Alloantibody Responses After Renal Transplant Failure Can Be Better Predicted by Donor-Recipient HLA Amino Acid Sequence and Physicochemical Disparities Than Conventional HLA Matching.

We have assessed whether HLA immunogenicity as defined by differences in donor-recipient HLA amino-acid sequence (amino-acid mismatch score, AMS; and eplet mismatch score, EpMS) and physicochemical properties (electrostatic mismatch score, EMS) enables prediction of allosensitization to HLA, and also prediction of the risk of an individual donor-recipient HLA mismatch to induce donor-specific antibody (DSA). HLA antibody screening was undertaken using single-antigen beads in 131 kidney transplant recipients returning to the transplant waiting list following first graft failure. The effect of AMS, EpMS, and EMS on the development of allosensitization (calculated reaction frequency [cRF]) and DSA was determined. Multivariate analyses, adjusting for time on the waiting list, maintenance on immunosuppression after transplant failure, and graft nephrectomy, showed that AMS (odds ratio [OR]: 1.44 per 10 units, 95% CI: 1.02-2.10, p = 0.04) and EMS (OR: 1.27 per 10 units, 95% CI: 1.02-1.62, p = 0.04) were independently associated with the risk of developing sensitization to HLA (cRF > 15%). AMS, EpMS, and EMS were independently associated with the development of HLA-DR and HLA-DQ DSA, but only EMS correlated with the risk of HLA-A and -B DSA development. Differences in donor-recipient HLA amino-acid sequence and physicochemical properties enable better assessment of the risk of HLA-specific sensitization than conventional HLA matching.

Use of Ex Vivo Normothermic Perfusion for Quality Assessment of Discarded Human Donor Pancreases.

A significant number of pancreases procured for transplantation are deemed unsuitable due to concerns about graft quality and the associated risk of complications. However, this decision is subjective and some declined grafts may be suitable for transplantation. Ex vivo normothermic perfusion (EVNP) prior to transplantation may allow a more objective assessment of graft quality and reduce discard rates. We report ex vivo normothermic perfusion of human pancreases procured but declined for transplantation, with ABO-compatible warm oxygenated packed red blood cells for 1-2 h. Five declined human pancreases were assessed using this technique after a median cold ischemia time of 13 h 19 min. One pancreas, with cold ischemia over 30 h, did not appear viable and was excluded. In the remaining pancreases, blood flow and pH were maintained throughout perfusion. Insulin secretion was observed in all four pancreases, but was lowest in an older donation after cardiac death pancreas. Amylase levels were highest in a gland with significant fat infiltration. This is the first study to assess the perfusion, injury, as measured by amylase, and exocrine function of human pancreases using EVNP and demonstrates the feasibility of the approach, although further refinements are required.

T cell requirements for the rejection of renal allografts bearing an isolated class I MHC disparity.

This study has examined the cellular and humoral responses underlying the rejection of rat renal allografts bearing an isolated RT1Aa class I MHC disparity. RT1Aa disparate kidneys were rejected promptly by high responder RT1u but not by low responder RT1c recipients (median survival time 10 d and greater than 100 d, respectively). The magnitude and phenotype of the cellular infiltrate were similar in rejecting and nonrejecting RT1Aa disparate kidneys. Paradoxically, graft infiltrating cells and spleen cells from RT1u recipients showed minimal ability to lyse donor strain lymphoblasts in vitro, whereas effector cells from RT1c recipients showed modest levels of cytotoxicity. Injection of RT1u rats with MRC OX8 mAb was highly effective at selectively depleting CD8+ cells from graft recipients but had no effect in prolonging the survival of RT1Aa disparate grafts despite the complete absence of CD8+ cells from the graft infiltrate, which included numerous CD4+ T cells and macrophages. RT1u, but not RT1c, recipients mounted a strong alloantibody response against RT1Aa disparate kidneys. Immune serum obtained from RT1u recipients that had rejected a RT1Aa disparate graft was able, when injected into cyclosporin-treated RT1u recipients, to restore their ability to reject a RT1Aa, but not a third-party RT1c, kidney. These results suggest that CD8+ cells in general and CD8+ cytotoxic effector cells in particular are unnecessary for the rapid rejection of RT1Aa class I disparate kidney grafts by high responder RT1u recipients. By implication, CD4+ T cells alone are sufficient to cause prompt rejection of such grafts and they may do so by providing T cell help for the generation of alloantibody.

