c-myb Proto-Oncogene Is Expressed by Quiescent Scleroderma Fibroblasts and, Unlike B-myb Gene, Does Not Correlate With Proliferation

Systemic sclerosis (scleroderma) is characterized by excessive deposition of extracellular matrix constituents. Although it has been proposed that tissue fibrosis is due to increased fibroblast synthesis of various collagen polypeptides, there is some experimental evidence that patients with systemic sclerosis have a defect in the control of fibroblast growth. The myb family of genes includes, among others, the c-myb proto-oncogene and the structurally related gene, B-myb, which are both implicated in the regulation of differentiation and/or proliferation of hematopoietic and nonhematopoietic cells. To elucidate the molecular basis responsible for scleroderma fibroblast proliferation, we therefore elected to investigate the expression of c-myb and B-myb genes in scleroderma and control cells. Using the reverse transcriptase polymerase chain reaction technique, we detected c-myb transcripts in scleroderma skin fibroblasts rendered quiescent by serum deprivation. Under the game experimental conditions, c-myb message was not found in normal skin fibroblasts, but, after serum stimulation, c-myb RNA was clearly evident from 3 to 72h in both normal and pathologic cells. Treatment of these cells with c-myb antisense oligonucleotides caused downregulation of c-myb expression, and the inhibition of scleroderma fibroblast proliferation was 42%, whereas in normal fibroblasts the inhibition was weaker (22%). In contrast to c-myb, in normal and scleroderma fibroblasts the level of expression of B-myb correlated with cell proliferation assessed by cell count, and densitometric analysis showed that B-myb message was 1.5–5 times higher in most of pathologic cells studied. The antisense B-myb oligonucleotides had a weaker antiproliferative effect compared with antisense c-myb, inhibiting scleroderma and normal fibroblasts by 23% and 13%, respectively. These data suggest that the B-myb and c-myb genes may play a role in scleroderma fibroblast proliferation and function

Complement Activation Determines the Therapeutic Activity of Rituximab In Vivo

Rituximab is an anti-CD20 chimeric mAb effective for the treatment of B-NHL. It can lyse lymphoma cells in vitro through both C- and Ab-dependent cellular cytotoxicity. The mechanism of action of rituximab in vivo is however still unclear. We have set up a new in vivo model in nonimmunodeficient mice by stable transduction of the human CD20 cDNA in the murine lymphoma line EL4. Animals injected i.v. with the EL4-CD20+ lymphoma cells died within 30 days with evident liver, spleen, and bone marrow involvement, confirmed by immunohistochemistry and PCR analysis. A single injection of rituximab or the murine anti-CD20 Ab 1F5, given i.p. 1 day after the tumor, cured 100% of the animals. Indeed, at week 4 after tumor cell inoculation, CD20+ cells were undetectable in all organs analyzed in rituximab-treated animals, as determined by immunohistochemistry and PCR. Rituximab had no direct effect on tumor growth in vitro. Depletion of either NK cells or neutrophils or both in tumor-injected animals did not affect the therapeutic activity of the drug. Similarly, rituximab was able to eradicate tumor cells in athymic nude mice, suggesting that its activity is T cell independent. In contrast, the protective activity of rituximab or the 1F5 Ab was completely abolished in syngeneic knockout animals lacking C1q, the first component of the classical pathway of C (C1qa−/−). These data demonstrate that C activation is fundamental for rituximab therapeutic activity in vivo

Platelet-Lysate-Expanded Mesenchymal Stromal Cells for the Treatment of Resistant GVHD

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Mesenchymal Stromal Cells Do Not Increase the Risk of Viral Reactivation Nor the Severity of Viral Events in Recipients of Allogeneic Stem Cell Transplantation

Mesenchymal stromal cells (MSC) are tested in clinical trials to treat graft versus host disease (GvHD) after stem cell transplantation (SCT). In vitro studies demonstrated MSC's broad immunosuppressive activity. As infections represent a major risk after SCT, it is important to understand the role of MSC in this context. We analyzed 24 patients (pts) receiving MSC for GvHD in our Unit between 2009 and 2011. We recorded viral reactivations as measured in whole blood with polymerase chain reaction for 100 days following MSC administration. In patients with a documented viral reactivation in the first 3 days following MSCs infusion the frequency of virus-specific IFNgamma-producing cells was determined through enzyme-linked immunospot assay. In our cohort of patients viral reactivation after MSC infusion occurred in 45% of the cases, which did not significantly differ from the incidence in a historical cohort of patients affected by steroid resistant GvHD and treated with conventional immunosuppression. No patient presented severe form of infection. Two cases could be checked for immunological response to viral stimulus and demonstrated virus specific T-cytotoxic lymphocyte activity. In our experience MSC infusion did not prove to trigger more frequent or severer viral reactivations in the post transplantation setting

