A drug targeting both the inflammatory initiators (lipopolysaccharide; LPS) and mediators [tumor necrosis factor-␣ (TNF-␣)]should have advantage over a "single-factor targeting strategy" in sepsis prevention trials. We have prepared conjugates of polymyxin B (PMB) and the cytokine binding protein ␣2-macroglobulin (A2M). The conjugate binds TNF-␣ as well as LPS as studied by electrophoresis and phase partitioning. Compared with free PMB, the conjugate is nontoxic to cells and does not affect the viability of human monocytes. The A2M-PMB conjugate binds to the A2M receptor (CD91/low-density lipoprotein receptor-related protein 1) with affinity similar to that of the nonmodified protein. Fluorescein isothiocyanate-labeled LPS in the presence of A2M-PMB is rapidly transported into fibroblasts for degradation via receptor-mediated endocytosis. In vitro, A2M-PMB demonstrated inhibition of LPS-induced secretion of TNF-␣ from isolated monocytes as well as in the whole blood assay. The efficacy of the drug was tested in mice after induction of acute inflammation (LPS model) and after induction of a polymicrobial sepsis by cecal ligation and puncture (CLP) model. Treatment of mice with A2M-PMB up to 250 g/g body weight was not toxic to the animal. When the drug was administered 30 min before or 30 min after the LPS challenge, a survival rate of 90 and 70%, respectively, was obtained compared with the placebo control group (5%). A2M-PMB also protected mice after induction of polymicrobial sepsis when administered 30 min before CLP. These results support our hypothesis that A2M-PMB acts as a polyvalent drug to target different host mediators as well as sepsis inducer at the same time.In the past several years, mortality in patients with sepsis syndrome has decreased somewhat, but in those patients with septic shock and multiple organ failure, mortality still exceeds 50% (Danai and Martin, 2005). This high mortality is observed despite ventilatory, hemodynamic, metabolic, and renal support. Some patients-in particular, patients with genetic polymorphisms associated with low or moderate TNF response-survive the ordeal more often, but it remains frustrating not being able to stop the downhill course leading to multiple organ failure and death in other patients that results from overwhelming inflammation. New therapies have been sought and tested, including those preventing the biological activity of pathogen-associated molecular patterns, such as endotoxin or the downhill host response, most notably TNF-␣. Based on a wealth of animal studies, anti-inflammatory strategies, such as neutralizing TNF-␣, have been advocated to provide adjunctive therapy to the patient who continues to deteriorate in the face of considerable support in the intensive care unit. Unfortunately, these anticytokine therapies have not dramatically reduced mortality in doubleblind, placebo-controlled trials involving thousands of patients, although there is a consistent but statistically non-
Background: Different molecular forms of prostatespecific antigen (PSA) have been used to differentiate between benign prostatic hyperplasia and prostate cancer. Detecting PSA bound to endogenous inhibitors such as ␣ 1 -antichymotrypsin (ACT) and ␣ 2 -macroglobulin (␣ 2 M) is often difficult because of epitope masking or sensitivity problems. Here we report the characterization of four novel mouse monoclonal antibodies (mabs) obtained by immunization with PSA-␣ 2 M complexes. Their ability to detect free PSA and PSA-inhibitor complexes was shown, and their epitopes were analyzed by phage display technology. Methods: The properties of the mabs were studied by competition and sandwich assays and by Western blotting. Epitope mapping was performed by screening of a phage display peptide library. Results: All four mabs recognized free PSA, PSA-ACT, and PSA-␣ 2 M complexes, but to various degrees. With different combinations of mabs in competition experiments, antibodies were identified that enhance binding of other mabs to PSA, forming the molecular basis of a very sensitive assay for the detection of PSA and PSA-ACT complexes. Mabs with highest reactivity for PSA-␣ 2 M were selected to establish an immunoassay for that complex. Western blot analysis revealed that all mabs recognized conformational epitopes of PSA. These findings were supported by phage display results demonstrating mimotopes in the PSA molecule.
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