MHC Class II Transactivator Is an In Vivo Regulator of Osteoclast Differentiation and Bone Homeostasis Co-opted From Adaptive Immunity.

The molecular networks controlling bone homeostasis are not fully understood. The common evolution of bone and adaptive immunity encourages the investigation of shared regulatory circuits. MHC Class II Transactivator (CIITA) is a master transcriptional co-activator believed to be exclusively dedicated for antigen presentation. CIITA is expressed in osteoclast precursors, and its expression is accentuated in osteoporotic mice. We thus asked whether CIITA plays a role in bone biology. To this aim, we fully characterized the bone phenotype of two mouse models of CIITA overexpression, respectively systemic and restricted to the monocyte-osteoclast lineage. Both CIITA-overexpressing mouse models revealed severe spontaneous osteoporosis, as assessed by micro-computed tomography and histomorphometry, associated with increased osteoclast numbers and enhanced in vivo bone resorption, whereas osteoblast numbers and in vivo bone-forming activity were unaffected. To understand the underlying cellular and molecular bases, we investigated ex vivo the differentiation of mutant bone marrow monocytes into osteoclasts and immune effectors, as well as osteoclastogenic signaling pathways. CIITA-overexpressing monocytes differentiated normally into effector macrophages or dendritic cells but showed enhanced osteoclastogenesis, whereas CIITA ablation suppressed osteoclast differentiation. Increased c-fms and receptor activator of NF-κB (RANK) signaling underlay enhanced osteoclast differentiation from CIITA-overexpressing precursors. Moreover, by extending selected phenotypic and cellular analyses to additional genetic mouse models, namely MHC Class II deficient mice and a transgenic mouse line lacking a specific CIITA promoter and re-expressing CIITA in the thymus, we excluded MHC Class II expression and T cells from contributing to the observed skeletal phenotype. Altogether, our study provides compelling genetic evidence that CIITA, the molecular switch of antigen presentation, plays a novel, unexpected function in skeletal homeostasis, independent of MHC Class II expression and T cells, by exerting a selective and intrinsic control of osteoclast differentiation and bone resorption in vivo. © 2014 American Society for Bone and Mineral Research.

Endothelial cells as key determinants of the tumor microenvironment: interaction with tumor cells, extracellular matrix and immune killer cells.

Besides tumor cells, the tumor microenvironment harbors a variety of host-derived cells, such as endothelial cells, fibroblasts, innate and adaptive immune cells. It is a complex and highly dynamic environment, providing very important cues to tumor development and progression. Tumor-associated endothelial cells play a key role in this process. On the one hand, they form tumor-associated (angiogenic) vessels through sprouting from locally preexisting vessels or recruitment of bone marrow-derived endothelial progenitor cells, to provide nutritional support to the growing tumor. On the other hand, they are the interface between circulating blood cells, tumor cells and the extracellular matrix, thereby playing a central role in controlling leukocyte recruitment, tumor cell behavior and metastasis formation. Hypoxia is a critical parameter modulating the tumor microenvironment and endothelial/tumor cell interactions. Under hypoxic stress, tumor cells produce factors that promote tumor angiogenesis, tumor cell motility and metastasis. Among these factors, VEGF, a main angiogenesis modulator, can also play a critical role in the control of immune tolerance. This review discusses some aspects of the role of endothelial cells within tumor microenvironment and emphasizes their interaction with tumor cells, the extracellular matrix and with immune killer cells. We will also address the role played by circulating endothelial progenitor cells and illustrate their features and mechanism of recruitment to the tumor microenvironment and their role in tumor angiogenesis.

Regulation of Rat and Human T-cell Immune Response by Pharmacologically Modified Dendritic Cells.

