Antibody-mediated depletion of select leukocyte subsets in blood and tissue of nonhuman primates.

Understanding the immunological control of pathogens requires a detailed evaluation of the mechanistic contributions of individual cell types within the immune system. While knockout mouse models that lack certain cell types have been used to help define the role of those cells, the biological and physiological characteristics of mice do not necessarily recapitulate that of a human. To overcome some of these differences, studies often look towards nonhuman primates (NHPs) due to their close phylogenetic relationship to humans. To evaluate the immunological role of select cell types, the NHP model provides distinct advantages since NHP more closely mirror the disease manifestations and immunological characteristics of humans. However, many of the experimental manipulations routinely used in mice (e.g., gene knock-out) cannot be used with the NHP model. As an alternative, the in vivo infusion of monoclonal antibodies that target surface proteins on specific cells to either functionally inhibit or deplete cells can be a useful tool. Such depleting antibodies have been used in NHP studies to address immunological mechanisms of action. In these studies, the extent of depletion has generally been reported for blood, but not thoroughly assessed in tissues. Here, we evaluated four depleting regimens that primarily target T cells in NHP: anti-CD4, anti-CD8α, anti-CD8ß, and immunotoxin-conjugated anti-CD3. We evaluated these treatments in healthy unvaccinated and IV BCG-vaccinated NHP to measure the extent that vaccine-elicited T cells - which may be activated, increased in number, or resident in specific tissues - are depleted compared to resting populations in unvaccinated NHPs. We report quantitative measurements of in vivo depletion at multiple tissue sites providing insight into the range of cell types depleted by a given mAb. While we found substantial depletion of target cell types in blood and tissue of many animals, residual cells remained, often residing within tissue. Notably, we find that animal-to-animal variation is substantial and consequently studies that use these reagents should be powered accordingly.

Geographic inequalities in need and provision of social prescribing link workers.

BACKGROUND: Long-term health conditions are major challenges for care systems. Social prescribing link workers have been introduced via Primary Care Networks (PCNs) across England since 2019 to address the wider determinants of health by connecting individuals to activities, groups, or services within their local community. AIM: To assess whether the rollout of social prescribing link workers was in areas with the highest need. DESIGN AND SETTING: A retrospective study of social prescribing link workers in England from 2019 to 2023. METHOD: We combined workforce, population, survey, and area-level data at the PCN-level from April 2020 to October 2023. We measured population need prior to the rollout of link workers using reported lack of support from local services in the 2019 GP Patient Survey. To assess if rollout reflected need, we used linear regression to relate provision of link workers (measured by full-time equivalent (FTE) per 10,000 patients) in each quarter to population need for support. RESULTS: Populations in urban, more deprived areas and with higher proportions of minority ethnicities had the highest reported lack of support. Geographically these were in the North West and London. Initially, there was no association between need and provision; then from July 2022, this became negative and significant. By October 2023, a 10-percentage point higher need for support was associated with a 0.035 (95%CI(-0.634 to -0.066)) lower FTE per 10,000 patients. CONCLUSION: Rollout of link workers has not been sufficiently targeted at areas with the highest need. Future deployments should be targeted at those areas.

Trends in full-time working in general practice: repeated cross-sectional study.

BACKGROUND: There is little evidence and no agreement on what constitutes full-time working for general practitioners (GPs). This is essential for workforce planning, resource allocation and accurately describing GP activity. AIM: To clarify the definition of full-time working for general practitioners, how this has changed over time and whether these changes are explained by GP demographics. DESIGN AND SETTING: Repeated cross-sectional national surveys between 2010 and 2021. METHOD: Comparison of three measures of working time commitments (hours and sessions per week and hours per session) plus a measure of workload intensity across survey years. Multiple regression to adjust the changes over time for age, sex, ethnicity, contract type, area deprivation, and rurality. Unadjusted hours and sessions per week were compared to definitions of full-time working. RESULTS: Average hours and sessions per week reduced from 40.5 (95% CI: 38.5, 42.5) to 38.0 (36.3, 39.6) and 7.3 (7.2, 7.3) to 6.2 (6.2, 6.3) respectively between 2010 and 2021. In 2021, 54.6% of GPs worked at least 37.5 hours per week and 9.5% worked at least 9 sessions. Hours per session increased from 5.7 (5.7, 5.7) to 6.2 (6.2, 6.3) between 2010 and 2021. Partners worked more hours, sessions and hours per session. Adjustments increased the increase in hours per session from 0.54 to 0.61. CONCLUSION: At the current average duration of sessions, six sessions per week aligns with the NHS definition of full-time hours. However, hours per week is a more consistent way to define full-time work for GPs.

