DOI: 10.31038/CST.2017251

Abstract

Investigations in immunomodulating therapies for cancer treatment over the past 20 years have flourished. Given the complex tumor microenvironment and differential signaling pathways a wide variety of potential targeting mechanisms have come to attention. Herein we review the immunomodulating potential of Chemokine 21 in pre-clinical and clinical studies, as well as examine the novel multi-faceted immune-based treatments in advanced head and neck cancer.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world, accounting for more than 300,000 deaths annually [1]. Despite advances in surgical techniques and chemo-/radiation treatment strategies, patients with advanced T3 or T4 HNSCC continue to demonstrate 20%-30% survival with failure of primary surgical or medical (radiation and chemotherapy) management. Salvage therapies, in recalcitrant or recurrent disease, are limited by systemic toxicity or operative morbidity owing to nearby vital structures (carotid artery, skull base). Immunotherapy and modulation in HNSCC is a promising venture given recent advances in cancer biology and investigations in the tumor microenvironment.

Historically studied in the contexts of inflammation and immune response, chemokines have increasingly been studied in the context of tumor proliferation [2, 3]. Chemokines are a family of small chemotactic cytokines involved in cellular signaling and inflammation. Through interactions within a subset of G-protein- coupled transmembrane receptors (GPCRs), secreted cellular chemokines regulate cellular homing, activation, and recruitment of a variety of diverse leukocytes and activated adhesion molecules [2, 4-6]. In the tumor microenvironment, responses to specific chemokines result in the migration of different immune cell subsets and regulate tumor immune responses; these are both pro- and anti- inflammatory in nature. Chemokines, have been directly associated with cancer immunity, proliferation, and metastases [7,8]. In this review, we address the role of Chemokine 21 (CCL-21) in tumor progression, its role as an immunotherapy platform, and the potential within head and neck oncology.

CCL-21 chemotaxis and lymphocyte recruitment

Divided into four subfamilies, chemokine nomenclature is related to the location of their first N-terminal cysteine (C) residues; classified as: C-, CC-, CXC-, and CX3C-chemokines [9]. CCL-21 (also known as Thymus-derived chemokine 4, 6Ckine, or Exodus-2) is a CC chemokine expressed in high endothelial post-capillary venules, T-cell stromal zones of the spleen, Peyer’s patches, and afferent peripheral lymph nodes that strongly attracts naïve T-cells and mature dendritic cells (DC) including NK T-cells [10-13]. The effects of CCL-21 on chemotaxis have been demonstrated in neutralization studies, where the homing of T-cells and DCs has been shown to be significantly reduced [14, 15]. Acting through the GPCR CCR7, a convergence of the immune response elements to sites of CCL-21 production have been demonstrated to co-stimulate the expansion of CD4+ and CD8+ T cells and induce Th1 polarization [16]. Additionally, chemokine bound DCs form T cell adhesions resulting in a hyper-responsive T-cell on subsequent exposure to CCL-21 and antigen-presenting cells [17].

Role in cancer immunotherapy

The cancer immunosuppressant microenvironment results in ineffective antigen processing and presentation; resulting in poor host response. The CCL-21/CCR-7 axis has been studied in efforts to create a more immunogenic environment through chemotaxis of DCs, NK cells, and lymphocytes. By recruiting host antigen presenting cells (APC) for tumor antigen presentation, T cells within lymphoid organs have the ability to prime anti-tumor specific activity. Of note, while CCL-21 aids in the polarization of Th1 lymphocytes, the secondary lymphoid chemokine has also been shown to have minimal effect on the proliferation of suppressor cell population; CD4+CD25+ T-regulatory cells are hypo responsive to CCL-21 induced migration and unresponsive to CCL-21 co-stimulation [16]. High levels of T-regulatory cells within the tumor microenvironment has demonstrated poor prognosis in many cancers, dependent on the type and location of neoplasm [18].

Pre-clinical animal models

The ability to process and present a high ratio of activated tumor-antigen-APCs has long been the goal of immunotherapy in the immunosuppressive cancer microenvironment. The first established model for the creation of a CCL-21 chemotactic gradient in restoring tumor-antigen presentation was undertaken by Sharma and Dubinett [19]. In an immune competent murine lung cancer model, intratumoral injection of CCL21 induced infiltration of CD4+ and CD8+ T cells and DC in both tumor and draining lymph nodes. Additionally, a potent antitumor response was observed as complete tumor eradication was found in 40% of treated mice [19]. Furthermore, the tumor microenvironment displayed a concomitant decrease in immunosuppressive molecules such as PGE-2 and TGF-ß [19]. When introduced to the afferent axillary lymph nodes, CCL-21 injections resulted in a marked infiltration of lymphocytes and DCs into the lung tumor microenvironment, associated with a significant reduction in tumor burden [20]. The anti-tumor efficacy of CCL-21 has further been confirmed in a variety of transgenic lung, ovarian, melanoma, and liver cancer models [21-25]. Despite the promising results, in vivo studies have utilized specific cancer cell lines, with greater success observed in low-malignant weakly metastatic clones as compared to highly-metastatic clones [26]. Further research on the efficacy within specific tumor phenotypes, locations, timing, and dosing of CCL-21 continues to be underway.

Antigen presenting cells as a platform

APCs are fundamental in the activation of specific immunity and have been investigated as adjuvants to cancer immunotherapy to stimulate tumor-specific antigen presentation for promotion of T cell activation and anti-cancer immunity [27, 28]. Prior investigations have generated DCs with enhanced immune stimulatory and T-cell activity through pulsed electroporation of antigens or viral transduction of cytokines [29-33].

A platform to transduce DCs with CCL-21 (DC-CCL21) through an adenovirus vector has also recently been developed demonstrating viable in vitro chemotaxis of activated lymphocytes [34, 35]. The anti- tumor efficacy of DC-CCL21 has been studied in the murine lung and melanoma cancer models. These studies demonstrated a significant reduction in melanoma tumor growth and complete eradication of lung tumor burden in 60% of mice with lung cancer [32, 34]. Clinical trials are currently underway in late stage non-small cell lung cancer patients based on the promising results of this preclinical data [36].

