Fecal microbiota transplantation (FMT) is an alternative method for the treatment of gastrointestinal diseases with a high recovery rate. Disadvantages are ethical concerns, high donor requirements and the low storability of stool samples. The cultivation of an in vitro microbiota in a continuous bioreactor was established as an alternative to FMT to overcome these problems. In this study, the influence of the system parameters and donor stool characteristics was investigated. Each continuous colonic fermentation system was inoculated with feces from three different donors until a stable state was established. The influence of the fermentation conditions on the system’s behavior regarding cell count, metabolic activity, short-chain fatty acid profile and microbiota composition as well as richness and diversity was assessed. Cultivation conditions were found to affect the microbial system: the number of cells and the production of short-chain fatty acids increased. The abundance of Actinobacteria and Firmicutes decreased, Bacteroidetes increased, while Proteobacteria and Verrucomicrobia remained largely unaffected. Diversity in the in vitro system decreased, but richness was unaffected. The cultivation of stool from different donors revealed that the performance of the created in vitro system was similar and comparable, but unique characteristics of the composition of the original stool remained. Due to the high retention time of food and, therefore, the high amount of nutrients, the colon has the highest abundance of microbial cells with up to 10 species ,3. One f species ,6.Clostridium difficile infections [The microorganisms in the human intestinal microbiota belong to the phyla Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia. Firmicutes and Bacteroidetes represent the most abundant phyla, each constituting 40 to 50% of total bacteria. Actinobacteria (around 2.5%), Proteobacteria 0.1–1%) and Verrucomicrobia (~0.1%) are less abundant –1% and V. Varietyfections ,17.Clostridium difficile infections (CDI) is treatment with antibiotics, such as vancomycin or metronidazole [The standard therapy for nidazole , but thinidazole ,20. Thennidazole ,22,23. Nnidazole ,25,26. Fnidazole .2, N2 or forming gas to ensure anaerobic conditions. The temperature is normally set to +37 °C. Nutrients are supplied by a complex growth medium adapted in accordance with Macfarlane and Gibson [This study is part of a concept aiming at the generation of transferrable material in vitro to overcome concerns and challenges related to traditional FMT. The expectation is that by mimicking gut conditions in vitro, the production of controlled and stable artificial colonic microbiota would be possible. In vitro models have already been applied successfully to reveal the mechanistic effects of probiotics, drug absorption and transport ,31,32,33d Gibson . To simud Gibson ,37,39, sd Gibson . The curd Gibson  to 400 md Gibson . For FMTd Gibson ,22. To dIn this study, we combined different parts of existing cultivation models to establish a less complex, easy to use system by choosing a single-stage cultivation bioreactor without cell retention with a higher vessel volume of 850 mL. This should enable the generation of larger amounts of infusion material for FMT by the continuous removal of cell suspension in a flow-through mode according to the feed volume flow at the system’s inlet to keep the volume constant over time. Nevertheless, it is unknown whether an amplification of the vessel volume and a simplification to a single-stage system without cell retention still generates a culturable and stable community with a similar richness and diversity. The approach was as follows: the impact of the cultivation conditions themselves as well as the individual characteristics of the donor stool were observed until the formation of a stable state in composition and number of cells was achieved. The open question was in which way the cultivation system itself, but also the individual donor stool characteristics, affects the microbiota characteristics in the continuously harvested cell suspension. The aim was to demonstrate the technical feasibility of the production of a culturable, stable community with a high diversity and richness of representative gut bacteria. Furthermore, the differences that may occur by the cultivation of three various stool samples were identified. The bioreactor system established in this study may be preferentially used for the cultivation of artificial colonic microbiota instead of direct FMT in later studies.For this study, stools from three different donors of one defecation each were used. The donors were chosen and tested regarding the criteria of Terveer et al. . To mini2O. Stock solution 2 and 3 were each mixed with 50 mL, and stock solution 4 was diluted in 100 mL. Solution 2 was subsequently filtrated with a 0.22 µm filter, whereas solution 1 and 4 were each autoclaved. For the preparation of solution 5, hemin (0.05 g) was dissolved in 1 mL 1 M NaOH and filled up to 100 mL with ultrapure H2O. Solution 6 was produced by mixing 0.1 g Vitamin K1 with 20 mL 95% ethanol. Solution 5 and 6 were stored in the refrigerator until use. Solution 3 was mixed, stirred and centrifuged using an Allegra X15R centrifuge . The supernatant was subsequently filtrated (0.22 µm) and joined with solutions 1, 2, 4, 5 and 6 under sterile, anaerobic conditions. The final medium was stored at +4 °C and used within 72 h.For the preparation of inoculum and the cultivation itself, a medium mimicking the chyme in the human colon was used. It was adapted according to Macfarlane and Gibson  and callw/v) fecal slurry was prepared by mixing 30 g of stool with 300 mL CFF medium and homogenizing it for 15 s . To remove large food residues, the mixture was centrifuged , and 120 mL of the resulting supernatant was used as the inoculum for each experiment of this study.For all experiments, the stool from one defecation of each donor was used. The preparation of inoculum was adapted in accordance with McDonald  and is sBioStat B bioreactor . Cultivation was started by transferring 120 mL of inoculum sterile into the glass vessel containing 730 mL of anaerobic CFF medium. Anaerobic conditions in the bioreactor were maintained by sparging continuously with 8 ccm forming gas. The process conditions were adapted to the human colon by setting the temperature to +37 °C. Furthermore, the pH was adjusted to 6.5 [−1) and harvesting cell broth in the same amount. As a result, the mean retention time was 28.3 h, which is comparable to the human colonic transit time [The set-up of the cultivation system was adapted in accordance with already existing systems with the focus on an easy set-up and handling as well as a sufficient generation of transferable material ,37,39. Td to 6.5  and regusit time . All culc is the sum of colonies of the subsequent dilutions with n1 the number of colonies in the less diluted solution, and n2 is the number of colonies in the more diluted solution.The numbers of anaerobic and facultative aerobic colony forming units (CFUs) were analyzed separately. Therefore, samples were collected and diluted with 0.25 strength Ringer’s solution and either plated on Wilkins–Chalgren Anaerobe agar plates (anaerobic cell count) or Plate Count agar plates (facultative aerobic cell count). For each point of time, up to three dilution factors and at least four plates per dilution were plated. The plates were incubated either aerobically or anaerobically for 48 h at +37 °C, and only plates with 30 to 300 colonies were included in the analysis. The number of CFUs N per mL of sample was calculated according to the equation (Equation (1)). Here, Aminex HPXH-87H ion exclusion column  and a refractive index detector G1362A . Separation was performed with 0.0005 mol L−1 H2SO4, 0.45 mL min−1 flow rate and 20 to 100 µL injection volume. Before injection, the samples were centrifuged , and the supernatant was used after an additional filtration (0.22 µm). The metabolites were identified and quantified using external standards  and the Agilent ChemStation Instrument 1 offline software .The concentration of sugars , metabolic intermediates (succinate and lactate), as well as the main short-chain fatty acids (SCFAs) acetate, propionate, butyrate and isovalerate, were identified and measured by means of high-performance liquid chromatography (HPLC). The HPLC unit  was equipped with an IMNGS pipeline [Rhea [The composition of the microbial community, richness and diversity were characterized in the stool samples and at several points of time during cultivation by sequencing the V3/V4 region of 16S rRNA. High-throughput 16S rRNA gene amplicon sequencing was performed by an external lab  according to the protocol described by Reitmeier et al. . After rpipeline . Before pipeline . Furtherne [Rhea ,51. The ix. The distribution of the values was calculated from the standard deviation s due to the random error. All data, graphs and tables in the following show arithmetic means ± standard deviations. Statistical significance was tested using a one-way ANOVA (p ≤ 0.05) followed by a Tukey post hoc analysis conducted with the OriginPro 2019 software .In this study, all analyses were repeated at least in triplicate. The arithmetic mean In this study, an in vitro system representing the human intestinal system was developed to cultivate the microbiota of three different donors until a stable state was formed. Therefore, each continuous colonic fermentation system was inoculated with feces from one of the three different donors that originated from one defecation each. After 24 h of processing time, the fermentation was switched to continuous mode by adding fresh medium on the one side and removing broth in the same amount. The whole system was run until a stable state according to the criteria described below was established. The influence of the cultivation parameters on the system’s behavior was investigated regarding cell count, metabolic activity and SCFA production. Additionally, microbiota composition, as well as the richness and diversity of the microbial community, was assessed.Further, the cultivation of all three donor samples was compared to reveal whether the obtained system is influenced by the process parameters, the donor characteristics or both.p ≤ 0.05) in anaerobic or aerobic cell count among the three donors  and 10 ± 9 × 105 CFU mL−1 (donor B). The high variation, especially in the stool of donor B, may be due the individual availability of aerobic growth of the microorganisms. The anaerobic cell count was more homogenous and between 2 ± 0.02 × 108 (donor B) and 4 ± 3 × 108 CFU mL−1 (donor A). Furthermore, the concentration of SCFAs in the stool was from 5.12 ± 0.14 (donor B) to 6.80 ± 0.47 mg mL−1 (donor A). The ratio for acetate: propionate: butyrate was comparable as well, with 3:1:1 for donor A, 3:2:1 for donor B and 3:1:2 for donor C. The concentration of propionate (2.00 ± 0.16 mg mL−1) in the stool of donor A was higher than for donors B and C. Higher values for propionate may indicate an overweight person, nonetheless, the BMI of donor A was normal [The cell count of facultative aerobes was between 0.7 ± 0.4 × 10s normal . NeverthBifidobacteria were abundant. Akkermansia represented