Informal caregiving and the allocation of time: implications for opportunity costs and measurement.

Informal care requires a considerable time investment from providers that inherently involves trade-offs against various uses of time. We examine what other uses of time are forgone when individuals provide informal care. We further consider how caregiving is linked to a range of rarely explored time use characteristics relating to multitasking, the fragmentation and the timing of activities. We use data from 5670 adults across 11003 diary days from the 2014/15 UK Time Use Survey. Using a 'doubly robust' approach of entropy balancing and regression adjustment, we find carers spend an additional 49.0 min on non-market work, 2.9 min on personal care, 5.8 min on leisure and 2.9 min on miscellaneous activities on weekdays. They spend 46.1 min less on market work and 14.4 min less on sleep. Carers report more time stress, more multitasking, and more fragmented time. We estimate with attribution factors that 16% and 11% of reported household task activity is due to caregiving on weekday and weekend days, respectively. These findings provide evidence on additional opportunity costs faced by carers and possible channels through which carer labour market and health outcomes are realised. The attribution factors we calculate can be applied to total reported caregiving time to avoid overestimation when this is incorporated into economic evaluations.

Protective intravenous BCG vaccination induces enhanced immune signaling in the airways.

Intradermal (ID) Bacillus Calmette-Guérin (BCG) is the most widely administered vaccine in the world. However, ID-BCG fails to achieve the level of protection needed in adults to alter the course of the tuberculosis epidemic. Recent studies in non-human primates have demonstrated high levels of protection against Mycobacterium tuberculosis ( Mtb ) following intravenous (IV) administration of BCG. However, the protective immune features that emerge following IV BCG vaccination remain incompletely defined. Here we used single-cell RNA-sequencing (scRNAseq) to transcriptionally profile 157,114 unstimulated and purified protein derivative (PPD)-stimulated bronchoalveolar lavage (BAL) cells from 29 rhesus macaques immunized with BCG across routes of administration and doses to uncover cell composition-, gene expression-, and biological network-level signatures associated with IV BCG-mediated protection. Our analyses revealed that high-dose IV BCG drove an influx of polyfunctional T cells and macrophages into the airways. These macrophages exhibited a basal activation phenotype even in the absence of PPD-stimulation, defined in part by IFN and TNF-α signaling up to 6 months following BCG immunization. Furthermore, intercellular immune signaling pathways between key myeloid and T cell subsets were enhanced following PPD-stimulation in high-dose IV BCG-vaccinated macaques. High-dose IV BCG also engendered quantitatively and qualitatively stronger transcriptional responses to PPD-stimulation, with a robust Th1-Th17 transcriptional phenotype in T cells, and augmented transcriptional signatures of reactive oxygen species production, hypoxia, and IFN-γ response within alveolar macrophages. Collectively, this work supports that IV BCG immunization creates a unique cellular ecosystem in the airways, which primes and enables local myeloid cells to effectively clear Mtb upon challenge.

Vaccine elicitation and structural basis for antibody protection against alphaviruses.

Alphaviruses are RNA viruses that represent emerging public health threats. To identify protective antibodies, we immunized macaques with a mixture of western, eastern, and Venezuelan equine encephalitis virus-like particles (VLPs), a regimen that protects against aerosol challenge with all three viruses. Single- and triple-virus-specific antibodies were isolated, and we identified 21 unique binding groups. Cryo-EM structures revealed that broad VLP binding inversely correlated with sequence and conformational variability. One triple-specific antibody, SKT05, bound proximal to the fusion peptide and neutralized all three Env-pseudotyped encephalitic alphaviruses by using different symmetry elements for recognition across VLPs. Neutralization in other assays (e.g., chimeric Sindbis virus) yielded variable results. SKT05 bound backbone atoms of sequence-diverse residues, enabling broad recognition despite sequence variability; accordingly, SKT05 protected mice against Venezuelan equine encephalitis virus, chikungunya virus, and Ross River virus challenges. Thus, a single vaccine-elicited antibody can protect in vivo against a broad range of alphaviruses.

