Type I interferon gene transfer sensitizes melanoma cells to apoptosis via a target activity on mitochondrial function

Our previous article reported that retroviral transduction of human type I consensus interferon-coding sequence into two human melanoma cells increased their susceptibility to cisplatin-induced apoptosis. Importantly, primary melanoma cells were significantly more sensitive to cisplatin-induced apoptosis with respect to metastatic melanoma cells. The aim of this study was to elucidate the subcellular mechanisms involved in this interferon-induced apoptotic proneness. Our results indicate that 1) cisplatin-induced apoptosis can be referred to as the type II apoptosis, ie, to the mitochondrially driven cascade; 2) treatment of interferon-producing melanoma cells with other type II apoptotic stimuli, such as radiation or staurosporine, also resulted in massive apoptosis, whereas type I stimuli, ie, anti-Fas, were ineffective; 3) interferon sensitization involved the caspase cascade in primary melanoma cells and the alternative pathway represented by cathepsin-mediated apoptosis in metastatic melanoma cells; 4) interferon production sensitizes cells to apoptosis by inducing, as the earliest event, mitochondrial membrane hyperpolarization. These results suggest that constitutive production of type I interferon by melanoma cells can act as an intracellular booster capable of increasing cell proneness to apoptosis by specifically modifying mitochondrial homeostasis and independently from the apoptotic cascade involved.     

Mutational landscape of patients with acute promyelocytic leukemia at diagnosis and relapse

Despite the high probability of cure of patients with acute promyelocytic leukemia (APL), mechanisms of relapse are still largely unclear. Mutational profiling at diagnosis and/or relapse may help to identify APL patients needing frequent molecular monitoring and early treatment intervention. Using an NGS approach including a 31 myeloid gene-panel, we tested BM samples of 44 APLs at the time of diagnosis, and of 31 at relapse. Mutations in PML and RARA genes were studied using a customized-NGS-RNA panel. Patients relapsing after ATRA-chemotherapy rarely had additional mutations (P = .009). In patients relapsing after ATRA/ATO, the PML gene was a preferential mutation target. We then evaluated the predictive value of mutations at APL diagnosis. A median of two mutations was detectable in 9/11 patients who later relapsed, vs one mutation in 21/33 patients who remained in CCR (P = .0032). This corresponded to a significantly lower risk of relapse in patients with one or less mutations (HR 0.046; 95% CI 0.011-0.197; P < .0001). NGS-analysis at the time of APL diagnosis may inform treatment decisions, including alternative treatments for cases with an unfavorable mutation profile.     

Action potential propagation in transverse-axial tubular system is impaired in heart failure

The plasma membrane of cardiac myocytes presents complex invaginations known as the transverse-axial tubular system (TATS). Despite TATS's crucial role in excitation-contraction coupling and morphological alterations found in pathological settings, TATS's electrical activity has never been directly investigated in remodeled tubular networks. Here we develop an ultrafast random access multiphoton microscope that, in combination with a customly synthesized voltage-sensitive dye, is used to simultaneously measure action potentials (APs) at multiple sites within the sarcolemma with submillisecond temporal and submicrometer spatial resolution in real time. We find that the tight electrical coupling between different sarcolemmal domains is guaranteed only within an intact tubular system. In fact, acute detachment by osmotic shock of most tubules from the surface sarcolemma prevents AP propagation not only in the disconnected tubules, but also in some of the tubules that remain connected with the surface. This indicates that a structural disorganization of the tubular system worsens the electrical coupling between the TATS and the surface. The pathological implications of this finding are investigated in failing hearts. We find that AP propagation into the pathologically remodeled TATS frequently fails and may be followed by local spontaneous electrical activity. Our findings provide insight on the relationship between abnormal TATS and asynchronous calcium release, a major determinant of cardiac contractile dysfunction and arrhythmias.     

International meeting on cancer vaccines: how can we enhance efficacy of therapeutic vaccines?

The major aims of the International Meeting on Cancer Vaccines were to review the state-of-the-art research on cancer vaccines, to compare different experimental approaches of therapeutic vaccination and to discuss critical issues and perspectives. The results from recent clinical trials in patients treated with different types of cancer vaccines were presented. Reasons for the limited response and possible modalities for enhancing efficacy of therapeutic vaccines were subjects of major discussion. A consensus was achieved on the need of combining cancer vaccines with other anticancer treatments. Of note, evidence stemming from studies in animal models pointed out new rationales for a selective combination of cancer vaccines with chemotherapy. In addition, some main presentations focused on new adjuvants (CpG oligonucleotides) and on the role of cytokines (i.e., type I IFN, interleukin 12, and interleukin 15) in promoting an antitumor immune response to vaccines. A considerable attention was given to regulatory T cells and to strategies for suppressing their function, thus enhancing vaccine efficacy. An entire session was devoted to the use of dendritic cells for the development of cancer vaccines. The results of clinical studies and the advantages of using new modalities for preparing dendritic cell-based vaccines were discussed.     

