Brain targeted delivery of rapamycin using transferrin decorated nanostructured lipid carriers

Introduction: Recent studies showed that rapamycin, as a mammalian target of rapamycin (mTOR) inhibitor, could have beneficial therapeutic effects for the central nervous system (CNS) related diseases. However, the immunosuppressive effect of rapamycin as an adverse effect, the low water solubility, and the rapid in vivo degradation along with the blood-brain barrier-related challenges restricted the clinical use of this drug for brain diseases. To overcome these drawbacks, a transferrin (Tf) decorated nanostructured lipid carrier (NLC) containing rapamycin was designed and developed. Methods: Rapamycin-loaded cationic and bare NLCs were prepared using solvent diffusion and sonication method and well characterized. The optimum cationic NLCs were physically decorated with Tf. For in vitro study, the MTT assay and intracellular uptake of nanoparticles on U-87 MG glioblastoma cells were assessed. The animal biodistribution of nanoparticles was evaluated by fluorescent optical imaging. Finally, the in vivo effect of NLCs on the immune system was also studied. Results: Spherical NLCs with small particle sizes ranging from 120 to 150 nm and high entrapment efficiency of more than 90%, showed ≥80% cell viability. More importantly, Tf-decorated NLCs in comparison with bare NLCs, showed a significantly higher cellular uptake (97% vs 60%) after 2 hours incubation and further an appropriate brain accumulation with lower uptake in untargeted tissue in mice. Surprisingly, rapamycin-loaded NLCs exhibited no immunosuppressive effect. Conclusion: Our findings proposed that the designed Tf-decorated NLCs could be considered as a safe and efficient carrier for targeted brain delivery of rapamycin which may have an important value in the clinic for the treatment of neurological disorders.

Avocado Seeds Relieve Oxidative Stress-Dependent Nephrotoxicity but Enhance Immunosuppression Induced by Cyclosporine in Rats

Cyclosporine A’s (CsA) immunosuppressive effect makes it an ideal drug for organ transplantation. However, CsA’s uses are restricted due to its side effects. We investigated the effects of avocado seed (AvS) powder on CsA-induced nephrotoxicity and immunosuppression in rats. The injection of CsA (5 mg/kg, subcutaneously, for 10 days) increased serum levels of creatinine, uric acid, and urea, and the renal levels of the malondialdehyde. It decreased creatinine clearance and the renal activity of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) and Na+/K+ ATPase. The administration of CsA also significantly downregulated the renal expression of interferon-gamma, tumor necrosis factor-alpha, interleukin 1 beta, monocyte chemotactic protein 1, intercellular adhesion molecule-1, and vascular cell adhesion molecule 1 genes, and increased renal DNA damage. Histopathological examination confirmed the biochemical and molecular alterations that accompanied CsA nephrotoxicity. All CsA-induced deleterious effects, except immunosuppression, were ameliorated by feeding rats on a basal diet supplemented with 5% AvS powder for 4 weeks. Importantly, AvS also maximized CsA’s immunosuppressive effect. These findings suggest a potential ameliorative effect of AvS on CsA-induced nephrotoxicity, and AvS enhances CsA’s immunosuppressive effect. Therefore, AvS might be used in combination with CsA in transplantation treatment to relieve the CsA-induced nephrotoxicity.

Nephrotoxicity Risk of Cyclophosphamide in Lupus Model

Cyclophosphamide is one of the standard therapies for lupus, especially lupus nephritis based on its immunosuppressive effect. However, cyclophosphamide is also known as a nephrotoxic agent. Therefore, this research was aimed to measure the effect of cyclophosphamide at the dose that comparable to the human dose of 1 mg/kg BW on the kidney of lupus mice induced by means of 2,6,10,14-tetramethylpentadecane (TMPD). In this research, the IL-6 as a pro-inflammatory cytokine was tested by using flow cytometry method. In addition, the structural damage of the kidney tissues was assessed by means of Moroni’s kidney organ scoring method for lupus. The result showed that cyclophosphamide reduced the IL-6 significantly with the value of 36.72±22.79% for the TMPD-treated group; 32.59±9.97% for the cyclophosphamide group; and 30.25±4.48% for the naïve group. Moreover, the damages of the kidney tissues on the cyclophosphamide group were more severe than the TMPD-treated group. In conclusion, despite its anti-inflammatory effect which is useful for lupus, cyclophosphamide has a severe nephrotoxic effect which harms the patient. The effects may be a cause of the long interval use of cyclophosphamide. It can be a consideration for the further research and the next revision of the guideline for lupus nephritis treatment.

