Abstract: BACKGROUND: Despite the fact that a great majority (>90%) of patients with systemic mastocytosis (SM) carry a common genetic lesion, the D816V KIT mutation, little is known regarding the molecular and biological pathways underlying the clinical heterogeneity of the disease. OBJECTIVE: We sought to analyze the gene expression profile (GEP) of bone marrow mast cells (BMMCs) in patients with SM and its association with distinct clinical variants of the disease. METHODS: GEP analyses were performed by using DNA-oligonucleotide microarrays in highly purified BMMCs from patients with SM carrying the D816V KIT mutation (n = 26) classified according to the diagnostic subtype of SM versus normal/reactive BMMCs (n = 7). Validation of GEP results was performed with flow cytometry in the same set of samples and in an independent cohort of 176 subjects. RESULTS: Overall, 758 transcripts were significantly deregulated in patients with SM, with a common GEP (n = 398 genes) for all subvariants of SM analyzed. These were characterized by upregulation of genes involved in the innate and inflammatory immune response, including interferon-induced genes and genes involved in cellular responses to viral antigens, together with complement inhibitory molecules and genes involved in lipid metabolism and protein processing. Interestingly, aggressive SM additionally showed deregulation of apoptosis and cell cycle-related genes, whereas patients with indolent SM displayed increased expression of adhesion-related molecules. CONCLUSION: BMMCs from patients with different clinical subtypes of SM display distinct GEPs, which might reflect new targetable pathways involved in the pathogenesis of the disease.
Abstract: A variable percentage of patients with systemic mast cell (MC) activation symptoms meet criteria for systemic mastocytosis (SM). We prospectively evaluated the clinical utility of the REMA score versus serum baseline tryptase (sBt) levels for predicting MC clonality and SM in 158 patients with systemic MC activation symptoms in the absence of mastocytosis in the skin (MIS).
Abstract: D816V KIT mutation of bone marrow (BM) mast cells (MC) is a common feature to systemic mastocytosis (SM) patients. Nevertheless, occurrence of the KIT mutation in BM cell compartments other than MC is associated with progression to more aggressive forms of the disease and poor outcome in indolent SM (ISM). Here, we assessed the potential association between the immunophenotype of MC and multilineage KIT mutation in the BM of SM patients through the investigation of the flow cytometric protein expression profile (PEP) of bone marrow mast cells (BMMC) from 70 control individuals and 206 SM patients, classified according to the WHO (World Health Organization), and the degree of involvement of BM hematopoiesis by the D816V KIT mutation; additionally, we developed a score-based class prediction algorithm for the detection of SM cases with multilineage mutation. Our results show that aberrant expression of CD25 with a FcÉRI(lo), FSC(lo), SSC(lo) and CD45(lo) immature phenotype of BMMC, in the absence of coexisting normal MC in the BM, was associated with multilineage involvement by the D816V KIT mutation, regardless of the diagnostic subtype of the disease (for example, indolent vs aggressive SM), which supports the utility of the immunophenotype of BMMC as a surrogate marker to screen for multilineage KIT mutation in ISM.
Abstract: Elite level athletes seem to be prone to illness especially during heavy training phases. The aim of this study was to investigate the influence of long term intensive training on the functional features of innate immune cells from high competitive level swimmers, particularly the production of inflammatory mediators and the possible relationship with upper respiratory symptoms (URS) occurrence. A group of 18 swimmers and 11 healthy non athletes was studied. Peripheral blood samples were collected from athletes after 36 h of resting recovery from exercise at four times during the training season and at three times from non athletes. Samples were incubated in the presence or absence of LPS and IFN-γ and the frequency of cytokine-producing cells and the amount produced per cell were evaluated by flow cytometry. In addition, plasma cortisol levels were measured and URS recorded through daily logs. The athletes, but not the controls, showed a decrease in the number of monocytes, neutrophils, and dendritic cell (DC) subsets and in the amount of IL-1β, IL-6, IL-12, TNF-α, and MIP-1β produced after stimulation, over the training season. Differences were most noticeable between the first and second blood collections (initial increase in training volume). Athlete's cortisol plasma levels partially correlated with training intensity and could help explain the reduced in vitro cell response to stimulation. Our results support the idea that long-term intensive training may affect the function of innate immune cells, reducing their capacity to respond to acute challenges, possibly contributing to an elevated risk of infection.
