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Background: Progressive injury that is refractory to conventional immunosuppression remains the major hurdle to indefinite survival of transplanted organs. Several clinical risk factors of chronic renal allograft rejection have been identified; although some (e.g., acute rejection) are direct manifestations of immunological injury, others (e.g., donor age) have been more difficult to conceptually link with graft dysfunction., Methods: We conducted formal multivariate statistical analyses to reveal associations between established clinical risk factors and allograft histopathology. In a multicenter protocol biopsy-controlled study, 17 clinical risk factors were studied in relation to either the composite Chronic Allograft Damage Index (CADI) score or, to each of eight individual histological indices, using multiple linear regression with forward selection., Results: Nine clinical risk factors were not significantly associated with any histopathological index. Four (donor age, acute rejection, recipient age, and cold ischemia time) were associated both with the total CADI score and, to varying extents, with the individual histopathological indices. In our analysis, clinical risk factors accounted for, at best, only about 60% of the interindividual variation in histopathological score., Conclusions: Our study reveals a missing link between specific clinical risk factors and early histopathological findings that are known to presage accelerated failure of clinically healthy grafts. Given the complex relationship between clinical risk factors, early histopathological changes, and graft outcome, we conclude that composite, quantitative histological indices are best suited to for evaluation of the histological status of the transplant.

Chronic allograft damage index (CADI) is a semi-quantitative histopathological score that predicts renal graft outcome. We aimed to develop an objective image analysis-based method for quantification of CADI parameters. Thirty-five kidney transplant biopsies were visually analyzed according to the original CADI criteria, and divided into normal, mildly, moderately and severely altered groups. Digital images of the same samples were then analyzed with IPLab software. Areas of inflammation and fibrosis measured using image analysis increased simultaneously with corresponding visual scores, although the difference between non-inflamed and mildly inflamed groups was not statistically significant. Area of normal tubuli decreased in the images of samples with visually mild/moderate tubular atrophy and tended to be even smaller in the group with severe tubular atrophy. Image analysis-based glomerular sclerosis score increased concurrently with increasing visual score. Mesangial matrix increase score in image analysis was greater in the samples with visually mild/moderate mesangial matrix increase score compared to those with normal glomeruli, and it was highest in the group with severe mesangial matrix increase. An image analysis-based CADI scoring of renal allograft biopsies could provide more precise data for scientific studies, and help pathologists in renal allograft biopsy scoring.

Following encouraging results from several single-center studies showing that early histological manifestations of chronic rejection are seen in the graft before a decline in transplant function, we tested this concept in a multicenter study and investigated whether protocol needle biopsy may be used as a surrogate to late graft survival in multicenter renal transplantation trials. During two mycophenolate mofetil trials, 621 representative protocol biopsies were obtained at baseline, 1 year, and 3 years. The samples were coded and evaluated blindly by two pathologists and a Chronic Allograft Damage Index (CADI) score was constructed. At 1 year only 20% of patients had elevated (>1.5 mg/100 mL) serum creatinine, whereas 60% of the biopsies demonstrated an elevated (>2.0) CADI score. The mean CADI score at baseline, 1.3 +/- 1.1, increased to 3.3 +/- 1.8 at 1 year and to 4.1 +/- 2.2 at 3 years. The patients at 1 year were divided into 3 groups, those with CADI <2, between 2 and 3.9, and >4.0, the first two groups having normal (1.4 +/- 0.3 and 1.5 +/- 0.6 mg/dL) and the third group pathological (1.9 +/- 0.8 mg/dL) levels of serum creatinine. At 3 years there were no lost grafts in the "low" CADI group, six lost grafts (4.6%) in the "elevated" CADI group, and 17 lost grafts (16.7%) in the "high" CADI group (P <.001). One-year histological CADI score predicts graft survival even when the graft function is still normal. This observation makes it possible to use CADI as a surrogate endpoint in prevention trials and to identify the patients at risk for intervention trials.

We investigated the effects of ileum allotransplantation and chronic graft rejection on the synthesis and absorption of cholesterol. Twenty pigs underwent intestinal transection or ileum transplantation, in which the distal half of the jejunoileum was replaced with an ileal autograft or allograft. Conventional triple therapy with cyclosporine (10 mg/kg per day), azathioprine and methylprednisolone was tapered to cyclosporine (5 mg/kg per day) after 10 weeks. Serum lathosterol and campesterol, respective markers of cholesterol synthesis and absorption, were determined and related to graft histology. When compared to transected controls, auto- and allotransplantation of ileum similarly increased ( P<0.05) lathosterol and decreased ( P<0.01) campesterol for 12 weeks, despite normal graft histology. Chronic graft rejection progressed between 12 and 18 weeks, when a further increase in lathosterol (+104%) and decrease in campesterol (-67%) was observed. Obliteration of mesenterial arteries in chronically rejecting grafts was associated with high cholesterol synthesis ( R=0.975, P=0.0512). Auto- and allotransplantation of the ileum similarly modulate synthesis and absorption of cholesterol in pigs with non-rejecting grafts. Chronic rejection of the ileal graft appears to markedly increase cholesterol synthesis, which may primarily result from impaired ileal reabsorption of bile acids due to gradual obliteration of mesenterial arteries (chronic rejection). Serial measurements of cholesterol synthesis and bile acid absorption may prove to be a useful tool in the diagnosis of chronic rejection-associated small intestinal graft dysfunction.

