Anaesthetic efficacy and postinduction hypotension with remimazolam compared with propofol: a multicentre randomised controlled trial.

Remimazolam, a short-acting benzodiazepine, may be used for induction and maintenance of total intravenous anaesthesia, but its role in the management of patients with multiple comorbidities remains unclear. In this phase 3 randomised controlled trial, we compared the anaesthetic efficacy and the incidence of postinduction hypotension during total intravenous anaesthesia with remimazolam vs. propofol. A total of 365 patients (ASA physical status 3 or 4) scheduled for elective surgery were assigned randomly to receive total intravenous anaesthesia with remimazolam (n = 270) or propofol (n = 95). Primary outcome was anaesthetic effect, quantified as the percentage of time with Narcotrend® Index values ≤ 60, during surgery (skin incision to last skin suture), with a non-inferiority margin of -10%. Secondary outcome was the incidence of postinduction hypotensive events. Mean (SD) percentage of time with Narcotrend Index values ≤ 60 during surgery across all patients receiving remimazolam (93% (20.7)) was non-inferior to propofol (99% (4.2)), mean difference (97.5%CI) -6.28% (-8.89-infinite); p = 0.003. Mean (SD) number of postinduction hypotension events was 62 (38.1) and 71 (41.1) for patients allocated to the remimazolam and propofol groups, respectively; p = 0.015. Noradrenaline administration events (requirement for a bolus and/or infusion) were also lower in patients allocated to remimazolam compared with propofol (14 (13.5) vs. 20 (14.6), respectively; p < 0.001). In conclusion, in patients who were ASA physical status 3 or 4, the anaesthetic effect of remimazolam was non-inferior to propofol.

Environmental Exposures-The Missing Link in Immune Responses After Transplantation.

In transplantation, immunosuppression has been directed at controlling acute responses, but treatment of chronic rejection has been ineffective. It is possible that factors that have previously been unaccounted for, such as exposure to inhaled pollution, ultraviolet light, or loss of the normal equilibrium between the gut immune system and the outside environment may be responsible for shifting immune responses to an effector/inflammatory phenotype, which leads to loss of self-tolerance and graft acceptance, and a shift towards autoimmunity and chronic rejection. Cells of the immune system are in a constant balance of effector response, regulation, and quiescence. Endogenous and exogenous signals can shift this balance through the aryl hydrocarbon receptor, which serves as a thermostat to modulate the response one way or the other, both at mucosal surfaces of interface organs to the outside environment, and in the internal milieu. Better understanding of this balance will identify a target for maintenance of self-tolerance and continued graft acceptance in patients who have achieved a "steady state" after transplantation.

Influence of intensive care treatment on the protein binding of sufentanil and hydromorphone during pain therapy in postoperative cardiac surgery patients.

BACKGROUND: Our objective was to evaluate the effect of intensive care treatment on the protein binding of sufentanil and hydromorphone in cardiac surgery patients during postoperative analgesia using a target-controlled infusion (TCI) and patient-controlled analgesia (PCA). METHODS: Fifty adult patients were enrolled in this prospective randomized study; of which, 49 completed the study (age range 40-81 yr). Sufentanil was administered as an analgesic intraoperatively, and hydromorphone was dosed after operation with TCI and PCA until 8 a.m. on the first postoperative day. Arterial plasma samples were collected for drug and protein concentration measurements up to 24 h after cardiac surgery. Corresponding patient data were collected from the electronic patient data system. After explorative data analysis with principal component analysis, multivariate regression analysis and non-linear mixed effects modelling was used to study the effect of treatment on protein binding. RESULTS: Data of 35 patients were analysed. The median protein binding of sufentanil and hydromorphone was 88.4% (IQ range 85.7-90.5%) and 11.6% (IQ range 9.5-14.3%), respectively. Free fraction of sufentanil increased towards the end of the study period, whereas hydromorphone free fraction remained nearly constant. The total sufentanil concentration and volume balance were identified as significant covariates for the protein binding of sufentanil. For the protein binding of hydromorphone, no significant covariate effects were found. CONCLUSIONS: Sufentanil protein binding was significantly dependent on changes in the total drug concentration and volume balance addressing the importance of adequate dosing and fluid-guided therapy. Hydromorphone protein binding was nearly constant throughout the study period. CLINICAL TRIAL REGISTRATION: EudraCT 2011-003648-31 and ClinicalTrials.gov: NCT01490268.

