Rapamycin nanoparticles increase the therapeutic window of engineered interleukin-2 and drive expansion of antigen-specific regulatory T cells for protection against autoimmune disease.

Interleukin-2 (IL-2) therapies targeting the high affinity IL-2 receptor expressed on regulatory T cells (Tregs) have shown promising therapeutic benefit in autoimmune diseases through nonselective expansion of pre-existing Treg populations, but are potentially limited by the inability to induce antigen-specific Tregs, as well as by dose-limiting activation of effector immune cells in settings of inflammation. We recently developed biodegradable nanoparticles encapsulating rapamycin, called ImmTOR, which induce selective immune tolerance to co-administered antigens but do not increase total Treg numbers. Here we demonstrate that the combination of ImmTOR and an engineered Treg-selective IL-2 variant (termed IL-2 mutein) increases the number and durability of total Tregs, as well as inducing a profound synergistic increase in antigen-specific Tregs when combined with a target antigen. We demonstrate that the combination of ImmTOR and an IL-2 mutein leads to durable inhibition of antibody responses to co-administered AAV gene therapy capsid, even at sub-optimal doses of ImmTOR, and provides protection in autoimmune models of type 1 diabetes and primary biliary cholangitis. Importantly, ImmTOR also increases the therapeutic window of engineered IL-2 molecules by mitigating effector immune cell expansion and preventing exacerbation of disease in a model of graft-versus-host-disease. At the same time, IL-2 mutein shows potential for dose-sparing of ImmTOR. Overall, these results establish that the combination of ImmTOR and an IL-2 mutein show synergistic benefit on both safety and efficacy to provide durable antigen-specific immune tolerance to mitigate drug immunogenicity and to treat autoimmune diseases.

Assessing the impact of a test and vaccinate or remove badger intervention project on bovine tuberculosis levels in cattle herds.

Bovine tuberculosis (bTB) is a chronic, zoonotic infection of domestic and wild animals caused mainly by Mycobacterium bovis. The Test and Vaccinate or Remove (TVR) project was a 5-year intervention (2014-2018) applied to Eurasian badgers (Meles meles) in a 100 km2 area of County Down, Northern Ireland. This observational study used routine bTB surveillance data of cattle to determine if the TVR intervention had any effect in reducing the infection at a herd level. The study design included the TVR treatment area (Banbridge) compared to the three adjacent 100 km2 areas (Dromore, Ballynahinch, and Castlewellan) which did not receive any badger intervention. Results showed that there were statistically lower bTB herd incidence rate ratios in the Banbridge TVR area compared to two of the other three comparison areas, but with bTB herd history and number of bTB infected cattle being the main explanatory variables along with Year. This finding is consistent with other study results conducted as part of the TVR project that suggested that the main transmission route for bTB in the area was cattle-to-cattle spread. This potentially makes any wildlife intervention in the TVR area of less relevance to bTB levels in cattle. It must also be noted that the scientific power of the TVR study (76%) was below the recommended 80%, meaning that results must be interpreted with caution. Even though statistical significance was achieved in two cattle-related risk factors, other potential risk factors may have also demonstrated significance in a larger study.

Mechanism and linkage specificities of the dual retaining ß-Kdo glycosyltransferase modules of KpsC from bacterial capsule biosynthesis.

