Purpose: Autoantibodies against NMDA receptors (NMDAR) in the cerebrospinal fluid (CSF) from anti-NMDAR encephalitis patients have been suggested to be pathogenic since in previous studies using patient CSF, NMDAR-dependent processes such as long-term potentiation (LTP) were compromised. However, autoantibodies may represent a family of antibodies targeted against different epitopes, and CSF may contain further autoantibodies. Here, we tested the specificity of the autoantibody by comparing NMDAR-dependent and NMDAR-independent LTP within the same hippocampal subfield, CA3, using CSF samples from four anti-NMDAR encephalitis patients and three control patients.Methods: We performed a stereotactic injection of patient-derived cell-free CSF with proven presence or absence of NMDAR-antibodies into the rat hippocampus in vivo. Hippocampal brain slices were prepared 1–8 days after intrahippocampal injection, and NMDAR-dependent LTP at the associational-commissural (A/C) fiber-CA3 synapse was compared to NMDAR-independent LTP at the mossy fiber (MF)-CA3 synapse.Results: The LTP magnitude at A/C fiber-CA3 synapses in slices from control-CSF-treated animals (168 ± 8% n = 54) was significantly higher than LTP in slices from NMDAR-CSF-treated animals , although there was some variation between the individual CSF samples. We found residual LTP in NMDAR-CSF-treated tissue which could be abolished by the NMDAR inhibitor D-AP5. Moreover, the CA3 field excitatory postsynaptic potential (fEPSP) was followed by epileptiform afterpotentials in 5% of slices (4/78) from control-CSF-treated animals, but in 26% of slices (12/46) from NMDAR-CSF-treated animals (P = 0.002). Application of the LTP-inducing paradigm increased the proportion of slices with epileptiform afterpotentials, but D-AP5 significantly reduced the occurrence of epileptiform afterpotentials only in NMDAR-CSF-treated, but not in control tissue. At the MF synapse, no significant difference in LTP values of control-CSF and in NMDAR-CSF-treated tissue was observed indicating that NMDAR-independent MF-LTP is intact in NMDAR-CSF-treated tissue.Conclusion: These findings indicate that anti-NMDAR containing CSF impairs LTP at the A/C fiber-CA3 synapse, although there is substantial variation among CSF samples suggesting different epitopes among patient-derived antibodies. The differential inhibition of LTP at this synapse in contrast to the MF-CA3 synapse suggests the specificity and underlines the pathophysiological role of the NMDAR-antibody. N-methyl D-aspartate receptors  is commonly associated with impaired hippocampus-dependent memory function; especially when patients harbor autoantibodies against Most, but not all forms of hippocampal LTP are NMDAR-dependent. In the CA3 area, two distinct afferent pathways converge onto the same pyramidal neurons. On the one hand, associational-commissural (A/C) fibers terminate on distal parts of CA3 apical dendrites displaying a gradient of NMDAR densities towards higher distal expression .LE patient derived CSF containing NMDAR-antibodies was stereotactically injected into the hippocampus on both sides All patients gave their written informed consent to use their CSF samples for scientific purposes. The animal procedures were performed according to national and international guidelines on the ethical use of experimental animals ; all efforts were made to minimize animal suffering and to reduce the number of animals used.n = 28; NMDAR-CSF: 2.65 ± 0.42 days, n = 26). Briefly, rats were decapitated in deep anesthesia with diethyl ether, the brains were rapidly removed and submerged into oxygenated ice-cold dissection solution containing (in mM) 125 NaCl, 26 NaHCO3, 3 KCl, 1.25 NaH2PO4, 0.2 CaCl2, 5 MgCl2 and 13 D-glucose . Then, 400 μm horizontal hippocampal brain slices were prepared using a vibratome , and stored in a holding chamber containing ACSF containing (in mM) 125 NaCl, 26 NaHCO3, 3 KCl, 1.25 NaH2PO4, 2.5 CaCl2, 1.3 MgCl2 and 13 D-glucose .Hippocampal slices were prepared 1–8 days after stereotactic surgery as described previously . Field excitatory postsynaptic potentials (fEPSP) were recorded in the CA3 “s.r.”  at double baseline stimulation intensity. We used this protocol since these synapses express NMDAR-dependent LTP  judged the number of afterpotentials without knowing the animal group. Afterpotentials were identified as local minima following the fEPSP that exceeded double peak-to-peak noise in 10 sweeps before mdBS  and 10 sweeps after mdBS . Then, we calculated the average number of epileptiform afterpotentials before and after the mdBS paradigm of LTP induction. In order to correlate epileptiform afterpotentials and LTP magnitudes, we calculated the fold change of the number of fEPSP peaks after mdBS to the number of fEPSP peaks under baseline conditions. To this end, we counted the number of fEPSP peaks  before and after mdBS and then calculated the ratio of these numbers.P < 0.05; and significant differences were indicated with the exact P values when applicable.Data are expressed as mean values ± the standard error of the mean (SEM). All data were tested for normal distribution (SigmaStat 3.5) and then evaluated using the appropriate statistical test as indicated. The level of significance was set to P = 0.062, two-way-ANOVA; PPR: 0.315, Mann-Whitney U-test; Figures n = 11; Figures n = 8; C1: 173 ± 9%, n = 12; C2: 198 ± 25%, n = 13; C3: 148 ± 12%, n = 18; Figures P = 0.168; Kruskal-Wallis H-test). Hence, under control conditions, A/C fiber LTP was on average 167 ± 8% (n = 62). NMDAR-dependance of A/C fiber LTP was confirmed using the NMDAR-inhibitor D-AP5 in naive , ACSF-injected  and control-CSF-injected animals  was significantly reduced as compared to the averaged control LTP . Following mdBS, however, afterpotentials were seen in all but two slices, and the number of afterpotentials increased significantly . In contrast, slices from the NMDAR-CSF-injected subgroup N2 never showed epileptiform afterpotentials , although the increased number of afterpotentials did not reach statistical significance .The data so far point to a deficit in NMDAR-dependent LTP at A/C fibers in the hippocampal subfield CA3 in animals injected with CSF containing NMDAR antibodies. As shown by the sample traces in Figure s Figure . In addiP = 0.011, χ2 test). On the contrary, D-AP5 almost prevented epileptiform afterpotentials in NMDAR-CSF-treated tissue . The LTP-inducing mdBS paradigm facilitated the occurrence of afterpotentials following the first fEPSP peak in both control and anti-NMDAR subgroups. However, the NMDAR blocker significantly reduced the proportion of slices with epileptiform afterpotentials only in tissue from NMDAR-CSF-treated animals, but not in control tissue. These findings indicate that the stereotactic injection of anti-NMDAR CSF exerts a facilitative effect on the presence of epileptiform afterpotentials and suggest that this facilitation is mediated by targeting NMDA receptors.In summary, under baseline conditions, epileptiform afterpotentials were present in 5 of 86 slices from ACSF and control-CSF-treated animals, but in 12 of 46 slices from NMDAR-CSF-treated animals , but there was no significant animal group effect  and slices from NMDAR-CSF-treated animals (195 ± 22%, n = 10; P = 0.653 vs. control, Mann-Whitney U-test; Figure Since MF fiber LTP is NMDAR-independent (Harris and Cotman, The present study was conducted to address the specificity of CSF from patients with anti-NMDAR encephalitis. To this end, we chose a hippocampal subfield where both NMDAR-dependent and NMDAR-independent forms of LTP are expressed in adjacently located synapses. In the CA3 region, the former is found at the A/C fiber synapse, the latter is expressed at the MF synapse. Our data demonstrate that, on average, A/C fiber LTP was significantly suppressed in slices from NMDAR-CSF-treated animals whereas MF LTP remained intact in these animals. Since A/C fiber LTP could be abolished by the NMDAR blocker D-AP5, but MF LTP was induced in the presence of NMDAR inhibition, our findings demonstrate that CSF from anti-NMDAR encephalitis patients selectively blocks NMDAR-dependent LTP but leaves NMDAR-independent LTP unaltered.The finding that NMDAR-dependent LTP at the A/C fiber synapse is reduced by NMDAR-CSF is consistent with a series of recent reports that have also demonstrated LTP impairment by both commercial and patient-derived NMDAR-antibodies (Zhang et al., While overall LTP at A/C fibers was depressed in tissue from anti-NMDAR-treated animals, the present study also revealed significant variation among the three NMDAR-CSF subgroups tested. Stereotactic application of CSF sample N3 failed to intervene with A/C fiber LTP, but interleaved experiments with D-AP5 also proved the NMDAR dependance of LTP in anti-NMDAR tissue from this subgroup. Although high CSF titers could generally be due to lack of antibody binding to the receptor, these data suggest that the presence of NMDAR-antibodies does not necessarily mean that all NMDARs are targets for these autoantibodies. On the molecular level, the epitope of NMDAR-antibodies has been clearly attributed to the N368/G369 residues of the GluN1 subunit (Kreye et al., But besides methodological aspects, an important issue may also be that patient-derived CSF samples may contain not yet known pathogenic components in addition to NMDAR-antibodies such as autoantibodies against other targets. In addition, treatment with EphrinB2, the ligand of the EphB2 receptor, reversed the effects of patient-derived NMDAR-antibodies (Planagumà et al., What is known about NMDAR subunit expression in CA3? Recently, the CA3 pyramidal neuron was found to express significantly more GluN2B at the A/C fiber synapse as compared to the MF synapse (Carta et al., Clinically, seizures are very common in anti-NMDAR encephalitis, but it is difficult to explain the epileptic condition by the commonly proposed mechanism of NMDAR hypofunction due to receptor cross-linking and internalization (Lynch et al., One attractive, but unresolved explanation for epileptic activity by NMDAR hypofunction is the action of NMDAR-antibodies at NMDARs on GABAergic interneurons, very likely within the hippocampus. In previous reports, either the epileptic phenotype has not been addressed (Dupuis et al., The CA3 interneuronal network has attracted less attention in the past, but in recent years, this network has come into the focus of several CA3 plasticity studies. On the one hand, the CA3 network is highly divergent and one CA3 pyramidal cell may even drive several CA3 interneuron classes. For instance, CA3 pyramidal cells were found to innervate parvalbumin-positive basket cells more extensively than axo-axonic cells, but the NMDAR-mediated EPSC component had been similar in both types of interneurons (Papp et al., NMDAR hypofunction of GABAergic interneurons should result in a disinhibited state of the CA3 network and thus displays an attractive mechanism for epileptogenesis in anti-NMDAR encephalitis. CA3 has recently been found to be the seizure onset zone in most animals following pilocarpine-induced status epilepticus (Behr et al., The LTP induction protocol significantly increased the prevalence of epileptiform afterpotentials in slices from both control-CSF- and NMDAR-CSF-treated animals, but also, albeit very rarely, in slices from naive rats. In most cases, however, only one additional component was obtained, and it is very likely that this reflected the fEPSP decay phase after a population spike rather than any epileptiform afterpotential. Nonetheless, even though we formally counted these components as epileptiform afterpotentials, it is of note that the NMDAR blocker D-AP5 lowered the prevalence of such components only in NMDAR-CSF-treated, but not in control-CSF-treated tissue. Intriguingly, this was also the case for the NMDAR-CSF subgroup N3 which did not block A/C fiber LTP at all. In summary, D-AP5 blocked both epileptiform afterpotentials and LTP. An unexpected finding, however, was that D-AP5 rather tended to raise the proportion of slices showing epileptiform afterpotentials. This might indicate that the most parsimonious explanation—simply diminishing the excitability by acting on remaining NMDARs following D-AP5 application—may not fully explain this unexpected finding. Therefore, the NMDAR specificity of the enhanced incidence of epileptiform potential components associated with LTP-inducing stimuli and may suggest that epitope accessibility of pyramidal cell-expressed and interneuron-expressed NMDAR complexes might be an issue in future.In summary, our data presented here showed that CSF containing NMDAR-antibodies impaired LTP at the A/C fiber-CA3 synapse, but left MF LTP unaltered. This demonstrates NMDAR specificity of patient-derived CSF, but beyond this, differential suppression of A/C fiber LTP and the newly-discovered facilitation of epileptiform activity suggest that autoantibodies may differentiate certain NMDAR subunit compositions and thereby excitatory and inhibitory neurons in CA3.RB, WB, XG, KP and TS performed experiments. CB, RK and TK contributed to the conception and design of the study. RB, WB and XG organized the database and contributed to manuscript preparation. RB, WB, XG and TK performed the statistical analysis. TK wrote the first draft of the manuscript. CB and RK wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the final version of this manuscript for submission.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.


