Lucia Cerundolo

Apoptosis in ischemia/reperfusion injury of human renal allografts.

BACKGROUND Ischemia/reperfusion injury of human renal allografts has a number of clinically significant consequences. A number of mechanisms of ischemia/ reperfusion injury have been elucidated, and there is evidence that apoptosis may be a contributing factor. METHODS To examine immediate posttransplant events, fixed tissue sections from paraffin-embedded wedge biopsy specimens taken before and after reperfusion of human renal allografts were stained using terminal deoxytransferase-mediated dUTP nick-end labeling to detect the DNA fragmentation characteristic of apoptosis. Thirty-six pairs of pre- and postreperfusion biopsy specimens were examined, 11 from living-related donor renal transplants and 25 from cadaveric donor transplants. RESULTS Quantitation of the terminal deoxytransferase-mediated dUTP nick-end labeling signal showed that significantly more apoptosis occurred in postreperfusion compared with prereperfusion biopsy specimens from cadaveric donor transplants, but a similar difference was not observed in living-related donor renal transplants. Furthermore, significantly more apoptosis was observed in postreperfusion biopsy specimens from cadaveric compared with living-related renal transplants. Postreperfusion biopsy specimens from kidneys that were cold preserved longer than 30 hr had a higher mean apoptosis score than those stored for less than 24 hr, but the result was not statistically significant. CONCLUSIONS Thus, apoptosis occurs predominantly as a result of reperfusion after cold preservation of cadaveric donor renal allografts and provides additional information regarding the extent of ischemia/ reperfusion injury in an organ. The clinical value of this information remains to be determined.

Leukocyte infiltration and inflammatory antigen expression in cadaveric and living-donor livers before transplant1

Background. There is evidence to indicate that organs obtained from cadaveric donors may be injured as a result of inflammatory events occurring at around the time of brain death. The aim of this study was to investigate whether there are differences in the expression of proinflammatory molecules between cadaveric and living-donor livers before transplant and to determine whether there is any association with donor factors and posttransplant graft function. Methods. A comparison of biopsies obtained before implantation from cadaveric (n=22) and living-related donor (LRD) (n=10) livers was performed. Cryostat tissue sections were stained with antibodies to leukocyte subpopulations, adhesion molecules, and human leukocyte antigen class II antigens. Results. Significantly higher levels of CD3+ lymphocytes (1.5%±0.8% vs. 0.5%±0.3%; P =0.00004), CD68+ monocytes and macrophages (4.0%±1.2% vs. 2.7%±0.6%; P =0.0003), and Fas-ligand staining (4.2%±2.6% vs. 1.5%±1.1%; P =0.0003) were detected in cadaveric livers compared with LRD livers before transplantation. Furthermore, higher levels of intercellular adhesion molecule-1 expression were detected in cadaveric donor livers and found to be associated with longer periods of ventilation (P =0.01), infection in the donor (P =0.013), and administration of dopamine (P =0.03). Although there were no differences in neutrophil infiltration between cadaveric and LRD livers, significantly higher levels were found in cadaveric donors with infection (P =0.01). Conclusion. This study demonstrates that inflammatory changes occur in cadaveric donor livers and are associated with events occurring during the period of intensive care. These proinflammatory changes did not seem to affect the short-term clinical outcome of cadaveric liver allografts but may contribute to alloimmune responses and impairment of graft function in the long term.

Ischemic preconditioning of cadaver donor livers protects allografts following transplantation.

Background. Ischemic preconditioning (IP) has been shown in animal models to protect livers against ischemia/reperfusion injury. The aim of this clinical study is to investigate whether IP of cadaver livers prior to retrieval confers protection on the allografts. Methods. Cadaveric donor livers were subjected to IP prior to retrieval by clamping of the hepatic pedicle for 10 min followed by reperfusion. Biopsies were obtained from the preconditioned (n=9) and control nonpreconditioned (n=14) liver transplants prior to and 2 hr following reperfusion. Cryosections were stained with antibodies against neutrophils and platelets. Results. IP livers were associated with significantly lower serum levels of aspartate aminotransferase (240±98 IU/L vs. 382±163 IU/L; P>0.016) and lactate (0.81±0.07 mmol/L vs. 1.58±0.9 mmol/L; P>0.018) 24 hr following transplantation. Furthermore, recipients of IP livers spent a significantly shorter time in the intensive care unit following transplantation compared to those given nonpreconditioned allografts (1 vs. 2.8±1.6 days; P=0.0008). Increases in neutrophil infiltration were detected in 6/14 (43%; P=0.022) and in CD41 deposition in 5/14 (36%; P=0.042) of nonpreconditioned livers. However, none of the IP allografts showed any change in the levels of platelets or neutrophil infiltration following transplantation. Conclusion. IP is an effective method of protecting cadaver donor allografts from cold ischemia and subsequent reperfusion injury. IP is also associated with a reduction in the nonspecific inflammatory response.

