Bovine alphaherpesviruses type 1 , type 5 , and bubaline herpesvirus type 1 (BuHV-1) induce highly, though not fully, cross-reactive antibody responses, but no antibody assay has, to date, been evaluated against all recognized types/subtypes of these viruses. Six hundred bovine field serum samples were screened in serum neutralization tests (SN) performed against the seven virus types/subtypes and tested in seven ELISAs prepared with each of the virus types/subtypes. A combined, multiple antigens ELISA (mAgELISA) was prepared by mixing five viral antigens. In comparison to SN, the mAgELISA sensitivity was 96.5% with 96.1% specificity . The findings reveal that the mAgELISA developed here is suitable for the detection of antibodies to BoAHV-1, BoAHV-5, and BuHV-1, with sensitivity and specificity comparable to SN, performed with all recognized types of BoAHV-1, BoAHV-5, and BuAHV-1 as challenge viruses.Bovine alphaherpesvirus 1 , type 5 , and bubaline herpesvirus 1 (BuHV-1) induce highly, though not fully cross-reactive serological responses. Most types and subtypes of these viruses circulate particularly in countries of the southern hemisphere, notably Brazil and Argentina. Therefore, the detection of infected animals is important in defining prevention and control strategies, particularly when flocks are destined for international trade. Identification of infected herds is most often achieved by assays that detect antibodies, such as enzyme immunoassays (ELISAs). However, to date, no ELISA has been evaluated in its capacity to detect antibodies to these alphaherpesviruses. Here, an ELISA was developed to detect antibodies to all currently recognized BoAHV-1, BoAHV-5, and BuAHV-1 types/subtypes, and its sensitivity and specificity were determined. Six hundred bovine sera were screened in serum neutralization tests (SN) against the seven viruses. ELISAs prepared with each of the viruses were compared to SN. Subsequently, a combined assay with multiple antigens LISA was prepared by mixing five viral antigens, chosen for their highest sensitivity in the preparative assays. In comparison to SN, the mAgELISA sensitivity was 96.5% with 96.1% specificity . The findings reveal that the mAgELISA developed here is highly suitable for the detection of antibodies, comparable in sensitivity and specificity to that of SN when performed with all known types and subtypes of bovine and bubaline alphaherpesviruses. Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus [Bubalus bubalis) farming worldwide. In water buffaloes, BuAHV-1 is believed to induce mostly inapparent infections, although it has been, on occasion, associated with reproductive and respiratory diseases [Bovine alphaherpesvirus 1 , bovine alphaherpesvirus 5 , and bubaline alphaherpesvirus 1  are members of the family llovirus ,3. BuAHVdiseases ,5,6. A mdiseases ,8,9.At the time of writing of this article, there are 51 complete genomes of BoAHV-1, 6 of BoAHV-5, and 1 BuAHV-1 available at Genbank NCBI. The degree of identity between the three virus types, both at the nucleotide and amino acid levels, is remarkable; BoAHV-1 and BoAHV-5 share a mean overall identity of up to 88% at the nucleotide/amino acid level, whereas BoAHV-5 and BuAHV-1 share a greater than 95% nucleotide/amino acid identity . The antRuminant alphaherpesviruses may cross the species barrier ,15,16,17No currently available serological assay has been evaluated to detect antibody responses to all of these virus types and subtypes. Here, an immunoassay was developed to detect cross-reactive antibodies to these seven virus types and subtypes, with optimized sensitivity and specificity. A multiple antigen assay (mAgELISA) was prepared with five selected viruses that were shown to display the highest sensitivity when compared to preparative immunoassays and SN performed with the seven distinct challenge viruses.All investigative work involving the collection of samples from animals and subsequent steps in the laboratory was approved by the Ethics Committee (ECAE), Veterinary Research Institute Desidério Finamor (acceptance code: 20/2014).Six hundred bovine serum samples were collected from herds in ten farms in the state of Rio Grande do Sul, Brazil. The history of vaccination and records on bovine or bubaline herpesvirus infections BoAHV-1 or BoAHV-5 were unknown. Sera were sent to the lab under refrigeration, inactivated at 56 °C for 30 min, and stored at –20 °C until testing.A pool of sera that reacted positively or negatively at SN in all assays with the seven virus types/subtypes were used as positive/negative controls throughout.Madin Darby bovine kidney cells , were multiplied and subcultured every 3–4 days following standard procedures . All celRepresentatives of all BoAHV-1, BoAHV-5, and BuAHV-1 types and/or subtypes were used as challenge viruses in serum neutralization assays (SN), and for the preparation of the ELISA antigens. Viral strains used were: BoAHV-1.1 strain Los Angeles, or LA (Genbank access no. MF421714.1) , BoAHV-12 atmosphere for five days and read by examining the presence of cytopathic effect (CPE) in cells. Appropriate negative and positive controls, prepared as described above, were included in each batch of assays.The SN tests were performed separately against each of the seven BoAHV-1, BoAHV-5, and BuAHV-1types/subtypes as reported previously . Plates 2 roller bottles. Each of the viruses was inoculated in nearly confluent monolayers of MDBK cells at a multiplicity of infection equal to 0.01. When the CPE was evident in 80% of the monolayers, the cells were scraped off the flasks, mixed with the supernatant, and centrifuged at 5000× g for 15 min. The medium was removed and cells overlaid with 2 mL 0.2% OGP (Sigma#08001) in PBS  and incubated on a rocking platform for 2 h at 4 °C. The suspension obtained was centrifuged at 5000× g for 5 min to remove cell debris. This suspension constituted the antigen, which was aliquoted and stored at −70 °C until use. Uninfected cell cultures were similarly prepared and used as control antigens. Viral antigens were prepared with each of the viruses separately.ELISA antigens were prepared in 850 cm2CO3, 0.6% NaHCO3, pH 9.6). Each antigen was tested in individual plates . The positive and negative serum control pools were diluted (1:2 to 1:4096) in dilution buffer  and incubated with each antigen preparation, individually. The peroxidase/anti-bovine IgG conjugate (Sigma-A5295) was titrated and incubated with each antigen preparation. The assays were optimized to provide the most intense differences in optical density (OD) readings between positive and negative sera. The optimum dilutions for the viral antigens to be used in the preparative single (sAg) and multiple antigens (mAg) ELISAs were established at 1:200 . Test sera and controls were diluted 1:2. The anti-bovine IgG/peroxidase conjugate was used at 1:1500. Tests were considered valid when the difference in OD readings between positive and negative controls was at least 0.7 OD units. The cut-off point of the ELISAs for each plate was defined at ≥45% of the optical density (OD) of the negative control when the difference between negative and strong positive controls was ≥0.7 OD units. Plates whose readings were not within these limits were repeated.Serial dilutions of each of the antigens (1:50 to 1:6400) were prepared in carbonate/bicarbonate buffer . All sera with OD readings close to the cut-off point were repeated until consistent results were obtained. A negative control antigen prepared with uninfected MDBK cells was added to each battery of assays.2PO4; 0.09% NaH2PO4; 0.8% NaCl; 0.02% KCl, pH 7.3). Subsequently, blocking was performed with 70 µL of 2% milk powder  for 1 h at 37 °C and plates washed as above. Test sera (diluted 1:2) were added to duplicate wells (50 µL/well) and incubated for 1 h at 37 °C. Subsequently, plates were washed again and incubated for 1 h at 37 °C with an appropriate dilution (1:1500 in dilution buffer) of rabbit/anti-bovine peroxidase/IgG conjugate (Sigma-A5295). After another round of washings, 50 µL of the substrate ABTS (Sigma-A1888) with 0.3% H2O2 was added to the wells. After 30 min of incubation at room temperature, the optical density (OD) was measured (405 nm), and the average OD of each serum was determined. Every batch of tests included positive and negative controls.Preliminary assays consisting of seven preparative, sAgELISAs, each one with a distinct antigen preparation, were carried out; test sera were evaluated separately with each of these. Polystyrene microplates (Greiner) were coated overnight at 4° C with 50 µL of the previously titrated antigens (1:200 in carbonate/bicarbonate buffer). After incubation, plates were washed three times with 100 µL PBST-20 , as well as 20 sera negative in all assays, were included. The cut-off point of the ELISAs was established for each plate by comparing the OD of negative controls and strong positive controls.The sensitivity of the SN and the seven sAgELISAs was defined by taking the sum of the sera with positive results as the reference. All statistical procedures performed here were calculated using the Dag Stat program .From the 259 neutralizing antibody-positive sera, 213 (82.2%) neutralized all of the seven viruses tested. However, the remaining 46 (17.8%) presented discrepant profiles of reactivity. Such variation was dependent on the virus used in the challenge at SN .The results of SN with all seven viruses, as well as those of the seven sAgELISAs, are shown in In the preparative sAgELISAs , 263/600In the comparison between the results of the seven preparative sAgELISAs and mAgELISA , the twoWith the mAgELISA, 263 of the total 600 samples give rise to positive results. When compared to the SN , the mAgSeveral ELISAs for antibodies to bovine alphaherpesviruses have been developed, most of those based on a single BoAHV-1 strain as the source of antigen ,28,29,30In serological diagnosis, the cross-reactivity of BuHV-1-induced antibodies with antibodies to bovine herpesviruses has been documented on several occasions ,31,33, aThe best combination of antigens for the mAgELISA was achieved by mixing five different antigens. The combined antigens considerably increase the sensitivity of the assay. However, antigen preparation is cumbersome and requires absolute care to avoid cross-contaminations. Nevertheless, the mAgELISA achieves the objective of detecting significantly more seropositive samples than any of the ELISAs prepared with antigens of each of the particular viruses.Some of the drawbacks raised during the development of the mAgELISA reported here must be commented on. The vast majority of the assays conducted in diagnostic laboratories in search for antibodies to bovine alphaherpesviruses—whether ELISAs or SN—are performed with one strain of BoAHV-1 as a source of antigen or as a challenge virus, often using one of the “classical” BoAHV-1.1 strains. As demonstrated here, depending on the strain used as a source of antigen , a varying number of false negative results may be obtained. However, maximum sensitivity should be required to minimize the chances of disseminating herpesviral infections, since seropositive animals are potential spreaders of the viruses and may compromise control or eradication efforts. Therefore, an assay capable of detecting most, if not all, seropositive animals becomes an important tool to prevent virus spread. As such, the mAgELISA reported here may become an exquisite tool to minimize the dissemination of BoAHV-1, BoAHV-5, and BuAHV-1.In addition, particularly in tropical countries, the number of buffalo breeding farms is increasing, in most instances close to, or in conjunction with, bovine cattle farming. The extensive antibody cross-reactivity induced by infections with any of the herpesviruses examined here has already been demonstrated, both here and in previous studies ,33,35. MHowever, as currently available serological assays do not discriminate between serological responses induced by these types/subtypes of viruses, determining the virus that induces a particular antibody response is still serologically unattainable.Another limitation of the assay introduced here is that it does not allow for differentiation between infected and vaccinated animals. This would be essential in countries where differential vaccines (DIVA) are in use. Nonetheless, in Brazil and many other countries, DIVAs are not licensed for BoAHV-1 control; as such, a serological assay that would be adequate for strategies based on the use of DIVAs would not be adequate for such countries. In fact, in the country of origin of the authors and many others, neither DIVAs nor differential antibody assays are licensed for sale. For that reason, we did not perform comparative assays with DIVA-related assays. As such, the assay reported here may not be useful in countries where DIVA-based eradication programs have been implemented. Nevertheless, examining DIVA-compatible assays in their capacity to recognize antibodies induced by infections with all seven herpesvirus types and subtypes, as performed in the present study, remains a future work to be carried out.In conclusion, it is shown here that the mAgELISA is suitable for the detection of antibodies to BoAHV-1, BoAHV-5, and BuHV-1, with sensitivity and specificity significantly compared to that of at least three SN tests performed with three different challenge viruses. The assay is capable of detecting antibodies to all known types and/or subtypes of BoAHV-1, BoAHV-5, and BuHV-1. To date, the mAgELISA is the first assay to be evaluated in its capacity to detect antibodies to all these alphaherpesviruses. It is expected that this test will become useful in providing support to control or eradicate programs for herpesviruses in ruminants. Moreover, the assay presented here may become useful in the trade of ruminants whenever serological testing of bovine/bubaline herpesviruses may be required.


