In vivo kinetics of eosinophils and mast cells in experimental murine Schistosomiasis

During the schistosomiasis infection there is a [quot ]dance of the cells[quot ], varying from site to site and related to the time of infection. 1 - Eosinophil levels exhibit a bimodal pattern, with the first peak related to the egg deposition and maturation and increased Kupfferian hyperplasia; the second peak precedes the death of some adult worms; 2 - The peritoneal eosinophilic levels are inversely proportional to the blood eosinophilic levels; 3 - Eosinopoiesis in the bone marrow begins at day 40, reaching the highest levels at day 50 and coincides with hepatic eosinophilic and neutrophilic metaplasia; 4 - Peritoneal mast cell levels present a bimodal pattern similar to the blood eosinophils, and inverse to the peritoneal eosinophils. They also show a cyclic behaviour within the hepatic and intestinal granulomas. Integral analysis of the events related to the eosinophils in the blood, bone marrow, peritoneal cavity and hepatic and intestinal granulomas allows the detection of two important eosinophilic phases: the first is due to mobilization and redistribution of the marginal pool and the second originates from eosinophilic production in the bone marrow and liver. The productive phase is characterized by an increase in the number of eosinophils and monocyte/macrophages, and a decrease in neutrophils and stabilization of megakariocytes and erithroid lineages.

Statin use and peripheral blood progenitor cells mobilization in patients with multiple myeloma.

PURPOSE: Statins have beneficial effects in patients after myocardial infarction and at least part of the benefit results from mobilization of marrow endothelial progenitors to repopulate damaged myocardial tissues. This study examines if statins may have the same effect in mobilizing marrow progenitors to be harvested and subsequently used in high-dose chemotherapy with progenitor cell rescue in multiple myeloma. METHODS: From 2006 to 2012, 86 patients with multiple myeloma were mobilized with the use of G-CSF and were retrospectively analyzed. Patients with other malignancies or mobilized with the use of chemotherapy or with plerixafor were excluded. RESULTS: The median age of the patients was 60 years. 72 patients had received one line of chemotherapy and 14 patients two or more lines of chemotherapy. Twenty patients were taking statins at the time of the harvest while 66 patients were not. In the group of patients taking statins the success rate of first leukapheresis (obtaining the target number of 4 × 10(6) CD34+ cells/kg) was 85 % while in the group not taking statins this rate was 63.6 %. Despite the comparatively small number of patients this difference approached statistical significance (χ (2) = 0.07). CONCLUSION: This retrospective analysis of 86 patients shows for the first time a possible benefit of statins for peripheral blood progenitor cells mobilization in patients with multiple myeloma. Larger studies would be required to clarify the issue. If their effectiveness is confirmed, statins could be a safe and cheaper addition to chemotherapy and plerixafor for peripheral hematopoietic stem cell mobilization.

Adipose-derived stem cells enhance peripheral nerve regeneration.

Traumatic injuries resulting in peripheral nerve lesions often require a graft to bridge the gap. Although autologous nerve auto-graft is still the first-choice strategy in reconstructions, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to create a favourable environment for nerve regeneration. We decided to test new fibrin nerve conduits seeded with various cell types (primary Schwann cells and adult stem cells differentiated to a Schwann cell-like phenotype) for repair of sciatic nerve injury. Two weeks after implantation, the conduits were removed and examined by immunohistochemistry for axonal regeneration (evaluated by PGP 9.5 expression) and Schwann cell presence (detected by S100 expression). The results show a significant increase in axonal regeneration in the group of fibrin seeded with Schwann cells compared with the empty fibrin conduit. Differentiated adipose-derived stem cells also enhanced regeneration distance in a similar manner to differentiated bone marrow mesenchymal stem cells. These observations suggest that adipose-derived stem cells may provide an effective cell population, without the limitations of the donor-site morbidity associated with isolation of Schwann cells, and could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

99mTc-sestamibi imaging and bone marrow karyotyping in the assessment of multiple myeloma and MGUS

