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.

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.

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.

Disentangling the innate immune responses of intestinal epithelial cells and lamina propria cells to Salmonella Typhimurium infection in chickens.

Salmonella enterica serovar Typhimurium (STm) is a major foodborne pathogen and poultry are a key reservoir of human infections. To understand the host responses to early stages of Salmonella infection in poultry, we infected 2D and 3D enteroids, the latter of which contains leukocytes, neurons, and mesenchymal cells that are characteristic of the lamina propria. We infected these enteroids with wild-type (WT STm), a non-invasive mutant lacking the prgH gene (ΔprgH STm), or treated them with STm lipopolysaccharide (LPS) and analyzed the expression of innate immune related genes by qPCR at 4 and 8 h. The localization of the tight junction protein, ZO-1, expression was disrupted in WT STm infected enteroids but not ΔprgH STm or LPS treated enteroids, suggesting a loss of epithelial barrier integrity. The innate immune response to LPS was more pronounced in 2D enteroids compared to 3D enteroids and by 8 hpi, the response in 3D enteroids was almost negligible. However, when STm adhered to or invaded the enteroids, both 2D and 3D enteroids exhibited an upregulation of inflammatory responses. The presence of lamina propria cells in 3D enteroids resulted in the unique expression of genes associated with immune functions involved in regulating inflammation. Moreover, 2D and 3D enteroids showed temporal differences in response to bacterial invasion or adherence. At 8 hpi, innate responses in 3D but not 2D enteroids continued to increase after infection with WT STm, whereas the responses to the non-invasive strain decreased at 8 hpi in both 2D and 3D enteroids. In conclusion, STm infection of chicken enteroids recapitulated several observations from in vivo studies of Salmonella-infected chickens, including altered epithelial barrier integrity based on ZO-1 expression and inflammatory responses. Our findings provide evidence that Salmonella-infected enteroids serve as effective models for investigating host-pathogen interactions and exploring the molecular mechanisms of microbial virulence although the 3D model mimics the host more accurately due to the presence of a lamina propria.

Malignant neoplasm of lacrimal gland with pulmonary metastasis.

Adenoid cystic carcinomas (ACC) make up 3%-5% of head and neck malignancies. They have a high propensity to metastasise, in particular to the lungs. A 65-year-old male diagnosed with a right lacrimal gland ACC T2N0M0 (surgically resected 12 years prior) presented with an incidentally noted 1.2 cm right lower lobe lung nodule seen on MRI liver. Subsequent imaging confirmed a non-FDG avid 1.6 cm solitary ovoid subpleural lesion, percutaneous biopsy confirmed adenocarcinoma. A surgical metastasectomy was performed and recovery was complete. Prognosis in ACC is improved with radical management of metastatic disease. Rather than a simple chest radiograph, more detailed imaging, such as MRI or CT scanning may increase the probability of early detection of pulmonary metastasis and, thereby facilitate radical treatment and improve survival.

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.

Chicken CSF2 and IL-4-, and CSF2-dependent bone marrow cultures differentiate into macrophages over time.

Chicken bone marrow-derived macrophages (BMMΦ) and dendritic cells (BMDC) are utilized as models to study the mononuclear phagocytic system (MPS). A widely used method to generate macrophages and DC in vitro is to culture bone marrow cells in the presence of colony-stimulating factor-1 (CSF1) to differentiate BMMΦ and granulocyte-macrophage-CSF (GM-CSF, CSF2) and interleukin-4 (IL-4) to differentiate BMDC, while CSF2 alone can lead to the development of granulocyte-macrophage-CSF-derived DC (GMDC). However, in chickens, the MPS cell lineages and their functions represented by these cultures are poorly understood. Here, we decipher the phenotypical, functional and transcriptional differences between chicken BMMΦ and BMDC along with examining differences in DC cultures grown in the absence of IL-4 on days 2, 4, 6 and 8 of culture. BMMΦ cultures develop into a morphologically homogenous cell population in contrast to the BMDC and GMDC cultures, which produce morphologically heterogeneous cell cultures. At a phenotypical level, all cultures contained similar cell percentages and expression levels of MHCII, CD11c and CSF1R-transgene, whilst MRC1L-B expression decreased over time in BMMΦ. All cultures were efficiently able to uptake 0.5 µm beads, but poorly phagocytosed 1 µm beads. Little difference was observed in the kinetics of phagosomal acidification across the cultures on each day of analysis. Temporal transcriptomic analysis indicated that all cultures expressed high levels of CSF3R, MERTK, SEPP1, SPI1 and TLR4, genes associated with macrophages in mammals. In contrast, low levels of FLT3, XCR1 and CAMD1, genes associated with DC, were expressed at day 2 in BMDC and GMDC after which expression levels decreased. Collectively, chicken CSF2 + IL-4- and CSF2-dependent BM cultures represent cells of the macrophage lineage rather than inducing conventional DC.

