MiRNA 126 as a New Predictor Biomarker in Venous Thromboembolism of Persistent Residual Vein Obstruction: A Review of the Literature Plus a Pilot Study.

Venous thromboembolism (VTE) is the third most common cardiovascular disease. Interleukins (ILs) and micro-ribonucleic acids (miRNAs) have been proposed as molecules able to modulate endothelial inflammation and platelet hyperactivity. At present, no early biomarkers are available to predict the outcome of VTE. We investigated in a pilot study a selected number of miRNAs and ILs as prognostic VTE biomarkers and reviewed literature in this setting. Twenty-three patients (aged 18-65) with a new diagnosis of non-oncological VTE and free from chronic inflammatory diseases were enrolled. Twenty-three age- and sex-matched healthy blood donors were evaluated as control subjects. Serum miRNAs (MiRNA 126, 155, 17.92, 195), inflammatory cytokines (IL-6, tumor necrosis factor-α, IL-8), and lymphocyte subsets were evaluated in patients at enrolment (T0) and in controls. In VTE patients, clinical and instrumental follow-up were performed assessing residual vein obstruction, miRNA and ILs evaluation at 3 months' follow-up (T1). At T0, IL-8, activated T lymphocytes, Treg lymphocytes, and monocytes were higher in patients compared with healthy controls, as were miRNA 126 levels. Moreover, miRNA 126 and IL-6 were significantly increased at T0 compared with T1 evaluation in VTE patients. Higher levels of MiR126 at T0 correlated with a significant overall thrombotic residual at follow-up. In recent years an increasing number of studies (case-control studies, in vivo studies in animal models, in vitro studies) have suggested the potential role of miRNAs in modulating the cellular and biohumoral responses involved in VTE. In the frame of epidemiological evidence, this pilot study with a novel observational approach supports the notion that miRNA can be diagnostic biomarkers of VTE and first identifies miRNA 126 as a predictor of outcome, being associated with poor early recanalization.

CD14+ CD16+ monocytes are involved in daratumumab-mediated myeloma cells killing and in anti-CD47 therapeutic strategy.

A deep elucidation of the mechanisms of action of anti-CD38 monoclonal antibodies (mAbs), such as daratumumab (DARA), is required to identify patients with multiple myeloma (MM) who are more responsive to this treatment. In the present study, an autologous ex vivo approach was established, focussing on the role of the monocytes in the anti CD38-mediated killing of MM cells. In bone marrow (BM) samples from 29 patients with MM, we found that the ratio between monocytes (CD14+ ) and MM cells (CD138+ ) influences the response to DARA. Further, the exposure of the BM samples to DARA is followed by the formation of a CD138+ CD14+ double-positive (DP) population, that quantitatively correlates with the anti-MM cells killing. These effects were dependent on the presence of a CD14+ CD16+ monocyte subset and on high CD16 expression levels. Lastly, the addition of a mAb neutralising the CD47/signal-regulatory protein α (SIRPα) axis was able to increase the killing mediated by DARA. The effects were observed only in coincidence with high CD14+ :CD138+ ratio, with a significant presence of the DP population and were correlated with CD16 expression. In conclusion, the present study underlines the critical role of the CD16+ monocytes in DARA anti-MM killing effects and gives a rationale to test the combination of an anti-CD47 mAb with anti-CD38 mAbs.

Dependence on glutamine uptake and glutamine addiction characterize myeloma cells: a new attractive target.

The importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138(+) cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138(+) cells. Gln-free incubation or treatment with the glutaminolytic enzyme l-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138(+) cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM.

CMV-Specific T-cell Responses at Older Ages: Broad Responses With a Large Central Memory Component May Be Key to Long-term Survival.

Cytomegalovirus (CMV) infection sometimes causes large expansions of CMV-specific T cells, particularly in older people. This is believed to undermine immunity to other pathogens and to accelerate immunosenescence. While multiple different CMV proteins are recognized, most publications on age-related T-cell expansions have focused on dominant target proteins UL83 or UL123, and the T-cell activation marker interferon-γ (IFN-γ). We were concerned that this narrow approach might have skewed our understanding of CMV-specific immunity at older ages. We have, therefore, widened the scope of analysis to include in vitro-induced T-cell responses to 19 frequently recognized CMV proteins in "young" and "older" healthy volunteers and a group of "oldest old" long-term survivors (>85 years of age). Polychromatic flow cytometry was used to analyze T-cell activation markers (CD107, CD154, interleukin-2 [IL-2], tumor necrosis factor [TNF], and IFN-γ) and memory phenotypes (CD27, CD45RA). The older group had, on average, larger T-cell responses than the young, but, interestingly, response size differences were relatively smaller when all activation markers were considered rather than IFN-γ or TNF alone. The oldest old group recognized more proteins on average than the other groups, and had even bigger T-cell responses than the older group with a significantly larger central memory CD4 T-cell component.