KpsC is a dual-module glycosyltransferase (GT) essential for "group 2" capsular polysaccharide biosynthesis in Escherichia coli and other Gram-negative pathogens. Capsules are vital virulence determinants in high-profile pathogens, making KpsC a viable target for intervention with small-molecule therapeutic inhibitors. Inhibitor development can be facilitated by understanding the mechanism of the target enzyme. Two separate GT modules in KpsC transfer 3-deoxy-ß-d-manno-oct-2-ulosonic acid (ß-Kdo) from cytidine-5'-monophospho-ß-Kdo donor to a glycolipid acceptor. The N-terminal and C-terminal modules add alternating Kdo residues with ß-(2→4) and ß-(2→7) linkages, respectively, generating a conserved oligosaccharide core that is further glycosylated to produce diverse capsule structures. KpsC is a retaining GT, which retains the donor anomeric carbon stereochemistry. Retaining GTs typically use an SNi (substitution nucleophilic internal return) mechanism, but recent studies with WbbB, a retaining ß-Kdo GT distantly related to KpsC, strongly suggest that this enzyme uses an alternative double-displacement mechanism. Based on the formation of covalent adducts with Kdo identified here by mass spectrometry and X-ray crystallography, we determined that catalytically important active site residues are conserved in WbbB and KpsC, suggesting a shared double-displacement mechanism. Additional crystal structures and biochemical experiments revealed the acceptor binding mode of the ß-(2→4)-Kdo transferase module and demonstrated that acceptor recognition (and therefore linkage specificity) is conferred solely by the N-terminal α/ß domain of each GT module. Finally, an Alphafold model provided insight into organization of the modules and a C-terminal membrane-anchoring region. Altogether, we identified key structural and mechanistic elements providing a foundation for targeting KpsC.

Evaluating the risk of bovine tuberculosis posed by standard inconclusive reactors identified at backward-traced herd tests in Northern Ireland that disclosed no reactors.

Bovine tuberculosis is a notifiable disease in Northern Ireland with the national eradication programme of compulsory testing and slaughter of reactor animals costing approximately £40 million per year. Backward tracing, known as Backward Check Tests (BCTs), of reactor animals is used to identify previous herds where the bTB positive animal has resided. The aim of this study was to quantify the bovine tuberculosis (bTB) risk posed by inconclusive reactors (ICs) at BCTs at both the individual animal and the herd level. ICs to the Comparative Intradermal Tuberculin Test (CITT) at a BCT, in which no reactors were found, were matched with CITT negative animals, based on age, sex, test ID and follow up period, in Northern Ireland between 1st January 2008 and 31st December 2017 (inclusive). A retrospective matched cohort study design was used with the outcome of interest being the bTB status of each animal and subsequent bTB herd breakdowns. After adjusting for herd size, IC animals at a BCT had 16 times the odds (95% confidence interval: 7.75 to 38.28, p < 0.001) of becoming bTB positive compared to CITT negative animals. The percentage population attributable risk was 0.0001%. The majority 75% (n = 71) of ICs that became bTB positive were identified at the 42 day retest. Of those that were not disclosed at the 42 day retest (n = 24), almost a third (29%) had moved to an unrestricted herd. However, after adjusting for herd size and type, herds that had ICs only identified at a BCT did not have an increased odds of a subsequent bTB herd breakdown compared to herds that had a CITT negative BCT. Given the increased risk posed by ICs at a BCT, it may be justifiable to remove them from the herd immediately or place them under lifetime movement restrictions to the herd where they were detected. However, further action regarding the herd of origin does not appear to be justified.

The regression of the bovine tuberculin reaction: Results from the Reactor Quality Assurance study in Northern Ireland.

Tuberculin skin tests remain widely used in the control of bovine tuberculosis (bTB) in cattle. Little is known about the rate of regression of tuberculin reactions after the comparative intradermal cervical test (CICT) in cattle. This study aimed to collect data to describe tuberculin regression in reactors following the CICT at 72 ± 4 h post injection. Reactors were also tested using the interferon gamma (IFN-γ) assay to establish if any pattern existed between these results and the CICT reaction regression. The data were derived from 108 herds, 112 herd-level CICTs and 1008 animals. A multivariable linear mixed model was built to explore the regression of the bovine tuberculin reaction over time and the influence of potential predictors. The results confirmed a proportional decline in the bovine tuberculin reaction occurred over time. The predictors in the final model demonstrated that regression of the tuberculin reaction differed between reactors according to their IFN-γ test results and whether visible lesions were present at slaughter. Follow-up measurement of tuberculin reactions and the serial use of the IFN-γ assay in large breakdowns has the potential to provide both a mechanism for quality assurance of the current CICT bTB surveillance and the identification of atypical breakdowns or reactors requiring further investigation.

