Background: The use of living-donor kidney allografts with multiple vessels continues to rise in order to increase the donor pool. This requires surgeons to pursue vascular reconstructions more often, which has previously been associated with a higher risk of developing early post-transplant complications. We therefore wanted to investigate the prognostic role of using living-donor renal allografts with a single artery (SA) vs. multiple arteries (MA) at the time of transplant.Methods: We retrospectively analyzed a cohort of 210 consecutive living-donor kidney transplants performed between January, 2008 and March, 2019, and compared the incidence of developing postoperative complications and other clinical outcomes between SA vs. MA recipients.Results: No differences were observed between SA (N = 161) and MA (N = 49) kidneys in terms of the incidence of developing a postoperative  complication, a urologic complication, hospital length of stay, delayed graft function, estimated glomerular filtration rate at 3 or 12 mo post-transplant, and graft survival.Conclusions: The use of live-kidney allografts with MA requiring vascular reconstruction shows excellent clinical outcomes and does not increase the risk of developing postoperative complications or other adverse outcomes when compared with SA renal allografts. Kidney transplantation has long been established as the optimal therapy for patients with end-stage renal disease (ESRD). Living kidney donation has increased substantially in the attempt to meet current demands, leading to a surge in transplantation of kidney allografts with multiple arteries. These allografts often require complex back-table reconstruction prior to transplantation, which has been linked with poorer post-transplant outcomes in their recipients, when compared with single artery kidney allografts –5. The aBetween February 2008 and March 2019, 210 patients underwent consecutive living-donor kidney transplantation by a single surgeon (G.C.) at our institution . A retrospective review of these 210 consecutively transplanted patients was approved by the University of Miami Institutional Review Board and follows the ethical principles (as revised in 2013) of the Helsinki Declaration. All patients gave written informed consent prior to enrollment. The donor vascular renal anatomy was evaluated with computed tomography angiography .In living-donor kidney transplantation, our strategy is to always use the donor kidney (left or right) considered to be the less favorable kidney, allowing the donor to retain the more favorable kidney. In cases where both the left and right donor kidneys are equally favorable, the choice of which donor kidney to use for transplant is then based on the ease in performing the donor nephrectomy, minimizing surgical risks to both the donor and recipient. For instance, a right donor kidney with a short renal vein would favor using the left kidney for transplant; however, the presence of multiple renal arteries in one kidney might favor the removal of the other kidney.Following hand-assisted laparoscopic extraction of the donor kidney, standard benching preparation was performed, and the graft was flushed with cold Histidine-tryptophan-ketoglutarate until the effluent was clear. The renal arteries and veins were dissected from the surrounding perivascular lymphatics and fat, and the side branches were ligated. The ureter with its blood supply and the periureteric tissue were preserved, and all remaining redundant perinephric fat was trimmed. In the instance of multiple renal vessels, different back-table techniques were adopted to reconstruct the renal arteries or veins depending on their lengths and calibers.N = 36, 33/36 with two renal arteries, 3/36 with three renal arteries; N = 6, upper pole branch in 4 cases, lower pole branch in 2 cases, 6/6 with two renal arteries) (Double-barrel side-to-side vascular reconstruction was most frequently used (rteries) . In the More complex vascular reconstructions were performed in the remaining six donor kidneys having two or three renal arteries. In the 1st case with three renal arteries , one shoOnce reperfusion of the graft was achieved, mobilization of the bladder with subsequent extravesical ureteroneocystostomy was carried out using two running 6-0 polydioxanone sutures. Finally, the detrusor muscle was closed over the anastomosis to create an anti-reflux tunnel with interrupted 4-0 PDS sutures .After surgery and prior to hospital discharge, patients had daily measurements of serum creatinine, BUN, and electrolytes, a complete blood count, and Doppler and gray scale renal ultrasonography as baseline.All recipients received immunosuppressant therapy according to protocols at our center, with induction consisting of intravenous antithymocyte globulin 1 mg/kg, basiliximab 20 mg, and methylprednisolone 500 mg administered intraoperatively before organ reperfusion. Maintenance immunosuppression included a steroid-free regimen consisting of tacrolimus and mycophenolate mofetil, starting on postoperative day 1 .At the Miami Transplant Institute (MTI)/Jackson Memorial Hospital (JMH), all patients are prospectively followed. When a kidney transplant patient is seen either as an inpatient in the hospital or as an outpatient in the transplant clinic, both the attending physicians (surgeon and transplant nephrologist) and transplant-dedicated nurses record detailed records of all medical history, procedures performed, drugs given, and clinical status of the patient. This information is updated daily while the patient is in the hospital (at JMH). These documented records are kept within 2 distinct databases at our center: Cerner (the JMH patient database), and OTTR (the MTI database). The first 4 co-authors of this manuscript  along with the senior author (GC) had retrospectively organized the relevant patient data into a clinical research EXCEL file. Any checks of the information recorded into this EXCEL file were made via re-review of the original information recorded in Cerner and OTTR. So, while all clinical outcomes were initially recorded  in the JMH and MTI databases by the attending physicians (and nurses) who saw the patients while hospitalized or as an outpatient in the transplant clinic, the co-authors of this manuscript reviewed the relevant clinical data on each patient and then transcribed it into an EXCEL file. The statistical analysis was then performed using data contained in this EXCEL file.t-tests were used to test the associations of categorical and continuous variables with the number of donor renal arteries (1 vs. >2), respectively. Stepwise linear (logistic) regression was performed to determine multivariable baseline predictors of the likelihood of requiring vascular reconstruction at the time of transplant . Time-to-event variables  were compared using the Kaplan-Meier technique and log-rank tests. P-values ≤ 0.05 were considered to be statistically significant.Data from patients were retrospectively organized according to the number of renal vessels or anomalies reported in the donor operative report and were entered into an EXCEL file. Analyzed baseline variables included date of transplant, recipient age, recipient gender, recipient race/ethnicity, recipient BMI, recipient pretransplant history of diabetes mellitus (no/yes), kidney retransplant status (no/yes), donor kidney location (left or right), number of donor arteries, number of donor veins, need for vascular reconstruction, living donor type (related/unrelated), double-J ureteral stent insertion (no/yes), operative time, cold ischemic time, and warm ischemic time. The primary outcome variable was the development of a post-operative  complication during the first 30 days (12 months) post-transplant. Secondary outcomes included length of hospital stay, development of delayed graft function (DGF) , postoperative creatinine, development of a urologic complication during the first 12 months post-transplant, and graft loss . Estimated glomerular filtration rate (eGFR) was calculated using the “Chronic Kidney Disease Epidemiology Collaboration Equation.” Percentages of patients having selected baseline characteristics were determined as well as means and standard errors for baseline continuous variables. Pearson (uncorrected) chi-square and ordinary (2-sided) The date of last follow-up for this study was March 20, 2020. Median follow-up among 195 patients who were alive with a functioning graft as of the last follow-up date was 26.2 (range: 12.0–128.4) months post-transplant. Of note, the minimum follow-up for all patients who were alive with a functioning graft was 12 months post-transplant.