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1.
J Hosp Infect ; 102(3): 332-336, 2019 Jul.
Article in English | MEDLINE | ID: mdl-30633945

ABSTRACT

BACKGROUND: Medico-legal conflicts arise when it is difficult to prove the cause of nosocomial infections. AIM: To report an outbreak of patient-to-patient transmission of hepatitis C virus (HCV) through the repeated use of a multi-dose saline flask during the rinsing of central venous catheters. METHODS: Blood samples were taken from each patient for the comparative analysis of their HCV RNA strains. No samples were available for one patient who died before the investigation started. Despite the known lability of HCV RNA, the body was exhumed four months after burial and postmortem samples were collected. HCV RNA was extracted successfully from liver and spleen samples. Genotyping of all the HCV strains was performed by sequence analysis of the 5'NC untranslated region, the E1 core conserved region and the E1/E2 hypervariable region. FINDINGS: Forensic investigators retraced the route used by two ward nurses, when saline catheter flushes were given to 14 patients with each nurse administering to seven patients. The comparative phylogenetic analysis of all case strains identified the deceased patient as the source of contamination to five patients. CONCLUSIONS: This study highlights the value of sequence analysis as a tool for solving medico-legal conflicts. The High Court of Justice found that a health worker's re-use of a contaminated needle resulted in the nosocomial transmission of HCV.


Subject(s)
Cross Infection/epidemiology , Cross Infection/transmission , Disease Transmission, Infectious , Hepacivirus/isolation & purification , Hepatitis C/epidemiology , Hepatitis C/transmission , Adult , Aged , Aged, 80 and over , Cross Infection/mortality , Exhumation , Female , Genotype , Genotyping Techniques , Hepacivirus/classification , Hepacivirus/genetics , Hepatitis C/mortality , Humans , Male , Molecular Epidemiology , RNA, Viral/genetics , RNA, Viral/isolation & purification , Sequence Analysis, DNA
2.
Clin Microbiol Infect ; 21(12): 1122.e1-10, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26235197

ABSTRACT

In recent years, West Nile virus (WNV) lineage 2 has been spreading and causing disease outbreaks in humans and animals in Europe. In order to characterize viral diversity, we performed full-length genome sequencing of WNV lineage 2 from human samples collected during outbreaks in Italy and Greece in 2013 and 2014. Phylogenetic analysis showed that these WNV lineage 2 genomes belonged to a monophyletic clade derived from a single introduction into Europe of the prototype Hungarian strain. Correlation of phylogenetic data with geospatial information showed geographical clustering of WNV genome sequences both in Italy and in Greece, indicating that the virus had evolved and diverged during its dispersal in Europe, leading to the emergence of novel genotypes, as it adapted to local ecological niches. These genotypes carried divergent conserved amino acid substitutions, which might have been relevant for viral adaptation, as suggested by selection pressure analysis and in silico and experimental modelling of sequence changes. In conclusion, the results of this study provide further information on WNV lineage 2 transmission dynamics in Europe, and emphasize the need for WNV surveillance activities to monitor viral evolution and diversity.


Subject(s)
Disease Outbreaks , RNA, Viral/genetics , West Nile Fever/epidemiology , West Nile virus/classification , West Nile virus/genetics , Amino Acid Substitution , Evolution, Molecular , Genome, Viral , Greece , Humans , Italy , Models, Molecular , Phylogeny , Phylogeography , Sequence Analysis, RNA , West Nile Fever/transmission
3.
J Cell Physiol ; 230(8): 1794-806, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25510868

ABSTRACT

Similar to phosphorylation, transient conjugation of ubiquitin to target proteins (ubiquitination) mediated by the concerted action of ubiquitin ligases and de-ubiquitinating enzymes (DUBs) can affect substrate function. As obligate intracellular parasites, viruses rely on different cellular pathways for their own replication and the well conserved ubiquitin conjugating/de-conjugating system is not an exception. Viruses not only usurp the host proteins involved in the ubiquitination/de-ubiquitination process, but they also encode their own ubiquitin ligases and DUBs. Here we report that an N-terminal variant of the herpes simplex virus (HSV) type-1 large tegument protein VP1/2 (VP1/2(1-767)), encompassing an active DUB domain (herpesvirus tegument ubiquitin specific protease, htUSP), and TSG101, a component of the endosomal sorting complex required for transport (ESCRT)-I, functionally interact. In particular, VP1/2(1-767) modulates TSG101 ubiquitination and influences its intracellular distribution. Given the role played by the ESCRT machinery in crucial steps of both cellular pathways and viral life cycle, the identification of TSG101 as a cellular target for the HSV-1 specific de-ubiquitinating enzyme contributes to the clarification of the still under debate function of viral encoded DUBs highly conserved throughout the Herpesviridae family.


