Analysis of Primary Chronic Lymphocytic Leukemia Cells' Signaling Pathways.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by a specific expansion of mature B-cell clones. We hypothesized that the disease has a heterogeneous clinical outcome that depends on the genes and signaling pathways active in the malignant clone of the individual patient. It was found that several signaling pathways are active in CLL, namely, NOTCH1, the Ikaros family genes, BCL2, and NF-κB, all of which contribute to cell survival and the proliferation of the leukemic clone. Therefore, we analyzed primary CLL cells for the gene and protein expression of NOTCH1, DELTEX1, HES1, and AIOLOS in both peripheral blood lymphocytes (PBLs) and the bone marrow (BM) of patients, as well as the expression of BCL2 and miRNAs to see if they correlate with any of these genes. BCL2 and AIOLOS were highly expressed in all CLL samples as previously described, but we show here for the first time that AIOLOS expression was higher in the PBLs than in the BM. On the other hand, NOTCH1 activation was higher in the BM. In addition, miR-15a, miR-181, and miR-146 were decreased and miR-155 had increased expression in most samples. The activation of the NOTCH pathway in vitro increases the susceptibility of primary CLL cells to apoptosis despite high BCL2 expression.

Expert Revision of Key Elements for Clinical-Grade Production and Qualification of Perinatal Derivatives.

Perinatal derivatives have been proposed as adjunct therapeutic strategies or innovative treatments. Undoubtedly, perinatal derivatives can offer the opportunity and source material to isolate multipotent stem cells, but both maternal- and fetal-derived tissues can be processed and transformed into engineered tissues or advanced biomedical devices, whose potential remains to be fully elucidated. Promising preclinical and clinical results collected so far clearly foresee an escalation of such novel treatments. Market forecasts predict exponential growth in such advanced medicinal products during the next decade, with a pragmatic innovation for medicine into a more advanced biomedical version, enlarging the portfolio for treating a wide range of congenital and acute conditions. However, all these promising and fascinating therapeutic possibilities cannot gain a solid and recognized role in established medical practice without rigid and harmonized manufacturing strategies. The implementation of strategies according to guidelines and directives compiled by Regulatory Agencies, in conformity to (European) Pharmacopoeia and for Good Manufacturing Practice -conforming production of such products, represent critical steps required to translate perinatal technologies into effective therapeutic approaches. During the past 5 years, a panel of European experts and developers, gathered under the umbrella of the COST Sprint Action, supported by the European Cooperation in Science and Technology action, had the opportunity to revise and summarize experience and recommendations for a fruitful and proficient generation of perinatal biomedical products. In order to facilitate the creation and potential commercialization of perinatal bioengineered and advanced pharmaceutical products and technologies, such a collection of data and recommendations is described and discussed here.

Expert Consideration on Regulatory Aspects for Perinatal Derivatives in Clinical Settings.

Perinatal derivatives (PnD) are drawing growing interest among the scientific community as an unrestricted source of multipotent stem cells, secretome, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options, but they require the development of regenerative approaches. With this development, the question of regulation of donation, processing, and distribution has therefore become more important. Within the European Cooperation in Science and Technology (COST) community, we compiled a group of international experts on PnD technologies, who revised and compared existing EU national regulations. Notably, despite clear European directives, each EU Country has developed their own implementation and standard levels for cell- and tissue-based therapies. To enable extended applications of PnD treatments within the EU community and worldwide, harmonization is highly recommended. This paper aims to provide an overview of the various options available to introduce PnD into clinical practice. For this purpose, the different aspects resulting from (1) the type of PnD, (2) the amount of available data, (3) the degree of manipulation, and (4) the intended application and the process toward a possible commercialization will be presented. In the future, it will be important to find a balance between regulatory requirements and the best medical quality of the PnD product.

Perinatal derivatives application: Identifying possibilities for clinical use.

