Primary Central Nervous System Lymphoma (PCNSL) remains a diagnostic challenge due to the variable clinical manifestations. Liquid biopsies, particularly those involving cell-free DNA (cfDNA) from plasma, are rapidly emerging as important and minimally invasive adjuncts to traditional biopsies. However, conventional pathology may be still essential to obtain a diagnosis.A 56-year-old woman presented with a progressive headache, dizziness, blurred vision, and lower limbs weakness with dysesthesia. Atypical clinical and radiological presentations, previous empirical treatment in another hospital, together with the patient's refusal to stereotactic brain biopsy made it challenging to diagnose. Her status deteriorated continuously during hospitalization.Lumber punctual was performed, and CSF cytological analysis revealed malignancy cells with a high nuclear-cytoplasmic ratio. However, these cells were too loose to perform immunohistochemical stains. Genetic aberrations detections with CSF and peripheral blood sample were also inconclusive. We made a “cell-block” using the sedimentary cells collected from CSF collected through multiple aspirations via an Omaya reservoir. We further performed cytopathological and immunohistochemical analysis using this “cell-block,” which finally confirmed the diagnosis of diffuse large-B cell PCNSL.Intracranial chemotherapy began afterwards .Unfortunately, this patient was dead 2 weeks later due to severe myelosuppression and secondary septic shock.We provided “cell-block” method, which collects cell components from large amount of CSF for cytology and immunohistochemical analysis. “Cell-block” cytology can be an alternative diagnostic method in diagnosis of PCNSL. Besides, these MRI findings can mimic high-grade gliomas, metastatic tumors, tumefactive demyelinating lesions, or infectious and granulomatous diseases.,6 Some recent studies suggest a role of advanced MRI and metabolic imaging such as fluorodeoxyglucose positron emission tomography (FDG-PET) in distinguishing PCNSL from these entities.,8 However, the histological analysis of biopsy material is still regarded as a gold standard. Unfortunately, major complications can arise during the procedure of a brain biopsy, such as intracranial hemorrhage or functional impairment.Primary Central Nervous System Lymphoma (PCNSL) is a rare extranodal non-Hodgkin lymphoma (NHL), which can involve the brain, eye leptomeninges, and rarely spinal cord without evidence of systemic disease. Liquid biopsy also is a real-time sampling of circulating tumor cells, nucleotides from biofluids, which offers a promising, less-invasive mean to obtain tumor genetics and monitor tumor evolution in patients with both primary and secondary CNS malignancies.,9–11 Presently, mutations in specific genes , dysregulation in signaling pathways , and some translocations and copy number alterations  has been identified as new biomarkers for PCNLS diagnosis, which further expand the diagnostic armamentarium.,9,12,13 Emerging data suggest a role for CSF molecular analysis in improving the sensitivity and specificity of liquid biopsies. Some biomarker may even predict prognosis and response to treatments.,9The NCCN guideline for PCNSL also suggests 15 to 20 mL spinal CSF sampling to increase diagnostic yield under a safe condition. CSF analysis should include flow cytometry and CSF cytology and possibly gene rearrangements. However, the predictive value is still very low due to the sparsity of malignant cells in the CSF. Presently, liquid biopsies often use cerebrospinal fluid (CSF) for cytomorphologic and flow cytometric analysis in PCNSL diagnosis.However, it is incapable of concluding diagnose purely based on molecular diagnosis. Thus, a special CSF procedure may need to provide conventional pathological supports. Here, we present a case, in which a PCNSL patient with atypical clinical and radiological presentations failed to conclude a diagnosis by gene sequencing. The final diagnosis was achieved by a “cell-block” method and subsequent classic histopathological analysis.2A 56-year-old woman presented with a progressive headache, dizziness, blurred vision, and lower limbs weakness with dysesthesia. Her symptoms initiated four months before this presentation, and she went to the local hospital for examination. Gadolinium-enhanced contrast MRI revealed multiple intracranial mass with ring enhancement. Metastatic malignancy was the primary consideration. She underwent FDG-PET scan afterward, which showed an increased FDG uptake in the callosal area with a maximal standardized uptake value (SUV) of 16.5, and areas abutting the fourth ventricle with a maximal SUV of 16.2 , and she was in largely remission when discharged.Two weeks before this presentation, she began to complain progressively intensifying low back pain, lower limb ache, and dysesthesia. Spinal MRI after administration of gadolinium showed multiple lesions occupied the whole spinal cord. The spinal spread of intracranial malignancy was considered. She was then referred to our department.Her medical history was notable for coronary heart disease, second-degree atrioventricular block, hyperlipidemia, pulmonary tuberculosis, chronic HBV infection, and chronic atrophic gastritis. Medications on presentation were atorvastatin, gabapentin, caltrate, and lactulose.She reported no fever, abdominal pain, chest pain, palpitations, or dyspnea.On examination, her vital sign was normal, and she was alert, her vision was blurred, the hearing of her left ear was deceased, her muscle weakness of her lower and upper limbs was Grade II and Grade III, respectively. The muscular tension was normal, as well as the tendon reflexes. There was no neck stiffness and other sign of meningeal irritation.Lumbar puncture was performed, and laboratory examination of the cerebrospinal fluids (CSF) reveals decreased chloride  and glucose , and increased protein , erythrocytosis (10/μL) and leukocytosis . CSF smear revealed malignancy cells with a high nuclear-cytoplasmic ratio, whereas these cells were too loose for immunohistochemical stains. On hospitalization, her condition was deteriorating continuously. She began to show multiple cranial nerve palsy, including right ptosis and dysphagia. Repeated cranial and spinal MRI revealed malignancy progression , we made a compact cell-block processed with paraffin wax just like processing normal biopsied tissue specimen, and further immunohistochemical stains were performed Fig. . The resAfterward, this patient began her intracranial chemotherapy . Unfortunately, she was dead 2 weeks later due to severe myelosuppression and secondary septic shock during the process of chemotherapy.3,14 The diagnosis of CNS lymphoma is always challenging. Presently, it is mainly based on clinical characteristics, imaging, CSF analysis, and brain biopsy.,13,15–17 Magnetic resonance imaging is the cornerstone for PCNSL lesions determination, which most typically enhance homogeneously on T1-weighted (MRI) and appear T2-hypointense, but high variability in MRI features is commonly encountered.Advanced MRI  and metabolic imaging, such as FDG-PET may help to distinguish PCNSL with other entities, such as high-grade gliomas, tumefactive demyelinating lesions, or infectious and granulomatous diseases.,16Multiple MRIs and PET-CT were applied in this case. However, the radiological features were atypical regarding both the manifestation and location , which was more consistent with metastatic CNS malignancy than PCNSL.,19Primary Central Nervous System Lymphoma is a rare extranodal NHL, involving the brain, eye, leptomeningitis, and rarely spinal cord. While brain biopsy provides histologic and genetic information, it is an invasive procedure with the risk of major complications. Brain biopsy is not conducive to obtaining multiple samples over time, and previous treatments may alter the diagnostic yield of biopsies, such as steroids and radiotherapies.,10,20,21 Alternatively, the liquid biopsy is a promising, less-invasive means to obtain the histologic diagnosis, from multi-analysis of the cytology, flow cytometry, molecular markers, and specific gene mutations.,13 Among these CSF analyses, cytology or cytopathology  entails the gold standard. Unfortunately, this method has a low diagnostic yield as often no malignant cell detected, or cells are too lose or too lytic for proper analysis.,13 Despite malignant cells were found in the initial CSF analysis in our case, the number was not abundant to perform the immunohistochemistry. As is often the case, diagnosis of PCNSL from CSF cytology occurs in only 10% to 20% patients, as malignant cells detected are too loosely or too lytic for proper analysis.,22 Flow cytometry, a method of immunophenotyping , can increase the sensitivity in the diagnosis of PCNSL, which usually works together with cytology.,24 In our case, flow cytometric analysis was not performed due to initial suspicion of metastatic malignancy. However, sensitivity is disappointing even for this analysis approach, and detection of clonal B-cell populations does not distinguish between lymphoma entities.For PCNSL, histological analysis through a brain biopsy or liquid biopsy of biopsy material is still regarded as the gold standard.,26 Multiple studies have tried to identify a molecular signature specific for PCNSL, using many different analysis strategies on tumor tissue. Interleukin (IL)-10 with its receptors, IL-6 and CXCL13 are shown to be upregulated in the CSF of patients with PCNSL, which are related to lymphoid cells growth and immunity regulation.,27 Osteopontin and neopterin are proinflammatory cytokines involved in immune cell activation and B-cell migration and proliferation, which are significantly higher in patients with PCNSL than in those with other CNS disorders.,28 microRNAs (miRNAs) are very promising biomarkers as well, not only for diagnostic purposes but also for monitoring therapy response. Furthermore, emerging data suggest a role for gene aberrations in predicting prognosis and response to treatments. Presently, genetic aberrations reported in PCNSL include CARD11, CD79B, CDKN2A , ETV6, MYD88, PIM1, PRDM1, TBL1XR1, TNFAIP3(A20), and TOX.,16 Half of the selected genes are involved in the NFκB pathway , while the other half is not . Unfortunately, none of these genetic aberrations were detected in this patient. The results of CNS specific gene mutation detection are often inconclusive and hard to interpret in clinical practice, on which the diagnostic criteria based still has a long way to validation.The advent of lymphoma-specific mutations and unique combinations of biomarkers  that can be tested in CSF further increase the diagnostic value of this liquid biopsy for PCNSL. Therefore, this “cell-block” method can improve the effectiveness of liquid biopsy, and especially useful in cases where the molecular analysis is unavailable or inconclusive.The final diagnosis of our case relied on the cytopathologic and combined immunohistochemistry analysis of the “cell-block,” which made from CSF cells collected from repeatedly aspirated patient's CSF. To the best of our knowledge, this is the first case demonstrating such a “cell-block” methods. As the major constraint is removed (the hypocellular composition of CSF), classical histopathological diagnostic methods can function effectively in liquid biopsy, such as cytopathology and immunohistochemistry analysis.However, it is not known if this methods help if PCNSL did not invade the CSF circulation system. As this is only a case report, further clinical studies should performed to address the sensitivity and specificity of this cell-block method.4This case demonstrated a safe, easy-practicing and cost-effective method for CSF procedure for PCNSL diagnosis. Presently, molecular diagnosis is still far from an essential diagnostic method for PCNSL; this “cell-block” method is especially useful in the scenario where brain biopsy, molecular analysis are unavailable or inconclusive.Data curation: Lei Zhou.Writing – original draft: Kaiyuan Huang.Writing – review & editing: Ying Tong.


