Gioacchin Iannolo

Construction, exploitation and evolution of a new peptide library displayed at high density by fusion to the major coat protein of filamentous phage

The amino-terminus of the major coat protein (PVIII) of filamentous phage can be extended, up to 6-7 residues, without interfering with the phage life cycle. We have constructed a library of approximately ten millions different phage each displaying a different octapeptide joined to the amino-terminus of the 2700 copies of PVIII. Most of the resulting clones are able to produce infective particles. This molecular repertoire constituted by the periodic regular decoration of the phage filament surface, can be utilized to search elements that bind proteins or relatively small organic molecules like the textile dye Cibacron blue. By sequential growth cycles we have performed a library evolution experiment to select phage clones that have a growth advantage in the absence of any requirement for binding a specific target. The consensus of the best growers reveals a Pro rich sequence with large hydrophobic residues at position 7 and Asn at position 1 of the random peptide insert. We propose that the assembly secretion process is favoured in phages displaying this family of peptides since they fit the groove between two adjacent PVIII subunits by making advantageous molecular contacts on the phage surface.

Antitumor activity of bortezomib alone and in combination with TRAIL in human acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant disease characterized by abnormal proliferation of clonal precursor cells. Although different strategies have been adopted to obtain complete remission, the disease actually progresses in about 60–70% of patients. Bortezomib has been used in multiple myeloma and other lymphoid malignancies because of its antitumor activity. Here we examined the sensitivity of bone marrow cells from AML patients (34 patients: 25 newly diagnosed, 4 relapsed, 5 refractory) to bortezomib alone or in combination with TRAIL, a member of the TNF family that induces apoptosis in tumor cells while sparing normal cells. Bortezomib induced cell death in blasts from each patient sample. The cytotoxic effect was dose- and time-dependent (concentration from 0.001 to 10 µM for 24 and 48 h) and was associated with a downregulation of Bcl-xL and Mcl-1, an upregulation of TRAIL-R1, TRAIL-R2, p21, activation of executioner caspases and a loss of the mitochondrial membrane potential. Moreover, low doses of bortezomib primed TRAIL-resistant AML cells for enhanced TRAIL-mediated killing. These results suggest that a combination of proteasome inhibitors and TRAIL could be effective for treating AML patients, even patients who are refractory to conventional chemotherapy.

NF-κB localization in multiple myeloma plasma cells and mesenchymal cells

Several reports demonstrated that the activation of Nuclear Factor-kappa B NF-κB is essential for the pathogenesis of multiple myeloma (MM). We analyzed the nuclear localization of NF-κB in MM-cells derived from 60 different patients with MM at presentation and in relapse, as well as in three myeloma cell lines. Nuclear localization (the active form) of NF-κB was detected in only one MM-sample from a refractory patient and in two samples from relapsed patients, while all the other samples, including the MM-cell lines, almost exclusively express the cytoplasmic (inactive) form of NF-κB. In mesenchymal cells from MM-patients NF-κB was clearly present in the nucleus. In addition, the proteasome inhibitor Bortezomib, which is described to antagonize NF-κB activity, had a consistent antitumor activity against both chemoresistant and chemosensitive MM-cells, regardless the NF-κB localization, thus suggesting the existence of other molecular targets of proteasome inhibitors in MM.

HCV replication in gastrointestinal mucosa: Potential extra-hepatic viral reservoir and possible role in HCV infection recurrence after liver transplantation

