Neuroblastoma cells inhibit the immunostimulatory function of dendritic cells

Purpose

Dendritic cells (DC) are critical for induction of antitumor immunity. Recent studies suggest that tumors may avoid immune destruction by inhibiting DC function. The authors investigated the effect of neuroblastoma (NB) on surface antigen expression and T cell activation by DCs.

Methods

DCs were generated in the presence of granulocyte and macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) from peripheral blood of healthy donors. On day 6 of culture, DCs were exposed to human NB cells and were analyzed by flow cytometry.

Results

The proinflammatory cytokine tumor necrosis factor alpha (TNF-α) failed to upregulate the expression of HLA-DR and costimulatory molecule CD86 by DCs that were cultured with NB. Conversely, upregulation was preserved when DCs were cultured in the absence of NB. Exposure to NB also led to apoptosis of DCs as shown by 2-fold increase in surface phosphatidylserine. It appears that direct contact was required to inhibit DC maturation, because DCs separated from NB cells using a transwell insert did not suppress surface antigen expression. Finally, DCs exposed to NB inhibited the proliferation of allogeneic T cells in mixed lymphocyte reactions.

Conclusions

These findings have significant implications for tumor-pulsed DC vaccines in the treatment of NB and suggest a mechanism by which NB escape rejection.     

Murine neuroblastoma attenuates dendritic cell cysteine cysteine receptor 7 (CCR7) expression

Background/Purpose

Dendritic cell (DC) migration from tumors to T-cell priming sites is critical in developing antitumor cytotoxicity. Cysteine cysteine receptor 7 (CCR7), a promigratory chemokine receptor, regulates DC recruitment to secondary lymphoid organs. Tumors may inhibit CCR7 expression to evade immunodetection. Previous work implicates impaired DC migration as a critical defect in immunity to neuroblastoma (NB). However, the mechanism has yet to be defined. We hypothesize that NB abrogates DC CCR7 expression and signaling, leading to decreased antitumor immunity.

Methods

A/J mice (N = 36) were injected with saline (control) or murine NB (TBJ) and bone marrow-derived DC were isolated at 7, 14, and 28 days. CCR7 expression was analyzed by polymerase chain reaction, Western blot, and flow cytometry. Cytometry data were analyzed using the paired Student's t test.

Results

Dendritic cells isolated from mice with NB had a 60% increase in CCR7 protein expression by flow cytometry compared with control mice at day 7. However, there was a 43% downregulation of CCR7 expression by DC from tumor-bearing mice compared with controls 2 weeks postinoculation (P < .005). These observations were confirmed by polymerase chain reaction and Western blot analysis.

Conclusion

Neuroblastoma initially upregulates CCR7 expression by DC. However, with tumor progression, this chemokine is downregulated, likely leading to impaired DC migration. Immunotherapeutic strategies to bypass or augment CCR7-dependent DC trafficking may improve survival for patients with aggressive disease.     

Induction of cytolytic T lymphocytes against pediatric solid tumors in vitro using autologous dendritic cells pulsed with necrotic primary tumor

Purpose

Effective and generally applicable methods for generating cancer vaccines in children have not been defined. Dendritic cells (DCs) are the most potent professional antigen-presenting cells capable of activating primary cytolytic T cells. We tested the ability of DCs generated from pediatric patients' peripheral blood monocytes and pulsed with a necrotic tumor to activate autologous tumor-specific cytolytic T cells.

Methods

Tumor and peripheral blood cells were obtained from pediatric patients undergoing biopsy or resection for advanced solid tumors according to an institutional research board-approved protocol and after acquiring informed consent from them. To generate DCs, we treated peripheral blood monocytes with granulocyte-macrophage colony stimulating factor and interleukin (IL)-4. Maturation was induced with a cytokine cocktail (CC) containing tumor necrosis factor-α, IL-6, IL-1β, and prostaglandin E₂. The DC phenotype was assayed using flow cytometry. Tumor necrosis was induced by exposure to UV-B irradiation (1000 mJ). Dendritic cells pulsed with a UV-B-treated primary tumor and matured with CC were used to stimulate autologous peripheral blood lymphocytes weekly. Tumor-specific cytolytic activity was assayed using 4-hour ⁵¹Cr release.

