Protocol core needle biopsy and histological chronic allograft damage index as surrogate endpoint for Long-Term graft survival.

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.

Expression of estrogen receptor sub-types alpha and beta in acute and chronic cardiac allograft vasculopathy.

BACKGROUND: The vasculoprotective effects of estrogen are well-established not only in women with age-related atherosclerosis, but also after experimental vascular injury and in chronic allograft vasculopathy. Evidence exists that the newly discovered estrogen receptor (ER) beta, rather than the classical ERalpha is related to the vasculoprotective effect. Here we investigate whether and to what extent the two ERs are expressed in cardiac allografts in the rat and human in native state and during acute and chronic rejection. METHODS: Rat cardiac allografts were performed from male DA (AG-B4, RT1(a)) to male WF (AG-B2, RT1(v)) strain and syngeneic transplants from DA to DA strain; human male-to-male heart allograft endomyocardial biopsies came from our biopsy files. RESULTS: Under in situ hybridization, ERbeta mRNA was prominently expressed in rat vessels and stroma, whereas ERalpha mRNA was present in low levels only. In immunohistochemistry, 2 ERbeta-specific antibodies stained rat and human vessels and stroma, whereas only a weak or no signal was obtained with 2 ERalpha-specific antibodies. Interestingly, the mRNA and protein expression levels in the rat carried only a weak correlation with the intensity of acute rejection, i.e., was not related to the intensity of inflammation. CONCLUSIONS: Our results demonstrate that ERbeta is the predominant ER in rat and human cardiac allografts, and suggest that, unless additional ERs are identified, the vasculoprotective effects of estrogen derivatives in cardiac allograft vasculopathy are mediated by ERbeta rather than by the classical ERalpha.

Estrogen receptor beta dominates in baboon carotid after endothelial denudation injury.

Increasing evidence, mainly from rodents, suggests that the predominant estrogen receptor (ER) in arteries is the newly-described ERbeta. We have investigated the expression of the two ERs in baboon carotid artery before and after denudation injury. Prior to denudation, both full length receptors were detected in semiquantitative RT-PCR; in addition two ERalpha but no ERbeta splicing variants were found. After denudation, ERbeta mRNA increased five-fold and declined, whereas ERalpha mRNA expression remained low. Prior to and after denudation, two ERalpha-specific antibodies showed no reaction with the vessel wall. Instead, two affinity purified antisera to ERbeta demonstrated a weak but distinct reaction over vascular smooth muscle cells with predenudation specimens, escalating post-denudation and declining thereafter. The results suggest that selective targeting to ERbeta should be attempted when designing estrogen-based vasculoprotective drug therapies devoid of uterotrophic side effects.

Structure of carotid artery in baboon and rat and differences in their response to endothelial denudation angioplasty.

BACKGROUND: Most studies dealing with vascular response to injury have been conducted using rodent and rabbit models, although it is expected that the response to injury in these species is dissimilar from man. AIMS: Here we compare the structure of native carotid artery in rat and baboon and the response of these vessels to endothelial denudation angioplasty. METHODS: In both species, the carotid is a musculoelastic artery. Only baboon carotid has a distinct intima, correlating in size with the weight of male baboons. Complete endothelial denudation of left carotid was performed on eight male baboons and 24 male rats by applying an equivalent pull force with a Fogarthy catheter. The animals were sacrificed prior to and 15 min and 2, 3, 4, 7, 14 and 28 days postinjury, one baboon and three rats per time point. RESULTS: Re-endothelialization in the baboon was complete already on day 4, whereas in the rat it was still incomplete on day 28. The proliferative response to injury was far smaller in the baboon than in the rat, the intimal area increased only by 5-fold in baboon compared with 25-fold in rat, and the number of intimal nuclei by 4-fold in baboon compared with 12-fold in rat. Complete compensatory remodelling of the lumen size occurred in the baboon, whereas in the rat remodelling remained incomplete. The cell types participating in the response were, however, similar: deposition of thrombocytes on denuded luminal surface, expression of alpha-actin by intimal cells, and lack of any significant white cell infiltration in the denuded intima. CONCLUSIONS: Baboon carotids are very different from rat carotids both in their native structure and in their response to injury. With the limited amount of information available from human vessels, baboon carotids closely resemble human carotids in both respects.

Quantitation of cell migration in a rat carotid artery balloon injury model. Indications for a perivascular origin of the neointimal cells.

UNLABELLED: Medial and neointimal smooth muscle cells differ in phenotype. Adventitial cells have been shown to migrate to the intima and this could partly explain this difference. OBJECTIVE: The aim of this study was to quantitate the kinetics of cell migration from the adventitial and medial layers to the intima after endothelial injury. METHODS: Labelled proliferating cells at different periods post-injury in a rat carotid artery balloon injury model, were used to calculate the kinetics of migration using the alteration in cell populations and the ratio of proliferating cells. RESULTS: The increase in the number of neointimal cells was greater than the level of proliferation during the 30-day follow-up. Changes in the number and percentage of proliferating cells remained low after the appearance of the first neointimal cells. 28% of the neointimal cells were labelled during the first wave of migration, and in reverse at least 72% had migrated there. Of these migrating cells, 74% were non-proliferating. The formation of neointima was efficiently blocked with cyclophosphamide and batimastat (metalloproteinase inhibitor), which resulted in a decrease in the number of medial and intimal cells. CONCLUSION: The increase in the number of neointimal cells is mostly due to cells migrating from the outer layers through the media. The majority of these cells are not proliferating, but migration can still be efficiently blocked with antiproliferative drugs.