Fasting blood glucose level was positively correlated with CSF Aβ42 level , CSF Aβ42/Aβ40 ratio , Aβ42/P-tau ratio , and Aβ42/T-tau ratio . Interaction analysis indicated that gender affected the correlations of FBG level with CSF Aβ40 (p < 0.001) and Aβ42/Aβ40 ratio (p < 0.001). This study raises additional questions about the role of blood glucose in the predisposition to AD and supports the possibility of targeting these processes in pre-symptomatic AD trials in non-diabetic elders.It is unclear how blood glucose levels mediate the pathology of Alzheimer's disease (AD). This study aimed to investigate whether fasting blood glucose (FBG) levels are associated with cerebrospinal fluid (CSF) biomarkers preferentially affected by AD in non-diabetic cognitively normal elders. A total of 499 non-diabetic cognitively normal elders were from the Chinese Alzheimer’s Biomarker and LifestyLE (CABLE) study. We detected the associations of FBG with individual CSF measures using multiple linear regression models controlling for age, sex, educational level, and  We also tested the interactions of gender and apolipoprotein E (APOE) ε4 status with FBG levels related to AD biomarkers in CSF.Our study aimed to explore the cross-sectional relationships between FBG and CSF biomarkers of neurodegeneration [CSF AβAPOE ε4 allele positivity (15.4%), and a mean Mini-Mental State Examination (MMSE) score of 27.89 ± 2.12.APOE ε4 status, higher FBG levels were associated with elevated CSF Aβ42 levels , CSF Aβ42/Aβ40 ratio , Aβ42/P-tau ratio , and Aβ42/T-tau ratio , but not associated with CSF Aβ40 (n = 496), P-tau (n = 499), or T-tau (n = 482) levels in the non-diabetic CN elders , but negatively related among the males . However, only among the males, FBG was significantly associated with higher CSF Aβ42/Aβ40 ratio  levels. These results are consistent with a previous cohort study among non-diabetic AD patients which found that CSF/plasma glucose ratio was inversely related with CSF Aβ42 level rather than CSF tau level [42 overproduction, as seen in familial AD [42/Aβ40 ratio could outperform CSF Aβ42 as a more accurate marker of brain amyloidosis, since it normalizes the CSF Aβ42 levels according to the total production of Aβ in the brain [42/Aβ40 ratio was discovered, suggesting that FBG may increase the incidence of Aβ aggregation (maybe through increased amyloidogenic APP processing) [42/P-tau ratio and Aβ42/T-tau ratio has been proposed to provide more accurate risk assessments for the development of AD [42), and neurodegeneration (tau) [42/P-tau ratio and Aβ42/T-tau ratios.In the present study, we discovered that FBG levels were positively associated with CSF Aβau level . The earilial AD , or defeilial AD  during tilial AD , 12. Morhe brain , 14. Thecessing) . Moreovent of AD –17. Theson (tau) . Higher Actually, most previous studies that have examined associations between glucose metabolism and neuropathological outcomes of AD have focused on diabetes, finding that T2DM poses a great known risk (2- to 4- fold) for developing AD. Both of the two diseases share common pathologies albeit they have been identified in the periphery with T2DM and in the brain with AD. These include an increase in inflammation, oxidative stress, adiponectin deficiency, plasma cholinesterase activity, vascular damage, dysregulation of glucose and insulin signaling –21. Alth40 and Aβ42/Aβ40 levels. Specifically, FBG was positively associated with CSF Aβ40 levels among the females, but negatively related among the males. Only among males, FBG was significantly associated with higher CSF Aβ42/Aβ40 ratio. This is the first study to detect the sex differences in the relationships between blood glucose and AD biomarkers in CSF. The exact mechanism remains unclear. Further studies are warranted to clarify the essence of gender differences and the underlying mechanisms.These positive relationships between FBG and CSF biomarkers were demonstrated in the CN elders. As it is nowadays contextualized, AD has a long asymptomatic period in which there is a silent accumulation and progression of pathology and brain structural changes. Symptoms appear when compensatory mechanisms have been overcome, initially as mild cognitive impairment (MCI) and ultimately as dementia , 23. ThiAPOE ε4 positive, we were unable to link the impact of the APOE ε4 risk allele on the association of FBG with levels of AD biomarkers. In addition, our measures of glucose. metabolism are limited to peripheral, rather than cerebral measures. However, this adds to the current literature by indicating that peripheral hyperglycemia, which is very easy to measure with noninvasive measures, is related to AD-related CSF biomarkers. Lastly, the level of fasting blood glucose can be influenced by many factors, including time related to meal, type of meal, and exercise pattern. Future studies regarding the correlations of plasma level of glycosylated hemoglobin (HbA1C), a more stable value related blood glucose status, and AD-related core biomarkers are needed.Some limitations should be acknowledged. Firstly, the present study is limited by its cross-sectional design, therefore limiting inferences of causality. Further longitudinal analyses will assist in establishing whether the associations support causality. Next, because of the smaller proportion of people who were Despite these limitations, we are the first to investigate the relationship between FBG and Alzheimer’s CSF biomarkers in the non-diabetic CN elders. Our finding is especially important when taking into consideration the public health impacts of AD coupled with epidemiological data showing that rates of this disease states are expected to dramatically increase. Our study advanced the association between AD and blood glucose level to the stage where no cognitive impairment occurred and diabetes did not develop. Among cognitively normal elders without diabetes, maintaining appropriate levels of hyperglycemia demonstrated a protective effect on AD pathology, whereas low glycemic level were adverse. Hypoglycemia could cause the pathogenesis of AD by the way described above. Older individuals are thought to have less brain reserve or brain plasticity , 25, and42 level, Aβ42/Aβ40 ratio, Aβ42/P-tau ratio, and Aβ42/T-tau ratio. Gender significantly modified the association between elevated blood glucose and AD pathology. However, the underlying pathological mechanisms warrant further investigation in the future.In summary, this study provides additional evidence for the relationship between higher fasting blood glucose levels and the predisposition to AD. It demonstrated that, in non-diabetic cognitively normal elders, higher FBG levels were associated with elevated CSF AβNon-diabetic cognitively normal northern Han Chinese participants were from the Chinese Alzheimer’s Biomarker and LifestyLE (CABLE) study. CABLE is an ongoing large-scale study majorly focused on risk factors and biomarkers for AD in Chinese Han population since 2017, aiming to determine the genetic and environmental modifiers of AD biomarkers and their utility in early diagnosis. Individuals were recruited at Qingdao Municipal Hospital, Shandong Province, China. All enrolled participants were Han Chinese aged between 40 to 90 years. The exclusion criteria include: (1) central nervous system infection, head trauma, epilepsy, multiple sclerosis or other major neurological disorders; (2) major psychological disorders; (3) severe systemic diseases ; (4) family history of genetic diseases. All participants gave written informed consent to participate in this study, which was approved by the Institutional Review Board of Qingdao Municipal Hospital and conducted in accordance with the principles of the Declaration of Helsinki. This study only included those without T2DM diagnosis.APOE ε4 genotypes.At study entry, detailed clinical assessment was performed including an interview with the care-giver and an extensive neurological examination. Standardized questionnaires were then evaluated. The China-Modified MMSE (CM-MMSE) and Montreal Cognitive Assessment (MoCA) scales were used to examine global cognition. The basic Activities of Daily Living score (ADL) was used to assess basic living ability; the Geriatric Depression Scale (GDS), Hamilton Rating Scale for Depression (HAMD) and Hamilton Rating Scale for Anxiety (HAMA) for behavioral or psychological symptoms. The GDS and HAMD scores of participants in CABLE database were less than seven. Moreover, vascular factors were assessed by Hachinski Inchemic Score (HIS). The HIS scores of participants in our study were less than four. Finally, this current study included 499 non-diabetic cognitively normal individuals with available data on CSF measures, FBG levels, and 42, Aβ40, T-tau, and P-tau levels were measured by enzyme-linked immunosorbent assays (ELISAs) using INNOTEST (Fujirebio Europe N.V.). All CSF samples were distributed randomly across plates and measured in duplicate. All the antibodies and plates were from a single lot in order to exclude variability between batches. Experiments were performed by experienced laboratory technicians blinded to clinical diagnosis and other clinical information. The mean intra-batch coefficient of variation (CV) was 4.86 ± 4.37% for Aβ42, 3.63 ± 2.84% for Aβ40, 2.34 ± 2.30% for P-tau, and 4.59 ± 3.49% for T-tau. The mean inter-batch CV was 9.60% for Aβ42, 8.12% for Aβ40, 11.04% for P-tau, and 12.15% for T-tau. CSF biomarker levels were corrected by inter-batch variation and corrected values were used for analyses.CSF sample collection and management was in accordance with the international consensus on standardization of CSF research . CSF wasA laboratory blood specimen to assess a patient's fasting blood level was obtained after the patient has abstained from eating for a minimum of 8 hours. Fasting blood glucose levels were measured by glucose hexokinase (HK) method using Glucose Reagent .APOE ε4 genotyping by a Multiplex SNaP shot. Participants were classified as APOE ε4 non-carriers (participants with no copies of the APOE ε4 gene), and APOE ε4 carriers . General demographic data, including age, sex, and educational level, were collected via a structured questionnaire.DNA extracted from 10 mL ethylene diamine tetraacetic acid (EDTA) blood samples using the QIAamp DNA Blood kit , was used for APOE ε4 genotype. All CSF variables in linear regression models were log-transformed to normalize the distributions and to facilitate comparisons between modalities. Models with interaction terms were performed in order to examine the modifying effect of gender and APOE ε4 genotype on the relationship between blood glucose level and CSF biomarkers. In case of significant interactions, stratified analyses were performed. All tests were 2-sided and statistical significance was set at p < 0.05. Analyses were performed using IBM SPSS Statistics 23.CSF values outlying the mean ± 3 SD were regarded as extremes and then excluded when analyzed. Continuous variables are presented as mean ± SD, and categorical variables as number (percentage). The associations of continuous FBG increment with individual CSF measures were analyzed using multiple linear regression models adjusting for age, sex, educational level, and


