Retinol-binding protein 4 (RBP4), a novel adipokine secreted by adipocytes and the liver, has elevated levels in type 2 diabetes mellitus (T2DM). However, its association with human metabolic diseases remains controversial. The present study was designed to investigate the associations of plasma RBP4 levels with oxidative stress, inflammatory markers, and metabolic syndrome (MetS) in a Chinese population.We evaluated plasma RBP4 levels in a cross-sectional sample of 1748 Chinese men and women aged 50 to 70 years in Guangzhou using an in-house developed and validated sandwich ELISA. Plasma glucose, insulin, lipid profile, serum adiponectin, adipocyte fatty acid-binding protein (A-FABP), 8-iso-prostaglandin F2α (8-iso PGF2α), 13-(S)-hydroxyoctadecadienoic acid (13-HODE), high-sensitivity C-reactive protein (hsCRP), interleukin 6 (IL6), monocyte chemotactic protein 1 (MCP1) and tumor necrosis factor α (TNFα) were all measured. MetS was defined according to the updated National Cholesterol Education Program Adult Treatment Panel III criteria for Asian Americans.r = 0.104, P < 0.001), 8-iso PGF2α , and 13-HODE  and were inversely correlated with HDL cholesterol . After multivariable adjustment, the RBP4 levels were strongly associated with MetS and its components. The ORs (95% CIs) for the comparisons of the extreme quartiles of RBP4 were 3.46  for MetS, 5.92  for hypertriglyceridemia, 1.42  for reduced HDL cholesterol, 1.87  for central obesity and 2.74  for hyperglycemia . When we further controlled for adipokines, markers of oxidative stress and proinflammatory response, the association of RBP4 with central obesity was abolished but not the association with other MetS components.Circulating RBP4 levels were positively correlated with A-FABP (Plasma RBP4 levels are associated with an adverse profile of oxidative stress and inflammatory markers and an increased risk of MetS in this Chinese population. These associations are independent of conventional risk factors. Metabolic syndrome (MetS) is a clustering of multiple metabolic abnormalities, including central obesity, dyslipidemia, elevated blood pressure, hyperglycemia and insulin resistance. MetS plays an important role in the origin of cardiometabolic diseases, including cardiovascular disease (CVD) and type 2 diabetes mellitus -3. MultiRetinol-binding protein 4 (RBP4), mainly secreted by adipocytes and the liver, was originally known as the specific carrier of retinol in circulation . Recent Therefore, we examined the association between RBP4 and MetS risk among a middle-aged and elderly Chinese population. We focused on the correlation of RBP4 with oxidative stress and inflammatory factors, both of which are established or proposed risk factors for metabolic disorders.The study population consisted of individuals who underwent the Nutrition and Health of Aging Populations in South China study, which investigated the associations of dietary and genetic factors, as well as their interactions, with aging-related chronic diseases. From March 2008 to March 2009, 2289 participants aged 50–70 years were recruited from Guangzhou if they had been residents in their respective cities for 10 years. For this study, participants were excluded if one or more of the following criteria were met: younger than 50 years old; had a history of cancer, diabetes, CVD, or stroke; or had missing data on one component of the MetS diagnostic. The study protocol was approved by the Institutional Review Board of the Sun Yat-sen University, and written informed consent was obtained from all participants.Baseline data were collected by trained interviewers via semi-structured questionnaires during face-to-face interviews. The questionnaire was designed based on the pilot surveys among this population. Information on socio-demographic factors, health status, and lifestyle practices  was included in the questionnaire. Standing height, body weight, and waist circumference were measured with the participants in light indoor clothing and without shoes. Body mass index was calculated as weight in kilograms divided by height in meters squared. Two researchers independently entered the baseline data from the questionnaires, and the data were further checked by a third researcher when differences were found. All of the subjects were examined in the morning after an overnight fast.Overnight fasting blood samples were collected in tubes containing liquid EDTA, centrifuged at 4°C, and stored at −80°C until analysis. Total blood cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides and glucose were measured enzymatically on a Hitachi 7180 Biochemistry Automatic Analyzer  using a commercial assay kit . Low-density lipoprotein (LDL) cholesterol was subsequently calculated using the Friedewald formula . Plasma The serum RBP4 concentrations were measured using an enzyme-linked immunosorbent assay , and the intra- and interassay CVs were 1.92–3.68% and 6.57–8.59%, respectively. Serum adiponectin concentrations were measured using an enzyme-linked immunosorbent assay , and the intra- and interassay CVs were 4.1–5.9% and 3.7–6.3%, respectively. Plasma RBP4 levels were measured in duplicate in plasma aliquots that had undergone 1 or 2 freeze-thaw cycles using a competitive enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions with purified human RBP4 standards . The ELISA samples were run in duplicate. The coefficient of variation for interassay replicate samples was less than 7%. The assay system was subsequently cross-validated by a Western blot analysis. The intraassay coefficient of variation was 1.8–7.6%, and that of the interassay was 3.7–8.8%. Plasma 8-iso PGF2α levels were quantitatively determined using an ELISA kit purchased from Enzo Life Sciences International . The assay sensitivity was 16.3 pg/ml . The intraassay coefficient of variation was 4.4–11%, and that of the interassay was 5.0–11%. Plasma 13-HODE concentrations were measured using a colorimetric competitive enzyme immunoassay kit . The assay sensitivity was 1.6 pmol/ml (range 0.7–100 pmol/ml). The intraassay coefficient of variation was 6.4–7.3%, and that of the interassay was 5.4–11.3%.Plasma CRP was measured using a particle-enhanced immunoturbidimetric assay  with microparticles coated with anti-human CRP antibodies. The precision of the method in the cut-off value of decision (1.8–2 μg/ml) is less than 5.5%. The serum levels of interleukin 6 (IL6), monocyte chemotactic protein 1 (MCP1), and tumor necrosis factor alpha (TNFα) were measured using a MILLIPLEX™ Human Cytokine/Chemokine panel . The intra- and interassay coefficients of variation were 8.1 and 11.6% for IL6, 6.1 and 12.0% for MCP1, and 10.5 and 15.9% for TNFα, respectively.MetS was defined using the updated National Cholesterol Education Program/Adult Treatment Panel III criteria for Asian Americans as having ≥3 of the following components: waist circumference ≥90 cm for men or ≥80 cm for women; triglycerides (TG) ≥1.7 mmol/L; high-density lipoprotein (HDL) cholesterol <1.03 mmol/L for men or <1.30 mmol/L for women; blood pressure ≥130/85 mmHg or current use of antihypertensive medications; or fasting glucose ≥5.6 mmol/L .P < 0.05 was considered statistically significant.The normally distributed data were expressed as the means ± SD, whereas variables with a skewed distribution were reported as the median (interquartile range) and log transformed to approximate normality before analysis. The categorical variables were represented by frequency and percentage. An analysis of covariance for the continuous variables and a multivariate logistic regression analysis for the categorical variables were applied for the comparison according to the RBP4 quartiles. An analysis of covariance was used to compare the RBP4 levels between genders and geographic locations. Correlation coefficients between RBP4 and metabolic features were calculated by a partial correlation analysis on ranks (Spearman correlation). The plasma RBP4 levels were depicted according to the number of MetS components using a linear regression model. Multivariate logistic regression models were used to estimate the odds ratios (ORs) for MetS and its components. Potential confounding variables, including age, gender, smoking, alcohol drinking, physical activity, educational level, self-reported CVD, family history of diabetes and CVD, CRP, adiponectin, homeostatic model assessment of insulin resistance (HOMA-IR), and body mass index (BMI), were controlled for in the regression models. The data management and statistical analyses were performed with SPSS 16.0 for Windows . A P < 0.001). However, no significant associations were observed between RBP4 and current drinking or smoking status.A total of 1748 individuals (men and women) were included in this study. The characteristics of the study population according to the quartiles of plasma RBP4 concentrations are summarized in Table P = 0.018) and HOMA-IR (P = 0.048), but no significant associations with insulin were found . A higher plasma RBP4 was significantly correlated with both higher LDL cholesterol (P < 0.001) and lower HDL cholesterol (P = 0.004). No significant associations were observed for blood pressure. RBP4 was also significantly associated with elevated A-FABP levels , but no correlation was observed between RBP4 and adiponectin . In addition, RBP4 was strongly correlated with not only oxidative markers, such as 8-iso PGF2α  and 13-HODE , but also with elevated proinflammatory molecules, including hsCRP , IL6 , MCP1  and TNFα .There were significant correlations between plasma RBP4 and plasma fasting glucose (The RBP4 levels increased gradually with the number of MetS components Figure . The RBPIn the present study, we observed strong positive associations between plasma RBP4 concentrations and the increased risk of MetS and its components among middle-aged and elderly Chinese people. Furthermore, a chronic inflammatory response was significantly associated with higher plasma RBP4 levels, and the participants with those conditions were more likely to have higher RBP4 levels. Additionally, higher RBP4 levels were associated with oxidative stress, as reflected by a higher concentration of the oxidative markers.RBP4, which is mainly secreted from adipocytes and hepatocytes, has been suggested to be a central regulator of insulin sensitivity. In animal models, the overexpression of human RBP4 or the injection of recombinant RBP4 induces insulin resistance in mice, whereas RBP4 knockout mice showed enhanced insulin sensitivity . Our reset al. [In humans, several studies have also shown positive associations between RBP4 and insulin resistance as well as features of MetS ,26. Howeet al.  reportedet al. . With a et al.  demonstrThe obese condition and MetS are accompanied by low-grade chronic inflammation, which is characterized by an increased expression of inflammatory cytokines and infiltration of immune cells in adipocytes . The infAccumulating evidence also supports the important role that oxidative stress plays in MetS-related manifestations . In thisSeveral limitations of this study need to be addressed. First, the cross-sectional nature of the study design cannot be translated into a clear cause–effect inference. Prospective studies and randomized clinical trials are needed. Second, as with any observational study, the role of unmeasured or residual confounding cannot be ruled out; however, the multivariate models did adjust for a wide range of risk factors that have been implicated in the development of MetS or its individual components. Finally, we did not distinguish among full-length and truncated forms of RBP4 that might have various biological activities with potential varying effects on metabolic risk factors and MetS. Future studies with multiple measurements may substantiate our findings.In conclusion, there was a strong positive association between plasma RBP4 with the risk of developing MetS in a middle-aged and elderly Chinese population: subjects with MetS or its components had a higher RBP4 status than those without these conditions. Moreover, the participants with high plasma RBP4 levels were also associated with the inflammatory markers and oxidative stress. Our results suggest a potential link between RBP4 and the incidence of MetS.BMI: Body-mass index; CI: Confidence intervals; CRP: High-sensitivity C-reactive protein; CVD: Cardiovascular disease; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; 8-iso PGF2α: 8-iso-prostaglandin F2α; MetS: Metabolic syndrome; MCP1: Monocyte chemotactic protein-1; OR: Odds ratio; RBP4: Retinol-binding protein 4; TG: Triglyceride; T2DM: Type 2 diabetes mellitus; WHR: Waist-to-hip ratio.The authors declare that they have no competing interests.YL and DW conducted the research, performed the statistical analyses and wrote the manuscript; YL, DW and MW participated in the data collection and checked the data; and MX participated in the design of this study and wrote the manuscript. All authors have read and approved the final manuscript.


