Search

Glioblastoma multiforme (GBM), a malignant tumor of the central nervous system, has a high mortality rate. No curative treatment is presently available, and the most commonly used chemotherapeutic drug, the alkylating agent temozolomide (TMZ), is only able to increase life expectancy and is often associated with drug resistance. Therefore, an urgent need does exist for novel drugs aimed at treating gliomas. In the present study, we obtained three major results using corilagin: (a) demonstrated that it inhibits the growth of U251 glioma cells through activation of the apoptotic pathway; (b) demonstrated that it is also active on TMZ-resistant T98G glioma cells; and (c) demonstrated that when used in combination with TMZ on T98G glioma cells, a higher level of proapototic and antiproliferative effects is observed. Our study indicates that corilagin should be investigated in more detail to determine whether it can be developed as a potential therapeutic agent. In addition, our results suggest that corilagin could be used in combination with low doses of other standard anticancer chemotherapeutic drugs against gliomas (such as TMZ) with the aim of obtaining enhanced anticancer effects.

Introduction: β-thalassemia is caused by autosomal mutations in the β-globin gene, which induce the absence or low-level synthesis of β-globin in erythroid cells. It is widely accepted that a high production of fetal hemoglobin (HbF) is beneficial for patients with β-thalassemia. Sirolimus, also known as rapamycin, is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopicusthat serves as a strong HbF inducer in vitroand in vivo. In this study, we report biochemical, molecular, and clinical results of a sirolimus-based NCT03877809 clinical trial (a personalized medicine approach for β-thalassemia transfusion-dependent patients: testing sirolimus in a first pilot clinical trial, Sirthalaclin).Methods: Accumulation of γ-globin mRNA was analyzed using reverse-transcription quantitative polymerase chain reaction (PCR), while the hemoglobin pattern was analyzed using high-performance liquid chromatography (HPLC). The immunophenotype was analyzed using a fluorescence-activated cell sorter (FACS), with antibodies against CD3, CD4, CD8, CD14, CD19, CD25 (for analysis of peripheral blood mononuclear cells), or CD71 and CD235a (for analysis of in vitrocultured erythroid precursors).Results: The results were obtained in eight patients with the β+/β+and β+/β0genotypes, who were treated with a starting dosage of 1 mg/day sirolimus for 24–48 weeks. The first finding of this study was that the expression of γ-globin mRNA increased in the blood and erythroid precursor cells isolated from β-thalassemia patients treated with low-dose sirolimus. This trial also led to the important finding that sirolimus influences erythropoiesis and reduces biochemical markers associated with ineffective erythropoiesis (excess free α-globin chains, bilirubin, soluble transferrin receptor, and ferritin). A decrease in the transfusion demand index was observed in most (7/8) of the patients. The drug was well tolerated, with minor effects on the immunophenotype, and an only side effect of frequently occurring stomatitis.Conclusion: The data obtained indicate that low doses of sirolimus modify hematopoiesis and induce increased expression of γ-globin genes in a subset of patients with β-thalassemia. Further clinical trials are warranted, possibly including testing of the drug in patients with less severe forms of the disease and exploring combination therapies.

Thermolysin (TLN) and other thermolysin-like zinc metalloproteinases (TLPs),are important virulence factors for pathogenesis of bacterial infections by suppressing the innate immune system of the host. Therapeutic inhibition ofTLPs is believed to be a novel strategy inthe development of a new generation antibiotics.In the present study inhibition of TLN and angiotensin I-converting enzyme (ACE) by small peptides were studied by in vitro binding assays and theoretical calculations. The capacity of the peptides to inhibitTLN induced cleavage ofthe transcription factor nuclear factor kappa beta (NF-B) was studied by electrophoretic mobility shift assays (EMSAs).Nine peptides inhibited ACE with IC50 values in the range 0.48 (IVY) to 1408 (HF) M, while seven inhibited TLN with IC50 values in the range 0.00034 (IY) to 95640 (FW) M. Calculations indicated that the peptides occupied the S1 and S2 subsites of ACE, and that IY, LW and IW occupiedthe S1 and S2 subsites, while FW, WL and WV occupiedthe S1 and S1 subsites of TLN. EMSA showed that peptides inhibited TLN induced cleavage of NF-B. The studied peptides may form as a basis for the design of new compoundstargeting TLN with a potential in the treatment of bacterial infections.