Results

Peripheral blood monocytes isolated from pediatric patients differentiated into immature DCs (CD14⁻, MHCII⁺ [major histocompatibility complex], CD80^low, CD86^low) in the presence of granulocyte-macrophage colony stimulating factor and IL-4. Cytokine cocktail induced maturation of DCs, as characterized by increased expressions of MHCII, CD83, CD80, and CD86. Patients' peripheral blood lymphocytes stimulated in vitro with DCs loaded with a necrotic primary tumor and matured with CC specifically lysed autologous neuroblastoma in 7 of 9 patients.

Conclusion

Dendritic cells generated from the peripheral blood of children with advanced solid tumors and pulsed with a necrotic primary tumor undergo maturation and effectively stimulate autologous tumor-specific cytolytic T cells in vitro. We describe a simple method for generating a vaccine capable of activating cytotoxic T cells against pediatric solid tumors that does not require the genetic identification of tumor-associated antigens.     

Advanced neuroblastoma impairs dendritic cell function in adoptive immunotherapy

Background/purpose

The authors previously described the complete regression of established neuroblastoma (NB) by the adoptive transfer of syngeneic interleukin-12 transduced dendritic cells (DC) from naive mice. However, some malignancies, like NB, abrogate DC immunostimulation. The authors hypothesize that IL-12 transduction of DC from NB-bearing mice will have the same antitumor properties.

Methods

A/J mice (n = 32) with established NB received peritumoral injection of 1 × 10⁶ DC (DC, IL-12 DC, day 7 IL-12 DC or day 14 IL-12 DC) on day 7. Tumor growth, phenotype, and ability to induce NK and T cell activity were measured.

Results

Vaccination with naive admIL-12 DC resulted in 100% tumor regression and prolonged survival. Transduced DC induced only partial responses in 75% (day 7) and 25% (day 14) of animals. No differences in phenotype or effector cell activation were noted between admIL-12DC in tumor-bearing or naive mice.

Conclusions

IL-12 DC from tumor-bearing animals have a decreased ability to induce antitumor activity against established murine NB. This decreased capacity appears to be related to the duration of exposure to tumor because day 14 transduced DC had less of an effect than day 7 DC, despite similar phenotypes and ability to activate immune effector cells.     

Dyslipidemia in human kidney transplant recipients receiving cyclosporine and tacrolimus is associated with different expression of CD36 on peripheral blood monocytes

Purpose

We studied the mechanisms by which immunosuppressants result, in dyslipidemia among human kidney transplant recipients.

Methods

Seventy-five living donor kidney transplant recipients were enrolled in our study with informed consent and the approval of out Institutional Ethics Committee. Each donor-recipient pair were relatives, there were no prisoners. The serum lipid profile, the expression of CD36 on peripheral blood monocytes, and the whole blood concentrations of cyclosporine (CsA) or tacrolimus (FK506) were determined at various times after transplantation.

Results

CsA significantly increased serum lipid concentrations. The CsA concentration correlated positively with low-density lipoprotein cholesterol (LDL) levels, whereas FK506 showed no significant effect on serum lipid level. There was a positive correlation between the CsA concentrations and the expression of CD36; FK507 showed no significant effect on CD36 expression.

Conclusions

Hyperlipidemia in kidney transplant recipients treated with CsA was associated with overexpression of CD36 on peripheral blood monocytes.     

EGF receptor signaling affects bcl-2 family gene expression and apoptosis after massive small bowel resection

Background

After massive small bowel resection (SBR), enterocyte apoptosis is elevated and inversely correlates with epidermal growth factor receptor (EGFR) signaling. The purpose of the current study was to determine whether EGFR manipulation affects the expression of specific bcl-2 family members.

Methods

A 50% proximal SBR or sham operation was performed in 3 groups of mice control, after exogenous EGF, or mutant mice with defective EGFR signaling (waved-2). Apoptotic index (no. of apoptotic bodies per crypt), and bax (pro-apoptosis) and bcl-w (anti-apoptosis) protein expression was measured in the remnant ileum after 12, 24, and 72 hours.

Results

Waved-2 mice with defective EGFR showed the greatest increase in apoptosis and altered the ratio of bax to bcl-w in favor of apoptosis after SBR. Conversely, EGF prevented the expected increase in apoptosis after SBR by shifting the ratio of bax to bcl-w in favor of cell survival.

