Bone marrow tryptase as a possible diagnostic criterion for adult systemic mastocytosis.

BACKGROUND: Mastocytosis is difficult to diagnose, especially when systemic mast cell activation symptoms are not present or involve only one extracutaneous organ. OBJECTIVE: The main objective was to evaluate the accuracy of the bone marrow tryptase level in the diagnosis of systemic mastocytosis in patients with a clinical suspicion of mastocytosis. METHODS: We included all adult patients evaluated in our centre between December 2009 and 2013 for suspected mastocytosis as part of a standardized procedure and who had a bone marrow and serum tryptase assay on the same day. The diagnosis of systemic mastocytosis was established on the basis of the World Health Organization criteria as the gold standard. The accuracy of the bone marrow tryptase level in the diagnosis of systemic mastocytosis was assessed by a receiver operating characteristics curve analysis. The different sensitivity and specificity values, corresponding to the set of possible bone marrow tryptase level cut-off values, were estimated with 95% confidence intervals. RESULTS: Seventy-three patients were included. The diagnosis of systemic mastocytosis was established in 43 patients (58.9%). The median bone marrow tryptase level was 423 µg/L [95% CI: 217-868] in the systemic mastocytosis group and 7.5 µg/L [95% CI: 4.6-17.1] in the non-systemic mastocytosis group (P < 0.001). A cut-off value of 50 µg/L for bone marrow tryptase identified systemic mastocytosis with a sensitivity of 93.0% [95% CI: 80.9-98.5%] and a specificity of 90.0% [95% CI: 73.5-97.9%]. CONCLUSION AND CLINICAL RELEVANCE: The bone marrow tryptase level appears to be a valuable diagnostic criterion for confirming systemic mastocytosis. If this diagnosis can reliably be excluded by evaluation of the bone marrow tryptase level, there would be no need to perform a bone marrow biopsy.

Immuno-metabolism and adipose tissue: The key role of hematopoietic stem cells.

The field of immunometabolism has come a long way in the past decade, leading to the emergence of a new role for white adipose tissue (WAT) that is now recognized to stand at the junction of immune and metabolic regulations. Interestingly, a crucial role of the abundant and heterogeneous immune population present in WAT has been proposed in the induction and development of metabolic diseases. Although a large body of data focused on mature immune cells, only few scattered studies are dedicated to leukocyte production, and the activity of hematopoietic stem cells (HSC) in these pathological states. Considering that blood cell production and the differentiation of HSCs and their progeny is orchestrated, in part, by complex interacting signals emanating from their microenvironment, it thus seems worth to better understand the relationships between metabolism and HSC. This review discusses the alterations of hematopoietic process described in metabolic diseases and focused on the emerging data concerning HSC present in WAT.

[Use of adipose tissue in regenerative medicine]. / Tissu adipeux et médecine régénératrice.

Adipose tissue is abundant and well known for its involvement in obesity and associated metabolic disorders. Its uses in regenerative medicine recently attracted many investigators, as large amounts of this tissue can be easily obtained using liposuction and it contains several populations of immature cells. The largest pool of such cells corresponds to immature stromal cells, called adipose-derived stromal cells (ADSCs). These cells are purified after proteolytic digestion of adipose tissue and selection by an adherent step. ADSCs display many common features with mesenchymal stem cells derived from bone marrow, including paracrine activity, but with some specific features, among which a greater angiogenic potential. This potential is now investigating at clinical level to treat critical ischemic hindlimb by autologous cells. Other potentials are also investigated and the treatment of fistula associated or not with Crohn's disease is reaching now phase III level.

Bone marrow mesenchymal stem cells are abnormal in multiple myeloma.

Recent literature suggested that cells of the microenvironment of tumors could be abnormal as well. To address this hypothesis in multiple myeloma (MM), we studied bone marrow mesenchymal stem cells (BMMSCs), the only long-lived cells of the bone marrow microenvironment, by gene expression profiling and phenotypic and functional studies in three groups of individuals: patients with MM, patients with monoclonal gamopathy of undefined significance (MGUS) and healthy age-matched subjects. Gene expression profile independently classified the BMMSCs of these individuals in a normal and in an MM group. MGUS BMMSCs were interspersed between these two groups. Among the 145 distinct genes differentially expressed in MM and normal BMMSCs, 46% may account for a tumor-microenvironment cross-talk. Known soluble factors implicated in MM pathophysiologic features (i.e. IL (interleukin)-6, DKK1) were revealed and new ones were found which are involved in angiogenesis, osteogenic differentiation or tumor growth. In particular, GDF15 was found to induce dose-dependent growth of MOLP-6, a stromal cell-dependent myeloma cell line. Functionally, MM BMMSCs induced an overgrowth of MOLP-6, and their capacity to differentiate into an osteoblastic lineage was impaired. Thus, MM BMMSCs are abnormal and could create a very efficient niche to support the survival and proliferation of the myeloma cells.

