Inhibitors of ADAM10 reduce Hodgkin lymphoma cell growth in 3D microenvironments and enhance brentuximab-vedotin effect.

Shedding of ADAM10 substrates, like TNFa or CD30, can affect both anti-tumor immune response and antibody-drug-conjugate (ADC)-based immunotherapy. We have published two new ADAM10 inhibitors, LT4 and MN8 able to prevent such shedding in Hodgkin lymphoma (HL). Since tumor tissue architecture deeply influences the outcome of anti-cancer treatments, we set up a new threedimensional (3D) culture systems to verify whether ADAM10 inhibitors can contribute to, or enhance, the anti-lymphoma effects of the ADC brentuximab-vedotin (BtxVed). In order to recapitulate some aspects of lymphoma structure and architecture, we assembled two 3D culture models: mixed spheroids made of HL lymph node (LN) mesenchymal stromal cells (MSC) and Reed Sternberg/Hodgkin lymphoma cells (HL cells) or collagen scaffolds repopulated with LN-MSC and HL cells. In these 3D systems we found that: i) the ADAM10 inhibitors LT4 and MN8 reduce ATP content or glucose consumption, related to cell proliferation, increasing lactate dehydrogenase release as a cell damage hallmark; ii) these events are paralleled by mixed spheroids size reduction and inhibition of CD30 and TNFa shedding; iii) the effects observed can be reproduced in repopulated HL LN-derived matrix or collagen scaffolds; iv) ADAM10 inhibitors enhance the anti-lymphoma effect of the anti-CD30 ADC BtxVed both in conventional cultures and in repopulated scaffolds. Thus, we provide evidence for a direct and combined antilymphoma effect of ADAM10 inhibitors with BtxVed, leading to the improvement of ADC effects; this is documented in 3D models recapitulating features of the LN microenvironment, that can be proposed as a reliable tool for anti-lymphoma drug testing.

Chronic lymphocytic leukemia cells impair osteoblastogenesis and promote osteoclastogenesis: role of TNFα, IL-6 and IL-11 cytokines.

Bone skeletal alterations are no longer considered a rare event in chronic lymphocytic leukemia (CLL), especially at more advanced stages of the disease. This study is aimed at elucidating the mechanisms underlying this phenomenon. Bone marrow stromal cells, induced to differentiate toward osteoblasts in osteogenic medium, appeared unable to complete their maturation upon co-culture with CLL cells, CLL-cell-derived conditioned media (CLL-cm) or CLL-sera (CLL-sr). Inhibition of osteoblast differentiation was documented by decreased levels of RUNX2 and osteocalcin mRNA expression, by increased osteopontin and DKK-1 mRNA levels, and by a marked reduction of mineralized matrix deposition. The addition of neutralizing TNFα, IL-11 or anti-IL-6R monoclonal antibodies to these cocultures resulted in restoration of bone mineralization, indicating the involvement of these cytokines. These findings were further supported by silencing TNFα, IL-11 and IL-6 in leukemic cells. We also demonstrated that the addition of CLL-cm to monocytes, previously stimulated with MCSF and RANKL, significantly amplified the formation of large, mature osteoclasts as well as their bone resorption activity. Moreover, enhanced osteoclastogenesis, induced by CLL-cm, was significantly reduced by treating cultures with the anti-TNFα monoclonal antibody infliximab. An analogous effect was observed with the use of the BTK inhibitor, ibrutinib. Interestingly, CLL cells co-cultured with mature osteoclasts were protected from apoptosis and upregulated Ki-67. These experimental results parallel the direct correlation between amounts of TNFα in CLL-sr and the degree of compact bone erosion that we previously described, further strengthening the indication of a reciprocal influence between leukemic cell expansion and bone structure derangement.

Calcified brain metastases may be more frequent than normally considered.