Loureirin B Exerts its Immunosuppressive Effects by Inhibiting STIM1/Orai1 and KV1.3 Channels

Loureirin B (LrB) is a constituent extracted from traditional Chinese medicine Resina Draconis. It has broad biological functions and an impressive immunosuppressive effect that has been supported by numerous studies. However, the molecular mechanisms underlying Loureirin B-induced immune suppression are not fully understood. We previously reported that Loureirin B inhibited KV1.3 channel, calcium ion (Ca2+) influx, and interleukin-2 (IL-2) secretion in Jurkat T cells. In this study, we applied CRISPR/Cas9 to edit KV1.3 coding gene KCNA3 and successfully generated a KV1.3 knockout (KO) cell model to determine whether KV1.3 KO was sufficient to block the Loureirin B-induced immunosuppressive effect. Surprisingly, we showed that Loureirin B could still inhibit Ca2+ influx and IL-2 secretion in the Jurkat T cells in the absence of KV1.3 although KO KV1.3 reduced about 50% of Ca2+ influx and 90% IL-2 secretion compared with that in the wild type cells. Further experiments showed that Loureirin B directly inhibited STIM1/Orai1 channel in a dose-dependent manner. Our results suggest that Loureirin B inhibits Ca2+ influx and IL-2 secretion in Jurkat T cells by inhibiting both KV1.3 and STIM1/Orai1 channels. These studies also revealed an additional molecular target for Loureirin B-induced immunosuppressive effect, which makes it a promising leading compound for treating autoimmune diseases.

Immunosuppressive effect of voacamine from Voacanga africana Stapf based on SPRi experiment

Purpose: To investigate the affinity of a bis-indole alkaloid - voacamine from Voacanga Africana Stapf for IL-2Rα - and its immunosuppressive effect on concanavalin A-induced T cell proliferation and lipopolysaccharide -induced B cell proliferation in vitro. Methods: Surface plasmon resonance imaging (SPRi) was used to screen the target protein of voacamine, while CCK-8 kit was used to evaluate cytotoxicity. Mitogen-induced proliferation assay was carried out to assess the inhibitory effect of voacamine on Con A-induced T cell proliferation and LPSinduced B cell proliferation. The binding characteristics of voacamine were investigated using a binding model with IL-2Rα constructed based on molecular docking simulation. Results: Voacamine had a high-affinity for IL-2Rα with an equilibrium dissociation constant (KD) of 1.85×10-8 M. Cytotoxicity data showed that voacamine did not exhibit cytotoxicity at concentrations lower than 0.32 µM. However, it exerted significant immunosuppressive effect on B cells at a lower concentration, but had no influence on proliferation of T cells. Autodock results indicate that voacamine has a good interaction with the enzyme active site. Conclusion: Voacamine and its analogues exert influence on the immune system.

A procedure for in vitro evaluation of the immunosuppressive effect of mouse mesenchymal stem cells on activated T cell proliferation

Background Mesenchymal stem/stromal cells (MSC) represent adult cells with multipotent capacity. Besides their capacity to differentiate into multiple lineages in vitro and in vivo, increasing evidence points towards the immunomodulatory capacity of these cells, as an important feature for their therapeutic power. Although not included in the minimal criteria established by the International Society for Cellular Therapy as a defining MSC attribute, demonstration of the immunomodulatory capacity of MSC can be useful for the characterization of these cells before being considered MSC. Methods Here we present a simple and reliable protocol by which the immunosuppressive effect of mouse bone marrow-derived MSC can be evaluated in vitro. It is based on the measuring of the proliferation of activated T cells cultured in direct contact with irradiated MSC. Results Our results showed that mouse MSC have a dose-dependent inhibitory effect on activated T cell proliferation, which can be quantified as a percentage of maximum proliferation. Our data shows that batch-to-batch variability can be determined within one or multiple experiments, by extracting the area under curve of T cell proliferation plotted against the absolute number of MSC in co-culture. Conclusions The validation of the immunosupressive capacity of MSC could be added to the characterization of the cells before being used in various MSC-based approaches to treat immunological diseases. Our results showed that mouse MSC have a dose-dependent inhibitory effect on activated T cell proliferation. The immunosuppressive properties of MSC vary between batches, but not between different passages of the same batch.