A tumor-associated heparan sulfate-related glycosaminoglycan promotes the generation of functional regulatory T cells.

Functional Tregs play a key role in tumor development and progression, representing a major barrier to anticancer immunity. The mechanisms by which Tregs are generated in cancer and the influence of the tumor microenvironment on these processes remain incompletely understood. Herein, by using NMR, chemoenzymatic structural assays and a plethora of in vitro and in vivo functional analyses, we demonstrate that the tumoral carbohydrate A10 (Ca10), a cell-surface carbohydrate derived from Ehrlich's tumor (ET) cells, is a heparan sulfate-related proteoglycan that enhances glycolysis and promotes the development of tolerogenic features in human DCs. Ca10-stimulated human DCs generate highly suppressive Tregs by mechanisms partially dependent on metabolic reprogramming, PD-L1, IL-10, and IDO. Ca10 also reprograms the differentiation of human monocytes into DCs with tolerogenic features. In solid ET-bearing mice, we found positive correlations between Ca10 serum levels, tumor size and splenic Treg numbers. Administration of isolated Ca10 also increases the proportion of splenic Tregs in tumor-free mice. Remarkably, we provide evidence supporting the presence of a circulating human Ca10 counterpart (Ca10H) and show, for the first time, that serum levels of Ca10H are increased in patients suffering from different cancer types compared to healthy individuals. Of note, these levels are higher in prostate cancer patients with bone metastases than in prostate cancer patients without metastases. Collectively, we reveal novel molecular mechanisms by which heparan sulfate-related structures associated with tumor cells promote the generation of functional Tregs in cancer. The discovery of this novel structural-functional relationship may open new avenues of research with important clinical implications in cancer treatment.

Mapping the neural systems driving breathing at the transition to unconsciousness.

After falling asleep, the brain needs to detach from waking activity and reorganize into a functionally distinct state. A functional MRI (fMRI) study has recently revealed that the transition to unconsciousness induced by propofol involves a global decline of brain activity followed by a transient reduction in cortico-subcortical coupling. We have analyzed the relationships between transitional brain activity and breathing changes as one example of a vital function that needs the brain to readapt. Thirty healthy participants were originally examined. The analysis involved the correlation between breathing and fMRI signal upon loss of consciousness. We proposed that a decrease in ventilation would be coupled to the initial decline in fMRI signal in brain areas relevant for modulating breathing in the awake state, and that the subsequent recovery would be coupled to fMRI signal in structures relevant for controlling breathing during the unconscious state. Results showed that a slight reduction in breathing from wakefulness to unconsciousness was distinctively associated with decreased activity in brain systems underlying different aspects of consciousness including the prefrontal cortex, the default mode network and somatosensory areas. Breathing recovery was distinctively coupled to activity in deep brain structures controlling basic behaviors such as the hypothalamus and amygdala. Activity in the brainstem, cerebellum and hippocampus was associated with breathing variations in both states. Therefore, our brain maps illustrate potential drives to breathe, unique to wakefulness, in the form of brain systems underlying cognitive awareness, self-awareness and sensory awareness, and to unconsciousness involving structures controlling instinctive and homeostatic behaviors.

Evaluation of the density spectral array in the Wada test: report of six cases.

Wada test is an invasive procedure used in the preoperative evaluation for epilepsy surgery to determine language lateralization, postoperative risk of amnesia syndrome, and to assess the risk of memory deficits. It involves injection of amobarbital into internal carotid artery of the affected hemisphere followed by the healthy hemisphere to shut down brain function. We performed an observational study evaluating the density spectral array (DSA) of the bilateral bispectral index VISTA™ Monitoring System (BVMS) in 6 patients with drug-resistant epilepsy undergoing Wada test. DSA revealed the presence of bifrontal alpha waves in absence of loss of consciousness in all patients.

Effect of dexmedetomidine on evoked-potential monitoring in patients undergoing brain stem and supratentorial cranial surgery.

