Ex Situ Left Ventricular Pressure-Volume Loop Analyses for Donor Hearts: Proof of Concept in an Ovine Experimental Model.

Ex situ heart perfusion (ESHP) has emerged as an important strategy to preserve donation after brain death (DBD) and donation after circulatory death (DCD) donor hearts. Clinically, both DBD and DCD hearts are successfully preserved using ESHP. Viability assessment is currently based on biochemical values, while a reliable method for graft function assessment in a physiologic working mode is unavailable. As functional assessment during ESHP has demonstrated the highest predictive value of outcome post-transplantation, this is an important area for improvement. In this study, a novel method for ex situ assessment of left ventricular function with pressure-volume loop analyses is evaluated. Ovine hearts were functionally evaluated during normothermic ESHP with the novel pressure-volume loop system. This system provides an afterload and adjustable preload to the left ventricle. By increasing the preload and measuring end-systolic elastance, the system could successfully assess the left ventricular function. End-systolic elastance at 60 min and 120 min was 2.8 ± 1.8 mmHg/mL and 2.7 ± 0.7 mmHg/mL, respectively. In this study we show a novel method for functional graft assessment with ex situ pressure-loop analyses during ESHP. When further validated, this method for pressure-volume assessments, could be used for better graft selection in both DBD and DCD donor hearts.

High throughput screening aids clinical decision-making in refractory acute myeloid leukaemia.

BACKGROUND: Despite advances in therapeutics for adverse-risk acute myeloid leukaemia (AML), overall survival remains poor, especially in refractory disease. Comprehensive tumour profiling and pre-clinical drug testing can identify effective personalised therapies. CASE: We describe a case of ETV6-MECOM fusion-positive refractory AML, where molecular analysis and in vitro high throughput drug screening identified a tolerable, novel targeted therapy and provided rationale for avoiding what could have been a toxic treatment regimen. Ruxolitinib combined with hydroxyurea led to disease control and enhanced quality-of-life in a patient unsuitable for intensified chemotherapy or allogeneic stem cell transplantation. CONCLUSION: This case report demonstrates the feasibility and role of combination pre-clinical high throughput screening to aid decision making in high-risk leukaemia. It also demonstrates the role a JAK1/2 inhibitor can have in the palliative setting in select patients with AML.

Determining the affinities of high-affinity antibodies using KinExA and surface plasmon resonance.

Accurate and efficient affinity measurement techniques are essential for the biophysical characterization of therapeutic monoclonal antibodies, one of the fastest growing drug classes. Surface plasmon resonance (SPR) is widely used for determining antibody affinity, but does not perform well with extremely high affinity (low picomolar to femtomolar range) molecules. In this study, we compare the SPR-based Carterra LSA and the kinetic exclusion assay (KinExA) for measuring the affinities of 48 antibodies generated against the SARS-CoV-2 receptor-binding domain. These data reveal that high-affinity antibodies can be generated straight from selections using high-quality in vitro library platforms with 54% correspondence between affinities measured using LSA and KinExA. Generally, where there was a 2-fold or greater difference between LSA and KinExA, KinExA reported that affinities were tighter. We highlight the differences between LSA and KinExA, identifying the benefits and pitfalls of each in terms of dynamic range and throughput. Furthermore, we demonstrate for the first time that single-point screening with KinExA can significantly improve throughput while maintaining a strong correlation with full binding curve equilibrium measurements, enabling the accurate rank-ordering of clones with exceptionally tight binding properties.

Three Decades Single Center Experience of Airway Complications After Lung Transplantation.

