Gastrointestinal malignant tumor is the fourth most common cancer in the world and the second cause of cancer death. Due to the susceptibility to lymphatic metastasis and liver metastasis, the prognosis of advanced tumor patients is still poor till now. With the development of tumor molecular biology, the tumor microenvironment and the cytokines, which are closely related to the proliferation, infiltration and metastasis, have become a research hotspot in life sciences. Colony stimulating factor-1 (CSF-1), a polypeptide chain cytokine, and its receptor CSF-1R are reported to play important roles in regulating tumor-associated macrophages in tumor microenvironment and participating in the occurrence and development in diversities of cancers. Targeted inhibition of the CSF-1/CSF-1R signal axis has broad application prospects in cancer immunotherapy. Here, we reviewed the biological characters of CSF-1/CSF-1R and their relationship with gastrointestinal malignancies. With the development of tumor molecular biology and related disciplines, tumor microenvironment and the involved cytokines, which are closely related to the occurrence, proliferation, infiltration and metastasis of GI malignant tumors, have become a research hotspot in life sciences Malignant tumors of the gastrointestinal (GI) tract are one of the common malignant tumors in China, among which gastric cancer is the fourth most common cancer in the world and the second cause of cancer death 5in vivo and a member of the hematopoietic growth factor family. It plays an important role in different stages of hematopoiesis in vitro, CSF-1 can stimulate different hematopoietic stem cells to form cells in semisolid media and stimulate bone marrow and spleen macrophage cell lines CSF-1 is an important cytokine CSF-1 is a glycoprotein composed of 174 amino acid residues CSF-1R belongs to the receptor tyrosine kinase signaling system. One of the characters of such a receptor is that when the ligand binds to the receptor recognition sites outside the cell membrane, the receptor will then aggregate and phosphorylate the tyrosine kinase-activated receptors within the cell membrane, finally leading to the phosphorylation of the tyrosine residues of effector proteins and the alteration of the biological activity of the effector . C-fms is located at 5q33-3 of the human chromosome. Human CSF-1R synthesizes a 130 kDa immature transmembrane glycoprotein (gp130c-fms) from the rough endoplasmic reticulum, which is then modified by N-terminal oligosaccharide chains to form a mature 150 kDa glycoprotein (gp150c-fms) during intracellular transport. CSF-1R is secreted from the Golgi to the cytoplasm and then localized to the cell membrane. It can be converted from gp130c-fms to gp150c-fms within 1 hour . The extracellular domain consists of five immunoglobulin-like rings for ligand binding. The intracellular domain contains tyrosine residues for self-phosphorylation, the kinase catalytic region contains three tyrosine self-phosphorylation sites , and the C-terminal region also has a tyrosine residue (809) for self-phosphorylation CSF-1R, also known as FMS kinase, is the coding product of the c-fms proto-oncogene 32When membrane receptors are abnormally activated, tyrosine residues can be phosphorylated to induce ligand conformational changes that stimulate continuous activation of tyrosine kinases, leading to cell growth and proliferation and eventually carcinogenesis Recent studies have shown that TAM is closely related to macrophage colony stimulating factor (M-CSF) The interaction between CSF-1 and CSF-1R could cause receptor dimerization, tyrosine phosphorylation, and the subsequent interaction with multiple intracellular signaling pathways such as Ras, MAPK, PI-3K, JAK, finally producing various biological effects in vivo and in vitro become an important inducement for the macrophage polarization. Abnormally high expression of CSF-1 is related to pathological processes such as tumors and inflammation According to their phenotypes and secreted cytokines, TAM is divided into two types of polarization: classically activated M1 type and selectively activated M2 type macrophages Previous studies have demonstrated that CSF-1 and interleukin 34 (IL-34) signal via CSF-1R play important roles in macrophage differentiation in several inflammatory and oncological preclinical models Fig. 1). Consistent with this, in metastatic PDAC, tumor-cell-derived CSF-1 induces macrophages to produce granulin, a secreted glycoprotein that promotes fibroblast activation and spurs tumor growth Alternative ligands of CSF-1R, including IL-34 et al. discovered a potent, highly selective, and orally bioavailable CSF1R inhibitor, IACS-9439, which was proposed as a potential therapy to reduce TAMs, especially the protumor, immune-suppressive M2 TAMs and promote macrophage polarization toward the M1 phenotype by targeting CSF-1R The CSF-1/CSF-1R signaling pathway modulates the production, differentiation, and function of TAMs. However, the discovery of selective CSF-1R inhibitors devoid of type III kinase activity has proven to be challenging in tumor treatment. Barbara Czako + and Ki67+ T cells infiltration and unfavorable clinical outcome in gastric cancer patients. Importantly, the authors provided evidence that tumor-associated macrophages-derived CXCL8 determines immune evasion through autonomous PD-L1 expression in gastric cancer, suggesting that it may be a promising strategy to block the CXCL8 pathway to increase anti-tumor immunity in gastric cancer In vitro studies have shown that CSF can also be produced by tumor cells and stromal cells, and it could promote tumor fitness and cell proliferation and metastasis CSF-1 is a hematopoietic growth factor that acts through the cfms/CSF-1R. The CSF-1/CSF-1R axis is considered to be involved in the invasion and development of various types of cancer +T-lymphocyte infiltration in “cold” colon cancer Recent works showed that serum CSF-1 has great value in the diagnosis and progression of colorectal cancer (CRC), making it an independent prognostic factor for the survival of patients with CRC It has been found that abnormal expression of CSF-1/CSF-1R axis can also occur in malignant meningiomas, hepatocellular carcinoma, and pancreatic cancer, where CSF-1/CSF-1R blockade reprograms tumor-infiltrating macrophages and improves response improves response to T cell checkpoint immunotherapy Fig. 2). A study investigated the effect of CSF/CSF-R on the progression of colon cancer and pancreatic cancer in a mouse model and the results showed that in the absence of CSF-R, macrophage-related tumor cytotoxicity was amplified, indicating that CSF/CSF-R is an important clinical application target for controlling tumor microenvironment and gastrointestinal tumor progression The tumor microenvironment includes the structure, function and metabolism of the tissues in which tumors are located, as well as the external environment in which tumors themselves and surrounding immune cells and immunoregulatory factors are formed Fig. 3). JAK/STAT is generally believed to be the main pathway for CSF signal transmission CSF-1R binding to the CSF-1 ligand on the surface of cancer cells can activate multiple intracellular signaling pathways to promote proliferation, invasion, metastasis, and angiogenesis Understanding the signaling mechanism of CSF-1 and CSF-1R in cancer and other diseases and taking appropriate measures to block CSF-1/CSF-1R signaling is a promising new immunotherapy with potential for future clinical application. Studies have found that CSF-1 is one of the most common proinflammatory cytokines, and it can cause various inflammatory diseases . CSF-1 and CSF-1R play a role through multiple signaling pathways by inhibiting the immune response of the body. Blocking the CSF-1/CSF-1R axis can not only directly treat tumor cells with CSF1R, as a targeted therapy, but also can promote the polarization of TAM and improve the tumor microenvironment, thereby exerting an anti-tumor effect. However, the mechanism of CSF-1 and CSF-1R in GI malignant tumors is still not clear, and the mechanism of how inflammatory immune cells participate in the occurrence and development of malignant tumors is not perfectly defined. Therefore, more in-depth exploration and clinical validation are needed to improve the effects of targeted drugs and comprehensive treatments and to help identify new markers of malignant tumors for early diagnosis and evaluation of prognosis.CSF is widely used in clinic to treat granulocytopenia patients after chemotherapy. Increasing research has shown that CSF-1 and CSF-1R are expressed in malignant tumors of the digestive tract and can promote the growth, migration and invasion of tumors


Blood-cerebrospinal fluid (CSF) barrier transporters affect the influx and efflux of drugs. The antiretrovirals tenofovir and emtricitabine may be substrates of blood-brain barrier (BBB) and blood-CSF barrier transporters, but data are limited regarding the pharmacogenetics and pharmacokinetics of their central nervous system (CNS) penetration.We investigated genetic polymorphisms associated with CSF disposition of tenofovir and emtricitabine.ABCC5, ABCG2, ABCB1, SLCO2B1, SCLO1A2, SLCO1B1 and ABCC4) and 782 met a linkage disequilibrium (LD)-pruning threshold.We collected paired plasma and CSF samples from 47 HIV-positive black South African adults who were virologically suppressed on efavirenz, tenofovir and emtricitabine. We considered 1846 single-nucleotide polymorphisms from seven relevant transporter genes  and for emtricitabine, it was ABCC5 rs11921035 (p = 1.4 × 10−3). None withstood correction for multiple testing.The geometric mean  values for tenofovir and emtricitabine CSF-to-plasma concentration ratios were 0.023 (0.021–0.026) and 0.528 (0.460–0.605), respectively. In linear regression models, the lowest No genetic polymorphisms were associated with plasma, CSF concentrations or CSF-to-plasma ratios for either tenofovir or emtricitabine. Data from small cohorts indicate that CSF concentrations of tenofovir and emtricitabine are 5% and 50% of plasma concentrations, respectively.5Tenofovir and emtricitabine are part of the current first-line antiretroviral therapy (ART) regimens for HIV-positive adults in resource-limited settings and both are widely used in high-income countries.5 Transporters in the BBB and blood–CSF barrier (BCB) affect the influx and efflux of drugs, including tenofovir and emtricitabine.7 Multidrug resistance protein-5  is ubiquitous and mediates the efflux of nucleoside reverse transcriptase inhibitors.8 Lower CSF emtricitabine exposure in females compared to males is hypothesised to reflect differential expression of MRP transporters at the BBB and BCB.3In vitro, tenofovir is a substrate of the breast cancer resistance protein , MRP-4 (encoded by ABCC4) and P-glycoprotein (encoded by ABCB1).11 A polymorphism in ABCG2 rs2231142 has been associated with 1.5-fold increased plasma tenofovir exposure and Thai patients carrying ABCC4 3463 AG or GG (rs1751034) had an 11% greater tenofovir clearance compared with AA.13However, higher CSF tenofovir concentrations and lower emtricitabine concentrations have been reported, which may be explained by polymorphisms in drug transporters or altered blood–brain barrier (BBB) permeability.ABCC4 polymorphisms have been associated with reduced clearance of tenofovir.14 In genome-wide analyses, SLC17A1 rs12662869 was associated with an increase in tenofovir clearance.15 It is possible that genetic polymorphisms that affect transporter function will affect tenofovir or emtricitabine CSF penetration. The pharmacogenetics of CSF penetration of tenofovir and emtricitabine have not been described.Loss-of-function 16 South Africa has the world’s largest ART programme, with most patients currently