Purpose Hepatitis C virus (HCV) predominantly infects hepatocytes, although it is known that receptors for viral entry are distributed on a wide array of target cells. Chronic HCV infection is indeed characterized by multiple non-liver manifestations, suggesting a more complex HCV tropism extended to extrahepatic tissues and remains to be fully elucidated. In this study, we investigated the gastrointestinal mucosa (GIM) as a potential extrahepatic viral replication site and its contribution to HCV recurrence. Methods We analyzed GIM biopsies from a cohort of 76 patients, 11 of which were HCV-negative and 65 HCV-positive. Of these, 54 biopsies were from liver-transplanted patients. In 29 cases, we were able to investigate gastrointestinal biopsies from the same patient before and after transplant. To evaluate the presence of HCV, we looked for viral antigens and genome RNA, whilst to assess viral replicative activity, we searched for the replicative intermediate minus-strand RNA. We studied the genetic diversity and the phylogenetic relationship of HCV quasispecies from plasma, liver and gastrointestinal mucosa of HCV-liver-transplanted patients in order to assess HCV compartmentalization and possible contribution of gastrointestinal variants to liver re-infection after transplantation. Results Here we show that HCV infects and replicates in the cells of the GIM and that the favorite hosts were mostly enteroendocrine cells. Interestingly, we observed compartmentalization of the HCV quasispecies present in the gastrointestinal mucosa compared to other tissues of the same patient. Moreover, the phylogenetic analysis revealed a high similarity between HCV variants detected in gastrointestinal mucosa and those present in the re-infected graft. Conclusions Our results demonstrated that the gastrointestinal mucosa might be considered as an extrahepatic reservoir of HCV and that could contribute to viral recurrence. Moreover, the finding that HCV infects and replicates in neuroendocrine cells opens new perspectives on the role of these cells in the natural history of HCV infection.

The PU.1 transcription factor induces cyclin D2 expression in U937 cells.

The PU.1 transcription factor is expressed in a wide variety of haematopoietic precursors including long-and short-term reconstituting stem cells, the common myeloid and lymphoid progenitors (CMP, CLP), granulocyte–monocyte progenitors (GMP), monocytes, neutrophils and B-lymphocytes. Compelling evidence gathered from both mouse models and human studies has demonstrated that PU.1 is a pivotal component of the intricate network regulating normal and neoplastic haematopoiesis. Physiologically, PU.1 expression contributes to the commitment of the CMPs to granulocytes and monocytes and to B-cell differentiation. These observations derive from studies on PU.1 knockout mice showing that PU.1-null animals present normal erythroid and megakaryocytic cells, but display embryonic/newborn lethality owing to the lack of monocytes, neutrophils and B-lymphocytes. More recently, the role of PU.1 in normal haematopoiesis has been further defined by conditional knockout mice demonstrating that PU.1 expression is critical to perpetuate the haematopoietic stem cell pool and to allow the generation of CMPs and CLPs. PU.1 disruption in CMPs or GMPs blocks their maturation, whereas PU.1 deficiency in CLPs does not affect their ability to differentiate in mature B cells. These results are not surprising considering that PU.1 induces the expression of multiple proteins critically involved in the commitment of the myeloid and lymphoid lineages, including the granulocyte/macrophage colony-stimulating factor receptor (CSF), the macrophage CSF receptor, the granulocyte CSF receptor and the interleukin-7 receptor. PU.1 is also directly involved in the pathogenesis of human leukaemias. Overexpression of PU.1 in mice exposed to the Friend virus results in the development of erythroleukaemia. Likewise, mice harbouring deletions of the upstream regulatory element of the PU.1 promoter that lead to reduced PU.1 expression develop acute myeloid leukaemia (AML) or T-cell lymphoma. Moreover, mutations impairing PU.1 transcriptional activity have been reported in 7% of 126 patients diagnosed with AML. Recent findings have also shown that both AML1/ETO and FLT3-ITD oncoproteins inhibit PU.1 activity, therefore reinforcing the notion that reduced PU.1 function (possibly coupled with mutation-induced haploinsufficiency) may contribute to the development of