In vitro assessment of mesenchymal stem cells immunosuppressive potential in multiple sclerosis patients

Mesenchymal stem cells (MSC) are promising for multiple sclerosis (MS) treatment. However, clinical results remain controversial, and no criteria are available for predicting the efficiency of MSC therapy. Using an in vitro model of lymphocytes and MSC cocultivation we revealed that the Index of MSC Suppression of myelin-induced memory T cells proliferation was stronger than that of PHA-stimulated proliferation and inversely correlated with patients’ EDSS score. In vitro expression of CD119 (IFNGR1) in mitogen/myelin-stimulated T cells increased in the presence of MSC being inversely correlated with T-lymphocytes proliferation. The Index of MSC Suppression and CD119 expression in T-lymphocytes may be useful when assessing MSC immunosuppressive potential in MS patients.     

Corticospinal excitability in patients with anoxic,traumatic, and non-traumatic diffuse brain injury

BackgroundTranscranial magnetic stimulation (TMS) have been frequently used to explore changes in motor cortex excitability in stroke and traumatic brain injury, while the extent of motor cortex reorganization in patients with diffuse non-traumatic brain injury remains largely unknown.Objective/HypothesisIt was hypothesized that the motor cortex excitability would be decreased and would correlate to the severity of brain injury and level of functioning in patients with anoxic, traumatic, and non-traumatic diffuse brain injury.MethodsTMS was applied to primary motor cortices of 19 patients with brain injury (5 traumatic and 14 non-traumatic causes; on average four months after insult), and 9 healthy controls. The test parameters included resting motor threshold (RMT), short intracortical inhibition (SICI), intracortical facilitation (ICF), and short latency afferent inhibition (SAI). Excitability parameters were correlated to the severity of brain injury measured with Glasgow Coma Scale and the level of functioning assessed using the Ranchos Los Amigos Levels of Cognitive Functioning Assessment Scale and Functional Independence Measure.ResultsThe patient group revealed a significantly decreased SICI and SAI compared to healthy controls with the amount of SICI correlated significantly to the severity of brain injury. Other electrophysiological parameters did not differ between the groups and did not exhibit any significant relationship with clinical functional scores.ConclusionsThe present study demonstrated the impairment of the cortical inhibitory circuits in patients with brain injury of traumatic and non-traumatic aetiology. Moreover, the significant correlation was found between the amount of SICI and the severity of brain injury.     

Enhancement of radiation effect on cancer cells by gold-pHLIP

Previous research has shown that gold nanoparticles can increase the effectiveness of radiation on cancer cells. Improved radiation effectiveness would allow lower radiation doses given to patients, reducing adverse effects; alternatively, it would provide more cancer killing at current radiation doses. Damage from radiation and gold nanoparticles depends in part on the Auger effect, which is very localized; thus, it is important to place the gold nanoparticles on or in the cancer cells. In this work, we use the pH-sensitive, tumor-targeting agent, pH Low-Insertion Peptide (pHLIP), to tether 1.4-nm gold nanoparticles to cancer cells. We find that the conjugation of pHLIP to gold nanoparticles increases gold uptake in cells compared with gold nanoparticles without pHLIP, with the nanoparticles distributed mostly on the cellular membranes. We further find that gold nanoparticles conjugated to pHLIP produce a statistically significant decrease in cell survival with radiation compared with cells without gold nanoparticles and cells with gold alone. In the context of our previous findings demonstrating efficient pHLIP-mediated delivery of gold nanoparticles to tumors, the obtained results serve as a foundation for further preclinical evaluation of dose enhancement.Gold is an inert and generally nontoxic material with unique properties suitable for many applications such as cancer diagnosis and treatment (1–7). Nanometer-size gold particles have recently been shown to increase radiation damage to tumors (2, 8–11). With enhanced radiation, the same level of tumor killing can be had with less radiation exposure for a patient, reducing the adverse effects of radiation treatments. Similarly, more tumor killing can be had while using the same levels of radiation that are currently given.The increase in radiation effectiveness with gold nanoparticles is largely a result of two causes. First, gold is capable of absorbing radiation at a significantly higher rate than tissue: up to about 100 times more for keV energies (2). Second, gold nanoparticles that interact with radiation can release extra electrons via the Auger effect. The Auger effect occurs when an atom releases electrons postionization. Multiple electrons, called Auger electrons, can be released per ionization. The Auger electrons usually have low enough energy that their effect is localized to the area surrounding the gold nanoparticles; see, for example, figure 1 in ref. 11. Thus, it is very important to effectively deliver gold nanoparticles to cancer cells in tumors and to locate them near DNA or other vital cellular structures and components.Specific delivery can be accomplished by conjugating gold (or other nanoparticles) to antibodies or ligands that target overexpressed proteins on cancer cell surfaces; this approach has been actively explored for many years for the delivery of small molecules. However, several recent studies have raised serious questions about the efficacy of targeting ligands on nanoparticle accumulation in tumor tissues. Multiple reports have shown that targeted nanoparticles did not lead to increased tumor accumulation over nontargeted controls, although increased cellular uptake was observed in each case (12–14). In addition, histologic studies showed that antibodies conjugated with gold nanoparticles do not penetrate deeply into tumors, but mostly stain peripheral tumor regions (15). The direct injection of micrometer-sized gold particles does not lead to tumor targeting, as particles stayed only at the injection site and were not able to diffuse even within a tumor, hindering tumor coverage (16).Our approach is based on the targeting of tumor acidity, which correlates with tumor malignancy (17–19). The pH-sensitive targeting agents we are developing are based on the action of a family of pHLIPs (pH Low-Insertion Peptides), which can “sense” acidity at the surface of cancer cells and deliver diagnostic and therapeutic molecules to tumors of different origins (20–25). It was shown that pHLIP can promote fusion of liposomes with cancer cells and cellular delivery of various payloads (26, 27), including small gold nanoparticles (26). Recently, pHLIP was successfully used for the targeting of various nanoparticles to tumors and other acidic diseased tissue (28–31).pHLIP has also been used to mediate pH-controlled delivery of both 13-nm water-soluble gold nanoparticles coated with luminescent europium into human platelets in vitro (32) and 1.4-nm gold nanoparticles to tumors (33). Intratumoral and i.v. administrations of both demonstrated a significant enhancement of tumor uptake of 1.4-nm gold nanoparticles conjugated with pHLIP. Statistically significant reduction of gold accumulation was observed in acidic tumors and kidney when pH-nonsensitive K-pHLIP was used as a vehicle, suggesting an important role of pH in the pHLIP-mediated targeting of gold nanoparticles.In this work, we made another important step toward clinical application of 1.4-nm gold nanoparticles conjugated with pHLIP. We show that pHLIP can deliver gold to cellular components in a pH-dependent manner and can enhance radiation damage in cells.     