Cerebrospinal fluid leaks (CSF) result from an abnormal communication between the subarachnoid space and the extracranial space. Approximately 90% of CSF leak at the anterior skull base manifests as rhinorrhea and can become life-threatening condition. Endoscopic sinus surgery (ESS) has become a common otolaryngologist procedure. The aim of this article is to consider our experience and to evaluate the outcomes in patients who underwent a purely endoscopic repair of CSF leaks of the anterior skull base.Retrospective chart review was performed of all patients surgically treated for CSF leaks presenting to the Section of Nasal and Sinus Disorders at the Service of ENT–Head and Neck Surgery, University Hospital Complex of Santiago de Compostela (CHUS), between 2004 and 2010. A total of 30 patients who underwent repair CSF leak by ESS. The success rate was 93.4% at the first attempt; only two patients (6.6%) required a second surgical procedure, and none of it was necessary to use a craniotomy for closure. Follow-up periods ranged from 4 months to 6 years.Identifying the size, site, and etiology of the CSF leak remains the most important factor in the surgical success. It is generally accepted that the ESS have made procedures minimally invasive, and CSF leak is now one of its well-established indications with low morbidity and high success rate, with one restriction for fistulas of the posterior wall of the frontal sinus should be repaired in conjunction with open techniques. CSF leaks of the anterior skull base present one of the more difficult challenges in Endonasal edoscopic surgery (EES), involving an area that is anatomically complicated and technically demanding to access. The challenge is to recreate the barrier between the cranial vault and the nasal cavity to prevent and eliminate cerebrospinal fluid (CSF) leaks and protect the brain from exposure to infectious sources. Approximately 90% of CSF leaks at the anterior skull base manifests as rhinorrhea and can become life-threatening condition [Cerebrospinal fluid leaks (CSF) result from an abnormal communication between the subarachnoid space and the extracranial spaceondition ,2. CSF londition . And anyondition . Evaluatondition -3.ESS has become a common otolaryngologist procedure. EES is a minimally invasive surgical technique, which provided a direct short-cut access to anterior and middle skull base without traversing any mayor neurovascular structures, which until recently accounted for a significant morbidity and a high-risk surgery  and CSF CSF leaks can have many etiologies including spontaneous, tumor-related, traumatic , or congenital leaks. Etiology affects the risk of recurrence and thus the method of repair by having an impact on the defect size, location, degree of dural involvement, the likelihood of elevated intracranial pressure (ICP), and the possibility of meningoencephalocele protrusion -7.The aim of this article is to consider our experience and to evaluate the outcomes in patients who underwent a purely endoscopic repair of CSF leaks of the anterior skull base.Retrospective chart review was performed of all patients surgically treated for CSF leaks of the anterior skull base presenting to the Section of Nasal and Sinus Disorders at the Service of ENT-Head and Neck Surgery, University Hospital Complex of Santiago de Compostela (CHUS), between 2004 and 2010. Data were collected according to the patient’s characteristics, CSF fistula, surgical techniques, materials used to repair the defect, and complications. We will consider the 30 patients who underwent repair CSF leaks by ESS. 16  men and 14  women . The study was approved by the University Hospital Complex of Santiago de Compostela (CHUS) medical ethics board.Preoperatively patients underwent various evaluations to confirm CSF leak and location, including thorough history and physical with nasal endoscopy and testing including magnetic resonance imaging (MRI), computed tomography (CT), β-trace testing and β-2 transferrin testing for confirmation of CSF rhinorrhea . In cases where the leak had stopped we relied on high resolution CT scan and CT cisternography to determine the leak and location. In three patients with ascending bacterial meningitis the high resolution CT and endoscopic examination showed the location of the bone defect.The precise location of site and size of fistula is the keystone for successful endoscopic closure and closure of the leak was performed by EES. The closure techniques depend on the size and location of cranial defect, the three forms of grafting are the underlay, overlay and combined. “Underlay” or “inlay” technique the intact dura is separated from the edge of the skull base defect to expose an adequate buttress for the stabilization of the graft. The free graft, or flap, should be designed in such a way that it can be pushed a few millimeters between the bone and the dura on all sides of the defect.Bone or cartilage underlay grafts are advocated for large, bony defects associated with herniating brain or meninges. “Overlay” or “onlay” technique , is a technique for small defects. The third approach is to place two separate grafts, one as an overlay and the other as an underlay.The size of the defect also has an impact on surgical planning for the type of grafting required, as smaller defects are more conducive to pliable overlay grafts, and larger (>3 mm) sites can accommodate an underlay graft or multilayer closure with both underlay and overlay grafts. Other options for situations requiring a stronger reconstruction (very large defects or elevated ICP that could dislodge a soft graft) include bony underlay grafts and soft overlay grafts with bony countersinking techniques -10.From the etiological point of view, dural defects were classified into three groups:Traumatic : 12 (40%) were iatrogenic CSF fistula, these patients had acquired leaks following traditional sinonasal surgical approaches or functional endoscopic sinus surgery (FESS) and 8  patients were caused by closed head injury. The iatrogenic (40%): 9 (75%) patients by FEES , 2  patients for removal a meningioma and 1  patient after septum and turbinate surgery . Of these, the CSF leaks were caused by the use of microdebrider blade in 3 (25%) patients, being identified and closed immediately in the same surgical procedure. In 6 (50%) patients the CSF leak was diagnosed after surgery, because they developed serious complications, they suffered bacterial meningitis and two developed a pneumocephalus . The closure of the CSF leak was made after resolving infectious pathology.Nontraumatic : this group included adults without any previous history of trauma or surgery .Tumor invading the skull base  patients: a sinus tumor invading the skull base and debuted with a CSF leaks.The most frequent site of the anterior skull base defect was the cribriform plate , followed by the ethmoid roof .The closure technique used depended on the size and location of the fistula. We use the used the underlay technique in 20  patients. The onlay technique was used in 10  patients. The most frequently used graft was mucoperichondrium combined with cartilage harvested from the nasal septum harvested either alone , or mucoperichondrium either alone .The success rate was 93.4% at the first attempt; only two patients (6.6%) required a second surgical procedure, and none of it was necessary to use a craniotomy for closure. Follow-up periods ranged from 4 months to 6 years.Confirmation of CSF at the nasal secretion was demonstrated by quantification of β2-transferrin testing in 24 (80%) patients and β-trace testing in 8 . The radiological diagnosis by CT cisternography confirmed the CSF leaks and location the defect in 18/30 (60%) patients. In 6 (20%) patients was not necessary because the fistula was identified and closed intraoperatively and the remaining 6 patients with bacterial meningitis, the high resolution CT and endoscopic examination showed the location of the bone defect.The leak size was determined by high resolution CT and measurement the defect with curettes in the surgical field, in 10  patients was less than 3 mm, and 20  patients greater than 3 mm.In all cases we use fibrin glue to improve adherence of the graft, and the graft is supported in place with layers of Surgicel®, to separate the graft from the packing material, to prevent avulsion of the graft or flap during its removal. One or two pieces of Merocel packs  into a sterile glove finger were placed in the nasal cavity at the end of the procedure. Packs were removed from the common nasal meatus on the third day. We use ceftriaxone as antibiotic coverage during the hospitalization period.The post-operative phase, conservative measures such as bed rest, avoidance of straining and Valsalva maneuvers, especially vomiting, blowing the nose or avoiding uneasy defecation, can be targeted as ways of avoiding rapid changes in intracranial pressure. The slow resumption of normal activities is mandatory. A post-operative follow-up visit is arranged at 1–2 weeks, with conservative management of crusting. Debridement is undertaken only at 3–4 weeks postoperatively to minimize the possibility of dislodging the graft. Regular follow-up continues weekly until the repair leak site is completely mucosalised and ventilation of paranasal sinuses is ensured.In 2  patients (the first two that were operated in our department) underwent lumbar puncture before surgery, which was intended to ensure that non-surgical closure of a seal failed. And in other 2  patients, lumbar puncture took place at the end of surgery by a large surgical defect. In others patients, the lumbar puncture was avoided when considering major postoperative morbidity associated and the conservative measures.In recent years we have seen the establishment of the ESS as a technique of choice for closure of CSF leak, seen as a less invasive technique with less morbidity and mortality, excellent view of the surgical field, and a higher success rate about 95%), replacing the usual techniques, such as transcranial and extracranial interventions that had a success rate of 70% with significant morbidity (anosmia was permanent sequel) 5%, repla.The presence of CSF rhinorrhea entails a significant risk to the patient’s life . The cliThe pneumocephalus is other major complication in the anterior skull base defects, and are due to direct contact between nasal cavity and the intracranial cavity. The rapid onset of headache and neurological signs, changes in mental status to coma should alert. The two patients who had a severe pneumocephalus, CT confirmed the presence of intracranial air and the fistulous tract through the ethmoid roof into the nasal cavity. It is caused by either a ball-valve mechanism that allows air to enter but not to exit, or by CSF leakage, which creates a negative pressure with subsequent air entry . The cloThe identification of the site is necessary for successful surgical repair. CT, with and without contrast, and nasal endoscopic exploration are the most common form of locating the fistula, and when not displayed properly, CT cisternography is a helpful test .In the surgical iatrogenic CSF fistula, need to clean and control the nasal bleeding that occurs in the surgical field to locate and properly close the fistula. It should be noted the risk that exists with the use of microdebrider blade in FESS, due to the fast and aggressive cut that exposes the skull base to iatrogenic injury, in this area is common to use less aggressive material but we must remember that the skull base lesions can occur with any instrument or technique . We noteThe observation in one of our patients with spontaneous CSF fistula the presence of idiopathic intracranial hypertension, has led us to consider the need for a previous study as the search for an empty sella syndrome . The preThere have been many materials used for sealing of the fistula, and we have resorted to many. The mucoperichondrium, and cartilage in our patients were able to seal the fistula, and we agree with Hegazy , which rThe controversy lies in the technique of graft placement. Onlay and underlay techniques are used depending on the size of the fistula, and both have similar results when used properly . DetermiWe do not use intrathecal fluorescein for serious complications that can arise. We believe that lumbar drainage may increase morbidity and hospital stay. To observe the leak during surgery, we encourage increased intracranial pressure by increasing the pressure at the abdomen that allows us to locate the CSF leak as a stream of clear liquid and transparent.Like others authors ,20, we rThe use of antibiotics in skull base surgery is controversial, however the penicillin and macrolides are used in the postoperative phase of endoscopic sinus surgery, and although the risk of meningitis must be counterbalanced with the risk of resistance to antibiotics, we recommend an antibiotic coverage in cases of iatrogenic fistulas. Like other authors we recommend the use of ceftriaxone . HospitaThe control of CSF leakage has been significantly improved through the development of the EES. The excellent exposure of the nasal cavity roof by endoscope offers the opportunity to identify the area of the fistula and allowing an adequate treatment plan. It is generally accepted that the ESS have made procedures minimally invasive, and (CSF) fistula is now one of its well-established indications with low morbidity and high success rate, with one restriction for fistulas of the posterior wall of the frontal sinus should be repaired in conjunction with open techniques.Identifying the size, site, and etiology of the CSF fistula remains the most important factor in the surgical success. The risk of bacterial meningitis, with a significant mortality rate, it’s high enough to consider surgical closure of the fistula. Provides excellent results, and allows us to address a problem that until recently was a serious medical conflict.The authors declare that they have no competing interests.CM-M, GM-C, RS-G and FE-R designed the study. CM-M coordinated sample and medical record data collection. All authors contributed to writing and reviewing the final manuscript. All authors read and approved the final manuscript.