Perinatal derivatives are drawing growing interest among the scientific community as an unrestricted source of multipotent stromal cells, stem cells, cellular soluble mediators, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options by means of developing regenerative approaches. In this paper, to generate a complete view of the state of the art, a comprehensive 10-years compilation of clinical-trial data with the common denominator of PnD usage has been discussed, including commercialized products. A set of criteria was delineated to challenge the 10-years compilation of clinical trials data. We focused our attention on several aspects including, but not limited to, treated disorders, minimal or substantial manipulation, route of administration, dosage, and frequency of application. Interestingly, a clear correlation of PnD products was observed within conditions, way of administration or dosage, suggesting there is a consolidated clinical practice approach for the use of PnD in medicine. No regulatory aspects could be read from the database since this information is not mandatory for registration. The database will be publicly available for consultation. In summary, the main aims of this position paper are to show possibilities for clinical application of PnD and propose an approach for clinical trial preparation and registration in a uniform and standardized way. For this purpose, a questionnaire was created compiling different sections that are relevant when starting a new clinical trial using PnD. More importantly, we want to bring the attention of the medical community to the perinatal products as a consolidated and efficient alternative for their use as a new standard of care in the clinical practice.

Inhibition of Notch Signaling Stimulates Osteoclastogenesis From the Common Trilineage Progenitor Under Inflammatory Conditions.

Osteoclasts, macrophages and dendritic cells (DCs) can be derived from a common trilineage myeloid progenitor of hematopoietic origin. Progenitor commitment is susceptible to regulation through Notch signaling. Our aim was to determine the effects of Notch modulation on trilineage progenitor commitment and functional properties of differentiated cells under inflammatory conditions. We used the conditional inducible CX3CR1CreERT2 mouse strain to achieve overexpression of the Notch 1 intracellular domain (NICD1) or to inhibit Notch signaling via deletion of the transcription factor RBP-J in a bone marrow population, used as a source of the trilineage progenitor (CD45+Ly6G-CD3-B220-NK1.1-CD11b-/loCD115+). Cre-recombinase, under the control of the CX3CR1 promoter, expressed in the monocyte/macrophage lineage, was induced in vitro by 4-hydroxytamoxifen. Differentiation of osteoclasts was induced by M-CSF/RANKL; macrophages by M-CSF; DCs by IL-4/GM-CSF, and inflammation by LPS. Functionally, DCs were tested for the ability to process and present antigen, macrophages to phagocytose E. coli particles, and osteoclasts to resorb bone and express tartrate-resistant acid phosphatase (TRAP). We found that Notch 1 signal activation suppressed osteoclast formation, whereas disruption of the Notch canonical pathway enhanced osteoclastogenesis, resulting in a higher number and size of osteoclasts. RANK protein and Ctsk gene expression were upregulated in osteoclastogenic cultures from RBP-J+ mice, with the opposing results in NICD1+ mice. Notch modulation did not affect the number of in vitro differentiated macrophages and DCs. However, RBP-J deletion stimulated Il12b and Cd86 expression in macrophages and DCs, respectively. Functional assays under inflammatory conditions confirmed that Notch silencing amplifies TRAP expression by osteoclasts, whereas the enhanced phagocytosis by macrophages was observed in both NICD1+ and RBP-J+ strains. Finally, antigen presentation by LPS-stimulated DCs was significantly downregulated with NICD1 overexpression. This experimental setting allowed us to define a cell-autonomous response to Notch signaling at the trilineage progenitor stage. Although Notch signaling modulation affected the activity of all three lineages, the major effect was observed in osteoclasts, resulting in enhanced differentiation and function with inhibition of canonical Notch signaling. Our results indicate that Notch signaling participates as the negative regulator of osteoclast activity during inflammation, which may be relevant in immune and bone diseases.

Key Factors for Thymic Function and Development.

