Localized myxedema histologically mimicking spindle cell lipoma.

In this report, a 55-year-old woman with Graves disease and exophthalmos had a recurrent nodule on the foot. Her initial biopsy and excision specimens were believed to be consistent with spindle cell lipoma, which aligned with her early tumor-like clinical morphology. Her tumor recurred after excision, which is not consistent with spindle cell lipoma. As her condition progressed, her clinical morphology became more consistent with localized myxedema and her biopsies were congruent, securing clinicopathologic correlation. With standard treatment for localized myxedema, she improved significantly. This case emphasizes how clinicians need to have high suspicion for localized myxedema in patients with history of Graves disease and exophthalmos. It also emphasizes how localized myxedema should be included in the histologic differential diagnosis for spindle cell lipoma with prominent myxoid stroma, particularly in those not responding to treatment as anticipated.

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.

Spatio-temporal epidemiology of Campylobacter jejuni enteritis, in an area of Northwest England, 2000-2002.

A total of 969 isolates of Campylobacter jejuni originating in the Preston, Lancashire postcode district over a 3-year period were characterized using multi-locus sequence typing. Recently developed statistical methods and a genetic model were used to investigate temporal, spatial, spatio-temporal and genetic variation in human C. jejuni infections. The analysis of the data showed statistically significant seasonal variation, spatial clustering, small-scale spatio-temporal clustering and spatio-temporal interaction in the overall pattern of incidence, and spatial segregation in cases classified according to their most likely species-of-origin.

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.

Human platelets contain a glycosylated estrogen receptor beta.

Platelets play an important role in the coronary thrombus formation that leads to myocardial ischemia and infarction. Gender differences in the development of coronary heart disease and its outcomes are partly regulated by estrogen and its receptors, but the roles of the latter in thrombogenicity are less well-defined. We previously demonstrated the presence of estrogen receptor (ER) beta in cells of the megakaryocytic lineage. In this study, we characterize human platelet ERbeta and its expression using biochemical and molecular biological techniques. Western immunoblotting showed that platelet ERbeta migrated with an apparent molecular mass approximately 3.7 kDa larger than ERbeta in a variety of cell lines (including those of prostate and breast origin). A rigorous investigation of platelet ERbeta mRNA by reverse transcriptase-polymerase chain reaction revealed normal transcripts and a single alternately spliced mRNA. However, this variant form was smaller, lacking exon 2, and could not account for the larger protein size seen in platelets. Treatment of ERbeta with N-glycosidase F, which removes core carbohydrate residues, caused a more rapid migration through polyacrylamide gels but had no effect on ERbeta from human cell lines. We conclude that the larger form of ERbeta in human platelets is not attributable to alternate mRNA splicing but primarily to tissue-specific glycosylation.

Mechanisms of adoptive immunotherapy: improved methods for in vivo tracking of tumor-infiltrating lymphocytes and lymphokine-activated killer cells.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) and lymphokine-activated killer cells has been demonstrated to mediate regression of tumors in murine models and in selected patients with advanced cancer. Improved methods for monitoring immune cell traffic, particularly to sites of tumor, are needed to elucidate mechanisms of antitumor activity and optimize treatment protocols. Traditional cell tracking methods such as fluorescent protein labeling and radiolabeling using 111In, 125I, or 51Cr are limited by isotope half-life, leakage or transfer of label from immune cells, and toxicity or altered cell function caused by the labeling process. Labeling with genetic markers allows long-term cell tracking but is laborious to perform and difficult to quantitate. We have used two recently described lipophilic cell tracking compounds (PKH26 and 125I-PKH95) which stably partition into lipid regions of the cell membrane to track immune cells in vivo. Concentrations of each tracking compound which had no adverse effects were determined for a variety of murine TIL and lymphokine-activated killer cell functions. Viability was unimpaired at labeling concentrations of up to 5 microM for PKH95 and 20 microM for PKH26. TIL proliferation was unaltered by labeling with up to 5 microM PKH95, 20 microM PKH26, or a combination of 15 microM PKH26 and 5 microM PKH95. In vivo cytotoxic effector function and in vivo therapeutic efficacy of lymphokine-activated killer cells and TIL were also unimpaired by labeling with 20 microM PKH26 or 1 microM 125I-PKH95. Subsequent studies in an adoptive transfer immunotherapy model used 125I-PKH95 to track the biodistribution of TIL in tumor and in non-tumor-bearing animals and PKH26 fluorescence to monitor microdistribution within tissues and distinguish TIL from host T-cells. The results suggest that differential accumulation, selective retention, or proliferation at the tumor site cannot account for the observed pattern of therapeutic efficacy. We hypothesize that a minimum number of TIL must reach the tumor site in order to achieve a demonstrable therapeutic effect.

Believe it or not: Moving non-biological stimuli believed to have human origin can be represented as human movement.

Does our brain treat non-biological movements (e.g. moving abstract shapes or robots) in the same way as human movements? The current work tested whether the movement of a non-biological rectangular object, believed to be based on a human action is represented within the observer's motor system. A novel visuomotor priming task was designed to pit true imitative compatibility, due to human action representation against more general stimulus response compatibility that has confounded previous belief experiments. Stimulus response compatibility effects were found for the object. However, imitative compatibility was found when participants repeated the object task with the belief that the object was based on a human finger movement, and when they performed the task viewing a real human hand. These results provide the first demonstration that non-biological stimuli can be represented as a human movement if they are believed to have human agency and have implications for interactions with technology and robots.

Loading with oxidised low density lipoprotein alters endocytic and secretory activities of murine macrophages.