AML. Although it is well established that PU.1 modulates the proliferation and differentiation of several haematopoietic precursors, the mechanisms responsible for these activities are still partially unclear. As D-type cyclins are critical modulators of cell proliferation favouring cell cycle transition from the G1 to the S phase, we investigated if PU.1 could regulate the levels of D-cyclins. We report that PU.1 induces the expression of cyclin D2 in the U937 promonocytic cell line. A preliminary sequence analysis of the promoter for human cyclin D2 (accession number U47284) revealed two GAGGAA consensus sites for PU.1, one of which is conserved in mice (accession number AFO15788) (Figure 1a, in bold). To determine if PU.1 is able to transactivate this sequence, we transiently expressed the full-length human cyclin D2 promoter (cloned in the PGL2 luciferase vector, gift of Professor M Eilers, Marburg, Germany) in human embryonic kidney (HEK) 293 cells either alone or with PU.1. Luciferase assays performed normalizing transfection efficiency with an expression vector for Renilla demonstrated a reproducible 2.7-fold increase in the activity of the cyclin D2 promoter in the presence of PU.1 (Figure 1b). However, as PU.1 is expressed exclusively in haematopoietic lineages, we wanted to ascertain if these results could be reproduced in a haematopoietic cell line. Previous evidence has shown that treatment of U937 cells with tetradecanoyl-phorbol-13 acetate (TPA) leads to the phosphorylation of PU.1 and induction of its transcriptional activity. Indeed, as had been reported previously, when we carried out an electrophoretic mobility shift assay on U937 cells incubated with 20 ng/ml TPA, we observed an increase in PU.1 transcriptional activity after 9–12 h (not shown). Interestingly, PU.1 phosphorylation by serine–threonine kinases can be monitored by immunoblot because hyperphosphorylated (active) PU.1 exhibits a supershift compared to the unphosphorylated (inactive) protein. We therefore performed an antiPU.1 Western blot on U937 cell lysates derived from cells incubated with TPA and noticed a reproducible increase of the hyperphosphorylated PU.1 protein after 9 h. This increase reached a plateau after a 21-h stimulation (Figure 1c, left panel). When we repeated the immunoblot employing an anticyclin D2 antibody, we found a marked increase in cyclin D2 levels starting from 12 h of TPA incubation, suggesting that in U937 cells activation of PU.1 leads to increased expression of cyclin D2 (Figure 1c, right panel). To exclude that PU.1-independent biological events may be responsible for the observed TPA-mediated induction of cyclin D2 expression, we retrovirally transduced U937 cells with a dominant-negative form of PU.1 (DNPU.1 provided by Professor R Kawara, University of Nebraska Medical Center, USA) that retains the DNA-binding domain of the wild-type protein, but is devoid of both the transactivation and the PEST domains. Control Western blots showed that DNPU.1-infected cells (lanes 5–8) but not mock-infected cells (lanes 1–4) expressed the dominant-negative form of the transcription factor (Figure 1d, left panel). Moreover, this experiment demonstrated that expression of DNPU.1 did not affect TPA-induced phosphorylation of the endogenous PU.1 as incubation with the drug caused PU.1 hyperphosphorylation both in the absence and in the presence of DNPU.1. However, when we analysed cyclin D2 expression levels, we found a marked difference in the two U937 cell populations. In mock-infected U937 exposure to TPA increased cyclin D2 expression as had been observed previously (Figure 1d, right panel, lanes 1–4). On the contrary, U937 cells expressing DNPU.1 did not present increased levels of cyclin D2 upon PU.1 phosphorylation (Figure 1d, right panel, lanes 5–8), indicating that the dominant-negative form of the transcription factor successfully antagonized the effect of the endogenous PU.1 on the cyclin D2 promoter. To further determine the biological effect of PU.1 regulation of cyclin D2 expression in U937 cells, we transduced this cell line with viral particles encoding for DNPU.1 and cyclin D2, alone or in combination. Immunoblotting experiments confirmed proper expression of the expected proteins in each experimental condition (Figure 1e, left panels). Fluorescentactivated cell sorting (FACS) analysis of these different cell populations after an 18-h treatment with TPA revealed that DNPU.1 did not alter the cell cycle distribution of these cells, as Letters to the Editor

