Quantifying Target Occupancy of Small Molecules Within Living Cells.

The binding affinity and kinetics of target engagement are fundamental to establishing structure-activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.

Can 60 days of feeding lead to increased fitness for transport in cull dairy cows?

The welfare of cull cows during transport to slaughter is a current concern in the Canadian dairy industry. Cull cows sold through auction often have a high prevalence of lameness, low BCS, hock lesions, and udder engorgement. To evaluate whether drying off and feeding cull dairy cows before transport can mitigate these challenges, 45 cows designated for culling were randomly assigned to either be fed for 60 d after being dried off (fed group; n = 24) or to serve as controls by being sent directly to slaughter (direct group; n = 21). Two fed group cows were removed for health reasons before completing the feeding period. Both fed group and direct group cows were assessed for locomotion (5-point scale), BCS (5-point scale), hock lesions (3-point scale), udder engorgement (3-point scale), and BW at the time of enrollment. Fed group cows, locomotion, BCS, hock, and udder engorgement scores were assessed weekly until slaughter. Weights of the fed group cows were measured again the day before slaughter. Mixed linear regression models were used to assess continuous outcomes BCS and weight. Mixed logistic regression models were used to assess dichotomous outcomes presence of hock lesions and lameness. Fed group cows gained an average of 116.9 kg over the feeding period (SE ± 8.20). Fed group cows had an average weight at slaughter of 834.2 kg, whereas direct group cows' average weight was 767.3 kg (SE ± 26.8). The fed group cows' average BCS at the start of the trial was 2.4, and at slaughter was 3.6, with an average gain of 1.2 BCS points. At slaughter, the proportion of udders involuted in the fed group was 45.1% (10/22) and in the direct group cows, was 0% (0/21). No differences were found in locomotion or hock lesions between the fed group and direct groups. It is important to weigh potential benefits for the fed group cows with the fact that direct group cows did not endure a drying off procedure, nor were they placed at risk of potential adverse health events. However, despite these potential limitations, due to the improved BCS and udder engorgement scores, cows fed for 60 d may be better prepared for the transportation to slaughter, as well as sell for a higher price due to increased BW and body condition.

Improving market (cull) dairy cows carcass traits and meat quality.

Despite a high volume of market (cull) dairy cows entering the food chain every year, beef from market dairy cows is largely considered insignificant when compared with beef from beef cattle in the market and is widely thought to be used for ground beef only. Our study aimed to evaluate the effects of feeding dairy market cows on a lactating TMR diet before slaughter on carcass and beef quality traits. Forty-three Holstein market cows were randomly assigned into 2 treatments: Fed or Direct. Fed cows (n = 22) were dried off, then fed a lactating cow TMR ration for approximately 60 d whereas Direct cows (n = 21) were sent directly to slaughter. Hot carcass weight (HCW) was used to calculate the dressing percentage (DP) together with the animals' body weight recorded when animals left the farm to go to the abattoir. At 24h post-mortem, the rib fat thickness (RFT) and rib eye area (REA) were measured by a certified grader. Afterward, rib samples were collected between the 12th -10th ribs from one side of the carcass and divided into 5 ribeye steaks. The first steak was used for intramuscular fat (IMF) content analysis by NIRS. The other 4 steaks were then aged for 7, 14, 21, or 28 d. Following aging, the steaks available were assessed for tenderness using the Warner-Bratzler Shear Force protocol. Data were analyzed by mixed linear regression to compare experimental groups for continuous outcomes. HCW of the Fed cows was higher than the Direct cows, with a mean HCW of 408.3 kg and 326.1 kg, respectively (SE ± 9.0). Average DP in Fed cows was 49.1%, compared with 42.5% in Direct cows (SE ± 0.69). The average percentage of IMF in Fed cows was 8.1% and in Direct cows was 5.0% (SE ± 0.43). Fed cows had a mean REA of 68.1 cm2 and Direct cows had a mean of 57.7 cm2 (SE ± 11.9). No differences were found for RFT. The steaks aged for 14 d resulted in meat with a mean shear force (SF) of 4.19 kg in Fed cows, while Direct cows had a mean SF of 6.28 kg (SE ± 0.29) Feeding market dairy cows for 60 d before slaughter enhanced carcass weight and yield, IMF content, and tenderness. These results indicate that approximately 60 d of feeding can improve the quality of market dairy cow meat and may help improve market cow value.

