Neurally Adjusted Ventilatory Assist (NAVA) improves the matching of diaphragmatic electrical activity and tidal volume in comparison to pressure support (PS) under non invasive ventilation

INTRODUCTION. Patient-ventilator asynchrony is a frequent issue in non invasivemechanical ventilation (NIV) and leaks at the patient-mask interface play a major role in itspathogenesis. NIV algorithms alleviate the deleterious impact of leaks and improve patient-ventilator interaction. Neurally adusted ventilatory assist (NAVA), a neurally triggered modethat avoids interferences between leaks and the usual pneumatic trigger, could further improvepatient-ventilator interaction in NIV patients.OBJECTIVES. To evaluate the feasibility ofNAVAin patients receiving a prophylactic postextubationNIV and to compare the respective impact ofPSVandNAVAwith and withoutNIValgorithm on patient-ventilator interaction.METHODS. Prospective study conducted in 16 beds adult critical care unit (ICU) in a tertiaryuniversity hospital. Over a 2 months period, were included 17 adult medical ICU patientsextubated for less than 2 h and in whom a prophylactic post-extubation NIV was indicated.Patients were randomly mechanically ventilated for 10 min with: PSV without NIV algorithm(PSV-NIV-), PSV with NIV algorithm (PSV-NIV+),NAVAwithout NIV algorithm (NAVANIV-)and NAVA with NIV algorithm (NAVA-NIV+). Breathing pattern descriptors, diaphragmelectrical activity, leaks volume, inspiratory trigger delay (Tdinsp), inspiratory time inexcess (Tiexcess) and the five main asynchronies were quantified. Asynchrony index (AI) andasynchrony index influenced by leaks (AIleaks) were computed.RESULTS. Peak inspiratory pressure and diaphragm electrical activity were similar in thefour conditions. With both PSV and NAVA, NIV algorithm significantly reduced the level ofleak (p\0.01). Tdinsp was not affected by NIV algorithm but was shorter in NAVA than inPSV (p\0.01). Tiexcess was shorter in NAVA and PSV-NIV+ than in PSV-NIV- (p\0.05).The prevalence of double triggering was significantly lower in PSV-NIV+ than in NAVANIV+.As compared to PSV,NAVAsignificantly reduced the prevalence of premature cyclingand late cycling while NIV algorithm did not influenced premature cycling. AI was not affectedby NIV algorithm but was significantly lower in NAVA than in PSV (p\0.05). AIleaks wasquasi null with NAVA and significantly lower than in PSV (p\0.05).CONCLUSIONS. NAVA is feasible in patients receiving a post-extubation prophylacticNIV. NAVA and NIV improve patient-ventilator synchrony in different manners. NAVANIV+offers the best patient-ventilator interaction. Clinical studies are required to assess thepotential clinical benefit of NAVA in patients receiving NIV.

Fas-associated death domain protein (FADD) and caspase-8 mediate up-regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP)-regulated pathway.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL- and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL- and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD- and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

Evaluation of melanoma vaccines with molecularly defined antigens by ex vivo monitoring of tumor-specific T cells.

Immunotherapy of melanoma is aimed to mobilize cytolytic CD8+ T cells playing a central role in protective immunity. Despite numerous clinical vaccine trials, only few patients exhibited strong antigen-specific T-cell activation, stressing the need to improve vaccine strategies. For a rational development, we propose to focus on molecularly defined vaccine components, and evaluate their immunogenicity with highly reproducible and standardized methods for ex vivo immune monitoring. Careful immunogenicity comparison of vaccine formulations in phase I/II studies allow to select optimized vaccines for subsequent clinical efficacy testing in large scale phase III trials.

Establishment of animal models to analyze the kinetics and distribution of human tumor antigen-specific CD8⁺ T cells.

Many patients develop tumor antigen-specific T cell responses detectable in peripheral blood mononuclear cells (PBMCs) following cancer vaccine. However, measurable tumor regression is observed in a limited number of patients receiving cancer vaccines. There is a need to re-evaluate systemically the immune responses induced by cancer vaccines. Here, we established animal models targeting two human cancer/testis antigens, NY-ESO-1 and MAGE-A4. Cytotoxic T lymphocyte (CTL) epitopes of these antigens were investigated by immunizing BALB/c mice with plasmids encoding the entire sequences of NY-ESO-1 or MAGE-A4. CD8(+) T cells specific for NY-ESO-1 or MAGE-A4 were able to be detected by ELISPOT assays using antigen presenting cells pulsed with overlapping peptides covering the whole protein, indicating the high immunogenicity of these antigens in mice. Truncation of these peptides revealed that NY-ESO-1-specific CD8(+) T cells recognized D(d)-restricted 8mer peptides, NY-ESO-181-88. MAGE-A4-specific CD8(+) T cells recognized D(d)-restricted 9mer peptides, MAGE-A4265-273. MHC/peptide tetramers allowed us to analyze the kinetics and distribution of the antigen-specific immune responses, and we found that stronger antigen-specific CD8(+) T cell responses were required for more effective anti-tumor activity. Taken together, these animal models are valuable for evaluation of immune responses and optimization of the efficacy of cancer vaccines.

