Treatment of pseudoarthrosis of the upper limb using expanded mesenchymal stem cells: a pilot study.

BACKGROUND: In orthopedic field is growing interest in the use of stem cells: this mesenchymal multipotent line (MSCs) can lead to differentiation into osteocytes and thus the formation of bone tissue. In literature applications of this line are described in injuries of tendons and ligaments, small bony avulsions, nonunion fractures and cartilage defects. AIM: Utilize MSCs expanded in laboratory in case of atrophic pseudoarthrosis of the upper limb. MATERIALS AND METHODS: We obtain the amount of cell necessary for the implant by the collaboration with the UO Haematological Department. For the procedure we make a blood sample from the iliac crest bone marrow and a subsequent phase of selection and cultivation of mesenchymal line for 3 weeks, to get a sufficient amount of tissue to be used, which is presented at the time of surgery on a scaffold made by autologous plasma gel and CaCl(2). We reassessed our experience in 8 different types of upper limb fractures result in pseudarthrosis and delayed of consolidation: 4 women and 4 men, average 44 years old followed with a follow-up of 50.3 months. In all cases the site of non-union has been revitalized (by microfractures and drilling) and a synthesis was performed with a rigid plate. So we fill the bone gap with autologous bone and mesenchymal stem cells expanded in the laboratory. RESULTS: We have a radiographic healing in 8 cases and no adverse events were highlighted. CONCLUSIONS: Using this cells line we obtained encouraging but certainly not conclusive impressions, according to the limited number of cases and lack of adequate comparative studies. In tissue engineering are also certainly needed further investigations and developments.

Mesenchymal cells inhibit expansion but not cytotoxicity exerted by gamma-delta T cells.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) exert a relevant immunosuppressive activity by inhibiting T- and B-lymphocytes, natural killer (NK) cells and dendritic cell expansion. Nevertheless, a possible activity on gamma/delta T cells has still not been evaluated. Gamma-delta T lymphocytes play an important role in the control of cancer and they have been shown to be implicated in graft-vs.-host disease. Thus, modulation of activation and proliferation of these cells could be relevant for therapeutic purposes. MATERIALS AND METHODS: Peripheral blood mononuclear cells from 21 healthy donors were used as source for gamma-delta T cells, expanded in presence of 10 IU mL(-1) interleukin-2 (IL-2) and 1 microM zoledronate. MSCs were recovered from patients undergoing routine total hip replacement surgery, and characterised by flow cytometry. Cytotoxicity on multiple myeloma and melanoma cell lines was assessed by measuring dilution of the carboxyfluorescein diacetate succinimydylester dye (CFSE). Gamma-delta T cells were then incubated with MSCs in contact cultures, and with addition of MSC-conditioned medium. RESULTS: In this article we confirmed that (1) in vitro expanded gamma-delta T cells play a significant anti-proliferative effect on multiple myeloma and melanoma cells and (2) multipotent mesenchymal stromal cells effectively suppress the ex vivo expansion of T cells carrying a specific T-cell receptor gene (TCR) rearrangement, Vgamma9/Vdelta2, induced by the combination of IL-2 and zoledronate, without interfering with their cytotoxic activity. DISCUSSION: These findings contribute to explain the activity of ex vivo expanded mesenchymal cells, suggesting that MSCs would interact with gamma-delta T lymphocytes. CONCLUSION: This effect could be relevant in separating graft-vs.-host from the graft-vs.-tumour effect, especially considering the possibility of modulating T-lymphocytes activity by the immunomodulating drugs now available.

Embedding methods for poly(L-lactic acid) microfiber mesh/human mesenchymal stem cell constructs.

Fiber mesh scaffolds were recently investigated in tissue engineering as possible support for stem cell growth and differentiation, in order to repair lesion areas in clinical practice. In particular, the literature is focused on fiber mesh scaffolds constituted of biocompatible and resorbable polymeric structures, like poly(L-lactic acid) (PLLA). However, as regards the study of constructs constituted of PLLA microfibers and cells, only quantitative and SEM analyses were reported, lacking histological analysis. Histological evaluation of these constructs could give important information about cellular distribution in the scaffold, cell-scaffold interactions and extracellular matrix production. The purpose of our study was to find a valid method to analyze PLLA microfiber/cell constructs from both histological and histochemical angles. Biodegradable non-woven fiber meshes were prepared using hollow microfibers, based on PLLA. We first evaluated different embedding methods useable for histological analysis and the results showed that among the paraffin, Killik, and acrylic resin the only suitable medium was the latter. Then we employed the acrylic resin to embed the constructs made up of PLLA microfibers and bone marrow-derived human mesenchymal stromal cells, which we then analyzed with Toluidine Blue, PAS and Alcian Blue staining. These constructs, previously analyzed for cell viability by MTT and CCK-8 tests, showed viable/proliferating cells until 6 weeks of culture. The stainings performed on constructs confirmed viability data obtained with SEM and MTT/CCK-8 and supplied other information on the cell behaviors such as the distribution and organization onto the scaffold and the production of extracellular matrix molecules. In conclusion, this methodological study mainly suggests a suitable method to analyze PLLA microfiber/cell constructs, at the same time confirming and enriching the literature data on the compatibility between PLLA microfibers and hMSCs.

PS-341 (Bortezomib) inhibits proliferation and induces apoptosis of megakaryoblastic MO7-e cells.