Cellular requirements for renal allograft rejection in the athymic nude rat.

This study has examined the ability of adoptively transferred CD4+ and CD8+ T cells to mediate rejection of a fully allogeneic DA renal graft in the PVG nude rat. Transfer, at the time of transplantation, of naive CD4+ T cells caused rapid graft rejection and primed CD4+ cells were several times more potent. In contrast, naive or specifically sensitized CD8+ cells were entirely ineffective at mediating renal allograft rejection. Whereas nonrejecting grafts showed only a mild cellular infiltrate, rejecting grafts in CD4+ reconstituted animals showed a substantial infiltrate and many of the infiltrating cells had a phenotype (MRC OX8+, MRC OX19-), consistent with NK cells. Experiments using a mAb (HIS 41) against an allotypic determinant of the leukocyte common antigen confirmed that the majority (greater than 80%) of the cellular infiltrate in rejecting grafts derived from the host rather than from the CD4+ inoculum. Infiltrating mononuclear cells, obtained from rejecting allografts 7 d after transplantation in CD4+-injected PVG nude hosts, showed high levels of in vitro cytotoxicity against not only kidney donor strain Con A blasts but also third-party allogeneic Con A blasts, as well as against both NK and LAK susceptible targets. When splenocytes from nontransplanted nude PVG rats were tested in vitro they also demonstrated high levels of lytic activity against both NK and LAK susceptible targets as well as allogeneic Con A blasts, which were not susceptible to lysis by spleen cells from euthymic rats. These findings suggest that injected CD4+ cells may cause renal allograft rejection by the recruitment of extrathymically derived, widely alloreactive cells into the kidney in this model of graft rejection.

Prolonged survival of actively enhanced rat renal allografts despite accelerated cellular infiltration and rapid induction of both class I and class II MHC antigens.

Administration of 1 ml of donor whole blood 7 d before renal transplantation produces long-term (greater than 100 d) graft survival in the DA (RT1a) into PVG (RT1c) rat strain combination. Using this model, the pattern and phenotype of infiltrating leukocytes were examined in rejecting and enhanced renal allografts, at days 1, 3, 5, and 7 after transplantation, by immunohistologic techniques. Paradoxically, enhanced grafts showed a more rapid and substantial leukocyte infiltrate, the phenotype of which was similar to that in rejecting grafts except for a reduced number of MRC OX-8+ cells and MRC OX-39+ cells. Graft infiltrating cells and splenocytes from transfused animals showed similar, although modest, levels of both nonspecific cytotoxicity and alloantigen-specific cytotoxicity. Immunohistologic analysis of MHC antigen distribution within the allograft revealed, unexpectedly, that enhanced grafts underwent an accelerated and extensive induction of both donor class I and class II MHC antigens. These findings were confirmed by allospecific quantitative absorption analysis, which showed severalfold increases in class I and class II MHC antigens by day 3 in enhanced grafts but not until day 5 in rejecting grafts. An additional observation was the more rapid disappearance of donor interstitial cells from enhanced grafts. These findings emphasize the overwhelming suppressive effect induced by an organ allograft after preoperative blood transfusion despite the associated induction of large numbers of potential effector cells and increased target antigen density within the graft.

Evaluating the perioperative safety of laparoscopic radical nephrectomy for large, non-metastatic renal tumours: a comparative analysis of T1-T2 with T3a tumours.

