Nicola Lo Buono

The CD157-Integrin Partnership Controls Transendothelial Migration and Adhesion of Human Monocytes

CD157, a member of the CD38 gene family, is an NAD-metabolizing ectoenzyme and a signaling molecule whose role in polarization, migration, and diapedesis of human granulocytes has been documented; however, the molecular events underpinning this role remain to be elucidated. This study focused on the role exerted by CD157 in monocyte migration across the endothelial lining and adhesion to extracellular matrix proteins. The results demonstrated that anti-CD157 antibodies block monocyte transmigration and adhesion to fibronectin and fibrinogen but that CD157 cross-linking is sufficient to overcome the block, suggesting an active signaling role for the molecule. Consistent with this is the observation that CD157 is prevalently located within the detergent-resistant membrane microdomains to which, upon clustering, it promotes the recruitment of β1 and β2 integrin, which, in turn, leads to the formation of a multimolecular complex favoring signal transduction. This functional cross-talk with integrins allows CD157 to act as a receptor despite its intrinsic structural inability to do so on its own. Intracellular signals mediated by CD157 rely on the integrin/Src/FAK (focal adhesion kinase) pathway, resulting in increased activity of the MAPK/ERK1/2 and the PI3K/Akt downstream signaling pathways, which are crucial in the control of monocyte transendothelial migration. Collectively, these findings indicate that CD157 acts as a molecular organizer of signaling-competent membrane microdomains and that it forms part of a larger molecular machine ruled by integrins. The CD157-integrin partnership provides optimal adhesion and transmigration of human monocytes.

ELOVL5 Mutations Cause Spinocerebellar Ataxia 38

Spinocerebellar ataxias (SCAs) are a heterogeneous group of autosomal-dominant neurodegenerative disorders involving the cerebellum and 23 different genes. We mapped SCA38 to a 56 Mb region on chromosome 6p in a SCA-affected Italian family by whole-genome linkage analysis. Targeted resequencing identified a single missense mutation (c.689G>T [p.Gly230Val]) in ELOVL5. Mutation screening of 456 independent SCA-affected individuals identified the same mutation in two further unrelated Italian families. Haplotyping showed that at least two of the three families shared a common ancestor. One further missense variant (c.214C>G [p.Leu72Val]) was found in a French family. Both missense changes affect conserved amino acids, are predicted to be damaging by multiple bioinformatics tools, and were not identified in ethnically matched controls or within variant databases. ELOVL5 encodes an elongase involved in the synthesis of polyunsaturated fatty acids of the ω3 and ω6 series. Arachidonic acid and docosahexaenoic acid, two final products of the enzyme, were reduced in the serum of affected individuals. Immunohistochemistry on control mice and human brain demonstrated high levels in Purkinje cells. In transfection experiments, subcellular localization of altered ELOVL5 showed a perinuclear distribution with a signal increase in the Golgi compartment, whereas the wild-type showed a widespread signal in the endoplasmic reticulum. SCA38 and SCA34 are examples of SCAs due to mutations in elongase-encoding genes, emphasizing the importance of fatty-acid metabolism in neurological diseases.

Generation of potent neutralizing human monoclonal antibodies against cytomegalovirus infection from immune B cells

BackgroundHuman monoclonal antibodies (mAbs) generated as a result of the immune response are likely to be the most effective therapeutic antibodies, particularly in the case of infectious diseases against which the immune response is protective.Human cytomegalovirus (HCMV) is an ubiquitous opportunistic virus that is the most serious pathogenic agent in transplant patients. The available therapeutic armamentarium (e.g. HCMV hyperimmune globulins or antivirals) is associated with severe side effects and the emergence of drug-resistant strains; therefore, neutralizing human mAb may be a decisive alternative in the prevention of primary and re-activated HCMV infections in these patients.ResultsThe purpose of this study was to generate neutralizing mAb against HCMV from the immunological repertoire of immune donors. To this aim, we designed an efficient technology relying on two discrete and sequential steps: first, human B-lymphocytes are stimulated with TLR9-agonists and IL-2; second, after both additives are removed, the cells are infected with EBV. Using this strategy we obtained 29 clones secreting IgG neutralizing the HCMV infectivity; four among these were further characterized. All of the mAbs neutralize the infection in different combinations of HCMV strains and target cells, with a potency ~20 fold higher than that of the HCMV hyperimmune globulins, currently used in transplant recipients. Recombinant human monoclonal IgG1 suitable as a prophylactic or therapeutic tool in clinical applications has been generated.ConclusionThe technology described has proven to be more reproducible, efficient and rapid than previously reported techniques, and can be adopted at low overall costs by any cell biology laboratory for the development of fully human mAbs for immunotherapeutic uses.

