Fine Tuning Mesenchymal Stromal Cells - Code For Mitigating Kidney Diseases.

Kidney Disease (KD), has a high global prevalence and accounts for one of the most prominent causes of morbidity and mortality in the twenty-first century. Despite the advances in our understanding of its pathophysiology, the only available therapy options are dialysis and kidney transplantation. Mesenchymal stem cells (MSCs) have proven to be a viable choice for KD therapy due to their antiapoptotic, immunomodulatory, antioxidative, and pro-angiogenic activities. However, the low engraftment, low survival rate, diminished paracrine ability, and delayed delivery of MSCs are the major causes of the low clinical efficacy. A number of preconditioning regimens are being tested to increase the therapeutic capabilities of MSCs. In this review, we highlight the various strategies to prime MSCs and their protective effects in kidney diseases.

Mesenchymal Stromal Cells Mediate Clinically Unpromising but Favourable Immune Responses in Kidney Transplant Patients.

BACKGROUND: Allograft rejection postkidney transplantation (KTx) is a major clinical challenge despite increased access to a healthcare system and improvement in immunosuppressive (IS) drugs. In recent years, mesenchymal stromal cells (MSCs) have aroused considerable interest in field of transplantation due to their immunomodulatory and regenerative properties. This study was aimed at investigating safety, feasibility, and immunological effects of autologous MSCs (auto-MSCs) and allogeneic MSCs (allo-MSCs) as a complement to IS drug therapy in KTx patients. METHODS: 10 patients undergoing KTx with a living-related donor were analysed along with 5 patients in the control group. Patients were given auto-MSCs or allo-MSCs at two time points, i.e., one day before transplant (D-0) and 30 days after transplant (D-30) at the rate of 1.0-1.5 × 106 MSCs per kg body weight in addition to immunosuppressants. Patients were followed up for 2 years, and 29 immunologically relevant lymphocyte subsets and 8 cytokines and important biomarkers were analysed at all time points. RESULTS: Patients displayed no signs of discomfort or dose-related toxicities in response to MSC infusion. Flow cytometric analysis revealed an increase in B regulatory lymphocyte populations and nonconventional T regulatory cells and a decrease in T effector lymphocyte proportions in auto-MSC-infused patients. No such favourable immune responses were observed in all MSC-infused patients. CONCLUSION: This study provides evidence that auto-MSCs are safe and well tolerated. This is the first ever report to compare autologous and allogeneic MSC infusion in KTx patients. Importantly, our data demonstrated that MSC-induced immune responses in patients did not completely correlate with clinical outcomes. Our findings add to the current perspective of using MSCs in KTx and explore possibilities through which donor/recipient chimerism can be achieved to induce immune tolerance in KTx patients.

Regulatory B Cells Are Reduced and Correlate With Disease Activity in Primary Membranous Nephropathy.

INTRODUCTION: Primary membranous nephropathy (PMN) is an autoimmune disease. Both T-regulatory cells (TREGs) and B-regulatory cells (BREGs) are decreased in patients with autoimmune disease. We evaluated the TREG and BREG population in patients of PMN treated with cyclical cyclophosphamide and steroid therapy (cCYC/GC). METHODS: Twenty-four patients with PMN resistant to a restrictive strategy and treated with cCYC/GC therapy and 10 healthy controls were enrolled. The proteinuria, serum creatinine, and serum albumin were tested at monthly intervals and blood samples were collected before starting cCYC/GC and at 6 and 8 (2 months wash out) months of therapy. The peripheral blood mononuclear cells (PBMCs) after staining with fluorochrome-conjugated antibodies were then subjected to flow cytometric analysis for detection of TREGs (CD3+CD4+CD25hiCD127loFoxP3+) and BREGs (CD19+CD5+CD1dhiIL10+). TREGs and BREGs are presented as the percentage of T and B cells, respectively. Cases with remission at month 18 were classified as responders, and those without any remission as nonresponders. RESULTS: Patients with PMN had a lower percentage of TREGs (P = 0.07) and BREGs compared with healthy controls (P = 0.0007). As compared with baseline, there was a significant increase in both BREGs (P = 0.001) and TREGs (P = 0.02) with the treatment (8 months). Patients who responded to therapy by 18 months had an increase in TREG (P = 0.05) and BREG (P = 0.0001) at month 8 compared with baseline. CONCLUSION: As compared with healthy controls, patients with PMN displayed a lower percentage of BREGs. Both TREGs and BREGs significantly improved with disease-specific therapy. BREGs had an association with clinical activity.