GDAP1 gene, whose mutations are known to be causative for CMT type 4A (CMT4A). Due to low penetrance and a rare occurrence limited to five patients from two Polish families affected by the CMT phenotype, there is doubt as to whether we are dealing with real pathogenic mutation. Thus, we aimed to study the pathogenic effect of the c.664G>A, p.Glu222Lys variant in its natural environment, i.e., the neuronal SH-SY5Y cell line. Additionally, we have checked the pathogenic status of p.Glu222Lys in the broader context of the whole exome. We also have analyzed the impact of GDAP1 gene mutations on the morphology of the transfected cells. Despite the use of several tests to determine the pathogenicity of the p.Glu222Lys variant, we cannot point to one that would definitively solve the problem of pathogenicity.Charcot–Marie–Tooth disorders (CMT) represent a highly heterogeneous group of diseases of the peripheral nervous system in which more than 100 genes are involved. In some CMT patients, a few weak sequence variants toward other CMT genes are detected instead of one leading CMT mutation. Thus, the presence of a few variants in different CMT-associated genes raises the question concerning the pathogenic status of one of them. In this study, we aimed to analyze the pathogenic effect of c.664G>A, p.Glu222Lys variant in the  The currently available gene databases contain the still rising number of variants of unknown significance (VUS), variants of uncertain effect, or variants characterized even by contradictory results. In the era of next-generation sequencing (NGS), a leading problem seems to be establishing the pathogenic status of sequence variants rather than detecting it. The missense variants resulting from single substitutions within SNPs are an especially challenging task in terms of the pathogenic status of variants.In 2015, nearly half of deposited sequence variants in the widely used ClinVar database were categorized as VUS. Five years later, in 2020, almost 75% of ClinVar variants were classified as VUS [The exponentially increasing number of variants of unknown clinical relevance has led to attempts to develop systems for the comprehensive assessment of the pathogenicity of mutations involving entire genes. Unfortunately, due to the diverse functions of proteins encoded even by the same gene, the creation of a common system for the functional assessment of the effects of mutations encounters quite serious difficulties. Different mutations of the same gene typically result in different disorders . Hence, there is a need to individualize the pathogenicity assessment of individual mutations. Some authors even propose the use of a summary assessment of the mutation effect, in which the results of various functional tests are taken into account—the integrated phenotypic score .GDAP1 gene (8q21) coding for the ganglioside differentiation-associated protein 1 was first characterized as the gene whose expression is tightly correlated with the differentiation of the neuroblastoma cells [GDAP1 gene mutations inherited in an autosomal dominant trait were reported [GDAP1 mutations. For many years, the hypothesis of the role of GDAP1 function of glutathione transferase was discussed. As the protein expressed in the outer mitochondrial membrane, GDAP1 was shown to be involved in the fusion/fission processes. Some authors have shown evidence of the GDAP1 role in intracellular calcium transport. For 23 years, no functional association between the neuronal differentiation process accompanied by GDAP1 and any molecular pathways have been established till now [GDAP1 was found. Interestingly, the c.664G>A, p.Glu222Lys variant has been shown to segregate with CMT phenotype both in dominant and recessive (together with p.Leu239Phe) traits. Till now, in the literature, there was no reported, not even one CMT -affected patient harboring the heterozygous p.Leu239Phe mutation (carrier) manifesting any CMT symptoms. However, the p.Leu239Phe mutation is a recurrent pathogenic variant occurring both in the homozygous and compound heterozygous state in the CMT-affected patients [The ma cells . In 2002ma cells ,5,6. In till now . RecentlAP1 gene q21 codinIn the dominant trait, Glu222Lys amino-acid substitution was associated with the extremely mild phenotype of peripheral neuropathy limited to the lower limbs beginning after the seventh decade of life . In the GDAP1 mutation, including that resulting in p.Glu222Lys in a yeast-based model in which we classified this mutation as weak/moderate [GDAP1 expression, cell viability, sensitivity to H2O2 and the differentiation process of the neuronal cell line (human neuroblastoma SH-SY5Y cell line).Due to extremely weak clinical manifestation and the lack of further CMT families, the pathogenic character of Glu222Lys amino-acid substitution could be considered at least disputable. Although the vast majority of GDAP1 mutations are inherited in recessive traits, there are some mutations that are dominant. However, the Glu222Lys amino-acid substitution is exceptional because it could be recessive as well as dominant. The double inheritance pattern occurs very rarely in inherited disorders. For such a reason, we decided to analyze the effects of Glu222Lys amino-acid substitution in a more detailed way. We could also not exclude the possibility that the pathogenic effect of the p.Glu222Lys variant depends on the ethnic or even intrafamilial context (mutation found in east European population in two families which could be related). Previously, we checked the pathogenic effect of moderate . The yeaGDAP1 gene was c.664G>A, p.Glu222Lys heterozygous variant. For details, see Kabzińska et al. [In family A a, the sia et al. .GDAP1 mutations, p. His123Arg and p.Ala156Gly were selected as the positive controls of the pathogenic effect of investigated p.Glu222Lys variant. The Gln218Glu substitution was also documented only in one Korean family and associated with a weak phenotype in two patients (father and daughter). We used p.Gln218Glu as a representative variant for weak mutations. The pathogenic variants resulting in His123Arg, Ala156Gly, and Gln218Glu amino-acid substitutions have been shown to segregate with CMT disease with an autosomal dominant trait of inheritance [Two eritance ,14,15.Ethics approval was obtained from the local ethic committee, as well as the informed concern from patients from families A and B. The study was conducted in accordance with the Declaration of Helsinki.Whole exome sequencing (WES) of the proband’s DNA was performed in line with the protocol from Illumina’s TruSeq Exome Enrichment Guide. A SureSelect Human All Exon 50 Mb Kit . Hybridized fragments were bound to streptavidin beads, while non-hybridized fragments were washed out. The library was then verified on an Agilent 2100 Bioanalyzer and sequenced on a HiSeq 2000 instrument (Illumina) from Intelliseq LLC .We have selected 154 genes  for the 2 atmosphere.Neuroblastoma cell line SH-SY5Y (gift from M. Szeliga MMRI PAS) was maintained in DMEM/F12  containing 10% FBS , 100 IU/mL penicillin, and 100 μg/mL streptomycin , +1% Non-Essential Amino Acids (NEAA)  and uridine 1 mg/mL  in a humidified tissue culture incubator at 37 °C and 5% COGDAP1 cDNA was subcloned from pCMV6-XL5 GDAP1 (NM_018972) Human Untagged Clone  to pIRES2-AcGFP1 Vector  using SacI and SalI enzymes .GDAP1 gene  was performed using Mut Express II Fast Mutagenesis Kit V2  according to the manufacturer’s instructions and verified by sequencing.Mutagenesis for mutations in the GDAP1 knockout was generated using the CRISPR method with GDAP1 sgRNA (spCas9) CRISPR Lentivector set , pLenti-U6-sgRNA-PGK-Neo from ABM . Plasmids were multiplying in TOP One Escherichia coli chemically competent cells then isolated with NucleoBond Xtra Midi EF kit . HEK293T cells  were seeded approximately 3 × 106 cells on 10 cm Ø plates containing a complete DMEM/F12 growth medium. Cells were growing overnight to approximately 70–80% confluence. All lentiviruses used for GDAP1 knockout  were generated in HEK293T cells via calcium phosphate transfection using two viral packaging plasmids ∆8.9 and VSVG . The viruses were collected after 24 h, and viral supernatants were filtered using a 0.45-μM filter and stored at −80 °C before SH-SY5Y cells transduction. The SH-SY5Y cells transduction was performed by adding 2 mL of viruses with different sgRNAs to 2 mL of complete DMEM/F12 growth medium on 6 cm Ø plates with ~50% confluent SH-SY5Y cells. The medium was changed 24 h after transfection and selected 48 h after transduction for Cas9 expression with 500 μg/mL G418  for two weeks. The mixture of T1, T2, and T3 sgRNA viruses characterized by the most effective reduction of the GDAP1 protein level was selected for further analyses.A SH-SY5Y cells were transfected using the pIRES2-AcGFP1 Vector with Viromer Red  according to the manufacturer’s recommendations with the standard transfection scale. In all cases, the medium was changed 48 h after transfection. The resulting transfectant SH-SY5Y cells were cultured in an appropriate medium with 500 μg/mL G418  for two weeks.v/v . Proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane Amersham Protran , and analyzed by Western blotting using rabbit polyclonal anti-GDAP1  and secondary anti-rabbit IgG horseradish peroxidase (HRP)-conjugated antibody  followed by enhanced chemiluminescence .Total protein cell extracts were prepared using a cell lysis buffer  with 1 mM PMSF and Protease Inhibitor Cocktail 1/200 6 cells/mL concentration. The 10 µL of 0.4% trypan blue  and 10 µL of cells suspension were mixed and incubated ~3 min at room temperature. After incubation, cells were immediately applied on disposable slides, and the percentages of viable cells were