Verrucomicrobia, whereas Escherichia and Shigella can be found within the phylum of Proteobacteria. Bacteroidetes were represented by Bacteroides, Parabacteroides, Prevotella and Alistipes. The phylum Firmicutes were comprised of several genera of Clostridium as well as other genera, such as Blautia, Dorea, Dialister, Faecalibacterium, Roseburia or Ruminococcus. Not all detected genera were abundant in all stool samples. Among the genera with an abundance >1%, differences were detected regarding Dialister, Eisenbergiella, Prevotella and Oscillibacter. Eisenbergiella and Oscillibacter were not abundant in donor A, but in donors B and C. Donor B instead lacked Dialister. Donor C showed a significantly higher abundance in Prevotella than donors A and B. How these differences affect the formation of a stable system and the fermentation outcome is described below. Since all donors represent a healthy intestinal microbiome that is typical for a Western diet [The different phyla were represented by several genera. Within the phylum of Actinobacteria, only ern diet ,53 and eIn the following, the establishment of the in vitro system is described by the cultivation of stool A. The same experiments were also performed for donors B and C but are not described in detail for the sake of clarity, since their behavior was comparable.6 CFU mL−1 anaerobes and 3 ± 3 × 104 CFU mL−1 facultative aerobes. After addition to the bioreactor and dilution with the medium, the cultivation cell broth contained a starting cell count level of 1 ± 0.6 × 106 CFU mL−1 anaerobes and 3 ± 3 × 103 CFU mL−1 facultative aerobes. The inoculum cell count level was 3 ± 0.7 × 108 CFU mL−1. The cell count then dropped slightly and stabilized subsequently. After 115.3 ± 8.9 h of cultivation, a stable number of 1 ± 0.5 × 108 CFU mL−1 was reached.The number of aerobic cells started to increase immediately after inoculation, and a peak was reached after 23.6 ± 1.3 h with a number of 6 ± 5 × 109 CFU mL−1 after 23.6 ± 1.3 h of processing time. At that point of time, it seemed that anaerobic cells had adapted to the conditions and then established a stable amount of 8 ± 2 × 109 CFU mL−1 after 115.3 ± 8.9 h of processing time. The evolution of cell count and beginning of growth is in accordance with the detected turbidity (data not shown). The internal optical sensor detected a measurable total microbial growth after 5 ± 1.7 h of inoculation. Although the number of anaerobes in the inoculum was higher than that of facultative aerobes, the ratio of both was balanced after the first 9.5 ± 2.2 h. This dominance of facultative aerobes in the beginning may be explained by the fact that traces of oxygen were transferred into the reactor by the inoculation. Compared to the inoculation of gut at birth, this behavior seems similar. In vivo, the microbiota is also dominated by facultative aerobes, such as Bifidobacteria and Lactobacilli, after birth [8 to 1011 anaerobic CFU mL−1 [In comparison, the number of anaerobic CFUs had a lag time of 5.0 ± 1.3 h in the beginning. After 9.5 ± 2.2 h, the cell count increased rapidly and reached 3 ± 2 × 10er birth . Regardi−1 of acetate was contained in the broth after inoculation. Growth was detected after 10 ± 1.8 h of processing time, when the SCFA concentration had increased to 1.02 ± 0.38 mg mL−1. The highest concentration of acetate in the broth was detected after a processing time of 24 to 36 h. Here, a concentration of 4.14 ± 0.59 mg mL−1 at the peak of acetate production occurred. The amount then dropped slowly and stabilized after 76 ± 0.8 h to a stable concentration of 3.50 ± 0.22 mg mL−1.In addition to cell count, the concentration of metabolic intermediates and products was determined throughout the processing time to provide further data on the molecular composition affecting microbial growth and the behavior of individual groups. When starting the cultivation, only trace amounts of SCFAs and intermediates were found in the cell broth originating from the inoculum prepared from stool. In accordance with cell growth, metabolic activity started to grow after 8 to 22 h. The development of concentration of the major SCFAs, acetate, butyrate, propionate and isovalerate, is described in the following. As −1), and growth was detectable later, after 22 h (0.47 ± 0.00 mg mL−1). In contrast, the maximum was reached earlier compared to the concentration of acetate. Here, a concentration of 1.64 ± 0.18 mg mL−1 was established after 28 ± 0.6 h of processing time. The concentration of propionate at the start of cultivation was 0.10 ± 0.02 mg mL−1. It increased continuously until a stable concentration of 2.94 ± 0.13 mg mL−1 was reached after 78.0 ± 1.7 h. For isovalerate, the concentration after inoculation was not detectable (<0.01 mg mL−1), but increased after 24.3 ± 0.3 h and reached a stable concentration of 0.25 ± 0.04 mg mL−1 after 77.3 ± 1.7 h. To evaluate the in vitro system, the concentration of SCFAs was compared to studies from the literature. Schwiertz et al. [In comparison, the concentration of butyrate  after inz et al.  detectedIn the human intestinal microbiota, the ratio of acetate, propionate and butyrate is a marker for human health. Rowland et al.  and MacfBifidobacteria [−1. The concentration was subsequently lowered to below 0.04 mg mL−1 after 77.4 ± 2.7 h. The progress was similar for lactate. First, a constant concentration of 0.12 ± 0.05 mg mL−1 was in the broth for the first 24 h until it dropped below a concentration of 0.02 mg mL−1. Facultative aerobic microorganisms, such as Lactobacilli and Bifidobacteria, are the main producers of succinate and lactate in the microbiome. Consequently, the production of metabolic intermediates is in accordance with the development of aerobic CFUs. The main growth occurs in the first 80 h. Afterwards, a stable system with a constant metabolic activity and lower levels intermediates is established.In addition to the main SCFAs, the metabolic intermediates succinate and lactate were analyzed. These substances are metabolized to acetate or butyrate, e.g., by bacteria . The proTo compare and evaluate the in vitro system, a control value had to be defined. During cultivation, the value for SCFAs changed in the beginning but then plateaued, indicating stable state conditions. In this study, the stability of fermentation performance was defined as the point in time when the values for acetate, butyrate as well as propionate did not vary by more than 1% per h of processing time. For donor A, the stable system was reached after 29.2 ± 1.9 (acetate), 76.2 ± 1.0 (propionate) and 31.5 ± 0.3 h (butyrate). Overall, a stable state was reached after a maximum of 77 h. To verify this, a cultivation was run for 180 h of processing time. Here, as well, no shift in the factors described above was detected after 77 h. For further comparison and evaluation, averaged values were used in the stable system of 77 to 120 h.The development of microbial composition, richness and diversity was determined by sequencing 16S rRNA gene amplicons. Stool A was from a healthy donor with a normal microbial distribution and diversity. In this stool, the major phyla were represented by Bacteroidetes and Firmicutes with a relative abundance of 46.89% and 48.52%, respectively. Actinobacteria (2.02%), Proteobacteria (0.78%) and Verrucomicrobia (1.79%) were present in lower relative abundance numbers. This human microbiome also hosted a richness of 100 and a diversity with a Shannon effective index of 21.42 . When prAlistipes and Bacteroides. They occur with a relative abundance of 6.41 ± 4.97% (Alistipes) and 68.17 ± 5.05% (Bacteroides).This is probably due to the oxygen and shear stress sensitivity of the different phyla. During the preparation of the inoculum, the exposure to oxygen was kept as low as possible but could not be fully avoided. Further to that, by mixing and centrifugation, shear stress occurred that may have had an impact on the abundance of various groups of cells. Consequently, the abundance of Bacteroidetes as anaerobic microorganisms decreased, but could be fully restored during cultivation. Although there was a drop after inoculation, the abundance increased after 24.6 ± 0.6 h to 37.91 ± 1.65% and finally established with a value of 75.99 ± 1.78% in the stable system. To identify the ongoing microbial changes at the genera level, we defined clusters of sequences representing single microbial entities, known as operational taxonomic units (OTUs) that share at least 97% of genetic similarity based on the 16S rRNA V3/V4 region. In the stable system, the most represented genera with >1% relative abundance within the Bacteroidetes phylum are Blautia, Clostridium XIVa, Dialister, Faecalibacterium, Gemmiger, Lachnospiracea, Roseburia and Ruminococcus. During cultivation, the abundance of Blautia, Gemmiger, Lachnospiracea, Roseburia and Ruminococcus lowered <1%. Clostridium XIVa, Dialister and Faecalibacterium were able to establish in higher abundances in the in vitro system. The abundance of Clostridium XIVa, represented with 1.95 ± 0.74% in the inoculum, was stable during cultivation (2.91 ± 0.42% in the stable system). The genera Dialister decreased in its abundance from 3.63 ± 0.15% in the inoculum to 1.06 ± 0.38% in the stable system. Faecalibacterium, which was reported to have a major influence on human health, was preserved [In comparison, the relative abundance of the phylum Firmicutes in the inoculum is way higher (94.82 ± 0.75%). After inoculation, the abundance started to decrease to a value of 49.20 ± 2.12% after 24.6 ± 0.6 h. The final relative abundance in the stable system was 16.92 ± 1.96%. In the inoculum, the main representing genera within the Firmicutes phylum were reserved . This geBifidobacterium, which was present with an abundance of 2.24 ± 0.55% in the inoculum but decreased to a value of 0.05 ± 0.02% in the stable system. The phylum Verrucomicrobia showed a contrary behavior. While only a low abundance of <0.05% was observed in the inoculum, it increased after 48.0 ± 1.3 h to 1.10 ± 0.76% and finally stabilized at a value of 2.22 ± 0.72%. This phylum is represented mainly by the genus Akkermansia. The phylum Proteobacteria showed a varying abundance during cultivation. After inoculation, the abundance increased from 0.12 ± 0.14 to 11.37 ± 7.69% after 8.3 ± 2.6 h. Then, a decrease and afterwards stabilization in the stable system at an abundance of 4.38 ± 2.42% was observed. This peak in abundance can be explained since Proteobacteria are mainly facultative aerobe microorganisms. The trend in abundance is similar to the development of aerobic CFUs. Further, the production of acetate followed a comparable development, from which it can be concluded that most of the Proteobacteria in the system produced acetate. Here, the main producers were the genera Escherichia and Shigella, which were represented in the stable system at 2.34 ± 0.59%. The progress of the different phyla shown in this study is comparable to the formation of the microbiota after birth [Bifidobacterium, in the beginning. Consequently, the formation of acetate was highest during the first 48 h. Afterwards, strict anaerobic, butyrate producing bacteria as representatives from the phylum Bacteroidetes started to grow and establish in the gut as well as in the