OBJECTIVE: With increasing surgeon experience, the use of laparoscopic radical nephrectomy (LRN) in large and locally advanced renal tumours (T3a) is gaining favour in urological practice. There are limited studies reporting surgical outcomes in such groups. The aim of this study was to review our experience with LRN in these patients. METHODS: Data was retrospectively collected on 201 consecutive patients who underwent LRN for renal cancer by a single surgeon. Perioperative parameters assessed were age, gender, American Society of Anaesthesiologists score (ASA), waist circumference, tumour size, specimen size, histological subtypes, anaesthetic duration, operative approach and technique, surgery duration, blood loss, pre and postoperative renal function, complication rate and duration of hospital stay. RESULTS: Of 201 patients undergoing LRN, 43 (21%) patients had T3a tumours (group 2). The remaining 158 (79%) patients had T1 tumours (group1). Mean tumour size in group 2 was 12.2 cm. Renal cell carcinoma (RCC) was more common in males than females (131/201; 65%). Patients with T3a disease were more likely to have an ASA score of 2 (37/201; 18%). In the majority of patients across both groups, LRN was completed using a 3-port approach (173/201; 86%). There were no significant differences between groups in terms of mean anaesthetic duration, average surgical time, average estimated blood loss, complication rate and mean hospital stay. CONCLUSION: Our study shows that LRN has equivalent perioperative outcomes and safety in larger and locally advanced renal tumours.

The Effect of Obesity and Increased Waist Circumference on the Outcome of Laparoscopic Nephrectomy.

Introduction. The prevalence of obesity is increasing worldwide. Obesity can be determined by body mass index (BMI); however waist circumference (WC) is a better measure of central obesity. This study evaluates the outcome of laparoscopic nephrectomy on patients with an abnormal WC. Methods. A WC of >88 cm for women and >102 cm for men was defined as obese. Data collected included age, gender, American Society of Anaesthesiologists (ASA) score, renal function, anaesthetic duration, surgery duration, blood loss, complications, and duration of hospital stay. Results. 144 patients were assessed; 73 (50.7%) of the patients had abnormal WC for their gender. There was no difference between the groups for conversion to open surgery, number of ports used, blood loss, and complications. Abnormal WC was associated with a longer median anaesthetic duration, 233 min, IQR (215-265) versus 204 min, IQR (190-210), p = 0.0022, and operative duration, 178 min, IQR (160-190) versus 137 min, IQR (128-162), p < 0.0001. Patients with an abnormal WC also had a longer inpatient stay, p = 0.0436. Conclusion. Laparoscopic nephrectomy is safe in obese patients. However, obese patients should be informed that their obesity prolongs the anaesthetic duration and duration of the surgery and is associated with a prolonged recovery.

A single centre experience of zero-ischaemia laparoscopic partial nephrectomy in Ireland.

BACKGROUND: Nephron-sparing surgery in the form of partial nephrectomy is increasingly becoming the standard of care in patients with small renal tumours. Oncological outcomes for partial nephrectomy are equivalent to radical nephrectomy, however, clamping of the hilar vessels to allow resection of tumours during partial nephrectomy may cause ischaemic damage to the kidney and result in long-term renal impairment. AIM: We carried out a retrospective review of 43 patients undergoing laparoscopic partial nephrectomy (LPN) and assessed functional and oncological outcomes. METHODS: The operative technique initially utilised a thulium laser, with later cases using the LigaSure™ vessel sealing device. All patients underwent preoperative cross sectional imaging and anatomical classification accordingly. RESULTS: Forty three patients underwent LPN in our unit from 2006 to 2014. The mean (range) tumour diameter on preoperative cross sectional imaging was 28.2 (12-49) mm. All cases had a warm ischaemia time of zero, as hilar vessels were not clamped in any case. The mean (range) preoperative estimated glomerular filtration rate (eGFR) was 73 (37 to >90) ml/min/1.73 m2 and was not significantly different to the post-operative mean (range) eGFR of 71 (31 to >90) ml/min/1.73 m2. 34 (79%) of the tumours were found to be malignant. Positive surgical margins were found in one case. The mean (range) follow-up time in our cohort was 61.6 (24-127) months and no patient has had a local or distant recurrence. CONCLUSION: Zero ischaemia laparoscopic partial nephrectomy appears to be a safe and oncologically satisfactory procedure for the management of small localised kidney tumours.

Immunohistological observations of rat fetal pancreas allografts transplanted into unmodified and cyclosporine-treated recipients.

