Inhibition Of Lymphocyte Proliferation Research Articles

HSP90β shapes the fate of Th17 cells with the help of glycolysis-controlled methylation modification.

Ulcerative colitis (UC) is a refractory inflammatory disease associated with immune dysregulation. Elevated levels of heat shock protein (HSP) 90 in the β but not α subtype were positively associated with disease status in UC patients. This study validated the possibility that pharmacological inhibition or reduction of HSP90β would alleviate colitis, induced by dextran sulfate sodium, in mice and elucidated its mechanisms. Histopathological and biochemical analysis assessed disease severity, and bioinformatics and correlation analysis explained the association between the many immune cells and HSP90β. Flow cytometry was used to analyse the homeostasis and transdifferentiation of Th17 and Treg cells. In vitro inhibition and adoptive transfer assays were used to investigate functions of the phenotypically transformed Th17 cells. Metabolomic analysis, DNA methylation detection and chromatin immunoprecipitation were used to explore these mechanisms. The selective pharmacological inhibitor (HSP90βi) and shHSP90β significantly mitigated UC in mice and promoted transformation of Th17 to Treg cell phenotype, via Foxp3 transcription. The phenotypically-transformed Th17 cells by HSP90βi or shHSP90β were able to inhibit lymphocyte proliferation and colitis in mice. HSP90βi and shHSP90β selectively weakened glycolysis by stopping the direct association of HSP90β and GLUT1, the key glucose transporter, to accelerate ubiquitination degradation of GLUT1, and enhance the methylation of Foxp3 CNS2 region. Then, the mediator path was identified as the "lactate-STAT5-TET2" cascade. HSP90β shapes the fate of Th17 cells via glycolysis-controlled methylation modification to affect UC progression, which provides a new therapeutic target for UC.

Adenosine 2A receptor antagonists promote lymphocyte proliferation in sepsis by inhibiting Treg expression of PD-L1 in spleen.

The spleen is essential for lymphocyte proliferation, which is associated to sepsis prognosis. Adenosine 2A receptor (A2AR) blocking promotes lymphocyte proliferation in sepsis, however the mechanism is uncertain. Our sepsis cecum ligation perforation model showed that blocking A2AR increased survival and CD4+ cell numbers in a spleen-dependent mechanism. The sequencing of the transcriptome of the spleen indicated alterations in the expression of genes involved in the control of lymphocyte proliferation by inhibiting A2AR, including a reduction in the expression of PD-L1. Flow cytometry analysis of PD-L1 expression intensity in splenic cell subpopulations revealed that the Treg cell subpopulation was the strongest PD-L1-expressing cell population, and Treg PD-L1 expression decreased after blocking A2AR. In vitro activation of A2AR was able to upregulate PD-L1 expression of Treg and boost Treg capacity to limit lymphocyte proliferation, while blockage of PD-L1 partly reduced A2AR-activated Treg's ability to inhibit lymphocyte proliferation. In addition, blocking CREB phosphorylation significantly inhibited A2AR-induced PD-L1 expression. According to the findings of our research, inhibiting A2AR improves the prognosis of sepsis by lowering the level of PD-L1 expression by Treg in the spleen and reducing the inhibition of lymphocyte proliferation.

A Novel, Closed-Line Mini Extracorporeal Photopheresis (ECP) Technique Using the Therakos TM CellEx TM Photopheresis System, Developed for Patients with Contraindications Against Conventional ECP Therapy

