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L Zentilin

Publications and source records attributed to L Zentilin.

At least 19 recordsLinked to original sources
PubMed2002

Recombinant AAV vector encoding human VEGF165 enhances wound healing.

Delivery of therapeutic genes represents an appealing possibility to accelerate healing of wounds that are otherwise difficult to treat, such as those in patients with metabolic disorders or infections. Experimental evidence indicates that in such conditions potentiation of neo-angiogenesis at the wound site might represent an important therapeutic target. Here we explore the efficacy of gene therapy of wound healing with an adeno-associated virus (AAV) vector expressing the 165 amino acid isoform of vascular endothelial growth factor-A (VEGF-A). By gene marker studies, we found that AAV vectors are highly efficient for gene transfer to the rat skin, displaying an exquisite tropism for the panniculus carnosus. Gene expression from these vectors is sustained and persistent over time. Delivery of VEGF165 to full thickness excisional wounds in rats resulted in remarkable induction of new vessel formation, with consequent reduction of the healing time. Histological examination of treated wounds revealed accelerated remodeling of epidermis and dermis, with formation of a thick granular layer, containing numerous newly formed capillaries, as well as vessels of larger size. These data underline the importance of neo-angiogenesis in the healing process and indicate that VEGF gene transfer might represent a novel approach to treat wound healing disorders.

Animals↗
PubMed2001

Involvement of cellular double-stranded DNA break binding proteins in processing of the recombinant adeno-associated virus genome.

Unlike postmitotic tissues in vivo, transduction of cultured cells is poor with recombinant adeno-associated virus (rAAV). The ability of rAAV to transduce cells is greatly enhanced by a variety of agents that induce DNA damage and is elevated in cells defective in the ataxia telangiectasia gene product (ATM), showing increased genomic instability. Here we show that DNA double-stranded break (DSB) repair pathways are involved in the regulation of rAAV transduction efficiency. By quantitative chromatin immunoprecipitation, we found that Ku86 and Rad52 proteins associate with viral DNA inside transduced cells. Both proteins are known to competitively recognize hairpin structures and DNA termini and to promote repair of DSBs, the former by facilitating nonhomologous end joining and the latter by initiating homologous recombination. We found that rAAV transduction is increased in Ku86-defective cells while it is inhibited in Rad52 knockout cells. These results suggest that binding of Rad52 to the rAAV genome might be involved in processing of the vector genome through a homologous recombination pathway.

Animals↗
PubMed2000

Variegation of retroviral vector gene expression in myeloid cells.

We have comparatively evaluated the efficiency of a series of retroviral vectors transducing the gp91-phox gene, whose defects are responsible for impaired production of superoxide anion (O2-) by phagocytic cells and lead to the X-linked form of chronic granulomatous disease (X-CGD). These vectors included four constructs based on the MoMuLV backbone and expressing gp91-phox from the viral long terminal repeat (LTR) or from internal promoters, and one construct based on the myelotropic FMEV vector. Expression of the therapeutic gene from the MoMuLV LTR was unsatisfactory after transduction of the PLB985 X-CGD knockout cell line and of primary CD34+ hematopoietic progenitors from X-CGD patients. The presence of either constitutive or inducible internal promoters did not result in important improvements in the efficiency of O2- production and lowered the titers of the viral preparations. In contrast, sustained levels of superoxide generation were obtained upon transduction with the FMEV vector. To analyze the efficiency of transgene expression at the single cell level, over 150 cellular clones were generated from bulk cultures of PLB985 X-CGD cells transduced with this vector, each one representative of an individual transduction event. These clones revealed a markedly heterogeneous pattern of gp91-phox expression, ranging from complete silencing to full restoration of superoxide production. Within each clone, expression of the therapeutic gene correlated with the number of expressing cells rather than with the average levels of expression from each cell, indicating that at the single cell level, the proviral promoter is regulated by a binary, on/off mechanism. Moreover, both transduced bulk and clonal cell populations displayed a tendency to a progressive extinction of expression over time, with a mechanism involving LTR methylation. The design of novel retroviral vectors escaping silencing is highly desirable for efficient gene therapy.

