Mesenchymal Stem Cells and Their Role in Neurodegenerative Diseases.

Mesenchymal stem cells (MSCs) have garnered significant interest in the field of regenerative medicine for their ability to potentially treat various diseases, especially neurodegenerative disorders [...].

Analogies and Differences Between Dental Stem Cells: Focus on Secretome in Combination with Scaffolds in Neurological Disorders.

Mesenchymal stem cells (MSCs) are well known for their beneficial effects, differentiation capacity and regenerative potential. Dental-derived MSCs (DSCs) are more easily accessible and have a non-invasive isolation method rather than MSCs isolated from other sources (umbilical cord, bone marrow, and adipose tissue). In addition, DSCs appear to have a relevant neuro-regenerative potential due to their neural crest origin. However, it is now known that the beneficial effects of MSCs depend, at least in part, on their secretome, referring to all the bioactive molecules (neurotrophic factors) released in the conditioned medium (CM) or in the extracellular vesicles (EVs) in particular exosomes (Exos). In this review, we described the similarities and differences between various DSCs. Our focus was on the secretome of DSCs and their applications in cell therapy for neurological disorders. For neuro-regenerative purposes, the secretome of different DSCs has been tested. Among these, the secretome of dental pulp stem cells and stem cells from human exfoliated deciduous teeth have been the most widely studied. Both CM and Exos obtained from DSCs have been shown to promote neurite outgrowth and neuroprotective effects as well as their combination with scaffold materials (to improve their functional integration in the tissue). For these reasons, the secretome obtained from DSCs in combination with scaffold materials may represent a promising tissue engineering approach for neuroprotective and neuro-regenerative treatments.

Lipid rafts mediate multilineage differentiation of human dental pulp-derived stem cells (DPSCs).

Cell outer membranes contain glycosphingolipids and protein receptors, which are integrated into glycoprotein domains, known as lipid rafts, which are involved in a variety of cellular processes, including receptor-mediated signal transduction and cellular differentiation process. In this study, we analyzed the lipidic composition of human Dental Pulp-Derived Stem Cells (DPSCs), and the role of lipid rafts during the multilineage differentiation process. The relative quantification of lipid metabolites in the organic fraction of DPSCs, performed by Nuclear Magnetic Resonance (NMR) spectroscopy, showed that mono-unsaturated fatty acids (MUFAs) were the most representative species in the total pool of acyl chains, compared to polyunsatured fatty acids (PUFAs). In addition, the stimulation of DPSCs with different culture media induces a multilineage differentiation process, determining changes in the gangliosides pattern. To understand the functional role of lipid rafts during multilineage differentiation, DPSCs were pretreated with a typical lipid raft affecting agent (MßCD). Subsequently, DPSCs were inducted to differentiate into osteoblast, chondroblast and adipoblast cells with specific media. We observed that raft-affecting agent MßCD prevented AKT activation and the expression of lineage-specific mRNA such as OSX, PPARγ2, and SOX9 during multilineage differentiation. Moreover, this compound significantly prevented the tri-lineage differentiation induced by specific stimuli, indicating that lipid raft integrity is essential for DPSCs differentiation. These results suggest that lipid rafts alteration may affect the signaling pathway activated, preventing multilineage differentiation.

10th Anniversary of Biomedicines-Advances in Mesenchymal Stem Cells.

Mesenchymal stromal cells (MSCs) are non-specialized adult stem cells (ASCs), cells that reproduce to provide specific cytotypes [...].

Role of a Novel Heparanase Inhibitor on the Balance between Apoptosis and Autophagy in U87 Human Glioblastoma Cells.

BACKGROUND: Heparanase (HPSE) is an endo-ß-glucuronidase that cleaves heparan sulfate side chains, leading to the disassembly of the extracellular matrix, facilitating cell invasion and metastasis dissemination. In this research, we investigated the role of a new HPSE inhibitor, RDS 3337, in the regulation of the autophagic process and the balance between apoptosis and autophagy in U87 glioblastoma cells. METHODS: After treatment with RDS 3337, cell lysates were analyzed for autophagy and apoptosis-related proteins by Western blot. RESULTS: We observed, firstly, that LC3II expression increased in U87 cells incubated with RDS 3337, together with a significant increase of p62/SQSTM1 levels, indicating that RDS 3337 could act through the inhibition of autophagic-lysosomal flux of LC3-II, thereby leading to accumulation of lipidated LC3-II form. Conversely, the suppression of autophagic flux could activate apoptosis mechanisms, as revealed by the activation of caspase 3, the increased level of cleaved Parp1, and DNA fragmentation. CONCLUSIONS: These findings support the notion that HPSE promotes autophagy, providing evidence that RDS 3337 blocks autophagic flux. It indicates a role for HPSE inhibitors in the balance between apoptosis and autophagy in U87 human glioblastoma cells, suggesting a potential role for this new class of compounds in the control of tumor growth progression.

