Excellent outcome following emergency deceased donor ABO-incompatible liver transplantation using rituximab and antigen specific immunoadsorption.

BACKGROUND: The acceptance of ABO-incompatible (ABOi) liver grafts will expand the donor pool for a patient in urgent need for a liver transplantation (LT). Here we report our results with emergency ABOi DD (deceased donor) LT using rituximab and antigen specific immunoadsorption. PATIENTS AND METHODS: 2009 to 2019 we performed 20 ABOi DD LTs (adults n = 17, children n = 3) for patients in urgent need for a LT. Immunosuppression consisted of rituximab (n = 20) and basiliximab (n = 15) or anti-thymocyte globuline (n = 4), intravenous immunoglobulin (IVIG; n = 6), tacrolimus, prednisolone and mycophenolate mofetil. Fifteen patients were treated with IA (n = 14) or both IA and plasmapheresis (PP; n = 1) pre-transplant and 18 patients were treated with IA (n = 15) or both IA and PP (n = 3) post-transplant. The median pre-transplant MELD- score was 40 (range 18-40). Patient and graft survival and complications were compared to a 1:4 case matched control group of ABO-identical or compatible (ABOid/c) DDLT. RESULTS: The 1-, 3- and 5-year patient and graft survival rates were 85, 85 and 78% for the ABOi recipients and not significantly different compared to ABOid/c controls. Only one ABOi patient developed antibody-mediated rejection. CONCLUSION: Patient and graft survival after emergency ABOi DDLT using rituximab and immunoadorption was equal to ABOid/DDLT. ABOi DD LT was a successful approach to expand the donor pool for patients in urgent need for a liver graft.

Homosynaptic frequency-dependent depression by release site inactivation at neonatal hippocampal synapses in the stratum lacunosum-moleculare.

When activated at low frequencies (0.1-1 Hz), second postnatal week synapses onto the most distal part of the apical dendritic tree (stratum lacunosum-moleculare) of rat hippocampal CA1 pyramidal cells display a frequency-dependent synaptic depression not observed for the more proximal (stratum radiatum) synapses. Depression in this frequency range is thought of as a possible contributor to behavioural habituation. In fact, in contrast to the proximal synapses, the distal synapses provide more direct sensory information from the entorhinal cortex as well as from thalamic nuclei. The use of antagonists showed that the activation of GABAA , GABAB , NMDA, mGlu, kainate, adenosine, or endocannabinoid receptors was not directly involved in the depression, indicating it to be intrinsic to the synapses themselves. While the depression affected paired-pulse plasticity in a manner indicating a decrease in vesicle release probability, the depression could not be explained by a stimulus-dependent decrease in calcium influx. Despite affecting the synaptic response evoked by brief high-frequency stimulation (10 impulses, 20 Hz) in a manner indicating vesicle depletion, the depression was unaffected by large variations in release probability. The depression was found not only to affect the synaptic transmission at low frequencies (0.1-1 Hz) but also to contribute to the depression evolving during brief high-frequency stimulation (10 impulses, 20 Hz). We propose that a release-independent process directly inactivating release sites with a fast onset (ms) and long duration (up to 20 s) underlies this synaptic depression.

Targeting CXCR1/2 Does Not Improve Insulin Secretion After Pancreatic Islet Transplantation: A Phase 3, Double-Blind, Randomized, Placebo-Controlled Trial in Type 1 Diabetes.

OBJECTIVE: Reparixin is an inhibitor of CXCR1/2 chemokine receptor shown to be an effective anti-inflammatory adjuvant in a pilot clinical trial in allotransplant recipients. RESEARCH DESIGN AND METHODS: A phase 3, multicenter, randomized, double-blind, parallel-assignment study (NCT01817959) was conducted in recipients of islet allotransplants randomized (2:1) to reparixin or placebo in addition to immunosuppression. Primary outcome was the area under the curve (AUC) for C-peptide during the mixed-meal tolerance test at day 75 ± 5 after the first and day 365 ± 14 after the last transplant. Secondary end points included insulin independence and standard measures of glycemic control. RESULTS: The intention-to-treat analysis did not show a significant difference in C-peptide AUC at both day 75 (27 on reparixin vs. 18 on placebo, P = 0.99) and day 365 (24 on reparixin vs. 15 on placebo, P = 0.71). There was no statistically significant difference between treatment groups at any time point for any secondary variable. Analysis of patient subsets showed a trend for a higher percentage of subjects retaining insulin independence for 1 year after a single islet infusion in patients receiving reparixin as compared with patients receiving placebo (26.7% vs. 0%, P = 0.09) when antithymocyte globulin was used as induction immunosuppression. CONCLUSIONS: In this first double-blind randomized trial, islet transplantation data obtained with reparixin do not support a role of CXCR1/2 inhibition in preventing islet inflammation-mediated damage.