Introduction: Conventional ECP has proven efficacy for treating several diseases, most significantly steroid-refractory chronic GVHD. Unfortunately, body weight and sufficient venous access limit the availability of this treatment modality. Children, in particular, usually have limited access to ECP. A simplified mini buffy coat ECP technique that allows treating small children and patients with apheresis contraindications has been tested in selected cases during the last 20 years. However, this method relies on an open-line system that requires a longer time and a cell processing laboratory, making this approach challenging. A new -closed-line- method, using the Therakos TM CellEx TM Photopheresis System, has been developed and evaluated in our Center. This method requires no processing laboratory or other special equipment and takes only 60-120 minutes, making the procedure potentially usable in routine practice. Design and methods: A 7-year-old girl had been treated at our Pediatric Bone Marrow Transplantation Unit with grade IV chronic steroid-refractory GVHD (skin, and less severe liver involvement). As all other options were exhausted, ECP became a desirable form of treatment. However, the patient's low body weight and serious venous access problems hindered conventional ECP treatment. We developed a solution that requires 200-250 ml whole blood collection from the patient, takes less than 2 hours, does not need special cell processing equipment, and allows the ECP treatment to commence in the form of a regular autotransfusion. Consent forms were obtained from the patient's caregivers as well as from the local Ethical Committe. The patient's age at the start of treatment was seven years, and her body weight was 28 kg. A total of 17 mini-ECPs were performed weekly up until the submission of this abstract, and the number of treatments per patient was 33. The duration of mini-ECP treatment has been five months. 5 to 8 mL/kg of whole blood was drawn from the patient. Separation of the white blood cell (WBC) rich buffy coat of the patient's blood - with donor blood product supplementation as needed - was performed through an individualized use of the CellEx TM photopheresis kit and blood prime program. The buffy coat was ultraviolet A (UVA)-irradiated after adding 8-methoxypsoralen (8-MOP) within the Therakos TM CellEx TM Photopheresis System and divided into two fractions under a laminar air flow cabinet. The autologous residual blood with the first fraction of the UVA irradiated buffy coat was transfused immediately. The second buffy coat fraction was stored at 4 °C and transfused the following morning through a peripheral vein. Results : The median volume of the collected whole blood was 250 mL (203-250 mL). The median volume was 72 ml (48-88 mL) for the first fraction of the irradiated buffy coat and 71 ml for the second fraction (48- 88 mL). Apoptosis and cell death were analyzed by annexin V staining; apoptosis and lymphocyte proliferation was measured by flow cytometry. UVA irradiation of the 8-MOP infused buffy coat resulted in significant induction of WBC apoptosis at 24 hours. No clinical side effects were observed during mini ECP treatment; all microbiological control tests were negative. Partial response was observed within three months (skin), and the tapering of systemic immunosuppression (methylprednisolone and ruxolitinib) began at five months. Conclusion: Mini whole blood ECP induces apoptosis and lymphocyte proliferation inhibition, both occurring after standard ECP. The closed-line method requires no special equipment and is not demanding for the patient, providing a viable solution to the technical difficulties children with the need for ECP treatment face.

TPN10475 alleviates concanavalin A-induced autoimmune hepatitis by limiting T cell development and function through inhibition of PI3K-AKT pathway

Autoimmune hepatitis (AIH) is an inflammatory liver disease in which the autoimmune system instigates an attack on the liver, causing inflammation and liver injury, and its incidence has increased worldwide in recent years. The mouse model of acute hepatitis established by concanavalin A (Con A) is a typical and recognized mouse model for the study of T-cell-dependent liver injury. In this study, we aimed to investigate whether the artemisinin derivative TPN10475 could alleviate AIH and its possible mechanisms. TPN10475 effectively inhibited lymphocyte proliferation and IFN-γ+ T cells production in vitro, alleviated liver injury by decreasing infiltrating inflammatory T cells producing IFN-γ in the liver and peripheral immune tissues, and demonstrated that TPN10475 weakened the activation and function of T cells by inhibiting PI3K-AKT signaling pathway. These results suggested that TPN10475 may be a potential drug for the treatment of AIH, and the inhibition of PI3K-AKT signaling pathway may provide new ideas for the study of the pathogenesis of AIH.

Androgen receptor agonist and antagonist reduce response of cytokine-induced killer cells on prostate cancer cells.

