Akkermansia muciniphila, Atopobium vaginae, Bacteroides fragilis, Lactobacillus paracasei, Odoribacter splanchnicus, Pseudomonas aeruginosa, Cutibacterium (Propionibacterium) acnes, Fusobacterium necrophorum, Porphyromonas gingivalis, and Streptococcus mutans. CSF responses from patients with demyelinating diseases  were compared to those with other neurological diseases  and controls (N = 13). Commercial positive and negative control CSF specimens were run with each assay. ELISA index values were derived for each specimen against each of the 10 bacterial lysates. CSF reactivity was significantly higher in the DD group compared to the controls against Akkermansia, Atopobium, Bacteroides, Lactobacillus, Odoribacter, and Fusobacterium. Four of the 11 tested DD group subjects had elevated antibody indexes against at least one of the 10 bacterial species, suggesting intrathecal antibody production. This CSF serological study supports the hypothesis that several of the previously identified MS candidate microbes contribute to demyelination in some patients.A panel of 10 IgG enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of anti-microbial immune responses in the cerebrospinal fluid (CSF) of patients with demyelinating diseases (DD). The anti-microbial ELISA assays follow on prior human brain tissue RNA sequencing studies that established multiple sclerosis (MS) microbial candidates. Lysates included in the ELISA panel were derived from A panel of 10 IgG enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of anti-microbial immune responses in the cerebrospinal fluid (CSF) of patients with demyelinating diseases, including multiple sclerosis and acute disseminated encephalomyelitis.CSF reactivity was significantly higher in the demyelination group compared to the controls against the bacteria Akkermansia, Atopobium, Bacteroides, Lactobacillus, Odoribacter, and Fusobacterium.Several of the demyelination subjects had elevated antibody indexes against at least one of the 10 antigens, suggesting at least limited intrathecal production of anti-bacterial antibodies.This CSF serological study supports the hypothesis that several of the previously identified MS candidate microbes contribute to demyelination in some patients.The online version contains supplementary material available at 10.1007/s00109-021-02085-z. A number of infections of the central nervous system (CNS) can lead to demyelination, including distemper (dogs), measles (humans), JC virus (human), influenza (humans), and now possibly SARS-CoV-2 , 2. The Microbes, particularly herpes viruses such as EBV and HHV6, have long been suspected as causative agents of the prototypic demyelinating disease multiple sclerosis (MS), based on the epidemiology of the disease including geographic patterns, isolated outbreaks, and migration studies , 4. HoweWe recently utilized next-generation unbiased RNA sequencing of demyelinated human brain samples from living subjects as a method for defining possible microbial contributors to MS and related demyelinating diseases  . RNA seqCollectively, these findings suggest that both exogenous bacterial pathogens and commensal species could directly or indirectly contribute to the development and progression of demyelinating disease. This study was designed to determine if some MS candidate microbes, discovered by sequencing of brain tissue, are also eliciting an immune response in the CSF of patients with demyelinating diseases, including MS. Ten organisms were selected for further study. Bacterial lysates were used to develop a panel of 10 novel dedicated ELISA assays. The ELISA panel was applied to CSF from patients with demyelinating disease (DD), other neurologic disease (OND), and controls. The data demonstrates significantly higher CSF reactivity among DD subjects against several of the panel components compared to controls.The study was approved by the University of Utah Health Sciences Institutional Review Board (IRB). Each subject either signed an IRB-approved informed consent or had a legally authorized representative sign for them. CSF was obtained from 14 patients with MS or other demyelinating diseases (DD) during the course of their regular clinical care. CSF was also collected from 8 patients with other neurologic diseases (OND) and 13 controls. Most of the OND subjects were enrolled in the study for suspicion of demyelinating disease, but the final neurologic diagnosis was something else . The controls were patients undergoing spinal fluid shunt placements or exchanges who did not have active infection at the time of collection. These control subjects did not have clinical tests performed on their CSF samples, but were expected to have normal CSF based on their stable clinical condition. Paired serum samples taken around the time of CSF collections were obtained from 11 DD and 8 OND subjects. Serum was not collected on the control cohort. Oligoclonal band OCB testing and albumin index determinations were performed (ARUP Laboratories) as part of regular clinical care in some of the DD and OND subjects. CSF and serum IgG concentrations were determined by nephelometry, also at ARUP laboratories.Akkermansia muciniphila, Atopobium vaginae, Bacteroides fragilis, Cutibacterium (Propionibacterium) acnes, Lactobacillus paracasei, Odoribacter splanchnicus, Porphyromonas gingivalis, Pseudomonas aeruginosa, Streptococcus mutans, and Fusobacterium necrophorum.Selection of microbial candidates was guided by the MS microbial candidate list . MS cand8 bacterial cells per milliliter and 1.93 × 107 bacterial cells per well in a flat-bottom 96-well plate (Corning Life Sciences Product 9018). Plates were read on a Gen5 2.08 ELISA plate reader at 600 nm. Cells were sonicated for 10 s with cycle time continuous, a duty cycle of 40%, and at position 4.5 with a Branson Sonifier 250. The sonicated bacterial lysates were aliquoted and stored at -80 °C for future use.Bacterial strains were obtained from ATCC as freeze-dried, lyophilized cultures. Cultures were rehydrated using the appropriate medium and incubation conditions specified on the ATCC product sheets. Organisms were allowed to come out of the lyophilized state, and once within the exponential phase of growth, stock cultures were stored at -80 °C in 10% glycerol. The cultured bacteria were harvested during the optimal growth phase as recommended by ATCC. Bacterial cells from broth cultures were washed in PBS at least twice before proceeding. Bacteria obtained from both plate and broth cultures were diluted in PBS to obtain a final OD of 1.0, equivalent to 3.9 × 10Flat-bottom 96-well ELISA plates were coated with 50 μl/well of harvested lysate (sonicated bacteria), covered, and rocked overnight at 4 °C. The plates were washed with phosphate buffered saline with 0.1% Tween 20 (PBST), blocked with Starting Blocker for 1 h at room temperature, and washed again before applying the appropriate primary antibody for 2 h. Secondary antibodies were applied for 1 h. The plates were developed with TMB for 5 min and stopped with 2 N HCL. Absorbance was read on a Gen5 2.08 ELISA plate reader at 450 nm.A series of steps were taken to optimize the 10 separate ELISA assays. Four commercial blocking agents were assessed for blocking efficiency including 5X ELISA/ELISPOT Diluent (Invitrogen), Blocker BLOTTO in Tris-buffered saline (ThermoScientific), Blocker Casein (ThermoScientific), and Starting Blocker (ThermoScientific). Starting Blocker proved to be optimal for most of the assays and was adopted for use with all 10 bacterial species. Three primary anti-bacterial antibodies were assessed as positive controls for assay performance validation: mouse monoclonal IgG3 anti-peptidoglycan , mouse monoclonal IgG2b anti-LPS , and rabbit polyclonal anti-pseudomonas . These positive control primary Abs were titrated to yield strong and weak (just above the background) positive signals. The polyclonal anti-Pseudomonas Ab PA1-73116 proved to be the best primary positive control antibody against all the 10 bacteria tested. This positive control Ab was used at dilutions between 1:200 and 1:6400 as strong (OD ~ 1.0) and between 1:400 and 1:102,400 as weak (OD > background) positive controls for each of the 10 assays. Secondary antibodies used in the study included anti-mouse IgG , anti-rabbit IgG , and anti-human . The antibodies and dilutions used for the study are detailed in Table Commercial human CSF  was used as a positive control. IgG-depleted commercial human CSF  was utilized as a negative  control. The “cut-off calibrator” method was chosen for each of the assays  8, 9]. , 9. 8, 9CSF:EIserum ratio or antibody index (AI). An AI greater than one shows excess Ab in the CSF compared to serum, suggesting that intrathecal Ab synthesis has taken place . We. We16]. Based on the commercial polyclonal and monoclonal positive control antibodies employed, the novel ELISAs demonstrated good specificity and sensitivity. Randox® CSF was chosen as a reference for patient CSF because it is commercially available and relatively well characterized. This commercial CSF preparation containing 10 mg/dl IgG was used as a positive control for each of the assays. Randox® CSF was also IgG-depleted for use as the negative or calibration control in each of the assays –9. This Pseudomonas aeruginosa antibody used as a positive control in all the assays. Cross-reactivity between Lyme and syphilis testing is well recognized among clinicians who employ these anti-bacterial serologic tests in clinical practice [Borrelia burgdorferi was not included as a candidate bacterium in this study, but clinical diagnostic testing showed clear intrathecal production of anti-B. burgdorferi antibodies. This patient did have an elevated albumin index suggesting a compromised BBB, so some anti-bacterial antibodies could have leaked into the CNS from the serum. Cross-reactivity could also be due to the commonly shared features of bacterial organisms such as the lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN).Within these ELISA assays, we suspect that there is considerable cross-reactivity, similar to that seen with the commercial polyclonal anti-practice . The resWe argue that the data presented here supports a pathogenic role for several of the organisms in some of the subjects. That is, there is evidence of intrathecal antibody production against several of the MS candidate microbes in several of the subjects. Specifically, there were AI’s > 1.0, evidence for intrathecal antibacterial antibody synthesis, in DD subjects 19, 80, 82, and 83. Among these, subjects DD-19, DD-82, and DD-83 were relatively young women at the time of diagnosis, the most common demographic for the development of MS and related diseases. Comparisons of EI’s with the albumin index analysis Fig.  suggesteLactobacillus paracasei among all the subjects tested. However, the AI against lactobacillus (0.86) was not elevated in this subject, arguing against intrathecal antibody production. However, intrathecal antibody production is notoriously difficult to prove in individual subjects, methods vary, and a patient with compatible clinical findings should be considered to have the disease being considered, with or without elevated AI [L. paracasei, encountered from prolonged exposure to a newborn calf.Testing of CSF from subject DD-03, a relatively young Idaho rancher, proved to be uniquely interesting. This subject and his family have been outspoken about his disease, both in the local media and as the subject of an ESPN feature video . A possivated AI , 23. In OCBs are a common clinical diagnostic feature of MS. For the DD cases, CSF was taken about a week after DD-03 s onset of neurologic symptoms, but DD-017, DD-019, and DD-021 had their CSF taken years after disease onset without any intervening attacks. DD-082 had her CSF collected only a few days after onset of disease, so it is possible that OCBs did not have time to develop. Additional CSF collections were not feasible in our population because, in general, the treating neurologists did not believe that they were necessary or helpful for clinical care. It would be ideal to collect CSF at the time of attack, again a few months later, and around the time of any MS relapses, to assess if the antibacterial CSF responses become more focused over time.Previously, one of the current authors (J.D.L.) demonstrated anti-peptidoglycan antibodies in CSF collected from patients with active MS . This grCampylobacter jejuni antibodies [C. jejuni infection [C. jejuni contains polysaccharides that resemble glycoconjugates of the human nerve tissues resulting in misguided attack on myelin and axons. Certain types of this disorder involve an immune response against gangliosides, which is suspected to originate due to molecular mimicry between gangliosides and lipopolysaccharides of C. jejuni [Similarly, for Guillain-Barré syndrome (GBS), serological studies demonstrated that most GBS patients who had anti-GBS antibodies also had anti-tibodies . GBS is nfection –32. The . jejuni .Akkermansia muciniphila was enriched in the stool of MS patients when compared to healthy controls and, when using a murine model for MS, altering the gut microbiome modulates CNS autoimmunity. A French group recently demonstrated that anti-Akkermansia muciniphila immunoglobulin G (IgG) was increased in CSF from people with MS compared to controls, congruent with our findings [Fusobacterium necrophorum or anti-Bacteroides fragilis antibodies in their CSF samples, while we did show an elevation in EIs in the DD group against these bacterial lysates.Due to the importance of the gut microbiome and immune function, Janji et al. assessed the gut microbiome within MS patients . They defindings . They alIn conclusion, the current study demonstrates that antibacterial antibodies can be detected in the CSF of subjects with demyelinating disease and that CSF reactivity in the DD and OND groups exceeds reactivity in the controls for several bacterial species. This supports, but does not prove, the hypothesis that microbes contribute to demyelination in persons with MS and related diseases.ESM 1(DOCX 385 kb)ESM 2(DOCX 18 kb)ESM 3(DOCX 21 kb)ESM 4(DOCX 23 kb)ESM 5(DOCX 26 kb)ESM 6(DOCX 21 kb)


