Asparagine transport through SLC1A5/ASCT2 and SLC38A5/SNAT5 is essential for BCP-ALL cell survival and a potential therapeutic target.

B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) blasts strictly depend on the transport of extra-cellular asparagine (Asn), yielding a rationale for L-asparaginase (ASNase) therapy. However, the carriers used by ALL blasts for Asn transport have not been identified yet. Exploiting RS4;11 cells as BCP-ALL model, we have found that cell Asn is lowered by either silencing or inhibition of the transporters ASCT2 or SNAT5. The inhibitors V-9302 (for ASCT2) and GluγHA (for SNAT5) markedly lower cell proliferation and, when used together, suppress mTOR activity, induce autophagy and cause a severe nutritional stress, leading to a proliferative arrest and a massive cell death in both the ASNase-sensitive RS4;11 cells and the relatively ASNase-insensitive NALM-6 cells. The cytotoxic effect is not prevented by coculturing leukaemic cells with primary mesenchymal stromal cells. Leukaemic blasts of paediatric ALL patients express ASCT2 and SNAT5 at diagnosis and undergo marked cytotoxicity when exposed to the inhibitors. ASCT2 expression is positively correlated with the minimal residual disease at the end of the induction therapy. In conclusion, ASCT2 and SNAT5 are the carriers exploited by ALL cells to transport Asn, and ASCT2 expression is associated with a lower therapeutic response. ASCT2 may thus represent a novel therapeutic target in BCP-ALL.

Ataluren improves myelopoiesis and neutrophil chemotaxis by restoring ribosome biogenesis and reducing p53 levels in Shwachman-Diamond syndrome cells.

Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, exocrine pancreatic insufficiency and skeletal abnormalities. SDS bone marrow haematopoietic progenitors show increased apoptosis and impairment in granulocytic differentiation. Loss of Shwachman-Bodian-Diamond syndrome (SBDS) expression results in reduced eukaryotic 80S ribosome maturation. Biallelic mutations in the SBDS gene are found in ~90% of SDS patients, ~55% of whom carry the c.183-184TA>CT nonsense mutation. Several translational readthrough-inducing drugs aimed at suppressing nonsense mutations have been developed. One of these, ataluren, has received approval in Europe for the treatment of Duchenne muscular dystrophy. We previously showed that ataluren can restore full-length SBDS protein synthesis in SDS-derived bone marrow cells. Here, we extend our preclinical study to assess the functional restoration of SBDS capabilities in vitro and ex vivo. Ataluren improved 80S ribosome assembly and total protein synthesis in SDS-derived cells, restored myelopoiesis in myeloid progenitors, improved neutrophil chemotaxis in vitro and reduced neutrophil dysplastic markers ex vivo. Ataluren also restored full-length SBDS synthesis in primary osteoblasts, suggesting that its beneficial role may go beyond the myeloid compartment. Altogether, our results strengthened the rationale for a Phase I/II clinical trial of ataluren in SDS patients who harbour the nonsense mutation.

Monocyte-macrophage polarization and recruitment pathways in the tumour microenvironment of B-cell acute lymphoblastic leukaemia.

B-cell acute lymphoblastic leukaemia (B-ALL) reprograms the surrounding bone marrow (BM) stroma to create a leukaemia-supportive niche. To elucidate the contribution of immune cells to the leukaemic microenvironment, we investigated the involvement of monocyte/macrophage compartments, as well as several recruitment pathways in B-ALL development. Immunohistochemistry analyses showed that CD68-expressing macrophages were increased in leukaemic BM biopsies, compared to controls and predominantly expressed the M2-like markers CD163 and CD206. Furthermore, the "non-classical" CD14+ CD16++ monocyte subset, expressing high CX3CR1 levels, was significantly increased in B-ALL patients' peripheral blood. CX3CL1 was shown to be significantly upregulated in leukaemic BM plasma, thus providing an altered migratory pathway possibly guiding NC monocyte recruitment into the BM. Additionally, the monocyte/macrophage chemoattractant chemokine ligand 2 (CCL2) strongly increased in leukaemic BM plasma, possibly because of the interaction of leukaemic cells with mesenchymal stromal cells and vascular cells and due to a stimulatory effect of leukaemia-related inflammatory mediators. C5a, a macrophage chemoattractant and M2-polarizing factor, further appeared to be upregulated in the leukaemic BM, possibly as an effect of PTX3 decrease, that could unleash complement cascade activation. Overall, deregulated monocyte/macrophage compartments are part of the extensive BM microenvironment remodelling at B-ALL diagnosis and could represent valuable targets for novel treatments to be coupled with classical chemotherapy.

