the d-Ct method. Taqman Gene Expression Assays (Life Technologies) for mouse Ccl19, Ccl20 Ccr7, Ccl2, Il23r and Csf2, and human RPLPO and CSF2 were used.For human biopsy samples, tissue was immediately placed in RNARag-/- background. Unless otherwise indicated, mice were sacrificed 7 days post αCD40 injection, and weight loss was monitored throughout the course of the experiment.To induce acute innate colitis, 25-–50 mg of αCD40 IgG2a mAb  was administered via i.p. injection to mice on a C57BL/6 Helicobacter hepaticus, Hh NCI-Frederick isolate 1A (strain 51449) was grown as previously described (Rag2-/- background were fed 3x on consecutive days with Hh 1A (1.0x108 CFU) by oral gavage. Animals were analysed at week 6 following infection.To induce colitis using escribed . Mice onHelicobacter hepaticus infection was administered i.p. 2x per week starting at day 0.To block specific cytokine or cellular activity in vivo, aCD40-treated mice received 0.375 mg IL-17A blocking mAb , 0.15 mg IL-22 blocking mAb , 0.8 mg IL-23R blocking mAb , 0.125 mg GM-CSF blocking mAb , 100 mg IL-17F blocking antibody  or isotype controls . Treatment started on day -1 and antibodies were injected i.p. at day -1 and day 3, except blocking mAb to IL-22 which was administered at days -1, 1, 3 and 5. Blocking mAb to GM-CSF during The following antibodies were purchased from eBioscience: fixable viability dye, GM-CSF-PE (MP1-22e9), CD127-FITC/PE/PECy5 (A7R34), Thy1.2-PECy7 (53.2.1), T-bet-eFluor660 (eBio4B10), RORγt-PE (AFKJS-9), B220-PerCP-Cy5.5 (RA3-6B2), CD11b-PerCP-Cy5.5 (M1/70), CD11c-PerCP-Cy5.5/APC (N418), Gr-1-PerCP-Cy5.5 (RB6-8C5), CD64-PE (X54-5/7.1), F4/80-PerCP-Cy5.5 (BM8), MHCII-AlexaFluor700 (M5/114.15.2), CCR7-PE (4B12), perforin-APC (eBioOMAK-D) and cKit-Alexa-Fluor700 (ACK2). The following antibodies were purchased from Biolegend : NKp46-FITC (29A1.4), CD49b-PerCP-Cy5.5 (DX5), Ly6C-PECy7 (HK1.4), Ly6G-BV570 (1A8), CCR6-BV421 (29-2L17), CD4-BV605 (RM4-5), CCR2-AF647 (SA203G11) and NK1.1-BV650 (PK136). The following antibodies were purchased from BD Horizon : NKp46-V450 (29A1.4), CD11b-V500 (M1/70), CD45-V500/BV650 (30-F11), SiglecF-BV421 (E5-2440) and NKG2D-PE CF594 (CX5). Lineage staining was performed using PerCP-Cy5.5-conjugated antibodies against B220, CD11b, CD11c, Gr-1, F4/80 and CD49b.The following antibodies were purchased from eBioscience: CD1a-FITC (HIH9), CD3-BV650 (OKT3), CD8-FITC (RPA-T8), CD19-FITC (HIB19), IL-17A-PE (eBio64DEC17), IL-22-PECy7 (22URT1). The following antibodies were purchased from Biolegend: CD3-FITC (UCHT1), CD4-FITC (OKT4), CD11c-FITC (B-LY63.9), CD14-FITC (MSE2), CD16-FITC (3G8), CD34-FITC (581), CD123-FITC (6H6), TCRαβ-FITC (IP26), TCRγδ-FITC (B1), CD45-AlexaFluor700 (HI30), CD56-PerCP-Cy5.5/BV510 (HCD56), CD127-BV421 (A019D5), GM-CSF-PerCP-Cy5.5 (BVD2-21C11), IL-5-APC (TRFK5) and IFNγ-BV605 (4S.B3). Lineage staining was performed using FITC-conjugated antibodies against CD1a, CD8, CD19, CD3, CD4, CD14, CD16, CD34, CD123, TCRαβ and TCRγδ.Single cell suspensions were prepared from mouse spleen or colonic LP or human LPMC or PBMC cells as described above. For intracellular cytokine staining mouse or human were directly cultivated for 3 or 4 hr in the presence of phorbol 12-myristate 13-acetate  (100ng/ml), ionomycin (Sigma-Aldrich) (1μg/ml), monensin (BD Biosciences) and brefeldin A (eBioscience). Where indicated, mouse and human cLP cells were stimulated overnight with 10 ng/ml of recombinant IL-12  or IL-23 , human - Peprotech), and then for the last 4 hr PMA, ionomycin and brefeldin A were added.Cells were incubated with CD16/32 (eBioscience) to block Fcγ receptors, prior to incubation with fixable viability dye , and mAb conjugates in 100 μl PBS containing 2% BSA and 1 mM EDTA (PBS-E) for 30 min. Cells were subsequently washed twice in PBS-E. For cytokine staining, cells were stained with antibodies for 1 hr using the Cytofix/Cytoperm kit (BD Biosciences). For transcription factor staining, cells were stained with transcription factor antibodies for 1 hr using the Foxp3 staining kit (eBioscience). Cells were washed twice in PBS-E. All labelled cells were then acquired on a LSRII SORP (BD Biosciences) and analysed using Flowjo software .To assess the severity of colitis, samples of the proximal colon were taken and immediately fixed in buffered 10% formalin . 4–5 μm paraffin-embedded sections were cut and stained with haematoxylin and eosin (H&E). Inflammation was assessed using previously published criteria . Each saet al. . Imaging proceeded immediately as described above.Intravital microscopy was analyzed using Imaris 8  after linear unmixing using Zen (Carl Zeiss). Images were drift corrected based on mucus or collagen signal. ILCs were tracked using autoregressive motion algorithm of the spot-tracking module. Motile ILCs were gated based on distance less than 75 μm from a cryptopatch, track duration greater than 5 min, and displacement greater than 14 μm (approximately two cell lengths). Images were smoothed using a Gaussian filter for display.oC. 6 μm frozen sections were cut and collected on frosted glass slides. For staining, slides were fixed in 2% formalin. For RORγt staining, slides were subsequently also treated with Fix-Perm buffer (eBioscience). Endogenous peroxidase activity was blocked with 1% H2O2 (Sigma-Aldrich) and 2% sodium azide and non-specific binding was blocked with 10% donkey serum. Sections were incubated with the following antibodies CD4  IL-7Rα  in 10% donkey serum or RORγt  in Perm buffer (eBioscience). Sections were then incubated with donkey anti-rat HRP secondary antibody . Tyramide signal amplification was the performed . Then sections were stained with CD11c  in 10% goat serum followed by goat anti-hamster Cy3, or with MHCII-FITC or AF647 .For immunofluorescence, samples of the proximal colon were taken and immediately embedded in OCT compound  and frozen in a bath of isopentane on dry ice prior to storage at -80Sections were mounted with Vectashield containing DAPI. Images were collected using a 710 microscope (Carl Zeiss), and analysed using ImageJ open access software.Rag1-/-Il23rgfp/+ mice were freshly isolated and maintained in room temperature PBS before imaging. Opened sections of colon were immobilized using Vetbond tissue adhesive  in dishes containing PBS. CPs were imaged from the muscularis side of the colon using a 20x water dipping objective and spectral detector on a Zeiss 780 upright multiphoton microscope. Images were analysed using the linear unmixing module of Zen (Carl Zeiss) and Imaris (Bitplane).The colons from C57BL/6 Statistical analysis and graphical representations were performed using Prism 5.0a software . For comparison of qPCR results and histopathological analysis, the nonparametric Mann-Whitney U test was used, and for multiple