Tribulus terrestris L. (TT) is a plant used in traditional Chinese medicine, Ayurvedic medicine, and sports nutrition to improve health and performance. However, no conclusive evidence exists about the potential beneficial effects of TT on sport and health biomarkers in physically active adults. Based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, and the modified McMaster Critical Review Form for methodological quality assessment, we systematically reviewed studies indexed in Web of Science, Scopus, and PubMed, to assess the effects of TT on immunological, hematological, biochemical, renal, lipidic, hormonal behavior, and anti-inflammatory response in physically active adult males. Among 340 records identified in the search, a total of 7 studies met the inclusion and exclusion criteria. Overall, participants supplemented with TT displayed significant improvements in lipid profile. Inflammatory and hematological biomarkers showed moderate beneficial effects with no significant changes on renal biomarkers. No positive effects were observed on the immune system response. Additionally, no TT-induced toxicity was reported. In conclusion, there was no clear evidence of the beneficial effects of TT supplementation on muscle damage markers and hormonal behavior. More studies are needed to confirm the benefits of TT due to the limited number of studies available in the current literature. Sports nutrition influences athletes’ health and sports performance . When thTribulus terrestris L. (TT) [Ergo nutritional supplement sales have annually increased in the U.S. market, and it is estimated an annual growing rate of 8.9% over the next years . One typ L. (TT) , has incTT is an herbaceous plant originally from India that belongs to the Zygophyllaceae family that comprises about 20 worldwide species. TT is known variously as “Tribulus”, “puncture vine”, “caltrop”, “goat head”, “puncture weed”, “Mexican sandbur”, “Texas sandbur” or “bullhead”, among other names. TT predominantly grows in the countries around the Mediterranean Sea and in sub-tropical regions around the world [TT is a popular in traditional Chinese medicine and it has been widely used in many countries for thousands of years. TT (fruits and roost) has been mainly used for its potential cardioprotective, antiurolithic, antidiabetic, anti-inflammatory, antitumor, and antioxidant effects [TT is composed of saponins, polyphenols, flavonoids, glycosides, alkaloids, and tannins [TT on sexual performance, coronary heart disease, avoidance of ischemia/reperfusion injury to the heart, and modulation of hypertension [he world . TT is a effects . TT is c tannins . Steroid tannins ,10. Saportension ,10.TT extracts are commonly used by numerous athletes and non-athletes to improve testosterone levels and strength performance [TT is attractive for athletes because of its potential ergogenic effects on sports performance [TT as a testosterone booster [TT [TT could exert an anabolic/androgenic influence by activating endogenous production of testosterone and luteinizing hormone (LH), which may stimulate muscle growth . The polyphenols and flavonoids contained in TT act as antioxidants because they effectively scavenge free radicals in a concentration-dependent manner [TT may reduce inflammation [Currently, products with formance . TT is aformance , improviformance , strengtformance  associat booster , and pro booster ,13. Thesster [TT . The sapgrowth 7,. The polt manner . Moreoveammation  by attenammation  and oxidammation .TT on sports biomarkers in healthy physically active adults. Biomarker monitoring may provide crucial information to adjust exercise workloads to maintain an optimal level of health. Thus, the purpose of this study was to systematically review current studies on the potential effects of TT supplementation on immunological, hematological, and biochemical biomarkers, hormonal behavior, lipid profile, renal function, and anti-inflammatory activity in physically active healthy adult males.Blood circulation is responsible for biochemical, metabolic, hormonal, and immunological processes, and for maintaining homeostasis in the body. Therefore, it is important to assess the physiological changes and adverse effects that may occur under the influence of sports activity or nutritional supplements used through biomarker analysis . AdditioPopulation: physically active healthy adult males; Intervention: supplementation with TT; Comparison: placebo/control group or pre/post comparison data group; Outcomes: immunological ; hematological ; biochemical ; hormonal ; lipidic ; renal ; inflammatory  biomarkers.We used the PICO model according to the standard methods proposed by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Guidelines (PRISMA)  as folloWe established a structured search via the databases Scopus, Web of Science (WOS), and Medline (PubMed) for studies published from database inception to 29 April 2022. The search strategy contained a mix of Medical Subject Headings (MeSH) and free words for key concepts related that included:  AND  AND . The search for published studies was independently performed by 2 authors (D.F.L. and J.M.A.) and disagreements about records were resolved by a third reviewer (J.S.-C.)TT supplementation administered alone in physically active adult males; (c) records with specific information on the dose of TT intake and intervention period; (d) studies with information about the type of pharmaceutical form used for the supplementation ; (e) records that examined at least one reported outcomes related to any of the hematological, inflammatory, antioxidant, and biochemical biomarkers before communicated. On the other hand, the exclusion criteria were the following: (a) studies conducted on animals, and/or in vitro studies; (b) studies in which TT was administered with other supplements or administered as a combination; (c) records in which the outcomes were not related to sports performance or health; (d) editorials, reviews, notes, and any other non-original studies; (e) studies conducted in participants with any cardiovascular, metabolic, musculoskeletal, or other chronic disorder.The following inclusion criteria were applied to select studies: (a) original records with randomized and non-randomized trials, double-blind or parallel controlled design; (b) records that evaluated the impact of The methodological quality assessment of the selected records was performed using the McMaster University Occupational Therapy Evidence-Based Practice Research Group . The aimThe following information was extracted from each study included in the systematic review: included name of the first author; publication year; country where the study was conducted; study design; sample size; participants’ sex and age; weight or body mass index (BMI); dosage, i.e., specific amount, number, frequency, and percentage of saponins), timing of the supplementation; duration of intervention; outcomes reported; and final results. Two investigators (D.F.-L. and J.M.A.) conducted the data extraction process using a spreadsheet. In case of disagreements related to the data extraction, a third author reviewer (J.S.-C.) was involved in the process.A total of 340 studies were identified, 334 studies were from 3 electronic databases WOS, SCOPUS, and PubMed, and 6 studies were retrieved from reference lists of selected electronic databases studies. After exclusion of 273 duplicates, a total of 61 articles identified in databases and registries were examined. After evaluation of the title and abstract, 17 articles were considered as potential registries. After review of the full text and evaluation of potential records from databases and registries as well as other sources, 7 ,23,24 stFour studies ,14,15,17The characteristics of the participants are shown in TT supplementation was used in 5 studies [TT supplementation varied from 1875 mg [TT supplementation well [ studies ,15,22,23 studies ,24 was s 1875 mg  mg, with 1875 mg  or in th 1875 mg ,22. Two  1875 mg ,15. No aion well ,22,23,24TT on immunological markers. In LYM, MON, and GRAN, no significant changes were observed when comparing the intervention group with respect to the control group. However, a significant increase in GRAN and a significant decrease in LYM were observed after 20 days of TT supplementation compared to baseline levels.One study  analyzedTT supplementation group with the control group. Only HCT levels showed a non-significant increase from baseline to the end of training in the TT group [Hematological biomarkers were evaluated by Milasius et al. . This stTT group .TT on CK was evaluated in 3 studies [TT group were moderately lower compared to its pre-exercise levels. Moreover, differences between the groups regarding post-exercise CK were not significant [The effect of  studies ,17,22. M studies  showed tTT group was significantly lower than in the placebo group in response to resistance exercise training [TT supplementation group with the control group and when examining changes from baseline [LDH level in the training . BIL conbaseline .TT on renal biomarkers, Cr, Ua, Ure, and BUN were evaluated in 2 studies [The impact of  studies ,22, obseTT intervention group compared with the control group [Chol and LDH-Chol levels were significantly reduced and HDL-Chol were significantly increased among participants in the ol group  and inveol group . Moreoveol group  reportedTT supplementation group with the control group [Inflammatory biomarker studies were examined in 2 studies ,22. IL-6ol group , howeverol group . One stuTT intervention group relative to the control [TT supplementation groups from baseline [Testosterone levels were examined in 5 studies ,22,23,24 control ,24. Exam control ,22 and obaseline ,22. IGF-baseline  showed aTT supplementation on sports biomarkers in physically active healthy adult males. A total of 7 studies met the inclusion/exclusion criteria. Participants supplemented with TT presented significant improvements in lipid profile. Inflammatory and hematological biomarkers showed moderate beneficial effects with no significant changes on renal biomarkers. However, TT seemed not to have a positive effect on the immune system. There was no clear evidence of the beneficial effects of TT supplementation on muscle damage markers and hormonal behavior. No TT-induced toxicity was reported.The purpose of this systematic review was to critically evaluate the effects of TT supplementation protocol. No significant abnormal changes in renal function biomarkers  were found [TT. Sleep disturbances, exhaustion and fatigue, hypertension, and elevated heart rate have been reported after administration of TT at doses ≥1000 mg per day [Scientific Committee of the Spanish Agency for Consumer Affairs, Food Safety and Nutrition (AECOSAN) reported that the recommended daily intake of TT would be between 250 and 9000 mg [TT has not been listed as a banned substance by the World