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.

Antigen Sampling CSF1R-Expressing Epithelial Cells Are the Functional Equivalents of Mammalian M Cells in the Avian Follicle-Associated Epithelium.

The follicle-associated epithelium (FAE) is a specialized structure that samples luminal antigens and transports them into mucosa-associated lymphoid tissues (MALT). In mammals, transcytosis of antigens across the gut epithelium is performed by a subset of FAE cells known as M cells. Here we show that colony-stimulating factor 1 receptor (CSF1R) is expressed by a subset of cells in the avian bursa of Fabricius FAE. Expression was initially detected using a CSF1R-reporter transgene that also label subsets of bursal macrophages. Immunohistochemical detection using a specific monoclonal antibody confirmed abundant expression of CSF1R on the basolateral membrane of FAE cells. CSF1R-transgene expressing bursal FAE cells were enriched for expression of markers previously reported as putative M cell markers, including annexin A10 and CD44. They were further distinguished from a population of CSF1R-transgene negative epithelial cells within FAE by high apical F-actin expression and differential staining with the lectins jacalin, PHA-L and SNA. Bursal FAE cells that express the CSF1R-reporter transgene were responsible for the bulk of FAE transcytosis of labeled microparticles in the size range 0.02-0.1 µm. Unlike mammalian M cells, they did not readily take up larger bacterial sized microparticles (0.5 µm). Their role in uptake of bacteria was tested using Salmonella, which can enter via M cells in mammals. Labeled Salmonella enterica serovar Typhimurium entered bursal tissue via the FAE. Entry was partially dependent upon Type III secretion system-1. However, the majority of invading bacteria were localized to CSF1R-negative FAE cells and in resident phagocytes that express the phosphatidylserine receptor TIM4. CSF1R-expressing FAE cells in infected follicles showed evidence of cell death and shedding into the bursal lumen. In mammals, CSF1R expression in the gut is restricted to macrophages which only indirectly control M cell differentiation. The novel expression of CSF1R in birds suggests that these functional equivalents to mammalian M cells may have different ontological origins and their development and function are likely to be regulated by different growth factors.

Avian Pathogenic Escherichia coli (APEC) Strain-Dependent Immunomodulation of Respiratory Granulocytes and Mononuclear Phagocytes in CSF1R-Reporter Transgenic Chickens.

Avian pathogenic Escherichia coli (APEC) cause severe respiratory and systemic disease in chickens, commonly termed colibacillosis. Early immune responses after initial infection are highly important for the outcome of the infection. In this study, the early interactions between GFP-expressing APEC strains of serotypes O1:K1:H7 and O2:K1:H5 and phagocytic cells in the lung of CSF1R-reporter transgenic chickens were investigated. CSF1R-reporter transgenic chickens express fluorescent protein under the control of elements of the CSF1R promoter and enhancer, such that cells of the myeloid lineage can be visualized in situ and sorted. Chickens were separately inoculated with APEC strains expressing GFP and culled 6 h post-infection. Flow cytometric analysis was performed to phenotype and sort the cells that harbored bacteria in the lung, and the response of the sorted cells was defined by transcriptomic analysis. Both APEC strains were mainly detected in CSF1R-transgeneneg (CSF1R-tgneg) and CSF1R-tglow MHC IIneg MRC1L-Bneg cells and low numbers of APEC were detected in CSF1R-tghigh MHC IIpos MRC1L-Bpos cells. Transcriptomic and flow cytometric analysis identified the APECposCSF1R-tgneg and CSF1R-tglow cells as heterophils and the APECposCSF1R-tghigh cells as macrophages and dendritic cells. Both APEC strains induced strong inflammatory responses, however in both CSF1R-tgneg/low and CSF1R-tghigh cells, many immune related pathways were repressed to a greater extent or less activated in birds inoculated with APEC O2-GFP compared to APEC O1-GFP inoculated birds. Comparison of the immune pathways revealed the aryl hydrocarbon receptor (AhR) pathway, IL17 and STAT3 signaling, heterophil recruitment pathways and the acute phase response, are modulated particularly post-APEC O2-GFP inoculation. In contrast to in vivo data, APEC O2-GFP was more invasive in CSF1R-tghigh cells in vitro than APEC O1-GFP and had higher survival rates for up to 6 h post-infection. Our data indicate significant differences in the responses induced by APEC strains of prevalent serotypes, with important implications for the design and interpretation of future studies. Moreover, we show that bacterial invasion and survival in phagocyte populations in vitro is not predictive of events in the chicken lung.

