This study investigated the three-dimensional (3D) cellular interactions and tunneling nanotubes (TNTs) during fetal mouse skin regeneration on embryonic days 13 (E13) and 15 (E15). We aimed to understand spatial relationships among cell types involved in skin regeneration and assess the potential role of TNTs. Full-thickness skin incisions were performed in E13 and E15 embryos. Wound sites were collected, embedded in epoxy resin, processed for 3D reconstruction (1 μm thickness sections), and subjected to whole-mount immunostaining. We conducted in vitro co-culture experiments with fetal macrophages and fibroblasts to observe TNT formation. To assess the effect of TNTs on skin regeneration, an inhibiting agent (cytochalasin B) was administered to amniotic fluid. Results revealed that E13 epidermal keratinocytes interacted with dermal fibroblasts and macrophages, facilitating skin regrowth. TNT structures were observed at the E13-cell wound sites, among macrophages, and between macrophages and fibroblasts, confirmed through in vitro co-culture experiments. In vitro and utero cytochalasin B administration hindered those formation and inefficient skin texture regeneration at E13 wound sites. This emphasizes the necessity of 3D cellular interactions between epidermal and dermal cells during skin regeneration in mouse embryos at E13. The prevalence of TNT structures indicated their involvement in achieving complete skin texture restoration.

Immunotherapy represents an innovative approach to cancer treatment, based on activating the body\'s own immune system to combat tumor cells. Among various immunotherapy strategies, dendritic cell vaccines hold a special place due to their ability to activate T-lymphocytes, key players in cellular immunity, and direct them to tumor cells. In this study, the influence of dendritic cells processed with tumor-derived vesicles on the viability of melanoma cells in vitro was investigated. Dendritic cells were loaded with tumor-derived vesicles, after which they were used to activate T-cells. The study demonstrated that such modified T-cells exhibit high activity against melanoma cells, leading to a decrease in their viability. Our analysis highlights the potential efficacy of this approach in developing immunotherapy against melanoma. These results provide new prospects for further research and the development of antitumor strategies based on the mechanisms of T-lymphocyte activation using tumor-derived vesicles.

Among perinatal stem cells of the umbilical cord, human Wharton\'s jelly mesenchymal stem cells (hWJ-MSCs) are of great interest for cell-based therapy approaches in regenerative medicine, showing some advantages over other MSCs. In fact, hWJ-MSCs, placed between embryonic and adult MSCs, are not tumorigenic and are harvested with few ethical concerns. Furthermore, these cells can be easily cultured in vitro, maintaining both stem properties and a high proliferative rate for several passages, as well as trilineage capacity of differentiation. Recently, it has been demonstrated that cytoskeletal organization influences stem cell biology. Among molecules able to modulate its dynamics, Cytochalasin B (CB), a cyto-permeable mycotoxin, influences actin microfilament polymerization, thus affecting several cell properties, such as the ability of MSCs to differentiate towards a specific commitment. Here, we investigated for the first time the effects of a 24 h-treatment with CB at different concentrations (0.1-3 μM) on hWJ-MSCs. CB influenced the cytoskeletal organization in a dose-dependent manner, inducing changes in cell number, proliferation, shape, and nanomechanical properties, thus promoting the osteogenic commitment of hWJ-MSCs, as confirmed by the expression analysis of osteogenic/autophagy markers.

Multiple sclerosis (MS) is an incurable, progressive chronic autoimmune demyelinating disease. Therapy for MS is based on slowing down the processes of neurodegeneration and suppressing the immune system of patients. MS is accompanied by inflammation, axon-degeneration and neurogliosis in the central nervous system. One of the directions for a new effective treatment for MS is cellular, subcellular, as well as gene therapy. We investigated the therapeutic potential of adipose mesenchymal stem cell (ADMSC) derived, cytochalasin B induced artificial microvesicles (MVs) expressing nerve growth factor (NGF) on a mouse model of multiple sclerosis experimental autoimmune encephalomyelitis (EAE). These ADMSC-MVs-NGF were tested using histological, immunocytochemical and molecular genetic methods after being injected into the tail vein of animals on the 14th and 21st days post EAE induction. ADMSC-MVs-NGF contained the target protein inside the cytoplasm. Their injection into the caudal vein led to a significant decrease in neurogliosis at the 14th and 21st days post EAE induction. Artificial ADMSC-MVs-NGF stimulate axon regeneration and can modulate gliosis in the EAE model.

