Eotaxin-3 but not eotaxin gene expression is upregulated in asthmatics 24 hours after allergen challenge.

Eotaxin is an important mediator of eosinophil recruitment and activation in the airways of asthmatics. Eotaxin-2 and eotaxin-3 are two recently identified chemokines with activity similar to that of eotaxin. Using quantitative polymerase chain reaction analysis, we determined the messenger RNA (mRNA) expression of eotaxin, eotaxin-2, and eotaxin-3 relative to GAPDH mRNA expression in bronchial biopsies and bronchoalveolar lavage fluid (BALF) cells obtained from subjects with mild asthma, asthmatic subjects 24 h after allergen challenge, and normal control subjects. In bronchial biopsies, gene expression was upregulated in asthmatic subjects as compared with control subjects for eotaxin (log median values 3.18 pg/microg, 95% confidence interval [CI]; 2.27 to 3.79 versus 4.37 pg/microg, 95% CI; 3.97 to 4.65, P = 0.003) and for eotaxin-2 (0.82 pg/microg, 95% CI; 0.08 to 1.72 versus 2.97 pg/microg, 95% CI; 1.97 to 3.45, P = 0.006), but no further increase was observed after allergen challenge. In contrast, eotaxin-3 mRNA expression was not increased in asthmatic compared with control subjects, but was dramatically enhanced 24 h after challenge (median log value 1.93 pg/microg, 95% CI; 0.74 to 3.92 versus 4.62 pg/microg, 95% CI; 3.05 to 6.23, P = 0.036). No significant difference between groups was observed in BALF cell gene expression for any of the chemokines examined. These data suggest that eotaxin-3 rather than eotaxin or eotaxin-2 may account for the ongoing eosinophil recruitment to asthmatic airways in the later stages (24 h) following allergen challenge.

Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.