Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep(+) hybrid amplicon vector.

Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep(+) hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep(+), rep(-) (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep(+) hybrid amplicon vector than with rep(-) or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep(+) hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep(-) or standard amplicon vectors resulted in random integrations of the entire rep(-) hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.

HSV-1 amplicon vector-mediated expression of ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype.

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and radiation sensitivity. Previous research has shown that it is possible to correct the hereditary deficiency A-T by DNA transfection in cell culture, but the large size of the ATM cDNA (9 kb) limits the use of many vector types for gene replacement. HSV-1 amplicon vectors provide a means to deliver large genes to cells efficiently and without toxicity. In this study, the FLAG-tagged cDNA for human ATM was inserted into an HSV-1 amplicon under control of the CMV promoter (designated as HGC-ATM). FLAG-ATM expression was confirmed in 293T/17 cells and human A-T fibroblasts (GM9607) after transduction, by immunoprecipitation, Western analysis, and immunocytochemistry. Functional recovery was assessed by two independent assays. First, in vitro kinase assay showed that vector-derived ATM in GM9607 cells could successfully phosphorylate wt p53 using recombinant GST-p53(1-101). Second, in A-T cells infected with the HGC-ATM vector, the extent of accumulation in G2/M phase at 24 h postirradiation was similar to that observed in cells with wild-type endogenous ATM and lower than that observed in A-T cells infected with a control vector. Thus, these vectors provide a tool to test the feasibility of HSV-amplicons as gene therapy vectors for A-T.

[Infantile acute hemorrhagic edema (IAHE). A report of a new clinical case]. / Edema Emorragico Infantile Acuto (EEIA). Descrizione di un nuovo caso clinico.

The authors describe a new case of infantile acute haemorrhagic oedema in a six-month-old child. Even being a vasculitis the authors do not retain to assimilate it to the Schönlein-Henoch syndrome.

[The risk of infection after a tick bite in an endemic area. The experience of the use of an effective protocol on a cohort of Tuscan children]. / Rischio di infezione dopo puntura di zecca in una zona endemica. Esperienza sull'applicazione di un protocollo operativo su una coorte di bambini toscani.

The authors suggest an executive protocol for the infections due to the sting of a tick in a child. They report the experience they had observing 92 children in the period between spring and autumn in the year 1996.

[Subdural effusion during bacterial meningitis]. / Versamento sottodurale in corso di meningite batterica.

Twelve children of age ranged from 4 to 34 months with Haemophilus influenzae type b meningitis treated at Meyer Hospital of Florence, were retrospectively reviewed. Eight patients had subdural effusion demonstrated with TC, RM and transfontanellar ultrasonography. All patients are cured without sequelae.

[Evaluation of pediatric patients hospitalized for acute diarrhea from 1990 to 1996]. / Valutazioni su pazienti pediatrici ricoverati per diarrea acuta dal 1990 al 1996.

Infectious diarrhea is a common disorder in children in Italy, which may lead to hospitalization especially during infancy. In order to obtain data about epidemiology and clinic pictures of acute diarrhea, the carts of 1295 paediatric outpatients, hospitalized for this pathology in the time between 1990-1996 at the Children's Hospital "Meyer" of Florence, were analyzed. An offending organism could be isolate in 43.3% of patients; Rotaviruses are the leading cause of diarrhea, followed by salmonella spp. Furthermore the role of Campylobacter as common bacterial pathogen worldwide has been clarified.

[The importance of C-reactive protein (CRP) in scarlet fever diagnosis (author's transl)]. / Importanza della proteina C reattiva (PCR) nella diagnostica della scarlattina.

Scarlet fever, especially in the mild and attenuated forms, proposes considerable diagnostic problems. The Authors, on the basis of their cases, remark the importance of C-reactive protein (CRP) positivity. After comparing the high initial positivity of this aspecific index of illness to the bacteriological pharyngeal findings and to the movement of antibodies against the Streptococcus, they think that CRP in scarlet fever may have a punctual significance, also for the ease and the quickness of this test.