R410 – Immunosuppressive Properties of Tonsil-Derived MSCs

2008 ◽  
Vol 139 (2_suppl) ◽  
pp. P180-P180
Author(s):  
Sasa Janjanin ◽  
Farida Djouad ◽  
Drago Prgomet ◽  
Rabie M Shanti ◽  
Gollapudi Kiran ◽  
...  
Keyword(s):  
Hematopoietic Cell Transplantation ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Immunosuppressive Activity ◽  
Peripheral Blood Mononuclear ◽  
Murine Splenocytes ◽  
Department Of State ◽  
Wide Range ◽  
Organ Repair ◽  
The Difference

Problem We have previously shown that stroma of human palatine tonsils contains mesenchymal stem cells (MSCs) that can be isolated and expanded in culture. These tonsil-derived MSCs (T-MSCs) show multipotent differentiation properties, i.e. can differentiate along multiple mesenchymal lineages, including osteoblasts, chondrocytes, adipocytes, and myocytes. Recent findings also show that MSCs display immunoregulatory properties. Although the exact immunosuppressive mechanisms are unknown, the capacity of MSCs to suppress T-cell proliferation stimulated by allogeneic lymphocytes, dendritic cells, and phytohemaglutinin (PHA) is well documented. This study explores immunosuppressive characteristics of T-MSCs and compares them with characteristics of bone marrow-derived MSCs (BM-MSCs), a well-characterized cell population. Methods The mixed lymphocyte reaction (MLR) using human peripheral blood mononuclear cells (PBMC) from healthy donors and xenogeneic murine splenocytes was used to test the immunosuppressive properties of T-MSCs and BMMSCs. Indoleamine 2,3-dioxygenase (IDO) enzyme activity was measured spectrophotometrically based on tryptophan-to-kynurenine conversion in the supernatant. Interferon (IFN)-g in culture supernatants was quantified using a commercially available ELISA kit. Results Addition of BM-MSCs and T-MSCs both inhibited the PHA-induced proliferative response of PBMC and xenogeneic splenocytes. The difference in immunosuppressive activity correlates with the level of cell surface interferon (IFN)-g receptor as well as the differential ability of IFN-g to stimulate of IDO activity by T-MSCs compared to BM-MSCs. Conclusion T-MSCs share similar immunosuppressive characteristics as BM-MSCs in MLR. The immunosuppressive activity is significant and dose-dependent, although at a lower level than that of BM-MSCs. Significance Owing to their ease of isolation, rapid proliferation in the culture and self-renewal capacity, MSCs to date are considered an attractive candidate cell type for the development of novel cell-based therapies. They could be relevant in a wide range of clinical applications, including tissue and organ repair, drug or gene delivery to diseased tissues, improvement of allogenic hematopoietic cell transplantation, and the management of graft-versus-host disease. Support Supported by NIAMS Intramural Research Program (NIH ZO1 AR 41131). Sasa Janjanin is a recipient of the Fulbright Scholarship of the U.S. Department of State.

scholarly journals TLR4-induced IFN-γ production increases TLR2 sensitivity and drives Gram-negative sepsis in mice

2008 ◽  
Vol 205 (8) ◽  
pp. 1747-1754 ◽  
Author(s):  
Stephan Spiller ◽  
Greg Elson ◽  
Ruth Ferstl ◽  
Stefan Dreher ◽  
Thomas Mueller ◽  
...  
Keyword(s):  
Antibiotic Therapy ◽  
Bacterial Infection ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Dependent Manner ◽  
Tlr2 Expression ◽  
Peripheral Blood Mononuclear ◽  
Murine Splenocytes ◽  
Gram Negative ◽  
Ifn Γ

Gram-negative bacterial infection is a major cause of sepsis and septic shock. An important inducer of inflammation underlying both syndromes is the cellular recognition of bacterial products through pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). We identified a novel antagonistic mAb (named 1A6) that recognizes the extracellular portion of the TLR4–MD-2 complex. If applied to mice before infection with clinical isolates of Salmonella enterica or Escherichia coli and subsequent antibiotic therapy, 1A6 prevented otherwise fatal shock, whereas application of 1A6 after infection was ineffective. In contrast, coapplication of 1A6 and an anti-TLR2 mAb up to 4 h after infection with Gram-negative bacteria, in combination with the start of antibiotic therapy (mimicking clinical conditions), provided robust protection. Consistent with our findings in mice, dual blockade of TLR2 and TLR4 inhibited TNF-α release from human peripheral blood mononuclear cells upon Gram-negative bacterial infection/antibiotic therapy. Both murine splenocytes and human PBMCs released IFN-γ in a TLR4-dependent manner, leading to enhanced surface TLR2 expression and sensitivity for TLR2 ligands. Our results implicate TLR2 as an important, TLR4-driven sensor of Gram-negative bacterial infection and provide a rationale for blockade of both TLRs, in addition to antibiotic therapy for the treatment of Gram-negative bacterial infection.

