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Background The relationship between intestinal epithelial integrity and the development of intestinal disease is of increasing interest. A reduction in mucosal integrity has been associated with ulcerative colitis, Crohn’s disease and potentially could have links with colorectal cancer development. The Ussing chamber system can be utilised as a valuable tool for measuring gut integrity. Here we describe step-by-step methodology required to measure intestinal permeability of both mouse and human colonic tissue samples ex vivo, using the latest equipment and software. This system can be modified to accommodate other tissues. Methods An Ussing chamber was constructed and adapted to support both mouse and human tissue to measure intestinal permeability, using paracellular flux and electrical measurements. Two mouse models of intestinal inflammation (dextran sodium sulphate treatment and T regulatory cell depletion using C57BL/6-FoxP3 DTR mice) were used to validate the system along with human colonic biopsy samples. Results Distinct regional differences in permeability were consistently identified within mouse and healthy human colon. In particular, mice showed increased permeability in the mid colonic region. In humans the left colon is more permeable than the right. Furthermore, inflammatory conditions induced chemically or due to autoimmunity reduced intestinal integrity, validating the use of the system. Conclusions The Ussing chamber has been used for many years to measure barrier function. However, a clear and informative methods paper describing the setup of modern equipment and step-by-step procedure to measure mouse and human intestinal permeability isn’t available. The Ussing chamber system methodology we describe provides such detail to guide investigation of gut integrity.

Background The landscape of cancer treatment has evolved rapidly within the last 50 years, and whilst radiotherapy, chemotherapy, and surgery remain the mainstay treatment options, there has been a shift towards using immunotherapy alone or in combination with other treatment modalities. There is an emerging paradigm that radiotherapy is immunogenic, driving stimulation of antigen-specific T cells capable of recognising tumour cells at distal sites to the treatment location. Methods Whole blood samples were collected from patients with primary and oligometastatic cancer before, during, and after treatment with stereotactic ablative radiotherapy (SABR). Using clinical full blood counts, multiparameter flow cytometry, Luminex, and ELISpot assays, this study explored the impact of SABR on systemic immune cell composition, inflammatory markers, and antigen-specific T cell responses. Results We identified striking systemic changes collectively indicating profound SABR-driven immunosuppression. Such changes were characterised by pronounced and sustained lymphopenia which included loss of CD4 + and CD8 + T cells, B cells, and natural killer (NK) cells accompanied by an overall decline in effector T cell responses to common recall and cancer antigens. This loss of lymphocytes drove a rise in the neutrophil-to-lymphocyte ratio (NLR), which was associated with poorer progression-free survival (PFS) if increased from baseline. A higher dosage of radiation and treatment to a larger area were both associated with more pronounced lymphocyte loss, a concomitant NLR increase, and poorer PFS, particularly in individuals with liver lesions. Conclusions These findings support a role for lymphocytes in preventing disease progression after SABR and suggest that a change to clinical practice to spare lymphocytes from the toxic effects of irradiation may have beneficial effects for patients.

A wealth of evidence obtained using mouse models indicates that CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) maintain peripheral tolerance to self-antigens and also inhibit anti-tumor immune responses. To date there is limited information about CD4(+) T cell responses in patients with colorectal cancer (CRC). We set out to measure T cell responses to a tumor-associated antigen and examine whether Treg impinge on those anti-tumor immune responses in CRC patients. Treg were identified and characterized as CD4(+)CD25(+)FOXP3(+) using flow cytometry. An increased frequency of Treg was demonstrated in both peripheral blood and mesenteric lymph nodes of patients with colorectal cancer (CRC) compared with either healthy controls or patients with inflammatory bowel disease (IBD). Depletion of Treg from peripheral blood mononuclear cells (PBMC) of CRC patients unmasked CD4(+) T cell responses, as observed by IFNgamma release, to the tumor associated antigen 5T4, whereas no effect was observed in a healthy age-matched control group. Collectively, these data demonstrate that Treg capable of inhibiting tumor associated antigen-specific immune responses are enriched in patients with CRC. These results support a rationale for manipulating Treg to enhance cancer immunotherapy.