Subject(s)
DNA-Binding Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Host-Parasite Interactions/physiology , Simplexvirus/pathogenicity , Transcription Factors/metabolism , Ubiquitin-Specific Proteases/metabolism , Viral Proteins/metabolism , Animals , Chlorocebus aethiops , Humans , Immunoprecipitation , Microscopy, Confocal , Real-Time Polymerase Chain Reaction , Simplexvirus/metabolism , Ubiquitination , Vero Cells
4.
Gene Ther ; 21(3): 272-81, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24430237

ABSTRACT

Corneal graft rejection is a major problem in chronic herpetic keratitis (HK) patients with latent infection. A new class of antiviral agents targeting latent and active forms of herpes simplex virus type 1 (HSV-1) is importantly required. Meganucleases are sequence-specific homing endonucleases capable of inducing DNA double-strand breaks. A proof-of-concept experiment has shown that tailor-made meganucleases are efficient against HSV-1 in vitro. To take this work a step forward, we hypothesized that the pre-treatment of human corneas in eye banks using meganuclease-encoding vectors will allow HK patients to receive a medicated cornea to resist the recurrence of the infection and the common graft rejection problem. However, this strategy requires efficient gene delivery to human corneal endothelium. Using recombinant adeno-associated virus, serotype 2/1 (rAAV2/1), efficient gene delivery of a reporter gene was demonstrated in human corneas ex vivo. The optimum viral dose was 3.7 × 10(11) VG with an exposure time of 1 day, followed by 6 days incubation in de-swelling medium. In addition, 12 days incubation can result in transgene expression in excess of 70%. Using similar transduction conditions, meganuclease transgene expression was detected in 39.4% of the endothelial cells after 2 weeks in culture. Reduction of the total viral load in the media and the endothelial cells of corneas infected with HSV-1 was shown. Collectively, this work provides information about the optimum conditions to deliver genetic material to the cornea, and demonstrates for the first time the expression of meganuclease in human corneas ex vivo and its antiviral activity. In conclusion, we demonstrate that the treatment of human corneas in eye banks before transplantation is a new approach to address the unmet clinical needs in corneal diseases.


Subject(s)
Cornea/metabolism , Deoxyribonuclease I/genetics , Viral Proteins/genetics , Deoxyribonuclease I/metabolism , Dependovirus/genetics , Dependovirus/metabolism , Gene Transfer Techniques , Genes, Reporter/genetics , Herpesvirus 1, Human/enzymology , Humans , In Vitro Techniques , Viral Proteins/metabolism
5.
Euro Surveill ; 18(38)2013 Sep 19.
Article in English | MEDLINE | ID: mdl-24084339

ABSTRACT

A human outbreak of West Nile virus (WNV) infection caused by WNV lineage 2 is ongoing in northern Italy. Analysis of six WNV genome sequences obtained from clinical specimens demonstrated similarities with strains circulating in central Europe and Greece and the presence of unique amino acid changes that identify a new viral strain. In addition, WNV lineage 1 Livenza, responsible for a large outbreak in north-eastern Italy in 2012, was fully sequenced from a blood donor during this 2013 outbreak.


