Renal pathological analysis using galactose-deficient IgA1-specific monoclonal antibody is a strong tool for differentiation of primary IgA nephropathy from secondary IgA nephropathy.

In several cases with IgA nephropathy (IgAN), differential diagnosis is difficult due to the complication with other systemic diseases which can induce secondary IgAN. Recently, we demonstrated that immunostaining with galactose-deficient IgA1-specific monoclonal antibody (KM55 mAb) specifically showed positive in primary IgAN cases. Here, we report four cases which we could make definitive diagnosis by immunohistological analysis using KM55 mAb. The underlying systemic diseases are rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), hepatitis C (HCV) and Crohn's disease (CD). Renal pathological findings in the four cases revealed mesangial proliferative glomerulonephritis with IgA and C3 deposits. Immunostaining with KM55 mAb was positive for three cases complicated with RA, SLE and CD, respectively. Thus, these three cases were diagnosed as primary IgAN and treated with tonsillectomy and steroid pulse therapy. These three cases finally achieved clinical remission. On the other hand, the case with HCV showed negative for KM55. Finally, we diagnosed as HCV-related nephropathy and successfully treated by antiviral agents. These cases suggested KM55 mAb is a strong tool to differentiate primary IgAN from secondary IgAN.

Targeted Delivery of LXR Agonist Using a Site-Specific Antibody-Drug Conjugate.

Liver X receptor (LXR) agonists have been explored as potential treatments for atherosclerosis and other diseases based on their ability to induce reverse cholesterol transport and suppress inflammation. However, this therapeutic potential has been hindered by on-target adverse effects in the liver mediated by excessive lipogenesis. Herein, we report a novel site-specific antibody-drug conjugate (ADC) that selectively delivers a LXR agonist to monocytes/macrophages while sparing hepatocytes. The unnatural amino acid para-acetylphenylalanine (pAcF) was site-specifically incorporated into anti-CD11a IgG, which binds the α-chain component of the lymphocyte function-associated antigen 1 (LFA-1) expressed on nearly all monocytes and macrophages. An aminooxy-modified LXR agonist was conjugated to anti-CD11a IgG through a stable, cathepsin B cleavable oxime linkage to afford a chemically defined ADC. The anti-CD11a IgG-LXR agonist ADC induced LXR activation specifically in human THP-1 monocyte/macrophage cells in vitro (EC50-27 nM), but had no significant effect in hepatocytes, indicating that payload delivery is CD11a-mediated. Moreover, the ADC exhibited higher-fold activation compared to a conventional synthetic LXR agonist T0901317 (Tularik) (3-fold). This novel ADC represents a fundamentally different strategy that uses tissue targeting to overcome the limitations of LXR agonists for potential use in treating atherosclerosis.

Oxygen levels in normal and previously irradiated human skin as assessed by EF5 binding.

The oxygen status of skin is a controversial topic. Skin is radiosensitive, suggesting it is well-oxygenated. However, it can be further sensitized with nitroimidazole drugs, implying that it is partially hypoxic. Skin oxygen levels are difficult to measure with either electrodes or the hypoxia-monitoring agent (3)H-misonidazole. For the latter, binding has previously been reported to be high in murine skin, but this could be attributed to either non-oxygen-dependent variations in nitroreductase activity, drug metabolism, and/or actual oxygen gradients. We obtained tumor and skin from patients given EF5, a 2-nitroimidazole tissue hypoxia monitor. We performed immunohistochemical studies using highly specific monoclonal antibodies for the hypoxia-dependent production of EF5 tissue adducts. Some tissue sections were counterstained using either Ki67 for proliferation or CD31 for vessels. We found that the human dermis is well-oxygenated, the epidermis is modestly hypoxic and portions of some sebaceous glands and hair follicles are moderately to severely hypoxic. Normal and irradiated skin had similar oxygenation patterns. Control studies demonstrated that these observations are not due to tissue variations in nitroreductase activity. The importance of the highly heterogeneous distribution of oxygen in skin requires further study, but recent investigations suggest that skin hypoxia may have important clinical ramifications including mediating cellular transformation.

Detection of individual hypoxic cells in multicellular spheroids by flow cytometry using the 2-nitroimidazole, EF5, and monoclonal antibodies.

PURPOSE: The purpose of this work was to evaluate EF5, a 2-nitroimidazole compound, and anti-EF5 antibodies as a method to quantify radiobiologically hypoxic cells. METHODS AND MATERIALS: Multicellular spheroids of EMT6 mammary sarcoma cells were used as a model to identify hypoxic cells that were resistant to radiation damage. This was accomplished by incubating the spheroids with the 2-nitroimidazole (EF5), which forms hypoxia-dependent adducts with cellular macromolecules that are detected by fluorescent monoclonal antibodies. RESULTS: Cells from spheroids grown for 2 days in sealed flasks had an increased surviving fraction following radiation as compared to fully reoxygenated spheroids, indicating the presence of radiobiological hypoxia. Treatment of the spheroids with EF5 and subsequent immunohistochemical staining of cryosections with an anti-EF5 fluorochrome conjugated monoclonal antibody allowed for the identification of EF5-adduct containing cells. Spheroids grown under hypoxic conditions in the presence of EF5 showed limited staining of the peripheral cell layers, intense staining of the interior, and an absence of staining within the necrotic center. In contrast, there was minimal staining in reoxygenated spheroids and no staining in control spheroids incubated in the absence of EF5. Flow cytometric analysis of single cells dissociated from spheroids allowed for the calculation of the percentage of stained cells, as well as the intensity of staining. A comparison of the intensity of staining of EF5 treated hypoxic spheroids with the intensity of staining of single cells incubated with EF5 under controlled oxygen concentrations was used to estimate the oxygen concentration range within spheroids. Selective dissociation of spheroids provided a direct demonstration that the cells containing the highest level of EF5 binding were also the cells with increased radiation resistance. CONCLUSION: This technique provides an excellent means of detecting and quantifying hypoxia, which should be directly applicable in tumors.

Detection of cysteine conjugate metabolite adduct formation with specific mitochondrial proteins using antibodies raised against halothane metabolite adducts.

Antibodies raised against halothane metabolite adducts cross-react with S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine metabolite adducts. Using these antibodies in immunohistochemical experiments, metabolite binding was localized to the damaged areas of the proximal tubule after treatment of male rats with TFEC. Immunoblot analysis of subcellular fractions of rat kidney tissue after in vivo treatment with TFEC revealed a high specificity for binding of metabolites to proteins of the mitochondrial fraction. These proteins may represent target molecules which play a role in cysteine conjugate induced nephrotoxicity.