The thymus is the organ responsible for T cell development and the formation of the adaptive immunity function. Its multicellular environment consists mainly of the different stromal cells and maturing T lymphocytes. Thymus-specific progenitors of epithelial, mesenchymal, and lymphoid cells with stem cell properties represent only minor populations. The thymic stromal structure predominantly determines the function of the thymus. The stromal components, mostly epithelial and mesenchymal cells, form this specialized area. They support the consistent developmental program of functionally distinct conventional T cell subpopulations. These include the MHC restricted single positive CD4+ CD8- and CD4- CD8+ cells, regulatory T lymphocytes (Foxp3+), innate natural killer T cells (iNKT), and γδT cells. Several physiological causes comprising stress and aging and medical treatments such as thymectomy and chemo/radiotherapy can harm the thymus function. The present review summarizes our knowledge of the development and function of the thymus with a focus on thymic epithelial cells as well as other stromal components and the signaling and transcriptional pathways underlying the thymic cell interaction. These critical thymus components are significant for T cell differentiation and restoring the thymic function after damage to reach the therapeutic benefits.

Design, Synthesis, and Biological Evaluation of Desmuramyl Dipeptides Modified by Adamantyl-1,2,3-triazole.

Muramyl dipeptide (MDP) is the smallest peptidoglycan fragment able to trigger the immune response. Structural modification of MDP can lead to the preparation of analogs with improved immunostimulant properties, including desmuramyl peptides (DMPs). The aim of this work was to prepare the desmuramyl peptide (L-Ala-D-Glu)-containing adamantyl-triazole moiety and its mannosylated derivative in order to study their immunomodulatory activities in vivo. The adjuvant activity of the prepared compounds was evaluated in a murine model using ovalbumin as an antigen, and compared to the reference adjuvant ManAdDMP. The results showed that the introduction of the lipophilic adamantyl-triazole moiety at the C-terminus of L-Ala-D-Glu contributes to the immunostimulant activity of DMP, and that mannosylation of DMP modified with adamantyl-triazole causes the amplification of its immunostimulant activity.

MiR-7 in Cancer Development.

MicroRNAs (miRNAs) are short non-coding RNA involved in the regulation of specific mRNA translation. They participate in cellular signaling circuits and can act as oncogenes in tumor development, so-called oncomirs, as well as tumor suppressors. miR-7 is an ancient miRNA involved in the fine-tuning of several signaling pathways, acting mainly as tumor suppressor. Through downregulation of PI3K and MAPK pathways, its dominant role is the suppression of proliferation and survival, stimulation of apoptosis and inhibition of migration. Besides these functions, it has numerous additional roles in the differentiation process of different cell types, protection from stress and chromatin remodulation. One of the most investigated tissues is the brain, where its downregulation is linked with glioblastoma cell proliferation. Its deregulation is found also in other tumor types, such as in liver, lung and pancreas. In some types of lung and oral carcinoma, it can act as oncomir. miR-7 roles in cell fate determination and maintenance of cell homeostasis are still to be discovered, as well as the possibilities of its use as a specific biotherapeutic.

Thymus Regeneration and Future Challenges.

Thymus regenerative therapy implementation is severely obstructed by the limited number and expansion capacity in vitro of tissue-specific thymic epithelial stem cells (TESC). Current solutions are mostly based on growth factors that can drive differentiation of pluripotent stem cells toward tissue-specific TESC. Target-specific small chemical compounds represent an alternative solution that could induce and support the clonal expansion of TESC and reversibly block their differentiation into mature cells. These compounds could be used both in the composition of culture media designed for TESC expansion in vitro, and in drugs development for thymic regeneration in vivo. It should allow reaching the ultimate objective - autologous thymic tissue regeneration in paediatric patients who had their thymus removed in the course of cardiac surgery.

The role of regulatory T lymphocytes in immune control of MC-2 fibrosarcoma.

