G. Oberhuber

Immunology of atherosclerosis: Cellular composition and major histocompatibility complex class II antigen expression in aortic intima, fatty streaks, and atherosclerotic plaques in young and aged human specimens

There is evidence that fatty streaks in arteries can transform into atherosclerotic plaques. Mononuclear cells, including both monocytes and lymphocytes, are among the first cells participating in the development of atherosclerosis of experimental animals. To investigate the roles of different cell types in human atherosclerosis, we enumerated and compared the cellular compositions of normal intima, the transition zone (the area between the normal intima and the core of fatty streaks), fatty streaks, and plaques in young (age 16-30 years) and aged (over 60 years) human specimens using double-staining immunofluorescence with a series of monoclonal and polyclonal antibodies. T lymphocytes, both T helper/inducer (70% of T cells) and T suppressor/cytotoxic (30%) phenotypes, were found in every stage of atherosclerosis, constituting 30 to 40% of total cells in fatty streaks and transition zones of young subjects, and occasionally even in normal intima. Seventy percent of these T cells were HLA-DR positive, which indicated that most of them were activated. Macrophages were most frequent in fatty streaks and around the necrotic core of plaques. Smooth muscle cells, increasing from 5 to 30% with lesion progression, were HLA-DR positive where activated T helper cells occurred in the vicinity. The intracellular presence of the invariant gamma chain confirmed that HLA-DR was actually synthesized by these smooth muscle cells. Endothelial cells were HLA-DR positive above those regions of the lesions where HLA-DR-positive cells had accumulated, but not in normal intima, again suggesting induction of HLA-DR expression by T-cell-derived gamma-interferon. Furthermore, most HLA-DR-positive cells were also identified as HLA-DP and HLA-DQ positive. This aberrant major histocompatibility complex class II antigen expression in smooth muscle and endothelial cells may participate in the perpetuation of the atherogenetic autoimmune reaction.

Histologic Pattern of Small Bowel Allograft Rejection in the Rat

Accessory small bowel transplantation was performed in a rat model, and the histologic sequence of acute rejection with and without immunosuppression with cyclosporine defined. The first rejection episodes occurred on the fifth postoperative day, defined as phase I. The mucosa and submucosa of the grafts were infiltrated by mononuclear and polynuclear leukocytes, and lymphocytes were scattered along the myenteric ganglia. Three to five days later (phase II) the infiltrate intensified and was associated with villous flattening and sloughing of epithelial cells. The muscular layer was invaded by numerous lymphocytes and neutrophils. At the end of the rejection episode complete destruction of the mucosa and the muscular layer developed (phase III). As no lesion other than lymphocytic infiltration could be detected in the mucosa in phases I and II of rejection, which may be considered reversible, and this change is known to be unspecific, biopsies of the mucosa alone are inadequate for a purely histologic diagnosis of small bowel allograft rejection.

Protective effects of various preservation solutions on cultured endothelial cells.

Vascular endothelium represents the first target in organ preservation and plays an important role in reperfusion injury. Bovine aortic endothelial cells were cultivated and the most commonly used preservation solutions, such as University of Wisconsin HTK (Brettschneiders histidine-tryptophane-ketoglutarate), and Euro-Collins solutions were tested on the endothelial monolayer. In addition, one group of cultivated cells was preserved with cold saline solution, and endothelial monolayers grown in culture medium were used as controls. The quality of preservation was assessed after 24, 48, and 72 hours of cold storage. Reperfusion was simulated and its effects were observed by reincubation in culture medium at 37 degrees C for 6 hours. The total number of cells, cell viability (determined using trypan blue exclusion), and morphologic alterations were determined. Prostacyclin release was evaluated as a biochemical marker. University of Wisconsin solution maintains more than 99% cell viability after rewarming after both 24 and 48 hours of cold storage. After 72 hours, 86.7% of cells were still viable. Preservation with HTK and Euro-Collins solution allowed cell survival for only 24 hours (96.7%, HTK; 49.9%, Euro-Collins), with no viable cells seen after 48 hours. The cold saline-preserved sample showed 57.8% viable cells after 24 hours and 29.7% after 48 hours. No viable cells were detectable after 72 hours. Light microscopy revealed several patterns of both structural damage and intracellular change (nucleus and cytoplasm) in the endothelial monolayer after preservation with HTK, Euro-Collins solution, and cold saline solution. No morphologic alterations were seen in the University of Wisconsin solution group for as long as 72 hours.(ABSTRACT TRUNCATED AT 250 WORDS)