Plasticity of adipose tissue: a promising therapeutic avenue in the treatment of cardiovascular and blood diseases?

The adipose tissue represents a large amount of adult tissues. For long time, it was considered as a poorly active overgrown and undesirable tissue even if its usefulness was demonstrated in reconstructive surgery. It was studied for its main involvement in energy metabolism and disorders as diabetes and obesity. More recently, its endocrine functions emerged and appeared to play a key role in many physiological situations such as inflammation and immunity. The presence of preadipocytes throughout life was demonstrated using primary culture technology from cells derived from adipose tissue. These cells can display a macrophagic or endothelial potential according to their environment and could be now considered as vascular progenitors. Differentiation of various adipose derived cell subsets towards functional cardiomyocytes, osteoblasts, haematopoietic and neural cells was also obtained in vitro. Altogether, these data emphasise the need to consider with a new look preadipocyte status and adipose tissue biology. These spectacular data, together with the fact that adipose tissue is easy to obtain lead to numerous and promising perspectives in regenerative medicine. They highlight the concept that progenitor cells from adipose tissue constitute an alternative for cells-based strategies designed for the treatment of cardiovascular diseases.

[Adipose tissue, plastic and reconstructive surgery: come back to sources]. / Tissus adipeux, chirurgie plastique et reconstructrice: le retour aux sources.

The adipose tissue represents a large amount of adult tissue. For long time, it was considered as a filling tissue and used in plastic and reconstructive surgery. It was always studied for its main involvement in energy metabolism and energy disorders as diabetes and obesity. More recently, its endocrine functions emerged and thus play a key role in many physiological functions as inflammation and immunity. The presence of preadipocytes throughout life was demonstrated using primary culture technology from cells derived from adipose tissue. In recent papers, cells derived from adipose tissue were used for haematopoiesis, vascularisation or skeletal muscle recovery. Differentiation into functional cardiomyocytes, osteoblasts and neural cells was obtained in vitro. These spectacular data, the fact that adipose tissue is easy to sample and the possibility to create cell or tissue banks open numerous and promising perspectives in regenerative medicine.

Inflammatory/haematopoietic cytokine production by human bone marrow adipocytes.

Adipocytes are now considered as secretory and endocrine cells. White and brown adipocytes synthesize and secrete a variety of cytokines, among a number of peptide and non-peptide products. Some of these cytokines, particularly IL-6 and TNF-alpha, appear multifunctional since they may be involved in the control of adipose mass, inflammatory response and haematopoiesis. Bone marrow adipocytes are another abundant type of adipocytes, but their precise role in humans is poorly understood. In the present study, we demonstrate that, in contrast to non-medullary adipocytes, human bone marrow adipocytes in primary culture secrete only trace amounts of IL-1 beta and TNF-alpha, but, they secrete significant and regulated levels of IL-6. These results reinforce the concept of functional heterogeneity of adipose tissues according to their anatomical localization, and indicate that bone marrow adipocytes may contribute to the complex network of cytokines involved in the control of haematopoiesis.

Regulation by cytokines of leptin expression in human bone marrow adipocytes.

Leptin is a hormone secreted by adipocytes. Besides controlling appetite and body weight, it has been suggested that leptin plays a role in inflammation and hemopoiesis. In this study we demonstrate that the pro-inflammatory/hemopoietic cytokines, IL-1beta, IL-6, TNF-alpha, and interferon-gamma, significantly inhibit gene expression and secretion of leptin by bone marrow adipocytes. These findings are in agreement with the data recently obtained from non-medullary adipose tissues. Within the bone marrow environment, leptin regulation by these pleiotropic cytokines could contribute to controlling the proliferation and differentiation of hemopoietic precursors as well as the maturation of stromal cells.

High concentration of leptin stimulates myeloid differentiation from human bone marrow CD34+ progenitors: potential involvement in leukocytosis of obese subjects.