Blockade of complement inhibits obliterative bronchiolitis in rat tracheal allografts.

The role of complement activation in the development of obliterative bronchiolitis, a manifestation of chronic lung allograft rejection, was investigated in the heterotopic rat tracheal allograft model. An increase in intragraft complement components C3 and C5b-9 (membrane attack complex) as well as IgM and IgG deposits were demonstrated during the progressive loss of respiratory epithelium and airway occlusion in nontreated allografts compared with syngeneic grafts. A 7-d treatment with recombinant human soluble complement receptor type 1 (sCR1; 20 mg/kg/d, intraperitoneal), an inhibitor of both the classic and alternative complement pathways, significantly decreased epithelial necrosis and intragraft neutrophil infiltration, and reduced obliterative changes by 40%. Immunohistochemical analysis of the grafts showed that sCR1 treatment significantly decreased early C5b-9 and IgG deposits, neutrophil chemoattractant IL-8 immunoreactivity, and ICAM-1 expression. Treatment with sCR1 was associated with increased staining for Th2 cytokines, in particular IL-10, with concomitant downregulation of IL-2 and TNF-alpha immunoreactivity. In contrast, sCR1 treatment did not affect the number of graft-infiltrating CD4(+) and CD8(+) T cells, CD45(+) B cells, ED1(+) and ED3(+) macrophages, or immune activation with expression of IL-2Ralpha or MHC class II. In conclusion, this is the first study to demonstrate that blockade of complement activation attenuates the development of OB and suggests that in addition to T cell-driven responses, humoral and antigen-independent immune responses also operate in the disease process. A blockade of complement activation renders the chemokine milieu unattractive to neutrophils and also modulates the alloimmune response toward Th2 cytokines, which may have an antiproliferative role in fibroproliferative disorders.

Differentiation and maturation of porcine fetal islet cells in vitro and after transplantation.

BACKGROUND: Porcine fetal pancreas is a potential source of beta cells for transplantation. The immaturity of the cells is a problem. We have defined the optimal conditions for in vitro propagation of this tissue before transplantation. METHODS: Porcine fetal pancreas tissue was obtained for tissue culture at various stages of development. Serum-containing and serum-free media and a variety of potential differentiation factors were tested. In vitro, the numbers of endocrine islet cells and their proliferation were quantified and functional maturity of the beta cells was assessed by perifusion. Growth and maturation of the cells was assessed 3 months after transplantation into nude mice. RESULTS: Highest beta cell mass was obtained from end-gestational, as compared with early fetal or neonatal, pancreas. Nicotinamide and sodium butyrate effectively increased the insulin content and the number of endocrine cells in culture. In combination, these factors led up to a 90-fold increase in the insulin content of islet-like cell clusters (ICC) as compared with untreated controls. However, a high level of cell death through apoptosis was observed in these maximally stimulated endocrine cells, and they did not survive as grafts when transplanted into nude mice. Instead, a serum-free culture medium containing 10 mM nicotinamide and 0.1 mM isobutylmethylxanthine was found to support both differentiation and proliferation of endocrine cells as loose ICCs. Insulin release from these ICCs was sensitive to glucose. When transplanted under the kidney capsule of normoglycemic nude mice, a high level of beta cell differentiation and function was evident only in the ICCs cultured in the serum-free medium, and in freshly isolated ICCs. When transplanted to hyperglycemic nude recipients, the cells cultured in serum-free medium for 3 weeks reversed hyperglycemia more consistently and rapidly than freshly isolated ICCs. CONCLUSIONS: Optimal maturation of porcine fetal pancreatic cells is obtained in serum-free medium supplemented with nicotinamide. Butyrate is a potent stimulus for beta cell differentiation but leads to increased apoptotic cell death.

Enhanced intimal proliferation upon injury to pre-existing neointima and resistance of neointimal cells to cell death.

Recent evidence supports a role for cell death and inflammation as etiologic factors in neointimal formation and restenosis after angioplasty. This study was undertaken to examine the pattern and intensity of the proliferative response, cell death, and activation of inflammatory, endothelial and smooth muscle cells (SMC) in a model of intimal reinjury. Two ballooning injuries were performed to rat aorta, the second one 14 days after the first injury. Our results demonstrate that ballooning injury to pre-existing neointima differs clearly from an injury to a normal aorta. First, ballooning injury to pre-existing neointima doubled the proliferative response of SMC and intimal thickening, but proliferation of SMC occurred only in the intima, and did not extend into the media. Second, within four hours after the first injury, the number of TUNEL-positive SMC in the media increased from 3% to 23%, but no such increase was found in the pre-existing neointima after the second injury. Third, the prompt proliferative response of intimal SMC after the second injury was linked with a significant increase in endothelial P-selectin and neointimal VCAM-1 immunoreactivity, compared to the first injury at corresponding time points, followed by high numbers of activated ED3+ macrophages and CD4+ T cells in the developing neointima. A balance in injury-induced cell death and proliferation obviously maintains stable cell numbers observed in the media, whereas in the neointima, the resistance of SMC to injury-induced cell death may contribute to a rapid lesion formation in restenosis.

Endothelial L-selectin ligands are likely to recruit lymphocytes into rejecting human heart transplants.

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.