Alessandra Gambacurta

A new clinical approach: Use of blood-derived stem cells (BDSCs) for superficial digital flexor tendon injuries in horses

AIMS In this study, we present an innovative therapy using stem cells that were obtained from the peripheral blood of racehorses affected by uninduced superficial digital flexor tendon (SDFT) injuries. MAIN METHODS Blood-derived stem cells (BDSCs) were generated from the blood samples of three horses in the presence of macrophage colony-stimulating factor (M-CSF). The racehorses received a single autologous BDSC treatment, which resulted in the successful repair of the tendons injuries. KEY FINDINGS The results demonstrated that the BDSCs injection into the damaged tendon stimulated the regeneration of normal tissue. Furthermore, a relationship may exist between the speed and the quality of new tissue formation and the welfare and management of the treated animals. SIGNIFICANCE This study demonstrates that stem cell technology offers new tools for tissue repair that in many cases is considered incurable, and provides additional evidence that BDScs injections increase the speed and quality of the regeneration process in different animal tissues.

Sphingosine kinase 1 overexpression is regulated by signaling through PI3K, AKT2, and mTOR in imatinib-resistant chronic myeloid leukemia cells

OBJECTIVE As a better understanding of the molecular basis of carcinogenesis has emerged, oncogene-specific cell-signaling pathways have been successfully targeted to treat human malignances. Despite impressive advances in oncogene-directed therapeutics, genetic instability in cancer cells often manifest acquired resistance. This is particularly noted in the use of tyrosine kinase inhibitors therapies and not more evident than for chronic myeloid leukemia. Therefore, it is of great importance to understand the molecular mechanisms affecting cancer cell sensitivity and resistance to tyrosine kinase inhibitors. MATERIALS AND METHODS In this study, we used continuous exposure to stepwise increasing concentrations of imatinib (0.6-1 μM) to select imatinib-resistant K562 cells. RESULTS Expression of BCR-ABL increased both at RNA and protein levels in imatinib-resistant cell lines. Furthermore, expression levels of sphingosine kinase 1 (SphK1) were increased significantly in resistant cells, channeling sphingoid bases to the SphK1 pathway and activating sphingosine-1-phosphate-dependent tyrosine phosphorylation pathways that include the adaptor protein Crk. The partial inhibition of SphK1 activity by N,N-dimethylsphingosine or expression by small interfering RNA increased sensitivity to imatinib-induced apoptosis in resistant cells and returned BCR-ABL to baseline levels. To determine the resistance mechanism-induced SphK1 upregulation, we used pharmacological inhibitors of the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin signaling pathway and observed robust downmodulation of SphK1 expression and activity when AKT2, but not AKT1 or AKT3, was suppressed. CONCLUSIONS These results demonstrate that SphK1 is upregulated in imatinib-resistant K562 cells by a pathway contingent on a phosphoinositide 3-kinase/AKT2/mammalian target of rapamycin signaling pathway. We propose that SphK1 plays an important role in development of acquired resistance to imatinib in chronic myeloid leukemia cell lines.

IL-25 prevents and cures fulminant hepatitis in mice through a myeloid-derived suppressor cell-dependent mechanism.

Fulminant hepatitis (FH) is a disease characterized by massive destruction of hepatocytes with severe impairment of liver function. The pathogenesis of FH is not fully understood, but hyperactivity of T cells and macrophages with excessive production of cytokines are important hallmarks of the condition. In this study, we investigated the role of interleukin (IL)−25 in FH. IL‐25 expression was evaluated in patients with FH and in livers of mice with FH induced by D‐galactosamine (D‐Gal) and lipopolysaccharide (LPS). Mice were treated with IL‐25 before D‐Gal/LPS‐induced FH and before or after concanavalin A (ConA)‐induced FH. Mononuclear cells were isolated from livers of mice treated with or without IL‐25 and analyzed for GR1+CD11b+ cells. CFSE‐labeled T cells were cocultured with GR1+CD11b+ cells and their proliferation was evaluated by flow cytometry. Mice were also treated with a depleting anti‐GR1 antibody before IL‐25 and D‐Gal/LPS administration. IL‐25 was constitutively expressed in mouse and human liver and down‐regulated during FH. IL‐25 prevented D‐Gal/LPS‐induced FH and this effect was associated with increased infiltration of the liver with cells coexpressing GR1 and CD11b. In vitro studies showed that GR1+CD11b+ cells isolated from mice given IL‐25 inhibited T‐cell proliferation. Consistently, in vivo depletion of GR1+ cells abrogated the protective effect of IL‐25 in experimental D‐Gal/LPS‐induced FH. IL‐25 was both preventive and therapeutic in ConA‐induced FH. Conclusions: IL‐25 expression is markedly reduced during human and experimental FH. IL‐25 promotes liver accumulation of GR1+CD11b+cells with immunoregulatory properties. (Hepatology 2013;58:1436–1450)

Blood derived stem cells: an ameliorative therapy in veterinary ophthalmology.