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.

The functions of the avian receptor activator of NF-κB ligand (RANKL) and its receptors, RANK and osteoprotegerin, are evolutionarily conserved.

A new member of the chicken TNF superfamily has recently been identified, namely receptor activator of NF-κB ligand (RANKL), as have its signalling receptor, RANK, and its decoy receptor, osteoprotegerin (OPG). In mammals, RANKL and RANK are transmembrane proteins expressed on the surface of Th1 cells and dendritic cells (DC) respectively, whereas OPG is expressed as a soluble protein from osteoblasts and DC. Recombinant soluble chicken RANKL (chRANKL) forms homotrimers whereas chicken OPG (chOPG) forms homodimers, characteristic of these molecules in mammals. ChRANKL, chRANK and chOPG are expressed at the mRNA level in most tissues and organs. ChRANKL is transcriptionally regulated by Ca(2+) mobilisation and enhances the mRNA expression levels of pro-inflammatory cytokines in bone marrow-derived DC (BMDC); this is inhibited by both chOPG-Fc and soluble chRANK-Fc. However, chRANKL does not enhance the expression of cell surface markers in either BMDC or BM-derived macrophages (BMM). Furthermore, chRANKL enhances the survival of APC similar to its mammalian orthologue.

Assessment of the feasibility and clinical value of further research to evaluate the management options for children with Down syndrome and otitis media with effusion: a feasibility study.

BACKGROUND: There is clinical uncertainty of the benefits and costs of different treatment options for children with Down syndrome who have glue ear. This study was designed to assess the extent of this lack of knowledge and determine if pursuing further information would be practical, beneficial and cost-effective. OBJECTIVES: To assess the level and practical effect of current uncertainty around treatment options for children with Down syndrome and glue ear. To assess the feasibility of studying the options for management of glue ear in children with Down syndrome via a randomised controlled trial (RCT) or multicentre prospective cohort study by evaluating the willingness of (1) parents to agree to randomisation for their children and (2) clinicians to recruit participants to a definitive study. To undertake value of information analyses to demonstrate the potential economic benefit from undertaking further research. DESIGN: A feasibility study exploring the views of parents of children with Down syndrome and professionals who have responsibility for the health and education of children with Down syndrome, on the participation in, and value of, future research into interventions for glue ear. Data were collected from parents via self-completed questionnaires, face-to-face interviews and focus groups and from professionals via online questionnaires and a Delphi review exercise. Development of economic models to represent clinical pathways of care and a RCT informed a value of information (VOI) analysis. SETTING: UK (professionals); East Midlands region of the UK (parents). PARTICIPANTS: Parents of children aged 1-11 years with Down syndrome (n = 156). Professionals including audiologists, ear, nose and throat surgeons, audiological physicians, speech and language therapists, and teachers of the deaf (n = 128). MAIN OUTCOME MEASURES: Quantitative and qualitative data on parental views and experiences of glue ear and its effects; interventions and treatment received; taking part in research and factors that would encourage or discourage participation; and the importance of various outcome domains to them and for their children. For professionals: information on caseloads; approaches to clinical management; opinions on frequency and significance of the consequences of glue ear for this population; importance of different outcome measures; opinions of interventions and their role in future research; views on health research; facilitators and barriers to recruitment, and participation in research involving RCTs. RESULTS: The complexity of the experience and individual characteristics of children with Down syndrome poses challenges for the design of any future research but these challenges were not considered by professionals to raise sufficient barriers to prevent it being undertaken. Parents were generally supportive of the need for, and value of, research but identified practical and emotional issues that would need addressing. Glue ear was considered to impact more on speech, language and communication than on hearing. Outcome measures for future research would need to evaluate these elements but measures should be designed specifically for the population. Parents and professionals identified randomisation as a significant barrier to participation. The VOI analyses identified lack of data as problematic but concluded that a future trial involving surgical intervention would be feasible at costs of < £650,000. CONCLUSIONS: Future research into the benefits of interventions for glue ear in children with Down syndrome would be feasible and could be cost-effective but should be carefully designed to facilitate and maximise participation from parents and professionals responsible for recruitment. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

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.