This study is an investigation of whether a protocol biopsy may be used as surrogate to late graft survival in multicenter renal transplantation trials. During two mycophenolate mofetil trials, 621 representative protocol biopsies were obtained at baseline, 1 yr, and 3 yr. The samples were coded and evaluated blindly by two pathologists, and Chronic Allograft Damage Index (CADI) score was constructed. At 1 yr, only 20% of patients had elevated (>l.5 mg/100 ml) serum creatinine, whereas 60% of the biopsies demonstrated an elevated (>2.0) CADI score. The mean CADI score at baseline, 1.3 +/- 1.1, increased to 3.3 +/- 1.8 at 1 yr and to 4.1 +/- 2.2 at 3 yr. The patients at 1 yr were divided into three groups, those with CADI <2, between 2 and 3.9, and >4.0, the first two groups having normal (1.4 +/- 0.3 and 1.5 +/- 0.6 mg/dl) and the third group pathologic (1.9 +/- 0.8 mg/dl) serum creatinine. At 3 yr, there were no lost grafts in the low CADI group, six lost grafts (4.6%) in the in the elevated CADI group, and 17 lost grafts (16.7%) in the high CADI group (P < 0.001). One-year histologic CADI score predicts graft survival even when the graft function is still normal. This observation makes it possible to use CADI as a surrogate end point in prevention trials and to identify the patients at risk for intervention trials.

We investigated whether determination in fine-needle aspiration biopsy (FNAB) specimens of cells expressing granzymes (Grs) and Fas ligand would provide a reliable, easy, and quantitative measure of rejection activity in the transplanted liver. Retrospectively, 13 FNAB specimens obtained during clinical acute rejection, 10 FNAB specimens obtained during subclinical rejection, 12 FNAB specimens obtained during cytomegalovirus (CMV) infection, and 26 FNAB specimens obtained in the absence of rejection or infection were included on the study. Cytospin preparations of FNAB and peripheral-blood specimens were immunocytochemically stained for Fas-ligand and Gr, and increments in the liver were calculated by subtracting frequencies of positive cells in blood from those in FNAB specimens. Only sporadically Fas ligand-expressing, but many Gr-expressing, cells were detected in FNAB specimens. Increments in Gr-positive (Gr(+)) cells were significantly greater in FNAB specimens obtained during clinical rejection (median, 70 Gr(+) cells; range, 0 to 312 Gr(+) cells; P = .006) and tended to be greater in FNAB specimens obtained during subclinical rejection (median, 62 Gr(+) cells; range, 5 to 113 Gr(+) cells; P = .09) compared with those obtained in the absence of rejection (median, 16 Gr(+) cells; range, 0 to 103 Gr(+) cells). Increments obtained during clinical or subclinical rejection did not differ from those obtained during CMV infection (median, 27 Gr(+) cells; range, 6 to 212 Gr(+) cells). With the exclusion of specimens obtained during CMV infection, the sensitivity of Gr determination in FNAB specimens for the diagnosis of acute rejection (either clinical or subclinical) was 70%, and specificity, 69%. In FNAB specimens obtained during clinical and subclinical acute rejection episodes after liver transplantation, increased numbers of Gr-expressing cells were present; in the absence of CMV infection, their quantification provides a measure for rejection activity with moderate accuracy.