Conclusions

After massive small bowel resection, inhibition of the EGFR accelerates the rate of apoptosis and modifies the expression of specific bcl-2 family members to favor apoptosis. These results further support a specific mechanistic pathway for the regulation of enterocyte apoptosis after SBR via EGFR signaling.     

Delivery of alloantigens via apoptotic cells generates dendritic cells with an immature tolerogenic phenotype

Background

Dendritic cells (DCs) are professional antigen-presenting cells able to induce immunity or tolerance. The interactions of immature DCs with naive T lymphocytes induce peripheral tolerance through mechanisms that include anergy or deletion of lymphocytes or the generation of regulatory T cells. Because of the central role of DCs in the immune response, they are potential targets for the induction of experimental tolerance. Thus, the generation of immature (tolerogenic) DCs able to capture and present alloantigens to T cells represents an important aim in our efforts to achieve better transplant acceptance.

Methods

In this work, we generated immature DCs by using vitamin D₃ (VD3) during the process of DC differentiation.

Results

The VD3-DCs showed an immature phenotype characterized by a low expression of major histocompatibility complex antigens of class II, CD86, and CD80 molecules and the secretion of a tolerogenic cytokine pattern. Furthermore, we showed that VD3-DCs phagocytose apoptotic allogeneic cells efficiently without inducing DC maturation or activation. Most important, our experiments demonstrated that mice treated with VD3 produce immature DCs in vivo, and that DCs from VD3-treated mice immunized with allogeneic apoptotic cells maintained their tolerogenic phenotype.

Conclusion

Our results show that allogeneic apoptotic cells in combination with VD3 generate DCs with tolerogenic characteristics that could be used to induce tolerance towards alloantigens.     

Single nucleotide polymorphism array analysis to predict clinical outcome in neuroblastoma patients

Purpose

Neuroblastoma (NB) is a heterogeneous tumor and demonstrates favorable or unfavorable outcomes. In Japan, a nationwide NB mass screening (MS) had been performed on 6-month-old infants for approximately 20 years, which might have detected almost all NB including regressing/maturing tumors. To clarify the heterogeneity of this tumor, we examined genetic alterations in the representative cases using genomewide microarrays.

Methods

Genomic DNA was extracted from 198 NB tissue samples and paired blood samples including 76 MS-detected cases and analyzed by single nucleotide polymorphism arrays.

Results

The single nucleotide polymorphism array classified the genetic aberrations into 4 types: whole gain/loss type, partial gain/loss type, MYCN-amplified type, and silent type. Most MS-detecting cases belonged to the whole gain/loss type, whereas unfavorable cases who died of disease showed partial gain/loss, MYCN-amplified, or silent types.

Conclusions

Genomewide genetic analysis is useful to predict the outcome of patients. Although the cases whose tumors showed whole gain/loss may respond well to contemporary therapy, sparing intensive surgery, current therapeutic strategy may be insufficient for the subgroups with partial gain/loss, MYCN-amplified, or silent type. Validation of these results would provide new tools to predict clinical outcome of children with NB.     

Oxygen inhalation improves survival time of mice with acute intra-abdominal hypertension and protects liver cells

Objective

The objectives of this study were to establish a mouse model of sustainable, stable acute intra-abdominal hypertension (IAH), seeking to observe the survival time and liver functions at different intra-abdominal pressures (IAP), and to investigate changes after oxygen therapy.

Methods

Sixty Kunming mice were assigned to 4 groups with an average of 15, 20, 30, or 40 cmH₂O IAP. The 40 cmH₂O group seemed to be appropriate for follow-up experiments. The 45 mice added to the cohort were assigned into 3 groups administered an average of 50%, 80%, and 100% O₂, respectively. Liver and blood samples were used to compare the rates of apoptosis using the TUNEL assay as well as alanine aminotransferase (ALT), aspartate aminotransferase (AST); Caspase-3, 9, MDA, and SOD concentrations.

Results

Most animals in the 15 and 20 cmH₂O groups survived 8 hours. The average survival for the 30 and 40 cmH₂O groups were 4.54 ± 0.54 hours and 2.04 ± 0.44 hours, respectively (P < .01). As the oxygen concentration increased, the survival time was prolonged among the 40 cmH₂O IAP group (P < .01), and the member of apoptotic hepatic cells decreased (P < .01), with a concomittent decrease in caspase 3 and 9 as well as malondialdehyde, although superoxide dismutase showed the opposite results.