Head and neck oncology

Recurrent and advanced HNSCC has been extraordinarily challenging to treat, with stagnant survival and cure rates over the past 20 years [37]. Depending on the tumor site, reoccurrence rates may range from 25%-50%, and the incidence of subsequent reoccurrences similarly fall within this broad range [38-40]. Morbidity from current salvage treatment strategies are unfortunately common and include chronic pain, respiratory distress, and dysphagia oftentimes resulting in tracheotomy, or, gastrostomy tube dependence [41]. Despite cancer outcomes, cosmetic and functional deficits may negatively impact the head and neck cancer patient’s quality of life [42-44]. Limited surgically by nearby structures and systemically by toxic effects, advancements in locoregional therapies in the tumor microenvironment are strongly desired.

Recent advances in material science, have developed biocompatible, functional, three dimensional polymer systems for molecular and cellular delivery in cancer care [45]. A novel implantable polymer- based system for the delivery of chemokines to interact directly with tumor cells, has the potential to integrate with a wide range of anti- cancer treatments. Recently, a biodegradable poly-ε-caprolactone (PCL), polylactide-co-glycolide (PLG), co-polymer seeded with DC- CCL21 and/or cisplatin has been tested in an animal model resembling unresectable head and neck squamous cell carcinoma (HNSCC) [46, 47]. Tumor cells from the squamous cell carcinoma (SCCA) VII/ SF were injected, grown, and debulked in the flanks of C3H/HeJ mice. The animals then underwent debulking surgery to replicate an unresectable cancer setting. The flexible seeded PCL/PLCL polymer was then applied to contour the cancer tissue bed. In the first study, the cisplatin-polymer group effectively reduced tumor volume by over 16-fold when compared to control polymer with intratumoral cisplatin injection groups [47]. When combined with radiation, the cisplastin-seeded polymer group enhanced the efficacy of radiation therapy by tumor volume reductions of 53% when compared to surgery and radiation alonel [47]. Given the well-established literature in the role of cytokines in tumor regression, the HNSCC murine polymer model was then seeded with DC-CCL21 using a fibrin gel delivery mechanism. After implantation to the partially resected tumor, DC-CCL21 secreting polymer significantly reduced SCC VII/ SF tumors by 41% as compared to control groups [46]. Additionally, the DC-CCL21 polymer resulted in increased CD4+ and CD11+ DCs as well as a marked decrease in T-regulatory cells within the tumor microenvironment.

Current studies are underway to assess the anti-tumor efficacy of DC-CCL21 with combination of cisplatin and radiation therapy in the HNSCC murine model. Additionally, the promising findings of APC and T cell recruitment provide the rationale for combination with immune checkpoint blockade therapy to enhance the frequency of activated T cells in the tumor micro-environment for improved patient outcome.

Conclusion

Research in the field of immunology has evolved tremendously as the complex immunosuppressive tumor microenvironment continues to unravel. The role of CCL-21 is critical in promoting DC homing and T-lymphocyte activation. Tumor antigen presentation is significantly amplified both locally and peripherally following introduction of CCL-21. Head and neck cancer therapies, previously limited by morbidity,
may now have an alternative for locoregional control in unresectable tumors. A multi-faceted approach to innovative cancer treatment is necessary in order to maximize the immunogenic potential for tumor cell death. As described, it is clear that CCL-21 is a potent chemokine in eliciting an immune response with substantial evidence supporting its use in future oncologic therapies. Collaborations with materials scientists, immunologists, and physicians can not be underestimated in the ongoing pursuit for cancer cure.

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DOI: 10.31038/CST.2017247

Abstract

Nothing makes sense except considering evolution, by which understanding how life originates it is important to be able to decipher the mysteries that lead to the abnormal behavior of the cancer cell. So far, the emergence of life is explained in religious terms, but in scientific terms, it was not possible. The various existing theories had gaps that could not be resolved. The discovery of unsuspected capacity of melanin transform light into chemical energy, such as chlorophyll in plants; It is a watershed in biology, it turns out that melanin is the missing link in the chain of events that leads to the emergence of life. Now the Outlook is different, we can advance faster in the study and treatment of diseases that currently constitute a scourge to mankind, such as cancer. We are on the threshold of a new era in biology and in medicine.

Introduction

The unsuspected bio-energetic role of melanin has been described previously [1]. Electromagnetic radiation, melanin and water are substances that do not present any data that allow us to think that they have some thought as an evolution process. You could say that its main physical and chemical characteristics are the same since the beginning of time.

What we call evolution, can realize to the biochemical scaffolding that nature has patiently developed over eons of years, to increasingly optimize the properties of light, melanin and water, which have not changed a whit.

Eukaryote cell is becoming more sophisticated, biochemical gear is increasingly complex, and what is most striking, the power supply remains the same since the beginning of time. This is: the first living entities, had similar amount of available chemical energy derived from the melanin that human beings today.

It is something that has not changed despite the passage of time. What, has changed continuously over time, is the number of molecules, organelles, enzymes, etc., have been bolted together to make increasingly more sophisticated, increasing the complexity of the cell. And the term sophisticated, applied to any system; means that it contains many parts.

Continues to call me the attention that the power source is still, on the one hand, the sunlight mainly, on the other hand, melanin, which has not changed in the slightest despite the course of millions of years; and so on the perfect substrate which is water.

It is interesting that one of the elements, melanin; is completely dark and we can’t see through it, either with the naked eye or with instruments, and to this we attribute the fact that its formula is not known. But, on the other hand; water is completely transparent, we observe it with the naked eye, and yet we don’t understand as the hydrogen and oxygen atoms that compose it are organized, due to diverse factors, i.e. because its arrangement is dynamic and not uniform.

The nature of light, we neither speak, don’t even understand if it is a wave or a particle, or both. So, from three natural phenomena which we know little or nothing, life originated. Melanin is the missing link between the different theories that try to explain how emerged those we named life.

Light, Melanin, and Water

The physical and chemical properties of light, melanin and water whether we understand them or not, they possess characteristics that seem to be immutable, they inner dynamic had not change in the slightest despite the passage of time. And it seems paradoxical that the union of these quite stable elements generates the very dynamic phenomenon that we call life; which is characterized by constant changes, in all times, everywhere.

Charles Darwin said that life originated in warm water, with low oxygen levels, and without any other form of present life. And it is all spent out there, because the amniotic fluid is warm water with low oxygen levels and without any other way of life.

The evolution of the human eukaryote cell, requiring billions of years of evolution, based on primitive cells, is repeated in each pregnancy, and reducing days. Thus, the initial cells during pregnancy are primitive cells, whose structure is not as complex as mature or specialized eukaryote cell.

But despite differences that may be, at least in appearance; different cell lines retain the same fundamental process of generation and distribution of energy from the melanin.