Anti-Doping Agency (WADA) [TT may increase the testosterone/epitestosterone ratio in urine above the WADA permitted limits (4:1) [TT supplementation [Supplementation doses administered in interventions varied from 2700  to 450 mre found ,22, and re found ,26, gynere found , nephrotre found ,30, hypere found , hepatot per day . In rats per day  and neph per day . The rep 9000 mg . TT has y (WADA) . Howeverts (4:1) , and mayts (4:1) . There aTT supplementation containing 100% saponins. Investigators observed a moderate increase of HCT and HB compared to control group, but did not have positive effect on RCB and MVC. TT does not directly affect erythropoietic activity in persons. This property may be explained because TT supplementation showed a trend in improvement of testosterone levels [TT, like Protodioscin, could have beneficial effects on physical fitness [The study by Milasius et al.  includede levels . Testoste levels . Another fitness , by impr fitness .TT had demonstrated immunostimulant activity on macrophage activity in vitro assays and activation of non-specific immunity in animal model [TT showed an enhancement of B cell activity with significant increases on serum antibody titers that are potentially effectors of the humoral response [TT for 20 days induces a change in leukogram transformed into granulocytes with substantial decrease in lymphocytes and significant increase of neutrophils, basophils, and eosinophiles in endurance athletes. In this regard, dose and/or the high percentage of steroid saponins in TT supplementation could be responsible for the immunosuppression, which could be like that of corticosteroids [Steroidal saponins contained in al model . In addiresponse . These fresponse , where tresponse  have repsteroids .TT-induced toxicity, but because TT is not able to restore the effects that exercise has on the immune system. In this sense, no immunosuppressive effect of TT has been reported to date [TT supplementation in athletes.However, it should be considered that intense and acute physical exercise is accompanied by responses that are remarkably similar in many respects to those induced by infection, sepsis, or trauma. The number of circulating leukocytes  and their magnitude are related to both the intensity and duration of exercise . For thi to date ,29. TherTT supplementation demonstrated a significant reduction for CK activity (during high-intensity training) [TT supplementation could be attributed to an antioxidant role [TT has been reported by its inhibitory action on cyclooxygenase-2 (COX-2) expression [TT could markedly reduce histamine and/or prostaglandin production by its inhibitory action on COX-2 [TT supplement administered. Perhaps, several bioactive components of TT such as flavonoids, alkaloids, phenols, and saponins [Intense and prolonged periods of physical activity increases circulating levels of LDH and CK, which negatively affect athletes by decreasing muscle performance . This syraining)  and LDH raining) . Furtherraining) , post-exraining) . Potentiant role . In thisant role  or inducant role . Previoupression . This pron COX-2  found sisaponins ,17 are nsaponins .TT showed a remarkable hepatoprotective activity, that seems to be related to inhibition of lipid peroxidation and increased levels of antioxidant enzymes, in addition to free radical scavenging action [TT’s flavonoids would enhance the viability and cellular leakage of transaminases  and could be responsible for protecting the liver against oxidative damage and tissue-damaging enzymatic activities. However, we must also consider that TT plant contains alkaloids, steroidal glycosides, and steroidal saponins [Another blood biomarker included in this study is BIL . In athlg action ,45. TT’ssaponins .TT extracts are known to have anti-inflammatory activities in in vivo and in vitro assays [TT ’s properties would include down-regulation of enzymes responsible for the production of cytokines and inflammatory mediators [TT, then 500 mg × day−1 for 2 weeks of TT supplementation was sufficient to induce a moderate but not statistically non-significant decrease in IL-6 and Hs-CRP [TT inhibits NF-κB a signaling pathways [TT to suppress leukocyte infiltration, activation, and maturation, as well as the production of proinflammatory mediators TNF-α and IL-4, at the focus of inflammation. Control of IL-6 levels could attenuate a wide range of inflammatory events that affect the homeostasis of the organism. These properties are similar to other nutritional supplements such as glycophosphopeptical AM3 [TT group. Maybe this is because the anti-inflammatory effect requires several active ingredients of TT and saponins alone would not be sufficient.It is known that exercise induces an increase in plasma levels of IL-6, which may be the indicator of a more intense inflammatory response . Increaso assays . Additioediators . These fd Hs-CRP . A plauspathways ; this prpathways , plus inpathways , allows ical AM3 . ESR is ical AM3 . One stuical AM3  showed mTT, as a nutraceutical, has been used in traditional Chinese medicine, in Ayurvedic medicine in India, as well as by modern herbalists as an adjuvant for its various anti-inflammatory properties in inflammation-mediated diseases such as diabetes, obesity, pancreatitis, cancer, inflammatory bowel disease, kidney disease, and arthritis [Overall, rthritis .TT’s treatment [TT supplementation in renal biomarkers [TT-induced renal toxicity. These results agree with those reported by Gandhi et al. [TT-induced toxicity. However, kidney injury after TT supplementation has been described [Cytoprotective power on the cells of the renal system would suggest improvements in renal function after reatment , but 2 somarkers ,22. All i et al.  who neitescribed ,30.TT group) [TT showed significant dose-dependent protection against elevation of biochemical parameters in urine. This premise would add new perspective of TT on the maintenance in physiological range of renal biomarkers that are increased after exercise.Concerning the Cr, Ua, Urea, and BUN (high-intensity training) levels, athletes of the studies integrated in this systematic review did non-significantly decrease when compared with baseline (T group) ,22. BUN T group)  and renaT group) , TT showModerate physical exercise is associated with a healthy plasma lipid profile and a lower risk of coronary artery disease and cardiovascular mortality . PhysicaTT has demonstrated lipid-lowering activity [TT supplementation in animal models leads to a decrease in Chol and LDL-Chol, and an increase in HDL-Chol levels in the blood [TT was demonstrated on diet-induced hyperlipidemia in mice by the decrease in Chol and LDL-Chol blood levels [TT has shown lipid-lowering activity [TT as a sport supplement to counterbalance and equilibrate lipid profile to create lipid level balances in situations of high physical stress or demanding exercise.activity . TT supphe blood ,58. Addid levels . These ractivity ,22,24. Pactivity . Phenoliactivity . Saponinactivity . These rTT has become popular for its anabolic properties that could potentially raise the blood testosterone level and stimulate hypertrophy of the skeletal muscles of Bulgarian weightlifters [Intensive exercise has been shown to induce a dysfunction of the hypothalamic-pituitary-testicular axis, especially testicular impairment by causing a suppression of testosterone secretion during the latter stages of exercise. Additionally, during intense prolonged exercise, Adrenocorticotropic Hormone (ACTH) concentrations increase, resulting in a significant release of cortisol . These atlifters .TT supplementation on testosterone levels [TT has shown to increase testosterone. The properties of TT may also influence the effects on testosterone levels because of their pleiotropic effects such as: (i) direct action of the pituitary gland that secretes more LH; (ii) increased levels of dehydroepiandrosterone (DHEA) molecules; (iii) suppression of aromatase that prevents estrogen synthesis; (iv) antioxidant effect that protects against endothelial dysfunction in the gonads; (v) anti-inflammatory properties [TT’s steroidal saponins, such as gitonin, protodioscin, and tribulosaponins A and B, would be responsible for stimulating testosterone production by these multiple pathways [TT significantly decreased. Moreover, TT provided no benefit on plasma testosterone levels in rugby players, with a marked decrease in the testosterone/epitestosterone ratio [TT was no more effective than placebo for serum total testosterone concentration [TT have been ratified by some investigators, as shown above, but are questioned by other studies.Investigators in 4 studies reported beneficial effects of e levels ,15,22,24e levels ,60,61,62e levels  in whichoperties ,46,64. Tpathways , includene ratio . Likewisntration . The tesTT supplementation [TT [TT on hypothalamic-pituitary-testicular axis.The indirect hormonal behavior of testosterone showed contradictory results. Poprzecki et al.  showed aentation . Increastion [TT ,11. PerhTT administration compared to control [TT supplementation group in both testosterone and DHT hormones. These contradictory findings may be explained by the different percentage of steroidal saponins, the dose or the bioavailability of TT, which were higher in the animal study [DHT is an active androgen of testosterone. Saponins, mainly protodioscin, induce the transformation of testosterone into DHT, through their intervention on the enzyme 5-α reductase . Previou control . However control  reportedal study .® athletes, the results showed a decrease in cortisol from baseline and when compared to the control group. In addition, these investigators [Two studies included in this review evaluated cortisol levels, showing opposite results. Milasius et al. , in endutigators  describeTT as an anabolic sports supplement to counteract and balance biomarker levels to create balances at the hormonal level.These results showed uncertainty about the use of TT supplementation rather than other supplements due to its potential anabolic effects, uncommon side effects [TT is not listed as a banned substance by the WADA [TT showed significant improvements in blood concentration of GH and IGF-1 [TT treatment may be a successful form of skeletal muscle protection and in accelerating skeletal muscle regeneration/repair. In opposition, supplementation with 1250 mg of TT extracts decreased the plasma level of IGFBP-3 and IGF-1 in male boxers after both types of training (volume/intensity) [TT on IGF-1 and GH, the goal of supplementation is met by improving athletic performance and muscle condition.Athletes may prefer to use  effects , and becthe WADA . IGF-1 ithe WADA . A 12-wend IGF-1 . These rTT could restore and modulate the hormonal levels diminished by age.Of particular interest for middle-aged adults are the results obtained in the study of Wilk et al.  on a popTT [TT. Regular physical exercise and sports supplementation induce physiological and metabolic adaptations, which influence sports performance [There are many published reviews on the phytochemical and/or pharmacological profile of TT ,64, howeformance . To moniformance . TherefoTT supplementation widely varied across studies. Despite these limitations, the strengths of this systematic review rely in the use of the PRISMA guidelines [Several limitations need to be acknowledged. First, the systematic review included a limited number of records. Moreover, most of these studies included a small sample size and only male participants. Second, the intervention characteristics of the studies such intensity and duration of exercise, timing and dose of idelines  and the idelines , the useTT supplementation is safe although side effects have been reported in other animal and human studies. Given the improvements in certain biomarkers, it may also benefit physically active adults. The pleiotropic effect of TT may act to counteract and modulate certain physiological biomarkers out of the normal range by intense and stressful physical exercise. In this regard, some examples are hematological, lipid, renal, and hepatic biomarkers. There are doubts about the potential effect on muscle damage, anti-inflammatory effect, and hormonal biomarkers. In addition, it lacks effect on the immune system. However, more research is needed to confirm the potential health benefits of TT supplementation in healthy adults without chronic diseases.The evidence presented in the studies in this systematic review showed that