Antibody-mediated depletion of select T cell subsets in blood and tissue of nonhuman primates.

Understanding the immunological control of pathogens requires a detailed evaluation of the mechanistic contributions of individual cell types within the immune system. While knockout mouse models that lack certain cell types have been used to help define the role of those cells, the biological and physiological characteristics of mice do not necessarily recapitulate that of a human. To overcome some of these differences, studies often look towards nonhuman primates (NHPs) due to their close phylogenetic relationship to humans. To evaluate the immunological role of select cell types, the NHP model provides distinct advantages since NHP more closely mirror the disease manifestations and immunological characteristics of humans. However, many of the experimental manipulations routinely used in mice (e.g., gene knock-out) cannot be used with the NHP model. As an alternative, the in vivo infusion of monoclonal antibodies that target surface proteins on specific cells to either functionally inhibit or deplete cells can be a useful tool. Such depleting antibodies have been used in NHP studies to address immunological mechanisms of action. In these studies, the extent of depletion has generally been reported for blood, but not thoroughly assessed in tissues. Here, we evaluated four depleting regimens that primarily target T cells in NHP: anti-CD4, anti-CD8α, anti-CD8ß, and immunotoxin-conjugated anti-CD3. We evaluated these treatments in healthy unvaccinated and IV BCG-vaccinated NHP to measure the extent that vaccine-elicited T cells - which may be activated, increased in number, or resident in specific tissues - are depleted compared to resting populations in unvaccinated NHPs. We report quantitative measurements of in vivo depletion at multiple tissue sites providing insight into the range of cell types depleted by a given mAb. While we found substantial depletion of target cell types in blood and tissue of many animals, residual cells remained, often residing within tissue. Notably, we find that animal-to-animal variation is substantial and consequently studies that use these reagents should be powered accordingly.

Effects of chain ownership and private equity financing on quality in the English care home sector: retrospective observational study.

BACKGROUND: the structure of care homes markets in England is changing with the emergence of for-profit homes organised in chains and financed by private equity. Previous literature shows for-profit homes were rated lower quality than not-for-profit homes when inspected by the national regulator, but has not considered new forms of financing. OBJECTIVES: to examine whether financing and organisation of care homes is associated with regulator assessments of quality. METHODS: retrospective observational study of the Care Quality Commission's ratings of 10,803 care homes providing services to older people as of January 2020. We used generalised ordered logistic models to assess whether ratings differed between not-for-profit and for-profit homes categorised into three groups: (i) chained ownership, financed by private equity; (ii) chained ownership, not financed by private equity and (iii) independent ownership. We compared Overall and domain (caring, effective, responsive, safe, well-led) ratings adjusted for care home size, age and location. RESULTS: all three for-profit ownership types had lower average overall ratings than not-for-profit homes, especially independent (6.8% points (p.p.) more likely rated as 'Requires Improvement/Inadequate', 95% CI: 4.7-8.9) and private equity chains (6.6 p.p. more likely rated as 'Requires Improvement/Inadequate', 95% CI: 2.9-10.2). Independent homes scored better than private equity chains in the safe, effective and responsive domains but worst in the well-led domain. DISCUSSION: private equity financing and independent for-profit ownership are associated with lower quality. The consequences of the changing care homes market structure for quality of services should be monitored.

Rapid and Facile Fabrication of Polyiodide Solid-State Dye-Sensitized Solar Cells Using Ambient Air Drying.

Dye-sensitized solar cells are promising candidates for low-cost indoor power generation applications. However, they currently suffer from complex fabrication and stability issues arising from the liquid electrolyte. Consequently, the so-called zombie cell was developed, in which the liquid electrolyte is dried out to yield a solid through a pinhole after cell assembly. We report a method for faster, simpler, and potentially more reliable production of zombie cells through direct and rapid drying of the electrolyte on the working electrode prior to cell assembly, using an iodide-triiodide redox couple electrolyte as a basis. These "rapid-zombie" cells were fabricated with power conversion efficiencies reaching 5.0%, which was larger than the 4.5% achieved for equivalent "slow" zombie cells. On a large-area cell of 15.68 cm2, over 2% efficiency was achieved at 0.2 suns. After 12 months of dark storage, the "rapid-zombie" cells were remarkably stable and actually showed a moderate increase in average efficiencies.