receiving efavirenz-based regimens that include the nucleos(t)ides tenofovir and emtricitabine.17 We previously reported on the pharmacogenetics of CSF penetration of efavirenz in black South Africans.18 Here, we characterise the associations between transporter gene polymorphisms and CSF penetration of tenofovir and emtricitabine in the same cohort.Africans are the most genetically diverse population worldwide.19 We also invited participants who were screened for that trial but were excluded based on cognitive impairment criteria. All participants provided written informed consent. This study was approved by the University of Cape Town Human Research Ethics Committee (HREC 071/2013).Adults (≥ 18 and ≤ 70 years of age) from a randomised control trial (PACTR201310000635418) that investigated lithium for HIV-associated neurocognitive impairment were invited to participate in the present study.We collected paired plasma and CSF samples for tenofovir and emtricitabine assays. Participants recorded the dosing time the night before and were admitted in the morning for pharmacokinetic sampling. Whole blood was collected within 45 min of CSF sampling and centrifuged within 1 h of collection. Plasma and CSF aliquots were stored at −80 °C until analysis.The analytical laboratory in the Division of Clinical Pharmacology at the University of Cape Town quantified total tenofovir and emtricitabine in plasma and CSF using validated liquid chromatography tandem mass spectrometry assays.The lower limits of quantification (LLQs) for plasma tenofovir and emtricitabine were 10.0 ng/mL and 37.5 ng/mL, respectively. For CSF, the LLQs for total tenofovir and emtricitabine were 0.5 ng/mL. Concentrations below the limits of quantification were treated as missing data.EX; Illumina, San Diego, CA, USA).We extracted DNA from the buffy coat using the QIAsymphony kit. Genotyping was performed using the Infinium® Expanded Multi-Ethnic Genotyping Array (MEGAABCB1 (301 polymorphisms), ABCC4 (630 polymorphisms), ABCC5 (225 polymorphisms), ABCG2 (164 polymorphisms), SLCO1A2 (406 polymorphisms) and SLCO2B1 (118 polymorphisms). Polymorphisms were excluded for genotyping efficiency less than 99%, minor allele frequency less than 5% and Hardy-Weinberg equilibrium p-values less than 0.00001. We also genotyped SLCO1B1 521T→C (rs4149056) and SLCO1B1 (rs4149032) using the MassARRAY iPLEX® Gold system .Polymorphisms that were not genotyped were imputed. Polymorphisms were extracted from seven genes ±50 kB: All genotyping was performed at Vanderbilt Technologies for Advanced Genomics (VANTAGE), by laboratory personnel with no knowledge of clinical data. All samples were genotyped in duplicate. The final dataset included 1846 polymorphisms from 47 participants.r correlation was used to assess the correlations between plasma and CSF concentrations. We performed statistical analysis using STATA version 15.0 . Graphs were created using GraphPad Prism version 7.03 for Windows .Pharmacokinetic data were not normally distributed so were expressed as median and interquartile ranges (IQRs) and geometric means . Pearson’s 10 transformed for association analyses. We used ratios of total concentrations without correcting for protein binding. Cerebrospinal fluid-to-plasma concentration ratios were calculated using raw concentrations and then log10 transformed. We performed genetic association analyses using PLINK version 1.9.20Associations with pharmacokinetic parameters were assessed by univariable analysis. Pharmacokinetic data were log2 threshold of 0.95 within a 50-kB window at 5-kB increments.For primary analyses, we conducted linkage disequilibrium (LD) pruned with an Rp = 0.05 divided by 782 polymorphisms). We generated an LD plot using Haploview (https://www.broadinstitute.org/haploview/haploview). We previously reported LD plots for these polymorphisms.18The final analysis included 782 polymorphisms that met the LD-pruning threshold. We used Bonferroni correction to adjust for multiple testing (All participants provided written informed consent. This study was approved by the University of Cape Town Human Research Ethics Committee (HREC 071/2013).n = 43) or efavirenz, tenofovir and lamivudine (n = 4). The median (IQR) values of the baseline characteristics were age 36 (IQR = 32–43) years, a CD4 T-cell count of 470 (IQR = 384–586) cells/mm3, a time on ART of 38 (IQR = 18–54) months and a body mass index (BMI) of 25.6 (IQR = 22.7–29.3) kg/m2. The concentrations of tenofovir (plasma and CSF) and emtricitabine (plasma and CSF) are presented in p < 0.0001, R2 = 0.53 and p < 0.0001, R2 = 0.45; respectively) (Appendix We studied 47 participants who self-identified as black South Africans (isiXhosa speaking), of whom 41 were female. All were virologically suppressed and were receiving efavirenz, tenofovir and emtricitabine . The remaining 1845 polymorphisms were in Hardy–Weinberg equilibrium based on a Bonferroni-adjusted p-value threshold of 6.4 × 10−5; 56 had unadjusted Bonferroni p-values of < 0.05. Minor allele frequencies for all polymorphisms are provided in Appendix Amongst the 47 participants, 1846 polymorphisms were successfully genotyped. Only ABCB1 rs1989830 . The emtricitabine CSF-to-plasma ratio was best predicted by a model that included ABCC5 rs11921035 , as shown in p-values below 0.01 for tenofovir and emtricitabine CSF-to-plasma ratios are shown in ABCG2, ABCC5, SLCO1A2 and ABCC4 for plasma and six in ABCB1, ABCG2, ABCC5, SLCO1A2 and ABCC4 for CSF had p-values less than 0.01 (data not shown). For absolute plasma and CSF emtricitabine concentrations, six polymorphisms in ABCC5, SLCO1A2, ABCC4 and SLCO2B1 for plasma and 12 in ABCB1, ABCG2, ABCC5, SLCO1A2 and ABCC4 had p-values less than 0.01 (data not shown). No associations with SLCO1B1 rs4149032 were found.In univariable linear regression analyses , the tenp-value for tenofovir CSF-to-plasma ratio was ABCB1 rs1989830 (p = 1.2 × 10−3), and for emtricitabine was ABCC5 rs11921035 (p = 1.4 × 10−3). None were significant after correcting for multiple testing. In addition, we found no significant associations with absolute CSF or plasma concentration after correcting for multiple testing.We characterised the associations between 782 genetic polymorphisms and CSF disposition of tenofovir and emtricitabine in black South African adults. The lowest ABCC4 4131T→G (genotype TG or GG) in 150 Thai HIV-infected adults.14ABCB1 rs3213619 was associated with increased tenofovir bioavailability in a predominantly African-American patient population (n = 45) and thought to be a result of decreased P-glycoprotein function.21 A genome-wide and candidate gene association analyses with tenofovir pharmacokinetics showed that SLC17A1 rs12662869 was associated with an increase in tenofovir clearance (p = 7.1 × 10−9) but failed to show significant associations in candidate genes  after correction for multiple comparisons.15Associations with tenofovir pharmacokinetics and genetic polymorphisms were found in other populations. An increase in tenofovir plasma concentrations were independently associated with 18 We may therefore have introduced a selection bias.Our study has limitations. With our sample size, we were underpowered to detect associations with small effect sizes. We could only detect associations with relatively frequent polymorphisms and with large effect sizes. Therefore, these data should be regarded as exploratory. Polymorphisms not genotyped in our study may be associated with tenofovir or emtricitabine disposition into CSF. Whilst we did not adjust for creatinine clearance, this should not be a confounder that affects drug disposition into CSF. We included 33 (70%) participants with mild to moderate neurocognitive impairment, as previously reported.In conclusion, we found no significant associations between any of the 782 polymorphisms and plasma concentrations, CSF concentrations or CSF-to-plasma ratios for either tenofovir or emtricitabine in univariate linear regression models after correcting for multiple testing.


Mycobacterium tuberculosis (Mtb) infections by enhancing GM-CSF biostability in serum. Specifically, albumin is very safe, stable, and has a long half-life, thereby enhancing the biostability of GM-CSF. In the lungs and draining lymph nodes, albGM-CSF is able to increase the numbers of dendritic cells, which are crucial for the activation of naive T cells and for eliciting potent immune responses. Subcutaneous administration of albGM-CSF alone reduced the mean lung bacillary burden in mice with chronic tuberculosis infection. While GM-CSF administration was associated with IL-1β release from Mtb-infected dendritic cells and macrophages, higher IL-1β levels were observed in albGM-CSF-treated mice with chronic tuberculosis infection than in mice receiving GM-CSF. Albumin fusion with GM-CSF represents a promising strategy for the control of chronic lung tuberculosis infections and serves as a novel therapeutic vaccination platform for other infectious diseases and malignancies.A long duration of treatment and emerging drug resistance pose significant challenges for global tuberculosis (TB) eradication efforts. Therefore, there is an urgent need to develop novel strategies to shorten TB treatment regimens and to treat drug-resistant TB. Using an albumin-fusion strategy, we created a novel albumin-fused granulocyte-macrophage colony-stimulating factor  molecule that harnesses albumin’s long half-life and targeting abilities to enhance the biostability of GM-CSF and direct it to the lymph nodes, where the effects of GM-CSF can increase dendritic cell populations crucial for eliciting a potent immune response. In this study, we demonstrate that albGM-CSF serves as a novel immunotherapy for chronic  Although this regimen has been shown to be efficacious, it requires proper provision and direct supervision, which can be taxing for health care systems, especially in TB-endemic regions. Inadequate treatment of TB leads to excess morbidity and mortality, continued transmission, and emergence of drug resistance.2 Therefore, novel strategies are needed to shorten the duration of curative TB treatment. In addition to novel antimicrobial agents, host-directed therapies represent attractive strategies to combat disease due to drug-susceptible and drug-resistant Mycobacterium tuberculosis (Mtb).3 In particular, host-directed therapies may reverse TB-related lung inflammation and/or augment innate and adaptive immune responses to accelerate mycobacterial clearance during anti-TB treatment.4Tuberculosis (TB) is currently the most common cause of death by a single infectious agent worldwide.5 Dendritic cells (DCs) are key antigen-presenting cells (APCs) that activate naive T cells by upregulating chemokine receptors and costimulatory molecules.6 Mature DCs are characterized by higher expression of surface MHC class II molecules and the integrin-αX chain, as well as CD11c and other costimulatory molecules.7 Adoptive transfer of antigen-pulsed DCs has been shown to significantly improve vaccination efficacy relative to control treatment, indicating that adequate antigen presentation in the lungs is one of the key factors for controlling Mtb infection.8Effective immunity against TB depends on antigen presentation by MHC class I or class II molecules, which occurs in the draining lymph nodes (dLNs) at the site of infection.11 Other myeloid lineage cells, including monocytes, macrophages, neutrophils, and eosinophils, are also activated by GM-CSF.9 Coadministration of GM-CSF during vaccination increases antigen-specific IFNγ-secreting T cells and enhances protection against various infectious agents.18 Conversely, deficiency of GM-CSF is associated with reduced T-cell responses after vaccination.19 Mice vaccinated with a bacillus Calmette–Guérin vaccine including cells expressing murine GM-CSF were found to have enhanced DC maturation in dLNs and increased protection against disseminated TB.16 In addition to its promotion of the maturation of DCs, GM-CSF is one of the key cytokines that promotes the differentiation of M1 macrophages, which are key effectors in controlling intracellular pathogens through the release of proinflammatory cytokines.22 GM-CSF enhances the ability of human macrophages to inhibit Mtb growth ex vivo.23 GM-CSF−/− mice are highly susceptible to Mtb infection, and anti-GM-CSF autoantibodies increase the risk of cryptococcal meningitis and pulmonary TB in patients.26 GM-CSF secreted by T cells has been shown to offer protection against Mtb infection in murine models.25 A greater proportion of GM-CSF+ multifunctional CD4+ T cells are present in latently infected individuals than in those with active TB.27 Furthermore, GM-CSF secretion was significantly reduced when CD3+ T cells were cocultured with myeloid-derived suppressor cells in patients with active TB.28 These observations indicate that GM-CSF plays an important role in innate immunity and initiating adaptive immunity, indicating the potential utility of this cytokine in anti-TB immunotherapy. Indeed, GM-CSF enhances the bactericidal activity of anti-TB drugs in both mouse models and in humans.31 However, the observed synergy of GM-CSF is relatively limited due to its side effects, short half-life of ~7 h, and reduced penetration into the lungs.32 Therefore, an alternative strategy is required to improve the bioavailability of GM-CSF in the lungs, which are the primary site of TB.Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor critical for DC generation, proliferation, and maturation.35 However, one limitation of this approach is the potential development of autoantibodies directed against the Fc region.34 Similar to immunoglobulin, albumin has an extended serum half-life of 3 weeks due to its size and its ability to undergo neonatal Fc receptor (FcRn)-mediated recycling, thus preventing intracellular degradation.33 Since albumin is very safe and stable and has a very long half-life, it has been frequently used for drug delivery. Currently, there are six albumin-based drugs that are commercially available, with many more being tested in clinical trials.36 Labeled human albumin has been used for LN identification and imaging, suggesting that albumin can traffic to LNs.37 We reasoned that these favorable properties of albumin may be exploited by fusing this protein to GM-CSF to enhance the serum levels of GM-CSF and augment its effect in the lungs and LNs, thus achieving organ-targeting vaccination. Specifically, we determined whether murine albumin conjugation was able to increase the effects of GM-CSF in mice. In our proof-of-concept studies, we show that this albumin-fusion strategy  enhances the serum levels of GM-CSF, leading to increased DC populations, cytokine secretion, and CD4+ T-cell activation, thus improving the control of chronic TB in mice.A fusion strategy using the fragment crystallizable region (Fc region) has been used to improve the biostability and half-life of proteins, as well as for mucosal targeting.GAGGCCAAGCCCACCGAGAAC-3′ and 5′-TTTCTCGAGGTCACCACCGGCCCCCTTGA-3′. The amplified product was then cloned into the EcoRI/XhoI sites of the PET28a vector . All plasmid constructs were confirmed by DNA sequencing. The AlbGM-CSF and albumin-GLuc  proteins were expressed using the Expi293F Expression System Kit  according to the manufacturer’s instructions. Expi293F cells were transfected with albGM-CSF and alb-GLuc, and the transfection efficiency was determined by the expression levels of the target protein. Proteins were purified by a HiTrap albumin column . GLuc was expressed in E. coli BL21  and purified by Ni+ affinity chromatography  according to the manufacturer’s protocol). Mouse GM-CSF was purchased from Genscript.To generate albumin-fused GM-CSF , mouse albumin was first amplified with PCR using the cDNA template of mouse albumin  and a set of primers, 5′-AAATCTAGAGCCACCATGAAGTGGGTAACCTTT-3′ and 5′-TTTGAATTCGGCTAAGGCGTCTTTGCATC-3′. The amplified product was then cloned into the XbaI/EcoRI sites of a pcDNA3 vector . Next, for the generation of pcDNA3-AlbGM-CSF, mouse GM-CSF was first amplified via PCR with a cDNA template of the mouse GM-CSF (NM_009969.4) gene synthesized from Genscript  and the following primers: 5′-TTTGAATTCGCACCCACCCGCTCACCCAT-3′ and 5′-AAACTTAAGTCATTTTTGGACTGGTTTTTTG-3′. The amplified product was then cloned into the EcoRI/Afl II sites of pcDNA3-Alb. For the generation of pcDNA3-albGLuc, Gaussia luciferase (GLuc) was first amplified via PCR with a cDNA template of phGLuc  and the primers 5′-AAAGAATTCATGGGAGTCAAAGTTCTGTTTG-3′ and 5′-TTTAAGCTTTTAGTCACCACCGGCCCCCTTG-3′. The amplified product was then cloned into the EcoRI/HindIII sites of pcDNA3-Alb. For the generation of pET28a-GLuc, GLuc was first amplified via PCR with a cDNA template of phGLuc and the following primers: 5′-AAAGAATTCn = 3–4)  or eight-to-ten-week-old FcRn-knock-out (KO) mice (n = 3–4)  received retro-orbital injections with either Gaussia luciferase (GLuc) (20 μg) or albGLuc (2.8 μg) in 20 μL of phosphate-buffered saline (PBS) following anesthesia by ketamine/xylazine intraperitoneal injection. Seventy-two hours after injection, mice were euthanized, and the serum, inguinal LNs, and lungs were removed and disrupted by bead-beating. GLuc activity was measured with coelenterazine-H (Regis) by a GloMax Luminometer . The total luminescence was normalized to tissue weight.Eight-to-ten-week-old female C57BL/6J . All animal procedures were performed according to the approved protocols and in accordance with the recommendations for the proper use and care of laboratory animals. To ensure that animal discomfort, distress, pain, and injury were kept to a minimum, a maximum of five mice were housed in the same cage. All animals were maintained and all experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee at the Johns Hopkins University School of Medicine.n = 3–4) received 20 μg of lipopolysaccharide (LPS) from E. coli O26:B6  via the intranasal route. The control group received PBS. One day later, both groups of mice received either GLuc (20 μg) or albGLuc (2.8 µg) by intranasal injection. To measure luciferase activity, sera from each group were collected the following day. Luciferase expression was taken to indicate transcytosis activity in the airway, as previously described.38Female C57BL/6J mice  supplemented with 10% oleic acid-albumin-dextrose-catalase (Difco), 0.1% glycerol, and 0.05% Tween-80 at 37 °C in a roller bottle.39Wild-type 40 To compare the efficacy of GM-CSF and albGM-CSF, bone marrow cells were incubated with either GM-CSF (0.625 µM) or albGM-CSF (0.625 µM) at 37 °C in 5% CO2 and RPMI media with 10% fetal bovine serum (FBS) for 7 days (Sigma-Aldrich). Antigen-containing supplemented media were replenished on day 3. The cells were collected to analyze the percentage of CD11c+ cells and CD11c+MHCII+ cells. For Mtb infection studies, bone marrow cells were incubated with GM-CSF  to obtain BMDCs or with macrophage colony-stimulating factor  to obtain BMDMs. A total of 2 × 105 BMDMs or 5 × 105 BMDCs were plated in a 24-well plate 1 day prior to infection. H37Rv was used to infect BMDCs at an MOI of 1:2.5 (1.25 × 106 bacteria) for 1 day or BMDMs (5 × 105 bacteria) for 2 days in 1 mL of complete RPMI medium  with 10% FBS (Sigma-Aldrich) and 0.625 µM GM-CSF or albGM-CSF.Bone marrow was harvested from C57BL/6J mice, as previously described.The levels of GM-CSF in sera and IL-1β in cell culture media were determined by ELISAs with mouse GM-CSF or IL-1β DuoSet ELISA kits from R&D Systems . The IL-1β levels of lung lysates were normalized to the lysate protein concentration using a Qubit protein assay kit (Thermo Fisher Scientific).42 To determine ESAT6 antigen-specific CD4+ or TB10 antigen-specific CD8+ T-cell responses, splenocytes were incubated with ESAT6 ((MTEQQW NFAGIEAAA) or TB10 (IMYNYPAM) peptides (Genscript) and GolgiPlug  overnight.43 After incubation, the cells were washed once with FACScan buffer and then stained with a PE-conjugated monoclonal rat anti-mouse CD4 antibody (BD Biosciences) and/or an APC-conjugated monoclonal rat anti-mouse CD8 antibody . Cells were permeabilized using the Cytofix/Cytoperm kit (BD Biosciences). Intracellular IFN-γ was stained using a FITC-conjugated rat anti-mouse IFN-γ antibody. Flow cytometry was performed on a FACSCalibur instrument, and the results were analyzed with FlowJo software  by esophageal gavage once daily (5 days/week). In the experimental groups, mice received 0.375 μM GM-CSF or albGM-CSF in 100 µL of PBS by retro-orbital or subcutaneous injection at predetermined time points. Mice were euthanized on days 30, 60, and 90 after aerosol challenge, lungs were homogenized, and the cells were plated for colony-forming unit assays to evaluate the potential synergistic effect of each therapeutic vaccine.44Female 6–8-week-old C57BL/6J mice were aerosol-infected with ~100 bacilli of wild-type t test for sample sizes >4, and a p value of < 0.05 was considered statistically significant.Mean differences between groups were compared using one-way analysis of variance with Tukey–Kramer post hoc analyses . If the data did not pass through the normality test by the D’Agostino–Pearson test, the differences of groups were compared by the Kruskal–Wallis test with post hoc analyses. Data from at least three biological replicates were used to calculate means and standard errors of the mean (SEMs) for graphing purposes. To compare differences between experimental and control groups, statistical analyses employed the Mann–Whitney test for sample sizes less than 4 or an unpaired Student’s 37 In the current study, we used murine albumin-fused GLuc  to determine the tissue distribution and serum levels of GLuc. Separate groups of female C57BL/6J mice received retro-orbital injections with either GLuc or albGLuc. In the experiments to analyze the enzymatic activity of the protein in different tissues, both albGLuc (0.028 µg/µL) and GLuc (0.2 µg/µL) had similar luminescence activities prior to injection . After injection of albGLuc or GLuc, the serum luciferase activity of the albGLuc group was 100-fold higher than that of the GLuc group 24 h after injection, and this significant difference in serum luciferase activity was maintained for at least 72 h after injection  T cells to control intracellular umn Fig. . We usedumn Fig. . It has CSF Fig. . Both alCSF Fig. . To inveCSF Fig. . SimilarCSF Fig. . Therefo50 To determine whether FcRn is required for albGM-CSF to mediate the enhancement of DC maturation in the LNs or lungs, FcRn-KO mice and C57BL/6J mice were subcutaneously injected with either albGM-CSF or GM-CSF. Four days after injection, the numbers of DCs observed in the dLNs of C57BL/6J mice receiving albGM-CSF were significantly increased compared with those observed in mice receiving GM-CSF alone  recycling and transport to LNs and prolongs the half-life of both albumin and IgG.one Fig. , but theone Fig. . Interesone Fig. .Mtb, female C57BL/6J mice were intravenously injected once with albGM-CSF or GM-CSF. One group of mice was treated daily (5 days/week) with isoniazid by esophageal gavage.42 The control group received no treatment. Four weeks after treatment initiation, the mice were euthanized to determine the lung bacterial burden. The lungs of the albGM-CSF-treated group had a significantly lower mean bacterial burden than those of the untreated and GM-CSF-treated groups  and pharmacodynamics of albGM-CSF under different administration routes.FcRn, a cell membrane-bound receptor, is expressed in endothelial cells66 GM-CSF can enhance intracellular bacterial killing in vitro47 and is an important mediator for controlling intracellular Mtb infection