When applying these methods to study other signaling molecules, it is important to design the study to analyze various forms and fractions of the molecules by using antibodies that can bind to multiple forms rather than one structural epitope. Normal T cells or autoimmune disease controls such as rheumatoid arthritis, Sjogren's syndrome, Still's disease, limited systemic sclerosis, mixed connective tissue disorder, antiphospholipid syndrome, and dermatositis can be used as controls for SLE. Similarly, when the size of the molecule of interest and endogenous controls are significantly different, it is reasonable to combine the antibodies when probing the immunoblots to reduce experimental time and too much stripping of blots. Samples taken on different dates can be compared by normalizing the data against a proper internal control.
Studies showed that, in addition to abnormal expression of key signaling molecules, the downstream signaling molecules that associate and participate in signaling function are altered in SLE T cells. Accordingly, it is important to include studies of downstream signaling molecules and their function in SLE T cells by TCR activation using antibodies or by non-receptor-mediated stimulation using phorbol-12-myristate 13-acetate/ionomycin. Abnormalities in signaling molecules should be correlated with the SLE disease activity index, current medication, and demographics (race, gender, age) to distinguish abnormalities intrinsic to the disease from those associated with disease activity. If possible, it is also a good idea to request that patients not take any medication at least 24 h before drawing the blood sample to reduce the effects of medication. To confirm the results further, the study should be designed to follow up the patients over a period of 3-4 yr, during which the SLE disease activity will improve considerably.
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