IL21R expressing CD14+CD16+ monocytes expand in multiple myeloma patients leading to increased osteoclasts.

Bone marrow monocytes are primarily committed to osteoclast formation. It is, however, unknown whether potential primary alterations are specifically present in bone marrow monocytes from patients with multiple myeloma, smoldering myeloma or monoclonal gammopathy of undetermined significance. We analyzed the immunophenotypic and transcriptional profiles of bone marrow CD14+ monocytes in a cohort of patients with different types of monoclonal gammopathies to identify alterations involved in myeloma-enhanced osteoclastogenesis. The number of bone marrow CD14+CD16+ cells was higher in patients with active myeloma than in those with smoldering myeloma or monoclonal gammopathy of undetermined significance. Interestingly, sorted bone marrow CD14+CD16+ cells from myeloma patients were more pro-osteoclastogenic than CD14+CD16-cells in cultures ex vivo Moreover, transcriptional analysis demonstrated that bone marrow CD14+ cells from patients with multiple myeloma (but neither monoclonal gammopathy of undetermined significance nor smoldering myeloma) significantly upregulated genes involved in osteoclast formation, including IL21RIL21R mRNA over-expression by bone marrow CD14+ cells was independent of the presence of interleukin-21. Consistently, interleukin-21 production by T cells as well as levels of interleukin-21 in the bone marrow were not significantly different among monoclonal gammopathies. Thereafter, we showed that IL21R over-expression in CD14+ cells increased osteoclast formation. Consistently, interleukin-21 receptor signaling inhibition by Janex 1 suppressed osteoclast differentiation from bone marrow CD14+ cells of myeloma patients. Our results indicate that bone marrow monocytes from multiple myeloma patients show distinct features compared to those from patients with indolent monoclonal gammopathies, supporting the role of IL21R over-expression by bone marrow CD14+ cells in enhanced osteoclast formation.

Functional Diversity of Cytomegalovirus-Specific T Cells Is Maintained in Older People and Significantly Associated With Protein Specificity and Response Size.

BACKGROUND: Parallel upregulation of several T-cell effector functions (ie, polyfunctionality) is believed to be critical for the protection against viruses but thought to decrease in large T-cell expansions, in particular at older ages. The factors determining T-cell polyfunctionality are incompletely understood. Here we revisit the question of cytomegalovirus (CMV)-specific T-cell polyfunctionality, including a wide range of T-cell target proteins, response sizes, and participant ages. METHODS: Polychromatic flow cytometry was used to analyze the functional diversity (ie, CD107, CD154, interleukin 2, tumor necrosis factor, and interferon γ expression) of CD4+ and CD8+ T-cell responses to 19 CMV proteins in a large group of young and older United Kingdom participants. A group of oldest old people (age >85 years) was included to explore these parameters in exceptional survivors. Polyfunctionality was assessed for each protein-specific response subset, by subset and in aggregate, across all proteins by using the novel polyfunctionality index. RESULTS: Polyfunctionality was not reduced in healthy older people as compared to young people. However, it was significantly related to target protein specificity. For each protein, it increased with response size. In the oldest old group, overall T-cell polyfunctionality was significantly lower. DISCUSSION: Our results give a new perspective on T-cell polyfunctionality and raise the question of whether maintaining polyfunctionality of CMV-specific T cells at older ages is necessarily beneficial.

Intense antiextracellular adaptive immune response to human cytomegalovirus in very old subjects with impaired health and cognitive and functional status.

