Catherine Booth

Acute gastrointestinal syndrome in high-dose irradiated mice.

Abstract The most detailed reports of the response of the gastrointestinal system to high dose acute radiation have focused mainly on understanding the histopathology. However, to enable medical countermeasure assessment under the animal rule criteria, it is necessary to have a robust model in which the relationship between radiation dose and intestinal radiation syndrome incidence, timing, and severity are established and correlated with histopathology. Although many mortality studies have been published, they have used a variety of mouse strains, ages, radiation sources, and husbandry conditions, all of which influence the dose response. Further, it is clear that the level of bone marrow irradiation and supportive care can influence endpoints. In order to create robust baseline data, the authors have generated dose response data in adult male mice maintained under identical conditions and exposed to either total or partial-body irradiation. Partial-body irradiation includes both extensive (40%) and minimal (5%) bone marrow sparing models, the latter designed to correlate with an established primate model and allow assessment of effects of any medical countermeasure on all three major radiation syndromes (intestinal, bone marrow, and lung) in the surviving mice. Lethal dose (LD30, LD50, and LD70) data are described in the various models, along with the impact of enteric flora and response to supportive care. Correlation with diarrhea severity and histopathology are also described. These data can be used to aid the design of good laboratory practice (GLP)-compliant Animal Rule studies that are reflective of the conditions following accidental radiation exposure.

Development of non-antibiotic macrolide that corrects inflammation-driven immune dysfunction in models of inflammatory bowel diseases and arthritis.

Inflammation-driven immune dysfunction supports the development of several chronic human disorders including inflammatory bowel diseases and rheumatoid arthritis. Macrolides are effective antibiotics endowed with immunomodulatory effects. In this study we report the chemical synthesis and the pharmacological characterization of CSY0073, a non-antibiotic derivative of azithromycin. CSY0073 was tested for efficacy in two experimental models of colitis induced by administering mice with dextran sulfate (DSS) and trinitrobenzene sulphonic acid (TNBS) and in collagen induced arthritis. Like azithromycin, CSY0073 improved clinical, macroscopic and histopathological scores in mice administered DSS (12.5μmol/kg/day p.o.) and TNBS (45μmol/kg/day p.o.). When administered to TNBS-treated mice, CSY0073 effectively attenuated influx of neutrophils and macrophages into the colonic mucosa and reduced the intestinal expression pro-inflammatory cytokines TNFα, IL-2 and IFNγ. CSY0073 (0.1 to 10μM) counter-regulated TNFα, IFNγ, IL-12 and IL-23 release caused by exposure of mouse spleen monocytes and CD11b+ cells isolated from the colonic lamina propria to endotoxin. CSY0073 (25μmol/kg/day) reduced clinical scores in the collagen induced murine model of rheumatoid arthritis. In myeloid cells, CSY0073 (10μM) prevented the nuclear translocation of the p65 subunit of NF-κB and its binding to canonical NF-κB responsive elements. In summary, we report a novel class of non-antibiotic 14-member macrocycles with anti-inflammatory and immune-modulatory effects. CSY0073, the prototype of this new class of macrolides exerts counter-regulatory activity on NF-κB signaling. This study suggests the exploitation of non-antibiotic macrolides in the treatment of inflammatory disorders characterized by immune dysfunction.

Evidence of delayed gastrointestinal syndrome in high-dose irradiated mice.

