Diabetes-resistant (DR)-BB rats with induced diabetes offer advantages over spontaneously diabetic diabetes-prone (DP)-BB animals. Untreated DR rats are not lymphopenic and their T cell populations are normal with respect to phenotype and function. Treatments to induce diabetes do not induce severe lymphopenia. In addition, T cells from diabetic DR-BB rats adoptively transfer IDDM with no requirement for mitogen activation (see Alternate Protocol 2). Another practical advantage is that cells can be obtained from cohorts of young DR-BB rats treated to become diabetic at approximately the same age. This simplifies experimental design and reduces costs, because, for example, DR-BB rats treated at 30 days old will become diabetic between 44 and 51 days of age, whereas DP-BB rats need to be housed and tested from 55 to 120 days of age to identify all potentially diabetic animals.
This protocol describes a method for treating 21- to 30-day-old nonlymphopenic DR-BB rats with a cytotoxic monoclonal antibody (MAb) against the T cell alloantigen RT6, together with poly(IC), to induce IDDM (Thomas et al., 1991). It is based on transient depletion of RT6+ T cells that are known to regulate the immune system and can prevent spontaneous IDDM (see Basic Protocol 2). Poly(IC) is used because IDDM occurs in DR-BB rats that have been infected with certain viruses. Poly(IC) is a synthetic double-stranded polyribonucleotide that elicits immune responses analogous to those observed during viral infection; specifically, it induces interferon production. This treatment induces IDDM in >90% of both male and female viral-antibody-free (VAF) DR-BB/Wor rats within 14 to 21 days.
Additional Materials (also see Basic Protocol 1)
Hybridoma DS4.23 cells (available from the Division of Diabetes, University of
Massachusetts Medical School, Worcester, Mass.) Complete RPMI medium (appendix2) supplemented with 2% (v/v) FBS, hybridoma serum supplement, or DR-BB rat serum (RPMI-2) Four 4- to 6-week-old DR-BB rats (available from Dennis Guberski; see Critical
Parameters and Troubleshooting) 21- to 30-day old DR-BB rats of either sex (available from Dennis Guberski) Polyinosinic:polycytidylic acid, sodium salt [poly(IC), Sigma], sterile PBS (appendix 2), sterile
Additional reagents and equipment for preparing MAb supernatants (unit 2.6), removal of lymphoid tissues (unit 1.9), immunofluorescence staining of single-cells suspensions (unit5.3), and screening and diagnosis of IDDM (Basic Protocol 1)
1. Prepare MAb supernatant from cultures of DS4.23 cells grown in complete RPMI-2 medium and filter sterilize (unit 2.6).
DS4.23 secretes rat Ig2b anti-rat RT6.1, the RT6 allele expressed in BB rats.
2. Verify the in vivo depleting activity of each MAb preparation by treating three 4- to 6-week-old DR-BB rats with 2 ml DS4.23 supernatant once daily for 3 days. Use an
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Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...