Cadaveric versus living-donor livers: differences in inflammatory markers after transplantation

Background. Prolonged cold storage of organs for transplantation may lead to inflammatory damage upon reperfusion. The aim of this study was to investigate whether organs from living donors experience less damage upon reperfusion than those retrieved from cadaver donors, where cold ischemia times are significantly longer. MethodS. Biopsies were obtained from cadaveric (n=23) and living-related donor (LRD) (n=10) liver transplants before and 2 hours after reperfusion. Cryosections were stained with antibodies against neutrophils, platelets, activated platelets, and endothelium. Results. LRD liver allografts showed minimal changes postreperfusion. In contrast, after reperfusion of cadaver allografts, neutrophil infiltration was detected in 22% and increased expression of von Willebrand factor (vWF), CD41, and P-selectin in 48%, 30%, and 13% of allografts, respectively. In cadaver allografts with deposition of activated platelets expressing either P-selectin or vWF, the cold ischemia time was significantly longer (885±123 min vs. 608±214 min, P =0.04; 776.8±171 min vs. 559.3±216 min, P =0.01, respectively). Increases in neutrophils and platelets after reperfusion were not significantly associated with clinical events posttransplant. However, in cadaver transplants that experienced early acute rejection, the mean cold ischemia time was significantly longer than in allografts with no rejection (732±174 min vs. 480±221 min, P =0.006). Conclusions. This study demonstrates that in the clinical situation, cold ischemia causes platelet deposition and neutrophil infiltration after reperfusion of cadaveric liver allografts. These early inflammatory events may contribute to make the graft more susceptible to acute rejection.

Expression of MHC class I-related Chain B (MICB) molecules on renal transplant biopsies.

Background. MICA and MICB (MHC class I-related chain A and B) are polymorphic genes that encode molecules related to MHC class I and are expressed on epithelial cells in response to stress. Incompatible donor MIC antigens can stimulate antibody production in transplant recipients. This study was designed to determine MICB expression in kidney pretransplant and any subsequent changes in expression following transplantation and to correlate changes with inflammatory markers and clinical events. Methods. Paired renal biopsies obtained from living donor (n=10) and cadaveric allografts (n=50) before and 7 days posttransplant were stained for MICB, leukocytic infiltration, and HLA class II antigens. Results. Variable tubular MICB expression was evident in donor biopsies [high 6/60 (10%), low/negative 13/60 (22%), intermediate 41/60 (68%)]. Following transplantation, MICB was up-regulated on renal tubules of 17/60 (28%) biopsies and was associated with MHC class II antigen induction (P=0.02) and leukocyte infiltration (P=0.01). Acute tubular necrosis leading to delayed graft function (DGF) and acute rejection (AR) cause cellular stress within the transplanted kidney. We found a strong association between up-regulation of MICB and cellular stress, 15/17 biopsies with up-regulated MICB expression had AR and/or DGF (P=0.003). Conclusions. This is the first study demonstrating variable levels of MICB expression in kidneys before transplantation and induction of MICB expression following renal transplantation. MICB expression is associated with HLA class II antigen induction, leukocytic infiltration of the graft and cellular stress in the transplanted kidney. Expression of MICB could contribute significantly to the alloimmune response in mismatched donors and recipients.

Non-heart-beating versus cadaveric and living-donor livers: differences in inflammatory markers before transplantation.