BACKGROUND: The central function of dendritic cells (DC) in inducing and preventing immune responses makes them ideal therapeutic targets for the induction of immunologic tolerance. In a rat in vivo model, we showed that dexamethasone-treated DC (Dex-DC) induced indirect pathway-mediated regulation and that CD4+CD25+ T cells were involved in the observed effects. The aim of the present study was to investigate the mechanisms underlying the acquired immunoregulatory properties of Dex-DC in the rat and human experimental systems. METHODS: After treatment with dexamethasone (Dex), the immunogenicity of Dex-DC was analyzed in T-cell proliferation and two-step hyporesponsiveness induction assays. After carboxyfluorescein diacetate succinimidyl ester labeling, CD4+CD25+ regulatory T-cell expansion was analyzed by flow cytometry, and cytokine secretion was measured by ELISA. RESULTS: In this study, we demonstrate in vitro that rat Dex-DC induced selective expansion of CD4+CD25+ regulatory T cells, which were responsible for alloantigen-specific hyporesponsiveness. The induction of regulatory T-cell division by rat Dex-DC was due to secretion of interleukin (IL)-2 by DC. Similarly, in human studies, monocyte-derived Dex-DC were also poorly immunogenic, were able to induce T-cell anergy in vitro, and expand a population of T cells with regulatory functions. This was accompanied by a change in the cytokine profile in DC and T cells in favor of IL-10. CONCLUSION: These data suggest that Dex-DC induced tolerance by different mechanisms in the two systems studied. Both rat and human Dex-DC were able to induce and expand regulatory T cells, which occurred in an IL-2 dependent manner in the rat system.

CD103 marks a subset of human CD34(+)-derived langerin(+) dendritic cells that induce T-regulatory cells via indoleamine 2,3-dioxygenase-1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive molecule expressed in some subsets of normal and neoplastic cells. Mature human dendritic cells (DCs) have been shown to express IDO1, but little is known about its expression and function during DC differentiation from bone marrow hematopoietic stem/progenitor cells (HSPCs). Here, we show that during in vitro differentiation along the myeloid DC lineage, CD34(+) HSPCs acquire IDO1 expression, which acts in a tolerogenic manner by inducing a population of fully functional CD4(+)CD25(+) FOXP3(+) T-regulatory cells. Phenotypically, CD1a(+)CD14(-) HPSC-derived DCs expressed IDO1, langerin, CD11b, and CD1c. Cell-sorting experiments demonstrated that IDO1 expression is found in a subset of CD1a(+)CD14(-)langerin(+) cells, expressing CD103, which is capable of inducing T-regulatory cells in an IDO1-dependent manner. In conclusion, DC differentiation from CD34(+) HSPCs results in the expression of a functionally active IDO1 protein in CD1a(+)langerin(+), CD103-expressing DCs. These data point toward IDO1 expression as part of a tolerogenic signature during DC development.

Cell-Cell interaction in erythropoiesis: role of human monocytes

Erythroid burst forming units (BFU-E) are proliferative cells present in peripheral blood and bone marrow which may be precursors of the erythroid colony forming cell found in the bone marrow. To examine the possible role of monocyte-macrophages in the modulation of erythropoiesis, the effect of monocytes on peripheral blood BFU-E proliferation in response to erythropoietin was investigated in the plasma clot culture system. Peripheral blood mononuclear cells from normal human donors were separated into four fractions. Fraction-I cells were obtained from the interface of Ficoll-Hypaque gradients (20-30% monocytes; 60-80% lymphocytes); fraction-II cells were fraction-I cells that were nonadherent to plastic (2-10% monocytes; 90-98% lymphocytes); fraction-III cells were obtained by incubation of fraction-II cells with carbonyl iron followed by Ficoll-Hypaque centrifugation (>99% lymphocytes); and fraction-IV cells represented the adherent population of fraction-II cells released from the plastic by lidocaine (>95% monocytes). When cells from these fractions were cultured in the presence of erythropoietin, the number of BFU-E-derived colonies was inversely proportional to the number of monocytes present (r = ¿0.96, P < 0.001). The suppressive effect of monocytes on BFU-E proliferation was confirmed by admixing autologous purified monocytes (fraction-IV cells) with fraction-III cells. Monocyte concentrations of ¿20% completely suppressed BFU-E activity. Reduction in the number of plated BFU-E by monocyte dilution could not account for these findings: a 15% reduction in the number of fraction-III cells plated resulted in only a 15% reduction in colony formation. These results indicate that monocyte-macrophages may play a significant role in the regulation of erythropoiesis and be involved in the pathogenesis of the hypoproliferative anemias associated with infection and certain neoplasia in which increased monocyte activity and monopoiesis also occur.

