Cerebrospinal fluid (CSF) shunt placement is frequently complicated by bacterial infection. Shunt infection diagnosis relies on bacterial culture of CSF which can often produce false-negative results. Negative cultures present a conundrum for physicians as they are left to rely on other CSF indices, which can be unremarkable. New methods are needed to swiftly and accurately diagnose shunt infections. CSF chemokines and cytokines may prove useful as diagnostic biomarkers. The objective of this study was to evaluate the potential of systemic and CSF biomarkers for identification of CSF shunt infection.We conducted a retrospective chart review of children with culture-confirmed CSF shunt infection at Children’s Hospital and Medical Center from July 2013 to December 2015. CSF cytokine analysis was performed for those patients with CSF in frozen storage from the same sample that was used for diagnostic culture.3 cells/mL. Those with shunt infection had a median CSF WBC of 332 cells/mL, median CSF protein level of 406 mg/dL, and median CSF glucose of 35.5 mg/dL. An interesting trend was observed with gram-positive infections having higher levels of the anti-inflammatory cytokine interleukin (IL)-10 as well as IL-17A and vascular endothelial growth factor (VEGF) compared to gram-negative infections, although these differences did not reach statistical significance. Conversely, gram-negative infections displayed higher levels of the pro-inflammatory cytokines IL-1β, fractalkine (CX3CL1), chemokine ligand 2 (CCL2), and chemokine ligand 3 (CCL3), although again these were not significantly different. CSF from gram-positive and gram-negative shunt infections had similar levels of interferon gamma (INF-γ), tumor necrosis factor alpha (TNF-α), IL-6, and IL-8.A total of 12 infections were included in this study. Patients with shunt infection had a median C-reactive protein (CRP) of 18.25 mg/dL. Median peripheral white blood cell count was 15.53 × 10This pilot study is the first to characterize the CSF cytokine profile in patients with CSF shunt infection and supports the distinction of chemokine and cytokine profiles between gram-negative and gram-positive infections. Additionally, it demonstrates the potential of CSF chemokines and cytokines as biomarkers for the diagnosis of shunt infection. Staphylococcus epidermidis and Staphylococcus aureus among the leading causes [Propionibacterium acnes, enterococci, and a variety of gram-negative bacteria including Pseudomonas aeruginosa and Escherichia coli commonly cause shunt infections [Cerebrospinal fluid (CSF) shunts are the most common treatment of hydrocephalus in the USA; however, they are frequently complicated by bacterial infection . Shunt ig causes –6. Apartfections –7.Shunt infections present unique diagnostic and treatment challenges, since they are biofilm rather than planktonic infections . BacteriStaphylococcus epidermidis and Staphylococcus aureus are the predominant organisms that are not included in this panel [While newer DNA-based detection methods are being instituted in the clinical microbiology laboratory, the gold standard method to diagnose shunt infection currently relies on isolating a pathogen from CSF culture –19. Whilis panel –6.Traditional CSF culture normally requires 24–48 h to isolate organisms, and some slower growing organisms may require several days of incubation, which can delay appropriate treatment . AdditioIdentifying biomarkers in the CSF or serum of patients with shunt infection may improve our ability to diagnose CSF shunt infections. Biomarkers are distinct biochemical, genetic, or molecular substances that characterize infection , 21. TheStreptococcus pneumoniae, Neiserria meningitidis, and Haemophilus influenzae based on levels of interferon gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), and the enzyme matrix metallopeptidase-9 (MMP-9) [Several studies have examined the possibility of using chemokines and cytokines as biomarkers for meningitis in the pediatric population. Elevated IL-8, IL-1β, TNF-α, and IL-6 have routinely been shown to be elevated in bacterial meningitis , 29, 30. (MMP-9) .Studies by Srinivasan et al.  in neonaS. epidermidis infection, which is the most common cause of shunt infection [To date, no study has examined CSF chemokine and cytokine expression exclusively in patients with CSF shunt infections. Animal models of shunt infection suggest that the inflammatory response in these biofilm infections may be attenuated in comparison to planktonic infections; thus, by extension, the same cytokine and chemokine responses may not be observed in shunt infections as in meningitis , 33–35. nfection . TherefoIn this retrospective study, we included all patients less than or equal to 21 years of age with culture-confirmed bacterial CSF shunt infection diagnosed between July 1, 2013, and December 31, 2015, at Children’s Hospital and Medical Center (CHMC) in Omaha, Nebraska. Patients were identified by International Classification of Disease, 9th revision coding, and confirmed by positive CSF culture results in accordance with the modified criteria for nosocomial infection of the US Centers for Disease Control and Prevention . CSF celPatients were excluded if their diagnostic CSF culture was performed at another institution or grew yeast or if no CSF culture data was available. All participants were screened for additional inflammatory infection by medical chart review and were excluded if there was an additional inflammatory condition documented in the medical record that would alter inflammatory markers, such as a concomitant viral upper respiratory infection.Data extracted from the patients’ chart included the following: patient age, peripheral white blood cell (WBC) count, peripheral C reactive-protein (CRP), CSF WBC count and differential, CSF glucose, CSF protein, and CSF culture results. Patient’s peripheral WBC, CRP, CSF WBC and differential, CSF glucose, and CSF protein were included if they were obtained within a 48-h window from the time of the CSF culture.3CL1), vascular endothelial growth factor (VEGF), TNF-α, IFN-γ, IL-1β, IL-6, IL-8, IL-10, and IL-17A.For those patients with CSF in frozen storage from the same sample that was used for diagnostic culture, additional cytokine analysis was conducted. The CSF was analyzed by multiplex analysis for cytokines and chemokines . The multiplex panels included the following: chemokine ligand 2 (CCL2), chemokine ligand 3 (CCL3), fractalkine , viridans group streptococci (1), coagulase-negative staphylococci (2), Enterococcus faecalis (1), Gemella haemolysans (1), Serratia marcescens (1), Pseudomonas aeruginosa (2), Escherichia coli (1), Enterobacter cloacae (1), and Stenotrophomonas maltophilia (1).A total of 11 individual patients with 12 separate shunt infections were included in this study (Table 3 cells/mL (5.97–29.71 × 103 cells/mL). Nine patients had CSF WBC within 48 h of their CSF culture with a median CSF WBC of 332 cells/mL (24–1914 cells/mL). The gram-positive infections appeared to have a neutrophil predominance in the CSF while gram-negative infections had a more varied cellular differential. Eight patients had results for CSF protein and CSF glucose. CSF protein had a median value of 406 mg/dL (24–600 mg/dL), and CSF glucose had a median value of 35.5 mg/dL (20–75 mg/dL).Of the eight patients with CRP, the median was 18.25 mg/dL (0.5–35.9 mg/dL). All but one patient had a peripheral WBC count performed within 48 h of their CSF culture, and the median WBC was 15.53 × 103CL1 (532.93 pg/mL vs 187.95 pg/mL), CCL2 (7094.50 pg/mL vs 5741.00 pg/mL), and CCL3 (665.74 pg/mL vs 183.06 pg/mL)  as well as IL-17A (193.95 pg/mL vs 14.05 pg/mL) and VEGF (303.84 pg/mL vs 45.37 pg/mL) (Fig. Prompt diagnosis of shunt infection can allow for earlier treatment, including surgical intervention, potentially averting damaging consequences such as seizures and reduced IQ , 40. HowPro-inflammatory mediators have been explored as a diagnostic strategy for bacterial meningitis. Multiple studies have demonstrated elevated levels of TNF-α, IL-6, and IL-8 in the CSF of patients with bacterial meningitis, especially in the pediatric population , 29, 30.The results of our study demonstrated similar levels of INF-γ, TNF-α, IL-6, and IL-8 in the CSF of patients with both gram-positive and gram-negative CSF shunt infections. This is consistent with results from Srinivasan et al. , which d3CL1 CCL2, and CCL3.In our cohort, there were trends of a distinct inflammatory response in gram-positive versus gram-negative shunt infections. CSF from patients with gram-positive infections had higher levels of the anti-inflammatory cytokine IL-10 as well as IL-17A and VEGF. Those with gram-negative infection had higher levels of the pro-inflammatory cytokines IL-1β, CXS. epidermidis CNS catheter infection [S. epidermidis infected compared to sterile catheters [Staphylococcus aureus blood stream infection had higher mortality. Interestingly, two of the patients included in our cytokine analysis cohort had coagulase-negative staphylococcal infections. The increase in IL-10 may be dampening pro-inflammatory mediators, such as the elevated IL-17A and VEGF seen in these patients, and serving a neuroprotective role. This is being further evaluated in animal models of shunt infection.The increase in IL-10 in the gram-positive patients is consistent with a murine model of nfection . This statheters . IL-10 iatheters , 44. It atheters , 45. Addatheters  demonstrStaphylococcus aureus brain abscess [In addition to increased IL-10, patients with gram-positive infections had higher levels of IL-17A and a neutrophil predominance in the CSF when compared to patients with gram-negative shunt infection. This is consistent with IL-17’s role as an inducer of neutrophil chemokines, including IL-8, and IL-17’s importance in a murine model of  abscess , 48. WhiStreptococcus pneumoniae meningitis as well as in tuberculous meningitis and other types of bacterial meningitis [In addition to elevated IL-10 and IL-17A, patients with gram-positive shunt infection had elevated levels of VEGF, a potent mediator of vascular permeability associated with cerebral edema and neutrophil migration in the CNS –51. VEGFningitis –54. VEGFOur ability to evaluate whether the gram-negative shunt infections have a Th1, Th2, or Th17 bias was limited due to sample availability. However, the data would suggest a potential skewing towards a Th1 immune response in gram-negative shunt infection with increased monocytes in the CSF of these patients compared to gram-positive infection as well as increase in IL-1β and a modest increase in INF-γ in gram-negative infection. While there is limited data on the type of immune response gram-negative bacteria create in the CNS, studies have demonstrated that gram-negative elicit Th1 immune responses in models of intestinal inflammatory disorders , 56.The CSF patterns reported in this study are likely reflective of differing immune pathways triggered by gram-positive versus gram-negative organisms . ImportaStaphylococcus aureus as the second most common cause [This pilot study is the first to characterize the CSF cytokine profile in patients with CSF shunt infection and has revealed distinct chemokine and cytokine profiles between gram-negative and gram-positive infections. While there is an interesting trend in the CSF cytokines that could greatly improve diagnosis of CSF shunt infection, there are several limitations with this pilot study. Epidemiologically, coagulase-negative staphylococci is the most common cause of CSF shunt infection, causing approximately two thirds of shunt infections, with on cause , 5, 33. Secondly, in this small cohort, which was not large enough to determine statistical significance, clear trends of chemokines and cytokines were evident in gram-positive versus gram-negative shunt infection. These trends demonstrate a potential Th17-predominant immune response to gram-positive shunt infection and Th1-predominant response in gram-negative shunt infection. Additionally, due to the restrictions of available CSF, there is not a control population as CSF is not obtained from the shunts of well children as there is the possibility of iatrogenic infection. To address these limitations, we are currently developing a rat model of CSF shunt infection to further delineate CSF and serum inflammatory profiles in a controlled manner that will allow for analysis of postoperative versus infective changes. However, these pilot findings serve as a valuable introduction to the potential of CSF chemokines and cytokines as biomarkers for the diagnosis of shunt infection. By improving our ability to diagnose these infections, we can provide earlier effective therapy in a disease process which remains difficult to diagnose and treat.


