Multi-color immune-phenotyping of CD34 subsets reveals unexpected differences between various stem cell sources.

Flow cytometric routine CD34 analysis enumerates hematopoietic stem and progenitor cells irrespective of their subpopulations although this might predict engraftment dynamics and immune reconstitution. We established a multi-color CD34 assay containing CD133, CD45RA, CD10, CD38 and CD33. We examined PBSC, donor bone marrow (BMd) and BM of patients 1 year after allografting (BM1y) regarding their CD34 subset composition, which differed significantly amongst those materials: the early CD45RA(-)CD133(+)CD38(low) subpopulations were significantly more frequent in PBSC than in BMd, and very low in BM1y. Vice versa, clearly more committed CD34 stages prevailed in BM, particularly in BM1y where the proportion of multi-lymphoid and CD38(++) B-lymphoid precursors was highest (mean 59%). CD33 was expressed at different intensity on CD45RA(±)CD133(±) subsets allowing discrimination of earlier from more committed myeloid precursors. Compared with conventional CD34(+) cell enumeration, the presented multi-color phenotyping is a qualitative approach defining different CD34 subtypes in any CD34 source. Its potential impact to predict engraftment kinetics and immune reconstitution has to be evaluated in future studies.

Relevance of flow cytometric enumeration of post-thaw leucocytes: influence of temperature during cell staining on viable cell recovery.

BACKGROUND AND OBJECTIVES: Our post-thaw cell recovery rates differed substantially in interlaboratory comparisons of identical samples, potentially due to different temperatures during cell staining. MATERIALS AND METHODS: Viable CD34(+) cells and leucocyte (WBC) subtypes were quantified by multiparameter single-platform flow cytometry in leucapheresis products collected from 30 adult lymphoma and myeloma patients, and from 10 paediatric patients. After thawing, cells were prepared for analysis within 30 min between thawing and acquisition, at either 4°C or at room temperature. RESULTS: For cell products cryopreserved in conventional freezing medium (10% final DMSO), viable cell recovery was clearly lower after staining at 4°C than at RT. Of all WBC subtypes analysed, CD4(+) T cells showed the lowest median recovery of 4% (4°C) vs. 25% (RT), followed by CD3, CD34 and CD8 cells. The recovery was highest for CD3γδ cells with 44% (4°C) vs. 71% (RT). In the 10 samples cryopreserved in synthetic freezing medium (5% final DMSO), median recovery rates were 89% for viable CD34 (both at 4°C and RT) and 79% (4°C) vs 68% (RT) for WBC. CONCLUSIONS: The post-thaw environment and, potentially, the cryoprotectant impact the outcome of cell enumeration, and results from the analysis tube may not be representative of the cells infused into a patient.

Human AdV-specific T cells: persisting in vitro functionality despite lethal irradiation.

Viral infections caused by human adenovirus (HAdV) or CMV remain life-threatening complications in immunocompromised patients undergoing allogeneic hematopoietic stem cell transplantation. Adoptive immunotherapy with virus-specific T cells showed impressive clinical results without or with only mild GvHD. However, because of high costs and high regulatory barriers, these protocols are accessible to only a few centers. The infusion of unmanipulated donor lymphocytes (DLIs) that contain virus-specific T cells is not feasible because of the risk of GvHD. Reports about three patients treated with irradiated granulocytes or DLIs that potentially comprised virus-specific T cells discussed an active role of virus-specific lymphocytes despite irradiation, but real evidence could not be provided. Therefore, we tested the effect of irradiation on HAdV-specific T cells, which had been expanded in vitro, by stimulating PBMCs with HAdV-peptide pools and IL-15 for 12 days. Cells were then irradiated with 30 Gy, as performed for normal granulocyte concentrates. Cell viability and polyfunctional activity were determined by flow cytometry. Even 48 h after irradiation, 15.6% of expanded HAdV-specific T cells were apparently viable and cytolytically active. Although the in vivo antiviral activity was not tested, these data support earlier assumptions about the potential role of irradiated cells in patients.

