Donor cytokine genotype influences the development of acute rejection after renal transplantation.

BACKGROUND: Acute allograft rejection remains an important cause of morbidity after kidney transplantation, and has been shown to be a crucial determinant of long-term graft function. Although rejection is mediated by recipient lymphocytes, both donor and recipient factors contribute to the local environment that influences the nature, severity, and duration of the rejection response. Cytokines are a major determinant of this milieu, and this study sought to explore the impact of donor cytokine and cytokine receptor gene polymorphisms on acute rejection after renal transplantation. METHODS: A total of 145 cadaveric renal allograft donors were selected for analysis according to the presence or absence of graft rejection in the first 30 days after transplantation. DNA was genotyped for 20 polymorphisms in 11 cytokine and cytokine receptor genes using the polymerase chain reaction with sequence specific primers. Associations were assessed using contingency table analysis and the chi2 test, using a two-set design. RESULTS: A polymorphism at position -174 of the donor IL-6 gene was associated with the incidence (P=0.0002) and severity (P=0.000007) of recipient acute rejection. This finding was independent of HLA-DR matching. Acute rejection was not influenced by recipient IL-6 genotype, or by donor-recipient matching of IL-6 genotype. CONCLUSION: This study identifies donor IL-6 genotype as a major genetic risk factor for the development of acute rejection after renal transplantation. This provides evidence that donor-derived cytokines play a major role in determining outcome after transplantation, and will contribute to the development of therapeutic algorithms to predict individuals at particularly high risk of acute rejection.

The impact of recipient cytokine genotype on acute rejection after renal transplantation.

BACKGROUND: Acute allograft rejection remains an important cause of morbidity after kidney transplantation, and has been shown to be a crucial determinant of long-term graft function. As cytokines are major regulators of the immune system, genetic variation in cytokine production or activity may influence susceptibility to acute rejection. This study sought to determine the impact of recipient cytokine and cytokine receptor polymorphisms on acute rejection after renal transplantation. METHODS: A total of 209 cadaveric renal transplant recipients were selected for analysis according to the presence or absence of graft rejection in the first 30 days after transplantation. DNA was genotyped for 22 polymorphisms in 11 cytokine and cytokine receptor genes using the polymerase chain reaction with sequence specific primers. Results were stratified by incidence and severity of rejection, and by HLA-DR mismatching. RESULTS: No association between any polymorphism and the incidence or severity of acute rejection was detected. In particular, no association was seen with tumor necrosis factor or interleukin-10 genotype, either alone or in combination. CONCLUSIONS: We have failed to demonstrate any association between recipient cytokine genotype and acute rejection after cadaveric renal transplantation. Although more extensive studies may disprove these findings, it would seem premature to use recipient cytokine genotyping to predict transplant outcome, or to guide immunosuppressive therapy after transplantation.

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.

Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis.

A polymerase chain reaction (PCR) amplification of 16S rDNA was developed to identify spirochaetes recovered from cases of intestinal spirochaetosis in humans and pigs; these bacteria belong to a distinct genetic group of spirochaetes, with the proposed name 'Anguillina coli'. The PCR incorporated a universal eubacterial 16S rDNA sequencing primer (1492r), and a 21-base forward primer designed to include a nucleotide sequence specific for 'A. coli'. The PCR was used to correctly identify DNA extracted from 43 isolates of 'A. coli' from humans and pigs, whilst no product was produced from Escherichia coli, or from other intestinal spirochaetes, including 38 isolates of Serpulina spp., and one each of Treponema succinifaciens and Brachyspira aalborgi. The amplification provided a rapid and simple means of identifying DNA from isolates of 'A. coli', and could be used on boiled whole 'A. coli' cells, with a detection limit equivalent to 2.5 x 10(2) cells. The reaction was used to detect and identify these spirochaetes from selective agar plates inoculated with stool specimens from infected pigs.

Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons.