Subject(s)
RNA, Viral/genetics , West Nile Fever/genetics , West Nile virus/classification , West Nile virus/genetics , Base Sequence , Disease Outbreaks , Genome , Humans , Italy/epidemiology , Molecular Epidemiology , Phylogeny , West Nile Fever/diagnosis , West Nile Fever/epidemiology , West Nile Fever/virology
6.
Clin Microbiol Infect ; 19(10): E428-34, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23573945

ABSTRACT

Accurate HPV typing is essential for evaluation and monitoring of HPV vaccines, for second-line testing in cervical cancer screening, and in epidemiological surveys. In this study, we set up and assessed in clinical samples a new HPV typing method based on 454 next-generation sequencing (NGS) of HPV L1 amplicons, generated by using a modified PGMY primer set with improved sensitivity for some HPV types that are not targeted by standard PGMY primers. By using a median 12 800-fold coverage, the NGS method allowed us to correctly identify all high-risk HPV types, in either single or multiple infections, with a sensitivity of 50 genome equivalents, as demonstrated by testing WHO LabNet EQA sample panels. Analysis of mixtures of HPV16- and HPV18-positive cell lines demonstrated that the NGS method could reproducibly quantify the proportion of each HPV type in multiple infections in a wide dynamic range. Testing of HPV-positive clinical samples showed that NGS could correctly identify a high number of HPV types in multiple infections. The NGS method was also effective in the analysis of a set of cervical specimens with discordant results at hybrid capture 2 and line probe assays. In conclusion, a new HPV typing method based on 454 pyrosequencing was set up. This method was sensitive, specific, quantitative and precise in both single and multiple infections. It could identify a wide range of HPV types and might potentially discover new HPV types.


Subject(s)
High-Throughput Nucleotide Sequencing/methods , Molecular Typing/methods , Papillomaviridae/classification , Papillomaviridae/genetics , Papillomavirus Infections/virology , Genitalia/virology , Genotype , Humans , Papillomaviridae/isolation & purification , Papillomavirus Infections/diagnosis , Reproducibility of Results , Sensitivity and Specificity
7.
Euro Surveill ; 17(36): 20260, 2012 Sep 06.
Article in English | MEDLINE | ID: mdl-22971328

ABSTRACT

In July-September 2012, one month earlier than in previous years, 13 confirmed human cases of West Nile virus infection were diagnosed in northern Italy, including five with neuroinvasive disease, three with West Nile fever, and five West Nile virus (WNV)-positive blood donors. In nine cases, the presence of the WNV lineage 1a Livenza strain, characterised in 2011, was ascertained. Symptomatic patients had prolonged viruria with high viral load.


Subject(s)
Disease Outbreaks , RNA, Viral/genetics , West Nile Fever/virology , West Nile virus/genetics , Blood Donors , Follow-Up Studies , Humans , Italy/epidemiology , Population Surveillance/methods , Real-Time Polymerase Chain Reaction , Sequence Analysis , Viral Load , West Nile Fever/epidemiology , West Nile Fever/genetics , West Nile virus/isolation & purification
8.
Clin Microbiol Infect ; 18(12): E541-4, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23004685

ABSTRACT

During 2008-2009, several human cases of WNV disease caused by an endemic lineage 1a strain were reported in areas surrounding the Po river in north-eastern Italy. Since 2010, cases have been recorded in nearby northern areas, where, in 2011, both lineage 1a and 2 were detected. We describe here two new WNV complete genome sequences from human cases of WNV infection occurring in 2011 in the Veneto Region. Phylogenetic analysis showed that both genome sequences belonged to lineage 1a and were related to WNV strains of the Western Mediterranean subtype. The novel WNV genomes had high nucleotide and amino acid sequence divergence from each other and from the WNV strain circulating in Italy in 2008-2009. The presence of different WNV strains in a relatively small geographical area is a novel finding with unpredictable impact on human disease that requires further investigation.


Subject(s)
Genome, Viral , RNA, Viral/genetics , Sequence Analysis, DNA , West Nile Fever/virology , West Nile virus/genetics , Genetic Variation , Genotype , Humans , Italy , Molecular Sequence Data , Phylogeny , West Nile virus/classification , West Nile virus/isolation & purification
9.
Euro Surveill ; 17(31)2012 Aug 02.
Article in English | MEDLINE | ID: mdl-22874456

ABSTRACT

We report here the first blood donation positive for West Nile virus (WNV) by nucleic acid amplification testing collected in north-eastern Italy in July 2012.Partial sequencing of the WNV RNA demonstrated identity with a WNV lineage 1a genome identified in the same area in 2011 and divergence from the strain responsible for the outbreak in northern Italy in 2008­09. These data indicate that WNV activity in northern Italy is occurring earlier than expected and that different WNV strains are circulating.