Post lung transplantation airway complications like necrosis, stenosis, malacia and dehiscence cause significant morbidity, and are most likely caused by post-operative hypo perfusion of the anastomosis. Treatment can be challenging, and airway stent placement can be necessary in severe cases. Risk factors for development of airway complications vary between studies. In this single center retrospective cohort study, all lung transplant recipients between November 1990 and September 2020 were analyzed and clinically relevant airway complications of the anastomosis or distal airways were identified and scored according to the ISHLT grading system. We studied potential risk factors for development of airway complications and evaluated the impact on survival. The treatment modalities were described. In 651 patients with 1,191 airway anastomoses, 63 patients developed 76 clinically relevant airway complications of the airway anastomoses or distal airways leading to an incidence of 6.4% of all anastomoses, mainly consisting of airway stenosis (67%). Development of airway complications significantly affects median survival in post lung transplant patients compared to patients without airway complication (101 months versus 136 months, p = 0.044). No significant risk factors for development of airway complication could be identified. Previously described risk factors could not be confirmed. Airway stents were required in 55% of the affected patients. Median survival is impaired by airway complications after lung transplantation. In our cohort, no significant risk factors for the development of airway complications could be identified.

Preening cups increase apparent wet preening behaviors, but have no impact on other behaviors, body condition, growth, or body morphometrics of grow-out Pekin ducks.

Preening cups may be a form of open water that would allow ducks to express preening behaviors. We set out to test the hypothesis that preening cups would not have detrimental effects on ducks or their environment. Control pens (N = 6, 65 ducks/pen) had nipple lines while experimental pens (N = 6, 65 ducks/pen) had the same nipple line plus one preening cup (PC). Body weights of 30 ducks per pen, and body condition scores on 50 ducks per pen were recorded weekly. On d 18 and 43, 5 ducks per pen were euthanized and their spleens, Bursas, liver, and uropygial glands were weighed. Behavior data were collected using scan sampling with video being recorded for 72 continuous hours at 4 different ages: 25 d, 30 d, 36 d, and 40 d. Body morphometrics were analyzed by 2-way ANOVA with repeated measures. Body condition scoring was analyzed by Pearson's chi-square. The GLIMMIX procedure (SAS 9.4) was used for behavioral analyses to examine treatment differences in the proportion of ducks performing dry preening, wet preening, eating, drinking, standing, and laying down. Feather pecking, feather picking, preening conspecifics (also known as allopreening), dunking head, and drinking from preening cup were analyzed using PROC LOGISTIC with the Firth bias correction for quasi-complete separation and odds ratios were calculated. More PC ducks housed with PC performed wet preening compared to control ducks (25 d: F1,26 = 6.90, P = 0.0143; 30, 36, and 40 d; F1,78 = 24.53, P < 0.0001). Ducks in the PC group were also more likely to lay down compared to controls (25 d: F1,33 = 4.95, P = 0.0330). No differences were observed for any other behavior, body condition score, body weight or morphometrics at any age. Although ducks in the preening cup group showed an increase in wet preening, our data suggest that open water is not necessary to maintain feather condition or uropygial gland size.

Insights into next generation sequencing guided antibody selection strategies.

Therapeutic antibody discovery often relies on in-vitro display methods to identify lead candidates. Assessing selected output diversity traditionally involves random colony picking and Sanger sequencing, which has limitations. Next-generation sequencing (NGS) offers a cost-effective solution with increased read depth, allowing a comprehensive understanding of diversity. Our study establishes NGS guidelines for antibody drug discovery, demonstrating its advantages in expanding the number of unique HCDR3 clusters, broadening the number of high affinity antibodies, expanding the total number of antibodies recognizing different epitopes, and improving lead prioritization. Surprisingly, our investigation into the correlation between NGS-derived frequencies of CDRs and affinity revealed a lack of association, although this limitation could be moderately mitigated by leveraging NGS clustering, enrichment and/or relative abundance across different regions to enhance lead prioritization. This study highlights NGS benefits, offering insights, recommendations, and the most effective approach to leverage NGS in therapeutic antibody discovery.

High throughput purification of monoclonal recombinant antibodies using a Protein-A coated membrane plate system.