Test and vaccinate or remove: Methodology and preliminary results from a badger intervention research project.

BACKGROUND: In the British Isles, it is generally accepted that the Eurasian badger (Meles meles) plays a role in the maintenance of bovine tuberculosis (bTB) in cattle. Non-selective culling is the main intervention method deployed in controlling bTB in badgers along with smaller scale Bacillus Calmette-Guérin (BCG) vaccination areas. This paper describes the use of selective badger culling combined with vaccination in a research intervention trial. METHODS: In Northern Ireland, a 100 km2 area was subjected to a test and vaccinate or remove (TVR) badger intervention over a 5-year period. Badgers were individually identified and tested on an annual basis. Physical characteristics and clinical samples were obtained from each unique badger capture event. RESULTS: A total of 824 badgers were trapped with 1520 capture/sampling events. There were no cage-related injuries to the majority of badgers (97%). A low level of badger removal was required (4.1%-16.4% annually), while 1412 BCG vaccinations were administered. A statistically significant downward trend in the proportion of test positive badgers was observed. CONCLUSION: This is the first project to clearly demonstrate the feasibility of cage side testing of badgers. The results provide valuable data on the logistics and resources required to undertake a TVR approach to control Mycobacterium bovis in badgers.

Biosynthesis of a conserved glycolipid anchor for Gram-negative bacterial capsules.

Several important Gram-negative bacterial pathogens possess surface capsular layers composed of hypervariable long-chain polysaccharides linked via a conserved 3-deoxy-ß-D-manno-oct-2-ulosonic acid (ß-Kdo) oligosaccharide to a phosphatidylglycerol residue. The pathway for synthesis of the terminal glycolipid was elucidated by determining the structures of reaction intermediates. In Escherichia coli, KpsS transfers a single Kdo residue to phosphatidylglycerol; this primer is extended using a single enzyme (KpsC), possessing two cytidine 5'-monophosphate (CMP)-Kdo-dependent glycosyltransferase catalytic centers with different linkage specificities. The structure of the N-terminal ß-(2→4) Kdo transferase from KpsC reveals two α/ß domains, supplemented by several helices. The N-terminal Rossmann-like domain, typically responsible for acceptor binding, is severely reduced in size compared with canonical GT-B folds in glycosyltransferases. The similar structure of the C-terminal ß-(2→7) Kdo transferase indicates a past gene duplication event. Both Kdo transferases have a narrow active site tunnel, lined with key residues shared with GT99 ß-Kdo transferases. This enzyme provides the prototype for the GT107 family.

Utilization of Fluorescently Tagged Synthetic Acceptor Molecules for In Vitro Characterization of a Dual-Domain Glycosyltransferase Enzyme, KpsC, from Escherichia coli.

The incorporation of fluorescent tags into synthetic acceptor molecules for in vitro biochemical assays allows quick and easy detection of enzyme activity. Reaction products can be separated via thin-layer chromatography and visualized under UV light for rapid detection of reaction progress. Subsequent structural analysis of these reaction products through the use of NMR spectroscopy and mass spectrometry allows for complete functional characterization of enzyme activity. Here we describe an application of this technique which was previously used to functionally characterize a dual-domain glycosyltransferase enzyme, KpsC, involved in capsular polysaccharide biosynthesis in Escherichia coli.

Reducing acute kidney injury incidence and progression in a large teaching hospital.

