Objectives: GM-CSF is a pro-inflammatory cytokine with multiple actions predominantly on myeloid cells. Enhanced GM-CSF expression by lymphocytes from patients with Ankylosing Spondylitis (AS) has recently been described, however, its potential pathogenic role(s) in AS are unknown.Methods: The effects of GM-CSF on TNF, IL-23, and CCL17 production by blood, PBMCs and isolated CD14+ monocytes from AS patients and healthy controls (HCs) were studied using ELISA. Serum CCL17 and GM-CSF and T cell GM-CSF production were studied in AS patients including pre-and on TNFi therapy.Results: GM-CSF markedly increased TNF production by LPS-stimulated whole blood, peripheral blood mononuclear cells (PBMC) and purified monocytes from AS patients, with 2 h GM-CSF exposure sufficient for monocyte “priming.” Blocking of GM-CSF significantly reduced the production of TNF by whole blood from AS patients but not HCs. GM-CSF priming increased IL-23 production from LPS-stimulated AS and HC whole blood 5-fold, with baseline and stimulated IL-23 levels being significantly higher in AS whole blood. GM-CSF also stimulated CCL17 production from AS and HC blood and CCL17 levels were elevated in AS plasma. GM-CSF could be detected in plasma from 14/46 (30%) AS patients compared to 3/18 (17%) HC.Conclusion: We provide evidence that GM-CSF primes TNF and IL-23 responses in myeloid cells from AS patients and HC. We also show CCL17 levels, downstream of GM-CSF, were elevated in plasma samples of AS patients. Taken together these observations are supportive of GM-CSF neutralization as a potential novel therapeutic approach for the treatment of AS. Ankylosing spondylitis (AS) is a common inflammatory rheumatic disorder and a prototype of Spondyloarthritis (SpA), characterized by axial skeleton and sacroiliac joint involvement . AS affeGM−CSF”) overlaps but is distinct from that of classical IL-17A-producing TH17 cells .Patients attending the Oxford University Hospitals National Health Service Foundation Trust were recruited. Blood samples were taken with informed written consent and their research use was ethically approved . Ankylosing Spondylitis (AS) patients met Assessment of Spondyloarthritis international society (ASAS) criteria for Axial Spondyloarthritis. Patients commencing TNFi therapy were deemed responders sustaining combined reduction of >4 points BASDAI and spinal pain. Patients with Rheumatoid Arthritis fulfilled 2010 ACR/EULAR criteria. Demographics of patients for plasma CCL17 comparison between AS, RA and HC are shown in One milliliter of whole blood from AS patients or healthy controls was incubated with 10 ng/ml human recombinant GM-CSF (PeproTech) or 10 μg/ml GM-CSF antibody (R&D) or IgG (R&D) at 37°C for 2 h, before being transferred into 2 ml tubes containing 1 ml of RPMI with or without 20 ng/ml LPS , and incubated at 37°C overnight. Supernatant was harvested for TNF, IL-23, or CCL17 ELISA.PBMCs were isolated from sodium-heparinized peripheral blood of AS patients or healthy control by density gradient centrifugation using Histopaque (sigma). Prior to isolation, whole blood was diluted 1:1 in RPMI 1640 medium . CD14+ monocytes were isolated from PBMCs of AS patients or healthy control using CD14 microbeads (Miltenyi Biotec).Pbmcs, CD14– PBMCs, or CD14+ monocytes were pre-incubated with 10 ng/ml human recombinant GM-CSF for 2 h, in 96-well plates (0.2 M per well), and incubated with LPS (10 ng/ml), at 37°C overnight. For GM-CSF priming condition, after 2 h incubation with GM-CSF (10 ng/ml), CD14+ monocytes were washed with PBS, and then stimulated with LPS and incubated overnight at 37°C. Pam3Csk4 (15 ng/ml), or RX848 (resiquimod 1 μg/ml) or CpGODN (10 μM) were also studied. ELISA was performed on supernatants according manufacturer's instructions; TNF, IL-23 both Thermo Fisher Scientific or CCL17 (R&D UK).2 Profiler PCR array, designed in 384-wells for the detection of 84 JAK/STAT related genes. After 2 h of incubation with GM-CSF, CD14+ monocytes were pelleted and resuspend in TRIzol. Total RNA was isolated and extracted using an RNA extraction kit . After RNA extraction and quality assessment (NanoDrop) cDNA synthesis was performed using RT2 First Strand kit (Qiagen). Samples were prepared with the Master Mix and Template Cocktail and loaded into the 384–well RT2 Profiler PCR array. RT2 Profiler PCR arrays were run on ABI ViiA7 thermal cycler using the SYBR Green dye. ΔΔCt values determined. CT values greater than 35 were excluded. Fold change in expression of each gene in GM-CSF-treated cells was compared to untreated control. Genes with fold change ≥ 1.5 were considered up-regulated, and ≥ −1.5 as down-regulated.CD14+ monocytes from AS and HC with or without GM-CSF treatment were assayed using the RTPaired samples from 9 AS patients with active disease  pre- and during TNFi treatment (3–6 months) were studied. PBMCs were isolated from fresh blood and cryopreserved at high cell density, 50–100 million/ml, in fetal bovine serum with 10% DMSO. This freezing condition was selected after comparing post-thawing viabilities of PBMCs frozen at different density and resulted in 80–90% viability. On the day of staining, PBMCs (isolated within 2 h of venesection and frozen in liquid nitrogen) were thawed and stimulated for 4 h at 37°C with phorbol myristate acetate  and ionomycin , in the presence of Golgiplug (Brefeldin A) and Golgistop (Monensin) (both from BD Biosciences). Staining antibodies and dilutions are shown in 0. Equalization beads (Fluidigm-Maxpar) were added at a concentration of 1:10 and the sample was acquired on a CyTOF-2 machine. Data analysis was carried out using Cytobank® software.One microliter to one milliliter of 103Rh intercalator (Fluidigm) was added to cells and incubated for a 20 min at 37°C. Cells were resuspended in 50 μl staining buffer containing surface antibody master mix .Levels of free GM-CSF were assessed using a GSK in house clinically validated Singulex bead-based immunoassay. Neat plasma samples were incubated in duplicate with beads bound with GSK3196165, an anti-GM-CSF monoclonal antibody, for 2 h. Beads were washed, and a fluorescent-labeled detection antibody was added and incubated for 1 h. Beads were washed again and the GM-CSF/fluorescent labeled complexes were eluted and transferred to a 384-well plate with buffer. The plate was read by the Singulex Erenna reader. GM-CSF concentrations were obtained by back-calculating signal data to a standard curve prepared with recombinant human GM-CSF in a filtered, low responding serum pool. Values below LLQ (lower level of quantification) were imputed to 0.5t-tests, unpaired t-tests, or one-way ANOVAs were used as denoted in the figure and 2 Profiler PCR array experiment  were performed using the QIAGEN data analysis website, p ≤ 0.05 was considered significant. The false discovery rate (FDR) was calculated using the Original FDR method of Benjamini and Hochberg. The CCL17 data presented in Figure 4B was not normally distributed and was therefore log-transformed prior to one-way ANOVA analysis.Prism was used for statistical analysis. Paired p = 0.001, p = 0.008). GM-CSF blockade reduced the production of TNF (but not IL-23) in response to LPS by whole blood from AS patients but not HCs  responsible for the GM-CSF enhancement of cytokine responses to LPS, we separated peripheral blood into peripheral blood mononuclear cells (PBMCs) and non-PBMCs (containing granulocytes). Little TNF was produced in response to LPS by PBMC-depleted whole blood (data not shown). GM-CSF potently promoted the production of TNF by PBMCs from AS patients and HCs with LPS stimulation . LPS-indMass cytometry (CyTOF) analysis confirmed high expression of GM-CSF receptor α (CD116) primarily on CD14+ monocytes . We therTo investigate whether GM-CSF can affect TNF production by other TLR agonists, CD14+ monocytes were cultured with Pam3Csk4 (TLR1 and 2 agonist), RX848 , and CpGODN (TLR9 agonist). IL4R, SOCS2, and SOCS3 in CD14+ monocytes .We next asked if short term exposure of GM-CSF is sufficient to increase the response of monocytes to stimulation. Two hours priming of monocytes with GM-CSF significantly promoted TNF production by LPS-stimulated monocytes from AS patients to levels similar to that seen with longer term GM-CSF exposure . SimilarRecent studies have shown that GM-CSF can promote CCL17 production via IRF4 gene induction . We therWe then asked if GM-CSF regulated PBMC CCL17 production directly through monocytes. Whilst GM-CSF indeed induced CCL17 release from isolated monocytes, comparison with experiments using whole blood and PBMCs suggested that other cell types may potentiate this effect . To deteGM-CSF-producing lymphocytes in paired PBMC samples from AS patients pre- and on TNFi treatment were quantified using intracellular staining flow cytometry. Although numbers were small (9 pairs), TNFi responders had a higher baseline percentage of GM-CSF-producing CD8 T cells than TNFi non-responders . TNFi trGM-CSF was detected by Singulex bead-based immunoassay in 3/18 HCs, 2/18 RA patients, 4/20 AS patients naïve for biologic treatment, 5/18 AS patients on biologic treatment, and 5/8 AS patients studied both pre- and on TNFi treatment, totalling 14/46 (30%) AS patients compared to 3/18 (17%) HC . GM-CSF We have previously described that patients with axial SpA have increased numbers of GM-CSF-secreting lymphocytes . We hereWe also show that GM-CSF potentiates IL-23 production by LPS-stimulated whole blood and PBMC from AS patients. This effect was not seen for isolated AS monocytes, implying involvement of other cell types. Lastly, we demonstrate that GM-CSF stimulates CCL17 production from AS and HC blood; CCL17 levels were elevated in AS plasma and, in a small cohort of patients, were reduced upon TNFi therapy. A cartoon model showing the potential pro-inflammatory the roles of GM-CSF in SpA is shown in AS is characterized by chronic  gut inflammation and Reactive Arthritis is a related form of SpA that often follows gastrointestinal infection. Both these scenarios might lead to increased gastrointestinal or systemic LPS exposure. Indeed AS patients have recently been reported to have impaired gut vascular barrier and increased circulating LPS levels . Whilst ex vivo in whole blood from AS patients but not healthy controls. This AS-specific effect of anti-GM-CSF strongly supports the increased levels of GM-CSF in AS blood and the therapeutic potential of blocking GM-CSF in AS. GM-CSF was also shown to potentiate LPS-stimulated IL-23 levels in whole blood from AS patients and HC by ~5-fold. Interestingly the levels of IL-23 stimulated by LPS were always significantly greater in AS patients compared to HC, whether GM-CSF was exogenously applied or not. We decided to investigate IL23 levels in these experiments as dysregulation of the IL-23/IL-17 pathway has been associated with AS pathology  of monocytes with GM-CSF is sufficient to enhance TNF cytokine production by LPS-stimulated monocytes. GM-CSF priming altered the transcriptome of monocytes and regulated the expression of IRF4, IRF5, and other cytokine regulatory genes, confirming and extending previous findings, with IRF4 shown previously to drive CCL17 production in human monocytes stimulated overnight with GM-CSF . The “GMex vivo. We also show that baseline CCL17 levels in AS plasma were elevated compared to healthy controls, and CCL17 levels may be reduced following anti-TNF treatment. Although our prospective cohort was small, our findings agree with two previous studies (GM-CSF has been shown to drive CCL17 expression and protein release from human monocytes via JMJD3 (a histone demethylase) and IRF4 and that absence/inhibition of these mediators ameliorates inflammatory arthritis and pain in mouse models . We here studies . Additio studies .It is interesting that blocking of GM-CSF reduced the LPS-induced TNF-alpha production in AS but not healthy control whole blood, and did not affect LPS-induced IL-23 production in AS or HC. A possible explanation is that whilst AS patients have higher concentrations of GM-CSF than healthy controls, the level is lower than that required for the enhancement of IL-23 production. Notably, lymphocytes from inflamed synovium have been shown to produce more GM-CSF than those from peripheral blood, and might result in tissue-localized high concentrations. Thus, it is plausible to predict that GM-CSF inhibition might reduce IL-23 as well as TNF production in inflamed joints and consequently provide additional therapeutic benefits over and above TNF inhibition alone.Circulating levels of CCL17 have been shown to be suppressed in RA subjects in the clinic following blockade of the GM-CSF receptor alpha subunit, and CCL17 was proposed to serve as a specific pharmacodynamic marker for GM-CSF pathway targeting . Our datOur data support an important role of GM-CSF in AS, suggesting pathogenic mechanisms that are likely active in AS including promotion of LPS-stimulated TNF and IL-23 cytokine release and direct effects on CCL17 levels. We also show CCL17 levels were elevated in plasma samples of AS patients. Taken together these observations are supportive of GM-CSF neutralization as a potential novel therapeutic approach for the treatment of AS. The efficacy of namilumab, a GM-CSF neutralizing monoclonal antibody, is to be assessed in a Phase II Proof-of-Concept clinical study in moderate-to-severe axial spondyloarthritis patients NCT03622658.All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by South Central - Oxford C Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.HS, LC, AR, IB, CC, and NZ performed the studies and take complete responsibility for the integrity of the data and the accuracy of the data analysis. PB, HS, and JS designed the experiments and had full access to all the data. PB and HS wrote the original draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version. All authors contributed to the article and approved the submitted version.The authors declare that this study received funding from GlaxoSmithKline (GSK). The funder had the following involvement with the study: partnering on analysis of samples, experimental design, interpretation of data, pre-submission review of this article and the decision to submit it for publication. CC and JS declare they are employees and stockholders of GSK.