via iNK T cells or keratinocyte growth factor.47 GM-CSF-deficient mice infected with Mtb had accelerated mortality and more extensive pulmonary necrosis than control Mtb-infected mice, and these findings were related to impaired Th1 responses in the former mice.67 During chronic TB infection in mice, GM-CSF expression is decreased relative to that during acute infection, and intratracheal administration of adenovirus encoding GM-CSF enhances Mtb control in acute and chronic infection.30 It has been shown that GM-CSF-producing T cells play an important role in controlling chronic TB.25 The utility of GM-CSF in controlling chronic TB has been shown in animal models,29 although limited efficacy was observed in a clinical trial,31 perhaps due to the suboptimal route and frequency of administration. In the current study, we used an albumin-fusion strategy to improve the bioavailability and targeting of GM-CSF to the lungs and dLNs of mice. During pulmonary TB, there is significantly increased transcytosis, supporting the potential role of this approach for targeting protein or drugs to the lungs.GM-CSF promotes bone marrow cell proliferation and the expansion of macrophages and granulocytes.11 as is its role in enhancing antigen presentation to T cells, thus supporting its potential utility as an adjuvant for TB vaccination.69 Its dual functions in both innate and adaptive immunity make GM-CSF a unique candidate in treating TB. Macrophages and DCs express the GM-CSF receptor, whereas T cells do not.53 We have shown that both GM-CSF and albGM-CSF can enhance bacterial control during macrophage infection ex vivo  by which GM-CSF exerts its antimycobacterial effects. Finally, more work is required in preclinical models to evaluate the potential utility of albGM-CSF as an adjunctive therapy in combination with the standard first-line regimen with the goals of shortening the duration of treatment for drug-susceptible and drug-resistant TB and improving TB-induced lung pathology.The ability of GM-CSF to differentiate bone marrow cells into DCs in vitro is well characterized,ivo Fig. . This efIn the current study, we used an albumin-fusion strategy to enhance the delivery of GM-CSF to the lungs and dLNs of mice. We found that albGM-CSF acted as an in situ vaccine, increasing the number of DCs in the dLNs and lungs and yielding a tuberculocidal effect in the lungs. Further studies are needed to evaluate the potential utility of albGM-CSF as an adjunctive therapy in combination with the standard first-line regimen to shorten the duration of treatment for drug-susceptible and drug-resistant TB and to improve TB-induced lung pathology. In addition to its potential utility for chronic pulmonary infections, this albumin-fusion strategy may represent a promising approach for developing novel adjuvants in cancer prevention and therapeutic vaccines.Supplemental Figure 1Supplemental Figure 2Supplemental Figure 3Summary


Since the early phase of the COVID-19 pandemic, a principal international panel of experts from European Society for Medical Oncology (ESMO) , have drIn managing febrile neutropenia (FN), supportive care indications have driven to an expanded use of recombinant human granulocyte colony-stimulating factors (rhG-CSFs) to accelerate absolute neutrophil count recovery; in particular, they have also recommended its use in patients with intermediate risk 10–20%) and all patients with FN antecedents without risk factors [0–20% andG-CSF is a glycoprotein that stimulates granulopoiesis and leads to proliferation, maturation and mobilisation of neutrophils . It is mIn this paper, the authors analyze the role of rhG-CSF. Eventhough, it was firstly emphasised, later it was considered with caution because of the risk of acute lung injury . HoweverBefore the COVID-19 pandemic, the prophylactic use of rhG-CSF  has beenet al. reported that rhG-CSF led to a sustained increase in lymphocyte, but they excluded patients with comorbidities such as cancer from the analysis, to minimise their potential adverse effects on the immune responses of the subjects [Some authors highlighted that the rhG-CSF administration may counteract with COVID-19-related lymphocytopenia and improve the outcome of the infection in patients without cancer . Cheng esubjects .We also found three case reports of agranulocytosis following COVID-19 infection responding to rhG-CSF without apparent adverse events . SpencerFinally, Hernandez described a case with FN successfully managed with a 7-day course of rhG-CSF 53 days from the beginning of COVID-19 infection . The favet al. illustrated the respiratory worsening of three cancer patients within 72 h after rhG-CSF administration [et al. reported a clinical deterioration within 24 h after rhG-CSF administration in a patient with COVID-19 and leukopenia [Soon after the onset of the pandemic, some authors described the possibility of worsening of clinical conditions in cancer patients following the use of rhG-CSF during FN . In partstration . These pstration . Taha etukopenia . Malek sukopenia .rhG-CSF-induced neutropenia recovery may trigger off respiratory deterioration due to acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in neutropenic cancer patients with viral pneumonia . ALI is Therefore, as remarked by Taha, rhG-CSFs therapy might worsen the overwhelming inflammatory reaction in COVID-19 and lead to worse outcomes .et al. have reported a higher risk of hospitalisation, respiratory failure and death in cancer patients recovered from neutropenia through the administration of rhG-CSF [Recently, Zhang  rhG-CSF . In part rhG-CSF .This paper has confirmed our conclusion in the early phase of pandemic  that theThe particular sequence of events, triggered by a hyperinflammatory mechanism, makes COVID-19 management particularly difficult and general rules often do not apply to COVID-19 patients. The complex interplay between cytokines and cellular response interaction with G-CSF metabolism as well as the use of rhG-CSF remains a dilemma in cancer patients with COVID-19 infection. The question remains as to whether it is better to force neutrophil count recovery or to run the risk of severe respiratory complications. The available data are not sufficient to confirm this.Answering this question may be the object of both biological and clinical studies. Identifying the patients' risk by using analytical quick technologies with more sensitive biomarkers of cellular activation is an ideal field for precision medicine research. Targeting G-CSF might be a therapeutic option, and knowledge of the serum level of G-CSF in patients with sepsis might indicate whether its administration may benefit patients with low G-CSF level or harmful for those with a higher G-CSF level . MoreoveAnother research goal is to study different ways of immune stimulation and G-CSF release following short-acting and long-acting rhG-CSF administration to establish which one has better safety profile.In this commentary, we would like to emphasise the importance of obtaining more clinical and preclinical data to understand the complexity of G-CSF and its interaction with the immune system in cancer patients with COVID-19 disease.We recognize as a limitation of this commentary that most of the references are anecdotal case reports. However, we would like to underline that in neutropenic cancer patients with COVID-19 infection, the possible risks versus the benefits of the rhG-CSF administration should be weighed carefully, as rhG-CSF may lead to worsening clinical and respiratory status.The balance between the potential increased use of rhG-CSF in patients with cancer receiving chemotherapy and the concerns raised by some of the studies quoted might be addressed by prospective trials by utilizing a more precision medicine approach.Therefore, big data analysis on a large number of patients may be useful to correlate ANC, inflammatory marker trend and disease outcome.


Digital PCR (dPCR) is the third-generation PCR that enables real-time absolute quantification without reference materials. Recently, global diagnosis companies have developed new dPCR equipment. In line with the development, the Lab On An Array (LOAA) dPCR analyzer (Optolane) was launched last year. The LOAA dPCR is a semiconductor chip-based separation PCR type equipment. The LOAA dPCR includes Micro Electro Mechanical System that can be injected by partitioning the target gene into 56 to 20,000 wells. The amount of target gene per wells is digitized to 0 or 1 as the number of well gradually increases to 20,000 wells because its principle follows Poisson distribution, which allows the LOAA dPCR to perform precise absolute quantification. LOAA determined region of interest first prior to dPCR operation. To exclude invalid wells for the quantification, the LOAA dPCR has applied various filtering methods using brightness, slope, baseline, and noise filters. As the coronavirus disease 2019 has now spread around the world, needs for diagnostic equipment of point of care testing (POCT) are increasing. The LOAA dPCR is expected to be suitable for POCT diagnosis due to its compact size and high accuracy. Here, we describe the quantitative principle of the LOAA dPCR and suggest that it can be applied to various fields. Digital PCR (dPCR) assay, a third-generation PCR method, is the latest PCR method capable of real-time absolute quantification of target genes without reference materials . CommercAs the coronavirus disease of 2019 (COVID-19) has now spread worldwide, diagnostic equipment capable of point of care testing (POCT) in various diagnostic spaces is required ,5. CurreIn 2019, the LOAA dPCR was approved as a class II medical device in South Korea. In addition, it was approved as an emergency use product that Viral Load 20K kit (Optolane) diagnose COVID-19. Here, we describe the principle of the LOAA dPCR, which can be quantified with high accuracy, and the POCT potential of this equipment with various advantages.The LOAA dPCR follows the semiconductor plate method using MEMS technology with excellent performance such as small size, light weight, rapid response, and precise measurement . The MEMMicro-statistics, based on Monte Carlo Simulation , which rIP means the average number of photons. E is the energy value of the photon. K stands for Boltzmann’s constant (1.38 × 10-23 J/K), and T refers to the absolute temperature. When E≫kT, it is expressed as ip represents the probability that there are i target genes per well.The shot noise distribution above can be described by the classical Poisson distribution shown below, where ip increases as the number of wells decreases  in 1 to 22 cycles is judged to be invalid .In the entire PCR cycle, the slope of available well data is generally between 20 and 50. Considering the noise data that occurs at the beginning of the run, the data is excluded if the slope exceeds 10 multiples (‒200 to 500) . In addiDetecting invalid well also involves filtering noise based on the algorithm written in Since the COVID-19 pandemic has occurred, many countries have come to require equipment capable of POCT diagnosis. POCT equipment requires two conditions: size and sensitivity. Compared with existing commercialized qPCR and dPCR devices in the diagnostic market, the LOAA dPCR has a relatively compact size. In addition, the LOAA dPCR has twice the detection sensitivity of DNA and three times the sensitivity of RNA compared to Bio-rad equipment, called gold-standard equipment . TherefoAccuracy and sensitivity of the LOAA dPCR may contribute to the advancement of GMO tests, drug resistance research, and personalized cancer treatment . LOAA dPThe LOAA dPCR has a compact size due to its structural specificity and has high accuracy based on the Poisson statistics. Based on these advantages, it is expected to be utilized in more various fields.