In eukaryotes normal cells, of any cell line; organelles are very similar, even in size; which reflected a surprisingly uniform pattern, which cannot be a result of chance. And that consistency comes from the origin of life, as both light, melanin, and water, are very dynamic elements but do not change, do not seem to evolve in the least; It seems that they reached perfection and do not need to go further.

What if it evolves, and does so constantly, all the time, is what we call life. And one of the features of the evolution, is the increasingly complex management, in many respects, from the chains of carbon mainly from photosynthesis-derived glucose, this is: a homogenously arrangement of carbon, oxygen, and hydrogen atoms, with C_nH_2nO_n as general formula, and whose most soluble example is glucose. While more advanced is a cell in the evolution, more efficient is the way in which atoms and carbon chains entwine each other forming increasingly intricate and complicated processes and structures, but in the end, reflects an increasingly efficient use of the energy that emanates from the melanin in the form of molecular hydrogen and high-energy electrons. A kind of energy astonishing accurate.

The proliferative cell

It is a deep-rooted dogma that normal differentiated cells, rely primarily on mitochondrial oxidative phosphorylation to generate energy needed to impel cellular processes.

So, it is not surprising that the metabolism of embryonic cells present findings consistent with the Warburg effect [2], or aerobic glycolysis. Which is an inefficient way to generate adenosine 5´-triphosphate (ATP), allowing to incorporate nutrient into biomass, rather than efficient and too much complicated, ATP production.

However, mitochondria and ATP biological functions are regulation of temperature and phosphate levels, but no energy production. Therefore, ATP biological function is regarding temperature control and phosphate levels, compounds that are chemically unstable but thermodynamically stables.

Thereby, embryonic cells require generating biomass so quickly, and gradually more and more complicated processes appear inside these cells, reflected, i.e.; by the increasing mitochondria number, whose function is the temperature control as much as possible, due to the chemical reactions inside normal differentiated cells are astonishing accurate, and temperature is no exception.

By other hand, uncontrolled proliferation is due to the cells that turn back in evolution, being the explanation due to the generation and distribution of melanin´s energy has been affected chronically.

Cancer cells are not generally controlled by normal regulatory mechanisms [3]. Thereby, the characteristic disordered tumor growth is dependent of difficult-to-understand mixture of local factors. A good example are the processes involved in angiogenesis and vascular remodeling implied in the vascularization of malignant tumors.

Total spectrum of morphogenic and molecular events required to form a neovascular network are way beyond our abstraction capacity, because casualness is a significant factor. And worst, those events are significantly different of normal vasculogenesis.

But in the light of the bio-energetic unsuspected role of melanin, we can reconsider such processes initiating the analysis as follows: the blood cannot carry energy, and on the other hand, the amount of chemical energy available in a cancer cell, is significantly lower than in a normal cell, therefore we have a series of events that reflect a step backwards in the evolution of the diseased cell and their chemical and anatomical, physical characteristics indicate that address.

While more evolved it is a cell, its operation is farther from random, and vice versa.

The intricacy of the processes that make up a specialized cell, they make these increasingly more and more precise, and one of the purposes of evolution is gradually reducing the interference of random in intracellular biochemical processes. Since any alteration in its sequence, temporality, or spatial location, would have devastating effects on perfectly than a specialized eukaryote cell represents.

Anti-vascular therapy of cancer

It has been over 30 years since Judah Folkman hypothesized that tumor growth is angiogenesis dependent [4]. But after years of clinical trials based in Dr. Folkman theory, results have been disappointing. Inhibition of VEGF, the main molecular mediator in capillary sprouting has been insufficient to halt tumor progression permanently in many cancer types.

The reasons can be diverse. i.e. the existence of multiple vascularization mechanisms and additional growth factor pathways. In our experience, the main cause of this failed therapeutic focus is that blood cannot carry on energy, just cell´s building blocks as glucose, aminoacids, lipids, etc., and CO₂.

Cancer cells

Most solid tumors display distinct aneuploid karyotypes (abnormal chromosomal numbers) and frequently miss-segregate whole chromosomes in a phenomenon called chromosomal instability (CIN). CIN positively correlates with poor patient prognosis, indicating that reduced mitotic fidelity contributes to cancer progression by increasing genetic diversity among tumor cells [5].

Mechanisms leading to the loss of mitotic fidelity in CIN are not known. A common mitotic defect in tumor cells with CIN is the persistence of erroneous attachments of chromosomes to spindle microtubules (merotely).

In normal diploid cells erroneous attachments arise spontaneously and are efficiently corrected to preserve genomic stability. Paradoxically, kinetochore microtubule attachments in cancer cells with CIN are more stable than those in normal diploid cells, thereby, accounting for the persistence of mal-attachments into anaphase, causing chromosome miss-segregation. Seems that cancer cells have a diminished capacity to correct erroneous kinetochore-microtubules attachments; a dysfunction with a widespread occurrence of CIN in tumors.

The mechanisms involved in ploidy protection and genomic integrity are highly complex, not understood, and astonishingly accurate. In example, initiation of a new round of DNA replications should be restricted until after completion of the previous mitosis. Thereby DNA replication depends of biochemical pathways that occur exactly in time, space, location, amount; etc., in an amazing way; but are largely unknown.

The failures in these poor-understood regulatory mechanisms are generalized. Therefore, the plausible explanation is energy. Other theories proposed in the scientific literature, trying to explain abnormalities observed in cancer cells, has been proved elusive, given the inherent complexity of specialized eukaryotic cell after four billion yeas of evolution.

However, we are in front of a new era in the molecular biology of cancer cells; thanks to unsuspected bio-energetic role of melanin. And the explanation seems as quite simple: a specialized eukaryotic cell whose levels of generation and distribution of energy (from melanin) are impaired by contaminated water, polluted air, pesticides, herbicides, fertilizers, transition metals, heavy metals, addictive drugs, solvents of diverse types, industrial waste, stress, etc., tends to turn back in evolution. Simply because the complicated biochemical cell scaffolding that gives vital and highly-ordered support to the poorly understood specialized eukaryotic cell, requires astonishingly accurate amounts of energy.

Insofar as the available quantities of energy inside the cell to reduce enough, for example, in time and form, the very complicated biochemical pathways start to collapse in a disorderly way. Apparently, keeping only the most basic functions; or at least the first that appeared during the evolution, which gives us an idea that cell not is being able to keep relatively recent occurrence mechanisms.

