Despite thymic deletion of cells with specificity for self-antigens, autoreactive T cells are readily detectable in the normal T-cell repertoire. In recent years, a population of CD4⁺ T cells that constitutively express the interleukin-2 receptor-α chain, CD25, has been shown to play a pivotal role in the maintenance of self-tolerance in rodent models. This study investigated whether such a regulatory population exists in humans. A population of CD4⁺CD25⁺ T cells, taken from the peripheral blood of healthy individuals and phenotypically distinct from recently activated CD4⁺ T cells, was characterized. These cells were hyporesponsive to conventional T-cell stimuli and capable of suppressing the responses of CD4⁺CD25⁻ T cells in vitro. Addition of exogenous interleukin-2 abrogated the hyporesponsiveness and suppressive effects of CD4⁺CD25⁺ cells. Suppression required cell-to-cell contact but did not appear to be via the inhibition of antigen-presenting cells. In addition, there were marked changes in the expression of Notch pathway molecules and their downstream signaling products at the transcriptional level, specifically in CD4⁺CD25⁺ cells, suggesting that this family of molecules plays a role in the regulatory function of CD4⁺CD25⁺ cells. Cells with similar phenotype and function were detected in umbilical venous blood from healthy newborn infants. These results suggest that CD4⁺CD25⁺ cells represent a population of regulatory T cells that arise during fetal life. Comparison with rodent CD4⁺CD25⁺ cells suggests that this population may play a key role in the prevention of autoimmune diseases in humans.

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

Objective Rituximab appears to be effective in many studies of systemic lupus erythematosus (SLE), with variable initial clinical response and time to relapse. However, results of a randomized controlled trial of rituximab were negative. This study was undertaken to evaluate the effectiveness of rituximab in SLE, using highly sensitive flow cytometry (HSFC), which can define B cell numbers 50–100 times lower than conventional techniques and predicts responses in rheumatoid arthritis. Methods Thirty‐nine patients with active SLE were started on a standard regimen of rituximab with intravenous and oral steroids. Clinical response and relapse were defined using the British Isles Lupus Assessment Group (BILAG) index with criteria for major clinical response, partial clinical response, and nonresponse. HSFC, including analysis of B cell subsets, was performed. Results There was a significant reduction from baseline in global BILAG score at all time points analyzed (P < 0.0001), and major clinical response and partial clinical response rates were 51% and 31%, respectively. Time to relapse was highly variable. Fifty percent of the patients relapsed after 6–18 months (earlier relapse); the remainder relapsed at a slower rate (later relapse). B cell depletion and repopulation were variable and were predictive of these clinical outcomes. There was a persistent B cell presence in 21 patients after 2 infusions of rituximab, which included all 7 patients with no response (P = 0.012 versus patients with complete depletion of B cells). Memory B cell (P = 0.02) and plasmablast (P < 0.001) repopulation after 26 weeks was markedly faster in patients with earlier relapse versus patients with later relapse. Conclusion Our findings indicate that rituximab is effective in SLE, and clinical responses are supported by close correlation with B cell numbers. HSFC is a valuable tool in the assessment and prediction of response in SLE.

Aging at the cellular level is a complex process resulting from accumulation of various damages leading to functional impairment and a reduced quality of life at the level of the organism. With a rise in the elderly population, the worldwide incidence of osteoporosis (OP) and osteoarthritis (OA) has increased in the past few decades. A decline in the number and “fitness” of mesenchymal stromal cells (MSCs) in the bone marrow (BM) niche has been suggested as one of the factors contributing to bone abnormalities in OP and OA. It is well recognized that MSCs in vitro acquire culture-induced aging features such as gradual telomere shortening, increased numbers of senescent cells, and reduced resistance to oxidative stress as a result of serial population doublings. In contrast, there is only limited evidence that human BM-MSCs “age” similarly in vivo. This review compares the various aspects of in vitro and in vivo MSC aging and suggests how our current knowledge on rejuvenating cultured MSCs could be applied to develop future strategies to target altered bone formation processes in OP and OA.

