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.

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.

The effect of a Rapid Access Prostate Cancer Clinic on prostate cancer patient and disease characteristics, primary treatment and surgical workload.

BACKGROUND: In 2009, Rapid Access Prostate Cancer Clinics (RAPC) were introduced to St. James's Hospital to improve the access and organisation of patients to prostate cancer investigations and treatment. AIMS: To observe the effects of the RAPC on prostate cancer diagnosis, primary treatment and overall workload. METHODS: Using a prospectively designed patient database, the records of all prostate cancer patients between 2007 and 2011 were retrieved and analysed. Data were obtained for age, PSA, biopsy Gleason score and primary treatment modality and charted for the observation and comparison of trends. RESULTS: Seven hundred and eighty-nine patients had a new diagnosis of prostate cancer between 2007 and 2011. The median PSA prior to the RAPC was 9.7-13.1 ng/ml, which decreased to 7.79-9 ng/ml after the RAPC. Prior to the RAPC, 77-81 biopsies were performed annually versus 149-271 in the post-RAPC era. Annual requirements for radical prostatectomy also increased from 12 to 27 in the post-RAPC era. Conversely, an initially increasing percentage of patients for radiotherapy was reversed in the post-RAPC period. An increasing trend for higher grade PCa (Gleason score 4 + 4 and higher) was also reversed. CONCLUSIONS: The introduction of a RAPC improves the overall pathological characteristics of patients with prostate cancer. However, RAPCs are also associated with a considerable increase in surgical workload. These are important considerations for units considering the incorporation of a similar facility in their institutions.

Audit of rapid access introduction reveals high prevalence of prostate cancer in Western Region.

INTRODUCTION: Men with symptoms suggestive of prostate cancer are now directly referred by their general practitioners to rapid access prostate assessment clinics (RAPACs). This service implements recommendations outlined by the National Cancer Control Programme. The RAPAC was introduced at Galway University Hospital, Galway, Ireland in June 2009, aiming to structure GP referral of patients with suspected prostate cancer to a urology service. AIMS: The aims of this study are to assess our initial experience with particular emphasis on access times, patient demographics, detection rates and treatment outcomes. METHODS: Data on all patients presenting to the RAPAC during the preliminary 2-year period have been gathered prospectively and analysed using standard parametric analysis methods. RESULTS: A total of 1,106 patients were reviewed at 278 clinic sessions during the initial 2-year period. The average waiting time to first clinic visit was 18 days (12-39 days). The mean age of referral to the clinic is 65 years (44-88 years). The mean PSA is 16.31 g/dL (0.4-845 g/dL). Of the 1106 patients undergoing TRUS biopsies, 503 (45.5 %) patients were diagnosed with prostate cancer. Further analysis patient demographics and cancer grading is presented in the article. Seventy-one patients (14.1 %) underwent radical retropubic prostatectomy. Sixty-seven patients (13.3 %) are being followed on an active monitoring programme, whilst 235 (56.7 %) received primary treatment with external beam radiotherapy and 68 (13.5 %) received brachytherapy. CONCLUSION: This data highlight the necessity of a RAPAC to streamline the provision of prostate cancer services in the west of Ireland.

Lentivirus-mediated gene transfer of viral interleukin-10 delays but does not prevent cardiac allograft rejection.

Human immunodeficiency virus (HIV)-based lentiviral vectors expressing viral interleukin-10 (vIL-10) were used to transduce rat cardiac allografts with the aim of extending graft survival. vIL-10 expression was first shown, by RT-PCR, to persist in transduced heart isografts for at least 28 days after transduction. Cardiac transplants were performed in a fully allogeneic rat strain combination (Lewis to DA); allografts transduced by vectors expressing vIL-10 showed significantly prolonged survival (14.5 vs 7.5 days median survival time). Mixed lymphocyte reactions (MLRs) were used to determine the influence, in vitro, of vIL-10 on alloantigen-induced T-cell proliferation. Bioactive vIL-10, produced by DA rat aortic endothelial cells transduced with HIV-PGK-vIL-10, was added to MLRs at different time points and lymphocyte proliferation was assessed by uptake of [3H]thymidine. T-cell proliferation was inhibited by >80% when vIL-10 was added to the MLR at day 1, 2 or 3 of coculture. The inhibitory effect was significantly decreased when addition of vIL-10 was delayed until day 4 or 5 (47 and 35% inhibition, respectively). The extended graft survival time is comparable to that using adenoviral vectors delivering vIL-10 in a similar rat strain combination. The limited improvement in survival may be due to lack of inhibition of the early phase of the alloimmune response as suggested by in vitro studies confirming that maximum suppression of the MLR by vIL-10 can only be achieved if the cytokine is present at the initiation of alloimmune recognition. The delay in expression of vIL-10 from the lentiviral vector means that protocols must be developed to suppress the early stages of alloimmune stimulation before vIL-10 is produced.