Currently, amino-terminal PEGylated human granulocyte colony stimulating factor (huG-CSF) is used to prevent and treat neutropenia. Although huG-CSF has been used as a drug for more than 20 years, it has three significant drawbacks: (i) it relies on PEG aldehyde for PEGylation of the alpha-amino group of the first amino acid, and this leads to non-specific PEGylation of the epsilon amino group of lysine residues within the G-CSF; (ii) longer-acting G-CSF variants are desirable to reduce the risk of chemotherapy-associated neutropenia; and (iii) G-CSF cannot be administered on the day of chemotherapy. In an attempt to overcome the above drawbacks, we engineered cysteine variants of G-CSF to facilitate the maleimide PEG-based site-specific PEGylation that leads to a highly homogenous PEGylated product. Importantly, we have demonstrated that 20 kDa thiol-reactive PEG conjugated by maleimide chemistry to the Cys2 G-CSF variant exhibits leukocyte proliferative activity similar to that of the commercially available G-CSF conjugated with aldehyde PEG in a neutropenia mice model. Moreover, we have demonstrated that PEGylation of the cysteine variant of huG-CSF with higher molecular weight PEGs, such as 30 kDa PEG and 40 kDa PEG, leads to significantly prolonged leukocyte proliferation activity compared to the variant conjugated with 20 kDa PEG. Importantly, even a half-dose of the engineered variant conjugated with 40 kDa PEG exhibited significantly longer biological activity than the commercially available 20 kDa PEGylated huG-CSF. Finally, we have demonstrated that administration of the engineered variant conjugated with 40 kDa PEG on the day of administration of cyclophosphamide for inducing neutropenia in mice can alleviate neutropenia through leukocyte proliferation. In summary, this study provides the design of site-specific PEGylated huG-CSF variants with improved therapeutic potential. It opens the possibility of long-acting and same-day prophylactic administration of G-CSF after chemotherapy drug regimens. These results may pave the way for the development of potential G-CSF derivatives possessing longer half-lives and favorable clinical attributes. Human granulocyte colony stimulating factor (huG-CSF) is a 19 kDa cytokine that stimulates the proliferation, maturation, and functional activation of the cells in the granulocyte lineage . HuG-CSFin vivo half-life of G-CSF, 20 kDa methoxy PEG propionaldehyde was conjugated to the alpha-amino group of the first amino acid residue of huG-CSF to make PEGylated G-CSF , known as PEGylation , conjugaed G-CSF . Althouged G-CSF . Notablyed G-CSF . Such heed G-CSF . Furthered G-CSF .in vivo half-life  and two disulfide bonds (Cys36-Cys42 and Cys64-Cys74). These disulfide bonds are critical for structural integrity and G-CSF activity; however, Cys17 could be mutated. Interestingly, Cys17 was also utilized for generating a PEGylated version of G-CSF . Since tIn the present study, we utilized a computational approach for identifying strategically defined solvent-accessible sites wherein cysteines could be introduced or substituted without affecting G-CSF activity. Solvent accessibility is essential for efficient and homogenous PEGylation. G-CSF PEGylation was carried out at these sites, using methoxy PEG maleimide moiety of different molecular weights. PEGylation and homogeneity of the reaction product were confirmed using various physicochemical methods. Finally, prolonged and enhanced activity was detected in the neutropenia mice model through neutrophils’ enumeration in mice blood.Escherichia coli strain BL21 (DE3) was procured from Novagen Inc. . The oligonucleotide primers used in the study for cloning and site-directed mutagenesis were custom synthesized from Integrated DNA Technologies (IDT), United States, and BioServe Biotechnologies (India) Pvt Ltd., G-CSF standards (09/136 and 12/188) were purchased from the National Institute for Biological Standards and Control (NIBSC). Commercially available G-CSF and its PEGylated versions were from Dr. Reddy’s Laboratories Ltd., Methoxy PEG maleimide reagents were purchased from JenKem Technology, United States. Methoxy PEG maleimide with catalog nos. A3115, A3116, and A3123 were used in this study. SP-Sepharose, Macro CAP SP, Capto SP ImpRes, Superdex-75 pg, and Superdex-200 pg resins and columns used for different chromatographic processes were procured from GE-Healthcare Life Science, Sweden. Zeba Spin Desalting Columns were purchased from Thermo Fisher Scientific, United States. Spectrophotometric analysis was performed on Perkin Elmer LAMBDA 35 UV/Vis spectrophotometer. All the reagents were of the highest available analytical grade. We used an AKTA Pure M (25M1) chromatographic system with Unicorn 7.3 software, version 7.3. Cyclophosphamide (C7397) was from Sigma-Aldrich. B.D. Vacutainer K3EDTA tubes were used to collect mice’s blood.All enzymes and reagents required for molecular biology were acquired from Fermentas . Purification of plasmid and DNA from agarose gel was performed using kits available from Qiagen . Expression host 1 using accession number M13008.1. This sequence was codon-optimized for expression in E. coli using the OptimumGeneTM algorithm from GenScript, United States. The codon-optimized cDNA sequence of human G-CSF was cloned at the NdeI and HindIII sites of the expression vector pET23a using primers IMTSD1 5’ ATG ACG CCG CTG GGT CCG 3’ and IMTSD2 5’ CGG CTG TGC CAG GTG AC 3’. Free cysteine at position 17 of G-CSF was replaced with serine by site-directed mutagenesis to prevent unwanted PEGylation using primers SDM17a 5’ CTG CTG AAG TCT CTG GAA CAA 3’ and SDM17b 5’ TTG TTC CAG AGA CTT CAG CAG 3’. The cysteine substitution and or insertion variants were derived from the G-CSF variant in which cysteine 17 has been changed to serine. G-CSF variants were created using standard protocols of site-directed mutagenesis or by PCR using primers with desired changes for insertion or substitution of cysteine at a particular position in the coding sequence, as reported earlier  was used to induce protein in the form of inclusion bodies, which were then solubilized in 2 M urea. Washed inclusion bodies (IB) were solubilized in urea containing buffer  for 60 min. After centrifugation at 11,000 rpm for 20 min at 4°C, the supernatant containing the solubilized IBs was collected. The pH was adjusted to 8.0 by using glacial acetic acid. Partially unfolded protein was further subjected to a two-step refolding procedure using refolding buffers  at 4°C with gentle stirring. Each refolding step is given 1–2 change of buffer for efficient refolding of the protein. Refolded protein was purified using cation exchange chromatography (CEC) on Capto SP ImpRes resins from GE-Healthcare Life Science, Sweden, equilibrated with 25 mM sodium acetate pH 4.5, 50 mM NaCl, and 5 mM EDTA. The G-CSF protein was eluted using 1 M Tris-Cl, pH 8.0.The G-CSF and cysteine variants cloned in the T7 RNA polymerase inducible promoter-based expression vector pET23a were used to transform the BL21 (DE3) strain of In the case of the cysteine variants of G-CSF, the cation exchange purified protein was used to conjugate PEG of varying sizes. The protein was dialyzed in PEGylation buffer, i.e., sodium phosphate buffer (pH 7.5). The 5-fold molar excess of thiol-specific PEG was used to conjugate the Cys2 G-CSF variant in PEGylation buffer. The reaction was allowed to proceed at 20°C for 12–16 h with slow stirring. The PEGylation reaction was dialyzed against 25 mM sodium acetate pH 4.5 and 5 mM EDTA. The PEGylated species were purified by CEC using MacroCap SP resins or Capto SP ImpRes resins , followed by size exclusion chromatography (SEC) using Superdex-75 pg or Superdex-200 pg column. The CEC purified PEGylated protein was loaded onto the Superdex column, preequilibrated with 25 mM sodium acetate pH 4.5, 50 mM NaCl and 5 mM EDTA and eluted in the same buffer. Fractions containing the purified protein were collected and desalted in formulation buffer  and stored at −20°C.All PEGylated and non-PEGylated derivatives were checked for their purity on 10.5% non-reducing SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). Barium iodide staining was also used to visualizing the PEGylated G-CSF. To examine the PEGylated protein, barium iodide staining was performed, where PEG moiety is stained based on the formation of a barium iodide complex with PEG. The gel was treated with 5% barium chloride solution for 10 min and then with 0.1 M iodine solution for 5 min .® 5600/5600+.The molecular mass of G-CSF, cysteine variants, and PEG conjugated variants were determined by Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF) on an ABI SCIEX machine, model TripleTOFCircular dichroism (CD) spectroscopy was performed to investigate the secondary structure of G-CSF, cysteine variants, and PEG-conjugated variants. Far-UV CD spectra of wild-type and modified variants were recorded from 195 to 250 nm on the Jasco J-815 spectro-polarimeter at 25°C. Measurements of all the samples were performed at concentrations 0.2 mg/ml, using cuvettes of 0.1 cm path length.TM) and by running on 10.5% non-reducing SDS-PAGE gel. Quantitation was done using ImageJ software.PEGylated cysteine variants were formulated in a formulation buffer consisting of 10 mM sodium acetate (pH 4.5), 5% Sorbitol, and 0.004% Tween 20, followed by filtration using a 0.22 μm filter. Protein samples were aliquoted for subsequent usage and stored at −20°C. Before administration in mice, samples were quantitated by NanoDrop .All the mice experiments were approved by the Institutional Animal Ethics Committee of Council of Scientific and Industrial Research-Institute of Microbial Technology . These experiments were performed according to the guidelines issued by the Committee for the Purpose of Supervision of Experiments on Animals (No.55/1999/CPCSEA) under the Prevention of Cruelty to Animals Act 1960 and amendments introduced in 1982 by the Ministry of Environment and Forest, Govt. of India. Mice were maintained and bred in the animal house facility of CSIR-Institute of Microbial Technology; 8–12-week-old male BALB/c mice were used in the current study for analyzing the biological activity of PEG-conjugated cysteine variants of G-CSF. Toward this, mice were acclimatized for a week, and neutropenia was induced in mice using an intraperitoneal injection of cyclophosphamide (200 mg/kg body weight of mice) as per standard procedures. Cyclophosphamide is an anti-cancer chemotherapy drug. This medication is classified as an alkylating agent and also used to induce neutropenia in laboratory animals . To conf∗indicates a P-value < 0.05, ∗∗indicates a P-value < 0.01, ∗∗∗indicates a P-value < 0.001, and ****indicates a P-value < 0.0001.GraphPad Prism 6 for Windows, Version 6.05, was used to plot animal experiment data. The plots represent scatter dot plot wherein data are means with SEM for three mice per group. Statistical significance was determined