The detection of α-synuclein (α-syn) in the cerebrospinal fluid (CSF) of patients with synucleinopathy has yielded promising but inconclusive results. The aim of the present study was to determine the diagnostic value of α-syn as a biological marker for Dementia with Lewy bodies (DLB) vs. normal subjects and patients with Alzheimer’s disease (AD), after strict control of several recognized confounders. Sixteen patients with DLB, 18 patients with AD and 22 age- and sex-matched normal controls (CTRL) were recruited. The levels of total α-syn in CSF were measured using a novel enzyme-linked immunosorbent assay. There was a significant increase of CSF α-syn levels in DLB patients as compared to the CTRL and AD groups (P= 0.049 and 0.01 respectively). ROC analysis revealed that increased α-syn was 81.8% specific for the discrimination of DLB vs. CTRL and 90% vs. AD. However, sensitivity was lower (56.2 % and 50% respectively). These findings provide evidence for a possible diagnostic role of α-syn as a surrogate biomarker for DLB. Early diagnosis of dementing neurodegenerative disorders such as Alzheimer’s disease (AD) and Dementia with Lewy Bodies (DLB) is considered necessary for a number of reasons, including treatment initiation at the earliest stage . CerebroDLB is considered to be the second most common type of neurodegenerative dementia in the elderly, characterized by fluctuating cognition, parkinsonism and psychiatric symptoms. Although the current clinical diagnostic criteria appear very specific, they are much less sensitive due to substantial clinical as well as pathological overlap mainly with AD and other dementia or parkinsonian syndromes. Patients with DLB typically do not exhibit tau pathology, but they often have Aβ pathology. The core protein involved in its pathogenesis is α-synuclein (α-Syn), which is the major component of Lewy bodies, also present in Parkinson’s disease (PD) . It has The discrepancy among these studies probably results from several factors, including variation in antibodies that might detect different species of α-Syn, limited numbers of patients and differences in the control group . In addition, there was inadequate control for important confounding factors, such as diurnal variation, rostro-caudal gradient within CSF, gender- or age-dependence and, importantly, blood contamination of CSF.The aim of the present study was to determine the diagnostic value of CSF α-Syn levels to discriminate DLB from healthy controls as well as AD , after strict implementation of the current proposed recommendations on standardized operating procedures (SOPs) for the CSF biomarkers . For thiThe study was performed according to the ethical guidelines of the 1964 Declaration of Helsinki. It was conducted in two stages and had the approval of the local committee of Eginition hospital. Both patients and controls gave written informed consent to participate in the study. Both patients and controls gave standard (written) informed consent to participate in the study. In cases that this was not feasible, the next of kin carer takers or guardians consented on the behalf of participants whose capacity to consent was compromised.During the 1st stage, a total of 19 control CSF samples from healthy subjects were used (CTRL batch 1). They came from our CSF Bank and had been collected during 2006. They comprised of otherwise healthy, elderly individuals that had undergone minor surgery (such as hernia repair or knee joint surgery) under spinal anesthesia. They suffered no neurological, psychiatric or other major disease and their cognitive function was within normal limits as detected by history, semi-structured interview and MiniMental State Examination (MMSE)  prior toAll patients underwent a detailed evaluation . Laboratory evaluation included complete blood count, serum electrolytes, blood urea nitrogen, creatinine, glucose, vitamin B12 and thyroid function tests; all results were within normal limits.Lumbar puncture was performed at the L4-S1 interspace, between 9–12 AM after overnight fasting. Samples were obtained in polypropylene tubes. Approximately 12 ml of CSF were drawn from each subject, in three fractions of 2, 5 and 5 ml. The first 2 ml-tube was used for routine diagnostic purposes. The following two tubes were immediately (within 30 min) centrifuged at 500xg for the removal of cells, aliquoted (0.75 ml) into several 1 ml polypropylene tubes and stored at –80°C until analysis. They were thawed only once, just before the assay. To avoid any potential variations arising from a rostro-caudal gradient, the same fractions of both patient and control group (2nd tube) were assessed. Samples with more than 50 RBCs in the first tube were rejected from the 2nd stage of the study (see below).The in-house ELISA for the accurate quantification of α-Syn concentration in biological fluids was developed by using two commercially available α-Syn-specific antibodies: the monoclonal Syn-1 (BD Transductions) as capture antibody and the polyclonal C-20 (Santa Cruz) as detection antibody, which was used after its covalent conjugation with HRP . Briefly To avoid misinterpretation of our results, we wanted to verify that CSF samples showing high levels of α-Syn but < 50 RBCs were not contaminated with RBCs lysed during sample collection and processing. For this reason, haemoglobin levels were determined using the Human haemoglobin ELISA Quantitation Kit (Bethyl Lab Inc) in two CSF samples from each CTRL group (Batch 1 and Batch 2) and three CSF samples from each disease group (DLB and AD) that showed the higher α-Syn levels. All samples analysed had heamoglobin levels <300 ng/ml. The assay was performed according to the manufacturer’s instructions.All numerical variables were checked for their distribution and for homogeneity of variances by the Shapiro-Wilk’s and Levene’s tests respectively. The levels of α-Syn did not follow the normal distribution, while their variances were heterogeneous. Logarithmic transformation restored the above violations, permitting the use of two-way analysis of covariance (two-way ANCOVA) for comparison among groups. Sex and diagnostic group were introduced as co-factors and age and MMSE as covariates. One-way ANCOVA and Kruskal-Wallis test were also performed when appropriate.P) or the Spearman rank correlation (RS) coefficients as appropriate.Correlation of α-Syn levels with clinical parameters or various parameters of the CSF (including RBCs) was performed by the parametric Pearson .The diagnostic value of α-Syn was analyzed by Receiver Operating Characteristic (ROC) curve analysis. Analysis of ROC curves, including cut-off values with the optimal combination of sensitivity and specificity, was performed by Medcalc® version 6.16 . Asymptotic Demographic, clinical and biochemical data of patients and controls are summarized in The optimal concentration of the capture antibody was determined by measuring the signal over background ratio (S/B) obtained for a low concentration of recombinant α-Syn (0.1 ng/ml) . ReplaceThe analytical characteristics of the α-Syn ELISA were determined using the established optimized protocol. The limit of quantitation (LOQ) of the assay was established by analyzing serial dilutions of recombinant human α-Syn . As demoThe intra-assay variation for 0.1-25 ng/ml of recombinant α-Syn ranged from 1 to 7%. The day-to-day reproducibility of the assay was assessed by performing similar calibration graphs during a period of 6 months (n=7 curves). The average of the S/B ratios for each concentration were estimated and plotted versus the input of recombinant α-Syn, where the error bars correspond to the reproducibility of the assay for that period . In addiP = 0.017). A post-hoc test for trend showed a statistical significant increase of α-Syn levels among the 3 subgroups with increasing RBC content , while Newman-Keuls post-hoc tests revealed that both the 50–200 and the >200 RBCs subgroups had higher α-Syn levels as compared to the <50 RBCs subgroup .A positive correlation was noted between α-Syn levels and RBC content of the CSF . This poS = 0.04, P = 0.88), CSF protein (RS = –0.06 P = 0.79) and MMSE . A positive correlation with age did not reach statistical significance . No significant difference was observed between males and females (P = 0.36).No correlation was noted between α-Syn and white blood cells , WBCs  and MMSE , while no difference was observed between males and females (P = 0.85). Positive correlations with age  and CSF protein  did not reach statistical significance.No correlation was noted between α-Syn levels and RBCs . The controls of batch 2 had lower levels as compared to the subgroups of batch 1 with 50-200 and >200 RBCs (Newman-Keuls post hoc tests P = 0.02 and 0.004 respectively). However, they did not differ significantly as compared to the subgroup of batch 1 having < 50 RBCs in CSF (P = 0.67).The levels of α-Syn in this second batch see  were comSince α-Syn levels in CTRL batch 2 were almost identical with the subgroup of CTRL batch 1 with <50 RBCs/μl, both were combined in a common CTRL group and compared to the levels of patients with AD and DLB . FinallyP = 0.0029). Age and MMSE did not affect the model significantly. Post hoc Neuman-Keuls tests revealed a significant increase of α-Syn levels in DLB patients as compared to the CTRL and AD groups (P = 0.049 and 0.01 respectively), while the AD group did not differ as compared to CTRL. Diagnostic group (but not sex or sex by group) showed a significant effect  probably reflects technical/methodological differences and/or different species of α-Syn measured, rather than an actual biological cause. In the majority of the studies, the control group comprised of patients suffering from other neurological diseases and only a few studies ,21,27,28Among various confounders of CSF α-Syn quantification, blood contamination has been widely recognized, as α-Syn is highly abundant in the blood. Especially in erythrocytes, its levels are markedly higher than those in plasma and/or serum . HoweverIn the present study, we have shown a very strong correlation of α-Syn levels with RBCs, which disappeared only when <50 RBCs/μl were present in the first collection tube. This suggests that in the second cell-free collection tube almost no RBCs were present, and contamination, if any, might have resulted by a few RBCs undergone hemolysis before centrifugation. We have also tried to eliminate other confounders which could influence the levels of α-Syn. Diurnal variation was avoided by performing lumbar puncture in the morning. Food intake was controlled, since samples were collected following overnight fasting. We have also avoided the appearance of a