calculated automatically.SH-SY5Y cells viability was measured using the trypan blue dye exclusion test in cells suspension by automated cell counter Countess II . SH-SY5Y cells were counted and diluted to target approximately 2 × 105 cells was suspended in 200 µL of PBS  or PBS with different amounts of 30% H2O2 to concentrations: 100, 200, 300, 400, 500, and 600 mM, respectively. Cells were incubated 10 min in a humidified tissue culture incubator at 37 °C and 5% CO2 atmosphere, spin down 3 min at 300 g, suspended in 100 µL of PBS, and tested for viability with the trypan blue dye.The amount of 4 × 105 and incubated in regular DMEM media containing 10% FBS at 37 °C, 5% CO2 for 168 h (7 days). Pictures were taken at 20× using Zeiss Axio Imager brightfield microscope and ZEN 3.1 (blue edition) software at 24 (Day 1), 48 (Day 2), 72 (Day 3), 120 (Day 5), and 168 (Day 7) hours after cells seeding. Scale bar = 100 μm.SH-SY5Y cells were plated on 6-well plates uncoated (plastic) in the density of 0.5 × 10https://www.graphpad.com/scientific-software/prism/, Accessed on 10 May 2022) using one-way ANOVA and Dunnett correction: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001. Data are presented as means ± standard deviations.Statistical analyses were performed using GraphPad Prism Software   of additional variants in ‘‘CMT genes’’ were found in the exome of the most seriously affected patient III:2 from the family A. In the much milder affected brother III:3, only two additional variants were found in the ‘‘CMT genes’’ a. The moDST c.8626A>T variant, the pathogenicity status is unknown because it has not been reported to the databases up until now. Similarly, the databases have not yet recorded the PRNP c.654C>T variant. In the case of family B, the genetic load seems to be lower/low, and both patients III:9 and III:11 have the same two additional sequence variants in DST c.8626A>T and PEX1 c.149G>A genes. The difference in the genetic load between the studied two families is interesting.In the case of the Due to the identity of mutations in patients III:9 and III:11 in family B, it can be presumed that they were inherited from one of the parents.As presented in this study of two families, we see that the number of CMT variants correlated with the clinical picture. Similarly, in the larger group of CMT families, we observed a positive correlation between sequence variants and the clinical picture of CMT. All the above-presented data strongly support the hypothesis that genetic background could have an additional impact on the clinical presentation of CMT.GDAP1 and c.664G>A, p.Glu222Lys and control variants with established pathogenicity c.652C>G, p.Gln218Glu; c.368A>G, p.His123Arg; and c.467C>G, p.Ala156Gly. This is due to the fact that a decrease in the level of GDAP1 has already been observed in the fibroblasts taken from the patients with the p.Leu239Phe/p.Arg273Gly and GDAP1 c.579+1G>A pathogenic variants [GDAP1 alleles. Additionally, the knockout GDAP1 mutant was used as a control of GDAP1 protein level reduction. The level of Glu222Lys protein in the SH-SY5Y cells, together with levels of three other GDAP1 protein variants, as a control, was analyzed by Western blot. The cells transfected with cDNA of the wild-type GDAP1 show the highest level of GDAP1. In the case of three pathogenic GDAP1 variants, i.e., p.Gln218Glu, p.His123Arg, and p.Ala156Gly, respectively, the level of GDAP1 was reduced. A highly reduced level of GDAP1 was observed for the analyzed p.Glu222Lys variant.We expected to answer the question of whether p.Glu222Lys is a leading mutation after WES, but our analysis identified mutations of unknown status or completely unknown. This does not allow us to answer the question if the p.Glu222Lys variant is a leading mutation. Is p.Glu222Lys one of many variants in CMT genes whose total effect we observe in the presented families? To more accurately answer these questions, we made a model to assess the pathogenicity of the p.Glu222Lys variant. The model is based on SH-SY5Y cells transfected with the empty vector or vector bearing cDNA of wild-type variants . The firGDAP1 overexpression increases the cellular level of GSH in the HT22 cells and stabilizes the mitochondrial membrane potential by decreasing the production of reactive oxygen species (ROS) [GDAP1 gene mutations by reduction of GDAP1 level  . In lighP1 level  could reGDAP1 gene variants, i.e., p.Gln218Glu, p.Glu222Lys, p.His123Arg and p.Ala156Gly. Interestingly, the overexpression of theoretically protective wild-type GDAP1 resulted in a slight reduction of SH-SY5Y cell viability  and Tyr124, and, additionally, this residue has van der Waals contacts to Leu239, the amino acid residue most frequently substituted in CMT4A [Not only do the various sequence variants contribute to the phenotype, but genetic interaction between two alleles of in CMT4A ,25.milieu of the human neuronal cells i.e., in the neuroblastoma SH-SY5Y cell line. We have shown that expression of some of the GDAP1 mutants analyzed by us resulted in reduced viability of the cells. The SH-SY5Y transfected cells were sensitive to the exposure to the H2O2 with the highest sensitivity displayed by cells having a knockout of the GDAP1 gene. These findings are in line with observations made previously by Noack et al. for other GDAP1 gene mutations [GDAP1 gene overexpression on survival in the presence of H2O2. However, we used the different cell lines to model the effect of mutations . The reduced viability upon H2O2 treatment has to be related to the level of GDAP1 protein variants. In contrast, we see that p.Glu222Lys and p.Gln218Glu, despite very low expression, result in a weaker sensitivity than GDAP1 overexpression, which results in high levels of GDAP1 protein. Moreover, the effect of p.Glu222Lys variant observed in the cell-line model of CMT4A disease does not correspond with the clinical severity of CMT4A. Within four dominantly inherited GDAP1 analyzed variants, the reduction of p.Glu222Lys protein level is the biggest one. If GDAP1 gene mutation will be acting in gene dosage fashion, the clinical phenotype for Glu222Lys amino-acid substitution should be the strongest one, but it is not. However, in general, the gene dosage effects are observed in CMT1A disease caused by duplication of the PMP22 gene and in CMT2S caused by the mutations in the IGHMBP2 gene [Since the studies of genetic load did not provide a clear explanation of the pathogenic nature of p.Glu222Lys variant, we have investigated the effect of the Glu222Lys amino acid substitution in the more natural utations . SurprisBP2 gene ,27.GDAP1 gene [GDAP1 expression and neural sprouting may be generalized because it is not limited to the primarily examined Neuro2a cells. It is of note that the strong effect of p.Glu222Lys variant observed for the morphology of SH-SY5Y cells could not be observed at the clinical level. In the light of these findings, everything shows that the process of neuronal differentiation is not a critical issue in the pathogenesis of CMT4 disease and that the other more pathogenic role of GDAP1 remains to be discovered. It is worth noting that a severe reduction in GDAP1 protein levels, as in the case of Glu222Lys amino-acid substitution, is accompanied by morphological changes. In recent studies by Miressi et al., the mutation resulting in p.Ser194* protein truncation was shown to be associated with a low level of protein, probably due to nonsense-mediated RNA decay. This also caused disturbances in the differentiation of motor neurons from hiPSCs [Due to the fact that, in the first study devoted to the GDAP1 protein, the authors showed the correlation between neurite -like differentiation and sprouting of the Neuro2a cells and expression of AP1 gene . Followim hiPSCs .Nevertheless, the morphological phenotype associated with the p.Glu222Lys variant may serve as the measure of the pathogenic effect of this variant but seems to be not serviceable in any attempts trying to establish the clinical prognosis of CMT-GDAP1 types. Interestingly, Glu222Lys substitution has a similar effect as mentioned above GDAP1 protein truncation.Our studies show that, even for a single gene, no general functional test of mutation pathogenicity can be established. We see that the mechanism of action of the p.His123Arg and p.Ala156Gly mutations are quite different from the mechanism for the p.Gln218Glu or p.Gly222Lys variants. In the case of p.His123Arg and p.Ala156Gly, we see a significant reduction of cell viability, which is not present for p.Gln218Glu and p.Glu222Lys. On the contrary, p.His123Asp and p.Ala156Gly do not affect cell sprouting, which does not exclude their obvious and proved pathogenicity, verified by other functional assays. We have shown that assessing the pathogenicity of mutations requires a highly individualized approach.milieu, which closely resembles the natural protein environment. For some weak sequence variants, even the combination of various pathogenicity approaches may not be sufficient to establish the causative role of mutation. Thus, especially in cases of weak variants, reporting of the clinical picture of unrelated patients harboring the same variant seems to be crucial in the process of assessing of causative role of the certain mutation.We assume that a proper test of sequence variant’s pathogenicity should be selected very carefully, taking into account the most impaired function or cellular process disturbed by mutation. The pathogenic status of certain sequence variants should be assessed in the cellular There is an urgent need to add the results of functional tests concerning the pathogenic effect of mutations to existing databases and correlate them with available clinical and genealogical data.