Binding of CD157 Protein to Fibronectin Regulates Cell Adhesion and Spreading

Background: Surface CD157 modulates leukocyte and ovarian cancer cell adhesion and migration through the interaction with an unknown ligand. Results: CD157 binds heparin-binding domains of numerous extracellular matrix proteins with high affinity. Conclusion: The interaction of CD157 with extracellular matrix proteins is instrumental in the regulation of cell adhesion. Significance: These findings provide valuable insights into the biological mechanism responsible for the nonenzymatic functions of CD157. CD157/BST-1 behaves both as an ectoenzyme and signaling receptor and is an important regulator of leukocyte trafficking and ovarian cancer progression. However, the molecular interactions underpinning the role of CD157 in these processes remain obscure. The biological functions of CD157 and its partnership with members of the integrin family prompted us to assume the existence of a direct interaction between CD157 and an unknown component of the extracellular matrix. Using solid-phase binding assays and surface plasmon resonance analysis, we demonstrated that CD157 binds fibronectin with high affinity within its heparin-binding domains 1 and 2. Furthermore, we found that CD157 binds to other extracellular matrix proteins containing heparin-binding domains. Finally, we proved that the CD157-fibronectin interaction occurs with living cells, where it elicits CD157-mediated cell responses. Indeed, knockdown of CD157 in Met-5A mesothelial cells changed their morphology and cytoskeleton organization and attenuated the activation of intracellular signaling pathways triggered by fibronectin. This led to impaired cell spreading and adhesion to selected extracellular matrix proteins. Collectively, these findings indicate a central role of CD157 in cell-extracellular matrix interactions and make CD157 an attractive therapeutic target in inflammation and cancer.

Cytotoxic activity of gemcitabine, alone or in combination with mitotane, in adrenocortical carcinoma cell lines.

We aimed at investigating in vitro the cytotoxic activity (determined using WST-1, apoptosis and cell cycle assays) of gemcitabine, alone or in combination with mitotane, in mitotane-sensitive H295R and mitotane-insensitive SW-13 cells. Results of these experiments were compared with drug-induced modulation of RRM1 gene, the specific target of gemcitabine. In H295R cells, mitotane and gemcitabine combinations showed antagonistic effects and interfered with the gemcitabine-mediated inhibition of the S phase of the cell cycle. By contrast, in SW-13 cells, except when mitotane was sequentially administered prior to gemcitabine, the combination of the two drugs was synergistic. Such opposite effects were associated with opposite expression profiles of the target gene, with significant up-modulation in H295R but not in SW-13 under gemcitabine and mitotane combination treatment.

Genome-wide expression profiling and functional characterization of SCA28 lymphoblastoid cell lines reveal impairment in cell growth and activation of apoptotic pathways

BackgroundSCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We performed a whole genome expression profiling using lymphoblastoid cell lines (LCLs) from four SCA28 patients and six unrelated healthy controls matched for sex and age.MethodsGene expression was evaluated with the Affymetrix GeneChip Human Genome U133A 2.0 Arrays and data were validated by real-time PCR.ResultsWe found 66 genes whose expression was statistically different in SCA28 LCLs, 35 of which were up-regulated and 31 down-regulated. The differentially expressed genes were clustered in five functional categories: (1) regulation of cell proliferation; (2) regulation of programmed cell death; (3) response to oxidative stress; (4) cell adhesion, and (5) chemical homeostasis. To validate these data, we performed functional experiments that proved an impaired SCA28 LCLs growth compared to controls (p < 0.005), an increased number of cells in the G0/G1 phase (p < 0.001), and an increased mortality because of apoptosis (p < 0.05). We also showed that respiratory chain activity and reactive oxygen species levels was not altered, although lipid peroxidation in SCA28 LCLs was increased in basal conditions (p < 0.05). We did not detect mitochondrial DNA large deletions. An increase of TFAM, a crucial protein for mtDNA maintenance, and of DRP1, a key regulator of mitochondrial dynamic mechanism, suggested an alteration of fission/fusion pathways.ConclusionsWhole genome expression profiling, performed on SCA28 LCLs, allowed us to identify five altered functional categories that characterize the SCA28 LCLs phenotype, the first reported in human cells to our knowledge.

A novel homozygous change of CLCN2 (p.His590Pro) is associated with a subclinical form of leukoencephalopathy with ataxia (LKPAT)

ClC-2 is a plasma membrane chloride channel with widespread expression in the human body, including the brain. Its function is still being studied, although it is thought to have a role in ion and water homoeostasis in the brain. ClC-2 is part of a complex containing GlialCAM and MLC1. Both these genes are associated with autosomal recessive human leukodystrophies with intramyelinic oedema. Biallelic mutations in CLCN2 , encoding the ClC-2 channel, have been reported in patients with a rare form of leukoencephalopathy with ataxia (LKPAT; MIM #615651). No peculiar neurological features have been reported for this disease, although slight visual impairment due to chorioretinopathy or optic atrophy, mild ataxia, learning disabilities, and headaches are recurrent symptoms in patients. However, MRI shows a typical diagnostic pattern that consists of white matter signal abnormalities in the posterior limbs of the internal capsules, cerebral peduncles, pontine pyramidal tracts and in the middle cerebellar peduncles, associated with lower apparent diffusion coefficient values in most cases. Specific anomalies of brainstem auditory evoked potentials (BAEP) have also been described.1–3 Here, we report on a 52-year-old Moroccan woman presenting with mild and asymptomatic bilateral optic atrophy detected at a routine ophthalmological examination for presbyopia. Best-corrected high-contrast visual acuity was 20/20 in both eyes. Anterior segment and intraocular pressures were normal, and pupillary reflexes were present. On fundus biomicroscopy, mild pallor and excavation of the optic …

Human canonical CD157/Bst1 is an alternatively spliced isoform masking a previously unidentified primate-specific exon included in a novel transcript

CD157/Bst1 is a dual-function receptor and β-NAD+-metabolizing ectoenzyme of the ADP-ribosyl cyclase family. Expressed in human peripheral blood neutrophils and monocytes, CD157 interacts with extracellular matrix components and regulates leukocyte diapedesis via integrin-mediated signalling in inflammation. CD157 also regulates cell migration and is a marker of adverse prognosis in epithelial ovarian cancer and pleural mesothelioma. One form of CD157 is known to date: the canonical sequence of 318 aa from a 9-exon transcript encoded by BST1 on human chromosome 4. Here we describe a second BST1 transcript, consisting of 10 exons, in human neutrophils. This transcript includes an unreported exon, exon 1b, located between exons 1 and 2 of BST1. Inclusion of exon 1b in frame yields CD157-002, a novel proteoform of 333 aa: exclusion of exon 1b by alternative splicing generates canonical CD157, the dominant proteoform in neutrophils and other tissues analysed here. In comparative functional analyses, both proteoforms were indistinguishable in cell surface localization, specific mAb binding, and behaviour in cell adhesion and migration. However, NAD glycohydrolase activity was detected in canonical CD157 alone. Comparative phylogenetics indicate that exon 1b is a genomic innovation acquired during primate evolution, pointing to the importance of alternative splicing for CD157 function.