Lack of tumor recognition by hTERT peptide 540-548-specific CD8(+) T cells from melanoma patients reveals inefficient antigen processing.

Telomerase is a ribonucleoprotein complex responsible for the maintenance of the length of the telomeres during cell division, which is active in germ-line cells as well as in the vast majority of tumors but not in most normal tissues. The wide expression of the human telomerase catalytic subunit (hTERT) in tumors makes it an interesting candidate vaccine for cancer. hTERT-derived peptide 540-548 (hTERT(540)) has been recently shown to be recognized in an HLA-A*0201-restricted fashion by T cell lines derived from peptide-stimulated peripheral blood mononuclear cells (PBMC) from healthy donors. As a first step to the inclusion of this peptide in immunotherapy clinical trials, it is crucial to assess hTERT(540)-specific T cell reactivity in cancer patients as well as the ability of hTERT-specific CD8(+) T lymphocytes to recognize and lyse hTERT-expressing target cells. Here, we have analyzed the CD8(+) T cell response to peptide hTERT(540) in HLA-A*0201 melanoma patients by using fluorescent HLA-A*0201/hTERT(540) peptide tetramers. HLA-A*0201/hTERT(540) tetramer(+) CD8(+) T cells were readily detected in peptide-stimulated PBMC from a significant proportion of patients and could be isolated by tetramer-guided cell sorting. hTERT(540)-specific CD8(+) T cells were able to specifically recognize HLA-A*0201 cells either pulsed with peptide or transiently transfected with a minigene encoding the minimal epitope. In contrast, they failed to recognize hTERT-expressing HLA-A*0201(+) target cells. Furthermore, in vitro proteasome digestion studies revealed inadequate hTERT processing. Altogether, these results raise questions on the use of hTERT(540) peptide for cancer immunotherapy.

Selective accumulation of differentiated FOXP3(+) CD4 (+) T cells in metastatic tumor lesions from melanoma patients compared to peripheral blood

Precise identification of regulatory T cells is crucial in the understanding of their role in human cancers. Here, we analyzed the frequency and phenotype of regulatory T cells (Tregs), in both healthy donors and melanoma patients, based on the expression of the transcription factor FOXP3, which, to date, is the most reliable marker for Tregs, at least in mice. We observed that FOXP3 expression is not confined to human CD25(+/high) CD4(+) T cells, and that these cells are not homogenously FOXP3(+). The circulating relative levels of FOXP3(+) CD4(+) T cells may fluctuate close to 2-fold over a short period of observation and are significantly higher in women than in men. Further, we showed that FOXP3(+) CD4(+) T cells are over-represented in peripheral blood of melanoma patients, as compared to healthy donors, and that they are even more enriched in tumor-infiltrated lymph nodes and at tumor sites, but not in normal lymph nodes. Interestingly, in melanoma patients, a significantly higher proportion of functional, antigen-experienced FOXP3(+) CD4(+) T was observed at tumor sites, compared to peripheral blood. Together, our data suggest that local accumulation and differentiation of Tregs is, at least in part, tumor-driven, and illustrate a reliable combination of markers for their monitoring in various clinical settings.

Melan-A/MART-1-specific CD8 T cells: from thymus to tumor.

The self-antigen Melan-A/MART-1 is frequently involved in T-cell responses against malignant melanoma. The use of fluorescent tetramers incorporating the immunodominant Melan-A/MART-1 peptide has provided new insights into HLA-A2-restricted T-cell responses against this antigen in cancer patients and in healthy individuals. Direct evidence has been provided that a large Melan-A/MART-1-specific CD8 T-cell pool is generated during thymic selection. Although several other examples of naive self-peptide-specific T-cell repertoires are known, this is the only one directly accessible to analysis in healthy individuals

Intravaginal and subcutaneous immunization induced vaccine specific CD8 T cells and tumor regression in the bladder.