AIM: The first pathogenetic step in multiple myeloma is the emergence of a limited number of clonal plasma cells, clinically known as monoclonal gammopathy of undetermined significance (MGUS). Patients with MGUS do not have symptoms or end-organ damage but they do have a 1% annual risk of progression to multiple myeloma or related malignant disorders. With progression of MGUS to multiple myeloma, complex genetic events occur in the neoplastic plasma cell. Karyotyping and fluorescence in-situ hybridization (FISH) were shown to be of prognostic value in patients with multiple myeloma. Tc-sestamibi imaging reflects myeloma disease activity in bone marrow with very high sensitivity and specificity predicting disease evolution. This study was undertaken to evaluate the role of Tc-sestamibi imaging and cytogenetic analysis in prognosis prediction of MGUS and multiple myeloma. METHODS: We enrolled 30 consecutive patients with a confirmed diagnosis of multiple myeloma or MGUS. Bone marrow biopsy and biochemical staging according to the International Staging System (ISS) were performed in all cases. Karyotype analysis and FISH were performed in 11 of 12 patients with MGUS and in 17 of 18 patients with multiple myeloma having adequate metaphases. RESULTS: The karyotype was abnormal in four of 11 MGUS and in six of 17 multiple myeloma. Abnormalities of chromosome 13 were present in one case of MGUS and in six cases of multiple myeloma whereas the involvement of immunoglobulin was observed in one case of multiple myeloma. An abnormal FISH panel was found in four MGUS and nine multiple myeloma patients. All patients with MGUS showed a normal MIBI scan (score 0). Among patients with multiple myeloma only three, all with ISS stage I, showed a normal scan while a positive scan was obtained in others (score range, 1-7). The MIBI uptake was strongly related to the bone marrow plasma cell infiltration and to cytogenetic abnormalities. Particularly, a MIBI uptake score above 5 identified patients with poor prognosis encompassing all stage III multiple myeloma and three of seven stage II multiple myeloma. On the other hand all stage I and II patients having a MIBI score less than 5 showed a good prognosis. CONCLUSION: Both cytogenetic analysis and a MIBI scan add no relevant prognostic information to the ISS in patients with stage I and III multiple myeloma. The MIBI scan was of prognostic value in stage II multiple myeloma patients. Additionally, MIBI imaging may be useful to guide bone marrow biopsy in order to obtain adequate samples for cytogenetic analysis.

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.

