Lung vascular targeting using antibody to aminopeptidase P: CT-SPECT imaging, biodistribution and pharmacokinetic analysis.

BACKGROUND/AIMS: Aminopeptidase P (APP) is specifically enriched in caveolae on the luminal surface of pulmonary vascular endothelium. APP antibodies bind lung endothelium in vivo and are rapidly and actively pumped across the endothelium into lung tissue. Here we characterize the immunotargeting properties and pharmacokinetics of the APP-specific recombinant antibody 833c. METHODS: We used in situ binding, biodistribution analysis and in vivo imaging to assess the lung targeting of 833c. RESULTS: More than 80% of 833c bound during the first pass through isolated perfused lungs. Dynamic SPECT acquisition showed that 833c rapidly and specifically targeted the lungs in vivo, reaching maximum levels within 2 min after intravenous injection. CT-SPECT imaging revealed specific targeting of 833c to the thoracic cavity and co-localization with a lung perfusion marker, Tc99m-labeled macroaggregated albumin. Biodistribution analysis confirmed lung-specific uptake of 833c which declined by first-order kinetics (t(½) = 110 h) with significant levels of 833c still present 30 days after injection. CONCLUSION: These data show that APP expressed in endothelial caveolae appears to be readily accessible to circulating antibody rather specifically in lung. Targeting lung-specific caveolar APP provides an extraordinarily rapid and specific means to target pulmonary vasculature and potentially deliver therapeutic agents into the lung tissue.

High cell density-mediated pericellular hypoxia is a crucial factor inducing expression of the intrinsic hypoxia marker CA IX in vitro in HeLa cells.

Oxygen plays a central role in respiration of the cells and thus in generation of energy by aerobic metabolism. The cells precisely detect oxygen level and changes in oxygen perfusion leads to induction of various responses enabling to adapt to unfavorable conditions. CA IX carbonic anhydrase is a hypoxia-inducible tumor-associated antigen which is overexpressed in dense HeLa cells. Presented study investigates the effects of oxygen tension on CA IX expression in HeLa cell culture. Using of an immunoradiometric assay to quantify CA IX protein, it was revealed that expression of CA IX correlates with increasing cell density, lactate production and medium acidification under normoxic conditions. These observations and hypoxia-inducibility of CA IX suggested a possible role of pericellular hypoxia in density-induced CA IX expression. To test this hypothesis, HeLa cells were incubated in normobaric hyperoxia (50% O2) or cell culture medium was convected to disturb oxygen deprivation. Both approaches completely abrogated CA IX expression in dense HeLa cell cultures and therefore confirmed the importance of decreased oxygen tension in high cell density-induced CA IX expression. In addition, HeLa cells exposed to hyperoxia retained inducibility of CAIX expression by transition metals and iron chelators, suggesting that they act independently of cell density mediated-pO2-gradient or at a downstream site from oxygen sensor. Observed data indicate that high cell density-lowered pericellular pO2 is a crucial factor inducing CA IX expression and influencing composition of metabolic micromilieu surrounding the dense HeLa cells.

Immunotargeting of human cervical carcinoma xenograft expressing CA IX tumor-associated antigen by 125I-labeled M75 monoclonal antibody.

The aim of our present study was to explore a potential use of 125I-labeled murine monoclonal antibody M75 that recognizes carbonic anhydrase IX (CA IX) in the immunotargeting of human cervical carcinoma xenografts in nude mice. CA IX is a hypoxia-inducible antigen, whose expression is significantly associated with carcinomas of the uterine cervix, whereas normal cervical tissue does not express CA IX protein. M75 monoclonal antibody was labeled with 125I and used to quantify hypoxic induction of CA IX expression in vitro in HeLa human cervical carcinoma cells by immunoradiometric assay. HeLa cells showed inducible expression of CA IX in vitro by hypoxia (0.1% O2) and various hypoxia mimicking agents (Co2+, Ni2+, Mn2+, desferrioxamine, o-phenanthroline and Na2S2O4). CA IX expression was also upregulated in the centre of HeLa multicellular clusters (spheroids) corresponding to the conditions of chronic hypoxia. For the immunotargeting study, 125I-M75 was intravenously injected into immunodeficient mice bearing HeLa cervical carcinoma xenografts. Biodistribution profile showed selective and preferential accumulation of 125I-M75 mAb in CA IX expressing HeLa xenografts in comparison with control unreactive 125I-T111 antibody. Specificity was also confirmed by low uptake in CA IX negative C33A xenografts. In addition, CA IX expression in cervical carcinoma xenografts was analyzed by immunohistochemistry with M75. Detailed immunohistochemical analysis of HeLa xenograft sections revealed perinecrotically intensified expression of CA IX. These results indicate that M75 mAb, recognizing CA IX antigen, has targeting properties which could be potentially useful in radioimmunodetection or radioimmunotherapy of human cervical carcinomas and derived metastases.

Effect of salt stress on fatty acid alterations in some strains of Dipodascopsis and Dipodascus spp.

The effect of salt stress (8% w/v NaCl) on fatty acid composition of eight strains of Dipodascus and Dipodascopsis spp. varied from being of slight influence only (Dipodascopsis uninucleata), to decreasing the content of 18:2 (D. reesii, D. tetrasperma and D. australiensis) and to decreasing both 18:1 and 18:2 (D. tothii and D. aggregatus) with a concomitant rise of 14:1 and 16:1. With the exception of D. aggregatus, NaCl inhibited lipid accumulation in all strains. Only trace amounts of fatty acids over C18 in chain length were found.