CSF-restricted OCBs positivity dropped from 94.6% to 86% but 26 patients (28%) had an increased number of OCBs at the follow-up. The baseline to follow-up EDSS and T2-LV were stable; a decrease in the relapse rate was consistent with a decrease in the CSF inflammatory markers and previous knowledge about the effectiveness of natalizumab. The average annualized brain volume loss during the follow-up was −0.50%  and was predicted by the baseline IgM index . Conclusions: Natalizumab is associated with a reduction of basic CSF inflammatory measures supporting its strong anti-inflammatory properties. The IgM index at the baseline predicted future brain volume loss during the course of natalizumab treatment.Background: There is a lack of knowledge about the evolution of cerebrospinal fluid (CSF) markers in multiple sclerosis (MS) patients undergoing natalizumab treatment. Aim: We aimed to evaluate the effect of natalizumab on basic inflammatory CSF and MRI measures. Methods: Together, 411 patients were screened for eligibility and 93 subjects with ≥2 CSF examinations ≤6 months before and ≥12 months after natalizumab initiation were recruited. The effect of natalizumab on CSF as well as clinical and paraclinical measures was analyzed using adjusted mixed models. Results: Natalizumab induced a decrease in CSF leukocytes ( Multiple sclerosis (MS) is a chronic, demyelinating autoimmune disease. It is multifactorial in nature due to an interaction between environmental factors and an underlying genetic susceptibility. The most common clinical course of MS is relapsing-remitting MS (RRMS). There have been great advances in the treatment of RRMS after the discovery of monoclonal antibodies and other disease modifying therapies (DMT). Among them, natalizumab (NTZ) is a humanized recombinant antibody against α4-integrin and is highly effective in reducing disability progression, relapses and radiological disease activity in RRMS ,2,3,4. IThe binding of NTZ is more marked for B cells than for T cells, leading to a disproportionate increase in B cells in the blood following treatment . Blood mConsidering that only a few small sample size studies have investigated the effects of NTZ on CSF measures, long-term longitudinal studies are needed to better understand the dynamics of the anti-inflammatory effect of NTZ in MS. Additionally, the association between NTZ-induced changes in the CSF and radiological disease activity on MRI has not been studied in detail. We hypothesized a strong effect of NTZ on the reduction of basic inflammatory CSF measures in RRMS patients. Conversely, the null hypothesis of the study was that there was no statistically significant relationship between NTZ treatment initiation and the reduction of inflammatory CSF measures.In this longitudinal retrospective study, we included relapsing-remitting MS patients treated with NTZ at the General University Hospital in Prague, Czech Republic. The inclusion criteria were as follows: a diagnosis of MS, age > 18 years, NTZ treatment, an assessment of the baseline CSF < 6 months before NTZ initiation, a follow-up of the CSF performed during NTZ treatment < 24 months after NTZ initiation and a follow-up of the CSF performed < 6 weeks after NTZ discontinuation. We excluded patients who had received high dose steroids < 30 days before the CSF assessment, had a relapse activity < 30 days before the CSF, were pregnant or had blood contamination of the CSF (number of erythrocytes > 50/mcL)  23]. Th. Th23]. ®, Biogen-Idec, Cambridge, MA, USA) during the first year and every 4–6 weeks afterwards. Relapses during the study were treated with 3–6 g of methylprednisolone.The study included clinical visits every 3–6 months during the follow-up in routine clinical practice. All patients started the treatment at the baseline with 300 mg of intravenous NTZ every 4 weeks . The MRI scans were performed at least 30 days after intravenous high dose steroids and not more than 120 days after or before their respective spinal tap . Axial bAll lumbar punctures were performed at the General University Hospital in Prague, Czech Republic. The mean time between the baseline CSF and NTZ initiation was 7.7 weeks . The mean time between the follow-up CSF and NTZ initiation was 151.4 weeks . A total of 24 patients had a follow-up CSF after NTZ discontinuation (mean 4.4 weeks). For the biochemical assessments, the CSF was drawn from the L5–S1, L4–5 or L3–4 interspace in an upright sitting or lying position in the morning hours using a standard sterile preparation and a 20G Sprotte atraumatic needle. A total of 10–15 mL of CSF and a 5 mL volume of blood were obtained. The total protein in the CSF was determined photometrically on a Beckman Coulter analyzer (Synchron LX 20) with a reaction with pyrogallol redmolybdate. Albumin, immunoglobulin G (IgG) and immunoglobulin M (IgM) concentrations were quantified in serum and the CSF by immunonephelometry on a Beckman Coulter analyzer (IMMAGE). The QAlb was defined as the ratio of the albumin concentration in the CSF divided by the albumin concentration in the blood serum /albumin in serum (mg/L))*1000). The identification of OCBs was performed by isoelectric focusing (IEF) with an ultra-sensitive immunofixation on Sebia (Hydrasys Focusing). All CSF examinations were analyzed in the same laboratory with identical procedures.http://www.R-project.org (accessed on 26 November 2021) and SPSS 22.0 . The normality of the distribution was assessed using the Kolmogorov–Smirnov method and a visual inspection of the histograms. Non-normally distributed variables were (log x + 1) transformed. The changes of brain MRI volumetric measures were annualized to adjust for different times between the MRI timepoints.All analyses were performed using R statistical software available online: The associations among the CSF and other clinical and paraclinical measures were analyzed using Spearman’s correlation. Considering repeated measurements on a single patient, the MRI measures at different timepoints within patients were compared using linear mixed models   with a fp-value < 0.05 was considered to be statistically significant, indicating that there was less than a 5% probability that the null hypothesis was correct. The Benjamini–Hochberg (BH) procedure with p < 0.05 was used to control the false discovery rate (FDR) when conducting multiple comparisons (the number in (brackets) refers to the number of comparisons). The p-values from the multivariate models were not corrected for the FDR.A We analyzed 186 paired CSF measurements from 93 RRMS patients. The average time between the baseline and follow-up spinal taps was 3.1 years . The majority of patients were treated with low efficacy DMT before NTZ initiation . p = 0.008), a shorter disease duration , a lower EDSS  and a higher BPF  had a higher number of CSF leukocytes (p = 0.027), a lower EDSS (p = 0.041) and a higher BPF (p = 0.028) were independent correlates of a higher number of CSF leukocytes at the baseline.Patients with a younger age , CSF protein levels (0.34 vs. 0.31 mg/L) and albumin quotients (4.76 vs. 4.34). In addition, NTZ also decreased the IgG and IgM quotients and indices and decreased the number of CSF-restricted OCBs  .In 9 (9.7%) patients, the OCBs disappeared (the positivity dropped from 94.6% to 86%). These patients had a lower IgG index at the baseline and after the follow-up; they did not differ in clinical or radiological disease activity at the baseline or over the follow-up compared with the other patients. On the other hand, in 26 (28%) patients, the number of OCBs increased over the follow-up. However, patients with an increase in OCBs did not differ in the basic characteristics in comparison with those with a stable or decreased number of OCBs at the follow-up (data not shown).p = 0.032) and a longer time on the NTZ treatment  were associated with a higher decrease of an NTZ-induced reduction of the albumin quotient. Otherwise, only the baseline levels of different CSF measures were associated with an extent of the decrease of CSF inflammatory measures during the NTZ treatment. Other variables such as age, disease duration, EDSS or MRI indices were not associated with changes of CSF measures following NTZ initiation  and BPF (n = 64) between the baseline and the follow-up   = 0.005) but not by the baseline IgG . In other words, a 10% higher level of the IgM index at the baseline was associated with a 0.046 greater annualized brain volume loss over time  = 0.10). The predictive values of the IgG and IgM indices also remained significant in the multivariable models adjusted for the baseline CSF leukocytes. An annualized change of T2-LV over the follow-up was not predicted by any baseline CSF measures (We found a reduction in the relapsing activity and stable EDSS over the follow-up . We did ctively) . The avever time . The premeasures .In this study, NTZ induced a reduction of a wide spectrum of basic CSF inflammatory measures such as the number of CSF leukocytes, CSF proteins, albumin quotients and IgG and IgM quotients and indices. Overall, NTZ led to the normalization of CSF measures in the majority of patients and, hence, the most obvious treatment effect on the biochemical parameters was observed in patients with the highest degree of inflammation before NTZ treatment initiation. Indeed, patients with a younger age, a shorter disease duration, a lower EDSS and a higher BPF had a higher number of CSF leukocytes at the baseline. This was consistent with recent research showing a negative correlation between a younger age and higher CSF leukocytes  but alson = 73) [n = 33 before and 49 after NTZ treatment) [n = 15) [n = 24) [Although the number of OCBs reduced after NTZ treatment, OCBs disappeared completely only in 9 patients (9.7%) and OCBs positivity dropped from 94.6% to 86.0%. This was consistent with previous studies reporting a decrease in OCB positivity from a 100% positivity at the baseline to an 84% positivity at the follow-up (n = 73) , from aneatment)  and a 93[n = 24) . Interes[n = 24) ,33. PatiSupporting previous research, we also found that NTZ treatment was also associated with relatively stable clinical (EDSS) and radiological (T2-LV and BPF) measures over the follow-up ,2,3,4,34Taken together, our results were in agreement with previous research showing the strong anti-inflammatory properties of NTZ. Given that NTZ targets mostly short-lived B cells (plasmablasts) rather than long-lived plasma cells , NTZ trAlthough NTZ is a highly effective and safe DMT, the risk of progressive multifocal leukoencephalopathy (PML) in human polyomavirus JC virus (JCV)-positive patients represents the most important drawback responsible for not initiating or discontinuing NTZ in a great proportion of patients . Hence,In conclusion, we demonstrated that NTZ treatment induced a significant reduction of a spectrum of CSF inflammatory measures and the stabilization of radiological disease activity. The anti-inflammatory effect of NTZ was most evident in younger patients and in those at the early stage of the disease who had the highest inflammatory activity. Our findings emphasize the need for effective treatment strategies in early disease stages when inflammatory disease activity tends to be the highest.