Subject(s)
Blood Donors , RNA, Viral/genetics , West Nile Fever/virology , West Nile virus/genetics , Endemic Diseases , Humans , Italy/epidemiology , Nucleic Acid Amplification Techniques , Phylogeny , Population Surveillance , Sequence Analysis , West Nile Fever/epidemiology , West Nile Fever/genetics
10.
Euro Surveill ; 16(33)2011 Aug 18.
Article in English | MEDLINE | ID: mdl-21871228

ABSTRACT

In 2010, for the third consecutive year, human cases of West Nile virus (WNV) infection, including three confirmed cases of neuroinvasive disease and three confirmed cases of West Nile fever, were identified in north-eastern Italy. While in 2008 and 2009 all human cases of WNV disease were recorded in the south of the Veneto region, cases of WNV disease in 2010 additionally occurred in two relatively small northern areas of Veneto, located outside those with WNV circulation in the previous years. WNV IgG antibody prevalence in blood donors resident in Veneto was estimated as ranging from 3.2 per 1,000 in areas not affected by cases of WNV disease to 33.3 per 1,000 in a highly affected area of the Rovigo province. No further autochthonous human cases of WNV disease were notified in Italy in 2010. The recurrence of human cases of WNV infection for the third consecutive year strongly suggests WNV has become endemic in north-eastern Italy.


Subject(s)
Antibodies, Viral/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , West Nile Fever/epidemiology , West Nile virus/isolation & purification , Adult , Aged , Animals , Antibodies, Viral/immunology , Blood Donors , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Italy/epidemiology , Male , Middle Aged , Population Surveillance , Prevalence , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Seroepidemiologic Studies , West Nile Fever/diagnosis , West Nile Fever/virology , West Nile virus/immunology
11.
Euro Surveill ; 16(10)2011 Mar 10.
Article in English | MEDLINE | ID: mdl-21435323

ABSTRACT

Following reports of West Nile neuroinvasive disease in the north-eastern area of Italy in 2009, all blood donations dating from the period between 1 August and 31 October 2009 in the Rovigo province of the Veneto region were routinely checked to exclude those with a positive nucleic acid test for West Nile virus (WNV). Only one of 5,726 blood donations was positive (17.5 per 100,000 donations; 95% confidence interval (CI): 0.4­97.3). In addition, a selection of 2,507 blood donations collected during the period from 20 July to 15 November 2009 were screened by ELISA for IgG and IgM antibodies against WNV. A positive result was received for 94 of them. The positive sera were further evaluated using immunofluorescence and plaque reduction neutralisation test (PRNT), in which only 17 sera were confirmed positive. This corresponds to a prevalence of 6.8 per 1,000 sera (95% CI: 4.0­10.9). In a case-control study that matched each of the 17 PRNT-positive sera with four negative sera with the same date of donation and same donation centre, we did not find a significant association with age and sex of the donor; donors who worked mainly outdoors were significantly more at risk to have a positive PRNT for WNV.


Subject(s)
Antibodies, Viral/blood , Blood Donors , Immunoglobulin G/blood , Immunoglobulin M/blood , West Nile Fever/epidemiology , West Nile virus/isolation & purification , Adult , Aged , Antibodies, Viral/immunology , Enzyme-Linked Immunosorbent Assay , Female , Fluorescent Antibody Technique , Hemolytic Plaque Technique , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Incidence , Italy/epidemiology , Male , Middle Aged , Neutralization Tests , Prevalence , Retrospective Studies , Sensitivity and Specificity , Seroepidemiologic Studies , West Nile Fever/diagnosis , West Nile Fever/virology , West Nile virus/immunology
12.
Infection ; 39(2): 127-33, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21327628