A novel role for thyroid hormone receptor beta in cellular radiosensitivity

Thyroid hormone receptors (THRs) widely govern cell growth, differentiation and metabolism acting in a ligand- and cofactor-dependent manner to modulate tissue-specific gene expression. Given a large variety of genes regulated by THRs and multiplicity of cellular processes potentially influenced by THRs, we addressed the role of THRB (thyroid hormone receptor beta) in cellular radiosensitivity. Wild-type and mutant THRB were overexpressed in several cell lines using an adenovirus-mediated gene delivery and their effects were examined after cell exposure to gamma-rays. Wild-type THRB decreased clonogenic survival of the cell lines with low levels of endogenous THRB, retarded their growth and synergized with radiation in decreasing proliferative potential and promoting cellular senescence. These changes were accompanied by the accumulation of p21 (CDKN1A, CIP1, WAF1) and p16 (CDKN2A, INK4a) inhibitors of cyclin-dependent kinases and by the decrease of Rb (retinoblastoma protein) phosphorylation. Mutant THRB produced a radioprotective effect, attenuated radiation-induced growth inhibition and cellular senescence. The results suggest that THRB may modulate cellular radiosensitivity and stress-induced senescence.     

pH-Sensitive Drug Delivery System Based on Chitin Nanowhiskers–Sodium Alginate Polyelectrolyte Complex

Polyelectrolyte complexes (PECs), based on partially deacetylated chitin nanowhiskers (CNWs) and anionic polysaccharides, are characterized by their variability of properties (particle size, ζ-potential, and pH-sensitivity) depending on the preparation conditions, thereby allowing the development of polymeric nanoplatforms with a sustained release profile for active pharmaceutical substances. This study is focused on the development of hydrogels based on PECs of CNWs and sodium alginate (ALG) for potential vaginal administration that provide controlled pH-dependent antibiotic release in an acidic vaginal environment, as well as prolonged pharmacological action due to both the sustained drug release profile and the mucoadhesive properties of the polysaccharides. The desired hydrogels were formed as a result of both electrostatic interactions between CNWs and ALG (PEC formation), and the subsequent molecular entanglement of ALG chains, and the formation of additional hydrogen bonds. Metronidazole (MET) delivery systems with the desired properties were obtained at pH 5.5 and an CNW:ALG ratio of 1:2. The MET–CNW–ALG microparticles in the hydrogel composition had an apparent hydrodynamic diameter of approximately 1.7 µm and a ζ-potential of −43 mV. In vitro release studies showed a prolonged pH-sensitive drug release from the designed hydrogels; 37 and 67% of MET were released within 24 h at pH 7.4 and pH 4.5, respectively. The introduction of CNWs into the MET–ALG system not only prolonged the drug release, but also increased the mucoadhesive properties by about 1.3 times. Thus, novel CNW–ALG hydrogels are promising carriers for pH sensitive drug delivery carriers.