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Multiple myeloma (MM) is linked to chronic NF-κB activity in myeloma cells, but this activity is generally considered a cell-autonomous property of the cancer cells. The precise extent of NF-κB activation and the contributions of the physical microenvironment and of cell-to-cell communications remain largely unknown. By quantitative immunofluorescence, we found that NF-κB is mildly and heterogeneously activated in a fraction of MM cells in human BMs, while only a minority of MM cells shows a strong activation. To gain quantitative insights on NF-κB activation in living MM cells, we combined advanced live imaging of endogenous p65 Venus-knocked-in in MM.1S and HS-5 cell lines to model MM and mesenchymal stromal cells (MSCs), cell co-cultures, microfluidics and custom microbioreactors to mimic the 3D-interactions within the bone marrow (BM) microenvironment. We found that i) reciprocal MM-MSC paracrine crosstalk and cell-to-scaffold interactions shape the inflammatory response in the BM; ii) the pro-inflammatory cytokine IL-1β, abundant in MM patients' plasma, activates MSCs, whose paracrine signals are responsible for strong NF-κB activation in a minority of MM cells; iii) IL-1β, but not TNF-α, activates NF-κB in vivo in BM-engrafted MM cells, while its receptor inhibitor Anakinra reduces the global NF-κB activation. We propose that NF-κB activation in the BM of MM patients is mild, restricted to a minority of cells and modulated by the interplay of restraining physical microenvironmental cues and activating IL-1β-dependent stroma-to-MM crosstalk., (© 2024. The Author(s).)

Objective: Trained immunity (TI) is a de facto memory program of innate immune cells, characterized by immunometabolic and epigenetic changes sustaining enhanced production of cytokines. TI evolved as a protective mechanism against infections; however, inappropriate activation can cause detrimental inflammation and might be implicated in the pathogenesis of chronic inflammatory diseases. In this study, we investigated the role of TI in the pathogenesis of giant cell arteritis (GCA), a large-vessel vasculitis characterized by aberrant macrophage activation and excess cytokine production., Methods: Monocytes from GCA patients and from age- and sex-matched healthy donors were subjected to polyfunctional studies, including cytokine production assays at baseline and following stimulation, intracellular metabolomics, chromatin immunoprecipitation-qPCR, and combined ATAC/RNA sequencing. Immunometabolic activation (i.e. glycolysis) was assessed in inflamed vessels of GCA patients with FDG-PET and immunohistochemistry (IHC), and the role of this pathway in sustaining cytokine production was confirmed with selective pharmacologic inhibition in GCA monocytes., Results: GCA monocytes exhibited hallmark molecular features of TI. Specifically, these included enhanced IL-6 production upon stimulation, typical immunometabolic changes (e.g. increased glycolysis and glutaminolysis) and epigenetic changes promoting enhanced transcription of genes governing pro-inflammatory activation. Immunometabolic changes of TI (i.e. glycolysis) were a feature of myelomonocytic cells in GCA lesions and were required for enhanced cytokine production., Conclusions: Myelomonocytic cells in GCA activate TI programs sustaining enhanced inflammatory activation with excess cytokine production., (© The Author(s) 2023. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In this protocol, we describe how to generate 3D culture surrogates of chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) bone marrow microenvironments. We detail the use of culturing scaffolds populated with BM stromal cells and tumor cells in the RCCS™ bioreactor. This 3D culture can efficiently recapitulate tumor-stroma crosstalk and allows the testing of drugs such as ibrutinib and bortezomib. Moreover, this protocol can be used for the generation of other and more complex tumor microenvironments. For complete details on the use and execution of this protocol, please refer to Belloni et al. (2018) and Barbaglio et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Therapeutic monoclonal antibodies (mAbs) are an emerging and very active frontier in clinical oncology, with hundred molecules currently in use or being tested. These treatments have already revolutionized clinical outcomes in both solid and hematological malignancies. However, identifying patients who are most likely to benefit from mAbs treatment is currently challenging and limiting the impact of such therapies. To overcome this issue, and to fulfill the expectations of mAbs therapies, it is urgently required to develop proper culture models capable of faithfully reproducing the interactions between tumor and its surrounding native microenvironment (TME). Three-dimensional (3D) models which allow the assessment of the impact of drugs on tumors within its TME in a patient-specific context are promising avenues to progressively fill the gap between conventional 2D cultures and animal models, substantially contributing to the achievement of personalized medicine. This review aims to give a brief overview of the currently available 3D models, together with their specific exploitation for therapeutic mAbs testing, underlying advantages and current limitations to a broader use in preclinical oncology.