At present, there is an increasing interest in the potential role of extracellular vesicles (EVs), acting as multi-signal messengers of the tumor stroma, in the development and progression of tumor. Tumor cell-derived EVs are considered a potential vector for the targeted delivery of antitumor agents due to the ability to fuse with parental cells through endocytosis and release their contents into the cytoplasm of the recipient cell. Tumor cell-derived EVs could be also used for priming immune cells and therapeutic vaccine development. It is also known that mesenchymal stem cells (MSCs) have a tropism toward tumor niches. It is believed that MSC migration to the tumor is due to its inflammatory signaling. Presumably, with the accumulation of MSCs at tumor sites, these cells differentiate into pericytes or tumor-associated fibroblasts, thereby forming a supporting tumor growth microenvironment. However, besides the ability to promote tumor progression, MSCs can also suppress its growth by inhibiting proliferation and cell cycle progression, and angiogenesis. Thus, the further studies of the MSC role in TME and MSC interaction with other cells of the tumor stroma, including through EVs, are of particular interest. To increase the yield of vesicles the isolation method based on pharmacological disorganization of the actin cytoskeleton induced by treating with cytochalasin B was used in this study. In this investigation the interaction of SH-SY5Y neuroblastoma cell-derived membrane vesicles, obtained using cytochalasin B (CIMVs), with human bone marrow-derived MSCs was analyzed using imaging flow cytometry. Using transmission electron microscopy, it was shown that CIMVs have a size similar to that of natural microvesicles, which is 100-1000 nm. Using imaging flow cytometry, it was shown that after 24 h of co-cultivation 6% of the MSCs contained a large number of CIMVs, and 42% of the MSCs contained a small amount of CIMVs. Cultivation of MSCs with SH-SY5Y cell-derived CIMVs also induced dose-dependent decrease in the expression of CD markers typical for MSCs. Thus, the internalization of SH-SY5Y cell-derived CIMVs within MSCs and the ability of the CIMVs to modulate immunophenotype of the recipient cells were shown. However, further studies are required to determine the effect of CIMVs on pro- or antioncogenic phenotype and function of MSCs.

Human adenoviruses (HAdV) cause a variety of infections in human hosts, from self-limited upper respiratory tract infections in otherwise healthy people to fulminant pneumonia and death in immunocompromised patients. Many HAdV enter polarized epithelial cells by using the primary receptor, the Coxsackievirus and adenovirus receptor (CAR). Recently published data demonstrate that a potent neutrophil (PMN) chemoattractant, interleukin-8 (IL-8), stimulates airway epithelial cells to increase expression of the apical isoform of CAR (CAREx8), which results in increased epithelial HAdV type 5 (HAdV5) infection. However, the mechanism for PMN-enhanced epithelial HAdV5 transduction remains unclear. In this manuscript, the molecular mechanisms behind PMN mediated enhancement of epithelial HAdV5 transduction are characterized using an MDCK cell line that stably expresses human CAREx8 under a doxycycline inducible promoter (MDCK-CAREx8 cells). Contrary to our hypothesis, PMN exposure does not enhance HAdV5 entry by increasing CAREx8 expression nor through activation of non-specific epithelial endocytic pathways. Instead, PMN serine proteases are responsible for PMN-mediated enhancement of HAdV5 transduction in MDCK-CAREx8 cells. This is evidenced by reduced transduction upon inhibition of PMN serine proteases and increased transduction upon exposure to exogenous human neutrophil elastase (HNE). Furthermore, HNE exposure activates epithelial autophagic flux, which, even when triggered through other mechanisms, results in a similar enhancement of epithelial HAdV5 transduction. Inhibition of F-actin with cytochalasin D partially attenuates PMN mediated enhancement of HAdV transduction. Taken together, these findings suggest that HAdV5 can leverage innate immune responses to establish infections.

The main advantage of extracellular vesicles (EVs) as a drug carrier system is their low immunogenicity and internalization by mammalian cells. EVs are often considered a cell-specific delivery system, but the production of preparative amounts of EVs for therapeutic applications is challenging due to their laborious isolation and purification procedures. Alternatively, mimetic vesicles prepared from the cellular plasma membrane can be used in the same way as natural EVs. For example, a cytoskeleton-destabilizing agent, such as cytochalasin B, allows the preparation of membrane vesicles by a series of centrifugations. Here, we prepared cytochalasin-B-inducible nanovesicles (CINVs) of various cellular origins and studied their tropism in different mammalian cells. We observed that CINVs derived from human endometrial mesenchymal stem cells exhibited an enhanced affinity to epithelial cancer cells compared to myeloid, lymphoid or neuroblastoma cancer cells. The dendritic cell-derived CINVs were taken up by all studied cell lines with a similar efficiency that differed from the behavior of DC-derived EVs. The ability of cancer cells to internalize CINVs was mainly determined by the properties of recipient cells, and the cellular origin of CINVs was less important. In addition, receptor-mediated interactions were shown to be necessary for the efficient uptake of CINVs. We found that CINVs, derived from late apoptotic/necrotic cells (aCINVs) are internalized by in myelogenous (K562) 10-fold more efficiently than CINVs, and interact much less efficiently with melanocytic (B16) or epithelial (KB-3-1) cancer cells. Finally, we found that CINVs caused a temporal and reversible drop of the rate of cell division, which restored to the level of control cells with a 24 h delay.