Heterogeneity of the bone marrow niche in patients with myeloproliferative neoplasms: ActivinA secretion by mesenchymal stromal cells correlates with the degree of marrow fibrosis.

Mesenchymal stromal cells (MSCs) represent an essential component of the bone marrow (BM) niche and display disease-specific alterations in several myeloid malignancies. The aim of this work was to study possible MSC abnormalities in Philadelphia-negative myeloproliferative neoplasms (MPNs) in relationship to the degree of BM fibrosis. MSCs were isolated from BM of 6 healthy donors (HD) and of 23 MPN patients, classified in 3 groups according to the diagnosis and the grade of BM fibrosis: polycythemia vera and essential thrombocythemia (PV/ET), low fibrosis myelofibrosis (LF-MF), and high fibrosis MF (HF-MF). MSC cultures were established from 21 of 23 MPN patients. MPN-derived MSCs did not exhibit any functional impairment in their adipogenic/osteogenic/chondrogenic differentiation potential and displayed a phenotype similar to HD-derived MSCs but with a decreased expression of CD146. All MPN-MSC lines were negative for the patient-specific hematopoietic clone mutations (JAK2, MPL, CALR). MSCs derived from HF-MF patients displayed a reduced clonogenic potential and a lower growth kinetic compared to MSCs from HD, LF-MF, and PV/ET patients. mRNA levels of hematopoiesis regulatory molecules were unaffected in MSCs from HF-MF compared to HD. Finally, in vitro ActivinA secretion by MSCs was increased in HF-MF compared to LF-MF patients, in association with a lower hemoglobin value. Increased ActivinA immunolabeling on stromal cells and erythroid precursors was also observed in HF-MF BM biopsies. In conclusion, higher grade of BM fibrosis is associated with functional impairment of MSCs and the increased secretion of ActivinA may represent a suitable target for anemia treatment in MF patients.

Cutaneous barrier leakage and gut inflammation drive skin disease in Omenn syndrome.

BACKGROUND: Severe early-onset erythroderma and gut inflammation, with massive tissue infiltration of oligoclonal activated T cells are the hallmark of Omenn syndrome (OS). OBJECTIVE: The impact of altered gut homeostasis in the cutaneous manifestations of OS remains to be clarified. METHODS: We analyzed a cohort of 15 patients with OS and the 129Sv/C57BL/6 knock-in Rag2R229Q/R229Q (Rag2R229Q) mouse model. Homing phenotypes of circulating lymphocytes were analyzed by flow cytometry. Inflammatory cytokines and chemokines were examined in the sera by ELISA and in skin biopsies by immunohistochemistry and in situ RNA hybridization. Experimental colitis was induced in mice by dextran sulfate sodium salt. RESULTS: We show that memory/activated T cells from patients with OS and from the Rag2R229Q mouse model of OS abundantly express the skin homing receptors cutaneous lymphocyte associated antigen and CCR4 (Ccr4), associated with high levels of chemokine C-C motif ligands 17 and 22. Serum levels of LPS are also elevated. A broad Th1/Th2/Th17 inflammatory signature is detected in the periphery and in the skin. Increased Tlr4 expression in the skin of Rag2R229Q mice is associated with enhanced cutaneous inflammation on local and systemic administration of LPS. Likewise, boosting colitis in Rag2R229Q mice results in increased frequency of Ccr4+ splenic T cells and worsening of skin inflammation, as indicated by epidermal thickening, enhanced epithelial cell activation, and dermal infiltration by Th1 effector T cells. CONCLUSIONS: These results support the existence of an interplay between gut and skin that can sustain skin inflammation in OS.

