Animal Models to Translate Phage Therapy to Human Medicine

Phagotherapy, the use of bacteriophages to fight bacterial infections as an alternative to antibiotic treatments, has become of increasing interest in the last years. This is mainly due to the diffusion of multi-drug resistant (MDR) bacterial infections that constitute a serious issue for public health. Phage therapy is gaining favor due to its success in agriculture and veterinary treatments and its extensive utilization for human therapeutic protocols in the Eastern world. In the last decades, some clinical trials and compassionate treatments have also been performed in the Western world, indicating that phage therapy is getting closer to its introduction in standard therapy protocols. However, several questions concerning the use of phages in human therapeutic treatments are still present and need to be addressed. In this review, we illustrate the state of art of phage therapy and examine the role of animal models to translate these treatments to humans

prox1 expression pattern and functional analysis in the CNS and lateral line of zebrafish embryo (Danio rerio)

Embryonic prox1 expression pattern and functional analyses in the CNS and lateral line of zebrafish (Danio rerio) Zebrafish prox1 is a divergent homeobox transcription factor whose homologues in Drosophila and mice regulate cell proliferation, fate determination and differentiation in Central Nervous System (CNS) and sensory organs. In an attempt to identify prox1 roles in zebrafish, we focused our attention on the CNS and the posterior lateral line (PLL), a fish sensorial system. By means of whole-mount in situ experiments, we evidenced prox1 expression in the posterior tuberculum (PT) a region of the basal diencephalon that houses a discrete population of nascent catecholaminergic (CA) neurons. Down-regulation of prox1 expression led to a marked decrease of the number of such CA neurons, and its over-expression resulted in PT supernumerary CA neurons. We also demonstrated that prox1 in the PT acts as a positive regulator of otp1 and finally, we searched for potential prox1 regulators affecting prox1 expression at the level of the PT. prox1 is also expressed in the migrating primordium of the PLL. Loss of function experiments showed that the gene is necessary for neuromast differentiation, while it plays little or no role in primordium migration and neuromast deposition. In order to understand prox1 role in PLL cell differentiation, we correlated the expression pattern of neurogenic ath1 and notch3 genes in wild type and prox1 knock-down embryos, concluding that prox1 could function as a mitotic suppressor in the proneural ath1 expressing cells, switching their fate from proliferative to differentiative

A peek inside the neurosecretory brain through Orthopedia lenses

The wealth of expression and functional data presented in this overview discloses the homeogene Orthopedia (Otp) as critical for the development of the hypothalamic neuroendocrine system of vertebrates. Specifically, the results depict the up-to-date portrait of the regulation and functions of Otp. The development of neuroendocrine nuclei relies on Otp from fish to mammals, as demonstrated for several peptide and hormone releasing neurons. Additionally, the activity of Otp is essential for the induction of the dopaminergic phenotype in the hypothalamus of vertebrates. Recent insights into the pathways required for Otp regulation have revealed the implication of the main extracellular signals acting during hypothalamic development. Alterations in these pathways are involved in several neuronal disorders, and the resultant downstream misregulation of Otp might impair the development of the hypothalamus, and be therefore responsible for the neuroendocrine dysfunctions that typify these diseases

Phages as immunomodulators and their promising use as anti-inflammatory agents in a cftr loss-of-function zebrafish model

Cystic Fibrosis (CF), one of the most frequent hereditary diseases due to mutations in the CFTR gene, causes mortality in humans mainly due to infection in the respiratory system. However, besides the massive inflammatory response triggered by chronic bacterial infections, a constitutive pro-inflammatory state associated with the most common CFTR mutations has been reported in paediatric cases before the onset of bacterial colonization. In previous works we isolated and characterized a mix of virulent bacteriophages (phage cocktail) able to efficiently counteract Pseudomonas aeruginosa infection in a zebrafish model with cftr loss-of-function (LOF), but also showing anti-inflammatory effects in zebrafish embryos not infected by bacteria. On these premises, in this work we demonstrated the anti-inflammatory role of the phage cocktail both in the wild-type (WT) and hyper-inflamed cftr LOF zebrafish embryos in terms of reduction of pro-inflammatory markers. We also dissect that only the virion proteinaceous components, but not the phage DNA, are responsible for the immune-modulatory effect and that this action is elicited through the activation of the Toll-like Receptor (TLR) pathway. In the cftr LOF zebrafish embryos, we demonstrated that phages injection significantly reduces neutrophil migration following acute inflammatory induction. The elucidation of the molecular interaction between phages and the cells of vertebrate immune system might open new possibility in their manipulation for therapeutic benefits especially in diseases such as cystic fibrosis, characterized by chronic infection and inflammation

Jagged blockade promotes tumor cells response to Bortezomib in a zebrafish model of multiple myeloma