samples, one-way ANOVA with Bonferroni’s post-test was used unless otherwise stated. Technical replicates are from the same sample, analysed on the same day. Biological replicates are from independent experiments.Sample size was computed using an estimate of colitis score between 6–8, to detect a score reduction by 50% in a treated group with a standard deviation of 30%, false positive of 0.05, and power of 0.80. Sample size was therefore calculated to be between 6–7. Where smaller changes were expected, sample sizes were increased accordingly. review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for submitting your work entitled "Pathogenic ILC3 GM-CSF production and mobilisation orchestrate acute intestinal inflammation" for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.This manuscript further elaborates the role ILCs play in the pathology of colitis induced by agonistic anti-CD40 antibody by showing that group 3 ILC-driven colitis in this model depends on GM-CSF. It also shows that ILCs traffic through gut cryptopatches under homeostatic conditions and that their egress is enhanced by anti-CD40 antibody, associated with increased tissue chemokine levels and development of inflammatory foci. However, before publication, the following issues would be important to address.1) While the manuscript purports to exclude roles for IL-17A and IL-22, combined neutralisation of these two cytokines has not been done, nor has a role for IL-17F been tested. Also, the data indicate that GM-CSF neutralisation may not entirely abrogate disease, and triple-neutralisation studies would be useful to examine if there are some distinct non-overlapping quantitative roles for these various cytokines.2) The determination of GM-CSF-producing cells is not precise. There is a significant GMCSF production by cells that weakly express lineage markers. CD127+ lineage- cells appear to contain some lineage+ cells. Have the lineage-dim cells been gated out when analyzing the ILC subsets producing GM-CSF? There is no negative control shown for IL7R staining , making 3) For mechanistic insights into the anti-CD40-mediated modulation of the ability of ILC3s to produce GM-CSF, it is useful to test if the levels of Rorc or IL-23R have been modulated by anti-CD40 treatment.Helicobacter hepaticus-induced colitis (a model in use by the authors) would be very helpful.4) Since a role for GM-CSF in inflammatory situations is not by itself novel , it is appropriate to expect some data attempting to examine possible mechanisms through which only GM-CSF appears to be non-redundantly required in a disease depending on cells that make all three potentially pro-inflammatory cytokines, IL-17, IL-22 and GM-CSF. Notably, the authors show data that GM-CSF appears to be required for the induction of a putative upstream mediator (of IL-17 and IL-22 as well as GM-CSF), IL-23, making a number of complex regulatory scenarios plausible and worth examination. Also, there is prior evidence that bacterial gut inflammation may require IL-22, at least, and comparative data on the relative roles of these inflammatory cytokines between non-microbial induction of colitis as used in the manuscript and, say, + ILC3 produce GM-CSF , and the present data do not identify the subsets of total ILCs that produce GM-CSF. While more ILCs from the peripheral blood of IBD patients appear to express GM-CSF, the authors suggest in the text that this is not so for IFNγ, while the data in 5) 6) With respect to the data on ILC migration , it woul7) The manuscript suggests that migration of innate-like lymphoid cells is essential for colitogenesis, and that one role of GM-CSF is in controlling this migration by inducing the expression of CCR7, the receptor for the chemokine CCL19. Yet, the manuscript shows no studies examining, for example, the balance of cryptopatch-egress in animals with GM-CSF blockade or with CCL19 or CCR7 blockade, or indeed examining possible modulations in the state of the villous-tip inflammatory foci by CCL19 blockade, which would help provide evidence for these putative cause-and-effect relationships between innate-like lymphocyte migration and inflammogenesis. This manuscript further elaborates the role ILCs play in the pathology of colitis induced by agonistic anti-CD40 antibody by showing that group 3 ILC-driven colitis in this model depends on GM-CSF. It also shows that ILCs traffic through gut cryptopatches under homeostatic conditions and that their egress is enhanced by anti-CD40 antibody, associated with increased tissue chemokine levels and development of inflammatory foci. However, before publication, the following issues would be important to address. 1) While the manuscript purports to exclude roles for IL-17A and IL-22, combined neutralisation of these two cytokines has not been done, nor has a role for IL-17F been tested. Also, the data indicate that GM-CSF neutralisation may not entirely abrogate disease, and triple-neutralisation studies would be useful to examine if there are some distinct non-overlapping quantitative roles for these various cytokines.We have now included IL-17A/IL-22 double blockade as well as IL-17F blockade . IL-17F 2) The determination of GM-CSF-producing cells is not precise. There is a significant GMCSF production by cells that weakly express lineage markers. CD127+ lineage- cells appear to contain some lineage+ cells. Have the lineage-dim cells been gated out when analyzing the ILC subsets producing GM-CSF? There is no negative control shown for IL7R staining , making -/- l15ra-/-Rag1we now show that lack of NK cells, while it does have an effect on weight loss, does not significantly affect colitis  For mechanistic insights into the anti-CD40-mediated modulation of the ability of ILC3s to produce GM-CSF, it is useful to test if the levels of Rorc or IL-23R have been modulated by anti-CD40 treatment.Il23r expression 6 hours after anti-CD40 treatment  Since a role for GM-CSF in inflammatory situations is not by itself novel , it is appropriate to expect some data attempting to examine possible mechanisms through which only GM-CSF appears to be non-redundantly required in a disease depending on cells that make all three potentially pro-inflammatory cytokines, IL-17, IL-22 and GM-CSF. Notably, the authors show data that GM-CSF appears to be required for the induction of a putative upstream mediator (of IL-17 and IL-22 as well as GM-CSF), IL-23, making a number of complex regulatory scenarios plausible and worth examination. Also, there is prior evidence that bacterial gut inflammation may require IL-22, at least, and comparative data on the relative roles of these inflammatory cytokines between non-microbial induction of colitis as used in the manuscript and, say, Helicobacter hepaticus-induced colitis (a model in use by the authors) would be very helpful.