BACKGROUND: Dexmedetomidine is used as adjuvant in total intravenous anaesthesia (TIVA), but there have been few studies concerning its effect on intraoperative neurophysiological monitoring (IONM) during cranial surgery. Our aim was to study the effect of dexmedetomidine on IONM in patients undergoing brain stem and supratentorial cranial surgery. METHODS: Two prospective, randomized, double-blind substudies were conducted. In substudy 1, during TIVA with an infusion of propofol and remifentanil, 10 patients received saline solution (SS) (PR group) and another 10 (PRD group) received dexmedetomidine (0.5 mcg/kg/h). Total dosage of propofol and remifentanil, intensity, latency and amplitude of motor-evoked potentials following transcranial electrical stimulation (tcMEPs) as well as somatosensory-evoked potentials (SSEP) were recorded at baseline, 15, 30, 45 minutes, and at the end of surgery. In order to identify differences in the same patient after dexmedetomidine administration, we designed substudy 2 with 20 new patients randomized to two groups. After 30 minutes with TIVA, 10 patients received dexmedetomidine (0.5 mcg/kg/h) and 10 patients SS. The same variables were recorded. RESULTS: In substudy 1, propofol requirements were significantly lower (P = .004) and tcMEP intensity at the end of surgery was significantly higher in PRD group, but no statistically significant differences were observed for remifentanil requirements, SSEP and tcMEP latency or amplitude. In substudy 2, no differences in any of the variables were identified. CONCLUSIONS: The administration of dexmedetomidine at a dosage of 0.5 mg/kg/h may reduce propofol requirements and adversely affect some neuromonitoring variables. However, it can be an alternative on IONM during cranial surgeries. REDEX EudraCT: 2014-000962-23.

Largest scale dissociation of brain activity at propofol-induced loss of consciousness.

The brain is a functional unit made up of multilevel connected elements showing a pattern of synchronized activity that varies in different states. The wake-sleep cycle is a major variation of brain functional condition that is ultimately regulated by subcortical arousal- and sleep-promoting cell groups. We analyzed the evolution of functional MRI (fMRI) signal in the whole cortex and in a deep region including most sleep- and wake-regulating subcortical nuclei at loss of consciousness induced by the hypnotic agent propofol. Optimal data were obtained in 21 of the 30 healthy participants examined. A dynamic analysis of fMRI time courses on a time-scale of seconds was conducted to characterize consciousness transition, and functional connectivity maps were generated to detail the anatomy of structures showing different dynamics. Inside the magnet, loss of consciousness was marked by the participants ceasing to move their hands. We observed activity synchronization after loss of consciousness within both the cerebral cortex and subcortical structures. However, the evolution of fMRI signal was dissociated, showing a transient reduction of global cortico-subcortical coupling that was restored during the unconscious state. An exception to cortico-subcortical decoupling was a brain network related to self-awareness (i.e. the default mode network) that remained connected to subcortical brain structures. Propofol-induced unconsciousness is thus characterized by an initial, transitory dissociated synchronization at the largest scale of brain activity. Such cortico-subcortical decoupling and subsequent recoupling may allow the brain to detach from waking activity and reorganize into a functionally distinct state.

Chronic pain after groin hernia repair: pain characteristics and impact on quality of life.

BACKGROUND: Chronic postsurgical pain (CPSP) after hernia repair research has mainly relied on unconfirmed self-reporting. We aimed to describe confirmed CPSP incidence, management, and quality of life (QoL) in a 2-year prospective study. METHODS: Multicenter study (GENDOLCAT) of 3890 patients undergoing 4 common surgical procedures in 23 hospitals to develop a risk model for CPSP; 2352 men underwent open hernia repair. Patients with pain were identified by telephone at 1 and 3 months and referred to the hospital 4 months after surgery for a physical examination to confirm CPSP. Three validated tools were used: the Brief Pain Inventory (BPI) for severity, analgesic use, and interference with activities; the SF-12 questionnaire for QoL (validated Spanish version), and the Douleur Neuropathique 4 (DN4). Patients with CPSP were called again at 1 and 2 years. RESULTS: In 1761 patients who underwent hernia repair and were eligible for physical examination for CPSP, the incidence of confirmed pain at 4 months was 13.6% (patient-reported pain, 6.2% at 1 year and 4.0% at 2 years). Neuropathic pain was diagnosed in 38.5% of the CPSP patients at 4 months. The incidences of neuropathic CPSP in patients with mesh or non-mesh repairs were similar (38.6 and 33.3%, respectively). SF-12 physical component scores changed little in all patients, whether or not they developed CPSP. The SF-12 mental component decreased significantly in all patients, but the decrease was clinically significant only in CPSP patients. CPSP interfered with activities (18%), work (15.6%), walking (15%) and mood (10.2%). At 2 years 52.1% of CPSP patients had moderate/intense pain and 28.2% took analgesics. CONCLUSION: CPSP affects QoL-related activities, and although it diminishes over the course of 2 years after surgery, many patients continue to have moderate/intense pain and take analgesics. CPSP and neuropathic pain rates seem to be similar after mesh and non-mesh repair. BPI and SF-12 mental component scores detect effects on QoL. TRIAL REGISTRATION: ClinicalTrials.gov NCT01510496.

