Author: ApoptosisCompoundLibrary

With CLL have a high prevalence of EBV and thus possess EBV-specific memory cells with a high frequency of CD4+ gp350- specific T cells, the incorporation of gp350 into exosomes has a dual function: it confers B-cell tropism and serves as an immunodominant viral CD4 antigen. The cellular immune system of cancer patients is usually impaired but virus-specific immune responses are detectable even in late-stage patients and T lymphocytes specific for herpes viruses are often present at high numbers. Given the fact that the vast majority of CLL patients are seropositive for EBV, gp350 and other EBV proteins have the potential as promising neo-antigens in B-CLL cells exploiting and redirecting the strong anti-viral cellular immune responses to leukemic cells. Based on these results, we propose here a new immunotherapeutic approach for B-CLL, based on the simultaneous targeted transfer of functional CD154 and the EBV protein gp350 onto malignant cells using exosomes. gp350 is the major envelope protein of EBV and confers viral B-cell tropism by interacting with the complement receptor 2, which is highly expressed on B lymphocytes. Here, we generated modified exosomes produced in 293 cells. As a result of gp350 incorporation, these particles have a profound B-cell tropism similar to wild-type EBV, so that gp350- carrying vesicles specifically and efficiently bind to B cells. In addition, gp350 serves as a viral neo-antigen in B-CLL cells. We also found that CD154 on exosomes from HEK293 cells is functionally active as demonstrated by the induction of immune accessory molecules on B target cells probably through the CD40 pathway. Taken together, our experiments suggest that leukemia cells WZ4002 treated with CD154+/gp350+ exosomes are efficiently stimulated and subsequently killed by autologous B-CLL and gp350-specific cytolytic T lymphocytes. In summary, our results demonstrate that modified exosomes carrying the EBV protein gp350 display a distinct tropism to normal and leukemic B cells and efficiently transfer CD154 as a functional protein onto these cells. Leukemic B cells treated with these particles acquire an activated phenotype and become potent stimulators of autologous T lymphocytes. Engineered exosomes can be easily generated and can readily be scaled up for clinical applications. In addition, they can be individually tailored to express additional accessory molecules like OX40L or the Fas ligand or alternative viral molecules to target other classes of cells like macrophages and DCs. The generation of modified exosomes is not limited to 293 cells, which we used in this proof-of-concept. Instead, other cell lines that are approved for human therapy, such as MRC-5 fibroblasts, should also be tested as an optional origin of exosomes to facilitate transition into clinical trials. Modified gp350-carrying exosomes can thus be regarded as powerful and promising tools for various immunotherapeutic approaches. Globins are small heme-proteins that have the ability to reversibly bind molecular oxygen. For a long time only two globin types have been known in vertebrates: hemoglobin and myoglobin. Most likely, Hb and Mb are the best-studied proteins in biological, biochemical, biophysical and medical sciences.

Temozolomide ARLTS1 belongs to the ADP-ribosylation factor -ARF-like family of the Ras protein superfamily. ARFs are guanine-nucleotide-binding proteins which are critical components of several different eukaryotic vesicle trafficking pathways. As with other members of the Ras superfamily, ARFs function as molecular switches by cycling between inactive GDP- and active GTP-bound conformations. ARLTS1 has been characterized as an intracellular protein having tissue specific expression in the lung and leucocytes. ARLTS1 variants, such as the nonsense polymorphism Trp149Stop have been suggested to have a role in different cancers. Prostate cancer is the most frequently diagnosed cancer in males in many countries, including Finland. Aging and improved diagnostics most evidently increase the number of new cases, but the incidence is influenced also by some unknown factors. Growing number of new cases create pressure to health care system and new tools for PCa diagnostics, prognostics and treatment are required, especially to avoid over treatment and unnecessary biopsies. During the last several years there has been extensive research in PCa etiology and genome-wide association studies have revealed several common low penetrance genetic alterations. The association of these variants with clinicopathologic features and prognosis remains unclear and results are lacking clinical implications. We recently showed a significant association withCys148Arg variant and the risk of PCa. Further evaluation of this variant is warranted to increase the power of the association and study the functional role of the variant in PCa. More samples are also needed to evaluate the implication of this variant to clinical outcome and a potential role in predictive biomarker of PCa. Besides the genetic variants, DNA copy number aberrations are one of the most frequently observed genetic changes in familial and sporadic PCa. In most of the cases target genes for the aberrations are not fully identified. Interestingly, allelic imbalance has been detected at 13q14.2-13q14.3, and it is an important event in the progression of localized PCa. Differences of 13q14 loss of heterozygosity in different PCa groups could also be used to distinguish clinically insignificant PCa. In this study we analyzed the role of ARLTS1 in more detail, especially the role of Cys148Arg in PCa risk. Chromosomal aberration in 13q14.3 was analyzed with aCGH to evaluate the ARLTS1 copy number changes in PCa xenografts and cell lines. The expression of ARLTS1 was studied in clinical tumor samples, BPH samples and also co-expression data form previously published data was analyzed. The ARLTS1 gene and ARLTS1 polymorphisms have been shown to have a role in the pathogenesis of many cancers. The nonsense polymorphism at the end of the coding region has been revealed to predispose to familial cancer. Functional analyses of the truncated protein have indicated that the Trp149Stop variant might affect apoptosis and tumor suppression. Another ARLTS1 variant, the missense polymorphism Cys148Arg, and especially the CC genotype, has been found to be significantly associated with high-risk familial breast cancer.