Trained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation., (© 2021 by The American Society of Hematology.)

Therapeutic strategies designed to tinker with cancer cell DNA damage response have led to the widespread use of PARP inhibitors for BRCA1/2-mutated cancers. In the haematological cancer multiple myeloma, we sought to identify analogous synthetic lethality mechanisms that could be leveraged upon established cancer treatments. The combination of ATR inhibition using the compound VX-970 with a drug eliciting interstrand cross-links, melphalan, was tested in in vitro, ex vivo, and most notably in vivo models. Cell proliferation, induction of apoptosis, tumor growth and animal survival were assessed. The combination of ATM inhibition with a drug triggering double strand breaks, doxorucibin, was also probed. We found that ATR inhibition is strongly synergistic with melphalan, even in resistant cells. The combination was dramatically effective in targeting myeloma primary patient cells and cell lines reducing cell proliferation and inducing apoptosis. The combination therapy significantly reduced tumor burden and prolonged survival in animal models. Conversely, ATM inhibition only marginally impacted on myeloma cell survival, even in combination with doxorucibin at high doses. These results indicate that myeloma cells extensively rely on ATR, but not on ATM, for DNA repair. Our findings posit that adding an ATR inhibitor such as VX-970 to established therapeutic regimens may provide a remarkably broad benefit to myeloma patients.

Background: Targeted therapy with BRAF and MEK inhibitors has improved the survival of patients with BRAF-mutated metastatic melanoma, but most patients relapse upon the onset of drug resistance induced by mechanisms including genetic and epigenetic events. Among the epigenetic alterations, microRNA perturbation is associated with the development of kinase inhibitor resistance. Here, we identified and studied the role of miR-146a-5p dysregulation in melanoma drug resistance., Methods: The miR-146a-5p-regulated NFkB signaling network was identified in drug-resistant cell lines and melanoma tumor samples by expression profiling and knock-in and knock-out studies. A bioinformatic data analysis identified COX2 as a central gene regulated by miR-146a-5p and NFkB. The effects of miR-146a-5p/COX2 manipulation were studied in vitro in cell lines and with 3D cultures of treatment-resistant tumor explants from patients progressing during therapy., Results: miR-146a-5p expression was inversely correlated with drug sensitivity and COX2 expression and was reduced in BRAF and MEK inhibitor-resistant melanoma cells and tissues. Forced miR-146a-5p expression reduced COX2 activity and significantly increased drug sensitivity by hampering prosurvival NFkB signaling, leading to reduced proliferation and enhanced apoptosis. Similar effects were obtained by inhibiting COX2 by celecoxib, a clinically approved COX2 inhibitor., Conclusions: Deregulation of the miR-146a-5p/COX2 axis occurs in the development of melanoma resistance to targeted drugs in melanoma patients. This finding reveals novel targets for more effective combination treatment. Video Abstract.

Erdheim-Chester disease (ECD) is a rare histiocytosis characterized by infiltration of multiple tissues by CD68 + foamy Mϕs (or 'histiocytes'). Clinical manifestations arise from mass-forming lesions or from tissue and systemic inflammation. ECD histiocytes harbor oncogenic mutations along the MAPK-kinase signaling pathway (BRAF V600E in more than half of the patients), and secrete abundant pro-inflammatory cytokines and chemokines. Based on these features, ECD is considered an inflammatory myeloid neoplasm, and is accordingly managed with targeted kinase inhibitors or immunosuppressive and cytokine-blocking agents. Evidence is emerging that maladaptive metabolic changes, particularly up-regulated glycolysis, represent an additional, mutation-driven feature of ECD histiocytes, which sustains deregulated and protracted pro-inflammatory activation and cytokine production. Besides translational relevance to the management of ECD patients and to the development of new therapeutic approaches, recognition of ECD as a natural human model of chronic, maladaptive Mϕ activation instructs the understanding of Mϕ dysfunction in other chronic inflammatory conditions., (©2020 Society for Leukocyte Biology.)

