It has been hypothesized that neuronal damage releasing antigens might trigger secondary immune response. The objective of the study was to investigate whether brain damage due to spontaneous subarachnoid haemorrhage (SAH) or intracerebral haemorrhage (ICH) induces reactivity against neuronal surface proteins.Brain autoimmunity has been reported in patients with preceding infection of the central nervous system by Patients with spontaneous SAH and ICH, who had cerebrospinal fluid (CSF) and serum sampling within 2 weeks after disease onset (baseline) and afterwards at least 10 days later (follow-up), were included. Antibodies against NMDA, GABA-B, AMPA-1/− 2 receptor, LGI1 and CASPR2 were determined by indirect immunofluorescence.A total of 43 SAH and 11 ICH patients aged 62 (±12) years  had simultaneous CSF/ serum sampling median 5 and 26.5 days after disease onset. At baseline, all CSF samples were collected via ventricular drainage, at follow-up 20 (37.0%) patients had CSF collection by lumbar puncture because ventricular drain had been already removed. All CSF and serum samples at baseline and follow-up tested negative for antibodies against NMDA, GABA-B, AMPA-1/− 2 receptor, LGI1 and CASPR2.Immunoreactivity against common neuronal surface proteins was not observed within the early disease course of spontaneous SAH and ICH. Spontaneous subarachnoid haemorrhage (SAH) and spontaneous intracerebral haemorrhage (ICH) are both severe neurological disorders that result in brain injury due to several mechanisms including primary mechanical tissue damage caused by the bleeding and secondary effects such as inflammatory processes, oxidative stress responses, brain edema and in case of SAH delayed cerebral ischemia , 2.herpesviridae [With cell injury and blood brain barrier disruption antigens are released into the intrathecal space and, therefore, abundantly presented to the immune system. This may result in breaking tolerance and in the formation of an immune response. Brain autoimmunity has been observed in patients with preceding infections of the central nervous system (CNS) by sviridae –8. In thsviridae . It has sviridae .In the present study, we aimed to investigate whether acute brain damage due to spontaneous SAH or ICH induces the formation of antibodies against neuronal surface proteins in the early and post-acute phase.Patients with spontaneous SAH and ICH admitted within 24 h of disease onset to the neuro-intensive care unit of Medical University of Innsbruck have been recorded in a computerized database since April 2010 and July 2011, respectively. Diagnoses were confirmed by computed tomography of the head and traumatic causes were excluded. Only patients who had ventricular cerebrospinal fluid (CSF) and serum sampling within 2 weeks after disease onset (baseline) and afterwards at least 10 days later (follow-up), were included into this retrospective analysis. At follow-up, also lumbar CSF analysis was eligible in case of lacking ventricular CSF sampling , α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-1, AMPA-2 receptor and against the voltage-gated potassium channel (VGKC)-associated proteins leucine-rich glioma inactivated 1 (LGI1) and contactin-associated protein 2 (CASPR2) were determined in CSF and serum at baseline and follow-up by commercially available cell-based indirect immunofluorescence assays .Antibodies against NMDA, γ-aminobutyric acid type B . Distribution of data was assessed by Kolmogorov-Smirnov test and, accordingly, data were displayed as mean ± standard deviation (SD) or as median and interquartile range (IQR).A total of 43 SAH and 11 ICH patients aged 62 (±12) years comprising 65% females had simultaneous CSF and serum sampling at baseline, median 5 (IQR 3–8) days after disease onset, and at follow-up after 26.5 (19–35.5) days. At baseline, all CSF samples were collected via ventricular drainage. At follow-up, 15 (34.9%) SAH and 5 (45.5%) ICH patients had CSF collection by lumbar puncture because ventricular drain had been already removed. Demographic and main sample characteristics for each disease group are displayed in Table B, AMPA-1, AMPA-2 receptor and against the VGKC-associated proteins LGI1 and CASPR2.All CSF and serum samples at baseline and follow-up were tested negative for antibodies against NMDA, GABAherpesviridae who developed secondary brain autoimmunity as shown by the detection of anti-NMDA receptor antibodies and other so far unknown neuronal surface antibodies within weeks after disease onset [B, AMPA-1, AMPA-2 receptors and against the VGKC-associated proteins LGI1 and CASPR2 in the CSF and serum of patients with spontaneous SAH and ICH. Within a median time period of approximately 4 weeks after disease onset, we did not obtain any positive result.According to recent reports on patients with CNS infection by se onset –8, one mherpesviridae encephalitis is still unknown [There might be several reasons for the absence of a secondary immune response in our cohort of SAH and ICH patients. First, the aetiology of the evolution of anti-NMDA receptor antibodies in patients with preceding  unknown . Althoug unknown , there mThere are some limitations of the study, e.g. the small number of patients. One could argue that autoantibodies against neuronal surface proteins might occur at a low frequency, and, therefore, were not detected. Although a higher patient number would strengthen the (negative) results, one has still to consider a false positive rate of the immunofluorescence test of up to 1%  , 11. FurFinally, antibodies against neuronal surface proteins cover only a small part of possible antigens against an immune response can be directed. Therefore, screening for antibodies against neuropil as well as determination of antibodies against intracellular epitopes has to be done in a further study.Immunoreactivity against common neuronal surface proteins was not observed within the early disease course of spontaneous SAH and ICH.


Schistosoma mansoni threatens hundreds of millions of people in >50 countries. Schistosomulae migrate through the lung and adult worms reside in blood vessels adjacent to the intestinal mucosa. Current candidate vaccines aren’t designed to elicit a mucosal response. We have repurposed an attenuated Salmonella enterica Typhimurium strain (YS1646) to produce such a vaccine targeting Cathepsin B (CatB), a digestive enzyme important for parasite survival. Promoter-Type 3 secretory signal pairs were screened for protein expression in vitro and transfected into YS1646 to generate candidate vaccine strains. Two strains were selected for in vivo evaluation (nirB_SspH1 and SspH1_SspH1). Female C57BL/6 mice were immunized twice, 3 weeks apart, using six strategies: i) saline gavage (control), ii) the ‘empty’ YS1646 vector orally (PO) followed by intramuscular (IM) recombinant CatB (20μg IM rCatB), iii) two doses of IM rCatB, iv) two PO doses of YS1646-CatB, v) IM rCatB then PO YS1646-CatB and vi) PO YS1646-CatB then IM rCatB. Serum IgG responses to CatB were monitored by ELISA. Three weeks after the second dose, mice were challenged with 150 cercariae and sacrificed 7 weeks later to assess adult worm and egg burden (liver and intestine), granuloma size and egg morphology. CatB-specific IgG antibodies were low/absent in the control and PO only groups but rose substantially in other groups (5898-6766ng/mL). The highest response was in animals that received nirB_SspH1 YS1646 PO then IM rCatB. In this group, reductions in worm and intestine/liver egg burden (vs. control) were 93.1% and 79.5%/90.3% respectively . Granuloma size was reduced in all vaccinated groups (range 32.9–52.8 x103μm2) and most significantly in the nirB_SspH1 + CatB IM group (34.7±3.4 x103μm2vs. 62.2±6.1 x103μm2: vs. control P < .01). Many eggs in the vaccinated animals had abnormal morphology. Targeting CatB using a multi-modality approach can provide almost complete protection against S. mansoni challenge. Schistosoma mansoni is the most widely distributed and is endemic in the Caribbean, South America, and Africa. It causes a chronic disease with severe negative effects on quality of life. Mass drug administration of praziquantel is the only available course of action due to a current lack of vaccines. However, praziquantel does not protect from reinfection. Therefore, a vaccine would be beneficial as a long-term solution to reduce morbidity and transmission of the disease. Our group has repurposed the attenuated YS1646 strain of Salmonella Typhimurium as an oral vaccine vector for the digestive enzyme Cathepsin B of S. mansoni. Oral vaccination followed by an intramuscular dose of recombinant Cathepsin B lead to significant reductions in parasite burden in mice. These animals had the highest titers in serum IgG and intestinal IgA antibodies. This multimodal vaccination approach also elicited both Th1 and Th2 cytokines as seen by the increases in IFNγ and IL-5. Finally, vaccinated mice had reductions in granuloma size along with a higher proportion of morphologically-abnormal eggs. This work demonstrates that a YS1646-based, multimodality, prime-boost immunization schedule can provide nearly complete protection against S. mansoni in a well-established murine model.Schistosomiasis is a parasitic disease that affects over 250 million people worldwide and over 800 million are at risk of infection. Of the three main species,  Schistosoma spp. These trematodes currently infect >250 million people worldwide and more than 800 million are at risk of infection , synthesized by GenScript  and inserted into the pUC57 plasmid with a 6x His tag at the 3’ end. Promoter-T3SS pairs were cloned upstream of the CatB gene and inserted separately into pQE30 . Parallel constructs were made with CatB gene replaced by eGFP to produce expression plasmids used for imaging studies. See S. enterica Typhimurium YS1646  was cultured in Lysogeny broth (LB) media and strains bearing each construct were generated by electroporation . Successfully transformed strains were identified using LB agar containing 50 μg/mL ampicillin . Aliquots of each transformed strain were stored in LB with 15% glycerol at -80°C until used in experiments.Gene segments of the Recombinant YS1646 strains were grown in LB broth with 50 μg/mL ampicillin at 37°C in a shaking incubator under aerobic or low oxygen  conditions. Bacterial lysates were prepared by centrifugation  then boiling the pellet (100°C x 10 min). Proteins from the culture supernatant were precipitated with 10% trichloroacetic acid for 1 hour on ice followed by centrifugation  and removal of the supernatant. Protein pellets were resuspended in NuPAGE LDS sample buffer and NuPAGE reducing agent according to the manufacturer’s instructions (Thermo Fisher). Immunoblotting was performed as previously described . Briefly6 cells/well in 12-well plates in Dulbecco's Modified Eagle's medium (DMEM) (Wisent Bioproducts) supplemented with 10% fetal bovine serum (FBS: Wisent Bioproducts). Transformed YS1646 were diluted in DMEM-FBS to give a multiplicity of infection of 100 and centrifuged onto the monolayer (110xg for 10 min) to synchronize the infection. After 1 hour at 37°C in 5% CO2, plates were washed three times with phosphate buffered saline (PBS: Wisent Bioproducts) and replaced in the incubator with DMEM-FBS containing 50 μg/mL gentamicin (Sigma-Aldrich) to kill any extracellular bacteria and prevent re-infection. After 2 hours, the cells were washed with PBS three times and the gentamicin concentration was lowered to 5 μg/mL. After 24 hours, the cells were harvested, transferred to Eppendorf tubes and centrifuged (400xg for 5 min). Pellets were prepared for western blotting as above. For imaging experiments, RAW 264.7 cells were seeded into 6-well chamber slides at 104 cells/well and cultured as above. After 24 hours, the cells were stained with 4',6-diamidino-2-phenylindole (DAPI) (Thermo Fisher), fixed with 4% paraformaldehyde in PBS and incubated for 10 min at RT. Images were obtained using a Zeiss LSM780 laser scanning confocal microscope and analyzed using ZEN software .Murine macrophage-like cells (RAW 264.7: ATCC-TIB 71) were seeded at 10S. mansoni CatB was cloned and expressed in Pichia pastoris as previously described [escribed . Briefly9 colony-forming units (CFUs)/dose). Intramuscular (IM) vaccinations were administered using a 25G needle in the lateral thigh (20μg rCatB in 50μL PBS). Each experiment included six groups with 8 mice/group: i) saline PO twice (Control) ii) YS1646 transformed with ‘empty’ pQE30 vector (EV) PO followed by rCatB IM (EV→IM), iii) CatB-bearing