Epitope mapping of the indirect T cell response to allogeneic class I MHC: sequences shared by donor and recipient MHC may prime T cells that provide help for alloantibody production.

Indirect allorecognition occurs when T cells recognize donor MHC presented as peptide epitopes by recipient APC, but the precise nature of the epitopes involved remains unclear. Rejection of rat MHC class I-disparate PVG.R8 (RT1.A(a)) grafts by PVG.RT1(u) (RT1.A(u)) recipients is mediated by indirectly restricted CD4 T cells that provide help for the generation of alloantibody. In this study, epitope mapping was performed using a functionally relevant readout (alloantibody production) to identify key peptides that prime an indirect alloimmune response, leading to graft rejection. PVG.RT1(u) rats were immunized with a series of overlapping 15-mer peptides (peptides 1-18) that spanned the alpha1 and alpha2 domains of the RT1.A(a) molecule. Several peptides were able to accelerate both the alloantibody response to the intact RT1.A(a) Ag and PVG.R8 heart graft rejection. An immunodominant epitope was identified within the hypervariable region of the alpha1 domain. Fine mapping of this region with a second series of peptides overlapping by single amino acids confirmed the presence of an eight-amino acid core determinant. Additional "subdominant" epitopes were identified, two of which were located within regions of amino acid homology between the RT1.A(a) and RT1.A(u) molecules and not, as had been expected, within other hypervariable regions. The contribution of self-epitopes to indirect allorecognition was emphasized by the demonstration that i.v. administration of a 15-mer peptide encompassing one of the subdominant self-determinants diminished the recipient's ability to mount an alloantibody response on challenge with intact A(a) alloantigen. Our findings suggest that cryptic self-epitopes recognized by autoreactive T cells may contribute to allograft rejection and should be considered when designing novel strategies for inducing tolerance to alloantigen.

Selective blockade of IL-15 by soluble IL-15 receptor alpha-chain enhances cardiac allograft survival.

IL-15 is a T cell growth factor that shares many functional similarities with IL-2 and has recently been shown to be present in tissue and organ allografts, leading to speculation that IL-15 may contribute to graft rejection. Here, we report on the in vivo use of an IL-15 antagonist, a soluble fragment of the murine IL-15R alpha-chain, to investigate the contribution of IL-15 to the rejection of fully vascularized cardiac allografts in a mouse experimental model. Administration of soluble fragment of the murine IL-15R alpha-chain (sIL-15Ralpha) to CBA/Ca (H-2k) recipients for 10 days completely prevented rejection of minor histocompatibility complex-mismatched B10.BR (H-2k) heart grafts (median survival time (MST) of >100 days vs MST of 10 days for control recipients) and led to a state of donor-specific immunologic tolerance. Treatment of CBA/Ca recipients with sIL-15Ralpha alone had only a modest effect on the survival of fully MHC-mismatched BALB/c (H-2d) heart grafts. However, administration of sIL-15Ralpha together with a single dose of a nondepleting anti-CD4 mAb (YTS 177.9) delayed mononuclear cell infiltration of the grafts and markedly prolonged graft survival (MST of 60 days vs MST of 20 days for treatment with anti-CD4 alone). Prolonged graft survival was accompanied in vitro by reduced proliferation and IFN-gamma production by spleen cells, whereas CTL and alloantibody levels were similar to those in animals given anti-CD4 mAb alone. These findings demonstrate that IL-15 plays an important role in the rejection of a vascularized organ allograft and that antagonists to IL-15 may be of therapeutic value in preventing allograft rejection.

Exogenous type-1 cytokines modulate mercury-induced hyper-IgE in the rat.