-/-Ragmice and in accumulation of inflammatory monocytes in the intestine. There was no analysis of the functional role of GM-CSF in colitis or movement of ILCs in that study, so our data provide important new mechanistic insight into the functions of GM-CSF in the colon. However both studies together strongly support a role for GM-CSF in monocyte/macrophage accumulation in the intestine.Our new data supports dual mechanisms for GM-CSF in initiation and perpetuation of inflammation. We now show that GM-CSF is required for the mobilization of ILCs from cryptopatches, implicating the cytokine in the pro-inflammatory initiation step of the disease. These data, coupled with a decrease in inflammatory monocytes with anti-GM-CSF blockade, newly implicate ILCs in the development of inflammatory foci within the tissue. While our manuscript was in revision, a new study appeared demonstrating a role for GM-CSF in the systemic anti-CD40 induced disease in H. hepaticus driven innate colitis associated cancer. This may reflect the differential involvement of epithelial derived signals in bacteria versus anti-CD40 induced colitis with IL-17 and IL-22, which act on the epithelium, having a more dominant role in the former. Importantly by contrast with the redundant role of IL-17 and IL-22 in anti-CD40 colitis, GM-CSF is required in both models and we have now included data showing anti-GM-CSF blockade ameliorates colitis in bacteria-driven innate colitis . However the presence of particular microbes in the RAG colony may alter IL-23 dependent down-stream effector mechanisms in anti-CD40 colitis. Indeed our own data supports this showing a non-redundant role for IL-23 driven IL-22 in  colitis . A more + ILC3 produce GM-CSF , and the present data do not identify the subsets of total ILCs that produce GM-CSF. While more ILCs from the peripheral blood of IBD patients appear to express GM-CSF, the authors suggest in the text that this is not so for IFN5) γ, while the data in γ-producing ILCs as well. Further, there are no data regarding GM-CSF expression in healthy versus IBD-inflamed colonic tissue. Thus, these data are correlative and say little about a potential role of GM-CSF in human IBD. These ambiguities need to be addressed explicitly.+ cells is higher in the colon than the blood  With respect to the data on ILC migration , it woul+ population. The question of ILC chemotaxis is therefore still an open topic, and further studies, beyond the scope of this report, will be required to elucidate what is likely a very complicated and possibly tissue-specific process.We have clarified that aggregates refer to accumulations in the villous tips. Data now included in 7) The manuscript suggests that migration of innate-like lymphoid cells is essential for colitogenesis, and that one role of GM-CSF is in controlling this migration by inducing the expression of CCR7, the receptor for the chemokine CCL19. Yet, the manuscript shows no studies examining, for example, the balance of cryptopatch-egress in animals with GM-CSF blockade or with CCL19 or CCR7 blockade, or indeed examining possible modulations in the state of the villous-tip inflammatory foci by CCL19 blockade, which would help provide evidence for these putative cause-and-effect relationships between innate-like lymphocyte migration and inflammogenesis.We thank the reviewers for this suggestion. We now show that blocking GM-CSF does return the cryptopatch balance of entrance and exit to steady-state levels . This so


Galactans ranging in length from one to five residues were prepared in a single step by treatment of the glycosyl donor 2,3,4-tri-O-benzoyl-β-D-galactopyranosyl fluoride with lanthanum perchlorate in the presence of an initiator alcohol. The product oligosaccharides were readily chromatographically separable. This oligomerization was used to synthesize a pentagalactan in a single step from monosaccharide building blocks in reasonable overall yields. Additionally, numerous other steps involving protecting group removal and installation are required to assure regio- and stereoselective glycan assembly. One-pot strategies for oligosaccharide synthesis can streamline the process by sequential iterative glycosylations, without the need for work-up and purification after the formation of each glycosidic bond, and have enabled the rapid preparation of complex glycans . We. We1 andentry 2, , even thentry 3, . Moreove2, which lacks a silyl protecting group on the primary hydroxy group, proved to be remarkably clean, providing products ranging from the monosaccharide to the tetrasaccharide in isolable yield (3 [153 and La(ClO4)3 might reduce self-oligomerization. However, addition of 2 via a syringe pump did not significantly improve the yields, nor did it shift the product distribution to longer oligomers.In an attempt to tune silyl ether deprotection rates and promote formation of longer oligomers, we examined the glycosylation of a bulkier TBDPS-protected donor. While this donor underwent oligomerization, the products were a mixture of the TBDPS capped and desilylated oligosaccharides and purification and isolation of pure products was challenging, even after resilylation of the oligosaccharide mixture. However, to our surprise, we observed that the reaction using donor le yield , entry 4yield (3 –1521. We4)3 as the promoter appears to be critical in this reaction. The use of the more Lewis acidic [3 resulted in a reduction in both overall yield and in the degree of oligomerization 3rization , entry 6additive ,23, but promoter  was comp5) (3. Glycosylation with 4-pentenol (6)  , entry 1enol (6) , entry 2>100 mg, , entry 5p-methoxyphenyl 2,4,6-tri-O-benzyl-β-galactopyranoside (11) as the acceptor provided only the product derived from a single glycosylation in modest yield. When we used disaccharide 12 as the acceptor (12 was recovered (45%) and only a small portion of the donor 2 (27%) reacted with the remaining 55% of the acceptor.Secondary alcohols such as cyclohexanol or the sterically more hindered menthol successfully underwent glycosylation to provide mono- and disaccharides, but longer oligomers were not observed. Moreover, very little of the donor is incorporated into the products, suggesting that self-oligomerization competes effectively with the desired initiation. The use of The results described here illustrate that oligogalactans as long as pentamers can be generated in a single reaction by the activation of a bifunctional galactosyl donor/acceptor in the presence of an initiating alcohol. While isolated yields of the tri-, tetra-, and pentasaccharides are modest, the obtained yields of 19%, 10%, and 4% , entry 5File 1Experimental details for Tables 1 and 2, experimental protocols, and characterization data.File 2NMR spectra for all new compounds.