Multiple myeloma develops primarily inside the bone marrow microenvironment, that confers pro-survival signals and drug resistance. 3D cultures that reproduce multiple myeloma-bone marrow interactions are needed to fully investigate multiple myeloma pathogenesis and response to drugs. To this purpose, we exploited the 3D Rotary Cell Culture System bioreactor technology for myeloma-bone marrow co-cultures in gelatin scaffolds. The model was validated with myeloma cell lines that, as assessed by histochemical and electron-microscopic analyses, engaged contacts with stromal cells and endothelial cells. Consistently, pro-survival signaling and also cell adhesion-mediated drug resistance were significantly higher in 3D than in 2D parallel co-cultures. The contribution of the VLA-4/VCAM1 pathway to resistance to bortezomib was modeled by the use of VCAM1 transfectants. Soluble factor-mediated drug resistance could be also demonstrated in both 2D and 3D co-cultures. The system was then successfully applied to co-cultures of primary myeloma cells-primary myeloma bone marrow stromal cells from patients and endothelial cells, allowing the development of functional myeloma-stroma interactions and MM cell long-term survival. Significantly, genomic analysis performed in a high-risk myeloma patient demonstrated that culture in bioreactor paralleled the expansion of the clone that ultimately dominated in vivo Finally, the impact of bortezomib on myeloma cells and on specialized functions of the microenvironment could be evaluated. Our findings indicate that 3D dynamic culture of reconstructed human multiple myeloma microenvironments in bioreactor may represent a useful platform for drug testing and for studying tumor-stroma molecular interactions., (Copyright© 2018 Ferrata Storti Foundation.)

Erdheim-Chester disease (ECD) is a rare histiocytosis, characterized by xanthogranulomatous tissue infiltration by foamy histiocytes. Fibrosis, a histologic hallmark of ECD, is responsible for lesion growth and clinical manifestations. Unraveling molecular fibrotic pathway in ECD would allow the identification of new pharmacologic targets. In this study, we evaluated serum and tissue samples from a large cohort of ECD patients focusing on two major pro-fibrotic mediators, TGF-β1 and chemokine ligand 18 (CCL18). We found a marked increase in CCL18 but not TGF-β1 levels in serum and lesions of ECD patients ( p < 0.001 ), independently of treatment status and consistently over time. Using a linear mathematical model, we also found that elevated CCL18 serum levels correlate with both number and severity of disease localizations. These findings suggest the involvement of CCL18-induced fibrosis in ECD pathogenesis, providing a rationale for exploring CCL18 inhibition as a treatment for progressive fibrosis in ECD.

Treatment of Erdheim-Chester disease (ECD), a rare non-Langerhans histiocytosis, relies on interferon-α, chemotherapeutic agents such as purine analogs, cytokine-blocking agents and BRAF inhibitors. Since interleukin (IL)-6 levels are elevated in serum and lesions of ECD patients, we evaluated the therapeutic efficacy and safety of IL-6 blockade with tocilizumab. We conducted an open-label, single-arm, phase II, prospective study of tocilizumab in three patients with multisystem ECD and poor tolerance/contraindications to IFN-α. Modifications of symptoms attributed to ECD represented the criteria for evaluation of clinical response. Changes at positron emission tomography scan, computed tomography scan, and magnetic resonance imaging at month 6 represented the main criteria for the evaluation of radiological response. Sustained complete clinical response and partial radiological improvement were observed in two patients, paralleled by modulation of systemic pro-inflammatory mediators. In spite of disease stabilization or improvement at extra-neurological sites, a third patient experienced a radiologic and clinical progression of central nervous system involvement, mirrored by a dramatic increase of circulating IL-6 and related cytokines. These findings indicate that IL-6 inhibition can be effective in ECD, but caution is advisable in patients with neurologic involvement. IL-6 emerges as a central mediator in ECD pathogenesis.