Is 1 mg/kg of sugammadex sufficient to reverse moderate neuromuscular block? A randomized clinical trial / ¿Es suficiente 1mg/kg de sugammadex para revertir el bloqueo neuromuscular moderado? Ensayo clínico aleatorizado

Abstract Background: Sugammadex has made it possible to reverse any type of rocuronium-induced block quickly and safely. The most frequent neuromuscular blockade is the moderate one where doses smaller than those recommended by the industry could get a full reversal. Objective: We aimed to evaluate the effectiveness of half the industry recommended dose of sugammadex to reverse a moderate neuromuscular block. Methods: Unicenter phase IV clinical trial that included 34 patients undergoing laparoscopic cholecystectomy. Intravenous general anesthesia was induced, with acceleromyographic monitoring of the neuromuscular block. After the intervention, the block was reversed with all or half the dose of sugammadex recommended for moderate blocks, using a blinded syringe. Results: Patient characteristics of the 2 groups were similar. Mean time to recovery was 3.6± 1.7minutes for the study group and 3.1 ± 1.7minutes for the control group (P=0.42). Reversal of the block was complete with a single dose of sugammadex in all patients. There was an important linear correlation between depth of block and time to recovery. Conclusion: Intraoperative monitoring is essential to allow us to individualize the dose of the neuromuscular blocking agent. To reverse a moderate block under neuromuscular monitoring, a dose of 1 mg/kg is sufficient in most cases and is equally safe and effective.

Resumen Introducción: El sugammadex permite revertir cualquier tipo de bloqueo inducido por rocuronio de forma rápida y segura. El bloqueo neuromuscular más frecuente es el moderado, en el cual dosis inferiores a las recomendadas por la industria podrían revertirlo completamente. Objetivo: Evaluar la efectividad de la mitad de la dosis de sugammadex recomendada por la industria para revertir un bloqueo neuromuscular moderado. Métodos: Ensayo clínico de fase IV en un unico centro que incluyó a 34 pacientes intervenidos de colecistectomía laparoscópica. Se realizó anestesia general intravenosa con monitoreo aceleromiográfico del bloqueo neuromuscular. Tras la intervención, el bloqueo se revirtió con la totalidad o con la mitad de la dosis de sugammadex recomendada para bloqueos moderados, utilizando una jeringa ciega. Resultados: Las características de los pacientes de los dos grupos fueron similares. La media de tiempo de recuperación fue de 3,6 ± 1,7 minutos para el grupo de estudio y de 3,1 ± 1,7 minutos para el grupo de control (p = 0,42). La reversión del bloqueo se completó con una dosis única de sugammadex en todos los pacientes. Hubo una correlación lineal importante entre la profundidad del bloqueo y el tiempo de recuperación. Conclusión: El monitoreo transquirúrgico es esencial para individualizar la dosis del agente de bloqueo neuromuscular. Para revertir un bloqueo moderado bajo monitoreo neuromuscular, una dosis de 1mg/kg es suficiente e igualmente segura y efectiva.

Density spectral array during the intra and postoperative of a left frontal craniotomy / Matriz de Densidad Espectral durante el intra y el posoperatorio de una craneotomía frontal izquierda

Abstract We present the case of an adult with an extensive left frontal meningioma. He was scheduled for resection by craniotomy. During the surgery we used the density spectral array (DSA) and asymmetry obtained from Bispectral Index VISTA Monitoring System Bilateral. We observed a power increase in low frequency (0.1-1 Hz) and alpha bands (8-12 Hz) in the left hemisphere, where the meningioma was located. In this case, DSA was useful during and after the surgery because it provided information about the hemisphere with maximum brain activity and its subsequent normalization, which may reflect the effectiveness of the surgery.

