Although the predictive power of demographic, clinical, genetic, and imaging features for amyloid positivity has previously been investigated, it is unknown whether these features differentially predict amyloid-β status based on PET or CSF or whether this differs by disease stage.Amyloid-β PET and CSF Aβn = 194), mild cognitive impairment , dementia  with amyloid-β PET and CSF Aβ42 measurement within 1 year. Ninety-seven (13%) patients had discordant PET/CSF amyloid-β status. We performed parallel random forest models predicting separately PET and CSF status using 17 patient features tau, cognitive performance, and MRI visual ratings) in the total patient group and stratified by syndrome diagnosis. Thereafter, we selected features with the highest variable importance measure (VIM) as input for logistic regression models, where amyloid status on either PET or CSF was predicted by (i) the selected patient feature and (ii) the patient feature adjusted for the status of the other amyloid modality.We included 768 patients , MCI , and dementia , but not for CSF-amyloid. APOE4  was associated with CSF-amyloid in SCD, while it was only predictive for PET-amyloid in MCI . Worse MMSE scores  were associated to CSF-amyloid status in SCD, whereas worse memory  only predicted PET positivity in dementia.APOE4, CSF tau, and p-tau had the highest VIM for PET and CSF in all groups. In the amyloid-adjusted logistic regression models, p-tau was a significant predictor for PET-amyloid in SCD (OR = 1.02 [1.01–1.04], Amyloid status based on either PET or CSF was predicted by different patient features, and this varied by disease stage, suggesting that PET-CSF discordance yields unique information. The stronger associations of both APOE4 carriership and worse memory z-scores with CSF-amyloid in SCD suggest that CSF-amyloid is more sensitive early in the disease course. The higher predictive value of CSF p-tau for a positive PET scan suggests that PET is more specific to AD pathology. As the median CSF Aβ42 values of our cohort have been gradually increasing over the years Florbetaben PIB Flutemetamol Florbetapir [n = 24, 3%). PET scans were rated as positive or negative based on visual read by an expert nuclear medicine physician (BvB). PET scans were performed, on average, within 54 (± 75) days of the lumbar puncture.Amyloid-β PET scanning is not part of standard diagnostic process in the Amsterdam Dementia Cohort. Patients underwent an amyloid-β PET for research purposes in the vast majority –44 or otrbetaben , 44 ) [n = 127 (17%)), and dementia (n = 447 (58%)). Within the dementia group, 309 (69%) patients had the diagnosis of Alzheimer’s disease, 66 (15%) a diagnosis within the frontotemporal dementia spectrum, 22 (5%) dementia with Lewy bodies, 6 (1%) vascular dementia, and 44 (10%) other dementia syndromes. Patient diagnosis was determined without knowledge of PET or CSF status. To reflect the information provided to the models in our analysis, we present patient group characteristics based on the binarized amyloid-β status on PET and CSF: concordantly positive (PET+/CSF+) or negative  or CSF (PET/CSF+).We stratified the patients based on syndrome diagnosis: subjective cognitive decline ) , mild cot tests, Wilcoxon rank-sum tests, and linear regression models with Bonferroni correction for group-wise testing. Cognitive scores were compared while adjusting for age, sex, education, and syndrome diagnosis.Statistical analysis was performed using R software (version 3.4.4) . When prmice library [APOE ε4 positivity, CSF tau, and p-tau), cognitive measures , and MRI scores . Accuracy, sensitivity, and specificity of the random forest models were evaluated using the mean out-of-bag (OOB) error estimates. Using this method, the performance of every tree in the random forest model is evaluated on the approximately 37% of observations that are not used for its training, allowing a means to train the model and perform analysis in the same dataset [All subsequent analyses were performed in the total patient group as well as in the syndrome diagnosis groups of SCD, MCI, and dementia. We first summarized the relative predictive power of every variable in predicting PET and CSF amyloid-β status using random forest modeling. We performed random forest modeling to (i) get an estimate of the predictive power of variables in a setting, where all variables are present in the model; (ii) compare the importance of variables between models predicting PET and CSF amyloid-β status; and (iii) select patient features for multivariable logistic regression models. As classifier models are affected by missing data, we accounted for missing values using multiple imputations -based permutation variable importance measure (VIM) to estimate the relative predictive power for every patient feature. This measure was selected because of its higher accuracy in datasets with an unbalanced outcome class  and we etj denotes the area under the curve computed in the OOB observations in the selected tree before permuting predictor j, and (3) t after randomly permuting predictor j [where (1) ntree denotes the number of trees in the forest whose OOB observations include observations from both outcome classes, (2) AUCdictor j . As the dictor j , 55, 56.For the second stage of the analysis, we selected patient features based on their predictive value in the random forest models. Similar to a previous study , we inclAPOE ε4 positivity, CSF tau, and p-tau, we compared these multivariable logistic regression models to a univariate logistic regression model, where PET or CSF status was predicted only by the status of the other amyloid modality. We calculated the difference in Akaike Information Criterion (AIC) between the two models to investigate the change in model fit. A decrease in AIC between models can be interpreted as some (0–2), considerable (4–7), or strong (> 10) evidence for gain in model fit in favor of the second model [Finally, as confirmation for our main findings for nd model .We calculated the odds ratios (OR) with corresponding 95% confidence intervals for every patient feature both in the original dataset and in the 25× imputed datasets. Non-overlapping confidence intervals were considered significantly different. We used the false discovery rate (FDR) correction with a significance level of 0.05 to account for multiple testing .n = 30), 13% in MCI (n = 17), and 11% in dementia (n = 50). Of the discordant group, 67% (n = 20/30) of SCD, 53% (n = 9/17) of MCI, and 72% (n = 36/50) of dementia were PET−CSF+.In total, 32 patients (4%) were discordantly amyloid-β positive based on PET and 65 (8%) based on CSF. The proportion of PET/CSF discordance was 15% in SCD  for the total group are shown in Fig. n = 589, Additional file Additionally, in a subanalysis in the total patient group excluding patients with concordantly negative amyloid status and MCI/dementia, CSF p-tau was the most important predictor for PET but not for CSF  decrease of AIC compared to univariate logistic regression models, where PET or CSF status was predicted only by the status of the other amyloid modality  whether these features have a different association with PET or CSF and (ii) whether this differs per disease stage. We found significant differences in the predictive strength of patient features for amyloid-β status based on PET or CSF. For example, CSF tau and especially CSF p-tau consistently showed a stronger association with amyloid-β status on PET. Additionally, the differential predictive pattern was influenced by the extent of cognitive impairment, as CSF tau was more important in SCD and MCI, while CSF p-tau became more important in the stage of dementia. Moreover, 42 production [42 [42 analysis might be more sensitive, as the decrease in the concentration of soluble isoforms might precede fibrillar amyloid-β plaque deposition detectable by PET [The idea to study differences in the predictive strength of patient features for PET/CSF amyloid-β status was based on the differences in characteristics of patients with discordant amyloid-β biomarkers, which have been theorized to be caused by various factors. Possible explanations for the discordance include individual variances in CSF Aβoduction , the comoduction , differeoduction , or a vaoduction , 63, inction [42 . It has e by PET . Overalle by PET . These re by PET .42 capturing an earlier stage amyloid-β preceding tau pathology. This was reflected by the predictive patterns in the multivariable logistic regression models: when predicting PET status by CSF status, CSF (p)tau adds information about the added burden of disease (including advancing from CSF+PET− to CSF+PET+). When predicting CSF amyloid-β positivity, however, the existence of amyloid-β pathology on PET already provides sufficient predictive power, of subjects already having reached a later stage in amyloid deposition. Overall, although the exact cause of this finding remains unclear, it supports the notion that PET detects more advanced stages of AD pathology, being in accordance with previous work by others [Our main finding was that CSF p-tau and tau had a stronger association to amyloid-β based on PET compared to CSF. If we assume that CSF is a more sensitive modality for amyloid-β pathology, then the weaker association with tau could be explained by CSF Aβy others . Althougy others , the resy others –68. Anoty others , as our ome predictive power for amyloid-β pathology for both PET and CSF. Of them, the biological factors APOE ε4 carriership, CSF tau, and p-tau were most consistent in having significant predictive ability amyloid-β status irrespective of the modality. These findings are not unexpected, as APOE ε4 carriership [Although we focus on the relative differences between PET and CSF, it should be emphasized that in the majority of cases these two modalities contain similar information. This was demonstrated by our finding that many of the selected patient features had similarly sriership , 70, 71 riership , 72 are riership .42 values, causing some loss of information, which could influence our results. Finally, this patient group did not have CSF Aβ40 values available, which have been shown to correct for the individual variation in the production of amyloid-β [The main strength of our study is the large number of patients with both amyloid-β modalities from a well-characterized cohort. Nevertheless, there were still a limited number of patients with discordant amyloid status, which could influence the reliability of our findings, especially when performing subgroup analysis. Another limitation is that due to the stratification by syndrome diagnoses, the outcome of amyloid-β positivity was not equally prevalent. Our results in the multivariable logistic regression models might be influenced by the high concordance rate between PET and CSF status, although the results are supported by similar findings in the random forest models and by the decrease in AIC compared to models using only the other amyloid modality as predictors. Additionally, the included patients underwent amyloid-β PET scans with four different radiotracers, allowing for variability in thresholds for amyloid-β positivity. However, this effect is likely reduced by all of the PET scans being visually rated by the same experienced nuclear medicine physician. As continuous measures for PET imaging were not available, we dichotomized CSF Aβmyloid-β , 75.Our findings can be summarized by a hypothetical model highlighting the relative predictive power of patient features towards amyloid-β status based on PET and CSF Fig. . This moIn this exploratory work, we demonstrated that although various patient features have general predictive value towards amyloid-β status, there are finer differences revealed by discordant cases between the predictive pattern for amyloid-β status based on PET and CSF. This indicates that PET-CSF discordance might include valuable information on underlying clinical and neuropathological differences.Additional file 1: Table S1. Proportion of missing values per patient feature. Table S2. Out-of-bag accuracy, sensitivity and specificity for random forest models predicting amyloid PET and CSF status. Figure S1. Relative predictive power of patient features for amyloid PET and CSF status when removing PET-CSF- MCI and dementia patients. Table S3. Predictive value of patient features for amyloid status based on PET or CSF. Table S4. Amyloid-adjusted predictive value of patient features for amyloid status based on PET or CSF.