Midazolam is known as a safe drug and is widely used as a sedative and an anesthetic adjuvant. Therefore, there is a lack of awareness that midazolam can cause anaphylaxis. Midazolam anaphylaxis is rare, and only a few cases have been reported, but such a risk is always present. In this study, we report a case of midazolam anaphylaxis by an intravenous injection, in the prone position, during general anesthesia.A 62-year-old woman was intravenously administered 1 mg midazolam during general anesthesia, and sudden severe hypotension, bronchospasm, decreased oxygen saturation, erythema, and diarrhea occurred.Midazolam anaphylaxis was presumptively diagnosed by clinical symptoms and was confirmed by an intradermal test after 9 weeks.The patient was treated with 100% oxygen, large volume of fluid, epinephrine, phenylephrine, ephedrine, dexamethasone and prednisolone, ranitidine, and flumazenil.Severe hypotension and decreased oxygen saturation were resolved within 20 minutes of the onset of anaphylaxis, and the patient was discharged after 3 days without any sequelae.Midazolam anaphylaxis is very rare, but it can happen always. Therefore, the possibility of anaphylaxis due to midazolam should be considered and always be prepared for treatment. Midazolam is known as a safe drug and is widely used as an anxiolytic, sedative, and adjuvant to general anesthetics in patients with drug allergy. Therefore, there is a lack of awareness that midazolam can cause anaphylaxis. Midazolam anaphylaxis is rare, and only a few cases have been reported.–9 But such a risk is always present. In this study, we report a case of anaphylactic shock caused by 1 mg midazolam that was intravenously administered as an adjuvant during general anesthesia.Anaphylaxis is an acute, potentially life threatening, systemic hypersensitivity reaction that occurs when a patient is re-exposed to a previously sensitized antigen.2A 62-year-old woman, weight 46 kg, height 153 cm, was scheduled for endoscopic discectomy due to the diagnosis of right L5S1 lateral stenosis.Her medical history showed that she underwent myomectomy 20 years ago and had taken alprazolam and diazepam per os for 2 months and underwent a colon polypectomy with midazolam 3 mg intravenous (IV) 2 months ago. She had no history of a drug or food allergy. The preoperative examination revealed no specific findings and the intradermal test for the antibiotic cefotetan injection was negative.2) 98%, Bispectral index 99, and body temperature 36.5 °C were recorded. Lidocaine 40 mg and 1% propofol 80 mg IV were administered to induce anesthesia, and tracheal intubation was performed after muscle relaxation with rocuronium 40 mg IV. Sevoflurane 1 to 2 vol%:O2:air was administered for maintenance of anesthesia. Artificial ventilation was maintained at a tidal volume of 400 mL, respiratory rate 11 breaths/min, and end tidal CO2 (ETCO2) 28 to 30 mmHg. Remifentanil 0.5 to 1 ng/mL was continuously infused using target-controlled infusion pump from the anesthetic induction. After the anesthetic induction, BP 145/65 mmHg and HR 80 beats/min were noted.On arrival to the operating room, electrocardiogram was normal and blood pressure (BP) 135/71 mmHg, heart rate (HR) 78 beats/min, peripheral oxygen saturation . Subsequently, we decided to inject midazolam 1 mg IV to prevent awareness of the patient without decrease of BP during general anesthesia.The patient was positioned in the prone position for surgery, and the vertebra level of the surgical site was checked with a C-arm x-ray. While simultaneously administering sevoflurane 2 vol% and remifentanil 0.5 ng/mL, BP 125/68 mmHg, HR 55 beats/min, and ETCO2 96%, and ETCO2 15 mmHg had declined and capnography showed an obstructive pattern. Wheezing sound in both lungs was noted on auscultation and some small erythema appeared on the back. Sevoflurane and remifentanil were discontinued and 100% O2 and ephedrine 10 mg IV were administered twice. However, BP 53/35 mmHg, HR 74 beats/min, and SpO2 91% were noted. SpO2 further reduced to 89% and BP was not detected. Ringer lactate solution at a high rate, 2 times of epinephrine 100 μg IV, and phenylephrine 100 μg IV were administered twice. BP 58/38 mmHg, HR 98 beats/min, and SpO2 92% were noted.One minute after administering midazolam 1 mg IV, Bispectral index decreased from 68 to 54. At the same time, BP 41/30 mmHg, HR 46 beats/min, SpO2 94%, and ETCO2 25 mmHg were noted, and phenylephrine 100 μg IV was administered. Vital signs improved to BP 86/61 mmHg, HR 110 beats/min, and SpO2 98% after 20 minutes of the anaphylactic shock. We then explained to the family the possibility of side effects due to midazolam and decided to postpone the operation. Glycopyrrolate 0.2 mg IV and pyridostigmine 10 mg IV were administered to reverse muscle relaxation. The wheezing sound disappeared and self-respiration of the patient gradually recovered. We performed tracheal extubation after 55 minutes of anesthesia induction. BP 121/60 mmHg, HR 110 beats/min, and SpO2 98% were noted at the time of extubation.The patient was changed to supine position, followed by Trendelenburg position. Based on the suspicion of anaphylaxis due to midazolam, we injected flumazenil 0.25 mg, dexamethasone 5 mg, methylprednisolone 125 mg, and ranitidine 100 mg intravenously. However, BP 73/48 mmHg, HR 104 beats/min, SpO2 99% were noted. The patient complained of diarrhea. After 15 minutes of arrival at the recovery room, BP and HR decreased again to 78/45 mmHg and 85 beats/min, respectively, and phenylephrine 100 μg IV was injected. On evaluation, BP was 88–92/48–50 mmHg and HR was 48 to 50 beats/min. Arterial blood gas analysis was performed, but there were no specific findings. After 80 minutes in the recovery room, BP, HR, and SpO2 were maintained at 92–100/51–52 mmHg, 51 beats/min, and 99%, respectively. The skin erythema disappeared, and the patient was transferred to the intensive care unit after 2 hours in the recovery room. The total amount of injected Ringer lactate solution was 1800 mL. Examinations for serum β-tryptase and serum immunoglobulin E were not performed.In the recovery room, oxygen 5 L/min was supplied and the patient's legs were elevated, and BP 90 to 100/53 to 55 mmHg, HR 85 to 100 beats/min, and SpOIn the intensive care unit, BP was maintained at 80/50 mmHg and HR at 60 beats/min. After 1 hour , the vital signs were recorded as BP 126/66 mmHg and HR 60 beats/min. The body temperature rose from 36.9 to 37.6 °C and was normalized after supportive care for 2 days. We examined Troponin-T, creatine kinase-MB isoenzyme, and echocardiogram to rule out the cardiovascular problem, which was normal.The day after the anaphylactic shock, the patient was transferred to the general ward. The main complaint, back pain, was treated with medication and physical therapy. After 2 days, the patient and her family were informed again about midazolam anaphylaxis and the patient was discharged without any sequelae. After 9 weeks of anaphylaxis, the patient showed a positive reaction for midazolam in the intradermal test.The study was approved by the Institutional Review Board of our University Hospital. Written informed consent was obtained from the patient for the publication of this case report.3 so proper diagnosis and treatment by the physician is very important.Anaphylaxis causes a very fast life-threatening condition and can involve multiple organs, In this case, the vena cava compression due to the prone position reduced the venous return and induced more severe hypotension. Therefore, it was recommended to change to a Trendelenburg position or supine position with leg elevation. The patient also developed fever on the day of anaphylaxis. This was due to the inflammatory reaction by newly formed proinflammatory phospholipid-derived mediators such as prostaglandin D2, leukotrienes, thromboxane A2, and platelet activating factor.Severe hypotension after the onset of anaphylaxis is due to vasodilation and increased vascular permeability by preformed mediators such as histamine, neutral protease , and proteoglycans (heparin) released from mast cells or basophils. Due to increased vascular permeability, 35% of the intravascular volume shifts to the interstitial space.2 agonist action, including bronchial dilatation, gastrointestinal smooth muscle relaxation, inhibition of further mediator release from mast cell and basophil, and inotropic/chronotropic effects.Treatments for anaphylactic reaction are administration of 100% oxygen, large volume of fluid, epinephrine, corticosteroid, and antihistamine. Epinephrine should be administered as early as possible and carefully. Epinephrine, in addition to vasoconstriction, has β2 decrease, abrupt drop in ETCO2, obstructive pattern on capnography, and erythema. Many drugs have been administered at various stages of anesthetic induction, but only midazolam was injected immediately before the anaphylactic response. We clinically suspected midazolam as the causative agent because anaphylactic signs appeared after 1 minute of IV injection of midazolam. An acute serum tryptase test within 2 hours after the onset of anaphylaxis was recommended. However, it is difficult to sample acute serum tryptase due to insufficient equipment or other practical limitations in urgent clinical situations. We also did not perform the test and it is a limitation of this case. Buka et al reported that, using a cutoff of 12.4 ng/mL (75th centile), sensitivity, specificity, positive predictive value, and negative predictive value of acute serum tryptase were 28%, 88%, 93%, and 17%, respectively. It means acute serum tryptase is a poor indicator for anaphylaxis. Nonetheless, acute serum tryptase is useful in clinical circumstances to distinguish from its mimics. In the cause analysis with the surgeon, they suspected deep anesthesia depth, heart disease, and other causes rather than midazolam anaphylaxis. We confirmed that there was a clear relationship between midazolam and anaphylaxis through skin test  performed after 9 weeks.The recognition of anaphylaxis during anesthesia is usually confusing because hypotension and bronchospasm can develop due to other reasons and because many drugs, including muscle relaxants and propofol, are administered during general anesthesia. In this case, the chief clinical factors of diagnosing anaphylaxis were sudden severe hypotension, bradycardia, SpOThis case clearly shows that midazolam is one of the causes of anaphylaxis.In conclusion, midazolam anaphylaxis is very rare, but it can happen always. Therefore, the possibility of anaphylaxis due to midazolam should be considered and always be prepared for treatment.Conceptualization: Yeon Su Jeon.Investigation: JinWoo Shim, Eun Hwa Jun, Seung Tae Choi.Writing – original draft: Yeon Su Jeon.Writing – review & editing: Hong Soo Jung.Yeon Su Jeon orcid: 0000-0002-2607-3107.JinWoo Shim: orcid: 0000-0002-9869-2812.Eun Hwa Jun orcid: 0000-0003-1342-0576.Seung Tae Choi orcid: 0000-0003-2041-5580.Hong Soo Jung orcid: 0000-0002-6812-1955.