One of the clinical features of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NF-kappaB interferes with the NF-kappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3–1 cells infected with Pseudomonas aeruginosaleads to a decrease occupancy of the Il-8 gene promoter by NF-kappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NF-kappaB decoy chimera on accumulation of pro-inflammatory mRNAs in P.aeruginosainfected IB3–1 cells reproduce that of decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis.

In this paper we applied the “macromolecular docking” procedure to perform molecular modeling with the aim of screening transcription factor sequences for possible interaction to the HIV-1 TAR-RNA, employing the software Hex version 4.2. The molecular modeling data were compared with electrophoretic mobility shift assays (EMSA) and surface plasmon resonance (SPR) based biospecific interaction analysis (BIA) using an optical biosensor. Finally the specific interactions between NF-κB and RNA have been calculated utilizing the AMBER-MM and FMO calculations. The results obtained clearly indicate that (a) NF-kB p50 transcription factor can bind TAR-RNA; (b) this binding efficiency is lower than that displayed by NF-kB factor in respect to DNA sequences; (c) other structured RNAs used as controls do not bind to NF-kB; (d) TAR-RNA is capable to bind pre-formed NF-kB/DNA complexes. Despite the fact that our data do not indicate whether NF-kB/TAR-RNA complexes play a role in the early steps of HIV-1 transcriptional activation, the results presented strongly indicate that interactions between transcription factors recruited at the level of HIV-1 LTR might interact with the TAR-RNA and deserve further studies aimed to determine its role in the HIV-1 life cycle.

Peptide nucleic acids (PNAs) are oligonucleotide mimics in which the sugar‐phosphate backbone has been replaced by a pseudo‐peptide backbone. Among PNA‐based molecules, PNA‐DNA conjugates characterized by tracts of DNA bound to N and/or C terminus of PNA are very soluble in aqueous media, are able to recognize exclusively single strands of DNA and RNA in antiparallel fashion, activate RNAse H, bind to transcription factors and are more stable than DNA to nucleases degradation. Very little information is available on chimeras constituted of alternating monomers of PNA and DNA. In this article, we describe a simple fully automated strategy for the synthesis of 6‐mer and 10‐mer alternate PNA‐DNA chimeras consisting of polythymine oligomers, stability assays in fetal calf serum, UV and CD studies of the single strand alternate chimeras and of alternate chimera/DNA and alternate chimera/RNA duplexes. Evidences supporting the formation of duplex hybrids were found. Furthermore, the ability of forming Hoogsteen base pairing with duplex DNA was investigated. Finally, we tested the ability of the PNA‐DNA alternates in (a) interfering with reverse transcription of eukaryotic mRNA and (b) inhibiting DNA‐protein interactions. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 815–822, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

A new series of compounds in which the 2-amino-5-chlorophenyl ring of phenstatin analogue 7 was replaced with a 2-amino-5-aryl thiophene was synthesized and evaluated for antiproliferative activity and for inhibition of tubulin polymerization and colchicine binding to tubulin. 2-Amino-3-(3‘,4‘/V‘-trimethoxybenzoyl)-5-phenyl thiophene (9f) as well as the p-fluoro-, p-methyl-, and p-methoxyphenyl substituted analogues (8H, j, and l, respectively) displayed high antiproliferative activities with IC50 values from 2.5 to 6.5 nM against the L1210 and K562 cell lines. Compounds 8H and j were more active than combretastatin A-4 as inhibitors of tubulin polymerization. Molecular docking simulations to the colchicine site of tubulin were performed to determine the possible binding mode of 8H. The results obtained demonstrated that antiproliferative activity correlated well with the inhibition of tubulin polymerization and the lengthening of the G2/M phase of the cell cycle. Moreover, a good correlation was found between these inhibitory effects and the induction of apoptosis in cells treated with the compounds.