Leveraging pleiotropic association using sparse group variable selection in genomics data.

BACKGROUND: Genome-wide association studies (GWAS) have identified genetic variants associated with multiple complex diseases. We can leverage this phenomenon, known as pleiotropy, to integrate multiple data sources in a joint analysis. Often integrating additional information such as gene pathway knowledge can improve statistical efficiency and biological interpretation. In this article, we propose statistical methods which incorporate both gene pathway and pleiotropy knowledge to increase statistical power and identify important risk variants affecting multiple traits. METHODS: We propose novel feature selection methods for the group variable selection in multi-task regression problem. We develop penalised likelihood methods exploiting different penalties to induce structured sparsity at a gene (or pathway) and SNP level across all studies. We implement an alternating direction method of multipliers (ADMM) algorithm for our penalised regression methods. The performance of our approaches are compared to a subset based meta analysis approach on simulated data sets. A bootstrap sampling strategy is provided to explore the stability of the penalised methods. RESULTS: Our methods are applied to identify potential pleiotropy in an application considering the joint analysis of thyroid and breast cancers. The methods were able to detect eleven potential pleiotropic SNPs and six pathways. A simulation study found that our method was able to detect more true signals than a popular competing method while retaining a similar false discovery rate. CONCLUSION: We developed feature selection methods for jointly analysing multiple logistic regression tasks where prior grouping knowledge is available. Our method performed well on both simulation studies and when applied to a real data analysis of multiple cancers.

Validation of multiplex PCR sequencing assay of SIV.

BACKGROUND: The generation of accurate and reproducible viral sequence data is necessary to understand the diversity present in populations of RNA viruses isolated from clinical samples. While various sequencing methods are available, they often require high quality templates and high viral titer to ensure reliable data. METHODS: We modified a multiplex PCR and sequencing approach to characterize populations of simian immunodeficiency virus (SIV) isolated from nonhuman primates. We chose this approach with the aim of reducing the number of required input templates while maintaining fidelity and sensitivity. We conducted replicate sequencing experiments using different numbers of quantified viral RNA (vRNA) or viral cDNA as input material. We performed assays with clonal SIVmac239 to detect false positives, and we mixed SIVmac239 and a variant with 24 point mutations (SIVmac239-24X) to measure variant detection sensitivity. RESULTS: We found that utilizing a starting material of quantified viral cDNA templates had a lower rate of false positives and increased reproducibility when compared to that of quantified vRNA templates. This study identifies the importance of rigorously validating deep sequencing methods and including replicate samples when using a new method to characterize low frequency variants in a population with a small number of templates. CONCLUSIONS: Because the need to generate reproducible and accurate sequencing data from diverse viruses from low titer samples, we modified a multiplex PCR and sequencing approach to characterize SIV from populations from non-human primates. We found that increasing starting template numbers increased the reproducibility and decreased the number of false positives identified, and this was further seen when cDNA was used as a starting material. Ultimately, we highlight the importance of vigorously validating methods to prevent overinterpretation of low frequency variants in a sample.

Is health research undertaken where the burden of disease is greatest? Observational study of geographical inequalities in recruitment to research in England 2013-2018.

BACKGROUND: Research is fundamental to high-quality care, but concerns have been raised about whether health research is conducted in the populations most affected by high disease prevalence. Geographical distribution of research activity is important for many reasons. Recruitment is a major barrier to research delivery, and undertaking recruitment in areas of high prevalence could be more efficient. Regional variability exists in risk factors and outcomes, so research done in healthier populations may not generalise. Much applied health research evaluates interventions, and their impact may vary by context (including geography). Finally, fairness dictates that publically funded research should be accessible to all, so that benefits of participating can be fairly distributed. We explored whether recruitment of patients to health research is aligned with disease prevalence in England. METHODS: We measured disease prevalence using the Quality and Outcomes Framework in England (total long-term conditions, mental health and diabetes). We measured research activity using data from the NIHR Clinical Research Network. We presented descriptive data on geographical variation in recruitment rates. We explored associations between the recruitment rate and disease prevalence rate. We calculated the share of patient recruitment that would need to be redistributed to align recruitment with prevalence. We assessed whether associations between recruitment rate and disease prevalence varied between conditions, and over time. RESULTS: There was significant geographical variation in recruitment rates. When areas were ranked by disease prevalence, recruitment was not aligned with prevalence, with disproportionately low recruitment in areas with higher prevalence of total long-term and mental health conditions. At the level of 15 local networks, analyses suggested that around 12% of current recruitment activity would need to be redistributed to align with disease prevalence. Overall, alignment showed little change over time, but there was variation in the trends over time in individual conditions. CONCLUSIONS: Geographical variations in recruitment do not reflect the suitability of the population for research. Indicators should be developed to assess the fit between research and need, and to allow assessment of interventions among funders, researchers and patients to encourage closer alignment between research activity and burden.