YS1646 PO twice (PO→PO), iv) rCatB IM twice (IM→IM), v) CatB-bearing YS1646 PO followed by rCatB IM (PO→IM), and vi) rCatB IM followed by CatB-bearing YS1646 PO (IM→PO). The number of animals that were used for each study outcome at each time-point is indicated in the figure legends.Female 6–8 week old C57BL/6 mice were purchased from Charles River Laboratories . All animals received two doses three weeks apart  at a 1:5 dilution (w/v) on ice until processed. Tissue was homogenized , centrifuged at 2500xg at 4°C for 30 minutes and the supernatant was collected. Supernatants were stored at -80°C until analyzed by ELISA.2SO4 after 15 min . Optical density (OD) was measured at 450 nm with an EL800 microplate reader . The concentration of CatB-specific IgG and IgA were calculated by extrapolation from the mouse IgG or IgA standard curves.Blood was collected from the saphenous vein at baseline (week 0) and at 3 and 6 weeks in microtainer serum separator tubes . Cleared serum samples were obtained following the manufacturer’s protocol and stored at −20°C until used. Serum CatB-specific IgG and intestinal CatB-specific IgA levels were assessed by ELISA as previously described . Briefly2SO4 after 15 min . Optical density (OD) was measured at 450 nm with an EL800 microplate reader (BioTek Instruments Inc.). The results are expressed as the mean IgG1/IgG2c ratio of the endpoint titers ± standard error of the mean. Endpoint titers refer to the reciprocal of the highest dilution that gives a reading above the cut-off calculated as previously described [Serum CatB-specific IgG1 and IgG2c levels were assessed by ELISA as previously described . Brieflyescribed .6 cells/well) in RPMI-1640 (Wisent Bioproducts) supplemented with 10% fetal bovine serum, 1 mM penicillin/streptomycin, 10 mM HEPES, 1X MEM non-essential amino acids, 1 mM sodium pyruvate, 1 mM L-glutamine , 0.05 mM 2-mercaptoethanol (Sigma-Aldrich). The cells were incubated at 37ºC in the presence of 2.5 μg/mL of rCatB for 72 hours after which the supernatant cytokine levels of IL-2, IL-4, IL-5 IL-10, IL-12p70, IL-13, IL-17, IFNγ, and TNF-α were measured by QUANSYS multiplex ELISA (9-plex)  following the manufacturer’s recommendations.In some experiments, some of the animals were sacrificed 4 weeks after the second vaccination. Spleens were collected and splenocytes were isolated as previously described with the following modifications . SplenocBiomphalaria glabrata snails infected with the S. mansoni Puerto Rican strain were obtained from the Schistosomiasis Resource Center of the Biomedical Research Institute  through NIH-NIAID Contract HHSN272201700014I for distribution through BEI Resources. Mice were challenged three weeks after the second immunization (week 6) with 150 cercariae by tail exposure and were sacrificed seven weeks post-challenge as previously described [3 μm2 ± SEM. Eggs were classified as abnormal if obvious shrinkage had occurred, if internal structure was lost or if the perimeter of the egg was crenelated and are reported as a percent of the total eggs counted (± SEM).escribed . Brieflyescribed , 34. BriStatistical analysis was performed using GraphPad Prism 6 software . In each experiment, reductions in worm and egg burden were expressed relative to the saline control group numbers. Results are represented from two separate experiments. Data were analyzed by one-way ANOVA and multiple comparisons were corrected using Tukey’s multiple comparison procedure. P values less than 0.05 were considered significant.S. mansoni CatB or eGFP. In monomicrobial culture, CatB expression was effectively driven by the nirB_SspH1, SspH1_SspH1 and SteA_SteA plasmids (in vivo testing.Thirteen expression cassettes were built and the sequences were verified (McGill University Genome Quebec Innovation Centre) . The proplasmids  with theplasmids . In infeplasmids  but onlyplasmids . These cP < .01 vs. control) but these titers were not significantly different from groups that had received either one (EV→IM) or two doses of rCatB (IM→IM) . IgG antibody titers were generally lower in all groups that received the YS1646 strain bearing the SspH1_SspH1 construct . The IM→PO group also had higher intestinal IgA titers than controls, but this increase did not reach statistical significance (259.8 ± 19.4 ng/g).Intestinal IgA levels in the saline, EV→IM, and IM→IM groups were all low (range 37.0–148.0 ng/g of tissue: P < .01) and the nirB_SspH1 PO→IM group  whereas the control group was below the limit of detection at 63.1 pg/mL (P < .05) . There were no significant differences in the levels of IL-2, IL-4, IL-10, IL-12p70, IL-13, IL-17 or TNF-α between vaccinated and control groups . Compare.1 pg/mL . Only thP < .05) .P < .01) reduction in worm numbers and multi-modality vaccination with this strain achieved 93.1% (P < .001) and 81.7% (P < .01) reductions in the PO→IM and IM→PO groups respectively.  and IM→IM groups (20.5%) across all studies. Overall, protection was better with nirB_SspH1_CatB schedules compared to SspH1_SspH1_CatB schedules. In the SspH1_SspH1 animals, reductions in worm numbers were similar to the IM→IM group: 17.2% with oral vaccination alone (PO→PO) and only 17.8% and 24.7% in the PO→IM and IM→PO groups respectively. In contrast, the PO→PO group vaccinated with the nirB_SspH1 YS1646 strain had an 81.7% (ctively. .P < .001). The greatest impact on hepatic and intestinal egg burden was seen in the nirB_SspH1 multi-modality groups: 90.3% (P < .0001) and 79.5% (P < .0001) respectively in the PO→IM group and 79.4% (P < .001) and 75.9% (P < .0001) respectively in the IM→PO group  and IM→PO groups (17.7% and 29.8% respectively). These apparent differences in egg burden between the two multi-modality groups did not parallel the reductions in worm numbers or the systemic anti-CatB IgG levels. Groups that received the nirB_SspH1 strain had more consistent and greater reductions in egg burden: the PO→PO group had 73.6% and 69.2% reductions in hepatic and intestinal egg numbers respectively (both PO group .3 μm2) and essentially all of the eggs in these granulomas had a normal appearance. The EV→IM and IM→IM groups had slightly smaller granulomas (52.0 ± 6.9 x103 μm2 and 52.8 ± 10.4 x103 μm2 respectively) with modest numbers of abnormal-appearing eggs  . In the groups that received the nirB_SspH1 strain, both the purely oral (PO→PO) and multi-modality strategies (PO→IM and IM→PO) resulted in even smaller granulomas . The large majority of the eggs in these granulomas had disrupted morphology . Overall, the greatest and most consistent reductions in both adult worm numbers and egg burdens in hepatic and intestinal tissues were seen in the animals that received oral dosing with the YS1646 bearing the nirB_SspH1_CatB construct followed 3 weeks later by IM rCatB.Hepatic granulomas were large and well-formed in the PBS-treated control mice (62.2 ± 6.1 x10ed edge)  but thesS. mansoni vaccine candidate capable of providing ≥40% protection [S. mansoni female worms can produce hundreds of eggs per day [S. mansoni-associated morbidity and mortality while reducing the intestinal egg burden would likely decrease transmission.In the mid-1990s, the Tropical Diseases Research (TDR) committee of the World Health Organization (WHO) launched the search for a  per day . While t per day . Eggs tr per day . ReducinSalmonella enterica Typhimurium strain YS1646 that had been used in a phase 1 clinical cancer trial at doses up to 3x108 IV was attractive for many reasons. Although S. enterica species replicate in a membrane-bound host cell compartment or vacuole [Salmonella enterica species, YS1646 has two distinct T3SS located in Salmonella pathogenicity islands 1 and 2 (SPI-I and SPI-II) [Salmonella-based vaccine development programs [Our group has previously described the protective efficacy of CatB-based vaccines delivered IM with adjuvants , 13. Usi vacuole , foreign SPI-II)  that are SPI-II) . The SPI SPI-II) . These Tprograms , 42.Salmonella enterica serovar Typhimurium in a heterologous prime-boost vaccination regimen. Compared to infected controls, vaccination with CatB IM followed by YS1646 bearing the nirB_SspH1 strain resulted in an 93.1% reduction in worm numbers and 90.3% and 79.5% reductions in hepatic and intestinal egg burdens respectively compared to the control group. These results not only surpass the WHO’s criterion for an effective S. mansoni vaccine by a considerable margin, they are a marked improvement on our own work using CatB delivered IM with adjuvants and are among the best results ever reported in similar murine models [In this study, we report the protective efficacy of CatB delivered by the attenuated strain YS1646 of e models , 13. Fore models , 44, IM e models  while IMe models , 46. Anoe models . It is ne models , particuSalmonella-vectored vaccines clearly leads to higher mucosal IgA responses than IM dosing [S. japonicum in a similar murine model [S. japonicum model targeting the long hydrophobic domain of the surface exposed membrane protein Sj23LHD and a host-parasite interface enzyme (glutathione S-transferase or GST), the PO→IM vaccination schedule led to important reductions in both worm numbers (51.4%) and liver egg burden (62.6%) [To what extent the surprising reductions in worm and egg burdens that we observed with the YS1646 can be attributed to the systemic or the local antibody response is currently unknown although it is likely that both contributed to the success of the combined schedules (ie: IM→PO and PO→IM). Oral administration of M dosing  and the M dosing . The migM dosing , 52. Admne model . Althougne model , it is i (62.6%) .overall pattern of immunity to S. mansoni and/or reduce the fitness of the eggs produced . Such effects could significantly extend the value of the combined PO→IM vaccination strategy, i.e.: more durable impact, reduced transmission, etc. Furthermore, prior work with IM vaccination with CatB alone revealed a Th2-type pattern of cytokine response in splenocytes  [S. mansoni infection after YS1646 infection and we have previously shown that CD4+ T cells and anti-schistosomula antibody-dependent cellular cytotoxicity (ADCC) contribute to protection after CatB immunization (± adjuvants) [+ or CD8+ T cells).In addition to the substantial overall reductions in worm numbers and egg burden in our animals that received multimodality vaccination, there were additional suggestions of benefit in terms of both hepatic granuloma size and possible reduced egg fitness . The sizd IL-13) . In the juvants) . StudiesS. mansoni target antigens that have shown promise in pre-clinical and/or clinical development  to generate a ‘cocktail’ vaccine. In this context, an attenuated Salmonella vector like YS1646 might be ideal because of its high ‘carrying capacity’ for foreign genes [Salmonellae have a good safety track-record in vaccination: eg: the Ty21a S. typhi vaccine and a wide range of candidate vaccines [8 bacteria could be delivered intravenously in these vulnerable subjects without causing serious side effects [Although the findings presented herein are promising, this work has several limitations. First, immune protection is likely to be relatively narrow when only a single schistosome antigen is targeted. In the long term, this limitation could be easily overcome by adding one or more of the many gn genes . Second,gn genes . Althougvaccines  despite vaccines . AlthougS. mansoni in a well-established murine model. The protection achieved against a range of parasitologic outcomes was the highest reported to date for any vaccine. These observations strongly support the continued development of this candidate vaccine for prophylactic and possibly even therapeutic use in the many hundreds of millions of people in low- and middle-income countries at risk of or already infected by S. mansoni.In summary, this work demonstrates that a YS1646-based, multimodality, prime-boost immunization schedule can provide nearly complete protection against S1 Table#Values were below the limit of detection.Supernatant levels of different cytokines after stimulating splenocytes with rCatB for 72 hours were measured by QUANSYS multiplex ELISA. These results represent 5–7 animals per group. Results are expressed as the mean ± the standard error of the mean. (TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S1 FigRepresentative images of H&E staining of granulomas from livers of vaccinated mice (A) and saline control mice (B). Panel A represents the PO → IM group for the nirB_SspH1 construct. Scale is set to 100 μm.(TIF)Click here for additional data file.


