A detailed understanding of the CSF dynamics is needed for design and optimization of intrathecal drug delivery devices, drugs, and protocols. Preclinical research using large-animal models is important to help define drug pharmacokinetics-pharmacodynamics and safety. In this study, we investigated the impact of catheter implantation in the sub-dural space on CSF flow dynamics in Cynomolgus monkeys. Magnetic resonance imaging (MRI) was performed before and after catheter implantation to quantify the differences based on catheter placement location in the cervical compared to the lumbar spine. Several geometric and hydrodynamic parameters were calculated based on the 3D segmentation and flow analysis. Hagen-Poiseuille equation was used to investigate the impact of catheter implantation on flow reduction and hydraulic resistance. A linear mixed-effects model was used in this study to investigate if there was a statistically significant difference between cervical and lumbar implantation, or between two MRI time points. Results showed that geometric parameters did not change statistically across MRI measurement time points and did not depend on catheter location. However, catheter insertion did have a significant impact on the hydrodynamic parameters and the effect was greater with cervical implantation compared to lumbar implantation. CSF flow rate decreased up to 55% with the catheter located in the cervical region. The maximum flow rate reduction in the lumbar implantation group was 21%. Overall, lumbar catheter implantation disrupted CSF dynamics to a lesser degree than cervical catheter implantation and this effect remained up to two weeks post-catheter implantation in Cynomolgus monkeys. A detailed understanding of cerebrospinal fluid (CSF) dynamics is needed for design and optimization of intrathecal drug delivery devices, drugs, and protocols. The intrathecal space, also termed the dural sac, is an anatomic region located between the spinal cord tissue and the dura mater that is filled with CSF. In intrathecal drug delivery, medications are introduced directly to the CSF using a drug delivery system. The most prevalent method of intrathecal drug administration is the externalized intrathecal catheter . One advCurrently approved medications for intrathecal administration by the U.S. Food and Drug Administration (FDA) include morphine, ziconotide, and baclofen. If the motivation for treatment is to decrease spasticity in body extremities, then the intrathecal catheter can be implanted in the rostral intrathecal space to potentially improve drug spread uniformity across the neuroaxis . IntrathPreclinical intrathecal drug delivery studies need to be performed using large-animal models  since these models have utility to define drug pharmacokinetic-pharmacodynamics and provide an important tool for assessment of drug safety. Preclinical studies also provide insight into the potential mechanisms of intrathecal drug delivery. Cynomolgus monkeys are a commonly used model for these studies because of their similarity to humans with regard to the pathophysiology of a variety of diseases and presumed similarity with regard to central nervous system anatomy and CSF hydrodynamics.Unfortunately, we do not know how prolonged intrathecal catheter implantation in nonhuman primates (NHP) or humans may impact CSF dynamics. This is important because the dynamic movement of CSF impacts drug spread along the neuroaxis , 9. AlsoTo our knowledge, no studies have investigated how catheter placement may impact cardiac-related CSF dynamics in Cynomolgus monkeys, or any other species. Our previous study developed a quantitative method to characterize CSF dynamics and geometry in non-human primates (NHPs) using a non-invasive MRI-based protocol . This meThis study was submitted to and approved by the local governing Institutional Animal Care and Use Committee at Northern Biomedical Research . This study did not unnecessarily duplicate previous experiments and alternatives to the use of live animals were considered. Procedures used in this study were designed with consideration of the well-being of the animals.This study complied with all applicable sections of the current version (at the time of study 2016) of the Final Rules of the Animal Welfare Act Regulations (9 CFR), and the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 8th edition. The animals were housed individually in stainless steel cages. The housing was in compliance with the Guide for the Care and Use of Laboratory Animals, DHHS, (NIH) No. 86–23, and the Animal Welfare Act (9 CFR 3). Non-contact absorbent wood chips were used to line the pans underneath the animal’s cage. Room temperatures was maintained at 74° ± 10°F. Twenty-five biscuits of PMI Certified Primate Diet #5048 were placed in the feeders daily during the study. More biscuits were provided based on the weight and caloric needs of the animal. Filtered municipal water (City of Norton Shores) was supplied ad libitum via an automatic watering system. All animals in the study were provided with environmental enrichment according to the facility Program of Animal Care that was accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. All animals were observed for morbidity, mortality, injury, and availability of food and water at least twice daily.For catheter implantation, the animals were pretreated with atropine sulfate as a subcutaneous injection at a dose of 0.04 mg/kg. At least 15 minutes later, an intramuscular dose of 8 mg/kg of ketamine HCl was provided to induce sedation. The animals were masked, intubated, and maintained on approximately 1 liter/min of oxygen and approximately 2.0% isoflurane. The anesthetic gases and mixtures were varied as required by the animal and recorded in the raw data. Prednisolone sodium succinate IV, 30 mg/kg, and flunixin meglumine intramuscular, 2 mg/kg, were administered prior to surgery. A hemilaminectomy was made in the L5 vertebra, the catheter tip was inserted and advanced intrathecally with the tip located near T12/L1 or C5. All of the catheters were terminated in a subcutaneous access port . The actual location of the catheters and catheter tips were verified by a myelogram with Iopamidol. Upon recovery from anesthesia, the animals were provided 0.05 mg/kg intramuscular butorphanol tartrate for analgesia and placed on 5.0 mg/kg post-surgical antibiotic ceftiofur sodium (one injection prior to surgery followed by three injections). A post-surgical recovery period of at least 10 days was taken prior to CSF flow imaging. Following this MRI study, animals were returned to the facility stock colony.Table 1). NHP 01 was male and all other NHPs were female (02–08). These animals were purpose-bred and experimentally naïve.Eight (NHP 01–08) healthy cynomolgus monkeys  were obtained from Charles River Research Models, Houston TX with an average weight of 4.4 ± 1.2 kg and age of 4.6 ± 0.4 years (mean ± standard deviation) . MRIPRE-1 and MRIPRE-2 were spaced 14 days apart prior to catheter placement. MRIPRE-1 and MRIPRE-2 were used in our previous publication [th and 28th days, catheter patency was confirmed through a visual inspection and ability to withdraw CSF from the catheter port. MRIPOST-1 was collected on day 31 to study the acute impact of implantation on CSF dynamics and geometry by comparison of results to MRIPRE-2. MRIPOST-2 was collected at day 45 to determine if the impact persisted after catheter implantation.Each NHP was scanned with an identical MRI protocol (see MRI methods) across all study time points .Thru-plane  CSF flow was measured by phase-contrast MRI (PC-MRI) images collected at six axial locations along the spine for each NHP. Axial locations were marked at the foramen magnum (FM), C2-C3, C5-C6, T4-T5, T10-T11, and L3-L4. The slice location for each scan was oriented roughly perpendicular to the CSF flow direction with slice planes intersecting vertebral discs . Frequent use of the manual segmentation tool was utilized in order to view the three orthogonal planes. The catheter was considered to be an empty region within the spinal SAS, because it was not possible to consistently identify within the MR images due to its small lumen diameter. Once the segmentation was complete, the 3D model  was exported in a stereo lithography format for subsequent analysis.The high-resolution T2-weighted anatomic MRI images were semi-automatically segmented using the free open-source ITK-snap software  , which pFig 2C) using GTFLOW software  by the segmentation procedure previously described by Khani et al. [PWV, was computed based on the slope of the arrival time of peak CSF flow along the spine [CSF flow was quantified at six axial locations along the spine , was based on cross-sectional area of the spinal cord at that location, Ac(z), and dura, Ad(z). The axial distribution of the catheter cross-sectional area for the lumbar and cervical catheters were subtracted for MRIPOST-1 and MRIPOST-2. Similarly, the hydraulic diameter, Dh(z) = 4Asas(z)/Psas(z), was determined based on the wetted perimeter, Psas(z), with the perimeter computed as the sum of the spinal cord, Pc(z), and dura, Pd(z), perimeters at each z-location. The axial distribution of the catheter perimeter for the lumbar and cervical catheters were added for MRIPOST-1 and MRIPOST-2. Axial distribution of CSF stroke volume was computed as SV(z) = ∫|Q|dt, where |Q| was the absolute value [Qsys(z) and Qdia(z), while the CSF flow rate amplitude was given by Qa(z) = Qdia(z)−Qsys(z). Spatial mean thru-plane velocity at peak systole was computed as Axial distribution of the SAS cross-sectional area, te value . Peak syν was the kinematic viscosity of CSF at body temperature, 0.693 mPa∙s [DNR, was given by the axial distribution of nerve roots diameters along the spine, ranging from 0.2 to 0.3 mm [ω was the angular velocity (ω = 2π/T) of the volume flow waveform with T is the average cardiac cycle time (T = 0.53 s). To assess possibility of instabilities in an oscillatory flow around cylinders, Keulegan-Carpenter number was computed as: K>2 under a given β as shown experimentally by Honji (1981) [Reynolds number based on hydraulic diameter was computed as 93 mPa∙s . Reynoldo 0.3 mm . Reynoldi (1981)  and theoi (1981) .Lsas measured for all NHPs. After normalization, the mean axial distribution for each parameter was computed for each group  at each MRI time point .Womersley number was computed as Dh(z), was the axial distribution of hydraulic diameter for MRIPRE-2, and Dh-w(z), was the predicted hydraulic diameter by taking into account the axial distribution of catheter area and perimeter for the lumbar and cervical groups. This flow reduction prediction is approximated based on the Hagen-Poiseuille equation for steady, incompressible, laminar pipe flow under the assumption that intracranial pressure pulsations, that drive CSF flow along the spine, are not affected by presence or absence of the catheter (i.e. Δp = constant).Catheter implantation could potentially reduce CSF flow due to increased hydraulic resistance. We estimated the potential flow reduction for the cervical and lumbar implantation group by: We hypothesized that implantation of the catheter would decrease CSF dynamics and geometry, and that these changes would be elevated for NHPs with cervical implantation compared to lumbar implantation. For each of the parameters investigated here, MRI measurements were taken at multiple locations along the spinal cord for each cynomolgus monkey. Since the NHPs were randomly selected from a population, we developed the following linear mixed-effects model:yi represented the geometric or hydrodynamic parameter of interest, xi1 stood for the location, while xi2 was the catheter location  or the MRI time point , xi3 and xi4 are the age and weight of each NHP, respectively. The random error, εi, has a normal distribution with mean 0 and variance σ2: εi~N. While β are fixed effect sizes, z represent the random-effect coefficients, which follow a multivariate normal distribution with mean of 0 and a symmetric variance-covariance matrix:fitlme” function in Matlab  to estimate the parameters in this linear mixed-effects model and test the hypothesis.We used the “This model treats the catheter location and the MRI time point as a fixed effect, with the corresponding coefficient indicating the effect size. We could further test whether