Reports on typical routine cerebrospinal fluid (CSF) findings are outdated owing to novel reference limits (RL) and revised diagnostic criteria of Multiple Sclerosis (MS).To assess routine CSF parameters in MS patients and the frequency of pathologic findings by applying novel RL.alb), intrathecal synthesis of immunoglobulins (Ig) A, M and G, oligoclonal IgG bands (OCB) were determined in patients with clinically isolated syndrome (CIS) and MS.CSF white blood cells (WBC), CSF total protein (CSF-TP), CSF/serum albumin quotient (Qalb were increased in 19% and 7% applying age-corrected RL as opposed to 34% and 26% with conventional RL. Quantitative intrathecal IgG, IgA and IgM synthesis were present in 65%, 14% and 21%; OCB in 95% of patients. WBC were higher in relapsing than progressive MS and predicted, together with monocytes, the conversion from CIS to clinically definite MS. Intrathecal IgG fraction was highest in secondary progressive MS.Of 541 patients 54% showed CSF pleocytosis with a WBC count up to 40/μl. CSF cytology revealed lymphocytes, monocytes and neutrophils in 99%, 41% and 9% of patients. CSF-TP and QCSF profile in MS varies across disease courses. Blood-CSF-barrier dysfunction and intrathecal IgA/IgM synthesis are less frequent when the novel RL are applied. Evidencynthesis .alb as indicator for the blood-CSF-barrier function, were based on reference limits that are outdated nowadays  and to assess the frequency of pathological findings by applying novel reference limits (RL).The objective of the present study was to describe routine CSF parameters in patients with MS, to assess differences with regard to the disease course  or MS according to the 2017 revised McDonald criteria  (n=88), Patients were categorized into following groups: CIS, McDonald MS, relapsing-remitting MS (RRMS), secondary-progressive MS (SPMS) and primary progressive MS (PPMS) applying the 2017 revised McDonald criteria , both atFurthermore, patients with a first clinical attack at baseline were stratified based on their initial MRI findings into following groups: only DIS, only DIT, DIS and DIT, no DIS and no DIT. The definition of DIS was fulfilled by one or more T2-hyperintense lesions characteristic of MS in two or more of four areas of the CNS: periventricular, cortical or juxtacortical, infratentorial brain regions, and the spinal cord. DIT was demonstrated by the simultaneous presence of gadolinium-enhancing and non-enhancing lesions .A subgroup of these patients, who were followed for at least one year, were eligible for stratification according to the occurrence of further relapse. MRI findings were analysed by two blinded independent investigators.CSF samples were collected by standard LP for routine diagnostic purposes. Blood samples were withdrawn simultaneously by peripheral venous puncture. Serum was isolated from blood by centrifugation after the blood samples were allowed to clot for ≥30 minutes.The routine parameters comprised CSF RBC count, CSF WBC count, CSF cellular differentiation, CSF total protein (CSF-TP) as well as albumin, IgG, IgA and IgM and oligoclonal IgG bands (OCB) in CSF and serum.CSF WBC and RBC were counted in a Fuchs-Rosenthal chamber, which has a volume of 3.2 μL . DivisioCSF-TP was determined by spectrophotometry after incubation of centrifuged CSF with 3% trichloroacetic acid . Albuminalb) as an established marker of the blood-CSF-barrier function was calculated as the ratio of CSF albumin/serum albumin   , Eq. (1)t (cURL) , Eq. (2)Frequency of increased CSF-TP concentrations was assessed using the cURL of 0.45 g/L , Eq. (3)Intrathecal synthesis of IgG, IgM and IgA were determined by the Reiber formula , Eq. (5)a, b, and c have to be applied depending on the Ig isotype. The previously determined constants for the Reiber formulae Q has been calculated, the relative intrathecal Ig fraction (IF) is calculated according to following formula:After th-95th percentile, as appropriate. Categorical variables were reported as frequency and percentage. Spearman coefficient was used for correlation analysis. For group comparisons, Mann-Whitney U, Kruskal-Wallis test and Pearson Chi-squared test were applied. Multivariate regression analyses were performed including age, sex, disease duration and – depending on the precise research question – further covariables that differed statistically significantly between the respective patient groups by univariate comparisons, e.g. disease course. P-values <0.05 were considered statistically significant. P values marked as p* were corrected for multiple comparisons by Bonferroni.Statistical analysis was performed by SPSS 26.0 . Distribution of data was assessed by Kolmogorov-Smirnov test. Non-parametric data were displayed as median and interquartile range (IQR) or 5www.ris.bka.gv.at., 2018) by anonymizing patients’ data. In accordance with Austria’s national regulations for retrospective analyses of already existing data obtained for routine diagnostic procedures, no ethical committee’s vote is needed.We adhered to the guidelines of the declaration of Helsinki as well as the instructions of the Austrian Data Safety Authority  after sample withdrawal. Eighty patients had the diagnosis CIS, 240 McDonald MS, 157 RRMS, 19 SPMS and 45 PPMS at the time of LP. Of 320 patients with a first clinical attack, 75 (23%) showed DIS only, 21 (7%) DIT only and 168 (53%) both DIS and DIT on brain MRI. A subgroup of 103 patients, other than those fulfilling DIS and DIT on MRI, were followed for median 9 years (IQR 5-12) for the occurrence of a second clinical attack. Details on demographics and main clinical characteristics of patients are shown in th percentile); the maximum WBC count was 65/μl. Visual inspection of CSF cytology identified cells in 403 (75%) of cases. Almost all patients (99%) showed lymphocytes, 41% monocytes and 9% neutrophils, while eosinophils and basophils were not detected  of 541 patients showed an elevated WBC count ≥5/μl. In those patients with CSF pleocytosis, WBC count had a median of 10/μl reaching an upper limit of 40/μl . All these findings were confirmed in multivariate linear regression analysis adjusting for sex, age, disease duration and disease course  as compared to progressive courses (SPMS and PPMS) Table S4In patients with LP at first clinical attack, elevation of WBC count  as well as the presence of monocytes/macrophages in CSF cytology  were predictive for the occurrence of a second clinical attack during follow-up  and was increased in 37 (7%) patients using the aURL as opposed to 138 (26%) using the cURL. CSF total protein was determined in 540 patients, showed a median of 390 mg/L (95th percentile: 680 mg/L) and was elevated in 103 (19%) patients using the aURL compared to 185 (34%) patients applying the cURL . In multivariate linear regression analysis, the effect of sex and age was confirmed, while disease course had no impact on Qalb levels  of 527 patients using the Auer & Hegen formula and in 357 (68%) applying the Reiber formula. The IgG index was elevated (>0.7) in 362 (69%) of patients Table 1BIntrathecal fraction of IgG significantly differed among the various disease courses with the lowest values in patients with CIS and the highest in SPMS Table S8Of 320 patients with LP at first clinical attack, 75 (23%) patients showed DIS on brain MRI. Of those, 72 (96%) had CSF-restricted OCB and, thus, finally fulfilled the diagnostic criteria for MS. In 43 patients with PPMS and available MRI data, 29 (67%) patients fulfilled only the criteria for DIS. Of those, 26 (90%) had CSF-restricted OCB, and, thus, were diagnosed as PPMS.In this study, we showed that CSF profiles differ between the various MS disease courses and that certain CSF abnormalities occur less frequently than previously reported. This might be due to several reasons, such as the revisions of MS diagnostic criteria as well as the novel CSF RL.alb in 7% of the whole population applying aURL as opposed to 26% using cURL. In multivariate analysis, we showed that absolute changes in Qalb are associated with patients’ age and sex, and not with the disease course  -associated disorders (MOGAD) (We observed blood-CSF-barrier disturbance as indicated by elevation of the Qe course Table S6with age . Previoupatients  are mostnostics) . As Qalb (MOGAD) , a correalb and CSF-TP are both elevated in case of blood-CSF barrier dysfunction  and CSF flow . With regard to the URL, previous studies have already shown that agreement between Qalb and CSF-TP is higher when aURL were used   . In line result) , 19 and  result) .In patients with a first demyelinating event, we could show that OCB were associated with the extent of brain MRI activity. While OCB were demonstrated in only 84% of patients not fulfilling both DIS and DIT by means of MRI, the highest frequency of positive OCB was found in patients with DIS and DIT (98%). This is of special interest, as OCB status does not add to diagnostic certainty in the second scenario because almost all patients who fulfilled MRI criteria were OCB positive. On the other hand, OCB was a significant contributor to diagnosis in patients not meeting MRI criteria. Of 75 patients with a first clinical event who showed only DIS on brain MRI, 72 (96%) had CSF-restricted OCB and, thus, fulfilled the diagnostic criteria for MS. Importantly, it has to be stated that patients with a first demyelinating event without DIS and DIT might not develop MS in future or even might suffer from a disorder other than MS.Similarly, of 29 patients with PPMS fulfilling DIS only, 26 (90%) had CSF-restricted OCB, and, thus, were diagnosed as PPMS. This substantiates CSF-restricted OCB as a cumulative marker for DIT being constantly detectable during disease course – as opposed to contrast-enhancing lesions which are only temporarily detectable on MRI. This concept has already been incorporated into the 2017 McDonald Criteria   or variable duration between MRI and LP cannot be excluded. At this point, we also want to state that the presence of certain leukocyte subtypes was retrieved from routine CSF reports. It cannot be excluded that the frequency of cell types that are infrequently found in the CSF of MS patients are underestimated. Due to this shortcoming, we grouped plasma cells in the same category as lymphocytes and macrophages in the same as monocytes. That is why an analysis of different states of cell activation was not feasible, e.g. to separate lymphocytes from activated lymphocytes and plasma cells. Whereas the differentiation of leukocyte subtypes is usually simple and the simple evaluation of their presence in a cytological preparation reliable; this is not the case for shadow cells. Shadow cells are supposed to reflect apoptotic cells and even though we referred to a common definition , their dIn the present work, we confirmed previous studies on the CSF profile in MS, including e.g. the upper limit of WBC count up to 40/μl) or the high sensitivity of OCB (reaching 95%) 0/μl or t. We alsoThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval were not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.KB has participated in acquisition and interpretation of data, and in drafting the manuscript. GB has participated in acquisition of data and in reviewing the manuscript for intellectual content. MA has participated in reviewing the manuscript for intellectual content. FP has participated in reviewing the manuscript for intellectual content. AZ has participated in reviewing the manuscript for intellectual content. TB has participated in reviewing the manuscript for intellectual content. FD has participated in reviewing the manuscript for intellectual content. HH has participated in the conception and design of the study, acquisition, statistical analysis and interpretation of the data, and in drafting the manuscript. All authors contributed to the article and approved the submitted version.KB has participated in meetings sponsored by and received travel funding from Roche and Biogen. GB has participated in meetings sponsored by, received speaker honoraria or travel funding from Biogen, Celgene, Lilly, Merck, Novartis, Sanofi-Genzyme and Teva, and received honoraria for consulting Biogen, Celgene, Merck, Novartis, Roche and Teva. MA received speaker honoraria and/or travel grants from Biogen, Merck, Novartis and Sanofi. FP has participated in meetings sponsored by, received honoraria  or travel funding from Bayer, Biogen, Merck, Novartis, Sanofi-Genzyme, Teva, Celgene and Roche. Her institution has received research grants from Roche. AZ has participated in meetings sponsored by, received speaking honoraria or travel funding from Biogen, Merck, Sanofi-Genzyme and Teva. TB has participated in meetings sponsored by and received honoraria  from pharmaceutical companies marketing treatments for MS: Allergan, Biogen, Biologix, Bionorica, Celgene, Eisei, MedDay, Merck, Novartis, Roche, Sanofi-Genzyme, Teva, UCB. His institution has received financial support in the past 12 months by unrestricted research grants  and for participation in clinical trials in multiple sclerosis sponsored by Alexion, Bayer, Biogen, Merck, Novartis, Roche, Sanofi-Aventis, Teva. FD has participated in meetings sponsored by or received honoraria for acting as an advisor/speaker for Almirall, Alexion, Biogen, Celgene, Genzyme-Sanofi, Merck, Novartis Pharma, Roche, and TEVA ratiopharm. His institution has received research grants from Biogen and Genzyme Sanofi. He is section editor of the MSARD Journal (Multiple Sclerosis and Related Disorders). HH has participated in meetings sponsored by, received speaker honoraria or travel funding from Bayer, Biogen, Merck, Novartis, Sanofi-Genzyme, Siemens, Teva, and received honoraria for acting as consultant for Teva and Biogen.