Despite many advances, prostate cancer (PCa) is still the second most frequently diagnosed cancer and fifth leading cause of cancer death in men worldwide. So far, the promising field of onco-immunology has not yet provided a satisfactory treatment option for PCa. Here we show that the ex vivo expansion and activation of cytokine-induced killer (CIK) cells isolated from primary peripheral blood mononuclear cells induce immune-mediated apoptosis in both human PCa LNCaP and C4-2 cells. Interestingly, pretreating LNCaP and C4-2 cells with either androgen or the androgen receptor (AR) antagonist enzalutamide mediates resistance to this immunogenic attack. This is associated with a reduction of both total cell loss and apoptosis levels suggesting one possible mechanism blunting onco-immunological activity. The data also suggest that secreted factors from AR ligand-treated PCa cell suppress lymphocyte proliferation. Further, we analysed immune-mediated killing activity using conditioned media from LNCaP and C4-2 treated cells. The obtained data suggest that the conditioned media from PCa treated cells does not influence a measurable lymphocyte-mediated apoptosis. However, analysing clonal expansion of activated lymphocytes, the androgen-derived conditioned media suppresses lymphocyte proliferation/expansion suggesting inhibition of onco-immunological activity by pretreatment of PCa cells with AR ligands.

Vit D Assessment in Patients with Covid-19 Virus

Abstract Background vitamin D may potentially play an important role in preventing or ameliorating the multi-system consequences of COVID-19 infections. Aim of the Work The aims of this study is to assess that there is a potential association between mean levels of vitamin D in the cases and mortality caused by COVID-19. Patients and Methods This was a case control study that was conducted on 80 Egyptian Patients attending at El medani hospital, El Obour specialized hospital, and the outpatient clinics at Ain shams University hospitals through six months. Results Vitamin D deficiency has been found to contribute to acute respiratory distress syndrome, a major cause of death associated with COVID-19. Vitamin D plays a role in strengthening the body‟s immunity by inducing monocyte differentiation and inhibiting lymphocyte proliferation. Regarding to the laboratory investigations: We have found that the number and percentage of WBC, LYM and NEU were significantly different between positive and negative RT-PCR cases for COVID-19/or SARS-CoV-2. In comparison to the normal range, we found low WBC and LYM counts in patients with positive RT-PCR COVID-19. Regarding to vitamin D: Our study showed statistically significant positive correlation between vit D and lymphocytes, and there was statistically significant negative correlation between vit D, neutrophil-lymphocyte ratio, CRP, BUN, S. creatinine, AST, ALT, FERRITIN, LDH and D-DIMER. Conclusion Vitamin D deficiency is a risk factor for infections as well as for insufficient innate immune system reactivity, therefore the winter time testing for serum vitamin D status (lowest levels expected) should be mandatory as part of regular health check status. The test is even more mandatory in patients with chronic diseases, where we expect vitamin D deficiency during all the year and an increased burden of risk factors for further disease severity and even mortality. In conclusion, this study confirms that vitamin D deficiency is associated with more severe lung involvement, longer disease duration, in COVID-19 patients.

Unveiling the immunomodulatory shift: Epithelial-mesenchymal transition Alters immune mechanisms of amniotic epithelial cells

Epithelial-mesenchymal transition (EMT) changes cell phenotype by affecting immune properties of amniotic epithelial cells (AECs). The present study shows how the response to lipopolysaccharide of cells collected pre- (eAECs) and post-EMT (mAECs) induces changes in their transcriptomics profile. In fact, eAECs mainly upregulate genes involved in antigen-presenting response, whereas mAECs over-express soluble inflammatory mediator transcripts. Consistently, network analysis identifies CIITA and Nrf2 as main drivers of eAECs and mAECs immune response, respectively. As a consequence, the depletion of CIITA and Nrf2 impairs the ability of eAECs and mAECs to inhibit lymphocyte proliferation or macrophage-dependent IL-6 release, thus confirming their involvement in regulating immune response. Deciphering the mechanisms controlling the immune function of AECs pre- and post-EMT represents a step forward in understanding key physiological events wherein these cells are involved (pregnancy and labor). Moreover, controlling the immunomodulatory properties of eAECs and mAECs may be essential in developing potential strategies for regenerative medicine applications.