concentration gradient between samples by measuring the same CSF fraction. Other processing details included use of polypropylene tubes to minimize possible tube-wall absorbance, filling of the tube by the same amount of CSF (3/4 in 1ml tubes) and immediate sample processing. Regarding the length of storage, α-Syn in CSF seems to be stable when samples are treated and stored under optimum conditions, since no differences in α-Syn levels were observed between old (6 years storage) vs. new (6 months) CTRL samples. This is the first study contacted with such strict adherence to the very recent recommendations .The measurements presented in this study have been performed using a new ELISA with high sensitivity and selectivity. Importantly, the antibodies utilized by this method (Syn1 and C-20) are both commercially available, with well-characterized, known epitopes, which ensure recognition of the α-Syn monomer. However, due to the topology of the epitopes, we have verified that α-Syn detected by this assay corresponds to the total levels of the protein (oligomeric and monomeric). Other monoclonal antibodies, such as 211 and 5C2, have been tested as capture antibodies in our assay format using C-20 as detection antibody. From these antibodies, 211 showed a preferential detection of oligomeric species probably due to the epitopes of the two antibodies being in close proximity, if not overlapping with each other. On the other hand, 5C2 antibody displayed almost no binding with the target protein. This lack of binding could either be due to limited accessibility of 5C2 epitope (which is located in the NAC domain of α-Syn) or to steric hindrance between 5C2 and C-20 antibodies. These findings highlight the importance of selecting the appropriate antibody for the development of a new ELISA assay in a way to allow detection of specific species or quantification of the total amount of target protein.No significant effect of age, sex, and MMSE on α-Syn was observed in CTRL or in the patient groups. Other authors have reported a decrease with age ,16,18 whThe main finding of the present study is the significant increase of α-Syn levels in DLB as compared to the CTRL and AD groups, while AD did not differ as compared to the CTRL group. This is the first study to our knowledge that reports an increase in CSF total α-Syn concentration as detected by our optimized ELISA. Previous studies, have reported either decreased -15 or unAccording to the published criteria for biological markers of the Reagan Research Institute of Alzheimer’s Association and the National Institute on Aging   in orderIt has been shown that under normal conditions full length α-Syn is detected in CSF ,26. InveAn inherent limitation in the majority of biomarker studies (the present study included) is the lack of pathological verification. Although our clinical sample is rather small, the main strength of our study is the implementation of standardized operating procedures and the use of a healthy control group for comparisons. Additionally, in order to minimise incorrect classification of patients as much as possible, extensive investigation, implementation of the latest clinical criteria, enhanced by determination of the already established CSF biomarkers, plus a short follow up period were applied. The importance of confirmation of these results in pathologically validated series in future studies is required.In conclusion, the results of the present study are compatible with an increase of CSF total α-Syn in DLB as compared to both CTRL and AD groups. Although specificity was high, sensitivity did not exceed 60%. However, this is the first study presenting the possible diagnostic value of CSF α-Syn in terms of sensitivity and specificity, thus rendering CSF α-Syn as a potential diagnostic tool for DLB. Further studies are required as to whether combination of α-Syn with other biomarkers may increase the diagnostic value not only from CTRL and AD, but from other synucleinopathies as well.Figure S1Scatterplots of CSF levels of total tau (τT), amyloid Aβ42 and phospho-tau (τP-181) in the studied groups. Horizontal bars indicate median values and horizontal lines indicate cut-off values of our laboratory .(TIF)Click here for additional data file.Figure S2Optimization studies for the α-Syn ELISA. (A) The optimal concentration for the capture antibody (0.5 µg/ml) determined by the measurement of 0.1 ng/ml recombinant α-Syn. (B) Use of 211 monoclonal antibody results in a 4-fold decrease in assay sensitivity. Capture antibodies were used at 0.5 µg/ml and the concentration of recombinant α-Syn measured was 0.1 ng/ml. (C) Determination of the time required for the optimal binding of α-Syn (0.3 ng/ml) to the capture antibody. (D) The assay buffer (TBST) was selected by the measurement of 0.1 ng/ml recombinant α-Syn.(TIF)Click here for additional data file.Figure S3aCSF behaves identically with TBST in our assay format. Recombinant α-Syn  was diluted either in TBST or aCSF and the calibration curves were presented in parallel.(TIF)Click here for additional data file.Table S1T), amyloid Aβ42 and phospho-tau (τP-181) in the studied groups.CSF levels of total tau (τ(DOC)Click here for additional data file.


TTR gene which are considered to influence the ability of the protein to carry its ligands were also analyzed. CSF TTR levels were significantly higher in LBD patients compared to controls. Post-hoc analysis demonstrated that this effect was driven by PDND patients. In addition, CSF TTR levels correlated negatively with CSF Abeta1–42, total tau and phospho-tau levels. Serum TTR levels did not significantly differ among the studied groups. There were no relevant associations between TTR levels and genetic, demographic and clinical data, respectively. These results suggest an involvement of the clearance protein TTR in LBD pathophysiology, and should motivate to elucidate TTR-related mechanisms in LBD in more detail.Parkinson’s disease (PD) without  and with dementia (PDD), and dementia with Lewy bodies (DLB) are subsumed under the umbrella term Lewy body disorders (LBD). The main component of the underlying pathologic substrate, i.e. Lewy bodies and Lewy neurites, is misfolded alpha-synuclein (Asyn), and - in particular in demented LBD patients - co-occurring misfolded amyloid-beta (Abeta). Lowered blood and cerebrospinal fluid (CSF) levels of transthyretin (TTR) - a clearance protein mainly produced in the liver and, autonomously, in the choroid plexus - are associated with Abeta accumulation in Alzheimer’s disease. In addition, a recent study suggests that TTR is involved in Asyn clearance. We measured TTR protein levels in serum and cerebrospinal fluid of 131 LBD patients  and 72 controls, and compared TTR levels with demographic and clinical data as well as neurodegenerative markers in the CSF. Five single nucleotide polymorphisms of the  The term “Lewy body disorders (LBD)” subsumes the three entities Parkinson’s disease (PD) without dementia (PDND), PD with dementia (PDD), and dementia with Lewy bodies (DLB) 1–42 production higher than its removal results in an enhanced presence of Abeta1–42 monomers, oligomers, insoluble fibrils and plaques in the central nervous system ApoE genotype 1–42. Similar mechanisms may lead to Asyn accumulation, at least for late onset PD although there is less evidence for this hypothesis Moreover, co-occurrence of amyloid-beta (Abeta) deposits with Asyn pathology is common, particularly in LBD patients with dementia transthyretin (TTR) gene is located at chromosome 18q11 and consists of four exons 1–421–42 aggregation and destroy already formed Abeta1–42 fibrils by proteolytic cleavage of the peptides 1–42-binding and -sequestering proteins in human cerebrospinal fluid (CSF) 1–42 toxicity, and reduced levels of the protein in blood TTR mutation carriers suffering from familial amyloidosis compared to healthy control individuals. Interestingly, Asyn levels in saliva of symptomatic TTR mutation carriers who underwent orthotropic liver transplantation  were comparable with those of controls.The TTR gene are differentially capable of altering the ability of TTR to carry and transport its ligands TTR single nucleotide polymorphisms (SNPs) rs13381522 and rs3764478 (both lie at the 5`end in the promoter region of the TTR gene), rs1800458 , rs723744 , and rs36204272  It has been demonstrated that not only the relatively rare mutations but also common variants of the The study was approved by the ethics committee of the Medical Faculty, University of Tuebingen, Germany, and was performed according to the principles expressed in the Declaration of Helsinki. All participants gave their written informed consent. In case of compromised capacity of the participants to consent (MMSE <18 points), or another person was named to make decisions on behalf of the person, responsible persons consented on the behalf of the participant.Demented DLB, PDD and non-demented LBD patients (PDND), and elderly neurodegeneratively healthy control individuals (HC) were recruited from the ward and the outpatient clinic of the Neurodegenerative Department of the University of Tuebingen. LBD patients were diagnosed based on clinical criteria by specialists in the field of neurodegenerative movement disorders . All PD patients fulfilled the UKPDS Brain Bank criteria All individuals underwent demographic analysis and clinical testing including a Hoehn&Yahr (H&Y) staging and a Mini-Mental State Examination (MMSE). http://www.hih-tuebingen.de/de/nd/biobank/for-researchers/), without any previous freeze-thaw cycle. Our biobank fulfills highest international standards with regard to collection, preparation and storage of biosamples. Samples for determination of TTR levels were centrifuged  and stored at −80°C within 60 minutes after collection until analysis. Only samples of patients with normal routine CSF diagnostics were included in the study, slightly increased CSF albumin levels (up to 450 mg/L) were tolerated.CSF and serum collection as well as determination of routine diagnostic parameters were performed according to standardized protocols . Samples were run in triplicate. We observed within run precision of <2.0% coefficient of variation (CV), and total precision of <5% CV.1–42, total tau and phospho-tau levels were determined using commercially available ELISA kits .CSF AbetaTTR gene was determined with Sanger-sequencing technique to identify the SNPs rs1800458 and rs36204272. SNPs rs13381522, rs3764478, and rs723744 which are located at the TTR intron or promoter region were analyzed by the SNaPshot method. Briefly, a PCR with specific SNaPshot primers was performed. After single base extension with fluorescent-labeled ddNTPs the SNaPshot product was analyzed by capillary gel electrophoresis with ABI Prism 3100 Genetic Analyzer sequencer . Fluorescence data were analyzed using Gene Mapper v3.5.Genomic DNA was extracted using standard protocols. The coding sequence of the ApoE genotypes were obtained by PCR amplification of exon 4 of the ApoE gene, and subsequent digestion with enzyme HhaI according to 2) was used as a measure for the proportion of response variation which was explained by the regressors in the model. Significance of each model effect was assessed by the likelihood ratio.Data were analyzed with JMP software . As dementia associated with LBD may per se be a relevant factor associated with TTR levels, PDD and DLB were not only considered as single groups but also comprised as a PDD/DLB group  were corrected for multiple testing  nor in serum . CSF TTR values were approx. 