Multilocus enzyme electrophoresis (MEE) analysis and comparisons of nearly complete 16S rRNA gene sequences (1416 nucleotide positions) were used to evaluate phylogenetic relationships among Serpulina hyodysenteriae strain B78T, S. innocens strain B256T, Brachyspira aalborgi strain 513AT, and eight uncharacterised strains of swine, avian, and human intestinal spirochaetes. From MEE analysis, nine strains could be assigned to five groups containing other intestinal spirochaetes (genetic distances between groups = 0.6-0.9). Chicken spirochaete strain C1 and B. aalborgi 513AT represented unique electrophoretic types and formed their own MEE groups. Despite MEE differences, the 11 strains had highly similar (96.3-99.9%) 16S rRNA sequences. These findings point out limitations of both MEE analysis and 16S rRNA sequence comparisons when used as solitary techniques for classifying intestinal spirochaetes related to Brachyspira/Serpulina species.

Collapse in an accident and emergency department.

Collapse is a common presenting complaint to accident and emergency (A & E) departments. This retrospective study of 4180 new attendances at a district general hospital A & E showed that this accounted for 2.9%. A wide disease spectrum was implicated. This patient group has a high admission rate (47.8%) and a high mortality rate (31.3%). The deaths occurred largely in the elderly and it is suggested that elderly patients should either be admitted for observation, or a careful screening carried out for underlying pathology. The diversity of disease precludes a standard management protocol.

Experimental infection of laying hens with Serpulina intermedia causes reduced egg production and increased faecal water content.

Serpulina intermedia strain HB60, isolated from an Australian hen with diarrhoea, was used to infect 10 individually caged 14-week-old laying hens. Another 10 birds were sham inoculated with sterile broth. Birds were kept for 16 weeks, and faecal water content, egg production and body weights recorded. Strain HB60 was isolated from the faeces of nine of the infected birds at irregular intervals throughout the experiment, and from their caeca at slaughter. Infected birds tended to be lighter and their faeces, on average, were significantly wetter (by 2.85%; P < 0.002) than those of the controls. Significant reductions in mean number of eggs laid (1.4/week; P < 0.002) and mean egg weights (1.16 g; P < 0.05) were recorded in infected birds. Colonization did not induce any characteristic pathological changes. S. intermedia is potentially an economically significant cause of reduced egg production, and wet faeces in layer and broiler breeder flocks.

Pathogenicity of human and porcine intestinal spirochetes in one-day-old specific-pathogen-free chicks: an animal model of intestinal spirochetosis.

One-day-old chicks were infected orally with two strains of weakly hemolytic spirochetes isolated from a human and a pig with intestinal spirochetosis. These spirochetosis both colonized birds, attached end-on to their cecal enterocytes, induced watery diarrhea, and significantly depressed growth rates. Cultures of Serpulina innocens failed to colonize the chicks.

Human intestinal spirochetes are distinct from Serpulina hyodysenteriae.

Twenty-nine intestinal spirochetes isolated from Australian aboriginal children and six strains from Italian adults (HRM1, -2, -4, -5, -7, and -14) were genetically examined at 15 enzyme loci by using multilocus enzyme electrophoresis. Results were compared with those previously obtained for 188 porcine intestinal spirochetes. DNA from human strain HRM7 and porcine strain Serpulina hyodysenteriae P18A were also radioactively labeled and hybridized with DNA from 12 other human and porcine intestinal spirochetes. Both the multilocus enzyme electrophoresis and hybridization techniques demonstrated that the human spirochetes were not S. hyodysenteriae. They belonged to another distinct genetic group of spirochetes that included P43/6/78, the bacterium recovered from the first recorded case of porcine intestinal spirochetosis. Bacteria in this distinct group also differed from Serpulina spp. in possessing only four, five, or occasionally six axial filaments, being slightly thinner, and having more pointed ends. These findings add further weight to the possibility that human intestinal spirochetes may act as enteric pathogens.