Urine-Derived Renal Epithelial Cells (URECs) from Transplanted Kidneys as a Promising Immunomodulatory Cell Population.

Kidney transplantation is a lifesaving procedure for patients with end-stage kidney disease (ESKD). Organs derived from donation after cardiac death (DCD) are constantly increasing; however, DCD often leads to ischaemia-reperfusion (IR) and Acute Kidney Injury (AKI) events. These phenomena increase kidney cell turnover to replace damaged cells, which are voided in urine. Urine-derived renal epithelial cells (URECs) are rarely present in the urine of healthy subjects, and their loss has been associated with several kidney disorders. The present study aimed to characterize the phenotype and potential applications of URECs voided after transplant. The results indicate that URECs are highly proliferating cells, expressing several kidney markers, including markers of kidney epithelial progenitor cells. Since the regulation of the immune response is crucial in organ transplantation and new immunoregulatory strategies are needed, UREC immunomodulatory properties were investigated. Co-culture with peripheral blood mononuclear cells (PBMCs) revealed that URECs reduced PBMC apoptosis, inhibited lymphocyte proliferation, increased T regulatory (Treg) cells and reduced T helper 1 (Th1) cells. URECs from transplanted patients represent a promising cell source for the investigation of regenerative processes occurring in kidneys, and for cell-therapy applications based on the regulation of the immune response.

Dynamics of Ex Vivo Mesenchymal Stromal Cell Potency under Continuous Perfusion.

Mesenchymal stromal cells (MSCs) are a candidate for cell immunotherapy due to potent immunomodulatory activity found in their secretome. Though studies on their secreted substances have been reported, the time dynamics of MSC potency remain unclear. Herein, we report on the dynamics of MSC secretome potency in an ex vivo hollow fiber bioreactor using a continuous perfusion cell culture system that fractionated MSC-secreted factors over time. Time-resolved fractions of MSC-conditioned media were evaluated for potency by incubation with activated immune cells. Three studies were designed to characterize MSC potency under: (1) basal conditions, (2) in situ activation, and (3) pre-licensing. Results indicate that the MSC secretome is most potent in suppressing lymphocyte proliferation during the first 24 h and is further stabilized when MSCs are prelicensed with a cocktail of pro-inflammatory cytokines, IFNγ, TNFα, and IL-1β. The evaluation of temporal cell potency using this integrated bioreactor system can be useful in informing strategies to maximize MSC potency, minimize side effects, and allow greater control for the duration of ex vivo administration approaches.

Treg-targeted efficient-inducible platform for collagen-induced arthritis treatment.

Regulatory T cells (Tregs) display great promise in rheumatoid arthritis (RA) therapy. However, their low number and differentiation rate limit their further application in the clinics. In the present study, we first optimized a combination of IL-2, TGF-β and cyclin dependent kinase inhibitor AS2863619 (IL-2/TGF-β/AS), which could induce Tregs with high efficiency in vitro. After the induced Tregs (iTregs) were confirmed to suppress lymphocyte proliferation and pro-inflammatory T help cells (Th1 and Th17) activation, a chitosan-stabilized nanoparticle drug delivery system (NDDS) was developed according to the optimized formula of IL-2/TGF-β/AS. In vivo study, the NDDS was injected into the knees of mice with collagen-induced arthritis (CIA). As a result, the NDDS remarkably reduced the pathological score of the CIA, alleviated the inflammatory cell infiltration and synovial hyperplasia, and minimized cartilage tissue damage in the knee joint of the CIA mice. Mechanically, the NDDS administration promoted Treg differentiation and decreased Th17 production, consequently reversing the ratio of Treg/Th17, and reducing the secretion of TNF-α in the sera, which facilitated to relieve the severity and progression of arthritis. In sum, NDDS capable of efficiently inducing Tregs were constructed successfully and provided a potential platform for treating RA by restoring the equilibrium of Treg/Th17 destroyed in RA.