The present works focuses on the development of a novel fully bio-based composite using a bio-based high-density polyethylene (Bio-HDPE) obtained from sugar cane as matrix and a by-product of extraction of chia seed oil (CO) as filler, with the objective of achieving a circular economy model. The research aims to revalorize an ever-increasing waste stream produced by the growing interest in vegetable oils. From the technical point of view, the chia seed flour (CSF) was chemically modified using a silane treatment. This treatment provides a better interfacial adhesion as was evidenced by the mechanical and thermal properties as well as field emission scanning electron microscopy (FESEM). The effect of silane treatment on water uptake and disintegration rate was also studied. On the other hand, in a second stage, an optimization of the percentage of treated CSF used as filler was carried out by a complete series of mechanical, thermal, morphological, colour, water absorption and disintegration tests with the aim to evaluate the new composite developed using chia by-products. It is noteworthy as the disintegration rate increased with the addition of CSF filler, which leads to obtain a partially biodegradable wood plastic composite (WPC) and therefore, becoming more environmentally friendly. A global polymer production of 368 million tons was recorded in 2019. In Europe 58 million tons were produced and almost 25% of plastic post-consumer wastes is directly deposited in landfills 22], PVC Salvia hispanica L.) in a polymeric matrix based on Bio-HDPE. The study of compatibilization as well as the optimization of the lignocellulosic filler percentage is the polymeric matrix were carried out. A complete characterization of the composites has been carried out by means of mechanical , thermal , thermo-mechanical (DTMA) and morphological techniques (Field Emission Scanning Electron Microscopy or FESEM) as well as water uptake, colourimetry and disintegration tests.Given the current situation in which the use of vegetable oils in the polymer and food industry is attracting great interest, it is necessary to provide technical solutions to the possible waste generated in order to find a solution that is based on the circular economy. Therefore, the main objective of the present work was the reuse of the lignocellulosic waste obtained after the extraction of chia seed oil . In the second stage, an optimization of percentage of filler has been carried out in samples with 10, 20, 30 and 40 wt.% of treated CSF with silane coupling agent.−3 and melt flow index 20 g·10 min−1 at 20 °C. As a lignocellulosic filler a residual cake obtained after cold pressed extraction of chia seed oil from entire chia seed (CS) supplied by Frutoseco . was used. The residual cake was milled using an ultra-centrifugal mill from Retsch GmbH  at 8000 rpm and equipped with 0.25 mm sieve. After milling, CSF was obtained for use as a lignocellulosic filler. As silane coupling agent 3-(2-aminoethylamine) propyl]trimethoxysilane (APS) provided by Sigma Aldrich  was employed.Commercial bio-based high-density polyethylene (Bio-HDPE) HA7260 grade has been employed as polymer matrix and was supplied by Braskem . This polymer presents a minimum bio-based content of 94%, being obtained from bioethanol derived from sugarcane. Bio-HDPE present a density of 0.955 g·cmCortaderia selloana (Pampa grass) -trimethoxysilane (APS) treatment were examined by comparing untreated and treated samples with 20 wt.%. The improvement of interfacial adhesion between treated CSF with APS and Bio-HDPE matrix was observed by FESEM. As a result of this better interaction, an enhancement in general mechanical properties was obtained compared to untreated CSF, with an improvement of 41% in elongation at break. Regarding water uptake, the sample with APS recorded a decrease of 17% compared to untreated sample, caused by the reduction of available hydroxyl groups in the lignocellulosic filler. Consequently, in the test of disintegration under composting conditions, a sample treated with APS recorded a reduction of the weight loss of 50% due to its more hydrophobic nature resulting from the silane treatment which hinders the penetration of water and microorganisms.In the second stage, an optimization of the amount of CSF filler has been carried out. The addition of 40 wt.% of CSF resulted in improvements of 22% and 18% in tensile and flexural modulus compared to neat Bio-HDPE, respectively. The addition of CSF caused a slight decrease of thermal stability due to the presence of lignocellulosic filler, but aesthetically, the characteristic brown colour of the composites developed made them suitable to for manufacturing furniture or food packaging. The degree of water uptake was similar to that of other wood plastic composites on the market, while the study of disintegration by composting shows a weight loss ratio 20% higher than that of Bio-HDPE, a positive aspect to be taken into account in the development of new composites for sectors with products with short life cycles.


According to the literature, the cerebrospinal fluid (CSF) in the cerebralventricles contains numerous neuron-derived physiologically active substancesthat can function as neurohormones and contribute to volume neurotransmissionin the periventricular region of the brain. This study was aimed at carryingout a comparative analysis of CSF and the blood levels of monoamines in ratsduring ontogenesis as an indicator of age-related characteristics of monoaminetransport to body fluids and their function as neurohormones in volumeneurotransmission in the periventricular region of the brain. We have shownthat CSF in the perinatal period and adulthood contains the most functionallysignificant monoamines: dopamine, noradrenaline, and serotonin. A comparison ofthe monoamine levels in the CSF and blood of animals of different age groupsrevealed that CSF contains monoamines of predominantly neuronal origin and almost no monoamines derived from the general circulation. We alsoestablished that monoamines are found in the CSF at physiologically activelevels that allow them to act as neurohormones in both reversible volumeneurotransmission in the adult brain and irreversible regulation of braindevelopment in the perinatal period. Monoamine dopamine (DA), noradrenaline (NA), and serotonin, which are synthesized in brain neurons, play animportant role in the regulation of brain function via volume neurotransmission and synaptic neurotransmission (actionin the synaptic area) . In adulThere is evidence that neuron-derived monoamines in the cerebrospinal fluid(CSF) in the cerebral ventricles enter the brain and participate asneurohormones in volume neurotransmission due to the absence of aCSF–brain barrier for them ,6. AlthDespite the abundance of evidence to the presence of physiologically activesubstances, including monoamines, in the CSF and blood, the pattern of changesin the monoamine level in these body fluids during ontogenesis has not beenelucidated yet. In addition, the monoamine level gradient at theCSF–blood border during various stages of ontogenesis has never beenassessed before. Considering our recent data on the absence of a CSF barrierfor monoamines during ontogenesis in rats , the levBased on the above, the purpose of our study was to perform a comparativeanalysis of the CSF and blood levels of monoamines in rats during ontogenesisas an indicator of age-related characteristics of monoamine transport to bodyfluids and their participation as neurohormones in volume neurotransmission inthe brain. To achieve this goal, the following tasks were set: (a) determinethe level of monoamines DA, NA, adrenaline (A), and 5-HT as an indicator of thesecretory activity of the corresponding neurons in rat CSF on embryonic day 18(E18) and postnatal days 5 (P5) and 30 (P30); (b) determine the plasma levelsof monoamines in these animals; (c) evaluate the ratio of CSF to the plasmalevels of the monoamines as an integrated index of the existence of barriersfor monoamines between the cerebral ventricles and the general circulation.AnimalsThe study was performed in female and male Wistar rats on E18 and male Wistarrats on P5 and P30 .To obtaAll animal procedures were performed under anesthesia with either chloralhydrate  at a dose of either 100 mg/kg on P5 and 400 mg/kg on P30and E18 or 1% isoflurane on P30 .Collection of rat CSF and blood on E18, P5, and P30CFS was collected from rats on E18 (n = 112), P5 (n = 30), and P30 (n = 20).On gest4 and3,4-dihydroxybenzylamine (DHBA) , which is an internal standard forthe determination of monoamines and their degradation products, were added tothe CSF samples to final concentrations of 0.1 M and 25 pM, respectively. CSFobtained from 14 fetuses was used as the E18 sample, and CSF of three animalswas used as P5 and P30 samples. CSF samples were frozen in liquid nitrogen andstored at –70°C prior to determination of monoamines DA, NA, and5-HT and their main degradation products 3,4-dihydroxyphenylacetic acid(DOPAC), 3-methoxytyramine (3-MT), homovanillic acid (HVA), and5-hydroxyindoleacetic acid (HIAA) (CSF was collected from the cisterna magna of rats on P5 and P30 according tothe previously described technique .For thid (HIAA) .2, and 1.2 mM CaCl2) was inserted intothe guide cannula. The probe was connected to a CMA 4004 Microdialysis Pump through Teflon tubes. Microdialysis was initiated 3 h after probeinsertion: lateral ventricles were perfused for 20 min at a flow rate of 2μl/min. The resulting dialysis sample was mixed with 4 μl of 1 NHClO4, frozen in liquid nitrogen and stored at –70°Cuntil determination of monoamines and their degradation products was inserted stereotaxically into the lateral ventricle of the brain inrats (n = 4) anesthetized with isoflurane based on coordinates calculatedaccording to the rat brain atlas  . Thecanproducts.4 and 25 pmol of DHBA. Plasma was centrifugedat 16,500 g and 4°C for 20 min, the supernatant was frozen in liquidnitrogen and stored at –70°C until determination of monoamines andtheir degradation products  and 10% sodium metabisulfite (Sigma). Samples were centrifuged at1,350 g and 4°C for 10 min, the supernatant (plasma) was collected andmixed with 10% 1 N HClOproducts .High-performance liquid chromatography with electrochemicaldetectionThe concentration of monoamines and their degradation products in CSF, plasma,and microdialysis samples was determined using high-performance liquidchromatography with electrochemical detection. CSF and plasma samples weredivided into two parts. One part was extracted by precipitating catecholaminesand their degradation products onto aluminum oxide, another part was notprecipitated but was used directly to measure the 5-HT and HIAA levels.Microdialysis samples were not precipitated.The test substances were separated using a 4 × 100-mm reversed-phaseReproSil-Pur ODS-3 column with a pore diameter of 3 μm  at 28°C and a mobile phase flow rate of 1 ml/min using aLC-20ADsp Liquid Chromatograph Pump  at a potential of 850 mV.Citrate-phosphate buffer  containing 0.3 mM sodiumoctanesulfonate, 0.1 mM EDTA, and 8% acetonitrile  was usedas the mobile phase. Monoamines were determined using a DECADE IIelectrochemical detector  with a glassy carbonworking electrode (0.85 V) and a silver chloride reference electrode. The peaksof monoamines and their metabolites were determined based on their retentiontime in a standard solution.Statistical data analysisA statistical analysis was performed using the GraphPad Prism 6.0 software(USA). Data are presented as the mean ± standard error of the mean (mean± SEM). Differences were considered statistically significant at p <0.05; 0.05 < p < 0.1 was regarded as a tendency to differences; thedifferences were considered insignificant at p > 0.1. The statisticalsignificance of the results was determined using the parametric