CSF flow disorders are frequently encountered in children. The advent of MR technology with the emergence of new pulse sequences allowed better understanding of CSF flow dynamics. In this pictorial review, we aim to conduct a comprehensive review of the MR protocol used to study CSF flow disorders and to discuss the utility of each pulse sequence in the adopted protocol. We will focus on the key anatomical structures that should be examined to differentiate hydrocephalus form ventricular dilatation ex-vacuo. The MR features of obstructive and communicating hydrocephalus will be discussed, in addition to the manifestations of CSF disorders associated with posterior fossa malformations . Moreover, the value of MRI in the assessment of patients following interventional procedures  will be addressed.The online version of this article (10.1186/s13244-019-0686-x) contains supplementary material, which is available to authorized users. MRI is the ideal modality to investigate CSF flow disorders in children.Our practical illustrated protocol based on 3D high-resolution T2-WI and phase contrast along with conventional sequences helps to recognize different entities of CSF flow disorders in children.Most cases of hydrocephalus are related to obstruction with the aqueduct of Sylvius being the commonest site.The advent in MRI technology has led to better understanding of CSF flow disorders which are frequently encountered in children , 2. FlowThe MRI protocol should include brain imaging with T2-weighting in axial and coronal planes, axial fluid attenuation inversion recovery (FLAIR), and sagittal T1-weighted image (T1-WI) Figs. . Post-coCine phase-contrast sequence can demonstrate CSF pulsatile flow throughout the cardiac cycle. It can be acquired in sagittal section to monitor the CSF flow through the aqueduct and basal subarachnoid spaces . Also, it can be acquired in axial section for quantification of CSF flow (quantitative assessment)  demonstrates CSF flow as a bright signal throughout the cardiac cycle. (MP4 418 kb)Additional file 2: Video S2. Cine phase-contrast sequence (phase images) demonstrates CSF flow changes from positive to negative and back to positive throughout the diastolic and systolic phases of the cardiac cycle. (MP4 392 kb)In the phase-contrast technique, two data sets with opposite sensitization are acquired. When the two data sets are subtracted, the signal of the stationary protons is eliminated and flowing nuclei accumulate a net phase proportional to their velocity. The anticipated maximum velocity must be entered into the pulse sequence protocol before phase-contrast data are acquired . After sOne of the ongoing challenges in neuroradiology is to differentiate hydrocephalus from ventricular enlargement associated with brain atrophy  especially in children, as the sizes of the ventricles and subarachnoid spaces have variable size over the first 2 years of life. Imaging findings should be interpreted in the context of clinical data in order to reach a firm diagnosis of active hydrocephalus. Knowledge of the infant’s head size is essential; a large head or too rapidly enlarging head suggests hydrocephalus, whereas a small or diminishing head circumference is more compatible with atrophy .Coronal T2-WI and high-resolution T2-WI (3D-DRIVE) can help differentiate hydrocephalus and ventriculomegaly ex-vacuo. In cases of hydrocephalus, coronal T2-WI demonstrates commensurate dilatation of the temporal horns with the lateral ventricles associated with rounding of the lateral angles and medial displacement of the hippocampus. Effacement of pericerebral CSF spaces above the level of obstruction is also demonstrated. However, in cases with ventriculomegaly ex-vacuo, the temporal horns  are small compared to the bodies of the lateral ventricles; they retain their normal shape and the hippocampi are not displaced medially Fig. . In addiA consensus classification of hydrocephalus has been proposed to relate most cases of hydrocephalus to obstruction of the CSF pathway, reflecting the surgical approach to hydrocephalus that aims to divert the CSF between the site of production and absorption , 18.Obstructive hydrocephalus can be related to purulent or tuberculous meningitis. In the acute stage, MR assessment targets the evidence of infection or its complication via demonstration of leptomeningeal enhancement after contrast administration Fig. . At the Midline and midline-compressing brain tumors can obstruct the CSF pathway as well. A contrast-enhanced study is indicated in these instances Fig. . Among bIntraventricular hemorrhage in premature infants is the leading cause of pediatric hydrocephalus. T2* demonstrates blood and blood residue. Blood in the acute phase and hemosiderin and scarring in the chronic phase obstruct the ventricles, basilar cisterns, and arachnoid granulation, impairing CSF flow and absorption , 21.Obstruction can be encountered in the aqueduct of Sylvius, fourth ventricular exit foramina, and foramen of Monro Fig.  or seconThe criteria suggesting aqueduct stenosis include a small fourth ventricle with dilated third and lateral ventricles out of proportion to cortical atrophy. However, these have been described as non-reproducible parameters . 3D-DRIVCommunicating hydrocephalus implies cases with no obstruction in the ventricles or the cisterns. Aqueductal CSF flow void is increased in communicating hydrocephalus plausibly because of decreased intracranial compliance Fig.  20]. Aq. Aq20]. In children, the apparently non-obstructive hydrocephalus has been attributed to an imbalance of CSF secretion and absorption. Two patterns have been identified: the benign idiopathic external hydrocephalus (which is more common) and the choroid plexus papilloma-associated hydrocephalus (which is much rarer) .Benign idiopathic external hydrocephalus is also known as benign enlargement of subarachnoid spaces in infancy. It is characterized by macrocephaly with dilatation of the anterior subarachnoid spaces and ventricular system to a lesser extent. The extra-axial CSF spaces should have CSF signal in all pulse sequences and be traversed by the bridging veins Fig. . These fChoroid plexus papilloma-related hydrocephalus is assumed to be related to high secretion of CSF that overwhelms the capacity of absorption. The tumor predominantly affects infants and young children. The tumor usually appears as an avidly enhancing mass floating within the ventricular atrium .Dandy-Walker complex represents a continuum spectrum of posterior fossa malformations that are characterized by a posterior fossa cyst associated with variable degrees of abnormal development of the cerebellar vermis. The classic Dandy-Walker malformation, Blake’s pouch cyst, posterior fossa arachnoid cyst, mega cisterna, and vermian hypoplasia represent steps on this continuum. Phase-contrast sequence has value in studying CSF flow between CSF spaces in the posterior fossa and the posterior cervical subarachnoid space .The typical Dandy-Walker malformation constitutes a large posterior fossa cyst communicating with the fourth ventricle in association with partial or complete agenesis of the cerebellar vermis. A phase-contrast study demonstrates no flow between the posterior fossa cystic space and the posterior cervical subarachnoid space Fig. . HydroceBlake’s pouch cyst appears as an infracerebellar or retrocerebellar arachnoid cyst associated with tetraventricular hydrocephalus. A phase-contrast study reveals lack of communication between the cyst and the posterior cervical subarachnoid space Fig.  3, 5, 2, 25, 24.A posterior fossa arachnoid cyst appears as a well-defined extra-axial cyst or fluid collection that is associated with a normal vermis. It may produce mass effect on the cerebellum or vermis with remodeling of the overlying skull bone. A phase-contrast study can demonstrate whether the cyst is communicating to the CSF spaces which is important in preoperative planning , 24.Mega cisterna magna represents focal enlargement of the subarachnoid space in the posterior and inferior portions of the subarachnoid space with a normal vermis, cerebellum, and fourth ventricle. Phase contrast reveals free communication with the fourth ventricle and posterior cervical subarachnoid space , 24.They represent hindbrain anomalies with caudal displacement of the cerebellar tonsil through the foramen magnum causing plugging of the CSF pathway  and can be associated with myelomeningocele . A phase-contrast study provides information about the extent of CSF flow blockage at the craniocervical junction Fig.  3, 4, 2, 24, 20.3 indicates effective operation with patient improvement. A decrease of stroke volume during follow-up could indicate ventriculostomy malfunction and clinical deterioration [Phase-contrast and 3D-DRIVE sequences have high confidence in the assessment of the flow through the floor of the third ventricle with proper identification of ventriculostomy malfunction The diagnosis of shunt malfunction depends mainly on CT of the cranium along with clinical symptoms  . HoweverMRI with the advent of high-resolution T2-WI (3D-DRIVE) and phase-contrast sequences is a crucial imaging modality for the assessment of CSF motion alterations in various disorders which is important for proper patient management.