using two-way ANOVA multiple comparisons of the data. in vivo half-life. Solvent accessibility was determined using webserver NetSurfP-2.0  and the mutagenesis of cysteine 17 to serine were confirmed using DNA sequencing. Cys2 variant was overexpressed in fication . SDS-PAGfication . Finallyfication .Purified Cys2 variant was conjugated with 20 kDa methoxy PEG maleimide using maleimide chemistry. The PEGylated species were purified by CEC using MacroCap SP resins or Capto SP ImpRes resins , followed by SEC . SDS-PAGrd, 6th, 9th, and 12th day. We observed that the PEGylated Cys2 variant conjugated with 20 kDa PEG possesses similar biological activity to that of the commercially available PEGylated huG-CSF  on day 0. After the induction and confirmation of neutropenia, sham (having buffer only), commercially available PEGylated huG-CSF, and the mono-PEGylated Cys2 variant were independently administered as the single subcutaneous dose  of the Cys2-40 kDa PEG conjugate could compare for biological activity with the full dose (1 mg/kg mice body weight) of the standard 20 kDa PEG conjugate. Toward this, neutropenia was induced in mice as described above, and mice were treated with sham, a full dose of standard PEGylated G-CSF conjugated with 20 kDa PEG (1 mg/kg mice body weight) and half-dose (0.5 mg/kg mice body weight) of the Cys2-40 kDa PEG conjugate. Importantly, we observed that on day 6, the mice receiving a half-dose of the Cys2-40 kDa PEG conjugate and a full dose of 20 kDa PEGylated G-CSF have a comparable TLC. Importantly, on day 9 and 12, the proliferation of leukocytes was significantly higher in the group receiving the half-dose of the Cys2-40 kDa PEG conjugate compared to the group receiving the full dose of the standard PEGylated G-CSF conjugated with 20 kDa PEG . These dOne of the challenges in prophylactic administration of G-CSF during chemotherapy is that it must be administered 24 h after dispensing chemotherapeutic drugs to achieve adequate prophylactic protection from neutropenia. A same-day G-CSF substitute will be beneficial and as well as convenient for patients. As the administration of Cys2 variant conjugated with 40 kDa PEG has resulted in much longer biological activity, we checked whether the administration of the Cys2 40 kDa PEG conjugated variant at the day of neutropenia induction (mimicking the G-CSF treatment at the day of chemotherapy) could facilitate the recovery of leukocyte count in a neutropenic mice model. Interestingly, the same-day administration of the Cys2 variant conjugated with 40 kDa PEG has improved leucocytic recovery at day 6, 9, and 12 as compared to the standard PEGylated G-CSF conjugated with 20 kDa PEG administrated 24 h after cyclophosphamide treatment .huG-CSF is a beneficial and widely used therapeutic protein for several clinical conditions, including neutropenia, leukopenia, AIDS, sepsis, and also in patients undergoing bone marrow transplantation . To incrin vitro activity as monitored through the use of M-NFS-60 cell line-based assays and increase in in vivo circulation half-life  1 day after the administration of chemotherapy. We believe that innovations leading to same-day administration of chemotherapeutic and prophylactic agents could tremendously improve the outcome and be convenient for patients. We would also like to emphasize that we observed that in mice receiving same-day administration of huG-CSF-40 kDa PEG, the proliferation of leukocytes in neutropenic mice was considerably lower at the third day when compared to the administration of 20 kDa PEGylated G-CSF.Another significant finding of the present study was that the same-day administration of the Cys2 variant conjugated with 40 kDa PEG effectively increased the leukocyte counts to desirable levels or more at the 6in vivo biological activity.The production process for the currently available PEG conjugated huG-CSF relies upon amine-reactive PEGs . Such PEWe have successfully designed G-CSF variants for site-specific PEGylation, which may provide a solution toward prolonged half-life and biological activity. The site-specific PEGylated variants were successfully expressed, purified, and characterized. Importantly, the administration of the engineered variant conjugated with 40 kDa PEG leads to improved and longer leukocyte proliferation in neutropenic mice, wherein neutropenia was induced by cyclophosphamide. Even at half the dose, the 40 kDa PEG conjugated G-CSF variant leads to enhanced and longer biological activity in the neutropenia mice model. Consequently, this variant could be administered at a lower dosage than the 20 kDa PEGylated G-CSF while retaining the same therapeutic efficacy. The site-specific higher-molecular-weight PEGylated variant also shows promise for the same-day administration of the G-CSF drug, which is a challenge in the current G-CSF therapy regimen in the treatment of chemotherapy-induced neutropenia. These engineered cysteine variants and their derivatives hold promise to become a next-generation G-CSF drug.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by IAEC, CSIR-IMTECH.SD conceived the study, helped in analysis of data, and wrote the manuscript. MK performed the experiments and analyzed the data. GS provided technical and administrative support to the project. He also helped in improving the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We identified 206 diagnostic metabolites in targeted analysis. In a subset of 20 high‐intensity metabolites and 10 biomarkers, 17 (57%) were age‐dependent. For each neurometabolic patient, ≥1 specific biomarker(s) could be identified in CSF, thus confirming the diagnosis. In two cases, age‐matching was essential for correct interpretation of the metabolomic profile. In conclusion, NGMS in CSF is a powerful tool in defining a diagnosis for neurometabolic disorders. Using our database with many (age‐dependent) features in CSF, our untargeted approach will facilitate biomarker discovery and further understanding of mechanisms of neurometabolic disorders.Timely diagnosis is essential for patients with neurometabolic disorders to enable targeted treatment. Next‐Generation Metabolic Screening (NGMS) allows for simultaneous screening of multiple diseases and yields a holistic view of disturbed metabolic pathways. We applied this technique to define a cerebrospinal fluid (CSF) reference metabolome and validated our approach with patients with known neurometabolic disorders. Samples were measured using ultra‐high‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometry followed by (un)targeted analysis. For the reference metabolome, CSF samples from patients with normal general chemistry results and no neurometabolic diagnosis were selected and grouped based on sex and age (0‐2/2‐5/5‐10/10‐15 years). We checked the levels of known biomarkers in CSF from seven patients with five different neurometabolic disorders to confirm the suitability of our method for diagnosis. Untargeted analysis of 87 control CSF samples yielded 8036 features for semiquantitative analysis. No sex differences were found, but 1782 features (22%) were different between age groups ( Next‐Generation Metabolic Screening in CSF will facilitate biomarker discovery and further understanding of mechanisms of neurometabolic disorders, taking age‐dependency into account.1Neurometabolic disorders are a group of inherited diseases that mainly affect the central nervous system. Even though the individual disorders are rare, collectively they represent an important category of severe but potentially treatable neurological disorders. The phenotypic manifestation of neurometabolic disorders can be very heterogeneous, for example, ranging from epilepsy to movement disorders to developmental delay. The common theme is presentation at a (very) young age. Coming to a correct diagnosis as soon as possible can be life saving for a child, as specific therapy can only be initiated once the metabolic defect is identified. Furthermore, understanding of the disturbed metabolic pathways of a disease may also lead to new opportunities for treatment.Currently, in parallel to genetic diagnostics, neurometabolic disorders are functionally evaluated by both imaging techniques (magnetic resonance imaging and spectroscopy) and biochemical analyses of a limited set of known (metabolite) biomarkers in body fluids. Specific metabolic perturbations for neurometabolic diseases can often only be detected in cerebrospinal fluid (CSF). However, this strategy prevents the identification of novel disorders affecting unexplored metabolic pathways. Therefore, there is a need for a more holistic approach to biochemical diagnostics for neurometabolic disorders. We recently described such an approach, called Next‐Generation Metabolic Screening (NGMS), applied to inborn errors of metabolism (IEMs) using plasma samples.As UHPLC‐QTOF‐MS is a semiquantitative technique, the detection of deviating metabolites relies on comparison to controls. Biological and preanalytical variation within the control group may hamper such comparisons. Therefore, we present the metabolomic profile of control CSF samples obtained from children aged 0 to 15 years , defining which metabolites can be identified using NGMS and the degree of variation in metabolite levels introduced by sex, age, and CSF fraction. Furthermore, we demonstrate the application of NGMS in CSF for the diagnosis of neurometabolic disorders, taking into account the variation of our control cohort.22.1Controls: leftover CSF samples (storage at −80°C) were selected based on the following criteria: (a) subject age 0 to 15 years old, (b) collected in 2016, 2017, or 2018, (c) erythrocyte and leukocyte count in CSF within reference range, (d) if available, routine CSF parameters hemoglobin, bilirubin, IgG production, glucose, lactate and protein within reference range, and (e) clinical data of the subject, as investigated by a pediatric neurologist, did not suggest the presence of a disease associated with abnormal CSF results. In accordance with local legislation, we only included samples of patients who did not reject to the anonymized use of their left‐over material for validation purposes. To ensure anonymity, all samples were labeled with a study number based on sex and age group .To study potential concentration gradients of metabolites, CSF was used from a 6‐year‐old patient with increased intracranial pressure of unknown origin (despite extensive diagnostic work‐up). 