Effect of glycerol supplementation in early lactation on metabolic health, milking activity, and production of dairy cows in automated milking system herds.

The objective of this study was to quantify the effects of supplementing early-lactation cows with a dry pure glycerol product, delivered through the automated milking system (AMS) concentrate, in the first 21 d in milk (DIM) on metabolic markers, milking behavior, and milk production. In 5 commercial AMS dairy herds, 389 dairy cows were randomly assigned, controlling for parity, 21 d before expected calving to 1 of 2 treatments, within farm: (1) control group (CON) receiving the standard AMS pellet (n = 213) from 1 to 150 DIM, or (2) glycerol group (GLY) receiving the treatment AMS pellet (n = 176) formulated to deliver 250 as fed g/d of glycerol product from 1 to 21 DIM (mean actual = 249 g/d dry matter [DM]), then they received the standard AMS pellet from 22 to 150 DIM. Across all farms, cows were fed partial mixed rations (PMR) that were similar in ingredient and nutrient composition. One prepartum blood sample and 5 postpartum blood samples were collected from each cow to determine serum nonesterified fatty acids (NEFA), blood ß-hydroxy butyrate (BHB), and blood glucose concentrations. Cow body condition score (BCS) was recorded every 21 d from -21 to 63 DIM. Data were collected and analyzed for the treatment period (1 to 21 DIM) and a follow-up period (22 to 150 DIM). There was no detected treatment effect on serum NEFA concentrations in the first week of lactation. There was a treatment by time interaction for blood BHB and blood glucose, where GLY cows tended to have increased BHB concentrations at 5 DIM and had decreased glucose concentrations at 9 and 12 DIM. There was an interaction of BCS with treatment on the incidence of BHB ≥1.2 mmol/L, whereby over-conditioned CON cows (BCS ≥3.5) were 3.5x more likely to have a high BHB test than CON cows with normal prepartum BCS. During the treatment period GLY cows had 0.1 ± 0.05 more successful milkings/d, were delivered 0.27 ± 0.05 DM kg/d more AMS concentrate and tended to yield 0.8 ± 0.47 kg/d more milk. During the follow-up period GLY cows had 0.1 ± 0.04 more successful milkings/d, were delivered 0.18 ± 0.06 DM kg/d more AMS concentrate, and yielded 1.5 ± 0.53 kg/d more milk than CON cows. Glycerol supplementation allowed cows to maintain better BCS, as GLY cows lost less BCS from calving to 63 DIM than CON cows. Overall, the results of this study demonstrate that supplementing pure glycerol through the AMS concentrate for the first 21 DIM can reduce BCS loss in early lactation, improve milking behavior, and increase milk yield to mid lactation.

Colostrum insulin supplementation to neonatal Holstein bulls affects small intestinal histomorphology, mRNA expression, and enzymatic activity with minor influences on peripheral metabolism.

The objectives of this study were to evaluate how varying colostral insulin concentrations influenced small intestinal development and peripheral metabolism in neonatal Holstein bulls. Insulin was supplemented to approximately 5× (70.0 µg/L; n = 16) or 10× (149.7 µg/L; n = 16) the basal colostrum insulin (12.9 µg/L; BI, n = 16) concentration to maintain equivalent macronutrient intake (crude fat: 4.1 ± 0.06%; crude protein: 11.7 ± 0.05%; and lactose: 1.9 ± 0.01%) among treatments. Colostrum was fed at 2, 14, and 26 h postnatal and blood metabolites and insulin concentration were measured at 0, 30, 60, 90, 120, 180, 240, 360, 480, and 600 min postprandial respective to the first and second colostrum meal. At 30 h postnatal, a subset of calves (n = 8/treatment) were killed to excise the gastrointestinal and visceral tissues. Gastrointestinal and visceral gross morphology and dry matter and small intestinal histomorphology, gene expression, and carbohydrase activity were assessed. Insulin supplementation tended to linearly reduce the glucose clearance rate following the first meal, whereas after the second meal, supplementation linearly increased the rate of glucose absorption and nonesterified fatty acid clearance rate, decreased the time to maximum glucose concentrations, and decreased the time to reach minimum nonesterified fatty acid concentrations. Additionally, insulin clearance rate was linearly increased by insulin supplementation following the second colostrum feeding. However, there were no overall differences between treatments in the concentrations of glucose, nonesterified fatty acids, or insulin in plasma or serum. With respect to macroscopic intestinal development, dry rumen tissue mass linearly decreased when insulin was supplemented in colostrum, and supplementation linearly increased duodenal dry tissue density (g dry matter/cm) while tending to increase duodenal dry tissue weight. Increasing the colostrum insulin concentration improved small intestinal histomorphological development in the distal small intestine, as ileal villi height and mucosal-serosal surface area index were increased by supplementing insulin. Lactase enzymatic activity linearly increased in the proximal jejunum while ileal isomaltase activity linearly decreased with insulin supplementation. These data indicate that changes in colostrum insulin concentrations rapidly affect gastrointestinal growth prioritization and carbohydrase activity. The changes in gastrointestinal ontology result in minor changes in postprandial metabolite availability and clearance.