Neuromyelitis optica (NMO), multiple sclerosis (MS) and autoimmune glial fibrillary acidic protein (GFAP) astrocytopathy are idiopathic inflammatory demyelinating diseases (IIDDs) that mainly present as encephalomyelitis. Heparan sulfate (HS) and hyaluronic acid (HA) are two components of glycocalyx, a carbohydrate-rich layer on the surface of blood vessels that mediates interaction with blood. Degradation of glycocalyx in NMO is poorly understood.To detect the serum and cerebrospinal fluid (CSF) levels of shed HS and HA and to correlate these levels with disease severity to determine their diagnostic value.via ELISA.We obtained serum and CSF samples from 24 NMO patients, 15 MS patients, 10 autoimmune GFAP astrocytopathy patients, and 18 controls without non-inflammatory neurological diseases. Soluble HS and HA, and IFNγ, IL17A, and matrix metalloproteinase (MMP) 1 were detected Serum and CSF levels of HS, HA and related cytokines but not of plasma MMP1 were significantly elevated in these diseases. Notably, HS and HA levels were positively correlated with Expanded Disability Status Scale scores.Our results indicate glycocalyx degradation and inflammation in NMO, MS and autoimmune GFAP astrocytopathy. Moreover, increased shedding of HS or HA may indicate a worse clinical situation. Furthermore, therapeutic strategies that protect glycocalyx may be effective in these diseases. Idiopathic inflammatory demyelinating diseases (IIDDs) are devastating neurological diseases that affect both neurological and psychiatric functions and lead to poor quality of life . NeuromyThe blood-brain barrier (BBB) has long been accepted to prevent entry of immune cells and antibodies into the CNS . In IIDDencephalomyelitis, and anti-NMDA receptor encephalitis , which can act on glycocalyx –26. ThusHerein, we focus on the role of glycocalyx in NMO, MS and autoimmune GFAP astrocytopathy. To assess the severity of BBB injury on the severity of these neurological disorders, and to identify potential factors affecting the integrity of BBB glycocalyx, we detected the levels of two glycocalyx molecules, HS and HA, in both CSF and serum. We then correlated these levels with CSF levels of pro-inflammatory factors IFNγ and IL17A, the sheddase, MMP1, and Expanded Disability Status Scale (EDSS)·scores. CSF and plasma concentrations of HS and HA, and levels of CSF IFNγ, IL17A, and MMP1 were significantly elevated in these three idiopathic inflammatory demyelinating diseases. More importantly, the levels of shed glycocalyx molecules in CSF positively affected the severity of NMO, MS and autoimmune GFAP astrocytopathy, and the degree of inflammation may aggravate the disruption of the blood-brain barrier. However, the dynamic concentrations of HS and HA and their relationships with the severity of these disorders remain unknown. Of note, CSF HS and HA may be reliable markers for the diagnosis of NMO. Furthermore, therapeutic strategies focused on preservation of glycocalyx may improve outcomes of these neurological disorders.All subjects were enrolled from the Department of Neurology of Nanfang Hospital, Southern Medical University, China, including 24 NMO patients, 15 MS patients, 10 patients with autoimmune GFAP astrocytopathy and 18 controls without inflammatory or autoimmune neurological diseases . Diagnoses of autoimmune encephalitis were confirmed by two doctors on the basis of diagnostic criteria , 27, 28.We obtained CSF and serum samples from all subjects within 3 days of admission and before immunotherapy therapies were commenced. All samples were centrifuged at 1,000 g for 10 min. Then the supernatant was packed into polypropylene tubes and stored at -80°C until the detection. Take one small tube for each test to avoid repeated freezing and thawing. Enzyme-Linked Immunosorbent Assay (ELISA) kits were used to measure the concentrations of HS , HA , IFNγ, IL17A and MMP1 . All detections were performed in accordance with the manufacturer’s instructions and every standard and sample were assayed in duplicate.All statistical analyses were conducted using SPSS version 24.0 . Data are displayed as the mean ± SD or the median with interquartile range according to normality test results. The Kruskal–Wallis test was used to analyze the differences in CSF HS, CSF HA, SE HS and SE HA levels among the subgroups. The Pearson’s test were used to evaluate the correlations between CSF HS, CSF HA, SE HS and SE HA levels and EDSS scores. The Spearman test was used to evaluate correlations between CSF glycocalyx molecules and other cytokine parameters. p<0.05 was taken as statistically significant. Graphs were plotted using GraphPad Prism 9 . All analyses were performed in a blinded manner.The data from patients with NMO (n = 24), MS (n = 15), autoimmune GFAP astrocytopathy (n=10) and 18 controls are presented in As shown in To assess possible links between CSF and plasma glycocalyx levels and the severity of these three disorders, we examined correlations among them Figure 2 can be found in the article/supplementary material.This study was approved by the Ethics Committee of the Nan Fang Hospital and all the above subjects have signed the informed consent. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.HW conceived this study and designed the experiments. QZ, SP, JC, YP, ZZ and ZW collected the samples and clinical data. QZ, JC, SP performed the experiments, analyzed the data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.Financial support came from the National Natural Science Foundation of China , Guangxi Natural Fund Project (No. 2019GXNSFAA245079), Guangdong Provincial Science and Technology plan projects (2017A020215182), Natural Science Foundation of Guangdong Province (2019A1515011434), and Guangzhou Science and Technology Plan Project (202102020434).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The kinetics of amyloid beta turnover within human brain is still poorly understood. We previously found a dramatic decline in the turnover of Aβ peptides in normal aging. It was not known if brain interstitial fluid/cerebrospinal fluid (ISF/CSF) fluid exchange, CSF turnover, blood-brain barrier function or proteolysis were affected by aging or the presence of β amyloid plaques. Here, we describe a non-steady state physiological model developed to decouple CSF fluid transport from other processes. Kinetic parameters were estimated using: (1) MRI-derived brain volumes, (2) stable isotope labeling kinetics (SILK) of amyloid-β peptide (Aβ), and (3) lumbar CSF Aβ concentration during SILK. Here we show that changes in blood-brain barrier transport and/or proteolysis were largely responsible for the age-related decline in Aβ turnover rates. CSF-based clearance declined modestly in normal aging but became increasingly important due to the slowing of other processes. The magnitude of CSF-based clearance was also lower than that due to blood-brain barrier function plus proteolysis. These results suggest important roles for blood-brain barrier transport and proteolytic degradation of Aβ in the development Alzheimer’s Disease in humans. To understand if brain interstitial fluid/cerebrospinal fluid (ISF/CSF) exchange, CSF turnover, blood-brain barrier function or proteolysis were affected by aging or the presence of β amyloid plaques, Elbert et al. develop a non-steady state physiological model using MRI-derived brain volumes, stable isotope labeling kinetics of Aβ, and lumbar CSF Aβ concentration. Their model suggests an important role for blood-brain barrier transport and proteolytic degradation of Aβ in the development Alzheimer’s Disease in humans. One weakness of the previous analysis was the assumption that the system was at a steady state. As such, the kinetic models could not account for the effects of secretase inhibitors6, or explain the rise in the lumbar cerebrospinal fluid (CSF) concentration of Aβ peptides observed during the 36–48 h SILK studies10. Other existing non-steady-state models do not address the concentration rise observed in SILK studies17.Stable isotope-labeling kinetics (SILK) in humans have previously revealed that: (1) the turnover rate of Aβ20. Ideally, brain tissue or ISF would be sampled. However, such procedures are highly invasive21. Our approach was to use subject-specific MRI-derived CNS compartment volumes to better model transport of CSF from the cranial subarachnoid space (SAS) to the lumbar SAS. Although we do not directly measure or model “glymphatic” fluxes , we can infer these fluxes via the model, because the flux of Aβ transferred from ISF to CSF will equal the flux of Aβ lost from the CSF. CSF is lost from the CNS via arachnoid granulations, dural lymphatics, at the cribriform plate, and down cranial or spinal nerves20. These exit processes, involving bulk fluid flow of CSF, affect the Aβ concentration throughout the SAS, including at the lumbar sampling site. Via the combination of kinetic measurements, MRI volumes and CSF concentration rises upon CSF withdrawal in humans, we can estimate the turnover of Aβ in CSF and thus infer the flux of Aβ from ISF to CSF and the rate of irreversible loss of Aβ from within the ISF.The goal of this approach is to more accurately model Aβ peptide kinetics in the central nervous system (CNS). Myriad processes affect Aβ turnover in the brain, including transport across the blood–brain barrier, enzymatic/lysosomal degradation, interaction and deposition into plaques, and the complex flow and exchange of CSF and interstitial fluid (ISF)22. Others argue that aquaporin-4 deletion does not affect dye penetration into the paravascular space and surrounding parenchyma23. Some have observed that transport through brain parenchyma is molecular-weight dependent suggesting a dominant role for molecular diffusion versus convective flow27. Correctly modeling the tortuous flow in the presence of cardiac-driven pressure fluctuations through the highly cellular cortex and dense extracellular matrix is daunting, and the current approach is desirable as it is largely if not entirely agnostic to the exact mechanism of the “glymphatic” process.Glymphatics is defined by Nedergaard et al. as “astrocyte-mediated transport of CSF and ISF that clears metabolic waste from the interstitial space of the brain parenchyma primarily during non-REM (nonrapid eye movement) sleep and states of high slow-wave activity. This process serves a pseudolymphatic function in the CNS”29. Each heartbeat and breath also induces substantial oscillatory CSF flow32. The complicated flow patterns of the CSF resulting from these pulsations are observable by MRI velocimetry and enhance mass transport in the CNS35. The presence of trabeculations and nerve roots passing through the SAS result in complicated, time-dependent recirculation patterns36. The trabeculations and nerve roots are similar to baffles, which are known in the field of reactor engineering to enhance fluid mixing37. Accurate flow analysis by computational fluid dynamics is impractical without higher-resolution imaging, so the model presented here assumes that oscillatory CSF flow results in the perfect mixing of fluids within distinct compartments in the SAS. This simplification is consistent with our previous compartmental model that fit the SILK data exceptionally well2. The model accounts for changes in lumbar