Gene Expression↗
PubMed1999

Effectiveness of IFN-gamma for liver abscesses in chronic granulomatous disease.

In chronic granulomatous disease, interferon-gamma (IFN-gamma) significantly reduces the incidence and severity of recurrent infections, but its effectiveness administered ex novo during acute infection has been reported in only one case. In this report, we describe two adult brothers with chronic granulomatous disease treated successfully with IFN-gamma for acute liver abscesses. Two brothers with severe recurrent infections of unknown origin were hospitalized for septic fever, malnutrition, and ultrasonographic evidence of liver abscess. Autosomal recessive chronic granulomatous disease was diagnosed based on lack of superoxide anion production by phagocytes and absence of p47-phox protein. An antibiotic regimen specifically directed against Staphylococcus aureus was ineffective, whereas treatment with 50 microg/m2 IFN-gamma s.c. thrice weekly induced complete healing with scarring within 3 months. No septic recurrence was observed during a 4-year follow-up period. In chronic granulomatous disease, IFN-gamma is effective not only in preventing but also in healing life-threatening acute infections.

Adolescent↗
PubMed1998

Nicotinamide-adenine dinucleotide phosphate oxidase assembly and activation in EBV-transformed B lymphoblastoid cell lines of normal and chronic granulomatous disease patients.

This paper deals with the mechanisms of activation of NADPH oxidase investigated using EBV-transformed human B lymphoblastoid cell lines (B cells) from normal subjects and from patients affected by X-linked chronic granulomatous disease (CGD). The results reported are as follows. 1) In normal B cells, the NADPH oxidase components p67phox, p40phox, p22phox, and gp91phox were less expressed than in polymorphonuclear neutrophils. 2) In normal B cells stimulated with PMA, p47phox, p67phox, and p40phox translocated to the membranes as occurs in polymorphonuclear neutrophils. 3) In CGD, B cells expressing p22phox in the absence of gp91phox, p47phox, p67phox, and p40phox did not translocate to the membranes after stimulation with PMA. 4) In PMA-stimulated B cells from an X91+ CGD patient in which p22phox was normally expressed and gp91phox was present but lacked five amino acids, translocation of p47phox to the membranes was unaffected, but p67phox and p40phox were poorly translocated, and the production of O2- was greatly reduced with respect to that by normal B cells. Taken together, these findings indicate that 1) a low expression of some NADPH oxidase components may represent the molecular basis of the low production of O2- in B lymphocytes; 2) the cytosolic components of NADPH oxidase cannot bind to p22phox on the membranes in the absence of gp91phox; 3) p47phox can translocate to the membranes independently of p67phox and p40phox; and 4) gp91phox may have a role in mediating and/or stabilizing the binding of p67phox and p40phox to the membranes of activated cells.

B-Lymphocytes↗
PubMed1998

Gene therapy of chronic granulomatous disease (CGD) by gene transfer into hematopoietic stem cells.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency characterized by severe recurrent bacterial and fungal infections of several organs. The disease is due to the inability of phagocytic leukocytes to generate reactive oxygen species upon phagocytosis. The defect arises as a consequence of mutations of the genes encoding for the subunits of a membrane NADPH oxidase, which catalyzes the production of superoxide anion (O2-). CGD represents an ideal candidate disorder for gene therapy, since the disease has a recessive inheritance, its phenotype is exclusively expressed in phagocytic cells, and a partial correction is likely to be effective. Given the short half-life of mature phagocytes, the optimal target cell population for gene transfer is the pluripotent hematopoietic stem cell. Transduction of CD34+ hematopoietic progenitors with retroviral vectors carrying the cDNA of the defective gene results in the correction of the enzymatic defect in myeloid cells differentiated in vitro. Still, the effective development of a clinical gene therapy protocol for this disease will await a substantial improvement in our current technology for the identification and manipulation of hematopoietic stem cells, and in our understanding of their biological and molecular properties.