18-fold lower than serum values (1.7 versus 30.1 mg/dl).TTR values in CSF and serum were normally distributed, and did not correlate. They were not age-related, neither in CSF . Serum TTR levels were significantly higher in males than in females  and were therefore calculated separately.Gender did not differ significantly between LBD patients and controls. CSF TTR values were comparable between females and males . Serum TTR levels were not significantly different between LBD patients and controls, and among LBD patients , nor with 1–42 , CSF total tau  and CSF phospho-tau . No significant correlations were detectable between serum TTR values and neurodegenerative markers.There were weak but significant negative correlations between CSF TTR values and respective levels of CSF AbetaTTR SNPs  showed a significant association with LBD occurrence. In addition, no relevant associations of the SNPs with demographical, clinical and biochemical parameters were detectable (data not shown).None of the analyzed 1–42 pathology decreased CSF TTR levels in disorders that are not primarily associated with inflammation  than had control individuals and, most interestingly, TTR mutation carriers who underwent orthotropic liver transplantation. This strongly indicates that TTR is associated with clearance of (misfolded) Asyn.Actually it is not entirely clear whether (misfolded) Asyn is a target protein of TTR. The latter mainly functions as a transport protein, and is able to transfer e.g. Abeta1–42 pathology in demented LBD patients. Together with the observation that PDND patients have increased CSF TTR levels one may hypothesize that demented LBD patients face two driving forces with regard to TTR-associated pathways: Asyn pathology which goes along with increased TTR expression, and Abeta1–42 pathology associated with decreased TTR expression. Based on the abovementioned associations between TTR levels and amyloid pathology, one can also hypothesize that increased TTR levels in the brain protect against Abeta1–42 pathology. Indeed, there is accumulating evidence from in-vitro and in-vivo studies that TTR has beneficial direct and indirect effects on Abeta-associated pathology , and TTR values. This may indicate that TTR, although obviously involved in amyloidogenic and clearance pathways in LBD, cannot serve as a marker of disease progression and severity.1–42-associated LBD pathologies. The obvious interaction of centrally produced TTR with pathophysiological mechanisms in these neurodegenerative processes should motivate to more in-depth analyses.In conclusion, data presented in this study argue for a role of TTR in both Asyn- and AbetaTable S1Post-hoc analyses of transthyretin levels between different groups(DOC)Click here for additional data file.


Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity We evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice.+Gr-1+ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8+ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation.We find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11bOur findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF. The advent of recombinant G-CSF and related factors able to induce myeloid cell expansion have rapidly revolutionized the treatment of cancer and other diseases-5. Firstin vivo means that the biological activity of administered G-CSF is transient and patients must receive repeated dosing to achieve a durable myeloid cell recovery. As a means of prolonging the half-life of G-CSF and improving its biological activity in vivo, it was found that, as with other cytokines[in vitro activity of many cytokines. Furthermore, recent findings suggest the existence of anti-PEG (polyethylene glycol) antibodies in up to 25% of healthy individuals[Despite the potency of G-CSF therapy, there are significant clinical limitations. Foremost, the half-life of recombinant G-CSF is relatively short (3.5 hours),16. Thisytokines,18, pegyytokines,19. Thusytokines. This efytokines. Becauseytokines, and it ytokines-28. It iividuals-29. Whilividuals, and couin vivo is pre-association with a cytokine-specific monoclonal antibody or a soluble receptor prior to injection. Thus, pre-association of IL-2, IL-3, IL-4, IL-6, IL-7, IFNα, or TNFα with specific cytokine-specific monoclonal antibodies dramatically improves biological activity in vivo[An alternative method for increasing the biological activity of a cytokine y in vivo-32. Simiy in vivo. While ty in vivo-39.in vivo, it is not clear whether cytokine complexes can universally act on all types of potential target cells[in vivo might favor their activity on some cell populations but not others. Alternatively, some cytokines may be inherently more amenable to enhanced activity when administered as a cytokine complex due to the mechanism by which they induce receptor signaling. Finally, it is possible that accessory cells necessary to facilitate the mechanism by which cytokine complexes act are not present after cytoreductive therapy. The latter would represent an obstacle limiting the use of cytokine complexes in many types of cancer therapy. Our purpose was to address these issues by the evaluation of cytokine-antibody complexes composed of G-CSF and anti-G-CSF mAb. Upon association, we find that these cytokine complexes are potent stimulators of neutrophils, a G-CSF-responsive cell type not previously shown to be responsive to cytokine complexes. Furthermore, our data demonstrate that these G-CSF/anti-G-CSF mAb complexes facilitate myeloid cell recovery after cytoreductive therapy without inducing a suppressive environment that compromises antigen-specific CD8+ T cell responses. These findings suggest a novel strategy for enhancing the clinical utility of recombinant G-CSF.Although cytokine complexes can effectively drive the expansion and activation of a wide range of cell populations et cells. The loc+Gr-1+ myeloid cells . We observed that G-CSF/anti-G-CSF mAb complexes induced a marked increase in the percent of splenic CD11b1-37G10. ulations,44, the +Gr-1+ myeloid cells, it was important to assess whether other cell populations were affected. To maximize any changes observed upon administration of cytokine complexes, we treated mice with G-CSF/anti-G-CSF mAb complexes every 48 hours for 20 days. We observed no significant differences in the frequencies or numbers of B cells (B220+), T cells (CD8+ or CD4+), or dendritic cells (CD11c+) among lymphocyte-gated cells  or vehicle alone, mice injected with G-CSF/anti-G-CSF mAb complexes demonstrated a roughly 100-fold reduction in bacterial counts within the spleen at day 3 post-infection Figure .+ tumor-specific T cell response[+Gr-1+ myeloid cells or other biological changes following administration of a high dose of G-CSF might suppress CD8+ T cell responses[+ T cells undergo an expansion, a contraction, and a memory phase following infection -induced immunosuppresions[+ T cells, and then given a prime/boost vaccination with the gp10025-33 peptide/poly I:C as outlined in Figure + T cells. The effectiveness of the G-CSF/anti-G-CSF mAb complexes was apparent from the potent expansion of CD11b+Gr-1+ myeloid cells observed in the peripheral blood at day 9 following CTX treatment  G-CSF (Neupogen) and PEG-G-CSF (Neulasta) were purchased from the UCSD pharmacy . Anti-hG-CSF mAb (clone BVD11-37G10) was purchased from SouthernBiotech . Cytokine complexes were generated by incubation of the cytokine and antibody for 20 minutes at 37°C and diluted at least 10-fold in phosphate-buffered saline before injection (i.p. or i.v.). G-CSF/anti-G-CSF mAb complexes were generated at a 1:5 ratio of cytokine to antibody (1 μg of cytokine pre-associated with 5 μg of antibody), a ratio that ensures all cytokine is pre-bound with antibody.-/-, OT-I, and pmel-1 mice were purchased from the Jackson Laboratory . All animal work was performed under institutional and federal guidelines outlined by the University of California, San Diego and the Medical University of South Carolina. Bacterial protection and the determination of colony forming units were performed using Listeria monocytogenes expressing ovalbumin (LM-OVA) as previously described[5 or 106 naive OT-I CD8+ T cells were injected i.v. one day before antigenic challenge. For infections, mice were injected i.v. with 105 plaque forming units of vesicular stomatitis virus expressing ovalbumin (VSV-OVA)[6 naïve pmel-1 CD8+ T cells were injected i.v. one day after treatment with 4 mg of cyclophosphamide . One and ten days after pmel-1 transfer, mice were immunized s.c. with 100 μg of EGSRNQDWL (gp10025-33) peptide (American Peptide) and 200 μg of poly I:C. For bone marrow transplantation experiments, mice were lethally irradiated (1400 rad) and adoptively transferred with 2x106 T- and B-depleted CD45.1 congenic bone marrow cells as previously described[C57BL/6 (B6), B6.Ly5.1 (CD45.1), RAG1escribed. For expVSV-OVA). For pepVSV-OVA). Brieflyescribed. For meaCells were analyzed by flow cytometry using standard techniques. Briefly, cells were washed in staining buffer  and stained with fluorescently labeled antibodies as indicated. The antibodies used in these studies were as follows: B220-FITC and- PerCP , CD3-PE-Cy5 ; CD4-FITC , CD8-FITC and –PerCP , CD11b-PE and –Pe-Cy5 , CD11c-PE and –APC ,CD25-FITC and phycoerythrin , CD44-allophycocyanin , CD122-PE , CD45.1-FITC, -PE, and –APC , CD45.2-FITC and –APC ,CD49b-PE and -APC ; c-kit-PE , Gr-1-FITC and PE-Cy5 , Ly6C- PE , Ly6G-biotin , NK1.1-PE , Sca-1-FITC , and TER-119-PE-Cy5.5 . Staining for intracellular bromodeoxyuridine (BrdU) was performed with reagents and according to the instructions outlined in the FITC BrdU flow kit . Flow cytometry was performed with a BD FACS Caliber and BD Accuri (BD Biosciences). Data were analyzed using FlowJo software .The