20 mL of CSF was withdrawn and divided into four subsequent fractions of 5 mL before storage at −80°C.l‐amino acid decarboxylase (AADC) deficiency (OMIM #608643). For all diagnoses except the latter, heparin plasma samples collected on the same date as the respective CSF sample were included as well. For each patient, the diagnosis was confirmed on both the genetic and the metabolic level. Moreover, the patients with NANS (patient 9)Patient CSF samples were collected from the local clinical archive. Informed consent for the use of these samples for research purposes had been obtained previously. Diagnoses included isovaleric acidemia , succinic semialdehyde dehydrogenase (SSADH) deficiency (OMIM #271980), N‐acetylneuraminic acid synthase (NANS) deficiency (OMIM #610442), dihydropyrimidinase (DHP) deficiency (OMIM #222748), and aromatic 2.2CSF samples were analyzed by the reversed‐phase UHPLC‐QTOF‐MS method previously described in detail for heparin‐anticoagulated plasma,2.3m/z), retention time (RT) and for each sample an intensity) were preprocessed by selecting those within an m/z range of 70 to 700, RT between 0.4 and 16 min, intensity in the PB lower than 10% of the mean intensity in the control cohort CSF samples, and with an intensity of ≥10 000 in at least one sample. For semiquantitative analyses, we averaged duplicate measurements. In the case of between‐run comparisons, we normalized feature intensities by dividing them by the average intensity in the pooled QC. Only features for which the coefficient of variation (CV) in the RPP sample was ≤20% after normalization were used in these between‐run comparisons.For untargeted analysis, raw data acquired from the UHPLC‐QTOF‐MS runs were aligned using the R package “xcms” , or m/z only (level 2). Features were matched to the IEM panel based on a <5 ppm deviation for mass accuracy and a <10% relative RT difference from the reference compound measurement (if available). Of note, only [M+H]+ and [M+Na]+ adducts for the positive mode and [M−H]− and [M+Cl]− adducts for the negative mode were included in this annotation. Initially, we matched features from the XCMS list obtained in untargeted analysis. In case a metabolite was not found, Agilent MassHunter Personal Compound Database and Library Manager Software version B.04.00, build 92.0 was used to further check its presence in the raw data. The 20 metabolites with the highest signal intensity as well as 10 metabolites that served as biomarkers for our patient samples (see section 2.5) were selected for semiquantitative analysis.Annotation was performed using a predefined panel of 322 metabolites known to be associated with IEMs  according to the procedure recommended by the manufacturer. Neurotransmitter metabolites in CSF were investigated by high‐performance liquid chromatography with fluorimetric detection as previously described.We then applied NGMS to samples of patients with a known neurometabolic diagnosis. Features that significantly differed from controls and could be annotated using the IEM panel were selected and checked for known biomarkers of the respective disease of the patient.2.6Z‐scores were calculated to assess variation within the control group. For the analysis of CSF fractions and for comparison of NGMS to targeted assays, we calculated R2 and the relative beta coefficient  using linear regression. All statistics were performed using R version 3.5.2.Sex‐related differences were tested by Wilcoxon signed‐rank test to all selected features, followed by Benjamini‐Hochberg correction with a false discovery rate (FDR) of 5%. Differences between age groups were tested by a Kruskal‐Wallis test with the same correction. For targeted analysis, we used the feature with the highest non‐saturated intensity for each metabolite for semiquantitative analysis. Of note, the Benjamini‐Hochberg correction is based on the number of comparisons within an analysis. Therefore, it is less stringent in targeted analysis (aimed at 30 features) than in untargeted analysis (aimed at thousands of features). t tests followed by Bonferroni‐Holm correction as described previously.Features significantly different between a single patient and controls were determined by 33.1q < 0.05).The demographics of the 87 selected control samples are specified in Table 3.2Z‐scores. Figure Z‐scores per age group together with the FDR‐corrected P‐values for each metabolite.Features extracted from the control cohort were matched against a predefined panel Table . This reβrel). For 12 of the 13 metabolites, we found a corresponding feature in the fractions for which R2 and βrel could be calculated. Figure We identified 13 metabolites which were previously reported to exist in a rostrocaudal concentration gradient in CSF  and neurotransmitter metabolites (n = 4) in 13 samples. This generally showed a good correlation (Table P < .05 (after correction). For two biomarkers, this was not the case: 3‐methoxytyrosine in one AADC deficiency patient, and 5‐hydroxytryptophan in the other. Since the concentrations of these metabolites were age‐dependent in our control cohort study, the analysis was repeated using an age‐matched control group. In the age‐matched reanalysis, concentrations of both 3‐methoxytyrosine and 5‐hydroxytryptophan were significantly increased in both patients , age‐matched controls were required to detect the complete expected biomarker pattern.Using XCMS, we initially extracted a total of 39 855 features from our CSF measurements. From our experience, plasma measurements usually yield approximately 80 000 features. This difference suggests that plasma contains more metabolites than CSF. A possible explanation is that plasma contains more exogenous metabolites, for example, metabolites originating from food that do not reach the CSF. However, biomarker research is usually aimed at elucidating the endogenous metabolism. Thus, the use of CSF may be advantageous due to less interference by exogenous metabolites that are not of direct interest. Furthermore, analysis of CSF rather than plasma is already known to be essential for the biochemical diagnosis of some neurometabolic disorders, as is the case with AADC deficiency.Previously, an extensive list of metabolites identified by “untargeted” metabolomics in CSF of patients aged 0 to 20 years has been published.Another study recently also presented an untargeted metabolomics approach in CSF for diagnosis of (neuro)metabolic disorders.As with all metabolomic approaches, we were not able to get a full coverage of the CSF metabolome. First, metabolites must be susceptible to ionization and be present in sufficiently high concentrations to be detected by QTOF‐MS. Furthermore, detection and (semi‐)quantification of polar metabolites is limited by the use of a reversed‐phase column. The latter is an obstacle in the assessment of sugars, which are generally highly polar and therefore only partially separated. Indeed, in our study, we were not able to discriminate between glucose and galactose, as they have the same retention time on the used column. Such problems may be improved by also performing measurements using a hydrophilic liquid interaction chromatography (HILIC) column,Our results with regard to the effect of sex and age are in line with daily practice of our diagnostic laboratory: while age‐dependent reference ranges are applied to several targeted assays, sex is usually not taken into account. A previous metabolomic study did find sex differences in the concentrations of several metabolites, but this was in an elderly population.When metabolites show a concentration gradient in CSF, the fraction that is used for measurement will influence the outcome. Therefore, we studied four consecutive 5 mL CSF fractions to confirm the gradient of a literature‐based selection of metabolites. While this 20 mL is only a small amount compared to the total CSF volume (estimated to be 150 mL in adults), larger amounts are not relevant for diagnostic purposes: if the gradient is too subtle to present in the first 20 mL, it will also not interfere with standard diagnostic testing. Moreover, it is not ethically justified to withdraw a larger volume because of the elevated risk of post‐dural‐puncture headaches. Several metabolites showed a high correlation among the four fractions, especially 5‐hydroxyindoleacetic acid, homovanillic acid, and methylimidazoleacetic acid. This fits with their presumed concentration gradients.As demonstrated by data from the AADC deficiency patients, knowledge on the age‐dependency of metabolites can be vital for making the correct diagnosis. Conversely, deviating levels of an age‐dependent metabolite can be mistakenly interpreted as perturbations caused by the disease if age is not considered when comparing patients to controls. This may especially apply to very young patients (<0.5 years old), since several metabolites appear at much higher level at this age—also demonstrated by several metabolites with high levels in the 0 to 2 years age group in our study Figure . TherefoOur current study shows that NGMS in CSF is a powerful tool for diagnosis of neurometabolic disorders. We have established a reference CSF metabolome using a large, well‐characterized control CSF cohort. Based on this reference CSF metabolome, we were able to define age‐dependency of different metabolites. Our validation study indicates that information on age‐dependency can be crucial for the correct diagnosis of neurometabolic diseases. Now that we have proven the diagnostic power of NGMS in CSF, this methodology will allow us to extend this approach to yet unsolved cases with a suspicion of neurometabolic disease. Untargeted metabolomics will facilitate biomarker discovery and aid in further understanding of disease mechanisms in the brain.The authors declare that they have no conflict of interest.Tessa M. A. Peters designed and executed the experiments, performed data analysis and interpretation, and drafted the manuscript. Udo F. H. Engelke contributed to experimental design, data analysis and data interpretation, and revised the manuscript. Siebolt de Boer performed analytical measurements, was involved in data processing and revised the manuscript. Ed van der Heeft set up and validated the method, aided in analytical measurements, and revised the manuscript. Cynthia Pritsch selected the samples and revised the manuscript. Purva Kulkarni wrote the scripts used for data analysis, aided in data analysis, and revised the manuscript. Ron A. Wevers contributed to experimental design and revised the manuscript. Michèl A. A. P. Willemsen contributed to experimental design, selected the samples, and revised the manuscript. Marcel M. Verbeek contributed to experimental design, aided in data interpretation, and revised the manuscript. Karlien L. M. Coene contributed to experimental design, aided in data interpretation and initial drafting of the manuscript, and revised the manuscript.Data supporting the findings of this study are available as electronic supplementary material.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000. The study has been approved by the Commissie Mensgebonden Onderzoek Radboudumc under file number 2016‐3011 and titles “Metabolomic characterization of reference cerebrospinal fluid in children” (for control samples) and “Metabolomics in cerebrospinal fluid of patients with known and uncharacterized neurometabolic disorders” (for patient samples). All patients (or their guardians) approved of the possible use of their left‐over samples for method validation purposes, in agreement with institutional and national legislation. This article does not contain any studies with animal subjects performed by the any of the authors.AppendixS1. Supporting Information.Click here for additional data file.