Beyond acute infections, group B coxsackieviruses (CVB) are also reported to play a role in the development of chronic diseases, like type 1 diabetes. The viral pathogenesis mainly relies on the interplay between the viruses and innate immune response in genetically-susceptible individuals. We investigated the interaction between CVB4 and macrophages considered as major players in immune response. Monocyte-derived macrophages (MDM) generated with either M-CSF or GM-CSF were inoculated with CVB4, and infection, inflammation, viral replication and persistence were assessed. M-CSF-induced MDM, but not GM-CSF-induced MDM, can be infected by CVB4. In addition, enhancing serum was not needed to infect MDM in contrast with parental monocytes. The expression of viral receptor (CAR) mRNA was similar in both M-CSF and GM-CSF MDM. CVB4 induced high levels of pro-inflammatory cytokines (IL-6 and TNFα) in both MDM populations. CVB4 effectively replicated and persisted in M-CSF MDM, but IFNα was produced in the early phase of infection only. Our results demonstrate that CVB4 can replicate and persist in MDM. Further investigations are required to determine whether the interaction between the virus and MDM plays a role in the pathogenesis of CVB-induced chronic diseases. Enteroviruses (EV) are small 20–30 nm) non-enveloped positive single-strand RNA viruses that belong to the Picornaviridae family. The genus Enterovirus is very important in medicine and includes several major human pathogens. Group B coxsackieviruses CVB 1–6) are classified within the Human Enterovirus B (HEV-B) species [–6 are cl0–30 nm nIn T1D patients, EV components have been detected more frequently in peripheral blood mononuclear cells (PBMC) ,7,8,9, aIn vitro, monocytes and monocytic cell lines are poorly permissive to CVB. However, these cells can be effectively infected when the virus is previously incubated with non-neutralizing serum or IgGs [ or IgGs ,12. The  or IgGs ,12,13,14 or IgGs .Monocytes are produced from progenitors in the blood marrow and usually circulate via the bloodstream to peripheral tissues. In the steady state or in response to inflammation, monocytes migrate into tissues, attracted by various cytokines or necrotic cells, and mature to replenish resident macrophages and dendritic cells (DC) . MacrophHuman monocyte-derived DCs can be infected by many viruses ,19,20,21Macrophages are primarily remarkable phagocytic cells, but they also have a great plasticity that allows them to respond to environmental cues and change their phenotype, depending on the activation state . MacrophIn addition to CD8 cytotoxic T lymphocytes, macrophages have been reported to be common in insulitis found in the pancreas of T1D, both at early and later stages of disease progression . TherefoIn vitro, several viruses can infect monocyte-derived macrophages (MDM) [es (MDM) ,27,28 wiIn this study, we describe the infection of human MDM by CVB4 and further investigate the inflammation induced by the virus, as well as its persistence in these cells.Monocytes were cultured for seven days in serum-free medium (SFM) containing either M-CSF or GM-CSF. Under these conditions, on Day 7, the cells appeared roughly similar under the microscope, but they were slightly larger in cultures treated with M-CSF a. The stMonocytes and monocyte-derived macrophages were inoculated with CVB4 or CVB4 mixed with non-neutralizing dilutions of human serum. In all of the experiments of this study, the multiplicity of infection (MOI) was one. The cultures were incubated; then, 24 and 72 h post-infection (pi), supernatants were collected for IFNα quantification, and the cells were washed five times with PBS and used for viral RNA quantification. As shown in As far as the quantification of intracellular enteroviral RNA was concerned, the levels were very low in monocytes inoculated with CVB4, 85 ± 47 and 146 ± 68 copies/ng of total RNA at 24 and 72 h pi, respectively, and increased significantly when monocytes were inoculated with CVB4 mixed with 100-fold diluted human serum, reaching 2070 ± 626 and 1215 ± 645 copies/ng of total RNA at 24 and 72 h, respectively see b. In M-CAltogether, these results show that CVB4 can infect MDM differentiated in culture medium containing M-CSF and that there is no human serum-dependent enhancement of CVB4 infection of these cells. In contrast, MDM differentiated in culture medium containing GM-CSF were not permissive to CVB4.We hypothesized that GM-CSF-induced MDM were not permissive to CVB4, compared to M-CSF-induced MDM, because of a reduced expression of CAR, the receptor for CVB4. CAR mRNA was then quantified in both cell populations collected after differentiation on Day 7. As shown in We previously observed that PBMCs were not infected when they were inoculated with CVB4; nevertheless, they can produce high levels of pro-inflammatory cytokines . TherefoIn so far as M-CSF-induced MDM were permissive to CVB4 infection, we investigated further the replication and the persistence of the virus in these cells. MDM were inoculated with CVB4, and after 2 h, the cells were extensively washed five times with PBS and then cultured in serum-free medium for 12 days. Supernatants and cells were collected every three days. The viability of cells was assessed during the follow-up a.High levels of IFNα were detected in supernatants on Day 3 pi. Afterwards, the levels of IFNα decreased dramatically and were almost undetectable on Day 6 pi b.50 determination on HEp-2 cells. The viral titers reached a maximum of 8.6 ± 2 log TCID50/100 µL on Day 3 and decreased progressively, but were still around 2.5 log TCD50/100 µL on Day 12  and was 3.6 ± 0.2 log copies/µL on Day 12 d.Intracellular viral RNA was also detected significantly during the follow-up. The highest levels were around 4 log copies/ng of total RNA and were reached between three and six days pi e.In this study, we described, for the first time, the infection of human primary macrophages by group B coxsackieviruses. We demonstrated that CVB4 can effectively infect M-CSF-induced MDM, but not GM-CSF-induced MDM. In addition to the production of viral particles, a persistence of viral RNA has been observed in M-CSF-induced MDM cultures.in vitro, as well as in vivo [Previous investigations by our team focused on peripheral blood mononuclear cells (PBMCs) and revealed that among these cells, only monocytes could be infected by CVB  in vivo . Monocyt in vivo ,13. The  in vivo ,13. Enha in vivo ,29.However, monocytes are not long-life cells and usually leave the bloodstream after 2–3 days to reach tissues and differentiate into mature cells, such as macrophages . Therefoin vitro studies on macrophages, several protocols have been described by researchers to differentiate macrophages from blood monocytes, and usually include the use of: (i) media containing human autologous or heterologous AB serum or fetal bovine serum; or (ii) media containing growth factors, namely M-CSF or GM-CSF [For r GM-CSF ,31.In this report, MDM were generated by treating monocytes with a serum-free medium containing either M-CSF or GM-CSF. The phenotypes of cells obtained in both conditions were similar as shown by immunological markers; but surprisingly, only cells treated with M-CSF could be infected by CVB4. It is important to note that the cells were treated for seven days, and then, after washings, they were maintained in culture medium without growth factors. Thus, the opposite patterns of data regarding the infection of MDM in our experiments can be due to some differences between M-CSF-treated cells and GM-CSF-treated cells.in vivo, while those induced with M-CSF tend to replicate alternatively-activated anti-inflammatory (M2) macrophages [in vitro approach considers MDM generated with M-CSF and GM-CSF