ENDOGENOUS INTERLEUKIN 1 RECEPTOR ANTAGONIST DURING HUMAN BONE MARROW TRANSPLANTATION: Increased Levels During Graft-versus-Host Disease, During Infectious Complications, and After Immunoglobulin Therapy1

In order to understand in more detail the role of endogenous interleukin 1 receptor antagonist (IL-1ra) during bone marrow transplantation, IL-1ra serum levels of 28 patients undergoing allogeneic (n=25) or autologous (n=3) bone marrow transplantation were measured with a commercially available ELISA. In addition, the impact of intravenous immunoglobulin (IVIG) was evaluated by analyzing IL-1ra serum levels before and 2, 5, and 24 hr after IVIG infusion. IL-1ra measurements revealed a nadir of circulating IL-1ra levels 3-5 days after bone marrow transplantation, with an increase during conditioning and hematological reconstitution. Circulating IL-1ra levels were significantly increased in patients with cytomegalovirus (CMV) disease, CMV reactivation, graft-versus-host disease (GVHD), or fever of unknown origin, when compared with time-matched controls without complications. Highest levels were observed in patients with CMV disease (1922+/-388 pg/ml), followed by patients with CMV reactivation (1575+/-435 pg/ml) and GVHD (1178+/-317 pg/ml). The magnitude of IL-1ra increase in GVHD was related to disease severity. Patients with grade III-IV GVHD developed higher IL-1ra levels than did patients with grade I-II GVHD. Lower but still significantly elevated IL-1ra levels were observed during fever of unknown origin (384+/-87 pg/ml). An increase of IL-1ra serum levels followed the administration of IVIG before transplantation and after hematopoietic reconstitution, but not during aplasia, pointing to the important role of hematopoietic cells in the production of IL-1ra. In conclusion, we show that IL-1ra release is related to conditioning regimen, hematopoietic reconstitution, complications of infectious and alloimmune etiology after bone marrow transplantation, and exogenously administered IVIG.

Preservation of Small Bowel Grafts – A Comparison with Two Standard Solutions

This study investigates the use of EuroCollins (EC) and University of Wisconsin (UW) solution, two major preservation fluids, for small bowel preservation. After in situ flushing, grafts were cold-stored at 4 degrees C with either EC for 30 min (group 1a), 6 h (group 1b) and 12 h (group 1c) or with UW for 30 min (group 2a), 6 h (group 2b) and 12 h (group 2c). Using UW, cold ischemia was extended to up to 18 h (group 2d). As a control, small intestines were flushed and stored for the same time periods in cold saline (group 3a-c). Survival in group 1b was 66% versus 100% in group 2b. After 12 h 80% survived in group 2c, but there were not survivors in group 1c. After 18 h of cold storage, survival was only observed in group 2d (25%). Saline was ineffective after 6 h of preservation. Histology at the end of preservation revealed characteristic changes for EC (intracellular vacuoles) and UW (amorphic granules). We conclude that with UW small bowel can be preserved for up to 12 h.

Increase in Intraepithelial Lymphocytes as an Early Marker of Rejection in a Fully Allogeneic Rat Small Bowel Transplantation Model

In a model of fully allogeneic heterotopic rat small bowel transplantation, the changes in intraepithelial lymphocyte (IEL) number and subpopulations were analysed. During early phases of rejection (5th postoperative day) a 4-fold increase in the number of IEL was observed when compared with native small bowel (4.05 vs. 15.84 IEL/100 epithelial cells). When cyclosporine was given, counts were still as high as 11.4 and 12.58 on the 5th and 10th postoperative days, respectively. The percentage of CD8+ IEL, constituting a major population (84%) in the untreated small bowel, was significantly decreased (46.4%) during early phases of rejection. At that time, the majority of intraepithelial mononuclear cells were both CD8- and CD4-. In cyclosporine-treated animals, this was not observed until the 10th postoperative day. Some 23% of IEL in untreated animals expressed MHC class I antigens of the host; 17.2% (5th postoperative day) and 19.8% (10th postoperative day) did so in the cyclosporine-treated animals. Transmission electron microscopy revealed lymphocytes that bore cytoplasmic buds and pseudopods protruding between the enterocytes. There was no morphological difference between the IEL of rejected allografts and native small bowel. Due to the unspecific histologic changes associated with rejection, interpretation of histopathologic findings in mucosa biopsies of the allograft can be rather troublesome. An increase in the number of IEL is therefore a welcome additional marker of rejection.