Kynurenic acid (KYNA) is a product of the tryptophan (TRP) metabolism via the kynurenine pathway (KP). This pathway is activated in neurodegenerative disorders, such as Alzheimer´s disease (AD). KYNA is primarily produced by astrocytes and is considered neuroprotective. Thus, altered KYNA levels may suggest an inflammatory response. Very recently, significant increases in KYNA levels were reported in cerebrospinal fluid (CSF) from AD patients compared with normal controls. In this study, we assessed the accuracy of KYNA in CSF for the classification of patients with AD, cognitively healthy controls, and patients with a variety of other neurodegenerative diseases, including frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and progressive supranuclear palsy (PSP). Averaged KYNA concentration in CSF was higher in patients with AD when compared with healthy subjects and with all the other differentially diagnosed groups. There were no significant differences in KYNA levels in CSF between any other neurodegenerative groups and controls. These results suggest a specific increase in KYNA concentration in CSF from AD patients not seen in other neurodegenerative diseases. This analysis made it possible to establish these parameters as clinical biomarkers indicating that reduced Aβ levels and increased t-tau levels in CSF are distinctive of AD [42 levels in the CSF and the brain is explained because Aβ42 in the AD brain is sequestered in the cerebral neuritic plaques, as it is their main component, with less Aβ being available to diffuse into the CSF, and therefore reduced levels of Aβ42 in CSF from AD patients (reviewed in [Alzheimer’s disease (AD) is a complex and progressive neurodegenerative disorder that represents one of the major health problems in the world . The maiiewed in ). In theiewed in ). Althouiewed in ,6,7,8. Niewed in . One coniewed in . KP is tiewed in . There iiewed in ,13. One iewed in . It was iewed in . Therefoiewed in ,17.N-methyl-D-aspartate (NMDA) and α7 nicotinic acetylcholine (α7nACh) receptor antagonist and is considered to be neuroprotective [TRP is an essential amino acid necessary for protein biosynthesis, and a precursor of several compounds with important biological functions, such as serotonin, melatonin, and kynurenines . TRP entotective ,21,22,23otective ,25. Incrotective ,27. Addiotective . Howeverotective ,33,34,35otective ,37. Howeotective . In thisn = 23); (ii) patients with mild cognitive impairment due to AD (MCI) (n = 24); (iii) probable mild AD (n = 41); (iv) moderate–severe AD (n = 20); (v) frontotemporal dementia (FTD) (n = 8); (vi) amyotrophic lateral sclerosis (ALS) (n = 8); and (vii) progressive supranuclear palsy (PSP) (n = 8). Subjects were recruited from the Neurology Service of three different geographical areas in Spain: 12 de Octubre University Hospital (Madrid); Donostia-Osakidetza University Hospital (San Sebastian); and Santa Creu i Sant Pau Hospital (Barcelona). Diagnoses were based on detailed clinical assessments, neuropsychological studies, neuroimaging, electromyography, and CSF Aβ42 and t-tau levels. An AD- indicative biomarker profile (Aβ+) was defined as CSF Aβ42 <550 pg/µL and t-tau/Aβ42 ratio >0.52, according to ELISA manufacture instructions and reported cut-offs [42 ≥550 pg/µL and t-tau/Aβ42 ratio ≤0.52 were defined as non-AD biomarker profiles (Aβ−). Diagnosis of MCI and dementia due to AD were established according to the National Institute on Aging and the Alzheimer’s Association guidelines [+ CSF biomarker profiles. Global cognition was assessed using the Mini Mental State Examination (MMSE) [+ CSF biomarker profile, while it was Aβ− in patients with other neurodegenerative diseases and healthy controls. Controls were usually recruited from among the patients’ partners when they fulfilled the age and health criteria. Inclusion criteria for old, cognitively-normal individuals were age over 50, no history or clinical signs of neurological or psychiatric disease, and absence of AD CSF biomarker profile. Exclusion criteria for patients were evidence of concomitant cerebrovascular disease or any neurological, psychiatric, or non-neurological medical conditions; or medications that could affect cognition or motor function. Consent was in accordance with the Declaration of Helsinki, and approval was obtained from the research ethics committee of the responsible institution ; Biodonostia Health Research Institute (22/2016 Date: 19 July 2017); Sant Pau Biomedical Research Institute (16/2013 Date: 14 January 2014)). Written informed consent was signed by all participants or representatives.In total, 132 subjects were included in this study: (i) elderly non-demented subjects classified as controls  mon (MMSE) , or the n (MMSE) . ALS andn (MMSE)  and Boxen (MMSE)  respectiCSF samples were collected from all subjects and processed according to standardized procedures by lumbar puncture in 15-mL sterile polypropylene tubes. Samples were then centrifuged at 3000 rpm at 4 °C for 10 min. Supernatant aliquots were stored at −80 °C into 0.5 mL polypropylene tubes with Protease Inhibitor Cocktail .Blood samples were obtained through antecubital vein puncture from patients and healthy subjects. Plasma was isolated from whole blood and collected in 7-mL-EDTA-2Na tubes. Whole blood was centrifuged at 2000 rpm for 10 min at room temperature. Supernatants were then collected in tubes with Protease Inhibitor Cocktail  and stored at −80 °C.APOE C112R and R158C polymorphisms were detected to identify the APOE ε2, ε3, and ε4 alleles, using LightCycler 480 II Instruments Kit  following manufacturer instructions.Genomic DNA was extracted from peripheral blood using QIAmp DNA Blood Mini Kit , according to the manufacturer instructions. Human 42 and t-tau levels in CSF samples were quantified using INNOTEST ELISAs , according to manufacturer’s instructions.Endogenous Aβ50 of 23.6 µg/L and a linear range (LR) from 2.8 to 100 µg/L for KYNA; and a LOD of 2.5 µg/L with an IC50 of 250 µg/L and a LR from 40–912 µg/L for TRP. The immunoreagents used for KYNA detection  and the enzyme tracer (HRP-SIA-IHSH)) were developed by the Nb4D group with the support of the ICTS “NANBIOSIS”; more specifically, by the Custom Antibody Service . Their preparation and characterization will be described elsewhere. The tryptophan hydroxylase polyclonal antibody (ref. PA5-19749) was purchased from Thermo Fisher Scientific, . A 96-well ELISA microplate was coated overnight at 4 °C with anti-KYNA and anti-TRP primary antibodies. Then, plates were washed with phosphate-buffered saline containing 0.05% Tween-20 (PBST). After washing, following standards and CSF samples (50 µL), 50 µL of HRP-bioconjugates (HRP-SIA-IHSH or HRP-L-5-HTP) were added to each well and the plate was incubated for 30 min at room temperature. Next, wells were washed and 100 µL of substrate solution were added. After 30 min incubation, absorbances were measured at 450 nm using a Fisher Multiskan Go microplate reader . Inter-assay variability for KYNA and L-5-HTP assays was estimated at 21.4% (n = 17) and 21% (n = 4), respectively. Intra-assay variability was estimated at 13.4% (n = 18) and 10.6% (n = 6) for KYNA and L-5-HTP, respectively.KYNA and 5-hydroxy-L-tryptophan  levels in CSF and plasma samples were quantified using an in-house competitive ELISA in triplo developed by the Bioelectrochemistry and Biosensors Group from the University of Alcalá in collaboration with the Nanobiotechnology for Diagnostics (Nb4D) group from IQAC-CSIC. Analytical parameters found in CSF were a limit of detection (LOD) of 0.26 µg/L with an ICp < 0.05. Statistical analysis was performed using STATA/IC 14.2 for Windows, Texas, USA.Numerical data are shown as means ± standard deviations (SD) and categorical data as a percentages. To compare demographic, clinical, and plasma and CSF biomarker data between groups, we used the non-parametric Kruskal–Wallis rank test and non-parametric pairwise comparisons adjusted by Bonferroni. Fisher’s exact test was used to compare the distribution of categorical data across groups. Correlations between biomarkers were assessed using Spearman rank correlation. Receiver operating characteristic (ROC) curves were used to determine the diagnostic accuracy of KYNA from CSF to differentiate between the diagnostic groups. The optimal cut-off point, sensitivity, specificity, and area under the curve (AUC) were calculated in all cases. Statistical significance was set out at 42 and t-tau levels in CSF are also shown in Clinical, demographic, and genetic profiles from all subjects are shown in p < 0.05), mild AD , and moderate–severe AD  patients compared with controls (3.9 ± 2.9 µg/L)  a. Althou.9 µg/L) . KYNA/TR+ or Aβ− profile), KYNA levels were still found higher in MCI/AD group (Aβ+) compared with healthy subjects (Aβ−)  , mild AD (n = 17), moderate–severe AD (n = 9), and healthy controls (n = 20). Neither KYNA and TRP levels nor the KYNA/TRP ratio showed any significant change between patients and controls  in peripheral fluids or tissues, such as blood. To evaluate whether KP was also altered in blood, plasma levels of KYNA and TRP were determined in a patient’s subgroup with MCI , and MCI/AD group -2 [KYNA levels in CSF may also be increased upon inflammatory conditions following immune activation. Elevated KYNA concentration in CSF was found in patients with inflammatory diseases, such as bacterial, viral, fungal, and parasitic infections; meningitis; autoimmune diseases; and septicemia, where autoimmune activation is present ,66,67. R Candida ,71. Infe (HSV)-2 ,73. TRP  (HSV)-2 . The rel (HSV)-2 .Among other chronic neurodegenerative disorders, a decrease in KYNA concentration was detected in brain tissue and CSF from Parkinson’s disease (PD) patients . However42 and tau). Along with our present findings and previous works [Here we report in a new and large cohort, increased KYNA levels in CSF from AD patients compared to healthy subjects, including prodromal stages of the disease. Our data clearly demonstrate that KYNA levels in CSF are specifically higher in AD patients but not in patients with other neurodegenerative disorders. We propose that high KYNA concentration in CSF may reflect a compensatory mechanism in AD patients, based on its neuroprotective feature, but also an inflammatory response following putative immune activation. Thus, we suggest that KYNA levels in CSF have potential as a diagnostic marker of AD in combination with the existing CSF biomarkers (Aβus works ,79, we s