>2 donor arteries was similar between those who received a left vs. right donor kidney, 23.8% (45/189) vs. 19.0% (4/21) (P = 0.62).Distributions of demographic characteristics and post-operative outcomes appear in N = 7), #2 only (N = 4), #3 only (N = 8), #1 combined with #3 (N = 1), and #2 combined with #3 (N = 1).There were three reasons for using the right donor kidney for transplant: (#1) the left donor kidney had multiple arteries, (#2) the right donor kidney was either smaller or had decreased function (or both), and (#3) the right donor kidney had one or more cysts (or kidney stones). Among the 21 recipients of a right donor kidney, reasons for using the right donor kidney were as follows: #1 only . Thus, in only this latter case (1/7) was the same iliac fossa used in placing the retransplanted kidney.>2 renal arteries had vascular reconstruction. Among the seven recipients of allografts having one renal artery but still requiring vascular reconstruction, three patients who received a right donor kidney had a short donor vein that required extension . In the other two cases, an interposition graft was used in a similar fashion from a deceased donor common iliac vein. Among the four other cases with a single renal artery that required vascular reconstruction, two patients required aneurysm repair, one patient received a right donor kidney with two small renal veins that were conjoined, and 1 7-year old patient required thrombectomy of the renal vein which was observed intra-operatively as a web-like membrane within the lumen, most likely representing a neonatal thrombosis.Overall, the percentage of recipients who required vascular reconstruction was 26.7% (56/210); 100% (49/49) of the patients who received a donor kidney with N = 1), c. difficile colitis/sepsis at 4 days post-transplant (N = 1), diverticulitis at 4 days post-transplant (N = 1), a suspected ureteral leak at 4 days post-transplant (N = 1), acute respiratory distress syndrome (ARDS) at 6 days post-transplant (N = 1), wound infection/necrosis at 2 weeks post-transplant (N = 1), wound infection at 6.6 months post-transplant (N = 1), and ureteral stricture at 9.0 months post-transplant (N = 1). The patient who developed c. difficile colitis/sepsis died of that infection (with a functioning graft) at 0.8 months post-transplant. None of the other seven patients who developed a post-operative  complication within 30 days (12 months) post-transplant experienced graft loss during subsequent follow-up. The Clavien-Dindo classification grades of these 8 post-operative complications were: III, V, III, III, IV, III, II, and III, respectively.None of the 210 patients (0.0%) developed DGF. Eight patients (3.8%) developed a post-operative  complication during the first 30 days (12 months) post-transplant, including: bladder leak at 2 days post-transplant (Among the three patients (1.4%) who developed a urologic complication, a bladder leak observed in one patient was caused by traumatic injury during insertion of a Jackson-Pratt drain. This patient's bladder leak was surgically repaired. A ureteral stricture that developed in another patient was successfully treated with a percutaneous nephro-ureteral stent placement. Similarly, a ureteral leak that was suspected to have developed in a third patient  was successfully treated with a percutaneous nephro-ureteral stent placement. One of the three patients who developed a urologic complication  had a ureteral stent placed at the time of transplant. Of note, none of these three patients suffered any long-term consequences of their urological complications.As of the last follow-up date, six patients (2.9%) have developed graft failure . The six causes and times-to-graft failure were as follows : Acute antibody mediated rejection (AMR) at 5.4 months, acute T-cell mediated rejection at 41.8 months, MPGN recurrence at 58.0 months, chronic allograft injury (CAI) at 67.7 months, CAI at 68.2 months, and acute AMR/Nonadherence at 86.7 months post-transplant. Nine patients have died with a functioning graft. The nine causes of death with a functioning graft and corresponding times-to-death were as follows: cardiovascular event in six patients , and infection/sepsis in three patients .>2) are shown in >2 renal arteries, 100.0% (49/49) vs. 4.3% (7/161) among recipients of a single artery kidney (P < 0.000001); (ii) mean cold ischemia time was significantly longer among recipients of a donor kidney with >2 renal arteries, 1.31 ± 0.07 vs. 0.72 ± 0.02 h among recipients of a single artery kidney (P < 0.000001); and (iii) mean operative time was significantly longer among of recipients of a donor kidney with >2 renal arteries, 5.03 ± 0.14 vs. 3.90 ± 0.08 h among recipients of single artery kidneys (P < 0.000001). There was no notable association of the number of donor arteries with any of the outcome variables, including development of a post-operative  complication, mean eGFR at various time points, and graft survival  and receiving a right donor kidney (P = 0.00002). Once these two variables were selected, no other variables contained additional predictive value (P > 0.05). In fact, none of the other baseline variables were associated in univariable analysis with the requirement of vascular reconstruction (P > 0.10).Stepwise linear regression to determine multivariable predictors of which patients required vascular reconstruction at the time of transplant yielded two significant predictors: receiving a donor kidney with P > 0.10). Of note, the observed percentage who developed a post-operative  complication for: (i) recipients of a donor kidney with 1 vs. 2–3 renal arteries was 3.7% (6/161) vs. 4.1% (2/49), respectively (P = 0.91), (ii) recipients of a left vs. right donor kidney was 4.2% (8/189) vs. 0.0% (0/21), respectively (P = 0.34), and (iii) recipients who did not require vs. required vascular reconstruction was 3.2% (5/154) vs. 5.4% (3/56), respectively (P = 0.48).Finally, stepwise logistic regression to determine multivariable predictors of developing a post-operative  complication (eight events) yielded no significant predictors (Living-donor kidney transplantation has evolved in an attempt to keep up with the increasing demand of patients requiring transplantation . Donor vN = 218) vs. 89.6, 83.2, and 71.8% for the MA group (N = 60). Furthermore, there was a higher percentage of graft loss from chronic allograft nephropathy in the MA group than in the SA group, and the presence of multiple arteries was an independent risk factor for both acute rejection and graft loss, validating their conclusion that laparoscopic procurement of living-donor kidneys with single arteries yields lower risks  patients requiring vascular reconstruction of either the renal arteries, renal veins, or both. Our results were in line with previous literature in which neither short nor long-term recipient outcomes were affected by initial allograft anatomy. This highlights the fact that a meticulous technique triumphs over vessel multiplicity, as the good results achieved in our study appear to be due, at least in part, to the careful vascular reconstructions that were performed. Clearly, the surgeon's experience in handling multiple renal vessels plays a role in determining the incidence of post-operative/surgical complications that may develop following living-donor kidney transplantation.Of special interest is the use of ureteral stents in living-donor kidney recipients at the time of transplant. Double-J stents have been described as crucial in preventing mechanical ureteral complications in renal transplantation, with definite indications for patients with neurogenic bladder or previous bladder surgery; however, their use remains controversial due to a potentially increased risk of developing hematuria and urinary tract infections as well as inconsistent evidence from previously reported randomized clinical trials –26. Our N = 127) vs. multiple (N = 21) vessel living-donor RAKT was performed in which the vascular reconstruction techniques performed on the back table were similar to ours reported here. In each of their cases, single arterial and venous anastomoses were performed after vascular reconstruction, and no unfavorable clinical outcomes for grafts with multiple vessels were found. Thus, both our data and this European study  has been successfully introduced in both living and deceased donor transplants –32. In oan study  reinforcLimitations of our study include the fact that this was a retrospective evaluation of consecutively transplanted patients performed at a single center by a single  transplant surgeon. Second, while our overall cohort size of 210 patients was reasonable, sample sizes for certain subgroups of patients were relatively small. Third, while our overall results were excellent, with only 3.8% (8/210) of patients developing a post-operative  complication post-transplant, statistical power to perform a multivariable analysis of this (primary) clinical outcome was limited due to the small number of events that occurred.Comparable results may be obtained in living-donor kidney transplantation when using allografts that require vascular reconstruction (on the back table), with no increased risks of developing DGF, post-operative  complications, poorer renal function, or graft loss. Using the right kidney with a short renal vein was also not associated with any increased risk of developing a post-transplant vascular complication.