as M0 macrophages, and then, M1 originate from M0 induced with GM-CSF or M-CSF in the presence of IFNγ and/or LPS, while M2 macrophages are triggered from M-CSF-induced cells by the presence of cytokines, such as IL-4 or IL-10 [It has been reported that GM-CSF-induced MDM share some transcriptional profiles of classically-activated pro-inflammatory (M1) cells rophages ,31,32, brophages ,34. A moor IL-10 ,36. In sThe discrepancy observed between M-CSF- and GM-CSF-induced MDM regarding CVB4 infection was not due to a reduced transcriptional expression of the specific viral receptor CAR in GM-CSF-treated cells, as shown by quantitative real-time RT-PCR. It remains to be determined whether the receptor is present at the same level at the surface of both M-CSF and GM-CSF cells. Furthermore, in non-polarized cells, it has been reported that a secondary receptor, such as decay-accelerating factor (DAF), is not mandatory for CVB infection, whereas other entry pathways and molecules seem to be critical for entry . WhetherIn this study, it has been observed that CVB4 induced the production of high levels of pro-inflammatory cytokines, such as IL-6 and TNα, in both M-CSF MDM and GM-CSF MDM cultures. Since GM-CSF MDM were not infected with CVB4, the production of cytokines was thought to be triggered mainly by surface sensors on these cells, which is in agreement with previous studies in our laboratory. Indeed, it has been shown that inactivated CVB4 can stimulate the production of IL-6, TNFα and IL-12 by monocytes , which sIn GM-CSF MDM inoculated with CVB4, a low level of viral RNA was detected by sensitive real-time RT-qPCR, but was not associated with the presence of IFNα in supernatants, suggesting that the infection was at a very low level in these cells or was abortive. This hypothesis is in agreement with the demonstration by another team that HIV replication was suppressed in GM-CSF-induced macrophages, but not in M-CSF-induced macrophages . A simili.e., no increase in FNα or intracellular viral RNA) was obtained in MDM when the virus was mixed with serum, although both monocytes and MDM were reported to express FcγRI and FcγRII [M-CSF MDM were infected with CVB4, whereas monocytes were infected when the virus was mixed with human non-neutralizing serum. In addition, no enhancement of CVB4 infection , while the cellular response, especially the production of IFNα, was limited to early steps (Day 3). This finding suggests that CVB4 probably overrides the cellular response to establish persistence in MDM. Further studies are needed to understand the mechanisms of interaction between CVB4 and macrophages.The micro-environment and the phenotype highly impact the physiology of macrophages. Therefore, the relationship between CVB4 and these cells in the pathogenesis of autoimmune diseases, such as T1D, can depend on a specific micro-environment. Indeed, for example, it has been suggested that GM-CSF plays a role in maintaining macrophage populations in a physiological steady state and in controlling the development of autoimmune diseases by regulating the immune response and the immunological tolerance .Whether the phenotype of macrophages recruited in pancreas plays a role in interactions with CVB and in the development of autoimmunity deserves further investigation.In conclusion, human primary M-CSF-induced MDM, but not GM-CSF-induced MDM, can be infected by CVB4. A productive infection can be obtained, and the virus can persist in these cells. An inflammatory reaction of the cells is observed during the acute phase of the infection. Macrophages can then be considered not only as target cells, but also as reservoirs for CVB4 in tissues, with implications in the pathogenesis of chronic diseases triggered by these viruses.CVB4 E2, the diabetogenic strain of coxsackievirus B4, was used in all experiments. It was kindly provided by Ji-Won Yoon  and was propagated in Hep-2 cells .Human blood collected from healthy donors was obtained at the Regional Blood Bank . This study was conducted in accordance with the rules of the Declaration of Helsinki of 1975, revised in 2008. Written consent was obtained from donors by the blood bank, and they were informed that the blood would be used for research purposes.TM PLUS , as described previously [7 cells per well (~1.5 million/cm2) was seeded in a Falcon® polystyrene six-well plate . Monocytes from the PBMCs were allowed to adhere to plastic for 2 h at 37 °C, 5% CO2, and non-adherent cells were subsequently removed by aspiration and extensive washing with PBS. Monocytes were cultivated in serum-free medium (SFM) containing either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF)  at 20 ng/mL. The medium was changed on Day 3. On Day 7, the cells were differentiated into macrophages. All media were purchased from Gibco® .Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient centrifugation using Ficoll-Hypaqueeviously . Cells wWhole blood was centrifugated at 2500 rpm for 15 min, and serum was aliquoted and stored at −20 °C. The level of anti-CVB4 antibodies was determined by the seroneutralization assay, as previously described [MDM were detached from the plates by incubation at 4 °C with PBS containing 5 mM EDTA followed by gentle scraping. Then, cells were plated on a slide by cytocentrifugation at 500 rpm for 5 min, and slides were air dried. Cells intended for May-Grunwald-Giemsa (MGG) staining were fixed with ethanol for 5 min and stained using the automated SP-10™ slide maker/stainer . For immunocytochemistry purposes, cells were fixed with acetone for 10 min. The FLEX monoclonal mouse anti-human CD68 (clone KP1) antibody was purchased from Dako , and anti-CD163 monoclonal antibody was supplied by DB Biotech . The staining was performed using the Ultra View Universal DAB Detection Kit, on the VENTANA® Benchmark XT fully-automated IHC/ISH staining instrument .Monoclonal antibodies directed against human surface markers CD14, CD80, CD83, CD86, CCR7  and HLA-DR  were used at the supplier’s recommended concentrations. The CD14 antibody was fluorescein isothiocyanate (FITC)-conjugated, whereas CD80, CD83, CD86, CCR7 and HLA-DR antibodies were phycoerythrin (PE)-conjugated. Monocytes and monocyte-derived cells were detached and washed once in PBS. Cell suspensions were incubated at 4 °C for 20 min with the appropriate antibodies. The cells were then fixed in 0.5% paraformaldehyde, washed once with PBS and analyzed by flow cytometry on a Navios Flow cytometer .IFNα was measured using the IFNα pan-specific ELISA kit  that allowed detection of subtypes 1/13, 2, 4, 5, 6, 7, 8, 10, 14, 16 and 17 of IFNα. TNFα, IL-6 and IL-10 were quantified with ELISA kits purchased from Peprotech®. Assays were performed according to the manufacturer’s instructions. The detection ranges were 7–700 pg/mL (IFNα), 23–1500 pg/mL (TNFα), 32–2000 pg/mL (IL-6) and 32–2000 pg/mL (IL-10).50).The viral titer in supernatants of infected cells was assessed using the end-point dilution assay, and the Spearman–Karber statistical method was used to determine the tissue culture 50% infectious dose . Extracted RNA was then dissolved in 50 μL of nuclease-free water, quantified with a Nanodrop® spectrophotometer (Thermo Fischer Scientific).A DNA enzymatic digestion was performed on RNA extracts. A volume of 10 μL of RNA was mixed in a tube with 2 μL of DNase RDD10× buffer , 0.2 μL of DNase I (Qiagen), 0.25 μL of RNase inhibitor (Invitrogen/Thermofischer Scientific) and 7.55 μL of nuclease-free water. The tube was incubated at 37 °C for 30 min and at 65 °C for 5 min. The tube was brought to 4 °C and then vortexed.The Affinityscript® QPCR cDNA Synthesis Kit  was used for the RNA retro-transcription step on a Perkin Elmer 2400 thermocycler. Quantitative RT-PCR for cDNA amplification was performed with the Brilliant II® QPCR Kit (Agilent) on the Mx3000p® (Stratagene/ Thermofischer Scientific). Oligonucleotides and RT-PCR conditions were previously described . EnterovCt−ΔΔ formula [CAR mRNA was quantified by real-time RT-qPCR using the SuperScript III Platinum One-Step Quantitative RT-PCR System (Life Technologies/Thermofischer Scientific). The oligonucleotides were already published . The qua formula .U test with the significance set at 0.05.Data were presented as the mean ± SEM. Graphs and analyses were performed with GraphPad Prism® V6.0 software. Comparisons were performed with the Mann–Whitney