We tested the hypothesis that circulating CXCL10 and IL-6 in donor after brain death provide independent additional predictors of graft outcome. From January 1, 2010 to June 30, 2012 all donors after brain death managed by the NITp (n = 1100) were prospectively included in this study. CXCL10 and IL-6 were measured on serum collected for the crossmatch at the beginning of the observation period. Graft outcome in recipients who received kidney , liver  and heart  was evaluated. Both CXCL-10 and IL-6 showed increased concentration in donors after brain death. The intensive care unit stay, the hemodynamic instability, the cause of death, the presence of risk factors for cardiovascular disease and the presence of ongoing infection resulted as significant determinants of IL-6 and CXCL10 donor concentrations. Both cytokines resulted as independent predictors of Immediate Graft Function. Donor IL-6 or CXCL10 were associated with graft failure after liver transplant, and acted as predictors of recipient survival after kidney, liver and heart transplantation. Serum donor IL-6 and CXCL10 concentration can provide independent incremental prediction of graft outcome among recipients followed according to standard clinical practice. Unfortunately, the ultimate goal of providing long-term graft survival has not been achieved, with a relatively stable rate of attrition, with a 5-year allograft survival of ~ 85%, ~ 75% and ~ 75% for kidney, liver and heart transplant, respectively6, i.e., 15–25% of graft loss within 5 years after transplantation. Whereas early immune-mediated injury is primarily responsible for graft dysfunction and failure, the influence of antigen-independent events may have been underestimated. This concept is supported by data showing similar survival rates for kidneys from living-unrelated donors and one-haplotype matched living-related donors9. Furthermore, organs from living donors, regardless of their relatedness to recipients, have consistently superior outcomes than those from donor after brain death11. An obvious difference between living and donor after brain death are the potential effects of brain death. During and after brain death, circulating leukocyte traffic through peripheral organs slows, and cells adhere to the vascular endothelium and infiltrate the tissues12. As a consequence, donor brain death promptly upregulates inflammatory mediators in peripheral organs with massive increase of major histocompatibility antigens, adhesion molecules, cytokines, and other acute-phase proteins16. This, in turn, may amplify host alloresponsiveness both early after transplant and in the long-term. The different cellular and molecular changes presumably occur secondary to the initial activity of catecholamines18 as well as circulatory cytokines originating from the injured brain and activation of systemic host responses21. As a result, organs from donors after brain death that are transplanted into unmodified allogeneic hosts experience function loss at an accelerated rate compared to those from living donors23. This concept is supported by the clinical finding of a consistently inferior outcome  of kidney allografts with delayed function plus acute rejection compared to organs with a single insult or no insult at all27. However, there is still a gap in knowledge regarding the specific pathways associated with inferior post-transplant outcomes28. The role of the “immune memory” of the transplanted organ in triggering the host immune response and the mediators involved in this process are still not fully elucidated37. Here, a prospective observational study to assess the predictive value of circulating CXCL10 and IL-6 in the donor after brain death for graft survival and function following allotransplantation is presented. We focused our analysis on these two immunological mediators because they have a common double advantage: to be extremely relevant for the immune response after transplantation and to be the target of drugs already available on the market  or in advanced experimental clinical phases in humans  (45)(46).Success of organ transplantation from deceased donor in the short term has progressively improved, with 1-year allograft survival of ~ 95%, ~ 85% and ~ 85% for kidney, liver and heart transplant, respectivelyFrom January 1, 2010 to June 30, 2012 1100 donors after brain death were prospectively included in the study: 533 (48.5%) from Lombardia, 78 (7.1%) from Liguria, 270 (24.5%) from Veneto, 87 (7.9%) from Friuli‐Venezia Giulia, 102 (9.3%) from Marche and 30 (2.7%) from the Autonomous Province of Trento. During the same period, 2869 patients underwent various types of transplant in 21 different centres, receiving organ from 1074 out of 1100 donors included in the study . The level of function of a graft in the immediate postoperative period was described to be correlated with long-term graft and patient survival. Confirming what is expected, even in our cohort the absence of IGF in the postoperative period was correlated with graft and recipient survival . In addition to the two cytokines, the following variables were included in the model: donor sex and age, recipient sex and age, donor cause of death , donor characteristics , donor inotrope administered , time spent on the waiting list, number of HLA mismatches , maximum panel-reactive antibody (PRA) level, immunologic risk and NITK3The association of donor IL-6 and CXCL10 levels with the recipient and graft survivals was performed by Cox Regression analysis including variables as for IGF analysis. In univariate analysis  or in advanced experimental clinical phases in humans . IL-6 is critical for the progression of naïve B cells transforming into plasmablasts and mature plasma cells as well as shaping T cell immunity and is also responsible for activating the production of IL-17 signalling, inhibiting Treg function44. The chemokine CXCL10 is a potent chemoattractant for macrophages, dendritic cells, NK cells, and activated T cell and its level rapidly rises following organ reperfusion and during early rejection of the heart, kidney and liver45. Second, and equally important, we were able to define the correlation between IL-6 and CXCL10 levels of donor after brain death and their baseline characteristics. The evidence that both IL-6 and CXCL10 levels were significantly different from those in healthy subjects confirmed the detrimental effect of brain death in modifying the homeostasis of the immune system within a short time. These changes presumably occur secondary to the initial activity of catecholamine as well as circulatory cytokines originating from the injured brain19 Collectively, the donor characteristics explained a relatively low percentage of the variance (12–14%) in IL-6 and CXCL10 values, underling that brain death itself is probably the major driver of the “inflammatory signature”. Unfortunately, for many of the characteristics it is not possible to determine whether the relationship is of cause-effect. Despite this, some results are of great interest. For example, variables related to intensive care duration and hemodynamic instability, including the use of inotropes and vasopressors like norepinephrine, resulted generally associated with higher levels of circulating IL-6 and CXCL10. On the other side, not both the cytokines have the same relationship with baseline characteristics. Different causes of death appeared to determine different effects on measured cytokines with a relevant impact on CXCL10 but not IL-6 concentration. Kidney and liver function tests showed strong positive correlation with both circulating cytokines, while white blood cells showed a negative correlation. Taken as a whole these data showed that the activation of the brain-dead donor inflammatory response is very complex, and the mediators do not always respond in an identical way. Third, we were able to demonstrate the independent role of donor IL-6 and CXCL10 on graft outcome. This suggests that treatment aimed to reduce the donor inflammatory response could have impact on graft outcome. The identification of donor mediators acting as “master predictor” of outcome for all transplants is a “conditio sine qua non” to start to treat donors with drugs able to inhibit specific molecular pathways with the intention to reduce the inflammatory response in multiorgan donors and thereby improving organ survivals. Until now, this type of study has not yet been carried out for various reasons, including the difficulty of identifying the best targets for different organ grafts, the unavailability of measurable biomarkers and the difficulty of identifying a timing of treatment compatible with the explant procedures. Our results could fill some of these gaps. In fact, our data suggest a framework in which the inhibition of IL-6 and/or CXCL10 at the beginning of the donor observation period could be measured and the results used to improve the outcome of transplantation. The delay (kidney and heart) or the absence (liver) of functional recovery after transplantation was documented as being consistently associated with the donor inflammatory state. Concordantly, preventing the delay or lack of functional recovery of transplanted organs could represent the primary end-point of a study aimed at inhibiting donor IL-6/CXCL10. Of note, in the long-term follow up, factors more closely related to the specificity of individual organs could be influenced in different ways in each specific organ.To our knowledge, this is the largest prospective study examining whether the inflammatory status of the donor after brain death provides independent additional prediction of graft outcome among recipients followed according to standard clinical practice. Experiments in animal models have previously demonstrated the relation between brain death and the rapid infiltration of leukocyte populations in peripheral organs with intense upregulation of their associated productsThe findings of our study must be also seen in light of some limitations. First, we observed our cohort after a median follow-up of about 5 years and therefore the study lacks information of possible long-term effects of donor cytokines/chemokines on outcome. Second, deep data about the incidence of rejection episodes and the causes of graft failure were not always available. This makes difficult to understand whether donor factors have prediction value for specific causes of graft loss . Third, the cytokine levels were measured from the serum specimen sent for tissue typing, and at only that point. The time between blood sampling for circulating CXCL10 and IL-6 determination and organ procurement was quite homogenous in the different intensive care units (6–12 h), and this variability should not represent a major limitation for the study. Despite this, it is well known that inflammatory changes after brain death are dynamic and very heterogeneous, and we cannot exclude that significant changes can occur between blood sampling and organ procurement. On the other hand, the choice to use the serum specimen obtained at the beginning of the observation period was the only one possible, considering the number of intensive care units involved and of donors analyzed. Furthermore, in a pragmatic application perspective, it is the most standardizable observation point in organizational terms and would allow obtaining results in time for the selection of donors and recipients. Fourth, we did not included other donor types as controls, e.g., donor after cardiac death or living-related donors. During our study (2010–2012), transplants from donors after cardiac death were rare (less than 10) and, therefore, were not included in our analysis. On the other hand, living related organ donors were excluded from the analysis because they represent a highly selected population (to meet the living donation criteria) which does not obviously include heart donors.In conclusion, this study was