Traditionally, medicine has held that some human body sites are sterile and that the introduction of microbes to these sites results in infections. This paradigm shifted significantly with the discovery of the human microbiome and acceptance of these commensal microbes living across the body. However, the central nervous system (CNS) is still believed by many to be sterile in healthy people. Using culture-independent methods, we examined the virome of cerebrospinal fluid (CSF) from a cohort of mostly healthy human subjects. We identified a community of DNA viruses, most of which were identified as bacteriophages. Compared to other human specimen types, CSF viromes were not ecologically distinct. There was a high alpha diversity cluster that included feces, saliva, and urine, and a low alpha diversity cluster that included CSF, body fluids, plasma, and breast milk. The high diversity cluster included specimens known to have many bacteria, while other specimens traditionally assumed to be sterile formed the low diversity cluster. There was an abundance of viruses shared among CSF, breast milk, plasma, and body fluids, while each generally shared less with urine, feces, and saliva. These shared viruses ranged across different virus families, indicating that similarities between these viromes represent more than just a single shared virus family. By identifying a virome in the CSF of mostly healthy individuals, it is now less likely that any human body site is devoid of microbes, which further highlights the need to decipher the role that viral communities may play in human health. For some time, many body surfaces were believed to be inhabited by few if any microorganisms and the exposure to bacteria, viruses, and other pathogens generally resulted in potentially life-threatening infections. The development of modern cultivation techniques  and nextMuch of the human microbiome research is focused on the bacterial component, yet a growing body of work is demonstrating the ubiquity of viral communities across the body. Studies have shown the presence of viral communities in saliva and dental plaque , lower gThe majority of the viruses found on human body surfaces may be bacterial viruses known as bacteriophages. On some of these surfaces, bacteriophages (phages for short) are persistent and ubiquitous, with some able to persist months and perhaps years . Membersin vitro epithelial barriers  from a cohort of 20 subjects with and without infections, and contrasted these with viromes obtained from a number of different body sites. Our goals were to: (1) demonstrate the presence of a virome unique to the CNS, (2) identify whether viruses found in CSF are similar to those found on other body surfaces, and (3) identify ecological trends in CSF viromes and compare with ecological trends observed on other body surfaces.Human subject involvement in this study was approved by the University of California, San Diego Administrative Panel on Human Subjects in Medical Research. The study was certified as category 4 exempt, which does not require informed consent on behalf of the study subjects. The research was categorized as exempt because it involved the collection or study of existing data, documents, records, pathological specimens, or diagnostic specimens that were recorded in such a manner that the subjects could not be identified, directly or through identifiers linked to the subjects. We sampled CSF from 20 human subjects, body fluids from 5 subjects, and plasma (the supernatant collected after spinning whole blood centrifuged for 15 min that was collected in EDTA-treated lavender top tubes) from 10 subjects . Each ofCSF specimens were collected under sterile conditions using standard protocols . Brieflydevoid of bacteria, we analyzed the CSF specimens for the presence of 16S rRNA. DNA was extracted from CSF using the Qiagen DNeasy Powersoil kit (Qiagen) and further concentrated using the Zymo gDNA Clean and Concentrate kit (Zymo). Purified and concentrated DNA was subjected to PCR using Kapa Hifi Hotstart Readymix (Kapa Biosystems) and PCR forward primer 5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG CCT ACG GGN GGC WGC AG-3′ and reverse primer 5′-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3′ to amplify the V3–V4 hypervariable segment of 16S rRNA. We used the following cycling parameters: 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final elongation step of 72°C for 5 min. Amplicons were purified with Ampure XP beads (Beckman-Coulter) and visualized using a High Sensitivity DNA Kit on a Bioanalyzer (Agilent Technologies). No amplification products could be visualized from CSF specimens from subjects not already known to be infected with bacteria.In an effort to verify that CSF specimens were relatively CSF and body fluid specimens were treated in an identical manner as we have developed for processing of saliva specimens . To dete–5) against the human reference database available at ftp://ftp.ncbi.nlm.nih.gov/genomes/H_sapiens/. Any reads with significant sequence similarities to human sequences were removed prior to further analysis using Ion Assist1. We also screened the virome reads for potential bacterial contamination by mapping reads with low stringency (80% similarity across 20% of the read length) against the database of bacterial genomes2 using CLC Genomics Workbench 9.5.3 (Qiagen). For any virome where >10% of the reads mapped to bacteria genomes, we manually examined the read mapping patterns. When viromes mapped across much of the bacteria genome, they were considered contaminated and were removed from the study even if bacteria were not detected by 16S rRNA amplification.To purify the CSF and body fluid viromes, the samples were filtered sequentially using 0.45 and 0.2 μm cellulose acetate filters  to remove cellular and other debris . CSF andAll reads were assembled using CLC Genomics Workbench 9.5.3 (Qiagen) based on 98% identity with a minimum of 50% read overlap, which were more stringent than those developed to discriminate between highly related viruses . BecauseE-value cutoff value of 10–5. Specific viral sequences were identified using Ion Assist3 by parsing BLASTX results for known viral genes including replication, structural, transposition, restriction/modification, hypothetical, and other genes previously found in viruses for which the E-value was at least 10–5. Each individual virome contig was annotated using this technique, however, if the best hit for any portion of the contig was to a gene with no known function, lower level hits were used as long as they had known function and still met the E-value cutoff. These BLASTX hits were used for comparisons of gene functions in viruses, but not for taxonomic analysis. ORF prediction was performed using FGenesV , and putative functions assigned by BLASTP homology against the NR database (Escore < 10–5). Virus types were determined by parsing the virus families from the TBLASTX best hits of each viral contig with an E-value < 10–20  against the NR database. Resulting virus structures were determined for ORF structures and directionality using MetaGeneMark , and these compiled data used to calculate beta diversity with Bray Curtis distances using QIIME (U-test using SPSS 25 (IBM). We also performed a permutation test  to assess whether viromes from different specimen types had significant overlap using Ion Assist4  between different contigs within the same specimen type. We accepted only high levels of homology that spanned at least 50% of the length of the shorter contig being compared, which would be a minimum length of 75 bp. All contigs in each specimen type were treated as nodes and those contigs that had high homology to other contigs in the same specimen type were added to a network by directing edges between the nodes. After evaluating homologies among all intra-specimen type contigs, networks formed from directed edges/nodes were assigned to individual viruses and nodes with no associations were considered singular viruses. For each resulting network, we added the number of reads assigned to each node on the network and the combined number of reads was used to represent the relative abundance of the virus represented by that network. The relative abundances of all viruses were calculated using this technique, and a new contig spectrum representing the viral population in each specimen type was formed. The contig spectrum from each specimen type then was used as a surrogate for population structures and input directly into the chao1 index . The technique is based on finding high levels of homology amongst contigs within viromes that likely belong to the same virus but were placed into separate contigs due to the limitations of the assembly process . Virome o1 index  to estimWe received approval from our Human Subjects Protections Program to obtain residual CSF specimens from a cohort of 20 subjects who had undergone lumbar punctures within the prior 72 h. Of those 20 individuals, four subjects were diagnosed with CNS infections based on their CSF white blood cell counts and the recovery of pathogens . We also4 VLPs per mL of CSF in the cohort (6 VLPs (devoid of bacteria.We performed epifluorescence microscopy on CSF specimens to identify the presence of virus-like particles (VLPs) prior to virome processing. We found an average of 10e cohort , compare  only a small percentage of the variation could be explained by the sample type , Siphoviridae (mid-sized dsDNA genomes with non-contractile tails), and Podoviridae   could not be assigned to viral families . Of thosl stubs) . Many bul stubs) . Phages Inoviridae. There were no obvious taxonomic distinguishing features of CSF or plasma viromes that could not be identified in the other specimen types.To delineate whether taxonomic trends seen in CSF and body fluid viromes may be similar to those from other human body sites, we compared data from previously sequenced viromes from feces , saliva p < 0.001, R2 = 0.37). However, most of the variation was observed along coordinate 1, where the viromes of urine, saliva, and feces shared similarities. CSF, plasma, and body fluid specimens also clustered similarly on coordinate 1. The breast milk specimens were located between these separate clusters, and clustered separately from all other specimen types along coordinate 3.We next examined beta and alpha diversity in the viromes of each specimen type. We visualized beta diversity using PCoA analysis and tested whether there were significant differences among the sample types with Adonis . The virWe also performed a permutation test to decipher whether the differences observed by PCoA could be further supported statistically . We founp < 0.001) contrast in the alpha diversity observed in the viromes of urine, saliva, and feces when compared to breast milk, body fluids, plasma, and CSF. Urine, saliva, and feces were of relatively high diversity, while breast milk, body fluids, plasma, and CSF were of relatively low diversity   based oniversity . The alpiversity . These dWe next examined