Obstruction to cerebrospinal fluid (CSF) flow in idiopathic normal pressure hydrocephalus (iNPH) results in reduced CSF total tau (t-tau) and amyloid-β 42 (Aβ42) protein concentrations . RestoraWe included 5 patients  with temporary shunt responsive iNPH in the study. CSF t-tau and Aβ42 protein levels were measured at the time of lumbar drainage prior to initial shunt insertion, during shunt insertion, shunt tap to investigate shunt function and finally at shunt revision. The changes in these biomarkers were calculated.The duration between the first sample and the last sample at shunt revision was 527.8 ± 99.7 days (mean±SD). The mean t-tau levels were 181.7pg/ml, 821.0pg/ml, 392.5pg/ml and 492.25pg/ml at the time of lumbar drainage, during first shunt insertion, shunt tap and during shunt revision, respectively. The corresponding mean Aβ42 levels were 465.0pg/ml, 407.8pg/ml, 646.75pg/ml and 472.25pg/ml, respectively.Our results demonstrated features suggestive of mild degenerative pathology distinct from Alzheimer's pattern. It remains to be proven if CSF drainage alters the biomarkers levels.


However, whereas the accumulation of TAU-protein is characteristic for Alzheimer’s disease and other tauopathies, glaucoma patients do not demonstrate an increase in the target protein versus controls [Worldwide, 67 million patients are affected by the neurodegenerative eye disease glaucoma. Glaucomatous optic neuropathy (GON) is the second leading cause of permanent vision loss. GON is a chronic degenerative process the onset of which is not possible to monitor by currently existing diagnostic tools. Early treatment has been reported to be highly beneficial for well-timed treatment measures to slow-down the disease progression . As revicontrols . A monitversus healthy controls, DNA from vasospastic individuals showed highly group-specific comet-patterns with the degree of damage intermediate between healthy controls and glaucoma patients.Comparative “Comet Assay” analysis revealed patterns of comets typical for glaucoma patients and some pattern similarities with vasospastic individuals, in contrast to controls as shown in Figure p53 (stress regulated gene) and down-regulation of XPGC  have been demonstrated ex vivo in CL of glaucoma patients [These findings indicate “comet assay” profiling of DNA-damage in CL as a potentially powerful tool for the non-invasive early/predictive molecular diagnostics of glaucoma disease in vasospastic individuals. Furthermore, unrepaired DNA-damage in vasospastic individuals can lead to several pathologies different from glaucomatous optic nerve degeneration. This predisposition should be thoroughly investigated and the specificity of “Comet Assay”-patterns of vasospastic individuals should be validated comparing with patterns of other degenerative and non-degenerative pathologies. Thus, “Comet Assay”-analysis as a suitable tool for biomarkers has also been suggested for another neurodegenerative disorder – Alzheimer’s disease . “Comet A potential predisposition of vasospastic individuals to related pathologies should be thoroughly examined. This examination requires innovative strategies to cover following aspects:• identification of possible similarities as well as dissimilarities in molecular pathways between healthy vasospastic individuals and potential related pathologies developed later in life• specificity for predictive diagnostics of glaucoma pathology in predisposed vasospastic individuals should be strictly validated against several control groups including other neuro/degenerative diseases• selection of molecular targets should be performed for vasospastic individuals in favour of non-invasive (blood test) diagnostic approaches followed by personalised treatment towards individual predisposition to single pathologies.


The presence of donor-specific alloantibodies (DSAs) against the MICA antigen results in high risk for antibody-mediated rejection (AMR) of a transplanted kidney, especially in patients receiving a re-transplant. We describe the incidence of acute C4d+ AMR in a patient who had received a first kidney transplant with a zero HLA antigen mismatch. Retrospective analysis of post-transplant T and B cell crossmatches were negative, but a high level of MICA alloantibody was detected in sera collected both before and after transplant. The DSA against the first allograft mismatched MICA*018 was in the recipient. Flow cytometry and cytotoxicity tests with five samples of freshly isolated human umbilical vein endothelial cells demonstrated the alloantibody nature of patient’s MICA-DSA. Prior to the second transplant, a MICA virtual crossmatch and T and B cell crossmatches were used to identify a suitable donor. The patient received a second kidney transplant, and allograft was functioning well at one-year follow-up. Our study indicates that MICA virtual crossmatch is important in selection of a kidney donor if the recipient has been sensitized with MICA antigens. In order to avoid the risk of AMR in kidney transplantation, a prospective complement-dependent cytotoxicity crossmatches (CDC) and/or flow crossmatches (FXM) are performed prior to renal transplant in many centers [4]. These experiments can detect alloantibodies against donor HLA-I and II antigens, but neither test detects alloantibodies against MHC class I related chain A (MICA), because the lymphocytes used in the test do not express MICA antigens on their cell surfaces . The polymorphic MICA molecules, which are expressed on the surface of vascular endothelial cells, might be antigenic targets for AMR [7]. Zou and his collaborators were the first to detect MICA alloantibodies in 14 patients with failed kidney allografts [8]. In a large blinded retrospective renal transplant observational study, pre-transplant MICA antibodies were associated with kidney allograft rejection and shorter graft survival [9]. As a well-documented case of acute rejection due to antibodies against MICA has not been reported, screening for MICA antibodies is not used in routine clinic testing prior to renal transplantation. In this report, we present a case of a patient who suffered early aggressive AMR in the presence of donor specific antibodies (DSA) against MICA after the first renal transplant. During donor selection for re-transplant, a donor with a negative MICA virtual crossmatch and negative lymphocyte CDC and FXM crossmatches was selected. The patient received the second kidney transplant, and her allograft was functioning well at one-year follow-up.Although selective immunosuppressive drugs and biologic agents generally alleviate acute rejection in the solid organ transplant patients, treatment of antibody-mediated rejection (AMR) remains challenging [rd Hospital of Xiangya Medical School (2014-S091). The kidney donor was a 34-year-old man killed in a car accident. Our organ procurement of organization (OPO) obtained the consent from the next kin. All other participants provided written informed consent to participate in this study.Human umbilical cord samples were obtained from the maternity ward at Xiangya Hospital in collaboration with the Department of Obstetrics according to the research