Obstructive apneas induce early activation of mesenchymal stem cells and enhancement of endothelial wound healing

Background: The aim was to test the hypothesis that the blood serum of rats subjected to recurrent airway obstructions mimicking obstructive sleep apnea (OSA) induces early activation of bone marrow-derived mesenchymal stem cells (MSC) and enhancement of endothelial wound healing. Methods: We studied 30 control rats and 30 rats subjected to recurrent obstructive apneas (60 per hour, lasting 15 s each, for 5 h). The migration induced in MSC by apneic serum was measured by transwell assays. MSC-endothelial adhesion induced by apneic serum was assessed by incubating fluorescent-labelled MSC on monolayers of cultured endothelial cells from rat aorta. A wound healing assay was used to investigate the effect of apneic serum on endothelial repair. Results: Apneic serum showed significant increase in chemotaxis in MSC when compared with control serum: the normalized chemotaxis indices were 2.20 +- 0.58 (m +- SE) and 1.00 +- 0.26, respectively (p < 0.05). MSC adhesion to endothelial cells was greater (1.75 +- 0.14 -fold; p < 0.01) in apneic serum than in control serum. When compared with control serum, apneic serum significantly increased endothelial wound healing (2.01 +- 0.24 -fold; p < 0.05). Conclusions: The early increases induced by recurrent obstructive apneas in MSC migration, adhesion and endothelial repair suggest that these mechanisms play a role in the physiological response to the challenges associated to OSA.

Innate Immune Sensing of Fusarium culmorum by Mouse Dendritic Cells.

Chronic inhalation of grain dust is associated with asthma and chronic bronchitis in grain worker populations. Exposure to fungal particles was postulated to be an important etiologic agent of these pathologies. Fusarium species frequently colonize grain and straw and produce a wide array of mycotoxins that impact human health, necessitating an evaluation of risk exposure by inhalation of Fusarium and its consequences on immune responses. Data showed that Fusarium culmorum is a frequent constituent of aerosols sampled during wheat harvesting in the Vaud region of Switzerland. The aim of this study was to examine cytokine/chemokine responses and innate immune sensing of F. culmorum in bone-marrow-derived dendritic cells and macrophages. Overall, dendritic cells and macrophages responded to F. culmorum spores but not to its secreted components (i.e., mycotoxins) by releasing large amounts of macrophage inflammatory protein (MIP)-1α, MIP-1β, MIP-2, monocyte chemoattractant protein (MCP)-1, RANTES, and interleukin (IL)-12p40, intermediate amounts of tumor necrosis factor (TNF), IL-6, IL-12p70, IL-33, granulocyte colony-stimulating factor (G-CSF), and interferon gamma-induced protein (IP-10), but no detectable amounts of IL-4 and IL-10, a pattern of mediators compatible with generation of Th1 or Th17 antifungal protective immune responses rather than with Th2-dependent allergic responses. The sensing of F. culmorum spores by dendritic cells required dectin-1, the main pattern recognition receptor involved in β-glucans detection, but likely not MyD88 and TRIF-dependent Toll-like receptors. Taken together, our results indicate that F. culmorum stimulates potently innate immune cells in a dectin-1-dependent manner, suggesting that inhalation of F. culmorum from grain dust may promote immune-related airway diseases in exposed worker populations.