OBJECTIVES: To verify the incidence of calcified brain metastases (CBM), illustrating the different presentation patterns and histology of primary tumor. METHODS: A series of 1002 consecutive brain computed tomography (CT) scans of patients with known primary tumors was retrospectively assessed. CBM were defined by the presence of calcification within intra-axial-enhancing lesions; identification of CBM was based on visual examination and ROI analysis (> 85 Hounsfield units). Also, calcifications in the primary tumor of all patients with brain metastases were evaluated. In CBM patients, we investigated the type of calcifications (punctate, nodular, cluster, ring, coarse), the histology of primary tumor, and if a previous RT was performed. RESULTS: Among 190 (18.9%) patients with brain metastatic disease, 34 presented with CBM (17.9%). Sixteen patients were previously treated with RT, while 18 presented calcifications ab initio (9.5% of all brain metastases). The majority of patients with CBM had a primitive lung adenocarcinoma (56%), followed by breast ductal invasive carcinoma (20%) and small cell lung carcinoma (11.8%). CBM were single in 44.1% of patients and multiple in 55.9%. With regard to the type of calcifications, the majority of CBM were punctate, without specific correlations between calcification type and histology of primary tumor. No patients with ab initio CBM had calcifications in primary tumor. CONCLUSION: In conclusion, our data show that CBM are more common than usually thought, showing an incidence of 9.5% ab initio in patients with brain metastases. This study underlines that neuroradiologists should not overlook intraparenchymal brain calcifications, especially in oncologic patients. KEY POINTS: • Among the differential diagnosis of brain intraparenchymal calcifications, metastases are considered uncommon and found predominantly in patients treated with radiotherapy (RT). • Our data show that CBM are more common than usually thought, showing an incidence of 9.5% ab initio in patients with brain metastases. • A proportion of intraparenchymal brain calcifications, especially in oncologic patients, might represent evolving lesions and neuroradiologists should not overlook them to avoid a delay in diagnosis and treatment.

Tracing CLL-biased stereotyped immunoglobulin gene rearrangements in normal B cell subsets using a high-throughput immunogenetic approach.

BACKGROUND: B cell receptor Immunoglobulin (BcR IG) repertoire of Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of quasi-identical BcR IG. These are observed in approximately 30% of patients, defined as stereotyped receptors and subdivided into subsets based on specific VH CDR3 aa motifs and phylogenetically related IGHV genes. Although relevant to CLL ontogeny, the distribution of CLL-biased stereotyped immunoglobulin rearrangements (CBS-IG) in normal B cells has not been so far specifically addressed using modern sequencing technologies. Here, we have investigated the presence of CBS-IG in splenic B cell subpopulations (s-BCS) and in CD5+ and CD5- B cells from the spleen and peripheral blood (PB). METHODS: Fractionation of splenic B cells into 9 different B cell subsets and that of spleen and PB into CD5+ and CD5- cells were carried out by FACS sorting. cDNA sequences of BcR IG gene rearrangements were obtained by NGS. Identification of amino acidic motifs typical of CLL stereotyped subsets was carried out on IGHV1-carrying gene sequences and statistical evaluation has been subsequently performed to assess stereotypes distribution. RESULTS: CBS-IG represented the 0.26% average of IGHV1 genes expressing sequences, were detected in all of the BCS investigated. CBS-IG were more abundant in splenic and circulating CD5+ B (0.57%) cells compared to CD5- B cells (0.17%). In all instances, most CBS IG did not exhibit somatic hypermutation similar to CLL stereotyped receptors. However, compared to CLL, they exhibited a different CLL subset distribution and a broader utilization of the genes of the IGHV1 family. CONCLUSIONS: CBS-IG receptors appear to represent a part of the "public" BcR repertoire in normal B cells. This repertoire is observed in all BCS excluding the hypothesis that CLL stereotyped BcR accumulate in a specific B cell subset, potentially capable of originating a leukemic clone. The different relative representation of CBS-IG in normal B cell subgroups suggests the requirement for additional selective processes before a full transformation into CLL is achieved.