CD8ß Depletion Does Not Prevent Control of Viral Replication or Protection from Challenge in Macaques Chronically Infected with a Live Attenuated Simian Immunodeficiency Virus.

We evaluated the contribution of CD8αß+ T cells to control of live-attenuated simian immunodeficiency virus (LASIV) replication during chronic infection and subsequent protection from pathogenic SIV challenge. Unlike previous reports with a CD8α-specific depleting monoclonal antibody (mAb), the CD8ß-specific mAb CD8ß255R1 selectively depleted CD8αß+ T cells without also depleting non-CD8+ T cell populations that express CD8α, such as natural killer (NK) cells and γδ T cells. Following infusion with CD8ß255R1, plasma viremia transiently increased coincident with declining peripheral CD8αß+ T cells. Interestingly, plasma viremia returned to predepletion levels even when peripheral CD8αß+ T cells did not. Although depletion of CD8αß+ T cells in the lymph node (LN) was incomplete, frequencies of these cells were 3-fold lower (P = 0.006) in animals that received CD8ß255R1 than in those that received control IgG. It is possible that these residual SIV-specific CD8αß+ T cells may have contributed to suppression of viremia during chronic infection. We also determined whether infusion of CD8ß255R1 in the LASIV-vaccinated animals increased their susceptibility to infection following intravenous challenge with pathogenic SIVmac239. We found that 7/8 animals infused with CD8ß255R1, and 3/4 animals infused with the control IgG, were resistant to SIVmac239 infection. These results suggest that infusion with CD8ß255R1 did not eliminate the protection afforded to LASIV vaccination. This provides a comprehensive description of the impact of CD8ß255R1 infusion on the immunological composition in cynomolgus macaques, compared to an isotype-matched control IgG, while showing that the control of LASIV viremia and protection from challenge can occur even after CD8ß255R1 administration.IMPORTANCE Studies of SIV-infected macaques that deplete CD8+ T cells in vivo with monoclonal antibodies have provided compelling evidence for their direct antiviral role. These studies utilized CD8α-specific mAbs that target both the major (CD8αß+) and minor (CD8αα+) populations of CD8+ T cells but additionally deplete non-CD8+ T cell populations that express CD8α, such as NK cells and γδ T cells. In the current study, we administered the CD8ß-specific depleting mAb CD8ß255R1 to cynomolgus macaques chronically infected with a LASIV to selectively deplete CD8αß+ T cells without removing CD8αα+ lymphocytes. We evaluated the impact on control of virus replication and protection from pathogenic SIVmac239 challenge. These results underscore the utility of CD8ß255R1 for studying the direct contribution of CD8αß+ T cells in various disease states.

Estimating Joint Health Condition Utility Values.

OBJECTIVES: To predict health state utility values (HSUVs) for individuals with up to 4 conditions simultaneously. METHODS: Person-level data were taken from the General Practice Patient Survey, a national survey of adult patients registered with general practices in England. Individuals reported whether they had any 1 of 16 chronic conditions and completed the 3-level EuroQol 5-dimensional questionnaire. Four nonparametric methods (additive, multiplicative, minimum, and the adjusted decrement estimator) and 1 parametric estimator (the linear index) were used to predict HSUVs for individuals with a joint health condition (JHC). Predicted and actual utility scores were compared for precision using root mean square error and mean absolute error. Bias was assessed using mean error. RESULTS: The analysis included 929,565 individuals, of which 30.5% had at least 2 conditions. Of the nonparametric estimators, the multiplicative approach produced estimates with the lowest bias and most precision for 2 JHCs. For populations with a long-term mental health condition within the JHC, the multiplicative approach overestimated utility scores. All nonparametric methods produced biased results when estimating HSUVs for 3 or 4 JHCs. The linear index generally produced unbiased results with the highest precision. CONCLUSIONS: The multiplicative approach was the best nonparametric estimator when estimating HSUVs for 2 JHCs. None of the nonparametric approaches for estimating HSUVs can be recommended with more than 2 JHCs. The linear index was found to have good predictive properties but needs external validation before being recommended for routine use.

