Cure Arthritis Naturally

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low- to moderate-responder strains. In general, gender makes little difference in arthritis susceptibility in rats, with possible exception of the BUF strain, in which males might be more susceptible than females (Cremer et al., 1994a). Young adult rats weighing 125 to 200 g provide the most consistent results. High-responder rats such as BB often remain arthritis-susceptible up to 3 or 4 months of age. The same can not be said with confidence regarding other inbred strains. Environmental conditions are important both for animal well-being and ensuring consistent results. Exposure to regular, intense stress can adversely affect CIA in rats (Rogers et al., 1980); however, gentle daily handling and weekly collections of <1 ml of blood do not noticeably affect the incidence or course of arthritis.

Arthritis has been induced in high-re-sponder strains of rats with CII purified from the cartilage of a number of species—e.g., human, chick, bovine, porcine, rabbit, guinea pig, and deer. With the unique exception of the DA rat (Griffiths et al., 1993), most strains respond poorly to rat CII compared to other mammalian and avian products (Griffiths, 1988; Griffiths et al., 1992; Cremer et al., 1994a). Arthritic responses to chick and bovine CII are often equivalent, with the notable exception of ACI and BN rats where disparate responses are seen. Studies by Griffiths et al. (1992) have also shown that the arthritis-producing potential of porcine CII is essentially identical to that of bovine and human CII.

Whereas the species of collagen plays a relatively minor role in determining arthrito-genicity in the rat, the tertiary structure of CII used for immunization is of critical importance. In sharp contrast to the mouse, where arthritis can be induced at a respectable incidence with highly purified a1(II) chains or cyanogen bromide fragments of the a1(II) chain, CII must be maintained in its native triple-helical conformation during preparation in order to induce arthritis in the rat. For this reason, CII solutions must be kept chilled at temperatures below 4°C to minimize the risk of denaturation.

The amount of CII used to induce arthritis varies broadly among investigators and to some degree depends on the strain being immunized. Doses ranging between 75 and 1000 pg per rat have been described as effective, with intermediate doses of 150 to 300 pg being used most commonly for high-responder inbred strains and outbred strains such as Wistar and Sprague-Dawley. Higher doses are unnecessary in susceptible strains and often ineffective in resistant ones. The use of a booster injection given 7 days after priming is often helpful in producing a more uniform time of onset and may also increase the incidence of disease in moderate- and poor-responder strains.

In the mouse, the H-2q (DBA/lLacJ, CIA-susceptible) proliferative response is dominated by recognition of the CII(260-270) determinant (Brand et al., 1994), whereas IFN-y production can be detected to both CII(260-270) and CII(181-210) (Myers et al., 1989, 1992, 1993). The dominant response in CIA-susceptible H-2r (B10.RIII) mice is directed to CII(610-618), although a second T cell determinant is also present in CB8, CII(442-456) (Miyahara et al., 1995; Myers et al., 1995). In the rat, T cell proliferative responses to native and denatured CII, as well as to collagen peptides (Cremer et al., 1994b), can also be measured. Collagen-specific antibody levels to both the immunogen and the autoantigen correlate very well with the presence or absence of arthritis within susceptible strains, and appear to be a major contributor to the overall pathology of the experimental disease.

Critical Parameters and Troubleshooting

Collagen preparation

Generally, the younger the animal from which fresh cartilage is obtained, the higher the yield of CII. However, when using fetal bovine cartilage as a source of CII, the fetus size should be at least 7 kg. Long-term freezing of cartilage prior to collagen extraction should be avoided as this will result in diminished yield. Extraction and solubilization of CII can be greatly facilitated by rendering the animals lathyritic, thereby preventing collagen cross-linking (Trelstad et al., 1970). Lathyrism is induced by administration of P-aminopropionitrile. Although this procedure requires added expense and time and is not practical for large animals, it is helpful when preparing collagen from rat or mouse cartilage. In addition, the use of a lathyrogen is essential in preparing CII from the Swarm rat chondrosarcoma (Smith et al., 1975; Miller and Rhodes, 1982). For animal use, 0.3% P-aminopropionitrile fumarate or maleate is simply added to the food or drinking water for 7 to 10 days. Higher doses or longer exposure offer little advantage and increase animal mortality and morbidity.