space volume due to the hourly withdrawal of CSF from the lumbar space and potential CSF leaks due to the indwelling catheter, which is perhaps the most important feature of the model.CSF flow in the CNS is generally directed from the choroid plexus to the superior sagittal sinus, although this is a simplification42 in the cortical interstitial fluid38. Thus, changes in the rate of clearance of Aβ42 from the cortical interstitial fluid may be a critical factor in the onset of plaque formation, just as differences in the rate of production of Aβ42 affect the age of onset of Alzheimer’s disease1. In this study, we estimate the rates of clearance of Aβ in cortical interstitial fluid due to different processes, such as transport across the blood–brain barrier, proteolysis, and deposition into plaques. Transport of Aβ42 across the blood–brain barrier is mediated by lipoprotein-related protein 1 (LRP-1)39, leading to the transfer of Aβ42 from the brain interstitial fluid to the blood40. Binding of Aβ40 and Aβ42 to LRP-1 is saturable and clearance across mouse brain capillaries is blocked by antibodies against LRP-141. Expression of LRP-1 declines in aging rats and is negatively correlated with the concentration of Aβ40 and Aβ42 in rat brain cortex homogenates42. Multiple enzymes contribute to the proteolysis of Aβ42 within the CNS, including secreted and membrane-bound enzymes such as an angiotensin-converting enzyme, neprilysin, insulin-degrading enzyme and various MMPs, in addition to degradation downstream of endocytosis or phagocytosis by cells, particularly microglia and monocyte-derived macrophages43. Transport across the BBB, enzymatic degradation and deposition into plaques within the brain will result in similar kinetics for isotope-labeled Aβ, as all processes likely exhibit close to first-order kinetics and occur within the same compartment. In contrast, transfer of Aβ from ISF to CSF will result in elimination of Aβ by processes governed by CSF turnover kinetics. CSF-based processes will affect the shape of the SILK curves differently from processes that occur within the ISF, and this provides the rationale for the current study.It is believed that the rate of nucleation and growth of amyloid plaques depends upon the concentration of Aβ2. Subject demographics, lumbar CSF tau and Aβ concentrations, and results from the previous steady-state model are shown in Supplementary Tables 47. Amygdala, hippocampus, and precuneus volumes declined most dramatically due to β-amyloidosis. Total ventricle volume was increased in amyloid-positive subjects, while the total gray volume decreased. The effects of normal aging on CNS volumes and thicknesses were assessed in amyloid-negative subjects to eliminate effects of β-amyloidosis 53. In addition, a range of CSF volumes was examined to discover the best fit to the observed change in lumbar concentration of Aβ during the course of the 36–48 h SILK experiment. Transfer between compartments was modeled as volumetric flows (mL/h), which were divided by the volumes of the compartments to obtain first-order rate constants for mass transfer between compartments.A new physiological model was developed to decouple transport of Aβ peptides within CSF from 1) transport across the blood–brain barrier, 2) changes in proteolytic degradation, and (3) deposition into plaques. The new physiological model Fig.  assumed  transpor changes QCSF. During the SILK study, 6 mL of CSF were withdrawn every hour from an indwelling lumbar catheter. The withdrawal occurred over 5–10 min. During the hourly CSF collection, the CSF withdrawal rate (QLP) was much greater than the model-predicted CSF production rate (QCSF) in all subjects. It was thus assumed that the volume of the lumbar space (VSP3) decreased during withdrawal 54. Qosc and Qglymph are bidirectional continuous flows that approximate the oscillatory flows due to the cardiac and respiratory cycles. For some subjects with large lumbar CSF Aβ concentration rises, the data were better fit assuming a leak due to catheterization (Qleak). Further details of the model are provided in Supplementary Methods CSF production was assumed to entirely arise from the choroid plexus, specified as the CSF production rate wal Fig. , which h10. However, sleep was not modeled in this study as the sleep-wake cycle was not controlled and was not apparent in 19 out of 96 subjects. In this study, the subjects arose early (how early was uncontrolled) to reach the hospital by 7 a.m. and the study was started at 8 a.m. They were instructed to remain mostly in bed for 36–48 h and could sleep/nap at will. Of the 77 subjects that showed a sleep effect, many showed abbreviated dips, double dips, or prolonged dips in the lumbar Aβ concentration. SILK and concentration-time courses for all 96 subjects are presented in Supplementary Data The lumbar CSF concentration rise of Aβ peptides was substantial Fig. , with a VCSF 100% curve in Fig. Qleak = 0 mL/h curve in Fig. Qleak, which represented continual leakage of CSF around the indwelling catheter. A leak was also assumed for the seven subjects with clinical records of a blood patch to seal a CSF leak. Qleak was not different by amyloid status but did significantly decrease with age  transport across the blood–brain barrier (“B”), (2) proteolytic degradation (“P”), and (3) permanent deposition into plaques (“D”). kBPD is similar to FTR from the steady-state model, but the effects of CSF transport are modeled explicitly, which should allow kBPD to more accurately represent processes that occur in the brain ISF. Like FTR, both kBPD38 and kBPD40 were highly correlated with age in amyloid-negative subjects  was negatively correlated with age in amyloid-positive subjects  but not in amyloid-negative subjects . This implies that changes in Aβ clearance in normal aging are driven by changes in blood–brain barrier transport or proteolysis . Aβ clearance in amyloid-positive subjects appears to be influenced by changes in CSF production, and potentially from the substantial decrease in brain parenchymal volumes and increase in ventricle volume , and  permanenkBPD38 and kBPD40 are significantly lower in amyloid-positive subjects after controlling for age .ge Table . However40 Table . This mits Table  or in amkex42) was significantly higher in amyloid-positive subjects  was determined relative to Vmax,γ40, with Vmax,γ40 fixed for all subjects to a literature-derived value57. Vmax,γ42 was thus normalized by Vmax,γ38 instead of Vmax,γ40. The production rate ratio of Aβ42 relative to Aβ38 by gamma-secretase was elevated in presenilin mutation carriers compared to amyloid negatives and amyloid positives  declined with age, perhaps related to the decline in total gray volume . Qosc is mathematically similar to a diffusion coefficient and higher values will tend to decrease the ISF/lumbar concentration gradient. It is known that the total cerebral blood flow decreases in aging, as does the cervical CSF stroke volume63. This suggests that the enhanced mixing of CSF that decreases the ISF/lumbar gradient in aging is due to biomechanical factors other than CSF pulsatility. The increase in Qosc with age may be similar to the increase in pulse wave velocity with age due to stiffening of the arteries64. Although ISF-to-lumbar gradients can be inferred from experiments, there is scant evidence of a ventriculo-lumbar gradient for Aβ65, in contrast to the negative gradient for brain proteins such as tau and the positive gradient for blood proteins such as albumin66.The physiological model accounts for the oscillatory flow of CSF driven by circulatory and respiratory pulsations. The oscillatory flow was modeled as a continuous bidirectional flow with magnitude ts Table . Qosc inge Table , consist68. Although the study protocol did not include cisternography, the model makes specific predictions about the distribution and disappearance of label within the CNS. This includes a prediction of a “cisternography half-life”, the half-life in the CNS of a labeled compound introduced into the lumbar space. Moriyama et al. studied the effects of age on cisternography half-life and developed a linear regression model67. The Moriyama et al. model predicted a mean cisternography half-life for the subjects in our study of 20 ± 2.4 h, compared to 21 ± 4.5 h predicted by the physiological model. The predicted cisternography half-life was strongly influenced by the exit of CSF from the CNS down spinal nerves and this was used to fix QSN at 10% of QCSF for all subjects. Higher or lower values of QSN resulted in unreasonable values for the predicted cisternography half-life 69. Although the observed increase in Qosc with age would tend to decrease the cisternography half-life, the increase in the total volume of CSF (VCSF) with age seems to negate this effect . Assuming a cellularity of the cortex of ~90%80, this suggests that cortical ISF concentrations are about 10× higher than concentrations measured in the homogenized cortex. The physiological model specifically incorporates the volume of gray matter ISF, and the mean rise in Aβ40 concentration from lumbar SAS to brain ISF ([Aβ40]ISF/[Aβ40]lumbar) was about tenfold and did not differ by amyloid status . However, using microdialysis, the concentration of Aβ in the brain ISF is about the same as in ventricular and lumbar CSF, and this is more than an order of magnitude lower than the value predicted by the physiological model82. Given the dramatic concentration rise observed during sustained withdrawal of lumbar CSF, some fluid within the CNS must have a higher concentration of Aβ and this is likely the ISF. This suggests that microdialysis measurements underreport the ISF concentration of Aβ.The concentrations and fluxes of Aβ peptides within ISF are difficult to measure in humans. The measured Aβ40 peptides from the CNS into blood was previously reported as 9.7 ng/min, which was found by measuring the venous–arterial Aβ concentration difference during inferior petrosal sinus sampling40. This flux should include Aβ transported to the venous system by CSF absorption at the superior sagittal sinus and Aβ transported across the blood–brain barrier. It should not include Aβ transported to the lymphatic system via CSF absorption at the dura, cribriform plate, or via perivascular transport20. The physiological model predicted a mean Aβ40 flux of 15 ± 13 ng/min due to CSF absorption in all subjects according to Eq.   83. The 40 peptides by transport across the BBB or via proteolysis is predicted by the physiological model to be 25 ± 28 ng/min . This i38 and Aβ40 but decreased for Aβ42  ng/h, while BBB + proteolysis flux was 5.5 (0.7) ng/h . CSF-based flux of Aβ40 was 14 (2) ng/h, while BBB + proteolysis flux was 31 (4) ng/h . CSF-based flux of Aβ42 was 2.2 (0.2) ng/h, while BBB + proteolysis flux was 4.5 (0.6) ng/h .The model predicts that the fluxes of Aβ peptides across the BBB and via proteolysis decrease dramatically in normal aging Fig. . However42 Table . The deccts Fig.  is likeles Table . The mag42, a decrease in the BBB/proteolysis fluxes of Aβ42, and an increase in deposition flux of Aβ42  were obtained hourly for the first 16 h and every other hour for the rest of the experiment (36 h). CSF (6 mL) was withdrawn hourly. The deposition of amyloid plaques was quantified by [11C]PIB-PET