Immortalized Canine Adipose-Derived Mesenchymal Stem Cells as a Novel Candidate Cell Source for Mesenchymal Stem Cell Therapy.

Mesenchymal stem cells are expected to be a cell source for stem cell therapy of various diseases in veterinary medicine. However, donor-dependent cell heterogenicity has been a cause of inconsistent therapeutic efficiency. Therefore, we established immortalized cells from canine adipose tissue-derived mesenchymal stem cells (ADSCs) to minimize cellular heterogeneity by reducing the number of donors, evaluated their properties, and compared them to the primary cells with RNA-sequencing. Immortalized canine ADSCs were established by transduction with combinations of the R24C mutation of human cyclin-dependent kinase 4 (CDKR24C), canine cyclin D1, and canine TERT. The ADSCs transduced with CDK4R24C, cyclin D1, and TERT (ADSC-K4DT) or with CDK4R24C and cyclin D1 (ADSC-K4D) showed a dramatic increase in proliferation (population doubling level >100) without cellular senescence compared to the primary ADSCs. The cell surface markers, except for CD90 of the ADSC-K4DT and ADSC-K4D cells, were similar to those of the primary ADSCs. The ADSC-K4DT and ADSC-K4D cells maintained their trilineage differentiation capacity and chromosome condition, and did not have a tumorigenic development. The ability to inhibit lymphocyte proliferation by the ADSC-K4D cells was enhanced compared with the primary ADSCs and ADSC-K4DT cells. The pathway analysis based on RNA-sequencing revealed changes in the pathways mainly related to the cell cycle and telomerase. The ADSC-K4DT and ADSC-K4D cells had decreased CD90 expression, but there were no obvious defects associated with the decreased CD90 expression in this study. Our results suggest that ADSC-K4DT and ADSC-K4D cells are a potential novel cell source for mesenchymal stem cell therapy.

Mitochondrial-linked de novo pyrimidine synthesis as a regulator of T cell responses

It has been well established that the metabolism of T cells is integral to their functionality. If a T cell cannot generate enough energy or building blocks, it will not be able to exert its cytotoxic properties to eliminate pathogens and cancer cells. Impairment of mitochondrial oxidative phosphorylation is a well-known disruptor of T cell activation. Dihydroorotate dehydrogenase (DHODH) is a rate-limiting component of the de novo synthesis of pyrimidines and its activity is dependent on functional mitochondrial oxidative phosphorylation. In this regard, DHODH inhibitors have long been used in clinical settings for the treatment of autoimmune diseases, as they potently inhibit lymphocyte proliferation. The exact mode-of-action of these inhibitors in T lymphocytes is not yet exactly understood. In this review, we briefly discuss the critical role of mitochondria in T cell functionality. We also describe how de novo pyrimidine biosynthesis is linked to mitochondrial activity. Finally, we summarize our current knowledge of how mitochondrial-linked de novo pyrimidine biosynthesis modulates T cell responses.

FTIR Characterization of Sulfated Polysaccharides Obtained from Macrocystis integrifolia Algae and Verification of Their Antiangiogenic and Immunomodulatory Potency In Vitro and In Vivo.