The Bone Marrow Niche in B-Cell Acute Lymphoblastic Leukemia: The Role of Microenvironment from Pre-Leukemia to Overt Leukemia.

Genetic lesions predisposing to pediatric B-cell acute lymphoblastic leukemia (B-ALL) arise in utero, generating a clinically silent pre-leukemic phase. We here reviewed the role of the surrounding bone marrow (BM) microenvironment in the persistence and transformation of pre-leukemic clones into fully leukemic cells. In this context, inflammation has been highlighted as a crucial microenvironmental stimulus able to promote genetic instability, leading to the disease manifestation. Moreover, we focused on the cross-talk between the bulk of leukemic cells with the surrounding microenvironment, which creates a "corrupted" BM malignant niche, unfavorable for healthy hematopoietic precursors. In detail, several cell subsets, including stromal, endothelial cells, osteoblasts and immune cells, composing the peculiar leukemic niche, can actively interact with B-ALL blasts. Through deregulated molecular pathways they are able to influence leukemia development, survival, chemoresistance, migratory and invasive properties. The concept that the pre-leukemic and leukemic cell survival and evolution are strictly dependent both on genetic lesions and on the external signals coming from the microenvironment paves the way to a new idea of dual targeting therapeutic strategy.

Pro-inflammatory cytokines favor the emergence of ETV6-RUNX1-positive pre-leukemic cells in a model of mesenchymal niche.

ETV6-RUNX1 (E/R) fusion gene, arising in utero from translocation t(12;21)(p13:q22), is the most frequent alteration in childhood acute lymphoblastic leukemia (ALL). However, E/R is insufficient to cause overt leukemia since it generates a clinically silent pre-leukemic clone which persists in the bone marrow but fails to out-compete normal progenitors. Conversely, pre-leukemic cells show increased susceptibility to transformation following additional genetic insults. Infections/inflammation are the most accredited triggers for mutations accumulation and leukemic transformation in E/R+ pre-leukemic cells. However, precisely how E/R and inflammation interact in promoting leukemia is still poorly understood. Here we demonstrate that IL6/TNFα/ILß pro-inflammatory cytokines cooperate with BM-MSC in promoting the emergence of E/R+ Ba/F3 over their normal counterparts by differentially affecting their proliferation and survival. Moreover, IL6/TNFα/ILß-stimulated BM-MSC strongly attract E/R+ Ba/F3 in a CXCR2-dependent manner. Interestingly, E/R-expressing human CD34+ IL7R+ progenitors, a putative population for leukemia initiation during development, were preserved in the presence of BM-MSC and IL6/TNFα/ILß compared to their normal counterparts. Finally, the extent of DNA damage increases within the inflamed niche in both control and E/R-expressing Ba/F3, potentially leading to transformation in the apoptosis-resistant pre-leukemic clone. Overall, our data provide new mechanistic insights into childhood ALL pathogenesis.

Functional Consequences of Low Activity of Transport System A for Neutral Amino Acids in Human Bone Marrow Mesenchymal Stem Cells.

In cultured human fibroblasts, SNAT transporters (System A) account for the accumulation of non-essential neutral amino acids, are adaptively up-regulated upon amino acid deprivation and play a major role in cell volume recovery upon hypertonic stress. No information is instead available on the expression and activity of SNAT transporters in human bone marrow mesenchymal stromal cells (MSC), although they are increasingly investigated for their staminal and immunomodulatory properties and used for several therapeutic applications. The uptake of glutamine and proline, two substrates of SNAT1 and SNAT2 transporters, was measured in primary human MSC and an MSC line. The amino acid analogue MeAIB, a specific substrate of these carriers, has been used to selectively inhibit SNAT-dependent transport of glutamine and, through its sodium-dependent transport, as an indicator of SNAT1/2 activity. SNAT1/2 expression and localization were assessed with RT-PCR and confocal microscopy, respectively. Cell volume was assessed from urea distribution space. In all these experiments, primary human fibroblasts were used as the positive control for SNAT expression and activity. Compared with fibroblasts, MSC have a lower SNAT1 expression and hardly detectable membrane localization of both SNAT1 and SNAT2. Moreover, they exhibit no sodium-dependent MeAIB uptake or MeAIB-inhibitable glutamine transport, and exhibit a lower ability to accumulate glutamine and proline than fibroblasts. MSC exhibited an only marginal increase in MeAIB transport upon amino acid starvation and did not recover cell volume after hypertonic stress. In conclusion, the activity of SNAT transporters is low in human MSC. MSC adaptation to amino acid shortage is expected to rely on intracellular synthesis, given the absence of an effective up-regulation of the SNAT transporters.