in vitro system. Overall, in the in vitro cultivation system, the relative abundance of the phyla differed from the abundance in the donor stool. The value of Actinobacteria and Firmicutes was lower since Bacteroidetes, Proteobacteria and Verrucomicrobia have a higher abundance in vitro than in vivo in contrast. The decrease in Firmicutes may have been caused by the higher redox potential exceeding normal physiological levels in the in vitro system, which was also observed by Zihler et al. [The phylum Actinobacteria decreased after inoculation and was established with an abundance of 0.09 ± 0.05% after 77 h. This phylum is represented by the genus r et al. . The char et al. . Consider et al. , who alsr et al. . In thesThe influence of the donor sample was tested by comparing all values in the stable system. For all samples, the stable system was defined as the change over cultivation time in concentrations of acetate, butyrate as well as propionate <1% per h of processing time. For donor A, the stable system was reached no later than 77 h. For donor B, the stable state was reached after 76 h, and for donor C after 74 h. Consequently, the averaged values of each factor of 77–120 h processing time were considered for a comparison.10 (donor A) and 1 ± 0.3 × 1010 CFU mL−1 (donor B) of anaerobic cells. For the concentration of SCFAs, an increase in the total amount from 8.29 ± 0.55 mg mL−1 for donor A to 9.22 ± 0.27 mg mL−1 for donor C was detected. In detail, no significant difference was observed for propionate. In contrast, the concentration of acetate, butyrate and isovalerate varied. For acetate, donor B showed a significantly higher value of 3.82 ± 0.14 mg mL−1. For butyrate, the concentration of 2.39 ± 0.15 mg mL−1 increased for donor C. In the in vitro microbiota of donor A, the concentration of isovalerate decreased significantly to 0.24 ± 0.04 mg mL−1. Under these conditions, the ratio of acetate:propionate:butyrate was 3:3:2 for donor A and 4:3:2 for donors B and C.For cell counts, no significant differences were observed. All donor samples resulted in between 0.9 ± 0.2 × 10p-value of 0.05 among the three donors. Contrary to this, the abundance of Firmicutes and Bacteroidetes was different. Donor C did not show different abundances compared to donors A and B. When comparing donors A and B, A showed a decrease in Firmicutes and an increase in Bacteroidetes.Diversity, represented by the Shannon effective index, was significantly higher in the system of donor B with a value of 24.40 ± 1.75. In terms of richness, all donors differed. Richness increased from 96.00 ± 7.07 (donor B) to 105.17 ± 5.71 (donor A) and 115.00 ± 2.83 (donor C). Bifidobacteria, Blautia, Faecalibacterium, Gemmiger, Lachnospiraceae, Parabacteroides, Roseburia and Ruminococcus showed no significant differences among the three donors. Some differences were due to the lack of genera in the donor stool. Dialister was neither abundant in the stool nor in the stable system of donor B. Donor A lacked Eisenbergiella, Oscillibacter and Prevotella. In contrast, donor C showed a high abundance of Prevotella, while donor B only had a low abundance of this genera. For donor C, Prevotella was also abundant in a high number in the stable system. In the stable system of donor B, the abundance of Akkermansia was reduced, while Clostridium XlVa increased. The abundance of Alistipes was significantly different between donors A and B, with A showing higher values. For Bacteroides as well as for Escherichia and Shigella, donor C showed a reduced abundance compared to donor B.At the genus level, the genera In general, different donor stools led to different in vitro microbiomes regarding several factors. For cell count, propionate, and the phyla of Actinobacteria, Verrucomicrobia and Proteobacteria no differences were observed. The abundance of Firmicutes and Bacteroidetes was indeed divergent among the donors. Therefore, the metabolization of acetate, butyrate and isovalerate was different as well as diversity and richness. Nevertheless, the produced systems were comparable to in vitro growth, as shown by the formation of the stable system. As 8 (facultative aerobe) and 0.9 ± 0.2 × 1010 CFU mL−1 (anaerobe). Regarding the metabolic profile, an increase was also observed. The amount of SCFAs in the broth was significantly higher with a value of 8.29 ± 0.55 mg mL−1 compared to the donor stool (6.80 ± 0.47 mg mL−1). These increases in cell count and metabolites are probably linked since more cells produce more metabolites. Regarding the composition of these cells, the abundance of Proteobacteria and Verrucomicrobia showed no significant difference between stool and cultivated broth. On the other hand, the phyla Actinobacteria, Bacteroidetes and Firmicutes differed significantly based on a p-value of 0.05 from the original sample. As can be seen in When cultivating the microbiota in vitro, a high similarity with the original stool is the aim. For donor A, the cell count in the cultivated stable system differed: it increased significantly to 1 ± 0.6 × 10In this study, differences among three donor stool samples were investigated. Three samples are considered as the scientific standard and commonly accepted in this field of research, as comparable studies used similar sample numbers. In other publications, the value ranges from four  and threWhen comparing the in vitro microbiota with the fecal inoculum , all phyIn the current study, a human in vitro intestinal microbiome of three different donors was developed, established and assessed. From the results obtained, we can conclude that the in vitro cultivation of a human intestinal microbiome is a valid alternative, but results in a slightly different system compared to in vivo. The number of anaerobic as well as facultative aerobic cells increased, as did the production of SCFAs. Regarding the microbial profile, the cultivation led to a decrease in the abundance of Actinobacteria. The abundance of Bacteroidetes increased, while Firmicutes decreased. Proteobacteria and Verrucomicrobia showed no significant changes. Diversity in the in vitro system decreased, but richness was not affected by cultivation. When cultivating stool from three different donors, the behavior of the created in vitro system was similar and comparable, but showed unique characteristics originating from the composition of the stool. The aim, however, was not to produce an exact copy of the in vivo system, but rather to demonstrate that a culturable, stable community with a high diversity and richness of representative gut bacteria can be produced. Further, the concentration and ratio of SCFAs were required to be comparable to the in vivo system to increase the chance of recovery when using the broth as an infusion material. As shown by this study, this can be reached, but the cultivation system and cultivation conditions have a major impact on the resulting microbiota profile. The produced in vitro microbiota can be influenced by choosing system parameters, which provide some degrees of freedom in producing a certain target microbiome, as well as the stool characteristics, with the cultivation parameters having a higher impact. Nevertheless, it should be considered that the donor stools used in this study shared a common social and environmental background. We cannot exclude that this fact might lead to a similar in vitro microbiota system and prevented the detection of differences based on the donor stool. Additionally, the sample volume of three was sufficient to validate the concept, but more samples will be needed to confirm the findings presented and conclusions drawn here. Consequently, further parameters, such as pH or stirring rate, as well as more and especially different stool samples from donors with different backgrounds, need to be cultivated and investigated to improve and enhance the system established here.Comparing stool and the created system in regard to the fecal microbiota transplantation concept, we can conclude that the in vitro microbiome shows a higher similarity with the donor stool than the inoculum that is normally used for therapy. Nevertheless, future studies in vivo need to confirm the results, since it is not clear whether cultivated bacteria have the same impact on patients’ recovery as FMT itself.


Thirty-eight patients with non-α-synuclein-related neurodegenerative and non-neurodegenerative disorders, including Alzheimer’s disease (n = 10), sporadic Creutzfeldt–Jakob disease (n = 10), progressive supranuclear palsy (n = 8), corticobasal syndrome (n = 1), fronto-temporal dementia (n = 3) and other neurological conditions (n = 6) were also included, as controls. All 81 patients underwent olfactory swabbing while CSF was obtained in 48 participants. At the initial blinded screening of olfactory mucosa samples, 38 out of 81 resulted positive while CSF was positive in 19 samples out of 48 analysed. After unblinding of the results, 27 positive olfactory mucosa were assigned to patients with probable dementia with Lewy bodies, five with prodromal dementia with Lewy bodies and three to patients with mixed dementia, as opposed to three out 38 controls. Corresponding results of CSF testing disclosed 10 out 10 positive samples in patients with probable dementia with Lewy bodies and six out of six with mixed dementia, in addition to three out of 32 for controls. The accuracy among results of real-time quaking-induced conversion assays and clinical diagnoses was 86.4% in the case of olfactory mucosa and 93.8% for CSF. For the first time, we showed that α-synuclein real-time quaking-induced conversion assay detects α-synuclein aggregates in olfactory mucosa of patients with dementia with Lewy bodies and with mixed dementia. Additionally, we provided preliminary evidence that the combined testing of olfactory mucosa and CSF raised the concordance with clinical diagnosis potentially to 100%. Our results suggest that nasal swabbing might be considered as a first-line screening procedure in patients with a diagnosis of suspected dementia with Lewy bodies followed by CSF analysis, as a confirmatory test, when the result in the olfactory mucosa is incongruent with the initial clinical diagnosis.In patients with suspected dementia with Lewy bodies, the detection of the disease-associated α-synuclein in easily accessible tissues amenable to be collected using minimally invasive procedures remains a major diagnostic challenge. This approach has the potential to take advantage of modern molecular assays for the diagnosis of α–synucleinopathy and, in turn, to optimize the recruitment and selection of patients in clinical trials, using drugs directed at counteracting α-synuclein aggregation. In this study, we explored the diagnostic accuracy of α-synuclein real-time quaking-induced conversion assay by testing olfactory mucosa and CSF in patients with a clinical diagnosis of probable ( Perra et al. detected α-synuclein aggregates in olfactory mucosa and CSF samples of patients with dementia with Lewy bodies, using real-time quaking-induced conversion assay. Positivity in unpaired olfactory mucosa and CSF samples was significantly concordant with the clinical diagnosis and nearly 100% in participants in whom paired samples were tested.  