Human aging is characterized by expanded and altered adaptive immune responses to human CMV (HCMV). It is unclear whether this expansion has its origins in age-related homeostatic disturbances or viral reactivation, whether anti-CMV immune surveillance may still be effective, and what are the consequences of this expanded immune response for health and longevity. We conducted an observational cross-sectional study in groups of HCMV-seropositive subjects aged >or=65 y of variable health status to compare the intensity of Ab responses against HCMV with those against EBV and with CD4(+) and CD8(+) T cell proinflammatory effector responses directed to HCMV-derived pp65 and immediate-early protein 1 synthetic peptides. Ab responses to HCMV, but not to EBV, and anti-HCMV CD4(+), but not CD8(+), T cell responses were more intense in elderly subjects aged >or=85 y in poor health and were inversely correlated with markers of functional activity and cognitive function. Therefore, humoral and CD4(+) T cell anti-HCMV responses were specifically intensified in advanced aging associated with comorbidity and cognitive and functional impairments. Such a distinctive pattern of adaptive immunity indicates that immune responses targeting the extracellular phase of HCMV are increased in these elderly subjects and could represent an indirect effect of localized and undetectable HCMV reactivation. This study demonstrates that the oldest subjects in poor health with physical and mental impairment express intense functional immune responses to extracellular HCMV and suggests that they may be at risk for direct pathogenic effects by HCMV reactivation as well as indirect pathogenic effects linked to proinflammatory anti-HCMV effector responses.

Immune response to SARS-CoV-2 mRNA vaccination and booster dose in patients with multiple myeloma and monoclonal gammopathies: impact of Omicron variant on the humoral response.

The humoral and cellular response to SARS-CoV-2 mRNA full vaccination and booster dose as well as the impact of the spike variants, including Omicron, are still unclear in patients with multiple myeloma (MM) and those with pre-malignant monoclonal gammopathies. In this study, involving 40 patients, we found that MM patients with relapsed-refractory disease (MMR) had reduced spike-specific antibody levels and neutralizing titers after SARS-CoV-2 vaccination. The five analyzed variants, remarkably Omicron, had a significant negative impact on the neutralizing ability of the vaccine-induced antibodies in all patients with MM and smoldering MM. Moreover, lower spike-specific IL-2-producing CD4+ T cells and reduced cytotoxic spike-specific IFN-γ and TNF-α-producing CD8+ T cells were found in MM patients as compared to patients with monoclonal gammopathy of undetermined significance. We found that a heterologous booster immunization improved SARS-CoV-2 spike humoral and cellular responses in newly diagnosed MM (MMD) patients and in most, but not all, MMR patients. After the booster dose, a significant increase of the neutralizing antibody titers against almost all the analyzed variants was achieved in MMD. However, in MMR patients, Omicron retained a negative impact on neutralizing ability, suggesting further approaches to potentiating the effectiveness of SARS-CoV-2 vaccination in these patients.

Report from the second cytomegalovirus and immunosenescence workshop.

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.

PD-L1/PD-1 Pattern of Expression Within the Bone Marrow Immune Microenvironment in Smoldering Myeloma and Active Multiple Myeloma Patients.

Background: The PD-1/PD-L1 axis has recently emerged as an immune checkpoint that controls antitumor immune responses also in hematological malignancies. However, the use of anti-PD-L1/PD-1 antibodies in multiple myeloma (MM) patients still remains debated, at least in part because of discordant literature data on PD-L1/PD-1 expression by MM cells and bone marrow (BM) microenvironment cells. The unmet need to identify patients which could benefit from this therapeutic approach prompts us to evaluate the BM expression profile of PD-L1/PD-1 axis across the different stages of the monoclonal gammopathies. Methods: The PD-L1/PD-1 axis was evaluated by flow cytometry in the BM samples of a total cohort of 141 patients with monoclonal gammopathies including 24 patients with Monoclonal Gammopathy of Undetermined Significance (MGUS), 38 patients with smoldering MM (SMM), and 79 patients with active MM, including either newly diagnosed or relapsed-refractory patients. Then, data were correlated with the main immunological and clinical features of the patients. Results: First, we did not find any significant difference between MM and SMM patients in terms of PD-L1/PD-1 expression, on both BM myeloid (CD14+) and lymphoid subsets. On the other hand, PD-L1 expression by CD138+ MM cells was higher in both SMM and MM as compared to MGUS patients. Second, the analysis on the total cohort of MM and SMM patients revealed that PD-L1 is expressed at higher level in CD14+CD16+ non-classical monocytes compared with classical CD14+CD16- cells, independently from the stage of disease. Moreover, PD-L1 expression on CD14+ cells was inversely correlated with BM serum levels of the anti-tumoral cytokine, IL-27. Interestingly, relapsed MM patients showed an inverted CD4+/CD8+ ratio along with high levels of pro-tumoral IL-6 and a positive correlation between %CD14+PD-L1+ and %CD8+PD-1+ cells as compared to both SMM and newly diagnosed MM patients suggesting a highly compromised immune-compartment with low amount of CD4+ effector cells. Conclusions: Our data indicate that SMM and active MM patients share a similar PD-L1/PD-1 BM immune profile, suggesting that SMM patients could be an interesting target for PD-L1/PD-1 inhibition therapy, in light of their less compromised and more responsive immune-compartment.