PURPOSE: Vaccines targeting tumor associated antigens are in development for bladder cancer. Most of these cancers are nonmuscle invasive at diagnosis and confined in the mucosa and submucosa. However, to our knowledge how vaccination may induce the regression of tumors at such mucosal sites has not been examined previously. We compared different immunization routes for the ability to induce vaccine specific antitumor CD8 T cells in the bladder and bladder tumor regression in mice. MATERIALS AND METHODS: In the absence of a murine bladder tumor model expressing a tumor antigen relevant for human use we established an orthotopic model expressing the HPV-16 tumor antigen E7 as a model. We used an adjuvant E7 polypeptide to induce CD8 T cell mediated tumor regression. RESULTS: Subcutaneous and intravaginal but not intranasal vaccination induced a high number of TetE7(+)CD8(+) T cells in the bladder as well as bladder tumor regression. The entry of vaccine specific T cells in the bladder was not the only key since persistent regression of established bladder tumors by intravaginal or subcutaneous immunization was associated with tumor infiltration of total CD4 and CD8 T cells. This resulted in an increase in TetE7(+)CD8(+) T cells and a decrease in T regulatory cells, leading to an increased number of effector interferon-γ secreting vaccine specific CD8 T cells in the regressing bladder tumor. CONCLUSIONS: These data show that immunization routes should be tailored to each mucosal tumor site. Subcutaneous or intravaginal vaccination may be of additional value to treat patients with bladder cancer.

Cytokine targeting in tumors using a bispecific antibody directed against carcinoembryonic antigen and tumor necrosis factor alpha.

The use of tumor necrosis factor alpha (TNFalpha) in cancer therapy is limited by its short circulatory half-life and its severe systemic side effects. To overcome these limitations, we evaluated the capability of a bispecific antibody (BAb) directed against carcinoembryonic antigen (CEA) and human TNFalpha to target this cytokine in tumors. A BAb was constructed by coupling the Fab' fragments from an anti-CEA monoclonal antibody (MAb) to the Fab' fragments from an anti-TNFalpha MAb via a stable thioether linkage. The double specificity of the BAb for CEA and TNFalpha was demonstrated using a BIAcoreTM two-step analysis. The affinity constants of the BAb for CEA immobilized on a sensor chip and for soluble TNFalpha added to the CEA-BAb complex were as high as those of the parental MAbs (1.7 x 10(9) M-1 and 6.6 x 10(8) M-1, respectively). The radiolabeled 125I-labeled BAb retained high immunoreactivity with both CEA and TNFalpha immobilized on a solid phase. In nude mice xenografted with the human colorectal carcinoma T380, the 125I-labeled BAb showed a tumor localization and biodistribution comparable to that of 131I-labeled anti-CEA parental F(ab')2 with 25-30% of the injected dose (ID)/g tumor at 24 h and 20% ID/g tumor at 48 h. To target TNFalpha to the tumor, a two-step i.v. injection protocol was used first, in which a variable dose of 125I-labeled BAb was injected, followed 24 or 48 h later by a constant dose of 131I-labeled TNFalpha (1 microg). Mice pretreated with 3 microg of BAb and sacrificed 2, 4, 6, or 8 h after the injection of TNFalpha showed a 1.5- to 2-fold increased concentration of 131I-labeled TNFalpha in the tumor as compared to control mice, which received TNFalpha alone. With a higher dose of BAb (25 microg), mice showed a better targeting of TNFalpha with a 3.2-fold increased concentration of 131I-labeled TNFalpha in the tumor: 9.3% versus 2.9% ID/g in control mice 6 h after TNFa injection. In a one-step injection protocol using a premixed BAb-TNFalpha preparation, similar results were obtained 6 h postinjection (3.5-fold increased TNFalpha tumor concentration). A longer retention time of TNFalpha was observed leading to an 8.1-fold increased concentration of TNFalpha in the tumor 14 h postinjection (4.4 versus 0.5% ID/g tumor for BAb-treated and control mice, respectively). These results show that our BAb is able, first, to localize in a human colon carcinoma and, there, to immunoabsorb the i.v.-injected TNFalpha, leading to its increased concentration at the tumor site.