Scedosporium and Fusarium are considered emerging opportunistic pathogens, causing invasive fungal diseases in humans that are known as scedosporiosis and fusariosis, respectively. These mold infections typically affect patients with immune impairment; however, cases have been reported in otherwise healthy individuals. Clinical manifestations vary considerably, ranging from isolated superficial infection to deep-seated invasive infection—affecting multiple organs—which is often lethal. While there have been a number of advances in the detection of these infections, including the use of polymerase chain reaction (PCR) and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS), diagnosis is often delayed, leading to substantial morbidity and mortality. Although the optimal therapy is controversial, there have also been notable advances in the treatment of these diseases, which often depend on a combination of antifungal therapy, reversal of immunosuppression, and in some cases, surgical resection. In this paper, we review these advances and examine how the management of scedosporiosis and fusariosis may change in the near future.Species of  Candida spp. and Aspergillus spp., while less common fungal pathogens, such as mucormycetes, Scedosporium spp., and Fusarium spp. are reported with increasing frequency [The past few decades have witnessed a remarkable increase in the prevalence and severity of invasive fungal infections (IFI) in children and adults with immune impairment, including patients with hematologic malignancies, stem cell and solid organ transplantations, and primary and acquired immunodeficiencies and preterm neonates ,2,3. Therequency ,5. DiagnScedosporium spp. usually relies on the detection of fungi from clinical samples by direct microscopic examination or histological analysis of the clinical specimen and culture on appropriate culture media [Scedosporium spp. from species of Fusarium or Aspergillus, as all of them present dichotomous branching, hyaline hyphae, and regular hyphal septation [The timely diagnosis of mycotic disease is an enduring challenge in clinical practice. A diagnosis of re media . Howevereptation ,7. GivenScedosporium, but the partial β-tubulin gene (BT2) is required to differentiate closely related species [Nucleotide sequence-based analysis is the current gold standard for fungal identification; rDNA internal transcribed spacer (ITS) sequencing appropriately identifies the main species in  species ,9. Matri species .Scedosporium, without the need for purification or extraction. However, this platform is expensive and is not routinely used at most centers; moreover, the database of filamentous fungi is relatively small, and the process of specimen preparation may be cumbersome [Scedosporium spp., as a delay in diagnosis is often associated with substandard treatment and poor outcomes.Polymerase chain reaction (PCR)/electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) combines 16 PCR assays using broad-range primers targeting nuclear or mitochondrial genes and T2 magnetic resonance (T2MR), which enables rapid determination of molecular weight and base composition in the amplicons after electrospray ionization and chromatographic separation ,12,13. Tmbersome ,15,16. TScedosporium, including Lomentospora prolificans (formerly S. prolificans), cause a wide range of clinical manifestations in humans from superficial infection to severe invasive disease, as well as colonization of the respiratory tract and allergic reactions [Scedosporium, including S. boydii, S. apiospermum, and S. aurantiacum are among the most commonly recovered molds from respiratory secretions of patients with chronic pulmonary diseases such as cystic fibrosis (CF) and may lead to invasive disease [Species of the pathogenic mold eactions ,17. Scedeactions . Given teactions . Species disease ,7,20,21.Scedosporium, with the azoles (such as itraconazole and voriconazole) typically having the lowest mean inhibitory concentration (MIC) among the antifungal drugs [Scedosporium species are typically resistant to polyenes, as well as to fluconazole and demonstrate reduced susceptibility to echinocandins [Scedosporium make these infections difficult to manage and susceptibility testing of isolates from patients with scedosporiosis is highly recommended [There are three major classes of antifungal agents approved for use in humans: (1) triazoles ; (2) polyenes (various formulations of amphotericin B); and (3) echinocandins  ,25,26,27al drugs . Scedospocandins ,30. The ommended ,31. Reveommended .S. apiospermum infection had successful responses. Patients with malignancy and hematopoietic cell transplant (HSCT) recipients had the worst outcomes [Although the optimal choice and duration of therapy for scedosporiosis is controversial and may depend on the immune status of the patient, a large retrospective study provides support for the use of voriconazole . In thatoutcomes .S. apiospermum and L. prolificans; however, these combinations have demonstrated variable outcome in the treatment of these infections in humans [L. prolificans and showed synergy in vitro, but the data in humans with scedosporiosis is exceedingly limited.Most international guidelines recommend voriconazole as first-line therapy; however, antifungal combination therapy (ACT) has emerged as a promising option, because therapeutic effect can be achieved at lower concentrations thereby reducing toxic side effects, improving safety and tolerability, and shortening the therapeutic effect while potentially preventing treatment failure ,34,35,36n humans ,37. CombIn March 2015, the FDA approved the extended-spectrum triazole isavuconazole for the treatment of invasive aspergillosis and mucormycosis ,39. The Scedosporium spp. [Scedosporium tested (S. prolificans and S. apiospermum) showed marked differences in susceptibility: isavuconazole showed good activity against Scedosporium apiospermum, with a MIC90 very similar to those for Aspergillus spp., whereas S. prolificans (L. prolificans) was relatively resistant. Subsequent work by Pfaller and colleagues has reinforced this finding [Guinea and collaborators examined the activity of isavuconazole against more than 1000 opportunistic fungi collected from 1986 to 2007, including 22 isolated of ium spp. . The two finding . Indeed,Scedosporium and Lomentospora isolates [S. apiospermum, S. boydii, and S. aurantiacum, with the MIC decrease ranging from 0.125 to 0.5 mg/L. Similarly, Wiederhold and collaborators had similar results with F901318 against S. apiospermum, S. aurantiacum, S. dehoogii, and S. boydii, with the MIC ranging from ≤0.008 to 0.25 [The novel compound F901318 represents a new class of antifungal drug called the orotomides, which inhibits dihydroorotate dehydrogenase, a key enzyme in pyrimidine biosynthesis . This drisolates . F901318 to 0.25 . ClinicaAlthough diagnosis may be suspected based on clinical phenomenon in high-risk patients, such as characteristic skin lesions in patients with acute myelogenous leukemia (AML), confirmatory diagnosis of fusariosis by histopathology and culture is strongly recommended by the European Confederation of Medical Mycology and the European Society of Clinical Microbiology and Infectious Diseases (ECMM/ESCMID) guidelines ,48. HoweFusarium species that can also distinguish clinical species complexes, such as Fusarium equiseti and F. sporotrichioides [Aspergillus, Candida, and F. oxysporum, but this method cannot distinguish between the Fusarium spp. that have closely-related ITS amplicons [Immunohistochemistry uses antibodies directed at cell antigens to identify fungal organisms visualized in situ. However, this approach is not widely used. Recently, DNA-based assays have emerged as a promising new approach to diagnosing fusariosis. A PCR platform based on the intergenic spacer (IGS) region has been developed for different chioides . A fluormplicons ,51,52.Fusarium isolates on the species level, and enzyme-linked immunosorbent assay (ELISA) is the preferred method in Fusarium to identify specific mycotoxins [Multi-locus sequence typing (MLST) is currently viewed as the best option for the identification of cotoxins ,53,54,55cotoxins ,57,58. HFusarium species cause a variety of diseases in humans, including superficial skin infections, keratitis, blood stream infections, and life-threatening end organ damage [Fusarium spp. are a frequent cause of corneal damage and may lead to endophthalmitis [n damage ,60. Infen damage ,62. Fusahalmitis ,63.Fusarium spp. may cause peritonitis, bloodstream infection, osteomyelitis (often after trauma), arthritis, otitis, sinusitis, and brain abscess [Less commonly,  abscess ,48. As wFusarium spp. often display high levels of resistance to existing antifungal agents and are some of the more difficult fungi to treat [F. solani, which is somewhat more susceptible to amphotericin B but less susceptible to voriconazole than other species, such as F. oxysporum [to treat ,64,65. Dto treat ,54,66,67xysporum ,69. Howexysporum . Essential for successful therapeutic outcome is the restoration of innate host defenses, particularly with recovery from neutropenia. GCSF may accelerate recovery from neutropenia. Granulocyte transfusions from GCSF-mobilized donors may stabilize infection until recovery from neutropenia in persistently neutropenic patients with hematological malignancies or in those with aplastic anemia. Fusarium spp. [Scedosporium and Fusarium species [Fusarium species [There are currently a number of novel antifungal agents in the pre-clinical pipeline that may have activity against human fusariosis . These iium spp. ,72,73. T species ,74. Gene species . We are Aspergillus filamentous fungi are less well understood than those against aspergilli. However, limited data with Fusarium spp. and Scedosporium spp. show certain similarities in pathogenesis and host defenses with Aspergillus fumigatus, with which most studies have been performed.Host defenses against filamentous non-S. apiospermun and S. prolificans [A. fumigatus. If the immune response of the host is compromised, spores can germinate to hyphae and invade to adjacent tissues. PMNs are the main immune cells causing damage to hyphae using oxygen-dependent  and oxygen-independent (cationic peptides such as defensins and cathelicidins) mechanisms [S. prolificans [The predominant line of host defenses against filamentous fungi is circulating polymorphonuclear (PMNs) and mononuclear leukocytes (MNCs), as well as monocyte-derived macrophages (MDMs) . MDMs inlificans ,77, compchanisms . PhagocyFusarium or Scedosporium spp. Of note, S. prolificans has been shown to induce significantly more TNF-α and IL-6 release by human MNCs as compared to Aspergillus spp., which could be associated with the virulence of the specific fungus [Fusarium and Scedosporium are likely linked to the frequency and severity of infections by some of these fungi.A variety of