Acute kidney allograft rejection is characterized by a lymphocyte infiltration. L-selectin on lymphocytes and its endothelial glycosylated ligands are instrumental in the initiation of lymphocyte extravasation to sites of inflammation. From more than 500 core biopsy specimens taken from kidneys after transplantation, 250 biopsies were graded to have signs of acute rejection. Of these, 52 biopsies with various grades of histologic signs of acute rejection were selected for the study. Controls were 15 biopsies taken within 30 min after revascularization and 10 specimens from well-functioning allografts showing no clinical or histologic evidence of rejection. Immunochemical stainings with monoclonal antibodies against functionally active decorated L-selectin ligands. i.e., sialyl-Lewis x (sLex, 2F3 and HECA-452) or sulfated lactosamine (MECA-79) were performed. Although no endothelial 2F3 and MECA-79 epitopes were detected in nonrejecting control specimens, the expression was induced at the onset and during acute allograft rejections. The level of expression (in semi-quantitative score) of 2F3 reactivity correlated with the severity of rejection (P<0.0001, grade I versus grade IIB), and the level of expression decreased as the rejection resolved. Kidney biopsies taken shortly after revascularization and thus undergoing reperfusion injury showed endothelial staining with another anti sLex antibody, HECA-452. This staining disappeared from well-functioning grafts and reappeared at the onset of an acute allograft rejection. These results suggest that expression of functionally active, properly glycosylated L-selectin ligands might have a role in reperfusion injury and in the initiation of acute rejections after human kidney allograft transplantation.

L-selectin-dependent lymphocyte extravasation is a hallmark of acute heart allograft rejection in rats. On screening over 600 endomyocardial biopsies (EMBs), taken at different time points after heart transplantation in man, we identified 91 samples with histological signs of acute rejection. Rejection and nonrejection EMBs were analyzed for the presence of properly glycosylated, ie, sulfated sialyl Lewis-x (sLex) decorated L-selectin ligands. Two anti-sLex (2F3 and HECA-452) and one anti-6- or 6'-sulfated and/or 6, 6'-bisulfation (MECA-79) monoclonal antibodies were used. Nonrejecting heart endothelium did not express, or expressed only weakly, sulfated and or sLex decorations of L-selectin ligands. On the contrary, these epitopes were readily detectable on endothelium of capillaries and venules at the onset and during acute rejection episodes. The more intense the sulfated sLex expression was, the more severe the rejection episode was in histological grading. The endothelial expression of L-selectin ligands decreased to background levels as the rejection resolved. Our data demonstrate a complete correlation between the level of expression of the sulfated sLex-decorated ligands on the one hand and the histological severity of acute heart allograft rejection on the other hand. These data suggest that functionally active endothelial L-selectin ligands are instrumental in lymphocyte extravasation into the human heart allografts at the onset and during acute rejection episodes.

Porcine small bowel allografts were followed for 18 weeks during immunosuppression with cyclosporine-A (CyA), azatioprine and prednisone. The mucosal alterations noted at the 12th week were epithelial vacuolation and loss of Goblet cells. Moderate infiltration of inflammatory cells, mainly lymphocytes, was found in the lamina propria. In addition, a few grafts exhibited oedema and fibrosis. Vessels already showed endothelial swelling and intimal proliferation at the 12th week. In the submucosa, the infiltration of inflammatory cells was not present till the 18th week. Further changes in the mucosa at the 18th week were the blunting of villi, cuboidal epithelium, crypt abscesses and epithelial atrophy. The histological alterations of mucosa and lamina propria existing in the full thickness biopsies were mostly also detectable in mucosal biopsies, provided that multiple biopsies were taken. Thus these parameters analyzed from mucosal biopsy material are suitable for the diagnosis and monitoring of chronic small bowel rejection. In autopsy, the most prominent features were in the mesenterial arteries: intimal proliferation, vasculitis, proliferation of media and endothelial alterations. The activity of the mucosal disaccharidases maltase and sucrase remained near the initial level till the 12th week and had decreased markedly by the 18th week.

Objective: To examine the changes in brush cytology during acute small bowel allograft rejection and to evaluate the usefulness of cytology in detecting acute rejection., Study Design: Heterotopic porcine small bowel allografts were followed by brush cytology and full-thickness biopsies during unmodified rejection., Results: The most prominent changes in cell differential counts were an increase in the proportion of granulocytes and decrease in the proportion of epithelial cells. In brushed epithelial fragments, infiltration of granulocytes and acidophilia increased, and cell cohesion was gradually lost with the degeneration of nuclei and necrosis of epithelium. The cell differential count was compared to the histologic acute rejection index, which was created on the basis of five semiquantitatively evaluated histologic parameters. The sensitivity of cell differentiation in detecting histologically moderate or severe rejection was 87% (13 of 15 cases) and the specificity 76% (5 false positives in 21 negative cases)., Conclusion: Suspicion of acute rejection can be assessed with reasonable reliability by cytology, but it cannot be detected earlier than by histology. Brush cytology complements mucosal biopsies in the evaluation of rejection and, as a rapid and cost-effective method, may even partly replace them. It may also prove valuable in detecting opportunistic bowel infections in immunosuppressed patients.