3D-dynamic culture models represent an invaluable tool for a better comprehension of tumor biology and drug response, as they accurately re-create/preserve the complex multicellular organization and the dynamic interactions of the parental microenvironment, which can affect tumor fate and drug sensitivity. Hence, development of models that recapitulate tumor within its embedding microenvironment is an imperative need. This is particularly true for multiple myeloma (MM), which survives almost exclusively in the bone marrow (BM). To meet this need, we have previously exploited and validated an innovative 3D-dynamic culture technology, based on the use of the Rotary Cell Culture System (RCCS ™) bioreactor . Here, we describe, step by step, the procedures we have employed to establish two human MM ex vivo models, i.e., the culture of human BM-derived isolated cells and of MM tissues from patients.

Erdheim-Chester disease (ECD) is a rare non-Langerhans cell histiocytosis (LCH) characterized by tissue infiltration with CD68(+) foamy histiocytes. TNF-related chronic inflammation and mutations in the MAP kinase signaling pathway in histiocytes are recognized as the two major pathogenic events. Among pleomorphic clinical manifestations, cardiovascular involvement is frequent and prognostically relevant. Evaluation of ECD clinical course and response to treatment is, however, still challenging. Taking advantage of the two largest cohorts of ECD patients worldwide, we investigated the relevance and the potential of circulating Chromogranin A (CgA), a pro-hormone involved in cardiovascular homeostasis and inflammation, as a biomarker of response to therapy in ECD. Consistent with other TNF-related inflammatory diseases, we found that not only TNF-α and soluble TNF-Receptors (sTNF-Rs), but also CgA plasma levels were significantly increased in ECD patients compared to controls. CgA, but not sTNF-Rs, discriminated cardiovascular involvement in ECD patients and correlated with pro-Brain Natriuretic Peptide (pro-BNP). In a single case, where a cardiac biopsy was available, CgA was found expressed by cardiomyocytes but not by infiltrating histiocytes. In four ECD patients, where serial determination of these parameters was obtained, the kinetics of sTNF-Rs and CgA paralleled response to therapy with anti-cytokine inhibitors; specifically, sTNF-Rs overlapped TNF-associated inflammation, while CgA, together with pro-BNP, closely mirrored response of cardiac disease. Our data indicate that both sTNF-Rs and CgA are linked to ECD pathophysiology. Moreover, CgA, in concert with pro-BNP, can be further exploited to fulfill the unmet clinical need of non-invasive reliable biomarkers of cardiac disease in these patients.

Hypoxia-inducible transcription factors (HIFs) regulate a wide array of adaptive responses to hypoxia and are often activated in solid tumors and hematologic malignancies due to intratumoral hypoxia and emerging new layers of regulation. We found that in chronic lymphocytic leukemia (CLL), HIF-1α is a novel regulator of the interaction of CLL cells with protective leukemia microenvironments and, in turn, is regulated by this interaction in a positive feedback loop that promotes leukemia survival and propagation. Through unbiased microarray analysis, we found that in CLL cells, HIF-1α regulates the expression of important chemokine receptors and cell adhesion molecules that control the interaction of leukemic cells with bone marrow and spleen microenvironments. Inactivation of HIF-1α impairs chemotaxis and cell adhesion to stroma, reduces bone marrow and spleen colonization in xenograft and allograft CLL mouse models, and prolongs survival in mice. Of interest, we found that in CLL cells, HIF-1α is transcriptionally regulated after coculture with stromal cells. Furthermore, HIF-1α messenger RNA levels vary significantly within CLL patients and correlate with the expression of HIF-1α target genes, including CXCR4, thus further emphasizing the relevance of HIF-1α expression to CLL pathogenesis., (© 2016 by The American Society of Hematology.)