Conclusion

The present work using a mouse model for acute IAH showed oxygen inhalation to improve host survival and protect liver cells.     

Neuroblastoma detection using serum proteomic profiling: a novel mining technique for cancer?

Background

Serum proteins in neuroblastoma (NB), such as neuron-specific enolase and lactate dehydrogenase, are used as nonspecific markers of disease severity. In this study, we have generated serum protein profiles that correlate with NB by applying proteomic technologies to uncover, at the protein level, serum polypeptide expression patterns in patients with NB.

Methods

Surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry was used to generate protein expression spectra in human NB (group I, n = 18) and healthy children (group II, n = 17) sera. Groups I and II mass spectral data were compared after baseline subtraction. Peaks with high signal-to-noise ratios were selected and grouped into bins with various intervals along mass-to-charge axis. Two-sample t test and 3-fold cross validation were used to identify differential biomarkers between groups I and II.

Results

Significant differentially expressed proteins were identified between groups I and II (P ≤ .05). The discriminatory features (proteomic patterns) of cancer from normal sera were successfully identified using the classification algorithm. The average classification performance after 3-fold cross validation was 87.26%.

Conclusion

Surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry is suitable for preliminary assessment of NB and could potentially provide a noninvasive diagnosis of NB. We propose that surface-enhanced laser desorption/ionization provides a novel method for NB diagnosis because direct observations of spectral differences between normal and NB sera are possible.     

Pancreatic alpha-cell differentiation by mesenchyme-to-epithelial transition: implications for cell-based therapies in children

Purpose

Stem cell-derived tissue may provide a curative treatment for children with type 1 diabetes. Using an avian model, we have previously shown that foregut mesenchyme is able to differentiate into insulin-positive β-cell islets (B islets). Successful clinical islet transplantation, however, is reliant on graft tissue containing both insulin- and glucagon-secreting cells. Therefore, in this study, we assessed the ability of foregut mesenchyme to differentiate into glucagon-positive α-cell islets (A islets).

Methods

Chimeric recombinants (n = 14) were constructed using chick pancreatic epithelium combined with quail stomach mesenchyme from day 4 avian embryos and then cultured in 3 dimensions for 7 days. Cryosectioned recombinants were analyzed using immunocytochemistry against glucagon, insulin, and the quail-specific nucleolar antigen. The A islets and B islets were determined to be of solely epithelial, solely mesenchymal, or mixed origin according to the coexpression of the quail-specific nucleolar antigen.

Results

Forty-eight A islets and 34 B islets were analyzed. Eighty-five percent of the A islets were solely derived from the epithelium, but, notably, 5% were solely derived from the mesenchyme and 10% were of mixed origin. A-islet differentiation from foregut mesenchyme was reduced as compared with B islets (P = .03).

Conclusion

We demonstrate that foregut mesenchyme is able to differentiate into both α and β cells, albeit with quantitative differences. These findings may have important implications for the derivation of islet tissue from mesenchymal stem cells to cure juvenile-onset diabetes.     

Graft serosectomy in adult small bowel transplantation without vascular reconstruction in rats improves graft survival by induction of vascular endothelial growth factor

Purpose

The aim of this study was to assess whether adult small bowel grafts (ASBGs) can survive transplantation without vascular reconstruction if graft serosectomy (SS) is performed.

Methods

Syngeneic ASBG transplants were performed in 85 Lewis rats. The entire serosa was removed just before transplantation in the SS group (n = 50) and left intact in the nonserosectomy group (n = 35). Transplanted ASBG was harvested 1, 3, 5, 7, 14, 21, or 28 days after transplantation and studied using staining with hematoxylin-eosin, immunohistochemistry for protein gene product 9.5, S-100, CD34 and vascular endothelial growth factor (VEGF), and quantification of VEGF messenger RNA (mRNA). Adult small bowel graft viability was assessed blindly using a mucosal surface expansion score (0, no mucosa; 1, mucosa on one fourth of graft; 2, mucosa on one half of graft; 3, mucosa on three fourths of graft; and 4, circumferential mucosa on graft).