the true effect size is significantly different from zero. If so, it means that there is a statistically significant difference between cervical and lumbar implantation, or between two MRI time points. This model treats the NHPs as random; this means that the multiple measurements from an NHP can form a curve, and that this curve may be different from one NHP to another.Using this linear mixed-effects model, we estimated the relative effect sizes of the following seven pairs: four pairs comparing time points , and three comparing cervical versus lumbar implantation . For each pair, we tested the statistical significance of the two groups being different and obtained a P value. Since we performed this analysis for 13 geometric and hydrodynamic parameters, we derived 13×7 = 91 P values. Many of these P values were dependent due to the strong dependence among several parameters of interest. We accounted for multiple comparison with Bonferroni correction by adjusting the threshold for P values to be 0.05/91 = 5.49e-4. This identified a highly conservative set of significant P values. Note that this approach assumes independence among P values. When two parameters of interest are highly correlated, they would lead to similar P values that are both identified to be significant after correction. In this case, we can only conclude that one or both parameters are significant, but we cannot pinpoint the truly significant parameter.PRE-2, MRIPOST-1, and MRIPOST-2 for the cervical (C) and lumbar (L) groups are shown in Table 2 (Mean ± STD). Statistical assessments revealed that multiple hydrodynamic parameters were statistically different across study groups and time points (Table 3). However, geometric parameters were largely unchanged.A summary of geometric and hydrodynamic parameter results obtained at MRIFig 3). Overall, 33 out of 42 geometric parameters did not change statistically across MRI measurement time points or depending on catheter location (Table 3).Results indicated that cervical catheter insertion altered spinal SAS geometry to a greater degree than lumbar catheter insertion . However, Psas  for the MRIPOST-1C and MRIPOST-2C groups increased significantly below the catheter tip after insertion (Table 3). Average CSF volume in the spinal SAS for all NHPs across all measurement time points  was 7.00 ml. Average cross-sectional area for spinal cord, dura and SAS for all NHPs was 15.43, 39.65 and 23.66 mm2, respectively. Average perimeter for spinal cord, dura and SAS was 13.88, 22.09, and 38.36 mm, respectively. Average SAS cross-sectional perimeter  in the thoracic region increased from 3 to 6 mm with cervical catheter implantation.Axial distribution of geometric parameters showed relatively small changes across the lumbar and cervical implantation groups for Fig 4). For example, MRIPOST-1C flow rate was lower than MRIPRE-2C for all axial locations. Catheter implantation was found to decrease CSF flow pulsations even 31 days after catheter insertion (MRIPOST-2C and MRIPOST-2L). These findings were supported by statistical analysis that showed changes in hydrodynamic parameters with cervical and lumbar catheter implantation to be highly significant for 40 out of 49 hydrodynamic parameters with p values < 0.05/91 (Table 3).Catheter implantation was found to decrease CSF flow pulsations along the entire spine and this impact was greater for cervical catheter implantation compared to lumbar implantation . CSF flow waveform showed a systolic peak at 100 to 150 ms in the cervical spine ranging from 0.2–0.6 (ml/s) for all NHPs. CSF flow rate at the C5-C6 for MRIPOST-1C and MRIPOST-2C was markedly smaller than both MRIPRE-2C and 2L, and MRIPOST-1L and MRIPOST-2L due to catheter placement within cervical SAS in those cases.CSF flow rate of each NHP group quantified along the spine had a similar waveform shape, and axial distribution . Pulse wave velocity magnitude was similar across the groups and ranged from 1.09–1.24 m/s. Maximum CSF flow rate occurred for the MRIPRE-2 measurement within the cervical spine. Catheter placement decreased the flow rate spatially and temporally below the catheter tip in both MRIPOST-1 and MRIPOST-2.Average spatial-temporal distribution of the CSF flow along the spine showed a relatively smooth decrease in amplitude with a caudally directed CSF pulse wave velocity . MRIPOST-2C had the lowest Re value of 28 due to the cervical catheter implantation. Maximum ReNR, Reδ and Keulegan–Carpenter number was 12.55, 14.31 and 5.12 (β = 2.79), respectively. Catheter implantation also decreased CSF flow rate amplitude  and stroke volume  at MRIPOST-1C and MRIPOST-2C compared to MRIPRE-2C and for MRIPOST-1L and MRIPOST-2L compared to MRIPRE-2L. Albeit, the changes in flow rate amplitude and stroke volume were greater under cervical implantation.Maximum Dh  and α  decreased a great degree with cervical catheter implantation and to a lesser degree with lumbar implantation. Maximum Dh and α was 4 and 8 located near the FM. The peak value of the mean velocity ranged from +1.8 to -2.9 cm/s in MRIPRE-2 and occurred at the C3-C4 level . Based on Hagen–Poiseuille equation, CSF flow reduction was predicted to be 48% after cervical implantation and 6% after lumbar implantation. These predictions were comparable to the MRI-measured Qpeak-sys reduction of 55% after cervical implantation and 21% after lumbar implantation (Table 2).To the best of our knowledge, the impact of intrathecal catheter implantation on spinal CSF dynamics in a cynomolgus monkey has not been reported in the literature. Our results showed that catheter implantation decreases spinal CSF dynamics and that the reduction was greater for cervical implantation compared to lumbar implantation. Also, that the decrease in spinal CSF dynamics was present immediately post-implantation and persisted two weeks after implantation. The MRI method used in this study to quantify intrathecal CSF dynamics in cynomolgus NHPs can be used to quantify intrathecal CSF dynamics and geometry in people with neurological disorders or healthy conditions. The technique provided for hydrodynamic parameter analysis could also aid development of emerging CSF deliverable therapeutics and be applied to better understand CSF-related pathologies.Figs 4 and 5). Additionally, nearly all measures of CSF dynamics were altered to a greater degree for cervical implantation compared to lumbar implantation (Table 2). These results were further supported by estimation of CSF flow reduction, based on the Hagen-Poiseuille equation, indicating that the CSF flow reduction was likely due to increased hydraulic resistance stemming from the catheter’s reduction in SAS hydraulic diameter .The potential impact of catheter implantation on intrathecal CSF dynamics should be considered when implanting spinal catheters in NHPs and potentially humans. Although catheter diameter was relatively small, our results showed that cervical catheter implantation reduced peak CSF flow by 55% compared to 21% for lumbar implantation . For example, the CSF flow waveform amplitude was smaller at the FM compared to C2–C3 and then decreased at T4–T5 . CSF flow oscillations are primarily produced from cardiac-related pulsation of the intracranial arteries [The distribution of CSF flow along the spine was non-uniform and showed local variations in waveform shape and magnitude at each axial location . This is a potential indicator that spinal compliance, and likely intracranial pressure, was not affected due to catheter implantation. CSF PWV was previously measured by our research group in NHPs and found to have a similar value at 1.13 m/s [PWV was measured to be 1.94 m/s [PWV within the spine in humans to potentially be different than NHPs.Average 1.13 m/s . However1.94 m/s , indicatPOST-2C with a maximum Re of 28, 100% less than MRIPRE-2C . Reδ, was less than 14.31, which is below the threshold of 550 for conditional turbulence in oscillatory flow [β and small K, and the critical Keulegan–Carpenter number for these limits has been calculated as: Kcr = 5.778β−1/4(1+0.205β−1/4) given by Hall [β = 2.79, Kcr has a value of 22.32. The value for the current results, K = 5.12, is below this threshold. Therefore, the occurrence of Honji-type instabilities [Re was computed to represent the ratio of steady inertial forces to viscous forces and help indicate whether laminar flow (<2300) was present at each phase-contrast slice location . A laminar CSF flow indicates that the flow is smooth with relatively little lateral mixing. This is different from a turbulent flow, where chaotic changes in pressure and velocity occur and can lead to a large increase in lateral mixing. Chaotic CSF velocity or pressure fluctuations are not expected to occur before or after catheter placement. However, it is possible that disease states that result in strongly elevated CSF flow velocities (jets) could result in turbulence [CSF flow remained laminar throughout the CSF flow cycle for all cases analyzed. Results showed that CSF dynamics were affected the most in the cervical spine near the C5 vertebral level in MRIory flow . Appearabilities  in the Srbulence .α varied along the spine in a similar fashion as Dh with a minimum and maximum value of 3.8 and 8.1 . α was computed to quantify the ratio of unsteady inertial forces to viscous forces that impact the CSF velocity profile shape [α<2, the CSF velocity profiles will be parabolic in shape and considered quasi-static. For 2<α<10 velocity profiles will be M-shaped and, for α>10, velocity profiles will be relatively flat (plug shaped) [α in the thoracic region decreased to ~4 after cervical catheter insertion. This concept implies that the CSF velocity profiles will show an M-shape throughout the spine. However, the upper cervical and lumbar spine had higher α indicating a relatively flat velocity profile within those regions. Our previous computational fluid dynamics NHP model without catheter implantation indicated a relatively blunt CSF velocity profile in the cervical spine [Fig 2C) as the MRI resolution was not fine enough to accurately capture the relatively thin boundary layer expected in a blunt or M-shaped flow profile.Inertial effects are expected to dominate the SAS CSF flow field for normal physiological flow rates, frequencies and CSF fluid properties. le shape . For α<2 shaped) . The maxal spine . It was Re in Fig 6A and Table 2), indicate that CSF velocities and streaming in the cervical spine were much greater than the lumbar spine. Thus, although catheter placement in the cervical spine could result in decreased CSF flow, drug delivery in this region may still allow more rapid mixing compared to the lumbar spine. Catheter implantation location may also need to be taken into account alongside potentially diminished CSF flow dynamics in healthy [Based on the statistical analysis, catheter implantation led to decreased CSF flow rate within the spinal SAS, most notably under cervical implantation. In principle, a lower CSF flow rate is expected to decrease solute transport in the spine. Thus, we predicted that cervical catheter implantation would decrease solute transport to a greater degree than lumbar implantation. However, previous research , and our healthy  or disea healthy . OptimalBased on the results, it can be hypothesized that the impact of catheter implantation on CSF dynamics would potentially be greater in cynomolgus monkeys compared to adult humans due to relatively smaller SAS cross-sectional area in NHPs compared to humans . Th2 and ~6 ml, respectively within the SAS in humans [This study provides quantitative measures to investigate the impact of catheter insertion on intrathecal CSF dynamics and geometry in cynomolgus monkeys. Further studies should quantify the potential variance of these parameters in a larger study size across NHP species, in addition to including more diverse variables such as age, sex, weight, and disease states. Geometric characterization did not take into account spinal cord nerve root surface area or volume, which may account for ~231 cmn humans . It was There were also a few limitations in relation to CSF flow dynamics. First, CSF flow coupling with the cardiovascular cycle was accounted for in the present study. However, CSF flow has been shown to be impacted by respiration under normal breathing, forced breathing, and other maneuvers , which wCatheter