The role of T regulatory lymphocytes (Treg) particularly in cancer is well known. The goal of the present study was to determine the contribution of these lymphocytes in the regulation of anti-tumor immunity of CBA/HZgr mice against MC-2 fibrosarcoma (4th generation of methylcholanthrene induced tumor). The levels of T lymphocytes (CD4+, CD8+ and CD4+CD25+) were determined 8 and 20 days after tumor transplantation. Further, the role of CD4+CD25+ (Tregs) in tumor-host interaction was evaluated in vitro and in vivo by using specific monoclonal antibodies. We found that splenocytes of both control and Treg depleted tumor bearing mice strongly but differently inhibited growth of tumor cells in vitro. While splenocytes of untreated mice exhibited significant decrease of this activity (from 74.4% to 62.6% and 32.95%), the splenocytes of Treg depleted mice showed increase of this activity (from 79.5% to 84.3% and 86.2%) from day 6 to day 13 and day 21 after tumor grafting, respectively. Further, upon i.v. injecting specific monoclonal anti-Treg antibody tumor immediately prior to tumor cell intracutaneous transplantation, the tumor was rejected after initial growth. In treated mice, the incidence of Treg cells was very low initially, reaching normal values two weeks later. These animals were shown to be resistant to tumor transplantation four months later.

Induction of Urokinase Activity by Retinoic Acid in Two Cell Lines of Neuronal Origin.

Retinoic acid is one of the most well-known agents able to induce differentiation in several types of tumours. Unfortunately, most of the tumours are refractive to the differentiation cues. The aim of this investigation was to analyse the effects of prolonged treatment with retinoic acid on two cell lines of neural origin refractive to differentiation. Cells were also treated with retinoic acid in combination with a poly(ADP-ribosyl) polymerase (PARP) inhibitor because PARP1 is a known chromatin modulator and can influence the process of differentiation. The main methods comprised tumour cell line culturing and treatment; analysis of RNA and protein expression after cell treatment; as well as analysis of urokinase activity, migration, and proliferation. Both cell lines continued to proliferate under the prolonged treatment and showed increase in urokinase plasminogen activator activity. Analysis of gene expression and cell phenotype revealed different mechanisms, which only in neuroblastoma H4 cells could indicate the process of epithelial-mesenchymal transition. The data collected indicate that the activity of the urokinase plasminogen activator, although belonging to an extracellular protease, does not necessary lead to epithelial-mesenchymal reprogramming and increase in cell migration but can have different outcomes depending on the intracellular milieu.

Design, synthesis and biological evaluation of immunostimulating mannosylated desmuramyl peptides.

Muramyl dipeptide is the minimal structure of peptidoglycan with adjuvant properties. Replacement of the N-acetylmuramyl moiety and increase of lipophilicity are important approaches in the preparation of muramyl dipeptide analogues with improved pharmacological properties. Mannose receptors present on immunocompetent cells are pattern-recognition receptors and by mannose ligands binding they affect the immune system. Here we present the design, synthesis and biological evaluation of novel mannosylated desmuramyl peptide derivatives. Mannose was coupled to dipeptides containing a lipophilic adamantane on N- or C-terminus through a glycolyl or hydroxyisobutyryl linker. Adjuvant activities of synthesized compounds were investigated in the mouse model using ovalbumin as an antigen. Their activities were compared to the previously described mannosylated adamantane-containing desmuramyl peptide and peptidoglycan monomer. Tested compounds exhibited adjuvant activity and the strongest enhancement of IgG production was stimulated by compound 21 (Man-OCH2-ᴅ-(1-Ad)Gly-ʟ-Ala-ᴅ-isoGln).

Effect of Notch and PARP Pathways' Inhibition in Leukemic Cells.

Differentiation of blood cells is one of the most complex processes in the body. It is regulated by the action of transcription factors in time and space which creates a specific signaling network. In the hematopoietic signaling system, Notch is one of the main regulators of lymphocyte development. The aim of this study was to get insight into the regulation of Notch signalization and the influence of poly(ADP-ribose)polymerase (PARP) activity on this process in three leukemia cell lines obtained from B and T cells. PARP1 is an enzyme involved in posttranslational protein modification and chromatin structure changes. B and T leukemia cells were treated with Notch and PARP inhibitors, alone or in combination, for a prolonged period. The cells did not show cell proliferation arrest or apoptosis. Analysis of gene and protein expression set involved in Notch and PARP pathways revealed increase in JAGGED1 expression after PARP1 inhibition in B cell lines and changes in Ikaros family members in both B and T cell lines after γ-secretase inhibition. These data indicate that Notch and PARP inhibition, although not inducing differentiation in leukemia cells, induce changes in signaling circuits and chromatin modelling factors.