Student’st-test (t-test) and the nonparametric Mann–Whitney U-test (U-test); theBonferroni correction was used for multiple testing.CSF levels of catecholamines, 5-HT, and their degradation products in thecisterna magna in rats during ontogenesisThe DA level is approximately 5 pmol/ml on E18; it increases twofold on P5 andreaches that of fetuses on P30.The NA level does not change during the entire period of ontogenesis andremains at a high level (~70 pmol/ml) in all age groups. In contrast to DA andNA, A was detected in the CSF during neither the prenatal nor postnatal period.In addition to catecholamines, their degradation products 3-MT (except for dayE18), DOPAC, and HVA are found in the CSF of rats of all age groups.AlthougNo 5-HT is detected in rat CSF on E18; however, a high CSF level of 5-HT (8pmol/ml) is noted on P5 and P30.The levMonoamines and their degradation products in the lateral ventricles ofrats on P30Of all monoamines and their degradation products, only DOPAC and HIAA weredetected by microdialysis in the lateral ventricles of the rat brain on P30. Atleast three measurements were made to establish the baseline level of the testsubstances. The following DOPAC and HIAA levels were detected in dialysissamples: 5.2 ± 1.1 and 105.7 ± 14.8 pmol/ml, respectively.Levels of catecholamines, 5-HT, and their degradation products in ratplasma during ontogenesisThe plasma level of DA in rats on E18 does not exceed 0.4 pmol/ml, it increasessixfold on P5, and decreases about twofold on P30. The patThe plasma level of 5-HT is approximately 5.5 pmol/ml onE18 . ItincrRatio of CSF to the plasma levels of monoamines and their degradationproducts in rats during ontogenesisThe CSF levels of DA, NA, and their degradation products are many times higherthan their plasma levels in all age groups.HoweverThe CSF levels of 5-HT and HIAA exceed those in plasma on P30 (for 5-HT), P5and P30 (HIAA) .It shouThe main goal of this work was to perform a comparative analysis of the CSF andblood levels of monoamines in rats during ontogenesis as an indicator ofage-related characteristics of monoamine release into the body fluids and theirrole as neurohormones in brain development and function. Special attention ispaid to the CSF as a body fluid that, on the one hand, receives monoamines fromthe brain and, on the other hand, releases monoamines to the periventricularregion of the brain in the absence of a CSF–brain barrier, which thenacts as neurohormones in volume neurotransmission. The discovery of theCSF-contacting neurons was the historical basis for hypothesizing the releaseof physiologically active substances from the brain to the CSF , 13.CSF monoamines acting as neurohormones are potential participants involume neurotransmission and the morphogenetic control of braindevelopmentDespite the fact that dozens of neurotransmitters and neuromodulators aresynthesized in brain neurons, monoamines DA, NA, and A are the most commonclassical neurotransmitters in the brain. These monoamines are involved in theregulation of the functional activity of target neurons in adult animals andthe regulation of neuron and brain development in the perinatal period , 3,4, 5Based on the concept of qualitative differences in monoamine action atdifferent stages of ontogenesis, the CSF level of monoamines was determined inrats at three age periods. The first age group included E18 rats, since thefollowing events take place by this time: (a) formation of neurons fromprogenitor cells is completed; (b) the differentiating neurons migrate to thesites of their final localization in the brain; (c) neurons express a specificphenotype; and (d) axons of the differentiating neurons reach brain ventriclesand blood vessels in the circumventricular organs, forming pathways forneurohormones to enter the CSF (axo-ventricular contacts) and blood vessels(axo-vascular contacts). The second age group included P5 rats. The followingevents take place by this time: (a) migration of differentiating neurons to thesites of their final localization is completed; (b) axo-ventricular andaxo-vascular contacts are established; and (c) afferent synaptic innervation ofneurons continues to form. The third age group included P30 rats. The followingevents take place by this time: (a) formation of synaptic contacts ends; (b)formation of the blood–brain barrier, which prevents penetration of mostnon-lipid neurotransmitters from the brain into the blood and vice versa, iscompleted , 16, 17.From the standpoint of the neurohormonal effect of CSF monoamines on the brainneurons, the CSF level of monoamines is considered the most importantfunctional parameter. Indeed, the previous, mainly in vitro, studiesdemonstrated that monoamines have a neurotransmitter effect on neurons at awide range of concentrations: from 10-11 to 10-8 M . MoreoveAt the first stage of our study, it was necessary to determine whether thequalitative composition and level of monoamines change with the CSF flow fromthe lateral ventricles, where CSF is formed as a result of plasma filtrationfrom the vessels of the choroid plexus to the cisterna magna: the caudal partof the ventricular system. For this, we compared the composition of monoaminesand their degradation products in rat CSF obtained from the lateral ventriclesby microdialysis and rat CSF of the cisterna magna collected on P30 using amicropipette. We found all monoamines and their degradation products detectedby us in the cisterna magna, while only some products of monoamine degradationwere observed in the lateral ventricles: DOPAC and HIAA. These data indicatethat monoamines enter the CSF not from the plasma of the choroid plexus vesselsand the nerve tissue surrounding the lateral ventricles but mainly from thenervous tissue caudal to the lateral ventricles of the brain.-8 M) on E18; it remains the same on P5 (6.3 ×10-8 M) and P30 (6.3 × 10-8 M). The obtained dataindicate that noradrenergic neurons secrete monoamines into the CSF during theprenatal and postnatal periods. This assumption is supported by the fact that,according to the in vitro data, NA at approximately the same level as that ofCSF can exert a neurotransmitter effect on neurons in adult rats [Further, the composition of CSF obtained from the cisterna magna only wasdetermined. Catecholamines DA and NA were found in the CSF of rats of all agegroups; however, changes in their levels were significant with age. Forinstance, NA is present in the CSF at a significant level  is at least an order of magnitude lower than that of NA (0.48 × 10-8 M)on E18. However, this does not reduce the possible morphogenetic effect of DAon target neurons. The CSF level of DA increases twofold (1 × 10-8 M) byP5, although it remains significantly lower than that of NA. It should be notedthat the level of DA could have increased in the perinatal period(E18–P5) much more, if the activity of DA breakdown enzymes, MAO andCOMT, had not increased simultaneously. This is evidenced by a significantincrease in the levels of the products of MAO and COMT enzymatic activity:DOPA, 3-MT, and HVA in the perinatal period. Nevertheless, an increase in theCSF level of DA on P5 significantly increases the probability of itsmorphogenetic effect on differentiating target neurons and brain development ingeneral . The secThe age-related pattern of the CSF level of 5-HT differs significantly fromthat of catecholamines. For instance, almost no 5-HT is detected in the CSF onE18, while its level almost reaches that of DA on P5. The CSF level of 5-HTcould have increased to an even greater extent on P5, if not for a significantincrease in the activity of the 5-HT degradation enzyme MAO by that period, asindicated by the high level of the product of its enzymatic breakdown: HIAA.The 5-HT level remains the same by P30. The above data indicates that the CSF5-HT can both participate in volume neurotransmission in the postnatal periodand regulate the development of target neurons and the brain in general in theearly postnatal period in rats.The brain is the only source of CSF monoamines during ontogenesisAs shown in our study, the CSF of rats on P5 and P30 contains monoamines atphysiologically active levels. Furthermore, in contrast to 5-HT, catecholaminesare also present in the CSF on E18. However, these data cannot serve as directevidence that the brain is the only source of CSF monoamines. Indeed, we cannotexclude that monoamines enter the CSF not only from the brain neurons but fromthe bloodstream as well in the absence of a blood–brain barrier formonoamines in the perinatal period ,29, 30,An answer to the question of whether the brain is the only source of monoaminesin the CSF can be found in a first approximation by calculating the integratedindex of permeability of all possible barriers on the way of monoamines fromthe blood to the CSF in the form of the ratio of CSF to blood levels ofmonoamines. Three options can be considered: (1) the permeability coefficientequals to unity, indicating the absence of a barrier between the CSF and theblood; (2) the permeability coefficient is greater than unity, which indicatesthe presence of a barrier for monoamine entry from the CSF to the blood; (3)the permeability coefficient is less than unity, which indicates the existenceof a barrier preventing monoamine entry from the blood to the CSF.To calculate the coefficient of permeability of barriers between the CSF andblood for monoamines, both the CSF and blood levels of monoamines were measuredin rats during ontogenesis. Age-related changes in the plasma levels of themonoamines DA, NA, and 5-HT were found to be similar. These monoamines weredetected in the blood at insignificant levels on E18, their levels increasedsignificantly by P5 and then decreased almost to the E18 level by P30. However,the levels of individual monoamines in each age group varied significantly. Forinstance, on P5, the plasma level of NA was 15 times higher than that of DA andmore than 150 times lower than that of 5-HT.Determination of the ratio of CSF to the blood levels of monoamines showed thatthe permeability coefficient for barriers between the CSF and blood on E18 is8.5 and 13 for NA and DA, respectively. This means that CSF catecholaminesoriginate from the brain. These calculations could not be carried out for 5-HT,since 5-HT is not detected in the CSF of the fetus at such a time point.Although the permeability coefficient for catecholamines is reducedsignificantly, it remains above unity in the postnatal period. This indicatesthat, during this period, catecholamines enter the CSF only from the brainneurons.In contrast to catecholamines, the permeability coefficient for 5-HT on P5 isonly slightly less than unity and, then, sharply increases by P30. Thisindicates that the barrier preventing the exchange of monoamines between theCSF and blood also exists for 5-HT. The most important evidence that monoaminescan penetrate the barriers on their way from the blood to the CSF is the highblood level of E in rats on P5 and P30 and its absence in the CSF in rats ofthe same age. The obtained data confirm the existence of barriers preventingthe entry of substances from the CSF into the blood and vice versa in the pre-and postnatal period , 35, 36.The following conclusions were made: (1) the cerebrospinal fluid of rats in theperinatal period and adulthood contains the most functionally significantmonoamines: dopamine, noradrenaline, and serotonin; (2) the cerebrospinal fluidcontains monoamines of predominantly neuronal  origin and almost nomonoamines derived from the general circulation; (3) monoamines are found inthe cerebrospinal fluid at physiologically active levels that allow them to actas neurohormones in volume neurotransmission: as morphogenetic factors inirreversible regulation of neuronal development in the perinatal period andreversible regulation of the functional activity of target neurons in adultanimals.