conducted to test the hypothesis that the inflammatory status of the heart beating multiorgan donor at the time of organ recovery provides independent incremental prediction of graft outcome among recipients followed according to standard clinical practice. The results confirmed the starting hypothesis. The characterization of the inflammatory signature may bring new therapeutic approaches in the transplant field. In fact, attenuating the donor inflammatory response before organ procurement may improve early and long-term outcomes after organ transplantation, and help maximize organ use from the available donor pool.The study population consists of recipients who received organs from deceased individuals from whom organs were procured from January 1, 2010 to June 30, 2012 in The Nord Italia Transplant program (NITp) area. NITp is an inter‐regional transplant agency comprising six Italian regions: Lombardia, Liguria, Veneto, Friuli‐Venezia Giulia, Marche and the Autonomous Province of Trento. This area has 129 intensive care and 43 transplant units  for a population of 19 million inhabitants. NITp manages waiting lists, performs pre-transplant immunological tests, allocates organs, collects and analyses data , organizes organ procurement, transport and transplant. A total of 1100 donors after brain death were considered during this period and their related recipients were prospectively included, obtaining a cohort made up of 2700 recipients with complete follow-up records. A non-diluted venous blood sample drawn from each donor at the procuring hospital was shipped to NITp central laboratory for crossmatch. The serum specimens were obtained in the participating intensive care units 6–12 h before procurement. An aliquot of 1 ml of serum was separated and stored at the Interregional Coordinating Center at − 80 °C for the measurements of inflammatory/immunological factors. The mean time from sampling and freezing was 3.67 h and during this time the sample was conserved at 4 °C. There was no evidence of significant change in IL-6 and CXCL10 levels related to the time from sample collection until freezing. Information on donor demographics and medical characteristics and the disposition of each organ that could potentially be used for transplantation therapy was obtained from the deceased donor registry data maintained by the NITp. All donor data were abstracted from the donor medical records and entered on standardized NITp donor data collection form by the transplant coordinator at the procuring hospital. Donor characteristics used in our analysis can be found in Table For the study we used a bead-based assay based on Luminex technology , that allowed to measure both CXCL10 and IL-6 using a low volume of serum (50 µl). To minimize inter-assay variation, donor sera was assayed at the end of the study using the same commercial lot. Appropriate pool of sera was used to estimate intra-assay and inter-assay coefficient of variation.46. Immediate Graft Function (IGF) was defined as the absence of early allograft dysfunction as previously defined after kidney47, liver48 and heart transplantation49. Kidney primary non function (PNF) was defined as the absence of a decrease in serum creatinine levels, which resulted in the need for dialysis 3 months after transplant. Acute and chronic kidney rejection were defined as biopsy proven or clinically evident rejection. Liver PNF was defined as graft failure resulting in death or retransplantation within 30 days of the index transplant when other causes of graft failure were excluded, i.e., vascular thrombosis, rejection, or recurrent disease. Acute and chronic liver rejection was defined according to the International Consensus Document on Terminology of Hepatic Allograft Rejection50. Heart Primary Graft Failure (PGF) was defined as previously reported49. Acute and chronic heart rejection was defined according to the International Consensus Document on nomenclature in the diagnosis of heart rejection51. NITK3 is an allocation algorithm established in 1997, which aims at ensuring quality, equity, transparency and traceability during all the phases of the allocation decision-making process52. NITK3 has been set up by the NITp Working Group on the basis of biological, medical and administrative criteria and it is periodically reviewed after the analysis of transplant results52.In Italy, organ donation and transplantation activities are coordinated by law by the CNT that, in collaboration with regional and interregional coordinating Centers, ensures the quality and traceability of the entire process all over the national territory. To this purpose, all transplants performed in Italy are recorded on the Transplant Information System (SIT). Transplant activity data registered in SIT were used to evaluate graft failure and recipient survival. Graft survival was defined as time from transplant to graft failure, censoring for death with a functioning graft and grafts still functioning at time of analysis. Patient survival was defined as time from transplant to patient death, censoring for patients still alive at time of analysis. Recipient at immunological risk included patients with high rate of antibodies (> 50% antibodies against panel), recipients who had lost their first graft due to early rejection, cross match positive or HLA incompatibility according to immunology service center criterionData are presented as mean ± standard deviation (SD) or median and interquartile range (IQR), according to their distribution. Class variables are given as numbers of individuals in percentages. Variables with a normal distribution were compared with one-way unpaired Student’s t test. Variables with a non-normal distribution were compared with Mann–Whitney U test. Categorical variables were compared using the chi-squared test or Fisher’s exact test as appropriate. Spearman correlation was used to study associations of continuous variables. Graft or patient survivals were estimated according to Kaplan–Meier. Association between variables and outcomes or survivals was assessed by Logistic or Cox proportional-hazard regression, respectively. For the regression models, cytokines concentrations were used as continuous variables after Log transformation or as preplanned binary variables: 0 if the value was ≤ 66th percentiles, 1 if the value was > 66th percentiles defining “high IL-6” category for concentration > 563 pg/ml (66th percentile), and “high CXCL10” category for concentration > 1748 pg/ml (66th percentile). The multivariate analysis was performed using variables significant at the p < 0.1 in the univariate analysis. All statistical analyses were performed using the SPSS statistical software, version 13.0 .Supplementary Information


Significant results were followed up in VSMCs in vitro. We demonstrate that 1,25(OH)2D can enhance the ability of PEx to induce SpA remodeling, via a mechanism associated with increased secretion of granulocyte-colony stimulating factor (G-CSF). G-CSF appears able to increase VSMC disorganization and phenotypic switching in both an ex vivo vascular model and in vitro VSMC cultures. The clinical relevance of these findings are still to be determined. G-CSF may have differential effects depending on dose and vascular bed, and vitamin D may play a role in potentiating these actions. G-CSF may be an interesting potential therapeutic target for facilitating physiological vascular remodeling for the prevention of adverse obstetric outcomes.Vitamin D deficiency is associated with complications of pregnancy such as pre-eclampsia, fetal growth restriction, and miscarriage, all of which are also associated with incomplete spiral artery (SpA) remodeling. We have previously shown that both uterine natural killer (uNK) cells and extravillous trophoblast cells (EVT) are required for successful SpA remodeling, but whether their activity in this process is modulated by vitamin D is not known. In the current study, we use a previously described chorionic plate artery (CPA)  Early iDRs) (2D . By contDRs)  are far reaching. Classically, severe vitamin D deficiency is recognized for its negative effect on bone mineralization, as manifested by rickets in children and osteomalacia in adults . In morevia a process involving separation and de-differentiation (phenotypic switching) of the vascular smooth muscle cells (VSMCs), and their eventual loss from the vessel wall via migration into the surrounding stroma and apoptosis . Samples for isolation of uNK cells (decidua) or placental explants  were processed immediately as described below (n = 10 decidua and placenta). Chorionic plate artery (CPA) segments, which were used as an in vitro model to analyze the effect of uNK cell and PEx products on VSMC morphology, were obtained from normal term placenta delivered by elective cesarean section. The study was approved by the Ethical Review Board of GWCMC. According to local guidelines it was not necessary to obtain written informed consent for the use of these tissues.Human placenta and decidua samples were obtained from women undergoing elective surgical termination of apparently uncomplicated pregnancies between 5–7 weeks gestation (determined by ultrasound) in Guangzhou Women and Children’s Medical Center (GWCMC). Placental and decidua samples were separated and washed thoroughly with sterile saline. Samples for immunohistochemistry (IHC) were fixed with 10% (v/v) formalin and embedded in paraffin  fetal calf serum, 1% (v/v) penicillin/streptomycin, 1% (v/v) amphotericin; all from Gibco, life Technologies, United States) containing 1,25(OH)2D  or 25OHD . Stock solutions (10 and 100 mM respectively) of 1,25(OH)2D and 25OHD were prepared in 100% ethanol and further dilutions prepared in phosphate buffered saline (PBS), controls included 0.001% ethanol as per the highest concentration of each agent. After 24 h the uNK-CM was removed, centrifuged and stored at −80°C. Cell purity and viability were tested routinely by immunocytochemical staining of cell smears and trypan blue exclusion, respectively; both were consistently >95%.uNK cell-enriched isolates were prepared by enzymatic disaggregation and positive immunomagnetic selection  as described previously . To prep2D  or 25OHD  in a 24 well plate coated with growth factor reduced Matrigel . Conditioned medium was harvested (PEx-CM) after 24 h, centrifuged and stored at −80°C.PEx were isolated as previously described  and appr6 uNK cells (from the same patient) in DMEM/F12 containing 1,25(OH)2D  or 25OHD  to a total volume of 1 ml  or 25OHD  (n = 5 each group); (B) granulocyte-colony stimulating factor (G-CSF)  (n = 6); (C) 20% (v/v) PEx-CM and anti-G-CSF  (n = 6); or (D) RPMI1640 complete medium control.Intact CPA segments (5 mm length)  were disThe medium was completely replaced every 48 h. CPAs were harvested after 120 h, fixed in 10% (v/v) neutral buffered formalin for 24 h and embedded in paraffin wax. 3 μm serial sections were stained with hematoxylin and eosin (H & E).2D  or 25OHD  (n = 5 each group) by Luminex multiplex assay according to the manufacturer’s instructions (Millipore). The analytes tested were EGF, IFN-γ, IL-1RA, IL-1β, IL-4, IL-6, MDC, PDGF-AA, PDGF-BB, VEGF-A, FGF-2, MCP-1, MCP-3, MIP-1α, MIP-1β, RANTES, TNF-α, PLGF, IP-10, G-CSF, GM-CSF, GRO-α, and Ang-2.Cytokine, chemokine, and angiogenic growth factor levels were tested uNK-CM, PEx-CM, uNK/PEx-dir-CM and uNK/PEx-indir-CM all with or without 1,25(OH)2 in air. VSMCs were used between passages three and six.Human Umbilical Artery Smooth Muscle Cells (HUASMC) were obtained from ScienCell  and cultured in smooth muscle cell medium  supplemented with 2% (v/v) fetal bovine serum , 1% (v/v) penicillin/streptomycin , 1% (v/v) smooth muscle cell growth supplement  maintained in a standard tissue culture incubator at 37°C, 5% CO2 in air.The RTCA utilizes specialized plates that contain interdigitated electrodes which can be used to