Filtering Crosstalk from Bath Non-Markovianity via Spacetime Classical Shadows.

From an open system perspective non-Markovian effects due to a nearby bath or neighboring qubits are dynamically equivalent. However, there is a conceptual distinction to account for: neighboring qubits may be controlled. We combine recent advances in non-Markovian quantum process tomography with the framework of classical shadows to characterize spatiotemporal quantum correlations. Observables here constitute operations applied to the system, where the free operation is the maximally depolarizing channel. Using this as a causal break, we systematically erase causal pathways to narrow down the progenitors of temporal correlations. We show that one application of this is to filter out the effects of crosstalk and probe only non-Markovianity from an inaccessible bath. It also provides a lens on spatiotemporally spreading correlated noise throughout a lattice from common environments. We demonstrate both examples on synthetic data. Owing to the scaling of classical shadows, we can erase arbitrarily many neighboring qubits at no extra cost. Our procedure is thus efficient and amenable to systems even with all-to-all interactions.

Owners' views of canine nutrition, weight status and wellbeing and their implications for the veterinary consultation.

OBJECTIVES: To investigate owners' views around canine nutrition and wellbeing, how these beliefs may differ according to the weight status of the dog and the implications for owner support approaches. MATERIALS AND METHODS: A researcher-mediated questionnaire collected quantitative and qualitative data via structured interviews with dog owners (n=147) attending a country park in the East Midlands, UK, with a specific focus on views around canine nutrition and wellbeing. RESULTS: Forty-four percent of owners cited "past experience" when determining what to feed, and only 9% of owners cited the veterinarian as a source of this information. When comparing chosen verbal description versus non-sequential body condition score images of the dog, only 22% of owners with overweight animals matched perceived verbal and visual appraisals, compared with 89% of owners of ideal weight dogs. Owners cited a good diet and regular exercise as important factors for canine wellbeing, but companionship with other dogs as the least important factor. CLINICAL SIGNIFICANCE: Owners report being aware of the important association between canine nutrition and wellbeing, but their considered importance of factors influencing dog wellbeing may be influenced by their perceived weight status of the animal. Veterinarians may need to reframe owner discourse such that there is more routine discussion around weight and nutrition at every consultation. Furthermore, the use of non-sequential body condition score images could be a useful tool for a more considered opinion of canine weight status by owners.

Demonstration of non-Markovian process characterisation and control on a quantum processor.

In the scale-up of quantum computers, the framework underpinning fault-tolerance generally relies on the strong assumption that environmental noise affecting qubit logic is uncorrelated (Markovian). However, as physical devices progress well into the complex multi-qubit regime, attention is turning to understanding the appearance and mitigation of correlated - or non-Markovian - noise, which poses a serious challenge to the progression of quantum technology. This error type has previously remained elusive to characterisation techniques. Here, we develop a framework for characterising non-Markovian dynamics in quantum systems and experimentally test it on multi-qubit superconducting quantum devices. Where noisy processes cannot be accounted for using standard Markovian techniques, our reconstruction predicts the behaviour of the devices with an infidelity of 10-3. Our results show this characterisation technique leads to superior quantum control and extension of coherence time by effective decoupling from the non-Markovian environment. This framework, validated by our results, is applicable to any controlled quantum device and offers a significant step towards optimal device operation and noise reduction.

Quantum Bath Control with Nuclear Spin State Selectivity via Pulse-Adjusted Dynamical Decoupling.

Dynamical decoupling (DD) is a powerful method for controlling arbitrary open quantum systems. In quantum spin control, DD generally involves a sequence of timed spin flips (π rotations) arranged to either average out or selectively enhance coupling to the environment. Experimentally, errors in the spin flips are inevitably introduced, motivating efforts to optimize error-robust DD. Here we invert this paradigm: by introducing particular control "errors" in standard DD, namely, a small constant deviation from perfect π rotations (pulse adjustments), we show we obtain protocols that retain the advantages of DD while introducing the capabilities of quantum state readout and polarization transfer. We exploit this nuclear quantum state selectivity on an ensemble of nitrogen-vacancy centers in diamond to efficiently polarize the ^{13}C quantum bath. The underlying physical mechanism is generic and paves the way to systematic engineering of pulse-adjusted protocols with nuclear state selectivity for quantum control applications.