We aimed to investigate the relationship between cerebrospinal fluid levels (CSF) of amyloid precursor protein (APP)‐derived peptides related to the amyloidogenic pathway, cortical thickness, neuropsychological performance, and cortical gene expression profiles in frontotemporal lobar degeneration (FTLD)‐related syndromes, Alzheimer’s disease (AD), and healthy controls.We included 214 participants with CSF available recruited at two centers: 93 with FTLD‐related syndromes, 57 patients with AD, and 64 healthy controls. CSF levels of amyloid β (Aβ)1‐42, Aβ1‐40, Aβ1‐38, and soluble β fragment of APP (sAPPβ) were centrally analyzed. We compared CSF levels of APP‐derived peptides between groups and, we studied the correlation between CSF biomarkers, cortical thickness, and domain‐specific cognitive composites in each group. Then, we explored the relationship between cortical thickness, CSF levels of APP‐derived peptides, and regional gene expression profile using a brain‐wide regional gene expression data in combination with gene set enrichment analysis.The CSF levels of Aβ1‐40, Aβ1‐38, and sAPPβ were lower in the FTLD‐related syndromes group than in the AD and healthy controls group. CSF levels of all APP‐derived peptides showed a positive correlation with cortical thickness and the executive cognitive composite in the FTLD‐related syndromes group but not in the healthy control or AD groups. In the cortical regions where we observed a significant association between cortical thickness and CSF levels of APP‐derived peptides, we found a reduced expression of genes related to synaptic function.APP‐derived peptides in CSF may reflect FTLD‐related neurodegeneration. This observation has important implications as Aβ1‐42 levels are considered an indirect biomarker of cerebral amyloidosis. Frontotemporal lobar degeneration (FTLD) is a neuropathological umbrella term encompassing multiple proteinopathies with common patterns of neurodegeneration in frontotemporal regions.However, several studies in patients with FTLD have shown substantial disagreement between CSF levels of Aβ1‐42 and amyloid positron emission tomography.Aβ peptides are generated by proteolytic cleavage from the amyloid precursor protein (APP) by sequential action of β‐ and γ‐secretases.in‐vivo identification of FTLD and its differentiation from other non‐neurodegenerative diseases and could also provide information about central pathophysiological mechanisms in FTLD. In this multimodal biomarker study, we compare the CSF levels of Aβ1‐42, Aβ1‐40, Aβ1‐38, and sAPPβ in FTLD‐related syndromes, AD, and healthy controls, and we explore their relationship with cortical thickness and neuropsychological measures of cognitive impairment. To further explore the mechanisms underlying the relationship between APP‐derived peptides and neurodegeneration in FTLD, we investigated the transcriptional architecture of cerebral regions where cortical thickness correlated with APP‐derived peptides in the FTLD group.However, it remains unknown whether Aβ1‐42 and other Aβ peptides generated in the amyloidogenic APP pathway are related to neurodegeneration in FTLD‐related syndromes or AD.Patients with AD and FTLD‐related syndromes were diagnosed at each center by experts in behavioral neurology according to current diagnostic criteria.Cognitive functioning was assessed using a previously described neuropsychological evaluationAll biomarkers were analyzed at the Sant Pau Memory Unit Laboratory with commercially available ELISA kits of Aβ1‐40, Aβ1‐38, and sAPPß  following previously reported methods and manufacturer’s instructions.http://surfer.nmr.mgh.harvard.edu) to compute individual cortical thickness maps, as previously reported.A subset of 135 participants had 3T magnetic resonance imaging (MRI) available for quantitative neuroimaging analyses. Of these, 88 participants were scanned on a 3T Philips Achieva, 7 participants were scanned on a different 3T Philips Achieva, and 40 participants were scanned on 3T Siemens 3T TrioTim. Briefly, surface‐based cortical reconstruction was performed using FreeSurfer v5.1 software package  and analysis of covariance (ANCOVA) analyses with age and APOEɛ4 status as covariates. For ANOVA and ANCOVA analyses, variables not following a normal distribution  were log‐transformed to achieve normality. For the study of the correlations within CSF biomarkers and between CSF biomarkers and cognitive measures, we applied simple correlations (Pearson’s coefficient) and partial correlations  with bootstrapping‐based 95% confidence intervals (bias corrected and accelerated for 1000 samples). To explore the significant differences between the magnitude of the correlations of CSF biomarkers between groups, we applied a R‐to‐R transform. All statistical tests were two‐sided and statistical significance was set at 5% (α = 0.05). Statistical analyses were performed with the IBM SPSS Statistics 25 (IBM corp.) software. For ANOVA and ANCOVA analyses, all P values were corrected for multiple comparisons (Bonferroni).We assessed normality of the variables by means of the Kolmogorov–Smirnov test. Between‐group differences in baseline characteristics were assessed using t‐test, ANOVA, or Kruskal–Wallis test for continuous variables, and χhttp://human.brain-map.org/). Data were processed according to previously published methods .Microarray data from a total of 3702 regional tissue sample from post‐mortem brain tissue of six individuals from the Allen Institute Human Brain Atlas were used to analyze cortical gene expression (https://string-db.org/).After the identification of cortical regions where cortical thickness showed a significant correlation with the CSF levels of APP‐derived peptides, we quantified the differences in gene expression between these areas and the background expression levels in the rest of the cortex, calculating the Cohen’s delta for each gene. The resulting Cohen’s deltas were first normalized using Z‐score and then used to build a histogram distribution of the differences between ROI and background expression. Then, a threshold of 2.58 × SD  was set to obtain the list of genes showing a differential expression. Then, we introduced these genes in the GO Enrichment AnalysisThe study was approved by the local ethics committee of each participant center and was conducted in accordance with the Declaration of Helsinki. All participants gave their written informed consent to participate in the study.The datasets analyzed during the current study are available from the corresponding author on reasonable request.APOEɛ4 carriers were overrepresented in the AD group compared to FTLD‐related syndromes and controls. In the FTLD‐related syndromes group, there were 11 cases with known pathogenic mutations in FTLD‐related genes .The main clinical and demographic characteristics of the study are shown in Table APOEɛ4. As expected, the lowest levels of Aβ1‐42 were observed in the AD group. However, Aβ1‐42 levels were also decreased in FTLD‐related syndromes when compared to healthy controls.CSF levels of Aβ1‐42, Aβ1‐40, Aβ1‐38, and sAPPβ were reduced in the FTLD‐related syndromes group when compared to the healthy control group  had low CSF levels of Aβ1‐42, below our validated threshold for cerebral amyloidosis.As reported in previous studies,APOEɛ4. The CSF levels of Aβ1‐40, Aβ1‐38, and sAPPβ were lower in the FTLD‐related syndromes group than in the AD and healthy control groups  and FTLD‐TDP . However, patients with C9orf72, GRN, and VCP mutations (n = 11) had lower levels of Aβ1‐42 than patients in the sporadic FTLD‐S group  = 2.158; P = 0.045).We did not find any significant differences in the CSF levels of APP‐derived peptides between participants with increased certainty of underlying FTLD‐Tau (APOEɛ4 positive (n = 18) and APOEɛ4 negative (n = 75) FTLD‐S cases. This finding together with the similar Aβ1‐42/Aβ1‐40 ratio in FTLD‐S cases and controls suggests that comorbid AD may not be the cause of the observed decrease in APP‐derived peptides in the FTLD‐S group.Of note, we did not observe statistically significant differences in the CSF levels of APP‐derived peptides between n = 28). We also found significant correlations in the semantic variant of primary progressive aphasia group despite the small sample size (n = 5). However, we were unable to obtain significant correlations in the nfaPPA (n = 14) and PSP‐CBS (n = 5) subgroups. Of note, these two subgroups were the ones with the lowest neurodegenerative burden. Importantly, we did not observe any correlation between APP‐derived peptides and cortical thickness in the AD and healthy control groups despite the existence of extensive areas of cortical thinning in regions related with neurodegeneration in both groups (data not shown). Of note, neither Aβ1‐42/Aβ1‐40 nor Aβ1‐42/Aβ1‐38 ratios correlated with cortical thickness in the FTLD‐related syndromes or the AD groups.As shown in Figure We next investigated the correlation between APP‐derived peptides and domain‐specific cognitive scores in each clinical group. Importantly, we only found correlations in the FTLD‐related syndromes group, and these correlations remained significant after accounting for age, sex, and education. As shown in Table We next explored whether the correlation between different APP‐derived peptides differed between groups. As shown in Table APP and other genes related to APP processing were overexpressed in the cortical regions with significant associations. We found that neither APP nor other APP‐related genes  activity is increased in sporadic AD,The transcriptional analyses performed in this study suggest that biological processes related to synaptic function could link neurodegeneration and APP processing in FTLD. Interestingly, there is strong evidence that APP is expressed at the synapse and that APP‐derived peptides are involved in multiple synaptic functions.The main strengths of this study are the inclusion of participants with a high probability of underlying FTLD and AD and the multimodal biomarker approach that allowed us to explore the structural correlates of APP‐derived peptides and their transcriptional architecture. However, this study has also some limitations. First, this study lacks neuropathological confirmation and misdiagnosis could have occurred. However, in all participants, the diagnosis of underlying FTLD and AD was supported by CSF cutoffs and longitudinal follow‐up. Although we did not find differences in APP‐derived peptides between specific clinical syndromes related to FTLD or participants with presumed Tau and TDP‐43 pathology in our study, further path‐confirmed studies should compare the CSF profile of APP‐derived peptides between the different neuropathological subtypes of FTLD. Moreover, additional studies should compare genetic and sporadic cases since results from this and other studies suggest the existence of different CSF biomarker profiles in genetic cases.In summary, we report that patients with FTLD show a global reduction of APP‐derived peptides in CSF and that this reduction correlated with FTLD‐related neurodegeneration and cognitive impairment. This pattern of APP‐derived peptides should be considered when evaluating the presence of AD pathophysiology in FTLD‐related syndromes. More studies are needed to advance our mechanistic understanding of the relationship between APP‐derived peptides and synapses.I.I.‐G. and A. Lleó contributed to the conception and design of the study; I.I.‐G., J.P., V.M., D.A., E.V., A.B., S.B.‐E, F.S., A.S., M.B.S.‐S., R.R.‐G., R.B., J.C., A.A., A. Lladó, R.‐S.‐V., J.F., and A. Lleó contributed to acquisition of data; I.I.‐G., J.P., V.M., E.V. A.B., F.S., and J.C. contributed to analysis of data and preparing the figures; I.I.‐G., J.P., D.A., J.C., and A. Lleó contributed to drafting the text.Nothing to report.Table S1. Analytical procedures.Table S2. List of genes with the highest confidence score of interaction with APP gene from STRING.Table S3. List of genes with differential expression at cerebral regions where cortical thickness correlated with APP‐derived peptides.Table S4. Gene enrichment analysisClick here for additional data file.