Results

No rejection was identified in any ASBG. Average mucosal surface expansion score and VEGF mRNA expression were significantly higher in the SS group (both P < .01). Vascular endothelial growth factor protein was detected in enterocytes from day 3 posttransplant in the SS group. Distribution of protein gene product 9.5 and S-100 was normal in SS-group ASBG.

Conclusions

Our results suggest that SS allows VEGF mRNA and, subsequently, VEGF protein in ASBG to be induced very soon after transplantation, which may contribute to the survival of ASBG transplanted without vascular reconstruction.     

Effects of raised-intensity phonation on inflammatory mediator gene expression in normal rabbit vocal fold

Objective

To investigate the hypothesis that a transient episode of raised-intensity phonation causes a significant increase in vocal fold inflammatory messenger RNA (mRNA) expression in vivo.

Study Design

Prospective animal study.

Setting

Laboratory.

Subjects and Methods

Ten New Zealand White breeder rabbits received 30 minutes of experimentally induced modal or raised-intensity phonation, followed by a 30-minute recovery period. A separate group of five rabbits served as sham controls. Real-time polymerase chain reaction was performed to investigate the mRNA expression of interleukin 1β (IL-1β), transforming growth factor β-1 (TGFβ1), and cyclooxygenase-2 (COX-2). Separate one-way analysis of variance (ANOVA) tests were used to investigate differences in gene expression across groups, with an appropriate alpha correction of 0.016 to control for type I error. Significant main effects were further examined using Fisher's least significant difference.

Results

ANOVA revealed that there were differences for IL-1β, TGFβ1, and COX-2 between sham control, modal phonation, and raised-intensity phonation (P < 0.0001). Pairwise comparisons revealed that the expression of IL-1β, COX-2, and TGFβ1 increased significantly during raised-intensity phonation, compared to modal phonation and sham control (P < 0.0001).

Conclusion

Results provided support for the hypothesis that a transient episode of raised-intensity phonation causes a significant increase in vocal fold inflammatory mRNA expression. Future studies will investigate the signal transduction pathways and mechanisms regulating the vocal fold inflammatory response. The long-term goal of these studies is to advance understanding of the molecular and cellular events underlying phonation-related tissue alterations.     

Keyhole limpet hemocyanin potentiates standard immunotherapy for melanoma

Introduction

Our hypothesis was that keyhole limpet hemocyanin (KLH) would augment the effects of standard immunotherapies for melanoma including interferon-alpha (AIFN) and interleukin (IL)-2.

Methods

The HTB68 melanoma cell line was treated with KLH, AIFN, and IL-2 as single and combined agents. Cell viability, apoptotic activity, and vascular endothelial growth factor levels were all evaluated.

Results

Cell growth was reduced with KLH (28%), AIFN (54%), and IL-2 (29%) (all P < .001). KLH and IL-2 combined exhibited a 47% inhibition of cell growth, whereas KLH and AIFN combined yielded a 67% reduction in cell growth (both P < .001). KLH and AIFN combined significantly increased both early (10%) and late (14%) apoptotic activity compared with controls (5% and 7%, P < .001).

Conclusions

The additive effects exhibited by the combination of KLH with AIFN or IL-2 are encouraging and support combination therapy as an effective treatment for this aggressive disease.     

Topoisomerase II-�� as a predictive factor of response to therapy with anthracyclines in locally advanced breast cancer

Background

Topoisomerase II-?? is a molecular target of anthracyclines; several studies have suggested that topoisomerase II-?? expression is related to response to anthracycline treatment. The objective of this study was to evaluate if topoisomerase II-?? overexpression predicts response to anthracycline treatment in locally advanced breast cancer patients.

Material and methods

Topoisomerase II-??, HER2, estrogen receptor (ER) and progesterone receptor (PR) expression were evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded breast tumors from 111 patients presenting with locally advanced breast cancer between 1995 and 2002. The prognostic value of these markers was analyzed using a multivariate proportional hazards regression model and an interaction analysis between topoisomerase II-?? status and dose intensity.