Cerebriforms cells in cerebrum: an unusual finding

Background. Mycosis Fungoides (MF) is the most common cutaneous T-cell lymphoma, and large cell trasformation (tMF) is an adverse prognostic event. Extra-cutaneous dissemination can occur in the course of the disease, but dissemination to the central nervous system (CNS) is uncommon. Moreover, CNS lymphomas are overall rare and most often of B-cell phenotype. We report a case of CNS large T-cell lymphoma presenting as multiple cerebral lesions in a patient with a history of MF. Methods. We report a case of a 33-year-old woman, known since the age of 16 for erythematous plaques thought to be atopic dermatitis, who developed, end 2012, multiple nodular skin lesions and peripheral adenopathies. Two skin lesions were biopsied simultaneously, and diagnosed as MF and tMF. A lymph node biopsy showed dermatopathic changes without lymphoma (Stage IIB). She received local treatment (UVB, PUVA and radiation therapy) and interferon therapy, and experienced almost complete remission. In 2015 neurological symptoms lead to evidence multiple cerebral lesions, suspicious for lymphoma, evaluated by stereotaxic biopsies. We compared histopathological and molecular features of these with previous skin specimens. After negative bone marrow staging biopsy, she was recently started on chemotherapy (MATRIX). Short follow-up shows rapidly worsening clinical conditions. Results. One of the initial skin biopsies showed atypical lymphoid cells with epidermotropism, Pautrier abcesses and CD4+ CD30- phenotype; the other revealed diffuse dermal infiltration by predominantly large cerebriform tumor cells with high proliferative fraction, and CD2−CD3 −CD4+/−CD7−CD30+ALK- EMA- non-cytotoxic immunophenotype. Altogether, these results led us to diagnose MF and tMF, respectively. The brain was infiltrated by large atypical lymphoid cells with cerebriform nuclei, somewhat anaplastic features and perivascular distribution. By immunohistochemistry, tumor cells were highly proliferative, with a CD2−CD3+CD5−CD7+CD30+ activated cytotoxic immunophenotype. A diagnosis of CD30+ cytotoxic peripheral T-cell lymphoma was retained. TRG and TRB clonality analyses revealed clonal rearrangements in skin and CNS biopsies, with identical patterns in both skin specimens but only minimally overlapping profiles when compared to the CNS sample. Der Pathologe 6 ? 2015 | 633 Conclusions. The reported case illustrates an uncommon finding of a CNS T-cell lymphoma in a patient with previous MF, questioning the clonal relationship between the two diseases and challenging the adequate classification of this CNS lymphoma as either a progression or a de novo lymphoma. Despite differences in immunophenotype and clonality patterns, this CNS lymphoma could possibly represent an aggressive divergent evolution of a primary cutaneous T-cell lymphoma. Additional sequencing is ongoing to try to solve the question.

Macroporous Scaffolds for Bone Engineering. Studies on Cell Culture and Ectopic Bone Formation

Bone engineering is a rapidly developing area of reconstructive medicine where bone inducing factors and/or cells are combined with a scaffold material to regenerate the structure and function of the original tissue. The aim of this study was to compare the suitability of different macroporous scaffold types for bone engineering applications. The two scaffold categories studied were a) the mechanically strong and stable titanium fiber meshes and b) the elastic and biodegradable porous polymers. Furthermore, bioactive modifications were applied to these basic scaffold types, and their effect on the osteogenic responses was evaluated in cell culture and ectopic bone formation studies. The osteogenic phenotype of cultured cell-scaffold constructs was heightened with a sol-gel derived titania coating, but not with a mixed titania-silica coating. The latter coating also resulted in delayed ectopic bone formation in bone marrow stromal cell seeded scaffolds. However, the better bone contact in early implantation times and more even bone tissue distribution at later times indicated enhanced osteoconductivity of both the coated scaffold types. Overall, the most promising bone engineering results were obtained with titania coated fiber meshes. Elastic and biodegradable poly(ε-caprolactone/D,L-lactide) based scaffolds were also developed in this study. The degradation rates of the scaffolds in vitro were governed by the hydrophilicity of the polymer matrix, and the porous architecture was controlled by the amount and type of porogen used. A continuous phase macroporosity was obtained using a novel CaCl2 • 6H2O porogen. Dynamic culture conditions increased cell invasion, but decreased cell numbers and osteogenicity, within the scaffolds. Osteogenic differentiation in static cultures and ectopic bone formation in cell seeded scaffolds were enhanced in composites, with 30 wt-% of bioactive glass filler.