Bovine pestivirus is a new alternative virus for multiple myeloma oncolytic virotherapy.

BACKGROUND: The oncolytic viruses have shown promising results for the treatment of multiple myeloma. However, the use of human viruses is limited by the patients' antiviral immune response. In this study, we investigated an alternative oncolytic strategy using non-human pathogen viruses as the bovine viral diarrhea virus (BVDV) that were able to interact with CD46. METHODS: We treated several human myeloma cell lines and non-myeloma cell lines with BVDV to evaluate the expression of CD46 and to study the effect on cell viability by flow cytometry. The possible synergistic effect of bortezomib in combination with BVDV was also tested. Moreover, we infected the bone marrow mononuclear cells obtained from myeloma patients and we checked the BVDV effect on different cell populations, defined by CD138, CD14, CD3, CD19, and CD56 expression evaluated by flow cytometry. Finally, the in vivo BVDV effect was tested in NOD-SCID mice injected subcutaneously with myeloma cell lines. RESULTS: Human myeloma cells were selectively sensitive to BVDV treatment with an increase of cell death and, consequently, of apoptotic markers. Consistently, bone marrow mononuclear cells isolated from myeloma patients treated with BVDV, showed a significant selective decrease of the percentage of viable CD138+ cells. Interestingly, bortezomib pre-treatment significantly increased the cytotoxic effect of BVDV in myeloma cell lines with a synergistic effect. Finally, the in vitro data were confirmed in an in vivo myeloma mouse model showing that BVDV treatment significantly reduced the tumoral burden compared to the vehicle. CONCLUSIONS: Overall, our data indicate, for the first time, a direct oncolytic effect of the BVDV in human myeloma cells suggesting its possible use as novel alternative anti-myeloma virotherapy strategy.

Fibrocytes in Chronic Periaortitis: A Novel Mechanism Linking Inflammation and Fibrosis.

OBJECTIVE: Chronic periaortitis (CP) is a rare disease characterized by periaortic and periiliac fibroinflammatory tissue. The pathogenic mechanisms leading to tissue accumulation and activation of fibroblasts are unclear. This study was undertaken to explore the role of fibrocytes, circulating precursors of tissue fibroblasts, in patients with CP. METHODS: We studied 44 patients with newly diagnosed CP and 30 healthy controls. Circulating fibrocytes were identified as Col1+CD45+ cells using flow cytometry. Retroperitoneal tissue biopsy samples from 9 CP patients were stained with anti-type I procollagen, anti-CXCR4, and anti-CD45 antibodies and analyzed by confocal microscopy to detect tissue-infiltrating fibrocytes. Circulating levels and tissue expression of CXCL12, a CXCR4 ligand that promotes fibrocyte homing, were investigated using enzyme-linked immunosorbent assay and immunohistochemistry, respectively. We also characterized T helper polarization in biopsy samples from CP patients and measured serum levels of a panel of cytokines that are hallmarks of T helper responses and capable of influencing fibrocyte differentiation. RESULTS: The frequency of circulating Col1+CD45+ fibrocytes was higher in patients than in controls (P = 0.0371). CD45+proCol1+ and CXCR4+proCol1+ cells were detected in all examined biopsy samples from CP patients. Serum levels of CXCL12 were also higher in CP patients than controls (P = 0.0056), and tissue-infiltrating inflammatory cells intensely expressed CXCL12. Increased serum levels of Th2 cytokines (e.g., interleukin-13 [IL-13] and IL-10) were found in patients, and immunohistochemistry revealed a dominant infiltration of GATA-3+ cells, also indicating Th2 polarization; Th2-skewed responses are known to promote fibrocyte differentiation. CONCLUSION: Our findings indicate that fibrocytes are enriched in the peripheral blood of CP patients and infiltrate target lesions. The accumulation of fibrocytes in the pathologic tissue might be driven by CXCL12, and Th2-skewed immune responses are likely to facilitate their differentiation.

Bone Marrow CX3CL1/Fractalkine is a New Player of the Pro-Angiogenic Microenvironment in Multiple Myeloma Patients.