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of Miami Institutional Review Board. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.LG and GC: research design, data collection, and manuscript preparation. NP and LB: data collection. JG: statistical analysis and manuscript preparation. GG: research design. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Microglial activation is a central player in the pathophysiology of Alzheimer’s disease (AD). The soluble fragment of triggering receptor expressed on myeloid cells 2 (sTREM2) can serve as a marker for microglial activation and has been shown to be overexpressed in AD. However, the relationship of sTREM2 with other AD biomarkers has not been extensively studied. We investigated the relationship between cerebrospinal fluid (CSF) sTREM2 and other AD biomarkers and examined the correlation of plasma sTREM2 with CSF sTREM2 in a cohort of individuals with AD and without AD. Participants were consecutively recruited from Asan Medical Center from 2018 to 2020. Subjects were stratified by their amyloid positivity and clinical status. Along with other AD biomarkers, sTREM2 level was measured in the plasma as well as CSF. In 101 patients with either amyloid-positive or negative status, CSF sTREM2 was closely associated with CSF T-tau and P-tau and not with Abeta42. CSF sTREM2 levels were found to be strongly correlated with CSF neurofilament light chain. The comparison of CSF and plasma sTREM2 levels tended to have an inverse correlation. Plasma sTREM2 and P-tau levels were oppositely influenced by age. Our results suggest that neuroinflammation may be closely associated with tau-induced neurodegeneration. Extracellular amyloid plaque and intraneuronal hyperphosphorylated tau accumulation are considered hallmarks of Alzheimer’s disease (AD) and many biomarkers have been developed to reflect these central pathophysiological events. By virtue of these biomarkers, we possess a valuable window into the changes occurring in the brain of individuals with Alzheimer’s disease and can identify the disease process at the earliest possible stage4. This theory is centered on microglial activation, which can be brought about by various stimuli, including beta-amyloid. Failure of activated microglia to wall off amyloid plaques from surrounding neurons and phagocytize them leads to the accumulation of amyloid plaques and neurofibrillary tangles6. Microglial activation is mediated by its membrane-bound receptor, triggering receptor expressed on myeloid cells 2 (TREM2)7. The activity of TREM2 can be measured by evaluating the levels of the soluble fragment of TREM2 (sTREM2), which is generated during the TREM2 cleavage process8. The concentration of sTREM2 in the cerebrospinal fluid (CSF) has been shown to be elevated in AD, raising the possibility that sTREM2 can serve as a reliable biomarker for AD11. It is important to investigate at the relationship of one biomarker with another to better understand the pathophysiology of AD. The role of microglial activation in the amyloid cascade is a matter of curiosity and requires further exploration12. However, the relationship between sTREM2 and other conventional biomarkers across the various stages of AD has not been extensively studied, particularly in patients with sporadic AD. Whether the change in CSF sTREM2 can be reflected in the plasma is also controversial14. If sTREM2-induced changes in the brain can be reflected in the plasma levels of sTREM2 akin to neurofilament light chain in multiple sclerosis, we will be able to elucidate neuroinflammation associated with AD throughout the course of the disease.Neuroinflammation has been deemed a secondary phenomenon, but is now emerging as a central player in the development of ADThe purpose of this study was to investigate the relationship between CSF sTREM2 and other AD biomarkers and to examine the plasma levels of sTREM2 and its correlation with CSF sTREM2 in a cohort population of AD and non-AD conditions.APOE ε4 allele was significantly higher in the amyloid-positive group than that in the amyloid-negative group . The frequency of diabetes mellitus was higher in the amyloid-negative group than that in the amyloid-positive group (P = 0.01). The amyloid-positive group had poor performances in K-MMSE compared to those of the amyloid-negative group (P = 0.029). The amyloid-positive group showed significantly higher serum LDL levels (P = 0.042) and albumin (P = 0.02). The amyloid-negative group showed higher serum ESR than did the amyloid-positive group (P = 0.025). Other demographic features, including sex distribution, CRP serum levels, frequency of hypertension (HTN), and hyperlipidemia, did not show significant differences between the two groups.The clinical characteristics of the participants are presented in Table 42, T-tau, and P-Tau S199 levels were compared between the two groups (amyloid-positive and amyloid-negative) to demonstrate that the cohort had typical patterns of AD core biomarkers. As expected, the CSF Aβ42 levels were significantly lower in the amyloid-positive group than those in the amyloid-negative group (P < 0.001)  . In contrast, the CSF sTREM2 was not significantly correlated with the plasma biomarkers  and age . Furthermore, NfL and age were strongly correlated with each other . In contrast, the leukocyte TL was negatively correlated with sTREM2 , plasma NfL , and age , showing that the aging and neurodegeneration factors were progressing together , CSF NfL , and plasma NfL . The plasma sTREM2 levels correlated positively with age  (Table In the amyloid-positive group (AD continuum), the association between CSF and plasma sTREM2 which was observed in the whole dataset disappeared. The CSF sTREM2 levels significantly correlated with CSF T-tau level , and Aβ42/P-tau S199 . The plasma sTREM2 levels correlated with CSF P-tau S199  and AβIn the amyloid-negative conditions, the CSF sTREM2 levels showed a positive correlation with the CSF P-tau S199  and T-tau  levels. No significant significance was found between the plasma sTREM2 and CSF biomarker levels. The plasma sTREM2 levels were correlated with the plasma NfL , TL , and age  , and amyloid-positive late-onset disease. In case of CSF P-Tau S199, T-tau levels, and age correlation, we found that the CSF P-tau S199 and T-tau levels were inversely influenced by age in the whole dataset, as previously noted Fig. . The tau42; second, the CSF sTREM2 levels were found to be strongly correlated with CSF NfL; third, comparison of the CSF sTREM2 and plasma sTREM2 levels tended to show an inverse correlation; fourth, the plasma sTREM2 and P-tau levels were influenced by age in an opposite way (higher levels of sTREM2 and lower levels of P-tau were associated with older age). Taken together, these results suggest that sTREM2 can serve as a reliable biomarker of AD and that neuroinflammation may be closely associated with tau-induced neurodegeneration.We collected both plasma and CSF samples from participants in this AD biomarker study to examine whether CSF sTREM2 can be mapped onto other AD biomarkers and also correlate with plasma sTREM2. The major findings of this study were as follows: first, we observed that CSF sTREM2 was closely associated with CSF T-tau and P-tau, but not so much with Aβ9. Their levels were closely associated with P-tau levels but not with Aβ42 levels. The role of sTREM2 in AD is contentious17. It could be either a byproduct of full-length TREM2 shedding or an active peptide playing a role in microglial modulation19. The increase in CSF sTREM2 levels in the early symptomatic stage of AD was interpreted to reflect a corresponding change in status of microglial activation in response to neurodegeneration induced by hyperphosphorylated tau aggregation8. Another cross-sectional study of sporadic AD patients showed that the levels of sTREM2 peaked at the early symptomatic phase of the disease and that the CSF sTREM2 levels were positively correlated with the concentrations of T-tau and P-tau in the CSF, suggesting the important role of sTREM2 in the development of AD pathology and neurodegeneration21. We can suggest that elevated levels of sTREM2 may have close relationship to inducing tau phosphorylation in neurites neighboring amyloid plaques23. Microglial clustering around amyloid plaques represented by increased sTREM2 levels play a role in initially providing a barrier function24. If it fails to wall off amyloid plaques from surrounding neurons and phagocytize them, this can lead to the propagation of amyloid plaques and formation of neurofibrillary tangles26. Dystrophic neurites containing hyperphosphorylated