Changes in total and unbound concentrations of sufentanil during target controlled infusion for cardiac surgery with cardiopulmonary bypass.

BACKGROUND: Target controlled infusion (TCI) with sufentanil is usually performed using the Gepts model, which was derived from patients undergoing general surgery. It is, however, known that pharmacokinetics of sufentanil can be changed during cardiopulmonary bypass (CPB). We tested whether TCI during coronary artery bypass surgery with CPB produces constant total, unbound sufentanil concentration-time course or both. METHODS: After IRB approval, written informed consent was obtained from 38 male patients (48-74 yr) undergoing coronary artery bypass surgery. Anaesthesia was managed with propofol and TCI of sufentanil, using the Gepts model, targeting plasma concentrations of 0.4 (n=18) or 0.8 ng ml(-1) (n=20). Arterial blood samples were taken before, during, and after CPB. Total and unbound sufentanil concentrations were measured by HPLC with tandem mass spectrometry. The accuracy of the TCI model was assessed by the prediction error, and a pharmacokinetic model was determined by population analysis. RESULTS: The median prediction error of the TCI with the Gepts model before, during, and after CPB was 59.6, 3.9, and -10.4%, respectively. The unbound sufentanil concentrations increased significantly during CPB. Pharmacokinetic modelling showed an increase in elimination and intercompartmental clearance after initiation of CPB. CONCLUSIONS: Neither total nor unbound sufentanil concentrations remained constant when performing a TCI with the Gepts model in coronary artery bypass surgery with CPB. A pharmacokinetic model derived from patients undergoing cardiac surgery with CPB might improve the performance of TCI in this population.

Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection in nonhuman primates.

CD154 is the ligand for the receptor CD40. This ligand-receptor pair mediates endothelial and antigen-presenting cell activation, and facilitates the interaction of these cells with T cells and platelets. We demonstrate here that administration of a CD154-specific monoclonal antibody (hu5C8) allows for renal allotransplantation in outbred, MHC-mismatched rhesus monkeys without acute rejection. The effect persisted for more than 10 months after therapy termination, and no additional drug was required to achieve extended graft survival. Indeed, the use of tacrolimus or chronic steroids seemed to antagonize the anti-rejection effect. Monkeys treated with antibody against CD154 remained healthy during and after therapy. The mechanism of action does not require global depletion of T or B cells. Long-term survivors lost their mixed lymphocyte reactivity in a donor-specific manner, but still formed donor-specific antibody and generated T cells that infiltrated the grafted organ without any obvious effect on graft function. Thus, therapy with antibody against CD154 is a promising agent for clinical use in human allotransplantation.

Precision and accuracy of a new device (CNAPTM) for continuous non-invasive arterial pressure monitoring: assessment during general anaesthesia.