Accurately identifying low-allelic fraction variants in single samples with next-generation sequencing: applications in tumor subclone resolution.

Current methods for resolving genetically distinct subclones in tumor samples require somatic mutations to be clustered by allelic frequencies, which are determined by applying a variant calling program to next-generation sequencing data. Such programs were developed to accurately distinguish true polymorphisms and somatic mutations from the artifactual nonreference alleles introduced during library preparation and sequencing. However, numerous variant callers exist with no clear indication of the best performer for subclonal analysis, in which the accuracy of the assigned variant frequency is as important as correctly indicating whether the variant is present or not. Furthermore, sequencing depth (the number of times that a genomic position is sequenced) affects the ability to detect low-allelic fraction variants and accurately assign their allele frequencies. We created two synthetic sequencing datasets, and sequenced real KRAS amplicons, with variants spiked in at specific ratios, to assess which caller performs best in terms of both variant detection and assignment of allelic frequencies. We also assessed the sequencing depths required to detect low-allelic fraction variants. We found that VarScan2 performed best overall with sequencing depths of 100×, 250×, 500×, and 1,000× required to accurately identify variants present at 10%, 5%, 2.5%, and 1%, respectively.

Mutation detection by clonal sequencing of PCR amplicons and grouped read typing is applicable to clinical diagnostics.

We describe a sensitive technique for mutation detection using clonal sequencing. We analyzed DNA extracted from 13 cancer cell lines and 35 tumor samples and applied a novel approach to identify disease-associated somatic mutations. By matching reads against an index of known variants, noise can be dramatically reduced, enabling the detection and quantification of those variants, even when they are present at less than 1% of the total sequenced population; this is comparable to, or better than, current diagnostic methods. Following the identification or exclusion of known variants, unmatched reads are grouped for BLAST searching to identify novel variants or contaminants. Known variants, novel variants, and contaminants were readily identified in tumor tissue using this approach. Our approach also enables an estimation of the per-base sequencing error rate, providing a confidence threshold for interpretation of the results in the clinic. This novel approach has immediate applicability to clinical testing for disease-associated genetic variants.

Diffuse noxious inhibitory control function in women with provoked vestibulodynia.

OBJECTIVES: The objective of the study was to assess diffuse noxious inhibitory control (DNIC) function in women with provoked vestibulodynia (PVD) compared with healthy controls through the use of 2 different methodologies. Furthermore, the study aimed to assess whether pain characteristics correlate with DNIC in women with PVD. METHODS: Twenty-three healthy control women and 23 women diagnosed with PVD by the study gynecologist participated in the study. To assess DNIC, heat pain tolerance, determined through an ascending method of limits and temporal summation of thermal pain were used as test stimuli and a cold water bath was used as the conditioning stimulus. Participants reported on pain characteristics as potential correlates with DNIC function. RESULTS: No significant group differences were found in the number of DNIC responders per group when using heat pain tolerance or temporal summation procedures to examine DNIC. The magnitude of the DNIC response was examined for the overall groups and for positive DNIC responders only. When all participants were included in the analyses with the heat pain tolerance procedure, women with PVD displayed a higher magnitude of DNIC responding. Correlations between pain variables and DNIC responding and magnitude were nonsignificant. DISCUSSION: Results support previous findings of intact DNIC function in women with PVD, using both an ascending method of limits and a temporal summation paradigm. Pain-related variables were not correlated with DNIC function in women with PVD, perhaps this unexpected finding is due to the possibility that central processes other than DNIC, such as descending facilitation, provoke or maintain this chronic pain condition.

To lube or not to lube: experiences and perceptions of lubricant use in women with and without dyspareunia.