Intrahepatic cholangiocarcinoma (iCCA) is a cancer arising from intrahepatic bile duct epithelium. An iCCA incidence is increasing worldwide; however, the outcome of the disease is dismal. The linkage between chronic inflammation and iCCA progression is well established, but the roles of granulocyte-macrophage colony-stimulating factor (GM-CSF) remain unrevealed. Thus, a better understanding of GM-CSF functions in CCA may provide an alternative approach to CCA treatment.GM-CSF and GM-CSFRα mRNA expressions in CCA tissues were investigated by Gene Expression Profiling Interactive Analysis (GEPIA) based on The Cancer Genome Atlas (TCGA) database. The protein expressions and localizations of GM-CSF and its cognate receptor (GM-CSFRα) in iCCA patients’ tissues were demonstrated by the immunohistochemistry (IHC) techniques. The survival analyses were performed using Kaplan-Meier survival analysis with log-rank test and Cox proportional hazard regression model for multivariate analysis. The GM-CSF productions and GM-CSFRα expressions on CCA cells were assessed by ELISA and flow cytometry. The effects of GM-CSF on CCA cell proliferation and migration were evaluated after recombinant human GM-CSF treatment. The relationship between GM-CSF or GM-CSFRα level and related immune cell infiltration was analyzed using the Tumor Immune Estimation Resource (TIMER).Differential GM-CSF and GM-CSFRα expressions were higher in CCA tissues than in normal counterparts, and high GM-CSFRα was related to the longer disease-free survival of the patients (p < 0.001). IHC analysis revealed that CCA cells differentially expressed GM-CSF, while GM-CSFRα was expressed on cancer-infiltrating immune cells. The patient whose CCA tissue contained high GM-CSF expressed CCA, and moderate to dense GM-CSFRα-expressing immune cell infiltration (ICI) acquired longer overall survival (OS) (p = 0.047), whereas light GM-CSFRα-expressing ICI contributed to an increased hazard ratio (HR) to 1.882 . In non-papillary subtype, an aggressive CCA subtype, patients with light GM-CSFRα-expressing ICI had shorter median OS  and the HR was elevated to 2.788 . Additionally, TIMER analysis demonstrated GM-CSFRα expression was positively correlated with neutrophil, dendritic cell, and CD8+ T cell infiltrations, though it was conversely related to M2-macrophage and myeloid-derived suppressor cell infiltration. However, the direct effects of GM-CSF on CCA cell proliferation and migration were not observed in the current study.GEPIA analysis indicated α-expressing ICI were suggested. Altogether, the benefits of acquired GM-CSFRα-expressing ICI and GM-CSF for CCA treatment are proposed herein and require elucidation.Light GM-CSFRα-expressing ICI was an independent poor prognostic factor for iCCA patients. Anti-cancer functions of GM-CSFR Opisthorchis viverrini, infection is one contributing factor to the high number of cases (Cholangiocarcinoma (CCA) is an epithelial cancer originating from the neoplastic transformation of bile duct lining cells, which the highest incidence has been reported in the Mekong subregion of Asia, including Thailand . Accordiof cases . Althougof cases , the lacof cases .α, and TGF-β, to promote cancer growth, metastasis, and immune evasion. Several studies target these cytokine-related signaling pathways as promising therapy (α) and a signaling subunit (β), is expressed on myeloid cells such as monocytes, macrophages, and dendritic cells (DC)  . In a phlls (DC) . It playlls (DC) . Regardills (DC) , whereaslls (DC) . In CCA,lls (DC) ; howeverlls (DC) . Altogetα expressions in patient iCCA tissues were investigated and correlated with the clinical parameters. The direct effects of GM-CSF on CCA cell proliferation and migration were assessed in vitro. The immune cell infiltration (ICI) profile related to GM-CSF and GM-CSFRα expressions in CCA was analyzed by a web-based tool, the Tumor Immune Estimation Resource (TIMER). Altogether, this study could better understand GM-CSF roles and their implications in iCCA.The GM-CSF and GM-CSFRNinety-six formalin-fixed paraffin-embedded tissues were obtained with informed consent from the specimen bank of Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand. Tissues were collected from 1998–2012 and were selected under the following criteria: (1) cancers were iCCA, (2) samples were from hepatic resection, (3) clinical data were available for analysis, and (4) patients had no known history of other cancers. Perioperative deaths or patients with an overall survival less than 30 days were excluded. The study protocol was approved by the Ethics Committee for Human Research of Khon Kaen University based on the Declaration of Helsinki (HE571283 and HE611034).2 at 37 °C. All cell culture-related reagents were obtained from Gibco .Four CCA cell lines, KKU-055, KKU-100, KKU-213A, and KKU-213B were obtained from the Japanese Collection of Research Bioresources Cell Bank  . Two metα was from Santa Cruz , anti-mouse and anti-rabbit EnVision-horseradish peroxidase (HRP)-conjugated antibodies were from DAKO , phycoerythrin (PE)-conjugated anti-GM-CSFRα was from BioLegend , PE-conjugated mouse IgG was from eBioscience . ELISA MAX™ Deluxe Set Human GM-CSF and recombinant human GM-CSF (rhGM-CSF) were from BioLegend. All chemicals were purchased from Sigma-Aldrich .The sources of antibodies were as follows: rabbit anti-GM-CSF antibody was from Novus , mouse anti-GM-CSFRGM-CSF and GM-CSFRα mRNA expressions in CCA tissues compared with their normal counterparts were investigated by Gene Expression Profiling Interactive Analysis , an online server for cancer and normal gene expression profiling analysis based on the Cancer Genome Atlas (TCGA) database , CM was collected at 24 h and determined GM-CSF concentration by GM-CSF ELISA kit following the manufacturer’s instruction. The absorbance was measured at 450 nm by an iMark microplate reader .α expressions on surface CCA cell lines were evaluated by flow cytometry . Cells were stained by PE-conjugated anti-GM-CSFRα. PE-conjugated mouse IgG1 was used as isotype control. Data were analyzed by FlowJo software V.10.7.2 . GM-CSFRα expression levels were calculated as mean fluorescence intensity (MFI) of anti-GM-CSFR α-stained cells/MFI of isotype control-stained cells and were expressed as the relative MFI . The absorbance at 540 nm was determined by a Sunrise microplate reader .The effects of GM-CSF on KKU-055 and KKU-213B cell numbers were determined by MTT assay. Cells were seeded at 2 × 10The scratch-wound assay was performed to determine the effect of GM-CSF on CCA migration. KKU-055 and KKU-213B were seeded into 24-well plates as a monolayer until confluent, and then the scratch was made using a sterile 200-µl tip, and cells were allowed to migrate in 0, 1, and 10 ng/ml rhGM-CSF containing media for 24 h. The images of wound closure area at 0, 6, 12, 18, and 24 h were taken by Nikon’s DS-Fi2 camera connected to a Nikon Eclipse inverted microscope  and then calculated by ImageJ software V.1.48 as previous described . The perhttp://timer.cistrome.org/), based on computational algorithms of the TCGA database , all macrophages, M2 macrophages, myeloid-derived suppressor cells (MDSCs), and CD8+ T cells were estimated and plotted as correlation scatter plots. The correlation coefficients between variables were presented as rho values.The ICI in CCA tissues was analyzed by a web-based tool, the Tumor Immune Estimation Resource , Kaplan–Meier survival curves were compared using the log-rank test, and multivariate analyses using Cox proportional hazard models were performed to identify independent prognostic factors for survival of patients, then the backward selection was tested to avoid confounding factors (t-test using GraphPad Prism (V.7.0) . A p-value less than 0.05 was considered of statistical significance.Statistical analyses were performed using SPSS (V.26.0)  as follows: the associations between categorical variables were determined by Pearson’s chi-square tests ( factors . The quath edition of the American Joint Committee in Cancer (AJCC) staging system  and longer disease-free (DF) survival times than those with low expressions   (These data prompted determination of the GM-CSF and GM-CSFRon (ICI) . GM-CSFRll cases , while Gs (3.1%) .Distinct histological subtypes of iCCA expressed GM-CSF differently . Therefoα-expressing ICI was observed in 62 cases (64.6%), and moderate to dense infiltration was observed in 34 cases  (α-expressing ICI were comparable between papillary and non-papillary subtypes (α-expressing ICI was detected in 22 cases (62.9%) of papillary iCCA while light GM-CSFRα was noted in ICI of 40 non-papillary cases (65.6%).Light GM-CSFR7 cases) . The dissubtypes . Light Gα expressions and clinical parameters of the iCCA patients using χ2 test. The results demonstrated that the papillary subtype expressed higher GM-CSF  compared to one of these high expressions (329 days) or those who had decreased expressions of both proteins  (α-expressing ICI were independent poor prognostic indicators (HR) = 2.130; 95% CI [1.046−4.337], p = 0.037; HR = 4.233, 95% CI [1.325–13.522], p = 0.015; and HR = 1.882, 95% CI [1.077–3.287], p = 0.026  . Multiva = 0.026 . It is wpatients .vs. 236 days, p < 0.001)  , while cancer stage III and lymph node metastasis increased risk of death 6.017 times, and 5.094 times in papillary iCCA   . Hence, = 0.002) . The mul= 0.023) .α, on CCA cells was assessed by flow cytometry. GM-CSFRα expressions on CCA cells varied from undetectable in KKU-213B to slight expression in KKU-213A and KKU-213L5 (1.11 and 1.12 times higher than MFI of isotype control). Moderate expressions were detected in KKU-055 and KKU-214L5 (2.44 and 2.17 times), while the highest expression was demonstrated in KKU-100 , but in KKU-213A and KKU-213B, there were 84 ± 6 and 100 ± 13 pg/ml and were higher in metastatic cells, KKU-213L5 and KKU-214L5 , all macrophages, M2 macrophages, and myeloid-derived suppressor cells (MDSCs), and CD8+ T cells, were selected  and 5B.  T cells . Similartrations .α, in CCA were investigated.To date, surgical resection with a free surgical margin is the only potentially curative treatment for CCA, but the number of candidates is limited due to high metastasis . AlthougGM-CSF and GM-CSFRα mRNA expressions in CCA tissues compared with normal counterparts by GEPIA analysis were first demonstrated. Patients with high GM-CSFRα appeared to have longer DFS. The levels of GM-CSF and GM-CSFRα in Thai iCCA patient tissues were determined by immunohistochemistry staining. GM-CSF was differentially expressed in CCA cells, whereas GM-CSFRα was observed primarily in ICI. The high expressions of GM-CSF in CCA and increased GM-CSFRα-expressed ICI seemed to benefit CCA patients, particularly the worse prognostic subtype, the non-papillary one, demonstrated by longer survival times .The elevated al times . It is wal times . Using me.g., cyclooxygenase-2 and inducible nitric oxide synthase). On the contrary, cancer-derived GM-CSF promoting immunosuppressive cell infiltration is reported in the iCCA mouse model  , prostatse model . The simse model . Thus, tα-expressing iCCA cell properties were not detected in the present study. These results implied GM-CSF functions on ICI in the CCA microenvironment. These results implied the indirect effects of GM-CSF on CCA progression. The effects on immune cells were speculated.The autocrine effect of GM-CSF on cancer growth promotion is also reported in GM-CSFR-expressing glioma cells , but theGM-CSF-expressed CCA was associated with DC macrophage, and CD8+ T cell infiltrations, and GM-CSFRα expression was allied to neutrophil, DC, and CD8+ T cell infiltrations. Increased GM-CSFRα was negatively correlated with the levels of M2-macrophage and MDSC in the CCA microenvironment. In CCA, the increased number of mature DCs correlates with T cell infiltration and a lower incidence of lymph node metastasis in patients  and papillary subtypes (C). Bar = 50 µm.The immunohistochemistry of GM-CSFRClick here for additional data file.10.7717/peerj.14883/supp-2Supplemental Information 2α expressions in papillary subtypes (C).Kaplan–Meier survival analysis of different GM-CSF expressions in non-papillary (A) and papillary subtypes (B) of iCCA, and GM-CSFRClick here for additional data file.10.7717/peerj.14883/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.14883/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.14883/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.14883/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.14883/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.14883/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.14883/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.14883/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.14883/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.14883/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.14883/supp-13Supplemental Information 13Click here for additional data file.