The DNA binding properties and the effects on protein/DNA interactions of a series of four hybrids prepared combining oligopyrrole minor groove binders and pyrrolo [2,1-c][1,4] benzodiazepine (PBD) were studied. In addition, the effects on in vitro and ex vivo transcription directed by the long terminal repeat (LTR) of the human immunodeficiency type 1 virus (HIV-1) were analysed and structure-activity relationships developed. These hybrids (compounds 2–5) contain from one to four pyrrole units, respectively. To investigate sequence-selectivity and stability of drugs/DNA complexes, DNase I foothyphen-printing, filter binding, and arrested polymerase-chain reaction (PCR) were performed on HIV-1 LTR gene sequences. The antiproliferative activity of the hybrids was tested in vitro on T-lymphoid Jurkat cell lines and compared to antiproliferative effects of the natural product distamycin A 1 and pyrrolo [2,1-c][1,4] benzodiazepine (PBD 6). The results obtained demonstrate that hybrids 2–5 exhibit different DNA-binding activity with respect to both distamycin A 1 and PBD 6. A direct relationship was found between number of pyrrole rings present in the hybrids 2–5 and stability of drugs/DNA complexes. With respect to antiproliferative effects, it was found that the increase in the length of the polypyrrole backbone leads to an increase of in vitro antiproliferative effects, i.e., the hybrid 5, containing the four pyrroles distamycin analogs, is more active than 2, 3 and 4 against the Jurkat cell lines. With respect to inhibition of HIV-1 LTR driven transcription, it was found that the hybrid 5 containing the four pyrroles distamycin analogs, is more active than 2, 3 and 4. Interestingly, the present results suggest that the hybrid 3 exhibits low antiproliferative activity on Jurkat cell lines, being still active in inhibiting HIV-1 LTR driven transcription both in vitro and ex vivo. Drug Dev. Res. 60:173–185, 2003. © 2003 Wiley-Liss, Inc.

The DNA binding properties and the effects on protein/DNA interactions of a series of four hybrids prepared combining oligopyrrole minor groove binders and pyrrolo [2,1‐c][1,4] benzodiazepine (PBD) were studied. In addition, the effects on in vitro and ex vivo transcription directed by the long terminal repeat (LTR) of the human immunodeficiency type 1 virus (HIV‐1) were analysed and structure‐activity relationships developed. These hybrids (compounds 2–5) contain from one to four pyrrole units, respectively. To investigate sequence‐selectivity and stability of drugs/DNA complexes, DNase I foothyphen‐printing, filter binding, and arrested polymerase‐chain reaction (PCR) were performed on HIV‐1 LTR gene sequences. The antiproliferative activity of the hybrids was tested in vitro on T‐lymphoid Jurkat cell lines and compared to antiproliferative effects of the natural product distamycin A 1and pyrrolo [2,1‐c][1,4] benzodiazepine (PBD 6). The results obtained demonstrate that hybrids 2–5exhibit different DNA‐binding activity with respect to both distamycin A 1and PBD 6. A direct relationship was found between number of pyrrole rings present in the hybrids 2–5and stability of drugs/DNA complexes. With respect to antiproliferative effects, it was found that the increase in the length of the polypyrrole backbone leads to an increase of in vitro antiproliferative effects, i.e., the hybrid 5, containing the four pyrroles distamycin analogs, is more active than 2, 3and 4against the Jurkat cell lines. With respect to inhibition of HIV‐1 LTR driven transcription, it was found that the hybrid 5containing the four pyrroles distamycin analogs, is more active than 2, 3and 4. Interestingly, the present results suggest that the hybrid 3exhibits low antiproliferative activity on Jurkat cell lines, being still active in inhibiting HIV‐1 LTR driven transcription both in vitro and ex vivo. Drug Dev. Res. 60:173–185, 2003. © 2003 Wiley‐Liss, Inc.