Abstract: Despite the good prognosis of pediatric mastocytosis, some patients suffer from severe mast cell (MC) mediator-associated symptoms. The aim of this study was to identify predictors for severe MC mediator release symptoms in children with mastocytosis in the skin (MIS).
Abstract: Aberrant expression of CD2 and/or CD25 by bone marrow, peripheral blood or other extracutaneous tissue mast cells is currently used as a minor World Health Organization diagnostic criterion for systemic mastocytosis. However, the diagnostic utility of CD2 versus CD25 expression by mast cells has not been prospectively evaluated in a large series of systemic mastocytosis. Here we evaluate the sensitivity and specificity of CD2 versus CD25 expression in the diagnosis of systemic mastocytosis. Mast cells from a total of 886 bone marrow and 153 other non-bone marrow extracutaneous tissue samples were analysed by multiparameter flow cytometry following the guidelines of the Spanish Network on Mastocytosis at two different laboratories. The 'CD25+ and/or CD2+ bone marrow mast cells' World Health Organization criterion showed an overall sensitivity of 100% with 99.0% specificity for the diagnosis of systemic mastocytosis whereas CD25 expression alone presented a similar sensitivity (100%) with a slightly higher specificity (99.2%). Inclusion of CD2 did not improve the sensitivity of the test and it decreased its specificity. In tissues other than bone marrow, the mast cell phenotypic criterion revealed to be less sensitive. In summary, CD2 expression does not contribute to improve the diagnosis of systemic mastocytosis when compared with aberrant CD25 expression alone, which supports the need to update and replace the minor World Health Organization 'CD25+ and/or CD2+' mast cell phenotypic diagnostic criterion by a major criterion based exclusively on CD25 expression.
Abstract: With the purpose of contributing to a better knowledge of the APCs functional activity in SLE, we evaluated the distribution and functional ability to produce pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-12) of peripheral blood (PB) monocytes and DC (tDC), particularly myeloid (mDC) and CD14(-/low)CD16(+) DC subpopulations comparing them with those obtained from healthy individuals. The study was performed in 34 SLE patients with diverse disease activity scores (SLEDAI) and 13 healthy age- and sex-matched controls (NC). Our results show an overall decrease in absolute number and relative frequency of tDC in SLE patients with active disease when compared to those with inactive disease and NC, although this decrease did not seem to have an effect on the distribution of PB DC subsets. The monocytes number in SLE patients was similar to those found in NC, whereas a higher frequency of monocytes producing cytokines as well as the amount of each cytokine per cell found without stimulation was particularly observed in those patients with active disease. After stimulation, we observed a higher frequency of IL-12-producing monocytes in active SLE patients. On the other hand, we found among DCs higher frequencies of cytokine-producing CD14(-/low)CD16(+) DCs and a higher amount of cytokines produced per cell, particularly in active disease. These findings support an increased production of inflammatory cytokines by APCs in active SLE, mostly associated with alterations in CD14(-/low)CD16(+) DC subset homeostasis that might contribute to explain the dynamic role of these cells in disease pathogenesis.
Abstract: Mastocytosis comprises a heterogeneous group of disorders characterized by the presence of clonal mast cells (MC) in organs such as skin, bone marrow (BM), and gastrointestinal tract, among other tissues. The clonal nature of the disease can be established in most adult patients by the demonstration of activating KIT mutations in their BM MC. When highly sensitive techniques capable of identifying cells present at very low frequencies in a sample are applied, BM MC from virtually all systemic mastocytosis patients display unique immunophenotypical features, particularly the aberrant expression of CD25. By contrast, large, multifocal BM MC aggregates (the only World Health Organization major criterion for systemic mastocytosis) are absent in a significant proportion of patients fulfilling at least three minor criteria for systemic mastocytosis, particularly in subjects studied at early stages of the disease with very low MC burden. Moreover, recent molecular and immunophenotypical investigations of BM MC from patients with indolent systemic mastocytosis have revealed a close association of some biological features (e.g., multilineage involvement of hematopoiesis by the KIT mutation and an immature mast cell immunophenotype) with an increased risk for disease progression. These observations support the fact that, although the current consensus diagnostic criteria for systemic mastocytosis have been a major advance for the diagnosis and classification of the disease, rationale usage of the most sensitive diagnostic techniques available nowadays is needed to improve the diagnosis, refine the classification, and reach objective prognostic stratification of adult mastocytosis.