Lipid-loaded macrophages were produced in vitro by incubation with acetylated or copper-oxidized LDL. In order to establish whether cellular membrane traffic is generally perturbed by such loading, we assessed endocytosis of fluid; cell surface binding, internalisation and degradation of a soluble ligand and of a particulate preparation; and exocytosis of lysosmal enzymes. Fluid-phase pinocytosis of sucrose was unaffected by either form of loading. Binding, uptake and degradation of soluble (mannosylated-BSA) and particulate (zymosan) ligands by these lipid-loaded and by non-loaded cells were compared. Loading with oxidized LDL decreased the processing of both ligands, while loading with acetylated LDL had little effect. Loading with oxidized LDL (Ox-LDL) also decreased zymosan binding at 4 degrees C; and the internalisation and degradation of ligands in Ox-LDL loaded and non-loaded cells reflected the extent of surface binding. Changes in binding and uptake of mannosylated-BSA and zymosan were not due to changes in viability or cell number. Zymosan stimulated release of lysosomal beta-N-acetyl-D-glucosaminidase from the cells. Loading with Ox- but not Ac-LDL decreased beta-N-acetyl-D-glucosaminidase secretion. After incubation with zymosan, intracellular levels of the enzyme were increased in the Ox-LDL loaded cells. Zymosan uptake and beta-N-acetyl-D-glucosaminidase secretion were correlated, but enzyme activity per culture rose more in the absence than in the presence of zymosan. We conclude that membrane traffic is perturbed in model foam cells, particularly those loaded with Ox-LDL.

In vivo administration of recombinant macrophage colony-stimulating factor induces macrophage-mediated antibody-dependent cytotoxicity of tumor cells.

Macrophage colony-stimulating factor (M-CSF) has been previously shown to facilitate the in vitro survival and differentiation of mononuclear phagocytes. We assessed whether M-CSF administration in vivo could induce macrophages capable of killing tumor via an antibody-dependent mechanism. C57BL/6 mice were given intraperitoneal M-CSF, and peritoneal macrophages were assayed for their ability to kill fluorochrome-labeled R1.1 thymoma cells in vitro in the presence or absence of target-specific antibody. Two-color flow cytometry was used in measuring tumor ingestion by macrophages; macrophages from M-CSF-treated mice eliminated greater than 90% of R1.1 thymoma target within 24 hours, while macrophages from saline-treated controls were ineffective. R1.1 tumor elimination by macrophages depended on the presence of target-specific antibody. These are the first studies that demonstrate the in vivo induction, by M-CSF, of macrophages directly capable of ingesting antibody-conjugated tumor cells.

Enhanced LDL oxidation by murine macrophage foam cells and their failure to secrete nitric oxide.

Foam cells were produced in vitro by incubation of mouse peritoneal macrophages with acetylated or copper-oxidized LDL. Nitric oxide synthesis was stimulated by exposure of the cells to IFN gamma and LPS. Nitric oxide production, detected by measurement of nitrite in the culture medium, was unchanged in Ac-LDL loaded cells as compared with non-loaded cells. However, Ox-LDL foam cells produced 68-99% less nitrite than non-loaded cells. Failure to detect nitric oxide synthase (NOS) products from macrophages previously loaded with Ox-LDL appeared to result from lack of NOS activity, as little active enzyme could be recovered from Ox-LDL loaded cells. However, addition of Ox-LDL to an active cell-free NOS preparation had no direct effect on enzymic activity. When native LDL was subsequently incubated with these various IFN gamma/LPS stimulated cells, cells pre-loaded with Ox-LDL promoted, on average, a 2-fold greater increase in oxidative modification of the LDL added than either non-loaded or Ac-LDL loaded cells. That is, there was an inverse correlation between NOS activity and the ability of the cells to promote LDL oxidation. Unstimulated Ox-LDL loaded foam cells also oxidized LDL better than unstimulated non-loaded or Ac-LDL loaded foam cells, and the extent of oxidative modification was generally greater than seen with the equivalent IFN gamma/LPS stimulated cells. This suggests that Ox-LDL loading also affects some additional factor(s) responsible for cell-mediated LDL oxidation.

Detection of antigen specific T lymphocytes by determination of intracellular calcium concentration using flow cytometry.

We present a method for the detection of lymphocytes with specific reactivity to antigens on stimulator cells using flow cytometry. Cultured human T lymphocytes were loaded with the intracellular fluorochrome indo-1 and were mixed with stimulator cells. Using flow cytometry we could detect a specific increase in intracellular calcium in the T lymphocytes as well as conjugation between the T cells and the stimulator cells. Examination of antigen-specific CD4+ and CD8+ T cell clones demonstrated that the vast majority of T cells which were conjugated to antigen-bearing stimulator cells manifested a rapid increase in intracellular calcium. In contrast T cells conjugated to stimulator cells which did not bear specific antigen demonstrated no such increase in calcium. A similar finding was observed when examining polyclonal tumor infiltrating lymphocytes obtained from patients with melanoma. Tumor infiltrating lymphocytes with specific antitumor reactivity demonstrated an increase in intracellular calcium when conjugated to autologous tumor but not to allogeneic melanoma. In contrast to the T cell clones, only a small subpopulation of tumor infiltrating lymphocytes manifested this specific signal upon conjugation with autologous tumor. This suggests that tumor infiltrating lymphocyte cultures contain T cells with varying reactivities to tumor or may also imply heterogeneity in the stimulating tumor cell lines. The method allows for the detection of specific T cells on an individual cell basis in real time. The procedure is not lethal to the cell and sorting and subculturing of reactive T cell populations can be readily performed. The method could also be used to sort stimulator cells based on their ability to elicit an increase in intracellular calcium in selected T cells.

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.