growth factors, cytokines, and chemokines play an important role in the interface of innate and adaptive immunities against filamentous fungal infections . Howeverc fungus . DiffereF. solani hyphae, but not by F. oxysporum hyphae. In contrast, the release of TNF-α was not affected by the use of IL-15 [S. prolificans, whereas release of TNF-α was not affected. In addition, the presence of IL-15 significantly enhanced PMN-induced hyphal damage and oxidative respiratory burst of S. prolificans but not S. apiospermum. This inability of IL-15 to exhibit enhanced damage of S. apiospermum hyphae is in concordance with this fungus’ greatest intrinsic virulence in humans. IL-15 may have species-specific enhancing effects on antifungal activities of PMNs against Scedosporium spp. and Fusarium spp. [A number of studies have assessed the immunomodulatory utility of cytokines in confronting fungal pathogens. Th1- and Th17-type cytokines have exhibited certain enhancing activities on antifungal phagocytic responses. For example, IL-15 increased interleukin-8 (IL-8) release from PMNs challenged by of IL-15 . Similarium spp. .Scedosporium spp. are interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulation factor (GM-CSF) [Scedosporium spp., enhancing PMN functions including oxidative burst in response to S. apiospermum hyphae [Among other cytokines studied that enhance PMN antifungal activity against (GM-CSF) . These c(GM-CSF) . GM-CSF (GM-CSF) ,85. Tream hyphae .Fusarium and Scedosporium spp. In vitro studies have shown that MDM activity against Fusarium spores [F. solani hyphae [S. prolificans and S. apiospermum [S. prolificans and S. apiospermum [Antifungal agents may also have differential immunomodulatory effects against m spores , oxidatii hyphae , and PMNospermum  can be mospermum . RegardlS. prolificans, demonstrated a modest efficacy of liposomal amphotericin B (LAMB) at 10 mg/kg/day combined with G-CSF [S. prolificans infection, posaconazole and GM-CSF had a combined effect in damaging S. prolificans hyphae. However, when posaconazole and GM-CSF were administered to mice with invasive infection due to S. prolificans, they had selective beneficial effects on the burdens in certain organs but offered no additional benefit to survival [No animal model studies have reported on the effects of cytokines, such as granulocyte colony stimulating factor (G-CSF), on the outcome of experimental fusariosis to date. Ortoneda et al., in an immunosuppressed murine model of invasive infection by th G-CSF . Subsequth G-CSF . Interesth G-CSF ,91. In asurvival .The principal therapy in fusariosis is early aggressive antifungal therapy. Immunomodulation with G-CSF or GM-CSF potentially combined with IFN-γ can be adjuvant therapeutic strategies together with source control. Over the last decades, efforts to reconstitute host defenses with granulocyte transfusion therapy have increased after the advances of the availability of recombinant hematopoietic growth factors and modern transfusion practices. In severely neutropenic patients suffering from fusariosis, treatment with G-CSF or GM-CSF and granulocyte transfusions may be considered ,95,96,97Scedosporium spp. infection, a favorable outcome was reported only in four of them [S. prolificans survived, and their survival coincided with hematologic recovery [S. prolificans infection was fatal in all neutropenic patients [The clinical evidence on the role of immunomodulation on the host response against scedosporiosis is limited, involving mainly haematopoietic growth factors (G-CSF or GM-CSF) and IFN-γ . Of note of them . In thes of them ,102. Witrecovery . Similarpatients .Pediatric pharmacokinetic, safety, and efficacy data are sparser than those in adult patients. As a consequence, the pace of guideline development for pediatric patients has been much slower than that for adults, and indeed, dedicated guidelines for treating infants and children with IFI do not exist. Extrapolating adult recommendations to pediatric practice should be done cautiously, as differences pertaining to epidemiological factors and pharmacokinetics of many antifungal agents exist between adults and children.Scedosporium spp. infections in children have been associated with brain abscess formation after near drowning in immunocompetent individuals [Pseudallescheria spp. [Scedosporium spp., has been proposed [ividuals . These iria spp. . The mosria spp. . Among aproposed .Fusarium spp. infections are between the third and fourth most common cause of IFI in children; F. solani and F. oxysporum species complex are the most commonly implicated in pediatric disease [. Clinical presentation of Fusarium spp. infections depends on the portal of entry, as well as the host’s immune status. In immunocompetent patients, infections are usually superficial  or limited to a single organ , whereas in immunosuppressed hosts, such as allogeneic hematopoietic stem cell recipients, patients with severe or prolonged neutropenia, or acute leukemia, fusariosis may be invasive and disseminated [ disease ,108,109.eminated ,110.Given the rarity of these infections, treatment guidelines are not based on randomized controlled clinical trials but on large uncontrolled case series mainly in adult patients . EspeciaThe FungiScope registry, a global fungal infection registry, found 10 children with invasive fusariosis between 2006 and 2015 . All theFusarium infections in immunocompromised children  showed considerable variation in the treatment regimens used: Amphotericin B was used in most cases (76%); combination treatment with  was used in 33%; G-CSF was used in 18%; granulocyte transfusion in 12%; and surgery was performed in 12% of the cases [A recent literature review of invasive he cases . In bothhe cases ,112.Posaconazole has been recommended as a salvage treatment option for invasive fusariosis in high-risk patients with hematologic malignancies . Data foFusarium spp., the severity of the infection, high mortality—especially in patients with multiple comorbidities requiring salvage treatments—and their uncertainty about the effectiveness and options in treating this serious infection.Data for combination antifungal therapy in children with IFI, including invasive fusariosis, are scant; however, it is frequently used in pediatrics as evidenced by recent pediatric cohort studies ,109,118.Scedosporium- or Fusarium-specific T cells [A promising approach for the fungal strains that are resistant to currently used antifungal agents or for patients with compromised host immunity is immune based treatments. While, until now, most immunotherapeutic approaches have aimed to augment the number of granulocytes through granulocyte transfusions, the infusion of growth factors , the administration of cytokines such as IFN-γ, and most recently, the use of adoptive T cell therapy, which was initiated for the treatment of cancer, seems to be a promising approach for the treatment of patients suffering from drug-resistant IFI . Even th T cells .Scedosporium spp and is considered first-line treatment. For fusariosis treatment, voriconazole, lipid formulations of amphotericin B, and various combinations should be considered as the optimal alternatives. Duration of treatment is usually individualized based on the site, the extent of the infection, and the immune status of the patient [In summary, the optimal treatment for scedosporiosis and fusariosis in children is unknown. Voriconazole demonstrates strong in vitro activity against  patient . In addiHuman scedosporiosis and fusariosis are emerging opportunistic infections that are difficult to diagnose and may be even more challenging to treat. In this paper, we have reviewed the challenges associated with diagnosis, which typically relies on labor-intensive histopathology, as well as recent advances in non-culture-based systems. We have also reviewed novel antifungal compounds, such as E1210, F901318, and hemafungin, which may one day play a role in treatment. However, these agents are still in development. For now, treatment of these two potentially-lethal conditions relies on early diagnosis, effective antifungal therapy, possible surgical excision, and reversal of immunosuppression when possible. In the coming years, the mycology community must make it a priority to design non-inferiority trials to evaluate these new agents to meet the needs of vulnerable patients.


Rana dybowskii between the summer and autumn groups of captive frogs and between wild and captive R. dybowskii, whereas the differences between juvenile and adult wild frogs were not significant. The dominant gut bacterial phyla in frogs from both captive and wild environments included Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Linear discriminant effect size (LEfSe) analysis showed that Bacteroidetes and Firmicutes were significantly enriched in captive and wild R. dybowskii, respectively linear discriminant analysis (LDA > 4). The core operational taxonomical units (OTUs) that were found in >90% of all frogs tested encompassed 15 core OTUs. The captive frogs exhibited 15 core OTUs in addition to the above overall core microbiota, whereas the wild frogs exhibited 19 core OTUs in addition to the above overall core microbiota. Predictions made using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) suggested that eleven KEGG pathways, such as infectious diseases, immune system diseases, metabolism, metabolism of other amino acids, metabolism of cofactors and vitamins, metabolism of terpenoids and polyketides, neurodegenerative diseases, and transport and catabolism, were enriched in captive frogs. The relative abundance of several red-leg-syndrome-related pathogens increased significantly in captive frogs compared with that in wild frogs. To our knowledge, this is the first study on the effects of individual seasons and captivity on the gut microbiota of frogs.The gut microbiota of amphibians is affected by exogenous and endogenous factors. We performed a comprehensive analysis using high-throughput sequencing technology and functional predictions and observed general changes in the gut microbiota of frogs in different growth stages, seasons, and growth environments. There were no significant differences in microbial richness and diversity between juvenile and adult wild frogs, between the summer and autumn groups of captive frogs, or between wild and captive frogs. There were significant differences in the gut microbiota community structure of  Vertebrates harbor and interact with diverse microbiotas that influence the physiology of the vertebrate hosts in many ways  and affect their overall health, development, reproduction and behavior . To dateRana dybowskii, including variations in temperature and the bacterial flora populations of terrestrial habitats, can be expected to influence the gut microbiota of this frog  or an intensive culture farm (captive group). The wild frogs were sampled at two development