whether there may be similar homologous viruses within the ecological clusters. To examine these similarities, we assembled contigs from all specimen types and then measured the proportion of the resulting assemblies that were derived from each specimen type. We found that the majority of the contigs assembled from body fluids also included contributions from CSF, breast milk, and plasma . Few of We next characterized the homologous viruses shared between specimen types to determine whether they were shared from a single family or across multiple different families. We found that there was no single virus family that was shared within or between the different ecological clusters; instead, we found all observed virus families shared across the specimen types . For exaWe characterized the structures of some of the shared homologous viruses among the different body sites to identify characteristics of some virus genomes that are shared within ecological clusters. Viral genomes were constructed by assembling contigs with high stringency among all of the different body sites. We identified Contig1516 that was assembled exclusively with contributions from urine, feces, and saliva . InteresWe also examined the CSF specimens to decipher whether there were viruses present in the CSF of all subjects studied. We created assemblies from the contigs of all study subjects using highly stringent criteria and examined those assemblies to decipher which study subjects contributed to each virus assembly. Of the 2429 CSF assemblies created, we found only 2 virus assemblies (0.08%) that were derived from all 20 subjects . The majWe noted that the specimens belonging to each ecological cluster seemed to correlate with sequencing modality, where the urine, feces, and saliva cluster were all sequenced by semiconductor sequencing , while tPrevailing clinical dogma suggests that the CNS and some other surfaces are generally sterile in healthy individuals. Here we prove otherwise using culture-independent methods to assess the microbial constituents of CSF samples to reveal a pervasive virome that appears to be partially shared with plasma, body fluids, and breast milk. Our discovery of a CSF virome suggests that the CNS is colonized by a somewhat diverse viral community and challenges the clinical dogma that the CNS is free of microbes in healthy individuals. While herpesviruses have been known for some time to inhabit the CNS of symptomatic individuals , the majWe recognize that contamination is of significant concern in identifying a virome present in a body site that previously was believed to be devoid of viruses in healthy people. The primary sources of any potential contamination in CSF samples would likely be derived from the skin, blood, or consumables used in the production of the viromes. We do not believe that the skin is a reasonable source of contamination because of the means by which the CSF is collected. The skin is first sterilized, and then a single puncture with a needle is used to collect the CSF. Because we examined the viromes only from Tube#3 , it is highly unlikely that there could be sufficient remaining skin-derived viruses present from a single skin puncture after 4–8 mL of CSF have already been collected. Often, there is some blood in CSF specimens because blood vessels can sometimes be punctured as CSF is collected. However, for 7/20 of the subjects in this study, no blood was detected in their CSF . We noteWe characterized the viromes of 7 different body surfaces and revealed two separate viral ecological groups dominated by phages with varying properties: a high diversity viral group that included feces, saliva, and urine, and a low diversity group made up of CSF, body fluids, plasma, and breast milk. The robust and diverse bacterial communities inhabiting feces, saliva, and urine might explain why these sample sites had high virome diversity. The low diversity viral group, including CSF, body fluids, and plasma, generally had few if any bacterial communities associated, which likely explains why few VLPs were observed and the low diversity. Interestingly, breast milk was associated with the low diversity cluster, but is also known to harbor relatively diverse bacterial communities , so the There are a number of limitations to the methods we used to isolate and sequence viruses that have been revealed in a series of recent studies . Those mIdentifying the source of the CSF virome is of substantial importance. Prior to performing this study, we hypothesized that the GI tract would be the primary source of viromes throughout the body due to transcytosis of viruses across the gut epithelium into the bloodstream , where tWhile we identified viruses in the CSF of this study population, we were not able to ascertain whether they were stable members of the CSF virome. Because this study was not longitudinal, we do not know whether the viruses we observed would be the same viruses we could identify if we examined the CSF virome of these same individuals days, weeks, or even months later. The invasive nature of obtaining CSF via lumbar puncture prohibited us from performing such an analysis. We believe that many of the bacteriophages found in the CSF arrived into the CNS via transcytosis and do not have viable host cells they are capable of infecting in the CSF. Thus, many of these viruses likely are transient members of the CSF virome.The discovery of a virome in the CSF and various body fluids suggests that there are no body surfaces that are free of microbes. To date, viromes have been identified in saliva, dental plaque, feces, skin surfaces, breast milk, urine, lungs, blood, the vagina, and now body fluids and CSF. While the contents of viromes may appear different based on the techniques used to characterize them, each of the viromes detailed in this study were characterized using the same methodology. This methodology reveals taxonomic differences amongst viromes, including an abundance of microviruses in the feces, siphoviruses in the mouth, and myoviruses in breast milk. The most striking feature amongst the viromes was the high and low diversity ecological clusters identified, where body surfaces with many bacteria generally had high virome diversity, while others such as CSF and body fluids had low diversity. Even in the CSF and body fluid of infected individuals, there was low virome diversity. Thus, differences in alpha diversity appear to be features of each body surface. Further study is necessary to determine the origin of the viruses on many of these surfaces and to decipher why certain surfaces have low viral diversity compared to their counterparts.Human subject involvement in this study was approved by the University of California, San Diego Administrative Panel on Human Subjects in Medical Research. The study was certified as category 4 exempt, which does not require informed consent on behalf of the study subjects. The research was categorized as exempt because it involved the collection or study of existing data, documents, records, pathological specimens, or diagnostic specimens that were recorded in such a manner that the subjects could not be identified, directly or through identifiers linked to the subjects.CG and DP conceived and designed experiments. MeL, JS, LS, KA, JC, and MaL performed the experiments. CG, JB, and DP analyzed the data. CG, JB, RS, and DP wrote and critically reviewed the manuscript.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 FDA approved drug granulocyte-colony stimulating factor (G-CSF) displays anti-apoptotic and immunomodulatory properties with neurogenesis and angiogenic functions. It is known to demonstrate neuroprotective mechanisms against ischemic global stroke. Autophagy is a method for the degradation of intracellular components and in particular, unrestrained autophagy may lead to uncontrolled digestion of affected neurons as well as neuronal death in cerebral ischemia. Mitochondrial dynamics is vital for the regulation of cell survival and death after cerebral ischemia and an early upstream event in neuronal death is mitochondrial fission. We examined the pro-survival mechanisms of G-CSF against apoptosis resulting from autophagy, mitochondrial stress and endoplasmic reticulum (ER) stress.Male Swiss Webster mice (20 weeks of age) were subjected to bilateral common carotid artery occlusion (BCAO) for 30 min. After occlusion, mice were injected with G-CSF (50 μg/kg) subcutaneously for 4 days. Behavioral analysis was carried out using the corner test and locomotor activity test before animals were sacrificed on day 4 or day 7. Key proteins in ER stress, autophagy and mitochondrial stress induced apoptosis were analyzed by immunoblotting.G-CSF improved neurological deficits and improved behavioral performance on corner and locomotor test. G-CSF binds to G-CSF receptors and its activation leads to upregulation of Akt phosphorylation (P-Akt) which in turn decreases levels of the ER stress sensor, GRP 78 and expression of proteins involved in ER stress apoptosis pathway; ATF6, ATF4, eIF2α, XBP1, Caspase 12 and CHOP. G-CSF treatment significantly decreased Beclin-1, an autophagy marker, and decreased mitochondrial stress biomarkers DRP1 and P53. G-CSF also up-regulated the mitochondrial fusion protein, OPA1 and anti-apoptotic protein Bcl-2 while down-regulating the pro-apoptotic proteins Bax, Bak and PUMA.G-CSF is an endogenous ligand in the CNS that has a dual activity that is beneficial both in reducing acute neuronal degeneration and adding to long-term plasticity after cerebral ischemia. G-CSF treatment exerts neuroprotective effects on damaged neurons through the suppression of the ER stress and mitochondrial stress and maintains cellular homeostasis by decreasing pro-apoptotic proteins and increasing of anti-apoptotic proteins. One of the leading causes of disability and death is stroke. More than 87% of strokes are ischemic and caused by obstruction of one or more cerebral arteries . An inadMitochondrial dynamics is vital for the regulation of cell survival and death; importantly, mitochondrial fission is an early upstream event in neuronal death after cerebral ischemia . While tAutophagy is a biological, ordered, and destructive mechanism of the cell that helps to abolish unwarranted or dysfunctional components . In addiThe mitochondrion plays crucial role in the cell death machinery because of its relationship to large list of apoptosis-related proteins . AccumulIn the ER, ischemia elicits accumulation of unfolded proteins, with the successive stimulation of the unfolded protein response (UPR). The UPR involves the detection of unfolded protein by the intraluminal ER chaperone; glucose regulated protein 78 (GRP 78), which then separates from the three distinct ER stress transmembrane sensors; double-stranded RNA-activated protein kinase-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring kinase 1 (IRE1), promoting their release . DownstrWith only limited progress in development of stroke treatments, it is therefore essential to acquire