Resumen Presentamos el caso de un paciente adulto con diagnóstico de un meningioma extenso a nivel frontal izquierdo, que fue programado para exéresis mediante craneotomía. Durante la cirugía se utilizó la Matriz de Densidad Espectral (MDE) y la asimetría obtenida del Sistema de Monitorización VISTATM del Índice Biespectral Bilateral (BVMS). Se observó un aumento de potencia en las bandas de baja frecuencia (0.1-1Hz) y en las bandas alfa (812 Hz) del hemisferio cerebral izquierdo, donde se encontraba el meningioma. En este caso la MDE demostró su utilidad durante y después de la cirugía, al proporcionar información sobre el hemisferio con registro de máxima actividad cerebral y su posterior normalización, reflejando así la efectividad de la cirugía.

Sedación con propofol y remifentanilo para la ultrasonografía endobronquial con punción-aspiración en tiempo real / Sedation with propofol and remifentanil for real-time endobronchial ultrasound needle aspiration

Introducción: Para la óptima realización de la ultrasonografía endobronquial (USEB) lineal es imprescindible que el paciente esté sedado para que se mantenga tranquilo, no tosa ni se mueva y el endoscopista trabaje cómodamente con un buen rendimiento de la exploración. Actualmente la técnica anestésica no está estandarizada y varía desde una anestesia general a una sedación. El objetivo del presente trabajo es conocer la dosificación, la seguridad y la satisfacción de la sedación endovenosa con propofol y remifentanilo e identificar los posibles factores predictivos de tos durante el procedimiento. Pacientes y métodos: Se estudió prospectivamente a los pacientes a quienes se realizó la USEB bajo sedación en un hospital de tercer nivel. Resultados: Se realizó la USEB a 90pacientes bajo sedación con remifentanilo y propofol, a una velocidad de infusión de 0,13 (0,09-0,17) ixgkg-¹ min^¹ y 2,34 (1,5-3,6) mgkg-¹ h_1, respectivamente. El 81% de los pacientes tosieron en algún momento de la exploración. En el 8% de los pacientes se interrumpió el procedimiento puntualmente por tos y desaturación. No se registraron complicaciones graves directamente relacionadas con la sedación. El nivel de satisfacción del neumólogo y del paciente con el procedimiento fue excelente o bueno en la mayoría de casos. No se observó relación estadísticamente significativa entre el número de episodios de tos durante la ecobroncoscopia y las variables test de la tos, ser tosedor habitual, hábito tabáquico o grado de severidad de la EPOC. Conclusiones: La sedación con remifentanilo y propofol realizada por un anestesiólogo permite realizar la USEB lineal en ventilación espontánea aunque con una elevada incidencia de tos y desaturación, siendo esta última la complicación más frecuente. No se encontraron factores predictivos de la tos durante el procedimiento.

Introduction: Optimal linear endobronchial ultrasound (EBUS) outcomes require sedation to ensure that the patient remains calm, immobile, and does not cough, and so that the bronchoscopist can work comfortably. The choice of anesthesia techniques, on a spectrum ranging from general anesthesia to sedation, is not standardized. The aims of this study were to determine doses, safety and satisfaction for intravenous sedation with propofol and remifentanil, and identify potential predictors of coughing during the procedure, and determine patient and bronchoscopist satisfaction with the procedure. Patients and methods: The prospective study included patients undergoing EBUS under sedation in a tertiary hospital. Results: A total of 90 patients underwent EBUS under sedation with remifentanil and propofol, at infusion rates of 0,13 (0,09-0,17) g kg-¹ min^¹ and 2.34 (1.5-3.6) mg kg-¹ Lr¹, respectively. Just over four fifths of the patients (81%) coughed at some point during the ultrasound procedure. In 8% of patients the procedure was promptly discontinued due to coughing and desaturation. There were no major complications directly related to sedation. Bronchoscopists and patients rated their satisfaction with the procedure as excellent or good in most cases. There was no statistically significant relationship between the number of coughing episodes during the procedure and any of the following variables: positive cough test, a habitual cough, tobacco dependence, or severity of chronic obstructive pulmonary disease. Conclusions: Remifentanil and propofol administered by an anesthesiologist enabled spontaneously breathing patients to undergo linear EBUS, although with a high incidence of coughing and particularly desaturation. No predictors for coughing during EBUS were identified.