We report in this paper that the DNA-binding drug mithramycin is a potent inducer of γ-globin mRNA accumulation and fetal hemoglobin (HbF) production in erythroid cells from healthy human subjects and β-thalassemia patients. Erythroid precursors derived from peripheral blood were grown in 2-phase liquid culture. In this procedure, early erythroid progenitors proliferate and differentiate during phase 1 (in the absence of erythropoietin) into late progenitors. In phase 2, in the presence of erythropoietin, the latter cells continue their proliferation and mature into Hb-containing orthochromatic normoblasts. Compounds were added on days 4 to 5 of phase 2 (when cells started to synthesize Hb), and cells were harvested on day 12. Accumulation of mRNAs for γ-globin, β-globin, α-globin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were measured by real-time quantitative reverse transcription–polymerase chain reaction (RT-PCR); induction of HbF was analyzed by high-performance liquid chromatography (HPLC) and, at cellular level, by flow cytometry. We demonstrated that mithramycin was able to up-regulate preferentially γ-globin mRNA production and to increase HbF accumulation, the percentage of HbF-containing cells, and their HbF content. Mithramycin was more effective than hydroxyurea, being, in addition, not cytotoxic. This was shown by the lack of cytotoxicity on erythroid and myeloid in vitro primary cell cultures treated with mithramycin at concentrations effective for HbF induction. These results are of potential clinical significance because an increase of HbF alleviates the symptoms underlying β-thalassemia and sickle cell anemia. The results of this report suggest that mithramycin and its analogs warrant further evaluation as potential therapeutic drugs.

We report in this paper that the DNA-binding drug mithramycin is a potent inducer of γ-globin mRNA accumulation and fetal hemoglobin (HbF) production in erythroid cells from healthy human subjects and β-thalassemia patients. Erythroid precursors derived from peripheral blood were grown in 2-phase liquid culture. In this procedure, early erythroid progenitors proliferate and differentiate during phase 1 (in the absence of erythropoietin) into late progenitors. In phase 2, in the presence of erythropoietin, the latter cells continue their proliferation and mature into Hb-containing orthochromatic normoblasts. Compounds were added on days 4 to 5 of phase 2 (when cells started to synthesize Hb), and cells were harvested on day 12. Accumulation of mRNAs for γ-globin, β-globin, α-globin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were measured by real-time quantitative reverse transcription–polymerase chain reaction (RT-PCR); induction of HbF was analyzed by high-performance liquid chromatography (HPLC) and, at cellular level, by flow cytometry. We demonstrated that mithramycin was able to up-regulate preferentially γ-globin mRNA production and to increase HbF accumulation, the percentage of HbF-containing cells, and their HbF content. Mithramycin was more effective than hydroxyurea, being, in addition, not cytotoxic. This was shown by the lack of cytotoxicity on erythroid and myeloid in vitro primary cell cultures treated with mithramycin at concentrations effective for HbF induction. These results are of potential clinical significance because an increase of HbF alleviates the symptoms underlying β-thalassemia and sickle cell anemia. The results of this report suggest that mithramycin and its analogs warrant further evaluation as potential therapeutic drugs.

The recent development of advanced analytical and bioseparation methodologies based on microarrays and biosensors is one of the strategic objectives of the so-called post-genomic. In this field, the development of microfabricated devices could bring new opportunities in several application fields, such as predictive oncology, diagnostics and anti-tumor drug research. The so called “Laboratory-on-a-chip technology”, involving miniaturisation of analytical procedures, is expected to enable highly complex laboratory testing to move from the central laboratory into non-laboratory settings. The main advantages of Lab-on-a-chip devices are integration of multiple steps of different analytical procedures, large variety of applications, sub-microliter consumption of reagents and samples, and portability. One of the requirement for new generation Lab-on-a-chip devices is the possibility to be independent from additional preparative/analytical instruments. Ideally, Lab-on-a-chip devices should be able to perform with high efficiency and reproducibility both actuating and sensing procedures. In this review, we discuss applications of dielectrophoretic(DEP)-based Lab-on-a-chip devices to cancer research. The theory of dielectrophoresis as well as the description of several devices, based on spiral-shaped, parallel and arrayed electrodes are here presented. In addition, in this review we describe manipulation of cancer cells using advanced DEP-based Lab-on-a-chip devices in the absence of fluid flow and with the integration of both actuating and sensing procedures.

