Acinetobacter baumannii is a pathogen described as an “urgent threat.” Infection with this bacterium manifests as different diseases such as community and nosocomial pneumonia, bloodstream infections, endocarditis, infections of the urinary tract, wound infections, burn infections, skin and soft tissue infections, and meningitis. In particular, nosocomial meningitis, an unwelcome complication of neurosurgery caused by extensively-drug resistant (XDR) A. baumannii, is extremely challenging to manage. Therefore, understanding how A. baumannii adapts to different host environments, such as cerebrospinal fluid (CSF) that may trigger changes in expression of virulence factors that are associated with the successful establishment and progress of this infection is necessary. The present in-vitro work describes, the genetic changes that occur during A. baumannii infiltration into CSF and displays A. baumannii’s expansive versatility to persist in a nutrient limited environment while enhancing several virulence factors to survive and persist. While a hypervirulent A. baumannii strain did not show changes in its transcriptome when incubated in the presence of CSF, a low-virulence isolate showed significant differences in gene expression and phenotypic traits. Exposure to 4% CSF caused increased expression of virulence factors such as fimbriae, pilins, and iron chelators, and other virulence determinants that was confirmed in various model systems. Furthermore, although CSF's presence did not enhance bacterial growth, an increase of expression of genes encoding transcription, translation, and the ATP synthesis machinery was observed. This work also explores A. baumannii’s response to an essential component, human serum albumin (HSA), within CSF to trigger the differential expression of genes associated with its pathoadaptibility in this environment.In a recent report by the Centers for Disease Control and Prevention (CDC), multidrug resistant (MDR)  Acinetobacter baumannii has emerged as an important pathogen due to its ability to resist multiple antibiotics, persist in hospital settings, and cause a wide variety of infections such as pneumonia, bacteremia, urinary tract infections, skin and soft-tissue infections, and meningitis, showing its capacity to infect diverse host environments 4. The acquisition of resistance to carbapenems by certain strains  increased the problematic nature of this pathogen6, which has been qualified as an “urgent” threat in a recent report by the Centers for Disease Control and Prevention7.10. Post neurosurgical A. baumannii meningitis is reported between ~ 10–17% of hospitalized inpatients13, can cause death or leave permanent sequelae, and is usually associated with high mortality rates reaching up to 40 to 70%15. Illustrative of these infections' dangerous nature is the recent reported case of the A. baumannii infection of a 39-year-old man treated with external ventricular drainage of cerebrospinal fluid (CSF)16. Although the strain was susceptible to colistin at the time of detection, it quickly acquired resistance while maintaining virulence16. This genetic plasticity, a consequence of its ability to acquire and integrate foreign DNA, gives A. baumannii a tremendous metabolic versatility that permits the bacterium to adapt and persist in harsh conditions21. A. baumannii's success in causing numerous infections, where it gets in contact with different body components and fluids, must be the result of its capabilities to not only capture adequate genetic determinants, but also regulate expression of the proper cell components24. Previously, we demonstrated that human serum albumin (HSA) and pleural fluid (HSA-containing fluid) affect A. baumannii behavior, triggering an adaptive response that modulates DNA uptake, cytotoxicity, immune evasion, stress responses and metabolism26.Bacterial meningitis, which is considered a medical emergency, is a serious infection that can cause permanent disabilities  or death if untreatedA. baumannii pathogenicity in relation to meningitis, we assessed in these experiments how gene expression changes when in contact with CSF. Our goal was to enhance our understanding of why this disease and pathogen are so problematic with the intent to develop better therapies for this fatal infection.Understanding the virulence determinants of this bacterium requires a thorough comprehension of the general genotypic and phenotypic responses when it is exposed to different human body fluids. As part of our studies on A. baumannii strains, A118 (low pathogenicity and high antibiotic susceptibility) and AB5075 (hypervirulent and multi-drug resistant), were exposed to CSF29. Transcriptomic analysis of A. baumannii A118, using a fold-change cutoff of log2 > 1 , showed 275 differentially expressed-genes (DEGs), 7.76% of the total genes in the A. baumannii A118 reference genome. However, statistically significant changes were not observed when A. baumannii AB5075, a hypervirulent and highly resistant strain, was exposed to CSF under the conditions tested. Previous observations have shown that A. baumannii’s response to different stimuli correlates with each particular strain’s degree of pathogenicity. Less pathogenic strains induced more changes in their phenotypic behavior to overcome the stressful environment and persist30.To uncover the impact of specific host environments at the transcriptional level, two different A. baumannii A118 DEGs revealed an increase in the expression of many genes involved in gene expression processes and energy production machineries , were overexpressed with a log2fold just below 1. However, the gene encoding the alpha subunit was also upregulated with a log2fold change of 1.48  displayed a threefold transcriptional increase in expression  of AbA118F_2664, a gene that encodes a CitMHS citrate-magnesium hydrogen complex symporter  and catabolic proteins  Table .A. baumannii’s adaptive responses to CSF. Consistent with the above mentioned DEGs, GO enrichment analysis revealed a statistically significant overrepresentation of the GO categories ATP synthesis coupled proton transport, translation, tricarboxylic acid cycle, and aerobic respiration by 13.8-, 8.8-, 4.9- and 4.3-fold, respectively . Other studies found that exposure of A. baumannii strains to amikacin, imipenem, and meropenem was associated with increased expression of genes involved in the tricarboxylic acid cycle, biosynthesis of amino acids, purines, and pyrimidines, as well as the operons related to ATP, RNA, and protein synthesis33. Taken together, these results suggest that various stressful environments (low nutrient availability and antibiotic treatment) induce expression of genes associated to energy production, protein synthesis, and metabolism in A. baumannii. As a consequence of these transcriptomic changes, the bacterial cell adapts to survive and accelerate metabolism rive in these hostile conditions.Gene ontology (GO) analysis was next undertaken to identify molecular functions and biological pathways associated to The CSF-mediated upregulation of genes coding for the elements necessary for transcription, translation, expression and ATP synthesis was not accompanied by a decrease in generation time Fig. . These r32. However, these growth differences were not evident in either of the A. baumannii strains in the presence of CSF. An attractive notion to explain this observation is that the increase in gene expression capabilities is channeled toward the synthesis of cell components necessary for survival in the human body, e.g., adhesins and pilins.There are numerous reports supporting the hypothesis that increasing the expression of enzymes involved in transcription, translation, and synthesis of ATP is correlated with faster growth rateA. baumannii metabolism are uncoupled from growth rate. This is not anticipated because bacterial cells are characterized by allocating resources to maximize growth according to the needs for each environmental condition34. Our data also suggest that in depleted medium such as CSF, A. baumannii may be allocating all possible resources towards metabolism using an uncoupled metabolism to optimize its survival.The data described in this set of investigations indicate that certain modifications in the A. baumannii A118 cultures induced an increase in the expression of a set of genes that code for virulence-associated functions such as type IV pili, iron uptake systems, the type VI secretion system (T6SS), and poly-N-acetylglucosamine (PNAG) production.We next observed that the addition of CSF to A. baumannii lacks flagellum-mediated motility, twitching and surface-associated motility was demonstrated in several strains36. Numerous studies on twitching and surface-associated motility in A. baumannii A118 showed dependence on changes in light and temperature27 as well as on the components of the growth media. In particular, addition of HSA resulted in increased motility and concomitantly upregulation of the cognate genes26.Type IV pili participate in microbial adherence as well as motility (gliding or twitching). While A. baumannii A118 to CSF produced an increase in the expression of pilW (log2fold change 1.22), pilJ (log2fold change 0.43), fimA (log2fold change 3.09), fimB (log2fold change 2.32), and the fimbrial protein precursor AbA118F_3133 (log2fold change 1.91) , the acinetobactin biosynthesis  and export (barB)40 40 Fig. . BesidesN-acetyl-d-glucosamine (PGA) or cellulose. Previous studies showed that functional production of PGA in Escherichia coli depends on the products of four genes, pgaABCD. pgaC and pgaD are essential for biosynthesis, and pgaB, which specifies a N-deacetylase, together with pgaA are needed to export the polysaccharide from the periplasm to the extracellular milieu43. All four homologs were significantly upregulated when A. baumannii A118 was cultured in the presence of CSF   Fig. . As expeA. baumannii’s virulence is the T6SS44. The transcriptomic data showed that nine out of the 14 A. baumannii A118 T6SS genes were significantly upregulated in the presence of CSF. These genes tssABCDHIKLM, code for essential components of the T6SS  and betI  were differentially upregulated by a log2fold change of -1.20 and -1.40, respectively. For genes associated with capsule formation (K-locus), pgm which encodes a phosphomannomutase was upregulated by 1.08 log2fold . Regarding the SOS response associated genes, any of them was differentially expressed  production, sed Fig. . OmpA isA. baumannii virulence was determined using cytotoxicity assays. Bacteria-free conditioned medium (BFCM) obtained from A. baumannii A118 and AB5075 cultured in LB with or without CSF was added to human embryonic kidney cells (HEK-293), and the cells were inspected after 1 h.An initial assessment of the effect of CSF on A. baumannii AB5075 cultures were significantly more cytotoxic than BFCM from cultures that lacked CSF. This increase in cytotoxicity was observed at all tested concentrations, 1.5% BFCM , 5% BFCM , and 50% BFCM . Conversely, BFCM obtained from A. baumannii A118 cultures containing CSF showed an increased cytotoxic effect only at the highest concentration tested (50%) . The results of these assays show that CSF induces the release of one or more cytotoxic substances by A. baumannii  is correlated with modifications of the expression of more than 1100 bauD and vgrG, were up-regulated in both, CSF (log2fold-change = 2.5247 and 1.3611 respectively) and HSA (log2fold-change = 0.8046 and 1.1063 respectively)26. On the other hand, the fimA gene was up-regulated with a log2 fold change of 3.0938 in the presence of LB supplemented with CSF but surprisingly, it was slightly down-regulated in medium supplemented with HSA (log2 fold change of 1.6434) 26. While the results obtained with bauD and vgrG seemed to uphold the hypothesis, those produced when assessing expression of fimA did not seem to support that HSA is responsible for CSF-induced upregulation.RNA-seq showed that A. baumannii A118 cells cultured in LB or LB supplemented with either CSF, HSA-depleted CSF (dCSF), or dCSF + 0.2% HSA  and dCSF . However, supplementation of LB with dCSF + 0.2% HSA, resulted in a robust increase of 6.065-fold  and 3.825-fold  with respect to LB or LB supplemented with dCSF, respectively  and 7.544-fold  in cells growing in LB supplemented with CSF or dCSF + 0.2% HSA, respectively. Moreover, a 15.847-fold increase  was observed in levels of expression of bauD