The role of Pten in the Hematopoietic System and the Hematopoietic Stem Cells

Résumé Identification, localisation et activation des cellules souches hématopoiétiques dormantes in vivo Les cellules souches somatiques sont présentes dans la majorité des tissus régénératifs comme la peau, l'épithélium intestinal et le système hématopoiétique. A partir d'une seule cellule, elles ont les capacités de produire d'autres cellules souches du même type (auto-renouvellement) et d'engendrer un ensemble défini de cellules progénitrices différenciées qui vont maintenir ou réparer leur tissu hôte. Les cellules souches adultes les mieux caractérisées sont les cellules souches hématopoiétiques (HSC), localisées dans la moelle osseuse. Un des buts de mon travail de doctorat était de caractériser plus en profondeur la localisation des HSCs endogènes in vivo. Pour ce faire, la technique "label retaining assay", se basant sur la division peu fréquentes et sur la dormance des cellules souches, a été utilisée. Après un marquage des souris avec du BrdU (analogue à l'ADN) suivi d'une longue période sans BrdU, les cellules ayant incorporés le marquage ("label retaining cells" LCRs) ont pu être identifiées dans la moelle osseuse. Ces cellules LCRs étaient enrichies 300 fois en cellules de phenotype HSC et, en utilisant de la cytofluorométrie, il a pu être montré qu'environ 15% de toutes les HSCs d'une souris restent dormantes durant plusieures semaines. Ces HSCs dormantes à long terme ne sont probablement pas impliquées dans la maintenance de 'hématopoièse. Par contre, on assiste à l'activation rapide de ces HSCs dormantes lors d'une blessure, comme une ablation myéloide. Elles re-entrent alors en cycle cellulaire et sont essentielles pour une génération rapide des cellules progénitrices et matures qui vont remplacer les cellules perdues. De plus, la détection des LCRs, combinée avec l'utilisation du marqueur de HSCs c-kit, peut être utilisée pour la localisation des HSCs dormantes présentes dans la paroi endostéale de la cavité osseuse. De manière surprenante, les LCRs c-kit+ ont surtout étés trouvées isolées en cellule unique, suggérant que le micro-environement spécifique entourant et maintenant les HSCs, appelé niche, pourrait être très réduit et abriter une seule HSC par niche. Rôles complexes du gène supresseur de tumeur Pten dans le système hématopoiétique La phosphatase PTEN disparaît dans certains cancers héréditaires ou sporadiques humains, comme les gliomes, les cancers de l'utérus ou du sein. Pten inhibe la voie de signalisation de la PI3-kinase et joue un rôle clé dans l'apoptose, la croissance, la prolifération et la migration cellulaire. Notre but était d'étudier le rôle de Pten dans les HSC normale et durant la formation de leucémies. Pour ce faire, nous avons généré un modèle murin dans lequel le gène Pten peut être supprimé dans les cellules hématopoiétiques, incluant les HSCs. Ceci a été possible en croissant l'allèle conditionnelle ptenflox soit avec le transgène MxCre inductible par l'interféron α soit avec le transgène Scl-CreERt inductible par le tamoxifen. Ceci permet la conversion de l'allèle ptenflox en l'allèle nul PtenΔ dans les HSCs et les autres types cellulaires hématopoiétiques. Les souris mutantes Pten développent une splénomégalie massive causée par une expansion dramatiques de toutes les cellules myéloides. De manière interessante, alors que le nombre de HSCs dans la moelle osseuse diminue progressivement, le nombre des HSCs dans la rate augmente de manière proportionnelle. Etrangement, les analyses de cycle cellulaire ont montrés que Pten n'avait que peu ou pas d'effet sur la dormance des HSCs ou sur leur autorenouvellement. En revanche, une augmentation massive du niveau de la cytokine de mobilisation G-CSF a été détéctée dans le serum sanguin, suggérant que la suppression de Pten stimulerait la mobilisation et la migration des HSC de la moelle osseuse vers la rate. Finallement, la transplantation de moelle osseuse délétée en Pten dans des souris immuno-déficientes montre que Pten fonctionnerait comme un suppresseur de tumeur dans le système hématopoiétique car son absence entraîne la formation rapide de leucémies lymphocytaires. Summary Identification, localization and activation of dormant hematopoietic stun cells in vivo Somatic stem cells are present in most self-renewing tissues including the skin, the intestinal epithelium and the hematopoietic system. On a single cell basis they have the capacity to produce more stem cells of the same phenotype (self-renewal) and to give rise to a defined set of mature differentiated progeny, responsible for the maintenance or repair of the host tissue. The best characterized adult stem cell is the hematopoietic stem cell (HSC) located in the bone marrow. One goal of my thesis work was to further characterize the location of endogenous HSCs in vivo. To do this, a technique called "label retaining assay» was used which takes advantage of the fact that stem cells (including HSCs) divide very infrequently and can be dormant for months. After labeling mice with the DNA analogue BrdU followed by a long BrdU free "chase", BrdU "label retaining cells" (CRCs) could be identified in the bone marrow. These CRCs were 300-fold enriched for phenotypic HSCs and by using flow cytometry analysis it could be shown that about 15% of all HSCs in the mouse are dormant for many weeks. Our results suggest that these long-term dormant HSCs are unlikely to be involved in homeostatic maintenance. However they are rapidly activated and reenter the cell cycle in response to injury signals such as myeloid ablation. In addition, detection of LRCs in combination with the HSC marker c-Kit could be used to locate engrafted dormant HSCs close to the endosteal lining of the bone marrow cavities. Most surprisingly, c-Kit+LRCs were found predominantly as single cells suggesting that the specific stem cell maintaining microenvironment, called niche, has limited space and may house only single HSCs. Complex roles of the tumor suppressor gene Pten in the hematopoietic system. The phosphatase PTEN is lost in hereditary and sporadic forms of human cancers, including gliomas, endometrial and breast cancers. Pten inhibits the PI3-kina.se pathway and plays a key role in apoptosis, cell growth, proliferation and migration. Our aim was to study the role of Pten in normal HSCs and during leukemia formation. To do this, we generated a mouse model in which the Pten gene can be deleted in hematopoietic cells including HSCs. This was achieved by crossing the conditional ptenflox allele with either the interferona inducible MxCre or the tamoxifen inducible Scl-CreERT transgene. This allowed the conversion of the ptenflox allele into a pterr' null allele in HSCs and other hematopoietic cell types. As a result Pten mutant mice developed massive splenomegaly due to a dramatic expansion of all myeloid cells. Interestingly, while the number of bone marrow HSCs progressively decreased, the number of HSCs in the spleen increased to a similar extent. Unexpectedly, extensive cell cycle analysis showed that Pten had little or no effect on HSC dormancy or HSC self-renewal. Instead, dramatically increased levels of the mobilizing cytokine G-CSF were detected in the blood serum suggesting that loss-of Pten stimulates mobilization and migration of HSC from the BM to the spleen. Finally, transplantation of Pten deficient BM cells into immuno-compromised mice showed that Pten can function as a tumor suppressor in the hematopoietic system and that its absence leads to the rapid formation of T cell leukemia.