C-X3-C motif chemokine ligand 1 (CX3CL1)/fractalkine is a chemokine released after cleavage by two metalloproteases, ADAM metallopeptidase domain 10 (ADAM10) and ADAM metallopeptidase domain 17 (ADAM17), involved in inflammation and angiogenesis in the cancer microenvironment. The role of the CX3CL1/ C-X3-C motif chemokine receptor 1(CX3CR1) axis in the multiple myeloma (MM) microenvironment is still unknown. Firstly, we analyzed bone marrow (BM) plasma levels of CX3CL1 in 111 patients with plasma cell disorders including 70 with active MM, 25 with smoldering myeloma (SMM), and 16 with monoclonal gammopathy of undetermined significance (MGUS). We found that BM CX3CL1 levels were significantly increased in MM patients compared to SMM and MGUS and correlated with BM microvessel density. Secondly, we explored the source of CX3CL1 in MM and BM microenvironment cells. Primary CD138⁺ cells did not express CXC3L1 but up-regulated its production by endothelial cells (ECs) through the involvement of tumor necrosis factor alpha (TNFα). Lastly, we demonstrated the presence of CX3CR1 on BM CD14⁺CD16⁺ monocytes of MM patients and on ECs, but not on MM cells. The role of CX3CL1 in MM-induced angiogenesis was finally demonstrated in both in vivo chick embryo chorioallantoic membrane and in vitro angiogenesis assays. Our data indicate that CX3CL1, present at a high level in the BM of MM patients, is a new player of the MM microenvironment involved in MM-induced angiogenesis.

Calcium metabolism and vitamin D in the extreme longevity.

Skeletal remodelling is a continuous process during life and is still active also in extreme senescence. In the elderly, bone resorption often prevails over bone formation, causing bone loss and fragility. Elderly subjects are exposed to the risk of fractures, and loss of self-sufficiency, if considering that the proximal femur is the most frequently involved site. Bone remodelling can maintain circulating calcium within physiological ranges, at the expense of a substantial loss of this ion from the skeleton, particularly during senescence. Calcium metabolism is regulated at cellular/molecular level by a network of cytokines, growth factors, systemic hormones that act on bone in paracrine/autocrine/systemic fashion. Among the molecules involved in bone metabolism, parathyroid hormone (PTH) and vitamin D present some peculiar aspects during senescence. The osteometabolic features in a consistent group of centenarians have been evaluated. It results that a severe hypovitaminosis D was present in 99 out of 104 centenarians (25-OH vitamin D below 5 nmol/L), and that it plays an important role as a factor inducing a vicious circle involving hypocalcemia, secondary hyperparathyroidism, together with biochemical features indicating a consistent bone loss. Serum C-terminal cross-linking telopeptide, a specific marker of bone resorption was elevated in 92% of these subjects. Moreover, it has been found that several femoral fractures had occurred after 90 years of age. These data offer a rational for the possible prevention of elevated bone turnover, bone loss and consequently the reduction of osteoporotic fractures and fractures-induced disability, in the oldest olds, through the simple supplementation with calcium and vitamin D.

A Paradigmatic Interplay between Human Cytomegalovirus and Host Immune System: Possible Involvement of Viral Antigen-Driven CD8+ T Cell Responses in Systemic Sclerosis.

Human cytomegalovirus (HCMV) is a highly prevalent opportunistic agent in the world population, which persists as a latent virus after a primary infection. Besides the well-established role of this agent causing severe diseases in immunocompromised individuals, more recently, HCMV has been evoked as a possible factor contributing to the pathogenesis of autoimmune diseases such as systemic sclerosis (SSc). The interplay between HCMV and immune surveillance is supposed to become unbalanced in SSc patients with expanded anti-HCMV immune responses, which are likely involved in the exacerbation of inflammatory processes. In this study, blood samples from a cohort of SSc patients vs. healthy subjects were tested for anti-HCMV immune responses (IgM, IgG antibodies, and T cells to peptide pools spanning the most immunogenic HCMV proteins). Statistically significant increase of HCMV-specific CD8+ T cell responses in SSc patients vs. healthy subjects was observed. Moreover, significantly greater HCMV-specific CD8+ T cell responses were found in SSc patients with a longer disease duration and those with higher modified Rodnan skin scores. Given the known importance of T cells in the development of SSc and that this virus may contribute to chronic inflammatory diseases, these data support a relevant role of HCMV-specific CD8+ T cell responses in SSc pathogenesis.

Expression of CD38 in myeloma bone niche: A rational basis for the use of anti-CD38 immunotherapy to inhibit osteoclast formation.