Adenoviridae↗
PubMed1996

Functional reconstitution of oxidase activity in X-linked chronic granulomatous disease by retrovirus-mediated gene transfer.

The feasibility of correction of the disease phenotype by gene gene transfer was investigated in cells of four patients with X-linked chronic granulomatous disease. These patients carry point mutations of the gp91-phox gene, encoding for the large subunit of the catalytic core of the phagocytic cell NADPH oxidase. A retroviral vector expressing the gp91-phox protein was constructed and used to transduce lymphoblastoid cell lines established from the patients. Several transduced lymphoblastoid cell clones were investigated for mRNA and protein expression, and for functional reconstitution of oxidase activity. Although extensive quantitative variability was detected among different clones, functional reconstitution of O2- production was obtained in most cases, with oxidase function within the same range as in B cell lines derived from normal individuals. The same vector was also used for transduction of hematopoietic precursors from bone marrow or peripheral blood either with or without enrichment for CD34+ cells. A comprehensive analysis was performed on differentiated myeloid colonies, to evaluate the efficiency of transduction, the levels of gp91-phox expression, and the extent of functional reconstitution of oxidase activity. A high efficiency of transduction was obtained in most experiments, with 60-100% of colonies containing proviral DNA. Among the transduced colonies, an extensive variability in the levels of expression of the transduced gene and of functional restoration of NADPH oxidase activity was observed. These results represent a step toward the development of a gene therapy protocol for these patients.

3T3 Cells↗
PubMed1996

Rapid retrovirus titration using competitive polymerase chain reaction.

A quantitative polymerase chain reaction (PCR) procedure has been developed for rapid retrovirus titration. This procedure, which is based on the simultaneous amplification of the sample with known amounts of a competitor DNA fragment (competitive PCR), was used for the quantification of viral RNA genomes in retrovirus-producing cell clone supernatants and of proviral DNA molecules formed at 24 h after infection of different reference cell lines. The results obtained from the analysis of several samples indicated that proviral DNA quantification is in complete agreement with the number of selectable colonies in a standard colony assay. Conversely, the number of viral RNA genomes in the producer cell clone supernatants is a poor predictor of the actual efficiency of infection. Repeated competitive PCR experiments for provirus copy number determination at different times after transduction indicated that the number of proviral DNA molecules remains stable over time, suggesting stable integration into the host genome. The developed procedure is rapid and simple, is applicable to retroviral constructs not containing a selectable gene and can be used to directly measure the efficiency of infection of any target cell type, thus overcoming the problem of the dependency of retroviral titer determination on the rate of expression of a selectable gene and on the efficiency of colony formation of a reference cell line.

3T3 Cells↗
PubMed1995

Gene amplification for c-erbB-2, c-myc, epidermal growth factor receptor, int-2, and N-myc measured by quantitative PCR with a multiple competitor template.

We recently proposed a quantitative PCR procedure for the absolute measurement of c-erbB-2 oncogene amplification, based on the simultaneous polymerase chain reaction (PCR) amplification of the target gene and of a competitor DNA molecule acting as internal standard. To increase the number of assayable oncogenes and the accuracy of the quantitative comparison of gene amplification degree within the same tumor, we have now constructed a single synthetic competitor for int-2, c-myc, N-myc epidermal growth factor receptor, and c-erbB-2 genes, and for the reference gene beta-globin. This competitor was constructed by a two-step recombinant PCR procedure and inserted as a 297-bp sequence in a plasmid. The order of primer insertion was designed to obtain competitors of comparable sizes to those of the respective genomic targets, but still easily recognizable from the latter ones by gel electrophoresis. The clone competitor was tested to evaluate the linearity range for each assay. The present application of quantitative PCR based on a multiple competitor represents the first approach for the achievement of a single reagent for the evaluation of a panel of genes potentially amplified in human tumors.