Evaluation of vegetable protein in canine diets: Assessment of performance and apparent ileal amino acid digestibility using a broiler model.

Recent technological advances in the human food industry with respect to meat processing have decreased the availability of animal proteins to the pet food industry which typically formulates diets with an excess of animal protein. In the long term, this is not sustainable; thus, alternative protein sources need to be investigated. This study examined three canine diets, comparing a typical animal protein-based diet (control) with two experimental diets where the animal protein was substituted in part with vegetable protein (formulated based either on total protein or amino acid content) using a broiler model. Each diet was fed to six cages each containing two birds from day 15, 18 cages in total (36 birds). Excreta were collected from days 19 to 21. On day 23, birds were euthanized and weighed, and their ileal digesta were collected and pooled for each cage. In addition, one leg per cage was collected for evaluation of muscle mass. Results showed no significant difference in animal performance (feed intake or live weight gain) or muscle to leg proportion across the diets. Birds fed the control diet and the diet balanced for amino acid content exhibited the greatest coefficients of apparent metabolizability for nitrogen (p < .001). Birds fed the diets that contained partial replacement of animal with vegetable protein generally had greater ileal digestibility of amino acids compared to birds fed the control (animal protein) diet. Analysis of excreta showed no dietary difference in terms of dry matter content; however, birds fed the diet balanced for total protein and the diet balanced for amino acid content had significantly greater excreta nitrogen than the control (p = .038). Overall, the study suggests vegetable proteins when formulated based on amino acid content are a viable alternative to animal proteins in canine diets.

Rapeseed napin and cruciferin are readily digested by poultry.

Rapeseed proteins have been considered as being poorly digestible in the gut of non-ruminants. The aim of the study was to assess the digestibility of napin and cruciferin in ileal digesta of broiler chickens, testing sixteen samples of rapeseed co-products with protein levels ranging from 293 g/kg to 560 g/kg dry matter. Each sample was included into a semi-synthetic diet at a rate of 500 g/kg and evaluated with broiler chickens in a randomised design. Dietary and ileal digesta proteins were extracted and identified by gel-based liquid chromatography-tandem mass spectrometry (LC-MS/MS). Three isomers of napin (a 2S albumin) and nine cruciferins (an 11S globulin) were identified in the rapeseed co-products, whereas six endogenous enzymes such as trypsin (I-P1, II-P29), chymotrypsin (elastase and precursor), carboxypeptidase B and α-amylase were found in the ileal digesta. It is concluded that as none of the rapeseed proteins were detected in the ileal digesta, rapeseed proteins can be readily digested by broiler chickens, irrespective of the protein content in the diet.

"Who's been a good dog?" - Owner perceptions and motivations for treat giving.

Complex relationships commonly exist between owners and their companion animals, particularly around feeding behaviour with an owner's affection or love for their animal most pronounced through the provision of food. It is notable that the pet food market is experiencing strong year-on-year growth in sales of dog and cat treats. Recognising the impact of treat giving in pet nutrition, the objective of the study was to investigate owner attitudes and motivations towards feeding treats (shop bought and other) to their dogs. A researcher-mediated questionnaire consisting of both quantitative and qualitative questions was used to interview dog owners (n=280) at two locations: an out-of-town retail park and a country park in the East Midlands. Owners almost unanimously viewed the word 'treat' within a nutritional context, as opposed to a new toy or other pleasure. The majority (96%) of owners interviewed reported feeding treats to their dog, with 69% feeding shop-bought treats on a daily basis. A wide range of treats was reportedly given by owners and the majority of owners interviewed fed multiple treat types. No association was found between owner age and frequency of shop-bought treats fed (P=0.659) nor between owner age and frequency of food given to the dog from the owner's plate (P=0.083). A wide range of foods which would not be considered balanced for the animal's nutritional requirements was viewed as a treat by some dog owners. A range of positive and negative views around the feeding of treats were expressed by dog owners, with some citing beneficial effects while others were clearly aware of the association between treat feeding and potential weight gain/obesity. Owner views included themes around positive reinforcement and responsibility but also reflected relational aspects of the human-animal bond. The results of the study show that treat giving is commonplace in feeding regimes and that treats are embedded in the feeding behaviour of many dog owners. However, the different views expressed around the motivations for, and feeding of, dog treats, reinforce the need to better understand owner psychology linked to this area, and the role this may play in the growing pet obesity epidemic.