BACKGROUND: Continuous non-invasive arterial pressure measured with CNAP (CNAP) has been shown to be superior to intermittent oscillometric measurements during procedural sedation and spinal anaesthesia. We assessed the performance of CNAP during general anaesthesia by analysis of agreement with invasive measurements of arterial pressure (AP). METHODS: Eighty-eight patients undergoing elective abdominal surgery, cardio-, or neurosurgery were included in the study. Systolic, diastolic, and mean AP measured by an intra-arterial catheter in the radial artery (IAP) were compared with those obtained by CNAP from the same arm. Data were analysed to determine the precision (i.e. measurement error) and accuracy (i.e. systematic error) of beat-to-beat CNAP values with respect to IAP. Also, we compared the frequency of fast changes in AP (FCAP) and hypotension (IOH) by both methods. RESULTS: CNAP precision of 4.5, 3.1, and 3.2 mm Hg (systolic, diastolic, and mean AP, respectively) was not significantly different from IAP precision, and CNAP accuracy was +6.7, -5.6, and -1.6 mm Hg. The frequency of AP pairs having a difference within the calculated limits of agreement was 81%, 64%, and 76% for systolic, diastolic, and mean AP, respectively. The calculated limits of agreement were +/-17.6, +/-11.4, and +/-12.0 mm, Hg, respectively. CNAP and IAP detected simultaneously to 82.1% FCAP and to 84.6% IOH. CONCLUSIONS: CNAP provides real-time estimates of arterial pressure comparable with those generated by an invasive intra-arterial catheter system during general anaesthesia.

BAFF is increased in renal transplant patients following treatment with alemtuzumab.

Alemtuzumab is a monoclonal antibody that depletes T and B cells and is used as induction therapy for renal transplant recipients. Without long-term calcineurin inhibitor (CNI) therapy, alemtuzumab-treated patients have a propensity to develop alloantibody and may undergo antibody-mediated rejection (AMR). In pursuit of a mechanistic explanation, we analyzed peripheral B cells and serum of these patients for BAFF (Blys) and BAFF-R, factors known to be integral for B-cell activation, survival, and homeostasis. Serum BAFF levels of 22/24 alemtuzumab-treated patients were above normal range, with average levels of 1967 pg/mL compared to 775 pg/mL in healthy controls (p = 0.006). BAFF remained elevated 2 years posttransplant in 78% of these patients. BAFF-R on CD19(+) B cells was significantly downregulated, suggesting ligand/receptor engagement. BAFF mRNA expression was increased 2-7-fold in CD14(+) cells of depleted patients, possibly linking monocytes to the BAFF dysregulation. Addition of recombinant BAFF to mixed lymphocyte cultures increased B-cell activation to alloantigen, as measured by CD25 and CD69 coexpression on CD19(+) cells. Of note, addition of sirolimus (SRL) augmented BAFF-enhanced B-cell activation whereas CNIs blocked it. These data suggest associations between BAFF/BAFF-R and AMR in alemtuzumab-treated patients.

CD4+ CD25+ FOXP3+ regulatory T cells increase de novo in kidney transplant patients after immunodepletion with Campath-1H.

Campath-1H (Alemtuzumab) is an effective immunodepletion agent used in renal transplantation. To evaluate its influence on T lymphocytes during repletion, we analyzed peripheral blood from Campath-1H-treated renal allograft recipients for the presence of FOXP3(+) regulatory T (Treg) cells. Flow cytometry demonstrated that CD4(+)CD25(+)FOXP3(+) lymphocytes increased significantly within the CD4(+) T-cell population, skewing Treg/Teff (T effector) ratios for up to several years. In contrast, Treg levels in patients treated with anti-CD25 (Basiliximab) and maintained on CsA demonstrated a sustained decrease. The increase in Tregs in Campath-1H treated patients developed independent of maintenance immunosuppression. Importantly, the increase in Tregs was not fully explained by their homeostatic proliferation, increased thymic output, or Treg sparing, suggesting de novo generation/expansion. Consistent with this, in vitro stimulation of PBMCs with Campath-1H, with or without anti-CD3, activation led to an increase in CD4(+)CD25(+)FOXP3(+) cells that had suppressive capabilities. Together, these data suggest that Campath-1H promotes an increase in peripheral Tregs and may act as an intrinsic generator of Tregs in vivo.

Unaltered graft survival and intragraft lymphocytes infiltration in the cardiac allograft of Cxcr3-/- mouse recipients.