Cellular metabolism is critical for the host immune function against pathogens, and metabolomic analysis may help understand the characteristic immunopathology of tuberculosis. We performed targeted metabolomic analyses in a large cohort of patients with tuberculous meningitis (TBM), the most severe manifestation of tuberculosis, focusing on tryptophan metabolism.We studied 1069 Indonesian and Vietnamese adults with TBM (26.6% HIV-positive), 54 non-infectious controls, 50 with bacterial meningitis, and 60 with cryptococcal meningitis. Tryptophan and downstream metabolites were measured in cerebrospinal fluid (CSF) and plasma using targeted liquid chromatography mass-spectrometry. Individual metabolite levels were associated with survival, clinical parameters, CSF bacterial load and 92 CSF inflammatory proteins.CSF tryptophan was associated with 60-day mortality from tuberculous meningitis  both in HIV-negative and HIV-positive patients. CSF tryptophan concentrations did not correlate with CSF bacterial load nor CSF inflammation but were negatively correlated with CSF interferon-gamma concentrations. Unlike tryptophan, CSF concentrations of an intercorrelating cluster of downstream kynurenine metabolites did not predict mortality. These CSF kynurenine metabolites did however correlate with CSF inflammation and markers of blood-CSF leakage, and plasma kynurenine predicted death . These findings were mostly specific for TBM, although high CSF tryptophan was also associated with mortality from cryptococcal meningitis.TBM patients with a high baseline CSF tryptophan or high systemic (plasma) kynurenine are at increased risk of mortality. These findings may reveal new targets for host-directed therapy. Hydrocephalus, stroke, seizures, focal neurological deficits, and loss of consciousness are common complications and lead to death in around 30% of patients, even when treated with anti-tuberculosis drugs and adjuvant corticosteroid therapy.1 Development of more effective host-directed therapy is hampered by a lack of knowledge on the biological pathways involved in the immunopathology of TBM.2Tuberculous meningitis (TBM) is the most severe manifestation of tuberculosis affecting approximately 160,000 adults each year.3 This study did not include HIV-infected patients and the association was not validated in other populations.5 Moreover, there is a need to investigate the downstream metabolites in the kynurenine pathway  metabolites could help unravel underlying biological mechanisms in TBM. Previously, using a large-scale metabolomics analysis, we found that lower cerebrospinal fluid (CSF) tryptophan concentrations were associated with survival of TBM patients in Indonesia. pathway , throughWe therefore sought to define and validate the relationship between tryptophan and its metabolites and survival from TBM in large, independent populations, including HIV-positive individuals. We aimed to confirm that a higher CSF tryptophan would predict higher mortality across different populations and we hypothesized that high tryptophan would be associated with a higher CSF bacterial load, more inflammation and lower downstream kynurenine metabolites. We lastly sought to investigate how systemic (plasma) metabolite concentrations linked to outcome.7 and Hasan Sadikin hospital in Indonesia between 2007–2019.5 TBM patients were defined as having ‘definite TBM’ if they had either microbial confirmation by Ziehl-Neelsen staining, positive CSF culture or GeneXpert. Based on previous studies,5 probable TBM was defined as clinically suspected TBM fulfilling at least 2 out of the following 3 criteria: CSF leukocytes ≥ 5 cells/μL, CSF/blood glucose ratio < 0.5, and CSF protein > 0.45 g/L. Patients were treated with antibiotics according to national guidelines for 180 days minimally, and received adjunctive dexamethasone starting at 0·3 mg/kg for grade I and 0·4 mg/kg for grade II or III tuberculous meningitis and tapered thereafter.8 Patients were followed-up clinically or by phone up until day 180 from admission. Primary outcome was 60-day survival, when most deaths attributable to TBM occur. As a secondary endpoint, earlier and later mortality were explored separately. We ensured equal power for both time windows by separating them by the median time to death for those patients who died during the total follow up of 180 days.Patients with subacute meningitis were included from the Hospital for Tropical Diseases and Pham Ngoc Thach Hospital for Tuberculosis and Lung Disease in Ho Chi Minh City, Vietnam between 2011–2014,Patients without an infection (non-infectious controls) were included from the same sites. In Indonesia, patients in this group had undergone a lumbar puncture for suspected central nervous system infection or subarachnoid bleeding, but infection was excluded by negative microscopy, GeneXpert and bacterial culture, and CSF leucocytes < 5 cells/μL and CSF/blood glucose ratio ≥ 0.5. In Vietnam, patients were included as controls if they had undergone a lumbar puncture, but an alternative, non-infectious, diagnosis was confirmed. In both sites, none of the non-infectious controls received anti-tuberculosis treatment. HIV-negative patients with microbiologically confirmed bacterial meningitis and HIV-positive patients with cryptococcal meningitis patients were included from the same sites.Ethical approval was obtained from the Ethical Committee of Hasan Sadikin Hospital, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia and from the Oxford Tropical Research Ethics Committee in the United Kingdom, the Institutional Review Boards of the Hospital for Tropical Diseases and Pham Ngoc Thach Hospital in Vietnam. Written (Vietnam) or oral (Indonesia) consent to be included in the study, for storage of surplus sample, and to obtain follow-up data was obtained from patients or close relatives of patients who were unconscious. The paper adheres to the STROBE methodology.9 CSF and plasma metabolites were measured using targeted a liquid chromatography tandem mass spectrometry (LC-MS) method with a system comprised of a 1290 Infinity II U-HPLC coupled to an Agilent 6495 Triple Quadrupole mass spectrometer . Metabolites were extracted from plasma or CSF (10 μL) using 90 μL of acetonitrile/methanol/formic acid (74.9:24.9:0.2 v/v/v) containing stable isotope-labeled internal standards . The samples were centrifuged , and the supernatants were injected directly onto a 150 × 2 mm, 3 μm Atlantis HILIC column . The column was eluted isocratically at a flow rate of 250 μL/min with 5% mobile phase A (10 mM ammonium formate and 0.1% formic acid in water) for 0.5 minute followed by a linear gradient to 40% mobile phase B (acetonitrile with 0.1% formic acid) over 10 minutes. Pairs of pooled samples generated using aliquots of all samples in the study were included every 20 samples correct for MS sensitivity drift and for quality control analyses. Sample stability over the 7 years study inclusion and 4-year storage time was checked by plotting metabolite levels of definite TBM patients against storage time. Tryptophan metabolites were measured using the following multiple reaction monitoring transitions: 3-hydroxyanthranilic acid (154.1 -> 136.0), 3-indoleacetic acid (176.1 -> 130.1), 3-methoxyanthranilate (168.1 -> 150.0), 5-hydroxyindoleacetic acid (192.1 -> 146.0), 5-methoxytryptophol (192.1 -> 130.0), kynurenic acid (190.1 -> 144.1), kynurenine (209.1 -> 94.0), tryptophan (205.1 -> 187.9), N-acetylserotonin (219.1 -> 160.0), niacinamide (123.1 -> 80.1), quinolinic acid (168.0 -> 149.9) and xanthurenic acid (206.1 -> 132.0). Absolute concentrations were determined using external calibration curves created via serial dilution of stable isotope-labeled compounds in CSF and plasma. These compounds were sourced from Cambridge Isotope Labs: 3-indoleacetic acid-d7 (DLM-8040), anthranilic acid-13C6 (CLM-701), 5-HIAA-13C6 (CLM-9936), kynurenic acid-d5 (DLM-7374), L-kynurenine-d6 (DLM-7842), L-tryptophan-13C11 (CLM-4290) and niacinamide-13C6 (CLM-9925). Peak abundances were manually integrated using the MassHunter software provided by the LC-MS manufacturer.CSF and blood samples were centrifuged for 15 minutes according to local protocols (865 – 3000 × g) and supernatants were stored at −80’C.10 and inferred from CSF M. tuberculosis culture. CSF inflammatory cytokines in 178 Indonesian HIV-negative TBM patients were measured using a multiplex proximity extension assay (Olink) in two batches. Olink uses a multiplex assay that simultaneously recognize 96 target proteins through specific paired-antibodies which are coupled with unique oligonucleotides, for quantitative PCR measurement.11 For each protein, overlapping samples from two batches were fitted in a linear regression model, where the linear components were subsequently extracted, and used as correction factors for batches normalization. In 304 Vietnamese HIV-negative patients, 10 human cytokines were measured in CSF with Luminex multiplex bead array technology .4 CSF total protein was used as proxy for blood-CSF barrier disruption as it showed a near-perfect correlation with the established marker CSF-serum albumin (r2=0.98).12The CSF mycobacterial load was inferred qualitatively by comparing patients with negative versus positive CSF culture, and semiquantitively from the GeneXpert Ct-values as described previously,13, using the R