Degradation and extraction of high molecular weight DNA from formaldehyde fixed tissues suitable for gene analysis are presented. We previously reported that DNase might play an important role in the degradation of DNA extracted from formaldehyde fixed tissues (Tokuda et al. 1990). In the present study, DNase activity of the supernatant from rat tissues fixed in buffered formaldehyde at room temperature was negligible within 3 hr. Analysis of DNA extracted from reconstituted chromatin revealed that the degradation increased in the absence of DNase depending on the duration of the formaldehyde fixation. Furthermore, high molecular weight DNA could be extracted from tissues devoid of DNase activity fixed in buffered formaldehyde containing EDTA. These results demonstrated that DNA degradation was due mainly to a mechanism other than DNAse which was inhibited by EDTA. For clinical application, v-H-ras gene was successfully detected by Southern blotting from rat spleen tissues fixed in buffered formaldehyde especially at 4 C. Fixation at low temperature is useful for gene analysis.
Diverse adhesion molecules participate in many important responses and thus would be implicated in the pathogenesis of various autoimmune diseases. However, there is little evidence for the role of these molecules in autoimmune insulin-dependent diabetes mellitus. Here we present several lines of evidence suggesting that leukocyte function-associated antigen-1 (LFA-1) and its counter-receptor intercellular adhesion molecules (ICAM-1), one of the most important pairs among these adhesion molecules, are involved in the development of autoimmune diabetes in the non-obese diabetic (NOD) mouse. Immunohistochemical study showed the hyperexpression of ICAM-1 on islet-infiltrating mononuclear cells and vascular endothelium in NOD pancreas. In vivo administration of anti-LFA-1 or anti-ICAM-1 mAb from 5 to 30 (or 12) weeks of age exerted a very strong preventative effect on the development of spontaneous diabetes with a marked reduction of insulitis, whereas both antibodies, even combined to use simultaneously, could not prevent cyclophosphamide-induced diabetes. Adoptive transfer of insulitis and diabetes to young NOD mice following the injection of islet-derived mononuclear cells from diabetic donors was completely blocked by administration of both antibodies to recipients. The present study, therefore, provides the first evidence that immunointervention to LFA-1-ICAM-1 interaction has a strong prophylactic effect on autoimmune diabetes in NOD mice.
The present study demonstrated that a short-term administration of mAbs against leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) at critical periods resulted in complete protection of autoimmune diabetes in non-obese diabetic (NOD) mice. When these mAbs were administered for only 6 days at 2 wk of age, neither diabetes nor insulitis was observed at 30 wk of age. It appears that the tolerance against beta cell Ag(s) was induced by this transient blockade of the LFA-1/ICAM-1 pathway. Protective suppressor activity was not enough to prevent diabetes because co-transfer of splenocytes from female NOD mice, which had received these mAbs at 2 wk of age, resulted in only a short delay of the diabetic onset caused by adoptive transfer of splenocytes from acutely diabetic NOD mice. Transfer of these splenocytes to young NOD mice could not also abrogate the spontaneous diabetes and insulitis. Furthermore, cyclophosphamide treatment could not abrogate the protection. When splenocytes from the treated NOD mice were transferred to NOD-SCID mice, none of the recipient mice developed significant insulitis and subsequent overt diabetes, suggesting the absence or the inactivation of diabetogenic effector T cells. However, splenic T cells from the insulitis-free NOD mice that had received the mAb treatment preserved proliferative responses to both islet cells and 65-kDa glutamic acid decarboxylase (GAD65) in vitro. These results suggest that a unique peripheral tolerance was induced by the transient blockade of the LFA-1/ICAM-1 pathway in an early age of NOD mice.
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