Prevention of high-fat diet-induced adipose tissue remodeling in obese diabetic mice by n-3 polyunsaturated fatty acids.

OBJECTIVE: Obesity is associated with reduced insulin sensitivity and extensive reorganization of adipose tissue. As polyunsaturated fatty acids (PUFA) appear to inhibit diabetes development, we investigated PUFA effects on markers of matrix remodeling in white adipose tissue. METHODS AND PROCEDURE: Male obese diabetic (db/db) mice were treated with either a low-fat standard diet (LF), or high-fat diets rich in saturated and monounsaturated fatty acids (HF/S), n-6 PUFA (HF/6) or the latter including marine n-3 PUFA (HF/3). White adipose tissue was analyzed for gene expression, fatty acid composition and by immunofluorescence. RESULTS: HF/S treatment increased adipose tissue expression of a number of genes involved in matrix degradation including matrix metalloproteinase (MMP)-12, -14 and cathepsin K, L and S compared with LF. MMP-12 gene was expressed in macrophages and adipocytes, and MMP-12 protein colocalized with both cell types. In addition, mean adipocyte area increased by 1.6-fold in HF/S-treated mice. Genes essential for collagen production, such as procollagen I, III, VI, tenascin C and biglycan were upregulated in HF/S-treated animals as well. N-3 PUFA supplementation resulted in enrichment of these fatty acids in adipose tissue. Moreover, n-3 PUFA inhibited the HF/S-induced upregulation of genes involved in matrix degradation and production I restored mean adipocyte area and prevented MMP-12 expression in macrophages and adipocytes. CONCLUSION: N-3 PUFA prevent high-fat diet-induced matrix remodeling and adipocyte enlargement in adipose tissue of obese diabetic mice. Such changes could contribute to diabetes prevention by n-3 PUFA in obese patients.

Liver X receptors in cardiovascular and metabolic disease.

Liver X receptors (LXRs) alpha and beta are nuclear oxysterol receptors and metabolic sensors initially found to regulate cholesterol metabolism and lipid biosynthesis. Recent studies have elucidated the importance of LXR in the development of cardiovascular diseases and metabolic disorders. LXR agonists prevent development of atherosclerosis by modulation of metabolic as well as inflammatory gene expression in rodent models. Moreover, LXR activation inhibits hepatic gluconeogenesis and lowers serum glucose levels, indicating possible application of LXR activation in the treatment of diabetes mellitus. However, first-generation LXR agonists elevate hepatic and serum trigylceride levels, making subtype-specific agonists and selective LXR modulators rather than unselective LXR agonists a potential pharmacological strategy. This review summarizes the multiple physiological and pathophysiological implications of LXRs and observations that identify LXRs as potential targets for therapeutic interventions in human cardiovascular and metabolic disease.

The novel immunosuppressant FK778 inhibits formation of the immunologic Synapse.

The malononitrilamide FK778 is a derivative of A77 1726, the active metabolite of the antirheumatic drug leflunomide. A77 1726 inhibits de novo pyrimidine synthesis and activity of Src-family kinases; thus, it may interfere with T-cell proliferation as well as with early T-cell signaling. Formation of a stable interaction between T cells and antigen-presenting cells (APC)--the immunologic synapse--has emerged to be of crucial importance for T-cell activation. Here in we show that FK778 inhibits formation of the immunologic synapse by blocking superantigen-stimulated relocalization of adhesion (LFA-1), and signaling molecules (CD3) to the T-cell/APC contact site. These data show that FK778 affects T-cell/APC interactions, particularly events crucial for T-cell adhesion and formation of stable conjugates underlying sustained and effective T-cell activation. Thus, in this model system close to physiologic T-cell stimulation, FK778 affects critical events in the course of T-cell-mediated immune responses earlier than T-cell proliferation, which may contribute to its immunosuppressive potential.