packages survival, tableone, dplyr, openxlsx, pheatmap, grid, and ggplot2. Correlation analyses between metabolites levels, and between metabolites levels and clinical and inflammatory parameters, were calculated using spearman-rank correlation. The impact of baseline CSF and plasma metabolite levels on 60-day survival was tested in a Cox-regression model, adjusted for sex, age, and HIV status as covariates. The model stratified by study site as mortality is known to be higher in the Indonesian5 than in the Vietnamese7 cohort. An analysis plan was made before the study, and correction for multiple testing using the Benjamini Hochberg method was done if multiple comparisons were done in primary analysis.Only metabolites and proteins with a coefficient of variation (CV) of the pooled samples < 30% and < 25% missing values, respectively among TBM patients were further included in the analysis. Remaining missing metabolite values after quality control were replaced with half of the minimum measured value of the corresponding metabolite, and log2-transformed subsequently. Statistical analyses were performed in R 4.0.414 The rate of mycobacterial confirmation was 64.1%. Sixty-day mortality, the primary endpoint in the analysis, was 21.6%. Patients who died within 180 days from admission did so after a median of 14 days. A 14-day cut-off was therefore used to distinguish early from late mortality as a secondary endpoint.We studied 1069 adults with TBM, 390 from Indonesia and 679 from Vietnam . Patient12 was higher in Indonesian  than Vietnamese  patients. CSF polymorphonuclear cell counts were higher in the Indonesian than in the Vietnamese patients where it showed a bimodal distribution associated to study site (There were some differences between the populations. Indonesian patients presented with more severe diseases (91.9% grade with grade II or III) than Vietnamese patients (62.2%). Also, CSF total protein, a proxy for blood-CSF barrier leakage,udy site  and straTen metabolites showed detectable levels in >75% of patients and passed quality control, while two metabolites, 3-methoxyanthranilate and 5-hydroxyindoleacetic acid, were detected in less than 75% of patients and excluded from further analysis. Metabolite measurements showed stable concentrations over and were not affected by year of patient inclusion and duration of sample .3 higher baseline CSF tryptophan levels predicted 60-day survival in patients with TBM , all analyses corrected for age, sex and HIV status, and stratified for cohort, 5 Baseline CSF tryptophan was associated with both early  and late  mortality  than culture positive  TBM patients. Similarly, among patients with CSF GeneXpert-confirmed tuberculous meningitis patients, we did not find a correlation between CSF tryptophan and quantitative PCR results  was much more marked than the decrease in CSF tryptophan , which is important for the upregulation of TNF among other pro-inflammatory cytokines in a mouse model,20 in line with our CSF findings. The increased CSF kynurenine levels we found in TBM have been reported before in bacterial meningitis22 and in cerebral malaria23 and in plasma from pulmonary TB patients.17 Of interest, nicotinamide can inhibit M. tuberculosis growth, and can compete with isoniazid for antimycobacterial effects.24 We did however not find a detrimental effect of a higher nicotinamide, possibly because of its complex biology, i.e. it can also be produced by M. tuberculosis when human dietary niacin intake is limited.25We examined whether higher CSF tryptophan concentrations reflected higher concentrations of downstream kynurenine metabolites that may have neurotoxic (quinolinic acid) or lower levels of the metabolites that may have neuroprotective (kynurenic acid) properties26 should be priorities as adjuvant therapeutic candidates for future personalized trials.Strengths of our study include the large numbers of clinically well-phenotyped patients from multiple independent study sites in Indonesia and Vietnam, including a significant proportion of HIV-positive patients. We moreover used a sensitive triple quadrupole (QQQ) mass spectrometry method specifically designed to accurately target the tryptophan metabolites. Absolute quantification of a subset of metabolites further facilitated interpretation. Due to differences in polarity of the downstream tryptophan metabolites, we could not measure the complete tryptophan pathway. The availability of CSF at baseline only, limits our ability to understand how changes in tryptophan metabolism influence mortality. And we infer our observations from lumbar CSF, which reflects biological processes from both the blood and the brain. The use of ventricular CSF and potentially animal models, could help establishing what processes in the brain parenchyma take place. Animal studies are warranted to see whether pharmacological induction of IDO1 (for instance with recombinant IFN-γ), or inhibitory tryptophan analoguesIn summary, we confirm the importance of CSF tryptophan to outcome from HIV-negative and HIV-positive adults with TBM, independent from downstream kynurenine metabolism, bacterial load and inflammation. We additionally show the potential importance of systemic kynurenine as a predictor of mortality. Better understanding of the metabolic pathways associated with TBM may lead to more targeted therapies, as adjuvant immunotherapy may modulate the aberrant metabolic pathways and thus improve outcome.The clinical metadata and LC-MS data before pre-processing is available as Supplement 1


Extracellular vesicles (EVs) are a versatile group of cell-secreted membranous nanoparticles present in body fluids. They have an exceptional diagnostic potential due to their molecular content matching the originating cells and accessibility from body fluids. However, methods for EV isolation are still in development, with size exclusion chromatography (SEC) emerging as a preferred method. Here we compared four types of SEC to isolate EVs from the CSF of patients with severe traumatic brain injury. A pool of nine CSF samples was separated by SEC columns packed with Sepharose CL-6B, Sephacryl S-400 or Superose 6PG and a ready-to-use qEV10/70 nm column. A total of 46 fractions were collected and analysed by slot-blot followed by Ponceau staining. Immunodetection was performed for albumin, EV markers CD9, CD81, and lipoprotein markers ApoE and ApoAI. The size and concentration of nanoparticles in fractions were determined by tunable resistive pulse sensing and EVs were visualised by transmission electron microscopy. We show that all four SEC techniques enabled separation of CSF into nanoparticle- and free protein-enriched fractions. Sepharose CL-6B resulted in a significantly higher number of separated EVs while lipoproteins were eluted together with free proteins. Our data indicate that Sepharose CL-6B is suitable for isolation of EVs from CSF and their separation from lipoproteins. Extracellular vesicles (EVs) are cell-originated nanoparticles ranging in size from roughly 30 to 1000 nm and containing a hydrophilic inner core ,3,4. ConDespite the remarkable progress achieved in EV research during the last decade, some technical issues still need to be resolved, particularly regarding the isolation of EVs from body fluids. Such isolation is challenging due to the inherent features of EVs, such as diversity in size, density, and molecular content on the surface, but also due to the characteristics of the body fluids, which differ in viscosity, pH, and in quantity and types of EVs, proteins, lipoproteins, and other nanoparticles. Depending on the isolation method, proteins and non-EV nanoparticles can co-isolate with EVs and thus impair the purity of the EV isolate. Consequently, diverse principles are applied to isolate EVs, focusing on EV size, density, and/or surface molecules. To date, the most commonly used method for isolating EVs is ultracentrifugation, followed by ultrafiltration and polymer-based precipitation, which have been extensively described in several reviews ,17,18,19Size exclusion chromatography (SEC) is currently less commonly used for EV isolation than the above-mentioned methods, but it might soon flourish, as it can yield intact and contaminant-free EVs ,23,24,25Being in direct contact with the central nervous system (CNS), CSF is a particularly valuable source of biomarkers for the ongoing processes in the nervous tissue ,33. CSF The objective of this study was to compare four gravity-based SEC methods in their ability to isolate EVs from intracranial CSF of patients with severe TBI. The SEC methods included three columns packed with Superose 6PG, Sephacryl S-400 or Sepharose CL-6B and the ready to use qEV10/70 nm column. The yield, purity and size distributions of the resulting isolated EVs were considered, as well as the simplicity and the duration of the separation process. Our results indicate that Sepharose CL-6B outperforms the other SEC methods applied here.The study enrolled 3 adult patients with severe traumatic brain injury (TBI) who received ventriculostomy as part of their therapeutic intervention for intracranial pressure (ICP) monitoring and management in the intensive care unit of the Clinical Hospital Centre Rijeka and Pula General Hospital in Croatia. The patients were included in the study after one of their family members signed an informed consent form previously approved by the Ethical Committee. Patients were eligible if they were 18 to 80 years of age and had no immunological, malignant or chronic inflammatory disease. The research was conducted in accordance with the Declaration of Helsinki and local laws and regulations.CSF samples were obtained from ICP catheters using sterile 10 mL syringes under aseptic conditions by an appropriately trained physician. After being collected every 24 h during the first 3 days post injury, the samples were aliquoted into low-protein-binding tubes  and stored at −80 °C. Sampling was conducted during 2020 and 2021. Samples had a maximum of two freezing–thawing cycles.Sepharose CL-6B , Sephacryl S-400 HR  and Superose 6 Prep Grade  were packed into separate 1.5 × 50 cm glass columns fitted with a 30 µm bottom frit and equipped with flow adaptors . Approximately 75 mL of each resin was packed by applying distilled water under gravity flow. Columns were washed with at least 3 bed volumes of distilled water and equilibrated with 2 bed volumes of sterile PBS  as running buffer prior to the first SEC run. Commercial qEV10/70 nm  column was washed and equilibrated in PBS as per the manufacturer’s instructions. The CSF-pool for SEC was created by combining equal volumes of 9 CSF samples from 3 severe TBI patients. The CSF-pool of 2.8 mL was loaded by a 5 mL sterile plastic syringe on each column and 50 fractions of 1.5 mL were collected in low-protein-binding tubes  for each run. SEC was performed at room temperature and by gravity flow with the running