In humans, aneuploidy is linked to pathological defects such as development abnormalities, mental retardation, or cancer, but the underlying mechanisms remain elusive. There are many types of aneuploidy whose origin remains unknown. A common response independent of the type of aneuploidy can be a novel target for cancer therapy.

Genomic stability requires that genetic material must be equally distributed between the two daughter cells during mitosis. An apparently straightforward process, by far beyond our abstraction capacity. By other side, an aberrant number of chromosomes, or aneuploidy, has been recognized as a feature of human malignancies for over a century, but until today without compelling evidence of causality.

Molecular logic underlying proper and aberrant chromosome segregation are extremely complex. It is undoubtedly that chromosome instability is detrimental for the fitness and survival of normal cells, being also the hallmark of cancer cells. Paradoxically, cells with an elevated proliferative potential, are also highly aneuploid.

Theoretically, Aneuploidy is a direct consequence of chromosome segregation errors in mitosis, while structural aberrations are caused by improperly repaired DNA breaks, but also exists a cross-talk between them. The long-lasting efforts to selectively inhibit proliferation of tumor cells has been infructuous.

Toxins against microtubules exert anti-tumor effects in some patients, but it is unclear its action´s mechanism. Apparently, these compounds acts interfering with microtubule dynamics during mitosis activates the spindle checkpoint, causing a prolonged mitotic arrest. Thereby, cells either then undergo death in mitosis or slippage; returning to interphase without dividing; but it is unclear what dictates the balance between these two fates.

Therapeutic inhibition of major mitotic kinases and kinesins has given a modest clinical activity at best.

Thereby, the maintenance of chromosomal stability involves the whole cell, not only genes. So, generation and distribution of energy from melanin has a fundamental role in cell biology (Figure 1).

The energy that melanin provides incessantly, night and day, in a quite consistently form, both in and outside the cell, explains finally, the origin of life.

In chlorophyll, the water dissociation is irreversible; but in melanin is reversible. The process is as follows:

2H₂O → 2H₂ + O₂ →2H₂O + 4e^–

The liquid water is sublimated by melanin into its gaseous components molecular Hydrogen (H₂) and Oxygen (O₂) in astonishing and accurate way, this is: always the product of the water dissociation is molecular hydrogen and molecular oxygen. Thereby, it is a quite precise process even in a diversity of environmental conditions, because melanin acts also as attemperator of surrounding sudden changes, for instance, a significant increase or decrease in the amount of light; keeping the output of H₂ and O₂ between narrow ranges.

The biochemical process of cell (and body) is amazing accurate, otherwise life is not possible; but this astonishing precise start since the very first step of life that is the energy production. Evolution can be interpreted as the increasing and gradual organization of the distribution of energy that is more and more exquisite, efficient; and precise as time pass by. Recall that is the same energy since the beginning of time, both in nature and amount.

The specialized eukaryotic cell does not generate more energy from melanin, because melanin energy generation has a top, a guarded proportion; unless more melanin is added to the system. Instead it is much more efficient in the distribution and thereby using of H₂ and e- that the melanin dissociation and re-formation of water produces constant and exactly during night and day.

Eukaryote cell, mature, specialized; It is a whole that works perfectly in every one of its parts, starting from the generation and distribution of energy; which has been patiently optimizing over millions of years of evolution. You could say that nothing or almost nothing of what happens inside a specialized cell is the result of chance.

In any system, not only in biology; energy, defined as everything that produces a change; It is the most sensitive segment as an alteration in this area produces widespread failures, such as that seen in cancer cells.

Hence how difficult that has been to develop a successful therapy in cancer cells, it seems virtually impossible, because when designing strategies that act on a certain part of the cell, for example microtubules; the cell simply changes its behavior, which is now largely influenced by random, totally opposed to a normal cell; and quickly becomes resistant to instituted treatment.

And the explanation is that simply the imbalance now is expressed differently, but the actual background of the problem doesn´t change, and the proof is that proliferation does not diminish. Many of the deviations observed in a cancer cell should be taken care at the same time, so cell tends to function normally, as it has millions of years, millions of times.

Experiments in this regard have shown that partial reparation of the altered intracellular processes is not the solution, it is necessary to address the entire e ideally at the same time.

This is not currently possible, except to increase the generation and distribution of energy that comes from the melanin, and since all the biochemical processes that occur inside the eukaryote cell depend entirely on the H₂ and e^–, to normalize these levels, all processes tend to operate normally, because ultimately, they are chemical reactions quite exact, that may not happen or inadequately occurs if the power level is not suitable.

QIAPI 1^® and Cell Proliferation

The following Tables (1-4) show results of experiments carried out in cultured human cells. The results are part of the development of medication implemented in our laboratory, and which is in phase of patent in several countries, which has already been granted in several of them.

Table 4. Inhibitory concentrations of QIAPI 1^®.

Cell line	Concentrations of  QIAPI 1 inhibit 50% proliferation[mg/ml]	Concentrations of  QIAPI 1 inhibit 99% proliferation[mg/ml]
WI-38	1,0	4,5
A549	2,0	5,5
HS-683	2,0	5,5

These results present them as a proof of concept, since the mechanism of action of QIAPI 1^® is intensifying the dissociation and reformed the water, by the melanin molecule. (Tables 1-4)

The similarity in the results, although cultures of different cell lines, support our theory that acting on the generation and distribution of energy, cells, regardless of human classifications, they tend to recover complex order that is required so that their behavior is normal, proper; as millions of years, has done millions of times.

QIAPI 1^® is a new therapeutic agent [6] that gradually will appear in the market, as legal requirements should be fitted.

Conclusion

The apparent chaos that reigns in efforts to decipher the bases of the anomalous behavior of cancer cells, appears to take a totally different direction when we do aside dogma that the main source of cell energy is glucose, then the discovery of unsuspected intrinsic property of melanin make visible and invisible light into chemical energy, such as chlorophyll in plants; through the dissociation of the water molecule, it is a watershed in cell biology.

We have a new view in the study of the biology of cancer, and while we move faster in that sense, the benefit that can give every day to numerous patients affected by the disease will be higher.

Acknowledgement: This work was supported by Human Photosynthesis® Research Center.