Improvement of saliva production in ENT cancer patients treated by IMRT

Intensity modulated radiotherapy (IMRT) is a conformal radiotherapy that produces concave and irregular target volume dose distributions. IMRT has a potential to reduce the volume of healthy tissue irradiated to a high dose, but this often at the price of an increased volume of normal tissue irradiated to a low dose. Clinical benefits of IMRT are expected to be most pronounced at the body sites where sensitive normal tissues surround or are located next to a target with a complex 3D shape. The irradiation doses needed for tumor control are often markedly higher than the tolerance of the radiation sensitive structures such as the spinal cord, the optic nerves, the eyes, or the salivary glands in the treatment of head and neck cancer. Parotid gland salivary flow is markedly reduced following a cumulative dose of 30 50 Gy given with conventional fractionation and xerostomia may be prevented in most patients using a conformal parotid-sparing radiotherapy technique. However, in cohort studies where IMRT was compared with conventional and conformal radiotherapy techniques in the treatment of laryngeal or oropharyngeal carcinoma, the dosimetric advantage of IMRT translated into a reduction of late salivary toxicity with no apparent adverse impact on the tumor control. IMRT might reduce the radiation dose to the major salivary glands and the risk of permanent xerostomia without compromizing the likelihood for cure. Alternatively, IMRT might allow the target dose escalation at a given level of normal tissue damage. We describe here the clinical results on postirradiation salivary gland function in head and neck cancer patients treated with IMRT, and the technical aspects of IMRT applied. The results suggest that the major salivary gland function can be maintained with IMRT without a need to compromise the clinical target volume dose, or the locoregional control.

Right coronary artery flow velocity and volume assessment with spiral K-space sampled breathhold velocity-encoded MRI at 3 tesla: accuracy and reproducibility.

PURPOSE: To evaluate accuracy and reproducibility of flow velocity and volume measurements in a phantom and in human coronary arteries using breathhold velocity-encoded (VE) MRI with spiral k-space sampling at 3 Tesla. MATERIALS AND METHODS: Flow velocity assessment was performed using VE MRI with spiral k-space sampling. Accuracy of VE MRI was tested in vitro at five constant flow rates. Reproducibility was investigated in 19 healthy subjects (mean age 25.4 +/- 1.2 years, 11 men) by repeated acquisition in the right coronary artery (RCA). RESULTS: MRI-measured flow rates correlated strongly with volumetric collection (Pearson correlation r = 0.99; P < 0.01). Due to limited sample resolution, VE MRI overestimated the flow rate by 47% on average when nonconstricted region-of-interest segmentation was used. Using constricted region-of-interest segmentation with lumen size equal to ground-truth luminal size, less than 13% error in flow rate was found. In vivo RCA flow velocity assessment was successful in 82% of the applied studies. High interscan, intra- and inter-observer agreement was found for almost all indices describing coronary flow velocity. Reproducibility for repeated acquisitions varied by less than 16% for peak velocity values and by less than 24% for flow volumes. CONCLUSION: 3T breathhold VE MRI with spiral k-space sampling enables accurate and reproducible assessment of RCA flow velocity.

Kinesin spindle protein SiRNA slows tumor progression.

The kinesin spindle protein (KSP), a member of the kinesin superfamily of microtubule-based motors, plays a critical role in mitosis as it mediates centrosome separation and bipolar spindle assembly and maintenance. Inhibition of KSP function leads to cell cycle arrest at mitosis with the formation of monoastral microtubule arrays, and ultimately, to cell death. Several KSP inhibitors are currently being studied in clinical trials and provide new opportunities for the development of novel anticancer therapeutics. RNA interference (RNAi) may represent a powerful strategy to interfere with key molecular pathways involved in cancer. In this study, we have established an efficient method for intratumoral delivery of siRNA. We evaluated short interfering RNA (siRNA) duplexes targeting luciferase as surrogate marker or KSP sequence. To examine the potential feasibility of RNAi therapy, the siRNA was transfected into pre-established lesions by means of intratumor electro-transfer of RNA therapeutics (IERT). This technology allowed cell permeation of the nucleic acids and to efficiently knock down gene expression, albeit transiently. The KSP-specific siRNA drastically reduced outgrowth of subcutaneous melanoma and ovarian cancer lesions. Our results show that intratumoral electro-transfer of siRNA is feasible and KSP-specific siRNA may provide a novel strategy for therapeutic intervention. J. Cell. Physiol. 228: 58-64, 2013. © 2012 Wiley Periodicals, Inc.

Tumor necrosis factor and transforming growth factor β regulate clock genes by controlling the expression of the cold inducible RNA-binding protein (CIRBP).

The circadian clock drives the rhythmic expression of a broad array of genes that orchestrate metabolism, sleep wake behavior, and the immune response. Clock genes are transcriptional regulators engaged in the generation of circadian rhythms. The cold inducible RNA-binding protein (CIRBP) guarantees high amplitude expression of clock. The cytokines TNF and TGFβ impair the expression of clock genes, namely the period genes and the proline- and acidic amino acid-rich basic leucine zipper (PAR-bZip) clock-controlled genes. Here, we show that TNF and TGFβ impair the expression of Cirbp in fibroblasts and neuronal cells. IL-1β, IL-6, IFNα, and IFNγ do not exert such effects. Depletion of Cirbp is found to increase the susceptibility of cells to the TNF-mediated inhibition of high amplitude expression of clock genes and modulates the TNF-induced cytokine response. Our findings reveal a new mechanism of cytokine-regulated expression of clock genes.