B-Cell Attracting chemokine1)) for MS diagnosis. 320 paired (CSF-serum) samples were collected from 160 patients with MS (n = 82) and non-MS diseases (n = 78). All patients benefited from IgG index determination, OCB detection, CSF CXCL13 and FLC (κ and λ) measurement in CSF and serum for metrics calculation ). CXCL13 and FLC metrics in CSF were higher in patients with MS and positive OCB. As expected, κFLC metrics—in particular, κFLC index and κFLC IF—had the highest accuracy for MS diagnosis. κ index showed the best performance (sensitivity 83% and specificity 91.7%) at a cut-off of 14.9. Most of the FLC-related parameters were positively correlated with IgG index and the level of CXCL13. In conclusion, the quantitative, standardizable, and technically simple CSF FLCκ metrics seem to be reliable for MS diagnosis, but could not replace OCB detection. CXCL13 appears to be an effective parameter reflecting the intrathecal B cell response. An optimized way for CSF testing combining the conventional and the new B cell-related parameters is proposed in this study.In multiple sclerosis (MS) disease, the importance of the intrathecal B cell response classically revealed as IgG oligoclonal bands (OCB) in cerebrospinal fluid (CSF) was reaffirmed again in the recently revised diagnostic criteria. We aimed to optimize Laboratory investigation by testing the performance of new B cell-related molecules in CSF (Ig free light chains (FLCκ and λ) and CXCL13 ( An auto-reactive cellular and humoral immune response responsible for multifocal myelin destruction throughout the CNS seems to be the major pathological process. Although MS is traditionally considered a predominantly T cell-mediated autoimmune disease, several lines of evidence have supported the idea that B cell may be a major contributor to the disease pathogenesis5. In particular, studies of the cerebrospinal fluid (CSF) have provided important highlights into the B cell response in MS. The first evidence was identified decades ago with the identification of unique immunoglobulin (Ig) G fractions in the CSF6 produced by intrathecal clonal B cell populations7 and revealed as oligoclonal bands (OCB) in up to 90% of all patients with MS8. This biological sign is considered as a diagnostic hallmark of the disease. Other findings have supported the role of B cells and intrathecal Ig in the pathogenesis of MS, which includes (i) detection of B-cell infiltrates in the CNS -like structures), in particular within lesions 10, (ii) elevated levels of antigen-experienced, class-switched memory B cells and antibody-producing plasma cells in the CSF (relative to their levels in the blood) of patients with MS (iii) Ig-related genes upregulation in cortical sections of patients with MS11, and (iv) efficacy of B cell-targeting therapies 13.The etiology of multiple sclerosis (MS), a common neuroinflammatory and neurodegenerative disorder of the central nervous system (CNS)16. It has been particularly reported that CSF levels of CXCL13 correlate with the clinical response to B cell-targeting therapies17.Furthermore, much evidence suggests that the inflamed CNS provides different factors supporting the long-term survival of B-lineage cells in the environment—such as TNF ligand superfamily member 13B, also known as BAFF or BLyS and chemoattractants—such as stromal cell-derived factor 1 ) and CXCL1319. According to the last 2017 revisions of the McDonald MS diagnostic criteria, the diagnosis is based on clinical symptoms, MRI imaging, and biological investigation, including cerebrospinal fluid (CSF) testing20.An early and accurate MS diagnosis is crucial for the clinical management of patients since recent therapeutic alternatives (disease-modifying therapies) effectiveness depends on the stage of disease diagnosis22, has gained more importance in these recently revised MS diagnostic criteria. This biological sign reflecting an intrathecal synthesis (IS) of IgG antibodies and considered an immunological hallmark of MS, is classically revealed using the gold-standard technique, which is the CSF Isofocusing test. Other quantitative tests  are also useful in this context. However, in routine practice, it is not rare to be faced with some confusing situations  and some technical issues related mainly to the standardization of OCB detection.From the Laboratory’s point of view, the detection of the IgG oligoclonal bands (OCB) in CSF, which has been reported since the 1970s29. These B cell-related parameters have been reported to be increased in the CSF of MS patients and were proposed as quantitative standardizable diagnostic and/or prognostic biomarkers for MS. In particular, κ-FLC level in CSF seems to be a more sensitive marker of IS of IgG compared with OCBs30.Recently, standard assays to measure other B cell-related biomarkers levels in CSF (the two free light chains (FLC) kappa (κ) and Lambda (λ) and the chemokine CXCL13 responsible for B cell recruitment within the CNS) have been studied, and seem to improve the detection of intrathecal B cells activityWe aimed to determine the additional value of testing the new B cell-related markers in CSF (FLC metrics and CXCL13) for the detection of the intrathecal humoral response and MS diagnosis in order to optimize the Laboratory investigation in this context.During the 4 years study period (January 2018–December 2021), patients from the Department of Neurology of the Habib Bourguiba Hospital  who are suspected of having an inflammatory disorder of the CNS were included. Lumbar puncture was performed for a diagnostic purpose (no disease-modifying drugs or glucocorticosteroids at the time of lumbar puncture). Paired CSF and serum samples were collected from patients and addressed to the Laboratory of Immunology of Habib Bourguiba Hospital for Immunological investigation . Patients’ clinical and radiological records have been retrospectively consulted. The MacDonald 2017 revised criteria of MS were used for the disease diagnosis. This diagnosis was made within 2 weeks of the Lumbar puncture for all of the patients. The degree of neurological impairment in patients with MS was evaluated using the expanded disability status scale (EDSS) at the time of sampling.Our study population consisted of 160 patients with neurological disorders  (CPP SUD No 0407/2022).Written informed consent was obtained from all subjects and/or their legal guardian(s).®, France): an isoelectric focusing on agarose gel and an immunofixation with peroxidase-labeled anti-IgG antiserum. The Isofocusing test profile allows the comparison of IgG distribution in both fluids (IgG OCB detection). A positive pattern (OCB+) is defined by the presence of at least two additional OCB in CSF in comparison with serum. Measurement of total IgG and albumin levels in sera and CSF for the calculation of Tibbling et Link IgG index using the formula: [(IgG CSF/IgG serum)/(Albumin CSF/Albumin serum)] was performed using the automatic nephelometric method . These 2 routine tests are used in such context to detect an IS of IgG  in CSF in comparison with serum and/or a value of IgG index > 0.7). We used Reiber Diagram31 to check the blood–brain barrier status.In our routine practice, we receive couples of samples (CSF specimen from a lumbar puncture and venous blood sample) for immunological investigation of patients with suspicion of inflammatory disorders of the CNS. The collected specimens are centrifuged, aliquoted, and frozen at − 80 °C. Each patient’s serum and CSF samples were analyzed in parallel, at the time of diagnosis using a CSF Isofocusing test which includes 2 steps according to the manufacturer’s instructions . The lower detection limit for the CXCL13 ELISA is 4.0 pg/mL.The concentrations of κFLC and λFLC in CSF and serum were measured according to the same nephelometric method. In the case of undetectable FLC concentrations, the corresponding detection limit  was used as the level of the parameter. Regarding the CXCL13 level in CSF, it was determined using an immunoenzymatic technique (ELISA) (Euroimmun[CSF κFLC (mg/L)/serum κFLC (mg L)]/[CSF albumin (mg/L)/serum albumin (mg/L)] and [CSF λFLC (mg/L)/serum λFLC (mg/L)]/[CSF albumin (mg/L)/serum albumin (mg/L)], respectively.To analyze the different FLC metrics, different formulas were used. The κFLC index and λFLC index were calculated according to the following formulas:We also calculated the FLC ratio in CSF (CSF κFLC/CSF λFLC), the κIgG index [(CSF κFLC (mg/L)/serum κFLC (mg/L)]/[CSF IgG (mg L)/serum IgG (g L) × 100)] and the λIgG index [(CSF λFLC (mg L)/serum λFLC (mg L)/CSF IgG (mg L)/serum IgG (g L) × 100)].We evaluated the sensitivity of a test as [true positives/] and the specificity as [true negatives/]. The positive predictive value (PPV) was calculated as [true positives/] and the negative predictive value (NPV) as [true negatives/].The Youden index (or Youd’s J statistic) is defined as J = sensitivity + specificity − 1.http://www.albaum.it) for FLCκ calculates the κloc (mean) and κFLC inthrathecal fraction IF (mean) values for the data taken from Excel tables. The percentage FLC IF is calculated by comparing QFLC to a previously empirically determined, albumin quotient-dependent reference limit. A graphical result could also be generated for FLCκ using the software.In addition, the software “FLC-κ statistics and graphic program” . Roc curves and Box plots were generated using SPSS software.Statistical analysis was performed using SPSS 20.0 software. To analyze quantitative variables, we calculated means and SEM (standard error of the mean) and performed a comparison using Student-T test. In case of a skewed distribution from the normality, medians (1st Quartile–3rd Quartile) were calculated and compared using the Mann–Whitney U test. Correlations were studied using the Spearman test. A In this study, in addition to the conventional tests  Table , we inveIn this study, the mean value of κFLC (but not λFLC) level in CSF was significantly higher in the MS group than in the control group. When comparing all patients according to the OCB positivity, both types of FLC were significantly higher in the OCB-positive group than in the OCB-negative group.We also noticed that κFLC and λFLC were undetectable in CSF of 26/160 (16.25%) and 62/160 (38.75%) included patients, respectively. Among the 26 patients with an undetectable level of κFLC in CSF, only 4 were MS patients, and all of them were OCB negative. It is interesting to note that for the remaining 22 non-MS patients with an undetectable level of κFLC, 19 were OCB-negative, 2 OCB-positive, and 1 with one band in CSF.p < 0.001) (Table p = 0.052).The calculated mean value of the κ/λ ratio in CSF was significantly higher in the MS group than in the non-MS group  and in OCB-positive than in OCB-negative patients 9 vs 1.3; 1) Table . The dis.3; 1 TabRegarding serum results, levels of the 2 FLC were slightly higher in the non-MS group and OCB-negative patients.p < 0.001). The λ FLC-related parameters (λFLC index and λIgG index) were also higher in the MS group, with a significant difference for the λFLC index  was significantly higher in presence of OCB.The comparative distribution of the κ index and λ index according to the status  and the OCB detection  is shown in Fig. The mean level of κ index in OCB-negative MS patients was 16.6 ± 22.1 (1.75–88.2). This mean was 25.92 ± 29.8 in OCB-positive non-MS patients. The λFLC index and the ratio were also higher in the non-MS group with OCB. However, κIgG index and λIgG index were lower in OCB-positive non-MS patients when compared to the OCB-negative MS group.A ROC curve analysis was created with data from both MS and non-MS diagnosed patients. A very high performance of the κ-index for the discrimination of MS diagnosis from non-MS disorders was reflected by an area under the curve (AUC) of 0.910 Fig. A. We alsAccording to the ROC curves relative to the discrimination of MS diagnosis Fig. A,C,D, weUsing the software “FLC-κ statistics and graphic program” for Free Light Chains κ, we generated a graphical result for MS and non-MS patients