Tag-based next generation sequencing: a feasible and reliable assay for EGFR T790M mutation detection in circulating tumor DNA of non small cell lung cancer patients.

BACKGROUND: The demonstration of EGFR T790M gene mutation in plasma is crucial to assess the eligibility of Non Small Cell Lung Cancer (NSCLC) patients, who have acquired resistance to first or second generation Tyrosine Kinase Inhibitors (TKIs), to receive a subsequent treatment with osimertinib. Since circulating tumor DNA (ctDNA) is present in very low amounts in plasma, high sensitive and specific methods are required for molecular analysis. Improving sensitivity of T790M mutation detection in plasma ctDNA enables a larger number of NSCLC patients to receive the appropriate therapy without any further invasive procedure. METHODS: A tag-based next generation sequencing (NGS) platform capable of tagging rare circulating tumor DNA alleles was employed in this study for the identification of T790M mutation in 42 post-TKI NSCLC patients. RESULTS: Compared to Real Time PCR, tag-based NGS improved the T790M detection rate (42.85% versus 21.4%, respectively), especially in those cases with a low median mutation abundance (i.e. 0.24, range 0.07-0.78). Moreover, the tag-based NGS identified EGFR activating mutations more efficiently than Real Time PCR (85.7% versus 61.9% detection rate, respectively), particularly of the L858R variant type (0.06-0.75 mutation abundance range). Patients in whom the T790M mutation was detected in plasma, achieved an objective response to osimertinib (9/14, 64.28%). CONCLUSIONS: Tag-based NGS represents an accurate and sensitive tool in a clinical setting for non-invasive assessment and monitoring of T790M variant in NSCLC patients.

Early interstitial macrophage infiltration with mild dysfunction is associated with subsequent kidney graft loss.

Macrophage infiltration is associated with unfavorable kidney graft outcome in protocol biopsies, but few studies have evaluated its impact on clinical practice. We therefore prospectively evaluated 37 kidney transplant recipients (KTRs) who underwent kidney biopsy due to slight increases in serum creatinine, or mild proteinuria (>0.3 g/24 hr), in the first post-transplant year. Banff score, CD68+ count (score 0-3) by immunohistochemistry, and 1-year DSA were assessed. DGF was reported in 10 (27%) patients, 6 (16%) had normal biopsy, 7 (19%) borderline lesions, 13 (35%) IFTA, and 11 (30%) other lesions. Fifteen KTRs had grade 3 CD68+ infiltration, and 47% developed de novo DSA. During a 6.2 ± 2.7 year follow-up, four patients (11%) suffered from biopsy-proven T-cell rejection, 17 KTRs (46%) lost their graft (12 in the grade 3 CD68+ group). Graft survival was lower in KTRs with grade 3 CD68+ infiltration (P = 0.0074; log-rank test). Grade 3 CD68+ infiltrate was an independent predictor of graft loss (HR 5.41, 95% CI 1.74-16.8; P = 0.003), together with more severe graft dysfunction at biopsy (HR 6.41, 95% CI 2.57-16; P < 0.001). We conclude that grade 3 CD68+ interstitial infiltration is associated with increased risk of subsequent graft loss independent of other factors.

Pharmacokinetics, pharmacodynamics and efficacy on pediatric tumors of the glioma radiosensitizer KU60019.