From cookies to carrots; the effect of inhibitory control training on children's snack selections.

Children consume too much sugar and not enough fruit and vegetables, increasing their risk of adverse health outcomes. Inhibitory control training (ICT) reduces children's and adults' intake of energy-dense foods in both laboratory and real-life settings. However, no studies have yet examined whether ICT can increase healthy food choice when energy-dense options are also available. We investigated whether a food-specific Go/No-Go task could influence the food choices of children aged 4-11, as measured by a hypothetical food choice task using healthy and unhealthy food images printed on cards. Participants played either an active game (healthy foods = 100% go, unhealthy foods = 100% no-go; Studies 1 & 2), a food control game (both healthy and unhealthy foods = 50% go, 50% no-go; Studies 1 & 2) or a non-food control game (sports equipment = 100% go, technology = 100% no-go; Study 2 only) followed by the choice task. In Study 2, food card choices were also measured before training to examine change in choices. A post-training real food choice task was added to check that choices made in the card-based task were representative of choices made when faced with real healthy and unhealthy foods. Overall, the active group chose the greatest number of healthy food cards. Study 2 confirmed that this was due to increases in healthy food card choice in this group only. Active group participants chose a greater number of healthy foods in the real food choice task compared to children in the non-food control group only. The results are discussed with reference to methodological issues and the development of future healthy eating interventions.

Exploring relationships between body condition score, body fat, activity level and inflammatory biomarkers.

Obesity is associated with inflammatory disorders in humans, including degenerative joint disease. While obesity is endemic in horses, its relationship to equine degenerative joint disease has not been explored. The current study sought to describe relationships between: body weight (BW), body condition score (BCS), lameness grade (AAEP), total body fat mass (kg; FM) and fat per cent (FP) [multifrequency bioelectrical impedance analysis (mfBIA)], age, gender, activity level (AL), synovial fluid (SF) and plasma (PL) PGE2 and glycosaminoglycan (GAG) in horses. During this field investigation, the BCS (of nine) of 54 horses at multiple farms in southern Ontario, Canada, was determined. Horses were categorized as thin (BCS=3/9; n = 6), moderate (BCS=4 or 5/9; n = 18), overweight (BCS=6 or 7/9; n = 19) or obese (BCS=8 or 9/9; n = 11). Total fat mass (kg) and body fat% was measured using mfBIA, lameness was assessed (AAEP lameness scale) and synovial fluid was collected via aseptic arthrocentesis from the left intercarpal joint for assessment of inflammatory biomarkers (PGE2 , GAG). Means were compared with a one-way ANOVA; correlation coefficients were calculated using a Spearman Rank Order Correlation to reveal correlations between variables. BCS was positively correlated with BW, FM, FP, AL and PL-PGE2 . BW was also significantly positively correlated with PL-PGE2 . It is concluded that BCS is significantly correlated with PL-PGE2 , due in part to the combined effect of AL and body condition. Net inflammatory effects of body fat on risk for joint disease require further study.

Quantification of CD4(+) T Cell Alloreactivity and Its Control by Regulatory T Cells Using Time-Lapse Microscopy and Immune Synapse Detection.