Abstract The acute effects of irradiation on the gastrointestinal (GI) system are well documented, but the longer-term effects are less well known. Increased incidence of adenocarcinoma has been noted, but apart from descriptions of fibrosis, the development of other pathologies specific to survivors of acute radiation is poorly understood. Samples were taken from C57BL/6 mice irradiated with partial-body irradiation where the thorax, head, and forelimbs were shielded (i.e., sparing 40% of the bone marrow). Tissue from age-matched controls was also collected. There were clear pathological changes in the intestine associated with DEARE (Delayed Effects of Acute Radiation Exposure) at doses greater than 12 Gy, with a dose-related increase in observed pathologies. Mice maintained on the synthetic antibiotic ciprofloxacin during the acute phase (days 4 to 20), however, had a lower or delayed incidence of symptoms. After 20 d, mice developed structures similar to early adenomas. Abnormally high levels of apoptotic and mitotic cells were present in some crypts, along with the early adenomas, suggesting tissue regeneration and areas of deregulated cell turnover. Over time, there was inhibited crypt cell proliferation in animals with advanced symptoms, a blunting of the crypts and villi, and an enlargement of villus girth, with an increasingly acellular and fibrotic extracellular matrix (a characteristic that has been demonstrated previously in aging mice). Together these changes may lead to a reduced functional surface area and less motile intestine. These observations are similar to those seen in geriatric animals, suggesting a premature aging of the GI tract.

HIV enteropathy: HAART reduces HIV-induced stem cell hyperproliferation and crypt hypertrophy to normal in jejunal mucosa

Objective To analyse the structural and kinetic response of small intestinal crypt epithelial cells including stem cells to highly active antiretroviral therapy (HAART). Design Crypt size and proliferative activity of transit and stem cells in jejunal mucosa were quantified using morphometric techniques. Methods Crypt length was measured by counting the number of enterocytes along one side of a number of crypts in each biopsy specimen and the mean crypt length was calculated. Proliferating crypt cells were identified with MIB-1 monoclonal antibody, and the percentage of crypt cells in proliferation was calculated at each cell position along the length of the crypt (proliferation index). Data were obtained from 9 HIV-positive test patients co-infected with microsporidia, 34 HIV-positive patients receiving HAART and 13 control cases. Results Crypt length was significantly greater in test patients than in controls, but crypt length in patients receiving HAART was normal. The proliferation index was greater in test subjects than in controls in stem and transit cell compartments, and was decreased in patients treated with HAART only in the stem cell region of the crypt. Conclusions Villous atrophy in HIV enteropathy is attributed to crypt hypertrophy and encroachment of crypt cells onto villi. HAART restores normal crypt structure by inhibition of HIV-driven stem cell hyperproliferation at the crypt bases.

The Delayed Effects of Acute Radiation Syndrome: Evidence of Long-Term Functional Changes in the Clonogenic Cells of the Small Intestine.

AbstractLong term or residual damage post-irradiation has been described for many tissues. In hematopoietic stem cells (HSC), this is only revealed when the HSC are stressed and required to regenerate and repopulate a myeloablated host. Such an assay cannot be used to assess the recovery potential of previously irradiated intestinal stem cells (ISC) due to their incompatibility with transplantation. The best approximation to the HSC assay is the crypt microcolony assay, also based on clonogen survival. In the current study, the regenerative capacity of intestinal clonogenic cells in mice that had survived 13 Gy irradiation (with 5% bone marrow shielding to allow survival through the hematopoietic syndrome) and were then aged for 200 d was compared to previously unirradiated age-matched controls. Interestingly, at 200 d following 13 Gy, there remained a statistically significant reduction in crypts present in the various small intestinal regions (illustrating that the gastrointestinal epithelium had not fully recovered despite the 200‐d interval). However, upon re-irradiation on day 196, those mice previously irradiated had improved crypt survival and regeneration compared to the age-matched controls. This was evident in all regions of the small intestine following 11–13 Gy re-exposure. Thus, there were either more clonogens per crypt within those previously irradiated and/or those that were present were more radioresistant (possibly because a subpopulation was more quiescent). This is contrary to the popular belief that previously irradiated animals may have an impaired/delayed regenerative response and be more radiosensitive.

The Intestine as a Model for Studying Stem-Cell Behavior

The intestine is lined by a simple columnar epithelium that folds into a number of cavities—the crypts of Lieberkuhn—embedded in the connective tissue. In the small intestine, there are also finger-like protrusions known as villi, which are covered in this epithelium. The villi are approximately ten times larger than the crypts, but much less common (4–7× fewer, depending upon the intestinal region) (1). The functional cells are located on the villus (or toward the top of the crypts of the large intestine), and are continuously sloughed off into the lumen. These cells are replaced by massive cell production within the bottom two-thirds of the crypt, where cells in the mouse are dividing approx twice daily. This crypt region is therefore frequently referred to as the proliferative zone.

