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.

A Standardized Method of Isolating Adipose-Derived Stem Cells for Clinical Applications.

White adipose tissue is the most abundant and accessible source of stem cells in the adult human body. In this paper, we present a standardised and safe method of isolating and maximizing the number of adipose-derived stem cells (ASCs) from conventional liposuction for clinical applications, which was carried out through both mechanical (centrifuge) and enzymatic (collagenase) means. Isolated cells were characterized through flow cytometry assay. Gathered data showed a greater amount (9.06 × 10(5) ASCs from 100 mL of adipose tissue) of isolated ASCs compared to previous protocol, also with high (99%) cell vitality; the procedure we presented is easy and fast (80 minutes), allowing collecting a significative number of mesenchymal stem cells, which can be used for clinical purposes, such as wound healing.

Enhancement of the uptake and cytotoxic activity of doxorubicin in cancer cells by novel cRGD-semipeptide-anchoring liposomes.

Novel liposemipeptides hanging cyclic azabicycloalkane-RGD or aminoproline-RGD terminals were synthesized and incorporated into liposomal nanoparticles cAba/cAmpRGD-LNP5 3C/3D. Liposomes with similar composition and lacking semipeptide conjugates were constructed for comparison (LNP, 3A), and physical encapsulation of the anticancer doxorubicin drug in both targeted and untargeted liposomes was accomplished. Microstructural analysis performed by dynamic light scattering (DLS), small-angle neutron scattering (SANS), and electron paramagnetic resonance (EPR) revealed that the conjugated nanoparticles presented an average size of 80 nm and were constituted by 5 nm thick unilamellar liposome bilayer. Flow cytometry and fluorescent microscopy studies showed that 3C-DOXO and 3D-DOXO efficiently delivered the drug into the nuclei of both quiescent and proliferating cells even in a high serum concentration environment. The uptake of doxorubicin when carried by liposomes was faster than that of the free drug, and 30 min incubation was sufficient to load cell nuclei with doxorubicin. Targeted liposomes significantly induced cell death of human breast adenocarcinoma MCF7 cells (IC50 = 144 nM, 3C-DOXO; IC50 = 274 nM, 3D-DOXO), about 2- to 6-fold more potent than free doxorubicin or 3A-DOXO controls (IC50 = 527 and 854 nM, respectively). These results suggest that cAba/cAmpRGD liposomal nanoparticles hold promise for the rapid and efficient delivery of chemotherapeutic agents to αVß3-expressing tumor cells.

Orthogonal proteogenomic analysis identifies the druggable PA2G4-MYC axis in 3q26 AML.

The overexpression of the ecotropic viral integration site-1 gene (EVI1/MECOM) marks the most lethal acute myeloid leukemia (AML) subgroup carrying chromosome 3q26 abnormalities. By taking advantage of the intersectionality of high-throughput cell-based and gene expression screens selective and pan-histone deacetylase inhibitors (HDACis) emerge as potent repressors of EVI1. To understand the mechanism driving on-target anti-leukemia activity of this compound class, here we dissect the expression dynamics of the bone marrow leukemia cells of patients treated with HDACi and reconstitute the EVI1 chromatin-associated co-transcriptional complex merging on the role of proliferation-associated 2G4 (PA2G4) protein. PA2G4 overexpression rescues AML cells from the inhibitory effects of HDACis, while genetic and small molecule inhibition of PA2G4 abrogates EVI1 in 3q26 AML cells, including in patient-derived leukemia xenografts. This study positions PA2G4 at the crosstalk of the EVI1 leukemogenic signal for developing new therapeutics and urges the use of HDACis-based combination therapies in patients with 3q26 AML.

Impact of the rs1024611 Polymorphism of CCL2 on the Pathophysiology and Outcome of Primary Myelofibrosis.

