Irradiation-Induced Dysbiosis: The Compounding Effect of High-Fat Diet on Metabolic and Immune Functions in Mice.

The negative impact of irradiation or diet on the metabolic and immune profiles of cancer survivors have been previously demonstrated. The gut microbiota plays a critical role in regulating these functions and is highly sensitive to cancer therapies. The aim of this study was to investigate the effect of irradiation and diet on the gut microbiota and metabolic or immune functions. We exposed C57Bl/6J mice to a single dose of 6 Gy radiation and after 5 weeks, fed them a chow or high-fat diet (HFD) for 12 weeks. We characterised their faecal microbiota, metabolic (whole body and adipose tissue) functions, and systemic (multiplex cytokine, chemokine assay, and immune cell profiling) and adipose tissue inflammatory profiles (immune cell profiling). At the end of the study, we observed a compounding effect of irradiation and diet on the metabolic and immune profiles of adipose tissue, with exposed mice fed a HFD displaying a greater inflammatory signature and impaired metabolism. Mice fed a HFD also showed altered microbiota, irrespective of irradiation status. An altered diet may exacerbate the detrimental effects of irradiation on both the metabolic and inflammatory profiles. This could have implications for the diagnosis and prevention of metabolic complications in cancer survivors exposed to radiation.

Formation and Glomerular Deposition of Immune Complexes in Mice Administered Human Antibodies: Evaluation of Dose, Frequency, and Biomarkers.

Administration of human protein-based drugs to animals often leads to formation of antidrug antibodies (ADAs) that may form circulating immune complexes (CICs) with the dosed protein. Circulating immune complexes can activate and bind complement (cCICs), and if large amount of CICs or cCICs is formed, the clearance mechanism potentially becomes saturated, which can lead to immune complex (IC) deposition and inflammation. To obtain a better understanding of the underlying factors, including the relationship between different dose regimes on IC formation and deposition and identification of possible biomarkers of IC deposition and IC-related pathological changes in kidneys, BALB/c and C57BL/6J mice were administered with human anti-tumor necrosis factor α (aTNFα, adalimumab) or a humanized anti-TNP (aTNP) antibody for 13 weeks. Particularly, ADA, CIC, cCIC formation, IC deposition, and glomerulonephritis were observed in C57BL/6J administered with aTNFα, whereas the immunologic response was minor in BALB/c mice administered with aTNFα and in BALB/c and C57BL/6J mice administered aTNP. Changing dose levels or increasing dosing frequency of aTNFα on top of an already-established CIC and cCIC response did not lead to substantial changes in CIC, cCIC formation, or IC deposition. Finally, no association between the presence of CICs or cCIC in plasma and glomerular IC deposition and/or glomerulonephritis was observed.

Formation and glomerular deposition of immune complexes in mice administered bovine serum albumin: Evaluation of dose, frequency, and biomarkers.

In preclinical toxicity studies, species-foreign proteins administered to animals frequently leads to formation of anti-drug antibodies (ADA). Such antibodies may form circulating immune complexes (CIC) with the administered protein. These CIC can activate the classical complement pathway, thereby forming complement-bound CIC (cCIC); if large of amounts of CIC or cCIC is formed, the clearance mechanism may become saturated which potentially leads to vascular immune complex (IC) deposition and inflammation. Limited information is available on the effect of different treatment related procedures as well as biomarkers of IC-related vascular disease. In order to explore the effect of different dose regimens on IC formation and deposition, and identification of possible biomarkers of IC deposition and IC-related pathological changes, C57BL/6J and BALB/c mice were dosed subcutaneously twice weekly with bovine serum albumin (BSA) for 13 weeks without adjuvant. After 6 and 13 weeks, CIC and cCIC were detected in plasma; after 13 weeks, IC deposition was detected in kidney glomeruli. In particular immunohistochemistry double-staining was shown to be useful for detection of IC deposition. Increasing dosing frequency or changing BSA dose level on top of an already established CIC and cCIC response did not cause changes in IC deposition, but CIC and cCIC concentrations tended to decrease with increased dose level, and increased cCIC formation was observed after more frequent dosing. The presence of CIC in plasma was associated with glomerular IC deposits in the dose regimen study; however, the use of CIC or cCIC as potential biomarkers for IC deposition and IC-related pathological changes, needs to be explored further.

An ELISA for detection of complement-bound circulating immune complexes in mice.

Measurements of complement-bound circulating immune complexes (cCICs) in pre-clinical studies may provide important information about the etiology of certain pathology findings suggestive of being immune complex mediated. This article describes the development and qualification of a universal methodology to measure cCIC in mice after dosing with species foreign proteins. The assay is a sandwich enzyme-linked immunosorbent assay - exclusively based on commercially available reagents - that could detect mouse IgG bound to complement C3 independent of the test-substance present in the plasma sample. Heat-aggregated serum was used as positive control. The assay was qualified by assessment of acceptance criteria, stability of positive control, precision, and specificity. Finally, the performance of the assay was tested using plasma from mice administered either of three different proteins, i.e bovine serum albumin (BSA), a fully human monoclonal antibody, and a humanized monoclonal antibody.

Generic immune complex assay for detection of murine anti-drug-antibodies in complex with human IgG.

Rapid and versatile methods are needed for evaluation of immunogenicity in early safety studies. The present work presents a generic, simple and easy to use sandwich enzyme-linked immunosorbent assay for quasi-quantitative measurement of circulating immune complexes (CICs) formed by anti-drug antibodies (ADAs) in complex with human IgG in mouse plasma. The assay is suitable for evaluating the presence of in vivo formed CICs in mice exposed to human IgG antibodies independent of target and IgG subtype. The assay is established using commercially available antibodies, and calibrated using CIC mimics based on bis(sulfosuccinimidyl)suberate conjugated human and mouse IgG. The development and qualification process of the generic methodology is described and include acceptance criteria, stability, sensitivity, drug tolerance, spike recovery, precision and cut point determination. In order to demonstrate assay performance, its use is exemplified by quantifying CICs in mice administered with a fully human anti-TNF-α IgG1 antibody (adalimumab) or a humanized anti-trinitrophenol (TNP) IgG4 antibody. Results show a well-qualified reproducible assay set-up with adequate sensitivity, easy discrimination between positive and negatives and quasi-quantitative measurement of ADA-human IgG CICs in mice administered with each of two different human/humanized IgG antibodies.