Angiogenesis has been postulated to be critical for the pathogenesis of multiple myeloma, a neoplastic disease characterized by abnormal proliferation of malignant plasma cells in the bone marrow (BM). Cleavage of the N- and C-terminal regions of circulating chromogranin A (CgA, CHGA), classically an antiangiogenic protein, can activate latent antiangiogenic and proangiogenic sites, respectively. In this study, we investigated the distribution of CgA-derived polypeptides in multiple myeloma patients and the subsequent implications for disease progression. We show that the ratio of pro/antiangiogenic forms of CgA is altered in multiple myeloma patients compared with healthy subjects and that this ratio is higher in BM plasma compared with peripheral plasma, suggesting enhanced local cleavage of the CgA C-terminal region. Enhanced cleavage correlated with increased VEGF and FGF2 BM plasma levels and BM microvascular density. Using the Vk*MYC mouse model of multiple myeloma, we further demonstrate that exogenously administered CgA was cleaved in favor of the proangiogenic form and was associated with increased microvessel density. Mechanistic studies revealed that multiple myeloma and proliferating endothelial cells can promote CgA C-terminal cleavage by activating the plasminogen activator/plasmin system. Moreover, cleaved and full-length forms could also counter balance the pro/antiangiogenic activity of each other in in vitro angiogenesis assays. These findings suggest that the CgA-angiogenic switch is activated in the BM of multiple myeloma patients and prompt further investigation of this CgA imbalance as a prognostic or therapeutic target. Cancer Res; 76(7); 1781-91. ©2016 AACR., (©2016 American Association for Cancer Research.)

Objectives: Erdheim-Chester disease (ECD) is a rare form of histiocytosis characterised by uncontrolled chronic inflammation. The oncogenic BRAF(V600E) 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. BRAF(V600E) 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: BRAF(V600E) 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 BRAF(V600E) 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., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Angiopoietin-2 (Ang-2) is involved in angiogenesis in both solid and hematological malignancies. In Multiple Myeloma (MM), serum Ang-2 correlates with disease progression and response to therapy. To address the patho-physiologic role of Ang-2 in MM associated angiogenesis, we used sera from patients with active MM, which contained significantly higher levels of the molecule, compared to those from patients with smoldering MM and Monoclonal Gammopathy of Undetermined Significance. MM Bone Marrow (BM) sera with high Ang-2 concentration specifically contributed to endothelial cell (EC) activation, while Ang-1 containing sera maintained EC stabilization. The functional dichotomy of Ang-1 and Ang-2 was confirmed by the triggering of distinctive signaling pathways down-stream the common Tie-2 receptor, i.e., the Akt or the ERK- phosphorylation pathway. Notably, Ang-2 but not VEGF serum levels correlated with BM micro-vessel density, further underscoring the key role of Ang-2 in angiogenesis. Western Blot, RT-PCR and immunocytochemistry identified MMEC as the major source of Ang-2, at variance with MM cells and CD14(+) BM monocytes. These data suggest that Ang-2 produced in the BM milieu may contribute to MM angiogenesis and suggest that the molecule can be further exploited both as angiogenesis biomarker and as a potential therapeutic target., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

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 patients'sera 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.

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., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Vgamma9 Vdelta2 T lymphocytes are involved in the immune response against hematological malignancies and certain pathogens through the recognition of nonpeptidic Ags expressed by tumors and infected cells. Being equipped with proinflammatory chemokine receptors, they participate to the early phases of inflammation acting as both effector and connector cells between innate and adaptive immunity. We show in this study that after initial TCR triggering short- and long-term cultured gammadelta lymphocytes differ in their susceptibility to activation-induced apoptosis and proinflammatory phenotype. Activation-induced apoptosis was triggered by anti-CD95 mAbs or by the gammadeltaTCR stimuli isopentenyl pyrophosphate and pamidronate, the latter in the presence of monocytes. In particular, short-term cultured cells are resistant to apoptosis and characterized by expression of anti-apoptotic cellular FLIP molecules and partial spontaneous caspase-8 activation. Linked to this behavior, short-term gammadelta cells display constitutive activation of the transcription factor NF-kappaB, which is functionally related to their apoptosis-resistant phenotype. Finally, they spontaneously secreted elevated amounts of the NF-kappaB-regulated chemokines CCL3, CCL4, and CCL5, which likely contributed to down-modulation of the inflammatory CCR5 receptor. Conversely, long-term cultured apoptosis-sensitive gammadelta cells displayed uncleaved caspase-8 and no constitutive NF-kappaB activation; moreover, they secreted CC chemokines only upon TCR triggering coupled to the re-expression of CCR5. The expression of members of the TNF receptor family, including CD30 and TNFRII, also varied according to the time in culture. Altogether our data support a link between resistance to apoptosis and a proinflammatory phenotype in gammadelta T lymphocytes, unraveling the crucial role of NF-kappaB in regulating the switch from resistance to apoptosis susceptibility.