MiR34 inhibition induces human heart progenitor proliferation

MiR34 involvement in myocardial injury repair and ageing has been well documented in mouse model. Our aim was to establish whether the inhibition of miR34 expression through locked nucleic acid (LNA) could be used as a pharmacological intervention to enhance human heart repair. Cardiac progenitor cells were obtained by right atrial specimen collection during intraoperative procedures. Our analysis revealed a direct correlation between miR34 expression and patient age, and its silencing by LNA promoted the cardiac progenitor growth rate up to twofold ( ± 0.8). Our results confirmed the relevance of miR34a in human heart ageing, as previously demonstrated in mouse. Moreover, the decrease of miR34 expression in the cardiac progenitor cell population indicates its role in maintaining an undifferentiated status and consequently in a lower proliferation rate with the involvement of genes such as Notch-1, Numb, and p63.

Numb Expression Contributes to the Maintenance of an Undifferentiated State in Human Epidermis.

The epidermis is a stratified epithelium with a stem cell subpopulation in the basal layer that constantly replicates and periodically detaches from the base, undergoing a differentiation process that involves various developmental signals and regulatory pathways. During the last 10 years, a number of studies tried to elucidate the intricate scenario that maintains the epithelial shield during the entire life span. In our study, we investigated the role of Numb in the skin compartment and, in particular, its involvement in stem cell maintenance. Numb expression in the skin compartment was assessed by immunofluorescence and immunohistochemistry analysis. We evaluated Numb expression in primary epithelial cells at various differentiative stages. Moreover, we overexpressed Numb in the isolated population enriched for undifferentiated progenitors to establish its involvement in in vitro differentiation. We demonstrated that Numb in high-proliferating epithelial undifferentiated progenitors contributes to the maintenance of an undifferentiated state. This regulation involves the E3 ligases Itch binding. Moreover, the analysis of a cohort of cutaneous carcinomas showed that Numb is highly expressed in squamous cell carcinoma (SCC), where we observed a direct correlation between the expression of Numb and Ki-67. Our data indicate for the first time that Numb is involved in the maintenance of the undifferentiated proliferating stem cell pool in the epithelial basal layer and its expression could become a new marker in skin cancer.

Carnosine protects pancreatic beta cells and islets against oxidative stress damage

Islet transplantation is a valid therapeutic option for type 1 diabetes treatment. However, in this procedure one of the major problems is the oxidative stress produced during pancreatic islet isolation. The aim of our study was to evaluate potential protective effects of L-carnosine and its isomer D-carnosine against oxidative stress. We evaluated the carnosine effect on cell growth, cell death, insulin production, and the main markers of oxidative stress in rat and murine stressed beta cell lines as well as in human pancreatic islets. Both isomers clearly inhibited hydrogen peroxide induced cytotoxicity, with a decrease in intracellular reactive oxygen and nitrogen species, prevented hydrogen peroxide induced apoptosis/necrosis, nitrite production, and reduced glucose-induced insulin secretion. In addition, NF-κB expression/translocation and nitrated protein induced in stressed cells was significantly reduced. Furthermore, both isomers improved survival and function, and decreased reactive oxygen and nitrogen species, and nitrite and nitrotyrosine production in human islets cultured for 1, 3, and 7 days. These results seem to indicate that both L and D-carnosine have a significant cytoprotective effect by reducing oxidative stress in beta cell lines and human islets, suggesting their potential use to improve islet survival during the islet transplantation procedure.

Detection of the IncX3 plasmid carrying blaKPC-3 in a Serratia marcescens strain isolated from a kidney–liver transplanted patient

Dissemination of resistance to carbapenems among Enterobacteriaceae through plasmids is an increasingly important concern in health care worldwide. Here we report the first description of an IncX3 plasmid carrying the blaKPC-3 gene in a strain of Serratia marcescens isolated from a kidney-liver transplanted patient at the transplantation centre ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione, Palermo, Italy). To localize the transposable element containing the resistance-associated gene Next-Generation Sequencing of the bacterial DNA was performed. S. marcescens was positive for blaKPC-3 and blaSHV-11 genes. The molecular analysis demonstrated that the blaKPC-3 gene of this bacterial strain was located in one copy of the Tn-3-like element Tn4401-a carried in a plasmid that is 53 392 bp in size and showed the typical IncX3 scaffold. Our data demonstrated the presence of a new blaKPC-3 harbouring the IncX3 plasmid in S. marcescens. The possible dissemination among Enterobacteriaceae of this type of plasmid should be monitored and evaluated in terms of clinical risk.

MARCH-I expression in cord blood CD34+KDR+ cells

Hematopoietic stem cells transplantation has been successfully used in the treatment of patients with hematological malignances. A better knowledge of the mechanisms beyond their ability to completely repopulate the entire hematopoietic system would help in the treatment of hematological diseases. For this reason we focused our studies on a cell population that has been demonstrated to have some peculiar characteristics among the stem cells: CD34+KDR+ cells. These cells, an extremely rare population among the CD34 (0.1%-0.5%) cells, have been demonstrated from different groups to have the potential to give rise to the hematopoietic and endothelial lineage. By a subtraction library approach we found different sequences more expressed in CD34+KDR+ than their CD34+KDR- counterpart. In particular, we found an open reading frame correspondent to a newly characterized E3 ligase, MARCH-I. This gene is part of a recently described family involved in immune response modulation through the proteosomal mediated degradation. MARCH-I expression in stem cells could be important for their intrinsic immune properties.