Valentina Franceschi

Hypoxia-inducible factor (HIF)-1α suppression in myeloma cells blocks tumoral growth in vivo inhibiting angiogenesis and bone destruction

Hypoxia-inducible transcription factor-1 (HIF-1α) is overexpressed in multiple myeloma (MM) cells within the hypoxic microenvironment. Herein, we explored the effect of persistent HIF-1α inhibition by a lentivirus short hairpin RNA pool on MM cell growth either in vitro or in vivo and on the transcriptional and pro-angiogenic profiles of MM cells. HIF-1α suppression did not have a significant impact on MM cell proliferation and survival in vitro although, increased the antiproliferative effect of lenalidomide. On the other hand, we found that HIF-1α inhibition in MM cells downregulates the pro-angiogenic genes VEGF, IL8, IL10, CCL2, CCL5 and MMP9. Pro-osteoclastogenic cytokines were also inhibited, such as IL-7 and CCL3/MIP-1α. The effect of HIF-1α inhibition was assessed in vivo in nonobese diabetic/severe combined immunodeficiency mice both in a subcutaneous and an intratibial MM model. HIF-1α inhibition caused a dramatic reduction in the weight and volume of the tumor burden in both mouse models. Moreover, a significant reduction of the number of vessels and vascular endothelial growth factors (VEGFs) immunostaining was observed. Finally, in the intratibial experiments, HIF-1α inhibition significantly blocked bone destruction. Overall, our data indicate that HIF-1α suppression in MM cells significantly blocks MM-induced angiogenesis and reduces MM tumor burden and bone destruction in vivo, supporting HIF-1α as a potential therapeutic target in MM.

Lower Rim Guanidinocalix[4]arenes: Macrocyclic Nonviral Vectors for Cell Transfection

Guanidinium groups were introduced through a spacer at the lower rim of calix[4]arenes in the cone conformation to give new potential nonviral vectors for gene delivery. Several structural modifications were explored, such as the presence or absence of a macrocyclic scaffold, lipophilicity of the backbone, length of the spacer, and nature of the charged groups, in order to better understand the factors which affect the DNA condensation ability and transfection efficiency of these derivatives. The most interesting compound was a calix[4]arene unsubstituted at the upper rim and having four guanidinium groups linked at the lower rim through a three carbon atom spacer. This compound, when formulated with DOPE, showed low toxicity and transfection efficiency higher than the commercially available lipofectamine LTX in the treatment of human Rhabdomiosarcoma and Vero cells. Most of the investigated compounds showed a tendency to self-aggregate in pure water or in the presence of salts, as evidenced by NMR and AFM studies, and it was found that the ability to condense DNA plasmids in nanometric globules is a necessary but not sufficient condition for transfection. The superiority of macrocyclic vectors over linear Gemini-type analogues and of guanidinium compared to other ammonium head groups in determining the biological activity of the vectors was also ascertained.

HOXB7 expression by myeloma cells regulates their pro-angiogenic properties in multiple myeloma patients

The deregulation of the homeobox genes as homeoboxB (HOXB)-7 has been previously associated to tumor progression and angiogenesis; here we investigated the potential role of HOXB7 in the pro-angiogenic properties of multiple myeloma (MM) cells. We found that HOXB7 was expressed in 10 out of 22 MM patients analyzed at the diagnosis related to high bone marrow angiogenesis and overexpressed in about 40% of myeloma cell lines compared with normal plasma cells. Enforced HOXB7 expression in MM cells by a lentiviral vector significantly modified their transcriptional and angiogenic profile, checked by combined microarray and angiogenesis PCR analyses, upregulating VEGFA, FGF2, MMP2, WNT5a and PDGFA and downregulating thrombospoindin-2. The pro- and anti-angiogenic HOXB7-related gene signature was also validated in a large independent dataset of MM patients. Accordingly, MM-induced vessel formation was significantly increased by HOXB7 overexpression both in vitro angiogenic and chorioallantoic membrane assays, as well as the HOXB7 silencing by small interfering RNA inhibited the production of angiogenic factors, and the pro-angiogenic properties of MM cells. Finally, in SCID-NOD mice we confirmed that HOXB7 overexpression by MM cells stimulated tumor growth, increased MM-associated angiogenesis and the expression of pro-angiogenic genes by microarray analysis supporting the critical role of HOXB7 in the angiogenic switch in MM.

Immunization of knock-out α/β interferon receptor mice against lethal bluetongue infection with a BoHV-4-based vector expressing BTV-8 VP2 antigen.