plan and protocol approved by Xiangya Hospital Ethnics Committee (EC201403157). Collection of clinic samples for the research was approved by the Ethnics Committee of the 310].Donor typing for HLA-A, HLA-B, and HLA-DR was performed using a PCR-SSP kit (Invitrogen). HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 typing for the patient and two donors were confirmed using PCR and sequence-specific oligonucleotide probes  according to the manufacturer’s protocol. MICA typing was performed using Sanger sequence-based typing as described previously [th International Histocompatibility and Immunogenetics MICA workshop [11]. Antibody specificity was based on normalized mean fluorescence intensity (MFI) greater than 2000. DSAs were identified based on the reaction of patient sera to the mismatched antigens for a given donor. Some serum samples were also tested for antibody-C1q binding using a commercially available kit .IgG antibodies against HLA class I  and class II  were detected using a single antigen bead array on a Luminex platform (Gen Probe) according to the protocol suggested by the manufacturer. MICA antibody testing was performed on patient serum samples using single antigen beads conjugated with recombinant MICA*001, *002, *004, *007, *008, *009, *012, *016, *017, *018, *019, and *045. This kit was prepared in our laboratory and validated using the reference sera obtained from the 152 in air. Cultured cells were harvested and washed with PBS and used for flow cytometry assays. Cultured HUVECs were used within five passages. The purity of isolated endothelial cells was determined by staining with anti-CD31-PE (BD Biosciences) to facilitate endothelial cell gating. MICA expression on the surface of endothelial cells was detected using MICA-specific monoclonal antibody (mAb) 6B3 as described previously [9]. Briefly, 50,000 cells were incubated with mAb 6B3 at 1.0 μg/ml at room temperature for 30 min. Goat anti-mouse IgG conjugated with FITC (Jackson ImmunoResearch Laboratories) was added after three washes, and cells were analyzed using a Gallios flow cytometer (Beckman).Umbilical cord veins were cannulated, washed with phosphate buffered saline (PBS) solution, and treated with 0.2% collagenase (Sigma) at 23°C for 20 min. Endothelial cells were collected and cultured for 3 to 5 days in medium prepared using Endothelial Media BulletKits (Lonza Inc.) at 37°C in a humid atmosphere of 5% CO6 cells with or without pronase treatment were incubated at room temperature for 30 min with 25 μl of patient serum diluted 1:4. Random endothelial FXM was performed using freshly isolated human umbilical cord vein endothelial cells (HUVECs). HUVECs were incubated with patient serum which had been treated by pooled normal human platelet absorption. Mouse monoclonal antibodies against human IgG conjugated with FITC (BD Biosciences) was added after three washes. Samples were analyzed using a Beckman Gallios flow cytometer. Cutoff values were set with 50 mean channel shift (MCS) for T cells, 70 MCS for B cells, and 60 MCS for HUVECs. The FXM results in IgG MFI values were converted to MCS values using the following formula: MCS value = 1024 * (log(lgG_MFI) + 1)/4.A standard flow cytometry crossmatch assay (FXM) was used to analyze the patient’s sera and donor’s T and B lymphocytes prepared from peripheral blood by three-color staining with anti-human CD3-PE, CD19-APC, and IgG-FITC mAbs (BD Biosciences). Retrospective FXM was performed using the donor T and B lymphocytes from donor spleen which had been stored at liquid nitrogen. Approximately 0.5 x 10The complement dependent cytotoxicity (CDC) crossmatch assay was used to evaluate cell death in the patient’s serum in the presence of complement. Random HUVEC crossmatches were performed by the modified Amos method (using three washes). Serum was treated using pooled human platelet absorption to exclude anti-HLA antibodies before addition to the endothelial cells. Briefly, 1 μl of negative control, positive control mAb, normal human serum, or patient treated serum was dispensed into individual wells of Terasaky microplates, followed by addition of 1 μl of HUVEC suspension (approximately 5000 cells), and samples were incubated for 1 hour at room temperature. After three washes with PRMI-1640 medium, rabbit complement (One Lambda) was added. After further 30-minute incubation at room temperature, the cells were stained with EB/AO and FluoroQuench (One Lambda) to enable detection of the killing ratio by fluorescence microscopy.Since T and B lymphocyte crossmatches do not response to the presence of anti-MICA antibodies, and it is difficult to obtain donor endothelial cells, MICA ‘virtual crossmatch’ was therefore used to detect sensitivity to MICA antigens. We determined whether the patient had MICA-DSA for a given donor by performing MICA genotyping of donor and recipient and testing recipient MICA antibodies using single antigen Luminex flow cytometry. The algorithm of MICA virtual crossmatch was based on the results of missed MICA allele or alleles of a given donor and the specificity of MICA antibodies in recipient’s serum.The patient is a 48-year-old Han female of blood type A with end-stage renal disease due to glomerulonephritis. She received a kidney transplant from a donor after cardiac death (DCD) in January 2013. Although the patient had HLA-I antibodies  before transplant, the transplant was performed because of the negative CDC and FXM crossmatch with the donor; importantly, the donor was zero-antigen mismatched to the recipient at HLA-A, HLA-B, and HLA-DR loci. The recipient’s serum creatinine levels decreased by 75% relative to pre-transplant levels one week after transplantation, but an acute rejection episode occurred after two weeks. The pathological tissue sections from renal biopsy were positive for C4d  and revest donor’s genotype is MICA*008/MICA*018  continue to be the major barrier for the successful renal transplantation. Antibodies against non-HLA antigens such as MICA/B antigens, vimentin [18]. According to the new Banff standard of AMR diagnosis, anti-donor specific alloantibodies (DSA) should be considered prior to transplantation. In the case reported here, the recipient was given a DCD kidney with a zero antigen mismatch, but this allograft was rejected one month after transplantation. Analyses indicated that kidney allograft failed due to AMR, even though there was no HLA-A, B, DR, DQ antigen mismatched to the first donor and negative prospective T and B cell crossmatches.Patients with high levels anti-HLA antibody wait longer for donor organs than those with lower levels unless a zero antigen mismatched donor is assigned. Even with a zero antigen mismatched donor, some of these patients will have been sensitized to non-HLA antibodies such as MICA antibodies [To determine the cause of the AMR in our patient, we performed a retrospective analysis of MICA genotyping, which revealed potential for MICA-DSAs in the patient. We used pooled normal human platelets to absorb HLA-class I antibodies from patient serum samples as previous described , and useRoutine T and B cell crossmatch is not able to detect donor-specific MICA alloantibodies. If a transplant patient has MICA-DSAs, donor kidney endothelial cells become the target of injury. The patient in our study had been sensitized to MICA antigens as well as HLA before transplantation. The patient’s HLA alloantibodies were not specific to the first kidney donor, but the MICA alloantibodies were. The titer of MICA-DSA was very high . Fortunately, the second renal transplant with a negative MICA virtual crossmatch has been successful. In conclusion, a virtual crossmatch for MICA, performed in addition to the HLA histocompatibility assay, will benefit organ transplant recipients.