valuable neuroprotective agents to effectively treat stroke. G-CSF controls the generation, proliferation, survival, and maturation of neutrophilic granulocytes and provokes their release from bone marrow to the peripheral blood , 24. G-CWe have used a mechanism based therapeutic approach for stroke to first examine the connection of mitochondrial, autophagy and endoplasmic reticulum stress inhibition in the protective action of G-CSF and second to analyze relevant ER stress pathways in the bilateral common carotid Artery occlusion (BCAO) model of stroke.All animal procedures were carried out in accordance with the guidelines for care and use of Animals and were approved by the institutional animal care and use committee (IACUC) of the Florida Atlantic University, Boca Raton. Male Swiss Webster mice (20 weeks of age) were obtained from Charles River laboratory. Mice were anesthetized with ketamine  plus xylazine . For maintenance of anesthesia, isoflurane concentration was used to 0.5%. Mice were breathing spontaneously via breathing mask throughout the surgical procedure. A rectal temperature probe was inserted. During surgery, mice were resting on a thermostat-controlled heating pad, ensuring a constant core temperature of 37.0 ± 0.5 °C.Anesthesia was maintained as described above. The mouse was placed on its back. The animal’s tail and paws were fixed to the heating pad using adhesive tape. A sagittal ventral midline incision (~ 1 cm length) was performed. Both salivary glands were carefully separated and mobilized to visualize the underlying both Common Carotid Artery (CCA). Both CCA’s were carefully separated from the respective vagal nerves and accompanying veins without harming these structures. Manipulations of the vagal nerves might lead to transient or permanent dysfunction of the parasympathetic nerve system, which has the potential for the occurrence of significant cardiac arrhythmia or even irreversible cardiac arrest. Therefore, it is crucial to avoid any manipulations of the vagal nerves. Both common carotid arteries (CCAs) were isolated, freed of nerve fibers, and occluded using non-traumatic aneurysm clips. Complete interruption of blood flow was confirmed under an operating microscope. After 30 min of ischemia, the aneurysm clips were removed from both CCAs and the incision was sutured closed. Post-surgery analgesics were administered, and animals could wake up within 10 to 20 min after surgery . All micn ≥ 18). Each mouse took part in ten trials, after which we calculated the percentage of turns to each side, recording only those turns involving full rearing along one of the boards [n = 4); we included mice that turned in either direction (n = 14) with a pretest score of 0.50 ± 0.08. Each mouse took part in ten trials for up to 4 days after BCAO.The corner test, which determines an animal’s asymmetric direction of turning when encountering a corner, is used as an indicator of brain injury. We used an experimental corner setup composed of two boards (with dimensions of 30 × 20 × 1 cm3) arranged to form a 30° corner; a small opening was left along the joint between the two boards. The mouse was placed 12 cm from the corner and allowed to walk into the corner, so that the vibrissae on both sides of the animal’s face contacted the two boards simultaneously . Before e boards . This stThe force-plate actometer is an ensemble of mechanical, electronic, and computing elements that embody mathematical and physical principles to produce measurement of whole-organism behavioral attributes of relevance to basic neuroscience research. Methods of calibration and details of data acquisition have been described elsewhere , 28. BriN = 15) and day 7 (N = 15) experiment. Thereafter, in experimental group , rhG-CSF  (50 μg/kg in 0.3 mL Dextrose 5%) was injected subcutaneously 30 min after occlusion and continue daily until the animal sacrificed on Day 4 and Day 7. In control groups , vehicle (0.3 ml Dextrose 5%) was injected subcutaneously 30 min after occlusion, followed by daily administration of the same dose for an additional 3 days. Mice were received vehicle for 4 days before sacrifice. Sham-operated group (N = 10) received the same surgical procedure without occlusion of common carotid artery. After surgery, animals could recover from the anesthesia and given food and water ad libitum. The animals were daily examined for body temperature and weight, and those who had body temperature more than 39 °C after 24 h were excluded from the experiment [Animals were randomly assigned for sham, control, and experimental groups, divide equally for day 4  and decapitated, and then brains were rapidly removed. Using an adult mouse brain matrix , brains were sectioned coronally into 2-mm coronal slices  and incubated for 5 min in a 2% (w/v) solution of 2,3,5-triphenyltetrazolium chloride  at 37 °C for staining followed by collecting samples for western blot , 31.0 C until use. Protein samples were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Western blot was carried out as described previously [Animals were deeply anesthetized by isoflurane (Phoenix) and decapitated, and then brains were rapidly removed. After sacrifice, while the brain was on ice , Brains eviously  with theeviously .P < 0.05 were considered statistically significant. At least three independent replicates were performed for each experiment.All data were expressed as the mean ± SEM. A computer program  was used for statistical analysis. The statistical significance of the data was determined with t-test or one-way ANOVA combined with Dunnett post-hoc or Tukey test for comparison between groups. Differences of G-CSF and its receptor are expressed by neurons in the CNS, and their expression is regulated by ischemia, which points to an autocrine protective signaling mechanism . G-CSF iLevels of G-CSFR expression were decreased by stroke to less than 50% relative to untreated Sham animals. G-CSF treatment can increase expression of G-CSFR by greater than 1.9 fold and 2.1 fold in the frontal brain and middle brain regions respectively relative to vehicle treated BCAO animals. Similarly, we see the effect of G-CSF treatment increases expression of G-CSF protein by 2.4 fold and 1.9 fold in frontal and middle brain regions respectively compared to vehicle treated BCAO.Phosphorylated Akt (P-Akt) is a well-established biomarker for cell protection , levels G-CSF can modulate the unfolded protein response and ER stress-induced apoptosis .In analyzing ER stress pathways, we found that G-CSF decreased expression of GRP78 to less than 70% and less than 60% in frontal and middle regions respectively relative to vehicle treated BCAO. Fig. a and b. We examined all three ER stress signaling pathways  on Day 7.Treatment with G-CSF can decrease cleaved ATF6 level by up to 60% in frontal brain and 40% in middle brain compared to vehicle treated BCAO group Fig. e. Our daWith respect to the IRE1 pathway, the results showed that XBP1 is highly expressed in the frontal & middle region of the brain in the BCAO mouse in comparison to the sham-operated group. Our data showed that G-CSF treatment decreased XBP-1 levels to less than 50 and 40% respectively relative to vehicle treated BCAO. Fig. h.Cleaved caspase − 12 protein expression was decreased in the GCSF treated group to less than 75% relative to vehicle treated BCAO  causing apoptosis . We founIn this study, pro-apoptotic P53  arranged to form a 300 corner. Normal mice were found to show a rate of 50 ± 8% (symmetric and without bias) before BCAO-30 min surgery. At 4 days after BCAO we observed a significant increase in asymmetric turning (~ 90% to one side) in BCAO mice. When facing a 30° corner. BCAO mice without treatment showed significant asymmetric turning (~ 90%) beginning one day after the procedure and persisting for at least four days  . This deays Fig. a and b. ays Fig. c and d.n = 5, p < 0.05) and day 7  treatment window for global ischemia, the initial dose of G- CSF was administered 24 h post-BCAO. This initial dose was then followed by a single application, of the same dose, for another 3 days resulting in a total of 4 days of G-CSF administration. Our previous paper reported that the initial administration of G-CSF (50 μg/kg body weight. s.c.) after 24 h post-MCAO in rat has beneficial effects on stroke with reduction of infarct volume .We have demonstrated that G-CSF was protective against ER stress elicited apoptosis by downregulating ER stress-induced apoptotic CHOP and downregulating the expression levels of ATF4, XBP1, eIF2α and cleaved ATF6, the down- stream targets in the ER stress sensor pathways: PERK, IRE1 and ATF6, respectively . GRP78 has several functions in the cell including protein folding in the ER, the UPR and inhibition of apoptosis , 37. GRPThe UPR disconnects GRP78 from its sensors  inside the ER lumen in response to buildup of unfolded proteins. Our previous study confirmed that G-CSF has beneficial effects on the protection against ER stress in the penumbra and core of the MCAO infarct . FollowiATF6 signaling is mostly pro-survival with little evidence linking it to cell death , 49. We In ER stress IRE1a signals to synthesize the mRNA for the transcription factor X box binding protein (XBP1). XBP1 is responsible for regulating a specific subset of UPR target genes, involved in folding and ER-associated degradation (ERAD) . IRE1a aUnder severe stress, the UPR pathways signal for apoptosis via the key transcription factor C/EBP homologous protein (CHOP), which differentially regulates transcription of genes encoding both pro- and anti- apoptotic Bcl-2 family members respectively  promotes neuroprotection during cerebral ischemia . We demoThe mitochondrion appears to be condensed at the point of contact with the ER, pointing to an essential role for the ER–mitochondria association in the initiation of mitochondrial fission . MitochoMitochondrial p53 is known to form inhibitory complexes with pro-survival Bcl-XL and Bcl-2 proteins, resulting in cytochrome c release from mitochondria  DRP1 (b) OPA1 (c) P53. Analysis was conducted comparing of the protein signals seen within the middle region of the cerebrum in mice brain using the Immunohistochemistry technique. a, b, c The signal of DRP1 protein was detected to be more frequent/stronger in the vehicle animals and least in the animal treated with G-CSF after 30-min BCAO. Sham animal is observed to be in-between. d, e, f The signal of OPA1 proteins are detected more frequent in animals treated with G-CSF in comparison to vehicle animals after 30-min BCAO. Sham animals are seen to have no OPA1 protein signal. g, h, i P53 protein signaling is detected more frequent in vehicle animals in comparison to animals treated with G-CSF after 30-min BCAO. Sham animals are seen to have no P53 protein signal. For all images, nuclei were counterstained with DAPI. DRP1, OPA 1 and Neu-N protein are labeled in red, P53 is labeled in green. a, d, g represents Sham; b, e, h represents BCAO and c, f, i represent BCAO + G-CSF. Scale bar = 20 μm (microns). (n = 3).