Single nucleotide polymorphisms (SNPs) can modify the individual pro-inflammatory background and may therefore have relevant implications in the MPN setting, typified by aberrant cytokine production. In a cohort of 773 primary myelofibrosis (PMF), we determined the contribution of the rs1024611 SNP of CCL2-one of the most potent immunomodulatory chemokines-to the clinical and biological characteristics of the disease, demonstrating that male subjects carrying the homozygous genotype G/G had an increased risk of PMF and that, among PMF patients, the G/G genotype is an independent prognostic factor for reduced overall survival. Functional characterization of the SNP and the CCL2-CCR2 axis in PMF showed that i) homozygous PMF cells are the highest chemokine producers as compared to the other genotypes; ii) PMF CD34+ cells are a selective target of CCL2, since they uniquely express CCR2 (CCL2 receptor); iii) activation of the CCL2-CCR2 axis boosts pro-survival signals induced by driver mutations via Akt phosphorylation; iv) ruxolitinib effectively counteracts CCL2 production and down-regulates CCR2 expression in PMF cells. In conclusion, the identification of the role of the CCL2/CCR2 chemokine system in PMF adds a novel element to the pathophysiological picture of the disease, with clinical and therapeutic implications.

Production of Wnt inhibitors by myeloma cells: potential effects on canonical Wnt pathway in the bone microenvironment.

Osteoblast impairment occurs within multiple myeloma cell infiltration into the bone marrow. Canonical Wnt signaling activation in osteoprogenitor cells is involved in osteoblast formation through the stabilization of dephosphorylated beta-catenin and its nuclear translocation. The effects of multiple myeloma cells on Wnt signaling in human mesenchymal/osteoprogenitor cells are unclear. In 60 multiple myeloma patients checked, we found that among the Wnt inhibitors, Dickkopf-1 and secreted frizzled-related protein-3 were produced by multiple myeloma cells. However, although multiple myeloma cells or multiple myeloma bone marrow plasma affected expression of genes in the canonical Wnt signaling and inhibited beta-catenin stabilization in murine osteoprogenitor cells, they failed to block the canonical Wnt pathway in human mesenchymal or osteoprogenitor cells. Consistently, Wnt3a stimulation in human osteoprogenitor cells did not blunt the inhibitory effect of multiple myeloma cells on osteoblast formation. Consequently, despite the higher Wnt antagonist bone marrow levels in osteolytic multiple myeloma patients compared with nonosteolytic ones, beta-catenin immunostaining was not significantly different. Our results support the link between the production of Wnt antagonists by multiple myeloma cells and the presence of bone lesions in multiple myeloma patients but show that myeloma cells do not inhibit canonical Wnt signaling in human bone microenvironment.

In human endothelial cells rapamycin causes mTORC2 inhibition and impairs cell viability and function.

AIM: Drug-eluting stents are widely used to prevent restenosis but are associated with late endothelial damage. To understand the basis for this effect, we have studied the consequences of a prolonged incubation with rapamycin on the viability and functions of endothelial cells. METHODS AND RESULTS: Human umbilical vein or aorta endothelial cells were exposed to rapamycin in the absence or in the presence of tumour necrosis factor alpha (TNFalpha). After a 24 h-incubation, rapamycin (100 nM) caused a significant cell loss associated with the increase of both apoptosis and necrosis, as quantified by propidium iodide staining, caspase 3 activity, and lactate dehydrogenase release. Rapamycin also impaired cell mobility, as assessed by a wound test, and promoted the formation of actin stress fibres, as determined with confocal microscopy. Moreover, the inhibitor prolonged TNFalpha-dependent E-selectin induction, inhibited endothelial nitric oxide synthase expression at both mRNA (quantitative real-time polymerase chain reaction) and protein level (enzyme-linked immunosorbent assay and western blot), and lowered bioactive nitric oxide output (RFL-6 reporter cell assay). Under the conditions adopted, rapamycin inhibited both mammalian target-of-rapamycin complexes (mTORC1 and mTORC2), as indicated by the reduced amount of raptor and rictor bound to mTOR in immunoprecipitates and by the marked hypophosphorylation of protein S6 kinase I (p70S6K) and Akt, determined by western blotting. The selective inhibition of mTORC1 by AICAR did not affect endothelial viability. CONCLUSION: A prolonged treatment with rapamycin impairs endothelial function and hinders cell viability. Endothelial damage seems dependent on mTORC2 inhibition.