Stem cell technology has evoked considerable excitement among people interested in the welfare of animals, as it has suggested the potential availability of new tools for several pathologies, including eye disease, which in many cases is considered incurable. One such example is ulcerative keratitis, which is very frequent in horses. Because some of these corneal ulcers can be very severe, progress rapidly and, therefore, can be a possible cause of vision loss, it is important to diagnose them at an early stage and administer an appropriate treatment, which can be medical, surgical, or a combination of both. The therapeutic strategy should eradicate the infection in order to reduce or stop destruction of the cornea. In addition, it should support the corneal structures and control the uveal reaction, and the pain associated with it, in order to minimize scarring. In this study, we address how stem cells derived from peripheral blood can be used also in ophthalmological pathologies. Our results demonstrate that this treatment protocol improved eye disease in four horse cases, including corneal ulcers and one case of retinal detachment. In all cases, we detected a decrease in the intense inflammatory reaction as well as the restoration of the epithelial surface of the central cornea. J. Cell. Physiol. 227: 1250–1256, 2012.

Cooperative homodimeric hemoglobin from Scapharca inaequivalvis. cDNA cloning and expression of the fully functional protein in E. coli.

The overexpression of the fully functional, cooperative homodimeric hemoglobin of the bivalve mollusc. Scapharca inaequivalvis, has been accomplished in E. coli from its cDNA. The latter was isolated by PCR amplification of total RNA and sequenced. The cDNA‐derived sequence differed by a single amino acid when compared to that previously obtained from purified protein. Interest in this hemoglobin resides in the unique assemblage of the two identical subunits, with the heme groups facing each other in the inside of the molecule, opposite to that occurring in vertebrate hemoglobins. The results presented here are the basis for future studies of structure/function relationships by site directed mutagenesis.

HERV-K activation is strictly required to sustain CD133+ melanoma cells with stemness features

BackgroundMelanoma is a heterogeneous tumor in which phenotype-switching and CD133 marker have been associated with metastasis promotion and chemotherapy resistance. CD133 positive (CD133+) subpopulation has also been suggested as putative cancer stem cell (CSC) of melanoma tumor. Human endogenous retrovirus type K (HERV-K) has been described to be aberrantly activated during melanoma progression and implicated in the etiopathogenesis of disease. Earlier, we reported that stress-induced HERV-K activation promotes cell malignant transformation and reduces the immunogenicity of melanoma cells. Herein, we investigated the correlation between HERV-K and the CD133+ melanoma cells during microenvironmental modifications.MethodsTVM-A12 cell line, isolated in our laboratory from a primary human melanoma lesion, and other commercial melanoma cell lines (G-361, WM-115, WM-266-4 and A375) were grown and maintained in the standard and stem cell media. RNA interference, Real-time PCR, flow cytometry analysis, self-renewal and migration/invasion assays were performed to characterize cell behavior and HERV-K expression.ResultsMelanoma cells, exposed to stem cell media, undergo phenotype-switching and expansion of CD133+ melanoma cells, concomitantly promoted by HERV-K activation. Notably, the sorted CD133+ subpopulation showed stemness features, characterized by higher self-renewal ability, embryonic genes expression, migration and invasion capacities compared to the parental cell line. RNA interference-mediated downregulation experiments showed that HERV-K has a decisive role to expand and maintain the CD133+ melanoma subpopulation during microenvironmental modifications. Similarly, non nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine were effective to restrain the activation of HERV-K in melanoma cells, to antagonize CD133+ subpopulation expansion and to induce selective high level apoptosis in CD133+ cells.ConclusionsHERV-K activation promotes melanoma cells phenotype-switching and is strictly required to expand and maintain the CD133+ melanoma cells with stemness features in response to microenvironmental modifications.