Robot-assisted rehabilitation is a growing field that can provide an intensity, quality, and quantity of treatment that exceed therapist-mediated rehabilitation. Several control algorithms have been implemented in rehabilitation robots to develop a patient-cooperative strategy with the capacity to understand the intention of the user and provide suitable rehabilitation training. In this paper, we present an upper-limb motion pattern recognition method using surface electromyography (sEMG) signals with a support vector machine (SVM) to control a rehabilitation robot, ReRobot, which was built to conduct upper-limb rehabilitation training for post-stroke patients. For poststroke rehabilitation training using the ReRobot, the upper-limb motion of the patient's healthy side is first recognized by detecting and processing the sEMG signals; then, the ReRobot assists the impaired arm in conducting mirror rehabilitation therapy. To train and test the SVM model, five healthy subjects participated in the experiments and performed five standard upper-limb motions, including shoulder flexion, abduction, internal rotation, external rotation, and elbow joint flexion. Good accuracy was demonstrated in experimental results from the five healthy subjects. By recognizing the model motion of the healthy side, the rehabilitation robot can provide mirror therapy to the affected side. This method can be used as a control strategy of upper-limb rehabilitation robots for self-rehabilitation training with stroke patients. Stroke is the leading cause of adult disability around the world  participated in the experiments. All subjects gave their informed consent before participation. The experimental procedures were conducted in accordance with the Declaration of Helsinki and approved by the Ethic Board of Medical School, South China University of Technology. Each subject performed five repetitions in accordance with five standard motions. Since stroke patients are mostly elderly people with lower motion abilities on their healthy side compared to young adults, the designed motions were imitated as movements of elderly stroke patients on their healthy side.Five healthy subjects  is the tth value of channel i, and sEMG is the sum of all channel signals.where The sample entropy can be calculated as follows:m is the dimension of the sEMG signal, r is the similar tolerance, L is the length of the muscle signal, and Bm (r) is the probability of the two signal sequences matching m points. In this study, we set m = 2 and r = 0.25*σ, where σ is the standard deviation of the sEMG signal.where d (d = 0.6) is the threshold and s(n) is the judgment function of the EMG signal. When s(n) = 1, it is the effective part of the action; when s(n) = 0, it is the invalid part of the action, as shown in where Because of the short-term stationarity of sEMG signals, the signals need to be divided into frames. To prevent spectrum leakage, window functions should be used for interception. Compared with window functions such as the rectangular window and triangular window, the Hanning window has the characteristics of fast side lobe attenuation and is suitable for non-stationary signals. Therefore, this paper adopts a Hanning window for framing. Hanning windows with window lengths ranging from 30 to 300 ms  is a machine learning method based on statistical learning theory. The characteristic behavior of SVM is to construct a high-dimensional hyperplane for small samples and non-linear models and to classify samples by calculating the maximum distance of training data points on the hyperplane  represents the training data of the kth window. ξi is a slack variable, which represents the magnitude of the classification error.where  were selected for shoulder flexion, abduction, pronation, and elbow flexion. Each action was performed five times for data classification and recognition.To test the feasibility of the genetic optimization algorithm, simulations were carried out using MATLAB. Five healthy subjects  that is able to activate the device as soon as the patient's motion intention is detected. By using the SVM system, the upper-limb movements of the healthy side can be detected and classified automatically through sEMG signals, and the ReRobot will then assist the impaired arm with that movement. The control scheme based on the SVM classification of sEMG signals was thus established, as shown in This platform acquires the test subject's sEMG signals of the upper limb on the healthy side, and the preprocessed EMG signals from one to eight channels are used for upper limb motion recognition using the SVM classification method. The recognized action label signals are sent to the robot for motion calculation, and then the robot actuates the subject's affected arm with the position control method to perform the corresponding actions based on a presupposed trajectory.When the patient feels discomfort in his or her arm or muscle abnormalities during the test, the whole system can be safely stopped by patient through the first clenching motion, and the sEMG signal from channel 9 is sent to the manipulator. At the same time, there is an emergency stop button in the test subject's hand throughout the test process to ensure safety.To test the feasibility of the upper-limb motion recognition method based on the SVM classification, simulations were carried out using MATLAB. Five movements of five normal people were collected 20 times. After processing feature extractions and label recognition of the collected data, 10 five-fold cross-validations were performed. The results are shown in Based on the chaotic matrix, three accuracy metrics  can be obtained  participated in the rehabilitation training experiments, and all five subjects were able to complete robot-assisted voluntary exercises, as shown in Five subjects (To verify the effectiveness of the safety switch based on the sEMG signal, a total of 25 tests of safety stops based on sEMG signals were performed. ReRobot had its cut power during each test, which proved the feasibility of using sEMG signals as an emergency stop in the rehabilitation system.The experimental results showed that SVM-based classification can provide good accuracy in upper-limb motion pattern recognition and enabled patients to choose actions actively for rehabilitation. It is possible to use sEMG signals as an emergency stop button in the upper-limb rehabilitation system to ensure safety.In this paper, we investigated the feasibility of SVM classifiers for intention identification of the upper limb from sEMG signals. A new human–machine interface for self-rehabilitation training with stroke patients was developed. The upper-limb rehabilitation robot, ReRobot, could adequately understand the desired upper-limb movement and facilitate its execution, thus providing active support to the impaired arm. Experiments with the ReRobot showed that the SVM classification based on sEMG signals can provide good accuracy in upper-limb motion pattern recognition when a time-dependent multifeature set was used.In future research, this method to extract upper-limb intention from sEMG signals will be tested by experiments with stroke patients. The application of this classifier to upper-limb rehabilitation robots will be implemented to achieve successful clinical verification.This study was carried out in accordance with the recommendations of SCUT Research Ethics Guidelines and Researcher's Handbook, Ethic Board of Medical school, South China University of Technology with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ethic Board of Medical school, South China University of Technology.SC, YC, and LX contributed conception and design of the study. SH carried out the experiments. SC and YC performed the formal analysis and methodology part. SC wrote the first draft of the manuscript. YC, SH, YW, HZ, XL, and LX wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


POLG disease.To describe the spectrum of movement disorders and cerebrospinal fluid (CSF) neurotransmitter profiles in paediatric patients with POLG disease, in whom CSF neurotransmitter analysis had been undertaken. Clinical data were collected retrospectively. CSF neurotransmitter levels were compared to both standardised age-related reference ranges and to non-POLG patients presenting with status epilepticus.We identified children with genetically confirmed POLG disease were identified. Almost 50% of the patients had documented evidence of a movement disorder, including non-epileptic myoclonus, choreoathetosis and ataxia. CSF neurotransmitter analysis was undertaken in 15 cases and abnormalities were seen in the majority (87%) of cases tested. In many patients, distinctive patterns were evident, including raised neopterin, homovanillic acid and 5-hydroxyindoleacetic acid levels.Forty-one patients with POLG mutations can manifest with a wide spectrum of abnormal movements, which are often prominent features of the clinical syndrome. Underlying pathophysiology is probably multifactorial, and aberrant monoamine metabolism is likely to play a role.Children with The online version of this article (10.1007/s10545-018-0227-7) contains supplementary material, which is available to authorized users. POLG, encoding the catalytic subunit of pol γ, result in MDDS with evidence of reduced mtDNA content or abnormal mtDNA (multiple mtDNA deletions or point mutations) in affected tissues  depletion syndromes (MDDS) are caused by defects in mtDNA maintenance due to mutations in nuclear genes which affect either mitochondrial deoxyribonucleoside triphosphate supply or components of the mtDNA replication machinery  is the most frequent clinical presentation  standardised proformas completed for diagnostic CSF and genetic testing and (ii) patient hospital records, where available (see Paediatric patients (16 years or younger) with confirmed biallelic able see .POLG patients admitted to a single paediatric intensive care unit (PICU) from August 1999 to November 2011 were reviewed. All patients who had neurotransmitter analysis secondary to non-POLG-related status epilepticus were included in the study.For comparative analysis, CSF neurotransmitter profiles of non-POLG gene sequencing was performed as previously described .Information regarding early clinical features was available for all 20 patients. Encephalopathy and/or status epilepticus was the most common mode of presentation (17/20 cases). Where CSF neurotransmitter analysis had also been performed, 11/15 patients presented either with status epilepticus or epilepsia partialis continua (EPC), preceded by an intercurrent infection in 2/15 cases. The remaining 4/15 patients  presented initially with a movement disorder, although all eventually developed status epilepticus/EPC in the ensuing weeks or months. Data regarding administered antiepileptic drugs (AEDs) were limited or absent in most cases , chorea and/or athetosis (7/15), and ataxia (5/15) were described most commonly, but tremor (3/15) and dystonia (3/15) were also reported , with bilateral symmetrical thalamic changes evident in 5/14  after initial neurological presentation. No patient had been administered levodopa prior to CSF sampling. Thirteen of these 15 patients had CSF neurotransmitter abnormalities . Three of 15 patients (P13–P15) had a suspected or proven mitochondrial disorder, with CSF showing high neopterin levels in 2/3. Additionally, 3/15 patients  had a suspected or proven central nervous system (CNS) infection, with elevated neopterin in all three cases. Overall, CSF neopterin was elevated in 6/11 cases, where data were available. Two of 15 patients had a raised CSF HVA, one of whom was on dopaminergic therapy, whilst 4/15 had low HVA levels. 5-HIAA levels were abnormal in 5/14 cases . CSF 5-MTHF levels, undertaken in 9/15 patients, were low in one patient (P8) , whereas neopterin levels were similarly elevated in both cohorts (p = 0.68)  Table  CSF HVA POLG mutations. POLG disease has previously been associated with a wide range of movement disorders. In adults and adolescents, ataxia, dystonia, chorea and myoclonus have been described but, overall, parkinsonism seems to be the most commonly encountered motor phenotype  had an initial presentation of status epilepticus and the majority (12/15) had neurotransmitter analysis performed during a period of increased seizure burden, often whilst in the PICU. Notably, children who presented with a movement disorder in the absence of seizures  had fewer neurotransmitter abnormalities than the POLG status epilepticus group , where the HVA:5-HIAA ratios are commonly above 5  and more markedly reduced in another (D13). Cerebral folate deficiency is reported in several types of mitochondrial disease  in a larger cohort of POLG patients. The mechanisms underpinning movement disorders in POLG disease are not fully understood; however, our report indicates that aberrant dopamine and serotonin metabolism may play a role.In conclusion, hyperkinetic movement disorders are frequently encountered in children with ESM 1(DOCX 399 kb)Below is the link to the electronic supplementary material.


