From Oxidative Stress Damage to Pathways, Networks, and Autophagy via MicroRNAs.

Oxidative stress can alter the expression level of many microRNAs (miRNAs), but how these changes are integrated and related to oxidative stress responses is poorly understood. In this article, we addressed this question by using in silico tools. We reviewed the literature for miRNAs whose expression is altered upon oxidative stress damage and used them in combination with various databases and software to predict common gene targets of oxidative stress-modulated miRNAs and affected pathways. Furthermore, we identified miRNAs that simultaneously target the predicted oxidative stress-modulated miRNA gene targets. This generated a list of novel candidate miRNAs potentially involved in oxidative stress responses. By literature search and grouping of pathways and cellular responses, we could classify these candidate miRNAs and their targets into a larger scheme related to oxidative stress responses. To further exemplify the potential of our approach in free radical research, we used our explorative tools in combination with ingenuity pathway analysis to successfully identify new candidate miRNAs involved in the ubiquitination process, a master regulator of cellular responses to oxidative stress and proteostasis. Lastly, we demonstrate that our approach may also be useful to identify novel candidate connections between oxidative stress-related miRNAs and autophagy. In summary, our results indicate novel and important aspects with regard to the integrated biological roles of oxidative stress-modulated miRNAs and demonstrate how this type of in silico approach can be useful as a starting point to generate hypotheses and guide further research on the interrelation between miRNA-based gene regulation, oxidative stress signaling pathways, and autophagy

FUS MUTATIONS IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS: CLINICAL AND GENETIC ANALYSIS

Fused in sarcoma (FUS) or translocation in liposarcoma (TLS), a DNA/RNA-binding protein, causes a dominant autosomal inherited form of amyotrophic lateral sclerosis (ALS), ALS 6. Its main role in neurodegeneration is highlighted by the presence of cytoplasmic accumulation of its mutant protein form in ALS patients. To further define the frequency and spectrum of FUS gene mutations, we have performed a molecular screening of a cohort of 327 Italian patients from Southern Italy with sporadic ALS (SALS). We identified 4 patients carrying 3 different missense mutations and several polymorphisms. Two different substitutions occurring in the same amino acidic position have been observed in 2 patients: R521G and R521C respectively; P525L mutation has been found in 2 additional cases. Most of the patients with FUS mutations showed early symptom onset and had short disease survival. We also detected 4 different polymorphic variants (3=-untranslated region [UTR] variant, c.*41G.A; c.52313ins[GAGGTG]; c.335-15del[TTTT]; and rs13331793) in 9 patients from within our cohort. This study underlines the importance of population-based mutation screening of newly identified genes. \ua9 2011 Elsevier Inc. All rights reserved

Gene profiling for the prediction of tumor response to treatment: the case of immunotherapy

Although anticancer immune responses can occur, the biological mechanisms responsible for them remain largely unexplained. Immunologists have extensively studied specific interactions between immune and cancer cells and have identified cofactors that may modulate the effectiveness of such interactions. In particular, as a result of the increasing molecular understanding of the basis for tumor/host interactions, their complexity has become manifest, leading to the conclusion that no single mechanism can model in humans the phenomenon of tumor rejection. It is likely that, due to human and disease heterogeneity, distinct trails lead to a final common pathway responsible for immune-mediated tumor regression. The synergy of the innate and adaptive immune response is likely to be required for successful tumor rejection. These two systems may act by enhancing and remodeling each of the functions by being recruited and activated at the tumor site by molecules with immune modulatory properties produced in the tumor micro-environment by cancer or tumor-associated normal cells. Such complexity could only be recently appreciated in its extent by high-throughput tools capable of providing a global view of biological processes as they occur. In this chapter, we will present selected examples of high-throughput gene expression profiling that may contribute to the understanding of anticancer immune responses

Adenovirus as a new agent for multiple myeloma therapies: Opportunities and restrictions.