Altered distribution of MHC class II antigens on enterocytes during acute small bowel allograft rejection in rats

Class II major histocompatibility complex (MHC) antigen induction was investigated on enterocytes of heterotopic rat small bowel allografts in the Lewis-Brown Norway strain combination and on isografts in the Lewis-Lewis strain combination. I a antigens were detected with monoclonal antibodies using an immunoperoxidase technique. Generally, MHC class II antigens were not exhibited in the isografted group, with the exception of two long-term isografts that presented the same pattern as normal small bowel. In these cases, I a was expressed in a patchy distribution predominantly in the villi, and only very few enterocytes stained positive in Lieberkühns crypts. Allografted rats showed a typical pattern of I a expression on the enterocytes during the rejection course. The initial expression was confined to the crypts, indicating a very early stage of rejection when compared to histological findings. More advanced stages of rejection were accompanied by increasing I a biosynthesis in the crypts and I a expression by the epithelium lining the villi. Cyclosporin (CyA) was not able to fully inhibit MHC class II antigen expression; however, the appearance of I a was delayed.

The pattern of rejection after combined stomach, small bowel, and pancreas transplantation in the rat

This study was designed to investigate whether in combined stomach, smallbowel, and pancreas transplantation allograft rejection occurs in the individual organs concomitantly and with the same intensity. Heterotopic enbloc transplantation of the stomach, small bowel, and pancreas was performed in a Lewis‐to‐Brown Norway rat combination. Group 1 animals received no immunosuppressive therapy while animals in group 2 were treated with cyclo‐sproin (10 mg/kg body weight, orally) daily. Grafts were histologically evaluated on the 5th (subgroups 1 a and 2a) and 10th (subgroups lb and 2b) postoperative days. The degree of rejection was defined as moderate, intermediate, or severe according to predefined criteria. The results indicate that the small bowel is more susceptible to rejection than either the stomach or the pancreas. Mucosal biopsies of the stomach are unlikely to provide a reliable guide to rej ection in the small bowel.