Abstract 96: A novel in vivo preclinical model for the study of human tumor angiogenesis with human vessels

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The preclinical assessment of anti-angiogenic drug efficacy is hampered by the lack of availability of suitable model systems to study anti-human therapies. Good in vitro assays exist for testing anti-human-specific therapies but these are let down by the lack of robust translatable in vivo models. Currently available preclinical models such as the matrigel plug assay and the window chamber assay focus on the angiogenesis of mouse vessels which may not be truly reflective of the influence of test items on human vasculature, and in vivo models to study the angiogenesis of human tumors by human vessels are unfortunately lacking. We have developed a sophisticated model to address this using adult human skin engrafted onto immunocompromised CB17-SCID mice. Angiogenesis of the skin follows, with human and host vessels anastomosing, resulting in a viable skin graft containing functioning human CD31-labelled vessels. This skin graft can be intradermally injected with a cell line of interest, such as the human melanoma cell line A375, and the ensuing tumors contain human vessels as evidenced by immunohistochemistry with a human-specific anti-CD31 antibody. Using the Aperio® Scanscope and FDA-approved microvessel analysis algorithm, 5-10 human vessels per mm2 of human tumor are observed. Furthermore, the model has the potential to be used in combination with non-invasive in vivo imaging techniques to assess tumor growth inhibition in response to therapies in a longitudinal fashion. This novel technique could potentially enable the rapid preclinical testing of anti-angiogenic anti-human therapies to help provide a go/no-go decision prior to costly clinical trials. Citation Format: Kirsty A. Holden, Michael A. Batey, Martyn Bottomley, Catherine Booth. A novel in vivo preclinical model for the study of human tumor angiogenesis with human vessels. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 96. doi:10.1158/1538-7445.AM2014-96

Abstract 1345: The use of optical imaging in a clinically relevant mouse intrafemoral model of leukaemia

One of the challenges facing preclinical development in non solid tumours, such as leukaemia, is the development and validation of clinically relevant models, both to better understand the biology of disease, and to provide reliable efficacy testing for novel agents. In conventional animal models of leukaemia, cells are implanted using intravenous injection, leading to inconsistent engraftment as cells are not introduced directly to their biological niche. Disease burden is measured using either terminal endpoints, such as hind limb paralysis, or by fluorescence activated cell sorting on bone marrow or peripheral blood samples. Neither of these strategies make it practical to identify animals in the early stages of leukemic disease development, limiting investigators’ ability to examine novel therapies in these models. The use of preclinical imaging strategies, such as in vivo optical imaging, allows disease progression to be followed at orthotopic sites and, in the case of non solid tumours, to monitor systemic disease burden longitudinally in an individual animal over time. Here we describe the use of an intrafemoral (i.f.) transplantation method, in which cells are implanted directly into the biological niche of interest, correlating with the environment in which disease develops in patients. Luciferase-labelled human leukaemia cell lines were injected into the bone marrow cavity of the femur of severely immunocompromised NOG (NOD/Shi-scid/IL-2Ry null ) mice via the knee joint. Animals were followed over a period of several weeks from initial transplantation, and disease burden monitored and quantified using bioluminescent detection. This model consistently gives rise to disease disseminating gradually from the site of initial transplantation into systemic leukaemia. The ability to quickly identify engraftment and the predictable development of disease allows for therapeutic intervention at an early stage and extends the window available for therapy. The model provides a clinically relevant environment for the preclinical assessment of novel therapeutics for non solid tumours. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1345. doi:1538-7445.AM2012-1345

Abstract 3763: Development of a plucked hair immunohistochemisty biomarker platform