buffer positioned 20 cm above the column. Columns were washed with at least two bed volumes of PBS between SEC runs.v/v), 10% SDS (w/v), 0.05% bromophenol blue (w/v), 2-mercaptoethanol) and boiled at 95 °C for 10 min. 25 µL of individual CSF and CSF-pool sample or 200 µL of SEC fractions was loaded onto 10% or 12% polyacrylamide gel and electrophoresed  in 1× running buffer  on 90 V to 150 V. Proteins were transferred to a 0.2 µm nitrocellulose membrane  at the constant voltage of 20 V for 35 min using a semi-dry transfer unit . Membranes were stained with Ponceau S (0.1% Ponceau S in 5% acetic acid), followed by blocking with 5% milk in Tris-buffer saline  for 15 min, and probed overnight at 4 °C in 5% bovine serum albumin /TBS-T (TBS supplemented with 0.1% Tween 20) with rabbit monoclonal antibodies against CD81 (#56039), CD9 (#13174), apolipoprotein E (#13366) or albumin (#4929), and mouse monoclonal antibody against apolipoprotein AI (#3350), diluted 1:1000 . Membranes were washed three times for 5 min in TBS-T and incubated with horseradish peroxidase-linked anti-rabbit (#7074) or anti-mouse (#7076) secondary antibodies  at room temperature for 30 min. After additional washing in TBS-T, the signal was visualised using SignalFire Plus ECL Reagent or SignalFire Elite ECL Reagent  and imaged with an imager .Individual CSF samples, CSF-pool or SEC fractions were mixed with reducing 5× Laemmli buffer  was soaked in distilled water and placed in the slot blot apparatus . SEC fractions in volume of 200 µL were loaded per slot and applied to the membrane by a vacuum pump. Each slot was then rinsed three times with 1 mL of PBS  before removing the membrane from the slot blot apparatus. Membranes were stained with Ponceau S, blocked, and blotted for CD81, CD9, ApoAI, ApoE, and albumin antibodies, as stated above. Imaging and quantification were performed using the imager and Image Studio Digits software . Arbitrary units obtained by quantification of single membrane were normalised against the highest value and plotted as percentages.Tunable resistive pulse sensing (TRPS) was used to measure nanoparticle concentration and size distribution in SEC fractions. The measurements were conducted using a qNano Gold instrument, NP400 nanopores and Control Suite 3.4 software according to the manufacturer’s instructions . Briefly, nanopores were wetted, coated, and equilibrated using the Izon reagent kit and calibration runs were performed with 350 nm carboxylated polystyrene beads at two pressure conditions: 6 mbar and 3 mbar. To ensure comparability between measurements, nanopore stretch and voltage were individually adjusted for each measurement session to detect particles in the 185–740 nm diameter range and maintain baseline current at approximately 135 nA. All samples were suspended at a 1:1 ratio in electrolyte solution provided in the Izon reagent kit and measured for 5 min at 6 mbar. If more than 500 particles were recorded in 5 min, an additional measurement of another 5 min was conducted at 3 mbar. To avoid cross-contamination, nanopore was flushed with electrolyte solution for at least 5 min at 20 mbar pressure between each sample measurement. The identical concentration fraction, i.e., the particle size range over which the particle concentration is calculated, from 185 to 740 nm, was applied for all measurements prior to any data analysis. Particle concentration and size distribution were determined only for TRPS-positive fractions whose measurements attained at least 500 nanoparticles at both pressure conditions after 5 min of recording. Recordings containing less than 500 nanoparticles in 5 min were categorised as negative, and corresponding fractions were considered as TRPS-negative fractions.The zeta potential of nanoparticles was determined in SEC fractions by applying TRPS measurement on a qNano Gold instrument. Only fractions with at least 500 nanoparticles detected under two pressures as described above have been used for zeta potential measurement. Measurements were performed on NP400 nanopores. Initial calibration with carboxylated polystyrene beads was conducted at 1 pressure condition and 3 applied voltages, generating a corresponding baseline current of 85 nA, 110 nA, and 135 nA, followed by an additional measurement at a second pressure condition at the highest voltage applied in the previous step. Sample measurements were completed at the highest calibration voltage and pressure. At least 500 particles were recorded per each measurement.The samples were suspended and fixed in an equal volume of 6% paraformaldehyde (PFA)/3% glutaraldehyde (GA) solution for 15 min at room temperature, resulting in the fixed sample in 3% PFA/1.5%GA. A 10 μL drop of fixed sample was then deposited on a formvar/carbon-coated, 200-mesh copper grid  that was placed on parafilm in a Petri dish for 60 min incubation on ice in a humidified atmosphere. After incubation, the excess liquid was removed by filter paper. The grids were incubated with 0.5% aqueous solution of uranyl acetate for a few seconds to contrast the sample. Then, the grids were consecutively transferred three times to 200 μL drops of distilled water and air dried. After drying, grids were observed with the 120-kV transmission electron microscope .p < 0.05.Statistical analysis was performed using GraphPad Prism 8 . The Shapiro–Wilk test was performed to assess the normality of data distributions, followed by one-way ANOVA with a post-hoc Tukey test. The statistical difference was accepted when To compare EV isolation by different SEC methods, we applied CSF samples from severe TBI patients. Our previous data showed that intracranial CSF collected during the first days after the injury contained EVs ,39. ThusBoth individual CSF samples and the resulting CSF-pool were screened for the presence of EVs, lipoproteins, and albumin by western blot analyses . The mosAfter showing that the CSF-pool contains albumin, EVs, and lipoproteins, we proceeded with the SEC separation by applying gravity flow through the following SEC stationary phases, respectively: Superose 6PG, Sephacryl S-400, and Sepharose CL-6B, which have been packed into columns, and the commercially available ready-to-use column qEV10/70 nm . To enabTo estimate the efficacy of SEC methods in separating CSF to nanoparticles and free proteins, 46 successive fractions were collected for each SEC method and individually analysed. First, a slot blot was performed followed by Ponceau staining to detect protein- enriched fractions . SepharoTRPS measurements were performed only in approximately the first half of fractions since the protein-enriched fractions, as detected by Ponceau staining, caused clogging of the nanopore and hampered the measurement. With the predefined conditions for a fraction to reach either the required number of detected nanoparticles or the maximal time of measurement, we were able to distinguish between nanoparticle-positive and nanoparticle-negative fractions. The TRPS particle size distributions of the nanoparticle-positive fractions were continuous and unimodal, while the distributions of the nanoparticle-negative fractions were fragmented and multimodal, with insufficient number of particles for a reliable statistical analysis .To compare the quality of SEC separations, TRPS measurements and quantified Ponceau staining of fractions were plotted together for each SEC method  was compared, the differences between SEC methods became more obvious b. SepharSince applied SEC methods showed a different efficacy in separation of nanoparticles from free proteins contained in CSF, we decided to further characterise nanoparticle-positive fractions by measuring zeta potential . The disAfter discovering that Sepharose CL-6B effectively separated nanoparticles from the majority of proteins in human CSF, our next goal was to determine the identity of the isolated nanoparticles. Apparent candidates were EVs and lipoproteins, since both types of nanoparticles were detected in the CSF-pool applied for SEC separation . MoreoveTo provide a better overview of the protein levels in individual fractions obtained with Sepharose CL-6B, we quantified the signals immunodetected on slot blot and plotted them on one graph a. All coExtracellular vesicles (EVs) from cerebrospinal fluid (CSF) are expected to contain molecular cargo that could reveal biochemical changes in nervous tissue. To enable the detection of such biomarkers, it is important to extract EVs from clinical CSF in the highest possible purity, quantity, and heterogeneity. These criteria for EV-isolation might be met by size exclusion chromatography (SEC)—a well-established method for protein purification but not yet a common and standardised approach for EV-isolation. Here we demonstrated the differences between four SEC methods in their ability to enrich EVs from human CSF.For this purpose, we analysed CSF samples obtained from adult severe traumatic brain injury (TBI) patients whose treatment included monitoring and management of intracranial pressure. Our study included intracranial CSFs drained during the first 3 days after injury. In the majority of individual CSFs, we detected high levels of albumin and apolipoproteins indicating blood content. This is consistent with the brain injury severity and the invasive procedure required for accessing the intracranial CSF . EV protWe applied the same volume of CSF-pool for four SEC columns based on different stationary phases: Sepharose CL-6B, Sephacryl S400, Superose 6PG and qEV10/70 nm. By this direct comparison we could show that all four analysed SEC methods were successful in separating nanoparticles of roughly 200 nm in size from free proteins, such as albumin. The importance of clear separation of EVs and free proteins has been recently demonstrated for the case of L1CAM, a transmembrane protein expressed on neural cells, which was thought to be suitable for immunoisolation of neuron-derived EVs . HoweverOur results further indicate that the SEC stationary phase can largely influence the quantity of isolated EVs. Sepharose CL-6B and Superose 6PG provided significantly higher levels of concentrated EVs in comparison to Sephacryl S400 and qEV10/70 nm, which resulted in up to two to four times lower amounts of enriched EVs, respectively b. MoreovIn conclusion, this study provides a protocol for gravity-based SEC that can be easily applied in any lab. We demonstrate that Sepharose CL-6B and Superose 6PG outperform Sephacryl S400 and qEV10/70 nm in enrichment of EVs from clinical CSF. The distribution of EVs in fractions is most likely affected by manual collection of fractions, and here applied gravity-based columns are not suitable for upgrading to large-scale analyses. Still, all compared SEC stationary phases were able to separate EVs from lipoproteins and free proteins and further improvements might be achieved by choosing and/or further developing stationary phase materials.