tau can be found around amyloid plaques during this process27. Why CSF sTREM2was not significantly associated with Aβ42 in our sample is unknown. Given the early appearance of Aβ42 and its plateau in the prodromal stage of AD, giving way to the increase in P-tau, sTREM2 may be closely linked with Aβ-triggered tau phosphorylation occurring after Aβ42 accumulation28. Tau aggregations might affect the microglial activation state, suppressing Aβ clearance and producing neurodegeneration30. sTREM2 could serve as a marker for the tau-dependent pathogenetic pathway of AD32.With regard to the relationship of sTREM2 to well-known AD biomarkers, a longitudinal study of the DIAN cohort revealed that CSF sTREM2 levels were higher in patients with MCI due to AD than in AD and control groups33. Increased levels of NfL are thought to represent axonal damage induced by neuroinflammatory conditions, typically exemplified by multiple sclerosis34. These two proteins moving in the same direction indicate that sTREM2 can reflect microglial activation and consequent neuroinflammation. The comparison of CSF sTREM2 and plasma sTREM2 levels revealed no definite relationship with AD continuum. The CSF sTREM2 levels showed a trend toward having an inverse correlation with the plasma sTREM2 levels. This disconnect between CSF sTREM2 and plasma sTREM2 is somewhat difficult to explain. In multiple sclerosis, there is a strong positive correlation between CSF NfL and plasma NfL, indicating the same direction for peripheral and central neuroinflammation35. In AD, however, the relationship between CSF sTREM2 and plasma sTREM2 is not straightforward, and previous reports on this are inconsistent38. One study performed on AD showed a significant association between peripheral sTREM2 and CSF sTREM2 levels13. The opposite results were reported in a study performed on AD and healthy subjects, where increased levels of CSF sTREM2 were observed in AD patients, particularly in the early stage; however, plasma levels were not different between the two groups, suggesting incorrect plasma sTREM2 levels39. In AD, the plasma sTREM2 levels may not necessarily reflect the CSF sTREM2 levels, and may not follow the same dynamics as CSF sTREM240. Further studies are required to clarify this.Plasma sTREM2 levels were found to be strongly correlated with CSF neurofilament light chain (NfL). These results are in accordance with those of prior studies43, however the different way that age impacts the CSF sTREM2 and tau levels is intriguing45. Higher levels of plasma sTREM2 were associated with older age, whereas lower levels of tau were correlated with older age. Aging per se is represented by a chronic and systemic low-grade inflammatory process. In this context, increased levels of plasma sTREM2, a potential neuroinflammatory biomarker, are not surprising in older subjects, but decreased levels of CSF P-tau and T-tau in the elderly are quite puzzling, even more so given the apparent positive correlation between the CSF sTREM2 and tau levels. These are the findings derived from the group as a whole across the amyloid and clinical status. When we focused on the AD continuum, however, the age effect on the CSF tau levels disappeared46. As we broke down the subjects into earlier onset (< 65 years) and late onset AD , and CSF analysis. Subjects were included based on the following criteria: (1) aged over 40 years and under 90 years; (2) no evidence of parenchymal lesions that could influence the cognitive function based on brain MRI. Three subjects were excluded from the dataset because of the withdrawal of consent and thus a total of 101 subjects were included in this study.We enrolled 104 participants prospectively from the memory clinic of Asan Medical Center, Seoul, Korea from June 2018 to July 2020. All participants or their proxies signed an informed consent form. All subjects underwent brain MRI, comprehensive neuropsychological testing, fluorine-18 -florbetaben PET scans. The RCTU scores were then condensed into a single three-grade scoring system for each PET scan (BAPL score): 1, no β-amyloid load; 2, minor β-amyloid load; 3, significant β-amyloid load. The final score was reached by consensus, with a BAPL score of 1 regarded as amyloid negative (Aβ-) and BAPL scores of 2 and 3 considered to be amyloid positive (Aβ+)56. All participants were classified into one of these two groups according to the PET results.All PET images were obtained using Discovery 690, 710, and 690 Elite PET/computed tomography scanners . Amyloid PET images were collected for 20 min, beginning 90 min after injection of 300 ± 30 MBq We divided subjects into amyloid positive and negative groups according to the amyloid status on PET irrespective of the cognitive status. In each group, therefore, there exists a cognitive continuum spanning from SMI to dementia.12, folate, and homocysteine serum level test; and thyroid function test. The apolipoprotein E (APOE) genotype was identified after extracting genomic DNA from the venous blood. All CSF samples were subjected to complete cell count, protein, and albumin tests.All of the participants underwent the following blood tests: complete blood count; lipid profile; erythrocyte sedimentation rate (ESR); vitamin BThis study was approved by the Institutional Review Board of Asan Medical Center, Republic of Korea (#2018–0614). All methods were carried out in accordance with relevant guidelines and regulations.Proximity ligation assay (PLA) buffer, comprising 1 mM D-biotin (Life Technologies), 0.1% purified BSA (Bovogen), 0.05% Tween-20 (Sigma), 100 nM goat IgG (Sigma), 0.1 μg/μL salmon sperm DNA (Life Technologies), and 5 mM EDTA in PBS (pH 7.4) (Sigma), was stored at – 20 ℃. Conjugate probe sequences were provided as below. Streptavidin conjugated oligonucleotide SLC1 (Streptavidin-5′-CGCATCGCCCTTGGACTACGACTGACGAAC CGCTTTGCCTGACTGATCGCTAAATCGTG-3′) and SLC2 (5′–TCGTGTCTAAAGTCC GTTACCTTGATTCCCCTAACCCTCTTGAAAAATTCGGCATCGGTGA-3′-streptavidin) were purchased from Solulink. The ligation was conducted by using polymerase chain reaction (PCR) primer 1 (reverse) 5′-GGGAATCAAGGTAACGGACTTTAG-3′, PCR primer 2 (forward) 5′-CATCGCCC TTGGACTACGA-3, and PCR primer 3 (splint) 5′-TACTTAGACACGACACGATTTAG TTT-3′.57. spPLA is able to detect proteins from 1 or 10 nM to femtomolar concentrations. sTREM2 concentrations in the CSF and plasma were measured using biotinylated anti-sTREM2 polyclonal antibody and recombinant TREM2 protein (R&D systems). Briefly, 1 mg/mL Dynabeads MyOne Streptavidin T1 (Invitrogen) was incubated with 50 nM biotinylated anti-sTREM2 polyclonal antibody (R&D systems) for 1 h at room temperature (RT) under rotation to immobilize the sTREM2 antibodies to the magnetic beads. The magnetic beads were washed twice with 0.05% Tween 20 in 1X PBS (washing buffer). During the reaction, plasma samples were diluted twofold in PLA buffer, and the recombinant TREM2 protein (R&D systems) was serially spiked in PLA buffer from 1 nM to 1 fM as a standard. The diluted plasma samples and serial dilution of the standards were then mixed with the antibody-conjugated magnetic beads and incubated for 90 min under rotation. Then, the beads were washed two times with washing buffer. The PLA probes were formed by separately incubating 50 nM of the streptavidin–oligonucleotide conjugates SLC1 and SLC2 with 50 nM biotinylated anti-sTREM2 antibody for 1 h at RT. Prior to use, the SLC1- and SLC2-anti-sTREM2 antibodies were mixed at an equal ratio and incubated for 5 min at RT. The final concentration of each probe was 500 pM. Finally, the magnetic beads were mixed with 1 nM of PLA probe mix and incubated for another 90 min at RT followed by washing twice with washing buffer. Real-time PCR mix  was prepared as previously described. Before the cycling stage, a heat incubation step was performed for 15 min at 95 °C, and application steps were performed at 40 cycles for 30 s at 94 °C, 1 min at 50 °C, and 1 min at 72 °C. A StepOnePlus real-time PCR instrument (Applied Biosystems) was used for the experiment and analysis. To investigate the performance efficiency of sTREM2 spPLA, LLOD, LLOQ, precision, and dilutional linearity were measured. The LLOD formula was CtLOD = CtN-2SN. CtN is the average of Ct value gained from the background noise and SN is the standard deviation of this value. sTREM2 spPLA had inter-assay precision < 17.5% and intra-assay precision < 2%. In terms of inter-assay precision, previous study explained that the relatively large coefficients of variation are the results of the PCR step, which can be highly variable at low target copy numbers. The sTREM2 spPLA showed LLOD 50.58 pg/mL, LLOQ 151.75 pg/mL and average 95.9% dilutional linearity.To detect sTREM2 in the CSF and plasma, a spPLA was conducted following the previous protocolTo measure the levels of the biomarkers, we conducted ELISA and SIMOA following the instructions provided from