Marginal Structural Models (MSMs) are novel methods to estimate causal effect in epidemiology by using Inverse Probability of Treatment Weighting (IPTW) and Stabilized Weight to reduce confounding effects. This study aimed to estimate causal effect of donor source on renal transplantation survival.In this cohort study, 1354 transplanted patients with a median 42.55 months follow-up in Namazee Hospital Transplantation Center, Shiraz from Mar 1999 to Mar 2009, were included to use marginal structural Cox regression, binomial logistic regression model to estimate causal effect of donor source on the survival of renal transplantation. IPTW and stabilized inverse probability of treatment weighting are used as weights.P=0.89) and , respectively. Using a marginal structural Cox regression model and by stabilized weight, the hazard ratios for live-unrelated donor and cadaveric donor were  and , respectively. There was no difference between estimated effect size from marginal structural Cox regression, marginal structural logistic regression, and marginal structural Weibull regression model.The un-weighted (crude) hazard ratios for live unrelated donor and deceased donor in comparison to live related donor as reference group was  has a better survival of renal transplantation than cadaveric donor. Renal transplantation is the best therapy for end-stage renal disease (ESRD) and in comparison to peritoneal dialysis and hemodialysis is of a better long-term survival and better quality of life that are also cost-effective for public health –5. The lGrafts and patients survival rates are favorable for those who receive kidneys from living kidney donors (related or unrelated)  but due The objectives of this research were to determine the causal effect of donor source on graft survival rate using stabilized inverse probability weighted estimators of marginal structural models.Study design has been reported in details in previously published papers , 4. ThisOverall, 1354 transplanted patients in this 10-yr period (with 60650.8 person months of follow up) were included in the study. Of those, 68 individuals were excluded due to lack of follow-up; remained participants were followed until Mar 2009 with a median 42.55 months follow-up.Potential confounder variables were donor’s and recipient’s age, donor’s and recipient’s gender , donor’s and recipient’s blood groups (ABO and same or different), recipient’s immunosuppressive drug regimen, recipient’s marital status, underlying cause of ESRD, recipient’s weight, creatinine level at discharge and the duration of dialysis therapy before transplantation.The exposure of interest was donor source of graft. Donor source was categorized into three groups of; live related donor, live unrelated donor and cadaveric donor (or deceased donor).All required data were collected through reviewing of patients’ hospital records. The organ survival and every patient’s needs to regular dialysis were assessed and determined by nephrologists, follow-up clinics, and related institutions such as “Management Center for Transplantation and Special Diseases” and “Renal Patients Support Society”.A marginal structural Cox regression model was used to measure the effect of donor source on graft survival rate quantified by HR (95% CI). A polytomous logistic regression model was used to estimate the probabilities of different categories of donor source. The reciprocal value of these probabilities, stabilized by multiplying the marginal probability of the exposure, they was used as stabilized weights in an unadjusted Cox regression analysis , 20–22.In the modeling of exposure (donor source), fractional polynomials of Royston  were useP-value of less than 0.05 was considered to be statistically significant.Proportionality of hazard that is the stability of effect of a covariate over time, is one of the most important assumptions in Cox regression model . This asThe results obtained from the preliminary analysis of baseline characteristics are presented in The mean (SD) weight of donors and recipients were 65.31 (11.98) and 57.73 (15.02) kg, respectively.Overall, 403 cases (29.76%) were live related donor, 440 cases (32.5%) were live unrelated donor, and 511 cases (37.74%) were deceased donor. In 53.91% of patients, cause of end-stage renal disease (ESRD) was not known, but among cases with known cause, glomerulonephritis was more frequent (12.78%).The rate of graft rejection was 1.76 (CI 95%: 1.46–2.13) cases per 1000 person-month follow-up. The rate for live related donor, live unrelated donor, and for deceased donor was 1.09 (CI 95%: 0.73–1.61), 1.17 (CI 95%:0.78–1.76), and 3.24 (CI 95%: 2.51–4.18) cases per 1000 person-month follow up respectively.PH assumption for Cox regression model was tested for donor source. The stabilized inverse probability weights had a mean of 1.01 SD= 2.26); the mean is about 1 as it should be, and SD is greater than the mean because the distribution of the weights was skewed to the right. In a sensitivity analysis, we truncated the weights at the first and 99th percentiles, but truncation does not change substantially the results  and 2..26; the P=0.89), and 2.69  respectively (P=0.16) and 3.72  respectively. The inverse probability-of-exposure-weighted hazard ratio from marginal Cox regression model for unrelated donor and in deceased donor was 1.08  and 3.63  respectively. Hazard of graft rejection in unrelated and deceased donor is 1.08 and 3.63 times more than related donor.Interestingly, the unadjusted hazard ratio for renal transplantation among unrelated donors and deceased donor compared to the related donor was 1.03 .Compared to the unrelated donor, the inverse probability-of-exposure-weighted hazard ratio in deceased donor was 3.35 , the outcome of renal transplantation has improved in recent years. The outcome has improved for both living (related and unrelated) donor and deceased donor recipients, but as our findings showed, living donor recipients have a better graft survival rate . A betteOur results showed that deceased donor has the worst graft survival rate. Cold ischemia time play an important role in the graft survival rate for deceased donor renal transplantation. “The association between extended cold ischemia time and inferior renal allograft outcomes are shown in deceased donor transplantation” . A high In a study among more than 150000 recipients transplanted from 1987 to 1997 at transplant centers, the best renal graft survival rate could be found among living donors whereas the worst survival rate could be found among cadaver donors . This diThe marginal structural model estimates the net effect of donor source on graft survival rate. According to our findings, marginal structural model is better than other adjustment methods such as stratification; nevertheless, this model has restrictions, such as the assumption of no unmeasured confounders .The main finding of this paper is that the marginal hazard ratio for unrelated donor was not significantly different than the ratio for related donors, but hazard ratio in deceased donor was 3.63 times higher than of ratio for related donors. In addition, the marginal hazard ratio for deceased donors was 3.35 higher than the ratio for unrelated donors. The live donors (related or unrelated) have a better survival of renal transplantation than deceased donors.As one of the main limitations of the study, we conducted a retrospective cohort study and in few cases we used registered phone number to ascertain the outcome. Large prospective cohort studies are recommended to confirm the results of our study.The live donor (related or unrelated) has a better survival of renal transplantation than cadaveric donor. There is no difference between related and unrelated donor source hazard; but hazard for cadaveric donor was 3.63 times of hazard for related donor and 3.34 times of it for unrelated donor.Ethical issues  have been completely observed by the authors.


Torkildsen operation is a ventriculo-cisternal shunt that diverts the cerebrospinal fluid flow from one of the lateral ventricles, via a ventricular catheter, to the cisterna magna of the posterior fossa. It is the first described operation of CSF diversion for the treatment of hydrocephalus  (Scarff,We present the case of a premature infant who presented with post hemorrhagic hydrocephalus who underwent several failed CSF diversion surgeries before Torkildsen shunt procedure. The patient overcame the irregularities of CSF circulation that were observed before the operation and his post-operative course was uneventful, in terms of CSF –related complications.We summarize the historical data and the technical aspects of the procedure, and we present a brief literature review of the indications and limitations associated with it.Torkildsen shunt can be effective in selected patients with hydrocephalus even in the modern era of computed tomography and magnetic resonance imaging. the procedure enables one to avoid a standard ventriculoperitoneal shunt, especially in cases it is not technically feasible. Arne Torkildsen, a Norwegian neurosurgeon, and reported in 1939 . It is cAlthough it seems that initially had been widely accepted as efficient surgical procedure for treating hydrocephalus, introducement of the VA shunt and later of the VP shunt procedure, was the cause that it was largely abandoned.In this paper, we report our experience using this historical procedure to a case of infantile hydrocephalus that seemed to be untreatable with modern therapeutic strategies.2We refer to a premature infant, 26 weeks of gestational age at birth, who was born via cesarean incision with sciatic projection. Amniocentesis was executed three days before birth in order to investigate the possibility of short extremities stature, after which mother suffered placental abruption. The infant’s gestational weight was 760 g and he was hospitalized in an incubator.Two days after birth, as a screening test, an initial ultrasound via the anterior fontanelle was performed, which verified the presence of intraventricular hemorrhage grade IV with accompanying porencephalic cysts that were communicating with the ventricular system. Clinical and neurological examination recognized only an upcoming fullness of the anterior fontanelle and a left posterior plagiocephaly.From the patient medical history, we have to mention that he suffered from necrotic enterocolitis and he was operated on and 10 cm of the small intestine, at a distance of 15 cm from the ileocaecal valve were removed. Besides that, a colostomy on the right side was performed. Necrotic enterocolitis and respiratory insufficiency syndrome due to the prematurity of the infant constituted the medical co-morbidities of our patient.Based on this findings, an initial CT scan was performed which verified the aforementioned finding, and an external ventricular drainage was inserted through the frontal horn of the right lateral ventricle, in order to manage the post hemorrhagic ventricular dilation.A few days after the operation, a CSF leak from the surgical wound was observed and the ventricular catheter was removed, the wound was revaluated and a new EVD was inserted due to fullness of the anterior fontanelle and new onset opisthotonos.An MRI was performed to evaluate the course of the post hemorrhagic ventricular dilation∙ it revealed significant dilation of the 3rd, 4th, and lateral ventricles bilaterally, as well as dilation of the anterior subarachnoid space, a finding that could be explained based on the age of the infant. Concurrently, hypoplasia of the pons and cerebellum were identified , Fig. A3Two weeks after, two months after birth, the EVD was removed and a VP shunt was introduced .Three weeks after the operation, the abdomen of the infant appeared to distend gradually and signs of increased intracranial pressure appeared, namely reappearance of opisthotonos and fullness of the anterior fontanelle. Abdominal ultrasound revealed significant amount of free floating, non-encapsulated fluid and compatible with dysfunction of the peritoneal segment of the VP shunt. Taking into consideration the medical history of the patient, it was attributed to decreased capacity of the peritoneal cavity to absorb the CSF, in the context of immaturity.After 