INTRODUCTION: There are few studies examining the relationship between lubricant use and sexual functioning, and no studies have examined this relationship in women with dyspareunia. Vaginal dryness is a prevalent complaint among women of all ages. There is an association between vaginal dryness and painful intercourse; therefore, women with dyspareunia represent a particularly relevant sample of women in which to investigate lubricant use. AIM: The aim of this study was to examine differences between women with and without dyspareunia in self-reported natural lubrication and attitudes toward and use of personal lubricants. METHODS: Respondents completed an online survey including questions on demographics, gynecological/medical history, sexual functioning, and lubricant use and attitudes. MAIN OUTCOME MEASURES: The main outcome measures used were the Female Sexual Function Index (FSFI) and questions regarding attitudes toward and use of lubricants. RESULTS: Controls scored higher on the lubrication subscale of the FSFI than women with dyspareunia (P < 0.001). Women with dyspareunia reported greater frequency of lubricant use during sexual activity over the last year (P < 0.01). They were also more likely to use lubricant prior to penetration (P < 0.05). The most common use for controls was to enhance sexual experiences. This was also a common answer for women with dyspareunia; however, in this group, the most common reason was to reduce/alleviate pain. Lubricants were rated as less effective among women with dyspareunia vs. controls across all reported reasons for use. Nevertheless, lubricant use was still rated as being moderately effective in alleviating pain for women with dyspareunia. CONCLUSIONS: Women with dyspareunia have more difficulty with natural lubrication; it is consequently not surprising that they reported using lubricant more frequently than control women. Women with dyspareunia reported using lubricants more often than controls to try to prevent or alleviate pain and reported this as being a moderately effective strategy, suggesting that it may be a useful tool for some women with dyspareunia.

Improving the quality of colon cancer surgery through a surgical education program.

PURPOSE: Recent evidence has demonstrated the importance of dissection in the correct tissue plane for the resection of colon cancer. We have previously shown that meticulous mesocolic plane surgery yields better outcomes and that the addition of central vascular ligation produces an oncologically superior specimen compared with standard techniques. We aimed to assess the effect of surgical education on the oncological quality of the resection specimen produced. METHODS: We received clinicopathological data and specimen photographs from 263 resections for primary colon cancer from 6 hospitals in the Capital and Zealand regions of Denmark before a national training program. Ninety-three cases were from Hillerød Hospital, where surgeons had previously implemented a surgical educational training program in complete mesocolic excision with central vascular ligation and adopted the procedure as standard practice. The specimen photographs were assessed for the plane of surgery and tissue morphometry was performed. RESULTS: Hillerød specimens had a higher rate of mesocolic plane surgery (75% vs 48%; P < .0001) compared with the other hospitals. The surgeons at Hillerød Hospital also removed a greater length of colon in both fresh (median, 315 vs 247 mm; P < .0001) and fixed (269 vs 207 mm; P < .0001) specimens with a greater distance between the tumor and the closest vascular tie in both fresh (105 vs 84 mm; P = .006) and fixed (82 vs 67 mm; P = .002) specimens. This resulted in the removal of more mesentery in both fresh (14,466 vs 8706 mm; P < .0001) and fixed (9418 vs 6789 mm; P < .0001) specimens and a greater median lymph node yield (28 vs 18; P < .0001). CONCLUSIONS: We have shown that adoption of complete mesocolic excision with central vascular ligation results in a change to the production of an oncologically superior specimen compared with standard techniques. This should improve outcomes toward those reported by centers that have long practiced meticulous colon cancer surgery.

Ceramic materials as bearing surfaces for total hip arthroplasty.

During the past decade, advances in total hip arthroplasty component design have produced implants with reliable clinical results in regard to fixation. The foremost unresolved challenge has been the development of bearing surfaces that can withstand the higher demands of younger and more active patients. New alternative bearings with superior wear characteristics that minimize debris include ceramic-on-ceramic, metal-on-metal, and highly cross-linked polyethylenes in combination with ceramic or metal. Alumina-on-alumina ceramic bearings are extremely hard and scratch resistant and provide superior lubrication and wear resistance compared with other bearing surfaces in clinical use. Survivorship revision for any reason for the alumina ceramic bearings at 10 years was significantly higher compared with metal-on-polyethylene. Bearings currently being studied because of their encouraging wear performance in the laboratory are an alumina matrix (82% alumina, 17% zirconia, 0.3% chromium oxide), zirconium oxide, and ceramic-on-cobalt-chromium.