Wilcoxon Rank-Sum test  was used to determine the statistically significant difference between the underlying distributions of two groups. For measurements taken over time we fitted a linear mixed model with a log-transformed outcome and the following effects: study group (treatment), time, and group-time interaction. Three variance-covariance structures were tested. The covariance that resulted in the smallest Akaike’s information criterion was used. In all cases it was the compound symmetry structure. If the overall group-time interaction showed to be significant, we further explored the differences in the levels of that interaction. We performed comparisons of the group levels within each timepoint. All the analyses were carried out using SAS 9.2 and R 3.0.1 software. Throughout the paper all P-values presented are two-sided with 0.05 or less considered significant.BrdU: Bromodeoxyuridine; CTX: Cyclophosphamide; LM-OVA: Listeria monocytogenes expressing ovalbumin; mAb: Monoclonal antibody; PEG: Polyethylene glycol; sIL-15Rα: Soluble IL-15 receptor α; OVAp: SIINFEKL peptide; VSV-OVA: Vesicular stomatitis virus genetically engineered to express ovalbumin.Authors  are inventors on a provisional patent application associated with the application of G-CSF/anti-G-CSF mAb complexes.MPR and MLS conceived the study, participated in the design, conducted experiments, wrote and edited the manuscript. DJC and AWG conceived the study, participated in the design, wrote and edited the manuscript. AD, CJM, and GLR participated in the design, conducted experiments, and edited the manuscript. CC performed statistical analysis and edited the manuscript. All authors read and approved the final manuscript.+Gr-1+ myeloid cells. Figure S2. Administration of G-CSF/anti-G-CSF mAb complexes induces CD11b+Gr-1+ myeloid cells with a Ly6G+Ly6Clow phenotype. Figure S3. Pre-association of anti-G-CSF mAb with pegylated G-CSF improves biological activity. Figure S4. Long-term administration of G-CSF/anti-G-CSF mAb complexes induces splenomegaly and dramatic expansion of CD11b+Gr-1+ myeloid cells. Figure S5. Administration of G-CSF/anti-G-CSF mAb complexes induces increased numbers of hematopoietic progenitor cells in the spleen and peripheral blood. Figure S6. Extended administration of G-CSF/anti-G-CSF mAb complexes does not affect hematocrit. Figure S7. G-CSF/anti-G-CSF mAb complexes induce the proliferation of CD11b+Gr-1+ myeloid cells. Figure S8. Activated CD8+ T cells proliferate normally when mixed with splenocytes from G-CSF/anti-G-CSF mAb complex-treated mice. Figure S9. G-CSF/anti-G-CSF mAb complexes expand CD11b+Gr-1+ myeloid cells after bone marrow transplantation. Figure S10. The combination of G-CSF/anti-G-CSF mAb complexes and IL-15/sIL-15Rα-Fc complexes induces more effective hematopoietic recovery following bone marrow transplantation. G-CSF/anti-G-CSF mAb complexes induce the expansion of CD11bClick here for file


The immunoadjuvant potential of cross-linked dextran microspheres (CDM) as absorption enhancer and Quillaja saponins (QS) as immunomodulator adjuvant was evaluated. CDM loaded or tetanus-mixed toxoid (TT) or Quillaja saponin (QS) were nasally administered to rabbits in dry powder form, three times in 2 weeks interval and serum IgG and nasal lavage sIgA titers were determined by ELISA.P< 0.001). Among nasally immunized groups, the highest serum IgG titer was induced by (TT+QS)CDM (P< 0.01). Mixing of CDM with TT+QS powders (CDM+TT+QS), could not induce the high serum IgG titers as (TT+QS)CDM (P< 0.01). CDM loaded with TT+QS induced higher IgG titers than CDM loaded with TT alone (P< 0.01). No significant difference was observed in nasal lavage sIgA titers of various groups. The highest serum IgG titer was induced by parenteral immunization through alum adsorbed TT (CDM microspheres loaded with TT+QS significantly increased serum anti-TT IgG titers, but mixing of CDM with TT+QS powder could not increase IgG titers. Both QS and CDM adjuvant could not significantly increase the lavage anti-TT IgA titers. Most of the present vaccines are administered parenteraly and encountered with several disadvantages. Mucosal delivery of vaccines could be a highly effective route for induction of local and systemic immunity  . Among t®) could be utilized to overcome the mucosal barriers and increase immune responses   has been shown in several studies. The purified Quillaja saponins (Quil A) are now approved for veterinary vaccines and these are commercially available vaccines, like bovine respiratory syncytial virus vaccine, adjuvanted with QS  as immunomodulator adjuvant were used for nasal immunization against a model antigen, tetanus toxoid. Powder formulations adjuvanted with CDM, QS or both were nasally administered in rabbits and their impacts on mucosal and systemic immune responses were evaluated. At the present study, CDM powder as an absorption enhancer adjuvant and MaterialsQuillaja saponin was purchased from Sigma . Cross-linked dextran microsphere (Sephadex® G-150) was from BioGene (Sweden). Tetanus toxoid (TT) 2700 Lf/ml and alum-adsorbed TT 50 Lf/ml were obtained from Razi Institute. . Each Lf of TT was equivalent to 5 µg protein, as determined by BCA protein assay. Anti-rabbit IgG and IgA antibodies were from Sigma  and Bethyl Laboratories Inc. .Purified White albino rabbits weighing 2–2.5 kg were provided by Pasteur Institute .Loading of CDM with TT and/or QSTen mg of CDM powder was added to 40 Lf TT and/or 20 µg QS solution and were mixed in ambient temperature for 10 min. The mixture was freeze-dried and the resulting powder was used for nasal immunization. In some of experimental groups, TT or TT+QS solution were first freeze-dried and resulting powder was mixed with CDM. Morphology and particle size determinationOptical microscope  was used for studying morphological features of CDM before and after freeze-drying. Scanning electron microscope  was used for studying the morphology and size of microspheres . For samNasal immunization studiesWhite albino rabbits weighing 1.5–2.5 kg  were nasally immunized with the following formulations in days 0, 14 and 28 of experiment. . When freeze-dried powder of TT was mixed with CDM microspheres, the IgG titer was higher than groups immunized with TT-loaded CDM microspheres (P< 0.01) and TT solution (P< 0.05). There was no significant difference between IgG titers induced with nasal administration of TT solution and freeze-dried powder of TT+QS and TT-loaded CDM microspheres (P> 0.05). Positive control group were intramuscularly injected with 10 Lf Alum-TT and showed the highest IgG titers (P< 0.001). Rabbits (n= 4) were nasally immunized with 40 Lf TT and 20 µg QS at weeks 0, 2 and 4 and were bled at weeks 3 and 6. Sera anti-TT IgG titers were determined by an ELISA method . Among nNasal lavage anti-TT IgA titersth week nasal lavage was collected, pooled and anti- TT IgA titers were determined by an ELISA method (P> 0.05). Following the above-mentioned immunizations, at the 6A method . No signA method   cells were thought to be the principal uptake site of particulate antigens. These cells which are found in the distal regions of the nose, the nasopharyngeal and palatine tonsils and bronchial associated lymphoid tissues (BALT) in the lung could absorb particles of 1-5 µm in diameter , 18. TheThe need to cold chain for storage and distribution is a restricting factor for massive vaccination, especially in developing countries. One strategy for resolving the problem is preparation of stable powder vaccines. Dry powder formulations are more stable against chemical and microbial destabilization and could eliminate the necessity of the cold chain .®, were examined as absorption enhancer and excipient for nasal formulations. CDM powder was loaded or mixed with TT as a model antigen and QS as an adjuvant its impact on immune responses was evaluated. At the present study CDM microspheres, commercially available as SephadexQuillaja saponin (QS) as an adjuvant and cross-linked dextran microspheres (CDM) as an absorption enhancer adjuvant could positively affect the systemic IgG titers. Intranasal administration of (TT+QS)CDM microspheres in powder form showed the highest serum IgG titers (P< 0.001). The end point titer (EPT) induced with this formulation was half of EPT induced with IM injection of alum-adsorbed TT (P< 0.001), which is indicative of a potent mucosal adjuvant system. When TT powder was mixed with CDM, induced higher IgG titers than TT+QS powder (P< 0.05), therefore the adjuvant potential of CDM is higher than QS. The results presented herein indicate that CDM was similar to CDM mixed with TT powder (P> 0.05). However, (TT+QS)CDM induced significantly higher IgG titers than CDM+TT+QS (P<0.001). This implies that loading of CDM microspheres with TT+QS could highly increase its adjuvant potential. Based on previous studies on CDM microspheres for nasal delivery of peptide and protein drugs, absorption of water by the microspheres from the mucus layer may Induce reversible shrinking of the epithelial cells and widening of the tight junctions for about 15 min. In this time, the transport of hydrophilic compounds could be increased ncreased , 20. Addncreased . It seemIn several studies the porous cross-linked dextran and starch microspheres have been used for mucosal delivery of insulin -25, HCG P< 0.0001). The serum IgG titers of the CDM+TT group was higher than the TT solution group (P< 0.05). The adjuvant potentials of CDM and CpG-ODN in inducing In our previous nasal immunization study in rabbits, the immunoadjuvant potential of CDM loaded with TT was compared with TT and TT+ CpG ODN solutions. Among the nasally immunized animals, the highest antitoxin titers was seen in group immunized with CDM+TT (P> 0.05) (IgG titers was not significantly different (P> 0.05) . CDM group. The lowest titers were seen in groups immunized with i.m. injection of Alum-TT and i.n. administration of TT sol. All differences seen in sIgA titers were not statistically significant. In our previous studies, PLGA nanospheres and alginate microspheres encapsulated with TT were mixed with CDM and administered as dry powder. CDM could increase the sIgA titers compared with lactose powders . The highest titer was seen in TT+QS+CDM and followed by (TT)  powders , 36. In  powders . It seemIn nasal delivery of vaccines, the residence time of antigen on the nasal mucosa is very important . MucoadhAfter nasal immunization, CDM microspheres loaded with TT+QS significantly increased serum anti-TT IgG titers, but mixing of CDM with TT+QS powder could not increase IgG titers. Neither QS nor CDM adjuvant could significantly increase the lavage anti-TT IgA titers.