Sodium Salicylate Inhibits Urokinase Activity in MDA MB-231 Breast Cancer Cells.

INTRODUCTION: Sodium salicylate (NaS) is a derivate of acetylsalicylic acid or aspirin, used as a nonsteroidal anti-inflammatory drug for centuries, for its analgesic and anti-inflammatory effects. It was found to modulate different signaling pathways, in a cell-specific way. Here, we explore the effect of NaS on cell growth and urokinase activity in MDA MB-231 breast cancer cells. MATERIALS AND METHODS: We analyzed the effect of NaS treatment on cell growth by flow cytometry and viability test. The transwell migration assay was used to study the migratory response of the cells. The gene expression was analyzed by qRT-PCR on RNA level and by Western blot analysis on protein level. Urokinase activity was assessed by caseinolysis. RESULTS: Sublethal concentrations of NaS decreased cell growth and inhibited urokinase activity. The latter was a consequence of decrease in urokinase expression and increase in expression of its inhibitors. Analysis of signaling molecules revealed activation of transforming growth factor-ß signaling, increase in master transcription factors for epithelial-mesenchymal transition and changes in integrin expression. CONCLUSIONS: We propose that NaS causes partial cellular reprogramming through transforming growth factor-ß signaling which, together with direct NaS influence, causes changes in expression in a set of genes involved in extracellular proteolysis. These data could be beneficial for the development of new therapeutic approaches in invasive breast cancer treatment.

Modulation of urokinase plasminogen activator system by poly(ADP-ribose)polymerase-1 inhibition.

The urokinase plasminogen activator (uPA) system is a complex regulator of extracellular proteolysis which is involved in various physiological and pathological processes. The major components of this system are the serine protease uPA, two inhibitors PAI-1 and PAI-2, and the receptor uPAR. It has been previously shown by several groups that the uPA system has an important role in cancer progression and therefore its possible prognostic and therapeutic value has been evaluated. The aim of this study is to tackle the role of poly(ADP-ribosyl)ation in the induction of uPA activity in a glioblastoma cell line, A1235. This cell line is sensitive to alkylation damage and is a model for drug treatment. The components of the uPA system and the level of DNA damage were analyzed after alkylation agent treatment in combination with poly(ADP-ribose)polymerase-1 (PARP-1) inhibition. Here we show that the increase in uPA activity results from the net balance change between uPA and its inhibitor at mRNA level. Further, PARP-1 inhibition exerts its influence on uPA activity through DNA damage increase. Involvement of several signaling pathways, as well as cell specific regulation influencing the uPA system are discussed.

Transplanted tumor growth and the incidence of T-lymphocyte populations in the spleen of newcastle virus-treated mice.

C3Hf/HZgr mice were transplanted with SCCVII carcinoma cells and treated with Newcastle disease virus (NDV). The treatment slows down the growth of transplanted tumor. Furthermore, by using specific monoclonal antibodies, the frequencies of CD4+, CD8+, and CD4+CD25+ T lymphocytes were determined in the spleen of tumorous mice at particular times following tumor transplantation and/or NDV application. The incidence of lymphocytes CD4+ and CD8+ decreased and of CD4+CD25+ increased in the spleen of mice during the time following tumor transplantation. However, the frequency of regulatory CD4+CD25+ T lymphocytes in the spleen is very low, while CD4+ and CD8+ increased to normal level following intraperitoneal (i.p.) NDV injection in tumor-bearing mice. Thus, besides directly destroying transplanted tumor, NDV seems to be involved against growing tumor by reducing the frequency of regulatory T lymphocytes maintaining the frequency of CD4+ and CD8+ T lymphocytes within the control values pointing to its role in immunomodulation.