The Importance of Stem Cells Isolated from Human Dental Pulp and Exfoliated Deciduous Teeth as Therapeutic Approach in Nervous System Pathologies.

Despite decades of research, no therapies are available to halt or slow down the course of neuro-degenerative disorders. Most of the drugs developed to fight neurodegeneration are aimed to alleviate symptoms, but none has proven adequate in altering the course of the pathologies. Cell therapy has emerged as an intriguing alternative to the classical pharmacological approach. Cell therapy consists of the transplantation of stem cells that can be obtained from various embryonal and adult tissues. Whereas the former holds notable ethical issue, adult somatic stem cells can be obtained without major concerns. However, most adult stem cells, such as those derived from the bone marrow, are committed toward the mesodermal lineage, and hence need to be reprogrammed to induce the differentiation into the neurons. The discovery of neural crest stem cells in the dental pulp, both in adults' molar and in baby teeth (dental pulp stem cells and stem cells from human exfoliated deciduous teeth, respectively) prompted researchers to investigate their utility as therapy in nervous system disorders. In this review, we recapitulate the advancements on the application of these stem cells in preclinical models of neurodegenerative diseases, highlighting differences and analogies in their maintenance, differentiation, and potential clinical application.

Different Chronic Stress Paradigms Converge on Endogenous TDP43 Cleavage and Aggregation.

The TAR-DNA binding protein (TDP43) is a nuclear protein whose cytoplasmic inclusions are hallmarks of Amyotrophic Lateral Sclerosis (ALS). Acute stress in cells causes TDP43 mobilization to the cytoplasm and its aggregation through different routes. Although acute stress elicits a strong phenotype, is far from recapitulating the years-long aggregation process. We applied different chronic stress protocols and described TDP43 aggregation in a human neuroblastoma cell line by combining solubility assays, thioflavin-based microscopy and flow cytometry. This approach allowed us to detect, for the first time to our knowledge in vitro, the formation of 25 kDa C-terminal fragment of TDP43, a pathogenic hallmark of ALS. Our results indicate that chronic stress, compared to the more common acute stress paradigm, better recapitulates the cell biology of TDP43 proteinopathies. Moreover, we optimized a protocol for the detection of bona fide prions in living cells, suggesting that TDP43 may form amyloids as a stress response.

Mitotic phosphorylation of Tau/MAPT modulates cell cycle progression in prostate cancer cells.

PURPOSE: Tau/MAPT (microtubule associated protein tau) protein is actively studied for the pathologic consequences of its aberrant proteostasis in central nervous system leading to neurodegenerative diseases. Besides its ability to generate insoluble toxic oligomers, Tau homeostasis has attracted attention for its involvement in the formation of the mitotic spindle. This evidence, in association with the description of Tau expression in extra-neuronal tissues, and mainly in cancer tissues, constitutes the rationale for a more in-depth investigation of Tau role also in neoplastic diseases. METHODS: In our study, we investigated the expression of phosphorylated Tau in prostate cancer cell lines with particular focus on the residue Thr231 present in microtubule binding domain. RESULTS: The analysis of prostate cancer cells synchronized with nocodazole demonstrated that the expression of Tau protein phosphorylated at residue Thr231 is restricted to G2/M cell cycle phase. The phosphorylated form was unable to bind tubulin and it does not localize on mitotic spindle. As demonstrated by the use of specific inhibitors, the phosphorylation status of Tau is under the direct control of cdk5 and PP2A, while cdk1 activation was able to exert an indirect control. These mechanisms were also active in cells treated with docetaxel, where counteracting the expression of the dephosphorylated form, by kinase inhibition or protein silencing, determined resistance to drug toxicity. CONCLUSIONS: We hypothesize that phosphorylation status of Tau is a key marker for G2/M phase in prostate cancer cells and that the forced modulation of Tau phosphorylation can interfere with the capacity of cell to efficiently progress through G2/M phase.