Currently, our understanding of mechanisms underlying cell-mediated immunity and particularly of mechanisms that promote robust T cell memory to respiratory viruses is incomplete. Interleukin (IL)-6 has recently re-emerged as an important regulator of T cell proliferation and survival. Since IL-6 is abundant following infection with influenza virus, we analyzed virus-specific T cell activity in both wild type and IL-6 deficient mice. Studies outlined herein highlight a novel role for IL-6 in the development of T cell memory to influenza virus. Specifically, we find that CD4+ but not CD8+ T cell memory is critically dependent upon IL-6. This effect of IL-6 includes its ability to suppress CD4+CD25+ regulatory T cells (Treg). We demonstrate that influenza-induced IL-6 limits the activity of virus-specific Tregs, thereby facilitating the activity of virus-specific memory CD4+ T cells. These experiments reveal a critical role for IL-6 in ensuring, within the timeframe of an acute infection with a cytopathic virus, that antigen-specific Tregs have no opportunity to down-modulate the immune response, thereby favoring pathogen clearance and survival of the host.

There is an established link between the inflammation process and the genesis of colorectal cancer. A recent study suggests that intestinal microbiota may have a mediatory role between the initial steps of tumorigenesis and the initiation of an immune response that influences clinical outcome. Colorectal cancer is a common cancer that leads to 600,000 deaths globally each year. The progression of colonic polyps from dysplastic adenomas to adenocarcinomas is defined histologically, in part, by invasion of the submucosal tissue through the muscularis mucosae. This progression is mirrored by a recognized series of mutations along a genetic pathway from the initial loss of the adenomatous polyposis coli ( APC ) tumor-suppressor gene to the activation of mutations in genes such as KRAS, PIK3CA, and TP53 . A synthesis of several aspects of the pathogenesis of colorectal cancer — epithelial genetic mutations, mucosal integrity, microbiota, and . . .

CD200 receptor (CD200R) negatively regulates peripheral and mucosal innate immune responses. Viruses, including herpesviruses, have acquired functional CD200 orthologs, implying that viral exploitation of this pathway is evolutionary advantageous. However, the role that CD200R signaling plays during herpesvirus infection in vivo requires clarification. Utilizing the murine cytomegalovirus (MCMV) model, we demonstrate that CD200R facilitates virus persistence within mucosal tissue. Specifically, MCMV infection of CD200R-deficient mice (CD200R(-/-)) elicited heightened mucosal virus-specific CD4 T cell responses that restricted virus persistence in the salivary glands. CD200R did not directly inhibit lymphocyte effector function. Instead, CD200R(-/-) mice exhibited enhanced APC accumulation that in the mucosa was a consequence of elevated cellular proliferation. Although MCMV does not encode an obvious CD200 homolog, productive replication in macrophages induced expression of cellular CD200. CD200 from hematopoietic and non-hematopoietic cells contributed independently to suppression of antiviral control in vivo. These results highlight the CD200-CD200R pathway as an important regulator of antiviral immunity during cytomegalovirus infection that is exploited by MCMV to establish chronicity within mucosal tissue.

Objective There is evidence that natural killer (NK) cells help control persistent viral infections including hepatitis C virus (HCV). The phenotype and function of blood and intrahepatic NK cells, in steady state and after interferon (IFN) α treatment has not been fully elucidated. Design We performed a comparison of NK cells derived from blood and intrahepatic compartments in multiple paired samples from patients with a variety of chronic liver diseases. Furthermore, we obtained serial paired samples from an average of five time points in HCV patients treated with IFNα. Results Liver NK cells demonstrate a distinct activated phenotype compared to blood manifested as downregulation of the NK cell activation receptors CD16, NKG2D, and NKp30; with increased spontaneous degranulation and IFN production. In contrast, NKp46 expression was not downregulated. Indeed, NKp46-rich NK populations were the most activated, correlating closely with the severity of liver inflammation. Following initiation of IFNα treatment there was a significant increase in the proportion of intrahepatic NK cells at days 1 and 3. NKp46-rich NK populations demonstrated no reserve activation capacity with IFNα treatment and were associated with poor viral control on treatment and treatment failure. Conclusions NKp46 marks out pathologically activated NK cells, which may result from a loss of homeostatic control of activating receptor expression in HCV. Paradoxically these pathological NK cells do not appear to be involved in viral control in IFNα-treated individuals and, indeed, predict slower rates of viral clearance.