We have recently reported that glioblastoma (GB)-initiating cells (GIC) with low expression and/or mutation of TP53 and high expression of PI3K ("responder" genetic profile) can be effectively and safely radiosensitized by the ATM inhibitor KU60019. We report here on drug's diffusion and elimination from the animal body and brain, its effects on orthotopic GB and efficacy toward pediatric GIC. Healthy mice were infused by convection enhanced delivery (CED) with KU60019 and the drug kinetics followed by high performance liquid chromatography-mass spectrometry. Already at the end of CED, KU60019 had diffused from the injection site to the ipsilateral and, to a lower extent, controlateral hemisphere. After 24 hr, no drug could be detected all over the brain or in other organs, indicating rapid draining and excretion. After intraperitoneal injection, traces only of KU60019 could be detected in the brain, indicating inability to cross the brain-blood barrier. Consistent with the induction of cell cycle progression previously observed in vitro, KU60019 stimulated proliferation of orthotopic GB cells with the highest effect observed 96 hr after drug delivery. Adult GIC with high expression of TP53 and low expression of PI3K could be radiosensitized by KU60019, although less promptly than GIC bearing the "responder" profile. Consistent with the kinetics of proliferation induction, the highest radiosensitizing effect was observed 96 hr after delivery of KU60019 to GIC. Pediatric GIC could be similarly radiosensitized after exposure to KU60019. The results indicate that ATM inhibition may allow to radiosensitize a wide range of adult and pediatric high-grade gliomas.

High ERp5/ADAM10 expression in lymph node microenvironment and impaired NKG2D ligands recognition in Hodgkin lymphomas.

Herein we describe that in classic Hodgkin lymphomas (cHL, n = 25) the lymph node (LN) stroma displayed in situ high levels of transcription and expression of the disulfide-isomerase ERp5 and of the disintegrin-metalloproteinase ADAM10, able to shed the ligands for NKG2D (NKG2D-L) from the cell membrane. These enzymes were detected both in LN mesenchymal stromal cells (MSCs) and in Reed-Sternberg (RS) cells; in addition, MIC-A and ULBP3 were present in culture supernatants of LN MSCs or RS cells. NKG2D-L-negative RS cells could not be killed by CD8(+)αßT or γδT cells; tumor cell killing was partially restored by treating RS cells with valproic acid, which enhanced NKG2D-L surface expression. Upon coculture with LN MSCs, CD8(+)αßT and γδT cells strongly reduced their cytolytic activity against NKG2D-L(+) targets; this seems to be the result of TGF-ß, present at the tumor site, produced in vitro by LN MSCs and able to down-regulate the expression of NKG2D on T lymphocytes. In addition, CD8(+)αßT and γδT cells from the lymph nodes of cHL patients, cocultured in vitro with LN MSCs, underwent TGF-ß-mediated down regulation of NKG2D. Thus, in cHL the tumor microenvironment is prone to inhibit the development of an efficient antitumor response.

Aminobisphosphonates prevent the inhibitory effects exerted by lymph node stromal cells on anti-tumor Vδ 2 T lymphocytes in non-Hodgkin lymphomas.

In this study, we analyzed the influence of mesenchymal stromal cells derived from lymph nodes of non-Hodgkin's lymphomas, on effector functions and differentiation of Vdelta (δ)2 T lymphocytes. We show that: i) lymph-node mesenchymal stromal cells of non-Hodgkin's lymphoma inhibit NKG2D-mediated lymphoid cell killing, but not rituximab-induced antibody-dependent cell-mediated cytotoxicity, exerted by Vδ2 T lymphocytes; ii) pre-treatment of mesenchymal stromal cells with the aminobisphosphonates pamidronate or zoledronate can rescue lymphoma cell killing via NKG2D; iii) this is due to inhibition of transforming growth factor-ß and increase in interleukin-15 production by mesenchymal stromal cells; iv) aminobisphosphonate-treated mesenchymal stromal cells drive Vδ2 T-lymphocyte differentiation into effector memory T cells, expressing the Thelper1 cytokines tumor necrosis factor-α and interferon-γ. In non-Hodgkin's lymphoma lymph nodes, Vδ2 T cells were mostly naïve; upon co-culture with autologous lymph-node mesenchymal stromal cells exposed to zoledronate, the percentage of terminal differentiated effector memory Vδ2 T lymphocytes increased. In all non-Hodgkin's lymphomas, low or undetectable transcription of Thelper1 cytokines was found. In diffused large B-cell lymphomas and in a group of follicular lymphoma, transcription of transforming growth factor ß and interleukin-10 was enhanced compared to non-neoplastic lymph nodes. Thus, in non-Hodgkin lymphomas mesenchymal stromal cells interfere with Vδ2 T-lymphocyte cytolytic function and differentiation to Thelper1 and/or effector memory cells, depending on the prominent in situ cytokine milieu. Aminobisphosphonates, acting on lymph-node mesenchymal stromal cells, can push the balance towards Thelper1/effector memory and rescue the recognition and killing of lymphoma cells through NKG2D, sparing rituximab-induced antibody-dependent cell-mediated cytotoxicity.