Scapharca inaequivalvis tetrameric hemoglobin A and B chains: cDNA sequencing and genomic organization

A and B globin cDNAs from the tetrameric hemoglobin of the bivalve molluscScapharca inaequivalvis were isolated by RT-PCR and sequenced. When compared with the biochemical data, the deduced protein sequences revealed only one amino acid substitution in the B chain. In order to investigate the genomic structure of these invertebrate globin genes, their intronic regions were amplified by PCR. The two genes showed the typical two-intron/three-exon organization found in vertebrates and seemed to reflect the ancestral gene structure, in accordance with the new globin gene evolution theory proposed by Dixon and Pohajadak (Trends Biochem. Sci. 17:486–488, 1992). The alternative hypothesis suggested by Go (Nature 291:90–92, 1981), that the central intron was lost during evolution, is also considered. In contrast to the related clamAnadara trapezia, S. inaequivalvis A and B globin genes were found to be present in multiple copies differing in intron size. In this study we report the complete sequences of the A (1,471 bp) and B (2,221 bp) globin genes, giving a detailed analysis of their intron features.

ATP regulation of the ligand-binding properties in temperate and cold-adapted haemoglobins. X-ray structure and ligand-binding kinetics in the sub-Antarctic fish Eleginops maclovinus.

The major haemoglobin of the sub-Antarctic fish Eleginops maclovinus was structurally and functionally characterised with the aim to compare molecular environmental adaptations in the O(2)-transport system of sub-Antarctic fishes of the suborder Notothenioidei with those of their high-latitude relatives. Ligand-binding kinetics of the major haemoglobin of E. maclovinus indicated strong stabilisation of the liganded quaternary T state, enhanced in the presence of the physiological allosteric effector ATP, compared to that of high-Antarctic Trematomus bernacchii. The activation enthalpy for O(2) dissociation was dramatically lower than that in T. bernacchii haemoglobin, suggesting remarkable differences in temperature sensitivity and structural changes associated with O(2) release and exit from the protein. The haemoglobin functional properties, together with the X-ray structure of the CO form at 1.49 Å resolution, the first of a temperate notothenioid, strongly support the hypothesis that in E. maclovinus, whose life-style varies according to changes in habitat, the mechanisms that regulate O(2) affinity and the ATP-induced Root effect differ from those of high-Antarctic Notothenioids.

A new T677C mutation of the aspartoacylase gene encodes for a protein with no enzymatic activity

OBJECTIVE To verify the effect of and to date the unknown T677C mutation of the human N-acetylaspartoacylase (hASPA) gene on the function of the mutated enzyme. DESIGN AND METHODS Wild type and I226T-mutated proteins were expressed and purified from a transformed Escherichia coli colony. Enzymatic activities were measured in the presence of varying substrate concentrations. RESULTS Whilst kinetic parameters of wild type hASPA were in line with data in literature, I226T-mutated hASPA showed no enzymatic activity. CONCLUSION Data indicated that this new mutation might be responsible in homozygosis for the phenotype corresponding to Canavan disease.

Blood-Derived Stem Cells (BDSCs) Plasticity: In Vitro Hepatic Differentiation

The limited availability of hepatic tissue suitable for the treatment of liver disease and drug research encourages the generation of hepatic‐like cells from alternative sources as support for the regenerative medicine. Human blood derived stem cells (BDSCs) express surface markers and genes characteristic of pluripotent stem cells and have the ability to differentiate into different cell types, including tissues of endodermal origin (i.e., liver). Therefore they can represent a valuable source of hepatocytes for medicine. In this investigation, we exploited a fast hepatic differentiation protocol to generate hepatocyte‐like cells from human BDSCs using only hepatocyte growth factor (HGF) and fibroblast growth factor‐4 (FGF‐4) as growth factors. The resulting cell population exhibited hepatic cell‐like morphology and it was characterized with a variety of biological endpoint analyses. Here, we demonstrate how human BDSCs can be reprogrammed in hepatocyte‐like cells by morphological, functional analysis, reverse transcriptase (RT)‐PCR, and Western Blot assay. This study defines a fast and easy reprogramming strategy that facilitates the differentiation of human BDSCs along a hepatic lineage and provides a framework for a helpful source in the stem cells therapy and liver disorders. J. Cell. Physiol. 228: 1249–1254, 2013.