ABSTRACT

BACKGROUND: Outbreaks of vancomycin-resistant Enterococcus faecium (VRE) strains is an emerging problem worldwide. Even if still relatively uncommon in European hospitals, infections caused by VRE have also been increasing recently in this continent. METHODS: In this study, we characterized 50 consecutive VRE and 23 vancomycin-sensitive E. faecium (VSE) isolates collected in an Italian hospital. The presence of the esp gene and that of genes encoding resistance to glycopeptides was investigated by polymerase chain reaction (PCR). All of the isolates were typed by multi-locus sequence typing (MLST), and a selection of them also by pulsed-field gel electrophoresis (PFGE). RESULTS: We found that all of the VRE and 18 (78%) of the VSE strains belonged to the single clonal complex-17 (CC17). The most represented sequence type (ST) was ST78 (34% of the isolates). When further analyzed by PFGE, ST78 isolates were subdivided into five pulsotypes, four of them closely related. The strong association between the esp gene and CC17 was confirmed. Interestingly, such an association was higher among vancomycin-resistant isolates. Most of the esp-positive isolates (34/46, 74%) encoded Esp4, a rare variant of this protein characterized by the absence of A repeats. CONCLUSIONS: Our findings underscore the role of the CC17 lineage in the nosocomial spread of VRE and VSE, and its rapid local evolution, underscoring the need for programs designed to provide early detection in order to prevent its spreading among the nosocomial population.


Subject(s)
Cross Infection/epidemiology , Enterococcus faecium/classification , Enterococcus faecium/genetics , Gram-Positive Bacterial Infections/epidemiology , Anti-Bacterial Agents/pharmacology , Bacterial Typing Techniques , Cluster Analysis , Cross Infection/microbiology , Electrophoresis, Gel, Pulsed-Field , Enterococcus faecium/drug effects , Enterococcus faecium/isolation & purification , Genes, Bacterial , Genotype , Gram-Positive Bacterial Infections/microbiology , Hospitals , Humans , Italy , Microbial Sensitivity Tests , Molecular Epidemiology , Multilocus Sequence Typing , Vancomycin/pharmacology
13.
Euro Surveill ; 14(44)2009 Nov 05.
Article in English | MEDLINE | ID: mdl-19941775

ABSTRACT

In 2009, six new human cases of West Nile neuroinvasive disease (WNND) were identified in Veneto region, following the six cases already reported in 2008. A human West Nile virus (WNV) isolate was obtained for the first time from an asymptomatic blood donor. Whole genome sequence of the human WNV isolate showed close phylogenetic relatedness to the Italy-1998-WNV strain and to other WNV strains recently isolated in Europe, with the new acquisition of the NS3-Thr249Pro mutation, a trait associated with avian virulence, increased virus transmission, and the occurrence of outbreaks in humans.


Subject(s)
Base Sequence , Genome , West Nile virus/genetics , West Nile virus/isolation & purification , Amino Acid Sequence , Disease Outbreaks , Humans , Italy , Molecular Sequence Data , Phylogeny , West Nile Fever/epidemiology
14.
Euro Surveill ; 14(31)2009 Aug 06.
Article in English | MEDLINE | ID: mdl-19660244

ABSTRACT

We report here an update on human cases of West Nile virus (WNV) infection in Veneto region, northeastern Italy. In addition to two cases of WNV neuroinvasive disease notified through a surveillance programme started in September 2008, further four cases were retrospectively identified (in May 2009) by investigating patients with aseptic meningoencephalitis of unknown aetiology occurring in Veneto region in June-September 2008. All six patients had symptom onset in August-September 2008 and were resident in a wetland area close to the Po river delta in Rovigo province. Further five cases of asymptomatic WNV infection, including four residents of the same area in Rovigo, were identified in a seroprevalence study in farm workers from Veneto region. To date, no human cases have been notified in 2009.


Subject(s)
Animal Diseases/virology , Meningoencephalitis/virology , Population Surveillance , West Nile Fever , West Nile virus/isolation & purification , Adult , Aged , Aged, 80 and over , Animal Diseases/epidemiology , Animals , Female , Horses , Humans , Italy/epidemiology , Male , Meningoencephalitis/cerebrospinal fluid , Meningoencephalitis/epidemiology , Middle Aged , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction , Seroepidemiologic Studies , West Nile Fever/blood , West Nile Fever/epidemiology , West Nile Fever/virology , West Nile virus/immunology , Young Adult
15.
Mycoses ; 51(6): 497-504, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18331444

