A 52 Week, Single Center, Open-label Study to Evaluate Neutrophil Function and Survival Effects of Tocilizumab (TCZ) in Patients With Active Rheumatoid Arthritis (RA) on Background Non-biologic DMARDs Who Have an Inadequate Response to Current Non-biologic DMARD and/or Anti-TNF Therapy

Mean Fluorescence Intensity of CD11b on Neutrophil Surface

时间窗: Visits 2, 3, and 5 (Baseline and Weeks 4 and 12)

Neutrophils were incubated with labeled antibodies against CD11b. Flow cytometry was used to determine the mean fluorescence intensity. Greater fluorescence correlates with greater adhesion, migration, and ingestion of complement-opsonized particles.

Mean Fluorescence Intensity of CD62L (L Selectin) on Neutrophil Surface

时间窗: Visits 2, 3 and 5 (Baseline and Weeks 4 and 12)

Neutrophils were incubated with labeled antibody against CD62L (L selectin). Flow cytometry was used to determine the mean fluorescence intensity. Greater fluorescence correlates with greater adhesion of neutrophils to vessel walls.

Mean Fluorescence Intensity of Membrane Bound Tumor Necrosis Factor Alpha (mTNFα) on Neutrophil Surface

时间窗: Visits 2, 3, and 5 (Baseline and Weeks 4 and 12)

Neutrophils were incubated with labeled antibody against mTNF. Flow cytometry was used to determine the mean fluorescence intensity. Greater fluorescence correlates to a greater density of membrane bound TNF.

Mean Chemiluminescence (Area Under the Concentration-time Curve [AUC]) of Neutrophil Reactive Species Production Using Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) Stimulation

时间窗: Visit 2, 3, 5, and 8 (Baseline and predose at Weeks 4, 12 and 24)

Using luminol as a substrate for reactive oxidants, a chemical reaction is produced resulting in photon emission (chemiluminescence). fMLP stimulation is mediated through the fMLP receptor on the cell surface. The fMLP response is only observed in primed neutrophils and response is a measure of in vivo priming. Measurements of reactive oxygen species are calculated as total chemiluminescence or the AUC.

Mean Percentage of Cells Staining Positive for Annexin V Binding in Apoptosis

时间窗: Visits 2, 3, 5, and 8 (Baseline and Weeks 4, 12 and 24)

Aging neutrophils translocate phosphatidylserine from the inner leaflet of the plasma membrane to the outer leaflet during the early stages of apoptosis. This translocation can be measured due to the affinity of fluorescein isothiocyanate (FITC)-labeled annexin V to bind exposed phosphatidylserine. Cells that stain positive to Annexin V binding are apoptotic. At 4 hours (hrs) and 20 hrs stimulated and control samples were analyzed for levels of apoptosis.

Mean Percentage of Cells Staining Positive for Annexin V Binding With Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF)

时间窗: Visits 2, 3, 5, and 8 (Baseline and Weeks 4, 12 and 24)

Aging neutrophils translocate phosphatidylserine from the inner leaflet of the plasma membrane to the outer leaflet during the early stages of apoptosis. This translocation can be measured due to the affinity of FITC-labeled annexin V to bind exposed phosphatidylserine. Cells that stain positive to Annexin V binding are apoptotic. At 4 hrs and 20 hrs stimulated and control samples were analyzed for levels of apoptosis. GM-CSF is an agent that delays apoptosis. Percentage of cells that stained positive for Annexin V binding in the presence or absence of GM-CSF (GM-CSF delayed or constitutive) were determined by flow cytometry.

Mean Fluorescence Intensity of CD18 on Neutrophil Surface

时间窗: Visits 2, 3, and 5 (Baseline and Weeks 4 and 12)

Neutrophils were incubated with labeled antibodies against CD18. Flow cytometry was used to determine the mean fluorescence intensity. Greater fluorescence correlates with greater adhesion, migration, and ingestion of complement-opsonized particles.

Mean Fluorescence Intensity of CD63 on Neutrophil Surface

时间窗: Visits 2, 3, and 5 (Baseline and Weeks 4 and 12)

Neutrophils were incubated with labeled antibody against CD63b. Flow cytometry was used to determine the mean fluorescence intensity. Greater fluorescence correlates with greater azurophilic degranulation, an indicator of greater microbe killing.

Mean Fluorescence Intensity of Interleukin-6 Receptor (Il-6R) on Neutrophil Surface

时间窗: Visits 2, 3, and 5 (Baseline and Weeks 4 and 12)

Neutrophils were incubated with labeled antibody against IL-6R. Flow cytometry was used to determine the mean fluorescence intensity. Greater fluorescence correlates with greater density of membrane bound IL-6 receptor.

Mean Chemiluminescence (AUC) of Neutrophil Reactive Species Production Using Phorbol 12-Myristate 13-Acetate (PMA) Stimulation

时间窗: Visit 2, 3, 5, and 8 (Baseline and predose at Weeks 4, 12 and 24)

Using luminol as a substrate for reactive oxidants, a chemical reaction is produced resulting in photon emission (chemiluminescence). PMA is a receptor-independent stimulator of the respiratory burst and the PMA response measures the total capacity of neutrophils to generate reactive oxidants. Measurements of reactive oxygen species are calculated as total chemiluminescence or the AUC.

Percentage of Neutrophils Positive for Propidium Iodide (PI)-Labeled Staphylococcus Aureus (S. Aureus) Uptake

时间窗: Visit 2, 3, 5, and 8 (Baseline and Weeks 4, 12 and 24)

S. aureus were heat killed then labeled with PI and opsonized with AB serum (SAPI). S. aureus was then incubated with the neutrophils for 30 minutes at 37 degrees Celsius. The neutrophils were washed, then the percentage of cells positive for the labeled S. aureus (that is, phagocytosed) was calculated via flow cytometry. A higher percentage represented more active phagocytosis.

Percentage of Neutrophils Positive for Dihydrorhodamine-123 (DHR) Oxidation

时间窗: Visit 2, 3, 5, and 8 (Baseline and Weeks 4, 12 and 24)

Phagocytosis can be measured by incubating neutrophils with PI-labeled heat killed S. aureus following incubation for 30 minutes. Neutrophils are co-incubated with DHR, which becomes oxidized by the products of the respiratory burst generated during phagocytosis. Fluorescence can then be measured by flow cytometry.