Immunomodulatory plasticity of mesenchymal stem cells: a potential key to successful solid organ transplantation.

Organ transplantation remains to be a treatment of choice for patients suffering from irreversible organ failure. Immunosuppressive (IS) drugs employed to maintain the allograft have shown excellent short-term graft survival, but, their long-term use could contribute to immunological and non-immunological risk factors, resulting in graft dysfunctionalities. Upcoming IS regimes have highlighted the use of cell-based therapies, which can eliminate the risk of drug-borne toxicities while maintaining efficacy of the treatment. Mesenchymal stem cells (MSCs) have been considered as an invaluable cell type, owing to their unique immunomodulatory properties, which makes them desirable for application in transplant settings, where hyper-activation of the immune system is evident. The immunoregulatory potential of MSCs holds true for preclinical studies while achieving it in clinical studies continues to be a challenge. Understanding the biological factors responsible for subdued responses of MSCs in vivo would allow uninhibited use of this therapy for countless conditions. In this review, we summarize the variations in the preclinical and clinical studies utilizing MSCs, discuss the factors which might be responsible for variability in outcome and propose the advancements likely to occur in future for using this as a "boutique/personalised therapy" for patient care.

Immunophenotyping of blood mononuclear cells from P. berghei (NK-65) infected and immunized BALB/c mice.

The present study demonstrates the alterations in the frequencies of helper, cytotoxic and suppressor T cells in blood of P. berghei infected and TPA immunized rodent host towards the induction of protective immune response. Statistically significant (p < 0.005) number of CD4+ T cells was recorded on D9 [Post Challenge (PC)] meanwhile CD4+ Treg cells were found to be declined in immunized mice as compared to infected. This increase in frequencies of T helper cells points towards the stimulation of strong Th2 immune response in immunized mice. Statistically significant (p < 0.005) decline was observed in the frequencies of CD8+ T cells on D9 (PC) in immunized mice. In infected controls cytotoxic T cells were also found to be increased after 24 h post infection. Due to strong Th2 immune response, evident from lower frequencies of CD4+ Treg cells in immunized mice, complete protection was obtained. T reg population was found to be higher in infected controls and all control mice died.

Safety and efficacy of autologous mesenchymal stromal cells transplantation in patients undergoing living donor kidney transplantation: a pilot study.

AIM: This pilot study assesses the safety and feasibility of autologous mesenchymal stromal cell (MSC) transplantation in four patients that underwent living donor renal transplantation, and the effect on the immunophenotype and functionality of peripheral T lymphocytes following transplantation. METHODS: All patients received low dose ATG induction followed by calcineurin inhibitor-based triple drug maintenance immunosuppression. Autologous MSCs were administered intravenously pre transplant and day 30 post-transplant. Patients were followed up for 6 months. The frequency of regulatory T cells and T cell proliferation was assessed at different time points. RESULTS: None of the four patients developed any immediate or delayed adverse effects following MSC infusion. All had excellent graft function, and none developed graft dysfunction. Protocol biopsies at 1 and 3 months did not reveal any abnormality. Compared to baseline, there was an increase in the CD4 + CD25+FOXP3+ regulatory T cells and reduction in CD4 T cell proliferation. CONCLUSION: We conclude that autologous MSCs can be used safely in patients undergoing living donor renal transplantation, lead to expansion of regulatory T cells and decrease in T cell proliferation. Larger randomized trials studies are needed to confirm these findings and evaluate whether this will have any impact on immunosuppressive therapy.