implantation may have had an impact on CSF production and/or absorption rate. Unfortunately, this study did not measure CSF production and/or absorption and, we are therefore unable to make any conclusion about this potential impact. Finally, CSF flow was measured at six axial locations and interpolated to generate a smooth distribution along the spine. The ideal study would minimize or eliminate interpolation as much as possible by adding more axial slice locations. CSF dynamics should also be quantified within the intracranial space to better understand the exact distribution of CSF flow disruption that a spinal catheter may produce. However, the MRI time limitation for each NHP did not allow more slice measurement locations. The focus of the present study was on the intrathecal space because this region is commonly accessed by lumbar puncture or other relatively minimally invasive procedures for drug delivery to the central nervous system. Injection of medications within the ventricular space of the brain or cortical SAS would also be impacted by nearby CSF dynamics within the ventricles and cisterns of the brain.Fig 3) indicating a strong dependence among geometric parameters. This means that if one parameter shows a significant difference between two conditions or two-time points, some of the other parameters should also display a significant difference. On the other hand, if only one parameter shows a significant difference, such significance may be due to experimental error and may not be reliable. Therefore, although nine of the 42 p values in Table 3 are significant, they are not consistent with the dependence among the parameters and therefore should be interpreted with caution.The axial distribution for all geometric parameters tended to have a similar trend  to quantify the differences that occur based on catheter implantation location in the cervical compared to the lumbar spine. The overall findings were: 1) catheter insertion decreases CSF dynamics within the spine, 2) changes in CSF dynamics were greater for cervical implantation compared to lumbar catheter implantation, and 3) decrease in CSF dynamics persisted up to two weeks post-catheter implantation. In combination, these results support that intrathecal catheter implantation can adversely impact CSF flow dynamics in the spinal SAS in cynomolgus monkeys.S1 TablePRE-2) and after catheter implantation (MRIPOST-1 and MRIPOST-2).Data for all eight NHPs measured before catheter implantation (MRI(XLSX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


One patient showed sustained, highly increased NFL levels  in the initial assessment when radiological signs of axonal injury were still absent. Anti-SSA(Ro)-antibodies were found in the serum of three patients, while two patients additionally presented intrathecal anti-SSA(Ro)-antibody production. Elevated CSF-NFL levels and intrathecal synthesis of anti-SSA(Ro)-antibodies were associated with a relapsing and treatment-resistant disease course. Conclusion: Inflammatory spinal cord lesions associated with pSS are a rare but serious disease leading to severe disability. NFL and anti-SSA(Ro)-antibodies in CSF might serve as prognostic biomarkers and should be routinely assessed in patients with pSS.Objective: To identify radiological and laboratory hallmarks in patients with primary Sjögren’s syndrome (pSS) presenting with spinal cord involvement. Methods: Clinical and laboratory routine parameters were analyzed in a retrospective multicenter case series of four patients who developed myelitis associated with pSS. Serological and cerebrospinal fluid (CSF) measurements of pSS associated anti-SSA(Ro)-antibodies were initiated, and CSF neurofilament light chain (NFL) levels were assessed. NFL values were compared with results from 15 sex- and age-matched healthy controls. Radiological assessment was performed using multi-sequence spinal cord magnetic resonance imaging. Results: Three of the four patients initially developed neurological signs suggestive of myelitis and were subsequently diagnosed with pSS. All patients presented a longitudinal spinal T2-hyperintense lesion in the cervical spinal cord, whereas only two patients showed pleocytosis and oligoclonal bands in the CSF. Median (range) CSF-NFL levels were significantly elevated in patients compared to controls  vs. 585 pg/mL (357–729),  Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease characterized by lymphocytic infiltrations in secretory glands and extraglandular neurological manifestations ,2. The aAnti-SSA(Ro)-antibodies can be found in approximately 50% of patients suffering from pSS. These antibodies have a high diagnostic value and seem to be associated with extraglandular disease manifestations, although seronegative patients with CNS involvement have been described ,7,8,9. IThe levels of neurofilament light chain (NFL) in serum or cerebrospinal fluid (CSF) are thought to reflect neuroaxonal damage and are proposed to have a predictive value concerning outcomes in patients with inflammatory and autoimmune diseases associated with myelitis ,12.Thus, we aimed to identify clinical, radiological, and laboratory hallmarks, including new potential biomarkers, in a detailed case series of patients with pSS presenting with spinal cord involvement.Patients were recruited at the Departments of Neurology of the University Hospitals Magdeburg, Münster, and Hannover, all located in Germany. CSF and serum samples from 15 sex- and age-matched healthy controls recruited in Magdeburg were included for comparison. All controls reported non-specific complaints and underwent lumbar puncture (LP) during a routine diagnostic examination conducted to rule out any neurological condition. None of the controls was diagnosed with a specific neurological disorder or showed any specific abnormalities during the neurological examination. In addition to the clinical classification, all controls fulfilled the following laboratory criteria defining a normal CSF: < 5 cells/μL, < 2.0 mmol/L lactate, no disruption of the blood-CSF-barrier function , no oligoclonal bands (OCB), and no intrathecal synthesis of the immunoglobulins (Ig) IgG, IgA, and IgM . In addiFor all patients, a LP was performed at each respective study site according to standard protocols. The LP for all controls was conducted in Magdeburg. At each study site, CSF cells were counted manually using a Fuchs-Rosenthal chamber. Concentrations of Albumin, IgG, IgM, and IgA in serum and CSF were measured by kinetic nephelometry. Intrathecal synthesis of IgG, IgA, and IgM was calculated according to Reiber’s revised hyperbolic function. Isoelectric focusing was applied to detect CSF specific oligoclonal bands. All samples for CSF-NFL and antibody measurements were stored at −80 °C and shipped on dry ice. CSF-NFL levels were determined in Magdeburg using commercially available ELISA kits . Each measurement was performed together with a blank and a commercial positive and negative control provided by the manufacturer. Anti-SSA(Ro)-antibodies both in sera and CSF were measured in Hannover using EliA . Samples were measured in serial procedures and not in batches.Multi-sequence MRI (1.5 Tesla) of the spinal cord was performed in all patients and included a sagittal T2-weighted turbo-spin echo, a short tau inversion recovery sequence, and a T1-weighted postcontrast sequence covering the cervical and upper thoracic cord. Additionally, axial T2-weighted turbo-spin echo sequences were used. Furthermore, multi-sequence MRI of the head was performed in patients 1, 3, and 4, and included an axial T1- (pre- and postcontrast), a T2-weighted turbo-spin echo sequence and an axial diffusion-weighted imaging sequence. Moreover, a 3D sagittal T2-FLAIR-weighted sequence was applied in patients 1 and 4 and an axial T2-FLAIR-weighted sequence in patient 3.This study was performed according to the Declaration of Helsinki and approved by the local ethics committees . All patients gave written informed consent.Demographic, clinical, and laboratory findings are provided in A 39-year-old Caucasian female with known hypothyroidism (without evidence of a Hashimoto thyroiditis), alopecia of undeterminable cause, and sicca symptoms developed progressive sensory ataxia of the lower limbs and a mild paraparesis in 10/2017. MRI examinations of the brain and spinal cord were inconspicuous in 10/2017 and 12/2017. Lower limb somatosensory evoked potentials (SEP) showed axonal affection, whereas motor and sensory neurographies provided no evidence of neuropathy. Repeated CSF investigations revealed a pleocytosis (12–45 cells/µL) and OCB in CSF A. Immuno2 body surface area). During therapy, an additional left-sided T2-hyperintense lesion, without T1-gadolinium enhancement, was detected in the posterior column of the cervical and thoracic spinal cord -antibodies under treatment with hydroxychloroquine and intermittent use of oral glucocorticosteroids (GCS), developed a mild dysesthesia of the upper and lower limbs and slight sensory ataxia in 2016. Lower limb SEP, peripheral nerve conduction studies of upper and lower limbs, and CSF analysis were inconspicuous. The cervical MRI identified a T2-hyperintense longitudinally extensive spinal cord lesion without T1-gadolinium enhancement in the posterior column as the cause for neurological symptoms C,D. DiseA 55-year-old Caucasian female with known primary biliary cholangitis developed a progressive dysesthesia of the upper and lower limbs and a mild tetraparesis in 2018. The spinal cord MRI showed a long segment T2 lesion without T1-gadolinium-enhancement in the lateral pyramidal tracts and lateral spinothalamic tracts of the cervical spine E,F. The 2 body surface), oral GCS, and finally with rituximab , under which disease progression could be stopped.Pathological changes in lower limb SEPs were recorded, whereas nerve conduction examinations of upper and lower limbs showed no impairment of motor and sensory nerves. CSF measurements did not reveal inflammatory signs. In line with the evidence of elevated anti-alpha-fodrin antibodies, objective xerophthalmia, and a pathological minor salivary gland histology, an inflammatory spinal cord lesion associated with pSS was diagnosed. Therapy was initiated with IV cyclophosphamide -antibodies in sera and CSF. An elevated antibody specificity index (ASI) was detectable in patients 1 (2.2) and 4 (5.1), while patient 2 showed no elevated ASI (1.0). We could not detect anti-SSA(Ro)-antibodies in the serum or CSF of patient 3.Although CSF-NFL levels in patient 2 were normal , the medDue to the aggressive disease course involving the CNS, all patients were extensively assessed, including radiological, serological, and CSF analysis.Previous reports have described a rare occurrence of spinal cord manifestations associated with pSS ,19,20. TTo date, the relationship between anti-SSA(Ro)-antibodies and neurological manifestations in pSS is not sufficiently understood, in particular since both antibody-negative courses and isolated antibody-positive associated myelitis have been described ,19,24,25CSF-NFL measurements have recently been discovered to possess biomarker qualities for different neurological disorders and can be applied to assess early axonal damage and reflect early disease activity , even inIn conclusion, physicians need to keep in mind that in rare cases, imaging findings can be absent, particularly in the early disease course, or difficult to detect since fine longitudinal tracts can be involved in an isolated manner. However, absent radiological findings should not prevent from further diagnostic procedures necessary to identify a possible pSS. Moreover, our case series implies that CSF-NFL and intrathecal synthesis of anti-SSA(Ro)-antibodies are reliable and early markers for severe spinal cord involvement associated with pSS. We therefore strongly suggest that both values are routinely assessed to predict pSS-related CNS involvement even in the absence of radiological findings or systemic disease activity. Nevertheless, this study is limited because of its low number of analyzed patients, which is a common problem in rare diseases. Further investigations in larger cohorts are needed to verify the pattern of spinal cord affection and to clarify the association between the clinical course, NFL levels and the intrathecal production of anti-SSA(Ro)-antibodies.