PET Imaging of Osteomyelitis. Feasibility of 18F-FDG, 68Ga-Chloride and 68Ga-DOTAVAP-P1 Tracers in Staphylococcal Bone Infections

Due to technical restrictions of the database system the title of the thesis does not show corretly on this page. Numbers in the title are in superscript. Please see the PDF-file for correct title. ---- Osteomyelitis is a progressive inflammatory disease of bone and bone marrow that results in bone destruction due to an infective microorganism, most frequently Staphylococcus aureus. Orthopaedic concern relates to the need for reconstructive and trauma-related surgical procedures in the fast grow¬ing population of fragile, aged patients, who have an increased susceptibility to surgical site infections. Depending on the type of osteomyelitis, infection may be acute or a slowly progressing, low-grade infection. Peri-implant infections lead to implant loosening. The emerging antibiotic resistance of com¬mon pathogens further complicates the situation. With current imaging methods, significant limitations exist in the diagnosing of osteomyelitis and implant-related infections. Positron emission tomography (PET) with a glucose analogue, 18F-fluoro¬deoxyglucose (18F-FDG), seems to facilitate a more accurate diagnosis of chronic osteomyelitis. The method is based on the increased glucose consumption of activated inflammatory cells. Unfortunately, 18F-FDG accumulates also in sterile inflammation regions and causes false-positive findings, for exam¬ple, due to post-operative healing processes. Therefore, there is a clinical need for new, more infection-specific tracers. In addition, it is still unknown why 18F-FDG PET imaging is less accurate in the detec¬tion of periprosthetic joint infections, most frequently due to Staphylococcus epidermidis. This doctoral thesis focused on testing novel PET tracers (68Ga-chloride and 68Ga-DOTAVAP-P1) for early detections of bone infections and evaluated the role of pathogen-related factors in the appli¬cations of 18F-FDG PET in the diagnostics of bone infections. For preclinical models of S. epidermidis and S. aureus bone/implant infections, the significance of the causative pathogen was studied with respect to 18F-FDG uptake. In a retrospective analysis of patients with confirmed bone infections, the significance of the presence or absence of positive bacterial cultures on 18F-FDG uptake was evalu¬ated. 18F-FDG and 68Ga-chloride resulted in a similar uptake in S. aureus osteomyelitic bones. However, 68Ga-chloride did not show uptake in healing bones, and therefore it may be a more-specific tracer in the early post-operative or post-traumatic phase. 68Ga-DOTAVAP-P1, a novel synthetic peptide bind¬ing to vascular adhesion protein 1 (VAP-1), was able to detect the phase of inflammation in healing bones, but the uptake of the tracer was elevated also in osteomyelitis. Low-grade peri-implant infec¬tions due to S. epidermidis were characterized by a low uptake of 18F-FDG, which reflects the virulence of the causative pathogen and the degree of leukocyte infiltration. In the clinical study, no relationship was found between the level of 18F-FDG uptake and the presence of positive or negative bacterial cul¬tures. Thus 18F-FDG PET may help to confirm metabolically active infection process in patients with culture-negative, histologically confirmed, low-grade osteomyelitis.

Biology and clinical utilization of mesenchymal progenitor cells

Within the complex cellular arrangement found in the bone marrow stroma there exists a subset of nonhematopoietic cells referred to as mesenchymal progenitor cells (MPC). These cells can be expanded ex vivo and induced, either in vitro or in vivo, to terminally differentiate into at least seven types of cells: osteocytes, chondrocytes, adipocytes, tenocytes, myotubes, astrocytes and hematopoietic-supporting stroma. This broad multipotentiality, the feasibility to obtain MPC from bone marrow, cord and peripheral blood and their transplantability support the impact that the use of MPC will have in clinical settings. However, a number of fundamental questions about the cellular and molecular biology of MPC still need to be resolved before these cells can be used for safe and effective cell and gene therapies intended to replace, repair or enhance the physiological function of the mesenchymal and/or hematopoietic systems.