in cells growing in LB supplemented with dCSF + 0.2% HSA with respect to those in cells growing in LB supplemented with dCSF  or dCSF , there was a decrease of 1.107-fold  when the medium was LB supplemented with dCSF + 0.2% HSA. A comparison between levels of expression in cells growing in LB supplemented with dCSF + 0.2% HSA or dCSF showed that the presence of HSA resulted in 1.370-fold decrease  52. Another example is the case of Pseudomonas aeruginosa, in which the presence of albumin is correlated with increased expression of iron-controlled genes (pvdS and regA)53. In summary, our observations, together with the evidence available from studies with other bacteria, suggest an important role of HSA as signal for expression of genes and systems essential for survival within the human body.Bacterial cells express genes that code for factors that allow growth in the hostile environments they encounter upon invading the human body. The results shown in this section indicate that HSA is one of the signals that triggers the expression of several A. baumannii is one of many causative agents of nosocomial bacterial meningitis, an infection associated with high morbidity and mortality rates. During the infection, bacteria can be found in CSF. In fact, of the several methods available for diagnosis of bacterial meningitis, CSF culture is the most favored54. This study describes changes in expression of numerous genes when A. baumannii is exposed to CSF  sample was acquired from Innovative Research, MI, which is a certified vendor that obtains human samples from Food and Drug Administration (FDA)-approved facilities. The samples were collected from normal healthy individual donors and pooled. Lysogeny Broth, LB, supplemented with 4% CSF was used for all CSF conditions. The following concentration was used since 4% of other human fluids has been used in previously studiesA. baumannii colonies (A118 and AB5075) were suspended in LB with or without 4% CSF and incubated with agitation for 18 h at 37 °C. Overnight cultures were then diluted 1:10 in fresh LB broth and incubated with agitation for 7 h at 37 °C. RNA was extracted from each strain using the TRI REAGENT Kit  as previously described26. Total RNA extractions were performed in two biological replicates for each condition..RNA sequencing was outsourced to Novogene  for mRNA-seq analysis, which includes rRNA depletion, library preparation following the protocols of the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) (New England Biolabs) and HiSeq 2500 paired-end 150 bp sequencingA. baumannii strain A118 and AB5075 exposed to LB or LB plus 4% CSF were analyzed as follows. Trimming of low-quality bases at the ends of the reads to a minimum length of 100 bp and removal of Illumina adaptor sequences was performed using Trimmomatic58. FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to assess the quality of the reads before and after trimming. Burrows-Wheeler Alignment software (BWA) was used to align the RNA-seq reads to sequences of the whole genome shotgun sequencing project of strain Acinetobacter baumannii A118F (DDBJ/ENA/GenBank accession VCCO01000000). FeatureCounts was used to calculate the read counts per gene, and differential expression analysis was performed using DEseq260. Principal component analysis (PCA) and gene expression heat map with clustering dendrograms of the RNA-seq data analysis of LB and CSF treatments are shown in Supplementary Fig. 2fold change > 1 were considered statistically significant.RNA-seq reads (GEO accession No GSE153967) corresponding to A. baumannii A118F genes. Using GO.db Bioconductor annotation data package in R language, GO terms and ancestor terms were assigned for all DEGs from this study. GO enrichment analysis was performed using custom-made scripts as described previously61. The enrichment factor was estimated as the ratio between the proportions of genes associated with a particular GO category present in the dataset under analysis, relative to the proportion of the number of genes in this category in the whole genome. P-values were calculated using the Fisher Exact Test and adjusted by the Benjamini–Hochberg method.GO terms were retrieved from UniProt for the best BLASTx hits to 600 nm was measured every 20 min using a Synergy 2 multi-mode plate reader  and Gen5 microplate reader software (BioTek). To study the effect of different carbon sources, both strains were culture overnight under different condition (LB broth and LB broth + 4% CSF).Growth curves of both strains, A118 and AB5075, were conducted on 96-well plates. Overnight cultures were subcultured 1:50 in LB or LB + 4% CSF and incubated for 18 h at 37 °C with medium shaking. OD26. A118 and AB5075 cells were cultured in LB broth with or without 4% CSF. Bacterial cells were incubated with agitation for 18 h at 37 °C. Experiments were performed in triplicate, with at least three technical replicates per biological replicate.Motility and biofilms assays were performed as previously described6 cells/mL in colorless DMEM were suspended in the well and intoxicated for 1 h at 37 °C, 5% CO2. CellTiter-Glo Reagent (100 μl) was added to each experimental and standard curve well and then placed on an orbital shaker for 2 min. Following mixing, plates were incubated at room temperature for 10 min to stabilize the luminescent signal. The viability of HEK cells was measured at room temperature using the “all” luminescence function of SpectraMax M3.In a Nunclon Delta Surface opaque 96-well microplate (ThermoScientific), we added colorless DMEM, 4% CSF, and A118 or AB5075 BFCM diluted in LB broth to make 50 μL of BFCM at final concentrations of 1.5%, 5%, and 50%. An additional 50 μL of ATCC HEK-293 cells at a concentration of 1 × 10A. baumannii with and without CSF in vivo, the G. mellonella insect model of infection was used62. Larvae weighing between 200 and 400 mg were maintained on wood chips in the dark at 4 °C. A. baumannii A118 was grown overnight in either LB or LB with 4% CSF. An equivalent of 1.0 OD600 unit of overnight culture was pelleted and resuspended in 1 mL of cold sterile 20 mM phosphate buffered saline, pH 7.4 (PBS). The cells were further diluted 1:10 in sterile PBS and used for injections. A Hamilton syringe was used to inject 5 μL of the diluted bacterial suspension via the left proleg of each larva. A control group of untreated larvae was used to assess overall larval viability for the duration of the assay. One hundred G. mellonella larvae were used in each condition and incubated at (37 °C) in a sterile Petri dish for 24 h intervals for 48 h total. Larvae viability was monitored by observing response to gentle prodding with a glass rod; those with no response were considered dead. Four replicates with 100 larvae per Petri dish were performed for each condition.To assess the virulence of 63. Plates were incubated at 28 °C in static incubator for up to 48hs. Results were recorded at 24 h with a Plugable USB 2.0 Digital Microscope.To study extracellular matrix (ECM) production, microcolony biofilm was used as model system. 5 μl of overnight cultures of A118 cultured in LB broth and LB broth + 4% CSF were inoculated on LB agar and supplemented with Congo red as previously describedHSA was depleted from CSF by placing 1 mL of CSF into a 30 kDa Amicon Ultra Centrifugal Filter  and the solution was centrifuged at 20,000 × g for 10 min. To identify HSA was successfully depleted, an SDS-PAGE was conducted that contained 4% CSF, depleted CSF (dCSF), and dCSF plus 0.2% HSA Fig. .A. baumannii strain A118 grown in LB, 4% CSF, 4% depleted CSF and 4% depleted CSF + 0.2% HSA, were synthesized to cDNA using the manufacturer protocol provided within the iScript Reverse Transcription Supermix for qPCR . The cDNA concentrations were measured with a DeNovix DS-11 + spectrophotometer; each sample was then diluted to a concentration of 50 ng/μl. qPCR was conducted using the iQ SYBR Green Supermix through the manufacturer’s instructions. At least three biological replicates of cDNA were used and were run in quadruplet. All samples were then run on the CFX96 Touch Real-Time PCR Detection System .Previously extracted and DNase-treated RNA from recA rRNA transcript levels for each cDNA sample. The relative quantification of gene expression was performed using the comparative threshold method 2−ΔΔCt. The ratios obtained after normalization were expressed as folds of change compared with cDNA samples isolated from bacteria cultures on LB. Asterisks indicate significant differences as determined by ANOVA followed by Tukey’s multiple comparison test (P < 0.05), using GraphPad Prism .The transcript levels of each sample were normalized to the P-value < 0.05 was considered statistically significant.All experiments were performed at least in technical and biological triplicate. Data was expressed as means ± standard deviation. Statistical analysis using Mann–Whitney test or ANOVA followed by Tukey’s multiple comparison test were performed using GraphPad Prism , and a All procedures performed in this study were in accordance with the CSUF Institutional Biosafety Committee Approval plan (DBH117-01) and are in compliance with the NIH, CDC, OSHA and other environmental and occupational regulations.Supplementary figuresSupplementary Tables


Cryptococcus deuterogattii in 2 kidney transplant recipients in Brazil and published information on other cases. Prompt reduction of immunosuppression and initiation of antifungal therapy was required to successfully control the fungal infections and preserve engraftment. We describe cases of donor-derived transmission of  Cryptococcus deuterogattii in Brazil. The donor in both of these cases was a 43-year-old man with an antemortem history of an unspecified brain tumor who had been declared brain dead after respiratory arrest. After antiretroviral therapy for HIV patients was introduced, solid organ transplant recipients became one of the major risk groups for developing cryptococcosis, possibly transmitted from donors  agglutination test result was positive (titer 1:8). Blood and urine cultures indicated Cryptococcus species, and the serum CrAg-latex agglutination test result was positive . Case-patient 1 was a 51-year-old man who received a kidney from the donor. Physicians initiated induction therapy with antithymocyte globulin and maintenance therapy with tacrolimus, prednisone, and azathioprine. On day 7 after the procedure, doctors performed a kidney biopsy after the patient experienced delayed graft function. Histopathology of the graft showed organisms consistent with 2O, 3 leukocytes/mm3, protein 35 mg/dL, and glucose 174 mg/dL. Results of India ink and CrAg-latex agglutination tests were positive (titer: 1:64). Blood cultures and a urine sample indicated Cryptococcus species. No fungal growth was detected in a cerebrospinal fluid sample. The serum CrAg-latex agglutination was positive . After the diagnosis of cryptococcosis in the first patient, a 59-year-old woman (case-patient 2) who had received a kidney from the same donor was contacted for evaluation. Physicians had initiated induction therapy with antithymocyte globulin and maintenance therapy with tacrolimus, prednisone, and mycophenolic acid. She was discharged on day 8 after the procedure and was asymptomatic when recalled on day 10. Clinicians performed blood and urine cultures, radiographs of the chest and the brain, and a lumbar puncture. The radiograph revealed no abnormalities. Results of lumbar puncture showed opening pressure of 15 cm HCryptococcus species that had not been detected previously. Results of a CrAg-latex agglutination test performed on a stored serum sample from the donor was positive . To rule out infection by Cryptococcus species in the organ recipients prior to the transplantations, CrAg-latex agglutination tests were performed on stored pretransplant serum samples from both recipients; results for both were negative. A revised histology of the brain biopsy of the donor showed yeasts of Both patients were treated with amphotericin B lipid complex (5 mg/kg 1×/d for 21 d) in combination with 5-fluorocytosine (25 mg/kg 4×/d for 14 d); dosages of immunosuppressive drugs were lowered. Therapy was switched to intravenous fluconazole (400 mg/d) after results of blood and urine cultures became negative. After clinical and microbiological remission of the infection, dosage was adjusted on the basis of renal function to 300 mg/d of oral fluconazole and was maintained for 2 years. Three years after stopping all antifungal therapy, no relapse of cryptococcosis was documented. C. deuterogattii genotype AFLP6/VGII. The revision of slides from formalin-fixed paraffin-embedded tissue blocks from donor brain biopsies showed Cryptococcus yeasts. We performed fungal DNA extraction and amplified parts of CAP59, GPD1, IGS1, LAC1, PLB1, SOD1, and URA5 loci  and sent to Canisius-Wilhelmina Hospital . We performed AFLP (amplified fragment length polymorphism) fingerprinting and multilocus sequencing typing, which showed that all isolates were ’s brain . We searched the literature for other cases of fungal infections from solid organ donors in transplant recipients and found 12 additional cases of presumed or confirmed donor-derived cryptococcosis . MoleculC. deuterogattii may be transmitted by infected allografts, representing a medical concern in countries to which C. gattii species complex is endemic. Cryptococcosis incidence might be reduced by excluding organ donor candidates with a history of neurologic disease without a clear definition of its etiology. Ensuring that clinicians are trained to recognize and treat this fungal infection would likely further reduce transmission from donors. In conclusion, we found that Cryptococcus spp. in solid organ transplantation.Demographic, clinical, and transplant data of donor-derived transmission of confirmed and presumed