in 77 subjects. Subjects with mean cortical binding potential (MCBP) score >0.18 were considered to be amyloid positive. If PET PIB was not available, a lumbar CSF concentration ratio Aβ42/Aβ40 < 0.12 was considered to be amyloid positive. PET PIB scans were performed within 3 years before or after the SILK tracer study date. MRI scans (3 T volumetric T1-weighted) were processed using FreeSurfer version 5.3 as previously described86. FDA, Center for Devices and Radiological Health, and IT’IS Foundation collected the MRI data used to generate the images in Fig. 87.SILK data collection procedures and PET PIB measurements were previously describedg l-U-13Cleucine w2. Briefly, Aβ peptides were immunoprecipitated with a mid-domain antibody, in the presence of isotope-labeled internal standard peptides. Concentrations of peptides and fractional isotope labeling were quantified by LC-MS/MS analysis on a Xevo TQ-S (Waters) or TSQ Vantage (ThermoScientific) mass spectrometer. Isotope-labeled leucine was captured from the blood by cation exchange chromatography. The N-heptafluorobutyryl n-propyl ester derivative was quantified by GCMS (Agilent 6890N GC and Agilent 5973N Mass Selective Detector).Sample processing and mass spectrometric measurements were previously describedModel equations were solved numerically in Julia as described in Supplementary Methods kBPD38, kBPD40, kBPD42, SF38, SF40, SF42, Qosc, QCSF, kex42. Other parameters were estimated as follows:QCSF, which was assumed to arise solely from the choroid plexus. Extravasation of fluid from the capillaries of the brain parenchyma was not considered in the model. It was assumed that the concentration of Aβ peptides in newly formed CSF was zero.The volumetric rate of CSF production was VISF represents the volume of brain gray matter ISF, which was 10% of MRI-derived total gray volume. Vcranial is the volume of the cranial SAS. VCV is the volume of the cisterns and ventricles, which are assumed to be in intimate communication due to oscillatory flow. Nominally, the cervical spinal volume is VSP1, the thoracic spinal volume is VSP2, and the lumbar spinal volume is VSP3.VLP/tCSF draw= 60 mL/h, where VLP is 6 mL and tCSF draw is 0.1 h. It was assumed that the net caudal flow could not exceed QCSF during CSF withdrawal. If VLP/tCSF draw exceeded QCSF, which it did for all subjects, it was assumed that the excess flow came exclusively from a decrease in volume of the lumbar space (VSP3), which has been observed in humans during lumbar puncture51. The parameter QLP represented net caudal flow during withdrawal and refill, while Qrefill was used to account for volumetric changes in the lumbar compartment.CSF was withdrawn from the lumbar catheter at the start of each hour and the model assumptions are summarized in Fig. QCSF was used to refill the lumbar volume to its natural dimensions. The time required to refill the lumbar space was ΔVSP3/QCSF.Following CSF withdrawal, it was assumed that the entirety of QLP = 0 for the rest of the hour. However, if a CSF leak was suggested by the lumbar Aβ concentration data, then QLP = Qleak, where Qleak is a constant during the entire 36–48 h SILK experiment, unless otherwise modified to better fit the concentration rise data. The maximum value for Qleak was assumed to be QCSF. During CSF withdrawal and lumbar space refilling, it was assumed that the drop in pressure halted the leak, i.e., Qleak = 0 during withdrawal and refilling.After the lumbar space was fully refilled, Qleak during the study. This might have occurred if the catheter shifted due to a change in the subject position. In these cases, an initial constant value of Qleak was changed to another constant value at a certain time to better fit the lumbar Aβ concentration data.Some concentration-time courses suggested step changes in QSN, was modeled as a net flow of CSF out of each spinal SAS compartment. It was assumed that QSN was divided equally between cervical, thoracic and lumbar compartments. The full flow entered the cervical space, but 1/3 QSN left down spinal nerves within the cervical space, while 2/3 QSN continued into the thoracic compartment. In the thoracic space, 1/3 QSN left down spinal nerves within the thoracic space, while 1/3 QSN continued into the lumbar space. The final 1/3 QSN left down spinal nerves in the lumbar space. During removal of CSF and refilling of the lumbar space, it was assumed that the drop in pressure completely halted loss of CSF down spinal nerves (QSN = 0). The model-predicted cisternography half-life was sensitive to QSN. QSN was varied between 5 and 20% of QCSF. The Moriyama et al. model predicted a mean half-life for all subjects of 20 ± 2.4 h67. The mean cisternography half-life predicted by the current model best agreed with the Moriyama et al. prediction when QSN equaled 10% of QCSF  was Qosc and Qglymph represent the oscillatory CSF flow generated by the cardiac and respiratory cycles but are modeled as steady bidirectional flows instead of pulsatile flows due to computational and MRI data constraints. In both cases, mass transport would only occur in the presence of a concentration difference between compartments.42 and was implemented as in the previous steady-state model3.The exchange compartment was only relevant to Aβ38, SF40, and SF42). These are believed to account for instrument calibration errors and were previously described3.The SILK isotope-labeling data were also scaled by three additional free parameters, the scaling factors , Vmax,gamma38 and Vmax,gamma42 were determined from solution of the steady-state equations. An example of labeling kinetics from plasma leucine to the lumbar SAS is shown in Supplementary Fig. The Michaelis–Menten rate constants for conversion of APP to C99 + C83 and C99 to Aβ79, which are both topologically extracellular. Because the Michaelis–Menten rate constants were determined in cultured cells in a relative manner56, and for beta-secretase kinetics in an absolute manner in human temporal cortex preparations57, it was assumed that all of the rate constant values were applicable to the neuronal plasma membrane found in cortical tissue. The volume of ISF was assumed to be 10% of the total gray volume80, and the total gray volume was determined for each subject by MRI . In pilot studies, when Qglymph was decreased from QCSF to QCSF/2, the lumbar-to-ISF concentration rise increased from 9.6 ± 6.9 to 14 ± 11. Further decreasing Qglymph by setting it equal to Qosc (slightly less than QCSF/2) resulted in a lumbar-to-ISF concentration rise of 71 ± 130. Given the similarity between Aβ concentrations measured in homogenized cortex and CSF, lower values of Qglymph were ruled out and Qglymph was set equal to QCSF for all subjects. The governing assumption was that soluble Aβ exists entirely in ISF and that homogenization of cortical tissue dilutes the ISF about tenfold with intracellular fluids, thus decreasing the measured concentration by a factor of 10. With this assumption, cortical ISF concentrations are predicted to be tenfold higher than those measured in the homogenized cortex. In the final model with Qglymph = QCSF, the mean lumbar-to-ISF concentration rise was 10 ± 6.6.The parameter SP3/dt), isotopically labeled and unlabeled peptides were modeled separately. The APP production rate (kf) was scaled by fLeu (the fraction of labeled leucine) for production of labeled APP, and scaled by (1 − fLeu) for production of unlabeled APP. It was assumed that the fraction of labeled leucine in ISF was the same as the fraction of labeled leucine in plasma, which was measured hourly for the first 13 h, then at 17 and 35 h. The tail of the plasma leucine labeling curve was fit to an exponential model2.For each equation shown (except for dVThe parameters that were fit to the model were: t test with unequal variances or ANOVA with Tukey post hoc analysis. Errors are standard deviations except where noted. Box plots show: center line = median; box limits = upper and lower quartiles; whiskers = 1.5× interquartile range; points = outliers. Confidence interval region = 95%. Measurements were taken from distinct samples. Replicates are biological replicates.Statistical analyses were performed in R and comparisons were by two-sided Further information on research design is available in the Supplementary MaterialsDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


The high misdiagnosis rate of asymptomatic neurosyphilis (ANS) has long challenged infectious disease clinicians. We aim to develop a model for diagnosing ANS in asymptomatic syphilis (AS) patients without CSF indicators.277 AS patients with HIV-negative and underwent lumbar puncture were enrolled in this horizontal study.The area under the curve for predicting ANS by CSF leukocytes and protein was 0.643 and 0.675 . Through LRM, the AUC increased to 0.806 , and the Youden's index was 0.430. If the score is ≤ 0.159, ANS can be excluded with a predictive value of 92.9%; we can identify ANS while the score is over 0.819, with a predictive value of 91.7% and a specificity of 99.25%. This study showed that the LRM can diagnose ANS in AS patients effectively.Given a large number of misdiagnosis ANS patients and CSF results' insufficiency, the model is more practical. Our research will help clinicians track suspected syphilis, especially those who cannot accept the CSF test. Treponema pallidum infection and is a serious global health issue , P < 0.001), gender , WBC , NEUT/LY , NEUT , NEUT% , LY% , MO , EO , EO% , MPV , serum TRUST titer  associated with ANS. In order to establish the diagnostic model for ANS in AS patients, collinearity diagnostic was performed on the mentioned indicators that are meaningful and differentiated. Results demonstrated that WBC, NEUT, NEUT%, EO, EO% have multicollinearity (Variance Inflation Factor > 5). After principal component analysis, dimensionality reduction processing, then all indicators were included in the bivariate regression analysis. The results show that age , MPV , s-TRUST , EO , and WBC  associated to the occurrence of ANS independently. Subsequently, we established the following LRM based on the above parameters: Y = 0. 037 × age + 0. 451 × MPV + 0. 038 × s-TRUST-9. 871 × EO + 0. 443 × WBC, the goodness-of-fit test of logistic regression model by Hosmer–Lemeshow shows that the model has a good fit with the observed values, P = 0.581. A forest plot was then produced to offer a visualization of the relationship among the ANS risk-related factors identified in the previous group analysis  positively , 33, 34.WBC count and protein concentration of CSF is the most used for syphilis nervous system infection. Similar to previous studies , our resDespite the large sample size, this research is restricted by its single-centre, retrospective design. In fact, we did not dynamically monitor changes and could not determine the syphilis staging of all events, although repeated syphilis was more likely to be asymptomatic . Future In summary, the long-term consequences of untreated ANS are high morbidity and mortality. Considering the limitations of CSF results and LP risks, it will benefit from using the convenient LMR in patients diagnosed with AS. This basis considers empirical therapy for patients with a high pre-test probability of neurosyphilis without routine CSF analysis.