The behavior of droplets trapped in geometric structures is essential to droplet manipulation applications such as for droplet transport. Here we show that directional droplet movement can be realized by a V-shaped groove with the movement direction controlled by adjusting the surface wettability of the groove inner wall and the cross sectional angle of the groove. Experiments and analyses show that a droplet in a superhydrophobic groove translates from the immersed state to the suspended state as the cross sectional angle of the groove decreases and the suspended droplet departs from the groove bottom as the droplet volume increases. We also demonstrate that this simple grooved structure can be used to separate a water-oil mixture and generate droplets with the desired sizes. The structural effect actuated droplet movements provide a controllable droplet transport method which can be used in a wide range of droplet manipulation applications. The dynamic behavior of droplets has drawn much attention in recent years due to its fundamental effect in many practical applications. Generally, droplets can be manipulated by a surface free energy gradient1234567911121415161920212223242526141516This study shows that the directional movement of droplets can be realized in a V-shaped groove and the movement direction can be controlled by adjusting the surface wettability of the groove inner wall and the cross sectional angle of the V-shaped groove. In contrast to the droplet transport direction in natural structures1415163031323334353637θ, and the cross sectional angle, β. The IM mode denotes a droplet that is immersed in the groove bottom while the SU mode denotes a droplet that is suspended in the groove center. Due to the interfacial free energy minimization, the droplet will remain in spherical shape with identical contact angles for the local contact lines. As a result, for the suspended droplet as demonstrated in θu and θd, will be equal to each other with θu = θd = θ. For the SU droplet, the half central angle of the lower meniscus, D, can be expressed as follows from geometry:The analysis starts from the basic resting states of droplets on various grooved structures with different cross sectional angles and surface wettabilities. To form a suspended droplet, a lower meniscus has to form so that the groove bottom is not entirely wetted by the droplet, suggesting that the half central angle of the lower meniscus has to be larger than zero. The following criterion can then be obtained:β < 2θ-π, as shown in Equation θ, advancing contact angle, θa, and receding contact angle, θr, are listed in θ, and the cross sectional angle, β. For smaller θ and larger β conditions, the droplets are immersed within the grooves in the Immersed (IM) mode. For other conditions with β < 2θ-π, the droplets are suspended in the groove center in the Suspended (SU) mode. These experimental results provide support for the criterion in Eq. The criterion in Eq. β changing from high-to-low and low-to-high (Methods). The resultant experimental images are shown in β = 106°  to superhydrophobic (Etched SAM groove).The resting mode transitions and directional droplet movements can be realized by dynamically adjusting the cross sectional angle of the grooves. The Etched SAM and Smooth SAM grooves were used in these experiments with β = 106° , while tβ = 109° . The resβ becomes smaller than 58.4° according to Eq. β . For the Etched SAM groove, the differences between θa, θr and θ are all small enough to ensure that the upper and lower meniscuses move upward almost simultaneously. As the droplet volume increases, the upper and lower meniscuses all move upward so that the droplet departs from the groove bottom and the resultant larger droplet is suspended higher in the groove . For the Etched SAM groove, the upper and lower contact lines move downwards almost simultaneously as during the droplet growth due to the relatively small contact angle hysteresis b1. For tns fixed b2.β. For a SU mode droplet, the droplet volume increase will first cause deformation of the meniscuses and the local contact angle will then increase. The local contact angles for the newly-deformed upper and lower meniscuses, θu′ and θd′, are related as  and the geometric parameters. A better understanding of the capillary effect between droplets and geometric structures is certainly helpful for the droplet manipulation applications.β < 2θ-π, with the suspending droplets formed in grooves with superhydrophobic inner walls and small cross sectional angles. The resting state also changes with the geometric parameters. For instance, the droplet in a superhydrophobic groove can translate from the immersed state to the suspended state as the cross sectional angle of the groove decreases. The IM-to-SU transition occurs because the droplet in SU mode will be energetically more favorable when β is decreased. The driving force for the IM-to-SU transition is provided by the deformation of the upper meniscus when the groove cross sectional angle is decreasing.The present work shows that the resting states of droplets in grooves can be predicted by a criterion of In addition, the directional movement of droplets can be realized in the V-shaped groove and the moving direction is also changeable. Our analyses demonstrate that the movement of droplet meniscuses are in stepwise manner and can be affected greatly by the contact angle hysteresis. In a superhydrophobic groove, the suspended droplet departs from the groove bottom as the droplet volume increases. This structural effect actuated droplet movements offer new insights into the interfacial phenomena of droplets trapped in geometric structures and provide a controllable droplet transport method which can be used in a wide range of droplet manipulation applications.β of the groove could be adjusted in real time to the desired values. The microsyringe system was used to generate droplets with the desired volumes for the wetting mode measurements and to adjust the droplet volume in real time for the droplet growth observations. The surface wettabilities, including the apparent contact angle θ, advancing contact angle θa, and receding contact angle θr, were measured by an OCAH200 contact angle system  with the results listed in The experimental setup was composed of a grooved structure, a microsyringe system, a mechanical stepping drive, and a microscopic CCD camera . The groThe droplet growth was simulated with a 2 μL water droplet placed in the groove with a microsyringe needle then inserted into the droplet to supply water at a constant flow rate of 0.06 μL/s. The effect of the inserted needle on the droplet movement can be neglected since its contact angle is ~90° and it has a relatively small diameter. In the evaporation experiments, a 4 μL water droplet was placed in the groove and then the droplet was allowed to evaporate by exposure to the ambient environment.The experimental images were captured by the CCD camera of the OCAH200 system with a resolution of 752 × 484 pixels and frame rate of 60 fps. The image overlapping in The V-shaped grooved structures were folded from a copper sheet. The groove surfaces  were treated with W0.5 abrasive paste polishing, 180# sandpaper grinding, and chemical etchingHow to cite this article: Xu, W. et al. Directional Movement of Droplets in Grooves: Suspended or Immersed? Sci. Rep.6, 18836; doi: 10.1038/srep18836 (2016).