Pillar 2: minimising bleeding and blood loss.

One of the main factors that contributes to the need for transfusion in the surgical patient is excessive blood loss. Pillar 2 of patient blood management (PBM) includes all the strategies to reduce bleeding and preserve the patient's own blood, designed with an aim to reducing or avoiding transfusion. Some of these strategies, such as identifying and planning the management of patients at high risk of bleeding, can be implemented as early as at the preoperative assessment visit. During the intra-operative period, local haemostasis is the most important factor in the control of bleeding; in this context, therefore, surgical technique and meticulous haemostasis are fundamental measures. However, there are also additional anaesthetic techniques that can help reduce blood loss and transfusion requirements, such as the use of pharmacological or haemostatic agents. After surgery, PBM continues in the postoperative recovery unit or on the ward with the monitoring and management of postoperative bleeding. The blood lost via drains can be filtered, washed and reinfused, if needed.

Renal cell carcinoma tumors induce T cell apoptosis through receptor-dependent and receptor-independent pathways.

Tumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumor gangliosides also participate, a notion consistent with reports indicating that, in some cell types, gangliosides can activate the intrinsic apoptotic pathway by stimulating reactive oxygen species production, cytochrome c release, and caspase-9 activation. In this study, we used normal peripheral blood T cells, as well as caspase-8-, caspase-9-, and Fas-associated death domain protein-deficient Jurkat cells, to assess whether the death ligands and gangliosides overexpressed by the renal cell carcinoma (RCC) cell line SK-RC-45 can independently stimulate T cell apoptosis as a mechanism of immune escape. Anti-FasL Abs and the glycosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) each partially inhibited the ability of SK-RC-45 to kill cocultured activated T cells; together, as purified molecules, RCC gangliosides and rFasL induced a more extensive mitochondrial permeability transition and greater levels of apoptosis than either agent alone, equivalent to that induced by the FasL- and ganglioside-expressing RCC line itself. rFasL-mediated apoptosis was completely inhibited in caspase-8- and Fas-associated death domain protein-negative Jurkat cells, though apoptosis induced by purified gangliosides remained intact, findings that correlate with the observed partial inhibition of SK-RC-45-induced apoptosis in the Jurkat lines with defective death receptor signaling. Western blot analysis performed on lysates made from wild-type and mutant Jurkat cells cocultured with SK-RC-45 revealed caspase activation patterns and other biochemical correlates which additionally supported the concept that tumor-associated gangliosides and FasL independently activate the caspase cascade in T cells through the intrinsic and extrinsic pathways, respectively.

GM1 and tumor necrosis factor-alpha, overexpressed in renal cell carcinoma, synergize to induce T-cell apoptosis.

The ability to induce T-cell apoptosis is one mechanism by which tumors evade the immune system, although the molecules involved remain controversial. We found that renal cell carcinoma (RCC)-induced T-cell apoptosis was inhibited by >50% when cocultures were performed with ganglioside-depleted tumor cells, caspase-8-negative lymphocytes, or anti-tumor necrosis factor-alpha (TNFalpha) antibodies, suggesting that tumor gangliosides synergize with signals delivered through TNFalpha death receptors to mediate T-cell killing. The synergy between tumor-derived TNFalpha and the RCC-overexpressed ganglioside GM1 for killing resting T cells is corroborated by studies using purified GM1 and rTNF alpha, which indicate that a 48-hour pretreatment with the ganglioside optimally sensitizes the lymphocytes to a TNFalpha-induced apoptotic death. However, activated T cells, which synthesize TNFalpha themselves, can be killed by exogenous GM1 alone. RelA-overexpressing lymphocytes are protected from GM1 plus TNFalpha-mediated apoptosis, a finding consistent with our previous studies indicating that gangliosides inhibit nuclear factor-kappaB activation. These results are clinically relevant because, similar to T-cells cocultured with GM1-overexpressing RCC lines, T cells isolated from the peripheral blood of patients with metastatic RCC are also heavily coated with that tumor-shed ganglioside. This population of patient cells, unlike T cells isolated from normal donors, is highly susceptible to apoptosis induced by rTNF alpha or by metastatic patient sera, which contain elevated levels of the cytokine. This report thus extends our previous studies by demonstrating that tumor-derived TNFalpha enhances RCC apoptogenicity not only by inducing ganglioside synthesis but also by initiating receptor-dependent apoptosis in T cells in which the nuclear factor-kappaB activation pathway has been inhibited by GM1.