Assessment of caecal parameters in layer hens fed on diets containing wheat distillers dried grains with solubles.

There is much interest in quantifying the nutritional value of UK wheat distillers dried grains with solubles (W-DDGS) for livestock species. A study was designed to evaluate caecal parameters (pH, short chain fatty acids (SCFAs) and bacterial diversity) in layer hens fed on balanced diets containing graded concentrations of W-DDGS. A total of 32 layer hens (Bovans Brown strain at 27 weeks of age) were randomly allocated to one of 4 dietary treatments containing W-DDGS at 0, 60, 120 or 180 g/kg. Each treatment was fed to 8 replicate individually housed layer hens over a 5-d acclimatisation period, followed by a 4-week trial. Individual feed intakes were monitored and all eggs were collected daily for weeks 2, 3 and 4 of the trial, weighed and an assessment of eggshell "dirtiness" made. All hens were culled on d 29 and caecal pH and SCFAs measured. Polymerase chain reaction denaturing gradient gel electrophoresis of the bacterial 16 S rDNA gene was used to assess total bacterial diversity of luminal caecal content from hens fed the 0 and 180 g W-DDGS/kg diets. Unweighted pair group method with arithmetic mean (UPGMA) dendrograms were generated from DGGE banding patterns. Increasing W-DDGS dietary concentrations resulted in a more acidic caecal environment. Caecal SCFAs were unaffected by diet aside from a quadratic effect for molar proportions of isobutyric acid. Diversity profiles of the bacterial 16S rRNA gene from luminal caecal contents were unaffected by W-DDGS inclusion. The results of the current study suggest that W-DDGS can be successfully formulated into nutritionally balanced layer diets (supplemented with xylanase and phytase) at up to 180 g/kg with no detrimental effects to the caecal environment.

Influence of the in vivo method and basal dietary ingredients employed in the determination of the amino acid digestibility of wheat distillers dried grains with solubles in broilers.

As distillers dried grains with solubles (DDGS) become increasingly available, it is important to determine their nutritional value for precise feed formulation. The accurate determination of digestibility is crucial, and it is known that the methods used will affect the values obtained. An experiment was designed to determine and compare the standardized ileal digestibility (SID) of amino acids from wheat DDGS using a semisynthetic diet and a difference method using 4 further diets based on corn, wheat, corn DDGS, and wheat DDGS. Eighty 1-d-old male broilers were fed a commercial starter diet until d 21. Between d 21 and 23, they were fed test diets in order to adapt to those diets before the trial took place between d 24 and 27. The trial period took place between d 24 and 27. Feed intake was measured, excreta collected, and at d 27, all birds were culled and ileal digesta was collected for the determination of apparent ileal digestibility and SID of amino acids. Values determined were similar to those reported elsewhere in the literature, although SID values for lysine were particularly low, being 0.26, 0.27, or 0.32, measured in semisynthetic, corn, or wheat diet backgrounds, respectively. It appeared that diet type employed was influential in the values obtained. The SID values for methionine, cysteine, methionine plus cysteine, and arginine were significantly lower (P < 0.05) when measured in semisynthetic diet backgrounds than wheat- or corn-based diets. It appears that dextrose and possibly purified starch have a detrimental impact on the broiler digestive tract. This may affect all digestibility methodologies in which such a diet base is used.