Dental Pulp Stem Cells (DPSCs) and Tissue Regeneration: Mechanisms Mediated by Direct, Paracrine, or Autocrine Effects.

Among mesenchymal stem cells, dental pulp stem cells (DPSCs) were discovered most recently [...].

Gangliosides and Their Role in Multilineage Differentiation of Mesenchymal Stem Cells.

Gangliosides (GGs) are a glycolipid class present on Mesenchymal Stem Cells (MSCs) surfaces with a critical appearance role in stem cell differentiation, even though their mechanistic role in signaling and differentiation remains largely unknown. This review aims to carry out a critical analysis of the predictive role of gangliosides as specific markers of the cellular state of undifferentiated and differentiated MSCs, towards the osteogenic, chondrogenic, neurogenic, and adipogenic lineage. For this reason, we analyzed the role of GGs during multilineage differentiation processes of several types of MSCs such as Umbilical Cord-derived MSCs (UC-MSCs), Bone Marrow-derived MSCs (BM-MSCs), Dental Pulp derived MSCs (DPSCs), and Adipose derived MSCs (ADSCs). Moreover, we examined the possible role of GGs as specific cell surface markers to identify or isolate specific stem cell isotypes and their potential use as additional markers for quality control of cell-based therapies.

Anticancer effects of ABTL0812, a clinical stage drug inducer of autophagy-mediated cancer cell death, in glioblastoma models.

Background: Glioblastoma multiforme (GBM) is the most malignant adult brain tumor. Current standard of care treatments have very limited efficacy, being the patients´ overall survival 14 months and the 2-year survival rate less than 10%. Therefore, the treatment of GBM is an urgent unmet clinical need. Methods: The aim of this study was to investigate in vitro and in vivo the potential of ABTL0812, an oral anticancer compound currently in phase II clinical stage, as a novel therapy for GBM. Results: We showed that ABTL0812 inhibits cell proliferation in a wide panel of GBM cell lines and patient-derived glioblastoma stem cells (GSCs) with half maximal inhibitory concentrations (IC50s) ranging from 15.2 µM to 46.9 µM. Additionally, ABTL0812 decreased GSCs neurosphere formation. GBM cells aggressiveness is associated with a trans-differentiation process towards a less differentiated phenotype known as proneural to mesenchymal transition (PMT). ABTL0812 was shown to revert PMT and induce cell differentiation to a less malignant phenotype in GBM cell lines and GSCs, and consequently reduced cell invasion. As previously shown in other cancer types, we demonstrated that the molecular mechanism of action of ABTL0812 in glioblastoma involves the inhibition of Akt/mTORC1 axis by overexpression of TRIB3, and the activation of endoplasmic reticulum (ER) stress/unfolded protein response (UPR). Both actions converge to induce autophagy-mediated cell death. ABTL0812 anticancer efficacy was studied in vivo using subcutaneous and orthotopic intra-brain xenograft tumor models. We demonstrated that ABTL0812 impairs tumor growth and increases disease-free survival and overall survival of mice. Furthermore, the histological analysis of tumors indicated that ABTL0812 decreases angiogenesis. Finally, we investigated the combination of ABTL0812 with the standard of care treatments for GBM radiotherapy and temozolomide in an orthotopic model, detecting that ABTL0812 potentiates the efficacy of both treatments and that the strongest effect is obtained with the triple combination of ABTL0812+radiotherapy+temozolomide. Conclusions: Overall, the present study demonstrated the anticancer efficacy of ABTL0812 as single agent and in combination with the GBM standard of care treatments in models of glioblastoma and supports the clinical investigation of ABTL0812 as a potential novel therapy for this aggressive brain tumor type.

Antioxidant Properties of Cerium Oxide Nanoparticles Prevent Retinal Neovascular Alterations In Vitro and In Vivo.