Interactions between macrophages and adipocytes are early molecular factors influencing adipose tissue (AT) dysfunction, resulting in high leptin, low adiponectin circulating levels and low-grade metaflammation, leading to insulin resistance (IR) with increased cardiovascular risk. We report the characterization of AT dysfunction through measurements of the adiponectin/leptin ratio (ALR), the adipo-insulin resistance index (Adipo-IRi), fasting/postprandial (F/P) immunometabolic phenotyping and direct F/P differential gene expression in AT biopsies obtained from symptom-free adults from the GEMM family study. AT dysfunction was evaluated through associations of the ALR with F/P insulin-glucose axis, lipid-lipoprotein metabolism, and inflammatory markers. A relevant pattern of negative associations between decreased ALR and markers of systemic low-grade metaflammation, HOMA, and postprandial cardiovascular risk hyperinsulinemic, triglyceride and GLP-1 curves was found. We also analysed their plasma non-coding microRNAs and shotgun lipidomics profiles finding trends that may reflect a pattern of adipose tissue dysfunction in the fed and fasted state. Direct gene differential expression data showed initial patterns of AT molecular signatures of key immunometabolic genes involved in AT expansion, angiogenic remodelling and immune cell migration. These data reinforce the central, early role of AT dysfunction at the molecular and systemic level in the pathogenesis of IR and immunometabolic disorders. AT dysfunction plays a key role and is at the core of IR development . IR is aThe recently developed adiponectin/leptin ratio (ALR) correlates with IR better than adiponectin or leptin alone. It is strongly associated with surrogate measures of IR: the homoeostatic model assessment (HOMA) and the hyperinsulinemic-euglycemic clamp . This emThe most relevant age-related chronic immunometabolic diseases (type 2 diabetes [T2D] and CVD) associated with increased mortality are slowly progressive, associated with traditional CVD risk factors  and withAlthough fasting and postprandial dysglycemia, lipid-lipoprotein abnormalities, excess of body fat, and elevated systolic/diastolic blood pressure  are wellThe GEMM family study  is a bi-national, multi-centre collaborative study of CVRIMO related to T2D and the risk of cardiovascular and immunometabolic disease. GEMM’s study design characterizes detailed dynamic and function-based metabolic and molecular phenotypes in fasting and fed states  in symptom-free volunteers. Data are acquired from the circulation and from F/P adipose tissue and skeletal muscle biopsies, key tissues to understand F/P metaflammation, insulin action and carbohydrate/lipid homoeostasis. All measurements in blood are taken over a time course of 5 h to allow fine-scale profiling of individual postprandial responses .The aim of this paper is to introduce the methodology to characterize early, key contributors triggering the pathogenesis of AT dysfunction with data collected from the GEMM cohort. We studied the immunometabolic characteristics in the symptom-free female volunteers and compared the relation and trends of the high vs. low ALR with their F/P insulin-glucose axis, lipid-lipoprotein metabolism, and systemic inflammatory markers. In addition, we analysed whether their plasma non-coding exosomal  microRNAThe GEMM study involves an ongoing recruitment from 10 University-based metabolic research units and their affiliated teaching hospitals across Mexico, as described previously . The goan = 80) to determine AT dysfunction at a systemic and at a molecular level . They also provided blood samples at defined timepoints over a 5 h time course, as well as subcutaneous adipose tissue biopsies under local anaesthesia at fasting and 180 min postprandium to represent fasting and peak of postprandial response. The fasting biopsies were taken after a two-week interval rather than on the same day to avoid post-surgical inflammatory effects on the postprandial gene and protein expression as it could happen if both biopsies are taken the same day  using an established pipeline in Partek Flow.Total RNA was isolated from adipose tissue using the RiboPure kit (Applied Biosystems) after homogenizing the RNALater-stabilized tissues in TRI Reagent. The quantity and quality of the RNA samples were determined using a NanoDrop ND-1000 spectrophotometer. Samples whose A260/A280 ratio deviate ±0.2 from the accepted ratio of 2.0 were excluded. The quality/integrity of the RNA was assessed using the Agilent RNA 6000 Nano LabChip Kit and an Agilent 2100 Bioanalyzer (Agilent Technologies), ensuring that the 28 S and 18 s ribosomal RNA species are intact and that significant degradation had not occurred. The concentration of the resulting RNA samples was determined by using the NanaoDrop ND-1000 spectrophotometer. For synthesis of cRNA, the Illumina TotalPrep RNA Amplification Kit (Applied Biosystems) was used. The quality of the cRNA was assessed using the Agilent RNA 6000 Nano LabChip Kit and an Agilent 2100 Bioanalyzer (Agilent Technologies). cRNA quantity was measured using a NanoDrop ND-1000 spectrophotometer.Human WG-6 y3.0 Expression BeadChips for whole genome expression analysis. These BeadChips contain six arrays, each with >48,000 probes. Each array provides genome-wide transcriptional coverage of well-characterized genes, gene candidates and splice variants. About 1.8 million beads are available to quantify mRNA levels for each sample providing on average a 30x redundancy and therefore a very high precision of detection. This system uses a ‘direct hybridization’ approach, whereby gene-specific probes attached to beads on the array are used to capture and detect labelled cRNAs. After hybridization, detection of cRNAs was achieved by using an Illumina BeadArray™ reader. Gene expression data were generated using the BeadStudio software package. This application reports quality of performance based on built-in controls that accompany each experiment. The resulting ~48,000 quantitative measures were entered into a phenotypic database and additional statistical quality control procedures were performed to prepare these phenotypes for statistical analysis.We used commercially available Illumina are presented as mean ± standard deviation (SD) unless otherwise indicated. Differences between groups were analysed by two-tailed unpaired Student’s t-tests, as appropriate. The calculations were performed using SPSS23  and GraphPad Prism 6 . Scatter plot mathematical diagrams using Cartesian coordinates to display two-dimensional data visualization using dots were applied to represent association between two different variables.± 0.56) and (H) (3.2 ± 1.3) ALR. Mean plasma concentrations of total fasting adiponectin and leptin were 29.9 ± 21.6 ug/mL and 9.6 ± 5.0 ng/mL in the (H)ALR group, and 15.1 ± 7.12 ug/mL and 18.4 ± 8.6 ng/mL in the (L)ALR, respectively.Mean values in (H) vs. (L)ALR for weight, BMI, waist circumference and body fat percentage showed that these anthropometric measurements were higher in the (L)ALR group. Age showed that the (L)ALR group was younger . Most meP-values were statistically non-significant.Proinflammatory fasting systemic biomarker levels between the (H) and (L)ALR groups are shown in p = 0.01), insulin and GLP-1 curves, except for the glucose curve. The area under the curve (AUC) for glucose in the (H) ALR group compared to the (L) ALG showed a 5% difference indicating that they were practically similar. On the contrary, the AUC for insulin and GLP-1 in the (H) ALR group compared to the (L) ALG, respectively, showed an 85% and 28% differences. Systemic lipid metabolism also showed an increase in the (L)ALR symptom-free females for NEFA. The adipose tissue-insulin resistance index (Adipo – IRi) (p = 0.01) was significantly higher in individuals with a (L)ALR. The fasting liver transaminases aspartate transaminase (AST/SGOT) (p = 0.01) and alanine transaminase (ALT/SGPT) (p = 0.01) were also significantly higher in individuals with a (L)ALR ALR subjects  for HOMAa (L)ALR .Figure For this paper, we decided to present results from fed and fasted samples that underwent shotgun lipidomics only for specific classes considered proinflammatory: Lysophos phatidylethanolamine (LPE), lysophosphatidylcholine (LPC) and Ceramide (Cer) as shown in Comprehensive fed/fasted circulating miRNA profiling was performed in this initial sample. We detected 2974 miRNAs as described in the methods. LEP, TNFaIP1, CD86, FABP4, TGFB1) and anti-inflammatory genes  at 0 min (fasted) and 180 min (fed). Values are shown in ANG (P = 0.03).We also quantified mRNA levels of F/P subcutaneous adipose tissue for whole genome expression analysis. We were able to directly measure and characterize key proinflammatory  IR and an overt metabolic syndrome. It has been stated that the body mass index (BMI) , waist cUsing deep phenotyping  such as A pattern of increased circulating leptin concentrations along with a decreased levels of adiponectin seems indicative of an impaired adipose tissue adipokinome, as it was found in the subgroup with a (L)ALR  41]. As. As41]. These constant elevated postprandial levels of insulin in the symptom-free females with a (L)ALR may explain why the postprandial glucose curve and the AUC results in the same group were normal, corroborating that in nondiabetic individuals, the β-cells can compensate for resistance to insulin-mediated glucose disposal and maintain normoglycemia at the expense of increased levels of insulin that, unfortunately, is prothrombotic . These fThe appearance of deleterious cardiovascular and immunometabolic risk phenotypes has been the main concern regarding body fat accumulation . HoweverMetabolically driven inflammation is a hallmark of CVD and T2D . TNF-α, i = plasma-free fatty acids (NEFA) x fasting plasma insulin [FPI] [mmol/L/pmol/L]) is calculated based on the linear relationship between the rise in the FPI level and inhibition of the rate of fasting plasma NEFA [i markedly elevated in the (L)LAR subjects when compared to the (H)LAR ones  have earned great deal of attention not only for their ability to regulate adipogenesis and adipose function, but also for their presence in circulating blood leading to potential tools as diagnostic biomarkers . Table 4pression . Mir-378pression . miR-375pression . miR-140pression . miR-33apression . Lower epression . The letpression .LEP, TNFAIP1, CD86, RBP4 and TGFβ and anti-inflammatory ADIPOR1, CD163, IL10, HIF1AN and ANG activity to directly measure the balance of the inflammatory/anti-inflammatory response during the development of cellular adaptive responses in early adipose tissue expansion and remodelling have not been fully elucidated [LEP and TNFAIP1 increased in the (L)ALR subjects. In activated macrophages, proinflammatory M1 and M2b are the main cell types expressing and secreting TNF‐α [CD86 gene expression was decreased in subjects with (L)ALR. Several studies have shown that CD86 is expressed in and used as a marker to identify M1 pro-inflammatory and polarized macrophages [RBP4) is secreted by adipocytes, is increased in obese and insulin resistant subjects and induces proinflammatory cytokines through the JNK and TLR-4 pathways in macrophages [RBP4 did not show any apparent change in expression in either (H) or (L)ALR participants. TGFβ is a master regulator and promoter of fibrosis in adipose tissue [TGFβ expression were found in fasting