Acute kidney injury (AKI) is a common syndrome that is associated with significant mortality and cost. The Quality Improvement AKI Collaborative at Salford Royal Foundation Trust was established to review and improve both the recognition and management of AKI. This was a whole-system intervention to tackle AKI implemented as an alternative to employing separate AKI nurses. Our aims were to reduce the overall incidence of AKI by 10%, to reduce hospital-acquired AKI by 25% and to reduce the progression of AKI from stage 1 to stage 2 or 3 by 50%. From 2014 to 2016, several multifaceted changes were introduced. These included system changes, such as inserting an e-alert for AKI into the electronic patient record, an online educational package and face-to-face teaching for AKI, and AKI addition to daily safety huddles. On 10 Collaborative wards, development of an AKI care bundle via multidisciplinary team (MDT) plan, do, study, act testing occurred. Results showed a 15.6% reduction in hospital-wide-acquired AKI, with a 22.3% reduction on the collaborative wards. Trust-wide rates of progression of AKI 1 to AKI 2 or 3 showed normal variation, whereas there was a 48.5% reduction in AKI progression on the Collaborative wards. This implies that e-alerts were ineffective in isolation. The Collaborative wards' results were a product of the educational support, bundle and heightened awareness of AKI. A number of acute hospitals have demonstrated impactful successes in AKI reduction centred on a dedicated AKI nurse model plus e-alerting with supporting changes. This project adds value by highlighting another approach that does not require a new post with attendant rolling costs and risks. We believe that our approach increased our efficacy in acute care in our front-line teams by concentrating on embedding improved recognition and actions across the MDT.

Further description of bovine tuberculosis trends in the United Kingdom and the Republic of Ireland, 2003-2015.

Extending on earlier work, trends in bovine tuberculosis (bTB) from 2003 to 2015 are described for the countries of the UK and the Republic of Ireland using standardised definitions and measures. Based on measures of animal and herd incidence, there remains a stable situation of extremely low prevalence in Scotland and the Low Risk Area of England, and a higher but ongoing reduction in prevalence in the Republic of Ireland. In Northern Ireland, there has been a rising bTB trend during 2010-2015, although not to levels experienced during 2002-2004. In the High Risk Area and Edge Area of England during 2010-2015, the rising bTB trends have continued but with some recent evidence of stabilisation. In Wales, prevalence has fallen subsequent to a peak in 2008. The paper considers country-level differences in the light of key policy changes, which are presented in detail. This work is unique, and will assist policymakers when critically evaluating policy options for effective control and eradication. Ongoing updates of this analysis would be useful, providing an evidence base for country-level comparison of bTB trends into the future. The use of multivariable analytical methods should be considered, but will rely on substantial sharing of raw data across the five countries.

Lipid content and fatty acid profile during lake whitefish embryonic development at different incubation temperatures.

Lipids serve as energy sources, structural components, and signaling molecules during fish embryonic development, and utilization of lipids may vary with temperature. Embryonic energy utilization under different temperatures is an important area of research in light of the changing global climate. Therefore, we examined percent lipid content and fatty acid profiles of lake whitefish (Coregonus clupeaformis) throughout embryonic development at three incubation temperatures. We sampled fertilized eggs and embryos at gastrulation, eyed and fin flutter stages following chronic incubation at temperatures of 1.8, 4.9 and 8.0°C. Hatchlings were also sampled following incubation at temperatures of 3.3, 4.9 and 8.0°C. Fertilized eggs had an initial high percentage of dry mass composed of lipid (percent lipid content; ~29%) consisting of ~20% saturated fatty acids (SFA), ~32% monounsaturated fatty acids (MUFA), ~44% polyunsaturated fatty acids (PUFA), and 4% unidentified. The most abundant fatty acids were 16:0, 16:1, 18:1(n-9c), 20:4(n-6), 20:5(n-3) and 22:6(n-3). This lipid profile matches that of other cold-water fish species. Percent lipid content increased during embryonic development, suggesting protein or other yolk components were preferentially used for energy. Total percentage of MUFA decreased during development, which indicated MUFA were the primary lipid catabolized for energy during embryonic development. Total percentage of PUFA increased during development, driven largely by an increase in 22:6(n-3). Temperature did not influence percent lipid content or percent MUFA at any development stage, and had inconsistent effects on percent SFA and percent PUFA during development. Thus, lake whitefish embryos appear to be highly adapted to low temperatures, and do not alter lipids in response to temperature within their natural incubation conditions.