Canine obesity: is there a difference between veterinarian and owner perception?

OBJECTIVES: The overall aim of this study was to examine the nature of the relationship between dog-ownership and canine obesity, explored in two cohorts of overweight/obese or non-overweight dogs (n=125). This paper concentrates on the owners' perception of dog weight. METHODS: A researcher-mediated questionnaire was used to collect data from dog owners attending a small veterinary practice. Interviewees (n=121) were asked qualitative and quantitative questions, designed to examine the owners' views of their relationship with their dogs. RESULTS: Although a high proportion of owners claimed to have discussed the dog's weight with their veterinarian, some discrepancies were apparent between owner perception of animal weight and the veterinarian's evaluation of body condition score. Owner disagreement was significantly greater for the veterinarian-defined overweight dogs (P=0·005). Owners often provide personal narratives to account for their dog's weight status. CLINICAL SIGNIFICANCE: This study confirms the important role of the vet in providing information about the issue of dog weight but also suggests that providing verbal information is sometimes insufficient. The study also indicates the potential value of qualitative research methods to further understand client perception of complex animal care issues and highlights the need for further in-depth research.

Physicochemical properties and nutritional quality of raw cereals for newly weaned piglets.

The digestibility of the starch component of raw cereals in newly weaned piglets is highly variable. Reasons for this must be elucidated if the most suitable cereals are to be used. A novel approach was employed, which consisted of assessing the physicochemical properties (rapid visco analysis, water absorption and solubility indices, particle size distribution and in vitro amylolytic digestion) of eight raw cereals contained within piglet diets and subsequently relating this in vitro data to the biological responses of weaned piglets. Trial 1 examined soft and hard wheat, trial 2 - soft wheat, barley, rye and triticale, and trial 3 - soft wheat, naked oats, whole oats and maize. The initial observation was that in vitro testing prior to animal trials is recommended in nutritional evaluation since it indicated fundamental differences between raw cereals, such as for example the levels of endogenous amylase in wheat. Starch and nitrogen digestibility differed between cereals (apparent digestibility coefficients at the 0.5 site of the small intestine ranged from 0.10 to 0.69 for starch and from 0.17 to 0.68 for nitrogen). There is also a probable relationship between the coefficients of ileal apparent starch digestibility, at approximately halfway from the gastric pylorus to the ileocaecal valve, and the presence of endogenous amylase (mean values of 0.53 and 0.62 in trials 2 and 3, respectively, for the higher amylase wheat; 0.38 for the low-amylase wheat used in trial 1). This additional variable (i.e. the unforeseen presence of endogenous amylase) in wheat made it more difficult to draw a firm conclusion about the nutritional suitability of the different cereals. All raw-cereal diets caused atrophy of the villi during the initial week following weaning, but the soft wheat was associated with the highest comparative villi height and might therefore be considered the best of all raw cereals in minimising the post-weaning growth check. For wheat, this might also suggest a possible interaction between villus architecture and endosperm texture in the immediate post-weaning period.

Physicochemical changes to starch granules during micronisation and extrusion processing of wheat, and their implications for starch digestibility in the newly weaned piglet.