in (H)ALR participants, decreasing in the fed state. TGFβ can also be anti-inflammatory and promote M2-like macrophage activity that localize to fibrotic areas of adipose tissue [Temporal gene expression changes during the fasted and fed state in proinflammatory ucidated . We measng TNF‐α . Unexpecrophages . Retinolrophages . RBP4 die tissue . Elevatee tissue .ADIPOR1 receptor expression showed a slight increase in the postprandial (L)ALR group. This and several other papers [CD163 and IL10 postprandial gene expression in the same group. Macrophages with CD163 expression are considered anti-inflammatory M2 macrophages [IL-10 is decreased in subjects with impaired glucose tolerance and obesity [ANG is an important group of vascular remodelling angiogenic factors expressed in adipose tissue that control vessel maturation, patterning, and stabilization [ANG in the postprandial (H)ALR subjects with apparently no change in the (L)ALR group.We also characterized the pattern of expression for AT anti-inflammatory genes . The ADIr papers  have shor papers . In addir papers . A modesrophages . IL-10 i obesity . This re obesity . ANG is lization . Local hlization . We founSome potential limitations of this study should be pointed out. First, due to the small number of subjects, these findings should be interpreted with caution and considered as hypotheses generating. These results should be confirmed by studies with a larger number of subjects. Second, as we used the ALR  to compaWe are beginning to understand the importance of the complex interactions between inflammation, the extracellular matrix (ECM), and angiogenesis in the context of AT dysfunction. Indeed, our study was carried out in a group of symptom-free normoglycemic, mainly normal-weight women with no history of age-related chronic diseases associated with immunometabolic abnormalities. Moreover, as IR can precede the dysglycemic states of prediabetes and type 2 diabetes mellitus (T2DM) by a number of years and is an early marker of risk for immunometabolic and cardiovascular disease, our early research findings raise several important questions: Does the ALR predict early cardiometabolic risk? Does the elevation of the inflammatory markers  in these symptom-free individuals relate to early risk for endothelial dysfunction and cardiovascular disease? Do the postprandial insulin-glucose axis abnormalities, HOMA-IR and adipo-IRi elevations reflect early risk for prediabetes? Could the correlations of systemic lipid species and microRNAs along with the direct molecular characterization of adipose tissue immunometabolic function lead to early biomarkers of risk for metabolic and cardiovascular disease before the development of frank insulin resistance? It is expected that concrete answers to this question will pave the way for the identification of novel biomarkers to diagnose adipose tissue dysfunction perhaps without the need to fully account for adipose tissue accumulation in a subgroup of symptom-free individuals.A valid take-home message relates to the 1988 American Diabetes Association Banting award lecture , where tIn conclusion, there is a major demographic and epidemiologic change taking place in the U.S. and worldwide. The preventive approach based on single diseases towards symptom-driven medicine is becoming out-of-date. A precision and personalized medicine linked to the identification of early risk and prevention instead of identification of curative pathological symptoms in immunometabolic and cardiovascular disease is rapidly taking place. Optimism to achieve success is in the horizon due to the overwhelming advancement of genomic medicine, particularly integrative systems biology through multi-OMICS technology and definitions of cardiovascular, metabolic and immune disease risk deep phenotypes for early detection of age-related chronic diseases associated with immunometabolic pathology.


In PD patients, there were no correlations between CSF NFL level and the measures of disease severity. The CSF NFL turned out to have a high discriminatory value (AUC 0.850) for differentiating between PD subjects and healthy controls, with 84% sensitivity and 85.2% specificity. The study indirectly demonstrates that axonal damage is present in early PD in addition to neuronal loss. Interestingly, white matter damage was observed in non-demented PD patients. In the light of the results of recent MRI studies which confirm early white matter damage in PD, our data may turn out to be potentially useful in the diagnosis of early, or even preclinical, stages of the disease.Parkinson’s disease (PD) is a chronic neurodegenerative disorder with various underlying pathological processes. Until now, no fluid biomarkers have been established for PD. Given recent biochemical and neuroimaging evidence for the presence of white matter damage in PD, which may even precede neuronal loss, we investigated whether neurofilament light (NFL) was increased in the cerebrospinal fluid (CSF) of PD patients in comparison to controls. NFL is located mainly in large myelinated axons, and increased CSF levels of this protein reflect axonal injury. CSF levels of NFL in 58 early PD patients and 28 controls were quantified by ELISA (Uman Diagnostics). Measures of PD severity included disease duration, UPDRS-III, and Hoehn-Yahr stage. Statistically significant differences in CSF NFL levels were found between PD patients and controls [median with interquartile range 524.82 (393.28–678.34) vs. 271.84 (198.09–335.24) ng/l;  Parkinson’s disease (PD) is a chronic neurodegenerative disorder with various underlying pathologies. Gray matter integrity loss in PD has been extensively investigated . Neurofilament (NF) proteins are highly phosphorylated proteins of the neuronal cytoskeleton. NF in the CNS is composed of four subunits (or chains): NFL, NF middle (NF-M), NF heavy (NF-H), and α-internexin  NFL has an established position as a biomarker in the differential diagnosis between PD and atypical parkinsonism  who were admitted to the Department of Neurology of the Medical University of Lublin, Poland, were enrolled. All PD patients who gave their informed consent were assessed on the UPDRS  and gender-matched healthy controls were collected. The control subjects were in-patients at the Department of Neurology, who required lumbar puncture mainly due to severe headache, to exclude subarachnoid hemorrhage or meningitis and had a pathological process in the CNS excluded.Also, CSF samples from 28 age-matched , and all study participants gave written informed consent for participation in the study.t-test were used, respectively, to compare gender and age between PD patients and controls. For comparisons of CSF NFL levels between groups, the U Mann–Whitney test was performed.Data with normal distribution were presented as means with standard deviation (SD); the minimum and maximum values were also shown. Because CSF NFL values were not normally distributed, the data for CSF NFL were presented as medians with interquartile ranges. Chi-square test and student’s The correlations between CSF NFL and age, disease severity, disease duration, and levodopa dose were assessed using rho Spearman correlation coefficients.The diagnostic accuracy of CSF NFL in differentiating between PD and healthy subjects was calculated based on the area under the curve (AUC) of the receiver operating characteristic curve.Cut-off values were calculated using Youden’s index, which maximizes the sum of sensitivity and specificity.p < 0.05. Statistical analysis was performed using Graph-Pad.Statistical significance was set at p > 0.05). Clinical and biochemical characteristics of the study population is presented in p < 0.05; The PD and healthy control groups were matched for gender and age, and no statistically significant differences between the investigated groups were found in terms of age (p = 0.943) or UPDRS part III (p = 0.951).In PD patients, no correlation was found between CSF NFL levels and disease severity assessed on the H-Y scale (p = 0.937), overall levodopa dose (p = 0.557), or levodopa equivalent (p = 0.735).Also, no correlations were found between CSF NFL levels and disease duration . Interestingly, this correlation was not observed in healthy controls. Correlation coefficients between CSF NFL and clinical variables are presented in the In the group of PD patients, there was a significant positive correlation between CSF NFL levels and age  between PD subjects and healthy controls, with 84% sensitivity and 85.2% specificity at a cut-off value of 350.66 ng/l .The fact that there exist established CSF biomarkers for Alzheimer’s disease  spurs interest in finding a specific CSF biomarker for PD to support the clinical diagnosis of this disease. Many studies have focused on the quantification of α-synuclein in CSF of patients with parkinsonian syndromes, but the results preclude its use as a biomarker of PD , as there is a high age-dependency of CSF NFL levels. Of course, we cannot exclude that the elevated CSF NFL levels reflect neuronal degeneration with the release of disrupted NFL from body cells. Normally, phosphorylated NF is localized mainly in the axons of healthy neurons. Nevertheless, in amyotrophic lateral sclerosis, another neurodegenerative disorder, pathological disruptions of the NFL, taking the form of spheroids, are localized also in proximal axons and body cells .In our cohort of PD patients, the CSF NFL level correlated positively with age, which suggests that age also contributes to some degree to axonal degeneration. This positive correlation was not observed in healthy controls. This could be explained by the fact that all our control subjects were above 50 years old, so subjects with limited age range were involved in this study (p > 0.05).A positive correlation between NFL and age in PD subjects was also found in other studies (Oosterveld et al., Further studies in a larger cohort of patients in different stages of the disease, using CSF and serum samples, should address the question of whether NFL levels may correlate with disease severity and disease progression.Our study has some limitations. Firstly, PD was diagnosed based on clinical symptoms and not a neuropathological examination. Secondly, our study involved a limited number of PD patients, and only ones with early-stage PD. This could be the reason for the lack of correlations between CSF NFL and the markers of disease severity.This study indirectly demonstrates that axonal damage is present in PD in addition to neuronal loss. Interestingly, we observed white matter damage in non-demented PD patients. In the light of recent MRI studies that also report early white matter damage in PD which may even precede neuronal loss, our data may turn out to be of potential use in diagnosing early or even preclinical stages of the disease.All datasets generated for this study are included in the article.The studies involving human participants were reviewed and approved by Ethics Commitee at the Medical University of Lublin, Poland. The patients/participants provided their written informed consent to participate in this study.EP: conceptualization, data collection, investigation and writing—original draft. EP and KR: formal analysis and methodology. KR: software and writing—review and editing.s The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intrathecal immunoglobulin M (IgM) synthesis has been demonstrated in the early disease stages of multiple