The altered TBI fecal microbiome is stable and functionally distinct.

Introduction: Patients who suffer a traumatic brain injury (TBI) often experience chronic and sometimes debilitating sequelae. Recent reports have illustrated both acute and long-term dysbiosis of the gastrointestinal microbiome with significant alterations in composition and predicted functional consequences. Methods: Working with participants from past research, metagenomic stability of the TBI- associated fecal microbiome (FMB) was evaluated by custom qPCR array comparing a fecal sample from 2015 to one collected in 2020. Metatranscriptomics identified differently expressed bacterial genes and biochemical pathways in the TBI FMB. Microbiota that contributed the largest RNA amounts identified a set of core bacteria most responsible for functional consequences of the TBI FMB. Results: A remarkably stable FMB metagenome with significant similarity (two-tail Spearman nonparametric correlation p < 0.001) was observed between 2015 and 2020 fecal samples from subjects with TBI. Comparing the 2020 TBI FMB metagenome to FMBs from healthy controls confirmed and extended the dysbiotic genera and species. Abundance differences between average TBI and healthy FMBs revealed Bacteroides caccae, B. uniformis, Blautia spp., Collinsella spp., Dialister spp., and Ordoribacter spp. were significantly different. Functionally, the Parabacteroides genus contributed the highest percentage of RNA sequences in control FMBs followed by the Bacteroides genus as the second highest contributor. In the TBI FMB, the Corynebacterium genus contributed the most RNA followed by the Alistipes genus. Corynebacterium and Pseudomonas were distinct in the top 10 contributing genera in the TBI FMB while Parabacteroides and Ruminococcus were unique to the top 10 in controls. Comparing RNA profiles, TBI samples had ∼1.5 fold more expressed genes with almost 700 differently expressed genes (DEGs) mapped to over 100 bacterial species. Bioinformatic analysis associated DEGs with pathways led identifying 311 functions in the average TBI FMB profile and 264 in the controls. By average profile comparison, 30 pathways had significantly different abundance (p < 0.05, t-test) or were detected in >80% of the samples in only one of the cohorts (binary distinction). Discussion: Functional differences between TBI and healthy control FMBs included amino acid metabolism, energy and carbon source usage, fatty acid metabolism, bacterial cell wall component production and nucleic acid synthesis and processing pathways. Together these data shed light on the functional consequences of the dysbiotic TBI FMB decades after injury.

Claudin-2 Expression Levels in Ulcerative Colitis: Development and Validation of an In-Situ Hybridisation Assay for Therapeutic Studies.

Ulcerative colitis is a chronic inflammatory disease affecting the colon and is characterized by epithelial damage and barrier dysfunction. Upregulation of the tight junction protein claudin-2 by cytokines is hypothesized to contribute to the dysregulation of the epithelial barrier. New therapeutic agents which block the action of cytokines are being investigated in patients with ulcerative colitis. In order to understand the potential of these therapies, it is important to have reliable assays that can assess downstream endpoints that reflect drug mechanism of action. The aim of the current study was therefore to establish & validate an assay to reproducibly assess the expression and distribution of claudin-2 in human colon biopsy samples. Initially, the potential to measure claudin-2 protein by immunohistochemistry (IHC) was investigated. To identify suitable reagents to develop an IHC assay, pre-established criteria were used to screen five commercial antibodies by Western blotting, immunofluorescence and immunohistochemistry on claudin-2 positive and negative cells and healthy and ulcerative colitis colon tissue. Despite some of these antibodies specifically detecting claudin-2 using some of these techniques, none of the antibodies showed the expected specific staining pattern in formalin fixed human colon samples. As an alternative method to detect claudin-2 expression and distribution in formalin fixed biopsy sections, an in situ hybridization assay was developed. This assay underwent a novel tiered approach of validation to establish that it was fit-for-purpose, and suitable for clinical deployment. In addition, to understand the possible relationship of claudin-2 in the context of disease severity, expression was compared to the Geboes score. Overall, the microscopical Geboes score correlated with the claudin-2 biomarker score for samples that retained crypt morphology; samples with the highest Geboes score were not specifically distinguished, probably due to crypt destruction. In summary, we have applied a strategy for identifying target-specific antibodies in formalin fixed biopsy samples and highlighted that (published) antibodies may not correctly identify the intended antigen in tissues fixed using this method. Furthermore, we have developed and, for the first time, validated an in situ hybridization assay for detection of claudin-2 mRNA, suitable for use as a supportative method in clinical trials. Using our validated assay, we have demonstrated that increased claudin-2 expression correlates with the severity of ulcerative colitis, where crypt destruction is not seen.