Two trials were performed to assess changes in the physicochemical properties of precisely processed (micronised v. extruded) wheats, prior to inclusion in piglet diets. The in vitro data obtained were subsequently related to biological responses of newly weaned piglets over 14 days. The effects of the severity of micronisation (Trial 1) or extrusion (Trial 2) on the nutritional value of two wheats (varying in endosperm texture) were examined. Extrusion, in contrast to micronisation, drastically disrupted the structural properties of wheat starch granules through melting of crystallites and macromolecular degradation of starch polysaccharides. These structural changes strongly improved the hydration characteristics of starch and its digestibility. The amount of starch digested in vitro was approximately 0.20, 0.70 and 0.90 for the unprocessed, micronised and extruded samples, respectively. This enhanced in vitro digestibility correlated well with, and helped to explain, the significant improvement in the apparent digestibility of starch at both the 0.5 region (mean coefficients for extruded wheat were 0.869 and 0.959 v. raw 0.392; P = 0.017) and 0.75 region (extruded 0.973 v. raw 0.809; P = 0.009) of the small intestine, when compared with piglets fed an unprocessed wheat diet. Extrusion and, to a lesser extent, micronisation lessened the reduction in apparent starch digestibility on day 4 post-weaning, typically seen at the 0.5 intestinal region in piglets fed an unprocessed wheat diet. Processing variables influenced both in vitro and in vivo data, with for instance, a positive relationship between specific mechanical energy (SME) input during extrusion and starch digestibility at the 0.5 region. The higher digestibility coefficient observed at the 0.5 region for the high SME diet suggests enhanced digestion and more rapid release of starch. However, it remains to be seen whether a diet containing rapidly digestible, as opposed to slowly digestible, starch is more beneficial for piglets. This rate of starch breakdown in the piglet is an important finding, which may have implications in helping to alleviate the post-weaning growth check, particularly in the absence of in-feed antibiotic growth promoters. Processing did not appear to offer any benefit over unprocessed wheat with regard to daily live-weight gain or the apparent digestibility of nitrogen in the small intestine over the 14-day period. Based on the enhanced in vivo starch digestibility, performance might be improved over a longer period, although future studies are required to confirm this. Precise processing variables for raw materials must be stated in all animal trials.

Improvements in circulating cholesterol, antioxidants, and homocysteine after dietary intervention in an Australian Aboriginal community.

BACKGROUND: Poor nutrition contributes to high rates of coronary heart disease among Australian Aboriginal populations. Since late 1993, the Aboriginal community described here has operated a healthy lifestyle program aimed at reducing the risk of chronic disease. OBJECTIVE: We evaluated the effectiveness of a community-directed intervention program to reduce coronary heart disease risk through dietary modification. DESIGN: Intervention processes included store management policy changes, health promotion activities, and nutrition education aimed at high-risk individuals. Dietary advice was focused on decreasing saturated fat and sugar intake and increasing fruit and vegetable intake. Evaluation of the program included conducting sequential, cross-sectional risk factor surveys at 2-y intervals; measuring fasting cholesterol, lipid-soluble antioxidants, and homocysteine concentrations; and assessing smoking status. Nutrient intakes were estimated from analysis of food turnover in the single community store. RESULTS: There was a significant reduction in the prevalence of hypercholesterolemia (age-adjusted prevalences were 31%, 21%, and 15% at baseline, 2 y, and 4 y, respectively; P < 0.001). There were significant increases in plasma concentrations of alpha-tocopherol, lutein and zeaxanthin, cryptoxanthin, and beta-carotene across the population. Retinol and lycopene concentrations did not change significantly. Mean plasma homocysteine concentrations decreased by 3 micromol/L. There was no significant change in smoking prevalence between the 2 follow-up surveys. There was an increase in the density of fresh fruit and vegetables and carotenoids in the food supply at the community store. CONCLUSION: This community-directed dietary intervention program reduced the prevalence of coronary heart disease risk factors related to diet.

Effectiveness of a community-directed 'healthy lifestyle' program in a remote Australian aboriginal community.