Once type I collagen contaminates a CII preparation, it is very difficult to remove it. All steps in this procedure must be performed at

<4°C unless otherwise indicated. This enhances the solubility of the collagen, ensures the retention of native conformation in the solu-bilized collagen, and reduces the risk of bacterial growth.

Collagen solubilized in 10 mM acetic acid may be lyophilized and stored in a desiccator at -20° or 4°C. Lyophilized collagen tends to become progressively insoluble with time, for reasons that are not well understood. Care should be taken to prevent ambient moisture from condensing on collagen each time the desiccator is removed from the refrigerator. It is important to allow the desiccator to equilibrate to room temperature before opening it. Lyophilized collagen is best dissolved in 100 or 10 mM acetic acid by stirring overnight in the cold. It should be emphasized that the acetic acid solution must be prechilled to 4°C before use to dissolve the collagen. When it is necessary to work with collagen in a neutral buffer, the collagen should be first dissolved in acetic acid and then dialyzed into the desired buffer in the cold.

CII solubilized in acetic acid may be stored frozen at -70°C. Under these conditions, CII remains stable for periods up to 3 years and possibly longer. This mode of storage has the advantage of avoiding the time required for solubilizing lyophilized collagen. CII stored in this manner retains arthritogenicity for long periods (years).

High concentrations of native CII—up to 6 mg/ml—are achievable, but at the expense of a highly viscous solution. Acetic acid concentrations >100 mM should be avoided as they only cause pain on injection and irritation at the injection site. If for some reason CII must be used in a neutral buffer, such as PBS or TBS, it should be first dissolved in acetic acid and then diluted or dialyzed in to the desired buffer.

Induction of CIA

As with most immunizations, complete emulsification of CII in either CFA or IFA is essential to establishing a high incidence of autoimmune arthritis. Additionally, care must be taken to ensure that the CII is kept cold throughout the emulsification procedure, and once emulsified, that it is kept cold until used. The use of Mycobacteria-containing adjuvants is not recommended in the rat model because complete Freund's adjuvant alone can induce an "adjuvant arthritis" in the rat (Pearson, 1963). Once arthritis has developed in either model, it is helpful to add food to the bottom of the cages as severely arthritic mice have difficulty feeding from the cage top.

Anticipated Results

CII preparation

Although yields of CII will vary widely with the species and tissue source, generally 1 % to 3% of the initial cartilage weight can be recovered as native CII.

Mouse and rat CIA

The incidence of arthritis in both the rat and mouse model following immunization with native CII should be >80%. In the mouse model, an incidence of 25% to 50% is expected when using either fragments of CII—e.g., CB11 or CB8 or a1(II) chains to induce arthritis. The absence of arthritis or the induction of only mild disease at a low incidence can usually be attributed either to inadvertent denaturation of the collagen, a poor quality emulsion, or poor injection technique. Booster injections do little to correct these problems.

Serum collected at the peak of arthritis—21 days after immunization in the rat model and 35 to 42 days after immunization in the mouse model—should yield positive results by ELISA at minimally 1:1000 dilution. High-responder rat strains (BB and WF) and mouse strains (DBA/1 and B10.RIII) frequently yield positive ELISA results at serum dilutions ranging from 1:10,000 to 1:100,000.

Time Considerations

Extraction and purification of CII from 100 to 400 g of cartilage will take minimally 4 weeks. Smaller quantities of starting material will not appreciably shorten this time.

Literature Cited

Brand, D.D., Myers, L.K., Whittington, K.B., Stuart, J.M., Kang, A.H., and Rosloniec, E.F. 1994. Characterization of the T cell determinants in the induction of autoimmune arthritis by bovine a1(II)-CB11 in H-2q mice. J. Immunol. 152:3088-3097.

Courtenay, J.S., Dallman, M.J., Dayan, A.D., Martin, A., and Mosedale, B. 1980. Immunisation against heterologous type II collagen induces arthritis in mice. Nature 283:666-668.

Cremer, M.A., Griffiths, M.M., Terato, K., and Kang, A.H. 1994a. Type XI and II collagen-induced arthritis in rats: Characterization of inbred rats for arthritis-susceptibility and immune-responsiveness to types XI and II collagen. Autoimmunity 20:153-161.

Animal Models for Autoimmune and

Inflammatory Disease

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