Despite promising expectations, enzyme-linked immunosorbent assay (ELISA) assays for the detection of disease-associated α-syn in the CSF showed poor specificity and sensitivity.,ante-mortem diagnosis.Dementia with Lewy bodies (DLB) is a progressive neurodegenerative disease characterized by fluctuating cognitive decline, visual hallucinations, parkinsonism, and rapid eye movement sleep behaviour disorder. Although the diagnosis of probable DLB is supported by biomarkers of downstream neurodegeneration, such as dopamine transporter SPECT imaging, I-123 metaiodobenzylguanidine myocardial scintigraphy, and polysomnography, the diagnostic accuracy of these tests for DLB is still limited, at early stages of the disease.,More recently, the real-time quaking-induced conversion (RT-QuIC) assay has been shown to reliably detect misfolded α-syn in CSF and other peripheral tissues of patients with α-synucleinopathies.,In human prion disorders, we provided evidence that prion RT-QuIC analysis of CSF and olfactory mucosa (OM) samples tested separately provided a 100% specificity and a sensitivity of 77–86% and 97–95%, respectively.Alpha-syn RT-QuIC assay on OM samples had been explored in a single study in patients with Parkinson’s disease (PD) or Multi System Atrophy (MSA) versus 18 controls with non-α-syn-related disorders.In this study, we collected OM samples using nasal swabbing from patients with probable, prodromal DLB and DLB/AD mixed dementia (DLB group) and with non-α-syn-related neurodegenerative or non-neurodegenerative disorders (non-α-syn NDs) and tested them by α-syn RT-QuIC. We also analysed CSF samples to further validate in another specimen the OM RT-QuIC results and to determine the diagnostic accuracy in each of the two tissues and in combination.The study was conducted according to the revised Declaration of Helsinki and Good Clinical Practice guidelines. Informed consent was given by study participants or the next of kin.Five neurology units participated in the study: University of Verona (UNIVR), University of Trieste (UNITS), University of Udine (UNIUD), University of Genova (UNIGE) and University of Florence (UNIFI). OM ad CSF sample collections were performed according to protocols approved by the Ethical Committees of each neurology unit. Written informed consent for OM brushing and lumbar puncture was obtained from each patient or from legal representative.n = 32), UNITS (n = 10), UNIUD (n = 10), UNIGE (n = 22), and UNIFI (n = 7) between Jan 2019 and July 2020. Clinical diagnosis was based on the established criteria. OM samples obtained from 43 patients with probable (n = 32) or prodromal DLB (n = 5) and DLB/AD mixed dementia (n = 6) and 38 controls with a clinical diagnosis of non-α-syn NDs, including patients with sporadic Creutzfeldt–Jakob disease (sCJD) (n = 10), AD (n = 10), progressive supranuclear palsy (PSP) (n = 8), corticobasal syndrome (CBS) (n = 1), fronto-temporal dementia (FTD) (n = 3)  and other neurological disorders (n = 6), including, psychosis (n = 2), Down syndrome (n = 1), vascular dementia (n = 1); mild cognitive impairment (n = 1) and Arnold–Chiari malformation type 1 (n = 1). In particular, the diagnosis of DLB follows the 2017 McKeith criteria,Eighty-one patients were enrolled in the study UNIVR , as described previously.https://www.youtube.com/watch?v=wYb9W3u6uMY&t=28s. Each nasal swabbing procedure took around 5 min per patients. Exclusion criteria included severe nasal cavity abnormalities, or infections. Coagulation disorder or anticoagulant/antiplatelet drug intake or other medical conditions were not exclusion criteria. One to four OM samples were collected from each individual, depending on the patient’s tolerance for the procedure. To minimize a potential bias in the pre-analytical step, tubes and nasal swabs for nasal swabbing procedure were provided by UNIVR. Preparation, processing and preservation of OM samples were standardized for all groups.Eighty-one patients were included in this study and underwent nasal swabbing. The procedure was well tolerated by all patients and no adverse events were reported. Nasal swabbing procedure was performed in all participants using flocked swabs  analyses using commercially available ELISA kits . CSF biomarkers were considered positive for AD pathology when the ratio of Aβ42 and p-tau was lower than 6.5 or when the ratio T-tau/Aβ42 was lower than 0.52.,Conversely, CSF samples were obtained by lumbar puncture from 48 patients which included 10 patients with probable DLB, 6 patients with DLB/AD mixed dementia and 32 controls with non-α-syn NDs . CSF samEscherichia coli BL21 (DE3). Cell cultures (1 L) were grown at 37°C to an OD 600 nm of 0.3–0.4 and the expression was induced with 0.1 mM isopropyl b-d-1-thiogalactopyranoside (IPTG) for 5 h. Cells were collected by centrifugation, re-suspended in 100 ml of osmotic shock buffer  and incubated for 10 min at room temperature. The cells were centrifuged at 12 000 rpm, re-suspended in 90 ml of cold water with 37.5 µl of saturated MgCl2 solution and, after 5 min incubation on ice, centrifuged again. The supernatant containing the periplasm proteins was boiled for 15 min and cleared by centrifugation. The soluble fraction, enriched in α-syn was subjected to ammonium sulphate precipitation followed by extensively dialysis against 20 mM Tris–HCl, pH 8.0. Further purification of α-syn was performed by anion exchange chromatography loading the sample on a Q-Sepharose column  equilibrated with the same buffer and eluted with a 0–500 mM linear gradient of NaCl. The purity of α-syn was checked by SDS–PAGE. The protein was then dialysed against 10 mM Sodium Phosphate buffer pH 7.4, and stored at −80°C until use.Recombinant α-syn was expressed and purified from the periplasmic fraction as reported.,We performed α-syn RT-QuIC assay as reported previously for CSF and OM except where indicated.For OM seeded reactions, we used 2 μl of diluted OM in 98 µl of Reaction Buffer composed of 100 mmol/l phosphate buffer (pH 8.2), 10 μmol/l ThT, and 0.05 mg/ml human recombinant full length (1–140 aa) α-syn and 37 ± 3 mg of 0.5-mm glass beads (Sigma). The plate was then sealed with a plate sealer film  and then incubated at 30°C in a BMG FLUOstar Omega plate reader with cycles of 1 min shaking  and 14 min rest. ThT fluorescence measurements  were taken every 45 min.In the case of CSF seeded reactions, 15 µl of undiluted CSF was added 85 µl of reaction mix composed of 100 mmol/l phosphate buffer (pH 8.2), 10 μmol/l ThT, 0.05 mg/ml human recombinant full length (1–140 aa) α-syn, 0.0075% sodium dodecyl sulphate (SDS) and 37 ± 3 mg of 0.5-mm glass beads (Sigma). The reaction plates were incubated at 30°C in a BMG FLUOstar Omega plate reader with cycles of 1 min shaking  and 14 min rest. ThT fluorescence measurements  were taken every 45 min.The criteria for discriminating positive versus negative RT-QuIC tests of CSF and OM are criteria similar to those previously described for prion RT-QuIC analyses.t-test or by Mann–Whitney U-test following ascertainment of normal distribution of data by Shapiro–Wilk normality test.RT-QuIC relative fluorescence responses were analysed and plotted using the software Graphpad Prism 8.3. We compared the mean relative ThT fluorescence and the lag phase responses in OM and CSF samples from DLB patients by either two-tailed unpaired We compared batch-to-batch difference in the mean maximum ThT fluorescence values obtained in OM and CSF from DLB group and non-α-syn NDs patients by Kruskal–Wallis test with Dunn's multiple comparisons after the ascertainment of normal distribution of data by Shapiro–Wilk normality test.P values < 0.05 were considered statistically significant. Agreement between OM, CSF and clinical diagnosis was tested for significant differences by McNemar test, and quantified with Cohen’s kappa (k).Data can be available upon request to the corresponding author.To establish the α-syn RT-QuIC assay for OM and CSF we run an initial set of experiments using a small subset of OM and CSF samples from cases of definite DLB and non-α-syn NDs. Seeding reactions were performed by testing different concentrations of human recombinant α-syn  and we chose 0.05 mg/ml as the optimal concentration for our current protocol. No increase in fluorescence was observed in OM and CSFs from control patients, while fluorescence above threshold occurred within ∼ 25 h for OM and CSF samples, in reactions seeded with DLB samples .In a first round, we performed blinded analyses of 81 OM samples. Thirty-nine OM samples gave a positive RT-QuIC response at 30 h while 42 remained negative until 80 h . AlthougIn a second round, 48 CSF samples were analysed by α-syn RT-QuIC. In 26 samples, the initial seeding reaction was observed at 25 h and the fluorescence gradually increased to 80 h while in 22 CSF samples did not exceed the designated positivity threshold . Seven Cκ = 0.729, 95% CI 0.582–0.876). The proportion of cases labelled as DLB did not differ between clinical and laboratory diagnosis (P = 0.227).Once all the OMs and CSFs were tested, the results were unblinded. We learned that 27 RT-QuIC positive OM samples were obtained from patients with probable DLB, five with prodromal DLB and three with DLB/AD mixed dementia while the remaining three positive samples were from patients with non-α-syn NDs . These iRT-QuIC analyses of CSF showed that the 19 out of 47 positive samples belonged to 10 patients with probable DLB and six with DLB/AD mixed dementia while the remaining three were from three patients with a clinical diagnosis of AD, PSP and FTD, respectively .κ = 0.866, 95% CI 0.721–1.000), that resulted in a similar proportion of identified cases (P = 0.250).Thus, RT-QuIC was positive in 16 out of 16 CSF samples from DLB group of patients and in three out of 31 from patients with non-α-syn NDs. with an accuracy of 93.8% (sensitivity 100% and specificity 90.6%). We observed a high agreement between clinical diagnosis and RT-QuIC CSF testing  or lag-time phase (P = 0.14 t-test) between OM and CSF samples from patients with DLB  albeit agreement was moderate .In 16 patients and 32 controls, both OM and CSF specimens were available, thus the diagnostic accuracy of RT-QuIC was analysed by combining the results obtained in tissues . Five OMAlthough the RT-QuIC sensitivity in CSF was 100%, three CSF specimens from patients with non-α-syn NDs tested positive but their OM samples were negative and included one patient with a clinical diagnosis of AD another with PSP and one with FTD .In conclusion, when the results of RT-QuIC in OM and CSF results were combined the diagnostic accuracy increased to 100%.Clinical diagnosis of DLB is still a challenge and the availability of diagnostic assays that detect pathological α-syn in biological specimens of patients with DLB have relevant clinical and potentially therapeutic implications.To date, several studies have demonstrated the presence of p-α-syn in skin and lip biopsies from patients with a clinical diagnosis of DLB by immunohistochemistry, showing a sensitivity ranging from 100% to 50% and a specificity from 100% to 97%. However, tissue biopsies are invasive, requiring multiple samplings and the accuracy of immunohistochemistry in detecting p-α-syn is variable among studies.,Of course, RT-QuIC assays have revolutionized