The recognition of active inflammation in the central nervous system (CNS) in the absence of infectious agents is challenging. The present study aimed to determine the diagnostic relevance of five selected chemo/cytokines in the recognition of CNS inflammation and in the context of traditional cerebrospinal fluid (CSF) biomarkers  and clinical diagnoses.C-C and C-X-C motif ligands  and interleukin (IL) 6 levels in the CSF and serum from 37 control and 87 symptomatic children with ten different (mostly noninfectious) inflammatory CNS disorders (16 of which had follow-up samples after recovery) were determined using Luminex multiple bead technology and software. Nonparametric tests were used; p < 0.05 was considered statistically significant. Receiver operating characteristic curves were constructed to analyze controls and 1) all symptomatic samples or 2) symptomatic samples without CSF pleocytosis.Compared with the control CSF samples, levels of all investigated chemo/cytokines were increased in symptomatic CSF samples, and only IL-6 remained elevated in recovery samples (p ≤ 0.001). CSF CXCL-13 levels (> 10.9 pg/mL) were the best individual discriminatory criterion to differentiate neuroinflammation (specificity/sensitivity: 97/72% and 97/61% for samples without pleocytosis), followed by CSF WBC counts (specificity/sensitivity: 97/62%). The clinical utility of the remaining CSF chemo/cytokine levels was determined in descending order of sensitivities corresponding to thresholds that ensured 97% specificity for neuroinflammation in samples without pleocytosis : IL-6 , CXCL8 , CXCL10  and CCL2 . Different diagnosis-related patterns of CSF chemo/cytokines were observed.The increased CSF level of CXCL13 was the marker with the greatest predictive utility for the general recognition of neuroinflammation among all of the individually investigated biomarkers. The potential clinical utility of chemo/cytokines in the differential diagnosis of neuroinflammatory diseases was identified. Neuroimmunological diseases represent a broad spectrum of rare but serious disorders. The recognition of active inflammation in the central nervous system (CNS) in the absence of infectious agents is challenging. Currently available cerebrospinal fluid (CSF) or serum biomarkers and magnetic resonance imaging (MRI) have limited sensitivity and specificity, and novel biomarkers of CNS inflammation are constantly being assessed –3.Under neuroinflammatory conditions, circulating immune cells in the peripheral blood gain access to the CNS, and CSF pleocytosis is a crucial hallmark of neuroinflammation . CSF whiBoth animal and human studies show that chemokines play an important role in (neuro)inflammation, as chemokines and their corresponding receptors are required for leukocyte migration and function –12. GliaCertain C-C and C-X-C motif ligand  chemokines are frequently investigated in patients with CNS disorders of different etiologies, but their clinical utility has yet to be clearly established . CXCL13,In addition to chemokines, IL-6, a pleiotropic cytokine with contradictory proinflammatory and neuroprotective functions, has also been frequently investigated in the context of neuroinflammation. Increased CSF IL-6 levels have been observed in patients with infectious meningitis and some noninfectious inflammatory CNS disorders, but not in patients with MS , 37.In the present study, we investigated the clinical utility of CCL2/MCP-1, CXCL8/IL-8, CXCL10, CXCL13, and IL-6 in the general recognition of neuroinflammation by evaluating their CSF and serum levels in a large cohort of pediatric patients with various (mostly noninfectious) inflammatory CNS disorders. We also compared these markers to traditional CSF neuroinflammatory biomarkers, such as WBC counts, oligoclonal bands (OCBs) or markers of a blood-brain barrier (BBB) failure. Finally, we outlined “disease-specific chemo/cytokine patterns” for selected diagnoses using multiparametric visualization tools.This study was approved by the Ethics Committee at Motol University Hospital. Informed written consent to a detection of chemo/cytokine levels in CSF and serum was obtained from parents or guardians of all pediatric participants. The data were analyzed anonymously.In total, 140 pairs of CSF and serum samples from 37 controls and 87 symptomatic children (patients) with various inflammatory CNS disorders (follow-up samples after recovery were obtained from 16 of these patients) were analyzed . All spePatients  were diagnosed with the following inflammatory CNS disorders: acute disseminated encephalomyelitis  and ADEM followed by optic neuritis , anti-N-methyl-D-aspartate receptor encephalitis , Rasmussen encephalitis , acute cerebellitis of unknown etiology , encephalitis of unknown etiology , clinically isolated syndrome , MS (n = 17), neuromyelitis optica spectrum disorders  and NB (n = 11). The clinical manifestations of CIS were optic neuritis (n = 15), acute myelitis (n = 2), brainstem syndrome (n = 2), and focal supratentorial syndrome (n = 2). The clinical manifestations of NB were meningitis (n = 8), meningoradiculitis (n = 1), meningoencephalitis (n = 1), and meningomyeloencephalitis (n = 1).Symptomatic inflammatory samples were collected from all patients at the time of presentation of their clinical symptoms, which were 1) acute , 2) progressive , and 3) relapsing . The majority of the symptomatic samples were collected before immunotherapy (72 of 87). The immunotherapy in 11 of 15 patients before sampling included steroids  and intravenous immunoglobulins ; three patients were treated with azathioprine and one with cyclosporine. Follow-up asymptomatic recovery samples were available from 16 of 87 patients. The absence of the initial and any new clinical symptoms defined a recovery. The time at which recovery samples were collected differed from patient to patient and diagnosis, and the median was 5 months after onset of the first disease symptoms (range 7 days to 2 years).Controls  were children with various symptoms for which neuroinflammation was initially considered, but ultimately excluded. Detailed examinations of these children also excluded a neurodegenerative etiology for their symptoms, and CSF, blood and MRI findings were within the normal range. The symptoms and/or final diagnoses were: headache (n = 13), different ophthalmologic impairments , different psychological/psychiatric manifestations , pavor nocturnus (n = 1), and trauma of the plexus brachialis (n = 1).All children in the study underwent an MRI, lumbar puncture and blood tests. Other specific examinations necessary for diagnosis were indicated in individual cases but those are not relevant for the current study.3/ serum albumin [mg/L]). CSF findings of a WBC count < 5 x 106 cells/L, OCB of 0–1, protein levels < 400 mg/L, and Qalb < 5 were considered normal  and occasionally anti-myelin-oligodendrocyte glycoprotein (MOG) IgG antibodies were performed using commercial kits based on indirect immunofluorescence technique .A routine analysis of CSF included a determination of the following clinically accepted traditional biomarkers: WBC count, OCB level via isoelectric focusing of immunoglobulin (Ig) G, protein levels, and CSF/serum albumin ratio . Our multiplex panel also included other cytokines that are associated with specific immune responses, lymphocytes functions, and immunoregulation . The methodological details including assay protocol, standards and sensitivity are available at the manufacturer’s website, http://www.thermofisher.com. All samples were measured undiluted and in doublets. The chemo/cytokine standards were assayed in the same manner as patient samples. The data were collected using a Luminex-100 system .The concentrations of CCL2/MCP-1, CXCL8/IL-8, CXCL10, CXCL13 and IL-6 were measured using Luminex multiple bead technology. We created our own multiplex panel by combining multiple simplex kits with a basic kit according to the manufacturer’s instructions  were determined using receiver operating characteristic (ROC) curves and by measuring the area under the ROC curve (AUC). ROC curves for combinations of biomarkers were constructed based on predictive models using 50% of patients; the remaining 50% of patients were used as a test group. The thresholds that provided an optimal trade-off between specificity and sensitivity as a criterion for discriminating CNS inflammatory processes were calculated . In addition to the optimal thresholds, the 97% specificity thresholds and their corresponding sensitivities were also derived from the ROC curves for each CSF chemo/cytokine .Chemokines CCL2/MCP-1, CXCL8/IL-8, CXCL10, CXCL13 and cytokine IL-6 were detected in the majority of the patients’ samples. Compared with controls, levels of these chemo/cytokines were significantly increased in symptomatic inflammatory CSF samples  and CCL2/MCP-1 levels showed no significant differences . In recoThe WBC counts, protein levels and CSF/serum albumin ratios in symptomatic inflammatory samples correlated with all CSF chemo/cytokine levels, except for CCL2/MCP-1. The strongest correlation with WBC counts was observed for CXCL13 . The presence of OCBs in the symptomatic inflammatory samples correlated with CSF CXCL13 and IL-6 levels .Traditional and chemo/cytokine biomarkers were evaluated individually and in selected combinations. ROC analyses using all symptomatic samples and controls were performed.6 cells/L, yielding a specificity of 97% and sensitivity of 62%. The combination of all four traditional biomarkers improved the predictive accuracy of the test by increasing the specificity to 100% and sensitivity to 87%  than sensitivity (50–72%) for differentiating CNS inflammation. The best individual discriminative criterion among the traditional biomarkers was the WBC count, with an optimal threshold of 2.165 x 10y to 87% .The individual AUCs for the CSF chemo/cytokine levels were all greater than 0.70. The best discriminative criterion among investigated chemo/cytokines for differentiating CNS inflammation was the CXCL13 level, with an optimal threshold of 10.9 pg/mL; compared with WBC counts, the corresponding specificity was similar (97%), and the sensitivity was higher (72%). In contrast to the traditional biomarkers and CXCL-13 levels, the optimal thresholds for the remaining chemo/cytokines showed lower specificity (range 59–68%) than sensitivity (71–84%). Combination of two chemo/cytokines with the highest individual AUCs (CXCL13 and CXCL8/IL-8) did not reach the individual specificity of CXCL13, but did increase the sensitivity of the test to 86%. Determination of the 97% specificity threshold generated a cut-off value for each chemo/cytokine that ensured a high probability (≥ 97%) of detecting the CNS inflammatory process, but modified a sensitivity. Equalizing of the specificity enabled to compare corresponding sensitivities and assess the clinical utility of the remaining chemo/cytokines for the recognition of CNS inflammation as follows: IL-6 (sensitivity 40%), CXCL10 (38%), CXCL8/IL-8 (31%) and CCL2/MCP-1 (12%) .The individual AUCs for serum chemo/cytokines levels did not exceed 0.64, which was the AUC for CCL2/MCP-1, yielding 62% specificity and 69% sensitivity .6/L). Moreover, in 23/52 samples, the other traditional CSF biomarkers also showed no noticeable abnormalities. In addition, no infectious agents were detected in these CSF samples. Thus, we performed a separate analysis of this subgroup; the optimal and 97% specificity thresholds were determined by ROC analyses using only symptomatic inflammatory CSF samples without pleocytosis and controls.In 52 symptomatic inflammatory samples (60%), the WBC counts were within the clinically accepted normal range  than sensitivity (72–90%) for differentiating neuroinflammation. According the AUC, CXCL8/IL-8 was the best discriminative criterion in this subgroup, followed by CXCL13. However, in contrast to CXCL13, the optimal threshold for CXCL8/IL-8 yielded a lower specificity (68% vs. 79%). Combinations of all five chemo/cytokines or the best two (CXCL8/IL-8 and CXCL13) improved the predictive accuracies of the test by increasing specificity to 94% and sensitivity up to 88%. The use of the 97% specificity threshold and its corresponding sensitivity helped us to assess the clinical utility for each chemo/cytokine for the recognition of neuroinflammation in patients without CSF pleocytosis as follows: CXCL13 (sensitivity 61%), IL-6 (34%), CXCL8/IL-8 (26%), CXCL10 (24%) and CCL2/MCP-1 (10%) .When we focused on CSF chemo/cytokines levels in the context of diagnoses and 97% specificity thresholds (derived from the ROC curve for samples without pleocytosis), we noted certain diagnosis-related differences . CXCL13 Finally, we created multiparametric graphs and displayed the sum of medians of investigated chemo/cytokine levels for those diagnoses for which at least five CSF samples each were available. Thus, we revealed the different proportional and quantitative chemo/cytokines involvement in each diagnosis . NB and Chemo/cytokines play a substantial role in