Suppression of IgE responses is a major goal for immunotherapy, especially in the field of allergy. The Th2 subset of helper T cells plays a vital role in class switching of B cells to IgE production by releasing IL-4. In susceptible rat strains, mercuric chloride (HgCl2) induces activation of Th2 cells, with enhanced expression of IL-4, polyclonal B cell activation and very high levels of circulating IgE. We have previously shown that spontaneous regulation of this response coincides with enhanced expression of Th1/type-1 cytokines, including interferon-gamma (IFN-gamma) and IL-12. We now report the effects of administration of exogenous type-1 cytokines on HgCl2-induced Th2 responses. At high doses, recombinant rat IFN-gamma markedly reduced serum IgE levels. Recombinant mouse IL-12 was less effective at suppressing the IgE response following HgCl2, although it caused marked up-regulation of IFN-gamma gene expression in the spleen. In Lewis rats, which are resistant to HgCl2-induced autoimmunity, a rise in serum IFN-gamma was observed after HgCl2, but administration of polyclonal anti-IFN-gamma antibodies did not render them susceptible to induction of a Th2 response by HgCl2. Our data show that individual type-1 cytokines are capable of suppressing the dramatic Th2 response induced by HgCl2 in the rat, even when they are not given until after starting HgCl2 administration. IFN-gamma is a pivotal cytokine in ameliorating the Th2 response and measures aimed at selective up-regulation of this cytokine may be of therapeutic value in suppression of unwanted IgE responses.

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.

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.

Indirect T cell allorecognition and alloantibody-mediated rejection of MHC class I-disparate heart grafts.

Recent studies in the rat have identified a role for T cell-dependent alloantibody in rejection of MHC class I-disparate allografts. RT1Aa-disparate PVG.R8 heart grafts are rejected acutely in naive, and hyperacutely in sensitized, PVG.RTIu recipients by CD4 T cell-dependent alloantibody. Here, we examined the T cell Ag recognition pathways responsible and show that direct injection into skeletal muscle of plasmid DNA, encoding a water-soluble form of the RT1Aa MHC class I heavy chain (pcmu-tAa), stimulates IgG2b cytotoxic alloantibody and markedly accelerates rejection of PVG.R8 heart grafts (median survival time 2 days). pcmu-tAa injection did not induce CTL to Aa, arguing against direct allorecognition of soluble Aa. Treatment with mAbs confirmed that the alloimmune response to pcmu-tAa injection depended on CD4, not CD8, T cells. Priming T cells for indirect allorecognition by injection of 15-mer peptides spanning the alpha1 and alpha2 domains of Aa failed to stimulate anti-Aa Ab but caused an accelerated Ab response to a PVG.R8 heart and a modest acceleration in graft rejection (median survival time 4 days). These results suggest that both soluble MHC class I and allopeptides prime CD4 T cells by the indirect pathway, but that soluble class I is a more effective immunogen for humoral alloimmunity because its tertiary protein structure provides B cell epitopes. We propose that priming humoral alloimmunity, like CTL priming, requires recognition of intact MHC on donor cells, but essential T cell help can be provided by CD4 T cells recognizing allogeneic class I exclusively by the indirect pathway.

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.

The reduction of alpha-tocopherolquinone by human NAD(P)H: quinone oxidoreductase: the role of alpha-tocopherolhydroquinone as a cellular antioxidant.

alpha-Tocopherolquinone (TQ), a product of alpha-tocopherol oxidation, can function as an antioxidant after reduction to alpha-tocopherolhydroquinone (TQH2). We examined the ability of human NAD(P)H:quinone oxidoreductase (NQO1) to catalyze the reduction of TQ to TQH2 in cell-free and cellular systems. In reactions with purified human NQO1, TQ was reduced to TQH2. Kinetic parameters for the reduction of TQ by NQO1 (Km = 370 microM; k(cat) = 5.6 x 10(3) min(-1); k(cat)/Km = 15 min(-1) x microM(-1)) indicate that NQO1 can efficiently reduce TQ to TQH2. A comparison of the rate of reduction of TQ and coenzyme Q10 by NQO1 showed that TQ is reduced more efficiently than coenzyme Q10. Experiments with either Chinese hamster ovary (CHO) cells stably transfected with human NQO1 or CHO cell sonicates demonstrated a correlation between NQO1 activity and TQ reduction to TQH2. CHO cells with elevated NQO1 generated and maintained higher levels of TQH2 after treatment with TQ relative to NQO1-deficient CHO cells. TQH2 generated from NQO1-mediated reduction of TQ prevented cumene hydroperoxide-induced lipid peroxidation in rat liver microsomes. In addition, cumene hydroperoxide-induced lipid peroxidation was inhibited more efficiently by TQ in CHO cell lines with elevated NQO1 activity. These data demonstrate that NQO1 can reduce TQ to TQH2 and that TQH2 can function as an efficient antioxidant. This work suggests that one of the physiological functions of NQO1 may be to regenerate antioxidant forms of alpha-tocopherol.

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.