Humoral innate immunity at the crossroad between microbe and matrix recognition: The role of PTX3 in tissue damage.

Innate immunity is involved in regulating inflammatory and tissue repair responses to injury. In particular, humoral innate immunity plays functions related to wound clearance from tissue debris, and regulation of macrophage and stromal cell activities. PTX3, a component of humoral innate immunity, orchestrates tissue repair by interacting with plasminogen and fibrin. Fluid-phase molecules of innate immunity interact with elements of the extracellular matrix, and some of the latter display opsonic activity against certain bacterial species. Thus, recognition of extracellular matrix and microbial components is a recurrent theme in the humoral arm of the innate immune system.

Shwachman-Diamond syndrome with clonal interstitial deletion of the long arm of chromosome 20 in bone marrow: haematological features, prognosis and genomic instability.

In Shwachman-Diamond syndrome (SDS), deletion of the long arm of chromosome 20, del(20)(q), often acquired in bone marrow (BM), may imply a lower risk of developing myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML), due to the loss of the EIF6 gene. The genes L3MBTL1 and SGK2, also on chromosome 20, are in a cluster of imprinted genes, and their loss implies dysregulation of BM function. We report here the results of array comparative genomic hybridization (a-CGH) performed on BM DNA of six patients which confirmed the consistent loss of EIF6 gene. Interestingly, array single nucleotide polymorphisms (SNPs) showed copy neutral loss of heterozygosity for EIF6 region in cases without del(20)(q). No preferential parental origin of the deleted chromosome 20 was detected by microsatellite analysis in six SDS patients. Our patients showed a very mild haematological condition, and none evolved into BM aplasia or MDS/AML. We extend the benign prognostic significance of del(20)(q) and loss of EIF6 to the haematological features of these patients, consistently characterized by mild hypoplastic BM, no or mild neutropenia, anaemia and thrombocytopenia. Some odd results obtained in microsatellite and SNP-array analysis demonstrate a peculiar genomic instability, in an attempt to improve BM function through the acquisition of the del(20)(q).

ActivinA: a new leukemia-promoting factor conferring migratory advantage to B-cell precursor-acute lymphoblastic leukemic cells.

B-cell precursor-acute lymphoblastic leukemia modulates the bone marrow (BM) niche to become leukemia-supporting and chemo-protective by reprogramming the stromal microenvironment. New therapies targeting the interplay between leukemia and stroma can help improve disease outcome. We identified ActivinA, a TGF-ß family member with a well-described role in promoting several solid malignancies, as a factor favoring leukemia that could represent a new potential target for therapy. ActivinA resulted over-expressed in the leukemic BM and its production was strongly induced in mesenchymal stromal cells after culture with leukemic cells. Moreover, MSCs isolated from BM of leukemic patients showed an intrinsic ability to secrete higher amounts of ActivinA compared to their normal counterparts. The pro-inflammatory leukemic BM microenvironment synergized with leukemic cells to induce stromal-derived ActivinA. Gene expression analysis of ActivinA-treated leukemic cells showed that this protein was able to significantly influence motility-associated pathways. Interestingly, ActivinA promoted random motility and CXCL12-driven migration of leukemic cells, even at suboptimal chemokine concentrations, characterizing the leukemic niche. Conversely, ActivinA severely impaired CXCL12-induced migration of healthy CD34+ cells. This opposite effect can be explained by the ability of ActivinA to increase intracellular calcium only in leukemic cells, boosting cytoskeleton dynamics through a higher rate of actin polymerization. Moreover, by stimulating the invasiveness of the leukemic cells, ActivinA was found to be a leukemia-promoting factor. Importantly, the ability of ActivinA to enhance BM engraftment and the metastatic potential of leukemic cells was confirmed in a xenograft mouse model of the disease. Overall, ActivinA was seen to be a key factor in conferring a migratory advantage to leukemic cells over healthy hematopoiesis within the leukemic niche.