The value of mucosal biopsies in evaluating small bowel rejection is controversial. In this study, the value of mucosal biopsies was estimated in unmodified porcine small bowel rejection. Ten animals received the distal half of the small bowel as a heterotopic loop (Thiry-Vella loop). The allografts were followed by proximally and distally harvested full-thickness and mucosal biopsies every other day, starting from the 3rd day and continuing until the grafts became necrotic. The histological parameters in both types of biopsies were semiquantitatively scored from 0 to 3 and compared with each other. The difference in mean values on the subsequent days was not remarkable, the results favoring slightly higher values in full-thickness than in mucosal biopsies. Our results suggest that multiple mucosal biopsies are adequate in monitoring morphological changes of small bowel grafts during rejection and that the proximal and distal ileum are similarly affected by acute rejection.

The change in activity of three disaccharidase enzymes (maltase, sucrase and lactase) was determined according to the method of Dahlqvist during acute rejection in non-immunosuppressed piglet small bowel grafts. In addition, two brush border enzymes, lactase and aminopeptidase, were stained with monoclonal antibodies. Diminishing disaccharidase activity was an early event during rejection. Diminution began 2 days before distinct morphological changes were seen in the mucosal biopsies. Evaluation of disaccharidase activity can thus be used as a confirmatory method in detecting rejection. Reduction in immunohistological staining of lactase and aminopeptidase with monoclonal antibodies and changes in mucosal morphology were observed to progress simultaneously.

Acute organ transplant rejection is characterized by a heavy lymphocyte infiltration. We have previously shown that alterations in the graft endothelium lead to increased lymphocyte traffic into the graft. Here, we demonstrate that lymphocytes adhere to the endothelium of rejecting cardiac transplants, but not to the endothelium of syngeneic grafts or normal hearts analyzed with the in vitro Stamper-Woodruff binding assay. Concomitant with the enhanced lymphocyte adhesion, the cardiac endothelium begins to de novo express sialyl Lewis(a) and sialyl Lewis(x) (sLea and sLex) epitopes, which have been shown to be sequences of L-selectin counterreceptors. The endothelium of allografts, but not that of syngeneic grafts or normal controls, also reacted with the L-selectin-immunoglobulin G fusion protein, giving further proof of inducible L-selectin counterreceptors. The lymphocyte adhesion to endothelium could be significantly decreased either by treating the lymphocytes with anti-L-selectin antibody HRL-1, or by treating the tissue sections with sialidase or anti-sLea or anti-sLex monoclonal antibodies. Finally, we synthetized enzymatically several members of the sLex family oligosaccharides and analyzed their ability to block lymphocyte adhesion to cardiac endothelium. The monovalent sLex (a tetramer), divalent sLex (a decamer), and tetravalent sLex (a 22-mer) could all significantly reduce lymphocyte binding, but the inhibition by the tetravalent sLex-construct was clearly superior to other members of the sLex family. The crucial control oligosaccharides, sialyl lactosamines lacking fucose but being otherwise similar to the members of sLex family, had no effect on lymphocyte binding.

The relevance of hyperlipidemia in allograft arteriosclerosis (chronic rejection) is controversial. Isolated hypercholesterolemia induced with cholesterol-cholic acid-diet (CC-diet) or hypertriglyceridemia induced with glycerol-diet (G-diet) had no or only a protective effect on aortic allograft arteriosclerosis in the rat. Combined hyperlipidemia with both diets (CC+G-diet) enhanced allograft arteriosclerosis by doubling intimal thickness and cellularity (P < .05) but had no effect on host arteries. Compared with normolipidemic controls, the CC+G-diet increased the total serum cholesterol concentration 4.8-fold (P < .05). Levels of VLDL2 and IDL increased 4.8- and 18.1-fold (P < .05), and their composition changed from triglyceride-rich to cholesterol-rich lipoproteins in an atherogenic direction. The CC+G-diet had no effect on the structure of inflammation in the vascular wall. Instead, significant lipid deposits were observed, and the expression of epidermal growth factor and insulin-like growth factor-1 was significantly elevated in the vascular wall. Thus, elevations in VLDL and IDL lipoprotein levels and their cholesterol content associate with the generation of allograft arteriosclerosis in rats. Deposition of lipids in the vascular wall seems to induce local synthesis of certain growth factors, which ultimately leads to the induction of smooth muscle cell replication.

Using nonimmunosuppressed piglet small bowel grafts, we performed a detailed histological study on acute rejection and analyzed altogether 45 different histological parameters throughout the bowel wall, in 10 allografts and 6 autografts. Heterotopic grafts were followed by full-thickness biopsies every other day beginning on the 3rd day after transplantation. The parameters were scored from 0 to 3 according to the severity of the change and the values of every variable were compared statistically between the 2 groups. The earliest significant changes, beginning on days 3-5, were infiltration of inflammatory cells and pyroninophilia among these cells in lamina propria; edema, fibrosis, and inflammation in submucosa and muscularis; vacuolar degeneration of myocytes; and endothelial and intimal changes in vessels. Villous blunting and cuboidal epithelium were the other early markers. On day 7, the epithelial and vascular changes became most significant. We want to emphasize especially the vascular parameters: endothelial swelling and proliferation, intimal thickening, intramural inflammation, and obliteration of the lumen. The dilatation of lymph vessels and changes of Peyer's patches had no value in the estimation of rejection. All allografts became necrotic by day 11 after transplantation.