10 days, another CSF leak was noticed across the surgical wound and the VP shunt was removed to avoid the possibility of infection. The wound edges were incised; skin and subcutaneous tissues were dissected and sutured again at healthy margins. A few days after, the anterior fontanelle was tense and ultrasound examination revealed that the ventricular size was increased, so a new EVD was inserted.A new MRI was executed, in order to monitor the radiological appearance of the ventricular system of the infant. We notice that the posterior fossa of the patient is relatively small, with concurrent hypoplasia of the pons and cerebellum. We noticed that the 4th ventricle and its foramens were dilated. The white matter of the cerebral hemispheres was diminished in size with accompanying dilation of the subarachnoid space in the vicinity of the cerebral hemispheres and the posterior fossa.th ventricles and also on the surface of the cerebellar hemispheres. These were of low signal intensity on T2 GRE sequence and are attributable to hemorrhage of the germinal matrix layer. Also, we noticed low signal intensity areas at the tentorium cerebelli and falx cerebri on T2 GRE sequence, attributed to subarachnoid hemorrhage . After three weeks, we realized that abdominal distension reappeared. Ultrasound of the abdomen verified that there was an excessive CSF collection intraperitoneally, which should be correlated with the inability to absorb the accumulating CSF. The neurological examination of the infant revealed hypotonia of the trunk, hypertonia of the lower extremities and opisthotonos.We externalized our VP shunt just as a temporary measure, until an ultimate operation was perform to address the abnormalities of CSF circulation. One alternative would be the insertion of a ventriculo-atrial shunt.Unfortunately, Hickman catheters were inserted to both internal cerebral veins, at different time periods, due to long hospital stay and prematurity of the infant. When an ultrasound of the internal cerebral veins was performed, to evaluate their patency, we identified that no blood flow could be documented to both of them, although a collateral venous network was visible.Consideration also could be given to performing a ventriculo-pleural shunt. Unfortunately, it is well known that due to connective tissue adhesions in the pleural cavity, repeated operations were performed to keep the tube shunting. Therefore, this procedure was discontinued and we were not convinced that it was the best option for our patient.Based on that evidence, we decided to perform a Torkildsen operation to our patient∙ size of the subarachnoid space at the cisterna magna and upper cervical level, and presence of any vascular anomalies or abnormal flow void were checked beforehand. We decided to follow the previously described therapeutic plan for our patient as in most of premature infants the absorptive capacity of the peritoneum is gradually regained and we can commonly resolve the problem of hydrocephalus by inserting a ventriculoperitoneal shunt. As soon as it was obvious that the peritoneum was unable to absorb the excess of CSF, we were convinced that Torkildsen shunt should be used as a salvage procedure.A bur hole at the right parieto-occipital region was performed and a ventricular catheter was inserted into the ipsilateral occipital horn. A limited suboccipital craniectomy was performed, the cisterna magna was identified, and the previously inserted ventricular catheter was subcutaneously guided in the vicinity of the cisterna magna. It was subsequently opened and the end of the ventricular catheter was inserted within it and secured for safety .Immediately after the operation, the episodes of CSF leak were stopped, opisthotonos disappeared and the anterior fontanelle was never under tension. We performed a postoperative CT scan at two weeks’ time, which verified the correct positioning of our catheter and a small reduction in the dimension of the ventricular system , Fig. D33Arne Torkildsen was a pioneering Norwegian neurosurgeon best known for developing ventriculocisternostomy (VCS), the first clinically successful procedure for shunting of CSF. It is widely accepted that Torkildsen developed a procedure that received international recognition and was successfully and safely executed by many neurosurgeons at a period of time when no other safe and effective treatment existed for patients with hydrocephalus . Indeed,The surgical procedure for the Torkildsen shunt is simple, but could be technically more demanding than VP shunt. The Torkildsen shunt is a simple tubing between two CSF spaces to bridge them. It does not require any special shunt valve to control draining pressure. Patency of the shunt is unable to confirm, demonstrated mild to moderately decrease in ventricular size within a few weeks after the surgery strongly support the Torkildsen shunt played a key role in improvement of clinical findings. One advantage of the Torkildsen shunt is that it enables drainage of CSF through a physiologic pathway, thus avoiding the complications of shunting CSF into the peritoneal cavity.The Torkildsen shunt can be withdrawn by body growth like a VP shunt. It should be reminded that the distance between head and neck is quite shorter than that between head and abdomen. The distance does increase as a child grows but the influence of body growth to the Torkildsen shunt would be less significant than that happens in children with VP shunt. If the Torkildsen shunt is installed in infants or young children, the shunt withdrawal which requires shunt revision would occur in the future.4Although this operation is largely abandoned in modern era, we can support the concept that the Torkildsen shunt is a valuable salvage surgical procedure in definite cases of hydrocephalus related to obstruction within the ventricular system . More exAll authors state that we do not have any financial and personal relationships with other people or organisations that could inappropriately influence (bias) our work.There are no sources of funding for our research. Also we declare that no sponsors have any role to our study.Our study is not considered to be Research study, so ethical approval is not applicable.We state that we have parental, written and signed, consent on behalf of the patient to publish this case report.•Concept of design.•Data collection.•Analysis and interpretation of data.•Writing and final approval of the manuscript.Dimitrios Panagopoulos, Corresponding Author, contributed to the paper as follows:•Concept of design.•Data collection.•Writing and final approval of the manuscript.Marios Themistocleous contributed to the paper as follows:•Data collection.•Analysis and interpretation of data.•Writing and final approval of the manuscript.Andreas Mitsios contributed to the paper as follows:•Data collection.•Analysis and interpretation of data.•Writing and final approval of the manuscript.Katerina Apostolopoulou contributed to the paper as follows:•Analysis and interpretation of data.•Writing and final approval of the manuscript.•Data collection.Georgios Sfakianos contributed to the paper as follows:Our case report did not did not involve human participants and it does not have to be registered in a publicly accessible database (This section is not applicable to our manuscript).I, Dimitrios Panagopoulos, the Corresponding Author of this manuscript, accept full responsibility for the work and the conduct of the study, had access to the data, and controlled the decision to publish.Not commissioned, externally peer reviewed.


Granulocyte-monocyte progenitor (GMP) cells play a vital role in the immune system by maturing into a variety of white blood cells, including neutrophils and macrophages, depending on exposure to cytokines such as various types of colony stimulating factors (CSF). Granulocyte-CSF (G-CSF) induces granulopoiesis and macrophage-CSF (M-CSF) induces monopoiesis, while granulocyte/macrophage-CSF (GM-CSF) favors monocytic and granulocytic differentiation at low and high concentrations, respectively. Although these differentiation pathways are well documented, the mechanisms behind the diverse behavioral responses of GMP cells to CSFs are not well understood. In this paper, we propose a mechanism of interacting CSF-receptors and transcription factors that control GMP differentiation, convert the mechanism into a set of differential equations, and explore the properties of this mathematical model using dynamical systems theory. Our model reproduces numerous experimental observations of GMP cell differentiation in response to varying dosages of G-CSF, M-CSF, and GM-CSF. In particular, we are able to reproduce the concentration-dependent behavior of GM-CSF induced differentiation, and propose a mechanism driving this behavior. In addition, we explore the differentiation of a fourth phenotype, monocytic myeloid-derived suppressor cells (M-MDSC), showing how they might fit into the classical pathways of GMP differentiation and how progenitor cells can be primed for M-MDSC differentiation. Finally, we use the model to make novel predictions that can be explored by future experimental studies. Hematopoietic stem cells differentiate into blood cells  in a finely regulated process called hematopoiesis. In this branching process, each branch point represents a cell differentiating into one of two alternative lineages. Stimulatory factors, such as cytokines, induce differentiation into one lineage over another, and cross-antagonistic transcription factors maintain commitment to the chosen lineage , 2. In tSPI1 gene in humans), early growth response proteins 1 and 2 (Egr-1 and Egr-2), interferon-regulatory factor 8 (IRF8), M-CSF receptor (M-CSFR), and GM-CSFR  and study their properties by dynamical systems theory. We first explore the polarization of GMP cells resulting from G-CSF and M-CSF signals. Next we explore the dynamics of the system in response to GM-CSF and propose a mechanism driving the complex behavior observed in GM-CSF experiments. We also explore how M-MDSCs may fit into this differentiation scheme, including the stability of the state and the nature of the phenotype itself. Finally, we evaluate the system's response to cytokine combinations and provide insight into the spectrum of behaviors induced by signaling crosstalk.PU.1 and C/EBPα are thought to be master regulators of myelopoiesis, as C/EBPα favors granulopoiesis and PU.1 favors monopoiesis , 34. In C/EBPβ gene was knocked in to the C/EBPα locus, no significant changes in hematopoiesis or gene expression were ascertained. Since these proteins have highly conserved C-terminal dimerization- and DNA-binding domains, it is reasonable to assume that they bind to the same promoter sites  has multiple potential C/EBP binding sites, and that C/EBPα can induce PU.1 expression by directly binding to the promoter to activate transcription  transduce cytokine signals to regulate transcription factor activity Figure . These tpromoter , 51. Thupromoter , 50, 52.promoter .IRF8 gene , 0 ≤ Xi(t) ≤ 1. The function Wi(Xj) accounts for all interactions within the network that directly affect the rate of change of Xi such that ωi, j quantifies the direction and strength of the affect that protein j exerts on protein i. Negative values represent inhibition while positive values represent activation. The time scale for the rate of change of Xi(t) is determined by 1/ρi. The value of Xi when it is not receiving stimulus from any Xj. One unit of the time variable, t, is roughly 2 h in our simulations.To convert the interaction diagram in Figure W, with steepness determined by the parameter σ. Many biological phenomena such as phosphorylation cascades and transcriptional regulation are characterized by sigmoidal response curves. Our sigmoidal function H(W) captures such behavior in a very convenient way. As an example, we show the case of C/EBP activity:The nonlinear function, T, and the concentration of the “free” form of the protein, [C/EBP]F. C/EBP is considered free when it's not bound to IRF8, therefore [C/EBP]F represents the active portion of [C/EBP]T, whereF + [IRF8]F ⇋ [C/EBP:IRF8] is at equilibrium. Thus,T as a function of [C/EBP]F and [IRF8]T:F as a function of [C/EBP]T and [IRF8]TT. In this case, the concentration