There are variations in recommendations from different guidelines regarding the indications for repeat lumbar puncture (LP) in young infants with the diagnosis of bacterial meningitis.To evaluate the frequency of repeat LPs and the characteristics of cerebrospinal fluid (CSF) parameters in repeated sampling and their predictive values for adverse outcomes in a national cohort.This cohort study included infants born January 1, 2013 through December 31, 2014, who had proven or suspected bacterial meningitis within the first 90 days of life at seven paediatric tertiary care hospitals across Canada, and who underwent a repeat LP at the discretion of the treating physicians.6/L, providing a sensitivity of 91% and specificity of 88%.Forty-nine of 111 infants (44%) underwent repeat LP at a median of 5  days after the LP that led to the diagnosis of bacterial meningitis. Those who had meningitis caused by gram negative bacilli were more likely to have repeat LP than those with gram positive bacteria . White blood cell (WBC) count on the second spinal tap yielded an area under the curve of 0.88 for predicting sequelae of meningitis at discharge from the hospital, with a cut-off value of 366 × 10In this multi-centre retrospective cohort study, infants with gram negative meningitis were more likely to have repeated LP. A high WBC on the second CSF sample was predictive of adverse outcome at the time of discharge from the hospital. Neonatal bacterial meningitis is a life-threatening disease with high rates of mortality and morbidity and leads to substantial long-term neuro-disability –3. EarlyRepeat lumbar puncture (LP) during the course of treatment of bacterial meningitis and prolongation of antimicrobials for persistently abnormal cerebrospinal fluid (CSF) parameters have been advocated in young infants , 6. HoweThe primary report on epidemiology, management, and outcomes of bacterial meningitis in this cohort of infants was previously published .This cohort study included infants born January 1, 2013 through December 31, 2014 with onset of proven or suspected bacterial meningitis within the first 90 days of life at seven paediatric tertiary care hospitals across Canada. Infants underwent repeat LP at the discretion of the treating physicians. Infants with meningitis caused by fungus or virus, related to neurosurgical procedures or CSF shunts were excluded.https://www.equator-network.org/reporting-guidelines/strobe/). Ethics approval was obtained from the seven participating hospital Research Ethics Boards for this retrospective study. The cases were identified according to the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision, and Canada Diagnostic Codes for Target Discharge [The study results are reported following the Strengthening the Reporting of Observational Studies in Epidemiology guidelines (ischarge .6/L white blood cells (WBC) and (1) < 100 × 106/L red blood cells (RBC), or (2) WBC:RBC ratio > 1:100 [Proven bacterial meningitis was defined as the detection of bacteria from CSF by culture or molecular techniques during life or at autopsy. Suspected meningitis was defined as the detection of bacteria known to cause central nervous system infection from blood or another normally sterile site (including urine) along with either sterile CSF pleocytosis or head imaging consistent with bacterial meningitis. CSF pleocytosis was defined as > 30 × 10 > 1:100 , 13. The > 1:100 .The data were collected by Research Electronic Data Capture. The data included demographic characteristics, microbiological results (including drug susceptibilities), CSF results, head imaging results , duration of antimicrobial therapy, complications during hospital stay and sequelae noted at discharge, including ongoing seizures, use of anticonvulsants despite no ongoing seizures, documented hearing loss, abnormal findings on ophthalmological examination, increased tone, decreased tone, monoplegia/hemiplegia/quadriplegia/or other sequelae.SPSS 26.0  was used to perform the statistical analyses. Descriptive statistics were used to characterize the patient demographics and CSF parameters along with the outcomes. Kolmogorov-Smirnov test or Shapiro-Wilk test was applied to test for normality. The Mann-Whitney U test or Kruskal-Wallis test was performed as appropriate. The χ2 test or Fisher exact test was performed to compare the proportions of the categorical variables. A receiver operating characteristic curve was constructed to determine the threshold of CSF WBC for predicting adverse outcomes. A p value < 0.05 was considered as statistically significant.After excluding infants with pre-existing CSF shunt (N = 1) and post-endoscopic ventriculostomy (N = 1), 111 infants with proven or suspected bacterial meningitis were included, with 44 (40%) born preterm . Thirty-There were no differences in gestational age (GA), birth weight (BW), sex and days of life (DOL) of diagnosis of meningitis with and without repeat LP . Those wEscherichia coli meningitis. The proportion of E. coli strains susceptible to ampicillin , gentamicin  and cefotaxime  showed no significant differences among infants with and without repeat CSF obtained. All infants with meningitis due to gram negative bacilli other than E. coli or Klebsiella sp. underwent repeat LP [The pathogens isolated from infants with proven bacterial meningitis (n = 61) are summarised in epeat LP .There were no differences of baseline demographic characteristics and microbiology between infants who underwent repeat LP with suspected (n = 12) and proven meningitis (n = 37), though the latter group had earlier repeat of CSF samples and higher median WBC in the second CSF samples .The median time chosen for repeat LP was 5 days  after the initial LP, with no difference between infants with proven or suspected bacterial meningitis caused by gram positive versus gram negative pathogens . The second sample was obtained < 7 days , 7 to 14 days  and > 14 days  after the initial LP. There was no significant variation of practice of CSF samplings beyond 14 days after the initial diagnosis among different centres (p = 0.444).E. coli (n = 2), GBS (n = 3), and one each of Klebsiella pneumoniae and Enterobacter cloacae. The CSF cell count in neither the initial nor second sample differentiated infants with persistent positive from sterile CSF cultures at repeat sampling (Seven of 37 (19%) infants with initial positive CSF culture had persistent positive bacterial cultures on the second CSF sample. This included infants with meningitis caused by sampling .E.coli (n = 8), GBS (n = 13) and other bacterial pathogens (n = 9) were 7, 4, and 5 days, respectively [Thirty out of 37 infants with bacterial growth on the initial CSF had sterile CSF on their second LP . Median ectively , with noAll infants survived to hospital discharge, so diagnosis was never made at autopsy. Twelve of 49 infants (24%) had at least one sequelae noted at the time of discharge, namely ongoing seizures (n = 10), hearing loss (n = 1), abnormal tone (n = 2) and/or others (n = 4). There was no significant difference in the proportion with sequelae between term  and preterm infants .6/L, providing a sensitivity of 91% and specificity of 88% [WBC in the first CSF sample or the magnitude of decrease in WBC in from the first to second CSF samples were not found to be predictive of sequelae at hospital discharge . Howevery of 88% .In this cohort, persistent positive culture on the second CSF samples was not predictive of sequelae at hospital discharge .6/L or above at the second LP was predictive of sequelae at the time of hospital discharge.In our cohort, almost half of infants with suspected or proven bacterial meningitis had CSF sampled at least twice during their treatment course with repeat sampling being more common with gram-negative than with gram-positive meningitis. Approximately 60% of repeat sampling was performed within 7 days of the initial sampling. There was no difference in the median days to document CSF sterilization among infants with meningitis caused by different pathogens. Our ROC analysis revealed that elevated CSF WBC of 366 x 10E. coli. [rd-generation cephalosporin-resistant Enterobacteriaceae, β-lactam-resistant Streptococcus pneumoniae) to monitor the clinical response and document clearance of the organism [Some experts recommend that all infants with bacterial meningitis undergo repeat LP 48 hours after initiation of therapy . For infE. coli. . In the organism , 16. In organism .However, the utility of repeat LP with meningitis due to pathogens that are not multidrug-resistant is less clear. In adult bacterial meningitis, repeat LP is not commonly performed, although it might be useful in selected cases to confirm the diagnosis, to exclude relapse or persistent infection, or for therapeutic purposes in communicating hydrocephalus . Durack Some experts recommend performing repeat LP routinely in all neonates when meningitis is caused by gram-negative bacilli at 48 hours, whereas others suggest repeating LP only if clinical improvement is not evident after 24 to 72 hours of antibiotics , 20. In We demonstrated that the WBC on the second CSF sample but not the first CSF sample was predictive of adverse outcomes at the time of discharge from the hospital. Greenberg et al. reported the presence of a positive culture in a repeat CSF was associated with increased mortality . StudiesTo the best of our knowledge, this is the first study evaluating the usefulness of repeat LP in predicting adverse neonatal outcomes other than mortality among infants with bacterial meningitis. The major limitation of the study is that long-term outcome data were inconsistently available and not collected in a standardized manner. The total number of subjects was relatively small. Details of brain imaging were not recorded systematically to allow meaningful analysis. Adverse outcomes could have been related to other factors rather than to bacterial meningitis . The timing of repeat LP was not consistent. The decision to repeat the LP was at the discretion of the treating physician and the rationale was typically not documented. The database did not capture the initial clinical presentation nor any failed repeat LP attempts. The relatively small number of subjects did not allow us to carry out further multivariate analysis to look at the contributing factors towards adverse sequelae. Having said that, our study indicated that repeat LP may provide insights on prognostication. Future prospective studies with large sample sizes should analyze the decision making process regarding repeat LP, the results and interpretation of CSF parameters and the relationship between CSF parameters and long-term outcomes in samples obtained with uniform timing (possibly 3 days after effective antibiotics were started).”In this multi-centre retrospective cohort study, we found that infants with gram negative meningitis were more likely to have repeated LP. A high WBC on the second CSF sample was predictive of adverse outcomes at the time of discharge from the hospital.


The cerebrospinal fluid (CSF) real-time quaking-induced conversion assay (RT-QuIC) is an ultrasensitive prion amyloid seeding assay for diagnosis of sporadic Creutzfeldt–Jakob disease (CJD) but several prion strains remain unexplored or resistant to conversion with commonly used recombinant prion protein (rPrP) substrates. Here, bank vole (BV) rPrP was used to study seeding by a wide range of archived post-mortem human CSF samples from cases of sporadic, acquired and various inherited prion diseases in high throughput 384-well format. BV rPrP substrate yielded positive reactions in 70/79 cases of sporadic CJD [Sensitivity 88.6% (95% CI 79.5–94.7%)], 1/2 variant CJD samples, and 9/20 samples from various inherited prion diseases; 5/57 non-prion disease control CSFs had positive reactions, yielding an overall specificity of 91.2% (95% CI 80.1–97.1%). Despite limitations of using post-mortem samples and our results’ discrepancy with other studies, we demonstrated for the first time that BV rPrP is susceptible to conversion by human CSF samples containing certain prion strains not previously responsive in conventional rPrPs, thus justifying further optimisation for wider diagnostic and prognostic use. In spite of this singular mechanism of disease, the human conditions exhibit striking phenotypic diversity even within sporadic Creutzfeldt–Jakob disease (CJD), which comprises 80–85% of cases. Aside from the classical triad of rapid cognitive decline, gait ataxia and/or myoclonus, distinct clinical phenotypes such as the Heidenhain variant, ataxic variant (Brownell–Oppenheimer), corticobasal syndrome and sporadic fatal insomnia have been observed2. This diversity is hypothesised to be imparted by prion strains through the propagation in the brain of different pathological conformational states3. There is evidence that this process is modified considerably by the polymorphic codon 129 of the prion protein gene (PRNP). Strains can be inferred by laboratory assays including by western blot5. Highly-penetrant disease-causing PRNP mutations cause diseases historically classified into Gerstmann-Straussler-Scheinker (GSS) syndrome, fatal familial insomnia (FFI) and familial CJD (fCJD)6, although this fails to encapsulate the diversity of phenotypes resulting from octapeptide repeat insertions (OPRIs) and the more recently described peripheral systemic amyloidosis secondary to the PRNP Y163X mutation8.Prion disease represents a heterogeneous group of rare, transmissible and fatal neurodegenerative conditions affecting humans and other mammalian species, unified biologically by autocatalytic templated-misfolding of the host-encoded prion protein (PrP) into disease-associated forms11. Though highly sensitive, the above are not seen exclusively in prion disease, and moreover are really surrogate markers of rapid axonal injury and gliosis; as such, histological examination endures in epidemiological case definitions as mandatory for definite diagnosis12.Cerebrospinal fluid (CSF) examination has long been a key clinical investigation in individuals suspected to have sporadic CJD, in particular the detection of protein 14.3.3, raised S100B and exceedingly high total tau levels  are all considered to be highly supportive14. The RT-QuIC exploits and accelerates the seeded amyloidosis of PrP through repeated cycles of mechanical agitation, coupled with Thioflavin T (ThT) fluorescence read-outs that can be monitored in real time. The typical RT-QuIC reaction mixture, composed vitally of a buffer, recombinant PrP (rPrP) and ThT, has been successfully seeded by a variety of mammalian tissue and bodily fluids18. The efficiency and sensitivity of the reaction can be further enhanced by altering conditions such as raising temperature, pH and addition of sodium dodecyl sulphate (SDS)19, or by using different rPrP constructs, giving rise to the so-called second generation IQ-CSF RT-QuIC21.More recently, real-time quaking-induced conversion (RT-QuIC) seeded by CSF has emerged as a rapid, sensitive (> 90%) and highly specific tool (≈100%) for ante mortem diagnosis in sCJDPRNP E200K. Save for a single study in CSF samples from patients with P102L and D178N mutations which successfully seeded RT-QuIC reactions using human rPrP22, the level of sensitivity has not been replicated in reactions seeded by CSF from GSS-causing IPD mutations  and variant CJD (vCJD) even with IQ-CSF RT-QuIC above. Other IPD mutations are simply too rarely encountered such that CSF may not be available for testing. Crucially, the lack of PrP sequence homology is expected to present a significant barrier to conversion, based on previous P102L and A117V human PrP transgenic mouse modelling experiments24. Thus, the expectation that a single rPrP type can serve as a ‘universal acceptor’ seemed improbable until some experiments showed efficient transmission of multiple prion strains in bank voles and in transgenic mice expressing bank vole rPrP (BV rPrP)26, indicating a lack of both strain and species barriers. BV rPrP as a 'universal acceptor' gained further traction when a series of RT-QuIC experiments seeded by brain homogenate (BH) not only demonstrated seeding activity in variant CJD and GSS cases due to a number of PRNP mutations which were hitherto undetectable, but also in other mammalian species15.The extraordinary success of RT-QuIC in clinical diagnosis is at present restricted to sCJD and sCJD-like IPD phenotypes, mainly 28.Here, we have explored the universality of BV rPrP as the substrate in RT-QuIC reactions seeded by CSF from a variety of human prion diseases and attempt to adapt the RT-QuIC technique to a 384-well microplate in place of the conventional 96-well format for higher throughput. The wealth of longitudinal clinical data gathered from the UK National Prion Monitoring Cohort (NPMC) since 2008 allowed us to then determine whether the RT-QuIC seeding dose in post-mortem CSF correlates with the rate of functional decline in patients with sCJDPRNP mutations in the IPD group encompassed P102L (n = 5), A117V (n = 2), Y163X (n = 1), D178N (n = 2), E196K (n = 1), E200K (n = 3), 4-OPRI (n = 2) and 6-OPRI (n = 4). Control PM CSF samples were obtained from 49 anonymised autopsy-proven non-prion disease cases. An further set of 8 non-prion PM CSF samples were identified from the NPMC cohort from patients suspected to have sCJD clinically in life but PM examination revealed other causes of illnesses and deaths; PM CSF control samples amounted to 57 in total. A summary of patient demographics, including the PRNP codon 129 genotype where applicable and molecular strain type5 is available in Table A total of 101 archived post-mortem (PM) CSF samples from autopsy-proven prion disease cases were identified; this included sCJD (n = 79), vCJD (n = 2) and IPD (n = 20) cases. The range of First, we compared the RT-QuIC assays using human (Hu) rPrP and BV rPrP as substrates in the standard 96-well plate format at 45 °C, seeded by sCJD n = 13) and vCJD (n = 1) CSFs against control CSFs. Under the same conditions, RT-QuIC reactions using BV rPrP were positive for all sCJD CSFs (13/13), as opposed to 12/13 for reactions using Hu rPrP. Furthermore, the RT-QuIC reaction with BV rPrP was successfully seeded by vCJD but not using Hu rPrP, albeit with a longer lag phase and lower maximal relative fluorescence units (rfu) in the former compared to sCJD  and 75 μL (CSF 11.25 μL) per well respectively in 384-well plates to see if the reaction kinetics are replicated in smaller well volumes. Both final well volumes of 50 μL and 75 μL produced consistently positive reactions for both sCJD and vCJD, but at lower maximal rfu.Finally, we then performed BV RT-QuIC reactions with 50 μL final volumes at 50 °C in 384-well plates. This showed enhanced maximal rfu for the positive reactions in wells seeded by sCJD and vCJD cases, replicating reaction kinetics seen in those conducted at final volumes of 100 μL in 96-well plates at 45 °C Fig. .Figure 2Once optimal conditions were determined  at 50 °C), BV RT-QuIC reactions were run in 384-well plates seeded by prion disease PM CSFs and non-prion non-neurological disease control PM CSFs  of BV rPrP RT-QuIC reactions seeded by serial dilutions of sCJD BH . In these 29 individuals, no significant correlation was found between prion seeding dose and functional decline  , r = 0.13, p = 0.73; MV (n = 15), r = − 0.34, p = 0.21; VV (n = 5), r = − 0.88, p = 0.05).The rate of decline in individual MRC Scale scores from RT-QuIC positive CSF samples were calculated according to the mixed effects model described in Mead et al41) Fig. A. This er = 0.44, p = 0.0004), i.e. the longer the duration of disease, the higher the seeding dose  with low molecular weight fragments on western blot, and D178N-FFI which had previously yielded negative results in reactions using hamster rPrP even with enhanced IQ-CSF conditions19. Furthermore, the failure of IQ-CSF RT-QuIC iterations (with either full length or truncated hamster rPrP) to uniformly replicate their enhanced sensitivity in non-fCJD PRNP mutation samples emphasises this unmet clinical need for a universal acceptor31.Interest in exploring the utility of BV rPrP in CSF RT-QuIC here followed a series of benchmark experiments carried out by Orrú et al. 2015 in which BV rPrP appeared to show potential as a 'universal' acceptor in a wide range  of mammalian prion species31. In our study, BV rPrP also appear to be successfully converted by other prion disease subtypes such as vCJD and a range of IPDs. While E200K, P102L and D178N CSF have been shown to seed RT-QuIC reactions in other studies using different rPrP including full length Hu rPrP, full length hamster rPrP, truncated Syrian hamster rPrP (90–231) and hamster-sheep chimeric rPrP32, our study here showed for first time that CSF from cases of 6-OPRI IPD can seed RT-QuIC reactions.In the largest series of sCJD PM CSF samples in the literature tested with BV rPrP RT-QuIC to date, both the sensitivity and specificity of 88.6% (95% CI 79.5–94.7%) and 91.2% (95% CI 80.7–97.1%) respectively in our study are below that reported in a number of recent large non-BV rPrPstudies  in which the reactions are seeded by ante mortem CSF15, and suggest that perhaps BV rPrP may not be a completely “universal acceptor” of prion strains. Moreover, we are aware of a selection of 19 CSF samples  ante mortem CSF) tested under BV rPrP RT-QuIC conditions similar to Orrù et al.15 which returned an even lower sensitivity relative to our study of 52.6%33. Possible explanations for our discrepancies may lie in the differential sample matrix and RT-QuIC conditions, and the presence of ‘conversion’ barriers, or a combination thereof.The lower sensitivity and specificity of CSF BV rPrP RT-QuIC in sCJD, and particularly its even lower sensitivity across IPD subtypes appear to be at variance with the findings in Orrù et al.6/L may quench the fluorescent signal sufficiently to return false negative results34; on the other hand, protein levels greater than 1.0 g/L and raised white cell count beyond 10 × 106/L are liable to cause false positive misinterpretations due to high and fluctuating baseline rfu measurements35. It is recognised that significant physicochemical changes take place in death, even in closed compartment fluids like CSF36; broadly speaking, leakage of impurities into PM CSF which follows cessation of active transport mechanisms that maintain the blood–brain barrier such as cell debris and proteins can inhibit amyloid seeding assays39. More specifically, CSF total protein level is known to increase significantly by about 20 times (mean 8.37 g/L) in agonal death durations greater than 6 h such as that observed in prion disease40, and perhaps further increased with longer death to autopsy intervals41. As for PM CSF white cell count, it increases progressively with post mortem interval, and autopsy studies indicate that CSF cell count would have exceeded 10 × 106/L by 10 h post mortem42.Firstly, the use of PM CSF in this pilot study is relatively unconventional as well-established CSF RT-QuIC protocols in use are honed chiefly for ante mortem CSF which has a different fluid matrix to PM CSF, and PM CSF is expected to have lower seeding activity compared to BH previously tested with BV rPrP RT-QuIC. However, given the large volumes of PM CSF that can be collected relative to ante mortem CSF, we judged it to be an appropriate biofluid medium to conduct an exploratory study in a reasonably large sample set. Key CSF composition factors identified to have profound effects on RT-QuIC reaction kinetics include red cell count (haemoglobin concentration), white cell count and protein level. The haem concentration in CSF samples with red cell counts exceeding 1250 × 106/L respectively. Indeed, all 5 of our positive control PM CSF samples were scored as such due to high and fluctuating baseline. These 5 reaction kinetics and curves were distinct from conventional positive results, and arguably should have been considered ‘negative’ from a qualitative perspective. These anonymised control samples were obtained from a different institution where only the ages of death and gender were provided; these patients died of non-neurological causes (and thus presumably non-prion), some of which involve trauma, but the precise cause of death for each individual was not provided. As a result, we did not have the resources to age- and gender-match all cases for this exploratory study.Cell counts and routine biochemistry were not performed on our PM CSF sample set in clinical laboratories for biohazard concerns, although visually pink or red samples indicating significant red cell contamination were discarded from analyses. In spite of this, it remains likely that the PM CSF samples used here  contain total protein levels and white cell counts (or its debris) greatly in excess of 1.0 g/L and 10 × 1015 and our study, other differences are summarised in Table 20. One may be tempted to raise the reaction temperature but BV rPrP RT-QuIC has tended to spontaneously fibrillise between the 60–70th hour even at a 42 °C15.Aside from the different seed matrices between Orrù et al.PRNP mutations with a fCJD phenotype, in particular the E200K mutation, can easily seed RT-QuIC reactions, but less so in others which produce the GSS or FFI phenotypes31. What this is down to is not entirely clear but this disparity seemed to be resolved by moving towards rPrP sequence homology where RT-QuIC conversion rates between different IPDs were shown to be comparable by using full length Hu rPrP22; Sano et al. in 2013 achieved positive RT-QuIC reactions from E200K (18/22), P102L (18/20) and D178N-FFI (10/12) CSF samples by using full length Hu rPrP22. However it ought to be pointed out that the reaction solution composition, temperature and microplate reader is significantly different to the RT-QuIC protocols employed by us and Orrù et al.15. Mouse modelling studies with GSS-related P102L and A117V prions where prions derived from the respective mutant protein can only be transmitted to transgenic mice expressing the corresponding mutant human PrP, and not to wild type mice or mice expressing wild type human PrP24, may give this further traction. It is thus possible that prions from certain IPD mutations may require mutant human rPrP to allow conversion in RT-QuIC reactions.One other key consideration is the possibility that there may still exist “conversion” barrier(s) with BV rPrP RT-QuIC if differential seeding activity and matricial compositional factors are insufficient to explain the result discrepancy. The collated literature does suggest that CSF from reporting” assay in which the outcome is binary, though attempts have been made at probing RT-QuIC kinetics and products to elucidate the prion seeding dose in test samples. RT-QuIC end-point dilution remains the most accurate way of measuring seeding-competent PrP conformers in a test sample in place of animal bioassay38, but the quantitative method proposed by Shi et al. (similar to that employed here) based on correlating dose against reaction lag times on a calibrated curve, appears to promise efficiency in terms of time and spatial requirements29. CSF is routinely only drawn once for diagnostic purposes, and regular CSF sampling at intervals during the illness is hard to justify both clinically and ethically. On the expectation that PM CSF contains the “final and maximal" CSF prion seeding dose, we explored if the extrapolated dose scorrelates with the rates of functional decline in these patients, measured by the percentage decline per day of the validated MRC Scale score obtained in life28. In this study, no clear correlation was found between seeding dose and rate of MRC Scale score  decline. A moderate positive correlation was counterintuitively observed between prion seeding dose and total illness duration across all patients; however, once segregated into codon 129 genotypes this relationship remained statistically significant only for patients with the MV genotype, and could be accounted for by a mere 2 outliers with extremely long durations of illness. This is perhaps unsurprising as it is not precisely known whether a single or multiple species of permissible conformational strains of prions are present within any single biofluid sample and which ones are capable of seeding RT-QuIC reactions43. Moreover, the two-phase kinetic model of prion propagation distinguishes infectious prions and neurotoxic PrP species whose production is uncoupled45. In other words, the abundance of the toxic species mediating functional decline may not be reflected in RT-QuIC seeding competency, and vice versa. Also, rate of decline data was only available from about a third of sCJD patients in the sample set, and as such the numbers may be too small to draw any firm conclusions at the present time.The RT-QuIC assay is largely perceived as a “15. This however does not necessarily invalidate BV rPrP’s potential as a “universal acceptor” of prions as the disparities may merely reflect differences in the matrices of test samples (and hence their seeding doses) and/or in the RT-QuIC conditions used. Nevertheless, we believe our results justifies further optimisation of this assay for wider diagnostic and prognostic use.Our pilot study demonstrated that BV rPrP RT-QuIC can be successfully seeded by PM CSF from a relatively large and diverse sample set derived from prion disease patients, and that it can be adapted to the 384-well format to increase throughput and conserve limited CSF volumes. Significant difference remains between its efficiency for sCJD and IPD groups, and as such is at variance with its previously shown “universality” with prion speciesCSF and brain tissue used in this study were obtained at post mortem from patients who were suspected to have prion disease while alive, and subsequently archived at the Medical Research Council Prion Unit at UCL. The study was performed with approval from the London—Queen Square Research Ethics Committee (Reference: 03/N022). Fully informed consent was obtained, and samples procured and used in accordance with the approved study and relevant national legislation and guidelines.46 the autopsies were largely performed at the National Hospital for Neurology & Neurosurgery on Queen Square or less frequently in local hospitals, both in rooms designated for high-risk autopsies. Full research consent was obtained for all samples used in this study; Scotland A Research Ethics Committee (NPMC) or the Eastern Research Ethics Committee (PRION-1). Prion disease patients enrolled into the NPMC and PRION-1 Trial had regular clinical assessments and their functional status recorded as the Medical Research Council Prion Disease Rating Scale  over time46.The majority of these patients were enrolled either into the National Prion Monitoring Cohort (NPMC) or the MRC PRION-1 TrialAt each autopsy, CSF was aspirated by pipette from the pontine cistern by access through the skull base; this is achieved sequentially by skull removal, separation of dura, lifting of frontal and temporal lobes, and separations of the optic chiasm from the skull base and pituitary gland from pituitary stalk respectively. All CSF samples were stored at − 80 °C prior to analysis. CSF from neuropathologically confirmed prion cases were obtained from MRC Prion Unit at UCL. Fully anonymised non-neurological control post-mortem (PM) CSF samples were obtained from Dr Esiri at the Oxford Brain Bank.6 and stored at − 80 °C. Aliquots of 10% w/v BH were thawed at room temperature freshly for each RT-QuIC assay. The BHs were diluted serially in artificial CSF  in the quantitative RT-QuIC assay.Brain homogenates  were prepared as previously described. 1999 with minor modifications47. Briefly, the DNA sequences encoding either rPrP in pTrcHisB were transformed into BL21 (DE3). BL21 cultures were grown in LB medium in the presence of 100 µg/ml Ampicillin. Expression of either rPrP was then induced using 1 mM IPTG, and purified from inclusion bodies under denaturing conditions using Nickle superflow resin with an AKTA Pure . Subsequently, the rPrP was eluted from the column using an imidazole gradient, and the eluted material was extensively dialysed against 20 mM Bis Tris pH 6.5. The rPrP Histidine tag was cleaved with the addition of 2.5 mM CaCl2 and 50U Thrombin (VWR). Following this, the rPrP was run through a second NiNTA on the AKTA Pure to remove the Histidine tag, and dialysed against 10 mM Bis Tris pH 6.5. The concentration of rPrP was determined by absorption at 280 nm with molar extinction coefficients of 61,880 M−1 cm−1 and 56,667 M−1 cm−1 for BV rPrP and Hu rPrP respectively. Recombinant PrP concentration was adjusted to 0.2–0.5 mg/mL by the addition of buffer. Aliquots were stored at − 80 °C until use. Prior to usage, aliquot(s) of rPrP was dialysed into 20 mM Sodium phosphate buffer (pH 5.8), filtered (0.22 μm syringe).Hu rPrP  and BV rPrP  were prepared according to Jackson et alThe RT-QuIC reaction buffer was composed of 10 mM phosphate buffer (pH 7.4), 300 mM NaCl, 0.1 mg/mL rPrP, 10 μM Thioflavin T (ThT), and 10 μM ethylenediaminetetraacetic acid (EDTA). Reactions were prepared in either 96-well or 384-well optical clear-bottomed plates . For 96-well plates, each well contained either 98 μL of reaction buffer seeded by 2 μL of BH or 85 μL of reaction buffer seeded by 15 μL CSF, bringing the total volume in each well up to 100 μL. The proportion of the components, and hence its concentrations were maintained when applied to different volumes in 384-well plate experiments e.g. 42.5 μL reaction buffer seeded by 7.5 μL CSF . Each CSF sample was run in quadruplicate, allowing for 5 negative controls (PM non-prion CSF) and 5 positive controls (definite sCJD CSFs).Thereafter, the plates were sealed and incubated in a BMG OPTIMA FLUOstar plate reader at either 45 °C or 50 °C for 90–120 h with cycles of intermittent shaking. Each cycle consisted of 60 s of double orbital shaking at 700 rpm followed by 60 s rest, with ThT fluorescence measurements  taken every 2 min. The plate reader measures ThT fluorescence in relative fluorescence units (rfu) with a maximum rfu at 260,000. A reaction was considered positive if the average rfu in 2 or more wells exceeded a threshold rfu defined as the average rfu from 5 negative controls plus 5 standard deviations, and within 90 h of start time.The sensitivity and specificity were calculated for sCJD and IPD groups, and statistical significance between groups was determined by Fisher's Exact Test . Mixed effect linear modelling of MRC Scale scores was undertaken with Stata, v15.1.


Elevation of the chemokine CXCL13 in CSF frequently occurs during active and acute CNS inflammatory processes and presumably is associated with B cell-related immune activation. Elevation levels, however, vary a lot and “leaking” of CXCL13 from blood across dysfunctional brain barriers is a possible source. The aim was to clarify the relation between CXCL13 concentrations in CSF, CXCL13 concentrations in serum and blood–CSF barrier (BCSFB) function for a correct interpretation of the intrathecal origin of CXCL13.We retrospectively analyzed CXCL13 of banked CSF/serum samples (n = 69) selected from patient records and categorized the CSF CXCL13 elevations as CXCL13 negative (< 30 pg/ml), low (30–100 pg/ml), medium (101–250 pg/ml), or high (> 250 pg/ml). CXCL13 concentrations in CSF and serum and the corresponding CSF/serum CXCL13 quotients (Qcxcl13) were compared to CSF/serum albumin quotients (QAlb) as a measure for BCSFB function. The CXCL13 negative category included two subgroups with normal and dysfunctional BCSFB.Serum CXCL13 concentrations were similar across categories with median levels around 100 pg/ml but differed between individuals (29 to > 505 pg/ml). Despite clear evidence in serum, CXCL13 was detectable only at trace amounts (medians 3.5 and 7.5 pg/ml) in CSF of the two CXCL13 negative subgroups irrespective of a normal or pathological QAlb. Moreover, we found no association between CSF and serum CXCL13 levels or between QAlb and CSF CXCL13 levels in any of the CSF CXCL13-delineated categories. CXCL13 apparently does not “leak” from blood into CSF. This implies an intrathecal origin also for low CSF CXCL13 levels and a caveat for analyzing the Qcxcl13, because higher serum than CSF concentrations arithmetically depress the Qcxcl13 resulting in misleadingly low CSF/serum quotients.We demonstrated that CXCL13 does not cross from blood into CSF, not even during severe BCSFB dysfunction. CSF CXCL13 elevations therefore most likely always are CNS-derived, which highlights their relevance as indicator of inflammatory CNS processes. We recommend data should not be corrected for BCSFB permeability (QAlb) and not to calculate CSF/serum quotients for CXCL13 as these may introduce error. The C-X-C motif ligand 13 (CXCL13) is a key homeostatic chemokine constitutively expressed in lymphoid organs and crucial for recruitment and compartmentalization of lymphocytes. Both B cells and follicular T helper cells, a special subgroup of CD4 T cells required for B cell activation, follow CXCL13 gradients towards B-T cell contact zones , 3.CXCL13, however, is not restricted to development and maintenance of lymphoid tissues but is also implicated in chronic inflammation via the formation of tertiary lymphoid structures (TLS) in a process called “ectopic lymphoneogenesis” . TLS resCXCL13 also occurs in CNS inflammation and is the focus of biomarker research for Lyme neuroborreliosis (LNB), CNS lymphoma, and multiple sclerosis (MS) . The broPeripheral blood is still another possible source, particularly in case of blood–CSF-barrier (BCSFB) dysfunction. Several studies use CSF/serum quotients of CXCL13 (Qcxcl13) and albumin (QAlb) for correcting for BCSFB permeability –20. ObseThis study included 69 samples collected 2017–2018 from patients assigned to lumbar puncture who gave informed consent (415-E/2286/7-2018) for inclusion of blood and CSF samples in the CSF Biobank Salzburg project, a collaboration of the Departments of Neurology and Laboratory Medicine. Sample processing and storage were in accordance to international standardization recommendations for CSF biomarker development .−3 [Patient samples were specifically selected based on CXCL13 concentration in CSF, which we retrieved along with the CSF to serum albumin concentration quotient (QAlb) from records, and categorized as CSF CXCL13 negative (< 30 pg/ml), low (30–99 pg/ml), medium (100–250 pg/ml) and high (> 250 pg/ml). The cut-offs between categories were selected according to recommendations of Euroimmun CXCL13 ELISA instructions and published literature –27. An a−3 .The CXCL13 negative CSF category comprised two subgroups, with normal QAlb (< 30/N) and with pathological QAlb (< 30/P). The subgroup < 30/N of the CXCL13 negative category contained samples from patients with back pain, headache, dementia, stroke and nonspecific symptoms. The subgroup < 30/P of the CXCL13 negative category contained samples from patients with aseptic and purulent meningitis, spondylodiscitis, polyradiculitis, Wernicke encephalopathy, normal pressure hydrocephalus and cervical myelopathy. The CXCL13 low and medium categories contained samples from patients with aseptic and purulent meningitis, multiple sclerosis, facial paralysis, polyneuropathy, stroke and seizures. The CXCL13 high category contained samples from patients with Lyme neuroborreliosis, aseptic and purulent meningitis, and autoimmune myelitis and encephalitis.The responsible ethics committee of the Country of Salzburg consented to this study (415-E/2430/3-2018).CXCL13 concentrations of paired CSF and serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s recommendation . This involved a 1:2 dilution of sera in a special serum-dilution buffer (provided in the kit). Since CSF was used undiluted in the assay, we multiplied the resultant serum concentration by factor 2. For quality control, we plotted CSF CXCL13 concentrations of the banked sample run against the corresponding diagnostic sample concentrations.Data consistency was checked, and data were screened for outliers and normal distribution by using Kolmogorov–Smirnov test. Data distributions deviated from normal distributions. We used medians and interquartile ranges to summarize CXCL13 concentrations in CSF and serum, the Qcxcl13 and QAlb of the CSF CXCL13-delineated categories and subgroups, the Kruskal–Wallis ANOVA and Mann–Whitney U test for comparisons between categories  and Wilcoxon’s rank sum test for comparing CSF versus serum CXCL13 concentrations within categories. For analyzing the associations of serum CXCL13 concentrations and QAlb with CSF CXCL13 concentrations regression analyses were done and Spearman correlations computed. Spearman’s rank-order correlation was used to test the relation of ELISA results between the diagnostic and banked CSF sample runs. All reported tests were two-sided, and p-values < 0.05 were considered as statistically significant. All statistical analyses and illustrations were done by use of SPSS Statistics 24.0 (IBM Germany GmbH), STATISTICA 13  and Microsoft Excel 2016 .The study sample included 22 CSF categorized as CXCL13 negative, 14 CSF categorized as CXCL13 low, 13 categorized as CXCL13 medium, and 20 categorized as CXCL13 high. The CXCL13 negative CSF category was divided into two subgroups, 10 samples with normal QAlb (< 30/N) and 12 samples with pathological QAlb (< 30/P). Table Importantly, we perfectly reproduced CXCL13 concentrations of the diagnostic run with the banked CSF samples   and low (p < 0.05) categories. In addition, they were lower than CSF levels of the medium (p < 0.001) and high (p < 0.001) categories , the other with a significant BCSFB dysfunction  were analyzed. Importantly, we detected only trace amounts of CXCL13 in CSF (medians 3.5 and 7.5 pg/ml), despite similar concentrations in serum (medians 97 and 105 pg/ml) and therefore independent of BCSFB function as measured by QAlb,  between the subgroup < 30/P of the CXCL13 negative category and the three categories with low, medium and high CSF CXCL13 elevations. Expectedly, the QAlb of subgroup < 30/P  was higher than in the CXCL13 low category (30–99 (L); median 7.7, range 3.5–16.4; p < 0.05) but similar to the QAlb of the CXCL13 medium (100–250(M); median 11.7, range 3.5–28.8) and CXCL13 high (> 250 (H); median 14.9, range 5.9–102.4) categories.Taken together, these data provide evidence for a compartment restriction because CXCL13 did not pass from blood to CSF even in case of BCSFB dysfunction.In the median and high CXCL13 categories, CSF levels exceeded serum levels with corresponding Qcxcl13 of median 2.6 and 3.8, clearly indicating an intrathecal origin of CXCL13. Whereas in the CXCL13 low category, serum levels exceeded CSF levels with a corresponding Qcxcl13 of median 0.5. Assuming diffusion dynamics across the BCSFB similar to albumin or immunoglobulins, a Qcxcl13 < 1, rather would suggest a blood-derived origin.However, our data argued against CXCL13 passing from blood into CSF, which means that also low CSF levels most likely were intrathecally produced also in case of Qcxcl13 < 1.Moreover, serum CXCL13 levels—although rather homogeneous across categories—strongly differed between individuals (range 29 to > 505 pg/ml). High serum concentrations arithmetically may depress the CSF/serum CXCL13 quotient and result in erroneously low Qcxcl13, as for instance very likely is the case in bacterial meningitis triggered by otitis media in Table To determine whether CXCL13 elevations in blood played a role or were otherwise related to CXCL13 elevations in CSF, we looked over serum CXCL13 data in more detail and retrieved diagnoses of those samples with serum CXCL13 concentrations > 250 pg/ml (Table Serum CXCL13 levels > 250 pg/ml occurred in nine patients (13%). Five of them had CXCL13 levels > 250 pg/ml in both serum and CSF. The associated diseases were LNB (n = 2), one suspected CNS lymphoma, one otitis media with bacterial meningitis and one SLE with myelitis transversa, all with evidence of both peripheral and CNS inflammatory foci. High serum but low levels in CSF (61 and 74 pg/ml) concurred with stroke in the context of arteritis temporalis and HSV2 meningitis.The last two samples had high serum CXCL13 levels (315 and > 505 pg/ml) but borderline respectively negative CXCL13 levels in CSF (3 and 34 pg/ml), in the presence of significant BCSFB dysfunction (QAlbs of 16.7 and 14.7). The diagnoses were Spondylodiscitis, and Wernicke encephalopathy in combination with peripheral infection of unclear focus , for example, specified mean serum values of 81.9 pg/ml (range 39.4-252) derived from “apparently healthy donors without medical history”. Another study reported serum values of median 64 pg/ml (range 26–507) from 300 blood donors versus 94 pg/ml (range 28–417) in aseptic meningitis/facial palsy patients and 70 pg/ml (range 40–417) in patients with different neurological diagnoses [The clinical relevance of serum CXCL13 in autoimmunity, infection, chronic inflammation and malignancy is widely apprehended and intensely investigated –35. EverOur finding that CXCL13 did not pass from blood into CSF, not even despite its small molecular size (~ 13 kDa) and not in cases of severe BCSFB dysfunction was somewhat unexpected. On the other hand, the role of CXCL13 as tissue-specific chemoattractant confined to lymph nodes and TLS, implicates the necessity for mechanisms restricting its diffusion and spatial distribution. The mechanisms for its tissue-specific local containment has recently been shown to rely on special molecular binding sites on CXCL13 for heparan sulfate side chains of proteoglycans on cells and within extracellular matrices . These iDemonstrating that the chemokine CXCL13 does not cross from blood into CSF highlights its usefulness as biomarker for neuroinflammation, in particular, regarding conditions related to B cell activation and/or active humoral immune processes. Moreover, evidence that CSF CXCL13 is CNS-derived and independent from BCSFB integrity and serum levels requires cautious data interpretation with regard to calculating CSF/serum CXCL13 quotients and correcting for BCSFB permeability as these are prone to error.In addition, we show that co-determination of serum CXCL13 has potential in allocating the focus of the inflammatory process, which may localize to the periphery or to the CNS or co-localize in both compartments with possible therapeutic consequences.


In experiments on cardiac fibers treated with the hypertrophic cardiomyopathy-related mutants E244D- and K247R-troponin-T, treatment with K247R-troponin-T did not recruit contracting actomyosin to a greater extent than wild-type-troponin-T, although a similar drop in the intensity of the troponin reflection occurred. Therefore, the conformational change in K247R-troponin-T was suggested to be unable to fully recruit actomyosin interaction, which may be the cause of cardiomyopathy.X-ray diffraction and tension measurement experiments were conducted on rat left ventricular skinned fibers with or without “troponin-T treatment,” which exchanges the endogenous troponin T/I/C complex with exogenous troponin-T. These experiments were performed to observe the structural changes in troponin-T within a fiber elicited by contractile crossbridge formation and investigate the abnormality of hypertrophic cardiomyopathy-related troponin-T mutants. The intensity of the troponin reflection at 1/38.5 nm Deciphering the regulatory mechanism from a structural aspect is important for understanding the pathology of striated muscle diseases related to mutations of regulatory proteins, including familial cardiomyopathy. As for troponin, which is involved in the pathogenesis of familial cardiomyopathy, pieces of structural evidence that suggest formation of contractile crossbridges is involved in the regulatory mechanism of contraction have been accumulated to date ,3,4,5,6.2+, since the inhibitory effect of troponin I (TnI), which can be released by Ca2+ binding to TnC under physiological conditions, is now removed by the exchange. First, we confirmed that the active actomyosin interaction caused by addition of ATP in TnT-treated fibers elicited a structural change in troponin-T in a cooperative manner, as has been reported with skeletal muscle [In this study, we performed X-ray diffraction experiments on carefully prepared cardiac skinned fibers devised to obtain detailed X-ray diffraction patterns. Furthermore, we applied the method of “troponin-T (TnT) treatment”  to thesel muscle ,13. Secol muscle  whereas l muscle ,16. Equa−1 on the meridional axis, troponin reflection was observed . In Diffraction patterns of cardiac muscle fibers with no treatment before and after addition of ATP are shown in Next, we examined the diffraction patterns from TnT-treated cardiac muscle fibers. Diffraction patterns obtained from cardiac fibers treated with wild-type TnT before and after the addition of ATP are shown in Finally, we examined the changes in the diffraction pattern of cardiac fibers treated with E244D-TnT and K247R-TnT mutants to obtain information about the pathological mechanism of HCM-related TnT mutations. The intensity profile of the troponin reflection along the equatorial axis and integrated intensity of TnT-treated cardiac fibers with E244D-TnT and K247R-TnT are shown in To evaluate the number of recruited crossbridges in the fibers after the TnT-treatment, the tension generating capacity and intensity ratio of the 1.1 and 1.0 equatorial reflections were analyzed. As seen in the representative tension trace after the TnT treatment in Significant intensity change of troponin reflection was induced by addition of ATP in cardiac muscle fibers after TnT-treatment, indicating that troponin-T undergoes a structural change triggered by formation of contractile crossbridges in the cardiac skinned fibers. It is known that regulation by the tropomyosin-troponin system is influenced by formation of active crossbridges ,13, implAs for the intensity drop, Matsuo et al. , from thRegarding this study, the intensity change of the 38.5 nm troponin reflection observed with TnT-treated fibers was considered to be due to a structural change of TnT because the remaining endogenous T/I/C complex is unlikely to cause a large intensity drop A,E. HoweThe relative 1.1/1.0 intensity ratio and tension measurement suggested that cardiac fibers treated with K247R-TnT exhibited a weaker property of contractile crossbridge recruitment, contrary to the anticipation based on reports by Matsumoto et al.  and FordNext arises the question of how this weaker property of contractile crossbridge recruitment causes K247R-TnT to induce cardiomyopathy. It is notable that K247R-TnT was considered as an HCM-causing mutant based on the first report of this mutation in HCM-patients by Garcia-Castro et al. ; howeverThe results of the 1.1/1.0 intensity ratio and tension development experiments imply that introduction of E244D-TnT would confer the trend to elicit larger crossbridge recruitment than the wild-type TnT B,D. The Matsuo et al. , based o−1), following which their hearts were rapidly excised and blood was discharged by beating in warmed and oxygenated artificial extracellular solution. Next, cardiac muscle fiber bundles were dissected from the left ventricle wall very carefully. Cell membrane was disrupted by treatment of the fibers for more than 3 h with artificial intracellular solution containing 0.5% triton-X 100.Male rats  were used with approval of the Institutional Animal Care and Use Committee of The Jikei University . The rats were deeply anesthetized by intraperitoneal injection of pentobarbiturates  for an average period of 5 h . Recombi2+, with ionic strength of 0.2 at pH 7.0, and in the ATP solution, which contained 3.5 mM MgATP and was nominally free of Ca2+,with ionic strength of 0.2 at pH 7.0, using an imaging plate system . The X-ray wavelength was 0.09 nm and the beam size was 250 µm in width and 150 µm in height. The specimen-to-detector distance was 1.8 m. The sarcomere length of each fiber bundle was adjusted to 2.3 µm on average by laser diffraction. Experiments were carried out at 20 °C.X-ray diffraction experiments were performed at BL45XU in SPring8  using a setup described elsewhere  with sli−4.4 M Ca2+, with ionic strength of 0.2 at pH 7.0, was used.Tension development in the cardiac muscle skinned fibers with or without TnT-treatment was measured using a tension transducer . To evaluate maximally activated tension of the fibers, Maximally-activating solution, containing 3.5 mM MgATP and 10−1 along the meridional axis was integrated within a slice of 0.00081 nm−1 width in the equatorial direction. Then, two Gaussian distributions along the meridional axis were fitted to the profile to obtain the troponin-derived reflection separately from that of the C-protein-derived reflection for each slice. This was repeated after shifting the slice by 0.00081 nm−1 in the equatorial direction.Diffraction patterns were analyzed using MATLAB R2016B. Each X-ray diffraction pattern was rotated around the center and averaged quadrantally. The intensity profiles of the 38.5 nm troponin reflection along the meridional axis were obtained as below. Intensity in 1/54.8–1/32.9 nm−1.The obtained intensity of the troponin reflection for each slice was plotted along the equatorial axis in the range of 0–1/47.4 nm−1 along the meridional axis was integrated within a slice of 0.010 nm−1 width in the equatorial direction and the peak area above the background line between the bottoms at either side of the peak was obtained for each slice. This procedure was repeated after shifting the slice by 0.010 nm−1 in the equatorial direction. The obtained intensity for each slice was integrated along the equatorial axis in the range of 0–1/4.9 nm−1. Intensity of the 38.5 nm meridional reflection of troponin was expressed relative to the total integrated intensity of the 5.9 nm actin layer line.The intensity profile of the 5.9 nm actin layer line was obtained as below. Intensity in 1/5.6–1/6.3 nm−1 in the meridional direction. Next, intensities of the 1.0 and 1.1 equatorial reflections were obtained by fitting two Gaussian distributions on an exponential background to the integrated profile.Equatorial intensity profiles were obtained by integrating intensities in the range of 0–1/61.7 nmThrough direct observation of structural changes in TnT within the cardiac fibers, the K247R-TnT mutation was suggested to have a lower recruitment capacity of contractile crossbridges, even though a similar level of intensity drop in the troponin reflection occurred. Thus, the alteration of the single residue, K247R, affected function of the tropomyosin-troponin regulatory system without a large conformational change in TnT.2+ sensitivity, is beyond the scope of this study. Future work on cardiac muscle fibers with mutant TnT and normal troponin I/C will further clarify the molecular pathology of TnT-related cardiomyopathy.Finally, it should be noted that this study is limited to the function of TnT. Its interaction with troponin I/C, which affects actomyosin kinetics and Ca


We tested the hypothesis whether there is a correlation between the echogenicity and calcium and water contents of carotid plaques.Ninety carotid befurcations from 45 deceased patients were removed during autopsy. Thirty-four plaques were categorized as homogenous echolucent (HEL), homogenous echogenic (HEG) and heterogenous (HE) plaques based on premortem B-mode image. Water content was expressed in % of wet weight. Ca was determined by proton-induced X-ray emission and expressed in ppm. Relative optical density of the B-mode images was analyzed offline using a computer program.P ≤ 0.001). HE plaques showed an intermediate calcium content  that was statistically not different from echolucent plaques. Water content of HEG plaques was statistically not different from HEL and HE . HEG plaques had the highest relative optical densities (196 [188–217%]). HEL and HE had similar relative optical densities . A significant positive correlation was found between the Ca content and relative optical density of plaques.HEL plaques had lower Ca content  compared to HEG , Echogenicity of carotid plaques increases along with their calcium content. Water content may be an important factor in differentiation of different plaques. The maip = 0.02), whereas 40.9% of symptomatic plaques are echogenic in comparison to 67.2% of asymptomatic plaques [For a long time, it was believed that the degree of carotid stenosis is of paramount importance, causing hemodynamic strokes , but lat plaques .Some authors have tried to compare the ultrasonic tissue characterization with biochemical factors that might play a role in echoreflexion . HoweverIn this study, we have used computer-assisted B-mode ultrasonography image analysis to measure the echostructure of carotid atherosclerotic atherosclerotic lesions and attempted to compare their calcium and water contents. The aim of the present study was to check whether a correlation exists between the echogenicity and calcium as well as the water content of carotid plaques with different echostructures. According to our hypothesis, testing the hypothesis that both the calcium and water contents of carotid plaques are the main factors in determining that echogenicity could increase the amount of information about plaque composition and stability gained by the B-mode ultrasonography.22.1The subjects of this investigation were 45 critically ill patients admitted to the Stroke Unit of the Department of Neurology, University of Debrecen, with the diagnosis of acute ischemic stroke, between the time period of November 2012 and February 2014. Subjects underwent B-mode carotid ultrasound examination as a part of the routine diagnostics. All patients had acute ischemic cerebrovascular lesions confirmed by computer tomography. Confirmed carotid stenosis on the B-mode ultrasound imaging were the main inclusion criteria for our study.Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration, and has been approved by the Ethics Committee at University of Debrecen (RKEB-TUKEB 218/2012 OEC).Informed consent: Informed consent has been obtained from all individuals included in this study.B-mode ultrasonography in 45 moribound patients admitted with the diagnosis of acute ischemic stroke as a part of the routine diagnosis. Storage and categorization of the lesions  seen on the B-mode image (see detailed description below).Removal of the carotid bifurcations and sampling the lesions that were seen on the B-mode image.Biochemical analysis of tissue samples for determining the Ca and water contents (see detailed description below).Off-line analysis of the relative optical density of carotid plaques (see detailed description below).Phases of the study2.2Ultrasound examinations were performed with a Hewlett-Packard Sonos Ultrasound equipped with a 10 MHz linear-array transducer. Common and internal carotid arteries and bifurcations were scanned longitudinally by the B-mode method for the presence of atherosclerotic plaque. In order to avoid variability in image acquisition between subjects, ultrasound gain was stepwise adjusted in each individual measurement from a bright image to the darker direction until the point where the appearance of the flowing blood just turned into total black.According to the modified proposal of Gray-Weale , visual Type 1: predominantly echolucentType 2: mixed echolucent/echogenicType 3: predominantly echogenicAll scans were performed by two experienced neurosonologists (BF and PS), who were not involved in the later phases of analysis .2.3in vivo measurement.Sampling of the tissues occurred during the routine autopsy following the patients’ death within 24 h post mortem. Carotid bifurcations were removed, dissected longitudinally and examined macroscopically. Removal and preparation of the samples for further analysis were performed by two independent members of the team (SF and ZG). Calcium and water contents have been measured only on those bifurcations where macroscopic lesions were unequivocally identical to the lesions observed on the ultrasound images. For matching images to histology sections, we have used the relative distance of plaque from the common carotid bifurcation as a landmark, expressed in millimeters. This was carried out by a software option of the duplex scanner, resulting in identifying of the lesion location on the basis of the 2.4During sampling, an attempt was made to remove plaques unimpaired.2.4.1W) with 0.0001 g precision. The samples were then dried at 100°C for 24 h and reweighed to obtain dry weight (D). Water content was calculated as follows: 100(W − D)/W and were expressed in percents.After excision, tissue samples were weighed on a chemical balance to obtain wet weight (Proton-Induced X-Ray Emission (PIXE) measurements were carried out at the Debrecen microprobe facility installed on the 0° beam line of the 5 MeV Van de Graaf accelerator at the Institute of Nuclear Research of the Hungarian Academy of Science. After pretreatment, dried samples were bombarded by a proton beam of 2 MeV. The characteristic X-ray spectra of the samples were measured by a silicium-semiconductor X-ray spectrometer both qualitatively and quantitatively. The PIXE analysis gives information about the absolute amounts of the elements present within the beam spot which, however, cannot be directly converted to tissue concentrations. Hence, the PIXE spectra were analyzed by personal computer using XRF-BIO, a self-made program, estimating Ca concentrations corresponding to parts per million (ppm). For the calibration procedure, NBS and IMEA standards were used. Tissue analysis was performed by the same physicist (JB) who had previous experience in the analysis of vessel segments [segments .2.5All B-mode images were stored and were later off-line processed with the software program Image-Pro Plus version 1.2 for Windows  as suggested by the methodology by Grønholdt et al. . Any are2.6P < 0.05. Statistica for Windows version 5.5  was used for data analysis.The normality of variables was checked by the Shapiro–Wilk test. Calcium, water and optical density were not normally distributed; therefore, median and quartile ranges were calculated. For any comparison where these data were involved, Wilcoxon Sum-of-Ranks (Mann–Whitney) test was used for comparing two unmatched samples. The Spearman’s rank-order correlation was used to describe the relationship between variables with nonnormal distribution. Statistical significance was assumed if 3n = 28), myocardial infarction (n = 7), pulmonary edema (n = 4) or pulmonary embolism (n = 6).Ninety bifurcations of 45 deceased patients were removed during autopsy. There were 29 male patients (64.4%) and 16 females (35.6%), with a mean age of 65.1 (±5.6) years. Clinical characteristics are summarized in Thirty-four plaques (16 homogeneous and 18 heterogeneous) were diagnosed, categorized by the B-mode ultrasound during the clinical treatment phase. After the death of the patients, the identical carotid bifurcations were removed and dissected during autopsy. All plaques were located at the proximal part of the internal carotid arteries. As described earlier, based on their ultrasound appearance, lesions were grouped into homogenous plaques with predominantly echolucent, or with predominantly echogenic structure and heterogenous plaques with mixed echogenicity/echolucency.3.1P ≤ 0.001). Heterogeneous plaques showed an intermediate calcium content  that was statistically not different from echolucent plaques. Data are depicted on Echolucent plaques had significantly lower Ca content  compared to echogenic plaques , respectively, Water content in echogenic plaques was statistically not significantly different from that of echolucent and heterogenous plaques . It has to be mentioned that there was a tendency of echogenic plaques to have lower water content, but the differences did not reach the level of significance, probably due to the sample size. Data are summarized in P ≤ 0.0001), i.e., the higher was the Ca content, the lower was the water content of plaques.A significant inverse correlation has been found between water and Ca contents of plaques . Echolucent and heterogeneous plaques had similar relative optical densities  .3.3P = 0.001). The water content of different lesions and their optical densities were negatively correlated .In general, the calcium content of different plaques significantly correlated with their relative optical density  echogenicity are in the intermediate position with regard to Ca and water contents. When the optical density of different lesions was plotted against their Ca content, a significant positive correlation could be detected between the two parameters.Previous histopathological studies revealed that echogenicity of the plaque is determined by its composition: hypoechoic heterogeneous plaques are associated with intraplaque hemorrhage (increased water content) and lipids, whereas hyperechoic homogeneous plaque is mostly fibrous or calcified. Elastin content has also been associated with echolucency ,19. The In our study, plaques were characterized by high calcium in comparison with normal vessel wall. Mineralization and lipid deposition are coincident in plaques ,23. The Computer-based densitometric analysis and mean pixel determination of carotid ultrasonic images offer the possibility of digitizing B-mode images, allowing enhanced differentiation of plaque composition; furthermore, they can be used to standardize images and make measurements of echogenicity objective ,14. HoweOur results show that homo- and heterogeneous plaques had higher calcium content and optical densities, explaining why plaques can be easier detected by the ultrasound. In spite of the fact that calcium accumulates continuously in the plaque, we have found no significant difference of calcium content and relative optical density between homogeneous and heterogeneous plaques, which could be explained by the complexity of intraplaque events during plaque ageing or by measuring the entire plaque area to express its relative optical density. It was not the objective of our study to define a threshold value of optical density for the distinction of different types of plaques. Our main purpose was to confirm the correlation between echogenicity and calcium and water contents in the atherosclerotic lesions. According to our results, calcium seems to be one of the major determinants of the optical density, with high calcium concentrations resulting in high optical densities, whereas water content has a definitive impact on plaque echogenicity.What is the clinical implication of the results? In previous studies, different factors have been identified that may lead to an increased TIA and stroke risk in patients with hemodynamically nonsignificant carotid stenosis: the length of the stenosis along with a consequently increased cross-sectional area of the plaque, thin or ruptured fibrous cap, lipid-rich necrotic core, and plaque hemorrhage . All theWe have to mention the limitations of our study. The most important limitation is related to the averaged relative optical density of the carotid lesion. In fact, pixel distribution would have provided better description of echogenicity of the lesions, pathological samples were taken from the macroscopic gross regions of the carotid arteries and an averaged water content and Ca content could be determined by further analysis as well. We believe that despite this limitation, these data may describe the nature of plaque maturation process.Our conclusions are that  relative


Advanced granulocyte colony‐stimulating factor (G‐CSF)‐producing lung tumours are generally refractory to platinum‐based chemotherapy and are associated with poor prognosis. However, therapeutic strategies for these tumours remain unknown. A 74‐year‐old man was diagnosed with non‐small cell lung cancer‐not otherwise specified (NSCLC‐NOS); the clinical stage was T4N0M1c stage IVb. Blood testing showed leucocytosis and aberrant G‐CSF expression. We chose single‐agent pembrolizumab as the initial treatment because PD‐L1 was highly expressed in the tumours. A clinically favourable response was achieved from seven courses of pembrolizumab with a total disease‐free survival of 10 months. During this period, the blood leucocyte count was concordant with the disease condition. These observations showed that pembrolizumab monotherapy may be an effective treatment for patients with advanced G‐CSF‐producing NSCLC‐NOS and that the monitoring of leucocyte count may be a useful biomarker for predicting the efficacy of pembrolizumab monotherapy. Here, we report a patient with non‐small cell lung cancer‐not otherwise specified (NSCLC‐NOS) with high PD‐L1 expression and aberrant granulocyte colony‐stimulating factor (G‐CSF) production, in whom monotherapy with the anti‐PD‐1 antibody pembrolizumab was effective as an initial treatment. Granulocyte colony‐stimulating factor (G‐CSF) is a cytokine and hormone that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream. Asano et al. demonstrated that G‐CSF was produced autonomously in the plasma of nude mice transplanted with human lung cancer cells . G‐CSF‐pA 74‐year‐old man whose chest X‐ray findings indicated an upper left lung tumour was admitted to our hospital Fig. . He had 18F‐FDG in the bone marrow in the PET scan, indicating glucose metabolism was enhanced with the promotion of haematopoietic ability [The diagnostic criteria for a G‐CSF‐producing malignant tumour are: 1) marked increase in WBC count without infection or other diseases; 2) elevated serum G‐CSF level; (3) reduction in WBC count after tumour resection; and (4) G‐CSF‐positive staining from immunohistochemical analysis of the tumour [ marked i elevatedSeveral previous case reports have shown the efficacy of systemic chemotherapy such as platinum‐combined chemotherapy with etoposide or paclitaxel and non‐platinum chemotherapy with gemcitabine and vinorelbine for G‐CSF‐producing lung tumours . HoweverCancer immunotherapy has recently been developed as a promising alternative strategy for treating advanced NSCLC patients. In the KEY‐NOTE 024 clinical study, pembrolizumab monotherapy significantly prolonged progression‐free survival of NSCLC patients with high PD‐L1 expression, compared to systemic chemotherapy [Neutrophils induce pro‐inflammatory cytokines and chemokines that lead to tumour proliferation, invasion, and angiogenesis. However, there is no evidence on abnormal neutrophils by G‐CSF‐producing tumours having functional roles in the tumour microenvironment. Further investigations are warranted to elucidate these roles.Serum WBC and neutrophil counts were more responsive to the abundance of tumour burden than tumour markers during immunotherapy and relapse of G‐CSF‐producing lung cancers. Therefore, monitoring leucocyte count may be a useful biomarker for predicting the efficacy of pembrolizumab monotherapy. Evidence accumulated from further cases are warranted to validate our observations.Appropriate written informed consent was obtained for publication of this case report and accompanying images.