Previous studies showed that absence of chemokine receptor Cxcr3 or its blockade prolong mouse cardiac allograft survival. We evaluated the effect of the CXCR3 receptor antagonist MRL-957 on cardiac allograft survival, and also examined the impact of anti-CXCR3 mAb in human CXCR3 knock-in mice. We found only a moderate increase in graft survival (10.5 and 16.6 days, p < 0.05) using either the antagonist or the antibody, respectively, compared to control (8.7 days). We re-evaluated cardiac allograft survival with two different lines of Cxcr3(-/-) mice. Interestingly, in our hands, neither of the independently derived Cxcr3(-/-) lines showed remarkable prolongation, with mean graft survival of 9.5 and 10.8 days, respectively. There was no difference in the number of infiltrating mononuclear cells, expansion of splenic T cells or IFN-gamma production of alloreactive T cells. Mechanistically, an increased other chemokine receptor fraction in the graft infiltrating CD8 T cells in Cxcr3(-/-) recipients compared to wild-type recipients suggested compensatory T-cell trafficking in the absence of Cxcr3. We conclude Cxcr3 may contribute to, but does not govern, leukocyte trafficking in this transplant model.

Pharmacodynamic modelling of the bispectral index response to propofol-based anaesthesia during general surgery in children.

BACKGROUND: This study describes a pharmacodynamic model during general anaesthesia in children relating the bispectral index (BIS) response to the anaesthetic dosing of propofol, fentanyl, and remifentanil. METHODS: BIS, heart rate, mean arterial pressure, sedation scores, and anaesthetic protocols from 59 children aged 1-16 yr undergoing general surgery were considered for the study. Anaesthesia was performed with propofol, fentanyl, and remifentanil. A sigmoid model assuming additive interaction of propofol, fentanyl, and remifentanil was fitted to individual BIS as effect variable. The pharmacodynamic parameters were estimated by non-linear regression analysis. The ability of BIS to predict anaesthetic drug effect was quantified by the prediction probability Pk. RESULTS: BIS started at a baseline of 90 (9), decreased during induction to 30 (14) and remained at 57 (10) during anaesthesia. BIS predicted the anaesthetic drug effect with a Pk of 0.79 (0.08). The EC(50 Propofol) and the k(e0 Propofol) were 5.2 (2.7) microg ml(-1) and 0.60 (0.45) min(-1), respectively. The k(e0 Propofol) decreased from approximately 0.91 min(-1) at 1 yr to 0.15 min(-1) at 16 yr. The EC(50 Remifentanil), k(e0 Remifentanil), EC(50 Fentanyl), and the k(e0 Fentanyl) were 24.1 (13.0) ng ml(-1), 0.71 (0.32) min(-1), 8.6 (7.4) ng ml(-1), and 0.28 (0.46) min(-1), respectively. CONCLUSIONS: The effect equilibration half-time of propofol in children was age dependent. The pharmacodynamics of fentanyl and remifentanil in children were similar to those reported in adults. The BIS showed a close relationship to the modelled effect-site concentration, and therefore, it may serve as a measure of anaesthetic drug effect in children older than 1 yr.

Pharmacokinetics and pharmacodynamics of GPI 15715 or fospropofol (Aquavan injection) - a water-soluble propofol prodrug.

Propofol (2,6-diisopropylphenol) is inadequably soluble in water and is therefore formulated as a lipid emulsion. This may have disadvantages when propofol is used to provide total intravenous anaesthesia or especially during long-term sedation. There has been considerable interest in the development of new propofol formulations or propofol prodrugs. GPI 15715 or fospropofol (Aquavan injection; Guilford Pharmaceutical, Baltimore, MD) is the first water-soluble prodrug that has been thoroughly studied in human volunteers and patients. GPI 15751 or fospropofol is cleaved by alkaline phosphatase to phosphate, formaldehyde and propofol. Formaldehyde is rapidly metabolised to formate. Although a formate accumulation is the principal pathomechanism responsible for the toxicity of methanol ingestion, so far there has been no report of toxicity due to the administration of fospropofol or other phosphate ester prodrugs, such as fosphenytoin. Fosphenytoin has been successfully introduced into the market for the treatment of status epilepticus in 1996. The main side-effects were a feeling of paraesthesia after rapid i.v. administration of GPI 15715 or fospropofol, which has also been described for fosphenytoin. The pharmacokinetics of GPI 15715 or fospropofol could be described by a combined pharmacokinetic model with a submodel of two compartments for GPI 15715 and of three compartments for propofol(G). The liberated propofol(G) compared to lipid-formulated propofol showed unexpected pharmacokinetic and pharmacodynamic differences. We found a significantly greater V(c), V(dss), significantly shorter alpha- and beta-half-life and a longer MRT (mean residence time) for propofol(G). The pharmacodynamic potency of propofol(G) appears to be higher than propofol when measured by EEG and clinical signs of hypnosis. In summary, GPI 15715 or fospropofol was well suited to provide anaesthesia or conscious sedation.