commercial suppliers. For p-tau S199 measurement, Tau (Phospho) pS199 Human ELISA Kit (Invitrogen) was performed with CSF samples. Briefly, standards and samples were spiked with standard diluent buffer at 1:1 ratio and added 96 wells plate coated with capture antibodies against P-Tau S199 for 2 h. Then, unattached antigens were washed out. The human P-Tau S199 detection antibody solution applied to the wells for 1 h following 30 min of incubation with anti-rabbit IgG HRP solution. Finally, the chromogen was added to each well and incubated for 30 min. The reaction was stopped with stop solution and read the absorbance at 450 nm. For total-tau in CSF, human tau  ELISA kit (Invitrogen) was conducted. The standards and samples were diluted with standard diluent buffer at 1:1 ratio and added to the wells coated with capture antibodies for 2 h at room temperature. After washing, human total tau biotin conjugate antibodies were added to each well for 1 h. Then, streptavidin-HRP solution was applied to the wells for 30 min. Finally, stabilized chromogen and stop solutions were added to each well. The level of total tau in CSF was read at 450 nm. In case of Aβ42 levels, the CSF was diluted threefold with sample diluent due to high endogenous levels. The standards and samples were added and incubated for 2 h at 2–8 °C. After 4 times of washing, human amyloid β (aa1-42) conjugate added to each well and incubated for another 2 h at at 2–8 °C. The wells were washed out and substrate was applied and reacted with for 30 min at room temperature. The level of Aβ42 was determined using a microplate reader (Biotek) set to 450 nm.57.For plasma neurogranin, Human Neurogranin ELISA (Lifespan Biosciences) was used. Samples were applied to the wells for 1 h. After washing step, detection reagent A and B was added to the wells for 1 h each at 37 °C. TMB substrate reaction was proceeded for 15 min and the optical density was determined at 450 nm. Finally, the level of NfL in CSF and plasma was detected using Simoa NF-light Advantage Kit. Plasma and CSF were diluted 4-folds and 100-folds, respectively. CSF and plasma NfL were measured in the DNA Link Laboratory, South Korea, on the Simoa-HD1 platform as previously describedDNA was extracted from the whole blood using D-DEX IIb RBC Lysis Buffer and D-DEX IIb Cell Lysis Buffer . DNA hydration was performed with 300 μL of DNA hydration solution . TL analysis was carried out using a nonradioactive TeloTAGGG TL Assay  according to the manufacturer’s instructions. Approximately 2–4 μg of DNA from each sample was digested with Hinf I/RsaI enzyme mix and isolated by gel electrophoresis. DNA fragments were transferred to a nylon membrane  by Southern transfer and hybridized to digoxigenin (DIG)-labeled probes specific for telomeric repeats. The membrane was incubated with DIG-specific antibodies conjugated to alkaline phosphatase, and the probe was visualized by chemiluminescence detection and an image analyzer . The mean telomeric repeat binding factor lengths were determined by comparing them to the molecular weight standards.2 test to compare group differences in dichotomous variables, such as sex, diabetes, hypertension, and dyslipidemia, between two groups including the effect of the APOE genotype. To compare the levels of CSF Aβ42, T-tau, and P-tau S199 between amyloid positive and negative groups, Mann–Whitney test was used. Linear regression analyses were used to demonstrate a correlation between the CSF and plasma biomarkers. We defined a p-value less than 0.05 was statistically significant .The statistical analyses included a χ


All patients were within the norm with regards to a careful neurological examination, a magnetic resonance imaging (MRI) of the brain, an electroencephalogram (EEG), and routine blood pathology. All CSF samples were autoantibody-negative. In three serum samples of individuals with FEP, we detected low-titer CASPR2 immunoglobulin (Ig) G antibodies  and non-IgG antibodies against NMDAR (n = 1) . However, the IgG titers were below the laboratory cut-off defined for positivity, and non-IgG antibodies are of no clinical relevance. This suggests that there were no cases of autoimmune encephalitis in our cohort. Our results highlight the importance and the high specificity of CSF analysis to reliably detect autoantibodies. They confirm the hypothesis that pure psychotic manifestations of antibody-associated autoimmune encephalitides without any additional neuropsychiatric findings are very rare. However, special attention must be paid to those presenting with atypical mental illnesses with additional neurological symptoms, evidence of clinically-significant cognitive involvement, profound sleep-wake perturbations, seizures, electroencephalographic, or magnetic resonance imaging pathologies to be able to identify cases with autoimmune-mediated psychiatric syndromes.The pathophysiological role of neural autoantibodies in acute psychotic disorders is receiving increased attention. However, there is still an ongoing debate, whether predominantly psychotic manifestations of autoimmune encephalitides exist that may remain undetected and, thus, untreated. Furthermore, it is discussed if such conditions can be diagnosed based on serum antibody results or if a reliable diagnosis requires additional cerebrospinal fluids (CSF) results. In this study, we screened pairs of serum and CSF samples from antipsychotic-naïve individuals with first-episode schizophrenic psychosis (FEP,  As Susannah Cahalan put it in her famous book about her own anti-NMDAR encephalitis: “If it took so long for one of the best hospitals in the world [to make this diagnosis], how many other people were going untreated, diagnosed with a mental illness or condemned to a life in a nursing home or a psychiatric ward?”  G autoantibodies against neural antigens  and are clinically characterized by transdiagnostic neurological and psychiatric symptoms . The leac ward?” .Experts in the field have emphasized that isolated psychiatric (predominantly psychotic) manifestations in definite autoimmune encephalitides are rare, e.g., only 4% in a sample of 571 cases with autoimmune encephalitis and NMDAR antibodies in cerebrospinal fluid (CSF) . ConsequSeveral groups studied series of patients with psychosis, often first-episode psychosis (FEP) without predominant neurological features, mostly in serum, and always found some autoantibody-positive patients; the positive results have recently been reviewed and summarized . One groTo clarify these issues, neural antibodies in serum and CSF should be examined in larger numbers of psychiatric patients . Ideallyn = 190) were recruited from two clinical sites with full community service obligations providing both inpatient units and outpatient services, including early recognition for psychosis units in Germany: The Department of Psychiatry and Psychotherapy, University of Cologne and the Department of Psychiatry and Psychotherapy, Central Institute of Mental Health (CIMH), Mannheim. The study protocol was approved by the Ethics Committee of the University of Cologne and the Ethics Committee II of the Medical Faculty Mannheim, Heidelberg University. All study participants provided written informed consent. Notably, the approved study protocol allowed for retrospective consent to the use of remaining CSF and serum samples obtained during a clinical CSF investigation, where individuals were able to consent to a CSF investigation prior to a lumbar puncture but could initially not consent to the research study. Procedures were performed in accordance with the code of ethics of the world medical association for experiments involving humans (Declaration of Helsinki).Participants  had no history of mental illness and were recruited within the same geographic area and population. Individuals with CHR states  met the following criteria: (1) attenuated positive symptoms, (2) brief, limited intermittent psychotic symptoms that spontaneously resolved within 1 week, and (3) a recent decline in function for at least 1 month, in combination with the existence of a first or second degree relative with a history of any DSM-IV  psychotic disorder or met the criteria for schizotypal personality disorder. Individuals defined as having FEP  met the DSM-IV criteria for schizophrenia. All individuals with FEP were antipsychotic-naïve, but some individuals with either FEP or CHR received low-dose benzodiazepines . Noteworthy, patients were only included in this study when a careful neurological examination, a magnetic resonance imaging (MRI) of the brain, an electroencephalogram (EEG), and a routine blood pathology were within the norm as recommended by several guidelines . As expected, Bonferroni post hoc tests revealed that individuals with CHR were significantly younger than individuals with FEP (P ≤ 0.001) but also than HV (P = 0.011). Furthermore, the proportion of males was higher