Plastic waste management has become a serious environmental and health concern owing to large amounts of plastic deposits globally. Recently, innovative and sustainable solutions have been introduced  to overcome the growing environmental threats. Hence, green marketers need to develop effective advertising campaigns to enhance the usage of bio-nanomaterial plastics. Past literature has suggested that cultural value-laden advertising appeals can give sustainable behavioral cues to consumers. Hence, this research unfolds the underlying cultural dimensions between the value-laden eco-friendly advertising appeals and intention to use bio-nanomaterial plastics (henceforth IBP). The present study proposes a moderating model in which two dimensions presented in the Global Leadership and Organizational Behavior Effectiveness (henceforth GLOBE) framework interact with the individuals’ perception of eco-friendly advertising appeals (henceforth IPEA) to drive bio-nanomaterial plastics usage. The model was tested by conducting an experimental study on a sample of 364 Pakistani consumers. Findings of structural equation modeling show a significant difference in the relationship between IPEA and IBP, which is moderated by the performance orientation (henceforth PO) and institutional collectivism (henceforth IC) dimensions with diverse intensity. These findings validate the effectiveness of PO and IC  and eco-friendly advertisements that can potentially promote the consumption of bio-nanomaterials plastic. In the recent decade, the management of plastic-based waste has emerged as an enormous global challenge . The devRecently, scientists are innovating in the development of sustainable and renewable materials to provide alternatives such as bio-nanomaterials . These sResearch suggests that promoting the usage of such environmentally friendly products can be attained through persuasive tools of communication such as advertising ,6. HenceTo address this issue, literature  recommenThe Similarity Attraction Paradigm model (henceforth SAP) suggests that ad appeals embedding values robust favorable responses between individuals . When inThus, it is assumed that the IBP in response to advertising appeals relies on the individual’s cultural deciphering of appeals. However, it is expected to be stronger when the perceived level of advertising appeals depicting a particular dimension match with the culturally endorsed level of that dimension . In otheHypothesis 1 (H1). Individual perception of a higher level of PO (H1a), and IC in an Advertising Appeal (H1b) will lead to a more positive IBP.The performance orientation is the extent to which a human community encourages and rewards, setting challenging goals, innovation, and performance improvement . PO as aPast literature shows that PO has been incorporated in advertising appeals . A persoInversely, in a cultural context that is lesser performance-oriented, where people have less reference worth concerning performance, very similar advertisements may be less regarded. Thus, the strength of the relation between the IPEA and IBP would be dependent on the culturally learned context of PO. In a nutshell, this is consistent with the adaptation-level theory and GLOBE model  that an Hypothesis 2 (H2). The effect of PO will be stronger among Pakistanis as individuals place greater importance on PO and thus positively moderate the relationship between IPEA and IBP.In the past, there has been a wide debate on the construct of collectivism in anthropology’s value theory , psycholFrom the persuasion theory standpoint, the similarity is the influential principle of persuasion because the similarity  surges likeability . ScholarAsian countries are ranked highly as collectivistic societies; thus, advertisers use collectivistic appeals in Asian countries . Past stThus, collectivistic culture like Pakistan has an expressive common emphasis on collectivism and people are more anxious about group desires; hence, they prefer collective goals. Collectivistic cultures such as Pakistan have shared principles and what matters is the collective goal of their group rather than individual gain . The alaHypothesis 3 (H3). The effect of IC will be stronger among Pakistanis as individuals place greater importance on IC and thus positively moderate the relationship between IPEA and IBP.Based on the review of mainstream literature, the hypothesized relationship in a conceptualized model of intentions to use bio-nanomaterial plastics is presented in The present study proposes to underpin the moderating effect of cultural values upheld by individuals in predicting the relationship between eco-friendly advertising appeal embedded cultural dimensions on the IBP. The fundamental idea behind the study is that a given eco-friendly advertising appeal is expected to be perceived and assessed in cultural context; the appeal relies on the degree of importance of individuals’ dwelling on a particular dimension  along with the level of a particular dimension in the society in which the individual belongs . Henceforth, the study clearly distinguishes among cultural dimensions on an individual level vs. cultural dimensions on a societal-level by using two-facet constructs of GLOBE as well as eco-friendly ad-stimuli reflecting/embedding the cultural dimension.To validate this in line with past researches ,45, an eThe participants were requested to gather at the on-campus facility. The participants were informed about the purpose of the study and asked to sign a consent to participate in the experiment. The 364 participants considered for the study were randomly assigned to the two experimental conditions n = 182). Therefore, participants were exposed to the two different eco-friendly ad-stimulus (ad-stimuli for one dimension) and experimented. After the exposure of the relevant eco-friendly ad-appeal participants were asked to fill the questionnaire to tap IBP as a result of the exposed eco-friendly ad-appeal . The sample size in this study is common for experimental design and in advertising research underpinning cultural values and ad-appeals 2. Theref.Two advertisements were used as the stimuli; these eco-friendly ads represented a high level of cultural orientation . To ensure relevant selection, the eco-friendly ad-stimuli and other instruments were subsequently pretested in two phases of the pilot studies. In the first phase, a pilot study was carried out with a group of 30 students at a major university in Pakistan. After reading the advertisements, the participants were asked to fill a questionnaire that involved queries on how much they perceived the advertisements as congruent with the dimensions . Changes were incorporated to improve the stimuli to achieve the desired level. In the second phase, comments from 5 advertising professors and 5 experts from advertising agencies were also solicited. Minor amendments were made to improve the clarity and relevance of the questionnaire. These pre-tests ensured that the experimental procedures and ad-stimuli were well understood.This study adopted and modified the 3-items scale of measuring individual perception of PO ad appeals from the literature  the seveThis study adopted the items of measuring two cultural dimensions based on the conceptualization of the GLOBE ,30, 4 itThis study used a total 5-items scale of measuring IBP from the literature ,47 on thThis study measured the socio-demographic characteristics namely; (1) sex, (2) education, (3) income, and (4) age as the control variables see .The descriptive analysis to screen the normality of the data was performed. The data revealed the normality based on the cut-off values of the recommended normality test and visual inspections of all constructs after deleting the outliers. The main analysis comprises n = 356 and n = 178 for each group after the deletion of the cases. Before proceeding, bivariate analysis was conducted separately for two assumed models containing the relevant ad-appeal  construct, cultural dimension, and IBP see .Additionally, an assessment of multicollinearity between the predictor variables was carried out by using the variance inflation factor (VIF) as well as the tolerance index (TI). Both VIF and TI were found to be within the acceptable threshold for collinearity (VIF is > 10 and TI > 0.84).The confirmatory factor analysis (CFA) was conducted using AMOS 23.0 to analyze the uniqueness of all constructs including cultural dimensions IPEA, IBP, PO, and IC.In total, four models based on all the possible factors (structure) were examined concerning the constructs of IPEA, IBP, PO and IC that include two separate models for each dimension: (a) One single-factor model, one for each dimension’s data by loading items of IPEA, IBP and the dimension in question on a single-factor to check CMV; and (b) one model of the three-factor models, by loading items of the IPEA, IBP, and the dimension in question on their parent factor. The results of the CFAs in Information related to the composite reliability, Cronbach’s alpha and average variance extracted for each variable is available in For testing the moderation of cultural norms, in total, four models were examined (two for each dimension). To do so, Model 1 examined the main effect of an individual’s perception of eco-friendly advertising appeals and cultural dimensions (PO and IC). To evaluate the possible influence of the control variable, a stepwise analysis approach was adopted. In this recommended approach all control variables of this study, namely, sex, age, income, and education were added in Model 1. However, based on the findings the null hypothesis of the control variables impact was rejected.β = 0.32, p < 0.001) and PO is also significant  and 52% of the variance was recorded for IBP. Therefore, H1 (a) is supported, as was expected that IPEA would be more positively related to IBP among Pakistanis .The moderation of PO Model 2 among sample also revealed the interaction of IPEA and PO and described 67% variance for IBP with an adequate model fitness: χ2 = 837.14, df = 97, χ2/df = 2.82; CFI = 0.94; IFI = 0.96; TLI = 0.93; SRMSR = 0.063; and RMSEA = 0.03. The model showed consistency with model fitness cut-offs ,49. The IPEA see  was withβ = 0.14, p < 0.001, R2 change = 0.15). It can then be concluded that, in the case of eco-friendlily advertising appeals and PO cultural value congruence, Pakistanis would have more IBP. Equally, the same approach was implemented for testing the moderation of the IC dimension. The main effect of IPEA on IBP was , and IC was  and H1(b) is supported. The interaction effect between IPEA and IC was  with R2 = 0.61. This supports H3; therefore, it is concluded that in the case of matched perceived level of IC eco-friendly advertising appeal with the individuals’ level of IC value, Pakistanis would have a more positive relationship between IPEA and IBP.Furthermore, the simple slopes were examined for analyzing the nature of the IPEA × PO) interaction ,51. The β = 0.32, p < 0.001) and H1(b) . These findings validate the past research that relates advertisement effectiveness with the cultural deciphering [This experimental study explains the effect of PO and IC manifested eco-friendly advertising appeals to understand the consumer decision on bio-nanomaterial plastic products. The current study addresses the novel question how similarity with consumers uphold cultural values PO and IC with the stimuli in question could influence consumers’ decisions on bio-nanomaterial plastic products. To do so, two separate experimental groups were selected and were exposed to two different ad stimuli containing PO and IC appeals. The empirical finding of CVB–SEM indicates that advertisements manifesting PO and IC, EC has a robust effect on IBP in Pakistan, and supported H1(a)  can be examined. This would give helpful directions to managers interested to promote sustainable products.


