Silke Lucke

Eignung spezifischer Färbemethoden für die Bestimmung des β-Zellvolumens im Rattenpankreas mit normalem and reduziertem Insulingehalt

Zusammenfassung Die Untersuchungen wurden an mit Streptozotocin (SZ) behandelten, weiblichen Wistarratten (einmalige i.v. Injektion von 30 mg/kg Korpermasse) Bowie citratpuffer-behandelten Kontrolltieren durchgefuhrt. Nach Farbung der pankreatischen β -Zellen mit Aldehydfuchsin (AF), Viktoriablau nach Ivic bzw. FITC-markiertem Antiserum (indirekt) wurde das β -Zellvolumen nach einem point-sampling Verfahren ermittelt. Bei den Kontrolltieren ergeben alle 3 genannten β -zellspezifischen Farbemethoden das gleiche β -Zellvolumen. Mit Reduktion des Insulingehaltes im Pankreas and in den β -Zellen durch SZ-Applikation nimmt die Empfindlichkeit der histochemischen Farbemethoden im Vergleich zum immunhistochemischen Nachweis ab. Normoglykamische SZ-behandelte Tiere (Pankreasinsulingehalt ca. 30 % der Kontrollwerte) zeigen nach der AF-Farbung ein β -Zellvolumen von 0,22 %, nach der Ivic-Farbung von 0,14 %. Bei hyperglykamischen Tieren (ca. 6 % des Pankreasinsulingehaltes der Kontrolle) sind mit der AF-Farbung 0,08 % and nach Ivic 0,03 % β -Zellvolumen mesbar. Mit der Immunfluorescenzmethode Bind die β -Zellvolumine beider SZ-behandelten Gruppen identisch (0,34 %). Die nach SZ-Behandlung auftretende Abnahme des Pankreasinsulingehaltes ist auf die Reduktion des β -Zellvolumens, vor allem aber auf die starke Abnahme des Insulingehaltes pro β -Zelle zuruckzufuhren.

Endocrine pancreas histology of congenic BB-rat strains with reduced diabetes incidence after genetic manipulation on chromosomes 4, 6 and X.

Congenic BB.SHR rat strains were established by crossing of spontaneously diabetic BB/OK rats and diabetes-resistant SHR rats. Chromosomal regions on which the genes Iddm 4 (BB.6s), Iddm6 (BB.Xs) and Iddm 2 (BB.LL) are located were exchanged. As a result of genetic manipulation diabetes incidence was markedly reduced from 80% in BB/OK to 50% in BB.SHR (Chr. X), to 14% in BB.SHR (Chr. 6) and to 0% in BB.LL rats. Pancreata of these newly generated BB.SHR rats were investigated histologically. In newly diagnosed diabetic rats of congenic strains pancreatic insulin content (BB.6s: p<0.05; BB.Xs p<0.01) and relative volume of insulin-positive cells (BB.Xs: p<0.001) were significantly higher than in BB/OK rats. The degree of insulitis was not different in 90-day-old and newly diagnosed diabetic animals. Surprisingly, in 30-day-old rats we observed an increase of the degree of insulitis with decreasing diabetes incidence. We suppose that by an earlier occurrence of the immunological β-cell destruction, a part of the animals is able to develop a secondary diabetes resistance. The exchange of the BB-lymphopenia gene by that of SHR-rats prevented the development of hyperglycaemia without altering the auto-reactive immune response, which could be observed in all animals investigated.

Immunotherapy of insulin-dependent diabetes mellitus: Tolerance after short-term anti—;IL-2 receptor/cyclosporine therapy in BB/OK rats

There is convincing evidence that the destruction of pancreatic B-cells leading to insulin-dependent diabetes mellitus (IDDM) is immunologically mediated1–3 and occurs in all mammals.4 Investigations of animal strains predominantly used in diabetes research, the NOD mouse and the BB rat, demonstrated that the acute onset of hyperglycemia is the final result of a chronic preclinical phase (prediabetes). Already during prediabetes and definitely at the onset of the disease, the number of pancreatic B-cells is reduced.5–11 In addition to the diminished number of B-cells, the pancreatic islets of prediabetic or newly diagnosed diabetic animals or patients are characterized by an invasion of immunocytes, so-called insulitis.5,6,9–14 The immunocytochemistry of invading mononuclear cells (MNC) reveals the presence of all major subsets, such as T-lymphocytes (CD4 and CD8 T-cells), B-lymphocytes, macrophages, and NK-cells.8,10–15 Electron microscopic investigations of inflafhed pancreatic islets demonstrated a close contact between immune cells and pancreatic B-cells. As a result of B-cell destruction, B-cell granules have been observed in the extracellular space.15–17 Although the molecular mechanism of B-cell killing is still not established, cytotoxic reactivities of CD4 T-cells,18 CD8 T-cells,19 NK cells,20 and macrophages21 against pancreatic B-cells have been demonstrated. In addition, it has been shown that the secretory products of immunocytes, the cytokines, may directly disrupt B-cells,22 or may induce surface proteins as MHC-antigens or adhesion molecules, which allow or facilitate cell-mediated killing of B-cells.23

Is the DNA Synthesis of Pancreatic Islets Influenced by the MHC and/or the Genetic Background?

SummaryNeonatal pancreatic rat islets were used to investigate the effect of MHC haplotype RT1u on islet replication measured by incorporation of3H-thymidine and autoradiography. The special interest in RT1u resulted from the fact that BB rats, which develop an insulin-dependent diabetes, belong to the RT1u haplotype. Thus, the RT1u, when influencing the replication, may be involved in the development of the B-cell deficiency observed in diabetic rats.The incorporation of labeled thymidine into islets obtained from rat strains carrying the RT1u or RTF haplotype revealed no differences. No matter whether the islets were isolated from Lewis, Wistar, or BB rats, no alteration of islet replication by RT1u islets was found. However, islets obtained from Wistar rats had a higher replicatory activity than islets from Lewis rats. This finding was confirmed by a decrease in DNA synthesis of BB rat islets, in which the Wistar background was substituted by the Lewis background. We conclude from our results that MHC has no influence on islet replication, whereas genetic background seems to be involved in the regulation of islet DNA synthesis.