CSF orexin A concentration showed a correlation with Mini-Mental State Examination scores, drug assumption, history of compulsive behaviour and extrapyramidal signs. Moreover, we found a relationship between CSF markers of neurodegeneration, total tau and Aβ1–42 and CSF orexin A concentrations. Our study provides evidence of an orexinergic dysfunction in bvFTD, correlating with several clinical symptoms. Further larger studies are needed to confirm our data.Orexins are hypothalamic neuropeptides that regulate several physiological functions, such as appetite, arousal, cognition, stress, sleep and metabolism. Emerging pieces of evidence suggest an orexinergic dysfunction in several neuropsychiatric disorders, including depression, anxiety and addiction. A syndromic overlap between behavioural variant frontotemporal dementia (bvFTD) and several psychiatric disorders was recently demonstrated. Therefore, we analysed cerebrospinal fluid (CSF) orexin A concentrations of 40 bvFTD and 32 non-demented patients, correlating neuropeptide concentrations with several clinical characteristics. A significant increase of orexin A concentrations was found in bvFTD patients when compared to controls (The online version contains supplementary material available at 10.1007/s10072-021-05250-x. Behavioural variant frontotemporal dementia (bvFTD) is the most common clinical variant within the spectrum of frontotemporal lobar degeneration syndromes and is characterized by personality changes, apathy, decline in socially appropriate behaviour, and empathy. These symptoms reflect progressive deregulation of the neural circuits involved in social cognition, emotion regulation, motivation and decision making .Orexins (orexin A and orexin B) are two neuropeptides synthesized by hypothalamic neurons with widespread projections throughout the central nervous system. Orexins regulate several physiological functions, like appetite, arousal, cognition, stress, sleep and metabolism. Several studies suggested a role for orexins in psychiatric disorders, including addiction, depression and anxiety . MoreoveStudies about orexins in bvFTD are few and not conclusive. A study with magnetic resonance imaging (MRI) of early-FTD patients and post-mortem analyses of bvFTD cases showed significant atrophy of the posterior hypothalamus with orexinergic neuron sparing . In a smOrexinergic system dysregulation could represent a neurobiological basis of certain clinical and behavioural features of patients with frontotemporal dementia. Thence, considering the above-mentioned observations, the purpose of this study was to better investigate the role of orexinergic system in bvFTD through the measurement of CSF orexin A concentrations and the evaluation of possible correlations with clinical symptoms.18fluorodeoxyglucose (18FDG-PET) were available. All patients underwent lumbar puncture with measurement of CSF levels of orexin A, Aβ1–42, p-tau and t-tau. Genetic variants in the MAPT, PGRN and C9orf72 genes were excluded. Cognitive functions were assessed by a standard neuropsychological evaluation, including Mini-Mental State Examination (MMSE). Detailed clinical history was recorded particularly focusing on cognitive and behavioural characteristics, drugs assumption and cardiovascular risk factors. A group of 32 cognitively healthy subjects  examined for neurological conditions other than dementia (e.g. suspected polyneuropathy or multiple sclerosis) was used as controls. A complete description of the above-mentioned clinical procedures is available in the A group of 40 bvFTD consecutive patients , evaluated and treated at the Department of Neuroscience of University Hospital “Città della Salute e della Scienza” of Torino and at the Department of Neurology and Neurorehabilitation of “S. Giuseppe” Hospital, Piancavallo, Italy, were recruited for the study. Diagnosis of probable bvFTD was made according to Rascovsky et al. criteria [All CSF samples were obtained by lumbar puncture, using an atraumatic needle, performed early in the morning (between 9.00 and 11.00 a.m.) after overnight fasting [t-Tau, p-Tau181 and Aβ1-42 levels were measured separately, in duplicate, using commercially available sandwich enzyme-linked immunosorbent assays (ELISA) kits  according to the manufacturer’s instructions. Intra-assay and inter-assay coefficients of variation were 3.2% and 11.5% for t-tau, 1.7% and 11.4% for p-tau, 4.6% and 7.8% for Aβ1–42. The CSF Orexin levels were detected with the commercially available EIA kit , based on the principle of competitive enzyme immunoassay. The intra-assay and inter-assay coefficients of variation were 7.5% and 5.8%, respectively. All samples were assayed in duplicate.The CSF t-test or its non-parametric variant (Mann-Whitney test), while differences of categorical variables by the Chi-square test with Yate’s correction. Correlation between continuous variables was analysed through Pearson or Spearman  correlation. Multivariate generalized linear models (GLMs) were performed to estimate the quantitative relationship between CSF orexin A concentration and other variables considered as independent predictors. All analyses were run with R software (www.r-project.org). The level of statistical significance was defined at p < 0.05.Continuous variables were described as mean and standard deviation or medians and range, and categorical ones as count and percentage. D’Agostino-Pearson’s test was used to assess the normality of included variables. Differences of continuous data between groups were studied by the unpaired p < 0.001) .We found increased CSF orexin A concentrations in bvFTD patients (mean: 0.226 ng/mL ± SD: 0.103) compared to controls (mean: 0.139 ng/mL ± SD: 0.058), and this resulted statistically significant (p < 0.001), while age at lumbar puncture and disease duration did not correlate with orexin A concentrations. In multivariate analysis, the presence of repetitive/compulsive behaviours as well as extrapyramidal signs was associated with increased CSF orexin A concentrations. Finally, neurodegenerative biomarkers of dementia, CSF t-Tau and Aβ1–42 concentrations co-variated with orexin A concentrations, though in the reciprocally opposite sense.In addition, several clinical and demographic variables emerged as significantly linked to CSF orexin A concentrations in the bvFTD group Table . We founn = 11) with controls. However, this study was conducted in patients with a diagnosis of FTD syndrome, while our study included only bvFTD patients according to Rascovsky criteria.In this study, we found that CSF orexin A concentrations are significantly increased in bvFTD patients in comparison to controls. In addition, we found that neuropeptide concentration correlated with several clinical characteristics of the disease, like repetitive/compulsive behaviour and extrapyramidal signs. Finally, a correlation between markers of neurodegeneration and CSF orexin A concentrations was found. Our data are in accordance with a previous study  showing Increased orexin A concentrations in bvFTD patients observed in our study may have several neurobiological explanations as well as important clinical implications. Interconnections between cortical areas and orexinergic neurons are increasingly under investigation, especially with concern to feeding and addiction disorders . The preWe found an intriguing correlation between repetitive/compulsive behaviour and CSF orexin A concentrations. Several animal-model studies showed suppression of compulsive behaviours linked to abuse substances by orexin receptor antagonism . Althoug1–42 and total tau. Preclinical and clinical studies suggested a role for Aβ1–42 in regulating amyloid clearance and, consequently, Alzheimer’s disease progression [t-tau reinforces a role for this neuropeptide in neurodegenerative processes. Further studies are warranted in order to evaluate the potential role of orexin A as a biomarker of bvFTD.Finally, we found that CSF orexin A concentrations correlated with markers of neurodegeneration like Aβgression . FurtherOur study certainly has some limitations. For example, the low number of enrolled patients must be acknowledged. However, even if preliminary, correlations between orexin A concentrations and cognitive status, as well as motor and psychiatric symptoms, deserve additional investigations. To our knowledge, this is the largest clinical series demonstrating an increase of orexin A concentrations in cerebrospinal fluid of bvFTD patients. Moreover, it was possible to report several associations with the clinical characteristics of the disease. Better cognitive performance appeared to be a predictor of lower orexin A, thus reinforcing the hypothesis of a deregulation of this neuropeptide in the disease. Regarding specific neuropsychiatric manifestations, we found significant orexin A variations when independently assessing for a history of compulsive behaviours or extrapyramidal signs. In both cases, a possible explanation is based on orexinergic physiological functions and interconnections with other neurotransmitter systems. Finally, also neurodegenerative process, as delineated by CSF markers, emerged as an independent predictor of orexin A variability in this study, suggesting a complex role of neuronal loss in deregulating the cognitive-behavioural functions related to orexinergic system.ESM 1(DOCX 25 kb)


Background: Perineal groove is a very rare congenital malformation that usually occurs in females. It has been described as a wet, un-epithelialized mucus tissue extending from the posterior vaginal fourchette to the anterior anal opening. It is generally asymptomatic and self-limited. Due to its rarity, it is often unfamiliar to clinicians, often leading to a missed diagnosis or unnecessary interventions. Methods: During the period from September 2017 to September 2020, four patients (two newborns and 2 infants) were diagnosed with perineal grooves. They were referred to the pediatric surgery clinic because of abnormal findings during their genital examinations. During the same period of time, the clinic received 12 other new patients with various forms of anorectal malformations. Results: All four patients were girls. During examinations by their respective pediatricians, an abnormality in the perineum was noted in each of the patients. The depth of the grooves varied between the four patients and extended from the anus to the vaginal fourchette. None of the patients experienced any other symptoms related to the malformation. All the cases were referred to the pediatric surgeon by their pediatricians as a diagnosed anal fissure or abnormal finding in the genitalia. In all four patients, the mothers did not take folic acid during the pregnancy. The four patients were observed, and no surgery was needed; in three of the cases, there was a complete spontaneous resolution while the fourth patient still remains under observation. Conclusions: Perineal groove is a rare malformation with a low incidence rate. It is more common in female infants and usually self resolves before the age of 2 years. The condition is diagnosed on clinical examination; however, it is easily misdiagnosed and may lead to unnecessary interventions and surgery. Perineal groove is a very rare congenital malformation that usually occurs in females. It is part of a broader category of malformations; the incidence rate of anogenital malformations remains underestimated. It has been described as a wet, un-epithelialized mucus tissue that extends from the posterior vaginal fourchette to the anterior anal opening. It is generally asymptomatic and self-limited. Due to its rarity, perineal groove is often unfamiliar to clinicians and can in turn lead to a missed diagnosis and even unnecessary interventions. The causes behind the malformation remain unclear, and few cases have so far been reported. In this study, we aim to highlight this problem by reviewing the literature and exploring the four cases of perineal groove diagnosis in the pediatric surgery clinic from September 2017 to September 2020.During the period from September 2017 to September 2020, four patients (two newborns and 2 infants) were diagnosed with perineal grooves. They were referred to the pediatric surgery clinic because of abnormal findings during their genital examination. During the same period, the clinic received 12 other new patients with various forms of anorectal malformations.All the four patients were girls  and an aThe four patients were observed, and no surgery was needed. Three of the patients experienced a complete spontaneous resolution while the fourth is still under observation.A 7-day old female patient was referred by her neonatologist with a suspicion of a congenital anal fissure. During examination we found a perineal groove. She was born to a 26-year-old gravida 1 para 1 woman who did not take folic acid during her pregnancy. The patient was not noted to have any other associated anomalies during her exam.She was followed up regularly for 2 years, and it was noted that the perineal groove spontaneously resolved.A 15-day old female also with no other associated medical issues was referred by her neonatologist with an abnormality of the anogenital area. We diagnosed the patient with perineal grove in our clinic. She was born to a 38-year-old woman gravida 3 para 2. The mother did not take folic acid during her pregnancy.We conducted follow-ups for 18 months, and it appeared that the groove underwent a spontaneous resolution.A 2-month-old female patient presented with a perineal groove. The patient was already known to have a mild ventricular septal defect (VSD). She was born to a 40-year-old mother gravida 2 para 2. The mother did not take folic acid during her pregnancy.We conducted follow-ups for 18 months. The VSD naturally closed at 9 months of age. The perineal groove also spontaneously resolved at 16 months of age.A 6-month-old female was referred to the clinic by her pediatrician due to abnormal findings in the genitalia.A perineal groove was found during examination. She was born to a 31-year-old woman gravida 3 para 2 who had not taken folic acid during her pregnancy.The patient had been observed for 6 months. The perineal groove has not yet been resolved; the patient remains under observation, though there is no plan for surgery at this point.A perineal groove is a rare entity of anorectal malformations that occurs nearly exclusively in female patients, with only two reported cases in male patients ,2. TherePerineal groove has been described as a wet red sulcus extending from the posterior vaginal fourchette to the anterior anal opening. Usually, it is an isolated finding, but it may be associated with other urological or genital defects such as imperforate anus, perineal ectopic anus, hypospadias or rectal prolapse .The occurrence of perineal groove in male infants points towards its development during the embryological stage. There are at least four different theories regarding its development: (1) failure in the developmental stage of the external genitalia, (2) abnormal fusion of the median genital fold, 3) remnant of the cloacal duct due to a defect in the urorectal septum ,5,6, (4) remnant The types of the perineal grooves have been divided into complete and incomplete. The complete perineal groove extends from the vaginal fourchette to the anus, while the incomplete type either extends from the vaginal fourchette to the middle of the perineum or from the anus to the middle of the perineum .Three criteria need to be met in order to define this malformation: (1) wet groove in the perineum; (2) normal anatomy of the vestibular organs: both vagina and urethra; 3) hypertrophy of minoral tails  hypertro,7.Patients with perineal grooves are generally asymptomatic ; howeverPerineal groove is a clinical diagnosis, with a biopsy only rarely performed. When conducted, the histological findings of the biopsy specimens exhibit non-keratinizing squamous epithelium with an intervening area of rectal type or transitional epithelium . While oIt is important to properly diagnose perineal groove as the main treatment is conservative observation . Within Perineal grooves are noted as anogenital malformations . HistoloThe benefits of folic acid during early pregnancy in the prevention of neural tube defects is well documented ,13. It iPerineal groove is a rare malformation with a low incidence rate. It predominantly affects female infants and usually self-resolves before the age of 2 years. The condition is diagnosed on clinical examination; however, it is easily misdiagnosed and can thus lead to unnecessary interventions and surgery.Further studies are needed to identify the relationship between a mother’s folic acid intake and a reduction in perineal groove incidences