CSF3 and its receptor (CSF3R) were expressed in porcine maternal reproductive tissues (oviduct and uterus), cumulus cells, and embryos and to investigate the effects of human recombinant G-CSF (hrG-CSF) supplementation during in vitro culture (IVC) on the developmental competence of pre-implantation embryos. To do this, we first performed reverse-transcription polymerase chain reaction (RT-PCR). Second, we performed parthenogenetic activation (PA), in vitro fertilization (IVF), and somatic cell nuclear transfer (SCNT) to evaluate the embryonic developmental potential after hrG-CSF supplementation based on various concentrations  and durations  of IVC. Finally, we examined transcriptional levels of several marker genes in blastocysts. The results of our study showed that CSF3 transcript was present in all samples we assessed. CSF3-R was also detected, except in cumulus cells and blastocysts from PA. Furthermore, 10 ng/mL and Days 0–7 were the optimal concentration and duration for the viability of in vitro embryonic development, especially for SCNT-derived embryos. The rate of blastocyst formation and the total cell number of blastocysts were significantly enhanced, while the number and index of apoptotic nuclei were significantly decreased in optimal condition groups compared to others. Moreover, the transcriptional levels of anti-apoptotis- (BCL2), proliferation- (PCNA), and pluripotency- (POU5F1) related genes were dramatically upregulated. In conclusion, for the first time, we demonstrated that CSF3 and CSF3R were expressed in porcine reproductive organs, cells, and embryos. Additionally, we determined that hrG-CSF treatment improved porcine embryonic development capacity in vitro.Granulocyte-colony stimulating factor (G-CSF), a pleiotropic cytokine, belongs to the hematopoietic growth factor family. Recent studies have reported that G-CSF is a predictive biomarker of oocyte and embryo developmental competence in humans. The aim of our study was to determine whether  However, despite extensive research in previous decades, conditions for the production of in vitro porcine embryos remain suboptimal .Blastocyst samples (approximately 10 blastocysts per group) were prepared as described above in the RT-PCR analysis section and stored at –80°C for further analysis. Gene expression  was anal and BAX . ReactioP < 0.05 was considered significant.Each experiment consisted of at least three replicates; for each replicate, oocytes were collected on the same day from the same group of slaughterhouse-derived ovaries. Statistical analysis was carried out using SPSS 17.0 . Embryonic development  was analyzed by the chi-squared test. The total cell number of blastocysts derived from PA and IVF were compared by one-way analysis of variance (ANOVA), followed by Duncan’s multiple range tests. T-tests were conducted to assess the apoptotic differences between cloned blastocysts and for relative gene expression levels. All data are presented as mean ± SEM. CSF3R was detected in cumulus cells and parthenogenetic blastocysts expressed neither CSF3 nor its receptor , the second 89 h (Days 4–7), and the entire 178 h (Days 0–7), respectively. No difference in parthenote development capacity was observed between hrG-CSF treatment groups. Nevertheless, blastocyst cell numbers were significantly increased in the 178 h-treated embryos (51.2 ± 2.1) compared to the first 89 h treated and non-treated embryos  enhanced in embryos treated with hrG-CSF compared to non-treated embryos , pluripotency (POU5F1), and anti-apoptosis (BCL2).Here we show for the first time that in vitro culture conditions [in vitro culture in humans [CSF3R in PA blastocysts. However, transcript levels of POU5F1 and BCL2 were found to be increased in all three types of hrG-CSF-treated blastocysts. These findings may be due to the expression of CSF3R in porcine oocytes, as we described previously [in vitro-derived embryos  such as DNA methylation [In previous studies, M-CSF and GM-CSF have been reported to increase murine, bovine, and porcine blastocyst formation in nditions , 18, 20.nditions . Moreoven humans  and miceeviously ; alternahylation , leadinghylation .CSF3 expression found in both follicular and luteal phase oviducts and uteri as well as cumulus cells.Considering previous reports, these molecules may be produced during specific periods of pre-implantation embryonic development. An example of such would be interleukin-1β, which is thought to exert its action on embryonic growth before Day 5 in bovines . GM-CSF in vitro, especially those derived from SCNT [POU5F1, which is important for establishing the distinct identity of the ICM in early embryogenesis [POU5F1 stealth siRNA-injected porcine embryos were arrested at 8 cell stage [POU5F1 and several POU5F1-related genes [POU5F1 in hrG-CSF-treated blastocysts. Overall, we speculated that hrG-CSF may overcome some of the reprogramming deficiencies and plays a positive role in the maintenance of sufficient pluripotency during porcine pre-implantation embryonic development.Defects in DNA modification and chromosome remodeling have been detected frequently in embryos cultured rom SCNT , 44. Theogenesis , 46; theogenesis . A previll stage  and the ll stage . Nuclearll stage . Incomplll stage . One caned genes . In thisin vitro can be affected by environmental stressors that are potential inducers of unplanned apoptosis. This apoptosis may lead to embryo arrest or influence embryo viability [BCL2 is associated with anti-apoptotic effect and has been primarily detected after embryonic genome activation [BCL2, while fragmented embryos exhibit low expression levels [BCL2 expression levels in PA, IVF, and SCNT-derived blastocysts. Accordingly, our study indicated that hrG-CSF may have anti-apoptotic effects.Embryos cultured iability . BCL2 istivation . In bovin levels . In our et al. suggested that G-CSF plays a role in promoting the proliferation of trophoblast cells [et al. showed that it does not affect the proliferation of ovarian cancer cell lines but does protect against apoptosis [PCNA is an essential component of the DNA replication and repair machinery [PCNA is only significantly enhanced in hrG-CSF-treated SCNT embryos, not in PA and IVF embryos. This is in-line with previous reports that embryos originating from different reproductive technologies display distinguishable global gene expression [in vitro blastocysts that are shown not only in the total cell number of blastocysts but also in the transcription level of PCNA.The role of G-CSF on proliferation according to different cell types remains controversial. Miyamay st cells . G-CSF hpoptosis . PCNA isachinery  and has achinery . Our finpression , 59. Thein vitro. In other words, G-CSF may play a role in porcine embryonic development by maintaining embryo pluripotency and exhibiting anti-apoptotic and proliferative effects. G-CSF is likely a maternal factor secreted by maternal reproductive tissues.In conclusion, we found that hrG-CSF enhanced porcine pre-implantation embryonic developmental competence S1 File(XLSX)Click here for additional data file.


Ambient temperature is likely to be playing a role in the aetiology of TT and TA in Scotland but not EO. Further study is warranted to explain underlying mechanisms.The role of ambient temperature in the aetiology of acute scrotal pain (ASP) remains uncertain. The most common causes of ASP are torsion of the testis (TT) or its appendages (TA) and epidymo-orchitis (EO). We undertook an ecological study of ASP in Scotland to determine whether a seasonal variation could be observed. Episode reports for TT, TA and EO in Scotland over 25 years were collated monthly. Statistical analyses were performed to determine whether changes in ambient temperature during the year could explain variations in monthly frequency. 7882 episodes of TT and TA (Group A), and 25,973 episodes of EO (Group B) were reported. There was significant variance in the frequency of Group A ( Acute scrotal pain requires urgent medical assessment and surgical exploration of the scrotum if testicular torsion cannot be ruled out clinically. TA can be difficult to diagnose without exploration, leading to a high rate of diagnosis at exploration2. EO is managed with appropriate antimicrobials, determined by the clinical history and results of microbiological culture.Acute scrotal pain (ASP) is a medical emergency. The most common causes of ASP are testicular torsion (TT), torsion of the testicular appendages (TA), and epididymo-orchitis (EO)3. It has been shown that decreasing temperature causes increased contraction of the cremasteric muscles5, and this may lead to an increase in the frequency of TT in winter months3. We performed a large ecological study of ASP in Scotland, which lies within the temperate latitudes, to determine if seasonality of presentation could be identified.Seasonal variations have been reported for several medical conditions, with a number of pathophysiological processes explaining such findings. Reports on the seasonality of ASP and TT have been contradictory, with some studies showing evidence of seasonality and other studies notp < 0.0001) with higher frequency in the colder months  with highest frequency in March, May and October , but no difference for Group B episodes (p = 0.58) , but no correlation with ambient temperature for Group B episodes   were TT and TA (Group A), and 25,973 (77%) were EO (Group B). May to October were the warmest (range 4.8–9.6 °C) and November to April the coldest months (range 0.1–2.5 °C). We found a significant variance in the frequency of Group A episodes by month , with higher frequency in the colder months, and an inverse correlation between monthly frequency and ambient temperature. There was increased frequency of EO in March, May and October but no correlation with temperature.6. Decreasing temperature causes increased contractility of the cremasteric muscles5, which may lead to an increase in the frequency of TT in colder months. Previous studies have yielded conflicting results3. Most of these studies reported case series of a relatively small number of patients (n = 39 to n = 2876). A large study from Brazil3 analysing 21,289 episodes of TT found seasonality of presentation with higher incidence in colder months, which was more significant in the more temperate regions than tropical regions of Brazil. A previous report from Dundee in Scotland showed an increased frequency of TT during the colder months from a series of 173 patients1. The present report is a larger study involving 33,855 episodes, of which 7882 had torsion events, and provides more robust evidence of seasonality of torsion.There has been interest in the seasonality of TT for many yearsSeasonal variation in the frequency of EO has not previously been reported to our knowledge. We were unable to explain the increased frequency of EO in March, May and October within our dataset. Further epidemiological study will be required to elucidate the reasons. Possibilities to consider include sexual behaviour patterns of the male population.7, and the ecological fallacy, meaning that it may not be appropriate to apply these generalised population-based findings to individual patient care.Limitations of this study include the use of data from a large public database with well reported advantages and disadvantages8.We do not suggest, based on our findings, that the threshold for surgical exploration be raised for patients with ASP presenting during warmer months. Public health measures could be considered, for example encouraging the wearing of warm clothing and undergarments by young males during colder months may reduce the frequency of TT and TA, as the style of clothing could have a direct effect on scrotal temperatureIn conclusion, the findings of this large ecological study provide further robust evidence of seasonality of ASP, with the frequency of torsion events correlating negatively with ambient temperature. Further study is required to explain monthly variations in presentation of EO.9 (Table All episode reports for the three most common causes of ASP namely torsion of the testis (TT), torsion of appendages (TA) and epididymo-orchitis (EO) in Scotland for the 25 year period from 1983 to 2007 was obtained from the Information Services Division (ISD) of the National Health Service (NHS), Scotland. There are currently 14 Health Boards in Scotland, covering well defined geographical areas with 2008 population figures ranging from 9,789 in Orkney to 573,021 in Greater Glasgow and ClydeStatistical analyses were performed to determine whether there was a seasonal variation in the presentation of Group A and Group B events, and whether differences in ambient temperature during the year could explain any variation observed. The Friedman test was used to analyse for variance in the monthly frequency of episodes. The Mann–Whitney test was used to compare the frequency of episodes between the colder and warmer months, and the Spearman test was used to determine if there was a correlation between the cumulative monthly frequency of episodes and average monthly ambient temperature. All statistical analyses were performed using GraphPad Prism 5 software .