Regeneration of lymph drainage following transplantation of the small intestine

SummarySince the resorption of long-chained fatty acids proceeds exclusively via the lymphatics, the regeneration of lymphatic flow from small bowel grafts takes on a significant role. An isogene, heterotopic small bowel transplant was performed in a total of 54 Lewis rats, whereby 27 animals received no immunosuppression (group A) and the remaining 27 rats received cyclosporin A (group B). The lymphatic regeneration was determined by instilling dye into the mesenterial lymph nodes of the graft between day 1 and day 42 after transplantation and measuring the interval between instillation and appearance in the retroperitoneal lymph channels of the host. Additionally, the severity of the lymph edema was examined in histological slides, and the lymph vessels of the villi were examined for the presence of chylomicrons. In group A the passage of the dye into the lymphatic system first occurred on day 3 after transplantation, namely after a mean of 11.8 min, while in group B the lymph channels of the retroperitoneum first became visible on day 7 after transplantation (mean 8 min after instillation). Passage of the dye out of the lymphatic system of the graft was prompt and unhindered from day 10 in group A and from day 14 after transplantation in group B. In all grafts there was a marked interstitial lymph edema during the first week, and it was less severe in both groups up to day 14. Chylomicrons in the villi were observed in group A as of day 10 and after two weeks in group B. Regeneration of the lymphatics in small bowel grafts thus appears to be adequate for enteral nutrition only after 14 days.ZusammenfassungDa die Resorption langkettiger Fettsäuren ausschließlich über den Lymphweg funktioniert, kommt der Regeneration des Lymphabflusses aus Dünndarmtransplantaten eine entscheidende Rolle zu. Bei insgesamt 54 Lewis Ratten erfolgte eine isogene, heterotope Dünndarmtransplantation, wobei 27 Tiere keine Immunosuppression (Gruppe A) und die restlichen 27 Ratten Cyclosporin A erhielten (Gruppe B). Die Regeneration des Lymphabflusses wurde dadurch ermittelt, daß zwischen dem ersten und dem 42. Tag nach Transplantation ein Farbstoff in die mesenterialen Lymphknoten des Transplantates instilliert und die zeitliche Verzögerung zwischen Instillation und Darstellung der retroperitonealen Lymphbahnen des Empfüngers gemessen wurde. Zusätzlich wurde an histologischen Schnitten der Schweregrad des Lymphoedems beurteilt und die Lymphgefäße des Zottenstromas auf das Vorhandensein von Chylomicronen untersucht. In Gruppe A floß der Farbstoff erstmals am dritten postoperativen Tag nach durchschnittlich 11,8 min ab, während in Gruppe B die Lymphbahnen des Retroperitoneums erst ab dem siebten Tag nach Transplantation zur Darstellung kamen (durchschnittlich 8 min nach Instillation). Ein prompter, ungehinderter Abfluß aus den Lymphbahnen des Transplantates fand in Gruppe A ab dem 10. und in Gruppe B ab dem 14. postoperativen Tag statt. Ein interstitielles Lymphoedem war wührend der ersten Woche in allen Transplantaten deutlich ausgeprügt und in beiden Gruppen bis zum 14. Tag nur noch schwach ausgebildet. Chylomikronen im Zottenstroma konnten in Gruppe A ab dem zehnten Tag und in Gruppe B erst nach zwei Wochen beobachtet werden. Die Regeneration des Lymphabflusses aus Dünndarmtransplantaten scheint somit erst nach 14 Tagen für eine enterale Belastung ausreichend zu sein.Since the resorption of long-chained fatty acids proceeds exclusively via the lymphatics, the regeneration of lymphatic flow from small bowel grafts takes on a significant role. An isogene, heterotopic small bowel transplant was performed in a total of 54 Lewis rats, whereby 27 animals received no immunosuppression (group A) and the remaining 27 rats received cyclosporin A (group B). The lymphatic regeneration was determined by instilling dye into the mesenterial lymph nodes of the graft between day 1 and day 42 after transplantation and measuring the interval between instillation and appearance in the retroperitoneal lymph channels of the host. Additionally, the severity of the lymph edema was examined in histological slides, and the lymph vessels of the villi were examined for the presence of chylomicrons. In group A the passage of the dye into the lymphatic system first occurred on day 3 after transplantation, namely after a mean of 11.8 min, while in group B the lymph channels of the retroperitoneum first became visible on day 7 after transplantation (mean 8 min after instillation). Passage of the dye out of the lymphatic system of the graft was prompt and unhindered from day 10 in group A and from day 14 after transplantation in group B. In all grafts there was a marked interstitial lymph edema during the first week, and it was less severe in both groups up to day 14. Chylomicrons in the villi were observed in group A as of day 10 and after two weeks in group B. Regeneration of the lymphatics in small bowel grafts thus appears to be adequate for enteral nutrition only after 14 days.

Small Bowel Transplantation in the Rat: Impact of Various Immunosuppressive Regimens on Graft-versus-Host Reaction

The effect of ciclosporin (CS) and methotrexate (MTX) on the development of graft-versus-host (GvH) disease was examined after small bowel allotransplantation in the rat. The drugs were tested either alone or in combination. Lewis small bowel allografts were transplantated into Brown Norway recipients in a heterotopic position. The native small bowel, spleen, liver, skin, mesenteric lymph nodes and the kidney of the recipients were examined histologically 5, 10 and 20 days after allotransplantation. Intraepithelial lymphocyte numbers were determined quantitatively in the native small bowel. The relative spleen weight of the host was determined after sacrifice for estimation of the severity of GvH disease. Grade I GvH reaction of the native small bowel occurred in the animals without immunosuppression, but graft rejection predominated in this group. Treatment with CS was effective in the early postoperative periods; after 10 and 20 days GvH lesions in the native small bowel were comparable to those observed in the allogeneic combinations. MTX had a detrimental effect on the allografts and the GvH reaction was augmented. When CS and MTX were combined, GvH lesions were comparable to those in the animals treated solely with CS. Animals, however, suffered from heavy side effects. The spleen, liver, lymph nodes and kidney exhibited only unspecific histologic changes, which could not unequivocally be recognized as a GvH reaction. This was true for all groups. As a conclusion it can be said that GvH reaction occurs in the early postoperative period in a fully allogeneic model and cannot be prevented by CS in the dosae used. MTX was not seen to be of any value in this regard.