Angiogenesis is recurrent function among the genes listed in Table 3, which is not surprising. As already mentioned, as a microtissue grows beyond a certain size, nutrient and oxygen depletion become limiting factors leading to the inhibition of cell proliferation and initiation of Enzalutamide 915087-33-1 angiogenic signaling. Oxygen concentration in 3D tissues depends on the balance between oxygen delivery and consumption. In vivo, this balance is tightly regulated by evenly distributed capillary networks but in vitro homotypic 3D microtissues lack vasculature and therefore develop a hypoxic core as their size increases. This event leads to the cells producing chemical signals for angiogenesis and is quite similar to the response occurring in normal hypoxic tissues where balanced signaling cascades lead to vascular remodeling and angioadaptation until the tissue oxygen concentration is back within its normal range. The up-regulation of VEGF-A and other genes with angiogenic function like ANGPTL7/CDT6, FGF5, NOV/IBP-9, and TGFA is pointing to a functional class of cytokines with great potential as three-dimensionality biomarkers. The above genes may play other roles besides being factors involved with hypoxia induced angiogenesis. For example, VEGFA has been shown to regulate neuronal development, survival, neurite growth and it also possesses gliotrophic properties. Also, it’s up-regulation is not limited to neuronal cells; it has been shown to be up-regulated in 3D cultures of a variety of cell and tissue types including human fetal lung fibroblasts, oral squamous cell carcinoma, glioblastoma, breast cancer, neonatal rat cardiomyocytes and neonatal mouse cardiomyocytes. This up-regulation in a non-cell type specific manner in 3D cultures of VEGF-A, and probably other members of the class, lends credibility to the notion of ubiquity of these potential biomarkers in different cells derived from different tissue types. Another recurrent gene function theme in Table 3 is terminal differentiation, a process by which cells commit to being part of a particular tissue or organ and perform a particular function. Terminal differentiation is preceded by inhibition of proliferation and cell cycle arrest. This exerts endoplasmic reticulum stress on the cell, but anti-apoptotic factors like Bcl-2 protect the cell from apoptosis and commit it to differentiation. In 3D, much like in organs in vivo, there is a spatial constraint on the microtissue, exerted by the defined pore size of the scaffold, which prevents them from proliferating freely, maintaining them in a quiescent state. Evidence in support of this comes from the fact that cells grown in 3D have shown lower proliferation rates than their 2D counterparts and higher expression of cyclin dependent kinase inhibitors p21. Also integrin mediated adhesion to the ECM leads to activation of Bcl-2 family of genes and makes the 3D cells more resistant to apoptosis. Such conditions are favorable for the cell to undergo terminal differentiation and this can be clearly seen by up-regulation in a number of differentiation and survival factor in 3D and NS compared to 2D, like PDGFB and STC1.