among the individuals with CHR compared to FEP or HV [2 × 3 Fisher's exact test  and subsequent z-test]. Individuals with CHR and FEP completed significantly fewer years of education than HV  and subsequent z-test), possibly due to a need for treatment during illness.Demographic and clinical information on age, gender, ethnicity, years of education, and symptom severity  is given in The median PANSS total scores of 93 in the FEP group and 70 in the CHR group were within the expected range of symptom severity in acute psychotic conditions for FEP and CHR individuals, with the latter not fulfilling diagnostic criteria for schizophrenia but presenting with substantial, mostly intermittent symptoms. As far as retrospectively assessable, the average duration of illness did only slightly differ from the expected range , indicatAll participants underwent lumbar puncture and venipuncture for the collection of CSF–serum pairs. Serum and CSF samples were stored at −80°C until being shipped to the Epilepsy Center Bethel, Krankenhaus Mara, Bielefeld, Germany.BR), glycine receptors . Their preparation follows an established method . These fixed cell-based assays (CBA) consist of human embryonic kidney (HEK 293) cells transfected with plasmids encoding the following antigens (fixation method in brackets): NMDAR consisting of NR1 subunits only, glutamic acid decarboxylase , leucine-rich glioma inactivated protein 1 (LGI1), contactin-associated protein-like 2 (CASPR2), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunit 1, AMPAR subunit 2, γ-aminobutyric acid-B receptors  with modifications (CGB); the datasheet instructions are given in brackets: serum samples were diluted to 1:16 as the screening dilution (Euroimmun: 1:10); buffer was PBS (Euroimmun: PBS-Tween); secondary antibody: goat-anti-human IgG (heavy and light chain [H+L]) conjugated with Alexa Fluor® 594 produced by Jackson ImmunoResearch, West Grove, PA, USA, Code No. 109-585-088, used at a dilution of 1:100, incubation 30 min at room temperature (RT) ; nuclear counterstaining with Hoechst 33342, 1:10,000 (Euroimmun: no nuclear staining); embedding with 1,4-Diazabicyclo[2.2.2]octan (Euroimmun: glycerol). Stained biochips were examined using a fluorescence microscope  equipped with adequate filters. One of two neurologists experienced in reading this assay  decided if an antibody was present using the signal of the surrounding (supposedly negative) fields as respective negative controls. The readers were blind to the participants' diagnosis. The codes were only opened when all analyses had been completed and the manuscript draft was written.Samples that reacted with the H+L secondary antibody were re-tested in the Laboratory Krone (after the facilities had been moved from The Mara to this lab in January 2016) by using a goat-anti-human antibody against the Fcγ fragment of IgG  at a dilution of 1:100, incubation 30 min at RT. Samples that were not reactive with the Fcγ antibody or gave a much weaker Fcγ than H+L signal were investigated with specific IgA secondary antibodies  and IgM secondary antibodies to clarify this discrepancy . H+L antibodies also bind to high titer IgA and IgM antibodies, whereas IgG-Fcγ antibodies are IgG specific but may not be as sensitive as the H+L antibodies. True IgG antibodies were endpoint titrated with the IgG-Fcγ antibody in multiples of 1:2 and rated by two investigators  independently. In cases of divergent ratings, the mean of the two ratings was recorded. The titration steps for serum were: 1:20, 1:40, 1:80, and so forth; CSF: neat, 1:2, 1:4, and so forth. In general, IgG antibodies were considered positive if serum titers were ≥1:16 or were found in CSF at any dilution starting from neat. For anti-CASPR2-encephalitides, this lab determined a serum titer cut-off of ≥1:512 for MRI-negative cases as included in this study .Sera were also tested for onconeural antibodies  by an immuno-dot-blot according to the manufacturer's recommendation .In this study on individuals with CHR or FEP and HV, three individuals harbored neural antibodies. These were only low-titer  CASPR2 sNone of the CSF samples, neither from individuals with CHR or FEP nor HV, harbored neural autoantibodies .This series of serum-CSF pairs of individuals with CHR or FEP and HV did not reveal CSF antibodies against a broad panel of neural intracellular and surface antigens, including the NMDAR. There were only two cases with serum IgG antibodies. Both were CASPR2 antibodies in serum only. Their titers were below the cut-off defined for positivity for autoimmune encephalitides in this laboratory , suggestThe study was not explicitly designed to identify IgA and IgM antibodies. In two serum samples, such antibodies were found when clarifying a mismatch of the non-IgG specific H+L secondary antibody and the IgG specific secondary antibody against the Fcγ fragment. These were likely the highest titer non-IgG antibodies in this series. Lower titer IgA or IgM antibodies may have gone undetected by the H+L secondary antibodies. This means there may be more non-IgG antibody positives in this series not revealed by our approach. Of note, non-IgG antibodies against the NMDAR have been found to be non-specific .Within a series of 121 people with the initial diagnosis of schizophrenia, one study finally found two individuals with antibodies against the NR1 subunit of the NMDAR in CSF—both were re-classified as anti-NMDAR encephalitis because they had the characteristic polymorphic presentation . AnotherApart from these cases, there have been several reports of patients with NMDAR antibodies in serum but not in CSF or of non-IgG NMDAR antibodies—both constellations are not disease-specific , 23. OneA recent meta-analysis found that the serum NMDAR IgG antibody prevalence in individuals with psychosis is associated with the assay type. More precisely, compared to a fixed CBA, a live CBA was associated with a significantly increased prevalence and significantly higher odds ratios when comparing patients and controls . The autNotably, the individuals with CHR and FEP included in this study were carefully assessed and fulfilled not only DSM-IV criteria but were also found to be within the normal range regarding a careful neurological examination, a brain MRI, an EEG, and extensive blood pathology. Therefore, our study results particularly highlight that isolated, psychotic manifestations of autoimmune encephalitides occur only sporadically. That is, clinicians need to be specifically vigilant when individuals present with additional neurological symptoms, evidence of clinically-significant cognitive involvement, profound sleep-wake perturbations, EEG or brain MRI pathologies . Expertsn = 190) is still relatively small to draw general conclusions on the relevance of the neural autoantibodies in general psychiatry settings.Since none of the healthy controls was younger than 18 years, these findings are not valid for pediatric and geriatric populations. Furthermore, despite a good clinical characterization of the participants presenting to two different clinical sites affiliated with universities, the overall sample size , thyroglobulin (TG), thyrotropin (TSH) receptor]. However, these systemic non-CNS  autoantibodies have also been reported to be associated with neuropsychiatric manifestations .Furthermore, we did not include samples from individuals with atypical psychotic syndromes. Future studies should compare the prevalence of neural autoantibodies in serum and CSF samples of individuals with schizophrenia, atypical psychotic syndromes, or atypical mood syndromes, and healthy controls to better understand the complex clinical picture of autoimmune-mediated psychiatric syndromes.atypical psychotic and mood syndromes. Therefore, it is essential to pay attention to those who present with polymorphic psychiatric symptoms, significant cognitive involvement, and neurological phenomena in daily clinical practice. Further, performing a careful neurological examination as well as a brain MRI scan and an EEG is essential to not miss neural antibody-associated autoimmune conditions. Consequently, in line with the majority of international guidelines, a comprehensive assessment is needed to detect autoimmune encephalitides. Our study highlights the importance of CSF analysis to reliably detect the involvement of immune processes and demonstrates the high specificity of positive CSF test results by the absence of detectable antibodies from our healthy controls.In summary, our study speaks against a major role of neural antibody-associated autoimmune encephalitides in the conditions studied here, namely typical presentations of CHR and FEP. However, there is emerging evidence of the involvement of various neural autoantibodies and systemic non-CNS autoantibodies in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the University of Cologne and Ethics Committee II of the