the diagnostic approach not only in human prion disorders but also in other proteinopathies and in particular in α-synucleinopathies.,Here, we show for the first time that OM samples are α-syn RT-QuIC positive in 35 out of 43 patients with a clinical diagnosis of DLB and in three with non-α-syn NDs with a sensitivity and specificity of 81.4% and 92.1%, respectively. Compared to the previously reported RT-QuIC results in OM of patients with PD and MSA, and iRBD, the RT-QuIC sensitivity in DLB is apparently higher.Notably, in all patients with prodromal DLB, OM RT-QuIC resulted positive providing evidence that the process of α-syn seeding aggregation might be revealed at early disease stage. However, given the relative low number of investigated patients, further studies are needed to confirm whether OM swabbing might be considered a reliable diagnostic assay in this group of patients.Three positive OMs were assigned to patients with non-α-syn NDs but a similar result was also reported in OM from patients with atypical parkinsonism.,We showed that RT-QuIC testing of CSF correctly identifies DLB patients with 100% sensitivity and 90.3% specificity, in line with previous studies.,In both OM and CSF, we found a considerable agreement between clinical diagnoses and RT-QuIC assay. In particular, the sensitivity of CSF was higher compared to OM (100% versus 81.4%) while the specificity was comparable in CSF versus OM (90.3% versus 92.1%). Indeed, the 100% concordant results of RT-QuIC in CSF samples analysis of DLB group argue that positivity in the three controls with AD, PSP and FTD might be related to the presence of coincident α-syn aggregates, as also observed in previous studies.Our strategy of “double tissue α-syn RT-QuIC testing” from the same patient, by testing of OM first and subsequently CSF, as a confirmatory analysis showed 100% concordance with the clinical diagnosis when both specimens are considered. However, we could perform this kind of combined analysis in approximately half of patients with DLB. In particular, a confirmatory CSF analysis could not be verified in three DLB patients with RT-QuIC negative OM samples because CSF samples were not available.intra-vitam diagnosis in patients with suspected DLB or DLB/AD mixed dementia, with the limitation that these analyses do not dissect Parkinson’s dementia from DLB diagnoses.Of course, lack of both OM and CSF specimens in all patients represents a limitation of the present study and additional investigations on a larger cohort of patients are needed. However, these preliminary data indicate that testing both OM and CSF would be a promising approach to improve the refinement of In conclusion, we demonstrated that a gentle procedure such as OM swabbing detects α-syn aggregates in OMs with a considerable diagnostic robustness. Conversely, CSF might be considered as an ancillary test to be analysed in the absence of a concordance between clinical diagnosis and RT-QuIC results. Therefore, we envision OM RT-QuIC testing as a technique that could be used to select patients for clinical trials aimed at developing drugs that target α-syn aggregation.Brain Communications online.This work was supported in part by Fondazione Cariverona: “Development and validation of a novel molecular assay for alpha-synuclein in patients with Parkinson’s disease and other alpha-synucleinopathies” to G.Z. and Brain Research Foundation Verona. This study was supported in part by the intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIAID/NIH).C.D.O. has a patent Provisional (US): 62/567,079 pending, a patent PCT: PCT/US2018/052968 pending, a patent Canada: 3074914 pending, a patent Europe: 18786583.7 pending, and a patent U.S.: 16/652,804 pending. All other authors report no competing interests.fcab045_Supplementary_DataClick here for additional data file.


Limited mobilization was allowed—patients were allowed to adjust their inclined beds, sit in chairs and walk under the supervision of a nurse or physical therapist. The primary outcome was the number of prematurely terminated drainage sessions. Results: Twenty-three patients were included. Drainage was terminated prematurely in eight (35%) patients. In three (13%) of these patients, drainage was terminated due to signs of hydrocephalus. Pressure-controlled drainage in patients with external lumbar drains (ELD) showed inaccurate pressure curves, which was solved by using volume-controlled drainage in ELD patients. Conclusion: The implementation of an automated CSF drainage system (LiquoGuard®7) for CSF drainage allows for early mobilization in a subset of patients with external CSF drains. External lumbar drains require volume-based drainage rather than differential pressure-dependent drainage.Background: Automated cerebrospinal fluid (CSF) drainage systems allow for the mobilization of patients with an external CSF drain. The aim of this study is to describe the implementation of an automated CSF drainage system in neurosurgical patients with external CSF drains. Methods: A feasibility study was performed using an automated CSF drainage system (LiquoGuard Temporary treatment of hydrocephalus using external lumbar or ventricular drainage is common in neurosurgical practice. Conventional hydrostatic pressure-controlled drainage with dripping chambers are the gold standard . HoweverIt has been shown that early mobilization decreases the complications associated with immobilization, results in a shorter length of hospital stay and seems to be safe for patients with an external cerebrospinal fluid (CSF) drain ,4,5,6,7.®7 , is an automated cerebrospinal fluid (CSF) drainage system that has been available since 2011. It acts as a self-adjusting, pressure-based resistor that allows CSF to drain in a controlled manner, and is thus intended to prevent over- or underdrainage [The LiquoGuarddrainage ,9,10. Ho®7 in mobilized hydrocephalus patients with external CSF drains.The aim of this study is to describe the implementation of the LiquoGuard® BACTISEAL® EVD Catheter, Johnson & Johnson, Raynham, MA, USA) or an external lumbar drain  were included.This feasibility study was performed in accordance with the ethical standards of the institutional board review (NL62883.091.17). From December 2017 until June 2020, adult neurosurgical patients (>18 years) treated with an external ventricular drain  were used. The device is an automated pressure-based CSF drainage system connected to the EVD or ELD catheter. It measures ICP by using a pressure sensor that is fixed by an electrocardiogram sticker on the skin over the temporal bone at the height of the foramen of Monro  and a nurse practitioner were available to answer remaining questions during the implementation and to provide assistance to daily caregivers. User manuals, in the form of flowcharts, were also attached to each system.®7 was installed if written and verbal informed consent was obtained from patients, or their legally authorised representatives. Patients were included in the study on a first-come, first-served basis, due to restricted availability of the system.The placement of the EVDs and ELDs was performed according to the institutional standard insertion protocol. At the intensive care unit (ICU), patients were treated with a conventional drainage system . After transfer to the neurosurgical ward, the LiquoGuard®7—pressure-regulated drainage and volume-regulated drainage. In this study, pressure-regulated drainage was used.Two drainage modes are available for the LiquoGuard®7 was set to 10 cmH2O, with an alarm range between −10 cmH2O and 20 cmH2O. In volume-regulated drainage, the system drains a pre-set volume of CSF per hour, regardless of intracranial pressure. According to the instructions for use, the diameter of the lumen of the EVD and ELD catheters used needed to be more than 0.7 mm in order to measure CSF pressure correctly.In pressure-regulated drainage, the system drains CSF as soon as intracranial pressure exceeds the pre-set pressure value. The device produces an audible alarm when pre-set alarm limits are exceeded. Our conventional system is calibrated at 10 cm above the foramen of Monro per institutional protocol. Accordingly, the used pre-set pressure value for the LiquoGuardMobilization was encouraged as much as was clinically possible. Patients received physical therapy once a day and were seated for at least thirty minutes, three times a day. Furthermore, patients were allowed to adjust their inclined bed and walk under the supervision of a nurse.®7. Reasons for termination were described. Complications were included as secondary outcome measures and were registered if they occurred between the placement of the first drain and 30 days after removal of the last drain.The primary outcome was the number of prematurely terminated drainage sessions with the LiquoGuardComplications were defined according to our earlier study regarding complications of external CSF drainage in aneurysmal subarachnoid haemorrhage (aSAH) , and werPatient-specific data were retrieved from the digital patient information system (Epic Systems Corporation (2014), Madison, WI, USA). Information regarding demographics, drainage period, drain type, length of hospital stay, destination after discharge and complications were prospectively registered.All data were analysed using IBM SPSS Statistics for Windows . Continuous data were presented as median and range. Categorical data were presented as counts and percentages.®7 (one patient received an ELD after the EVD was removed). The median time from drain placement until mobilization (connection to the LiquoGuard®7) was three (0–20) days, with a median mobile period of six days (Twenty-three patients were included . Nineteesix days .Twenty-eight complications occurred in 14 patients (61%). A total of nine direct complications and 19 indirect complications occurred in these patients, with a median of one (0–4) complication per patient , Table 2®7. LiquoGuard®7′s tubing set occluded twice. In these cases, the LiquoGuard®7 was removed instead of being replaced.One drain dislodgement occurred while connected to the LiquoGuard®7 was disconnected prematurely in eight patients (35%) (The LiquoGuardts (35%) . The firAlthough the lumen of the ELD tubing set used was more than 0.7 mm, it was observed that the pressure curves were not accurate in the first patient with an ELD. No pulsation was measured, and the pressures shown were constantly negative, even in supine position. As a result, the device did not drain the desired amount of CSF, which lead to underdrainage. After contacting a technical expert from the manufacturer, the drainage mode in patients treated with an ELD was set to volume-regulated with a pre-set volume of 10 mL/h, which solved the problem.®7  , future ®7) for CSF drainage allows early mobilization in a subset of patients with an external CSF drain. External lumbar drains require a volume-based drainage rather than a differential pressure-dependent drainage.The implementation of an automated CSF drainage system (LiquoGuard