New professional roles and patient satisfaction: Evidence from a European survey along three clinical pathways.

This paper reports the results of an empirical analysis exploring the impact of new professions (eg a physician associate) and new professional roles on patient experiences of and satisfaction with care. A sub set of data from a patient survey conducted as part of the MUNROS programme of work was used. The overall survey aim was to describe and quantify the use of new professionals and new roles for established health care professionals other than medical doctors, in primary and secondary care sectors in three care pathways in nine European countries Ordered logit models were used to investigate the association between: (1) patient satisfaction with the last visit; (2) with their care provider; (3) with the information provided and a set of covariates explaining the involvement of new professional roles in three clinical pathways: type 2 diabetes, heart disease and breast cancer. For patients with breast cancer, high levels of satisfaction are associated with the involvement of new professions/professional roles in the provision of conditions specific education and monitoring. For patients with heart disease, the involvement of new professions/professional roles is likely to have a negative impact on satisfaction. For patients with Type 2 diabetes results are ambivalent. Patients belonging to countries experiencing innovative models of healthcare delivery and with high levels of involvement of new professions/professional roles are generally more satisfied. In conclusion, the introduction of new professions does not affect patient satisfaction negatively, therefore introducing new health professional roles is a pursuable strategy from a patient satisfaction perspective, at least for breast cancer and type 2 diabetes.

Acute-Phase CD4+ T Cell Responses Targeting Invariant Viral Regions Are Associated with Control of Live Attenuated Simian Immunodeficiency Virus.

We manipulated SIVmac239Δnef, a model of major histocompatibility complex (MHC)-independent viral control, to evaluate characteristics of effective cellular responses mounted by Mauritian cynomolgus macaques (MCMs) that express the M3 MHC haplotype, which has been associated with poor control of pathogenic simian immunodeficiency virus (SIV). We created SIVΔnef-8x to test the hypothesis that effective SIV-specific T cell responses targeting invariant viral regions can emerge in the absence of immunodominant CD8+ T cell responses targeting variable epitopes and that control is achievable in individuals lacking known "protective" MHC alleles. Full-proteome gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assays identified six newly targeted immunogenic regions following SIVΔnef-8x infection of M3/M3 MCMs. We deep sequenced circulating virus and found that four of the six newly targeted regions rarely accumulated mutations. Six animals infected with SIVΔnef-8x had T cell responses that targeted at least one of the four invariant regions and had a lower set point viral load than two animals that did not have T cell responses that targeted any invariant regions. We found that MHC class II molecules restricted all four of the invariant peptide regions, while the two variable regions were restricted by MHC class I molecules. Therefore, in the absence of immunodominant CD8+ T cell responses that target variable regions during SIVmac239Δnef infection, individuals without protective MHC alleles developed predominantly CD4+ T cell responses specific for invariant regions that may improve control of virus replication. Our results provide some evidence that antiviral CD4+ T cells during acute SIV infection can contribute to effective viral control and should be considered in strategies to combat HIV infection.IMPORTANCE Studies defining effective cellular immune responses to human immunodeficiency virus (HIV) and SIV have largely focused on a rare population that express specific MHC class I alleles and control virus replication in the absence of antiretroviral treatment. This leaves in question whether similar effective immune responses can be achieved in the larger population. The majority of HIV-infected individuals mount CD8+ T cell responses that target variable viral regions that accumulate high-frequency escape mutations. Limiting T cell responses to these variable regions and targeting invariant viral regions, similar to observations in rare "elite controllers," may provide an ideal strategy for the development of effective T cell responses in individuals with diverse MHC genetics. Therefore, it is of paramount importance to determine whether T cell responses can be redirected toward invariant viral regions in individuals without protective MHC alleles and if these responses improve control of virus replication.