ABSTRACT

A reliable diagnosis of invasive aspergillosis (IA) is hampered by the difficulty in obtaining suitable tissue samples. To evaluate the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the LightCycler PCR for the diagnosis of IA, 536 blood samples were collected over a 22-month period from 62 paediatric patients (median age 10 years, range 1-18) considered at risk of IA. The galactomannan antigen (GM) and fungal DNA were assessed on serial blood samples. IA was diagnosed in eight of 62 patients (13%): proven, five, probable, three. Sensitivity, specificity, PPV and NPV of LightCycler PCR varied according to the number of positive samples used to define positivity: 88%; 37%; 17% and 95% for single sample positivity; and 63%, 81%, 33% and 94% for serial sample positivity respectively. The concordance between positivity of LightCycler PCR assay and the diagnosis of IA was 79%. The single positivity of LightCycler PCR assay showed a good sensitivity for the diagnosis of IA in paediatric patients. The high NPV makes LightCycler PCR a promising tool in addition to GM testing to design a strategy of pre-emptive antifungal therapy, although further validation studies are needed.


Subject(s)
Aspergillosis/diagnosis , Hematologic Neoplasms/complications , Polymerase Chain Reaction/methods , Adolescent , Child , Child, Preschool , DNA, Fungal/blood , Female , Galactose/analogs & derivatives , Humans , Infant , Male , Mannans/blood , Predictive Value of Tests , Sensitivity and Specificity
16.
Croat Med J ; 42(4): 473-7, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11471203

ABSTRACT

Gene therapy of cancer has become a major interest of medical research since more than 60% of the ongoing gene therapy protocols today involve cancer patients. To increase the therapeutic index of cancer gene therapy, targeting strategies have been developed to ensure that the expression of therapeutic genes is restricted exclusively to the tissue of interest. An attractive approach lies in the possibility to control the expression of therapeutic genes at the transcriptional level by the introduction of tissue-specific or tumor-specific enhancers/promoters offers. We have developed transcriptionally targeted vectors for gene therapy of solid tumors, including malignant gliomas and epithelial thyroid tumors. The choice of these tumor types relies on their clinical impact, ie, morbidity and mortality, the lack of effective conventional therapeutic strategies, and the ability of these tumors to express tissue/tumor-specific genes, whose transcriptional control elements (enhancer/promoter) may be used for achieving selective transgene expression. Here we report our clinical and preclinical experience in gene therapy of brain and thyroid tumors, and review the literature published on this topic.


Subject(s)
Brain Neoplasms/therapy , Gene Targeting , Genetic Therapy , Glioblastoma/therapy , Brain Neoplasms/genetics , Glioblastoma/genetics , Humans , Thyroid Neoplasms/genetics , Thyroid Neoplasms/therapy , Transcription, Genetic
17.
Gene Ther ; 6(3): 330-7, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10435083

ABSTRACT

Retrovirus-mediated gene therapy is a particularly attractive approach for glioblastoma multiforme (GBM), given the poor prognosis of this tumour and its localized proliferation in post-mitotic tissue. In this study we assessed, for the first time in humans, the therapeutic potential of a newly designed bicistronic Moloney vector (pLIL-2-TK), combining the expression of a suicide gene (thymidine kinase, tk) with an immunomodulatory gene (human interleukin 2, IL-2). Evidence of transgene activity in the treated tumours is presented.


Subject(s)
Brain Neoplasms/therapy , Genetic Therapy/methods , Glioblastoma/therapy , Interleukin-2/genetics , Thymidine Kinase/genetics , Transfection/methods , Adult , Brain/pathology , Brain Neoplasms/pathology , Female , Gene Expression , Genetic Vectors/administration & dosage , Glioblastoma/pathology , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Pilot Projects , Retroviridae/genetics
18.
Gene Ther ; 5(7): 1003-7, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9813672

ABSTRACT

Gene-based therapeutic strategies for cancer mainly include augmentation of immunotherapeutic and chemotherapeutic approaches. In this study we report the design and functional assay of a novel bicistronic Moloney-based retroviral vector expressing human interleukin-2 (IL-2) and herpesvirus thymidine kinase (tk) through a cap-dependent translation and an internal ribosome entry site (IRES)-regulated translation, respectively. This construct has the potential for allowing combination of cytokine and suicide gene therapy, especially in areas such as the brain, composed of post-mitotic cells refractory to transduction by type C retroviral vectors. Accordingly, human glioma cells were used as targets for gene transfer after selecting a packaging cell clone that produced a reasonable titer of recombinant virus and expressed high levels of IL-2 and tk transcripts. Although transduction efficiency was reduced in glioma cells as compared with murine NIH 3T3 cells, transgene expression was effectively achieved. Transduced glioma cells were sensitive to ganciclovir and secreted around 1000 U/ml IL-2 in the culture supernatants. Simultaneous production of IL-2 and tk in vivo by genetically treated tumor cells would hopefully potentiate the effect of gangiclovir-induced metabolic suicide, possibly by boosting the immune response associated with tumor debulking or by amplifying the bystander response.


