Helen A. Papadaki

Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2

Mice lacking the transcriptional repressor oncoprotein Gfi1 are unexpectedly neutropenic. We therefore screened GFI1 as a candidate for association with neutropenia in affected individuals without mutations in ELA2 (encoding neutrophil elastase), the most common cause of severe congenital neutropenia (SCN; ref. 3). We found dominant negative zinc finger mutations that disable transcriptional repressor activity. The phenotype also includes immunodeficient lymphocytes and production of a circulating population of myeloid cells that appear immature. We show by chromatin immunoprecipitation, gel shift, reporter assays and elevated expression of ELA2 in vivo in neutropenic individuals that GFI1 represses ELA2, linking these two genes in a common pathway involved in myeloid differentiation.

Immunohistochemical profile of endometrial adenocarcinoma: a study of 61 cases and review of the literature.

The differences in immunohistochemical expression of p53, bcl-2, bax, estrogen receptor (ER), and progesterone receptor (PR) were evaluated in 40 endometrioid and 21 papillary serous carcinomas of endometrium and correlated with known predictors of survival, such as grade and stage. Uterine papillary serous adenocarcinomas (UPSA) showed significantly higher p53 expression than did uterine endometrioid adenocarcinomas (UEA) (76.2% versus 35%), whereas both ER and PR were more often positive in endometrioid than in serous tumors (p =.005 and.0005). No significant difference was found in bcl-2 and bax expression between both histologic types. However, there was definite decrease in intensity of bcl-2 in UPSA compared with UEA. In endometrioid carcinoma, p53 overexpression was associated with high-grade and advanced-stage tumors (p =.0006 and.006), whereas ER and PR expression was associated with low-grade and early-stage tumors (p =.0006 and.0001; p =.003 and.0006). Bcl-2 immunopositivity was more common in low-grade, early-stage rather than in high-grade, advanced-stage adenocarcinomas, but the difference was not statistically significant (p =.24 and.07). Bax immunopositivity was associated with well-differentiated (p =.04) and early-stage tumors. Furthermore, a significant inverse relationship between bax and p53 reactivity was defined (p =.05), especially in tumors of endometrioid type. Bax and PR immunoexpression correlated near the limit of statistical significance (p =.08), whereas no relationship was found among bax, bcl-2, and ER immunopositivity. Our results indicate that the differences in immunohistochemical profiles of endometrioid and serous carcinomas support the existence of different molecular pathways of their development. The correlation of immunohistochemical findings with histologic grade and clinical stage could help in predicting biologic behavior and planning treatment in patients who are diagnosed as having these tumors.

Functional, molecular and proteomic characterisation of bone marrow mesenchymal stem cells in rheumatoid arthritis

Objective: Bone marrow (BM) mesenchymal stem cells (MSCs) are being considered as potential therapeutic agents in various inflammatory autoimmune diseases for their tissue-repair and anti-inflammatory tissue-protective properties. This study investigates the reserves and function, the molecular and proteomic profile and the differentiation potential of BM MSCs in patients with active rheumatoid arthritis (RA). Methods: We evaluated the frequency of MSCs in the BM mononuclear cell fraction using a limiting dilution assay, the proliferative/clonogenic potential and the capacity of cells to differentiate towards the osteogenic/chondrogenic/adipogenic lineages using appropriate culture conditions. We also assessed the molecular and proteomic characteristics in terms of inflammatory cytokine gene and protein expression, the relative telomere length and the survival characteristics of BM MSCs. Results: MSCs from patients with RA (n = 26) and age- and sex-matched healthy individuals (n = 21) were similar in frequency, differentiation potential, survival, immunophenotypic characteristics, and protein profile. Patient MSCs, however, had impaired clonogenic and proliferative potential in association with premature telomere length loss. Transcriptome analysis revealed differential expression of genes related to cell adhesion processes and cell cycle progression beyond the G1 phase. Previous treatment with methotrexate, corticosteroids, anti-cytokine and biological agents or other disease-modifying anti-inflammatory drugs did not correlate with the clonogenic and proliferative impairment of BM MSCs. Conclusion: In spite of some restrictions related to the impaired clonogenic and proliferative potential, our findings support the use of autologous BM MSCs in RA and may have important implications for the ongoing efforts to repair tissue injury commonly seen in the course of the disease.

Mesenchymal stem cells derived from Wharton's Jelly of the umbilical cord: biological properties and emerging clinical applications.

In recent years there seems to be an unbounded interest concerning mesenchymal stem cells (MSCs). This is mainly attributed to their exciting characteristics including long-term ex vivo proliferation, multilineage potential and immunomodulatory properties. In this regard MSCs emerge as attractive candidates for various therapeutic applications. MSCs were originally isolated from the bone marrow (BM) and this population is still considered as the gold standard for MSC applications. Nevertheless the BM has several limitations as source of MSCs, including MSC low frequency in this compartment, the painful isolation procedure and the decline in MSC characteristics with donors age. Thus, there is accumulating interest in identifying alternative sources for MSCs. To this end MSCs obtained from the Whartons Jelly (WJ) of umbilical cords (UC) have gained much attention over the last years since they can be easily isolated, without any ethical concerns, from a tissue which is discarded after birth. Furthermore WJ-derived MSCs represent a more primitive population than their adult counterparts, opening new perspectives for cell-based therapies. In this review we will at first give an overview of the biology of WJ-derived UC-MSCs. Then their potential application for the treatment of cancer and immune mediated disorders, such graft versus host disease (GVHD) and systemic lupus erythematosus (SLE) will be discussed, and finally their putative role as feeder layer for ex vivo hematopoietic stem cell (HSC) expansion will be pointed out.

Bone Marrow Mesenchymal Stem Cells: Biological Properties and Their Role in Hematopoiesis and Hematopoietic Stem Cell Transplantation

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that are present in practically all tissues as a specialized population of mural cells/pericytes that lie on the abluminal side of blood vessels. Originally identified within the bone marrow (BM) stroma, not only do they provide microenvironmental support for hematopoietic stem cells (HSCs), but can also differentiate into various mesodermal lineages. MSCs can easily be isolated from the BM and subsequently expand in vitro and in addition they exhibit intriguing immunomodulatory properties, thereby emerging as attractive candidates for various therapeutic applications. This review addresses the concept of BM MSCs via a hematologist’s point of view. In this context it discusses the stem cell properties that have been attributed to BM MSCs, as compared to those of the prototypic hematopoietic stem cell model and then gives a brief overview of the in vitro and vivo features of the former, emphasizing on their immunoregulatory properties and their hematopoiesis-supporting role. In addition, the qualitative and quantitative characteristics of BM MSCs within the context of a defective microenvironment, such as the one characterizing Myelodysplastic Syndromes are described and the potential involvement of these cells in the pathophysiology of the disease is discussed. Finally, emerging clinical applications of BM MSCs in the field of hematopoietic stem cell transplantation are reviewed and potential hazards from MSC use are outlined.

Rituximab therapy reduces activated B cells in both the peripheral blood and bone marrow of patients with rheumatoid arthritis: depletion of memory B cells correlates with clinical response

IntroductionBone marrow (BM) is an immunologically privileged site where activated autoantibody-producing B cells may survive for prolonged periods. We investigated the effect of rituximab (anti-CD20 mAb) in peripheral blood (PB) and BM B-cell and T-cell populations in active rheumatoid arthritis (RA) patients.MethodsActive RA patients received rituximab (1,000 mg) on days 1 and 15. PB (n = 11) and BM (n = 8) aspirates were collected at baseline and at 3 months. We assessed B-cell and T-cell populations using triple-color flow cytometry.ResultsRituximab therapy decreased PB (from a mean 2% to 0.9%, P = 0.022) but not BM (from 4.6% to 3.8%, P = 0.273) CD19+ B cells, associated with a significant reduction in the activated CD19+HLA-DR+ subset both in PB (from 55% to 19%, P = 0.007) and in BM (from 68% to 19%, P = 0.007). Response to rituximab was preceded by a significant decrease in PB and BM CD19+CD27+ memory B cells (P = 0.022). These effects were specific to rituximab since anti-TNF therapy did not reduce total or activated B cells. Rituximab therapy did not alter the number of activated CD4+HLA-DR+ and CD4+CD25+ T cells.ConclusionsRituximab therapy preferentially depletes activated CD19+HLA-DR+ B cells in the PB and BM of active RA patients. Clinical response to rituximab is associated with depletion of CD19+CD27+ memory B cells in PB and BM of RA patients.

Increased apoptosis of bone marrow CD34+ cells and impaired function of bone marrow stromal cells in patients with systemic lupus erythematosus

The changes in bone marrow (BM) stem cell reserve and function and stromal cell function in patients with active systemic lupus erythematosus (SLE) were investigated. The study was carried out on seven SLE patients and 28 healthy controls using flow cytometry and in vitro cell culture assays. We found that patients had low CD34+ cells, compared with the control group, reflecting the decrease of both CD34+/CD38− and CD34+/CD38+ cells. Patient CD34+/Fas+ but not CD34−/Fas+ cells were significantly increased. Apoptotic (7AADdim) cells were higher among CD34+/Fas+ than among CD34+/Fas− cells, and individual values of apoptotic CD34+ cells strongly correlated with the number of CD34+/Fas+ cells. These findings are suggestive of a Fas‐mediated apoptosis accounting for the low CD34+ cells in SLE patients. Moreover, we found that patients had low numbers of granulocyte‐macrophage colony‐forming units (CFU‐GM) and erythroid burst‐forming units (BFU‐E), compared with the control group, and that the generation of colony‐forming cells in long‐term BM cultures was significantly reduced. Patient BM stroma failed to support allogeneic progenitor cell growth. In one patient, CD34+ cells were increased, apoptotic CD34+/Fas+ cells were normalized and defective stromal cell function was restored after autologous stem cell transplantation. We concluded that defective haemopoiesis in SLE patients is probably caused, at least in part, to the presence of autoreactive lymphocytes in BM.

Comparative proteomic analysis of human mesenchymal and embryonic stem cells: Towards the definition of a mesenchymal stem cell proteomic signature

Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2‐DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo‐natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.

Interleukin-7 deficiency in rheumatoid arthritis: consequences for therapy-induced lymphopenia

We previously demonstrated prolonged, profound CD4+ T-lymphopenia in rheumatoid arthritis (RA) patients following lymphocyte-depleting therapy. Poor reconstitution could result either from reduced de novo T-cell production through the thymus or from poor peripheral expansion of residual T-cells. Interleukin-7 (IL-7) is known to stimulate the thymus to produce new T-cells and to allow circulating mature T-cells to expand, thereby playing a critical role in T-cell homeostasis. In the present study we demonstrated reduced levels of circulating IL-7 in a cross-section of RA patients. IL-7 production by bone marrow stromal cell cultures was also compromised in RA. To investigate whether such an IL-7 deficiency could account for the prolonged lymphopenia observed in RA following therapeutic lymphodepletion, we compared RA patients and patients with solid cancers treated with high-dose chemotherapy and autologous progenitor cell rescue. Chemotherapy rendered all patients similarly lymphopenic, but this was sustained in RA patients at 12 months, as compared with the reconstitution that occurred in cancer patients by 3–4 months. Both cohorts produced naïve T-cells containing T-cell receptor excision circles. The main distinguishing feature between the groups was a failure to expand peripheral T-cells in RA, particularly memory cells during the first 3 months after treatment. Most importantly, there was no increase in serum IL-7 levels in RA, as compared with a fourfold rise in non-RA control individuals at the time of lymphopenia. Our data therefore suggest that RA patients are relatively IL-7 deficient and that this deficiency is likely to be an important contributing factor to poor early T-cell reconstitution in RA following therapeutic lymphodepletion. Furthermore, in RA patients with stable, well controlled disease, IL-7 levels were positively correlated with the T-cell receptor excision circle content of CD4+ T-cells, demonstrating a direct effect of IL-7 on thymic activity in this cohort.

Isolation of Human Bone Marrow Mesenchymal Stem Cells Using Different Membrane Markers: Comparison of Colony/Cloning Efficiency, Differentiation Potential, and Molecular Profile

Bone marrow (BM) mesenchymal stem cells (MSCs) represent an interesting field of research for their in vitro properties and the in vivo therapeutic applications. In the present study, we compared the clonogenic and differentiation capacity of MSCs present in three BM-derived populations-namely, the CD105(+)/CD45(-) cells, the glycophorin A (GlycoA)(-)/CD45(-) cells, and the BM mononuclear cells (BMMCs)-by growing/expanding clones from single colony-forming unit fibroblasts (CFU-F). We also quantified the Oct-4 and Nanog mRNA in the CD105(+)/CD45(-) and GlycoA(-)/CD45(-) cells to define the fraction containing more immature MSCs. We found that basic-fibroblast growth factor (bFGF) favors the long-term survival and growth of the more immature MSCs but has no significant effect on MSC clonogenic potential. CFU-F number and clone recovery were higher in CD105(+)/CD45(-) compared to GlycoA(-)/CD45(-) (p < 0.0001 and p = 0.0364, respectively) cells or BMMCs (p < 0.0001 and p = 0.0007, respectively). The relative mRNA expression of Oct-4 and Nanog was significantly increased in CD105(+)/CD45(-) compared to GlycoA(-)/CD45(-) cells (p < 0.0001 and p < 0.0001, respectively). No significant difference was found in the immunophenotypic characteristics and differentiation potential of clones derived from all three cellular sources. These data suggest that the CD105(+)/CD45(-) BM cell fraction is enriched in immature MSCs and, accordingly, represents an appropriate source for MSC culture initiation.