Multiple myeloma (MM) is the second most frequently diagnosed hematologic tumor and remains still incurable, mainly due to the development of drug resistance that leads to relapse and contributes to the fatal outcome of this disease. MM cells accumulate in the bone marrow (BM) and establish a complex interplay with the neighboring normal cells, inducing them to assume a pro-tumor behavior. In this process, a key role is played by the two Notch ligands Jagged1 and 2, whose dysregulated expression promotes an aberrant activation of the Notch pathway both in MM cells and the normal cells of the BM milieu. Our preliminary results indicate that Jagged1 and 2 blockade reduces MM cell ability to induce Notch activation in BM stromal cells, causing a decrease in their capability to sustain MM resistance to Bortezomib. Results obtained in vitro on MM cell lines were further validated on co-cultures of primary CD138+ cells and BMSCs from newly diagnosed MM patients. The zebrafish embryo was recently validated as a complementary in vivo model for MM and allows the rapid screening of the outcome of different pharmacologic treatments. Moreover, key growth factor such as IL6 and SDF1 produced by zebrafish are biologically active also for human cells and support MM cells growth and survival. Thus, we aimed to validate our in vitro findings using a zebrafish xenograft model of MM that allows a rapid and reliable screening of the outcome of Jagged1 and 2 knockdown on MM cells response to Bortezomib. Jagged1 and 2 expression was inhibited transiently in MM cell lines using specific siRNAs. MM cells were stained with the CM-Dil vital dye, resuspended in PBS + 3% polyvinyl pyrrolidone and injected in the yolk area of 2dpf zebrafish embryos. Injected embryos were treated with 10\ub5M Bortezomib or DMSO for 48h and MM cells growth in zebrafish was evaluated by fluorescence microscopy. The analysis performed on xenotransplanted embryos showed that the treatment with 10nM Bortezomib caused a decrease of about the 50% in tumor growth in comparison to DMSO-treated controls, with no effect on embryos viability. Jagged inhibition alone has a comparable effect to Bortezomib, while the combination of Bortezomib and Jagged knockdown results in a stronger decrease in tumor growth of approximately 80% in comparison to the vehicle-controls. In conclusion, our findings demonstrate that Jagged inhibition represents a suitable strategy to increase MM cell sensitivity to Bortezomib and support the use of zebrafish embryos as an innovative and feasible tool to assess tumor cell sensitivity to standard of care drugs

Jagged1/2 inhibition promotes tumor cells response to Bortezomib in a zebrafish model of multiple myeloma

Background: Multiple myeloma (MM) is the second most frequently diagnosed hematological malignancy and today is still incurable, mainly due to the development of drug resistance that causes relapse and contributes to the fatal outcome of this disease. MM cells accumulate in the bone marrow (BM) and establish complex interactions with the surrounding normal cells, forcing them to assume a pro-tumor behavior. In this process, a key role is played by the two Notch ligands Jagged1 and 2, whose dysregulated expression causes an aberrant activation of the Notch pathway both in MM cells and in the BM niche cells. Aims: We aimed to validate the effect of Jagged1/2 silencing on MM cells resistance to the standard-of-care drug Bortezomib by: i) in vitro, on co-culture of MM cell lines and BM stromal cells (BNSC); ii) ex vivo, on co-culture of primary cells from MM patients and BMSC; iii) in vivo, using a zebrafish xenograft model of MM that allows a rapid and reliable screening of MM cells response to chemotherapics. Methods: Jagged1/2 expression was inhibited transiently in MM cell lines using specific siRNAs and constitutively in primary MM cells using a lentiviral vector that encodes specific shRNAs. Cells were cultured alone or co-cultured with BMSC and treated with 8nM Bortezomib. Apoptosis was evaluated by Annexin V staining and flow cytometry. For in vivo experiments cells were stained with the CM-Dil vital dye, resuspended in PBS + 3% polyvinyl pyrrolidone and injected in the yolk area of 2dpf (days post fertilization) zebrafish embryos. Injected embryos were treated with 10\u3bcM Bortezomib or DMSO for 48h. MM cells growth in zebrafish was evaluated by fluorescence microscopy and tumor area were calculated using ImageJ and normalized on tumor volume at the time of injection. Results: Jagged1/2 blockade reduces MM cells ability to induce Notch activation in BMSC, causing a decrease in their capacity to sustain MM resistance to Bortezomib. Results obtained in vitro on MM cells lines were further validated on co-culture of primary CD138+ cells and BMSC from newly diagnosed MM patients. The analysis performed on xenotransplanted embryos showed that the treatment with 10\u3bcM Bortezomib caused a decrease of about the 50% in tumor growth in comparison to DMSO-treated controls, with no effect on embryos viability. Jagged1/2 knockdown alone has a comparable effect to Bortezomib, while the combination of Bortezomib and Jagged1/2 inhibition results in a stronger decrease in tumor growth of about the 75% in comparison to the vehicle-controls. Summary/Conclusion: Our findings demonstrate that Jagged1/2 inhibition represents a suitable strategy to promote MM response to the standard of care drug Bortezomib, contrasting BM-induced drug resistance

Notch signaling promotes drug resistance in multiple myeloma through the regulation of the CXCR4/SDF1α axis