Abstract: Eisenia andrei was exposed, for 56 days, to a contaminated soil from an abandoned uranium mine and to the natural reference soil LUFA 2.2. The organisms were sampled after 0, 1, 2, 7, 14 and 56 days of exposure, to assess metals bioaccumulation, coelomocytes DNA integrity and cytotoxicity. Radionuclides bioaccumulation and growth were also determined at 0 h, 14 and 56 days of exposure. Results have shown the bioaccumulation of metals and radionuclides, as well as, growth reduction, DNA damages and cytotoxicity in earthworms exposed to contaminated soil. The usefulness of the comet assay and flow cytometry, to evaluate the toxicity of contaminants such as metals and radionuclides in earthworms are herein reported. We also demonstrated that DNA strand breakage and immune cells frequency are important endpoints to be employed in the earthworm reproduction assay, for the evaluation of soil geno and cytotoxicity, as part of the risk assessment of contaminated areas. This is the first study that integrates DNA damage and cytotoxicity evaluation, growth and bioaccumulation of metals and radionuclides in a sub lethal assay, for earthworms exposed to soil contaminated with metals and radionuclides.
Abstract: The impact of pregnancy on mast cell (MC)-related symptoms and newborn outcome in women with mastocytosis is not well described. We report a series of 30 women who had 45 pregnancies.
Abstract: Diagnosis and classification of mastocytosis is currently based on the World Health Organization (WHO) criteria. Here, we evaluate the utility of the WHO criteria for the diagnosis and classification of a large series of mastocytosis patients (n=133), and propose a new algorithm that could be routinely applied for refined diagnosis and classification of the disease. Our results confirm the utility of the WHO criteria and provide evidence for the need of additional information for (1) a more precise diagnosis of mastocytosis, (2) specific identification of new forms of the disease, (3) the differential diagnosis between cutaneous mastocytosis vs systemic mastocytosis, and (4) improved distinction between indolent systemic mastocytosis and aggressive systemic mastocytosis. Based on our results, a new algorithm is proposed for a better diagnostic definition and prognostic classification of mastocytosis, as confirmed prospectively in an independent validation series of 117 mastocytosis patients.
Abstract: Basophil activation tests (BATs) have been demonstrated to be useful in detecting IgE-mediated sensitization by measuring basophil activation surface markers (CD63 and CD203c). Hymenoptera venom is one of the best known mediators-release trigger in patients with systemic mastocytosis (SM). The aim of this study was to investigate the use of BATs as an additional diagnostic tool in patients with mastocytosis suffering from hymenoptera venom anaphylaxis (HVA).
Abstract: To evaluate dermoscopic features in a group of 127 patients with mastocytosis in the skin and to investigate the relationship between different dermoscopic patterns and other clinical and biological characteristics of the disease.
Abstract: To compare frequency and functional activity of peripheral blood (PB) Th(c)17, Th(c)1 and Treg cells and the amount of type 2 cytokines mRNA we recruited SLE patients in active (n=15) and inactive disease (n=19) and healthy age- and gender-matched controls (n=15). The study of Th(c)17, Th(c)1 and Treg cells was done by flow cytometry and cytokine mRNA by real-time PCR. Compared to NC, SLE patients present an increased proportion of Th(c)17 cells, but with lower amounts of IL-17 per cell and also a decreased frequency of Treg, but with increased production of TGF-beta and FoxP3 mRNA. Iotan active compared to inactive SLE, there is a marked decreased in frequency of Th(c)1 cells, an increased production of type 2 cytokines mRNA and a distinct functional profile of Th(c)17 cells. Our findings suggest a functional disequilibrium of T-cell subsets in SLE which may contribute to the inflammatory process and disease pathogenesis.
Abstract: Allergic rhinoconjunctivitis induced by pollen is a highly prevalent chronic inflammatory disease in Europe. Parietariajudaica is a frequent trigger in the Mediterranean area. The function of regulatory T cells (Treg cells) in allergy has recently been investigated, but further data are necessary to better understand their role and to find new strategies to treat allergic diseases such as allergic rhinoconjunctivitis.