Compelling experimental evidence confirms that the robustness and longevity of mixed chimerism (MC) relies on the persistence and availability of donor-derived hematopoietic stem cell (HSC) niches in recipients. Based on our prior work in rodent vascularized composite allotransplantation (VCA) models, we hypothesize that the vascularized bone components in VCA bearing donor HSC niches, thus may provide a unique biologic opportunity to facilitate stable MC and transplant tolerance. In this study, by utilizing a series of rodent VCA models we demonstrated that donor HSC niches in the vascularized bone facilitate persistent multilineage hematopoietic chimerism in transplant recipients and promote donor-specific tolerance without harsh myeloablation. In addition, the transplanted donor HSC niches in VCA facilitated the donor HSC niches seeding to the recipient bone marrow compartment and contributed to the maintenance and homeostasis of stable MC. Moreover, this study provided evidences that chimeric thymus plays a role in MC-mediated transplant tolerance through a mechanism of thymic central deletion. Mechanistic insights from our study could lead to the use of vascularized donor bone with pre-engrafted HSC niches as a safe, complementary strategy to induce robust and stable MC-mediated tolerance in VCA or solid organ transplantation recipients. The establishment of mixed chimerism (MC), as a result of donor-specific bone marrow transplantation, is one of the few approaches that have been reliably associated with “permanent,” rejection-free solid organ allograft acceptance in different species, notably in human renal transplantation –4. Howevin vivo depend on their specific microenvironment, which has been historically called the hematopoietic inductive microenvironment  has highlighted its benefits for restoration of complex injuries or defects , 31. Thec), LEW (RT1Al), Wistar-Furth (RT1Au) rats were purchase from Harlan Laboratories . The green fluorescent protein (gfp) transgenic SD rats were provided by Dr. Noriko Murase (University of Pittsburgh). Male B6 (H2b), B10.A (H2k), BALB/c (H2d), B6 nude (B6.Cg-nuFoxn1/J), BALB/c nude (CBy.Cg-nuFoxn1/J), B6.SCID (B6.CB17-Prkdcscid/SzJ), B6.Rag1 (B6.129S7-Rag1tm1Mom/J) mice were purchased from The Jackson Laboratories . All animals were maintained under specific pathogen-free condition and procedures were performed according to the guidelines of the Institutional Animal Care and Use Committee at the University of Pittsburgh.Male BN ; 3) skin/muscle/bone (VSMB) allograft ; 4) hind-limb allograft . For secondary skin grafting, full-thickness skin grafts were transplanted onto the dorsum of recipients  and mice (weighing 25-30g) were performed as previously described –37. 1) vemur BMC , injectecipients , 39. Forcipients , 41. We 8) BMT (Rat VCA recipients were treated as follows: 0.5ml anti-rat lymphocytes serum (ALS), intraperitoneal injection (i.p.) on postoperative day (POD) -4 and 0, cyclosporine A (0.6mg/kg/day i.p.) for the first 7 days after transplantation, followed by rapamycin  for 14 days. Mouse VCA recipients were treated with co-stimulation blockade plus rapamycin protocol based on our previous study, in which we demonstrated a stable MC and skin allograft tolerance could be established across MHC barriers by a noncytotoxic, irradiation-free approach using costimulation blockade plus rapamycin treatment and high dose (2 x 108) BMT .The transplanted allografts were inspected daily for clinical signs of rejection. Graft rejection was defined as epidermolysis/desquamation of the donor skin. Allografts displaying a healthy skin component with normal hair growth > 120 days post transplantation were considered signs of graft acceptance , 42. Hisd, CD19, CD3, CD4, CD8, CD11b, CD11c, NK1.1, Ter119, B220, TCRγδ, IgM, IgG, Vβ5.1/5.2, Vβ8, and Vβ11 for flow cytometry were purchased from AbD Serotec , BD Pharmingen , and eBioscience . At the time points noted after transplantation, cells harvested from host rats or mice were analyzed using LSRII flow cytometer  and FlowJo software . For analysis of multilineage chimerism, transplant recipients were assessed serially for the presence of donor hemopoietic cell lineages in peripheral blood (PB), bone marrow, thymus, lymph nodes, and spleen by detecting anti-rat MHC-class I antibody (RT1Ac) or gfp, and linage-specific cell surface markers. For analysis of T cell receptor (TCR) Vβ families, PB was obtained from B6 recipients that received B10.A hind-limb allografts at different time points after transplantation. The proportion of CD4+ T cells expressing each Vβ was determined  or anti-DEC205 from Santa Cruz , followed by fluorescent secondary antibodies. DAPI (Sigma-Aldrich) was used to stain the nucleus. The slides were evaluated in a blinded fashion.t-test for two groups or by one-way ANOVA test for multiple comparisons. The allograft survival times between groups were calculated and compared using a Logrank test. ‘p’ values < 0.05 were considered significant for all statistical tests.Values are expressed as mean ± SEM. Differences between experimental groups were analyzed by Student’s In BN to LEW rat VCA, with the same immunosuppressive treatment  up to 24 weeks post transplantation (+ cell chimerism (4-6%) persisted for over 24 weeks in the PB of both VSMB and hind-limb recipients, while only 0.5-1% of donor gfp+ cells were detected in either VSM or VSMB+BMC recipients during the 2-8 weeks post transplantation and disappeared thereafter (+ cell profile (percentage of chimeric subpopulations) in PB at 6 weeks , mesenteric lymph nodes , spleen (11%), thymus (9%), and bone marrow from adjacent bone marrow (5%) as well as remote bone marrow (5.5%) (+ cells (94%) in thymus were CD4+CD8+ cells, a transition status of T-cell differentiation during its maturation, demonstrating the undergoing maturation of donor T cell progenitors in recipient thymus  were detected in PB of B6.SCID recipients with VSMB at 3 weeks and increased to 50% at 8 weeks post transplantation (n=4) (+ T-cells in PB of VSM+BMC recipients (n=4) were only detected at 3 weeks, but not 8 weeks, post transplantation , then resumed quickly to 6.0-12.0% and persisted at 4.5-5.5% for > 24 weeks (60% of recipients). In some recipients (30%), the donor cell chimerism reached 90%. No chimeric recipients demonstrated signs of graft versus host disease (GVHD). All remaining skin/muscle allografts maintained their engraftment permanently (>150 days) and the tolerance was confirmed by acceptance of secondary donor skin grafts (data not shown).To assess whether the donor bone component of VCA is imperative for maintenance of stable MC, we removed the bone component from recipients that received VSMB of gfp-SD rats at 1, 2, 4, 20 weeks after transplantation. The remaining skin and muscle tissue were reconstructed as vascularized myocutaneous allografts. As shown in + and osterix+, and donor HSCs were also detected by co-staining of gfp+ cells with c-kit+ near osteoblasts . In agreement with previous studies , 44–47, + cells with CD11c+ and DEC205+ (DC markers) as well as cytokeratin (CK)5+ and CK8+ (TEC markers) cells (Since both dendritic cells (DC) and thymic epithelia cells (TEC) have been found to critically contribute to physiological self-tolerance by mediating T cell central deletion –51, we s+ and Vβ11+, but not irrelevant Vβ8+, T cells were significantly reduced in PB of tolerant MC recipients at POD 120 after transplantation , as compared with naïve B6 controls  and BALB/c (MST=11 days) skin grafts. In contrast, B6.Rag1-/- mice receiving tolerant CD4+CD25- T cells accepted B10.A grafts (MST >120 days) and rejected BALB/c skin grafts (MST=10 days). Moreover, throughout a follow-up >12 weeks period after skin transplantation, partial deletion of Vβ5.1/5.2+ and Vβ11+ CD4 T cells was observed in Rag1-/- mice receiving tolerant CD4+CD25- T cells  or age-matched naïve B6 mice were transplanted underneath the kidney capsule of the B6.nude mice , only exhibited transient, lower-level MC in periphery blood, and failed to prevent allograft acute rejection. Similar studies were reported by multiple groups using bone graft transplantation , 52, 53 + cells (94%) in thymus are CD4+CD8+ double positive cells, a transition status of T cell differentiation during its maturation, indicate the homing, proliferating, differentiating and maturating of donor derived HSC in the recipients. Furthermore, at 20 weeks post transplantation, the bone marrow from the excised donor bone grafts still contained 40% - 60% gfp+ cells with typical bone marrow cell phenotype, supporting the hypothesis that the vascularized bone component of VCA bears HSC niches to facilitate a stable MC.Moreover, multilineage chimerism is evidenced by the similar gfp+ cell profile in peripheral blood in comparison with gfp- cell profile in durable chimeric recipients and the fact of most of gfp6 BMC from nude mice as control. Indeed, we found CD3+ T cells in the PB of SCID recipients receiving nude VCA at 3 weeks post nude VCA transplant, which steadily increased to 50% at 8 weeks post transplantation. Moreover, these VCA SCID recipients were capable of rejecting skin allograft after 4 weeks of transplantation, indicating the CD3+ T cells were functional active. In contrast, only transient T cells were detected in the PB of SCID recipients receiving VSM+BMC from nude mice, and they accepted skin allograft after VSM transplantation. These results provided convincing evidences that the donor bone marrow HSC niche of VCA plays a role in establishing a stable MC after transplantation.We designed a novel VCA mouse model, in which hind limb allografts from athymic nude mice donor were transplanted to allogeneic SCID mice to directly test the roles of donor bone marrow HSC niche of VCA in facilitating stable MC. Nude mice have a spontaneous FOXN1 gene deletion resulted in a non-functional thymic cortical defect – athymic, but have functional bone marrow hematopoietic progenitors , 55; whi+ cells in PB of VCA recipients after the bone component being removed at 2, 4, 20 weeks post VCA, transiently decreased, then resumed quickly and persisted at previous level for > 24 weeks (60% of recipients) suggested that donor HSC niches were integrated into the recipient immune system for maintaining the homeostasis of stable MC. It was surprising and fascinating result that the donor cells chimerism reach to 90% in 30% recipients after the bone component being removed at 4 weeks post VCA and without any sign of GVHD. The mechanisms and prospective of these results and the complex interplay between HSC niches in the bone allograft and recipient immune system merits further investigation.We further sough to determine the