Marina Ferrarini

Consensus guidelines for the diagnosis and clinical management of Erdheim-Chester disease

Erdheim-Chester disease (ECD) is a rare, non-Langerhans histiocytosis. Recent findings suggest that ECD is a clonal disorder, marked by recurrent BRAFV600E mutations in >50% of patients, in which chronic uncontrolled inflammation is an important mediator of disease pathogenesis. Although ∼500 to 550 cases have been described in the literature to date, increased physician awareness has driven a dramatic increase in ECD diagnoses over the last decade. ECD frequently involves multiple organ systems and has historically lacked effective therapies. Given the protean clinical manifestations and the lack of a consensus-derived approach for the management of ECD, we provide here the first multidisciplinary consensus guidelines for the clinical management of ECD. These recommendations were outlined at the First International Medical Symposium for ECD, comprised of a comprehensive group of international academicians with expertise in the pathophysiology and therapy of ECD. Detailed recommendations on the initial clinical, laboratory, and radiographic assessment of ECD patients are presented in addition to treatment recommendations based on critical appraisal of the literature and clinical experience. These formalized consensus descriptions will hopefully facilitate ongoing and future research efforts in this disorder.

Human γδ T cells: A nonredundant system in the immune-surveillance against cancer

Abstract Down-regulation of expression of MHC alleles, as well as tumor-specific antigens, is observed frequently during tumor progression, resulting in an impairment of MHC-restricted, αβ-T-cell-mediated, tumor-specific immunity. Given the unique set of antigens recognized and the lack of requirement for classical antigen-presenting molecules, γδ T cells might, therefore, represent a nonredundant system in anticancer surveillance, as proposed for the immune response against pathogens. Evidence that γδ and αβ T cells make distinct contributions to anticancer surveillance has been provided recently in mice. Here, we discuss the potential role played by resident Vδ1 + and circulating Vδ2 + T cells in the defense against solid tumors and hematological malignancies.

Autocrine Nitric Oxide Modulates CD95-induced Apoptosis in γδ T Lymphocytes

γδ T lymphocytes play an important early role in the defense against pathogens. Their function is terminated by acquisition of susceptibility to CD95-triggered apoptosis. Here we show that the regulation of this process depends on the activity of the endothelial NO synthase expressed by γδ T lymphocytes, which is modulated in an activation-dependent way. The effects of nitric oxide thus generated, mediated via cGMP generation, are exerted at at least two sites along the CD95 signaling cascade: one at, or upstream, and the other downstream of ceramide generation. At either site, nitric oxide/cGMP action is sufficient for protection from apoptosis. The effect of NO is selective for apoptosis induced by CD95 cross-linking, since it does not affect apoptotic program triggered by other stimuli. The evidence here reported demonstrates a new physiological role for nitric oxide, acting as a survival factor for T lymphocytes.

Macrophages exposed to Mycobacterium tuberculosis release chemokines able to recruit selected leucocyte subpopulations: focus on γδ cells

Granuloma is a typical feature of tuberculosis. We evaluated the chemotaxis of selected human leucocyte subsets induced by macrophages incubated with Mycobacterium tuberculosis (MT)‐derived products in vitro. The release of monocyte chemotactic protein 1 (MCP‐1) and interleukin‐8 (IL‐8) correlated with the specific induction of strong chemotaxis towards monocytes and polymorphonuclear leucocytes (PMNs). γδ and T helper type 1 (Th1) αβ lymphocytes were chemoattracted, while T‐resting, IL‐2‐activated and Th2 lymphocytes were unaffected. Activation with mycobacterium‐derived, phosphate‐containing components, modulated the chemokine receptor profile of γδ T lymphocytes as well as their pattern of cyto‐chemokine production, disclosing a potential for their active participation in granuloma formation. In particular, CXCR3 and IP‐10, which we found to be released by MT‐pulsed alveolar macrophages, seem to represent the receptor–counter‐receptor pair implicated in the chemotaxis of γδ lymphocytes. Immunohistochemical analysis and in situ hybridization revealed the in vivo presence of IL‐8, MCP‐1 and IL‐10 in lymph node and lung tuberculous granulomas. Our results underscore the role of MT extracts in the induction of macrophage‐derived chemokines responsible for the orchestrated recruitment of PMNs, monocytes, and Th1 and γδ T cells, as well as in the regulation of γδ function.