References

• Solis-Herrera, A. Arias-Esparza, MC. Solis-Arias, RI. Solis-Arias, PE. Solis Arias, Martha P (2010) The unexpected capacity of melanin to dissociate the water molecule, fills the the gap between the life before and after ATP. Biomed Res 21: 224-227.
• Vander Heiden, Matthew Cantley, Lewis, Thompson, Craig B (2009) Understanding the Warburg effect: The Metabolic Requirements of Cell Proliferation. Science 324: 1029-1033.
• Döme, Balázs Hendrix, Mary JC, Paku, Sändor Tóvári, József, Timár, Jozsef (2013) Biological perspectives. Alternative Vascularization Mechanisms in Cancer. Pathology and Therapeutic implications. Am J Path.
• Folkman J (1971) Tumor angiogenesis: therapeutic implications. N Engl J Med 285: 1182-1186. [crossref]
• Compton, Duane (2013) Mechanism of chromosomal instability in human tumor cells. Geisel School of Medicine at Dartmouth, Hanover, USA.
• Solis-Herrera, A Ashraf, Ghulam M, Arias-Esparza, MC Solis-Arias, et al. (2015) Biological Activities of QIAPI 1 as Melanin Precursor and its therapeutic effects in Wistar Rats exposed to Arsenic Poisoning. CNS Agen in Med Chem 2: 99-109.

Abstract

In chronic lymphocytic leukemia, overexpression of CTLA-4 may be associated with a good outcome, whereas the Wnt/β-catenin-regulated transcription factor LEF1 is a pro-survival factor and is markedly overexpressed compared to normal B cells. In this study, peripheral blood B cells from 20 patients with CLL were purified and a strong correlation between gene expression levels of CTLA-4 and LEF-1 was found. This suggests that CTLA-4 expression in CLL may be a target of Wnt/β-catenin signalling.

Keywords:

CLL; CTLA-4; Wnt/β-catenin pathway; LEF1; CD38

Highlights

Percentage surface expression of CD38 and CTLA-4 and gene expression levels of CTLA-4, CCND1, LEF1 and STAT3 were measured in 20 patients with chronic lymphocytic leukemic. A strong positive correlation was found between gene expression levels of CTLA-4 and LEF-1.

Targeting of the Wnt/β-catenin pathway in CLL may result in unwanted effects on CTLA-4 expression and function.

Introduction

Chronic lymphocytic leukemia (CLL) is a clonal proliferation of mature CD5+ CD19+ CD23+ B lymphocytes, characterized by progressive accumulation of leukemic cells in peripheral blood, bone marrow and lymphoid tissues. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4, CD152) is a member of the CD28 receptor family and is mainly expressed on CD4+ T-cells. In CLL, overexpression of the CTLA-4 gene is associated with lower CD38-expression and, therefore, perhaps a better outcome [1]. CLL cells also exhibit aberrantly active Wnt signaling and Wnt/β-catenin-regulated transcription factor lymphoid enhancer binding factor-1 (LEF1) has been shown to be a pro-survival factor in CLL [2]. In this study, we wished to further investigate the relationship between CD38 and CTLA-4 in CLL and their potential relationship with transcription factors LEF1, signal transducer and activator of transcription 3 (STAT3) and cyclin D1. In purified peripheral blood B cells from 20 patients with CLL, a strong positive correlation between gene expression levels of CTLA-4 and LEF1 was found, suggesting that CTLA-4 expression in CLL may well be a target of Wnt/β-catenin signalling.

Material and methods

After ethical approval and signed written consent, 20 patients with CLL (9 previously treated, 11 untreated) donated peripheral blood for this study. No patient had active therapy for CLL in the 3 months prior to blood donation. All patients had FISH analysis performed. CD19+ B lymphocytes were isolated using a magnetic bead separation technique (Invitrogen-Dynabeads). The percentage surface expression of CD38 and CTLA-4 was measured by flow cytometry. Total RNA was isolated from the B cells by the RNeasy Mini Kit (QIAGEN). Gene expression levels of CTLA-4, cyclin D1 (CCND1), LEF1 and STAT3 were measured using RT-PCR (ABI 7500 Fast-Applied Biosystems). GAPDH was used as a reference gene. Statistical analyses of data were performed using Spearman rank correlation and Mann-Whitney U tests. Differences of P < 0.05 were considered statistically significant.

Results

Median (range) CD19+ B cell purity was 93.8 (84.8-98.5) %, with CD19+ B cell purity > 90% in 19/20 cases. Median (interquartile range) percentage surface expression of CD38 and CTLA-4 was 8.36 (26.45) % and 43.32 (50.22) % respectively. Median (range, interquartile range) ∆CT gene expression levels of CCND1, CTLA-4, LEF1 and STAT3 were 11.89 (1.81), 4.79 (2.35), 4.82 (0.89) and 9.12 (0.75) respectively. Gene expression of LEF1 showed significant positive correlations with gene expression levels of CTLA-4 (rs=0.572, p=0.008), CCND1 (rs=0.61, p=0.004) and STAT3 (rs=0.587, p=0.006). There was also a significant positive correlation between gene expression of CCND1 and of STAT3 (rs =0.486, p=0.03). No significant correlations were found between percentage surface expression of CTLA-4 and gene expression levels of either CTLA-4 or of LEF1. Although we found a negative correlation between percentage surface expression of CTLA-4 and CD38, this was not statistically significant. Comparing untreated and previously treated patients or comparing patients with poor risk cytogenetics (17p or 11q deletions: n = 6) to those without, there was no significant difference in gene expression levels of CTLA-4, CCND1, LEF1 and STAT3 or in surface expression of CTLA-4 and CD38.

Discussion

The Wnt signalling pathway has been shown to be activated in CLL cells and uncontrolled Wnt/β-catenin signalling contributes to defective apoptosis in CLL [3]. Importantly, Wnt pathway activation leads to upregulation of β-catenin and subsequently LEF1 activation, which is markedly overexpressed in CLL compared to normal B cells [4] and appears to play an essential role in the leukaemogenesis of CLL [2]. Furthermore, cyclin D1, a downstream target of LEF-1, is overexpressed in CLL. Targeting of LEF-1 has been shown to induce apoptosis in CLL cells both in vitro and in vivo [5].