roles of the donor bone marrow HSC niche of VCA in maintaining a stable MC. Interestingly we found that only two weeks were required for the donor vascularized bone component of VCA to establishing a MC in this rodent VCA model. In this study donor-derived osteoblasts and HSC were detected homing closely to the endosteal region in trabecular bone of chimeric VSMB recipient by immunofluorescence staining, which are in line with the features ascribed to endosteal HSC niche , 57–59 s+CD25- effective T cells from tolerant VCA recipients into Rag1-/- mice failed to reject donor strain skin allograft, but were capable to reject third party skin allografts suggesting the role of clonal deletion of alloreactive cells in the induction and maintenance of donor-specific tolerance in these VCA models. Furthermore, the immuno-fluorescence staining on the thymus sections of tolerant VSMB recipients shows that both donor-derived DC and donor-derived TEC are present in thymic cortico-medullary junction field, as evidenced by co-staining of gfp+ cells with CD11c+ and DEC205+ (DC markers) as well as CK5+ and CK8+ (TEC markers) cells. Since DC and TEC are essential in T cell thymic clonal deletion of self-tolerance (In parallel with the development of stable MC, the donor-specific tolerance to composite tissues, including highly immunogenic skin allografts, was achieved in both mouse and rat bone-containing VCA models as the chimera accepted secondary donor skin allografts, but not third-party skin allografts without immunosuppression. The adoptive transfer of CD4olerance , 60, ourolerance , strongl+ T cells in B6.nude recipients receiving chimeric thymus grafts from B6 tolerant donors will be the result of the migration, differentiation and maturation of nude HSC in the chimeric thymus. The nude mice bearing naïve-thymus acutely rejected both B10.A and BALB/c skin grafts. In contrast, the nude mice bearing tolerant MC thymus acutely rejected BALB/c skin grafts, while demonstrated a prolonged engraftment for donor-specific B10.A skin allografts with 50% of them survived infinitely. Since the frequency of donor-derived DC and/or TEC in the donor thymus was not defined and the peripheral tolerance could not be overlooked, this experiment alone is not sufficient to indicate the extent of the role of central deletion in VCA tolerance. Nevertheless, it is reasonable to speculate that the chimerism in the donor thymus is inducing central tolerance. In addition, by using Vβ5, Vβ11, and Vβ8 TCR probes we evidenced specific deletion of alloreactive T cell clones in peripheral blood of chimeric thymus recipients. These results provided favorable evidence in a hind-limb VCA model that the thymus plays an important role in MC mediated allograft tolerance through a mechanism of intrathymic central deletion of allo-reactive T cell populations.We designed a unique thymus transplant mouse model to directly test the role of the chimeric thymus in MC-mediated transplant tolerance in which the chimeric thymus from tolerant VCA B6 recipients were transplanted into syngeneic B6.nude mice recipients, while the thymus from naïve B6 mice were used as control. Since nude mice are athymic and consequently lack T cells, any detectable functional CD3This study supports the hypothesis that vascularized bone grafts bear donor HSC niches for sustained donor derived HSC/mesenchymal stem cells production, homing and renewal, provide a unique biologic opportunity to facilitate stable MC and transplant tolerance without harsh myeloablation.In summary, this study provides a mechanistic insight into the MC-mediated transplantation tolerance Figure 6The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Institutional Animal Care and Use Committee at the University of Pittsburgh.XZ and WZ designed experiments, analyzed data and interpreted results. WZ, YW, FZ, RS, and C-HL performed animal experiments, immunologic assays and data acquisition. WZ and XZ wrote the manuscript and prepared the figures. VG, GB, and MS provided critical scientific input and reviewed and edited the manuscript. XZ coordinated and directed the project. All authors contributed to the article and approved the submitted version.


Central nervous system (CNS) symptoms may occur in patients with acute COVID-19. The role of CSF examination in these patients remains to be established.A systematic review of CSF findings relating to COVID-19 was carried out.CSF parameters, including cytological and biochemical analyses, SARS-CoV-2 RT-PCR and other CSF markers, were recorded and analyzed among patients with acute COVID-19 and one of the following CNS syndromes: stroke, encephalopathy, encephalitis, inflammatory syndromes, seizure, headache and meningitis.Increased white blood cells and/or increased protein concentration were found in 52.7% of the patients with encephalitis, 29.4% of the patients with encephalopathy and 46.7% of the patients with inflammatory syndromes (P < 0.05). CSF RT-PCR for SARS-CoV-2 was positive in 17.35% of the patients with encephalitis and less than 3.5% of the patients with encephalopathy or inflammatory syndromes (P < 0.05). Intrathecal production of immunoglobulins was found in only 8% of the cases. More than 85% of the patients had increased CSF cytokines and chemokines. Increased CSF neurofilament light chain (NfL) and CSF Tau were found in 71% and 36% of the cases, respectively.Non-specific inflammatory CSF abnormalities were frequently found in patients with COVID-19 CNS syndromes. The increase in neurodegeneration biomarkers suggests that neuronal damage occurs, with long-term consequences that are still unknown. The new coronavirus (SARS-CoV-2) appeared in Wuhan, China, and quickly evolved into a pandemicCerebrospinal fluid (CSF) analysis is essential in making diagnoses of infections of the central nervous system (CNS), since it can provide information about the inflammatory response and can identify the etiological agentIn this article, we present a systematic review of CSF findings among patients with COVID-19 and acute CNS symptoms and discuss the potential findings in different CNS syndromes. We also discuss the potential contribution of CSF analysis in understanding the underlying mechanisms in CNS manifestations associated with acute COVID-19.MEDLINE (accessed from PubMed) was systematically searched from January 1, 2020, to April 30, 2021. The search strategy included the key words: “COVID 19” AND “Cerebrospinal Fluid”. Articles written in English, Spanish, Portuguese and French were included in this search. We included the following study types: case reports, descriptive retrospective studies and longitudinal studies. Review articles were not included. We only included studies that reported CSF findings. Studies reporting CSF findings from patients with COVID-19 and peripheral nervous system (PNS) manifestations were not included in the present analysis. We also excluded reports on multisystem inflammatory syndrome in children, associated with COVID-19. Cases in which an infectious etiology other than COVID-19 was identified to explain the neurological condition, either through conventional microbiology or through molecular methods, were also excluded. -We retrieved the results from CSF analysis in the different clinical syndromes that were reported in the studies: stroke, encephalitis, encephalopathy, inflammatory syndromes, meningitis, seizures and headache. The clinical syndrome definitions used had some variation between studies but, in general, encephalopathy was defined as alteration of one or more brain functions that was attributed to systemic disease without structural brain lesionsThe following CSF parameters were sought and registered from the studies: opening CSF pressure; CSF white blood cells (WBC); CSF protein concentration; CSF SARS-CoV-2 RT-PCR; intrathecal immunoproduction ; CSF specific antibodies ; CSF interleukins (Ils); chemokines (CXCLs); tumor necrosis factor alpha (TNF-α); and neurodegeneration CSF biomarkers . We statistically compared the CSF data from encephalitis, encephalopathy and inflammatory syndrome cases. The statistical comparisons were carried out using the chi-square test or Fisher’s exact test, depending on the number of observations in the contingency table. The significance level was set at P < 0.05. A total of 222 articles were identified. Among these, we selected 75 articles in accordance with the search criteria. The study types were case report (37 studies), retrospective (38 studies) and longitudinal (one study). A total of 663 patients were included in these 75 studies. The clinical diagnoses of CNS syndromes among the patients reported in the studies were the following: hemorrhagic stroke , ischemic stroke , encephalitis , encephalopathy , headache , other inflammatory syndromes , meningitis  and seizures . The seizure types were described as motor (tonic-clonic) generalized onset seizures (2), focal non motor onset with impaired awareness (2) and unknown (13). The clinical syndrome was not defined by the authors of the studies in 159 cases. The findings of CSF parameters were as follows: -Opening CSF pressure: this parameter was reported in 59 cases, which were 14 cases of headache, 13 cases of inflammatory syndromes, 30 cases of encephalitis and two cases of encephalopathy. Among these, 22% presented increased opening CSF pressure3. In two cases of encephalitis there were more than 100 cells/mm3, in one case the CSF WBC was 115 cells/mm3 and in one case there was marked pleocytosis with 1920 cells/mm3. Overall, there was a predominance of lymphomononuclear cells-CSF WBC: CSF WBC was reported in 349 cases. Increased CSF WBC was found in 86 cases (24.64%). Most cases with pleocytosis had less than 100 cells/mm,,-,,,-,,,-,-CSF protein concentration: This information was reported in 281 cases. Increased CSF protein concentration was found in 93 patients (33.09%). A mild increase in CSF protein concentration (< 100 mg/dl) was found in 269 patients, moderate increase (100-200 mg/dl) in nine cases and high increase (> 200 mg/dl) in three cases. The highest concentration was found in a patient with a CNS inflammatory syndrome with CSF protein concentration of 803 mg/dl,,-,-,-,-,-SARS-CoV-2 CSF RT-PCR: A search for SARS-CoV-2 genetic material in CSF samples by means of RT-PCR was reported in 243 cases. Thirteen patients (5.34%) were found to be positive,,,,,,,,,,Intrathecal immunoproduction and specific CSF antibodies: OCBs were found in 20% of the 138 patients on whom this test was performed. The most frequent pattern was type 4, with identical OCBs in CSF and serum, thus indicating systemic inflammation. The IgG index was negative in all 12 cases tested. Among the specific antibodies tested in CSF, anti-MOG