Immunosuppressive effects of an HLA class I-derived peptide in a rat cardiac allograft model.

B7.75-84, a 10-amino-acid peptide derived from the HLA-B7 molecule, prolongs rat heterotopic cardiac allograft survival time (GST) when used with cyclosporine in the Lewis-to-ACI strain combination. We evaluated the ability of B7.75-84 to prolong GST in other strain combinations without cyclosporine and studied the effect of B7.75-84 on the immune response in the Wistar-Furth (WF)-to-ACI strain combination. GST was markedly prolonged in most low-responder (ACI) recipients but only slightly prolonged in the high-responder (Lewis) recipient. Cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) limiting dilution assays (LDA) were performed 10 days after cardiac allografts from WF donors were placed in ACI recipients treated with B7.75-84. HTL-LDA assays at 10 days posttransplant showed a slight decrease in HTL precursor frequency and a decrease in their IL-2 production in B7.75-84 treated recipients with prolonged GST in response to donor antigen as well as third-party (Lewis) antigen. CTL-LDA assays at day 10 showed no difference in CTL precursor frequency among treated recipients but did show a significant decrease in CTL killing activity against donor cells in recipients with prolonged GST. No significant difference in CTL killing activity was seen against third- party cells. Antibody analysis was performed at day 8 in treated recipients. Serum from B7.75-84-treated recipients with prolonged graft survival generally showed no detectable IgG antibody response against donor MHC class I antigen. All B7.75-84 treated recipients showed a strong IgM response against donor antigen regardless of allograft outcome. Our results suggest that the immunosuppressive effect of B7.75-84 in rats is greater using a low-responder RT1 haplotype. Furthermore, B7.75-84 induces a nonspecific decrease in HTL function while producing a donor-specific decrease in CTL function and a diminished antidonor MHC class I IgG response.

Activation of T lymphocytes for adhesion and cytokine expression by toxin-conjugated anti-CD3 monoclonal antibodies.

BACKGROUND: Immunosuppressive drugs that target T cells are useful for prolonging allograft survival. The anti-CD3 immunotoxin FN18-CRM9 has been shown to effectively prolong renal allograft survival in a rhesus monkey model of transplantation. However, immunotoxin-treated monkeys showed increased levels of inflammatory cytokines and produced antibodies to donor proteins. To better understand the role of FN18-CRM9 in the production of cytokines and anti-donor antibodies in the monkey model, we examined whether this immunotoxin elicits functional responses in T cells. METHODS: Purified normal rhesus monkey T cells (>98% purity) were incubated with immunotoxin FN18-CRM9 or the unconjugated anti-CD3 monoclonal antibodies and then examined for changes in protein tyrosine phosphorylation, adhesion to fibronectin, gene expression, and proliferation in the presence or absence of anti-CD28 monoclonal antibodies (mAb) and interleukin-2. RESULTS: Immunotoxin treatment of T cells in vitro increased protein tyrosine phosphorylation, cell adhesion to the extracellular matrix, and expression of the inflammatory cytokines interferon-gamma and tumor necrosis factor-alpha. These immunotoxin effects were similar in magnitude to those induced by the unconjugated mAb. In contrast, immunotoxin-induced T cell proliferation was markedly less than that induced by the unconjugated mAb. Interestingly, the mitogenic molecules IL-2 and anti-CD28 mAb did not prevent immunotoxin-induced inhibition of cell proliferation. CONCLUSIONS: The activation of T cells for protein phosphorylation, adhesion, and cytokine expression strongly suggests that the actions of FN18-CRM9 in vivo are not limited to the inhibition of protein synthesis.