Fig. A and we According to the ROC curve, diagnostic accuracy (AUC: 0.926) of κ-FLC IF was slightly higher than the κFLC index. For a cut-off of 54.9%, the sensitivity was as high as 92.3%, with a specificity of 75%. With a higher cut-off of 80%, the specificity reached 90.7%, with a sensitivity of 8.5%.In OCB-negative MS patients, 5/19 (26.3%) cases had a combination of high κ index (cut-off: 14.9) and high IgG index (cut-off: 0.7). This profile (double increased indexes) was not found in any OCB-negative non-MS patients (n = 51).In the second step of the study, the CXCL13 level was measured in the CSF of the study population (n = 160). To evaluate the comparative distribution in the presence and absence of MS diagnosis and an IS of IgG, Means, medians, and quartiles of CXCL13 levels in CSF have been calculated. The same comparison has been performed between different subgroups of MS patients Table .Table 4Qp = 0.029)  Table , Fig. 7AConsidering the total population, the level of this chemokine was significantly higher in patients with a detected intrathecal humoral immune response  or an IS of IgG (Reiber diagram)) than the corresponding negative group Table . The comIn the MS group, under clustering according to the previous parameters ), the statistical significance was found only between subgroups with high and low mean of IgG index. On the other hand, the CXCL13 level in CSF was not correlated with the clinical disability evaluated by the score EDSS in the MS group.Results of the correlation analysis of the studied B cell-related parameters  were summarized in Table 4. To reveal these IS of affinity-maturated IgG antibodies considered a hallmark of the disease, MS biological investigation is mainly based on the detection of IgG OCB by parallel CSF and serum samples testing using an isoelectric focusing technique, known as the gold standard for CSF assessment in this context32. Recently, OCB detection has been included in the revised criteria of McDonald 2017 for MS, which has permitted the substitution of the dissemination in time criterion.Currently, B cells are reported to be major contributors to the pathogenesis of MS, an inflammatory neurodegenerative disorder of the CNS characterized by an intrathecal humoral immune response33, the need for some expertise to perform the technique—a relatively laborious method, partially automatized—and the lack of standardization34 are some reported issues for the practice of the test and the validation of its results. In addition, OCBs detection is not specific enough for MS diagnosis35 and can be missed in a subgroup of authentic MS36. Taking all of this into account, an additional indicator may be needed to improve the biological diagnosis of MS. Recently, standard assays to measure other B cell-related biomarkers in CSF—the κ and λ FLC and the chemokine CXCL13 responsible for B cell recruitment within the CNS—have been studied and seem to improve the detection of the intrathecal B cells activity. In general, the detection of high levels of the polyclonal form of FLC was observed in many inflammatory and auto-immune disorders, which has led to testing their relevance as a biomarker of disease activity and as an attractive therapeutic target. Polyclonal FLC overproduction can occur when there is an excess production of Ig, as a consequence of chronic immune stimulation. During the inflammatory process, FLC seem to inhibit the apoptosis of inflammatory immune cells such as neutrophils and bind to a broad range of cell membranes (high affinity with monocytes).However, some limitations of OCBs detection are still reported in clinical practice. From the laboratory's point of view, the imprecise number of bands in the CSF defining positive results37. To the best of our knowledge, this is the first study interested in the diagnostic value of the recently described B cell-related parameters  in Tunisian and North African population. Such findings should be very helpful for MS clinicians treating diverse patient populations.Therefore, in this study, we aimed to optimize the diagnostic algorithm using routinely available and recently reported Laboratory tests to reveal the IS of IgG and to discriminate MS patients from non-MS patients with symptoms of central neurological disorders. Indeed, in our laboratory, we have started performing FLC and CXCL13 measurements within the routine CSF investigation for central neuroinflammation since January 2018. In the present study, we tried to compare the performance and the usefulness of different CSF parameters related to B cell activity  for the detection of IS and MS diagnosis, in a cohort of Tunisian patients. We also tested the κFLC data interpretation using the new hyperbolic reference range in quotient diagramsAs expected, our main findings revealed that the κFLC-related parameters, in particular the κFLC index and the intrathecal fraction κFLCIF, which consider serum FLC levels as well as BBB function, reached the highest accuracy in MS diagnosis and IS detection. Regarding CXCL13 measurement in CSF, it seems to be an effective parameter reflecting the inflammatory intrathecal B cell response and may be suggested to take part in the diagnostic but mainly the prognostic investigation in MS disease..29; where authors found that 160/252 (63.8%) patients had undetectable κ-FLC levels in CSF (turbidimetric technique), of whom 157 did not have MS. These results lead them to conclude that it is justified to obviate OCB testing in patients with suspicion of MS having undetectable κ-FLC levels in CSF unless there is strong clinical suspicion. Our findings comfort this proposed strategy, which should allow saving reagent costs and labor time for unnecessary OCB testing.When considering our global results of FLC measurement in CSF, 26/160 patients had an undetectable level of κFLC with our nephelometric technique, only 4 of them were diagnosed with MS and they were all OCB-negative MS patients. These findings are in line with those reported in the study of Sanz Diaz et al48. It has been described that after B cell activation, plasma cells secrete an excess of κ and λ FLC, in addition to intact Ig production. FLC do not appear to derive from the degradation of the whole IgG49. These parameters may therefore represent a potential quantitative tool to detect the IS of IgG and support MS diagnosis.When comparing our CSF findings relative to each type of FLC, and as expected, κ production in CSF appears to be a much more interesting quantitative parameter than λFLC; Indeed, κFLC is associated with the IS of IgG (OCB) and with MS disease, while λ level in CSF seems to be only related to the intrathecal activity of B Lymphocytes and not specifically to the disease. Indeed, several studies from different non-African continents have indicated that during MS, abnormal intrathecal production of these FLC occur, resulting in increased κ and, to a lesser extent, λ FLC levels in the CSF of patients50. Interestingly, the same study demonstrated that the high κ/λFLC ratio in CSF (good prediction of disability accumulation) seems to result from a selective expansion of κ light chain-expressing plasmablasts revealed within the CSF of MS patients and is not an epiphenomenon of FLC measurement. Other authors reported that the CSF FLC ratio may predict the conversion from CIS  to MS disease38.Another interesting result in our CSF findings is that the mean value of the κ/λ ratio in CSF was significantly higher in the MS group than in the non-MS group and in OCB-positive than in OCB-negative patients. In addition, our study revealed a trend for a significant correlation between the score of clinical disability (EDSS) and this ratio. In fact, it has been demonstrated that CSF κ/λ ratios, determined at the time of diagnostic lumbar puncture, may predict the progression of MS disease (EDSS at 5 years postdiagnosis) and thus may be useful as a prognostic biomarker for early therapeutic stratification51.Overall, κFLC metrics were more performant for MS diagnosis and detection of the IS than λFLC-related parameters. Results of the comparative sensitivity, specificity, PPV, and NPV of the κ and λFLC metrics for MS diagnosis in our Tunisian population confirmed the reported vast superiority of κFLC-related parameters described in other populations. In particular, the κFLC index and κFLC IF showed the best accuracy for MS diagnosis. The higher clinical relevance of κFLC in MS could be relatively explained by the fact that the κ chain is rearranged first during Ig production and is consequently quantitatively more dominant in the human body. In addition, λFLC are usually found in a dimeric form while κFLC are mostly monomeric52. It is therefore necessary to differentiate the locally synthesized intrathecal fraction of FLC from the blood-derived fraction. Two approaches to studying the intrathecal fraction of κFLC have been established and evaluated: the known linear approach  by calculating the FLC index (for both types of FLC) with reference to a determined cut-off, and a non-linear function for the κ chain  by coupling each QFLC (CSF FLC/serum FLC) to its corresponding Qalb dependent upper normal limit (Qlim FLC). In the Two approaches, Qalbis considered a well-established parameter for BBB status and function, and the inter-individual variability of serum FLC levels is taken into account52. Non-linear functions are in general considered more representative of the physiological conditions and appear to be superior in case of barrier dysfunction54. However, the linear IgG index formula is relatively simpler to perform in routine.On the other hand, FLC, as proteins detected in the CSF, originated in part from the blood by diffusion across the blood–brain barrier (BBB) but could also be produced within the CNS during pathological conditions, such as MS disease55. Evaluation of κFLC with this hyperbolic reference range in quotient diagrams seems to decrease the number of false-positive/-negative cases in the presence of low/high albumin quotients, which allows a most reliable interpretation of the data56. Other authors reported that both linear and non-linear approaches have a similar performance in patients with normal or only slightly elevated Qalb57 and that diagnostic accuracy does not differ between the κFLC index and κFLCIF60. Therefore, we can consider κFLC IF as a very promising parameter in the context of MS suspicion, having at least the same diagnostic accuracy as the κ index.In our study, and according to the ROC curves analysis, κ metrics were overall more accurate than the classical Total IgG index for MS diagnosis. In particular, κFLC IF (AUC: 0.926) accuracy was slightly higher than the κFLC index (AUC: 0.910) and than IgG index (AUC:0.874). This finding is in line with the results of previous studies where κFLC IF has been found to be the more accurate metric compared to κ index and conventional parameters61, which can reflect that the contribution of blood-derived FLC to total CSF FLC concentration is very low in MS cases with intrathecal synthesis. This observation has led some authors to use QFLC (without considering BBB function)64, or even the absolute CSF FLC concentrations65 for MS laboratory investigation.Another interesting finding in our study is the high percentage of the intrathecal fraction IF detected using the diagram in our MS patients (mean 86.87%). Indeed, it has been described that this intrathecal fraction of CSF FLC is greater than 80% in most MS patients67. Interestingly, both sensitivity and specificity of the κFLC index were higher than OCB detection (using the Isofocusing test) for MS diagnosis in our same Tunisian population 68. By focusing on our MS group, there were 6 cases of OCB-positive patients with a high κ index (> 14.9)/normal IgG index and no MS cases with the inverse profile. This could reflect, in the context of MS suspicion, the higher sensitivity and the more reliability of the κ index as a quantitative test for the detection of intrathecal antibody production. In the OCB-negative MS subgroup, 5/19 (26.3%) cases had a