PCRctic is an innovative assay based on 16S rDNA PCR technology that has been designed to detect a single intact bacterium in a specimen of cerebro-spinal fluid (CSF). The assay’s potential for accurate, fast and inexpensive discrimination of bacteria-free CSF makes it an ideal adjunct for confident exclusion of bacterial meningitis in newborn babies where the negative predictive value of bacterial culture is poor. This study aimed to stress-test and optimize PCRctic in the “field conditions” to attain a clinically useful level of specificity.The specificity of PCRctic was evaluated in CSF obtained from newborn babies investigated for meningitis on a tertiary neonatal unit. Following an interim analysis, the method of skin antisepsis was changed to increase bactericidal effect, and snap-top tubes (Eppendorf™) replaced standard universal containers for collection of CSF to reduce environmental contamination.The assay’s specificity was 90.5% in CSF collected into the snap-top tubes – up from 60% in CSF in the universal containers. The method of skin antisepsis had no effect on the specificity. All CSF cultures were negative and no clinical cases of neonatal bacterial meningitis occurred during the study.A simple and inexpensive optimization of CSF collection resulted in a high specificity output. The low prevalence of neonatal bacterial meningitis means that a large multi-centre study will be required to validate the assay’s sensitivity and its negative predictive value. The clinical signs of neonatal meningitis are very non-specific  and the Based on clinical and microbiological data –14, the 2–103 colony-forming units per milliliter [CFU/ml]) [A recent Cochrane review highlighted limitations of the reported molecular assays for neonatal sepsis . MultiplCFU/ml])  and are The sensitivity of standard 16S rDNA PCR is limited due to the universal detection of free bacterial DNA contaminating samples and reagents . To addrThe broad range and high sensitivity of the assay promise an unprecedented scope for the evaluation of the bacterial carriage in clinical specimens but also make it vulnerable to false positive results due to the ubiquitous environmental bacteria. This feasibility study of PCRctic was conducted to establish the operating procedures that result in a clinically useful level of specificity.From October 2016 to September 2017 we recruited newborn babies of at least 34 weeks post-menstrual age undergoing lumbar puncture to exclude meningitis on either the postnatal wards or on the neonatal unit at the Rosie Hospital, Cambridge University Hospitals NHS Trust. Informed consent was obtained before CSF was analysed by PCRctic. UK National Research Ethics Committee and UK Health Research Authority (HRA) approved the study.All babies recruited into the study had their CSF tested by bacteriological culture and in PCRctic. The study had two phases. In the first, at least five drops of CSF were collected into each of three sterile universal containers . CSF in two of these was tested using the standard laboratory assessment for meningitis, and, following parental consent, CSF in the third container was tested in PCRctic in the clinical microbiology laboratory, Addenbrookes Hospital, Cambridge University Hospitals NHS Trust. Whilst the 30 ml sterile universal containers are routinely used across the UK for CSF collection, special care is needed to avoid contamination through the handling of the screw-on tops. In the second phase, CSF for PCRctic was collected into 1.5 ml sterile individually sealed snap-top tubes . Lumbar puncture technique was as standard. Unisept  solution of 0.05% chlorhexidine was used in the first phase of the study for skin antisepsis. This was changed to ChloraPrep , containing 2% chlorhexidine and 70% alcohol for the second phase of the study.Results from the initial 39 samples suggested possible environmental contamination and the study protocol was amended to include individually sealed sterile snap-top tubes and ChloraPrep skin antisepsis (as above). The amendment was approved by HRA.The primary outcome measure was the rate of false positive results. As this was a feasibility study, the results had no effect on patient care.PCRctic Fig.  uses prig. After carefully removing the supernatant with a sterilized gel-loading tip, 10 μl of a previously-frozen PCR mastermix  were added. The closed PCR tubes were then illuminated as above to photoactivate EtA. The samples were then amplified on a quantitative PCR (qPCR) system  followed by a melting curve analysis.Contamination from free bacterial DNA was eliminated by the use of ethidium bromide monoazide (EtA) . ExposurE. coli , S. aureus (ATCC 29213), S. agalactiae (NCTC 8181) or L. monocytogenes (NCTC 7973). In CSF samples spiked with bacteria, PCRctic reliably detected as few as 1.5 CFUs . Bacterial strains used were ATCC 2921, S. agalFUs Fig. . PositivA nonparametric Mann-Whitney U test was used for the significance of the difference between the studied groups. Bayesian statistics were used to estimate the significance of the positive results under different prevalence, specificity, and sensitivity conditions.In total, CSF from 73 babies was tested in PCRctic in parallel with standard microbiological testing. All babies received antibiotics before lumbar puncture. All bacterial cultures (blood and CSF) were negative, and all babies, including those with CSF white cell count of ≥20 per microliter Table , had no The first 39 samples were collected into universal containers and were from babies where Unisept was used for skin antisepsis. Sixteen of these were positive in PCRctic 41%). Sequencing revealed a diverse group of Gram-positive and Gram-negative microorganisms, some of which belonged to bacterial families associated with neonatal meningitis  – this change was serendipitous as it reflected the comprehensive change of the unit’s practice for all procedures requiring aseptic technique.P = 0.04 two-tailed). Of the 23 negative controls only one was positive in PCRctic . There was no statistically significant relationship between PCRctic result and the number of CSF white cells  or red cells .Skin antisepsis with ChloraPrep but using universal containers  [The sensitivity of the detection can be increased many-fold by PCR, which has already been widely used for diagnosis of infections (including meningitis) . PCR assia coli) –14, 35, ia coli) , 39–48.In contrast, a broad-range PCR that targets the DNA region highly conserved between different types of bacteria (16S rDNA PCR), can, with few exceptions, detect bacteria of any type . Until rBased on 16S rDNA PCR technology, PCRctic can detect virtually any type of bacterium. Through elimination of free bacterial DNA it can fully realize its sensitivity and detect single numbers of intact bacteria in the specimen – just as a well-designed bacterium-specific PCR can . These tWhereas skin antisepsis with either Unisept or ChloraPrep resulted in the same rate of positive results in universal containers (about 40%), the collection into snap-top tubes significantly reduced the level to 9.5%. Thus a simple and low-cost optimization of CSF handling increased the specificity of the assay from ≈60%  to 90.5% . The format therefore addresses both types of environmental contamination: eliminates contaminating free bacterial DNA (ethidium azide) and reduces the risk of contamination with environmental bacteria (snap-top tubes). The performance of the negative controls (one positive out of 23) indicates that the limit of the assay’s specificity is around 96%. The assay is inexpensive: the cost of reagents is significantly less than $1, the cost of a sterile snap-top tube is $0.23. The feedback from the clinical staff was that the snap-top tubes were easy to use and no handling-related issues were reported.Application of Bayesian analysis shows that with these false-positive rates , the chance that a positive result is truly positive  will always be less than 50% Table . Thus, oIn this feasibility study, an optimization of neonatal CSF collection resulted in a high specificity 16S rDNA PCR output. This study was underpowered to test the assay’s sensitivity. In fact, based on the significance level of ≤5 and 80% power, 12 cases of meningitis will be required to demonstrate a 40% improvement in sensitivity against bacterial CSF culture, and 49 cases to demonstrate a 20% improvement . With th


Eleven young males performed, on two separated occasions, a RI test either in normoxia  or hypoxia . Oxygen uptake (V˙O2), and [HHb] signal from the vastus lateralis muscle were continuously measured. PeakV˙O2 (2.98 ± 0.36 vs. 3.39 ± 0.26 L min−1) and PO (282 ± 29 vs. 310 ± 19 W) were lower in HYPO compared to NORM condition, respectively. TheV˙O2 and PO associated with RCP and [HHb]bp were lower in HYPO  when compared to NORM  (p < .05). Within the same condition, theV˙O2 and PO associated with RCP and [HHb]bp were not different (p > .05). Bland–Altman plots mean average errors between RCP and [HHb]bp were not different from zero in HYPO (0.01 L min−1 and 1.1 W) and NORM (0.00 L min−1 and 3.6 W) conditions. The intra‐individual changes between thresholds associated withV˙O2 and PO in HYPO from NORM were strongly correlated . Therefore, breathing a lower FiO2 during a RI test resulted in proportional reduction in the RCP and the [HHb]bp in terms ofV˙O2 and PO, which further supports the notion that these physiological responses may arise from similar metabolic changes reflecting a common phenomenon.This study investigated the effect of reduced inspired fraction of O 2 during a RI test resulted in proportional reduction in the RCP and the [HHb]bp in terms ofV˙O2 and PO, which further supports the notion that these physiological responses may arise from similar metabolic changes reflecting a common phenomenon.A lower FiO At approximately 80% ofV˙O2max, two physiological responses become discernible: at the pulmonary level the respiratory compensation point (RCP) , a breakpoint in the near‐infrared spectroscopy (NIRS)‐derived deoxygenated hemoglobin signal ([HHb]bp) is observed  test to exhaustion, the rate of O2), which causes the arterial tension of CO2 to fall  arising from the accelerated glycolytic rate within the active musculature  during RI tests  and critical power (CP)  of trained cyclists changed uniformly during the course of a competitive season  during incremental exercise  during a RI test would also affect the RCP and the [HHb]bp by reducing theV˙O2 and PO at which they occur with respect to normoxia. Indeed, previous findings have shown a reduction in the PO associated with the [HHb]bp in hypoxia compared to normoxia  during a RI test to exhaustion. It was hypothesized that a reduction in both theV˙O2 and PO at the RCP and the [HHb]bp would occur as a consequence of the decreased FiO2, and that these changes would be proportional on an individual basis.Therefore, this study explored the changes in the22.1Eleven healthy males were recruited . Participants gave informed written consent to participate in this study after completing the physical activity readiness questionnaire (PAR‐Q+) and being cleared for exercise. All participants were free of any medical condition that could have altered their normal cardiovascular responses to exercise. The study was approved by the Conjoint Health Research Ethics Board at the University of Calgary. This study was part of a larger project designed to answer different research questions  or hypoxia . The FiO2 for hypoxia was chosen with reference of previous investigations demonstrating that this specific FiO2 was effective in altering arterial and muscle O2 saturation during exercise . Each exercise session was preceded by a 5 min wash‐in period with the predetermined FiO2 (either hypoxia or normoxia), during which participants rested seated on the bike. All exercise testing was performed on an electro‐magnetically braked cycle ergometer .Participants reported to the laboratory on two separate occasions at similar time of the day (±1 hr). The visits were separated by at least 48 hr, during which participants performed a RI test to exhaustion either in normoxia . To control the FiO2 during HYPO, inspired air was delivered by a gas‐mixing device  connected with a tube to the T‐valve to provide the desired concentrations of O2. During NORM, the same system was used to deliver room air in order to blind participants from the experimental condition.A breath‐by‐breath metabolic cart system , which was calibrated before each test as per the manufacturer's recommendations, was used to measure ventilatory and gas exchange variables. The expiratory and inspiratory air volume rates and the concentrations of inspired and expired O2.3.22 saturation index (StO2) (%) signals of the VL muscle were measured using a two‐channel frequency‐domain NIRS device  at a sampling rate of 2 Hz, as described elsewhere  and tissue O2.3.32) was measured via a pulse oximetry pod (ADInstruments) from the forefinger of the right hand at a 1 Hz sampling rate and at wavelengths of 660 and 910 nm for red and infrared lights, respectively. The oximeter was connected to the acquisition apparatus  linked to a computer software .The pulse oxygen saturation (SpO2.3.4−]) were measured with a portable lactate analyzer . A lancet was used to perform a pinprick after wiping the finger with an alcohol swab, and a 2 µl capillary sample of whole blood was collected and immediately analyzed.Within 1 min after the RI task failure, capillary blood lactate concentrations  from the local mean were removed. Data were then linearly interpolated to 1 s intervals.V˙O2max was defined as the highestV˙O2 computed from a 20‐s rolling average. Gas exchange threshold (GET) and RCP were identified by three independent investigators by examining raw respiratory data. Briefly, GET corresponded to the breakpoint in theV˙O2‐to‐V˙CO2 relationship (i.e. V‐slope method) concomitant with an increase in the ventilatory equivalent of O2 and a leveling off of end‐tidal pressure of CO2 (PCO2)  was superimposed on theV˙O2 versus PO relationship. Thereafter, the MRT corresponded to the difference between (a) the PO equivalent to the abscissa of the intersection between theV˙O2 and the linear fit versus PO, and (b) the steady‐stateV˙O2 corresponding to 80 W (i.e. measured during the step transition) , as previously described  is the time coordinate corresponding to the interception of the two regression lines (i.e. [HHb]bp), i1 and i2 are the intercepts of the first and second linear function, respectively and s1 and s2 are the slopes . Model parameter estimates were determined by linear least‐square regression analysis in which the best fit was defined by minimization of the residual sum of squares (RSS) and highest coefficient of determination (R2). The “double‐linear” fit was performed plotting the [HHb] data against PO with the fit starting at the onset of the systematic increase in the [HHb] signal until the last data point corresponding to the end of the RI test. Aberrant data that were 3 ± SD from the local mean were removed. The piecewise equation was selected to fit this specific range of data where the equation parameters  were not previously fixed and were automatically selected by the mathematical software. Thereafter, theV˙O2 associated with the [HHb]bp was identified after having “left‐shifted” theV˙O2 data  were utilized to compare the effectiveness of distinct FiO2 in altering the O2 saturation at the arterial and muscle level, respectively. Figure bp were identified.where 2.5SD). Data normal distribution was tested by Shapiro–Wilk test and Q‐Q plots. End‐exercise PPO,V˙O2max, SpO2, StO2, and [Lac−] were compared using paired samples t tests. TheV˙O2 and the PO associated with the RCP and the [HHb]bp variables  and the [HHb] NIRS‐derived slopes before and after the [HHb]bp  were compared utilizing a two‐way repeated measures ANOVA. When F values were significant, Tukey post hoc were used to determine the loci of significant differences. The effect size was computed as partial eta squared (ηp2) for ANOVA comparisons     between the RCP and [HHb]bp at each FiO2 condition .Descriptive data are presented as mean ± standard deviation  compared to NORM (3.39 ± 0.26 L min−1) . Similarly, PPO was lower in HYPO (282 ± 29 W) than in NORM (310 ± 19 W) . [Lac−] was not different between conditions . SpO2 and StO2 in HYPO  were lower than in NORM   at the last 10% of the RI test. The slope of the [HHb] NIRS signal before the [HHb]bp  was greater than after the [HHb]bp   but there was no difference between conditions . The “double‐linear” fitting parameters for [HHb]bp identification showed high values for goodness‐of‐fit within the data set, in accordance with a previous study  and NORM  conditions.3.2bp associated with absolute and relativeV˙O2 and PO values are shown in Table V˙O2 associated with the RCP and the [HHb]bp were lower in HYPO compared to NORM condition  but there was no difference between variables within the same condition . The %V˙O2max associated with the RCP and the [HHb]bp were neither different between conditions  nor variables within the same condition . The absolute values for the PO associated with the RCP and the [HHb]bp were lower in HYPO when compared to NORM , but there was no difference between the RCP and the [HHb]bp within the same condition . The %PPO associated with RCP and [HHb]bp in HYPO were lower compared to NORM  but there was no difference between the RCP and the [HHb]bp within the same condition . The MRT obtained for HYPO (37 ± 10 s) and NORM (39 ± 13 s) conditions were not different .The RCP and the [HHb]3.3V˙O2 and the PO associated with the RCP and the [HHb]bp within the HYPO and NORM conditions are shown in the Bland–Altman plots       . The main and novel findings were that: (a) the RCP and [HHb]bp were equally reduced during hypoxia in terms of bothV˙O2 and PO; (b) these changes were highly correlated, showing small and nonsignificant biases within each individual in each experimental condition (i.e. NORM and HYPO). Taken together, these results indicate that breathing a lower FiO2 during a RI test results in a proportional reduction in both theV˙O2 and PO associated with the RCP and the [HHb]bp. These data further support the notion that these indices of intensity reflect a similar physiological response that is linked to metabolic changes within the active muscles.This study investigated whether the strong relationship between the RCP and the [HHb]2 and StO2, and lowerV˙O2max and PPO throughout and at the end of RI test, respectively, compared to NORM. Most importantly, the earlier development of metabolic perturbations associated with exercise in hypoxia is evident in the attenuatedV˙O2 and PO at which RCP and [HHb]bp occurred. Acute changes in FiO2 have detrimental effects on exercise performance and maximal aerobic capacity due to reduced O2 delivery and diffusive driving pressure of O2 at the muscular level  is not different either before or after the [HHb]bp  nor in HYPO conditions (0.01 L min−1)  and HYPO (i.e. 1.1 W), and similar to previously reported findings  Figure . SimilarORM i.e. .6 W and ORM i.e. .6 W and bp continued to occur at the same metabolic rate, independently of the FiO2, provides further support to the idea that these events might be triggered by common physiological responses occurring at the systemic and muscular level  as a result of its uncoupling in relation to V̇CO2. Even though the exact mechanisms underpinning the hyperventilatory response throughout a RI test, and more specifically above the RCP, are still under debate  in thebp during a RI test occur at the same metabolic rate and PO, even under distinct FiO2 conditions that alter the absolute values at which these intensity indices occur during the test. Furthermore, and most importantly, the magnitudes of the changes in both the RCP and the [HHb]bp from normoxic to hypoxic conditions were proportional on an individual basis. Taken together, data from the present study reinforce the idea that the RCP and the [HHb]bp parameters represent a similar physiological response and that the mechanisms underpinning their occurrence are likely linked to metabolic changes that take place when surpassing the heavy‐to‐severe boundary of exercise intensity.In conclusion, the present study demonstrated that the RCP and the [HHb]5bp during a RI test under distinct FiO2 conditions (i.e. normoxia and hypoxia). It was found that theV˙O2 and PO associated with the RCP and the [HHb]bp were not different within conditions but both reduced under hypoxia condition. Most importantly, the changes in hypoxia, in terms of theV˙O2 and PO, negatively affected the RCP and the [HHb]bp in a similar magnitude compared to normoxia. Thus, the present data reinforce the idea that both markers of intensity represent a similar physiological response that delimitates the metabolic rate associated with the transition from the heavy to the severe intensity domain. These findings have important implications to further our understanding on the physiological underpinnings of these markers.This study is the first to evaluate the correspondence between the RCP and the [HHb]The authors declare no conflict of interest.