The aim of this study was to evaluate the antiangiogenic and immunomodulatory potential of sulfated polysaccharides from the marine algae Macrocystis integrifolia characterized by FTIR. The cytotoxicity of sulfated polysaccharides was evaluated using the 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay. Antiangiogenic activity was evaluated using the chicken chorioallantoic membrane (CAM) assay. Immunomodulatory activity was determined on macrophage functionality and allergic response. The results showed that sulfated polysaccharides significantly decreased angiogenesis in chicken chorioallantoic membranes (p < 0.05). Likewise, they inhibited in vivo chemotaxis and in vitro phagocytosis, the transcription process of genes that code the enzymes cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and nitric oxide synthase-2 (NOS-2) and the nuclear factor kappa-light chain enhancer of activated B cells (NF-κB), showing immunomodulatory properties on the allergic response, as well as an in vivo inhibitory effect in the ovalbumin-induced inflammatory allergy model (OVA) and inhibited lymphocyte proliferation specific to the OVA antigen in immunized mice. Finally, these compounds inhibited the histamine-induced skin reaction in rats, the production of immunoglobulin E (IgE) in mice, and the passive response to skin anaphylaxis in rats. Therefore, the results of this research showed the potential of these compounds to be a promising source for the development of antiangiogenic and immunomodulatory drugs.

Overcoming apoptotic resistance afforded by Bcl-2 in lymphoid tumor cells: a critical role for dexamethasone

Bcl-2 is an anti-apoptotic protein that promotes cell survival and resistance to cell death. Predictably, Bcl-2 as well as other anti-apoptotic Bcl-2 family members have been found to be overexpressed in a variety of human cancers. Approaches to overcome apoptotic resistance afforded by Bcl-2 in cells include anti-sense oligonucleotides, drugs that inhibit Bcl-2 function, and BH3 mimics have not been universally effective; thus, the need to understand the underlying mechanism of this resistance is vital. Glucocorticoids are stress hormones that act through their cognate receptors to control the transcription of numerous target genes, and in turn regulate a diverse array of biological processes. Synthetic glucocorticoids, such as dexamethasone, are prescribed in many chemotherapy protocols for neoplasms of lymphoid origin based on their ability to inhibit lymphocyte proliferation and promote apoptosis. However, lymphoid cells expressing Bcl-2 are resistant to glucocorticoid-induced cell death. We observed both pro- and anti-apoptotic characteristics in lymphoid cells expressing Bcl-2 following glucocorticoid treatment. These cells exhibited a profound change in their intracellular ionic composition, but a limited apoptotic ion flux and the absence of cell death. Provocatively, mimicking the loss of intracellular potassium using a low dose of a microbial toxin that acts as a potassium ionophore in combination with dexamethasone overcame the resistance afforded by Bcl-2 and killed the cells. Extending our study using other potassium ionophores revealed that direct depolarization of the mitochondria membrane potential coupled with prior treatment with glucocorticoids is the key mechanism for activating the cell death program and bypassing the resistance afforded by Bcl-2 in lymphoid cells. Finally, we show that the duration of dexamethasone pre-treatment is critical for regulating distinct genes and signaling pathways that sensitize the cells to die.

MONOCYTIC MYELOID-DERIVED SUPPRESSOR CELL EXPANSION AFTER CARDIAC SURGERY WITH CARDIOPULMONARY BYPASS INDUCES LYMPHOCYTE DYSFUNCTION.

Cardiac surgery with cardiopulmonary bypass (CPB) is associated with an immune paresis that predisposes to the development of postoperative infections and sepsis. Among factors responsible for CPB-induced immunosuppression, circulating myeloid-derived suppressor cells (MDSCs) have been found to induce early lymphocyte apoptosis and lymphocyte proliferation inhibition. However, the mechanisms involved are not fully understood. In this study, we found that the main lymphocyte subsets decreased significantly 24 h after cardiac surgery with CBP. As expected, cardiac surgery with CPB induced a monocytic MDSC expansion associated with an increased T-cell apoptosis and decreased proliferation capacity. Noteworthy, granulocytic MDSCs remain stable. Myeloid-derived suppressor cell depletion restored the ability of T-cell to proliferate ex vivo . After CPB, indoleamine 2,3-dioxygenase activity and IL-10 plasma level were increased such as programmed death-ligand 1 monocytic expression, whereas plasma level of arginine significantly decreased. Neither the inhibition of indoleamine 2,3-dioxygenase activity nor the use of anti-programmed death-ligand 1 or anti-IL-10 blocking antibody restored the ability of T-cell to proliferate ex vivo . Only arginine supplementation restored partially the ability of T-cell to proliferate.

