Objective. To determine whether the plasma levels of a range of inflammatory proteins have utility as biomarkers of disease activity in rheumatoid arthritis (RA) patients.Methods. Plasma proteins (n ‫؍‬ 163) were profiled in 44 patients with RA diagnosed according to the American College of Rheumatology 1987 criteria (22 with active and 22 with quiescent disease) and in 16 ageand sex-matched healthy controls. The utility of a subset of differentially expressed proteins as predictors of RA disease activity was investigated using partial leastsquares discriminant analysis, and their response to therapeutic intervention was evaluated in plasma from an additional cohort of 16 patients with active RA treated with anti-tumor necrosis factor ␣ (anti-TNF␣).Results. The protein profiling study identified 25 proteins that were differentially expressed in plasma samples from patients with active RA (P for the false discovery rate < 0.01) compared with those with quiescent RA, including the previously described interleukin-6 (IL-6), oncostatin M, and IL-2, and the 5 less-established markers macrophage colonystimulating factor (M-CSF), tumor necrosis factor receptor superfamily member 9, CCL23, transforming growth factor ␣, and CXCL13. Systemic levels of these 5 markers correlated with the C-reactive protein level, erythrocyte sedimentation rate, rheumatoid factor level, tender joint count in 68 joints, and Disease Activity Score in 28 joints (DAS28), and their combined plasma levels were shown to be good predictors of disease activity ( ‫؍‬ 0.64). In anti-TNF␣-treated RA patients, plasma levels of CXCL13 were reduced after 1 and 7 days of therapy, and levels of CCL23, M-CSF, and CXCL13 showed a statistically significant positive correlation with the DAS28 score.

Replication of human papillomavirus (HPV) DNA requires the viral proteins E1 and E2. Amino acid similarities to SV40 large-T antigen had suggested that E1 is a DNA helicase/ATPase involved in initiating viral DNA replication, and this has recently been shown for bovine papillomavirus type 1 (BPV-1) E1 protein. However, in vitro analysis of HPV E1 has been hampered by the inability to produce purified protein using heterologous expression systems. We have succeeded in demonstrating ATPase and DNA helicase activities in purified HPV E1, expressed in E. coli as a maltose-binding protein fusion (MBP-E1), for the first time. As further confirmation that the ATPase and DNA helicase activities are due to E1 and not contaminating E. coli enzymes, we have shown that a fusion protein containing an amino acid change (E1 Pro-479 to Ser), predicted to inactivate ATP-binding, has impaired activities. We have carried out a structure prediction analysis which suggests that E1 may form two domains: a relatively open N-terminal domain (residues 1-125), and a highly structured C-terminal domain (170-649), with an intermediate region (125-170) predicted to form an inter-domain linker. This is consistent with the proteolytic susceptibility of MBP-E1 at a site 15-20 kD from the N-terminus of E1, and the accumulation of a 58 kD C-terminal fragment of E1. We speculate that the N-terminal domain is involved in DNA-binding, while the C-terminal 58 kD may constitute a distinct enzymatic domain. HPV E1 is of interest as a therapeutic target and the availability of pure enzyme will be invaluable in the search for antiviral compounds.

The discovery of antiviral compounds against human papillomaviruses (HPV) has been hindered by the difficulties in culturing virus in vitro or assaying stable HPV DNA replication. However, plasmids containing the HPV replication origin replicate transiently upon co-transfection with HPV E1 and E2 expression vectors. We have adapted this assay using secreted alkaline phosphatase (SAP) as a reporter for rapid analysis of DNA copy number. Use of the SV40 early promoter in controlling SAP expression was critical in ensuring both a strong signal and copy number dependence: the stronger beta-actin promotor inhibited replication, while the weaker SV40 late promoter yielded very low levels of SAP. The precise configuration of the E1 and E2 expression vectors also was critical, most pre-existing vectors did not support efficient replication and SAP secretion. The extent of DNA replication and SAP secretion were both proportional to the amount of E1/E2 vector used in transfections; under optimal conditions SAP increased 100-fold during replication. The assay has been developed for compound screening in 96-well plates and several inhibitors have been identified. Quantitative Southern blot analysis has shown that most of these inhibit HPV DNA replication rather than SAP accumulation or activity, and several are under test in models of viral replication. The assay also provides a rapid system for functional analysis of the HPV E1, E2 genes and the replication origin.

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