Mapping and identifying service models for community-based services for children with intellectual disabilities and behaviours that challenge in England.

BACKGROUND: One in five children with an intellectual disability in the UK display behaviours that challenge. Despite associated impacts on the children themselves, their families, and services, little research has been published about how best to design, organise, and deliver health and care services to these children. The purpose of this study was to describe how services are structured and organised ("service models") in England for community-based health and care services for children with intellectual disability who display behaviours that challenge. METHODS: Survey data about services were collected from 161 eligible community-based services in England. Staff from 60 of these services were also interviewed. A combination of latent class and descriptive analysis, coupled with consultation with family carers and professionals was used to identify and describe groupings of similar services (i.e., "service models"). RESULTS: The latent class analysis, completed as a first step in the process, supported a distinction between specialist services and non-specialist services for children who display behaviours that challenge. Planned descriptive analyses incorporating additional study variables were undertaken to further refine the service models. Five service models were identified: Child and Adolescent Mental Health Services (CAMHS) (n = 69 services), Intellectual Disability CAMHS (n = 28 services), Children and Young People Disability services (n = 25 services), Specialist services for children who display behaviours that challenge (n = 27 services), and broader age range services for children and/or adolescents and adults (n= 12 services). CONCLUSIONS: Our analysis led to a typology of five service models for community health and care services for children with intellectual disabilities and behaviours that challenge in England. Identification of a typology of service models is a first step in building evidence about the best provision of services for children with intellectual disabilities who display behaviours that challenge. The methods used in the current study may be useful in research developing service typologies in other specialist fields of health and care. STUDY REGISTRATION: Trial Registration: Current Controlled Trials ISRCTN88920546, Date assigned 05/07/2022.

Establishment of bovine 3D enteroid-derived 2D monolayers.

Three-dimensional (3D) intestinal enteroids are powerful in vitro models for studying intestinal biology. However, due to their closed structure direct access to the apical surface is impeded, limiting high-throughput applications of exogenous compounds and pathogens. In this study, we describe a method for generating confluent 2D enteroids from single-cell suspensions of enzymatically-dissociated ileum-derived bovine 3D enteroids. Confluent monolayers were first achieved using IntestiCult media but to establish a defined, cost-effective culture media, we also developed a bovine enteroid monolayer (BEM) medium. The monolayers cultured in BEM media proliferated extensively and formed confluent cell layers on both Matrigel-coated plastic plates and transwell inserts by day 3 of culture. The 2D enteroids maintained the epithelial cell lineages found in 3D enteroids and ileum tissue. In addition, the monolayers formed a functional epithelial barrier based on the presence of the adherens and tight junction proteins, E-cadherin and ZO-1, and electrical resistance across the monolayer was measured from day 3 and maintained for up to 7 days in culture. The method described here will provide a useful model to study bovine epithelial cell biology with ease of access to the apical surface of epithelial cells and has potential to investigate host-pathogen interactions and screen bioactive compounds.

Novel chicken two-dimensional intestinal model comprising all key epithelial cell types and a mesenchymal sub-layer.

The intestinal epithelium plays a variety of roles including providing an effective physical barrier and innate immune protection against infection. Two-dimensional models of the intestinal epithelium, 2D enteroids, are a valuable resource to investigate intestinal cell biology and innate immune functions and are suitable for high throughput studies of paracellular transport and epithelial integrity. We have developed a chicken 2D enteroid model that recapitulates all major differentiated cell lineages, including enterocytes, Paneth cells, Goblet cells, enteroendocrine cells and leukocytes, and self-organises into an epithelial and mesenchymal sub-layer. Functional studies demonstrated the 2D enteroids formed a tight cell layer with minimal paracellular flux and a robust epithelial integrity, which was maintained or rescued following damage. The 2D enteroids were also able to demonstrate appropriate innate immune responses following exposure to bacterial endotoxins, from Salmonella enterica serotype Typhimurium and Bacillus subtilis. Frozen 2D enteroids cells when thawed were comparable to freshly isolated cells. The chicken 2D enteroids provide a useful ex vivo model to study intestinal cell biology and innate immune function, and have potential uses in screening of nutritional supplements, pharmaceuticals, and bioactive compounds.

Visualisation and characterisation of mononuclear phagocytes in the chicken respiratory tract using CSF1R-transgenic chickens.