Multiple myeloma is a malignancy of B-cells that is characterized by the clonal expansion and accumulation of malignant plasma cells in the bone marrow. This disease remains incurable, and a median survival of 3-5 years has been reported with the use of current treatments. Viral-based therapies offer promising alternatives or possible integration with current therapeutic regimens. Among several gene therapy vectors and oncolytic agents, adenovirus has emerged as a promising agent, and it is already being used for the treatment of solid tumors in humans. The main concern with the clinical use of this vector has been its high immunogenicity; adenovirus is often able to induce a strong immune response in the host. Furthermore, new limitations in the efficacy of this therapy, intrinsic to the nature of tumor cells, have been recently observed. For example, our group showed a strong antiviral phenotype in vitro and in vivo in a subset of tumors, shedding new insights that may explain the partial failure of clinical trials based on this promising new therapy. In this review, we describe novel therapeutic approaches that implement viral-based treatments in hematological malignancies and address the novelty as well as the possible limitations of these new therapies, especially in the context of the use of adenoviral vectors for treating multiple myeloma

Evolution of tumor infiltrating lymphocyte culture followed by HLA/epitope tetrameric complexes (tHLA)

Melanoma differentiation antigens, such as MART-1/MelanA and gp100/PMel17, frequently are observed as targets of tumor infiltrating lymphocytes (TIL) originated from HLA-A*0201-expressing patients with melanoma. Furthermore, particular clinical relevance was attributed to gp100/pMel17 based on the impression that the adoptive transfer of gp100-recognizing TIL was associated with clinical responses in a small group of patients. However, the actual frequency of specific T cells for these melanoma differentiation antigens has never been directly enumerated in ex vivo or in vitro expanded TIL cultures. Here, we enumerated melanoma differentiation antigen-specific T-cell precursor frequency in TIL using tetrameric HLA/epitope complexes, functionally characterizing their responsiveness to cognate epitope by cytokine release assay. T-cell precursor frequencies were enumerated in 11 fresh-tumor preparations and 17 TIL adoptively transferred into patients bearing HLA-A*0201. MART-1 or gp100-specific T cells could be detected respectively in 5 and 2 of the 11 fresh preparations and in 5 and 2 of the 17 adoptively transferred TIL. With one exception, melanoma differentiation antigen-specific T-cell precursor frequency in fresh material and TIL ranged between 5,000 to 21,000/10(6) CD8+ T cells. T-cell precursor frequency was not significantly higher in TIL whose administration was associated with clinical response. These data provide direct enumeration of MART-1/MelanA and gp100/pMel17 reactivity ex vivo and in vitro in the context of HLA-A*0201

A global approach to tumor immunology

Biological and clinical advances in the understanding of tumor immunology suggest that immune responsiveness of human tumors is a complex biological phenomenon that could be best studied by a real-time comparison of tumor/host interactions in the tumor microenvironment through a high-throughput discovery-driven approach. This conclusion is derived from our recognition that too many hypotheses or, in other words, no solid single hypothesis exist, based on experimental results, to further drive experimentation in human subjects. Functional genomic studies entertained during the last few years consolidated the belief that in humans the interactions between tumor and immune cells are too complex to be approached exclusively with a hypothesis driven method. We believe that immune cells suit cancer cells in a Yin and Yang balance by opposing and yet mutually depending on each other. Indeed, immune infiltration in tumors may play a dual role modulating in different circumstances cancer cell growth or destruction through a physiological modulation of inflammation. It is reasonable to question what induces inflammation at the tumor site. We hypothesize that inflammation is primarily driven by the phenotype of tumor cells that can modulate their microenvironment through cell-to-cell interactions or the secretion of soluble factors. Thus, in analogy the observation of immune cells within tumors parallels the presence of paramedics, police and firemen at the scene of an accident, which is reactive to and not causative of the occurrence. In this review we will explore this hypothesis by reporting and summarizing most of our recent work in the frame of available literature on the subject

Modulation of vaccine-specific T cell frequency and TCR repertoir during epitope-based vaccination