Estimating the number of people with trachomatous trichiasis allows managers to plan surgical services and obtain the resources needed to eliminate this painful condition.Chlamydia trachomatis infection eventually leads to trachomatous trichiasis (TT), in which one or more eyelashes are misdirected so that they rub on the eyeball.,,,-,In some people living in trachoma-endemic areas, repeated conjunctival This paper, intended for programme managers and their supporters, explains the concepts involved in estimating TT prevalence and discusses programmatic applications of that information. We hope it will help these stakeholders to use their TT data with greater confidence.1prevalence of a disease is the percentage of people in a defined population who are affected by that disease at a particular time. In this definition, it is important to clearly identify the defined population. The prevalence of TT can be estimated for a single community,The 2-district has different meanings in different contexts. Because of this, and because the administrative level surveyed may change from one round of surveys to the next, we will subsequently refer here instead to evaluation units (EUs), a generic term for the population unit surveyed, regardless of size – even though we used ‘district-level’ (for readability) in the title of this article.For trachoma elimination purposes, the World Health Organization (WHO) defines districts as “the normal administrative unit for health care management,” which “for purposes of clarification, consists of a population unit between 100,000 and 250,000 persons”.Regardless of the administrative level at which EUs are framed, data should be interpreted and applied at that same level; in other words, local data should inform local action.3To estimate TT prevalence, population-based surveys are recommended.(first-stage clusters) from the set of all communities in the EU. The second stage, undertaken within each selected community, involves selecting a fixed number of households  All qualifying individuals living in selected households are asked to participate  in each community varies. Communities in which a greater number of ≥15-year-olds are examined should not contribute more weight to the EU-level prevalence.To compensate, partially, for the first problem , standard trachoma survey analyses adjust the first-stage cluster data by gender and age in five-year age bands. This can be conceptualised as filling in missing data from individuals who were resident in selected households but not examined, using the assumption that their risk of trichiasis was similar to that of residents of similar age and gender who were examined. To compensate for the second problem , the age- and gender-adjusted first-stage cluster-level TT percentages are averaged to generate the EU-level TT prevalence. This gives equal weight to each of the firststage clusters, as if the same number of ≥15-year-olds had been examined in each one.5Estimates generated through sampling are subject to two types of error: bias and chance. Bias is present if the people included in the sample are systematically different to the EU population as a whole: under- representation of adults with jobs that result in absence on the day of the survey, for example. Gender- and age-adjustment, as described above, attempts to partially correct for this problem, but cannot fully compensate for it. Chance affects prevalence estimates through sampling variation: if a different sample of 2000 ≥15-year-olds living in the EU had been examined, a different prevalence estimate might have been generated. The chance-induced uncertainty of an estimate produced through sampling can be quantified: it is expressed as a confidence interval. A 95% confidence interval suggests that, based on the observed data, if surveys using the same methodology were repeated multiple times in the EU, in 95% of instances the prevalence estimate would fall between the confidence interval's lower and upper bounds. Other factors being equal, larger sample sizes will produce narrower confidence intervals.6In some programmes, TT case-finding is undertaken through house-to-house searches.7,http://tropicaldata.knowledgeowl.com/help/demo-project—expanded-trichiasis-report) requirements for selected consumables and operating theatre time. The expanded trichiasis report uses data on the proportion of survey subjects who had bilateral disease to provide an estimate of the number of eyes, as well as the number of people, with TT.12No: not unless those people were managed within a few weeks of the survey and no further time has passed. As months and years elapse after a survey, new (incident) cases of TT develop; determining that the TT prevalence is below the TT prevalence threshold for elimination of trachoma as a public health problem13Each of these terms has been used in different ways by different stakeholders, such that their usefulness as labels has been completely eroded. We do not recommend that these terms be used.14C. trachomatis transmission,,Yes. Unlike active trachoma – where the dynamics of Vision loss from TT is avoidable. Together, we must do everything we can to consign it to history.The authors alone are responsible for the views expressed in this article and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated


Neutrophil traps, followed by macrophage traps, were the most prominent types in the autopsy series of atherothrombotic plaques, including the adventitia adjacent to thrombosed plaques. In contrast, macrophage traps were more numerous than neutrophil traps in intact plaques (lipid cores) and organised thrombi. Mast cell and eosinophil extracellular traps were also present, but sparse in all instances. In conclusion, not only neutrophils but also macrophages, eosinophils, and mast cells are sources of etosis involved in evolving coronary thrombosis. Neutrophil traps dominate numerically in early thrombosis and macrophage traps in late (organising) thrombosis, implying that together they span all the stages of thrombus progression and maturation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Extracellular traps generated by neutrophils contribute to thrombus progression in coronary atherosclerotic plaques. It is not known whether other inflammatory cell types in coronary atherosclerotic plaque or thrombus also release extracellular traps. We investigated their formation by macrophages, mast cells, and eosinophils in human coronary atherosclerosis, and in relation to the age of thrombus of myocardial infarction patients. Coronary arteries with thrombosed or intact plaques were retrieved from patients who died from myocardial infarction. In addition, thrombectomy specimens from patients with myocardial infarction were classified histologically as fresh, lytic or organised. Neutrophil and macrophage extracellular traps were identified using sequential triple immunostaining of CD68, myeloperoxidase, and citrullinated histone H3. Eosinophil and mast cell extracellular traps were visualised using double immunostaining for eosinophil major basic protein or tryptase, respectively, and citrullinated histone H3. Single‐ and double‐stained immunopositive cells in the plaque, adjacent adventitia, and thrombus were counted. All types of leucocyte‐derived extracellular traps were present in all thrombosed plaques, and in all types of the  Extracellular traps (ETs) are thread‐like structures of decondensed DNA decorated with proteins from cytoplasmic granules Recent research has shown that ETs can also be generated by cells other than neutrophils n = 6) or plaque erosion underlying the thrombus (n = 6).Paraffin blocks containing coronary plaques obtained at autopsy from patients with acute MI were derived from the pathology archives of the Academic Medical Center, Amsterdam. For the purpose of this study, six plaques with intact endothelial surface without thrombus and 12 thrombosed plaques were selected, of which the latter had either a fibrous cap rupture , CD68 for macrophages , tryptase for mast cells , EMBP (eosinophil major basic protein) for eosinophils ; and CitH3 (citrullinated histone‐3) for ETs . NETs and METs were identified using sequential triple staining of anti‐CD68, MPO, and CitH3 , thrombus and adjacent plaque (thrombosed plaques), and adventitia (both plaque types); whereas for coronary thrombi, these were quantified in areas of interest according to the distinct features of each histological thrombus age. The surface areas of the selected regions were measured in mmt‐test or a Mann–Whitney test for comparison between thrombosed and intact plaques, and either Kruskal Wallis or ANOVA for different thrombus ages with a post hoc test when the results were significant .Statistical analysis was conducted using SPSS 24.00 . According to the normal or non‐normal distribution of the data, we performed either a p < 0.05, Figure The occurrence of NETs, METs, MCETs, and EETs was noticed in all 12 coronary segments with thrombosed plaques. They were present in different locations: not only in the plaque but also in adjacent thrombus and in surrounding perivascular fat  and lytic thrombi (136 per mm2), followed by METs in fresh (45 per mm2) and in lytic thrombi (99 per mm2). In organised thrombi, on the other hand, METs exceeded NETs in number  in atherosclerotic plaques and to a much larger extent in the coronary thrombus of MI patients. NETs and METs are the two most prominent contributors to etosis in coronary atherothrombosis, although low numbers of mast cell‐ and eosinophil‐derived traps (MCETs and EETs) can also be encountered in the lesions. When the age of a thrombus was taken into consideration, NETs appeared to dominate in the early (fresh) stages, whereas METs are significantly more numerous in the late (organising) stages of thrombus formation  in glomerular MPO‐containing macrophages. In all those studies, similar antibodies were applied to those we used in the present sudy, to visualise histones as the key elements in the structure of extracellular traps Recent studies have shown that macrophages also are capable of releasing extracellular DNA in response to micro‐organisms and inflammatory mediators In intact plaques, we observed significantly higher numbers of METs compared with the other types of ETs; they were mostly located around and inside the lipid core, which also contained immunostainable remnants of dead macrophages. Macrophage cell death, generally attributed to necrosis and apoptosis, is considered an important contributor to the growth of lipid cores of atherosclerotic plaques. Death of (foamy) macrophages leads to extracellular spill of lipids and, as a consequence, volume expansion of the lipid core  Still, in thrombosed coronary atherosclerotic plaques (be they ruptured or eroded), the numbers of METs were significantly increased compared with intact plaques, not only in the plaque or overlying thrombus but also in the periadventitial tissues surrounding these complicated plaques. As yet, it remains unknown whether METs contribute to the onset of thrombus formation or otherwise aggravate the process of progression of a thrombus, for example by forming a scaffold for fibrin network or stimulating other pro‐thrombotic and pro‐coagulant mediators. The significant number of METs that we found in the perivascular fat tissue around thrombosed plaques follows the