Results

Tumors from 40 patients (36%) showed topoisomerase II-?? overexpression, 62 patients (56%) for ER, 39 (35%) for PR and 26 (23%) for HER2. There were no significant correlations between topoisomerase II-?? expression and response to therapy, progression-free survival (PFS) or overall survival (OS). Anthracycline dose intensity had a significant impact on PFS and OS in patients overexpressing topoisomerase II-?? (P = 0.010 and 0.027, respectively). Negative PR (P = 0.041), positive HER2 (P = 0.013) were identified as risk factors in the multivariate model. The multivariate analysis in patients topoisomerase II-?? negative shown no significance (HR = 0.92, IC 95% 0.39-2.15, P = 0.839) while the multivariate analysis in topoisomerase II-?? positive, dose intensity shown to be statistically significant (HR = 2.725, IC 95% 1.07-6.95, P = 0.036).

Conclusions

Our data do not support a correlation between topoisomerase II-?? expression in breast cancer patients and improved clinical benefit with anthracycline therapy. However, they do suggest that tumors overexpressing topoisomerase II-?? may experience better clinical benefit with higher anthracycline dose intensity.     

Intestine as Source of Cytokines and Growth Factors

Background

The activation status of intestinal immune system cells is much higher than that of analogous peripheral cells. Increased serum concentrations of proinflammatory cytokines have been reported in various pathologic conditions; however, the source of these mediators has not been elucidated.

Objective

To assess the role of the human intestine and its lymphatic system in production of growth factors and proinflammatory cytokines.

Material and Methods

Twenty liver transplant recipients and 20 donors were included in the study. Blood samples were obtained from the artery supplying the intestine, the portal vein, and a peripheral vein during liver harvesting in donors and after transplantation in recipients. An enzyme-linked immunosorbent assay was used to assess serum concentrations of IL-6, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), and hepatocyte growth factor (HGF).

Results

In transplant recipients, IL-6 concentration in arterial blood was lower than that in portal blood (P < .049), whereas in donors, there was no significant difference in these concentrations. Neither recipients nor donors demonstrated significant differences in arterial or portal blood concentrations of TNF-α, TGF-β, or HGF.

Conclusions

In healthy human beings, the intestine is not a substantial source of IL-6, TNF-α, TGF-β, or HGF. However, in patients with liver cirrhosis, the intestine is an important source of IL-6 but not of the other studied growth factors and cytokines.     

Remifentanil for the insertion and removal of long-term central venous access during monitored anesthesia care

Study Objective

To determine the analgesic efficacy of three different rates of remifentanil infusion in patients undergoing insertion or removal of long-term central venous access devices during monitored anesthesia care and local anesthetic field infiltration.

Design

Double-blinded, randomized, controlled study.

Setting

Operating theatre of an University hospital.

Patients

44 unpremedicated, ASA physical status 1 and 2 patients, aged 18-65 years, undergoing insertion or removal of a Port-a-Cath or Hickman catheter.

Interventions

Patients sedated with a propofol target-controlled infusion were randomly allocated to three groups: Group R25 (n = 14), Group R50 (n = 15), and Group R75 (n = 15), to receive remifentanil 0.025, 0.05, and 0.075 μg/kg/min, respectively. Rescue remifentanil 0.5 μg/kg was administered for pain scores > 3. The remifentanil infusion rate was maintained constant unless respiratory and/or cardiovascular unwanted events occurred, whereupon the rate was adjusted in 0.01 μg/kg/min decrements as necessary.

Measurements

Pain scores (primary outcome), sedation, and movement scores (secondary outcomes) were assessed during local anesthetic infiltration of the anterior chest wall and 5 other procedural steps.

Main Results

All infusion rates had equal analgesic efficacy, as shown by comparable pain scores, number of rescue boluses, and number of patients requiring rescue analgesia. Excessive sedation was associated with the highest remifentanil rate such that Group R75 patients were significantly more sedated than Groups R25 or R50 at selective procedural steps (P < 0.05). More Group R75 patients (6/15) required remifentanil rate reduction than did patients from Group R50 (1/15) or Group R25 (0/14), P < 0.01, most commonly because of respiratory depression.

Conclusions

For the insertion or removal of long-term central venous access devices, all three remifentanil infusion rates proved to be equally analgesic-efficient. However, the excessive sedation and tendency to respiratory and cardiovascular events associated with the highest remifentanil infusion rate renders such a rate less desirable for this purpose.