A model of cellular dosimetry for macroscopic tumors in radiopharmaceutical therapy.

PURPOSE: In the radiopharmaceutical therapy approach to the fight against cancer, in particular when it comes to translating laboratory results to the clinical setting, modeling has served as an invaluable tool for guidance and for understanding the processes operating at the cellular level and how these relate to macroscopic observables. Tumor control probability (TCP) is the dosimetric end point quantity of choice which relates to experimental and clinical data: it requires knowledge of individual cellular absorbed doses since it depends on the assessment of the treatment's ability to kill each and every cell. Macroscopic tumors, seen in both clinical and experimental studies, contain too many cells to be modeled individually in Monte Carlo simulation; yet, in particular for low ratios of decays to cells, a cell-based model that does not smooth away statistical considerations associated with low activity is a necessity. The authors present here an adaptation of the simple sphere-based model from which cellular level dosimetry for macroscopic tumors and their end point quantities, such as TCP, may be extrapolated more reliably. METHODS: Ten homogenous spheres representing tumors of different sizes were constructed in GEANT4. The radionuclide 131I was randomly allowed to decay for each model size and for seven different ratios of number of decays to number of cells, N(r): 1000, 500, 200, 100, 50, 20, and 10 decays per cell. The deposited energy was collected in radial bins and divided by the bin mass to obtain the average bin absorbed dose. To simulate a cellular model, the number of cells present in each bin was calculated and an absorbed dose attributed to each cell equal to the bin average absorbed dose with a randomly determined adjustment based on a Gaussian probability distribution with a width equal to the statistical uncertainty consistent with the ratio of decays to cells, i.e., equal to Nr-1/2. From dose volume histograms the surviving fraction of cells, equivalent uniform dose (EUD), and TCP for the different scenarios were calculated. Comparably sized spherical models containing individual spherical cells (15 microm diameter) in hexagonal lattices were constructed, and Monte Carlo simulations were executed for all the same previous scenarios. The dosimetric quantities were calculated and compared to the adjusted simple sphere model results. The model was then applied to the Bortezomib-induced enzyme-targeted radiotherapy (BETR) strategy of targeting Epstein-Barr virus (EBV)-expressing cancers. RESULTS: The TCP values were comparable to within 2% between the adjusted simple sphere and full cellular models. Additionally, models were generated for a nonuniform distribution of activity, and results were compared between the adjusted spherical and cellular models with similar comparability. The TCP values from the experimental macroscopic tumor results were consistent with the experimental observations for BETR-treated 1 g EBV-expressing lymphoma tumors in mice. CONCLUSIONS: The adjusted spherical model presented here provides more accurate TCP values than simple spheres, on par with full cellular Monte Carlo simulations while maintaining the simplicity of the simple sphere model. This model provides a basis for complementing and understanding laboratory and clinical results pertaining to radiopharmaceutical therapy.

Correlations between imatinib pharmacokinetics, pharmacodynamics, adherence, and clinical response in advanced metastatic gastrointestinal stromal tumor (GIST): An emerging role for drug blood level testing?

Imatinib is the standard of care for patients with advanced metastatic gastrointestinal stromal tumors (GIST), and is also approved for adjuvant treatment in patients at substantial risk of relapse. Studies have shown that maximizing benefit from imatinib depends on long-term administration at recommended doses. Pharmacokinetic (PK) and pharmacodynamic factors, adherence, and drug-drug interactions can affect exposure to imatinib and impact clinical outcomes. This article reviews the relevance of these factors to imatinib's clinical activity and response in the context of what has been demonstrated in chronic myelogenous leukemia (CML), and in light of new data correlating imatinib exposure to response in patients with GIST. Because of the wide inter-patient variability in drug exposure with imatinib in both CML and GIST, blood level testing (BLT) may play a role in investigating instances of suboptimal response, unusually severe toxicities, drug-drug interactions, and suspected non-adherence. Published clinical data in CML and in GIST were considered, including data from a PK substudy of the B2222 trial correlating imatinib blood levels with clinical responses in patients with GIST. Imatinib trough plasma levels <1100ng/mL were associated with lower rates of objective response and faster development of progressive disease in patients with GIST. These findings have been supported by other analyses correlating free imatinib (unbound) levels with response. These results suggest a future application for imatinib BLT in predicting and optimizing therapeutic response. Nevertheless, early estimates of threshold imatinib blood levels must be confirmed prospectively in future studies and elaborated for different patient subgroups.