Kidney allograft rejection is an inflammatory process dominated by lymphocytes. During rejection lymphocytes preferentially adhere to the peritubular capillary endothelium (PTCE), which acquires morphological features common to high endothelium. These observations indicate that PTCE is the site of lymphocyte entry into the rejecting renal allograft. Of the identified endothelial adhesion molecules, ICAM-1 was already expressed on the endothelium of normal kidneys, and its expression was strongly enhanced during rejection without site-specific restriction. VCAM-1 was not expressed on the endothelium of normal or syngeneic kidneys, but its expression was induced during allograft rejection not only in PTCE, but occasionally also on the endothelium of larger vessels. Sialyl Lewisx (sLex) showed a very restricted pattern of expression; endothelium was sLex-negative both in control and syngeneic kidneys. On the other hand, PTCE reacted strongly with anti-sLex antibody in allografts. When kidney frozen sections were treated with sialidase the binding of lymphocytes decreased by 70%. Low-dose chymotrypsin treatment of lymphocytes, known to remove L-selectin from the lymphocyte surface, decreased their binding to PTCE by 60%. Likewise lymphocyte adhesion to PTCE was inhibited by 70% by anti-sLex- and anti-L-selectin-antibodies and by sLex tetrasaccharide. Finally PTCE in the allografts, but not in syngeneic grafts or normal kidneys, bound an L-selectin-IgG fusion protein, indicating that ligands for L-selectin were induced during rejection.

Chronic rejection is the most common reason for late loss of a transplant. The molecular mechanism of chronic rejection is not known and there is no treatment for this disorder. The characteristic histological feature in chronic rejection is increased smooth muscle cell replication in the vascular wall, leading to allograft arteriosclerosis. In this study we demonstrate that nonimmunosuppressed rat aortic allografts undergoing chronic rejection synthesize increased quantities of several smooth muscle cell growth-promoting substances in the vascular wall including interleukin-1, eicosanoids, and several peptide growth factors. Administration of a stable somatostatin analog lanreotide, BIM 23014, strongly inhibits myocyte proliferation in the allograft in vivo. It has no inhibitory effect on the proliferation of smooth muscle cells in vitro. Concomitantly, the locally produced peptide growth factors, i.e., epidermal growth factor, insulin-like growth factor 1, and BB-isomer of platelet-derived growth factor, but not other mediators of inflammation, are significantly reduced. The results suggest that growth factors are the main effector molecules leading to myocyte proliferation in allograft arteriosclerosis and that allograft arteriosclerosis (chronic rejection) may be specifically inhibited by lanreotide administration.

Rat aortic allografts from the DA (RT1a) to the WF (RT1v) strain but not syngeneic DA-to-DA control grafts develop arteriosclerotic changes in the vascular wall that are virtually identical to human allografts during chronic rejection. A more prominent medial cell destruction in the rat aorta, leading ultimately to complete medial necrosis, is the major difference between rat and human allografts. If the adventitia of syngeneic grafts is exposed to starch before transplantation, these grafts also develop an inflammatory reaction in the adventitia and an extensive intimal thickening at the site of the granulomas, but the medial smooth muscle cells are preserved. In both types of transplants with an intact endothelium as determined by light microscopy, adventitial inflammatory cell proliferation was accompanied by smooth muscle cell replication in the media and thickening of the intima. We therefore propose that an adventitial proliferative response is a prerequisite for intimal thickening to occur. In the allograft but not in starch-exposed syngeneic grafts there was also a notable lymphoid activation in the adventitia, which was accompanied by medial necrosis. We suggest that the medial necrosis in the allograft is linked to a toxic effect of activated lymphoid cells on medial myocytes and is not a prerequisite for intimal proliferation. Instead, intimal proliferation and medial necrosis in the allograft seem to be independently regulated.