Troglitazone metabolism and transporter effects in chimeric mice: a comparison between chimeric humanized and chimeric murinized FRG mice.

1. The biotransformation, hepatic transporter and blood chemistry effects of troglitazone were investigated following 7 days of dosing at 600 mg/kg/day to chimeric murinized or humanized FRG mice, Mo-FRG and Hu-FRG mice, respectively. 2. Clinical chemistry and histopathology analysis revealed a significant drop in humanization over the time course of the study for the Hu-FRG mice but no significant changes associated with troglitazone treatment in either the Mo-FRG or the Hu-FRG models. No changes in transporter expression in livers of these mice were observed. Oxidative and conjugative metabolic pathways were identified with a 15- to 18-fold increase in formation of troglitazone sulfate in the Hu-FRG mice compared with the Mo-FRG mice in blood and bile, respectively. This resembles the troglitazone metabolism in human and these data are comparable with the formation of this metabolite in the chimeric uPA(+/+)/SCID mice. 3. However, larger amounts of troglitazone glucuronide were also observed in the Hu-FRG mouse compared with the Mo-FRG mouse which may be an effect of the drop in humanization of the Hu-FRG mouse during the study. 4. Highly humanized mice have a considerable potential in providing a useful first insight into circulating human metabolites of candidate drugs metabolized in the liver.

Determination of the safety and efficacy of therapeutic neutralization of tumor necrosis factor-α (TNF-α) using AZD9773, an anti-TNF-α immune Fab, in murine CLP sepsis.

OBJECTIVE AND DESIGN: TNF-α neutralization is associated with increased mortality in mouse cecal ligation puncture (CLP) models. AZD9773 is an ovine polyclonal human TNF-α immune Fab, with pharmacological properties that differ from previously studied anti-TNF-α agents. We explored the safety and efficacy of therapeutically administered AZD9773 in mouse CLP sepsis. METHODS: A moderate/severe-grade CLP model resulting in 20-30 % 5-day survival and a mild-grade CLP model resulting in ~70 % 5-day survival were established in human TNF-α transgene/murine TNF null (Tg1278/-/-) mice. TREATMENT: Mice received saline resuscitation and imipenem administration every 12 h (0-72 h post-CLP). AZD9773 (or DigiFab control) was dosed 24, 36, 48 and 60 h post-CLP. RESULTS: Therapeutic dosing of AZD9773 in moderate/severe-grade CLP resulted in significantly increased survival (>70 %) compared with DigiFab (27 %, P < 0.05). Therapeutic dosing of AZD9773 in mild-grade CLP did not significantly affect survival outcome compared with DigiFab or imipenem alone (~60-70 % survival). CONCLUSIONS: These data demonstrate that TNF-α neutralization can improve survival in moderate/severe CLP sepsis. TNF-α suppression in mild-grade models was not associated with survival benefit and did not increase 5-day mortality. These findings suggest that therapeutic benefit following TNF-α attenuation in models of sepsis may depend on model severity.

Quantitative histopathological assessment of retardation of islets of langerhans degeneration in rosiglitazone-dosed obese ZDF rats using combined insulin and collagens (I and III) immunohistochemistry with automated image analysis and statistical modeling.

Islets of Langerhans represent a heterogeneous population in insulin resistant and diabetic animals and humans as histological appearances and function vary substantially. Mathematical representation that reflects this morphological diversity will assist in assessment of degeneration and regeneration, enabling comparisons between species, strains, and experimental investigations. Our investigative approach used a model of islet degeneration in diabetic male obese Zucker Diabetic Fatty (ZDF) rats and evaluated its prevention using rosiglitazone treatment. Immunohistochemical staining (insulin and collagens I/III) with automated image analysis reliably measured numbers, area, clustering, and staining intensity of ß-cells and degree of islet fibrosis. Finite mixture mathematical modeling for the joint probability distribution of seven islet parameters to represent islet numerical data variation provided an automatic procedure for islet category allocations as normal or abnormal. Allocations for obese ZDF controls and rosiglitazone-treated animals were significantly different, with no significant difference between the latter and lean ZDF controls, indicative of differences within islet populations of individual animals, between lean and obese rat strains and following drug treatment. Islet morphology showed clear association with mathematical characterization. Information on islet morphology obtained by histopathological assessment of single pancreatic tissue sections was confirmed by this method showing drug-induced retardation of islet of Langerhans degeneration.

Explant culture of gastrointestinal tissue: a review of methods and applications.