Genetic and phenotypic characterization of intestinal spirochetes colonizing chickens and allocation of known pathogenic isolates to three distinct genetic groups.

Infection with intestinal spirochetes has recently been recognized as a cause of lost production in the poultry industry. Little is known about these organisms, so a collection of 56 isolates originating from chickens in commercial flocks in Australia, the United States, The Netherlands, and the United Kingdom was examined. Strength of beta-hemolysis on blood agar, indole production, API ZYM enzyme profiles, and cellular morphology were determined, and multilocus enzyme electrophoresis was used to analyze the extent of genetic diversity among the isolates. The results were compared with those previously obtained for well-characterized porcine intestinal spirochetes. The chicken isolates were genetically heterogeneous. They were divided into 40 electrophoretic types distributed among six diverse genetic groups (groups b to g), with a mean genetic diversity of 0.587. Strains in two groups (groups d and e) may represent new species of Serpulina, and the groups contained only strains isolated from chickens. Three genetic groups contained isolates previously shown to be pathogenic for chickens. These corresponded to the proposed species "Serpulina intermedius," to an unnamed group (group e), and to Serpulina pilosicoli. Two of the chicken isolates (one "S. intermedius" and one S. pilosicoli isolate) were strongly beta-hemolytic, two (both "S. intermedius") had an intermediate level of beta-hemolysis, and the rest were weakly beta-hemolytic. Fourteen isolates of "S. intermedius" produced indole, as did one isolate from group d. Isolates identified as S. pilosicoli resembled porcine isolates of this species, having four to six periplasmic flagella inserted subterminally in a single row at each end of the cell, and had tapered cell ends. All other spirochetes were morphologically similar, having seven or more periplasmic flagella and blunt cell ends. The identification of three genetic groups containing pathogenic isolates provides an opportunity for more detailed epidemiologic studies with these pathogens and for the development of improved diagnostic tests.

Chronic allograft failure in human renal transplantation: a multivariate risk factor analysis.

OBJECTIVE: To identify potential risk factors for the development of chronic renal allograft failure. SUMMARY BACKGROUND DATA: Chronic allograft failure (CAF) is the leading cause of late graft loss in renal transplantation. The authors studied the risk factors for the development of CAF in a single center during a period in which a consistent baseline immunosuppression regimen (cyclosporine, azathioprine, and prednisolone) was used. METHODS: Data from the Oxford Transplant Center Database were assessed on 862 renal allografts during a 10-year period. Risk factors were identified using multivariate logistic regression analysis. RESULTS: Biopsy-proven CAF occurred in 77 patients (9.2%) in the entire group. Multivariate risk factor analysis revealed that early and late acute rejection episodes, proteinuria, and serum triglycerides were significant factors. Acute rejection after 3 months was more important than early acute rejection. Serum triglyceride level and proteinuria at 1 year were both elevated in the CAF group. Male sex provided a protective effect. Serum creatinine levels at 6 months after the transplant were not predictive of the risk of developing CAF. CONCLUSIONS: These results from the largest single-center review to date suggest that both antigen-dependent and -independent factors are involved in the pathogenesis of CAF. Acute rejection at all time points has a significant impact on the development of CAF.

Cadaver versus living donor kidneys: impact of donor factors on antigen induction before transplantation.