In vitro cytotoxicity of the LDE: daunorubicin complex in acute myelogenous leukemia blast cells

Acute myelogenous leukemia (AML) blast cells show high-affinity degradation of low-density lipoprotein (LDL), suggesting an increased expression of cellular LDL receptors. LDE is a lipid microemulsion easily synthesized in vitro which is known to mimic the metabolic pathway of LDL. We used LDE as a carrier for daunorubicin and assayed the cytotoxicity of the complex using AML blast cells since RT-PCR analysis showed that AML cells express LDL receptor mRNA. The LDE:daunorubicin complex killed 46.7% of blast cells and 20.2% of normal bone marrow cells (P<0.001; Student t-test). Moreover, this complex destroyed AML blast cells as efficiently as free daunorubicin. Thus, LDE might be a suitable carrier of chemotherapeutic agents targeting these drugs to neoplastic cells and protecting normal tissues.

Differential expression of integrin subunits on adherent and nonadherent mast cells

Mast cell progenitors arise in bone marrow and then migrate to peripheral tissues where they mature. It is presumed that integrin receptors are involved in their migration and homing. In the present study, the expression of various integrin subunits was investigated in three systems of adherent and nonadherent mast cells. Mesentery mast cells, freshly isolated bone marrow-derived mast cells (BMMC) and RBL-2H3 cells grown attached to tissue culture flasks are all adherent mast cells and peritoneal mast cells, and cultured BMMC and RBL-2H3 cells grown in suspension represent nonadherent mast cell populations. Pure populations of mast cells were immunomagnetically isolated from bone marrow, mesentery and peritoneal lavage using the mast cell-specific monoclonal antibody AA4. By immunomicroscopy, we could demonstrate that all of these mast cells expressed alpha4, alpha5, alpha6, ß1 and ß7 integrin subunits. The expression of the alpha4 integrin subunit was 25% higher in freshly isolated mesentery mast cells and BMMC. Consistent with the results obtained by immunomicroscopy, mesentery mast cells expressed 65% more mRNA for the alpha4 integrin subunit than peritoneal mast cells. In vitro studies were also conducted using the rat mast cell line RBL-2H3. RBL-2H3 cells grown attached to the tissue culture flasks or as suspension cultures expressed the same integrin subunits identified in bone marrow, mesenteric and peritoneal mast cells ex vivo. Similarly, the expression of alpha4 integrin was higher in adherent cells. Therefore, alpha4 integrins may play a critical role in the anchorage of mast cells to the extracellular matrix in bone marrow and in peripheral tissues.

Asynchronous expression of myeloid antigens in leukemic cells in a PML/RARalpha transgenic mouse model

Acute promyelocytic leukemia (APL) is characterized by the expansion of blasts that resemble morphologically promyelocytes and harbor a chromosomal translocation involving the retinoic acid receptor a (RARa) and the promyelocytic leukemia (PML) genes on chromosomes 17 and 15, respectively. The expression of the PML/RARa fusion gene is essential for APL genesis. In fact, transgenic mice (TM) expressing PML/RARa develop a form of leukemia that mimics the hematological findings of human APL. Leukemia is diagnosed after a long latency (approximately 12 months) during which no hematological abnormality is detected in peripheral blood (pre-leukemic phase). In humans, immunophenotypic analysis of APL blasts revealed distinct features; however, the precise immunophenotype of leukemic cells in the TM model has not been established. Our aim was to characterize the expression of myeloid antigens by leukemic cells from hCG-PML/RARa TM. In this study, TM (N = 12) developed leukemia at the mean age of 13.1 months. Morphological analysis of bone marrow revealed an increase of the percentage of immature myeloid cells in leukemic TM compared to pre-leukemic TM and wild-type controls (48.63 ± 16.68, 10.83 ± 8.11, 7.4 ± 5.46%, respectively; P < 0.05). Flow cytometry analysis of bone marrow and spleen from leukemic TM identified the asynchronous co-expression of CD34, CD117, and CD11b. This abnormal phenotype was rarely detected prior to the diagnosis of leukemia and was present at similar frequencies in hematologically normal TM and wild-type controls of different ages. The present results demonstrate that, similarly to human APL, leukemic cells from hCG-PML/RARa TM present a specific immunophenotype.