Administration of CsA (15 mg/kg/day) prolonged the survival of DA (RT1a) rat fetal pancreas transplanted to the renal subcapular site of both PVG (RT1c) and Lewis (RT1(1] recipients. Sections of fetal pancreas examined 40 days after transplantation into allogeneic CsA-treated recipients showed growth and development of the fetal pancreas tissue, and the presence of numerous insulin-containing islets. CsA treatment prevented the induction of MHC antigen within allografts. Whereas at day 4, both rejecting and CsA treated grafts showed donor class I MHC expression on duct epithelium and islet cells, only rejecting grafts displayed class I MHC induction on acinar cells. Rejecting grafts showed strong induction of class II MHC antigen expression on duct epithelium from day 4 onward but this was completely prevented by CsA treatment. Islet cells in both rejecting and CsA treated allografts remained class II-negative throughout. CsA also resulted in a reduction in the day 6 cellular infiltrate of allografts (median area leukocyte infiltrate reduced from 43% to 10%) with a marked decrease in the number of MRC OX-8-positive cells. These results show a favorable effect of CsA on rat fetal pancreas allografts with a reduction in MHC antigen expression within the graft and prolonged survival of insulin-rich endocrine tissue.

Reduction of alloantibody response to class I major histocompatibility complex by targeting synthetic allopeptides for presentation by B cells.

BACKGROUND: PVG.RT1(u) rats develop a strong CD4 T cell-dependent alloantibody response to class I major histocompatibility complex (MHC) A(a) antigen, during which CD4 T helper cells recognize and respond to A(a)-derived peptides presented by recipient class II MHC (indirect allorecognition). On the basis of evidence that CD4 T cells that encounter antigen presented by resting B cells become tolerant, we have targeted synthetic A(a)-derived allopeptides for in vivo presentation to class I MHC-disparate CD4 T cells by resting recipient B cells. METHODS: PVG.RT1(u) rats were treated with two peptides, P1 and P2, corresponding to the alpha-helical regions of A(a) (residues 57-80 and 143-163), which were conjugated via an N-terminal cysteine residue to monovalent Fab fragments of OX60 monoclonal antibody, which labels membrane IgD-positive B cells. RESULTS: RT1(u) rats primed with free (nonconjugated) P1 or P2 emulsified in complete Freund's adjuvant produced strong peptide-specific antibody responses and a heightened anti-A(a) antibody response to an A(a)-disparate PVG.R8 heart graft, confirming that each peptide encompasses one or more major T cell determinant for B cell help. Pretreatment of PVG.RT1(u) rats with a mixture of OX60-Fab-P1/P2 conjugates markedly reduced their ability to mount an A(a) antibody response when challenged with either A(a)-disparate blood transfusion or an A(a)-disparate heart graft, although PVG.R8 heart graft survival was not prolonged. CONCLUSIONS: In this report, we show that synthetic A(a)-derived allopeptides are able, when targeted for in vivo presentation to CD4 T cells by resting B cells, to impair the ability of RT1(u) rats to mount an antibody response to A(a) antigen. All subclasses of IgG anti-A(a) alloantibody were profoundly reduced, suggesting that the responsible mechanism is more likely to be CD4 T helper cell unresponsiveness rather than Th1/Th2 T cell polarization.

Alloantibody and intragraft cellular response to MHC class I-disparate kidney allografts in recipients tolerized by donor-specific transfusion and cyclosporine.

Congenic PVG.RT1u rats rapidly reject Aa class I-disparate kidney allografts from recombinant PVG R8 donors and we recently demonstrated that anti-class I MHC alloantibody plays a critical role in effecting acute rejection in this experimental model. In this article, we show that PVG.RT1u recipients can be rendered permanently and specifically tolerant to R8 kidney allografts by administration of four weekly donor-specific transfusions (DST) combined with a 7-day course of cyclosporine given with the first DST. Tolerance induction correlated with abrogation of a cytotoxic alloantibody response by thymus-independent, i.e., peripheral mechanisms; IgM and all IgG subclasses of anti-class I alloantibody were abolished. In contrast, nonrejecting kidney allografts in tolerant rats and rejecting grafts from unmodified recipients were similarly infiltrated by mononuclear cells, and intragraft transcripts for interleukin (IL)-2, interferon-gamma, and IL-13 were readily detected by reverse transcriptase polymerase chain reaction with no apparent quantitative difference between the two groups. Messenger RNA for IL-4 and IL-10 was present in rejecting grafts but barely detectable in grafts from tolerant animals. These results suggest that tolerance induction by DST and cyclosporine is, in this experimental model, associated with a selective impairment in humoral alloimmunity.