stages: juvenile and adult. The captive brown frogs were sampled in the summer and autumn at a culture farm. Differences in the gut microbiota were compared (1) between juveniles and adults of wild R. dybowskii from the same habitat, (2) between the summer and autumn in R. dybowskii from the same population in the farming environment, and (3) between captive frogs and wild frogs.The aims of this study are to investigate the effects of season, development and captivity on the intestinal microbiota of The wild brown frogs were sampled from a natural distribution zone, and they prefer to live in deciduous broad-leaved forests, or coniferous and broad-leaved forests dominated by deciduous broad-leaved forests, and their associated forest edge thickets, forest creeks, and forest swamps. Brown frogs mainly eat insects, followed by earthworms, mollusks and spiders.The captive brown frogs were sampled from an intensive culture farm. The captive brown frogs derived from wild frog eggs, and wild frog eggs came from the same area as the wild frogs. Wild frog eggs were brought to the farm and hatched, and the tadpoles and young frogs were raised in the captive environment.2 in area. The ground inside the breeding enclosure consisted of land that was not completely bare and was planted with sparse low vegetation. The air humidity in the pens was generally stable and was controlled at 60–80% by spraying water, and the ground-level humidity of the pens was controlled at 25–35%. The farming density of the 2-year-old frogs was 40/m2. Disinfection with iodine volts was conducted two to three times a week. When the average temperature was above 10°C (when the frogs had rested inside the breeding fence for approximately a week) and the frogs were observed to be moving, we provided living yellow mealworms (Tenebrio molitor). In the experiment, the frogs were fed two times a day at 09:00 and 16:00, and the total amount fed was approximately 4% of their mean body weight.The breeding pens were greenhouse facilities enclosed by a sealed fence, equipped with a sprinkler and planted with low vegetation. All pens were made of the same construction materials and displayed the same physical properties. Each pen was approximately 80 mAeromonas hydrophila, A. sobria, Citrobacter freundii, Chryseobacterium indologenes, Edwardsiella tarda, Proteus mirabilis, P. vulgaris, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Streptococcus iniae , Heilongjiang, China. Captive R. dybowskii were captured from a frog farm in Huanan County , Heilongjiang Province, China.Brown frogs of the wild and captive groups were sampled from a natural distribution zone  and an intensive culture farm . Wild The two sample groups in captive frogs were designated summer group (CS group) and the autumn group (CA group). Captive frogs captured on 5 June and 15 September corresponded to the summer group (CS group) and the autumn group (CA group), respectively. The 2-year-old frogs that were sampled, which were re-fed in captivity after artificial wintering, had been fed for 20 days on 5 June and 120 days on 15 September. The two sample groups were designated CS and CA, and their body weights were 4.65 ± 0.31 g and 22.36 ± 2.65 g, respectively. The samples of the CA  group were numbered C01–C07, and the male to female ratio was 3:4. The samples of the CS  group were numbered C08–C12, and the male to female ratio was 2:3.R. dybowskii frogs generally weighed 3–5 g and did not reproduce. All male and female frogs weighing more than 20 g typically pair and spawn after hibernation.The two sample groups of wild frogs were designated the juvenile group (W1) and adult group (W2). The wild frogs captured on June 5 were divided into sexually immature young frogs and sexually mature adults. The samples of the juvenile group (W1) were numbered W01–W06, and the male to female ratio was 3:3. The samples of the adult group (W2) were numbered W07–W13, and the male to female ratio was 3:4. The body weights of the juvenile and adult wild frogs were 5.12 ± 0.57 g and 25.65 ± 3.54 g, respectively. It is difficult to distinguish between males and females in frogs of less than 10 g; anatomical observation of gonad development can be used to distinguish sex as well as sexual maturity. After hibernation, the 2-year-old All of the frogs were lively and vigorous. The frogs were first washed with tap water and then with sterilized water. The frogs were examined by intestinal dissection under euthanasia. The contents of the gut samples were collected from the gut within 20 min after euthanasia. We used a fresh pair of sterilized tweezers for the collection of each sample. The digestive tract was then carefully isolated from the body, and a portion of the gut was collected, extending from (but excluding) the stomach to the anus of the digestive tract. The contents of each gut were emptied into a sterile vial and immediately stored at −80°C.® Spin Kit for Soil  according to the manufacturer’s protocol, and the DNA concentration was measured on a NanoDrop 2000 spectrophotometer . The V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′)  according to the manufacturer’s protocol.Total genomic DNA was extracted from each sample with a FastDNATAAT-3′)  in a thePRJNA422729 (CA group), PRJNA509081 (CS group), PRJNA509086 (W2 group) and PRJNA428920 ].Purified amplicons were pooled in equimolar amounts subjected to paired-end sequencing (2 × 300) on the Illumina MiSeq platform  according to standard protocols. The raw reads were deposited in the NCBI Sequence Read Archive (SRA) database (Accession Number: 1), and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed with the RDP Classifier algorithm2 against the Silva (SSU128/16 s) 16S rRNA gene database using a confidence threshold of 70%  were clustered with a 97% similarity cutoff using UPARSE . A Venn diagram was generated using the R package  to show unique and shared OTUs. The core gut microbiota of the frogs was assigned if it was found in 90 or 100% of the samples and represented >0.1% of the reads. We used the linear discriminant analysis (LDA) effect size (LEfSe) to identify significant associations between bacterial taxa and different groups . The relative abundance of potential opportunistic pathogens in the gut of brown frogs between the C and W groups was compared via a zero-inflated Gaussian mixture model (metagenomeSeq) using the R stats package and the Python SciPy package .Rana species were predicted using PICRUSt, which can both predict the most recent Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog (KO) functional profiles of microbial communities via 16S rRNA gene sequences  were shared between C  and W  groups, however, the unique OTUs of the C group was 276, which was more than 150 OTUs of the W group . The totP > 0.05). Firmicutes (69.9%) was the most dominant bacterial phylum, followed by Bacteroidetes (12.23%), Proteobacteria (9.14%), and Actinobacteria (7.08%) in wild frogs .There were no significant differences in richness and diversity between juvenile and adult wild frogs  but not the weighted UniFrac dissimilarity matrix .There were no significant differences in richness and diversity between the summer and autumn groups . At the family level, Bacteroidetes, Lachnospiraceae, Enterobacteriaceae, Porphyromonadaceae, and Ruminococcaceae were the most abundant bacterial families in both the summer and autumn groups , Firmicutes , Proteobacteria , and Actinobacteria  . There wn groups .P < 0.05, P < 0.05, Linear discriminant analysis effect size showed that no phylum was significantly enriched in either the summer or autumn group . Among these 42 families, 30 were more abundant in the summer group, and 12 were more abundant in the autumn group (P < 0.05)  , S4.P > 0.05, Wilcoxon rank-sum test and multiple test correction with Benjamini-Hochberg FD, Predicted functional analysis of the gut microbiota of the summer and autumn groups by PICRUSt revealed 39 level-2 KO groups, as shown in P > 0.05; r = 0.569, P = 0.001; weighted UniFrac r = 0.632, P = 0.001).There were no significant differences in richness or diversity between the wild and captive groups . At the family level, Bacteroidetes (21.87 vs. 3.12% in wild frogs) and Lachnospiraceae (13.66 vs. 24.21% in wild frogs) were the most abundant families in captive frogs, and Lachnospiraceae (24.21 vs. 13.66% in captive frogs) and Ruminococcaceae (22.84 vs. 6.46% in captive frogs) were the most abundant families in wild frogs  were significantly enriched in wild frogs . Among the 113 detected genera, 55 and 58 were more abundant in captive and wild frogs, respectively, while amonvg the 156 species, 79 and 77 were more abundant in captive and wild frogs, respectively  was significantly higher than that in autumn (September 15)  . Temperamber 15) . In addimber 15) . The froNeophoca cinerea, Grus japonensis, Salmo salar L. and Paralichthys olivaceus, intensive culturing has been shown to significantly affected the gut microbiota composition in R. dybowskii , a carotenoid-enriched diet, rather than a carotenoid-free diet, improves the species richness and abundance of skin bacteria .The effect of the food composition on the gut microbiota is pronounced. Brown frogs feed on live food, so farms must supply frogs with live insects, such as an hosts . For redbacteria . CompareT. molitor) for cultured frogs, and the diet (T. molitor) was not sequenced along with the frog, while wild frogs have dozens of kinds of food. Captive frogs live on ground (soil) without vegetation, and food is placed on the ground. When captive frogs ingest T. molitor in their habitat, their gut microbiota is affected by the soil flora. In contrast, wild frogs capture more flying insects; therefore, their gut microbiota is less affected by the soil flora. The variations in the gut microbiota or core microbiota of captive frogs may be due to exposure to antibiotics  was detected in both captive and wild frogs. Many Bacillus species  that were previously confirmed as potential probiotics were unclassified in our study, and their presence should be confirmed in the future. To date, there have been few reports on the effects of Bacillus in frog culture, and it is difficult to deem Bacillus an effective probiotic, due to the high proportions of Bacillus in the guts of frogs.When grown to an appropriate number, probiotics can benefit living microbes of the host . ProbiotA. hydrophila, C. indologenes, C. meningosepticum, C. freundii, P. mirabilis, and P. aeruginosa . All experiments were performed in accordance with approved guidelines and regulations.QT, JB, ZH, and XL: data collection, data analysis and interpretation, and drafting of the manuscript. QT, HW, and JZ: conception or design of the work. QT, JD, and XL: sample collection. QT and ZH: writing and critical revision of the manuscript. JZ: final approval of the version submitted.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.