Rat aortic allografts transplanted across histoincompatible strains develop arteriosclerotic alterations in the vascular wall that are virtually indistinguishable from those observed in human heart allografts during chronic rejection. In this study we have investigated whether hypercholesterolemia in the recipient rat accelerates allograft arteriosclerosis. Hypercholesterolemia was induced by a 4% cholesterol and 0.5% cholic acid diet, added to the normal rat diet. The cholesterol and cholic acid diet increased the level of serum total cholesterol from 1.3 +/- 0.0 to 4.8 +/- 0.9 (+/- SD) mmol/L and the level of low-density lipoprotein cholesterol from 0.3 +/- 0.0 to 2.6 +/- 1.0 mmol/L (p < 0.05) but caused no change in the level of high-density lipoprotein cholesterol, 1.0 +/- 0.1 versus 0.7 +/- 0.3 mmol/L. The level of plasma triglycerides remained also unchanged. Quantitation of two major chronic rejection-associated eicosanoids from the allograft vascular wall showed a significant increase in the synthesis of thromboxane B2 in the hyperlipidemic animals from 6.0 +/- 5.0 to 8.0 +/- 5.0 ng/mg dry weight and a slight reduction in the synthesis of 6-keto-prostaglandins F1 alpha. In vivo labeling of the recipient rat with tritiated thymidine and autoradiography showed that hypercholesterolemia did not affect the proliferation of inflammatory cells in the allograft adventitia, slightly increased the proliferation of smooth muscle cells in the media from 23 +/- 14 cells to 34 +/- 13 cells (+/- SEM) per cross section (p = ns), but slightly reduced the proliferation of smooth muscle cells in the intima from 13 +/- 6 to 6.2 +/- 1.5 (p = ns). Hypercholesterolemic recipients did not show any significant enhancement but, in fact, showed a delay in the generation of arteriosclerotic changes in the allograft intima. We conclude that although hypercholesterolemia, in the absence of hypertriglyceridemia, induces significant alterations in the eicosanoid metabolism and minor alterations in smooth muscle cell proliferation in the transplant vascular wall, it does not enhance arteriosclerotic alterations in chronically rejecting rat aortic allografts.

We have previously demonstrated that rat aortic allografts from the DA (RT1a) to the WF (RT1v) strain develop chronic arteriosclerotic changes in the vascular wall after a short spontaneously reversible acute rejection episode. These changes, which are lacking in syngeneic DA-to-DA control grafts, are virtually identical with those observed in human allografts during chronic rejection. In this study we have investigated whether eicosanoids are involved in the generation of arteriosclerotic changes. Incubation of aortic wall rings in vitro and immunochemical assays demonstrated that the arteriosclerotic allografts synthesize significantly more thromboxane B2 (TxB2) but not 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) or leukotriene B4. The increased synthesis of TxB2 in the allografts persisted for at least 5 months after transplantation. Separate incubation of the two major components of the vascular wall, after microdissection of the intima and (media plus) adventitia, demonstrated that most of the synthesis of TxB2 during chronic rejection was due to the outer layer of aorta, presumably the inflammatory cells of the adventitia. In contrast, most of the 6-keto-PGF1 alpha was synthesized by the inner layer of the aorta, presumably the endothelial cells and the smooth muscle cells of the intima. Administration of 1 mg.kg-1 x day-1 of a specific TxA2 receptor inhibitor, GR32191B, to the recipient rat reduced the proliferative response of inflammatory cells in the adventitia by 30%, as detected by in vivo tritiated-thymidine (3H-TdR) labeling and autoradiography, but did not reduce the proliferative response of smooth muscle cells in the media and intima.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic allograft rejection is the major reason why allografts are lost. While only 2%-3% of all allografts are lost during the first year to irreversible acute rejection, approximately 6%-7% are lost during each subsequent year to chronic rejection. The major manifestation of chronic rejection in all organs is persistent perivascular inflammation and allograft arteriosclerosis. Bearing this in mind, we have developed a model to investigate the pathophysiology of allograft arteriosclerosis using aortic transplantations between inbred rat strains. The results obtained thus far indicate that several different inflammatory cascades are operative within the vascular wall during allograft arteriosclerosis. The relative importance of these different cascades, and particularly the role of growth factors as final effectors, has not yet been defined. Attempts to suppress allograft arteriosclerosis under experimental conditions have already met with some success: under conditions where no immunosuppression is provided we have been able to delay the process by at least 3 months, though we have not been able to block it indefinitely. It may be expected, however, that once the inflammatory cascades leading to smooth muscle cell replication in the allograft media and their influx into the intima are better defined, more specific approaches to the inhibition of allograft arteriosclerosis will be developed.