Chronic human skin graft rejection in severe combined immunodeficient mice engrafted with human PBL from an HLA-presensitized donor.

Mice with severe combined immunodeficiency (C.B-17 scid [SCID]) accepted xenografts of adult human peripheral blood leukocytes injected intraperitoneally as evidenced by production of human immunoglobulin (IgG and IgM), and circulation of human leukocytes in peripheral blood. SCID mice also accepted human split-thickness skin xenografts. Passenger leukocytes present in small numbers in such skin grafts could also recirculate in host peripheral blood and make detectable levels of human immunoglobulin. To test the immunocompetence of the transferred human PBL, SCID mice received a human skin xenograft from a second donor (HLA-mismatched with the PBL donor) either before (n = 6) or after (n = 23) xenografting of PBL. Skin was monitored daily for signs of rejection, and rejection was scored by histology 3-4 weeks after the second graft (PBL or skin) was placed. Of 19 SCID injected with PBL from an HLA presensitized patient (L.G.), 7/19 (37%) rejected a subsequent HLA-mismatched skin xenograft. Two of six SCID (33%) rejected a previously established skin xenograft when PBL were administered afterward. The rejection of the human skin was chronic, of relatively late onset (3-4 weeks), and was characterized grossly by contraction, glassy surface, and thickening. Histopathologic examination showed lymphocyte infiltration into the dermis with endothelial cell cuffing and destruction of capillaries, as well as lymphocyte tagging of the basal epidermis, hyperkeratosis, lymphocyte exocytosis and single epidermal cell necrosis. Immunostaining with monoclonal antibody to human CD2 or mouse CD3 revealed that human, but not mouse T lymphocytes were tagging the dermis/epidermis junction and infiltrating the epidermis of rejecting skin grafts. We conclude that a form of human skin graft rejection may be reproduced in an SCID mouse. The immune status of the transferred cells (sensitized vs. normal) and the lymphocytes ability to recirculate in SCID peripheral blood appear to be factors limiting the rejection process.

Immunotoxin FN18-CRM9 induces stronger T cell signaling than unconjugated monoclonal antibody FN18.

BACKGROUND: The T-cell receptor (TCR)/CD3 complex is the target of therapeutic strategies aimed at prolonging allograft survival. The immunotoxin FN18-CRM9, composed of the anti-CD3 monoclonal antibody FN18 and the mutated diphtheria toxin CRM9, is useful for prolonging allograft survival in preclinical models of transplantation. To explore the influence of conjugation of the mutated diphtheria toxin on functional activation of the TCR/CD3 complex, we compared the effects of FN18-CRM9 and unconjugated FN18 on protein tyrosine phosphorylation and ligand/receptor internalization in purified monkey peripheral blood T cells. METHODS: Purified normal rhesus monkey T cells were incubated with unconjugated FN18 or conjugated FN18-CRM9 and examined for differences in antibody binding, tyrosine phosphorylation, and CD3 internalization. RESULTS: Binding cross-inhibition studies demonstrated that both compounds were able to inhibit fluorescein isothiocyanate-FN18 binding to CD3 with similar efficacy and potency. However, FN18-CRM9 was more potent than FN18 in triggering the phosphorylation of several proteins on tyrosine residues and in inducing CD3 internalization. The tyrosine kinase inhibitor genistein blocked FN18-CRM9-induced protein tyrosine phosphorylation and CD3 internalization, suggesting that tyrosine phosphorylation is involved in the internalization of the immunotoxin. Interestingly, in FN18-CRM9- but not FN18-treated cells, there was a gradual decrease in cellular CD3 protein levels within 24 and 48 hr; such a decrease was not observed with the control protein Csk. CONCLUSIONS: Our findings suggest that the conjugation of the mutated diphtheria toxin CRM9 to FN18 modulates the monoclonal antibody-mediated cross-linking of the TCR/CD3 complex, leading to a stronger protein tyrosine phosphorylation and CD3 internalization. This may in turn contribute to the greater efficacy of the immunotoxin in prolonging allograft survival.