Originally believed to be primarily a disorder of T-cell signaling, evidence shows that macrophage-lineage cells also contribute to the pathogenesis of Crohn’s disease (CD). Colony stimulating factor-1 (CSF-1) is a key regulator of the macrophage lineage, but its role in CD has not been well established. We examined transcriptional data from CD mucosa for evidence of CSF-1 pathway activation and tested JNJ-40346527 (PRV-6527), a small molecule inhibitor of CSF-1 receptor kinase (CSF-1R), for its ability to inhibit disease indices in murine colitis.ex vivo with CSF-1 and compared to a TNFα-induced gene set generated from epithelial-lineage cells. Gene set variation analysis was performed using existing Crohn’s mucosa microarray data comparing patients who either responded or failed to respond to anti-TNFα therapy. Commencing day 14 or day 21, mice with T-cell transfer colitis were treated with vehicle or JNJ-40346527 until study termination (day 42). Endpoints included colon weight/length ratios and histopathology scores, and macrophage and T cells were assessed by immunohistochemistry. Mucosal gene expression was investigated using RNAseq.A CSF-1 pathway gene set was created from microarray data of human whole blood cultured + mononuclear cell and CD3+ lymphocyte numbers. RNAseq analysis confirmed the CSF-1 gene set was sharply reduced in treated mice, as were gene sets enriched in “M1” inflammatory and “M0” resident macrophages and in activated T cells.Both the CSF-1 and the TNFα gene sets were enriched in the colonic mucosal transcriptomes of Crohn’s disease and in mouse colitis, and expression of both gene sets was highest in patients who did not respond to anti-TNFα therapy. In these patients neither set was reduced by therapy. In the mouse model, JNJ-40346527 inhibited the increase in colon weight/length ratio by ∼50%, reduced histological disease scores by ∼60%, and reduced F4/80CSF-1 biology is activated in Crohn’s disease and in murine T cell transfer colitis. Inhibition of CSF-1R by JNJ-40346527 was associated with attenuated clinical disease scores and reduced inflammatory gene expression in mice. These data provide rationale for testing JNJ-40346527 (PRV-6527) in human inflammatory bowel disease. Crohn’s disease (CD) is a chronic inflammatory bowel disorder believed to result from loss of tolerance to gut derived commensal bacteria in the context of genetic susceptibility and an environmental trigger. It was generally believed that these factors led to disordered T-cell signaling resulting in the direct and indirect release of high levels of inflammatory mediators such as TNFα. As such, antibodies including infliximab and adalimumab that block TNFα have become an important therapeutic strategy for mitigating disease. However, only 40–50% of patients realize adequate response to this therapy with even fewer achieving full remission, and over time some patient populations will lose response. Thus, additional treatment options are required.More recently, cells of the macrophage lineage have been implicated in in driving disease pathology. Macrophages are present constituently in all bodily tissues where they mediate multiple processes in both health and disease. In general, these resident “M0” macrophages function as sentinels to defend against pathogens. They are essential for tissue homeostasis, acting as non-inflammatory scavengers of commensal bacteria and senescent cells, assisting in the maintenance of regulatory T cells, and promoting tissue cell renewal by production of growth factors. Resident macrophages are present in particularly high numbers in tissues in contact with the external environment such as the lung and gastrointestinal tract where analysis of their secreted mediator profile indicates that they produce high levels of IL-10 and exhibit an anti-inflammatory phenotype. However,+ CD14lo macrophages form a network at the subepithelial lamina propria.-1H-imidazole-2-carboxamide (abbreviated JNJ527), was prepared at Janssen as described. JNJ-4034CNTO5048 is an IgG2a anti-mouse TNFα monoclonal antibody that binds soluble and transmembrane TNFα and prevents interaction of TNFα with its receptor. CNTO6601, an IgG2ak anti-tumor associated epithelial membrane protein (EMP) monoclonal antibody was used as an isotype control. Both were designed and generated at Janssen R&D . The antibodies were formulated at 0.3 mg/ml in sterile phosphate buffered saline and dosed at 10 ml/kg (3 mg/kg) by intraperitoneal (IP) injection.To identify CSF-1 induced genes, whole blood from three healthy donors was drawn into EDTA vacutainer tubes. Participant blood samples acquired for generation of the CSF-1 gene signature were obtained subsequent to IRB approval from an independent ethics review board, Quorum Review IRB, Cambridge, MA, according to the principles of the Declaration of Helsinki. All subjects provided written informed consent.2. Aliquots (100 μL) were collected from all tubes at 4 and 24 hr, and RNA isolated using a MagMAX total RNA isolation kit (ThermoFisher Scientific) according to the manufacture’s protocol. cDNA was generated and aliquoted onto GeneChip Human Transcriptome Array 2.0 (ThermoFisher) and analyzed via the manufacturer’s online proprietary software. The CSF-1 gene set was comprised of 57 genes  with geometric means >2-fold relative to unstimulated control conditions (p≤0.05) at either 4 or 24 hrs. The microarray data is available in GEO DataSets under accession no. GSE136440.Two aliquots of 1 ml of blood were transferred to sterile 2 ml Eppendorf tubes. Recombinant human CSF-1 (PeproTech) was added to one of the two replicate tubes to a final concentration of 50 ng/ml, with the second replicate tube retained as an unstimulated control sample. Tubes were affixed to a tube rotator and incubated at 37°C, 5% COTo create a TNFα-induced gene set, triplicate cultures of A549, HT-29, HEK293, and primary human keratinocytes were cultured without (unstimulated control) or with 10 ng/ml human recombinant TNFα (PeproTech) for 4-to-24-h and gene expression analyzed by Affymetrix GeneChip® HG-U133+2 array or HG-U133+PM array plate. TNFα-induced gene set was comprised of 25 genes  with geometric means ≥1.5-fold relative to unstimulated control conditions (p<0.05) in at least two cell types. The microarray data is available in GEO DataSets under accession no GSE136439.Transcriptomic profiles of individual RNA samples from Crohn’s disease and healthy control mucosal biopsies have been previously published (NCBI/NIH GEO DataSet GSE16879).  Raw cel ad libitum. Mice were acclimated to animal facility housing for a minimum of 5 days prior to initiation of the study. All animal handling and housing procedures were approved and conducted in accordance with Bolder BioPath Institutional Animal Care and Use Committee guidelines [IACUC Protocol #BP12-100].The in-life portions of the murine colitis model studies were performed by Bolder BioPATH, Boulder Colorado. Female Balb/C mice (12 weeks of age) and female Fox Chase C.B-17 SCID mice (5–6 weeks of age) were obtained from Harlan, Inc., Indianapolis, Indiana. Animals were maintained under a 12 hr light/dark cycle and were provided with standard rodent chow and water + separator kit (Miltenyi Biotech). Naïve T cells were then isolated by FACS sorting using a specific antibody against CD45RB . Female Fox Chase C.B-17 SCID, which lack B and T cells, were inoculated intraperitoneally (IP) with a minimum of 4×105 CD4+CD45RBhigh naïve T cells. Animals so treated develop progressive and unrelenting inflammatory colitis. Two dosing paradigms were employed to determine the efficacy of JNJ-40346527: “Prophylactic” dosing was initiated on day 14, a time when naïve T cells are expected to have fully engrafted, but before clinical signs of disease  are manifest  daily  through day 41. SCID mice without T cell transfer (naïve) were included as disease-free controls.SCID mice underwent T cell transfer on day 0. On study day 14, all mice were randomized by body weight into treatment groups, and prophylactic treatment was initiated with 15 mg/kg JNJ-40346527 or vehicle. A third group of mice received vehicle until day 21 at which time therapeutic dosing of compound was initiated. Dosing was administered PO, QID through day 41. As a positive control, other groups of mice were treated intraperitoneally (IP), every third day (Q3D) commencing day 21 with either PBS, or with 3 mg/kg CNTO5048 (anti-mouse TNFα),or 3 mg/kg isotype control. Study 2 included additional parallel groups of mice designated for RNAseq analysis, treated under therapeutic dosing conditions. Specifically, additional SCID mice with adoptive T cell transfer were dosed PO, QD with vehicle or 15 mg/kg JNJ40346527, or IP, Q3D with PBS, 3 mg/kg CNTO5048 or 3 mg/kg isotype control. Also included for RNAseq analysis were naïve SCID mice (no T cell transfer) and a group of immunocompetent Balb/c mice.All mice were weighed on study day 0 and then every other day commencing on day 14. Final body weights were determined on day 41 with subsequent tissue harvest on day 42 following an overnight fast. Percent change in body weight relative to day 14 (Study 1) or day 0 (Study 2) was reported. At necropsy, following an overnight fast, the entire colon was harvested from each animal, inspected visually, measured for length, and weighed. Results were reported as weight/length ratio. Colons were fixed in 10% neutral buffered formalin (NBF) for 48 hours, transferred to 70% ethanol and embedded in paraffin within 1 week of fixation. Two paraffin blocks were prepared from each animal: 1 block containing 3 pieces of proximal colon, and 1 block containing 3 pieces of distal colon (~1 cm long each). Paraffin embedded blocks were sectioned, processed for H&E and scored for pathological changes by a trained pathologist (Bolder BioPATH). Four parameters  were scored and summed to arrive at a sum