Universitat Leipzig

University of Leipzig

Ulf Wagner

Manuela Rossol

The invention relates to a method for prognostic evaluation of the disease progression of rheumatoid arthritis, in particular prognostic evaluation of the disease progression during treatment, and for the diagnosis and/or activity determination of rheumatoid arthritis by analysing cytokines from a human full blood sample. In the method according to the invention, a volume of a full blood sample of a human is transferred into at least one test tube containing a stimulating agent. As control samples, the same volume of a full blood sample of the human in each case is transferred into an empty test tube as a negative control and a test tube containing lipopolysaccharide as a positive control respectively. After incubation, the concentration of at least one proinflammatory cytokine is determined from the cell-free residue of each test tube. By way of an altered concentration of the at least one cytokine in the at least one test tube comprising the stimulating agent, the prognostic evaluation of the disease progression or the diagnosis is subsequently made. The invention further relates to an associated diagnostic kit and to the use thereof. The invention is applied in medical diagnostics and medical research.

Method and kit for cytokine analysis from a human whole blood sample

The present invention is based on two important experimental observations: The first observation is that increased extracellular concentrations of ionized calcium are found in erosive arthritis and stimulate monocytic IL-1&#x3b2; release via the CaSR and GPRC6A. Simultaneous stimulation of monocytes with calcium ions and selected TLR ligands results in a 20-fold increased IL1&#x3b2; response compared to lipopolysaccharide (LPS) alone. During the crosstalk between GPCR and TLR signaling, phospholipase C is activated, which triggers calcium dependent potassium channels, resulting in potassium efflux, caspase-1 activation and IL-1&#x3b2; release. The amplification of IL1&#x3b2; secretion at sites of locally increased calcium ion concentrations aggravates rheumatoid arthritis. The second important observation is that both CaSR and GPRC6A, are highly expressed in the synovial membrane of patients with rheumatoid arthritis, but expression of GPRC6A, but not of CaSR, is lower in patients with osteoarthritis (s. FIG.). The latter is generally not accompanied by inflammation. Thus, expression of GPRC6A appears to be upregulated in chronic inflammatory situations. Based on these experimental observations the invention provides a method and compositions for the identification of agents that have a potential effect against chronic inflammatory conditions, in particular erosive arthritis and atherosclerosis.

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Leipzig, Germany

Ulf Wagner