Clinical and immunologic effects of subcutaneously administered interleukin-12 and interferon alfa-2b: phase I trial of patients with metastatic renal cell carcinoma or malignant melanoma.

PURPOSE: Interleukin-12 (IL-12) and interferon alfa-2b (IFN-alpha-2b) are pleiotropic cytokines with activity in renal cell carcinoma (RCC) and malignant melanoma (MM) as single agents. Preclinical studies suggest concurrent administration may have synergistic antitumor effects. We conducted a phase I trial of concurrent subcutaneous (SC) administration of IL-12 and IFN-alpha-2b in patients with metastatic RCC or MM to determine toxicity, maximum-tolerated dose, preliminary efficacy, and effects on chemokine/cytokine gene expression in peripheral blood mononuclear cells (PBMCs). PATIENTS AND METHODS: Cohorts of three to six patients were treated with escalating doses of IL-12 (dose I, 100 ng/kg; dose II, 300 ng/kg; dose III, 500 ng/kg; dose IV, 500 ng/kg SC) given twice weekly and IFN-alpha-2b (dose I, 1.0 MU/m(2); dose II, 1.0 MU/m(2); dose III, 1.0 MU/m(2); dose IV, 3.0 MU/m(2) SC) three times weekly in 4-week cycles. Effects on gene expression were assessed by reverse transcriptase polymerase chain reaction. RESULTS: Twenty-six patients (19 with RCC, seven with MM) were accrued at dose levels I (n = 3), II (n = 3), III (n = 13), and IV (n = 7). Dose-limiting toxicity included grades 3 and 4 hepatotoxicity and neutropenia/leukopenia. Patients received a median of three cycles of treatment. Two patients with RCC and one patient with MM had partial responses. Median survival was 13.8 months. Reverse transcriptase polymerase chain reaction on PBMCs revealed induction of IP-10, Mig, B7.1 (CD80), interleukin-5, and interferon gamma in selected patients. CONCLUSION: Concurrent SC administration of IL-12 and IFN-alpha-2b is possible at the dose levels utilized. Recommended doses for phase II trials are 500 ng/kg IL-12 and 1.0 MU/m(2) IFN-alpha-2b. Consistent induction of IP-10 and Mig, as well as variable induction of B7.1, interleukin-5, and interferon gamma expression was noted in PBMCs.

Degradation of NF-kappa B in T cells by gangliosides expressed on renal cell carcinomas.

T cells from cancer patients are often functionally impaired, which imposes a barrier to effective immunotherapy. Most pronounced are the alterations characterizing tumor-infiltrating T cells, which in renal cell carcinomas includes defective NF-kappaB activation and a heightened sensitivity to apoptosis. Coculture experiments revealed that renal tumor cell lines induced a time-dependent decrease in RelA(p65) and p50 protein levels within both Jurkat T cells and peripheral blood T lymphocytes that coincided with the onset of apoptosis. The degradation of RelA/p50 is critical for SK-RC-45-induced apoptosis because overexpression of RelA in Jurkat cells protects against cell death. The loss of RelA/p50 coincided with a decrease in expression of the NF-kappaB regulated antiapoptotic protein Bcl-xL at both the protein and mRNA level. The disappearance of RelA/p50 protein was mediated by a caspase-dependent pathway because pretreatment of T lymphocytes with a pan caspase inhibitor before coculture with SK-RC-45 blocked RelA and p50 degradation. SK-RC-45 gangliosides appear to mediate this degradative pathway, as blocking ganglioside synthesis in SK-RC-45 cells with the glucosylceramide synthase inhibitor, PPPP, protected T cells from tumor cell-induced RelA degradation and apoptosis. The ability of the Bcl-2 transgene to protect Jurkat cells from RelA degradation, caspase activation, and apoptosis implicates the mitochondria in these SK-RC-45 ganglioside-mediated effects.

