Improved adalimumab dose decision with comprehensive diagnostics data.

OBJECTIVES: Monoclonal antibodies are important in the treatment of rheumatoid arthritis (RA). This is the first trial to monitor the effect of adalimumab dose escalation in persistently active RA. The aim of this study was to identify the response to adalimumab to improve the basis for making decision in relation to actual drug capacity in serum. METHODS: The disease activity of RA patients was assessed with CDAI and DAS28 before administration of additional 40 mg adalimumab one week after standard injection. Serum samples were analysed using the recoveryELISA technology, a combination of sandwich ELISA and competitive assay. The recoveryELISA measure the concentrations of free TNF-α, drug level, and the remaining active adalimumab in the patients' sera. An adalimumab concentration of 5.0-10.0 g/mL was defined as the targeted therapeutic window. RESULTS: Five of 8 patients achieved moderate EULAR response by dose escalation. The results of the free adalimumab and TNF-α neutralisation measurements allowed a separation of the cohort (n=17) into three groups. Group 1 represents 18% of the patients with free adalimumab level higher 30.0 µg/mL and TNF-α neutralisation above 95%. Group 2 (47%) consists of patients within the therapeutic window with balanced free adalimumab and TNF-α neutralisation values. Group 3 contains 35% of the cohort with low concentrations of free adalimumab and lowest remaining TNF-α-neutralisation capacity. Anti-drug antibodies were detected in four patients but did not prevent response to treatment. CONCLUSIONS: Drug and antigen monitoring using recoveryELISA may support dose decision to avoid unnecessary switch in medication or possible overtreatment.

Stu1 inversely regulates kinetochore capture and spindle stability.

The Saccharomyces cerevisiae CLASP (CLIP-associated protein) Stu1 is essential for the establishment and maintenance of the mitotic spindle. Furthermore, Stu1 localizes to kinetochores. Here we show that, in prometaphase, Stu1 assembles in an Ndc80-dependent manner exclusively at kinetochores that are not attached to microtubules. Stu1 relocates to microtubules when a captured kinetochore reaches a spindle pole. This relocation does not depend on kinetochore biorientation, but requires a functional DASH complex. Stu1 at detached kinetochores facilitates kinetochore capturing. Furthermore, since most of the nuclear Stu1 is sequestered by one or a few detached kinetochores, the presence of detached kinetochores prevents Stu1 from localizing the spindle, and therefore from stabilizing the spindle. Thus, the sequestering of Stu1 by detached kinetochores serves as a checkpoint that keeps spindle poles in close proximity until all kinetochores are captured. This is likely to facilitate kinetochore biorientation. In agreement with the findings described above, a kinetochore mutant (okp1-52) that fails to release Stu1 from the kinetochore displays a severe spindle defect upon spindle pole body separation, and this defect can be rescued by destroying the okp1-52 kinetochore.

Circadian activity and abundance rhythms of the Neurospora clock transcription factor WCC associated with rapid nucleo-cytoplasmic shuttling.

The Neurospora clock protein FREQUENCY (FRQ) inhibits its transcriptional activator WHITE COLLAR COMPLEX (WCC) in a negative feedback loop and supports its accumulation in a positive loop. We show that positive feedback is a delayed effect of negative feedback underlying the same post-translational mechanisms: DNA-binding-competent active WCC commits rapidly to degradation. FRQ-dependent phosphorylation of WCC, which interferes with DNA binding (negative feedback), leads to reduced turnover and slow accumulation of newly expressed WCC (positive feedback). When DNA binding of WCC is compromised by mutation, its accumulation is independent of FRQ. Cycles of FRQ-dependent inactivation and PP2A-dependent reactivation of WCC occur in the minute range and are coupled to obligate rapid cycles of nucleo-cytoplasmic shuttling. WCC shuttling and activity cycles are modulated by FRQ in circadian fashion.