Low-dose mycophenolate mofetil improves survival in a murine model of Staphylococcus aureus sepsis by increasing bacterial clearance and phagocyte function.

Regulators of TLRs signaling pathways play an important role in the control of the pro-inflammatory response that contributes to sepsis-induced tissue injury. Mycophenolate mofetil, an immunosuppressive drug inhibiting lymphocyte proliferation, has been reported to be a regulator of TLRs signaling pathways. Whether MMF used at infra-immunosuppressive doses has an impact on survival and on innate immune response in sepsis is unknown.C57BL/6J mice were infected intraperitoneally with 108 CFU Staphylococcus aureus, and treated or not with low-dose of MMF (20mg/kg/day during 4 days). Survival rate and bacterial clearance were compared. Cytokine levels, quantitative and qualitative cellular responses were assessed. S. aureus – infected mice treated with MMF exhibited improved survival compared to non-treated ones (48% vs 10%, p<0.001). With the dose used for all experiments, MMF did not show any effect on lymphocyte proliferation. MMF treatment also improved local and systemic bacterial clearance, improved phagocytosis activity of peritoneal macrophages resulting in decreased inflammatory cytokines secretion. MMF-treated mice showed enhanced activation of NF-κB seemed with a suspected TLR4-dependent mechanism. These results suggest that infra-immunosuppressive doses of MMF improve host defense during S. aureus sepsis and protects infected mice from fatal outcome by regulating innate immune responses. The signaling pathways involved could be TLR4-dependent. This work brings new perspectives in pathogenesis and therapeutic approaches of severe infections.

Quantitation of mycophenolic acid and metabolites by UPLC-MS/MS in renal transplant patients

Mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS), specific inhibitors of lymphocyte proliferation, are commonly used as adjuvant therapy with calcineurin inhibitor agents after kidney transplantation. After administration, MMF and EC-MPS are hydrolyzed to mycophenolic acid (MPA), the active form of the drug, which must be monitored due to its narrow therapeutic window, drug–drug interactions, and large intra-and inter-individual pharmacokinetic variability despite a fixed dose. Monitoring plasma MPA level is recommended to maintain the drug within the therapeutic window, optimize its efficacy, and minimize side effects. This study aims to develop a method for quantifying MPA and its major metabolites (mycophenolic acid glucuronide [MPAG]) using on-line solid phase extraction (SPE) coupled with an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in kidney transplant patients. The linearity of MPA and MPAG were 0.3–13.6 μg/mL and 2.6–232.9 μg/mL, respectively (r2 > 0.999). The relative error of accuracy was < 15%. The within-run and between-run imprecision was < 5.8%. No carryover, ion suppression, or ion enhancement were observed. This method was used to analysis of 351 plasma samples from renal transplant patients after MMF or EC-MPS using this method showed large pharmacokinetic variability between patients. Analysis of the same samples by

Phenotypic and Functional Characterizations of Mesenchymal Stem/Stromal Cells Isolated From Human Cranial Bone Marrow.