Morphometric analysis of cellular infiltration assessed by monoclonal antibody labeling in sequential human renal allograft biopsies.

The mononuclear cell infiltrate in a total of 279 human renal allograft biopsies was determined by a panel of monoclonal antibodies using an indirect immunoperoxidase technique. Two hundred and seventy-two biopsies were obtained from 83 patients randomly allocated to receive short-term cyclosporine (CsA) or conventional azathioprine and low-dose prednisolone (AP). Biopsies were obtained routinely at days 0 (control biopsies), 7, 21, 90, and 365, as well as at other times when clinically indicated. A further 7 patients on AP therapy were biopsied several years after transplantation (median: 6 years 1 month). Morphometric analysis of cryostat tissue sections using a point-counting technique has shown that the infiltration in rejecting grafts is significantly greater than in grafts with stable function. However, significant infiltration also occurs within the first week after transplantation in grafts with stable function. While this infiltrate diminishes with time, it remains significant even in grafts biopsied several years after transplantation. The infiltration with CsA treatment is significantly less than with AP therapy. The magnitude of the infiltrate therefore varies with time, graft status, and immunosuppression. In contrast the phenotypic composition of the infiltrate remains relatively constant in all biopsies after transplantation with T lymphocytes (CD3+), accounting for approximately 35% of infiltrating cells and CD8+ cells more common than CD4+. Monocytes and macrophages account for most of the remainder of the infiltrate.

Renal allograft rejection in CD4+ T cell-reconstituted athymic nude rats. The origin of CD4+ and CD8+ graft-infiltrating cells.

PVG-rnu/rnu nude rats reject a fully allogeneic DA renal allograft after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many CD8+ leukocytes within the graft. In order to clearly establish the provenance of these CD8+ cells infiltrating rejecting kidney allografts, nude recipients (PVG-RT7a) were injected with CD4+ T cells from the PVG-RT7b congenic strain bearing an allotypic variant of the leukocyte-common antigen. Dual fluorescence and immunohistochemistry demonstrated that approximately 75% of the total infiltrate was host-derived; the donor-derived RT7b population was almost entirely (92-99%) CD4+, CD5+, CD3+, and alpha beta TCR+. At least 97% of the CD8+ cells were of nude origin. There was no evidence of donor-derived CD8+ cells or of a CD4+8+ double-staining population. Unexpectedly, nearly half of the alpha beta TCR+ cells from the grafts were of nude origin.

Allograft rejection in CD4+ T cell-reconstituted athymic nude rats--the nonessential role of host-derived CD8+ cells.

PVG-rnu/rnu nude rats reject fully allogenic renal (DA) and skin (BN, AO) allografts after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many nude-derived CD8+ leukocytes within the graft. In addition, mononuclear cells infiltrating the rejecting renal grafts in these animals display cytotoxic activity in vitro against specific and third-party alloantigens. In this investigation we have treated CD4+ T cell-restored nude rats bearing renal or skin allografts with the mAb MRC OX8 to deplete the host of CD8+ cells. In vivo treatment with OX8 completely eliminated CD8+ cells from rejecting grafts of both kidney and skin, but it did not prevent graft rejection, nor did OX8 treatment abolish the cytotoxic effector cells found in nude rat spleen or in graft-infiltrating cells (GIC) of rejecting renal allografts. The nature of the cytotoxic activity was examined with anti-CD3 mAb 1F4, which was shown to block conventional CD8+ Tc killing in vitro but did not inhibit allogeneic target cell lysis by spleen cells from nude rats. The cytotoxic activity found in GIC of rejecting allografts was not inhibited by anti-CD3 mAb, suggesting that these cytotoxic effector cells were CD3-CD8- and were of extrathymic origin. We conclude that non-thymus-derived CD8+ GIC are not essential for allograft rejection in CD4+ T cell-restored nude rats.

Skin allograft rejection in mice lacking inducible nitric oxide synthase.