In CLL, CTLA-4 expression is higher on the leukemic cells that on their normal B cell counterparts. A recent study has shown that CTLA-4 inhibits the proliferation/survival of CLL cells via regulation of the expression/activation of STAT1, NFATC2, Fos, Myc and Bcl-2 [6] and CTLA-4 blockade induces pro-survival signals in leukemic cells from CLL patients exhibiting high CTLA-4 expression [7]. However, CTLA-4 expression was also found to be the most highly induced gene following treatment with recombinant Wnt-3a in melanoma cell lines and CTLA-4 expression appeared to be directly regulated by the Wnt/β-catenin pathway as the β-catenin responsiveness of CTLA-4 promoter region required a T-cell factor-1/LEF-1 consensus site [8]. In our study, CTLA-4 and LEF-1 gene expression levels were strongly correlated, suggesting that CTLA-4 expression in CLL may well also be a direct target of Wnt/β-catenin signalling. Although the relationship between CTLA-4 and the Wnt/β-catenin pathway in CLL requires further study, the findings of this study suggest that targeting of the Wnt/β-catenin pathway in CLL may result in unwanted effects on CTLA-4 expression and function.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflicts of interest: none

References

1. Joshi AD, Hegde GV, Dickinson JD (2007) ATM, CTLA4, MNDA and HEM1 in high versus low CD38 expressing B-cell chronic lymphocytic leukemia. Clin Cancer Res 13: 5295–5304. [Crossref]
2. Gutierrez Jr A, Tschumper RC, Wu X (2010) LEF-1 is a prosurvival factor in chronic lymphocytic leukemia and is expressed in the preleukemic state of monoclonal B-cell lymphocytosis. Blood 116: 2975–2983. [Crossref]
3. Lu D, Zhao Y, Tawatao R (2004) Activation of the Wnt signaling pathway in chronic lymphocytic leukemia. Proc Natl Acad Sci USA. 101: 3118–3123. [Crossref]
4. Gandhirajan RK, Poll-Wolbeck SJ, Gehrke I, Kreuzer KA (2010) Wnt/β-catenin/LEF-1 signaling in chronic lymphocytic leukemia (CLL): a target for current and potential therapeutic options. Curr Cancer Drug Targets 10: 716–727. [Crossref]
5. Gandhirajan RK, Staib PA, Minke K (2010) Small molecule inhibitors of Wnt/β-carenin/Lef-1 signaling induces apoptosis in chronic lymphocytic leukemia cells in vitro and in vivo. Neoplasia 12: 326–335. [Crossref]
6. Mittal AK, Chaturvedi NK, Rohlfsen RA (2013) Role of CTLA4 in the proliferation and survival of chronic lymphocytic leukemia. PLoS ONE 8: e70352. [Crossref]
7. Ciszak L, Frydecka I, Wolowiec D, Szteblich A, Kosmaczewska A (2016) Patients with chronic lymphocytic leukemia (CLL) differ in the pattern of CTLA-4 expression on CLL cells: the possible implications for immunotherapy with CTLA-4 blocking antibody. Tumour Biol 37: 4143–4157. [Crossref]
8. Shah KV, Chien AJ, Yee C, Moon RT (2008) CTLA-4 is a direct target of Wnt/β-catenin signaling and is expressed in human melanoma tumors. J Invest Dermatol 128: 2870–2879. [Crossref]

DOI: 10.31038/CST.2017246

Commentary

The “biogenea pharmaceuticals” being the first biotech company in Southeast Europe, has the task to systematically promote the Science of Pharmaceutical Biotechnology, including the production of advanced therapy medicinal products ATMPs as defined by the European Medicines/Agency EMEA. On behalf of “biogenea pharmaceuticals” and its scientific board we are pleased to announce our innovative biotechnological services Nanostemogenea™ for the collection, processing, cryopreservation and therapeutic use of our advanced biotechnology applications utilizing next generation biocompatible complexes of stem, immune cells and nanoparticles in the field of magnetic guided regenerative medicine. Our GENEA cells ™ are totally safe and obtained from different sources of the human body for autologous cell therapy purposes in Renaissance medicine.

Biogenea Pharmaceuticals™ is the first Inter-Balkan Pharmaceutical Biotechnology Company since leading the way since 2005 in Red Biotechnology applications, in Cryobiology and in Autologous Cellular Therapy of Degenerative Diseases (cardiological diseases, neurological conditions and metabolic disorders).

Biogenea Pharmaceuticals focuses

• On the collection, processing, cryopreservation and cGMP (according to Good Manufacturing Practice) production -for solely autologous use – of cellular therapeutical solutions from blood (bone marrow, peripheral blood, cord blood) or blood compounds for human use.
• In collaboration with Regenetech on stem cell expansion technologies, which were created in the research laboratories of NASA (National Aeronautics and Space Administration).
• On the cGMP production of advanced medicinal products (1394/2007/ΕC) for solely autologous use from skin, dental pulp, cord tissue). (In preclinical-research phase: 2008-2009).
• On certified genetic analyses in collaboration with International Referral Centers.
• On copyright protection according to the American and/or European Copyright Agency. The “biogeneapharmaceuticals” is the only European bank that has been recognized by the European Medicines Agency EMEA as a pharmaceutical company and has the possibility of cryopreservation of hematopoietic stem cells and conducting clinical trials (EMEA/Qualification of an enterprise as an SME – GrigoriadisBros – Biogenea- Cellgenea Ltd, with registration number: EMA/SME/084/10).

In “biogenea pharmaceuticals” taking advantage of the unique properties of super paramagnetic nanoparticles as this high magnetic moment and susceptibility but also the existence yperparagnitikis behavioural development multitude biological applications agglomerate formation with these petidika molecular and different kinds STEM CELL for autologous use in the Renaissance medicine.

Biogenea Pharmaceuticals Ltd combines excellent trained scientific personnel with the most modern techniques, concerning cell expansion, that are used today in the field of biotechnology and have been developed by NASA. Thus our company is able to verge into the demanding field of clinical trials concerning stem cell treatments. One can understand the big potential of these stem cells to play a role in Regenerative Medicine by looking at the amount of clinical trials all over the world that use stem cells as a way to treat an increasing number of diseases in a supportive manner.

The “biogenea pharmaceuticals” provides the following services:

• Cardiogenea™: Autologous intracoronary, intraarterial or intracardiac injection of autologous blood, bone marrow and heart stem cells derive active cardiopoeitic spheres coated with superparamagneticnanoparticles for the restoration of myocardial infarction.
• Dendrigenea™: Autologous adjuvant immune hybridomatic therapy for cancer patients using advanced complexes of superparamagnetic iron oxide nanoparticles coated mature dendritic and dendritic tumor cell fusions as a cancer ‘cell vaccine’.
• Cartigenea™: Autologous and exvivo expanded chondrogenic cells coated with super paramagnetic nanoparticles for their intended use in Cartilage Defects.