was positive in one of two cases (50%), anti-NMDA was positive in three of 17 cases (11%) and anti-cardiolipin was positive in two of 11 cases (18%),,,,,,,,,Inflammatory mediators: The complete list of cytokines and chemokines tested and their results is shown in ,,,,Neurodegeneration CSF biomarkers: The most frequently tested CSF neurodegeneration biomarkers were Tau protein, NfL and GFAP, in 33, 51 and 33 cases, respectively. Increased levels were found in 36%, 71% and 18%, respectivelyStatistical analyses: Mild inflammatory changes in conventional CSF analyses were frequently found in patients with central neurological manifestations associated with COVID-19, and were seen more frequently in cases of encephalitis. The SARS-CoV-2 genome was found in a small percentage of cases, more commonly among those with encephalitis than in those with encephalopathy or other inflammatory syndromes. Most patients, regardless of the clinical syndrome, presented increased CSF cytokines and chemokines. CSF neurodegeneration markers were increased in several cases. These data suggest that CSF analysis is useful in the clinical evaluation of these patients and may contribute to better understanding of the CNS neurological manifestations associated with COVID-19. However, with the present data, it is not yet possible to define the precise mechanisms involved in neuronal injury and in generation of neurological symptoms. ,,,,The number of patients with ischemic and hemorrhagic stroke on whom CSF analysis was done was too small to allow any definitive conclusion about the role of CSF in these conditions. There were no abnormalities in the conventional CSF analysis in cases of ischemic stroke. The levels of Il-6, IL-8 and TNF-α were increased in all cases of ischemic and hemorrhagic stroke that were tested. It is still uncertain whether the inflammatory process revealed by these CSF measurements has a role in the pathogenesis of vascular injury associated with these clinical syndromes9. Future studies comparing cases with and without detection of the viral genome and correlating CSF data with neuroimaging findings may help to elucidate the pathophysiology of COVID-19 encephalitis and perhaps may contribute to a more refined definition of these cases according to these findings.  Most patients with encephalitis had mild pleocytosis with mild to moderate increases in CSF protein concentration. Only in 17.14% of the encephalitis cases was the SARS-CoV-2 genome detected in CSF. Previous studies have shown that SARS-CoV-2 can invade the CNS, via the olfactory nerve or through hematogenous spread, which thus explains why RT-PCR was positive in some cases,,-,-,-,-Changes to the conventional CSF analysis were found in a minority of cases of encephalopathy. The viral genome was found in the CSF in a small percentage of these patients. It is possible that systemic inflammation is a determining factor in these cases; however, this cannot be definitively stated since systemic inflammation was not evaluated in the present study with regard to encephalitis,Patients with other inflammatory syndromes presented with conventional CSF changes similar to what is normally seen in patients with ADEM due to other etiologies, i.e. normal CSF or CSF with mild inflammatory changes. The SARS-CoV-2 genome was found in only one case of CIS and was not found in cases of ADEM. ADEM cases can have transient OCBs in the CSF and not in the serum. In the present review, no cases with SARS-CoV-2-associated ADEM had intrathecal immunoproduction, but it is important to mention that only nine cases of inflammatory syndromes underwent this analysis. It is possible that an autoimmune disorder triggered by SARS-CoV-2 infection occurred in these cases. One case that was positive for anti-MOG antibodies and two cases with anti-cardiolipin antibodies suggest the possibility of an immunological cross-reaction,,,, The number of cases of patients with seizures and meningitis was too small for an accurate analysis on CSF findings in these groupsIncreased levels of neurodegeneration markers were found in more than 40% of the patients with encephalitis in whom these markers were tested. One study showed higher levels of NfL in patients with encephalitis than in patients with encephalopathyThis study had limitations that deserve to be mentioned. The most important concerns the definition of cases. The distinction between encephalopathy and encephalitis is often difficult to ascertain, as both conditions can lead to lowered consciousness. The presence of epileptic seizures can be part of the context of either encephalopathy or encephalitis, so it is difficult to assess this manifestation in isolation. Considering the non-uniformity of criteria for defining cases between the different studies, it is possible that this influenced the interpretation of CSF data in these different syndromes. Other limitations included the fact that the studies were retrospective or case reports. The extensive period of review, taking into account the period of occurrence of the pandemic, and the analysis of a wide CSF database are strengths of this study.In conclusion, the present review shows that CSF analysis has a role in evaluation of COVID-19-associated CNS disorders. CSF may also contribute to better understanding of the relationship between SARS-CoV-2 infection and the CNS. Prospective studies including assessment of CSF inflammatory and neurodegeneration biomarkers may possibly contribute to better understanding of the determinants of the neurological prognosis after acute COVID-19.


Testicular ectopia refers to abnormal positioning of testis, which includes a wide variety of variants. An ectopic testis is located off the normal path of male gonadal descent, unlike conventional undescended testis.A 37‐year‐old man presented with the complaint of a palpable lesion in the scrotum. Magnetic resonance imaging of the scrotum revealed bilateral testes on the respective opposite sides of the scrotum with bilateral spermatic cords crossing under the base of the penis. Accordingly, he was diagnosed as “left‐right reversal of the testes within the scrotum.” In retrospect, the “palpable” lesion was thought to be the spermatic cords crossing above the testes. Semen analysis identified deteriorated sperm motility, suggesting possible male infertility.This case of left–right reversal of the testes within the scrotum is probably a new variant of testicular ectopia that has never been reported. Testicular ectopia refers to abnormal positioning of testis and includes a wide variety of variants. An ectopic testis is located off the path of normal descent, unlike conventional undescended testis. We report a case of left–right reversal of the testes within the scrotum, whereby bilateral testes were located on the respective opposite sides of the scrotum with bilateral spermatic cords crossing under the base of the penis. The present case might be a new variant of testicular ectopia that has never been reported.Testicular ectopia is a generic term used to refer to the abnormal positioning of the testis. It accounts for approximately 10% of extrascrotal testes and is sometimes contrasted with undescended testis or cryptorchidism, the commonest genital malformation in boys.A 37‐year‐old man who had a past medical history of proteinuria  and was otherwise healthy visited our department with the complaint of a palpable lesion in the scrotum. He was suspected having left spermatocele by ultrasonography at another clinic 8 years previously but had undergone no further examination. A physical examination of the external genitalia demonstrated no abnormal findings. Laboratory blood tests did not detect any abnormal values, including serum total testosterone (6.71 ng/mL).Ultrasonography of the scrotum showed bilateral spindle‐shaped low‐echoic lesions located above the respective bilateral testes and epididymides, which had blood‐flow signals inside. It also detected normal but slightly small bilateral testes  Fig. . Based oScreening semen analysis identified normal semen volume and sperm concentration and morphology but deteriorated sperm motility , suggesting possible male infertility Table . HoweverThe present report describes a case of left–right reversal of the testes within the scrotum, whereby each testis moves to the opposite side of the scrotum with bilateral spermatic cords crossing under the base of the penis. To the best of our knowledge, this condition has never been described in the literature and might thus be a new variant of testicular ectopia. The patient also presented with deteriorated sperm motility with slightly small testes, suggesting possible male infertility, although its association with his testicular ectopia was unclear.before entering the inguinal canal in transverse testicular ectopia, bilateral testes and spermatic cords move across the midline after leaving the respective inguinal canals and cross each other in the present case, suggesting different underlying mechanisms.Testicular ectopia refers to abnormal positioning of the testis and is distinguished from conventional undescended testis or cryptorchidism by the fact that an ectopic testis is located off the normal path of male gonadal descent.The descent of testes through inguinal canals in the fetus occurs at 26 to 28 weeks as the gubernaculum pulls down the testes into the scrotum, after which the testes are fixed at the bottom of the scrotum at 35 to 40 weeks.Surgical correction is generally recommended for testicular ectopia as well as undescended testis, since it may optimize testicular function, facilitate diagnosis of testicular cancer, provide cosmetic benefits, and prevent complications such as hernia or torsion.In summary, we report for the first time a case of left–right reversal of the testes within the scrotum, which is presumably a new variant of testicular ectopia.Kenjiro Kishitani: Conceptualization; data curation; formal analysis; writing – original draft. Kenji Yoda: Conceptualization; data curation; formal analysis; project administration; writing – review and editing. Satoru Taguchi: Conceptualization; data curation; formal analysis; project administration; writing – original draft. Hikaru Suyama: Data curation; writing – review and editing. Hayato Hoshina: Data curation; writing – review and editing. Yoshiyuki Akiyama: Data curation; writing – review and editing. Yuta Yamada: Data curation; writing – review and editing. Yusuke Sato: Data curation; writing – review and editing. Daisuke Yamada: Data curation; writing – review and editing. Haruki Kume: Project administration; supervision; writing – review and editing.The authors declare no conflict of interest.Not applicable.Not applicable.Not applicable.