neuroinflammation . HoweverIn contrast to many previous studies, we did not primarily focus on a particular disease or group of diseases, such as MS –36. We tThe proper evaluation of chemo/cytokine levels critically depends on the controls . Individth percentile of the control values was used as a cut-off to analyze patient samples [Clinicians need reliable markers and cut-off values that indicate neuroinflammatory conditions with a high probability . In some samples . A ROC a samples . Thus, dTaken together, our data generally demonstrate the insufficient sensitivity of currently available traditional CSF biomarkers in discriminating noninfectious inflammatory CNS disorders and highlight CXCL13 as the best individual biomarker of neuroinflammation of various etiology. CXCL13 levels were increased in patients with all diagnoses, and consistent with other reports, CXCL13 levels correlated with the traditional biomarkers , 50, 54.We had to specifically focus on disease-specific chemo/cytokine CSF levels to better compare our findings with previously published data in pediatric and adult patients. The recent review shows congruent results in certain CNS disorders of either pediatric- or adult-onset . Thus siUsing multiparametric graphs, we finally utilized the potential of our data to visualize the different proportional and quantitative involvement of chemo/cytokines in certain diagnoses. All these disease-related findings supported the hypothesized additional clinical utility of the investigated chemo/cytokines in the differential diagnosis of neuroinflammatory conditions. Nevertheless, we are aware that further studies are needed to identify disease-specific chemo/cytokine patterns that may have diagnostic relevance in the future.Our study provided unique data on the levels of five chemo/cytokines using the same assay method simultaneously in a pediatric cohort of patients with one of ten different inflammatory (mostly noninfectious) CNS disorders. The increased CSF level of CXCL13 was the biomarker with the greatest predictive utility for the general recognition of neuroinflammation among all of the individually investigated biomarkers. The results of our study also revealed the potential clinical utility of the investigated chemo/cytokines in the differential diagnosis of certain neuroinflammatory diseases.S1 FigComparison of chemo/cytokine levels in paired symptomatic and recovery samples (n = 16) using the Wilcoxon signed-rank test and comparisons between recovery samples (n = 16) and controls (n = 37) using unpaired Mann-Whitney tests are displayed, the statistical significance is indicated.(TIF)Click here for additional data file.S1 TableTable A. Clinical and laboratory characteristics of the samples stratified according to diagnosis. Table B. Number of patients with increased CSF chemo/cytokines levels exceeding the “97% specificity threshold” stratified according to diagnosis.(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(XLSX)Click here for additional data file.


This study collected behavioral data for testing how regional contrast changes due to the addition of cosmetics would affect attractiveness ratings. In addition, we used an established model of asymmetry to look for a correlation between changes in attractiveness related to the application of cosmetics to specific regions of the face and changes in symmetry. Using this asymmetry model we compared female faces with and without makeup. Specifically, we used a highly controlled set of grayscale faces in which makeup application was standardized to explore these issues from a perceptual perspective. The human data showed that adding upper eye makeup significantly increased attractiveness ratings. In contrast, increases in contrast to the lower eyes and lips did not lead to increases in attractiveness ratings; application of cosmetics to the lower eyes led to a significant decrease in attractiveness. We found that for the makeup condition that led to increased attractiveness, asymmetry did not change significantly when makeup was applied to the female faces. This suggests a role for mechanisms other than symmetry related to increases in attractiveness related to makeup use in females. Attractiveness is a trait that may influence behavior across the lifespan, as data indicate that even very young infants discriminate between attractive and unattractive faces . Specifically, he manipulated the contrast difference between both the mouth and eye regions and the immediately surrounding skin of both these areas. Russell found that making a face appear more feminine, by increasing the contrast in both the mouth and eye regions, led to an increased perception of attractiveness for female faces  which are costly and permanent, makeup provides non-permanent alterations that tend to be simple and cost-efficient, and thus are more common. Despite this, cosmetic use has the potential to substantially change a person’s appearance by changing facial contrast, skin quality and color. Other studies have reported significant increases in attractiveness when female faces are shown with cosmetics . Cash etThe general finding that makeup enhances female attractiveness applies outside the laboratory as well. For example, In this study, we tested a face database consisting of 30 individual females. They were recruited from the human participant research pool of the Psychology Department at California State University Fullerton. The participants were given course credit for participation and gave permission for their images to be used in experiments.CF), as used by CF values were similar across all three regional applications of makeup . A one-way ANOVA showed that there was no significant difference among these CF values, F = 0.126, p > 0.05. Sample images from our database are shown in Figure We chose to use a more ecologically valid method of applying cosmetics directly to the faces (rather than a computer manipulation of contrast or morphing). We had an experimenter apply the makeup so that the application would be consistent across this particular set of faces. The faces were collected using a standardized method, holding constant the lighting and distance of the camera. Participants were required to have no previously applied makeup on their faces prior to adding cosmetics and their hair was pulled back from the face to ensure a clear view of the entire face. The same colors of makeup were applied to each participant by an experimenter to one of three areas of interest: lipstick applied to mouth region (lips); eye shadow and mascara applied to the upper eye region; eye shadow applied to the lower eye region Figure . The speA summary of our database appears in Table Each image was aligned and normalized according to the steps mentioned in Our experiment consisted of three stages:(1)Computing quantified asymmetry measures from these faces to study how asymmetry varies with the application of different types of artificial makeup.(2)Human participant rating of these faces on attractiveness, for both the original faces (no makeup) and the same faces with makeup applied to one region.(3)Comparing attractiveness ratings with asymmetry metrics to study the relationship between these two metrics, both with and without makeup.In this study we explored whether changes in the perception of facial attractiveness due to a standardized application of cosmetics use were related to changing the symmetry of the face. It is possible that makeup is able to mask or offset asymmetries in the face, serving as at least one possible contributing factor in the specific mechanism for how makeup increases attractiveness.Once a face midline is determined via alignment and normalization, each point in the normalized face image has a unique corresponding point on the other side of the image (given an even number of columns in the image). We used a coordinate system in a normalized face image, with the X-axis being perpendicular to the face midline and the Y-axis coinciding with the face midline. “I” denotes a normalized face, and “I”’ denotes its vertically reflected image. The asymmetry measurement is then defined as the pixel intensity variation between the two halves of the face (in a similar way as introduced in Density Difference (D-face):Dx, Dy. Note that the Dx features span along the x-axis on the coordinate system, thus from the middle of the face to the side of the face. The Dy features, on the other hand, span along the y-axis of the coordinate system, thus from the forehead to the chin. Each dimension is called a feature, so we have two related but differently quantified measures representing facial asymmetry. These features were computed for each image in our database. The means for D-face calculations are show in Table Figure We would like to note here that there are several other ways to measure facial asymmetry, for example methods based on edge orientations . ThornhiThirty-four human participants (different from those used to create the face database) were recruited using the CSUF psychology department research participant pool. Participants were either given course credit or paid $7 for their participation. Participants rated the 60 facial stimuli (30 faces shown both with and without makeup). The images were completely randomized within a single block. Participants rated the faces using a 7-point Likert-like scale on which “1” indicated “very unattractive” and “7” indicated “very attractive.” Trials started with a fixation cross shown for 500 ms, followed by a face image. The face image was shown until the participant had responded or 5000 ms had elapsed.Dx graph clearly shows that asymmetry of the face gradually decreases from the side of the face towards the center, on average. For Dy, on the other hand, the highest amount of asymmetry seems to be near the nose bridge area and then it decreases as one moves along towards the forehead and also towards the chin. Comparing the asymmetry values across the two dimensions, we find that the nasal area has the highest value, thus indicating that the area around the nose has maximum asymmetry. This is consistent with prior studies that observed that the asymmetry of the nose bridge is significant and it plays an important role in face recognition .It is clear that the overall pattern of asymmetry in both dimensions remain unchanged as a result of makeup application, regardless of whether it was applied to the upper eyes, lower eyes, or lips. Another thing that we observed is that application of makeup on the lips decreases asymmetry whereas the makeup in the eye region increases asymmetry. Moreover, the nose area has the highest amount of asymmetry in the whole face in all cases  = -1.87; p = 0.0471] whereas the other types of makeup did not . The effect size (Cohen’s d) for the significant t-test for the difference of asymmetry between faces with no makeup and makeup under the eyes was calculated as 0.65 which shows a moderate effect. We thus conclude that cosmetics applied to the upper eye region and lips did not significantly alter the average asymmetry of the face, whereas under eye makeup led to a significant increase in asymmetry.Table F = 39.67, p < 0.0001, η2 = 0.0135. A significant main effect of contrast level was also found, F = 12.15, p < 0.001, η2 = 0.006. The interaction between contrast level and regions was also significant, F = 26.16, p < 0.0001, η2 = 0.024. To explore this interaction further, we used planned contrasts to test for differences between the same faces with no cosmetics applied  and the different regional makeup applications. Faces with makeup applied to the upper eyes  were rated as significantly more attractive than original faces , t(66) = -2.15, p = 0.04. Faces with makeup applied under eyes  were rated as significantly less attractive than original faces , t(66) = -7.94, p < 0.001. Finally, for faces with lip makeup applied there was no significant difference between faces with lip makeup  and original faces , t(66) = -1.43, p > 0.05.We analyzed attractiveness ratings using an ANOVA with a two [contrast level: original  vs. changed (with makeup added)] x three  design to test for effects in responding. The dependent variable was the attractiveness rating. An overall significant main effect of region was found in attractiveness ratings, The findings suggest that for these images of female faces, an increase in attractiveness ratings was found only for the application of upper eye cosmetics. In contrast, female faces with makeup applied to the under eye region were found to be significantly less attractive than the same female faces with no makeup. Female faces with only added makeup to the lip region were not rated different from the same faces without makeup  whereas under eyes makeup increases facial asymmetry. Given the perceived notion that higher asymmetry is associated with lower attractiveness, based on these measures we could conclude that upper eye and lip makeup slightly increased facial attractiveness (as measured by symmetry measures) while make up under the eyes decreased attractiveness.D-face