Once-weekly hypofractionated radiotherapy for breast cancer: First results of a phase II clinical trial.

Hypofractionated radiotherapy (HF) in 15 or 16 daily fractions is well established as an alternative in early breast cancer after breast-conserving surgery. Evidences for a whole-breast treatment even shorter, in 5-10 fractions, are still scarce. Women 50 years or older, with early breast tumor (pT1-2pN0), after breast-conserving surgery were eligible to enter in this phase II trial and received whole breast once-weekly hypofractionated radiotherapy (wHF-RT) to a total dose of 30 Gy, in 5 fractions of 6 Gy. During treatment and in post-treatment follow-up the toxicity was assessed and graduated according to the "Common Terminology Criteria for Adverse Events" (CTCAE), v3.0. Breast pictures for esthetic comparison were taken in 5 timepoints and 2 breast surgeons independently graduated the cosmetics changes. The trial was registered with ClinicalTrials.gov, number NCT01965483. From October 2013 to November 2015, 44 patients were enrolled in the trial and treated according to the protocol of wHF-RT. The median age was 70.5 years (51-88 years), and the median follow-up was 22 months (9-33 months). Skin erythema was the most common acute adverse event. At the end of radiation, 30 patients (68.2%) had any grade of radiation dermatitis. Concerning cosmetic appearance, there was no significant difference between pretreatment and 1 year assessments. The 2-year overall survival and disease-free survival were, respectively 96.8% and 97.7%. There was only one distant recurrence and no local or regional recurrence. Once-weekly hypofractionated radiotherapy is a feasible and well tolerated alternative for early breast cancer adjuvant management with acceptable acute toxicity and esthetic outcomes.

Mesenchymal stromal cells from Shwachman-Diamond syndrome patients fail to recreate a bone marrow niche in vivo and exhibit impaired angiogenesis.

Shwachman-Diamond syndrome (SDS) is a rare multi-organ recessive disease mainly characterised by pancreatic insufficiency, skeletal defects, short stature and bone marrow failure (BMF). As in many other BMF syndromes, SDS patients are predisposed to develop a number of haematopoietic malignancies, particularly myelodysplastic syndrome and acute myeloid leukaemia. However, the mechanism of cancer predisposition in SDS patients is only partially understood. In light of the emerging role of mesenchymal stromal cells (MSCs) in the regulation of bone marrow homeostasis, we assessed the ability of MSCs derived from SDS patients (SDS-MSCs) to recreate a functional bone marrow niche, taking advantage of a murine heterotopic MSC transplant model. We show that the ability of semi-cartilaginous pellets (SCPs) derived from SDS-MSCs to generate complete heterotopic ossicles in vivo is severely impaired in comparison with HD-MSC-derived SCPs. Specifically, after in vitro angiogenic stimuli, SDS-MSCs showed a defective ability to form correct networks, capillary tubes and vessels and displayed a marked decrease in VEGFA expression. Altogether, these findings unveil a novel mechanism of SDS-mediated haematopoietic dysfunction based on hampered ability of SDS-MSCs to support angiogenesis. Overall, MSCs could represent a new appealing therapeutic target to treat dysfunctional haematopoiesis in paediatric SDS patients.

Ataluren-driven restoration of Shwachman-Bodian-Diamond syndrome protein function in Shwachman-Diamond syndrome bone marrow cells.