The therapeutic use of monoclonal antibodies (mAbs) ranges from cancer treatment to immune-mediated conditions, covering infectious and cardiovascular disorders, among others. The development of improved methods for therapeutic antibody discovery has accelerated the identification of numerous mAbs: a discovery campaign can be deeply mined, resulting in hundreds, even thousands, of potential antibody leads for a given target of interest. High throughput mAb expression and purification methods are required for the rapid validation of those leads. In this work, we describe the implementation of a Protein-A coated membrane plate system, the Purexa™ AHT membrane plate, for robust preparative purification of hundreds of recombinant mAbs, without the need for automation. The high efficiency (>80%) recovery generated sufficient mAb for downstream screening analyses such as ELISA and surface plasmon resonance (SPR). This new system allows the functional validation of hundreds of lead antibodies from discovery campaigns in a timely manner regardless of operational size.

Environmental sampling methods' influence on detection of pathogens in cage-free aviary housing.

The environmental sampling of layer housing systems is essential to identifying potential pathogens that are of concern to human health. To identify the natural occurrence of pathogens (Listeria, Campylobacter, and Salmonella) at various locations in a cage-free aviary housing system, swabs were collected when hens were 22 to 39 wks of age. Duplicate environmental swabs were taken and inoculated with a low dose (101 cfu) Salmonella Enteritidis (SE) and examined for the recovery of SE from environmental samples. Detection of Listeria (P < 0.0001) and Campylobacter (P < 0.0001) varied between the environmental sample types taken: concrete dust, drag swabs, egg belt dust, manure belt scraper swabs, and wall dust. Detection of Listeria (P < 0.0001) was the highest (70.0%) at the beginning of the study (22 wk) and decreased over time. Detection of Campylobacter (P < 0.001) was also the highest at 22 wk, however the decrease over time was more gradual. Interestingly, detection of Campylobacter (P < 0.0001) was the greatest in concrete dust samples (96.25%), which can be attributed to the presence of rodent excreta in the samples. Drag swabs and manure belt scraper swabs were the best sampling types for high detection of Listeria and Campylobacter. It should be noted that Listeria recovered was not of human health concern. No naturally occurring Salmonella was identified in this study. The recovery of the SE inoculum increased over time, reaching the greatest recovery in drag (81.25%; P < 0.0001), egg belt dust (100.00%; P < 0.0001) and wall dust swabs (100.00%; P < 0.0001) by 39 wk. This high rate of SE recovery occurred just before US mandatory SE environmental monitoring at 40 to 45 wks of age. Based on this study, the use of drag and manure belt scraper swabs are effective in detecting Listeria and Campylobacter in cage-free aviary housing. Along with good pest management, the occurrence of pathogens could be monitored and reduced in laying hen flocks.

Spatial Stochastic Model of the Pre-B Cell Receptor.

Survival and proliferation of immature B lymphocytes requires expression and tonic signaling of the pre-B cell receptor (pre-BCR). This low level, ligand-independent signaling is likely achieved through frequent, but short-lived, homo interactions. Tonic signaling is also central in the pathology of precursor B acute lymphoblastic leukemia (B-ALL). In order to understand how repeated, transient events can lead to sustained signaling and to assess the impact of receptor accumulation induced by the membrane landscape, we developed a spatial stochastic model of receptor aggregation and downstream signaling events. Our rule- and agent-based model builds on previous mature BCR signaling models and incorporates novel parameters derived from single particle tracking of pre-BCR on surfaces of two different B-ALL cell lines, 697 and Nalm6. Live cell tracking of receptors on the two cell lines revealed characteristic differences in their dimer dissociation rates and diffusion coefficients. We report here that these differences affect pre-BCR aggregation and consequent signal initiation events. Receptors on Nalm6 cells, which have a lower off-rate and lower diffusion coefficient, more frequently form higher order oligomers than pre-BCR on 697 cells, resulting in higher levels of downstream phosphorylation in the Nalm6 cell line.

Enhancing their quality of life: environmental enrichment for poultry.