of histopathology scores. Neutrophil infiltrate fraction was estimated separately. Details of the scoring method and the method to estimate neutrophil infiltrate fraction are provided in Immunohistochemical staining for F4/80 and CD3 was performed at HistoTox Labs, Boulder, CO. Detection of staining was accomplished by capturing digital images from three tissue sections at 40X using Aperio ScanScope XT . Staining was quantified using Aperio GENIE® pattern recognition software which detects the number and intensity of positively stained pixels and the total tissue area. Results were reported as total positive pixels normalized to tissue area.On study day 42 mice were euthanized and mucosa from the middle 2/3 of the colon was harvested by separating the mucosa from the muscle layer as previously described. Tissues GSVA was performed using the CSF-1 and TNFα-induced gene lists (after translation to mouse homologues) and gene lists enriched for M0 and M1 macrophages and activated Th1 T cells. The cell-type lists were derived from a matrix of genes reported to have low/limited expression outside the hematopoietic system but to have high differential expression in 24 subsets of lymphoid and myeloid cells. For use Statistical analyses of clinical indices were performed using Robust Analysis of Variance (ANOVA). The analysis included homogeneity of variance testing (Levine’s test) for assessing whether ANOVA should be performed on transformed values to stabilize variance. Following variance assessment, data was robust transformed to adjust for extreme and outlying values using the Huber-Bickel-Tukey method (a form of M-estimation). The robust transformed data was then fitted by ANOVA and Dunnett’s multiple comparison post hoc testing to evaluate significance of mean differences between select treatments. GSVA scores were analyzed by one-way ANOVA followed by the two-stage linear step-up procedure of Benjamin, Krieger and Yekutieli to determine false discovery rate (FDR) with cutoff at p≤0.05. Discrete data or data consisting of score counts was analyzed by Kruskal-Wallis and Dunn’s multiple comparison test, both nonparametric alternatives to ANOVA.-10).To correlate CSF-1R-driven biology in disease datasets, a CSF-1 gene set was derived by identifying fifty-seven genes induced >2-fold by CSF-1 in ex vivo whole human blood cultures  was published previously. Using thTNFα released from immune cells acts on a wide variety of cell types including epithelial cells during mucosal inflammation. GSVA was similarly performed using a TNFα-induced gene set consisting of twenty-five genes significantly up-regulated by TNFα in multiple epithelial cell lines . The CSFTransfer of naive T cells into the severe combined immunodeficiency (SCID) mice provides a highly reproducible and easily manipulated model of colitis that has characteristics similar to IBD in humans. To asses+ macrophages and CD3+ T cells were investigated by immunohistochemistry in Study 2. F4/80+ macrophages were significantly increased in the proximal and distal colon of mice with TCT-colitis compared to naïve SCID mice  and in the mouse dataset described herein. At the pathway level, there was strong concordance between the mouse model and human disease . FurtherNext, GSVA was employed to assess CSF-1 pathway strength in the model . Mild el+ cells suggested a reduction in macrophages, it may also reflect reduced expression of the F4/80 antigen as CSF-1 is reported to induce F4/80 protein expression.[Although the loss of F4/80pression. To examipression. From thiA similar approach was used to examine enrichment of human M1 and M0 macrophage genes in the aforementioned CD colon mucosa dataset. M1 and M0 gene signatures were elevated in CD patients and was elevated greatest in patients who did not respond to infliximab . Treatme+ T cells staining. IPA analysis of the TCT-colitis and CD gene signatures indicated good concordance at the level of biological pathways. JNJ-40346527 restored those pathways in TCT colitis toward normal similar to pathway restoration in CD patients responding to anti-TNFα therapy. Overall, the results support the ongoing clinical and translational evaluation of JNJ-40346527 (PRV-6527) in CD.In this manuscript we report the enrichment of CSF-1-inducible genes in CD colonic mucosa. Enrichment in CD was mirrored by enrichment of CSF-1-inducible genes in murine TCT colitis, wherein gene sets for M1 and M0 macrophages were also enriched. In TCT colitis, the CSF-1R inhibitor, JNJ-40346527, effectively restored the CSF-1 and macrophage gene sets to baseline. Reduction in CSF-1 and macrophage signal strength correlated with diminished disease activity and histological parameters, and in reduced enrichment of a Th1 T-cell associated gene set and reduced CD3We demonstrated the utility of a novel gene set induced by CSF-1 in human blood. While the gene set includes genes not previously known to be regulated by CSF-1, IPA functional curation confirmed it to be highly enriched for genes significant to macrophage biology. Robust suppression of the gene set in mice treated with CSF-1R inhibitor further supported its use as an indicator of CSF-1R mediated biology. Using the gene set with GSVA provided strong, contextual evidence of CSF-1R-mediated biology in CD, consistent with prior data indicating increased expression of CSF-1R and its ligands in CD., 30 Ther+/- mice were reported to be less susceptible to dextran sulfate sodium (DSS)-induced colitis,[Efficacy of JNJ-40346527 in TCT colitis was consistent with previous investigations of the role of CSF-1/CSF-1R in a second IBD model. Specifically, CSF-1-deficient mice and male CSF-1R colitis,, 18 and  colitis,in situ with little contribution from the circulating monocytes.[+ Ly6Chi bone marrow-derived monocytes.[hi CX3CR1hi cells produced IL-10 and adopted the anti-inflammatory phenotype associated with resident macrophages. However; during colitis, macrophage numbers in the colon increased several fold.[+ Ly6Chi monocytes that replenish resident macrophages in the healthy intestine. These newly recruited cells, described as F4/80int CX3CR1int, become the dominant subset and support key pathogenic mechanisms in colitis through robust expression of TNFα and IL-23 and efficient activation of T cells through MHCII. In the context of an inflamed tissue micro-environment, this population fails to transition efficiently to F4/80hi CX3CR1hi homeostatic cells, [Macrophage lineage cells in IBD and in healthy intestinal mucosa have been intensively investigated, , 31, 32 onocytes.–36 Howevonocytes. Shortly onocytes. but, overal fold. The incrc cells, , 8, 32 a+ cells in mice treated with JNJ-40346527. The conclusion that M1 and M0 macrophages were reduced was supported by sharp reductions in GSVA enrichment scores for gene sets indicative of these populations. Meanwhile, the strong reduction in CCR2 and Ly6c mRNA expression in the mucosa suggests attenuated recruitment of monocytes. CSF-1 is known to induce expression of CCL2 and CCL7[M1 and M0 macrophages express F4/80 antigen, as do monocytes, and IHC of TCT colitis mucosa indicated a deep reduction in F4/80 and CCL7. These a and CCL7–40 altho and CCL7hi in mice and CD14+ CD16- in humans. Bone marrow production of these “classical” monocytes is not CSF-1R-dependent, and although they are precursors of inflammatory macrophages in colitis, they are also precursors of a minor (~10%) population of “nonclassical” monocytes that are Ly6Clo in mice and CD14lo CD16+ in humans and that have increased potential to express TNFα. The maturation step yielding nonclassical blood monocytes is CSF-1R-dependent and inhibited in mice and in humans by anti-CSF-1R antibodies and by JNJ-40346527.[+ macrophages in CD mucosa are CD16+.[The majority (~90%) of blood monocytes are Ly6C40346527., 41 A sure CD16+. Whether re CD16+. MDSC mayin vitro[Whilst resident macrophages are considered protective in healthy mucosa, their function during ongoing inflammation has received less attention. Mature macrophages exhibit a high degree of phenotypic plasticity. This wasin vitro and reviin vitro Thus, itIPA analysis of the TCT colitis and the CD disease signatures revealed striking concordance at the pathway level. In CD, anti-TNFα therapy reversed the disease-associated pathways in responding patients, and the effect of JNJ-40346527 in mice was similar to that achieved by anti-TNFα in humans. Together, the analyses indicate CSF-1R inhibition may be effective in treating CD. Application of the CSF-1 and TNFα gene sets to the TCT colitis expression data revealed both differences as well as similarities in the mechanistic impact of JNJ-40346527 and anti-TNFα. GSVA indicated that the CSF-1 gene set was enriched in TCT colitis, only weakly and non-significantly impacted by anti-TNFα therapy, but robustly normalized following treatment with JNJ-40346527. At the same time, suppression of the M1 and M0 gene sets by JNJ-40346527 and by anti-TNFα indicate their mechanisms of action may intersect by addressing inflammatory macrophages, albeit through different biological mechanisms. Infliximab and CNTO5048 bind to both soluble and transmembrane TNFα to prevent interaction of TNFα with its receptor and the induction of proinflammatory signaling. In additTaken together, our findings suggest that inhibition of CSF-1/CSF-1R signaling by JNJ-40346527 may be effective in managing inflammatory bowel disease. CSF-1 and TNFα may mediate pathogenic processes that exhibit different degrees of dominance in some patient subsets, and this compound may be of particular benefit for patients who fail anti-TNFα therapy. To further investigate the hypothesis, JNJ-40346527 (PRV-6527) is currently being tested in a randomized, double-blind, placebo-controlled Phase 2 clinical trial in Crohn’s disease (EudraCT number: 2017-003017-25).S1 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.