Background. Liver transplantation from non–heart-beating donors (NHBD) has been reintroduced into clinical practice to increase the donor pool; however, little is known about the immune status of NHBD livers. The aim of this study was to assess intragraft cell populations and inflammatory markers in NHBD and to compare the findings with cadaveric and living-related donor (LRD) livers. Methods. Biopsy specimens were obtained from controlled NHBD (n=9), conventional cadaveric (n=22), and living-donor (n=10) livers at the end of cold storage. Cryostat sections were stained for monocytes-macrophages, T lymphocytes, and intercellular adhesion molecule (ICAM)-1. Results. The levels of leukocyte infiltration in NHBD reflected those found in conventional cadaver donors and were significantly higher than in LRD livers. Similar levels of CD68+ monocytes-macrophages were detected in cadaver (4.0±1.2%) and NHBD livers (4.6±1.2%) and were significantly greater than in the LRD livers (2.6±0.5%, P <0.01). Furthermore, the levels of T lymphocytes in NHBD (1.1±0.6%) and cadaver donors (1.5±0.8%) were similar, and were higher than in LRD (vs. 0.47±0.3%, P <0.05). Twelve of 22 (60%) cadaver livers had high levels of ICAM-1 expression (grade 3), compared with only 1 of 10 (10%) LRD livers (P =0.02). Four of nine (44%) controlled NHBD livers expressed high levels of ICAM-1. Conclusions. The results demonstrate that livers obtained from controlled NHBD before transplantation are similar to conventional cadaver donors regarding the level of leukocyte infiltration. Nevertheless, lower levels of ICAM-1 were detected in NHBD, suggesting less exposure to inflammatory mediators than conventional cadaver donor livers.

Role of anti-vimentin antibodies in renal transplantation.

Background The role of non-HLA antibodies in rejection is not clear. We investigate whether antibodies to vimentin are made after renal transplantation and if production is associated with interstitial fibrosis and tubular atrophy (IFTA). Methods In this retrospective study, sera from 70 recipients of renal allografts (40 controls, 30 IFTA) were studied. The biopsy diagnosis of interstitial fibrosis and tubular atrophy (IFTA) was based on random, cause-indicating biopsies. Sera were collected pretransplant and at 3 monthly intervals up to 5 years posttransplant or diagnosis of IFTA and assayed by ELISA for IgM and IgG anti-vimentin antibodies (AVA) and HLA antibodies. Results Mean titers of IgM AVA were higher at every year after transplantation compared with pretransplant for both IFTA and controls groups (P<0.001). There was no difference in the mean level of IgM AVA achieved by IFTA and control groups. The mean pretransplant levels of IgG AVA in the IFTA and control group were 18.2±11.7 and 11.0±8.1, respectively (P=0.001). There was a significant increase between the pretransplant mean levels of IgG AVA and the levels at years 1 to 4 in the IFTA group (years 1-3, P<0.0001, year 4 P=0.003) but not in the controls. There was no significant difference between the numbers of IFTA or control patients achieving a positive value (mean+2SD of pretransplant antibody titers) of IgM AVA (50% versus 37.5%, respectively) or IgG AVA (26.6% versus 12.5%, respectively). There was no association between production of HLA and AVA antibodies. Conclusion Posttransplant production of IgM AVA is not associated with IFTA. The production of IgG AVA by a minority of IFTA patients suggests that in some individuals, IgG AVA may be involved in the pathology of IFTA.

Cross‐species reactivity of a panel of antibodies with monkey and porcine tissue

The continuing shortage of organs available for transplantation limits the number of patients able to benefit from this highly successful form of therapy. Interest in alternative sources of organs has now turned towards the pig because of its physiological similarity to human. There is a requirement therefore for reagents not only for research purposes but possibly for studying xenotransplants in the clinical situation in the future. In this study, we have concentrated on determining the cross‐species reactivity of a large panel of antibodies directed against human leukocyte markers, testing peripheral blood leukocytes and also including renal tissue to determine non‐leukocyte cross‐reactivity. A total of 63 out of 127 antibodies cross‐reacted with cynomolgus monkey cells. Twenty of these antibodies stained similar populations of leukocytes to human, whereas the remaining 43 reacted with clearly different populations. The majority of antibodies (108/127) were unreactive with porcine leukocytes, reflecting the evolutionary differences between pig and man. Of the 19 antibodies cross‐reactive with porcine cells, seven reacted with similar proportions of leukocytes to human, whereas the remaining 12 antibodies stained entirely different populations. The most interesting, and potentially most useful, antibodies were four that reacted with human, cynomolgus monkey and porcine tissue in a similar manner, suggesting that the epitopes recognized are present on similar molecules. These antibodies were directed against CD29 (MEM101A, K20) and CD18 (BU87, 7E4), the common β1‐ and β2‐integrin subunits respectively. This study demonstrates that there are antigens common to cynomolgus monkey, pig and man that react with currently available antibodies. Nevertheless, when determining cross‐species reactivity of human antibodies, it is important to consider the possibility that there may be additional non‐leukocyte reactivity in other tissues.