The respiratory tract is a key organ for many avian pathogens as well as a major route for vaccination in the poultry industry. To improve immune responses after vaccination of chickens through increased uptake of vaccines and targeting to antigen presenting cells, a better understanding of the avian respiratory immune system is required. Transgenic MacReporter birds were used expressing a reporter gene (eGFP or mApple) under the control of the CSF1R promoter and enhancer in cells of the mononuclear phagocyte (MNP) lineage to visualize the ontogeny of the lymphoid tissue, macrophages and dendritic cells, in the trachea, lung and air sac of birds from embryonic day 18-63 weeks of age. Small aggregates of CSF1R-transgene+ cells start to form at the openings of the secondary bronchi at 1 week of age, indicative of the early development of the organised bronchus-associated lymphoid tissue. Immunohistochemical staining revealed subpopulations of MNPs in the lung, based on expression of CSF1R-transgene, CD11, TIM4, LAMP1, and MHC II. Specialised epithelial cells or M cells covering the bronchus-associated lymphoid tissue expressed CSF1R-transgene and type II pneumocytes expressed LAMP1 suggesting that these epithelial cells are phagocytic and transcytose antigen. Highly organised lymphoid tissue was seen in trachea from 4 weeks onwards. Throughout the air sacs at all ages, CSF1R-transgene+ cells were scattered and at later stages, CSF1R-transgene+ cells lined capillaries. These results will serve as a base for further functional characterization of macrophages and dendritic cells and their role in respiratory diseases and vaccine responses.

Detection of somatic mutations in tumors using unaligned clonal sequencing data.

Most cancers arise and evolve as a consequence of somatic mutations. These mutations influence tumor behavior and clinical outcome. Consequently, there is considerable interest in identifying somatic variants within specific genes (such as BRAF, KRAS and EGFR) so that chemotherapy can be tailored to the patient's tumor genotype rather than using a generic treatment based on histological diagnosis alone. Owing to the heterogeneous nature of tumors, a somatic mutation may be present in only a subset of cells, necessitating the use of quantitative techniques to detect rare variants. The highly quantitative nature of next-generation sequencing (NGS), together with the ability to multiplex numerous samples, makes NGS an attractive choice with which to screen for somatic variants. However, the large volumes of sequence data present significant difficulties when applying NGS for the detection of somatic mutations. To alleviate this, we have developed methodologies including a set of data analysis programs, which allow the rapid screening of multiple formalin-fixed, paraffin-embedded samples for the presence of specified somatic variants using unaligned Illumina NGS data.

Distribution and spatiotemporal development of organised lymphoid tissues in the chicken intestinal tract.

Chickens exhibit a distinct immune architecture characterised by the absence of draining lymph nodes and the presence of a well-developed mucosa-associated lymphoid tissue. The structure and spatiotemporal development of chicken lymphoid tissues in the intestine are poorly documented. The macroscopically indistinct structure of chicken Peyer's patches has impeded studies into their development. The generation of CSF1R-eGFP reporter transgenic chickens enables visualisation of the development, organisation and extent of chicken lymphoid tissues by unique macroscopic views. CSF1R-eGFP reporter transgenic chickens were used to investigate the distribution and spatiotemporal development of PP and caecal tonsils in embryonic day 18 to 8-week-old chickens. Peyer's patch anlagen are present at ED18 with a similar frequency and distribution pattern observed in 2- and 8-week-old chickens. These findings can support in ovo and post-hatch mucosal vaccination strategies and the development of vaccine delivery systems targeted to the specialized epithelium overlying the Peyer's patches.

Delineation of chicken immune markers in the era of omics and multicolor flow cytometry.

Multiparameter flow cytometry is a routine method in immunological studies incorporated in biomedical, veterinary, agricultural, and wildlife research and routinely used in veterinary clinical laboratories. Its use in the diagnostics of poultry diseases is still limited, but due to the continuous expansion of reagents and cost reductions, this may change in the near future. Although the structure and function of the avian immune system show commonalities with mammals, at the molecular level, there is often low homology across species. The cross-reactivity of mammalian immunological reagents is therefore low, but nevertheless, the list of reagents to study chicken immune cells is increasing. Recent improvement in multicolor antibody panels for chicken cells has resulted in more detailed analysis by flow cytometry and has allowed the discovery of novel leukocyte cell subpopulations. In this article, we present an overview of the reagents and guidance needed to perform multicolor flow cytometry using chicken samples and common pitfalls to avoid.

Discrimination of distinct chicken M cell subsets based on CSF1R expression.