Base Sequence↗
PubMed1994

Fine mapping of a replication origin of human DNA.

A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of mammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA fragments distributed along a given genomic region. This procedure was utilized for mapping the start site of DNA replication in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 cells. Within this region, DNA replication initiates in a 474-bp area corresponding to the 3' noncoding end of the lamin B2 gene and the nontranscribed spacer between this gene and the 5' end of another highly transcribed one. This localization was obtained both in aphidicolin-synchronized and in exponentially growing HL-60 cells.

Animals↗
PubMed1994

Measuring c-erbB-2 oncogene amplification in fresh and paraffin-embedded tumors by competitive polymerase chain reaction.

We present an original application of competitive polymerase chain reaction (PCR) for measuring oncogene amplification in DNA from human tumors by simultaneous PCR amplification of genomic DNA with fixed amounts of an internal standard (competitor DNA). Competitors share the same sequence as the target genes but contain an additional 15- to 20-base-pair insert, which allows resolution of the amplified products after polyacrylamide gel electrophoresis and ethidium bromide staining. The gene copy number is derived from the ratio between the intensities of the bands corresponding to the amplified products. Using this procedure, we measured c-erbB-2 amplification in breast and bladder carcinomas in both fresh tumor tissues and paraffin-embedded tissue samples and assessed the precision, sensitivity, and accuracy of the assay. Competitive PCR is a simple, reliable, and accurate method for the evaluation of c-erbB-2 amplification and is potentially suitable for use in the clinical laboratory.

Base Sequence↗
PubMed1993

Searching for replication origins in mammalian DNA.

The attempts at identifying precise replication origins (ori) in mammalian DNA have been pursued mainly through physico-chemical and biochemical approaches, in view of the essential failure of the search for autonomously replicating sequences in cultured cells. These approaches involve the mapping of short stretches of nascent DNA, the identification of the regions where either leading or lagging strands switch polarity, or the localization of replication intermediates by two-dimensional gel electrophoresis. Due to the complexity of animal cell genomes, most of these studies have been performed on amplified domains and with the use of synchronization procedures. The results obtained have been controversial. In order to avoid the use of experimental procedures potentially affecting the physiological mechanism of DNA replication, we have developed a method for the localization of ori in single-copy loci in exponentially growing cells. This method entails the absolute quantification of the abundance of selected DNA fragments along a genomic region within samples of newly synthesized DNA by competitive polymerase chain reaction (PCR); the latter is immune to all the uncontrollable variables which severely affect the reproducibility of conventional PCR. The application of this method to SV40 ori-driven plasmid replication precisely identifies the known ori localization. Using the same approach, we have mapped an ori for bi-directional DNA replication in a 13.7-kb locus of human chromosome 19 encoding lamin B2.

Animals↗
PubMed1993

Inhibition of heterologous DNA replication by the MVMp nonstructural NS-1 protein: identification of a target sequence.

The nonstructural protein NS-1 of minute virus of mice (MVMp), an autonomous parvovirus, trans-inhibits the replication of a chimeric plasmid containing the SV40 origin of replication (ori) embedded in the MVMp genome. It appears that a 157-bp 5' proximal sequence of MVMp DNA is sufficient, in the presence of NS-1, to cause the inhibition of DNA replication driven by the SV40 ori placed on the same molecule. This effect is not dependent on the orientation of the MVMp target sequence and results from both a reduced level of utilization of SV40 ori and the blockage of progressing replication forks at the level of the target. Furthermore, replication driven by Epstein-Barr virus origin (oriP) is trans-inhibited by MVMp but this inhibition does not require the presence of parvoviral sequences in cis. On the basis of sequence homologies between EBV oriP and MVMp 5' terminal sequence, it is proposed that the direct or indirect interaction of NS-1 with parvovirus-like sequences present in heterologous viral and possibly also cellular genomes may result in an inhibition of DNA replication.

Animals↗
PubMed1992

A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates.