Selection of T cell-directed immunization strategies is based extensively on discordant information derived from preclinical models. We characterized the kinetics of T cell selection in response to repeated antigenic challenge. By enumerating with epitope/HLA tetrameric complexes (tHLA) vaccine-elicited T cell precursor frequencies (Tc-pf) in melanoma patients exposed to the modified gp100 epitope gp100:209-217 (g209-2M) we observed in most patients that the Tc-pf increased with number of immunizations. One patient's kinetics were further characterized. Dissociation kinetics of g209-2M/tHLA suggested enrichment of T cell effector populations expressing TCR with progressively higher affinity. Furthermore, vaccine-elicited T cells maintained the ability to express IFN-gamma ex vivo and proliferate in vitro. Thus, repeated exposure to immunogenic peptides benefited immune competence. These results provide a rationale for immunization strategies

Phase I study of transgenic B lymphocyte immunization (TLI) against telomerase in androgen-indepedent prostate cancer (PC)

Background: Human telomerase reverse transcriptase (hTRT) is expressed in most human tumor cells (>90% of PC) but absent from most normal adult cells, identifying it as a potential immunotherapeutic target (PNAS 97:4796, 2000). We developed a new approach to induce T cell responses in vivo based on the injection of primary B lymphocytes genetically programmed to serve as antigen presenting cells with the dual function of antigen synthesis and presentation. This is done ex vivo by exploiting the phenomenon of spontaneous B cell transgenesis (Gene Therapy 11:42, 2003), using a plasmid with a modified immunoglobulin heavy chain gene incorporating 3 inserts: two HLA-A2-restricted hTRT peptides: p540 (PNAS 97:4796, 2000) in CDR 3, p572 (PNAS 99:12275, 2002) in CDR1; and a 12mer malarial peptide acting as a Th cell determinant in CDR2. Methods: HLA-A2(+) subjects had androgen-independent PC with (14) or without (1) overt metastases, PS6 months, no current steroid treatment. Median pretreatment PSA=124. Prior treatment, except immunotherapy, was allowed. Mononuclear cells were separated from peripheral blood and incubated overnight with plasmid DNA. Nine subjects (3 in each of 3 cohorts) were reinfused once with 104, 105, or 106 autologous transgenic B cells. Six additional subjects were reinfused twice at a 4 week interval with 5x105 cells (3 fresh and 3 frozen boosters). Results: 12/15 subjects remain alive with followup of 17-69 weeks. No infusion-related events or dose-limiting toxicities were seen. No clinical or PSA responses were seen. Tetramer staining of hTRT-specific CD8 T lymphocytes was seen in all patients. Three patients developed CD8(+) T cells capable of killing hTRT expressing tumor cells in-vitro. Conclusions: single and double-dose TLI is feasible, safe and capable of inducing a T cell response against hTRT. Attempts to translate this immune response into clinical benefit by inhibiting T regulatory cells and targeting earlier stage patients are planned

Next Generation Sequencing: new tools in immunology and hematology

One of the hallmarks of the adaptive immune system is the specificity of B and T cell receptors. Thanks to somatic recombination, a large repertoire of receptors can be generated within an individual that guarantee the recognition of a vast number of antigens. Monoclonal antibodies have limited applicability, given the high degree of diversity among these receptors, in BCR and TCR monitoring. Furthermore, with regard to cancer, better characterization of complex genomes and the ability to monitor tumor-specific cryptic mutations or translocations are needed to develop better tailored therapies. Novel technologies, by enhancing the ability of BCR and TCR monitoring, can help in the search for minimal residual disease during hematological malignancy diagnosis and follow-up, and can aid in improving bone marrow transplantation techniques. Recently, a novel technology known as next generation sequencing has been developed; this allows the recognition of unique sequences and provides depth of coverage, heterogeneity, and accuracy of sequencing. This provides a powerful tool that, along with microarray analysis for gene expression, may become integral in resolving the remaining key problems in hematology. This review describes the state of the art of this novel technology, its application in the immunological and hematological fields, and the possible benefits it will provide for the hematology and immunology community. \ua9 2013 Korean Society of Hematology