Shwachman-Diamond syndrome (SDS) is a rare inherited recessive disease mainly caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene, which encodes for the homonymous protein SBDS, whose function still remains to be fully established. SDS affects several organs causing bone marrow failure, exocrine pancreatic insufficiency, skeletal malformations, and cognitive disorders. About 15% of SDS patients develop myelodysplastic syndrome (MDS) and are at higher risk of developing acute myeloid leukemia (AML). Deficiency in SBDS expression has been associated with increased apoptosis and lack of myeloid differentiation in bone marrow hematopoietic progenitors. Importantly, most SDS patients carry nonsense mutations in SBDS. Since ataluren is a well-characterized small molecule inhibitor that can suppress nonsense mutations, here, we have assessed the efficacy of this drug in restoring SBDS expression in hematopoietic cells obtained from a cohort of SDS patients. Remarkably, we show that ataluren treatment readily restores SBDS protein expression in different cell types, particularly bone marrow stem cells. Furthermore, ataluren promotes myeloid differentiation in hematopoietic progenitors, reduces apoptotic rate in primary PBMCs, and brings mammalian target of rapamycin phosphorylation levels back to normal in both lymphoblasts and bone marrow mesenchymal stromal cells (BM-MSCs). Since a specific therapy against SDS is currently lacking, these results provide the rationale for ataluren repurposing clinical trials.

Mesenchymal stromal cell-secreted chemerin is a novel immunomodulatory molecule driving the migration of ChemR23-expressing cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent cells characterized by broad immunomodulatory properties exploited for the treatment of inflammatory disorders. However, the efficacy of MSC-based therapy is highly variable and tightly linked to MSC culture conditions and treatment schedule. Thus, the identification of novel key molecules regulating MSC immunomodulatory activities in vivo might constitute a crucial step toward the optimization of currently available clinical protocols. In this regard, herein, we sought to determine whether the newly identified chemotactic protein, chemerin, plays a role in MSC-mediated regulation of inflammation. METHODS: Chemerin production by human MSCs was investigated under different culture conditions using enzyme-linked immunosorbent assay (ELISA). After purification, MSC-secreted chemerin was identified using mass spectrometry analysis and the biological activity of secreted isoforms was evaluated using migration assay. RESULTS: Bone marrow-derived MSCs secrete chemerin and express its receptors ChemR23 and CCRL2. Chemerin production is dependent on culture conditions and increases upon stimulation with inflammatory cytokines. In particular, platelet lysate (PL)-MSCs produce higher levels of chemerin compared with fetal bovine serum (FBS)-MSCs. Furthermore, chemerin is secreted by MSCs as an inactive precursor, which can be converted into its active form by exogenous chemerin-activating serine and cysteine proteases. DISCUSSION: Our data indicate that, in response to various inflammatory stimuli, MSCs secrete high amounts of inactive chemerin, which can then be activated by inflammation-induced tissue proteases. In light of these initial findings, we propose that further analysis of chemerin functions in vivo might constitute a crucial step toward optimizing MSC-based therapy for inflammatory diseases.

Treatment of graft versus host disease with mesenchymal stromal cells: a phase I study on 40 adult and pediatric patients.

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow-derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 10(6)/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100.

Protumoral role of monocytes in human B-cell precursor acute lymphoblastic leukemia: involvement of the chemokine CXCL10.

Myelomonocytic cells play a key role in the progression of many solid tumors. However, very little is known about their contribution to the progression of hematopoietic cancers. We investigated the role of monocytes in the progression of human B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We demonstrated that coculturing human monocytes in vitro with CD19+ BCP-ALL blasts from patients "conditioned" them to an inflammatory phenotype characterized by significant up-regulation of the chemokine, CXCL10. This phenotype was also observable ex vivo in monocytes isolated from BCP-ALL patients, which show elevated CXCL10 production compared with monocytes from healthy donors. Functionally, the "conditioned" monocytes promoted migration and invasive capacity of BCP-ALL cells. Increased invasion was mediated by matrix metalloproteinase 9 expression and activity in the BCP-ALL cells induced by the monocyte-derived CXCL10. However, neither the "conditioned" monocytes nor the CXCL10 produced by these cells had any effect on the proliferation/viability of BCP-ALL cells and angiogenesis. Collectively, our results strongly suggest a protumoral role for human monocytes in BCP-ALL, orchestrated by CXCL10 and its effect on tumor cell migration and invasion. These observations highlight the importance of the CXCL10/CXCR3 chemokine circuit in BCP-ALL progression.