of the receptor:cytokine complex, [R:L], is given by the function:Kd is the dissociation constant of the receptor:cytokine complex. The cytokine concentrations are “inputs” to the model, the total receptor concentrations are dynamic variables of the model.Note that this ODE distinguishes between two concentrations of C/EBP: its “total” concentration, [C/EBP]Parameter values were hand-tuned so that the behavior of the system in response to cytokines aligns with experimental observations. For a more detailed discussion on parameter tuning, a table of parameter values and the complete set of equations constituting our mathematical model, see the All quantitative simulations were computed using the deterministic ODE solver, ode45, in MATLAB. To simulate a population of GMP cells, we generate a set of cells with stochastically varying initial conditions, taking the steady state concentrations of all variables in a naïve GMP cell (with no cytokine stimulation) and varying each initial concentration by a random factor drawn from a normal distribution with mean = 1 and standard deviation = 0.2.T. To do so, we demand that the other six variables are in pseudo-steady state by setting the right-hand-sides of those six ODEs = 0. Looking at the structure of those six ODEs , these concentrations are functions of the stimulus parameters, [GMCSF] and [GCSF], and of the pseudo-steady state values of [GMCSFR]T and [GCSFR]T, which in turn are functions, respectively, of [PU.1] and [C/EBP]F and of [PU.1], [C/EBP]F, and [Gfi1]. From Equation (7), we can express [C/EBP]T as a function of [C/EBP]F and [IRF8]T. By this line of reasoning, we can reduce the 8-dimensional system of ODEs to three variables, [PU.1], [C/EBP]T, and [Gfi1].Although our model consists of eight nonlinear ODEs, we characterize its behavior in a pseudo-phase plane spanned by only two variables: [PU.1] and [C/EBP]s in the , we see T. We cannot provide an explicit representation of this function but we can find it by an iterative numerical approach. For any combination of values of [C/EBP]F and [PU.1], we subdivide the interval [Gfi1] ∈  into 100 subintervals of length 0.01 and compute the value of d[Gfi1]/dt at the ends of each segment  value(s) of [Gfi1] for the given pair of [PU.1] and [C/EBP]F values. We solve for [Gfi1]pss with a tolerance of 10−9 .To reduce this system to two variables, we must express the pseudo-steady state concentration of Gfi-1 as a function of [PU.1] and [C/EBP]F, we calculate solutions for the PU.1 nullcline over a range of [C/EBP]F values. Stringing these solutions together, we acquire the desired nullcline. We can find the [PU.1] nullcline solutions for any [C/EBP]F value, as we did for finding [Gfi1]pss, by generating a subdivision of [PU.1] values between 0 and 1. For each [PU.1] value we solve for [Gfi1]pss as above. As mentioned previously, given a set of [C/EBP]F, [PU.1], and [Gfi1] concentrations we can numerically solve for every other variable's steady state solution. Thus, we are able to calculate d[PU.1]/dt, and then to find the nullcline for [PU.1], where d[PU.1]/dt = 0. In this case, we use a tolerance of 10−7 rather than 10−9. Similarly, we solve for the [C/EBP]F nullcline using the same method with a tolerance of 10−8.  Once we have acquired our pseudo-nullclines for the [C/EBP]F–[PU.1] phase plane, we convert [C/EBP]F to [C/EBP]T and plot the nullclines in the [C/EBP]T–[PU.1] phase plane. While unconventional and computationally taxing, our methods yield smoother, more accurate curves than we were able to obtain using XPPAUT, and we could do all our computations within MATLAB rather than switching between MATLAB and some other bifurcation software.Since each point along the PU.1 pseudo-nullcline has a fixed value of [C/EBP]F and [PU.1] that minimize the objective function ((d[C/EBP]F/dt)2 + (d[PU.1]/dt)2)1/2 given that all other variables are at steady state (To find [Gfi-1]ss we use the same iterative technique specified earlier). Once a solution for [C/EBP]F and [PU.1] is found, we use that solution as an initial guess for the next step  and search for the new solution. Close to saddle-node bifurcation points, where the derivative of [PU.1]ss with respect to the bifurcation parameter becomes very large, we hold the bifurcation parameter constant, and take a small step in the value of [PU.1]. We then use fminsearch to find the solution of [C/EBP]F and the bifurcation parameter at that given [PU.1] value. To evaluate stability, we use a typical eigenvalue analysis.Bifurcation diagrams were generated in a similar, non-conventional fashion. Bifurcation diagrams are based on steady state solutions of all 8 variables. Given a set of approximate steady state values, we use MATLAB's fminsearch function to find values of [C/EBP]To construct heat maps, we simulate 500 stochastically generated cells, using the method specified earlier, under each cytokine condition. Using the differentiated population's ratios, each pixel was assigned an RGB value determined by the following equation:https://github.com/bronsonweston/GMP-Modeling, which includes all algorithms previously mentioned and provides a script, “FigureGenerating.m,” to easily reproduce any of our results. This code can also be used as an example script to conduct alternative simulations not explored in this study.For those interested in exploring our model further, we provide two resources for utilizing our model and conducting simulations of your own. The supplementary code provides an ODE file, a stochastic simulation function and a user friendly MATLAB script, “MainScript.m,” to produce time course simulations and figures as well as stochastic simulations under user specified conditions. A more extensive resource is provided online at As with any model, we have made several simplifying assumptions to avoid unnecessary complexity. First, we ignore autocrine feedback loops of the GMP lineage. We maintain constant cytokine concentration(s) in order to evaluate the effects of the stimulus input, rather than accounting for how the cell may change external conditions. We assume that the cytokine production of an individual cell has a negligible impact on the initial decision-making process of GMP differentiation. Additionally, we assume that all protein isoforms function similarly in the context of our network. For example G-CSFR has seven isoforms, four of which are involved in granulopoiesis . We assuH(Wi) = 1/(1+eWi−σ), adequately capture the cumulative non-linear effects of these molecular mechanisms.The GMP differentiation network has many mechanisms for generating sigmoidal nonlinearities, such as dimerization of receptor subunits and cooperativity of transcription factor binding to DNA promoter sites. We assume that our sigmoidal functions, R = ρTF/10). It is hard to know for sure what timescales these proteins are functioning on. While transcription factors are often functional immediately after synthesis, receptors must be trafficked to the periphery of the cell, diffuse within the cell membrane, assemble with other subunits, and bind to cytokines before a signal can be transduced back into the cell, after which the signal itself may take some time to get to its downstream target. At the very least, we would expect a significant time delay between production of the receptor and its impact on the expression of down-stream genes. For these reasons, we justify using a slower timescale for receptors than for transcription factors.In addition, we assume that all transcription factors function at the same time scale, and all receptors function at the same, ten-fold slower time scale . The purpose of their model was to demonstrate that mutual antagonism between Gfi-1 and Egr can be a mechanism for inducing commitment of the monocytic and granulocytic lineages. While achieving its intended purpose, the model's forced symmetry and its neglect of critical regulatory mechanisms limit its predictive capacity and its ability to explain more complex phenomena of myelopoiesis. We improve upon the Laslo model with new, biologically relevant interactions, including a fifth transcription factor, IRF8, as well as new signaling pathways, CSF receptors, and regulatory mechanisms for these receptors. These additional interactions break the symmetry of Laslo's model but extend the range of behaviors we can model. Rather than modifying the equations of Laslo's model, we derive a new set of equations based on our standard-component modeling approach. Justification for these changes can be found in the methods section.Our motif for GMP cell differentiation is depicted in Figure d[PU.1]/dt = 0, and the C/EBP nullcline is the locus of points where d[C/EBP]T/dt = 0. Where these nullclines intersect lie steady states of the full eight-dimensional set of ODEs. With no cytokine stimulation, these nullclines intersect five times to form three stable steady states (nodes) and two saddle points. The stable steady state with low levels of both C/EBP and PU.1 corresponds to a naïve GMP cell, whereas the other two stable steady states correspond to granulocyte and monocyte progenitor cells, depending on whether C/EBP or PU.1 is elevated, respectively. For the case of no cytokine stimulation, the GMP cell will sit indefinitely in the naïve state.On the phase plane Figure , we plotlow, C/EBPlow), the granulocyte progenitor state as , and the monocyte state as . It is also important to note that, while PU.1 expression is elevated in neutrophils, PU.1 remains low in early granulocyte progenitors  and granulocyte progenitor  states remain, Figures We begin our investigation of external signaling by exploring how nullclines shift in response to M-CSF stimulation. Comparing Figures T ≈ 0.15 (the naïve GMP cell), whereas, for [MCSF] > 0.5, there are only two stable steady states, corresponding to monocytes and granulocyte progenitors. Thus, M-CSF induced monopoiesis is irreversible, as the monocyte state remains stable even if the M-CSF concentration is decreased to zero after the transition is made  phase plane Figure  more draThe bifurcation diagram Figure  is in agWhile experiments suggest that G-CSF induces granulopoiesis, the dynamical changes during this process of differentiation are not well documented. Our model Figure  suggestsInterestingly, when comparing the differentiation time of M-CSF induced monopoiesis and G-CSF induced granulopoiesis Figures , we findAn important question we wish to address in this paper is: what possible mechanism can explain the concentration-dependent behavior of GM-CSF induced differentiation? GM-CSF signals upregulate C/EBP, which in turn promotes PU.1 and Gfi-1 transcription. However, Gfi-1 and PU.1 are mutually antagonistic, and PU.1 suppresses C/EBP activity via IRF8 Figure . Thus, CAt low levels of GM-CSF Figures , both C/At higher concentrations of GM-CSF Figures , C/EBP iWe find that, even though the differentiation times of GM-CSF and G-CSF induced granulopoiesis are very similar Figure , during Intriguingly, our model suggests that GM-CSFR expression decreases slightly after granulocytic commitment, and remains at lower levels than the monocytic lineage. Experimental evidence shows that, indeed, GM-CSFR expression is higher in monocytes than in granulocytes, despite the fact that higher concentrations of GM-CSF favor granulopoiesis over monopoiesis , 74, 75.Our results suggest that in both monopoiesis and granulopoiesis the GM-CSFR signaling capacity changes significantly after the cell has already committed to one lineage over another. If this is true, then the high concentration of GM-CSFR in monocytes relative to granulocytes must serve an alternative function than lineage commitment. One possibility is that GM-CSFR signaling, or lack thereof, is crucial for regulating proteins not accounted for by this model. Alternatively, GM-CSF signaling may function to upregulate C/EBP in the monocytic lineage, since it is necessary for AP-1 to bind with C/EBP to promote monocytic genes . It is ahigh C/EBPhigh . Naturally, the question arises, is there a myeloid cell that fits this description? Indeed, M-MDSCs fit this profile, as these monocytic cells presumably express high levels of PU.1 and are known to highly express C/EBPβ . As one might expect, our model predicts