Biogenea Pharmaceuticals Ltd is in the process of developing novel therapies for the effective treatment of several diseases. These therapies make use of Stem Cells (ADSCs) and a special class of nanoparticles, referred to as Super paramagnetic Iron Oxide Nanoparticles (SPIONs). SPIONs have recently attracted the interest of the scientific community, due to several attributes, such as their paramagnetism and their potential us in a number of therapeutic approaches. SPIONs are small synthetic γ-Fe2O3 (maghemite), Fe3O4 (magnetite) or α-Fe2O3 (hermatite) particles with a core ranging from 10 nm to 100 nm in diameter. In addition, mixed oxides of iron with transition metal ions such as copper, cobalt, nickel, and manganese, are known to exhibit superparamagneticproperties and also fall into the category of SPIONs. However, magnetite and maghemite nanoparticles are the most widely used SPIONs in various biomedical applications. SPIONs have an organic or inorganic coating so that they can be tolerated by cells and tissues.

Our Personalized cancer therapy is determined by the needs and specificities of a particular oncology patient to provide the optimum desired therapeutic effect with minimal toxicity, strengthening the immune system of cancer patients with the use TARGETED complex dendritic cells and super-paramagnetic nanoparticles in simultaneous increase the concentration of magnetic particles into the tumor.

Our technique serves the philosophy of personalized cancer treatment based on tumor pluripotent cells (cancer stem cells), consisting of four specialized areas in the prevention, diagnosis, treatment and rehabilitation of cancer patients while capitalizing on the therapeutic effects of magnetic hyperthermia the implementation of magnetic field for the local heating of the cancer cells with a view to the disaster.

Preventive Cryopreservation Insurance of Primordial Cells & Therapeutic Applications

• Continuous update and prompt training of interested Donors and Clinicians of Public and Private Health Centers and Hospitals, about the progenitor cells applications.
• Immediate availability of the stored samples 365 days a year, 7 days a week, 24 hours a day.
• Complete attunement with the specifications set by the European Accreditation Organization for cellular therapy (FACT/JACIE/ NETCORD), the American Association of Blood Banks (AABB) and the relevant Greek.
• Presidential Decretal 2004/23 for the tissue/cell banks, with the use of cGMP methods.
• Control of Plasticity of progenitor cells based on the detailed Validation Master Plan of the Standard Operating Procedures (SOPs) regarding their hematopoietic origin (Methocult).
• Fully Automated Viability Control of the cryopreserved progenitor cells with automatic luminometric device, which increases the reproducibility and the accuracy of the results in contrast to the common laboratory techniques for detecting dead cells microscopically (Trypan Blue staining).
• Validated Molecular Diagnostics service provision (detection of HIV1/2, HBV, HCV, CMV, Syphillis, Toxoplasma) applying Real- Time PCR technology (Roche LightCycler).
• Validated ex-vivo cellular and/or tissue expansion of the recently processed cells/tissues, as well as of the cryopreserved cell/ tissues in advanced technology Bioreactors, created in the NASA research laboratories, offering unique micro-gravity conditions in alternating electromagnetic field! Cellgenea is the UNIQUE company in Greece able to expand and to use ex-vivo expanded cord blood and adult progenitor cells for therapeutic reasons and clinical trials thanks to the relevant know-how transfer from the research laboratories of NASA and Regenetech Biotechnology Company.
• Storing of progenitor cells in two-chambered bags and cryovials in complete (24 hours a day) controlled and automated liquid nitrogen tanks.
• Continuous control and cross-tracking of the cryopreserved samples with validated LIS-ERP software which ensures the ability to track down and to identify the sample during all the steps of its supply, the processing, the control, the storage and the distribution. The tracking is also used for controlling and identifying all the relevant data about the products and the materials that come in contact with these samples (2004/23/ΕΚ).
• Immediate availability and distribution of the sample inside a validated cryotank, in case of therapeutic application.
• Strict security concerning personal data, confidentiality and safety according to the Personal Data Protection Agency.
• Complete bacteriological and serological control of the samples using the automatic analyzers BacT/ALERT and Architect i1000 without any extra rate.
• Life insurance provided to all the members of the different programs of preventive cryopreservation (Cellgenea, Dentogenea, Dermigenea, Angiogenea, DΝΑgenea, Neurogenea, Cardiogenea) in cooperation with insurance companies.
• DNA & pharmacogenetic control services for personalized treatment without side effects.
• Offer of validated prenatal and DNA/RNA control for chromosomal anomalies screening in a variety of biological materials (adult peripheral blood, cord blood, tissue biopsies) in cooperation with the Technological Park of Ioannina.
• Continuous training and schooling of the scientific staff on the new

Nanuristemogenea™

Advanced biotechnology applications utilizing next generation biocompatible complexes of stem and hybridoma immune cells with nanoparticles in the field of magnetic guided regenerative medicine.

The “biogenea pharmaceuticals” being the first biotech company in Southeast Europe, has the task to systematically promote the Science of Pharmaceutical Biotechnology, including the production of advanced therapy medicinal products ATMPs as defined by the European Medicines/Agency EMEA.

On behalf of “biogenea pharmaceuticals” and its scientific board we are pleased to annpounce our innovative biotechnological services Nanostemogenea™ for the collection, processing, cryopreservation and therapeutic use of our advanced biotechnology applications utilizing next generation biocompatible complexes of stem, immune cells and nanoparticles in the field of magnetic guided regenerative medicine. Our GENEAcells™ are totally safe and obtained from different sources of the human body for autologous cell therapy purposes in Renaissance medicine.

Biogenea Pharmaceuticals™ is the first Inter-Balkan Pharmaceutical Biotechnology Company since leading the way since 2005 in Red Biotechnology applications, in Cryobiology and in Autologous Cellular Therapy of Degenerative Diseases (cardiological diseases, neurological conditions and metabolic disorders).