The invariant TCR expressed by iNKT cells recognizes lipid antigens presented in the context of the MHC class I-like LDN-193189 molecule CD1d. Invariant NKT cells, which have been extensively studied in mice, are, comparatively, quite rare in humans. For example, in mice 30–40% of T cells in the liver are iNKT cells; in humans only,1% of hepatic T cells are iNKT cells. Our lab, and others, have shown that in mice, PLZF controls the development of essentially all of the innate-like features of NKT cells. For example, PLZF-deficient NKT cells do not acquire the typical “activated” phenotype characterized by high expression of CD44 and CD69. PLZF deficient NKT cells also do not constitutively express granzyme B or the mRNA transcript for IL-4 and fail to acquire the capacity to secrete multiple cytokines upon primary stimulation. Furthermore, the frequency of NKT cells is substantially reduced in PLZF-deficient mice and the cells accumulate in the lymph nodes and spleen rather than in the thymus and liver. Overall, the phenotype of PLZF-deficient NKT cells is highly reminiscent of naı¨ve, conventional CD4 T cells. In contrast, ectopic expression of PLZF in conventional T cells results in the acquisition of innate T cell-like characteristics such as an activated phenotype, the rapid secretion of Th1 and Th2 cytokines in response to an initial stimulus and homing to non-lymphoid tissues. Recent studies have shown that PLZF expression is not strictly limited to invariant NKT cells in mice, but can also be found in a specific subset of cd T cells that express a Vc1.1Vd6.3 TCR. This subset of “NKT” cd T cells functionally resemble invariant NKT cells in that they co-secrete both IFN-c and IL-4 upon primary activation. Importantly, PLZF has been shown to be required for the innate T cell-like characteristics of NKT cd T cells. These studies, together with the findings in NKT cells, highlight an essential and non-redundant role for PLZF in the development of innate T cell effector functions. In addition to directly controlling the function of the cells it is expressed within, PLZF impacts immune function in trans. Of great interest, studies show that the IL-4 produced by these PLZFexpressing cells profoundly alters the CD8 T cell compartment. In mice with an expanded PLZF-expressing T cell compartment, CD8 T cells were found to take on an innate-like phenotype, represented by increased expression of CD44, Eomes and an enhanced capacity to secrete IFN-c. Such mice also harbor increased numbers of germinal center B cells and high serum levels of IgE, in concordance with their heightened Th2 responses. These data show that innate-like T cells, such as NKT cells, have a broad impact on the immune response. The role of NKT cells in disease is complex and appears to be dependent on both the NKT cell subtype and the microenvironmental context. In mice, NKT cells have been shown to be important in the suppression of solid tumors as a consequence of interactions with dendritic cells and other lymphocytes. In contrast, the immunomodulatory activity of NKT cells can also influence the immune response against autoantigens.

These changes were linked to perfusion deficits in solid tumors, which came from rapid tumor growth and profoundly disorganized vasculature. It has been suggested that the tumor microenvironment is a unique setting for tumor progression, which requires genetic adaptations in cancer cells for further survival and proliferation. Cell stresses induced by the microenvironment, especially hypoxia and reoxygenation, might cause these genetic changes. Regions of hypoxia are a common feature in solid tumors. Oxygen is a limiting factor because of the imbalance between O2 delivery and consumption. The O2 VE-821 deficiency is attributed to insufficient vasculatures and oxygen depletion in successive cell layers distal to the vessel lumen; simultaneously, there is an increase in O2 consumption due to the high metabolic rate of tumor cells. Many studies have reported that hypoxic tumors were more malignant and resistant to therapy, and thus had a worse prognosis. This phenomenon has been demonstrated in many tumor types. Moreover, the oxygen concentration within a hypoxic region is highly variable. Since tumor vasculatures are highly inefficient and unstable, red blood cells flux to the hypoxic regions, resulting in reperfusion or reoxygenation. Reoxygenation not only increases oxygen supply but also induces oxidative stress in the cells. This oxidative stress could cause damage to cellular macromolecules and lead to increased genomic instability. If tumor cells survive after exposure to hypoxia/reoxygenation insults, they may demonstrate increases in malignancy, DNA over-amplification, drug resistance, and metastatic potential. Cellular adaptation to hypoxia is well documented, but little is known about adaptive mechanisms to reoxygenation. Therefore, we used genome-wide expression microarrays to investigate the dynamics of transcriptional profiling during reoxygenation in MCF-7 breast cancer cells. Our microarray results showed that NMYC down-regulated gene 1 had the maximal response after reoxygenation. Therefore, we focused on investigating its functional role in reoxygenation. The functional assays revealed that cell migration of breast cancer cells during reoxygenation was driven by down-regulation of NDRG1. Lastly, the regulatory model of NDRG1 using in silico analysis was proposed for further investigation. Several studies have reported that tumor cells display increased drug resistance and metastatic potential after exposure to hypoxia/reoxygenation insults. Although cellular adaptations to hypoxia are well documented, little is known about adaptive mechanisms to reoxygenation. Here, we examined the dynamics of genome-wide gene expression during reoxygenation, and found that the differentially expressed genes were involved in the HIF-1-alpha transcription factor network and C-MYC transcriptional activation. In this study, principal component analysis of the oxygenresponsive genes showed high reproducibility over time. Based on the number of O2-responsive genes at different time points, the active period of transcription in response to reoxygenation appears to be between 8 and 12 hours.