Haploidentical hematopoietic stem cell transplantation in adults using the αßTCR/CD19-based depletion of G-CSF-mobilized peripheral blood progenitor cells.

In patients with hematological malignancies at high risk for relapse, a mismatched hematopoietic stem cells transplants can be offered with no undue delay between decision-making and transplantation as virtually all patients have a full-haplotype mismatched member who could serve immediately as a donor. Using a T-cell depletion approach, these patients can benefit from a graft-vs-leukemia effect in the absence of both acute and chronic graft-vs-host disease. Over the past decade, efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the posttransplant immunological recovery. The innovative strategy based on the selective depletion of alpha/beta-positive T lymphocytes from G-CSF-mobilized peripheral blood precursor cells has shown very promising results in the setting of the pediatric transplantation. This paper reports the outcome in adult patients with hematological malignancies.

A translocation t(4; 13)(q21;q14) as single clonal chromosomal abnormality in a parathyroid adenoma.

Most of the information about the genetic composition of parathyroid tumors has been obtained by comparative genomic hybridization (CGH) and loss of heterozygosity (LOH) studies, whereas only few conventional cytogenetic investigation results are available. We have performed cytogenetic analysis of short-term cultures from 3 parathyroid adenoma tissue samples. Two cases showed a normal karyotype in all the metaphases obtained from independent primary cultures. In one case 5 metaphases (in a total of 25) from 2 independent cultures showed a nonrandom translocation t(4;13)(q21;q14), which was therefore accepted as clonal. To our knowledge this is the second clonal translocation described in this tumor type. Further conventional cytogenetic analysis of more parathyroid tumor specimens would be necessary to identify other specific abnormalities and the involved genes with a potential important role in the diagnosis, prognosis and pathogenesis of parathyroid tumors.

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.

Cutaneous localization in multiple myeloma in the context of bortezomib-based treatment: how do myeloma cells escape from the bone marrow to the skin?

The skin is a possible site of extramedullary localization in multiple myeloma (MM) patients; however, the mechanisms involved in this process are poorly understood. We describe the case of a refractory MM patient who developed a cutaneous localization under bortezomib treatment and we further expanded observations in other eight MM patients. We focused on the expression of genes involved in plasma cell skin homing, including CCR10, which was highly expressed. Moreover, we observed a lack of CXCR4 surface expression and the down-regulation of ICAM1/CD54 throughout the progression of the disease, suggesting a possible mechanism driving the escape of MM cells from the bone marrow into the skin.

Immune tolerance induction by nonmyeloablative haploidentical HSCT combining T-cell depletion and posttransplant cyclophosphamide.

The establishment of safe approaches to attain durable donor-type chimerism and immune tolerance toward donor antigens represents a major challenge in transplantation biology. Haploidentical hematopoietic stem cell transplantation (HSCT) is currently used for cancer therapy either as a T-cell-depleted megadose HSCT following myeloablative conditioning or with T-cell-replete HSCT following nonmyeloablative conditioning (NMAC) and high-dose posttransplant cyclophosphamide (PTCY). The latter approach suffers from a significant rate of chronic graft-versus-host disease (GVHD), despite prolonged immunosuppression. The use of T-depleted grafts, although free of GVHD risk, is not effective after NMAC because of graft rejection. We now demonstrate in mice conditioned with NMAC that combining the power of high-dose PTCY with T-cell-depleted megadose HSCT can overcome this barrier. This approach was evaluated in 2 patients with multiple myeloma and 1 patient with Hodgkin lymphoma. The first myeloma patient now followed for 25 months, exhibited full donor-type chimerism in the myeloid and B-cell lineages and mixed chimerism in the T-cell compartment. The second myeloma patient failed to attain chimerism. Notably, the low toxicity of this protocol enabled a subsequent successful fully myeloablative haploidentical HSCT in this patient. The third patients was conditioned with slightly higher total body irradiation and engrafted promptly. All patients remain in remission without GVHD. Both engrafted patients were able to control cytomegalovirus reactivation. Enzyme-linked immunospot analysis revealed immune tolerance toward donor cells. Our results demonstrate a novel and safer nonmyeloablative haplo-HSCT offering a platform for immune tolerance induction as a prelude to cell therapy and organ transplantation.