Assays designed to select transplant recipients for immunosuppression withdrawal have met with limited success, perhaps because they measure events downstream of T cell-alloantigen interactions. Using in vitro time-lapse microscopy in a mouse transplant model, we investigated whether transplant outcome would result in changes in the proportion of CD4(+) T cells forming prolonged interactions with donor dendritic cells. By blocking CD4-MHC class II and CD28-B7 interactions, we defined immunologically relevant interactions as those ≥500 s. Using this threshold, T cell-dendritic cell (T-DC) interactions were examined in rejection, tolerance and T cell control mediated by regulatory T cells. The frequency of T-DC contacts ≥500 s increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. Regulatory T cells reduced prolonged T-DC contacts. Importantly, this effect was replicated with human polyclonally expanded naturally occurring regulatory T cells, which we have previously shown can control rejection of human tissues in humanized mouse models. Finally, in a proof-of-concept translational context, we were able to visualize differential allogeneic immune synapse formation in polyclonal CD4(+) T cells using high-throughput imaging flow cytometry.

Phase I study of safety and pharmacokinetics of the anti-MUC16 antibody-drug conjugate DMUC5754A in patients with platinum-resistant ovarian cancer or unresectable pancreatic cancer.

BACKGROUND: MUC16 is a tumor-specific antigen overexpressed in ovarian (OC) and pancreatic (PC) cancers. The antibody-drug conjugate (ADC), DMUC5754A, contains the humanized anti-MUC16 monoclonal antibody conjugated to the microtubule-disrupting agent, monomethyl auristatin E (MMAE). PATIENTS AND METHODS: This phase I study evaluated safety, pharmacokinetics (PK), and pharmacodynamics of DMUC5754A given every 3 weeks (Q3W, 0.3-3.2 mg/kg) or weekly (Q1W, 0.8-1.6 mg/kg) to patients with advanced recurrent platinum-resistant OC or unresectable PC. Biomarker studies were also undertaken. RESULTS: Patients (66 OC, 11 PC) were treated with DMUC5754A (54 Q3W, 23 Q1W). Common related adverse events (AEs) in >20% of patients (all grades) over all dose levels were fatigue, peripheral neuropathy, nausea, decreased appetite, vomiting, diarrhea, alopecia, and pyrexia in Q3W patents, and nausea, vomiting, anemia, fatigue, neutropenia, alopecia, decreased appetite, diarrhea, and hypomagnesemia in Q1W patients. Grade ≥3-related AE in ≥5% of patients included neutropenia (9%) and fatigue (7%) in Q3W patients, and neutropenia (17%), diarrhea (9%), and hyponatremia (9%) in Q1W patients. Plasma antibody-conjugated MMAE (acMMAE) and serum total antibody exhibited non-linear PK across tested doses. Minimal accumulation of acMMAE, total antibody, or unconjugated MMAE was observed. Confirmed responses (1 CR, 6 PRs) occurred in OC patients whose tumors were MUC16-positive by IHC (2+ or 3+). Two OC patients had unconfirmed PRs; six OC patients had stable disease lasting >6 months. For CA125, a cut-off of ≥70% reduction was more suitable for monitoring treatment response due to the binding and clearance of serum CA125 by MUC16 ADC. We identified circulating HE4 as a potential novel surrogate biomarker for monitoring treatment response of MUC16 ADC and other anti-MUC16 therapies in OC. CONCLUSIONS: DMUC5754A has an acceptable safety profile and evidence of anti-tumor activity in patients with MUC16-expressing tumors. Objective responses were only observed in MUC16-high patients, although prospective validation is required. CLINICAL TRIAL NUMBER: NCT01335958.

CD4 cell count at initiation of ART, long-term likelihood of achieving CD4 >750 cells/mm3 and mortality risk.