values and the mean attractiveness ratings in order to obtain a baseline, and a negative correlation of -0.16 was observed. Although this conforms to our prediction, note that the association is weak, and not statistically significant (p > 0.05). The scatterplot . Figure D-face values and the mean attractiveness ratings, for faces with and without any makeup, and they both depicted a slightly negative linear association.For the original faces without any cosmetics, we computed the average Facial asymmetry is a strong biometric, with the potential of explaining how makeup affects attractiveness. The relationship between facial symmetry and facial attractiveness is well-established. Thus, it seems possible that cosmetics may at least in part increase attractiveness by decreasing asymmetry. In this paper we explored this via a study of quantitative asymmetry metrics and human behavioral attractiveness ratings.Our results suggest that for human participants, the addition of cosmetics to some regions increased attractiveness ratings (upper eye makeup), whereas other cosmetics applications decreased attractiveness ratings (lower eye makeup), and still other types of cosmetics applications did not change perceived attractiveness ratings (lips). These data suggest that changes in contrast due to cosmetics added to the mouth and eye regions separately are not equivalent. This refines the results reported by ®). It is interesting to note that across cultures the use of black or brown makeup to darken eyelashes is relatively universal, whereas other colors are not commonly used. This is not true of other types of makeup, in which color choices are much more variable. On the other hand, darkening the under eye region may have indicated poor health, lack of sleep, or disease  or a smaller effect . A similar type of effect might be found for individuals rated as very unattractive. The symmetry analyses of this set of faces show that the addition of makeup sometimes increased symmetry, but at other times it decreased symmetry. These changes in symmetry were consistent with what we predicted would be the most attractive makeup applications, such that upper eye and lip cosmetics led to increased symmetry  and under eye makeup led to decreased symmetry . Most importantly, the changes in symmetry due to the addition of cosmetics were unrelated to differences in attractiveness ratings. These results suggest that our makeup application did not increase human attractiveness ratings due to a decrease in asymmetry. These results are somewhat surprising, as it seems logical to predict that adding contrast to the eye and lip regions could as a natural consequence make those regions more symmetrical. Even if that is sometimes the case, this change in this case was not big enough to drive the increase in attractiveness we measured.Facial asymmetry is not only important for attractiveness perceptions, it has also been used in classification algorithms for performing human identification, particularly under external variations caused by expression and illumination changes . The resAnother limitation of our study is that, in order to increase control, the cosmetics were applied uniformly to all the participants’ faces by an experimenter. It is possible that when a person applies their own makeup they may apply it in a way that significantly decreases asymmetry (whether consciously or unconsciously). However, it is the case that makeup applied to the upper eyes increased attractiveness. Therefore, cosmetics applied this way were still effective in increasing attractiveness, but did not similarly affect facial symmetry. In addition, we did not use color images, in order to limit this exploration to changes in contrast. Many studies have found that color plays an important role in how makeup affects attractiveness , and we In this study we found that increases in contrast affected attractiveness only for the upper eye region, suggesting that the effects reported by This study protocol was approved by the Institutional Review Committee of California State University Fullerton. Human participants gave written informed consent in accordance with the Declaration of Helsinki.AK and JP conceived of the idea for the behavioral study. SM and JP conceived of the idea for the asymmetry analyses. AK collected the behavioral data. SM designed the asymmetry model and conducted the asymmetry analyses. AK and JP conducted the behavioral analyses. All authors contributed to the writing of this manuscript.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.


Hematopoietic cell transplantation (HCT) activity is increasing at an unprecedented pace with > 50,000 allogeneic transplants occurring annually worldwide. Establishing a functional HCT donor registry can be very challenging with respect to ethnicities, financial, technical, and geopolitical issues. Extensive planning steps are essential to overcome the expected challenges while establishing the registry, and to maintain its functionality. A few strategies can help move past those challenges and push the development of such registries forward. Authorities involved in HCT donor registry establishment will have to balance the advantages and costs of such a project and accommodate the emerging alternatives such as cord blood or related haploidentical transplants. Miscalculations and incomplete understanding of the various aspects of the process can have tremendous impact on the optimization of a HCT donor registry especially in developing countries. Herein we present some challenges in establishing such a registry and present potential solutions. This highlights the importance of local registries because generally ethnic groups share unique common haplotypes [Hematopoietic cell transplantation (HCT) is a well-established curative therapy for certain types of cancers, blood disorders, genetic diseases, immunodeficiencies, and autoimmune conditions. The success of allogeneic HCT  depends in large part on the degree of human leukocyte antigen (HLA) matching between recipient and donor. The ideal donor for allo-HCT is a matched sibling donor (MSD) but the overall chance of finding a MSD in each family is only ~ 25–30%  in the wplotypes –8 and thCancer is one of the major causes of morbidity and mortality around the world; statistical models predict that in 2020 there will be 15 million new cancer cases, and that much of the cancer burden  will occur in the developing world . Fig. 1 P = 0.02), with a clear association between transplant rates and Gross National Income per/capita (P < 0.01) [1. Funding: maintaining a functional registry is expensive. Each step requires financial backup , ancillary tests , laboratory maintenance, wages, donor collection facilities, software, etc.), plus the costs of maintaining contact with and communication with an increasing number of potential donors represent a significant financial burden for developing countries. This has led to the preferential and broad use of MUD HCT by high-income countries and rapid growth of related haploidentical transplantation as an alternative transplantation modality in lower income countries , 16. A r < 0.01) . Fundinghttps://statistics.wmda.info). However, the chance of finding a MUD at 10/10 low resolution level is only 19%, whereas the chances of finding a donor at high-resolution 10/10 level is even less [2. Donor issues: emerging registries aim to improve donor availability for the country/region with its unique HLA phenotype spectrum. The needed registry size will depend on multiple factors including target population size, homogeneity of the population, and haplotype frequency distribution. India, for example, has a large, non-homogeneous population with > 300 distinct ethnic groups and 438 languages. In India, there are five organizations listing donors for international recipients: BMST India as an intermediary of DKMS Registry , Be The Cure Registry-Jeevan Foundation (6449 donors), Datri Blood Stem Cells Registry , GeneBandhu , and the Marrow Donor Registry India (MDRI) .  [3. Haplotype frequency: HLA genes are very polymorphic with more than 18,000 alleles (ex.html) . HLA locex.html) , 23. Theex.html) . On the ex.html) . Anotherex.html) . In summex.html) . Furtherex.html) .Fig. 5Pr4. HLA typing: HLA typing by PCR testing was introduced by Mullis and Faloona in the 1980s . Several5. Software: information technology (IT) is necessary to run donor searches, to analyze HLA-haplotype frequencies and to predict the chances of finding a donor for each patient, who usually desperately needs a transplant in the shortest possible timeframe. Historically, large international registries developed their own software with their internal IT staff and/or external software companies and some may have used blood bank software as their starting point. These in-house built programs are typically complex and tightly tailored to the local needs. They are not available commercially and thus not available for emerging registries. Building an in-house program needs a lot of effort, expertize, time, and expenses. This is particularly challenging for small and medium-sized emerging registries. Few commercial software packages for donor registries are now available in the market. Partnering with an established registry that has already developed an IT system is another way to acquire a software. Each of these models  has advantages and disadvantages Table . The pla6. Emerging alternatives: outcomes after HCT from related haploidentical donors  have improved significantly over the past few years, with results approaching those of MUD and MSD transplants. The use of haploidentical HCT will extend the availability of allo-HCT to virtually all patients in need, as almost all patients will have an available haploidentical related donor. This advantage, in addition to the low cost and immediate availability, is particularly important for countries with no donor registries and where cost is prohibitive for acquiring unrelated donor cells from international registries or for establishing a national registry. All of these facts suggest that in countries with a relatively small population and some ethnic and genetic diversity, establishing an alternate donor registry and making matched grafts available for a very limited number of patients per year may not be cost effective unless the country’s registry becomes part of an effective regional or international network for unrelated donors. Improvements in haploidentical HCT outcomes in the past decade, in particular using post-transplant cyclophosphamide, is making this alternative HCT modality preferred, over other alternative donors. Haploidentical family donor transplants are more affordable and readily available and therefore broadly used in many centers worldwide . The disadvantages of using UCB are mainly the low yield of stem cells, resulting in delayed recovery, and the cost of graft acquisition from the international network of cord blood banks.7. Cord blood banks: Umbilical cord blood (UCB) banks provide an attractive, off the shelf solution for patients in need of an HCT. Recent studies showed comparable rates of acute graft-versus-host disease (GVHD) and significantly lower rates of chronic GVHD with cord blood transplant as compared to conventional matched donor transplant –41. Desp8. Utilization of artificial intelligence: artificial intelligence (AI) particularly with machine learning (ML) technology is revolutionizing the field of HCT in the selection of donors, and predicting outcomes after allo-HCT and even in GVHD –44. The Although there has been a significant increase in the use of haploidentical related donors and unrelated cord blood  as the stem cell source, the role of HCT from unrelated donors is well established. Establishing a functional and sustainable donor registry in a constantly evolving world is challenging, especially in the setting of limited resources and competition from emerging alternatives. Some existing registries are neither efficient nor able to provide suitable donors. Different causes of inefficiency exist, and all converge on the fact that resources may not be appropriately directed leading to poor performance and low yield of suitable donors. Analyzing the various steps in the process of recruitment, testing, retention until requested for donation may help in preventing donor attrition and thus increase the chance of building a successful registry. Educating the public may result in committed donors with low attrition rate, having a smart and easy-to-update software especially utilizing artificial intelligence algorithms, will ensure new HLA types are recorded and new search algorithms are built. Development of registries with widespread certified donation facilities will ease up the donation process. All of these are essential components for a functional registry. However, what remains both crucial and critical is the decision to build a registry or not, and that decision depends on the needs of the target population and the accessibility to alternative therapeutic options.