pattern that we reported also for NETs previously in vitro and in vivo studies have described the presence of MCETs and EETs mainly in infectious, allergic, and autoimmune diseases. In vitro, MCETs were first recognised on human mast cell‐1 (HMC‐1) infected with several bacteria In vivo, MCETs were identified in psoriatic lesions and in skin explant cultures treated with IL‐23 and/or IL‐1β by using immunofluorescence with tryptase and DAPI Several In thrombosed coronary atherosclerotic plaques (be they ruptured or eroded), we found only a relatively small number of MCETs and EETs, in line with the usually small number of mast cells and eosinophils, respectively, that have been reported in plaques, including the lesions of MI patients Furthermore, investigations into the underlying mechanisms of etosis other than netosis are currently less numerous In conclusion, not only neutrophils but also macrophages, and to a lesser extent also mast cells and eosinophils, generate ETs after the onset of coronary plaque complications. In turn, the formation of ETs spans all stages of coronary thrombosis evolution. Different types of leucocytes and their ETs participate in orchestrating thrombus organisation and maturation towards stability in a time‐dependent manner. More knowledge about the role of ETs during atherothrombotic disease is important, since it may provide new strategies in the treatment of cardiovascular disease.KRP, OJB, and ACW designed the study. KRP and XL prepared and assessed the specimens. KRP and CM performed experiments. KRP, CM, and DP analysed the data. KRP, DP, and OJB contributed to manuscript design. KRP wrote the manuscript, while DP, OJB, RDW, and ACW critically revised the manuscript. OJB and ACW supervised this study. All authors had final approval of the submitted versions.Supplementary figure legendsFigure S1. Triple immunohistochemical staining to visualise NETs and METs simultaneously in a coronary thrombusFigure S2. Control slides for immunohistochemical stainingFigure S3. Extracellular traps in different types of thrombus specimenSupplementary figure legendsClick here for additional data file.Figure S1. Triple immunohistochemical staining to visualise NETs and METs simultaneously in a coronary thrombus. These figures show the same section stained sequentially with anti‐MPO (A), anti‐CD68 (B) and anti‐CitH3 (C) antibodies, with an elution step between staining rounds. (D) false‐colour image showing NETs as the colocalization of MPO+CD68−CitH3+ (in yellow) and METs as the colocalization of CD68+MPO−CitH3+ (in cyan). Scale bar in (A): 5 μmClick here for additional data file.Figure S2. Control slides for immunohistochemical staining. Representative examples of positive and negative control slides for CitH3 immunostaining, performed after an elution step to remove the previous dye and immune‐complexes, in two parallel sections of a coronary thrombus specimen. (A) Section stained with anti‐CitH3 antibody as positive control, showing positive cells in dark red. (B) Section stained with the omission of anti‐CitH3 antibody (substituted with antibody diluent only) as negative control, showing negative staining. Scale bar in (A): 50 μmClick here for additional data file.Figure S3. Extracellular traps in different types of thrombus specimen. Representative examples of neutrophil, macrophage, mast cell, and eosinophil extracellular traps  in all stages of thrombus evolution: fresh, lytic and organized. Boxed areas in H&E stains  show the regions of interest for higher magnification of false‐colour images to show the co‐localization of cell‐specific markers (in red) with CitH3+ (in green). Colocalization appears in yellow in all false‐colour images.  NETs as MPO+CitH3+;  METs as CD68+CitH3+;  MCETs as tryptase+CitH3+; : EETs as EMBP+CitH3+. Scale bar in H&E overview (A): 100 μm and in high power detail (B): 25 μmClick here for additional data file.


Objective: To evaluate safety, tolerability and feasibility of long-term treatment with Granulocyte-colony stimulating factor (G-CSF), a well-known hematopoietic stem cell factor, guided by assessment of mobilized bone marrow derived stem cells and cytokines in the serum of patients with amyotrophic lateral sclerosis (ALS) treated on a named patient basis.Methods: 36 ALS patients were treated with subcutaneous injections of G-CSF on a named patient basis and in an outpatient setting. Drug was dosed by individual application schemes  over a median of 13.7 months (range from 2.7 to 73.8 months). Safety, tolerability, survival and change in ALSFRS-R were observed. Hematopoietic stem cells were monitored by flow cytometry analysis of circulating CD34+ and CD34+CD38− cells, and peripheral cytokines were assessed by electrochemoluminescence throughout the intervention period. Analysis of immunological and hematological markers was conducted.Results: Long term and individually adapted treatment with G-CSF was well tolerated and safe. G-CSF led to a significant mobilization of hematopoietic stem cells into the peripheral blood. Higher mobilization capacity was associated with prolonged survival. Initial levels of serum cytokines, such as MDC, TNF-beta, IL-7, IL-16, and Tie-2 were significantly associated with survival. Continued application of G-CSF led to persistent alterations in serum cytokines and ongoing measurements revealed the multifaceted effects of G-CSF.Conclusions: G-CSF treatment is feasible and safe for ALS patients. It may exert its beneficial effects through neuroprotective and -regenerative activities, mobilization of hematopoietic stem cells and regulation of pro- and anti-inflammatory cytokines as well as angiogenic factors. These cytokines may serve as prognostic markers when measured at the time of diagnosis. Hematopoietic stem cell numbers and cytokine levels are altered by ongoing G-CSF application and may potentially serve as treatment biomarkers for early monitoring of G-CSF treatment efficacy in ALS in future clinical trials. Amyotrophic lateral sclerosis (ALS) is a life threatening neurodegenerative disorder characterized by premature loss of upper and lower motoneurons in the adult brain and spinal cord . The lifIn view of the great heterogeneity of disease etiology, neuronal damage likely results from many different pathologic changes, including neuroinflammation . Neurode+ hematopoietic stem cells prior to bone marrow transplantation. G-CSF enhances immunocompetence and has systemic anti-inflammatory effects  is a 20-kDa glycoprotein and a well characterized growth factor that plays a key role in production, mobilization, and differentiation of hematopoietic stem cells , 9. It i effects . G-CSF i effects . Aside f effects , 13. G-C effects . G-CSF i effects , 14, it  effects , 15. Sys effects , 17, whe effects . Observi effects  and indi effects . Small t effects –25, with effects , and pos effects . In summDue to the paucity of available treatment options we provided individual, off-label G-CSF treatment to ALS patients. G-CSF, considering its multimodal systemic and CNS effects, may be a promising treatment option in view of the etiopathological and clinical heterogeneity of ALS. Biomarkers are measurable indicators of disease and/or intervention and may be useful in monitoring long-term degenerative or reparative processes within the CNS. In view of the above-discussed complexity of ALS, it seems unlikely that a single biomarker can sufficiently reflect treatment effects on disease progression. We therefore used a panel of pro- and anti-inflammatory blood parameters, angiogenic factors, as well as hematopoietic stem cell markers. Monitoring pro-differentiation and -mobilization effects on hematopoietic stem cells may serve as a proxy for G-CSF activity on neural stem cells in individual patients and/or reflect direct and indirect beneficial effects of mobilized hematopoietic stem cells. Observing a panel of peripheral cytokines may reveal system wide immune and inflammatory status relevant for peripheral-CNS crosstalk.G-CSF is known to be a safe stem-cell mobilizing agent. We investigated whether the number of mobilized hematopoietic stem cells is different in G-CSF treated ALS patients of longer versus shorter survival. Secondly, we were interested in whether baseline cytokine levels are associated with survival of G-CSF treated ALS patients. Lastly, we sought to explore hematopoietic stem cells and cytokine level alterations during G-CSF treatment.Treatment with G-CSF was offered to 36 patients seen at the University of Regensburg with definite or probable ALS according to the revised El Escorial criteria . As thisALS patients were treated with subcutaneous injections of recombinant human G-CSF (Filgrastim) on an outpatient basis. Dose and application modes were adapted individually upon initiation and over time Figure . AdaptioPatient safety was analyzed at baseline (initiation of treatment) followed by monthly control visits with clinical examinations, blood counts, cytokines, blood smears and estimation of bone marrow function. We conducted baseline spleen sonography with follow-ups upon dose escalation. Clinical ALS progression was monitored using the established ALSFRS-R . If patiChanges in pro- and anti-inflammatory immune profiles were evaluated at baseline, at 3 months, and then every 6 months throughout treatment by multiplex electrochemoluminescence with the panel assay V-PLEX Human Biomarker 40-Plex Kit . This industry standard panel has been validated in different immune related and non-immune diseases (manufacture's information). In patients receiving G-CSF on five consecutive days, evaluations of cytokine levels in the peripheral blood were conducted twice a month, before (day 0) and after G-CSF application (day 7). In patients receiving G-CSF twice a week or every second day, analyses were conducted on a monthly basis 1 day after application. Peripheral blood serum was collected during regular visits at the hospital and immediately stored at −20°C for cytokine assays. For each assay, 25 μl of serum samples were used and test carried out in duplicates, according to the manufacturer's instructions.