The main histological features for 'chronic' rejection of rat renal allograft are ongoing perivascular and interstitial inflammation, vascular intimal hyperplasia and glomerular sclerosis. In this communication we have investigated the ultrastructural features of renal parenchyma by comparing chronically rejecting rat renal allografts immunosuppressed initially with cyclosporine to similarly immunosuppressed syngeneic transplants. Electron microscopy confirmed that the primary arterial change was smooth muscle cell proliferation in media, thickening and focal destruction of the internal elastic lamina, and influx of smooth muscle cells and their proliferation in the intima, leading to a nearly complete occlusion of the lumen. In the glomeruli, the most prominent features were mesangial matrix increase, narrowing of capillary lumen, and basement membrane thickening with a double contour appearance in capillary walls. A thin membrane, similar to lamina densa, appeared beneath the endothelium in some segments. Mesangial interposition, formation of new basement membrane and mesangial widening due to increase of basement membrane-like material was nearly always present. As none of these vascular and glomerular changes were observed in similarly immunosuppressed syngeneic transplants, we consider these ultrastructural alterations characteristic for chronic rejection in a rat renal allograft.

Chronic allograft rejection is both a clinical and a histopathological diagnosis. Until recently, the histological definition of chronic renal allograft rejection was based on clinical diagnostic biopsies, where the evidence was partially obscured by recurrence of the original renal disease, and/or by administration of immunosuppressive drugs. In this communication, we present an experimental rat model for chronic renal allograft rejection, devoid of recurrence of the original disease. By comparing allografts to similarly immunosuppressed syngeneic transplants, we define which histological features should be attributed to chronic rejection and which to cyclosporin nephrotoxicity. Rat renal transplants were performed from DA (Ag-B4, RT1av1) to WF strain (Ag-B2, RT1u) or, for control, to DA strain, and immunosuppressed for 2 or 3 weeks with cyclosporin using a variety of different dosages. The animals were monitored weekly for serum creatinine levels and for blood cyclosporin concentrations, and core needle biopsies were performed on the grafts at regular intervals. At 3 months post-transplantation the animals were sacrificed and a complete histopathological evaluation was performed. Thirty-one histological variables were scored blindly by two investigators and separately for the graft interstitium, glomeruli, tubuli, and the graft vasculature. The following histological alterations were significantly more prominent in allografts than in similarly immunosuppressed syngeneic transplants: the intensity of interstitial inflammation, particularly the degree of pyroninophilia within the inflammatory cell population; the extent of glomerular mesangial matrix increase, basement membrane thickening, and glomerular sclerosis; the increase in the vascular intimal thickness affecting in particular the first and second order branches of the renal artery; and the obliteration of the graft vasculature. These alterations were considered as being primarily due to chronic rejection. In contrast, the extent of interstitial fibrosis and the extent of tubular changes, including tubular epithelial vacuolation, epithelial atrophy, and tubular basement membrane changes, were not significantly different in the allografts as compared to the syngeneic controls. These alterations were attributed primarily to cyclosporin nephrotoxicity. Serial monitoring of the grafts by needle biopsies clarified the sequence of events in the development of the chronic alterations in the transplant. The first event, as expected, was tubulointerstitial pyroninophilic inflammation, resembling that of acute episodes of rejection. This was significantly stronger and appeared earlier in allografts immunosuppressed for 2 rather than for 3 weeks. Vascular alterations developed next. The last to develop were the glomerular lesions.

The effect of prolonged ischemia time on the generation of chronic rejection was investigated in long-surviving rat renal allografts. Three groups of rats were compared: allografts with a 30-min ischemia time, allografts with a 60-min ischemia time, and syngeneic grafts with a 60-min ischemia time. All transplants were initially immunosuppressed with 5 mg/kg/day of cyclosporine i.m. for 3 weeks postoperatively. The CsA trough levels were similar in the three rat groups. All groups demonstrated an initial rise and fall in serum creatinine; a simultaneous biopsy confirmed acute CsA toxicity. Thereafter the serum creatinine level remained on the normal control level both in the 30-min ischemia allografts and in the 60-min ischemia syngeneic grafts. After the initial decline, the serum creatinine level steadily increased in the 60-min ischemia allograft group and was 181 +/- 64 mumol/L at the end of the observation period, 12 weeks postoperatively, compared with 85 +/- 21 mumol/L in the 30-min ischemia allografts and 89 +/- 25 mumol/L in the 60-min ischemia syngeneic grafts. Quantitative histology that was performed upon autopsy demonstrated that the 60-min ischemia allografts showed persistent inflammation, inflammatory cell pyroninophilia, vascular arteriosclerosis and obliteration, and glomerular sclerosis, which were significantly stronger than in similarly immunosuppressed syngeneic transplants. Reduction of the ischemia time from 60 to 30 min significantly ameliorated the vascular and glomerular changes in the allografts but not the inflammatory alterations. This experimental study confirms previous clinical observations and demonstrates that prolonged ischemia contributes to chronic rejection in renal allografts. The results suggest that the effect of prolonged ischemia on chronic rejection is directed primarily to the parenchymal components of the graft, possibly to the graft vascular endothelium. As prolonged ischemia did not significantly affect the inflammation in the transplants, we conclude that the effect is not directed to the intensity of the host antigraft immune response.

