press-5.qxd 11/28/07 3:01 PM Page 402 402 B. Mosheimer et al.: Recombinant A1PI and granulocyte migration Polymerization of rhA1PI has not been reported so far; consequently, induction of migration by rhA1PI if poly- merized remains speculative. The present results indi- cate, however, that at high concentrations, a crude preparation of vehicle (MOCK) induced human neu- trophil, but not eosinophil migration to some extent, suggesting the potential of eventual contaminants to modify migration. Since eosinophils did not respond to the vehicle, the contaminating compound(s) may be specific and involve molecular mechanisms. As the rhA1PI was unable to induce the same effect, it may be that the dose equivalence between the two types of sam- ples was not given. Another possibility is that the immunomodulatory activity of rhA1PI on leukocytes heterogeneously cross-deactivates chemotactic contam- ination of the vehicle, such as that described between non-chemotactic tumor necrosis factor-alpha and chemokines [24]. Despite the application of good man- ufacturing practice, contamination of the yeast-derived rhA1PI formulation may thus potentially induce inflam- matory responses after aerosol inhalation. Host responses to yeast are mediated by Toll-like receptor 2 (TLR2), which is normally expressed by a variety of human cells. Cell-surface TLR2 activation is known to induce neutrophil activation. In vivo indirect chemotac- tic effects may be exerted by induced mediators that are released from respiratory cells, including IL-8, which is a potent granulocyte attractant responsible for leuko- cyte accumulation in lung airways [8, 13]. Thus for the evaluation of safety, direct and indirect mechanisms by which aerosol inhalation of yeast-derived rhA1PI for- mulations may adversely affect lung inflammation have to be taken into account. Given the concerns related to the limited supply of Fig. 2. Human peripheral blood eosinophil migration toward the chemoattractant RANTES or α(1)-anti- trypsin (rAAT). Mean distances of ran- dom migration were 62, 68, 69, and 72 µ m for rAAT-1, rAAT-2, rAAT-3, and MOC, respectively. *p<0.05, Mann- -Whitney U-test versus medium after multiple group comparison by using the Kruskal-Wallis test. pooled human plasma and the potential for the trans- mission of infectious agents, some manufacturers are particularly investigating alternative sources of augmen- tation therapy. Transgenic or recombinant sources could be used in inhalation devices. Yeast expression systems are well suited for the production of rhA1PI. Yeast rhA1PI manufactured in Saccharomyces cerevisiae (baker’s or brewer’s yeast) [4] was tested in a clinical trial when administered by aerosol inhalation to patients with rhA1PI deficiency associated with hereditary emphysema. According to the sponsors’ information, results from this phase 1A human study evaluating safe- ty and immunogenicity showed that rhA1PI was well tol- erated and a phase 1B/2A clinical trial has been said to be underway to evaluate its effects [1]. rhA1PI from yeast does not induce in vitro chemo- taxis of granulocytes in micropore filter assays. Contents in the preparation’s vehicle from yeast may still have the potential to induce migration of leukocytes because TLR-2 on other airway cells may indirectly contribute to leukocyte accumulation by release of leukocyte attrac- tants such as IL-8. In consequence, the proinflammato- ry potential of rhA1PI aerosol therapy will have to be assessed clinically in order to assure its safety. There remains the need for high standards for purification of recombinant proteins. Strategies to evaluate immune-mediated toxic effects of preparations of transgenic protein should be established to ensure the development of safe and effec- tive therapies. To evaluate the safety of sheep-derived transgenic rhA1PI, aerosolized sheep-derived trans- genic rhA1PI was administered in two clinical studies [23]. Systemic antibody responses to non-human protein that was present in very low concentrations in a sheep-