We have investigated the effects of aromatic polyamidines on HIV-1 transcription. We found a block to Tat-induced HIV-1 transcription assessed by inhibition of CAT activity in HL3T1 cells at a concentration lower than the IC50 value, suggesting that molecules with three (TAPB) and four (TAPP) benzamidine rings could be useful against HIV-1. In contrast, aromatic polyamidines with only two benzamidine rings (DAPP) did not block Tat-induced transcription. We reasoned that this effect could be due to binding of TAPB and TAPP to HIV-1 TAR RNA. By EMSA and filter binding assays, we studied possible interactions of aromatic polyamidines with HIV-1 TAR RNA. Wild-type TAR RNA or TAR RNA with mutations in the stem or bulge sequences, but retaining the stem-loop structure, was used to define the RNA-binding activities of these compounds. Our data suggest that aromatic polyamidines with two (DAPP) and four (TAPP) benzamidine rings, respectively, do not bind to TAR RNA or bind without sequence selectivity. Interestingly, an aromatic polyamidine with three benzamidine rings (TAPB) recognizes the wild-type TAR RNA in a specific manner. Furthermore, we found that introduction of one halogen atom into the benzamidine rings strongly increases the RNA-binding activity of these compounds.

AbstractPeptide nucleic acids (PNA) have recently been proposed as alternative reagents in experiments aimed to the control of gene expression. In PNAs, the pseudopeptide backbone is composed of N-(2-aminoethyl)glycine units and therefore is stable in human serum and cellular extracts. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and giving rise to highly stable (PNA)2-RNA triplexes with RNA targets. Therefore, antisense and antigéne PNAs have been synthetized and characterized.The major issue of the present paper is to describe some computational procedures useful to compare the behaviour of PNA double stranded molecules and PNA/DNA hybrids with the behaviour of regular DNA duplexes in generating complexes with DNA-binding proteins. The performed computational analyses clearly allow to predict that the lack of charged phosphate groups and the different shape of helix play a critical role in the binding efficiency of NF-κB transcription factors. These computational analyses are in agreement with competitive gel shift and UV-cross linking experiments. These experiments demonstrate that NF-κB PNA/PNA hybrids do not interact efficiently with proteins recognizing the NF-KB binding sites in genomic sequences. In addition, the data obtained indicate that the same NF-κB binding proteins recognize both the NF-κB DNA/PNA and DNA/DNA hybrids, but the molecular complexes generated with NF-κB DNA/PNA hybrids are less stable than those generated with NF-κB target DNA/DNA molecules.

ABSTRACTThe X-box element present within the promoter region of genes belonging to the major histocompatibility complex (MHC) plays a pivotal role in the expression of class II molecules, since it contains the binding sites for several well-characterized transcription factors. We have analyzed a randomly selected compilation of viral genomes for the presence of elements homologous to the X box of the HLA-DRA gene. We found that human immunodeficiency virus type 1 (HIV-1) shows the highest frequency of X-like box elements per 1,000 bases of genome. Within the HIV-1 genome, we found an X-like motif in the TAR region of the HIV-1 long terminal repeat (LTR), a regulative region playing a pivotal role in Tat-induced HIV-1 transcription. The use of a decoy approach for nuclear proteins binding to this element, namely, XMAS (X-like motif activator sequence), performed by transfection of multiple copies of this sequence into cells carrying an integrated LTR-chloramphenicol acetyltransferase construct, suggests that this element binds to nuclear proteins that enhance Tat-induced transcription. In this report we have characterized two proteins, one binding to the XMAS motif and the other to the flanking regions of XMAS. Mobility shift assays performed on crude nuclear extracts or enriched fractions suggest that similar proteins bind to XMAS from HIV-1 and the X box of the HLA-DRA gene. Furthermore, a UV cross-linking assay suggests that one protein of 47 kDa, termed FAX (factor associated with XMAS)-1, binds to the XMAS of HIV-1. The other protein of 56 kDa was termed FAX-2. In a decoy ex vivo experiment, it was found that sequences recognizing both proteins are required to inhibit Tat-induced HIV-1 LTR-driven transcription. Taken together, the data reported in this paper suggest that XMAS and nearby sequences modulate Tat-induced HIV-1 transcription by binding to the X-box-binding proteins FAX-1 and FAX-2. The sequence homology between XMAS and X box is reflected in binding of a common protein, FAX-1, and similar functional roles in gene expression. To our knowledge, this is the first report showing that transcription factors binding to the X box of the MHC class II genes enhance the transcription of HIV-1.

