Impaired coronary microcirculation in type 2 diabetic patients is associated with elevated circulating regulatory T cells and reduced number of IL-21R⁺ T cells.

BACKGROUND: Low-grade systemic inflammation is considered to participate in the progression of type 2 diabetes (T2D) and in diabetic complications. METHODS: To determine if circulating leukocytes were abnormally regulated in T2D patients, 8-color flow-cytometry (FACS) analysis was performed in a cross-sectional study of 37 T2D patients and 16 controls. Data obtained from the FACS analysis were compared to coronary flow reserve (CFR), assessed by Rb(82)-PET-imaging, to uncover inflammatory signatures associated with impaired CFR. RESULTS: Presence of T2D was associated with T cell attenuation characterized by reduced overall T cell, Th17, IL-21R(+), Treg's and TLR4(+) T cells, while the monocyte population showed enhanced TLR4 expression. Further, our data revealed reduced M1-like CD11c expression in T2D which was associated with impaired CFR. In contrast, we show, for the first time in T2D, increased TLR4 expression on CD8 T cells, increased Treg cell number and Treg maturation and reduced IL-21R expression on CD8 T cells to be functionally associated with impaired CFR. CONCLUSIONS: Our demonstration that HbA1c inversely correlates to several T cell populations suggests that T cells may play disease modulating roles in T2D. Further, the novel association between impaired CFR and regulatory T cells and IL-21R(+) T cells imply an intricate balance in maintaining tissue homeostasis in vascular diabetic complications.

Flow Cytometry Approaches to Obtain Medulloblastoma Stem Cells from Bulk Cultures.

Cancer stem cells are considered the reservoir cancer cells that are resistant to most of the forms of cancer therapies and cause relapse of the tumor. Medulloblastoma (MB), a primary CNS tumor, is a very fast-growing tumor affecting younger population. In order to characterize medulloblastoma cancer stem cells or studying the drug resistance in MB mediated through the cancer stem cells, it becomes essential to isolate and study them. Isolation and characterization of tumor cells is a critical step in understanding the cancer progression and to devise novel approaches against them as drug targets. Typically, characterization of stem cells is done through surface marker analysis and with the advent of flow cytometry based techniques, this has become incredibly straightforward. Flow cytometry employs a uniformly linear flow of cells created by complex hydraulics of the flow cytometer followed by illuminating flow path with a LASER beam. This gives very valuable information about cell composition in forward scatter (FSC) and side scatter (SSC). The surface molecules of the cells can further be stained with various florescent dyes which upon excitation with the LASER beam will give the signal that will be detected by the instrument. Flow cytometer is high-throughput equipment and requires careful operation to get valuable information about the samples. In this chapter, we describe how from a bulk cell sample of medulloblastoma cells, cancer stem cells are isolated.