In this study, we investigated whether cerium oxide nanoparticles (CeO2-NPs), a promising antioxidant nanomaterial, may contrast retinal vascular alterations induced by oxidative damage in vitro and in vivo. For the in vivo experiments, the light damage (LD) animal model of Age-Related Macular Degeneration (AMD) was used and the CeO2-NPs were intravitreally injected. CeO2-NPs significantly decreased vascular endothelial growth factor (VEGF) protein levels, reduced neovascularization in the deep retinal plexus, and inhibited choroidal sprouting into the photoreceptor layer. The in vitro experiments were performed on human retinal pigment epithelial (ARPE-19) cells challenged with H2O2; we demonstrated that CeO2-NPs reverted H2O2-induced oxidative stress-dependent effects on this cell model. We further investigated the RPE-endothelial cells interaction under oxidative stress conditions in the presence or absence of CeO2-NPs through two experimental paradigms: (i) treatment of human umbilical vein endothelial cells (HUVECs) with conditioned media from ARPE-19 cells, and (ii) coculture of ARPE-19 and HUVECs. In both experimental conditions, CeO2-NPs were able to revert the detrimental effect of H2O2 on angiogenesis in vitro by realigning the level of tubule formation to that of the control. Altogether, our results indicate, for the first time, that CeO2-NPs can counteract retinal neovascularization and may be a new therapeutic strategy for the treatment of wet AMD.

Hypoxia Induces DPSC Differentiation versus a Neurogenic Phenotype by the Paracrine Mechanism.

As previously described by several authors, dental pulp stem cells (DPSCs), when adequately stimulated, may acquire a neuronal-like phenotype acting as a favorable source of stem cells in the generation of nerves. Besides, it is known that hypoxia conditioning is capable of stimulating cell differentiation as well as survival and self-renewal, and that multiple growth factors, including Epidermal Growth factor (EGF) and basic fibroblast growth factor (bFGF), are often involved in the induction of the neuronal differentiation of progenitor cells. In this work, we investigated the role of hypoxia in the commitment of DPSCs into a neuronal phenotype. These cells were conditioned with hypoxia (O2 1%) for 5 and 16 days; subsequently, we analyzed the proliferation rate and morphology, and tested the cells for neural and stem markers. Moreover, we verified the possible autocrine/paracrine role of DPSCs in the induction of neural differentiation by comparing the secretome profile of the hypoxic and normoxic conditioned media (CM). Our results showed that the hypoxia-mediated DPSC differentiation was time dependent. Moreover, conditioned media (CM derived from DPSCs stimulated by hypoxia were able, in turn, to induce the neural differentiation of SH-SY5Y neuroblastoma cells and undifferentiated DPSCs. In conclusion, under the herein-mentioned conditions, hypoxia seems to favor the differentiation of DPSCs into neuron-like cells. In this way, we confirm the potential clinical utility of differentiated neuronal DPSCs, and we also suggest the even greater potential of CM-derived-hypoxic DPSCs that could more readily be used in regenerative therapies.

Anti-ß2-GPI Antibodies Induce Endothelial Cell Expression of Tissue Factor by LRP6 Signal Transduction Pathway Involving Lipid Rafts.

In this study we analyzed whether anti-ß2-GPI antibodies from patients with APS induce the endothelial cell expression of Tissue Factor (TF) by a LRP6 signal transduction pathway involving lipid rafts. HUVEC were stimulated with affinity purified anti-ß2-GPI antibodies. Both LRP6 and ß-catenin phosphorylation, as well as TF expression, were evaluated by western blot. Results demonstrated that triggering with affinity purified anti-ß2-GPI antibodies induced LRP6 phosphorylation with consequent ß-catenin activation, leading to TF expression on the cell surface. Interestingly, the lipid rafts affecting agent methyl-ß-cyclodextrin as well as the LRP6 inhibitor Dickkopf 1 (DKK1) partially reduced the anti-ß2-GPI antibodies effect, indicating that the anti-ß2-GPI effects on TF expression may depend on a signalling transduction pathway involving both lipid rafts and LRP6. An interaction between ß2-GPI, LRP6 and PAR-2 within these microdomains was demonstrated by gradient fractionation and coimmunoprecipitation experiments. Thus, anti-ß2-GPI antibodies react with their target antigen likely associated to LRP6 and PAR-2 within plasma membrane lipid rafts of the endothelial cell. Anti-ß2-GPI binding triggers ß-catenin phosphorylation, leading to a procoagulant phenotype characterized by TF expression. These findings deal with a novel signal transduction pathway which provides new insight in the APS pathogenesis, improving the knowledge of valuable therapeutic target(s).