Diabetes mellitus (DM) has detrimental effects on the function of microvascular beds, resulting in blood–brain barrier (BBB) dysfunction. The objective of the study was to investigate whether DM affects the brain physiology through composition of cerebrospinal fluid (CSF) and compare gas tension and electrolyte levels in CSF between the diabetic and nondiabetic populations.Patients aged between 20 and 70 years scheduled for elective orthopedic or urologic surgery requiring spinal anesthesia were enrolled. They were assigned to either of the two groups (control or type 2 DM). Gas tension and electrolytes in the CSF and whole blood samples were measured in both groups.+ and Mg2+ in the blood were significantly lower in the DM group than those in the control. The levels of pCO2 and All 49 enrolled patients (24 in the control and 25 in the DM group) completed the study. The concentrations of NaThe results show that there were some homeostatic changes in blood and CSF in patients with DM. It is a major component of the extracellular fluid in the central nervous system (CNS). Due to its special location and interaction with the brain parenchyma, CSF has many important roles in brain function. An analysis of CSF composition may assist clinicians identify patients with CNS disorders ,2.Diabetes mellitus (DM) is a major public health issue worldwide. It is a metabolic disorder that results from defects in insulin production  and insulin resistance . DM has detrimental effects on the function of vascular beds, resulting in various peripheral and CNS complications. Recent evidence from clinical and experimental studies suggested that prolonged hyperglycemic status induced tissue damage and elicited a progressive injury of the brain through several different pathogenetic mechanisms ,4. It haCSF is mainly produced by the choroid plexus via active transport instead of passive ultrafiltration . It regu22.1Forty-nine patients with American Society of Anesthesiologists physical status I or II scheduled for elective urologic or orthopedic surgery and receiving spinal anesthesia were enrolled in this controlled study. Of the 49 patients, while 25 had a history of type 2 DM and were taking oral hypoglycemic agents, the other 24 (control group) had no history of type 2 DM. All patients were between 20 and 70 years. Patients with contraindications for spinal anesthesia, severe cardiovascular dysfunction, renal dysfunction, type 1 DM, any history of neuropathy or neurodegenerative diseases, CNS infection, sepsis, or known allergies to any medications used in this study were excluded.Informed consent: Informed consent has been obtained from all individuals included in this study.Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies, and in accordance with the tenets of the Helsinki Declaration and has been approved by the Institutional Review Board of Chang Gung Memorial Hospital, Taoyuan, Taiwan (IRB: 1602240003).2.2® Critical Care Xpress Analyzer .According to DM status, the patients were assigned to the two groups (control or type 2 DM). All fasted for at least 8 h before sampling blood and CSF. In the operating room, standard monitoring, including noninvasive arterial blood pressure, electrocardiography, and pulse oximetry, was applied. The patient was positioned in the lateral decubitus position. Spinal anesthesia was performed at the L3–4 or L4–5 interspace with a 25-gauge Quincke-type needle through the midline. After discarding an initial volume of approximately 1 mL, another 1 mL of CSF was sampled, following which 15 mg of 0.5% isobaric bupivacaine was administered by an experienced anesthesiologist. After the regional anesthesia was complete, the patient was restored in the supine position. A whole blood sample (2.5 mL) was also collected through arterial puncture from the femoral or dorsalis pedis artery before the operation. CSF and whole blood samples were collected using commercial blood gas syringes with dry heparin preparation. Next, blood gas and electrolyte analyses of CSF and whole blood samples were performed using a StatProfile2.3t-test, the Mann–Whitney U-test was employed for nonparametric data. Multivariate logistic regression was applied with adjustment for confounding factors, including age, sex, weight, and height. Differences were considered statistically significant at p < 0.05.All data analyses were performed using SPSS version 17.0 . Data were assessed for normality using the Kolmogorov-Smirnov test. Categorical data were reported as numbers and percentages and analyzed using the chi-square test with the Yates correction. Continuous data were expressed as the mean ± standard deviation. While the parametric data were analyzed using an independent 3All 49 enrolled patients (24 in the control and 25 in the DM group) scheduled for elective urologic or orthopedic surgery and receiving spinal anesthesia completed the study. CSF and whole blood samples were collected from all the patients. The percentiles for the age categories are shown in 3.1p = 0.006). As compared to the control group, there were more males in the DM group (p = 0.001). In the DM group, weight and height of patients were higher as compared with those in the control group . There was no significant difference in the preoperative blood pressure, heart rate, and oxyhemoglobin saturation between the two groups.The characteristics of patients included in the two groups are depicted in 3.2+ and Mg2+ in blood was significantly lower in the DM group . The glucose level was markedly increased in the DM group compared with that in the control group . Additionally, no significant differences in the levels of K+, Cl−, Ca2+, and lactate were observed between the two groups.The results of the gas tension analysis, electrolyte variables, and blood glucose levels in the two groups are presented in 3.32 and p = 0.012; 21.24 ± 2.37 vs 23.48 ± 2.32 mEq/L, p = 0.023, respectively). In both groups, the higher pH of the CSF is attributed to its lower pCO2, reflecting a decreased bicarbonate buffering capacity. As regard the electrolyte values of CSF, there was no significant difference in the levels of Na+, K+, Cl−, Ca2+, Mg2+, and lactate between the two groups. As expected, the glucose level was significantly higher in the DM group than that in the control group .The values of gas tension analysis, electrolyte variables, and glucose in the lumbar CSF from the two groups are shown in 3.4In the DM group, nine of 25 patients were between 31 and 50 years, and the age of the remaining 16 ranged from 51 to 70 years . We compared the gas tension and biochemical variables in the blood and lumbar CSF in the two age subgroups . It was 3.5+ CSF/blood ratio and blood glucose is depicted in r = 0.299 and 0.113, respectively; p < 0.05).A linear correlation between the Na4+ and Mg2+ was lower in the blood of the DM group than that in the control group. In addition, there was a marked increase in the glucose level in both the blood and CSF in the DM group.In this study, CSF samples for profile analysis of patients requiring spinal anesthesia were collected. We analyzed the gas tension and electrolyte parameters of blood and CSF in DM and non-DM adult patients, and some differences between the two groups were seen. It was seen that the concentration of Nap = 0.006); approximately 60% of the patients were above 51 years, which is in line with the age distribution pattern in a previous meta-analysis study. Moreover, there were more males than females in the DM group. This may be because more patients scheduled for the urologic surgery and enrolled in the study were males. Therefore, for comparison, we adjusted the data for age and sex. Furthermore, in another previous study, the values of serum Na+ and K+ revealed a significant positive correlation with the age of patients [Diabetes is one of the largest epidemics worldwide. Globally, the number of people with type 2 DM has doubled in the past 20 years. With respect to age, a recent meta-analysis study found that the prevalence rate of type 2 diabetes was seven times higher in the 55–74 age group than that in the 20–34 group for Chinese adults, and similar prevalence rates were observed in both women and men . In our patients . However2, but not to hydrogen or 2 levels. The central canal of the spinal cord also has lining neurons that are sensitive to both acidic and alkaline pH, which intrinsically regulates the pH of the CSF [p = 0.415) in the CSF pH was observed between the two groups, although both were slightly alkaline. The higher pH in CSF contributes to its lower pCO2 and compensates for the decreased buffering capacity of the bicarbonate.Maintaining the pH of the CSF in a narrow range near 7.4 is important for homeostasis because the neurons in the CNS are highly susceptible to small changes in acid-base balance . Many co the CSF . In our + were very close in the blood and CSF in the control group, but not in the DM group. Likewise, the concentrations of K+ and Ca2+ were higher in the blood, whereas the concentrations of Cl− and Mg2+ were lower than those in the CSF.CSF is mainly produced by the choroid plexus. The respective distributions of main ions between blood and CSF are similar as seen in our study. We also observed that the respective concentrations of Na+/K+ ATPase, Na+/K+/2Cl− cotransporter, and channels for the selective secretion of K+ and +, Cl−, and +/K+ ATPase plays a major role in pumping sodium ions into the CSF and is important for the production of CSF. It also creates electrochemical gradients to drive Cl−, and + in the blood was significantly lower in the DM group (p = 0.005). Patients with DM usually have abnormal serum sodium concentrations. This dilutional hyponatremia is caused by an increased serum osmolality due to a high glucose level, which results in shifting of water from cells to extracellular fluid [+/K+ ATPase plays a vital role in maintaining the homeostasis of Na+ ions in the CSF. In an animal model of induced DM, the expression of Na+/K+ ATPase in the brain was shown to be associated with age and sex. In older animals, there seemed to be a significant loss of enzyme activity from diabetic insults. In addition, the activity of Na+/K+ ATPase was significantly lower in the brain of male diabetic animals than in the female group [+ in the CSF between the two groups was not observed. Further studies are needed to investigate the activities of Na+/K+ ATPase in patients with DM compared to the control. We also found that patients with higher blood glucose levels had higher Na+ CSF/blood ratios  in the blood Mg2+ concentration between the two groups.Increasing evidence suggests that Call death ,24. Ca2+activity . A higheactivity . In our an brain . Anotherrtension . The regGlucose is the primary metabolic energy source for the mammalian brain. In the CNS, glucose crosses the BBB into the extracellular space of the brain through the glucose transport (GLUT) family, mainly GLUT-1 and GLUT-3. GLUT1 plays an important role in brain glucose uptake and is largely distributed in the BBB and astrocytes . Previou5Diabetes-induced alterations in blood glucose levels may cause physiological changes. Our results showed that there are some homeostatic disorders of ions, acid-base, and sugar in the blood and CSF of patients with DM. A detailed analysis of BBB function, expression, and activity of glycated proteins that could induce endothelial damage or other solutes such as amino acids, inflammatory mediators, or metabolic products in CSF in DM patients is further indicated.