A method is described for the absolute quantification by polymerase chain reaction (PCR) of nucleic acids present in low abundance. The method entails the addition to the sample of competitor DNA molecules that share the same sequence as the amplified target (including primer recognition sites), except for a 20-bp insertion in the middle, which allows easy resolution by gel electrophoresis (competitive PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial rate of targets, rendering the reaction independent of the number of amplification cycles. An easy and reliable method for the construction and quantification of competitive templates obtained as recombinant PCR products was developed. The technique was used for the absolute quantification of human genomic DNA with primers from a single copy, subtelomeric region of chromosome 19.

Base Sequence↗
PubMed1992

A human DNA replication origin: localization and transcriptional characterization.

A single-copy 13.7 kb human DNA region (L30E) located on Ch. 19 p13.3 contains an origin of DNA replication in myeloid HL-60 cells. The origin was localized, by means of quantitative PCR within approximately 3000 bp, in a highly transcribed region containing at least two closely spaced genes with the same polarity of transcription, one encoding lamin B2 and the other an unidentified protein. The origin region overlaps an undermethylated "CpG island" at the 5'-end of the second transcription unit. A binding site (CACGTG) for basic helix-loop-helix (bHLH) DNA binding proteins such as USF/MLTF or MYC-MAX was located by DNase I footprinting analysis in the promoter of the second gene. DMSO differentiation of HL-60 cells, that completely shuts off replication, also drastically reduces the transcription of L30E region. On the other hand such treatment does not modify the methylation pattern of the CpG island and does not abolish the DNase I protection of the bHLH binding site.

Base Sequence↗
PubMed1992

The gene for a novel human lamin maps at a highly transcribed locus of chromosome 19 which replicates at the onset of S-phase.

A previously described human DNA fragment which is replicated early in S-phase of HL-60 cell DNA (C. Tribioli, G. Biamonti, M. Giacca, M. Colonna, S. Riva, and A. Falaschi, Nucleic Acids Res. 15:10211-10232, 1987) was used to screen a genomic library in lambda Ch28. A clone which contained a 13.7-kb insert (L30E) found to code for several transcripts was isolated. The transcription of L30E DNA exhibited a complex pattern and a tissue-specific and proliferation-dependent type of regulation. The data were consistent with two tandemly arranged transcription units, the 3' end of one separated from the 5' end of the other by a sequence of about 600 bp containing an active promoter. The isolation and sequencing of L30E-specific cDNAs permitted identification of two genes, one of which encoded a B-type human lamin (analogous to mouse lamin B2). L30E DNA was mapped by in situ hybridization at the G-negative subtelomeric band p13.3 of chromosome 19. Interestingly, in synchronized HL-60 cells, L30E DNA is replicated in the first minute of S-phase. Replication of the lamin gene early in S-phase may reflect a coupling between early replication and transcription of genes for S-phase-specific proteins such as lamins.

Amino Acid Sequence↗
PubMed1990

Isolation in Europe of 69 M-like (serotype 8) human rotavirus strains with either subgroup I or II specificity and a long RNA electropherotype.

During an epidemiological study on the prevalence of human rotavirus (HRV) serotypes 1-4 in Europe, we found that some strains could not be typed. However, when a monoclonal antibody directed to serotype 8 HRV was included in the typing assay, we detected seven 69 M-like (serotype 8) strains, six from Finland and one from Italy. The previously reported serotype 8 HRV strains, 69 M, B 37, and B 38 isolated in Indonesia, were of subgroup I specificity and presented a peculiar "super short" RNA electropherotype. In contrast, all the seven European strains possessed a long RNA pattern, and one of them had subgroup II specificity. Three of these strains were adapted to growth in cell cultures and were further characterized by neutralization and by Northern blot hybridization. They appeared to be closely related to serotype 8 HRV strain 69 M by neutralization, but showed partial homology with several human and animal strains by hybridization. The epidemiological importance of these serotype 8 strains circulating in Europe should be investigated, in view of their possible inclusion in a rotavirus vaccine.

Blotting, Northern↗