Objective There is evidence that natural killer (NK) cells help control persistent viral infections including hepatitis C virus (HCV). The phenotype and function of blood and intrahepatic NK cells, in steady state and after interferon (IFN) α treatment has not been fully elucidated. Design We performed a comparison of NK cells derived from blood and intrahepatic compartments in multiple paired samples from patients with a variety of chronic liver diseases. Furthermore, we obtained serial paired samples from an average of five time points in HCV patients treated with IFNα. Results Liver NK cells demonstrate a distinct activated phenotype compared to blood manifested as downregulation of the NK cell activation receptors CD16, NKG2D, and NKp30; with increased spontaneous degranulation and IFN production. In contrast, NKp46 expression was not downregulated. Indeed, NKp46-rich NK populations were the most activated, correlating closely with the severity of liver inflammation. Following initiation of IFNα treatment there was a significant increase in the proportion of intrahepatic NK cells at days 1 and 3. NKp46-rich NK populations demonstrated no reserve activation capacity with IFNα treatment and were associated with poor viral control on treatment and treatment failure. Conclusions NKp46 marks out pathologically activated NK cells, which may result from a loss of homeostatic control of activating receptor expression in HCV. Paradoxically these pathological NK cells do not appear to be involved in viral control in IFNα-treated individuals and, indeed, predict slower rates of viral clearance.

The inherent resistance of cancer stem cells (CSCs) to existing therapies has largely hampered the development of effective treatments for advanced malignancy. To help develop novel immunotherapy approaches that efficiently target CSCs, an experimental model allowing reliable distinction of CSCs and non‐CSCs was set up to study their interaction with non‐MHC‐restricted γδ T cells and antigen‐specific CD8+ T cells. Stable lines with characteristics of breast CSC‐like cells were generated from ras‐transformed human mammary epithelial (HMLER) cells as confirmed by their CD44hi CD24lo GD2+ phenotype, their mesenchymal morphology in culture and their capacity to form mammospheres under non‐adherent conditions, as well as their potent tumorigenicity, self‐renewal and differentiation in xenografted mice. The resistance of CSC‐like cells to γδ T cells could be overcome by inhibition of farnesyl pyrophosphate synthase (FPPS) through pretreatment with zoledronate or with FPPS‐targeting short hairpin RNA. γδ T cells induced upregulation of MHC class I and CD54/ICAM‐1 on CSC‐like cells and thereby increased the susceptibility to antigen‐specific killing by CD8+ T cells. Alternatively, γδ T‐cell responses could be specifically directed against CSC‐like cells using the humanised anti‐GD2 monoclonal antibody hu14.18K322A. Our findings identify a powerful synergism between MHC‐restricted and non‐MHC‐restricted T cells in the eradication of cancer cells including breast CSCs. Our research suggests that novel immunotherapies may benefit from a two‐pronged approach combining γδ T‐cell and CD8+ T‐cell targeting strategies that triggers effective innate‐like and tumour‐specific adaptive responses.

Defolliculated (Gsdma3Dfl/+) mice have a hair loss phenotype that involves an aberrant hair cycle, altered sebaceous gland differentiation with reduced sebum production, chronic inflammation, and ultimately the loss of the hair follicle. Hair loss in these mice is similar to that seen in primary cicatricial, or scarring alopecias in which immune targeting of hair follicle stem cells has been proposed as a key factor resulting in permanent hair follicle destruction. In this study we examine the mechanism of hair loss in GsdmA3Dfl/+ mice. Aberrant expression patterns of stem cell markers during the hair cycle, in addition to aberrant behavior of the melanocytes leading to ectopic pigmentation of the hair follicle and epidermis, indicated the stem cell niche was not maintained. An autoimmune mechanism was excluded by crossing the mice with rag1-/- mice. However, large numbers of macrophages and increased expression of ICAM-1 were still present and may be involved either directly or indirectly in the hair loss. Reverse transcriptase-PCR (RT-PCR) and immunohistochemistry of sebaceous gland differentiation markers revealed reduced peroxisome proliferator-activated receptor-γ (PPARγ), a potential cause of reduced sebum production, as well as the potential involvement of the innate immune system in the hair loss. As reduced PPARγ expression has recently been implicated as a cause for lichen planopilaris, these mice may be useful for testing therapies.