N-glycan sample preparation approaches to develop a more sensitive method. These methods, one with an increased amount of buffer solution during the N-glycan release step with a lower amount of sample volume and the other with Filter-Aided N-Glycan Separation (FANGS), were compared with recent work to demonstrate their effectiveness. It was demonstrated that an increased amount of buffer solution showed higher intensity in comparison to the previously published method and FANGS. This suggested that digestion efficiency during the N-glycan release step was not in an optimal condition from the previously published method, and that there is a substantial loss of sample with FANGS when preparing N-glycans from CSF.Cerebrospinal fluid (CSF) contains valuable biological and neurological information. However, its glycomics analysis is hampered due to the low amount of protein in the biofluid, as has been demonstrated by other glycomics studies using a substantial amount of CSF. In this work, we investigated different  In the last years, omics science have become the major field of study in cellular and molecular systems providing a better understanding of human disease . Each tyN-acetylglucosamine (GlcNAc) and core fucose glycans, which are structures known as “brain type N-glycans” [Among the major changes observed in CSF when a disease develops, alterations in the protein glycosylation process generate differences in the glycome that in many cases can be directly related with a disease stage . A number of neurodegenerative studies based on CSF glycomics have described significant changes in abundances for the bisecting glycans” ,26,27,28glycans” ,22,29. Aglycans” ,31. In tglycans”  and highglycans” .N-Glycan Separation (FANGS) [N-glycome.In this work, we present a sensitive CSF glycan analytical strategy that used as little as 15 μL of starting material; as far as we know, there is no other analytical application using such a small sample volume with reproducible results. The sample preparation was complemented with a SPE-C18 (solid phase extraction) glycan purification to avoid undesirable matrix components . This wa (FANGS) ,36. We aThe nomenclature of glycan structures is described in N-glycome profiling was investigated using the previously published method by Cho et al. [N-glycans were prepared from 50 μL of CSF, but the injection amount was equivalent to 10 μL of CSF. In this study, the injection amount was halved to assess the possibility of performing N-glycan profiling of CSF with less material. Initially, the attempt to reduce the injection amount of CSF required to perform o et al.  as a benN-glycans derived from CSF from both experiments were conducted as shown in Predictably, reducing the injection amount to 5 µL resulted in a decrease of the signal by nearly half. A relative quantitation comparison of N-glycome profiling with an injection amount of 5 µL CSF was comparable to a 10 µL CSF injection. Since N-glycome profiling with an injection amount of 5 µL CSF can be accomplished as effectively as an injection amount of 10 µL CSF, two different sample preparation methods were investigated using the previously published method by Cho et al. as the benchmark, where a total of 50 µL of CSF was used to prepare the sample but only 10 µL of 50 µL CSF was injected. Even though the injection amount was decreased by half, the linear correlation of relative quantitation between the two different injection amounts suggested that CSF N-glycans released from CSF by each method described in N-Glycomics (FANGS) platform, which was adopted from Hecht et al. [N-glycan release by PNGase F and using 10k MWCO filters to pre-concentrate the proteins prior to the N-glycan release to facilitate the removal of the matrix. It should also be noted that although the use of C18 cartridges with graphitized carbon SPE is routinely performed in glycan analysis, graphitized carbon SPE steps were omitted in this workflow, since all three methods tested in this work have utilized online purification steps where trap column was used to desalt permethylated glycans prior to separation, which have been shown to enhance sensitivity compared to other purification techniques such as liquid–liquid extraction and offline C18 SPE [Unlike other biofluids such as serum or plasma, CSF is a unique biofluid due to its direct contact with the central nervous system. What is more particular about CSF in terms of proteome or glycome analyses is the fact that CSF has significantly less protein content than serum or plasma. As a result of this, previous glycomics studies of CSF have used substantial amounts of the material to overcome the lack of signal generated by the deficient amount of analyte. This brings a unique challenge especially in glycan analysis, because a lower amount of protein equates to a smaller number of glycoproteins and thus a lack of analyte: the glycans. The previous work by Cho et al.  used 50 t et al. . The lat C18 SPE . To evaluate these techniques, we first compared a 10 µL injection from a 50 µL sample preparation and a 5 µL injection from the same sample. This was to examine whether the instrument could provide an adequate signal of glycans even though the injection amount had been halved. As shown in N-glycomics study of CSF, two different sample preparation methods were tested against the benchmark method with a 5 µL injection, as shown in N-glycans. Test Method 2 involved FANGS, which incorporates 10,000 or 30,000 MWCO (Molecular Weight Cut-Off) regenerated cellulose filter units to pre-concentrate the proteins and remove the matrix prior to the enzymatic digestion. This methodology was chosen first in order to not only remove the unwanted biological matrix but also to remove salts that could not be removed with the C18 glycan purification step; second, since the matrix had been removed, further purification steps using C18 cartridges or porous graphitized carbon sorbents could be avoided, reducing the potential sample loss. Since the 5 µL injection from the benchmark method provided a sufficient signal for an Interestingly, Test Method 1, an in-solution digestion with a smaller enzyme to substrate ratio, showed high sensitivity versus both the benchmark and Test Method 2, as shown in A more startling result was the fact that Test Method 2, where FANGS was utilized, not only demonstrated a lower amount of total glycan abundance but also showed a biased ratio of glycan abundances against the benchmark method, as shown in N-glycans of CSF. The method was compared against the benchmark method, which was previously published and against FANGs, an in-filter digestion method. We report that profiling of N-glycans in CSF using only 15 µL of CSF and injecting glycans derived from 5 µL of CSF showed higher sensitivity compared to the benchmark method as well as the FANGs technique, despite the fact that the protein concentration of CSF is considerably lower than those of other biofluids such as serum or plasma. Here, in this work, we demonstrated an improved method for profiling ® C18 (EC) cartridges were purchased from Biotage  and 10k Amicon Ultra-0.5 mL Centrifugal Filters were purchased from Millipore Sigma . Microspin columns were purchased from Harvard Apparatus . N-glycosidase F enzyme (PNGase F) was acquired from New England Biolabs . Solvents, including high-performance liquid chromatography (HPLC)-grade water, acetonitrile, methanol, and dimethyl sulfoxide were purchased from Fisher Scientific . Pooled CSF was acquired from Golden West Biologicals, Inc. .Iodomethane, sodium hydroxide beads, acetic acid, and ammonium borane complex were purchased from Sigma Aldrich . IsoluteCSF samples of 15 and 50 μL were diluted to a total volume of 100 μL with 50 mM ammonium bicarbonate buffer (ABC buffer pH ≈ 7.5) and denatured in boiling water for 15 min. After the samples had cooled to room temperature, 1 μL of PNGase F (1000 U) was added, and the samples were incubated at 37 °C for 20 h. After incubation, the samples were dried using a Labconco CentriVap benchtop vacuum concentrator .Dried samples were resuspended with 300 μL of 5% acetic acid. The SPE-C18 cartridges were washed with 3 mL of methanol and then equilibrated with 3 mL of 5% acetic acid. Resuspended samples were applied to the SPE-C18 cartridges and washed with 300 μL of 5% acetic acid three times while all flow-through was collected and dried using the vacuum concentrator. The FANGS method was adopted from Hecht et al. with minor modifications. CSF samples of 15 μL were diluted to a total volume of 100 μL with ABC (Ammonium Bicarbonate) buffer and denatured in boiling water for 15 min. The denatured samples were loaded onto the previously washed filter membrane and equilibrated with 500 μL of water and 100 μL of ABC buffer, respectively. The samples were washed two times with 100 μL of ABC buffer and then resuspended in another 80 μL of the same buffer. One microliter of PNGase F (1000 U) was added to the sample solution and incubated at 37 °C for 20 h. After enzymatic digestion, the samples were eluted from the filter by centrifugation, the sample solution was recovered in a 1.5 mL tube, the filter was washed twice with 100 μL of ABC buffer, and the solution was recovered in the sample tube. The total collected sample was dried using the vacuum concentrator. Digested and purified samples from SPE-C18 and FANGS protocols were reduced and permethylated according to the following procedure. The glycan reduction was accomplished by the addition of 10 μL of 10 μg/μL ammonium borane solution, which was followed by incubation at 60 °C for 1 h. After incubation, the residual borane was removed by the addition of methanol, generating methyl borate that was evaporated while drying in the vacuum concentrator.N-glycans were subjected to solid-phase permethylation. Dried glycans were resuspended in 30 μL of dimethyl sulfoxide (DMSO), 1.2 μL of water, and 20 μL of iodomethane. The solution was applied into a microspin column packed with sodium hydroxide beads that were subsequently incubated in darkness at room temperature for 25 min. After the initial incubation period, 20 μL of iodomethane was applied to the spin column, and the reaction was allowed to procced for an additional 15 min. Permethylated N-glycans were dried and resuspended in aqueous mobile phase for LC-MS analysis.Reduced N-glycans were separated using a C18 Acclaim PepMap column . Samples were desalted using the online system with 3 μL/min of flow rate with 0.1% formic acid and 2% acetonitrile in water for 10 min. Mobile phase A consisted of 0.1% formic acid in water, while mobile phase B was 0.1% formic acid in acetonitrile. The chromatographic gradient was as follows: mobile phase B was held at 20% with 350 nL/min of flow rate for the first 10 min, at which point it was increased to 42%. Next, mobile phase B was increased from 42% to 55% from 10 to 50 min, then increased to 90% over 5 min, and finally reduced to 20% over 5 min. The mass spectrometer was set to full-scan mode, with a scan range from 700 to 2000 m/z and a mass tolerance within 10 ppm with the mass resolution set at 50,000.A Dionex Ultimate 3000 nanoLC system  was coupled with an Exactive mass spectrometer . Permethylated glycans were first desalted with an online purification system using a C18 Acclaim PepMap 100 trapping column . Purified permethylated N-glycans in the samples, relative quantitation was chosen to observe full profiles of N-glycans expressed in the CSF samples. The m/z values of target glycan structures, as well as their corresponding adducts, were applied to generate the extracted ion chromatograms (EICs). The areas under the curve of these EICs were integrated, and the generated data were used to perform a relative glycan quantitation. Statistical analysis was performed using GraphPad Prism.The analysis of the raw data was performed using Xcalibur 4.2 (Thermo Scientific) software; extracted ion chromatograms of each glycan structure, including all possible sodium and ammonium adducts, were generated. To profile