that, when paired with low levels of GM/M-CSF, G-CSF can induce GMP cells to favor granulopoiesis over monopoiesis Figure . Howeverin vivo or in growth serums. Regardless, this calculation suggests that M-CSF paired with GM-CSF makes for a much stronger inducer of the M-MDSC phenotype than either cytokine alone.It has been well documented that both M-CSF and GM-CSF can contribute to M-MDSC development , 83. SinFinally, we evaluate GMP behavior when M-CSF is paired with equal concentrations of G-CSF and GM-CSF (GM/G-CSF) Figure . We findIn consideration of the crucial roles played by cells of the GMP lineage in human health and disease, we have proposed a molecular regulatory network for the differentiation of GMP cells Figure , based oSPI1 gene (encoding PU.1) to promote granulopoiesis. Alternatively, our model predicts that C/EBP has a positive impact on PU.1 in GM-CSF induced monopoiesis, as a result of a slower increase in C/EBP, allowing PU.1 enough time to upregulate itself and establish dominance. Second: PU.1's indirect antagonism of C/EBP and Gfi-1 is essential to commit the cell toward monopoiesis. Third: GM-CSFR signaling forms positive feedback loops with PU.1 and C/EBP. When stimulated, GM-CSFR transmits a signal to C/EBP to increase both C/EBP and PU.1 expression. These proteins, in turn, upregulate GM-CSFR resulting in a stronger GM-CSF signal, which results in even greater stimulation of C/EBP.Investigating the concentration-dependent response of GMP cells to GM-CSF, we uncovered three main features of the response. First: the dual regulatory effects of C/EBP on PU.1; C/EBP induces PU.1 directly by promoter stimulation and inhibits PU.1 indirectly through stimulation of Gfi-1 –43. The These feedback loops create a sensitive, switch-like response of gene expression to GM-CSFR stimulation. We find that the lower the GM-CSF concentration is the longer it takes for the switch to kick in. In GM-CSF-induced granulopoiesis, the switch kicks in early, to allow sufficient upregulation of C/EBP and Gfi-1. In GM-CSF-induced monopoiesis, the switch is delayed, to allow PU.1 to upregulate itself and repress C/EBP and Gfi-1. In this way, we propose that these three dynamic features of the control system work synergistically to produce the unique behaviors associated with GM-CSF-induced differentiation.Given that our model successfully captures the endpoints of GMP differentiation induced by G-, M-, and GM-CSF, we propose that our model can also offer significant insights into the different temporal patterns of protein concentrations during the differentiation processes. For example, GM-CSF induced monopoiesis exhibits a significant spike in C/EBP and Gfi-1 concentrations in its early stages, followed by suppression of both proteins, whereas M-CSF induced monopoiesis does not exhibit such a spike. It is possible that these differences could influence downstream transcription factors not accounted for in our model, perhaps resulting in different subtypes of monocytes.  Nonetheless, our model predicts that the final concentration of C/EBP in monocytes is dependent on the signaling strength of GM-CSF . We acknowledge that in vivo other cytokines that are similar to GM-CSF (such as IL-3) may play comparable roles in M-MDSC differentiation . For example, direct positive feedback loops of C/EBP and PU.1 are crucial for switch-like behavior and are required for the stability of the granulocyte and monocyte phenotypes, respectively. Additional positive feedbacks loops exist within the mutually antagonistic architecture of the network. As C/EBP can antagonize PU.1 through Gfi-1, PU.1 forms two positive feedback loops by inhibiting C/EBP through IRF8 and by inhibiting Gfi-1 through Egr-2. These positive feedback loops are crucial to the stability of the monocytic phenotype. In contrast, C/EBP forms a positive feedback loop by activating Gfi-1, which in turn prevents PU.1 from upregulating IRF8 and inhibiting C/EBP. Thus C/EBP can exist at high concentrations by suppressing PU.1 through this positive feedback loop. Furthermore, Gfi-1 has a positive feedback mechanism by inhibiting PU.1 and Egr-2, which in turn would inhibit Gfi-1. These feedback loops are critical to the irreversibility of the granulocyte phenotype. More positive feedback mechanisms exist between receptors and transcription factors. For example, activated M-CSFR stimulates PU.1 and PU.1 stimulates the expression of M-CSFR. These types of positive feedback loops make the system more responsive to cytokine stimuli. Finally, C/EBP forms a negative feedback loop by activating PU.1 which in turn activates IRF8. This negative feedback loop is necessary for GM-CSF induced monopoiesis, as C/EBP must be suppressed, but only after it has activated PU.1.Ultimately, the behavior of the model is a consequence of the network topology, i.e., multiple feedback and feedforward loops in the reaction mechanism Figure  and the Feedforward loops also make significant contributions to the behavior of the system. For instance, GM-CSFR activates C/EBP both directly and indirectly (by inhibiting IRF8), thus forming a coherent-feedforward loop. The inhibition of IRF8 by GM-CSF is important for GM-CSF induced differentiation of GMPs into M-MDSCs . Another example is the incoherent-feedforward loop by which C/EBP activates PU.1 directly and inhibits PU.1 indirectly through Gfi-1. This incoherent-feedforward loop is crucial to the concentration dependent nature of GM-CSF induced differentiation, as we discussed previously.While our model makes several intriguing predictions, we acknowledge that the model neglects many genes and proteins that play important roles in hematopoiesis. Therefore, in interpreting our models results, we must be aware of its limitations. We designed the model specifically to capture the initial decision-making stages of GMP differentiation, rather than the terminal stages of granulopoiesis and monopoiesis. We propose that transcription factors downstream of our network will play large roles in the maturation of granulocyte progenitor and monocyte cells, but only subtly effect the initial dynamics of lineage commitment.Additionally, our model is limited to qualitative predictions. Although experiments often report quantitative measurements, it is impossible to compare these quantitative experimental results with our simulations for a variety of reasons. First of all, our calculations are made in dimensionless units, and the “real life” equivalent of 1 unit of M-CSF is not necessarily equivalent to 1 unit of G-CSF. Secondly, laboratory experiments typically utilize cytokine-enriched serum, with undefined serum components apparently necessary for cell survival and growth. These serum components are not accounted for in our model and may drastically impact how cells differentiate . FurtherWe have presented a novel model of GMP cell differentiation and explored the molecular control system's dynamics to provide insight into experimental observations and to make new predictions. We investigated the concentration-dependent nature of GM-CSF-induced differentiation, and proposed a mechanism that can explain its mysterious behavior. We explored the dynamics of CSF signaling crosstalk and found that, while G-CSF may encourage monopoiesis under some conditions, it is likely that M-CSF always has an inhibitory effect on granulopoiesis. Furthermore, our model demonstrates how both GMP cells and monocytes may differentiate into M-MDSCs, providing new insight into how this bizarre phenotype fits into classical GMP cell differentiation. We found that G-CSF, M-CSF, and GM-CSF may all favor M-MDSC development under different conditions. Moreover, we proposed that, among the CSFs, GM-CSF is the most potent inducer of this phenotype.As for any “model” of a cellular control system, our model has limitations and potential sources of inaccuracy. For example, our model is not suitable for making quantitative predictions or capturing terminal states of GMP differentiation. Nonetheless, we are confident that our results have utility, as the dynamic processes captured by our model align with numerous experimental observations. Therefore, we welcome experimental evaluation of any of the qualitative predictions we have made.JT, BW, and LL were all involved in the project's conceptual development. BW designed and ran all computational simulations. BW developed all figures and wrote the first draft of the manuscript. All authors contributed to revising this draft into final form.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Clostridium difficile infection (rCDI). The use of freeze-dried, encapsulated donor material for FMT (cap-FMT) allows for an easy route of administration and remains clinically effective in the majority of rCDI patients. We hypothesized that specific shifts in the microbiota in response to cap-FMT could predict clinical outcome. We further evaluated the degree of donor microbiota engraftment to determine the extent that donor transfer contributed to recovery.Fecal microbiota transplantation (FMT) is an effective treatment for recurrent P < 0.05) observed among patient’s pre-FMT samples was restored following cap-FMT. At 1 week post-FMT, community composition varied by clinical outcome (ANOSIM P < 0.001), with similar abundances among families  in responder and donor samples. Families that showed differential abundances by outcome (response vs. recurrence) from samples collected 7 days following cap-FMT were used to construct a regression tree-based model to predict recurrence. Results showed a training accuracy of 100% to predict recurrence and the model was 97% accurate against a test data set of samples collected 8–20 days following cap-FMT. Evaluation of the extent of engraftment using the Bayesian algorithm SourceTracker revealed that approximately 50% of the post-FMT communities of responders were attributable to donor microbiota, while an additional 20–30% of the communities were similar to a composite healthy microbiota consisting of all donor samples.In total, 89 patients were treated with 100 separate cap-FMTs, with a success rate (no rCDI 60 days post cap-FMT) of 80%. Among responders, the lower alpha diversity  contains supplementary material, which is available to authorized users. Clostridium difficile infection (CDI) remains a common cause of hospital and community-acquired infection  who enrolled in the University of Minnesota donor program, as described previously . Any sinperience . The priC. difficile infection, failed to respond to antibiotic therapies, and were C.-difficile-toxin-B-positive by PCR, at least 3 months prior to treatment [C. difficile toxin (toxin B PCR) through a 2-month clinical follow-up [Patient inclusion and exclusion criteria, as well as a subset of patient demographics, were described previously . Patientreatment . This woreatment . The FMTollow-up . PatientPatient samples were collected in single-use toilet hats and transferred by the patients to 30 ml polystyrene fecal specimen containers . Samples were stored in the patients’ freezers prior to transport to the laboratory on dry ice, where they were stored at − 20 to − 80 °C prior to DNA extraction.DNA was extracted from 250 to 500 mg of thawed donor and patient fecal samples using the DNeasy® PowerSoil® Kit , according to the manufacturer’s instructions. Fecal material from donor 42 was not available for DNA extraction and sequencing. The V5-V6 hypervariable regions of the 16S rRNA gene were amplified using the BSF784/1064R primer set . AmplifiSequence processing and analyses were performed using mothur software ver. 1.35.1 , and theTo determine potential structure-function relationships and to examine changes in abundances of traits, functional inferences were made using the PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) software ver. 1.1.3 . Analyse1, α2, and β Dirichlet hyperparameters . To assess how differences in donor pools might affect SourceTracker results, engraftment was calculated by (1) specifying all donor lots as a composite source, with all patient samples as a sink (termed “composite donor”); (2) specifying each donor lot as a unique source, with all patient samples as a sink (termed “specific donor”); and (3) separating samples based on donor lot, where a single donor lot was the source and only samples from patients who received that lot were sinks .SourceTracker ver. 0.9.8 was used to assess engraftment, as the percent of a patient (sink) community that could be attributed to a donor (source) community using a Bayesian inference approach . For allThe Shannon index of alpha diversity was calculated by using mothur software. Analysis of similarity (ANOSIM), to assess differences in community composition , and ordA regression tree approach was used to determine whether it was possible to build a predictive model for the detection of recurrence. In this approach, the exhaustive chi-squared automatic interaction detection (CHAID) procedure  was utilpost hoc test, and Spearman rank correlations were also performed using XLSTAT. All statistics were evaluated at α = 0.05 with Bonferroni correction for multiple comparisons, where applicable.Chi-squared tests, analysis of variance (ANOVA), followed by Tukey’s Additional file 1:Supplemental figures and tables. (DOCX 292 kb)Additional file 2:Batch file for sequencing processing. (TXT 12 kb)