MICA Expressed by Multiple Myeloma and Monoclonal Gammopathy of Undetermined Significance Plasma Cells Costimulates Pamidronate-Activated γδ Lymphocytes

Amino-biphosphonates (like pamidronate) activate human Vgamma9/Vdelta2 T lymphocytes and promote their cytotoxicity against multiple myeloma cells. T-cell receptor (TCR)-mediated effector functions of gammadelta cells are enhanced upon triggering of the activating receptor NKG2D by MICA, a stress-inducible antigen expressed by epithelial and some hematopoietic tumors, including multiple myeloma. Here we show that MICA was expressed not only by myeloma cell lines and by 6 of 10 primary multiple myeloma cells from patients but also by bone marrow plasma cells from all (six of six) patients with preneoplastic gammopathy (monoclonal gammopathy of undetermined significance, MGUS), a direct precursor of multiple myeloma. Moreover, compared with multiple myeloma plasma cells, MICA was expressed by MGUS plasma cells at significantly (P < 0.05) higher levels. MICA expressed by myeloma cell lines contributed to killing and IFN-gamma production by Vgamma9/Vdelta2 cells only upon pamidronate treatment, suggesting a dual interaction between Vgamma9/Vdelta2 lymphocytes and multiple myeloma plasma cells involving both TCR triggering and NKG2D-mediated signals. Finally, MICA enhanced killing of freshly derived, pamidronate-treated multiple myeloma cells from patients by gammadelta cells, as indicated by the significantly (P < 0.05) higher gammadelta cytotoxicity against MICA-positive rather than MICA-negative multiple myeloma cells. Our results indicate that MICA expressed by monoclonal plasma cells is functional and correlates with disease stages, suggesting a role for the molecule in the immune surveillance against multiple myeloma. Moreover, pamidronate-activated Vgamma9/Vdelta2 lymphocytes can be exploited in the immune therapy of early stages multiple myeloma and possibly of premalignant disease.

BRAFV600E-mutation is invariably present and associated to oncogene-induced senescence in Erdheim-Chester disease

Objectives Erdheim-Chester disease (ECD) is a rare form of histiocytosis characterised by uncontrolled chronic inflammation. The oncogenic BRAFV600E mutation has been reported in biopsies in 19 out of 37 patients with ECD from the largest published cohort, but never found in the patients’ peripheral blood. Also, the role of the mutation in the pathogenesis of the disease has not been elucidated yet. BRAFV600E has been associated with oncogene-induced senescence (OIS), a protective mechanism against oncogenic events, characterised by the induction of proinflammatory pathways. Methods We verified the BRAF status in biopsies and peripheral blood from 18 patients with ECD from our cohort and matched controls by means of immunohistochemistry and of an ultrasensitive assay, based on the combination of a locked nucleic acid PCR and pyrosequencing. Droplet digital PCR was used to confirm the findings. We also evaluated the presence of senescence markers in ECD histiocytes. Results BRAFV600E mutation was present in all the biopsy and peripheral blood samples from patients with ECD and in none of the controls. ECD histiocytes and a fraction of circulating monocytes from patients with ECD showed signs of a constitutive activation of the MAPK pathway. Moreover, BRAF-mutated histiocytes expressed markers of OIS. Conclusions The oncogenic BRAFV600E mutation is present in biopsies and in the peripheral blood from all patients with ECD who were evaluated and is associated with OIS. These findings have significant implications for the pathogenesis, diagnosis and treatment of ECD.

Bortezomib induces autophagic death in proliferating human endothelial cells.

The proteasome inhibitor Bortezomib has been approved for the treatment of relapsed/refractory multiple myeloma (MM), thanks to its ability to induce MM cell apoptosis. Moreover, Bortezomib has antiangiogenic properties. We report that endothelial cells (EC) exposed to Bortezomib undergo death to an extent that depends strictly on their activation state. Indeed, while quiescent EC are resistant to Bortezomib, the drug results maximally toxic in EC switched toward angiogenesis with FGF, and exerts a moderate effect on subconfluent HUVEC. Moreover, EC activation state deeply influences the death pathway elicited by Bortezomib: after treatment, angiogenesis-triggered EC display typical features of apoptosis. Conversely, death of subconfluent EC is preceded by ROS generation and signs typical of autophagy, including intense cytoplasmic vacuolization with evidence of autophagosomes at electron microscopy, and conversion of the cytosolic MAP LC3 I form toward the autophagosome-associated LC3 II form. Treatment with the specific autophagy inhibitor 3-MA prevents both LC3 I/LC3 II conversion and HUVEC cell death. Finally, early removal of Bortezomib is accompanied by the recovery of cell shape and viability. These findings strongly suggest that Bortezomib induces either apoptosis or autophagy in EC; interfering with the autophagic response may potentiate the antiangiogenic effect of the drug.