ActivinA modulates B-acute lymphoblastic leukaemia cell communication and survival by inducing extracellular vesicles production.

Extracellular vesicles (EVs) are a new mechanism of cellular communication, by delivering their cargo into target cells to modulate molecular pathways. EV-mediated crosstalk contributes to tumor survival and resistance to cellular stress. However, the role of EVs in B-cell Acute Lymphoblastic Leukaemia (B-ALL) awaits to be thoroughly investigated. We recently published that ActivinA increases intracellular calcium levels and promotes actin polymerization in B-ALL cells. These biological processes guide cytoskeleton reorganization, which is a crucial event for EV secretion and internalization. Hence, we investigated the role of EVs in the context of B-ALL and the impact of ActivinA on this phenomenon. We demonstrated that leukemic cells release a higher number of EVs in response to ActivinA treatment, and they can actively uptake EVs released by other B-ALL cells. Under culture-induced stress conditions, EVs coculture promoted cell survival in B-ALL cells in a dose-dependent manner. Direct stimulation of B-ALL cells with ActivinA or with EVs isolated from ActivinA-stimulated cells was even more effective in preventing cell death. This effect can be possibly ascribed to the increase of vesiculation and modifications of EV-associated microRNAs induced by ActivinA. These data demonstrate that ActivinA boosts EV-mediated B-ALL crosstalk, improving leukemia survival in stress conditions.

The effects of propofol and ketamine on the cytokine levels of children with acute lymphoblastic leukemia.

The immune system of children with acute lymphoblastic leukemia (ALL) is affected by both the underlying disease and the chemotherapy. Children with ALL receive sedation for diagnostic and therapeutic procedures, which may contribute to immune competence alteration. The effects of propofol-ketamine combination on the immune system of children with ALL have not been investigated. This cohort study was designed to assess the immunomodulatory activity of the propofol-ketamine combination on proinflammatory and anti-inflammatory cytokines of children with ALL undergoing painful procedures. We enrolled 20 children with ALL undergoing bone marrow aspiration (BMA) and lumbar puncture with methotrexate. All children received sedation with IV ketamine (0.5 mg/kg) and propofol (3±2 mg/kg). Plasma concentration of cytokines interleukin (IL)-1ß, IL-2, IL-6, IL-10, IL-8, IL-12p70, and interferon-γ before sedation for BMA was represented as T0, during lumbar puncture with methotrexate sedation 6 hours after T0 was represented as T1, and 24 hours after BMA was represented as T2. Sedation with propofol-ketamine combination did not modify the plasma concentration of the most measured cytokines and the T helper 1/2 ratio in children with ALL. There was a significant reduction in IL-8 concentration 24 hours after BMA associated with the concomitant administration of steroids and methotrexate. These data suggest that sedation with propofol-ketamine combination may not affect the immediate outcome of children with ALL.

[Sub-acute thyroiditis in a patient on immunosuppressive treatment]. / Tiroidite subacuta in un paziente in trattamento immunosoppressivo.

Sub-acute thyroiditis or De Quervain's thyroiditis is a viral, inflammatory disease which causes the serum release of thyroidal hormones and hyperthyroidism. The pathogenesis of thyroid follicle damage is unclear because the exclusive viral action or a concomitant autoimmune component, determined by the lymphoid infiltrate remain to be assessed. We describe the case of a patient under immunosuppressive treatment, who developed sub-acute thyroiditis with hormone release and hyperthyroidism. The patient, while was under immunosuppressive treatment for kidney transplant, exhibited a clinical picture and hormonal profile of hyperthyroidism. Thyroid scintiscan exhibited an extremely low uptake. Fine-needle cytologic diagnosis was granulomatous sub-acute thyroiditis (De Quervain's thyroiditis). This case suggests the primary or even exclusive role of the viral infection in hormone release and hyperthyroidism in sub-acute thyroiditis, excluding an autoimmune component.