Annemarie H. Ralston

Alpha-1-antitrypsin inhibits human immunodeficiency virus type 1

Several observations suggest the existence of potent endogenous suppressors of human immunodeficiency virus type 1 (HIV‐1) production, and inhibitors of serine proteases may participate in this effect. Alpha‐1‐antitrypsin (AAT) is the most abundant circulating serine protease inhibitor. Physiological AAT concentrations inhibited HIV‐1 production in chronically infected U1 monocytic cells, reduced virus replication in freshly infected peripheral blood mononuclear cells, and blocked infection of permissive HeLa cells. In U1 cells, AAT suppressed activation of the HIV‐1‐inducing transcription factor NF‐κB. Similar results were obtained using CE‐2072, a synthetic inhibitor of host serine proteases. HIV‐1 did not replicate in blood obtained from healthy volunteers, but marked replication was observed in blood from individuals with hereditary AAT deficiency. These results identify AAT as a candidate circulating HIV‐1 inhibitor in vivo. Two different mechanisms of AAT‐induced HIV‐1 inhibition were identified, including reduced HIV‐1 infectivity and blockade of HIV‐1 production. A novel host pathogen interaction is suggested, and an alternative strategy to treat HIV‐1‐related disease may be possible.—Shapiro, L., Pott, G. B., Ralston, A. H. Alpha‐1‐antitrypsin inhibits human immunodeficiency virus type 1. FASEB J. 15, 122–115 (2001)

Expression and Purification of Functional Human α‐1‐Antitrypsin from Cultured Plant Cells

Human α‐1‐antitrypsin (AAT), the most abundant protease inhibitor found in the blood, was expressed in rice embryonic tissue suspension cell culture. This was accomplished by cloning the codon‐optimized AAT gene into a vector containing the rice RAmy3D promoter and its signal sequence. The synthetic gene incorporates codons synonymous with those found in highly expressed rice genes. Approximately 1000 stable transformed calli were produced by particle bombardment mediated transformation and were screened for high AAT expression using a porcine elastase inhibitory activity assay. The band shift assay also confirmed that rice‐derived AAT is functional regarding its binding capability to the elastase substrate. Time course studies were conducted to determine the optimum, postinduction expression levels from cell culture. AAT expression equivalent to 20% of the total secreted proteins was achieved, and a purification scheme was developed that yielded active AAT with purity greater than 95%. The potential applications of purified plant‐derived AAT for treatments of various AAT‐deficient diseases are discussed.

Alpha-1-antitrypsin inhibits nitric oxide production

NO is an endogenously produced gas that regulates inflammation, vascular tone, neurotransmission, and immunity. NO production can be increased by exposing cells to several endogenous and exogenous proinflammatory mediators, including IFN‐γ, TNF‐α, IL‐1β, and LPS. As AAT has been shown to inhibit cell activation and suppress cytokine production associated with proinflammatory stimulation, we examined AAT for NO‐suppressive function. In RAW 264.7 murine macrophagic cells, physiological AAT concentrations significantly inhibited combined LPS‐ and IFN‐γ‐induced NO synthesis, and NO synthesis inhibition was associated with decreased expression of iNOS, suppressed NF‐κB activation, and reduced translocation of extracellular AAT into the interior of RAW 264.7 cells. CE‐2072, a synthetic inhibitor of serine proteases, also suppressed NO production, iNOS expression, and NF‐κB activation. However, AAT did not alter activation of intracellular MAPKs. In subjects with genetic AAT deficiency, exhaled NO was increased significantly compared with exhaled NO in healthy controls. These in vitro and in vivo studies suggest that AAT is an endogenous inhibitor of NO production. Administering AAT or AAT‐like molecules may have use as a treatment for diseases associated with excessive NO production.

Large-Scale Production and Properties of Immunoaffinity-Purified Human Activated Protein C Concentrate

Activated protein C (APC) is a highly specific serine proteinase which functions as an important naturally occurring antithrombotic enzyme. APC also has antiinflammatory properties. We have developed a large‐scale process for the production of APC for therapeutic use starting with cryoprecipitate‐poor human plasma. This report describes the process, its performance at the pilot plant scale, and the characteristics of immunoaffinity‐purified human APC concentrate referred to as APC (human). The process consists of three chromatographic steps, an enzymatic conversion step, and incorporates a solvent/detergent treatment step for the inactivation of lipid‐enveloped viruses. Solvent/detergent was shown to rapidly inactivate spiked HIV‐1, as well as three marker viruses to nondetectable levels under process conditions. The immunoaffinity‐purified protein C (PC) intermediate was enriched 13,600‐fold over plasma and had a specific activity of 231 U/mg. The overall yield of the process following enzymatic conversion of the PC intermediate to APC and its processing by anion exchange chromatography was 36%. APC (human) was shown to be highly purified, functional and stable.