The interleukin-12 (IL-12) receptor (R) B2 gene acts as tumor suppressor in human acute and chronic B-cell leukemias/lymphomas and IL-12rb2-deficient mice develop spontaneously localized plasmacytomas. With this background, we investigated the role of IL-12R beta 2 in multiple myeloma (MM) pathogenesis. Here we show the following: (1) IL-12R beta 2 was expressed in primary MM cells but down-regulated compared with normal polyclonal plasmablastic cells and plasma cells (PCs). IL-6 dampened IL-12R beta 2 expression on polyclonal plasmablastic cells and MM cells. (2) IL-12 reduced the proangiogenic activity of primary MM cells in vitro and decreased significantly (P = .001) the tumorigenicity of the NCI-H929 cell line in SCID/NOD mice by inhibiting cell proliferation and angiogenesis. The latter phenomenon was found to depend on abolished expression of a wide panel of proangiogenic genes and up-regulated expression of the antiangiogenic genes IFN-gamma, IFN-alpha, platelet factor-4, and TIMP-2. Inhibition of the angiogenic potential of primary MM cells was related to down-regulated expression of the proangiogenic genes CCL11, vascular endothelial-cadherin, CD13, and AKT and to up-regulation of an IFN-gamma-related antiangiogenic pathway. Thus, IL-12R beta 2 directly restrains MM cell growth, and targeting of IL-12 to tumor cells holds promise as new therapeutic strategy.

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 eIF2alpha-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.

Angiogenesis is a complex, orchestrated process that plays a critical role in several conditions and has special relevance in the progression of cancer. Hypoxia is the major stimulus for angiogenesis, and hypoxia-inducible transcription factor-1 alpha (HIF-1alpha) is its key mediator. We set up a novel in vitro model of HIF-1alpha up-regulation by treating human umbilical vein endothelial cells (HUVECs) with the hypoxia-mimicking deferoxamine (DFO) and found that this condition was sufficient to promote angiogenesis, like the well-known HUVEC model cultured under low pO(2.) The proteasome inhibitor bortezomib, which induces strong apoptosis in cancer cells, abrogated proliferation and angiogenesis of HUVECs when used at a high concentration (100 nM), yet promoted both functions at a low dosage (10 nM). This double-edged effect appeared to be mediated by differential effects exerted by the different concentrations of bortezomib on 2 master regulators of tumor-associated angiogenesis, HIF-1alpha and nuclear factor kappa B (NF-kB). Significantly, when HUVECs were induced to express HIF-1alpha prior to bortezomib treatment, proliferative and angiogenic responses were abolished, and a greatly enhanced proapoptotic effect was promoted with both concentrations of the drug. These findings indicate that HIF-1alpha up-regulation may sensitize endothelial cells to the antiangiogenic and proapoptotic effects of bortezomib and might be exploited to target tumor-associated vessels in the course of antiangiogenic therapies.

Objective: Erdheim-Chester disease (ECD) is a rare form of non-Langerhans' cell histiocytosis (LCH) of unknown etiology, characterized by diffuse histiocyte infiltration of bones and soft tissue. The purpose of this study was to assess cell proliferation and expression of cytokines, chemokines, and chemokine receptors that may potentially be important in histiocyte accumulation in ECD lesions., Methods: Biopsies were performed on 3 patients with ECD. The diagnosis of the disease was based on clinical signs including typical radiologic osteosclerosis, and on the detection of foamy CD68+,CD1a- non-Langerhans' cell histiocytes on histologic examination. The expression of the proliferation marker Ki-67 as well as of selected chemokine/chemokine receptor pairs and cytokines was analyzed by immunohistochemistry., Results: In all samples, Ki-67 was undetectable in CD68+ histiocytes. Conversely, these cells expressed the chemokines CCL2 (monocyte chemotactic protein 1), CCL4/macrophage inflammatory protein 1beta (MIP-1beta), CCL5/RANTES, CCL20/MIP-3alpha, and CCL19/MIP-3beta, and their counter-receptors CCR1, CCR2, CCR3, CCR5, CCR6, and CCR7. Moreover, ECD histiocytes expressed interferon-gamma-inducible 10-kd protein (CXCL10), which is specifically induced by interferon-gamma, and interleukin-6 and RANKL, which are both implicated in bone remodeling. Finally, all cases showed a Th1-type lymphocyte infiltrate., Conclusion: Our data indicate that, similar to LCH, ECD lesions are characterized by a complex cytokine and chemokine network, which may orchestrate histiocyte activation and accumulation through an autocrine loop and contribute to the pathogenesis of the disease.