The ubiquity of cellular phone (cellphone) use in young people’s daily lives has emerged as a priority area of concern for youth mental health.This study measured the prevalence of depression and its association with high cellphone use among youth in Soweto and Durban, South Africa.We analysed cross-sectional, baseline survey data among youth aged 16–24 who participated in a dual-site cohort study, ‘AYAZAZI’, conducted from 2014 to 2017. The primary outcome was depression using the 10-item Center for Epidemiologic Studies Depression Scale, with a score of ≥ 10 indicating probable depression. Cellphone use was measured via self-reported average number of hours of active use, with ‘high cellphone use’ defined as daily usage of ≥ 8. Multivariable logistic regression models assessed the independent relationship between high cellphone use and probable depression, adjusting for potential confounders.P = .004). Nearly all (94.6%) owned a cellphone. About one-third (29.5%) reported spending ≥ 8 hours per day using their cellphone . In the overall adjusted model, youth reporting high daily cellphone use had higher odds of probable depression . In gender-stratified models, high daily cellphone use was associated with probable depression among women , but not among men .Of 425 participants with a median age of 19 years (IQR = 18–21), 59.5% were young women. Overall, 43.3% had probable depression, with a higher prevalence among women . The findings indicate a need for intersectoral initiatives focused on meaningful mental health support for South African youth to support positive growth and development. Globally, mental health disorders account for a significant proportion of disease burden and disability among adolescents and young adults, and depression is associated with increased morbidity and suicide . While mGlobally, youth have rapidly integrated cellular phone (cellphone) use into their daily lives ,14. CurrReporting of the relationship between social media use and poor mental health outcomes has been inconsistent, with no data available from South Africa. A study by Hoare et al. investigated the relationship between social media use among Australian adolescents and showed that spending ≥ 7 hours a day using the internet was associated with depression in females and psychological distress in males . AlthougResearchers distinguish between excessive and problematic cellphone use . AlthougSeveral studies have found associations between depression, anxiety and psychological distress with time spent on the internet or cellphone ranging from one to nine or more hours per day ,23,33–36To date, research on cellphone use in the South African context has focused primarily on technology uptake and cellphone-based HIV-related interventions, including their use for study recruitment, health promotion, and HIV care and prevention . A few SThe name ‘AYAZAZI’ is derived from Zulu  – ‘aya’ meaning adolescents and young adults and ‘zazi’ meaning knowing themselves. AYAZAZI was a prospective longitudinal cohort study from November 2014 to April 2017, aimed at linking socio-behavioural, clinical and biomedical data from youth aged 16–24 years. Participants were recruited from Soweto and Durban, South Africa, and were followed for a total of 18 and 12 months, respectively, with scheduled visits every six months. Data from the baseline visit were used for the present analysis.Soweto and Durban are densely populated South African cities in which most youth have access to cellphones ,46, but TM software) at baseline. Questionnaires were delivered in English, isiZulu, or Sesotho in Soweto and in English or isiZulu in Durban, according to each participant’s preference. Questionnaires were designed in English with input from the adolescent community advisory boards. Thereafter, questionnaires were translated into isiZulu and Sesotho by a local translation company and reviewed by the adolescent community advisory boards. Questionnaires took approximately 60 minutes to complete.Trained youth interviewers administered a structured online questionnaire  [ES-D-10) , a depreES-D-10) , and demES-D-10) . Each itES-D-10) .How much time in a day do you spend actively using a cellphone? This includes listening to music/radio, SMS, making phone calls, playing games and accessing the internet.’ Response options were: 0–1 hours, 2–4 hours, 5–7 hours and ≥8 hours per day. ‘High cellphone use’ was based on these categories already used in the survey and defined as active daily usage ≥8 hours in comparison with <8 hours. Participants were asked additional questions about their cellphone use, including: ‘How do you get airtime? (prepaid and contract)’; and ‘Who pays for the prepaid/contract airtime?’The exposure of interest was high cellphone usage. We asked participants whether they owned a cellphone (yes vs. no) and, if they answered yes, what activities they used it for. Participants could select from the multiple options: general communication (including sending and receiving SMSes); making phone calls and e-mail; mobile entertainment ; for emergencies only; cellphone banking, and an ‘other’ option. We then asked participants, ‘In the past 6 months, have you had access to the internet?’ and ‘When you are on the internet, what do you do or search for?’ For the latter question, participants selected all options given: downloading music and videos, research for school projects, looking up health information, looking up information about sex, apps, dating sites, getting directions to places  and looking at maps, finding out about parties/DJ events, internet banking, using social media sites, software, e-mail, selling and buying sites, and other.Internet access and usage were assessed through the following questions, which were analysed separately: ‘These variables were: selected based on known relationships with depression and cellphone use ,39,46,49We first used descriptive statistics to summarise the sample. Continuous measures were summarised descriptively using the medians, Q1-Q3 already defined. Frequencies and percentages were determined for categorical variables. Next, we assessed the associations between depressive symptoms and socio-demographics and cellphone use, stratified by gender. Both continuous and categorical variables were stratified by gender and symptoms of probable depression (yes vs. no). To test for statistical significance for categorical measures stratified by gender and probable depression, Pearson’s chi-square analysis or Fisher’s exact test was used. The medians and interquartile range (IQR) were compared between groups using the Kruskal-Wallis test.p-value ≤ 0.2 at univariate level thought to be explanatory variables based on a priori knowledge were included in the full multivariable model. Thereafter, a backward selection procedure was used to determine the final model with the lowest Akaike information criterion (AIC). The Hosmer-Lemeshow statistic was used to measure model diagnostics/fit in the final multivariate model. Regression results are presented as Odds Ratio (OR) in the univariate and adjusted OR (aOR) in the final multivariate model. All statistical analysis was conducted in SAS Enterprise Guide 7.1  using SAS/STAT procedures PROC FREQ, PROC NPAR1WAY and PROC LOGISTIC.We dichotomised the exposure variable into high (≥8 hours) and low (<8 hours). Using crude and adjusted logistic regression analyses overall and by gender, we assessed the independent association between high cellphone use and depressive symptoms, controlling for potential confounding. Variables with a The AYAZAZI study was approved by the University of the Witwatersrand, Johannesburg, South Africa, and Simon Fraser University, Burnaby, Canada. Written informed consent was obtained for participants 18 years and older. Written assent was obtained from minors younger than 18 years, together with written informed consent from a parent or legal guardian. Participants received a 150 ZAR (~$12 USD) reimbursement per scheduled visit to compensate for transportation and time.Of the 425 participants included in this analysis, 59.3% (n = 253) were women . Most paP = .04). Conversely, men were more likely than women to live in formal housing  and have had consensual  sex .Relative to men, women were more likely to receive an average income of ≤1600ZAR  and spent ≥8 hours using their cellphone daily . In the preceding six months all participants had used their cellphones primarily to search for apps on the internet  (P = .01), Instagram , to pay for their own prepaid airtime , download music and videos  and search for information about sex using their phones .Of the 425 participants, 94.6% (n = 402) owned a cellphone. Those with access used cellphones for general communication , mobile entertainment , WhatsApp , Facebook , Twitter  and Instagram . Most received airtime on prepaid  and paid for their own airtime . A significantly higher proportion of women than men owned a cellphone  . RelativP = .004). Youth with probable depression were likely to: spend ≥ 8 hours using a cellphone daily ; receive an average income of more than 1600ZAR ; have at least one dependent ; and search for apps in the past 6 months  (P = .045).Just under half  had probable depression based on their CES-D-10 scores. Women were more likely to have probable depression than men  . A higheP = .01) was independently associated with probable depression. In addition, probable depression was significantly associated with: being a woman , earning an average income of at least 1601ZAR  and having one or more financial dependents . Model fit was assessed using the Hosmer-Lemeshow goodness-of-fit statistic that indicated a good fit (P = 0.51).In the multivariate model , spendinP = .001), earning an average of more than 1601ZAR  and having at least one financial dependent . Model fit for women suggested a good fit under the Hosmer-Lemeshow statistics (P = .96)  . UnadjusOur study examined the relationship between cellphone use and probable depression among South African youth. Of concern is that nearly half of the sample met the CES-D-10 criteria for having significant depressive symptoms or probable depression, with a higher prevalence of probable depression among young women than young men. Cellphone ownership and high cellphone use were significantly higher in young women. High cellphone use was independently associated with probable depression among women but not among men.Our study contributes to a better understanding of depression post-apartheid in marginalised youth . HoweverNearly all participants in our study owned a cellphone, which is consistent with national cellphone ownership data among adults in South Africa  and prevWe did not find a direct association between general social network usage and probable depression in participants. In bivariate analyses, young people who used Instagram were less likely to be depressed or it could be that those with a higher socio-economic status were able to download and use Instagram. A 2015 study by Lup and Rosenthal found that although Instagram was associated with depression, the relationship with depression was tied to social comparison and following strangers, whereas positive associations were observed when individuals followed family and friends . AlthougOur study found that participants with probable depression were less likely to use their phones to download and use apps. In addition, socio-demographic factors may influence the association between high cellphone use and mental health and may be applicable only to South African youth who can afford to pay for enough data to download apps and spend extended periods on their phones. In South African adults, depression is directly associated with limited material wealth . Our datA limitation of this paper is that we did not include a validated scale to measure high cellphone usage, indicating a need to investigate the threshold at which cellphone is use is deemed excessive in South African populations. As the analysis used cross-sectional data, the direction of effect between depression and excessive cellphone use cannot be determined. However, our regression analysis does provide some guidance about the direction that we investigated. For our study, the dependent or exposure variable was frequency of daily cellphone use.Having surveyed individuals in a predominantly lower socio-economic area who probably experienced limitations in data access also affected our assessment of excessive usage, including social media, and thus may not be representative of the general population. However, nearly one-third of participants reported using their phones for ≥8 hours a day indicating some accessibility to cellphone usage despite socio-economic status. Future research should explore the association between increased screen time as well as problematic use, resilience and coping, as a means to better understand the high levels of dependency on cellphone technology in young South Africans.Our study found high prevalence of depressive symptoms and high cellphone use in youth, with high cellphone use associated with probable depression in women but not in men. The high proportion of participants reporting significant symptoms of depression indicates a need for intersectoral initiatives focused on meaningful mental health support for youth living in vulnerable communities, to support positive growth and development.