Tumor cells are particularly adept at exploiting the immunosuppressive potential of neutrophils as a strategy to achieve uncontrolled proliferation and spread. Recruitment of neutrophils, particularly those of an immature phenotype, known as granulocytic myeloid-derived suppressor cells, is achieved via the production of tumor-derived granulocyte colony-stimulating factor (G-CSF) and neutrophil-selective chemokines. This is not the only mechanism by which G-CSF contributes to tumor-mediated immunosuppression. In this context, the G-CSF receptor is expressed on various cells of the adaptive and innate immune systems and is associated with induction of T cell polarization towards the Th2 and regulatory T cell (Treg) phenotypes. In contrast to the potentially adverse effects of sustained, endogenous production of G-CSF by tumor cells, stringently controlled prophylactic administration of recombinant (r) G-CSF is now a widely practiced strategy in medical oncology to prevent, and in some cases treat, chemotherapy-induced severe neutropenia. Following an overview of the synthesis, structure and function of G-CSF and its receptor, the remainder of this review is focused on: (i) effects of G-CSF on the cells of the adaptive and innate immune systems; (ii) mechanisms by which this cytokine promotes tumor progression and invasion; and (iii) current clinical applications and potential risks of the use of rG-CSF in medical oncology. Colony-stimulating factors (CSFs) are hematopoietic growth factors that are released in response to infection or inflammation to stimulate hematopoietic stem cells to proliferate and generate colonies of differentiated progeny such as neutrophils or macrophages. There are four distinct types of CSFs, each with specific types of receptors, with the occurrence of cross-talk between these receptors, if any, being largely unknown. The CSFs are named after the major types of colonies that they initiate, specifically, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and multipotential colony-stimulating factor (most commonly termed interleukin-3) .G-CSF, the focus of the current review, stimulates the proliferation and differentiation of neutrophil precursors, to maintain the number of circulating mature and functional neutrophils.In the therapeutic setting, major applications of administration of recombinant (r) G-CSF include: (i) attenuation of the magnitude and duration of chemotherapy-induced neutropenia in cancer patients , describNeutrophils are continuously produced from bone marrow-derived pluripotent hematopoietic stem cells via the process of granulopoiesis in which G-CSF plays a key role not only by regulating the production of neutrophils, but also the activity of these cells ,10. G-CSEndogenous production of G-CSF is largely triggered by infection and tissue damage in response to production of several pro-inflammatory stimuli such as the cytokines interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNFα), as well as bacterial lipopolysaccharides (LPS) ,13. G-CSThe importance of G-CSF in the regulation of granulopoiesis became clear following the findings of two early studies based on mice deficient in expression of either G-CSF or the G-CSF receptor (G-CSFR). These mice displayed chronic neutropenia with a corresponding decrease in granulocytic precursors in the bone marrow ,16. DespAccording to Toghraie et al. (2019), four different mRNA isoforms resulting from alternative splicing have been reported for G-CSF  . TranscrThe biological activities of G-CSF are mediated via the G-CSFR, which belongs to the class I cytokine receptor superfamily . This reThe G-CSFR is a single-chain membrane protein composed of extracellular, transmembrane and intracellular regions ,19. The The intracellular region of the G-CSFR comprises three distinct box regions. Boxes 1 and 2 are required for activation of proliferation signals, while box 3 promotes the differentiation of myeloid progenitor cells . In addiG-CSF binds to the extracellular domains of the G-CSFR, triggering receptor homodimerization, leading to the activation of intracellular signaling cascades involving the Janus kinase-signal transduction and activator of transcription (JAK/STAT), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK/ERK); these signaling pathways initiate the transcriptional changes that promote proliferation differentiation, migration and survival ,20,21.According to Mehta et al. (2014), seven mRNA isoforms encoding the G-CSFR have been isolated with only class 1  and class IV (differentiation-defective) detectable in hematopoietic cells .Recombinant G-CSF appears to affect T cell homeostasis by multiple mechanisms. Most importantly, these include induction of T cell polarization toward the T helper 2 (Th2) phenotype, as well as inhibition of T cell proliferation, and enhancement of T cell apoptosis. This cytokine may affect T cells either directly or indirectly, by regulating the activities of various cell types such as dendritic cells, monocytes, myeloid-derived suppressor cells (MDSCs) and others .The proliferative responses of T lymphocytes to both allogeneic and mitogenic stimulation are profoundly reduced in patients, as well as healthy stem cell donors, following treatment with rG-CSF ,24,25. Tifng gene , as well as the inhibitory killer immunoglobulin-like receptors (KIRs) KIR2DL1 and 2 . AdditioK cells) .Administration of rG-CSF to humans prior to autologous stem cell transplantation is associated with decreased platelet counts . In thisThe immunomodulatory effects of rG-CSF, which presumably mimic those of endogenously generated G-CSF, are summarized in Various types of advanced, solid malignancies produce G-CSF and also express its receptor, enabling autocrine proliferation of tumor cells, as well as conferring a strategy to intensify the immunosuppressive milieu of the tumor microenvironment (TME) via recruitment of immature and mature neutrophils that function as MDSCs . ApproxiParticularly high levels of expression (~90%) of the G-CSFR have been reported in human gastric and colon cancers, a setting in which G-CSF originates not only from tumor cells per se, but also from stromal myofibroblasts/fibroblasts in the tumor microenvironment (TME) . In the Enhancement of tumor cell proliferation and immunosuppression represent the predominant mechanisms of G-CSF-driven tumor progression, while G-CSF-mediated pro-metastatic activity is achieved via several mechanisms that include augmentation of release of pro-angiogenic factors by various cell types in the TME, as well as via the involvement of neutrophil extracellular traps (NETs).The mechanisms by which G-CSF drives tumor progression vary according to the type of malignancy. For example, in the case of aggressive tumors with high-level expression of G-CSF/G-CSFR, such as breast cancer, gastric and colon cancers, squamous cell cancers of the head and neck and neuroblastoma, data derived from pre-clinical in vitro studies and animal models of tumorigenesis, as well as from histological/genotypic analysis of biopsy specimens from patients, have revealed the existence of autocrine mechanisms of tumor proliferation ,85,86,87Alternative mechanisms, independent of G-CSFR-driven autocrine tumor cell proliferation, appear to promote G-CSF-mediated tumor progression in other types of malignancies such as experimental metastatic bone tumors  and EwinWith respect to ES, analysis of biopsy material from 83 patients with this vascular malignancy revealed expression of G-CSF and G-CSFR protein and mRNA in all biopsy specimens, as well as in four ES cell lines . TreatmeNotwithstanding the myelopoiesis-independent mechanisms of G-CSF-mediated immunosuppression mentioned in an earlier section of this review, proliferation and mobilization of MDSCs, specifically those of granulocytic leukocyte origin, represent the most probable mechanisms by which tumor-derived G-CSF promotes tumor progression . In thisHigh-level production of anti-proliferative, cytotoxic reactive oxygen species (ROS) ;Depletion of arginine and tryptophan via extracellular release of the enzymes arginase and indoleamine-2,3-dioxygenase ,101;Expansion of Tregs via production of IL-10 ;Interference with the recognition of tumor antigens via nitrosative modification of the T cell receptor for antigen ;Physically impede the access of TILs to tumor cells in the TME ;Via potentiation of neutrophil extracellular trap (NET) formation within the TME, which also obstructs the access of TILs to tumor cells .Mechanisms utilized by MDSCs to suppress the functions of TILs have recently been described in detail elsewhere  and are In addition to promoting immunosuppression-associated tumor progression, the G-CSF/MDSC axis also contributes to tumor invasion and metastasis by driving NETosis within the TME.In addition to NETosis, other pro-invasive mechanisms involving the G-CSF/MDSC axis include MMP-9-driven epithelial-to-mesenchymal transition  and prodNETosis is a highly regulated sequence of events that leads to cell death in response to various microbial and inflammatory stimuli. As a result of NETosis, neutrophils extrude their chromatin material leading to the formation of NETs . ProductChemotherapy-induced febrile neutropenia (FN) is a life-threatening after-effect of cancer treatment that poses a significant risk for development of infection-related morbidity and mortality, as well as substantial dose-limiting toxicity. The risk of infection increases with the severity and duration of neutropenia.Patients developing grades 3 and 4 neutropenia  or FN during chemotherapy commonly require dose reductions and/or delayed administration of their chemotherapy, resulting in decreased relative dose intensity (RDI). Treatment outcomes may be compromised, mostly when the goal of treatment intent is either curative or to prolong survival. Accordingly, prophylactic use of rG-CSF is a therapeutic strategy for maintaining the RDI during treatment by decreasing the incidence of severe neutropenia and FN, thereby improving treatment outcomes and quality of life. Most commonly, rG-CSF is utilized to increase circulating neutrophil counts in patients facing severe and prolonged neutropenic episodes following chemotherapy. Additionally, as mentioned earlier, rG-CSF is used in patients and donors to mobilize peripheral blood stem cells for harvesting in the settings of autologous and allogeneic stem cell transplantation, respectively, as well as to support post-transplantation neutrophil recovery. The American Society of Clinical Oncology (ASCO), the National Comprehensive Cancer Network (NCCN) and the European Society for Medical Oncology (ESMO) have developed evidence-based guidelines defining the appropriate use of rG-CSF in cancer patients ,128,129.Primary prophylaxis is defined as the usage of rG-CSF in the first and subsequent cycles of chemotherapy. Most rG-CSF guidelines recommend primary prophylaxis for patients receiving chemotherapy regimens with an overall risk of FN that is greater than 20%. Patients receiving myelotoxic chemotherapy with curative or radical intent (including adjuvant/neoadjuvant settings) are included in this category. Additionally, primary prophylaxis should be considered for patients receiving myelotoxic chemotherapy with a documented FN incidence rate of 10–20% and who have one or more of the risk factors listed in Primary prophylaxis is supported by the fact that approximately half of the neutropenic episodes occur during the first cycle of chemotherapy .Secondary prophylaxis is defined as the usage of rG-SCF in subsequent cycles after initial episodes of severe neutropenia and/or FN. This strategy may be considered if dose reduction or treatment delay may result in a decrease in disease-free or overall survival. Examples of types of malignancies in this category include adjuvant breast cancer, non-Hodgkin’s lymphoma, Hodgkin’s disease, testicular cancer and other germ cell tumors, as well as patients undergoing neoadjuvant chemotherapy with curative intention. For patients undergoing chemotherapy with palliative intent, dose modifications are considered a reasonable alternative.Most guidelines do not recommend the use of rG-CSF for the treatment of patients with low-risk or uncomplicated FN, or afebrile neutropenia. Recombinant G-CSF is recommended for patients with severe or complicated FN as summarized in Short-acting and long-acting rG-CSF are approved for the prophylaxis of FN . AlthougAdditional clinical indications for the therapeutic usage of rG-CSF include idiosyncratic drug-induced neutropenia and agranulocytosis, congenital neutropenic syndromes and neutropenia-associated primary immunodeficiency disorders ,137,138.Colony-stimulating factors should be avoided in patients receiving concurrent chemotherapy and radiation therapy, particularly involving the mediastinum. In the absence of chemotherapy treatment, therapeutic use of CSFs may be considered in patients receiving radiation therapy alone if prolonged delays secondary to neutropenia are expected .Hematopoietic syndrome is a clinical diagnosis given to people who present with equal or more than one new-onset cytopenia in the background of acute radiation exposure . RecombiFinally and reassuringly, long-term follow-up of healthy donors who had received rG-CSF in the setting of allogeneic stem cell transplantation failed to reveal a causal link with future development of hematological malignancies , underscLike many other cytokines, G-CSF is a rather enigmatic protein. On the one hand, controlled and appropriate production of G-CSF plays a critical role in anti-infective host defense, while on the other hand, inappropriate production by tumor cells appears to contribute to tumor growth and invasion. In the latter scenario, of which many clinicians may be unaware, the pro-tumorigenic effects are achieved via a dual mechanism of immunosuppression, seemingly by directing T cell polarization towards the Th2 and Treg phenotypes, and, most prominently, via induction of production of MDSCs. Given its key role in host defense, the most effective strategies to neutralize the pro-tumorigenic effects of endogenous G-CSF in the setting of adjunctive anti-cancer therapy are likely to involve pharmacologic targeting of the CXCR1 and CXCR2 chemokine receptors. The involvement of endogenously produced, tumor-derived G-CSF in tumorigenesis contrasts with the beneficial administration of rG-CSF in the prevention of chemotherapy-induced severe and life-threatening infection. Recent innovations in this setting include prophylactic use of rG-CSF, rather than delayed therapeutic administration following the onset of FN, thereby alleviating development of infection and treatment disruption. Although this strategy does not appear to pose the risk of development of hematological and other types of malignancies, ongoing vigilance is necessary, particularly in the context of increasing prophylactic utilization of rG-CSF and the availability of more cost-effective biosimilars. Future studies should be undertaken to monitor the emergence of MDSCs and/or alterations in the levels of their soluble, systemic mediators of immunosuppression in patients with cancer who receive prolonged/frequent administration of rG-CSF.