Sustained improvement in left ventricular function after bone marrow derived cell therapy in patients with acute ST elevation myocardial infarction. A 5-year follow-up from the Stem Cell Transplantation in Ischaemic Myocardium Study.

BACKGROUND: Intracoronary injection of autologous bone marrow-derived mononucleated cells (BM-MNC) may improve LV function shortly after acute ST elevation myocardial infarction (STEMI), but little is known about the long-term durability of the treatment effect. METHODS: In a single-centre trial a total of 60 patients with acute anterior STEMI, successful reperfusion therapy and a left ventricular ejection fraction (LVEF) of <50% were screened for the study. 23 patients were actively treated with intracoronary infusion of BM-MNC within a median of 3 days. The open-label control group consisted of 19 patients who did not consent to undergo BM-MNC treatment but agreed to undergo regular clinical and echocardiographic follow-up for up to 5 years after AMI. RESULTS: Whereas at 4 months there was no significant difference between the increase in LVEF in the BM-MNC group and the control group (+7.0%, 95%CI 3.6; 10.4) vs. +3.9%, 95%CI -2.1; 10), the absolute increase at 5 years remained stable in the BM-MNC but not in the control group (+7.95%, 95%CI 3.5; 12.4 vs. -0.5%, 95%CI -5.4; 4.4; p for interaction between groups = 0.035). DISCUSSION: In this single-centre, open-labelled study, intracoronary administration of BM-MNC is feasible and safe in the short term. It is also associated with sustained improvement of left ventricular function in patients with acute myocardial infarction, encouraging phase III studies to examine the potential BM-MNC effect on clinical outcome.

Maturation of marginal zone and follicular B cells requires B cell activating factor of the tumor necrosis factor family and is independent of B cell maturation antigen.

B cells undergo a complex series of maturation and selection steps in the bone marrow and spleen during differentiation into mature immune effector cells. The tumor necrosis factor (TNF) family member B cell activating factor of the TNF family (BAFF) (BLyS/TALL-1) plays an important role in B cell homeostasis. BAFF and its close homologue a proliferation-inducing ligand (APRIL) have both been shown to interact with at least two receptors, B cell maturation antigen (BCMA) and transmembrane activator and cyclophilin ligand interactor (TACI), however their relative contribution in transducing BAFF signals in vivo remains unclear. To functionally inactivate both BAFF and APRIL, mice transgenic for a soluble form of TACI were generated. They display a developmental block of B cell maturation in the periphery, leading to a severe depletion of marginal zone and follicular B2 B cells, but not of peritoneal B1 B cells. In contrast, mice transgenic for a soluble form of BCMA, which binds APRIL, have no detectable B cell phenotype. This demonstrates a crucial role for BAFF in B cell maturation and strongly suggests that it signals via a BCMA-independent pathway and in an APRIL-dispensable way.