OBJECTIVE AND DESIGN: As well as its involvement in control of adipose mass and body energy balance, several reports suggest a link between leptin and hemopoiesis. To test its putative role in human hemopoiesis, we developed a homologous system, ie recombinant human leptin treatment of purified CD34+ progenitors from adult human bone marrow. RESULTS: Leptin (50-100 ng/ml) significantly stimulated the appearance of granulocyte-macrophage colonies in the presence or absence of erythropoietin. The concentration of leptin required for this effect was rather high but within the range of plasma leptin levels observed in obesity. Two results further support the hypothesis that leptin may be involved in the leukocytosis associated with obesity: (i) leptin concentrations in bone marrow and plasma of subjects studied were highly correlated; (ii) leptin and leukocyte count were correlated only in obese subjects. Paracrine effects of locally released leptin from bone marrow adipocytes could also be involved in the regulation of hemopoiesis, a hypothesis supported by marrow immunocytochemistry revealing the close association of CD34+ cells with adipocytes and by previous demonstration that leptin is secreted at a high level by these cells. CONCLUSION: These results indicate that leptin acts on human multilineage CD34+ cells and that high plasma leptin levels associated with obesity could participate in the differentiation of granulocytes from hemopoietic progenitors.

Evaluation of the Vega haematology analyser in a university hospital setting.

The performance of the ABX Vega haematology analyser was compared with that of the Sysmex NE-8000, with specific attention to flagging performance and ergonomics. Eight hundred routine samples underwent precision and interinstrument variability studies and 168 samples corresponding to various blood disorders were studied meanwhile. Results from the two instruments gave excellent correlation (r > 0.900) for most parameters except MCHC (r = 0.114), basophil and monocyte percentages (r = 0.617 and 0.552, respectively). The reproducibility, repeatability, linearity, carry-over and stability of the Vega were satisfactory; 'flagging' occurred in 31% of routine samples with sensitivity 88.8%, specificity 41.3% and positive predictive value 85.7%. Various flags appeared in 91% (42/46) of cases where blast cells were microscopically identified. In the four remaining cases, CBC anomalies would themselves have justified microscopic examination of a smear. On 'CBC only' mode reagent consumption was significantly reduced. In the laboratory the analyser was best appreciated for its user-friendliness.

A role for preadipocytes as macrophage-like cells.

Several lines of evidence have supported a link betweeen adipose tissue and immunocompetent cells. This link is illustrated in obesity, where excess adiposity and impaired immune function have been described in both humans and genetically obese rodents. In addition, numerous factors involved in inflammatory response are secreted by both preadipocytes and macrophages. Here we show that proliferating preadipocytes in cell lines and primary cultures, develop phagocytic activity toward microorganisms. This is demonstrated by phagocytosis assays and confocal microscopy. This function disappears when preadipocytes stop proliferating and differentiate into adipocytes. After phagocytosis, preadipocytes show microbicide activity via an oxygen-dependent mechanism. In addition, preadipocytes as well as adipocytes are stained with MOMA-2, a marker of monocyte-macrophage lineage, but are negative for specific mature macrophage markers (F4/80 and Mac-1). These results suggest that preadipocytes could function as macrophage-like cells and raise the possibility of a potential direct involvement of adipose tissue in inflammatory processes.

Atypical neutrophil alkaline phosphatase associated with impaired neutrophil functions.

We report the case of a healthy young man presenting with atypical neutrophil alkaline phosphatase (NAP) and reduced neutrophil chemotactic activity, but with no susceptibility to infection. NAP activity was low, kinetic parameters were modified and immunoreactive properties and subcellular distribution were abnormal. Neutrophil morphology was normal. A similar pattern was observed in the patient's healthy brother. The profile of the observed anomalies offers some similarity to that previously described in patients with chronic myelogenous leukaemia. However, in the present case, the NAP deficiency with impaired neutrophil function was present in two brothers with no haematological symptoms and is probably related to a non-acquired neutrophil abnormality. This observation of a primary NAP variant reinforces the hypothesis of a direct link between NAP activity and functional properties of neutrophils.

High expression of leptin by human bone marrow adipocytes in primary culture.

Adipocytes participate in the microenvironment of the bone marrow (BM), but their exact role remains to be determined. It has recently been shown that leptin, a hormone secreted from extramedullary adipocytes, could be involved in hematopoiesis. Therefore we have developed a primary culture system of human BM adipocytes to characterize their differentiation and determine whether leptin is also secreted from these adipocytes. BM cells were cultured with fetal calf and horse sera. In the presence of dexamethasone, cells with vesicles containing lipids appeared within 15 days. They expressed glycerol phosphate dehydrogenase activity and a lipolytic activity in response to isoproterenol, but expressed neither the adrenergic beta3 receptor nor the mitochondrial uncoupling protein UCP1. The addition of insulin alone to the culture media did not promote adipocyte differentiation. Leptin was expressed and secreted at high levels during adipocyte differentiation. Acute exposure of differentiated adipocytes to insulin had little effect on leptin expression whereas forskolin strongly inhibited it. These results show that although human BM adipocytes differ from extramedullary adipose tissues in their sensitivity to different effectors, they are a secondary source of leptin production. They suggest that BM adipocytes could contribute to hematopoiesis via the secretion of leptin in the vicinity of hematopoietic stem cells.