Francisella species. The organ donor, a middle-aged man from the southwestern United States, had been hospitalized for acute alcohol withdrawal syndrome, pneumonia, and multiorgan failure. F. tularensis subsp. tularensis (clade A2) was cultured from archived spleen tissue from the donor and blood from both kidney recipients. Whole-genome multilocus sequence typing indicated that the isolated strains were indistinguishable. The heart recipient remained seronegative with negative blood cultures but had been receiving antimicrobial drugs for a medical device infection before transplant. Two lagomorph carcasses collected near the donor’s residence were positive by PCR for F. tularensis subsp. tularensis (clade A2). This investigation documents F. tularensis transmission by solid organ transplantation.In July 2017, fever and sepsis developed in 3 recipients of solid organs (1 heart and 2 kidneys) from a common donor in the United States; 1 of the kidney recipients died. Tularemia was suspected only after blood cultures from the surviving kidney recipient grew  Francisella tularensis. Natural transmission to humans occurs through a variety of routes, including tick and deerfly bites, direct handling of infected tissues, ingestion of contaminated water or tissues, or inhalation of infective materials  was notified that blood cultures from 2 solid organ transplant recipients in different US states, both of whom had sepsis, yielded a small, gram-negative organism suspected to be In July 2017, septic shock developed in 2 patients who had received a kidney and 1 patient who had received a heart from a common donor. The clinical course of disease, antimicrobial drug regimens, and immunosuppressive medications for these patients are summarized in The patient was a middle-aged man who had received a kidney transplant for IgA nephropathy and focal segmental glomerulosclerosis. Four days after transplantation, fever (39.4°C), hypoxemia, and wheezing developed. The next day, despite empiric treatment with vancomycin and meropenem, disseminated intravascular coagulation and septic shock developed, and the patient died. The organ procurement organization was alerted and immediately notified the physicians for the other 2 solid organ recipients.https://www.asm.org/Articles/Policy/Laboratory-Response-Network-(LRN)-Sentinel-Level-C), the hospital laboratory discontinued identification procedures and referred the isolate to the local public health laboratory, a Laboratory Response Network (LRN) biological facility  developed, and she required mechanical ventilation and vasopressor support. Six days after transplantation, after the healthcare team was notified that the other kidney recipient had died of sepsis, doxycycline was added to the antimicrobial drug regimen. The patient eventually recovered.This patient was a woman in her 60s who had received a kidney transplant for diabetes mellitus–induced end-stage renal disease. Four days after transplantation, she experienced fever (39.4°C). Clinical samples were collected for culture, and treatment with vancomycin, ceftriaxone, and metronidazole was empirically initiated. She remained febrile; anemia and thrombocytopenia , hypotension, and septic shock developed. He received vancomycin, meropenem, and cefepime, and fever resolved 5 days after transplantation. Results for cultures of blood collected during the septic episode, when the patient was receiving systemic antimicrobial drugs, were negative. Eleven days after transplantation, the clinical team was notified that the organ donor possibly had tularemia. The patient was empirically administered a 10-day course of oral ciprofloxacin and discharged home 27 days after transplantation.The heart recipient was a middle-aged man with a history of nonischemic cardiomyopathy. Before transplantation, he had received ciprofloxacin and ceftriaxone for recurrent 3). Soon thereafter, the patient became hypoxic but did not require mechanical ventilation.In June 2017, a middle-aged alcoholic man was evaluated at an emergency department for obtundation, bloody emesis, fever (39.6°C), and respiratory distress. He had abruptly ceased alcohol intake 5 days before admission and experienced a nonproductive cough, nausea, headache, and conjunctivitis 3 days before admission. Chest radiographs revealed right upper and lower lobe infiltrates . Platelet count was low , bloody emesis, and melena progressively worsened. Over 7 days, he received 25 blood product transfusions. Chest radiographs demonstrated worsening interstitial opacities , urine, endotracheal aspirate, and bronchoalveolar lavage were all negative. Computed tomography of the chest performed after brain death revealed focal consolidation and a 1-cm nodule in the right perihilar region . BronchoWe reviewed medical records of the organ donor and recipients and interviewed families and the 2 surviving patients about potential exposures to tularemia. In addition, we asked the organ donor’s family about the donor’s course of illness before hospitalization and illnesses of anyone who had been in close contact with him.F. tularensis by direct fluorescent antibody (DFA) testing. DNA was extracted from organ donor and kidney recipient cultures and lagomorph bone marrow by using the QIAamp DNA MiniKit  and tested with real-time TaqMan PCR by using F. tularensis multitarget type A and type B assays, then A1 and A2 subtyping assays (F. tularensis by using the microagglutination assay (>1:128 to be positive.Recovered isolates were cultured at CDC on cysteine heart agar with 9% chocolatized sheep blood and confirmed as PmeI restriction enzyme (http://www.applied-maths.com). Whole-genome sequencing was performed by using Illumina V2 300 cycle reagents (F. tularensis A2 strain WY96-3418 as a reference (GenBank accession no. NC_009257·1) (Pulsed-field gel electrophoresis (PFGE) typing of isolates was performed with the F. tularensis by serology.To determine whether infection may have been transmitted from an asymptomatic blood donor to the organ donor, we investigated the sources of all blood products transfused to the organ donor. Blood donors were asked about any potential exposures to tularemia and whether they had experienced febrile illness during the 2-week period after blood donation. Donors who reported potential exposures or febrile illness were tested for We investigated the organ donor’s community—including the vicinity of the donor’s residence, neighboring homes, nearby fields, and a river swimming area—to identify animal carcasses or other evidence of recent rodent or lagomorph die-off. Presence of potential arthropod vectors such as deer flies was noted informally during the assessment; however, arthropods were not systematically collected as part of the investigation.We also evaluated the residential water supply as a possible source of infection. Records from standard municipal water testing results were reviewed, and the source well for the donor’s residence was examined for evidence of compromise or animal contamination.F. tularensis by PCR and culture . Results of DFA and direct PCR testing of spleen tissue were negative. No isolate was recovered from lymph node tissue. Isolates from kidney recipients 1 and 2 were confirmed as F. tularensis by DFA testing and determined to be F. tularensis subsp. tularensis (clade A2) by PCR genotyping.Testing of archived organ donor blood (collected in acid citrate dextrose and EDTA tubes 13 days after admission and stored at room temperature) and frozen spleen and lymph node tissues (collected 14 days after admission) produced the following results. Blood samples were negative for  culture . F. tulaF. tularensis (titer 1:128). A serum sample from the heart recipient collected 11 days after transplant was negative for F. tularensis antibodies (titer <1:4).Organ donor plasma from the archived EDTA blood specimen collected 13 days after admission was positive for antibodies to F. tularensis A2 strains from the donor and both kidney recipients found all 3 to be indistinguishable from one another by 2 methods (PmeI PFGE banding patterns for the 3 strains were the same. Comparison of this banding pattern to a larger PFGE database of PmeI patterns for A2 strains from throughout the western United States (n = 30) demonstrated that the pattern was unique. Genome sequencing followed by wgMLST analysis revealed no nucleotide differences across 1.5 megabases of compared genome sequences between the 3 strains.Molecular typing of the  methods . PmeI PFF. tularensis risk. One donor reported right-sided cervical lymphadenopathy; follow-up commercial laboratory testing of blood from this donor was negative for antibodies to F. tularensis.Two blood suppliers provided the blood products transfused to the organ donor. Further blood donation by the donors who provided these products was temporarily deferred until the traceback and further serologic testing were completed. We identified and interviewed 43 of the blood donors. A review of blood supplier records confirmed that all blood donors met applicable regulations and standards at the time of donation. Telephone interviews of donors asked 9 questions specific to Interviews and medical record reviews of the organ recipients and their families revealed no known risk factors for tularemia. The organ donor resided on tribal lands in the southwestern United States. Family interviews revealed no known contact with sick animals, arthropod bites, household pets, or other noteworthy exposures. One family member reported observing groundhogs and rabbits near the donor’s residence but denied any apparent animal die-offs. The donor was unemployed and had not traveled outside the region for several months before his death.F. tularensis subsp. tularensis (clade A2).No deer flies or other arthropod vectors were observed near the organ donor’s residence. Two lagomorph carcasses were found and collected ≈150 feet and 500 feet from the donor’s residence. No organs or soft tissue from the carcasses were available for testing; however, PCR testing of DNA from femur bone marrow followed by genotyping indicated that both lagomorphs were positive for Maintenance records of the community water source, a municipal well and spring, did not indicate a breakdown of the chlorination process. Investigation of the well site revealed an intact well cover and functioning system with no evidence of animal entry.F. tularensis infection in the donor and both kidney recipients was confirmed. Use of PFGE and wgMLST demonstrated that the F. tularensis isolates recovered from the donor and both kidney recipients were indiscernible from each other and distinct from other A2 strains, thereby corroborating transmission of F. tularensis A2 by solid organ transplant. Clinicians and organ procurement organizations evaluating potential organ donors who died of an unknown febrile illness should carefully assess risk factors for organ transplant–transmissible infectious diseases, including tularemia, and consider additional diagnostic testing if indicated  and subspecies holarctica (type B)  , as was done in this investigation. Generally speaking, recipients’ risks for infection from any pathogen must be balanced with the growing shortage of organs (F. tularensis continues to affect public health because of ongoing naturally acquired infections, potential to cause laboratory-acquired infections, status as a Tier 1 bioterrorism threat, and newly described human-to-human transmission. Clinicians should be aware of the possibility of F. tularensis infection in patients receiving organ transplants. When evaluating potential organ donors with febrile illnesses, clinicians should consider risk factors for tularemia such as recent contact with animal carcasses, arthropod bites, landscaping activities, residence in a rural area, and white or Native American race (