sclerosis (MS) as a predictor factor of a worsening disease course. Similarly, increased cerebrospinal fluid (CSF) molecules related to B-cell intrathecal activity have been associated with a more severe MS progression. However, whether CSF levels of IgM are linked to specific inflammatory and clinical profile in MS patients at the time of diagnosis remains to be elucidated.Using customized Bio-Plex assay, the protein levels of IgG, IgA, IgM, and of 34 other inflammatory molecules, related to B-cell, T-cell, and monocyte/macrophage activity, were analyzed in the CSF of 103 newly diagnosed relapsing–remitting MS patients and 36 patients with other neurological disorders. CSF IgM levels were also correlated with clinical and neuroradiological measures [advanced 3-T magnetic resonance imaging (MRI) parameters], at diagnosis and after 2 years of follow-up.p = 0.013). CSF IgM levels correlated with higher CSF levels of CXCL13 (p = 0.039), CCL21 (p = 0.023), interleukin 10 (IL-10) (p = 0.025), IL-12p70 (p = 0.020), CX3CL1 (p = 0.036), and CHI3L1 (p = 0.048) and were associated with earlier age of patients at diagnosis (p = 0.008), white matter lesion (WML) number (p = 0.039) and disease activity (p = 0.033) after 2 years of follow-up.A 45.6% increase in CSF IgM levels was found in MS patients compared to controls  and recruitment (CXCL13 and CCL21) and to macrophage/microglia activity  suggests possible correlation between humoral and innate intrathecal immunity in early disease stage. Furthermore, the association of IgM levels with WMLs and MS clinical and MRI activity after 2 years supports the idea of key role of IgM in the disease course. The presence of oligoclonal bands (OCBs) in the cerebrospinal fluid (CSF), but not within the serum, is a strong indicator of intrathecal antibody synthesis  and has in vitro , stronglin vitro , such asin vitro . The relin vitro . More rein vitro . These ein vitro . HoweverThe objective of this study was to evaluate the presence of IgM, and others immunoglobulins (IgG and IgA) in the CSF of both MS patients and controls with other neurological disorders. Furthermore, we investigated the existence of specific correlations between CSF IgM levels and the CSF inflammatory profiling at the time of diagnosis and the clinical and MRI activity in addition to baseline, even after 2 years of follow-up in a cohort of MS patients.We recruited 103 treatment-naive RRMS patients , followed at the MS Centre at Verona University Hospital , who received a diagnosis of MS from January 2012 to December 2019. All MS patients underwent at the time of diagnosis (T0) a detailed neurological evaluation including the Expanded Disability Status Scale (EDSS) assessment  and the Demographic, clinical, and MRI data of MS patients are reported in Thirty-six age- and sex-matched available patients affected by other neurological diseases, who underwent neurological evaluation and CSF examination at the time of the diagnosis, were included in the study. This group included 21 individuals with non-inflammatory neurological diseases, NIND , and 15 subjects with other inflammatory neurological diseases, OIND .The Ethics Committee of the University of Verona approved the study, and informed consent was obtained from all participants.CSF samples were obtained at least 1 month after the last relapse and within 2 months from the MRI acquisition, according to the Consensus Guidelines for CSF and Blood Biobanking . Once coAt the time of diagnosis, 3-T MRI was performed in all MS patients, and this was repeated 2 years after diagnosis on 70 of these patients. MRI sequences were acquired at the Radiology Unit of the University Hospital of Borgo Trento  using a Philips Achieva 3T MR Scanner  as previously described . The fol•3D T1-weighted turbo field echo [repetition time (TR)/echo time (TE) = 8.4/3.7 ms, voxel size of 1 mm × 1 mm × 1 mm), total acquisition time of 5:51 min;•3D double inversion recovery (DIR)  TI1/TI2 = 525/2,530 ms voxel size of 1 mm × 1 mm × 1 × mm), turbo spin echo (TSE) readout with an optimal variable flip angle scheme and number of excitations  , same TSE readout as the DIR sequence, number of excitations 1, with total acquisition time 5:44 min.The number of T2 hyperintense white matter lesions (WMLs) and CLs were identified on FLAIR and DIR images by an observer with a large experience on MS. The number of CLs was assessed following the recommendations for CLs scoring in patients with MS . Owing tU test was used to test differences between MS patients and control group, as well as differences between MS patients stratified by CL number at diagnosis  and by the presence or not of the EDA after 2 years of follow-up. Analysis of variance (ANOVA) followed by post hoc pairwise comparison using the Tukey test was used to evaluate difference among immunoglobulin CSF levels in MS patients.Mann–Whitney U test. p < 0.05 was considered statistically significant. GraphPad (version 5.0) and R software (version 3.5.3) were used to perform the analysis.Pairwise univariate Spearman rank index was used to evaluate the correlation between CSF IgM levels and demographic and clinical MRI parameters (both at diagnosis and after 2 years of follow-up) and several inflammatory/immune-mediated pathways. Differences between males and females and between those patients with or without OCBs were tested by Mann–Whitney Intrathecal levels of IgG, IgA, and IgM were investigated in the CSF of 103 RRMS patients and 36 controls with other neurological disorders .p < 0.001; p < 0.001; p = 0.013; p = 0.360; p = 0.700; R = −0.12, p = 0.270) between CSF IgM levels and blood–brain barrier alteration, suggesting that IgM intrathecal levels were not related to peripheral ones.CSF IgG levels in MS patients were significantly higher compared to IgA  were significantly higher in patients with EDA compared to NEDA , to obtain a simultaneous, sensitive, and reproducible evaluation of immunoglobulins and several other inflammatory mediators in the CSF of MS patients. Despite further studies are needed to confirm these preliminary results, the sensitivity and reproducibility of this easily performed procedure could allow extending such a detailed CSF proteomic analysis to the clinical practice.This study is not without limitations. The low number of patients recruited, compared to the number of parameters analyzed, and the limited MS phenotypes suggest that these data need to be further validated, considering an independent validation cohort, and confirmed by other studies. Moreover, only 2 years of follow-up have been analyzed at the moment in order to select only MS patients treated by first-line therapies, in order to avoid further confounding factor of the correlation analysis. All together, these limitations suggest that this study needs to be confirmed by further analysis of a larger sample size and longer follow-up and paired serum samples.In this study, we used a comprehensive, advanced proteomic approach for quantitative and qualitative CSF protein analyses in combination with clinical and radiological assessment of MS patients, demonstrating that intrathecal IgM levels are increased in the CSF of treatment-naive MS patients compared to controls and that there is significant correlation between CSF IgM levels and further CSF molecules related to B-cell, macrophage, and microglia activity. Moreover, we found an association between CSF IgM production and the number of new WMLs and MRI activity after 2 years of follow-up, suggesting that CSF IgM levels might reflect the relationship between humoral and innate intrathecal immune response in MS and might represent an early biomarker of underling disease activity.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The Ethics Committee of the University of Verona approved the study. The patients/participants provided their written informed consent to participate in this study. Biological material and associated data were obtained from MSBioB Biological bank – A.O.U.I., Verona . Biological material was obtained from voluntary donors in compliance with the Legislative Decree 196/2003 “Personal Data Protection Code.”RM and MC contributed to the conception and design of the work. RM, VM, LM, AP, DM, AT, and SR contributed to the acquisition, analysis, and interpretation of data for the work. RM, VM, LM, AP, DM, and FC contributed to the data interpretation. RM, VM, LM, AP, DM, AT, and MC contributed to writing the manuscript. RM, VM, DM, FC, AP, AT, and MC contributed to revise the manuscript. All authors provided approval for publication of the final content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ultrasound examinations of atherosclerotic carotid plaques can be used to calculate risk markers associated with plaque vulnerability. Recent studies demonstrate significant inter-frame variability in risk markers. Here, we investigate risk marker variability in symptomatic plaques and its impact on reclassification of plaque vulnerability, as well as its association with the shape of the temporal variation over the cardiac cycle.56 patients with symptomatic carotid stenosis were included in this study. 88 plaques were identified and the plaque risk markers size (area), echogenicity  and heterogeneity (coarseness) were measured in all frames of ultrasound B-mode image sequences. Inter-frame variability was quantified using the coefficient of variation (CV).2), whereas random pattern was found in larger plaques. In addition, hypoechoic plaques (GSM < 25) were associated with cyclic variation pattern, independent of their size.Inter-frame variabilities of the risk markers were area CV 5–8%; GSM CV 4–7%; coarseness CV 8–15% and was in general significantly lower in large as compared to smaller plaques. The variability in GSM risk marker caused a reclassification of vulnerability in 30 to 38% of the plaques. Temporal variations in GSM with a heart rate periodic or drift/trending pattern were found in smaller plaques (< 26 mmRisk marker variability causes substantial reclassification of plaque vulnerability in symptomatic patients. Inter-frame variation and its temporal pattern should be considered in the design of future studies related to risk markers. As one of the leading causes of death and disability worldwide, stroke constitutes a major threat to public health . About 8Ultrasonographic techniques are commonly used methods for detection and grading of carotid stenosis in clinical investigation , 4. In oAlthough proven clinically relevant, the risk markers have known issues with sensitivity and specificity for risk prediction , and in Previous research has shown significant inter-frame variability in GSM (and other risk markers) in ultrasound image sequences during the cardiac cycle –12 in boHowever, this risk re-classification remain to be explored for symptomatic plaques . Also, while significant differences in GSM have been shown between systolic and diastolic phases , 12, theThe aim of this study was to investigate risk marker variability in participants with symptomatic carotid plaques and assess the impact of variability on risk re-classification, and the association between variability and temporal characteristics of the risk markers (e.g. cyclic or random). In addition, we evaluated the influence of size and echogenicity on the risk marker variability.In this study, patients were retrospectively selected from the prospective cohort study of ANSYSCAP  . We seleThe ANSYSCAP-study, making