Background Plucked hair is a valuable surrogate tissue to monitor pharmacodynamic (PD) responses to treatment. Hair collection is minimally invasive, simple and permits frequent sampling. We have previously demonstrated the feasibility, and utility of monitoring mRNA expression in individual plucked human scalp hairs to correlate targeted gene expression changes in response to a range of oncology treatments. RNA analysis permits the examination of downstream events resulting from changes in target activation. To directly monitor target response to treatment, immunohistochemistry (IHC) protocols have now been developed. Methods Scalp hairs were plucked from healthy, normal volunteers (aged 18-42 yrs) and fixed and processed for IHC. IHC protocols were developed for whole mount and sectioned hairs (using fluorescence and DAB detection methods) for a range of markers of drug action, including markers of cell proliferation and growth factor receptor phosphorylation. Levels of inter- and intra-donor variation were also assessed by labeling for Ki67 and analyzing 7 anagen hairs from each donor. Once labeled, hairs were imaged and the label quantified by a range of methods, including manual counting of labeled nuclei and semi-automated analysis of number of labeled nuclei by Image-Pro Plus® software. Results Results showed that for a variety of antigens in plucked hair, the IHC method developed was a reproducible technique for both whole mounts and sectioned hair. One standard protocol could be used for each antibody, with adjustments made to primary antibody concentrations and the region of the hair analyzed. From each set of 7 scalp hairs approximately 5 were suitable for analysis of IHC labeling. The number of cells labeled for Ki67 was quantified manually from 10 volunteers (5 hairs per donor) in a 0.4mm length of the outer root sheath and this gave a percentage variation of 38% and 23% for all volunteers and male only volunteers, respectively. This inter-donor variation was higher than the intra-donor variation which was 27% and 20% for all volunteers and male only volunteers, respectively. This analysis was performed in parallel using Image-Pro Plus® which has the advantage of increased speed of analysis and removal of potential error introduced by using different human scorers. Values were comparative with manual counting with inter-donor variation of 37% (all donors), 19% (only male donors), and intra variation of 29% (all donors) and 23% (only male donors). Hairs processed 6 weeks after fixation retained 60-80% of original antigenicity dependant on the antigen analyzed. Conclusion The results presented demonstrate that IHC labeling and quantification protocols can be developed for plucked human hairs. The range of inter- and intra-donor variation was established. This provides a simple, acceptable technique for collecting and analyzing surrogate epithelial tissue to monitor direct cellular response to drug treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3763.

Abstract 5546: Improved gastrointestinal repair post irradiation with AEOL 10150

Background Gastrointestinal toxicity, often manifested as ulceration and diarrhoea (mucositis) occurs following exposure to radiation and many chemotherapeutics. Potential treatments include prophylactic damage limitation or therapeutic/mitigatory wound repair dosing regimes. Treatments that are effective mitigators also have biodefence applications. In both the clinic or following nuclear terrorism effective agents may be given in combination with haematological therapies, in addition to standard supportive care agents. Methods AEOL 10150 [5,10,15,20-tetrakis(1,3-diethylimidazolium-2-yl) porphyrinato] manganese (III) pentachloride was administered to C57BL/6 male mice s.c. daily from 24hrs following a single dose of 13Gy total body X-irradiation. AEOL 10150 was administered at a 2x initial loading dose of 40 or 20mg/kg and then subsequently each day at 20 or 10mg/kg respectively. Animals were euthanized 4 and 6 days post irradiation and the number of regenerating small intestinal crypts measured. In a parallel study the levels diarrhoea and animal viability were measured animals exposed to 15Gy partial body X-irradiation (partially lead shielded to protect the bone marrow). Results Treatment with AEOL 10150 increased the number of surviving regenerating crypt observed in histological cross sections of the small intestine 4 days post irradiation. This was highly significant at both doses (p Conclusion AEOL 10150 demonstrated efficacy in both histological and physiological readouts of radiation induced gastrointestinal toxicity. Studies are currently ongoing to investigate whether other dosing schedules can further improve gastrointestinal repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5546.