BACKGROUND: During allograft rejection, up-regulation of cytokine-inducible nitric oxide synthase (iNOS) leads to the production of large amounts of nitric oxide (NO). The net effect of NO on the alloimmune response is, however, difficult to predict because of its diverse biological effects, which include potentially opposing roles as an effector and immunoregulatory molecule. METHODS: In this study, the role of iNOS on the in vitro and in vivo alloimmune response was defined using mutant mice that lack a functional iNOS gene. The ability of spleen cells obtained from iNOS-deficient mutants to proliferate and to produce cytokines in response to irradiated BALB/c stimulator cells was determined, and the rate at which iNOS-deficient mice were able to reject BALB/c skin allografts was observed. RESULTS: Spleen cells from homozygous iNOS-deficient (129xMF1)F1 mice, when compared with cells from heterozygous control mice, showed an increased in vitro proliferative response and produced substantially higher levels of interferon-gamma, and also more interleukin-2 and interleukin-12, in response to allogeneic stimulation. The kinetics of BALB/c skin graft rejection were comparable in heterozygous control animals and iNOS-deficient mice. Moreover, no net effect of iNOS on skin allograft rejection was apparent in mice treated with depleting monoclonal antibodies (mAb) to CD4 or CD8 T cells, either alone or in combination, or in mice treated with both anti-CD8 mAb and a neutralizing anti-tumor necrosis factor mAb. CONCLUSIONS: These results show that iNOS has an immunomodulatory effect on the in vitro alloimmune response but lack of iNOS has no net influence on the kinetics of murine skin allograft rejection in either unmodified recipients or recipients in which the early contribution of T-cell subsets and tumor necrosis factor-alpha to graft rejection has been abrogated.

Thymus-dependent, anti-CD4-induced tolerance to rat cardiac allografts.

BACKGROUND: Treatment with anti-CD4 monoclonal antibodies (mAbs) leads to induction of transplant tolerance in rodent models, but the cellular mechanisms responsible are poorly defined. In this study, we used a rat model of cardiac transplantation to examine the contribution of the thymus gland to anti-CD4 mAb-induced tolerance. METHODS: Pretransplant administration of OX38 mAb partially depletes peripheral CD4 T cells and induces tolerance to fully allogeneic Lewis (RT1l) heterotopic cardiac allografts in DA (RT1a) recipients. Using this experimental model, the contribution of the adult thymus gland and of recent thymic emigrants to tolerance induction was assessed, and the cellular and humoral alloimmune responses accompanying tolerance defined. RESULTS: OX38 mAb selectively depleted mature CD4 T cells but spared CD4 T cells that had recently emerged from the thymus. Pretransplant thymectomy abrogated tolerance induction, but the data suggested a role for recent thymic emigrants rather than for the thymus gland per se. Both nonrejecting cardiac allografts in OX38-treated recipients and rejecting grafts in control animals were infiltrated to a similar extent by mononuclear cells, including activated T cells. Intragraft mRNA transcripts for interleukin (IL)-2, interferon-gamma, IL-4, IL-10, and IL-13 were similar in non-rejecting and rejecting allografts although, with the exception of IL-2, there was a trend towards reduced cytokine transcripts in tolerant grafts. CD4 T cells from long-term tolerant recipients proliferated normally to donor alloantigen in vitro, and produced IL-2, interferon-gamma, and IL-4 in amounts comparable to normal CD4 T cells. Tolerant recipients also developed a strong alloantibody response comprising both IgG1 (Th2-dependent) and IgG2b (Th1-dependent) subclasses. CONCLUSIONS: The results of this study suggest that the thymus, through the production of recent thymic emigrants, plays an important role in facilitating the induction of transplant tolerance after anti-CD4 mAb. Tolerant animals displayed strong cell-mediated and humoral alloimmune responses with no evidence of selective deviation from a Th1 to a Th2-like cytokine pattern.

Role of NAD(P)H:quinone oxidoreductase (DT-diaphorase) in cytotoxicity and induction of DNA damage by streptonigrin.