Balance between somatostatin and D2 receptor expression drives TSH-secreting adenoma response to somatostatin analogues and dopastatins.

CONTEXT: First-line therapy for thyrotropin-secreting pituitary adenomas (TSHomas) is neurosurgery, while medical treatment rests mainly on somatostatin analogues. Clinically available sst(2) -preferring analogues, octreotide and lanreotide, induce normalization of hormone levels in approximately 90% of patients and tumour shrinkage in 45%. OBJECTIVE: We evaluated somatostatin 1, 2, 3 and 5 and dopamine D2 receptor expression in tumour samples from three TSHomas, and the relationships between receptor expression, in vitro antiproliferative response and clinical data, including octreotide test and three months of therapy with octreotide long-acting repeatable (LAR). TSHoma cell proliferation was tested in vitro using octreotide, cabergoline and two chimeric compounds, BIM-23A760 and BIM-23A387. RESULTS: All patients showed significant TSH lowering to acute octreotide test, but a hormonal response to long-term treatment was observed in only two patients, showing a high sst(5) /sst(2) ratio. Patient 2, characterized by high expression of sst(2) and sst(1) and a relative lower expression of sst(5) , experienced tachyphylaxis after prolonged octreotide treatment. In vitro, the somatostatin/dopamine receptor agonist BIM-23A760 caused the highest antiproliferative effect among those tested. Combined treatment with octreotide and cabergoline displayed an additive effect of magnitude comparable to that of the other chimeric compound (BIM-23A387). Octreotide resistance was confirmed in cells isolated from the nonresponder patient, although it could be overcome by treatment with the chimeric compounds. CONCLUSIONS: A high sst(5) /sst(2) ratio might be predictive of a positive outcome to long-term treatment with somatostatin analogues in TSHomas. Moreover, combined somatostatin and D(2) receptor targeting might be considered as a potential tool to improve the response rate in octreotide-resistant tumours.

[Hypocomplementemic tubulointerstitial nephritis in IgG4-related disease]. / Nefrite tubulo-interstiziale ipocomplementemica in IgG4-related disease.

A novel lymphoproliferative disorder producing plasma cell expansion in the affected organ with fibrotic or sclerosing changes, known as ''IgG4-related disease'', was defined in Japan by Umehara's group in 2010. We present the first case reported in Italy. In 2007, a 63-year-old man presented with epigastric pain and elevated serum lipase levels. Computed tomography of the abdomen revealed a Kuttner's tumor of the pancreas. The patient underwent a biliary-enteric anastomosis, and biopsy of the pancreas revealed massive infiltration of lymphocytes and plasma cells. The patient was diagnosed with chronic sclerosing pancreatitis. After one year, he began to show signs of sicca syndrome and at the same time developed progressive renal failure. Immunological tests revealed hypocomplementemia, and the renal biopsy specimen showed diffuse interstitial inflammation. The infiltrate was composed of lymphocytes, while infiltrating plasma cells showed immunoreactivity to IgG-4. Sialography using a radioisotope revealed severe involvement of the salivary glands, and Schirmer's test gave a positive result. This led us to diagnose hypocomplementemic tubulointerstitial nephritis in IgG4-related disease. Corticosteroid treatment resulted in rapid improvement including disappearance of the sicca syndrome and progressive amelioration of renal function. After six months, we discontinued steroid administration and started mycophenolate mofetil to maintain a low degree of immunosuppression. Follow-up after two years showed that this therapy continued to be quite effective in our patient.