In mammals, a subset of follicle-associated epithelial (FAE) cells, known as M cells, conduct the transcytosis of antigens across the epithelium into the underlying lymphoid tissues. We previously revealed that M cells in the FAE of the chicken lung, bursa of Fabricius (bursa), and caecum based on the expression of CSF1R. Here, we applied RNA-seq analysis on highly enriched CSF1R-expressing bursal M cells to investigate their transcriptome and identify novel chicken M cell-associated genes. Our data show that, like mammalian M cells, those in the FAE of the chicken bursa also express SOX8, MARCKSL1, TNFAIP2 and PRNP. Immunohistochemical analysis also confirmed the expression of SOX8 in CSF1R-expressing cells in the lung, bursa, and caecum. However, we found that many other mammalian M cell-associated genes such as SPIB and GP2 were not expressed by chicken M cells or represented in the chicken genome. Instead, we show bursal M cells express high levels of related genes such as SPI1. Whereas our data show that bursal M cells expressed CSF1R-highly, the M cells in the small intestine lacked CSF1R and both expressed SOX8. This study offers insights into the transcriptome of chicken M cells, revealing the expression of CSF1R in M cells is tissue-specific.

Chicken intestinal organoids: a novel method to measure the mode of action of feed additives.

There is a rapidly growing interest in how the avian intestine is affected by dietary components and feed additives. The paucity of physiologically relevant models has limited research in this field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The development of complex 3D intestinal organoids or "mini-guts" has created ample opportunities for poultry research in this field. A major advantage of the floating chicken intestinal organoids is the combination of a complex cell system with an easily accessible apical-out orientation grown in a simple culture medium without an extracellular matrix. The objective was to investigate the impact of a commercial proprietary blend of organic acids and essential oils (OA+EO) on the innate immune responses and kinome of chicken intestinal organoids in a Salmonella challenge model. To mimic the in vivo prolonged exposure of the intestine to the product, the intestinal organoids were treated for 2 days with 0.5 or 0.25 mg/mL OA+EO and either uninfected or infected with Salmonella and bacterial load in the organoids was quantified at 3 hours post infection. The bacteria were also treated with OA+EO for 1 day prior to challenge of the organoids to mimic intestinal exposure. The treatment of the organoids with OA+EO resulted in a significant decrease in the bacterial load compared to untreated infected organoids. The expression of 88 innate immune genes was investigated using a high throughput qPCR array, measuring the expression of 88 innate immune genes. Salmonella invasion of the untreated intestinal organoids resulted in a significant increase in the expression of inflammatory cytokine and chemokines as well as genes involved in intracellular signaling. In contrast, when the organoids were treated with OA+EO and challenged with Salmonella, the inflammatory responses were significantly downregulated. The kinome array data suggested decreased phosphorylation elicited by the OA+EO with Salmonella in agreement with the gene expression data sets. This study demonstrates that the in vitro chicken intestinal organoids are a new tool to measure the effect of the feed additives in a bacterial challenge model by measuring innate immune and protein kinases responses.

Phenotypic and genomic characterisation of performance of tropically adapted chickens raised in smallholder farm conditions in Ethiopia.

Background: In sub-Saharan Africa, 80% of poultry production is on smallholder village farms, where chickens are typically reared outdoors in free-ranging conditions. There is limited knowledge on chickens' phenotypic characteristics and genetics under these conditions. Objective: The present is a large-scale study set out to phenotypically characterise the performance of tropically adapted commercial chickens in typical smallholder farm conditions, and to examine the genetic profile of chicken phenotypes associated with growth, meat production, immunity, and survival. Methods: A total of 2,573 T451A dual-purpose Sasso chickens kept outdoors in emulated free-ranging conditions at the poultry facility of the International Livestock Research Institute in Addis Ababa, Ethiopia, were included in the study. The chickens were raised in five equally sized batches and were individually monitored and phenotyped from the age of 56 days for 8 weeks. Individual chicken data collected included weekly body weight, growth rate, body and breast meat weight at slaughter, Newcastle Disease Virus (NDV) titres and intestinal Immunoglobulin A (IgA) levels recorded at the beginning and the end of the period of study, and survival rate during the same period. Genotyping by sequencing was performed on all chickens using a low-coverage and imputation approach. Chicken phenotypes and genotypes were combined in genomic association analyses. Results: We discovered that the chickens were phenotypically diverse, with extensive variance levels observed in all traits. Batch number and sex of the chicken significantly affected the studied phenotypes. Following quality assurance, genotypes consisted of 2.9 million Single Nucleotide Polymorphism markers that were used in the genomic analyses. Results revealed a largely polygenic mode of genetic control of all phenotypic traits. Nevertheless, 15 distinct markers were identified that were significantly associated with growth, carcass traits, NDV titres, IgA levels, and chicken survival. These markers were located in regions harbouring relevant annotated genes. Conclusion: Results suggest that performance of chickens raised under smallholder farm conditions is amenable to genetic improvement and may inform selective breeding programmes for enhanced chicken productivity in sub-Saharan Africa.