We determined whether peptide nucleic acids (PNAs) are able to interact with NF-κB p52 transcription factor. The binding of NF-κB p52 to DNA-DNA, DNA-PNA, PNA-DNA, and PNA-PNA hybrid molecules carrying the NF-κB binding sites of human immunodeficiency type 1 long terminal repeat was studied by (i) biospecific interaction analysis (BIA) using surface plasmon resonance technology, (ii) electrophoretic mobility shift, (iii) DNase I footprinting, and (iv) UV cross-linking assays. Our results demonstrate that NF-κB p52 does not efficiently bind to PNA-PNA hybrids. However, a DNA-PNA hybrid molecule was found to be recognized by NF-κB p52, although the molecular complexes generated exhibited low stability. From the theoretical point of view, our results suggest that binding of NF-κB p52 protein to target DNA motifs is mainly due to contacts with bases; interactions with the DNA backbone are, however, important for stabilization of the protein-DNA complex. From the practical point of view, our results suggest that DNA-PNA hybrid can be recognized by NF-κB p52 protein, although with an efficiency lower than DNA-DNA NF-κB target molecules; therefore, our results should encourage studies on modified PNAs in order to develop potential agents for the decoy approach in gene therapy.

Although many potential applications in early clinical diagnosis have been proposed, the use of a surface plasmon resonance imaging (SPRI) technique for non-invasive prenatal diagnostic approaches based on maternal blood analysis is confined. Here, we report a nanoparticle-enhanced SPRI strategy for a non-invasive prenatal fetal sex determination based on the detection of a Y-chromosome specific sequence (single-gene SRY) in cell-free fetal DNA from maternal plasma. The SPR assay proposed here allows for detection of male DNA in mixtures of 2.5 aM male and female genomic DNAs with no preliminary amplification of the DNA target sequence, thus establishing an analytical protocol that does not require costly, time-consuming, and prone to sample contamination PCR-based procedures. Afterward, the developed protocol was successfully applied to reveal male cell-free fetal DNA in the plasma of pregnant women at different gestational ages, including early gestational ages. This approach would pave the way for the establishment of faster and cost-effective non-invasive prenatal testing.

In the Shwachman-Diamond syndrome (SDS), mutations of the gene encoding for the SBDS protein causes several hematological disorders, including neutropenia and myelodysplastic syndrome (MDS), with high risk of acute myeloid leukemia (AML).MicroRNAs (miRNAs) are small noncoding RNAs regulating gene expression by sequence-selective targeting of mRNAs, leading to translational repression or mRNA degradation. Low miRNA expression is associated with the accumulation of target mRNAs; high miRNA content causes low expression of target mRNAs. In almost all hematological disorders microRNAs were found dysregulated, and often these alterations of miRNAlevels are associated with the phenotype of the disease and tumor onset and progression.

Bezzerri: Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents & Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome". Cipolli:Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents & Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome".

Bezzerri: Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents & Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome". Cipolli:Marco Cipolli, Valentino Bezzerri, Baroukh Maurice Assael: Patents & Royalties: WO2018/050706 A1 "Method of treatment of Shwachman-Diamond syndrome".

We have developed a microencapsulation procedure for the entrapment and manipulation of IB3-1 cystic fibrosis cells. The applied method is based on generation of monodisperse droplets by a vibrational nozzle. Different experimental parameters were analyzed, including frequency and amplitude of vibration, polymer pumping rate and distance between the nozzle and the gelling bath. We have found that the microencapsulation procedure does not alter the viability of the encapsulated IB3-1 cells. The encapsulated IB3-1 cells were characterized in term of secretomic profile, analyzing the culture medium by Bio-Plex strategy. The experiments demonstrated that most of the analyzed proteins, were secreted both by the free and encapsulated cells, even if in a different extent. In order to determine the biotechnological applications of this procedure, we determined whether encapsulated IB3-1 cells could be induced to pro-inflammatory responses, after treatment with TNF-α. In this experimental set-up, encapsulated and free IB3-1 cells were treated with TNF-α, thereafter the culture media from both cell populations were collected. As expected, TNF-α induced a sharp increase in the secretion of interleukins, chemokines and growth factors. Of great interest was the evidence that induction of interleukin-6 and interleukin-8 occurs also by encapsulated IB3-1 cells.