Longitudinal trajectories of amyloid-β positivity were tracked simultaneously for CSF and PET biomarkers. In the longitudinal cohort (n = 289), we found that participants with normal CSF/PET amyloid-β biomarkers progressed more frequently toward CSF/PET discordance than to full CSF/PET positivity (χ2(1) = 5.40; p < 0.05). Progression to CSF+/PET+ status was ten times more frequent in cases with discordant biomarkers, as compared to csf−/pet− cases (χ2(1) = 18.86; p < 0.001). Compared to the CSF+/pet− group, the csf−/PET+ group had lower APOE-ε4ε4 prevalence (χ2(6) = 197; p < 0.001; n = 867) and slower rate of brain amyloid-β accumulation  = 12.76; p < 0.001; n = 608). These results demonstrate that biomarker discordance is a typical stage in the natural history of amyloid-β accumulation, with CSF or PET becoming abnormal first and not concurrently. Therefore, biomarker discordance allows for identification of individuals with elevated risk of progression toward fully abnormal amyloid-β biomarkers, with subsequent risk of neurodegeneration and cognitive decline. Our results also suggest that there are two alternative pathways (“CSF-first” vs. “PET-first”) toward established amyloid-β pathology, characterized by different genetic profiles and rates of amyloid-β accumulation. In conclusion, CSF and PET amyloid-β biomarkers provide distinct information, with potential implications for their use as biomarkers in clinical trials.Mismatch between CSF and PET amyloid-β biomarkers occurs in up to ≈20% of preclinical/prodromal Alzheimer’s disease individuals. Factors underlying mismatching results remain unclear. In this study we hypothesized that CSF/PET discordance provides unique biological/clinical information. To test this hypothesis, we investigated non-demented and demented participants with CSF amyloid-β All participants gave written informed consent, as approved by local ethics committees and in accordance with the Declaration of Helsinki. For up-to-date information, see www.adni-info.org.Data used in the preparation of this article were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database . A total of n = 608 cases had a second [18F]Florbetapir PET scan performed after approximately 2 years (Δt = 2.01 ± 0.68 years) and n = 348 cases had a third scan after 4 years (Δt = 4.28 ± 0.63 years) from the baseline scan. A total of n = 305 had a second CSF amyloid-β42 measurement after ~2 years from baseline (Δt = 2 ± 0.11 years).Data were downloaded from the ADNI database on October 24, 2018. Our cohort consisted of 42 measurement was performed based on the multiple xMAP Luminex platform (Luminex Corp) with INNO-BIA AlzBio3 immunoassay kit (Innogenetics) Florbetapir PET acquisition and analysis are thoroughly described elsewhere . FollowiAdditional baseline and longitudinal measures of neurodegeneration, tau pathology, cognition, and neuropsychiatric symptomatology were available; genetic information was also retrieved. Number of participants with additional data available, at baseline and at follow-ups, is reported in the next paragraphs.n = 857 participants (n = 417 at 2-year follow-up). Data were retrieved from the ADNIMERGE.csv dataset.FDG-PET acquisition and analysis are thoroughly described elsewhere . A compon = 799 participants . Volumes of target regions were corrected for total intracranial volume. Data were retrieved from ADNIMERGE.csv dataset.T1-weighted MRI image acquisition and analysis are thoroughly described elsewhere . Hippocan = 818 participants Florbetapir PET SUVr , and whether baseline [18F]Florbetapir PET SUVr predicted the rate of change in CSF amyloid-β42 . CSF amyloid-β42 and [18F]Florbetapir PET were included in the regression models as continuous variables. Sex, age, number of APOE-ε4 alleles and clinical group were entered as nuisance covariates. The assumptions of normal distribution and homoscedasticity of residuals, as well as the absence of multicollinearity between predictors, were tested and met in all regression models.We fitted linear, quadratic, and cubic regression models, testing whether baseline CSF amyloid-β42 and [18F]Florbetapir PET biomarkers. CSF amyloid-β42 measurements were classified as abnormal (CSF+) when <192 pg/ml. This cutoff was initially established to allow optimal discrimination between healthy controls and Alzheimer’s disease cases confirmed at autopsy Fl autopsy , then va autopsy , and aga autopsy .42 and [18F]Florbetapir PET biomarkers, stratified by csf−/pet−, csf−/PET+, CSF+/pet− or CSF+/PET+ subgroups, we selected all participants (n = 289) with CSF and PET amyloid-β biomarkers available both at baseline and at the 2-year follow-up examinations, and obtained within 90 days. Differences in prevalence of longitudinal outcomes across subgroups were tested by means of χ2 tests.To investigate the simultaneous longitudinal changes in CSF amyloid-βχ2 tests were applied. Cox regression analysis was used for estimation of hazard risks. When the assumption of proportionality of hazards was not met, we run a stratified model. Sex, age, number of APOE-ε4 alleles, and clinical group were included as nuisance covariates.Group comparisons for continuous variables were performed via general linear modeling. Post-hoc tests were performed using Bonferroni correction for multiple comparisons. The assumptions of normality of residuals and homoscedasticity were tested; when not met, the estimates were bootstrapped with 1000 replicates, generating bias-corrected and accelerated 95% confidence intervals. For categorical variables, p < 0.05 (two tailed). Graphical renderings were performed in RStudio , using ggplot2 v.2.2.1.All statistical analyses were performed in PASW Statistics 18 (SPSS Inc.), setting the significance level at n = 867).Table n = 305) and PET  measurements, confirmed both our predictions, i.e.,: (i) baseline CSF amyloid-β42 values significantly predicted the rate of change in [18F]Florbetapir PET SUVr, as computed over a 2-year  = 12.11, p < 0.001; n = 608; Fig. F = 15.11, p < 0.001; n = 348; data not shown) time interval. Thus, CSF amyloid-β42 levels linearly predicted [18F]Florbetapir PET accumulation, with lower CSF amyloid-β42 levels associated with faster brain [18F]Florbetapir PET accumulation. Quadratic and cubic terms did not reach statistical significance in the respective models. (ii) Neither linear, quadratic nor cubic regression models significantly predicted the 2-year rate of change in CSF amyloid-β42 values as a function of baseline [18F]Florbetapir PET SUVr  = 1.06, p = 0.374; F = 0.92, p = 0.471; F = 0.95, p = 0.462, respectively; n = 305; Fig. Testing of our hypothesis by means of standard regression models in the whole cohort, considering participants with longitudinal amyloid-β CSF (hange in 8FFlorbethange in 8FFlorbet42 and [18F]Florbetapir PET biomarker profiles (n = 867).Table 42 and [18F]Florbetapir PET biomarkers in participants with both amyloid-β CSF and PET measurements available, obtained within 90 days, both at baseline and at 2-year follow-up  were more likely to progress to isolated CSF amyloid-β42 positivity or to isolated [18F]Florbetapir PET positivity than to progress directly to full biomarker positivity  = 5.40; p < 0.05). The frequency of progression to full biomarker positivity was significantly higher in both CSF+/pet− and csf−/PET+ groups (25.8% [n = 8/31]), compared to the csf−/pet− group (2.6% [n = 3/115]) (χ2(1) = 18.69; p < 0.001; χ2(1) = 9.97; p < 0.005, respectively), with no significant differences between CSF+/pet− and csf−/PET+ groups (χ2(1) = 0.26; p = 0.613). Finally, CSF+/PET+ participants were highly stable, with full biomarker positivity representing the endpoint of amyloid-β biomarkers changes for 99.3% [n = 142/143] of all cases.The assessment of simultaneous 2-year longitudinal changes in both CSF amyloid-βn = 867), when compared to the remaining groups, the groups with discordant amyloid-β biomarkers presented with “intermediate” levels of the pathological amyloid-β biomarker, i.e., (i) the CSF+/pet− group had significantly lower CSF amyloid-β42 levels than those with normal CSF amyloid-β42 values (both csf−/pet− and csf−/PET+), but significantly higher CSF amyloid-β42 levels as compared to the CSF+/PET+ group; (ii) the csf−/PET+ group had significantly higher [18F]Florbetapir PET retention compared to either the csf−/pet− or CSF+/pet− groups but significantly lower as compared to the CSF+/PET+ level  and 4-year (n = 348) follow-up time intervals. Significant [18F]Florbetapir PET accumulation rates were reported in CSF+/PET+  and CSF+/pet− cases , while minimal to null accumulation was present in csf−/pet−  and csf−/PET+  cases , we found that:When systematically comparing discordant and concordant groups considering (i) biomarkers for tau pathology and neurodegeneration, (ii) clinical and neuropsychological measures, (iii) genetic assessments and (iv) confounding factors (data available in up to 181) and neurodegeneration , and faster rate of neurodegeneration as compared to the csf−/pet− group (Supplementary Table (i) A systematic comparison of biomarkers for tau pathology and neurodegeneration among CSF/PET groups revealed no significant differences between the CSF+/pet− and csf−/PET+ groups. Only the CSF+/PET+ group presented with increased baseline tau pathology  as compared to the csf−/pet− group, and in contrast with the CSF+/PET+ group . Discordant groups presented no difference in either baseline levels or rate of cognitive decline as measured by MMSE, ADAS-cog11 and RAVLT-learning subscale, when compared to the csf−/pet− group. Only the CSF+/PET+ group presented with significant rates of cognitive decline compared to all other groups  = 2.75, p < 0.05) (Supplementary Table (ii) Groups with discordant biomarkers were not at increased risk of clinical progression  = 197; p < 0.001; Table APOE-ε4ε4 genotype than expected . Isolated PET positivity was associated with female sex  = 13.83; p < 0.005; Table (iii) Genetic assessment revealed a significant association between number of 42 changes Florbetapir injection and start of image acquisition was not increased in cases with discordant amyloid-β biomarkers  = 1.40, p = 0.242); (d) occurrence of infarcts (χ2(3) = 2.69, p = 0.442) as well as volume of white matter hyperintensities  = 0.57, p = 0.634) within the SUVr reference region did not differ between groups. Finally, lack of significantly decreased CSF amyloid-β40 levels in csf−/PET+ individuals , together with presence of relevant, but not severe, levels of occipital amyloid-β retention  makes it unlikely that csf−/PET+ cases are due to cerebral amyloid angiopathy.(iv) When examining a series of confounding factors in amyloid-β fluid and molecular imaging assessments . This evidence, coupled with the finding that about 30% of CSF+/pet− cases progressed to CSF+/PET+, qualifies isolated CSF positivity as a preferential pathway from fully normal to fully pathological amyloid-β biomarkers, consistent with previous reports Florbetapir PET became abnormal first, while levels of CSF amyloid-β42 were still normal (3.5% of csf−/pet− cases progressed to csf−/PET+). In addition, about 20% of the participants with isolated pathological [18F]Florbetapir PET progressed to CSF+/PET+. These longitudinal data suggest that cases with isolated [18F]Florbetapir PET positivity are part of an alternative (PET-first) pathway toward established amyloid-β pathology . Although we used all longitudinal CSF/PET data available from ADNI at the time of our study, joint CSF/PET examinations were available at two longitudinal time points only, thus future studies incorporating additional longitudinal CSF/PET time points will be valuable to confirm our findings.This study has some limitations. We acknowledge that the sample size of discordant biomarker groups was relatively limited  toward established amyloid-β pathology, characterized by different genetic profiles and rates of amyloid-β accumulation. In conclusion, CSF and PET amyloid-β biomarkers provide complementary rather than redundant information, with potential implications for their use as biomarkers in clinical trials.Supplementary Figure 1Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5