Abstract: In recent years, mesenchymal stem cells (MSC) have been attracting the greatest interest in the regeneration of injured tissues, autoimmune diseases, and transplantation of hematopoietic progenitor cells. Bone marrow (BM) represents the major source of MSC; however, umbilical cord blood (UCB) MSC has some advantages over BM, such as the higher differentiation capability and noninvasive collection methods. We sought to establish a 7-color, single-tube flow cytometric assay to quantify MSC in fresh tissues, namely BM and UCB, based on phenotypic markers of these cells. Moreover, we evaluated the differential expression of these markers in BM and UCB MSC. We used 5 UCB samples and 5 BM samples obtained from individuals without hematologic disease. To characterize MSC we used the following combination of monoclonal antibodies: CD71-FITC; CD105-PE; CD184-PE-Cy5; CD34-PE-Cy7; CD133-APC; CD45-APC-H7; CD44-Pacific blue, acquiring at least 1 million nucleated cells. We observed a greater number of BM MSC when compared with UCB MSC as well as some differences in the expression of some MSC antigens, particularly CD105 and CD44. Based on our preliminary results, phenotypic identification of MSC by flow cytometry is possible using a 7-color, single-tube assay. However, culture assays after sorting of cells characterized in this study are required to prove that they correspond to MSC.
Abstract: Umbilical cord blood (UCB) has been used as an alternative source of haematopoietic progenitors for transplantation presenting advantages over bone marrow (BM) that are related with known shortages of newborns' immune system at adaptive and innate levels. Using flow cytometry, we studied the expression of Toll-like receptors (TLRs) and chemokine receptors (CKRs) and the production of pro-inflammatory cytokines by monocytes and CD14(-/low)/CD16(+)DCs from peripheral blood (PB; n=10), and umbilical cord blood (UCB; n=10). CKRs and cytokines were studied before and after stimulation of cells with LPS plus IFN-gamma. We also identified the two populations in normal bone marrow samples (BM; n=5). BM presented lower frequencies of both studied populations when compared to UCB and PB. CD14(-/low)/CD16(+)DCs presented a pattern of TLR expression different from mature monocytes reflecting distinct functions for these two populations. UCB cells presented reduced expression of TLR-4 and lower capability to produce cytokines prior stimulation. The populations studied presented different patterns of CKR expression reflecting distinct migratory pathways. Moreover, UCB cells presented higher expressions of CXCR4 and CCR7 that may be involved in immune system maturation and stem cell homing. Monocytes and CD14(-/low)/CD16(+)DCs present functional and phenotypical characteristics that may contribute to the lower incidence and severity of GVHD.
Abstract: Chemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response, namely in allergic contact dermatitis (ACD). In this work, we investigated by flow cytometry the effect of the contact sensitizers 2,4-dinitrofluorobenzene (DNFB), 1,4-phenylenediamine (PPD) and nickel sulfate (NiSO(4)), on the surface expression of the chemokine receptors CCR6 and CXCR4 in DC. As an experimental model of a DC we used a fetal skin-derived dendritic cell line (FSDC), which has morphological, phenotypical and functional characteristics of skin DC. Our results show that all the skin sensitizers studied decreased the membrane expression of the chemokine receptors CCR6 and CXCR4. In contrast, 2,4-dichloronitrobenzene (DCNB), the inactive analogue of DNFB without contact sensitizing properties, was without effect on the surface expression of these receptors. Lipopolysaccharide (LPS), which induces the maturation of DC, also reduced surface CCR6 and CXCR4 expression.
Abstract: Mastocytosis is a term used to designate a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow (BM), liver, spleen, and lymph nodes, among others. Recent advances in our understanding of mast cell biology and disease resulted in the identification of important differences in the expression of mast cell surface antigens between normal and neoplastic mast cells. Most notably, detection of aberrant expression of CD25 and CD2 on the surface of neoplastic mast cells but not on their normal counterparts lead to the inclusion of this immunophenotypic abnormality in the World Health Organization diagnostic criteria for systemic mastocytosis. Aberrant mast cell surface marker expression can be detected in the bone marrow aspirate by flow cytometry, even in patients lacking histopathologically detectable aggregates of mast cells in bone marrow biopsy sections. These aberrant immunophenotypic features are of great relevance for the assessment of tissue involvement in mastocytosis with consequences in the diagnosis, classification, and follow-up of the disease and in its differential diagnosis with other entities. In this chapter, we provide the reader with information for the objective and reproducible identification of pathologic MCs by using quantitative multiparametric flow cytometry, for their phenotypic characterization, and the criteria currently used for correct interpretation of the immunophenotypic results obtained.