The Bcl-2 transgene protects T cells from renal cell carcinoma-mediated apoptosis.

PURPOSE: Tumors induce T-cell apoptosis as a mechanism of inhibiting antitumor immunity. Using coculture experiments, it has been shown that tumor lines stimulate T-cell apoptosis by a pathway involving a mitochondrial permeability transition and cytochrome c release. Activated T cells express abundant levels of Bcl-2, an antiapoptotic molecule that would be expected to confer resistance to such tumor-mediated killing. We examined the mechanism by which Bcl-2 is dysregulated in T cells exposed to the renal tumor line SK-RC-45, and we determined whether overexpressing Bcl-2 protects T cells from tumor-mediated apoptosis. EXPERIMENTAL DESIGN: Activated T lymphocytes and Jurkat cells transfected or not transfected with Bcl-2 were exposed to SK-RC-45 for 48-72 h. After coculture, lymphocytes were analyzed for Bcl-2 expression using Western analysis and for tumor-induced apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling. The role of SK-RC-45-stimulated caspase activation in degrading T-cell Bcl-2 was assessed using a pan-caspase inhibitor, as well as a specific inhibitor of caspase-9. RESULTS: The renal cell carcinoma cell line SK-RC-45 sensitizes peripheral blood activated T lymphocytes and Jurkat cells to apoptosis by a mechanism that involves degradation of the antiapoptotic protein Bcl-2. The SK-RC-45-induced modulation of lymphocyte Bcl-2 levels was largely caspase independent because pretreatment of T cells with pan-caspase inhibitor III or an inhibitor of caspase-9 had minimal or no effect on stabilizing the protein, although it did provide protection against apoptosis. Overexpression of Bcl-2 protected Jurkat cells from tumor-mediated killing. CONCLUSIONS: Bcl-2 inhibition is a mechanism by which tumors may render lymphocytes sensitive to other tumor-derived, proapoptotic stimuli.

Phase I trial of consensus interferon in patients with metastatic renal cell carcinoma: toxicity and immunological effects.

PURPOSE: The purpose of our study was to determine the maximum tolerated dose (MTD), dose-limiting toxicities, and effects on chemokine/cytokine gene expression in peripheral blood mononuclear cells (PBMCs) of consensus IFN (CIFN). EXPERIMENTAL DESIGN: Cohorts of three to six patients with metastatic renal cell carcinoma (RCC) were treated with escalating doses of CIFN (dose level I, 9.0 microg/m(2); dose level II, 15.0 microg/m(2); dose level III, 21.0 microg/m(2)) given s.c. three times weekly in 4-week cycles until progression. The cohort treated at the maximum tolerated dose was expanded to further define toxicity. An additional three patients were treated with i.v. CIFN (15.0 microg/m(2)) to evaluate route-related differences in gene expression. Cytokine and chemokine gene expression in PBMCs was assessed by reverse transcription-PCR. RESULTS: A total of 25 patients (18 men and 7 women) were enrolled between January 28, 1999, and November 1, 2000, at dose levels I (n = 4), II (n = 14), and III (n = 7). Dose-limiting toxicity occurred at dose level III (21 microg/m(2)) and included grade-3 or -4 respiratory distress/failure (n = 3) and hypocalcemia (n = 1) occurring within the first cycle of treatment. Other severe toxicities included grade-3 neutropenia, thrombocytopenia, fatigue, and nausea/vomiting. Studies of cytokine and chemokine gene expression in PBMCs from eight patients revealed induction of IFN-gamma, IP-10, and Mig. I.V. administration was associated with a faster induction, but of shorter duration. There were no responses; however, 24 patients had stable disease of variable duration (4-32 weeks) and received a median of three cycles of treatment (range, 1-8 cycles). Overall median survival was 13.5 months, and was 12.7 months in the previously treated patients. CONCLUSION: CIFN was safely administered s.c. three times weekly at doses up to 15.0 microg/m(2). Although there were no responses, the median survival was longer than expected in a previously treated patient population with metastatic RCC.