The measured CD26 level was consistent with the presence of T cell produced virus. We found no significant differences in ART concentrations between CSF escape and fully suppressed individuals in CSF or blood, and did not observe a clear association with drug resistance mutations in CSF virus which would allow HIV to replicate. Hence, CSF HIV in the face of ART may at least partly originate in CD4+ T cell populations.HIV cerebrospinal fluid (CSF) escape, where HIV is suppressed in blood but detectable in CSF, occurs when HIV persists in the CNS despite antiretroviral therapy (ART). To determine the virus producing cell type and whether lowered CSF ART levels are responsible for CSF escape, we collected blood and CSF from 156 neurosymptomatic participants from Durban, South Africa. We observed that 28% of participants with an undetectable HIV blood viral load showed CSF escape. We detected host cell surface markers on the HIV envelope to determine the cellular source of HIV in participants on the first line regimen of efavirenz, emtricitabine, and tenofovir. We confirmed CD26 as a marker which could differentiate between T cells and macrophages and microglia, and quantified CD26 levels on the virion surface, comparing the result to virus from  The brain may be a site where HIV persists despite antiretroviral therapy (ART). Persistence can manifest as cerebrospinal fluid (CSF) escape, where HIV is detectable in the CSF but not the blood in some individuals. The reasons for CSF escape are incompletely understood. Evidence from individuals mostly on second line protease inhibitor-based ART indicates that detectable HIV in this compartment may have acquired drug resistance. In this work we investigated HIV in blood and CSF of 156 participants from Durban, South Africa. We observed a very high prevalence of CSF escape of 28%. We aimed to find the cell type responsible for producing HIV in CSF escape and whether replication occurred because of lower CSF drug levels or because the virus has developed resistance to therapy. We found that at least some of the CSF HIV was produced by T cells, and that drug resistance was not always present. This suggests that at least part of the CSF HIV reservoir may be generated by either an infection mode not requiring drug resistance for viral replication, or by latently infected CD4+ T cells trafficking to and releasing HIV in the CSF without extensive viral replication taking place. HIV persistence in the face of ART necessitates lifelong adherence to treatment. The CNS may serve as one reservoir for HIV persistence . HIV infPotential reasons for a CNS reservoir include reduced drug levels. Drug levels of efavirenz (EFV), emtricitabine (FTC), and tenofovir (TFV) in the CSF are reduced approximately 200-fold, 2-fold, and 20-fold, respectively in the CSF relative to blood –19. SincThere is evidence for compartmentalized HIV infection in the CNS, indicating that CNS specific cell subtypes may be involved –27. HIV T cells are also present in the CNS. CSF contains trafficking T cells, mostly CD4+ memory cells, which enter across the choroid plexus . T cell-Here we aimed to determine the cellular source of HIV in the brain and whether lower ART levels relative to fully suppressed individuals account for CSF escape in individuals from Durban, South Africa. This is the first time in our knowledge where CSF escape in Sub-Saharan Africa has been investigated in a relatively large number of participants on ART . Since aCSF and matched blood were obtained from participants indicated for lumbar puncture enrolled at Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital in Durban, South Africa after written informed consent . Discarded tissue from the field of neurosurgery was obtained from two participants enrolled at Inkosi Albert Luthuli Central Hospital indicated for neurosurgery after written informed consent . Blood for PBMC, CD4+ and CD14+ cell isolation was obtained from adult healthy volunteers after written informed consent . Lymph nodes were obtained from the field of surgery of participants undergoing surgery for diagnostic purposes and/or complications of inflammatory lung disease. Informed consent was obtained from each participant, and the study protocol approved by the University of KwaZulu-Natal Institutional Review Board .Data is described with the non-parametric measures of median and interquartile range, and significance determined using the non-parametric Mann-Whitney U test for pairwise comparisons, Fisher exact test for pairwise comparisons of frequencies, and the Kruskal-Wallis test with multiple comparison correction by the Dunn Method for comparisons involved more than two populations. All tests were performed using Graphpad Prism 8 software.μl CSF was sent for viral load (Molecular Diagnostic Services) directly after the spin. Plasma was processed by spinning the whole blood for 10 min at 1300 g, no brake. The top layer was removed and stored in 1 mL aliquots at -80°C.Fresh CSF and matching blood samples were transported to the laboratory and processed immediately. Two separate EDTA tubes of 4 mL blood were sent for testing in parallel: one for a CD4/CD8 count at an accredited diagnostic laboratory  and one for viral load at an accredited diagnostic laboratory . CSF samples were spun for 10 min at 1000 g to remove debris. CSF supernatant was frozen in 1 mL aliquots at -80°C. One aliquot of 100 μg/mL PHA (Sigma-Aldrich). Monocyte-derived macrophages were grown in RPMI 1640 supplemented with 10% human serum (Sigma) with added L-glutamine, sodium pyruvate, HEPES, and non-essential amino acids (Lonza), and differentiated with 20 ng/mL M-CSF (Peprotech) for 10 days.The following reagents were obtained through the AIDS Research and Reference Reagent Program, National Institute of Allergy and Infectious Diseases, National Institutes of Health: the antiretrovirals EFV, FTC, and TFV and the plasmid for the macrophage tropic pNL4–3(AD8) HIV molecular clone. NL4–3 and NL4–3(AD8) HIV stocks were produced by transfection of HEK293 cells with the molecular clone plasmids using TransIT-LT1 (Mirus) transfection reagent. Supernatant containing released virus was harvested two days post-transfection and filtered through a 0.45 micron filter (GVS) and stored in 0.5 mL aliqouts at -80°C. The number of HIV RNA genomes in viral stocks was determined using the RealTime HIV-1 viral load test . RevCEM-E7 cells were generated as previously described . Cell cuμL staining buffer and acquired on a FACSCalibur machine (BD Biosciences). Results were analyzed using FlowJo software. Staining of LN cells: LN from the field of indicated cardiothoracic surgery were cellularized by gentle mechanical dissociation and cryopreserved. For staining, LN cells were thawed, washed once in PBS-/-, then incubated with the following fluorescently conjugated antibody mix for 30 min: CD45-HV500, CD19-BV785, CD3-PE-CF594, CD4-AF700 HLA-DR-BV605 , CD26-FITC, CD36-PE (Biolegend), and the LIVE/DEAD Fixable Near-IR Dead Cell Stain (ThermoFisher Scientific). Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization kit (BD Biosciences) according to the manufacturer’s instructions. Cells were then stained with CD68-APC antibodies (Biolegend). Cells were washed, fixed in 2% formaldehyde and acquired on a BD ARIA Fusion flow cytometer (BD Biosciences).Staining of MDM and PBMC T cells: MDM and CD4+ PBMCs were generated as described above. PBMCs were washed once in PBS-/-. MDM were washed once in PBS-/- then incubated in 5 mM EDTA in PBS-/- for 30 minutes on ice. Macrophages were collected by pipetting vigorously and the remaining attached cells were removed by gentle scraping. Cells were then incubated with either CD3-APC and CD8-Bv500 (PBMC) or CD68-APC (MDM) and CD26-FITC and CD36-PE (Biolegend) fluorescently labelled antibodies in staining buffer (PBS-/- with 3%FCS) for 30 min on ice. The samples were then washed, resuspended in 400 μL staining buffer, and acquired on a FACS Fortessa (BD Biosciences). Results were analyzed using FlowJo software.Meninges discarded tissue samples were transported immediately to the laboratory for processing. The tissue was dissociated . The dissociated cells underwent myelin removal  and CD11b+ cells were further purified . All kits were used according to the manufacturers instructions. The cells were then surface stained with fluorescently conjugated antibodies for microglial markers: CD11b-APC, CD45-Bv605 and P2RY12-Bv421, CD26-FITC, and CD36-PE (Biolegend). The cells were incubated with antibodies for 30 min on ice, washed, resuspended in 500 6 RNA copies/mL NL4–3(AD8) for 24 hours. Cells were then washed 4 times in growth medium to remove the input viral stock and incubated for 4 days (approximately two full virus cycles) for a total infection time of 5 days. Virus containing supernatant was collected, centrifuged at 300 g for 5 minutes then filtered through a 0.45 micron syringe filter (GVS) to remove cells and cellular debris. The number of virus genomes in the virus stock was determined using the RealTime HIV-1 viral load test (Molecular Diagnostic Services). To generate monocyte-derived macrophage virus, CD14+ monocytes were isolated and differentiated as described above. Cells were then infected with with 2 × 107 RNA copies/mL NL4–3(AD8) for 24 hours. Cells were washed 6 times with RPMI to remove input virus, and growth media was replaced. Half the volume of media was replaced every 3 days for 14 days for a total infection time of 15 days. Virus containing supernatant was collected, centrifuged and filtered, and viral load determined as for PBMC virus. We used 7 different donors blood donors: 6009, 6013, 6017, 6019, 6026, 6033 and 6049.To generate PBMC origin HIV, PBMCs isolated and activated as described above were infected with 2 × 10μMACS Streptavidin Kit protocol (Miltenyi): 1 μg of biotinylated antibodies to CD26 or CD36 (Ancell) were added to 1 mL of virus, mixed and incubated for 30 min at room temperature. Next, 30 μL of strepavidin MicroBeads (Miltenyi) were added per sample, mixed and incubated at room temperature for 10 min. The samples were then loaded onto a μColumn, washed three times and bound virus eluted. Clinical virus samples were centrifuged for 13,000 g for 30 seconds to clear debris before addition of antibodies. To avoid overloading columns, in vitro generated virus stocks from either PBMCs or macrophages were diluted to approximately 104 RNA copies/mL in PBS before incubation with antibodies. We used the 7 donors above. Virus was precipitated once for six of the donors, whereas 6049 was done in duplicate. The number of virus genomes in elutions from μColumns was determined using the RealTime HIV-1 viral load test (Molecular Diagnostic Services). Out of 22 CSF escape samples, 11 had sufficient volume for the assay (2 mL). One sample which showed neither detectable CD26 nor CD36 was excluded from the analysis due to possible degradation of the virus.The following protocol was adapted from the pNL4–3(AD8) was used as the source of the macrophage tropic HIV ENV which was excised from pNL4–3(AD8) using BamHI and EcoRI restriction enzymes (NEB) and ligated using T4 ligase (Invitrogen) into the pNL4–3-YFP vector (gift from David Levy), replacing the NL4–3 X4 specific HIV envelope gene between the unique EcoRI-BamHI restriction sites.