Critically ill patients require sedation to tolerate the interventions necessary to facilitate their care. There is growing evidence, however, that use of sedatives, such as the benzodiazepine midazolam, is associated with delirium and other complications that can lead to prolonged ICU stay and increased mortality. The pharmacokinetics of midazolam in healthy populations has been well characterized, and pharmacodynamic studies demonstrate a predictable dose-response relationship. However, in critical illness, where midazolam is often administered as a continuous infusion, the pharmacokinetic properties are often altered. We sought to investigate whether analysis of midazolam plasma concentrations in combination with electroencephalography (EEG) will better define the effect of critical illness on the pharmacokinetics and clinical response to midazolam, while providing a method to assess the adequacy of sedation thereby minimizing the risks associated with prolonged or over-sedation.For this observational study, patients admitted to the ICU with a diagnosis of sepsis and receiving a continuous infusion of midazolam were screened for inclusion. Upon enrollment, a continuous subhairline EEG was applied and blood samples were collected daily for plasma midazolam quantification. Clinical data and laboratory parameters were followed. Plasma midazolam levels were quantified using liquid chromatography with tandem mass spectroscopy.Data were available for nine patients. Midazolam clearance demonstrated wide intersubject variability  although average clearance among all patients (418 ml/minute) was comparable to that of healthy controls. Mean midazolam concentrations for patients with coma were significantly higher than for patients without coma (218 ± 185 ng/ml vs. 106 ± 107 ng/ml). The plasma midazolam concentration inversely correlated with EEG frequency, with maximal slowing in the delta (≤4 Hz) range.Midazolam concentrations while on continuous infusion were associated with EEG tracings suggestive of deep sedation. Although clearance was relatively preserved, it varied over a wider range than found in healthy populations. The apparent lower threshold for onset of coma may be a reflection of illness severity, concomitant medication use, and variable clearance during the course of illness. These preliminary results suggest that the combination of continuous bedside EEG and therapeutic drug monitoring may be useful for titrating midazolam infusions and to guide tapering to avoid prolonged coma in patients with variable clearance of midazolam.


Mol. Cell. Biol.18, 6779). PKC inhibitors did not affect CSF-1 induced Ras and A-Raf activity but markedly reduced MEK and Erk activity, implying that PKCζ regulated the CSF-1-Erk pathway at the level of MEK. PKCζ has been implicated in activating the NF-κB pathway. However, CSF-1 promoted proliferation in an NF-κB independent manner. We established stable 32D.R cell lines that overexpressed PKCζ. Overexpression of PKCζ increased the intensity and duration of CSF-1 induced Erk activity and rendered cells more responsive to CSF-1 mediated proliferation. In contrast to 32D.R cells, PKCζ inhibition in BMMs had only a modest effect on proliferation. Moreover, PKCζ -specific and pan-PKC inhibitors induced a paradoxical increase in MEK-Erk phosphorylation suggesting that PKCs targeted a common negative regulatory step upstream of MEK. Our results demonstrated that CSF-1 dependent Erk activation and proliferation are regulated differentially in progenitors and differentiated cells.Colony stimulating factor-1 (CSF-1 or M-CSF) is the major physiological regulator of the proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. CSF-1 binds to a receptor tyrosine kinase, the CSF-1 receptor (CSF-1R). Multiple pathways are activated downstream of the CSF-1R; however, it is not clear which pathways regulate proliferation and survival. Here, we investigated the role of atypical protein kinase Cs (PKCζ) in a myeloid progenitor cell line that expressed CSF-1R (32D.R) and in primary murine bone marrow derived macrophages (BMMs). In 32D.R cells, CSF-1 induced the phosphorylation of PKCζ and increased its kinase activity. PKC inhibitors and transfections with mutant PKCs showed that optimal CSF-1-dependent Erk activation and proliferation depended on the activity of PKCζ. We previously reported that CSF-1 activated the Erk pathway through an A-Raf-dependent and an A-Raf independent pathway (Lee and States,  CSF-1R, c-Kit and Flt3 all play pivotal roles in hematopoiesis. The importance of CSF-1-CSF-1R signaling in vivo is revealed by the pleiotropic functional defects of the CSF-1 null (op/op) and CSF-1R null mice Colony stimulating factor-1 (CSF-1 or M-CSF) is a growth factor secreted by numerous cell types, whose synthesis is often increased in response to different stimuli such as those causing inflammation CSF-1 binding initiates dimerization of the CSF-1R and results in kinase activation in vitro autokinase activity of a catalytic fragment of PKCδ but activated PKCζ was not detected in that assay The Ras-Raf-MEK-Erk pathway is evolutionarily conserved and controls many aspects of cell activities Herein we tested the hypothesis that PKCζ may mediate the A-Raf independent pathway to activate MEK-Erk in response to CSF-1 in myeloid cells: 32D.R myeloid progenitors and primary bone marrow derived macrophages (BMMs). We found that CSF-1 increased PKCζ Thr 410 phosphorylation and kinase activity in 32D.R cells. Pharmacologic inhibition and transfection studies demonstrated that atypical PKCs but not conventional or novel PKCs contributed towards CSF-1 induced MEK-Erk activity in a c-Raf-1 and A-Raf-independent fashion. While PKCζ kinase inhibition reduced CSF-1 supported mitogenesis in 32D.R cells, overexpression of PKCζ increased CSF-1 mitogenic responsiveness. However, PKCζ's promotion of mitogenic signaling in 32D.R cells was independent of NF-κB. In BMMs, PKCζ inhibition had a more modest effect on CSF-1 dependent mitogenesis, and, pan-PKC inhibition had a paradoxically enhancing effect on MEK-Erk phosphorylation. Thus the importance of PKCζ in the control of CSF-1 mediated MEK-Erk activity and mitogenesis depends on differentiation stage.Cell culture reagents and media were from Life Technologies  or Sigma-Aldrich . GF109203X was from EMD Chemicals  or Enzo Life Sciences , Ro-31-8220 was from Axxora  and Go 6983 was from EMD Chemicals. Myelin basic protein (MBP) was from Life Technologies, PKCε pseudosubstrate peptide  as phosphorylation substrate and myristoylated PKCζ pseudosubstrate peptide were from Enzo Life Sciences. Recombinant human CSF-1 was a gift of Genetics Institute , recombinant murine interleukin-3 (IL-3) was from Life Technologies, and phorbol 12-myristate 13-actetate (PMA) was from EMD Chemicals.Polyclonal antibodies against c-Raf-1, A-Raf, Erk2, were from Santa Cruz Biotechnology . Antibodies against PKCα, PKCβ, PKCγ, PKCδ and PKCε were from Life Technologies. We used a rabbit polyclonal antibody against PKCζ for immunoprecipitations or a monoclonal antibody for immunoblotting (both from Santa Cruz). The following monoclonal antibodies were used: MEK1 from BD Transduction Labs , Myc (9E10) from Santa Cruz, hemagglutinin (HA) antibody from BAbCo , and Ras Ab-4 from EMD Chemicals. Phosphospecific antibodies that recognize Erk or MEK were from Cell Signaling Technology  and an antibody that recognizes Thr 410 of PKCζ was a gift from Alex Toker  or purchased from Santa Cruz.2 asphyxiation followed by cervical dislocation.A colony of C57BL/6 mice was housed in a specific pathogen-free environment. The Animal Welfare Committee at the University of Texas Health Science Center, Houston approved all animal protocols  and studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were sacrificed by CO4, obtained from Peter Parker , is a dominant-negative PKCζ with Thr→Ala substitutions at the activation loop phosphorylation sites PKC constructs utilized in this study were as follows: PKCζ (T/A)Recombinant bacterially-produced His-tagged kinase-dead MAPK, MEK and GST-RBD, containing the Ras binding domain of Raf (residues 51–131) fused to GST  were expressed and purified as described The IL-3-dependent myeloid progenitor cell line expressing CSF-1R (32D.R) has been described previously in vitro kinase assay in the presence of 10 µCi of γ32P ATP, 50 µM ATP and 50 µM PKCε peptide as substrate. Reaction proceeded for 8 min at 30°C and was spotted onto P81 phosphocellulose paper as described previously 32P incorporation was quantified using a Storm PhosphorImager (Molecular Dynamics). Immunoblots were detected using ECL or ECL plus . To quantify band intensities on immunoblots, multiple exposures of each blot were obtained and band intensities determined using the NIH Image 1.43 software except where noted in the figure legend.Protein analysis methods have been extensively described in previous publications 7/ml in HB  supplemented with protease and phosphatase inhibitors and homogenized with a Dounce homogenizer until cell breakage (monitored by phase-contrast microscopy and Trypan Blue staining) was >90%. Nuclei and unbroken cells were removed by centrifugation at 1000×g for 5 min and the supernatant re-centrifuged at 100,000×g for 1 hr. The supernatant was adjusted to contain a final concentration of 0.5% NP40, 0.1% sodium deoxycholate and 0.1% Brij35 and after spinning, designated the S100 (cytosolic) fraction. The pellet after the 100,000×g spin was Dounce homogenized and extracted with HB containing the above detergents, centrifuged at 16,000×g for 15 min to remove insoluble material and the supernatant designated the P100 fraction. Variation in the ratio of total cytosolic to membrane protein amongst the samples was 3% indicating equivalent fractionation and extraction. Purity of the fractions was determined by assaying for the activity of a cytosolic marker, lactate dehydrogenase, and for the presence of Ras, by Western blotting. Less than 10% of the LDH activity was found in the membrane (P100) fraction and no Ras was detected in the cytosolic (S100) fraction (data not shown).Cells were resuspended at a density of 8×10Proliferation was assessed using the MTS tetrazolium assay  as described Luciferase activity was assayed using the Luciferase Reporter Assay (Promega). 