FN18-CRM9 immunotoxin promotes tolerance in primate renal allografts.

BACKGROUND: Transplant tolerance, rather than immunity, may be favored in the setting of a lower mature lymphoid mass in the recipient induced by anti-T cell agents. A novel immunosuppressive agent, FN18-CRM9, known to specifically kill T cells with great potency, was evaluated in a transplant model. METHODS: In order to ablate recipient T cells, the immunotoxin FN18-CRM9 was administered to rhesus monkey recipients of MHC-mismatched renal allografts. Donor lymphocytes were injected intrathymically into some animals. RESULTS: All monkeys with T-cell depletion by immunotoxin had prolonged allograft survival, and tolerance confirmed by skin grafting has been confirmed in five of six long-surviving recipients. CONCLUSIONS: In this clinically relevant model, profound but transient T-cell depletion by a single agent substantially promotes tolerance.

Microchimerism linked to cytotoxic T lymphocyte functional unresponsiveness (clonal anergy) in a tolerant renal transplant recipient.

A patient was found to be functionally tolerant of a maternal kidney allograft as evidenced by good graft function 5 years after cessation of all immunosuppressive drug therapy. Despite normal in vitro proliferative and IL-2 responses, patient anti-donor 1 degree MLR cultures yielded little donor-specific CTL activity in either bulk or limiting dilution analysis (LDA) cultures. Using polymerase chain reaction, the patient's PBL and skin were found to contain donor-derived Bw6+ cells. Removal of Bw6+ donor cells from the patient PBL with mAb and immunomagnetic beads before stimulation with donor PBL on day 0 failed to restore donor-specific CTL in either bulk 1 degree MLR or LDA cultures. Restimulation of 1 degree cultures with donor stimulator cells plus exogenous IL-2, however, completely restored anti-donor HLA class I-specific CTL, indicating class I-specific CTL precursors were not clonally deleted. Fresh patient PBL, as well as donor cell-enriched fractions, when added at the initiation of 3 degrees MLR cultures, inhibited the generation of anti-donor CTL, whereas donor cell-depleted fractions did not. The inhibition was cell dose-dependent, was specific for the anti-donor response, and was radioresistant (1200 rad). Thus, the clinical tolerance observed in patients with microchimerism may be due to the presence of veto cells within the circulating donor cell pool.

Analysis of primate renal allografts after T-cell depletion with anti-CD3-CRM9.

BACKGROUND: FN18-CRM9 is a CD3-specific immunotoxin that is capable of depleting CD3+ T cells. Pretreatment of rhesus monkeys with this agent before transplantation can induce donor-specific tolerance and "split tolerance" to renal allografts. METHODS: Heterotopic renal transplants were performed on monkeys that received posttransplant FN18-CRM9. Histological and immunohistological staining, as well as analysis of the intragraft cytokine profile by reverse transcriptase polymerase chain reaction, was performed on percutaneous allograft biopsies. RESULTS: Experimental monkeys had significant prolongation of allograft survival. Although an interstitial, mononuclear cell infiltrate was seen in all of the renal transplants, there was minimal evidence of acute cellular rejection. Histological evidence of alloantibody-mediated damage was detected 3 to 5 months after transplantation in the monkeys treated with FN18-CRM9. Immunohistology demonstrated the reappearance of CD3+ and CD4+ T cells, as well as CD20+ B cells, in the grafts. Cytokine analysis demonstrated expression of interferon-gamma. An intact anti-donor IgG response was seen. CONCLUSION: Treatment of monkeys with FN18-CRM9 immediately after transplantation significantly prolongs renal allograft survival. Allograft biopsies demonstrate a lack of acute cellular rejection; however, alloantibody-mediated graft damage and rejection occur, with an intact anti-donor IgG response. The intragraft expression of the interferon-gamma may reflect this ongoing humoral rejection. These data suggest that even a brief period of T-cell allosensitization may lead to humorally mediated allograft damage. Efforts to achieve tolerance with posttransplant FN18-CRM9 will require modification of the protocol to deplete T cells before allosensitization exposure or to supplement the posttransplant immunomodification strategy.