Mesenchymal stem/stromal cells (MSCs) are adult stem cells that were originally isolated from bone marrow. In contrast to long bone-derived MSCs that have been extensively characterized, our knowledge regarding to MSCs isolated from flat bones (e.g., cranial bones) remain less clear. In this study, MSCs were purified from human cranial bone marrow (CB-MSCs) and their transdifferentiation capacity and immunomodulatory functions were further characterized. Phenotypic analysis of CB-MSCs demonstrated high expression of CD73, CD90, and CD105 while negative for CD14, CD34, and HLA-DR. Further in vitro differentiation assay shown that CB-MSCs capable of differentiating into cell types of mesenchymal origin (i.e., adipocytes, osetoblasts, and chondrocytes) and collectively, these results indicated that cells isolated from cranial bone marrow in this study are bona fide MSCs according to the minimal criteria proposed by the International Society for Cellular Therapy. Following in vitro expansion, single colony-derived CB-MSCs (scCB-MSCs) were obtained and confocal microscopy analysis further revealed functional heterogeneity within primary CB-MSCs. Specifically, obtained scCB-MSCs exhibited GABA progenitor features, as determined by olig2 and nestin. As expect, scCB-MSCs were readily induced to differentiate into GABAergic neuron-like cells. Furthermore, immunomodulatory roles of scCB-MSCs were evaluated following co-culture with human peripheral blood lymphocytes and results shown that co-culturing with scCB-MSCs significantly suppressed lymphocyte proliferation and promoted differentiation of lymphocytes into regulatory T cells but not Th1/Th17 phenotype. Overall, our results indicated that CB-MSCs exhibited clonal heterogeneity with marked propensity to differentiate into neural-like cells and this might represent promising candidates for the treatment of neurodegenerative diseases.

Use of X-rays to prevent transfusion-induced graft-versus-host disease

Transfusion associated graft versus host disease (TA-GvHD) is a rare complication of transfusion treatment with blood products containing viable lymphocytes. The risk of developing TA-GvHD depends on number and sustainability of lymphocytes in blood product, host’s immune system reactivity and the difference in human leukocyte antigen (HLA) system between donor and recipient. Standard method of TA-GvHD prevention is irradiation of blood products with ionizing radiation and absorbed dose of 25-50 Gy. The aim of irradiation is inactivation of viable lymphocytes in blood product containing cells (red blood cells, platelets, granulocytes) and lymphocyte proliferation inhibition in blood product recipient. In the last two decades, gamma irradiation has been extensively abandoned and replaced with specially designed cabinet machines using X-ray irradiation which are suitable for hospital and blood bank use. This reduces the risk of accidental or deliberate misuse of radioactive source, solves radioactive waste disposal issues and reduces ionizing irradiation exposure of employees in blood establishments. In Blood Bank of Croatian Institute of Transfusion Medicine, X-ray irradiation of blood products has been carried out since 2014 with a year rate of 20,000 irradiated cell blood products used in treatment of patients at risk of developing TA-GvHD.

HLA-G and CD152 Expression Levels Encourage the Use of Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells as an Alternative for Immunosuppressive Therapy.

Mesenchymal stromal cells (MSCs) have been used in immunosuppressive therapy due to their therapeutic effects, with the HLA-G molecule seeming to play a fundamental role. This work evaluated alternative MSC sources to bone marrow (BM), namely, umbilical cord tissue (UC), adipose tissue (AD) and dental pulp tissue (DP), and the influence of interferon-γ (IFN-γ) and hypoxia on the cultivation of these cells for use in immunosuppression therapies. Expression of costimulatory markers CD40, CD80 and CD86 and immunosuppressive molecules CD152 and HLA-G was analyzed. Lymphocyte inhibition assays were also performed. Sequencing of the HLA-G gene from exons 1 to 5 was performed using next-generation sequencing to determine the presence of alleles. UC-derived MSCs (UCMSCs) expressed higher CD152 and HLA-G1 under standard cultivation. UCMSCs and DP-derived MSCs (DPSCs) secreted similar levels of HLA-G5. All MSC sources inhibited the proliferation of peripheral blood mononuclear cells (PBMCs); growth under regular versus hypoxic conditions resulted in similar levels of inhibition. When IFN-γ was added, PBMC growth was inhibited to a lesser extent by UCMSCs. The HLA-G*01:04:01:01 allele appears to generate a more efficient MSC response in inhibiting lymphocyte proliferation. However, the strength of this conclusion was limited by the small sample size. UCMSCs are an excellent alternative to BM in immunosuppressive therapy: they express high concentrations of inhibitory molecules and can be cultivated without stimuli, which minimizes cost.