Mutations decreasing function of the Fas death receptor cause the autoimmune lymphoproliferative syndrome (ALPS) with autoimmune manifestations, spleen/lymph node enlargement, and expansion of CD4/CD8-negative T cells. Dianzani Autoimmune Lymphoproliferative Disease (DALD) is a variant lacking this expansion. Perforin is involved in cell-mediated cytotoxicity and its biallelic mutations cause familial hemophagocytic lymphohistiocytosis (HLH). We previously described an ALPS patient carrying heterozygous mutations of the Fas and perforin genes and suggested that they concurred in ALPS. This work extends the analysis to 14 ALPS, 28 DALD, and 816 controls, and detects an N252S amino acid substitution in 2 ALPS, and an A91V amino acid substitution in 6 DALD. N252S conferred an OR = 62.7 (P = .0016) for ALPS and A91V conferred an OR = 3 (P = .016) for DALD. Copresence of A91V and variations of the osteopontin gene previously associated with DALD conferred an OR = 17 (P = .0007) for DALD. In one N252S patient, NK activity was strikingly defective in early childhood, but became normal in late childhood. A91V patients displayed lower NK activity than controls. These data suggest that perforin variations are a susceptibility factor for ALPS/DALD development in subjects with defective Fas function and may influence disease expression.

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.

We describe an immunocompetent 61-year-old woman who was negative for human immunodeficiency virus and who had recurrent human herpesvirus 8 (HHV-8) infection associated with a relapsing systemic inflammatory syndrome characterized by fever, lymphadenopathy, splenomegaly, edema, arthrosynovitis, and rash. Kaposi's sarcoma developed 10 months after the initial clinical presentation. A correlation was documented between the recurrent clinical manifestations and the HHV-8 load in plasma and peripheral-blood mononuclear cells. Histologic examination of an enlarged lymph node heavily infected with HHV-8 revealed an atypical lymphoproliferative disorder characterized by paracortical hyperplasia and collapsed primary and secondary follicles., (Copyright 2005 Massachusetts Medical Society)

A 27-year-old man with the autoimmune lymphoproliferative syndrome and a large-B-cell lymphoma had heterozygous mutations in the Fas and perforin (Prf1) genes. The Fas mutation was inherited from his healthy father and was also carried by his healthy brother, whereas the Prf1 mutation was inherited from his healthy mother. The combined effect of the two mutant genes may have contributed to the development of the autoimmune lymphoproliferative syndrome and lymphoma in this patient., (Copyright 2004 Massachusetts Medical Society)

We studied whether signaling through CD30, a member of the TNF receptor family, affected acute infection with HIV-1, encompassing its entire replicative cycle. Several non-Hodgkin cell lines, targets of CXCR4-dependent (X4) HIV-1 infection, were positive for CD30 expression. CD30 ligation induced up-regulation of viral replication only in certain CD30+ cell lines. Enhancement of X4 virus replication by CD30 engagement inversely correlated with both CD30 surface density and constitutive NF-kappaB activation. Conversely, expression of CD30, but not of other members of the TNF receptor family, was proportional to constitutive NF-kappaB binding. Concomitantly, secretion of soluble (s) CD30 increased in all cell lines by CD30 ligation. sCD30 release was enhanced by engagement of CD30 alone and, to a greater extent, by co-engagement of CD3 also in primary gamma delta T lymphocytes, along with complementary modulations of their surface CD30 expression. sCD30-containing supernatant specifically inhibited HIV-1 expression induced by CD30 engagement in chronically infected ACH-2 T cells; thus sCD30 may act as a negative feed-back molecule. In conclusion, we have delineated novel features of CD30 biology and underline the peculiar link of CD30 expression to constitutive NF-kappaB activation which is pivotal to both HIV replication and cell survival.

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). gammadelta and T helper type 1 (Th1) alphabeta 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 gammadelta 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 gammadelta 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 gammadelta T cells, as well as in the regulation of gammadelta function.