The metabolism, cytotoxicity, and genotoxicity of streptonigrin (SN) w ere determined in two human colon carcinoma cell lines: HT-29 with high NAD(P)H:quinone oxidoreductase (EC 1.6.99.2, DTD) activity and BE with undetectable DTD activity. Dicumarol-sensitive oxidation of NADH was observed with HT-29 cytosol, but not with BE cytosol. Oxygen consumption was also observed using HT-29 cytosol, but was absent with BE cytosol. Dicumarol inhibited oxygen consumption with HT-29 cytosol, but deferoxamine had no effect, suggesting that divalent metal cations were not necessary for efficient auto-oxidation of SN hydroquinone. In cytotoxicity studies, SN was much more toxic to the DTD-rich HT-29 cells than to the DTD-deficient BE cells. Deferoxamine decreased toxicity in both cell lines, implicating hydroxyl radicals produced during Fenton-type reactions as the toxic species. In the genotoxicity assay, SN induced a much higher incidence of DNA strand breaks in HT-29 cells than in BE cells, and deferoxamine protected against DNA strand breaks in both cell lines. Some evidence of DNA repair was also observed in the two cell lines. These results support an important role for DTD in the cytotoxicity of SN in the high DTD HT-29 colon carcinoma cell line.

Adhesion molecule expression (ICAM-1, VCAM-1, E-selectin and PECAM) in human kidney allografts.

Expression of the cellular adhesion molecules ICAM-1, VCAM-1, E-selectin and PECAM in human kidney allografts was assessed by immunoperoxidase labelling of cryostat sections. Biopsies from 10 kidneys immediately prior to transplantation and 58 biopsies from 51 kidney transplants with graft dysfunction were studied. Allograft dysfunction was due to acute tubular necrosis (n = 5), acute rejection (n = 30), cyclosporin A (CyA) nephrotoxicity (n = 6), acute pyelonephritis (n = 3), recurrent glomerulonephritis (n = 4) and chronic rejection (n = 10). There was variability in the distribution of ICAM-1, VCAM-1 and E-selectin expression in pretransplant kidneys but the principal observation was a marked increase in the expression of ICAM-1 and VCAM-1 by the renal vasculature and the proximal tubules during acute rejection. By contrast, grafts with dysfunction not attributed to rejection showed a pattern of ICAM-1 and VCAM-1 expression similar to that observed prior to transplantation. E-selectin was expressed only weakly by occasional intertubular capillaries during acute rejection but the three grafts with pyelonephritis displayed strong expression of E-selectin on intertubular capillaries. There was no change in the pattern of PECAM expression following transplantation. The induction of ICAM-1 and VCAM-1 during rejection may contribute to the recruitment of mononuclear cells and render endothelial and tubular renal cells more susceptible to cell-mediated injury.

Phenotypic analysis of graft infiltrating cells and major histocompatibility complex expression in actively enhanced rat renal allografts.

In this study donor specific blood transfusion of PVG recipients prevented rejection of DA strain kidneys but, paradoxically, failed to prevent the rapid and progressive accumulation of large numbers of mononuclear cells within enhanced grafts. Morphometric analysis showed that the percentage cellular infiltrate at day 3 was significantly greater in enhanced than in rejecting grafts but a notable feature in the phenotypic analysis of day 5 infiltrates was a markedly reduced number of MRC OX8 positive cells (Tc/s and NK cells) in enhanced grafts. Both rejecting and enhanced allografts showed a marked induction not only of class I but also of class II MHC antigens, and quantitative absorption analysis of donor class I MHC antigens indicated that induction occurred more rapidly in enhanced grafts. Taken together, these findings suggest that blood transfusion sensitizes the recipient, resulting in a more rapid allograft response, but that even in the presence of massive MHC/antigen induction and large numbers of infiltrating cells, immunoregulatory mechanisms are able to suppress the rejection response.

Neutralising IL-12 activity as a strategy for prolonging allograft survival and preventing graft-versus-host disease.

Interleukin-12 (IL-12) is a key immunoregulatory cytokine which promotes the development of Thl-dependent, cell-mediated immune responses. Acute allograft rejection after organ transplantation and acute graft-versus-host disease (GVHD) after bone-marrow transplantation are generally attributed to cell-mediated immune mechanisms and, therefore, potentially susceptible to immunological intervention at the level of IL-12. Recent data from murine models of transplantation have highlighted the potential of IL-12 as a selective target for immunotherapy. Neutralising endogenous IL-12 for a brief period at the time of transplant promotes long-term deviation from a Th1 to a polarised Th2 alloimmune response. This confers lasting protection from GVHD but is less effective at preventing acute rejection, possibly because Th2-dependent immune responses are also capable of effecting graft rejection.