The gastrointestinal (GI) tract is an important target organ for the toxicity of xenobiotics. The toxic effects of xenobiotics on this complex, heterogeneous structure have been difficult to model in vitro and have traditionally been assessed in vivo. The explant culture of GI tissue offers an alternative approach. Historically, the organotypic culture of the GI tract proved far more challenging than the culture of other tissues, and it was not until the late 1960s that Browning and Trier described the means by which intestinal tissues could be successfully cultured. This breakthrough provided a tool researchers could utilise, and adapt, to investigate topics such as the pathogenesis of inflammatory intestinal diseases, the effect of growth factors and cytokines on intestinal proliferation and differentiation, and the testing of novel xenobiotics for efficacy and safety. This review considers that intestinal explant culture shows much potential for the application of a relatively under-used procedure in future biomedical research. Furthermore, there appear to be many instances where the technique may provide experimental solutions where both cell culture and in vivo models have been unable to deliver conclusive and convincing findings.

The development of an immunohistochemical method to detect the autophagy-associated protein LC3-II in human tumor xenografts.

Autophagy is believed to be an important process during tumorgenesis, and in recent years it has been shown to be modulated in response to a number of conventional anticancer agents. Furthermore, the development of targeted small molecule inhibitors, such as those to the PI3K-AKT-mTOR pathway, has presented a molecular link between the disruption of this signalling cascade and the process of autophagy. The cellular consequence of stimulating or inhibiting autophagy in cancer cells is not completely understood, so it is important that this process be monitored, along with antiproliferative and apoptotic biomarkers, in the preclinical setting. The field of autophagy is still evolving, and there is a constantly changing set of criteria for the assessment of the process in cells, tissues, and organs. The gold standard technique for analyzing autophagy in mammalian cells remains transmission electron microscopy, which has many limitations and is often difficult to perform on in vivo tissue including human tumor xenografts. In order to monitor autophagy in human tumor xenogaft tissue, we have taken the approach to develop an immunohistochemical (IHC) method for the detection of the autophagosome-associated protein, microtubule-associated protein 1 light chain 3 (LC3), in human tumor xenografts. After synthesis, LC3 is cleaved to form LC3-I, and upon induction of autophagy, LC3-I is conjugated to the lipid phosphatidylethanolamine to form LC3-II, which is tightly bound to the membrane of the autophagosome. It is thought that detection of endogenous LC3-II by IHC could be difficult because of the relatively low level of expression of the protein. Here we present the validation of an IHC method to detect LC3 in human tumor xenografts that we believe is able to distinguish LC3-I from LC3-II. It is hoped that this assay can become a useful tool for the detection of autophagy in preclinical xenograft models and determine the effects of anticancer therapies on the autophagic process.

Time-course study of the immunotoxic effects of the anticancer drug chlorambucil in the rat.

In 2005, the International conference on harmonization (ICH) recommended that all new human pharmaceuticals be tested for unintended immunomodulatory potential via a tiered approach. Included in this approach is a semiquantitative description of changes in the separate compartments of lymphoid tissue (also called enhanced histopathology). Chlorambucil was administered to Hanover Wistar rats at regular time points, followed by a treatment-free (recovery) period. Groups of treated and control animals were sacrificed regularly during both the treatment and recovery periods. Selected tissues were removed, weighed fresh and fixed in formalin, processed, and stained with hematoxylin and eosin. Blood samples and bone marrow smears were also obtained. With the use of enhanced histopathology, a description of the changes in lymphoid tissues and bone marrow was used as a means of assessing the susceptibility, and recovery, of the different lymphoid cell populations over time. A correlation with organ weights, flow cytometry data, and bone marrow cytology was achieved. The administration of chlorambucil in the Hanover Wistar rat provided a useful tool to examine the rate and sequence of changes in the lymphoid organs and bone marrow during treatment with, and the recovery from the effects of, a potent immunosuppressive agent.

Urinary retention and cystitis associated with subcutaneous estradiol pellets in female nude mice.

Unexpected deaths occurred in studies involving a nude mouse model of mammary cancer that required subcutaneous implantation of 0.5 mg twenty-one-day release estrogen pellets for growth of the estrogen-dependent mammary tumor xenograft BT474c. Early deaths occurred in female nude mice and were associated with urinary retention, frequently with cystitis. Drug treatment had no effect on the incidence or severity of cystitis. Histological findings did not alter significantly over various time points following pellet implantation. Changes were not seen in males or in females receiving lower doses of estradiol even when the duration of administration was prolonged, suggesting that a threshold level was required for the onset of urinary retention. Because of the influence of estrogen on micturition, immunohistochemistry for estrogen receptor alpha (ERalpha) in the urinary bladder was carried out, which did not demonstrate any differences between females implanted with 0.5 mg twenty-one-day release estrogen pellets and nonimplanted females. Although previous publications have concentrated on possible mechanisms of action, this paper describes the histopathological changes seen in the urinary bladder of female nude mice resulting from exposure to high levels of estradiol.