combination of high κ (cut-off: 14.9) index and IgG index. No OCB-negative non-MS patients (n = 51) showed this profile, which could reveal the usefulness of the combination of κ and IgG indexes in case of strong MS suspicion with a negative result in the OCB detection test.With a cut-off of 14.9, the κFLC index showed a sensitivity of 83%, a specificity of 91.7%, and a positive predictive value (PPV) of 91.8%. These values are within the correspondent range of values reported for this parameter in different populations69. MRZ reaction has been proposed as a very specific-second line test for CSF investigation, especially when OCB is negative or equivocal and/or when the clinical context is confusing. In the present comparative study, the κFLC index had similar specificity to the MRZ reaction with much better sensitivity.Regarding MRZ Reaction testing in our population, it was found as a very specific test (91.9%) with a very good PPV (93.9%) but a relatively moderate sensitivity (65%) in another previous study performed in our laboratoryAs mentioned above, the intrathecal fraction κ-FLC IF determined using the software “FLC-κ statistics and graphic program” for κFLC showed a slightly higher diagnostic accuracy than the κFLC index. For a cut-off of 54.9%, the sensitivity reached 92.3% (specificity: 75%). With a higher cut-off of 80%, the specificity was 90.7% (sensitivity: 80.6%).70. CSF concentrations of FLC and IgG are known to be increased in MS patients, but they have never been used in the same algorithm. Our findings showed that the κIgG index (and not the λIgG index) has a diagnostic accuracy (AUC: 0.890) close to that of the κFLC index (AUC: 0.910) and is significantly higher in patients groups with MS and with positive OCB in CSF. This new tested parameter also showed interesting sensitivity and specificity for MS diagnosis when considering a cut-off of 2.4. In the study of Gudowska-Sawczuk et al.70, authors reported a significant difference in κIgG index and λIgG index values between MS patients and controls having other neurological disorders, with a higher diagnostic significance for κIgG index compared with the λIgG index in this context. Regarding OCB, in the same study, κIgG appeared to be useful in pattern differentiation. This parameter was significantly higher in patients with type 2 patterns in comparison with those with patterns type 1 and 4. Only the values of this parameter were markedly higher in patients with pattern type 3 than patterns type 4 and 1. Taking into account all these data, this parameter could be considered a good candidate for further studies with a larger patient group size.In addition to κFLC index and κFLC IF, the concentrations of FLC and IgG have been evaluated in our study using the same formula for the diagnosis of MS (κIgG index and λIgG index), as an innovative approach which has been just described and testedB-Cell Attracting chemokine 1 (BCA-1) or B-Lymphocyte chemoattractant (BLC). This chemokine plays an important role in B cell recruitment within the CNS during inflammatory disorders. It has a crucial role in the development of secondary lymphoid nodules and the regulation of lymphocyte migration. In these conditions, CXCL13 is mainly expressed by monocytes/macrophages/microglia but can also be produced by follicular dendritic cells. The Receptor for this molecule (CXCR5/BLR1) is found mainly on B cells and T-cells (about 20% of CSF TCD4+) and monocytes- macrophages72.In the last part of our study, we analyzed another B cell-related parameter in CSF, which is the molecule CXCL13, also called 28 and NMO conditions73, while very high CSF levels have been described in MS mimics, such as CNS lymphoma, neuroborreliosis (where CXCL13 is described as a specific biomarker) and viral miningitis74. The CSF levels of CXCL13 in our MS group seem slightly higher than in other studies76. Indeed, in our study, most of the patients had a RRMS and all of them were in relapse at the time of sampling. It has been reported that during MS relapse, Levels of CXCL13 are higher and the positive correlations between these levels and other molecules that are essential for B-cell recruitment, survival, and maturation (B-cell activating factor (BAFF), IL-6, and IL-10) seem to be crucial to driving the B-cell response76. Regarding levels in the non-MS group, there is a big heterogeneity of findings since it depends on the type of other inflammatory or non-inflammatory diseases included, where production of CXCL13 in the CNS could vary greatly76.In our study, the CXCL13 level in CSF was significantly higher in the MS group in comparison with the non-MS group (mostly inflammatory conditions of CNS). In fact, several studies reported CXCL13 as a potential marker of inflammation in CSF and high levels of this molecule have been reported in CSF during MS 77.On the other hand, considering the total population of our study, CXCL13 was significantly higher in patients with a positive intrathecal humoral immune response  or an IS of IgG according to the Reiber diagram)) than the corresponding negative group. In addition, CSF levels of this chemokine were in positive correlation with CSF markers of intrathecal B cell activity, in particular, IgG index, κFLC metrics , and κ/λ ratio. These findings were consistent with the results of other studies where CXCL13 levels were demonstrated to correlate strongly with the IS of Ig  as well as the presence of B cells, plasmablasts and T cells (or the number of inflammatory cells)78. In addition, as a factor that attracts and maintains B and T cells in inflamed CNS lesions, both CXCL13 and its receptor CXCR5 have been suggested as therapeutic targets in MS72.Some authors also mentioned that in the MS group, patients with IS of IgG had higher CXCL13 levels than patients without IgG synthesis, which was observed in our study with a significant difference. Therefore, CXCL13 seems to be a marker of intrathecal inflammation rather than a specific parameter for MS diagnosis. It is an important mediator in the inflammatory cascade associated with the polyspecific intrathecal B cell response classically revealed by IgG index or OCB. This chemokine could therefore be a potential candidate biomarker to evaluate the efficiency of B-cell targeting therapy during MS. This hypothesis has been studied in Rituximab-treated MS patients, where it has been shown that CXCL13 levels in the CSF decrease significantly under treatment76. In our study, no correlation with EDSS results was found, which can be explained by the relatively small size of the MS group. In the study of Di Sano et al., the CXCL13 index was calculated, and this parameter was also reported to be an excellent candidate prognostic biomarker for predicting MS disease activity79.Regarding MS prognosis, much evidence demonstrated that CXCL13 could be considered an effective marker of severity and activity of the disease. This chemokine has shown an association with disease exacerbations and unfavorable prognosis in RRMS. A significant positive correlation between CXCL13 level in CSF and relapse rate, clinical disability (EDSS), and the number of MRI lesions have been described. In addition, a high level of this molecule was predictive of CIS conversion to MS75.Regarding patient stratification, the CXCL13 level in CSF could identify an MS subgroup with higher CSF leukocyte counts and early evidence of cortical thinning reflecting the disease severity80.In clinically and radiologically stable patients, it has been reported that a significant proportion still showed increased CSF levels of CXCL13 (in addition to neurofilament) indicating residual disease activity. Thus, this chemokine seems to be more sensitive than clinical and MRI features in revealing disease activityWe propose that CSF CXCL13 measurement, as an effective parameter reflecting the inflammatory intrathecal B cell response, can be part of the diagnostic but mainly the prognostic work-up in MS. It has been particularly reported as a helpful tool in future treatment decisions.81 where both biomarkers have been evaluated in CSF during rituximab treatment, however, this study included only 9 treated MS patients, and the statistical analysis was very limited with no relevant results regarding these 2 parameters. However, this study presents some limitations, such as its monocentric characteristic and the relatively small number of patients included.To the best of our knowledge, this is the first study interested in the diagnostic value of FLC in CSF and their related parameters in the Tunisian and North African populations. In addition, our study has interestingly tested concomitantly almost all the κ and λ FLC metrics described so far , in addition to the recently reported software “FLC-κ statistics and graphic program”. The performance of these parameters has been studied comparatively, and they have also been compared to the performance of routine tests  reported in our previous studies including the same populations. Furthermore, our study is one of the very rare publications interested in the concomitant evaluation of CXCL13 (responsible for B cell recruitment within the CNS) and FLC levels in CSF during MS. There is a single study69 and present findings and the above literature data, we suggest that:Initial measurement of automatized κ-FLC in CSF allows obviating OCB testing in case of undetectable levels in patients with suspicion of MS unless there is strong clinical suspicion.Complete Calculation of IgG index, κ-index values, and evaluation of FLC-κ Reibergram (κFLC IF) as the next automatized, relatively simple, rapid, and standardizable step of effective screening for IS detection and therefore, MS diagnosis to obtain the highest sensitivity and accuracy.This CSF investigation is followed by the CSF Isofocusing test (OCB detection) if available, with a combined interpretation of all previous parameters  and the clinical context.As an optional additional step, in remaining confusing cases, MRZ Reaction could be performed (highest specificity for MRZ Reaction with a good sensitivity when combined with OCB detection).CXCL13 measurement in CSF can take part in the armamentarium for the work-up in MS, especially for prognostic evaluation (good prognostic biomarker for MS activity and prediction of treatment response).Finally, we aimed to optimize the algorithm of routine CSF investigation for MS diagnosis and prognosis by adding the new effective, simple, available, and standardizable laboratory parameters. Based on our previousIn conclusion, this study highlights the usefulness and accuracy of CSF examination in the diagnosis of MS, by demonstrating the high performance of the newly described CSF B cell-related parameters  in this context, in addition to the well-established  routine laboratory tests.Overall, the determination of FLC metrics in CSF seems to have many advantages over the established CSF parameters detecting the IS of IgG. They appear to be valuable and supportive additional quantitative parameters in CSF investigation for MS diagnosis but may not replace the gold standard OCB detection. These clinically reliable parameters are technically simple, rapid, standardizable, and offer objective results. In particular, κ-related metrics are more sensitive than the other quantitative and qualitative tests for IS detection with good specificity. The calculation of κ index and the interpretation of κ-FLC data using a diagram with a hyperbolic reference range (considering the BBB status) showed the best accuracy for MS diagnosis. However, FLC-related parameters do not inform about the clonality of the IS, which is one of the advantages of OCB detection using the Isofocusing test. On the other hand, CXCL13 appears as an established mediator of the inflammatory cascade associated with the polyspecific B cell response and it has been reported to be promising in prognosis evaluation and prediction . A CSF testing combining conventional and new biological tools (quantitative standardizable tests) may certainly improve the performance of MS investigation.