CXCR3 and its binding chemokines in myeloma cells: expression of isoforms and potential relationships with myeloma cell proliferation and survival.

BACKGROUND AND OBJECTIVES: The chemokine receptor CXCR3, involved in chemotaxis, is expressed on normal and malignant B cells and plasma cells. Recent data suggest that CXCR3-binding chemokines may also regulate proliferation and survival in endothelial cells through the interaction with two distinct isoforms of CXCR3 (CXCR3-A and CXCR3-B). DESIGN AND METHODS: We evaluated the potential expression of CXCR3 isoforms in myeloma cells, also investigating whether CXCR3 expression is affected by cell cycle and apoptosis. Furthermore, we assessed the effect of CXCR3 activation on myeloma cell proliferation and survival. RESULTS: We found that CXCR3 is widely expressed on human myeloma cell lines and freshly purified myeloma cells. The presence of both CXCR3 isoforms, CXCR3-A and CXCR3-B, was observed in myeloma cells with different ratios of expression. Interestingly, we found that CXCR3 expression in myeloma cell was cell cycle dependent and that myeloma growth factors inhibited CXCR3 expression in myeloma cells. On the other hand, we found that FAS (CD95)-mediated apoptosis up-regulated CXCR3 expression. A similar behavior was observed for the CXCR3-binding chemokines. Finally we found that the activation of CXCR3 on myeloma cells by CXCL10/IP-10 partially blunted FAS-mediated apoptosis in myeloma cells that express CXCR3-A and that high concentrations of CXCL10/IP-10 inhibit myeloma cell proliferation. INTERPRETATION AND CONCLUSIONS Our data indicate that myeloma cells express the CXCR3 system with patterns correlated to cell cycle and apoptosis and that CXCR3 activation may affect myeloma cell survival and proliferation.

Adipose-derived Stem Cells Added to Platelet-rich Plasma for Chronic Skin Ulcer Therapy.

INTRODUCTION: Adipose-derived stem cells (ASCs) hold great promise for regenerative medicine applications due to their ability to promote the healing process through in situ differentiation and secretion of paracrine factor. The aim of this paper is to present a clinical adjunct for chronic skin wound therapy based on ASCs added to platelet-rich plasma (PRP), to obtain an enhanced PRP (e-PRP). MATERIALS AND METHODS: For 18 months, 24 control-group patients with 31 chronic skin ulcers were treated with standard wound care, while 16 experimental-group patients with 21 chronic skin ulcers were treated with standard wound care and 1 e-PRP injection. The patients were randomly assigned to the control or experimental group. Outpatients had weekly follow-up visits where they were subjected to standard treatment and the wound healing process was assessed. RESULTS: At the end of the study, the control and experimental groups had similar healing rates but wound closure rates were significantly different (P = 0.0257): 0.0890 cm(2) x day and 0.2287 cm(2) x day respectively, resulting in a faster recovery for the group treated with e-PRP. No side effects were reported. CONCLUSION: In the authors' experience, e-PRP significantly enhanced wound closure rates when compared to standard wound care, without causing any serious complications. This finding highlights e-PRP as a valuable resource for chronic wound treatment.

A novel and effective strategy for the isolation of adipose-derived stem cells: minimally manipulated adipose-derived stem cells for more rapid and safe stem cell therapy.

Adipose-derived stem cells are an ideal mesenchymal stem cell population for regenerative medical application. The isolation procedure is performed by mechanical isolation under a laminar air flow bench without using serum or animal-derived reagents; cells were characterized by flow cytometric analysis. Cell availability is improved compared with enzymatic digestion procedures. The adipose-derived stem cell mechanical isolating procedure presented here is easier, safer, cheaper, and faster than traditional currently performed enzymatic procedures.