The Botanical Drug PBI-05204, a Supercritical CO2 Extract of Nerium Oleander, Is Synergistic With Radiotherapy in Models of Human Glioblastoma.

Glioblastoma multiforme (GBM) is the most common as well as one of the most malignant types of brain cancer. Despite progress in development of novel therapies for the treatment of GBM, it remains largely incurable with a poor prognosis and a very low life expectancy. Recent studies have shown that oleandrin, a unique cardiac glycoside from Nerium oleander, as well as a defined extract (PBI-05204) that contains this molecule, inhibit growth of human glioblastoma, and modulate glioblastoma patient-derived stem cell-renewal properties. Here we demonstrate that PBI-05204 treatment leads to an increase in vitro in the sensitivity of GBM cells to radiation in which the main mechanisms are the transition from autophagy to apoptosis, enhanced DNA damage and reduced DNA repair after radiotherapy (RT) administration. The combination of PBI-05204 with RT was associated with reduced tumor progression evidenced by both subcutaneous as well as orthotopic implanted GBM tumors. Collectively, these results reveal that PBI-05204 enhances antitumor activity of RT in preclinical/murine models of human GBM. Given the fact that PBI-05204 has already been examined in Phase I and II clinical trials for cancer patients, its efficacy when combined with standard-of-care radiotherapy regimens in GBM should be explored.

Carbamylation of ß2-glycoprotein I generates new autoantigens for antiphospholipid syndrome: a new tool for diagnosis of 'seronegative' patients.

OBJECTIVES: Antiphospholipid syndrome (APS) is a prothrombotic condition defined by recurrent thrombosis, pregnancy complications and circulating antiphospholipid antibodies (aPL), including anti-ß2-glycoprotein I (ß2-GPI). In clinical practice it is possible to find patients with APS persistently negative for the aPL tests according to Sydney criteria ('seronegative APS', SN-APS). Recently, several autoimmune responses have been described as a consequence of post-translational modifications of their target autoantigens. This study was undertaken to test carbamylated-ß2-GPI (Carb-ß2-GPI) as a new autoantigen of APS. METHODS: ß2-GPI was carbamylated by potassium cyanate and used to investigate its effect on monocyte-derived dendritic cell (moDC) phenotype and function. Sera from 114 SN-APS patients, 60 APS, 20 patients with RA, 20 non-APS thrombosis and 50 healthy donors were analysed for anti-Carb-ß2-GPI by ELISA. RESULTS: Carb-ß2-GPI is able to activate moDCs, inducing upregulation of CD80, CD86 and CD40, activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase and nuclear factor-κB, and IL-12p70 release. Serological results showed that both 37/114 SN-APS (32.46%) and 23/60 APS (38.33%) patients resulted positive for anti-Carb-ß2-GPI. Interestingly, SN-APS patients who tested positive for anti-Carb-ß2-GPI showed a higher prevalence of thrombocytopenia (P = 0.04, likelihood positive ratio of 3.9). CONCLUSION: Data obtained from both functional tests on moDCs and immunological approaches prompted identification of Carb-ß2-GPI as a 'new' antigenic target in APS. In particular, anti-Carb-ß2-GPI revealed a potential usefulness in identification of a significant proportion of SN-APS patients. Moreover, since patients who tested positive for anti-Carb-ß2-GPI reported a high risk of thrombocytopenia, this test may be considered a suitable approach in the clinical evaluation of SN-APS.

ATX-101, a Peptide Targeting PCNA, Has Antitumor Efficacy Alone or in Combination with Radiotherapy in Murine Models of Human Glioblastoma.

Cell proliferation requires the orchestrated actions of a myriad of proteins regulating DNA replication, DNA repair and damage tolerance, and cell cycle. Proliferating cell nuclear antigen (PCNA) is a master regulator which interacts with multiple proteins functioning in these processes, and this makes PCNA an attractive target in anticancer therapies. Here, we show that a cell-penetrating peptide containing the AlkB homolog 2 PCNA-interacting motif (APIM), ATX-101, has antitumor activity in a panel of human glioblastoma multiforme (GBM) cell lines and patient-derived glioma-initiating cells (GICs). Their sensitivity to ATX-101 was not related to cellular levels of PCNA, or p53, PTEN, or MGMT status. However, ATX-101 reduced Akt/mTOR and DNA-PKcs signaling, and a correlation between high Akt activation and sensitivity for ATX-101 was found. ATX-101 increased the levels of γH2AX, DNA fragmentation, and apoptosis when combined with radiotherapy (RT). In line with the in vitro results, ATX-101 strongly reduced tumor growth in two subcutaneous xenografts and two orthotopic GBM models, both as a single agent and in combination with RT. The ability of ATX-101 to sensitize cells to RT is promising for further development of this compound for use in GBM.