Providing environmental enrichments that increase environmental complexity can benefit poultry welfare. This Poultry Science Association symposium paper is structured around four themes on 1) poultry preferences and affective states 2) species-specific behavior, including play behavior and the relationship between behavior, activity level and walking ability, 3) environmental enrichment and its relationship with indicators of welfare, and 4) a case study focusing on the application of enrichments in commercial broiler chicken production. For effective enrichment strategies, the birds' perspective matters most, and we need to consider individual variation, social dynamics, and previous experience when assessing these strategies. Play behavior can be a valuable indicator of positive affect, and while we do not yet know how much play would be optimal, absence of play suggests a welfare deficit. Activity levels and behavior can be improved by environmental modifications and prior research has shown that the activity level of broilers can be increased, at least temporarily, by increasing the environmental complexity. However, more research on impacts of enrichments on birds' resilience, on birds in commercial conditions, and on slow(er)-growing strains is needed. Finally, incorporating farmers' expertise can greatly benefit enrichment design and implementation on commercial farms.

Pandemic's silver lining.

The intense international focus on the COVID-19 pandemic has provided a unique opportunity to use a wide array of novel tools to carry out scientific studies on the SARS-CoV-2 virus. The value of these comparative studies extends far beyond their consequences for SARS-CoV-2, providing broad implications for health-related science. Here we specifically discuss the impacts of these comparisons on advances in vaccines, the analysis of host humoral immunity, and antibody discovery. As an extension, we also discuss potential synergies between these areas.Abbreviations: CoVIC: The Coronavirus Immunotherapeutic Consortium; EUA: Emergency Use Authorization.

Simultaneous affinity maturation and developability enhancement using natural liability-free CDRs.

ABBREVIATIONS: CDR: complementarity determining region; FACS: fluorescence-activated cell sorting; ka: association rate; kd: dissociation rate; KD: dissociation constant; scFv: single-chain variable fragment; SPR: surface plasmon resonance.

Differences in carcass composition and meat quality of conventional and slow-growing broiler chickens raised at 2 stocking densities.

The fast growth rate of broiler chickens is a welfare concern that has increased consumer interest in chicken from slower growing (SG) broilers. Replacing conventional (CONV) broilers with SG broilers will reduce chicken supply and SG broilers require different management practices than CONV. This study evaluated the effects of 2 stocking densities on the carcass composition and meat quality of CONV broilers that reach market weight at 42 d and SG broilers that reach market weight at 63 d. Male broilers from each strain were exclusively stocked into 16 pens at a density of either 29 kg/m2 or 37 kg/m2. Live body, carcass without giblets (WOG), and part weights were recorded and used to calculate yield. Initial and 24-hour pH, color (L*, a*, and b*), cooking loss, and Warner Bratzler shear force of the breast and thigh muscles were measured. Birds from both strains reached similar live body and carcass WOG weights. CONV had 3.4%, 13.0%, and 2.8% greater (P ≤ 0.002), carcass WOG, breast, and tenderloin yields, while SG had 2.7%, 5.0%, 7.5%, and 1.2% greater (P < 0.0001) wings, leg quarters, frame, and skin yields, respectively. CONV breast 24-h pH and cooking loss were greater (P ≤ 0.04) than SG. While SG thigh shear force was greater than CONV (P = 0.008), breast shear force was the lowest for CONV stocked at 29 kg/m2 and the greatest for SG stocked at 37 kg/m2 (P = 0.04). SG had a paler breast than CONV, while CONV had a yellower breast than SG (P < 0.0001). While SG had a redder thigh than CONV (P = 0.002), SG stocked at 29 kg/m2 had a redder breast than SG stocked at 37 kg/m2, with both CONV groups intermediate (P = 0.04). These results indicate that differences in male broiler meat quality were affected more by strain than by stocking density. Compared with CONV, SG broiler meat quality was more affected by stocking density in this study.

A pandemic-enabled comparison of discovery platforms demonstrates a naïve antibody library can match the best immune-sourced antibodies.