Standard toxicity tests might not be fully adequate for evaluating nanomaterials since their unique features are also responsible for unexpected interactions. The in vitro cytokinesis-block micronucleus (CBMN) test is recommended for genotoxicity testing, but cytochalasin-B (Cyt-B) may interfere with nanoparticles (NP), leading to inaccurate results. Our objective was to determine whether Cyt-B could interfere with MN induction by TiO2 NP in human SH-SY5Y cells, as assessed by CBMN test. Cells were treated for 6 or 24 h, according to three treatment options: co-treatment with Cyt-B, post-treatment, and delayed co-treatment. Influence of Cyt-B on TiO2 NP cellular uptake and MN induction as evaluated by flow cytometry (FCMN) were also assessed. TiO2 NP were significantly internalized by cells, both in the absence and presence of Cyt-B, indicating that this chemical does not interfere with NP uptake. Dose-dependent increases in MN rates were observed in CBMN test after co-treatment. However, FCMN assay only showed a positive response when Cyt-B was added simultaneously with TiO2 NP, suggesting that Cyt-B might alter CBMN assay results. No differences were observed in the comparisons between the treatment options assessed, suggesting they are not adequate alternatives to avoid Cyt-B interference in the specific conditions tested.

Owing to their self-renewal and multi-lineage differentiation capability, mesenchymal stem cells (MSCs) hold enormous potential in regenerative medicine. A prerequisite for a successful MSC therapy is the rigorous investigation of their function after in vitro cultivation. Damages introduced to mitochondria during cultivation adversely affect MSCs function and can determine their fate. While it has been shown that microtubules and vimentin intermediate filaments are important for mitochondrial dynamics and active mitochondrial transport within the cytoplasm of MSCs, the role of filamentous actin in this process has not been fully understood yet. To gain a deeper understanding of the interdependence between mitochondrial function and the cytoskeleton, we applied cytochalasin B to disturb the filamentous actin-based cytoskeleton of MSCs. In this study we combined conventional functional assays with a state-of-the-art oxygen sensor-integrated microfluidic device to investigate mitochondrial function. We demonstrated that cytochalasin B treatment at a dose of 16 μM led to a decrease in cell viability with high mitochondrial membrane potential, increased oxygen consumption rate, disturbed fusion and fission balance, nuclear extrusion and perinuclear accumulation of mitochondria. Treatment of MSCs for 48 h ultimately led to nuclear fragmentation, and activation of the intrinsic pathway of apoptotic cell death. Importantly, we could show that mitochondrial function of MSCs can efficiently recover from the damage to the filamentous actin-based cytoskeleton over a period of 24 h. As a result of our study, a causative connection between the filamentous actin-based cytoskeleton and mitochondrial dynamics was demonstrated.

Two new isosarcophine derivatives, cherbonolides M (1) and N (2), were further isolated from a Formosan soft coral Sarcophyton cherbonnieri. The planar structure and relative configuration of both compounds were established by the detailed analysis of the IR, MS, and 1D and 2D NMR data. Further, the absolute configuration of both compounds was determined by the comparison of CD spectra with that of isosarcophine (3). Notably, cherbonolide N (2) possesses the unique cembranoidal scaffold of tetrahydrooxepane with the 12,17-ether linkage fusing with a γ-lactone. In addition, the assay for cytotoxicity of both new compounds revealed that they showed to be noncytotoxic toward the proliferation of A549, DLD-1, and HuCCT-1 cell lines. Moreover, the anti-inflammatory activities of both metabolites were carried out by measuring the N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced generation of superoxide anion and elastase release in the primary human neutrophils. Cherbonolide N (2) was found to reduce the generation of superoxide anion (20.6 ± 6.8%) and the elastase release (30.1 ± 3.3%) in the fMLF/CB-induced human neutrophils at a concentration of 30 μM.