Dormant and self-renewing hematopoietic stem cells and their niches.

In the mouse, over the last 20 years, a set of cell-surface markers and activities have been identified, enabling the isolation of bone marrow (BM) populations highly enriched in hematopoietic stem cells (HSCs). These HSCs have the ability to generate multiple lineages and are capable of long-term self-renewal activity such that they are able to reconstitute and maintain a functional hematopoietic system after transplantation into lethally irradiated recipients. Using single-cell reconstitution assays, various marker combinations can be used to achieve a functional HSC purity of almost 50%. Here we have used the differential expression of six of these markers (Sca1, c-Kit, CD135, CD48, CD150, and CD34) on lineage-depleted BM to refine cell hierarchies within the HSC population. At the top of the hierarchy, we propose a dormant HSC population (Lin(-)Sca1(+)c-Kit(+) CD48(-)CD150(+)CD34(-)) that gives rise to an active self-renewing CD34(+) HSC population. HSC dormancy, as well as the balance between self-renewal and differentiation activity, is at least, in part, controlled by the stem cell niches individual HSCs are attached to. Here we review the current knowledge about HSC niches and propose that dormant HSCs are located in niches at the endosteum, whereas activated HSCs are in close contact to sinusoids of the BM microvasculature.

Bone-marrow haematopoietic-stem-cell niches.

Adult stem cells hold many promises for future clinical applications and regenerative medicine. The haematopoietic stem cell (HSC) is the best-characterized somatic stem cell so far, but in vitro expansion has been unsuccessful, limiting the future therapeutic potential of these cells. Here we review recent progress in characterizing the composition of the HSC bone-marrow microenvironment, known as the HSC niche. During homeostasis, HSCs, and therefore putative bone-marrow HSC niches, are located near bone surfaces or are associated with the sinusoidal endothelium. The molecular crosstalk between HSCs and the cellular constituents of these niches is thought to control the balance between HSC self-renewal and differentiation, indicating that future successful expansion of HSCs for therapeutic use will require three-dimensional reconstruction of a stem-cell-niche unit.

Effect of Platinum-Based Chemoradiotherapy on Cellular Proliferation in Bone Marrow and Spleen, Estimated by 18F-FLT PET/CT in Patients with Locally Advanced Non-Small Cell Lung Cancer.

Historically, it has been difficult to monitor the acute impact of anticancer therapies on hematopoietic organs on a whole-body scale. Deeper understanding of the effect of treatments on bone marrow would be of great potential value in the rational design of intensive treatment regimens. 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) is a functional radiotracer used to study cellular proliferation. It is trapped in cells in proportion to thymidine-kinase 1 enzyme expression, which is upregulated during DNA synthesis. This study investigates the potential of (18)F-FLT to monitor acute effects of chemotherapy on cellular proliferation and its recovery in bone marrow, spleen, and liver during treatment with 2 different chemotherapy regimens.

Cutting edge: an essential role for Notch-1 in the development of both thymus-independent and -dependent T cells in the gut.

Whereas most T cells arise in the thymus, a distinct lineage of extrathymically derived T cells is present in the gut mucosa. The developmental origin of extrathymic T cells is poorly understood. We show here that Notch-1, a transmembrane receptor involved in T cell fate specification of bipotential T/B precursors in the thymus, is absolutely required for the development of extrathymic (as well as thymus-derived) mature T cells in the intestinal epithelium. In the absence of Notch-1, CD117(+) T cell precursors are relatively more abundant in the gut than the thymus, whereas immature B cells accumulate in the thymus but not the gut. Collectively, these data demonstrate that Notch-1 is essential for both thymic and extrathymic T cell fate specification and further suggest that bipotential T/B precursors that do not receive a Notch-1 signal adopt a B cell fate in the thymus but become developmentally arrested in the gut.

Protection from radiation-induced colitis requires MHC class II antigen expression by cells of hemopoietic origin.