A dual-label technique for the immunohistochemical demonstration of T-lymphocyte subsets in formalin-fixed, paraffin-embedded rat lymphoid tissue.

Immunotoxicology has developed into an integral regulatory requirement of the toxicological assessment of xenobiotics. Histopathological assessment of lymphoid tissues can provide genuine insight into perturbations of lymphoid cell populations. To facilitate retrospective examination of lymphoid organs should concerns over immunotoxicity be raised, we have endeavored to develop a panel of immunohistochemical techniques to demonstrate T-cells and T-cell subsets in formalin-fixed, paraffin-embedded rat lymphoid tissues. We were successful in developing methods for CD3 and CD8 but failed to arrive at a satisfactory technique for the direct demonstration of CD4 in these tissues. Taking the assumption that the majority of mature T-cells are either CD4+ or CD8+, we have combined our methods for CD3 and CD8 in a novel dual-labeling IHC method to simultaneously demonstrate CD3, CD8, and, by implication, CD4 in rat spleen, thymus, lymph node, and Peyer's patch.

Rosuvastatin: characterization of induced myopathy in the rat.

Rosuvastatin is a relatively new member of the statin family (HMG-CoA reductase inhibitors), with superior lipid-lowering effects and a pattern of clinical side effects, including a low incidence of myopathy, similar to other widely prescribed statins. This article describes investigations of myopathy in the rat following administration of very high doses of rosuvastatin. The nature of the changes were found to be entirely consistent with those seen with other statins, including a differential sensitivity of muscle fibers (with glycolytic fibers [type IIB] the most sensitive and oxidative fibers [type I] the least), a delay of approximately 10 days after the start of oral dosing before necrosis was apparent, and ultrastructural alterations appearing first in mitochondria. In addition, the development of myopathy was prevented by coadministration of mevalonate, the product of HMG-CoA reductase. The findings illustrate a pattern of induced myopathy in the rat directly attributable to inhibition of HMG-CoA reductase that is entirely consistent between the various statins, with the oral dose required to produce the changes being a differentiating feature (based on these new data and a previously reported study from the same laboratory): cerivastatin dose less than simvastatin, and simvastatin dose less than rosuvastatin.

The demonstration of immunohistochemical biomarkers in methyl methacrylate-embedded plucked human hair follicles.

Plucked human hair follicles have been proposed as a potential surrogate for tumour tissue for measuring the effect of drugs on pharmacodynamic biomarkers in drug intervention studies. We describe a new technique of embedding plucked hair follicles in the acrylic resin, methyl methacrylate, and the immunohistochemical demonstration of six potential biomarkers (Ki67, EGFR, phospho-p27, phospho-histone H3, phospho-MAPK and phospho-Rb) in de-plasticised sections. The advantages of this technique over those that have been used in support of clinical drug trials, such as skin and tumour biopsies, whole blood and whole hair samples is discussed.

Statin-induced muscle necrosis in the rat: distribution, development, and fibre selectivity.

Simvastatin and cerivastatin have been used to investigate the development of statin-induced muscle necrosis in the rat. This was similar for both statins and was treatment-duration dependent, only occurring after 10 days had elapsed even if the dose was increased, and still occurring after this time when dosing was terminated earlier as a result of morbidity. It was then widespread and affected all areas of the muscular system. However, even when myotoxicity was severe, particular individual muscles and some types of fibres within affected muscles were spared consistently. Fibre typing of spared muscles and of acutely necrotic fibres within affected muscles indicated a differential fibre sensitivity to statin-induced muscle necrosis. The fibres showed a necrotic response to statin administration that matched their oxidative/glycolytic metabolic nature: Least sensitive --> I < - > IIA < - > IID < - > IIB <-- most sensitive. Type I and IIB fibres represent metabolic extremes of a continuum of metabolic properties through the fibre types with type I fibres most oxidative in metabolism and type IIB fibres most glycolytic. In addition, in some (nonnecrotic) glycolytic fibres from muscles showing early multifocal single fibre necrosis the only subcellular alterations present in isolation of any other changes were mitochondrial. These changes were characterised by an increased incidence of vacuolation and the formation of myelinoid vesicular bodies that accumulated in the subsarcolemmal areas. These findings suggest an important early involvement of mitochondria in selective glycolytic muscle fibre necrosis following inhibition of the enzyme HMG-CoA reductase.