PS-341 (Bortezomib) is a dipeptide boronic acid proteasome inhibitor with antitumor activity that induces apoptosis in different human cancer cell lines. We investigated effects of PS-341 (Bortezomib) on cell proliferation, cell cycle progression, induction of apoptosis and differentiation in a megakaryoblastic (MO7-e) cell line. PS-341 was able to retain NF-kappaB in the cytoplasm and inhibit cell growth (IC(50)=22.5 nM), in a dose/time-dependent way. This anti-proliferative activity resulted to be lineage-specific, because other leukemic cell lines (KG1a, K562/R7, HL60/DNR) were unaffected by the PS-341 treatment. Moreover, PS-341 in MO7-e induced a significant pro-apoptotic effect from 10 nM concentration (40% versus 12% in the control, p<0.05). On the other hand, at lower concentration (5 nM), Bortezomib blocked cell cycle in the G2 phase. Finally, this compound was able to down-regulate WT1 expression. No significant effects on cell differentiation were found. Because a spontaneous NF-kappaB activation has been reported in megakaryocytes from patients affected by myeloproliferative disorders, Bortezomib would so be an attractive therapeutic tool for these malignancies, including essential thrombocythemia or idiopathic myelofibrosis. Preliminary data show an inhibiting activity of Bortezomib in the megakaryocytic colonies formation. Finally, also down-regulation of the WT1 gene Bortezomib-driven could be relevant, because of the role that this gene would play in the pathogenesis of acute and chronic myeloproliferative disorders.

Cell clonality in hypereosinophilic syndrome: what pathogenetic role?

OBJECTIVE: Idiopathic hypereosinophilic syndrome (HES) is a heterogeneous disorder, including either a myeloproliferative or a lymphoproliferative variant (l-HES). In l-HES, T-lymphocytes could be involved in the pathogenesis through several cytokines, including IL5. METHODS: We assayed both TCR Beta- and delta-rearrangements by fluorescent PCR, characterizing 14 patients affected by HES. Lyn activation (a src-kinase involved in the IL5 pathway) was also tested in 6 cases. RESULTS: FIP1L1-PDGFRa was detected in 4 cases (28.6%); a clonal TCR was found in 10 cases (71.4%), including cases FIP1L1-PDGFRalpha-positive; four cases did not show any molecular marker. In this series, levels of IL5, IL4, IL2 and gammaIFN were measured, without any significant difference among different subgroups. All pathological samples tested did not show Lyn activation. Immunophenotype was also characterized: only one case showed an atypical CD3-/CD4+ population in the bone marrow. CONCLUSION: This study would suggest that a real distinction between m- and l-HES is not wholly convincing and that clonal T-cell expansion could not be the "primum movens" but an epiphenomenon in HES.

Synergistic effects of alpha interferon and 1,25 dihydroxyvitamin D3: preliminary evidence suggesting that interferon induces expression of the vitamin receptor.

BACKGROUND: To active metabolite of vitamin D3-1,25(OH)2D3-is a well-known differentiation inducer. The addition of this metabolite to sensitive cell cultures inhibits proliferation and induces monocytic-macrophagic differentiation. Alpha interferon may also inhibit proliferation and increase the expression of some surface antigens in some neoplastic cells. In the present report, we describe the synergistic activity of these two drugs on U-937 and on cultured cells from a leukemic patient. METHODS: Proliferation was studied by 3H-thymidine incorporation; differentiation markers were evaluated immunologically by monoclonal antibodies and by cytochemical tests. Phagocytosis and NBT reduction test were also performed in order to confirm the differentiating properties of these drugs. Finally, the expression of the 1,25(OH)2D3 receptor was evaluated by immunochemical methods. RESULTS: After culturing these cells for 72 hours in the presence of 1,25(OH)2D3, cell proliferation was reduced and the expression of some phenotypic and functional markers suggested monocytic-macrophagic differentiation. Alpha interferon and 1,25(OH)2D3 synergistically inhibit the proliferation of U-937 cells. Alpha interferon increased the expression of the 1,25(OH)2D3 receptor in U-937 cells. CONCLUSIONS: The reported results confirm the synergistic activity of INF and 1,25(OH)2D3 on cell proliferation in monoblastic cells. The possible role of the increased expression of the vitamin receptor in cells cultured in the presence of INF is discussed.

Excretion of aminoglycosides in a rat kidney model.

Dactimicin is a new aminoglycoside antibiotic with an interesting low nephrotoxicity in animal experimental models. In order to establish if a correlation exists between the nephrotoxic effect and renal pharmacokinetic behaviour of aminoglycosides, tobramycin, sisomicin and dactimicin were studied in a model of isolated and perfused rat kidney. Concentrations in venous and urinary effluent during a continuous perfusion of a constant concentration of drugs were evaluated. The influence of active transport was studied comparing excretion data obtained with perfusion buffer with or without glucose. In the present experimental model the renal excretions of the aminoglycosides tested are quite similar and cannot account for the different nephrotoxicity observed in animals.

Experimental studies on diacerhein: effects on the phagocytosis of neutrophil cells from subcutaneous carrageenan-induced exudate.

Diacerhein (DAR), a new drug which is particularly suitable for the treatment of osteoarthritis, was studied for its interference with the phagocytic capacity of cells coming from exudates of subcutaneous carrageenan oedema and from the peripheral blood of Sprague-Dawley rats. DAR was found to inhibit phagocytosis in both types of cells examined. This finding indicates that DAR may exert its action by means of a direct effect on the cells involved in the inflammatory process.