+ and CD34+CD38− hematopoietic stem and progenitor cells (HSPC) were analyzed by flow cytometry as earlier described by our group , CD34-APC  and CD38-PE  monoclonal antibodies. Analysis was performed on a Becton Dickinson CALIBUR flow cytometer .We analyzed white blood cells including cell differentiation, platelet and red blood cell counts, and hemoglobin levels with an automatic cell counter . Peripheral blood smears were done on a 3-month basis by light microscopy. Peripheral blood CD34ur group . In shorFindings of immune parameters from three time points, baseline (initiation of treatment), 3 months and 6 months were selected for analysis. As patients did not always visit the outpatient clinic on the exact days of the given time points, the time points had to be defined as time periods. When assessing the ALSFRS-R at baseline, data from day of treatment initiation ±28 days were included. For baseline measures of blood counts, stem cell mobilization parameters and cytokines, only data obtained before the first G-CSF application were selected. The 3-month time point was defined as day 45–134 and the 6-month time point as ranging from day 135 to 224. If patients visited more than once during these time periods, the day closest to the intended time point was selected.n = 7). Patients who were alive and had not yet been observed for over 30 months were not considered for this analysis (n = 3). The same censoring was applied for correlation analysis. We then retrospectively analyzed baseline levels of cytokines, hematopoietic stem cells and blood counts in the long and short survivor groups and further correlated survival with cytokines upon treatment initiation.The immediate effects of G-CSF treatment on peripheral levels of cytokines, hematopoietic stem cells and blood counts were assessed by comparing respective levels 2 days before and 1 day after a 5-day treatment course with G-CSF. We then explored different patterns of immune responses depending on individual survival. Survival time was defined as time elapsed from day of diagnosis to day of death or day of last observation in the case of censoring. For this purpose, G-CSF treated patients were divided into two groups based on their survival being longer or shorter than 30 months from diagnosis, as this was a time point that separated the patients into two equal-sized groups. At the point of database closure, patients who were still alive were censored and included in the “long survival” group if they had been observed for over 30 months (2) and p-value. Comparisons were made with Mann-Whitney test and paired Wilcoxon test. Data were considered significant at p ≤ 0.05. A trend was noted at p ≤ 0.1. Comparisons were corrected for multiple testing by false discovery rate approach  at 10%) and considered a discovery at FDR-adjusted p-value (q) < 0.1. We used an Area Under the Curve (AUC) approach to estimate mobilization of hematopoietic stem cells after G-CSF treatment over time. Stem cell measurements before and after G-CSF dosing were available for patients on the 5-day treatment scheme. For better comparability regarding long and short survival times after diagnosis, we selected patients with ongoing 5-day treatment over the first 4 months. All patient measurements were used in the calculation. If patients had fewer data points, their mean AUC value calculated from all data points was applied (in the case of one patient). The AUC value was calculated with the auc function of the R-package “flux” . The baseline value at day 0 or from any day prior to treatment start was used as threshold for the calculation.R or GraphPad Prism 7 was employed for statistical analysis and graph design. Correlations were analyzed using two-tailed Pearson correlation and presented with correlation coefficient (r), coefficient of determination  were treated with G-CSF in addition to riluzole treatment. We here report on individual treatment on a named patient basis—consequently, treatment schemes were heterogeneous. Dose and application modes were adapted individually upon initiation and over time Table . In summLong-term outpatient treatment with G-CSF was generally well tolerated in ALS patients and compliance was excellent. Minor adverse events were mild to moderate bone pain after G-CSF injection and leukocytosis. One patient experienced an episode with heat sensation, lightheadedness, and 15 min. of dyspnea on 1 day of drug application 39 months into G-CSF treatment. Due to the possibility of drug-related intolerance or mild allergic reaction, G-CSF was discontinued in this patient; antibodies against G-CSF were not detectable. This patient was switched from Filgrastim to Pegfilgrastim, a PEGylated form of recombinant human G-CSF, from his 46th to 53rd month after initiation, and then ended the off-label treatment without further adverse reactions. As expected, mild to moderate splenomegaly evolved during ongoing G-CSF treatment in most patients. Without any further symptoms or complications, the mean spleen width increased from 4.3 to 4.9 cm and length from 10.7 to 12.1 cm during treatment. There were no severe adverse events (SAE), and no signs for pre-malignant transformation in peripheral blood smears.+ and CD34+CD38− hematopoietic stem and progenitor cells (HSPC) from the bone marrow into the peripheral blood. Leukocyte counts increased significantly in all treated patients, from an initial mean of 6.9 × 103/μl to 48.2 × 103/μl  after G-CSF application. A predicted increase in the average percentage of neutrophils  was accompanied by a relative decrease in lymphocytes , monocytes  and eosinophils  as well as a small decrease in red blood cell count , hemoglobin level  and hematocrit  during monitoring .Baseline hematology showed no abnormalities in our patients. G-CSF mobilizes neutrophil leukocytes as well as CD34deceased patients was 24.2 months from diagnosis . For further analysis, patients were divided into two equally sized groups by survival being longer or shorter than 30 months from diagnosis. Patients, who were alive at the time of database closure, were considered for this analysis had they been observed for at least 30 months. The mean (median) survival differed in the two survival groups: 46.59 (39.55) months, SD 16.34 and 17.04 (18.30) months, SD 8.16 . The ALSFRS-R slope over time was significantly flatter in longer surviving patients  and had a longer latency between diagnosis and treatment onset . Their clinical function upon treatment initiation was not significantly different (mean ALSFRS-R 38.6/48 vs. 37.3/48). Further, longer surviving patients were less frequently female (18.8 vs. 47.1%), but had similar occurrence of bulbar onset of disease (18.8 vs. 17.6%)  and 1 day after (day 7) a 5-day treatment course with G-CSF at baseline, 3 months and 6 months. G-CSF led to a sustained increase of CD34+ and CD34+CD38− HSPC at all time points   in Table Survival in months from diagnosis was negatively correlated with baseline serum levels of the cytokine TNF-beta. MCP-1 and INF-gamma were, as a trend, negatively correlated with survival as well. IL-16 baseline levels displayed a positive correlation with survival. MDC, IL-8, IL-17A, and PIGF were, as a trend, positively correlated with survival Table . We thenThe direct effects of G-CSF on cytokine levels were evaluated by comparing cytokine levels 2 days prior to and 1 day after ongoing 5-day G-CSF application in a subgroup of patients allowing this analysis. These immediate effects were determined at three different time points . Due to individual application modes, 5-day G-CSF applications with corresponding blood samples were available for 18 patients at baseline, for 17 patients at 3 months, and for 14 patients at 6 months of ongoing G-CSF treatment.We found G-CSF to have an immediate effect on the level of various cytokines Table . The serLong term and individually adapted off-label treatment with G-CSF in 36 ALS patients was well tolerated and safe. The number of G-CSF-mobilized hematopoietic stem cells, as measured by AUC, was associated with longer survival. Initial levels of serum cytokines, such as MDC, TNF-beta, IL-7, IL-16, and Tie-2 were significantly associated with survival, indicating the potential of prognostic application of these immune markers in G-CSF treated ALS patients. Continued application of G-CSF led to persistent alterations in various serum cytokines and ongoing measurements revealed the multifaceted effects of G-CSF.ALS has been recognized as a multifactorial disease. Neurodegenerative processes trigger neuroinflammation and vice versa. Neuroinflammation with microglial activation, infiltration of peripheral immune cells into the CNS, and alterations in cytokine levels are pathological features in ALS. Cytokines are mediators of the immune communication that may cross the blood-brain barrier (BBB) and provide a mechanism by which the peripheral immune system may directly influence the CNS . In a reALS is a multifactorial disease. Targeting common pathologic features such as neuro-inflammation and -degeneration may thus be beneficial for all ALS patients. Although G-CSF is an established, well-tolerated and safe growth factor for mobilization of hematopoietic stem and precursor cells , there ideceased patients and leave those still alive out, then the mean survival of these 26 patients at 25.5 months from diagnosis indicates no harm by G-CSF in ALS.Application of G-CSF in oncological indications is usually limited to treatment cycles, and the only clinical experience with life-long G-CSF therapy has accumulated with patients suffering from severe congenital neutropenia and cyclic neutropenia , 39. To + and CD34+ CD38−) into the peripheral blood. Interestingly, we found an association between stem cell mobilization and survival. Patients who survived longer than 30 months from diagnosis mobilized more CD34+CD38− hematopoietic stem cells than patients with shorter survival, as measured by Area Under the Curve after G-CSF treatment up to 1 year  levels negatively correlated with survival and were found at higher levels in shorter surviving ALS patients upon treatment initiation. G-CSF application led to reduction in TNF-beta, a pro-inflammatory cytokine and common cell death effector found to be increased in ALS sera . TNF-alpOn the other hand, the pro-inflammatory marker IL-16 was positively correlated with survival and increased after G-CSF application. IL-16 also holds an immunomodulatory role by expansion of regulatory T cells (Treg) , that atThe following 18 cytokines were significantly altered by G-CSF, however, not associated with survival. As known from healthy donors , IL-10 wIn ALS, a short time delay for diagnosis is associated with inferior prognosis as these patients are likely to have a more aggressive disease . AccordiPossible direct effects of G-CSF upon the CNS were not assessed, as we did not obtain post-mortem analysis of deceased patients. Neuroimaging studies conducted on our G-CSF treated patient cohort  did not This retrospective analysis has several limitations. Firstly, we have not conducted a controlled clinical trial and thus, there was no placebo-arm. Rather, the aim of the intervention was to offer individual ALS patients a potentially beneficial off-label treatment with G-CSF. Evaluation of respiratory function was driven by clinical indication and not systematically assessed. Hence, we did not regularly screen for respiratory deficits upon treatment initiation. The same applied to assessment of cognitive function. In addition, we did not systematically analyze for ALS-specific gene mutations. Such factors have predictive value concerning prognosis , the lacAltogether, we found that long term G-CSF treatment is feasible and safe for ALS patients. G-CSF efficiently mobilized hematopoietic stem cell into peripheral blood, and the amount of mobilized stem cells was associated with longer survival. Thus, stem cell mobilization could be a potential biomarker to monitor treatment response to G-CSF. Peripheral cytokines are relevant in the course of disease in ALS. We identified TNF-beta, MDC, IL-16, IL-7, and Tie-2 as cytokines whose baseline levels may predict G-CSF treatment response and survival. Additionally, long term G-CSF treatment led to sustained alterations of multiple cytokines in peripheral blood. Thus, cytokines represent potential biomarkers for survival prediction and for early monitoring of G-CSF treatment in ALS, all of which need further validation in a prospective controlled randomized trial.SJ care for ALS patients, conception of intervention, analysis, interpretation, wrote the manuscript. UB clinical responsibility for intervention. UB, WS-M, and VS care for ALS patients, conception of intervention and analysis, revision of the manuscript. T-HB conception of intervention and analysis, revision of the manuscript. BB contribution to analysis, revision of the manuscript. A-LM care for ALS patients, revision of the manuscript. TK organization of care and disposition of patient material. SP and EW performed cytokine experiments, revision of the manuscript. SI and JG performed and analyzed HSPC experiments, revision of the manuscript. AS and WH revision of the manuscript.UB holds patents for clinical application of G-CSF in ALS, Orphan Drug Status is granted for EU and US by EMA and FDA–all within NeuroVision Pharma GmbH, Murnau, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The detection of antigen-specific cytotoxic T lymphocytes after Wilms’ tumor 1-pulsed DC vaccination was higher in patients treated with rhG-CSF than those who were not, based on immune monitoring using tetramer analysis. Our study is the first to report that DC vaccines for cancer immunotherapy primed with low-dose rhG-CSF are expected to achieve higher acquired immunogenicity.Significant recent advances in cancer immunotherapeutics include the vaccination of cancer patients with tumor antigen-associated peptide-pulsed dendritic cells (DCs). DC vaccines with homogeneous, mature, and functional activities are required to achieve effective acquired immunity; however, the yield of autologous monocyte-derived DCs varies in each patient. Priming with a low dose of recombinant human granulocyte colony-stimulating factor (rhG-CSF) 16–18 h prior to apheresis resulted in 50% more harvested monocytes, with a significant increase in the ratio of CD11c Despite significant advances in cancer therapy, such as surgical techniques, radiotherapy, and systemic therapy including immune checkpoint inhibitors ,3,4,5,6,+ and CD8+ T cells and promotive expression of CD4+CD25+FoxP3+ regulatory T cells [®, Dendreon Corporation, Seattle, WA, USA) as autologous DC-based immunotherapy for patients with metastatic hormone-refractory prostate cancer, providing a new personalized cancer immunotherapy treatment option [Manufacturing technology for antigen-presenting cell (APC)-based immunotherapy is being developed using active dendritic cells (DCs), the most potent APCs of the immune system, for therapeutic vaccination against cancer. APC-based immunotherapy with active DCs has been reported for the induction of effective immunity against cancer antigens . DCs exp T cells ,10, whict option . DC vacct option , pancreat option ,14, lungt option , high-grt option , and pedt option ,18. The t option ,15,19. TLarge-scale preparation of DC vaccines with homogeneous, mature, and functional profiles are a prerequisite for achieving efficacious cancer immunotherapy . HoweverThe factors affecting the induction of tumor antigen-specific cytotoxic T cells (CTLs) using DC vaccination have yet to be fully elucidated. The objective of this study was to address the effectiveness of rhG-CSF administration and how it might affect the manufacture of DC-based vaccines for clinical use in cancer immunotherapy.+ cells were simultaneously analyzed in 20 evaluable patients who received rhG-SCF.Cancer patients were enrolled in the DC vaccination study after obtaining their informed consent. The application requirements and conditions for the DC vaccination study were described previously . We admiThere were 54 patients displaying human leukocyte antigen (HLA)-A*24:02, in whom immune monitoring using tetramer analysis was evaluated after one course of the DC vaccination study. These patients had cancer of the colon/rectum (12), lung (8), biliary tract (8), pancreas (18), and stomach (8). The first 15 patients were consecutively enrolled into the group without rhG-CSF priming, and then the next 43 patients were enrolled in the group that was treated with rhG-CSF 16–96 h before the apheresis session. Thirty-nine out of 43 patients who were primed with rhG-CSF (75 μg filgrastim) 16–18 h prior to apheresis included those with colon/rectum (10), lung (5), biliary tract (5), pancreatic (13), and stomach (6) cancers. In the untreated (control) group of 15 patients, the types of cancer included colon/rectum (2), lung (3), biliary tract (3), pancreatic (5), and stomach (2) cancers.7 in each lot. Immature DCs were generated by culturing adherent cells in AIM V medium  containing 50 ng/mL GM-CSF  and 50 ng/mL IL-4  for 5 days using mononuclear cell-rich fractions isolated via apheresis as previously described [mDCs were generated in compliance with Good Gene, Cellular, and Tissue-based Products Manufacturing Practice (GCTP) according to the Act on the Safety of Regenerative Medicine introduced in Japan on 25 November 2014 . The finescribed . mDCs we+, CD14−, HLA-DR+, HLA-ABC+, CD80+, CD83+, CD86+, CD40+, and CCR7+ using flow cytometry. The criteria for DC vaccine administration were as follows: purity (defined as >90% of CD11c+ CD14− CD86+ HLA-DR+ cells), >80% viability, mDC phenotype, negative for bacterial and fungal infection after 14 days, presence of endotoxin ≤ 0.05 EU/mL, and negative for mycoplasma [The antigenic profiles of the mDCs were determined as CD11ccoplasma .235–243 peptide (CYTWNQMNL) compatible with HLA-A*24:02 by substitution of the second amino acid  with tyrosine (Y) can induce CTLs to be more effective than the wild-type peptide [7 DCs with 1–2 KE of OK-432 intradermally injected at bilateral axillar and inguinal areas per session in accordance with previously described protocols for clinical use as per GCTP [A modified WT1 peptide ,28,29. Fper GCTP ,30.+ cells were analyzed using FITC-labeled anti-human CD34  to assess the effect of priming with rhG-CSF (filgrastim) for 16–18 h.A BD FACSCalibur™ flow cytometer  was used to detect the surface molecules expressed on the obtained DCs. The following antibodies were applied to viable DC populations, excluding lymphocytes, in the cryopreserved samples: fluorescein isothiocyanate (FITC)-labeled anti-human CD14 , CD40 , CD80 , HLA-ABC , CD3 , phycoerythrin (PE)-labeled anti-human CD11c , CD83 , CD86 , CD19 , and HLA-DR . CD34TM II (BD Biosciences). The presence of WT1 antigen-specific CTLs was defined according to the following criteria: (i) >0.02% WT1-positive CD8+ T cells in a population of 50,000–100,000 lymphocytes with no evidence of false-positive cells and (ii) WT1-positive population described as clustered and not diffuse [Next, to prove the hypothesis of an increase in acquired immunity using G-CSF-primed DC vaccines, we used tetramer analysis to assess the ratio and degree of the immune response to DC vaccination both with and without rhG-SCF 16–18 h prior to apheresis. Freshly isolated peripheral blood mononuclear cells were stained with PE-conjugated human immunodeficiency virus/HLA-A*24:02 tetramer as a negative control or with PE-conjugated WT1-modified peptide/HLA-A*24:02 tetramer . Other stains included allophycocyanin-conjugated anti-CD3 monoclonal antibody and FITC-conjugated anti-CD8 monoclonal antibody prior to analysis by flow cytometry on a BD FACSCanto diffuse .t-test. A paired t-test was utilized to assess the normal distribution of quantitative differences between the numbers of monocytes and CD34+ cells in each patient treated with rhG-CSF 24 h prior to apheresis. The acquisition of WT1 antigen-specific CTLs in patients with HLA class-A*24:02 was analyzed in G-CSF-primed and non-primed groups to compare distributions and medians without following normal distribution using the Mann–Whitney U-test. The Chi-square test was used to qualitatively analyze the distribution of G-CSF-primed and non-primed patients according to the types of cancers. A p-value < 0.05 was considered statistically significant. All analyses were performed using IBM SPSS Advanced Statistics 23.0 .Spearman’s rank-order correlation was used to analyze the processes between apheresis of the monocytes and the manufacture of mDCs. Differences in the peripheral blood, apheresed monocyte counts, the number of DCs, and the DC ratio for each factor were determined via an unpaired r = 0.828, p < 0.0001, n = 108). The statistical significance of the mDC product was dependent on the number of apheresed monocytes because the DC yield varied from a few DCs to a large amount, even when the standard operating procedure was applied at a single institute. Therefore, it was considered that some factors other than the number of apheresed monocytes also influenced the qualitative and quantitative yield of the DC product . The mean number of mDCs and the viability of the cells prior to cryopreservation were 20.2 ± 9.6 × 107 and 95.6% ± 4.3%, respectively  than in the 24–96-h G-CSF-treated group  and the non-G-CSF group . The ratio of DCs to apheresed monocytes was significantly higher in the 24-h (20.0% ± 7.5%) than in the 24–96-h G-CSF-treated group  and the non-G-CSF-treated group   b.p = 0.004), while CD14 was expressed to a lower degree in the 24-h G-CSF-treated group (p = 0.040). CD80+ DCs in the 24-h G-CSF-treated group were compared with the 24–96-h G-CSF-treated and non-treated groups  (The intensity of the yielded DCs with CD11c was increased in the 24-h G-CSF-treated group compared with the non-treated group (ctively) c.+ cells were present to a lower degree in 20 available cases treated with a low-dose exposure of G-CSF for 24 h (p = 0.0001), being 0.401 ± 0.273/μL and 0.169 ± 0.056/μL (p < 0.001), respectively, following the administration of rhG-CSF (75 μg filgrastim)  and 24 h after (586 ± 238/μL) the subcutaneous administration of rhG-CSF (75 μg filgrastim) in 40 available cases was statistically significant a. CD34+ grastim) b.p = 0.868). The administered DCs were not significantly different between G-CSF-primed (9.13 ± 2.42 × 107) and non-primed (8.64 ± 2.81 × 107) groups (p = 0.320). The induction ratio of WT1-CTLs (tetramer+/CD8+T cells) by tetramer analysis was 46/54 patients (85.2%), while a significant increase was found in 36/39 (92.3%) patients treated with DC vaccine in vivo primed with G-CSF compared with that of 10/15 (66.7%) patients without priming (p = 0.019). The median percentage of WT1-CTLs after one course of DC vaccination was 0.05% (range: 0.01%–0.33%) in the G-CSF-treated group and 0.06% (range: 0.01%–0.38%) in the non-G-CSF group (p = 0.712). The median growth rate of WT1-CTLs between the 1st and 7th sessions was 0.03% (range: −0.02%–0.31%) and 0.02% (range: −0.03%–0.38%) in the G-CSF-treated and non-G-CSF groups, respectively (p = 0.462). These findings suggest the immune effectiveness of in vivo G-CSF-primed DC vaccines by an increase in the ratio of acquired immunity as detected by the positive levels of WT1-specific CTLs.WT1-specific CTLs were detected using WT1-tetramer analysis . The disIn the era of the field of personalized cancer therapies, feasible, well-tolerated, and promising immunologic and multimodal therapies for various types of cancers are expected. Quality-verified DC vaccines are required to achieve the expected level of effectiveness . The rel+ stem cells were not mobilized by the dose of rhG-CSF and rather decreased in numbers , and Y.H. are inventors of the patent for the manufacturing of a DC vaccine using G-CSF (PCT/JP/2014/053676). H.S. is the inventor of the WT1 patent (PCT/JP02/02794 and PCT/JP04/16336).


Cerebrospinal fluid (CSF) can be collected into ethylenediaminetetraacetic acid (EDTA) or plain tubes. The EDTA content presumably contributes to a better cell preservation. EDTA, however, is reported to cause a false elevation in the total protein concentration and to dilute the CSF sample, thereby affecting the diagnostic interpretation. To the authors’ knowledge, no validated studies support this view. The aim of this study was therefore to determine if the choice of tube (EDTA or plain) influences the results of the standard CSF analysis.Thirty-two paired EDTA stabilised and plain CSF samples were included. There was no statistically significant difference in the semi-quantitative protein concentrations when comparing CSF samples from EDTA and plain plastic tubes (P > 0.99). The total nucleated cell count did not differ significantly between EDTA and plain tube samples (P = 0.85). There were no significant differences in the differential cell counts between the two tubes when evaluating polymorphonuclear cells (P = 0.90), lymphocytes (P = 0.84) and monocytes/macrophages (P = 0.86). Also, there was no significant difference in the preservation of cell morphology when evaluating cytological preparations from EDTA stabilised and plain tube samples (P = 0.45).The collection of CSF into EDTA tubes does not influence the result of the standard CSF analysis. However, a presumed positive effect of EDTA on cell preservation could not be shown in the present study. Cerebrospinal fluid (CSF) analysis is pivotal in the diagnostic work-up of patients with central nervous system (CNS) signs, and CSF should be collected and analysed whenever an infectious or inflammatory disease, neoplasia or degenerative disorder of the brain or spinal cord is suspected , 2. BecaA standard CSF analysis includes a total nucleated cell (TNC) count, a protein concentration measurement, a cytological evaluation and differential cell count . In healCerebrospinal fluid samples may be collected into either ethylenediaminetetraacetic acid (EDTA) or plain tubes. EDTA is primarily used for haematological examinations due to its anticoagulant effect . It has,The purpose of this study was to determine if the choice of tube (EDTA or plain) influences the outcome of the standard CSF analysis in terms of protein concentration, cell count, and cell morphology. We hypothesized that there was no difference in the protein concentration or cell count in CSF samples collected in EDTA versus plain tubes, and that the preservation of cell morphology was better in EDTA stabilised samples.The study was completed at the University Hospital for Companion Animals, University of Copenhagen between February and July 2016. CSF samples were collected prospectively from dogs presenting to the University Hospital’s Neurology Service for diagnostic purposes only. In addition, samples were collected from dogs presenting to the hospital for euthanasia for varying reasons, in order to obtain a larger sample size within the study period.Samples were only included if sufficient volumes for both EDTA and plain tubes were available and if the samples could be processed and analysed within 30 min of collection. Written owner consent was collected in all cases. The study was approved by the Local Administrative and Ethics Committee, Department of Veterinary Clinical Sciences, University of Copenhagen.Cerebrospinal fluid samples were obtained from the cerebellomedullary cistern upon aseptic preparation of the puncture site in all dogs. In dogs presenting for diagnostic purposes, CSF was collected with the dog placed in lateral recumbency under general anaesthesia. Pre-medication was chosen based on the clinical status of the dog, and in all dogs, anaesthesia was maintained on either isoflurane or sevoflurane. In dogs that presented for euthanasia, samples were collected immediately after death had occurred. CSF was collected in both EDTA  and plain plastic tubes , EDTA before plain tubes. Each tube was filled with 0.5–1 mL of CSF, taking the dog’s size into account. All samples were analysed within 30 min of collection. The CSF analysis was undertaken according to the laboratory’s standard protocol and included a macroscopic evaluation (colour and viscosity), a semi-quantitative protein concentration measurement, manual red blood cell (RBC) and TNC count, and a differential cell count.® 10 SG, Siemens, Erlangen, Germany), which is considered a valid screening method for measurement of protein concentration in CSF in dogs [The protein concentration in each CSF sample was determined using urinary dipsticks  for 2 to 4 min before further processing. Manual RBC and TNC counts were performed using a Neubauer improved  or a Bürker-Türk  haemocytometer. The cell counts were evaluated by a technician. The technician was not blinded as to whether the CSF came from EDTA or plain tubes, but was blinded to the purpose of the study. Ten microliter aliquots of undiluted CSF were placed onto each side of the haemocytometer chamber. The cells were allowed to settle for 2 min in a humidified environment, before being placed under the microscope. RBC and TNC counts were determined by counting all cells in the centre square and four corner squares in both chambers. The total number of cells counted, equalled the number of cells/μL. If the cell count deviated more than 10% when comparing the two chambers, the procedure was repeated.®, Beckman Coulter, GA, USA) along with a counterweight. The samples were centrifuged at 1300 rpm (93×g) for 8 min. The slides were manually stained with Hemacolor® . A differential nucleated cell count was performed on 200 cells or all cells, if fewer than 200 cells were present on the slides. Leukocytes were classified as polymorphonuclear (neutrophils and eosinophils), lymphocytes or monocytes/macrophages.Cytocentrifuged preparations were made for differential cell counts. Two-hundred microliter of CSF were transferred with a pipette to a cytocassette, which was placed in the cytocentrifuge  with vast experience with cytological evaluations. This evaluation was blinded, with the assessor not knowing if the preparations were made from CSF from EDTA or plain plastic tubes.Based on results of the D’Agostino-Pearson normality test, data was not considered consistent with a Gaussian distribution. For descriptive statistics of variables pertaining to CSF, median, interquartile range and total range were reported. A Chi square test for trend was used to examine the difference in the semi-quantitatively measured protein concentration between CSF samples in EDTA and plain tubes. When comparing RBC, TNC and differential cell counts between CSF in EDTA and plain tubes, a Wilcoxon matched pair’s test was used. For evaluating the difference between satisfactory and unsatisfactory cell morphology on slides made from EDTA stabilised CSF and CSF from plain tubes, a Fisher’s exact test was used. A P-value of < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 7.Thirty-two paired CSF samples were included in the study, 14 samples from dogs with neurological diseases and 18 samples from dogs being euthanized for various reasons.The group of dogs that presented for a neurological work-up included three Labrador retrievers, two large mixed breed dogs and one of each of the following: Beagle, Border terrier, Cavalier King Charles spaniel, Cocker spaniel, French bulldog, Lhasa apso, a medium mixed breed, Standard poodle and Welsh Springer spaniel. Five dogs were diagnosed with idiopathic epilepsy, three dogs with intervertebral degenerative disc disease, one with idiopathic facial paresis, one with Lafora disease, one with steroid responsive meningitis arteritis, one with degenerative myelopathy, one with suspected structural epilepsy and one with idiopathic myoclonic episodes.The group of dogs that presented for euthanasia included four Labrador retrievers, three Golden retrievers, two German shepherd dogs and one of each of the following: Bichon havanais, Border collie, Boxer, Cavalier King Charles spaniel, Danish spitz, Danish Swedish farm dog, Flat coated retriever, Rottweiler and Shih tzu. Six dogs were euthanized due to behavioural problems, four due to neoplastic disease, four due to degenerative joint disease, two dogs due to old age, one due to idiopathic chylothorax and one dog due to a chronic dermatological problem.There was no statistically significant difference in the semi-quantitative protein concentration measurement when comparing CSF samples from EDTA and plain plastic tubes (P > 0.99).The median manual TNC count was 2/μL  from CSF samples in EDTA and 2/μL  from CSF in plain tubes. There was no statistically significant difference in the median TNC count between CSF in EDTA and plain tubes (P = 0.85).The median manual RBC count was 3/μL  from CSF samples in EDTA and 1/μL  from CSF in plain tubes. There was no statistically significant difference in the median RBC count between CSF in EDTA and plain tubes (P = 0.18). Table There was no statistically significant difference in the proportionate differential cell count from CSF in EDTA and plain tubes when looking at polymorphonucleated cells (P = 0.9), lymphocytes (P = 0.84) and monocytes/macrophages (P = 0.86). The results from the statistical analysis of differential cell counts from CSF in EDTA and plain tubes are presented in Table In 13 of 32 cytological preparations (40.6%) made from EDTA stabilised CSF, the cell morphology was evaluated as satisfactory according to the quality assessment standards defined for the present study. When looking at preparations made from CSF from plain plastic tubes the cell morphology in 16 of 32 preparations (50%) was evaluated as satisfactory. There was no statistically significant difference in the quality of cell morphology between preparations from EDTA and plain plastic tubes (P = 0.45).According to the findings in this study, the collection of CSF into EDTA tubes does not influence the result of the standard CSF analysis in terms of the semi-quantitatively measured protein concentrations, TNC counts and differential cell counts. It should, however, be noted that CSF should be collected into an appropriate transport media or sterile tube if culturing of the fluid is anticipated .It is commonly stated that EDTA will contribute to a false high protein concentration, thus potentially altering the interpretation of the CSF analysis , 11, 12.Another concern commonly expressed when using EDTA tubes for CSF sampling is that the EDTA content will dilute the CSF sample, thereby falsely lowering the TNC count , 11, 12.Two of the CSF samples showed differences in the TNC count that could be clinically relevant. In one sample the TNC count was seven cells/µL in CSF from the EDTA tube and four cells/µL in CSF from the plain tube, this sample was taken from a dog diagnosed with idiopathic epilepsy. The second sample was collected from a dog euthanized due to non-CNS related neoplastic disease. In this sample the TNC count was three cells/µL in CSF from the EDTA tube and seven cells/µL in CSF from the plain tube. These findings are most likely incidental and not dependant on the choice of collection tube. The findings, however, emphasise the importance of using information from signalment, history, physical and neurological examination and imaging studies for proper interpretation of CSF changes for accurate diagnosis in the individual case , 15.The majority of CSF samples (68.8%) included in the study were considered normal based on a TNC count under five cells/µL. This may complicate extrapolation to CSF samples with higher cell counts. We, however, believe that the distribution of sample cell counts reflects the picture we see clinically where many dogs with neurological diseases have normal TNC counts. Another limitation in this study is that the dipstick readings and cell counts were not blinded, even though the technicians performing the tests were blinded to the purpose of the study. More reliable results would be achieved with the dipstick being read by a machine or performing quantitative protein measurements.® stained slide was assigned a morphology score (satisfactory or unsatisfactory) to try and quantify cell preservation, and we hypothesised that slides made from EDTA stabilised CSF samples would be superior with regards to the preservation of cell morphology. Surprisingly, this hypothesis could not be confirmed as we found no significant difference in the quality of cell morphology between the two groups. A possible explanation for this may be that all cytological preparations, both plain and EDTA stabilised, were made within 30 min of CSF collection. It is well known that cells in CSF degenerate quickly, possibly due to the hypotonicity and the low protein content of CSF, and that CSF samples therefore should be processed as soon as possible after collection, as in this study [In our cytological evaluations, each Hemacoloris study –18. Althis study , 10, 19,is study . This suAccordingly, EDTA stabilised CSF may be superior to plain CSF if the sample needs to be stored before an analysis can take place. Several reports recommend altering the CSF sample when it cannot be analysed as soon as possible after collection , 18. TheIn this study a semi-quantitative protein concentration measurement technique was chosen, using urine reagent strips. This has previously been reported to be a valid initial screening method to estimate the CSF protein concentration . The metThe collection of CSF into EDTA tubes did not influence the result of the standard CSF analysis, neither with regards to the semi-quantitatively measured protein concentration nor TNC count. A presumed positive effect of EDTA on cell preservation could however not be shown. The potential role of EDTA as an additive to prevent leukocyte degeneration over time remains elusive.