P53 staining index and zonal staining patterns in actinic keratoses.

Actinic keratoses (AKs) are common dysplastic lesions resulting from chronic excessive ultraviolet exposure. Neither the clinical grade of thickness nor the histological grade of dysplasia seems valid predictors of aggressive potential of AKs. Instead, the mutational status in AKs appears to predict well the clinical course. TP53 gene mutations result in a non-functional protein resistant to degradation, thus immunohistochemical staining for p53 can suggest mutation status. Increased p53 was associated with progression from AK to squamous cell carcinoma. To investigate how the intensity of p53 staining (p53 staining index) varies according to body site, histological subtype and grade dysplasia of AKs. Secondly, we sought to investigate the distribution in the epidermal layers of non-functional p53 (zonal staining patterns). p53 staining index was greater than 50% in 90.7% of AKs. p53 staining index was significantly higher in older age (p < 0.0093) and in facial AKs compared to other body areas (p = 0.03). A significant correlation between p53 staining index and grade of dysplasia was observed (p = 0.006) and between p53 staining index and zonal p53 staining pattern (p = 0.003). No significant differences in p53 staining index among the various histological AK types were observed. No correlation between clinical and histological grade. All AKs, independently from their clinical appearance, should be treated but special attention is required for AKs on severely photodamaged skin on the face and in older patients.

Post-Transplant Nivolumab Plus Unselected Autologous Lymphocytes in Refractory Hodgkin Lymphoma: A Feasible and Promising Salvage Therapy Associated With Expansion and Maturation of NK Cells.

Immune checkpoint inhibitors (CI) have demonstrated clinical activity in Hodgkin Lymphoma (HL) patients relapsing after autologous stem cell transplantation (ASCT), although only 20% complete response (CR) rate was observed. The efficacy of CI is strictly related to the host immune competence, which is impaired in heavily pre-treated HL patients. Here, we aimed to enhance the activity of early post-ASCT CI (nivolumab) administration with the infusion of autologous lymphocytes (ALI). Twelve patients with relapse/refractory (R/R) HL (median age 28.5 years; range 18-65), underwent lymphocyte apheresis after first line chemotherapy and then proceeded to salvage therapy. Subsequently, 9 patients with progressive disease at ASCT received early post-transplant CI supported with four ALI, whereas 3 responding patients received ALI alone, as a control cohort. No severe adverse events were recorded. HL-treated patients achieved negative PET scan CR and 8 are alive and disease-free after a median follow-up of 28 months. Four patients underwent subsequent allogeneic SCT. Phenotypic analysis of circulating cells showed a faster expansion of highly differentiated NK cells in ALI plus nivolumab-treated patients as compared to control patients. Our data show anti-tumor activity with good tolerability of ALI + CI for R/R HL and suggest that this setting may accelerate NK cell development/maturation and favor the expansion of the "adaptive" NK cell compartment in patients with HCMV seropositivity, in the absence of HCMV reactivation.

Overexpression of stromal cell-derived factor 1 and its receptor CXCR4 induces autocrine/paracrine cell proliferation in human pituitary adenomas.