Biochemical Characterization of Bifunctional 3-Deoxy-ß-d-manno-oct-2-ulosonic Acid (ß-Kdo) Transferase KpsC from Escherichia coli Involved in Capsule Biosynthesis.

3-Deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an essential component of bacterial lipopolysaccharides, where it provides the linkage between lipid and carbohydrate moieties. In all known LPS structures, Kdo residues possess α-anomeric configurations, and the corresponding inverting α-Kdo transferases are well characterized. Recently, it has been shown that a large group of capsular polysaccharides from Gram-negative bacteria, produced by ATP-binding cassette transporter-dependent pathways, are also attached to a lipid anchor through a conserved Kdo oligosaccharide. In the study reported here, the structure of this Kdo linker was determined by NMR spectroscopy, revealing alternating ß-(2→4)- and ß-(2→7)-linked Kdo residues. KpsC contains two retaining ß-Kdo glycosyltransferase domains belonging to family GT99 that are responsible for polymerizing the ß-Kdo linker on its glycolipid acceptor. Full-length Escherichia coli KpsC was expressed and purified, together with the isolated N-terminal domain and a mutant protein (KpsC D160A) containing a catalytically inactivated N-terminal domain. The Kdo transferase activities of these proteins were determined in vitro using synthetic acceptors, and the reaction products were characterized using TLC, mass spectrometry, and NMR spectroscopy. The N- and C-terminal domains were found to catalyze formation of ß-(2→4) and ß-(2→7) linkages, respectively. Based on phylogenetic analyses, we propose the linkage specificities of the glycosyltransferase domains are conserved in KpsC homologs from other bacterial species.

Genetic diversity of ruminant Pestivirus strains collected in Northern Ireland between 1999 and 2011 and the role of live ruminant imports.

BACKGROUND: The genus pestivirus within the family Flaviviridae includes bovine viral diarrhoea virus (BVDV) types 1 and 2, border disease virus (BDV) and classical swine fever virus. The two recognised genotypes of BVDV are divided into subtypes based on phylogenetic analysis, namely a-p for BVDV-1 and a-c for BVDV-2. METHODS: Three studies were conducted to investigate the phylogenetic diversity of pestiviruses present in Northern Ireland. Firstly, pestiviruses in 152 serum samples that had previously tested positive for BVDV between 1999 and 2008 were genotyped with a RT-PCR assay. Secondly, the genetic heterogeneity of pestiviruses from 91 serum samples collected between 2008 and 2011 was investigated by phylogenetic analysis of a 288 base pair portion of the 5' untranslated region (UTR). Finally, blood samples from 839 bovine and 4,437 ovine animals imported in 2010 and 2011 were tested for pestiviral RNA. Analysis of animal movement data alongside the phylogenetic analysis of the strains was carried out to identify any links between isolates and animal movement. RESULTS: No BVDV-2 strains were detected. All of the 152 samples in the first study were genotyped as BVDV-1. Phylogenetic analysis indicated that the predominant subtype circulating was BVDV-1a (86 samples out of 91). The remaining five samples clustered close to reference strains in subtype BVDV-1b. Out of the imported animals, 18 bovine samples tested positive and 8 inconclusive (Ct ≥36), while all ovine samples were negative. Eight sequences were obtained and were defined as BVDV-1b. Analysis of movement data between herds failed to find links between herds where BVDV-1b was detected. CONCLUSION: Given that only BVDV-1a was detected in samples collected between 1968 and 1999, this study suggests that at least one new subtype has been introduced to Northern Ireland between 1999 and 2011 and highlights the potential for importation of cattle to introduce new strains.