Visfatin is a multifaceted protein that plays an important role in regulating a variety of physiological and pathological processes, including obesity, diabetes and cardiovascular disease. However, circulating visfatin levels in atherosclerosis plaque progression in patients with type 2 diabetes, or its association with the vascular territory affected remain unclear.We evaluated the relationship between visfatin levels and carotid or femoral artery atherosclerosis in Chinese patients with type 2 diabetes. Serum levels of visfatin were measured by enzyme-linked immunosorbent assay (ELISA) in 97 inpatients with type 2 diabetes. Carotid and/or femoral atherosclerotic plaques were detected by B-mode ultrasound.P = 0.0002]. Patients with carotid plaques showed higher visfatin levels than those with or without femoral plaques. Pearson’s correlation analysis showed that serum visfatin levels were positively correlated with waist circumference , waist-hip ratio , TG  and number of plaques . Logistic regression analysis showed that a higher serum visfatin level was an independent predictor for the presence of atherosclerotic plaques.Serum visfatin levels were elevated in the group with atherosclerotic plaques compared to the control group without plaques [0.68 (0.46–1.58) versus 0.45 (0.23–0.76) ng/mL, respectively, In conclusion, among patients with T2DM, serum visfatin levels were elevated in those with atherosclerotic plaques, especially in patients with carotid atherosclerotic plaques. Serum visfatin may serve as a predictor of atherosclerotic plaques in patients with T2DM.The online version of this article (10.1186/s13098-019-0455-5) contains supplementary material, which is available to authorized users. Cardiovascular disease is the leading cause of death in China , 2. AtheVisfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), or pre-B cell colony-enhancing factor (PBEF), has been identified as a novel and multifaceted protein, which plays an important role in regulating a variety of physiological and pathological functions . In the The aim of this study was to explore the association between circulating visfatin and carotid, femoral artery atherosclerosis in a Chinese population with type 2 diabetes and to analyze the difference of visfatin levels between patients with carotid and femoral plaques.This was an observational cross-sectional study of a single-center cohort conducted at the department of endocrinology in Changhai Hospital . From June 2017 to December 2017, a total of 97 inpatients with type 2 diabetes were consecutively recruited. The diagnostic criteria of diabetes were based on the American Diabetes Association standards. We excluded patients with type 1 diabetes, acute complications of diabetes such as ketoacidosis and hyperosmolar state, hepatic and renal failure, liver cirrhosis, acute or chronic inflammatory diseases, malignancy, pregnancy or lactation, and autoimmune diseases. The study was approved by the ethics committee of the Changhai Hospital. Written informed consent was obtained before the data collection and analysis.Basic anthropometric measurements of all subjects, including height, body weight, waist circumference, hipline circumference, were collected according to standard protocols. Laboratory data was measured from blood samples, which were taken in the morning after an overnight fast of at least 10 h. Blood cell analysis (including white blood cell (WBC) count, total neutrophils, lymphocytes, and platelet count) was performed on the Sysmex XN-9000 analyzer . Plasma glucose, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), bilirubin, creatinine, uric acid, lipid profile, including total cholesterol, triglycerides, HDL cholesterol (HDL-c), LDL cholesterol (LDL-c) were measured using HITACHI 7600-120 automatic biochemical analyzer . Endogenous creatinine clearance rate (Ccr) was calculated using the following formula: Urine Cr (mg/dL) × 24 h urine volume (dL)/Serum Cr (mg/dL) × 1440 (min/day). Hemoglobin A1c (HbA1c) was measured by high-pressure liquid chromatography. Fasting blood glucose (FBG), and 2 h postprandial blood glucose (PBG), were measured using the glucose oxidase method. Insulin and C-peptide levels were measured on an automatic analyzer . Insulin resistance status was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR) according to the following formula: fasting serum insulin (mIU/L) × fasting plasma glucose (mmol/L)/22.5.Serum levels of visfatin were measured using the commercially available enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions . The intra-assay and inter-assay coefficients of variation were less than 10% in enzyme immunoassays.B-mode ultrasound of the carotid/femoral arteries was performed using the Philips rE33 Ultrasound System . A single experienced reader blinded to clinical follow-up data performed the analysis of the presence of atheromatous plaques. We defined plaques as having an IMT ≥ 1.3 mm and/or a focal protrusion into the lumen with a thickness of at least 50% more than the adjacent intima-media complex.Normally distributed data were expressed as mean ± standard deviation (SD), and data with skewed distribution were shown as medians (Q1–Q3). Differences between groups were evaluated with Student’s t test or Mann–Whitney U test. The association between serum visfatin levels and other variables were evaluated with Spearman’s correlation analysis. Logistic regression analysis was performed to evaluate the odds ratio of Atherosclerotic Plaques. Two-tailed P values < 0.05 were considered to be statistically significant.The sample size calculations for the comparison between the mean of 2 independent groups were performed using PASS 11 . According to these calculations, the minimal sample size required for each group was 19 subjects.Overall, our study included 97 patients with T2DM who were admitted to our hospital. The demographic and clinical characteristics of the recorded patients are given in Additional file P = 0.0002). Moreover, in both male and female subgroup, serum levels of visfatin were significantly higher in patients with atherosclerotic plaques compared to patients without plaques  versus 0.48(0.27–0.76) ng/mL, P = 0.005; female subgroup: 0.71(0.52–0.90) versus 0.39(1.18–0.64) ng/mL, P = 0.008). We also analyzed serum visfatin levels according to the type of vascular territory affected . Interestingly, as shown in Table According to the findings of the B-mode ultrasonic examination of carotid and femoral arteries, all patients were divided into two groups: plaque group (56 subjects) and no plaque group 41 subjects). As shown in Table  subjectsr = 0.226, P = 0.029), waist-hip ratio , TG  and number of plaques .To find out the related factors influencing serum visfatin levels, Pearson’s correlation analysis of clinical and biochemical parameters with visfatin were undertaken. As shown in Table In order to assess which factors were independent predictors of the presence of atherosclerotic plaques, multivariable logistic regression analysis was performed. Since many covariates  could affect visfatin levels, the relevant confounding variables  were entered in the full regression model. As shown in Table In the current study, we demonstrated that serum visfatin levels are significantly elevated in type 2 diabetic patients with atherosclerotic plaques compared to those without plaques. Interestingly, the elevation was most obvious in men with atherosclerotic plaques. Moreover, visfatin was higher in patients with T2DM with carotid plaques compared to those with femoral atherosclerotic plaques or without plaques.Growing evidence suggests that visfatin is expressed in a wide range of cell types  –15 and cControversial findings on visfatin levels have been reported across many metabolic diseases. Several studies confirmed an increased level of circulating visfatin in obesity, type 2 diabetes, and metabolic syndrome . On the In the last decade, there is growing interest in the potential role of visfatin in the pathogenesis of metabolic related cardiovascular complications. Increased visfatin serum levels were reported to be correlated with carotid plaque vulnerability in patients with carotid stenosis , and visP < 0.01). Therefore, different from another adipose tissue-secreted hormone, leptin, which has confirmed gender-based difference [Few previous epidemiologic studies have addressed the possible gender differences in visfatin-concentrations. There was no difference in serum visfatin concentration between male subjects and female subjects in the entire cohort. In both male and female subproup, serum levels of visfatin were significantly higher in patients with atherosclerosis compared to patients without atherosclerosis (fference , the actfference –28. In lAnother important finding of our study is the difference in visfatin concentrations observed depending on the vascular territory affected. Visfatin was higher in patients with T2D with carotid plaques compared with those with femoral atherosclerotic plaques or without plaques. One possible explanation is that the differences in visfatin serum concentration between patients with carotid and femoral plaques could be attributed to differences in the cellular composition of plaques. Compared with femoral plaques, carotid plaques are characterized by a higher inflammatory cell content and lower fibrotic content . In factAs a pro-inflammatory cytokine, visfatin can upregulate the production of IL-1β, IL-6 and TNF-α in human monocytes and vascular endothelial cells . Higher Nevertheless, beyond being just a clinical marker for cardiovascular diseases, visfatin plays an important role in regulating vascular inflammation and atherosclerosis. On the one hand, extracellular visfatin can promote proliferation of human vascular smooth muscle cells through a NAMPT-dependent mechanism . On the Our study has several limitations that need to be discussed. Firstly, the study was based on data obtained cross-sectionally with a relatively small sample size of Chinese T2DM patients at a single center. Prospective studies in larger samples and in other ethnic groups are needed to determine the role of serum visfatin in atherosclerotic plaque in patients with type 2 diabetes. Secondly, single measurements of serum visfatin level may underestimate the strength of the associations in cohorts. Furthermore, most patients with diabetes were kept on their current hypoglycaemic medications, which could affect the potential relationships under investigation. Finally, atherosclerosis is a slow, progressive disease with a complex pathological process, therefore a single biomarker would not be sufficient to detect or predict atherosclerotic plaques. Serum visfatin levels could provide additional information about the risk factors of developing cardiovascular disease.Our study demonstrated that among patients with T2DM, serum visfatin levels were elevated in those with atherosclerotic plaques, and especially those with carotid atherosclerotic plaques. Serum visfatin may serve as a predictor of atherosclerotic plaques in patients with T2DM.Additional file 1. The demographic and clinical characteristics of the recorded patients.