IFN-alpha2a induces IP-10/CXCL10 and MIG/CXCL9 production in monocyte-derived dendritic cells and enhances their capacity to attract and stimulate CD8+ effector T cells

Type I IFNs are immunomodulatory factors that possibly influence the properties of tissue-resident dendritic cells. Here, we have investigated the capacity of IFN-alpha2a to enhance DC chemoattractive and stimulatory capacity toward CD8+ T lymphocytes. Phenotypically, IFN-alpha2a-treated DC (IFN-DC) showed an increased expression of costimulatory and antigen-presenting molecules, maintained even after withdrawal of the cytokine. IFN-alpha2a enhanced DC stimulatory capacity toward CD8+ T cells, as assessed by increased MLR responses and induction of MART-1(26-35)-specific CTLs in vitro. No functional CCR7 chemokine receptor could be induced. Instead, high amounts of IP-10/CXCL10 and MIG/CXCL9 chemokines were produced. Freshly isolated CD8+RO+ cells and PHA-activated CD8+ T cells migrated efficiently in response to IFN-DC-conditioned medium, and the migration could be inhibited by neutralizing the CXCR3 receptor on responder cells. These results suggest that type I IFNs could enhance the elicitation of class I-restricted effector functions in vivo in the periphery by modulating DC chemoattractive properties.     

Gene therapy of cancer with interferon: lessons from tumor models and perspectives for clinical applications

Cytokine gene transfer is a current approach in studies of gene therapy of cancer IFNs represent valuable cytokines for these studies, since they exert multiple biological effects, including anti-tumor activities. Early studies have been focused on IFN-gamma. Recently, several reports have shown that the transfer of type I IFN (especially IFN-alpha) genes represents a powerful approach for inducing tumor suppression. Recent studies have underscored new IFN-induced activities on immune cells. This knowledge adds a further rationale for the use of IFN-alpha in strategies of gene therapy of cancer and can be exploited for the design of more selective and effective anticancer treatments.     

Immunization of stage IV melanoma patients with Melan-A/MART-1 and gp100 peptides plus IFN-alpha results in the activation of specific CD8(+) T cells and monocyte/dendritic cell precursors

The use of IFN-alpha in clinical oncology has generally been based on the rationale of exploiting its antiproliferative and antiangiogenic activities. However, IFN-alpha also exhibits enhancing effects on T-cell and dendritic cell functions, which may suggest a novel use as a vaccine adjuvant. We have carried out a pilot phase I-II trial to determine the effects of IFN-alpha, administered as an adjuvant of Melan-A/MART-1:26-35(27L) and gp100:209-217(210M) peptides, on immune responses in stage IV melanoma patients. In five of the seven evaluable patients, a consistent enhancement of CD8(+) T cells recognizing modified and native MART-1 and gp100 peptides and MART-1(+)gp100(+) melanoma cells was observed. Moreover, vaccination induced an increase in CD8(+) T-cell binding to HLA tetramers containing the relevant peptides and an increased frequency of CD45RA(+)CCR7(-) (terminally differentiated effectors) and CD45RA(-)CCR7(-) (effector memory) cells. In all patients, treatment augmented significantly the percentage of CD14(+) monocytes and particularly of the CD14(+)CD16(+) cell fraction. An increased expression of CD40 and CD86 costimulatory molecules in monocytes was also observed. Notably, postvaccination monocytes from two of the three patients showing stable disease or long disease-free survival showed an enhanced antigen-presenting cell function and capability to secrete IP10/CXCL10 when tested in mixed leukocyte reaction assays, associated to a boost of antigen and melanoma-specific CD8(+) T cells. Although further clinical studies are needed to show the adjuvant activity of IFN-alpha, the present data represent an important starting point for considering a new clinical use of IFN-alpha and new immunologic end points, potentially predictive of clinical response.     

Vaccination with inactivated murine gammaherpesvirus 68 strongly limits viral replication and latency and protects type I IFN receptor knockout mice from a lethal infection

Human gammaherpesviruses such as Epstein-Barr virus (EBV) cause lifelong infections and associated diseases, including malignancies, and the development of an effective vaccine against this class of viral infections is of considerable interest. The murine herpesvirus 68 (MHV-68) model provides a useful experimental setting to investigate the immune response to gammaherpesvirus infections and to evaluate the efficacy of vaccination strategies. In this study, we tested a heat-inactivated MHV-68 vaccine in immunocompetent mice as well as in B cell-deficient or type I IFN receptor knockout mice. Vaccination with heat-inactivated MHV-68 protected immunocompetent mice from the acute MHV-68 infection in the lung and strongly reduced the expansion of latently infected cells in the spleen and the development of splenomegaly. A similar inhibition of the acute viral replication in the lung was also observed in vaccinated B cell-deficient mice. Of note, the inactivated MHV-68 vaccine completely protected type I IFN receptor knockout mice from the infection with a lethal dose of MHV-68.     

The relation between sarcomere energetics and the rate of isometric tension relaxation in healthy and diseased cardiac muscle

Journal of Muscle Research and Cell Motility - Full muscle relaxation happens when [Ca2+] falls below the threshold for force activation. Several experimental models, from whole muscle organs and...