determine cell proliferation, migration, and invasion in real time . For all3 VSMCs in 150 μl complete SMCM medium containing 0, 0.1, or 3 ng/ml G-CSF were added to duplicate wells of an E-Plate. Recordings were taken every 15 min for a total of 48 h (n = 3).For proliferation assays 5 × 102 in air cell culture incubator. Complete SMCM medium was added to the lower chamber prior to attachment of the upper chamber and addition of 2 × 104 VSMCs in serum free SMCM medium containing G-CSF . Recordings were taken every 15 min for a total of 48 h (n = 3).For invasion assays the upper chamber of a CIM-16-Plate was coated with 20 μl growth factor reduced Matrigel diluted 1:30 with serum free SMCM medium and allowed to set for 4 h at 37°C in a 5% CO2O2 for 10 min. Samples were stained with specific antibodies overnight at 4°C after 30 min blocking with 10% (v/v) goat serum. Antibodies to human alpha smooth muscle Actin , osteopontin , anti-G-CSF (ab204989), and anti-G-CSF Receptor (ab126167) were purchased from Abcam. All antibodies were used in a 1:200 dilution for IHC, while negative control was performed by replacing the primary antibody with PBS or the appropriate IgG control. After washing, the slides were incubated with DAKO RealTM EnVisionTM Detection System, Peroxidase/DAB, Rabbit/Mouse  and detected by 3,3-diaminobenzadine (DAB). Analysis of each section using a modified “quickscore” method taking into account both percentage of cells stained  and the staining intensity for each percentage . The percentage and intensity scores were then multiplied and summed to give a possible score range of 0–12 , 85% (v/v), 75% (v/v) ethanol, and dH2O (5 min each step). According to the antibody datasheets antigen retrieval was performed in either citrate buffer (pH6.) or EDTA buffer (pH8.0) and heat. After samples were cooled to room temperature sections were incubated in 3% (v/v) H of 0–12 .TM secondary antibody Alexa Fluor 555  or goat anti-mouse IgG (H + L) superclonalTM secondary antibody Alexa Fluor 488  for 1 h and then DAPI  for 10 min. Images were captured using a Leica fluorescent microscope using a 40X objective and staining intensity analyzed using Image J software. At least three images were analyzed per condition over three separate experiments.The VSMCs were cultured up to 80% confluence in an 8-well Nunc Lab-Tek Chamber Slide , medium removed, rinsed with PBS and fixed with 4% (v/v) paraformaldehyde for 30 min. The cells were permeabilized with 0.5% (v/v) Triton-X100 for 15 min and then blocked in 10% (v/v) goat serum for 30 min. The slides were incubated overnight at 4°C with primary antibodies to αSMA , OPN , and KLF4 . After washing, the slides were incubated with goat anti-rabbit IgG (H + L) superclonalvia a Bio-Rad ZOE Fluorescent Cell Imager using a 20X objective. F-actin structures in individual cells were observed and categorized into three types , medium removed, rinsed with PBS and fixed with 4% (v/v) paraformaldehyde for 30 min. The cells were permeabilized with 0.5% (v/v) Triton-X100 for 15 min and then blocked in 10% (v/v) goat serum for 30 min. The slides were incubated with Texas Red-X Phalloidin  for 1 h prior to DAPI  staining at room temperature. For each treatment, 5 images were taken randomly within each chamber ee types . Quantifee types . Brieflyg at 4°C for 30 min and supernatant collected and stored at −80°C until required for analysis. Protein concentrations were determined by BCA protein assay kit . 20 μg total protein was mixed with 5X loading buffer and denatured at 95°C for 8 min before being separated on SDS-PAGE, and electro-transferred onto polyvinylidene difluoride (PVDF) membranes . Membranes were blocked with 5% (w/v) skimmed milk for 2 h, and incubated with target antibodies on a shaking bed overnight at 4°C. Primary antibodies used in this study were: Anti-SMA, Cat: ab32575, rabbit, 1:1,000; anti-OPN, Cat: ab69498, mouse, 1:1,000; anti-GAPDH, Cat: ab181602, rabbit, 1:10,000 . After removing the primary antibodies, membranes were washed with TBST 3 × 10 min. The membranes were then incubated with HRP-conjugated secondary antibodies on a shaking bed for 2 h at room temperature. After washing three times with TBST, chemiluminescent substrate was used to develop and signals were captured by ChemiDoc XRS+ imaging system . Data were analyzed by image J software . Each experiment was repeated three times.Control and G-CSF (3 ng/ml) treated VSMCs were washed twice with cold phosphate-buffer saline (PBS) and then protein extracted with RIPA buffer  containing PMSF  and protease inhibitor cocktail . Cell lysates were centrifuged at 12,000 post hoc test or Student’s t-test as appropriate. A P value of <0.05 was considered significant.Data are presented as mean ± SD. Statistical significance was determined using Statview by one-way ANOVA with Fisher’s P < 0.0001)  treated PEx CM induced VSMC disorganization compared to control and PEx CM alone  and 25OHD (100 nM) induced VSMC disorganization in the CPA model (data not shown). But since treatment of PEx with 1 nM 1,25(OH)2D treated PEx induced disorganization we conclude that this is a specific PEx mediated response and not due to residual vitamin D in the conditioned medium.To determine the effect of vitamin D treated uNK cell, PEx or co-culture CM on VSMC organization a CPA model of vascular remodeling was used . As prev 0.0001) , howevernization . PEx CM nization . HoweverCM alone . CM fromPA model . Both 1,2D altered PEx CM so that is was able to induce VSMC disorganization in the CPA model; we performed a wide panel Luminex multiplex array. Treatment with 1,25(OH)2D did not alter the secretion profile of uNK cells or uNK cell/PEx co-cultures (direct and indirect) (2D increased secretion of granulocyte colony stimulating factor (G-CSF) by PEx in a dose dependent manner ndirect) . TreatmeTo determine the spatial distribution of G-CSF and its receptor, G-CSFR, IHC was performed on first trimester decidua and placental tissue. In first trimester placenta G-CSF and G-CSFR were highly expressed by cytotrophoblast, syncytiotrophoblast and EVT of the cell columns . In the 2D treated PEx CM induction of VSMC disorganization, the CPA model was used under the following treatment conditions (i) G-CSF alone, (ii) 1,25(OH)2D treated PEx CM, and (iii) 1,25(OH)2D treated PEx CM pre-treated with G-CSF neutralizing antibody inhibitor. G-CSF induced VSMC disorganization in a dose dependent manner 2D treated PEx CM to induce VSMC disorganization P = 0.0002; OPN P = 0.005; 2D treated PEx CM induced VSMC phenotypic switching  and 48 h (0.1 ng/ml P = 0.04 and 3 ng/ml P = 0.04) time-points  to mediate the initial stages of vascular remodeling, via a mechanism associated with G-CSF secretion. In addition, we demonstrate a direct role for G-CSF in VSMC phenotype switching from a contractile to synthetic state.Remodeling of the uterine spiral arteries is one of the key maternal adaptations required for the establishment of a successful hemochorial pregnancy, with failure of this process leading to a number of serious complications of pregnancy, increasing both maternal and fetal mortality and morbidity. In epidemiological studies, maternal vitamin D deficiency has also been associated with a risk of developing pregnancy complications, although the etiology of this association is not well understood. This current study demonstrates that active 1,25 (OH)via migration and then apoptosis 2D-treated uNK cells identified no significant difference in cytokine, vessel remodeling or angiogenic transcript expression comparative to paired 1,25(OH)2D-treated pNK cells following immune activation 2D increased secretion of G-CSF by the placental explants, but no other differences were observed in any culture condition. Few other studies have investigated secretion of G-CSF after vitamin D treatment, despite its proposed immunomodulatory role. In agreement with the current study, treatment with vitamin D increased G-CSF secretion by peripheral blood NK cells  via a mechanism associated with Rac1 signaling 2D treated PEx in the ex vivo model suggesting that G-CSF was responsible for the observed effects and not any residual 1,25(OH)2D in the conditioned medium. However, more research in different vascular beds and in scenarios of physiological remodeling, as seen in early pregnancy, and pathological remodeling, as seen after vascular injury, are required to fully understand the role of G-CSF in these processes.Granulocyte-colony stimulating factor is a 30 kDa glycoprotein that is expressed by a range of cell types and is predominantly involved in release of hemopoietic progenitor cells from the bone marrow and their differentiation into granulocytes within the circulation . More res effect . Very fes effect . In addiignaling . These dignaling . Increasvia an increase in G-CSF secretion. In addition, vitamin D has previously been shown to stimulate EVT invasion (2D and not 25(OH)D, the main circulating marker of vitamin D status. This may simply reflect the relatively low levels of 1,25(OH)2D synthesis from added 25(OH)D in the explant model used for these experiments. However, in future studies it will be interesting to assess the effects of higher levels of 25(OH)D for longer incubation periods. A few studies have investigated the association of serum G-CSF with pre-eclampsia, one found increased, one decreased and one unaltered levels either at the time of delivery or in the second trimester, therefore it is not clear whether circulating levels contribute to complications of pregnancy (Vitamin D deficiency has been associated with a number of serious complications of pregnancy, and we originally hypothesized that it may alter the ability of different cells to mediate SpA remodeling, a key step in the establishment of a successful pregnancy. Our data suggests that vitamin D can enhance the ability of EVT to induce SpA remodeling invasion . Thereforegnancy . Howeverregnancy .in vitro tissue and cellular models, and as such it is not known if the results can be fully extrapolated into the in vivo situation. The vessel model makes use of the easily accessible chorionic plate arteries from term placenta. These vessels contain several layers of VSMCs and can be obtained in a non-invasive manner without requirement for unnecessary patient procedures. The model has been validated against sections of spiral arteries dissected from non-pregnant myometrium after hysterectomy (The current study relies on the use of erectomy . Howevervia a mechanism associated with increased secretion of G-CSF that in turn is able to increase VSMC disorganization and phenotypic switching in both an ex vivo vascular remodeling model and in vitro VSMC cultures. This is one of the first studies showing a direct effect of G-CSF on VSMC biology, although one previous study has also reported increased VSMC migration in response to G-CSF. The clinical relevance of these results is still to be determined as it appears that G-CSF may have differential effects depending on dose and vascular bed. However, it may be an interesting potential therapeutic target for facilitating physiological vascular remodeling for the prevention of adverse obstetric outcomes.In conclusion, in the current study we demonstrate that vitamin D can enhance the ability of EVT to induce vascular remodeling, The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Guangzhou Women and Children’s Medical Center Ethical Review Board. The patients/participants provided their written informed consent to participate in this study.JZ and PW conceived the study and performed the experiments and data analysis. DC, FN, and QL performed the experiments. XQ, JT, MH, GL, and MK conceived the study, obtained funding, critically assessed the data, and aided in interpretation. GL wrote the manuscript. All authors edited the manuscript and approved its final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.