Subject(s)
Genetic Engineering , Genetic Therapy/methods , Genetic Vectors , Interleukin-2/genetics , Moloney murine sarcoma virus , Neoplasms/therapy , Thymidine Kinase/genetics , Antimetabolites/pharmacology , Ganciclovir/pharmacology , Gene Expression , Gene Transfer Techniques , Glioma , Humans , Interleukin-2/metabolism , Tumor Cells, Cultured/drug effects
19.
Stereotact Funct Neurosurg ; 68(1-4 Pt 1): 245-51, 1997.
Article in English | MEDLINE | ID: mdl-9711724

ABSTRACT

Four patients affected by glioblastoma recurrence were treated with a gene therapy-immunotherapy protocol consisting of intratumoral injections of culture cells producing a retroviral vector which expresses human interleukin-2 and the herpes simplex virus thymidine kinase genes. Seven to 14 days after implantation, the patients were treated with ganciclovir at standard doses. Anatomopathological and immunohistochemical data confirm the efficacy of transduction. From the clinical point of view, gene therapy combined with immunotherapy demonstrated safety and a short-range but clearcut oncolytic effect.


Subject(s)
Brain Neoplasms/therapy , Genetic Therapy , Glioma/therapy , Brain Neoplasms/pathology , Brain Neoplasms/surgery , Clinical Protocols , Combined Modality Therapy , Female , Ganciclovir/therapeutic use , Gene Expression , Gene Transfer Techniques , Glioblastoma/immunology , Glioblastoma/pathology , Glioblastoma/therapy , Glioma/pathology , Glioma/surgery , Herpes Simplex/enzymology , Herpes Simplex/genetics , Humans , Immunotherapy , Injections, Intralesional , Interleukin-2/administration & dosage , Interleukin-2/immunology , Male , Middle Aged , Recurrence , Stereotaxic Techniques , Thymidine Kinase/genetics , Thymidine Kinase/metabolism , Treatment Outcome , Tumor Cells, Cultured/transplantation
20.
Gene Ther ; 4(11): 1216-24, 1997 Nov.
Article in English | MEDLINE | ID: mdl-9425445

ABSTRACT

HIV-1 infection results in the loss of CD4+ T helper lymphocytes which make up the immune repertoire. This leads to opportunistic infections that define AIDS. Here, we show that CD4 T cell lines from normal donors with specificity for different antigens can be rendered resistant to HIV-1 replication by retroviral transduction with an antisense vector directed to the HIV-1 tat gene. The genetic treatment did not affect the properties of antigen-specific CD4 lymphocytes such as proliferative response, lymphokine production and phenotypic markers. The HIV-1 challenge dose that resulted in productive infection was two to four logs higher for transduced cells as compared with control cells. Resistance was shown with the HXB2 strain, whose tat sequence was used to design the antisense gene, and with the SF2 strain, whose targeted tat sequence carries five nucleotide mismatches. Retroviral transduction was also performed on a Candida-specific T cell line from a seropositive individual. This line, derived from T cells infected in vivo, produced infectious virus when stimulated in vitro with antigen, but was no longer productive after transduction. In addition, a four log higher HIV-1 challenge dose was needed for a productive superinfection of this T cell line. The production of antigen-specific CD4 T cells resistant to HIV-1 replication to be used in adoptive immunotherapy of opportunistic infections may represent a new form of gene therapy of AIDS.


Subject(s)
AIDS-Related Opportunistic Infections/therapy , Adoptive Transfer/methods , CD4-Positive T-Lymphocytes/immunology , Epitopes , Gene Products, tat/genetics , Genetic Therapy/methods , Cell Line , Gene Transfer Techniques , Genetic Vectors , HIV/physiology , Humans , RNA, Antisense , RNA, Transfer , Retroviridae , Virus Replication , tat Gene Products, Human Immunodeficiency Virus
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