OBJECTIVES: We sought to evaluate associations between CD4 at ART initiation (AI), achieving CD4 >750 cells/mm(3) (CD4 >750), long-term immunological recovery and survival. METHODS: This was a prospective observational cohort study. We analysed data from ART-naive patients seen in 1996-2012 and followed ≥3 years after AI. We used Kaplan-Meier (KM) methods and log-rank tests to compare time to achieving CD4 >750 by CD4 at AI (CD4-AI); and Cox regression models and generalized estimating equations to identify factors associated with achieving CD4 >750 and mortality risk. RESULTS: Of 1327 patients, followed for a median of 7.9 years, >85% received ART for ≥75% of follow-up time; 64 died. KM estimates evaluating likelihood of CD4 >750 during 5 years of follow-up, stratified by CD4-AI <50, 50-199, 200-349, 350-499 and 500-750, were 20%, 25%, 56%, 80% and 87%, respectively (log-rank P < 0.001). In adjusted models, CD4-AI ≥200 (versus CD4-AI <200) was associated with achievement of CD4 >750 [adjusted HR (aHR) = 4.77]. Blacks were less likely than whites to achieve CD4 >750 (33% versus 49%, aHR = 0.77). Mortality rates decreased with increasing CD4-AI (P = 0.004 across CD4 strata for AIDS causes and P = 0.009 for non-AIDS death causes). Among decedents with CD4-AI ≥50, 56% of deaths were due to non-AIDS causes. CONCLUSIONS: Higher CD4-AI resulted in greater long-term CD4 gains, likelihood of achieving CD4 >750, longer survival and decreased mortality regardless of cause. Over 80% of persons with CD4-AI ≥350 achieved CD4 >750 by 4 years while 75% of persons with CD4-AI <200 did not. These data confirm the hazards of delayed AI and support early AI.

The influence of age and weaning on permeability of the gastrointestinal tract in Holstein bull calves.

Fourteen Holstein bull calves were used in a randomized complete block design to investigate the effect of calf age and weaning on permeability of the gastrointestinal tract (GIT). Calves were randomly assigned to 1 of 2 treatments: (1) a weaning protocol that was initiated on d 35; WN; n=7), or (2) a control treatment where calves were not weaned (CON; n=7). Calves were bottle-fed milk replacer (150 g/L), in 3 equal portions/d targeting 15% of their body weight (BW) in liquid milk intake [approximately 21.1g/kg of BW/d, dry matter (DM) basis]. On d 35, the amount of milk replacer offered to WN calves was reduced to 7.5% of BW for 7 d before calves were weaned on d 42. On d 14, 28, and 42, calves were orally dosed with 500 mL of Cr-EDTA (179 mM Cr-EDTA solution) and housed in a metabolism crate to enable total urine collection and determination of total urinary Cr recovery as an indicator of total-tract permeability. On d 44, calves were killed and tissues from the rumen, omasum, duodenum, jejunum, ileum, cecum, and proximal and distal colon were collected, rinsed, and transported in buffer solution (pH 7.4 at 38.5°C). Tissues were incubated in Ussing chambers under short-circuit conditions with buffer solutions designed to mimic the mucosal and serosal energy source that would be available in vivo (glucose for tissues from the small intestine and short-chain fatty acids for tissues that would be exposed to fermentation; rumen, omasum, and large intestinal tissues). The serosal to mucosal flux of (14)C-mannitol and (3)H-inulin was measured for each region. Although we detected treatment × period interactions for BW and starter intake, dietary treatments did not differ within a week. Overall, the time that ruminal pH was <5.5 was less before weaning than after weaning. We observed a differential response for the appearance of Cr in urine for WN and CON calves, where the appearance of Cr (mg/48 h) in urine decreased for both treatments from d 14 to 28, but increased from d 28 to 42 for WN, whereas Cr appearance continued to decrease for CON. The flux of mannitol and inulin did not differ between treatments but did differ among region of the GIT, with rumen, duodenum, and jejunum having the greatest permeability. These data suggest that permeability of the GIT decreases with age but weaning may disrupt this process. The rumen, duodenum, and jejunum appear to be the regions with greatest permeability.

Threat-related learning relies on distinct dorsal prefrontal cortex network connectivity.

Conditioned changes in the emotional response to threat (e.g. aversive unconditioned stimulus; UCS) are mediated in part by the prefrontal cortex (PFC). Unpredictable threats elicit large emotional responses, while the response is diminished when the threat is predictable. A better understanding of how PFC connectivity to other brain regions varies with threat predictability would provide important insights into the neural processes that mediate conditioned diminution of the emotional response to threat. The present study examined brain connectivity during predictable and unpredictable threat exposure using a fear conditioning paradigm (previously published in Wood et al., 2012) in which unconditioned functional magnetic resonance imaging data were reanalyzed to assess effective connectivity. Granger causality analysis was performed using the time series data from 15 activated regions of interest after hemodynamic deconvolution, to determine regional effective connectivity. In addition, connectivity path weights were correlated with trait anxiety measures to assess the relationship between negative affect and brain connectivity. Results indicate the dorsomedial PFC (dmPFC) serves as a neural hub that influences activity in other brain regions when threats are unpredictable. In contrast, the dorsolateral PFC (dlPFC) serves as a neural hub that influences the activity of other brain regions when threats are predictable. These findings are consistent with the view that the dmPFC coordinates brain activity to take action, perhaps in a reactive manner, when an unpredicted threat is encountered, while the dlPFC coordinates brain regions to take action, in what may be a more proactive manner, to respond to predictable threats. Further, dlPFC connectivity to other brain regions (e.g. ventromedial PFC, amygdala, and insula) varied with negative affect (i.e. trait anxiety) when the UCS was predictable, suggesting that stronger connectivity may be required for emotion regulation in individuals with higher levels of negative affect.

Harnessing regulatory T cells for clinical use in transplantation: the end of the beginning.

Owing to the adverse effects of immunosuppression and an inability to prevent chronic rejection, there is a pressing need for alternative strategies to control alloimmunity. In three decades, regulatory T cells (Tregs) have evolved from a hypothetical mediator of adoptively transferred tolerance to a well-defined population that can be expanded ex vivo and returned safely to patients in clinical trials. Herein, we review the historical developments that have permitted these advances and the current status of clinical trials examining Tregs as a cellular therapy in transplantation. We conclude by discussing the critical unanswered questions that face this field in the coming years.

Demethylation of the TSDR is a marker of squamous cell carcinoma in transplant recipients.

Malignancy is an important cause of death in transplant recipients. Cutaneous squamous cell carcinoma (cSCC) causes significant morbidity and mortality as 30% of transplant recipients will develop cSCC within 10 years of transplantation. Previously we have shown that high numbers of regulatory T cells (Tregs) are associated with the development of cSCC in kidney transplant recipients (KTRs). Demethylation analysis of the Treg-specific demethylated region (TSDR) provides a more accurate association with cSCC risk after transplantation. Age, gender and duration of immunosuppression matched KTRs with (n=32) and without (n=27) cSCC, were re-analyzed for putative clinical and immunological markers of cancer risk. The proportion of FOXP3+ CD4+ cells was higher in the population with a previous SCC. Major T cell subsets remained stable over time; although B cell, CD8 and CD4 subpopulations demonstrated age-related changes. TSDR methylation analysis allowed clarification of Treg numbers, enhancing the association of high Treg levels in KTRs with cSCC compared to the cSCC-free cohort. These data validate and expand on previous findings in long-term KTRs, and show that immune markers remain stable over time. TSDR demethylation analysis provides a more accurate biomarker of cancer posttransplantation.

Novel GMP-compatible protocol employing an allogeneic B cell bank for clonal expansion of allospecific natural regulatory T cells.

The adoptive transfer of natural regulatory T cells (nTreg) is a new option to reshape undesired immune reactivity in autoimmunity and transplantation toward "tolerance." The first clinical trials using adoptive transfer of polyclonal nTreg demonstrated safety and hints of efficacy. However, the low frequencies of antigen-specific cells among the pool of polyclonal nTreg and their broad antigen nonspecific suppression are limitations of this approach regarding efficacy and safety. Recently, the isolation and expansion of (allo)antigen-specific nTreg have successfully been achieved by using Treg-specific activation markers but the yield is relatively low. Here, we describe a novel good manufacturing practice (GMP)-compatible expansion protocol of alloantigen-specific nTreg based on the stimulation of nTreg by allogeneic activated B cells. Their functionality and specificity are superior compared to polyclonal nTreg both in vitro and in vivo. Employing an allogeneic B cell bank, designed to cover the majority of HLA types, allows fast GMP-compliant manufacturing for donor-specific nTreg for clinical application in organ and stem cell transplantation. TCR repertoire analyses by next generation sequencing revealed impressive expansion by several log-steps of even very low-abundance alloantigen-specific nTreg clones. This novel method offers a simple approach for expanding antigen-specific nTreg and is characterized by high replicability and easy transferability to full GMP standards.

The role of donor-specific HLA alloantibodies in liver transplantation.

The impact of donor-specific HLA alloantibodies (DSA) on short- and long-term liver transplant outcome is not clearly defined. While it is clear that not all levels of allosensitization produce overt clinical injury, and that liver allografts possess some degree of alloantibody resistance, alloantibody-mediated adverse consequences are increasingly being recognized. To better define the current state of this topic, we assembled experts to provide insights, explore controversies and develop recommendations for future research on the consequences of DSA in liver transplantation. This article summarizes the proceedings of this inaugural meeting. Several insights emerged. Acute antibody-mediated rejection (AMR), although rarely diagnosed, is increasingly understood to overlap with T cell-mediated rejection. Isolated liver allograft recipients are at increased risk of early allograft immunologic injury when preformed DSA are high titer and persist posttransplantation. Persons who undergo simultaneous liver-kidney transplantation are at risk of renal AMR when Class II DSA persist posttransplantation. Other under-appreciated DSA associations include ductopenia and fibrosis, plasma cell hepatitis, biliary strictures and accelerated fibrosis associated with recurrent liver disease. Standardized DSA testing and diagnostic criteria for both acute and chronic AMR are needed to distil existing associations into etiological processes in order to develop responsive therapeutic strategies.

Predicting the response of a long-distance migrant to changing environmental conditions in winter.

Access to high-quality food is critical for long-distance migrants to provide energy for migration and arrival at breeding grounds in good condition. We studied effects of changing abundance and availability of a marine food, common eelgrass (Zostera marina L.), on an arctic-breeding, migratory goose, black brant (Brant bernicla nigricans Lawrence 1846), at a key non-breeding site, Bahía San Quintín, Mexico. Eelgrass, the primary food of brant, is consumed when exposed by the tide or within reach from the water's surface. Using an individual-based model, we predicted effects of observed changes (1991-2013) in parameters influencing food abundance and availability: eelgrass biomass (abundance), eelgrass shoot length (availability, as longer shoots more within reach), brant population size (availability, as competition greater with more birds), and sea level (availability, as less food within reach when sea level higher). The model predicted that the ability to gain enough energy to migrate was most strongly influenced by eelgrass biomass (threshold January biomass for migration = 60 g m-2 dry mass). Conversely, annual variation in population size (except for 1998), was relatively low, and variation in eelgrass shoot length and sea level were not strongly related to ability to migrate. We used observed data on brant body mass at Bahía San Quintín and annual survival to test for effects of eelgrass biomass in the real system. The lowest observed values of body mass and survival were in years when biomass was below 60 g m-2, although in some years of low biomass body mass and/or survival was higher. This suggests that the real birds may have some capacity to compensate to meet their energy demands when eelgrass biomass is low. We discuss consequences for brant population trends and conservation.

Delayed anti-CD3 therapy results in depletion of alloreactive T cells and the dominance of Foxp3+ CD4+ graft infiltrating cells.

The engineered Fc-nonbinding (crystallizable fragment-nonbinding) CD3 antibody has lower mitogenicity and a precise therapeutic window for disease remission in patients with type 1 diabetes. Before anti-CD3 can be considered for use in transplantation, the most effective timing of treatment relative to transplantation needs to be elucidated. In this study anti-CD3F(ab')2 fragments or saline were administered intravenously for 5 consecutive days (early: d1-3 or delayed: d3-7) to mice transplanted with a cardiac allograft (H2(b)-to-H2(k); d0). Survival of allografts was prolonged in mice treated with the early protocol (MST = 48 days), but most were rejected by d100. In contrast, in mice treated with the delayed protocol allografts continued to survive long term. The delayed protocol significantly inhibited donor alloreactivity at d30 as compared to the early protocol. A marked increase in Foxp3(+) T cells (50.3 ± 1.6%) infiltrating the allografts in mice treated with the delayed protocol was observed (p < 0.0001 vs. early (24.9 ± 2.1%)) at d10; a finding that was maintained in the accepted cardiac allografts at d100. We conclude that the timing of treatment with anti-CD3 therapy is critical for inducing long-term graft survival. Delaying administration effectively inhibits the alloreactivity and promotes the dominance of intragraft Foxp3(+) T cells allowing long-term graft acceptance.