its inclusion of patients between 1 August 2007 and 31 December 2009, was reviewed by the regional ethics committee at Umeå University before it was conducted. They concluded that the study did not require committee approval since it was strictly observational.The examinations were performed by experienced ultrasonographers, following the protocol of standard US clinical settings, using a conventional system  with a 8L5 linear transducer, projecting towards the plaque in a longitudinal view. The mean duration of the B-mode sequences was 2.0 s and the frame rate ranged from 12 to 26 Hz.Based on the framework originally described by Nicolaides et al. , a plaquIn short, a plaque was manually delineated in an B-mode image, and then two region of interests (ROIs) were manually selected in the darkest area of the lumen and the brightest area of the adventitia, respectively. The image was then automatically normalized to 190 in the adventitia and 0 in the lumen based on the intensities of the two ROIs, and pixel density was standardized to 20 pixels/mm , 13. TheThe risk markers chosen for variability analysis were measurements of echogenicity (GSM), size (area), plaque type and heterogeneity (coarseness). The risk markers were analyzed in longitudinal images, according to guidelines , and theIn addition, an un-normalized GSM was calculated , 11 as tStatistical analysis was carried out using MATLAB R2015a .Risk marker variability was quantified by computing the coefficient of variation (CV) for every risk marker in all of the frames constituting a plaque image sequence. Further, the temporal pattern of the variability was assessed using the median frequency descriptor (MDF), calculated as the frequency that divides the power spectrum of the GSM variability signal in equal halves see Fig. g and hasU-test was used to assess whether the CV of the risk markers differed between the dichotomized groups.Furthermore, to determine how the variability is influenced by plaque size, echogenicity and temporal pattern, the collection of values for area, GSM and MDF, respectively, were each divided at the median into dichotomized groups. Then, an unpaired, nonparametric Mann–Whitney p value was set to 0.003 by Bonferroni correction (0.05/16) because of multiple comparisons.Lastly, the effect of variability on vulnerability reclassification was investigated by counting the number of plaques that changed from plaque type ≤ 2 to ≥ 3 or crossed specific threshold levels in GSM. Consequently, for analysis of plaque type the cut-off value was ≤ 2 , and theIn order to assess inter-rater reliability, two operators (E.S. and E.N.) independently carried out analysis of 20 randomly selected plaque image sequences. Intra-rater reliability, on the other hand, was evaluated by the intra-class correlation coefficient (ICC). With a confidence interval of 95%, ICC was measured by letting one operator analyse 20 randomly selected sequences twice. The time that had passed before reanalysis was six months.Characteristics of the 56 included subjects are summarized in Table 2, GSM from 5.6 to 130.9, un-normalized GSM from 8.0 to 93.7, coarseness from 2.8 to 26.5 and MDF from 0.4 to 5.9 Hz. The variabilities for area, GSM, un-normalized GSM and coarseness were, on average, 6.6%, 11.8%, 5.6% and 11.6% respectively.Plaque area ranged from 8.9 to 78.0 mm2, 8%) than for large plaques  (p < 0.001). No significant differences in the CV for GSM were found between small and large plaques. However, for un-normalized GSM, the CV value was significantly higher for small plaques (7%) than for large plaques (4%) (p = 0.001) . Regarding coarseness, however, the mean value was higher for echogenic plaques (16) than for echolucent plaques (11) (p < 0.001). Moreover, the CV for coarseness was higher for echolucent plaques (15%) than for echogenic plaques (8%) (p < 0.001)  than for echogenic plaques  (1) Table .2, p < 0.001).Plaques with higher median frequency  had significantly larger area as compared with plaques with MDF < 1.3 Hz, corresponding to slow changes in the temporal patterns  Table .Moreover, hypoechoic plaques with GSM < 25 had a MDF of their temporal variation pattern in the range 1–2 Hz independent of size, as compared to plaques with GSM > 25 , 0.93 (95% CI 0.88–0.96) and 0.83 (95% CI 0.71–0.91), respectively. The inter-rater reliability ICCs for area, GSM and coarseness were 0.71 (95% CI 0.50–0.81), 0.72 (95% CI 0.49–0.87) and 0.83 (95% CI 0.68–0.92), respectively.We investigated the frame-by-frame variations of risk markers for carotid plaques in symptomatic patients and its influence on plaque reclassification and shape of temporal variations . The variability of plaque risk markers within the ultrasound sequences was found to range from 6 to 12% and caused reclassification of plaque vulnerability in 30% or 38% of cases, depending on cutoff value.The impact of the variability in GSM on risk classification showed that 30% or 38% of plaques were reclassified, having at least one frame with GSM value crossing the classification cut-off value. This is similar to a previous study by our group on asymptomatic plaque variability causing 16–25% reclassification . To quanRapid changes in temporal variation (high MDF) with lower amplitude  was found in large plaques as compared with small plaques that had slower temporal variations and higher amplitude Table . This inHypoechoic plaques with GSM < 25 had a low MDF independent of their size. The cutoff value originates from the modified Geroulakos classification , which iOur results show that smaller plaques had a higher variability in plaque area than larger plaques. This is similar to the results of a previous study by Nyman et al.  but contMoreover, the difference in variability for un-normalized GSM could not be seen for normalized GSM. This could be caused by the propagation of errors during the many steps included in the risk marker assessment .The results of this study on symptomatic plaques together with a previous study by our group on asymptomatic plaques , as wellp = 0.003 compared to limit p = 0.0025), echolucent plaques had a higher variability in GSM than echogenic plaques. This could be explained by the fact that echogenic plaques are less elastic due to their composition of fibrotic and calcified tissues, and echolucent plaques are more elastic due to the lipid-rich composition and may deform easier by variations in blood pressure during the cardiac cycle [Although there was not a strict significant difference  is indicated to be related to plaque vulnerability. Inter-frame variation and its temporal pattern should be considered in the design of future studies related to risk markers.


Escherichia coli and is PEGylated at N-terminal. Besides neutropenia, G-CSF is also used in bone marrow transplantation for the mobilization and maturation of peripheral blood stem cells. Considering the requirement of producing G-CSF therapeutic in large quantities, construct designing for high expression is critical for the biopharmaceutical and industrial application. Earlier studies have employed approaches such as codon optimization, use of strong promoters, employment of protein tags, secretion signals, optimization of protein folding, etc., for increasing expression and yield of therapeutic proteins. In this study, it was observed that mRNA transcribed from the native human cDNA of G-CSF and the codon-optimized variant leads to low protein expression in E. coli. To understand the underlying reasons, the mRNA secondary structure of the 5′ end of the G-CSF transcript was analyzed. This analysis revealed the presence of stable secondary structures at the 5′ end of the G-CSF transcript, arising from the native human gene and even from the codon-optimized sequence. These secondary structures were disrupted through translationally silent mutations within the first 24 nucleotides of the transcript without affecting the protein sequence. Interestingly, through this approach, the G-CSF protein expression was increased 60 folds as compared to native G-CSF construct. We believe that these findings create a roadmap for optimization of G-CSF transcript for enhanced expression in E. coli and could be employed to increase the expression of other therapeutic proteins.The human granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor used to prevent and treat neutropenia. G-CSF stimulates the bone marrow to produce infection-fighting granulocytes. Food and Drug Administration of the United States approved G-CSF in 1991 and its PEGylated version in 2002 as a prophylactic and therapeutic measure against neutropenia. Recombinant human G-CSF is produced in surrogate host  Human granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that plays a critical role in the stimulation, proliferation, mobilization, maturation and activation of granulocytes, including neutrophils . NeutropEscherichia coli is utilized. The recombinant G-CSF differs from the native human G-CSF through the addition of N-terminal methionine for facilitating the expression in the surrogate host, and it lacks inherent glycosylation at Threonine-133 as observed in humans. However, the recombinant G-CSF possesses similar biological activity as that of the native G-CSF  using forward primer SD1 and reverse primer SD2 (primer sequences are provided in E. coli BL21 (DE3) from Novagen.The human G-CSF cDNA, codon-optimized gene sequence and cloning details are as provided earlier . BrieflyE. coli. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce protein expression. The bacterial pellet was disrupted by sonication. After centrifugation, the supernatant (soluble fraction) and the pellet (insoluble fraction) were analyzed using 12.5% polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Brilliant Blue. G-CSF was purified using the method reported earlier in The G-CSF constructs used in the study were cloned in pET 23a which is a T7 RNA polymerase inducible promoter based expression vector and were transformed in the BL21 (DE3) strain of TM cDNA Synthesis Kit from Bio-Rad. Different primer sets covering full, 5′ and 3′ regions were used to check mRNA expression levels.The RNA was extracted after IPTG induction using the RNeasy Kit from Qiagen (Cat No. 74104). The RNA extracted was also treated with RNase-Free-DNase from Qiagen (Cat. No. 79245). cDNA from different constructs were synthesized using the iScriptPurified G-CSF was analyzed using 15% SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). Western blot analysis was performed to confirm the identity of the purified G-CSF protein. After running purified recombinant G-CSF protein and the commercially available product on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the gel was electro-transferred onto the PVDF membrane. The membrane was blocked with 2% milk powder or 1% BSA in 1X PBS  for 1–2 h. The blocking solution was removed, and the blots were incubated for 1 h with a monoclonal antibody (mAb) against human G-CSF at 1:1000 dilution in 1X PBS-T. The intact molecular mass of G-CSF and the control was determined using liquid chromatography-mass spectrometry (LC-MS) Agilent 6550 system. Circular dichroism (CD) spectrometry was performed, as mentioned earlier on the Jasco J-815 spectropolarimeter .in vitro biological activity of G-CSF was determined by measurement of cell proliferation assay using the M-NFS-60 cell line (ATCC CRL- 1838). The cells were treated with various concentrations of WHO