Medical Faculty Mannheim, Heidelberg University. The patients/participants provided their written informed consent to participate in this study.FML, CGB, and CR conceived and designed the study. FML, JKM, and DK enrolled the participants and obtained data and body fluids. CGB and CIB analyzed the CSF and serum samples and the data. CGB, FML, and CR drafted the manuscript. All authors contributed to the final manuscript preparation and have read and approved the final manuscript.FML was a shareholder of curantis UG (Ltd.) and receives research support from the German Federal Ministry of Education and Research (BMBF), the Moyira Elizabeth Vine Fund for Research into Schizophrenia, and the DVCR research fund of the University of Sydney. CGB receives research support from the Deutsche Forschungsgemeinschaft  and Gerd-Altenhof-Stiftung . The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Inflammation-associated disorders are significant causes of morbidity in horses. Equine single-donor mesenchymal stromal cells (sdMSCs) hold promise as cell-therapy candidates due to their secretory nonprogenitor functions. This has been demonstrated by mononuclear cell suppression assays (MSAs) showing that sdMSCs are blood mononuclear cell (BMC) suppressive in vitro. sdMSCs derived from umbilical cord blood are of clinical interest due to their ease of procurement, multipotency, and immunomodulatory ability. Due to the inherent donor-to-donor heterogeneity of MSCs, the development of robust and easily deployable methods of potency assessment is critical for improving MSCs' predictability in treating inflammatory diseases. This study focuses on the development of robust in vitro potency assays and the assessment of potential sdMSC therapeutic end products generated from pooled sdMSCs (pMSCs). We hypothesized that, compared to MSA using only one donor, MSA using pooled BMCs (pBMCs) is a more robust sdMSC potency assay due to reduced donor BMC heterogeneity. pBMCs were generated by pooling equine BMCs isolated from peripheral blood of five donors in equal ratios. pBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stored in liquid nitrogen until use. Similarly, pooling sdMSCs from multiple equine donors in equal ratios generated pMSCs. sdMSC cultures were assessed with pBMCs in MSA using Bromodeoxyuridine ELISA and CFSE. Proliferation assessment of BMCs from individual donors revealed varied responses to concanavalin A (ConA) stimulation. MSA using BMCs from single donors further demonstrated BMC donor variability. Utilizing this assay, we have also found that the immunosuppressive potencies of pMSCs are at least equal, if not more, than the calculated mean of individual cultures. MSA based on pBMCs provides a consistent and reproducible equine sdMSC potency assay. This knowledge could be used in production monitoring of cellular potency and as release criteria before clinical use. Horses are valued in sports and recreation; they also serve as preclinical models for human conditions. Inflammation-related disorders, such as osteoarthritis and inflammatory airway disease, are significant causes of morbidity in the equine industry, and these diseases affect equine species similarly to humans . CurrentsdMSCs' immunosuppressive capabilities have been well documented in both humans and horses. Human sdMSCs have lymphocyte suppressive abilities in a dose-dependent manner . Similar+ and CD8+ lymphocytes increased and CD4+/CD8+ double-positive lymphocytes decreased [+ and CD8+ lymphocytes may be attributed to the increase in CD4+ helper T cells and CD8+ cytotoxic T cell populations. The presence of CD4+/CD8+ double-positive T cells is associated with several autoimmune conditions, such as autoimmune thyroiditis, systemic sclerosis, and in the fluid of joints from rheumatoid arthritis patients. Furthermore, higher CD4+/CD8+ double-positive T cell numbers have been shown to be associated with Kawasaki disease [Furthermore, when equine sdMSCs were administered in lipopolysaccharide induced synovitis in vivo, CD4ecreased . The inc disease .One of the main challenges for the clinical implementation of sdMSC therapeutics is the development of robust, consistent, and easily deployable end products . EffortssdMSCs are a heterogeneous population of cells with differential functional capabilities that depend on the health status of the donor, tissue of origin, isolation methods, and culture expansion methodologies ,10. ThusThe mononuclear cell suppression assay (MSA) can be used for evaluating sdMSCs' immunosuppressive potency in vitro. The most common MSA uses responder BMCs from one donor and either allogeneic stimulator cells, mitogens such as concanvalin A (ConA), or CD3/CD28 beads as stimulation for BMC proliferation . The addFor this study, we hypothesized that, compared to MSA using only one donor, MSA using pooled BMCs (pBMCs) is a more robust sdMSC potency assay due to reduced donor BMC heterogeneity. Furthermore, the pooling of sdMSCs from multiple donors allows the generation of standardized effective MSC products. In this study, we used a robust potency assay using pBMC and explored the immunosuppressive potency of pooled sdMSCs (pMSCs).The University of Guelph Animal Care Committee specifically approved this study with regards to the procedures of equine peripheral blood lymphocytes  and equine umbilical cord blood . Additional research conducted using specimens of this kind does not require review by the Animal Care Committee . Peripheral blood and cord blood collection were conducted as add-on procedures to the routine care of the horse. No animals were harmed or sacrificed during this study.The horse owners/agents provided informed consent in writing. Cord blood was collected after the umbilical cord had been clamped and detached from the foal. Access to research horses is granted after the approval of the animal care protocol by investigators. Peripheral blood was collected from adult horses at the Equine Research Station managed by the University of Guelph in partnership with the Ontario Ministry of Agriculture, Forestry, and Rural Affairs. Peripheral blood was collected by Dr. Koch. Horses were under mild sedation , and blood was collected from the jugular vein following which manual pressure was applied for several minutes to aid hemostasis.4CL, 100 mM KHCO4, 1 mM EDTA, pH 7.3; Sigma, Oakville, Canada) was added to cord blood in 3:1 ratio and mixed for 10 min at room temperature. Then the mixture was centrifuged for 10 min at 400 g. The supernatant was discarded, and the cell pellet was washed in PBS (Sigma) and centrifuged. The supernatant was discarded, and cells were resuspended in sdMSC isolation media consisting of DMEM low glucose , 10% fetal bovine serum (FBS) , 100 nM dexamethasone (Sigma), 2 mM l-glutamine (Sigma), and 100 U penicillin–streptomycin (Invitrogen) and seeded in culture flasks at density 1 × 106 cells cm−2, incubated at 38°C and 5% CO2.Umbilical cord blood-derived sdMSCs were isolated from newborn foals as previously described . Brieflyl-glutamine (Sigma), and 100 U penicillin–streptomycin (Invitrogen). Cells were passaged at 70% confluency and seeded at 5000 cells/cm2.Media were changed every 24 h for the first 3 days in culture and every 3 days thereafter. After the formation of putative sdMSC colonies, the adherent cells were detached with 0.25% trypsin (Sigma) and plated again in sdMSC expansion media consisting of DMEM low glucose (Lonza), 10% FBS (Invitrogen), 2 mM g for 30 min with no break. The BMC containing fraction was removed, pooled, and washed with PBS. The remaining pellet after supernatant removal was washed again with PBS and resuspended in Roswell park memorial institute (RPMI) 1640 medium supplemented with penicillin , streptomycin , and 10% FBS (Invitrogen), and a live cell count was performed with the NC-100 automatic cell counter .Peripheral blood was obtained from the jugular vein of adult horses. BMCs were isolated using Ficoll-density gradient. Fifteen milliliters of Ficoll-Paque Plus  was loaded with 35 mL of undiluted whole blood. Gradients were centrifuged at 300 ® (STEMCELL technologies) at a concentration of 6 × 106 cells/mL. BMCs were frozen and stored in liquid nitrogen until use.For carboxyfluorescein succinimidyl ester (CFSE) labeled BMCs, the cells were labeled with CellTrace™ CFSE cell proliferation Kit (ThermoFisher) following the manufacturer's protocol. BMCs were resuspended in CryoStor), CD44 , CD45 , CD73 , CD90 , MHC I , and MHC II .Cord-blood-sdMSCs were chemically detached with Accumax (STEMCELL Technologies) and washed with flow buffer . sdMSCs were characterized with flow cytometry for antigens: CD4 , CD8 , CD11a/18 , CD24 (clone: 4B4- Fluorescein isothiocyanate (FITC), Beckman Coulter, Mississauga, CanadaCells were incubated at 4°C in the dark for 15 min, followed by a wash and secondary antibody incubation at 4°C for 15 min in the dark, rat