32D.R cells stably expressing an NF-κB reporter were washed extensively and starved in serum-free medium +Ly6C+CD11b−) that are consistent with an immature myeloid phenotype Fig. S1, and 32D.R cells express cell surface markers (CD31in vitro assays 50 of ≈7.5 µM whereas the IC50 for Gö 6983 was between 2.5 and 5 µM (50 of ≈7.5 µM for GF109203X is consistent with a role for PKCζ yet at the concentration that is reported to inhibit PKCζ (1 µM), Gö 6983 was less potent. However published IC50 values are based on inhibition of recombinant PKC isoenzymes and do not reflect accessibility to intracellular stores of these enzymes, or duration of inhibition which in turn would depend on factors such as reversibility of inhibition, half life of the inhibitor and protein turnover. We also used a myristoylated PKCζ pseudosubstrate peptide to directly investigate the role of PKCζ. A dose dependent effect was observed and the IC50 was ≈20 µM, which is in the range of what has been reported in published work To assess the role of PKCs in CSF-1 dependent growth we used the MTS assay and 5 µM Fig. 1B.50 measured using purified proteins. We then determined the effect of PKC inhibition on A-Raf and c-Raf-1 kinase activity using recombinant kinase-dead MEK as substrate. Neither GF109203X nor Gö 6983 significantly influenced A-Raf activity whereas their effect was more pronounced on c-Raf-1  but it also suppressed PMA-induced ERK activity by an equivalent amount  and sequestration of upstream activators We further investigated the involvement of PKCζ by determining the effect of transient expression of a dominant-negative PKCζ 4 Fig. 5A.In another approach to investigate if PKCζ could contribute to ERK activation, we used HA-tagged constitutively active PKCζ containing only the catalytic domain (PKCζcat). First, we determined the kinase activity of transfected HA-tagged wildtype (WT) and PKCζcat. As shown Fig. 5B,Since PKCζ appeared to play a role in CSF-1 mediated mitogenesis and PKCζ has been shown to activate the NF-κB pathway which regulates genes involved in cell proliferation 50 from 0.58 nM in vector-transfected cells to 0.36 nM in WT- PKCζ overexpressing cells (p = 0.0005) indicating enhanced sensitivity to CSF-1 provoked mitogenesis. Importantly, CSF-1 induced Erk phosphorylation was also increased in cells overexpressing WT- PKCζ and this enhancement was observed at 5 min and beyond  as assessed by MTS activity and cell counts Fig. 9C.PKCs play pleiotropic roles in many cellular processes. How PKCs contribute to CSF-1 dependent functions is not well characterized. In this study, we addressed two questions using the 32D.R myeloid progenitor cell line and primary mouse BMMs : 1) Is PKC an activator of the MEK-Erk pathway; 2) Does PKC contribute towards CSF-1 dependent proliferation? In 32D.R cells, we presented evidence in support of a role for the atypical PKC, PKCζ, in CSF-1 dependent activation of MEK-Erk and mitogenesis. (1) Downregulation of PKCα and PKCδ by chronic phorbol ester treatment had no effect on CSF-1 mediated MEK-Erk activation, excluding a role for these enzymes. (2) The novel PKC, PKCε, which was expressed in these cells, was partially downregulated by chronic phorbol ester treatment but was not stimulated by CSF-1 in a membrane translocation assay and was unlikely to be involved. (3) PKCζ kinase activity and Thr 410 phosphorylation were increased by CSF-1. (4) At concentrations of GF109203X and Gö 6983 that should inhibit the kinase activity of PKCζ, CSF-1 induced MEK-Erk activity was significantly reduced. This conclusion was supported by the results of transient overexpression of dominant-negative or constitutively active PKCζ on Erk activity. Moreover, stable overexpression of WT- PKCζ increased the magnitude and duration of Erk activation. (5) At concentrations of PKC inhibitors that reduced CSF-1-supported Erk activation, mitogenesis was also suppressed. This was corroborated using a PKCζ specific inhibitory peptide and by stable overexpression of WT- PKCζ. In contrast, in BMMs, PKC inhibition increased CSF-1 dependent MEK-Erk activation indicating a different role for PKCs compared to immature cells. PKCζ was not a major contributor to proliferation but conventional/novel PKCs appeared to collaborate with the Erk and PI3K pathways to support mitogenesis.Previously, we showed in 32D.R cells that CSF-1 activated MEK-Erk using both an A-Raf-dependent and an A-Raf independent pathway that did not involve c-Raf-1 In BMMs, inhibitors that target conventional/novel/atypical PKCs all appeared to upregulate CSF-1 supported MEK-Erk activity. Our observations are consistent with PKC inhibition relieving negative regulation of MEK-Erk signaling. MAPKs and MEK1/2 are dephosphorylated by dual specific phosphatases (DUSPs) and protein phosphatases 2A respectively Our data show that PKCζ contributes towards CSF-1 mediated mitogenesis in 32D.R cells, in part through the MEK-Erk pathway. Since PKCζ is known to activate the NF-κB pathway In summary, we have identified PKCζ as a crucial activator of MEK-Erk in the CSF-1 signaling pathway in myeloid progenitors. In mature macrophages, PKCζ and other PKC members appear to target a common negative regulator of CSF-1-dependent MEK-Erk activity. These findings indicate that the cellular context of CSF-1 signaling must be considered and the role of different PKC subclasses in macrophages warrants an in-depth investigation.Figure S132D.R cells have an immature immunophenotype that is different from bone marrow derived macrophages (BMM). 32D.R cells were cultured in the presence of CSF-1 or IL-3 before analysis. Day 7 BMMs (see Methods) were used. (A) Forward and side scatter profile. (B) Staining of 32D.R with isotype control antibodies (isotype), or with CD31 and CD11b. (C) Staining of 32D.R with isotype control antibodies (isotype), or with CD31 and Ly6C. (D) Staining of BMMs with CD31, CD11b or with CD31, Ly6C.(TIF)Click here for additional data file.Methods S1Supplementary methods.(DOC)Click here for additional data file.


Spigelian hernia (SH) is rarely seen in pediatric age group and is usually associated with cryptorchidism on the same side; termed as a syndromic association of the defect in the Spigelian fascia and absence of gubernaculum and inguinal canal. The absence of the inguinal canal has surgical implication as to placement of the undescended testis into the scrotum. A 3-month-old baby presented with spigelian hernia and ipsilateral impalpable testis. The spigelian hernia was repaired and undescended testis which was present in abdominal wall layers was brought to scrotum with cord structures anterior to external oblique muscle. SH is rare in children with only 59 pediatric cases reported up until 2012 since its first description in 1935.[1-4] The anatomical basis of SH is weakness in the SH belt arising from a transversus abdominis aponeurosis between the lateral edge of the rectus sheath and the semilunar line that stretches from the ninth rib to the pubic tubercle. Whereas adult SHs are considered to be acquired because of trauma or increased intra-abdominal pressure, pediatric cases are suspected to be congenital. Among male infants with SHs, 75% are associated with cryptorchidism.[6] The association of ipsilateral undescended testis and SH is being debated whether it is a cause or effect relationship. Raveenthiran suggested that a defect in the Spigelian fascia combined with ipsilateral cryptorchidism, may be a part of new syndrome.[7] Rushfeldt and others suggested that the additional lack of a gubernaculum and inguinal canal in similar cases, may be considered as two additional and closely connected elements of this new syndrome.[2] We have managed a 3 months old male child who presented with SH and ipsilateral undescended testis along with all the features of this new syndrome.A 3-month-old male baby was admitted with a swelling on the right side of abdomen and absence of testis on the same side. The swelling increased in size on crying. The swelling occupied right lower quadrant of the abdomen and was non-tender. On genital examination, the left testis was normally descended and right one was impalpable in scrotal pouch and inguinal area . Ultrasonography of the swelling revealed an echogenic tissue consistent with testis in the layers of abdominal wall along with small gut loops echoes. At operation a defect found on the lateral border of rectus abdominis muscle around the arcuate line with a hernial sac containing testis without any inguinal canal and gubernaculum .The sac was opened and the testis was separated from the sac. The defect in the abdominal wall was repaired by absorbable sutures and testis placed in the right sub-dartos pouch by placing the cord structures anterior to the external oblique muscle (subcutaneous route). In the post operative period child remained well and discharged home after 3 days.Cryptorchidism in children presenting with SH is considered to be a congenital condition. There has been considerable speculation and controversy regarding the possible mechanism of cryptorchidism in SH. According to the general viewpoint, SH is the primary defect and the undescended testis takes the path of least resistance to descend and lie in the hernial sac accounting for this association. Raveenthiran speculated an ectopic location of the testis is the primary abnormality, and it leads to formation of SH by dragging a peritoneal sac along with it.[7] Rushfeldt et al believed in failure of development of gubernaculum as an inciting event; [2] thus inguinal canal could not develop and the testis remain in their intra-abdominal position.Ultrasonography helps in assessing the size of the defect and contents of the hernia sac. The management of this anomaly includes repair of the hernia and scrotal placement of the undescended testis. While undertaking surgical repair of SH, one should look for an undescended testis, and expect it to be in the hernia sac. Furthermore, since an inguinal canal may not be found, one should be prepared to perform an orchidopexy either via a subcutaneous route or a new internal ring may be created through the abdominal wall medial to the inferior epigastric vessels and just lateral to the pubic tubercle.[4] However one should be careful in orchidopexy by creating neo internal ring as testicular atrophy has been reported in one patient.[4] The possible reported explanations for such an outcome include vascular damage, tension and compression of the cord and scrotal infection.Source of Support: NilConflict of Interest: None declared