Biogenea Pharmaceuticals focuses

• On the collection, processing, cryopreservation and cGMP (according to Good Manufacturing Practice) production -for solely autologous use – of cellular therapeutical solutions from blood (bone marrow, peripheral blood, cord blood) or blood compounds for human use.
• In collaboration with Regenetech on stem cell expansion technologies, which were created in the research laboratories of NASA (National Aeronautics and Space Administration).
• On the cGMP production of advanced medicinal products (1394/2007/ΕC) for solely autologous use from skin, dental pulp, cord tissue). (In preclinical-research phase: 2008-2009).
• On certified genetic analyses in collaboration with International Referral Centers.
• On copyright protection according to the American and/or European Copyright Agency. The “biogeneapharmaceuticals” is the only European bank that has been recognized by the European Medicines Agency EMEA as a pharmaceutical company and has the possibility of cryopreservation of hematopoietic stem cells and conducting clinical trials (EMEA/Qualification of an enterprise as an SME – GrigoriadisBros – Biogenea- Cellgenea Ltd, with registration number: EMA/SME/084/10).
• In “biogenea pharmaceuticals” taking advantage of the unique properties of super paramagnetic nanoparticles as this high magnetic moment and susceptibility but also the existence yperparagnitikis behavioral development multitude biological applications agglomerate formation with these petidika molecular and different kinds STEM CELL for autologous use in the Renaissance medicine.
• Biogenea Pharmaceuticals Ltd combines excellent trained scientific personnel with the most modern techniques, concerning cell expansion, that are used today in the field of biotechnology and have been developed by NASA. Thus our company is able to verge into the demanding field of clinical trials concerning stem cell treatments. One can understand the big potential of these stem cells to play a role in Regenerative Medicine by looking at the amount of clinical trials all over the world that use stem cells as a way to treat an increasing number of diseases in a supportive manner.
• The “biogenea pharmaceuticals” provides the following services:
• Cardiogenea™: Autologous intracoronary, intra-arterial or intracardiac injection of autologous blood, bone marrow and heart stem cells derive active cardiopoeitic spheres coated with super paramagnetic nanoparticles for the restoration of myocardial infarction. 
• Dendrigenea™: Autologous adjuvant immune hybridomatic therapy for cancer patients using advanced complexes of super paramagnetic iron oxide nanoparticles coated mature dendritic and dendritic tumor cell fusions as a cancer ‘cell vaccine’. 
• Cartigenea™: Autologous and ex-vivo expanded chondrogenic cells coated with super paramagnetic nanoparticles for their intended use in Cartilage Defects.

Biogenea Pharmaceuticals Ltd is in the process of developing novel therapies for the effective treatment of several diseases. These therapies make use of Stem Cells (ADSCs) and a special class of nanoparticles, referred to as Super paramagnetic Iron Oxide Nanoparticles (SPIONs). SPIONs have recently attracted the interest of the scientific community, due to several attributes, such as their paramagnetism and their potential us in a number of therapeutic approaches. SPIONs are small synthetic γ-Fe2O3 (maghemite), Fe3O4 (magnetite) or α-Fe2O3 (hermatite) particles with a core ranging from 10 nm to 100 nm in diameter. In addition, mixed oxides of iron with transition metal ions such as copper, cobalt, nickel, and manganese, are known to exhibit super paramagnetic properties and also fall into the category of SPIONs. However, magnetite and maghemite nanoparticles are the most widely used SPIONs in various biomedical applications. SPIONs have an organic or inorganic coating so that they can be tolerated by cells and tissues.

Our Personalized cancer therapy is determined by the needs and specificities of a particular oncology patient to provide the optimum desired therapeutic effect with minimal toxicity, strengthening the immune system of cancer patients with the use TARGETED complex dendritic cells and super-paramagnetic nanoparticles in simultaneous increase the concentration of magnetic particles into the tumor.

Our technique serves the philosophy of personalized cancer treatment based on tumor pluripotent cells (cancer stem cells), consisting of four specialized areas in the prevention, diagnosis, treatment and rehabilitation of cancer patients while capitalizing on the therapeutic effects of magnetic hyperthermia the implementation of magnetic field for the local heating of the cancer cells with a view to the disaster.

Preventive Cryopreservation Insurance of Primordial Cells & Therapeutic Applications

• Continuous update and prompt training of interested Donors and Clinicians of Public and Private Health Centers and Hospitals, about the progenitor cells applications.
• Immediate availability of the stored samples 365 days a year, 7 days a week, 24 hours a day.
• Complete attunement with the specifications set by the European Accreditation Organization for cellular therapy (FACT/JACIE/ NETCORD), the American Association of Blood Banks (AABB) and the relevant Greek Presidential Decretal 2004/23 for the tissue/cell banks, with the use of cGMP methods.
• Control of Plasticity of progenitor cells based on the detailed Validation Master Plan of the Standard Operating Procedures (SOPs) regarding their hematopoietic origin (Methocult).
• Fully Automated Viability Control of the cryopreserved progenitor cells with automatic luminometric device, which increases the reproducibility and the accuracy of the results in contrast to the common laboratory techniques for detecting dead cells microscopically (Trypan Blue staining).
• Validated Molecular Diagnostics service provision (detection of HIV1/2, HBV, HCV, CMV, Syphillis, Toxoplasma) applying Real- Time PCR technology (Roche Light Cycler).
• Validated ex-vivo cellular and/or tissue expansion of the recently processed cells/tissues, as well as of the cryopreserved cell/tissues in advanced technology Bioreactors, created in the NASA research laboratories, offering unique micro-gravity conditions in alternating electromagnetic field! Cellgenea is the UNIQUE company in Greece able to expand and to use ex-vivo expanded cord blood and adult progenitor cells for therapeutic reasons and clinical trials thanks to the relevant know-how transfer from the research laboratories of NASA and Regenetech Biotechnology Company.
• Storing of progenitor cells in two-chambered bags and cryovials in complete (24 hours a day) controlled and automated liquid nitrogen tanks.
• Continuous control and cross-tracking of the cryopreserved samples with validated LIS-ERP software which ensures the ability to track down and to identify the sample during all the steps of its supply, the processing, the control, the storage and the distribution. The tracking is also used for controlling and identifying all the relevant data about the products and the materials that come in contact with these samples (2004/23/ΕΚ).
• Immediate availability and distribution of the sample inside a validated cryotank, in case of therapeutic application.
• Strict security concerning personal data, confidentiality and safety according to the Personal Data Protection Agency.
• Complete bacteriological and serological control of the samples using the automatic analyzers BacT/ALERT and Architect i1000 without any extra rate.
• Life insurance provided to all the members of the different programs of preventive cryopreservation (Cellgenea, Dentogenea, Dermigenea, Angiogenea, DΝΑgenea, Neurogenea, Cardiogenea) in cooperation with insurance companies.
• DNA & pharmacogenetic control services for personalized treatment without side effects.
• Offer of validated prenatal and DNA/RNA control for chromosomal anomalies screening in a variety of biological materials (adult peripheral blood, cord blood, tissue biopsies) in cooperation with the Technological Park of Ioannina.
• Continuous training and schooling of the scientific staff on the new scientific developments.