Inactivation of Human White Blood Cells in Red Blood Cell Products Using Mirasol® Pathogen Reduction Technology (PRT).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4135-4135
Author(s):  
Loren D. Fast ◽  
Susanne Marschner ◽  
Gilbert DiLeone ◽  
Suzann K. Doane ◽  
Raymond P. Goodrich
Keyword(s):  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
White Blood Cells ◽  
Platelet Concentrates ◽  
Peripheral Blood Mononuclear ◽  
Immunological Responses ◽  
Treatment Conditions ◽  
Wide Range ◽  
Lower Light ◽  
Pathogen Reduction

Abstract The transfusion of blood products can result in transmission of pathogens and immunological consequences such as transfusion associated graft-versus-host disease and alloimmunization. Previous studies have shown that exposure of platelet concentrates to riboflavin and light (Mirasol PRT treatment) causes irreparable modification of nucleic acids that results in inactivation of a wide range of pathogens as well as inhibition of the immunological responses mediated by the WBC present in platelet concentrates. Initial studies investigated the effects of Mirasol PRT treatment of RBC concentrates on WBC function. Human peripheral blood mononuclear cells (PBMNC) were purified by Ficoll-Hypaque discontinuous centrifugation from control or test non-leukoreduced RBC units exposed to riboflavin and varying doses of light. These PBMNC were tested for their ability to be activated in response to PMA, to proliferate in response to PHA or allogeneic stimulator cells, and to stimulate proliferative responses of allogeneic responder cells. While lower light doses were sufficient to inhibit activation and proliferation, higher energies (30J/ml RBC) were required for inhibition of antigen presentation. These Mirasol treatment conditions did not induce crossmatch incompatibility, methemoglobin (MetHb) levels stayed within an acceptable range (2.6% + 1.5) and hemolysis was below 1% during storage. Interestingly, MetHb levels decreased to background levels during storage at 4°C, suggesting that the activity of the enzyme NADH methemoglobin reductase was not compromised by the exposure to riboflavin and light. These treatment conditions also reduced levels of Gram positive and negative bacteria to the limits of detection and reduced infectivity of both enveloped and non-eveloped viruses by > 4 logs on average in packed, washed RBC units. In summary, Mirasol PRT is able to functionally inactivate WBCs and pathogens in washed RBC products without adversely affecting the quality of the RBCs. This novel pathogen reduction therapy for RBC products complements the Mirasol PRT treatment for platelets and offers inactivation of pathogens in cellular blood components using a single technology.

scholarly journals Fluorescent Cell Barcoding As New Flow Cytometric Technique for Multiplexed Phenotyping and Signaling Profiling in Hematologic Patients

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5033-5033 ◽  
Author(s):  
Valentina Giudice ◽  
Xingmin Feng ◽  
Sachiko Kajigaya ◽  
Angelique Biancotto ◽  
Neal S. Young
Keyword(s):  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Fold Increase ◽  
Good Resolution ◽  
Peripheral Blood Mononuclear ◽  
Fluorescent Cell ◽  
Cytokine Detection ◽  
Antibody Staining ◽  
Healthy Control ◽  
The Difference

Abstract Fluorescent cell barcoding (FCB) is a high-throughput multiplexing technique, in which samples from one or more donors can be combined, minimizing staining variability, antibody consumption, and decreasing sample volumes needed. In this study, we optimized FCB technique for routine application in immunophenotyping, phosphoFlow, and intracellular cytokine detection. Human peripheral blood mononuclear cells (PBMCs) from healthy controls and patients were used for optimization, and CBD450 and CBD500, Pacific Orange succinimidyl ester, DyLight 350, and DyLight 800 were tested for barcoding 6 to 36 samples. Working concentrations ranging from 0 to 125 µg/mL were tested, and viability dye staining was also optimized to increase data robustness. We first measured fluorescence intensity (MFIs) of six serial dilutions (0, 0.75, 3.25, 12.5, 62.5 and 125 µg/mL) and the difference in intensity separating them. Second, a 3x3 matrix was prepared using three concentrations of two different dyes, and PMBCs were barcoded with nine different combinations of FCB dye concentrations. When gated with the dyes, nine lymphocyte populations were detected. A 4x(3x3) matrix was also designed using three different FCB dyes (CBD450, DyLight 800, and Pacific Orange) at various concentrations to barcode 36 samples simultaneously. Thus, each lymphocyte population was clearly identified. The separation of each population or purity of deconvolution is based on the distance between MFIs and CVs. When we calculated the purity of deconvolution in our experiments, human PBMCs displayed CVs of 10 - 25%. When MFIs were separated by 2-fold increase, barcoded populations could be identified with a good resolution, higher when MFIs were separated by 3- or more fold increase. The use of viability dyes as LIVE/DEAD Fixable Aqua viability dye together with the FCB showed increase of data robustness due to exclusion of dead cells from gating strategy. In combination with viability dye, we successfully performed a six-color phosphoFlow and a simple four-color stainings using the same FCB dye combinations and cytometer voltages, and also combining one healthy control and two different patient samples. Our methods using FCB dye alone and/or in combination with antibody staining should be useful to efficiently perform multiplex drug screening and lymphocyte characterization. FCB minimizes batches and technical variations, and also increases data robustness, thus improving the immune phenotyping of patients. Disclosures Young: GSK/Novartis: Research Funding.

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