OBJECTIVE: To assess the sustainability and effectiveness of a community-directed program for primary and secondary prevention of obesity, diabetes and cardiovascular disease in an Aboriginal community in north-west Western Australia. METHOD: Evaluation of health outcomes (body mass index, glucose tolerance, and plasma insulin and triglyceride concentrations) in a cohort of high-risk individuals (n = 49, followed over two years) and cross-sectional community samples (n = 200 at baseline, 185 at two-year and 132 at four-year follow-ups), process (interventions and their implementation) and impact (diet and exercise behaviour). RESULTS: For the high-risk cohort, involvement in diet and/or exercise strategies was associated with protection from increases in plasma glucose and triglycerides seen in a comparison group; however, sustained weight loss was not achieved. At the community level, significant reductions were observed in fasting insulin concentration but no change in prevalence of diabetes, overweight or obesity. Weight gain remained a problem among younger people. Sustainable improvements were observed for dietary intake and level of physical activity. These changes were related to supportive policies implemented by the community council and store management. CONCLUSIONS: Community control and ownership enabled embedding and sustainability of program, in association with social environmental policy changes and long-term improvements in important risk factors for chronic disease. IMPLICATIONS: Developmental initiatives facilitating planning, implementation and ownership of interventions by community members and organisations can be a feasible and effective way to achieve sustainable improvements in health behaviours and selected health outcomes among Aboriginal people.

Class II MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition.

T cell expression of class II MHC/peptide complexes may be important for maintenance of peripheral self-tolerance, but mechanisms underlying the genesis of class II MHC glycoproteins on T cells are not well resolved. T cell APC (T-APC) used herein were transformed IL-2-dependent clones that constitutively synthesized class II MHC glycoproteins. When pulsed with myelin basic protein (MBP) and injected into Lewis rats, these T-APC reduced the severity of experimental autoimmune encephalomyelitis, whereas unpulsed T-APC were without activity. Normal MBP-reactive clones cultured without APC did not express class II MHC even when activated with mitogens and exposed to IFN-gamma. However, during a 4-h culture with T-APC or macrophage APC, recognition of MBP or mitogenic activation of responder T cells elicited high levels of I-A and I-E expression on responders. Acquisition of class II MHC glycoproteins by responders was resistant to the protein synthesis inhibitor cycloheximide, coincided with transfer of a PKH26 lipophilic dye from APC to responders, and resulted in the expression of syngeneic and allogeneic MHC glycoproteins on responders. Unlike rested I-A- T cell clones, rat thymic and splenic T cells expressed readily detectable levels of class II MHC glycoproteins. When preactivated with mitogens, naive T cells acquired APC-derived MHC class II molecules and other membrane-associated proteins when cultured with xenogeneic APC in the absence of Ag. In conclusion, this study provides evidence that APC donate membrane-bound peptide/MHC complexes to Ag-specific T cell responders by a mechanism associated with the induction of tolerance.

An autologous self-antigen differentially regulates expression of I-A glycoproteins and B7 costimulatory molecules on CD4- CD8- T helper cells.

During inflammation, T helper cells transiently express class II major histocompatibility complex (MHC) glycoproteins and present antigens to other T cells. To assess involvement of self-antigens in the generation of T cell antigen-presenting cell (T-APC) activity, rat (R) myelin basic protein (MBP) was used to stimulate a rat CD4-CD8- T cell clone. RMBP induced T cell surface expression of class II MHC glycoproteins and T-APC activity, although RMBP did not elicit interleukin (IL-2) production or proliferation. When added to culture with the strong agonist guinea pig (GP) MBP, RMBP acted as a partial antagonist and inhibited responses of IL-2 production, proliferation, and T cell expression of B7.1. RMBP did not, however, efficiently antagonize GPMBP-induced I-A expression on T cells. These findings indicate that the self-antigen RMBP specifically induces accumulation of I-A/peptide complexes at signaling thresholds that inhibit pathogenic autoimmune responses. Overall, this study suggests a role for self-antigens in the generation of B7-deficient T-APC activity as a mechanism of tolerance in experimental autoimmune encephalomyelitis.

Partial agonism elicits an enduring phase of T-cell-medicated antigen presentation.