Non-immunosuppressed rat aortic allografts from DA (RT1av1) to WF (RT1u) strain develop, after a short reversible acute rejection episode, chronic arteriosclerotic changes in the vascular wall, which are indistinguishable from those seen in human allografts during chronic rejection. Incubation of the aortic wall segments in vitro and immunochemical assays demonstrated that the allografts synthesized increased amounts of TxB2, but not 6-keto-PGF1alpha, or LTB4, compared to syngenic or normal aortas. The two major cellular components of the vascular wall, intima and adventitia, were incubated separately after microdissection. TxB2 was produced in the adventitia, whereas most of the 6-keto-PGF1alpha was synthesized in the intima. Administration of a specific TxA2 receptor inhibitor to the recipient rat reduced significantly the proliferation of adventitial inflammatory cells and the intimal smooth muscle cells. Nevertheless, it only delayed but did not inhibit the overall sclerosis of the intima.

Rat aortic allografts immunosuppressed with cyclosporin--but not with azathioprine or steroids--develop an early inflammatory lesion in the subendothelial space. This "endothelialitis" is followed by an influx of proliferating smooth muscle cells into the intima, resulting in intimal thickening and accelerated arteriosclerosis. Administration of azathioprine and steroids largely ameliorates the development of the accelerated lesion. Similar endothelialitis and accelerated arteriosclerosis have been observed previously in the autopsy material of cardiac transplant recipients. Our results confirm the suggestion that the development of accelerated allograft arteriosclerosis is most likely linked to cyclosporin administration.

Chronic rejection has several histological appearances, depending on the type of organ graft. Common to all of them is transplant arteriosclerosis associated with an ongoing inflammatory response in the transplanted graft. To the contrary of classical atherosclerosis, in which the manifestations are mostly focal, proximal, and asymmetric, transplant arteriosclerosis is generalized, and the intimal thickening is concentric. In this article, we describe an experimental animal model whereby transplant arteriosclerosis may be investigated in the inbred rat. Aortic allografts were transplanted from DA (RTIa) to major histocompatibility complex-incompatible WF (RTIv) rats or, for control, to rats of the DA strain. Transplantation was followed by an acute inflammation episode in the aortic adventitia of the allograft, largely lacking in the syngeneic graft, with a prominence of lymphoid activation markers (Cd25) in the cells of the inflammatory infiltrate. The inflammation episode peaked at 2 months after transplantation, became attenuated, and was followed by a proliferative response of myocytes in the allograft media. An increase in the migration of myocytes to the subendothelial space (presumably through small breaks generated in the internal elastic lamina) was observed thereafter, and myocyte proliferation continued in the intima with some intermingled macrophages. Finally, necrosis and disappearance of myocytes and their replacement by fibrous tissue were observed in the media. These alterations are virtually identical with the vascular lesion of chronically rejecting parenchymal organ transplants in human subjects. We suggest that aortic allografts exchanged between histoincompatible rat strains may be used as an experimental model for transplant arteriosclerosis.

We have estimated the frequency of B cells secreting antibodies against donor MHC antigens in rats rejecting histoincompatible renal allografts. In a major plus minor antigen-incompatible DA-to-WF combination on day 4 post-transplantation, reverse protein A plaque assay demonstrated that in the graft the frequency of lymphoid cells secreting Ig was 1:850. A major locus-incompatible and minor locus-compatible, congeneic LBN-to-Lewis strain combination was then applied to estimate the specificity of the secreted antibody. The lymphoid inflammatory cells were fused with mouse myeloma cells, cultured under limiting dilution conditions, and assayed by ELISA to donor and irrelevant strain spleen cells. Among cells infiltrating the graft, the fusion frequency was 1:172 x 10(3) and the frequency of Ig-producing hybrids 1:400 x 10(3) (i.e., this assay was approximately three log orders less sensitive than the reverse pA assay). The frequency of hybridomas secreting specifics antibodies against donor MHC antigens was 1:720 x 10(3) (i.e., every second hybridoma deriving from inflammatory population produced specific Ig). In addition, there was at least one obviously polyspecific population of hybridomas, detectable only in the spleen and reactive with all rat strains tested with a frequency of 1:700 x 10(3). The inflammatory cells were also cultured directly under limiting dilution conditions, and the frequency of Ig-secreting cells was determined by ELISA. The frequency of inflammatory lymphocytes secreting detectable amounts of immunoglobulin in the supernatant was 1:14 x 10(3) in the graft (i.e., this assay was approximately one log order less sensitive than the reverse protein A plaque assay).(ABSTRACT TRUNCATED AT 250 WORDS)