Patients with acute SRMA had significantly higher levels of NGAL in CSF than neurologically normal controls (p < 0.0001). Serum NGAL concentrations were significantly higher in dogs with SRMA than in patients with myelopathy and intracranial neoplasia (p < 0.0001). NGAL levels in CSF were strongly positively associated with IgA concentration , TNCC  and protein content  in CSF. It can be measured in CSF of healthy and diseased dogs. Higher concentrations in canine patients with SRMA as well as positive association with TNCC in CSF suggest an involvement in pro-inflammatory pathways and chemotaxis in SRMA. High serum levels of NGAL in serum of SRMA patients in different stages of disease might reflect the systemic character of the disease.Neutrophil gelatinase-associated Lipocalin (NGAL) is a glycoprotein involved in inflammation acting as an acute phase protein and chemokine as well as a regulator of iron homeostasis. NGAL has been shown to be upregulated in experimental autoimmune encephalomyelitis (EAE) in mice. Increased NGAL concentration in cerebrospinal fluid (CSF) and expression in central nervous system (CNS) has been described in human neuroinflammatory disease such as multiple sclerosis and neuropsychiatric lupus as well as in bacterial meningitis. We aimed to investigate involvement of NGAL in spontaneous canine neuroinflammation as a potential large animal model for immune- mediated neurological disorders. A commercially available Enzyme-linked Immunosorbent Assay (ELISA) for detection of canine NGAL was validated for use in canine CSF. Concentration in CSF and serum of canine patients suffering from steroid- responsive meningitis- arteriitis (SRMA), Meningoencephalitis of unknown origin (MUO), different non- inflammatory CNS disease and control dogs were compared. Relationship between NGAL concentration in CSF and serum and inflammatory parameters in CSF and blood , protein content) as well as association with erythrocytes in CSF, duration of illness, plasma creatinine and urinary leucocytes were evaluated. In dogs with SRMA and MUO, CSF concentration of NGAL was significantly higher than in dogs with idiopathic epilepsy, compressive myelopathy, intracranial neoplasia and SRMA in remission ( Neutrophil gelatinase-associated Lipocalin (NGAL) or Lipocalin-2 (LCN2), is a versatile glycoprotein acting as an acute-phase protein as well as part of the innate immune system mainly by its function as a siderophore-binding protein . It is pIncreased NGAL concentration was found in cerebrospinal fluid (CSF) of human patients with bacterial meningitis , 21 but Until now, data regarding concentration of NGAL in CSF of healthy dogs and canine patients with inflammatory and non-inflammatory CNS disease is lacking. Further investigation of spontaneous inflammation of the central nervous system in dogs –28 couldThis study aimed to test the hypothesis that NGAL concentrations in CSF would be higher in steroid-responsive meningitis-arteriitis (SMRA) and MUO  than in non-inflammatory  CNS disease and neurological healthy dogs. We also hypothesized that there would be a correlation with the total nucleated cell count (TNCC) in CSF, possibly due to chemotaxis.The overall goal of the study is improvement of differentiation of CNS diseases and amelioration of the understanding of the etiopathology of suspected autoimmune canine CNS disease like SRMA and MUO by detection of a new biomarker in canine CSF. Better understanding of chemokine signaling pathways and modulation of neuroinflammation could help to develop more specific treatment strategies for aforementioned pathologies , 29, 30.Paired serum and CSF samples were collected for routine diagnostic workup between December 2011 and May 2018 at the Department for Small Animal Medicine and Surgery of the University of Veterinary Medicine, Hannover, Germany, from client–owned dogs presented to the neurology group and healthy, university-owned beagles . The specimens were aliquoted and stored at −20°C prior to measurement of NGAL concentrations. The study was conducted according to the University's ethical guidelines.Table 2).The dogs underwent general and neurological examination, complete blood cell count (CBC), blood chemistry, CSF analysis including IgA measurement and individual combination of further specific testing  to obtain a definitive or highly likely diagnosis (Table 2).Dogs were retrospectively assigned to different groups regarding diagnosis: SRMA acute (untreated), SRMA recurrence, SRMA in remission (under therapy with glucocorticosteroids), MUO .Tissue samples were obtained following routine necropsy after spontaneous death or euthanasia on owner's request due to poor prognosis and were conducted in accordance with the German Animal Welfare Act within the law of animal welfare, Germany, and following the ethical guidelines of the University of Veterinary Medicine Hannover. No dogs were euthanized for this particular study; sample aliquots were obtained for clinical diagnostics or previously attained during other studies .Information regarding duration of clinical signs, comorbidities, signalment, and course of disease as well as blood and urine parameters and if available biopsy or necropsy results were retrospectively evaluated using the digital clinical data software . If possible, follow up was conducted via control appointments or phone calls to owners or local practitioners  established for canine NGAL in urine, plasma, serum, tissue extracts, or culture media –40 was uConcentration of NGAL in paired serum and CSF samples was afterwards measured following manufacturer's instructions. The mean minimum detectable value given by the manufacturer was 0.56 pg/mL. If the value exceeded the highest value of the standard curve (400 pg/mL), the sample was diluted and measured again. All samples were analyzed in duplicates and mean values were calculated.IgA in aliquoted serum and CSF samples was measured prior to this study as part of CSF analysis in-house using an ELISA as previously described .post hoc test and Wilcoxon two-sample test were performed. Wilcoxon two-sample test was used to compare all groups pairwise . As stability of NGAL in frozen canine samples has not been reported, linear regression analysis by groups was conducted to identify possible influence of sampling year and storage period on NGAL concentration in canine CSF and serum. Scatter graphs from the obtained data were created using GraphPad software .Data was analyzed using the statistical software SAS Enterprise Guide 9.3 . The Kolmogorov–Smirnov-Test was used to analyze the data for normal distribution. This was repeated after identification and exclusion of outliers. Because data did not conform to Gaussian distribution, non-parametric tests consisting of Kruskal–Wallis test, Dunn's-wise see . Values Recovery rate of the four CSF samples spiked with calibrator fluid is shown in In total, 163 CSF and 157 paired serum samples of 140 individual patients were included . Twenty-NGAL was detected in all available CSF and serum samples of patients of the different groups. NGAL concentrations in CSF and Serum of the disease groups and controls are shown in 2(8) = 95.9672; p < 0.0.0001] between the different groups. Wilcoxon rank sum test results are shown in p < 0.0001) between acute SRMA patients and the group with dogs displaying SRMA in remission under corticosteroid therapy, the group with idiopathic epilepsy, intracranial neoplasia, compressive myelopathy and the control group. NGAL concentrations in CSF were significantly higher in dogs with MUO than in SRMA patients in remission under therapy, idiopathic epilepsy patients, the compressive myelopathy group as well as than in dogs suffering from intracranial neoplasia  = 36.4667; p < 0.0001]. Using Wilcoxon-ranked sum test, in dogs with acute SRMA and SRMA in remission, serum levels of NGAL were significantly higher than in serum of dogs with compressive myelopathy  = 3.197].Kruskal- Wallis test identified significant differences for NGAL serum concentration when comparing the nine groups [χAs shown in There was a weak, negative association between duration of illness in inflammatory disease and level of NGAL in CSF. Duration of illness in inflammatory disease  showed a moderate negative association with NGAL concentration in canine serum.A weak, positive association was detected between NGAL serum levels and concentration of creatinine in blood plasma and leucocytes in urine.p > 0.05).Linear regression analysis by group did not identify significant linear regression coefficients of NGAL in CSF or serum through year of sampling  due to sample availability and rare occurrence of relapses in our SRMA population. Therefore, explanatory power is questionable for this group. MUO on the other hand represents a very heterogenous group because different entities are covered by this umbrella term (NGAL in canine CSF was shown to be elevated in dogs with inflammatory disease (SRMA and MUO) in comparison to non-inflammatory CNS disease categories . A positive association between NGAL concentration in CSF and TNCC in CSF was found, which could indicate enhanced chemotaxis as described in experimental studies –17 and illa term , 28, as lla term . For furThe strong, positive association of NGAL levels in CSF and protein content in CSF could be caused by aforementioned blood brain barrier disruption and influx of proteins from the periphery or, on the other hand, be caused by increased intrathecal immunoglobulin synthesis as well as NGAL synthesis in neuroinflammation. The strong positive association of NGAL and IgA in CSF most probably reflects the local synthesis of immunoglobulins in the CNS  and of NNGAL in CSF of clinically normal dogs with SRMA in remission under corticosteroid therapy, which were presented for CSF examination several weeks after initiation of therapy, did show significant decrease of levels compared to acute, untreated SRMA. Interestingly, in those dogs, serum NGAL levels remained high in the course of the disease as it has also been described for IgA levels in dogs , 56. Thipost-hoc test also identified a significant difference between serum samples of acute SRMA and intracranial neoplasia. This could as well-represent the systemic nature of SRMA in contrast to intracranial neoplasia as only one of the included patients suffered from metastatic neoplastic disease (prostatic carcinoma). However, this result should be considered with caution, as there was detectable overlap of concentrations in those two groups as can be seen in NGAL levels in serum differed significantly between acute SRMA cases and SRMA patients in remission when compared to compressive myelopathy patients, but this was not found in MUO patients. In contrast to MUO, SRMA is considered a systemic disease and is associated with neutrophilic pleocytosis in CSF and peripheral blood , 27, 41.n = 2, diagnosed by histopathology) and none of the patients showed increased plasma creatinine at time of sampling for CSF and serum. Sixteen dogs had clinical signs of urinary tract infection (UTI) at time of sampling . In the MUO group, the patient with highest NGAL concentration was euthanized directly after diagnosis on owner's request and an unusual, marked sterile pyogenic meningoencephalitis was found in necropsy. Nevertheless, the other two outliers had a good or satisfying outcome. One patient with very high NGAL CSF levels in the group with compressive myelopathy initially had a good outcome but developed another intervertebral disc herniation at another location 4 months later and the other outlier was a patient who had undergone hemilaminectomy 1 year prior. One of the patients with very high NGAL levels in CSF in the neoplasia group had a suspected choroid plexus tumor, an interesting fact, as the choroid plexus has been described as one of the main intrathecal areas of NGAL synthesis  or were lost to follow up . No remarkable features were detectable in the other outliers regarding NGAL in serum in the myelopathy and idiopathic epilepsy group, who had either a good outcome or were lost to follow up.A correlation of high serum NGAL concentration with negative outcome in patients with sepsis as well as traumatic brain injury has been described , 65. TheTreatment of SRMA and MUO consists of long- term immunosuppressant monotherapy with glucocorticosteroids or with additional immunomodulatory or chemotherapeutic drugs. Treatment so far is unspecific and bears risk of adverse effects as well as insufficient response , 61. TheIn conclusion, NGAL can be detected in canine CSF using commercially available ELISA. It is increased in CSF of dogs with SRMA compared to controls and non-inflammatory disorders and also is higher in dogs with MUO compared to dogs with idiopathic epilepsy, compressive myelopathy and intracranial neoplasia. Concentration of NGAL in CSF of dogs with SRMA in remission under therapy with corticosteroids is significantly lower than in acute SRMA- and MUO- patients. NGAL levels in canine serum were significantly higher in patients with SRMA compared to those suffering from myelopathy reflecting unspecific systemic disease, but not necessarily indicating renal failure as proposed before. NGAL is a promising biomarker in canine CSF and serum, potentially adding another piece of the puzzle to the etiopathology of autoimmune neuroinflammation.The datasets generated for this study are available on request to the corresponding author.Ethical review and approval was not required for the animal study because it is a retrospective study using data and samples acquired previously. Tissue samples were obtained following routine necropsy after spontaneous death or euthanasia on owner's request due to poor prognosis and were conducted in accordance with the German Animal Welfare Act within the law of animal welfare, Germany, and following the ethical guidelines of the University of Veterinary Medicine Hannover. No dogs were euthanized for this particular study; sample aliquots were obtained for clinical diagnostics or previously attained during other studies . Written informed consent was obtained from the owners for the participation of their animals in this study.AT conceived, designed, and supervised the study. NM assisted with study design, collected and analyzed data, performed the experiments, and wrote the first draft of the manuscript. KR gave substantial contribution to the statistical analysis of data. RC conceived in part the study and gave substantial contributions to acquisition, analysis and interpretation of the data. All authors contributed to the critical revision of the manuscript for important intellectual content and have read and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.