Ulcerative colitis, an inflammatory bowel disease, is believed to result from a breakdown of dominant tolerance mechanisms that normally control intestinal immunity. Although CD4+ T lymphocyte subpopulations and expression of MHC class II molecules have been shown to play a role in the pathogenesis of the disease, the nature of the responsible mechanisms remains unclear. In this paper we describe a novel mouse model for inflammatory bowel disease, radiation-induced colitis, that occurs with complete penetrance 6-8 wk postinduction. A combination of high dose gamma-irradiation and lack of MHC class II expression on cells of hemopoietic origin results in development of colitis in C57BL/6 mice. Because of its versatility (due to susceptibility of mice of the widely genetically manipulated C57BL/6 background), high reproducibility, and 100% penetrance, radiation-induced colitis will be a useful mouse model for colitis and a significant tool to study dominant immunological tolerance mechanisms. Moreover, our data imply that tolerization to enteric Ags requires MHC class II mediated presentation by APC of hemopoietic origin.

Fluid flow regulates stromal cell organization and CCL21 expression in a tissue-engineered lymph node microenvironment.

In the paracortex of the lymph node (LN), T zone fibroblastic reticular cells (TRCs) orchestrate an immune response by guiding lymphocyte migration both physically, by creating three-dimensional (3D) cell networks, and chemically, by secreting the chemokines CCL19 and CCL21 that direct interactions between CCR7-expressing cells, including mature dendritic cells and naive T cells. TRCs also enwrap matrix-based conduits that transport fluid from the subcapsular sinus to high endothelial venules, and fluid flow through the draining LN rapidly increases upon tissue injury or inflammation. To determine whether fluid flow affects TRC organization or function within a 3D network, we regenerated the 3D LN T zone stromal network by culturing murine TRC clones within a macroporous polyurethane scaffold containing type I collagen and Matrigel and applying slow interstitial flow (1-23 microm/min). We show that the 3D environment and slow interstitial flow are important regulators of TRC morphology, organization, and CCL21 secretion. Without flow, CCL21 expression could not be detected. Furthermore, when flow through the LN was blocked in mice in vivo, CCL21 gene expression was down-regulated within 2 h. These results highlight the importance of lymph flow as a homeostatic regulator of constitutive TRC activity and introduce the concept that increased lymph flow may act as an early inflammatory cue to enhance CCL21 expression by TRCs, thereby ensuring efficient immune cell trafficking, lymph sampling, and immune response induction.

Thogoto virus infection induces sustained type I interferon responses that depend on RIG-I-like helicase signaling of conventional dendritic cells.

Type I interferon (IFN-α/β) induction upon viral infection contributes to the early antiviral host defense and ensures survival until the onset of adaptive immunity. Many viral infections lead to an acute, transient IFN expression which peaks a few hours after infection and reverts to initial levels after 24 to 36 h. Robust IFN expression often is conferred by specialized plasmacytoid dendritic cells (pDC) and may depend on positive-feedback amplification via the type I IFN receptor (IFNAR). Here, we show that mice infected with Thogoto virus (THOV), which is an influenza virus-like orthomyxovirus transmitted by ticks, mounted sustained IFN responses that persisted up to 72 h after infection. For this purpose, we used a variant of THOV lacking its IFN-antagonistic protein ML, an elongated version of the matrix (M) protein [THOV(ΔML)]. Of note, large amounts of type I IFN were also found in the serum of mice lacking the IFNAR. Early IFN-α expression seemed to depend on Toll-like receptor (TLR) signaling, whereas prolonged IFN-α responses strictly depended on RIG-I-like helicase (RLH) signaling. Unexpectedly, THOV(ΔML)-infected bone marrow-derived pDC (BM-pDC) produced only moderate IFN levels, whereas myeloid DC (BM-mDC) showed massive IFN induction that was IPS-1-dependent, suggesting that BM-mDC are involved in the massive, sustained IFN production in THOV(ΔML)-infected animals. Thus, our data are compatible with the model that THOV(ΔML) infection is sensed in the acute phase via TLR and RLH systems, whereas at later time points only RLH signaling is responsible for the induction of sustained IFN responses.