Longitudinal tracking of triple labeled umbilical cord derived mesenchymal stromal cells in a mouse model of Amyotrophic Lateral Sclerosis

The translational potential of cell therapy to humans requires a deep knowledge of the interaction between transplanted cells and host tissues. In this study, we evaluate the behavior of umbilical cord mesenchymal stromal cells (UC-MSCs), labeled with fluorescent nanoparticles, transplanted in healthy or early symptomatic transgenic SOD1G93A mice (a murine model of Amyotrophic Lateral Sclerosis). The double labeling of cells with nanoparticles and Hoechst-33258 enabled their tracking for a long time in both cells and tissues. Whole-body distribution of UC-MSCs was performed by in-vivo and ex-vivo analyses 1, 7, 21 days after single intravenous or intracerebroventricular administration. By intravenous administration cells were sequestered by the lungs and rapidly cleared by the liver. No difference in biodistribution was found among the two groups. On the other hand, UC-MSCs transplanted in lateral ventricles remained on the choroid plexus for the whole duration of the study even if decreasing in number. Few cells were found in the spinal cord of SOD1G93A mice exclusively. No migration in brain parenchyma was observed. These results suggest that the direct implantation in brain ventricles allows a prolonged permanence of cells close to the damaged areas and makes this method of tracking reliable for future studies of efficacy

The CCL3 Family of Chemokines and Innate Immunity Cooperate In Vivo in the Eradication of an Established Lymphoma Xenograft by Rituximab

The therapeutic mAb rituximab induced the expression of the CCL3 and CCL4 chemokines in the human lymphoma line BJAB following binding to the CD20 Ag. Induction of CCL3/4 in vitro was specific, was observed in several cell lines and freshly isolated lymphoma samples and also took place at the protein level in vitro and in vivo. To investigate the role of these beta-chemokines in the mechanism of action of rituximab, we synthesized a N-terminally truncated CCL3 molecule CCL3(11\u201370), which had antagonist activity on chemotaxis mediated by either CCL3 or BJAB supernatant. We also set up an established s.c. BJAB tumor model in athymic mice. Rituximab, given weekly after tumors had reached 250 mm2, led to complete disappearance of the lymphoma within 2\u20133 wk. Treatment of mice with cobra venom factor showed that complement was required for rituximab therapeutic activity. Treatment of BJAB tumor bearing mice every 2 days with the CCL3(11\u201370) antagonist, starting 1 wk before rituximab treatment, had no effect on tumor growth by itself, but completely inhibited the therapeutic activity of the Ab. To determine whether CCL3 acts through recruitment/activation of immune cells, we specifically depleted NK cells, polymorphonuclear cells, and macrophages using mAbs, clodronate treatment, or Rag2\u2013/\u2013c\u2013/\u2013 mice. The data demonstrated that these different cell populations are involved in BJAB tumor eradication. We propose that rituximab rapidly activates complement and induces beta-chemokines in vivo, which in turn activate the innate immunity network required for efficient eradication of the bulky BJAB tumor

Platelet-lysate-Expanded Mesenchymal Stromal Cells as a Salvage Therapy for Severe Resistant Graft-versus-Host Disease in a Pediatric Population

Despite advances in graft-versus-host-disease (GVHD) treatment, it is estimated that overall survival (OS) at 2 years for hematopoietic cell transplantation (HCT) recipients who experience steroid-resistant GVHD is 10%. Among recent therapeutic approaches for GVHD treatment, mesenchymal stromal cells (MSCs) hold a key position. We describe a multicenter experience of 11 pediatric patients diagnosed with acute or chronic GVHD (aGVHD, cGVHD) treated for compassionate use with GMP-grade unrelated HLA-disparate donors' bone marrow-derived MSCs, expanded in platelet-lysate (PL)-containing medium. Eleven patients (aged 4-15 years) received intravenous (i.v.) MSCs for aGVHD or cGVHD, which was resistant to multiple lines of immunosuppression. The median dose was 1.2 × 10 6 /kg (range: 0.7-3.7 × 10 6 /kg). No acute side effects were observed, and no late side effects were reported at a median follow-up of 8 months (range: 4-18 months). Overall response was obtained in 71.4% of patients, with complete response in 23.8% of cases. None of our patients presented GVHD progression upon MSC administration, but 4 patients presented GVHD recurrence 2 to 5 months after infusion. Two patients developed chronic limited GVHD. This study underlines the safety of PL-expanded MSC use in children. MSC efficacy seems to be greater in aGVHD than in cGVHD, even after failure of multiple lines of immunosuppression

The polo-like kinase 1 (PLK1) inhibitor NMS-P937 is effective in a new model of disseminated primary CD56+ acute monoblastic leukaemia

CD56 is expressed in 15–20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56+ monoblastic AML (M5a). The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting (MST = 28, 36, 62 days for vehicle, cytarabine and NMS-P937, respectively). Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56+ AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML