Marijn W. M. Kruijsen

The mouse patella assay

SummaryAn easy method of quantitating articular cartilage chondrocyte function in mice is described, using a defined and anatomically intact cartilage structure, the patella. To avoid damage to the cartilage before incubation, 35S incorporation studies were performed leaving the patellae surrounded by a minimal area of non-cartilaginous connective tissue. The patellae were then punched out so that the 35S activity incorporated into the cartilage could be measured. Incorporation of 35S was almost completely blocked by 0.1 mM cycloheximide. Patellar cartilage from left and right knee joints of individual mice shows identical incorporation of 35S, as indicated by a right/left ratio of 1.01 in vitro and 0.99 in vivo, respectively. 35S incorporation values of patellae of mice of different ages do show considerable variation, but within properly age-matched groups of mice the incorporation values are comparable. A known suppressive effect of acute joint inflammation on proteoglycan synthesis was reliably quantitated in the zymosan-induced arthritis model, as indicated by 39% in vitro and 35% in vivo inhibition of 35S incorporation. The patella assay can be used for quantitative measurement of chondrocyte metabolism in normal and pharmacologically altered cartilage.

Azathioprine induced fever, chills, rash, and hepatotoxicity in rheumatoid arthritis.

Within one year three of 25 patients with rheumatoid arthritis treated with azathioprine 100 mg daily developed the following adverse reactions less than two weeks after starting treatment: patient one showed fever with chills, rash, and severe liver function abnormalities suggestive of cholestasis; the second patient had fever, nausea, diarrhoea, and moderately raised liver enzymes; the third patient showed very high fever and severe chills. In two patients the drug was rechallenged, with more rapidly arising and more severe symptoms. In one case raised liver enzymes persisted until seven months after discontinuation of azathioprine. Hypersensitivity reactions and hepatotoxicity of azathioprine are discussed.

Detection and quantification of experimental joint inflammation in mice by measurement of99mTc-pertechnetate uptake

We adapted the method of99mTc-pertechnetate (99mTc) uptake measurements to the mouse knee for detection and quantification of arthritis because clinical assessment of mouse knee-joint arthritis is not reliable. The main points to ensure reproducibility of measurements were proper fixation and positioning of the knee and careful shielding of the rest of the body from the gamma-radiation detector.99mTc uptake was calculated as the mean of three countings. The variation coefficient of these countings ranged from 0.007 to 0.082 in non-arthritic joints and from 0.025 to 0.081 in arthritic joints. Arthritis was scored as the ratio of the99mTc uptake in the right knee versus that in the left knee. This ratio averated 1.06 (S.D. 0.05) in non-arthritic mice 20 minutes after99mTc administration i.p. On the second day after induction of arthritis in the right knee, this ratio ranged from 1.44 to 1.96; this was significantly higher (P<0.005) than in non-arthritic mice, and the increase remained significant during at least 20 days.99mTc uptake measurements seem to be a useful method to detect and quantify arthritis of the knee joint in mice.

Side effects of azathioprine treatment in Rheumatoid Arthritis: analysis of 10 years of experience

Our experience with azathioprine in the treatment of rheumatoid arthritis covers ten years, during which 91 rheumatoid patients (66 female and 25 male) received this drug, with a median treatment period of 36 months. Total follow-up experience, during and after treatment, was 399 person years. Twelve patients died. The principal causes of death were malignant neoplasm (six patients) and cardiovascular diseases (three patients). The mortality in our patients was compared to that of the general Dutch population by the Standardised Mortality Ratio (SMR). In the male patient group a significant excess of both total mortality and mortality from malignancy was observed. The female patients showed no differences from the general population. In this follow-up study, no lymphoreticular tumours occurred during or after azathioprine therapy.

In vivo studies on the influence of antigen induced joint inflammation on patellar hyaline articular cartilage

Loss of articular cartilage is a major complication of chronic joint inflammation, leading to joint dysfunction and deformity. Enzymatic degradation of proteoglycans1 and collagen2 is currently thought to play an important role in cartilage destruction. Recent data have also implicated altered chondrocyte function as a possible contributing factor, since cartilage slices from rabbits with antigen induced arthritis were shown to have a decreased rate of proteoglycan synthesis in vitro 3. Whether this inhibition of the synthetic capacity of articular chondrocytes also occurs in vivo and contributes to loss of hyaline articular cartilage remains to be verified.

Influence of antigen-induced arthritis on connective tissue cell metabolism

Loss of hyaline articular cartilage during chronic joint inflammation may be due to enzymatic breakdown of cartilage proteoglycans and inhibition of proteoglycan biosynthesis. In vivo study in the mouse on the influence of antigen-induced arthritis on articular cartilage chondrocyte function revealed that proteoglycan synthesis was severely inhibited during active joint inflammation. In addition, autoradiographs showed that inhibition of chondrocyte synthetic function and chondrocyte death at later stages of the arthritis were most pronounced in the central part of patellar hyaline articular cartilage without pannus tissue being present nearby.