A reciprocal influence exists between mycobacteria and HIV: HIV-infected individuals are more susceptible to mycobacterial infections and, on the other hand, mycobacterial infection results inacceleration of HIV disease progression. Vgamma9/Vdelta2 T lymphocytes are known to participate in the defense against intracellular pathogens, including Mycobacterium tuberculosis. Indeed, they kill mycobacteria-infected macrophages and, upon recognition of mycobacterial Ag, release TNF-alpha and IFN-gamma, which are also up-regulators of HIV expression. To assess whether mycobacteria-activated gamma delta T lymphocytes contribute to the enhancement of HIV replication, we established an in vitro model mimicking HIV and mycobacteria co-infection with the latently HIV-infected promonocytic U1 cell line and Vgamma9/Vdelta2 peripheral lymphocytes stimulated with mycobacterial Ag. gamma delta T cell activation determined two distinct, but connected effects, namely U1cell death and HIV expression. Both effects were mainly mediated by release of TNF-alpha and IFN-gamma from activated gamma delta lymphocytes, although Fas-FasL interaction also contributed to U1 apoptosis. The final outcome on U1 survival, and thus, on HIV expression, highly depended on mycobacterial Ag concentration coupled to the differential secretory potency of gamma delta cells. In particular, the induction of viral expression prevailed at low Ag concentration and with lower cytokine production by mycobacteria-activated gamma delta cells. Notably, during the course of HIV infection, Vgamma9/Vdelta2 lymphocytes are reported to be functionally impaired and may thus indirectly influence the progression of HIV disease. In addition, a predominant inhibition of viral replication was encountered when mycobacteria-activated gamma delta T cells were co-cultured with primary HIV-infected macrophages. Thus, we suggest that specific recognition of mycobacterial Ag by gamma delta T lymphocytes in co-infected individuals may modulate viral replication through the complex array of soluble factors released.

Human Vgamma9/Vdelta2(+) T lymphocytes participate in the immune response against intracellular pathogens through the secretion of type-1 cytokines and chemokines and by killing of infected cells. Little is known of the effects by type-2 differentiation of gamma delta cells on these functions. Here, we report that bona fide naive cord blood-derived gamma delta lymphocytes expanded in vitro with the mycobacterial antigen isopentenyl pyrophosphate (IPP) can be differentiated as either type-1 or type-2 cells, in the presence of an appropriate cytokine milieu. Instead, peripheral gamma delta cells from PPD-negative healthy adults displayed a type-1 cytokine profile, i.e. IPP-stimulated secretion of IFN-gamma, but not of IL-4 and IL-10. Moreover, they released the macrophage inflammatory protein (MIP)-1beta, but not IL-8 nor the Th2 chemoattractants I-309 and TARC (thymus and activation-regulated chemokine). This cytokine profile was not significantly affected by in vitro culture in Th2 polarizing conditions. Only in one case out of seven were peripheral gamma delta cells fully differentiated to type-2 lymphocytes, characterized by sustained IL-4 and IL-10 production, along with secretion of substantial amounts of IL-8, I-309 and TARC. Type-2 gamma delta T lymphocytes preferentially expressed the co-stimulatory molecule CD30; conversely, no skewing in chemokine receptor expression was observed. Both polarized populations displayed high levels of CXCR3 in the absence of CCR3, CCR4 and CCR5. Finally, type-1, but not type-2, gamma delta T lymphocytes killed IPP-pulsed U937 cells and displayed elevated perforin content. Overall, our data suggest that type-2 differentiation of gamma delta T lymphocytes profoundly affects both their effector functions and their potential to recruit the appropriate leukocyte subsets to the sites of inflammation.

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, alphabeta-T-cell-mediated, tumor-specific immunity. Given the unique set of antigens recognized and the lack of requirement for classical antigen-presenting molecules, gammadelta T cells might, therefore, represent a nonredundant system in anticancer surveillance, as proposed for the immune response against pathogens. Evidence that gammadelta and alphabeta T cells make distinct contributions to anticancer surveillance has been provided recently in mice. Here, we discuss the potential role played by resident Vdelta1(+) and circulating Vdelta2(+) T cells in the defense against solid tumors and hematological malignancies.