Previous studies have shown that the anti-CD4 mAb W3/25 strongly enhances T cell APC (T-APC) activity. In this study, single positive CD4+ and double negative (DN) (CD4-CD8-) T-helper cells specific for the 55-69 or 72-86 sequence of guinea pig (GP) myelin basic protein (GPMBP) were used to study CD4 regulation of T-APC activity. Clones were cultured with irradiated SPL and GPMBP or rat (R) MBP for 2-3 days, were propagated in IL-2 for another 1-3 days, were irradiated, and were used as T-APC. DN T cells specific for GP55-69 effectively presented GPMBP and were superior APC compared to other CD4+ T cells for presentation of this antigen. In contrast, DN T cells specific for the dominant encephalitogenic 72-86 determinant did not effectively present the agonist GPMBP but potently presented the partial agonist RMBP. The heightened APC activity of DN T cells reflected the lack of CD4 because the anti-CD4 mAb W3/25 promoted T-APC activity of CD4+ T cells to those levels expressed by DN T cells. Overall, T cells with potent reactivity to GPMBP or RMBP were subsequently unable to present that antigen, whereas T cells exhibiting partial or low antigen reactivities were highly effective APC for presentation of that antigen. The unrelated antigen conalbumin was presented by MBP-specific clones only when added to culture with a specific partial agonist. Together, these data indicate that partially agonistic MHC ligands promote prolonged expression of T-APC activity and that DN T cells may be specialized to mediate postactivational antigen presentation.

Class II MHC/peptide complexes on T cell antigen-presenting cells: agonistic antigen recognition inhibits subsequent antigen presentation.

Previous studies have shown that tolerogenic anti-CD4 (W3/25) and anti-LFA-1 mAb (LRTC1) which block T cell activation paradoxically enhance T cell-mediated antigen presentation. Lasting T cell APC (T-APC) activity requires and initial exposure of T cells to these mAb in the presence of professional APC and antigen. This study revealed a central mechanism regulating the duration of T-APC activity. T cell recognition of class II MHC complexes of T-APC catalyzed a rapid decay in the presentation of agonistic antigens, whereas partial agonistic signals decayed at a shower rate. Likewise, blockade of agonistic T-T cell autorecognition by these mAb led to the persistence of agonistic MHC/antigen on T-APC. The best predictor of T-APC activity was related to the ability of clonal T cells to respond to antigen presented by neighboring T cells. Strong responders were inefficient T-APC, whereas inefficient responders were strong T-APC. Addition of irradiated myelin basic protein (MBP0-specific responders to T-APC cultures specifically inhibited the subsequent presentation of MBP but not conalbumin, and vice versa. T-APC presentation of antigen to responder T cells also resulted in reduced surface expression of class II MHC I-A glycoproteins on T-APC. These findings indicate that agonistic recognition of antigen of T-APC specifically inhibits subsequent presentation of that antigen, whereas antagonistic MHC/antigen complexes are preserved for an enduring T-APC activity.

Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence.

Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is consistent with the proposed recombination events. To provide a convenient vector production system that circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for production of large-scale preparations of Ad-based vectors lacking replication-competent variants.

T-helper lymphocytes specific for myelin basic protein: low-density activation prolongs a postactivation refractory phase marked by decreased pathogenicity and enhanced sensitivity to anergy.

Regulation of experimental autoimmune encephalomyelitis (EAE) in Lewis rats may involve activation-dependent negative feedback pathways of T-helper cells. Previous studies have shown that T-helper cells specific for myelin basic protein exhibit a postactivation refractory phase during which antigenic restimulation elicits proliferation without production of IL-2. Herein, we show that postactivation refractoriness inhibits regeneration of EAE transfer activity and is manifest by a lack of IL-2 mRNA accumulation despite induction of normal levels of IL-4 mRNA. Preactivated refractory T cells were substantially more susceptible than resting T cells to the induction of anergy. Low-density T cell activation or subcloning prolonged the duration of the refractory phase and engendered long-term desensitization of T cells marked by a blockade of IL-2 production and by enhanced susceptibility to anergy. Overall, these results support the concept that postactivation refractoriness controls the pathogenicity and differentiation of T-helper cells.