Multiple myeloma (MM) represents the 13% of the hematologic tumors and its social burden is increasing in western countries due to the progressive aging of the population. Nowadays, MM is still incurable due to the presentation as high-risk form or to the interactions of myeloma cells with the bone marrow (BM) milieu. Indeed, MM cells establish complex interactions with BM stromal cells (BMSCs), which provide a source of pro-survival and growth factors that promotes drug resistance, finally causing patients' relapse. In MM the Notch oncogenic pathway is dysregulated due to the overexpression of the two Notch ligands Jagged1 and Jagged2 by tumor cells. This causes Notch hyperactivation in both the neighboring MM cells and in the normal cells of the BM niche. To test how Jagged1/2 overexpression affects MM cells response to chemotherapeutics, we performed double-silencing of Jagged1/2 (J1/2KD) in myeloma cell lines (HMCLs) and assessed their response to standard-of-care drugs (i.e. bortezomib, melphalan and lenalidomide), both in single culture and in a co-culture system composed of HMCLs and the human BMSC line HS5. Our results showed that J1/2 KD promotes MM cells sensitivity to chemotherapeutics. Moreover, while HS5 are able to protect MM cells from drug-induced apoptosis, J1/2KD revert this effect, decreasing the ability of BMSCs to rescue MM cells. At molecular level, while MM cells are normally able to promote Notch activation in co-cultured BMSCs, this effect is lost after J1/2KD. These variations in Notch activation correlated with HS5 ability to produce one of the key chemokine in MM, SDF1\u3b1. This is particularly relevant since our data indicate that SDF1\u3b1 boosts the expression of the anti-apoptotic genes Bcl2, ABCC1 and Survivin in MM cells, improving their resistance to therapies. Of note, J1/2KD in MM cells causes a decrease in the expression of SDF1\u3b1 receptor, CXCR4, further potentiating the anti-tumor effect. The improvement in drug-response promoted by J1/2KD can be observed also in ex-vivo co-cultures of cells from MM patients, that further validate the key role of J1/2 in this process. Finally, an in vivo zebrafish xenograft model of MM allowed us to confirm that J1/2KD causes increase in MM cells response to bortezomib in vivo, decreasing tumor burden. These results provide the proof-of-concept for a Jagged1/2-tailored therapy in MM, that may allow to restore the response to standard drugs and to improve patient\u2019s overall survival

Notch signaling promotes bone marrow-induced drug resistance in multiple myeloma through the regulation of the CXCR4/CXCL12 system

Multiple myeloma (MM) is a diffuse hematologic tumor that is still incurable, despite the development of innovative therapies. One of the most relevant obstacles to currently available treatments is the protection from drug-induced apoptosis caused by the crosstalk of MM cells and bone marrow stromal cells (BMSCs), which provides a source of pro-survival and growth factors that promotes drug resistance, that causes patients' relapse and contribute to MM fatal outcome. The overexpression of the two Notch ligands Jag1 and Jag2 by tumor cells causes the hyperactivation of the Notch signaling both in the neighboring MM cells and in the cells of the tumor microenvironment. To test if Jag1/2 double-silencing (Jag1/2KD) in MM cells prevented BMSC-mediated drug resistance, we established a co-culture system composed of human myeloma cell lines (HMCLs) and the human BMSC line HS5, and compared the efficacy of commonly-used drugs, i.e. bortezomib, melphalan and lenalidomide between control and Jag1/2KD co-cultures

Mutational screening and functional analyses in the zebrafish model of GIGYF2 as a candidate gene for Parkinson disease

Several loci (PARK1 to 13) for familial Parkinson Disease (PD) have been described, but for some of them the specific gene remains elusive. GIGYF2 (Grb10-Interacting GYF Protein-2) has been recently proposed to be the causative gene at the PARK11 locus as a total of 10 different genetic variants were identified in this gene in 123 Italian and 126 French familial PD cases, being absent in 318 controls. Although GIGYF2 represents a good candidate gene for PD, being known to modulate the IGF-1 (insulin-like growth factor 1) signaling through its interaction with the Grb10 protein, further studies did not confirm its association with the disease. Therefore, to elucidate the role of GIGYF2 in the pathogenesis of familial and sporadic PD, we screened GIGYF2 in a large Italian population of familial and sporadic PD patients and controls. Moreover, we analyzed the function of GIGYF2 in vivo, using the zebrafish model. Mutational screening of GIGYF2 was performed by a combination of high-resolution melting analysis and direct DNA sequencing. Exons containing previously reported mutations were analyzed in 552 cases (243 familial, 309 sporadic) and 552 controls. Thereafter, a subset of 184 familial PD cases and an equal number of controls were subjected to a full screening of all 27 coding exons. This analysis identified 7 different missense variations in a total of 8 individuals (3 PD patients and 5 controls). Abrogation of gigyf2 function in zebrafish embryos by morpholino oligonucleotide injection did not lead to a drastic loss of neurons in the diencephalic dopaminergic (DA) neuron clusters, neither caused a change in the expression levels of otp1 and prox1 genes, two of the major players in diencephalic DA neurons development, suggesting that gigyf2 is not required for proper DA neuron differentiation. These data, together with those recently reported by other groups, suggest that GIGYF2 is unlikely to be the PARK11 gene