As a result of the SARS-CoV-2 pandemic numerous scientific groups have generated antibodies against a single target: the CoV-2 spike antigen. This has provided an unprecedented opportunity to compare the efficacy of different methods and the specificities and qualities of the antibodies generated by those methods. Generally, the most potent neutralizing antibodies have been generated from convalescent patients and immunized animals, with non-immune phage libraries usually yielding significantly less potent antibodies. Here, we show that it is possible to generate ultra-potent (IC50 < 2 ng/ml) human neutralizing antibodies directly from a unique semisynthetic naïve antibody library format with affinities, developability properties and neutralization activities comparable to the best from hyperimmune sources. This demonstrates that appropriately designed and constructed naïve antibody libraries can effectively compete with immunization to directly provide therapeutic antibodies against a viral pathogen, without the need for immune sources or downstream optimization.

Listing criteria for heart transplantation in the Netherlands.

The updated listing criteria for heart transplantation are presented on behalf of the three heart transplant centres in the Netherlands. Given the shortage of donor hearts, selection of those patients who may expect to have the greatest benefit from a scarce societal resource in terms of life expectancy and quality of life is inevitable. The indication for heart transplantation includes end-stage heart disease not remediable by more conservative measures, accompanied by severe physical limitation while on optimal medical therapy, including ICD/CRT­D. Assessment of this condition requires cardiopulmonary stress testing, prognostic stratification and invasive haemodynamic measurements. Timely referral to a tertiary centre is essential for an optimal outcome. Chronic mechanical circulatory support is being used more and more as an alternative to heart transplantation and to bridge the progressively longer waiting time for heart transplantation and, thus, has become an important treatment option for patients with advanced heart failure.

A single donor is sufficient to produce a highly functional in vitro antibody library.

Antibody complementarity determining region diversity has been considered to be the most important metric for the production of a functional antibody library. Generally, the greater the antibody library diversity, the greater the probability of selecting a diverse array of high affinity leads. According to this paradigm, the primary means of elevating library diversity has been by increasing the number of donors. In the present study we explored the possibility of creating an in vitro antibody library from a single healthy individual, showing that the number of lymphocytes, rather than the number of donors, is the key criterion in the production of a diverse and functional antibody library. We describe the construction of a high-quality phage display library comprising 5 × 109 human antibodies by applying an efficient B cell extraction protocol from a single donor and a targeted V-gene amplification strategy favoring specific antibody families for their improved developability profiles. Each step of the library generation process was followed and validated by next generation sequencing to monitor the library quality and diversity. The functionality of the library was tested using several therapeutically relevant targets for which a vast number of different antibodies with desired biophysical properties were obtained.

Waiting list mortality and the potential of donation after circulatory death heart transplantations in the Netherlands.

BACKGROUND: With more patients qualifying for heart transplantation (HT) and fewer hearts being transplanted, it is vital to look for other options. To date, only organs from brain-dead donors have been used for HT in the Netherlands. We investigated waiting list mortality in all Dutch HT centres and the potential of donation after circulatory death (DCD) HT in the Netherlands. METHODS: Two different cohorts were evaluated. One cohort was defined as patients who were newly listed or were already on the waiting list for HT between January 2013 and December 2017. Follow-up continued until September 2018 and waiting list mortality was calculated. A second cohort of all DCD donors in the Netherlands (lung, liver, kidney and pancreas) between January 2013 and December 2017 was used to calculate the potential of DCD HT. RESULTS: Out of 395 patients on the waiting list for HT, 196 (50%) received transplants after a median waiting time of 2.6 years. In total, 15% died while on the waiting list before a suitable donor heart became available. We identified 1006 DCD donors. After applying exclusion criteria and an age limit of 50 years, 122 potential heart donors remained. This number increased to 220 when the age limit was extended to 57 years. CONCLUSION: Waiting list mortality in the Netherlands is high. HT using organs from DCD donors has great potential in the Netherlands and could lead to a reduction in waiting list mortality. Cardiac screening will eventually determine the true potential.