Tumor-infiltrating mucosal-associated invariant T (MAIT) cells retain expression of cytotoxic effector molecules.

Mucosal-associated invariant T (MAIT) cells all express a semi-invariable T cell receptor recognizing microbial metabolites presented on the MHC class I-like molecule MR1. Upon activation, they rapidly secrete cytokines and increase their cytotoxic potential. We showed recently that MAIT cells with Th1 phenotype accumulate in human colon adenocarcinomas. Here, we investigated the cytotoxic potential of tumor-infiltrating MAIT cells in colon adenocarcinomas, and to what extent it may be affected by the tumor microenvironment. Activation of MAIT cells from tumors induced increased Granzyme B, and to a lesser extent, perforin expression. Degranulation was assessed by surface expression of CD107a, and was also seen in response to cognate antigen recognition. The cytotoxic potential of tumor-associated MAIT cells was very similar to that of MAIT cells from unaffected colon. MAIT cells were also identified by immunofluorescence in direct contact with tumor cells in sections from colon cancer specimens. To summarize, tumor-associated MAIT cells from colon tumors have strong cytotoxic potential and are not compromised in this regard compared to MAIT cells from the unaffected colon. We conclude that MAIT cells may contribute significantly to the protective immune response to tumors, both by secretion of Th1-associated cytokines and by direct killing of tumor cells.

The Small and Dynamic Pre-primed Pool at the Release Site; A Useful Concept to Understand Release Probability and Short-Term Synaptic Plasticity?

Advanced imaging techniques have revealed that synapses contain nanomodules in which pre- and post-synaptic molecules are brought together to form an integrated subsynaptic component for vesicle release and transmitter reception. Based on data from an electrophysiological study of ours in which release from synapses containing a single nanomodule was induced by brief 50 Hz trains using minimal stimulation, and on data from such imaging studies, we present a possible modus operandi of such a nanomodule. We will describe the techniques and tools used to obtain and analyze the electrophysiological data from single CA3-CA1 hippocampal synapses from the neonatal rat brain. This analysis leads to the proposal that a nanomodule, despite containing a number of release locations, operates as a single release site, releasing at most a single vesicle at a time. In this nanomodule there appears to be two separate sets of release locations, one set that is responsible for release in response to the first few action potentials and another set that produces the release thereafter. The data also suggest that vesicles at the first set of release locations are primed by synaptic inactivity lasting seconds, this synaptic inactivity also resulting in a large heterogeneity in the values for vesicle release probability among the synapses. The number of vesicles being primed at this set of release locations prior to the arrival of an action potential is small (0-3) and varies from train to train. Following the first action potential, this heterogeneity in vesicle release probability largely vanishes in a release-independent manner, shaping a variation in paired-pulse plasticity among the synapses. After the first few action potentials release is produced from the second set of release locations, and is given by vesicles that have been recruited after the onset of synaptic activity. This release depends on the number of such release locations and the recruitment to such a location. The initial heterogeneity in vesicle release probability, its disappearance after a single action potential, and variation in the recruitment to the second set of release locations are instrumental in producing the heterogeneity in short-term synaptic plasticity among these synapses, and can be seen as means to create differential dynamics within a synapse population.

Calcium: A Crucial Potentiator for Efficient Enzyme Digestion of the Human Pancreas.

BACKGROUND: Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential because they synergize with collagenase for effective pancreatic digestion. The activity of these enzymes is critically dependent on the presence of Ca2+ ions at a concentration of 5-10 mM. The present study aimed to determine the Ca2+ concentration during human islet isolation and to ascertain whether the addition of supplementary Ca2+ is required to maintain an optimal Ca2+ concentration during the various phases of the islet isolation process. METHODS: Human islets were isolated according to standard methods and isolation parameters. Islet quality control and the number of isolations fulfilling standard transplantation criteria were evaluated. Ca2+ was determined by using standard clinical chemistry routines. Islet isolation was performed with or without addition of supplementary Ca2+ to reach a Ca2+ of 5 mM. RESULTS: Ca2+ concentration was markedly reduced in bicarbonate-based buffers, especially if additional bicarbonate was used to adjust the pH as recommended by the Clinical Islet Transplantation Consortium. A major reduction in Ca2+ concentration was also observed during pancreatic enzyme perfusion, digestion, and harvest. Additional Ca2+ supplementation of media used for dissolving the enzymes and during digestion, perfusion, and harvest was necessary in order to obtain the concentration recommended for optimal enzyme activity and efficient liberation of a large number of islets from the human pancreas. CONCLUSIONS: Ca2+ is to a large extent consumed during clinical islet isolation, and in the absence of supplementation, the concentration fell below that recommended for optimal enzyme activity. Ca2+ supplementation of the media used during human pancreas digestion is necessary to maintain the concentration recommended for optimal enzyme activity. Addition of Ca2+ to the enzyme blend has been implemented in the standard isolation protocols in the Nordic Network for Clinical Islet Transplantation.