PURPOSE: Hypothalamic or locally produced growth factors and cytokines control pituitary development, functioning, and cell division. We evaluated the expression of the chemokine stromal cell-derived factor 1 (SDF1) and its receptor CXCR4 in human pituitary adenomas and normal pituitary tissues and their role in cell proliferation. EXPERIMENTAL DESIGN: The expression of SDF1 and CXCR4 in 65 human pituitary adenomas and 4 human normal pituitaries was determined by reverse transcription-PCR, immunohistochemistry, and confocal immunofluorescence. The proliferative effect of SDF1 was evaluated in eight fibroblast-free human pituitary adenoma cell cultures. RESULTS: CXCR4 mRNA was expressed in 92% of growth hormone (GH)-secreting pituitary adenomas (GHoma) and 81% of nonfunctioning pituitary adenomas (NFPA), whereas SDF1 was identified in 63% and 78% of GHomas and NFPAs, respectively. Immunostaining for CXCR4 and SDF1 showed a strong homogenous labeling in all tumoral cells in both GHomas and NFPAs. In normal tissues, CXCR4 and SDF1 were expressed only in a subset of anterior pituitary cells, with a lower expression of SDF1 compared with its cognate receptor. CXCR4 and SDF1 were not confined to a specific cell population in the anterior pituitary but colocalized with discrete subpopulations of GH-, prolactin-, and adrenocorticorticotropic hormone-secreting cells. Conversely, most of the SDF1-containing cells expressed CXCR4. In six of eight pituitary adenoma primary cultures, SDF1 induced a statistically significant increase in DNA synthesis that was prevented by the treatment with the CXCR4 antagonist AMD3100 or somatostatin. CONCLUSIONS: CXCR4 and SDF1 are overexpressed in human pituitary adenomas and CXCR4 activation may contribute to pituitary cell proliferation and, possibly, to adenoma development in humans.

Kidney graft loss associated with JC polyomavirus nephropathy.

This is the first case of documented treatment failure of JCV nephritis, despite a reduction of immunosuppressive agents and the use of antiviral therapy. We consistently detected high levels of JCV viremia, which correlated with the progressive deterioration of the graft and caused the final loss of the kidney, suggesting that JCV plays an etiological role in the onset of severe nephropathy in kidney transplant patients.

Author Correction: Faithful animal modelling of human glioma by using primary initiating cells and its implications for radiosensitization therapy.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Mesenchymal stem cells protective effect in adriamycin model of nephropathy.

Mesenchymal stem cells (MSCs) may be of value in regeneration of renal tissue after damage; however, lack of biological knowledge and variability of results in animal models limit their utilization. We studied the effects of MSCs on podocytes in vitro and in vivo utilizing adriamycin (ADR) as a model of renal toxicity. The in vivo experimental approach was carried out in male Sprague-Dawley rats (overall 60 animals) treated with different ADR schemes to induce acute and chronic nephrosis. MSCs were given a) concomitantly to ADR in tail vein or b) in aorta and c) in tail vein 60 days after ADR. Homing was assessed with PKH26-MSCs. MSCs rescued podocytes from apoptosis induced by ADR in vitro. The maximal effect (80% rescue) was obtained with MSCs/podocytes coculture ratio of 1:1 for 72 h. All rats treated with ADR developed nephrosis. MSCs did not modify the clinical parameters (i.e., proteinuria, serum creatinine, lipids) but protected the kidney from severe glomerulosclerosis when given concomitantly to ADR. Rats given MSCs 60 days after ADR developed the same severe renal damage. Only a few MSCs were found in renal tubule-interstitial areas 1-24 h after injection and no MSCs were detected in glomeruli. MSCs reduced apoptosis of podocytes treated with ADR in vitro. Early and repeated MSCs infusion blunted glomerular damage in chronic ADR-induced nephropathy. MSCs did not modify proteinuria and progression to renal failure, which implies lack of regenerative potential in this model.

ATR kinase inhibitors NVP-BEZ235 and AZD6738 effectively penetrate the brain after systemic administration.