Assessment of anti-donor T cell proliferation and cytotoxic T lymphocyte-mediated lympholysis in living donor kidney transplant patients.

Organ transplant recipients are at risk of allograft rejection, and remain dependent on lifelong immunosuppressive agents, with the attendant risks of infections, cancers, and drug toxicities. Mesenchymal stromal cells (MSCs) have emerged as an alternative to the current pharmacologic immunosuppressive therapy as these cells are immune privileged and possess immunomodulatory properties. However, clinical data are incomplete regarding the efficacy of MSC therapy to control alloimmune response of the transplant recipients. Coordinated efforts should now be directed towards assays for monitoring anti-donor T cell response of MSC-treated patients to establish the pro-tolerogenic potential of MSC-based therapy. Here, we describe two useful tools to monitor MSC-mediated immunomodulation: the assessment of T cell proliferation by carboxyfluorescein succinimidyl ester (CFSE) dilution assay and the evaluation of cytotoxic T lymphocyte (CTL)-mediated lysis of (51)Cr-labeled target cells (cell-mediated lympholysis; CML) following mixed lymphocyte cultures of peripheral blood mononuclear cells (PBMCs) from kidney donors and transplant recipients.

Inhibition of grade dependent autophagy in urothelial carcinoma increases cell death under nutritional limiting condition and potentiates the cytotoxicity of chemotherapeutic agent.

PURPOSE: We evaluated the status of autophagy in different grades of urothelial carcinoma and explored autophagy modulators as a potential adjunctive therapeutic agent for urothelial carcinoma. MATERIALS AND METHODS: The study was performed in tumor tissue from patients with low and high grade urothelial carcinoma, in normal urothelial tissue and in the T24 cell line. Autophagic vesicles and the expression of various autophagic proteins were studied in tissue samples by transmission electron microscopy and Western blot, respectively. The effect of autophagy induction and inhibition was evaluated by measuring AMPK and mTOR expression, cell viability and mitochondrial membrane potential. The therapeutic implication of autophagy was studied using cisplatin alone or combined with an autophagy inhibitor. RESULTS: High grade urothelial carcinoma showed a higher number of autophagic vesicles and significantly higher expression of autophagic proteins. Upon starvation cells cultured from high and low grade urothelial carcinoma demonstrated significant autophagy induction associated with AMPK activation and mTOR inhibition. AMPK inhibition decreased the autophagic response and increased cell death. Autophagy inhibition by wortmannin, 3-methyladenine and chloroquine increased mitochondrial hypopolarization as well as caspase-9 and 3 dependent cell death. Combined treatment with cisplatin and an autophagy inhibitor resulted in greater cell death than cisplatin treatment alone. CONCLUSIONS: Autophagy is related to urothelial carcinoma grade and regulated via the AMPK pathway for tumor cell survival. Autophagy inhibition leads to cancer cell death through an intrinsic apoptotic pathway. The potential application of autophagy inhibitors as an adjunct to chemotherapy for urothelial carcinoma must be explored.

Influence of low cholesterol eggs enriched with vitamin-E and omega-3 fatty acid on blood lipid profile of Wistar rats.

In the recent past, low cholesterol eggs enriched with vitamin-E and omega-3 fatty acid have been developed and are marketed under different brands claiming them as heart friendly. The influence of these eggs (smart eggs) on lipid profile of rats was evaluated in comparison to that of the standard eggs. Data of 4 week dietary treatment revealed that total plasma cholesterol, low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol increased only 22% in rats fed on diet containing 4 smart eggs per kg of semi-synthetic diet in contrast to the increase of more than 100 % when fed on diet containing standard eggs. The results suggest that it is not the low cholesterol content alone but also vitamin E and omega-3 fatty acids present in smart eggs that act synergically to prevent a substantial change in blood lipid profile and impose no serious risk to the health of the consumers.