It is known that multiple myeloma (MM) cells express CD38 and that a recently developed human anti-CD38 monoclonal antibody Daratumumab mediates myeloma killing. However, the expression of CD38 and other functionally related ectoenzymes within the MM bone niche and the potential effects of Daratumumab on bone cells are still unknown. This study firstly defines by flow cytometry and immunohistochemistry the expression of CD38 by bone marrow cells in a cohort of patients with MM and indolent monoclonal gammopathies. Results indicate that only plasma cells expressed CD38 at high level within the bone niche. In addition, the flow cytometry analysis shows that CD38 was also expressed by monocytes and early osteoclast progenitors but not by osteoblasts and mature osteoclasts. Indeed, CD38 was lost during in vitro osteoclastogenesis. Consistently, we found that Daratumumab reacted with CD38 expressed on monocytes and its binding inhibited in vitro osteoclastogenesis and bone resorption activity from bone marrow total mononuclear cells of MM patients, targeting early osteoclast progenitors. The inhibitory effect was not observed from purified CD14+ cells, suggesting an indirect inhibitory effect of Daratumumab. Interestingly, all-trans retinoic acid treatment increased the inhibitory effect of Daratumumab on osteoclast formation. These observations provide a rationale for the use of an anti-CD38 antibody-based approach as treatment for multiple myeloma-induced osteoclastogenesis.

Lenalidomide increases human dendritic cell maturation in multiple myeloma patients targeting monocyte differentiation and modulating mesenchymal stromal cell inhibitory properties.

The use of Lenalidomide (LEN), to reverse tumor-mediated immune suppression and amplify multiple myeloma-specific immunity is currently being explored. Particularly, LEN effects on dendritic cells (DCs) are still unclear. In this study, we investigated the potential effect of LEN on DC differentiation and activity. DCs were differentiated either from CD14+ cells obtained from patients with multiple myeloma or from a human monocytic cell line. LEN, at the concentration range reached in vivo, significantly increased the median intensity expression of HLA-DR, CD86 and CD209 by DCs derived from both bone marrow and peripheral myeloma monocytes and enhanced the production of Interleukin-8, C-C motif chemokine ligand (CCL) 2, CCL5 and tumor necrosis factor-α. Consistently, LEN pre-treated DCs showed an increased ability to stimulate autologous CD3+ cell proliferation. LEN effect on dendritic differentiation was associated with the degradation of the Cereblon-related factors Ikaros and Aiolos. Moreover, we showed that LEN also blunted mesenchymal stromal cell inhibitory effect on dendritic differentiation, inhibiting Casein Kinase-1α levels. Finally, in vitro data were confirmed in ex vivo cultures obtained from relapsed myeloma patients treated with LEN, showing a significant increase of DC differentiation from peripheral blood monocytes. In conclusion, LEN increased the expression of mature dendritic markers both directly and indirectly and enhanced DC ability to stimulate T cell proliferation and to release chemokines. This suggests a new possible mechanism by which LEN could exert its anti-myeloma activity.

Osteoprotegerin and receptor activator of nuclear factor-kappa B ligand modulation by enamel matrix derivative in human alveolar osteoblasts.

BACKGROUND: Bone regeneration techniques increasingly rely on the use of exogenous molecules able to enhance tissue formation in pathologic and traumatic defects. An enamel matrix derivative (EMD) has been largely used to promote tooth ligament regeneration within periodontal pockets. Recent evidence suggests that EMD may contribute to inducing osteoblast growth and differentiation. We investigated the effects of EMD on growth and osteogenic marker modulation in human mandibular osteoblasts. METHODS: We focused our attention on cell growth by 3-(4,5-dimethyl[thiazol-2-yl]-3,5-diphery)tetradium bromide (MTT) assay, cell differentiation, mineralized nodule formation, and, in particular, the expression of receptor activator of nuclear factor-kappa B ligand (RANKL), the main osteoclast differentiation factor, and its decoy receptor, osteoprotegerin (OPG), by enzyme-linked immunosorbent assay. RESULTS: Cell growth was significantly increased by EMD. Similarly, a significantly higher quantity of OPG and a lower amount of RANKL were detectable in groups treated with 50 and 100 microg/ml at weeks 1, 2, and 3, and alkaline phosphatase activity and osteocalcin production were enhanced in cultures treated with 50 and 100 microg/ml at weeks 2 and 3. Mineralized nodules appeared bigger and more numerous in cultures treated with 50 and 100 microg/ml EMD. CONCLUSIONS: EMD was able to enhance osteoblast cell growth and the expression of markers of osteoblastic phenotype and differentiation. EMD also seemed able to create a favorable osteogenic microenvironment by reducing RANKL release and enhancing osteoblastic OPG production.