Increased glomerular deposits of von Willebrand factor in chronic, but not acute, rejection of primate renal allografts.

BACKGROUND: In our previously described primate renal allograft model, T cell ablation leads to long-term graft survival. The role of endothelial cell alteration in chronic rejection was examined in our model. METHODS: Renal transplants were performed in rhesus monkeys using a T cell- depleting immunotoxin, FN18-CRM9. Sections from 10 rejected kidneys (5 acute and 7 chronic rejection) were examined after immunohistochemical staining for expression of endothelium-related proteins [von Willebrand factor (vWF), CD62P, and CD31], fibrinogen, and a macrophage marker (CD68). Glomerular staining for each antigen was graded on a semiquantitative scale. RESULTS: Intense staining for vWF was consistently observed in glomerular endothelium, subendothelium, and mesangium in all kidneys removed due to chronic rejection. vWF staining was weak in kidneys showing acute rejection. The difference in glomerular staining was statistically significant. Staining for vWF in extraglomerular vessels was nearly identical in kidneys showing acute and chronic rejection. Expression of CD62P was increased in extraglomerular vessels in allografts with chronic rejection, but the glomeruli showed little or no staining. There was no significant difference in the glomerular staining for CD62P or CD31 in organs showing acute and chronic rejection. Fibrinogen staining of glomerular mesangium was seen in kidneys with chronic rejection. Macrophages (CD68+) infiltrating glomeruli were more numerous in kidneys showing chronic rejection. CONCLUSION: Increased glomerular deposition of vWF in renal allografts showing chronic rejection, without increased staining for CD62P or CD31, suggests increased constitutive secretion of vWF from endothelial cells as a component of the mechanism of chronic rejection in our model.

Split tolerance induced by immunotoxin in a rhesus kidney allograft model.

BACKGROUND: Renal allografts were performed in rhesus monkeys using FN18-CRM9, a potent immunotoxin capable of depleting T cells to less than 1% of baseline levels in blood and lymph nodes, as a preparative agent. We have recently reported that animals pretreated with FN18-CRM9 1 week before transplantation without further immunosuppression had prolonged graft survival time compared with control animals, and frequently became tolerant. METHODS: This report examines the alloimmune responses of recipient monkeys to the donor, including cytotoxic T lymphocyte precursor (CTLp) frequency, mixed lymphocyte response, and antidonor IgG response. RESULTS: CTLp frequencies declined significantly (P<0.01) after FN18-CRM9 treatment and renal transplantation. This decline in CTLp was initially nonspecific, as CTLp frequencies against third-party animals also declined (P<0.01). The decrease in CTLp was maintained in five of five animals tested 6 months after transplant. However, unresponsiveness was limited to the CTL arm of the immune response as antidonor IgG was detected in four of four animals tested, and the 5-day mixed lymphocyte response stimulation index and relative response were not significantly different before and after transplant. In long-term survivors (>150 days), an increase in anti-third-party CTLp was detected 1 month after grafting with third-party skin. No change was seen in the antidonor CTLp frequency after donor skin grafting, indicating that a specific defect in the antidonor CTL response had developed. CONCLUSIONS: These data suggest that FN18-CRM9 treatment of rhesus monkeys allows the development of specific down-regulation of antidonor CTL activity in renal allograft recipients.