Prions and Neurodegenerative Diseases: A Focus on Alzheimer's Disease.

Specific protein misfolding and aggregation are mechanisms underlying various neurodegenerative diseases such as prion disease and Alzheimer's disease (AD). The misfolded proteins are involved in prions, amyloid-ß (Aß), tau, and α-synuclein disorders; they share common structural, biological, and biochemical characteristics, as well as similar mechanisms of aggregation and self-propagation. Pathological features of AD include the appearance of plaques consisting of deposition of protein Aß and neurofibrillary tangles formed by the hyperphosphorylated tau protein. Although it is not clear how protein aggregation leads to AD, we are learning that the cellular prion protein (PrPC) plays an important role in the pathogenesis of AD. Herein, we first examined the pathogenesis of prion and AD with a focus on the contribution of PrPC to the development of AD. We analyzed the mechanisms that lead to the formation of a high affinity bond between Aß oligomers (AßOs) and PrPC. Also, we studied the role of PrPC as an AßO receptor that initiates an AßO-induced signal cascade involving mGluR5, Fyn, Pyk2, and eEF2K linking Aß and tau pathologies, resulting in the death of neurons in the central nervous system. Finally, we have described how the PrPC-AßOs interaction can be used as a new potential therapeutic target for the treatment of PrPC-dependent AD.

Multiple Antitumor Molecular Mechanisms Are Activated by a Fully Synthetic and Stabilized Pharmaceutical Product Delivering the Active Compound Sulforaphane (SFX-01) in Preclinical Model of Human Glioblastoma.

Frequent relapses and therapeutic resistance make the management of glioblastoma (GBM, grade IV glioma), extremely difficult. Therefore, it is necessary to develop new pharmacological compounds to be used as a single treatment or in combination with current therapies in order to improve their effectiveness and reduce cytotoxicity for non-tumor cells. SFX-01 is a fully synthetic and stabilized pharmaceutical product containing the α-cyclodextrin that delivers the active compound 1-isothiocyanato-4-methyl-sulfinylbutane (SFN) and maintains biological activities of SFN. In this study, we verified whether SFX-01 was active in GBM preclinical models. Our data demonstrate that SFX-01 reduced cell proliferation and increased cell death in GBM cell lines and patient-derived glioma initiating cells (GICs) with a stem cell phenotype. The antiproliferative effects of SFX-01 were associated with a reduction in the stemness of GICs and reversion of neural-to-mesenchymal trans-differentiation (PMT) closely related to epithelial-to-mesenchymal trans-differentiation (EMT) of epithelial tumors. Commonly, PMT reversion decreases the invasive capacity of tumor cells and increases the sensitivity to pharmacological and instrumental therapies. SFX-01 induced caspase-dependent apoptosis, through both mitochondrion-mediated intrinsic and death-receptor-associated extrinsic pathways. Here, we demonstrate the involvement of reactive oxygen species (ROS) through mediating the reduction in the activity of essential molecular pathways, such as PI3K/Akt/mTOR, ERK, and STAT-3. SFX-01 also reduced the in vivo tumor growth of subcutaneous xenografts and increased the disease-free survival (DFS) and overall survival (OS), when tested in orthotopic intracranial GBM models. These effects were associated with reduced expression of HIF1α which, in turn, down-regulates neo-angiogenesis. So, SFX-01 may have potent anti-glioma effects, regulating important aspects of the biology of this neoplasia, such as hypoxia, stemness, and EMT reversion, which are commonly activated in this neoplasia and are responsible for therapeutic resistance and glioma recurrence. SFX-01 deserves to be considered as an emerging anticancer agent for the treatment of GBM. The possible radio- and chemo sensitization potential of SFX-01 should also be evaluated in further preclinical and clinical studies.