New effective tools for vaccine strategies are necessary to limit the spread of bluetongue, an insect-transmitted viral disease of domestic and wild ruminants. In the present study, BoHV-4-based vector cloned as a bacterial artificial chromosome (BAC) was engineered to express the bluetongue virus (BTV) immune-dominant glycoprotein VP2 provided of a heterologous signal peptide to its amino terminal and a trans-membrane domain to its carboxyl terminal (IgK-VP2gDtm), to allow the VP2 expression targeting to the cell membrane fraction. Based on adult α/β interferon receptor knockout (IFNAR(-/-)) mice, a newly generated bluetongue laboratory animal model, a pre-challenge experiment was performed to test BoHV-4 safety on such immune-compromised animal. BoHV-4 infected IFNAR(-/-) mice did not show clinical signs even following the inoculation of BoHV-4 intra-cerebrally, although many areas of the brain got transduced. IFNAR(-/-) mice intraperitoneally inoculated twice with BoHV-4-A-IgK-VP2gDtm at different time points developed serum neutralizing antibodies against BTV and showed a strongly reduced viremia and a longer survival time when challenged with a lethal dose of BTV-8. The data acquired in this pilot study validate BoHV-4-based vector as a safe and effective heterologous antigen carrier/producer for the formulation of enhanced recombinant immunogens for the vaccination against lethal bluetongue.

Bovine endometrial stromal cells display osteogenic properties

The endometrium is central to mammalian fertility. The endometrial stromal cells are very dynamic, growing and differentiating throughout the estrous cycle and pregnancy. In humans, stromal cells appear to have progenitor or stem cell capabilities and the cells can even differentiate into bone. It is not clear whether bovine endometrial stromal cells exhibit a similar phenotypic plasticity. So, the present study tested the hypothesis that bovine endometrial stromal cells could be differentiated along an osteogenic lineage. Pure populations of bovine stromal cells were isolated from the endometrium. The endometrial stromal cell phenotype was confirmed by morphology, prostaglandin secretion, and susceptibility to viral infection. However, cultivation of the cells in standard endometrial cell culture medium lead to a mesenchymal phenotype similar to that of bovine bone marrow cells. Furthermore, the endometrial stromal cells developed signs of osteogenesis, such as alizarin positive nodules. When the stromal cells were cultured in a specific osteogenic medium the cells rapidly developed the characteristics of mineralized bone. In conclusion, the present study has identified that stromal cells from the bovine endometrium show a capability for phenotype plasticity similar to mesenchymal progenitor cells. These observations pave the way for further investigation of the mechanisms of stroma cell differentiation in the bovine reproductive tract.

Double immunization strategy with a BoHV-4-vectorialized secreted chimeric peptide BVDV-E2/BoHV-1-gD.

A bovine herpesvirus 4 was isolated from the milk cell fraction of a healthy cow and his full genome cloned as a bacterial artificial chromosome. So cloned viral genome was used as a vector platform to deliver in vitro and in vivo an optimized secreted chimeric peptide obtained by the fusion of the bovine viral diarrhoea virus glycoprotein E2 ectodomain with the bovine herpesvirus 1 glycoprotein D ectodomain. Recombinant virus infected cells robustly expressed and secreted the chimeric peptide into the culture medium and inoculated animals with the recombinant virus successfully responded toward antigens, gE2 and gD. Thus, this work has implications for the development of safe and effective polyvalent vaccines.

The Chemokine IL8 Is Up-Regulated in Bovine Endometrial Stromal Cells by the BoHV-4 IE2 Gene Product, ORF50/Rta: A Step Ahead Toward a Mechanism for BoHV-4 Induced Endometritis

Postpartum infections of the endometrium and metritis are common causes of delayed conception and infertility in cattle. These infections are characterized by inflammation of the endometrium and secretion of the chemokine interleukin 8 (IL8), which attracts granulocytes to the endometrium. Bovine herpesvirus 4 (BoHV-4) is tropic for the endometrium and the only virus consistently associated with postpartum metritis. The BoHV-4 Immediate Early 2 (IE2) gene is the first viral gene transcribed by host cells after infection, and the IE2 gene product, ORF50/Rta, transactivates host cell genes. The present study tested the hypothesis that ORF50/Rta transactivates the IL8 gene promoter during BoHV-4 infection of bovine endometrial stromal cells (BESCs). Infection of primary BESCs with BoHV-4 stimulated IL8 gene promoter activity and IL8 protein secretion. However, IL8 production was dependent on the transcription of viral genes, because psoralen/ultraviolet cross-linking of the viral DNA abrogated the response to BoHV-4 infection. Furthermore, IL8 promoter serial deletion analysis revealed a specific region responsive to ORF50/Rta. These observations may represent an endometrial defense mechanism against viral infection or a virulence mechanism by which viral replication stimulates chemokine secretion to attract more susceptible host cells to the endometrium.

Isolation and characterization of bovine herpesvirus 4 (BoHV-4) from a cow affected by post partum metritis and cloning of the genome as a bacterial artificial chromosome.

BackgroundBovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus with a Worldwide distribution in cattle and is often isolated from the uterus of animals with postpartum metritis or pelvic inflammatory disease. Virus strain adaptation to an organ, tissue or cell type is an important issue for the pathogenesis of disease. To explore the mechanistic role of viral strain variation for uterine disease, the present study aimed to develop a tool enabling precise genetic discrimination between strains of BoHV-4 and to easily manipulate the viral genome.MethodsA strain of BoHV-4 was isolated from the uterus of a persistently infected cow and designated BoHV-4-U. The authenticity of the isolate was confirmed by RFLP-PCR and sequencing using the TK and IE2 loci as genetic marker regions for the BoHV-4 genome. The isolated genome was cloned as a Bacterial Artificial Chromosome (BAC) and manipulated through recombineering technologyResultsThe BoHV-4-U genome was successfully cloned as a BAC, and the stability of the pBAC-BoHV-4-U clone was confirmed over twenty passages, with viral growth similar to the wild type virus. The feasibility of using BoHV-4-U for mutagenesis was demonstrated using the BAC recombineering system.ConclusionThe analysis of genome strain variation is a key method for investigating genes associated with disease. A resource for dissection of the interactions between BoHV-4 and host endometrial cells was generated by cloning the genome of BoHV-4 as a BAC.

Cyclodextrin- and calixarene-based polycationic amphiphiles as gene delivery systems: a structure–activity relationship study

Multi-head/multi-tail facial amphiphiles built on cyclodextrin (CD) and calixarene (CA) scaffolds are paradigmatic examples of monodisperse gene delivery systems. The possibility to precisely control the architectural features at the molecular level offers unprecedented opportunities for conducting structure-activity relationship studies. A major requirement for those channels is the design of a sufficiently diverse ensemble of compounds for parallel evaluation of their capabilities to condense DNA into transfection nanoparticles where the gene material is protected from the environment. Here we have undertaken the preparation of an oriented library of β-cyclodextrin (βCD) and calix[4]arene (CA4) vectors with facial amphiphilic character designed to ascertain the effect of the cationic head nature (aminothiourea-, arginine- or guanidine-type groups) and the macrocyclic platform on the abilities to complex plasmid DNA (pDNA) and in the efficiency of the resulting nanocomplexes to transfect cells in vitro. The hydrophobic domain, formed by hexanoyl or hexyl chains, remains constant in each series, matching the overall structure found to be optimal in previous studies. DLS, TEM and AFM data support that all the compounds self-assemble in the presence of pDNA through a process that involves initially electrostatic interactions followed by formation of βCD or CA4 bilayers between the oligonucleotide filaments. Spherical transfectious nanoparticles that are monomolecular in DNA are thus obtained. Evaluation in epithelial COS-7 and human rhabdomyosarcoma RD-4 cells evidenced the importance of having primary amino groups in the vector to warrant high levels of transfection, probably because of their buffering capacity. The results indicate that the optimal cationic head depends on the macrocyclic core, aminothiourea groups being preferred in the βCD series and arginine groups in the CA4 series. Whereas the transfection efficiency relationships remain essentially unchanged within each series, irrespective of the cell type, the optimal platform (βD or CA4) strongly depends on the cell type. The results illustrate the potential of monodisperse vector prototypes and diversity-oriented strategies on identifying the optimal candidates for gene therapy applications.

Bovine Endometrial Stromal Cells Support Tumor Necrosis Factor Alpha-Induced Bovine Herpesvirus Type 4 Enhanced Replication

ABSTRACT Bovine uterine infections are the most important cause of economic losses in the cattle industry. Although the etiology of uterine diseases is mainly ascribed to bacterial infection, they can also be associated with viral infection, such as bovine herpesvirus 4 (BoHV-4), which is often a secondary agent following bacteria. Besides microbial infection, many inflammatory molecules belonging to the innate immune response orchestrate the outcome of the infection. In the present study, the interaction between BoHV-4-infected bovine endometrial stromal cells and tumor necrosis factor alpha (TNF-alpha) was investigated. Bovine herpesvirus 4 possesses a special tropism toward endometrial stromal cells. For this reason, a simian virus 40 (SV40) immortalized endometrial stromal cell line (SV40BESC) was established; it was proven that it was stable, it expressed toll-like receptors (TLRs; from 1 to 10) and TNF-alpha receptors I and II, and it was responsive to exogenous TNF-alpha. Further, an increase of BoHV-4 replication and cytopathic effect was observed in BoHV-4-infected and TNF-alpha-treated SV40BESCs. This increase of viral replication was associated with BoHV-4 immediate early 2 (IE2) gene promoter trans-activation through the interaction of the nuclear factor KB (NFKB) with the putative NFKB-responsive elements found within BoHV-4 IE2 gene promoter, and this interaction was abolished when NFKB-responsive elements were deleted. These data shed light on two important and rather controversial issues: the role of TNF-alpha receptor, which is weakly expressed in the stromal layer of the bovine uterus, as well as the possible interactions between proinflammatory molecules, viral replication, and chronic uterine disease.