Establishment and characterization of a human T-lymphocyte cell line immortalized by SV40 and with abnormal expression of TCR/CD3.

Human lymphocytes derived from the peripheral blood of a healthy woman were transfected with a plasmid carrying the simian virus 40 (SV40) large T antigen. The successfully transformed cells contained SV40 large T DNA and were negative for Epstein-Barr virus (EBV) and human T-cell leukaemia virus (HTLV)-1 genomes. The immortalized cell line was assigned to the T-lymphocyte lineage on the basis of morphological, immunological and cytochemical criteria. While the cells expressed CD1a and CD4 at the cell surface, the CD3 complex was solely intracytoplasmic. Immunoprecipitation studies indicated that these cells lacked T-cell receptor (TCR) alpha-chains but not beta-chains. They were negative for activation markers such as CD25, CD69 and major histocompatibility (MHC) class II molecules. In addition, the transformed cells exhibited a complete growth independency towards interleukin-2 (IL-2). However, after phorbol ester stimulation, CD25 and CD69 markers were expressed and IL-2 was secreted. This new human immortalized T-lymphocytic cell line, which is cell-surface TCR/CD3-negative, may be useful as an in vitro model for studying TCR/CD3 assembly, expression and signal transduction.

Kupffer cells are a dominant site of uncoupling protein 2 expression in rat liver.

The mechanisms underlying thermogenesis in liver are not well understood. They may involve proteins related to the mitochondrial uncoupling protein (UCP1) of brown adipocytes. In this paper, it is demonstrated that UCP1 is not expressed in any liver cell type of rat while UCP2, a recently cloned homologue of UCP1, is expressed at a very high level in Kupffer cells but not in hepatocytes. This high level of expression of UCP2 in Kupffer cells allowed cross immunoreactivity with antibodies directed against UCP1. This cross reactivity was confirmed by the detection of UCP2 with anti-UCP1 antibody, in western blotting analysis of transfected yeasts expressing rat UCP2. The high level expression of UCP2 in Kupffer cells suggests a particular function of UCP2 in macrophages.

Isolation of bone marrow plasma cells by negative selection with immunomagnetic beads.

In order to isolate bone marrow plasma cells from patients presenting with multiple myeloma or monoclonal gammopathy of undetermined significance, we developed a method for purifying these cells by negative selection using monoclonal antibodies and immunomagnetic beads. The results presented here were obtained from 75 procedures. Purity was extremely variable (2-100%) and was dependent on the percentage of plasma cells in the original bone marrow sample with a 10% cut-off, beyond which purity was over 96% in all cases. The mean yield was about 20%. The cells collected were viable and suitable for immunophenotyping, semi-quantitative studies of oncoproteins, and PCR.

[Computer-assisted validation system applied to hematology: Valab-hemato]. / Système de validation assistée par ordinateur appliqué à l'hématologie: Valab-hémato.

Validation of laboratory reports is the ultimate step before transmission of results to the clinician. The biologist checks the intrinsic consistency of the data as well as their possible medical value that is liable to lead to other investigations. Such a policy, when performed on all the data, is time-consuming, boring and uncertain. This step may be simplified by the use of a computerized expert system. The computer assisted validation system presented here concerns routine haematology data (Valab-haemato). Like its predecessor devoted to clinical chemistry (Valab-Biochem) it is based on the performance of a powerful inference engine which generates a decision-making tree for each report according to the data. This adaptability gives the system a capacity very close to human reasoning. In its haematology version the system deals with many variables including sex, age, origin of the patient (hospital ward), and the haematological data (blood cell count, differential, reticulocyte count, various information drawn from microscope examination of the blood smear as well as any report concerning the blood sample, erythrocyte sedimentation rate). Previous data are also taken into account, as well as the normal ranges, the values beyond which no result can be automatically validated and the delta-check. Some information definitely prevents validation of the results, others can be validated if they have been previously approved. Whereas the method of reasoning is fixed, all items are changeable in order to adapt the system to the type of activity of the laboratory.(ABSTRACT TRUNCATED AT 250 WORDS)