G-CSF standard and E8 G-CSF for 48 h and metabolic activity was assessed using standard XTT assay protocol. The cells were grown in RPMI-1640 medium containing 10% fetal bovine serum, 1X penicillin and streptomycin, 0.05 mM β-mercaptoethanol and 62 ng/ml of human recombinant macrophage colony-stimulating factor (M-CSF) as recommended by ATCC. M-NFS-60 cells  were seeded into a 96-well flat-bottom plate containing 2% FBS and serum-starved for 24 h. Varying concentrations of GCSF and G-CSF standard  were added to each well. G-CSF formulation buffer was used as a control. Each concentration was set up in triplicate. After 48 h of incubation, XTT was added to each well, and the cells were kept for incubation at 37°C for 4 h. The optical density of the 96 well plate was measured at 490 nm and at 650 nm to normalize the blank reading using an ELISA reader. Data were plotted using GraphPad Prism 6 for Windows, Version 6.05. The standard error of the mean (SEM) was calculated.The E. coli. Toward this, the cDNA sequence of human G-CSF was retrieved from NCBI GenBank using accession number M13008.1. This sequence was codon-optimized and synthesized  for expression in E. coli. Both the native and the codon-optimized sequences were PCR amplified and cloned in pET-23a. The constructs were transformed in E. coli BL21 (DE3) strain to analyze the protein expression level. Protein expression from both the constructs was analyzed using SDS-PAGE analysis. It is important to note that the expression of recombinant G-CSF using native human sequence was extremely low, and even the codon-optimized sequence results in low expression  with eight translationally silent modifications for further analyses . The traanscript . No signanscript .The E8 variant, along with the native G-CSF and codon-optimized constructs, were compared for the protein expression levels to determine the effect of translationally silent mutations on the translational efficiency. Interestingly, a substantial increase in the protein expression from the modified transcript was observed. The enhanced G-CSF protein expression was observed in the E8 variant . To quanThe purified protein was analyzed using SDS-PAGE . Westernin vitro biological activity of G-CSF protein using a G-CSF responsive M-NFS-60 cell line. The treatment of M-NFS-60 cells to increasing concentration of G-CSF induces a dose-dependent proliferation of M-NFS-60 cells. In this study, the WHO G-CSF standard obtained from National Institute for Biological Standards and Control (NIBSC) was used as a reference. The results demonstrate that E8 possesses similar cell proliferative activity/potential as that of the G-CSF standard  PLysS strain was utilized. The productivity was increased 1.5 folds  of the modified sequence (in vitro biological activity was also studied and was comparable with WHO standard of G-CSF protein. Thus the transcript engineering involving translational silent mutations results in enhancing the protein expression and biologically active protein.In the light of this literature, this study was initiated with the objective of engineering G-CSF transcript for enhanced recombinant G-CSF expression in sequence . The ratThis study has suggested that the protein expression from the construct having native human cDNA for G-CSF and the codon-optimized gene for recombinant G-CSF is low to moderate due to the formation of stable secondary structures at the 5′ end of the G-CSF transcript. Translationally silent mutagenesis within the first 24 bp in the G-CSF construct could dramatically disrupt the stable secondary structures to increase translational efficiency and G-CSF yield. This study suggests that such an approach could also be employed to increase the yield of other therapeutic proteins, wherein even engineering of the first few nucleotides in the transcript could result in a substantial increase in protein yield.Thus study suggests that the protein yield from the construct with native human cDNA G-CSF and codon-optimized sequence for recombinant G-CSF is low due to stable secondary structures at the 5′ end of the transcript. Translationally silent mutagenesis within the first 24 nucleotides of the G-CSF construct was employed to disrupt the stable secondary structures. This led to a dramatic increase in translational efficiency and enhanced protein expression. Interestingly, the free energy value of the first 24 bp thermodynamic ensemble is crucial in predicting translational efficiency. This study suggested that such an approach could also be employed to increase the yield of other therapeutic proteins and proteins involved for research purposes. Importantly, even engineering of the first few nucleotides in the transcript could substantially increase protein yield.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.SD conceived the idea, performed the experiments, interpreted the data, and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pythium insidiosum. Reports of both human and animal pythiosis are on the rise worldwide. Prognosis of the pythiosis patients relies on early diagnosis and prompt treatment. There are needs for an immunodiagnostic test that can detect the disease in both humans and animals. This study aims at reporting an optimized protocol for the development of a protein A/G-based enzyme-linked immunosorbent assay (ELISA) for the detection of anti-P. insidiosum antibody in multiple host species.Pythiosis is a deadly infectious disease caused by P. insidiosum antibodies in serum samples of both humans and animals. It is a versatile, feasible-to-develop, and functional immunodiagnostic assay for pythiosis.A total of 25 pythiosis and 50 control sera, obtained from humans, horses, dogs, cats, and cows, were recruited for the assay development. With a proper ELISA cutoff point, all pythiosis sera can ultimately be distinguished from the control sera. The successfully-developed protein A/G-based ELISA can detect the anti- Pythium insidiosum  and 1.5% NaCl . The plate was washed 4 times with the washing buffer , blocked with 250 µl of 0.5% bovine serum albumin  in PBS at 37 °C for 60 min, and washed 4 times again. Each serum (1 µl) was added to 9 ul of PBS. Then, the diluted serum (5 µl) was mixed with 795 µl of PBS. The resulting serum sample  was added to each well and incubated at 37 °C for 60 min. After washing (as above), 100 µl of protein A/G conjugated with peroxidase   was added to each well and stored at 37 °C for 60 min. The ELISA plate was washed once again. Chromogenic substrate  was applied to each well (100 µl/well) and incubated at room temperature for 3 min in a dark chamber. Sulfuric acid  was used to stop the enzymatic reaction. Optical density (OD) of each serum sample was measured at the 450-nm wavelength.We developed a multiple host-specific ELISA by modifying the human-specific ELISA protocols of Chareonsirisuthigul et al.  and LohnP. insidiosum by culture and molecular analysis [P. insidiosum antibodies by ICT [P. insidiosum antibodies by ICT. Positive (from a known pythiosis patient) and negative  control sera were analyzed in parallel. All serum samples were leftover specimens obtained from the Faculty of Medicine, Ramathibodi Hospital, Mahidol University, and the Faculty of Veterinary Medicine, Kasetsart University. Each serum sample was tested in duplicate by the protein A/G-based ELISA. ELISA value (EV) was an OD value of a serum sample divided by an OD value of the negative control serum. ELISA cutoff value was derived from the mean EV of the control sera plus 2, 3, 4, or 5 standard deviations (SD). Diagnostic performance  were calculated as described previously [P. insidiosum antibodies) of the pythiosis (n = 25) and control (n = 50) sera from both humans and animals was shown in Fig. P. insidiosum antibodies . This assay can detect the anti-P. insidiosum antibodies only in the sera from humans (EV range 19.8–72.0) and a dog , but not horses (EV range 6.1–6.6), with pythiosis. The result indicates that the goat anti-human IgG antibody could strongly bind human antibodies and slightly cross-react with dog antibodies. Based on the same serum samples from humans and animals, the anti-human IgG antibody-based ELISA showed equivalent detection specificity (100%), but markedly-lower detection sensitivity (84% vs. 100%), when compared with the protein A/G-based ELISA  , 23. TheEV range 9.8–72.0 P. insidiosum antibodies in a single animal species, but also in humans and various animals  those are the most affected hosts of pythiosis. A key feature of our ELISA is the use of protein A/G (rather than a species-specific IgG) as a conjugate, which can bind the anti-P. insidiosum immunoglobulins from humans and other animal species. Recently, Intaramat et al. have developed the protein A/G-based ICT, as a rapid and user-friendly assay for diagnosis of pythiosis in humans and animals [With a proper cutoff point, all pythiosis sera can ultimately be distinguished from the control sera by the protein A/G-based ELISA Table ; Fig. 1a animals . They pe animals  and the  animals , 13, 15 P. insidiosum antibodies in multiple host species, including the most affected hosts: humans, horses, and dogs. Other investigators could adopt our protocol to develop their own in-house ELISA. However, there are several limitations in association with the development and performance evaluation of the assay that need to be addressed:P. insidiosum strain Pi-S was used to prepare the coating antigen. Although this strain has been studied extensively [P. insidiosum strains could be used to prepare the coating antigen as well.The ensively , 22, 27,In the evaluation of our protein A/G-based ELISA, only pythiosis serum samples from 4 horses and a dog were tested, due to the low prevalence of the disease in animals in Thailand.The current study focuses on reporting an optimized protocol for the development of protein A/G-based ELISA. Thus, the diagnostic performance  of the assay was not extensively investigated, using a variety of serum samples from humans and animals with pythiosis and other diseases.P. insidiosum’s crude protein extract. In the future, if a purified P. insidiosum-specific protein is available for coating the ELISA plate, then an undiluted serum could be directly tested.Like the previously-established ELISAs, one extra step to perform the assay is to make a serum dilution (our assay requires 1:1600 dilution). This step is necessary for reducing the background ELISA signals from the bindings of non-specific antibodies and P. insidiosum is an indicator of an active infection. Isolation of P. insidiosum from a clinical specimen is difficult and challenging. Antibody detection is an alternative method, providing indirect diagnostic evidence for the P. insidiosum infection. Like the other established immunodiagnostic tests for pythiosis, the protein A/G-based ELISA cannot selectively detect an acute-phase infectious marker, such as IgM. Therefore, it cannot directly differentiate between acute, chronic, and previous P. insidiosum infections. However, in a patient with the typical features of pythiosis, an elevated or high level of the serum anti-P. insidiosum antibodies detected by ELISA suggests an active infection.Pythiosis patients usually present with a chronic infection, as the mean duration of symptoms before receiving medical care is several months . Direct The newly-developed protein A/G-based ELISA overcame the limitation of the previously-established ELISAs –15, as i