Different therapeutic modalities have been implicated in managing neonatal HIE, though translation of most of these regimens into clinical practices is still limited. Therapeutic hypothermia, for instance, is the most widely used standard treatment in neonates with HIE as studies have shown that it can inhibit many steps in the excito-oxidative cascade including secondary energy failure, increases in brain lactic acid, glutamate, and nitric oxide concentration. Granulocyte-colony stimulating factor (G-CSF) is a glycoprotein that has been implicated in stimulation of cell survival, proliferation, and function of neutrophil precursors and mature neutrophils. Extensive studies both in vivo and ex vivo have shown the neuroprotective effect of G-CSF in neurodegenerative diseases and neonatal brain damage via inhibition of apoptosis and inflammation. Yet, there are still few experimentation models of neonatal HIE and G-CSF’s effectiveness, and extrapolation of adult stroke models is challenging because of the evolving brain. Here, we review current studies and/or researches of G-CSF’s crucial role in regulating these cytokines and apoptotic mediators triggered following neonatal brain injury, as well as driving neurogenesis and angiogenesis post-HI insults.Hypoxic-ischemic encephalopathy (HIE) is an important cause of permanent damage to central nervous system (CNS) that may result in neonatal death or manifest later as mental retardation, epilepsy, cerebral palsy, or developmental delay. The primary cause of this condition is systemic hypoxemia and/or reduced cerebral blood flow with long-lasting neurological disabilities and neurodevelopmental impairment in neonates. About 20 to 25% of infants with HIE die in the neonatal period, and 25-30% of survivors are left with permanent neurodevelopmental abnormalities. The mechanisms of hypoxia-ischemia (HI) include activation and/or stimulation of myriad of cascades such as increased excitotoxicity, oxidative stress, N-methyl- Birth a post-HI –8, categ post-HI , 9.Fig.The clinical manifestations of neonatal HIE are insidious because the immature brain is more resistant to injury from hypoxia-ischemia (HI) events due to lower cerebral metabolic rate, plasticity of immature central nervous system (CNS), and immaturity in the development of balance in the functional neurotransmitters . Thus, nPotential neuroprotective strategies targeting different pathways leading to neuronal cell death in response to hypoxic-ischemic insult have been investigated, including hypothermia, erythropoietin, magnesium, allopurinol, xenon, melatonin, growth factors , barbiturates, statins, and stem cells in various animal models of neonatal HIE , 16–18. . G-CSF and its receptors are expressed by neurons, and their expression is regulated by ischemia, which points to an autocrine protective signaling mechanism [Granulocyte-colony stimulating factor (G-CSF) is a 20-kDa protein that readily crosses the blood-brain barrier (BBB), which is a member of the hematopoietic growth factor family, that promote the proliferation, differentiation, and survival of hematopoietic stem cells with the obvious protective effect on neurons in peripheral and central nervous system –30. The echanism . Extensiechanism , 54, Alzechanism , and strechanism –58, and echanism , 60. Recechanism , diagnosechanism , 15, treechanism , and emeThere are numerous studies that have shown the neuroprotective effect of G-CSF via inhibition of apoptosis and inflammation as well as by stimulating angiogenesis and neurogenesis both in adults and neonatal animal HI models –64. IndePrior studies have shown that interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) are early response cytokines in neuronal injury . IncreasThe effect of G-CSF is demonstrated by preventing neuronal and glial pro-inflammatory cytokine expression. This is evidenced by preventing an overactivation of monocytes and lymphocytes by reducing the release of pro-inflammatory mediators while simultaneously activating the anti-inflammatory defense neutrophils . Our preCertain other studies have reported mixed results about the effectiveness of G-CSF treatment in ischemic stroke in terms of improving neurodegenerative and neurobehavioral outcomes, especially during the hyperacute and acute stage of HI injury , 78–80. As some of these results and findings are mainly focused on adult ischemic stroke, it should be noted that differences do exist between the adult and neonate reaction to HI-induced brain insults, possibly due to immaturity of neonatal neurons. Specifically, there are differences in functional BBB response to acute experimental stroke between neonates and adults, as well as in gene expression of cerebral endothelial cells . Other sApoptosis can be triggered via the extrinsic pathway, which involves activation of cell surface death receptors, or the intrinsic pathway, which requires mitochondrial outer membrane permeabilization (MOMP) , which lStudies have shown that neonatal HI injury is associated with Bax translocation to mitochondria with a concomitant decrease in BCL-2, resulting in activation of caspase-3 leading to apoptotic cell death that peaks from 24 to 72 h post HI brain injury . BAD is G-CSF administration downregulates GSK-3ß activity, resulting in reduced neuronal cell death, apoptosis, and infarct volume, as well as upregulating anti-apoptotic protein Bcl-2 expression levels , 77. OurNeurogenesis is an important process for the reconstruction of neural networks and recovery of functional outcomes that are believed to continue throughout adulthood. It mainly occurs in the subventricular zone (SVZ) and subgranular layer of the hippocampal dentate gyrus, where the local environment tightly regulates neurogenesis . NonetheAngiogenesis is a physiological process by which new blood vessels are formed from pre-existing blood vessels. It is a complex and highly ordered process that relies upon extensive signaling networks both among and within endothelial cells (ECs) and their associated cells such as vascular endothelial growth factor (VEGF) proteins and angiopoietin-1 (Ang-1), which are required for angiogenesis . HypoxiaG-CSF administration increases local VEGF expression, which is necessary for vascular angiogenesis , thus coRecent reports have advocated for combination strategies in neonatal HI brain injury and that it is more effective than G-CSF monotherapy. For instance, Yu and colleagues  demonstrDoycheva et al.  also conG-CSF + hUCB can also decrease the number of MHCII+ cells not only in the corpus callosum and fornix but also in the cerebral peduncle. As G-CSF crosses the BBB, it acts upon neurons and glial cells through the G-CSF receptor. Indeed, glial cell activation has been demonstrated to downregulate expression of proinflammatory cytokines and to enhance neurogenesis . One stuRecently, Griva et al.  reportedStimulation of G-CSF by its receptor activates many downstream signaling pathways such as the Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the Ras/mitogen-activated protein kinase (MAPK), and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathways  thereby cell nucleus, resulting in the activation of genes through transcriptional processes [The JAK-STAT signaling pathway is a chain of interactions between protein cells and is involved in many cellular processes that communicate information from chemical signals outside of a cell to the rocesses . The neurocesses , 94. It rocesses , 93, 127rocesses , 94. G-Crocesses . In shorThe PI3K/AKT pathway is a key signaling pathway that participates in various cellular processes such as cell proliferation, survival, differentiation, and apoptosis , 130. G-The mammalian target of rapamycin/p70 ribosomal S6 protein kinase (mTOR/p70S6K) pathway has been implicated in neurogenesis as well, by decreasing the expression of PICs , as wellpCREBSer-133) serves an important role in neurological regulation of ion channel function, neuronal differentiation and maturation, and the processes of learning and memory. This transcription factor is decreased during HI insult, while treatment of G-CSF was shown to significantly increase the expression of pRaf-pERK1/2-pCREBSer-133 pathway in neonates exposed to perinatal hypoxia [The PI3K/AKT/mTOR pathway is an intracellular signaling pathway important in regulating the cell cycle. Therefore, it is directly related to cellular quiescence, proliferation, cancer, and longevity. PI3K activation phosphorylates and activates AKT, localizing it in the plasma membrane , 141. G- hypoxia . One stu hypoxia . Activat hypoxia . G-CSF d hypoxia .Thus, G-CSF plays diverse roles in neonatal HIE through diverse signaling pathways. Modi et al.  summarizThere are numerous studies that have shown the important role G-CSF plays in neurodegenerative diseases, ischemic stroke, and traumatic brain injury both using in vivo and ex vivo models. Recent research has specifically focused on its neuroprotective effect in neonatal HIE with both positive and mixed results. Indeed, G-CSF exerts a pivotal role in the control of immune response and acts as an anti-inflammatory cytokine, preventing an overactivation of monocytes and lymphocytes by reducing the release of pro-inflammatory cytokines as well as stabilizing the BBB and modulating neuroinflammation; inhibits pro-apoptotic mediators; enhances concentration of neurotrophic factors and facilitates bone marrow cell mobilization thereby driving neurogenesis; increases local VEGF expression necessary for vascular angiogenesis; upregulates Ang-1 that reduces vascular solute permeability; and contributes to vascular maturation and BBB stabilization. Despite these progresses being made, there are still few experimentation models of neonatal HIE and G-CSF’s neuroprotectiveness either directly or through signaling pathways, and extrapolation of adults’ stroke models is challenging due to the evolving neonatal brain. Also, there are mixed results about the effectiveness of G-CSF in improving HI-induced brain injury in neonates, specifically in the area of initiation of treatment, dosage, route of administration, and duration of treatment; this calls for more in-depth research to elucidate its underlying mechanisms and pertinent role in neuroprotection, as well as its long-term effect in neurological and behavioral outcomes.Recent studies have focused on combinational treatment in neonatal HI brain injury and that it is more effective than G-CSF monotherapy. Therapeutic hypothermia has also been advocated for with other agents such as EPO, melanin, xenon, stem cells, and anticonvulsants with promising results. As most of these agents have controversial effectiveness in improving HI-induced brain injury and most of them are under clinical trials for efficacy and safety, at present, it appears that combination of these therapeutic agents with TH could be the promising intervention strategies to treat newborns suffering from HIE, while awaiting the outcomes of both preclinical and clinical trials that are under investigation.In this review, we highlighted recent researches in the role of G-CSF in regulating cytokines and inhibition of apoptotic mediators, as well as promoting neurogenesis and angiogenesis thereby enhancing cell survival and proliferation, and modulation of inflammatory responses in the injured neonatal brain through activation of multistep signaling pathways.