Objective . Our aim was to test the hypothesis that there is a deficit in the CD4 ⁺ CD25 ^high regulatory T-cell population in early rheumatoid arthritis (RA), either in size or functional activity. Methods . Peripheral blood mononuclear cells were examined from subjects with early active RA who had received no previous disease-modifying therapy ( n = 43), from individuals with self-limiting reactive arthritis ( n = 14), from subjects with stable, well-controlled RA ( n = 82) and from healthy controls ( n = 72). The frequencies of CD4 ⁺ CD25 ^high T-cells were quantified using flow cytometry, and function was assessed by the ability to suppress proliferation of CD4 ⁺ CD25 ⁻ T-cells. Paired blood and synovial fluid was analysed from a small number of RA and reactive arthritis patients. Results . There was a smaller proportion of CD4 ⁺ CD25 ^high T-cells in the peripheral blood of early active RA patients (mean 4.25%) than in patients with reactive arthritis or in controls (mean 5.90 and 5.30%, respectively, P = 0.001 in each case). Frequencies in stable, well-controlled RA (mean 4.63%) were not significantly different from early active RA or controls. There were no differences in suppressor function between groups. Higher frequencies of CD4 ⁺ CD25 ^high T-cells were found in synovial fluid than blood in both RA and reactive arthritis. Conclusions . These data demonstrate a smaller CD4 ⁺ CD25 ^high regulatory T-cell population in peripheral blood of individuals with early active RA prior to disease-modifying treatment. This may be a contributory factor in the susceptibility to RA and suggests novel approaches to therapy.

Background: To study the biology of rare bone marrow (BM) multipotent mesenchymal stromal cells (MSCs), recognized protocols are needed. Colony‐forming unit‐fibroblast (CFU‐F) assays have historically been used for the enumeration of MSCs. However, the need to isolate and further analyze MSCs requires new strategies based on cell surface markers. The purpose of this work was to verify the phenotype of BM MSCs in vivo and to develop flow cytometry‐based methods for their evaluation.Methods: Pre‐enrichment with D7‐FIB‐conjugated microbeads, cell sorting for CD45^lowD7‐FIB⁺LNGFR⁺ cells, and CFU‐F assay were used to confirm the phenotype of BM MSCs in vivo. Further phenotypic characterization of MSCs was performed using three‐color flow cytometry following pre‐enrichment or by direct four‐color flow cytometry. The sensitivity of direct flow cytometry/rare event analysis for the accurate enumeration of MSCs was validated using 85 samples from patients with neoplastic BM diseases.Results: In normal BM, a significant correlation was found between the frequencies of CFU‐Fs and CD45^lowD7‐FIB⁺LNGFR⁺ cells (n = 19, R = 0.719, P = 0.001). Following cell sorting, ˜15% of these cells were clonogenic. The same cells were enriched using LNGFR‐based positive selection, CD45/Glycophorin A‐based depletion, or plastic adherence. CD45^lowD7‐FIB⁺LNGFR⁺ cells expressed classic makers of cultured MSCs CD73/SH3 and CD105/SH2 and markers of stromal reticular cells CD106/VCAM and alkaline phosphatase. Novel markers were identified including leukemia inhibitory factor receptor and gp130. CD45^lowD7‐FIB⁺LNGFR⁺ cells were increased fourfold in the floating fat fraction of normal BM aspirates. Their frequency was decreased in chronic lymphocytic leukemia (threefold, n = 13, P = 0.049) and chronic myelogenous leukemia (ninefold, n = 11, P = 0.001) compared with that in age‐matched controls (n = 26 and n = 31, respectively).Conclusions: This study demonstrates the usefulness of flow cytometry‐based methods for the detection, enumeration and further phenotypic analysis of BM MSCs. These findings have broad applications for the future evaluation of BM MSCs in health and disease. © 2006 International Society for Analytical Cytology