High miR-100 expression is associated with aggressive features and modulates TORC1 complex activation in lung carcinoids

Purpose Mammalian target of rapamycin (mTOR) is a promising therapeutic target in advanced lung carcinoid patients. However, the mechanisms of mTOR modulation and of responsiveness to mTOR inhibitors are largely unclear. Our aim was to analyze the expression and functional role of specific miRNAs in lung carcinoids as an alternative mechanism targeting mTOR pathway. Experimental design Seven miRNAs, selected by bioinformatic tools and literature search, were analyzed in 142 lung neuroendocrine neoplasms (92 carcinoids and a control group of 50 high grade neuroendocrine carcinomas), and compared with mTOR mRNA expression and clinical/pathological parameters. Tissue results were validated in vitro in two lung carcinoid cell lines by specific RNA interference and biological/pharmacological tests. Results Tissutal expression of five miRNAs (miR-99b, miR-100, miR-155, miR-193a-3p, miR-193a-5p) was inversely correlated with mTOR mRNA expression, supporting their role in the negative regulation of mTOR transcription. High expression of miR-100, miR-193a-3p and miR-193a-5p was associated with aggressive features and, for the former two, with shorter time to progression. In H727 and UMC11 lung carcinoid cells, miR-100 modulated mTOR RNA and TORC1 complex protein expression, positively promoted cell migration and negatively influenced cell proliferation. Moreover, miR-100 directly influenced responsiveness of H727 and UMC11 cells to rapamycin. Conclusions MiR-100 actively participates to the regulation of mTOR expression in lung carcinoids and represents a novel candidate prognostic biomarker for this tumor type; moreover, inhibition of its expression is associated to increased responsiveness to mTOR inhibitors and might represent a novel strategy to sensitize lung carcinoids to these target agents.

Ectoenzymes in Epithelial Ovarian Carcinoma: Potential Diagnostic Markers and Therapeutic Targets

Ovarian cancer is one of the most lethal among the gynaecological malignancies, affecting 12% of women in developed countries (Cannistra, 2004). The lethality of ovarian cancer is primarily attributable to our current inability to detect the disease at an early stage, when it is still limited to the ovary. Therefore, the majority of patients are diagnosed when they have advanced-stage disease. Despite progresses in cytotoxic therapies, only 30% of patients with advanced-stage ovarian cancer survive 5 years after diagnosis. The insidious nature of ovarian cancer stems from its unique biological behaviour: ovarian carcinoma can spread by direct extension to adjacent organs, and exfoliated tumour cells can be transported in peritoneal fluid (Naora et al., 2005). Subsequent implants are characterised by their adhesion to mesothelial cells, migration throughout and invasion of the tumor cells into the omentum and peritoneum. This seeding of the peritoneal cavity is frequently associated with ascites formation. Only secondarily and rather late during the disease progression, are pelvic and para-aortic lymph nodes involved. However, the local peritoneal disease cannot be controlled and remains a factor leading to death (Feki et al., 2009). The cellular processes that lead to local and distant dissemination of ovarian cancer are not fully understood, and the mechanisms of interaction between cancer cells and mesothelium need to be further elucidated to achieve novel information on the biology of this highly aggressive form of cancer and possibly, to identify new potential targets for selective therapeutic strategies. The combined effort of clinicians and researchers has led to the identification of a number of molecules that might facilitate screening, diagnosis, prognosis and monitoring response to treatment or relapse during follow-up. These new molecules might provide specific targets for anti-tumour therapy with antibody-directed treatments, gene therapy or specific inhibitory molecules. An unexpectedly high number of these newly identified molecules have turned out to be cell surface-expressed ectoenzymes. Ectoenzymes are a large, heterogeneous class of membrane proteins whose catalytically active sites face the extracellular environment. The products of their catalytic activities can influence the extracellular environment (for example, several of these products can function as second messengers or regulate the recruitment of cells). Moreover, many ectoenzymes can function