MAB3864 Sigma-AldrichAnti-DNA/Histone H1 Antibody

In vivo implantation of sterile materials and devices results in a foreign body immune response leading to fibrosis of implanted material. Neutrophils, one of the first immune cells to be recruited to implantation sites, have been suggested to contribute to the establishment of the inflammatory microenvironment that initiates the fibrotic response. However, the precise numbers and roles of neutrophils in response to implanted devices remains unclear. Using a mouse model of peritoneal microcapsule implantation, we show 30-500 fold increased neutrophil presence in the peritoneal exudates in response to implants. We demonstrate that these neutrophils secrete increased amounts of a variety of inflammatory cytokines and chemokines. Further, we observe that they participate in the foreign body response through the formation of neutrophil extracellular traps (NETs) on implant surfaces. Our results provide new insight into neutrophil function during a foreign body response to peritoneal implants which has implications for the development of biologically compatible medical devices.

Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction.

To improve the clinical potential of monoclonal antibodies (MAbs), new methods are required to augment antibody uptake in the tumor while minimizing binding in normal tissues. Our laboratory has pioneered the use of chemical modification to accomplish this goal. Using three chimeric MAbs, chTNT-1, chTNT-2, and chTNT-3, which target solid tumors by binding to common antigens found in the central necrotic core, we now demonstrate the potential of chemical modification to improve the pharmacokinetic characteristics of these unique MAbs. To identify optimal modification conditions, TNT MAbs were reacted with biotin at various ratios and tested by clearance and biodistribution analyses. The biodistribution results revealed that the numbers of biotin molecules per MAb yielding optimal tumor uptake were 3:1 for chTNT-1, 5:1 for chTNT-2, and 8:1 for chTNT-3. Biotinylated MAbs were found to have faster whole body clearance times and better biodistribution profiles compared to unmodified antibodies. Although chTNT-2 showed only a modest improvement after biotinylation, biodistribution results indicated that this MAb had the highest uptake in tumor. By reducing the charge of the antibody molecule, chemical modification appears to be a useful method for improving the pharmacokinetics and biodistribution of TNT antibodies directed to the necrotic region of solid tumors.

Direct and indirect radioimmunoassay (RIA) procedures to determine the amount of binding of a mouse monoclonal antibody (MoAb) reactive with an intracellular antigen present in human cells are described. In these RIAs, mouse IgG2a MoAb, designated as Tumor Necrosis Treatment (TNT-1) antibody, paraformaldehyde/acetone fixed cells, and Sephadex beads were used to standardize the assay conditions. In the direct RIA, 83% of the 125I-labeled TNT-1 MoAb was bound to the target cells within 30 min after the addition of reagents. The amount of binding of the MoAb was directly proportional to the amount of antigen present in the assay. When the direct RIA was carried out using different types of target cells, 125I-labeled TNT-1 MoAb showed greater than 70% binding. In the indirect RIA, the amount of binding of secondary 125I-labeled goat anti-mouse IgG antibody to the target cells was linear. These results suggest that the indirect RIA can be used to estimate the immunoreactivity of the unlabeled TNT-1 MoAb present in crude protein preparations. Based on the results of RIAs the following two conclusions are drawn: 1) the direct RIA can be used to estimate rapidly the amount of immunoreactive TNT-1 MoAb present in 125I-labeled antibody preparations and 2) the indirect RIA which estimates the amount of immunoreactivity of unlabeled TNT-1 MoAb can be used to monitor the purification and study the characteristics of the MoAb present in crude protein preparations. These methods enable the quantitative measurement of MoAbs reactive against intracellular antigens using standard RIA procedures.

Autoradiography was utilized to explore the patterns of distribution of two different monoclonal antibodies (Lym-1 and TNT-1) in tumor-bearing nude mice. Lym-1 is an antibody against a cell surface B-cell antigen. In comparison, TNT-1 represents a novel approach and is an antibody against an intracellular (nuclear) antigen that is selectively revealed in degenerating tumor cells. Experimentally iodine-125-(125I) labeled Lym-1 or TNT-1 was injected intravenously into nude mice bearing either the Raji lymphoma or the ME-180 human cervical carcinoma. Qualitative autoradiographic analyses performed after injection revealed that Lym-1 accumulated at the periphery of the target tumor where vascular permeability is marked and where Lym-1 positive cells are first encountered. By contrast, TNT-1 lost its initial peripheral distribution and demonstrated progressive concentration in the center of the tumor where binding to its nuclear antigen is facilitated by the presence of cell degeneration and necrosis. These studies confirm the ability of TNT-1 to bind areas deep within tumor that traditionally are considered inaccessible to antibodies administered for imaging and therapy.