Ataxia Telangiectasia and Rad3 related protein (ATR) is a central mediator of the response to DNA damage that may cause the quiescent resistance of cancer initiating cells to genotoxic radiotherapy. NVP-BEZ235 is a dual PI3K/mTOR inhibitor that also effectively targets ATR with IC50 = 21 × 10- 9 M in cells. AZD6738 does not target significantly PI3K/mTOR-related kinases but specifically inhibits ATR with IC50 = 74 × 10- 9 M in cells. Both drugs have been proposed as radiosensitizers of different tumors including glioblastoma (GB), the most malignant brain tumor. In order to study the radiosensitizing properties of ATR inhibitors NVP-BEZ235 and AZD6738 towards GB, we have preliminarily investigated their capacity to penetrate the brain after systemic administration. Tumor-free CD-1 mice were inoculated i.p. with 25 mg/Kg body weight of NVP-BEZ235 or AZD6738. 1, 2, 6 and 8 h later, blood was collected by retro-orbital bleeding after which the mice were euthanized and the brains explanted. Blood and brain samples were then extracted and NVP-BEZ235 and AZD6738 concentrations determined by High Performance Liquid Chromatography/Mass Spectrometry. We found for NVP-BEZ235 and especially for AZD6738, elevated bioavailability and effective brain penetration after intraperitoneal administration. Albeit low drug and radiation dosages were used, a trend to toxicity of NVP-BEZ235 followed by ionizing radiation (IR) towards mice bearing primary glioma initiating cells (GIC)-driven orthotopic tumors was yet observed, as compared to AZD6738 + IR and vehicle+IR. Survival was never improved with median values of 99, 86 and 101 days for vehicle+IR, NVP-BEZ235 + IR and AZD6738 + IR-treated mice, respectively. Although the present results indicate favorable pharmacokinetics properties of ATR inhibitors NVP-BEZ235 and AZD6738, they do not lend support to their use as radiosensitizers of GB.

Case of Spinal Osteoblastoma in Elderly: Is It Really a Young Patient's Disease?

BACKGROUND: Osteoblastoma is a benign bone-forming tumor, sometimes locally invasive, that may involve any bone. The highest incidence is between 20 and 30 years of age, and there are no cases described in the elderly. METHODS: We report a case of an elderly patient with a lesion in the lumbar spine in which osteoblastoma diagnosis was made. CONCLUSIONS: Osteoblastoma is a rare tumor older than 50 years of age, but it should be considered in the differential diagnosis of bone lesions of the spine in adulthood and in the elderly, to avoid a delay in the treatment.

IL-25 dampens the growth of human germinal center-derived B-cell non Hodgkin Lymphoma by curtailing neoangiogenesis.

Interleukin (IL)-25, a member of the IL-17 cytokine superfamily, is produced by immune and non-immune cells and exerts type 2 pro-inflammatory effects in vitro and in vivo. The IL-25 receptor(R) is composed of the IL-17RA/IL-17RB subunits. Previous work showed that germinal centre (GC)-derived B-cell non Hodgkin lymphomas (B-NHL) expressed IL-17AR, formed by IL-17RA and IL-17RC subunits, and IL-17A/IL-17AR axis promoted B-NHL growth by stimulating neoangiogenesis. Here, we have investigated expression and function of IL-25/IL-25R axis in lymph nodes from human GC-derived B-NHL, i.e. Follicular Lymphoma (FL,10 cases), Diffuse Large B Cell Lymphoma (6 cases) and Burkitt Lymphoma (3 cases). Tumor cells expressed IL-25R and IL-25 that was detected also in non-malignant cells by flow cytometry. Immunohistochemical studies confirmed expression of IL-25R and IL-25 in FL cells, and highlighted IL-25 expression in bystander elements of the FL microenvironment. IL-25 i) up-regulated phosphorylation of NFkBp65, STAT-1 and JNK in B-NHL cells; ii) inhibited in vitro proliferation of the latter cells; iii) exerted anti-tumor activity in two in vivo B-NHL models by dampening expression of pro-angiogenic molecules as VEGF-C, CXCL6 and ANGPT3. In conclusion, IL-25, that is intrinsically pro-angiogenic, inhibits B-NHL growth by reprogramming the angiogenic phenotype of B-NHL cells.