BACKGROUND: It is widely recognized that living-related donor (LRD) renal allografts have a higher overall graft survival than cadaver donor transplants. We tested the hypothesis that part of this is attributable to LRD kidneys being obtained under optimal conditions from healthy donors, whereas cadaveric kidneys may have experienced injury as a result of inflammatory events around the time of brain death. METHODS: We have performed a comparative immunohistochemical analysis of pretransplant donor biopsies from cadaveric (N = 65) and LRD (N = 29) kidneys to determine any differences that may predispose them to subsequent damage. Cryostat sections were stained with antibodies to leukocytes, adhesion molecules, and human leukocyte antigen (HLA)-DR antigens, and the expression was assessed semiquantitatively. RESULTS: High levels of endothelial E-selectin and proximal tubular expression of HLA-DR antigens, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 were detected in biopsies from cadaveric kidneys, whereas expression of these markers was markedly reduced in LRD kidneys. High levels of tubular antigen expression were significantly associated with traumatic death, prolonged ventilation, and episodes of infection in cadaver donors. Furthermore, the expression of pretransplant tubular antigens in cadaver donor kidneys was significantly associated with early acute rejection following transplantation, suggesting that such kidneys are predisposed to subsequent immune-mediated attack following transplantation. CONCLUSIONS: These results may explain, in part, the superior outcome of LRD allografts compared with cadaver renal allografts.

Delayed graft function: risk factors and the relative effects of early function and acute rejection on long-term survival in cadaveric renal transplantation.

Delayed graft function (DGF) and acute rejection have both been associated with reduced renal allograft survival. In some studies, they have been shown to have an interactive effect. We studied the risk factors for DGF and the relative impact of DGF and rejection on both short- and long-term survival in recipients of cadaveric renal transplants. Data from the Oxford Transplant Centre Database were assessed on 710 cadaver allografts over a 10-yr period, during which time all recipients received cyclosporin-based immunosuppressive protocols. The interaction between DGF and acute rejection was examined using logistic and Cox multivariate regression. Long cold ischaemia time (CIT), sensitisation and older donor age were found to be independent predictors of DGF. The occurrence of DGF resulted in a reduced 5-yr survival (56 vs. 75%). However, the effect of DGF was confined to the first year post-transplant, as there was no significant difference in survival, as measured by half-life (t1/2) of grafts functioning at 1 yr, with DGF alone and a group with good early function (t1/2 = 21.3 vs. 20.0 yr). There was no increase in acute rejection in grafts with DGF. However, the combination of DGF and acute rejection resulted in the worst short-term graft survival (68% at 1 yr, compared to 92.3% in those grafts with no DGF or acute rejection) and this continued over the long term (t1/2 = 10.5 yr). These data suggest that early function is critical to the success of renal transplantation. The effects of DGF are limited to the first year post-transplant. Long-term graft survival may be improved by efforts to limit CITs, particularly for grafts from older donors and sensitised recipients.

Adhesion molecule polymorphisms in chronic renal allograft failure.

BACKGROUND: Chronic allograft failure (CAF) is a major cause of late graft loss in renal transplantation. Up-regulation of adhesion molecules has been demonstrated in renal allograft biopsies during both acute and chronic rejection, and these molecules are known to regulate leukocyte migration into the graft. METHODS: A single-center retrospective study was performed between 1985 and 1996 on renal transplant recipients who developed CAF. Genotyping was performed for five polymorphisms in intercellular adhesion molecule-1 (ICAM-1), E-selectin, and L-selectin. Frequency data for the polymorphisms in the CAF group (N = 62) and their matched donors, where available (N = 33), were compared with a group of recipients with graft survival of more than 10 years (N = 110) and a group of United Kingdom (UK) controls (N = 101). RESULTS: A variant allele in exon 4 of ICAM-1 (R241) was more common in the CAF recipients compared with both long-term survivors and UK controls (19.4 vs. 10.0 and 9.4%, P = 0.015 and 0.025). In addition, stratification by time to graft failure caused by CAF revealed more rapid failure in the presence of another ICAM-1 variant in the recipient (E469) in exon 6 (P = 0.033). CONCLUSIONS: ICAM-1 polymorphisms may represent a predetermined genetic risk factor for CAF. The polymorphism in exon 4 is in the Mac-1 binding site, and that in exon 6 is in the fifth immunoglobulin-like domain. Potential mechanisms of action of ICAM-1 variants in CAF include an alteration of activity as an adhesion molecule, altered costimulation, or a minor histocompatibility antigen.