The bidirectional pulsatile movement of cerebrospinal fluid (CSF), instead of the traditionally believed unidirectional and constant CSF circulation, has been demonstrated. In the present study, the structure and parameters of the CSF compartments were revisited in our comprehensive and validated central nervous system (CNS)-specific, physiologically based pharmacokinetic (PBPK) model of healthy rats (LeiCNS-PK3.0). The bidirectional and site-dependent CSF movement was incorporated into LeiCNS-PK3.0 to create the new LeiCNS-PK“3.1” model. The physiological CSF movement rates in healthy rats that are unavailable from the literature were estimated by fitting the PK data of sucrose, a CSF flow marker, after intra-CSF administration. The capability of LeiCNS-PK3.1 to describe the PK profiles of other molecules was compared with that of the original LeiCNS-PK3.0 model. LeiCNS-PK3.1 demonstrated superior description of the CSF PK profiles of a range of small molecules after intra-CSF administration over LeiCNS-PK3.0. LeiCNS-PK3.1 also retained the same level of predictability of CSF PK profiles in cisterna magna after intravenous administration. These results support the theory of bidirectional and site-dependent CSF movement across the entire CSF space over unidirectional and constant CSF circulation in healthy rats, pointing out the need to revisit the structures and parameters of CSF compartments in CNS-PBPK models. Cerebrospinal fluid (CSF) is a physiological medium filling the cerebral ventricles and subarachnoid space (SAS) of the brain and spinal cord. It functions to (a) protect the brain from mechanical damage by reducing its effective weight; (b) act as a drainage pathway for the removal or dilution of waste products by the “sink” effect ,2; (c) sHowever, in spite of their close contact with each other, CSF drug concentrations do not necessarily provide a reliable measure for predicting drug concentrations in brain extracellular fluid (ECF), a target site for most CNS drug candidates . To overCSF physiology, especially the direction and rate of CSF flow between CSF compartments, is crucially important in predicting CNS PK profiles using CNS-PBPK models, given that drug movement by diffusion is much slower than CSF flow . In the In order to address the need to revisit and scrutinize the structure and parameters of CSF compartments in the LeiCNS-PK3.0 model, the model’s capability to predict PK profiles in CSF compartments in healthy rats after intra-CSF (intracerebroventricular (ICV), intracisternal (IC) or intrathecal ) administration was checked. Next, the model’s structure and parameters were updated through adopting a theory of site-dependent, bidirectional CSF movement to more accurately describe the observed CSF PK profiles. The present study shows that a new model with bidirectional CSF movement can describe CSF PK profiles after intra-CSF administration far more accurately than the original model with unidirectional CSF flow.uu,ECF/CSF, the ratio of the unbound drug concentration in brain ECF or CSF to that in plasma at steady state, after IV administration, was either available from the literature or calculated using the area under the unbound concentration–time curve in ECF or CSF and plasma.A literature search on PK data after ICV, IC or IT administration was performed using the National Library of Medicine PubMed database with search queries such as “(intraventricular [tiab] OR intracerebroventricular [tiab] OR intraventricularly [tiab] OR intracerebroventricularly [tiab] OR ICV [tiab] OR i.c.v [tiab]) AND (pharmacokinetics [tiab] OR pharmacokinetic [tiab]) AND (rats [tiab] OR rat [tiab])”. Large molecules such as proteins and nucleic acids were excluded from the dataset for the present analysis as the reported correlation between the aqueous diffusivity and molecular weight (MW), according to which the LeiCNS-PK3.0 model calculates the aqueous diffusivity of molecules, has the upper limit of MW 5000 Da . In addiWebPlotDigitizer version 4.5  was usedPlasma PK models of unbound molecules were either available from the literature or developed using plasma PK data after IV administration with a nonlinear mixed-effects modeling approach, where one-, two- or three-compartment models were evaluated. Interindividual variability (IIV) was tested for acetaminophen PK data reported by de Lange et al.  using exa) and the strongest basic group (pKb) and lipophilicity (logPo/w), were collected from DrugBank version 5.1.9 sucrose reported by Okura et al. [CSF in 3H]sucrose is reported, was performed.In order to assess the possible improvement in the description of CSF PK profiles by adopting altered CSF flow rates, further simulations were performed, allowing the site-dependent CSF flow rates. In addition, application of the correction factor to paracellular permeability (PPA) across the BBB and BCSFB was considered based on the previous finding that the LeiCNS-PK3.0 model may overestimate PPA in some cases . PK dataa et al.  was selea et al. , not beia et al. , which sLV/TFV (via the BCSFB) compartments. This structure allows ICV-administered molecules to traverse the BCSFB and immediately enter the brain ECF across the BBB, which is unlikely from an anatomical point of view. Simulations actually showed initial spikes in brain ECF concentrations of acetaminophen and atenolol immediately after ICV administration (MV) compartment was separated from the whole brain MV compartment, as shown in MV were collected from the literature as follows; CP weight—4 mg/rat (250 g of body weight) sucrose in CM and SAS after ICV administration [14C]sucrose in CM and plasma after IC administration sucrose and [14C]inulin concentrations reported in the literature sucrose in CM and SAS were compared to the observed ones. At 60%, the elimination rate of [3H]sucrose from CM was largely overpredicted , was estimated to be faster than ventricular CSF movement . Interestingly, the U/D ratio was estimated to be higher than 1, indicating an upward  net CSF flow. As shown in 3H]sucrose in CM and SAS after ICV administration were quite reasonably described by the LeiCNS-PK3.1 model, especially their similar elimination rates, which were impossible to describe with the unidirectional CSF flow model, and those of [14C]sucrose in CM and plasma after IC administration. Furthermore, when three parameter values estimated using sucrose data were fixed as the drug-independent physiological CSF parameters, the PK profiles of inulin, another CSF flow marker molecule DTPA concentrations in the right LV after IT administration cannot be explained by its re-entry to CSF across the BCSFB after first being absorbed into systemic circulation given the poor BCSFB penetration of DTPA-chelates [99mTc]DTPA against the traditional CSF flow direction.Importantly, bidirectional pulsatile CSF movement allows substances in the CSF to mix and spread in both directions. This is clearly demonstrated in the study reported by Smith et al. , where 1chelates ; thus, i3H]sucrose in CM and SAS after ICV administration, especially their similar elimination rates, as well as that of [14C]sucrose in CM after IC administration, in comparison with the original LeiCNS-PK3.0 model, which had unidirectional and constant CSF flow  was estimated to be four times higher than that from LV to TFV and from TFV to CM   . This orSecond, the U/D ratio was estimated to be higher than 1, indicating an upward  net CSF flow throughout all CSF compartments. Although this is difficult to intuitively understand from the viewpoint of the classical CSF physiology, some clinical results support this finding. Reported U/D ratio of CSF movement per heartbeat at the cerebral aqueduct was around 1.4 , close tImportantly, the LeiCNS-PK3.1 model showed the same level of predictability of CM PK profiles after IV administration as the original LeiCNS-PK3.0 model . Given t3H]sucrose in lumbar CSF after ICV administration reported by Okura et al.  ca ca22] caFurthermore, overestimation of PPA based on molecular aqueous diffusivity was indicated for the BCSFB, as with the previous study for the BBB . This isCSF), which is treated as a drug-independent passive CL in other CNS-PBPK models, with the exception of applying the lymph reflection coefficient for antibodies [CSF is currently impossible to predict from physicochemical properties, in vitro transporter studies or in vivo Kpuu,ECF/CSF values, and can be estimated only with in vivo PK profiles and model fitting.As far as we know, this is the first modeling study using a CNS-PBPK model to show the drug-dependent transfer CL from SAS to the systemic circulation (sasQtibodies . This istibodies ,64,65,66tibodies ,67. HencCSF of GSA was estimated to be below 0 (CSF value. Accordingly, additional datapoints are necessary to estimate this value with assurance.When estimating using in vivo data, the selection of timepoints is important. For example, the sasQ below 0 , indicatThis study pointed out the importance of considering the bidirectional pulsatile CSF movement whose rate is not necessarily balanced with the traditional CSF-formation rate, instead of classical unidirectional and constant CSF flow, in the CSF compartments of CNS-PBPK models. Given the relatively rich data on pulsatile CSF movement and imaging data with various contrast agents, most of which are considered to have characteristics as CSF flow markers, in clinical situations, similar analyses of human CSF compartments will be a significant and meaningful subject of future research.The LeiCNS-PK3.1 model, with bidirectional and site-dependent CSF movement, demonstrated superior performance in describing the PK profiles of small molecules after intra-CSF administration in comparison with the original LeiCNS-PK3.0 model, with unidirectional and constant CSF flow. Given the clearly demonstrated bidirectional movement of CSF in humans and the new concept of CSF physiology, the structures and parameters of the CSF compartments in CNS-PBPK models need to be revisited.