Neurochemical Aftermath of Repetitive Mild Traumatic Brain Injury.

IMPORTANCE: Evidence is accumulating that repeated mild traumatic brain injury (mTBI) incidents can lead to persistent, long-term debilitating symptoms and in some cases a progressive neurodegenerative condition referred to as chronic traumatic encephalopathy. However, to our knowledge, there are no objective tools to examine to which degree persistent symptoms after mTBI are caused by neuronal injury. OBJECTIVE: To determine whether persistent symptoms after mTBI are associated with brain injury as evaluated by cerebrospinal fluid biochemical markers for axonal damage and other aspects of central nervous system injury. DESIGN, SETTINGS, AND PARTICIPANTS: A multicenter cross-sectional study involving professional Swedish ice hockey players who have had repeated mTBI, had postconcussion symptoms for more than 3 months, and fulfilled the criteria for postconcussion syndrome (PCS) according to the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) matched with neurologically healthy control individuals. The participants were enrolled between January 2014 and February 2016. The players were also assessed with Rivermead Post Concussion Symptoms Questionnaire and magnetic resonance imaging. MAIN OUTCOMES AND MEASURES: Neurofilament light protein, total tau, glial fibrillary acidic protein, amyloid ß, phosphorylated tau, and neurogranin concentrations in cerebrospinal fluid. RESULTS: A total of 31 participants (16 men with PCS; median age, 31 years; range, 22-53 years; and 15 control individuals [11 men and 4 women]; median age, 25 years; range, 21-35 years) were assessed. Of 16 players with PCS, 9 had PCS symptoms for more than 1 year, while the remaining 7 returned to play within a year. Neurofilament light proteins were significantly increased in players with PCS for more than 1 year (median, 410 pg/mL; range, 230-1440 pg/mL) compared with players whose PCS resolved within 1 year (median, 210 pg/mL; range, 140-460 pg/mL) as well as control individuals (median 238 pg/mL, range 128-526 pg/mL; P = .04 and P = .02, respectively). Furthermore, neurofilament light protein concentrations correlated with Rivermead Post Concussion Symptoms Questionnaire scores and lifetime concussion events (ρ = 0.58, P = .02 and ρ = 0.52, P = .04, respectively). Overall, players with PCS had significantly lower cerebrospinal fluid amyloid-ß levels compared with control individuals (median, 1094 pg/mL; range, 845-1305 pg/mL; P = .05). CONCLUSIONS AND RELEVANCE: Increased cerebrospinal fluid neurofilament light proteins and reduced amyloid ß were observed in patients with PCS, suggestive of axonal white matter injury and amyloid deposition. Measurement of these biomarkers may be an objective tool to assess the degree of central nervous system injury in individuals with PCS and to distinguish individuals who are at risk of developing chronic traumatic encephalopathy.

Labile glutamate signaling onto CA1 pyramidal cells in the developing hippocampus depends mechanistically on input pathway.

The apical dendrite of hippocampal CA1 pyramidal cells receives information from the entorhinal cortex via the dentate gyrus and CA3 (Schaffer-collateral (SC) input) proximally within the stratum radiatum (SR) and directly from the entorhinal cortex/thalamus distally within the stratum lacunosum-moleculare (SLM). During the early postnatal development, very low/low frequency (0.033-1Hz) activation of previously non-stimulated (naïve) SC synapses (SR-CA1 synapses) results in a stimulus-, but not frequency-, dependent depression which is explained by postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) silencing. This lability of AMPA signaling has been suggested to play a role in the activity-dependent organization of the SR synaptic input. Compared with the SR region the SLM region is matured earlier and may become organized in a different manner. Here, using field recordings of synaptic activity in hippocampal slices from 2nd postnatal week rats, we found that SLM-CA1 synapses are also readily depressed by activity in the very low/low frequency range (0.033-1Hz). The depression was, however, quite distinct from that of SR-CA1 synapses, being frequency dependent and reversing faster following stimulus interruption. Bath application of an AMPA receptor agonist produced only a small (-8%) post-application (10min) depression of SLM-CA1 synapses in contrast to that of SR-CA1 synapses (-33%). The SLM-CA1 synapses did also exhibit less long-term depression than the SR-CA1 synapses. We conclude that in the developing hippocampus the labile glutamate signaling onto CA1 pyramidal cell depends mechanistically on input pathway. The labile AMPA signaling at SLM-CA1 synapses is most likely explained by a presynaptic mechanism with limited amount of postsynaptic AMPA silencing.