anti-mouse IgM-FITC or goat anti-mouse IgG1-FITC (Abcam) for CD90 were used as secondary antibodies. Ammonium chloride (Sigma-Aldrich) red blood cell lysis was performed on peripheral and cord blood for leukocyte isolation followed by a wash with flow buffer. sdMSCs were stained for CD4, CD8, CD11a/18, CD45, CD90, MHC I, and MHC II surface expression. A minimum of 10,000 events was acquired for each antibody with CellQuest software (Becton Dickinson), and data were analyzed with FlowJo software . Gates to identify leukocytes or sdMSC populations were consistent throughout all experiments.MSA and pooled MSA (pMSA) were used to evaluate sdMSCs for their ability to suppress BMC proliferation in vitro . Positiv2. After 3 days of coculturing, cells were stained with Bromodeoxyuridine (BrdU) for 18 h and assessed with a BrdU ELISA Kit  following the manufacturer's protocol. Fold changes of BMC proliferation are calculated using the formula below:The culture media for all groups in MSA and pMSA consisted of RPMI 1640 basal media supplemented with penicillin , streptomycin , and 10% FBS (Invitrogen). Cocultures were performed in 48-well plates, and reactions were incubated for 4 days at 38°C in 5% COFor CFSE pMSA, sdMSCs were collected and washed once with PBS and twice with flow buffer . Subsequently, 7-aminoactinomycin D (Sigma) was added for 5 min. Samples in triplicates were run and analyzed on a BD Accuri C6 cytometer unit .P < 0.05 (* indicates P < 0.05).Results were analyzed using GraphPad Prism Software Version 8.0 . Data are presented as mean ± standard error of the mean (SEM). MSA and pMSC data were analyzed using one-way ANOVA and corrected using Tukey's post-hoc test, at n = 7) were cocultured with BMCs from two donors. As expected, the positive controls, where BMCs were stimulated with ConA and cultured without sdMSCs, proliferated to different extents for each donor: by a fold change of 15 for donor A and by a fold change of 6 for donor B.To determine whether donor-to-donor variability exists between BMC donors, we quantified the proliferation of BMCs isolated from five donors after stimulation with ConA. Our results show that the extent of BMC proliferation differs significantly between BMC donors, demonstrating that BMCs' response to stimuli is donor dependent . To deteAll seven sdMSC cultures were able to suppress BMCs relative to the positive control. sdMSC cultures 1705, 1706, and 1707 were more immunosuppressive than 1701, 1702, 1703, and 1704 when tested against donor A. However, all seven sdMSC cultures showed similar immunosuppressive potencies when tested against donor B . The difBMCs were pooled from five donors in equal ratios to minimize BMC donor-to-donor variability. Then we tested whether sdMSCs were able to suppress pBMCs as they did when cocultured with single donor BMCs. ConA was added to all cocultures except for the negative control; thus, in addition to mixed lymphocyte stimulation between BMC cultures, mitogen stimulation was also present.As a preliminary test, four sdMSC cultures were added to pBMCs, and proliferation was assessed with BrdU ELISA. All four sdMSC cultures were able to suppress pBMC proliferation . We repeTo determine if pMSCs also suppress pBMCs, sdMSC cultures used in each of the experiments were pooled in equal ratios and their immunosuppressive potencies were assessed. It should be noted that sdMSCs were individually expanded and harvested before being pooled in equal numbers, as opposed to being pooled and then culture expanded before inclusion in the MSA as reported by others . This appMSCs were able to suppress pBMCs in all three experiments. Interestingly, pMSCs suppressed pBMC proliferation more than the calculated mean of individual cultures in the experiments using four sdMSC cultures  and 12 sOur data demonstrated that BMC donor-to-donor heterogeneity does exist; therefore, evaluating sdMSC potency using only one BMC donor could confound potency readouts. In this study, we have developed an in vitro potency assay using equine pBMCs that minimize biases that result from BMC donor-to-donor variations. Furthermore, we used this assay to evaluate the potency of pMSCs and demonstrated that they are equally or more immunosuppressive than individual sdMSC cultures. pMSC may, therefore, represent a therapeutic product with improved consistency and decreased production time.Our observation that substantial donor-to-donor variability was present in the unfractionated BMCs collected from individual donors agrees with several previous studies ,17. ThisThe concept of using pBMCs to minimize variability has been documented by other immunological assays . Unlike Surprisingly, even though both BrdU ELISA and CFSE allowed the reliable quantification of pBMC proliferation, these techniques yielded different results for the relative suppressive potency between sdMSC cultures. This discrepancy could be attributed to the different time frames within the coculture duration that each proliferation test captures. BrdU was added on day 3 of coculture, and pBMC proliferation was only measured between day 3 and 4. In contrast, pBMCs were prelabeled with CFSE before coculturing, and proliferation during the entire process was captured by CFSE. Thus, it is possible that the sdMSC cultures with the highest pBMC suppressive potency on day 3 may not be the same as those with the highest potency throughout the entire coculture period. This suggests that labeling with CFSE may provide a broader view of sdMSCs' immunosuppressive potency.CFSE is superior to BrdU ELISA in terms of deployability and flexibility, providing additional advantages. The CFSE method allows prelabeling and cryopreserving of pBMCs until use, which can be tested off-the-shelf for allo-stimulated, mitogen-driven proliferation assay. If further analysis of BMC subpopulations is required, CFSE provides the flexibility of simultaneous labeling with multiple markers.The results demonstrating that pMSCs are capable of suppressing pBMC proliferation agreed with Ketterl and colleagues 2015) with one exception: in 15 with oIn addition, Kuçi et al. (2016) demonstrated that patients treated with clinical end products generated by pooled bone marrow mononuclear cell (BM-BMC) derived sdMSCs had a higher overall survival rate than patients treated with sdMSCs from single donors. Intriguingly, their study also showed that sdMSCs derived from pooled bone marrow mononuclear cells suppressed BMC proliferation more than sdMSC cultures pooled upon coculturing . The poopMSCs have several advantages over sdMSCs. First, multiple studies have shown that sdMSCs exhibit donor-specific variability ; poolingSecond, due to the high cell number required for sdMSC therapy, culture extensive expansion is required; however, previous studies have reported that long-term in vitro culture expansion increases sdMSCs' genetic instability . ImportaOne limitation of this study is that the assessment of pMNC proliferation does not provide mechanistic insights to sdMSCs' immunosuppression; information such as the cytokines involved or suppression of subpopulations cannot be determined through quantifying the proliferation alone. Thus, we propose that future studies on elucidating the cytokine changes induced by pMSA will be necessary. In addition to offering mechanistic insights, cytokine analysis also increases the sensitivity of the assay, as small changes that are not detectable by measuring proliferation can be detected by measuring changes in cytokine levels.Even though in vitro pMSA offers faster and more economical solutions , preclinThus, future research avenues could consider administering the sdMSC cultures with the poorest and the best performance assessed by pMSA in preclinical models and utilize changes in cytokine levels and gene expression to elucidate the association between in vitro sdMSC potency determined with pMSA and in vivo efficacy. If a predictive panel of cytokines can be determined for the most potent sdMSC culture or pMSC batch, then it will be possible to create a faster and more sensitive in vitro potency test.Our results demonstrate that equine BMC donor-to-donor heterogeneity exists; thus, in vitro sdMSC potency tests based on one donor may be difficult to interpret reliably. pMSA based on pBMCs offers a more standardized potency assessment solution through minimizing BMC donor-to-donor heterogeneity. Importantly, using pMSA, we have shown that pMSCs have immunosuppressive potential. Compared to sdMSCs from single donors, pMSCs are superior in minimizing sdMSC donor-to-donor variability and are more predictable because it allows the generation of sdMSC banks which can be used for multiple treatment. Furthermore, pMSCs also require less culture expansion. If future studies could demonstrate that pMSCs have strong potency in vivo, then pMSCs can become promising therapeutic products for treating inflammatory diseases.