Notch affects the prodifferentiating effect of retinoic acid and PMA on leukemic cells.

Notch proteins determine cell fate decisions in the development of diverse tissues. Notch has been initially found in T-ALL but its role has been also studied in myelopoiesis and myeloid leukemias. Studies in different model systems have led to a widespread controversy as to whether Notch promotes or blocks myeloid differentiation. In this work, we evaluated the influence of Notch activation on leukemic cell differentiation along the monocytic and myelocytic pathway induced by phorbol 12-myristate 13-acetate (PMA) or all-trans retinoic acid (ATRA). We observed that differentiation of the human myeloblastic cell line HL-60 can be retarded or blocked by Delta/Notch interaction. ATRA induces complete remission in patients with acute promyelocytic leukemia, but it cannot completely eliminate the leukemic clone and to be effective it should be combined with chemotherapy. Our findings suggest that Notch signaling may contribute to the incomplete elimination of the leukemic cells after PMA or ATRA treatment and the blockage of Notch pathway may be beneficial in the treatment of myeloid leukemia. © 2014 International Society for Advancement of Cytometry.

Gene expression in formalin-fixed paraffin-embedded lymph nodes.

Elucidation of molecular pathways involved in development of human lymphoma requires efficient methods for tackling gene expression in lymph nodes. Expression studies of transcription factors in these malignancies facilitate understanding the changes occurring in neoplastic transformation and lymphoma development. Excised lymph nodes are routinely fixed in formalin and embedded in paraffin for diagnosis and stored in many hospitals' pathology archives. These tissues represent a precious resource for research since they allow retrospective studies to cover a broad range of human lymphoma even the less frequent types. Reverse transcription polymerase chain reaction (RT-PCR) is a commonly used method for gene expression analysis and a reproducible protocol for RNA isolation from lymph nodes is an inevitable requirement for these studies. However, formalin fixation and paraffin-embedding interfere with the quality of RNA especially when isolated from lymph nodes being the most fragile lymphatic tissues. We present here a simple and fast method for RNA isolation from formalin-fixed paraffin-embedded lymph nodes that can be successfully applied for RT-PCR as well as for quantitative RT-PCR analysis. We tested diverse isolation reagents and combined a range of factors in order to get a high quality RNA for retrospective studies of gene expression in human lymphoma samples. Our modified method of RNA extraction from FFPE provides superior yields and purity based on qPCR data.

Analysis of Ikaros family splicing variants in human hematopoietic lineages.

Transcription factors from the Ikaros family are involved in lymphocyte differentiation and have a critical role at specific check points of the haemopoietic pathway. However, how developmentally regulated changes are reflected in gene expression programs of lymphocyte differentiation is not well understood. It has been suggested that disregulation of transcription factors from the Ikaros family is associated with the development of different human leukemias. In this work we analyzed the state of Ikaros family members in different leukemic cells with the aim to explore the transcriptional control of human hematopoietic lineages and shed some new light on our understanding of transcription factor significance in human leukemias. By means of RT-PCR and specific primers we investigated the expression of Ikaros, Aiolos and Helios transcription factors and their splicing variants in seven leukemia cell lines derived from different types of leukemia (ALL, CML, AML) and lymphoma (histiocytic lymphoma, Burkitt lymphoma and anaplastic large cell lymphoma). In all of the cell lines examined Ikaros was present in dominant Ik1 to Ik4 isoforms and small Ik6 isoform was absent. Aiolos was expressed in the majority of the cell lines, of both, B and T origin, in the form of the full length Aio1. Helios was also present only in two long isoforms Hel1 and Hel2, and was absent in one third of the lines. Similar distribution of positive and negative expression of Aiolos and Helios found in various types of leukemias could implicate common pathways of their regulation.