μL of plasma or CSF. 50 μL of water and 50 μL of ISTD solution was added and the sample was briefly mixed. 150 μL of acetonitrile was subsequently added to facilitate protein precipitation, vortex mixed and centrifuged at 16000 g for 10 min at 4°C. 170 μL of the clear supernatant was then transferred to a clean micro-centrifuge tube and dried down using a SpeedVac dryer set at 40°C. The dried samples were then reconstituted in 100 μL of 0.02% sodium deoxycholate (Sigma) in Millipore filtered water, vortex mixed, briefly centrifuged, placed in a small insert vial, capped, placed in the auto sampler compartment (maintained at 4°C) and analyzed using LC-MS/MS. The analytes were separated on an Agilent Zorbax Eclipse Plus C18 HPLC column using gradient elution. The column oven was set at 40°C, a sample volume of 2 μL was injected and the flow rate was set to 0.2 mL/min. Mobile phase A consisted of water with 0.1% formic acid and B consisted of acetonitrile with 0.1% formic acid. The drug analytes were monitored using multiple-reaction monitoring mode for positive ions except for efavirenz which was monitored in the negative ion scan mode. Analyst software, version 1.6.2 was used for quantitative data analysis. Blanked values for EFV, FTC and TFV were in the range of 3 ng/mL and this was set as the detection limit.Sample analysis was performed using an Agilent High Pressure Liquid Chromatography (HPLC) system coupled to the AB Sciex 5500, triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) TurboIonSpray source. The LC-MS/MS method was developed and optimised for the quantitation of tenofovir, emtricitabine, efavirenz, lopinavir, ritonavir, nevirapine, zidovudine, lamivudine, abacavir, atazanvir and dolutegravir in the same sample. A protein precipitation extraction method using acetonitrile was used to process clinical plasma and CSF samples. The procedure was performed using 50 n = 156) clinically indicated for lumbar puncture as part of their diagnostic workup in Durban, South Africa (n = 80 or 51%) of participants showed an undetectable HIV viral load (VL) in the blood. Out of the subgroup of successfully ART suppressed individuals in the blood, 22 (28% of those with an undetectable VL in the blood) had detectable viremia in the CSF (CSF escape). The remainder of the participants  had detectable blood viremia either because they were treatment naïve (n = 34 or 45% of the viremic group) or failing ART (n = 42 or 55% of the viremic group). We note that the CSF escape detected in this study is likely neurosymptomatic CSF escape, leading to clinically indicated lumbar puncture . Otherwise, the CSF escape group was very similar to the suppressed group, except for the VL in the CSF. The groups did not differ in either median CD4 count , years on ART , fraction of individuals with diagnosed co-infections  or the ratio between males and females . Pleocytosis, abnormally high white blood cell concentrations in the CSF usually indicating co-infection with another pathogen in the CNS, occurred in 45% of CSF escape participants and 33% of suppressed participants. However, the trend of higher pleocytosis in CSF escape was not statistically significant . Surprisingly, median CSF VL in CSF escape participants was not significantly different from that of viremic participants . However, when compared to the treatment naïve and ART experienced viremic participant subgroups separately, the CSF escape participants showed a significantly lower median CSF VL compared to viremic naïve  and significantly higher VL compared to viremic participants on ART .The CSF escape group was significantly older compared to the suppressed group  the number of CD26 antibody bound virions after washing off unbound virions, and 2) the VL of the virus suspension added to the column  in virus originating from PBMC relative to MDM  assay as follows. We coupled CD26 antibodies to magnetic beads and added the sample containing either virus from CSF or plasma samples, or e column . We norme column , indicate to MDM .in vivo derived CSF escape virus for CSF samples with sufficient volume (11 samples excluded due to insufficient volume (2 mL minimum) for the assay and one excluded based on poor sample quality .We next used the CD26 normalized by input VL to infer the cellular origin of  quality ). We comand PBMC . The medIf the number of virions bound to the anti-CD26 antibodies from the CSF is very small, it may be difficult to accurately estimate the ratio of virions expressing CD26 relative to input. We therefore also examined the absolute numbers of anti-CD26 bound and input virions . To ensuin vitro by MDM . We compared drug concentrations between viremic participants on ART, suppressed participants, and CSF escape participants. Confirming previous results , 60, 61,p = 0.83 and p > 0.99 for plasma and CSF respectively, Kruskal-Wallis test with Dunn multiple comparison correction). The same was true of FTC (p > 0.99 and p > 0.99 for plasma and CSF respectively) and TFV (p > 0.99 and p > 0.99 respectively). This indicates that there is no reduction in CSF drug levels in CSF escape individuals relative to individuals with full suppression.We compared drug levels between groups, and included participants reported to be treatment naïve . We obsehttps://hivdb.stanford.edu/).One possible explanation for CSF escape is accumulation of drug resistance mutations (DRMs) from previous gaps in adherence or other factors. We therefore sequenced CSF virus. In participant samples where CSF virus sequencing was successful , we obselog10 or greater of plasma VL [https://hivdb.stanford.edu/) was detected. An additional participant (PID 0168) on an EFV based regimen showed a CSF viral load. The level of EFV in the CSF was near background for this participant. However, FTC was above the median concentration for suppressed and CSF escape participants. No DRMS were detected in CSF virus from this participant. These results may indicate that DRMs may not be necessary for a detectable VL in the CSF in the face of ART.One participant (PID 0172) showed no detectable ART and, as expected, showed no CSF DRMs. HIV from one of two participants with CSF escape also contained DRMs (PID 0213), while HIV sequenced from the second participant (PID 0161) did not. The CSF escape participant with detected CSF DRMs was on a lopinavir/ritonavir protease inhibitor based therapy as detected by LC-MS/MS. DRMs included M184V, V106M, and G190A, the latter mutations a possible result of first line EFV regimen failure. In contrast, PID 0161, who was on EFV based therapy, had no CSF DRMs detected. Two additional participants (PID 0148 and 0164) showed CSF discordance, defined as a CSF VL about 0.5 lasma VL . Both weHere, we examined the cellular source of viremia in study participants with CSF escape in Durban, South Africa, and whether drug levels in the CSF are lower in individuals with CSF escape versus those suppressed both in the blood and CSF. Detection of the CD26 T cell host surface marker on the viral envelope was consistent with the presence of at least some T cell origin virus in our study population, where the overwhelming majority of people living with HIV are infected with clade C virus . Our conin vitro infection of macrophages showed no CD26 expression. Given that CD26 is absent in myeloid lineage cells, a reasonable conclusion is that these cells did not generate virus with the CD26 host marker on its surface. This does not exclude the presence of virus produced in myeloid lineage cells in CSF escape, since about three-quarters of virions in the CSF did not express CD26  Gating for PBMC T cells (left two panels) and MDM (right two panels). Cells were stained with APC conjugated anti-CD3 (PBMC) or anti-CD68 (MDM) antibodies. (B) Gating for lymph node T cells. (C) Gating for lymph node macrophages. Representative result from one of 3 LN donors (024–09-0257).(TIF)Click here for additional data file.S2 Fig7 RNA copies/mL YFP-NL4–3(AD8). 2 days post-infection, cells were collected and stained with CD3 and CD14 antibodies, then analyzed for infection by detection of YFP positive cells in the CD3+ and CD14+ populations using flow cytometry. Shown are median and IQR for different blood donors. The median fraction of infected CD3+ gated CD4+ PBMC was 2.4% (IQR 1.7–3.2). No infected CD14+ monocytes were detected. The difference was significant . Brown circles denote blood donor 0020, red circles donor 0019, green donor 0049, and blue donor 0051.PBMC were infected with 2 × 10(TIF)Click here for additional data file.S3 Fig4 HIV RNA copies/mL. Half the diluted supernatant was applied to CD26 binding columns and the other half to CD36 binding columns to quantify the number of CD26 and CD36 expressing virions. Shown are median and IQR for different blood donors. Macrophage values: CD26 median 208 HIV RNA copies/mL (85–440 copies/mL), CD36 median 5403 copies/mL (3076–9263 copies/mL). PBMC values: CD26 median 6600 copies/mL (5749–11185 copies/mL), CD36 median 5862 copies/mL (3309–5862 copies/mL). p-values are: *< 0.05; **< 0.01; ****< 0.0001 by Kruskal-Wallis non-parametric test with Dunn multiple comparisons correction.MDM or PBMCs were infected with NL4–3(AD8) macrophage tropic HIV able to infect both cell types. Supernatant from the infected cells was collected and diluted to 10(TIF)Click here for additional data file.S4 FigA viral load assay was performed on the CSF escape samples. Samples were then added to a column with anti-CD26 bead bound antibodies for immuno-capture of virus expressing CD26. Captured virus was then eluted and viral load assay performed. Red dotted line represents limit of assay detection. GM: geometric mean of n = 10 participants for each condition. Geometric mean was 5874 HIV RNA copies/mL for total virus, and 1591 copies/mL for virus with CD26 surface expression.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.