Recipient Hyperbilirubinemia May Reduce Ischemia-Reperfusion Injury but Fails to Improve Outcome in Clinical Liver Transplantation.

Background. Exogenous bilirubin may reduce experimental ischemia-reperfusion injury (IRI) due to its antioxidant properties. We studied if early graft exposure to high bilirubin levels in the recipient affects the early IRI and outcomes after liver transplantation (LTx). Methods. In 427 LTx patients, the AUROC curve based on bilirubin and AST at day 1 identified a cutoff of 2.04 mg/dL for the recipient pretransplant bilirubin. Recipients were grouped as having low (group L, n = 152) or high (group H, n = 275) bilirubin. Both groups had similar donor-related variables (age, preservation time, donor BMI > 28, and donor risk index (DRI)). Results. Alanine (ALT) and aspartate (AST) aminotransferase levels were higher in group L at day 1; ALT levels remained higher at day 2 in group L. LTx from high risk donors (DRI > 2) revealed a trend towards lower transaminases during the first two days after transplantation in group H. One month and 1-year patient survival were similar in groups L and H. High preoperative bilirubin did not affect the risk for early graft dysfunction (EGD), death, or graft loss during the first year after transplantation nor the incidence of acute rejection. LTx using donors with DRI > 2 resulted in similar rates of EGD in both groups. Conclusion. Increased bilirubin appears to reduce the early IRI after LTx yet this improvement was insufficient to improve the clinical outcome.

Evaluation of Perfluorohexyloctane/Polydimethylsiloxane for Pancreas Preservation for Clinical Islet Isolation and Transplantation.

This study aimed to evaluate a 50:50 mix of perfluorohexyloctane/polydimethylsiloxane 5 (F6H8S5) preservation of pancreases in a clinical setting compared with standard solutions for 1) cold ischemia time (CIT) 10 h and 2) an extended CIT 20 h. Procured clinical-grade pancreases were shipped in either F6H8S5 or in standard preservation solutions, that is, University of Wisconsin (UW) or Custodiol. F6H5S5 was preoxygenated for at least 15 min. Included clinical-grade pancreases were procured in UW or Custodiol. Upon arrival at the islet isolation laboratory, the duodenum was removed followed by rough trimming while F6H8S5 was oxygenated for 1520 min. Trimmed pancreases were immersed into oxygenated F6H8S5 and stored at 4C overnight followed by subsequent islet isolation. Pancreas preservation using F6H8S5 proved as effective as UW and Custadiol when used within CIT up to 10 h, in terms of both isolation outcome and islet functionality. Preservation in F6H8S5 of pancreases with extended CIT gave results similar to controls with CIT 10 h for both isolated islet functionality and isolation outcome. This study of clinically obtained pancreases indicates a clear benefit of using F6H8S5 on pancreases with extended CIT as it seems to allow extended cold ischemic time without affecting islet function and islet numbers.

Renal function three years after early conversion from a calcineurin inhibitor to everolimus: results from a randomized trial in kidney transplantation.

In a 36-month, open-label, multicenter trial, 202 kidney transplant recipients were randomized at week 7 post-transplant to convert to everolimus or remain on cyclosporine: 182 were analyzed to month 36 (92 everolimus, 90 controls). Mean (SD) change in measured GFR (mGFR) from randomization to month 36 was 1.3 (14.0) ml/min with everolimus versus -1.7 (15.4) ml/min in controls (P = 0.210). In patients who remained on treatment, mean mGFR improved from randomization to month 36 by 7.9 (11.5) ml/min with everolimus (n = 37) but decreased by 1.4 (14.7) ml/min in controls (n = 62) (P = 0.001). During months 12-36, death-censored graft survival was 100%, patient survival was 98.9% and 96.7% in the everolimus and control groups, respectively, and 13.0% and 11.1% of everolimus and control patients, respectively, experienced mild biopsy-proven acute rejection (BPAR). Protocol biopsies in a limited number of on-treatment patients showed similar interstitial fibrosis progression. Donor-specific antibodies were present at month 36 in 6.3% (2/32) and 18.0% (9/50) of on-treatment everolimus and control patients with available data (P = 0.281). During months 12-36, adverse events were comparable, but discontinuation was more frequent with everolimus (33.7% vs. 10.0%). Conversion from cyclosporine to everolimus at 7 weeks post-transplant was associated with a significant benefit in renal function at 3 years when everolimus was continued.

Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation.

UNLABELLED: Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential as they synergize with collagenase for effective pancreas digestion. The presence of tryptic-like activity has been implicated in efficient enzyme blends and the present study aimed to evaluate if addition of clostripain, an enzyme with tryptic-like activity, could improve efficacy of the islet isolation procedure. METHODS: Clostripain was added to the enzyme blend just before pancreas perfusion. Islets were isolated per standard method and numerous isolation parameters, islet quality control, and the number of isolations fulfilling standard transplantation criteria were evaluated. Two control organs per clostripain organ were chosen by blindly matching against body mass index, cold ischemia time, hemoglobin A1c, donor sex, and donor age. RESULTS: There were no differences in pancreas weight, dissection time, digestion time, harvest time, percent digested pancreas, or total pellet volume before islet purification between control or clostripain pancreases. Glucose-stimulated insulin release results were similar between groups. Total isolation islet equivalents, purified tissue volume and islet equivalents/g pancreas as well as fulfillment of transplantation criteria favored clostripain processed pancreases. CONCLUSIONS: The addition of clostripain to the enzyme blend soundly improved islet yields and transplantation rates. It gently aided pancreas digestion and maintained proper islet functionality. The addition of clostripain to the enzyme blend has now been implemented into standard isolation protocols at the isolation centers in Uppsala and in Oslo.

Silent synapses onto interneurons in the rat CA1 stratum radiatum.

Glutamate transmission to gamma-aminobutyric acid (GABA)ergic interneurons and to principal neurons differs in various important respects. Whether these differences exist from an early developmental stage, or result from differential development from a more common state, is unclear. In the hippocampal CA1 area, glutamate transmission to the developing, but not to the adult, principal neurons is characterized by the presence of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) silent synapses and of AMPA silencing induced by test pulse stimulation (0.03-1 Hz). In the present study, we examined whether this developmental difference in AMPA signaling is also true for glutamate transmission to CA1 stratum radiatum interneurons. We found that AMPA silent synapses onto these interneurons also exist, and that they can be generated by test pulse stimulation. In marked contrast to AMPA silencing in principal neurons, AMPA silencing in interneurons was not developmentally restricted, but was observed to the same extent after the first postnatal month as in the second postnatal week. In addition, we found that glutamate synapses onto these interneurons can also be N-methyl-d-aspartate (NMDA)-silent, that is, only AMPA-signaling. After test pulse stimulation, the AMPA-silent, the NMDA-silent and the AMPA/NMDA-signaling synapses onto the developing interneurons were estimated to be about equally frequent. These results highlight a diversity of glutamate signaling to CA1 stratum radiatum interneurons, and they indicate that the glutamate synapses onto pyramidal neurons and to interneurons can mature differentially.

The in-vivo effect of pyrrolidine dithiocarbamate on hepatic parenchymal microcirculation and oxygenation of the rat liver.

OBJECTIVE: Pyrrolidine dithiocarbamate has been shown to be a potent inducer of haemeoxygenase-1. This study investigated its in-vivo effects on systemic and hepatic microcirculatory perfusion. METHODS: Male Sprague-Dawley rats (n=12) were administered intravenously with pyrrolidine dithiocarbamate (10, 20 and 50 mg/kg body weight) or vehicle (0.2 ml physiological saline) served as control. Systemic and hepatic haemodynamics including arterial oxygen saturation, heart rate, mean arterial blood pressure and portal blood flow were monitored. Microcirculation in skeletal muscle and liver was measured by laser Doppler flowmetry and intravital fluorescence microscopy, whereas hepatic tissue oxyhaemoglobin and cytochrome oxidase CuA redox state, which is an indicative of extracellular and intracellular oxygenation were measured by near infrared spectroscopy. RESULTS: Pyrrolidine dithiocarbamate induced a dose-dependent increase in mean arterial blood pressure and skeletal muscle microcirculation. The hepatic parenchymal microcirculation was significantly improved and an increase in sinusoidal diameter and reduction in RBC velocity were observed. Pyrrolidine dithiocarbamate also showed beneficial effect on hepatic tissue oxygenation showed by an increase in oxyhaemoglobin and cytochrome oxidase CuA redox state as well. CONCLUSION: Pyrrolidine dithiocarbamate improves hepatic parenchymal microcirculation and tissue oxygenation, suggesting that it may be used as a potential agent in pharmacological preconditioning in the liver.