Mycobacterium tuberculosisexploits the CD95/CD95 ligand system of γ δ T cells to cause apoptosis

Vγ9/Vδ2+ T cells specifically recognize Mycobacterium tuberculosis in vitro and are precociously recruited in early mycobacterial lesions. Even if γ δ T cells are only fortuitously detected in granulomas or bronchoalveolar lavages of patients with active pulmonary tuberculosis, a role in shaping the mature α β T cell response against M. tuberculosis is substantiated. Here we provide a molecular explanation for this paradox: the engagement of the γ δ TCR by mycobacterial antigens induced the expression of CD95 ligand (CD95L) by chronically activated CD95+ /CD95L− γ δ T lymphocytes. The receptor was functional, as CD95/CD95L interaction triggered the bystander death of CD95+ cells by apoptosis. Cell death was abolished by CD95‐blocking antibodies. The transient accumulation at the site of infection of CD95L+ γ δ lymphocytes, capable of interacting with CD95+ leukocytes attracted by the response towards the pathogen, may determine the characteristics of the ensuing granulomatous disease.

Redox homeostasis modulates the sensitivity of myeloma cells to bortezomib

The use of proteasome inhibitors have been a major advance in the treatment of multiple myeloma (MM), but their mechanisms of action remain largely unclear. A better understanding of the cellular events downstream of proteasome inhibition is essential to improve the response and identify new combination therapies for MM and other malignancies. This study analysed the relationships between redox homeostasis and bortezomib treatment in MM cells. Our data showed that decreasing intracellular glutathione through buthionine sulfoximine treatment strongly enhances bortezomib toxicity, whilst antioxidants protect MM cells from bortezomib‐mediated cell death. Bortezomib treatment decreases intracellular glutathione both in MM cell lines and in malignant plasma cells obtained from MM patients. Glutamate‐cysteine ligase (GCLM) and haem‐oxygenase‐1 (HMOX1), two genes involved in the Nrf‐2‐mediated antioxidant response, as well as two eIF2α‐downstream transcription factors, activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP), are upregulated, indicating that redox‐related adaptive responses are initiated in bortezomib‐treated MM cells. These findings demonstrate tight links between sensitivity to proteasome inhibition and redox homeostasis in MM cells and have potential implications for treatment.

Ex-Vivo Dynamic 3-D Culture of Human Tissues in the RCCS™ Bioreactor Allows the Study of Multiple Myeloma Biology and Response to Therapy

Three-dimensional (3-D) culture models are emerging as invaluable tools in tumor biology, since they reproduce tissue-specific structural features and cell-cell interactions more accurately than conventional 2-D cultures. Multiple Myeloma, which depends on myeloma cell-Bone Marrow microenvironment interactions for development and response to drugs, may particularly benefit from such an approach. An innovative 3-D dynamic culture model based on the use of the RCCS™ Bioreactor was developed to allow long-term culture of myeloma tissue explants. This model was first validated with normal and pathological explants, then applied to tissues from myeloma patients. In all cases, histological examination demonstrated maintenance of viable myeloma cells inside their native microenvironment, with an overall well preserved histo-architecture including bone lamellae and vessels. This system was then successfully applied to evaluate the cytotoxic effects exerted by the proteasome inhibitor Bortezomib not only on myeloma cells but also on angiogenic vessels. Moreover, as surrogate markers of specialized functions expressed by myeloma cells and microenvironment, β2 microglobulin, VEGF and Angiopoietin-2 levels, as well as Matrix Metalloproteases activity, were evaluated in supernatants from 3D cultures and their levels reflected the effects of Bortezomib treatment. Notably, determination of β2 microglobulin levels in supernatants from Bortezomib-treated samples and in patientssera following Bortezomib-based therapies disclosed an overall concordance in the response to the drug ex vivo and in vivo. Our findings indicate, as a proof of principle, that 3-D, RCCS™ bioreactor-based culture of tissue explants can be exploited for studying myeloma biology and for a pre-clinical approach to patient-targeted therapy.